Design and synthesis of pyrrolidine-5,5-trans-lactams (5-oxohexahydropyrrolo[3,2-b]pyrroles) as novel mechanism-based inhibitors of human cytomegalovirus protease. 2. Potency and chirality

Volume 45, Number 1

J anuary 3, 2002

Articles

Design a n d Syn th esis of P yr r olid in e-5,5-tr a n s-la cta m s

(5-Oxoh exa h yd r op yr r olo[3,2-b]p yr r oles) a s Novel Mech a n ism -Ba sed In h ibitor s

of Hu m a n Cytom ega lovir u s P r otea se. 2. P oten cy a n d Ch ir a lity

Alan D. Borthwick,* Andrew J . Crame, Peter F. Ertl,^†Anne M. Exall, Terry M. Haley,^‡Graham J . Hart,^§

Andrew M. Mason, Andrew M. K. Pennell, Onkar M. P. Singh,^‡Gordon G. Weingarten, and J ames M. Woolven^‡

Department of Medicinal Chemistry CVU UK, GlaxoSmithKline Research and Development, Medicines Research Centre,

Gunnels Wood Road, Stevenage, Herts SG1 2NY, U.K.

Received May 18, 2001

The stereospecific synthesis of a series of R-methylpyrrolidine-5,5-trans-lactam inhibitors of

human cytomegalovirus (HCMV) protease is described. Examination of the SAR in this series

has defined the size and chirality of the R-substituent, optimized the acyl substituent on the

lactam nitrogen, and defined the steric constraint of this functionality. The SAR of the

functionality on the pyrrolidine nitrogen of the trans-lactam has been investigated, and this

has led to the discovery of potent serine protease inhibitors that are highly selective for the

viral enzyme over the mammalian enzymes elastase, thrombin, and acetylcholine esterase.

The mechanism of action of our lead compounds has been established by mass spectrometry,

and enzymatic degradation of HCMV δAla protease acylated with these inhibitors showed that

Ser 132 is the active site nucleophile. The crystal structure of HCMV protease was obtained

and used to model the conformationally restricted, chiral (S)-proline-R-methyl-5,5-trans-lactams

into the active site groove of the enzyme, enabling us to direct and rationalize the SAR in this

series. The activity against HCMV δAla protease is the greatest with inhibitors based on the

dansyl-(S)-proline R-methyl-5,5-trans-lactam template, which have low nanomolar activity

against the viral enzyme.

In tr od u ction

and ganciclovir, together with the emergence of mutants

resistant to acyclovir, there is a need for a new class of

antiherpes compounds based on a novel mechanism.

Human herpes viruses cause a range of diseases:

HSV-1 (cold sores), HSV-2 (genital herpes), VZV (chicken

pox, shingles), and HCMV (retinitis, pneumonitis). The

current treatment of these diseases uses nucleoside

(acyclovir, ganciclovir) and phosphate (PFA) substrate

analogues. Because of the toxicity associated with PFA

Human herpes viruses encode a serine protease,

which is essential for viral replication.¹Recent X-ray

structures of the serine proteases of HCMV, HSV-1,

HSV-2, and VZV revealed that these enzymes belong

to a novel class of serine proteases where the active site

is composed of the His, His, Ser triad.^3-6Substrate

cleavage sites across all the herpes virus family are

unique and highly conserved, and these enzymes have

become attractive molecular targets for the design of

novel antiviral drugs.^1,2We recently reported on the

* To whom correspondence should be addressed. Telephone: +44

(0)1438 763422. E-mail: adb3028@glaxowellcome.co.uk. Fax: +44 1438

768483.

^†Department of Molecular Immunology.

^‡Department of Biomolecular Structure.

^§Department of Enzyme Pharmacology.

Published on Web 12/05/2001

2

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

Borthwick et al.

Sch em e 1

design and synthesis of a novel class of mechanism-

based inhibitors of human cytomegalovirus protease,^7,8

based on the R-methylpyrrolidine-5,5-trans-lactam tem-

plate incorporating the natural substrate requirements

of the consensus sequence of HCMV protease (Scheme

1). ESI-MS studies have shown that these inhibitors can

bind covalently and reversibly in a time-dependent

manner by a mechanism that is consistent with acyla-

tion of HCMV δAla protease at the active site nucleo-

phile Ser 132.⁷SAR in this series of pyrrolidine-5,5-

trans-lactams has defined the relative stereochemistry

of the methyl substituent adjacent to the lactam car-

bonyl and the trend in activity of the functionality on

the lactam nitrogen as acyl > ester > sulfonyl >

carbamate against HCMV δAla protease.

We now report on work to define further the require-

ments of the substituent adjacent to the lactam carbonyl

that accesses the S1 pocket and on work to exploit the

substituents on the nitrogen atoms of this template that

have the potential to access the conserved S1′ and S3

pockets of the protease that has given inhibitors with

low nanomolar potency against the HCMV δAla pro-

tease.

group. This proceeded in a lower yield (49%) to give 14

and was taken forward to the acetoxyacetyl derivative

16 via 15 in the usual manner (Scheme 3).

We recently showed that the gem-dimethyl-trans-

lactam 19 could be prepared by alkylation of the

â-methyl isomer 18 but not by alkylation of the corre-

sponding R-isomer 20.¹⁸Deprotection of 19 with TFA

followed by acylation of 21 with acetyl chloride or

acetoxyacetyl chloride gave 22 and 23 in 76% and 82%

yield, respectively (Scheme 4).

Functionalized acyl derivatives on the lactam nitrogen

in the R-methyl-5,5-trans-lactam series (Scheme 5) were

prepared in 40-87% yield by acylating the anion of

lactam 24, with the corresponding mixed anhydride or

acid chloride. The extended hydroxyethyl analogue 37

was similarly prepared via the silyl-protected derivative

36. The formyl compound 25 was prepared in 54% yield

by reacting 24 with sodium hydride followed by addition

of DMF in ethylene glycol dimethyl ether in the pres-

ence of triflic anhydride.

Compounds with functionality other than ketones and

amides were prepared by reacting the anion, generated

by treating the lactam 24 with LHMDS, with a variety

of electophiles (Scheme 6). Thus, 1,2-epoxy-3-phenoxy-

propane gave alcohol 38 in 41% yield and 2,4-dinitro-

fluorobenzene gave the dinitrophenyl derivative 39 in

70% yield. The N-pyrrole amide 40 was similarly

prepared in 72% yield by reacting the lactam anion with

pyrrole-1-carboxylic acid anhydride.¹⁰The phosphonate

derivative 41 and the methylthio analogue 42 were also

prepared by treating the lactam anion with dimethyl

chlorophosphate and methyl methanethiolsulfonate,

respectively. However, the corresponding reactions with

diethyl chlorophosphate and dimethylphosphinic chlo-

ride failed. The conjugated ketone 43 was prepared in

30% yield by reaction of vinyl methyl ketone with lactam

24 in the presence of a Pd catalyst (PdCl₂(MeCN)₂)

under an atmosphere of oxygen,¹¹while alcohol 44 was

prepared in 67% yield by treating 24 with paraformal-

dehyde in THF in the presence of potassium carbonate

at room temperature.

Ch em istr y

We previously reported⁷that an R-methyl group at

the 6 position and a carbonyl function on the nitrogen

at the 2 position was optimal for maximum potency in

the novel pyrrolidine-5,5-trans-lactam inhibitors (1 and

2) of HCMV protease (Scheme 1). To investigate the

absolute configuration at the 6 position, the racemic

mixture 1 was separated by chiral HPLC into the

isomers 3 (RSR) and 4 (SRS), and their structures were

confirmed by synthesis (Scheme 2). The racemic amine

5 was resolved using (+)-di-O,O′-p-toluyl-D-tartaric acid.

The (S)-amine salt 6 was crystallized from the mixture

of diastereomeric salts in ethanol and then recrystal-

lized from ethanol, and the free amine was regenerated

by base to give 7 in 98% yield. The amino ester 7 had

previously been synthesized⁹from L-glutamine, confirm-

ing the chirality of the primary amine as (S). Cyclization

of the amino ethyl ester 7 using tert-butylmagnesium

chloride in THF gave the translactam 8 in 78% yield

and 96% ee. Boc protection and methylation followed

by deprotection gave 11 in 55% overall yield. Acylation

of 11 with acetyl chloride gave 4 in 81% yield and 96%

ee and was shown by chiral HPLC to be identical to the

active isomer obtained by separation of the enantiomers.

Similarly acylation of 11 with acetoxyacetyl chloride

gave 12 in 75% yield.

Exploration of the substituents on the pyrrolidine

nitrogen required to access the S2-S3 space has been

done with a variety of linkers, namely, methyl ketone,

sulfonamide, ketoamide, and amide (Scheme 7). The

sulfonamide 46 was prepared in 53% yield by reacting

the pyrrolidine 45 with the corresponding phenethyl-

sulfonyl chloride at room temperature in the presence

of triethylamine, whereas the reaction of pyrrolidine 45

with benzyl bromomethyl ketone required heating under

reflux in acetonitrile to furnish the benzyl ketone 47 in

29% yield. The majority of amides were prepared using

We have introduced the larger allyl group into 13

using the same methodology developed for the methyl

Inhibitors of Human Cytomegalovirus Protease

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

3

Sch em e 2^a

a

Reagents and conditions: (a) RCO₂H ) (+)-di-O,O′-p-toluyl-D-tartaric acid, ethanol; (b) aqueous K₂CO₃, EtOAc; (c) Bu^tMgCl, THF;

(d) LHMDS (1.2 equiv)/THF, -78 °C, then 0 °C, 20 min, then (Boc)₂O (2.8 equiv)/THF, -78 °C; (e) LHMDS (1.3 equiv)/THF, -78 °C, then

MeI (17 equiv); (f) TFA, 1 h, room temp; (g) LHMDS (1.2 equiv)/THF, -78 °C, then 0 °C, 30 min, then MeCOCl, -78 °C; (h) LHMDS (1.2

equiv)/THF, -78 °C, then 0 °C, 30 min, then MeCO₂CH₂COCl, -78 °C.

Sch em e 3^a

a

Reagents and conditions: (a) LHMDS (1.3 equiv)/THF, -78 °C, then allyl I (17 equiv); (b) TFA, 2 h, room temp; (c) LHMDS (3 equiv)/

THF, -78 °C, then MeCO₂CH₂COCl.

the coupling conditions O-benzotriazol-1-yl-N,N,N′,N′-

tetramethyluronium tetrafluoroborate/1-hydroxyben-

zotriazole (TBTU/HOBT) in DMF at room tempera-

ture. Reacting racemic 45 with tosyl-(S)-proline gave the

(S-SRS) amide 51 (38%) and the (S-RSR) diastereoiso-

mer 52 (30%). Similarly, reaction of racemic 45 with

benzoyl-(S)-proline or dansyl-(S)-proline gave the cor-

responding diastereoisomers 53 and 54, or 55 and 56,

respectively.

The phenoxyacetamide 48, the ketoamide 50, the

indolacetamide 49, and the nitrophenylsulfonyl-(S)-

proline 57 derivatives were similarly prepared in 51%,

45%, 56%, and 23% yield, respectively.

Reacting the racemic Boc-protected trans-lactam 58

with dansyl-(S)-proline gave the two diastereoisomers

59 and 60 in 30% and 38% yield, respectively (Scheme

8).

4

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

Borthwick et al.

Sch em e 4^a

a

Reagents and conditions: (a) LiHMDS (3 equiv)/THF, -78 °C then (Boc)₂O; (b) LiHMDS (1.3 equiv)/THF, -78 °C, then MeI ((17

equiv); (c) TFA, 2 h, room temp; (d) LHMDS (1.2 equiv)/THF, -78 °C, then 0 °C, 30 min, then MeCOCl, -78 °C; (e) LHMDS (1.2 equiv)/

THF, -78 °C, then 0 °C, 30 min, then MeCO₂CH₂COCl, -78 °C.

Sch em e 5^a

a

Reagents and conditions: (a) LHMDS (1.4 equiv)/THF, -78 °C, then Me₃CCO₂COCH₂CH₂OSiMe₂Bu^t; (b) AcOH/H₂O/THF, 10/3/1,

room temp, 8 h; (c) LHMDS (1.4 equiv)/THF, -78 °C, then RCOCl or RCO₂COCMe₃.

Deprotection of 60 with TFA gave the lactam 61 in

95% yield, which reacted with cyclopropane carbonyl

chloride in the presence of LHMDS to give 62 in 80%

yield. Comparison of the CD spectra of 62 and 4 showed

that the configuration of the trans-lactam ring was the

same (SRS) in both compounds.

Resu lts a n d Discu ssion s

The chromogenic assay developed earlier^12,17based on

the 7-mer peptide p-nitroanilide (RESYVKA-pNA) of the

release site and HCMV δAla protease has been devel-

oped further into a microtiter assay and is comparable

to the HPLC assay used previously⁷(Table 1). IC₅₀data

for compounds are determined by preincubating the

enzyme with inhibitor for 15 min. We showed recently⁷

that the preferred stereochemistry of the methyl group

adjacent to the lactam carbonyl was R for activity

against HCMV δAla protease. We have investigated the

steric space at this position by making the R-allyl

derivative 16 (Scheme 2) and the gem-dimethyl ana-

logues 23 and 22 (Scheme 3). The R-allyl derivative 16

was less active (51% inhibition at 500 µM) than the

methyl derivative 1 (96% inhibition at 500 µM) with the

same stereochemistry. The gem-dimethyl analogues 23

and 22 were virtually inactive in this series with 2%

and 8% inhibition, respectively, at 500 µM. Thus, we

are confident that we have defined the stereochemistry

and size of the substituent at S1.

Deprotection of 28 with hydrogen in the presence of

palladium gave the amine, which was isolated as the

hydrochloride 63 in quantitative yield. Reaction of 63

with dansylsarcosine in the presence of TBTU/HOBT

and DIPEA gave the amide 64. Similarly, reaction of

63 with dansyl-(S)-alanine and (R,S)-pipecolic acid gave,

after separation from their corresponding diastereoiso-

mers, the (S-SRS) isomers 65 and 66 in 30% and 14%

yield, respectively (Scheme 9). Reaction of racemic 63

with Cbz-(S)-proline gave a diastereomeric mixture of

amides from which the (S-SRS) amide 67 was isolated

in 38% yield. Hydrogenolysis of 67 gave the amine 68

in quantitative yield. Reaction of 68 with 3-[(dimeth-

ylamino)carbonyl]benzenesulfonyl chloride, 3-(1-meth-

ylethoxy)benzenesulfonyl chloride, and 3-[(dimethylami-

no)carbonyl]benzenemethanesulfonyl chloride in the

presence of triethylamine gave the chiral sulfonamides

69, 70, and 71 in 32%, 43%, and 22% yield, respectively.

To define the chirality of the preferred substituent

at S1, both enantiomers (4-SRS and 3-RSR) of 1 were

Inhibitors of Human Cytomegalovirus Protease

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

5

Sch em e 6^a

a

Reagents and conditions: (a) LHMDS (1.4 equiv)/THF, -78 °C, then ClP(O)OMe₂; (b) LHMDS (1.5 equiv)/THF, -78 °C, then MeSSO₂Me

(2 equiv) -78 to 0 °C, 2 h; (c) LHMDS (1.1 equiv)/THF, -78 °C, then (pyrrole-CO)₂O; (d) LHMDS (1.4 equiv)/THF, -78 °C, then FC₆H₃(NO₂)₂;

(e) LHMDS (1.5 equiv)/THF, -78 °C, then 2-epoxy-3-phenoxypropane (1.5 equiv); (f) CuCl, DME, PdCl₂(MeCN)₂, O₂, MeCOCHdCH₂; (g)

(CH₂O)_n, K₂CO₃, THF, room temp.

Sch em e 7^a

a

Reagents and conditions: (a) H₂, Pd/C, i-PrOH, room temp, then HCl/ether; (b) RSO₂Cl (1.1-1.5 equiv), Et₃N, MeCN, room temp; (c)

PhCH₂COCH₂Br (1.5 equiv) Et₃N, MeCN, reflux; (d) RCO₂H, TBTU, HOBT, i-Pr₂EtN, DMF, room temp.

separated by chiral HPLC. Only one of these (4-SRS)

was shown to be active against HCMV δAla protease

(Table 1). The activity was predicted to be in the isomer

where the Me group was the same configuration (S) as

the Ala side chain of the natural peptide substrate for

HCMV protease (Scheme 10). This was confirmed by

synthesis (Scheme 2). The corresponding acetoxyacetyl

analogue (12-SRS) was also prepared and found to be

more active than the racemate 2 our previous lead

compound (Table 1).

Additional acyl derivatives were prepared in an effort

to increase potency in this series (Table 2). The hy-

droxyethylcarbonyl compound 37 was prepared in an

attempt to mimic the Ser hydroxyl at S1′, but it did not

show any improvement over the acetyl compound 1 in

terms of potency. Increasing the size of this acyl

functionality to the isopropyl and tertiary butyl ana-

logues 26 and 27, respectively, caused a loss in potency

(Table 2), indicating that the steric bulk adjacent to the

carbonyl at the S1′ site is not tolerated.

6

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

Borthwick et al.

Sch em e 8^a

a

Reagents and conditions: (a) H₂, Pd/C, i-PrOH, room temp, then HCl/ether; (b) dansyl-(S)-proline, TBTU, HOBT, i-Pr₂EtN, DMF,

room temp; (c) TFA, 2 h, room temp; (d) LHMDS (1.4 equiv)/THF, -78 °C, then cyclopropyl-COCl.

Sch em e 9^a

a

Reagents and conditions: (a) H₂, Pd/C, i-PrOH, room temp, then HCl/ether; (b) R′CO₂H, TBTU, HOBT, i-Pr₂EtN, DMF, room temp;

(c) Cbz-proline, TBTU, HOBT, i-Pr₂EtN, DMF, room temp; (d) H₂, Pd/C, i-PrOH, room temp, then HCl/ether; (e) R′SO₂Cl, (1.1-1.5 equiv),

Et₃N, MeCN, room temp.

Interestingly, some bulk is allowed in that both the

cyclopropylcarbonyl compound 28 and the cyclobutyl-

carbonyl compound 33 are more active than the acetyl

compound 1. Additional cyclic acyl derivatives were

prepared in an attempt to increase potency further in

this class. Increasing the steric bulk on the cyclopropyl

ring showed a decrease in potency from the monomethyl

derivative 31 via the dimethyl derivative 32 to the

tetramethyl derivative 30. While the monomethyl de-

rivative 31 is equipotent to the cyclopropylcarbonyl

compound 28, the tetramethyl derivative 30 is es-

sentially inactive. Interestingly the cis-(Z)-2,3-dimethyl

derivative 32 is more active than the trans-(E)-2,3-

dimethyl derivative 29, showing the subtle steric re-

quirements at this position. The arylcarbonyl compound

34 is essentially inactive; however, the benzocyclobutane

compound 35 with an aryl ring more distant from the

carbonyl function is as active as the acetyl compound

Inhibitors of Human Cytomegalovirus Protease

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

7

Sch em e 10

12

Ta ble 1

Ta ble 3

HCMV protease

HPLC assay⁷

pNA assay¹²

compd

R

protease IC₅₀, µM protease IC₅₀, µM

compd

R

IC₅₀, µM

1

3

4

2

12

rac- COMe

RSR COMe

SRS COMe

rac- COCH₂OCOMe

SRS COCH₂OCOMe

110

>1000

79

40

25

44

38

43

41

42

39

CO-N-pyrryl

CHO

CH₂OH

CH₂CH(OH)CH₂OPh

CHdCHCOCH₃

PO(OMe)₂

250

398

>500

∼500

13

6

11

12

SMe

C₆H₃-2,4-(NO₂)₂

Ta ble 2

enzyme at the S3 position. Comparison of several

compounds bearing linkages of a phenyl group three

atoms from the pyrrolidine nitrogen of 1, 47, 48, and

46 indicate that none has a particular advantage in

terms of potency (Table 4).

HCMV protease

R

In an effort to increase the potency of 1, we have used

array technology to make a wide range of substituents

on the pyrrolidine nitrogen to thoroughly span the S2-

S3 space, incorporating an isopropyl group and other

functionality to mimic the conserved valine required for

S3 in the natural substrate. This produced three lead

compounds with increased potency, the 2-methyl-5-

fluoroindole-3-acetamide 49, N-methylpyrrole-2-keto-

amide 50, and the N-tosyl-(S)-proline derivative 51

(Table 4). Replacing the ketocarbonyl of the N-meth-

ylpyrrole-2-ketoamide 50 with methylene, changing the

heterocycle, or removing the N-methyl function resulted

in lost activity, as did replacing the fluorine substituent

and changing the heterocycle in the 2-methyl-5-fluoro-

indole-3-acetamide 49. Lead optimization of the proline

derivative 51 was successful and showed that this

arylsulfonyl derivative is more active than the arylcar-

bonyl analogue 53 (Table 5). Also both the (S-SRS)

isomers 51 and 53 are more active than the correspond-

ing (S-RSR) isomers 52 and 54. While the p-nitro

analogue 57 has a potency similar to that of the

p-methyl analogue 51, both of these monocyclic deriva-

tives are less active than the bicyclic dansyl-(S)-proline

analogue 55 (IC₅₀) 0.54 µM). The corresponding

cyclopropyldansyl-(S)-proline analogue 62 is even more

potent (IC₅₀) 0.34 µM), and both are ∼2 orders of

compd

IC₅₀, µM

2

37

1

COCH₂OCOMe

COCH₂CH₂OH

COMe, log P ) 2.21

COCHMe₂

COCMe₃

CO-cyclobutyl

CO-benzocyclobutyl

CO-cyclopropyl

CO-2-Me-cyclopropyl

CO-(Z)-2,3-diMe-cyclopropyl

CO-(E)-2,3-diMe-cyclopropyl

CO-tetra-Me-cyclopropyl

CO-C₆H₄-4OMe

11

133

40

126

>500

32

26

27

33

35

28

31

32

29

30

34

11.5

9

10

72

>100

>500

1, indicating again that the bulk adjacent to this

functionality is not tolerated.

A series of other S1′ substituents was also prepared

in an effort to improve activity and to mimic the serine

hydroxyl at this position in the natural substrate (Table

3). Both alcohols 38 and 44 were inactive as were the

vinyl ketone 43, the dimethylphosphonate 41, and the

thiomethyl analogue 42. However, the dinitrophenyl

derivative 39 showed some activity at ∼500 µM and the

formyl compound 25 and acyl pyrrole 40 were weakly

active.

Having optimized the S1 and S1′ positions, we turned

our attention to the other conserved pocket in this

8

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

Borthwick et al.

12

Ta ble 4

Ta ble 7

HCMV protease

thrombin

acetylcholine

elastase

IC₅₀

,

K_i,

compd IC₅₀, µM esterase IC₅₀, µM IC₅₀, µM

µM

nM

55

62

>200

>100

>10

0.54

0.34

34

20

HCMV protease

IC₅₀

,

compd

R

µM

1

47

48

46

50

49

51

PhCH₂OCO

PhCH₂COCH₂

PhOCH₂CO

PhCH₂CH₂SO₂

N-Me-2-pyrrolyl-COCO

5-F-2-Me-3-indolyl-CH₂CO

N-tosyl-(S)-prolinyl

40

79

63

45

5

6

14

12

Ta ble 5

HCMV protease

R

compd

IC₅₀, µM

F igu r e 1.

51

52

53

54

57

55

56

4-MePhSO₂N-pyrrolidine-2-CO (S-SRS)

4-MePhSO₂N-pyrrolidine-2-CO (S-RSR)

PhCON-pyrrolidine-2-CO (S-SRS)

PhCON-pyrrolidine-2-CO (S-RSR)

4-NO₂PhSO₂N-pyrrolidine-2-CO (S-RSR)

5-NMe₂-naph-SO₂N-pyrrolidine-2-CO (S-SRS)

14

159

50

dansyl function are important for potent activity against

HCMV δAla protease. These two dansyl-(S)-proline

derivatives 55 and 62 are the most active compounds

of this series (Tables 5 and 6), having potencies of K_i)

34 and 20 nM, respectively, and show selectivity for the

viral HCMV protease over acetylcholine esterase and

the mammalian proteases elastase and thrombin (Table

7).

>500

11

0.54

5-NMe₂-naph-SO₂N-pyrrolidine-2-CO (S-RSR) >500

12

Ta ble 6

X-ray crystal structures of various HCMV protease

mutants are known.^3-6We have crystallized the recom-

binant HCMV protease mutant (V141Y, V207Y, and

A209N) and determined its X-ray crystal structure at

2.2 Å resolution.¹³We have used the X-ray coordinates

of this HCMV protease in a modeling study¹⁴with the

semirigid trans-lactam 62 and compared it to a similarly

docked substrate analogue model. Figure 1 shows a view

of the active site of (V141Y, V207Y, and A209N) HCMV

protease incorporating 62 in what could be considered

as an initial binding complex. The R-Me in the (S)

configuration extends into the S1 specificity pocket as

expected from the substrate requirements of a conserved

alanine at this position for this enzyme. The C-1 trans-

lactam carbonyl is situated in the oxyanion pocket

formed by the backbone amide NH of arginine 165 and

arginine 166 and is in position for attack by the hydroxyl

of serine 132, which is part of the active site catalytic

triad formed by serine 132, histidine 63, and histidine

157. The lactam substituent extends into the prime sites

of the enzyme. The model shows that R-Me in the (S)

configuration is easily accommodated in the S1 pocket

and allows us to visualize why the (R) configuration for

the R-Me is less favorable. There is clearly insufficient

room for the (R) configuration to fit and preserve the

complementarity of the other portions of the molecule

to the active site. The R-allyl in the (S) configuration

found in 16 is also unable to access the S1 pocket.

The (S)-proline carbons C-4′ and C-5′ fit into the S3

pocket, and the rigidity of the proline ring can be seen

as important because the flexibility of the partial

HCMV protease

compd

R

IC₅₀, µM

0.34

>100

62

64

65

66

69

71

70

5-NMe₂-naph-SO₂N-pyrrolidine-2-CO (S-SRS)

5-NMe₂-naph-SO₂NMe-CH₂CO (SRS)/(RSR)

5-NMe₂-naph-SO₂NH-CHMeCO (S-SRS)

5-NMe₂-naph-SO₂N-piperidine-2-CO (S-SRS)

3-NMe₂COPhSO₂N-pyrrolidine-2-CO (S-SRS)

3-NMe₂PhCH₂SO₂N-pyrrolidine-2-CO (S-SRS)

3-Me₂CHOPhSO₂N-pyrrolidine-2-CO (S-SRS)

7

13

1.4

2.2

65

magnitude more potent than Cbz compound 1. The

corresponding (S-RSR) diastereoisomer 56 of 55 is

inactive, again indicating that the stereochemistry of

the translactam ring is crucial for activity.

To see if the dansyl-(S)-proline was essential for good

activity, several other (S) amino acid linkers and partial

structures of the dansyl ring in the dansyl-(S)-proline

cyclopropyl analogue 62 were prepared (Table 6). How-

ever, the partial proline structures of the glycine 64 and

alanine 65 analogues were much less active, as was the

six-membered pipecolic analogue 66, indicating that the

rigid (S)-proline ring was optimal for activity.

Compounds 69-71 (Table 6), containing partial struc-

tures of the dansyl ring, have less activity than 62. It

seems that both chiral dansyl-(S)-proline analogues 55

and 62 are conformationally restricted, semirigid com-

pounds, where both the (S)-proline ring and the bicyclic

Inhibitors of Human Cytomegalovirus Protease

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

9

propyl ring substituted with one methyl group 31 is

tolerated, increasing this to two adjacent methyls (32

and 29) was less well tolerated and the tetramethyl

analogue 30 is essentially inactive because of unavoid-

able clashes with the enzyme regardless of what con-

formation this functionality takes up.

The semirigid nature of 62 prearranges the molecule

in a conformation that mimics the shape adopted by

peptide substrates bound in serine proteases, allowing

the inhibitor to fit snugly in the active site channel/

groove (Figure 2). This has been shown by a comparison

of our docked molecules with a model of a substrate

analogue iodo-Y-V-N-A-S-aldehyde recently shown³in

an X-ray crystal structure bound in the active site of

HCMV protease. We see good register between the R-Me

groups in P1, and also between the valine side chain of

the substrate analogue and the proline ring carbons of

62 residing in P3.

To investigate the mechanism of action of the dansyl-

(S)-proline-derived trans-lactam 55 interaction with

δAla, HCMV protease was studied by liquid chroma-

tography (LC) coupled to electrospray ionization mass

spectrometry (ESI-MS). The complexes formed between

the enzyme and the compound were analyzed over a

time course, aliquots taken initially at 20 min and 1, 2,

4, and 48 h, to enable assessment of the potency and

duration of inhibition as a result of any covalent

modification (Figure 3). In a detailed study additional

time points were introduced, 30 s and 1, 2, 3, 5, 8, 12,

and 18 min, specifically to investigate the early stages

of interaction. Once acylation of the enzyme had been

confirmed, the key residues in the catalytic triad were

identified using LC-ESI-MS and ESI-MS-MS tech-

niques in conjunction with enzymatic degradation of the

acylenzyme species, as previously described.¹²

F igu r e 2. Me probe of HCMV protease active site and

inhibitor 62.

structures 64 and 65 and the more flexible six-

membered ring 66 do not allow access to this pocket and

they are all >10-fold less active. Also, in the rigid (S)-

proline analogue 62, the dansyl ring is held in a position

to make a favorable hydrophobic interaction with the

enzyme, which is less efficiently accessed with the more

flexible partial structures. Both rings of the dansyl

function are necessary for this hydrophobic interaction

because the more flexible partial structures 71 and 69,

which make less of an interaction, are much less active.

While only one sulfonamide oxygen is located within

hydrogen-bonding distance of the backbone amide NH

of Ser 135, both sulfonamide oxygens are within hydro-

gen-bonding distance of this residue in the tetrahedral

transition state, which might explain some of the

beneficial effects of the sulfonamide over the amide at

this position.

The HCMV δAla protease, unmodified average mo-

lecular weight (M_av

) determined as 27 824 Da, was fully

acylated after only 1 min of incubation with compound

55. The M_avof the complex was determined as 28 336

Da for 55, corresponding to the M_avof the enzyme plus

the molecular weight of the inhibitor. Further to this,

complete acylation was preserved for at least 4 h

following addition. Samples analyzed after 48 h con-

tained only unmodified HCMV δAla protease, indicating

The cyclopropylcarbonyl function extends into the S′

prime sites of the enzyme. However, while the cyclo-

F igu r e 3. MS characterization of the acylation of HCMV δAla protease by R-methyl- pyrrolidine-5,5-trans-lactams.

10 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

Borthwick et al.

F igu r e 4.

Intersil M column ODS2. Standard conditions were eluent

system A (H₂O, 0.1% H₃PO₄) and system B (95% MeCN/H₂O,

0.1% H₃PO₄): gradient was 0% B at 2 min, 0-100% B at 40

min, 100% B at 10 min; flow rate was 1 mL/min; λ ) 215 nm.

Retention times (t_R) are given in minutes. All NMR spectra

were run on a Bruker 250 MHz instrument generally as

solutions in CDCl₃unless otherwise stated. IR spectra were

recorded on a Bio-rad FTS7 spectrometer from thin films on

NaCl plates, from a KBr mix, or from solutions in the solvent

specified. Mass spectra were run by an electrospray Hewlett-

Packard 5989B instrument. CD spectra were recorded in

acetonitrile on a J asco J -720A spectropolarimeter. Optical

rotations were taken with a Perkin-Elmer model 241 pola-

rimeter. Enantiomeric excess (% ee) was determined by chiral

HPLC analysis using a Chiracel OJ or Chiral Pak AD464

column with a UV detector at λ ) 215 nm and with eluents

and flow rates as indicated in each case. Final organic solutions

were dried over MgSO₄before filtration and evaporation using

a Buchi rotavapor. Ambient temperature was 20 °C. All

solvents used were Fisons analytical reagents except for

pentane (Aldrich Chemical Co.) and anhydrous THF (Fluka

Sureseal). All other reagents were obtained from Aldrich,

Fluka, or Lancaster. Elemental microanalyses were deter-

mined by the Microanalytical Laboratory, GlaxoSmithKline

Stevenage.

(2R,3S)-3-Am in o-2-eth oxyca r bon ylm eth ylp yr r olid in e-

1-ca r boxylic Acid Ben zyl Ester (2S,3S)-Bis(4-m eth yl-O-

ben zyloxy)su ccin a te Sa lt (6). To a solution of trans-3-

amino-2-ethoxycarbonylmethylpyrrolidine-1-carboxylic acid

benzyl ester⁷5 (168.9 g, 0.55 mol) in ethanol (2500 mL) was

added a solution of (+)-di-O-p-toluyl-^D-tartaric acid (213 g, 0.55

mol) in ethanol (2500 mL), and the solution was allowed to

stand overnight. The resulting solid was collected by filtration,

washed with ethanol, and then recrystallized from boiling

ethanol (3500 mL) to give the 6 as a white solid, 93.7 g. A

further recrystallization gave a white solid, mp 184-185 °C.

HPLC analysis using a Chiracel OJ column showed it to be

97.5% ee (ethanol/heptane (3:7), flow rate ) 1 mL/min, λ )

215 nm, t_R) 7.7 min)). Anal. (C₃₆H₄₀N₂O₁₂‚0.2EtOH) C, H, N.

(2R,3S)-3-Am in o-2-eth oxyca r bon ylm eth ylp yr r olid in e-

1-ca r boxylic Acid Ben zyl Ester (7). The intermediate salt

6 (131.8 gm, 190 mmol) was suspended in water/ethyl acetate

(1:1) (1500 mL), and solid potassium carbonate was added (63

g, 457 mmol). After 15 min, the phases were separated and

the aqueous phase was extracted with ethyl acetate (3 × 200

mL). The organic portions were combined and washed with

water and brine, dried, and evaporated to give 7 as a colorless

oil (57.8 g, 98%): ¹H NMR (CDCl₃) δ 7.37 (5H, m, C₆H₅), 5.15

(2H, m, PhCH₂), 4.15 (2H, m, CO₂CH₂CH₃), 3.91 (d, J ) 9.4

Hz, 1H) and 3.67 (m, 1H) and 3.49 (m, 2H) (CH₂CHNH₂, CH₂-

NCbz and CHNCbz), 2.99 (d, J ) 16.3 Hz, 0.5H) and 2.79 (d,

J ) 16.3 Hz, 0.5H) (EtOCOCHH), 2.33 (dd, J ) 10.0 Hz, J )

15.7 Hz, 1H, EtOCOCHH), 2.10 (m, 1H, CH₂CHHCHNH₂),

1.70 (m, 1H, CH₂CH₂CHNH₂), 1.38 (s, 2H, NH₂), 1.25 (m, 3H,

CH₃); MS (thermospray) m/z 307 (MH⁺), 613 (2M + H⁺); [R]_D

-11.3° (c 1.33, MeOH). Anal. (C₁₆H₂₂N₂O₄) C, H, N.

turnover of the compound within this period. LC-ESI-

MS analysis of the products of a tryptic digestion of the

acylated complex formed with the enzyme and 55

revealed a peptide having an increased mass relative

to that expected, which corresponded to the molecular

weight of peptide plus 55. ESI-MS-MS analysis of this

peptide revealed serine 132 to be the active site hydroxyl

nucleophile. This is in agreement with published crystal

structures^3-6and site-directed mutagenesis and affinity

labeling studies.^2,15

Con clu sion s

Having previously defined the stereochemistry⁷of the

substituent next to the lactam carbonyl (for accessing

the S1 specificity site) required for activity against

HCMV δAla protease, we have now defined its size and

shape and have shown the preferred chirality of this

R-Me substituent to be (S). A stereospecific synthesis

has been developed for the chiral (SRS)-R-methylpyr-

rolidine-5,5-trans-lactam template required for potent

inhibitors of human cytomegalovirus (HCMV) protease.

Optimization of the substituent on the lactam nitrogen

has shown that activity against HCMV δAla protease

is in the order CO-cyclopropyl > COMe > CO₂Me > SO₂-

Me > CONHMe. Optimization of the functionality on

the pyrrolidine nitrogen gave the highly potent dansyl-

(S)-proline derivatives 55 and 62 (with K_i’s in the low

nanomolar range against HCMV δAla protease), which

are highly selective over the mammalian enzymes

elastase, thrombin and acetylcholine esterase. Mecha-

nism of action studies using ESI-MS together with

enzymatic degradation of the acylated HCMV protease

showed that these inhibitors acylate HCMV protease

at the active site serine (Ser 132) in a time-dependent

and reversible manner. The crystal structure of HCMV

protease was obtained and used to model the confor-

mationally restricted, chiral (S)-proline-R-methyl-5,5-

trans-lactams into the active site groove of the enzyme,

enabling us to direct and rationalize the SAR in this

series. In summary, using SAR, we have developed the

racemic defined template 2 (Figure 4) with micromolar

activity against HCMV protease into the chiral proline

trans-lactam 62 with low nanomolar potency against the

viral enzyme.

Exp er im en ta l Section

Gen er a l P r oced u r es. Melting points were obtained using

an electrothermal digital melting point apparatus and are

uncorrected. All purifications by flash chromatography were

performed using Kieselgel 60, Merck 9385 silica gel. Monitor-

ing of reactions by TLC used Merck 60 F₂₅₄silica gel glass

backed plates (5 cm × 10 cm), and the products were eluted

with mixtures of ethyl acetate and cyclohexane and visualized

by UV light, followed by heating with aqueous phosphomo-

lybdic acid. Analytical HPLC measurements were run on a

Hewlett-Packard 1090 HPLC instrument equipped with an

(3a S,6a R)-5-Oxoh exa h yd r op yr r olo[3,2-b]p yr r ole-1-ca r -

boxylic Acid Ben zyl Ester (8). To 7 (52.9 g, 173 mmol) in

tetrahydrofuran (550 mL), under nitrogen and in an ice-salt

bath was added dropwise a solution of tert-butylmagnesium

chloride (554 mL of a 1 M solution in tetrahydrofuran, 554

mmol), keeping the temperature at <1 °C. The mixture was

warmed to room temperature over 1 h and 15 min, then

Inhibitors of Human Cytomegalovirus Protease

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 11

quenched with saturated ammonium chloride while cooling in

an ice bath. The phases were separated, and the aqueous phase

was extracted with ethyl acetate. The combined organics were

washed with water and brine, dried, and evaporated to give

the title compound as a cream solid, 43.5 g (97%). A portion of

the solid was purified by flash column chromatography using

ethyl acetate as the eluting solvent to give the lactam 8 as a

a golden oil. The crude residue was purified by flash column

chromatography, eluting with cyclohexanes/ethyl acetate (3:

1) to yield 10 (526 mg, 83%) as a white foam: IR (KBr) ν_max

1789, 1765, 1712 cm^-1; ¹H NMR (CDCl₃) δ 7.35 (s, 5H, C₆H₅),

5.12 (m, 2H, PhCH₂), 3.87 (m, 1H, NCHHCH₂), 3.68 (m, 2H,

NCHHCH₂and NCHCH₂), 3.44 (dd, J ) 6.6 Hz, J ) 11 Hz,

1H, NCHCHMe), 3.05 (br m, 1H, CHMe), 2.51 (m, 1H,

NCH₂CHH), 1.99 (m, 1H, NCH₂CHH), 1.54 (s, 9H, C(CH₃)₃),

1.12 (bs, 3H, CH₃); MS (thermospray) m/z 375 (MH⁺); HPLC

99.5% (t_R) 28.27 min); [R]_D-88.6° (c 1.1, MeOH); chiral HPLC

(Chiral Pak AD 464) 96.1% ee (propan-2-ol/heptane (2:23), flow

rate ) 1 mL/min, t_R) 20.85 min (3S,3aR,6aS), t_R) 3.71 min

(3R,3aS,6aR)). Anal. (C₂₀H₂₆N₂O₅) C, H, N.

white solid, mp 157-159 °C: IR (KBr) 3277, 1699, 1690 cm^-1

;

¹H NMR (CDCl₃) δ 7.35 (m, 5H, C₆H₅), 5.99 (s, 1H, NH), 5.12

(ABq, J ) 12.5 Hz, 2H, PhCH₂), 3.87 (dd, J ) 10.4 Hz, J )

12.4 Hz, 1H, NCHHCH₂), 3.71 (m, 1H, NCHHCH₂), 3.34 (m,

2H, NCHCH₂and NCHCH₂CO), 2.81 (m, 1H, CHHCO), 2.45

(m, 1H, CHHCO), 2.23 (m, 1H, NCH₂CHH), 1.87 (m, 1H,

NCH₂CHH); HPLC 97.5% (t_R) 18.56 min); MS (thermospray)

m/z 261 (MH⁺), 278 (MNH₄⁺); [R]_D-68.4° (c 1.28, MeOH);

chiral HPLC (Chiral Pak AD464) 95.9% ee (propan-2-ol/

heptane (1:4), flow rate ) 1 mL/min, t_R) 9.42 min (3aS,6aR),

t_R) 8.47 min (3aR,6aS)). Anal. (C₁₄H₁₆N₂O₃‚0.05CH₂Cl₂) C,

H, N.

The following compounds were similarly prepared.

r el-(3S,3a R,6a S)-3-Allyl-2-oxoh exa h yd r op yr r olo[3,2-b]-

p yr r ole-1,4-d ica r boxylic Acid 4-Ben zyl Ester 1-ter t-Bu tyl

Ester (14). Compound 13 was reacted with allyl iodide as

described for 10 to give 14 (40%) as a white foam: ¹H NMR

(CDCl₃) δ 7.35 (s, 5H, C₆H₅), 5.9-5.7 (bs, 1H, CH₂CHdCH₂),

5.3-4.9 (m, 4H, PhCH₂and CH₂CHdCH₂), 3.90-3.60 (m, 3H,

NCH₂CH₂and NCHCH₂), 3.50 (dd, J ) 6.6 Hz, J ) 11 Hz,

1H, NCHCHallyl), 3.2-2.7 (br m, 1H, CHallyl), 2.6-2.2 (m,

3H, CH₂CHdCH₂and NCH₂CHH), 2.00 (m, 1H, NCH₂CHH),

1.6 (s, 9H, C(CH₃)₃); MS (thermospray) m/z 419 (MNH₄⁺), 401

(MH⁺); HRMS calcd for C₂₂H₂₈N₂O₅(MH⁺) 401.207 647, found

401.207 134; HPLC 98% (t_R) 30.64 min).

(3a R,6a S)-2-Oxoh exa h yd r op yr r olo[3,2-b]p yr r ole-1,4-

d ica r boxylic Acid 4-Ben zyl Ester 1-ter t-Bu tyl Ester (9).

To 8 (43.2 g, 166 mmol) in tetrahydrofuran (1200 mL) at -75

°C under nitrogen was added 1 M LHMDS solution in

tetrahydrofuran (216 mL, 216 mmol) dropwise, keeping the

temperature at -70 °C. After 10 min, a solution of di-tert-butyl

dicarbonate (54.3 g, 249 mmol) in tetrahydrofuran (350 mL)

was added, keeping the temperature at -70 °C. The reaction

mixture was stirred at -75 °C for 2.5 h, and then the reaction

was quenched with saturated ammonium chloride. Then the

mixture was allowed to warm to room temperature, water was

added, and the phases were separated. The aqueous phase was

extracted with ethyl acetate, and the combined organic phase

was washed with water and brine, dried, and evaporated to

give the title compound as an orange-red semisolid. This

residue was purified by trituration under diethyl ether to give

the title compound 9 (42.5 gm, 71%) as a pale-cream solid. A

sample was crystallized from diethyl ether to give a white

tr a n s-3,3-Dim eth yl-2-oxoh exa h yd r op yr r olo[3,2-b]p yr -

r ole-1,4-d ica r boxylic Acid 4-Ben zyl Ester 1-ter t-Bu tyl

Ester (19). Compound 18 was reacted with methyl iodide as

described for 10 to give 19 (72%) as a white solid: IR (KBr)

ν_max1790, 1764, 1713 cm^{-1 1}H NMR (CDCl₃) δ 7.35 (s, 5H,

;

C₆H₅), 5.15 (q, 2H, PhCH₂), 3.9 (m, 1H, NCHHCH₂), 4.0-3.6

(m, 3H, NCH₂CH₂and NCHCH₂), 3.15 (d, J ) 10 Hz, 1H,

NCHCMe₂), 2.55 (m, 1H, NCH₂CHH), 1.95 (m, 1H, NCH₂-

CHH), 1.6 (s, 9H, C(CH₃)₃), 1.2 (bs, 6H, 2CH₃); IR (KBr) 1790,

1764, 1713 cm^-1. MS (thermospray), m/z 406 (MNH₄⁺), 389

(MH⁺); HPLC 99.67% (30.72 min). Anal. (C₂₁H₂₈N₂O₅) C, H,

N.

1

solid: mp 101-103 °C: IR (KBr) 1791, 1765, 1713 cm^-1; H

NMR (CDCl₃) δ 7.35 (s, 5H, C₆H₅), 5.12 (ABq, J ) 12.5 Hz,

2H, PhCH₂), 3.86 (m, 1H, NCHHCH₂), 3.75 (m, 1H, NCH-

HCH₂), 3.46 (m, 1H, NCHCH₂), 3.33 (m, 1H, NCHCHMe), 2.93

(m, 1H, CHHCO), 2.55 (m, 2H, NCH₂CHH and CHHCO), 2.01

(m, 1H, NCH₂CHH), 1.54 (s, 9H, C(CH₃)₃); MS (thermospray)

m/z 378 (MNH₄⁺); HPLC 100% (t_R) 27.2 min); [R]_D-45.6° (c

1.13, MeOH); chiral HPLC (Chiral Pak AD464) 96.0% ee

(propan-2-ol/heptane (2:5), flow rate ) 1 mL/min, t_R) 9.54

min (3aS,6aR), t_R) 6.79 min (3aR,6aS)). Anal. (C₁₉H₂₄N₂O₅)

C, H, N.

(3a S,6S,6a R)-6-Meth yl-5-oxoh exa h yd r op yr r olo[3,2-b]-

p yr r ole-1-ca r boxylic Acid Ben zyl Ester (11). To 10 (486

mg, 1.3 mmol) was added trifluoroacetic acid (6 mL), and the

mixture was stirred at room temperature for 40 min and then

evaporated to give a brown oil. This was dissolved in ethyl

acetate (6 mL) and was washed with saturated sodium

bicarbonate solution (2 × 3 mL), water (3 mL), and brine (3

mL), dried (MgSO₄), and evaporated to give 11 (340 mg, 95%)

as a pale-beige solid. The solid was recrystallized from diethyl

ether to give a white solid (202 mg, 56%), mp 112-113 °C: IR

(CDCl₃) 3273, 1710, 1698 cm^-1; ¹H NMR (CDCl₃) δ 7.35 (s, 5H,

C₆H₅), 5.98 (bs, 1H, NH), 5.13 (ABq, J ) 12.5 Hz, 2H, PhCH₂),

3.89 (br m, 1H, NCHHCH₂), 3.60 (m, 3H, NCHHCH₂, NCHCH₂,

and NCHCHMe), 2.90 (br m, 1H, CHMe), 2.22 (m, 1H,

NCH₂CHH), 1.86 (m, 1H, NCH₂CHH), 1.10 (br s, 3H, CH₃);

MS (thermospray) m/z 275 (MH⁺); HPLC 100% (t_R) 19.82

min); chiral HPLC (Chiral Pak AD 464) 96.00% ee (propan-

2-ol/heptane (2:25), flow rate ) 1 mL/min, t_R) 20.91 min

(3aS,6S,6aR), t_R) 18.85 min (3aR,6R,6aS)). Anal. (C₁₅H₁₈N₂O₃)

C, H, N.

The following compounds were similarly prepared.

r el-(3R,3a R,6a S)-3-Meth yl-2-oxoh exa h yd r op yr r olo[3,2-

b]p yr r ole-1,4-d ica r boxylic Acid 4-Ben zyl Ester 1-ter t-

Bu tyl Ester (18). Compound 17⁷was reacted with 1 M

LHMDS and then treated with di-tert-butyl dicarbonate as

described for 9 to give 18 (93%) as a white solid: mp 69-71

°C: ¹H NMR (CDCl₃) δ 7.35 (m, 5H, C₆H₅), 5.13 (m, 2H,

PhCH₂), 3.91-3.66 (m, 1H, NCH₂CH₂), 3.51-3.38 (m, 1H,

NCHCH₂), 3.05-2.94 (m, 1H, NCHCHMe), 2.71-2.45 (m, 2H,

CHMe and NCH₂CHH), 2.02-1.83 (m, 1H, NCH₂CHH), 1.56-

1.32 (m, 12H, C(CH₃)₃and CH₃); IR (KBr) 1787, 1766, 1713

cm^-1; MS (thermospray), m/z 392 (MNH₄⁺), 375 (MH⁺); HPLC

99.53% (29.46 min). Anal. (C₂₀H₂₆N₂O₅‚0.5H₂O) C, H, N.

(3S,3a R,6a S)-3-Meth yl-2-oxoh exa h yd r op yr r olo[3,2-b]-

p yr r ole-1,4-d ica r boxylic Acid 4-Ben zyl Ester 1-ter t-Bu tyl

Ester (10). Compound 9 (606 mg, 1.68 mmol) was dissolved

in tetrahydrofuran (6 mL) and cooled, under nitrogen, to -75

°C. A 1 M LHMDS solution in tetrahydrofuran (2.2 mL, 2.2

mmol) was added, keeping the temperature below -70 °C.

After 10 min, methyl iodide was added (1.8 mL, 28.9 mmol).

After the mixture was stirred for a further 45 min, the reaction

was quenched with saturated aqueous ammonium chloride and

then the mixture was allowed to warm to room temperature.

Water was added, and then the aqueous phase was extracted

with ethyl acetate. The combined organic phase was washed

with water and brine, dried (MgSO₄), and evaporated to give

The following compounds were similarly prepared.

r el-(3a S,6S,6a R)-6-Allyl-5-oxoh exa h yd r op yr r olo[3,2-b]-

p yr r ole-1-ca r boxylic Acid Ben zyl Ester (15). Compound

14 was reacted with trifluoroacetic acid as described for 11 to

give 15 (69%) as a white foam: ¹H NMR (CDCl₃) δ 7.40 (s,

5H, C₆H₅), 6.1 (bs, 1H, NH), 5.9-5.7 (bs, 1H, CH₂CHdCH₂),

5.2-4.9 (m, 4H, PhCH₂, CH₂CHdCH₂), 3.90 (m, 1H, NCH-

HCH₂), 3.7-3.5 (m, 3H, NCHHCH₂, NCHCH₂, and NCHCHal-

lyl), 3.0-2.7 (bm, 1H, CHallyl), 2.5-2.2 (m, 3H, CH₂CHdCH₂,

NCH₂CHH), 1.85 (m, 1H, NCH₂CHH); MS (thermospray) m/z

318 (MNH₄⁺), 301 (MH⁺); HRMS calcd for C₁₇H₂₀N₂O₃(MH⁺)

301.155 218, found 301.155 627; HPLC 98.13% (t_R) 22.21

min).

tr a n s-6,6-Dim eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr -

r ole-1-ca r boxylic Acid Ben zyl Ester (21). Compound 19

was reacted with trifluoroacetic acid as described for 11 to give

12 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

Borthwick et al.

21 (90%) as a white solid, mp 170 °C: ¹H NMR (CDCl₃): δ

7.35 (m, 5H, C₆H₅), 6.00 (s, 1H, NH), 5.14 (ABq, J ) 12 Hz,

2H, PhCH₂), 3.88 (br t, 1H, J ) 10.4 Hz, NCHHCH₂), 3.70 (m,

1H, NCHHCH₂), 3.46 (m, 1H, NCHCH₂), 3.20 (br d, J ) 10

Hz, NCHCMe₂), 2.21 (m, 1H, NCH₂CHH), 1.81 (m, 1H,

NCH₂CHH), 1.7-1.0 (br m, 6H, 2CH₃); MS (thermospray) m/z

289 (MH⁺), 577 (2M + H⁺); HPLC 99.5% (t_R) 21.85 min).

Anal. (C₁₆H₂₀N₂O₃) C, H, N.

of 4, the following compounds listed in Schemes 2-5 and 8

were prepared from 11, 15, 21, 24, or 61.

tr a n s-4-Acet yl-6,6-d im et h yl-5-oxoh exa h yd r op yr r olo-

[3,2-b]p yr r ole-1-ca r boxylic Acid Ben zyl Ester (22). 22 was

derived from 21 and acetyl chloride to give a pale-yellow gum

(76%): ¹H NMR (CDCl₃) δ 7.35 (m, 5H, C₆H₅), 5.13 (ABq, J )

12 Hz, 2H, PhCH₂), 3.87 (m, 1H, NCHHCH₂), 3.75-3.55 (m,

2H, NCHCH₂and NCHCH₂), 3.15 (br d, J ) 10 Hz, NCH-

CMe₂), 2.90 (m, 1H, NCH₂CHH), 2.70 (m, 1H, NCH₂CHH), 2.45

(s, 3H, COCH₃), 1.5-1.0 (br m, 6H, 2CH₃); MS (thermospray)

m/z 331 (MH⁺); HPLC 99.4% (t_R) 27.43 min). Anal. (C₁₈H₂₂N₂-

O₄‚0.1C₆H₁₂‚0.1EtOAc) C, H, N.

(3S,3aR,6aS)-4-[((2S)-1-{[5-(Dim eth ylam in o)-1-n aph th yl]-

su lfon yl}p yr r olid in -2-yl)ca r bon yl]-3-m eth ylh exa h yd r o-

p yr r olo[3,2-b]p yr r ol-2-on e (61). Deprotection of 60 (1.12 g,

1.96 mmol) with trifluoroacetic acid, using the same procedure

as for the preparation of 11, afforded 61 (870 mg, 95%): IR

(KBr) ν_max3280, 1713, 1666 cm^-1; ¹H NMR (CDCl₃) δ 8.53 (d,

J ) 8.5 Hz, 1H, dansyl-2H), 8.42 (d, J ) 8.5 Hz, 1H, dansyl-

4H), 8.28 (dd, J ) 1.2 Hz, J ) 7.3 Hz, 1H, dansyl-8H), 7.60-

7.48 (m, 2H, dansyl-3H, dansyl-7H), 7.18 (d, J ) 7.3 Hz, 1H,

dansyl-6H), 5.79 (s, 1H, CONH), 4.74 (dd, J ) 4.3 Hz, J ) 7.9

Hz, 1H, NCHCO), 4.29 (t, J ) 9.5 Hz, 1H, NCHHCH₂CH₂),

3.85-3.74 (m, 1H, NCHHCH₂CH₂), 3.65-3.34 (m, 4H, NCH₂-

CH₂, NCHCH₂, NCHCHMe), 3.10-2.98 (m, 1H, CHMe), 2.87

(s, 6H, NMe₂), 2.33-1.84 (m, 6H, NCH₂CH₂CH₂, NCH₂CH₂),

1.06 (d, J ) 7.3 Hz, 3H, CHCH₃); ¹³C NMR (CDCl₃) δ 179.5,

155.8, 136.2, 128.6, 128.2, 128.0, 67.2, 63.4, 60.5, 49.3, 39.6,

(3a S,6S,6a R)-4-Acet oxya cet yl-6-m et h yl-5-oxoh exa h y-

d r op yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid Ben zyl Ester

(12). 12 was derived from 11 and acetoxyacetyl chloride to give

a white gum (75%): IR (CDCl₃) ν_max1750, 1715, 1706 cm^-1

;

¹H NMR (CHCl₃) δ 7.36 (m, 5H, C₆H₅), 5.13 (ABq, J ) 11 Hz,

2H, PhCH₂), 5.01 (m, 2H, COCH₂O), 3.95-3.62 (m, 3H, NCH₂-

CH₂and NCHCH₂), 3.51 (dd, 1H, NCHCHMe), 3.10 (br m, 1H,

COCHCH₃), 2.72 and 2.02 (2m, 2H, NCH₂CH₂), 2.18 (s, 3H,

COCH₃), 1.18 (br m, 3H, CHCH₃); HPLC 98.86% (t_R) 25.41

min); MS (thermospray) m/z 375 (MH⁺); chiral HPLC (Chiral

Pak AD464) 96.3% ee (propan-2-ol/heptane (2:5), flow rate )

1 mL/min, t_R) 10.56 min (3aS,6S,6aR), t_R) 3.015 min

(3aR,6R,6aS)). Anal. (C₁₉H₂₂N₂O₆‚0.1 cyclohexane) C, H, N.

r el-(3a S,6S,6a R)-4-Acetoxya cetyl-6-a llyl-5-oxoh exa h y-

d r op yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid Ben zyl Ester

(16). 16 was derived from 15 and acetoxyacetyl chloride to give

a white foam (78%): ¹H NMR (CDCl₃) δ 7.40 (s, 5H, C₆H₅),

5.9-5.6 (bs, 1H, CH₂CHdCH₂), 5.2-4.9 (m, 6H, PhCH₂, CH₂-

OAc, and CH₂CHdCH₂), 3.90 (m, 1H, NCHHCH₂), 3.78 (m,

1H, NCHCH₂), 3.70 (m, 1H, NCHHCH₂), 3.60 (m, 1H, NCH-

CHallyl), 3.3-2.9 (bm, 1H, CHallyl), 2.77 (m, 1H, NCH₂CHH),

2.50 (bs, 1H, CHHCHdCH₂), 2.35-2.15 (bs, 1H, CHHCHd

CH₂), 2.20 (s, 3H, OAc), 2.00 (m, 1H, NCH₂CHH); MS (ther-

mospray) m/z 418 (MNH₄⁺), 401 (MH⁺); HRMS calcd for

C₂₁H₂₄N₂O₆(MH⁺) 401.171 262, found 401.171 325; HPLC 94%

(t_R) 27.93 min).

27.7; MS (thermospray) m/z 471 (MH⁺); HPLC: 99.2% (t_R

18.3 min). Anal. (C₂₄H₃₀N₄O₄S‚0.4EtOAc) C, H, N, S.

)

(3a S,6S,6a R)-4-Acet yl-6-m et h yl-5-oxoh exa h yd r op yr -

r olo[3,2-b]p yr r ole-1-ca r boxylic Acid Ben zyl Ester (4). The

lactam 11 (50 mg, 0.18 mmol) in tetrahydrofuran (2.5 mL)

stirred under nitrogen at -75 °C was treated with 1 M

LHMDS solution in tetrahydrofuran (0.22 mL, 0.22 mmol). The

solution was stirred and allowed to warm to -15 °C over 15

min, recooled to -75 °C, and treated with acetyl chloride (39

µL, 0.55 mmol). The solution was stirred and allowed to warm

to -15 °C over 1.5 h, and then the reaction was quenched with

saturated aqueous ammonium chloride (2.5 mL). After warm-

ing to room temperature over 1 h, the mixture was diluted

with water (2.5 mL) and extracted with ethyl acetate (3 × 2.5

mL). The combined organic extracts were washed with brine

(2.5 mL), dried, and evaporated to give a yellow gum (62 mg).

This gum was purified by flash column chromatography over

silica using cyclohexanes/ethyl acetate 2:1 as the eluting

solvent to give 4 (47 mg, 81%) as a white foam: IR (KBr) ν_max

1747, 1712, 1698 cm^-1; ¹H NMR (CDCl₃) δ 7.36 (s, 5H, C₆H₅),

5.13 (ABq, J ) 11 Hz, 2H, PhCH₂), 3.87-3.62 (m, 3H, NCH₂-

CH₂and NCHCH₂), 3.45 (dd, J ) 7.5 Hz, J ) 12 Hz, 1H,

NCHCHMe), 3.10 (br m, 1H, CHMe), 2.74 (m, 1H, NCH₂CHH),

2.46 (s, 3H, COCH₃), 1.97 (m, 1H, NCH₂CHH), 1.13 (br m, 3H,

CH₃CH); HPLC 99.48% (t_R) 24.72 min); MS (thermospray)

m/z 317 (MH⁺); circular dichroism (CH₃CN) λ_max211.2 nm,

dE -25.20, E16538, λ_max238.2 nm, dE 18.10, E1538; chiral

HPLC (Chiral Pak AD464) 96.2% ee (ethanol/heptane (2:5),

flow rate ) 1 mL/min, t_R) 12.12 min (3aS,6S,6aR), t_R) 19.6

min (3aR,6R,6aS)). Anal. (C₁₇H₂₀N₂O₄) C, H, N.

tr a n s-4-Acet oxya cet yl-6,6-d im et h yl-5-oxoh exa h yd r o-

p yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid Ben zyl Ester

(23). 23 was derived from 21 and acetoxyacetyl chloride to give

a white solid (82%), mp 108-109 °C: ¹H NMR (CDCl₃): δ 7.35

(m, 5H, C₆H₅), 5.20-4.95 (m, 4H, PhCH₂and COCH₂O), 4.95-

3.60 (m, 3H, NCH₂CH₂and NCHCH₂), 3.20 (br d, NCHCMe₂),

2.70 (m, 1H, NCH₂CHH), 1.97 (m, 1H, NCH₂CHH), 1.7-1.0

(br, 9H, 3CH₃); MS (thermospray) m/z 389 (MH⁺), 406 (MNH₄⁺);

HPLC 99.2% (t_R) 27.9 min). Anal. (C₂₀H₂₄N₂O₆‚0.12C₆H₁₂

C, H, N.

)

r el-(3a S,6S,6a R)-4-Isob u t yr yl-6-m et h yl-5-oxoh exa h y-

d r op yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid Ben zyl Ester

26. 26 was derived from 24 and isobutyryl chloride to give a

gum (57%): ¹H NMR (CDCl₃) δ 7.45 (s, 5H, C₆H₅), 5.2 (ABq, J

) 11 Hz, 2H, PhCH₂), 4.0-3.4 (3m, 5H, (CH₃)₂CH, NCH₂CH₂,

NCHCH₂, and NCHCHMe), 3.3-2.8 (2bm, 1H, CHMe), 2.74

(dt, J ) 6 Hz, 1H, NCH₂CHH), 1.97 (2t, J ) 11 Hz, 1H, NCH₂-

CHH), 1.3-1.0 (m, 3H, (CH₃)₂CH); MS (thermospray) m/z 345

(MH⁺); HPLC 95% (t_R) 29.84 min). Anal. (C₁₉H₂₄N₂O₄‚0.4H₂O)

C, H, N.

(3aS,6S,6aR)-4-Acetyl-6-m eth yl-5-oxoh exah ydr opyr r olo-

[3,2-b]p yr r ole-1-ca r boxylic Acid Ben zyl Ester (4) a n d

(3a R,6R,6a S)-4-Acetyl-6-m eth yl-5-oxoh exa h yd r op yr r olo-

[3,2-b]p yr r ole-1-ca r boxylic Acid Ben zyl Ester (3). The

racemic trans-lactam 1⁷was separated by preparative chiral

HPLC into the two enantiomers of 4: chiral HPLC (Chiral Pak

AD464) 99.5% (t_R) 12.12 min), identical in all respects to

r el-(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth yl-5-

oxoh exa h yd r op yr r olo[3,2-b]p yr r ole-1-ca r b oxylic Acid

Ben zyl E st er (28). 28 was derived from 24 and cyclopro-

panecarbonyl chloride to give as an oil (87%) that crystallized

to a white solid on standing, mp 101-102 °C: IR (KBr) ν_max

compound 4 above and 3; IR (KBr) ν_max1747, 1712, 1697 cm^-1

;

¹H NMR (CDCl₃) δ 7.37 (s, 5H, C₆H₅), 5.14 (q, J ) 11 Hz, 2H,

PhCH₂), 3.86 and 3.7 (m, 3H, NCH₂CH₂and NCHCH₂), 3.45

(dd, J ) 7.5 Hz, J ) 12 Hz, 1H, NCHCHMe), 3.1 (bm, 1H,

CHMe), 2.74 (m, 1H, NCH₂CHH), 2.46 (s, 3H, COCH₃), 1.97

(m, 1H, NCH₂CHH), 1.14 (br, 3H, CH₃CH); HRMS calcd for

1

1747, 1713, 1704, 1693, 1681 cm^-1; H NMR (CDCl₃) δ 7.35

(s, 5H, C₆H₅), 5.15 (AB q, J ) 12.5 Hz, 2H, PhCH₂), 4.0-3.60

and 3.50 (m, 4H, NCH₂CH₂, NCHCH₂, and NCHCHMe), 3.2

and 3.0 (br d, 1H, CHMe), 2.92 (br, 1H, COCHCH₂CH₂), 2.70

(m, 1H, NCH₂CHH), 1.95 (m, 1H, NCH₂CHH), 1.3-0.9 (m, 7H,

COCHCH₂CH₂and CH₃CH); MS (thermospray) m/z 343

(MH)⁺; HPLC 99.4% (t_R) 28.7 min). Anal. (C₁₉H₂₂N₂O₄) C,

H, N.

C

₁₇H₂₀N₂O₄(MH⁺) 317.150 132, found 317.150 109; chiral

HPLC (Chiral Pak AD464) 99.8% (t_R) 19.6 min); circular

dichroism (CH₃CN) λ_max211.0 nm, dE 25.40, E17153, λ_max

238.2 nm, dE -18.30, E1946.

P r ep a r a tion of 12, 16, 22, 23, 26, 28, 33, 34, a n d 62. By

use of essentially the same procedure as for the preparation

(3S ,3a R ,6a S )-1-(C y c lo p r o p y lc a r b o n y l)-4-[((2S )-1-

{[5-(d im e t h yla m in o)-1-n a p h t h yl]su lfon yl}p yr r olid in -

2-yl)car bon yl]-3-m eth ylh exah ydr opyr r olo[3,2-b]pyr r ol-2-

Inhibitors of Human Cytomegalovirus Protease

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 13

on e (62). 62 was derived from 61 and cyclopropanecarbonyl

MS (thermospray) m/z 461 (MH⁺) , 483 (MNa⁺); HPLC 95%

(t_R) 37 min). Anal. (C₂₄H₃₆N₂O₅Si) C, H, N.

chloride (3 equiv) to give 62 (80%) as a green-yellow foam: IR

(KBr) ν_max1747, 1689, 1682, 1667, 1651 cm^{-1 1}H NMR

;

r el-(3a S,6S,6a R)-4-(3-Hyd r oxyp r op a n oyl)-6-m eth yl-5-

oxoh exa h yd r op yr r olo[3,2-b]p yr r ole-1-ca r b oxylic Acid

Ben zyl Ester (37). The trans-lactam 36 (75 mg, 0.16 mmol)

was treated with acetic acid/water/tetrahydrofuran (10:3:1, 7

mL), and the solution was stirred at room temperature. After

8 h, solid NaHCO₃was added and the reaction mixture was

diluted with ethyl acetate (15 mL) and water (10 mL). The

organic phase was washed with saturated sodium hydrogen

carbonate solution until the solution was neutral and then was

washed with water (3 × 15 mL) and then dried (MgSO₄).

Evaporation of the solvent in vacuo gave a yellow oil (54 mg)

that was purified by flash column chromatography using

cyclohexanes/ethyl acetate as the eluting solvent to give 37

as a colorless gum (49 mg, 88%): IR (KBr) ν_max1749, 1705,

(CDCl₃): δ 8.54 (d, J ) 8.5 Hz, 1H, dansyl-2H), 8.42 (d, J )

8.5 Hz, 1H, dansyl-4H), 8.28 (dd, J ) 1.2 Hz, J ) 7.3 Hz, 1H,

dansyl-8H), 7.61-7.49 (m, 2H, dansyl-3H, dansyl-7H), 7.18 (d,

J ) 7.3 Hz, 1H, dansyl-6H), 4.75 (dd, J ) 4.9 Hz, J ) 7.9 Hz,

1H, NCHCO), 4.25 (t, J ) 9.5 Hz, 1H, NCHHCH₂CH₂), 3.87-

3.36 (m, 5H, NCHHCH₂CH₂, NCH₂CH₂, NCHCHMe, NCHCH₂),

3.27 (m, 1H, CHMe), 2.99-2.83 (m, 7H, NMe₂, COCHCH₂CH₂),

2.81-2.68 (m, 1H, NCH₂CHH), 2.29-1.81 (m, 5H, NCH₂CH₂-

CH₂, NCH₂CHH), 1.28-0.87 (m, 7H, CHMe, COCHCH₂CH₂);

MS (thermospray) m/z 539 (MH⁺), 471 (MH-CO-cyclopropyl⁺);

HPLC 100% (t_R) 26.3 min). Anal. (C₂₈H₃₄N₄O₅S‚0.5H₂O) C,

H, N.; circular dichroism λ_max200.8 nm, dE 0.01, λ_max217.8

nm, dE -23.80, λ_max238.4 nm, dE 15.10.

r el-(3a S,6S,6a R)-4-(Cyclob u t ylca r b on yl)-6-m et h yl-5-

oxoh exa h yd r op yr r olo[3,2-b]p yr r ole-1-ca r b oxylic Acid

Ben zyl E st er ) (33). 33 was derived from 24 and cyclobu-

tanecarbonyl chloride to a colorless gum (82%): IR (KBr) ν_max

1749, 1710, 1694 cm^-1; ¹H NMR (CDCl₃) δ 7.37 (m, 5H, C₆H₅),

5.13 (ABq, J ) 12.5 Hz, 2H, PhCH₂), 3.88 and 3.71 (2m, 4H,

NCH₂CH₂, NCHCH₂, and CH₂CH₂CH₂CHCO), 3.45 (dd, J )

7.5 Hz, J ) 12 Hz, 1H; NCHCHMe), 3.06 (br d, 1H, CHMe),

2.75 and 1.95 (2m, 4H, NCH₂CH₂and CH₂CH₂CH₂), 2.26 (m,

4H, CH₂CH₂CH₂), 1.12 (br, 3H, CH₃); MS (thermospray) m/z

357 (MH⁺); HPLC 99.6% (t_R) 30.24 min). Anal. (C₂₀H₂₄N₂O₄‚

0.1EtOAc) C, H, N.

1700 cm^-^{1 1}NMR (CDCl₃) δ 7.4 (s, 5H, C₆H₅), 5.1 (d, 2H,

;

PhCH₂), 4.0-3.8 (m, 3H, NCH₂CH₂, NCHCH₂), 3.8-3.6 (m,

2H, NCHCHMe, CHHOH), 3.5 (m, 1H, CHHOH), 3.3-2.9 (m,

3H, CHMe, COCH₂), 2.7 (m, 1H, NCH₂CHH), 2.4 (m, 1H, OH),

1.99 (m, 1H, NCH₂CHH), 1.2 (br s, 3H, CH₃); MS (thermo-

spray) m/z 347 (MH⁺); HRMS calcd for C₁₈H₂₂N₂O₅(MH⁺)

347.160 697, found 347.160 451; HPLC 98% (t_R) 21.6 min).

r el-(3a S,6S,6a R)-4-F or m yl-6-m eth yl-5-oxoh exa h yd r o-

p yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid Ben zyl Ester

(25). To a stirred solution of trans-lactam 24 (52 mg, 0.19

mmol) in dry dimethylformamide (22.6 mL) and dry dimeth-

oxyethane (1.3 mL) at 0 °C under nitrogen was added sodium

hydride (60% dispersion in mineral oil; 12.5 mg, 0.31 mmol).

After 30 min, the reaction mixture was cooled to -78 °C and

trifluoromethanesulfonic anhydride (96 µL, 0.57 mmol) was

added. After 3 min, the reaction mixture was rapidly warmed

to room temperature. The reaction was quenched by addition

of a saturated ammonium chloride solution and extracted with

ethyl acetate (3 × 25 mL). The combined organic extract was

washed with saturated sodium hydrogen carbonate solution

(20 mL) and brine (20 mL) and dried over MgSO₄. The solvent

was evaporated to give a pale-yellow gum (63 mg), which was

purified by flash column chromatography using cyclohexane/

ethyl acetate as the eluting solvent to give 25 as a colorless

r el-(3a S,6S,6a R)-4-(4-Meth oxyben zoyl)-6-m eth yl-5-oxo-

h exa h yd r op yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid Ben -

zyl Ester (34). 34 was derived from 24 and 4-methoxybenzoyl

chloride to give a clear oil (54%): IR (KBr) ν_max4199, 1753,

1747 cm^-1; ¹H NMR (CDCl₃) δ 7.75 (d, J ) 9 Hz, 2H, COCCH),

7.5-7.3 (m, 5H, C₆H₅), 6.92 (d, J ) 9 Hz, 2H, MeOCCH), 5.15

(m, 2H, PhCH₂), 4.1 (s, 3H, MeO), 4.0-3.75 (br m, 1H,

NCHHCH₂), 3.7-3.4 (m, 3H, NCHHCH₂, NCHCH₂, and NCH-

CHMe), 3.2-2.7 (br m, 1H, CHMe), 2.8-2.6 (br m, 1H,

NCH₂CHH), 2.1-1.8 (br m, 1H, NCH₂CHH), 1.3-1.0 (br, 3H,

CH₃); HPLC 99.2% (t_R) 28.9 min). Anal. (C₂₃H₂₄N₂O₅‚

0.2CHCl₃) C, H, N.

gum (31 mg, 54%): IR (KBr) ν_max1759, 1699 cm^{-1 1}NMR

;

r el-(3a S,6S,6a R)-4-(3-{[ter t-Bu tyl(d im eth yl)silyl]oxy}-

p r op a n oyl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr -

r ole-1-ca r boxylic Acid Ben zyl Ester (36). To a solution of

3-[[(1,1-dimethylethyl)dimethylsilyl]oxy]propanoic acid¹⁶(0.257

g, 1.3 mmol) in tetrahydrofuran (7 mL), cooled in an ice bath,

was added triethylamine (0.168 mL, 1.2 mmol) and trimethyl-

acetyl chloride (0.148 mL, 1.2 mmol). The resulting white

suspension of mixed anhydride was stirred under nitrogen for

3 h. Meanwhile, to a solution of trans-lactam 24 (0.113 g, 0.41

mmol) in anhydrous tetrahydrofuran (7 mL) cooled to -78 °C

under nitrogen was added 1 M LHMDS solution in tetrahy-

drofuran (0.494 mL, 0.49 mmol) over ca. 2 min. The solution

was stirred at -78 °C for 20 min, then stirred at 0 °C for 10

min and then recooled to -78 °C. The suspension of mixed

anhydride was added to the trans-lactam solution, keeping the

temperature below -70 °C, and the solution was stirred at

-70 °C for a further 35 min. Saturated aqueous ammonium

chloride (20 mL) was added, the cooling bath was removed,

and the mixture was stirred for 10 min. Ethyl acetate (75 mL)

and water (40 mL) were added to the stirred mixture, and the

organic phase was separated. The aqueous phase was re-

extracted with ethyl acetate (40 mL). The combined organic

phase was washed with saturated ammonium chloride solution

(40 mL) and brine (40 mL), dried (MgSO₄), and evaporated to

give a yellow oil. This was purified by flash column chroma-

tography using cyclohexanes/ethyl acetate as the eluting

solvent to give 36 as a pale-yellow gum (111 mg, 59%): IR

(CDCl₃) δ 9.1 (s, 1H, CHO), 7.4 (s, 5H, C₆H₅), 5.1 (d, 2H,

PhCH₂), 4.0-3.9 (br.m, 1H, NCHHCH₂), 3.8-3.6 (m, 2H,

NCHHCH₂, NCHCH₂), 3.5 (dd,1H, NCHCHMe), 3.3-2.9 (br.s,

1H, CHMe), 2.7 (m, 1H, NCH₂CHH), 2.1 (m, 1H, NCH₂CHH),

1.2 (m, 3H, CHMe); MS (thermospray) m/z 320 (MNH₄⁺), 303

(MH⁺). Anal. (C₁₆H₁₈N₂O₄) C, H, N.

P r ep a r a tion of 27, 29-32, 35. By use of essentially the

same procedure as for the preparation of 36, the following

compounds listed in Scheme 5 were prepared from 24.

r el-(3a S,6S,6a R)-4-(2,2-Dim eth ylp r op a n oyl)-6-m eth yl-

5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid

Ben zyl Ester (27). 27 was derived from 24 and the mixed

anhydride prepared from 2,6-dimethylbenzoic acid and tri-

methylacetyl chloride to give a white solid (70%): IR (KBr)

1

ν_max1747, 1712, 1683 cm^-1; H NMR (CHCl₃) δ 7.36 (m, 5H,

C₆H₅), 5.13 (q, J ) 11 Hz, 2H, PhCH₂), 3.92-3.60 (m, 3H,

NCH₂CH₂and NCHCH₂), 3.49 (dd, J ) 7.5 Hz, J ) 12 Hz,

1H; NCHCHMe), 3.06 (br d, 1H, CHMe), 2.65 and 1.81 (2m,

2H, NCH₂CH₂), 1.30 (s, 9H, Bu^t), 1.13 (br, 3H, CH₃); MS

(thermospray) m/z 359 (MH⁺); HPLC 99.7% (t_R) 32.07 min).

Anal. (C₂₀H₂₆N₂O₄) C, H, N.

r el-(3a S,6S,6a R)-6-Met h yl-4-[(2-m et h ylcyclop r op yl)-

ca r b on yl]-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ole-1-ca r -

boxylic Acid Ben zyl Ester (31). 31 was derived from 24 and

the mixed anhydride prepared from 2-methylcyclopropane-

carboxylic acid and trimethylacetyl chloride to give a white

solid (73%): IR (KBr) ν_max1747, 1708, 1685 cm^{-1 1}H NMR

;

(KBr) ν_max1749, 1712 cm^{-1 1}HNMR (CDCl₃) δ 7.3 (s, 5H,

;

(CHCl₃) δ 7.36 (m, 5H, C₆H₅), 5.13 (ABq, J ) 12.5 Hz, 2H,

PhCH₂), 3.95-3.60 (m, 3H, NCH₂CH_2,NCHCH₂), 3.48 (m, 1H,

NCHCHMe), 3.10 (br m, 1H, CHMe), 2.67 and 1.95 (2m, 3H,

COCHCH₂CH₂and NCH₂CH₂), 1.50, 1.31 and 0.84 (3m, 3H,

COCHCH₂CHMe), 1.16 (m, 6H, 2 × CH₃); MS (thermospray)

m/z 357 (MH⁺); HPLC 98.4% (t_R) 29.74 min); chiral HPLC

C₆H₅), 5.1 (m, 2H, PhCH₂), 3.9-3.7 (m, 3H, NCH₂CH₂,

NCHCH₂), 3.7-3.6 (m, 2H, NCHCHMe, CHHOSiMe₂Bu^t), 3.4

(m, 1H, CHHOSiMe₂Bu^t), 3.1-2.9 (m, 3H, CHMe, CH₂CO-

(CH₂)₂), 2.7 (m, 1H, NCH₂CHH), 1.9 (m, 1H, NCH₂CHH), 1.2-

1.0 (m, 3H, CHMe), 0.82 (s, 9H, t-BuSi), 0.06 (s, 6H, Me₂Si);

14 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

(run in ethanol/heptane (2:3), 1 mL/min, room temp), t_R

5.415 min 16.36%, t_R) 5.656 min, 22.13%, t_R) 5.844 min,

29.86%, t_R) 6.173 min, 29.14%; chiral HPLC (run in ethanol/

heptane (3:97), 0.5 mL/min, temp of 40 °C), t_R) 29.487 min,

20.06%, t_R) 31.162 min, 50.5%, t_R) 34.396 min, 23.1%. Anal.

(C₂₀H₂₄N₂O₄‚0.1EtOAc) C, H, N.

Borthwick et al.

)

r el-(3a S,6S,6a R)-4-(Dim et h oxyp h osp h or yl)-6-m et h yl-

5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid

Ben zyl Ester (41). 41 was derived from 24 and dimethyl

chlorophosphate (3.5 equiv) to give a colorless gum 45%): IR

(KBr) ν_max1739, 1709 cm^-1; H NMR (CDCl₃) δ 7.36 (s, 5H,

1

C₆H₅), 5.20-5.05 (ABq, J ) 12.2 Hz, 2H, PhCH₂), 3.96-3.45

(br m, 10H, NCH₂CH₂, NCHCH₂, NCHCHMe, 2 × OCH₃),

3.30-2.75 (br m, 1H, CHMe), 2.59-2.47 (m, 1H, NCH₂CHH),

2.10-1.85 (m, 1H, NCH₂CHH), 1.27-1.05 (br m, 3H, CH₃);

MS (thermospray) m/z 400 (MNH₄⁺), 383 (MH⁺); HPLC 92%

(t_R) 22.2min). Anal. (C₁₇H₂₃N₂O₆P) C, H, N.

r el-(3a S,6S,6a R)-4-(cis-2,3-Dim eth ylcyclop r op a n eca r -

bon yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ole-1-

ca r boxylic Acid Ben zyl Ester (32). 32 was derived from 24

and the mixed anhydride prepared from cis-2,3-dimethylcy-

clopropane carboxylic acid and trimethylacetyl chloride to a

colorless gum (40%): IR (KBr) ν_max1746, 1713, 1682 cm^-1; ¹H

NMR (CHCl₃) δ 7.36 (m, 5H, C₆H₅), 5.13 (ABq, J ) 12.5 Hz,

2H, PhCH₂), 3.93-3.58 (m, 3H, NCH₂CH_2,NCHCH₂), 3.48 (m,

1H, NCHCH), 3.08 (br m, 1H, CHMe), 2.82 (m, 1H, COCH-

CHMeCHMe), 2.70 and 1.91 (2m, 2H, NCH₂CH₂), 1.61 (m, 2H,

CHMeCHMe), 1.30 and 1.18 (2m, 9H, 3 × CH₃); MS (thermo-

spray) m/z 371 (MH⁺). HRMS calcd for C₂₁H₂₆N₂O₄(MH⁺)

371.197 083, found 371.197 289.

r el-(3a S,6S,6a R)-4-(tr a n s-2,3-Dim eth ylcyclop r op ylca r -

bon yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ole-1-

ca r boxylic Acid Ben zyl Ester (29). 29 was derived from 24

and the mixed anhydride prepared from trans-2,3-dimethyl-

cyclopropanecarboxylic acid and trimethylacetyl chloride to

give a colorless gum (69%): ¹H NMR (CHCl₃) δ 7.36 (m, 5H,

C₆H₅), 5.13 (ABq, J ) 12.5 Hz, 2H, PhCH₂), 3.96-3.58 (m, 3H,

NCH₂CH₂, NCHCH₂), 3.47 (m, 1H, NCHCH), 3.09 (br m, 1H,

CHMe), 2.83 and 2.46 (2m, 2 × 0.5 H, COCHCHMeCHMe),

2.71 and 1.94 (2m, 2H, NCH₂CH₂), 1.54-1.02 (m, 11H,

COCHCHMeCHMe and 3 × CH₃); MS (thermospray) m/z 371

(MH⁺); HRMS calcd for C₂₁H₂₆N₂O₄(MH⁺) 371.197083, found

371.197412; HPLC 97.86% (t_R) 30.77 min).

r el-(3a S,6S,6a R)-4-(2-Hyd r oxy-3-p h en oxyp r op yl)-6-m e-

t h yl-5-oxoh e xa h yd r op yr r olo[3,2-b]p yr r ole -1-ca r b ox-

ylic Acid Ben zyl Ester (38). 38 was derived from 24 and

1,2-epoxy-3-phenoxypropane (1.3 equiv) to give a white solid

1

(41%): IR (KBr) ν_max3420, 2930, 1701-1682 cm^-1; H NMR

(CDCl₃) shows rotamers, δ 7.40 - 7.25 (m, 7H, aryl), 7.03 -

6.85 (m, 3H, aryl), 5.20-5.05 (m, 2H, PhCH₂O), 4.23-3.30 (m,

10H, PhOCH₂, CHOH, NCH₂CH₂, NCHCH₂, NCHCHMe,

NCH₂CHOH, OH), 3.20-2.80 (2m, 1H, CHMe), 2.32-2.17 (n,

1H, NCH₂CHH), 1.98-1.70 (m, 1H, NCH₂CHH), 1.30-1.00 (m,

3H, CHMe); MS (thermospray) 425 (MH⁺); HPLC 99% (t_R

)

26.4 min). Anal. (C₂₄H₂₈N₂O₅) C, H, N.

r el-(3aS,6S,6a R)-6-Meth yl-4-(m eth ylth io)-5-oxoh exa h y-

d r op yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid Ben zyl Ester

(42). 42 was derived from 24 and methyl methanethiolsul-

fonate (1.3 equiv) to give a clear oil (67%): IR (KBr) ν_max2961,

1715, 1454 cm^-1; ¹H NMR (CDCl₃) shows rotamers, δ 7.35 (s,

5H, C₆H₅), 5.20-5.05 (m, 2H, PhCH₂O), 3.99-3.82 (m, 1H,

NCHHCH₂), 3.73-3.40 (m, 3H, NCHHCH₂, NCHCH₂, and

NCHCHMe), 3.20-2.85 (2m, 1H, CHMe), 2.42-2.30 (m, 4H,

SMe, NCH₂CHH), 2.00-1.80 (m, 1H, NCH₂CHH), 1.20-1.00

(m, 3H, CHMe); MS (thermospray) 321 (MH⁺); HPLC 100%

(t_R) 24.68 min). Anal. (C₁₆H₂₀N₂O₃S) C, H, N, S.

r el-(3a S,6S,6a R)-6-Meth yl-5-oxo-4-(2,2,3,3-tetr a m eth yl-

cyclop r op ylca r bon yl)h exa h yd r op yr r olo[3,2-b]p yr r ole-1-

ca r boxylic Acid Ben zyl Ester (30). 30 was derived from 24

and the mixed anhydride prepared from 2,2,3,3-tetramethyl-

cyclopropanecarboxylic acid and trimethylacetyl chloride to

r el-(3a S,6S,6a R)-6-Meth yl-5-oxo-4-(1H-p yr r ol-1-ylca r -

bon yl)-h exah ydr opyr r olo[3,2-b]pyr r ole-1-car boxylic Acid

Ben zyl Ester (40). 40 was derived from 24 and pyrrole-1-

carboxylic acid anhydride¹⁰(2 equiv) to give a white foam

give a white solid (66%): IR (KBr) ν_max1745, 1714, 1683 cm^-1

;

¹H NMR (CDCl₃δ 7.5-7.3 m, 5H, C₆H₅), 5.15 (m, 2H, PhCH₂),

4.0-3.75 (br m, 1H, NCHHCH₂), 3.7-3.4 (m, 3H, NCHHCH2,

NCHCH2 and NCHCHMe), 3.2-2.8 (br m, 1H, CHMe), 2.7

(br m, 1H, NCH₂CHH), 2.33 (s, 1H, COCHCMe₂CMe₂), 2.1-

1.8 (br m, 1H, NCH₂CHH), 1.4-1.0 (br, 15H, 5 x CH₃); MS

(thermospray) m/z 399 (MH⁺); HPLC 93% (t_R) 34 min). Anal.

(C₂₃H₃₀N₂O₄) C, H, N.

(72%): IR (KBr) ν_max1769, 1719,1701, 1686 cm^{-1 1}H NMR

;

(CDCl₃): δ 7.37 (s, 5H, C₆H₅), 7.20-7.16 (m, 2H, NCH)CH),

6.32-6.27 (m, 2H, NCHdCH), 5.23-5.07 (ABq, J ) 12.2 Hz,

2H, PhCH₂), 4.07-3.61 (m, 4H, NCH₂CH₂, NCHCH₂, and

NCHCHMe), 3.35-2.93 (br m, 1H, CHMe), 2.63-2.51 (m, 1H,

NCH₂CHH), 2.01-1.81 (m, 1H, NCH₂CHH), 1.32-1.10 (br m,

3H, CH₃); MS (thermospray) m/z 368 (MH⁺), 385 (MNH₄⁺);

HPLC 100% (t_R) 29.91 min). Anal. (C₂₀H₂₁N₃O₄) C, H, N.

r el-(3a S,6S,6a R)-4-(Bicyclo[4.2.0]oct a -1,3,5-t r ien -7-yl-

car bon yl)-6-m eth yl-5-oxoh exah ydr opyr r olo[3,2-b]pyr r ole-

1-ca r boxylic Acid Ben zyl Ester (35). 35 was derived from

24 and the mixed anhydride prepared from 1-benzocyclobutene

carboxylic acid and trimethylacetyl chloride to give a colorless

r el-(3a S,6S,6a R)-4-(H yd r oxym et h yl)-6-m et h yl-5-oxo-

h exa h yd r op yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid Ben -

zyl Ester (44). To a solution of the trans-lactam 24 (50 mg,

0.18 mmol) in dry THF (3 mL) was added paraformaldehyde

(56 mg, 1.9 mmol) and potassium carbonate (25 mg, 0.18

mmol). The heterogeneous reaction mixture was stirred at

room temperature for 43 h, more potassium carbonate (9 mg,

0.065 mmol) was added, and the mixture wasstirred for a

further 3 h. Saturated ammonium chloride solution (40 mL)

was added, and the mixture was extracted with ethyl acetate

(3 × 30 mL). The combined organic extract was washed with

water (25 mL) and dried, and the solvent was evaporated to

give a white residue (50 mg), which was purified by flash

column chromatography using cyclohexanes/ethyl acetate as

the eluting solvent to give 44 as a colorless gum (37 mg,

gum (61%). IR (KBr) ν_max1749, 1706, 1696 cm^{-1 1}H NMR

;

(CDCl₃) δ 7.37 (s, 5H, C₆H₅), 7.28-7.00 (m, 4H, C₆H₄), 5.23-

5.01 (m, 3H, PhCH₂, cyclobutaneCH), 3.9-3.4 (m, 6H, NCH-

CHMe, NCHCH₂, NCH₂CH₂, cyclobutaneCH₂), 3.3-3.0 (br m,

1H, CHMe), 2.79-2.65 (m, 1H, NCH₂CHH), 2.07-1.85 (m, 1H,

NCH₂CHH), 1.2 (br d, 3H, CH₃); MS (thermospray) m/z 405

(MH⁺); HPLC 99% (t_R) 32.2 min). Anal. (C₂₄H₂₄N₂O₄‚

0.4EtOAc) C, H, N.

P r ep a r a tion of 38-42. By use of essentially the same

procedure as for the preparation of 4, the following compounds

listed in Scheme 6 were prepared from 24.

67%): IR (KBr) ν_max1721, 1710, 1910, 1679 cm^{-1 1}H NMR

;

r el-(3a S,6S,6a R)-4-(2,4-Din itr op h en yl)-6-m eth yl-5-oxo-

h exa h yd r op yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid Ben -

zyl Ester (39). 39 was derived from 24 and 2,4-dinitrofluo-

robenzene (3 equiv) to give a yellow foam (70%): IR (KBr) ν_max

1711, 1605, 1542, 1531 cm^-1; ¹H NMR (CDCl₃) δ 8.83 (d, J )

2.4 Hz, 1H, C₆H₃(NO₂)₂), 8.49 (dd, J ) 2.4 Hz, J ) 9.2 Hz, 1H,

C₆H₃(NO₂)₂), 7.43-7.35 (m, 6H, C₆H₅and C₆H₃(NO₂)₂), 5.23-

5.11 (ABq, J ) 12.2 Hz, 2H, PhCH₂), 4.18-3.96 (m, 2H, NCH₂-

CH₂), 3.89-3.74 (m, 2H, NCHCH₂, NCHCHMe), 3.4-3.0 (br

d, 1H, CHMe), 2.47-2.35 (m, 1H, NCH₂CHH), 2.17-1.98 (m,

1H, NCH₂CHH), 1.30-1.10 (br m, 3H, CH₃); MS (thermospray)

m/z 458 (MNH₄⁺), 442 (MH⁺); HPLC 100% (t_R) 29.2 min).

Anal. (C₂₁H₂₀N₄O₇) C, H, N.

(CDCl₃) δ 7.36 (s, 5H, C₆H₅), 5.20-5.05 (m, 3H, PhCH₂,

NCHHOH), 4.58-4.45 (br m, 1H, NCHHOH), 4.02-3.85 (br

m, 1H, NCHHCH₂), 3.77-3.63 (m, 2H, NCHHCH₂and NCH-

CHMe), 3.53-3.43 (m, 1H, NCHCH₂), 3.15-2.77 (br m, 2H,

CHMe, OH), 2.38-2.25 (m, 1H, NCH₂CHH), 1.97-1.77 (m, 1H,

NCH₂CHH), 1.20-1.00 (br m, 3H, CH₃); MS (thermospray) m/z

305 (MH⁺), 610 (2MH⁺); HPLC 98% (t_R) 19.4 min). Anal.

(C₁₆H₂₀N₂O₄) C, H, N.

r el-(3aS,6S,6aR)-6-Meth yl-5-oxo-4-[(1E)-3-oxobu t-1-en yl]-

h exa h yd r op yr r olo[3,2-b]p yr r ole-1-ca r boxylic Acid Ben -

zyl Ester (43). A mixture of PdCl₂(MeCN)₂(24 mg, 0.09

mmol), CuCl (10 mg, 0.09 mmol), and 24 (500 mg, 1.82 mmol)

Inhibitors of Human Cytomegalovirus Protease

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 15

was stirred in dry dimethylformamide (5 mL) under an

atmosphere of oxygen at 60 °C. Methyl vinyl ketone (450 µL,

5.4 mmol) was added in portions over 4 h to the reaction

mixture, and then stirring at 60 °C was continued for 17 h.

The reaction mixture was diluted with ether at room temper-

ature and then filtered. The filtrate was evaporated, and the

residue was purified by flash column chromatography, eluting

with ethyl acetate/cyclohexane (2:1) to give 43 (188 mg, 33%)

45 (30 mg, 0.14 mmol) and triethylamine (78 uL, 56.6 mg, 0.56

mmol) in acetonitrile (2 mL) under nitrogen. The resulting

mixture was stirred at room temperature for 18 h, the solvent

was evaporated, and the residue was partitioned between ethyl

acetate (5 mL) and 1 N HCl (5 mL). The aqueous phase was

separated and extracted with ethyl acetate (2 × 5 mL), and

the combined organic phase was washed with water (5 mL)

and brine (5 mL) and dried over MgSO₄. The residue (33 mg)

was purified by flash column chromatography, eluting with

cyclohexanes/ethyl acetate (2:1) to give the title compound 46

(25 mg, 51%) as a solid, mp 147-149 °C: IR (KBr) ν_max1749,

1700 cm^-1; ¹H NMR (CDCl₃) δ 7.41-7.17 (s, 5H, C₆H₅), 3.90-

3.70 (m, 2H, NCH₂CH₂), 3.64-3.40 (m, 2H, NCHCH₂and

NCHCHMe), 3.32-3.31 (m, 2H, PhCH₂CH₂), 3.21-3.12 (m,

2H, PhCH₂), 3.12-2.95 (m, 1H, CHMe), 2.83-2.69 (m, 1H,

NCH₂CHH), 2.47 (s, 3H, COCH₃), 2.14-1.92 (m, 1H, NCH₂-

CHH), 1.28 (br, 3H, CH₃CH). Anal. (C₁₇H₂₂N₂O₄S‚0.1Et₂O) C,

H, N, S.

as a solid: IR (KBr) ν_max3000, 1741, 1705 cm^{-1 1}H NMR

;

(CDCl₃) δ 7.89 (d, J ) 15 Hz, 1H, NCHdCH), 7.37 (m, 5H,

C₆H₅), 5.75 (d, J ) 15 Hz, 1H, NCHdCH), 5.21-5.07 (ABq, J

) 12 Hz, 2H, PhCH₂), 4.05-3.90 (br m, 1H, NCHHCH₂), 3.84-

3.56 (m, 3H, NCHHCH₂, NCHCH₂, and NCHCHMe), 3.34-

2.90 (br m, 1H, CH₃CH), 2.64-2.53 (br m, 1H, NCH₂CHH),

2.28 (s, 3H, CH₃CO), 2.21-2.04 (br m, 1H, NCH₂CHH), 1.28-

1.06 (br, m, 3H, CHCH₃); MS (thermospray) 343 (MH⁺). Anal.

(C₁₉H₂₂N₂O₄‚0.4EtOAc) C, H, N.

r el-(3S,3a R,6a S)-1-Acetyl-3-m eth ylh exa h yd r op yr r olo-

[3,2-b]p yr r ol-2-on e (45). A solution of 1 (3.4 g, 10.7 mol) in

2-propanol (150 mL) containing 1 M HCl in ether (13 mL, 13

mmol) was added to the palladium catalyst (10% Pd/C,

Degussa type, E101, NE/W, 50% H₂O, 1.5 g) under an

atmosphere of nitrogen. The resulting mixture was then stirred

vigorously under an atmosphere of hydrogen for 3.5 h, filtered,

and evaporated to give 45 (2.369 g, 99%) as a white foam: IR

(KBr) ν_max1754 cm^-1; ¹H NMR (MeOD): δ 3.97-3.66 (m, 3H,

NCH₂CH₂and NCHCH₂), 3.61-3.51 (m, 1H, NCHCHMe),

3.11-2.97 (m, 1H, CHMe), 2.81-2.68 (m, 1H, NCH₂CHH), 2.44

(s, 1H, MeCO), 2.18-1.96 (m, 1H, NCH₂CHH), 1.30 (d, J )

7.3 Hz), 1.14 (d, J ) 6.1 Hz) (3H, CHCH₃); MS (thermospray)

m/z 183 (MH - HCl⁺); HPLC 97.2% (t_R) 3.45 min). Anal.

(C₉H₁₄N₂O₄‚HCl‚H₂O‚0.5C₃H₈O₂) C, H, N.

r el-(3S,3a R,6a S)-1-Acetyl-3-m eth yl-4-(2-oxo-3-p h en yl-

p r op yl)h exa h yd r op yr r olo[3,2-b]p yr r ol-2-on e (47). To a

solution of the trans-lactam 45 (30 mg, 0.137 mmol) in

anhydrous acetonitrile (2.5 mL) was added triethylamine (19

µL, 0.138 mmol), followed by a solution of the benzyl bromo-

methyl ketone (34.4 mg, 0.204 mmol) in anhydrous acetonitrile

(0.25 mL). The reaction mixture was stirred at room temper-

ature for almost 3 h, heated at 88 °C for 2 h, and then was to

stirred at room temperature overnight. The reaction mixture

was diluted with ethyl acetate (20 mL) and was washed with

saturated sodium hydrogen carbonate (10 mL). The aqueous

layer was re-extracted with ethyl acetate (2 × 15 mL). The

combined organic layers were washed with brine (10 mL) and

dried, and the solvent was evaporated. The yellow residue that

was purified by flash column chromatography using cyclohex-

anes/ethyl acetate as the eluting solvent gave 47 (12 mg, 29%)

d as a yellow gum: ¹H NMR (CDCl₃) δ 7.4-7.2 (m, 5H, C₆H₅),

3.8-3.5 (m, 4H, PhCH₂, NCHHCH₂, NCHCH₂), 3.5 (m, 1H,

NCHHCH₂), 3.2 (m, 1H, NCHCHMe), 2.8-2.5 (m, 4H, NCH₂-

CHH, COCH₂, CHMe), 2.4 (s, 3H, COCH₃), 2.9-1.8 (m, 1H,

NCH₂CHH), 1.1 (m, 3H, CHMe); MS (thermospray) m/z 315

(MH⁺); HRMS calcd for C₁₈H₂₂N₂O₃(MH⁺) 315.170 868, found

315.170 765.

The following compounds were similarly prepared.

r el-(3S,3a R,6a S)-3-Meth yl-2-oxoh exa h yd r op yr r olo[3,2-

b]p yr r ole-1-ca r boxylic Acid ter t-Bu tyl Ester Hyd r och lo-

r id e (58). Compound 20⁷was deprotected with hydrogen in

the presence of Pd/C as described for 45 to give 58 (88%) as a

white solid: IR (KBr) ν_max2974, 2697, 1786 cm^{-1 1}H NMR

;

(DMSO-d₆) δ 9.44 (br.s, 1H, NH), 3.93-3.29 (m, 4H, NCH₂-

CH₂, NCHCH₂, NCHCHMe), 2.91-2.75 (m, 1H,CHMe), 2.46-

2.30 (m, 1H, NCH₂CHH), 2.12-1.92 (m, 1H, NCH₂CHH), 1.46

(s, 9H, Bu^t), 1.20 (d, J ) 7.3 Hz), 1.04 (d, J ) 6.1 Hz) (3H,

CHCH₃); MS (thermospray) m/z 241 (MH⁺), 481 (2MH⁺), 258

(MNH₄⁺); HPLC 98.6% (t_R) 8.94 min). Anal. (C₁₂H₂₀N₂O₃‚

HCl‚0.25C₃H₈O) C, H, N.

(3S,3a R,6a S)-1-Acet yl-4-{[(2S)-1-b en zoylp yr r olid in -2-

yl]ca r b on yl}-3-m et h ylh exa h yd r op yr r olo[3,2-b]p yr r ol-

2(1H)-on e (53) a n d (3R,3a S,6a R)-1-Acetyl-4-{[(2S)-1-ben -

zoylp yr r olid in -2-yl]ca r b on yl}-3-m et h ylh exa h yd r op yr -

r olo[3,2-b]p yr r ol-2-on e (54). To a stirred solution of the

N-benzoyl-(S)-proline (30 mg, 0.14 mmol) in dimethylforma-

mide (1 mL) at room temperature was added a solution of

TBTU (36 mg, 0.11 mmol) in dimethylformamide (0.25 mL)

and a solution of HOBT‚H₂O (18 mg, 0.12 mmol) in dimeth-

ylformamide (0.25 mL). The reaction mixture was stirred at

room temperature for 20 min before addition of diisopropyl-

ethylamine (38 µL, 218 µmol) and the pyrrolidine 45 (31 mg,

0.14 mmol) in a solution of acetonitrile/dimethylformamide (1:

1) (0.6 mL). The reaction mixture was left at room temperature

for 20 h before it was diluted with dichloromethane (10 mL)

and washed with water (7 mL). The aqueous phase was back-

extracted with dichloromethane (10 mL), and the combined

organic extract was washed with 2 N HCl (10 mL), water (10

mL), and saturated sodium hydrogen carbonate (10 mL). It

was then dried, and the solvent was evaporated to give a

yellow residue (44 mg). This was purified by flash column

chromatography using cyclohexanes/ethyl acetate as the elut-

ing solvent to give the less polar diastereomer 53 (15 mg, 28%)

as an off-white foam: IR (KBr) ν_max1747, 1699, 1661, 1627

cm^-1; ¹H NMR (CDCl₃) δ 7.62-7.52 (m, 2H, C₆H₅) 7.46-7.35

(m, 3H, C₆H₅), 4.75-4.67 (m, 1H, NCHCO), 4.66-4.56 (m, 1H,

NCHHCH₂CH₂), 3.92-3.66 (m, 3H, NCH₂CH₂, NCHCH₂),

3.63-3.52 (m, 2H, NCHCHMe and NCHHCH₂CH₂), 3.38-3.22

(m, 1H, NCH₂CHH), 2.88-2.76 (m, 1H, NCH₂CHH), 2.48 (s,

3H, COCH₃), 2.36-1.81 (m, 5H, NCH₂CH₂CH₂and CHMe),

1.16 (d, J ) 7.3 Hz, 3H, CHMe); MS (thermospray) m/z 384

(MH⁺); HRMS calcd for C₂₁H₂₅N₃O₄(MH⁺) 384.192 332, found

384.192 158; HPLC 96% (t_R) 19.0 min).

r el-(3a S,6S,6a R)-1-Cyclop r op a n eca r b on yl-3-m et h yl-

h exa h ydr opyr r olo[3,2-b]p yr r ol-2-on e h yd r och lor ide (63).

Compound 28 was deprotected with hydrogen in the presence

of Pd/C as described for 45 to give 63 (91%) as a white solid:

¹H NMR (MeOD) δ 3.86-3.40 (m, 4H, NCH₂CH₂, NCHCH₂,

and NCHCHMe), 3.03-2.97 (m, 2H, NCH₂CHH and COCHCH₂-

CH₂), 2.65-2.53 (m, 1H, CHMe), 2.00-1.81 (m, 1H, NCH₂-

CHH), 1.21 (d, J ) 7.3 Hz, 3H, CH₃), 1.02-0.85 (m, 4H,

COCHCH₂CH₂); MS (thermospray) m/z 209 (MH⁺); HRMS

calcd for C₁₁H₁₆N₂O₂(MH⁺) 209.129 003, found 209.129 312;

HPLC 93% (t_R) 7.3 min).

r el-(3S,3a R,6a S)-1-(Cyclop r op ylca r bon yl)-3-m eth yl-4-

[(2S)-p yr r olid in -2-ylca r bon yl]h exa h yd r op yr r olo[3,2-b]-

p yr r ol-2-on e Hyd r och lor id e (68). Compound 67 was depro-

tected with hydrogen in the presence of Pd/C as described for

45 to give 68 (94%) as a white foam: ¹H NMR (MeOD) δ 4.56-

4.48 (m, 1H, NCHCO), 4.06-3.77 (m, 4H, NCH₂CH₂CH₂and

NCH₂CH₂), 3.68-3.60 (m, 1H, NCHCH₂), 3.53-3.14 (m, 7H,

NCHCHMe and CHMe + H₂O), 3.01-2.88 (m, 1H, COCHCH₂-

CH₂), 2.79-2.67 (m, 1H, NCH₂CHH), 2.61-2.45 (m, 1H, NCH₂-

CHH), 2.19-1.90 (br m, 4H, NCH₂CH₂CH₂and NCH₂-

CH₂CH₂), 1.20-0.95 (m, 7H, CHMe and COCHCH₂CH₂); MS

(thermospray) m/z 306 (MH⁺); HRMS calcd for C₁₆H₂₃N₃O₃

(MH⁺) 306.181 767, found 306.181 634; HPLC 98% (t_R) 10.8

min).

r el-(3S,3a R,6a S)-1-Acetyl-3-m eth yl-4-[(2-p h en yleth yl)-

su lfon yl]h exa h yd r op yr r olo[3,2-b]p yr r ol-2-on e (46). A so-

lution of the phenethylsulfonyl chloride (44 mg, 0.22 mmol)

in acetonitrile (1 mL) was added to a solution of hydrochloride

16 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1

Borthwick et al.

Further elution with cyclohexanes/ethyl acetate gave the

more polar diastereomer 54 (18 mg, 34%) as an off-white

foam: ¹H NMR (CDCl₃) shows rotameric forms present, δ

7.62-7.52 (m, 2H, C₆H₄), 7.46-7.35 (m, 3H, C₆H₄), 4.75-4.09

(3m, 2H, NCHCO and NCHHCH₂CH₂), 3.91-3.44 (m, 5H,

NCH₂CH₂, NCHCH₂, NCHCHMe, and NCHHCH₂CH₂), 3.37-

3.24 (m, 1H, NCH₂CHH), 2.97-2.68 (m, 1H, NCH₂CHH), 2.48

(s, 3H, COCH₃), 2.35-1.81 (m, 5H, NCH₂CH₂CH₂and CHMe),

1.30 and 1.22 (2d, J ) 7.3 Hz, 3H, CHMe); MS (thermospray)

m/z 384 (MH⁺); HRMS calcd for C₂₁H₂₅N₃O₄(MH⁺) 384.192 332,

found 384.192 019; HPLC 99% (t_R) 18.0 min.).

¹H NMR (CDCl₃) δ 7.76 (d, J ) 7.9 Hz, 2H, tosyl-2H, 6H), 7.31

(d, J ) 7.9 Hz, 2H, tosyl-4H, 5H), 4.52 (dd, J ) 4.3 Hz, J )

7.3 Hz, 1H, NCHCO), 4.30 (t, J ) 9.5 Hz, 1H, NCHHCH₂CH₂),

3.93-3.24 (m, 6H, NCH₂CH₂, NCHCH₂, NCHCHMe, NCHH-

CH₂CH₂, and NCH₂CHH), 2.90-2.78 (m, 1H, NCH₂CHH), 2.49

(s, 3H, COCH₃), 2.43 (s, 3H, tosylCH₃), 2.20-1.73 (m, 5H,

NCH₂CH₂CH₂and CHMe), 1.13 (d, J ) 7.3 Hz, 3H, CH₃); MS

(thermospray) m/z 434 (MH⁺), 392 (M - COCH₃⁺); HPLC 100%

(t_R) 23.3 min). Anal. (C₂₁H₂₇N₃O₅S‚0.2CH₂Cl₂) C, H, N, S.

The more polar diastereomer 52 (30%) was isolated as a

white solid: IR (KBr) ν_max1751, 1702, 1663 cm^{-1 1}H NMR

;

The following compounds were similarly prepared.

(CDCl₃) shows rotamers, δ 7.76 and 7.66 (2d, J ) 7.9 Hz, 2H,

tosyl-2H, 6H), 7.31 (d, J ) 7.9 Hz, 2H, tosyl-4H, 5H), 4.47-

4.20 (2m, 1H, NCHCO), 4.10-3.23 (m, 7H, NCH₂CH₂CH_2,

NCH₂CH₂, NCHCH₂, NCHCHMe, and NCH₂CHH), 2.95-2.70

(2m, 1H, NCH₂CHH), 2.48 (s, 3H, COCH₃), 2.43 (s, 3H,

tosylCH₃), 2.18-1.71 (m, 5H, NCH₂CH₂CH₂and CHMe), 1.21

and 1.17 (2d, J ) 7.3 Hz, 3H, CH₃); MS (thermospray) m/z

434 (MH⁺), 392 (M - COCH₃⁺); HPLC 95% (t_R) 22.9 min).

Anal. (C₂₁H₂₇N₃O₅S‚0.15CH₂Cl₂) C, H, N, S.

(3S,3a R,6a S)-1-Acetyl-3-m eth yl-4-({(2S)-1-[(4-n itr op h e-

n yl)su lfon yl]pyr r olidin -2-yl}car bon yl)h exah ydr opyr r olo-

[3,2-b]p yr r ol-2-on e (57). Similarly prepared as 53 using 1-[(4-

nitrophenyl)sulfonyl]-(S)-proline, the less polar diastereomer

57 (23%) was isolated as a white solid: IR (KBr) ν_max1749,

1694, 1656, 1539 cm^-1; ¹H NMR (CDCl₃) δ 8.39-8.32 (m, 2H,

C₆H₄), 8.14-8.07 (m, 2H, C₆H₄), 4.69 (dd, J ) 4.9 Hz, J ) 7.9

Hz, 1H, NCHCO), 4.24 (t, J ) 9.5 Hz, 1H, NCHHCH₂CH₂),

3.90-3.22 (m, 6H, NCH₂CH₂, NCHCH₂, NCHCHMe, NCHH-

CH₂CH₂, and NCH₂CHH), 2.92-2.80 (m, 1H, NCH₂CHH), 2.49

(s, 3H, COCH₃), 2.31-1.89 (m, 5H, NCH₂CH₂CH_2,+CHMe),

1.12 (d, J ) 7.3 Hz, 3H, CH₃); MS (thermospray) m/z 465

(MH⁺) 482 (MNH₄⁺); HPLC 97% (t_R) 23.3 min). Anal.

(C₂₀H₂₄N₄O₇S‚0.2H₂O) C, H, N.

r el-(3S,3a R,6a S)-1-Acet yl-3-m et h yl-4-[(1-m et h yl-1H -

p yr r ol-2-yl)(oxo)a cetyl]h exa h yd r op yr r olo[3,2-b]p yr r ol-

2-on e (50). Similarly prepared as 53, 1-methyl-pyrrole-2-

glyoxylic acid was used to give 50 (45%) as a white foam: IR

(KBr) ν_max1748, 1702, 1654, 1650, 1629 cm^-1; ¹H NMR (CDCl₃)

shows rotameric forms present, δ 7.23-7.20 and 7.08-7.05

(2m, 1H, pyrrole-4H), 6.98 (br s, 1H, pyrrole-3H), 6.22 (dd, J

) 2.4 Hz, J ) 4.3 Hz, 1H, pyrrole-5H), 4.17-3.34 (m, 7H,

NCH₃, NCH₂CH₂, NCHCH₂, and NCHCHMe), 2.96-2.75 (m,

2H, NCH₂CHH and CHMe), 2.50 and 2.47 (2s, 3H, COCH₃),

2.15-1.92 (m, 1H, NCH₂CHH), 1.32 and 0.93 (2d, J ) 7.3 Hz,

3H, CHMe); MS (thermospray) m/z 318 (MH⁺); HPLC 98% (t_R

) 19.4 min). Anal. (C₁₆H₁₉N₃O₄‚0.1CH₂Cl₂) C, H, N,

(3S,3a R,6a S)-1-Acet yl-4-[((2S)-1-{[5-(d im et h yla m in o)-

1-n a p h th yl]su lfon yl}p yr r olid in -2-yl)ca r bon yl]-3-m eth yl-

h exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H)-on e (55) a n d (3R,-

3a S,6a R)-1-Acetyl-4-[((2S)-1-{[5-(d im eth yla m in o)-1-n a p h -

th yl]su lfon yl}p yr r olid in -2-yl)ca r bon yl]-3-m eth ylh exa h y-

d r op yr r olo[3,2-b]p yr r ol-2(1H)-on e (56). Similarly prepared

as 53, using dansyl-(S)-proline, the less polar diastereomer 55

(26%) was obtained as a yellow-green foam: IR (KBr) ν_max

1749, 1700, 1687 cm^{-1 1}H NMR (CDCl₃) δ 8.54 (d, J ) 8.5

;

Hz, 1H, dansyl-2H), 8.42 (d, J ) 8.5 Hz, 1H, dansyl-4H), 8.28

(dd, J ) 1 Hz, J ) 7.3 Hz, 1H, dansyl-8H), 7.61-7.47 (m, 2H,

dansyl-3H, dansyl-7H), 7.18 (d, J ) 7.3 Hz, 1H, dansyl-6H),

4.74 (dd, J ) 4.9 Hz, J ) 7.9 Hz, 1H, NCHCO), 4.27 (t, J )

9.5 Hz, 1H, NCHHCH₂CH₂), 3.89-3.17 (m, 5H, NCHHCH₂-

CH_2,NCH₂CH₂, NCHCH₂, NCHCHMe), 2.91-2.74 (m, 8H,

NMe₂, NCH₂CHH, CHMe), 2.47 (s, 3H, COCH₃), 2.30-1.82 (m,

5H, NCH₂CH₂CH₂, NCH₂CHH), 1.11 (d, J ) 7.3 Hz, 3H,

CHCH₃); TLC R_f) 0.37 (ethyl acetate/cyclohexane, 7:3); MS

(thermospray) m/z 513 (MH⁺), 471 (M - COCH₃⁺); HPLC 100%

(t_R) 23.54 min). Anal. (C₂₆H₃₂N₄O₅S‚0.2H₂O) C, H, N, S.

r el-(3S,3a R,6a S)-1-Acet yl-4-[2-(5-flu or o-2-m et h yl-1H -

in d ol-3-yl)a cetyl]-3-m eth ylh exa h yd r op yr r olo[3,2-b]p yr -

r ol-2-on e (49). Similarly prepared as 53, 2-(5-fluoro-2-methyl-

1H-indol-3-yl)acetic acid was used to give 49 (56%) as a white

The more polar diastereomer 56 (30%) was isolated as a

yellow foam: ¹H NMR (CDCl₃) δ 8.54 (d, J ) 8.5 Hz, 1H,

dansyl-2H), 8.41 (d, J ) 8.5 Hz, 1H, dansyl-4H), 8.27 (broad

d, J ) 7.3 Hz, 1H, dansyl-8H), 7.60-7.46 (m, 2H, dansyl-3H,

dansyl-7H), 7.18 (d, J ) 7.3 Hz, 1H, dansyl-6H), 4.01-3.14

(m, 7H, NCHCO, NCHHCH₂CH₂, NCHHCH₂CH₂, NCH₂CH₂,

NCHCH₂, NCHCHMe), 2.93-2.76 (m, 8H, NMe₂, NCH₂CHH,

CHMe), 2.45 (s, 3H, COCH₃), 2.34-1.88 (m, 5H, NCH₂CH₂CH₂,

NCH₂CHH), 0.93 (d, J ) 7.3 Hz, 3H, CHCH₃); TLC R_f) 0.22

(ethyl acetate/cyclohexane, 7:3); MS (thermospray) m/z 513

[MH]⁺, 471 (M - COCH₃⁺); HRMS calcd for C₂₆H₃₂N₄O₅S

(MH⁺) 513.217 167, found 513.217 025; HPLC 97% (t_R) 23.41

min).

1

amorphous solid: IR (CHBr₃) ν_max1751, 1708 cm^-1; H NMR

(CDCl₃) δ 7.91 (bs, 1H, NH), 7.21-7.09 (m, 2H, indolyl-H4 and

H-7), 6.90-6.80 (m, 1H, indolyl-H6), 3.94-3.42 (m, 6H, NCH₂-

CH₂, NCHCH₂, indolylCH₂, and NCHCHMe), 3.38-3.23 (m,

1H, CHMe), 2.85-2.70 (m, 1H, NCH₂CHH), 2.47 (s, 3H,

COCH₃), 2.40 (s, 3H, indolylCH₃), 2.12-1.93 (m, 1H, NCH₂-

CHH), 1.16 (d, J ) 7.3 Hz, 3H, CH₃CH); TLC R_f) 0.63 (ethyl

acetate); MS (thermospray) m/z 372 (MH⁺); HPLC 98.1% (t_R

) 22.69 min). Anal. (C₂₀H₂₂FN₃O₃‚2H₂O‚0.4C₄H₈O₂) C, H, N,

F.

r el-(3S,3a R,6a S)-1-Acetyl-3-m eth yl-4-(p h en oxya cetyl)-

h exa h yd r op yr r olo[3,2-b]p yr r ol-2-on e (48). Similarly pre-

pared as 53, phenoxyacetic acid was used to give 48 (51%) as

(3S,3aR,6aS)-4-[((2S)-1-{[5-(Dim eth ylam in o)-1-n aph th yl]-

su lfon yl}pyr r olidin -2-yl)car bon yl]-3-m eth yl-2-oxoh exah y-

d r op yr r olo[3,2-b]p yr r ole-1-ca r b oxylic Acid ter t-Bu t yl

Ester (60) a n d (3R,3a S,6a R)-4-[((2S)-1-{[5-(Dim eth yla m i-

n o)-1-n aph th yl]su lfon yl}pyr r olidin -2-yl)car bon yl]-3-m eth -

yl-2-oxoh exah ydr opyr r olo[3,2-b]pyr r ole-1-car boxylic Acid

ter t-Bu tyl Ester (59). Similarly prepared as 53, using dansyl-

(S)-proline and 58, the less polar (3S,3aR,6aS) diastereomer

60 (38%) was isolated as a yellow foam: IR (KBr) ν_max1787,

1665 cm^-1; ¹H NMR (CDCl₃) δ 8.55 (d, J ) 8.5 Hz, 1H, dansyl-

2H), 8.42 (d, J ) 8.5 Hz, 1H, dansyl-4H), 8.28 (d, J ) 7.3 Hz,

1H, dansyl-8H), 7.61-7.47 (m, 2H, dansyl-3H, dansyl-7H), 7.18

(d, J ) 7.3 Hz, 1H, dansyl-6H), 4.74 (dd, J ) 4.3 Hz, J ) 7.9

Hz, 1H, NCHCO), 4.28 (t, J ) 9.5 Hz, 1H, NCHHCH₂CH₂),

3.89-3.12 (m, 5H, NCHHCH₂CH₂, NCH₂CH₂, NCHCH₂, NCH-

CHMe), 2.88 (s, 6H, NMe₂), 2.64-2.51 (m, 1H, CHMe), 2.28-

1.82 (m, 6H, NCH₂CH₂CH₂, NCH₂CH₂), 1.54 (s, 9H, CO₂C-

(CH₃)₃), 1.11 (d, J ) 7.6 Hz, 3H, CHCH₃); ¹³C NMR (CDCl₃) δ

179.5, 155.8, 136.2, 128.6, 128.2, 128.0, 67.2, 63.4, 60.5, 49.3,

1

a pale-yellow gum: IR (KBr) ν_max1748, 1698, 1673 cm^-1; H

NMR (CDCl₃) δ 7.38-7.24 (m, 2H, phenoxy-2H, phenoxy-6H),

7.08-6.88 (m, 3H, phenoxy-3H, phenoxy-4H, and phenoxy-5H),

4.64 (bs, 2H, PhOCH₂CO), 3.95 (m, 2H, NCH₂CH₂), 3.55 (m,

2H, NCHCH₂and NCHCHMe), 3.32 (m, 1H, CHMe), 2.81 (m,

1H, NCH₂CHH), 2.47 (s, 3H, COCH₃), 2.06 (m, 1H, NCH₂-

CHH), 1.11 (m, 3H, CH₃CH); TLC R_f) 0.63 (ethyl acetate);

MS (thermospray) m/z 317 (MH⁺); HPLC 97.4% (t_R) 20.74

min). Anal. (C₁₇H₂₀N₂O₄) C, H, N. HRMS calcd for C₁₇H₂₀N₂O₄

(MH⁺) 317.150 132, found 317.150 142.

(3S,3aR,6aS)-1-Acetyl-3-m eth yl-4-({(2S)-1-[(4-m eth ylph e-

n yl)su lfon yl]pyr r olidin -2-yl}car bon yl)h exah ydr opyr r olo-

[3,2-b]p yr r ol-2-on e (51) a n d (3R,3a S,6a R)-1-Acet yl-3-

m eth yl-4-({(2S)-1-[(4-m eth ylp h en yl)su lfon yl]p yr r olid in -

2-yl}ca r bon yl)h exa h yd r op yr r olo[3,2-b]p yr r ol-2-on e (52).

Similarly prepared as 53, using N-tosyl-(S)-proline, the less

polar diastereomer 51 (38%) was isolated as a white solid, mp

213.7-215.3 °C: IR (KBr) ν_max1751, 1738, 1693, 1658 cm^-1

;

Inhibitors of Human Cytomegalovirus Protease

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 17

39.6, 27.7; MS (thermospray) m/z 571 (MH⁺) 417 (M - Boc⁺);

HPLC 99.98% (t_R) 27.53 min). Anal. (C₂₉H₃₈N₄O₆S‚0.3EtOAc)

C, H, N, S.

mmol) in dry acetonitrile (1.6 mL) was added triethylamine

(0.032 mL, 0.23 mmol) followed by a solution of 3-[(dimethyl-

amino)carbonyl]benzenesulfonyl chloride¹⁹(44 mg, 0.18 mmol)

in acetonitrile (0.5 mL). After 45 min, the reaction was

quenched with 2-propanol (0.01 mL) and evaporated to dry-

ness. The residue was partitioned between water (15 mL) and

ethyl acetate (20 mL). The organic phase was washed with

water (15 mL), saturated sodium hydrogen carbonate solution

(15 mL), and water (15 mL) and dried (MgSO₄). The solvent

was evaporated to give a colorless gum, which was purified

by preparative TLC on silica gel, eluting with ethyl acetate to

give 69 (20 mg, 38%) as white solid: IR (KBr) ν_max1747, 1666,

1634 cm^-1; ¹H NMR (CDCl₃) δ 7.99-7.91 (m, 2H, C₆H₄), 7.70-

7.54 (m, 2H, C₆H₄), 4.55 (dd, J ) 4.9 Hz, J ) 7.9 Hz, 1H,

NCHCO), 4.25 (t, J ) 9.8 Hz, 1H, NCHHCH₂CH₂), 3.89-2.73

(m, 14H, NCHHCH₂CH_2,NCH₂CH₂, NCHCH₂, NCHCHMe,

CHMe, NMe₂, COCHCH₂CH_2,NCH₂CHH), 2.22-1.77 (m, 5H,

NCH₂CHH, NCH₂CH₂CH₂), 1.31-0.95 (m, 7H, CHMe, COCH-

CH₂CH₂); MS (thermospray) m/z 517 (MH⁺); HRMS calcd for

C₂₅H₃₂N₄O₆S (MH⁺) 517.212 108, found 517.212 135; HPLC

98% (t_R) 21.1 min).

The more polar (3R,3aS,6aR) diastereomer 59 (30%) was

isolated as a yellow foam: ¹H NMR (CDCl₃) δ 8.54 (d, J ) 8.3

Hz, 1H, dansyl-2H), 8.40 and 8.31 (2d, J ) 8.5 Hz, 1H, dansyl-

4H), 8.28 and 8.17 (2d, J ) 7.3 Hz, 1H, dansyl-8H), 7.59-7.48

(m, 2H, dansyl-3H, dansyl-7H), 7.18 (d, J ) 7.5 Hz, 1H, dansyl-

6H), 4.71-4.66 and 4.38-4.34 (2m, 1H, NCHCO), 4.04-3.34

(m, 6H, NCH₂CH₂CH₂, NCH₂CH₂, NCHCH₂, NCHCHMe),

3.23-3.14 (m, 1H, CHMe), 2.87 (s, 6H, NMe₂), 2.62-1.80 (m,

6H, NCH₂CH₂CH₂, NCH₂CH₂), 1.53 (s, 9H, CO₂C(CH₃)₃), 1.10

and 0.96 (2d, J ) 7.3 Hz, 3H, CHCH₃); MS (thermospray) m/z

571 (MH⁺), 417 (M - Boc⁺). Anal. (C₂₉H₃₈N₄O₆S‚0.1CHCl₃) C,

H, N.

N-{(1S)-2-[r el-(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-

m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-yl]-1-

m et h yl-2-oxoet h yl}-5-(d im et h yla m in o)n a p h t h a len e-1-

su lfon a m id e (65). Compound 63 was reacted with dansyl-

(S)-alanine as described for 53 to give 65 (30%) as a pale-yellow

foam: ¹H NMR (CDCl₃) shows rotamers, δ 8.59-8.49 and

8.32-8.20 (2m, 2H, dansyl-2H, dansyl-4H), 7.65-7.46 and

7.29-7.15 (2m, 4H, dansyl-8H, dansyl-3H, dansyl-7H, dansyl-

6H), 5.76-5.65 (m, 1H, NH), 4.10-2.60 (m, 14H, NCH(Me)-

CO, NCH₂CH₂, NCHCHMe, NCHCH₂, NMe₂, COCHCH₂CH₂,

CHMe, NCH₂CHH), 1.93-1.75 (m, 1H, NCH₂CHH), 1.34-0.92

and 0.39-0.33 (2m, 10H, NCH(Me)CO, CHMe, COCHCH₂CH₂);

MS (thermospray) m/z 513 (MH⁺); HRMS calcd for C₂₆H₃₂N₄O₅S

(MH⁺) 513.217 214, found 513.216 973; HPLC 97% (t_R) 24.3

min).

N-{2-[r el-(3aS,6S,6aR)-4-(Cyclopr opylcar bon yl)-6-m eth -

yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-yl]-2-oxo-

et h yl}-5-(d im et h yla m in o)-N-m et h yln a p h t h a len e-1-su l-

fon am ide (64). Compound 63 was reacted with dansylsarcosine

as described for 53 to give 64 (9%) as a yellow foam: ¹H NMR

(CDCl₃) shows rotamers, δ 8.59-8.53 and 8.37-8.23 (2m, 3H,

dansyl-2H, dansyl-4H, dansyl-8H), 7.60-7.49 (m, 2H, dansyl-

3H, dansyl-7H), 7.20-7.15 (m, 1H, dansyl-6H), 4.20-3.18 (m,

6H, N(Me)CH₂CO, NCH₂CH₂, NCHCHMe, NCHCH₂), 3.01-

2.73 (m, 12H, CHMe, NMe₂, NMe, COCHCH₂CH₂, NCH₂CHH),

2.12-1.92 (m, 1H, NCH₂CHH), 1.30-0.82 (m, 7H, CHMe,

COCHCH₂CH₂); MS (thermospray) m/z 513 (MH⁺); HRMS

calcd for C₂₆H₃₂N₄O₅S (MH⁺) 513.217 214, found 513.217 167;

HPLC 98% (t_R) 25.7 min).

The following compounds were similarly prepared.

(3S,3a R,6a S)-1-(Cyclop r op ylca r b on yl)-4-({(2S)-1-[(3-

isop r op oxyp h en yl)su lfon yl]p yr r olid in -2-yl}ca r bon yl)-3-

m eth ylh exa h yd r op yr r olo[3,2-b]p yr r ol-2-on e (70). Simi-

larly prepared as 69, using 3-(1-methylethoxy)benzenesulfonyl

chloride as the sulfonylating agent, product 70 was obtained

as a white solid (43%): IR (KBr) ν_max1754, 1674, 1661 cm^-1

;

¹H NMR (CDCl₃) δ 7.44-7.36 (m, 3H, C₆H₄), 7.11-7.04 (m,

1H, C₆H₄), 4.68-4.49 (m, 2H, OCHMe₂, NCHCO), 4.29 (t, J )

9.2 Hz, 1H, NCHHCH₂CH₂), 3.90-3.22 (m, 6H, NCHHCH₂-

CH₂, NCH₂CH₂, NCHCHMe, NCHCH₂, CHMe), 3.0-2.9 (m,

1H, COCHCH₂CH₂), 2.84-2.72 (m, 1H, NCH₂CHH), 2.20-1.76

(m, 5H, NCH₂CHH, NCH₂CH₂CH₂), 1.36 and 1.34 (2d, J )

6.1 Hz, 6H, OCHMe₂), 1.20-0.96 (m, 7H, CHMe, COCHCH₂-

CH₂); MS (thermospray) m/z 504 (MH⁺); HPLC 100% (t_R

28.3 min). Anal. (C₂₅H₃₃N₃O₆S‚0.2CH₂Cl₂) C, H, N, S.

)

3-{[((2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r b on yl)-6-

m et h yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]-

ca r bon yl}p yr r olid in -1-yl)su lfon yl]m eth yl}-N,N-d im eth -

ylben za m id e (71). To a suspension of 3-(dimethylamino)ben-

zenemethanesulfonic acid (43 mg, 0.175 mmol) in dichloro-

methane (2 mL) stirred at room temperature under nitrogen

was added DMF (0.003 mL) followed by triphosgene (38 mg,

0.127 mmol) and triethylamine (0.024 mL, 0.175 mmol). The

solution was stirred at room temperature for 2 h before a

solution of the trans-lactam 68 (30 mg, 0.088 mmol) and DBU

(0.026 mL, 0.175 mmol) in dichloromethane (2 mL) was added.

The mixture was stirred for 1.5 h at room temperature before

the reaction was quenched with 2-propanol (0.01 mL) and the

mixture was evaporated to dryness in vacuo. The residue was

partitioned between ethyl acetate (20 mL) and water (15 mL),

and the organic phase was washed with saturated sodium

hydrogen carbonate solution (15 mL) and brine (15 mL) and

dried (MgSO₄). The solvent was evaporated in vacuo to give a

white residue that was purified by preparative TLC on silica

gel, eluting with chloroform/methanol (95:5) to give 71 (10 mg,

22%) as a white solid: IR (KBr) ν_max1747, 1688, 1681, 1661,

r el-(3S,3a R,6a S)-1-(Cyclop r op ylca r bon yl)-4-[(1-{[5-(d i-

m et h yla m in o)-1-n a p h t h yl]su lfon yl}p ip er id in -2-yl)ca r -

bon yl]-3-m eth ylh exah ydr opyr r olo[3,2-b]pyr r ol-2(1H)-on e

(66). Compound 63 was reacted with ^D,L-pipecolic acid as

described for 53 to give 66 (14%) as a yellow foam: ¹H NMR

(CDCl₃) shows rotamers, δ 8.58-8.49, 7.60-7.47 and 7.30-

7.14 (3m, 6H, dansyl-2H, -4H, -8H, -3H, -7H, -6H), 4.87-4.65

and 4.10-2.66 (2m, 16H, NCHCO, NCH₂CH₂CH₂CH₂, NCH₂-

CH₂, NCHCHMe, NCHCH_2,CHMe, NMe₂, COCHCH₂CH₂,

NCH₂CHH), 2.07-0.80 (m, 14H, NCH₂CHH, CHMe, NCH₂CH₂-

CH₂CH₂, COCHCH₂CH₂); MS (thermospray) m/z 553 (MH⁺);

HRMS calcd for

C

₂₉H₃₆N₄O₅S (MH⁺) 553.248 468, found

553.248 712; HPLC 100% (t_R) 29.4 min).

Ben zyl (2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-

6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-yl]-

ca r bon yl}p yr r olid in e-1-ca r boxyla te (67). Compound 63

was reacted with Cbz-(S)-proline as described for 53 to give

67 (37%) as a white amorphous solid: IR (KBr) ν_max1747, 1701,

1664 cm^-1; ¹H NMR (CDCl₃) shows rotamers, δ 7.38-7.27 (m,

5H, C₆H₅), 5.20-4.95 (m, 2H, PhCH₂O), 4.48-4.44 and 4.37-

4.29 (2m, 2H, NCHCO, NCHHCH₂CH₂), 3.81-3.49 (m, 5H,

NCHHCH₂CH₂, NCH₂CH₂, NCHCHMe, NCHCH₂), 3.37-3.20

(m,1H,CHMe),3.08-1.82(m,7H,COCHCH₂CH₂,NCH₂CH₂CH₂,

NCH₂CH₂), 1.21-0.95 (m, 7H, CHMe, COCHCH₂CH₂); HPLC

100% (t_R) 24.9 min); MS (thermospray) m/z 440 (MH⁺); Anal.

(C₂₄H₂₉N₃O₅‚0.3H₂O) C, H, N.

1

1651, 1633 cm^-1; H NMR (CDCl₃) δ 7.7-7.6 (m, 2H, C₆H₄),

7.5-7.4 (2H, m, C₆H₄), 4.7-4.3 (m, 3H, PhCH₂, NCHCO), 4.1-

3.9 (m, 2H, NCH₂CH₂CH₂), 3.8-3.3 (m, 5H, NCH₂CH₂, NCH-

CHMe, NCHCH₂, CHMe), 3.2-2.9 (7H, m, NMe₂, COCHCH₂-

CH₂), 2.86-2.74 (m, 1H, NCH₂CHH), 2.4-1.8 (m, 5H, NCH₂-

CHH, NCH₂CH₂CH₂), 1.4-0.8 (m, 7H, CHMe, COCHCH₂CH₂);

MS (thermospray) m/z 531 (MH⁺); HPLC 98% (t_R) 21.2 min).

Anal. (C₂₆H₃₄N₄O₆S) C, H, N, S.

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J M0102203

Article Doi

Design and synthesis of pyrrolidine-5,5-trans-lactams (5-oxohexahydropyrrolo[3,2-b]pyrroles) as novel mechanism-based inhibitors of human cytomegalovirus protease. 2. Potency and chirality

DOI: 10.1021/jm0102203

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