Discovery of non-peptidic P2-P3 butanediamide renin inhibitors with high oral efficacy

Bioorganic & Medicinal Chemistry 7 (1999) 489±508

Discovery of Non-peptidic P₂±P₃Butanediamide Renin Inhibitors

with High Oral Ecacy

Bruno Simoneau,^a,* Pierre Lavallee,^aPaul C. Anderson,^aMurray Bailey,^a

Gary Bantle,^aSylvie Berthiaume,^aCatherine Chabot,^aGulrez Fazal,^aTed Halmos,^a

William W. Ogilvie,^aMarc-Andre Poupart,^aBounkham Thavonekham,^aZhili Xin,^a

Diane Thibeault,^aGordon Bolger,^aMaret Panzenbeck,^bRaymond Winquist^b

and Grace L. Jung^a,y

a

Â

Boehringer Ingelheim (Canada) Ltd., Bio-Mega Research Division, 2100 Cunard Street, Laval, Quebec, Canada H7S 2G5

^bBoehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, P.O. Box 368, Ridge®eld, CT 06877, USA

Received 3 September 1998; accepted 3 November 1998

AbstractÐA new series of non-peptidic renin inhibitors having a 2-substituted butanediamide moiety at the P₂and P₃positions has

been identi®ed. The optimized inhibitors have IC₅₀values of 0.8 to 1.4 nM and 2.5 to 7.6 nM in plasma renin assays at pH 6.0 and

7.4, respectively. When evaluated in the normotensive cynomolgus monkey model, two of the most potent inhibitors were orally

active at a dose as low as 3 mg/kg. These potent renin inhibitors are characterized by oral bioavailabilities of 40 and 89% in the

cynomolgus monkey. Inhibitor 3z (BILA 2157 BS) was selected as candidate for pre-development. # 1999 Elsevier Science Ltd. All

rights reserved.

Introduction

in the RAS, AII receptor antagonism has also been

evaluated. The discovery of small and non-peptidic

angiotensin II antagonists from high throughput

screening led to the rapid identi®cation of a series of

candidates for development.⁵Although high levels of

circulating AII resulting from receptor antagonism and

the existence of receptor subtypes raised some con-

cerns,⁵Losartan (Dup-753) was the ®rst compound of

this class to be approved for clinical use.⁶In contrast,

no small molecule inhibitor of renin has been identi®ed

from screening. A rational design approach was there-

fore used to generate potent renin inhibitors.^7±9As a

result of the renin substrate requirements, these com-

pounds have typically been quite large and peptidic.

Most renin inhibitors have a h₀igh molecular weight

since they usually cover the S₄±S₁region of the enzyme

in order to reach in vitro potency levels (low nM)

required for reasonable in vivo ecacy. Both their pep-

tidic character as well as their size have limited the oral

bioavailability of these compounds. So far, a renin

inhibitor has yet to be approved for use in humans.

Nevertheless, potent renin inhibitors with improved oral

bioavailability have been identi®ed. For instance, good

oral bioavailabilities have been reported in the cyno-

molgus monkey for A-72517 (8%),¹⁰FK906 (13%),¹¹

FK744 (30±50%)¹²and CP-108,671 (>27%)¹³as well as

in the beagle dog for PD-134672 (CI-992, 18%)^7,14and

SC-53315 (30%)¹⁵which indicate signi®cant progress in

this area. Besides good bioavailability, a commercially

The renin±angiotensin system (RAS) plays an important

role in the regulation of blood pressure and in the

maintenance of ¯uid volume.¹Renin, an aspartyl pro-

tease, is involved in the ®rst and rate-limiting step of

this enzymatic cascade. Renin cleaves the N-terminus of

the circulating glycoprotein angiotensinogen to generate

the decapeptide angiotensin I. Angiotensin-converting

enzyme (ACE) next removes the carboxy terminal

dipeptide of angiotensin I to give angiotensin II (AII),

one of the most potent vasoconstrictors known. Inhibi-

tion of ACE proved to be an eective therapy for the

treatment of hypertension and congestive heart failure.²

However, ACE is a non-speci®c enzyme that also

cleaves bradykinin as well as other substrates and this

lack of speci®city may contribute to certain side-eects

such as the persistent dry cough often observed during

ACE inhibitor therapy.^3,4Considering that renin has

only one known natural substrate, angiotensinogen, it

has been suggested that renin inhibitors could have an

improved side-eect pro®le. A third site of intervention

Key words: Antihypertension; renin inhibitors; peptido-mimetics; oral

activity.

*Corresponding author. Tel.: +1 450-682-4640; fax: +1 450-682-4189;

_ye-mail: bsimoneau@bio-mega.boehringer-ingelheim.ca

Present address: Nortran Pharmaceuticals Inc., 3650 Wesbrook

Mall, Vancouver, BC, Canada, V6S 2L2.

PII: S0968-0896(98)00265-X

490

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

viable potent renin inhibitor would have to be produced

economically in order to compete against several anti-

hypertensive drugs already on the market including the

newly introduced AII antagonists. To address these

issues, much eort has been directed toward the reduction

in size and peptidic character of renin inhibitors^9,16±20

and smaller inhibitors with good levels of potency have

been recently reported.^15,21±24

accessible 2-substituted butanediamide eliminates one

stereogenic center thereby contributing to the simpli®-

cation of this portion of the molecule. Finally, the P₃±P₄

appendages were amenable to a simple P₃±P₄amine

moiety. The synthesis, structure±activity±relationship

studies as well as biological properties of the novel

butanediamide 3 are discussed.

We recently described the identi®cation of small and

potent renin inhibitors 1 and 2 (IC₅₀=20 nM).²⁵

Unfortunately, these inhibitors did not have the

physico-chemical properties required for oral ecacy.

Although these compounds were very small (MW 503),

the reduction in size was achieved using synthetically

complex molecules.

Chemistry

The inhibitors of general structure 3 were made starting

in a convergent fashion from three synthons, the P₃±P₄

amine, the succinyl core and the transition state analo-

gue, by formation of two amide bonds. The P₃±P₄

amines were either commercially available amines or

easily accessible N-substituted glycine analogues. The 2-

(benzylamino)acetamides 5±8 were prepared from Boc-

N-benzylglycine (4)²⁷in two steps: amide formation

followed by the acidic cleavage of the Boc protective

group (Scheme 1). Reduction of 5 with LAH gave the

reduced analogue N,N,N⁰-trisubstituted 1,2-ethanedia-

mine 9. For the preparation of the P₃±P₄amines that did

not have an N-benzyl substituent, the direct alkylation

of the P₃amine with N,N-dimethyl 2-bromoacetamide

(11) in the presence of triethylamine was used (Scheme

2). Although polyalkylation reduced the eciency of the

sequence, this method produced the desired secondary

amines 12,13,16±18 in an expeditious fashion (26±43%).

In the case of more hindered amines such as 1(R)- and

1(S)-phenylethylamine, the yield of secondary amines 14

and 15 increased to 70±76%. In the case of P₃±P₄amines

27 and 29, a very ecient but longer procedure was used

in order to simplify the handling and puri®cation of the

intermediates and the polar amines (Scheme 3). The

sequence started with the alkylation of the appropriate

amine with ethyl 2-bromoacetate (19) in the presence of

triethylamine. This occurred smoothly at room tem-

perature to give the expected secondary amines 20 and

23, respectively, in good yield. In these cases, only a

trace (ca. 5%) of the corresponding tertiary amines was

detected. The alkylation of cyclohexanemethylamine

was signi®cantly more ecient with 2-bromoacetate

19 (66%) than with 2-bromoacetamide 11 (30%). The

secondary amines 20 and 23 were next converted into

the corresponding Boc derivatives, 21 and 24, and the

Returning to the drawing board using angiotensinogen

as template, our objectives were to design small, syn-

thetically less complex and potent renin inhibitors. The

focus was put on the P₂±P₃±P₄segment of the inhibitors

while maintaining a known transition state analogue²⁶

0

at P₁±P₁. The rationale behind the proposed P₂±P₃

butanediamide inhibitors 3 is outlined in Figure 1. A 2-

substituted butanediamide could replace the P₂±P₃

dipeptide usually found in most renin inhibitors. The

substituent at position 2 could play the role of the P₂

side chain. The P₂amino acid amide NH, not involved

in an important hydrogen bond, was simply replaced by

a methylene. The carbonyl of the P₃amino acid, which

is involved in a critical hydrogen bond with the enzyme,

was maintained via the carbonyl of the N⁴,N⁴-di-

substituted amide moiety of the butanediamide. As a

result, one group on the retro-amide could act as the P₃

side chain and the other as the P₄side chain. The pro-

posed replacement of the P₂±P₃dipeptide by a readily

.

Scheme 1. Reagents and conditions: (a) R₁R₂NH, BOP PF₆, i-Pr₂NEt,

DMF, 25 ^ꢀC; (b) 4 N HCl/dioxane, 25 ^ꢀC, 76±86%; (c) LAH, THF,

65 ^ꢀC, 100%.

Figure 1. Proposed P₂±P₃butanediamide inhibitors.

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

491

an amide using the P₃±P₄amine. Alternatively, the ester

(at C-4) of 41±46 could be ®rst transformed into the

amide, the chiral auxiliary removed, and the resulting

acid coupled to the transition state analogue moiety to

give the ®nal inhibitors. The ®rst approach gave rise to

advanced intermediates for SAR studies at the P₃±P₄

positions. The second approach was useful for the SAR

of the transition state analogue portion as well as for the

synthesis of speci®c inhibitors since the more complex

portion of the inhibitor is introduced at a later stage in

the synthesis. Using the ®rst approach strategy, the

chiral auxiliary present in 41±46 was removed using

LiOOH²⁹to give the corresponding acids which were

coupled with 2(S)-amino-1-cyclohexyl-6-methyl-3(R),

Scheme 2. Reagents and conditions: (a) Et₃N, MeOH, 25 ^ꢀC to re¯ux,

26±76%.

4(S)-heptanediol (47)^30,31using BOP PF₆reagent to give

.

amides 48±53. Upon treatment of 48±50 and 52, 53 with

TFA in CH₂Cl₂and selective hydrogenolysis of 51, the

corresponding acids 54±59 were obtained and used in

the last amide bond formation reaction with the P₃±P₄

amines to generate inhibitors 3a±3t generally in good

yields. The observed yields, not optimized, were low (19

to 24%) for the 2- and 4-thiazolyl derivatives 52 and 53.

Because of even lower yields in the case of 2-amino-4-

thiazolyl derivatives, the second approach was used

instead. First, the key succinyl intermediates bearing a

4-thiazolylmethyl substituent at position 2 were pre-

pared starting with the opening of succinic anhydride

with the lithium salt of (1-methylethyl)-2-oxazolidinone

which provided acid 60 (Scheme 5). This acid was

transformed into bromomethylketone 61 in the usual

way, via the reaction of the corresponding acyl chloride

with CH₂N₂and treatment of the resulting diazo-

methylketone with HBr. The elaboration of the hetero-

cycle was achieved using Hantzsch synthesis conditions.

Thus treatment of bromomethylketone 61 with thio-

formamide gave 4-thiazolyl analogue 62. The reaction

of 61 with thiourea aorded the 2-amino-4-thiazolyl

analogue that was isolated as the 2,2,2-trichloroethyl

carbamate derivative 63. Alkylation of the sodium eno-

late of 62 and 63 with tert-butyl 2-bromoacetate gave,

respectively, succinyl intermediates 46 and 64. In the

case of 64, the tert-butyl ester was ®rst cleaved with

TFA to give the corresponding acid that was immedi-

ately coupled with the P₃±P₄amines (5, 15, 16, 18, 29, or

27) to aord amides 65±70 in good yields. The chiral

auxiliary was next cleaved using either LiOOH (for 65±

ester moiety was saponi®ed to give the acids 22 and 25.

Finally, coupling with [2-(methylamino)ethyl]pyridine

and subsequent cleavage of the BOC protective group

gave the P₃±P₄amines 27 and 29.

The succinyl core portion of the inhibitors was prepared

using Evans' alkylation method.²⁸The required acids or

acyl chlorides bearing the P₂side chain (30±34) were

readily available and were converted into the corres-

ponding acyloxazolidinones 35±40 (Scheme 4). Alkyla-

tion of the lithium or sodium enolate of 36±40 with

tert-butyl or benzyl 2-bromoacetate provided the succinyl

intermediates 41±45 in good yields. These compounds

were found to be quite versatile intermediates and could

be utilized in two dierent routes. For the synthesis of

the proposed inhibitors, the chiral auxiliary could ®rst

be removed to liberate the acid function (at C-1) and the

transition state analogue portion could then be intro-

duced via coupling. The ester (at C-4) could next be

transformed into the acid and this acid converted into

32

67) or, in 2 steps, with Mg(OMe)₂followed by aqu-

eous NaOH saponi®cation (for 68±70) to deliver the

corresponding acids. These acids were coupled to 47 in

the usual manner to give butanediamides 71±76. In the

last coupling, a substantial amount (5±10%) of the epi-

meric 2(S)-substituted butanediamides was observed.

The presence of this epimer made chromatographic

separation and puri®cation of the desired 2(R) deriva-

tives rather dicult. Finally, the 2,2,2-trichloroethyl

carbamate protective group was removed with zinc in

aqueous HCl to aord inhibitors 3u±3z.

Scheme 3. Reagents and conditions: (a) Et₃N, THF, 25 ^ꢀC, 62±66%;

Results and Discussion

(b) (Boc)₂O, CH₂Cl₂, 25 ^ꢀC, 98±100%; (c) aq NaOH, _ꢀ25 ^ꢀC, 96±99%;

The SAR studies were initiated at the P₃and P₄posi-

tions of the butanediamide inhibitor of general formula

.

(d) 2-Pyr(CH₂)₂NHMe, BOP PF₆, i-Pr₂NEt, DMF, 25 C, 79±89%; (e)

4 N HCl/dioxane, 25 ^ꢀC, 95±96%.

492

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

Scheme 4. Reagents and conditions: (a) Me₃CCOCl, Et₃N, THF, 78 to 0 ^ꢀC, then Li±X_c, 78 to 0 ^ꢀC, 55±99%; for 31, Li±X_c, 78 ^ꢀC, 92%; (b)

CH₂N₂, Pd(OAc)₂, Et₂O, 0 ^ꢀC, 89% from 30; (c) _ꢀNaHMDS (LDA, DMPU for 36), BrCH₂CO₂R₂, THF, 78 ^ꢀC, 64±89%; (d) LiOOH, THF, H₂O,

ꢀ

.

0 to 25 C; _ꢀ(e) 47, BOP PF₆, i-Pr₂NEt, DMF, 25 C, 52±77%; (f) for R₂=t-Bu: TFA, CH₂Cl_ꢀ₂, 0 to 25 C, 46±100%; for R₂=Bn: H₂(1 atm), Pd/C,

MeOH, 48%.

.

EtOAc, 25 C, 99%; (g) BnNH₂, BnNHMe, 5±10 or 12±18), BOP PF₆, i-Pr₂NEt, DMF, 25 C, 19±78%; and for 57 only, H₂(1 atm), Pd(OH)₂/C,

3, to determine the requirements at these positions. The

exercise started with a 2(R)-(cyclopropylmethyl)butane-

diamide core. The cyclopropylmethyl substituent was

selected as a starting point since it turned out to be a

very good replacement for the histidine side chain in a

previous series of inhibitors (e.g. 1 and 2). The N-benzyl

amide 3a and the N-benzyl-N-methyl amide 3b were

the ®rst members of this butanediamide series to be

prepared. These small inhibitors demonstrated poor

potency against renin with IC₅₀values of 640 and

455 nM, respectively (Table 1). They contain only one

sizeable substituent that can occupy the S₃or S₄pockets

considering the geometry of this terminal amide. In

addition to the N-benzyl substituent, the introduction

of a second substituent such as an N,N-disubstituted

2-acetamide group onto the nitrogen increased the

potency of the resulting inhibitors by ca. tenfold which

quickly demonstrated the potential of this new series of

inhibitors. For instance, the N,N-dimethyl 2-acetamide

analogue 3c had an IC₅₀value of 56 nM. Other types

of terminal tertiary amide such as morpholino (3d) or

N-(2-pyridinyl)ethyl-N-methyl (3e) also aorded potent

renin inhibitors having IC₅₀values of 65 and 33 nM,

respectively. Note that the P₃±P₄portion of inhibitors

3c±3e constitutes a peptoid unit.³³

requirement in order to generate very potent inhibitors.

While the present work was well underway a patent

application by Terumo³⁴reporting a few renin inhibi-

tors related to 3d was published. However, the examples

described were only restricted to N⁴-(1-naphthyl-

methyl)butanediamide derivatives. To further probe the

importance of the carbonyl at P₄, the corresponding

tertiary amine 3f of N,N-dimethyl amide 3c was made

and exhibited an IC₅₀value of 655 nM. Moreover, the

tertiary amide was essential since the corresponding

N-methyl amide 3g as well as the methyl ester 3h proved

to be signi®cantly less potent than 3c. These ®ndings are

in complete agreement with observations made for other

renin inhibitors having a N-terminal amide at the P₄

position.⁷Because of the presence of a non-symmetrical

tertiary amide (N⁴,N⁴-disubstituted butanediamide), a

1

mixture of rotamers was easily detected by H NMR.

So, the P₄and P₃side chains are de®ned as s-cis and

s-trans respectively as drawn in 3 and are related to the

nature and the requirements of the corresponding S₄

and S₃pockets of the enzyme as well as the observed

binding mode of these butanediamide inhibitors.³⁵

Having de®ned the requirements of the P₄position, we

sought to optimize the P₃position next. Since the P₃

side chain is linked to the nitrogen of an amide a variety

of possibilities for the nature of this substituent were

available. An N⁴-phenylmethyl substituent on the butane-

diamide was ®rst used, a substitution based on the

The carbonyl of the acetamide seems to match the one

of the P₄proline residue in the natural renin substrate.

The presence of this extra carbonyl was therefore a

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

493

Scheme 5. Reagents and conditions: (a) THF, 0 ^ꢀC, 67%; (b) (i) (COCl)₂,CH₂Cl₂, 0 ^ꢀC; (ii) CH₂N₂, Et₂O, 0 ^ꢀC then HBr/AcOH, 0 ^ꢀC, 89%; (c) for

62: HCSNH₂, i-PrOH,_ꢀÁ, 55% from 61; for 63: (i) (NH₂)₂CS, i-PrOH, Á (ii) ClCO₂CH₂Cl₃, i-Pr₂NEt, DMAP, CH₂Cl₂, 25 ^ꢀC, 59% from 61; (d)

ꢀ

.

NaHMDS, THF, 78 C, 59±86%;_ꢀ(e) TFA, CH₂Cl₂, 0 to 25 C; (f) amine (15, 16, 18, 27, 29), BOP PF₆, i-Pr₂NEt, DMF, 25 C, 51±94%; (g) for

ꢀ

.

65±67: LiOOH, THF, H₂O, 0 to 25 C; for 68±70: (i) Mg(OMe)₂, MeOH, 0 C; (ii) aq NaOH; (h) 46, BOP PF6, i-Pr₂NEt, DMF, 25 C, 24±48%; (i)

Zn, 1 N aq HCl, dioxane, 10 ^ꢀC, 50±85%.

Table 1. In vitro potency of P₂±P₃butanediamide renin inhibitors

Compd no.

R₃

R₂

R₁

IC₅₀(nM)^a

3a

3b

3c

3d

3e

3f

3g

3h

3i

PhCH₂

Me

H

PhCH₂

PhCH₂CH₂

2-PyrCH₂

Cyclopropyl

Ethyl

640

455

56

65

33

655

350

385

52

145

375

43

Me₂NCOCH₂

MorpholinoCOCH₂

2-PyrCH₂CH₂NMeCOCH₂

Me₂NCH₂CH₂

MeNHCOCH₂

MeOCOCH₂

Me₂NCOCH₂

3j

3k

3l

(R)-PhCHMe

(S)-PhCHMe

CyclohexylCH₂

(S)-CyclohexylCHMe

(1-OH-Cyclohexyl)CH₂

(S)-PhCHMe

3m

3n

3o

3p

3q

3r

3s

3t

11

12

16

35

2-Thienyl

11

1H-Imidazol-4-yl

2-Thiazolyl

4-Thiazolyl

7.3

3.7

2.9

1.1

3u

2-Amino-4-thiazolyl

^aDetermined using a human plasma renin assay at pH 6.0.

494

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

observation of the side chain of phenylalanine being

found at the P₃position of most renin inhibitors.

Replacement of the N⁴-phenylmethyl by N⁴-(2-phenyl-

ethyl) substituent provided inhibitor 3i, which was

equipotent to 3c. As expected, a basic side chain such as

2-pyridinylmethyl was not well tolerated as inhibitor 3j

was three times less potent than 3c. The introduction of

a methyl group onto the benzylic position led to an

equipotent inhibitor in the case of the S isomer (3l)

whereas the R isomer (3k) was nine times less potent.

The objective of the introduction of this stereogenic

center was to probe for a possible bene®cial orientation

in the space of the P₃side chain considering the planarity

of the amide to which the benzyl is linked. Most likely,

the benzylic hydrogen (C±H bond) would be in the same

plane as the amide (allylic strain). So, the eect of the

stereogenic center would be to orient the phenyl ring

either on the bottom face (S con®guration) or to the top

face (R con®guration) of the inhibitor related to the plane

of the amide. Although the presence of the S-methyl

showed no signi®cant improvement in vitro, it proved to

be very important in vivo (vide infra). It was found that

the S-methyl group was projected into the solvent based

on the X-ray analysis of a crystal structure of renin in

complex with 3u, an inhibitor similar to 3l.³⁵However,

our observations contrast with ®ndings reported for

SC-51106¹⁵, an (S)-b-methylhydrocinnamide (P₃) renin

inhibitor lacking a P₄residue. In that case, a sevenfold

increase in potency was observed in comparison with

the non-substituted hydrocinnamide derivative, while

the R-epimer was inactive. A new binding site (S₃aux)

was proposed to explain these results. The replacement

of the N⁴-phenylmethyl by a N⁴-cyclohexylmethyl group

provided the ®rst signi®cant improvement in potency

leading to inhibitor 3m which exhibited an IC₅₀value of

11 nM. Close analogues of 3m such as the 1(S)-cyclo-

hexylethyl and the (1-hydroxycyclohexyl)methyl deriva-

tives 3n and 3o showed similar levels of potency.

of His287 through a water molecule. The P₄carbonyl

and the amide of Tyr220 complete the tetrahedral

coordination of the water molecule. The sulfur atom

may also give rise to stronger van der Waals interac-

tions with surrounding hydrophobic residues. By the

sequential optimization of the P₄, P₃, and P₂side

chains, the potency of the inhibitors was quickly

improved by 580-fold (cf. 3a and 3u), an enhancement

attributed to the presence of: (a) an amide carbonyl

at P₄; (b) N⁴-cyclohexylmethyl or N⁴-1(S)-phenylethyl

substituents as the P₃side chain and; (c) a 2-amino-4-

thiazolylmethyl substituent as the P₂side chain.

Having obtained highly potent inhibitors, we evaluated

some in conscious sodium depleted cynomolgus monkeys.

The renin inhibitors were given orally at a dose of

10mg/kg (po). The mean arterial blood pressure (MABP)

was monitored continuously for 3 h. Of the inhibitors (3l,

3p, 3r, 3s, and 3u) evaluated, only 3u produced a statis-

tically signi®cant decrease in the MABP. The observed

ca. 15% decrease of MABP was sustained at the end of

the 3 h experiment. Plasma angiotensin II levels ( 30%

change) also decreased signi®cantly. The nature of the

P₂side chain represents the sole structural dierence

among these inhibitors. For inhibitors 3l and 3p which

were signi®cantly less potent than 3u (35-fold), the lack

of oral ecacy could easily be explained based on the

weak potency. However, since inhibitors 3r and 3t were

only slightly less potent than 3u (twofold to sixfold),

their dierences in absorption and/or hepatic extraction

pro®le probably account for their poor oral ecacy. It

was already reported that inhibitors with a basic histi-

dine side chain at P₂had poor oral ecacy due to rapid

biliary elimination.^10,36We had also noticed that inhi-

bitors possessing thiazolylmethyl P₂side chains were

metabolized much faster by liver microsomes from

cynomolgus monkeys than those compounds having a

2-amino-4-thiazolylmethyl side chain. These results

clearly demonstrate that the 2-amino-4-thiazolylmethyl

group at the P₂side chain is critical for oral activity.

Using inhibitor 3l as a starting point, the P₂side chain

was next investigated (R₁). The inhibitor 3p having an

n-propyl side chain instead of a cyclopropylmethyl

showed similar potency to 3l with an IC₅₀value of

35 nM. It was quickly found that a ®ve-membered ring

heterocycle at this position was by far preferable for this

series of butanediamide inhibitors. The 2-thienylmethyl

analogue 3q (IC₅₀=11 nM) was four times more potent

than the cyclopropylmethyl containing inhibitor 3l.

Inhibitor 3r, which possesses the imidazolylmethyl side

chain of the P₂histidine present in the renin substrate,

was even more potent with an IC₅₀value of 7.3 nM. At

this point, a series of less basic imidazolyl surrogates

were evaluated. The 2- and 4-thiazolylmethyl analogues

3s and 3t exhibited IC₅₀values of 3.7 and 2.9 nM,

respectively. The (2-amino-4-thiazolyl)methyl analogue,

inhibitor 3u, proved to be the most potent inhibitor of

this series with an IC₅₀of 1.1 nM. We believe that the 2-

amino-4-thiazolyl heterocycle has stronger interactions

with the enzyme than the imidazole ring. The crystal

structure of renin-3u complex³⁵showed that the amino

group was hydrogen-bonded to the hydroxyl group of

Ser222 and the carbonyl of Tyr220. The ring nitrogen of

the thiazolyl was hydrogen bonded to the imidazole ring

The next step was the optimization of 3u based on in

vivo activity. Using 3u as our starting point, combined

modi®cations at the P₄and P₃positions based on the

previous exercise as well as other transition state mimics

were investigated. So far, the cyclohexylamidodiol descri-

0

bed by Abbott scientists²⁶had been used at the P₁±P₁

portion of the inhibitors. Although other known transi-

tion state mimics were investigated, only the amido-

lactam,³⁷shown in inhibitor 77, produced a potent

inhibitor with an IC₅₀value of 2.1 nM. Despite being

equipotent to 3u, 77 was found to be orally inactive when

tested in the cynomolgus monkey model at 10 mg/kg.

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

495

This result con®rmed the choice of the transition state

analogue unit for the P₂±P₃butanediamide renin inhibi-

tors. At the P₃position, a simple N⁴-benzyl substituent

gave inhibitor 3v (Table 2) that exhibited an IC₅₀value

of 2.8 nM, slightly less potent than 3u as expected (cf. 3c

and 3l). To our surprise, inhibitor 3v was inactive at

10 mg/kg po in the monkey model. The dierence in

potency was certainly an important element. In addi-

tion, the S-methyl substituent in 3u could play a role in

the metabolic stabilization of the N⁴-benzyl position vis-

a-vis oxidation/de-alkylation processes leading to the

costly loss of the P₃side chain. Inhibitor 3w, possessing

a cyclohexylmethyl substituent at the P₃side chain,

showed a pro®le similar to 3u, in vitro and in vivo. A

simple introduction of a hydroxyl group at position 1 of

the cyclohexyl, giving 3x, had again a signi®cant impact

in vivo. Inhibitor 3x exhibited an IC₅₀value of 1.2 nM

but was found to be orally inactive in the cynomolgus

monkey model. Finally, a modi®cation at the P₄position

brought the desired improvement in vivo. Replacement

of the terminal N,N-dimethyl amide by an N-methyl-N-

2-(2-pyridinyl)ethyl amide generated 3y that was as

potent as 3u in the plasma renin assay but was orally

active at a dose as low as 3 mg/kg in the cynomolgus

monkey model. The corresponding analogue of 3w,

inhibitor 3z had a pro®le very similar to 3y. The eect of

oral doses of 10, 3, and 1 mg/kg of inhibitor 3z on

MABP of cynomolgus monkeys are shown in Figure 2.

Statistically signi®cant decreases in MABP were

observed for doses of 10 and 3 mg/kg in a dose-related

fashion. Decreases in plasma angiotensin II levels were

observed for doses of 10 and 3 mg/kg. Inhibitors 3y

and 3z had oral ecacies comparable to that of one

of the most potent renin inhibitors reported to date,

A-72517.¹⁰

Figure 2. Maximum changes in mean arterial blood pressure (MABP)

after oral administration of 3z in conscious cynomolgus monkeys.

inhibitors. Obviously, these analogues possess dierent

physicochemical properties that impact on their oral

bioavailability pro®les. The oral bioavailabilities in

cynomolgus monkey (iv and oral doses of 1 and 10 mg/

kg, respectively) for the orally active renin inhibitors

3u, 3w, 3y, and 3z were 176%, 128%, 8953%,

and 4023%, respectively. After the oral dose, these

compounds exhibited peak concentrations (C_max) of

16854, 13165, 865470, 710350 ng/mL, respec-

tively, while their values for area under the curve

(AUC) were 510191, 365260, 23301570, 2500

1650 ng h/mL, respectively. These compounds are

among the most bioavailable renin inhibitors reported

to date. It is interesting to note that the sole dierence

between 3u, 3w, and 3y, 3z is the addition of a 2-pyr-

idinylmethyl moiety at the P₄position. This heterocycle

does not interact with the enzyme, the pyridine being

exposed to the solvent.³⁵The introduction of the 2-pyr-

idinylmethyl substituent had obviously no impact on in

vitro potency but had a bene®cial contribution to

absorption and oral bioavailability. Although the addi-

tion of this moiety increased the molecular weight of

the inhibitors by 91 mass units (MW of 643 and 635 to

734 and 726), the oral bioavailability was signi®cantly

improved.

All the inhibitors generated in the optimization of 3u

exhibited IC₅₀values similar (0.8±2.8 nM) to that of 3u

(IC₅₀=1.1 nM). In some cases, small chemical mod-

i®cations had profound eects on the oral activity of the

Table 2. In vitro and in vivo potency of P₂±P₃2-[(2-amino-4-thiazolyl)-

methyl]butanediamide renin inhibitors

Although a human plasma assay at pH 6.0 (optimal pH

for renin) was employed as the primary screen, in vitro

potencies at physiological pH (7.4) were also considered

during compound evaluation. It would be ideal to iden-

tify inhibitors with high potencies irrespective of pH. A

comparison of IC₅₀values at pH 6.0 and 7.4 for a

selection of inhibitors is shown in Table 3. Large shifts

in the IC₅₀values were observed for inhibitors (3l±3p)

possessing a cyclopropylmethyl or a n-propyl group at

the P₂position. For instance, since 3l and 3p have IC₅₀

values of 760 and 300 nM at pH 7.4, it was not surpris-

ing to discover their lack of oral activity. Inhibitors

having basic residues at the P₂side chain shifted the

Compd

no.

R₁

R₂

IC₅₀Oral activity^b

(nM)^amg/kg po

3u

3v

3w

3x

3y

3z

Me

(S)-PhCHMe

PhCH₂

CyclohexylCH₂

1.1 Active at 10

2.8 Inactive at 10

0.8 Active at 10

(1-OH-Cyclohexyl)CH₂1.2 Inactive at 10

2-PyrCH₂CH₂

(S)-PhCHMe

CyclohexylCH₂

0.9

1.4

Active at 3

^aDetermined using a human plasma assay at pH 6.0.

^bDetermined using sodium depleted normotensive cynomolgus mon-

keys.

496

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

Table 3. In vitro potency of P₂±P₃butanediamide inhibitors for renin

at pH 6.0 and 7.4 and for cathepsin D

Table 4. Yields and analytical properties of 3a±k, 3m±o, and 3v±3y

Compd

no.

Yield^a

(%)

MS FAB

m/z (MH)⁺C, H, N0.4%

Elemental analysis^b

Compd IC₅₀(nM) renin,

no.

IC₅₀(nM) renin,

plasma pH 7.4

IC₅₀(nM)

cathepsin D

plasma pH 6.0

3a

3b

3c

3d

3e

3f

3g

3h

3i

15

37

65

71

57

14

70

82

75

72

66

59

70

77

10

22

19

26

487

501

572

614

663

558

559

586

573

586

578

592

594

630

636

652

735

C₂₉H₄₆N₂O₄

C₃₀H₄₈N₂O₄

C₃₃H₅₃N₃O₅

3l

43

11

12

16

760

110

100

94

300

11

33

24

7.6

18

53

30

9

50

3m

3n

3o

3p

3r

C₃₅H₅₅N₃O₆

C₃₉H₅₈N₄O₅+0.76% H₂O^c

C₃₃H₅₅N₃O₄+0.74% H₂O^c

C₃₂H₅₁N₃O₅

35

66

0% inh. at 1 uM

7.3

3.7

2.9

1.1

2.8

0.8

1.2

0.9

1.4

3s

3t

24

57

C₃₂H₅₀N₂O₆

C₃₄H₅₅N₃O₅

C₃₂H₅₂N₄O₅+0.78% H₂O^c

C₃₄H₅₅N₃O₆+0.99% H₂O

C₃₃H₅₉N₃O₅+0.74% H₂O

C₃₄H₆₁N₃O₆

3u

3v

3w

3x

3y

3z

810

600

190

595

2650

540

3j

3k

3m

3n

3o

3v

3w

3x

3y

3.3

5.4

4.7

2.5

C₃₃H₅₉N₃O₆+1.92% H₂O

C₃₃H₅₁N₅O₅S+1.03% H₂O

C

₃₃H₅₇N₅O₅S+1.86% H₂O

C₃₃H₅₇N₅O₆S+2.00% H₂O

₄₀H₅₈N₆O₅S

C

least and retained their potency against renin at pH 7.4.

For inhibitor 3r, the IC₅₀value did not change with pH.

This inhibitor has an imidazolylmethyl P₂side chain

identical to the histidine side chain that is present in the

renin substrate. Inhibitors 3s±3z that possess basic sur-

rogates of the imidazolyl ring, remain highly potent

inhibitors at pH 7.4. For the most potent inhibitors, the

ranking based on IC₅₀values remains more or less the

same at both pH 6.0 and 7.4. All the orally active inhi-

bitors were potent at both pHs. Potency at pH 7.4

seemed to be one of the requirements for oral activity.

^aCompounds 3a±o were prepared using the method described for 3l.

Compounds 3v, 3w and compounds 3x, 3y were synthesized from 64

according to the method described for 3u and 3z, respectively.

^bThe amount of H₂O (% w/w) was determined using the Karl Fisher

method.

^cCorrect elemental analyses could not be obtained for these com-

pounds. The homogeneity was determined to be >98% by RP-HPLC

in two solvent systems.

as the P₂side chain. The orally active inhibitors (3u, 3w,

3y, 3z) had IC₅₀values of 0.8 to 1.4 nM and 2.5 to

7.6 nM at pH 6.0 and 7.4, respectively. The presence of

the 2-amino-4-thiazolyl heterocycle was critical for oral

activity. From a synthetic standpoint, we believe that

the introduction of the P₂±P₃butanediamide segment

contributed to the simpli®cation of the P₂±P₃±P₄portion

of these renin inhibitors. Only one stereogenic center

remains in this section of the molecule. This simpli®ca-

tion had only a small impact on the level of potency

obtained by the optimized inhibitors in comparison with

more peptidic inhibitors. The P₂±P₃butanediamide

inhibitors contain no amino acid residue other than the

N-terminal glycinamide. The optimized P₂±P₃butane-

diamide inhibitors demonstrated good oral bioavail-

ability in cynomolgus monkeys. The N,N-dimethyl

amide derivatives 3u and 3w had oral bioavailabilities of

11.5 and 17%, molecular weights of 643 and 635,

respectively, and were orally active at a dose of 10 mg/

kg in the monkey model. Interestingly, the correspond-

ing N-methyl N-2-(2-pyridinyl)ethyl derivatives 3y and

3z were orally active at 3 mg/kg in the monkey model,

had oral bioavailabilities of 89 and 40%, respectively,

values higher than 3u and 3w despite the fact that

molecular weights were increased to 734 and 726. After

further evaluation of the most potent orally active inhi-

bitors 3y and 3z as well as close analogues, inhibitor

3z (BILA 2157 BS) was selected as a candidate for

pre-development.

The speci®city of our inhibitors for renin was assessed

using three other human aspartyl proteases: pepsin,

gastricsin and cathepsin D. For pepsin and gastricsin,

the activity observed was never a concern since it was

only modest for all the inhibitors (a few % inhibition at

1 mM). In the case of cathepsin D, the situation was

dierent. Inhibitors 3l±3p had IC₅₀values very similar

for cathepsin D and renin at pH 6.0 (Table 3). Others

had only a small preference for renin (e.g. 3s, 3t). It is

apparent that the nature of the P₂side chain had a sig-

ni®cant impact on the speci®city of the inhibitors. The

presence of the imidazolylmethyl substituent at P₂(3r)

eliminated completely the anity of the compound for

cathepsin D. Inhibitors possessing the 2-amino-4-thia-

zolylmethyl side chain (3u±3z) had a good window of

selectivity, a good compromise between speci®city,

potency against renin at both pHs and in vivo proper-

ties. The nature of the P₃side chain also had an impact

on the speci®city pro®le of this class of inhibitors.

Cathepsin D seemed to prefer N⁴-(cycloalkyl)alkyl over

N⁴-phenylalkyl side chains (cf. 3u and 3w, 3y and 3z). In

general, bulkier and neutral lipophilic groups at P₂and

P₃positions were preferred by cathepsin D as predicted

by X-ray data.³⁸

Conclusions

We have identi®ed a new series of potent renin inhibi-

tors having a 2-substituted butanediamide moiety at the

P₂and P₃positions. The most potent inhibitors possess

an N⁴-2-(disubstituted-amino)-2-oxoethyl group at P₄

position and a (2-amino-4-thiazolyl)methyl substituent

Experimental

All reactions were performed under a nitrogen atmos-

phere. Reagents and anhydrous solvents were obtained

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

497

from commercial sources and were used without further

puri®cation or treatment. In order to remove traces of

solvents, the inhibitors were dissolved in a mixture of

water and acetonitrile, frozen and then lyophilized.

Melting points were determined on an electrothermal

Buchi 510 apparatus and are uncorrected. IR and FTIR

spectra were recorded on a Perkin±Elmer 781 and ATI

Mattson research series I, respectively. NMR spectra

were recorded on a Bruker AMX400 spectrometer.

Optical rotations were measured on a Perkin±Elmer 241

polarimeter. Electrospray ionization (ESI) mass spectra

were determined on a Micromass Quattro II apparatus.

Fast atom bombardment (FAB) mass spectra were

determined on a VG AutoSpec-Q or Kratos M550

apparatus at the Universite de Montreal. Microanalyses

were performed on a Carlo Erba 1100 or Fisons EA

1108 instruments. The residual amount of water was

quanti®ed using the Karl Fisher method with a

Metrohm 652 KF-Coulometer. RP-HPLC analyses

were performed on a Waters 600-MS (A: Supelcosil²

LC-ABZ; 10±80% MeCN+0.10% TFA/H₂O+0.10%

TFA) and a Waters 625 system (B: Symmetry²C₈col-

umn; 10±80% MeCN/50 mM NaH₂PO₄pH 4.4) using a

photodiode array detector.

N-Methyl-2-[(phenylmethyl)amino]acetamide (8). Pale

yellow solid (77%): IR (®lm) n_max3300, 3220,

1670 cm ¹; ¹H NMR (CDCl₃) d 7.37±7.27 (m, 5H), 7.21

(broad s, 1H), 3.80 (s, 2H), 3.34 (s, 2H), 2.83 (d, J=

5.1 Hz, 3H), 2.41 (broad s, 1H); MS (CI) m/z 179

(MH)⁺.

N,N-Dimethyl-N⁰-(phenylmethyl)-1,2-ethanediamine (9).

.

The reduction of 5 HCl with LiAlH₄gave 9 (132 mg,

ꢁ100%) as a yellowish oil which was used without pur-

1

i®cation: IR (®lm) n_max3380, 3330, 1670 cm

;

¹H

NMR (CDCl₃) d 7.38±7.23 (m, 5H), 3.83 (s, 2H), 2.73 (t,

J=6.1 Hz, 2H), 2.47 (t, J=6.1 Hz, 2H), 2.29 (broad s,

1H), 2.23 (s, 6H); MS (CI) m/z 179 (MH)⁺.

Methyl 2-[(phenylmethyl)amino]acetate (10).³⁹

Typical procedure for preparation of amines 12±18. A

solution of 11 (500 mg, 3.01 mmol), a primary amine

(1 equiv, 3.01 mmol) and Et₃N (610 mg, 6.02 mmol) in

MeOH (6.0 mL) was stirred at 25 ^ꢀC for 5 min then was

heated to re¯ux for 30 min. The cooled mixture was

diluted with EtOAc (100 mL) and the resulting solution

was washed with saturated aqueous NaHCO₃(30 mL),

brine (30 mL) before being dried (MgSO₄), ®ltered

and concentrated under reduced pressure. The residue

was puri®ed by ¯ash chromatography to give the amines

12±18.

Procedure for the preparation of amines 5±8. A solution

.

of 4 (531 mg, 2.00 mmol), R₁R₂NH or R₁R₂NH HCl

(2.40±3.00 mmol), i-Pr₂NEt (776 mg, 6.00 mmol) and

.

BOP PF₆(885 mg, 2.00 mmol) in DMF (8 mL) was stir-

red at 25 ^ꢀC for 4±16 h. The reaction mixture was dilu-

ted with EtOAc and the resulting solution was

successively washed with aqueous 1 N HCl (not for 7),

H₂O, saturated aqueous NaHCO₃and brine, and then

dried (MgSO₄), ®ltered and concentrated under reduced

pressure. The crude BOC derivative was treated with

excess anhydrous 4 N HCl in dioxane at 25 ^ꢀC for 1±2 h.

The mixture was concentrated under reduced pressure

and the residue partitioned between aqueous 5%

Na₂CO₃and EtOAc. The organic layer was dried

(MgSO₄), ®ltered and concentrated under reduced pres-

sure to give the amines 5±8 that were used without any

puri®cation.

N,N-Dimethyl-2-[(2-phenylethyl)amino]acetamide (12).

Using phenethylamine, 12 (36%) was obtained as a yel-

lowish oil after puri®cation by ¯ash chromatography

(EtOAc:MeOH, 3:1): IR (®lm) n_max3540, 3320,

1655 cm ¹; ¹H NMR (CDCl₃) d 7.30±7.17 (m, 5H), 3.44

(s, 2H), 2.95 (s, 3H), 2.93 (s, 3H), 2.94±2.50 (m, 2H),

2.87±2.83 (m, 2H), 2.75 (broad s, 1H); MS (CI) m/z 207

(MH)⁺.

N,N-Dimethyl-2-[(2-pyridinylmethyl)amino]acetamide di-

hydrochloride (13). Using (2-aminomethyl)pyridine and

the procedure described above, 13 could not be extrac-

ted from the NaHCO₃solution (soluble). The aqueous

layer was therefore concentrated under reduced pressure

and the residue was treated with (BOC)₂O (1 equiv) in

CH₂Cl₂(0.6 M) at 25 ^ꢀC for 2 h. The BOC derivative was

puri®ed by ¯ash chromatography (CHCl₃:EtOH, 15:1).

Upon treatment of the BOC derivative with excess 5 M

N,N-Dimethyl-2-[(phenylmethyl)amino]acetamide (5).

Colorless oil (77%): IR (®lm) n_max3520, 3330,

1650 cm ¹; ¹H NMR (CDCl₃) d 7.39±7.26 (m, 5H), 3.87

(s, 2H), 3.45 (s, 2H), 2.97 (s, 3H), 2.91 (s, 3H); MS (CI)

m/z 193 (MH)⁺.

HCl/dioxane for 2 h, 13 (26%) was obtained as a yellow

1

solid: FTIR (KBr) n_max3850±3540, 1652 cm

;

¹H

4-[2-[(Phenylmethyl)amino]acetyl]morpholine (6). Yellow-

1

NMR (DMSO-d₆) d 9.43 (broad s, 1H), 8.65 (ddd,

J=5.1, 1.6, 1.0 Hz, 1H), 7.93 (ꢁtd, J=7.9, 7.5, 1.6 Hz,

1H), 7.58 (broad d, J=7.9 Hz, 1H), 7.47 (ddd, J=7.5,

5.1, 1.0 Hz, 1H), 6.79 (broad s, 2H), 4.34 (s, 2H), 4.11 (s,

2H), 2.93 (s, 3H), 2.90 (s, 3H); MS (FAB) m/z 194

(MH)⁺.

ish oil (78%): IR (®lm) n_max3520, 3340, 1650 cm ¹; H

NMR (CDCl₃) d 7.38±7.25 (m, 5H), 3.85 (s, 2H), 3.74±

3.63 (m, 6H), 3.44 (s, 2H), 3.37±3.35 (m, 2H), 2.86

(broad s, 1H); MS (CI) m/z 235 (MH)⁺.

N-Methyl-2-[(phenylmethyl)amino]-N-[2-(2-pyridinyl)-

ethyl]acetamide (7). Pale yellow oil (86%): IR (®lm)

n_max3460, 3330, 1650 cm

d 8.48, 8.44 (2d, J=4.3 Hz, 1H), 7.69 (t, J=7.6 Hz,

1H), 7.34±7.20 (m, 7H), 3.73, 3.64 (2s, 2H), 3.64, 3.59

(2t, J=7.3 Hz, 2H), 3.40, 3.29 (2s, 2H), 2.95, 2.91 (2t,

J=7.3 Hz, 2H), 2.84 (s, 3H); MS (FAB) m/z 284

(MH)⁺.

N,N-Dimethyl-2-[[1(R)-phenylethyl]amino]acetamide (14).

From 1(R)-phenylethylamine, 14 (76%) was obtained

as a yellowish oil after puri®cation by column chroma-

1

;

¹H NMR (DMSO-d₆)

tography (EtOAc:MeOH, 4:1): [a]²_d⁵+52.4^ꢀ(c 1.28

1

MeOH); IR (®lm) n_max3320, 1655 cm

;

¹H NMR

(CDCl₃) d 7.36±7.30 (m, 4H), 7.24 (broad t, J=6.7 Hz,

1H), 3.81 (q, J=6.3 Hz, 1H), 3.27 (s, 2H), 3.05 (broad s,

498

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

1H), 2.94 (s, 3H), 2.81 (s, 3H), 1.42 (d, J=6.3 Hz, 3H);

MS (CI) m/z 207 (MH)⁺.

under reduced pressure to give 21 (533 mg, 98%): IR

1

(®lm) n_max1746, 1701 cm ¹; H NMR (CDCl₃) d (1:1

mixt. of rotamers) 4.12, 4.11 (2q, J=7.0 Hz, 2H), 3.86,

3.77 (2s, 2H), 3.05, 3.02 (2d, J=7.3 Hz, 2H), 1.69±1.55

(m, 5H), 1.48±1.40 (m, 1H), 1.40, 1.35 (2s, 9H), 1.21,

1.20 (2t, J=7.0 Hz, 3H), ꢁ1.23±1.07 (m, 3H), 0.90±0.78

(m, 2H); MS (FAB) m/z 300 (MH)⁺.

N,N-Dimethyl-2-[[1(S)-phenylethyl]amino]acetamide (15).

Using 1(S)-phenylethylamine, 15 (70%) was obtained

as a yellowish oil: [a]²_d⁵50.4^ꢀ(c 0.98 MeOH); IR and

¹H NMR identical to enantiomer 14; MS (CI) m/z 207

(MH)⁺.

1,1-Dimethylethyl N-(carboxymethyl)-N-(cyclohexyl-

methyl)carbamate (22). A heterogeneous solution of 21

(510mg, 1.70mmol) and 2 N NaOH (2.6 mL, 5.10mmol)

in MeOH (2.6 mL) was stirred at 25 ^ꢀC for 16 h. The

MeOH was removed under reduced pressure and the

residual aqueous suspension was diluted with H₂O

(70 mL). The resulting solution was washed with Et₂O

(2Â20 mL), rendered acidic (pH<3) by addition of 1 N

HCl and extracted with EtOAc (2Â50 mL). The com-

bined organic layers were washed with brine, dried

(MgSO₄), ®ltered and concentrated under reduced pres-

sure to give 22 (443 mg, 96%) as a colorless oil: IR (®lm)

2-[(Cyclohexylmethyl)amino]-N,N-dimethylacetamide (16).

From cyclohexanemethylamine, 16 (30%) was obtained

as a yellowish solid after puri®cation by ¯ash chromato-

graphy (CHCl₃:EtOH, 9:1 to 6:1): IR (KBr) n_max3320,

1

1645 cm ¹; H NMR (CDCl₃) d 3.43 (s, 2H), 2.97 (3H,

s), 2.96 (s, 3H), 2.88 (broad s, 1H), 2.47 (d, J=6.7 Hz,

2H), 1.73±1.63 (m, 5H), 1.54±1.47 (m, 1H), 1.30±1.12

(m, 3H), 0.99±0.90 (m, 2H); MS (CI) m/z 199 (MH)⁺.

2-[[1(S)-Cyclohexylethyl]amino]-N,N-dimethylacetamide

(17). From 1(S)-cyclohexylethylamine, 17 (42%) was

obtained as a yellowish oil after puri®cation by ¯ash

chromatography (CHCl₃:EtOH, 10:1): [a]²_d⁵+13.8^ꢀ(c

1

n_max1693 (broad) cm ¹; H NMR (DMSO-d₆) d 3.79,

3.77 (2s, 2H), 3.01 (broad d, J=7.3 Hz, 2H), 1.67±1.45

(m, 6H), 1.38, 1.34 (2s, 9H), 1.28±1.09 (m, 3H), 0.90±

0.82 (m, 2H); MS (FAB) m/z 272 (MH)⁺.

1

0.98 MeOH); IR (®lm) n_max3320, 1655 cm ¹; H NMR

(CDCl₃) d 3.53 (broad d, J=15.7 Hz, 1H), 3.39 (broad

d, J=15.7 Hz, 1H), 2.97 (broad s, 6H), 2.54±2.45 (m,

1H), 1.81±1.61 (m, 5H), 1.46±1.34 (m, 1H), 1.31±1.00

(m, 8H); MS (CI) m/z 213 (MH)⁺.

Ethyl 2-[[1(S)-phenylethyl]amino]acetate (23). 1(S)-Phe-

nylethylamine (3.03 g, 25.0 mmol) was converted into 23

using the procedure described for 20. Puri®cation by

¯ash chromatography (hexane:EtOAc, 2:1) gave 23 as

a colorless oil (3.20 g, 62%): ¹H NMR (DMSO-d₆)

d 7.33±7.29 (m, 4H), 7.24±7.20 (m, 1H), 4.06 (q, J=

7.2 Hz, 2H), 3.74 (q, J=6.6 Hz, 1H), 3.17 (d, J=

17.2 Hz, 1H), 3.06 (d, J=17.2 Hz, 1H), 2.35 (broad s,

1H), 1.25 (d, J=6.6 Hz, 3H), 1.16 (t, J=7.2 Hz, 3H);

MS (ESI) m/z 208 (MH)⁺.

N,N-Dimethyl-2-[[(1-hydroxycyclohexyl)methyl]amino]-

acetamide (18). Using 1-(aminomethyl)cyclohexanol, 18

(43%) was obtained as a beige solid after puri®cation by

¯ash chromatography (CH₂Cl₂:EtOAc, 85:15 to 3:1):

1

IR (KBr) n_max3410, 3370, 1655, 1630 cm ¹; H NMR

(DMSO-d₆) d 4.02 (broad s, 1H), 3.35 (s, 2H), 2.91 (s,

3H), 2.83 (s, 3H), 2.42 (s, 2H), 2.34±2.22 (broad s, 1H),

1.60±1.29 (m, 9H), 1.27±1.16 (m, 1H); MS (EI) m/z 215

(MH)⁺.

1,1-Dimethylethyl N-(2-ethoxy-2-oxoethyl)-N-[1(S)-phen-

ylethyl]carbamate (24). Using the procedure described

for 21, amine 23 (2.89 g, 13.9 mmol) and (Boc)₂O

(2.95 g, 13.5 mmol) gave carbamate 24 (4.14 g, 100%) as

Ethyl 2-[(cyclohexylmethyl)amino]acetate (20). A solu-

tion of ethyl 2-bromoacetate (19) (500 mg, 3.00 mmol),

cyclohexanemethylamine (340 mg, 3.00 mmol) and Et₃N

(607 mg, 6.00 mmol) in THF (6.0 mL) was stirred at

25 ^ꢀC for 1.5 h. The reaction mixture was diluted with

EtOAc (100 mL) and the solution was washed with

saturated aqueous NaHCO₃(40 mL), brine (40 mL) and

then dried (MgSO₄), ®ltered and concentrated under

reduced pressure. The residue was puri®ed by ¯ash

chromatography (hexane:EtOAc, 4:1 to 2:1) to give 20

(394 mg, 66%) as a pale yellow oil: IR (®lm) n_max

1

a colorless oil: H NMR (CDCl₃) d (1.7:1.0 mixt. of

rotamers) 7.35±7.26 (m, 5H), 5.62, 5.33 (2q, J=6.6 Hz,

1H), 4.11 (q, J=7.1 Hz, 2H), 3.92, 3.70 (2d, J=17.0,

17.6 Hz, 1H), 3.55, 3.44 (2d, J=17.0, 17.6 Hz, 1H),

1.53±1.42 (m, 12H), 1.22 (t, J=7.1 Hz, 3H); MS (FAB)

m/z 308 (MH)⁺.

1,1-Dimethylethyl N-(carboxymethyl)-N-[1(S)-phenyl-

ethyl]carbamate (25). Using the procedure described for

22, 24 (3.07 g, 10.0 mmol) was transformed into acid 25

(2.45 g, 99%), isolated as a thick colorless oil: [a]_d²³

65.7^ꢀ(c 0.63 MeOH); ¹H NMR (DMSO-d₆) d (2:1

mixt. of rotamers) 7.36 (m, 5H), 5.36, 5.09 (2q, J=

6.6 Hz, 1H), 3.83, 3.67 (2d, J=17.3 Hz, 1H), 3.67, 3.49

(2d, J=17.3 Hz, 1H), 1.47, 1.43 (2d, J=6.6 Hz, 3H),

1.38, 1.32 (2s, 9H); MS (FAB) m/z 280 (MH)⁺. Anal.

calcd for C₁₅H₂₁NO₄: C, 64.50; H, 7.58; N, 5.01. Found:

C, 64.42; H, 7.61; N, 4.97.

1

1736 cm ¹; H NMR (DMSO-d₆) d 4.08 (q, J=7.2 Hz,

2H), 3.27 (s, 2H), 2.33 (d, J=6.7 Hz, 2H), 2.04 (broad s,

1H), 1.72±1.60 (m, 5H), 1.38±1.28 (m, 1H), 1.24±1.07

(m, 3H), 1.19 (t, J=7.2 Hz, 3H), 0.89±0.80 (m, 2H); MS

(FAB) m/z 200 (MH)⁺.

1,1-Dimethylethyl N-(cyclohexylmethyl)-N-(2-ethoxy-2-

oxoethyl)carbamate (21). A solution of 20 (372 mg,

1.87 mmol) and (Boc)₂O (396 mg, 1.81 mmol) in CH₂Cl₂

(3.7 mL) was stirred at 25 ^ꢀC for 2.5 h. The reaction

mixture was diluted with EtOAc (75 mL) and the

resulting solution was washed with 1 N HCl (30 mL),

saturated aqueous NaHCO₃(30 mL), brine (30 mL) and

then was dried (MgSO₄), ®ltered and concentrated

1,1-Dimethylethyl N-(cyclohexylmethyl)-N-[2-[methyl[2-

(2-pyridinyl)ethyl]amino]-2-oxoethyl]carbamate (26).

.

BOP PF₆(713mg, 1.61mmol) was added to a solution of

22 (417mg, 1.53mmol), 2-[2-(methylamino)ethyl]pyridine

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

499

(220 mg, 1.61 mmol) and i-Pr₂NEt (397 mg, 3.07 mmol)

in DMF (6.0 mL) at 25 ^ꢀC. The mixture was stirred at

25 ^ꢀC for 3 h. The reaction mixture was diluted with

EtOAc (100 mL) and the resulting solution was washed

successively with saturated aqueous NaHCO₃(30 mL),

H₂O (2Â20 mL) and brine (30 mL) before being dried

(MgSO₄), ®ltered and concentrated under reduced pres-

sure. The residue was puri®ed by ¯ash chromatography

(EtOAc:MeOH, 20:1) to yield 26 (548 mg, 89%) as a

colorless oil: IR (®lm) n_max3600±3260, 1675 (broad)

was added (5 min) to an ice-cold solution of 4-pentenoic

acid (12.0 g, 120 mmol) and N-methylmorpholine (14.2 g,

140 mmol) in THF (100 mL). The mixture was stirred at

0 ^ꢀC for 30 min then was cooled to 78 ^ꢀC. A cold

( 78 ^ꢀC) solution of the lithium salt of 4(S)-(1-methyl-

ethyl)-2-oxazolidinone (100mmol) [prepared using 1.6 M

n-BuLi/hexane (62.5 mL, 100 mmol)] in THF (150 mL)

was added via cannula to the mixed anhydride and the

reaction mixture was stirred at 78 ^ꢀC for 30 min.

Saturated aqueous NH₄Cl was then added, the mixture

was allowed to warm to 25 ^ꢀC and was poured into

H₂O. The phases were separated and the aqueous layer

extracted with EtOAc. The combined organic extracts

were washed with brine, dried (MgSO₄), ®ltered and

concentrated under reduced pressure to give 35. Crude

35 was added to an ice-cold ether solution of CH₂N₂

(0.4 M, 175 mL) in a 4 L Erlenmeyer ¯ask followed by

the addition of Pd(OAc)₂(112 mg, 0.5 mmol). After the

vigorous reaction stopped, additional amounts of

Pd(OAc)₂(3Â112 mg) and CH₂N₂solution (3Â175 mL)

were added in sequence. The reaction mixture was ®l-

tered through a pad of Celite¹and concentrated under

reduced pressure. The residue was puri®ed by distilla-

tion (bulb-to-bulb, 100 ^ꢀC, 0.05 mm Hg) to yield 36

(22.4 g, 99%) as a colorless oil: IR (®lm) n_max1785,

1705 cm ¹; ¹H NMR (CDCl₃) d 4.42±4.38 (m, 1H), 4.24

(ꢁdd, J=9.1, 8.3 Hz, 1H), 4.17 (dd, J=9.1, 3.0 Hz, 1H),

3.05 (dt, J=16.4, 7.4 Hz, 1H), 2.93 (dt, J=16.4, 7.4 Hz,

1H), 2.34 (sept d, J=7.0, 3.8 Hz, 1H), 1.53 (ꢁq, J=

7.4 Hz, 2H), 0.88 (d, J=7.0 Hz, 3H), 0.84 (d, J=7.0 Hz,

3H), 0.76±0.67 (m, 1H), 0.41±0.38 (m, 2H), 0.06±0.02

(m, 2H); MS (CI) m/z 226 (MH)⁺.

1

cm ¹; H NMR (DMSO-d₆) d (ꢁ1:1:1:1 mixt. of rota-

mers) 8.52, 8.48 (2d, J=4.4, 4.1 Hz, 1H), 7.75±7.67 (m,

1H), 7.31, 7.27±7.19 (d and m, J=7.9 Hz, 2H), 3.92,

3.88, 3.79 (3s, 2H), 3.64±3.58 (m, 2H), 3.01±2.84 (m,

4H), 2.89, 2.82, 2.81 (3s, 3H), 1.70±1.43 (m, 6H), 1.38,

1.36, 1.31, 1.29 (4s, 9H), 1.19±1.09 (m, 3H), 0.90±0.76

(m, 2H); MS (FAB) m/z 390 (MH)⁺.

2-[(Cyclohexylmethyl)amino]-N-methyl-N-[2-(2-pyridin-

yl)ethyl]acetamide (27). A solution of 26 (518 mg,

1.33 mmol) in 4 N anhydrous HCl in 1,4-dioxane

(5.0 mL, 20 mmol) was stirred at 25 ^ꢀC for 1 h. The

mixture was concentrated under reduced pressure. The

residue was dissolved in H₂O and treated with aqueous

5% Na₂CO₃before the resulting solution was extracted

with EtOAc (5Â50 mL). The combined organic layers

were dried (MgSO₄), ®ltered and concentrated under

reduced pressure to give 27 (365 mg, 95%) as a colorless

;

¹H NMR

1

oil: IR (®lm) n_max3500±3220, 1645 cm

(DMSO-d₆) d (1:1 mixt. of rotamers) 8.50, 8.48 (2 broad

d, J=3.8, 4.8 Hz, 1H), 7.71, 7.69 (2td, J=7.3, 1.6 Hz,

J=7.6, 1.9 Hz, 1H), 7.30±7.19 (m, 2H), 3.64±3.59 (m,

2H), 3.26, 3.11 (2s, 2H), 2.97, 2.89 (2t, J=7.0, 7.0 Hz,

2H), 2.86, 2.82 (2s, 3H), 2.30, 2.16 (2d, J=6.7, 6.7 Hz,

2H), 1.74±1.57 (m, 5H), 1.40±1.08 (m, 4H), 0.91±0.75

(m, 2H); MS (ESI) m/z 290 (MH)⁺.

3-(1-Oxopentyl)-4(S)-(phenylmethyl)-2-oxazolidinone (37).

Valeryl chloride (3.74 g, 31.0 mmol) was added (2 min)

to a solution of the lithium salt of 4(S)-(phenylmethyl)-

2-oxazolidinone (28.2 mmol) [prepared using 1.6 M

n-BuLi/hexane (17.2 mL, 28.2 mmol) in THF at 78 ^ꢀC.

The mixture was stirred at 78 ^ꢀC for 15 min, allowed

to warm to 0 ^ꢀC and stirred 2 h at this temperature.

Saturated aqueous NaHCO₃was added and the mixture

was extracted with CH₂Cl₂(3Â). The combined organic

layers were washed with 5% aqueous Na₂CO₃and

brine, then dried (MgSO₄), ®ltered and concentrated

under reduced pressure. The residue was puri®ed by

¯ash chromatography (hexane:EtOAc, 4:1) to yield 37

(6.75 g, 92%) as a colorless oil: [a]²_d⁵+103.4^ꢀ(c 2.05

1,1-Dimethylethyl N-[2-[methyl[2-(2-pyridinyl)ethyl]-

amino]-2-oxoethyl]-N-[1(S)-phenylethyl]carbamate (28).

Acid 25 (2.39 g, 8.56 mmol) was converted into 28

using the procedure described for 26. Puri®cation by

¯ash chromatography (EtOAc:MeOH, 20:1) gave 28

(3.13 g, 79%) as a colorless oil: IR (®lm) n_max3615±

3310, 1674 (broad) cm ¹; ¹H NMR (DMSO-d₆) d (mixt.

of rotamers) 8.48, 8.38 (d and broad s, J=4.4 Hz, 1H),

7.72±7.66 (m, 1H), 7.36±7.09 (m, 7H), 5.29, 4.92 (2

broad s, 1H), 4.17±3.30 (m, 4H), 3.30±2.81 (m, 5H),

1.38±1.26 (m, 12H); MS (ESI) m/z 398 (MH)⁺.

1

MeOH); IR n_max1780, 1700 cm ¹; H NMR (CDCl₃) d

7.37±7.21 (m, 5H), 4.71±4.66 (m, 1H), 4.23±4.15 (m,

2H), 3.31 (dd, J=13.3, 3.5 Hz, 1H), 3.03±2.87 (m, 2H),

2.78 (dd, J=13.4, 9.9 Hz, 1H), 1.73±1.65 (m, 2H), 1.43

(sext, J=7.3 Hz, 2H), 0.97 (t, J=7.3 Hz, 3H); MS (CI)

m/z 262 (MH)⁺. Anal. calcd for C₁₅H₁₉NO₃: C, 68.94;

H, 7.33; N, 5.36. Found: C, 68.89; H, 7.39; N, 5.35.

N-Methyl-2-[[1(S)-phenylethyl]amino]-N-[2-(2-pyridinyl)-

ethyl]acetamide (29). Using the procedure described for

27, 28 (1.27 g, 3.19 mmol) was transformed into 29

(916 mg, 96%), isolated as a colorless oil: [a]²_d³40.5^ꢀ(c

1

1.61 MeOH); IR (®lm) n_max3550±3280, 1644 cm ¹; H

NMR (DMSO-d₆) d (1:1 mixt. of rotamers) 8.47, 8.37 (2

broad d, J=3.8, 4.1 Hz, 1H), 7.69, 7.65 (2td, J=7.6,

1.9 Hz, J=7.6, 1.6 Hz, 1H), 7.33±7.13 (m, 7H), 3.71±3.55

(m, 2H), 3.49, 2.88 (2t, J=7.0, 7.3 Hz, 2H), 3.13, 3.01 (2s,

2H), 2.88±2.84 (m, 1H), 2.80, 2.73 (2s, 3H), 1.25, 1.21

(2d, J=6.7, 6.7 Hz, 3H); MS (ESI) m/z 298 (MH)⁺.

4(S)-(1-Methylethyl)-3-(1-oxo-3-(2-thienyl)propyl)-2-oxa-

zolidinone (38). Trimethylacetyl chloride (4.64 g,

38.5 mmol) was added dropwise to a cold ( 78 ^ꢀC)

solution of 32⁴⁰(6.01 g, 38.5 mmol) and Et₃N (4.25 g,

42.0 mmol) in THF (155 mL). The mixture was allowed

to warm to 0 ^ꢀC, stirred at 0 ^ꢀC for 30 min then cooled

to 78 ^ꢀC. A cold ( 78 ^ꢀC) solution of the lithium salt

of 4(S)-(1-methylethyl)-2-oxazolidinone (35.0 mmol)

3-(3-Cyclopropyl-1-oxopropyl)-4(S)-(1-methylethyl)-2-

oxazolidinone (36). Pivaloyl chloride (14.5 g, 120 mmol)

500

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

[prepared from n-BuLi/hexane (21.9 mL, 35.0 mmol)] in

THF (140 mL) was added via cannula (10 min) to the

reaction mixture. After stirring at 78 ^ꢀC for 30 min

and then at 0 ^ꢀC for 45 min, saturated NH₄Cl (25 mL)

was added. The mixture was poured into H₂O (600 mL)

and the suspension was extracted with EtOAc

(2Â300 mL). The combined organic layers were washed

with saturated NaHCO₃(100 mL) and brine (100 mL),

then dried (MgSO₄) and concentrated under reduced

pressure. Puri®cation of the residue by ¯ash chromato-

graphy (hexane:EtOAc, 3:1) gave 38 (7.07 g, 69%) as a

colorless oil: [a]_d²⁵+72.9^ꢀ(c 2.19 MeOH); IR (®lm) n_max

Procedure for the preparation of 42±46. A solution of 1

M NaHMDS in THF (1.1 equiv) was added (ꢁ10 min)

to a cold ( 78 ^ꢀC) solution of the acyl oxazolidinone in

THF (0.2±0.25 M). The reaction mixture was stirred at

78 ^ꢀC for 45 min. A solution of tert-butyl (or benzyl)

2-bromoacetate (2.0 equiv) in THF (1/1, v/v) was next

added dropwise and the reaction mixture was stirred at

the same temperature for 1.5±2 h. Saturated aqueous

NH₄Cl was then added, the mixture was allowed to

warm to 25 ^ꢀC, poured into diluted aqueous NH₄Cl and

extracted with EtOAc (3Â). The combined organic lay-

ers were washed with saturated aqueous NaHCO₃and

brine, dried (MgSO₄), ®ltered and concentrated under

reduced pressure.

1

1780, 1700 cm ¹; H NMR (CDCl₃) d 7.12 (dd, J=5.1,

1.3 Hz, 1H), 6.9 (dd, J=5.1, 3.2 Hz, 1H), 6.85 (dd,

J=3.2, 1.3 Hz, 1H), 4.45±4.41 (m, 1H), 4.26 (ꢁdd,

J=8.9, ꢁ8.3> Hz, 1H), 4.20 (dd, J=8.9, 3.2 Hz, 1H),

3.41±3.27 (m, 2H), 3.26±3.15 (m, 2H), 2.37 (sept d,

J=7.0, 4.1 Hz, 1H), 0.91 (d, J=7.0 Hz, 3H), 0.85 (d,

J=7.0 Hz, 3H); MS (FAB) m/z 268 (MH)⁺. Anal. calcd

for C₁₃H₁₇NO₃S: C, 58.40; H, 6.41; N, 5.24. Found: C,

58.19; H, 6.40; N, 5.24.

3-[2(R)-[2-(1,1-Dimethylethoxy)-2-oxoethyl]-1-oxopentyl]-

4(S)-(phenylmethyl)-2-oxazolidinone (42). Starting with

37 (1.20 g, 4.60 mmol), 42 (1.35 g, 78%) was obtained as

a white solid after puri®cation by ¯ash chromatography

(hexane:EtOAc, 8:1): mp 90±92 ^ꢀC; [a]_d²⁵+72.3^ꢀ(c 1.06

1

MeOH); IR (®lm) n_max1749, 1717 cm

;

¹H NMR

(DMSO-d₆) d 7.34±7.23 (m, 5H), 4.67±4.61 (m, 1H),

4.33 (ꢁdd, J=8.7, 8.3 Hz, 1H), 4.15 (dd, J=8.7, 2.4 Hz,

1H), 4.06±3.99 (m, 1H), 3.00 (dd, J=13.5, 3.0 Hz, 1H),

2.86 (dd, J=13.5, 7.8 Hz, 1H), 2.62 (dd, J=16.5,

10.2 Hz, 1H), 2.48 (dd, J=16.5, 4.8 Hz. 1H), 1.60±1.51

(m, 1H), 1.39 (s, 9H), 1.39±1.23 (m, 3H), 0.84 (t, J=

7.1 Hz, 3H); MS (FAB) m/z 376 (MH)⁺. Anal. calcd for

C₂₁H₂₉NO₅: C, 67.18; H, 7.79; N, 3.73. Found: C,

67.19; H, 7.93; N, 3.65.

3-[1-Oxo-3-(2-thiazolyl)propyl]-4(S)-(phenylmethyl)-2-

oxazolidinone (40). Using the method described for 38,

acid 34⁴⁰(15.9 g, 89.9 mmol) gave, after puri®cation by

¯ash chromatography (hexane:EtOAc, 1.5:1), 40 (15.4,

55%) as a white solid: [a]²_d⁵+108.5^ꢀ(c 1.055 MeOH);

1

IR (®lm) n_max1781, 1694 cm ¹; H NMR (CDCl₃) d

7.69 (d, J=3.2 Hz, 1H), 7.36±7.27 (m, 3H), 7.22±7.20

(m, 3H), 4.73±4.67 (m, 1H), 4.25±4.17 (m, 2H), 3.59±

3.43 (m, 4H), 3.30 (dd, J=13.4, 3.2 Hz, 1H), 2.80 (dd,

J=13.4, 9.5 Hz, 1H); MS (CI) m/z 317 (MH)⁺. Anal.

calcd for C₁₆H₁₆N₂O₃S: C, 60.74; H, 5.10; N, 8.85.

Found: C, 60.49; H, 5.00; N, 8.82.

3-[4-(1,1-Dimethylethoxy)-1,4-dioxo-2(S)-(2-thienylmethyl)-

butyl]-4(S)-(1-methylethyl)-2-oxazolidinone (43). Using

38 (4.00 g, 15.0 mmol), 43 (4.51 g, 79%) was obtained as

a white solid after trituration of the crude residue with

ether (75 mL): [a]_d²⁵+91.8^ꢀ(c 0.51 CHCl₃); IR (KBr)

3-[2(R)-(Cyclopropylmethyl)-4-(1,1-dimethylethoxy)-1,4-

dioxobutyl]-4(S)-(1-methylethyl)-2-oxazolidinone (41). A

solution of 36 (20.0 g, 88.8 mmol) in THF (40 mL) was

added (45 min) to a solution of LDA [prepared from

i-Pr₂NH (10.8 g, 106 mmol) and 1.4 M n-BuLi/hexane

(70.0 mL, 96.0 mmol)] in THF (150 mL) cooled to

78 ^ꢀC. The solution was stirred at 78 ^ꢀC for 1 h.

DMPU (25.0 g, 195 mmol) was added (5 min) followed

by a solution of tert-butyl 2-bromoacetate (18.2 g,

93.2 mmol) (added over 10 min) in THF (20 mL). The

reaction mixture was stirred at 78 ^ꢀC for 1.5 h then

saturated aqueous NH₄Cl was added and the mixture

allowed to warm to 25 ^ꢀC. The reaction mixture was

diluted with EtOAc and the resulting solution was

washed with 5% aqueous citric acid, saturated aqueous

NaHCO₃, brine and then dried (MgSO₄), ®ltered and

concentrated under reduced pressure. The yellow oil

obtained was puri®ed by crystallization (EtOAc:hexane)

at 20 ^ꢀC to yield 41 (21.7 g, 72%) as white crystals: mp

104±105 ^ꢀC; [a]²_d⁵+52.8^ꢀ(c 1.02 CHCl₃); ¹H NMR

(DMSO-d₆) d 4.37±4.27 (m, 3H), 4.21±4.14 (m, 1H), 2.64

(dd, J=16.5, 10.5 Hz, 1H), 2.54 (dd, J=16.5, 4.4 Hz,

1H), 2.18 (sept d, J=7.0, 3.2 Hz, 1H), 1.43 (dt, J=13.8,

7.0 Hz, 1H), 1.36 (s, 9H), 1.29 (dt, J=13.8, 7.0 Hz, 1H),

0.84 (d, J=7.0Hz, 3H), 0.82 (d, J=7.0Hz, 3H), 0.69±0.65

(m, 1H), 0.40±0.34 (m, 2H), 0.04±0.01 (m, 2H); MS (CI)

m/z 340 (MH)⁺. Anal. calcd for C₁₈H₂₉NO₅: C, 63.69;

H, 8.61; N, 4.13. Found: C, 63.67; H, 8.72; N, 4.10.

1

n_max1785, 1730, 1705 cm ¹; H NMR (CDCl₃) d 7.13

(dd, J=5.1, 1.3 Hz, 1H), 6.91 (dd, J=5.1, 3.5 Hz, 1H),

6.86 (dd, Jꢁ3.5, 1.3 Hz, 1H), 4.50±4.42 (m, 1H), 4.36±

4.32 (m, 1H), 4.16 (dd, J=8.9, 2.9 Hz, 1H), 4.12 (ꢁdd,

J=8.9, 8.3 Hz, 1H), 3.18 (dd, J=14.3, 6.7 Hz, 1H), 2.91

(dd, J=14.3, 7.9 Hz, 1H), 2.78 (dd, J=16.5, 9.8 Hz,

1H), 2.44 (dd, J=16.5, 4.8 Hz, 1H), 2.35 (sept d, J=7.0,

4.1 Hz, 1H), 1.40 (s, 9H), 0.91 (d, J=6.7 Hz, 3H), 0.89

(d, J=7.0 Hz, 3H); MS (FAB) m/z 382 (MH)⁺. Anal.

calcd for C₁₉H₂₇NO₅S: C, 59.82; H, 7.13; N, 3.67.

Found: C, 59.57; H, 7.18; N, 3.60.

3-[1,4-Dioxo-4-(phenylmethoxy)-2(S)-[(1-triphenylmethyl-

1H-imidazol-4-yl)methyl]butyl]-4(S)-(1-methylethyl)-2-

oxazolidinone (44). From 39²⁵(8.06 g, 16.3 mmol) and

after puri®cation by ¯ash chromatography (EtOAc:

hexane, 2:1), 44 (6.68 g, 64%) was obtained as a white

solid: [a]²_d⁵+55.3^ꢀ(c 0.45 MeOH); IR (KBr) n_max1785,

1

1735, 1690 cm

;

¹H NMR (CDCl₃) d 7.33±7.30 (m,

15H), 7.13±7.11 (m, 6H), 6.60 (broad s, 1H), 5.07 (s, 2H),

4.53±4.46 (m, 1H), 4.40±4.36 (m, 1H), 4.15±4.13 (m, 2H),

2.98 (dd, J=16.8, 10.2 Hz, 1H), 2.89 (dd, J=14.3,

6.4 Hz, 1H), 2.75 (dd, J=14.3, 7.0 Hz, 1H), 2.60 (dd, J=

16.8, 4.4 Hz, 1H), 2.33 (sept d, J=7.0, 3.8 Hz, 1H), 0.88

(d, J=7.0 Hz, 3H), 0.86 (d, J=7.0 Hz, 3H); MS (FAB)

m/z 642 (MH)⁺. Anal. calcd for C₄₀H₃₉N₃O₅: C, 74.86;

H, 6.13; N, 6.55. Found: C, 74.98; H, 6.24; N, 6.56.

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

501

3-[4-(1,1-Dimethylethoxy)-1,4-dioxo-2(S)-(2-thiazolyl-

methyl)butyl]-4-(S)-(phenylmethyl)-2-oxazolidinone (45).

The alkylation of 40 (6.25 g, 19.8 mmol) gave 45

(7.60 g, 89%) as a white solid after puri®cation by ¯ash

chromatography (hexane:EtOAc, 2:1): [a]²_d⁵+102.2^ꢀ(c

1.005 MeOH); IR (®lm) n_max1765, 1714 cm ¹; ¹H NMR

(CDCl₃) d 7.67 (d, J=3.5 Hz, 1H), 7.36±7.26 (m, 5H),

7.23 (d, J=3.5 Hz, 1H), 4.71±4.65 (m, 1H), 4.64±4.57

(m, 1H), 4.20±4.16 (m, 2H), 3.43 (dd, J=14.7, 6.5 Hz,

1H), 3.36 (dd, J=13.4, 3.2 Hz, 1H), 3.24 (dd, J=14.7,

7.3 Hz, 1H), 2.92 (dd, J=16.6, 9.5 Hz, 1H), 2.77 (dd,

J=13.4, 10.0 Hz, 1H), 2.59 (dd, J=16.6, 4.9 Hz, 1H),

1.44 (s, 9H); MS (FAB) m/z 431 (MH)⁺. Anal. calcd for

C₂₂H₂₆N₂O₅S: C, 61.38; H, 6.09; N, 6.51. Found: C,

61.26; H, 6.10; N, 6.46.

d 7.66 (d, J=9.2Hz, 1H), 4.70 (d, J=6.4Hz, 1H), 4.60 (d,

J=3.8 Hz, 1H), 4.12±4.06 (m, 1H), 3.10±3.04 (m, 1H),

2.94±2.90 (m, 1H), 2.82±2.74 (m, 1H), 2.46 (dd, J=16.2,

9.2 Hz, 1H), 2.31 (dd, J=16.2, 5.7 Hz, 1H), 1.77±1.30

(m, 10H), 1.38 (s, 9H), 1.22±1.08 (m, 6H), 0.93±0.85 (m,

1H), 0.86 (d, J=7.0 Hz, 3H), 0.80±0.74 (m, 1H), 0.75 (d,

J=6.7 Hz, 3H), 0.68±0.63 (m, 1H), 0.40±0.35 (m, 2H),

0.05±0.00 (m, 2H); MS (CI) m/z 454 (MH)⁺.

N-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-methyl-

hexyl]-2(R)-[2-(1,1-dimethylethoxy)-2-oxoethyl]pentan-

amide (49). From 42, 49 (0.80 g, 66%) was obtained as

a white solid after puri®cation by ¯ash chromatography

(hexane:EtOAc, 6:1): [a]²_d⁵45.1^ꢀ(c 1.0 MeOH); ¹H

NMR (DMSO-d₆) d 7.67 (d, J=9.0 Hz, 1H), 4.71 (d,

J= 6.3 Hz, 1H), 4.64 (d, J=3.3 Hz, 1H), 4.11±4.05 (m,

1H), 3.10±3.04 (m, 1H), 2.93±2.90 (m, 1H), 2.72±2.67

(m, 1H), 2.41 (dd, J=15.9, 8.8 Hz, 1H), 2.21 (dd,

J=15.9, 5.7 Hz, 1H), 1.80±1.41 (m, 9H), 1.38 (s, 9H),

0.93±0.74 (m, 2H), 0.87 (d, J=6.6 Hz, 3H), 0.83 (t,

J=7.0 Hz, 3H), 0.78 (d, J=6.6 Hz, 3H); MS (FAB) m/z

442 (MH)⁺.

3-[4-(1,1-Dimethylethoxy)-1,4-dioxo-2(R)-(4-thiazolyl-

methyl)butyl]-4-(S)-(1-methylethyl)-2-oxazolidinone (46).

Starting with 62 (825 mg, 3.07 mmol) and after puri®ca-

tion by ¯ash chromatography (hexane:EtOAc, 2:1), 46

(1.02 g, 86%) was obtained as a white solid: [a]_d²⁵

+144.0^ꢀ(c 1.01 MeOH); IR (®lm) n_max1784, 1705 cm

;

1

¹H NMR (CDCl₃) d 8.78 (d, J=2.1 Hz, 1H), 7.16 (d,

J=2.1 Hz, 1H), 4.59±4.52 (m, 1H), 4.43 (dt, J=8.3,

ꢁ3.5 Hz, 1H), 4.25 (ꢁdd, J=8.9, 8.3 Hz, 1H), 4.20 (dd,

J=8.9, 3.5 Hz, 1H), 3.19 (dd, J=14.2, 6.2 Hz, 1H), 3.07

(dd, J=14.2, 7.3 Hz, 1H), 2.83 (dd, J=16.5, 9.9 Hz,

1H), 2.49 (dd, J=16.5, 4.8 Hz, 1H), 2.37 (sept d, J=7.0,

3.5 Hz, 1H), 1.41 (s, 9H), 0.93 (d, J=7.0 Hz, 3H), 0.92

(d, J=7.0 Hz, 3H); MS (CI) m/z 383 (MH)⁺. Anal.

calcd for C₁₈H₂₆N₂O₅S: C, 56.53; H, 6.85; N, 7.32.

Found: C, 56.20; H, 6.84; N, 7.24.

N-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-methyl-

hexyl]-4-(1,1-dimethylethoxy)-4-oxo-2(S)-(2-thienylmethyl)-

butanamide (50). Using 43, 50 (0.52 g, 73%) was

obtained as a white solid after puri®cation by ¯ash

chromatography (hexane:EtOAc, 3:1): [a]²_d⁵27.3^ꢀ(c

0.62 MeOH); IR (KBr) n_max3460, 3270, 1730, 1705,

1

1635 cm

;

¹H NMR (CDCl₃) d 7.18 (dd, J=5.1,

1.3 Hz, 1H), 6.93 (dd, J=5.1, 3.5 Hz, 1H), 6.86 (ꢁdd,

J=3.5, 1.3 Hz, 1H), 5.62 (d, J=8.6 Hz, 1H), 4.23 (td,

J=8.6, 4.8 Hz, 1H) 3.27±3.16 (m, 1H), 3.08 (dd, J=8.6,

1.3 Hz, 1H), 3.00 (dd, J=8.9, 3.2 Hz, 1H), 2.98±2.90 (m,

2H), 2.70 (dd, J=17.2, 9.2 Hz, 1H), 2.45 (dd, J=17.2,

4.1 Hz, 1H), 1.92±1.82 (m, 1H), 1.75±1.59 (m, 6H),

1.51±1.10 (m, 9H), 1.46 (s, 9H), 0.95±0.79 (m, 2H), 0.94

(d, J=6.7 Hz, 3H), 0.88 (d, J=6.7 Hz, 3H); MS (FAB)

m/z 496 (MH)⁺. Anal. calcd for C₂₇H₄₅NO₅S: C, 65.42;

H, 9.15; N, 2.82. Found: C, 65.27; H, 9.32; N, 2.76.

Procedure for the preparation of 48±53. To an ice-cold

solution of 41±46 in THF:H₂O (ꢁ3:1, 0.05±0.2 M) was

added 30% (w/%) hydrogen peroxide (3±6 equiv) fol-

.

lowed by LiOH H₂O (1±3 equiv). The mixture was

allowed to warm to 25 ^ꢀC and was stirred at this tem-

perature until disappearance (TLC) of the starting

material (1.5±16 h). The mixture was cooled to 0 ^ꢀC and

the excess peroxide was destroyed by addition of 1.5 M

aqueous Na₂SO₃. Most of the THF was removed under

reduced pressure. The residual aqueous solution was

diluted with H₂O and saturated aqueous NaHCO₃and

the resulting solution was washed with CH₂Cl₂(3±4Â).

The aqueous layer was rendered acidic (pH 3) with 10%

aqueous or solid citric acid and extracted with EtOAc

(3Â). The combined organic layers were washed with

brine, dried (MgSO₄), ®ltered and concentrated under

reduced pressure to give the corresponding acids. A

N-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-methyl-

hexyl]-4-oxo-4-(phenylmethoxy)-2(R)-[[(1-triphenylmethyl)-

1H-imidazol-4yl]methyl]butanamide (51). Starting with

44 and using the inseparable mixture of the acid and the

chiral auxiliary in the coupling reaction, 51 (6.08 g,

77%) was obtained as a white solid after puri®cation

by ¯ash chromatography (EtOAc then EtOAc:MeOH,

10:1): [a]²_d⁵19.6^ꢀ(c 0.70 MeOH); IR (KBr) n_max3380,

1

1735, 1645 cm

;

¹H NMR (CDCl₃) d 7.39±7.26 (m,

.

solution of the acid, 47 HCl (1.05 equiv), i-Pr₂NEt (3±

15H), 7.10±7.08 (m, 6H), 6.56 (s, 1H), 6.51 (d,

J=9.5 Hz, 1H), 5.09 (ABq, Án=16.8 Hz, J=12.2 Hz,

2H), 4.43±4.37 (m, 1H), 4.46±4.17 (broad s, ꢁ2H), 3.29

(t, J=8.3 Hz, 1H), 3.15 (d, J=8.6 Hz, 1H), 3.09±3.03

(m, 1H), 2.94 (dd, J=16.5, 8.6 Hz, 1H), 2.86 (dd, J=

15.3, 7.6 Hz, 1H), 2.68 (dd, J=15.3, 3.5 Hz, 1H), 2.38

(dd, J=16.5, 5.7 Hz, 1H), 1.88±1.12 (m, 14H), 0.97±0.80

(m, 2H), 0.88 (d, J=6.7 Hz, 3H), 0.72 (d, J=6.7 Hz,

3H); MS (FAB) m/z 756 (MH)⁺.

.

4 equiv) and BOP PF₆(1.05 equiv) in DMF (0.2±0.4 M)

was stirred at 25 ^ꢀC for 1.5±6 h. The reaction mixture

was diluted with EtOAc and washed successively with

10% aqueous citric acid or 1 N HCl, H₂O, saturated

aqueous NaHCO₃and brine, then was dried (MgSO₄),

®ltered and concentrated under reduced pressure.

N-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-methyl-

hexyl]-2(R)-(cyclopropylmethyl)-4-(1,1-dimethylethoxy)-

4-oxobutanamide (48). Starting with 41, 48 (3.74 g,

76%) was obtained as a white solid after crystallization

N-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-methyl-

hexyl]-4-(1,1-dimethylethoxy)-4-oxo-2(S)-(2-thiazolyl-

methyl)butanamide (52). Starting with 45, 52 (9.20 g,

(EtOAc:hexane): mp 138±139 ^ꢀC; H NMR (DMSO-d₆)

1

502

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

52%) was isolated as a white solid after puri®cation by

¯ash chromatography (hexane:EtOAc, 1.5:1): [a]²_d⁵29.1^ꢀ

(c 1.0 MeOH); IR (®lm) n_max3462, 3263, 1703,

1634 cm ¹; ¹H NMR (CDCl₃) d 7.67 (d, J=3.4 Hz, 1H),

7.24 (d, J=3.4 Hz, 1H), 6.29 (broad s, 1H), 4.23 (ꢁtd,

J=9.2, 5.1 Hz, 1H), 3.42 (dd, J=15.4, 9.5 Hz, 1H),

3.33±3.24 (m, 1H), 3.14 (dd, J=15.4, 2.5 Hz, 1H), 3.09

(d, J=8.3 Hz, 1H), 2.98 (ꢁtd, J=10.2, 2.5 Hz, 1H), 2.70

(dd, J=17.0, 8.6 Hz, 1H), 2.42 (dd, J=17.0, 5.4 Hz,

1H), 1.79±1.47 (m, 8H), 1.39 (s, 9H), 1.39±1.05 (m, 6H),

0.92±0.70 (m, 2H), 0.83 (d, J=6.7 Hz, 3H), 0.69 (d,

J=6.4 Hz, 3H); MS (FAB) m/z 497 (MH)⁺. Anal. calcd

for C₂₆H₄₄N₂O₅S: C, 62.87; H, 8.93; N, 5.64. Found: C,

62.73; H, 9.16; N, 5.60.

aqueous NaHCO₃(100 mL) and brine (100 mL), then

dried (MgSO₄), ®ltered and concentrated under reduced

pressure. The residue was puri®ed by ¯ash chromato-

graphy (EtOAc) to give 3l (1.43 g, 78%) as a white solid:

[a]²_d⁵59.0^ꢀ(c 1.14 MeOH); IR (KBr) n_max3400, 1655,

1

1640 cm ¹; H NMR (CDCl₃) d (1:1 mixt. of rotamers)

7.40±7.25 (m, 5H), 6.90 (d, J=8.3 Hz, 1H), 6.10, 5.23

(2q, J=7.0, 6.7 Hz, 1H), 4.35, 3.81, 3.27 (d, AB_qand d,

J=16.2 Hz, Án=31.2 Hz and J=17.8 Hz, J=16.2 Hz,

2H), 3.41±3.32 (m, 1H), 3.21±3.18 (m, 1H), 2.96, 2.94,

2.92, 2.91 (4s, 6H), 2.96±2.86 (m, 2H), 2.60±2.56 (m,

1H), 1.95±1.10 (m, 16H), 1.62, 1.37 (2d, J=7.0, 6.7 Hz,

3H), 0.98±0.85 (m, 2H), 0.94, 0.93, 0.86 (3d, J=6.7, 6.7,

6.7 Hz, 6H), 0.73±0.70 (m, 1H), 0.49±0.45 (m, 2H),

0.14±0.09 (m, 2H); MS (FAB) m/z 586 (MH)⁺. Anal.

calcd for C₃₄H₅₅N₃O₅+0.62% (w/w) H₂O: C, 68.82; H,

9.59; N, 7.05. Found: C, 69.28; H, 9.49; N, 7.13.

N-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-methyl-

hexyl]-4-(1,1-dimethylethoxy)-4-oxo-2(R)-(4-thiazolyl-

methyl)butanamide (53). Using 46, 53 (365 mg, 72%)

was isolated as a white solid after puri®cation by ¯ash

chromatography (hexane:EtOAc, 1:1): [a]²_d⁵26.9^ꢀ(c 1.02

MeOH); ¹H NMR (DMSO-d₆) d 9.00 (d, J=1.7 Hz,

1H), 7.78 (d, J=8.9 Hz, 1H), 7.37 (d, J=1.7 Hz, 1H),

4.71 (d, J=6.7 Hz, 1H), 4.60 (d, J=4.1 Hz, 1H), 4.08

(broad td, J=8.9, 4.1 Hz, 1H), 3.17±3.10 (m, 1H), 3.03

(dd, J=14.2, 6.5 Hz, 1H), 3.02±2.99 (m, 1H), 2.93±2.89

(m, 1H), 2.79 (dd, J=14.2, 8.0 Hz, 1H), 2.46 (dd,

J=16.3, 9.2 Hz, 1H), 2.14 (dd, J=16.3, 4.9 Hz, 1H),

1.77±1.50 (m, 7H), 1.47±1.35 (m, 2H), 1.25 (s, 9H),

1.20±1.07 (m, 5H), 0.92±0.75 (m, 2H), 0.85 (d, J=

6.7 Hz, 3H), 0.74 (d, J=6.7 Hz, 3H); MS (CI) m/z 497

(MH)⁺, 423 (M-OC(CH₃)₃)⁺. Anal. calcd for C₂₆H₄₄

N₂O₅S: C, 62.87; H, 8.93; N, 5.64. Found: C, 62.61; H,

9.08; N, 5.58.

N¹-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-meth-

ylhexyl]-N⁴-[2-(dimethylamino)-2-oxoethyl]-N⁴-[1(S)-phen-

ylethyl]-2(R)-propylbutanediamide (3p). TFA (3.4 mL)

was added to an ice-cold solution of 49 (750 mg,

1.70 mmol) in CH₂Cl₂(3.4 mL). The solution was stirred

at 0 ^ꢀC for 2 h, at 25 ^ꢀC for 1 h then was concentrated

under reduced pressure. The residue was coevaporated

with Et₂O. The residue was triturated with hexane to

.

give acid 55 (585 mg, 90%) as a white solid. BOP PF₆

(2.34 g, 5.29 mmol) was added to a solution of 55

(1.70 g, 4.41 mmol), 15 (1.09 g, 5.29 mmol) and i-Pr₂NEt

(1.14 g, 8.82 mmol) in DMF (22 mL) at 25 ^ꢀC. After

stirring for 5 h, the mixture was diluted with EtOAc

(300 mL). The solution was washed with 1 N aqueous

HCl, saturated aqueous NaHCO₃, H₂O and brine then

dried (MgSO₄), ®ltered and concentrated under reduced

pressure. The residue was puri®ed by ¯ash chromato-

graphy (hexane:EtOAc:EtOH, 15:10:2) to give 3p

4-[[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-methyl-

hexyl]amino]-3-(R)-(cyclopropylmethyl)-4-oxobutanoic

acid (54). A solution of 48 (2.00 g, 4.41 mmol) and

TFA (7.0 mL) in CH₂Cl₂(10 mL) was stirred at 0 ^ꢀC for

7 h. The mixture was concentrated under reduced pres-

sure. The residue was dissolved in EtOAc (25 mL) and

the resulting suspension was ®ltered to give acid 54

(1.77 g, 70%) as a white solid: IR (KBr) n_max3380,

1650 cm

1

;

¹H NMR (DMSO-d₆) d (1.8:1.0 mixt. of

rotamers) 7.64, 7.55 (2d, J=9.0, 9.0 Hz, 1H), 7.54±7.24

(m, 5H), 5.78, 5.26 (2q, J=6.9, 6.9 Hz, 1H), 4.71, 4.66 (2

broad s, 1H), 4.62, 4.58 (2d, J=6.3, 6.3 Hz, 1H), 4.25,

4.20, 3.82, 3.46 (4d, J=16.5, 18.1, 18.1, 16.5 Hz, 2H),

4.11±4.08 (m, 1H), 3.09 (broad q, J=8.7 Hz, 1H), 2.92±

2.76 (m, 2H), 2.88, 2.85, 2.79, 2.76 (4s, 6H), 2.58, 2.49,

2.36, 2.29 (4dd, J=16.2, 8.1 Hz, J=16.2, 6.0 Hz, J=16.2,

7.0 Hz, J=16.2, 6.6 Hz, 2H), 1.78±1.03 (m, 18H), 1.50,

1.32 (2d, J=6.9, 6.9 Hz, 3H), 0.91±0.74 (m, 11H); MS

(FAB) m/z 574 (MH)⁺. Anal. calcd for C₃₃H₅₅N₃O₅: C,

69.07; H, 9.66; N, 7.32. Found: C, 68.68; H, 10.10; N, 7.12.

(1.22 g, 70%) as a white solid: IR (KBr) n_max3400,

3290, 3140±2500, 1715, 1615, 1580 cm

1

;

¹H NMR

(DMSO-d₆) d 7.65 (d, J=9.2 Hz, 1H), 4.68 (d, J=

6.4 Hz, 1H), 4.61 (d, J=3.2 Hz, 1H), 4.10 (td, J=9.2,

4.7 Hz, 1H), 3.10±3.05 (m, 1H), 2.92 (broad t, J=

7.8 Hz, 1H), 2.83±2.76 (m, 1H), 2.48 (dd, J=16.5,

8.6 Hz, 1H), 2.33 (dd, J=16.5, 5.7 Hz, 1H), 1.79±1.08

(m, 16H), 0.93±0.74 (m, 2H), 0.86 (d, J=6.7 Hz, 3H),

0.74 (d, J=6.4 Hz, 3H), 0.70±0.63 (m, 1H), 0.40±0.35

(m, 2H), 0.08±0.01 (m, 2H); MS (FAB) m/z 398 (MH)⁺.

N¹-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-meth-

ylhexyl]-N⁴-[2-(dimethylamino)-2-oxoethyl]-N⁴-[1(S)-phen-

ylethyl]-2(S)-(2-thienylmethyl)butanediamide (3q). A

Typical procedure for the preparation of inhibitors 3a±3o

(0.1±0.38 mmol scale). N¹-[1(S)-(Cyclohexylmethyl)-2(R),

3(S)-dihydroxy-5-methylhexyl]-2(R)-(cyclopropylmethyl)-

N⁴-[2-(dimethylamino)-2-oxoethyl]-N⁴-[1(S)-phenylethyl]

solution of 50 (490 mg, 0.99 mmol) and TFA (2.0 mL) in

CH₂Cl₂(2.0 mL) was stirred at 0 ^ꢀC for 75 min and at

25 ^ꢀC for 75 min. The mixture was concentrated under

reduced pressure. EtOAc (10 mL) was added and the

acid was obtained by ®ltration. The ®ltrate was con-

centrated and the process repeated using EtOAc (2Â)

and ether (2Â) to give 56 (369 mg, 86%). A solution

of 56 (200 mg, 0.46 mmol), 15 (117 mg, 0.57 mmol),

.

butanediamide (3l). BOP PF₆(1.53 g, 3.45 mmol) was

added to an ice-cold solution of 54 (1.25 g, 3.14 mmol),

15 (811mg, 3.93mmol) and i-Pr₂NEt (691mg, 5.34mmol)

in DMF (15.7 mL). The reaction mixture was stirred at

25 ^ꢀC for 5.5 h. The mixture was diluted with EtOAc

(400mL) and the resulting solution was washed with 10%

aqueous citric acid (100 mL), H₂O (100 mL), saturated

.

i-Pr₂NEt (100 mg, 0.77 mmol) and BOP PF₆(221 mg,

0.50 mmol) in DMF (2.3 mL) was stirred at 25 ^ꢀC for

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

503

5.5 h. The reaction mixture was treated as described in

the previous example. Puri®cation by ¯ash chromato-

graphy (EtOAc) yielded 3q (222 mg, 78%) as a white

(15 mL) was added TFA (7.5 mL). The solution was

allowed to warm to 25 ^ꢀC and was stirred at 25 ^ꢀC for

5 h. The reaction mixture was concentrated under

reduced pressure. The residue was puri®ed by ¯ash

chromatography (CHCl₃:EtOH, 4:1) to aord 58

(940 mg, 46%) as a white solid. A solution of 58

(500 mg, 1.13 mmol), 15 (222 mg, 1.07 mmol), i-Pr₂NEt

solid: [a]²_d⁵57.0^ꢀ(c 0.99 MeOH); IR n_max3400, 3290,

1

1655, 1640 cm

;

¹H NMR (DMSO-d₆) d (1.6:1.0

mixt. of rotamers) 7.77, 7.75 (2d, J=8.6, ꢁ8.6 Hz, 1H),

7.36±7.22 (m, 6H), 6.91, 6.89±6.86, 6.82 (dd, m and d,

J=5.1, 3.2 Hz, J=3.2 Hz, 2H), 5.78, 5.18 (2q, J=7.3,

7.0 Hz, 1H), 4.59 (broad s, 1H), 4.55 (d, J=6.4 Hz, 1H),

4.24, 4.14, 3.80, 3.47 (4d, J=16.0, 18.3, 18.3, 16.0 Hz,

2H), 4.11±4.03 (m, 1H), 3.18±2.62 (m, 6H), 2.88, 2.82,

2.78, 2.74 (4s, 6H), 2.43±2.32 (m, 1H), 1.79±1.01 (m,

14H), 1.47, 1.33 (2d, J=7.0, 7.3 Hz, 3H), 0.92±0.69 (m,

2H), 0.86, 0.85 (2d, J=6.6, 6.7 Hz, 3H), 0.76, 0.75 (2d,

J=6.3, 6.0 Hz, 3H); MS (FAB) m/z 628 (MH)⁺; RP-

HPLC, 100% (system A), 100% (system B).

.

(439 mg, 3.40 mmol) and BOP PF₆(502 mg, 1.13 mmol)

in DMF (6 mL) was stirred at 25 ^ꢀC overnight. The

mixture was diluted with EtOAc and the resulting solu-

tion was washed with H₂O, saturated aqueous NaHCO₃

(2Â) and brine then was dried (MgSO₄), ®ltered and

concentrated under reduced pressure. The residue was

puri®ed by ¯ash chromatography (hexane:EtOAc:

EtOH, 5:5:1) to give 3s (130 mg, 19%) as a white solid:

[a]²_d⁵66.6^ꢀ(c 1.05 MeOH); IR n_max3420, 1650 (broad)

1

cm ¹; H NMR (DMSO-d₆) d (1.6:1.0 mixt. of rota-

N¹-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-meth-

ylhexyl]-N⁴-[2-(dimethylamino)-2-oxoethyl]-2(R)-[(1H-

imidazol - 4-yl)methyl]-N⁴-[1(S) -phenylethyl] butane-

diamide (3r). A solution of 51 (6.08 g, 8.04 mmol) in

EtOH (80 mL) was stirred under an H₂atmosphere

(1 atm) in presence of Pd/C (600 mg) for 2.5 h. The

reaction mixture was ®ltered through a pad of Celite¹

and the ®ltrate was concentrated under reduced pres-

sure. The residue was coevaporated with Et₂O (2Â) to

give the expected acid 57 (5.30 g, 99%) as a white solid.

A solution of 57 (4.30 g, 6.46 mmol), 15 (1.66 g,

mers) 7.81, 7.77 (2d, J=8.9, 8.9 Hz, 1H), 7.66, 7.63 (2d,

J=3.2, 3.4 Hz, 1H), 7.55, 7.53 (2d, J=3.2, 3.4 Hz, 1H),

7.36±7.22 (m, 5H), 5.79, 5.21 (2q, J=7.0, 7.0 Hz, 1H),

4.65±4.42 (m, 2H), 4.25, 4.21, 3.81, 3.49 (4d, J=16.2,

18.0, 18.0, 16.2 Hz, 2H), 4.10±4.05 (m, 1H), 3.4±3.31

(hidden m, 1H), 3.28 (d, J=14.8, 7.8 Hz, 1H), 3.11±2.95

(m, 2H), 2.88±2.57 (m, 2H), 2.88, 2.83, 2.78, 2.75 (4s,

6H), 2.46±2.39 (m, 1H), 1.76±1.00 (m, 14H), 1.48, 1.33

(2d, J=7.0 Hz, 3H), 0.90±0.68 (m, 2H), 0.85 (broad d,

J=6.7 Hz, 3H), 0.75, 0.74 (2d, J=6.4, 6.4 Hz, 3H); MS

(FAB) m/z 629 (MH)⁺; RP-HPLC, 98.8% (system A),

98.9% (system B).

.

8.07 mmol, i-Pr₂NEt (1.42 g, 11.0 mmol) and BOP PF₆

(3.00 g, 6.78 mmol) in DMF (32 mL) was stirred at 25 ^ꢀC

for 5 h. The reaction mixture was treated as described

for 3p. Puri®cation by ¯ash chromatography (EtOAc:

MeOH, 10:1) yielded the corresponding amide (3.60 g,

65%) as a yellowish solid. The trityl protective group

was removed by hydrogenolysis in a Parr shaker (50 psi)

using Pd(OH)₂/C (1.44 g) in EtOH (42 mL) and AcOH

(0.84 mL) for 41 h. The catalyst was removed by ®ltra-

tion through a pad of Celite¹. The ®ltrate was con-

centrated under reduced pressure and the residue was

dissolved in EtOAc (100 mL). The solution was washed

with 5% aqueous Na₂CO₃(25 mL) and brine (25 mL),

then was dried (MgSO₄), ®ltered, and concentrated

under reduced pressure. Puri®cation of the residue by

¯ash chromatography (EtOAc:MeOH, 4:1) ®nally gave

3r (1.24 g, 48%) as a white solid: [a]²_d⁵59.1^ꢀ(c 1.02

N¹-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-meth-

ylhexyl]-N⁴-[2-(dimethylamino)-2-oxoethyl]-N⁴-[1(S)-phen-

ylethyl]-2(R)-(4-thiazolylmethyl)butanediamide (3t).

Using conditions identical to those described above for

3s, 53 (3.70 g, 7.45 mmol) was ®rst converted into acid

59 (100%). The coupling of acid 59 (2.00 g, 4.54 mmol)

and amine 15 (1.21 g, 5.90 mmol) yielded 3t. Puri®cation

of the crude residue by ¯ash chromatography (hexane:

EtOAc:EtOH, 5:5:1) gave 3t (650 mg, 23%) as a white

solid: [a]²_d⁵55.9^ꢀ(c 1.05 MeOH); IR n_max3400, 1650

1

(broad) cm ¹; H NMR (DMSO-d₆) d (1.6:1 mixt. of

rotamers) 9.01, 8.98 (2s, 1H), 7.76, 7.67 (2d, J=8.4,

9.0 Hz, 1H), 7.37±7.23 (m, 6H), 5.78, 5.18 (2q, J=6.7,

7.0 Hz, 1H), 5.48±4.57 (m, 1H), 4.53 (d, J=6.6 Hz, 1H),

4.25, 4.16, 3.81, 3.46 (4d, J=16.3, 18.1, 18.1, 16.3 2H),

4.12±4.02 (m, 1H), 3.08±2.60 (m, 5H), 2.88, 2.84, 2.78,

2.75 (4s, 6H), 2.60±2.31 (m, 2H), 1.76±1.01 (m, 14H),

1.47, 1.33 (2d, J=6.7, 7.0 Hz, 3H), 0.92±0.72 (m, 2H),

0.84, 0.73 (2d, J=6.6, 6.3 Hz, 6H); MS (CI) m/z 629

(MH)⁺. Anal. calcd for C₃₄H₅₂N₄O₅S+1.15% (w/w)

H₂O: C, 64.19; H, 8.38; N, 8.81. Found: C, 64.14; H,

8.33; N, 8.77.

1

MeOH); IR n_max3380, 1650 (broad) cm ¹; H NMR

(DMSO-d₆) d (1.4:1.0 mixt. of rotamers) 7.64, 7.54

(2d, J=9.3, 9.3 Hz, 1H), 7.52, 7.48 (2 broad s, 1H),

7.36±7.23 (m, 5H), 6.82, 6.76 (2 broad s, 1H), 5.76, 5.15

(2q, J=7.2, 6.7 Hz, 1H), 4.77±4.50 (m, 2H), 4.25, 4.09,

3.79, 3.42 (4d, J=16.2, 18.2, 18.2, 16.2 Hz, 2H), 4.11±

4.06 (m, 1H), 3.13±3.01 (m, 2H), 2.92±2.75 (m, 2H),

2.87, 2.82, 2.78, 2.75 (4s, 6H), 2.67±2.44 (m, 2H), 2.35

(broad d, J=6.9 Hz, 1H), 1.78±1.04 (m, 14H), 1.46, 1.32

(2d, J=6.7, 7.2 Hz, 3H), 0.92±0.70 (m, 2H), 0.85 (d,

J=6.6 Hz, 3H), 0.74 (d, J=6.6 Hz, 3H); MS (FAB) m/z

612 (MH)⁺; RP-HPLC, >99% (system A), 99.1%

(system B).

3-(3-Carboxy-1-oxopropyl)-4(S)-(1-methylethyl)-2-oxazo-

lidinone (60). A solution of succinic anhydride (22.1 g,

220 mmol) in THF (400 mL) was added (30 min) to a

cold ( 78 ^ꢀC) solution of the lithium salt of 4(S)-(1-

methylethyl)-2-oxazolidinone (232mmol) [prepared using

1.6 M n-BuLi/hexane (148 mL, 237 mmol)] in THF

(460 mL). The reaction mixture was allowed to warm to

25 ^ꢀC and was stirred at this temperature for 5 h. Ether

(300 mL) and 1 N HCl (150 mL) were added and the

phases were separated. The organic layer was extracted

N¹-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-meth-

ylhexyl]-N⁴-[2-(dimethylamino)-2-oxoethyl]-N⁴-[1(S)-phen-

ylethyl]-2(S)-(2-thiazolylmethyl)butanediamide (3s). To

an ice-cold solution of 52 (2.30 g, 4.63 mmol) in CH₂Cl₂

504

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

with saturated aqueous NaHCO₃(2Â200 mL). The

combined aqueous layers were rendered acidic (pH 3)

by addition of solid citric acid and were extracted with

ether (3Â). The combined organic layers were washed

with H₂O and brine then were dried (MgSO₄), ®ltered

and concentrated under reduced pressure to give 60

(35.6 g, 67%) as a pale yellow oil: [a]²_d⁵+89.7^ꢀ(c 1.01

MeOH); IR (®lm) n_max3540, 3180 (broad), 1785, 1745,

1710 cm ¹; ¹H NMR (CDCl₃) d 4.44 (ꢁddd, J=8.3, 4.0,

3.2 Hz, 1H), 4.31 (ꢁdd, J=9.2, 8.3 Hz, 1H), 4.23 (dd,

J=9.2, 3.2 Hz, 1H), 3.29 (dt, J=18.6, 6.4 Hz, 1H), 3.21

(dt, J=18.6, 6.4 Hz, 1H), 2.73 (t, J=6.4 Hz, 2H), 2.38

(sept d, J=7.0, 4.0 Hz, 1H), 0.92 (d, J=7.0 Hz, 3H),

0.89 (d, J=7.0 Hz, 3H); MS (CI) m/z 230 (MH)⁺.

re¯ux for 30 min. The resulting suspension was cooled

to room temperature and poured into Et₂O (75 mL).

The salt obtained by ®ltration was suspended in 5%

aqueous Na₂CO₃(100 mL) and extracted with EtOAc

(2Â150 mL). The combined organic layers were washed

with brine (75 mL), dried (MgSO₄), ®ltered and con-

centrated under reduced pressure to give the 2-amino-4-

thiazolyl derivative (4.23 g). A solution of this derivative

(4.23 g, ꢁ14.9mmol), ClCO₂CH₂CCl₃(4.74 g, 22.4 mmol),

i-Pr₂NEt (2.12 g, 16.4 mmol) and DMAP (182 mg,

1.49 mmol) in CH₂Cl₂(50 mL) was stirred at 0 ^ꢀC for

45 min then at 25 ^ꢀC for 16 h. The mixture was diluted

with CH₂Cl₂(150 mL) and the resulting solution was

successively washed with saturated aqueous NaHCO₃

(100 mL), H₂O (100 mL) and brine (100 mL), then dried

(MgSO₄), ®ltered and concentrated under reduced pres-

sure. Puri®cation by ¯ash chromatography (hexane:

EtOAc, 2:1) yielded 63 (4.90 g, 61%) as a white foam:

[a]²_d⁵+46.0^ꢀ(c 0.97 MeOH); IR (KBr) n_max1785, 1740,

1705 cm ¹; ¹H NMR (CDCl₃) d 6.65 (s, 1H), 4.91 (Abq,

dn=6.4 Hz, J=11.9 Hz, 2H), 4.45±4.41 (m, 1H), 4.28

(ꢁdd, J=9.0, 8.6 Hz, 1H), 4.21 (dd, J=9.0, 3.0 Hz, 1H),

3.35±3.30 (m, 2H), 3.09 (t, J=7.5 Hz, 2H), 2.36 (sept d,

J=7.0, 4.0 Hz, 1H), 0.91 (d, J=7.0 Hz, 3H), 0.86 (d,

J=7.0 Hz, 3H); MS (FAB) m/z 458/460/462/464 (MH)⁺.

Anal. calcd for C₁₅H₁₈N₃O₅SCl₃: C, 39.27; H, 9.16; N,

3.95. Found: C, 38.77; H, 9.11; N, 3.83.

3-(5-Bromo-1,4-dioxopentyl)-4(S)-(1-methylethyl)-2-ox-

azolidinone (61). (COCl)₂(3.11 g, 24.5 mmol) was added

to an ice-cold solution of 60 (4.68 g, 20.4 mmol) in

CH₂Cl₂(14 mL). The reaction mixture was stirred at 0 ^ꢀC

for 10 min, at 25 ^ꢀC for 1.5 h then was heated to re¯ux

for 30 min. The crude acyl chloride obtained by con-

centration was diluted with Et₂O (15 mL) and the solu-

tion was added (5 min) to an ice-cold solution of CH₂N₂

in Et₂O (ꢁ0.6 M, 100 mL). The reaction mixture was

stirred at 0 ^ꢀC for 45 min then a solution of HBr in

AcOH (45% w/v, 10 mL) was added (3 min). The mix-

ture was stirred at 0 ^ꢀC for 1 h then was poured into a

saturated solution of NaHCO₃(350 mL) containing also

solid NaHCO₃(42 g) and Et₂O (50 mL). The phases

were separated. The aqueous layer was extracted with

Et₂O (100 mL). The combined organic layers were dried

(MgSO₄), ®ltered and concentrated under reduced pres-

sure to give 61 (5.60 g, 89%) as a brown oil which soli-

3-[4-(1,1-Dimethylethoxy)-1,4-dioxo-2(R)-[[2-[[(2,2,2-tri-

chloroethoxy)carbonyl]amino]-4-thiazolyl]methyl]butyl]-

4(S)-(1-methylethyl)-2-oxazolidinone (64). Starting with

63 and using the general procedure described for 42±46,

64 (2.95 g, 59%) was obtained, after puri®cation by

¯ash chromatography (hexane:EtOAc, 3:1) and recrys-

tallization, as white ¯akes: mp 98±101 ^ꢀC; [a]_d²⁵+55.2^ꢀ

(c 0.99 MeOH); IR (KBr) n_max1775, 1745, 1730,

1

di®ed slowly: H NMR (CDCl₃) d 4.40±4.37 (m, 1H),

4.29 (ꢁdd, J=9.1, 8.3 Hz, 1H), 4.21 (dd, J=9.1, 3.0 Hz,

1H), 3.99 (s, 2H), 3.28±3.24 (m, 2H), 2.99±2.94 (m, 2H),

2.33 (sept d, J=6.8, 3.8 Hz, 1H), 0.90 (d, J=6.8 Hz,

3H), 0.88 (d, J=6.8 Hz, 3H). The compound was used

directly in the subsequent reaction.

1

1700 cm ¹; H NMR (CDCl₃) d 6.71 (s, 1H), 4.95 (d,

J=12.1 Hz, 1H), 4.87 (d, J=12.1 Hz, 1H), 4.53±4.48

(1H, m), 4.42 (ꢁddd, J=7.0, 4.1, 3.8 Hz, 1H), 4.23±4.17

(m, 2H), 3.04 (dd, J=14.5, 6.0 Hz, 1H), 2.94 (dd,

J=14.5, 7.3 Hz, 1H), 2.78 (dd, J=16.6, 9.7 Hz, 1H),

2.45 (dd, J=16.6, 4.8 Hz, 1H), 2.34 (sept d, J=7.0,

3.8 Hz, 1H), 1.41 (s, 9H), 1.33±1.25 (m, 3.5H), 0.92±0.87

(m, 9.5H); MS (FAB) m/z 572/574/576/578 (MH)⁺.

Anal. calcd for C₂₁H₂₈N₃O₇SCl₃.0.5 C₆H₁₄: C, 46.80;

H, 5.73; N, 6.82. Found: C, 46.27; H, 5.63; N, 6.78.

3-[1-Oxo-3-(4-thiazolyl)propyl]-4(S)-(1-methylethyl)-2-

oxazolidinone (62). A solution of 61 (12.5 g, 40.8 mmol)

and freshly prepared thioformamide (5.00 g, 81.7 mmol)

in THF (120 mL) was stirred at 25 ^ꢀC overnight. The

reaction mixture was diluted with EtOAc and the

resulting solution was washed with saturated aqueous

NaHCO₃and brine then was dried (MgSO₄), ®ltered

and concentrated under reduced pressure. The residue

was puri®ed by distillation (bulb to bulb, ꢁ250 ^ꢀC)

under reduced pressure (ꢁ1 mm Hg) to give 62 (6.00 g,

55%) as a pale yellow oil: [a]²_d⁵+77.1^ꢀ(c 1.57 MeOH);

3-[4-[[2-(Dimethylamino)-2-oxoethyl][1(S)-phenylethyl]-

amino]-1,4-dioxo-2(R)-[[2-[[(2,2,2-trichloroethoxy)carbon-

yl]amino]-4-thiazolyl]methyl]butyl]-4(S)-(1-methylethyl)-

2-oxazolidinone (65).

A solution of 64 (894 mg,

1

IR (®lm) n_max1785, 1705 cm ¹; H NMR (CDCl₃) d

1.45 mmol) and TFA (2.9 mL) in CH₂Cl₂(2.9 mL) was

stirred at 0 ^ꢀC for 30 min and at 25 ^ꢀC for 3 h. The mix-

ture was concentrated under reduced pressure. The

residue was dissolved in C₆H₆(3Â25 mL) and the solu-

tion was concentrated under reduced pressure to give

the corresponding acid. To a solution of the crude

acid, 15 (359 mg, 1.74 mmol) and i-Pr₂NEt (319 mg,

8.77 (d, J=1.9 Hz, 1H), 7.06 (dt, J=1.9, 0.9 Hz, 1H),

4.44 (ꢁddd, J=8.3, 3.8, 3.2 Hz, 1H), 4.28 (ꢁdd, J=9.1,

8.3 Hz, 1H), 4.21 (dd, J=9.1, 3.2 Hz, 1H), 3.49±3.33 (m,

2H), 3.23 (t, J=7.5 Hz, 2H), 2.37 (sept d, J=7.0, 3.8 Hz,

1H), 0.91 (d, J=7.0 Hz, 3H), 0.87 (d, J=7.0 Hz, 3H);

MS (CI) m/z 269 (MH)⁺.

.

2.47 mmol) in DMF (5.8 mL) was added BOP PF₆

4(S)-(1-Methylethyl)-3-[1-oxo-3-[2-[[(2,2,2-trichloroethoxy)-

carbonyl]amino]-4-thiazolyl]propyl]-2-oxazolidinone (63).

A solution of 61 (5.38 g, 17.6 mmol) and thiourea

(1.34 g, 17.6 mmol) in i-PrOH (35 mL) was heated to

(705 mg, 1.60 mmol) at 25 ^ꢀC. The solution was stirred

at 25 ^ꢀC for 25 h then was diluted with EtOAc (150 mL).

The solution was successively washed with aqueous 10%

citric acid (30 mL), H₂O (30 mL), saturated aqueous

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

505

NaHCO₃(30 mL), brine (30 mL) and then dried (MgSO₄),

®ltered and concentrated under reduced pressure. The

residue was puri®ed by ¯ash chromatography (EtOAc:

hexane, 3:1) to yield 65 (751 mg, 73%) as a white solid:

[a]²_d⁵+13.7^ꢀ(c 0.92 MeOH); IR (KBr) n_max1780, 1740,

successive puri®cations by ¯ash chromatography ((a)

hexane:i-PrOH, 5:1; (b) EtOAc) gave 71 (252 mg, 32%)

as a white solid: [a]_d²⁵47.3^ꢀ(c 0.89 MeOH); IR (KBr)

1

n_max3400 (broad), 1745, 1655, 1640 cm

;

¹H NMR

(CDCl₃) d (1.2:1.0 mixt. of rotamers) 7.36±7.20 (m,

5H), 7.11, 6.88 (2d, J=8.6, 8.9 Hz, 1H), 6.79 (s, 1H),

6.03, 5.16 (2q, J=7.0, 7.0 Hz, 1H), 4.89±4.82 (m, 2H),

4.40, 3.97, 3.77, 3.24 (4d, J=16.2, 17.6, 17.6, 16.2 Hz,

2H), 4.36±4.29, 4.28±4.21 (2m, 1H), 3.52±3.35, 3.30±

3.16 (2m, ꢁ4H), 2.97±2.66 (m, 4H), 2.94, 2.91, 2.86,

2.84 (4s, 6H), 2.55 (dd, J=16.5, 6.7 Hz, 1H), 1.93±1.86

(m, 1H), 1.75±1.07 (m, 13H), 1.56, 1.38, (2d, J=7.0,

7.0 Hz, 3H), 0.96±0.83 (m, 2H), 0.92, 0.87, 0.83 (3d,

J=6.4, 6.7, 6.7 Hz, 6H); MS (FAB) m/z 818/820/822/

824 (MH)⁺.

1

1700, 1650 cm ¹; H NMR (CDCl₃) d (1.7:1.0 mixt. of

rotamers) 7.39±7.22 (m, 5H), 6.88, 6.79 (2s, 1H), 5.89,

5.22 (2q, J=7.0, 6.7 Hz, 1H), 4.89±4.85 (m, 2H), 4.67±

4.35 (m, 2H), 4.37, 3.81, 3.28 (d, AB_qand d, J=16.2 Hz;

Án=20.3 Hz, J=17.6 Hz; J=16.2 Hz, 2H), 4.31±4.24

(m, 1H), 4.21±4.16 (m, 1H), 3.16, 3.13 (2dd, J=14.5,

5.1, J=14.5, 5.1 Hz, 1H), 3.06 (dd, J=16.4, 8.1 Hz,

ꢁ1H), 2.94±2.85 (m, 1H), 2.90, 2.78, 2.71 (3s, 6H), 2.78±

2.69 (m, 1H), 2.43±2.32 (m, 1H), 1.60, 1.41 (2d, J=6.7,

7.0 Hz, 3H), 0.95±0.87 (m, 6H); MS (FAB) m/z 704/706/

708/710 (MH)⁺.

N⁴-(Cyclohexylmethyl)-N¹-[1(S)-(cyclohexylmethyl)-2(R),

3(S)-dihydroxy-5-methylhexyl]-N⁴-[2-[methyl[2-(2-pyridi-

nyl)ethyl]amino]-2-oxoethyl]-2(R)-[[2-[[(2,2,2-trichloro-

ethoxy)carbonyl]amino]-4-thiazolyl]methyl]butanediamide

(76). A solution of 0.8 M Mg(OMe)₂in MeOH (122 mL,

97.4 mmol) was added (15 min) to an ice-cold solution

of 70 (38.4 g, 48.7 mmol) in MeOH (500 mL). The reac-

tion mixture was stirred at 0 ^ꢀC for 1 h. Saturated aqu-

eous NH₄Cl (500 mL) was then added and the resulting

mixture was poured into H₂O (1 L). The solution was

extracted with EtOAc (3Â350 mL). The combined

organic layers were washed with H₂O (2Â500 mL),

saturated aqueous NaHCO₃(300 mL) and brine

(300 mL), dried (MgSO₄), ®ltered and concentrated

under reduced pressure. The residue was puri®ed by

¯ash chromatography (EtOAc:MeOH, 20:1) to give the

corresponding methyl ester (30.3 g, 90%) as a white

solid. A solution of this ester (28.7 g, 41.6 mmol) in

THF:MeOH:H₂O (415 mL:40 mL:120 mL) was treated

with aqueous 2 N NaOH (52 mL, 104 mmol) at 25 ^ꢀC

for 14 h. The reaction mixture was concentrated under

reduced pressure and 1 N HCl (100 mL) and EtOAc

(300 mL) were added. The phases were separated, and

the aqueous layer was extracted with EtOAc (200 mL).

The combined organic layers were washed with brine

(100 mL), dried (MgSO₄), ®ltered and concentrated

under reduced pressure to give the corresponding acid

(27.9, 99%). A solution of this acid (25.7 g, 38.0 mmol),

47 (9.24 g, 38.0 mmol), i-Pr₂NEt (9.82 g, 76.0 mmol) and

3-[4-[(Cyclohexylmethyl)[2-[methyl[2-(2-pyridinyl)ethyl]-

amino]-2-oxoethyl]amino]-1,4-dioxo-2(R)-[[2-[[(2,2,2-tri-

chloroethoxy)carbonyl]amino]-4-thiazolyl]methyl]butyl] -

4(S)-(1-methylethyl)-2-oxazolidinone (70). Using a pro-

cedure similar to the one described for 65, 64 (14.0 g,

24.4 mmol) was ®rst transformed into the corresponding

acid. The coupling of the crude acid with amine 27

(28.1 mmol) followed by the puri®cation by ¯ash chro-

matography (EtOAc:MeOH, 40:1) of the residue gave

70 (16.96 g, 88%) as a white solid: [a]²_d³+40.9^ꢀ(c 0.574

1

MeOH); IR (KBr) n_max1769, 1650 cm

;

¹H NMR

(CDCl₃) d (2:2:1:1 mixt. of rotamers) 8.67, 8.59, 8.56,

8.51 (4d, J=4.4, 4.4, 4.8, 5.1 Hz, 1H), 7.72±7.61 (m,

1H), 7.34±7.16 (m, 2H), 6.71, 6.68, 6.65 (3s, 1H), 4.89±

4.80 (m, 2H), 4.49±4.57, 4.42±4.36 (2m, 2H), 4.26±4.18

(m, 3H), 3.94±3.57 (m, 3H), 3.33, 3.19±2.77, 2.71±2.60,

2.54±2.45 (dd, 3m, J=13.8, 8.1 Hz, 8H), 2.93, 2.86 (2s,

3H), 2.40±2.32 (m, 1H), 1.77±1.42 (m, 6H), 1.26±1.13

(m, 3H), 0.91±0.82 (m, 8H); MS (ESI) m/z 787/789/801/

803 (MH)⁺.

N¹-[1(S)-(Cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-meth-

ylhexyl]-N⁴-[2-(dimethylamino)-2-oxoethyl]-N⁴-[1(S)-phen-

ylethyl]-2(R)-[[2-[[(2,2,2-trichloroethoxy)carbonyl]amino]-

4-thiazolyl]methyl]butanediamide (71). To an ice-cold

solution of 65 (679 mg, 0.96 mmol) in THF:H₂O

(3:1, 19mL), was added 30% (w/%) H₂O₂(0.55 mL,

.

4.81mmol) followed by LiOH H₂O (80.8 mg, 1.92 mmol).

The reaction mixture was stirred at 0 ^ꢀC for 1.5 h and at

25 ^ꢀC for 3.5 h. The mixture was cooled to 0 ^ꢀC, diluted

with H₂O (20 mL) and aqueous 1.5 M Na₂SO₃was

added to decompose the excess of peroxide. The result-

ing mixture was diluted with H₂O (50 mL) and rendered

acidic (pH 3) with aqueous citric acid. The mixture was

extracted with EtOAc (3Â100 mL). The combined

organic layers were washed with brine, dried (MgSO₄),

®ltered and concentrated under reduced pressure to give

a mixture of chiral auxiliary and the corresponding acid.

To a solution of the crude acid, 47 (234 mg, 0.96 mmol)

and i-Pr₂NEt (249 mg, 1.93 mmol) in DMF (4.8 mL)

BOP PF₆(17.6 g, 38.0 mmol) in DMF (152 mL) was

.

stirred at 25 ^ꢀC for 3.5 h. The reaction mixture was

poured into H₂O (1 L) and saturated aqueous NaHCO₃

(100 mL). The resulting solution was extracted with

EtOAc (2Â300 mL). The combined organic layers were

washed with saturated aqueous NaHCO₃(200 mL), a

mixture of H₂O and brine (3:1, 2Â300 mL), brine

(250 mL), and then dried (MgSO₄), ®ltered and con-

centrated under reduced pressure. Puri®cation of the

residue by ¯ash chromatography (EtOAc:MeOH, 50:1)

gave a mixture of 76 and its epimer (92:8 mixture,

4.1 g 12%) and pure diastereomer 76 (18.4 g, 54%) as a

white solid: [a]²_d³16.8^ꢀ(c 0.655 MeOH); IR (KBr)

.

was added BOP PF₆(447 mg, 1.01 mmol). The solution

was stirred at 25 ^ꢀC for 3.5 h. The reaction mixture was

diluted with EtOAc (125 mL) and the resulting solution

was washed with saturated aqueous NaHCO₃(30 mL),

H₂O (2Â30mL), and brine (30 mL), then dried (MgSO₄),

®ltered, and concentrated under reduced pressure. Two

n_max3400±3150, 1738, 1645 cm ¹; H NMR (CDCl₃) d

1

(3:3:1:1 mixt. of rotamers) 10.31 (broad s, 1H), 8.64,

8.54, 8.51 (3d, J=4.8, 4.1, 4.8 Hz, 1H), 7.67±7.58 (m,

1H), 7.23±7.11 (m, 2H), 6.76, 6.73, 6.71±6.69 (2d and m,

J=5.7, 5.7 Hz, 1H), 6.69, 6.61, 6.59 (3s, 1H), 4.90±4.79

506

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

(m, 2H), 4.50±4.36 , 4.26±4.13 (2m, 3H), 4.07, 3.94, 3.87,

3.80±3.61 (3d and m, J=17.5, 17.5, 17.8 Hz, 3H), 3.44±

2.82 (m, 9H), 2.92, 2.92 (2s, 3H), 2.67, 2.62±2.48, 2.37,

2.20, dd, m and 3 dd, J=16.2, 7.3 Hz, J=16.2, 5.7 Hz,

J=16.8, 5.7 Hz, 2H), 1.90±1.82 (m, 1H), 1.75±1.05 (m,

23H), 0.97±0.72 (m, 4H), 0.91, 0.84, 0.81 (3d, J=6.7,

6.7, 6.4 Hz, 6H); MS (ESI) m/z 901/903/905/907 (MH)⁺.

2H), 6.77, 6.73 (2s, 2H), 6.18, 6.17, 6.10, 6.06 (4s, 1H),

4.64±4.61, 4.59, 4.56 (m and 2d, J=6.6, 6.6 Hz, 2H),

4.18, 4.17, 4.11, 4.09±4.01, 3.88, 3.85 (3d, m and 2d,

J=17.8, 16.2, 16.5, 17.8, 16.2 Hz, 3H), 3.69±3.51 (m,

2H), 3.10±2.80 (m, 7H), 2.90, 2.89, 2.83, 2.80 (4s, 3H),

2.74±2.62 (m, 1H), 2.51±2.32, 2.24, 2.14, 2.02 (m and

3dd, J=15.8, 7.8 Hz, J=16.1, 5.7 Hz, J=15.8, 5.7 Hz,

3H), 1.78±1.05 (m, 23H), 0.90±0.69 (m, 4H), 0.86 (d,

J=6.9, 3H), 0.77 (d, J=6.6 Hz, 3H); MS (FAB) m/z 727

(MH)⁺. Anal. calcd for C₃₉H₆₂N₆O₅S: C, 64.42; H,

8.60; N, 11.56. Found: C, 64.15; H, 8.77; N, 11.49.

2(R)-[(2-Amino-4-thiazolyl)methyl]-N¹-[1(S)-(cyclohexyl-

methyl)-2(R),3(S)-dihydroxy-5-methylhexyl]-N⁴-[2-(di-

methylamino)-2-oxoethyl]-N⁴-[1(S)-phenylethyl]butane-

diamide (3u). To a solution of 71 (1.91 g, 2.34 mmol) in

aqueous 1 N HCl (5.8 mL) and 1,4-dioxane (11.7 mL)

was added Zn powder (19.0 g, 0.29 mol). The reaction

mixture was stirred at 25 ^ꢀC for 3 h. Saturated aqueous

NaHCO₃was added, the suspension was ®ltered and the

aqueous phase extracted with EtOAc. The organic layer

was washed with saturated aqueous NaHCO₃(2Â),

H₂O, brine (2Â), and then was dried (MgSO₄), ®ltered

and concentrated under reduced pressure. The residue

was partially puri®ed by ¯ash chromatography (EtOAc:

hexane:EtOH, 6:3:1) to give impure 3u which was com-

bined with another batch of the same material obtained

from a similar reaction (starting with 4.14 g, 5.07 mmol

of 71). A second puri®cation by ¯ash chromatography

(CHCl₃:MeOH:AcOH, 23:2:1) gave 3u (2.40 g, 50%) as

2(R)-[(2-Amino-4-thiazolyl)methyl]-N¹-[1(S)-(cyclohexyl-

methyl)-2(S)-hydroxy-2-(1,5,5-trimethyl-2-oxopyrrolidin-

3(S)-yl)ethyl]-N⁴-[2-(dimethylamino)-2-oxoethyl]-N⁴-[1(S)-

phenylethyl]butanediamide (77). Compound 77 was pre-

pared using a method similar to the one described to

prepare 3u from 71. The coupling of the acid (0.61 g,

1.05 mmol) obtained from 65 and the amine obtained

via treatment of 1,1-dimethylethyl N-[1(S)-(cyclohexyl-

methyl)-2(S)-hydroxy-2-(1,5,5-trimethyl-2-oxopyrrolidin-

3(S)-yl)ethyl]carbamate (0.40 g, 1.05 mmol) with TFA

gave the corresponding amide (0.42 g, 47%) after pur-

i®cation by ¯ash chromatography (CHCl₃:MeOH, 97:3

to 93:7). Upon treatment of this compound (0.53 g,

0.62 mmol) with Zn (0.40 g, 6.17 mmol) in 1,4-dioxane

(10 mL) and 1 N HCl (6 mL) for 3 h as described for 3u,

77 (0.20 g, 55%) was obtained as a white solid after

puri®cation by ¯ash chromatography (CHCl₃:MeOH:

a white solid: [a]_d²⁵58.8 (c 1.0 MeOH); IR (KBr) n_max

1

3560±3100, 1635 (broad) cm

;

¹H NMR (CDCl₃)

d (1.5:1.0 mixt. of rotamers) 7.38±7.22 (m, 5H), 6.77,

6.64 (2d, J=8.3, 10.2 Hz, 1H), 6.27, 6.23 (2s, 1H), 6.05,

5.18 (2q, J=7.0, 6.7 Hz, 1H), 5.39±5.27 (m, 2H), 4.62±

4.50 (m, 1H), 4.28±4.21 (m, 1H), 4.38, 3.79, 3.22 (d,

ABq, d, J=16.4 Hz, Án=16.7 Hz and J=17.5 Hz, J=

16.4 Hz, 2H), 3.64±3.12 (m, 3H), 2.94, 2.91, 2.88, 2.82

(4s, 6H), 3.00, 2.95±2.50 (dd and m, J=14.6, 7.0 Hz,

5H), 1.97±1.85 (m, 1H), 1.80±1.48 (m, 7H), 1.59, 1.38

(2d, J=6.7, 7.0 Hz, 3H), 1.43±1.08 (m, 6H), 0.98±0.80

(m, 2H), 0.94, 0.90, 0.86 (3d, J=6.7, 6.7, 6.7 Hz, 6H);

MS (FAB) m/z 644 (MH)⁺. Anal. calcd for C₃₄H₅₃

N₅O₅S+2.58% (w/w) H₂O: C, 61.79; H, 8.39; N, 10.60.

Found: C, 61.47; H, 8.31; N, 10.57.

1

AcOH, 10:2:1): H NMR (CDCl₃) d 7.40±7.29 (m, 5H),

6.17 (s, 1H), 6.03, 5.35 (2m, 1H), 5.64, 5.67 (2 broad s,

2H), 5.45 (m, 1H), 4.33, 3.79 (2d, J=15.9, 15.9 Hz, 1H),

4.17±4.02 (m, 2H), 3.94±3.76 (m, 3H), 3.22±3.12 (m,

2H), 2.93±2.90 (2s, 6H), 2.87±2.76 (m, 2H), 2.74 (s, 3H),

2.70±2.61 (m, 3H), 2.28±2.18 (m, 1H), 2.10±1.70 (m, 6H),

1.64, 1.43 (2d, J=6.7, 7.0 Hz, 3H), 1.32, 1.19 (2s, 6H),

1.35±1.05 (m, 4H), 1.00±0.82 (m, 1H), 0.82±0.68 (m,

1H); MS (FAB) m/z 683 (MH)⁺; RP-HPLC, 95% (sys-

tem A), 91.9% (system B).

Human plasma renin assay pH 6.0 and 7.4⁴¹

2(R)-[(2-Amino-4-thiazolyl)methyl]-N⁴-(cyclohexylmethyl)-

N¹-[1(S)-(cyclohexylmethyl)-2(R),3(S)-dihydroxy-5-methyl-

hexyl]-N⁴-[2-[methyl[2-(2-pyridinyl)ethyl]amino]-2-oxo-

ethyl]butanediamide (3z). Zinc powder (36.0 g, 0.55 mol)

was added in one portion to a cold solution (10 ^ꢀC) of

76 (16.58 g, 18.37 mmol) in 1,4-dioxane (185 mL) and 1

N HCl (184 mL, 184 mmol). The mixture was stirred at

10 ^ꢀC for 45 min. The mixture was cooled to 0 ^ꢀC, satu-

rated aqueous NaHCO₃(350 mL) and EtOAc (1.3 L)

were added and the heterogeneous mixture was stirred

at 25 ^ꢀC for 15 min. The salts were removed by ®ltration

through a pad of Celite¹. The phases were separated

and the organic layer was washed with brine (200 mL),

dried (MgSO₄), ®ltered and concentrated under reduced

pressure. The residue was puri®ed by ¯ash chromato-

graphy (EtOAc:MeOH, 10:1) to give 3z (9.38 g, 70%) as

a white solid: mp 93±96 ^ꢀC; [a]²_d⁵24.8^ꢀ(c 1.0 MeOH);

IR (KBr) n_max3350 (broad), 1645 (broad) cm ¹; ¹H NMR

(DMSO-d₆) d (ꢁ1:1:1:1 mixture of rotamers) 8.54±8.51,

8.48 (m and d, J=4.8 Hz, 1H), 7.75±7.67 (m, 1H), 7.59,

7.56, 7.53 (3d, J=9.0, 9.9, 9.6 Hz, 1H), 7.33±7.19 (m,

Human plasma (Biological Specialty Corporation,

Lansdale, PA) was used as the source of both the

enzyme, renin, and the substrate, angiotensinogen.

Angiotensin I was quanti®ed using a commercially

available radioimmunoassay kit from NEN-Dupont

(Cat. No. NEA-105). The assay (pH 6.0) was performed

in 0.27 M MES (4-morpholineethanesulfonic acid), 1%

HSA (human serum albumin), pH 5.85 containing 13

(mL/mL dimercaprol solution, 13 mL/mL 8-hydroxy-

quinoline sulfate solution and 1% DMSO. The dimer-

caprol and 8-hydroxyquinoline sulfate solutions were

added just before use and were provided with the

radioimmunoassay kit. For the pH 7.4 assay, the assay

buer was 0.15 M HEPES, 6 mM EDTA, 1% HSA, pH

7.4 containing 30 mL/mL 8-hydroxyquinoline sulfate

solution and 1% DMSO. Each assay was carried out in

a ®nal volume of 100 mL in 1.0 mL polypropylene mini-

tubes. To 50 mL of serial dilution of the test compounds

in assay buer or 50 mL of assay buer only (37 ^ꢀC and

4 ^ꢀC controls) was added 50 mL of human plasma to

initiate the reaction. After addition of the human

B. Simoneau et al. / Bioorg. Med. Chem. 7 (1999) 489±508

507

plasma, the assay mixture was incubated at 37 ^ꢀC for 60

to 90 min (pH 6.0 assay) or 120 to 150 min (pH 7.4

assay) in order to achieve an angiotensin I generation of

2±3 ng/mL. In 37 ^ꢀC controls, no inhibitor was added in

order to determine the maximum angiotensin I level

generated with the human plasma pool used. In 4 ^ꢀC

controls, no angiotensin I was generated, tubes being

kept on ice-cold water during the incubation. These

served to determine the background level of angiotensin

I. When the incubation was completed, tubes were

quickly returned to ice-cold water. The generation of

angiotensin I was then quanti®ed using the radio-

immunoassay kit from NEN-Dupont according to the

manufacturer instructions except that 2% PEG 8000

was added to the angiotensin I second antibody solu-

tion. IC₅₀values were generated from the inhibition

curves using the SAS statistical software system (SAS

Institute Inc., Cary, NC) and a non-linear curve ®t

using the Hill model.

samples were collected for the evaluation of plasma

angiotensin II (AII, measured by HPLC followed by

RIA).

Pharmacokinetic studies

Animal dosing and blood sampling were performed at

TSI Mason Laboratories in Worcester, Massachusetts.

Three male cynomolgus monkeys were fasted for 12 to

16 h prior to oral and intravenous administration of the

compound. On the ®rst day of the experiment, animals

received the renin inhibitor (1.0 mg/kg) via intravenous

injection. On the 15th day, the monkeys received the

inhibitors (10 mg/kg) by gavage. Blood samples were

obtained by peripheral venipuncture before the experi-

ment and at 5 (iv treatment only), 15, 30, 45 min and 1,

1.5, 2, 3, 4, 6, 8, and 24 h after dosing. Plasma was

recovered, frozen and stored at 20 ^ꢀC until analysis.

Plasma samples (0.5 mL) were alkalinized with 1.5 N

NaOH (0.05 mL) and were shaken for 20 min with Et₂O

(5 mL). The ether extracts were transferred to glass

tubes (13Â100 mm) and evaporated to dryness under

N₂at 40 ^ꢀC. The residues were each dissolved in 50 mM

KH₂PO₄: MeCN (40:60; 100 mL) and aliquots (75 mL)

were analyzed by HPLC.

Cathepsin D enzymatic assay

The enzyme activity was determined using the aspartyl

protease speci®c chromophoric substrate H-Pro-Thr-

Glu-Phe-Phe(NO₂)-Arg-Leu-OH.⁴²Cathepsin D pur-

i®ed from human liver (Medor, Munchen, Germany)

was dissolved at a concentration of 25 mg/mL in

100 mM HEPES, 300 mM KCl, 1 mM EDTA (pH

adjusted to 7.5 with NaOH) and stored at 20 ^ꢀC. The

enzyme assay was conducted in a total of 520 mL. The

assay volume consisted of 500 mL of 0.28 mM chromo-

phoric substrate dissolved in 100 mM sodium formate

buer pH 3.0 (pH adjusted with NaOH), 10 mL of pur-

i®ed cathepsin D and 10 mL of DMSO in presence or

absence of compound. The assay was initiated by addi-

tion of enzyme and incubation at 37 ^ꢀC was carried out

for 30 min. The degradation of the substrate was quan-

ti®ed by measuring the decrease in absorbance of the

reaction mixture at 310 nm (Hewlett Packard diode

array spectrophotometer, model HP 8452A). IC₅₀

values were generated from the inhibition curve using

the BMDP Statistical Software package for nonlinear

regression (BMDP Statistical Software, Los Angeles,

CA). For reference and comparison, pepstatin and

A-72517 exhibited IC₅₀values of 3 and 575 nM, respec-

tively, in this assay.

Acknowledgements

The authors would like to thank Karl Grozinger and

Larry Nummy of the process chemistry research

laboratory for supplying a valuable intermediate; Bill

Adams, Jennifer Ahlberg, and Christopher Sorge for the

determination of oral bioavailabilities; and Colette

Boucher, Sylvain Bordeleau, and Serge Valois of the

analytical chemistry group for providing analytical

support.

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Article Doi

Discovery of non-peptidic P2-P3 butanediamide renin inhibitors with high oral efficacy

DOI: 10.1016/S0968-0896(98)00265-X

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