Int. J . Protein Research III, 1971, 347-350 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

SYNTHESIS OF AN ACTINOMYCIN ANALOG WITH SUB STIT UT ED LACTO N E OXYGEN S

[ 1 ’, 1 ’-Bis (L-a, p-Diaminopropionic Acid)] -Actinornycin D (CI)*

JOHANNES MbrENHOFER and RAVINDRA P. PATEL

Children’s Cancer Research Foundation and Department of Biological Chemistry, Harvard Medical School,

Boston, Massachusetts 021 15, U.S.A.

Received 8 June 1971

A synthesis of a Iactam analog of actinomycin D is described in which the threonine residues have been replaced by L-x, &diaminopropionic acid. The key reaction, cyc- lization of’ the pentapeptide intermediate, was carried by a nitrophenyl ester conden- sation between the proline and sarcosine residues. [l’, 1’- Bis (L-x, P-diaminopropio- nic acid)]-actinomycin D exhibited potent antibacterial activity.

C11 NH.-- 2 -

0 CH, CH

CH, CH3 CH, \ / / \

CH 0 CHZ CHZ 0 CHB I I ! 1 ’ 1

0 CH3<> - / I

NH- kH- C-N---CH-C- N-CH,-C-N--CH-C--0

1’ 2’ 3‘ 4‘ 5‘ N / \ \Y

0

\ C-N H-CH-C-NH-CH-C- N--CH-C-N- -CH,-C- N--CH-C -- 0 CH9\-//-l, i I/ I I1 /I ‘ I .P \ o CH o 6~~ CH, o &, o ~ H , C H

CH, CH NH2

CHy CH3 CHZ

[v,/&Dpr-~-Val-Pro-Sar-MeVal] FIGURE 1 Structure of [ I ’,l’-bis(L-z,b-diaminopropionic acid)]-actinomycin D, the 1 ’,l’-bisfw-desmethy1)lactam analog of actinomycin D (4).

Actinomycins with improved therapeutic indices are needed for cancer chemotherapy (1). The im- portance of the peptide lactone moieties for acti- nomycin action (2) led us t o prepare peptide

analogs by total synthesis (3) in a search for clinically useful modifications.

This communication concerns a synthesis of [ 1 ’, 1 ’-bis(L- (1, P-diaminopropionic acid)]-actino-

Contribution V in the series “Syntheses of Actinornycin and Analogs”. Part IV by MEIENHOFER, J., SANO, Y., and PATEL, R. P., appeared in Peptides: Chemistry and Biochemistry, ed. WEINSTEIN, B., and LANDE, S. (M. Dekker, Inc., New York, N.Y., 1970), pp. 419434. * Abbreviations follow the rules of the IUPAC-IUB Commission on Biochemical Nomenclature, in Biochern- isfry, 5, 1445, 2485 (1966); 6, 362 (1967); J . Eiol. Chem., 241, 2491 (1966); Dpr, L-r,b-diaminopropionic acid.

Designation D is according to VINING, L. C., and WAKSMAN, S. A., Science, 120, 389 (1945); designation C, is according to BROCKMANN, H., and GRONE, H., Nuturwissenschuften, 41, 65 (1954).

347

JOHANNES MEIENHOFER AND RAVINDRA P. PATEL

I CI Nl14011 SOCI,-CH,OH MA. Boc-MeVal--

Z-Dpr-OMe I

Z-Ala-011 ---+ Z-Dpr-01-1 - - - - ---+ Z-Dpr-Ohle -----+

11 I11 IV V

Boc-Sar-McVal M.A. - 1 - H-_D-Val-Pro-ONp

-vIII Z-[)pr-O]I + Z-Dpr-Q-Val-Pro-ONp - 1 Roc-Sar-MeVal

NaOH 2-Dpr-OMc - -f

1 Boc-Sar-hkVal- a) TFA b) M.A. I

FIGURE 2 Synthesis of [ l , 1 '-bis(L-t(,p-dianiinopropionic acid)]-actinomycin D. - - - Dpr, L-r,/J-diaminopropionic acid ; M.A., Mixed anhydride synthesis; TFA, trifluoroacetic acid.

inycin D (I) (Figure l), an analog with substituted lactonc oxygens in which the threonine residues of actinomycin D (4) have been replaced by L- a, /j-diaminopropionic acid. The synthetic route, characterized by peptide cyclization between the proline and sarcosinc residues (3), is shown in Figure 2.

The required Na-protected L- (1, b-diaminopro- pionic acid intermediate was prepared from L- serine methyl ester hydrochloride (5 ) through the intermediate L- u-amino- $-chloropropionic acid methyl ester hydrochloride. Conversion to the acid (using 8 N HCl) and carbobenzoxylation gave the known L-ci-benzyloxycarbonylamiiio- @-chloropropionic acid [benzyloxycarbonyl-L-8- chloroalanine] (I[), (7). Treatmcnt with concen- trated ammonia for 3 days at 40' gave Nz-ben- zyloxycarbonyl-L- 0, ,&diaminopropionic acid (111) 30%, colorless needles, mp. 243-245", [ ( I ] ; !

-14.2' (c 1, glacial acetic acid). Esterification with methanol-thionyl chloride (8) afforded the crystalline Na-benzyloxycarbonyl-L- a, 3-diamino- propionic acid methyl ester hydrochloride (IV) 87%, mp. 165-167", [u]: 42.5"(c 1,methanol). Anal. calcd. for C,,H,,N,O,CI (288.7): C, 50.0; H, 5.90; N, 9.72; CI, 12.3. Found: C, 49.8; H, 6.12; N, 9.54; CI, 12.6.

Treatment of IV with the mixed anhydride (9) from tert-butyloxycarbonyl-L-N-methylvalinc* and isobutyl chloroforinate gave N"-bcnzyloxy- carbonyl-ND-(tert-butyloxycarbonyl-L- N -methyl- \alyl)-L- (1, P-diaminopropionic acid methyl ester (V) as an oil which was purified by column chro- matography on Sephadex LH-20 in ethanol, 86%, [a]: 70.8" (c 1, methanol). Anal. calcd. for C,,H,,N,O, (465.4): C, 59.3; H, 7.52; N, 9.03. Found: C, 59.0; H, 7.40; N, 8.82. Removal of the tert-butyloxycarbonyl protecting group from V by treatment with trifluoroacetic acid (12) and condensation of the product with tert-butyloxy- carbonylsarcosine (1 1) through the mixed anhy- dride method afforded Na-benzyloxycarbonyl- NB-(tert-butyloxycarbony lsarcosyl-L-N-methyha- lyl)-L-u, 8-diaminopropionic acid methyl ester (VI) as an oil which was purified as dewibed for V, 84%, [u]: - 83.0" (c 1, methanol). Anal. calcd. for C,,H,,N,O, (536.5): C, 58.2; H, 7.46; N, 10.4. Found: C, 58.2; H, 7.13; N, 10.2. Sapo- nification of V1, dissolved in acetone, with 1 N

* Prepared from tert-butyloxycarbonyl azide (10) with pH-stat controlled (pH 10.5) addition of 4 N LiOH, according to Schnabel (11): oil, 50-60'%,; dicyclo; hexylammonium salt: mp. 107-1 lo", [x]5 ' -54.1 (c 1, methanol).

~ . .-

348

ACTlNOMYCIN ANALOG

NaOH gave the corresponding acid (VII) as a colorless powder, 97%, [a]: -78.5" (c 1, me- thanol). Anal. calcd. for C,,H,,N,O, (522.6) : C, 57.6; H, 7.33: N, 10.7. Found: C, 57.7; H, 7.54; N, 10.7.

The required intermediate p-nitrophenyl D- valyl-L-prolinate hydrobromide (VIII) [colorless needles, mp. 145", [a]: -51.0" (c 0.25, diniethyl- formarnide). Anal. calcd. for C,,H,,N,O,Br (416.3): C, 46.2; H, 5.33; N, 10.1. Found: C, 46.5; H, 5.35; N, 9.851 was prepared from benzyloxy- carbonyl-D-valyl-L-proline (3) through treatment with di-p-nitrophenyl sulfite in pyridine (13) to give the p-nitrophenyl ester derivative followed by the removal of the N-protecting group with HBr in acetic acid (14). Condensation of the mixed anhydride obtained from VII and isobutyl chloroformate with VIII afforded Na-benzyloxy- carbonyl-Nj-(tert-butyloxycarbonylsarcosyl-L-N - methylvalyl) -L- u, $-diaminopropionyl-D-valyl-L- proline p-nitrophenyl ester (IX) which was puri- fied by column chromatography on Sephadex LH-20 in ethyl acetate to give a pale yellow oil, 87%, [a]: - -63.7' (c 1, methanol). Anal. calcd. for C4,H,,N,0,,(839.9): C, 58.6; H, 6.84; N, 11.7. Found: C, 58.2; H, 7.12; N, 11.5. The N-protect- ing tert-butyloxycarbonyl group was removed from IX by treatment with anhydrous trifluoro- acetic acid (12). The product was converted into the hydrochloride and then dissolved in a small volume of dimethylformamide-acetic acid (9 : 1). The cyclization was carried out at high dilution (c about 0.05) in pyridine in the presence of N- methylmorpholine for 6 hours at 60" (15). The solvent was replaced with ethyl acetate and the solution was washed successively with 1 M NaHCO,, 1 N HC1, and water and dried (MgS04). Evaporation of the solvent gave an off-white powder which was purified by twice repeated column chromatography on Sephadex LH-20 in ethanol. Evaporation of the fractions from the first eluting material afforded Na-benzyloxycar- bonyl-L- a , /3-diaminopropionyl-D-valyl-L-prolyl- sarcosyl-L-N-methylvaline (N P-diaminopropionic acid)lactam (X) as a colorless powder, 3574, [a]'$ --60.0' (c 1, methanol). Anal. calcd. for C,H,,N,O, (600.7): C, 60.0; H, 7.38; N, 14.0. Found: C, 60.1; H, 7.62; N, 13.6. Removal of the benzyloxycarbonyl group by catalytic hydro- genation (16) followed by condensation of the product with 2-nitro-3-benzyloxy-4-methylben- zoyl chloride (17) gave, after work-up and puri- fication as described for X, NOL-(2-nitro-3-benzyl-

oxy-4-1iiethylbenzoyl)-r-a, B-diaminopropionyl-D- valyl-L-prolyl-sarcosyl-L-N-methylvaline (N P-di- aminopropionic acid) lactam (XI) 69 %, which was crystallized from benzene-hexane as colorless needles, mp. 155-160", [u]: -12.6" (c 0.5, me- thanol). Anal. calcd. for C,,H,,N,O, (735.8) : C, 60.4; H, 6.71; N, 13.3. Found: C, 60.7; H, 6.95; N, 13.5. Catalytic hydrogenation of XI in the presence of palladium black followed by oxidative formation of the phenoxazinone moiety with the use of potassium ferricyanide (18) in a mixture (1 : 1) of methanol and MI15 phosphate buffer at pH 7.1, as described for actinomycin D (3), gave [ 1 ',1 '-bis(L- a, $-diaminopropionic acid)]- actinomycin D, I , as an orange-red powder, nip. 238-242", UV max (methanol) 240 m p and 442 mp. Microbiological assays (19), using Lacto- bacillus arabinosus (ATCC 8014) and L. ferrnenti (ATCC 9388) in pantothenate- and thiamine- dependent systems respectively, showed that I possessed potent antibacterial activities (ID5o, 0.2-0.6 ,ug/ml). Preliminary toxicity and anti- tumor tests in AKD,-F, mice bearing Ridgway osteogenic sarcoma (20) indicated that I is ap- proximately half as active as actinomycin D.

ACKNOWLEDGMENTS

We wish to thank Dr. G. E. Foley and Miss B. L. Brown for the in vitro and in vivo biological assay data, respectively; Dr. S . Sengupta for a gift of 2- nitro-m-cresotic acid; and Mrs. E. Judkins for tech- nical help. This work was supported in part by Public Health Service research grants C-6516 from the Na- tional Cancer Institute and FR-05526 from the Divi- sion of Research Facilities and Resources, National Institutes of Health.

1. 2.

3.

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5 .

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