45 Phosphonate Inhibitors of Carboxypeptidase A NEIL E. JACOBSEN and PAUL A. BARTLETT
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Department of Chemistry, University of California, Berkeley,CA94720
Stable compounds which resemble the transition-state structure of a substrate in an enzymatic reaction are expected to behave as potent reversible inhibitors (1). Based on the X-ray crystallographic structure of the active site of carboxypeptidase A (CPA) (2), a mechanism was proposed in which a water molecule adds directly to the scissile carbonyl group of the substrate to give the tetrahedral intermediate 1, which collapses to products (3). We proposed to mimic this tetrahedral intermediate, similar to the transition state, with the stable tetrahedral phosphonic acid derivatives 2, 3, 4, and 5.
N-[[[(Βenzyloxycarbonyl)amino]methyl]hydroxyphosphinyl]-L -phenylalanine, dilithium salt, 2 (4), was prepared in six steps from dimethyl phthalimidomethylphosphonate (5). The pure dilithium salt,
0097-6156/81/0171-0221$05.00/ 0 © 1981 American Chemical Society Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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obtained by anion-exchange chromatography, showed potent competitive inhibition of CPA with K = 8.9 x 10 M. Inhibition by 2 is rapid and reversible and added zinc chloride does not diminish the inhibitory activity. The hydrolysis of 2 is not catalyzed by CPA. The preparation of N-[amino[[(benzyloxycarbonyl)amino]methyl]-phosphinyl]-L-phenylalanine, 3, was approached via the methyl ester 8a. The model diamides 8b, 8c, and 8d were prepared in the same manner. Treatment of 8a with NaOH gave only a small amount of 3, which decomposed during work-up and anion-exchange chromatography. -8
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i
The model diamide 8b was unchanged under the same conditions, but gave (by H- and P-NMR) a 1:1 mixture of the desired diamide 9 and the rearranged product 10. Under the reaction conditions, 9 slowly hydrolyzes to give the dianion 11. At lower pH in buffered solution, the decomposition of 9 to give 11 is accelerated, with t½ less than 0.3 min at pH 7.5. 1
31
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
45.
JACOBSEN AND BARTLETT
Phosphonate
223
Inhibitors
Thus i t a p p e a r s t h a t t h e s t a r t i n g m e t h y l e s t e r §g u n d e r g o e s b a s e - c a t a l y z e d c y c l i z a t i o n t o g i v e t h e i n t e r m e d i a t e Xg, w h i c h i s o p e n e d b y h y d r o x i d e a t t a c k a t p h o s p h o r u s t o g i v e 10. The d e s i r e d d i a m i d e |, f o r m e d e i t h e r b y d i r e c t h y d r o l y s i s o f |g o r b y o p e n i n g o f t h e i n t e r m e d i a t e Ig a t t h e c a r b o n y l c a r b o n , l o s e s ammonia w i t h a s s i s t a n c e from t h e f r e e c a r b o x y l a t e t o g i v e 11. When t h e r e a c t i o n i s c a r r i e d o u t w i t h c a t a l y t i c m e t h o x i d e i n m e t h a n o l , t h e p r e s u m e d i n t e r m e d i a t e 12 i s o p e n e d b y m e t h a n o l t o give, q u a n t i t a t i v e l y , the rearranged product 1 J . Hydrolysis o f 1 J w i t h aqueous NaOH i s e x t r e m e l y r a p i d a n d g i v e s o n l y t h e p h e n y l a l a n i n e amide d e r i v a t i v e 10. Ο CH Ph K C0 II I — - — C H o -P-NH-CH-CONH? CH3OH ^ |
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2
2
8c
OH"
3
1
H 0
~* 10
2
O C H 3
13 When t h e d i a m i d e 9 was f o r m e d d i r e c t l y b y h y d r o g e n o l y s i s o f t h e b e n z y l e s t e r 8 d , o n l y t h e s o l v o l y s i s p r o d u c t s 12 o r 24 w e r e o b s e r v e d . Thus i t a p p e a r s t h a t t h e f r e e c a r b o x y l a t e o f e i t h e r J o r g, n e c e s s a r y f o r b i n d i n g t o CPA, p a r t i c i p a t e s i n t h e s o l v o l y t i c l o s s o f ammonia a n d p r e v e n t s t h e a s s e s s m e n t o f t h e s e compounds a s i n h i b i t o r s a t n e u t r a l pH. The m o d e l p h o s p h o n a m i d o t h i o a t e 1 § , i n c o r p o r a t i n g t h e e s s e n t i a l f e a t u r e s o f t h e d e s i r e d i n h i b i t o r 4, was p r e p a r e d f r o m m e t h y l p h o s p h o n o t h i o i c d i c h l o r i d e i n two s t e p s . The two d i a s t e r e o m e r s o f t h e d i e s t e r 15 w e r e s e p a r a t e d b y h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y and i n d i v i d u a l l y d e p r o t e c t e d w i t h b a s e t o g i v e p u r e 16a a n d 16b. S
S ÇH Ph a,b II I - CH -P-NH-CH-C0 CH
0
2
Il
CH -P-C1
2
3
3
2
OCH CH CN 2
15a,b
2
c 3
H0CH CH CN, 2
2
2
3
2
S_ 16a : K = 5.3 μΜ i
K = 8.5 μΜ i
Et N ; 3
b: L - P h e - 0 C H , E t N ; 3
I
^ CH -P-NH-CH-C0
16b: a:
ÇH Ph
II
3
c : NaOH
I n i t i a l e n z y m a t i c s t u d i e s show t h a t 16a a n d 16b b i n d t o CPA some w h a t l e s s s t r o n g l y t h a n t h e c o r r e s p o n d i n g p h o s p h o n a m i d a t e 11 (K^ o f 11 = 1.2 μ Μ ) . The p h o s p h o n a t e 5 , p r e p a r e d f r o m L - B - p h e n y l l a c t i c a c i d a n d the phosphonochloridate 6g, b i n d s t o CPA e q u a l l y a s t i g h t l y a s does t h e more l a b i l e p h o s p h o n a m i d a t e 2.
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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PHOSPHORUS
CHEMISTRY
We are c u r r e n t l y pursuing the p r e p a r a t i o n and t e s t i n g o f the phosphonamidothioates 4 and the p r e p a r a t i o n o f i n h i b i t o r s analogous t o 2-5 f o r the z i n c peptidases thermolysin and collagenase. ÇH Ph
Ο
2
a
HO-CH-C0 H 2
,
b
-
ÇH Ph 2
Cbz-NH-CH -P-0-CH-C0 CH 2
2
OCH a: C H I K C 0 , 18-crown-6; _ d: NaOH, H 0 3
2
f
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Λ Τ Τ
3
ττ Λ
d
»•
5
3
b: 6c, E t N ; " 3
° '
3
c: t-BuNH , Δ (see réf. 6) ; 2
2
LITERATURE CITED 1. Wolfenden, R. Ann. Rev. Biophys. Bioeng., 1976, 5, 271. 2. Lipscomb, W.N. Tetrahedron 1974, 30, 1725. 3. Breslow, R.; Wernick, D.L. Proc. Natl. Acad. Sci. U.S. 1977, 74, 1303. 4. Jacobsen, Ν.Ε.; Bartlett, P.A. J. Am. Chem. Soc. 1981, 103, 654. 5. Seyferth, D.; Marmor, R.; Hilberg, P. J. Org. Chem. 1971, 36, 1379. 6. Gray, M.; Smith, D. Tetrahedron Letters 1980, 859. RECEIVED
June 30, 1981.
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
