J. Med. Chem. 1994, 37, 1865-1873
`
`1865
`
`New Thiol Inhibitors of Neutral Endopeptidase EC 3.4.24.11: Synthesis and
`Enzyme Active-Site Recognition
`Isabel Gomez-Monterrey,7 Ann Beaumont, Patrick Nemecek,7 Bernard P. Roques,* * and
`Marie-Claude Fournie-Zaluski
`Unité de Pharmacochimie Moléculaire et Structurale, U266 INSERM - URA D1500 CNRS, Université René Descartes,
`UFR des Sciences Pharmaceutiques et Biologiques, 4, avenue
`de I’Observatoire, 75270 Paris Cedex 06, France,
`and Rhdne Poulenc Rorer, Centre de Recherche de Vitry-Alforville, 13, quai Jules Guesde, 94403 Vitry/Seine, France
`Received May 17, 1993*
`Selective, as well as mixed, inhibitors of the two zinc metallopeptidases, neutral endopeptidase
`(NEP) and angiotensin converting enzyme (ACE), are of major clinical interest in the treatment
`of hypertension and cardiac failure. New thiol inhibitors, corresponding to the general formula
`HS-CH(Ri)-CH2-CH(R2)-CONH-CH(R3)-COOH, were designed in order to explore the putative
`Si subsite of the active site of NEP. The inhibitors were also tested on ACE and the most
`representative on thermolysin (TLN) for comparison. The relatively low inhibitory potencies
`exhibited by these compounds (IC50S in the 10™7 M range for NEP and in the 10-6 M range for ACE)
`as compared to that of thiorphan (IC50S 2.10 X 10-9 M on NEP and 1.40 X 10-7 M on ACE) clearly
`indicate the absence of the expected energetically favorable interactions with the active site of both
`peptidases. A 100-fold loss of potency for these inhibitors was also observed for thermolysin as
`compared to thiorphan. Using the mutated Glu102-NEP, it was possible to demonstrate that the
`inhibitors do not fit the Si subsite of NEP but interact with the S'i and S'2 subsites through binding
`of their Ri and R2 residues and that the C-terminal amino acid is located outside the active site.
`These results seem to indicate that these thiol inhibitors are not well adapted for optimal recognition
`of the Si subsite of NEP, and probably ACE, and that other zinc-chelating moieties such as
`carboxylate or phosphonate groups may be preferred for this purpose.
`
`Introduction
`Neutral endopeptidase (EC 3.4.24.11, NEP) is a met-
`allopeptidase involved in the metabolism of a variety of
`physiologically important peptides and its inhibition offers
`several interesting new therapeutic possibilities (see review
`in ref 1). Thus, in the brain, the opioid peptides enke-
`phalins are inactivated by the common action of two zinc
`metallopeptidases, NEP and aminopeptidase N (APN).
`Mixed inhibitors of both enzymes have been shown to
`induce potent analgesic responses without the major side
`effects of morphine.2-4 In the intestine, the enkephalins,
`acting at   opioid receptors, are mainly involved in the
`regulation of fluid secretion.5 The demonstration that
`their intestinal actions can be blocked by NEP inhibitors
`has led to the marketing of thiorphan (HS-CH2-CH-
`(CH2<$>)-CONH-CH2-COOH),6 under its prodrug form
`acetorphan, as an antidiarrheal agent designated tiorfan.7·8
`In addition, the atrial natriuretic peptide ANP is cleaved
`inhibitors
`by NEP into inactive fragments, and numerous
`have been shown to produce natriuresis and diuresis in
`animals and humans (see review in ref 9). The association
`of an NEP inhibitor such as retrothiorphan with an
`angiotensin converting enzyme inhibitor such as enalapril
`led to a potentiation of the antihypertensive action of
`the ACE inhibitor,10 and mixed inhibitors of the two
`metallopeptidases could therefore represent a new ap-
`proach in the treatment of hypertension and cardiac
`failure.11
`The primary sequence of NEP has been deduced from
`cDNA clones from various species (rabbit,12 rat,13 and
`
`* To whom correspondence should be addressed. Tel:
`(33) 43-25-50-
`45. FAX:
`(33) 43-26-69-18.
`1 Present address: Instituto de Química Medica, Juan de la Cierva, 3
`Madrid, Spain.
`* Centre de Recherche de Vitry-Alforville.
`* Abstract published in Advance ACS Abstracts, May 1, 1994.
`
`0022-2623/94/1837-1865$04.50/0
`
`human14), but its three-dimensional structure remains
`its mechanism of action and its
`unknown. However,
`subsite specificity are well documented, due to its overall
`analogy with thermolysin (TLN), a bacterial metalloen-
`dopeptidase whose structure was established from X-ray
`crystallography.15 These analogies include not only the
`hydrophobicity of the binding subsites surrounding the
`catalytic domain16-19 and the three zinc coordinating
`ligands20-22 but also the amino acids involved in the
`stabilization of the Michaelis complex.23-26 However, the
`main difference between NEP and TLN is the presence
`in the S'2 subsite of the former of an arginine residue
`(Arg102) which was shown to interact with the free carboxyl
`group of substrates such as the enkephalins.6 In spite of
`large similarities in the active site of both enzymes, the
`potency of a large number of inhibitors is about 2 or 3
`orders of magnitude lower for TLN than for NEP. This
`is particularly evident for inhibitors interacting with the
`S'i and S'227 subsites of both enzymes. For example, the
`Ki of (S)-thiorphan (HS-CH2-CH(CH2i)-CONH-CH2-
`COOH) is 1.8 X 10-6 M for TLN and 1.9 X 10-9 M for
`NEP.28 However, the difference is smaller for inhibitors
`able to interact with the Si, S'i, and S'2 subsites of each
`enzyme. For example, the K\ of (l-carboxy-3-phenylpro-
`pyl)Leu-Trp is 5 X 10-8 M for TLN,29 and an analogous
`compound such as SCH 39370 ((l-carboxy-3-phenylpro-
`pyl)Phe-|3-Ala) has a K¡ of 1.1 X 10-8 M for NEP.30 Taken
`together, these results seem to indicate that the occupancy
`of the Si subsite is more
`important in TLN than in NEP
`for strong inhibition of the enzyme.
`The Si subsite of NEP has been investigated with various
`carboxyalkyl dipeptides30-33 or related compounds, such
`as UK 69578,34 constructed on the model of enalapril,35
`and the results obtained seem to indicate that the Si subsite
`corresponds to a hydrophobic domain. However, the great
`majority of these compounds have IC50S in the 10-8 M
`&copy; 1994 American Chemical Society
`
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`

`1866 Journal of Medicinal Chemistry, 1994, Vol. 37, No. 12
`Scheme 1. Scheme for the Synthesis of Inhibitors 17-27“
`S./COOH
`(a),
`(b)
`
`^   
`
`2a-c
`
`ch3o
`
`R1
`
`la-c
`
`CH30
`
`Gomez-Monterrey et al.
`
`R1
`
`(c)
`(d)
`
`CHsO
`
`Jgr-S^rCOOCH3
`Rj COOCH3
`3a-c
`
`(e)
`
`R1'/SV°
`
`6a-e
`
`(h)
`
`(g)
`
`ch3o
`
`(i)
`
`Rl
`
`R2
`
`2
`
`/§rw COOH
`
`R,
`
`r2
`
`5a-e
`
`(f)
`
`COOCH,
`j^f-Sr^-cOOCH3
`ch3o
`Ri
`4a-e
`
`R2
`
`"COOK
`
`7a-e
`“ (a) SOCl2, MeOH; (b) NaBH4, LiCl; (c) Nal, (CH3)3SiCl; (d) NaH, CH2(COOCH3)2; (e) NaH, R2X; (f) DMSO-20% KOH; (g) CF3COOH,
`reflux; (h) NaOH/EtOH, 02; (i) H2N-CH(R3)-COR, BOP, DIEA; (j) OH* then H30+.
`range, whatever the degree of the hydrophobicity of the
`Pi moiety, a result which does not permit a clear
`characterization of the Si subsite.
`Recently, we have reported two new series of NEP
`inhibitors containing a thiol group as the zinc-chelating
`agent and designed to interact with the Si, S'i, and S'2
`subsites of the enzyme.36 Some of these compounds were
`found to be very efficient, essentially those corresponding
`to the general formula HS-CH(Ri)-CH(R2)-CONH-CH-
`(Rsl-COOH with IC50 values in the nanomolar range for
`the most active stereoisomer. Nevertheless, none of these
`inhibitors is more active than thiorphan (HS-CH2-CH-
`(CH2$)-CONH-CH2-COOH),6 which interacts only with
`the S'i and S'2 subsites of NEP. Several hypotheses have
`been proposed to account for this result. The inhibitors
`might be unable to interact with the Si subsite for steric
`reasons, or the interaction of the Ri chain in the Si subsite
`might decrease the strength of the zinc complexation. On
`the other hand, the Si subsite of NEP might not be well
`defined, leaving the inhibitor Ri side chain to be located
`in the aqueous medium or at the surface of the enzyme
`active site.
`In order to test the hypothesis that the potency of the
`previously discussed series is limited by steric hindrance,
`we have developed new compounds of general formula
`HS-CH(Ri)-CH2-CH(R2)-CONH-CH(R3)-COOH, in which
`a methylene spacer has been introduced between the Ri
`and R2 chains. For Ri, R2, and R3, hydrophobic chains
`were chosen which have previously been found to fit well
`into the active site of NEP. For comparison,
`these
`compounds have also been tested on ACE and some of
`them on TLN.
`Results
`(1) Synthesis. The compounds 17-27 were prepared
`following the scheme summarized in Scheme 1. The
`protected 2-mercapto alkanoic acids la-c were obtained
`under optically pure forms from the corresponding a-amino
`acids through desamino halogenation of D-amino acids
`following the Fischer procedure,37 yielding the corre-
`sponding 2-bromo analogs with retention of configuration.
`The subsequent nucleophilic substitution of the halogen
`
`by the potassium salt of p-methoxybenzyl sulfide gave
`intermediates la-c with inversion of configuration. These
`protected  -mercapto acids were reduced by lithium boro-
`hydride to the corresponding /3-mercapto alcohols 2a-c.
`The following synthetic pathway involved three key
`steps. The first was
`the formation of the malonate
`derivatives 3a-c, which necessitated the transformation
`of the alcohols 2a-c to iodinated derivatives38 before
`reaction with the sodium salt of dimethyl malonate. The
`second critical step was the decarboxylation of the hindered
`intermediates obtained, 4a-e, which was achieved only by
`heating these compounds in DMSO-20% KOH to give
`5a-e. The third difficult step was the deprotection of the
`thiol group, which could not be performed at the last step
`of the synthesis either by the classical Hg(CF3COO)2 and
`H2S treatment or by reflux in CF3COOH39 without major
`degradation of the final compounds. This problem was
`bypassed in an intermediate step of the synthesis. Thus,
`the thiol deprotection was easily obtained from 5a-e by
`refluxing the compounds in CF3COOH, owing to the high
`stability of the thiolactones 6a-c which were spontaneously
`formed during this step. The synthesis was continued by
`opening the cyclic structures 6a-e by alkaline treatment
`and with bubbling O2 to obtain the disulfides 7a-e. The
`synthesis was achieved by condensation of  -amino esters
`or  -amino amides of defined stereochemistry using BOP
`reagent and final deprotection. The final compounds were
`isolated as mixtures of diastereoisomers in about equal
`proportions as shown by NMR and, in some
`cases, by
`HPLC. This is due to a racemization of the carbon bearing
`the protected thiol group, which occured probably during
`the drastic treatment with KOH of the intermediates
`3a-c.
`Compound 32 was obtained by a different pathway
`(Scheme 2), using the procedure leading to the preparation
`of a-/3-substituted /3-mercapto acids.36 Thus, condensation
`of triethyl 2-benzyl phosphonoacetate with phenylacetal-
`dehyde led to a mixture of  -ß and ß-y unsaturated esters
`(compounds 28 and 29). Saponification of this mixture
`yielded only the ß-y unsaturated acid 30 by transposition
`of the double bond. Addition of thiolacetic acid to 30
`gave 31 as a mixture of four stereoisomers in equal
`
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`

`New Thiol Inhibitors of NEP
`Scheme 2. Scheme for the Synthesis of Inhibitor 32
`
`Journal of Medicinal Chemistry, 1994, Vol. 37, No. 12
`
`1867
`
`COAla-OH 1) Ala-OMe, DCC/HOBt
`2) OH 7H +
`
`*
`
`Table 1.
`
`Inhibitory Potency of Compounds 17-32 on NEP and ACE Activities
`_ICso (µ )°_
`NEP6_ACEe
`4.0 ± 0.5d
`0.044 ± 0.012d
`
`compound
`
`B
`
`IC50 (µ )°
`ACE'
`NEP6
`0.018'
`
`compound
`o
`
`Of
`
`HOOC
`
`o
`
`0.19 ± 0.07
`
`3.8 ± 0.8
`
`0.38 ± 0.02
`
`3.4 ± 0.6
`
`0.29 ± 0.05
`
`3.5 ± 1.5
`
`0.36 ± 0.02
`
`4.0 ± 1.0
`
`0.32 ± 0.06
`
`3.6 ± 0.5
`
`0.27 ± 0.02
`
`4.4 ± 0.4
`
`0.15 ± 0.06
`
`2.4 ± 0.7
`
`0.32 ± 0.05
`
`8.0 ± 0.6
`
`0.22 ± 0.03
`
`0.8 ± 0.05
`
`0.35 ± 0.05
`
`3.5 ± 0.5
`
`0.35 ± 0.05
`
`2.8 ± 0.6
`
`0.22 ± 0.05
`
`2.3 ± 0.6
`
`0.13 ± 0.05
`
`3.5 ± 0.5
`
`0.25 ± 0.06
`
`4.0 ± 0.6
`
`0.26 ± 0.04
`
`2.9 ± 0.5
`
`7--7/^NH/x'COOH
`
`[oj
`
`24
`
`o
`
`“ Values are the mean ± SEM from three independent experiments computed by log probit of five inhibitor concentrations. 6 Concentration
`inhibiting 50% of NEP activity using 20 nM [3H]-D-Ala2-Leu-enkephalin as substrate.' Concentration inhibiting 50% of ACE activity using
`50 µ  N-Cbz-Phe-His-Leu as substrate. d From ref 36.' From ref 40.
`proportions, and the final compound 32 was obtained by
`condensation of alanine methyl ester followed by alkaline
`hydrolysis of both protective groups.
`(2) Inhibitory Activity. The inhibitory potencies of
`the various compounds synthesized were measured on
`NEP and ACE as mixtures of stereoisomers and are
`
`summarized in Table 1. The effects of five structural
`investigated (i) amidification of the
`modifications were
`free C-terminal carboxyl group (compound 26 versus
`17
`or 27S versus 18); (ii) modification of the C-terminal amino
`acid with successive introduction of an alanine (compound
`19), a jS-alanine (compound 20), a proline (compound 21),
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`

`1868 Journal of Medicinal Chemistry, 1994, Vol. 37, No. 12
`Chart 1. Schematic Representation of NEP Active Site"
`
`Gomez-Monterrey et al.
`
`s,vL
`
`9
`
`H
`
`?
`J1
`NH
`HN.
`HN\^NH
`•N^·
`Are 747
`Are 747
`° (A) Complex NEP-thiorphan as deduced from X-ray analysis of the complex TLN-thiorphan. (B) Possible model of interaction of inhibitors
`17-32 with NEP active site. In this model, the Ri, R2, and R3 side chains fit the Si, S'i, and S'2 subsites of the enzyme, respectively. (C) Possible
`model of interaction of inhibitors 17-32 with NEP active site deduced from experiments with the Glu102-mutated enzyme. In this model, the
`Ri and R2 side chains fit the S'i and S'2 subsites of NEP and R3 is outside the active site.
`In contrast to these results, all the compounds described
`or a tyrosine (compound 22) moiety; (iii) change in the
`stereochemistry of the C-terminal amino acid with 27S
`here exhibited surprisingly similar IC50S for NEP, which
`were in the 10™7 M range (Table 1). These data show a
`and 27fi; (iv) replacement of the benzyl group, assumed
`to interact with the S'i subsite by an isobutyl chain
`significant decreased efficiency of these compounds in
`binding NEP as compared to that of the thiol inhibitor
`(compounds 22 and 23 or 26 and 27); and finally (v)
`exploration of the putative Si subsite using successively
`In contrast, the
`A36 or the dibenzylglutaryl derivative B.
`IC50 values for ACE, which were found to be in the 10-6
`a phenyl (compound 32 versus 19), a benzyl, and phenethyl
`M range for all compounds, are not significantly different
`and isobutyl chains (compounds 23, 25, and 26).
`from those reported for A (Table 1) and derivatives.36 In
`Regarding NEP inhibition, all compounds showed
`similar potencies, with IC50S in the 1-4 X   -7 M range,
`order to understand in terms of active-site occupancy, the
`whatever the chemical modifications introduced in these
`decreased activity of these new compounds as compared
`to that of other thiol
`molecules. For ACE inhibition, the same potency was
`inhibitors such as thiorphan or
`also obtained for all compounds studied with IC50S in the
`compound A and its analogs, the activities of 19, 21, 23,
`micromolar range. Furthermore, the synthetic intermedi-
`26, 27S, and 27R, chosen as models for this series, were
`ates 6b and 7b were also tested on both enzymes, and
`checked on TLN (data not shown). Their IC50S (in the
`their activities were found to be in the 10-7 M range on
`10*4 M range) were increased by 3 orders of magnitude as
`NEP and in the micromolar range on ACE. The potencies
`regard to their IC50S for NEP (  -7 M range). This ratio
`of the compounds tested on TLN were found in the 10-4
`already found for thiorphan (IC50 on NEP 1.9 X   9 M,
`M range.
`IC50 on TLN 1.8 X ID™6 M) or
`for constrained thiols
`such as HS-CH(CH2CHiPr)-CH(CH2Ph)-CONHCH(CH3)-
`Discussion
`COOH (IC50 on NEP 3.6 X   "9 M, IC50 on TLN 4.0 X 10"6
`M) indicates again that the TLN active site can be used
`With the aim of improving NEP active-site recognition,
`as a model to investigate recognition of the active site of
`a new series of thiol inhibitors expected to interact with
`NEP. From the crystallographic data of the complex
`the Si, S'i, and S'2 subsites of the enzyme was synthesized
`TLN-thiorphan,41 the assumed interactions of this in-
`(Table 1). The development of these compounds was based
`hibitor with the active site of NEP are shown in Chart 1 A.
`on the structure of a recently described36 thiol inhibitor
`This model allows two possible types of binding to be
`(compound A, Table 1) and of NEP inhibitors of general
`proposed. Firstly, the inhibitors bind to the active site of
`formula HOOC-CH(CH2Ph)-CH2-CH(CH2Ph)-CO-AA
`NEP as shown in Chart IB, i.e., with the three side chains
`(compound B, Table l).40 Some of these latter compounds
`In this case,
`were shown to have IC50S for NEP in the nanomolar range,
`the
`fitting the Si, S'i, and S'2 subsites.
`suggesting that their interaction with the three subsites
`decreased affinity of these compounds, related to the
`(Si, S'i, and S'2) of the enzyme are optimized.
`dibenzylglutaryl derivatives, could be explained by the
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`

`New Thiol Inhibitors of NEP
`interaction of the three side chains with their respective
`inhibiting the perfect alignment of the thiol
`subsites,
`groups with the zinc atom, required for optimal coordina-
`tion.42 In the case of dibenzylglutaryl derivatives, such as
`B, this problem could be overcome by the enhanced degree
`of freedom of the carboxylate group and its possibility of
`interacting with the zinc atom, by using one or two of its
`oxygen atoms, as amply demonstrated by crystallographic
`analysis of complexes between various carboxyl inhibitors
`and TLN.15
`However, another explanation may account for the data
`reported in Table 1. For both compound A and its
`analogs,36 or for compound B and its analogs,40 the nature
`of the C-terminal residue is of great importance for the
`In Table 1 it can be seen that IC50S
`inhibitory activity.
`for NEP were the same for compounds 17 and 26 or 18 and
`27S, which differed by the presence of a free carboxylate
`in 26 and 27S and an amide group in 17 and 18. However,
`it is now well known that, due to the presence of Arg102
`in the active site of NEP, lower IC50S are obtained for
`inhibitors containing a free C-terminal carboxylate rather
`than a carboxamide group.26 More significant was the
`fact that compounds 21 and 24, which contain a C-terminal
`proline, have the same IC50 for NEP as 19 or 20 which are
`terminated by an alanine or a ß-alanine residue, as it has
`also been demonstrated that the S'2 subsite of NEP poorly
`recognizes a proline ring.16·43 Moreover, the IC50 values of
`27R and 27S on both NEP and ACE were almost identical
`(Table 1), although the S'2 subsite of these enzymes has
`a 100-1000 times better affinity for amino acids of S
`configuration.17’44-45
`Likewise, the comparison of the inhibitory potencies of
`compounds 20 and 21 on ACE surprisingly showed that
`they are identical, although it is also well known that ACE
`does not accept a /3-amino acid in its S'2 subsite, due to
`its strong carboxy dipeptidase nature.32 Finally, com-
`pound 7b and its cyclic analog 6b, which is transformed
`to 7b during the incubation time at pH 7.4, also have the
`same inhibitory potencies on NEP and ACE, although
`they do not contain a C-terminal amino acid. All these
`arguments seem to favor the mode of interaction with the
`active site of NEP (and ACE) in which the residues Ri
`and R2 interact with the S'i and S'2 subsites of the enzyme
`as shown schematically in Chart 1C, with the C-terminal
`In this model, the
`amino acid outside the active site.
`stabilizing hydrogen bonds between the amide group of
`the inhibitor and the side chains of Arg747 and Asn542,
`which are assumed to be important for the formation of
`stable complexes with NEP’s active site, do not occur.
`This proposal seems to be confirmed by experiments
`performed with 7b and 25 on the mutated enzyme Glu102-
`NEP25’26 that we have previously used as an index of
`inhibitor positioning in the enzyme's active site.46,36 As
`shown in Chart 1, Arg102 is thought to be positioned to
`interact with the C-terminal carboxyl of a P'2 residue.
`When Arg102 was replaced by Glu, the K¡ of thiorphan for
`2 orders of
`the mutated enzyme increased by over
`magnitude, due to the ionic repulsion between the two
`negatively charged groups. Under the same conditions,
`the Ki of thiorphan amide which has no free carboxylate
`was increased only by a factor of 6. The IC50S of 7b and
`25 on the mutated enzyme were
`respectively 3 X 10-5 and
`2 X   -6 M. These values correspond to a loss of affinity,
`when compared to the wild-type NEP, of about 120-fold
`for 7b and 7-fold for 25. Taken together, these results
`
`Journal of Medicinal Chemistry, 1994, Vol. 37, No. 12
`1869
`strongly suggest that both compounds interact with S'i
`and S'2 subsites of NEP and that the C-terminal tyrosine
`of 25 is outside the active site of the enzyme.
`In conclusion, the new series of thiol inhibitors described
`in this paper was not able to clarify the nature of the Si
`Indeed, it seems that both the
`subsite of NEP and ACE.
`hydrophobic character of the S'i subsite and the high
`tendency of the thiol group to optimize the complexation
`of the Zn2+ ion direct the positioning of the inhibitors in
`such a way that these two interactions are greatly privileged
`to the prejudice of any other stabilizing interactions, i.e.,
`with Arg747, Asn542, or Arg102. Consequently, it seems more
`easy to explore the Si subsite of NEP with inhibitors which
`do not contain a thiol group as a zinc ligand. Carboxylates
`have been intensively used, but hydroxamates or phos-
`phoryl groups may also be useful.
`Experimental Section
`I. Biological Tests.
`[3H]Tyr-D-Ala2-Leu-enkephalin (52 Ci/
`mmol) was obtained from CEA (Saclay, France). N-Cbz-Phe-
`His-Leu was from Bachem (Bubbendorf, Switzerland). Neutral
`endopeptidase was purified to homogeneity from rabbit kidney
`as previously described.47 Recombinant human angiotensin
`converting enzyme obtained as described48 was a generous gift
`from Prof. Corvol (Collége de France, Paris, France).
`Assay for Neutral Endopeptidase Activity.
`ICeo values
`were determined as previously described.36 NEP (final concen-
`tration 1 pmol/100 mL, specific activity for [3H]-D-Ala2-Leu-
`enkephalin 0.3 nmol/mg/min) was preincubated for 15 min at 25
`°C with or without increasing concentrations of inhibitor in a
`total volume of 100 µ , in 50 mM Tris-HCl buffer, pH 7.4.
`[3H]-
`D-Ala2-Leu-enkephalin (Km = 30 µ ) was added to a final
`concentration of 20 nM, and the reaction was stopped after 30
`min by adding 10 µ1* of 0.5 M HC1. The tritiated metabolites
`formed were separated on polystyrene beads. The mutated
`enzyme Glu102-NEP was obtained as previously described.26 The
`inhibitory potency of the inhibitors tested on this enzyme was
`determined by the method described for NEP.
`Assay for Angiotensin Converting Enzyme Activity.
`Enzymatic studies on ACE were performed using N-Cbz-Phe-
`His-Leu as substrate ikm = 50 mM) as previously described.49
`ACE (final concentration 0.02 pmol/100 µ , specific activity on
`Cbz-Phe-His-Leu 13 nmol/mg/min) was preincubated for 15 min
`at 37 °C with various concentrations of the tested inhibitors in
`50 mM Tris-HCl buffer, pH 8.0. N-Cbz-Phe-His-Leu was added
`to a fined concentration of 0.05 mM. The reaction was stopped
`after 15 min by adding 400 µ  of 2 M NaOH. After dilution with
`3 mL of water, the concentration of His-Leu weis determined
`following the fluorimetric assay described by Cheung et ed.60 with
`a MPF 44 A Perkin-Elmer spectrofluorimeter (excitation 365
`nm, emission 495 nm). The cedibration curve for His-Leu wets
`obtained by addition of increetsing concentrations of His-Leu
`into 0.1 mL of 5.0 M Tris-HCl buffer, pH 8.0, containing the
`denaturated enzyme.
`As the compounds tested were synthesized under their disulfide
`forms, 100 equiv of DTT was added to the stock solutions of the
`inhibitors before dilution.
`II. Chemistry. The amino acids were from Bachem (Bubben-
`dorf, Switzerland). Chlorotrimethylsilane, dimethyl malonate,
`benzyl bromide, 2-iodobutane, and (4-methoxyphenyl) methyl
`mercaptan, were from Aldrich (Strasbourg, France). BOP reagent
`was from Propeptide (Vert le Petit, France).
`from SDS (Peypin,
`The solvents (Normapur label) were
`France). The purity of the synthesized compounds was checked
`by thin-layer chromatography on silica gel plates (Merck 60F
`254) in the following solvent systems (v/v): A, EtOAc/hexane =
`1/1; B, EtOAc/hexane = 1/4; C, CHaCL/MeOH = 14/1; D, EtOAc/
`hexane = 1/2; E, CHCWMeOH/HOAc = 9/1/0.1; F, CHCla/MeOH
`= 9/1; G, EtOAc/hexane = 2/3. Plates were revealed with UV,
`iodine vapor, or ninhydrin. The purity of the final compounds
`was checked by HPLC on a reverse-phase Nucleosil Ce column
`(SFCC) with CH3CN/TFA 0.05 % as the mobile phase. The eluted
`The structure of all the
`peaks were monitored at 210 nm.
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1005, p. 005
`
`

`

`1870 Journal of Medicinal Chemistry, 1994, Vol. 37, No. 12
`compounds was confirmed by   NMR spectroscopy (Brüker
`AC 270 MHz) in [2He]DMSO using HMDS as an internal
`reference. Melting points of the crystallized compounds were
`determined on an Electrothermal apparatus and are reported
`uncorrected.
`The following abbreviations are used, EtOAc, ethyl acetate;
`HOAc, acetic acid; MeOH, methanol; EtOH, ethanol; DMF,
`dimethylformamide; THF, tetrahydrofuran; DMSO, dimethyl
`sulfoxide; HMDS, hexamethyldisiloxane; EtgN, triethylamine;
`HOBt, 1-hydroxybenzotriazole; DCC, dicyclohexylcarbodiimide;
`DCU, dicyclohexylurea; Et¡¡0, diethyl ether; TFA, trifluoroacetic
`acid; BOP, benzotriazol-l-yloxytris(dimethylamino)phospho-
`nium hexafluorophosphate; DIEA, diisopropylethylamine.
`General Procedure for the Preparation of Compounds 1.
`Procedure I. To a solution of CR)-2-bromoalkanoic acid
`(prepared from the corresponding (fi)-a-amino acid87) in DMF
`was added at 0 °C a solution of potassium (4-methoxybenzyl)-
`thiolate (prepared from 4-methoxybenzyl mercaptan and KOH
`in DMF (5 equiv)). After stirring overnight at room temperature,
`the mixture was evaporated in vacuo and the residue dissolved
`in water. The aqueous layer was washed (3X) by EtOAc, acidified
`to pH 2 by 3 M HC1, and extracted by EtOAc. The organic layer
`was washed, dried (Na2S04), and evaporated in vacuo.
`la (Ri = CH2Ph): oily product (98%), Rf (G) 0.55;   NMR
`  2.78 and 3.00 (CH2(Ph)), 3.35 (CH(COOH)), 3.70 (CH30), 3.80
`(CH2S), 6.8 and 7.10, 7.29 (Ph), 12.50 (COOH).
`lb (Ri =
`(CH2)2Ph): oily product (76%), Rf (B) 0.60;   NMR
`  1.77 and 2.00 (CHa8), 2.51 (0 2 ), 3.00 (CH(COOH)), 3.69
`(CHgO), 3.70 (CHS), 6.80, 7.00-7.18 (Ph), 12.46 (COOH).
`lc (Ri = CH(CH3)CH2CH3): oily product (92%), Rf (A) 0.77;
`  NMR   0.72 (CH3(CH2)), 0.85 (CH3(CH)), 1.10,1.30, and 1.70
`(CHiCH), 2.9 (CH(COOH)), 3.7 (CHaO + CH2S), 6.8 and 7.15
`((p-MeO)Ph), 12.43 (COOH).
`General Procedure for the Preparation of Compounds 2.
`Procedure II. The carboxylic acids 1 were dissolved in MeOH,
`and SOCl2 (1.7 equiv) was added dropwise at 0 °C. The mixtures
`were stirred for 1 h at room temperature and 40 min at 80 °C.
`The solvents were evaporated. The residues were dissolved in
`EtOAc, washed with water, 2% NaHC03, and brine, dried (Na2-
`S04), and evaporated in vacuo. The crude esters were dissolved
`in a mixture of THF and EtOH. NaBH4 (4 equiv) and LiCl (4
`equiv) were added at 0 0 C, and the mixtures were stirred overnight
`at room temperature. After evaporation of the solvents, the
`residues were taken off in water and extracted by EtOAc. The
`organic layers were washed, dried (Na2S04), and evaporated in
`vacuo. The crude alcohols were purified by chromatography on
`silica gel using EtOAc/hexane = 1/4 as eluents.
`2a (Ri = CH2Ph): colorless solid, 3.9 g (89%), mp 59-60 °C,
`Rf (A) 0.47;   NMR (DMSO)   2.6 and 2.95 (CH2(Ph)), 2.70
`(CHS)), 3.30-3.45 (CH2O), 3.55 (CHS), 3.68 (CH3O), 4.8 (OH),
`6.75-7.15 (Ph + (p-MeO)Ph). Anal.
`(ChHmOjS) C, H, S.
`(CH2)2Ph): colorless oil, 1.27 g (67%), R¡ (A) 0.5;  
`2b (Ri =
`NMR (DMSO)   1.5 and 1.9 (CHyS), 2.49-2.60 (CH2Ph + CH-
`(S)), 3.42-3.52 (CH2O), 3.68 (CHS), 3.72 (CH30), 4.75 (OH),
`6.80-7.15 (Ph + (p-MeO)Ph). Anal.
`(Ci8H2202S) C, H, S.
`2c (Rj = CH(CH3)CH2CH3): colorless oil, 4.1 g (83%), R¡ (A)
`0.61;   NMR (DMSO)   0.65 (2CH3), 1.2 (CH2(CH3)), 1.72
`(CHCH3), 2.52 (CHS), 3.35-3.50 (CH2O), 3.62 (CHS), 3.68
`(CHaO), 4.68 (OH), 6.S-7.2 ((p-MeO)Ph). Anal.
`(C14H2202S) C,
`H, S.
`General Procedure for the Preparation of Compounds 3.
`Procedure III. To a solution of alcohol 2 in MeCN at 0 °C
`under argon was added Nal (1 equiv) followed by dropwise
`addition of chlorotrimethylsilane (1.2 equiv) over 5 min. The
`mixture was stirred for 90 min, poured into water, and extracted
`with ether. The organic layer was washed with 5% aqueous
`Na2S203 and brine, dried (Na2S04), and evaporated to give the
`crude derivative. This compound in 1,2-dimethoxyethane at 0
`°C was added to a solution of the sodium salt of dimethyl
`malonate, prepared from dimethyl malonate (1.5 equiv) and NaH
`(1.5 equiv) in 1,2-dimethoxyethane at 0 °C under argon for 10
`min. The mixture was stirred for 40 h at room temperature,
`evaporated, and partitioned between water and EtOAc. The
`organic phase was washed with H20 and brine, dried (Na2S04),
`evaporated, and purified by flash column chromatography
`(EtOAc/hexane 1/7).
`
`Gomez-Monterrey et al.
`
`3a (Ri = CH2Ph): colorless solid, 4.4 g (83%), mp 64-65 °C,
`Rf (B) 0.29;   NMR (DMSO)   1.7 and 2.0 (CH2), 2.61 (CH-S),
`2.71-2.82 (CH2(Ph)), 3.45-3.65 (3CHsO + CH2S + CH(malonate)),
`6.8-7.15 (Ph + (p-CH30)Ph).
`(CH2)2Ph): colorless oil, 1.4 g (80%), Rf (B) 0.26; lH
`3b (Ri =
`NMR (DMSO)   1.60-1.72 (CHS), 1.90-2.10 ((CH)-CH2(CH)),
`2.38 (CH-S), 2.50-2.60 (CH2Ph), 3.49-3.69 (3CH30 + CH2S +
`CH (malonate)), 6.80-7.12 (Ph + (p-MeO)Ph).
`3c (Ri = CH(CH3)CH2CH3): colorless oil, 4.0 g (79%), Rf (B)
`0.36;   NMR (DMSO)   0.65-0.79 (2CH„), 1.10-1.35 (CH2(CH3)),
`1.55 (CH(CH3)), 1.76-2.08 (CH2), 2.31 (CH-S), 3.55-3.69 (3CH30
`+ CH2S + CH(malonate)), 6.82 and 7.12 ((p-MeO)Ph).
`General Procedure for the Preparation of Compounds 4.
`Procedure IV. To HNa (1 equiv) in 1,2-dimethoxyethane at 0
`°C under argon was added compound 3 (1 equiv). After the
`mixture was stirred for 15 min at 0 °C, a solution of R2X (1.3
`equiv) in 1,2-dimethoxyethane was added. Stirring was continued
`at room temperature for 18 h, the solvent was removed, and the
`residue was diluted with EtOAc and washed with H20 and brine.
`After drying (Na2S04), the solvent was removed at reduced
`pressure and the residue chromatographed (EtOAc/hexane 1/8).
`4a (Ri = R2 = CH2Ph): colorless solid, 4.5 g (85%), mp 96-97
`°C, Rf (B) 0.35;   NMR (DMSO)   1.86 (CH2), 2.62 (CH-S),
`2.82-3.10 (2CH2-Ph), 3.48-3.62 (3CH30 + CHS), 6.62-7.15 (Ph
`+ (p-OCHg)Ph).
`4b (Ri = CH2Ph, R2 = CH(CH3)CH2CH3): colorless oil, 2.8 g
`(56.7%),fí,(B) 0.42;   NMR (DMSO)   0.41-0.70 (2CH„), 0.75-
`0.98 (CH2(CH3)), 1.6 (CH(CH3)), 1.89 and 2.15 (CH2), 2.6 (CH2-
`Ph), 2.80 (CH-S), 3.45-3.62 (3CHsO + CH2S), 6.75-7.12 (Ph +
`(p-MeO)Ph).
`4c (Ri = (CH2)2Ph, R2 = CH(CH3)CH2CH3): colorless oil, 0.51
`g (89%), Rf (B) 0.39   NMR (DMSO)   0.75 (2CH3), 0.90-1.40
`(CH2(CH3) + CH(CHg)), 1.65 (CH-S + CHS), 1.92-2.15 (CH2),
`2.60 (CH2(Ph)), 3.50-3.68 (3CH30 + CH2S), 6.8-7.18 (Ph + (p-
`MeO)Ph).
`4d (Ri = CH(CH3)CH2CH3, R2 = CH2Ph): colorless oil, 1.75
`g (83%), Rf (B) 0.4;   NMR (DMSO)   0.65-0.78 (2CH3), 1.02-
`1.40 (CH2(CH3) + CH(CH3)), 1.80 (CH2), 2.72 (CH-S) + CH2-
`(Ph), 3.54-3.68 (3CH30 + CHS), 6.82-7.20 (Ph + (p-OMe)Ph).
`4e (Ri = R2 = CH(CH3)CH2CH3): colorless oil, 1.2 g (52%),
`Rf (B) 0.54;   NMR (DMSO)   0.60-0.80 (4CH3), 0.85-1.80 (2CHr
`(CH3) + 2CH(CH3)), 1.8 and 2.15 (CH2), 2.55 (CH-S), 3.52-3.65
`(3CH30 + CH2S), 6.78 and 7.1 ((p-MeO)Ph).
`General Procedure for the Preparation of Compounds 5.
`Procedure V. A solution of 4 in DMSO was treated with 20%
`KOH (3 equiv), and the mixture was stirred for 90 min at 60-70
`°C. The mixture was diluted into H20 and washed with ether.
`The aqueous layer was acidified with 3 N HC1 to pH 3 and
`extracted with EtOAc. The organic layer was washed with H20
`and brine, dried (Na2S04), evaporated, and purified by flash
`column chromatography (EtOAc/hexane 3/2).
`5a (Ri = R2 = CH2Ph): colorless oil, 2.8 g (77%), R/ (C) 0.53;
`  NMR (DMSO)   1.40-1.72 (CH¡¡), 2.5-2.82 (2CH-CH2), 3.45
`(CHaS), 3.68 (CH30), 6.8-7.12 (2Ph + (p-MeO)Ph), 12.12 (COOH).
`Anal.
`(CaeHagOgS) C, H, S.
`5b (Ri = CH2Ph, R2 = CH(CH3)CH2CH3): colorless oil, 0.72
`g (56%), Rf (C) 0.5;   NMR (DMSO)   0.55-0.68 (2CH3), 1.00-
`1.31 (CH(CH3) + CH2(CH3)), 1.52-1.68 (CH2), 2.25 (CH(COOH)),
`2.60-2.78 (S-CH-CH2(Ph)), 3.5 (CH2S), 3.68 (CH30), 6.80-7.10
`(Ph + (p-