Dual metalloprotease inhibitors. I. constrained peptidomimetics of mercaptoacyl dipeptides

Abstract: A series of benzo-fused Iactams were incorporated as conformationally restricted dipeptide mimetks of Ala-Pro in dual-acting ACE#NEP inhibitors I and 2. The result sf this modification bd to compounds possessing excellent ~~bi~~ patency versus ACE; and NEP both i+ V&Dand in viva. Angiatensin convetig

enzyme (ACE) is a dipeptidyl carboxypeptidase that catalyzes the hydrolysis of

the decapeptide angiotensin Z (AI) to the octapeptide angiotensin El (AU). AHI is a potent vasoconstri~~ which also tiggers the release of aldoste~~ne, a sodium-retaining steruid. T’bus ACE raises blood pressure by increasing both vascuk

resistance and fluid volume.

Consequently

ACE inhibitsrs

have gained wide

acceptance in the treatment of hypertension and congestive heart failure @ITElF). * Neutral endopeptidase WP), a membrane bound zinc metallaprotease present in high concentration irr be brush border of the renal prckmd tubule, is responsible in huge part fur the d~~~a~~~

of a&id natriuretk peptidc (ANP). ANP is a 28 aminu

acid peptide hormone secreti by the heart in response to atria1 distention and causes natiuresis, vasudilatation, and suppression of renin and aldosterone secretion .2 In pkents with CHF, ANP levels are elevated but its beneficial effecti a~ blunted via rapid degradation by NEP, As a rest& much effort has been directed toward developing NEP ~~~~~rs as new clinical agents for ~~~~si~~ Sirwe vascular resistance

and aldosterune

and CHF+3

secretion are affected oppositely by AII and AMP,

simultaneous inhibition of AII formation and ANP degradation should be synergistic. premise, ph~~~logi~~

studies involving the ~~*~rn~~s~ti~n

In support of this

of sekctive ACE and NEP inhibitors have

shown a beneficial synergistic effect over that of either an ACE QI NEP inhibitor alune in models of both h~~~~~4

and heart faWe.~

Recently a number of groups have reported their efforts in the deveIoprnent of dual-acting ACWNEP inhibitors.6

Our interest in this field arose with the discoveq

m~apt~pr~p~uy~

of mercaptaacyl dipeptides 1 and 2.7 The

dipeptide 2 is a potent inhib&r of both enzymes in vi@~, Hovirever, 2 displays weak

activity versus ACE in vies in the AI-induced presso~ response assay in the ~~~~~ns~ve was found to be short aetig (tlfi = 10 mirmtes @ 5 w&kg, de~ns~a~

a s~~~ti~

rat and in addition

iv>. In contrast the mercaptuacetyl dipeptide I

increase in both potency and duration @ID = 45 minutes @ 0.5 ~rn~~~~ iv) with versus ACE. Unfortunately I is a pour inhibitor of NEP E’n

respect to 2 despite diminished in V&IBativity V&U.

Herein we describe the utilization of canformationaUly restricted dipeptide surrogates as replacements for the alanyl-proline portion of 1 and 2, leading to compounds of w

3, Our goal was to enhance the in vitro

activity of the memaptoacetyl containing inhibitors against both ACE and especially NEP while retaining their

J. A. ROBL et al.

1790

potency in viva

We were also curious to see if this type of modification would confer improved in vivo

activity to the mercaptopropionyl class of inhibitors.

During this program we have incorporated both known

and novel dipeptide surrogates into 1 and 2 by drawing extensively from the SAR generated during the last ten years on conformationally

constrained analogs of the ACE inhibitor enalapril.*

The work described herein

focuses on the generation of mercaptoacyl benzo-fused azepinones 3 as constrained analogs of 1 and 2. The effect of this modification on in vitro and in vivo potency was determined.

In a recent and independent

discovery, others have also reported utilizing the mercaptoacetyl pharmacophore conformationally

in the generation of a

restricted peptidomimetic inhibitor of both ACE and NEP.& A biological comparison of

their inhibitor (MDL 100,173) is made with compounds of type 3.

Figure 1

> ACE1,=3OnM

ACE 1,=4nM

NEP150-400nM

NEPIJo=6.6nM

whemx=cH.2,o,s RofR=Ma;

The synthesis of benzazepinones@e (X = CHd, benzthiazepinonestivgj

n=O,l;m=l.2

(X = S), and

benzoxazepinones@vi (X = 0) related to compound 4 (where R = R’ = H and m = l), has The generation of $-methyl substituted been described in the literature. benzoxazepinones

9a and 9b essentially followed the procedure utilized for the des-

methyl analog and is depicted in Scheme 1, Reaction of N-Boc-allu-L-threonine N-Boc-L-threonine

+&N

NT-l)m

% 0

@a) or

W#

4

(5b) with 1-fluoro-2-nitrobenzene

products 6a and 6 b respectively.

and NaH afforded the SNAr Reduction of the nitro group followed by water soluble carbodiimide

mediated cyclization gave benzoxazepinones followed

W: x ,-/ A Q

by removal

7a,b in good yields.

of the Boc protecting

group afforded

Subsequent lactam nitrogen alkylation the desired

C-4 methyl

substituted

benzoxazepinones 9a and 9b quantitatively.

1. HP, w/C 40 psi, solvwlt c 2. EDAC, DMF, rt

NaH, dvent I (78%), b (90%)

H

a (71%),

H

I4

b(87%)

60

0 7&b

SaR=Me,R’=H SbR=H,R’=Me

NaH, THF HCI, dioxane

ercycc$a @ (97%),

t

b(88%)

0 Wb

CO@

Qa,b

A survey of the SAR of benzazepinone based ACE inhibitors related to enalapril suggests that potent inhibitory activity may be obtained with both acetic (m = 1) and propionic (m = 2) acid side chain analogs.&

Dual metalloprotease inhibitor-1

1791

Since it has been observed that B-amino acid containing NBP inhibitors are often more potent than those possessing an CZ-amino acid residue at the carboxy terminus, utilization of a propionic acid side chain may boost the intrinsic activity versus NBP. Consequently this type of modification was applied to the benzo-fused azepinones used in this study.

Equations l-3 outline the synthesis of benzo-fused azepinone dipeptide

mimetics possessing a propionic acid side chain.

Lactam N-alkylation under conditions similar to that described earlier (NaH, I(Br)C!I-I2CH2CO2R)gave the desired adducts in poor yield. It was subsequently found that treatment of the lactams with ethyl acrylate and t-BuOK in TI-IWt-BuOH afforded the desired alkylation products in much higher yields.

The protected intermediates were then converted to their corresponding

amines and used directly in the subsequent coupling reactions.

9\ /

$0 Y

Boa.

N

+r

t-B&K 4MHCl dbxane,

pCO*Et

rt

NH

H

0

THF,t-BuOH a (89%).

(2:l)

91%

b&Q%)

7aR=Ma,R’=H lQbR=R’-H

9 4r s \I

--N

NH

I+ ia

t-BuOK 3U%HBr HOAC. rt

FCqEt c THF, t-BuOH (2:l)

cbz.

0

57% (2 steps)

HBr.HZN

t-BuOK @

I+. FWC

CoIEt

THF, t&OH

EtOH (211)

(3)

100%

75%

Coupling of the requisite amines 4, or their hydrohalide benzenepropanoic yields.

acid9 19 or (S)-3-(acetylthio)-2benzylpropionic

salts, with either (S)a-(acetylthio)-2acidlo 20 afforded 21 generally in good

The tendency of acid 19 to partially racemize upon coupling made the choice of coupling reagent

critical. BOP reagent”

effected the desired amide formation with minimal (4%) racemization whereas the

use of other coupling agents (EDAC, HOBT, HOBT-mesylate; cyanuric fluoride; BOP-Cl) led to substantial amounts (5-15%) of racemization product. Saponification of 21 in the strict absence of oxygen, followed by acidification, afforded the desired inhibitors 3. &hem0 2

1792

3. A. ROBL et at.

Compounds 3 were tested for their ability to inhibit both ACE and NEP in vitro.12 ACE activity in vivo was determined from plots of percent maximal inhibition of the AI induced pressor response versus dose after intravenous (iv) administration in the normotensive rat. 13 Compounds which displayed acceptable activity in the AI pressor assay and possessed reasonable potency versus NFP in vitro were tested in the 1K-DOCA salt hypertensive rat assay.14 Selective NEP inhibitors have been shown to lower mean arterial pressure in this assay whereas ACE inhibitors are usually ineffective. Table 1 lists the in vitro data and ACE EDso values for dual-acting inhibitors 3, as well as the dual-acting ACE/NEP inhibitor MDL 100,173.~

Table 1 Compound

X

1 2 3a 3b :

CH, CHP s SO

30 31 !z

n

m

R

R

0 1

i 1 1

H Ii HH

H H

0 I: OIIHH 0 0

1

!

!

1

Me H

Me H

NEP

ACE

(b nM)

(150nM)

400 6.6 6.1 2.1 641.0

30 4 12 4.8 13

3.1 0.6 118 5.2

;

1

4.4 36 13

ik:

Cp CH2

0 1

2

H

H

;l; 2.4

;36

31 3m

::

0 0

2 2

H H MsH

1.2 83

31 18

9

32

UM lQQ,173*

Al Pressor *EDSo cmovke, iv)

0.13

cnai lasted;‘ble&~rre vdws”NEP Ki - 0.08 nM,ACEKi - 0 11 nM

Conformationally restricted mercaptopropanoyl (n = 1) inhibitors 3b, 3d, and 3f showed a 3-l 1 fold gain in potency versus NEP in vitro without significantly effecting activity against ACE as compared to their Ala-Pro analog 2 . These compounds displayed exceptional

activity versus both enzymes, especially

compound 3f which is one of the most potent dual-acting inhibitors discovered in our program. Unfortunately, despite their low nanomolar activity versus ACE, all of the m~apto~opanoyls activity in vim in the AI pressor assay. The results indicate that ~nzo-~s~ added in vivu potency to the class of mercaptop~panoyl

inhibitors.

were found to exhibit poor lactam mimetics do not confer

Utiiization of the confor~tionally

restricted dipeptide surrogates for Ala-Pro in the mercaptoacetyl class of inhibitors (n = 0) led to dramatic enhancements in in vitro potency versus NEP (compare 1 with 3a, 3c, 3e, and 3h). For example, compound 3a is = 60-fold more potent versus NEP in vitro as compared to 1. In addition, potency versus ACE was enhanced in vitro. As was seen in the comparison of 1 and 2, a large difference in the ACE in vivo activity of the

mercaptoacetyl and mercaptopropanoyl class of compounds was observed. Despite similar in vitro potencies versus ACE, the mercaptoacetyl inhibitors were generally 25fold more potent as ACE inhibitors in vivo as compared to their related m~aptoprop~oyl induction

counterparts (compare 3a with 3b, 3c with 3d, and 3e with 30.

of a methyl substituent to 3e to give 3g boosted ACE activity 2-fold in vitro and 17-fold in

vivo. Unfortunately a large decrease in in vitro activity versus NFP was also observed. The isomeric methyl

Dual me~opro~

inhibito-I

1793

compound 3h was essentially equipotent to the dcs-methyl analog 3e in vitro, but was IO-fold less potent in vim In general, replacement of the acetic acid side chain in compounds 3a-g with a ~opio~c

acid group ied

to modest increases in inhibitory potency versus NIZP without significantly affecting activity against ACE in vitru. The most dramatic example is observed upon the comparison of 3e (NEP 1% = 64 nM) with its propionic this modification invariably diminished the in vivu

acid counterpart 3k (NEP Ijo = 2.2 nM). Unf~n~ly

performanceof the propionic acid analogs as compared to their acetic acid counterparts. A select number of compounds were evaluated for NEP inhibition in viva in the IK-DOCA salt rat assay, a low-rcnin model of hypertension,

Compounds were given as bolus injections at 100 umohkg iv and

mean arterial pressure (MAP) was measured versus time over a 24 hour period. The data in Graph 1 represents the compiled area-over-the-curve (AOC) during this time period. As expected, the ACE inhibitor captopril was ineffective in this assay. Selective NEP ~ibitors

SQ 28,60315 and ~doxa~atl$

displayed modest activity

as did inhibitors 3e and 3e, In contrast to the results in the AI pressor assay, good activity in the IK-DOCA rat assay was not limited exclusively to the class of mercaptoacetyl inhibitors (compare 3a, 3b, and 3k). It is evident though that these ~nfo~tion~y

restricted inhibitors d~ons~a~ activity that is comparable to or better than that of the established single acting NEP inhibitors. Since the antihypertensive activity of selective

NEP inhibitors are not potentiated by the addition of ACE inhibitors in this assay,4athe ability of compounds 3 to lower MAP is attributed to their intrinsic ability to singularly inhibit NFJPin vivo.

e

'@.o5vs3a

-loo,

.,

., 0

60

., 120

. 130

FosiBapril

. 240

.,

. 300

'( 360

Time (s&n)

Based on both in vitro potency and in vivo performancein the Al pressor assay and the lKDOCA rat assay, compound 3a was selected as the most promising lead in this series. A comparison was made between 3a and other ~e~~utica~y i~ibi~r

proven ACE inhibitors (captopril and foamy)

in addition to dual-acting

MDL 100,173 and its S-acetyl pro-drug MIN., 100,24#I with respect to oral activity in the AI pressor

assay. The data in Graph 2 shows that while compound 3a is not as efficacious as fosinopril (once-a-day ACE inhibitor) at a comparative dose of 5 umol/kg, its potency and duration of action equals that of captopril. The MDL com~unds

were less effective than 3a in this assay.

1794

J. A. ROBL et al.

In conclusion, a series of benzo-fused lactams were utilized as conformationally restricted surrogates for Ala-pro in an effort to generate mercaptoacyl containing inhibitors of both ACE and NEP. Compound 3s was demonstrated to be a good inhibitor of both me~lopro~s

in vitro

and in vivu and served as an excellent

lead compound in our pursuit of a clinical candidate in this program. Studies focusing on the SAR of 3a @MS 182,657) and the utilization of the mercaptoacetyl pharmacophore in conjunction with other conformationally restricted dipeptide surrogates are described in the following ~o~unication. Acknowledgment: We are grateful for the technical assistance provided by Maxine Fox, Mary Giancarli, Balkrushna Panchal, and Hong Sun Cheung. Referencessod Nntes: Chatter@& K. Heart Qis. Stroke W92,1,128. :: (a) Schwartz, J. C.; Gros, C.; Lecomte, J. M.; Bralet, J. Life Sci. 1990,47,1279. (b) Brenner, B. M.; BaUerman, B. J.; Gunning, M. E.: ZeideI, M. L. Physiol. Rev. 199@,70,665. (c) ~~~u~, M. C. C. R. Sot. &at. 1992,186,612. (d) Roques, B. P.; Nobfe, F.; Dauge, V.; Fournie-Zaluski, M-C.; Beaumont, A. Pharm. Rev. 1993,45,87. (e)Wi~, M. R.; Unwin, R J.; Kenny, A. J. Kidrtey Internat. 1993,43,273. (f) Welchea, W. R.: Brosnihan, K. B.; Ferrario, C. M. tife Sci. 1993,52,1461 3. Recent work incbrdes: (a) Chxkalamannil, S.; Wang, Y.; Haslanger, M. F. Bioorg. Med. Chem. ht. 1992,2,1003. (b) Fournie-Wuski, M. C.; Chic, P.; Turcaud, S.; Lucas, E.: Noble, F.; Maldonado, R.; Roques, B. P. J. Med. Chem 1992,35, 2473. (c) Miiura, T.; Nakamum, Y.; Nishino, J.; Sawayama, T.; Komiya,T. Deguchi, T.; Kita, A.; Nakamura, I-I.; tumor J. J. Med. Cftem. 1992,35,602. (d) aeon, L, J.; Bayburt, E. K.; Capparelli, M. P.; Bohacek, R. S.; Clarke, F. H.; Ghai, R. D.: Satiane,Y.; Berry, C. J.: Peppard, J. V.; Trapani, A. I. f. Med. Chem. 1993.363821. (e) Neustadt, B, R. Cur? Opin. Invest. Drags 1993,2, 1175. (f) Gomez-Montetrey, I.: Turcaud, S.: Lucas, E.; Bruetschy, L.; Roquea, B. P.; Four&-ZaIuski, M. C. J. Med. Chem. 1993,36,87. 4. (a) Seymour, A. A.; Swerdel, J. N.; Abboa-Offei, B. J. Cardiovusc. Pharm. 1991,17,456. (b) K&an, C.; Ghai, R. D.; Lappe, R W.; Webb, R. L. FASEB J 1993.7, A247. (c) Pham, I.; Gonzdiez, W.; El Amraui, A.-I. K.: Fouruie-Zaluski, M.-C.; Philippe, M.; Laboukmdine, I.; Roques, B. P.; Michel, J.-B. J. Phartnacol. Exp. Ther. 1993,265,1339. 5. (a) TrIpnodo, N. C.; Fox, M. : Natamjan, V.: PanchaI, B. C.: Dorso, C. R.; Asaad, M. M. J. Pharmacot. Exp. Tirer. 1993,267, 108. (~jSe~~, A. A.: Assad, M. hf.; Lanuce, V. M; ~gen~her, K. w Fennell, S. A.; Rogers, W. L. J. Phar~coZ. J&D. Ther 1993.266.872. 6. CajGros, c.; No&N:; Souque, A.: Schwartz, J.-C.; Danvy, D.; Ptaquevent, J.-C.; Duhamel, L.; l&thame~, P.; ~e~~mte, J.-M.; Bralet, J. I%C Nati Acad. Sci. USA 1991.88,4210. (b) Roqnea, B. P.; Biochem. Sot. Trutts. 1993,21,678. (a) Flynn, G. A.; Beight, D. W.: Mehdi, S.; Koehl, J. R.; Giroux, E. L.: French.3. F.; Hake,P. W.; Dage, R. C. J. Med. C/rem. 1993,X, 2420. td) Sm@u, J. L.; Speti, D. M.; Trapani, A. J.; Cote, D.; Sakane, Y.; Berry, CJ.; Ghai, R.D. J. Med. Chem. 1993,36,3829. (e) Fo~ie-~~~, M-C.: Coric, P.: Tumaud, S.; Rousselet, N.; GonsaIes, W.; Barbe, B.; Pham, I.; J&an, N.: Michei, J-3.; Roques, B. P. J. hfed Chem. 1994.37.1070. 7. Delaney, N.G.: Banish, J.C.; Neubeck, R.: Natarajan, S.1.: Rovnyak, G.C.; Huber, G.; Murugesan, N.; Giroua, R; Sieber~ McMaster, E.; Robl. J.A.; Asad, M.; Cheung. H.S.; Bird, E.; W&Iron, T.; Pettillo, E.W. preceding paper in this issue. 8. (a) wywatt, M. J.: Tischler, M. H.; lkeler, T. J.; Springer. J. P.; Tristram, E. W.; Patchett, A. A. Pept Struct. Funct., Proc. Am Peut. Svm., Rth, 1993.1X93.551. (b) Thorsett. E. D.: Harris. E. E.: Aster. S. D.: Peterson. E. R.: Tristram. E. W.: Snvder. J. P.; SprinI+~ J. P.; Pntchett, A. A. Pep,.: Sttuct. Funct,. Proc. Am Pept. Symp., 8th: 1993,1983, SS$. (c) Parson, W.‘K.;* Davidson, J, L.: Taob, D.; Aster, S. D.; Thorsett, E. D.: Patcbett, A. A. Biochem. Biophys. Res. Comm. 1983, II?, 108. (d) SIade, J.: Stanton, J. L.; Bert-David. D.: Mazzenaa G. C. J. Med Cheat. 19#.28,1517. 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R.; Evin, G.; Selve, C. Tetrahedron Lea. 1975,1219. 12. The in vitro assays for inhibition of ACE and NEP are described in reference 7. 13. Rubin, B.; Laffim, R.J.; Kotter, D.G.; OXeefe, E.H.; DeMaio, D.A.; Goldberg, ME. J. Pharmacol. Exp. Ther. 1978,2@#,27l. 14. lb lK-DOCA rat assay followed that described in Syberta, E.J.; Chiu, PJS.; V~u~, S.; Pitts, B.; Foster, CJ.; Watkbts, R.W.: Bamett, A.; Ha&urger, M.F. J. Pascal. Eap. Ther. 1989,250,624 with Me foIIowIug rn~~o~: (i) ~ng water consisted of 0.9% NaCI and 0.2% KCI; (ii) the aorta rather than the caudal artery was used to measure btood pressure: (iii) compounds were given intraveneously rather than subcutaneousIy. 15. Seymour, A. A. Cardiovas. Drug Rev. 1991.9.285. 16. N&l-triage, D. B.; Alabaster, C. T.; Connell, I. M. C.: Dilly, S. G.; Lever, A. F.; JardIne, A. G.; Barclay, P. L.; Dargie, H. J.; Fiidlay, I. W.; SamueIs, G. M. R. .&meet ii 1989,591.

(Received

ipa UsA 5 April 1994; accepted 19 May

1994)