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GB2581394A - Pyrrolobenzodiazepines and conjugates thereof - Google Patents

Pyrrolobenzodiazepines and conjugates thereof Download PDF

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Publication number
GB2581394A
GB2581394A GB1902182.3A GB201902182A GB2581394A GB 2581394 A GB2581394 A GB 2581394A GB 201902182 A GB201902182 A GB 201902182A GB 2581394 A GB2581394 A GB 2581394A
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methyl
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alkyl
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Wilson Howard Philip
Christopher Kemp Gary
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MedImmune Ltd
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MedImmune Ltd
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
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    • A—HUMAN NECESSITIES
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68035—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a pyrrolobenzodiazepine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6863—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from stomach or intestines cancer cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

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Abstract

A compound of formula I: and salts and solvates thereof, wherein R2, R2’, R30, R31, R40, R41, R6, R6’, R7’, R9, R9’, R’’, Y, Y’ are as defined therein and where the bonds linking C2/C3 and C2’/C3’ may be single or double bonds; R7 is of formula A1: wherein Z is a C1-3 alkylene group and RL is a linker for connection to a cell binding agent as defined therein. Also disclosed are conjugates comprising the above compound, the cell binding agent being an antibody or an active fragment thereof.

Description

PYRROLOBENZODIAZEPINES AND CONJUGATES THEREOF
The present invention relates to pyrrolobenzodiazepines (PBDs), and their inclusion in targeted conjugates. The PBDs of the present invention are dimers linked to a targeting agent by via the C7 position.
Background to the invention Pyrrolobenzodiazepines
Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, at aL, J Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, at al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)).
Family members include abbeymycin (Hochlowski, et al., J Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et al., J Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58180 487; Thurston, at al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et a/., J Antibiotics, 41, 1366-1373 (1988)), prothracarcin (Shimizu, et a/, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al., J Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et aL, J. Am. Chem. Soc., 110, 29922993 (1988)) and tomamycin (Arima, at al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure: They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N=C), a carbinolamine(NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral Dila position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III.
Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc.
Chem. Res., 19, 230-237 (1986)). Their ability to form an adduct in the minor groove, enables them to interfere with DNA processing, hence their use as antitumour agents.
A particularly advantageous pyrrolobenzodiazepine compound is described by Gregson et all (Chem. Commun. 1999, 797-798) as compound 1, and by Gregson et al. (J. Med. Chem. 2001, 44, 1161-1174) as compound 4a. This compound, also known as SG2000, is shown below:
O
OMe Me0 SG2000 WO 2007/085930 describes the preparation of dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody. The linker is present in the bridge linking the monomer PBD units of the dimer.
Dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody, are described in WO 2011/130598. The linker in these compounds is attached to one of the available N10 positions, and are generally cleaved by action of an enzyme on the linker group. More recently, the warhead: has been used in drug linkers and antibody-drug conjugates. WO 2014/057074 discloses: H N 0 The drug-linker in the above conjugate is termed tesirine, and conjugates comprising it are undergoing clinical trials.
A number of disclosures, such as US2014/155590, W02014/159981, W02014/140862, and WO 2016/038383 disclose PBD dimers linked from an aromatic group in the tether, such as compound 33 in WO 2014/159981 as well as intermediates in their synthesis.
Other disclosures, such as W02011/130613, W02011/130616, W02013/053783 and W02014/057073 disclosure PBD dimers linked from the C2 position to an antibody. One of the conjugates described in W02011/130613 that comprises: (termed SGN-CD33A, where the drug linker is known as tairine) is also undergoing clinical trials.
W02016/067644 discloses PBD dimers linked to antibodes through the C7 position on one of the PBD moieties. Two particular formulae of linker are taught by this publication:
O n H (A1)
0 O N.0° -**---/
H
CB: \ -L I \\'' 1 \ Oh ?)
Q -N
H (A2)
Summary of the Invention
In a general aspect the present invention provides a conjugate comprising a PBD compound with a linker for connecting to a cell binding agent, wherein the linker is attached in a cleavable manner to the C7 position of the one PBD units. The cell binding agent is preferably an antibody. The invention also provides the PBD compound with the linking unit attached, and intermediates for their synthesis.
The linker used at the C7 position differs from A2 of W02016/067644 by having a methyl group on the amido moiety closest to the PBD. On cleavage of the linker, the payload is more protonatable, and thus more hydrophilic.
A first aspect of the present invention comprises a compound with the formula I: R2 and salts and solvates thereof, wherein: R7 is of formula Al: Ry0..,,tyN"'"-z' 0 (A1) wherein Z is a C1.3 alkylene group and RI-is a linker for connection to a cell binding agent, which is selected from: (ia): H 0
GL la
wherein Q is: cr=9- QX
NH-Z
H
0, where Qx is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue; X is:
N -b H a d
C
where a = 0 to 5, b = 0 to 16, c = 0 or 1, d = 0 to 5; GL is a linker for connecting to a Ligand Unit; OW: lb NO2] where RI-1 and RI-2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group; and e is 0 or 1; R" is a C3.12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. 0, S, NR' (where R"2 is H or C1_4 alkyl), and/or aromatic rings, e.g. benzene or pyridine; Y and Y' are selected from 0, S, or NH; when there is a double bond present between C2 and C3, R2 is selected from the group consisting of: (ha) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1.7 alkyl, C3.7 heterocyclyl and bis-oxy-Cis alkylene; (iib) Ci.5 saturated aliphatic alkyl; (iiC) C3-6 saturated cycloalkyl; R12 (iid) R11 wherein each of R11, R12 and., n r<13 are independently selected from H, C1.3 saturated alkyl, C2.3 alkenyl, C2.3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2 group is no more than 5; R15b 15a (iie) , wherein one of R15a and R1' is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and R14 where R14 is selected from: H; C1-3 saturated alkyl; 02_3 alkenyl; 02-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2 and 03, R16a 6b R2R2 is H or R, where IR1" and Rl" are independently selected from H, F, C1.4 saturated alkyl, C2_3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C14 alkyl ester; or, when one of IR16a and IRlab is H, the other is selected from nitrile and a C14 alkyl ester; when there is a double bond present between C2' and C3', R2' is selected from the group consisting of: (ilia) Cs_ici aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1_7 alkyl, C3_7 heterocyclyl and bis-oxy-C14 alkylene; (iiib) 01-5 saturated aliphatic alkyl; (iiic) C3_6 saturated cycloalkyl; R22 7..fR23, wherein each of R21, R22 and R23 are independently selected from H, (Hid) R21 Ci.3 saturated alkyl, 02-3 alkenyl, 02-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2' group is no more than 5; R25b (Hie) ,R25a, wherein one of R25° and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and 2 4 (iiif) , where R24 is selected from: H; C1-3 saturated alkyl; C24 alkenyl; C24 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2' and C3', R26 a R2' is H or R26b, where R26a and R26b are independently selected from H, F, C1.4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1_4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester; R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, N RR', nitro, Me3Sn and halo; where R and R' are independently selected from optionally substituted 01-12 alkyl, C420 heterocyclyl and C5-20 aryl groups; R7' is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR', nitro, Me3Sn and halo; and either (a) R4° is H, and R41 is OH, ORA, where R" is C1-4 alkyl; (b) R4° and R41 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; (c) R4° and R41 are both H; or (d) R4° is H and R41 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation, wherein if R41 and R31 are SOzM, M may represent a divalent pharmaceutically acceptable cation; R6', R9', R3° and R31 are selected from the same groups as R6, R9, R4° and R41 respectively.
A second aspect of the present invention provides a method of making a compound of the first aspect of the invention, comprising at least one of the method steps set out below.
In a third aspect, the present invention relates to Conjugates comprising dimers of PBDs linked to a Ligand unit/targeting agent, wherein the PBD dimer is a derivative of formula I, or a pharmaceutically acceptable salt or solvate thereof.
In some embodiments, the Conjugates are of formula IV: L -(1J-)p (IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L is a Ligand unit (i.e., a targeting agent), DL is a Drug Linker unit that is a PBD dimer of formula Ill: wherein R2, R6, R9, Y, R", Y', R2', R6', RT, R30, R31, R" and R41 are as defined in the first aspect of the invention; R7L is of formula A2:
O (A2)
where Z is as defined in the first aspect and RLL is a linker connected to the Ligand unit selected from (ia'): R31 30 R9' R9 R40 / I R41
N N
---R"--- III R2 0 C3 C3' GLL la' where Q and X are as defined in the first aspect and GLL is a linker connected to a Ligand Unit; and (ib'): where RL1 and RL2 are as defined in the first aspect; and p is an integer of from 1 to 20.
Accordingly, the Conjugates comprise a Ligand unit covalently linked to at least one Drug 20 unit by a Linker unit. The Ligand unit, described more fully below, is a targeting agent that binds to a target moiety. The Ligand unit can, for example, specifically bind to a cell component (a Cell Binding Agent) or to other target molecules of interest. Accordingly, the present invention also provides methods for the treatment of, for example, various cancers and autoimmune disease. These methods encompass the use of the Conjugates wherein the Ligand unit is a targeting agent that specifically binds to a target molecule. The Ligand unit can be, for example, a protein, polypeptide or peptide, such as an antibody, an antigen-binding fragment of an antibody, or other binding agent, such as an Fc fusion protein.
The drug loading is represented by p, the number of drug molecules per Ligand unit (e.g., an antibody). Drug loading may range from 1 to 20 Drug units (D) per Ligand unit (e.g., Ab or mAb). For compositions, p represents the average drug loading of the Conjugates in the composition, and p ranges from 1 to 20.
A fourth aspect of the present invention provides the use of a conjugate of the third aspect of the invention in the manufacture of a medicament for treating a proliferative disease. The fourth aspect also provides a conjugate of the third aspect of the invention for use in the treatment of a proliferative disease.
One of ordinary skill in the art is readily able to determine whether or not a candidate compound treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below.
A fifth aspect of the present invention provides a compound of formula V, which may be used in the preparation of the drug-linkers and conjugates of the invention: R30 R31 N Y' _Y R7P C3'
C
V
wherein R2, R6, R9, Y, R", R2', R6', RT, R9', R30, R31, Rao and R41 are as defined in the first aspect of the invention; IR' is of formula A3: H2N 0 z Z wherein Z is as defined in the first aspect of the invention.
A further aspect of the invention provides compounds with the formula REL: R31 30 Ry, R9 R40 R41 V' C3' R7R C2
R
REL C3
and salts and solvates thereof, wherein R2, R6, R9, Y, R", Y', R2', R6', R7', Rw, R30, R'', R4° and R41 are as defined in the first aspect of the invention; IR' is of formula A4: wherein Z is as defined in the first aspect of the invention.
Definitions Pharmaceutically acceptable cations Examples of pharmaceutically acceptable monovalent and divalent cations are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), which is incorporated herein by reference.
The pharmaceutically acceptable cation may be inorganic or organic.
Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not limited to, alkali metal ions such as Na* and Kt Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca' and Mg2+. Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NHe) and substituted ammonium ions (e.g. NH3R*, NH2R2+, NHR3*, NR4*). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CI-13)4+.
The phrase "optionally substituted" as used herein, pertains to a parent group which may be unsubstituted or which may be substituted.
Unless otherwise specified, the term "substituted" as used herein, pertains to a parent group which bears one or more substituents. The term "substituent" is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.
Examples of substituents are described in more detail below.
C1.12 alkyl: The term "C1.12 alkyl" as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). The term "Ci, alkyl" as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to n carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.
Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (Ca), butyl (04), pentyl (Cs), hexyl (CO and heptyl (07).
Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (CO, n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (Cs), n-hexyl (C6) and n-heptyl (C7).
Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4), tert-butyl (C4), iso-pentyl (C5), and neo-pentyl (C5).
C2.12 Alkenyl: The term "C2_12 alkenyl" as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.
Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3), 2-propenyl (ally!, -CH-CH=CH2), isopropenyl (1-methylvinyl, -C(CH3)=CH2), butenyl (04), pentenyl (C5), and hexenyl (06).
02-12 alkynyl: The term "02_12 alkynyl" as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.
Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-CECH) and 2-propynyl (propargyl, -CH2-CECH) 03-12 cycloalkyl: The term "C3.12 cycloalkyl" as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.
Examples of cycloalkyl groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbon compounds: cyclopropane (C3), cyclobutane (C4), cyclopentane (05), cyclohexane (06), cycloheptane (07), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (06), dimethylcyclopentane (07) and methylcyclohexane (07); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C3), cyclobutene (C4), cyclopentene (Cs), cyclohexene (06), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (Cs), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7) and methylcyclohexene (07); and saturated polycyclic hydrocarbon compounds: norcarane (07), norpinane (C7), norbornane (07).
C3.20 heterocyclyl: The term "C3_20 heterocyclyl' as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
In this context, the prefixes (e.g. C3-20, 03-7, 05-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C5_6heterocycly1", as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.
Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived 10 from: Ni: aziridine (C3), azetidine (Ca), pyrrolidine (tetrahydropyrrole) (Cs), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (Cs), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (07); 1: oxirane (03), oxetane (Ca), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5), oxane (tetrahydropyran) (06), dihydropyran (06), pyran (C6), oxepin (07); Si: thiirane (03), thietane (04), thiolane (tetrahydrothiophene) (Cs), thiane (tetrahydrothiopyran) (C6), thiepane (07); 2: dioxolane (Cs), dioxane (C6), and dioxepane (C7); 3: trioxane (CO; N2: imidazolidine (Cs), pyrazolidine (diazolidine) (Cs), imidazoline (Cs), pyrazoline (dihydropyrazole) (Cs), piperazine (06); N101: tetrahydrooxazole (Cs), dihydrooxazole (Cs), tetrahydroisoxazole (Cs), dihydroisoxazole (Cs), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (06); NIS,: thiazoline (C5), thiazolidine (Cs), thiomorpholine (C6); N201: oxadiazine (COI OiSi: oxathiole (Cs) and oxathiane (thioxane) (06); and, N10131: oxathiazine (Cs).
Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (Cs), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C6), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
C5.20 aryl: The term "C5-20 aryl", as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. The term "05-7 aryl", as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 7 ring atoms and the term "C5.10 aryl", as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 10 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.
In this context, the prefixes (e.g. 03.20, C5.7, C5.6, C5_10, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C5_6 aryl" as used herein, pertains to an aryl group having 5 or 6 ring atoms.
The ring atoms may be all carbon atoms, as in "carboaryl groups".
Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C6), naphthalene (Co), azulene (Cio), anthracene (C14), phenanthrene (Cm), naphthacene (Cis), and pyrene (C16).
Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H-indene) (Cs), indene (Cs), isoindene (CO, tetraline (1,2,3,4-tetrahydronaphthalene (C10), acenaphthene (012), fluorene (013), phenalene (C13), acephenanthrene (Cis), and aceanthrene (Cie).
Alternatively, the ring atoms may include one or more heteroatoms, as in "heteroaryl groups". Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from: Ni: pyrrole (azole) (Cs), pyridine (azine) (C6); 01: furan (oxole) (Cs); Si: thiophene (thiole) (05); N101: oxazole (C5), isoxazole (C5), isoxazine (Cs); N201: oxadiazole (furazan) (Cs); N301: oxatriazole (Cs); N1S1: thiazole (Cs), isothiazole (Cs); N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (06), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6); N3: triazole (Cs), triazine (Cs); and, N4: tetrazole (C5).
Examples of heteroaryl which comprise fused rings, include, but are not limited to: C3 (with 2 fused rings) derived from benzofuran (01), isobenzofuran (01), indole (N1), isoindole (N1), indolizine (N1), indoline (Ni), isoindoline (N1), purine (N4) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N101), benzisoxazole (N101), benzodioxole (02), benzofurazan (N201), benzotriazole (N3), benzothiofuran (Si), benzothiazole (N151), benzothiadiazole (N2S); Cio (with 2 fused rings) derived from chromene (01), isochromene (00, chroman (01), isochroman (01), benzodioxan (02), quinoline (Ni), isoquinoline (Ni), quinolizine (Ni), benzoxazine (N101), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4); (with 2 fused rings) derived from benzodiazepine (N2); Ci3 (with 3 fused rings) derived from carbazole (Ni), dibenzofuran (01), dibenzothiophene (Si), carboline (N2), perimidine (N2), pyridoindole (N2); and, Ci4 (with 3 fused rings) derived from acridine (Ni), xanthene (01), thioxanthene (Si), oxanthrene (02), phenoxathiin (01S1), phenazine (N2), phenoxazine (N101), phenothiazine (NISI), thianthrene (52), phenanthridine (N1), phenanthroline (N2), phenazine (N2).
The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.
Halo: -F, -CI, -Br, and -I.
Hydroxy: -OH.
Ether: -OR, wherein R is an ether substituent, for example, a C1J alkyl group (also referred to as a C1-7 alkoxy group, discussed below), a C3.25 heterocyclyl group (also referred to as a C3.25 heterocyclyloxy group), or a C5_20 aryl group (also referred to as a C5.20 aryloxy group), preferably a Ci_7alkyl group.
Alkoxy: -OR, wherein R is an alkyl group, for example, a Ci-7 alkyl group. Examples of Ci_2 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n-propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy).
Acetal: -CH(OR1)(OR2), wherein R1 and R2 are independently acetal substituents, for example, a C1.7 alkyl group, a 03_20 heterocyclyl group, or a 05_20 aryl group, preferably a 01-7 alkyl group, or, in the case of a "cyclic" acetal group, R1 and R2, taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, -CH(OMe)2, -CH(OEt)2, and -CH(OMe)(OEt).
Hemiacetal: -CH(OH)(0R1), wherein R1 is a hemiacetal substituent, for example, a C1-7alkyl group, a C3.20heterocycly1 group, or a C5.20 aryl group, preferably a C1_7 alkyl group.
Examples of hemiacetal groups include, but are not limited to, -CH(OH)(OMe) and -CH(OH)(0Et).
Ketal: -CR(OR1)(OR2), where R1 and R2 are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C1.7 alkyl group, a C3_20 heterocyclyl group, or a 05.20 aryl group, preferably a C1_7 alkyl group. Examples ketal groups include, but are not limited to, -C(Me)(OMe)2, -C(Me)(OEt)2, -C(Me)(OMe)(OEt), -C(Et)(OMe)2, -C(Et)(OEt)2, and -C(Et)(OMe)(OEt).
Hemiketal: -CR(OH)(0R1), where R1 is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a 01.7 alkyl group, a 03_20 heterocyclyl group, or a C5.20 aryl group, preferably a 01-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, -C(Me)(OH)(OMe), -C(Et)(OH)(OMe), -C(Me)(OH)(OEt), and -C(Et)(OH)(OEt).
Oxo (keto, -one): =0.
Thione (thioketone): =S.
Imino (imine) =NR, wherein R is an imino substituent, for example, hydrogen, 01.7 alkyl group, a 03.20 heterocyclyl group, or a C5.20 aryl group, preferably hydrogen or a 01.7 alkyl group. Examples of ester groups include, but are not limited to, =NH, =NMe, =NEt, and 35 =NPh.
Formyl (carbaldehyde, carboxaldehyde): -C(=O)H.
Acyl (keto): -C(=O)R, wherein R is an acyl substituent, for example, a 01-7 alkyl group (also referred to as C1.7 alkylacyl or C1-7 alkanoyl), a C3-20 heterocyclyl group (also referred to as C3.20 heterocyclylacyl), or a C5.20 aryl group (also referred to as C5-20 arylacyl), preferably a alkyl group. Examples of acyl groups include, but are not limited to, -C(=0)CH3 (acetyl), -C(=O)CH2CH3 (propionyl), -C(=0)C(CH3)3 (t-butyryl), and -C(=O)Ph (benzoyl, phenone).
Carboxy (carboxylic acid): -C(=O)OH.
Thiocarboxy (thiocarboxylic acid): -C(=S)SH.
Thiolocarboxy (thiolocarboxylic acid): -C(=O)SH.
Thionocarboxy (thionocarboxylic acid): -C(=S)OH.
Imidic acid: -C(=NH)OH.
Hydroxamic acid: -C(=NOH)OH.
Ester (carboxylate, carboxylic acid ester, oxycarbonyl): -C(=O)OR, wherein R is an ester substituent, for example, a CiJalkyl group, a C3_23heterocycly1 group, or a C5.2oaryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, -C(=0)0CH3, -C(=0)0CH2CH3, -C(=0)0C(CH3)3, and -C(=O)OPh.
Acyloxy (reverse ester): -0C(=0)R, wherein R is an acyloxy substituent, for example, a 01-7 alkyl group, a C3.20 heterocyclyl group, or a C5_20aryl group, preferably a C1-7 alkyl group. Examples of acyloxy groups include, but are not limited to, -0C(=0)CH3 (acetoxy), -0C(=0)CH2CH3, -0C(=0)C(CH3)3, -0C(=0)Ph, and -OC(=O)CH2Ph.
Oxycarboyloxy: -OC(=O)OR, wherein R is an ester substituent, for example, a C1_7 alkyl group, a C3.20 heterocyclyl group, or a C5.20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, -0C(=0)0CH3, -0C(=0)0CH2CH3, -0C(=0)0C(CH3)3, and -OC(=O)OPh.
Amino: -NR1R2, wherein R1 and R2 are independently amino substituents, for example, hydrogen, a C1-7 alkyl group (also referred to as C1Jalkylamino or di-C1Jalkylamino), a C3-20 heterocyclyl group, or a 05-20 aryl group, preferably H or a C1_7alkyl group, or, in the case of a "cyclic" amino group, R1 and R2, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (-NH2), secondary (-NHR1), or tertiary (-NHR1R2), and in cationic form, may be quaternary (-11NR1R2R3). Examples of amino groups include, but are not limited to, -NH2, -NHCH3, -NHC(CH3)2, -N(CH3)2, -N(CH2CH3)2, and -NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C(=O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -C(=O)NHCH2CH3, and -C(=O)N(CH2CH3)2, as well as amido groups in which R1 and R2, together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.
Thioamido (thiocarbamyl): -C(=S)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=S)NH2, -C(=S)NHCH3, -C(=S)N(CH3)2, and -C(=S)NHCH2CH3.
Acylamido (acylamino): -NR1C(=O)R2, wherein R1 is an amide substituent, for example, hydrogen, a C1_, alkyl group, a C3_20heterocycly1 group, or a C5_20 aryl group, preferably hydrogen or a ClJalkyl group, and R2 is an acyl substituent, for example, a C1_7alkyl group, a C3_20 heterocyclyl group, or a C5_20aryl group, preferably hydrogen or a C1Jalkyl group. Examples of acylamide groups include, but are not limited to, -NHC(=0)CH3, -NHC(=0)CH2CH3, and -NHC(=O)Ph. R1 and R2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:
N
Nr° °tNr° succinimidyl maleimidyl phthalimidyl Aminocarbonyloxy: -0C(=0)NR1R2, wherein IR1 and 52 are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, -OC(=O)NH2, -OC(=O)NHMe, -OC(=O)NMe2, and -OC(=O)NEt2.
Ureido: -N(R1)CONR2R3 wherein 52 and 53 are independently amino substituents, as defined for amino groups, and R is a ureido substituent, for example, hydrogen, a Cl_Talkyl group, a C3.20 heterocyclyl group, or a C5.20 aryl group, preferably hydrogen or a C1.7 alkyl group. Examples of ureido groups include, but are not limited to, -NHCONH2, -NHCONHMe, -NHCONHEt, -NHCONMe2, -NHCONEt2, -NMeCONH2, -NMeCONHMe, -NMeCONHEt, -NMeCONMe2, and -NMeCONEt2.
Guanidino: -NH-C(=NH)NH2.
Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom, lH
N
II
-N
!mina =NR, wherein R is an imino substituent, for example, for example, hydrogen, a C1_7 alkyl group, a C3.20 heterocyclyl group, or a C5_20 aryl group, preferably H or a CiJalkyl group.
Examples of imino groups include, but are not limited to, =NH, =NMe, and =NEt.
Amidine (amidino): -C(=NR)NR2, wherein each R is an amidine substituent, for example, hydrogen, a Cij alkyl group, a C3-20 heterocyclyl group, or a C5_20aryl group, preferably H or a C1-7 alkyl group. Examples of amidine groups include, but are not limited to, -C(=NH)NH2, 25 -C(=NH)NMe2, and -C(=NMe)NMe2.
Nitro: -NO2.
Nitroso: -NO.
Azido: -N3.
Cyano (nitrile, carbonitrile): -CN.
Isocyano: -NC.
Cyanato: -OCN.
Isocyanato: -NCO.
Thiocyano (thiocyanato): -SCN.
Isothiocyano (isothiocyanato): -NCS.
Sulfhydryl (thiol, mercapto): -SH.
Thioether (sulfide): -SR, wherein R is a thioether substituent, for example, a 01.7 alkyl group (also referred to as a C1.7alkylthio group), a C3.20 heterocyclyl group, or a C5.20 aryl group, preferably a C1-7 alkyl group. Examples of C1.7 alkylthio groups include, but are not limited to, -SCH3 and -SCH2CH3.
Disulfide: -SS-R, wherein R is a disulfide substituent, for example, a C1.7 alkyl group, a C3.20 heterocyclyl group, or a C5_20 aryl group, preferably a ClJalkyl group (also referred to herein as 01.7 alkyl disulfide). Examples of 01.7 alkyl disulfide groups include, but are not limited to, -SSCH3 and -SSCH2CH3.
Sulfine (sulfinyl, sulfoxide): -S(=O)R, wherein R is a sulfine substituent, for example, a C1.7 alkyl group, a C3.20 heterocyclyl group, or a C5_20 aryl group, preferably a C1.7 alkyl group.
Examples of sulfine groups include, but are not limited to, -S(=O)CH3 and -S(=O)CH2CH3.
Sulfone (sulfonyl): -S(=O)2R, wherein R is a sulfone substituent, for example, a C1.7 alkyl group, a C3.20 heterocyclyl group, or a C5.20 a ry I group, preferably a 01.7 alkyl group, including, for example, a fluorinated or perfluorinated C1.7 alkyl group. Examples of sulfone groups include, but are not limited to, -S(=0)2CH3 (methanesulfonyl, mesyl), -S(=0)2CF3 (triflyl), -S(-0)2CH2CH3 (esyl), -S(-0)2C4F0 (nonaflyl), -S(=O)2CH2CF3 (tresyl), -S(=O)2CH2CH2NH2 (tauryl), -S(=O)2Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).
Sulfinic acid (sulfino): -3(=0)0H, -502H.
Sulfonic acid (sulfo): -S(=0)20H, -SO3H.
Sulfinate (sulfinic acid ester): -S(=O)OR; wherein R is a sulfinate substituent, for example, a Ci.7 alkyl group, a C3_20 heterocyclyl group, or a C5_20 aryl group, preferably a Ci.7 alkyl group.
Examples of sulfinate groups include, but are not limited to, -S(=O)OCH3 (methoxysulfinyl; methyl sulfinate) and -S(=0)OCH2CH3 (ethoxysulfinyl; ethyl sulfinate).
Sulfonate (sulfonic acid ester): -S(=0)20R, wherein R is a sulfonate substituent, for example, a C1-7 alkyl group, a C3_20 heterocyclyl group, or a C5.20 aryl group, preferably a Cl_7alkyl group. Examples of sulfonate groups include, but are not limited to, -S(=0)20CH3 (methoxysulfonyl; methyl sulfonate) and -S(=O)2OCH2CH3 (ethoxysulfonyl; ethyl sulfonate).
Sulfinyloxy: -OS(=O)R, wherein R is a sulfinyloxy substituent, for example, a C1_7 alkyl group, a C3_20 heterocyclyl group, or a C5_20 aryl group, preferably a C1.7 alkyl group. Examples of sulfinyloxy groups include, but are not limited to, -OS(=O)CH3 and -OS(=O)CH2CH3.
Sulfonyloxy: -08(=0)2R, wherein R is a sulfonyloxy substituent, for example, a C1-7 alkyl group, a C3.20 heterocyclyl group, or a C5.20 aryl group, preferably a C1-7 alkyl group.
Examples of sulfonyloxy groups include, but are not limited to, -OS(=O)2CH3 (mesylate) and -0S(=0)2CH2CH3 (esylate).
Sulfate: -03(=0)20R; wherein R is a sulfate substituent, for example, a Ci_7 alkyl group, a C3.20 heterocyclyl group, or a C5_20 aryl group, preferably a C1_7 alkyl group. Examples of sulfate groups include, but are not limited to, -0S(=0)20CH3 and -SO(=O)2OCH2CH3.
Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): -S(=0)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, -S(=O)NH2, -S(=O)NH(CH3), -S(=O)N(CH3)2, -S(=0)NH(CH2CH3), -S(=O)N(CH2CH3)2, and -S(=O)NHPh.
Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): -S(=0)2NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, -S(=O)2NH2, -S(=O)2NH(CH3), -S(=O)2N(CH3)2, -S(=0)2NH(CH2CH3), -S(=O)2N(CH2CH3)2, and -S(=0)2NH Ph.
Sulfamino: -NR1S(=0)20H, wherein R1 is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, -NHS(=0)20H and -N(CH3)S(=0)20H.
Sulfonamino: -NR1S(=O)2R, wherein S1 is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a C1_, alkyl group, a C3-20 heterocyclyl group, or a C5_20 aryl group, preferably a C1.7alkyl group. Examples of sulfonamino groups include, but are not limited to, -NHS(=O)2CH3 and -N(CH3)S(=O)2C6H5.
Sulfinamino: -NR1S(=O)R, wherein R1 is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C1.7alkyl group, a C3.20 heterocyclyl group, or a C5_20 aryl group, preferably a C1.7 alkyl group. Examples of sulfinamino groups include, but are not limited to, -NHS(=O)CH3 and -N(CH3)S(=O)C6H5.
Phosphino (phosphine): -PR2, wherein R is a phosphino substituent, for example, -H, a C1-7 alkyl group, a C3.20 heterocyclyl group, or a C3_20 aryl group, preferably -H, a 01_7 alkyl group, or a 00-20 aryl group. Examples of phosphino groups include, but are not limited to, -PH2, -P(CH3)2, -P(CH2CH3)2, -P(t-Bu)2, and -P(Ph)2.
Phospho: -P(=0)2.
Phosphinyl (phosphine oxide): -P(=O)R2, wherein R is a phosphinyl substituent, for example, a 01.7 alkyl group, a C3.20 heterocyclyl group, or a C3.20 aryl group, preferably a C1_7 alkyl group or a 03-20 aryl group. Examples of phosphinyl groups include, but are not limited to, -P(=O)(CH3)2, -P(=O)(CH2CH3)2, -P(=O)(t-Bu)2, and -P(=O)(Ph)2.
Phosphonic acid (phosphono): -P(=O)(OH)2.
Phosphonate (phosphono ester): -P(=O)(OR)2, where R is a phosphonate substituent, for example, -H, a 01.7 alkyl group, a 03_20 heterocyclyl group, or a 03_20 aryl group, preferably -H, a C1.7 alkyl group, or a C5.20 aryl group. Examples of phosphonate groups include, but are not limited to, -P(=O)(OCH3)2, -P(=0)(OCH2CH3)2, -P(=O)(O-t-Bu)2, and -P(=0)(0Ph)2.
Phosphoric acid (phosphonooxy): -OP(=O)(OH)2.
Phosphate (phosphonooxy ester): -OP(=O)(OR)2, where R is a phosphate substituent, for example, -H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably -H, a 01_7 alkyl group, or a 05-20 aryl group. Examples of phosphate groups include, but are not limited to, -OP(=O)(OCH3)2, -OP(=O)(OCH2CH3)2, -OP(=O)(O-t-Bu)2, and -OP(=O)(OPh)2.
Phosphorous acid: -OP(OH)2.
Phosphite: -OP(OR)2, where R is a phosphite substituent, for example, -H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5_20 aryl group, preferably -H, a C1_7 alkyl group, or a C5_20 aryl group. Examples of phosphite groups include, but are not limited to, -0P(OCH3)2, -OP(OCH2CH3)2, -OP(O-t-Bu)2, and -0P(OPh)2.
Phosphoramidite: -0P(OR1)-NR22, where R1 and R2 are phosphoramidite substituents, for example, -H, a (optionally substituted) 01.7 alkyl group, a 03-20 heterocyclyl group, or a C5-20 aryl group, preferably -H, a C1_7 alkyl group, or a C5_20 aryl group. Examples of phosphoramidite groups include, but are not limited to, -OP(OCH2CH3)-N(CH3)2, -0P(OCH2CH3)-NO-Pr)2, and -0P(OCH2CH2CN)-NO-Pr)2.
Phosphoramidate: -OP(=O)(0R1)-NR22, where R1 and R2 are phosphoramidate substituents, for example, -H, a (optionally substituted) C1.7 alkyl group, a 03_20 heterocyclyl group, or a 05.20aryl group, preferably -H, a 01-7alkyl group, or a C5_20 aryl group. Examples of phosphoramidate groups include, but are not limited to, -0P(=0)(OCH2CH3)-N(CH3)2, -OP(=O)(OCH2CH3)-N(i-Pr)2, and -OP(=O)(OCH2CH2CN)-NO-Pr)2.
Alkylene 03_12 alkylene: The term "C3.12 alkylene", as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term "alkylene" includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.
Examples of linear saturated 03_12 alkylene groups include, but are not limited to, -(CH2), where n is an integer from 3 to 12, for example, -CH2CH2CH2-(propylene), -CH2CH2CH2CH2-(butylene), -CH2CH2CH2CH2CH2-(pentylene) and -CH2CH2CH2CH-2CH2CH2CH2-(heptylene).
Examples of branched saturated C3-12 alkylene groups include, but are not limited to, -CH(CH3)CH2-, -CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH(CH3)CH2CH2-, -CH(CH2CH3)-, -CH(CH2CH3)CH2-, and -CH2CH(CH2CH3)CH2-.
Examples of linear partially unsaturated C3_12 alkylene groups (03_12 alkenylene, and alkynylene groups) include, but are not limited to, -CH=CH-CH2-, -CH2-CH=CH2-, -CH=CH-CH2-CH2-, -CH=CH-CH2-CH2-CH2-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-CH2-CH2-, -CH=CH-CH2-CH=CH-, -CH=CH-CH2-CH2-CH=CH-, and -CH2-CEC-CH2-.
Examples of branched partially unsaturated C3.12alkylene groups (03.12 alkenylene and alkynylene groups) include, but are not limited to, -C(CH3)=CH-, -C(CH3)=CH-CH2-, -CH=CH-CH(CH3)-and -05C-CH(CH3)-.
Examples of alicyclic saturated C3-12 alkylene groups (C3_12 cycloalkylenes) include, but are not limited to, cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).
Examples of alicyclic partially unsaturated C3.12 alkylene groups (03_12 cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g. 4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien1,4-ylene).
Bis-oxy-C1.3 alkylene: The term "Bis-oxy-C14 alkylene", as used herein, pertains to a bidentate moiety of formula -0-(CH2)q-0-, where q is from 1 to 3, i.e. -OCH2CH2CH2O-, -OCH2CH20-, -OCH2O-.
Carbamate nitrogen protecting group: the term "carbamate nitrogen protecting group" pertains to a moiety which masks the nitrogen in the imine bond, and these are well known in the art. These groups have the following structure: F' OHO wherein R'1° is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T.W. and Wuts, G.M., Protective Groups in Organic Synthesis, 3ffi Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Hemi-aminal nitrogen protecting group: the term "hemi-aminal nitrogen protecting group" pertains to a group having the following structure: R'10-0 wherein R'1° is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T.W. and Wuts, G.M., Protective Groups in Organic Synthesis, 3ffi Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
The groups Carbamate nitrogen protecting group and Hemi-aminal nitrogen protecting group may be jointly termed a "nitrogen protecting group for synthesis".
Includes Other Forms Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (-COON) also includes the anionic (carboxylate) form (-COOS), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N+HR1R2), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-0), a salt or solvate thereof, as well as conventional protected forms. Salts
It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).
For example, if the compound is anionic, or has a functional group which may be anionic (e.g. -COOH may be -COOS), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na* and Kt, alkaline earth cations such as Cat* and Mg2*, and other cations such as Al'. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4') and substituted ammonium ions (e.g. NH3R., NH2R2t, NHIR3t, NR4t). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
If the compound is cationic, or has a functional group which may be cationic (e.g. -NH2 may be -NH3'), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic acid and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
Solvates It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term "solvate" is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc. The invention includes compounds where a solvent adds across the imine bond of the PBD moiety, which is illustrated below where the solvent is water or an alcohol (RAOH, where RA is C1_4 alkyl):
H H
v OH V ORA
N N H2O H
RAOH
These forms can be called the carbinolamine and carbinolamine ether forms of the PBD (as described in the section relating to Rl° above). The balance of these equilibria depend on the conditions in which the compounds are found, as well as the nature of the moiety itself.
These particular compounds may be isolated in solid form, for example, by lyophilisation.
Isomers Certain compounds of the invention may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis-and trans-forms; E-and Z-forms; c-, t-, and r-forms; endo-and exo-forms; R-, S-, and meso-forms; D-and L-forms; d-and I-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal-and anticlinal-forms; a-and 3-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").
The term "chiral" refers to molecules which have the property of non-superimposability of the mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner.
The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
"Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.
"Enanfiomers" refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.
Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and I or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or I meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers", as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, metachlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. Ci.7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and paramethoxyphenyl).
The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
I 0° ,OH 1-1÷ -C-C\ / C=C
N /C=C\ 1-1÷
keto enol enolate The term "tautomer" or "tautomeric form" refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some of the bonding electrons.
Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D), and 3H (T); C may be in any isotopic form, including 12C, 13C, and 14C; 0 may be in any isotopic form, including 160 and 160; and the like.
Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as, but not limited to 2H (deuterium, D), 3H (tritium), 11 C, 13C, 14C, 15N, 18F, 31p, 32p, 35n, 0 36a, and 1251. Various isotopically labeled compounds of the present invention, for example those into which radioactive isotopes such as 3H, 13C, and 14C are incorporated. Such isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. Deuterium labelled or substituted therapeutic compounds of the invention may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism, and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements. An 18F labeled compound may be useful for PET or SPECT studies. Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. Further, substitution with heavier isotopes, particularly deuterium (i.e., 2H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index. It is understood that deuterium in this context is regarded as a substituent. The concentration of such a heavier isotope, specifically deuterium, may be defined by an isotopic enrichment factor. In the compounds of this invention any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom.
Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
Ligand Unit The Ligand Unit may be of any kind, and include a protein, polypeptide, peptide and a nonpeptidic agent that specifically binds to a target molecule. In some embodiments, the Ligand unit may be a protein, polypeptide or peptide. In some embodiments, the Ligand unit may be a cyclic polypeptide. These Ligand units can include antibodies or a fragment of an antibody that contains at least one target molecule-binding site, lymphokines, hormones, growth factors, or any other cell binding molecule or substance that can specifically bind to a target.
The terms "specifically binds" and "specific binding" refer to the binding of an antibody or other protein, polypeptide or peptide to a predetermined molecule (e.g., an antigen).
Typically, the antibody or other molecule binds with an affinity of at least about 1x107 and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule.
Examples of Ligand units include those agents described for use in WO 2007/085930, which is incorporated herein.
In some embodiments, the Ligand unit is a Cell Binding Agent that binds to an extracellular target on a cell. Such a Cell Binding Agent can be a protein, polypeptide, peptide or a non-peptidic agent. In some embodiments, the Cell Binding Agent may be a protein, polypeptide or peptide. In some embodiments, the Cell Binding Agent may be a cyclic polypeptide. The Cell Binding Agent also may be antibody or an antigen-binding fragment of an antibody. Thus, in one embodiment, the present invention provides an antibody-drug conjugate (ADC).
Cell Binding Agent A cell binding agent may be of any kind, and include peptides and non-peptides. These can include antibodies or a fragment of an antibody that contains at least one binding site, lymphokines, hormones, hormone mimetics, vitamins, growth factors, nutrient-transport molecules, or any other cell binding molecule or substance.
Peptides In one embodiment, the cell binding agent is a linear or cyclic peptide comprising 4-30, preferably 6-20, contiguous amino acid residues. In this embodiment, it is preferred that one cell binding agent is linked to one monomer or dimer pyrrolobenzodiazepine compound.
In one embodiment the cell binding agent comprises a peptide that binds integrin a436. The peptide may be selective for avp6 over XYS.
In one embodiment the cell binding agent comprises the A2OFMDV-Cys polypeptide. The A2OFMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC. Alternatively, a variant of the A2OFM DV-Cys sequence may be used wherein one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are substituted with another amino acid residue. Furthermore, the polypeptide may have the sequence NAVXXXXXXXXXXXXXXXRTC.
Antibodies The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies and antibody fragments, so long as they exhibit the desired biological activity (Miller et a/ (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) lmmuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin.
"Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab1)2, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, US 4816567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459).
The monoclonal antibodies herein specifically include chimeric antibodies, humanized antibodies and human antibodies.
Examples of cell binding agents include those agents described for use in WO 2007/085930, which is incorporated herein.
Tumour-associate antigens and cognate antibodies for use in embodiments of the present invention are listed below, and are described in more detail on pages 14 to 86 of WO 2017/186894, which is incorporated herein.
(1) BMPR1B (bone morphogenetic protein receptor-type IB) (2) E16 (LAT1, SLC7A5) (3) STEAP1 (six transmembrane epithelial antigen of prostate) (4) 0772P (CA125, MUC16) (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin) (6) Napi3b (NAPI-3B, NPTIlb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b) (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, 25 sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B) (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene) (9) ETBR (Endothelin type B receptor) (10) MSG783 (RNF124, hypothetical protein FLJ20315) (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein) (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation 5 channel, subfamily M, member 4) (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor) (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792) (15) CD79b (CD79B, CD79(3, IGb (immunoglobulin-associated beta), B29) (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein la), SPAP1B, SPAP1C) (17) HER2 (ErbB2) (18) NCA (CEACAM6) (19) MDP (DPEP1) (20) IL20R-alpha (1L2ORa, ZCYTOR7) (21) Brevican (BCAN, BEHAB) (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyros) (23) ASLG659 (B7h) (24) PSCA (Prostate stem cell antigen precursor) (25) GEDA (26) BAFF-R (B cell -activating factor receptor, BLyS receptor 3, BR3) (27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814) (27a) CD22 (CD22 molecule) (28) CD79a (CD79A, CD79alpha), immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation), pl: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19q13.2).
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a 10 role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pl: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, (30) HLA-DOB (Beta subunit of MHC class II molecule (la antigen) that binds peptides and presents them to CD4+ T lymphocytes); 273 aa, pl: 6.56, MW: 30820.TM: 1 [P] Gene Chromosome: 6p21.3) (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa), pl: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3).
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2); 359 aa, pl: 8.66, MW: 40225, TM: 1 5 [P] Gene Chromosome: 9p13.3).
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis); 661 aa, pl: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12).
(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte 20 differentiation); 429 aa, pl: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22) (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies); 977 aa, pl: 6.88, MW: 106468, TM: 1 [P] Gene Chromosome: 1q21) (36) TEN B2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane 35 proteoglycan, related to the EGF/heregulin family of growth factors and follistatin); 374 aa) (37) PSMA -FOLH1 (Folate hydrolase (prostate-specific membrane antigen) 1) (38) SST ( Somatostatin Receptor; note that there are5 subtypes) (38.1) SSTR2 (Somatostatin receptor 2) (38.2) SSTR5 (Somatostatin receptor 5) (38.3) SSTR1 (38.4) SSTR3 (38.5) SSTR4 AvB6 -Both subunits (39+40) (39) ITGAV (Integrin, alpha V) (40) ITGB6 (Integrin, beta 6) (41) CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5) (42) MET (met proto-oncogene; hepatocyte growth factor receptor) (43) MUC1 (Mucin 1, cell surface associated) (44) CA9 (Carbonic anhydrase IX) (45) EGFRvIII ( Epidermal growth factor receptor (EGFR), transcript variant 3, (46) CD33 (CD33 molecule) (47) CD19 (CD19 molecule) (48) IL2RA (Interleukin 2 receptor, alpha); NCBI Reference Sequence: NM_000417.2); (49) AXL (AXL receptor tyrosine kinase) (50) CD30 -TNFRSF8 (Tumor necrosis factor receptor superfamily, member 8) (51) BCMA (B-cell maturation antigen) -TNFRSF17 (Tumor necrosis factor receptor superfamily, member 17) (52) CT Ags -CTA (Cancer Testis Antigens) (53) CD174 (Lewis Y) -FUT3 (fucosyltransferase 3 (galactoside 3(4)-L-fucosyltransferase, Lewis blood group) (54) CLEC14A (C-type lectin domain family 14, member A; Genbank accession no. NM175060) (55) GRP78 -HSPA5 (heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) 35 (56) CD70 (CD70 molecule) L08096 (57) Stem Cell specific antigens. For example: * 5T4 (see entry (63) below) * CD25 (see entry (48) above) * CD32 * LGR5/GPR49 * Prominin/CD133 (58) ASG-5 (59) EN PP3 (Ectonucleotide pyrophosphatase/phosphodiesterase 3) (60) PRR4 (Proline rich 4 (lacrimal)) (61) GCC -GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor) (62) Liv-1 -SLC39A6 (Solute carrier family 39 (zinc transporter), member 6) (63) 5T4, Trophoblast glycoprotein, TPBG -TPBG (trophoblast glycoprotein) (64) CD56 -NCMA1 (Neural cell adhesion molecule 1) (65) CanAg (Tumor associated antigen CA242) (66) FOLR1 (Folate Receptor 1) (67) GPNMB (Glycoprotein (transmembrane) nmb) (68) TIM-1 -HAVCR1 (Hepatitis A virus cellular receptor 1) (69) RG-1/Prostate tumor target Mindin -Mindin/RG-1 (70) B7-H4 -VTCN1 (V-set domain containing T cell activation inhibitor 1 (71) PTK7 (PTK7 protein tyrosine kinase 7) (72) CD37 (CD37 molecule) (73) CD138 -SDC1 (syndecan 1) (74) CD74 (CD74 molecule, major histocompatibility complex, class II invariant chain) (75) Claudins -CLs (Claudins) (76) EGFR (Epidermal growth factor receptor) (77) Her3 (ErbB3) -ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian)) (78) RON -MST1R (macrophage stimulating 1 receptor (c-met-related tyrosine kinase)) (79) EPHA2 (EPH receptor A2) (80) CD20 -MS4A1 (membrane-spanning 4-domains, subfamily A, member 1) (81) Tenascin C -TNC (Tenascin C) (82) FAP (Fibroblast activation protein, alpha) (83) DKK-1 (Dickkopf 1 homolog (Xenopus laevis) (84) CD52 (CD52 molecule) (85) CS1 -SLAMF7 (SLAM family member 7) (86) Endoglin -ENG (Endoglin) (87) Annexin Al -ANXA1 (Annexin Al) (88) V-CAM (CD106) -VCAM1 (Vascular cell adhesion molecule 1) The cell binding agent may be labelled, for example to aid detection or purification of the agent either prior to incorporation as a conjugate, or as part of the conjugate. The label may be a biotin label. In another embodiment, the cell binding agent may be labelled with a radioisotope.
Methods of Treatment The compounds of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate of formula II. The term "therapeutically effective amount' is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.
A conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a conjugate of formula I, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
The Conjugates can be used to treat proliferative disease and autoimmune disease. The term "proliferative disease" pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Other cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin. Any type of cell may be treated, including but not limited to, lung, gastrointestinal (including, e.g. bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.
Examples of autoimmune disease include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, SjOgren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal failure.
In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjogren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve disorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmunie disorder is a T cell-mediated immunological disorder.
Methods of Treatment The conjugates of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate compound of the invention. The term "therapeutically effective amount" is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.
A compound of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs, such as chemotherapeutics); surgery; and radiation therapy.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy.
Examples of chemotherapeutic agents include: erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.), trastuzumab (HERCEPT1NO, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0] nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine, NOLVADEX®, ISTUBALO, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2, HPPD, and rapamycin.
More examples of chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASARTM, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRATm, Johnson & Johnson), ABRAXANETM (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, II), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa and cyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, calicheamicin gammall, calicheamicin omegal1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ('Ara-C"); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBI NE0); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.
Also included in the definition of "chemotherapeutic agent" are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON®(toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKINO rIL-2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH; (ix) antiangiogenic agents such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above.
Also included in the definition of "chemotherapeutic agent" are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, lmclone); panitumumab (VECTIBIXO, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab (OMNITARGTM, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).
Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents in combination with the conjugates of the invention include: alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and visilizumab.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a conjugate compound, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Formulations While it is possible for the conjugate compound to be used (e.g., administered) alone, it is often preferable to present it as a composition or formulation.
In one embodiment, the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a conjugate compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
In one embodiment, the composition is a pharmaceutical composition comprising at least one conjugate compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
In one embodiment, the composition further comprises other active agents, for example, other therapeutic or prophylactic agents.
Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts.
See, for example, Handbook of Pharmaceutical Additives 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA). Remington's Pharmaceutical Sciences 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients 2nd edition, 1994.
Another aspect of the present invention pertains to methods of making a pharmaceutical composition comprising admixing at least one [11q-radiolabelled conjugate or conjugate-like compound, as defined herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound.
The term "pharmaceutically acceptable," as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.
The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.
The formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient.
Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active ingredient in the liquid is from about 1 ng/ml to about 10 pg/ml, for example from about 10 ng/ml to about 1 pg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
Dosage It will be appreciated by one of skill in the art that appropriate dosages of the conjugate compound, and compositions comprising the conjugate compound, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
In general, a suitable dose of the active compound is in the range of about 100 ng to about 25 mg (more typically about 1 tag to about 10 mg) per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.
In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 100 mg, 3 times daily.
In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 150 mg, 2 times daily.
In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 200 mg, 2 times daily.
However in one embodiment, the conjugate compound is administered to a human patient according to the following dosage regime: about 50 or about 75 mg, 3 or 4 times daily.
In one embodiment, the conjugate compound is administered to a human patient according to the following dosage regime: about 100 or about 125 mg, 2 times daily.
The dosage amounts described above may apply to the conjugate (including the PBD moiety and the linker to the antibody) or to the effective amount of PBD compound provided, for example the amount of compound that is releasable after cleavage of the linker.
For the prevention or treatment of disease, the appropriate dosage of an ADC of the invention will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 Egg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 µg/kg to 100 mg/kg or more, depending on the factors mentioned above. An exemplary dosage of ADC to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. An exemplary dosing regimen comprises a course of administering an initial loading dose of about 4 mg/kg, followed by additional doses every week, two weeks, or three weeks of an ADC. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
Drug loading The drug loading (p) is the average number of PBD drugs per cell binding agent, e.g. antibody. Where the compounds of the invention are bound to cysteines, drug loading may range from 1 to 8 drugs (D) per cell binding agent, i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attached to the cell binding agent. Compositions of conjgates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 8. Compositions of conjgates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 20, 1 to 10 or 1 to 8.
The average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis. The quantitative distribution of ADC in terms of p may also be determined. By ELISA, the averaged value of p in a particular preparation of ADC may be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852). However, the distribution of p (drug) values is not discernible by the antibody-antigen binding and detection limitation of ELISA. Also, ELISA assay for detection of antibody-drug conjugates does not determine where the drug moieties are attached to the antibody, such as the heavy chain or light chain fragments, or the particular amino acid residues. In some instances, separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.
For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached. Higher drug loading, e.g. p >5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates.
Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with the Drug Linker. Only the most reactive lysine groups may react with an amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety. Most cysteine thiol residues in the antibodies of the compounds exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP, under partial or total reducing conditions. The loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of Drug Linker relative to antibody, 00 limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.
Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues). US 7521541 teaches engineering antibodies by introduction of reactive cysteine amino acids.
Cysteine amino acids may be engineered at reactive sites in an antibody and which do not form intrachain or intermolecular disulfide linkages (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; W02009/052249). The engineered cysteine thiols may react with linker reagents or the drug-linker reagents of the present invention which have thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form ADC with cysteine engineered antibodies and the PBD drug moieties. The location of the drug moiety can thus be designed, controlled, and known. The drug loading can be controlled since the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or drug-linker reagents in high yield. Engineering an IgG antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain gives two new cysteines on the symmetrical antibody. A drug loading near 2 can be achieved with near homogeneity of the conjugation product ADC.
Where more than one nucleophilic or electrophilic group of the antibody reacts with a drug-linker intermediate, or linker reagent followed by drug moiety reagent, then the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to an antibody, e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate compounds in the mixture by drug loading value. Preparations of ADC with a single drug loading value (p) may be isolated, however, these single loading value ADCs may still be heterogeneous mixtures because the drug moieties may be attached, via the linker, at different sites on the antibody.
Thus the antibody-drug conjugate compositions of the invention include mixtures of antibody-drug conjugate compounds where the antibody has one or more PBD drug moieties and where the drug moieties may be attached to the antibody at various amino acid residues.
In one embodiment, the average number of dimer pyrrolobenzodiazepine groups per cell binding agent is in the range 1 to 20. In some embodiments the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4, and 4 to 8.
In some embodiments, there is one dimer pyrrolobenzodiazepine group per cell binding agent.
General synthetic routes The synthesis of PBD compounds is extensively discussed in the following references, which discussions are incorporated herein by reference: a) WO 00/12508 (pages 14 to 30); b) WO 2005/023814 (pages 3 to 10); 25 c) WO 2004/043963 (pages 28 to 29); and d) WO 2005/085251 (pages 30 to 39).
Conjugates of formula IV may be synthesised from corresponding drug-linker compounds of formula I by reacting them with cell binding agents under appropriate conditions.
The conditions, as described above, will depend on the type of bond being formed between the drug-linker compound and the cell binding agent, which itself will reflect the nature of the binding site on the cell binding agent.
Drug-linker compounds of formula I may be synthesised from corresponding compounds of formula V. Drug-linker compounds where RI-is of formula la may be synthesied from compounds of formula V, by reaction with a compound of formula El: El in an appropriate solvent, where Q' is
OH
in the presence of an amide coupling reagent.
Alternatively, the group formed by El may be added in two steps, adding compound Fl (or a protected version thereof) in an appropriate solvent, in the presence of an amide coupling reagent, followed by F2 in an appropriate solvent, in the presence of an amide coupling reagent. If a protected version of Fl is used, it will be deprotected before F2 is added. H 0 Q'
HOLGLL Fl F2
Compound of formula V can be made from the corresponding compound of formula G: 2 R2 by reaction with a compound of formula Hl: H N N /Br H1 ProtN/ 0 where Prot" is an amine protecting group, such as Alloc, in the presence of tetrabutylammonium iodide and potassium carbonate, followed by deprotection of the amine under standard conditions. The protecting group used should be orthogonal to any other protecting groups in the compound.
Alternativelty, compounds of formula V may be synthesised by reacting a compound of formula G with a compound of formula H2: 02N O N /Br H2 in the presence of tetrabutylammonium iodide and potassium carbonate, followed by reduction of the nitro group under standard conditions.
The synthesis of PBD dimer compounds containing two imine moieties is extensively discussed in the following references, which discussions are incorporated herein by 15 reference: a) WO 00/12508 (pages 14 to 30); b) WO 2005/023814 (pages 3 to 10); c) WO 2004/043963 (pages 28 to 29); d) WO 2005/085251 (pages 30 to 39); and e) WO 2011/130598 (pages 126 to 150).
The disclosure of WO 2005/085259 discussed above is also relevant to the synthesis of compounds of formula G which comprise two PBD moieties. The synthesis methods disclosed therein may be modified to include an orthogonally protected hydroxyl group at C7 (i.e.group RA in Scheme 4).
Alternatively, compounds of formula G may be synthesised as described in the above references, but starting from a dimer core of formula J: R19 02N NO2 1:t11 R17
J
where Prot° is a hydroxyl protecting group. Such compounds of formula J may be made by methods analogous to those in the examples of the present application.
In other methods, compounds of formula K:
HO
OH
I
Prot0 R6 H Q-N Br
N Prot (K)
may be reacted with G, followed by conversion to drug linkers of formula B, by deprotection and addition of a compound of formula F2.
Amine protecting groups Amine protecting groups are well-known to those skilled in the art. Particular reference is made to the disclosure of suitable protecting groups in Greene's Protecting Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pages 696-871.
Hydroxyl protecting groups Hydroxyl protecting groups are well-known to those skilled in the art. Particular reference is made to the disclosure of suitable protecting groups in Greene's Protecting Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pages 16-298.
Further Preferences The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.
R6' and Rz are selected from the same groups as R6 and R9. In some embodiments, R6', RT, Rz, and Y' are the same as R6, R2, R9 and Y respectively.
In some embodiments, Rz is the same as R2.
Dimer link In some embodiments, Y and Y' are both 0.
In some embodiments, R" is a 03_7 alkylene group with no substituents. In some of these embodiments, R" is a C3, C5 or C7 alkylene. In particular, R" may be a C3 or C5 alkylene.
In other embodiments, R" is a group of formula: where r is 1 or 2. In some of these embodiments r is 1.
The phenylene group may be replaced by a pyridylene group.
R6 to R9 In some embodiments, R9 is H. In some embodiments, R6 is selected from H, OH, OR, SH, NH2, nitro and halo, and may be selected from H or halo. In some of these embodiments R6 is H. These embodiments and preferences apply to R6' and Rz respectively.
In some embodiments, PT is selected from H, OH, OR, SH, SR, NH2, NHR, NRR', and halo. In some of these embodiments RT is selected from H, OH and OR, where R is selected from optionally substituted C1.7 alkyl, C3-10 heterocyclyl and 05.10 aryl groups. R may be more preferably a C1.4 alkyl group, which may or may not be substituted. A substituent of interest is a C5_6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7-position are OMe and OCH2Ph. Other substituents of particular interest are dimethylamino (i.e. -NMe2); -(0C2H4)60Me, where q is from 0 to 2; nitrogen-containing Ca heterocyclyls, including morpholino, piperidinyl and N-methyl-piperazinyl. R2
When there is a double bond present between C2 and 03, R2 is selected from: (a) C5.15 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, 01.7 alkyl, 03.7 heterocyclyl and bis-oxy-Ci_3 alkylene; (b) Ci.s saturated aliphatic alkyl; (c) C3_6 saturated cycloalkyl; R12 (d) Ar.Ri a, wherein each of IR", R12 and R13 are independently selected from H, Ci_3 saturated alkyl, C2.3 alkenyl, 02.3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2 group is no more than 5; R1 5b (e) R15a, wherein one of R1" and R1' is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl; and (f) R14, where R14 is selected from: H; Ci_3 saturated alkyl; 02.3 alkenyl; 02-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.
When R2 is a C6.10 aryl group, it may be a C5.7 aryl group. A C5.7 aryl group may be a phenyl group or a 05_7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R2 is preferably phenyl. In other embodiments, R2 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
When R2 is a 05.10 aryl group, it may be a 08-10 aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4y1, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4y1, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred.
When R2 is a 05.10 aryl group, it may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position.
Where R2 is 05.7 aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably p or y to the bond to the remainder of the compound. Therefore, where the C5_7 aryl group is phenyl, the substituent is preferably in the meta-or para-positions, and more preferably is in the paraposition.
Where 52 is a 08_10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).
R2 substituents, when R2 is a C5_10 aryl group If a substituent on 52 when 52 is a C5_10 aryl group is halo, it is preferably F or CI, more preferably Cl.
If a substituent on R2 when R2 is a C5_10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a 01_7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a 05-7 aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino).
If a substituent on R2 when R2 is a C5-10 aryl group is C1_7 alkyl, it may preferably be a 01-4 alkyl group (e.g. methyl, ethyl, propryl, butyl).
If a substituent on R2 when R2 is a C6.10 aryl group is C3.7 heterocyclyl, it may in some embodiments be Co nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by Ci.4 alkyl groups.
If the 06 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.
If a substituent on R2 when R2 is a C6_10 aryl group is bis-oxy-C1.3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.
If a substituent on R2 when R2 is a Cs_ir, aryl group is ester, this is preferably methyl ester or ethyl ester.
Particularly preferred substituents when R2 is a C5.10 aryl group include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl. Other particularly preferred substituents for R2 are dimethylaminopropyloxy and carboxy.
Particularly preferred substituted R2 groups when R2 is a C5.10 aryl group include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R12 group is 4-nitrophenyl. R12 groups of particular interest include 4-(4-methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.
When R2 is Ci-s saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.
When R2 is C3.6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl. R12 ii
When R2 is R, each of R", R12 and R13 are independently selected from H, C1_3 saturated alkyl, C24 alkenyl, 02.3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2 group is no more than 5. In some embodiments, the total number of carbon atoms in the R2 group is no more than 4 or no more than 3.
In some embodiments, one of R11, R12 and Kn13 is H, with the other two groups being selected from H, Ci_s saturated alkyl, C2.3 alkenyl, C2.3 alkynyl and cyclopropyl.
In other embodiments, two of 1511, 512 and rs n13 are H, with the other group being selected from H, C1_3 saturated alkyl, 02-3 alkenyl, 02-3 alkynyl and cyclopropyl.
In some embodiments, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that re not H are methyl.
In some embodiments, IR" is H. In some embodiments, 512 is H. In some embodiments, R13 is H. 20 In some embodiments, Ril and R12 are H. In some embodiments, R11 and 513 are H. In some embodiments, R12 and 513 are H. An 52 group of particular interest is: R15b When 52 is, one of 515a and IR' is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
When R2 is R, IR14 is selected from: H; Ci_3 saturated alkyl; 024 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
In some embodiments, R14 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R14 is selected from H and methyl.
When there is a single bond present between C2 and C3, R16a Arfib R2 is H or R, where R16a and IR16b are independently selected from H, F, C1.4 saturated alkyl, 02-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from 01_4 alkyl amido and C1_4 alkyl ester; or, when one of R16a and IR16b is H, the other is selected from nitrile and a C1_4 alkyl ester.
In some embodiments, R2 is H. In some embodiments, R2 is In some embodiments, it is preferred that R16a and Rleb are both H. In other embodiments, it is preferred that R16a and IR16b are both methyl.
In further embodiments, it is preferred that one of Risa and 51' is H, and the other is selected from C1-4 saturated alkyl, C23 alkenyl, which alkyl and alkenyl groups are optionally substituted. In these further embodiment, it may be further preferred that the group which is not H is selected from methyl and ethyl. tip R16a R2
The above preferences for R2 apply equally to R2'.
R" and R" In some embodiments, is R40 4 s n and R41 is OH. In other embodiments, R4° is H, and R41 is ORA. In some of these embodiments, RA is methyl.
In other embodiment, R4° and R41 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.
In other embodiment, R4° and R41 are both H. In other embodiments, R4° is H and R41 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation.
R" and R31 The above preferences for R4° and R41 apply equally to IR°° and IR31.
R30, R31, R4° and R41 In some embodiments, R" and R41 are both H and: (a) R30 is H, and R31 is OH, ORA, where RA is Ci_4 alkyl; (b) R3° and R31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (d) IR' is H and R31 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically
acceptable cation.
In other embodiments, IR' and IR31 are both H and: (a) R40 is H, and R41 is OH, ORA, where RA is C1.4 alkyl; (b) R4° and R41 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (d) R4° is H and R41 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation.
In other embodiments: (a) IR3° is H, and 531 is OH, ORA, where RA is C1.4 alkyl; (b) R3° and R31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (d) R3° is H and IR31 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; and (a) 540 is H, and 541 is OH, OR", where RA is 01-4 alkyl; (b) 540 and 541 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (d) R4° is H and R41 is SON, where z is 2 or 3 and M is a monovalent pharmaceutically
acceptable cation.
M and z It is preferred that M is a monovalent pharmaceutically acceptable cation, and is more preferably Nat.
z is preferably 3.
Linker In some embodiments, Z is methylene. In some embodiments, Z is ethylene. In some embodiments, Z is propylene.
In some embodiments, RL is of formula la. In some embodiments, 5LL is of formula la'. Qx
In one embodiment, Q is an amino acid residue. The amino acid may a natural amino acids or a non-natural amino acid.
In one embodiment, Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline.
In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
In one embodiment, Q is selected from: NH -Phe-Lys-', NH Val-Ala-0=0, NH Val-Lys-0=0, NH Ala-Lys-0=0, NH-Val-CI1-C=C), NH-Phe-Cit-c=°, NH-Ile-Cit-c=°, NH-Phe-Arg-', and NH-rp
--_
I Cit-0=0; where Cit is citrulline.
Preferably, Q is selected from: NH-Phe-Lys-0=0, NH-Val-Ala-0=0, "-Val-Lys-=c NH-Ala-Lys-0=0, and Most preferably, Q is selected from NH-Phe-Lys-C=O, NH-Val-Cit-0=0 or NH-Val-Ala- Other dipeptide combinations of interest include: NH -Gly-Gly-NH -Pro-Pro-0'0, and NH -Val-Glu-c=c).
Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.
In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide then is a recognition site for cathepsin.
In one embodiment, the amino acid side chain is chemically protected, where appropriate.
The side chain protecting group may be a group as discussed above. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog, and as described above.
GL
GL may be selected from (GLi-1) 0 (GL6) 0 O //
N o
d'ra o (GL1-2) 1011 (GL') Br Ar jr \ (GL2) 0 (GL8) \\,.
N0,N_A
O
(GL3-1) >k (G L9) N3\ S.>.. / 'N +/
(N 02) where the NO2 group is optional (G L3-2) >4 (G Ll 0) ii H S-S ---r
H
where (NO2) the NO2 group is optional (GI-3-3) s-s)-1 (G L11 Ni_ jN 02 -where the NO2 group is optional (GL3-4) S-S (G Ll 2) ,-/ 02N 4.
where the NO2 group is optional (GL4) 0 (G L13) N= N X- , / Hal N
H
Where Hal = I, Br, CI (GI') 0 Hal-// O-1 where Ar represents a C5.6 arylene group, e.g. phenylene, and X represents C1-4 alkyl.
In some embodiments, GL is selected from GL" and GL1-2. In some of these embodiments, GL is GL1-1.
GL
GLL ka may be selected from: (GLL1-1) CBA 0 N (GLL8-1) CBA \ ./,' ,,, N\ 0 N NN crt (GLL1-2) CBA o -- )5? G LL8-2) N CBA 0 ( *
N
___
(GLL2) o (G L191) ii., N o N--* N
CBA
(G LL3-1) A >1/4, (G LL9-2) N S N1'2 \ N-A
CBA
(G LL3-2) GL10 ca, N---, vo*
H
(GLL-4) cBA GL11 -
HN \ i f
H N
(G"5) WA1 0 GL12 N HN _ 1 7 s
X <'
0-1 01 (G LL6) 0 GL13 H
CEA
(G LL7) CBA where Ar represents a C5.6 arylene group, e.g. phenylene and X represents C1_4 alkyl.
In some embodiments, GLL is selected from GLL andGLL1-2. In some of these embodiments, GLL is Ga1-1
X X is: b N
H d c
where a = 0 to 5, b = 0 to 16, c = 0 or 1, d = 0 to 5.
a may be 0, 1, 2, 3, 4 or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 0.
b may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b is 0 to 12. In some of these embodiments, b is 0 to 8, and may be 0, 2, 4 or 8.
c may be 0 or 1 d may be 0, 1, 2, 3, 4 or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 1 or 2. In further embodiments, d is 2.
In some embodiments of X, a is 0, c is 1 and d is 2, and b may be from 0 to 8. In some of these embodiments, b is 0, 4 or 8.
In some embodiments, RL is of formula lb. In some embodiments, RLL is is formula lb'.
IRL1 and RL2 are independently selected from H and methyl, or together with the carbon atom to which they at bound form a cyclopropylene or cyclobutylene group.
In some embodiments, both RL' and RL2 are H. In some embodiments, RL' is H and RL2 is methyl.
In some embodiments, both RI' and RL2 are methyl.
In some embodiments, RI-1 and RL2 together with the carbon atom to which they are bound form a cyclopropylene group.
In some embodiments, RI-1 and RL2 together with the carbon atom to which they are bound form a cyclobutylene group.
In the group 111b, in some embodiments, e is 0. In other embodiments, e is 1 and the nitro group may be in any available position of the ring. In some of these embodiments, it is in the ortho position. In others of these embodiments, it is in the para position.
In some embodiments of the first aspect of the present invention the compounds are of formula la, lb, Ic, Id, le of If:
N
O la lb Ic le Id If
where Rea and Rea' are the same and are selected from: (a) Me0 (b) ; (c) (d) (e) (d) R7a' is selected from methoxy and benzyloxy; R' is as defined above; m is 1 or 3.
In some of the above embodiments, R' is of formula Ala: (g) and (Ala) RL/ wherein RL is a linker for connection to a cell binding agent, which is selected from: (ia): H GL la wherein Q is: NH-2, where Qx is such that Q is a dipeptide residue, such as Val-c(=cy
H
Ala; X is: 0 a N H d where a = 0, b = 0 to 16, c =1, d = 2; GL is a linker for connecting to a Ligand Unit, such as GL-1: (ib): lb where RI' is methyl and RL2 is H and methyl, and e is 1.
In some embodiments of the third aspect of the present invention, the drug linkers (DL) are of formula IIla,111b,111c,111d, Ille or Illf: IIlb Illc IIId IIle IIIf
O
O
O IIla
where Rea and Rea' are the same and are selected from: (a) Me° (b) ; (c) ; (h) R78' is selected from methoxy and benzyloxy; R7L is as defined above; m is 1 or 3.
In some of the above embodiments, R7L is of formula A2a: (g) and 0 (A2a) RLL / wherein RLL is a linker for connection to a cell binding agent, which is selected from: (ia'): GLL la'
X
wherein Q is: c(=cy NH-2, where Qx is such that Q is a dipeptide residue, such as Val-
H Ala; X is:
0 b N H
GL d
where a = 0, b = 0 to 16, c =1, d = 2; GLL is a linker for connecting to a Ligand Unit, such as G'1: CBA; and (ib'): lb' where IRL1 is methyl and RL2 is H and methyl, and e is 1.
Examples
General methods Flash chromatography was performed using a Biotage® IsoleraTM 1 and fractions checked for purity using thin-layer chromatography (TLC). TLC was performed using Merck Kieselgel 60 F254 silica gel, with fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light or iodine vapour unless otherwise stated. Extraction and chromatography solvents were bought and used without further purification from VWR U.K. The analytical LC/MS conditions were as follows: Positive mode electrospray mass spectrometry was performed using a Shimadzu Nexera®/Prominence® LCMS-2020. Mobile phases used were solvent A (H2O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic acid).
Gradient for 3-minute run: initial composition 5% B held over 25 seconds, then increased from 5% B to 100% B over a 1 minute 35 seconds' period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 minutes. Flow rate was 0.8 mdminute.
Gradient for 15-minute run: Initial composition 5% B held over 1 minute, then increased from 5% B to 100% B over a 9 minute period. The composition was held for 2 minutes at 100% B, then returned to 5% B in 10 seconds and held there for 2 minutes 50 seconds. The total duration of the gradient run was 15.0 minutes. Flow rate was 0.6 mL/minute.
Detection was at 254 nm. Columns: Waters Acquity U PLC® BEH Shield RP18 1.7pm 2.1 x mm at 50 °C fitted with Waters Acquity UPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7pm, 2.1 mm x 5 mm (routine 3-minute run); and ACE Excel 2 C18-AR, 2 p, 3.0 x 100mm (15-minute run).
Synthesis of Ally! (2-hydroxyethyl)(methyhcarbamate 2 H Alloc N..,.."...-.,OH _N.,,z^,OH 1 2 2-Methylaminoethanol 1 (25 mL, 0.311 Mol) was dissolved in a mixture of H20/Dioxane 1:1 (500 mL) and cooled to 0°C. NaHCO3 (26.124 g, 0.311 Mol) was added, followed by allyl chloroformate (33.078 mL, 0.311 Mol) and the mixture as allowed to warm to room temperature over 1h. At this point, TCL analysis indicated the reaction was complete. The reaction mixture was concentrated in vacuo to about 100 mL, before being diluted with H2O and extracted with EtOAc (100 mL x 3). The combined organics were washed with brine, dried with MgSO4, filtered and concentrated in vacuo. The crude product was purified by isolera to give pure material 2 as a clear oil (42.33 g, 86% yield).
Synthesis of Methyl (S)-4-methylene-114-pyrrolidinium-2-carboxylate chloride 9 Me0 0 BocN CIS 'N.-, H2N 7 8 9 a) 1-(terf-butyl) 2-methyl (S)-4-methylenepyrrolidine-1,2-dicarboxylate 8.
Acid 7 (20 g, 88 mMol) and K2CO3 (36.48 g, 264 mMol) were dissolved in dry DMF (400 mL) and the mixture was stirred for 20 min. Methyl iodide (17 mL, 272.8 mMol) was subsequently added and the reaction allowed to stir overnight at room temperature. The mixture was concentrated in vacuo to give an orange oil which was taken up in EtOAc before being washed with H2O. The aqueous were extracted back with EtOAc. The combined organics were dried with MgSO4, filtered and concentrated in vacuo. The crude mixture is used as such in the next step.
b) Methyl (S)-4-methylene-114-pyrrolidinium-2-carboxylate chloride 9.
Ester 8 (assumed 100%, 88 mMol) was dissolved in dioxane (20 mL) and 4M HCloco in dioxane (60 mL) was added dropwise over 10 min. The reaction mixture was stirred for 1h at room temperature and the product precipitated. The precipitate was filtered, washes with Et20 and allowed to dry in a vacuum over at 40°C, giving product 9 as a white solid (10.3 g, 66%).
Synthesis of tert-Butyl (11S,11aS)-7-(2-(((allyloxy)carbonyl)(methyl)amino)ethoxy)-11-((tert- butyldimethylsilyboxy)-8-hydroxy-2-methylene-5-oxo-2,3,11, 11a-tetrahydro-1H-benzoielpyrrolo11,2-41,41diazepine-10(5H)-carboxylate 18 0 NO2 HO NO2 TIPSO NO2 TIPSO NO2 NAlloc ^ NAlloc ^ NAlloc
O
TIPSO NO2 \>--0Me TIPSO NO2 OH TIPSO NO2 _ OTBS 0 N l 0 H 10 H 11 0 H 12 0 a) Allyl (2-(5-formy1-2-hydroxy-4-nitrophenoxy)ethyl)(methyl)carbamate 4 6-nitrobenzo[d][1,3]dioxole-5-carbaldehyde 3 (29.04 g, 0.151 Mol) was dissolved in anhydrous DMSO (200 mL). Allyl (2-hydroxyethyl)(methyl)carbamate 2 (43.17 mL, 0.301 Mol) and Sodium (6.93 g, 0.301 Mol) were subsequently added and the mixture was allowed to stir at 35°C until complete (reaction followed by LCMS). Upon completion, the reaction mixture was diluted with H2O, then neutralised with conc. HCI before being extracted with
H
OH 6 0
NAlloc NAlloc ""NAlloc NAlloc NAlloc Boc TIPSO NH2 OTBS TIPSO NH --OTBS TIPSO NAlloc NAlloc Et20. The organics were washed with H2O and brine, before being dried with MgSO4, filtered and concentrated in vacuo. The crude mixture was purified by isolera (ultra cartridge 340 g) using EtOAc in Hexane (12% to 80% over 11 CV) to give pure material 4 as a brown solid (15.46g, 32%). LC/MS, 3 min method, 1.46 min (ES-) m/z = 323 [M -H]-.
b) Allyl (2-(5-formy1-4-nitro-2-((triisopropylsilyl)oxy)phenoxy)ethyl)(methyl) carbamate 5. Phenol 4 (15.44 g, 47.6 mMol), TIPS-CI (12.23 mL, 75.1 mMol) and imidazole (6.47 g, 95.2 mMol) were heated to 90°C (with initial addition of CH2Cl2 (100 mL) allowed to boil off) for 30 min at which point the reaction is complete. The reaction mixture was taken up in CH2Cl2 and washed with H2O, before being dried with MgSO4, filtered and concentrated in vacuo.
The crude mixture was loaded on isolera cartridge (ultra 100g) for purification using EtOAc in Hexane (4% to 40% over 11 CV) to give pure material 5 as a brown oil (16.9g, 74%). LC/MS, 3 min method, 2.04 min (ES+) m/z = 481.15 [M + H]'..
c) 5-(2-(((allyloxy)carbonyl)(methypamino)ethoxy)-2-nitro-4-( (triisopropylsily1)oxy)benzoic acid 6 Aldehyde 5 (16.5 g, 34.3 mMol) was dissolved in THE (100 mL) and NaCIO2 (7.76 g, 85.8 mMol) and Na2H2PO4 (6.81 g, 48 mMol) dissolved in H2O (150 mL) were added. 30% H202 (38.9 mL, 340 mMol) were subsequently added, followed by 1M HCl(aq.) (until reaching pH=6). The reaction mixture turned bright orange at this point. After 15 min, the reaction is complete. The reaction mixture was diluted with 1M HCI and extracted with EtOAc. The organics were washed with brine before being dried with MgSO4, filtered and concentrated in vacuo. The crude mixture is used as such in the next step. LC/MS, 3 min method, 1.98 min (ES+) m/z = 497.25 [M + d) Methyl (S)-1-(5-(2-(((allyloxy)carbonyl)(methypamino)ethoxy)-2-nitro-4-( (triisopropylsilypoxy)benzoy1)-4-methylenepyrrolidine-2-carboxylate 10 Acid 6 (12.038 g, 24.27 mMol) was dissolved in CH2Cl2 (100 mL) and stirred at room temperature before adding oxalyl chloride (6.674 mL, 26.697 mMol) and dry DMF (100 pL).
A lot of effervescence is observed. The reaction mixture was stirred with a calcium drying tube for a further 10 min at which point the reaction is complete (aliquot quenched by Me0H and LCMS analysis shows methyl ester formation).
Separately, C-ring 9 (4.752 g, 26.697 mMol) was dissolved in CH2Cl2 (100 mL) and TEA was added (13.524 mL, 97.08 mMol). The reaction mixture was cooled to -40°C and the acid chloride solution was slowly added via a cannula. The mixture was allowed to warm to room temperature over 30 min at which point the reaction was complete. The mixture was diluted with further CH2Cl2 and washed with 0.1 M HCI(aq.), conc. NaHCO3(aq.) and brine before being dried with MgSO4, filtered and concentrated in vacuo. The crude mixture was purified by isolera (ultra cartridge, 100g) using EtOAc in Hexane (12% to 100% over 11 CV) to give pure material 10 as a yellow oil (10.245 g, 68%). LC/MS, 3 min method, 1.989min (ES+) m/z = 620.2 [M + H]'..
e) Allyl (S)-(2-(5-(2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-4-nitro-2- ((triisopropylsilyhoxy)phenoxy)ethyl)(methyl)carbamate 11 Ester 10 (11.598 g, 18.7 mMol) was dissolved in dry THE (145 mL) and cooled to 0°C. LiBH4 (1.223 g, 56 mMol) was added and the mixture was allowed to warm to room temperature over 1h. At this point the reaction was complete (followed by LCMS). The reaction was diluted with ice cold water and neutralised with an aqueous solution of citric acid to pH=5. The mixture was extracted with CH2Cl2 twice. The organics were dried with MgSO4, filtered and concentrated in vacuo. The product was used as crude in the next step. LC/MS, 3 min method, 1.93min (ES+) m/z = 592.30 [M + H]'..
f) Allyl (S)-(2-(5-(2-(((tert-butyldimethylsilyl)oxy)methyl) -4-methylenepyrrolidine-1-carbonyl)-4-nitro-2-((triisopropylsilyl)oxy) phenoxy)ethyl)(methyl)carbamate 12 Alcohol 11 (assumed 100%, 18.7 mMol) was dissolved in CH2Cl2 (150 mL). Imidazole (2.426 g, 35.6 mMol) and TBDMS-CI (4.028 g, 26.7 mMol) were subsequently added and the mixture was stirred for 1h at which point the reaction was complete (followed by LCMS). The imidazole salt was filtered off and the filtrate concentrated in vacuo. The crude mixture was purified by isolera (ultra cartridge, 100g) using EtOAc in Hexane (8% to 57% over 10 CV) to give pure material 12 as a yellow oil (10.616 g, 84%). LC/MS, 3 min method, 2.26 min (ES+) m/z = 706.30 [M + H]*..
g) Allyl (S)-(2-(4-amino-5-(2-(((rett-butyldimethylsilyhoxy)methyl) -4-methylenepyrrolidine-1-carbony1)-2-((triisopropylsilyhoxy)phenoxy) ethyl)(methyl)carbamate 13 Nitro compound 12 (10.616 g, 15.03 mMol) was dissolved in 5% formic acid in Me0H (150 mL) and Zinc (24.5 g, 0.376 Mol) was added portion wise making sure the exotherm does not exceed 35°C. Upon completion, the reaction mixture was filtered through a pad of celite. The pad was washed thoroughly with EtOAc. The organics were washed with sat. NaHCO3(aq.), brine and were dried with MgSO4, filtered and the volatiles removed in vacuo to give crude product 13 as a yellow oil (10.0 g). LC/MS, 3 min method, 2.21 min (ES+) m/z = 676.3 [M + H]*..
h) Allyl (S)-(2-(4-((tert-butoxycarbonybamino)-5-(2-(((tert-butyldimethylsilyboxy) methyl)-4-methylenepyrrolidine-1-carbonyl)-2-((triisopropylsilyboxy) phenoxy)ethyl)(methypcarbamate 14 Amine compound 13 (assumed 100%, 15.037 mMol) and Boc2O (3.547 g, 16.272 mMol) were dissolved in THE (200 mL) and the mixture was left to stir at 85°C over 2 days. The reaction was not quite complete so further Boc2O (350 mg) was added and the reaction mixture left to stir until complete. The mixture was concentrated in vacuo and the crude mixture was purified by isolera (ultra cartridge, 100 g) using EtOAc in Hexane (5% to 37% over 10 CV) to give pure material 14 as a pale-yellow oil (11.3 g, 99% over 2 steps). LC/MS, 3 min method, 2.36 min (ES+) m/z = 798.4 [M + Na]' i) Allyl (S)-(2-(4-((terf-butoxycarbonyDamino)-5-(2-(hydroxymethyl) -4-methylenepyrrolidine-1-carbony1)-2-((triisopropylsilyboxy)phenoxy) ethyl)(methyl)carbamate 15 Amine compound 14 (11.3 g, 14.562 mMol) was solubilised in a mixture of AcOH:THF:MeOH:H20 (7:1:1:2, 110 mL) and the mixture was stirred at 40°C for 3h at which point it was complete. The reaction mixture was diluted with EtOAc and washed with sat NaHCO3 (aq.), brine, dried over MgSO4, filtered and the volatiles removed in vacuo. The crude mixture was purified by isolera (ultra cartridge, 100 g) using EtOAc in Hexane (8% to 67% over 10 CV) to give pure material 14 as a pale-yellow oil (7.285 g, 76%). LC/MS, 3 min method, 2.05 min (ES+) m/z = 662.3[M + H]'..
j) fert-Butyl (11S,11aS)-7-(2-(((allyloxy)carbonyl)(methyl)amino)ethoxy) -11-hydroxy-2-methylene-5-oxo-8-((triisopropylsily0oxy)-2,3,11, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 16 DMSO (1.95 mL, 27.457 mMol) was added dropwise to a solution at -78°C of oxalyl chloride (6.59 mL, 13.180 mMol) in CH2Cl2 (70 mL). After 10 min, a solution of amine 15 (7.27 g, 10983 mMol) in CH2Cl2 (100 mL) was added slowly to the aforementioned solution (still at 78°C). After 15 min, TEA (7.665 mL, 54.915 mMol) was added dropwise and the reaction mixture was allowed to slowly warm to room temperature. Upon completion, the reaction was diluted with CH2Cl2, washed with 0.1M HCI(aq.), sat. NaHCO3(aq.) and brine, before being dried with MgSO4, filtered and the volatiles removed in vacuo. The crude mixture was purified by isolera (ultra cartridge, 100 g) using EtOAc in Hexane (12% to 100% over 11 CV) to give pure material 16 as a pale-yellow oil (7.08 g, 98%). LC/MS, 3 min method, 1.95 min (ES+) m/z = 660.25 [M + H]'..
k) tert-Butyl (11S,11aS)-7-(2-(((allyloxy)carbonyl)(methyl)amino)ethoxy)-11-( (tert-butyldimethylsilyboxy)-2-methylene-5-oxo-8-((triisopropylsilyl)oxy) -2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H) -carboxylate 17 Alcohol 16 (7.086 g, 10.733 mMol) was dissolved in CH2Cl2 (100 mL). 2,6-Lutidine (5 mL, 42.932 mMol) was added and the mixture cooled to 0°C. After stirring for 10 min, TBS-OTf (7.395 mL, 32.2 mMol) was added and the reaction allowed to warm to room temperature for 111 at which point it was complete. The mixture was diluted with CH2Cl2 and washed with H2O, sat. NaHC0300 and brine, before being dried with MgSO4, filtered and the volatiles removed in vacuo. The crude mixture was purified by isolera (ultra cartridge, 100 g) using EtOAc in Hexane (12% to 60% over 11 CV) to give pure material 17 as a pale-yellow oil (6.875 g, 81%). LC/MS, 3 min method, 2.36 min (ES+) m/z = 774.35 [M + H]+ . I) tert-Butyl (11S,11aS)-7-(2-(((allyloxy)carbonyl)(methyDamino)ethoxy)-11-( (tertbutyldimethylsilyl)oxy)-8-hydroxy-2-methylene-5-oxo-2,3, 11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H) -carboxylate 18 Compound 17 (6.875 g, 8.882 mMol) was dissolved in a mixture of H20/DMF (0.2/99.8, 40 mL). LiOAc (906 mg, 8.882 mMol) was added and the mixture was stirred at room temperature for 16h. The mixture was diluted with EtOAc and washed with an aqueous solution of citric acid, H2O. and brine, before being dried with MgSO4, filtered and the volatiles removed in vacuo. The crude mixture was purified by isolera (ultra cartridge, 100 g) using EtOAc in Hexane (8% to 85% over 18 CV) to give pure material 18 as a yellow foam (5.445 g, 99%). LC/MS, 3 min method, 1.93 min (ES+) m/z = 618.3 [M + H]' . Synthesis of tert-Butyl (11S,11aS)-84(3-(bromomethyl)benzypoxy)-11-((tert-butyldimethylsilyfloxy) -7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzolelpyrrolor1, 2-a111,41diazepine-10(5H)-carboxylate 56 HO NO2 TIPSO NO2 TIPSO NO2 Me0 Me0 Me0 OH O 21 0 TIPSO NO2 OTBS TIPSO NO2 --OH TIPSO NO2 \\*.-0Me MeO Nc22 MeO MeO 23 o Boc Boc TIPSO NH --OTBS TIPSO NH (OH
TIPSO MeO MeO 27 0
TIPSO MeO 28 0 Boc
HO OTBS MeO 0
1,5-Diiodopentane Boc /OTBS a,u,'-dibromo-m-xylene a) 4-Hydroxy-5-methoxy-2-nitrobenzaldehyde 19 Sodium (6.19 g, 0.269 Mol) was slowly added to dry methanol (250 mL) in a flask under argon and the mixture was left to stir until all the solid has dissolved. 6-nitrobenzo[d][1,3]dioxole-5-carbaldehyde 3 (25 g, 0.128 Mol) was added and the solution was heated to 90°C for 1.5h before being allowed to cool back to room temperature. The reaction mixture was acidified to pH=4 with a 1M HCI(aq) solution before being extracted with EtOAc (3x200 mL). The combined organics were washed with H2O (3x100 mL) and brine, before being dried with MgSO4, filtered and concentrated in vacuo. The crude mixture was used as such in the next step (19.9 g, dark brown solid). LC/MS, 3 min method, 1,26 min (ES-) m/z = 195.5 [M -H] . b) 5-Methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzaldehyde 20 Phenol 19, (19.9 g, crude) was dissolved in CH2Cl2 (100 mL). TIPS-CI (25.9 mL, 0.121 Mol) and TEA (28.1 mL, 0.202 Mol) were added and the mixture stirred at room temperature for 1 h at which point the reaction was complete. The mixture was concentrated to dryness and the crude mixture was directly loaded on isolera cartridge (ultra 100g) for purification using EtOAc in Hexane (5% to 60% over 12 CV) to give pure material 20 as a dark brown oil (30.8 g, 87% yield). LC/MS, 3 min method, 2.07 min (ES+) m/z = 368.15 [M + H2O].
c) 5-Methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid 21 Aldehyde 20 (30.5 g, 86.28 mMol) was dissolved in THE (200 mL) and NaCI02(19.507 g, 215 mMol) and Na2H2PO4 (17.152 g, 120.79 mMol) dissolved in H2O (200 mL) were added. 30% H202 (48.9 mL, 431 mMol) were subsequently added followed by 1M HC10,0(until reaching pH=6). The reaction mixture turns bright orange at this point. After 15 min the reaction is complete. The reaction mixture was diluted with 1M HCI(aq.) and extracted with EtOAc. The organics were washed with brine before being dried with MgSO4, filtered and concentrated in vacuo. The crude mixture (orange oil) is used as such in the next step. LC/MS, 3 min method, 1.99 min (ES-) m/z = 367.9 [M - d) Methyl (S)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoy1) -4-methylenepyrrolidine- 2-carboxylate 22 Acid 21 (15.83 g, 42.85 mMol) was dissolved in CH2Cl2 (200 mL) and stirred at room temperature before adding oxalyl chloride (23.571 mL, 47.14 mMol) and dry DMF (200 pL). A lot of effervescence was observed. The reaction mixture was stirred with a calcium drying tube for a further 10 min at which point the reaction is complete (aliquot quenched by Me0H and LCMS analysis shows methyl ester formation).
Separately, C-ring 9 (8.374 g, 47.14 mMol) was dissolved in CH2Cl2 (150 mL) and TEA was added (23.881 mL, 171.42 mMol). The reaction mixture was cooled to -40°C and the acid chloride solution was slowly added via a canula. The mixture was allowed to warm to room temperature over 30 min at which point the reaction was complete. The mixture was diluted with further CH2Cl2 and washed with 0.1 M HC100., conc. NaHCOS(aq.) and brine before being dried with Mg304, filtered and concentrated in vacuo. The crude mixture was purified by isolera (ultra cartridge, 100g) using EtOAc in Hexane (8% to 50% over 12 CV) to give pure material 22 as a yellow oil (14.327 g, 66%). LC/MS, 3 min method, 2.0 min (ES+) m/z = 515.2 [M + Na]'.
e) (S)-(2-(hydroxymethyl)-4-methylenepyrrolidin-1-y1)(5-methoxy-2-nitro-4-( (triisopropylsilyboxy)phenyl)methanone 23 Ester 22 (14.31 g, 29 mMol) was dissolved in dry THE (150 mL) and cooled to 0°C. LiBH4 (1.899 g, 87 mMol) was added and the mixture was allowed to warm to room temperature over 1 h. At this point the reaction was complete (followed by LCMS). The reaction was diluted with ice cold water and neutralised with an aqueous solution of citric acid to pH=5. The mixture was extracted with CH2Cl2 twice. The organics were dried with MgSO4, filtered and concentrated in vacuo. The product was used as crude in the next step. LC/MS, 3 min method, 1.93 min (ES+) m/z = 465.15 [M + H]'..
f) (S)-(2-(((tert-butyldimethylsilyboxy)methyl)-4-methylenepyrrolidin-1-y1) (5-hydroxy-2-nitro-4-((triisopropylsily0oxy)phenyOmethanone 24 Alcohol 23 (12.16 g, 26 mMol) was dissolved in CH2Cl2 (180 mL). Imidazole (3.558 g, 52.3 mMol) and TBDMS-CI (5.9 g, 31.2 mMol) were subsequently added and the mixture was stirred for 1h at which point the reaction was complete (followed by LCMS). The imidazole salt was filtered off and the filtrate concentrated in vacua. The crude mixture was purified by isolera (ultra cartridge, 100g) using EtOAc in Hexane (5% to 30% over 10 CV) to give pure material 24 as a yellow oil (13.57 g, 90%). LC/MS, 3 min method, 2.3 min (ES+) m/z = 601.55 [M + Na]'..
g) (S)-(2-amino-5-methoxy-4-((triisopropylsilypoxy)phenyl)(2-(( (terfbutyldimethylsilyboxy)methyl)-4-methylenepyrrolidin-1-Amethanone 25 Nitro compound 24 (13.57 g, 23.44 mMol) was dissolved in 5% formic acid in Me0H (200 mL) and Zinc (38.33 g, 586 mMol) was added portion wise making sure the exotherm does not exceed 35°C. Upon completion, the reaction mixture was filtered through a pad of celite.
The pad was washed thoroughly with EtOAc. The organics were washed with sat. NaHCO3 (a9.), brine and were dried with MgSO4, filtered and the volatiles removed in vacuo to give crude product 25 as a yellow oil. LC/MS, 3 min method, 2.27 min (ES+) m/z = 549.25 [M + H]* h) tert-Butyl (S)-(2-(2-(((tert-butyldimethylsily0oxy)methyl) -4-methylenepyrrolidine-1-carbonyI)-4-methoxy-5-((triisopropylsi ly0oxy)phenyl)carbamate 26 Amine compound 25 (7.995, 14.565 mMol) and Boc2O (3.497 g, 16.022 mMol) were dissolved in THE (30 mL) and the mixture was left to stir at 85°C over 2 days. The mixture was concentrated in vacuo and the crude mixture was used as such in the next step. LC/MS, 3 min method, 2.42min (ES+) m/z = 671.3 [M + H]'.
i) tert-Butyl (S)-(4-hydroxy-2-(2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-5-( (triisopropylsilypoxy)phenyficarbamate 27 Amine compound 26 (assumed 100%) was solubilised in a mixture of AcOH:THF:MeOH:H20 (7:1:1:2, 70 mL) and the mixture was stirred at 40°C for 3h at which point it was complete. The reaction mixture wsa diluted with EtOAc and washed with sat NaHCOZ(aq.), brine, dried over MgSO4, filtered and the volatiles removed in vacuo. The crude mixture was purified by isolera (ultra cartridge, 50 g) using EtOAc in Hexane (8% to 60% over 10 CV) to give pure material 27 as a yellow solid (6.133 g, 79%). LC/MS, 3 min method, 2.07 min (ES+) m/z = 535.3 [M + H]'..
j) tent-Butyl (11S,11aS)-11-hydroxy-7-methoxy-2-methylene-5-oxo-8-( (triisopropylsily0oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1, 4]diazepine-10(5H)-carboxylate 28 DMSO (0.639 mL, 9 mMol) was added dropwise to a solution of oxalyl chloride (2.16 mL, 4.32 mMol) at -78°C in CH2Cl2 (60 mL). After 10 min, a solution of amine 27 (1.925 g, 3.6 mMol) in CH2Cl2 (10 mL) was added slowly to the aforementioned solution (still at -78°C). After 10 min, TEA (2.512 mL, 18 mMol) was added dropwise and the reaction mixture was allowed to slowly warm to room temperature. Upon completion, the reaction was diluted with CH2Cl2, washed with 0.1M HCl(aq.), sat. NaHCOS(aq.) and brine, before being dried with MgSO4, filtered and the volatiles removed in vacuo. The crude mixture was purified by isolera (ultra cartridge, 100 g) using EtOAc in Hexane (8% to 60% over 11 CV) to give pure material 28 as a pale-yellow oil (1.27 g, 66%). LC/MS, 3 min method, 1.9 min (ES+) m/z = 532.25 [M + H]*.
k) tert-Butyl (11S,11aS)-11-((tert-butyldimethylsilypoxy)-7-methoxy-2-methylene-5-oxo-8- ((triisopropylsily0oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1, 4]diazepine-10(5H)-carboxylate 29 Alcohol 28 (1.28 g, 2.36 mMol) was dissolved in CH2Cl2 (40 mL). 2,6-Lutidine (1.1 mL, 9.4 mMol) was added and the mixture cooled to 0°C. After stirring for 10 min, TBS-OTf (1.63 mL, 7.09 mMol) was added and the reaction allowed to warm to room temperature for 1h at which point it has completed. The mixture was diluted with CH2Cl2 and washed with H2O, sat. NaHCO3(aq.) and brine, before being dried with MgSO4, filtered and the volatiles removed in vacuo. The crude mixture was purified by isolera (ultra cartridge, 50 g) using EtOAc in Hexane (5% to 30% over 12 CV) to give pure material 29 as a colourless solid (1.58 g, 86%). LC/MS, 3 min method, 2.33 min (ES+) m/z = 647.4 [M + H]'.
I) tert-Butyl (11S,11aS)-11-((tert-butyldimethylsilyl)oxy) -8-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e] pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 30. Compound 29 (1.57 g, 2.427 mMol) was dissolved in a mixture of H20/DMF (0.2:99.8; 10 mL). LiOAc (248 mg, 2.427 mMol) was added and the mixture was stirred at room temperature for 16h. The mixture was diluted with EtOAc and washed with an aqueous solution of citric acid, H2O. and brine, before being dried with MgSO4, filtered and the volatiles removed in vacuo to give crude product 30 (1.445 g). LC/MS, 3 min method, 1.81 min (ES+) m/z = 491.25 [M + H]' . m) tert-Butyl (11S,11aS)-11-((tert-butyldimethylsilyl)oxy)-8-((5-iodopentyl)oxy) -7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazepine-10(5H)-carboxylate 31 Alcohol 30 (535 mg, 1.09 mMol) was dissolved in CH2Cl2 (8 mL) before adding K2CO3 (150 mg, 1.09 mMol) and 1,5-diiodopentane (811 pL, 5.45 mMol). The reaction mixture was stirred at 60°C for 1h before allowing to cool to room temperature. The solution was filtered on a pad of celite and the pad washed with acetone. The filtrated was concentrated in vacuo and the crude solid was purified by isolera (ultra cartridge, 25 g) using EtOAc in Hexane (16% to 85% over 10 CV) to give pure material 31 as a pale yellow solid (513 mg, 86%). LC/MS, 3 min method, 2.09 min (ES+) m/z = 687.4 [M + H]' . n) tert-Butyl (11S,11aS)-84(3-(bromomethyObenzypoxy)-11-((tert-butyldimethylsily0oxy) -7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1, 2-41,4]diazepine10(5H)-carboxylate 56 Alcohol 30 (208 mg, 0.424 mMol) and a,a'-dibromo-m-xylene (560 g, 2.12 mMol) were dissolved in DMF (2.5 mL) and K2CO3 (59 mg, 0.424 mMol) was added. The reaction mixture was stirred at 35°C for 30 min before allowing to cool to room temperature. The reaction mixture was concentrated in vacuo and the crude solid was purified by isolera (ultra cartridge, 10 g) to give pure material 56 as a colourless solid (249 mg, 87%). LC/MS, 3 min method, 2.03 min (ES+) m/z = 696.85 [M + Na]' . Synthesis of tert-Butyl (S)-84(3-(bromomethyl)benzyl)oxy)-7-methoxy-2-methylene-5-oxo2,3,11, 11a-tetrahydro-1H-benzotelpyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 50 Boo a) tert-butyl (S)-7-m ethoxy-2-methylene-5-oxo-8-((triisopropylsilyl)oxy)-2,3,11, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 42 DIAD (2.582 mL, 13.117 mMol) was added slowly to a stirred solution of 27 (3.188 g, 5.962 mMol) and PPh3 (4.686 g, 17.886 mMol) in fry THE (60 mL). The reaction mixture was stirred at 40°C for 1 h at which point the reaction was complete. The volatiles were removed in vacuo and the crude mixture was purified by isolera (ultra cartridge, 100 g) using EtOAc in Hexane (8% to 60% over 11 CV) to give material 42 as a yellow oil (3.424 g, >100% some DIAD leftover). LC/MS, 3 min method, 2.11 min (ES+) m/z = 539.4 [M + Na]*.
b) tert-Butyl (S)-8-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1Hbenzo[e] pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 43 Compound 42 (assumed 100%, 5.962 mMol) was dissolved in a mixture of H20/DMF (0.2:99.8; 30 mL). LiOAc (608 mg, 5.962 mMol) was added and the mixture was stirred at room temperature for 16h. The mixture was diluted with EtOAc and washed with an aqueous solution of citric acid, H2O. and brine, before being dried with MgSO4, filtered and the volatiles removed in vacuo. The crude solid was purified by isolera (ultra cartridge, 25 g) using 20%MeOH/CH2Cl2 and CH2Cl2 (6% to 33% over 7 CV) to give pure material 43 (1.885 g, 88%). LC/MS, 3 min method, 1.44 min (ES-) m/z = no ionisation.
H ck,"'-dibromo-m-xylene Boc MeO 1,5-Diiodopentane Boc Boc Boc
I OH
TIPSO NH / MeO 27 0
TIPSO MeO
HO MeO
c) tert-Butyl (S)-8-((5-iodopentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,11, 11a-tetrahydro1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 44 Alcohol 43 (873 mg, 2.422 mMol) was dissolved in CH2Cl2 (8 mL) before adding K2CO3 (335 mg, 2.422 mMol) and 1,5-diiodopentane (1.802 mL, 12.11 mMol). The reaction mixture was stirred at 60°C for lh before allowing to cool to room temperature. The solution was filtered on a pad of celite and the pad washed with acetone. The filtrated was concentrated in vacuo and the crude solid was purified by isolera (ultra cartridge, 25 g) using EtOAc in Hexane (16% to 85% over 10 CV) to give pure material 44 as a pale yellow solid (731 mg, 54%). LC/MS, 3 min method, 1.75 min (ES+) m/z = 579.2 [M + Na].
d) tert-Butyl (S)-8-((3-(bromomethyl)benzyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,11, 11atetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 50 Alcohol 43 (303 mg, 0.841 mMol) and a,a'-dibromo-m-xylene (1.11 g, 4.204 mMol) were dissolved in DMF (3 mL) and K2CO3 (116 mg, 0.841 mMol) was added. The reaction mixture was stirred at 35°C for 30 min before allowing to cool to room temperature. The reaction mixture was concentrated in vacuo and the crude solid was purified by isolera (ultra cartridge, 25 g) using EtOAc in Hexane (16% to 100% over 10 CV) to give pure material 50 as a colourless solid (225 mg, 49%). LC/MS, 3 min method, 1.7 min (ES+) m/z = 565.1 [M + Na].
Synthesis of Ally! ((S)-3-methy1-1-WS)-1-((4-(a((4-nitrobenzynoxy)carbonyl)oxy)methyl) phenynamino)-1-oxoprooan-2-ynamino)-1-oxobutan-2-yl)carbamate 34 OH 0y0 N O2 NO2 Alcohol 62 (400 mg, 1.06 mMol) and carbonate 63 (1.056 g, 3.179 mMol) were dissolved in DMF (5 mL) before adding DIPEA (554 pL, 2.179 mMol). The reaction mixture was stirred for 16h at room temperature before the volatiles were removed in vacuo. The resulting solid was triturated with Et20 yielding pure product 34 as a pale yellow solid (495 mg, 84%). LC/MS, 3 min method, 1.6 min (ES+) m/z = no ionisation.
Example 1: 4-((2S 5S)-37-(2 5-dioxo-2 5-dihydro-1H-pyrrol-1-y1)-5-isopropy1-2-methyl-4 7 35- trioxo-10 13 16 19 22 25 28 31-octaoxa-3 6 34-triazaheptatriacontanamido)benzyl (2-(((S)-8- ((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5, 11a-tetrahydro-1H-benzofelpyrrolo11,2- a-1[1,41cliazepin-8-yboxy)pentyboxy)-2-methylene-5-oxo-2,3,5, 11a-tetrahydro-1Hbenzolelpyrrolort 2-a111,41diazepin-7-yboxy)ethyl)(methyl)carbamate Exl a) tert-Butyl (11S,11aS)-7-(2-(((allyloxy)carbonyl)(methyDamino)ethoxy)-8-((5-(((11S, 11aS)-10-(tett-butoxycarbony1)-11-((tert-butyldimethylsilypoxy)-7-m ethoxy-2-m ethylene-5-oxo2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-41,4] diazepin-8-yl)oxy)pentyl)oxy)-11- ((tett-butyldimethylsilyl)oxy)-2-methylene-5-oxo-2,3,11, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 32 Alcohol 18 (411 mg, 0.66 mMol) and monomer 31 (457 mg, 0.66 mmol) were dissolved in butanone (10 mL) and K2CO3 (102 mg, 0.74 mMol) was added. The reaction mixture was stirred at 80°C for 1h before allowing to cool to room temperature. The reaction mixture was concentrated in vacuo and the crude solid was purified by isolera (ultra cartridge, 25 g) using EtOAc in Hexane (5% to 100% over 20 CV) to give pure material 32 as a colourless solid (619 mg, 79%). LC/MS, 3 min method, 2.27 min (ES+) m/z = 1198.5 [M + Na].
:ns0 Ecc b) tert-Butyl (11S,11aS)-8-((5-(((11S,11aS)-10-(tert-butoxycarbonyI)-11-( (tert-butyldimethylsilypoxy)-7-(2-(methylamino)ethoxy) -2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1, 4]d iazepin-8-yhoxy)pentypoxy)-11-((tert-butyldimethylsilypoxy) -7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazepine-10(5H)-carboxylate 33 Dimer 32 (610 mg, 0.518 mMol) was dissolved in dry CH2Cl2 (7 mL) and pyrrolidine (86 pL, 1.037 mMol) was added. After 3 min of stirring, Pd(PPh3)4 (6 mg, 0.005 mMol) was added and the mixture was left to stir for 30 min at which point LCMS analysis indicated completion. The mixture was diluted with CH2Cl2 and washed with a saturated solution of NH4C1(aq.), brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo to give crude product 33 (511 mg). LC/MS, 3 min method, 1.67 min (ES+) m/z = 1092.5 [M + H]*..
c) tert-Butyl (115,11aS)-7-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino) -3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(methyl)amino)ethoxy) -8-((5- (((115,11aS)-10-(tert-butoxycarbony1)-11-((tert-butyldimethylsilyhoxy) -7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo [1,2-a][1,4]diazepin-8-yhoxy)pentyhoxy)-11-((tert-butyldimethylsily1)oxy) -2-methylene-5-oxo-2,3,11,11a-tetrahydro1H-benzo[e]pyrrolo[1,2-41, 41diazepine-10(5H)-carboxylate 35 Amine 33 (175 mg, 0.16 mMol) and carbamate 34 (89 mg, 0.16 mMol) were dissolved in dry THE (5 mL) and the mixture was stirred at room temperature for 2h. The reaction mixture was concentrated in vacua and the crude solid was purified by isolera (ultra cartridge, 10 g) using EtOAc in Hexane (16% to 100% over 25 CV) to give pure material 35 as a colourless solid (216 mg, 90%). LC/MS, 3 min method, 2.23 min (ES+) m/z = 1496.55 [M + H]'..
d) tert-Butyl (11S,11aS)-7-(2-((((44(S)-24(S)-2-amino-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)(methyl)amino)ethoxy)-8-( (5-(((11S,11aS)-10-(tert-butoxycarbonyI)-11-((tert-butyldimethylsilyl)oxy) -7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo [1,2-a][1,4]diazepin-8-yhoxy)pentyhoxy)-11-((tert-butyldimethylsilyl)oxy) -2-methylene-5-oxo-2,3,11,11a-tetrahydro- 1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 36 Dimer 35 (212 mg, 0.142 mMol) was dissolved in dry CH2Cl2 (3 mL) and pyrrolidine (18 pL, 0.213 mMol) was added. After 3 min of stirring, Pd(PPh3)4 (1.6 mg, 0.001 mMol) was added and the mixture was left to stir for 30 min at which point LCMS analysis indicated completion. The mixture was diluted with CH2Cl2 and washed with a saturated solution of NH4Cloo, brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo to give crude product 36 (193 mg). LC/MS, 3 min method, 1.72 min (ES+) m/z = 1412.4 [M + H]'.
e) tert-Butyl (11S,11aS)-8-((5-(((11S,11aS)-10-(tert-butoxycarbonyI)-11-( (tertbutyldimethylsilypoxy)-7-(2-((((4-((2S,5S)-37-(2,5-dioxo-2, 5-dihydro-1H-pyrrol-1-y1)-5-isopropy1-2-methy1-4,7,35-trioxo-10,13,16,19, 22,25,28,31-octaoxa-3,6,34- triazaheptatriacontanamido)benzyl)oxy)carbonyl)(methyl)amino)ethoxy) -2-methylene-5-oxo- 2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-Aoxy) pentypoxy)-11-((tett-butyldimethylsily1)oxy) -7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1Hbenzo[e]pyrrolo[1, 2-a][1,4]diazepine-10(5H)-carboxylate 37 Amine 36 (57 mg, 0.04 mMol), Mal-dPEGs-OH (24 mg, 0.04 mMol) and EDCI.HCI (8 mg, 0.04 mMol) were dissolved in dry CH2Cl2 (3 mL) and the reaction mixture was left to stir at room temperature for 30 min. The mixture was diluted with CH2Cl2 and washed with H2O, brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo. The crude material was purified by isolera (ultra cartridge, 10 g) using 10% Me0H/CH2C12 and CH2Cl2 (25% to 80% over 10 CV) to give pure material 37 as a colourless solid (59 mg, 75%).
LC/MS, 3 min method, 2.14 min (ES+) m/z = 1986.6 [M + f) tert-Butyl (115,11aS)-8-((5-(((11S,11aS)-10-(tert-butoxycarbony1) -11-hydroxy-7-methoxy2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo [e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentypoxy)-7-(2-((((44(2S,5S)-37- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y1)-5-isopropy1-2- methy1-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6, 34-triazaheptatriacontanamido)benzyl)oxy)carbonyl)(methyl)amino)ethoxy) -11-hydroxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazepine-10(5H)-carboxylate 38 Dimer 37 (89 mg, 0.044 mMol) was dissolved in THE (3 mL). TBAF (179 pL, 0.179 mMol) and acetic acid (11 pL, 0.179 mMol) were pre-mixed and added to the aforementioned solution. The reaction was left to stir over the weekend before being taken up in CHCI3 and washed with H2O, NaHCO3 and brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo to give crude product 39. LC/MS, 3 min method, 1.6 min (ES+) m/z = 1780.05 [M + Na].i g) 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y1) -5-isopropy1-2-methyl-4,7,35-trioxo10,13,16,19,22,25,28,31-octaoxa-3,6, 34-triazaheptatriacontanamido)benzyl (2-(((S)-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2- a][1,4]d azep n-8-y0oxy)pentyl)oxy)-2-m ethyle ne-5-oxo-2,3,5, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)oxy)ethyl) (methyl)carbamate Exl Dimer 38 (assumed 100%, 0.044 mMol) was cooled to 0°C. Separately, the solution of TFA 95% in H2O (0.85 mL) was cooled to 0°C before adding to the dimer. The reaction mixture was stirred at 0°C for 40 min before pouring onto a 1:1 solution of CHCI3/NaHCO3 (40 mL) at 0°C. The organic layer was separated, dried on MgSO4 before being filtered and the volatiles removed in vacuo. The crude material was purified by preparative HPLC yielding product Exl as a yellow solid (17 mg, 25%). LC/MS, 3 min method, 1.47 min (ES+) m/z = 1522.55 [M + Hr.
Example 2: 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y1) -5-isopropy1-2-methyl-4,7,35- trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido) benzyl (2-WS)-8- ((5-faS)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo [elpyrrolo11,2- a][1,41diazepin-8-yboxy)pentyl)oxy)-2-methylene-5-oxo-2,3,5, 11a-tetrahydro-1Hbenzofelpyrrolor1,2-a111,41diazepin-7-yl)oxy)ethyl) (methyl)carbamate Ex 2 B tit Bet 0 TES Hid Hoc
OTBS
CMS
a) tert-Butyl (11S,11aS)-7-(2-(((allyloxy)carbonyl)(methyl)amino)ethoxy)-8-((5-(((S)-10- (tert-butoxycarbony0-7-methoxy-2-methylene-5-oxo-2,3,5,10,11, 11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepi n-8-y0oxy)pentyl)oxy)-11-((tert-butyldimethylsilypoxy) -2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4] diazepine-10(5H)-carboxylate 45 Alcohol 18 (462 mg, 0.748 mMol) and monomer 44 (416 mg, 0.748 mMol) were dissolved in butanone (10 mL) and K2CO3 (103 mg, 0.748 mMol) was added. The reaction mixture was stirred at 80°C for 1h before allowing to cool to room temperature. The reaction mixture was concentrated in vacuo and the crude solid was purified by isolera (ultra cartridge, 25 g) using EtOAc in Hexane (16% to 100% over 20 CV) to give pure material 45 as a colourless solid (764 mg, 94%). LC/MS, 3 min method, 2.07 min (ES+) m/z = 1068.4 [M + Na]'-.
11 11 * '11 14 1111-'11111110Wi. 47 t -vaWe N tl fix2 31-t a b) tert-Butyl (11S,11aS)-8-((5-(((S)-10-(tett-butoxycarbony1) -7-methoxy-2-methylene-5-oxo2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo [1,2-41,4]diazepin-8-y1)oxy)pentyl)oxy)-11-((tert-butyldimethylsilypoxy) -7-(2-(methylamino)ethoxy)-2-methylene-5-oxo-2,3,11,11atetrahydro-1H-benzo [e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 46 Dimer 45 (722 mg, 0.69 mMol) was dissolved in dry CH2Cl2 (8 mL) and pyrrolidine (63 pL, 0.759 mMol) was added. After 3 min of stirring, Pd(PPh3)4 (80 mg, 0.069 mMol) was added and the mixture was left to stir for 30 min at which point LCMS analysis indicated completion. The mixture was diluted with CH2Cl2 and washed with a saturated solution of NH4Cloo, brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo. The crude solid was purified by isolera (ultra cartridge, 10 g) using 20%MeOH in CH2Cl2 and CH2Cl2 (12% to 70% over 20 CV) to give pure material 46 as a colourless solid (387 mg, 58%). LC/MS, 3 min method, 1.49 min (ES+) m/z = 962.4 [M + H]' . c) tert-Butyl (113,11aS)-7-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino) -3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(methyl)amino)ethoxy) -8-((5-(((S)-10-(tert-butoxycarbonyI)-7-methoxy-2-methylene-5-oxo-2,3,5, 10,11,11a-hexahydro-1Hbenzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl) oxy)-11-((tert-butyldimethylsily0oxy)-2-methylene-5-oxo-2,3,11, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 47 Amine 46 (144 mg, 0.15 mMol) and carbamate 34 (83 mg, 0.15 mMol) were dissolved in dry THE (5 mL) and the mixture was stirred at room temperature for 2h. The reaction mixture was concentrated in vacuo and the crude solid was purified by isolera (ultra cartridge, 10 g) using 20% Me0H in CH2Cl2 and CH2Cl2 (6% to 18% over 10 CV) to give pure material 47 as a colourless solid (163 mg, 80%). LC/MS, 3 min method, 2.03 min (ES+) m/z = 1388.05 [M + Na].
d) tent-Butyl (11S,11aS)-7-(2-((((4-((S)-2-((S)-2-amino-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)(methyl)amino)ethoxy)-8-( (5-(((S)-10-(tert-butoxycarbonyI)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11, 11a-hexahydro-1H- benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-( (tert-butyldimethylsilyl)oxy)-2-methylene-5-oxo-2,3,11, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 48 Dimer 47 (160 mg, 0.117 mMol) was dissolved in dry CH2Cl2 (3 mL) and pyrrolidine (15 pL, 0.175 mMol) was added. After 3 min of stirring, Pd(PPh3)4 (1.5 mg, 0.001 mMol) was added and the mixture was left to stir for 30 min at which point LCMS analysis indicated completion.
The mixture was diluted with CH2Cl2 and washed with a saturated solution of NH4Cloo, brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo to give crude product 48 (154 mg). LC/MS, 3 min method, 1.55 min (ES+) m/z = 1282.25 [M + H]+.
e) tent-Butyl (11S,11aS)-8-((5-(((S)-10-(tert-butoxycarbony1) -7-methoxy-2-methylene-5-oxo- 2,3,5, 10, 11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl) oxy)-11- ((tert-butyldimethylsilyl)oxy)-7-(2-((((4-((2S,5S)-37-(2,5-dioxo-2, 5-dihydro-1H-pyrrol-1-y1)-5-isopropy1-2-methy1-4,7,35-trioxo-10,13,16,19, 22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzypoxy)carbonyl) (methyl)amino)ethoxy)-2-methylene-5-oxo- 2,3, 11, 11a-tetrahyd ro-1H-benzo[e]pyrrol,2-a][1,4]cliazepine-10(5H)-carboxylate 49 Amine 48 (80 mg, 0.062 mMol), Mal-dPEGa-OH (37 mg, 0.062 mMol) and EDCI.HCI (12 mg, 0.062 mMol) were dissolved in dry CH2Cl2 (4 mL) and the reaction mixture was left to stir at room temperature for 30 min. The mixture was diluted with CH2Cl2 and washed with H2O, brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo to give crude material 49 as a colourless solid (237 mg, crude).LC/MS, 3 min method, 1.93 min (ES+) m/z = 1878.5 [M + Na].
f) 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y1) -5-isopropy1-2-methyl-4,7,35-trioxo10,13,16,19,22,25,28,31-octaoxa-3,6, 34-triazaheptatriacontanamido)benzyl (2-(((S)-84(5-WS)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11, 11a-hexahydro-1H-benzo[e]pyrrolo[1,2- a][1,4]cliazepin-8-y0oxy)pentypoxy)-2-methylene-5-oxo-2,3,5, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepi n-7-yl)oxy)ethyl)(methyl)carba mate Ex 2 Dimer 49 (90 mg, 0.0485 mMol) was cooled to 0°C. Separately, the solution of TFA 95% in H2O (1.5 mL) was cooled to 0°C before adding to the dimer. The reaction mixture was stirred at 0°C for 40 min before pouring onto a 1:1 solution of CHCI3/NaHCO3 (40 mL) at 0°C. The organic layer was separated, dried on MgSO4 before being filtered and the volatiles removed in vacuo. The crude material was purified by preparative HPLC yielding product Ex2 as a yellow solid (9 mg, 12%). LC/MS, 3 min method, 1.45 min (ES+) m/z = 761.65 [M + 21-]2+i.
Example 3: 44(2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y1) -5-isopropy1-2-methy1-4,7,35-trioxo-10 13 16 19 22 25 28 31-octaoxa-3 6 34-triazaheptatriacontanamido)benzyl (2-(aS)-8- ((3-M(S)-7-methoxy-2-methyl ene-5-oxo-2, 3, 5,11a-tetrahyd ro-1H-benzoiel pyrrol o[1,2- alit 41cliazepi n-8-yboxy)methyl) benzyl)oxy)-2-methylene-5-oxo-2,3, 5, 11a-tetrahydro-1H-benzoielpyrrolor1,2-411,41diazepin-7-y0oxy)ethyl) (methyl)carbamate Ex3 Br HO CI 18,NAlloc Boc l GIBS
H
a) tert-Butyl (11S,11aS)-7-(2-(((allyloxy)carbonyl)(methyl)amino)ethoxy)-8-((3-((((115, 11aS)-10-(tert-butoxycarbony1)-11-((tert-butyldimethylsily0oxy) -7-methoxy-2-methylene-5-oxo2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo [1,2-a][1,4]diazepin-8-yfioxy)methyl)benzyfioxy)-11-( (tert-butyldimethylsilyl)oxy)-2-methylene-5-oxo-2,3,11, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 57 Alcohol 18 (228 mg, 0.37 mMol) and monomer 56 (249 mg, 0.37 mMol) were dissolved in DMF (4 mL) and K2CO3 (51 mg, 0.37 mMol) was added. The reaction mixture was stirred at 35°C for 30 min before allowing to cool to room temperature. The reaction mixture was concentrated in vacuo. The crude solid was purified by isolera to give pure material 57 as a colourless solid (374 mg, 83%). LC/MS, 3 min method, 2.25 min (ES+) m/z = 1232.90 [M + Na]' b) tert-Butyl (11S,11aS)-8-((3-((((11S,11aS)-10-(tert-butoxycarbonyI)-11 -((tert-butyldimethylsily0oxy)-7-(2-(methylamino)ethoxy) -2-methylene-5-oxo-2,3,5,10,11,11a- hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yfioxy)methyObenzypoxy) -11-((tert-butyldimethylsilypoxy)-7-methoxy-2-methylene-5-oxo-2,3,11, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 58 Dimer 57 (374 mg, 0.309 mMol) was dissolved in dry CH2Cl2 (4 mL) and pyrrolidine (51 pL, 0.618 mMol) was added. After 3 min of stirring, Pd(PPh3)4 (3.5 mg, 0.003 mMol) was added and the mixture was left to stir for 30 min at which point LCMS analysis indicated completion.
The mixture was diluted with CH2Cl2 and washed with a saturated solution of NH4C1(aco, brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo. The crude solid was purified by isolera (ultra, 10g) to give pure material 58 as a colourless solid (226 mg, 76%). LC/MS, 3 min method, 1.66 min (ES+) m/z = 1126.55 [M + H]* . c) tert-Butyl (11S,11aS)-7-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino) -3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(methyl)amino)ethoxy) -8-((3-((((11S,11aS)-10-(tert-butoxycarbony1)-11-( (tert-butyldimethylsilyfloxy)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11, 11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl) benzyl)oxy)-11-((tert-butyldimethylsily0oxy)-2-methylene-5-oxo-2,3,11, 11atetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepi ne-10(5H)-ca rboxylate 59 Amine 58 (126 mg, 0.112 mMol) and carbamate 34 (62 mg, 0.112 mMol) were dissolved in dry THE (4 mL) and the mixture was stirred at room temperature for 4h. Some starting material remained so DIPEA (5pL, 0.025 mMol) was added and the mixture stirred overnight. The reaction mixture was concentrated in vacuo and the crude solid was purified by isolera (ultra cartridge, 10 g) to give pure material 59 as a yellow solid (114 mg, 67%). LC/MS, 3 min method, 2.22 min (ES+) m/z = 1529.5 [M + Hy.
d) tert-Butyl (115,11aS)-7-(2-((((44(S)-2-((S)-2-amino-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)(methyl)amino)ethoxy)-8-( (3- ((((11S,11aS)-10-(tert-butoxycarbony1)-11-((tert-butyldimethylsily1)oxy) -7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo [1,2-a][1,4]diazepin-8-Aoxy)methypbenzypoxy)-11-((tert-butyldimethylsily1) oxy)-2-methylene-5-oxo-2,3,11,11atetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4] diazepine-10(5H)-carboxylate 60 Dimer 59 (114 mg, 0.074 mMol) was dissolved in dry CH2Cl2 (4 mL) and pyrrolidine (5.5 pL, 0.089 mMol) was added. After 3 min of stirring, Pd(PPh3)4 (1 mg, 0.001 mMol) was added and the mixture was left to stir for 30 min at which point LCMS analysis indicated completion. The volatiles were simply removed in vacuo to give crude product 60. LC/MS, 3 min method, 1.71 min (ES+) m/z = 1446.4 [M + H]+.
e) tert-Butyl (115,11aS)-8-((3-((((115,11aS)-10-(tert-butoxycarbony1)-11-( (tertbutyldimethylsily0oxy)-7-(2-((((4-((2S,5S)-37-(2,5-dioxo-2, 5-dihydro-1H-pyrrol-1-y1)-5-isopropy1-2-methyl-4,7,35-trioxo-10,13,16, 19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl)oxy) carbonyl)(methyl)amino)ethoxy)-2-methylene-5-oxo- 2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy) methyl)benzyl)oxy)-11-((tert-butyldimethylsilyl)oxy) -7-methoxy-2-methylene-5-oxo2,3, 11, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 61 Amine 60 (assumed 100%, 0.074 mMol), Mal-dPEG8-OH (44 mg, 0.074 mMol) and EDCI.HCI (14 mg, 0.074 mMol) were dissolved in dry CH2Cl2 (4 mL) and the reaction mixture was left to stir at room temperature for 30 min. The mixture was diluted with CH2Cl2 and washed with H2O, brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo. The crude solid was purified by isolera to give pure material 61 (99 mg, 66%). LC/MS, 3 min method, 2.12 min (ES+) m/z = 1032.95 [M + 2Na]2+..
f) 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y1) -5-isopropy1-2-methyl-4,7,35-ftioxo- 10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl (2-(((S)-8-((3-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-y0oxy)methyl) benzypoxy)-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1Hbenzo[e]pyrrolo[1,2-a] [1,4]diazepin-7-y1)oxy)ethyl)(methyl)carbamate Ex3 Dimer 61 (96 mg, 0.047 mMol) was cooled to 0°C. Separately, the solution of TFA 95% in H2O (1 mL) was cooled to 0°C before adding to the dimer. The reaction mixture was stirred at 0°C for 40 min before pouring onto a 1:1 solution of CHCI3/NaHCO3 (30 mL) at 0°C. The organic layer was separated, dried on MgSO4 before being filtered and the volatiles removed in vacuo. The crude material was purified by preparative HPLC yielding product Ex3 (24.2 mg, 32%). LC/MS, 3 min method, 1.44 min (ES+) m/z = 1555.35 [M + H]'..
Example 4: (R)-2((3-nitropyridin-2-yl)disulfaneyl)proPYI (2-(((S)-84(5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5, 11a-tetrahydro-1H-benzo[elpyrrolo[1,2-a.111,41diazepin-8- Y110xY)Pentyl)oxy)-2-methylen e-5-oxo-2, 3, 5, 11a-tetrahydro-1 H-benzo[el pyrrol 0[1,2- ri Aid azep i n-7-yboxy)ethyl)(methyl)carbam ate Ex4 Ex4 a) tert-Butyl (11S,11aS)-84(5-(((11S,11aS)-10-(tert-butoxycarbony1)-11-( (tertbutyldimethylsilypoxy)-7-(2-(methylWR)-2-( (3-nitropyridin-2-ybdisulfaneyl)propoxy)carbonyl)amino)ethoxy) -2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1, 4]diazepin-8-yboxy)pentyboxy)-11-Wert-02N N butyldimethylsilyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,11, 11a-tetrahydro-1Hbenzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 41 Amine 33 (42 mg, 0.038 mMol) and pyridine (3.5 pL, 0.042 mMol) were dissolved in CH2Cl2 (2 mL) before adding chloroformate 40 (12 mg, 0.038 mMol) in CH2Cl2 (1 mL) dropwise. The mixture was stirred at room temperature for 1h. A little bit more chloroformate 40 was added to push the reaction to completion. The mixture was concentrated in vacuo and the crude solid was purified by isolera (ultra cartridge, 10 g) using 10%MeOH/CH2Cl2 and CH2Cl2 EtOAc in Hexane (16% to 30% over 3 CV) to give pure material 41 (36 mg, 69%). LC/MS, 3 min method, 2.3 min (ES+) m/z = 1386.95 [M + Na]'.
b) (R)-2-((3-nitropyridin-2-yl)disulfaneyl)propyl (2-(aS)-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy) -2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4] diazepin-7-y0oxy)ethyl)(methyl)carbamate Ex4 Dimer 41 (36 mg, 0.026 mMol) was cooled to 0°C. Separately, the solution of TFA 95% in H2O (0.7 mL) was cooled to 0°C before adding to the dimer. The reaction mixture was stirred at 0°C for 40 min before pouring onto a 1:1 solution of CHCI3/NaHCO3 (20 mL) at 0°C. The organic layer was separated, dried on MgSO4 before being filtered and the volatiles removed in vacuo. The crude material was purified by preparative H PLC yielding product Ex4 as a yellow solid (3.02 mg, 13%). LC/MS, 3 min method, 1.57 min (ES+) m/z = 900.25 [M + H]'..
Example 5: 44(23,53)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y1)-5-isopropy1-2-methyl4, 7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6, 34-triazaheptatriacontanamido)benzyl (2-(f(S)-8-a3-M(S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11, 11a-hexahydro-1Hbenzoielpyrrolo[1,2-4-1,41diazepi n-8-yl)oxy)methyl)benzyl)oxy)-2-methylene-5-oxo- 2,3,5,11a-tetrahydro-1H-benzofelpyrrololl,2-a111,41diazepin-7-yboxy)ethyl) (methyl)carbamate Ex5 Boo NI 0 Br HO r Boo
OTBS
H
O
0 OMe 0 H18 NAlloc poc 0 Boo t OTBS 0 O ome a) tert-Butyl (11S,11aS)-7-(2-(((allyloxy)carbonyl)(methyl)amino)ethoxy)-8-((3-((((S) -10-(tert-butoxycarbonyI)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11, 11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepi n-8-yl)oxy)methyl)benzyl)oxy)-11-((tertbutyldimethylsilypoxy) -2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4] diazepine-10(5H)-carboxylate 51 Alcohol 18 (256 mg, 0.414 mMol) and monomer 50 (225 mg, 0.414 mMol) were dissolved in butanone (10 mL) and K2CO3 (57 mg, 0.414 mMol) was added. The reaction mixture was stirred at 35°C for 30 min before allowing to cool to room temperature. The reaction mixture was concentrated in vacuo and the crude solid was purified by isolera (ultra cartridge, 25 g) using EtOAc in Hexane (16% to 100% over 20 CV) to give pure material 51 as a colourless solid (468 mg, >100%). LC/MS, 3 min method, 2.05min (ES+) m/z = 1080.45 [M + Hr.
b) tert-Butyl (11S,11aS)-8-((3-((((S)-10-(tert-butoxycarbonyI) -7-methoxy-2-methylene-5-oxo2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo [1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzyl)oxy)-11-( (tert-butyldimethylsily0oxy)-7-(2-(methylamino)ethoxy) -2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-41,4] diazepine-10(5H)-carboxylate 52 Dimer 51 (assumed 100%, 0.414 mMol) was dissolved in dry CH2Cl2 (4 mL) and pyrrolidine (68 pL, 0.828 mMol) was added. After 3 min of stirring, Pd(PPh3)4 (5 mg, 0.004 mMol) was added and the mixture was left to stir for 30 min at which point LCMS analysis indicated completion. The mixture was diluted with CH2Cl2 and washed with a saturated solution of NELICloq.), brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo to give crude material 52 as a colourless solid (389 mg). LC/MS, 3 min method, 1.49 min (ES+) m/z = 996.4 [M + c) tert-Butyl (11S,11aS)-7-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino) -3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(methyl)amino)ethoxy) -8-((3-((((S)-10-(tert-butoxycarbonyI)-7-methoxy-2-methylene-5-oxo-2,3,5, 10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy) methyl)benzyl)oxy)-11-((tert- butyldimethylsilyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo [e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 53 Amine 53 (166 mg, 0.167 mMol) and carbamate 34 (93 mg, 0.167 mMol) were dissolved in dry THE (5 mL) and the mixture was stirred at room temperature for 4h. Some starting material remained so DIPEA (5pL, 0.025 mMol) was added and the mixture stirred overnight. The reaction mixture was concentrated in vacuo and the crude solid was purified by isolera (ultra cartridge, 10 g) using 20% Me0H in CH2Cl2 and CH2Cl2 (6% to 18% over 10 CV) to give pure material 53 as a pale yellow solid (169 mg, 73%). LC/MS, 3 min method, 2.02 min (ES+) m/z = 1399.4 [M + H]'.
d) tert-Butyl (11S,11aS)-7-(2-((((4-((S)-2-((S)-2-amino-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)(methyl)amino)ethoxy)-8-( (3-((((S)-10- (tert-butoxycarbonyl)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11, 11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl) benzyl)oxy)-11-((tert-butyldimethylsilypoxy)-2-methylene-5-oxo-2,3,11, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 54 Dimer 53 (169 mg, 0.121 mMol) was dissolved in dry CH2Cl2 (3 mL) and pyrrolidine (13 pL, 0.157 mMol) was added. After 3 min of stirring, Pd(PPh3)4 (1.5 mg, 0.001 mMol) was added and the mixture was left to stir for 30 min at which point LCMS analysis indicated completion. The mixture was diluted with CH2Cl2 and washed with a saturated solution of NH4Cloq.), brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo to give crude product 54. LC/MS, 3 min method, 1.55 min (ES+) m/z = 1316.15 [M + e) tert-Butyl (115,11aS)-8-((3-((((S)-10-(tert-butoxycarbony1) -7-methoxy-2-methylene-5-oxo2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo [1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzyl)oxy)-11-( (tert-butyldimethylsilypoxy)-7-(2-((((4-((2S,5S)-37-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-y1)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16, 19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl)oxy) carbonyl)(methyl)amino)ethoxy)-2-methylene-5-oxo-2,3,11, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 55 Amine 54 (assumed 100%, 0.121 mMol), Mal-dPEG3-OH (72 mg, 0.121 mMol) and EDCI.HCI (23 mg, 0.121 mMol) were dissolved in dry CH2Cl2 (4 mL) and the reaction mixture was left to stir at room temperature for 30 min. The mixture was diluted with CH2Cl2 and washed with H2O, brine, then dried with MgSO4, filtered and the volatiles were removed in vacuo. The crude solid was purified by isolera to give pure material 55 (157 mg, 69%). LC/MS, 3 min method, 1.91 min (ES-) m/z = 1889.2 [M - f) 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y1) -5-isopropy1-2-methyl-4,7,35-trioxo10,13,16,19,22,25,28,31-octaoxa-3,6, 34-triazaheptatriacontanamido)benzyl (2-(((S)-8-((3-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11, 11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-y0oxy)methyl) benzyl)oxy)-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazepin-7-yl)oxy)ethyl)(methyl)carbamate Ex5 Dimer 55 (152 mg, 0.08 mMol) was cooled to 0°C. Separately, the solution of TFA 95% in H2O (1 mL) was cooled to 0°C before adding to the dimer. The reaction mixture was stirred at 0°C for 40 min before pouring onto a 2:3 solution of CHCI3/NaHCO3 (25 mL) at 0°C. The organic layer was separated, dried on MgSO4 before being filtered and the volatiles removed in vacuo. The crude material was purified by preparative HPLC yielding product Ex5 as a yellow solid (43 mg, 34%). LC/MS, 3 min method, 1.47 min (ES+) m/z = 1557.45 [M + H]'.
Conjuations Conivagtes 1, 2, 3 and 5 Site-specific Tratuzumab (30 mg) was loaded onto solid support and reduced, reoxidised, conjugated to the drug linkers listed below, purified, released from the resin and formulated onto 25 mM Histidine, 200 mM Sucrose, Tween-20 0.02%, pH 6.0 according to patent # US 2014/038041 Al.
UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm mm column eluting with a gradient of water and acetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm (PBD specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of drug linker. The drug-per-antibody ratio (DAR) measured is listed below.
UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgeI SuperSW mAb HTP 4 pm 4.6 x 150 mm column (with a 4 pm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ADC at 280 nm shows the monomer purity. UHPLC SEC analysis gives a concentration of final ADC, an obtained mass as listed below, from which the yield (listed below) can be calculated.
Drug Linker DAR Purity (%) Obtained mass (mg) Yield (%) Conji Ex 1 1.73 >91 13.3 44 Conj2 Ex2 1.79 >94 19.4 65 Conj3 Ex 3 1.77 >94 18.8 63 Conj5 Ex 5 1.77 >94 18.2 61 Conjugate 4 A 50 mM solution of DTT (Dithiothreitol) in phosphate-buffered saline pH 7.4 (PBS) was added (80 molar equivalent/antibody, 13.3 micromoles, 0.27 mL) to a 4.7 mL solution of site-specific Tratuzumab (25 mg, 0.166 pmol) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 5 mg/mL. The reduction mixture was incubated at room temperature for 4 hours with gentle (60 rpm) shaking. The reduced antibody was buffer exchanged against PBS, 1mM EDTA to remove excess DTT by 50 KDa MWCO spin filter. A 50 mM solution of dehydroascorbic acid (DHAA, molar equivalent/antibody, 2.49 pmol, 48 pL) in DMSO was added and the reoxidation mixture was allowed to react for 16 hours at room temperature with gentle (60 rpm) shaking. The reoxidation mixture was then sterile-filtered; 20 mg of antibody (8.7 mL as 2.3 mg/mL solution, 0.133 pmol) was added 0.7 mL of 1M sodium bicarbonate (NaHCO3) which resulted in pH of 8.5. Compound Ex4 was added as a DMSO solution (10 molar equivalent/antibody, 1.33 pmol in 0.8 mL DMSO) to the antibody solution. The resultant conjugation mixture was incubated for 4 days at 37°C under gentle shaking (60 rpm). Conjugation mixture was centrifuged, sterile-filtered and loaded onto superdex 5200 preparative SEC column using PBS as loading and elution buffer. Fractions with monomer > 98% were collected, pooled, concentrated using 50 KDa MWCO spin filter to yield the antibody drug conjugate.
UHPLC analysis on a Shimadzu Prominence system using a TSKgeI Butyl-NPR 4.6 mm ID35mm L column eluting with a gradient of 25mM Sodium phosphate-1.25M Ammonium sulphate-pH5.5 and 25mM sodium phosphate-25% isopropanol on a neat sample of ADC at 214 nm and 330 nm (PBD specific) reveals a drug-per-antibody ratio (DAR) of 1.79.
UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 pm 4.6 x 150 mm column (with a 4 pm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ADC at 280 nm shows a monomer purity greater than 100 %. UHPLC SEC analysis gives a concentration of final ADC at 1.50 mg/mL in 11.0 mL, obtained mass of ADC is 16.6 mg (86% yield).
In vitro cytotoxicity The concentration and viability of cells from a sub-confluent (80-90% confluency) T75 flask are measured by trypan blue staining, and counted using the LUNA-II TM Automated Cell Counter. Cells were diluted to 2x105/ml, dispensed (50 pl per well) into 96-well flat-bottom plates.
A stock solution (1 ml) of antibody drug conjugate (ADC) (20 tag/ml) was made by dilution of filter-sterilised ADC into cell culture medium. A set of 8x 10-fold dilutions of stock ADC were made in a 24-well plate by serial transfer of 100 pl into 900 pl of cell culture medium. ADC dilution was dispensed (50 pi/ well) into 4 replicate wells of the 96-well plate, containing 50 pl cell suspension seeded the previous day. Control wells received 50 pi cell culture medium.
The 96-well plate containing cells and ADCs was incubated at 37°C in a 002-gassed incubator for the exposure time.
At the end of the incubation period, cell viability was measured by MTS assay. MTS (Promega) was dispensed (20 pl per well) into each well and incubated for 4 hours at 37°C in the CO2-gassed incubator. Well absorbance was measured at 490 nm. Percentage cell survival was calculated from the mean absorbance in the 4 ADC-treated wells compared to the mean absorbance in the 4 control untreated wells (100%). IC50 was determined from the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose response, X is log(concentration).
MDA MB 468 NCI N87 ADC Incubation Time 4 days 7 days Cell growth medium RPMI 1640 with glutamax, 10% (v/v) HyCloneTM Fetal Bovine Serum RPMI 1640 with glutamax, 10% (v/v) HyCloneTM Fetal Bovine Serum Results ICH (pg/mL) MDA MB 468 NCI N87 Conji 0.3275 0.00014 Conj2 2.674 0.00089 Conj3 0.3634 0.001125 Conj4 1.194 0.5531 Conj5 688 0.00331 In vivo cytotoxicity Mice Female severe combined immunodeficient mice (Fox Chase SCID®, CB17/IcrPrkdcsmd/lcolcr0r1, Charles River) were nine weeks old with a body weight (BW) range of 16.0 to 22.0 grams on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm CI), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enricho'cobsTM Laboratory Animal Bedding in static microisolators on a 12-hour light cycle at 2022 °C (68-72 °F) and 40-60% humidity. CR Discovery Services specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.
Tumor Cell Culture Human NCI-N87 gastric carcinoma lymphoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, 100 pg/mL streptomycin sulfate and 25 pg/mL gentamicin. The cells were grown in tissue culture flasks in a humidified incubator at 37°C, in an atmosphere of 5% CO2 and 95% air.
In Vivo Implantation and Tumor Growth The NCI-N87 cells used for implantation were harvested during log phase growth and resuspended in phosphate buffered saline (PBS) containing 50% MatrigelTM (BD Biosciences). On the day of tumor implant, each test mouse was injected subcutaneously in the right flank with 1 x 10' cells (0.1 mL cell suspension), and tumor growth was monitored as the average size approached the target range of 100 to 150 mm3. Seventeen days later, designated as Day 1 of the study, mice were sorted according to calculated tumor size into thirteen groups each consisting of ten animals with individual tumor volumes ranging from 108 to 172 mm3 and group mean tumor volumes of 120-122 mm3 (see Appendix A). Tumors were measured in two dimensions using calipers, and volume was calculated using the formula: Tumor Volume (mm3) = (w2 x 0 / 2 where w = width and I= length, in mm, of the tumor. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume.
Treatment On Day 1 of the study, female SCID mice bearing established NCI-N87 xenografts were sorted into thirteen groups (n = 10), and dosing was initiated according to the treatment plan summarized in Table 1. All agents were administered i.v. via tail vein injection once on Day 1. The dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal. A vehicle-treated group served as the control group for efficacy analysis. Tumors were measured using calipers twice per week, and each animal was euthanized when its tumor reached the endpoint volume of 800 mm3 or at the end of the study (Day 82), whichever came first.
Treatment outcome was determined from percent tumor growth delay (%TGD), defined as the percent increase in median TTE for treated versus control mice, with differences between groups deemed statistically significant at P 0.05 using logrank survival analysis. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume was 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm3 for one or more of these three measurements. In a CR response, the tumor volume was less than 13.5 mm3 for three consecutive measurements during the course of the study.
Treatment tolerability was assessed by body weight measurements and frequent observation for signs of treatment-related side effects. Treatment tolerability was assessed by body weight measurements and frequent observation for signs of treatment-related side effects.
All regimens were acceptably tolerated. The median TTE for vehicle-treated controls was 57.0 days, establishing a maximum possible TGD of 25.0 days (44%) for the 85-day study.
All ADC treatments produced TGD outcomes that were significantly different from the vehicle-treated controls (P < 0.01). The minimally effective doses for the ADCs appeared to be 1 mg/kg for Conj3 and Conj5.
Table 1
ADC Dose % TGD PR CR TFS Conj3 0.3 mg/kg 44 0 0 0 Conj3 1 mg/kg 44 1 0 0 Coni5 1 mg/kg 44 0 0 0 Coni5 3 mg/kg 44 2 8 8
Statements of Invention
A compound of formula I: R31 R30R9 R R40 R41 R2 C3' O rc rt O C3 and salts and solvates thereof, wherein: R7 is of formula Al: °-i(Al) RL/ 11 wherein Z is a 01-3 alkylene group and RL is a linker for connection to a cell binding agent, which is selected from: (ia): GL la wherein Q is: cr=7 QX
NH-Z
H
0, where Qx is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue; X is:
O a --d
N b H
where a = 0 to 5, b = 0 to 16, c = 0 or 1, d = 0 to 5; GL is a linker for connecting to a Ligand Unit; (ib): RL2 lb NO2] where RL1 and RL2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group; and e is 0 or 1; R" is a 03.12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. 0, S, NRN2 (where RN2 is H or 01.4 alkyl), and/or aromatic rings, e.g. benzene or pyridine; Y and Y' are selected from 0, S, or NH; when there is a double bond present between C2 and C3, R2 is selected from the group consisting of: (ha) 03.10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, 01.7 alkyl, C3.7 heterocyclyl and bis-oxy-C1_3 alkylene; (iib) C1.5 saturated aliphatic alkyl; (iiC) Cm saturated cycloalkyl; R12 R13 (iid) RII wherein each of Rli, R12 and R13 r< are independently selected from H, 01.3 saturated alkyl, C2.3 alkenyl, 02.3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2 group is no more than 5; R15b 15a (iie) , wherein one of R" and R" is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and R14 where R14 is selected from: H; C1-3 saturated alkyl; 02_3 alkenyl; 02-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2 and 03, R16a 6b R2R2 is H or R, where IR1" and R' are independently selected from H, F, C1.4 saturated alkyl, C2_3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C14 alkyl ester; or, when one of IR16a and Rlab is H, the other is selected from nitrile and a C14 alkyl ester; when there is a double bond present between C2' and C3', R2' is selected from the group consisting of: (ilia) Cs_ici aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1_7 alkyl, C3_7 heterocyclyl and bis-oxy-C14 alkylene; (iiib) 01-5 saturated aliphatic alkyl; (iiic) C3_6 saturated cycloalkyl; R22 7..fR23, wherein each of R21, R22 and R23 are independently selected from H, (Hid) R21 Ci.3 saturated alkyl, 02-3 alkenyl, 02-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2' group is no more than 5; R25b (Hie) ,R25a, wherein one of R25° and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and 2 4 (iiif) , where R24 is selected from: H; C1-3 saturated alkyl; C24 alkenyl; C24 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2' and C3', R26 a R2' is H or R26b, where R26a and R26b are independently selected from H, F, 01-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1_4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester; R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, N RR', nitro, Me3Sn and halo; where R and R' are independently selected from optionally substituted C1-12 alkyl, C420 heterocyclyl and Cs-20 aryl groups; R7' is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR', nitro, Me3Sn and halo; and either (a) R4° is H, and R41 is OH, ORA, where R" is C1-4 alkyl; (b) R4° and R41 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; (c) R4° and R41 are both H; or (d) R4° is H and R41 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation, wherein if R41 and R31 are SOzM, M may represent a divalent pharmaceutically acceptable cation; R6', R9', R3° and R31 are selected from the same groups as R6, R9, R4° and R41 respectively.
The compound according to statement 1, wherein Y and Y' are both 0.
3. The compound according to either statement 1 or statement 2, wherein R" is a C3-7 alkylene group with no substituents.
4. The compound according to statement 3, wherein R" is a C3 or Cs alkylene group.
5. The compound according to either statement 1 or statement 2, wherein R" is a group of formula: where r is 1 or 2.
6. The compound according to statement 5, wherein r is 1.
7. The compound according to any of of statements 1 to 6, wherein R9 is H. 8. The compound according to any of of statements 1 to 7, wherein R6 is selected from H, OH, OR, SH, NH2, nitro and halo.
9. The compound according to statement 8, wherein R6 is selected from H and halo.
10. The compound according to statement 9, wherein R6 is H. 11. The compound according to any of of statements 1 to 10, wherein IR9' is H. 12. The compound according to any of of statements 1 to 11, wherein IR6' is selected from H, OH, OR, SH, NH2, nitro and halo.
13. The compound according to statement 12, wherein IR6' is selected from H and halo.
14. The compound according to statement 13, wherein IR6' is H. 15. The compound according to any of of statements 1 to 14, wherein 57' is selected from H, OH, OR, SH, SR, NH2, NHR, NRR', and halo.
16. The compound according statement 15, wherein R7' is selected from H, OH and OR, where R is selected from optionally substituted C1.7 alkyl, 03.10 heterocyclyl and C5.10 aryl groups.
17. The compound according statement 15, wherein R7' is selected from OMe and OCH2Ph.
18. The compound according to statement 17, wherein RT is OMe.
19. The compound according to any one of statements 1 to 18, wherein there is a double bond between C2 and C3, and R2 is a C5-7 aryl group.
20. The compound according to statement 19, wherein R2 is phenyl.
21. The compound according to any one of statements 1 to 18, wherein there is a double bond between C2 and C3, and R2 is a Ca-10 aryl group.
22. The compound according to any one of statements 19 to 21, wherein R12 bears one to three substituent groups.
23. The compound according to statement 22, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.
24. The compound according to any one of statements 1 to 18, wherein there is a double bond between C2 and C3, and R2 is a C1.5 saturated aliphatic alkyl group. 20 25. The compound according to statement 24, wherein R2 is methyl, ethyl or propyl.
26. The compound according to any one of statements 1 to 18, wherein there is a double bond between C2 and C3, and R2 is a C3_6 saturated cycloalkyl group. 25 27. The compound according to statement 26, wherein R2 is cyclopropyl.
28. The compound according to any one of statements 1 to 18, wherein there is a double bond between C2 and C3, and R2 is a group of formula: R12 R11 29. The compound according to statement 28, wherein the total number of carbon atoms in the R2 group is no more than 4.
30. The compound according to statement 29, wherein the total number of carbon atoms in the R2 group is no more than 3.
31. The compound according to any one of statements 28 to 30, wherein one of R11, R12 and R13 is H, with the other two groups being selected from H, C1_3 saturated alkyl, C2_3 alkenyl, C2.3 alkynyl and cyclopropyl.
32. The compound according to any one of statements 28 to 30, wherein two of R11, R12 and R13 are H, with the other group being selected from H, C1_3 saturated alkyl, C2_3 alkenyl, C2-3 alkynyl and cyclopropyl.
33. The compound according to any one of statements 1 to 18, wherein there is a double bond between C2 and C3, and R2 is a group of formula: R15b R15a 34. The compound according to statement 33, wherein R2 is the group: 35. The compound according to any one of statements 1 to 18, wherein there is a double bond between C2 and C3, and R2 is a group of formula: R14 36. The compound according to statement 35, wherein R14 is selected from H, methyl, ethyl, ethenyl and ethynyl.
37. The compound according to statement 36, wherein R14 is selected from H and methyl.
38. The compound to any one of statements 1 to 18, wherein there is a single bond 1S1--6b R16a between C2 and C3, and R2 is 39. The compound according to statement 38, wherein R162 and R166 are both H. 40. The compound according to statement 38, wherein R162 and R166 are both methyl.
41. The compound according to statement 38, wherein one of R1' and R16b is H, and the other is selected from C1_4 saturated alkyl, C2_3 alkenyl, which alkyl and alkenyl groups are 42. The compound according to statement 38, wherein the group of R162 and R166 which is not H is selected from methyl and ethyl.
43. The compound to any one of statements 1 to 18, wherein there is a single bond between C2 and C3, and R2 is H. 44. The compound according to any one of statements 1 to 43, wherein there is a double bond between C2' and C3', and Rz is a 05-7 aryl group.
45. The compound according to statement 44, wherein Rz is phenyl.
46. The compound according to any one of statements 1 to 43, wherein there is a double bond between C2' and C3', and Rz is a C8_11) aryl group.
47. The compound according to any one of statements 44 to 46, wherein R2' bears one to three substituent groups.
48. The compound according to statement 47, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.
49. The compound according to any one of statements 1 to 43, wherein there is a double bond between C2' and C3', and Rz is a Ci-s saturated aliphatic alkyl group.
50. The compound according to statement 49, wherein R2' is methyl, ethyl or propyl.
51. The compound according to any one of statements 1 to 43, wherein there is a double bond between C2' and C3', and R2' is a 03_6 saturated cycloalkyl group.
52. The compound according to statement 51, wherein R2' is cyclopropyl.
53. The compound according to any one of statements 1 to 43, wherein there is a double bond between C2' and C3', and R2' is a group of formula: R22 R23 54. The compound according to statement 53, wherein the total number of carbon atoms in the R2' group is no more than 4.
55. The compound according to statement 53, wherein the total number of carbon atoms in the Rz group is no more than 3.
56. The compound according to any one of statements 53 to 55, wherein one of R21, R22 and R23 is H, with the other two groups being selected from H, C1_3 saturated alkyl, C2-3 alkenyl, 02.3 alkynyl and cyclopropyl.
57. The compound according to any one of statements 53 to 55, wherein two of R21, R22 and R23 are H, with the other group being selected from H, C1_3 saturated alkyl, C2_3 alkenyl, 02.3 alkynyl and cyclopropyl.
58. The compound according to any one of statements 1 to 43, wherein there is a double bond between C2' and C3', and IR2' is a group of formula: R25b R25a 59. The compound according to statement 58, wherein R2' is the group: 60. The compound according to any one of statements 1 to 43, wherein there is a double bond between C2' and C3', and R2' is a group of formula: R24 61. The compound according to statement 60, wherein R24 is selected from H, methyl, ethyl, ethenyl and ethynyl.
62. The compound according to statement 61, wherein R24 is selected from H and methyl.
63. The compound to any one of statements 1 to 43, wherein there is a single bond R26a between C2' and C3', and R2' is R2613 64. The compound according to statement 63, wherein R26a and R26b are both H. 65. The compound according to statement 63 wherein R26a and R26b are both methyl.
66. The compound according to statement 63, wherein one of R268 and R26b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
67. The compound according to statement 66, wherein the group of R26a and R26b which is not H is selected from methyl and ethyl.
68. The compound according to any one of statements 1 to 43, wherein there is a single bond between C2' and C3', and R2' is H. 69. The compound according to any one of statements 1 to 68, wherein R4° is H, and R41 is OH or ORA.
70. The compound according to statement 69, wherein R41 is OH.
71. The compound according to any one of statements 1 to 68, wherein R4° and R41 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.
72. The compound according to any one of statements 1 to 68, wherein R4° and R41 are both H. 73. The compound according to any one of statements 1 to 68, wherein R4° is H and R41 is SOzM, and z is 3 and M is Nat.
74. The compound according to any one of statements 1 to 73, wherein R3° is H, and R31 is OH or ORA.
75. The compound according to claim 74, wherein R31 is OH.
76. The compound according to any one of statements 1 to 73, wherein R3° and R31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.
77. The compound according to any one of statements 1 to 73, wherein R3° and R31 are both H. 78. The compound according to any one of statements 1 to 73, wherein R3° is H and R31 is SOZM, and z is 3 and M is Nat. 30 79. The compound according to any one of statements 1 to 78, wherein Z is methylene.
80. The compound according to any one of statements 1 to 78, wherein Z is ethylene.
81. The compound according to any one of statements 1 to 78, wherein Z is propylene.
82. The compound according to any one of statements 1 to 81, wherein RL is of formula la.
83. The compound according to statement 82, wherein Q is an amino acid residue. 5 84. The compound according to statement 83, wherein Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, lle, Arg, and Trp.
85. The compound according to statement 82, wherein Q is a dipeptide residue. 10 86. The compound according to statement 85, wherein Q is selected from: NH -Phe-Lys-c=°, "" "" -Val-Lys-0=0, "" Ala-Lys-c=°, "H-Val-Cit-0=0, NH-Phe-Cit-c=°, NH-Leu-Cit-c=°, NH-Ile-Cit-c=°, NH-Phe-Arg-c-°, and "H-Trp-Cit-c=°.
87. The conjugate according to statement 86, wherein Q is selected from NH-Phe-LysC=0 NH-Val-Oft-0'0 and "H-Val-Ala-c=°.
88. The compound according to statement 82, wherein Q is a tripeptide residue.
89. The compound according any one of statements 82 to 88, wherein a is 0 to 3.
90. The compound according to statement 89, wherein a is 0 or 1.
91. The compound according to statement 89, wherein a is 0.
92. The compound according any one of statements 82 to 91, wherein b is 0 to 8.
93. The compound according to statement 92, wherein b is 0.
94. The compound according to statement 92, wherein b is 4.
95. The compound according to statement 92, wherein b is 8.
96. The compound according any one of statements 82 to 95, wherein c is 0.
97. The compound according any one of statements 82 to 95, wherein c is 1.
98. The compound according any one of statements 82 to 97, wherein d is 0 to 3.
99. The compound according to statement 98, wherein d is 1 or 2.
100. The compound according to statement 99, wherein d is 2. 15 101. The compound according any one of statements 82 to 88, wherein a is 0, c is 1 and d is 2, and b is 0, 4 or 8.
102. The compound according to any one of statements 82 to 101, wherein GL is selected 20 from (GLi.1) 0 (GL6) o O 4
N o
o Ni 0 (GL1-2) 0+11 (GL7) Br A r \ \O (GL2) 0 (GL8) \\,, NI-0,___.\ \.,,, 0
O
(GL3-1) >', (GL9) N3 S-S Sl'a
N
(N 02) where the NO2 group is optional (G L3-2) >i, (G Ll 0) "--_,,
S-S H
where (NO2) the NO2 group is optional (G L3-3) s-sI (GLii) " , , ,,__ - ____, 02 hiN where the NO2 group is optional (G L3-4) 02N S-S)--1 (G L12) / where 4.
the NO2 group is optional (GL4.) Hal 0 (G u; ) ..--Where H, <// /
NH H
Hal = I, Br, Cl k Hal
H
where Ar represents a C5.6 arylene group, and X represents C1.4 alkyl.
103. A compound according to statement 102, wherein GL is selected from GL1-land GL1-2.
104. A compound according to statement 102, wherein GL is GL11.
105. The compound according to any one of statements 1 to 81, wherein RL is of formula lb. 106. The compound according to statement 105, wherein both RL1 and RL2 are H.
LI
107. The compound according to statement 105, wherein R is H and RL2 is methyl.
108. The compound according to statement 105, wherein both RL1 and RL2 are methyl.
109. The compound according to statement 105, wherein RL1 and RL2 together with the carbon atom to which they are bound form a cyclopropylene group.
110. The compound according to statement 105, wherein RL1 and RL2 together with the carbon atom to which they are bound form a cyclobutylene group.
111. The compound according to any one of statements 105 to 110, wherein e is O. 112. The compound according to any one of statements 105 to 110, wherein e is 1.
113. A conjugate of formula IV: L -(D9 (IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L is a Ligand unit, DL is a Drug Linker unit of formula III: R31 R30 Ry, R9 R40 R41 R2' C3' C2 R2 0 C3 wherein R2, R6, R9, Y, Y', R2', R6', RT, R9', R30, R31, R40 and rs n41 are as defined in any one
of statements 1 to 78;
R7L is of formula A2: where Z is as defined in any one of statements 1 and 79 to 81 and RLL is a linker connected to the Ligand unit selected from (ia'): GLL la' where Q and X are as defined any one of statements 1 and 83 to 101 and GLL is a linker connected to a Ligand Unit; and (ib'): RL1 RL2 sA lb' where RL1 and RL2 are as defined in any one of statements 1 and 106 to 110; and p is an integer of from 1 to 20.
114. The conjugate according to statement 113, wherein GLL is selected from: (Gai-1) CBA O \ (GLL8-1) CBA N, NI' "N (GLL1-2) cBA 0 Ar, 2 (G LLB-2) N CBA N"- y NC% '. o
(GLL2) CBA 0..., 0 (GLL9-1) TA...,N \N N 0 CBA o (G LL3-1) cal S>1/4, (G LL9-2) N:: NNN-A
CBA
(G LL3-2) s GL10 ak
N ---N, ,H
N
H
(GLL-4) WAS H GL11 A _, \ H N,* \ ry / (GLL5) 0 GL12 4/ 1 al \ 0 1, t
X
(G LLB) 0 GL13 H
N-
WI c CEA (G LL7) CBA where Ar represents a C5.6 arylene group and X represents C1.4 alkyl.
115. The conjugate according to statement 114, wherein GLL is selected from GLL" and GLL1-2.
116. The conjugate according to statement 115, wherein GLL is 117. The conjugate according to any one of statements 113 to 116, wherein the cell binding agent is an antibody or an active fragment thereof.
118. The conjugate according to statement 117, wherein the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen.
119. The conjugate according to statement 118, wherein the antibody or antibody fragment is an antibody which binds to one or more tumor-associated antigens or cell-surface receptors selected from (1)-(88): (1) BMPR1B; (2) E16; (3) STEAP1; (4) 0772P; (5) MPF; (6) Napi3b; (7) Sema 5b; (8) PSCA big; (9) ETBR; (10) MSG783; (11) STEAP2; (12) TrpM4; (13) CRIPTO; (14) CD21; (15) CD79b; (16) FcRH2; (17) HER2; (18) NCA; (19) MDP; (20) IL20R-alpha; (21) Brevican; (22) EphB2R; 20 (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R; (27) CD22; (28) CD79a; (29) CXCR5; (30) HLA-DOB; (31) P2X5; (32) CD72; (33) LY64; (34) FcRH1; (35) IRTA2; (36) TENB2; (37) PSMA -FOLH1; (38) SST; (38.1) SSTR2; (38.2) SSTR5; (38.3) SSTR1; (38.4)SSTR3; (38.5) SSTR4; (39) ITGAV; (40) ITGB6; (41) CEACAM5; (42) MET; (43) M UC1; (44) CA9; (45) EGFRvIll; (46) CD33; (47) CD19; (48) IL2RA; (49) AXL; (50) CD30 -TNFRSF8; (51) BCMA -TNFRSF17; (52) CT Ags -CTA; (53) CD174 (Lewis Y) -FUT3; 20 (54) CLEC14A; (55) GRP78 -HSPA5; (56) CD70; (57) Stem Cell specific antigens; (58) ASG-5; (59) ENPP3; (60) PRR4; (61) GCC -GUCY2C; (62) Liv-1 -SLC39A6; (63) 5T4; (64) CD56 -NCMA1; (65) CanAg; (66) FOLR1; (67) GPNMB; (68) TIM-1 -HAVCR1; (69) RG-1/Prostate tumor target Mindin -Mindin/RG-1; (70) B7-H4 -VTCN1; (71) PTK7; (72) CD37; (73) CD138 -SDC1; (74) CD74; (75) Claudins -CLs; (76) EGFR; (77) Her3; (78) RON -MST1R; (79) EPHA2; (80) CD20 -MS4A1; (81) Tenascin C -TNC; (82) FAP; (83) DKK-1; (84) CD52; (85) CS1 -SLAMF7; (86) Endoglin -ENG; (87) Annexin Al -ANXA1; (88) V-CAM (CD106) -VCAM1.
120. The conjugate according to any one of statements 117 to 119, wherein the antibody or antibody fragment is a cysteine-engineered antibody.
121. The conjugate according to any one of statements 117 to 120, wherein the drug loading (p) of drugs (D) to antibody (Ab) is an integer from 1 to about 8.
122. The conjugate according to statement 121, wherein p is 1, 2, 3, or 4.
123. A mixture of conjugates according to any one of statements 113 to 122, wherein the average drug loading per antibody in the mixture of antibody-drug conjugate compounds is about 2 to about 5.
124. The conjugate or mixture according to any one of statements 113 to 123, for use in therapy.
125. A pharmaceutical composition comprising the conjugate or mixture of any one of statements 113 to 123 and a pharmaceutically acceptable diluent, carrier or excipient.
126. The conjugate or mixture according to any one of statements 113 to 123, or the pharmaceutical composition according to statement 125, for use in the treatment of a proliferative disease in a subject.
127. The conjugate or mixture according to statement 126, wherein the disease is cancer.
128. Use of a conjugate or mixture according to any one of statements 113 to 123, or the pharmaceutical composition according to statement 125 in a method of medical treatment. 10 129. A method of medical treatment comprising administering to a patient the pharmaceutical composition of statement 125.
130. The method of statement 129 wherein the method of medical treatment is for treating cancer.
131. The method of statement 130, wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate.
132. Use of a conjugate or mixture according to any one of statements 113 to 123 in a method of manufacture of a medicament for the treatment of a proliferative disease.
133. A method of treating a mammal having a proliferative disease, comprising administering an effective amount of conjugate or mixture according to any one of statements 113 to 123, or the pharmaceutical composition according to statement 125.
134. A compound of formula V: R31 R30 Ry, R9 R40 R41 C2 R7P R2 C3' 0 C3
N
V
wherein R2, R6, R9, Y, Y', R2', R6', R7', R9', R30, R31, R40 and rc n41 are as defined in any one
of statements 1 to 78;
R7 is of formula A3: I-12N wherein Z is as defined in any one of statements 1 and 79 to 81.
135. A compound of formula REL: R31 R30 R9 Rs R40 R41 Y' Y
REL 7 C2 R2
C3' O rt rt O C3 and salts and solvates thereof, wherein R2, R6, R9, Y, R", Y', R2', Rs!, RT, Rw, R30, R31, Rao and R41 are as defined in any one of statements 1 to 78; R7R is of formula A4:
H
wherein Z is as defined in any one of statements 1 and 79 to 81.
GB1902182.3A 2019-02-18 2019-02-18 Pyrrolobenzodiazepines and conjugates thereof Withdrawn GB2581394A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022218973A3 (en) * 2021-04-12 2022-12-22 Medimmune Limited Pyrrolobenzodiazepine conjugates
WO2023173096A1 (en) * 2022-03-11 2023-09-14 Sigma-Aldrich Co. Llc Pyrrolobenzodiazepine intermediates and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140174A1 (en) * 2013-03-13 2014-09-18 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
WO2016037644A1 (en) * 2014-09-10 2016-03-17 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140174A1 (en) * 2013-03-13 2014-09-18 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
WO2016037644A1 (en) * 2014-09-10 2016-03-17 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022218973A3 (en) * 2021-04-12 2022-12-22 Medimmune Limited Pyrrolobenzodiazepine conjugates
WO2023173096A1 (en) * 2022-03-11 2023-09-14 Sigma-Aldrich Co. Llc Pyrrolobenzodiazepine intermediates and uses thereof

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