J. Med. Chem.
`
`1985,28,57-66
`
`57
`
`mM substrate in a total volume of 2.5 mL. At various times up
`to 30 min, aliquots (0.3 mL) were removed and quenched with
`MeOH (4.7 mL). After centrifugation (5000g, 10 min), the samples
`were diluted (1:l) with 0.01 M KH2P04 and 20 pL of the resulting
`solution was injected directly onto the HPLC column for analysis.
`and V,, Determinations. The KM(app)-Vmax for the
`K M ( ~ ~ ~ )
`hydrolysis of p-NP-glc and p-NP-gal were determined with use
`of pooled cecal homogenates (200 mL) as described above. A range
`of substrate concentrations (56-1000 pM, final volume 2.25 mL),
`spanning their apparent K M , was used for each reaction. The
`amount of cecal homogenate used was 0.04 mL. Reaction mixtures
`were incubated, in duplicate at 37 "C in a shaking water bath,
`and the reaction was stopped by addition of 0.2 N NaOH (0.25
`mL) after 15 min. Release of p-nitrophenol was measured
`spectrophotometrically at 403 nm. Eadie-Hofstee plots were used
`to determine the KM(app) (pM) and V,,
`(pmol mi& g-') of both
`reactions. The wet weight (g), measured immediately after re-
`moval and pooling, was used throughout.
`The KM(app) and V,,
`were also measured for the hydrolysis
`of glycoside prodrugs 1,2, 5, 7, and 9-12. Again, cecal contents
`from four rats were pooled, weighed, diluted (100 mL, 0.01 pM
`phosphate buffer, pH 7.0), and homogenized. A range of substrate
`concentrations (0.5-48 pM, final volume 2.5 mL) spanning the
`apparent KM was used for each reaction. The amount of cecal
`homogenate used was 0.8 mL. Reactions were run, in duplicate,
`at 37 "C in a shaking water bath. After 15 min, the reactions were
`stopped by removing aliquots (0.3 mL) and quenching them with
`MeOH (4.7 mL). Following centrifugation (5000g, 10 min), the
`samples were diluted (1:l) with 0.01 M KHzPOl and 20 pL of the
`resulting solution was injected directly onto the HPLC column
`for analysis. Eadie-Hofstee plots were used to determine the
`K ~ ( a p p ) and Vrnw
`Determination of Apparent Partition Coefficients. The
`partitioning of prodrugs and free steroids between 1-octanol and
`
`an aqueous phase (0.01 M phosphate buffer, pH 7.0) were de-
`termined at 37 "C. Both octanol and buffer were saturated with
`the relevant aqueous or organic phase before use. Equal volumes
`(1.0 mL) of both phases were used and agitated for 30 min. The
`initiaI concentration of glycoside was 10 mM, dissolved in the
`aqueous phase. The initial concentration of steroid was 10 mM
`dissolved in the organic phase. The amount of glycoside and free
`steroid in the aqueous phase at equilibrium was measured
`spectrophotometrically at 239 nm for the dexamethasone and
`fludrocortisone compounds and 242 nm for the prednisolone and
`hydrocortisone compounds. The concentration of glycoside or
`free steroid in the octanol phase was determined by difference.
`Note Added in Proof: After this manuscript was ac-
`cepted, the authors learned of an earlier publication de-
`scribing the synthesis of steroid glycoside prodrugs for
`release in the synovial fluid of arthritis victims (Hirsch-
`mann, R., Strachan, R. G.; Buchschacher, P.; Sarett, L. H.;
`Steelman, S. L.; Silber, R. J. Am. Chem. SOC. 1964,86,
`3903).
`Acknowledgment. This work was supported by Na-
`tional Institutes of Health Training Grant GM07379,
`National Science Foundation Grant PCM19105, and the
`Cancer Research Coordinating Committee of the Univer-
`sity of California, Berkeley.
`Registry No. 1,88158-43-4; 2,88158-44-5; 3,50-02-2; 4,50-24-8;
`5,92901-21-8; 6,50-23-7; 7,92901-22-9; 8,127-31-1; 9,92901-23-0;
`10,92901-24-1; 11,92901-25-2; 12,92901-26-3; 13,92901-27-4; 16,
`92901-28-5; 17, 92901-29-6; 18, 92901-30-9; 19, 92901-31-0; 20,
`92937-53-6; 21, 92901-32-1; 22, 92901-33-2; 23, 572-09-8; 24,
`3068-32-4; 25, 14227-66-8; 0-D-glucosidase, 9001-22-3; 0-D-
`galactosidase, 9031-1 1-2.
`
`Angiotensin-Converting Enzyme Inhibitors. New Orally Active Antihypertensive
`(Mercaptoalkanoy1)- and [ (Acylthio)alkanoyl]glycine Derivatives'
`
`John T. Sub,*+ Jerry W. Skiles,' Bruce E. Williams,t Raymond D. Youssefyeh,+ Howard Jones,+ Bernard Loev,f
`Edward S. Neiss,t Alfred Schwab,' William S. Mann,B Atul Khandwala,* Peter S. Wolf,$ and Ira Weinrybt
`Departments of Medicinal Chemistry, Biochemistry, and Pharmacology, Revlon Health Care Group, Tuckahoe,
`New York 10707. Received February I , 1984
`
`A variety of N-substituted (mercaptoalkanoy1)- and [ (acylthio)alkanoyl]glycine derivatives was synthesized and their
`ability in inhibiting the activity of angiotensin-converting enzyme (ACE) was examined in vitro and in vivo. The
`acylthio derivatives prepared are assumed to act as prodrugs since they are much less active than the corresponding
`free SH compounds in vitro and can be expected to act in vivo only after conversion to the free sulfhydryl compounds.
`A number of these compounds are potent ACE inhibitors that lowered blood pressure in Na-deficient, conscious
`spontaneously hypertensive rats (SHR), a high renin model. One of the most active members of the series was
`(REV 3659-(S), pivopril).
`(S)-N-cyclopentyl-N-[3- [ (2,2-dimethyl-l-oxopropyl)thio]-2-methyl-l-oxopropyl]glycine
`Structure-activity relationships are discussed.
`
`CH, R'
`R ~ - ~ ,
`, c o o ~
`
`H
`
`~
`
`A
`
`~ O ,
`
`~
`
`~
`
`
`
`0
`
`2 (captopril)
`"v
`
`Hs&
`
`#-COOH
`
`0
`
`system is an im-
`The renin-angiotensin-aldosterone
`portant humoral mechanism involved in the regulation of
`
`blood p r e s s ~ r e ~ - ~ and renal f ~ n c t i o n . ~ In particular, the
`development of antihypertensive drugs that act selectively
`by inhibiting angiotensin-converting enzyme6?' (ACE) has
`received much attention in recent years. Recently orally
`active ACE inhibitors have been reported to show prom-
`ising clinical antihypertensive propertie~.~-l~ We now
`report the design and synthesis15 of an orally active novel
`
`known inhibitors such as of captopril generic
`
`
`formula series of substituted 1. Unlike the (mercaptoalkanoy1)glycines
`(2)6a,b and enalapril (3),7e which embody a C-terminal
`* Department of Biochemistry.
`Department of Medicinal Chemistry.
`s Department of Pharmacology.
`0022-2623/85/1828-0057$01.50/0
`0 1984 American Chemical Society
`
`0
`
`1
`N
`
`\:F$H
`
`3 (enalapril)
`[ tiapronin I
`4
`"v
`"v
`proline, this series of compounds contains exclusively the
`nonchiral amino acid glycine.
`
`Mylan Exhibit 1029, Page 1
`
`

`
`58 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 1
`During the course of our investigation we observed that
`~-(2-mercaptopropionyl)glycine (4; tiopronin)l6 is a mod-
`erately active inhibitor (ICm = 1.9 p M ) of rabbit lung ACE
`in vitro, but the inhibitory activity is diminished in serum
`or in the presence of other peptidases. This is presumably
`due to the instability of the unsubstituted amide of tio-
`pronin (4) to undergo cleavage by other hydrolytic en-
`zymes. With this hypothesis in mind, a series of potent
`ACE inhibitory compounds was designed and synthesized
`in which the amide nitrogen was substituted by various
`alkyl and aromatic functionality. The compounds of in-
`
`This paper has been presented in part as a communication;
`see: Schwab, A.; Weinryb, I.; Macerato, R.; Rogers, W.; Suh,
`J. T.; Khandwala, A. Biochem. Pharmacol. 1983, 32, 1957.
`Khosla, M. C.; Page, I. H.; Bumpus, M. F. Biochem. Pharma-
`col. 1979, 28, 2867.
`Swales, J. D. Pharmacol. Ther. 1979, 7, 173.
`Haber, E. Kidney Int. 1979, 15, 427.
`Laragh, J. H. Prog. Cardiovasc. Dis. 1978,21, 159.
`Mercapto-containing ACE inhibitors: (a) Ondetti, M. A.; Ru-
`bin, B.; Cushman, D. W. Science 1977,196,441. (b) Cushman,
`D. W.; Cheung, H. S.; Sabo, E. F.; Ondetti, M. A. Biochemistry
`1977,16, 5484. (c) Klutchko, S.; Hoefle, M. L.; Smith, R. D.;
`Essenburg, A. D.; et al. J. Med. Chem. 1981,24,104. (d) Mita,
`I.; Iwao, J.; Masayuki, 0.; Chiba, T.; Iso, T. Chem. Pharm.
`Bull. 1978,26, 1333. (e) Sugie, A.; Katsube, J. Chem. Pharm.
`Bull. 1979,27,1708. (0 Kim, D. H. J. HeterocycE. Chem. 1980,
`17, 1647. (g) Petrillo, E. W.; Spitzmiller, E. R. Tetrahedron
`Lett. 1979,4929. (h) Oya, M.; Matsumoto, J.; Takashina, H.;
`Iwao, J.; Funae, Y. Chem. Pharm. Bull. 1981,29,63. (i) Oya,
`M.; Matsumoto, J.; Tskashina, H.; Watanabe, T.; Iwao, J.
`Chem. Pharm. Bull. 1981, 29, 940. 6) Oya, M.; Kato, E.;
`Matsumoto, J.; Kawashima, Y.; Iwao, J. Chem. Pharm. Bull.
`1981,29,1203. (k) Condon, M. E.; et al. J. Med. Chem. 1982,
`25, 250. (1) McEvoy, F. J.; Lai, F. M.; Albright, J. D. J. Med.
`Chem. 1983,26,381. (m) Kim, D. H.; et al. J. Med. Chem.
`1983,26,394. (n) Stanton, J. L.; et al. J. Med. Chem. 1983,26,
`1267.
`Non-mercapto-containing ACE inhibitors: (a) Ondetti, M. A.;
`Williams, N. J.; Sabo, E. F.; Pluscec, J.; Weaver, E. R.; Kocy,
`0. Biochemistry 1971,10, 4033. (b) Holmquist, B.; Vallee, B.
`L. Proc. Natl. Acad. Sci. U.S.A. 1979, 76,6216. (c) Cheung,
`H. S.; Wang, F. L.; Ondetti, M. A.; Sabo, E. F.; Cushman, D.
`W. J. Bid. Chem. 1980,255,401. (d) Galardy, R. E. Biochem.
`Biophys. Res. Commun. 1980,97,94. (e) Patchett, A. A.; et al.
`Nature (London) 1980,288, 280. (f) Hangauer, D. G. Tetra-
`hedron Lett. 1981,22, 2439. (9) Thorsett, E. D.; et al. Proc.
`Natl. Acad. Sci. U.S.A. 1982, 79, 2176. (h) Vincent, M.; Re-
`mond, G.; Portevin, B.; Serkiz, B.; Laubie, M. Tetrahadron
`Lett. 1982,23,1677. (i) Meyer, R. F.; Essenburg, A. D.; Smith,
`R. D.; Kaplan, H. R. J. Med. Chem. 1982,25, 441. 6) Alm-
`quist, R. G.; et al. J. Med. Chem. 1982,25,1292. (k) Almquist,
`R. G.; Christie, P. H.; Chac, W. R.; Johnson, H. L. J. Pharm.
`Sci. 1983, 72,63. (1) Gruenfeld, N.; et d. J. Med. Chem. 1983,
`26, 1277. (m) Sybertz, E. J.; et al. J. Cardiouasc. Pharmacol.
`1983, 5, 643.
`Ferguson, R. K.; Turini, G. A.; Brunner, H. R.; et al. Lancet
`I1977,775.
`Gavras, H.; Brunner, H. R.; et al. N. Engl. J. Med. 1984,291,
`817.
`Biollaz, J.; Burnier, M.; Turini, G. A.; Brunner, D. B.; et al.
`Clin. Pharmacol. Ther. 1981, 29, 665.
`Gavras, H.; et al. Lancet 1981, 543.
`Gavras, H.; Brunner, H. R.; et al. N. Engl. J. Med. 1978,298,
`991.
`Biollaz, J.; Brunner, H. R.; Gavras, I.; Waeber, B.; Gavras, H.
`J. Cardiouasc. Pharmacol. 1982, 4, 966.
`Solomon, T. A.; Caruso, F. S.; Vukovich, R. A. Clin. Pharma-
`col. Ther. 1983,33, 231.
`(a) Suh, J. T.; Skiles, J. W.; Williams, B. E.; Schwab, A. U.S.
`Patent 4 256 761, 1981. (b) Suh, J. T.; Skiles, J. W.; Williams,
`B. E.; Schwab, A. U.S. Patent 4304771, 1981.
`(a) Mita et al. U.S. Patent 3246025, 1966. (b) Funae, Y.;
`Komori, T.; Sasaki, D.; Yarnamoto, K. Jpn. J. Pharmacol.
`1978, 28, 925.
`
`Suh et al.
`
`Scheme I. Syllthesis of N-Substituted
`(3-Merca~to-2-methylpropanoyl)glycines~
`RNH' & HN -COIR'
`I
`
`R'
`
`5
`"v
`
`6 R'= t . 8 ~ or Et
`rv
`
`Rza,+oR'
`R3 As +y
`
`b,c,d,c,_
`f
`
`CH,
`
`la, R' = H : R' = CH,
`w
`l b , R' : C I : R'z CH,
`m
`ic , R' : H : R' -- cp,
`m
`l d , R' = C I : R' : C,H,
`w
`
`A c o o R '
`
`CHl R'
`
`Ea, R' : t.Bu; R' : CH,
`rl"
`3, R' : H ; R3 : CH,
`8c, Rz : t-8u; R3 : C,H,
`w
`Ed, R': H; R3 = C,H,
`w
`
`9 *
`a Reagents: a, BrCH,CO,RZ, b, 'la-toluene-SOC1,-
`pyridine or DMF to give 7b or 7c-CHzC1,-SOCI,-DMF to
`give 7d; c, 7a-6-CH2Cl,-M=C or 7b-6-CH,C1,-Et3N
`to
`to give 8c; e, 8a-TFA-
`give 8a; d, 7d-6-CHzC1,-Et,N
`anisole or 8a-( CH,),SiI-CH,Cl,
`to give 8b; f , 8c-TFA-
`anisole t o give 8d; g, 8b or 8d-anhydrous NH,-CH,OH.
`
`Scheme 11.
`Synthesis of Hindered Thio Esters of
`N-Substituted (3-Mercapto-2-methylpropanoyl)gly cinesa
`
`rl"
`
`10
`w
`
`"v R' : t.Bu
`l l a ,
`13, R' : H
`a Reagents: a, 8a or 8c-anhydrous NH,-CH,OH; b,
`R3COC1-CH,Cl,-Et,N
`to give I l a ; c , lla-(CH3)3SiI-
`CH,Cl,.
`
`terest are exemplified by the generic formula 1. Our study
`differs from the design and synthesis of ACE inhibitors
`by Ondetti and co-workers, who reported that C-terminal
`proline was the amino acid that provided the maximum
`ACE inhibitory activities.6atbJ7
`Chemistry. The compounds of Table I were conven-
`
`(17) (a) Cushman, D. W.; Ondetti, M. A. Prog. Med. Chem. 1980,
`17,41. (b) Cushman, D. W.; et al. Experientia 1973,29,1032.
`(c) Cushman, D. W.; Cheung, S. H.; Sabo, E. F.; Ondetti, M.
`A. Prog. Cardiovasc. Dis. 1978,21, 176.
`
`Mylan Exhibit 1029, Page 2
`
`

`
`Angiotensin-Converting Enzyme Inhibitors
`
`Journal of Medicinal Chemistry, 1985, Vol. 28, No. 1 69
`
`iently prepared as illustrated in Scheme I in an analogous
`manner to that reported by Cushman and OndettiGb in
`which 3-(acetylthio)-2-methylpropionic acid (7a) was re-
`acted with naturally occurring a-amino acids with use of
`dicyclohexylcarbodiimide (DCC) as the amide-generating
`reagent. In our study, non-naturally-occurring N-substi-
`tuted glycines 6 were utilized. The appropriately substi-
`tuted glycine esters 6 were prepared by treatment of known
`primary amines 5 with either tert-butyl bromoacetate or
`ethyl bromoacetate in a polar solvent such as ethanol or
`acetonitrile. The glycine esters 6 were normally obtained
`as oils which were used directly and were characterized by
`NMR, MS, and TLC analysis. In a manner similar to that
`previously described,16J8 3-(acetylthio)-2-methylpropionic
`acid (7a) was prepared by the addition of thiolacetic acid
`to methacrylic acid in a Michael fashion. The corre-
`sponding acid chloride 7b15 was prepared conveniently in
`toluene in the presence of thionyl chloride witb a few
`added drops of pyridine or DMF as initiator, The ap-
`propriately substituted amino acid esters 6 were condensed
`with 7a in CH2C12 or EtzO with DCC as the amide-gen-
`erating reagent to give 8a. Alternatively the amides 8a
`were also prepared with use of the acid chloride 7b under
`standard Schotten-Baumann acylating conditions. In
`general, the crude amides 88 were converted directly to
`the free carboxylic acids 8b without further purification.
`In those instances in which 8a were purified, the general
`method was high-performance LC using the solvent system
`of AcOEt/n-C6H1, (595). The tert-butyl esters 8a were
`deprotected with either trimethylsilyl iodide ( (CH3),SiI)
`in CH2Clz or by means of trifluoroacetic acid (TFA) in
`anisole, both at room temperature. In the case of the ethyl
`esters 8a, treatment with ethanolic KOH gave directly the
`mercapto acids 9. In general, the pure acids 8b were ob-
`tained by high-performance LC over silica gel with the
`solvent system of n-C6H14/AcOEt/AcOH (6040:l) as
`eluent. All acids 8b were fuuy characterized by NMR, MS,
`and elemental analysis. In the case where the acids 8b are
`liquids or low melting, the elemental analyses were gen-
`erally performed on the corresponding dicyclohexylamine
`(DCHA) or benzathine salts. The free mercaptans 9 were
`generated from the thio esters 8b by treatment with an-
`hydrous NH, in CH30H followed by ion-exchange chro-
`matography (AG-50W-X2, Bio-Rad Laboratories) using
`CH,OH as the eluting solvent. The mercaptans 9 were
`fully characterized by means of NMR, MS, and combus-
`tion microanalysis.
`In a few selected cases, hindered thio esters l l b , such
`as neopentylcarbonyl and pivaloyl, were prepared in order
`to increase in vivo plasma stability and to decrease nu-
`cleophilic displacement of the thio ester carbonyl. These
`hindered esters were prepared as outlined in Scheme 11.
`The thio esters 8a were treated with anhydrous NH, in
`CHBOH to give the mercaptans 10. Alternatively, optically
`active amides 8c were conveniently prepared by conversion
`of commercially available D- (-)-3-(benzoylthio)isobutyric
`acid (7c) to its corresponding acid chloride 7d by means
`of SOCl2 followed by treatment with the appropriately
`substituted glycine ester 6. The thiobenzoyl ester 8c was
`treated with anhydrous ammonia in CH,OH to give the
`optically active thiol 10. After purification the mercaptans
`10 were treated with the appropriate acid chloride under
`standard Schotten-Baumann acylating conditions to give
`the hindered thio esters lla. The tert-butyl esters I l a
`were deesterified in CHzC12 at room temperature by
`
`(18) Fredga, A.; Martensson, 0. Ark. Kemi., Mineral. Geol. 1942,
`16B, 1.
`
`treatment with (CH3),SiI to afford the acids l l b .
`The mercapto acids 9 and the corresponding thio esters
`8b and 1 l b which were synthesized and evaluated for ACE
`inhibition are listed in Table I. Of the over 400 variants
`of structure 1 prepared, we report hereto approximately
`70 representative alkanoylglycines in which the glycine
`nitrogen is alkylated with various substituents including
`alkyl, cycloalkyl, bicycloalkyl, aryl, alkynyl, and hetero-
`cyclic groups.
`Results and Discussion
`The compounds presented in Table I represent an im-
`portant novel class of N-substituted glycines that are very
`potent and specific competitive inhibitors of ACE in vitro
`and in vivo. This series of compounds has demonstrated
`potential as therapeutic agents for hypertension14 and
`congestive heart failure. The in vitro IC60 values of the
`most active mercaptans, 17,21,23,25,27,29, 37,57, 59,
`63, and 68, are in the range of 0.0050-0.035 pM. These
`values are similar to the ICm obtained in our laboratories
`for captopril (2), IC50 = 0.017 p M .
`In order to increase the in vitro potency of tiopronin
`(4),l6 which is an (a-mercaptoalkanoyl)glycine, we pro-
`ceeded to systematically design a series of (P-mercapto-
`alkanoy1)glycines. It was previously noted by Cushman
`and Ondetti that (P-mercaptoalkanoy1)prolines are much
`more potent inhibitors of ACE than their a
`Upon preparation and evaluation of the glycine analogue
`12 in vitro, an IC60 of 0.21 p M was obtained. This is to
`be compared with an IC60 of 1.9 pM for tiopronin (4).
`Upon proceeding to substitute the nitrogen of 12 by various
`alkyl functionalities, 13-15,39, and 40, the ACE inhibitory
`ICm values proceeded to decrease from 0.21 p M for 12 to
`0.072 pM for the isopropyl analogue 15 and to 0.055 pM
`for the thio ether 40. The isopropyl analogue 15 appeared
`promising and gave us the incentive to prepare the cy-
`clopropyl analogue 17. The IC, of 17 (0.030 pM) relative
`to that of 15 (0.072 pM) decreased by a factor of 2-3. With
`this encouraging result, a series of N-substituted mono-
`cycloalkyl analogues 17,21,23a, 25, and 27 was prepared
`in which the ring varied from cyclopropyl to cycloheptyl.
`In this series the maximum activity appeared to reside in
`the cyclobutyl21 and cyclopentyl23a ring systems. The
`next logical course of action to follow in our systematic
`design was to prepare a series of N-bicycloalkyl-substituted
`analogues: 29-36. Suprisingly it was found that the
`exo-norbornyl thio ester 30 was a potent inhibitor of pu-
`rified rabbit lung ACE having an average IC, of 0.020 p M
`over many different experiments. This is to be compared
`with an IC60 of 0.032 pM for the thiol 29. This result was
`unlike the other analogues of our series in which the acetyl
`thio esters were a factor of 10 or more less potent than their
`respective mercaptans when tested in purified rabbit lung
`ACE.
`A series of heterocycloalkyl derivatives, 42, 44, 46, 48,
`and 50, was prepared that exhibited little or no substantial
`increases in inhibitory potency over the unsubstituted
`glycine analogue 12 or any of the other substituted ana-
`logues. The thienyl derivative 46 had the greatest potency
`in this series (IC60 = 0.055 pM).
`A series of N-aryl derivatives, 53,55,57,59, 61, 63, 65,
`66,68, 70, and 72, was prepared and evaluated. This series
`was very fruitful in producing the most active member of
`the compounds prepared by us. The in vitro IC50 values
`of this series ranged from a low of 0.30 p M for the N-
`phenyl analogue 53 to 0.0050 p M for the p-tolyl analogue
`59. The p-tolyl derivative 59 exhibited the maximum in
`vitro potency of all of the inhibitors of generic formula 1
`prepared by us.
`
`Mylan Exhibit 1029, Page 3
`
`

`
`60 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 1
`
`Suh et al.
`
`Table I. N-Substituted Mercaptopropanoylglycines and Inhibition of ACE in Vitro
`
`R'
`
`compda
`H
`12
`H
`13
`H
`14
`H
`15
`CH3CO
`16
`H
`17
`CH3CO
`18
`H
`19
`CH3CO
`20
`H
`21
`CH&O
`22
`23a (R + S) H
`23b (S)j
`H
`24
`CHsCO
`25
`H
`26
`CH&O
`27
`H
`28
`CHSCO
`2S0
`H
`
`30°
`
`31*
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`38
`
`39
`40
`41
`42
`
`43
`
`CH&O
`
`CHSCO
`
`H
`
`CH3CO
`
`H
`
`CH&O
`
`CHaCO
`
`H
`
`CH&O
`
`H
`H
`CH&O
`H
`
`CH&O
`
`procedured
`
`- .-
`remarks
`
`formulae
`yield," %
`C6HllNO3S
`92
`I
`C7H13N03S
`I
`90
`C8H16NO8 DCHA'
`I
`93
`DCHAf
`C9HIINO3S
`I
`95
`B, D, F
`72
`C11H19N04S
`C9H15N03S
`I
`84
`A, D, F
`61
`C11H11N04S
`C10H17N03S
`I
`72
`B, D, F
`C12HlBN04S
`42
`C10H17N03S
`I
`82
`B, E, G
`72
`C12HlBN04S
`CllH18N03S
`I
`87
`89 (82) B, E, G, I (M, N) CllHuNO3S
`B, E, G
`C13H21N04S
`75
`I
`C12H21N03S
`92
`f4 E, G
`83
`C14H23N04S
`88
`C13H23N03S
`B, E, G
`C12H26N04S
`86
`96
`I
`C13H21N03S
`
`DCHA8
`DCHAg
`
`DCHA'
`calcium salt
`calcium salt
`DCHAg
`DCHA'
`DCHA'
`
`DCHAg
`DCHAg
`
`R2
`
`R3 mp,b OC
`H
`115-117
`H
`71-73
`H
`131-132
`H
`159-160
`H
`104-105
`H
`89-91
`H
`68-70
`CH3 129-130.5
`CH, 83-85
`H
`liquid
`H
`162.5-164.5
`H
`173-176h
`H
`186-188
`H
`172-174
`H
`158-160k
`H
`142-144
`H
`oil*
`H
`116-117
`H
`120-122
`
`H
`
`H
`
`125-12P
`
`116
`
`H
`
`glass
`
`ICKo! U M
`0.21
`0.13
`0.075
`0.072
`5.9
`0.030
`0.54
`0.079
`0.22
`0.018
`0.22
`0.018'
`0.016
`0.082
`0.035'
`0.27
`0.031"
`0.088
`0.032
`
`0.020
`
`0.052
`
`0.44
`
`0.085
`
`0.16
`
`0.14
`
`86
`
`21
`
`87
`
`B, D, F
`
`B, D, F
`
`CigH23NO4S DCHA'
`
`CigH23NO4S DCHAg
`
`I
`
`C17H29N03S
`
`H
`
`117
`
`77
`
`C, D, F
`
`C19H31N04S
`
`H
`
`glass
`
`84
`
`I
`
`H
`
`120
`
`84
`
`C, D, F
`
`H
`
`134-138
`
`63
`
`C, D, F
`
`Ci7H27N04
`
`DCHA'
`
`0.045
`
`H
`
`186-188'
`
`95
`
`I
`
`C15HlsN03S DCHA'
`
`0.031*
`
`H
`H
`H
`H
`
`H
`
`129-132
`122-128
`120-121
`128-130
`
`138-140
`
`90
`91
`82
`64
`
`80
`
`B, E, G
`I
`B, E, G
`I
`
`B, D, F
`
`CgH17N04S DCHA'
`CloHleNOsSz DCHA'
`C12HziN04S2 DCHA'
`CiIHlgN04S DCHA'
`
`C13H21N05S DCHA'
`
`0.34
`
`0.095
`0.055
`0.90
`0.13
`
`1.9
`
`Mylan exhibit 1029, Page 4
`
`

`
`Angiotensin-Converting Enzyme Inhibitors
`-
`compd'
`44
`
`Journal of Medicinal Chemistry, 1985, Vol. 28, No. 1 61
`
`yield," %
`80
`
`procedured
`
`remarks
`formulae
`CllH1sN04S DCHA'
`
`IC,,f%
`0.17
`
`R3 mp,b 'C
`H
`150-153
`
`R2
`
`I
`H2cy7J
`I
`
`R'
`
`H
`
`45
`
`46
`
`47
`
`48
`
`49
`
`50
`
`51
`52
`53
`54
`55
`56
`57
`58
`59
`60
`61
`62
`63
`
`64
`
`CH3CO
`
`H
`
`CHSCO
`
`H
`
`CH&O
`
`H
`
`H
`CH&O
`H
`CH&O
`H
`CH&O
`H
`CHSCO
`H
`CHSCO
`H
`CH&O
`H
`
`CH&O
`
`H
`
`H2c%
`
`I
`
`n2c*
`I
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H2C% x
`0 6
`HzC '4
`L,,
`
`140-141
`
`122-128
`
`44
`
`82
`
`149.5-150.5
`
`49
`
`38-40
`
`90
`
`191-193
`
`85
`
`120-122
`
`57
`
`0.70
`
`0.055
`
`0.75
`
`0.28
`
`0.28
`
`0.64
`
`0.27
`4.5
`0.30''
`0.30
`0.12
`0.55
`0.019
`0.075
`0.0050
`0.13
`0.044
`0.044
`0.033
`
`CHECHCH~
`CHsCHCHZ
`CaHr
`
`H
`H
`H
`H
`H
`H
`H
`H
`H
`H
`
`164-166
`154-166
`168-170t
`94-94.5
`97-101
`128-130
`121-122
`104-105
`134-137
`146-148
`125-126
`89-92
`164-167
`
`91
`62
`90
`66
`89
`91
`93
`87
`95
`84
`96
`90
`92
`
`CgH13NOaS DCHA'
`CI1H16NO4S DCHA'
`C14H17N04S
`C14H17N04S
`C13H1,N03S Bemu
`C16HlgN04S BenzU
`C13HlTN03S Benz"
`Cl6Hl9NO4S BenzU
`C13H17N03S Bend'
`CI6HlsNO4S Bemu
`C14H1SN03S
`C16H21N04S
`C16HlsN03S Bemu
`
`117-118
`
`82
`
`Cl7HZ1NO4S Benzu
`
`0.048
`
`CH&O
`CH3CO
`CHSCO
`H
`CHBCO
`H
`4-(n-C4H&$H4' H
`CYaCO
`CHQCO
`4-(i-CaH~)CsH4 H
`(CH3)&CHZCO c-C5HS
`H
`(CH3)3CCO
`c-C~HS
`H
`(CH3)3CCO
`C-C&
`H
`(CH3)3CCO
`c-C~HS
`H
`
`103-105
`110-112
`oil
`155w
`oil
`137-145y
`151-153
`144
`85-87
`140-142
`156
`155-156
`
`80
`72
`68
`92
`82
`88
`66
`86
`82
`80
`62
`75.1
`
`Cl6HlSNO5S bemu
`ClsHlsNO4S2 Benz"
`C14H16FN04S
`C12H14FN03S
`C14H16FN04S
`C18H23N03S Benz"
`Cl8HZ6No4S BenzU
`Cl7HZ3NO4S Bemu
`C17H29N04S
`Cl6H27N04S
`C16H27N04S
`C16H27N04S
`
`0.11
`0.075
`0.051
`0.023J
`0.60
`0.191
`0.064
`0.060
`15
`3.70
`>loo
`3.60
`
`0.017"d
`8.0ae
`1.9af
`
`65
`66
`67
`68
`69
`70
`71
`72
`73
`74aaa
`74b(R)Osb
`74C(S)"C
`(pivopril)
`2 (captopril)
`3 (enalapril)
`4 (tiopro-
`nin)
`'Except where indicated all compounds are racemic. bUncorrected. 'Yield refers to the last step in each synthetic sequence. dSee
`Experimental Section. cAII compounds had satisfactory C, H, and N microanalyses and were within 0.4% of theoretical values. All com-
`pounds exhibited IR, 'H NMR, and MS spectra consistant with the assigned structures. 'Concentration inhibiting 50% of the activity of
`rabbit lung ACE at pH 8.3 in 0.10 M potassium phosphate buffer containing 0.30 M NaCl with the substrate Hip-His-Leu at a concentration
`of 2 mM. 'Dicyclohexylamine (DCHA) salt. hLiteraturesn mp (DCHA) 143-144 OC.
`'Literaturesn ICso = 0.007 p M . 'Corresponds to S
`isomer, [a]D -12.50'
`'Literatures" IC,, = 0.0075 pM. "Literature'" mp (DCHA)
`(c 1.0, CHC1,). kLiteraturesn mp (DCHA) 160-162 'C.
`143-145 "C. "Literaturesn ICw = 0.0071 pM. DCorresponds to exo isomer. PCalcium salt mp 157-161 OC. PCorresponds to endo isomer.
`'Literaturee" mp (DCHA) 180-183 'C.
`'Literaturesn IC,, = 0.012 pM. tLiteratuFes" mp 170-171 'C.
`"Literatureen IC60 = 0.013 pM.
`" Benzathine salt, N,"-dibenzylethylenediamine.
`Literaturea" mp 163-165 'C.
`Literaturesn IC60 = 0.011 pM. Y Literature'" mp (DCHA)
`ObCorresponds to R
`124-126 'C.
`'Literaturesn IC, = 0.056 pM. 'OCorresponds to a 1:l mixture of the R and S isomers, REV 3659.
`'CCorresponds to S isomer, [cy],,
`-104.64'
`(c 1.0, CHC13).
`isomer, [a]D +111.05'
`(c 1.0, CHC13).
`Corresponds to REV 3659-(S).
`ndLiteraturesb IC,, = 0.023 pM. neLiterature7e IC, = 1.2 pM.OfLiteratureGb IC, = 1.7 pM.
`
`Mylan Exhibit 1029, Page 5
`
`

`
`62 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 1
`
`~~
`
`Table 11. Angiotensin-Converting Enzyme (ACE) Inhibition of
`Selected Compounds in Conscious Normotensive Ratsa
`compd
`mg/kg,po
`compd
`IDSo,* mg/kg,po
`30
`17
`0.30
`0.06
`37
`18
`0.20
`0.15
`41
`21
`0.30
`1.5
`54
`23a
`0.15
`0.2
`58
`24
`0.15
`0.15
`25
`0.10
`0.15
`60
`64
`27
`0.05
`1.5
`2 (captopril)
`28
`0.06
`0.1oc
`3 (enalamil)
`29
`0.10
`0.08d
`'See Experimental Section. bDose (mg/kg, PO) required to in-
`hibit 50% of the angiotensin I induced vasopressor response in
`normotensive conscious rats. Literaturez3 IDm = 0.015 mg/kg, iv.
`Literature7e IDSo = 0.014 mg/ kg, iv.
`The very potent in vitro ACE inhibitory activities of the
`N-substituted alkanoylglycines (see Table I) were equally
`confirmed by oral administration in rats as shown in Table
`11. The oral ID, values of 0.05-1.5 mg/kg were exhibited
`by the in vitro active thiols 17,21,23a, 25,27, 29, and 37
`as well as the thio esters 18,22,24,26, 28,30, 41,54, 58,
`60, and 64. The compounds 23a (ID, = 0.15 mg/kg, PO),
`24 (IDS0 = 0.15 mg/kg, PO), and 28 (IDSO = 0.06 mg/kg,
`PO) were among the most active and their oral activities
`are comparable to captopril (2) (ID50 = 0.10 mg/kg, PO)
`when compared in our laboratories.
`The most common side effects accompanying the clinical
`use of captopril are rashes and loss of taste, both of which
`usually clear on withdrawal or reduction of dose.lg In the
`search for clinically useful potent and specific inhibitors
`of ACE lacking a free mercapto functionality, which has
`been reported to be associated with. these common side
`effects, we selected the N-cyclopentyl compound 23a for
`further development. A large number of analogues was
`prepared by this laboratory in which the free thiol of 23a
`was acylated and alkylated by a wide range of functionality
`in order that relatively low levels of the free SH compound
`be generated in vivo while maintaining inhibition of ACE.
`Most of these prodrugs are much less active than the
`corresponding free SH compound in vitro and can be ex-
`pected to act in vivo only after conversion to the free
`sulfhydryl compounds. Of the many analogues of 23a
`prepared by us, the pivaloyl thio ester 74c was selected for
`extensive preclinical and clinical development. The hin-
`dered pivaloyl thio ester 74a was prepared from 8a or 8c
`according to Scheme 11. The ability to inhibit the pressor
`response to angiotensin I for the pivaloyl thio ester 74a
`and its corresponding free mercaptan 23a in rats and dogs
`is presented in Table 111.
`A stereospecific synthesis of the S and R enantiomers
`of 23a was efficiently achieved by the method described
`in Scheme I utilizing optically pure thio ester 7a or 7ca20
`As shown in Table I11 the pivaloyl thio ester 74c with S
`configuration is considerably more potent both in vitro and
`in vivo in inhibiting ACE than the corresponding thio ester
`74b with R configuration but less potent than the free SH
`form 23a.
`In order to obtain information on the potential stability
`of an ACE inhibitor in vivo, an aliquot of inhibitor solution
`(20 pM) in MezSO was diluted 20-fold with rat and human
`
`(19) (a) Atkinson, A. B.; Robertson, J. I. S. Lancet I Z 1979,836. (b)
`Parfrey, P. S.; Clement, M.; Vandenburg, M. J.; Wright, P. Br.
`Med. J. 1980, 281, 194. (c) Editorial Lancet 1980, 2, 129.
`(20) (a) Optically pure S and R isomers of acetyl-P-mercaptoiso-
`butyric acid (7a) were obtained from Chemical Dynamics
`Corp., South Plainfield, NJ 07080. (b) Optically pure (S)-
`3-(Benzoylthio)isobutyric acid was obtained from Chemical
`Dynamics Corp., South Plainfield, NJ 07080.
`
`Suh et al.
`
`Table 111. ACE Inhibitory Activities of
`N-Cyclopentylalkanoylglycines and Comparison with Captopril
`(2) and Enalapril (3) in Vitro and in Vivo
`
`IDSO,'
`mg/kg, PO, mg/kg, PO,
`normoten-
`normoten-
`sive rats
`sive does
`~-
`0.15
`
`ICm," p M
`in vitro
`comDd
`23a (R + S)
`0.018
`23b (S)
`0.018
`73
`1.2
`15
`74a(R + S)d
`0.15
`3.70
`25
`74b(R)
`100
`7445') (pivopril)
`0.058
`0.17
`3.60
`o.id
`2 (captopril)
`0.057
`0.027e
`3 (enalapril)
`0.08'
`0.1GJ
`8.0h
`'Concentration inhibiting 50% of the activity of rabbit lung
`ACE at pH 8.3 in 0.10 M potassium phosphate buffer containing
`0.30 M NaCl with the substrate Hip-His-Leu at a concentration of
`2 mM. bDose (mg/kg, PO) required to inhibit 50% of the angiot-
`ensin I induced vasopressor response in normotensive conscious
`rats. cDose (mg/kg, PO) required to inhibit 50% of the angiotensin
`I induced vasopressor response in normotensive conscious dogs.
`dCorresponds to REV 3659. eLiteratureab ICso = 0.023 NM.
`fLiteraturez6 IDS0 = 0.015 mg/kg, iv. gLiteratureZ6 ID,,, = 0.044
`
`mg/kg, PO.
`ICSo = 1.2 FM. ' L i t e r a t ~ r e ~ ~ IDm = 0.014
`mg/kg, iv. JLiteratureIe IDw = 0.278 mg/kg, iv.
`
`plasma incubated at 37 "C. At various times, 0.05-mL
`aliquota were removed and assayed immediately for thiol
`content. Thiol concentrations were determined at pH 7.4
`according to a procedure described in the literature.21 In
`vitro the thio ester 74a was stable to hydrolysis by rat and
`human plasma (22 h, 37 "C) and rat gastric juice (24 h, 37
`"C). These results tend to suggest that the sites of lib-
`eration of the active drug 23a from the parent thio ester
`74a may be affected in a novel manner by the resistance
`to plasma and gastric juice enzymes. A detailed com-
`parison of the thio ester 74a with its possible metabolite
`23a is described elsewhere.22a
`The new ACE inhibitor (S)-N-cyclopentyl-N-[3-[(2,2-
`dimethyl-1-oxopropyl) thio]-2-methyl-l-oxopropyl]glycine
`(74c) has been given the generic name of pivopril and
`corresponds to REV 365943). This compound has shown
`promise both preclinically and clinically to be a potent and
`specific inhibitor of ACE while also being a antihyper-
`tensive. Herein we report some of pivopril's (74c) bio-
`logical properties.
`A comparison was made between pivopril (74c) and
`captopril (2) to determine their abilities to inhibit ACE
`in vivo, as judged by the inhibition of angiotensin I pressor
`responses in the conscious normotensive rat.22b Both pi-
`vopril and captopril inhibited angiotensin I pressor re-
`sponses within 20 min after oral dosing in a dose-related
`fashion. Oral ID50 values for pivopril and captopril were
`0.058 and 0.10 mg/kg, respectively. The intensity and
`duration of action for both pivopril and captopril were dose
`related and similar to each other except at 0.3 mg/kg.
`After intravenous administration pivopril had similar
`potency, and onset of action, as it did after oral admin-
`istration. At 100 mg/kg, PO, neither pivopril (74c) nor
`captopril(2) affected the pressor responses to angiotensin
`I1 or norepinephrine or lowered arterial pressure.
`From the above study it is concluded that pivopril(74c)
`is a rapidly absorbed, orally effective inhibitor of angiot-
`ensin I pressor responses in the normotensive rat. Its
`
`(21) Ellman, G.; Lysko, H. Anal. Biochem. 1979,93,98.
`(22) (a) Schwab, C.; Weinryb, I.; Macerata, R.; Rogers, W.; Suh, J.
`T.; Khandwala, A. Pharmacologist 1983,25,241. (b) Wolf, P.
`S.; Mann, W. S.; Perone, M.; Suh, J. T.; Smith, R. D. Phar-
`macologist 1982, 25, 176.
`
`Mylan Exhibit 1029, Page 6
`
`

`
`Angiotensin-Converting Enzyme Inhibitors
`
`mechanism of action is