Inhibitors of the hepatitis C virus NS3 protease with basic amine functionality at the P3-amino acid N-terminus: Discovery and optimization of a new series of P2-P4 macrocycles

4820 J. Med. Chem. 2009, 52, 4820–4837

DOI: 10.1021/jm900372w

Inhibitors of the Hepatitis C Virus NS3 Protease with Basic Amine Functionality at the P3-Amino

Acid N-Terminus: Discovery and Optimization of a New Series of P2-P4 Macrocycles

Steven Harper,* Marco Ferrara, Benedetta Crescenzi, Marco Pompei, Maria Cecilia Palumbi, Jillian M. DiMuzio,^†

Monica Donghi, Fabrizio Fiore, Uwe Koch, Nigel J. Liverton,^†Silvia Pesci, Alessia Petrocchi, Michael Rowley,

Vincenzo Summa, and Cristina Gardelli

IRBM (Merck Research Laboratories Rome), Via Pontina km 30,600, 00040 Pomezia, Rome, Italy. ^†Current address: Merck West Point,

Sumneytown Pike, 19486 West Point, PA.

Received March 24, 2009

In a follow-up to our recent disclosure of P2-P4 macrocyclic inhibitors of the hepatitis C virus (HCV)

NS3 protease (e.g., 1, Chart 1), we report a new but related compound series featuring a basic amine at

the N-terminus of the P3-amino acid residue. Replacement of the electroneutral P3-amino acid capping

group (which is a feature of almost all tripeptide-like inhibitors of NS3 reported to date) with a basic

group is not only tolerated but can result in advantageous cell based potency. Optimization of this new

class of P3-amine based inhibitors gave compounds such as 25 and 26 that combine excellent cell based

activity with pharmacokinetic properties that are attractive for an antiviral targeting HCV.

Introduction

excellent target for drug intervention. Subsequently related

noncovalent inhibitors⁷(e.g., ITMN-191 and TMC435350)

and serine-trap based covalent compounds⁸(e.g., VX-950 and

Sch503034) have elicited strong clinical reductions in viral

titers, further illustrating the potential benefit to human

health from development of drugs against NS3. Toward this

goal, we have recently described the design and initial evalua-

tion of a novel series of P2-P4 macrocyclic inhibitors of

the HCV NS3/NS4A protease.⁹These compounds (e.g., 1,

Chart 1) demonstrated strong inhibition of the NS3 enzyme,

had robust cell-based potency, and showed promising pre-

liminary pharmacokinetic profiles. The evolution of this

compound class to bismacrocyclic analogues (e.g., 2) and to

a clinical candidate MK-7009 has also been disclosed.¹⁰As

part of our continuing effort in this area, we report here an

exploration of structure-activity relationships in the linker

group that tethers the nitrogen atom of the P3-amino acid to

the heterocyclic group attached to the hydroxyproline residue

in P2.¹¹This work led to the finding that basic alkylamine

functionality at the N-terminus of active-site P1-P3 peptido-

mimetic inhibitors of NS3 is not only tolerated but can be

beneficial in terms of enhanced cell based potency or im-

proved pharmacokinetic properties.

Hepatitis C virus (HCV^a) is a human pathogen from the

flaviviridae family of positively stranded RNA viruses and infects

around 200 million individuals worldwide.¹Chronic infections of

HCV are initially asymptomatic but can ultimately lead to

serious liver disorders such as hepatocellular carcinoma and

cirrhosis. HCV is consequently a leading cause of liver trans-

plantation.²The current standard of care for HCV infections is

based on subcutaneous injection of a modified interferon-R

usually combined with ribavarin. Such treatment regimens are

inconvenient, poorly tolerated, and only successful at curing

HCV infection in around 50% of patients. New therapies

targeting virally encoded enzymes are under investigation,^3,4

but to date, no direct-acting antiviral agent has reached the

market.

Replication of HCV occurs predominantly in hepatocytes

where the genome is translated into a polyprotein that is

subsequently cleaved to produce the structural components of

HCV, as well as the nonstructural proteins that constitute the

viral replication machinery. Four out of the five polyprotein

cleavages that occur in the nonstructural region are mediated

by the virally encoded heterodimeric NS3/NS4A (NS3) en-

zyme.⁵This enzyme is a serine protease thathas been shown to

be essential for viral replication and that has been studied

intensively as a target for direct antivirals. BILN-2061, a

reversible P1-P3 macrocyclic “product-like” inhibitor⁶of

NS3 was the first compound for which clinical proof of

concept was achieved, validating the NS3 protease as an

Biology

Compounds were assessed for activity (K_i) against recom-

binant NS3 protease using a time-resolved fluorescence as-

say.¹²Inhibition of subgenomic HCV RNA replication was

measured in HUH-7 cells using modifications¹³of the proce-

dure described by Bartenschlager;¹⁴reported data were gen-

erated using cellular HCV RNA as the assayreadout and were

confirmed (results not shown) in an independent assay using a

monoclonal antibody against the viral NS3 protease as the

readout. Cell-based activity (EC₅₀) in the replicon assay was

measured routinely in the presence of 10% fetal bovine serum;

high serum conditions refer to data collected in the presence

*To whom correspondence should be addressed. Phone: þ39 06

91093444. Fax: þ39 06 91093225. E-mail: steven_harper@merck.com.

^aAbbreviations: HCV, hepatitis C virus; NS, nonstructural; HATU,

azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;

TBTU O-benzotriazole-1-yl-N,N,N⁰,N⁰-tetramethyluronium tetrafluoro-

borate; NOESY, nuclear Overhauser enhanced spectroscopy; RCM, ring

closing metathesis; RP-HPLC, reversed phase high performance liquid

chromatography; po, per os; iv, intravenous.

r

pubs.acs.org/jmc

Published on Web 07/22/2009

2009 American Chemical Society

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4821

Chart 1. P2-P4 Macrocyclic Inhibitors of the HCV NS3 Pro-

tease Enzyme

Table 1. P2-P4 Macrocyclic Amine Inhibitors Containing a Trifluoro-

ethylamine Amide Isostere (12a,b) and Early Structural Analogues

(13-15)

compd

R

R₁

R₂

K_i(nM)^a

EC₅₀(nM)^b

3

See Chart 1.

0.04 ( 0.01

1.1 ( 0.1

5.7 ( 0.3

0.27 ( 0.07

1.7 ( 0.5

0.51 ( 0.2

0.35 ( 0.01

12

18

470

1

12a

12b

13a^c

13b^c

14

H

F

CF₃

H

CF₃

H

Me

H

Me

H

40

7

H

15

H

14

^aK_ivalues in this and subsequent tables are quoted as the arithmetic

mean ( standard error (or half-range where n=2) for two to nine inde-

pendent determinations. ^bEC₅₀values are measured in the presence

of 10% fetal bovine serum. ^cThe absolute stereochemistry of com-

pounds 13a and 13b is assigned by analogy to 12a,b (see ref 21 for

further details).

of 50% normal human serum. Enzyme inhibition data and

replicon potencies were generated against genotype 1b HCV.

Preliminary pharmacokinetic studies were conducted through

oral administration of compounds to male Sprague-Dawley

rats at 5 mg/kg using PEG₄₀₀as vehicle. In these studies

plasma exposure was determined from the area under the

plasma concentration/time curve between 0 and 4 h, and liver

tissue concentrations were determined at the 4 h time point.

Follow-up pharmacokinetic studies were conducted for com-

pound 25 in both male Sprague-Dawley rat and male beagle

dog. The dosing parameters used were the following: dog,

intravenous (iv) 1 mg/kg (DMSO/PEG₄₀₀/H₂O, 20%:60%:

20% as vehicle); dog, per os (po) 5 mg/kg (PEG₄₀₀/H₂O,

80%:20% as vehicle); rat, iv 5 mg/kg (DMSO/PEG₄₀₀/H₂O

20%:60%:20% as vehicle); rat, po 5 mg/kg (PEG₄₀₀as vehi-

cle). Pharmacokinetic parameters were determined from the

area under the plasma concentration/time curve between

0 and 24 h (or to the last point at which compound levels

were quantifiable). Liver tissue concentrations were deter-

mined (in rat) at the 24 h time point.

Table 2. Most Significant NOE²³Intensities for Compounds 12a and

12b in Comparison with Predicted Interatomic Distances from Molec-

ular Modeling

12a (12-S)

12b (12-R)

proton

H₁₂-H₁₀

H₁₂-H_1(centroid)

calculated (A) measured^acalculated (A) measured^a

Chemistry

2.9

3.5

s

3.6

6.2

m

nd

m

The P2-P4 macrocyclic amine compounds described

(Tables 1, 3, and 4) were assembled using the modular

approach outlined in Figure 1. This route relied on ring

closing metathesis (RCM) methodology to construct the

central P2-P4 macrocyclic ring (10) followed by installation

of the P1-P1⁰functionality by amide coupling with the

known amino acid acylsulfonamide fragment 11.¹⁵The

RCM precursors 9 were available through amide coupling

between a trans-4-hydroxyproline methyl ester derivative (4-

7) functionalized with the required P2-heterocyclic group, and

the N-alkylated P3-amino acid derivatives 8. A vinyl group on

the P2-heterocycle in compounds 4-7 served as a partner for

the RCM step with the terminal olefin in the functionalized

amino acids 8.

Scheme 1 illustrates the synthesis of the P3-amino acid

derivatives 8 that were employed herein. Treatment of the

known oxazolidine 27¹⁶with 6-bromohex-1-ene afforded a

mixture of the amino alcohols 28a,b. The diastereomeric ratio

was 7:1, and the individual isomers were separated by reversed

phase high performance liquid chromatography (RP-HPLC).

^aNOE intensities determined from NOESY experiments: w = weak;

m = medium; s = strong; nd = not detected.

Oxidation of these compounds with Jones reagent furnished

the corresponding carboxylic acids 8a,b. The remaining

P2-P4 linking fragments 8c-j were prepared by reductive

amination using the appropriate amino ester derivative. Thus,

reaction of 7-octen-2-one 29 with the methyl esters of valine,

cyclopentylglycine, or cyclohexylglycine afforded (after

methyl ester hydrolysis) the functionalized amino acids

8c-e. The reductive amination step proceeded with a diaster-

eomeric ratio of around 1:1, and compounds 8c-e were used

as mixtures in the subsequent coupling reactions (see

Scheme 3). A reductive amination/ester hydrolysis sequence

also proceeded in efficient yield with the aldehydes 30, 31, and

32¹⁷giving 8f-j. 2,2-Difluoro-6-heptenal 31 (which was used

in the preparation of 8g) was obtained by fluorination of 30

with N-fluoro-N-(phenylsulfonyl)benzenesulfonamide using

racemic proline as the organocatalyst.¹⁸

4822 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Harper et al.

Table 3. Structural Changes to the P3-Amino Acid Residue and Their Effect on Rat Pharmacokinetic Properties

compd

bond

P₃

Val

R

K_i(nM)

EC₅₀(nM)^a

AUC_po(μM h)^b

Liver (μM)^c

3

13a

17a

17c

18a

19

single

double^d

single

H

0.27 ( 0.07

0.29 ( 0.07

0.1 ( 0.03

0.06 ( 0.01

0.35 ( 0.01

1

<0.1

<0.1^e

<0.1

<0.1^e

<0.1

0.26

C₅H₉

C₆H₁₁

H

0.5

0.7

2

H

<0.1^e

1.04

H

Me

3

0.56^e

^aEC₅₀values were measured in the presence of 10% fetal bovine serum. ^bArea under the plasma concentration/time curve between 0 and 4 h follo-

wing oral administration of the compounds (5 mg/kg) to Sprague-Dawley rats. ^cRat liver concentrations are measured 4 h after oral administration

(5 mg/kg) to Sprague-Dawley rats. ^dE-Olefin geometry. ^e4 mg/kg oral dose.

Scheme 2 illustrates the construction of compound 12a.

Thus, TBTU mediated amide coupling between 4¹⁹and

8a furnished the RCM precursor 9a as a single diastereo-

isomer, and this material was smoothly cyclized to 10a using

Zhan I²⁰as catalyst. Direct hydrogenation of the crude

product from the RCM reaction gave the saturated macro-

cyclic compound sat-10a (62% yield from 9a). The synthesis

Thus, coupling of 4 with the P3 fragments 8f or 8g gave the

RCM precursors 9f,g and Zhan I mediated RCM furnished

the olefins 10f,g. Hydrogenation, proline ester deprotection,

and amide coupling with 11 again completed the synthesis.

The macrocyclic amine compounds 20-26 were prepared

(Scheme 5) from the trans-4-hydroxyproline heterocyclic

derivatives 5,⁹6,¹⁰and 7²²together with the P3-amino acid

fragments 8h-j. HATU mediated coupling of 8h or 8i with

6 gave the ring closing metathesis precursors 9h,i from

which Zhan I catalyzed RCM generated the trans-olefins

10h,i. Following removal of the proline methyl ester, the

corresponding carboxylic acids were elaborated either to

22/24 by direct coupling with 11 or to 23/25 by a hydrogena-

tion/coupling sequence. Likewise, reaction of 8j with 5 or

of 8h with 7 afforded the macrocycles 20-21 or 26, respec-

tively.

was completed by LiOH H₂O mediated hydrolysis of the

3

proline methyl ester followed by TBTU mediated amide

coupling with 11. Compound 12a was isolated following

purification by RP-HPLC. In identical fashion as outlined

in Scheme 2, the inhibitor 12b (which is a diastereoisomer of

12a, Table 1) was accessed from 4 and 8b. The intermediates

(not shown) were 9b (RCM precursor) and 10b (P2-P4

macrocycle).

The construction of compounds 13a,b, 17a-c, and 18a,b is

shown in Scheme 3. Although the P3 fragments 8c-e used

contain both a free amine and a carboxylic acid group (which

have the potential to undergo a homocoupling reaction), their

reaction with4 was successfully accomplished using HATU as

the peptide reagent. The RCM precursors 9c-e were isolated

in 43-76% yield. The amine functionality in 9c-e was

deactivated as a TFA salt and in this form did not impede

the Zhan I mediated RCM reaction (45-73% yield). Macro-

cycles 10c-e (each a mixture of diastereoisomers at the chiral

center adjacent to the P3-N atom) were purified by RP-HPLC

to give the individual 12-R and 12-S isomers; only E-olefins

were isolated from the ring closing metathesis reaction. The

individual diastereoisomers²¹(R)-10c-e and (S)-10c-e were

elaborated separately through the final steps of the synthesis.

Thus, hydrolysis of the proline methyl ester and a hydrogena-

tion step set up the final TBTU-mediated coupling with 11.

Compounds 13a, 17c, and 18a (from (R)-10c, (R)-10d, and

(R)-10e, respectively) and compounds 13b and 18b (from

(S)-10c and (S)-10e, respectively) were isolated following

RP-HPLC purification. In a similar vein 17a and 17b were

obtained from (R)-10d and (S)-10d by omitting the catalytic

hydrogenation step. Compound 19 was prepared by direct

reductive-formylation of 18a, which was smoothly achieved

(55% yield following RP-HPLC purification) in the presence

of a Lewis acid (ZnCl₂) using NaBH₃CN as the reducing

agent.

Having been unable to obtain functionalized amino

acids 8 through reductive amination of aldehydes such

as 30 with sterically hindered amino acids, compound

16 was accessed as outlined in Scheme 6. Coupling of 4 with

(^D,L)-N-Boc-trifluoromethylglycine followed by N-Boc depro-

tection furnished 33. Reductive amination between this

compound and 30 was possible but gave 9l as a single

diastereoisomer in modest yield (17%). Following elabora-

tion of 9l to 16 as has previously been described, the P3-amino

acid residue was confirmed to have the desired (R)-configura-

tion by ¹H,¹H NOESY experiments.²³

The acyclic tripeptide analogues 38 and 39 were prepared as

outlined in Scheme 7. The intermediates 35a,b²⁴containing

the P2-proline residue from BILN-2061 were elaborated

either at the N-terminus to furnish the P2-P3 fragment

37 or at the carboxylic acid to give the P1-P2 dipeptide

analogue 36. Coupling of 36 with 34 (which was available

following reductive amination of cyclohexylglycine methyl

ester and 3,3-dimethylbutanal followed by routine protecting

group manipulations) led after N-Boc-deprotection to the

amine 38. TBTU mediated coupling of 37 with 11 provided

the urethane capped tripeptide analogue 39.

Results and Discussion

Peptidomimetic inhibitors that span the P1-P3 region (or

P1⁰-P3 region) of the NS3 protease enzyme are ubiquitous in

the HCV literature. Compound series based upon both acyclic

structures²⁵and several classes of P1-P3 macrocycles^7,8,26

An analogous reaction sequence as has already been de-

scribed was used to access compounds 14 and 15 (Scheme 4).

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4823

Table 4. Activity and Preliminary Rat Pharmacokinetic Profile for

P2-P4 Macrocyclic Amine Inhibitors: SAR at the P2 Heterocyclic

Group and Evaluation of an Acyclic Analogue 38

have been reported. More recently, P2-P4 macrocycles^9,10,27

have been shown to be a viable class of tripeptide mimetics

that strongly inhibit HCV replication. A structural feature of

almost all HCV inhibitors from these classes²⁸is an electro-

neutral capping group at their P3 N-terminus, most usually an

alkyl (or cycloalkyl) amide, urea, or urethane. The role of the

capping group may be seen as twofold; the lipophilic alkyl

group generates binding energy through interaction toward

the S4 subsite of NS3, while the P3-amidic NH group can

engage in a hydrogen bonding interaction with the backbone

carbonyl of Ala157.²⁹In light of these considerations we were

attracted to a recently reported³⁰trifluoroethylamine isostere

for an amide bond and questioned whether in the context of

our P2-P4 macrocyclic inhibitors (e.g., 3, Chart 1) this might

provide a viable alternative to the urethane functionality

found in this compound class to date. We envisioned that

the alkyl chain of the tether between the P2 and P3 residues

might still engage S4, while the nonbasic amine group of the

P3-amino acid would retain the potential to act as a hydrogen

bond donor. The suitability of this hypothesis was borne out

by molecular modeling studies³¹on 3 and the trifluoroethyl-

amine analogues 12a,b (Table 1) which have opposite config-

uration at the CF₃substituted carbon R to the P3-N atom.

Superposition of 3 with 12b (which has R-configuration at the

R-CF₃group) suggested thata stericclashbetween the enzyme

and the CF₃group would result in significant distortion of the

P2-P4 region of the ligand and ultimately diminish interac-

tion with the NS3 enzyme. In contrast, the isomer based on

(S)-configuration at the R-CF₃group (12a) overlaid well with

urethane 3. For this isomer the CF₃group is oriented away

from the enzyme surface, allowing the ligand to interact with

NS3 in a manner similar to that of the (predicted) bioactive

conformation of 3.

Compounds 12a,b were prepared as is outlined in Scheme 2

and then tested in our NS3 enzyme and cell based replicon

assays. Results showed thatalthough both compoundscaused

somewhat lower inhibition of the NS3 enzyme than the

urethane 3,³²they nonetheless retained potency in the low

nanomolar range (Table 1). Furthermore, in the cell based

replicon assay a comparable response for the more active

trifluoroethylamine 12a (EC₅₀= 18 nM) as for urethane 3

(EC₅₀=12 nM) was measured, highlighting the suitability of a

trifluoroethylamine based linker in the context of a P2-P4

macrocycle.

The absolute stereochemistry at C₁₂for 12a and 12b was

assessed by NMR analysis. Significantly different interatomic

distances between H₁₂and both H₁₀and the H₁centroids

were expected for the two diastereoisomers on the basis of

molecular modeling, and these predictions were confirmed by

¹H,¹H-NOESY experiments²³performed on both com-

pounds. As reported in Table 2, a strong match was observed

between the calculated distances and the measured NOE

intensities for both epimers, indicating (S)-configuration at

C₁₂for the more active isomer 12a and (R)-configuration for

12b. Both compounds had similar Z-conformations at their

proline amide bonds. Thus, the stronger biochemical potency

that is predicted (and observed) for compound 12a with

respect to 12b together with measured NMR data and molec-

ular modeling make a strong case for the assignment of (12S)-

configuration at the R-CF₃group in 12a.

^aEC₅₀values were measured in the presence of 50% normal human

serum. ^bArea under the plasma concentration/time curve between 0 and

4 h following oral administration of the compounds (5 mg/kg) to

Sprague-Dawley rats. ^cRat liver concentrations are measured 4 h after

oral administration (5 mg/kg). ^dnd=not determined.

A feature that distinguishes 12a from previously reported

cathepsin K inhibitors that incorporate a trifluoroethylamine

isostere of an amide is the pK_aof the amine functionality β to

the CF₃group. The predicted³³pK_aof this N-atom in 12a is

4824 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Harper et al.

Figure 1. General retrosynthetic analysis of P2-P4 macrocyclic amine inhibitors 12-26.

6.3, around 3 log units higher than that reported for trifluoro-

ethylamineamide isosteres inthe cathepsin K area. Thestrong

enzyme inhibition shown by 12a was notable given that a

precipitous drop in activity (of around 3 orders of magnitude)

was observed in the cathepsin area when an amine with pK_a

above 6 was employed as the amide isostere.^30bAlthough 12a

would be expected to be around 90% unprotonated at the

physiologicalpHofthe biological assays, asignificant amount

of the protonated form would still be present in the assay

media. To assess the contribution to enzyme inhibition from

this protonated species and to further probe the role of amine

pK_a, the R-methyl branched compounds 13a,b (which aside

from amine pK_ahave strong structural similarity to 12a,b)

were evaluated. Remarkably, the more active of these

R-methyl diastereoisomers, 13a,²¹had enzyme inhibition that

was enhanced over the CF₃compound 12a. While it cannot be

excluded that modification of the CF₃group to the sterically

less demanding CH₃may be favorable, the presence of a basic

amine likely improves potency through electrostatic interac-

tion with the acidic residue D168 which lies close to the P3

amino acid (Figure 2C). A further attraction of amine 13a was

that it elicited a much stronger cell based response (EC₅₀=1 nM)

than either 12a or the original urethane 3. The cell based

potency of 13a was especially attractive when the replicon

assay was conducted in the presence of human plasma. Under

these conditions, which may be considered to be more phy-

siologically relevant,³⁴12a and 13a had EC₅₀values of 320

and 3 nM, respectively. The improved cell-based potency of

13a is in line with reduced affinity for serum albumin when a

basic amine (which renders the inhibitor formally

zwitterionic) is present.³⁵In vitro binding experiments with

12a and 13a showed that their free fractions in human plasma

are 1.9% and 10.6%, respectively.

efficacy to the R-branched analogue 13a. Two simplified

analogues of 12a, both of which retained a P3 N-terminal

nonbasic amine, were also evaluated. The P3-trifluoromethyl-

glycine compound 16 (Scheme 6) retained enzyme inhibition

comparable to 12a (K_ivalues for 16 and 12a are 1.8 and

1.1 nM, respectively) but showed much reduced efficacy in the

cell based assay (EC₅₀of 160 nM vs 18 nM). In contrast, the

gem-difluoro compound 15 had somewhat improved bio-

chemical potency over the prototype trifluoroethylamine

inhibitor 12a and demonstrated comparable cell based effi-

cacy aseither 12a or 14. However, the absence of a basic amine

group in 15 again resulted in a less favorable serum shift than

for 13a or 14; when assayed in the presence of human serum,

15 was around a 100 nM inhibitor of HCV replication.

Rat pharmacokinetic studies were performed on all com-

pounds (12a-15) in Table 1. After oral administration con-

sistently low exposure of these compounds in rat plasma was

measured. AUC values ranged from zero (compound 14 was

undetectable in rat plasma following administration at 4 mg/kg)

to 0.05 μM h (compound 12a). Low compound levels were

3

also detected in rat liver tissue 4 h post-administration,³⁶the

highest concentration being measured for compound 13b

(0.22 μM). The combination of low plasma and liver levels

was viewed as a limitation for these macrocyclic amines given

that their main target is the suppression of HCV replication in

hepatocytes. The low observed plasma exposure and liver

tissue concentration likely reflects modest absorption and

uptake of the inhibitors from the gastrointestinal tract. It

has been reported⁹that in the carbamate series a change of

side chain in the P3 R-amino acid residue had a profound

effect on uptake, suggesting that an approach to improving

pharmacokinetics of the current inhibitors might be to eval-

uate changes at P3. The results in Table 3 reflect our efforts in

this direction and further emphasize that for P2-P4 macro-

cycles very subtle changes in the structure of the P3-amino

acid side chain can strongly influence the pharmacokinetic

profile of the inhibitor. Thus, little benefit came from the

presenceofa cyclopentylglycine residueinP3, withcompound

17c showing a profile similar to that of its direct P3-valine

analogue 13a. Somewhat improved liver tissue levels were

recorded when a benzylic double bond was present in the

On the basis of these initial findings, a research campaign to

optimize P2-P4 linked macrocyclic amine inhibitors was

initiated. The first target was evaluation of structurally sim-

plified inhibitors devoid of the chiral center resulting from the

branching functionality R to the P3-amine group. Compound

14 fulfills this goal and demonstrates that removal of the

branch can be achieved without significant loss of biological

activity; 14 showed similar intrinsic potency and cell based

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4825

Scheme 2^a

Scheme 1^a

^aReagents: (i) TBTU, CH₂Cl₂, DIEA; (ii) Zhan I, DCE, TFA then

RP-HPLC; (iii) H₂(g), Pd/C; (iv) LiOH.H₂O, THF/H₂O; (v) TBTU, 11,

DIEA, DMAP, then RP-HPLC.

were conducted on compounds from across a range of sub-

series differing in structure at the P3 amino acid residue, the

P2-P3 linker group, and the P2-heterocycle) was subse-

quently performed with a view to establishing trends. Figure 3

illustrates an interrogation of this early rat pharmacokinetic

data set aimed at establishing relationships between oral

plasma exposure and liver levels as a function of structural

changes to the P2 heterocyclic group. This plot of dose

normalized plasma vs dose normalized liver levels makes a

clear illustration that the choice of P2-heterocycle alone is

insufficient to ensure either strong plasma exposure or liver

levels in the micromolar range. However, a trend suggesting

that ether linked 2-isoquinoline P2-heterocyclic compounds

(indicated in yellow) tend to be more profitable emerges, and

the presence of additional functionality on the isoquinoline

ring, such as a methoxy group at the 6-position (blue), can

apparently bring a further improvement in plasma exposure.

In contrast, while compounds based on the isoindoline car-

bamate P2-heterocycle (which was a feature of our early

macrocyclic amine compounds and which are indicated in

grey) do on occasion achieve high liver concentrations (>10 μM),

they consistently fail to provide plasma exposure above

0.5 μM h.

3

^aReagents: (i) 6-bromohex-1-ene, Mg, Et₂O; (ii) RP-HPLC; (iii) Jones

reagent, Me₂CO; (iv) NaBH₃CN, ZnCl₂, MeOH; (v) LiOH H₂O, THF/

H₂O; (vi) (PhSO₂)₂NF, D,L-proline, THF.

Changes to the P2-heterocyclic group were consequently

explored as an approach toward remedying the poor rat

pharmacokinetics of early P2-P4 macrocyclic amine inhibi-

tors. Disappointingly, and for reasons not clearly understood,

inhibitors based on an unsubstituted 1,7-linked 2-isoquinoline

P2-heterocycle (Table4, 20, 21) tended to lack potencyagainst

the NS3 enzyme. Macrocyclic amine inhibitors based on

this P2-heterocycle are also modest inhibitors in the cell

based assay (e.g., 21 EC₅₀=410 nM), and compounds of this

type were not further pursued. In contrast 1,7-linked

3-phenyl-4-quinoline based inhibitors showed excellent po-

tency. Compounds 22 and 23 highlight this, both showing

subnanomolar enzyme blockade and strong efficacy in the

replicon assay when it was conducted under high serum

conditions (EC₅₀values were in the 20 nM range). The

presence of a 6-OMe substituent on the P2 heterocycle of 22

and 23 (a structural feature that had appeared to favor plasma

exposure in a related series; see Figure 3) did not strongly

benefit PK properties; no improvement in plasma exposure

or rat liver levels was achieved with respect to previous

3

P2-P4 linker group (compound 17a), but a stronger improve-

ment was noted for ring expanded cyclohexyl analogue 18a.

The liverlevelsof this compound wereatleast 100 times higher

than for initial macrocyclic amine inhibitors such as 13a. The

N-methylated compound 19, which interestingly demon-

strated that tertiary amine functionality at the P3 N-terminus

of these inhibitors is tolerated, showed a similarly improved

concentration in rat liver. Ultimately, however, although liver

concentrations were improved for the cyclohexyl analogues

18a and 19, the lack of plasma exposure remained anissuethat

could not be addressed via changes to the P3 residue alone.

As a result of the unpredictable effect of minor changes in

chemical structure on pharmacokinetic parameters, the early

optimization of all our P2-P4-macrocyclic compound series

relied heavily on in vivo studies run on closely related analo-

gues. Analysis of the data generated from these studies (which

4826 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Harper et al.

Scheme 3^a

^aReagents: (i) TBTU, CH₂Cl₂, DIEA; (ii) Zhan I, DCE, TFA, then RP-HPLC; (iii) RP-HPLC; (iv) LiOH H₂O, THF/H₂O; (v) H₂(g), Pd/C; (vi)

TBTU, 11, DIEA, DMAP, then RP-HPLC; (vii) HCHO, NaBH₃CN, ZnCl₂, MeOH, then RP-HPLC.

3

P3-cyclohexylglycine compounds such as 18a. However, a

strong improvement was achieved by installation of gem-

dimethyl functionality into the linker group that joins the

P2 and P3 amino acids. This change again highlights how

subtle structural features can dramatically impact pharma-

cokinetics; compounds 24 and 25 were among our first

macrocyclic amine inhibitors that following oral administra-

tion to rat achieved plasma exposure significantly higher

(compare 21 and 26). Compound 26 had excellent plasma

exposure in rat (7 μM h following a 5 mg/kg dose), and

although the liver concentration of this inhibitor was not as

high as had been achieved with compounds 24 and 25, it was

nonetheless detected at a level more than 2 orders of magni-

tude above its replicon EC₅₀.

Although there have been occasional reports in the patent

literature relating to tripeptide based inhibitors of NS3

that feature an amine group at their N-terminus,²⁸the in-

hibitors described here are to the best of our knowledge

the first potent compound series to be based on this

feature. The rareness of protease inhibitors featuring an

amine at P3 is probably a reflection of early work in the

NS3 area, where enzyme bound crystal structures were used

toward the design of smaller and more druglike inhibitors.

3

(∼0.5 μM h) than detection limits together with high com-

3

pound concentrations in liver (5.6 and 13.7 μM for 24 and

25, respectively). As projected on the basis of the analysis in

Figure 3, further augmented plasma levels came from inhibi-

tors based on a 6-methoxy-2-isoquinoline heterocycle at

P2. Gratifyingly this change also restored biochemical and

cell based potency to a more attractive, low nanomolar level

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4827

The presence of a hydrogen bonding interaction between

Ala157 and the (neutral) P3 NH group of the ligand is

a feature in these X-ray structures, and retention of this

structural element during subsequent phases of optimization

is understandable.³⁷However, the compounds described

show that neutral functionality is not a strong requirement

at the N-terminus, and in fact, benefits in terms of cell based

potency or pharmacokinetic properties can come from its

replacement with basic amine functionality. Thus, com-

pound 13a showed around a 100-fold stronger cell based

response (when assayed under high serum conditions) than

the closely related P3-urethane compound 3. During the

optimization of 13a a degree of cell based potency could

be sacrificed to favor inhibitors with pharmacokinetic proper-

ties that are attractive in an antiviral targeting HCV. Com-

pound 25 (Table 4) highlights this approach. Although

the cell-based potency of this inhibitor is not superior to

the carbamate analogue 40 (Chart 1; the compounds are

equipotent in our cell based assays), it does reach some-

what higher levels in rat liver (13.7 μM vs 9.9 μM) follow-

ing oral administration of a 5 mg/kg dose and also has

improved plasma exposure (0.45 μM h vs 0.15 μM h).

Scheme 4^a

3

Compound 25 was 44% bioavailable in rat, with moderate

plasma clearance (27 mL/min/kg); the half-life was 3.3 h.

Plasma levels were not quantifiable 24 h after oral adminis-

tration, but the compound was still detected in liver at a

concentration (0.3 μM) well above its replicon EC₅₀, suggest-

ing at least the potential for prolonged suppression of

HCV replication in the target organ. Significantly higher

exposure was also measured in dog for the amine based

inhibitor 25 vs 40 (68.8 μM h vs 5.7 μM h). The overall

^aReagents: (i) TBTU, CH₂Cl₂, DIEA; (ii) Zhan I, DCE, TFA, then

RP-HPLC; (iii) H₂(g), Pd/C; (iv) LiOH H₂O, THF/H₂O; (v) TBTU, 11,

DIEA, DMAP, then RP-HPLC.

3

Scheme 5^a

^aReagents: (i) HATU, CH₂Cl₂, DIEA; (ii) Zhan I, DCE, TFA; (iii) LiOH H₂O, THF/H₂O; (iv) H₂(g), Pd/C; (v) TBTU, 11, DIEA, DMAP, then

RP-HPLC.

3

4828 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Harper et al.

Scheme 6^a

^aReagents: (i) (D,L)-N-Boc-trifluoromethylglycine, HATU, NMM;

(ii) 4 N HCl in 1,4-dioxane; (iii) 30, NaBH₃CN, then RP-HPLC; (iv)

Zhan I, DCE, TFA; (v) LiOH H₂O, THF/H₂O; (vi) TBTU, 11, DIEA,

3

DMAP, then RP-HPLC.

Scheme 7^a

Figure 2. Superposition of urethane inhibitor 3 (green) with tri-

fluoroethylamine analogues 12a (cyan, A) and 12b (pink, B) and the

proposed binding orientation for compound 12a (cyan, C). Inhibi-

tors were minimized and docked into the active site of the genotype

1b NS3/peptide4A crystal structure. The enzyme surface and in-

dividual amino acid residues are shown in white (with the exception

of the side chain of residue D168 which is shown in red in parts

A and B). Potential hydrogen bonding interactions between the

ligand and enzyme are shown as broken lines.

To demonstrate that the tolerance for amine functionality

at the P3 N-terminus is not limited to P2-P4 macrocyclic

protease inhibitors (where constraint of the P3-capping group

into a ring may render it more permissive toward mod-

ification), compounds 38 and 39 that are based on the

P2 heterocycle found in BILN-2061 were prepared. Amine

analogue 38 showed marginally weaker inhibition (4-fold)

than the corresponding P3-urethane analogue 39 but none-

theless retained subnanomolar inhibition of the NS3 enzyme.

In line with the results described for the P2-P4 macrocyclic

compounds, amine 38 showed a similar response as urethane

39 in the cell-based assay. These results confirm that the

tolerance for simple amine functionality at the P3-amino acid

N-terminus is not confined to P2-P4 macrocyclic inhibitors

of the NS3 protease.

^aReagents: (i) NaBH₃CN, ZnCl₂, MeOH; (ii) Boc₂O, Et₃N, MeOH;

(iii) LiOH H₂O, THF/dioxane/H₂O; (iv) TBTU, DIEA, DMAP; (v)

3

TFA/CH₂Cl₂, then RP-HPLC; (vi) Boc-Chg-OH, HATU, Et₃N; (vii)

11, TBTU, DIEA, DMAP.

pharmacokinetic profile of 25 in dog was attractive, with the

compound showing low clearance (1 mL/min/kg), a reason-

able plasma half-life (3 h), and 92% bioavailability.

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4829

and were judged >95% pure by peak area (UV detection at

254 nm); for the purity of specific compounds, see below. RP-

HPLC was performedon WatersAlliance instruments (analytical

scale) or Waters preparative-scale instrument equipped for auto-

mated mass or UV-triggered collection. In all cases mixtures of

MeCN and H₂O (each buffered with 0.1% TFA) were used as the

mobile phase. The following conditions were employed. Condi-

tions A: stationary phase, Waters Symmetry C₁₈, 7 μm, 19 mm ꢀ

300 mm; flow rate of 20 mL/min; gradient of 40% MeCN for

4 min and thenfrom40%to 90%MeCN over 16 min. Conditions

B: same as for conditions A but using the following gradient,

20% MeCN for 4 min and then from 20% to 70% MeCN over

16 min. Conditions C: stationary phase, Atlantis C₁₈, 5 μm,

3 mmꢀ150 mm; flow rate of 1 mL/min; gradient of 20% MeCN

for 1 min and then from 20% to 80% MeCN over 7 min.

Conditions D: stationary phase, X-Bridge 5 μm, 4.6 mm ꢀ

50 mm; flow rate of 1 mL/min; gradient of 20% MeCN for

1 min and then from 20% to 100% over 4 min. Conditions E:

stationary phase, X-Bridge C₁₈, 5 μm, 19mm ꢀ 100mm; flowrate

of 20 mL/min; gradient of 20% MeCN for 1 min and then from

20% to 80% MeCN over 16 min. Low resolution mass spectro-

metry data were generated on Waters ZQ micromass systems.

High resolution data were collected on Waters Synapt Q-tof

instruments.

Methyl N-[(1S)-1-(Trifluoromethyl)hept-6-en-1-yl]-^L-valyl-(4R)-

4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-^L-pro-

linate (9a). A solution of 8a (82 mg, 0.29 mmol) in CH₂Cl₂(7 mL)

was treated with DIEA (112 μL, 0.64 mmol) and TBTU (112 mg,

0.35 mmol). The mixture was stirred for 5 min and then treated

with 4 (113 mg, 0.32 mmol). The mixture was stirred for 14 h and

then diluted with aqueous HCl (1 N). The organic layer was

separated and washed with saturated aqueous NaHCO₃and brine

and then dried. Removal of the volatiles gave a residue that was

purified by flash chromatography on silica gel (EtOAc/petroleum

ether gradient from 5:95 to 15:85) to furnish the title compound

(50 mg, 30%) as a white foam. ¹H NMR (DMSO-d₆, 600 MHz;

9:1* mixture of rotamers about the proline amide bond and a 1^#:1^##

mixture of rotamers about the isoindoline urethane bond) δ 7.48

(dd, J = 7.8, 2.4 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.26^#and 7.14^##

(d, J=7.8 Hz, 1H), 6.73^##and 6.62^#(dd, J=17.5, 11.1 Hz, 1H),

5.82-5.68 (m, 1H), 5.76 (d, J = 17.5 Hz, 1H), 5.38^#and 5.33^##(d,

J=11.1 Hz, 1H), 5.30-5.26 and 5.22-5.17* (m, 1H), 5.04-4.86

(m, 2H), 4.83-4.77* and 4.55-4.48 (m, 1H), 4.74-4.56 (m, 4H),

3.91 and 3.86* (d, J=11.9 Hz, J*=12.1 Hz, 1H), 3.79-3.75 and

3.58-3.50* (m, 1H), 3.69* and 3.64 (s, 3H), 3.26 and 2.97* (d, J =

7.8 Hz, 1H), 2.90-2.82* and 2.81-2.74 (m, 1H), 2.61-2.54* and

2.49-2.42 (m, 1H), 2.41-2.36* and 2.19-2.12 (m, 1H), 2.07-1.90

(m, 3H), 1.73-1.67 (m, 1H), 1.44-1.35 (m, 3H), 1.29-1.19 (m,

3H), 0.91 (d, J=6.8 Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H); ¹⁹F NMR

(376 MHz, DMSO-d₆, two rotamers with relative intensity 9:1* are

observed) δ -77.0, -77.5*; MS (ES^þ) m/z 580 (M þ H)^þ.

Figure 3. Pharmacokinetic data for P2-P4 macrocyclic com-

pounds measured following oral administration to Sprague-Daw-

ley rats at doses between 3 and 5 mg/kg. Plasma exposure (x-axis,

μM h) was measured as the area under the plasma concentration/

3

time curve between 0 and 4 h; liver concentrations (y-axis, μM) were

measured at 4 h. Data are colored according to the heterocycle

attached to the P2-hydroxyproline residue, as indicated below the

graph. Exposure values and liver concentrations for compounds

that were administered at doses below 5 mg/kg are normalized by

linear adjustment to 5 mg/kg.

Conclusion

In summary we have described the discovery and optimiza-

tion of a series of P2-P4 macrocyclic amine inhibitors of the

HCV NS3 protease. The starting point for this work was 12a/

13a, which showed promising cell based potency but had poor

pharmacokinetic properties in rat. Through optimization of

the P2 heterocycle, the P3 amino acid, and the P2-P3 linking

group, these issues were addressed to generate inhibitors with

attractive properties for anti-HCV agents. Basic amine func-

tionality at the P3 N-terminus of HCV NS3 inhibitors has

been shown to begenerally tolerated, andoptimizedinhibitors

that incorporate this structural feature have cell-based po-

tency and pharmacokinetic profiles that compare favorably

with their related P3-N-capped (urethane) analogues.

Methyl N-[(1R)-1-(Trifluoromethyl)hept-6-en-1-yl]-^L-valyl-(4R)-

4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-^L-prolinate

(9b). Following the procedure described for 9a, treatment of 8b

(250 mg, 0.89 mmol) with 4 furnished the title compound (89 mg,

17%) as a solid. ¹H NMR (600 MHz, DMSO-d₆; a 1:1* mixture of

rotamers about the isoindoline urethane bond and a >95:5 mix-

ture of rotamers about the proline amide bond were observed; only

the major isomer is assigned) δ 7.47 (d, J=7.8 Hz, 1H), 7.30 (t,

J = 7.8 Hz, 1H), 7.25 and 7.17* (d, J = 7.8 Hz, 1H), 6.73 and 6.63*

(dd, J=17.5, 11.1 Hz, 1H), 5.80-5.71 (m, 2H), 5.37 and 5.35-

5.25* (d and m*, J= 11.1 Hz, 1H), 5.32-5.25 (m, 1H), 4.97 (d, J=

17.2 Hz, 1H), 4.92 (d, J=10.1 Hz, 1H), 4.74-4.41 (m, 5H), 3.92

and 3.90* (d, J = 12.0 Hz, 1H), 3.75-3.68 (m, 1H), 3.63 and 3.62*

(s, 3H), 3.51 and 3.46* (dd, J=8.0, 5.3 Hz, 1H), 3.19-3.09 (m, 1H),

2.47-2.38 (m, 1H), 2.14-2.05 (m, 2H), 2.04-1.91 (m, 2H), 1.78-

1.68 (m, 1H), 1.51-1.28 (m, 6H), 0.95 and 0.94* (d, J = 6.8 Hz,

3H), 0.80 and 0.78* (d, J=6.8 Hz, 3H); ¹⁹F NMR (376 MHz,

DMSO-d₆; two rotamers with relative ratio 1:1 are observed)

δ -74.3, -74.4; MS (ES^þ) m/z 580 (M þ H)^þ.

Experimental Section

Solvents were obtained from commercial suppliers (Fluka,

puriss. grade) and were used without further purification. Or-

ganic extracts were dried over anhydrous Na₂SO₄(Merck). Flash

chromatography purifications were performed on Biotage pre-

packed silica gel columns using Biotage Horizon and SP2

instruments. NMR spectra were recorded on Bruker Avance

spectrometers and, unless otherwise stated, were acquired at

300 K. Proton chemical shifts are reported in parts per million

(δ) and are referenced to the residual proton signal of the

deuterated solvent (DMSO-d₆at 2.50 ppm, CDCl₃at 7.26 ppm,

CD₃OD at 3.31 ppm). Carbon chemical shifts are referenced to

the solvent signal of DMSO-d₆at 39.5 ppm. Compounds were

assessed for purity under two independent RP-HPLC conditions

4830 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Harper et al.

Methyl N-[(2R and 2S)-Oct-7-en-2-yl]-L-valyl-(4R)-4-{[(4-

ethenyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-L-prolinate

Trifluoroacetate (9c). Following the general procedure de-

scribed for 9a, treatment of 8c (467 mg, 2.06 mmol) with

4 furnished a solid that was further purified by RP-HPLC

(conditions B) to give a 3:2 mixture of diastereoisomers of

the title compounds (999 mg, 76%) as a white powder. MS

(ES^þ) m/z 526 (M þH)^þ.

Methyl 3,3,3-Trifluoro-N-(hept-6-en-1-yl)-^L-alanyl-(4R)-

4-{[(4-ethenyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-L-pro-

linate (9l). A solution of 34 (730 mg, 1.65 mmol) in THF (3 mL)

was treated with 30 (186 mg, 1.65 mmol) and NaCNBH₃

(312 mg, 4.96 mmol). The mixture was stirred for 1 h, and

then the volatiles were evaporated in vacuo. The resulting

residue was purified by RP-HPLC (conditions B) to furnish

the title compound (155 mg, 17%) as a white solid. MS (ES^þ)

m/z 538 (M þ H)^þ.

(3R,5S)-1-{(2S and 2R)-2-Cyclopentyl-2-[oct-7-en-2-ylamino]-

acetyl}-5-(methoxycarbonyl)pyrrolidin-3-yl 4-ethenyl-1,3-dihy-

dro-2H-isoindole-2-carboxylate (9d). Following the general pro-

cedure described for 9a, treatment of 8d (324 mg, 1.28 mmol)

with 4 furnished the title compounds (301 mg, 43%) as an oil. ¹H

NMR (400 MHz, DMSO-d₆, 1:1* mixture of diastereoisomers)

δ 7.54-7.43 (m, 1H), 7.36-7.24 (m, 2H), 7.36-7.24 and 7.20*

(m and t*, J* = 9.7 Hz, 1H), 6.80-6.61 (m, 1H), 5.84-5.63 (m,

1H), 5.42-5.19 (m, 2H), 5.02-4.82 (m, 2H), 4.77-4.36 (m, 6H),

4.01-3.87 (m, 1H), 3.83-3.67 (m, 1H), 3.65 (s, 3H), 3.31-3.17

(m, 1H), 2.33-0.96 (m, 19H), 0.79 (br d, J = 6.9 Hz, 3H); MS

(ES^þ) m/z 552 (M þ H)^þ.

Methyl (5R,7S,10S,12S,17E)-10-Isopropyl-3,9-dioxo-12-(tri-

fluoromethyl)-6,7,9,10,11,12,13,14,15,16-decahydro-1H,5H-2,

22:5,8-dimethano-4,2,8,11-benzoxatriazacycloicosine-7-carboxy-

late (10a). General Procedure for Ring Closing Metathesis. A

solution of 9a (100 mg, 0.17 mmol) in DCE (10 mL) was treated

with Zhan catalyst I (17 mg, 0.026 mmol), and the mixture was

heated under reflux for 1 h. The solution was cooled and filtered,

and then the volatiles were evaporated under reduced pressure. The

residue obtained contained the title compound and was used

without further purification in the following reaction step. ¹H

NMR (600 MHz, DMSO-d₆) δ 7.27 (t, J = 7.5 Hz, 1H), 7.21 (d,

J = 7.5 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 6.40 (d, J = 16.1 Hz,

1H), 5.88 (ddd, J = 16.1, 7.4, 6.7 Hz, 1H), 5.42 (t, J = 3.2 Hz, 1H),

4.77 (d, J = 15 Hz, 1H), 4.70-4.60 (m, 2H), 4.55 (d, J = 15 Hz,

1H), 4.49 (dd, J = 10.6, 7.3 Hz, 1H), 3.97 (dd, J = 11.7, 1.4 Hz,

1H), 3.73 (dd, J = 11.7, 3.1 Hz, 1H), 3.62 (s, 3H), 3.26 (t, J = 8 Hz,

1H), 2.91-2.82 (m, 1H), 2.41-2.35 (m, 1H), 2.28-2.16 (m, 2H),

2.11 (br s, 1H), 2.05 (ddd, J = 13.4, 10.8, 3.9 Hz, 1H), 1.73-1.66

(m, 1H), 1.64-1.59 (m, 2H), 1.57-1.50 (m, 1H), 1.47-1.39 (m,

1H), 1.38-1.30 (m, 1H), 1.26-1.18 (m, 1H), 0.91 (d, J = 6.6 Hz,

6H); ¹⁹F NMR (376 MHz, DMSO-d₆) δ -78.2; MS (ES^þ) m/z 552

(M þ H)^þ.

(3R,5S)-1-[(2S and 2R)-2-Cyclohexyl-2-(oct-7-en-2-ylamino)-

acetyl]-5-(methoxycarbonyl)pyrrolidin-3-yl 4-ethenyl-1,3-dihy-

dro-2H-isoindole-2-carboxylate (9e). Following the general pro-

cedure described for 9a, treatment of 8e (1.51 g, 5.0 mmol) with

4 furnished the title compound (1.28 g, 45%) as an oil. MS (ES^þ)

m/z 566 (M þ H)^þ.

Methyl N-(Hept-6-en-1-yl)-^L-valyl-(4R)-4-{[(4-ethenyl-1,3-di-

hydro-2H-isoindol-2-yl)carbonyl]oxy}-^L-prolinate (9f). Follow-

ing the general procedure described for 9a, treatment of

8f (990 mg, 3.96 mmol) with 4 furnished the title compound

(1.82 g, 90%) as a white solid. MS (ES^þ) m/z 512 (M þ H)^þ.

Methyl N-(2,2-Difluorohept-6-en-1-yl)-L-valyl-(4R)-4-{[(4-

ethenyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-^L-prolinate

(9g). Following the general procedure described for 9a (HATU

used in place of TBTU), treatment of 8g (286 mg, 1.15 mmol)

with 4 furnished the title compound (247 mg, 39%) as a foam.

MS (ES^þ) m/z 548 (M þ H)^þ.

Methyl (5R,7S,10S,12R or S,17E)-10-Isopropyl-3,9-dioxo-

12-(trifluoromethyl)-6,7,9,10,11,12,13,14,15,16-decahydro-1H,

5H-2,22:5,8-dimethano-4,2,8,11-benzoxatriazacycloicosine-7-

carboxylate (10b). Treatment of 9b (89 mg, 0.154 mmol) as

described in the general procedure for 10a furnished the title

compound that was used directly in the subsequent step. MS

(ES^þ) m/z 552 (M þ H)^þ.

Methyl (4R)-1-[(2S)-2-Cyclohexyl-2-(hept-6-en-1-ylamino)-

acetyl]-4-[(7-methoxy-2-phenyl-6-vinylquinolin-4-yl)oxy]-^L-pro-

linate (9h). Following the general procedure described for 9a (HA-

TU used in place of TBTU), treatment of 8h (269 mg, 1.06 mmol)

with 6 (375 mg, 0.925 mmol) furnished the title compound (512 mg,

35%) as an oil. MS (ES^þ) m/z 640 (M þ H)^þ.

(5R,7S,10S,12R,17E)-10-Isopropyl-7-(methoxycarbonyl)-12-

methyl-3,9-dioxo-6,7,9,10,11,12,13,14,15,16-decahydro-1H,5H-

2,22:5,8-dimethano-4,2,8,11-benzoxatriazacycloicosin-11-ium

Trifluoroacetate ((R)-10c). A sealed vial was charged with a

solution of 9c (448 mg, 0.70 mmol) and Zhan catalyst I (93 mg,

0.14 mmol) in CH₂Cl₂(45 mL). The mixture was heated under

microwave irradiation at 100 °C for 40 min and then cooled and

filtered. The volatiles were removed under reduced pressure

and the residue was purified by RP-HPLC (conditions B)

to furnish in the first fractions (t_R=14.58 min) the title com-

Methyl (4R)-1-{(2S)-2-Cyclohexyl-2-[(4,4-dimethylhept-6-en-

1-yl)amino]acetyl}-4-[(6-ethenyl-7-methoxy-2-phenylquinolin-4-

yl)oxy]-^L-prolinate (9i). Following the general procedure de-

scribed for 9a (HATU used in place of TBTU), treatment of

8i (293 mg, 1.04 mmol) with 6 (402 mg, 0.99 mmol) furnished

the title compound (308 mg, 47%) as an oil. MS (ES^þ) m/z 668

(M þ H)^þ.

1

pound¹⁷(160 mg, 37%) as a white solid. H NMR (400 MHz,

CDCl₃) δ 7.30-7.23 (m, 1H), 7.16-7.09 (m, 2H), 6.36 (d, J =

16.2 Hz, 1H), 5.82 (dt, J = 16.2, 5.7 Hz, 1H), 5.32-5.27 (br s,

1H), 4.83-4.65 (m, 4H), 4.59 (d, J = 14.6 Hz, 1H), 4.11-4.06

(br s, 1H), 3.96-3.81 (m, 2H), 3.78 (s, 3H), 2.93 (dd, J = 15.1,

8.3 Hz, 1H), 2.87-2.78 (m, 1H), 2.58-2.09 (m, 4H), 1.86-0.97

(m, 12H), 1.47 (d, J = 6.3 Hz, 3H); MS (ES^þ) m/z 498 (M þ H)^þ.

The later fractions (t_R=15.12 min) were freeze-dried to give

(5R,7S,10S,12S,17E)-10-isopropyl-7-(methoxycarbonyl)-12-

methyl-3,9-dioxo-6,7,9,10,11,12,13,14,15,16-decahydro-1H,

5H-2,22:5,8-dimethano-4,2,8,11-benzoxatriazacycloicosin-

11-ium trifluoroacetate ((S)-10c) (155 mg, 36%) as a white pow-

der. ¹H NMR (400 MHz, CDCl₃) δ 7.30-7.22 (m, 2H), 7.15 (d,

J = 7.1 Hz, 1H), 6.22 (d, J = 16.4 Hz, 1H), 5.95 (dt, J = 16.4, 6.3

Hz, 1H), 5.62-5.57 (br s, 1H), 4.82-4.70 (m, 3H), 4.64 (d, J = 14.7

Hz, 1H), 4.49 (d, J = 14.7 Hz, 1H), 4.11-4.07 (br m, 1H), 3.92-

3.79 (m, 2H), 3.79 (s, 3H), 2.88-2.80 (m, 1H), 2.66 (dd, J = 14.2,

7.8 Hz, 1H), 2.60-2.51 (m, 1H), 2.38-2.13 (m, 3H), 1.88-1.73 (m,

2H), 160-0.97 (m, 4H), 1.40 (d, J = 6.6 Hz, 3H), 1.21 (d, J=7.0

Hz, 3H), 1.10 (d, J = 6.6 Hz, 3H); MS (ES^þ) m/z 498 (M þ H)^þ.

Methyl (5R,7S,10S,12R,17E)-10-Cyclopentyl-12-methyl-3,9-

dioxo-6,7,9,10,11,12,13,14,15,16-decahydro-1H,5H-2,22:5,8-di-

Methyl (4R)-1-[(2S)-2-Cyclopentyl-2-(hept-6-en-1-ylamino)-

acetyl]-4-[(7-ethenylisoquinolin-1-yl)oxy]-^L-prolinate (9j). Follow-

ing the general procedure described for 9a (HATU used in place

of TBTU), treatment of 8j (263 mg, 1.10 mmol) with 5 (345 mg,

1.15 mmol) furnished the title compound (466 mg, 82%) as an oil.

¹H NMR (400 MHz, CDCl₃) δ 7.97 (br s, 1H), 7.94 (d, J = 5.9 Hz,

1H), 7.81 (br d, J = 8.5 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.22 (d,

J = 5.9 Hz, 1H), 6.83 (dd, J = 17.4, 10.9 Hz, 1H), 5.89-5.85 (m,

1H), 5.89 (d, J = 17.4 Hz, 1H), 5.74-5.63 (m, 1H), 5.38 (dd, J =

10.9, 1.2 Hz, 1H), 4.94-4.85 (m, 3H), 4.17 (d, J = 11.8 Hz, 1H),

4.00 (br d, J = 11.8 Hz, 1H), 3.79 (s, 3H), 3.12 (d, J = 8.5 Hz, 1H),

2.79-2.71 (m, 1H), 2.39-2.30 (m, 2H), 2.09-1.98 (m, 3H),

1.88-1.71 (m, 4H), 1.59-1.24 (m, 5H), 1.19-1.07 (m, 4H),

1.01-0.81 (m, 2H); MS (ES^þ) m/z 520 (M þ H)^þ.

Methyl (4R)-1-[(2S)-2-Cyclohexyl-2-(hept-6-en-1-ylamino)-

acetyl]-4-[(6-methoxy-7-vinylisoquinolin-1-yl)oxy]-^L-prolinate

(9k). Following the general procedure described for 9a (HATU

used in place of TBTU), treatment of 8h (222 mg, 0.88 mmol)

with 7 (320 mg, 0.88 mmol) furnished the title compound

(280 mg, 57%) as an oil. MS (ES^þ) m/z 564 (M þ H)^þ.

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4831

methano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-carboxylate

trifluoroacetate ((R)-10d). Following the procedure described for

10c, treatment of 9d (300 mg, 0.54 mmol) and TFA (54 μL, 0.71

mmol) gave a residue that was purified by RP-HPLC (condi-

tions B) to afford in the first fractions (t_R=14.81 min) the title

compound¹⁷(93.2 mg, 27%) as a white powder. MS (ES^þ) m/z

524 (M þ H)^þ. The later fractions (t_R=15.37 min) were freeze-

dried to give methyl (5R,7S,10S,12S,17E)-10-cyclopentyl-12-

methyl-3,9-dioxo-6,7,9,10,11,12,13,14,15,16-decahydro-1H,

5H-2,22:5,8-dimethano-4,2,8,11-benzoxatriazacycloicosine-

7(3H)-carboxylate trifluoroacetate ((S)-10d) as a white pow-

der (101.5 mg, 29%). MS (ES^þ) m/z 524 (MþH)^þ.

cin-8-ium trifluoroacetate (251 mg, 65% from 9h) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 13.30-12.60 (br s, 1H), 9.00-

8.73 (br m, 2H), 8.28-8.18 (m, 2H), 7.99 (s, 1H), 7.70-7.48 (m,

5H), 6.72 (d, J = 15.7 Hz, 1H), 6.14-6.03 (m, 1H), 5.75 (br s, 1H),

4.74-4.64 (m, 1H), 4.60-4.50 (m, 2H), 3.99 (s, 3H), 3.95-3.87 (m,

1H), 3.29-3.16 (m, 1H), 2.89-2.77 (m, 1H), 2.74-2.60 (m, 1H),

2.46-2.29 (m, 2H), 2.22-2.11 (m, 1H), 2.05-1.05 (m, 17H); MS

(ES^þ) m/z 598 (M þ H)^þ.

(2R,4S,7S,14E)-7-Cyclohexyl-23-methoxy-4-(methoxycarbo-

nyl)-12,12-dimethyl-6-oxo-20-phenyl-3,4,6,7,8,9,10,11,12,13-

decahydro-2H-16,18-etheno-2,5-methanopyrido[3,4-r][1,5,8]oxa-

diazacyclononadecin-8-ium Trifluoroacetate (10i). Treatment of

9i (307 mg, 0.46 mmol) and TFA (48 μL, 0.62 mmol) as

described in the general procedure for 10c afforded a residue

containing the title compound. MS (ES^þ) m/z 640 (MþH)^þ.

(5R,7S,10S,12R)-10-Cyclohexyl-7-(methoxycarbonyl)-12-

methyl-3,9-dioxo-6,7,9,10,11,12,13,14,15,16-decahydro-1H,5H-

2,22:5,8-dimethano-4,2,8,11-benzoxatriazacycloicosin-11-ium tri-

fluoroacetate ((R)-10e). Following the procedure described for

10c, treatment of 9e (1.39 g, 2.05 mmol) gave a residue that was

purified by RP- HPLC (conditions B) to afford in the first

fractions the title compound¹⁷(410 mg, 31%) as a white solid.

¹H NMR (DMSO-d₆) δ 8.60 (br s, 2H), 7.35-7.15 (m, 3H), 6.27

(d, J=16.4 Hz, 1H), 6.07 (dt, J = 16.4, 5.7 Hz, 1H), 5.42 (br s,

1H), 4.89-4.54 (m, 4H), 4.46 (d, J = 14.8 Hz, 1H), 4.40-4.31

(m, 1H), 4.26 (d, J = 12.2 Hz, 1H), 3.76-3.60 (m, 4H), 2.95-

2.77 (m, 1H), 2.47-2.40 (m, 1H, partially obscured by residual

DMSO signal), 2.30-2.03 (m, 3H), 2.01-1.71 (m, 6H), 1.70-

0.97 (m, 14H); MS (ES^þ) m/z 538 (M þ H)^þ. The later fractions

were freeze-dried to afford (5R,7S,10S,12S)-10-cyclohexyl-

7-(methoxycarbonyl)-12-methyl-3,9-dioxo-6,7,9,10,11,12,13,14,

15,16-decahydro-1H,5H-2,22:5,8-dimethano-4,2,8,11-ben-

zoxatriazacycloicosin-11-ium trifluoroacetate (S)-10e (190 mg,

Treatment of this material with LiOH H₂O according to step 2

3

of the general procedure described for 12a (vide infra) followed

by evaporation of the volatiles and trituration of the resulting

residue with MeCN afforded lithium (2R,4S,7S,14E)-7-cyclo-

hexyl-23-methoxy-12,12-dimethyl-6-oxo-20-phenyl-3,4,6,7,8,9,

10,11,12,13-decahydro-2H-16,18-etheno-2,5-methanopyrido-

[3,4-r][1,5,8]oxadiazacyclononadecine-4-carboxylate (233 mg,

80% from 9i) as a white solid. ¹H NMR (400 MHz, CD₃OD þ

TFA) δ 8.26 (s, 1H), 8.10-8.04 (m, 2H), 7.79-7.70 (m, 3H), 7.64

(s, 1H), 7.54 (s, 1H), 6.82 (d, J = 15.8 Hz, 1H), 6.43 (dt, J = 15.8,

7.9 Hz, 1H), 5.88 (br s, 1H), 4.75 (t, J = 8.8, 1H), 4.66 (d,

J = 12.4 Hz, 1H), 4.48 (d, J = 4.8 Hz, 1H), 4.11 (s, 3H), 4.11-

4.05 (m, 1H), 3.20 (td, J = 11.7, 5.5 Hz, 1H), 2.99-2.90 (m, 2H),

2.57-2.48 (m, 1H), 2.28-2.22 (m, 2H), 2.11-1.70 (m, 8H),

1.47-1.21 (m, 7H), 1.00 (s, 3H), 0.99 (s, 3H); MS (ES^þ) m/z 626

(MþH)^þ.

1

14%) as a white solid. H NMR (DMSO-d₆) δ 8.46 (br s, 2H),

7.33-7.15 (m, 3H), 6.35 (d, J = 16.2 Hz, 1H,), 6.04 (dt, J = 16.2,

5.8 Hz, 1H), 5.19 (br s, 1H), 4.80 (d, J=15.5 Hz, 1H), 4.73-4.50 (m,

3H, partially obscured by residual water), 4.49-4.39 (m, 1H), 4.34

(d, J= 11.7 Hz, 1H), 3.74 (dd, J= 12.4, 4.0 Hz, 1H), 3.69-3.60 (m,

3H), 2.87 (bs, 1H), 2.74-2.58 (m, 1H), 2.42-2.02 (m, 3H), 2.01-

1.46 (m, 7H), 1.45-0.94 (m, 14H). MS (ES^þ) m/z 538 (M þ H)^þ.

Methyl (5R,7S,10S,17E)-3,9-Dioxo-10-(propan-2-yl)-6,7,9,10,

11,12,13,14,15,16-decahydro-1H,5H-2,22:5,8-dimethano-4,2,8,

11-benzoxatriazacycloicosine-7(3H)-carboxylate trifluoroacetate

(10f). Treatment of 9f (2.24 g, 3.58 mmol) as described in the

general procedure for 10a furnished a residue that was crystal-

lized (petroleum ether/EtOAc, 8:2) to furnish the title com-

pound (1.3 g, 78%) as a pale solid. MS (ES^þ) m/z 484 (M þ H)^þ.

Methyl (5R,7S,10S,17Z)-13,13-Difluoro-10-isopropyl-3,9-di-

oxo-6,7,9,10,11,12,13,14,15,16-decahydro-1H,5H-2,22:5,8-di-

methano-4,2,8,11-benzoxatriazacycloicosine-7-carboxylate (10g).

Following the procedure described for 10c, treatment of 9g

(247 mg, 0.45 mmol) gave a residue that was purified by flash

chromatography on silica gel (EtOAc/petroleum ether, 2:8) to

furnish the title compound (154 mg, 66%) as a foam. ¹H NMR

(400 MHz, CDCl₃) δ 7.25 (t, J = 7.5 Hz, 1H), 7.19 (d, J = 7.5 Hz,

1H), 7.13 (d, J = 7.5 Hz, 1H), 6.30 (d, J = 16.3 Hz, 1H), 5.95 (dt,

J = 16.3, 6.1 Hz, 1H), 5.39 (t, J = 3.3 Hz, 1H), 4.83-4.55 (m, 5H),

3.94 (d, J = 11.1 Hz, 1H), 3.78-3.71 (m, 1H), 3.76 (s, 3H), 3.34 (d,

J = 6.6 Hz, 1H), 3.03 (ddd, J = 26.2, 13.9, 5.5 Hz, 1H), 2.87-2.66

(m, 2H), 2.51-1.72 (m, 8 H), 1.05 (d, J = 6.4 Hz, 3H), 1.03 (d, J =

6.4 Hz, 3H); MS (ES^þ) m/z 520 (M þ H)^þ.

(2R,4S,7S,14E)-7-Cyclopentyl-4-(methoxycarbonyl)-6-oxo-3,

4,6,7,8,9,10,11,12,13-decahydro-2H-16,18-etheno-2,5-methano-

pyrido[3,2-r][1,5,8]oxadiazacyclononadecin-8-ium Trifluoroace-

tate (10j). Treatment of 9j (350 mg, 0.673 mmol) and TFA

(70 μL, 0.91 mmol) as described in the general procedure for 10c

afforded a residue containing the title compound that was used

in the following step without further purification. MS (ES^þ) m/z

492 (M þ H)^þ.

(2R,4S,7S,14E)-7-Cyclohexyl-23-methoxy-4-(methoxycarbo-

nyl)-6-oxo-3,4,6,7,8,9,10,11,12,13-decahydro-2H-16,18-etheno-

2,5-methanopyrido[3,2-r][1,5,8]oxadiazacyclononadecin-8-ium

Trifluoroacetate (10k). Treatment of 9k (280 mg, 050 mmol) and

TFA (35 μL, 0.50 mmol) as described in the general procedure

for 10a afforded a residue containing the title compound that

was purified by RP-HPLC (conditions E) to give the title

compound (35 mg, 10%) as a white solid. MS (ES^þ) m/z 536

(MþH)^þ.

Methyl (5R,7S,10R,17E)-3,9-Dioxo-10-(trifluoromethyl)-6,7,

9,10,11,12,13,14,15,16-decahydro-1H,5H-2,22:5,8-dimethano-4,2,

8,11-benzoxatriazacycloicosine-7(3H)-carboxylate (10l). Follow-

ing the procedure described for 10c, treatment of 9l (150 mg,

0.28 mmol) gave a residue that was purified by RP-HPLC

(conditions B). The fraction with t_R= 1.72 min was freeze-dried

to furnish the title compound (17 mg, 12%) as a white solid. ¹H

NMR (500 MHz, DMSO-d₆) δ 7.30 (d, J = 7.6 Hz, 1H), 7.24 (t,

J = 7.6 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 6.24 (d, J = 16.4 Hz,

1H), 6.14 (ddd, J = 16.4, 5.6, 5.6 Hz, 1H), 5.80-5.70 (m, 1H),

5.36 (br t, J = 3.4 Hz, 1H), 4.85-4.72 (m 2H), 4.63 (s, 2H), 4.61

(d, J = 16.0 Hz, 1H), 4.38 (d, J = 12.4 Hz, 1H), 3.68 (dd, J =

12.4, 3.4 Hz, 1H), 3.65 (s, 3H), 3.10 (ddd, J = 11.9, 11.9, 5.2 Hz,

1H), 2.87 (ddd, J = 11.9, 11.9, 5.0 Hz, 1H), 2.56 (dd, J = 13.9, 8.5

Hz, 1H), 2.27-2.17 (m, 3H), 1.85-1.65 (m, 2H), 1.60-1.45 (m,

2H), 1.40-1.25 (m, 2H). MS (ES^þ) m/z 510 (MþH)^þ.

(2R,4S,7S,14E)-7-Cyclohexyl-23-methoxy-4-(methoxycarbonyl)-

6-oxo-20-phenyl-3,4,6,7,8,9,10,11,12,13-decahydro-2H-16,18-

etheno-2,5-methanopyrido[3,4-r][1,5,8]oxadiazacyclononadecin-

8-ium Trifluoroacetate (10h). Treatment of 9h (350 mg, 0.55 mmol)

and TFA (57 μL, 0.74 mmol) as described in the general procedure

for 10c afforded a residue containing the title compound. MS (ES^þ)

m/z 612 (M þ H)^þ. Treatment of this material according to step 2 of

the general procedure described for 12a (vide infra) followed by

RP-HPLC purification (conditions B) gave the corresponding

carboxylic acid (2R,4S,7S,14E)-4-carboxy-7-cyclohexyl-23-meth-

oxy-6-oxo-20-phenyl-3,4,6,7,8,9,10,11,12,13-decahydro-2H-16,

18-etheno-2,5-methanopyrido[3,4-r][1,5,8]oxadiazacyclononade-

(5R,7S,10R,12R)-N-{(1R,2S)-1-[(Cyclopropylsulfonyl)carba-

moyl]-2-ethenylcyclopropyl}-3,9-dioxo-10-(propan-2-yl)-12-(tri-

fluoromethyl)-6,7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,

5H-2,22:5,8-dimethano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-

carboxamide (12a). Step 1: General Procedure for Catalytic Hy-

drogenation. Methyl (5R,7S,10S,12S)-10-Isopropyl-3,9-dioxo-

4832 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Harper et al.

12-(trifluoromethyl)-6,7,9,10,11,12,13,14,15,16,17,18-dodecahy-

dro-1H,5H-2,22:5,8-dimethano-4,2,8,11-benzoxatriazacycloico-

sine-7-carboxylate (sat-10a). A solution of 10a (120 mg,

0.22 mmol) in MeOH (20 mL) was treated with Pd/C (10%,

20 mg). The mixture was evacuated and then stirred under an

atmosphere of hydrogen for 6 h. The solution was filtered and

the filtrate was concentrated in vacuo to give a residue that

was purified by flash chromatography on silica gel (EtOAc/

petroleum ether, gradient from 1:9 to 3:7) to afford the title

compound (106 mg, 62%) as a white foam. MS (ES^þ) m/z 554

(M þ H)^þ.

11.9 Hz, 1H), 3.75 (dd, J = 11.9, 3.6 Hz, 1H), 3.70-3.65 (m, 1H),

3.12 (d, J = 8.1 Hz, 1H), 2.95-2.85 (m, 1H), 2.80 (br s, 1H), 2.59

(dd, J = 12.9, 6.4 Hz, 1H), 2.40-2.35 (m, 1H), 2.26 (dd, J = 13.4,

6.7 Hz, 1H), 2.19 (q, J = 8.8 Hz, 1H), 2.09 (ddd, J = 13.4, 10.9, 3.6

Hz, 1H), 1.89-1.80 (m, 1H), 1.73 (dd, J = 7.9, 5.1 Hz, 1H), 1.63-

1.50 (m, 2H), 1.49-1.33 (m, 6H), 1.31-1.22 (m, 2H), 1.15-0.97

(m, 5H), 0.85 (d, J = 6.7 Hz, 3H), 0.83 (d, J = 6.7 Hz, 3H); ¹⁹

F

NMR (376 MHz, DMSO-d₆) δ -75.2. Analytical RP-HPLC t_R

(conditions C) 5.81 min (peak area 98%), t_R(conditions D) 4.45

min (peak area 99%); HRMS calculated for C₃₆H₄₉F₃N₅O₇S

(MþH)^þ752.3305, found 752.3306.

Step 2: General Procedure for Proline Ester Hydrolysis.

(5R,7S,10S,12S)-10-Isopropyl-3,9-dioxo-12-(trifluoromethyl)-6,

7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,5H-2,22:5,8-di-

methano-4,2,8,11-benzoxatriazacycloicosine-7-carboxylic Acid.

A solution of the preceding compound (105 mg, 0.19 mmol) in

a 2:1 (v/v) mixture of THF and H₂O (6 mL) was treated with

(5R,7S,10R,12R)-N-{(1R,2S)-1-[(Cyclopropylsulfonyl)carba-

moyl]-2-ethenylcyclopropyl}-12-methyl-3,9-dioxo-10-(propan-2-

yl)-6,7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,5H-2,22:5,

8-dimethano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-carboxa-

mide Trifluoroacetate (13a). Treatment of (R)-10c (192 mg,

0.31 mmol) as described in the general procedure reported for

12a gave the title compound (22%) as a white powder. ¹H NMR

(600 MHz, DMSO-d₆) δ 10.90 (br s, 1H), 8.73 (br s, 1H), 8.50-

8.25 (m, 2H), 7.26 (t, J = 7.4 Hz, 1H), 7.18 (d, J = 7.4 Hz, 1H),

7.14 (d, J = 7.4 Hz, 1H), 5.56 (ddd, J = 17.1, 10.2, 9.2 Hz, 1H),

5.41 (br s, 1H), 5.22 (d, J = 17.1 Hz, 1H), 5.11 (d, J = 10.2 Hz,

1H), 4.67 (m, 2H), 4.62-4.49 (m, 3H), 4.40-4.34 (m, 1H), 4.18 (d,

J = 12.2, 1H), 3.74 (dd, J = 12.2, 2.5 Hz, 1H), 2.94-2.82 (m, 2H),

2.52-2.43 (m, 2H, obscured by residual DMSO signal), 2.42-2.34

(m, 1H), 2.23 (m, 1H), 2.15 (q, J = 8.7 Hz, 1H), 2.06 (m, 1H), 1.74

(dd, J = 7.6, 5.5 Hz, 1H), 1.71 (m, 1H), 1.63-1.44 (m, 3H), 1.41-

0.97 (m, 20H). Analytical RP-HPLC t_R(conditions C) 5.96 min

(peak area 97%), t_R(conditions D) 4.31 min (peak area 98%);

HRMS calculated for C₃₆H₅₂N₅O₇S (M þ H)^þ698.3587, found

698.3579.

LiOH H₂O (24 mg, 0.57 mmol). The mixture was stirred at

3

20 °C for 14 h and then cooled and acidified to pH 6 by addition

of aqueous HCl (1 N). The mixture was diluted with EtOAc, and

the organic layer was separated and dried. Removal of the

solvent gave a residue that was used without further purification

in the subsequent step. MS (ES^þ) m/z 540 (M þ H)^þ.

Step 3: General Procedure for Amide Couplings with 11.

(5R,7S,10R,12R)-N-{(1R,2S)-1-[(Cyclopropylsulfonyl)carbamoyl]-

2-ethenylcyclopropyl}-3,9-dioxo-10-(propan-2-yl)-12-(trifluoro-

methyl)-6,7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,5H-2,

22:5,8-dimethano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-car-

boxamide (12a). To a stirred mixture of the preceding com-

pound (89 mg, 0.165 mmol) in CH₂Cl₂(10 mL) were added

DIEA (69 μL, 0.396 mmol), DMAP (10 mg, 0.082 mmol), and

TBTU (64 mg, 0.198 mmol). The solution was stirred for 5 min

and then treated with 11¹⁸(53 mg, 0.198 mmol). The resulting

mixture was stirred at 20 °C for 14 h and then diluted with

saturated aqueous NaHCO₃and EtOAc. The organic layer was

separated, washed with brine, and dried. Removal of the

volatiles gave a residue that was purified by RP-HPLC

(conditions B) to furnish the title compound (42 mg, 34%) as

(5R,7S,10R,12S)-N-{(1R,2S)-1-[(Cyclopropylsulfonyl)carba-

moyl]-2-ethenylcyclopropyl}-12-methyl-3,9-dioxo-10-(propan-2-

yl)-6,7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,5H-2,22:5,

8-dimethano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-carbox-

amide Trifluoroacetate (13b). Treatment of (S)-10c (281 mg,

0.46 mmol) as described in the general procedure reported for

12a gave the title compound (7%) as a white powder. ¹H NMR

(600 MHz, DMSO-d₆) δ 10.89 (br s, 1H), 8.77 (br s, 1H), 8.39-

8.23 (m, 2H), 7.24 (t, J = 7.4 Hz, 1H), 7.18 (d, J = 7.4 Hz, 1H),

7.12 (d, J = 7.4 Hz, 1H), 5.58 (ddd, J = 17.2, 10.3, 9.2 Hz, 1H),

5.34 (br s, 1H), 5.24 (d, J = 17.2 Hz, 1H), 5.12 (d, J = 10.3 Hz,

1H), 4.71 (d, J = 14.6 Hz, 1H), 4.64-4.58 (m, 3H), 4.48 (t, J =

8.8 Hz, 1H), 4.38-4.36 (m, 1H), 4.28 (d, J = 10.6 Hz, 1H), 3.76

(d, J = 10.6 Hz, 1H), 3.07-2.99 (br m, 1H), 2.95-2.89 (m, 1H),

2.58-2.45 (obscured by residual DMSO, 2H), 2.42-2.36 (m,

1H), 2.33-2.25 (m, 1H), 2.17 (q, J = 8.7 Hz, 1H), 2.13-2.05 (m,

1H), 1.78-1.71 (m, 2H), 1.73-1.56 (m, 1H), 1.49-1.19 (m, 9H),

1.28 (d, J = 6.3 Hz, 3H), 1.12 (d, J = 6.7 Hz, 3H), 1.15-0.96 (m,

4H), 0.97 (d, J = 6.8 Hz, 3H). Analytical RP-HPLC t_R

(conditions C) 6.01 min (peak area 97%), t_R(conditions D)

4.33 min (peak area 96%); HRMS calculated for C₃₆H₅₂N₅O₇S

(M þ H)^þ698.3587, found 698.3580.

1

a white solid. H NMR (600 MHz, DMSO-d₆, 294 K) δ 10.58

(br s, 1H), 9.22 (br s, 1H), 7.24 (t, J = 7.4 Hz, 1H), 7.17 (d, J = 7.4

Hz, 1H), 7.11 (d, J=7.4 Hz, 1H), 5.67 (ddd, J=17.1, 10.2, 9.2 Hz,

1H), 5.42 (br t, J = 3.4 Hz, 1H), 5.28 (dd, J = 17.1, 1.7 Hz, 1H),

5.12 (dd, J = 10.2, 1.7 Hz, 1H), 4.65 (s, 2H), 4.64-4.58 (m, 2H),

4.40 (dd, J = 11.0, 6.7 Hz, 1H), 3.96 (d, J = 11.9 Hz, 1H), 3.76 (dd,

J =11.9, 3.4 Hz, 1H), 3.30 (d, J=6.5 Hz, 1H), 3.00-2.95 (m, 1H),

2.90-2.85 (m, 1H), 2.51-2.44 (m, 2H), 2.22 (dd, J = 13.4, 6.7 Hz,

1H), 2.16 (q, J = 8.8 Hz, 1H), 2.05 (ddd, J = 13.4, 11.0, 3.4 Hz,

1H), 1.97 (br s, 1H), 1.79-1.71 (m, 2H), 1.56-1.48 (m, 3H), 1.44-

1.18 (m, 7H), 1.09-0.99 (m, 4H), 0.91 (d, J = 6.7 Hz, 3H), 0.82 (d,

J = 6.7 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ -75.8. ¹³

C

NMR (150 MHz, DMSO-d₆) δ 174.1, 172.2, 169.2, 152.9, 137.1,

136.2, 135.0, 133.4, 127.9, 127.6, 126.8 (q, J = 228 Hz), 120.3,

117.9, 73.5, 64.8, 59.0, 58.9 (q, J = 26 Hz), 53.2, 52.1, 50.7, 40.8,

34.6, 33.8, 31.4, 31.2, 30.7, 28.9, 28.5, 26.4, 26.0, 23.5, 22.8, 19.1,

17.9, 5.7, 5.4. Analytical RP-HPLC t_R(conditions C) 5.84 min

(peak area 98%), t_R(conditions D) 4.42 min (peak area 98%);

HRMS calculated for C₃₆H₄₉F₃N₅O₇S (M þ H)^þ752.3305, found

752.3325.

(5R,7S,10S,12R)-N-{(1R,2S)-1-[(Cyclopropylsulfonyl)carba-

moyl]-2-ethenylcyclopropyl}-12-methyl-3,9-dioxo-10-(propan-2-

yl)-6,7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,5H-2,22:5,

8-dimethano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-carboxa-

mide Trifluoroacetate (14). Treatment of 10f (180 mg, 0.35 mmol)

as described in the general procedure reported for 12a gave the title

compound (23%) as a white powder. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.74 (s, 1H), 8.74 (s, 1H), 8.70-8.56 (m, 2H), 7.25

(t, J = 7.4 Hz, 1H), 7.17 (d, J = 7.4 Hz, 1H), 7.13 (d, J = 7.4 Hz,

1H), 5.56 (ddd, J = 17.1, 10.3, 9.3 Hz, 1H), 5.39 (br s, 1H), 5.22 (d,

J = 17.1 Hz, 1H), 5.11 (d, J = 10.3 Hz, 1H), 4.78-4.48 (m, 5H),

4.27 (br s, 1H), 3.74 (dd, J = 11.6, 2.5 Hz, 1H), 2.95-2.89 (m, 2H),

2.69-2.66 (m, 1H), 2.38-2.32 (m, 1H, partially obscured by

residual DMSO signal), 2.21-2.06 (m, 3H), 1.73 (dd, J = 9.7,

6.9 Hz, 2H), 1.69-1.55 (m, 4H), 1.32-1.26 (m, 1H), 4.49-4.62 (m,

7H), 1.15-1.09 (m, 1H), 1.04 (dd, J = 9.7, 6.9 Hz, 9H). Analytical

RP-HPLC t_R(conditions C) 5.86 min (peak area 99%), t_R

(5R,7S,10S,12R)-N-((1R,2S)-1-{[(Cyclopropylsulfonyl)mino]-

carbonyl}-2-vinylcyclopropyl)-10-isopropyl-3,9-dioxo-12-(trifluoro-

methyl)-6,7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,5H-2,

22:5,8-dimethano-4,2,8,11-benzoxatriazacycloicosine-7-carbox-

amide (12b). Treatment of 10b (88 mg, 0.160 mmol) as described in

the general procedures for 12a furnished the title compound (12

mg, 10%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆, 294 K)

δ 10.73 (br s, 1H), 9.32 (br s, 1H), 7.25 (t, J = 7.4 Hz, 1H), 7.18 (d,

J = 7.4 Hz, 1H), 7.12 (d, J = 7.4 Hz, 1H), 5.71 (ddd, J = 17.1,

10.2, 9.2 Hz, 1H), 5.40 (br t, J=3.6 Hz, 1H), 5.30 (dd, J=17.1, 1.7

Hz, 1H), 5.13 (dd, J = 10.2, 1.7 Hz, 1H), 4.73 (d, J = 14.2 Hz, 1H),

4.67-4.55 (m, 3H), 4.31 (dd, J = 10.9, 6.7 Hz, 1H), 4.05 (d, J =

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4833

(conditions D) 4.24 min (peak area 99%); HRMS calculated for

C₃₅H₅₀N₅O₇S (M þ H)^þ684.3431, found 698.3431.

6.01 (dt, J = 16.2, 6.1 Hz, 1H), 5.58 (ddd, J = 17.2, 10.4, 9.1 Hz,

1H), 5.22(d, J= 17.2 Hz, 1H), 5.22-5.16 (m, 1H), 5.12 (d, J= 10.4

Hz, 1H), 4.83 (d, 14.7 Hz, 1H), 4.71 (d, 14.7, 1H), 4.65-4.53 (m,

3H), 4.41 (br t, J = 9.0 Hz, 1H), 4.29 (br d, J = 12.3 Hz, 1H), 3.76

(dd, J = 12.3, 3.4 Hz, 1H), 2.96-2.83 (m, 2H), 2.72-2.64 (m, 1H),

2.46-0.98 (m, 25H), 1.28 (d, J= 6.3 Hz, 3H). Analytical RP-HPLC

t_R(conditions C) 5.97 min (peak area 99%), t_R(conditions D)

4.41 min (peak area 99%); HRMS calculated for C₃₈H₅₂N₅O₇S

(MþH)^þ722.3587, found 722.3593.

(5R,7S,10S)-N-((1R,2S)-1-{[(Cyclopropylsulfonyl)amino]carbo-

nyl}-2-vinylcyclopropyl)-13,13-difluoro-10-isopropyl-3,9-dioxo-6,

7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,5H-2,22:5,8-di-

methano-4,2,8,11-benzoxatriazacycloicosine-7-carboxamide (15).

Treatment of 10g (22 mg, 0.158 mmol) according to the general

procedure described for 12a gave the title compound (15%) as a

white powder. ¹H NMR (400 MHz, DMSO-d₆) δ 10.66 (s, 1H),

8.97 (br s, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.17 (d, J = 7.5 Hz, 1H),

7.13 (d, J = 7.5 Hz, 1H), 5.61 (ddd, J = 17.2, 10.4, 9.3 Hz, 1H),

5.38 (br s, 1H), 5.25 (d, J = 17.2 Hz, 1H), 5.11 (d, J = 10.4 Hz,

1H), 4.73-4.54 (m, 4H), 4.45 (dd, J = 10.1, 7.3 Hz, 1H), 4.04 (d,

J = 12.1 Hz, 1H), 3.75 (dd, J = 12.0, 3.2 Hz, 1H), 3.50-3.02 (m,

3H), 2.96-2.85 (m, 1H), 2.45-2.34 (m, 3H, partially obscured by

residual DMSO), 2.24-1.67 (m, 6H), 1.66-1.48 (m, 2H), 1.49-

1.20 (m, 5H), 1.14-0.91 (m, 10H). Analytical RP-HPLC t_R

(conditions C) 8.03 min (peak area >99%), t_R(conditions D)

5.60 min (peak area 98%); HRMS calculated for C₃₅H₄₈F₂N₅O₇S

(MþH)^þ720.3243, found 720.3237.

(5R,7S,10S,12R)-10-Cyclopentyl-N-{(1R,2S)-1-[(cyclopropyl-

sulfonyl)carbamoyl]-2-ethenylcyclopropyl}-12-methyl-3,9-dioxo-

6,7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,5H-2,22:5,8-

dimethano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-carboxamide

Trifluoroacetate (17c). Treatment of (R)-10d (93.2 mg, 0.146

mmol) as described in the general procedure reported for 12a gave

the title compound (26 mg, 46%) as a white powder. ¹H NMR (400

MHz, DMSO-d₆) δ 10.76 (br s, 1H), 8.72 (br s, 1H), 8.63-8.34 (m,

2H), 7.26 (t, J = 7.5 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 7.14 (d, J =

7.5 Hz, 1H), 5.57 (ddd, J = 17.2, 10.3, 9.1 Hz, 1H), 5.42-5.38 (br s,

1H), 5.22 (d, J = 17.2 Hz, 1H), 5.11 (d, J = 10.3 Hz, 1H), 4.72-

4.41 (m, 6H), 4.18 (br d, J = 11.0 Hz, 1H), 3.75 (br d, J = 11.0 Hz,

1H), 2.96-2.79 (m, 2H), 2.54-2.02 (m, 6H, partially obscured by

residual DMSO signal), 1.93-0.99 (m, 24H), 1.23 (d, J = 6.4 Hz,

3H). Analytical RP-HPLC t_R(conditions C) 6.13 min (peak area

92%), t_R(conditions D) 4.34 min (peak area 93%); HRMS

calculated for C₃₈H₅₄N₅O₇S (MþH)^þ724.3744, found 724.3748.

(5R,7S, 10S, 12R)-10-Cyclohexyl-N-{(1R,2S)-1-[(cyclo-

propylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}-12-methyl-3,

9-dioxo-6,7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,5H-2,

22:5,8-dimethano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-carbox-

amide Trifluoroacetate (18a). Treatment of (R)-10e (170 mg,

0.26 mmol) as described in the general procedure reported for

12a gave the title compound (20%) as a white powder. ¹H NMR

(400 MHz, DMSO-d₆) δ 10.85 (br s, 1H), 8.73 (br s, 1H), 8.43 (br

s, 1H), 8.34-8.20 (m, 1H), 7.26 (t, J = 7.5 Hz, 1H), 7.18 (d, J =

7.5 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 5.58 (ddd, J = 17.3, 10.4,

9.1 Hz, 1H), 5.41 (br s, 1H), 5.21 (d, J = 17.3 Hz, 1H), 5.11 (d,

J = 10.4 Hz, 1H), 4.72-4.45 (m, 5H), 4.48-4.39 (m, 1H), 4.17

(d, J = 12.1 Hz, 1H), 3.76 (dd, J = 12.9, 2.3 Hz, 1H), 2.97-2.78

(m, 2H), 2.47-2.34 (partially obscured by residual DMSO, 2H),

2.19-2.03 (m, 2H), 1.99-1.42 (m, 11H), 1.41-0.93 (m, 20H).

Analytical RP-HPLC t_R(conditions C) 6.39 min (peak area

96%), t_R(conditions D) 4.51 min (peak area 97%); HR-

MS calculated for C₃₉H₅₆N₅O₇S (M þ H)^þ738.3900, found

738.3925.

(5R,7S,10S,17E)-N-{(1R,2S)-1-[(Cyclopropylsulfonyl)carba-

moyl]-2-ethenylcyclopropyl}-3,9-dioxo-10-(trifluoromethyl)-6,7,

9,10,11,12,13,14,15,16-decahydro-1H,5H-2,22:5,8-dimethano-4,

2,8,11-benzoxatriazacycloicosine-7(3H)-carboxamide (16). Treat-

ment of 10l (17 mg, 0.034 mmol) as described in the general

procedure reported for 12a gave the title compound (35%) as a

white powder. ¹H NMR (600 MHz, DMSO-d₆þ TFA, 294 K) δ

10.98 (bs, 1H), 8.55 (bs, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.27 (t, J =

7.6 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 6.24 (d, J = 16.4 Hz, 1H),

6.14 (ddd, J = 16.4, 5.5, 5.5 Hz, 1H), 5.61 (m, 1H), 5.51 (ddd, J =

17.1, 10.2, 9.0 Hz, 1H), 5.33 (br t, J = 3.3 Hz, 1H), 5.22 (dd, J =

17.1, 1.5 Hz, 1H), 5.11 (dd, J = 10.2, 1.5 Hz, 1H), 4.71-4.60 (m,

4H), 4.57 (dd, J = 9.7, 8.2Hz, 1H), 4.36 (d, J = 12.2 Hz, 1H), 3.70

(dd, J = 12.2, 3.3 Hz, 1H), 3.07 (ddd, J = 12.0, 12.0, 4.7 Hz, 1H),

2.96-2.85 (m, 1H), 2.77 (ddd, J = 12.0, 12.0, 5.4 Hz, 1H), 2.56

(dd, J = 14.3, 8.2 Hz, 1H), 2.30-2.15 (m, 2H), 2.14-2.07 (m,

2H), 1.80-1.65 (m, 3H), 1.60-1.45 (m, 2H), 1.42-1.27 (m, 2H),

1.24 (dd, J = 9.3, 5.2 Hz, 1H), 1.10-0.95 (m, 4H); ¹⁹F NMR (376

MHz, DMSO-d₆) δ -73.0. Analytical RP-HPLC t_R(conditions

C) 6.28 min (peak area 99%), t_R(conditions D) 4.53 min (peak

area 99%); HRMS calculated for C₃H₄₁F₃N₅O₇S (M þ H)^þ

708.2679, found 708.2702.

(5R,7S,10S,12R,17E)-10-Cyclopentyl-N-{(1R,2S)-1-[(cyclo-

propylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}-12-methyl-3,9-

dioxo-6,7,9,10,11,12,13,14,15,16-decahydro-1H,5H-2,22:5,8-di-

methano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-carboxamide

Trifluoroacetate (17a). Treatment of (R)-10d (42 mg, 0.068 mmol)

as described in the general procedure reported for 12a (steps 2 and

3) gave the title compound (37.5 mg, 59%) as a white powder. ¹H

NMR (400 MHz, DMSO-d₆) δ 10.72 (br s, 1H), 8.78-8.68 (m,

2H), 8.64 (br s, 1H), 8.57-8.47 (m, 2H), 7.32-7.22 (m, 3H), 6.24

(d, J = 16.3 Hz, 1H), 6.07 (dt, J = 16.3, 5.7 Hz, 1H), 5.56 (ddd,

J = 17.1, 10.3, 9.5 Hz, 1H), 5.42-5.38 (m, 1H), 5.21 (d, J = 17.1

Hz, 1H), 5.11 (d, J = 10.3 Hz, 1H), 4.72-4.54 (m, 4H), 4.51 (dd, J

= 10.0, 7.7 Hz, 1H), 4.41 (br t, J = 8.3 Hz, 1H), 4.23 (br d, J =

10.7 Hz, 1H), 3.73 (br d, J = 10.7 Hz, 1H), 2.95-2.82 (m, 2H),

2.54-2.01 (m, 6H, partially obscured by residual DMSO signal),

1.96-1.28 (m, 16H), 1.27 (d, J = 6.6 Hz, 3H), 1.13-0.99 (m, 4H).

Analytical RP-HPLC t_R(conditions C) 6.03 min (peak area 97%),

t_R(conditions D) 4.31 min (peak area 98%); HRMS calculated for

C₃₈H₅₂N₅O₇S (M þ H)^þ722.3587, found 722.3599.

(5R,7S,10S,12S)-10-Cyclohexyl-N-{(1R,2S)-1-[(cyclopropylsul-

fonyl)carbamoyl]-2-ethenylcyclopropyl}-12-methyl-3,9-dioxo-6,

7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,5H-2,22:5,8-di-

methano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-carboxamide

Trifluoroacetate (18b). Treatment of (S)-10e (180 mg, 0.29 mmol)

as described in the general procedure reported for 12a gave the

title compound(37%) as a white powder. ¹H NMR(DMSO-d₆) δ

10.70 (s, 1H), 8.85-8.73 (m, 1H), 8.41 (br s, 1H), 8.15 (br s, 1H),

7.24 (t, J = 7.4 Hz, 1H), 7.17 (d, J = 7.4 Hz, 1H), 7.12 (d, J = 7.4

Hz, 1H), 5.65-5.55 (m, 1H), 5.36 (br s, 1H), 5.23 (d, J = 17.1 Hz,

1H), 5.12 (d, J = 10.3 Hz, 1H), 4.76-4.53 (m, 4H), 4.48 (t, J =

9.3 Hz, 1H), 4.36-4.21 (m, 2H), 3.81 (d, J = 9.5 Hz, 1H), 3.05 (br

s, 1H), 2.91 (m, 1H), 2.60-2.53 (m, 1H, partially obscured by

residual DMSO), 2.46-2.38 (m, 1H), 2.22-2.10 (m, 2H), 2.00-

1.87 (m, 1H) 1.86-1.71 (m, 5H), 1.70-1.54 (m, 3H), 1.53-1.20

(m, 15H) 1.19-0.98 (m, 6H). Analytical RP-HPLC t_R

(conditions C) 6.39 min (peak area 94%), t_R(conditions D)

4.52 min (peak area 96%); HRMS calculated for C₃₉H₅₆N₅O₇S

(MþH)^þ738.3900, found 738.3915.

(5R,7S,10S,12R,17E)-10-Cyclopentyl-N-{(1R,2S)-1-[(cyclo-

propylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}-12-methyl-3,9-

dioxo-6,7,9,10,11,12,13,14,15,16-decahydro-1H,5H-2,22:5,8-di-

methano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-carboxamide

Trifluoroacetate (17b). Treatment of (S)-22d (102 mg, 0.160 mmol)

as described in the general procedure reported for 12a (steps 2 and 3)

(5R,7S,10S,12R)-10-Cyclohexyl-N-{(1R,2S)-1-[(cyclopropyl-

sulfonyl)carbamoyl]-2-ethenylcyclopropyl}-11,12-dimethyl-3,9-

dioxo-6,7,9,10,11,12,13,14,15,16,17,18-dodecahydro-1H,5H-2,

22:5,8-dimethano-4,2,8,11-benzoxatriazacycloicosine-7(3H)-car-

boxamideTrifluoroacetate(19). A solution of NaCNBH₃(5.2 mg,

0.083 mmol) and ZnCl₂(5.7 mg, 0.042 mmol) in MeOH (4 mL)

1

gave the title compound as a white powder (29.5 mg, 22%). H

NMR (400 MHz, DMSO-d₆) δ 10.86 (br s, 1H), 8.66 (br s, 1H),

8.51-8.33 (m, 2H), 7.31-7.18 (m, 3H), 6.38 (d, J = 16.2 Hz, 1H),

4834 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Harper et al.

was added dropwise to a solution of 18a (59 mg, 0.069 mmol)

and formaldehyde (37% in H₂O, 0.18 mL, 2.42 mmol) in MeOH

(2 mL). The mixture was stirred for 14 h, and then the volatiles

were evaporated under reduced pressure. The residue was pur-

ified by RP-HPLC (conditions B) to furnish the title compound

as a white powder (33mg, 55%). ¹H NMR (400 MHz, DMSO-d₆)

δ 10.88 (s, 1H), 8.73 (br s, 1H), 8.65 (br s, 1H), 7.26 (t, J = 7.5 Hz,

1H), 7.19 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 7.5 Hz, 1H), 5.60 (ddd,

J = 17.2, 10.3, 9.5 Hz, 1H), 5.37 (br s, 1H), 5.20 (d, J = 17.2 Hz,

1H), 5.11 (d, J = 10.3 Hz, 1H), 4.77-5.55 (m, 4H), 4.56-4.39 (m,

2H), 3.92 (d, J = 12.1 Hz, 1H), 3.80 (dd, J = 12.1, 2.5 Hz, 1H),

2.97-2.80 (m, 2H), 2.62 (d, J = 4.0 Hz, 3H), 2.47-2.36 (m, 3H,

partially obscured by residual DMSO signal), 2.15(q, J = 8.9 Hz,

1H), 2.10-1.93 (m, 2H), 1.92-1.41 (m, 12H), 1.40-1.81 (m,

11H), 1.17-0.94 (m, 6H). Analytical RP-HPLC t_R(conditionsC)

6.24 min (peak area 97%), t_R(conditions D) 4.46 min (peak area

98%); HRMS calculated for C₄₀H₅₈N₅O₇S (MþH)^þ752.4057,

found 752.4050.

99%), t_R(conditions D) 3.91 min (peak area 99%);

HRMS calculated for C₄₅H₅₆N₅O₇S (MþH)^þ810.3900, found

810.3911.

(2R,4S,7S)-7-Cyclohexyl-N-{(1R,2S)-1-[(cyclopropylsulfonyl)-

carbamoyl]-2-ethenylcyclopropyl}-23-methoxy-6-oxo-20-phenyl-3,

4,6,7,8,9,10,11,12,13,14,15-dodecahydro-2H-16,18-etheno-2,5-

methanopyrido[3,4-r][1,5,8]oxadiazacyclononadecine-4-carbox-

amide Trifluoroacetate (23). Treatment of 10h (120 mg, 0.166 mmol)

as described in the general procedure reported for 12a gave the title

1

compound (58 mg, 38% from 9h) as a white powder. H NMR

(400 MHz, DMSO-d₆) δ 10.84 (br s, 1H), 8.81-8.58 (m, 3H),

8.27-8.17 (m, 2H), 7.82 (s, 1H), 7.70-7.54 (m, 4H), 7.52-7.44 (m,

1H), 5.86 (br s, 1H), 5.57 (ddd, J = 17.1, 10.2, 9.1 Hz, 1H), 5.21 (d,

J = 17.1 Hz, 1H), 5.12 (d, J = 10.2 Hz, 1H), 4.61 (t, J = 8.8 Hz,

1H), 4.49 (br d, J = 12.1 Hz, 1H), 4.40-4.32 (m, 1H), 3.99 (s, 3H),

3.97-3.89 (m, 1H), 3.07-2.82 (m, 3H), 2.80-2.59 (m, 3H), 2.35-

2.24 (m, 1H), 2.14 (q, J = 8.7 Hz, 1H), 1.98-1.49 (m, 11H), 1.39-

0.97 (m, 16H). Analytical RP-HPLC t_R(conditions C) 5.49 min

(peak area 97%), t_R(conditions D) 3.92 min (peak area 99%);

HRMS calculated for C₄₅H₅₈N₅O₇S (Mþ H)^þ812.4057, found

812.4067.

(2R,4S,14E)-7-Cyclopentyl-4-({(1R,2S)-1-[(cyclopropylsul-

fonyl)carbamoyl]-2-ethenylcyclopropyl}carbamoyl)-6-oxo-3,4,6,

7,8,9,10,11,12,13-decahydro-2H-16,18-etheno-2,5-methanopyri-

do[3,2-r][1,5,8]oxadiazacyclononadecin-8-ium Trifluoroacetate (20).

Treatment of 10j (408 mg, 0.673 mmol) as described in the general

procedure for 12a (steps 2 and 3) gave the title compound (92 mg,

17%) as a white powder. ¹H NMR (400 MHz, DMSO-d₆) δ 10.72

(br s, 1H), 8.96-8.69 (m, 2H), 8.59 (br s, 1H), 8.09 (m, 1H), 7.99 (d,

J = 5.8 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.64 (br d, J = 8.4, 1.5

Hz, 1H), 7.41 (d, J = 5.8 Hz, 1H), 6.69 (d, J = 15.7 Hz, 1H), 6.02

(dt, J = 15.7, 7.3 Hz, 1H), 5.78 (br s, 1H), 5.56 (ddd, J = 17.2, 10.2,

9.6 Hz, 1H), 5.18 (d, J = 17.2 Hz, 1H), 5.09 (d, J = 10.2 Hz, 1H),

4.60-4.48 (m, 2H), 4.43 (dd, J = 10.3, 7.5 Hz, 1H), 3.85 (dd, J =

12.1, 2.3 Hz, 1H), 3.35-3.23 (m, 1H), 2.95-2.87 (m, 1H), 2.79-

2.58 (m, 2H), 2.38-2.06 (m, 5H), 1.96-1.83 (m, 2H), 1.79-1.36

(m, 12H), 1.33-1.20 (m, 2H), 1.15-0.95 (m, 4H). Analytical RP-

HPLC t_R(conditions C) 6.20 min (peak area 98%), t_R(conditions

D) 4.42 min (peak area 98%); HRMS calculated for C₃₇H₅₀N₅O₆S

(MþH)^þ690.3325, found 690.3322.

(2R,4S,7S,14E)-7-Cyclohexyl-N-{(1R,2S)-1-[(cyclopropylsul-

fonyl)carbamoyl]-2-ethenylcyclopropyl}-23-methoxy-12,12-di-

methyl-6-oxo-20-phenyl-3,4,6,7,8,9,10,11,12,13-decahydro-2H-

16,18-etheno-2,5-methanopyrido[3,4-r][1,5,8]oxadiazacyclononade-

cine-4-carboxamide Trifluoroacetate (24). Treatment of 10i

(224 mg, 0.297 mmol) as described in the general procedure

reported for 12a (steps 2 and 3) gave the title compound (104 mg,

37% from 9i) as a white powder. ¹H NMR (400 MHz, DMSO-

d₆) δ 10.80 (br s, 1H), 8.90-8.63 (m, 3H), 8.29-8.22 (m, 2H),

8.11 (s, 1H), 7.66-7.53 (m, 4H), 7.49-7.44 (m, 1H), 6.73 (d, J =

15.8 Hz, 1H), 6.28 (dt, J = 15.8, 7.8 Hz, 1H), 5.76 (br s, 1H), 5.56

(ddd, J = 17.1, 10.5, 9.1 Hz, 1H), 5.18 (d, J = 17.1 Hz, 1H), 5.08

(d, J = 10.5 Hz, 1H), 4.63 (br d, J = 11.9 Hz, 1H), 4.50-4.39 (m,

2H), 4.00 (s, 3H), 3.97-3.90 (m, 1H), 3.22-3.11 (m, 1H), 2.94-

2.86 (m, 1H), 2.80-2.67 (m, 2H), 2.27-2.06 (m, 4H), 2.00-1.61

(m, 9H), 1.32-0.96 (m, 12H), 0.92 (s, 3H), 0.91 (s, 3H).

Analytical RP-HPLC t_R(conditions C) 5.70 min (peak area

>99%), t_R(conditions D) 3.92 min (peak area >99%); HRMS

calculated for C₄₇H₆₀N₅O₇S (M þ H)^þ838.4213, found

838.4221.

(2R,4S,7S)-7-Cyclohexyl-N-{(1R,2S)-1-[(cyclopropylsulfonyl)-

carbamoyl]-2-ethenylcyclopropyl}-23-methoxy-12,12-dimethyl-6-

oxo-20-phenyl-3,4,6,7,8,9,10,11,12,13,14,15-dodecahydro-2H-16,

18-etheno-2,5-methanopyrido[3,4-r][1,5,8]oxadiazacyclononade-

cine-4-carboxamide Trifluoroacetate (25). Treatment of 10i

(699 mg, 0.93 mmol) as described in the general procedure

reported for 12a gave the title compound (301 mg, 34% from

9i) as a white powder. ¹H NMR (400 MHz, DMSO-d₆) δ 10.83

(br s, 1H), 8.79 (s, 1H), 8.65-8.55 (br s, 1H), 8.51-8.41 (br s,

1H), 8.28-8.20 (m, 2H), 7.83 (s, 1H), 7.67-7.54 (m, 5H), 7.47 (s,

1H), 5.85 (br s, 1H), 5.58 (ddd, J = 17.0, 10.3, 9.5 Hz, 1H), 5.22

(d, J = 17.0 Hz, 1H), 5.12 (d, J = 10.3 Hz, 1H), 4.63 (dd, J =

9.2, 8.2 Hz, 1H), 4.41 (d, J = 12.1 Hz, 1H), 4.38-4.31 (br m,

1H), 3.98 (s, 3H), 4.01-3.89 (m, 1H), 2.97-2.84 (m, 3H), 2.80-

2.73 (m, 1H), 2.68-2.57 (m, 2H), 2.35-2.25 (m, 1H), 2.14 (q,

J = 8.7 Hz, 1H), 2.01-1.90 (m, 1H), 1.81-0.99 (m, 22H), 0.85

(s, 3H), 0.77 (s, 3H). Analytical RP-HPLC t_R(conditions C) 5.81

min (peak area >99%), t_R(conditions D) 4.14 min (peak area

>99%); HRMS calculated for C₄₇H₆₀N₅O₇S (M þ H)^þ

840.4370, found 840.4381.

(2R,4S,7S)-7-Cyclopentyl-4-({(1R,2S)-1-[(cyclopropylsulfonyl)-

carbamoyl]-2-ethenylcyclopropyl}carbamoyl)-6-oxo-3,4,6,7,8,9,10,

11,12,13,14,15-dodecahydro-2H-16,18-etheno-2,5-methanopyrido-

[3,2-r][1,5,8]oxadiazacyclononadecin-8-ium Trifluoroacetate (21).

Treatment of 10j (408 mg, 0.67 mmol) as described in the general

procedure reported for 12a gave the title compound (140 mg,

26%) as a white powder. ¹H NMR (400 MHz, DMSO-d₆) δ 10.66

(br s, 1H), 8.85 (br s, 1H), 8.85-8.61 (m, 2H), 7.98 (d, J = 5.8 Hz,

1H), 7.86 (d, J = 8.4 Hz, 1H), 7.85-7.82 (m, 1H), 7.67 (dd, J =

8.4, 1.5 Hz, 1H), 7.41 (d, J = 5.8 Hz, 1H), 6.05 (br s, 1H), 5.57 (dt,

J = 17.1, 9.9 Hz, 1H), 5.23 (dd, J = 17.1, 1.5 Hz, 1H), 5.12 (dd, J

= 9.9, 1.5 Hz, 1H), 4.66 (dd, J = 10.2, 7.6 Hz, 1H), 4.40-4.30 (m,

2H), 3.88 (dd, J = 11.9, 2.9 Hz, 1H), 2.98-2.83 (m, 3H), 2.77-

2.58 (m, 3H), 2.28-2.12 (m, 3H), 1.89-0.95 (m, 24H). Analytical

RP-HPLC t_R(conditions C) 6.40 min (peak area 99%), t_R

(conditions D) 4.55 min (peak area 99%); HRMS calculated

for C₃₇H₅₀N₅O₆S (MþH)^þ692.3482, found 692.3475.

(2R,4S,7S,14E)-7-Cyclohexyl-N-{(1R,2S)-1-[(cyclopropylsul-

fonyl)carbamoyl]-2-ethenylcyclopropyl}-23-methoxy-6-oxo-20-

phenyl-3,4,6,7,8,9,10,11,12,13-decahydro-2H-16,18-etheno-2,

5-methanopyrido[3,4-r][1,5,8]oxadiazacyclononadecine-4-carbox-

amide Trifluoroacetate (22). Treatment of 10h (350 mg, 0.55 mmol)

as described in the general procedure reported for 12a (steps 2

and 3) gave the title compound (43 mg, 35% from 9h) as a white

1

powder. H NMR (400 MHz, DMSO-d₆) δ 10.86 (br s, 1H),

(2R,4S,7S,14E)-7-Cyclohexyl-4-({(1R,2S)-1-[(cyclopropylsul-

fonyl)carbamoyl]-2-ethenylcyclopropyl}carbamoyl)-23-methoxy-6-

oxo-3,4,6,7,8,9,10,11,12,13-decahydro-2H-16,18-etheno-2,5-metha-

nopyrido[3,2-r][1,5,8]oxadiazacyclononadecin-8-ium Trifluoroa-

cetate (26). Treatment of 10k (90 mg, 0.14 mmol) as described

in the general procedure reported for 12a (steps 2 and 3) gave the

title compound (25%) as a white powder. ¹H NMR (DMSO-d₆)

δ 11.12 (s, 1H), 8.90-8.71 (m, 2H), 8.58 (s, 1H), 7.96-7.90 (m,

2H), 7.33 (d, J = 5.9 Hz, 1H), 7.30 (s, 1H), 6.70 (d, J = 15.6 Hz,

8.83-8.73 (br m, 2H), 8.60 (s, 1H), 8.29-8.22 (m, 2H), 7.92 (s,

1H), 7.65-7.54 (m, 4H), 7.47-7.41 (m, 1H), 6.73 (d, J = 15.5 Hz,

1H), 5.99 (dt, J = 15.5, 7.2 Hz, 1H), 5.76 (br s, 1H), 5.57 (ddd,

J = 17.2, 10.3, 9.2Hz, 1H), 5.16 (d, J = 17.2Hz, 1H), 5.08(d, J =

10.3 Hz, 1H), 4.63 (br d, J = 10.9 Hz, 1H), 4.50-4.42 (m, 1H),

4.41 (dd, J = 10.9, 7.9 Hz, 1H), 3.98 (s, 3H), 3.97-3.88 (m, 1H),

3.33-3.19 (m, 1H), 2.94-2.77 (m, 2H), 2.64-0.95 (m, 28H).

Analytical RP-HPLC t_R(conditions C) 5.70 min (peak area

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4835

1H), 5.92 (dt, J = 15.6, 7.6 Hz, 1H), 5.74 (br s, 1H), 5.58 (ddd,

J = 17.0, 10.2, 9.1 Hz, 1H), 5.18 (d, J = 17.0 Hz, 1H), 5.09 (d,

J = 10.2 Hz, 1H), 4.52 (d, J = 11.6 Hz, 1H), 4.48-4.38 (m, 2H),

3.91 (s, 3H), 3.70 (dd, J = 12.4, 3.5 Hz, 1H), 3.21 (br s, 1H), 2.91

(m, 1H), 2.74 (dd, J = 14.1, 7.6 Hz, 1H), 2.60 (br s, 1H), 2.36-

2.24 (m, 1H), 2.23-2.07 (m, 3H), 1.99-1.60 (m, 9H), 1.57-1.34

(m, 3H), 1.33-0.93 (m, 11H). Analytical RP-HPLC t_R

(conditions C) 6.29 min (peak area 98%), t_R(conditions D)

4.45 min (peak area 98%); HRMS calculated for C₃₉H₅₂N₅O₇S

(MþH)^þ734.3587, found 734.3578.

(4R)-1-{(2S)-2-Cyclohexyl-2-[(3,3-dimethylbutyl)amino]acetyl}-

N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinyl-

cyclopropyl)-4-({2-[2-(isopropylamino)-1,3-thiazol-4-yl]-7-meth-

oxyquinolin-4-yl}oxy)-^L-prolinamide (38). Treatment of 36 (27 mg,

0.08 mmol) according to the general procedure described for 12a

(step 3 using 34 in place of 11, no RP-HPLC purification) gave a

residue thatwas taken up ina 4:1(v/v) mixture ofCH₂Cl₂/TFA(5

mL). The mixture was stirred for 2 h, and then the volatiles were

removed. The residue was purified by RP-HPLC to furnish the

1

title compound (7.5 mg, 12%) as a white solid. H NMR (400

MHz, DMSO-d₆, mixture of two conformers in a ratio 3:1; only

signals for the major conformer are reported) δ 10.86 (s, 1H), 8.90

(s, 1H), 8.64 (br s, 2H), 8.33 (s, 1H), 8.12 (br s, 1H), 8.04 (d, J =

9.2 Hz, 1H), 7.91 (d, J = 2.1 Hz, 1H), 7.82 (s, 1H), 7.31 (dd, J =

9.2, 2.1 Hz, 1H), 5.87 (br s, 1H), 5.61 (ddd, J = 17.1, 11.0, 9.2 Hz,

1H), 5.22 (d, J = 17.1 Hz, 1H), 5.12 (d, J = 11.0 Hz, 1H), 4.64

(dd, J = 9.7, 7.6Hz, 1H), 4.42 (d, J = 12.0 Hz, 1H), 4.32-4.25 (br

s, 1H), 4.22-4.13 (m, 2H), 3.96 (s, 3H), 2.98-2.88 (m, 1H), 2.86-

2.70 (m, 2H), 2.63-2.35 (m, 2H, partially obscured by residual

DMSO signal), 2.43-2.30 (m, 1H), 2.16 (q, J = 8.8 Hz, 1H),

1.97-1.69 (m, 7H), 1.69-1.60 (m, 1H), 1.50-1.40 (m, 2H), 1.38-

1.30 (m, 1H), 1.26 (d, J = 6.3 Hz, 3H), 1.25 (d, J = 6.2 Hz, 3H),

1.21-0.99 (m, 7H), 0.64 (s, 9H). Analytical RP-HPLC t_R

(conditions C) 5.05 min (peak area 95%), t_R(conditions D)

4.14 min (peak area 91%); HRMS calculated for C₄₄H₆₂N₇O₇S₂

(MþH)^þ864.4152, found 864.4182.

Methyl 3,3,3-Trifluoroalanyl-(4R)-4-{[(4-ethenyl-1,3-dihydro-

2H-isoindol-2-yl)carbonyl]oxy}-^L-prolinate (33). A solution of

4 (1.00 g, 2.83 mmol) in CH₂Cl₂(20 mL) was treated with (D,L)-

N-Boc-trifluoromethylglycine (827 mg, 3.40 mmol) and NMM

(686 μL, 6.24 mmol). HATU (1.51 g, 3.40 mmol) was added, and

the mixture was stirred for 14 h. The solution was diluted with

EtOAc and aqueous HCl (1 N), and then the layers were

separated. The organic layer was washed with saturated aqu-

eous NaHCO₃and brine and then dried. Removal of the

volatiles gave a residue (MS (ES^þ) m/z 542 (MþH)^þ) that was

taken up in a solution of HCl in dioxane (4 N, 5 mL). The

mixture was stirred for 4 h, and then the volatiles were removed

in vacuo to afford a residue that was triturated with Et₂O to

furnish the title compound (1.3 g, 59%) as a solid that was used

directly in the subsequent step. MS (ES^þ) m/z 442 (MþH)^þ.

(2S)-[(tert-Butoxycarbonyl)(3,3-dimethylbutyl)amino](cyclo-

hexyl)acetic Acid (34). Following the general procedure for re-

ductive amination described for 10c, treatment of methyl (2S)-

amino(cyclohexyl)acetate (400 mg, 1.92 mmol) and 3,3-dimethyl-

butanal (424 mg, 4.24 mmol) furnished (2S)-cyclohexyl[(3,3-di-

methylbutyl)amino]acetate (490 mg, 100%). MS (ES^þ) m/z 256

(Mþ H)^þ. This material was taken up in CH₂Cl₂(35 mL) and

treated with Et₃N (0.54 mL, 3.84 mmol) and di-tert-butyl dicarbo-

nate (544 mg, 2.49 mmol). The mixture was stirred for 14 h and then

diluted with EtOAc and washed with aqueous HCl (1 N) and

brine. The dried organic layer was concentrated to afford a

residue that was purified by flash chromatography on silica gel

(EtOAc/petroleum ether, gradient from 0:100 to 5:95) to give

(2S)-[(tert-butoxycarbonyl)(3,3-dimethylbutyl)amino](cyclohexyl)-

acetate (678 mg, 100%) as a white solid. MS (ES^þ) m/z 356

(MþH)^þ. This material was dissolved in a 2:1 (v/v) mixture of

tert-Butyl {(1S)-1-Cyclohexyl-2-[(2S,4R)-2-({(1R,2S)-1-[(cyclo-

propylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}carbamoyl)-4-

({7-methoxy-2-[2-(propan-2-ylamino)-1,3-thiazol-4-yl]quinolin-

4-yl}oxy)pyrrolidin-1-yl]-2-oxoethyl}carbamate Trifluoroacetate

(39). Treatment of 37 (100 mg, 0.15 mmol) as outlined in the

general procedure for 12a (steps 2 and 3) gave the title compound

(100 mg, 80%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ

10.42 (s, 1H), 9.05 (s, 1H), 8.21 (br s, 1H), 8.18 (d, J = 9.2 Hz, 1H),

8.04 (br s, 1H), 7.84 (br s, 1H), 7.75 (s, 1H), 7.28 (d, J = 9.2 Hz,

1H), 7.11 (d, J = 6.9 Hz, 1H), 5.77 (br s, 1H), 5.62 (ddd, J = 17.1,

10.9, 9.2 Hz, 1H), 5.25 (d, J = 17.1 Hz, 1H), 5.12 (d, J = 10.9 Hz,

1H), 4.67 (d, J = 12.1 Hz, 1H), 4.41 (t, J = 7.0 Hz, 1H), 4.22-4.08

(m, 1H), 3.98 (s, 3H), 3.97-3.93 (m, 1H), 3.88-3.80 (m, 2H),

2.99-2.89 (m, 1H),2.70-2.56 (m, 1H), 2.40-2.24 (m, 1H), 2.14 (q,

J = 8.8 Hz, 1H),1.91-1.48 (m, 7H), 1.34-1.28 (m, 1H), 1.26 (d,

J = 5.8 Hz, 6H), 1.16 (s, 9H), 1.13-0.8 (m, 8H). Analytical RP-

HPLC t_R(conditions C) 7.19 min (peak area 97%), t_R(conditions

D) 5.75 min (peak area 97%); HRMS calculated for

C₄₃H₅₈N₇O₉S₂(MþH)^þ880.3737, found 880.3731.

THF/H₂O (15 mL) and treated with LiOH H₂O (240 mg, 5.74

3

mmol). The mixture was heated at 50 °C for 3 h and then cooled and

diluted with EtOAc. The organic layer was washed with aqueous

HCl (1 N) and brine and then dried. Removal of the volatiles in

vacuo afforded the title compound (500 mg, 77%) as a white solid.

¹H NMR (400 MHz, DMSO-d₆, a mixture of two conformers in a

ratio 55:45*) δ12.51 (br s, 1H), 4.13 and 3.72* (d, J=9.9HzandJ*

= 8.9 Hz, 1H), 3.24-2.99 (m, 2H), 1.91-1.08 (m, 11H), 1.40 and

1.36* (s, 9H), 0.98-0.83 (m, 2H), 0.87 (s, 9H); MS (ES^þ) m/z 342

(MþH)^þ.

Supporting Information Available: Synthetic procedures and

characterization of compounds 8a-j, 28a,b, and 31; rat and dog

PK curves and parameters for compound 25. This material is

available free of charge via the Internet at http://pubs.acs.org.

(4R)-N-{(1R,2S)-1-[(Cyclopropylsulfonyl)carbamoyl]-2-ethenyl-

cyclopropyl}-4-({7-methoxy-2-[2-(propan-2-ylamino)-1,3-thiazol-

4-yl]quinolin-4-yl}oxy)-^L-prolinamide (36). Treatment of 35a

(123 mg, 0.16 mmol) according to the general procedure de-

scribed for 12a (step 3, no RP-HPLC purification) gave a residue

that was taken up in an 8:2 (v/v) mixture of CH₂Cl₂/TFA

(10 mL). The mixture was stirred for 2 h, and then the volatiles

were removed in vacuo. Trituration of the residue with Et₂O

afforded the title compound (122 mg, 97%) as a solid. MS (ES^þ)

m/z 641 (MþH)^þ.

Acknowledgment. We thank Vincenzo Pucci and Fabio

Bonelli for bioanalytical data and protein binding assays; Claudio

Giuliano for HRMS data; Sergio Altamura, Nadia Gennari,

Steven S. Carroll, and Mark Stahlhut for cell based assays.

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glycine analogues; the Z-amide is preferred for the 10-R epimer,

while the E-amide is preferred for the 10-S. NOESY experiments

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only with Z-conformation around the proline amide bond and

support R-configuration at C₁₀

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ꢀ

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(31) The conformational analysis of 12a and 12b was performed in the

absence of the protein using Macromodel and MMFFs as imple-

mented in Maestro7.5. Low energy conformations were sampled,

and the backbone atoms of each were first superimposed to a

covalent inhibitor from a cocrystal structure and subsequently

minimized.

(32) Full details of this compound class will be published elsewhere.

McCauley, J. A.; McIntyre, C. M.; Rudd, M. T.; Nguyen, K. T.;

Article Doi

Inhibitors of the hepatitis C virus NS3 protease with basic amine functionality at the P3-amino acid N-terminus: Discovery and optimization of a new series of P2-P4 macrocycles

DOI: 10.1021/jm900372w

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Article abstract of DOI:10.1021/jm900372w

Full text of DOI:10.1021/jm900372w

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