J. Med. Chem. 1995, 38, 1689-1700
`
`1689
`
`Dicarboxylic Acid Dipeptide Neutral Endopeptidase Inhibitors
`Gary M. Ksander,* Raj D. Ghai, Reynalda deJesus, Clive G. Diefenbacher, Andrew Yuan, Carol Berry,
`Yumi Sakane, and Angelo Trapani
`Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, 556 Morris Avenue, Summit, New Jersey 07901
`Received August 19, 1994s
`
`The synthesis of three series of dicarboxylic acid dipeptide neutral endopeptidase 24.11 (NEP)
`the amino butyramide 21a exhibited potent NEP
`inhibitors is described.
`In particular,
`inhibitory activity (IC50 = 5.0 nM) in vitro and in vivo. Blood levels of 21a were determined
`using an ex vivo method by measuring plasma inhibitory activity in conscious rats, mongrel
`dogs, and cynomolgus monkeys. Free drug concentrations were 10-1500 times greater than
`the course of a 6 h experiment. A good correlation of free
`the inhibitory constant for NEP over
`drug concentrations was obtained when comparing values determined by the ex vivo analysis
`to those calculated from direct HPLC measurements. Plasma atrial natriuretic
`factor
`(exogenous) levels were elevated in rats and dogs after oral administration of 19a. Urinary
`volume and urinary sodium excretion were also potentiated in anesthetized dogs treated with
`21a.
`
`Atrial natriuretic factor (ANF)1 is a potent diuretic,
`natriuretic, and vasorelaxant hormone. These proper-
`ties have led many investigators to speculate that this
`peptide might be effective for treating hypertension,
`congestive heart failure, and renal diseases.2 To cir-
`cumvent the inherent problems with the development
`of a peptide as a therapeutic agent, several approaches
`can be taken including the identification of nonpeptidic
`the clearance of the
`agonists or agents that affect
`is generally accepted that there are
`It
`two
`peptide.
`mechanisms responsible for the clearance of ANF.
`receptor-mediated internalization and deg-
`These are
`radation by so-called C-receptors3 and enzymatic hy-
`drolysis.4 Numerous groups have independently dem-
`onstrated that kidney membrane preparations degrade
`ANF enzymatically. Furthermore inactivation and the
`loss of biological activity in vivo,5 at least in part, occur
`via cleavage of the Cys7—Phe8 peptide bond by neutral
`endopeptidase 24.II6 (NEP; EC 3.4.24.11). Although
`the relative importance of NEP in the metabolism of
`endogenous ANF remains to be determined conclusively,
`NEP inhibitors have been shown to elicit ANF-like
`responses in animal models.7a-g Despite these encour-
`aging experimental results, recent clinical trials have
`In
`shown, at best, moderate pharmacologic activity.
`these clinical studies711-? several NEP inhibitors includ-
`ing sinorphan, SCH34826, and candoxatril have pro-
`duced no or modest antihypertensive effects. Somewhat
`superior, albeit moderate, effects of these agents have
`been observed in patients with congestive heart failure.
`Since these poor clinical outcomes may arise from
`inferior pharmacokinetics or potency, we have sought
`to identify novel NEP inhibitors with superior pharma-
`cologic properties.
`Chemistry
`The preparation of racemic unsymmetrical glutaric
`acids, Table 1, is outlined in Scheme 1. p-Phenylbenz-
`converted in successive steps to the
`aldehyde was
`oxetane 4 by condensation with dimethyl malonate/
`NaOMe, hydrogenation, and LAH reduction to the diol
`
`® Abstract published in Advance ACS Abstracts, April 1, 1995.
`0022-2623/95/1838-1689$09.00/0
`
`followed by monotosylation and cyclization with n-
`butyllithium. The oxetane can be converted to a 2,4-
`alkyl-substituted y-hydroxy ester (5) after boron tri-
`fluoride etherate-catalyzed condensation with various
`ierZ-butyl ester enolates at -78 to -90 °C. A variety of
`aliphatic, aryl-substituted, and heterosubstituted ester
`enolates8 as well as phosphates readily react with
`2-methylene biphenyl oxetane 4 to give diastereomeric
`mixtures separable by flash chromatography. However,
`less tedious. The y-hy-
`separation at compound 7 was
`droxy ester 5 was oxidized with pyridinium dichromate
`to give the 2,4-disubstituted mixed glutaric acid ester
`6. Activation of the carboxylic acid and coupling with
`a protected amino acid ester followed by sodium hy-
`droxide and/or TFA hydrolysis gave the diacids 8.
`The symmetrical glutaramides 10, Table 2, were
`prepared9® by heating /3-alanine or cis-4-aminocyclohex-
`anecarboxylic acid in a mixture of methylene chloride/
`pyridine with the appropriate irons-2,4-disubstituted
`glutaric acid anhydrides, Scheme 2.
`Preparation of the chiral amino butyramides 20
`(Table 3) is outlined in Scheme 3. N-Boc-D-tyrosine
`is converted to the triflate 11 which
`methyl ester
`undergoes Suzuki coupling reaction with phenylboric
`acid to give 12 in good yield. Conversion of the acid to
`the hydroxymate followed by lithium aluminum hydride
`reduction gave the aldehyde 15. Wittig condensation
`of aldehyde 15 with (carbethoxyethylidene)triphe-
`nylphosphorane gave the olefin 16 as one geometric
`In the analogous series, aldehyde 15 was
`isomer.
`condensed with (carbethoxymethylene)triphenylphos-
`phine leading to compound 21i. Palladium-catalyzed
`hydrogenation of 16 gave a 6:1 diastereomeric mixture
`removed with HC1
`17. The Boc protecting group was
`and condensed with succinic anhydride affording the
`mixed acid ester 19. The diastereomers are
`readily
`separated by flash chromatography after treatment of
`19 with JV,/V-dimethylforamide di-ieri-butyl acetal to
`give the mixed ieri-butyl/ethyl ester 20. Removal of the
`ier/-butyl group with TEA gave the chiral prodrugs 19
`which are readily converted to the chiral diacids 21 with
`base treatment. Compounds 21e,f were obtained fol-
`© 1995 American Chemical Society
`
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`

`

`1690 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 10
`Scheme Ia
`
`Ksander et al.
`
`d
`
`° (a) Dimethyl malonate, piperidine; (b) H2, Pd/C; (c) LÍBH4; (d) TsCl, nBuLi; (e) ester enolate, BF30EÍ2; if) pyridinium dichromate; (g)
`/9-alanine ieri-butyl ester, HOBT, EDC1; (h) TFA.
`Scheme 2
`
`amino acid, pyridine
`
`HOjC— (CH2)„—N
`
`9
`
`10
`
`sequence of reactions except glutamic
`lowing the same
`substituted for succinic
`and adipic anhydride were
`anhydride.
`In Vitro Structure-Activity Discussion
`A series of glutaramides9® were published several
`years ago describing very potent compounds as assessed
`by an in vitro assay using Leu enkephalin as substrate.
`Changing to our present assay using the synthetic
`substrate GAAP, we discovered that
`the previously
`potent compounds 10a and 21j were
`no longer active
`in the nanomolar concentrations, 8 and 2 nM, respec-
`tively, but displayed activity in micromolar concentra-
`
`tions. The weak in vitro activity was consistent with
`the poor pharmacology observed with these compounds;
`however, many factors could lead to this observation.
`felt that this class of compounds should
`Therefore it was
`be reinvestigated.
`Table 1 lists the series of racemic unsymmetrical
`glutaramides. The activity of other series of carboxy-
`alkyl dipeptides/glutaric acids inhibitors711'9 has been
`reported previously. The Pi' substituent, biphenyl-
`methyl, remained constant while the P2' substituent (R1)
`interchanged between /3-alanine and isoserine. The
`was
`Pi substituent was modified with alkyl, aralkyl, alkoxy,
`and arlyoxy groups. Potency improved by a modest
`3— 5-fold by changing /3-alanine to isoserine (8a—d).
`When /3-alanine and biphenylmethyl were
`kept con-
`stant, very little change in inhibitory activity was
`observed between alkyl, aralkyl, alkoxy, and aryloxy Pi
`modifications. However, in the absence of a Pi sub-
`(81), the activity was decreased approximately
`stituent
`4- fold. The stereochemistry is predictably important.
`Comparing the two pairs of phenoxy (8e,f) and methoxy
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1006, p. 002
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`

`

`Dicarboxylic Acid Dipeptide NEP Inhibitors
`Scheme 3°
`
`Journal of Medicinal Chemistry, 1995, Vol. 38, No. 10
`
`1691
`
`BocHN
`
`BocHN
`
`d
`
`BocHN
`
`h
`
`tBu02C
`
`“ (a) Triflic anhydride; (b) phenylboronic acid, tetrakis(triphenylphosphine)palladium(0); (c) NaOH; (d) HNCH3OCH3, EDC1, HOST;
`(e) L1AIH4; (f) (carbethoxyethylidene)triphenylphosphorane; (g) H/Pd/C; (h) HC1; (i) succinic anhydride; (j) M^-dimethylformamide di-
`ierf-butyl acetal; (k) NaOH.
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1006, p. 003
`
`

`

`1692 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 10
`In Vitro NEP Inhibition of Unsymmetrical
`Table 1.
`Glutaramides
`
`0
`
`O
`
`Table 3.
`
`In Vitro NEP Inhibition of Amino Butyramides
`
`Ksander et al.
`
`compd
`8a
`8b
`8c
`8d
`8e
`8f
`8g
`8h
`8i
`
`8j
`8k
`
`81
`8m
`
`R'
`/3-Ala
`isoserine
`/3-Ala
`isoserine
`/3-Ala
`/3-Ala
`/3-Ala
`/3-Ala
`/3-Ala
`
`/3-Ala
`/3-Ala
`
`/3-Ala
`/3-Ala
`
`R
`
`CH2-Ph(3,4-OMe)
`CH2-Ph(3,4-OMe)
`nBu
`nBu
`OPh
`OPh (erythreo)
`OCHs
`OCHs (erythreo)
`S-_
`
`CH2—Ph^(x j
`ch2ch2och3
`
`H
`CH3
`
`IC5o (nM)
`54
`11
`66
`21
`44
`>1000
`42
`>1000
`19
`
`46
`54
`
`155
`41
`
`Table 2.
`In Vitro NEP Inhibition of Symmetrical
`Glutaramides
`
`O
`
`0
`
`R^T^0H
`
`ch2 ch2
`R
`R
`
`compd
`10a9a
`10b
`10c
`
`lOd
`
`lOe
`
`lOf
`10g
`
`lOh
`
`Ri
`
`/3-Ala
`/3-Ala
`/9-Ala
`
`/3-Ala
`
`/9-Ala
`
`/3-Ala
`HaN—C^/)- c°2H
`h2n—co2h
`
`R
`
`Ph
`Ph-Ph
`Ph-N^
`S—
`Ph—(\ jN —
`/
`Ph~<
`Ph-O-Ph
`Ph~<2
`Ph
`
`IC50 (nM)
`
`1200
`49
`36
`
`>1000 (43% at luM)
`
`489
`
`203
`515
`
`52% at 10 µ 
`
`(8g,h) diastereomers, greater than a 100-fold difference
`in activity was observed.
`Table 2 lists a series of racemic symmetrical glutara-
`mides. The biaryl derivatives 10b,c (IC50 = 49, 36 nM,
`the most potent. This effect
`is
`respectively) were
`governed entirely by the aryl—aryl Pi' substituent. The
`chiral /3-alanine dibenzyl derivative 10a is a relatively
`in the GAAP assay with an IC50 of 1.2
`weak inhibitor
`µ . Substituting the monophenyl derivative in the
`para position with an isopropyl group (lOe) or a phenoxy
`substituent (lOf) improves activity as compared to the
`parent unsubstituted phenyl derivative 10a. However,
`a 2—3-fold decrease in activity is observed when com-
`paring these derivatives to the biphenylmethyl com-
`pound 10b. The phenylthiazole lOd, theoretically ster-
`ically compatible with the Pi' pocket on the basis of the
`
`FV
`
`compd
`21a (R,S isomer)
`21b (S,R isomer)
`21c (R,R isomer)
`21d (S,S isomer)
`21e
`21f
`21g
`21h
`21i
`21j9a
`
`R"
`R'
`R
`Ph
`C0(CH2)2C02H
`CHS
`Ph
`ch3
`C0(CH2)2C02H
`Ph
`C0(CH2)2C02H
`CH3
`ch3
`Ph
`C0(CH2)2C02H
`ch3
`Ph
`C0(CH2)sC02H
`Ph
`C0(CH2)4C02H
`CH3
`coch2co2h
`Ph
`CHs
`Ph OCHs
`C0(CH2)sC02H
`H
`Ph
`C0(CH2)2C02H
`C0(CH2)2C02H H
`CH2ph
`
`IC50 (nM)
`5
`190
`700
`27
`90
`324
`92
`49
`99
`4000
`
`inhibitory activity of 10b,c, was considerably weaker
`than other aryl—aryl derivatives. Comparison of 8i,
`Table 1 (19 nM), with lOd (>1 µ ) implies that one of
`the heteroatoms, possibly the nitrogen, is adversely
`affecting the enzyme interaction in the Pi' pocket.
`The inhibitory activities of the aminobutyr amides are
`compiled in Table 3. On the basis of the data generated
`in the two previous series (Tables 1 and 2), we assumed
`aliphatic or aralkyl modifications at the Pi site would
`not significantly alter the inhibitory activity.
`In addi-
`the Pi' biphenylmethyl substituent should be
`tion,
`nearly optimum for this series. Therefore, a limited
`number of compounds were prepared (Table 3) with Pi
`substituents being methyl and methoxy and Pi' sub-
`stituents as biphenylmethyl, while the P2' functionality
`varied from malonyl
`to
`to succinyl
`to butyryl
`was
`glutaryl to adipyl acids.
`The most active compound was 21a, IC50 = 5 nM. All
`four diastereomers, f?,S-21a, S,7?-21b, R,R-21c, and
`S,S-21d, were prepared, and IC50 values of 5,190, 700,
`and 27 nM were determined, respectively. The succinic
`acid in the P2' site appears to be optimal since extension
`of the carboxylic acid chain by one (21e) and two (21f)
`In
`methylene units decreased activity 18- and 65-fold.
`addition decreasing the chain length by one methylene
`(21g) also showed an 18-fold decrease in activity.
`Although in series 1 there was
`no difference in activity
`between the Pi methyl and methoxy substituents, a 10-
`observed in the amino-
`fold change in activity was
`butyramide series (21a,h). As expected the Pi desm-
`ethyl derivative 21i was considerably less potent than
`21a.
`Comparison of the Pi' benzyl substituent 21j with
`other derivatives in Table 3 demonstrates the effects of
`a Pi' biphenylmethyl group. The biphenyl effect was
`also evident in the previous examples, shown in Table
`2. This effect has been reported in the amino carboxylic
`acid9b and amino phosphonic acid10 series. However,
`replacement of a benzyl group with the biphenylmethyl
`In the
`does not always result in a potency increase.
`thiol series,113 thiorphan (Pi' = benzyl, IC50 = 4 nM) is
`similar in potency to the biphenylmethyl compound (Pi'
`In addition, a 50-fold
`= biphenylmethyl, IC50 = 3 nM).
`decrease in activity has been reported for a thiol sulfonic
`acid seriesllb with the same Pi' biphenyl modification.
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1006, p. 004
`
`

`

`Dicarboxylic Acid Dipeptide NEP Inhibitors
`Table 4.
`Inhibitory Constants of NEP Inhibitors Determined
`from Different Substrates
`
`Selectivity of these compounds for NEP over other Zn
`metalloproteases, e.g., stromelysin, endothelin convert-
`ing enzyme, is not known; however, these compounds
`inactive (<50% inhibition) in ACE at a concentra-
`were
`tion of 10 µ .
`In Vitro Assays. Leu enkephalin, glutaryl-Ala-Ala-
`Phe-jS-naphthylamide, and ANF are used as substrates
`56, 37, and 18, respectively) to identify
`(Kcat/Km =
`inhibitors of NEP. We have compared the potencies of
`three different classes of NEP inhibitors, thiols, amino
`in these three
`phosphonic acid, and carboxylic acid,
`assays, Table 4. The IC50 values determined in these
`assays for thiorphan (a thiol), an amino phosphonate,10
`and 21a (a dicarboxylic acid) were similar, although the
`Leu-ENK assay gave somewhat
`lower values, and
`predictive of functional potency in vivo. However, the
`correlation of inhibitory activity between assays was not
`always constant. Specifically within the dicarboxylic
`acid series of compounds, for example, 10a and 81 gave
`differences in potencies of greater than 2 orders of
`magnitude when tested against GAAP and Leu-ENK
`as substrates. A possible explanation could be differ-
`
`Journal of Medicinal Chemistry, 1995, Vol. 38, No. 10
`1693
`in the kinetics of individual compounds in each
`ences
`assay. Needless to say, the IC50 values for the dicar-
`boxylic acid series of compounds vary significantly
`depending upon the assay used in determining these
`values and may not be predictive of in vivo functional
`responses.
`Pharmacokinetic Profile
`A pharmacokinetic profile was determined for each
`compound with adequate in vitro potency. Since phar-
`macokinetic measurements using HPLC techniques are
`labor intensive. The plasma concentrations of NEP
`inhibitors unbound to plasma proteins were determined
`the inhibi-
`by ex vivo analysis. This method measures
`that all activity is
`tory activity in plasma and assumes
`from the
`produced by the administered substance or
`active substance released via a suitable prodrug. This
`total plasma levels of active
`method does not measure
`substance but only the concentration of free inhibitor
`(i.e., not bound to plasma proteins). The total plasma
`concentration of compound can be calculated after the
`plasma protein binding is determined.
`The pharmacokinetic profile of 19a, the ethyl ester
`prodrug of 21a, was determined using the ex vivo
`method by measuring plasma inhibitory activity of 21a
`in conscious rats, anesthetized mongrel dogs, and
`In conscious
`conscious cynomolgus monkeys (Figure 1).
`rats, administration of 19a at 30 mg/kg po produced free
`plasma levels of the active NEP inhibitor 21a ranging
`from 0.64 to 0.05 µ 
`of the 6 h
`the course
`over
`experiment. These values are 10-100 times greater
`than the inhibitory constant of 21a (IC50 = 5 nM) for
`Intraduodenal administration of 19a at 30 mg/
`NEP.
`kg in anesthetized mongrel dogs produced plasma levels
`of 21a which ranged from 2.8 to 0.08 µ  at 90 min and
`In cynomolgus monkeys, dosing at 30
`6 h postdosing.
`mg/kg po gave concentration values of 8.51 and 0.21 µ 
`at 1 and 6 h, respectively. These concentrations of free
`inhibitor 21a were 1500 and 38 times higher than its
`IC50 value for NEP. The apparent elimination half-life
`of 21a was 4.6 ± 0.4 h in cynomolgus monkeys.
`The accuracy of the ex vivo method was next exam-
`ined using the monkey blood samples. Total drug levels
`of the active inhibitor 21a were determined by HPLC.15
`The values obtained at 30, 60, 120, 180, 240, 300, 360,
`and 1440 min were 41, 98, 50, 21, 9.0, 4.3, 3.2, and 0.2
`µ , respectively. The diacid 21a was determined to be
`94% plasma protein bound, and the free plasma con-
`centrations of 21a calculated from the HPLC data were
`in good agreement with those determined by ex vivo
`analysis (Figure 2).
`ANF Potentiation Assay. Plasma ANF concentra-
`tions were determined in animals infused with exog-
`enous ANF before and after administration of NEP
`inhibitors. Figure 3 shows the effects of 19a and (±)-
`candoxatril16 administered at 10 mg/kg po on plasma
`ANF levels in conscious rats. Plasma ANF levels are
`expressed as a percent of those measured in vehicle-
`treated animals which received the infusion of exog-
`enous ANF. ANF levels were
`increased significantly at
`all timé points (30—240 min) after the administration
`of 19a.
`In contrast, the same
`dose of (i)-candoxatril
`produced a marked increase in plasma ANF levels
`initially, but this effect progressively diminished and
`was not significant 3 h after dosing. 19a produced dose-
`
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`
`

`

`1694 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 10
`
`Ksander et al.
`
`3 5
`
`1
`
`I
`
`0
`
`1
`
`I
`
`12
`
`1
`
`1
`
`1
`
`I
`3
`
`1
`
`I
`
`4
`
`1
`
`I
`5
`
`I
`6
`
`I—1—I—'—I—'—I—1—I—1—I—1—I
`4
`0
`3
`2
`6
`
`1
`
`6
`
`Time (hr)
`Time (hr)
`Figure 1. The pharmacokinetics of 21a after oral administration of 19a (30 mg/kg po) in conscious DOCA-salt rats, anesthetized
`dogs, and conscious monkeys (left panel). Plasma levels of 21a shown in the figure were determined by the ex vivo method which
`the course of
`measures NEP inhibitor activity and does not directly detect 21a. The ratio of plasma 21a concentration/ICso over
`the experiment is also shown (right panel).
`
`• Free (21a] - calculated
`
`I-1-1-1-1-1-1—
`0
`2
`1
`
`5
`
`// -1
`24
`
`6
`
`4
`3
`Time (hr)
`Figure 2. Comparison of free plasma concentrations of 21a
`determined by the indirect ex vivo method and the value
`calculated from the total concentration obtained from HPLC
`measurements. The data were obtained from four cynomolgus
`monkeys.
`
`increases in plasma ANF concentrations
`dependent
`after intraduodenal administration in anesthetized dogs
`(Figure 4). At 30 mg/kg id, 19a increased ANF levels
`by a maximum of 107%; this effect diminished to 55%
`by 5 h after dosing. Substantial, albeit smaller, effects
`of 19a on plasma ANF levels were observed at lower
`doses.
`ANF-induced Diuresis and Natriuresis.
`In anes-
`thetized rats intraduodenally administered 19a (30 mg/
`kg) significantly increased ANF-induced natriuresis
`without affecting diuresis. Prior to the administration
`of ANF, there were
`no significant differences in mean
`arterial pressure, urine flow, or urinary sodium excre-
`tion when rats treated with 19a were
`compared to
`In vehicle-treated rats, ANF increased urinary
`controls.
`sodium excretion from 0.72 ± 0.25 to 3.26 ± 0.63 /zequiv/
`kg/min. This effect was potentiated in animals which
`received 19a ((0.63 ± 0.22)—(8.84 ± 2.13) /¿equiv/kg/
`min).
`The effects of 21a (10 mg/kg iv) on ANF-induced
`diuresis and natriuresis in mongrel dogs is shown in
`
`I-'-1-1-1-1-1-1-1
`0
`12
`
`3
`
`4
`
`Time After Inhibitor (hr)
`Figure 3. Effect of 19a and (±)-candoxatril administered
`orally at 10 mg/kg on plasma ANF concentrations in conscious
`rats infused with exogenous ANF. Values are the mean ± SEM
`for six and three rats treated with 19a and (±)-candoxatril,
`respectively.
`In vehicle-treated dogs, ANF increased uri-
`Figure 5.
`nary sodium excretion from 17.3 ± 3.6 to 199.5 ± 18.4
`/¿equiv/kg/min. This effect was potentiated significantly
`in animals which received 21a ((20.8 ± 4.2)-(289.2 ±
`28.8) /¿equiv/kg/min. Urinary volume was also potenti-
`ated in animals which received an iv administration of
`(0.09 ± .01H1.07 ± 0.09) mL; 21a
`21a (control,
`potentiation, (0.10 ± 0.01)-(1.59 ± 0.21) mL).
`in vitro data are presented for three
`In summary,
`series of NEP inhibitors. The pharmacokinetic profile
`of 19a/21a was determined in three species. The
`prodrug 19a was also shown to increase exogenous
`levels of ANF and enhance ANF’s natriuretic
`and
`diuretic activity. Although these experiments do not
`prove 21a will enhance endogenous ANF levels and
`natriuretic and diuretic activity, it does demonstrate the
`potential to elicit these activities.
`Experimental Section
`  NMR spectra were
`General Procedures.
`recorded on
`a Varían XL 400 MHz, Varían VR 300 MHz, and/or Broker
`
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`

`

`Dicarboxylic Acid Dipeptide NEP Inhibitors
`
`Time (hr)
`Figure 4. Effect of 19a on plasma ANF concentrations in
`anesthetized dogs following intraduodenal administration at
`3 {  = 2), 10 (  = 3), and 30 ( 
`= 2) mg/kg. Values are the
`mean ± SEM.
`
`Figure 5. Effect of 21a administered at 10 mg/kg iv on ANF-
`induced diuresis and natriuresis in anesthetized mongrel dogs.
`Values are the mean ± SEM for 11 and 15 dogs treated with
`21a and vehicle, respectively. The experiment consisted of two
`control periods (Ci, C2), a period of intrarenal arterial ANF
`infusion alone (  ), and three points of ANF infusion with or
`without 21a (  -A3) followed by two periods of recovery
`(termination of ANF infusion; Ri, R2). Urine volume (UV) and
`urinary sodium excretion were determined for each of these
`collection periods.
`AC 250 MHz spectrometer with tetramethylsilane as internal
`Infrared spectra were
`recorded on a Nicolet 5SXFT
`standard.
`spectrometer. Optical rotations were measured with a Perkin-
`Elmer Model 241 polarimeter. Melting points were
`taken on
`a Thomas-Hoover melting point apparatus and are
`uncor-
`rected.
`Dimethyl 2-([ 1,1 '-Biphenyl]-4-ylmethylene)-1,3-pro-
`panedioate (1). A solution of dimethyl malonate (34.7 g, 0.26
`mol), 4-biphenylcarboxaldehyde (30 g, 0.16 mol), benzoic acid
`(2.0 g, 16 mmol), and piperidine (2.1 g, 25 mmol) was refluxed
`in 100 mL of toluene. The water was
`removed azeotropically
`with a Dean-Stark trap for 1 h. The reaction mixture was
`cooled, diluted with ether/ethyl acetate, washed with 3 x 1 N
`
`1695
`
`Journal of Medicinal Chemistry, 1995, Vol. 38, No. 10
`HC1, 3 x NaHCOs, and brine, dried (MgSO*), filtered, and
`concentrated to give 60 g of 1 as a yellow oil. The crude
`product which contained excess dimethyl malonate was used
`as is in the next step.
`Dimethyl 2-([l,l'-Biphenyl]-4-ylmethyl)-l,3-propanedio-
`ate (2). Crude 1 was dissolved in 200 mL of hot THF/CH3OH
`and cooled, and 2 g of 5% Pd/C was added. The mixture was
`hydrogenated at room temperature and pressure for 20 h. The
`mixture was filtered through a pad of Celite, concentrated, and
`distilled using a Kugelrohr apparatus. The dimethyl malonate
`removed between 100-150 °C, while the product was
`was
`redistilled which
`collected at 180—230 °C. The product was
`solidified upon standing to a sticky solid. The solid was
`heated; hexanes were added and cooled to 0 °C. The product
` 
`was collected and dried to give 49.5 g of 2, mp 72-73 °C:
`NMR (CDCI3) ó 7.56 (d, 2H), 7.50 (d, 2H), 7.40 (t, 2H), 7.30 (t,
`1H), 7.27 (d, 2H), 3.70 (t, 1H), 3.69 (s, 6H), 3.24 (d, 2H).
`2- ([ 1, l'-Biphenyl] -4-ylmethyl)-1,3-propanediol (3). To
`a solution of 2 (29 g, 97 mmol) in 70 mL of THF was added 68
`mL of 2.0 M lithium borohydride. TLC indicated the presence
`of 3 after refluxing for 5 h. Therefore, an additional 20 mL of
`lithium borohydride was added and refluxing continued an-
`other 2 h. The mixture was cooled, poured into ice—water,
`extracted three times with ether/EtOAc, dried (MgSCL), fil-
`tered, concentrated, and slurried with ether. The solid was
`  NMR
`collected affording 18.9 g of 3 melting at 97-99 °C:
`(CDCI3) <5 7.6-7.2 (m, 9H), 3.8 (m, 2H), 3.7 (m, 2H), 2.68 (d,
`2H), 2.4 (s, 2H), 2.1 (m, 1H).
`3- ([ 1,l'-Biphenyl]-4-ylmethyl)oxetane (4). To a 0 °C
`solution of 3 (19.6 g, 81.0 mmol) in 600 mL of THF was added
`32.4 mL of 2.5 M nBuLi. The thick suspension was kept at 0
`°C and stirred for 30 min. To this suspension was added tosyl
`chloride (15.4 g, 81.0 mmol) in 75 mL of THF. The mixture
`was stirred for 1 h followed by the addition of a second
`equivalent of nBuLi (32.4 mL, 81.0 mmol). The mixture was
`heated to 60 °C for 4 h, cooled, poured into ice-water, and
`extracted three times with ether. The organic extract was
`washed with brine, dried (MgSO*), concentrated, and chro-
`matographed on silica gel eluting with ether:hexane (1:4) to
`  NMR (CDCI3)   7.57
`give 10.1 g of 4 melting at 79—80 °C:
`(d, 2H), 7.52 (d, 2H), 7.42 (1, 2H), 7.34 (t, 1H), 7.2 (d, 2H),
`4.82 (dd, 2H), 4.5 (1, 2H), 3.35 (septet, 1H), 3.06 (d, 2H).
`1.1 -Dimethylethy 1 (oR,yS)-a-Butyl-y-(hydroxymethyl)-
`[l,l'-biphenyl]-4-pentanoate (5). A solution of iert-butyl
`hexanoate (3.69 g, 21.4 mmol) in 20 mL of dry THF was added
`to a -78 °C solution of LDA (21.4 mmol) in 20 mL of THF and
`stirred for 1 h. The enolate was cooled to —110 °C to which
`the oxetane 4 (0.96 g, 4.3 mmol) in 20 mL of THF was added.
`To this mixture was added boron trifluoride etherate (2.91 g,
`21 mmol) in 20 mL of THF. The reaction temperature was
`raised to -40 °C and the mixture stirred for an additional 90
`min. The reaction was quenched with 20 mL of 3:1 (THF:
`HOAc) and the mixture diluted with EtsO and washed with 2
`x H2O, 2 x 1 N HC1, 2 x NaHCOs, and brine. The organic
`layer was dried (MgSO*), filtered, concentrated, and chromato-
`graphed eluting with ether:hexane (1:4) to give 1.4 g of 5 as a
`mixture of diastereomers. The diastereomers can be separated
`by chromatography. However, the separation was less tedious
`at a later stage (compound 7).
`1.1 -DimethylethyI (aR,yS)-a-Butyl-y-carboxy[l,l'-bi-
`phenyl]-4-pentanoate (6). To a solution of pyridinium
`dichromate (9.0 g, 23.9 mmol) in 125 mL of DMF was added 5
`(1.9 g, 4.79 mmol) in 25 mL of DMF, and the mixture was
`stirred for 28 h. The reaction mixture was poured into 500
`mL of water and extracted with ether. The organic layer was
`washed with 4 x H2O, 2 x NaHCOs, and brine, dried (MgSO*),
`filtered, and concentrated to give 1.8 g of 6 as a brown oil.
`1.1 -Dimethylethyl (aR,yS)-a-Butyl-y-[[[3-(phenylmeth-
`oxy)-3-oxopropyl] amino] carbonyl] [1,1 '-biphenyl] -4-pen-
`tanoate (7a). To a solution of 6 (0.76 g, 1.85 mmol), /3-alanine
`iert-butyl ester hydrochloride (0.673 g, 3.72 mmol), HOBT (0.50
`g, 3.72 mmol), and triethylamine (0.47 g, 4.64 mmol) in 8 mL
`of methylene chloride was added EDCI (0.88 g, 4.6 mmol). The
`mixture was stirred for 17 h, diluted with 200 mL of ether/
`ethyl acetate, washed with 1 N HC1, H2O, NaHCOs, and brine,
`dried (MgSO.4), and concentrated. The mixture was chromato-
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1006, p. 007
`
`

`

`1696 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 10
`graphed on silica gel eluting with hexane:ethyl acetate (9:1)
`to give 190 mg of the desired diastereomer 7a as a colorless
`oil, 300 mg of mixed fractions (mostly the desired diastere-
`omer), and 445 mg of the undesired diastereomer 7b.
`  NMR (CDC13)   7.56 (d, 2H), 7.46 (t,
`Compound 7a:
`2H), 7.38 (d, 2H), 7.3 (t, 1H), 7.17 (d, 2H), 6.01 (t, 1H), 3.48
`(m, 2H), 2.95 (dd, 1H), 2.7 (dd, 1H), 2.4-2.1 (m, 4H), 1.9-1.6
`(m, 2H), 1.4 (s, 9H), 1.3 (s, 9H), 1.2 (m, 6H), 0.86 (t, 3H).
`  NMR (CDC13)   7.56 (d, 2H), 7.46 (t,
`Compound 7b:
`2H), 7.38 (d, 2H), 7.3 (t, 1H), 7.17 (d, 2H), 5.84 (t, 1H), 3.4 (m,
`2H), 2.85 (m, 2H), 2.3 (m, 2H), 2.2-1.9 (m, 2H), 1.7-1.5 (m,
`2H), 1.49 (s, 9H), 1.83 (s, 9H), 1.25 (m, 6H), 0.88 (t, 3H).
`(oJZ,yS)-a-Butyl-y- [ [ (2-carboxyethyl)amino]carbonyl] -
`11 , -biphenyl]-4-pentanoic Acid (8c). To a solution of 7
`(150 mg, 0.28 mmol) in 2 mL of methylene chloride was added
`5 mL of trifluoroacetic acid. The mixture was stirred for 4 h
`and concentrated, toluene was added, and the mixture was
`concentrated and solidified when slurried with ether. The
`solution was collected and dried to give 110 mg of 8c, mp 151-
`  NMR (DMSO-de) ó 12.1 (br, 2H), 7.89 (t, 1H), 7.6
`152 °C:
`(d, 2H), 7.5 (d, 2H), 7.42 (t, 2H), 7.30 (t, 1H), 7.22 (d, 2H), 3.16
`(m, 2H), 2.25 (m, 1H), 2.26 (m, 1H), 2.22 (t, 2H), 2.10 (m, 1H),
`1.55 (t, 2H), 1.35 (m, 2H), 1.2 (m, 5H), 0.82 (t, 3H). Anal.
`(C25H31NO5) C,H,N.
`The following compounds were prepared similarly.
`Compound 8a: mp 144-145 °C;   NMR (DMSO-de)   12.0
`(br, 2H), 7.88 (t, 1H), 7.60 (d, 2H), 7.50 (d, 2H), 7.40 (t, 2H),
`7.30 (t, 1H), 7.18 (d, 2H), 6.79 (d, 1H), 6.68 (s, 1H), 6.60 (d,
`2H), 3.68 (s, 3H), 3.67 (s, 3H), 3.16 (m, 2H), 2.S-2.4 (m, 6H),
`2.24 (t, 2H), 1.6 (m, 2H). Anal.
`(C25H31NO5) C,H,N.
`Compound 8b: mp 118-120 °C;   NMR (DMSO-d6) ó 12.3
`(br, 2H), 7.92 (t, 1H), 7.63 (d, 2H), 7.52 (d, 2H), 7.41 (t, 2H),
`7.30 (t, 1H), 7.18 (d, 2H), 6.79 (d, 1H), 6.68 (s, 1H), 6.60 (d,
`2H), 5.35 (br, 1H), 3.98 (m, 1H), 3.68 (s, 3H), 3.67 (s, 3H), 3.2-
`2.6 (m, 8H), 1.65 (m, 2H). Anal.
`(C25H31NO5) C,H,N.
`Compound 8d: mp 220-230 °C (mixture of diastereomers);
`  NMR (CD3OD)   7.55 (d, 2H), 7.5 (d, 2H), 7.4 (t, 2H), 7.38
`(t, 1H), 7.25 (d, 2H), 4.1 (m, 1H), 3.5 (m, 2H), 2.9 (m, 1H), 2.7
`(m, 1H), 2.6 (m, 1H), 2.8 (m, 1H), 1.75 (m, 2H), 1.6 (m, 1H),
`1.4 (m, 1H), 1.3 (m, 5H), 0.9 (t, 3H). Anal.
`(C25H29NNa206·
`2H20) C,H,N.
`Compound 8e: mp 70-75 °C;   NMR (CDC13)   7.52 (d,
`2H), 7.47 (d, 2H), 7.38 (t, 2H), 7.3-7.1 (m, 5H), 6.95 (t, 1H),
`6.84 (d, 2H), 6.1 (t, 1H), 5.6 (H20 plus exchangeable protons),
`4.67 (m, 1H), 3.29 (m, 2H), 2.96 (m, 1H), 2.80 (m, 1H), 2.75
`(m, 1H), 2.45 (m, 1H), 2.2 (m, 3H). Anal.
`(C27H27NO6-0.75H2O)
`C,H,N.
`  NMR (CDC13)
`  8.4
`Compound 8f: mp 75-78 °C;
`(exchangeable protons), 7.50 (d, 2H), 7.49 (d, 2H), 7.35 (t, 2H),
`7.3-7.1 (m, 5H), 6.90 (t, 1H), 6.80 (d, 2H), 6.4 (t, 1H), 4.65 (t,
`1H), 3.84 (m, 1H), 3.70 (m, 1H), 2.9 (m, 1H), 3.75 (m, 2H), 2.4
`(m, 2H), 2.25 (m, 1H), 2.1 (m, 1H). Anal.
`(C27H27N06) C,H,N.
`Compound 8g: mp 142—146 °C;   NMR (DMSO-de)   12.0
`(2H), 8.0 (t, 1H), 7.62 (d, 2H), 7.54 (d, 2H), 7.40 (t, 2H), 7.32
`(t, 1H), 7.25 (d, 2H), 3.47 (m, 1H), 3.3 (m, 2H), 3.17 (s, 3H),
`2.8 (m, 1H), 2.7 (m, 1H), 2.55 (m, 1H), 2.31 (t, 2H), 1.89 (t,
`1H), 1.47 (t, 1H). Anal.
`(C22H25NO6) C,H,N.
`Compound 8h: mp 120-125 °C;   NMR (DMSO-de)   12.0
`(br, 2H), 7.86 (t, 1H), 7.62 (d, 2H), 7.54 (d, 2H), 7.40 (t, 2H),
`7.30 (t, 1H), 7.2 (d, 2H), 3.65 (m, 1H), 3.20 (s, 3H), 3.15 (m,
`2H), 2.9—2.6 (m, 3H), 2.26 (t, 2H), 1.8 (m, 1H), 1.7 (m, 1H).
`Anal.
`(C22H25NO6) C,H,N.
`Compound 8i: mp >100 °C dec.;   NMR (CD3OD)   7.86
`(m, 3H), 7.55 (m, 3H), 7.50 (d, 2H), 7.39 (t, 2H), 7.28 (d, 2H),
`7.27 (m, 1H), 7.20 (d, 2H), 3.0-2.5 (m, 6H), 2.35 (m, 2H), 1.84
`(m, 2H). Anal.
`(CsiHso^OeS-ll^O) C,H,N.
`Compound 8j: mp 160-162 °C;   NMR (CDC13)   12.0
`(br, 2H), 7.52 (d, 2H), 7.46 (d, 2H), 7.37 (t, 2H), 7.30 (t, 1H),
`7.20 (d, 2H), 6.2 (t, 1H), 3.72 (m, 1H), 3.38 (t, 2H), 3.25 (s,
`3H), 3.15 (m, 1H), 3.0 (m, 1H), 2.67 (m, 1H), 2.57 (m, 1H), 2.4
`(m, 3H), 1.9 (m , 2H), 1.7 (m, 2H). Anal.
`(C24H29NO6) C,H,N.
`Compound 8k: mp 65-70 °C;   NMR (DMSO-de)   12.1
`(br, 2H), 7.91 (t, 1H), 7.62 (d, 2H), 7.54 (d, 2H), 7.40 (t, 2H),
`7.31 (t, 1H), 7.17 (d, 2H), 3.14 (m, 2H), 2.80 (m, 1H), 2.6 (m,
`2H), 2.25 (t, 3H), 1.6 (m, 10H), 1.5-1.0 (5H). Anal.
`(C28H35-
`N06) C,H,N.
`
`Ksander et al.
`Compound 81: mp 165-167 °C;   NMR (DMSO-de)   12.0
`(br, 2H), 7.92 (t, 1H), 7.60 (d, 2H), 7.50 (d, 2H), 7.40 (t, 2H),
`7.31 (t, 1H), 7.20 (d, 2H), 3.20 (dd, 2H), 2.8 (m, 1H), 2.6 (m,
`1H), 2.45 (m, 1H), 2.28 (t, 2H), 2.10 (t, 2H), 1.6 (m, 2H). Anal.
`(C21H23NO5O.5H2O) C,H,N.
`Compound 8m: mp 59-65 °C;   NMR (DMSOde)   12.1
`(br, 2H), 7.92 (t, 1H), 7.62 (d, 2H), 7.5 (d, 2H), 7.42 (t, 2H),
`7.32 (t, 1H), 7.2 (d, 2H), 3.19 (m, 2H), 2.80 (m, 1H), 2.6 (m,
`2H), 2.22 (m, 3H), 1.6 (m, 2H), 1.1 (d, 3H). Anal.
`(C22H25-
`NOe) C,H,N.
`(aR,yR)-y-[[(2-Carboxyethyl)amino]carbonyl]-a-([l,l-
`biphenyl]-4-ylmethyl)[l,l'-biphenyl]-4-pentanoic Acid
`(10b). A solution of /9-alanine (300 mg, 3.37 mmol) and 9b
`(150 mg, 0.34 mmol) in 30 mL of methylene chloride:pyridine
`(1:1) was stirred at room temperature for 2 days. The mixture
`was concentrated, redissolved in ether:ethyl acetate:toluene
`(1:1:1), washed with 2 N HC1, 3 x H20, and brine, dried
`(MgSCh), and concentrated. The solid was collected and dried
`  NMR (DMSO-de)
`to give 140 mg of 10b, mp 205-208 °C:
`  12.1 (br, 2H), 7.88 (t, 1H), 7.61 (d, 2H), 7.53 (d, 2H), 7.40 (t,
`2H), 7.31 (t, 1H), 7.21 (d, 2H), 3.2 (m, 2H), 2.7 (m, 3H), 2.6-
`2.4 (m, 3H), 2.25 (m, 2H), 1.7 (m, 2H). Anal.
`fC23H33N05)
`C,H,N.
`The following compounds were prepared similarly.
`Compound 10c: mp 201-203 °C; 1H NMR (DMSO-d8)  
`12.1 (br, 2