US3271439A - 3-cyclohexene-1-glyoxylic acid, salts and esters - Google Patents

Description

United States Patent 3,271,439 3-CYCLOHEXENE-l-GLYOXYLIC ACID, SALTS AND ESTERS Walter D. Celmer, New London, Conn, assignor to Chas. Pfizer & Co., Inc., New York, N.Y., a corporation of Delaware No Drawing. Filed Mar. 5, 1963, Ser. No. 262,829 5 Claims. (Cl. 260468) This invention relates to organic compounds which are useful as antibiotic substances. More particularly, it is concerned with 3-cyclohexene-l-glyoxylic acid, with alkali metal and alkaline metal salts thereof and with its ester derivatives. The invention also contemplates processes, both fermentative and synthetic, for the prep aration of the valuable new glyoxylates.

This invention relates specifically to compounds of the formula:

wherein R is selected from the group consisting of hydrogen, alkali metals, alkaline earth metals and alkyl groups containing from 1 to 3 carbon atoms.

It will be recognized that the ring carbon atom to which the glyoxylic group is attached is asymmetric, that is, it can be said to have four different radicals or atoms attached to it. By virtue of this, the compounds contemplated by the instant invention exist either as optically active forms, the so-called stereoisomeric 3-cyclohexenel-glyoxylates, or as mixtures thereof. It has now been found that those optically-active stereoisomers of the compounds of the instant invention which turn the plane of polarized light to the right, that is, clockwise and which are hereinafter referred to as the dextrorotatory forms, or the (+)-forms, possess antibiotic activity. This antibiotic activity is particularly pronounced against Gramnegative organisms and is accompanied by a substantial lack of acute toxic manifestations.

Particularly effective as an antibiotic is dextrorotatory (+)-3-cyclohexene l-glyoxylic acid, the compound of the formula above wherein R is hydrogen. This useful activity against a variety of organisms permits the application of the microbiologically-active stereoisomeric forms of the instant compounds in therapeutics, in veterinary medicine and in agriculture. The new antibiotic compounds are also of use in industrial fermentations to prevent contamination by sensitive organisms. They have activity in medical diagnostic techniques and, in addition, are useful in separating and classifying organisms.

With respect to the counterparts of the dextrorotatory compounds of the instant invention, those which, in contrast, turn the phase of polarized light to the left, that is, counterclockwise and which are hereinafter referred to as the levorotatory forms, or the ()-forms, these are useful as intermediates in the preparation of the microbiologically-active dextrorotatory forms. Thus the levorotatory form may be racemized, that is, converted by physical or by chemical means, to a mixture of the (-1-)- and ()-forms, conveniently, for example, by being treated with a basic reagent such as potassium hydroxide, and then the active (+)-form may be recovered from the mixture by the commonly employed technique known as resolution. Resolution contemplates, in essence, the separation of a racemic mixture into its optically-active components. Such separations may be effected by, for example, fractional crystallizations which are based on differences in solubility between the stereoisomers or derivatives thereof.

3,271,439 Patented Sept. 6, 1966 The compound D,L-3-cyclohexene-l-glyoxylic acid may be readily obtained by synthesis. For example, the following outlined route is especially convenient:

The starting material is the well-known and readilyavailable adduct of butadiene and acrolein. This is caused to react with ammonium carbonate and sodium cyanide, Step (1), and the intermediate formed thereby is treated with barium hydroxide and Water, Step (2), to form 3-cyclohexene-1-glycine, substantially in accordance with the procedure set out by Edelson et al., in volume 80, Journal of the American Chemical Society, pages 2698 et seq, 1958.

With respect to step (3), 3-cyclohexene-1-glycine is converted to 3-cyclohexene-l-glyoxylic acid by treatment with pyridoxal monohydrochloride. It has been found to be particularly convenient to use a modification of the method of Metzler and Snell, volume 74, Journal of the American Chemical Society, page 979, 1952, wherein a phosphate buffer is substituted for the acetate buffer described therein for similar transamination reactions. For example, pyridoxal monohydrochloride, dissolved in about 500 parts of water, is treated with an equivalent amount of 3-cyclohexene-l-glycine and equivalent of aluminum ammonium sulfate dodecahydrate per 10 equivalents of pyridoxal originally taken, is added. The solution is stirred at about 25 C. for about 15 minutes, then the pH, which is about 3.3, is increased to about 5.0 by the addition of an appropriate amount of 0.75 ammonium hydroxide solution. The resulting mixture is stirred and is heated to about C. during about 1 hour. The mixture is cooled to about 25 C. and is treated with enough 10% aqueous phosphoric acid to lower the pH to about 2.5, then is extracted with an equal volume of methylene chloride. The aqueous layer is adjusted to pH 5.0 by the addition of an appropriate amount of 5 N sodium hydroxide solution, is concentrated in vacuo to about /3 volume and then is refluxed for 16 hours. The reaction mixture is cooled to about 25 C. and is extracted with an equal volume of methylene chloride. The aqueous layer is adjusted to about pH 2.0 with an appropriate quantity of 50% phosphoric acid and the solution is extracted with an equal volume of methylene chloride. The organic layer is separated, is dried with anhydrous sodium sulfate, the drying agent is removed by filtration and the solvent is removed by distillation. The product, racemic 3 cyclohexene'l-glyoxylic acid remains as a white, crystalline residue, M.P. 45-50 C. The product can be further purified by recrystallization from heptane, from which it is deposited as rectangular plates, M.P. 5556 C.

In addition to the hereinbefore described synthetic process, the dextrorotatory form of 3-c-yclohexene-lglyoxylic acid may be obtained by'a fermentation process. Thus, the present invention contemplates a process for the preparation of (+)-3-cyclohexene-1-g1yoxylic acid which comprises cultivating Streptomyces antibiotic-us, strain ATCC 14890, in an aqueous nutrient medium containing a carbohydrate and a source of organic nitrogen and inorganic salts under submerged aerobic conditions until substantial anti-microbial activity is imparted to said 3 medium and recovering said acid from the fermentation broth. 7 1

The organism which is employed in the valuable fermentation process of the present invention has been identified to be a new strain of a species of microorganism known as Streptomyces antibioticus. Identification was made by planting and testing a culture of the organism on media normally used for such identifications and the media were incubated at controlled temperatures for 14 days. Readings were made at intervals and final observations were recorded after two weeks of incubation. A culture of this strain has been deposited in the American Type Culture Collection, Washington, DC, and has been assigned the number ATCC 14890.

The description of this microorganism, designated FD 13219 in the culture collection of Chas. Pfizer & Co., Inc. of Groton, Connecticut, was supplied by Dr. J. B. Routien. The cultural characteristics observed and recorded for this strain are set forth in Table I ((R) refers to Ridgways Color Standards and Nomenclature):

genetic recombination or some other genetical procedure using a nucleic acid or an equivalent material from the herein described species whereby it has acquired the ability to produce the elaboration product described herein and claimed.

Strain ATCC 14890 will grow on many media used for the propagation of Streptomycetes. A satisfactory medium is Pridhams Yeast Extract Agar: A mixture of 4.0 g. of yeast extract, 10.0 g. of malt extract and 4.0 g. of dextrose in 1000 ml. of water is adjusted to pH 7.3 with sodium hydroxide, 20.0 g. of agar is added, the mixture is steamed for 15 to minutes, dispensed, and then is sterilized for 15 minutes at 121 degrees C. The culture should be incubated at 28 degrees C.

(+)-3-cyclohexene-l-glyoxylic acid and its alkali metal and alkaline earth metal salts and its ester derivatives are remarkably effective in treating a number of infections. They exhibit significant activity against a wide variety of microorganisms. However, they are particularly noteworthy in their action on Gram-negative or- TABLE I.CULTURAL CHARACTERISTICS OF FD-13219 Medium Amount of Growth Aerial Mycelium and Spores Soluble Pigment Remarks Glucose asparagine agar. Good Good, nearLight Mouse Light Brown Reverse pale yellow at edge Gray (R). and becoming brown in center. Synthetic agar Very poor Very thin, pale cinnamon. Lacking Reverse colorless. Gauzesinorganic medium Good. Good, Light Mouse Gray Gray Brown Reverse brown; chains of N0. 1. to Mouse Gray (R). spores straight to flexuous and in loops, scattered along hyphae', spores broadly elilptical, 1.3-24) X 0.7-1.0 G. Calcium lnalatc agar Poor, flat Poor, nearMouse Gray Lacking Reverse light yellow;

(B). calcium malate weakly digested. Cellulose Good. Abundant, nearuNlouse .--..do

skimmed milk Potato plug Nutrient agar.. Glucose agar... Emerson's agar Pridhams yeast extract agar. Hickey dz 'Iresners agar. Gelatin Starch agar Peptone iron agar Moderate in brown ring.

Good Peer to moderate..

Excellent Good Moderate, flat Poor, fiat G ray (R). Lacking Good, gray Poor, grayish white. Good, grayish white Good, light grayish white...

Moderate, near Olive Gray to Mouse Gray Dark brownin upper hydrolyzed portion; in lower part near Ridgways Sayal Brown.

Gray to black Pale yellowish brown Dark brown No coagulation; hydrolysis partial; pH change from 6.5 to 6.9.

Vegetative rnycelium yellow Reverse whitish.

Reverse grayish tan.

Reverse grayish tan, vegetative inycelium yellow.

Reverse yellow.

' like.

In connection with the description of this organism, it has been observed occasionally that there is a tendency for the colors imparted to some media to vary somewhat. This is especially pronounced after a number of transfers have occurred and the reasons for this are not clearly understood at this time. However, this variation in appearance does not have a marked effect on the ability of the organisms to produce the antibiotic substance of the present invention.

It is to be understood that for the production of 3-cyclohexene-l-glyoxylic acid, the present invention is not limited to this particular strain or to a strain fully answering the above description, which is given only for illustrative purposes. It is especially desired and intended to include the use of mutants produced from the described organism by various means such as X-radiation, ultraviolet radiation, nitrogen mustards and the It is contemplated also to include any organism regardless of its appearance or physiological behavior,

that may be developed by transformation, transduction,

ganisms. While they demonstrate some activity against Gram-positive organisms, this activity is generally of a somewhat lower level.

The invitr-o activity of (+)-3-cyclohexene-1-glyoxylic acid against a group of microorganisms which cause various diseases is demonstrated against strains of Escherichia coll, Proteus vulgaris, A. aerogenes and Salmonella typhosa. In vivo, it demonstrates the ability to protect mice against death after being infected with E. coli; for example, (-1-)-3-cyclohexene-1-glyoxylic acid protected 50% (five out of ten, PD mice infected IP with E. coli strain 462 after a multiple oral dosage /2, 4, 24, 48 hours post infection) of 800 rng/k After administration by the subcutaneous route, the corresponding PD was found to be 500 mg./kg. The potassium salt of (+)-3-cyclohexene-1-glyoxylic acid exhibits low acute toxicity in rats; no deaths are observed after dosages of 2000 mg./kg. orally or 300 rug/kg. intravenously. The

intravenous LD was 400 mg./kg.

It is to be understood that for the purpose of treatmerit of infections with the valuable compounds of the instant invention, either the pure materials or one of the crude forms of the antibiotic may be used. This is to include either a filtered fermentation broth, as produced from, for example, strain ATCC 14,890, or a solid or liquid concentrate prepared therefrom. For administration to man and animals, a non-toxic carrier is, of course, selected, toxicity for this purpse being defined as an adverse effect on the treated host at the level of ordinary use. Either liquid or solid pharmaceutical carriers may be employed, including water, aqueous ethanol, isotonic saline or glucose, starch, lactose, calcium phosphate, animal feed stuffs, or mixtures of various materials as occur in a filtered fermentation broth. Either oral or parenteral administration is satisfactory, although the parenteral route is perhaps preferable until a satisfactory regimen adapted to the patient is established. For this purpose, solutions of suspensions in water, oils, such as peanut oil, or other pharmaceutically acceptable solvents or vehicles may be employed. Solid preparations for extemporaneous dilution can be prepared containing various buffering agents, local anaesthetics, and other medicinal agents including antibiotics, hypnotics, analgesics, as well as inorganic salts to afford desirable pharmaceutical properties to the composition.

The present invention embraces the process for growing Streptomyces antibioticus, ATCC 14890, under controlled conditions to produce (+)-3-cyclohexene-1-glyoxylic acid. The culture is grown submerged in an aqueous nutrient medium containing a source of nitrogen, a carbohydrate and minerals.

A satisfactory medium contains 10 g./l. of Cerlose (dextrose hydrate), g./l. of Curbay-BG (molasses residue), g./l. of cornstarch, 10 g./l. of soybean meal, and 5 g./l. of sodium chloride. The medium is adjusted to pH 7.0 with 1 N potassium hydroxide, calcium carbonate, 10 g./l., is added and broth is sterilized at 121 C. for minutes. It is inoculated with 27% of a -60 hour old culture grown in the same medium. Per- 8 tate, methyl isobutyl ketone and methylene chloride, respectively.

The crystalline antibiotic may be further purified by recrystallization of its potassium salt from a mixture of methanol and isopropanol. The less pure acid is dissolved in about 10 parts by Weight of methanol to give a 1.34.4 molar acid solution. Then 4 N methanolic potassium hydroxide, in an amount equivalent to the acid originally taken, is added and the precipitated solid is removed by filtration. The filtrate is concentrated to about one-half volume and 1.5-2.0 volumes of isopropanol is added, with stirring. The potassium salt which is precipitated is removed by filtration and is dried. A second crop is obtained by concentration of the mother liquor.

It has been surprisingly found that the crystalline antibiotic of the instant invention is also produced by the strain ATCC 11891 of Streptomyces antibioticus, which strain was taught in US. 2,757,123, as producing the basic antibiotic, oleandomycin, described and claimed therein; the strain disclosed in the said patent is being maintained, on public deposit, in the American Type Culture Collection, Washington, DC. It is now found that strain ATCC 11891, in addition to producing oleandomycin, also produces the crystalline acidic antibiotic of the instant invention. In contrast, the first mentioned strain of the instant application, ATCC 14890, produces only (+)-3-cyclohexene-1-glyoxylic acid and no oleandomycin. Co-production of the new antibiotic by the old strain ATCC 11891 has not been observed before, since the strain has been used to produce a basic antibiotic and the acidic antibiotic of the instant invention has been discarded in the sewer liquor without having been characterized or isolated.

In addition to the inability for strain ATCC 14890 to produce oleandomycin, there are other significant differences in growth behavior between it and the oleandornycin-producing S. antibioticus ATCC 11891 described in the above-mentioned patent; these differences are summarized in Table II:

DIFFERENCES BETWEEN ATCC 11891 AND ATCC 14890 Medium I ATCC 11891 ATCC 142390 Potato plu Emersous Stare-IL." Calcium inalate agar Gauzes inorganic medium No.

Vegetative inycelium yellow. Colonies with grayish white spores. No rings of growth.

Weak digestion of malate.

Soluble pigment dark brown. Reverse brown.

mentation can be carried out at about 26-30 C. in 4 liter stirred glass jars, mechanically aerated, for from about to about 130 hours. The progress of the fermentation can be followed by standard plate assay methods using the activity of the broth against Aerobacter aerogenes.

After a satisfactory level of antibiotic activity has been obtained, the active substances may be isolated by procedures well known to those skilled in the art. A particularly satisfactory procedure is outlined as follows: The broth is filtered and then is adjusted to a pH of 25:1 and is extracted with one-third to one-half volume of methylisobutyl ketone or ethyl acetate. The organic layer is partitioned against aqueous sodium phosphate solution, the aqueous layer is separated and is extracted with an equal volume of methylene chloride. The methylene chloride layer is separated and is treated with an equal volume of cyclohexene or heptane and concentration of the resulting solution to about one-fourth volume causes the precipitation of crystalline (+)-3-cyclohexenel-glyoxylic acid. The term substantially water-immiscible organic solvent as used in the appended claims contemplates those dissolving )-3-cyclohexene-1-glyoxylic acid and being of the type indicated, for example, esters, ketones and chlorinated hydrocarbons such as ethyl ace- In order to recover the valuable new acidic antibiotic from broths produced by the fermentation of strain ATCC 11891, the sewer liquors, which have up until now been discarded after removing oleandomycin, are subjected to the following procedure: The sewer liquor broth is adjusted to a pH of from about 1 to about 5 by the addition of phosphoric acid. About A; volume of methylisobutyl ketone is added and stirring is continued for about 15 minutes, then the aqueous layer is drawn off and is discarded. The organic layer is extracted with 6 one-fourth volume portions of 0.35% disodium phosphate solution and the organic layer is discarded. The aqueous layer is extracted with about onetenth volume of methylene chloride, the organic layer is separated and the aqueous layer is adjusted to pH 2 with phosphoric acid. The aqueous layer is then extracted with about one tenth volume of methylene chloride and the methylene chloride solution is distilled in vacuo until free of solvent. The residue is then dissolved in about 50 parts by weight of methanol, and the solution is adjusted to pH 6710.1 with 2 N methanolic potassium hydroxide, then the solvents are removed by distillation in vacuo. The crystalline residue is triturated with isopropanol, the crystals are removed by filtration and are dried.

As has been mentioned hereinbefore, advantage is taken of the interconvertibility of optically-active forms in the synthesis of the microbiologically active (+)-3- cyclohexene-l-glyoxylic acid and its derivatives. Thus the microbiologically inactive forms are racemized and the desired isomer is isolated by resolution of the mixture. The stereoisomers, though very stable at neutral and acidic pHs, are racemized with increasing rapidity as the alkalinity of the solution increases. For example, at the following pH values, there remains the respective percentages of original rotation after 19 hours: pH 7, 99%; pH 8.9, 86%; pH 9.5, 78%; pH 10.4, 45%; and pH 11.5, At pH 12.0, all optical activity is lost in about 30 minutes; all measurements were made on the potassium salt in buffered phosphate aqueous solutions at C., except for the unbuffered measurement at pH 12.

For purposes of preparing the (+)-form from the ()-form, it is especially convenient to racemize the potassium salt in sodium hydroxide solution and to separate the mixture of and ()-forms, which results, by fractional crystallization of salts with optically active basic alkaloids by techniques well known in the art. Thus, the potassium salt of the optically-active form is dissolved in about parts of water and the solution is rendered basic to pH 12.2 by addition of ION aqueous sodium hydroxide. Progress of the racemization is readily followed in a polarimeter and, after about 1.5 hours at 25 C., the solution has lost its rotation. After an additional half-hour the racemate may be isolated by adjusting the solution to pH of about 2 with 50% phosphoric acid, extracting with an equal volume of methylene chloride, separating the organic layer, drying the organic layer and evaporating the solvent therefrom. The racemic product remains as a crystalline residue which can be separated into pure and ()-forms by successive slow crystallizations of salts with optically active basic alkaloids, the mother liquor being removed for further crystallization followed by regeneration of the optically active acids.

Of course, in addition to the synthesis of (+)()-3- cyclohexene-l-glyoxylic acid from 3-cyclohexene-1-glycine, it will be obvious to those skilled in the art that alternative methods are available for its preparation. Among these are, for example:

The reaction of butadiene and certain selected acids in accordance with the following sequences *Instead of this compound, the Mannich Base,

ReNCHzCHzC O) C0211 or Mannich Base N-oxide, R2N( O) CHaCHzC (O) COaH, may be employed,

The following examples are given by way of illustration and are not to be construed as limitations of this invention, many variations of which are possible without departing from the spirit or scope thereof.

Example I A solution of 1018 mg., 5 meq., of pyridoxal monohydrochloride (Nutritional Biochemical Corporation, Cleveland, Ohio) in 500 ml. of water is treated with 3-cyclohexene-l-glycine, 775 mg, 5 meq., prepared by the process of Edelson et al., volume 80, ].A.C.S., page 698, 1958 (substituting a phosphate buffer for the corresponding acetate buffer), and 227 mg., 0.5 meq., of aluminum ammonium sulfate dodecahydrate. The resulting mixture is stirred at 25 C. for 15 minutes, then the pH is adjusted from an initial 3.3 to a final 5.0 by the addition of 6.6 ml. of 0.75 N ammonium hydroxide solution. The mixture is then heated to C. during 60 minutes, with stirring. The mixture is cooled to about 25 C., then is adjusted to a pH of 2.5 by the addition of 10 ml. of 10% phosphoric acid. The acidic mixture is extracted with 500 ml. of methylene chloride and the organic layer is separated. The aqueous phase is adjusted to pH 5.0 with 5 N sodium hydroxide, is evaporated to about one-half volume in vacuo to remove residual methylene chloride, then is refluxed for 16 hours. The resulting solution is cooled to 25 C. and is extracted with an equal volume of methylene chloride; the methylene chloride layer is separated and is discarded. The aqueous phase is adjusted to pH 2.0 with 50% phosphoric acid and is extracted with an equal volume of methylene chloride. The solvent layer is collected, is dried with sodium sulfate, the sodium sulfate is removed by filtration and the solvent is distilled leaving the product, 3-cyclohexene'l-glyoxylic acid, as a white, crystalline residue, 210 mg., M.P. 4550 C. The product is recrystallized from heptane and is deposited as rectangular plates, M.P. 55-56 C. D,L-3-cyclohexene-1- glyoxylic acid is converted to its potassium salt by dissolving the acid in methanol to afford a 1.3 molar concentration. Then is added 4 N methanolic potassium hydroxide until the pH is 7. A small amount of precipitated solid is removed by filtration, then the filtrate is concentrated to one-half volume and 1.5 volumes of isopropanol is added thereto. The potassium salt which precipitates is removed by filtration and is dried.

Example II A slant of S. anribioticus ATCC 14890 on Emerson agar is cultivated to develop spores for the purpose of inoculating a nutrient medium of the following composition:

' Grams Cerelose (dextrose hydrate) 10 Curbay-BG (molassess residue) 5 Cornstarch 10 Soybean meal 10 Sodium chloride 5 This mixture of nutrients is diluted to a volume of one liter with water, is adjusted to a pH of 7 with potassium hydroxide and is sterilized at 121 C. for thirty minutes. The medium is then cooled and the spores are added aseptically. The organism is cultivated in Fernbach flasks on a reciprocating shaker at 28 C. for two days. The mixture of broth and mycelium formed thereby is transferred to 20 volumes of a sterile fermentation medium having the same composition, adjusted to pH 7. After seeding the medium with the organisms from the Fernbach flasks, the mixture is agitated and aerated at 28 C. for 108 hours. The potency of the broth is measured against A. aerogenes. The mycelium is removed by filtration and the filtrate is adjusted to pH 2 with phosphoric acid. The acidic solution is extracted with one-fourth volume of iethylisobutyl ketone and the separated organic layer is extracted four times with equal-volume portions of a 0.35% di-sodium phosphate buffer solution. The buffer extracts are separated, are combined and are washed with one-twelfth volume of methylene chloride; the methylene chloride layer is separated and is discarded. The Washed bufier is adjusted to pH 2 with phosphoric acid and is extracted with an equal volume of fresh methylene chlo ride, then the methylene chloride is separated and is concentrated to dryness. The crystalline residue is triturated with cyclohexene and the crystalline (+)-3-cyclohexene-lglyoxylic acid is removed by filtration and is dried. The crystalline product, M.P. 5556 C. [alpha] =|60i5 (in Water), is found to be highly effective against a variety of gram-negative microorganisms, both in vivo and in vitro as is indicated in the description hereinbefore.

The potassium salt of the antibiotic [alpha] --+50i-5 (in water), is prepared by the procedure described in Example I.

Example [11 The procedure of Example II is repeated, substituting for strain ATCC 14890, strain ATCC 11891. After the (+)-3-cyclohexene-l-glyoxylic acid is removed by extraction at acidic pH, the aqueous phase is adjusted to a pH of about 9 and is extracted with chloroform. The chloroform extract is concentrated to a sirupy consistency and, after standing, crystalline oleandomycin chloroform solvate (cf. Celmer et al., Antibiotics Annual, 1957-1958, p. 478) formed. A methanol solution of the chloroform solvate is distilled to remove chloroform azeotropically and the halogen-free concentrate is then cautiously treated with water until crystallization of the free oleandomycin base is induced. This is removed by filtration and is identical to the product of US. 2,757,123. This antibiotic in contrast to the crystalline (+)-3-cyclohexene-1-glyoxylic acid is highly effective against gram-positive microorganisms, as is indicated in the cited patent.

Example IV Salts of 3-cycl0lzexene-I-gly0xylic acid.-A stirred solution of crystalline (+)-3-cyclohexene1glyoxylic acid, 1.0 g., in isopropanol, 20 ml., is treated dropwise with 1 N methanolic sodium hydroxide, 6 ml. The crystalline sodium salt which forms is recovered by filtration and is dried, yield, 560 mg. In a similar manner, the sodium salt of D,L-3-cyclohexene-l-glyoxylic acid of Example 1 is prepared.

A stirred solution of l g. of crystalline potassium (4-)- 3-cyclohexene-l-glyoxylate in 10 ml. of water is treated with a saturated aqueous solution of calcium acetate, 2 ml. The crystalline calcium salt which forms is removed by filtration and is dried, yield, 660 mg. This procedure is repeated With D,L-3-cyclohexene-1-glyoxylic acid and the corresponding racemic acid salt is formed. The procedure is repeated with stoichiometrically equivalent amounts of magnesium acetate and barium acetate; the corresponding magnesium and barium salts of (+)-3-cyclohexene-1 glyoxylic acid are obtained.

Example V Esters of 3-cycl0lrexene-Iglyoxylic acirL-Crystalline (+)-3-cyclohexene-1-glyoxylic acid, 40 g., is dissolved in ml. of methylene chloride and is treated With 40 ml. of methanol and 0.8 ml. of concentrated sulfuric acid. The solution is refluxed for 2 hrs., is cooled and filtered and the solvents are removed by distillation at 25 C. at a pressure of 1515 mm. of mercury. The residue is then distilled by raising the bath temperature to about C., whereupon a first fraction, 11.4 g., refractive index, 1.4735, and a second fraction, 21.3 g., refractive index, 1.4740 are obtained at approximately 100 C. (head temperature). By the same procedure, the methyl ester of D,L-3-cyclohexene-1-glyoxylic acid is obtained.

The procedure is repeated substituting for methanol, equivalent volumes of ethanol and n-propanol. There are obtained, respectively, the ethyl and n-propyl esters of 3- cyclohexene-l-glyoxylic acid.

What is claimed is:

1. A compound having the formula wherein R is selected from the group consisting of hydrogen, alkali metal, alkaline earth metal and alkyl of from 1 to 3 carbon atoms.

2. D,L-3-cyclohexene-1-glyoxylic acid.

3. (+)-3-cyclohexene-1-glyoxylic acid.

4. Potassium (+)-3-cyclohexene-l-glyoxylate.

5. Methyl (+)-3-cyclohexene-l-glyoxylate.

References Cited by the Examiner UNITED STATES PATENTS 2,734,018 2/1956 Minieri et al -180 2,771,487 11/1956 Morris et a1. 260-514 2,776,243 1/1957 McGhee et al. 195-180 2,808,363 10/1957 Hoeksema et a1 195-180 2,847,460 8/1958 Trapp et al. 260-514 LORRAINE A. WEINBERGER, Primary Examiner. A. LOUIS MONACELL, Examiner.

D. M. STEPHENS, R. K. JACKSON,

Assistant Examiners.

Claims (1)

1. A COMPOUND HAVING THE FORMULA