3,704,246 United States Patent Office Patented Nov. 28, 1972 2 (I) R 3,704,246 AMNO ACD DERVATIVES ( CH).--N Miklos Bodanszky, Shaker Heights, Ohio, assignor to R2-C YoH-R E. R. Squibb & Sons, Inc., New York, N.Y. No Drawing. Continuation-in-part of application Ser. No. R-i-o-c6 798,790, Feb. 12, 1969, which is a continuation-in-part wherein R is the residue of an a-, R1 is hydro of abandoned application Ser. No. 451,609, Apr. 28, gen, lower alkyl, hydroxy-substituted lower alkyl, or 1965. This application Dec. 16, 1970, Ser. No. 98,924 lower alkyl lower alkoxy; R and R3 each is hydrogen, int. C. C07d 87/54 lower alkyl, hydroxy-substituted lower alkyl, lower alkyl U.S. C. 260-333 9 Claims 10 lower alkoxy, or R and R3 together with the carbons to which they are joined form a phenyl ring which may be unsubstituted or substituted by halogen or nitro, and m ABSTRACT OF THE DESCLOSURE is an integer from 0 to 3. New lactone intermediates useful in the synthesis of 5 DETAILED DESCRIPTION OF THE INVENTION are prepared by reacting an a-amino acid with Any a-amino acid may be treated according to this an active carbonyl compound which forms a Schiff's base. invention. This includes the naturally occurring ox-amino The latter is treated with a condensing agent so that acids see for example, Hackh's Chemical Dictionary, cyclization occurs and a lactone is formed from which a 3rd ed. (1944), pages 44-45 as well as synthetic ox may be produced by reaction with an amino acid 20 amino acids. Some examples are monoaminocarboxylic ester removal of the protecting groups. aliphatic acids, e.g., glycine, a-aminoheptylic acid, o aminocaprylic acid, a-aminononylic acid, a-aminodecylic acid, a-aminoundecylic acid, alanine, , , threonine, methionine, valine, leucine, isoleucine; aro This application is a continuation-in-part of copending 25 matic monoaminomonocarboxylic acids, e.g., phenylala application Ser. No. 798,790, filed Feb. 12, 1969, now nine, tyrosine, dihydroxyphenylalanine, 3,5-dibromotyro abandoned, which in turn is a continuation-in-part of sine, 3-iodotyrosine, 3,5-diiodotyrosine, 3,5,3'-triiodo application Ser. No. 451,609, filed Apr. 28, 1965, now thyronine, and thyroxime; monoaminodicarboxylic acids abandoned. and their amides, e.g., aspartic acid, glutamic acid, aspar This invention relates to new intermediates useful in 30 agine and glutamine; diaminomonocarboxylic acids, e.g., the synthesis of peptides. More particularly, the invention lysine, hydroxylysine, and ; heterocyclic relates to amino acid derivatives, especially derivatives amino acids, e.g., histidine, tryptophane, proline, hy of a-amino acids, which are simultaneously protected and droxyproline, thiolhistidine; diaminocarboxylic acids, e.g., activated for reaction with other amino acids in the syn , and djenkolic; as well as cc-amino thesis of peptides. 35 acids of the same type found in special sources, e.g., In the usual synthesis of peptides, two amino acids are phenylglycine, butyrine, , homocystine, hypo joined "head to tail,” i.e., the carbonyl group of the acid glycin A, S-methylcysteine sulfoxide, allin and canava moiety of one amino acid is joined to the amino group of C. the second amino acid. It is conventional in such a syn 40 Other amino acids are aminomalonic acid, o-amino thesis to protect the reactive amino group of one amino adipic acid, oz-aminopimelic acid, oz-aminosuberic acid, acid with a readily removable protective group such as oz-aminosebacic acid, 5-hydroxyaspartic acid, cystine-6,6- a benzyloxycarbonyl, t-butyloxycarbonyl, phthalyl, tosyl dicarboxylic acid, 3-hydroxyglutamic acid, (3-methylaspar group, etc., so that the amino group does not enter into tic acid, 6-methyl-6-hydroxyaspartic acid, 8-methylglu the reaction. Then the carbonyl group of the same amino 45 tamic acid, (3,6-dimethylaspartic acid, y-hydroxyglutamic acid is activated with another group, e.g., with an azide acid, 6,y-dihydroxyglutamic acid, 6-phenylglutamic acid, or with an ester group such as the p-nitrophenyl ester, so ty-methyleneglutamic acid, oz-aminotricarballyic acid, 3 that the carbonyl group will react with or actylate the aminoalanine, y-aminobutyrine, homoarginine, homo amino group of the second amino acid in the presence of citrulline, o,a'-diaminosuccinic acid, c,c'-diaminoglutaric the protecting group. 50 acid, a,c'-diaminoadipic acid, oz,a'-, This procedure, it is evident, requires two distinct re o,a'-diamino-3-hydroxypimelic acid, a,c-diaminosuberic actions with two separate compounds in preparation of acid, or,a'-diaminoazelaic acid, cz,a'-diaminosebacic acid, the one amino acid for the ultimate reaction with the c,c'-diaminodecanedicarboxylic acid, c,c'-diaminododec second amino acid. It further requires the combination of anedicarboxylic acid, y-methylproline, pipecolic acid, two proper groups which will give the desired protection 55 baikiain, 5-hydroxypipecolic acid, azetidine-2-carboxylic and activation of the particular amino acid involved. acid, 6-phenylserine, , y-oxalysine, y-hydroxy ornithine, and 2-hexosaminic acids. A comprehensive dis BRIEF SUMMARY OF THE INVENTION cussion of amino acids, including those mentioned above is to be found in Chemistry of the Amino Acids, Green It has now been found that cy-amino acids can be both 60 stein et al., vol. III, Wiley, 1961. Other amino acids in protected and activated by reacting the amino acid with a addition to those listed are to be found in this volume but single compound and then forming a lactone. This method for sake of brevity are not specifically mentioned herein, and the novel lactones formed thereby form the essence but are included by reference. It is to be understood that of this invention. These lactones readily react with ce the present invention is applicable to any amino acid. amino acid esters to form condensation products from 65 The amino acid of the formula which the protecting-activating moiety is readily removed to leave the desired peptide. NH, Briefly, the method comprises reacting an a-amino acid R-CE-CO2H with an active carbonyl compound which forms a Schiff's wherein R is the residue of the amino acid is reacted with base and then the Schiff's base is treated with a condens 70 a carbonyl compound to produce a Schiff's base. These ing agent to induce cyclization and yield a lactone of the carbonyl compounds are aldehydes or ketones having the formula structural formula 3,704,246 4. wherein R is hydrogen, halo, especially chloro or bromo, or nitro, and n is 1, 2 or 3. Most preferred are salicyl aldehydes having a nitro or chloro group with the sub stituent meta or para but especially meta to the aldehyde group. The amino acid is reacted with the aldehyde or ketone in an inert Solvent, preferably an inert organic solvent wherein R is hydrogen or a straight or branched chain such as dimethylformamide, dioxane, pyridine or the like lower alkyl radical of from 1 to 4 carbon atoms; R and under conventional conditions for forming a Schiff's base. Reach is hydrogen, a lower alkyl radical, a hydroxy O Then, after the Schiff's base has formed, without isolating substituted lower alkyl radical, a lower alkyl lower alkoxy the base unless desired, a condensing agent is added to radical, each of said radicals being straight or branched the reaction mixture to form the lactone. An alcohol may chain of from 1 to 4 carbon atoms; R and R3 taken also be used as the reaction medium in which the Schiff's together with the carbon atoms to which they are joined base is formed, but in this case the solvent must be re are a phenyl ring which may be unsubstituted or which 5 moved before adding the condensing agent. may be substituted by halogen or nitro; m is an integer Any condensing agent which promotes the formation from 0 to 7 when the carbonyl compound is aliphatic of acyl esters may be used. These include, for example, and an integer from 0 to 3 when the carbonyl compound dicycloalkylcarbodiimides, e.g., dicyclohexylcarbodiimide; is aromatic. dialkylcarbodiimides, e.g., dipropylcarbodiimide; diaryl 20 carbodiimides, e.g., diphenylcarbodiimide; alkoxyacetyl Examples of suitable aliphatic carbonyl compounds are enes, e.g., ethoxyacetylene, diphenylketene, etc. Dicyclo the enol forms of the following compounds: hexylcarbodiimide and ethoxyacetylene are preferred. malondialdehyde About one mol or more of condensing agent per mol of 3-ketobutyra Schiff's base may be used. A mol ratio of about 1:1 may succindialdehyde 25 be used when the condensing agent is a carbodiimide. 4-ketopentanal An excess of the condensing agent is preferable when glutardialdehyde other condensing agents are used. 2,4-pentanedione Sufficient time must elapse for the Schiff's base to form 4-ketohexanal before the condensing agent is introduced. The time period adipdialdehyde 30 may vary within rather broad limits depending upon the 2,4-hexanedione particular reactants, but a period of about one hour to 2,5-hexanedione about twenty-four hours is generally adequate. Stirring 2-methyl-3-ketopentanal and/or heating will of course accelerate the reaction. 2-methyl-4-ketopentanal Temperatures in the range of about 15 to 60° C. may 3-methylpentanedial 35 be used. 2-ethylsuccindialdehyde The cyclization reaction which occurs upon addition 3-methyl-2,4-pentanedione of the condensing agent must be effected in an anhydrous 2,4-heptanedione medium and the solvent must be one which has no reac 2-methyl-3,5-hexanedione tive hydroxy or carboxy group. Media such as those 2-methyl-4-ketohexanal 40 named above, except alcohols, may be used for the cy 3-methyl-2,4-hexanedione clization as well. Room temperature up to about 60° C. 3-ethyl-2,4-pentanedione may be used, with a temperature in the lower end of the 2,2-dimethyl-3-ketopentanal range preferred for carbodiimides and a temperature at 3,3-dimethyl-2,4-pentanedione the higher end of the range for alkoxyacetylenes. octanedial 45 According to one modification, the amino acid and decandial aldehyde are dissolved in the solvent and stirred for about 1-ethoxy-2,4-pentanedione one hour to twenty-four hours. Then, without isolating 1-methoxy-3-ethoxymethyl-2,4-pentanedione the Schiff's base, or after merely concentrating the solu 1-methoxy-2,4-hexanedione tion, the condensing agent is added. 2-methyl-4-ketohexa-3-ene-6-al 50 The protected-activated lactones which are formed by 2,4-diketohexanol the procedure described have the general Formula I given 3-methyl-2,4-diketopentanol above wherein the symbols have the same meaning as 2,4-diketo-6-hydroxyheptanol previously defined. 3-ethyl-2,4-diketopentanol Preferred are the lactones of the formula 55 H Examples of aromatic carbonyl compounds are the foll (-N lowing: / N salicylaldehyde (R) C -R 3-chlorosalicylaldehyde 5-chlorosalicylaldehyde 60 o-c6 3-bromosalicylaldehyde wherein R, R and n have the meaning described previ 4-bromosalicylaldehyde ously and especially those wherein R is lower alkyl, R' 5-bromosalicylaldehyde is nitro and n is 1. 5-iodosalicylaldehyde 65 The active lactones of this invention will then react with 3-nitrosalicylaldehyde another amino acid, preferably a simple ester thereof, 5-nitrosalicylaldehyde such as a lower alkyl ester whereupon the ring opens and o-hydroxyphenylacetaldehyde the amino acid is acylated. Treatment with aqueous acid, o-hydroxyphenylpropionaldehyde e.g., with hydrochloric acid, dilute sulfuric acid or the like, Preferred are benzaldehydes of the formula 70 removes the protecting groups and the desired peptide is obtained. CEO It will be appreciated that the process of this inven (R) tion is not limited to the formation of dipeptides from O monomeric amino acids but applies as well to the forma tion of polypeptides from reactants having more than one 3,704,246 5 6 amino acid moiety, e.g., the formation of tripeptides, tetrapeptides, pentapeptides, hexapeptides, octapeptides, decapeptides, etc. When the amino acid used has another E=N-H-CH-(X reactive functional group elsewhere in the molecule, this ce do may have to be independently protected in the conven N tional manner. The following examples illustrate the invention without, CO/ however, limiting the same thereto. All temperatures given Example 4 are in degrees centigrade. 0. L-serine (0.105 g.) is reacted with ot-formyl-N-hydroxy Example 1-2-hydroxy-5-nitrobenzylidene-L-leucine and succinimide (0.15 g.) as described in Example 3. The its lactone Schiff base is obtained by removing the solvent and then L-leucine (2.62 g.) and 5-nitrosalicylaldehyde (5.0 g.) reacting with ethoxyacetylene to obtain the active actone are added to a mixture of absolute ethanol (750 ml.) and (IR 5.6) having the formula methanol (50 ml.). The mixture is stirred at room tem 5 CH-OE perature for several hours. When all the leucine is dis solved, the solvents are removed in vacuo. The residue CH-N-H extracted with ether, the ether extracts concentrated and &H- CO O diluted with hexane. A crystalline solid separates. It is Y-) filtered and washed with hexane. The crude Schiff base 20 (dec. at ca. 175-180 completely melting at ca. 200) is &H-c6 used in the cyclization step without additional purifica Example 5 tion. The benzylidene derivative (1.40 g.) is dissolved in tetrahydrofuran (40 ml.) and dicyclohexylcarbodiimide L-glutamine (1.5 g) and 4-acetyl-N-hydroxypiperidine (1.03 g.) is added to the solution. After about two hours 25 (1.5 g.) are stirred in ethanol (100 ml.) for 24 hours. at room temperature, the completion of the reaction is The solvent is removed in vacuo and the residue is treated checked with the IR spectrum of a sample (disappearance with dicyclohexylcarbodiimide (2.1 g.) in tetrahydrofuran of the strong band at 4.8pc, corresponding to the CN bond (20 ml.). After 3 hours at room temperature, the precipi of the diimide, and appearance of the carbonyl band of tated N,N'-dicyclohexylurea is removed by centrifugation the active ester at 5.65u. The by-product, mainly N,N'-di 30 and the supernatant solution, which contains the active cyclohexyl urea, is removed by filtration and washed with lactane having the structural formula tetrahydrofuran (20 ml.) The filtrate and washings are concentrated in vacuo to dryness, the residue is dissolved Cs CONE in ether, and the solution is diluted with hexane. The ac tive ester is obtained in good yield in the form of crystals 35 with strong double refraction. The product has a poorly defined melting point. The lactone obtained above and glycine ethyl ester are reacted in chloroform. Dilute hidrochloric acid is added to the reaction mixture and L-leucyl glycine ethyl ester is 40 Ö-bo obtained as the product. is used for the synthesis of glutaminyl derivatives. Example 2-2-hydroxy-5-chlorobenzylidene-L-alanine Example 6 lactone To a solution of the Schiff base from 2,4-pentadione 5-chlorosalicylaldehyde (0.157 g.) and L-alanine (0.91 45 g.) are added to dimethylformamide (10 ml.). After 24 and methionine having the formula hours at room temperature. dicyclohexylcarbodiimide C-CeN (0.206 g.) is added to the solution. The formation of a CO band at 5.65p, reaches its maximum in about 2 hours. &H YoH-CH=CH-S-CH, The precipitated dicyclohexylurea is removed by centrifu 50 c6on gation and the active lactone which has the structural CEs-C-OE formula (23.2 g.) in dimethylformamide (200 ml.), dicyclohexyl Cl -C=N-C-CE carbodiimide (21 g.) is added. After two hours at room 55 temperature, the precipitated N,N'-dicyclohexylurea is fil O-CO tered off and the filtrate which contains the active lac tone (IR 5.56p.) having the formula is precipitated by the addition of ether (50 ml.) to the decanted Supernatant solution (IR 5.65p). C-CN Example 3 60 CH-CH-CH-S-CH3 L-phenylalanine (0.165 g.) and 3-formyl-N-hydroxy phthalimide (0.19 g.) are suspended in methanol and the mixture is stirred until complete solution occurs. The is used in peptide synthesis. methanol is removed in vacuo, tetrahydrofuran (3 ml.) 65 and ethoxyacetylene (3 ml.) are added to the residue and Examples 7-26 the resulting solution is heated to boiling under reflux condenser for one hour. The solvent and the excess Following the procedure of Example 3, but protecting ethoxy-acetylene are removed in vacuo, tetrahydrofuran reactive side chains where necessary and substituting for (3 ml.) and ethoxyacetylene (3 ml.) are added to the 70 L-phenylalanine the amino acid indicated in column I, residue and the resulting solution is heated to boiling un and for 3-formyl- N - hydroxyphthalimide the carbonyl der a reflux condenser for one hour. The solvent and the compound indicated in column II, there is obtained the excess ethoxy-acetylene are removed in vacuo and the compound (or mixture of compounds) of Formula I residue is triturated with ether to yield the active acetone wherein the substituents R, R, R2, R3 and n are as in (IR5.6p.) having the formula 75 dicated in column III: 3,704,246

I I III Example Amino acid Carbonyl compound R R1 R2 R3 m 7------L-glycine.------Malondialdehyde------H -H -H -H o 8.------L-alanine------3-ketobutyral.------{3E EH, SH 8 9------L-serine------CH2OH88: -H-CH3 -HH. -CHC." O 10------L-glycine------Succindialdehyde------H -H Sh 11------do------4-ketopentanal.------in, SH 12------L-cysteine------Glutardialdehyde------CH2SH -H -H -H 2 - H-CH, -H -H -CHCH 18------L-threonine----- 4-ketohexanal.------ÖH Same as above.------CH, CH, -E -H 14------L-glycine.------Adipaldehyde------H -H -H -H 3 CH3 -CH2CH3 - -CH O -off 15.------L-valine------2,4-hexanedione.------N Chs Same as above.------CH -H -CHCH O 16------L-isoleucine.----- 2,6-hexanedione.------H-CH-CH, -CH3 -E -C 1. CH 17------L-aspartic------2-methyl-3-ketopentanal.------{3:38 EH, C SE HCH, 18------L-glycine.------2-methyl-4-ketopentanal.-- {{ 6H, 3E 19------do------3-methylpentanedial------H -E -H -E 2 20 L-alanine------Octanedial------CH3 - -E -H s 21------do------Pentanedial------CH3 -H -H -H 7 22------L-glycine------2-methyl-4-ketohexa-3-ene-6-a- - -H -E C3 O =C- cé CE 23------L-alanine------2,4-diketohexanol.------CH3 -CHCH - -CHOE 0. 24------do------3-methyl-2,4-diketopentanol----- 8. SoH. g 8. OE 8 25------do------2,4-diketo-6-hydroxyheptanol-...-- SE gigHOH-C-H. SEEhoh-oh, 9 26.------do------lethoxy-2,4-pentanedione.------3E SE-oCH-CH, i -SHOCH-CH, 8

Examples 27-37 What is claimed is: Following the procedure of Example 1, but protecting 1. A compound of the formula the reactive side chains where necessary and substituting R1 for L-leucine the amino acid indicated in column I and - sch)--N for 5-nitrosalicylaldehyde the carbonyl compound indi R2-C CH-R cated in column II, there is obtained the compound of the following formula R-b-o-c6 wherein R is the residue of an a-amino acid, R is hy drogen, a straight or branched chain lower alkyl radi cal, a hydroxy-substituted or alkoxy-substituted lower al 5 5 kyl radical, R and R3 are hydrogen, a straight or 4 O-C-CHR branched chain lower alkyl radical, or a hydroxy-sub 3 stituted or alkoxy-substituted lower alkyl radical, each of said radicals being straight or branched chain of from wherein the substituent R is the residue of the amino acid 1 to 4 carbon atoms; or R2 and R3 taken together with indicated in column I and wherein the substituent in the 60 the carbon atoms to which they are joined complete a 3-, 4- or 5-position is the same as that of the starting phenyl ring which may be unsubstituted or substituted by salicylaldehyde, and n has the value indicated in col halogen or nitro, provided that when R and R3 com umn III. plete a phenyl ring, R is hydrogen; m is an integer from 0 to 7 when R and R3 are not phenyl or an integer from I I 65 0 to 3 when R2 and R3 are phenyl. Example: 2. A compound according to claim 1 having the for 27------L-lysine------Salicylaldehyde------0. 28------hydroxylysine---- 3-chlorosalicylaldehyde-- 0 mula 29--- - L-cysteine---. --- 5-chlorosalicylaldehyde-- 0. 30--- - 3-bromoSalicylaldehyde------0 H 31--- - 4-bromosalicylaldehyde-- - 0 32--- - 5-bromosalicylaldehyde-...---- 0 ?y-(CH) --N 33------L-histidine.------5-iodosalicylaldehyde.------0 70 34------L-.-- 3-nitrosalicylaldehyde. --- 0 RO 35------I glycine.------5-nitrosalicylaldehyde------0 36------I-alanine- o-yoxyphenylacetalde- 1. ycie. S-o--but 37------do------o-Hydroxyphenylpropion- 2 wherein R is as defined in claim 1, m is an integer from aldehyde. 0 to 3 and wherein R is hydrogen, halogen or nitro. 3,704,246 3. A compound according to claim 2 having the name 2-hydroxy-5-nitrobenzylidene-L-leucine lactone. 4. A compound according to claim 2 having the name 2-hydroxy-5-chlorobenzylidene-L-alanine lactone. 5. A compound according to claim 2 wherein R is lower alkyl. 6. A compound according to claim 2 wherein R is lower alkyl and R4 is nitro. 7. A compound according to claim 2 wherein R is lower alkyl and R4 is halogen. O 8. A compound according to claim having the for mula wherein R, R2, R8 and m are as defined in claim 1, in CEs-C=N an inert organic solvent at a temperature in the range of about 15 to 60° C. and then adding a carbodiimide or &H YoH-CHCH-S-CH, 5 alkoxyacetylene condensing agent under anhydrous condi tions at room temperature up to about 60° C. to the CH-5-64o Schiff's base to form a lactone. 9. A method for forming a compound of claim 1 com No references cited. prising reacting an a-amino acid of the formula 20 N NORMA S. MILESTONE, Primary Examiner R-E-COH U.S. C. X.R. wherein R is the residue of the amino acid with an alde 260-112.5, 309, 326.14 T, 326.85,519, 534 R, 534 M, hyde or ketone having the formula 25 534 S, 534 C, 534 E, 534 G, 534 L s KXC PO-050 UNITED STATES PATENT OFFICE . (5/69) (5/69 CERTIFICATE OF CORRECTION Patent No. 3, 704, 246 Dated November 28, 1972 Inventor(s). Miklos Bodanszky It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below: - - Column l, line 21, "ester removali" should read --ester and removal--. Column l, line 47, "actylate" should read -acylate--. Column 2, line 34, "diaminocarboxylic" should read --diaminodicarboxylic--. w Column 5, line 31, "cyclohexyl urea" should read --cyclohexylurea--. Column 6, line 31, "lactine should read --lactone--. Example 25, column II R.", "-CH 2 -CHOH-C-H." should read a 40 -CH-CHOH-CH-. - Signed and sealed this 29th day of May 1973.

(SEAL) Attest : EDWARD M.FLETCHER, JR. ROBERT GOTTSCHALK Attesting Officer 8 Commissioner of Patents