WO2009060215A1

WO2009060215A1 - Polyamides

Info

Publication number: WO2009060215A1
Application number: PCT/GB2008/003767
Authority: WO
Inventors: Philip Howard; Luke Masterson
Original assignee: Spirogen Limited
Priority date: 2007-11-09
Filing date: 2008-11-07
Publication date: 2009-05-14
Also published as: GB0722087D0

Abstract

A polyamide moiety comprising at least one unit of formula I: -β1-X1-Y1-X3-X4-β3- (I) wherein: Y1 is either X2-β2 or β2-X2; β1 is -Rβ -CH2-NH- βa-, wherein βa is a β-alanine residue and Rβ is chosen from optionally substituted C1-7 alkylene, C1-7 alkenylene and C1-7 alkynylene groups; β2 and β3 are β-alanine residues; and X1, X2, X3 and X4 are independently fragments of formula (II): wherein E is an optionally substituted C5-6 heteroarylene group.

Description

POLYAMIDES

The present invention relates to polyamides, i.e. compounds comprising a number of amide linkages, their synthesis and binding pattern with certain DNA sequences.

Background to the invention

Aberrant Wnt Signalling has been identified as an important factor in a variety of human tumour types. The family of Wnt secreted factors control numerous biological processes including proliferation, differentiation and morphogenesis. Canonical Wnt signalling ultimately regulates the activity of β-catenin-Tcf/LEF transcriptional complexes. Specifically Wnt controls the availability of β-catenin for its transcriptional binding partners (Tcf/Lef) through an Axin-APC (axin-adenomatous polyposis coli) complex that controls the ubiquitination and proteosomal degradation of free intracellular β-catenin. In the absence of Wnt signals GSK-3β is highly active, leading to the phosphorylation dependent degradation of β-catenin via the Axin-APC destruction complex. However, in the presence of a Wnt signakGSK-ββ is disabled allowing β-catenin to escape degradation, enter the nucleus and form trans-activation complexes with Tcf/LEF DNA binding proteins. Once bound to β-catenin these transcription factors enable the expression of a suite of genes that block differentiation and encourage proliferation. The suite includes the known proto-oncogenes c-Myc, Cyclin D1 and Ets. More recently, three Tcf/Lef binding elements (TBEs 1 , 2 and 3) have been found in the androgen receptor (AR) promoter. In addition, the Tcf and LEF genes themselves contain TBEs allowing autoregulation to take place. Both Tcf and LEF genes contain dual promoter regions, the second of which is located in intronic DNA. The secondary promoters are responsible for the transcription of truncated versions of the transcription factor which are still capable of binding to DNA but are unable to capture β-catenin. In healthy cells these truncated proteins act as natural dominant negatives opposing the action of the full length transcription factors. Significantly LEF-1 is not observed in normal colon cells (even in stem cells in the crypt), however in cancerous cells Tcf-4/β-catenin binds to the primary promoter, whilst the second is repressed, aberrantly producing full length transcriptionally active Lef-1. The result of this disregulation is to enlarge the pool of active transcription factors (for binding to targets such as the Myc promoter) as well as increasing nuclear levels of β-catenin, thus establishing an oncogenic feed-back loop. Wnt itself is rarely mutated in cancer, however mutations of APC, β-catenin and (to a lesser extent) Axin are found in colon, liver, prostate, melanoma and thyroid tumours (to name but a few).

A feature of the Wnt signalling pathway is the lack of drugable kinase targets, with signal transduction being mediated through protein-protein interactions. Since the pathway lacks obvious kinase targets, there remains a need for pathway modulators.

Disclosure of the invention

The present inventors have realised that the ultimate effectors of the pathway are the sequence selective minor groove binding transcription factors of the Tcf/Lef family. As such, the inventors have considered that minor groove binding agents (such as heterocyclic polyamides and pyrrolobenzodiazepines (PBDs)) have the potential to occupy the minor groove at the appropriate sequences and block transcription factor binding.

The inventors have developed novel minor groove binding agents which can effectively bind to TCF binding elements. In TCF binding element 3 (TBE3), TCF-4 can be displaced by Lef-1. The Lef-1/β-catenin transcription complex cannot be dismantled and deactivated via TGF-β signalling (unlike TCF-4/β-catenin). TBE3 has the sequence 5'- CTCCTCTCCTCTTCTTTG ATC-3¹. The novel compounds of the present invention may bind to this sequence in competition with TCF-4 and LeM .

These compounds may block transcription factor binding and may have therapeutic use, for example in colon cancer. For example, in the case of colon cancer, studies have shown that blocking c-Myc expression with dominant negative Tcfs causes the tumour to differentiate, abolishing the tumour phenotype [Clevers, Cancer Cell, 5(1), 91-102, Jan 2004; van de Werering et al, Cell, 111(2), 241-250, Oct 2002].

In a first aspect, the invention provides a polyamide moiety of formula I: -P₁-X₁-Y₁-X₃-X₄-P₃. (i) wherein:

Yi is either X₂-β₂ or β₂-X₂; βi is -R^β-CH₂-NH-β_a-, wherein β_a is a β-alanine residue and R^p is chosen from optionally substituted Ci_-7 alkylene, Ci_-7 alkenylene and Ci_-7 alkynylene groups; β₂ and β₃ are β-alanine residues; and Xi_ι X₂, X₃ and X₄ are independently fragments of formula II:

wherein E is an optionally substituted C₅-₆ heteroarylene group.

A second aspect of the invention provides a polyamido moiety of formula III:

-P₁-X₁-Y₁-X₃-X₄-P₃-Y₂ (IM) wherein Y₂ is X₅, X₅-X₆ or X₅-X₆-P₄. Y₂ is preferably X₅-X₆ or X₅-X₆-P₄ and most preferably X₅-X₆-P₄. X₅ and X₆ are independently fragments of formula II:

wherein E is an optionally substituted C_5-6 heteroarylene group. β₄ is a β-alanine residue.

A third aspect provides a polyamido moiety of formula IV:

-P₁ -Xi -Yi -X₃-X₄-P₃-Y₂-B (IV) wherein B is a capping group of formula V:

.X ^M

In one set of embodiments, Z is an optionally substituted C_5-2O cyclic group. In preferred embodiments of this set, Z is optionally substituted C_5-20 heterocyclyl or heteroaryl. In a second set of embodiments, Z is an optionally substituted pyrrolobenzodiazepine (PBD) moiety of formula Via or VIb:

wherein: the dotted lines indicate the optional presence of a double bond between C1 and C2 or C2 and C3; R² is selected from -H₁ -OH, =0, =CH₂, -CN₁ -R, OR, halo, =CH-R, 0-SO₂-R, CO₂R and

COR;

R³ is selected from -H, -OH, =0, =CH₂, -CN, -R, OR, halo, =CH-R, 0-SO₂-R, CO₂R and

COR; R⁸ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR', nitro, Me₃Sn and halo;

R⁶, R⁷ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR', nitro, Me₃Sn and halo; where R and R' are independently selected from optionally substituted Ci_-7 alkyl, C_3-20 heterocyclyl and C_5-20 aryl groups; or R⁶ and R⁷ together form a group -O-(CH₂)_P-O-, where p is 1 or 2; R¹⁰ and R¹¹ either together form a double bond, or are selected from H and YR^Y, where

Y is selected from O, S and NH and R^γ is H or C_1-7 alkyl or H and SO_xM, where x is 2 or

3, and M is a monovalent pharmaceutically acceptable cation;

R^2L is X², where X² is C_1-7 alkylene or C_1-7 alkenylene;

R^8L is Q-X⁸, where Q is selected from O, S, NH or a single bond and X⁸ is C_1-7 alkylene or C_1-7 alkylene-CONH-C_1-7 alkylene.

In some embodiments, the moiety of formula Via or VIb may be in a protected form, wherein R¹⁰ is a nitrogen protecting group and R¹¹ is 0-R¹², wherein R¹² is H or a hydroxyl protecting group.

A fourth aspect provides a polyamido the moiety of formula VII:

A-P₁-X₁-Y₁-X₃-X₄-P₃-Y₂- (VII) wherein A is a capping group.

A, taken with -R^β-CH₂-NH-, is preferably an optionally substituted amino or amido capping group. More preferably it forms a group of formula IX:

wherein R^D is an optionally substituted C_1-7 alkyl group or a group of formula Via or VIb.

In one set of embodiments, when taken with -R^P-CH₂-NH-, A forms a dimethylaminopropylamino (Dp) group.

A fifth aspect of the invention provides a compound of formula VIII: A-P₁-X₁-Y₁-X₃-X₄-Ps-Y₂-B (VIII) wherein A and B are as described above. It is preferred that only one of A and B contains a PBD moiety.

A sixth aspect of the invention provides a compound comprising a polyamido moiety according to the first, second, third or fourth aspect.

A seventh aspect provides a pharmaceutical composition containing a compound of the fifth or sixth aspect, as well as a compound of the fifth or sixth aspect for use in a method of medical treatment.

An eighth aspect provides the use of a compound according to the fifth or sixth aspect in the manufacture of a medicament for treating a proliferative disease, as well as a compound of the fifth or sixth aspect for use in a method of treatment of a proliferative disease.

A ninth aspect provides a method of treatment of a proliferative disease, comprising administering to a subject in need of treatment a therapeutically effective amount of a compound according to the fifth or sixth aspect, preferably in the form of a pharmaceutical composition.

Figures

Figure 1 shows the footprint of four of the compounds of the invention (compounds 6, 7, 11 and 12) against TBE3 of the human MYC gene promoter. Lanes are labelled according to the identity and concentration of compound tested.

Figure 2 shows the footprint of a further four of the compounds of the invention (compounds 15, 19, 18 and 20) against TBE3 of the human MYC gene promoter. Lanes are labelled according to the identity and concentration of compound tested.

Figure 3 shows the footprint of a further two of the compounds of the invention (compounds 25 and 26) against TBE3 of the human MYC gene promoter. Lanes are labelled according to the identity and concentration of compound tested. Definitions Points of connection

When used in structures shown herein, asterisks (*) represent the positions of bonds which connect to further chemical moieties or groups.

β-Alanine residue

As used herein, the term "β-Alanine residue" refers to a moiety of formula:

Cs-20 Cyclic, C^₂₀ Cyclyl

As used herein, the terms "C5-20 cyclic" and "C_5-2O cyclyl" refer to groups which form a complete ring in their structure. The prefix "C_5-2o" refers to the number of ring atoms, whether they are carbon (carbocyclic, carbocyclyl) or heteroatoms (heterocyclic, heterocyclyl). Such cyclic or cyclyl groups may be saturated or unsaturated (partially or fully). Thus the terms "cyclic" and "cyclyl" include, but are not limited to, the subclasses carbocyclic, aryl and heterocyclic discussed below.

Cj_-7 alkylene

The term "C_1-7 alkylene" as used herein, pertains to a monovalent moiety obtained by removing two hydrogen atoms from carbon atoms of a hydrocarbon compound having from 1 to 7 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term "alkylene" includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.

Examples of saturated alkylene groups include, but are not limited to, methylene (Ci), ethylene (C₂), propylene (C₃), butylene (C₄), pentylene (C₅), hexylene (C₆) and heptylene (C₇).

Examples of saturated linear alkylene groups include, but are not limited to, methylene (Ci), ethylene (C₂), n-propylene (C₃), n-butylene (C₄), n-pentylene (C₅), n-hexylene (C₆) and n-heptylene (C₇). Examples of saturated branched alkylene groups include iso-propylene (C₃), iso-butylene (C₄), sec-butylene (C₄), tert-butylene (C₄), iso-pentylene (C₅), and neo-pentylene (C₅).

C_5-6 heteroarylene

The term "C_5-6 heteroarylene", as used herein, pertains to a monovalent moiety obtained by removing two hydrogen atoms from aromatic ring atoms of a heteroaromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefix (C_5-6) denotes the number of ring atoms, whether carbon atoms or heteroatoms. The term "C_5-6 heteroarylene" as used herein therefore pertains to a heteroarylene group having 5 or 6 ring atoms.

Examples of monocyclic C_5-6 heteroarylene groups include, but are not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);

O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅); N₁Oi: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅);

N₃O₁: oxatriazole (C₅);

N₁Si: thiazole (C₅), isothiazole (C₅);

N₂: imidazole (1 ,3-diazole) (C₅), pyrazole (1 ,2-diazole) (C₅), pyridazine (1 ,2-diazine) (C₆), pyrimidine (1 ,3-diazine) (C₆) (e.g., cytosine, thymine, uracil), pyrazine (1 ,4-diazine) (C₆);

N₃: triazole (C₅), triazine (C₆); and,

N₄: tetrazole (C₅).

Building blocks In the polyamide moieties of the present invention, some example chains are composed of numerous standard chemical blocks. For ease of reference, these are referred to in abbreviated form. Several of these standard blocks form fragments of the formula II. The abbreviations used herein are Dp, β, Py, Im and 5M4Tz. These abbreviations represent the following structures when used herein:

Dp Py

Im 5M4Tz

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 K⁺. Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca²⁺ and Mg²⁺. Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NH₄ ⁺) 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(CH₃J₄ ⁺.

Nitrogen protecting groups

Nitrogen protecting groups are well known in the art. Preferred nitrogen protecting groups are carbamate protecting groups that have the general formula: R'¹⁰-O '.^y -<3-

wherein R'¹⁰ is an optionally substituted alkyl (e.g. C₁.₂0 alky!), aryl (e.g. C_5-2O aryl) or heteroaryl (e.g. C_3-20 heterocyclyl) group.

A large number of possible carbamate nitrogen protecting groups are listed on pages

503 to 549 of Greene, T.W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.

Particularly preferred protecting groups include Alloc, Troc, Teoc, BOC, Doc, Hoc, TcBOC, Fmoc, 1-Adoc and 2-Adoc.

Also suitable for use in the present invention are nitrogen protecting groups which can be removed in vivo (e.g. enzymatically, using light) as described in WO 00/12507, which is incorporated herein by reference. Examples of these protecting groups include:

, which is nitroreductase labile (e.g. using ADEPT/GDEPT);

which are photolabile; and

* which is glutathione labile (e.g. using NPEPT).

Protected hydroxy! groups (Z')

Protected hydroxyl groups are of the formula -O-Prot, where Prot is an oxygen protecting group as discussed below. Oxygen protecting groups

Oxygen protecting groups are well known in the art. A large number of suitable groups are described on pages 23 to 200 of Greene, T.W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.

Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, benzoates, carbonates, and sulfonates.

Substituents

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.

C_1-7 alkyl: The term "C_1-7 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 7 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 (C₃), butyl (C₄), pentyl (C₅), hexyl (C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limited to, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl (amyl) (C₅), 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). C_2-7 Alkenyl: The term "C₂-₇ 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 (allyl, -CH-CH=CH₂), isopropenyl (1- methylvinyl, -C(CH₃)=CH₂), butenyl (C₄), pentenyl (C₅), and hexenyl (C₆).

C_2-7 alkynyl: The term "C_2-7 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 (ethinyl, - C≡CH) and 2-propynyl (propargyl, -CH₂-C=CH).

C_3-7 cycloalkyl: The term "C_3-7 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 (C₅), cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄), dimethylcyclopropane (C₅), methylcyclobutane (C₅), dimethylcyclobutane (C₆), methylcyclopentane (C₆), dimethylcyclopentane (C₇) and methylcyclohexane (C₇); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅), cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆), methylcyclopentene (C₆), dimethylcyclopentene (C₇) and methylcyclohexene (C₇); and saturated polycyclic hydrocarbon compounds: norcarane (C₇), norpinane (C₇), norbornane (C₇).

C_3-20 heterocyclyl: The term "C_3-20 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. C_3-2O, C_3-7, C_5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C_5-6 heterocyclyl", 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 from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole) (C₅), pyrroline (e.g.,

3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆), dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);

O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole (dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆), pyran (C₆), oxepin (C₇);

S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅), thiane

(tetrahydrothiopyran) (C₆), thiepane (C₇);

O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);

O₃: trioxane (C₆); N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline (C₅), pyrazoline

(dihydropyrazole) (C₅), piperazine (C₆);

N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (C₆); N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);

N₂O₁: oxadiazine (C₆);

O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,

N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C₅), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose. C_5-2O aryl: The term "C_5-2O 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. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C_3-2O, C_5-7, C_5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C_5-6 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 (C₁₀), azulene (Ci₀), anthracene (Ci₄), phenanthrene (Ci₄), naphthacene (Ci₈), and pyrene (Ci₆).

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-d i hydro- 1H- indene) (C₉), indene (C₉), isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (Ci₀), acenaphthene (Ci₂), fluorene (Ci₃), phenalene (Ci₃), acephenanthrene (Ci₅), and aceanthrene (Ci₆).

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:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆); O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

Niθi: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂Oi: oxadiazole (furazan) (C₅);

N₃O₁: oxatriazole (C₅); N₁S₁: thiazole (C₅), isothiazole (C₅);

N₃: triazole (C₅), triazine (C₆); and,

N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are not limited to: C₉ (with 2 fused rings) derived from benzofuran (O₁), isobenzofuran (Oi), indole (N₁), isoindole (N₁), indolizine (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g., adenine, guanine), benzimidazole (N₂), indazole (N₂), benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂), benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁), benzothiazole (N₁S₁), benzothiadiazole (N₂S);

C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁), benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂), quinazoline (N₂), cinnoline (N₂), phthalazine (N₂), naphthyridine (N₂), pteridine (N₄); C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂);

C₁₃ (with 3 fused rings) derived from carbazole (N₁), dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂), perimidine (N₂), pyridoindole (N₂); and,

C₁₄ (with 3 fused rings) derived from acridine (N-i), xanthene (O₁), thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁), phenoxazine (N₁O₁), phenothiazine (N₁S₁), thianthrene (S₂), phenanthridine (N₁), phenanthroline (N₂), phenazine (N₂).

C_2-7 Alkenylene: The term "C_2-7 alkenylene" as used herein, pertains to an alkylene group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenylene groups include, but are not limited to, ethenylene (vinylene, -CH=CH₂), 1-propenylene (-CH=CH-CH₃), 2-propenylene (allylene, -CH-CH=CH₂), isopropenylene (1-methylvinylene, -C(CH₃J=CH₂), butenylene (C₄), pentenylene (C₅), and hexenylene (C₆).

C_2-7 alkynylene: The term "C_2-7 alkynylene" as used herein, pertains to an alkylene group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynylene groups include, but are not limited to, ethynylene (ethinylene, -C=C-) and 2-propynylene (propargylene, -CH₂-C≡C-).

C_3-7 cycloalkylene: The term "C_3-7 cycloalkylene" as used herein, pertains to an alkylene group which is also a cyclyl group; that is, a monovalent moiety obtained by removing two hydrogen atoms from alicyclic ring atoms of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkylene groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbon compounds: cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅), cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄), dimethylcyclopropane (C₅), methylcyclobutane (C₅), dimethylcyclobutane (C₆), methylcyclopentane (C₆), dimethylcyclopentane (C₇) and methylcyclohexane (C₇); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅), cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆), methylcyclopentene (C₆), dimethylcyclopentene (C₇) and methylcyclohexene (C₇); and saturated polycyclic hydrocarbon compounds: norcarane (C₇), norpinane (C₇), norbornane (C₇).

C_3-2O heterocyclylene: The term "C_3-2O heterocyclylene" as used herein, pertains to a monovalent moiety obtained by removing two hydrogen atoms from ring atoms 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. C_3-2O, C_3-7, C_5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C_5-6 heterocyclylene", as used herein, pertains to a heterocyclylene group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclylene groups include, but are not limited to, those derived from:

3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆), dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇); d: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole (dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆), pyran (C₆), oxepin (C₇);

Si: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅), thiane

(tetrahydrothiopyran) (C₆), thiepane (C₇);

O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇); O₃: trioxane (C₆); N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);

N₁Oi: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (C₆);

NiS₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆); N₂Oi: oxadiazine (C₆);

OiSi: oxathiole (C₅) and oxathiane (thioxane) (C₆); and, N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclylene groups include those derived from saccharides, in cyclic form, for example, furanoses (C₅), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.

C_5-2O arylene: The term "C_5-20 arylene", as used herein, pertains to a monovalent moiety obtained by removing two hydrogen atoms from aromatic ring atoms of an aromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C_3-20, C_5-7, C_5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C_5-6 arylene" as used herein, pertains to an arylene group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in "carboarylene groups". Examples of carboarylene groups include, but are not limited to, those derived from benzene (i.e. phenylene) (C₆), naphthalene (C₁₀), azulene (C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), and pyrene (C₁₆).

Examples of arylene 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-1 H-indene) (C₉), indene (C₉), isoindene (C₉), tetraline (1 ,2,3,4-tetrahydronaphthalene (C₁₀), acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene (C₁₅), and aceanthrene (C₁₆). Alternatively, the ring atoms may include one or more heteroatoms, as in "heteroarylene groups". Examples of monocyclic heteroarylene groups include, but are not limited to, those derived from: N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);

O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅); N₃O^ OXaWaZoIe (C₅);

N₁S₁: thiazole (C₅), isothiazole (C₅);

N₃: triazole (C₅), triazine (C₆); and, N₄: tetrazole (C₅).

Examples of heteroarylene which comprise fused rings, include, but are not limited to:

C₉ (with 2 fused rings) derived from benzofuran (O₁), isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g., adenine, guanine), benzimidazole (N₂), indazole (N₂), benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂), benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁), benzothiazole (N₁S₁), benzothiadiazole (N₂S);

C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁), benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂), quinazoline (N₂), cinnoline (N₂), phthalazine (N₂), naphthyridine (N₂), pteridine (N₄);

C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂);

C₁₃ (with 3 fused rings) derived from carbazole (N₁), dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂), perimidine (N₂), pyridoindole (N₂); and, C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene (O₁), thioxanthene

(S₁), oxanthrene (O₂), phenoxathiin (O₁S₁), phenazine (N₂), phenoxazine (N₁O₁), phenothiazine (N₁S₁), thianthrene (S₂), 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 the groups listed above and the additional substituents listed below.

Halo: -F, -Cl, -Br, and -I.

Hydroxy: -OH.

Ether: -OR, wherein R is an ether substituent, for example, a Ci_-7 alkyl group (also referred to as a C_1-7 alkoxy group, discussed below), a C_3-2O heterocyclyl group (also referred to as a C_3-2O heterocyclyloxy group), or a C_5-20 aryl group (also referred to as a C_5-20 aryloxy group), preferably a d_-7alkyl group.

Alkoxy: -OR, wherein R is an alkyl group, for example, a C_1-7 alkyl group. Examples of C_1-7 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(OR¹J(OR²), wherein R¹ and R² are independently acetal substituents, for example, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 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)(OR¹), wherein R¹ is a hemiacetal substituent, for example, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, -CH(OH)(OMe) and - CH(OH)(OEt).

Ketal: -CR(OR¹J(OR²), where R¹ and R² are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 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)(OR¹), where R¹ is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a C_1-7alkyl group, a C_3-2O heterocyclyl group, or a C_5-2O aryl group, preferably a Ci_-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.

lmino (imine): =NR, wherein R is an imino substituent, for example, hydrogen, Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably hydrogen or a Ci_-7 alkyl group. Examples of ester groups include, but are not limited to, =NH, =NMe, =NEt, and =NPh.

Formyl (carbaldehyde, carboxaldehyde): -C(=O)H.

Acyl (keto): -C(=O)R, wherein R is an acyl substituent, for example, a Ci_-7 alkyl group (also referred to as Ci_-7 alkylacyl or Ci_-7 alkanoyl), a C_3-20 heterocyclyl group (also referred to as C_3-20 heterocyclylacyl), or a C_5-20 aryl group (also referred to as C_5-20 arylacyl), preferably a Ci_-7 alkyl group. Examples of acyl groups include, but are not limited to, -C(=0)CH₃ (acetyl), -C(=O)CH₂CH₃ (propionyl), -C(=O)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(=0)SH.

Thionocarboxy (thionocarboxylic acid): -C(=S)OH.

lmidic 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 Ci_-7 alkyl group, a C_3-2o heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of ester groups include, but are not limited to, -C(=O)OCH₃, -C(=O)OCH₂CH₃, -C(=O)OC(CH₃)₃, and -C(=O)OPh.

Acyloxy (reverse ester): -OC(=O)R, wherein R is an acyloxy substituent, for example, a Ci_-7 alkyl group, a C_3-2O heterocyclyl group, or a C_5-2O aryl group, preferably a Ci_-7 alkyl group. Examples of acyloxy groups include, but are not limited to, -OC(=O)CH₃ (acetoxy), -OC(=O)CH₂CH₃, -OC(=O)C(CH₃)₃, -OC(=O)Ph, and -OC(=O)CH₂Ph.

Oxycarboyloxy: -OC(=O)OR, wherein R is an ester substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-7 alkyl group. Examples of ester groups include, but are not limited to, -OC(=O)OCH₃, -OC(=O)OCH₂CH₃, -OC(=O)OC(CH₃)₃, and -OC(=O)OPh.

Amino: -NR¹R², wherein R¹ and R² are independently amino substituents, for example, hydrogen, a Ci_-7 alkyl group (also referred to as Ci_-7 alkylamino or di-Ci_-7 alkylamino), a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably H or a Ci_-7 alkyl 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 (-⁺NR¹R²R³). Examples of amino groups include, but are not limited to, -NH₂, -NHCH₃, -NHC(CH₃)₂, -N(CH₃J₂, -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 C_1-7 alkyl group, a C_3-2O heterocyclyl group, or a Cs_-2O aryl group, preferably hydrogen or a C_1-7 alkyl group, and R² is an acyl substituent, for example hydrogen, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a Cs-^aryl group, preferably hydrogen or a C_1-7 alkyl group. Examples of acylamide groups include, but are not limited to, -NHC(=O)CH₃ , -NHC(=O)CH₂CH₃, and -NHC(=O)Ph. R¹ and R² may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:

succinimidyl maleimidyl phthalimidyl

Aminocarbonyloxy: -OC(=O)NR¹R², wherein R¹ and R² 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 R² and R³ are independently amino substituents, as defined for amino groups, and R¹ is a ureido substituent, for example, hydrogen, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably hydrogen or a C_1-7 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,

N^{^N} Imino: =NR, wherein R is an imino substituent, for example, for example, hydrogen, a C₁-₇ alkyl group, a C_3-2O heterocyclyl group, or a C_5-2o aryl group, preferably H or a C_1-7alkyl 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 C_1-7 alkyl group, a C_3-2O heterocyclyl group, or a C_5-20 aryl group, preferably H or a C_1-7 alkyl group. Examples of amidine groups include, but are not limited to, -C(=NH)NH₂, -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.

lsothiocyano (isothiocyanato): -NCS.

Sulfhydryl (thiol, mercapto): -SH.

Thioether (sulfide): -SR, wherein R is a thioether substituent, for example, a C_1-7 alkyl group (also referred to as a C_1-7alkylthio group), a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of C_1-7 alkylthio groups include, but are not limited to, -SCH₃ and -SCH₂CH₃. Disulfide: -SS-R, wherein R is a disulfide substituent, for example, a Ci_-7 alkyl group, a C3-20 heterocyclyl group, or a C_5-2O aryl group, preferably a Ci_-7 alkyl group (also referred to herein as Ci_-7 alkyl disulfide). Examples of Ci_-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 Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-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 Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group, including, for example, a fluorinated or perfluorinated C_1-7 alkyl group. Examples of sulfone groups include, but are not limited to, -S(=O)₂CH₃ (methanesulfonyl, mesyl), -S(=O)₂CF₃ (triflyl), -S(=O)₂CH₂CH₃ (esyl), -S(=O)₂C₄F₉ (nonaflyl), -S(=O)₂CH₂CF₃ (tresyl), -S(=O)₂CH₂CH₂NH₂ (tauryl), -S(=O)₂Ph (phenylsulfonyl, besyl), A- methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenylsulfonyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): -S(=O)OH, -SO₂H.

Sulfonic acid (sulfo): -S(=O)₂OH, -SO₃H.

Sulfinate (sulfinic acid ester): -S(=O)OR; wherein R is a sulfinate substituent, for example, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of sulfinate groups include, but are not limited to, -S(=O)OCH₃ (methoxysulfinyl; methyl sulfinate) and -S(=O)OCH₂CH₃ (ethoxysulfinyl; ethyl sulfinate).

Sulfonate (sulfonic acid ester): -S(=O)₂OR, wherein R is a sulfonate substituent, for - example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-7 alkyl group. Examples of sulfonate groups include, but are not limited to, -S(=O)₂OCH₃ (methoxysulfonyl; methyl sulfonate) and -S(=O)₂OCH₂CH₃ (ethoxysulfonyl; ethyl sulfonate). Sulfinyloxy: -OS(=O)R, wherein R is a sulfinyloxy substituent, for example, a Ci_-7 alkyl group, a C_3-2O heterocyclyl group, or a C_5-2O aryl group, preferably a Ci_-7 alkyl group. Examples of sulfinyloxy groups include, but are not limited to, -OS(=O)CH₃ and -OS(=O)CH₂CH₃.

Sulfonyloxy: -OS(=O)₂R, wherein R is a sulfonyloxy substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-7 alkyl group. Examples of sulfonyloxy groups include, but are not limited to, -OS(=O)₂CH₃ (mesylate) and -OS(=O)₂CH₂CH₃ (esylate).

Sulfate: -OS(=O)₂OR; wherein R is a sulfate substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of sulfate groups include, but are not limited to, -OS(=O)₂OCH₃ and -SCK=O)₂OCH₂CH₃.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): -S(=O)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(=O)NH(CH₂CH₃), -S(=O)N(CH₂CH₃)₂, and -S(=O)NHPh.

Sulfonamide (sulfinamoyl; sulfonic acid amide; sulfonamide): -S(=O)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(=O)₂NH(CH₂CH₃), -S(=O)₂N(CH₂CH₃)₂, and -S(=O)₂NHPh.

Sulfamino: -NR¹S(=O)₂OH, wherein R¹ is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, -NHS(=O)₂OH and -N(CH₃)S(=O)₂OH.

Sulfonamino: -NR¹S(=O)₂R, wherein R¹ is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl 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 Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-2O aryl group, preferably a Ci_-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 C_1-7 alkyl group, a C_3-2O heterocyclyl group, or a C_5-20 aryl group, preferably -H, a Ci_-7 alkyl group, or a C_5-20 aryl group. Examples of phosphino groups include, but are not limited to, -PH₂, -P(CH₃J₂, -P(CH₂CH₃)₂, -P(t-Bu)₂, and -P(Ph)₂.

Phospho: -P(=O)₂.

Phosphinyl (phosphine oxide): -P(=O)R₂, wherein R is a phosphinyl substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-7 alkyl group or a C_5-20 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 Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably -H, a Ci_-7 alkyl group, or a C_5-20 aryl group. Examples of phosphonate groups include, but are not limited to, -P(=O)(OCH₃)₂, -P(=O)(OCH₂CH₃)₂, -P(=O)(O-t-Bu)₂, and -P(=O)(OPh)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 C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably -H, a Ci_-7 alkyl group, or a C_5-20 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 C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably -H, a C_1-7 alkyl group, or a C5-20 aryl group. Examples of phosphite groups include, but are not limited to, -OP(OCH₃)₂, -OP(OCH₂CHs)₂, -OP(O-t-Bu)₂, and -OP(OPh)₂.

Phosphoramidite: -OP(OR¹)-NR² ₂, where R¹ and R² are phosphoramidite substituents, for example, -H, a (optionally substituted) C_1-7 alkyl group, a C_3-2O heterocyclyl group, or a C_5-20 aryl group, preferably -H, a Ci_-7 alkyl group, or a C_5-2O aryl group. Examples of phosphoramidite groups include, but are not limited to, -OP(OCH₂CH₃)-N(CH₃)₂, -OP(OCH₂CH₃)-N(i-Pr)₂, and -OP(OCH₂CH₂CN)-N(J-Pr)₂.

Phosphoramidate: -OP(=O)(OR¹)-NR² ₂, where R¹ and R² are phosphoramidate substituents, for example, -H, a (optionally substituted) Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably -H, a Ci_-7 alkyl group, or a C_5-20 aryl group. Examples of phosphoramidate groups include, but are not limited to, -OP(=O)(OCH₂CH₃)-N(CH₃)₂, -OP(=O)(OCH₂CH₃)-N(i-Pr)₂, and -OP(=O)(OCH₂CH₂CN)- N(J-Pr)₂.

Proliferative Diseases

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.

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, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. 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.

Capping Groups

As used herein, the term "capping group" refers to any group which terminates one end of a polyamide chain. A complete chain has two capping groups.

Flanking Sequence A "flanking sequence" as used herein describes a short sequence of DNA which is positioned next to a transcription unit. Usually the flanking sequence itself is not transcribed.

Methods of Treatment As described above, the present invention provides as an eighth aspect the use of a compound in a method of therapy. Also provided as a ninth aspect is a method of treatment, comprising administering to a subject in need of treatment a therapeutically- effective amount of a compound of the fifth or sixth aspect, preferably in the form of a pharmaceutical composition, which is the seventh aspect of the present 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 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 compound of the sixth aspect, 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.

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 (-COOH) also includes the anionic (carboxylate) form (-COO^'), 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.

Isomers, Salts and Solvates

Certain compounds 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; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

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, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. d_-7alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

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.

keto enol enolate

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; O may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

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. Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below.

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 -COO^"), 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 K⁺, alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂(V, 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(CH₃J₄ ⁺.

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, 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. 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.

Solvates of particular relevance to the present invention are those where the solvent adds across the imine bond present in some PBD moieties of formula Via or VIb, which is illustrated below where the solvent is water or an alcohol (R^AOH, where R^A is an ether substituent as described above):

These forms can be called the carbinolamine and carbinolamine ether forms of the PBD. The balance of these equilibria depends on the conditions in which the compounds are found, as well as the nature of the moiety itself.

In general any nucleophilic solvent is capable of forming such solvates as illustrated above for hydroxylic solvents. Other nucleophilic solvents include thiols and amines.

These solvates may be isolated in solid form, for example, by lyophilisation.

Chemical Abbreviations

In this application several abbreviations are used when referring to well known chemical fragments and reagents.

EDCI: 1-ethyl-3(3'-dimethylaminopropyl)-carbodiimide

DMAP: 4-dimethylaminopyridine

Boc: tertiary-butyl carbamate

Ala: alanine, or a derivative fragment thereof

Su: succinimide or succinimidyl (succinimidyl being the deprotonated derivative of succinimide)

DCM: dichloromethane

cr a DMF: N,N-dimethylformamide

DMSO: dimethylsulfoxide

O

DIPEA: N.N-diisopropylethylamine

DIC: 5-(3,3-dimethyl-1-triazenyl)-imidazole-4-carboxamide

HOBt: 1-hydroxybenzotriazole

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

DTT: dithiothreitol

EDTA: ethylenediaminetetraacetic acid

APS: ammonium persulfate

TEMED: tetramethylethylenediamine

,N.

^{^}N^' 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.

The group βi is -R^p-CH₂-NH-β_a-, β_a being a β-alanine residue. R^β is chosen from optionally substituted Ci_-7 alkylene, C_1-7 alkenylene and C_1-7 alkynylene groups. R^p is prefereably and optionally substituted Ci_-7 alkylene groups, more preferably optionally substituted C₃ alkylene. Most preferably, R^p is substituted by a tertiary amine group.

In groups of formula II, E is an optionally substituted C_5-6 heteroarylene group. This group is preferably an optionally substituted C₅ heteroarylene group. The group preferably has one or two ring heteroatoms, which are preferably selected from N, S and O. If the heteroarylene group is substituted, it is preferably substituted with an optionally substituted Ci_-7 alkylene group, most preferably with a methyl group. If the heteroarylene group is substituted with a methyl group, the substitution is preferably on the atom β to that which is bonded to the acyl carbon of the fragment of formula II.

In some embodiments of the invention, Xi is a pyrrole (Py) based residue.

In some embodiments, Xi, X₂, X₃, X₄, Xs and X₆ are selected from:

Preferably, X₁, X₂, X₃, X₄, X₅ and X₆ are Pyrrole (Py) or Imidazole (Im) based residues. X₁ and X₃ are preferably Py. X₂, X₄, X₅ and X₆ are preferably Im.

In the capping groups of formula V, when Z is an optionally substituted C_5-20 cyclic group, the cyclic group preferably has one or more ring heteroatoms (that is, it is preferably a heterocyclic group). More preferably, the optionally substituted heterocycle has one or two heteroatoms. The heteroatoms are preferably chosen from N, O and S. Z can be made up of any number of optionally substituted fused ring systems, for example bicyclic or tricyclic. If the optionally substituted cyclic or heterocyclic group is substituted, it is preferably substituted by one or more Cw alkyl substituents.

If Z is an optionally substituted heterocyclic group, it is preferably an optionally substituted C_5-2O heterocyclic group, more preferably an optionally substituted C_5-I₀ heterocyclic group. Preferably Z comprises an optionally substituted C₅ or C₆ heterocycle optionally fused to a further optionally substituted C₅ or C₆ cyclic group. Most preferably, Z is an optionally substituted C₅ or C₆ heterocyclic group fused to a further optionally substituted C₆ cyclic group. If the fused ring is substituted, it is preferably substituted with -NRR', more preferably -N(C_1-7 alkyl)₂.

B is most preferably selected from the following groups (which are shown with abbreviated names):

2Im 4Im 2Fr 2BzFr

2Qinox 2Qin 4Tz 2M4Tz

3M2Pic 2Py 2ABz 7DMAC B is more preferably selected from the following groups (which are shown with abbreviated names):

2Fr 2BzFr 2BzIm 2Pic

2Qinox 2Qin

2BZIn 2Pyz

2ABz

4Tz 2M4Tz 3M2Pic

7DMAC

The most preferred groups for B are 2Pic and 2Pyz.

If Z is based around an optionally substituted pyrrolobenzodiazepine (PBD) moiety of formula Via or VIb, Q is preferably O and X⁸ is preferably optionally substituted Ci_-7 alkylene. R² is preferably H or optionally substituted Ci_-7 alkyl, most preferably H.

R⁸ is preferably OR, more preferably optionally substituted 0-Ci_-7 alkyl, most preferably OCH₃. R^2L is preferably optionally substituted Ci_-7 alkylene or optionally substituted Ci_-7 alkenylene, most preferably optionally substituted Ci_-7 alkenylene.

R⁷ R⁷ is preferably selected from H, OH, OR, SH, SR, NH₂, NHR, NRR", and halo, and more preferably independently selected from H, OH and OR, where R is preferably selected from optionally substituted Ci_-7 alkyl, C_3-I0 heterocyclyl and C_5-10 aryl groups.

Particularly preferred substituents at the 7- position are OMe and OCH₂Ph.

R³

R³ is preferably H or optionally substituted Ci_-7 alkyl, most preferably H.

R⁹ R⁹ is preferably H.

R^β

R⁶ is preferably selected from H, OH, OR, SH, NH₂, nitro and halo, and is more preferably H or halo, and most preferably is H.

R²

In some embodiments, R² is H.

In some embodiments, R (and particularly R²) is an optionally substituted C_5-2O aryl group. It may be selected from: an optionally substituted C_5-7 aryl group, for example phenyl; an optionally substituted C_9-I2 aryl group, for example for example naphthyl (e.g napthy-1-yl, napth-2-yl) and quinolinyl (e.g. quinolin-2-yl, quinolin-3-yl,quinolin-6-yl); an optionally substituted C_5-7 heteroaryl group, for example furanyl (e.g. furan-2-yl, furan-3- yl), thiophenyl (e.g. thiophen-2-yl, thiophen-3-yl) and pyridyl (e.g. pyrid-2-yl, pyrid-3-yl).

The C_5-20 aryl group may bear any substituent group. It may bear from 1 to 3, 1 to 2 or 1 substituent groups. C_5-20 aryl substituents, particularly for phenyl, include, but are not limited to: halo (e.g. F, Cl, Br); Ci_-7 alkoxy (e.g. methoxy, ethoxy); C_1-7 alkyl (e.g. methyl, trifluoromethyl, ethyl, propyl, t-butyl); bis-oxy-alkylene (e.g. bis-oxy-methylene, -0-CH₂- O-).

C_5-20 aryl groups of particular interest, include, but are not limited to, phenyl, 4-methyl- phenyl, 4-methoxy-phenyl, 3-methoxyphenyl, 4-fluoro-phenyl, 3,4-bisoxymethylene- phenyl, 4-triflouoromethylphenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, thiophen-2-yl, napth-2-yl, quinolin-3-yl and quinolin-6-yl. In some embodiments, R is =CH₂.

In some embodiments, R² is =CH-R. In these embodiments, R may be Ci_-7 alkyl (e.g. methyl, ethyl). The C_1-7 alkyl group may contain one or more unsaturated bonds conjugated to the double bond bound to the C-ring. Thus, in this case R² may be, for example, =CH-CH=CH₂, =CH-CH=CH-CH₃, =CH-C≡CH, =CH-CH=CH-CH=CH₂. Alternatively in these embodiments, R may be C_5-2O aryl, in particular C_5-6 aryl (e.g. phenyl, pyridyl, thiophenyl, furanyl).

In some embodiments where there is a double bond between the C2 and C3 positions of the C-ring, R² may be Ci_-7 alkyl containing one or more unsaturated bonds conjugated to the double bond in the C-ring. Thus, in this case R² may be, for example, -CH=CH₂, - CH=CH-CH₃, -C=CH, -CH=CH-CH=CH₂.

In some embodiments, R⁶ and R⁹ are H, R⁷ is OMe or OCH₂Ph, and R² is selected from the options set out above, in particular, when there is no double bond between C2 and C3: H, optionally substituted, C_5-7 aryl, =CH₂, -CH-CH₃, =CH-CH=CH₂, and when there is a double bond between C2 and C3: -CH=CH₂, -CH=CH-CH₃ and -C≡CH.

R¹⁰ and R¹¹

R¹⁰ and R¹¹ preferably together form a double bond between N10 and C11.

In the group SO_ZM, z is preferably 3. M is preferably Na⁺.

Most preferably, B is selected from the following groups (which are shown with abbreviated names):

LN4PBD PBDC2LN3

Most preferably, the polyamido moiety of formula I is: -βrPy-lm-β-Py-lm-β-

Examples

General Synthetic Routes Pyrrolobenzodiazepine moieties

The syntheses of pyrrolobenzodiazepine moieties are described in WO 00/12506. Protection at the C11 position can be readily introduced.

Reaction Schemes

2a X = CH, R = Me 3a X = CH 2b X = N, R = Et 3b X = N

10

11 12

16

17-26

NaOH VrV — HO M-¹' O see Fig 1 and 2 for structures

27-36

17 18

20

19

21 22

23 24

25 26

Figure 1

31 32

33 34

36 36

Figure 2

General methods for ester hydrolysis Method A An 0.5M solution of NaOH (2eq) was added to a solution of the ester in 1 ,4-dioxane. The reaction mixture was allowed to stir at room temperature until reaction was complete. The 1 ,4-dioxane was evaporated and the residue, diluted with water as necessary, was acidified to pH 3 with 1M citric acid. The product was collected by filtration, purified as necessary and dried. Method B

NaOH solution (excess) was added to a solution of the ester in MeOH. The reaction mixture was allowed to stir at 50-60⁰C until complete. The MeOH was evaporated and the residue, diluted with water as necessary was acidified to pH 3 with 1 M citric acid solution. The product was collected by filtration, purified as necessary and dried.

General methods for heterocvcle couplings Coupling Method A

A solution of the acid, amine, EDCI and DMAP in DCM was stirred at room temperature until reaction was complete. The solution was washed with 1M citric acid (x 2), sat.

NaHCO₃ (x 2), H₂O (x 1), brine (x 1), dried (MgSO₄) and evaporated. The product was either used without further purification or was purified by standard techniques.

Coupling Method B A solution of the acid, amine, EDCI and DMAP in DMF was stirred at room temperature until reaction was complete. The reaction mixture was poured onto ice and extracted with DCM (x 3). The solution was washed with either 1 M citric acid (x 2), sat. NaHCO₃ (x 2), H₂O (x 1), brine (x 1), dried (MgSO₄) and evaporated. The product was either used without further purification or was purified by standard techniques.

Building Block Synthesis

4-(3-tert-Butoxycart>onylamino-propionylamino)- 1-methyl-1 H-pyrrole-2-cart)oxylic acid methyl ester (2a)

Triethylamine (15.99 g, 22 ml, 158.4 mmol, 1.1 eq) was added to a solution of amine 1a HCI salt (27.44 g, 144 mmol, 1 eq) and Boc Ala succinimide (45.4 g, 158.4 mmol, 1.1 eq) in DCM (400 ml). The solution was stirred at room temperature for 1.5 hours. The reaction mixture was washed with H₂O (2 x 250 ml), 1 M citric acid (2 x 200 ml), saturated NaHCO₃ (2 x 200 ml), H₂O (250 ml) and brine (250 ml). Dried (MgSO₄) and evaporated to give the product as an off white solid (46.8 g, 100%). ¹H NMR (DMSO-Cf₆) δ 9.87 (S₁ 1 H)₁ 7.345 (d, J = 1.6 Hz, 1 H)₁ 6.81 (m, 1 H)₁ 6.725 (d, J = 2.0 Hz₁ 1 H)₁ 3.83 (s, 3H)₁ 3.74 (S₁ 3H)₁ 3.2 (dt, J = 6.8 Hz₁ 6 Hz₁ 2H)₁ 2.4 (t, J = 7.2 Hz₁ 2H)₁ 1.39 (s, 9H); MS (ES^') mlz 326.37 ([M + H]^", 40).

Compound 2b was synthesised according to the following literature procedure: Seio, K., et al., Journal of Organic Chemistry, 70(25), 10311-10322 (2005).

^(S-tert-Butoxycarbonylamino-propionylaminoyi-methyl-IH-pyrrole-Σ-carboxylic acid (3a)

4-(3-terf-Butoxycarbonylamino-propionylamino)-1-methyl-1H-pyrrole-2-carboxylic acid methyl ester 2a (13.53 g, 41.6 mmol, 1 eq) in 1 ,4-dioxane (200 ml) was hydrolysed (Method A) to give the product as a white solid (10.29 g, 79%).¹H NMR (DMSO-CZ₆) δ 12.14 (bs, 1H), 9.83 (S₁ 1 H)₁ 7.3 (d, J = 1.6 Hz₁ 1 H)₁ 6.81 (t, J = 5.06 Hz₁ 1 H)₁ 6.67 (d, J =

1.96 Hz, 1H)₁ 3.81 (s, 3H)₁ 3.2 (dt, J = 7.2 Hz, 6 Hz, 2H), 2.39 (t, J = 7.2 Hz, 2H), 1.39 (s, 9H); MS (ES^") mlz 310.35 ([M-H]^", 100).

Compound 3(b) is available from commercial sources.

4^[4-(3-tert-Butoxycaώonylamino-pmpionylamino)-1-methyl-1H-imidazole-2-carbonylJ- amino}-1-methyl-1H-pyrrole-2-carboxylic acid methyl ester (4)

A solution of the acid 3b (11.07 g, 35.4 mmol, 1 eq), amine 1a HCI salt (8.113 g, 42.56 mmol, 1.2 eq), EDCI (13.59 g, 70.9 mmol, 2.0 eq) and DMAP (10.84 g, 88.7 mmol, 2.5 eq) in DMF (200 ml) was stirred for 18 hours. Work up (Coupling Method B) gave the product as an off white solid. Trituration with Et.₂θ gave the product as a white solid (13.95 g, 88 %).¹H NMR (DMSO-cfe) δ 10.23 (s, 1 H)₁ 10.09 (s, 1H), 7.53 (d, J = 2 Hz, 1 H), 7.46 (S, 1 H), 7.01 (d, J = 2 Hz, 1 H), 6.795 (m, 1 H), 3.96 (s, 3H), 3.86 (s, 3H), 3.76 (S₁ 3H), 3.21 (dt, J = 7.2 Hz, 6 Hz, 2H), 2.49 (t, J = 7.2 Hz, 2H), 1.39 (s, 9H); MS (ES⁺) m/z 449.38 ([M + H]⁺, 50).

^{^-(S-tert-Butoxycarbonylamino-propionylaminoyi-methyl-IH-imidazole-Σ-carbonyl]- amino}-1-methyl-1H-pyrrole-2-caιϋoxylic acid (5)

The ester 4 (13.95 g, 31.1 mmol, 1 eq) in 1 ,4-dioxane (300 ml) was hydrolysed (Method A) to give the product as a white solid (12.84 g, 95 %). MS (ES⁺) m/z 457.27 ([M + Na]⁺, 50). 4-{[4-(3-tert-Butoxycart)onylamino-pmpionylamino)-1-methyl-1H-pyrτOle-2-cartonyl]- amino}-1-methyl-1H-imidazole-2-caιtoxylic acid ethyl ester (6)

A solution of the acid 3a (8.0 g, 25.7 mmol, 1.2 eq), amine 1b (3.62 g, 21.4 mmol, 1 eq), EDCI (8.2 g, 42.8 mmol, 2.0 eq) and DMAP (6.5 g, 53.5 mmol, 2.5 eq) in DCM (150 ml) was stirred for 72 hours. Work up (Coupling Method A) gave the product as a yellow foam (9.04 g, 91%). ¹H NMR (DMSOd₆) δ 10.7 (s, 1 H), 9.87 (s, 1H), 7.67 (s, 1H), 7.308 (d, J = 1.7 Hz, 1 H), 6.98 (d, J= 1.8 Hz, 1 H), 6.80 (t, J = 5.0 Hz, 1 H), 4.3 (q, J = 7.1 Hz, 2H), 3.95 (s, 3H), 3.85 (s, 3H), 3.20 (dt, J = 6.8 Hz, 6.4 Hz, 2H), 2.42 (t, J = 7.3 Hz, 2H), 1.4 (s, 9H), 1.32 (t, J = 7.1 Hz, 3H); MS (ES⁺) m/z 463.29 ([M+H]⁺, 100).

4^[4-(3-tert-Butoxycarbonylamino-pmpionylamino)-1-methyl-1H-pyrrole-2-carbonyl]- amino}-1-methyl-1H-imidazole-2-carboxylic acid (7)

The ester 6 (11.65 g, 25.2 mmol, 1 eq) in 1 ,4-dioxane (100 ml) was hydrolysed (Method A) to give the product as a tan solid (8.89 g, 81%).¹H NMR (DMSO-Cf₆) δ 10.56 (bs, 1H), 9.87 (bs, 1 H), 7.54 (bs, 1 H), 7.28 (s, 1 H), 6.96 (s, 1 H), 6.81 (m, 1 H), 4.5 - 3.8 (bs 1 H), 3.95 (s, 3H), 3.85 (s, 3H), 3.21 (dt, J = 6.9 Hz, 6.3 Hz, 2H), 2.42 (t, J = 7.2 Hz, 2H), 1.4 (S, 9H); MS (ES ) mlz 433.4 ([M-H]^", 100). 2-{[4-(3-teιi-Butoxycarbonylamino-propionylamino)-1-methyl-1H-pynOle-2-carbonyl]- amino}-5-methyl-thiazole-4-caώoxylic acid methyl ester (9)

A solution of the acid 3a (11.5 g, 36.94 mmol, 1.2 eq), 2-amino-5-methyl-thiazole-4- carboxylic acid methyl ester 8 (5.3 g, 30.78 mmol, 1.0 eq), EDCI (11.8 g, 61.56 mmol, 2.0 eq) and DMAP (9.79 g, 76.95 mmol, 2.5 eq) in DMF (300 ml) was stirred for 72h. Work up (Coupling Method B) gave a yellow oil. Trituration with Et₂O gave the product as a white solid (7.96 g, 55%). MS (ES ) m/z 464.45 ([M-H]^", 100).

2-{[4-(3-tert-Butoxycarbonylamino-propionylamino)-1-methyl-1H-pyrrOle-2-carbonyl]- amino}-5-methyl-thiazole-4-carboxylic acid (10)

The ester 9 (3.9 g, 8.4 mmol, 1 eq) in MeOH (100 ml) was hydrolysed (Method B) at room temperature to give the product as an off white solid (2.7 g, 71%).¹H NMR (DMSO- d₆) δ 12.7 (bs, 1H), 12.5 (bs, 1 H), 10.03 (s, 1H), 7.51 (s, 1 H), 7.3 (s, 1H), 6.9 (s, 1H), 3.98 (s, 3H), 3.3 (m, 2H), 2.72 (s, 3H), 2.61 (m, 2H), 1.49 (s, 9H); MS (ES^") m/z 450.61 ([M-H]^", 100). 2-(3-tert-Butoxycarbonylamino-propionylamino)-5-methyl-thiazole-4-carboxylic acid methyl ester (11)

1 ,3-Diisopropylcarbodiimide (15.14 g, 18.6 mL, 120 mmol, 2 eq) was added to a solution of Boc-β-Alanine (45.4 g, 240 mmol, 4 eq) in DCM (350 ml). The reaction mixture was allowed to stir at room temperature for 30 minutes. 2-Amino-5-methyl-thiazole-4- carboxylic acid methyl ester 8 (10.33 g, 60 mmol, 1 eq) was added portionwise followed by DIPEA (7.75 g, 9.73 ml, 60 mmol, 1 eq), and DMAP (2.2 g, 18 mmol, 0.3 eq) and the reaction mixture was allowed to stir at room temperature for 18 hours. The mixture was filtered and extracted with 1M citric acid (3 x 200 ml), sat. NaHCO₃ (3 x 200 ml), water (200 ml), brine (200 ml), dried (MgSO₄) and evaporated under reduced pressure to give a yellow foam. The product was contaminated with dicyclohexylcarbodiimide and was used without further purification assuming 100% yield.

2-(3-tert-Butoxycarbonylamino-propionylamino)-5-methyl-thiazole-4-carboxylic acid (12)

The ester 11 (20.6 g, 60 mmol, 1.0 eq) in 1,4 dioxane (250 ml) was hydrolysed (Method A) to give the product as an off white solid (15.83 g, 88%). ¹H NMR (DMSO-d₆) δ 12.3 (bs, 2H), 6.87 (m, 1H), 3.24 (dt, J = 6.8 Hz, 6 Hz, 2H), 2.61 (s, 3H)₁ 2.56 (t, J = 6.8 Hz, 2H), 1.38 (s, 9H); MS (ES^") m/z 328.24 ([M-H]^", 30), 284.31 ([M-CO₂]-, 100).

4-{[4-(3-tert-Butoxycaώonylamino-pfOpionylamino)-1-methyl-1H-imidazole-2-caιtonyl]- amino}-1-methyl-1H-imidazole-2-carboxylic acid ethyl ester (13)

A solution of the acid 3b (1.83 g, 5.85 mmol, 1.2 eq), amine 1 b (0.825 g, 4.88 mmol, 1.0 eq), EDCI (1.87 g, 9.75 mmol, 2.0 eq) and DMAP (1.49 g, 12.2 mmol, 2.5 eq) in DMF (50 ml) was stirred for 18 hours. Work up (Coupling Method B) gave a pale yellow solid. Trituration with Et₂O gave the product as a white solid (1.85 g, 68 %). MS (ES⁺) mlz 364.29 (100), 486.16 ([M + Na]⁺, 20) .

4-{[4-(3-tert-Butoxycaιtonylamino-propionylamino)-1-methyl-1H-imidazole-2-carbonyl]- amino}-1-methyl-1H-imidazole-2-carboxylic acid (14)

The ester 13 (1.85 g, 4.0 mmol, 1.0 eq) in 1 ,4 dioxane (30 ml) was hydrolysed (Method A) gave an off white solid. Trituration with Et₂O gave the product as a white solid (1.34 g, 77%). MS (ES⁺) mlz 436 ([M + H]⁺, 50). 4-[(4-{[4-(3-tert-Butoxycarbonylamino-piOpionylamino)-1-methyl-1H-imidazole-2- carbonylj-amino}- 1-methyl-1H-pyrrole-2-carbonyl)-amino]- 1 -methyl- 1 H-pyrrole-2- carboxylic acid methyl ester (15)

A solution of the acid 5 (1.52 g, 3.5 mmol, 1.0 eq), amine 1a HCI salt (0.799 g, 4.2 mmol, 1.2 eq), EDCI (1.34 g, 7.0 mmol, 2.0 eq) and DMAP (1.07 g, 8.7 mmol, 2.5 eq) in DMF (75 ml) was stirred for 18 hours. Work up (Coupling Method B) gave the product as an off white foam (1.9 g, 95 %). MS (ES⁺) mlz 593.32 ([M + Na]⁺,40), 471.22 (100).

4-[(4-{[4-(3-tert-Butoxycarbonylamino-propionylamino)- 1-methyl-1H-imidazole-2- carbonylj-amino}- 1 -methyl- 1 H-pyrrole-2-carbonyl)-amino]- 1 -methyl- 1 H-pyπrole-2- carboxylic acid (16)

The ester 15 (1.89 g, 3.3 mmol, 1.0 eq) in 1 ,4 dioxane (40 ml) was hydrolysed (Method A) to give an off white solid. Trituration with Et₂O gave the product as a white solid (1.78 g, 97%). MS (ES⁺) mlz 557.47 ([M + H]⁺, 10), 457.36 (100). ^[(Furan-Σ-carbonylj-aminoJ-i-methyl-IH-imidazole-Σ-carboxylic acid ethyl ester (17)

A solution of Furan-2-carboxylic acid (0.26 g, 2.1 mmol, 1.0 eq), amine 1b (0.43 g, 2.53 mmol, 1.2 eq), EDCI (0.8 g, 4.2 mmol, 2.0 eq) and DMAP (0.64 g, 5.3 mmol, 2.5 eq) in DCM (10 ml) was stirred for 72 hours. Work up (Coupling Method A) gave the product as a yellow solid (0.52 g, 95 %). MS (ES⁺) mlz 264 ([M + H]⁺, 70), 286.2 ([M + Na]⁺, 100).

^[(Benzofuran-Σ-carbonyQ-aminoJ-i-methyl-IH-imidazole-Σ-carboxylic acid ethyl ester (18)

A solution of Benzouran-2-carboxylic acid (0.25 g, 1.58 mmol, 1.0 eq), amine 1b (0.32 g, 1.89 mmol, 1.2 eq), EDCI (0.605 g, 3.2 mmol, 2.0 eq) and DMAP (0.48 g, 3.95 mmol, 2.5 eq) in DCM (10 ml) was stirred for 72 hours. Work up (Coupling Method A) gave the product as a brown crystalline solid (0.49 g, 89 %). MS (ES⁺) mlz 314.18 ([M + H]⁺, 100), 336.19 ([M + Na]⁺, 30).

i-MethyH-Kpyridine-Σ-carbony^-aminoJ-IH-imidazole-Σ-carboxylic acid ethyl ester (19)

A solution of Picolinic acid (0.25 g, 2.02 mmol, 1.0 eq), amine 1b (0.41 g, 2.42 mmol, 1.2 eq), EDCI (0.775 g, 4.0 mmol, 2.0 eq) and DMAP (0.62 g, 5.06 mmol, 2.5 eq) in DCM (10 ml) was stirred for 72 hours. Work up (Coupling Method A) gave the product as a yellow solid (0.465 g, 84 %). MS (ES⁺) mlz 275.20 ([M + H]⁺, 100). 1-Methyl-4-[(pyrazine-2-carbonyl)-amino]-1H-imidazole-2-carboxylic acid ethyl ester (20)

Coupling (Method A) gave the product as yellow solid (0.18 g, 85 %). MS (ES⁺) m/z 276.43 ([M + H]⁺, 100).

i-MethyM-tfS-methyl-pyridine-Σ-carbonyty-aminoJ-IH-imidazole-Σ-carboxylic acid ethyl ester (21)

Coupling (Method A) gave the product as an orange solid (0.19 g, 85 %). MS (ES⁺) m/z 289.44 ([M + H]⁺, 100)

4-[(1H-Benzimidazole-2-carbonyl)-amino]- i-methyl-IH-imidazole-2-carboxylic acid ethyl ester (22)

Coupling (Method A) gave the product as a white solid (0.117 g, 48 %). MS (ES⁺) m/z 314.43 ([M + H]⁺, 100).

1 -Methyl-4-[(1 -methyl- 1 H-imidazole-4-carbonyl)-amino]- 1 H-imidazole-2-carboxylic acid ethyl ester (23)

Coupling (Method A) gave the product as a white solid (0.241 g, 88 %). MS (ES⁺) m/z 278.27 ([M + H]⁺, 100). i-MethyM-tfthiazole-^carbonylj-aminoJ-IH-imidazole-Σ-carboxylic acid ethyl ester (24)

Coupling (Method A) gave the product as a white solid (0.196 g, 60 %). MS (ES⁺) m/z 281.31 ([M + H]⁺, 100).

i-MethyM-tfΣ-methyl-thiazole-^carbonyQ-aminoJ-IH-imidazole^-carboxylic acid ethyl ester (25)

Coupling (Method A) gave the product as a white solid (0.302 g, quant). MS (ES⁺) m/z 295.28 ([M + H]⁺, 100).

i-Methyl-^tfi-methyl-IH-pynOle^-carbonyQ-aminoJ-IH-imidazole-Σ-carboxylic acid ethyl ester (26)

Coupling (Method B) gave the product as a yellow solid (1.59 g, 98 %). MS (ES⁺) m/z ([M + H]⁺, 100). 4-[(Furan-2-carbonyl)-amino]-1-methyl-1 H-imidazole-2-carboxylic acid (27)

The ester 17 (0.515 g, 1.96 mmol, 1.0 eq) in 1 ,4 dioxane (20 ml) was hydrolysed (Method A) to give the product as an off white solid (0.31 g, 67%). MS (ES⁺) m/z 236.10 ([M + H]⁺, 100).

4-[(Benzofuran-2-caιtonyl)-amino]- 1 -methyl- 1 H-imidazole-2-carboxylic acid (28)

The ester 18 (0.43 g, 1.37 mmol, 1.0 eq) in 1 ,4 dioxane (20 ml) was hydrolysed (Method A) to give the product as a white solid (0.33 g, 85%). MS (ES⁺) m/z 286.08 ([M + H]⁺, 100).

1-Methyl-4-[(pyridine-2-carbonyl)-amino]- 1 H-imidazole-2-carboxylic acid (29)

The ester 19 (0.45 g, 1.66 mmol, 1.0 eq) in 1 ,4 dioxane (20 ml) was hydrolysed (Method A) to give the product as a white solid (0.12 g, 29%). MS (ES⁺) m/z 247.18 ([M + H]⁺, 100).

1 -Methyl-4-[(pyrazine-2-carbonyl)-amino]- 1 H-imidazole-2-carboxylic acid (30)

Hydrolysis of ester 20 (Method A) gave the product as a yellow solid (0.175 g, quant). MS (ES)⁺ m/z 248.33 ([M + H]⁺, 100). 1-Methyl-4-[(3-methyl-pyridine-2-carbonyl)-aminoJ- 1 H-imidazole-2-carboxylic acid (31)

Hydrolysis of ester 21 (Method A) gave the product as a yellow solid (0.15 g, 88 %). MS (ES)⁺ m/z 261.34 ([M + H]⁺, 70).

4-[(1H-Benzimidazole-2-carbonyl)-amino]-1-methyl-1 H-imidazole-2-carboxylic acid (32)

Hydrolysis of ester 22 (Method A) gave the product as a white solid (0.063 g, 59 %)). MS (ES)⁺ m/z 286.33 ([M + H]⁺, 100).

1-Methyl-4-[(1-methyl-1H-imidazole-4-carbonyl)-amino]- 1 H-imidazole-2-carboxylic acid (33)

Hydrolysis of ester 23 (Method A) gave the product as a white solid (0.106 g, 49 %). MS (ES)⁺ m/z 250 ([M + H]⁺, 50).

1 -Methyl-4-[(thiazole-4-carbonyl)-amino]- 1 H-imidazole-2-carboxylic acid (34)

Hydrolysis of ester 24 (Method A) gave the product as an orange solid (0.049 g, 28 %). MS (ES)⁺ m/z 253 ([M + H]⁺, 30). i-MethyM-ftΣ-methyl-thiazole-^carbonylj-aminoJ-IH-imidazole-Σ-carboxylic acid (35)

Hydrolysis of ester 25 (Method A) gave the product as a white solid (0.124 g, 45 %). MS (ES)⁺ m/z 267.23 ([M + H]⁺, 90).

1-Methyl-4-[(1-methyl-1 H-pyrnole-4-carbonyl)-amino]-1 H-imidazole-2-carboxylic acid (36)

The ester 26 (1.8 g, 6.5 mmol, 1.0 eq) in 1,4 dioxane (50 ml) was hydrolysed (Method A) to give the product as a white solid (1.59 g, 98 %). MS (ES⁺) m/z 249.23 ([M + H]⁺, 50).

General Coupling Methods

Synthesis of core polyamides (coupling and Boc deprotection)

A 1 M solution of DIC (2 eq) was added to a solution of the acid (1.25 - 1.5 eq) in DMF. The solution was stirred/shaken for 10 minutes and a 1 M solution of HOBt (2.5 eq) was added. After a further 10 minutes a solution of the amine (1 eq) in DMF was added. The reaction was stirred/shaken until complete by LCMS (typically 18 hours). The reaction mixture was loaded onto an lsolute SCX-2 acidic ion-exchange resin cartridge that had been pre-equilibrated with MeOH (1 vol). The cartridge was washed successively with volumes of DMF, MeOH, DMF and MeOH. The cartridge was then washed further with 4M HCI in 1 ,4 dioxane (0.5 vol) and MeOH several volumes. The product was eluted with 2M NH₃ in MeOH (2 vol), evaporation under reduced pressure gave the product which was used without further purification.

Capping of core polyamides

A 1 M solution of DIC (2 eq) was added to a solution of the acid (1.25 - 1.5 eq) in DMF. The solution was stirred/shaken for 10 minutes and a 1 M solution of HOBt (2.5 eq) was added. After a further 10 minutes a solution of the core polyamide amine (1 eq) in DMF was added. The reaction was stirred/shaken until complete by LCMS (typically 18 hours). The reaction mixture was loaded onto an lsolute SCX-2 acidic ion-exchange resin cartridge that had been pre-equilibrated with MeOH (1 vol). The cartridge was washed successively with volumes of DMF₁ MeOH, DMF and MeOH. The product was eluted with 2M NH₃ in MeOH (2 vol), evaporation under reduced pressure gave the product which was purified by preparative HPLC.

Example Compounds

The following polyamides were synthesised using the standard amide coupling protection/deprotection methodology described above.

9 base pairs

10 base pairs

12 base pairs

Examples

General methods - footprinting assay

The compound to be tested and buffer (20 mM HEPES pH 7.9, 20 mM NaCI, 2 mM MgCI₂, 1 mM DTT, 10% glycerol; 388 μl) were mixed with 2 μl of ³²P-radiolabelled DNA (128 base pairs containing TBE3 and flanking sequences of the human MYC gene promoter) and incubated overnight at room temperature. Each compound was tested at five different concentrations: 0.0003μM, 0.001 μM, 0.003μM, 0.01 μM and 0.03μM. Digestion was initiated by the addition, at timed intervals, of 10 μl of DNase I solution (200 mM NaCI₁ 20 mM MgCI₂, 20 mM MnCI₂; containing 0.02 U/μl DNase I [Promega]). Each reaction was stopped after exactly eight minutes by the addition of 40 μl of DNase I Stop solution (2.25 M NaCI, 150 mM EDTA₁ pH 8.0, 0.57 μg/μl glycogen and 19.3 ng/μl poly(dl-dC)»poly(dl-dC) DNA). Subsequently, 1.3 ml of absolute ethanol was added to the reactions and the precipitate was collected by centrifugation at 14,000 rpm for 30 minutes at 4^CC. Supernatant was carefully removed before the pellets were washed three times with ice-cold 70% ethanol and then air-dried for 5 minutes. The DNA was resuspended in 4 μl loading dye (80% formamide, 1x TBE, 0.1% bromophenol blue dye). Samples were prepared for gel electrophoresis by heating at 94⁰C for 4 minutes and immediately cooling on wet ice.

Single-stranded DNA fragments were resolved to a difference of one nucleotide by denaturing polyacrylamide gel electrophoresis. Denaturing gels (0.4 mm thick, 10%) were prepared using a National Diagnostics Sequencing Gel Kit according to the manufacturer's instructions. The gel was set by the addition of 300 μl of 10% APS (w/v) and 30 μl of TEMED. Electrophoresis was performed for around 150 minutes at 90 W (~ 2000-2250 V) in 1x TBE buffer until the bromophenol blue marker dye had run out of the gel, thus resolving the DNA sites 20-120 nucleotides from the labeled end. Gels were fixed by soaking in 10% acetic acid for 15 minutes and then dried onto Whatman 3MM paper under vacuum at 8O⁰C for 90 minutes.

Dried gels were apposed at room temperature to GE Healthcare phosphor storage screens for a minimum period of 48 hours. Data were collected from exposed screens using a Storm 840 Phosphorlmager (GE Healthcare) and transferred to ImageQuant TL V2003.03 software (GE Healthcare) for visual inspection.

As described herein, the target site in the MYC promoter comprises 5'-AAAGAAG AG-3'. However, due to degeneracy, the generic target sequence for the molecules as a whole comprises 5'-WWWGWWG WG-3', where W = A or T. Reference herein to a 'match' or a 'mismatch' site is with respect to this generic target.

Results discussion

Compounds targeted to TBE3 of the human MYC gene promoter were subjected to DNase I footprinting. Thirteen compounds designed to span nine base pairs, eleven designed to span ten base pairs and two designed to span twelve base pairs were included in the study. Every compound that spanned nine base pairs bound preferentially to the predicted match site (5'-AAAGAAGAG-S') with approximate C₅o values between 1-30 nM, as shown in the table below:

The footprints for four of these compounds are shown in Figure 1.

Secondary binding at an inverse-orientated, double mismatch site (5'-CGGAAGTAA-3') was observed at concentrations roughly three- to ten-fold greater than the C₅₀.

The compounds with the highest apparent affinity were 7, 8 and 9 representing C2- linked benzofuran, pyridine and pyrazine variations respectively. These three compounds exhibited inhibition of DNase l-mediated cleavage at the predicted match site at ~1 nM. In contrast, the standard pyrrole-imidazole molecule (1) bound to the same site at ~10 nM. The remaining nine compounds in this class (spanning nine base pairs) had apparent C₅₀ values in the range 3-30 nM.

Both 8 and 9 exhibited high levels of selectivity. On the fragment of the human MYC gene promoter footprinted, both compounds yielded a selectivity ratio of 50-100. This value is calculated as C50_Sec/C50p_rim where C50_sec is the concentration required to inhibit 50% of DNase l-mediated cleavage at the secondary (non-match) drug-binding site and C50p_ri_m is the equivalent at the primary (match) site. In this case, it is important to remember that the secondary binding site is an inverse-orientation, double mismatch site and thus, the selectivity ratio does not provide a full indication of sequence-selectivity. By way of comparison, the selectivity ratio of 1 (a typical pyrrole-imidazole polyamide) on the same fragment was 10-30.

The eleven compounds that were designed to span ten base pairs bound to their match site (5'-AAAGAAGAG G-3'); they exhibited C₅₀ values between 3-30 nM, as shown in the table below:

14, the pyrrole-imidazole 'parent' of the set, had an approximate C₅o of 3-10 nM. The C2- linked pyridine- and pyrazine-containing compounds (20 and 21 respectively) showed no easily discernible differences from 14.

The footprints of four of these compounds are shown in Figure 2.

The secondary binding sites of 14 were different to those of the nine base pair-spanning compounds, presumably due to the additional heterocycle causing a shift in sequence preference. 14 bound to an A₇ tract at -30 nM (selectivity ratio of 10).

25 and 26 were designed to bind over an eleven base pair region of DNA. Both compounds bound to the predicted preferred site ((5'-AAAGAAGAGAG-3') at 0.3-1 nM, as shown in the table below:

The mismatched molecule 26 (which contains a pyrrole unit juxtaposed to a guanine) exhibited the slightly higher affinity. From the images of footprinting gels, it is clear that as the molecules are extended from spanning nine base pairs to twelve base pairs, that the visible footprint widens accordingly. Both 25 and 26 bind to lengthy regions of DNA at concentrations above their C₅o values. Secondary binding over much of the studied fragment was apparent at 30 nM.

The footprints of these two compounds are shown in Figure 3.

The favourable binding affinity of these two compounds compared to the ten base pair- spanning molecules is most likely due to the increased number of non-covalent bonding interactions yielded by the additional β-alanine and heterocyclic units.

Claims

1. A polyamide moiety comprising at least one unit of formula I:

-P₁-X₁-Y₁-X₃-X₄-P₃. (i)

wherein:

Y₁ is either X₂-β2 or β₂-X₂; βi is -R^β-CH₂-NH-β_a-, wherein β_a is a β-alanine residue and R^p is chosen from optionally substituted Ci_-7 alkylene, Ci_-7 alkenylene and Ci_-7 alkynylene groups; β₂ and β₃ are β-alanine residues; and Xi, X∑, X3 and X₄ are independently fragments of formula II:

wherein E is an optionally substituted C_5-6 heteroarylene group.

2. A polyamide moiety according to claim 1 wherein the unit of formula I is of the formula III:

-P₁-X₁-Y₁-X₃-X₄-P₃-Y₂- (III)

wherein Y₂ is X₅, X₅-X₆ or X₅-X₆^₄;

X₅ and X₆ are independently fragments of formula II:

wherein E is an optionally substituted C_5-6 heteroarylene group; and β₄ is a β-alanine residue.

3. A polyamide according to claim 2, wherein the polyamide of formula III is of the formula IV: -P₁-X₁-Y₁-X₃-X₄-P₃-Y₂-B (IV)

wherein B is a capping group of formula V:

wherein Z is an optionally substituted C_5-2O cyclic group.

4. A polyamide according to claim 3, wherein Z is C_5-2O heteroaryl having one or two heteroatoms chosen from N, O and S.

5. A polyamide according to claim 2, wherein the polyamide of formula III is of the formula IV:

-P₁-X₁-Y₁-X₃-X₄-P₃-Y₂-B (IV)

wherein B is a capping group of formula V:

wherein Z is an optionally substituted pyrrolobenzodiazepine (PBD) moiety of formula Via or VIb:

wherein: the dotted lines indicate the optional presence of a double bond between C1 and C2 or C2 and C3; R² is selected from -H, -OH, =0, =CH₂, -CN, -R, OR, halo, =CH-R, 0-SO₂-R, CO₂R and

COR;

R⁶, R⁷ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR", nitro, Me₃Sn and halo; where R and R' are independently selected from optionally substituted Ci_-7 alkyl, C_3-20 heterocyclyl and C_5-20 aryl groups; or R⁶ and R⁷ together form a group -0-(CH₂)_p-0-, where p is 1 or 2; R¹⁰ and R¹¹ either together form a double bond, or are selected from H and YR^Y, where

3, and M is a monovalent pharmaceutically acceptable cation;

R^2L is X², where X² is Ci_-7 alkylene or Ci_-7 alkenylene;

R^8L is Q-X⁸, where Q is selected from O, S, NH or a single bond and X⁸ is C_1-7 alkylene or Ci_-7 alkylene-CONH-Ci_-7 alkylene.

6. A polyamide according to claim 1 , wherein the moiety of formula I is of formula VII:

wherein A, taken with -R^β-CH₂-NH-, is an optionally substituted amino or amido capping group, or a group of formula IX:

wherein R^D is an optionally substituted Ci_-7 alkyl group or a group of formula Via or VIb as defined in claim 5.

7. A polyamide according to claim 7 wherein 6 is a dimethylaminopropylamino group.

8. A polyamide according to any one of claims 3 to 5, wherein the moiety of formula IV is of formula VIII: A-P₁-X₁-Y₁-X₃-X₄-P₃-Y₂-B (VIII)

wherein A, taken with -R^P-CH₂-NH-, is an optionally substituted amino or amido capping group, or a group of formula IX:

wherein R^D is an optionally substituted C_1-7 alkyl group or a group of formula Via or VIb as defined in claim 5.

9. A polyamide according to any of the preceding claims, wherein E is optionally substituted Cs heteroaryl.

10. A polyamide according to claim 9, wherein E has one or two heteroatoms.

11. A polyamide according to claim 10, wherein the heteroatoms are selected from N, O and S.

12. A polyamide according to any of the preceding claims, wherein Y₂ is X₅-X₆-P₄.

13. A polyamide according to any of the preceding claims, wherein X₁, X₂, X3, X₄, X5 and X₆ are Pyrrole (Py) or Imidazole (Im) based residues.

14. A polyamide according to any of the preceding claims wherein X₁ and X₃ are Py.

15. A polyamide according to any of the preceding claims wherein X₂, X₄, X₅ and X₆ are Im.

16. A polyamide according to claim 1 , wherein the polyamido moiety of formula I is:

-P₁-Py-Im-P-Py-Im-P-

17. A compound comprising a polyamido moiety according to any of the preceding claims.

18. A compound according to claim 17 for use in a method of therapy.

19. A pharmaceutical composition containing a compound of claim 17 and a pharmaceutically acceptable carrier or diluent.

20. Use of a compound according to claim 17 in the manufacture of a medicament for treating a proliferative disease.

21. Use according to claim 20 wherein the disease is colon cancer.