Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-length NS3 (protease-helicase/NTPase): A comparative study of different C-terminals

Bioorganic & Medicinal Chemistry 11 (2003) 2551–2568

Acyl Sulfonamides as Potent Protease Inhibitors of the Hepatitis

C Virus Full-Length NS3 (Protease-Helicase/NTPase):

A Comparative Study of Diﬀerent C-Terminals

Anja Johansson,^aAnton Poliakov,^bEva Akerblom,^aKarin Wiklund,^a

b

Gunnar Lindeberg,^aSusanne Winiwarter,^aU.Helena Danielson,

Bertil Samuelsson^cand Anders Hallberg^a,*

^aDepartment of Medicinal Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden

^bDepartment of Biochemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden

^cMedivir AB, Lunastigen 7, SE-141 44 Huddinge, Sweden

Received 17 January 2003; accepted 13 March 2003

Abstract—Synthesis and inhibitory potencies of three types of protease inhibitors of the hepatitis C virus (HCV) full-length NS3

(protease-helicase/NTPase) are reported: (i) inhibitors comprising electrophilic serine traps (pentaﬂuoroethyl ketones, a-keto acids,

and a-ketotetrazoles), (ii) product-based inhibitors comprising a C-terminal carboxylate group, and (iii) previously unexplored

inhibitors comprising C-terminal carboxylic acid bioisosteres (tetrazoles and acyl sulfonamides).Bioisosteric replacement with the

tetrazole group provided inhibitors equally potent to the corresponding carboxylates, and substitution with the phenyl acyl sulfo-

namide group yielded more potent inhibitors.The hexapeptide inhibitors Suc-Asp- d-Glu-Leu-Ile-Cha-Nva-NHSO₂Ph and Suc-

Asp-d-Glu-Leu-Ile-Cha-ACPC-NHSO₂Ph with K_ivalues of 13.6 and 3.8 nM, respectively, were approximately 20 times more

potent than the corresponding inhibitors with a C-terminal carboxylate and were comparable to the carboxylate-based inhibitor

containing the native cysteine, Suc-Asp-d-Glu-Leu-Ile-Cha-Cys-OH (K_i=28 nM).The acyl sulfonamide group constitutes a very

promising C-terminal functionality that allows for prime site optimization.

Introduction

The HCV genome is a single stranded RNA of positive

polarity, which encodes the polyprotein NH₂-C-E1-E2-

p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH, that is

subsequently processed into ten mature viral proteins by

proteolytic enzymes.^8ꢀ10The non-structural protein 3

(NS3) is a bi-functional enzyme, of which the ﬁrst N-

terminal 180 amino acids possess protease activity and

the remaining 450 amino acids RNA helicase/NTPase

activity.The NS3 protease is responsible for auto-

catalytic cis cleavage at the NS3-NS4A junction and for

trans cleavages at the NS4A/NS4B, NS4B/NS5A and

NS5A/NS5B junctions.The consensus sequence for the

trans processing is Asp/Glu-x-x-xCys # Ser/Ala.The P1

Cys residue has been shown to be of major importance

for active site recognition in substrates.^8,11Enzyme

inhibitors that prevent the liberation of NS proteins

could ultimately inhibit the replication of the HCV

virus.Therefore, the NS3 protease, classiﬁed as a

chymotrypsin-like serine protease, has been studied

It is estimated that the Hepatitis C Virus (HCV), ﬁrst

identiﬁed in 1989, has infected more than 170 million

people globally, which is almost ﬁve times as many as

infected by the HIV virus.^1,2Acute HCV infection

results in chronic hepatitis in approximately 80% of the

cases, and can eventually progress into liver cirrhosis,

hepatocellular carcinoma or liver failure.^2ꢀ4Thus, HCV

is the primary reason for liver transplantation in the

western countries.^4,5Approved therapies are based on

interferon-a (IFN-a) and more recently pegylated IFN-

a in combination with ribavirin.There is an urgent need

for more eﬀective and safe therapies, especially as no

vaccine is yet on the market.^6,7

*Corresponding author.Tel:. +46-18-4714284; fax: +46-18-4714474;

e-mail: anders.hallberg@bmc.uu.se

doi:10.1016/S0968-0896(03)00179-2

2552

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

intensively and represents one of the primary targets in

the search for new eﬀective antiviral agents against

HCV.^6,7,10,12,13

Results

Chemistry

We aimed to synthesize penta- and hexapeptides con-

taining C-terminal pentaﬂuoroethyl ketones, a-keto

acids, a-ketotetrazoles, carboxylates, tetrazoles, and

acyl sulfonamides.We decided to utilize a direct

method, previously reported by us as a means of peptide

assembly in the N to C (inverse) direction on solid-

phase, which thus allows for C terminal modiﬁcations.⁴⁵

Classical solid-phase peptide synthesis (SPPS) based on

the work of Merriﬁeld is executed in the C to N direc-

tion, with the C-terminal consequently attached to the

solid support.⁴⁶The strategy used herein for the pre-

paration of the target compounds relied on the pre-

paration of various P1 building blocks for ultimate

attachment to resin bound peptides in a combinatorial

fashion.The synthesis of the P1 building blocks and the

corresponding HCV NS3 protease inhibitors, evaluated

in the full-length NS3 assay (Table 1), are outlined in

Schemes 1–7.

To date, several types of inhibitors of the HCV NS3

serine protease have been discovered,^7,13ꢀ15of which the

classical serine-trap inhibitors represent one category.

The electrophilic groups, forming reversible covalent

bonds with the active site serine hydroxyl group,

employed in these inhibitors are, for example, alde-

hydes, a-keto amides, a-keto esters, a-diketones, boro-

nic acids, and a-keto acids.^16ꢀ29The second category of

HCV NS3 protease inhibitors is derived from the

observation that the HCV NS3 protease is signiﬁcantly

inhibited by its N-terminal hexamer cleavage products

comprising a C-terminal carboxylate.^30,31Such product

inhibition, to the extent demonstrated in the NS3 pro-

tease case, is rare for serine proteases.The binding

exhibited by the carboxylate in the active site seems to

be a non-covalent interaction with the oxyanion cavity

(NH of Gly137 and Ser139) and with the catalytic his-

tidine as deduced from a 3-D-structure.³²Several struc-

turally optimized product-based inhibitors have been

prepared, which have led successively to inhibitors

possessing less peptide-like properties.^{7,13,15,22,33ꢀ35}

The product-based NS3 protease inhibitors display

high speciﬁcity for the HCV NS3 serine protease over

other serine proteases,²⁷and the ﬁrst drug-like product-

based HCV NS3 protease inhibitor, currently under-

going extensive clinical evaluation has now been

disclosed.³⁶

The building blocks for the preparation of inhibitors

with a C-terminal pentaﬂuoroethyl ketone function

were obtained from the commercially available Boc-

protected amino acids, Boc-l-norvaline (Boc-Nva-OH)

1, Boc-1-aminocyclopropane-1-carboxylic acid (Boc-

ACPC-OH) 2, and Boc-l-2-aminobutyric acid (Boc-

Abu-OH) 3, respectively, as is illustrated in Scheme 1.

The acids were converted to the corresponding penta-

ﬂuoroethyl ketones 4–6 using a standard procedure

through the synthesis of Weinreb amides, as described

by Peet and coworkers.^47ꢀ49Addition of penta-

ﬂuoroethyl lithium (generated in situ) delivered the

pentaﬂuoroethyl ketones 4–6, in acceptable total yields

(>63%).Notably, the ﬁnal yields were aﬀected con-

siderably by the tendency of the compounds 4–6 (and 7–

12, vide infra) to sublimation, which made handling

diﬃcult.Direct coupling of Boc deprotected penta-

ﬂuoroethyl ketone building blocks to the resin bound

peptide 37a using either N-[(1H-benzotriazole-1-yl)-

(dimethylamino)methylene] - N - methylmethanaminium

tetraﬂuoroborate N-oxide (TBTU) and N,N-diiso-

A literature survey reveals that most of the inhibi-

tors of the NS3 protease have been evaluated in an

artiﬁcial

truncated

NS3

(protease

domain)

assay.^{16,18ꢀ23,25ꢀ27,29ꢀ31,33,34,37,38}Previously, we have

compared results from a truncated NS3 assay to a full-

length NS3 (protease-helicase/NTPase) assay that we

considered to be more relevant to use for evaluation of

inhibitors, since the latter assay should better mimic the

in vivo situation.^39,40Diﬀerent structure–activity rela-

tionships were encountered, in addition to retained

inhibitory potencies of shorter product-based peptide

inhibitors in the full-length NS3 assay.³⁹As indicated

by molecular modelling, potential binding interactions

with the helicase domain might explain the data

obtained.⁴⁰In this context, it should be noted that full-

length NS3 assays have been utilized more frequently in

recent HCV related patent applications, with a few

examples cited herein.^41ꢀ44Hence, we felt prompted to

examine protease inhibitors with diﬀerent characteristic

features, with a special focus on the C-terminals, as

inhibitors of the full-length NS3 (protease-helicase/

NTPase) protein.

Herein, we report results for three types of HCV NS3

protease inhibitors as assessed in the full-length NS3

assay: (i) inhibitors comprising electrophilic serine traps,

(ii) product-based inhibitors with

a

C-terminal

carboxylate group, and (iii) previously unexplored

inhibitors comprising C-terminal carboxylic acid bio-

isosteres.Incorporation of a C-terminal phenyl acyl

sulfonamide group provided the most potent inhibitors.

Scheme 1.

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

2553

propylethylamine (DIEA), or by the acid ﬂuoride

method failed.This forced us to reduce the ketones to

the corresponding alcohols prior to the couplings, which

were executed subsequently in quantitative yields (vide

infra).Thus, reduction of the ketones 4–6 using sodium

borohydride gave the alcohols 7⁵⁰to 9 as diastereomeric

mixtures in 63–91% yield,⁵¹which upon Boc deprotec-

tion followed by pH adjustment gave the free amines

10–12 in good yields.

involves a one-step transformation of an amide into a

protected tetrazole.⁵⁹The cyanoethyl amides 23 and 24

were prepared from the corresponding Boc-protected

amino acids 1 and 2 using aminopropionitrile, N,N⁰-

dicyclohexylcarbodiimide (DCC) and N-hydroxy-

benzotriazole (HOBt) in 83 and 85% yields, respectively.

The amides were exposed to diethylazodicarboxylate

(DEAD), triphenylphosphine and trimethylsilyl azide to

generate the corresponding cyanoethyl protected tetra-

zoles, which were deprotected subsequently to yield the

tetrazoles 25 and 26.Tetrazole formation is highly

dependent on the steric hindrance around the neigh-

boring carbon, which was reﬂected in the yields of the

two derivatives.⁵⁹Thus, the total yield of the ACPC-

based derivative 26 possessing a quaternary neighboring

carbon was 15%, in comparison to 56% of the Nva-based

derivative 25.Boc deprotection ﬁnally aﬀorded the hydro-

chloride salt of the tetrazoles 27 and 28 in high yields.

Synthesis of the building blocks for the preparation of

the a-ketotetrazole inhibitors is outlined in Scheme 2,

and commenced with the generation of the aldehydes

13^52,53and 14,^54,55via the corresponding Weinreb

amides, and subsequent reduction using diisobutylalu-

minium hydride (DIBAL).^49,56Reaction of the alde-

hydes 13 and 14 with potassium cyanide aﬀorded the

cyanohydrins,⁵⁷which were subsequently trapped by

acetylation with acetic anhydride in the presence of

pyridine to yield the cyano compounds 15 and 16.The

equilibrium between the aldehyde and the correspond-

ing cyanohydrin in the case of Boc-Nva-H was forced

almost to completion, in contrast to the case with the

more sterically hindered Boc-ACPC-H, where approxi-

mately 50% conversion was achieved.The generation of

the tetrazoles 17 and 18 was accomplished readily in

almost quantitative yields using the protocol adopted

from Koguro et al.,⁵⁸by heating sodium azide and 15 or

16 in toluene, in the presence of triethylamine hydro-

chloride, followed by simple extraction.Subsequent

deacetylation using potassium carbonate gave the alco-

hols 19 and 20, and Boc deprotection ﬁnally yielded the

alcohols 21 and 22 as hydrochloride salts in excellent

yields.

Scheme 3.

As an alternative to the tetrazoles, we replaced the C-

terminal carboxyl group with the bioisosteric acyl sul-

fonamide group.The synthesis of the unnatural amino

acids comprising a phenyl acyl sulfonamide group is

shown in Scheme 4.The commercially available Boc-

protected amino acids 1 and 2 were coupled to benze-

nesulfonamide using 1,1-carbonyldiimidazole (CDI)

and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to aﬀord

the acyl sulfonamides 29 and 30 in 64 and 90% yield,

respectively.⁶⁰Boc deprotection gave the hydrochlorides

31 and 32 in high yields (95 and 90%), which were

transformed subsequently into the corresponding zwit-

terions 33 and 34 by propene oxide.^45,61

Scheme 2.

The synthesis of the tetrazole analogues, 27 and 28, of

the amino acids Nva and ACPC, where the carboxyl

group is replaced by the bioisosteric 5-tetrazolyl group

are presented in Scheme 3.The synthesis relies on the

method previously reported by Duncia et al., which

Scheme 4.

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A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

The general method for the synthesis of the resin bound

tetra- and pentapeptides 37a,b is described in Scheme 5.

A TentaGel based solid support with a photolabile lin-

ker, that is, TentaGel-P-Linker-2 35, was used as start-

ing resin.^62,63The capping group, succinic acid, was

introduced eﬃciently through coupling of succinic

anhydride in presence of DIEA to yield 36. A full conver-

sion was achieved as conﬁrmed by ¹³C NMR.The peptides

were assembled by coupling of the amino acid tri-tert-

butoxysilyl (Sil) esters using N-[(dimethylamino)-1H-1,2,3-

triazolo-[4,5-b]pyridin-1-yl-methylene]-N-methylmethan-

aminium hexaﬂuorophosphate N-oxide (HATU) and

2,4,6-trimethylpyridine (TMP), without any preactiva-

tion.⁴⁵The Sil esters were cleaved by using 5% tri-

ﬂuoroacetic acid (TFA) in dichloromethane.The base

labile ﬂuorenylmethyl (Fm) ester side chain was selected

to protect Asp and Glu.Peptide assembly in the N to C

direction is normally associated with a higher risk of

epimerization, since the amino acid residue being acti-

vated is N-acylated.As shown by us previously, epi-

merization can be suppressed when fast (HATU

promoted) coupling methods are used and preactivation

avoided.⁴⁵Accordingly, HPLC analysis after cleavage

of analytical samples of 37a,b shows the desired product

as the dominant peak.

were liberated from the resin as major products by

photolysis at 350 nm, and were ﬁnally puriﬁed by RP-

HPLC.The resin bound pentaﬂuoroethyl alcohols were

oxidized readily using Dess–Martin periodinane^64,65and

were cleaved thereafter from the resin to smoothly pro-

vide the desired peptide ketones 38–40a,b as the pre-

dominant products, which were puriﬁed subsequently

by RP-HPLC.The same oxidation and cleavage proce-

dure was used initially for the preparation of the

a-ketotetrazoles 41 and 42.As deduced by HPLC-MS

analysis of the crude reaction mixtures, complex mixes

of compounds had been formed.Only the a-keto-

tetrazoles 42a,b, based on ACPC, could be isolated after

puriﬁcation by RP-HPLC.No products of the Nva

derivatives 41 could be traced after oxidation and pho-

tolysis.As an alternative method, the alcohol product

(after coupling of the tetrazole containing alcohol 21 to

37b) was released from the solid support prior to oxi-

dation.According to the HPLC-MS analysis only par-

tial coupling of 21 had been achieved.The mixture was

mainly composed of the desired alcohol product, the

starting material and the dimethylamide of 37b (the

dimethylamine group most probably originating from

HATU and not from DMF, since the dimethylamide

side-product was formed to a greater extent when

dichlormethane was used as solvent).The hydroxyl-

peptide was puriﬁed by RP-HPLC and was oxidized

subsequently in solution using the Dess–Martin reagent.

Thus, the a-ketotetrazole inhibitor 41b could be isolated

after RP-HPLC puriﬁcation.

In some cases, the crude products after coupling of the

P1 building blocks (Scheme 6) contained isomers which

had the same molecular weight as the predominantly-

formed target compound.A partial epimerization of the

Cha residue presumably accounts for the side-product

formation.The major products were evaluated in the

bioassay only after separation from the epimer, unless

otherwise stated.Apart from the epimerization of the

Cha residue it is well recognized that epimerisation a

to the activated carbonyl function of electrophilic

inhibitors, such as in the pentaﬂuoroethyl ketones 38a,b

and 40a,b, is diﬃcult to avoid.^48,51Furthermore, in aque-

ous solution, hydration of the electrophilic ketones

38–40a,b occurs.⁴⁸Hence, a mixture of the ketone/hydrate

form and possibly epimers were used in the biological

evaluation.

Scheme 5.

The preparation of the a-keto acid inhibitor 49b is out-

lined in Scheme 7.The a-hydroxy-b-amino acid com-

pound 48 was prepared according to established

methods using a modiﬁed isolation procedure.^52,66,67

Thus, the aldehyde 14 was transformed into the corre-

sponding cyanohydrin (see the discussion above con-

cerning cyanohydrins from Boc-ACPC-H in the

synthesis of 16) by potassium cyanide.The crude cya-

nohydrin was hydrolyzed by concentrated hydrochlo-

ride acid in dioxane under reﬂux conditions.Isolation of

the zwitterionic product 47, in a total yield of 41%, was

achieved smoothly by removal of the hydrochloride salt

by propene oxide in an ethanolic solution,^45,61where-

upon the product precipitated.The Fmoc protected

a-hydroxy-b-amino acid 48 was obtained by reaction

For generation of the inhibitors 38–46a,b the P1 build-

ing blocks, described in Schemes 1–4, were coupled in a

parallel fashion to aliquots of 37a and 37b using HATU

as coupling reagent, as outlined in Scheme 6.TMP was

used as base for the coupling of the free amines 10–12,

33 and 34.To eﬀect the coupling of the hydrochlorides

21, 22, 27, and 28 the stronger base N,N-diisopropyl-

amine (DIEA) was employed.The resins were treated

subsequently with 25% piperidine in DMF and 2.5%

TFA in CH₂Cl₂to remove the Fm groups and the

formed piperidine salt, respectively.The peptide target

compounds comprising the C-terminal tetrazole 43a,b

and 44a,b and acyl sulfonamide group 45a,b and 46a,b

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

2555

Scheme 6. Method I: (a) 10, 11 or 12, HATU, TMP; (b) 25% piperidine/DMF; (c) 2.5% TFA/CH₂Cl₂; (d) Dess–Martin periodinane, (e) hv (350

nm), MeOH.Method II: (a) 21, HATU, DIEA; (b) 25% piperidine/DMF; (c) 2.5% TFA/CH₂Cl₂; (d) hv (350 nm), MeOH; (e) Dess–Martin peri-

odinane.Method III: (a) 22, HATU, DIEA; (b) 25% piperidine/DMF; (c) 2.5% TFA/CH₂Cl₂; (d) Dess–Martin periodinane; (e) hn (350 nm),

MeOH.Method IV: (a) 27, 28, HATU, DIEA; (b) 25% piperidine/DMF; (c) 2.5% TFA/CH₂Cl₂; (d) hn (350 nm), MeOH.Method V: (a) 33, 34,

HATU, TMP; (b) 25% piperidine/DMF, (c) 2.5% TFA/CH₂Cl₂, (d) hn (350 nm), MeOH.

Protease inhibition of full-length HCV NS3 (protease-

helicase/NTPase)

with Fmoc-Cl and aqueous sodium carbonate in diox-

ane in a rather poor yield (34%) and was attached sub-

sequently to a polystyrene-based 2-chlorotrityl chloride

resin in presence of DIEA, using a slightly modiﬁed lit-

erature procedure.⁶⁸The loading of the resin could be

monitored readily by IR analysis of the resin through

the appearance of a carbonyl stretch vibration band at

1732 cm^ꢀ1.The resin bound derivative was then sub-

jected to classical SPPS according to the standard

Fmoc/t-Bu protocol using TBTU as coupling reagent

and DIEA as base to deliver desired peptide sequence.

Treatment with TFA released the alcohol-containing

peptide from the resin.Oxidation using the Dess–Mar-

tin reagent in solution ﬁnally gave the a-keto acid 49b in

reasonable 43% yield, calculated from the weight of the

crude peptide used in the oxidation.

The inhibitors 38–40a,b, 41b, 42–46a,b, and 50–54a,b

(Table 1) were evaluated in an in vitro assay comprising

the full-length NS3 protein (amino acids 1027–1657 of

the HCV polyprotein) and the central part of NS4A

(2K-NS4A) as cofactor.^39,40,69The enzyme, cofactor

and inhibitor were pre-incubated for 10 min, whereafter

the reaction was initiated by addition of the substrate.

As a consequence, potentially slow kinetics of binding

and dissociation of the electrophilic inhibitors was not

studied herein.The reference compound Ac-Asp- d-Gla-

Leu-Ile-Cha-Cys was examined in parallel, yielding an

average inhibition of 70Æ11% at 0.104 mM.The inhi-

bitors are divided into three categories: A, electrophilic;

B, product-based; and C, carboxylic acid bioisosteres,

and their K_ivalues are presented in Table 1.

The remaining peptide inhibitors which possess a

C-terminal carboxylic acid, that is 50–54a,b were syn-

thesized using standard SPPS conditions.

Discussion

The penta- and hexapeptide inhibitors employed for the

assessment of the impact of diﬀerent C-terminal resi-

dues (P1 building blocks) contain two or three carboxyl

groups in the N-terminal region, while the central part

of the peptides encompass identical lipophilic amino

acid residues.As is shown in Table 1, all P1-building

blocks provided penta- and hexapeptidic protease inhi-

bitors of the full-length NS3 (protease-helicase/NTPase)

protein with K_ivalues in the micro- to nanomolar range.

Peptides with the native Cys in the P1 position were

included in the series as controls.The thiol side-chain

was superior to all lipophilic side chain substitutes as

apparent from a comparison of the product-based inhi-

bitors 50–54a,b (category B, Table 1).This observation is

in reasonable agreement with previous results using the

protease domain assay.^27,33Thus, the cysteine peptides

50a,b were approximately ten times more active than the

Nva-, ACPC- and allylglycine-based inhibitors 51–53a,b,

Scheme 7.

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A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

Table 1. Inhibition of the NS3 protease activity of the full-length

NS3 (protease-helicase/NTPase)

which in turn were more than ten times more active than

the inhibitors with Phe in P1 (54a,b).

Evaluation of the electrophilic inhibitors (category A,

Table 1) shows that the pentaﬂuoroethyl ketones of Nva

38a,b (and Abu 40a,b) as inhibitors were equally or

slightly more active than the corresponding acids

(51a,b), However the combination of the penta-

ﬂuoroethyl ketone and the cyclopropane ring of 39a,b

was deleterious, possibly due to unfavorable steric

interactions.The postulated tetrahedral hemiketal

formed with the active site serine hydroxyl might not be

combined successfully with a neighboring disubstituted

a-carbon as present in 39a,b.The a-ketotetrazole inhib-

itors 41b and 42a,b were primarily designed as bio-

isosteres to the very potent a-keto acid inhibitors

discovered by Narjes et al.²¹Therefore, the a-keto acid

49b was made as a reference substance.In the full-

length NS3 assay 49b exhibited a 20-fold higher potency

compared to the corresponding a-ketotetrazole 42b.

This result correlates with a recent observation by

Narjes’ group who studied tripeptide inhibitors with

diﬂuoroAbu in the P1 postion.²⁰However, the Nva-

based inhibitor 41b was fairly active, again demonstrat-

ing that serine-trap carbonyl groups are not compatible

with the disubstituted a-carbon found in the ACPC

based inhibitors in the present series (39a,b, 42a,b com-

pared to 38a,b and 41b).

Inhibitor type

Compd

P1

K_i(mM)ÆSD

A: Electrophilic

40a

40b

2.0

0.081Æ0.007

38a

38b

2.9Æ0.3

0.12Æ0.02

39a

39b

10% (83 mM)^a

1.8Æ0.1

49b

0.098Æ0.006

0.30Æ0.03

41b^b

42a

42b

13Æ2

1.9Æ0.4

Bioisosteric replacements in lead substances have fre-

quently been utilized in order to retain or enhance

potencies and to simultaneously improve pharmacoki-

netic properties.^70ꢀ72The inhibitors 43–46a,b (category

C, Table 1) were prepared as bioisosteres to the corre-

sponding product-based inhibitors with a C-terminal

carboxylate.Two common carboxylic acid bioisosteres,

the tetrazole and the acyl sulfonamide, with calculated

pK_a-values similar to that of the carboxyl group were

explored.⁷²It was found that both the tetrazole and the

acyl sulfonamide were suitable substitutes for the

C-terminal carboxylate (see compounds 43–46a,b, 51a,b

and 52a,b, Table 1).It is noteworthy that the acyl sul-

fonamide group had a pronounced positive impact on

the inhibitory potencies.Thus, both the Nva and ACPC

based inhibitors 45a,b and 46a,b exhibited approxi-

mately 10-fold lower K_ivalues than the carboxyl analo-

gues 51a,b and 52a,b.The acyl sulfonamide comprising

hexapeptide inhibitors 45b and 46b (K_i=13.6 and 3.8

nM, respectively) were indeed as potent as the inhibitor

that encompasses the native Cys (50b, K_i=28 nM).

Bearing in mind that these inhibitors have a non-opti-

mal P1 side chain, it is tempting to postulate that novel

interactions of the acyl sulfonamide group at the active

site might account for the improved inhibitory potency.

This hypothesis was supported by computer modeling,

where the 3-D-structure of full-length NS3 (as a scNS3-

NS4A construct) disclosed by Yao et al.,³²was used as a

starting point for docking of the inhibitor 45a by the

procedure recently reported.⁴⁰Diﬀerent binding modes

seem possible, of which two typical examples are

explored in Figure 1a and b.Thus, one of the oxygen of

the sulfone group might be engaged in hydrogen bond-

ing with the catalytic His57 (1a) or with the oxyanion

B: Product-based

50a

50b

0.12Æ0.01

0.028Æ0.004

51a

51b

1.9Æ0.1

0.22Æ0.03

52a

52b

1.8Æ0.2

0.091Æ0.005

53a

53b

3.0Æ0.2

0.12Æ0.01

54a

54b

30Æ3.5

1.5Æ0.2

C: Carboxylic

acid bioisosteres

43a

43b

5.3Æ0.6

0.156Æ0.005

44a^c

44b

10.2Æ1.1

0.19Æ0.01

45a

45b

0.21Æ0.04

0.0136Æ0.0005

46a

46b

0.56Æ0.1

0.0038Æ0.0004

^aPercent inhibition at 83 mM.

^bPurity=91%.

^c5:3 mixture of l-/d-Cha diastereomers.

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

2557

Figure 1. Stereoview of two possible binding modes (a and b) of Suc-d-Glu-Leu-Ile-Cha-Nva-NHSO₂Ph 45a docked into the protease active site of

the full-length scNS3–NS4A protein.Protease (3–181) residues depicted in pink, helicase (182–631) residues in blue, and the inhibitor (only the P2 –

P1 part of the inhibitor shown) color-coded with all carbons in green, oxygens in red, nitrogens in blue, and hydrogens in gray.Only residues close to

the inhibitor (within 3.0 A) are shown.Hydrogen bonds are indicated.

cavity (NH of Gly137 and Ser139) (1b).Furthermore

the carbonyl of the acyl sulfonamide group could act as

a hydrogen bond acceptor of the NH hydrogen of

Gly137 and of the OH hydrogen of the catalytic Ser139

(1a), or with the ﬂexible Lys136 (1b).Additionally, there

is suﬃcient space to accommodate the phenyl moiety of

the acyl sulfonamide (1a and b) allowing for favorable

hydrophobic interactions with the closely situated

Phe43 in one case (1a).Two hydrogen bonds from the

helicase residues Gln526 (1a and b) and His 528 to the

NH of the P2 residue and the carbonyl of the P4 resi-

due, respectively, were established in the model (the

latter not shown).Very recently, while this paper was in

preparation, Campbell et al.disclosed a patent applica-

tion covering potent HCV NS3 protease inhibitors with

P1 acyl sulfonamides.⁴⁴We are convinced that the acyl

starting point for reﬁnements which might ultimately

yield more drug-like inhibitors.

Conclusions

The inhibitory potencies of three types of HCV NS3

protease inhibitors assessed in the relevant full-length

NS3 (protease-helicase/NTPase) assay have been com-

pared, and consist of: (i) inhibitors comprising electro-

philic serine traps, (ii) product-based inhibitors with a

C-terminal carboxylate, and (iii) previously unexplored,

inhibitors with C-terminal carboxylic acid bioisosteres.

To summarize, bioisosteric replacement of the C-term-

inal carboxylic acid of the product-based inhibitors of

the HCV NS3 protease with tetrazoles and, especially,

acyl sulfonamide groups was successful.In fact, the

phenyl acyl sulfonamide residue produced product-

based inhibitors up to 20-fold more potent than the

corresponding carboxylates.In addition to possessing

better pharmacokinetic properties than a carboxylate, the

aryl acyl sulfonamide group also allows for structural

elongations and modiﬁcations.Thus, the bioisosteric acyl

sulfonamide group constitutes

a

very promising

C-terminal residue that allows for prime site optimiza-

tion with the phenyl group serving as a handle.Pep-

tides, in particular those containing multiple

carboxylates, as preferred by the HCV NS3 protease,

are unlikely to be orally bioavailable.However, the

bioisostere approach presented here could serve as a

2558

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

sulfonamide group has great potential in such inhibitors

and will be explored further in our on-going drug dis-

covery eﬀorts.

determined from N-analysis), prepared by coupling of 4-

acetyl-2-methoxy-5-nitrophenoxy acetic acid to Tenta-

Gel-S-NH₂(RAPP Polymer), followed by reduction with

NaBH₄.^62,63The amino acid tri-tert-butoxysilyl (Sil)

esters used in the solid-phase syntheses in the inverse

direction were prepared as described by us previously.⁴⁵

2-chloro-tritylchloride, H-Cys(Trt)-2-chlorotrityl and

Fmoc-Phe-Wang resins were obtained from Alexis Cor-

poration, Senn Chemicals and Nova Biochem, respec-

tively.The N-Fmoc protected amino acids, Boc-Nva-

OH 1, Boc-ACPC-OH 2 and Boc-Abu-OH 3 were pur-

chased from Senn Chemicals, Alexis Corporation, Nova

Biochem or Bachem. N-[(1H-benzotriazole-1-yl)-(dime-

thylamino)methylene]-N-methylmethanaminium tetra-

ﬂuoroborate N-oxide (TBTU) and N-[(dimethylamino)-

1H-1,2,3-triazolo-[4,5-b]pyridin-1-yl-methylene]-N-

methylmethanaminium hexaﬂuorophosphate N-oxide

(HATU) were obtained from Richelieu Biotech and

Millipore or Applied Biosystems, respectively.The

bases 2,4,6-trimethylpyridine (TMP, collidine) (Merck,

p.a) or N,N-diisopropylethylamine (DIEA), redistilled,

99.5% from Aldrich were used in the peptide synth-

esis.DMF (Aldrich, 99+. %, HPLC grade) was

stored over molecular sieves (4 A); dichloromethane

(Riedel-deHaen) was freshly distilled from calcium

hydride, and THF from sodium and benzophenone.

Pentaﬂuoroethyl iodide, min 97%, was obtained from

Stream Chemicals and Dess–Martin periodinane [1,1,1-

tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-[1H]-one],

97% from Aldrich or Lancaster Synthesis.Other mate-

rials were obtained from commercial sources and used

without further puriﬁcation.

Experimental

Chemistry. General methods

¹H and ¹³C NMR spectra were recorded on a JEOL

JNM-EX270 spectrometer at 270 (68) MHz or a JEOL

JNM-EX400 at 400 (100) MHz at ambient temperature.

Chemical shifts are reported as d values (ppm) indirectly

referenced to TMS by the solvent signal (CHCl₃) d 7.26,

(CDCl₃) d 77.23, (methanol-d₃) d 3.31, (methanol-d₄) d

49.15, (DMSO-d₅) d 2.50 and (DMSO-d₆) d 39.51. IR

spectra were recorded on a Perkin–Elmer Model 1605

FT-IR and are reported as n_max(cm^ꢀ1), and for

measurement of neat solids the IR spectrometer was

equipped with a Microfocus Beam Condenser with

ZnSe lenses in a Diasqueeze Plus Diamond Compressor

Cell (Graseby Specac Inc., Smyrna, USA). Column

chromatography was executed on Merck silica gel 60

(40–63 mm).Elemental analyses were performed by

Mikro Kemi AB, Uppsala, Sweden or Analytische

Laboratorien, Lindlar, Germany.Optical rotations were

recorded on a Perkin–Elmer Model 241 polarimeter at

ambient temperature.Speciﬁc rotations ([ a]_D) are

reported in deg/dm, and the concentration (c) is given

in g/100 mL in the speciﬁed solvent.Cleavage of the

peptides from the photolabile resin was performed in a

RPR-200 Rayonet Chamber Reactor equipped with the

Rayonet Merry-Go-round Model-RMA-500 (22 sam-

ples) working as a photoreactor.All samples were rota-

ted past the light source at a speed of 5 RPM.The

stirring in each vial was conducted using a rebuilt

magnetic stirrer placed underneath the reactor.The

photoreactor was equipped with 16 25W lamps emit-

ting 350 nm UV-light.The distance to the sample

was 1.7 cm. The Quest 210^TMorganic synthesizer

(Argonaut Technologies) was utilized in the peptide

syntheses.RP-HPLC-MS was performed on a Gilson-

Finnigan ThermoQuest AQA system in ESI mode, on

a Zorbax Stable Bond C8 column (20Â50 mm or

4.6Â50 mm), using an acetonitrile/H₂O gradient with

50 mM NH₄OAc (pH 6.3) (method A) or an aceto-

nitrile/H₂O gradient with 0.05% HCOOH (method B),

using UV (214, 255 nm) and MS detection.RP-HPLC

was performed on an analytical LiChrosphere C18

column (0.4Â25 cm, 5 mm particles, 100 A pores)

using an acetonitrile/H₂O gradient with 0.1% TFA

(method C), or an analytical Vydac C18 column

(0.46Â15 cm, 10 mm particles, 300 A pores).Amino

acid analyses were performed at the Department of

Biochemistry, Uppsala University, Sweden, on 24 h

hydrolyzates (on oxidized samples of Cys containing

peptides) with an LKB 4151 alpha plus analyser using

ninhydrin detection.

tert-Butyl (1S)-3,3,4,4,4-pentaﬂuoro-2-oxo-1-propylbutyl-

carbamate (4). Boc-Nva-OH 1 was transformed into

the corresponding Weinreb amide⁵²by standard proce-

dures^48,49(data for the Weinreb amide, tert-butyl (1S)-1-

{[methoxy(methyl)amino]carbonyl}butylcarbamate: ¹H

NMR (400 MHz, CDCl₃) d 5.13 (br d, 1H, NH), 4.68

(m, 1H), 3.77 (s, 3H), 3.20 (s, 3H), 1.73–1.58 (m, 2H),

1.57–1.30 (m, 2H), 1.44 (s, 9H), 0.92 (dd, J=7.3, 7.3

Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) d 173.5, 155.7,

79.6, 61.7, 50.3, 35.3, 32.2, 28.5, 18.8, 13.9. IR (neat)

3326, 1716, 1663 cm^ꢀ1.Anal.calcd for C ₁₂H₂₄N₂O₄: C,

55.4; H, 9.3; N, 10.8. Found: C, 55.1; H, 9.4; N, 10.7).

Condensed pentaﬂuoroethyl iodide (9.0 g, 37 mmol)

was added to freshly distilled ether (150 mL) in a dried

three-necked ﬂask under a stream of N₂, at ꢀ78 ^ꢁC.A

solution of the Weinreb amide (3.30 g, 12.7 mmol) in

freshly distilled ether (50 mL) was added, followed by

dropwise addition of 1.5 M methyl lithium–lithium bro-

mide complex in ether (24 mL, 36 mmol), at a rate that

maintained the temperature below ꢀ62 ^ꢁC.The reaction

mixture was stirred for 1.5 h under N₂atmosphere at

ꢀ65 to ꢀ73 ^ꢁC.Cooling was halted and the reaction

mixture allowed to warm to 0 ^ꢁC.Quenching was per-

formed by addition of water (200 mL), followed by 1 M

aqueous NaHSO₄.The organic layer was separated and

the aqueous layer extracted with ether (100 mL).The

combined organic layers were washed with saturated

aqueous NaHCO₃, dried (MgSO₄), ﬁltered and con-

centrated under reduced pressure.The crude product

was puriﬁed by column chromatography on silica gel

Materials and abbreviations

The resin used in the inverse SPPS was the photolabile

TentaGel-P-linker-2 35 (loading capacity: 0.167 mmol/g,

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

2559

(5% EtOAc/pentane) to yield 4 as a white solid (2.55 g,

63%), with a tendency to sublime, which aﬀected the

yield.An analytical sample was further puriﬁed by

sublimation. ¹H NMR (270 MHz, CDCl₃) d 5.08–4.90 (br

d, 1H, NH), 4.88–4.72 (m, 1H), 1.92–1.73 (m, 1H), 1.61–

1.36 (m, 3H), 1.43 (s, 9H), 0.95 (dd, J=7.2, 7.2 Hz, 3H).

¹³C NMR (100 MHz, CDCl₃) d 194.8 (dd, J=26, 26 Hz),

155.2, 118.0 (qdd, J=288, 34, 34 Hz), 107.4 (ddq, J=268,

268, 36 Hz), 80.8, 56.1, 32.9, 28.4, 18.9, 13.1. IR (neat)

3274, 1757, 1712 cm^ꢀ1.Anal.calcd for C ₁₂H₁₈F₅NO₃: C,

45.1; H, 5.7; N, 4.4. Found: C, 45.0; H, 6.1; N, 4.4.

mmol) was dissolved in a mixture of THF (27 mL) and

ꢁ

MeOH (7 mL).The solution was cooled to 0 C, sodium

borohydride (0.360 g, 9.52 mmol) was added and the

resulting reaction mixture was stirred for 1.5 h. The

mixture was diluted with ether (60 mL) and washed with

2% aqueous citric acid solution (2Â15 mL).The com-

bined aqueous layers were extracted with ether (3Â15

mL).The combined ether layers were successively

washed with saturated aqueous NaHCO₃(15 mL),

water (20 mL), brine (20 mL), dried (MgSO₄), ﬁltered,

and concentrated under reduced pressure to yield crude

7⁵⁰as a 1:4 mixture of diastereomers (A/B) (1.03 g,

93%).The crude product was further puriﬁed by col-

umn chromatography on silica gel (15% EtOAc/iso-

hexane).Separation of the two diastereomers (A, B) was

achieved.Total yield 06.99 g (63%) In this case, the

product also sublimed, which aﬀected the yield. ¹H

NMR (270 MHz, CDCl₃) d (A) 5.03 (br d, 1H), 4.81 (br

d, 1H), 4.21–3.81 (m, 1H), 3.69 (m, 1H), 1.89–1.24 (m,

4H), 1.44 (s, 9H), 0.94 (dd, J=7.2, 7.2 Hz, 3H). ¹³C

NMR (68 MHz, CDCl₃); (B) 4.74 (br d, 1H), 4.62 (br d,

1H), 4.17 (m, 1H), 3.92 (m, 1H), 1.70–1.21 (m, 4H), 1.45

(s, 9H), 0.95 (dd, J=7.2, 7.2 Hz, 3H). ¹³C NMR

(68 MHz, CDCl₃) d (A) 157.4, 119.2 (qdd, J=287, 36,

36 Hz), 114.0 (ddq, 263, 257, 36 Hz), 80.9, 70.7 (dd,

J=26, 20.5 Hz), 50.8, 33.4, 28.4, 19.5, 13.8; (B) 157.6,

119.2 (qdd, J=287, 35, 35 Hz), 114.5 (ddq, 263, 257,

35.5 Hz), 81.0, 71.7 (dd, J=26, 20 Hz), 53.5, 32.4,

28.4,19.9, 13.8. IR (neat) 3550-3150, 1684 cm^ꢀ1.Anal.

calcd for C₁₂H₂₀F₅NO₃: C, 44.9; H, 6.3; N, 4.4. Found

C, 44.9; H, 6.5; N, 4.3.

tert-Butyl 1-(2,2,3,3,3-pentaﬂuoropropanoyl)cyclopropyl-

carbamate (5). Compound 5 was prepared according to

the procedure described for 4 using the Weinreb amide⁷³

(1.60 g, 6.55 mmol) prepared from Boc-ACPC-OH 2,

pentaﬂuoroethyl iodide (16 g, 65 mmol) and 1.5 M

methyl lithium–lithium bromide complex in ether (37

mL, 56 mmol), with the exception that no column

chromatography was needed.Concentration under

reduced pressure was carried out carefully and not to

complete dryness, to minimize sublimation, to yield 5 as

white solid (2.24 g, 113%, including ether). An analy-

tical sample was further puriﬁed by sublimation. ¹H

NMR (270 MHz, CDCl₃) d 5.28 (br s, 1H, NH), 1.82–

1.74 (m, 2H), 1.64–1.40 (m, 2H), 1.44 (s, 9H). ¹³C NMR

(100 MHz, CDCl₃) d 192.6 (t, J=24.5 Hz), 155.5, 118.0

(qt, J=285, 34 Hz), 108.0 (tq, J=269, 38 Hz), 81.1,

40.6, 28.3, 22.8. IR (neat) 3354, 1734, 1690 cm^ꢀ1.Anal.

calcd for C₁₁H₁₄F₅NO₃: C, 43.6; H, 4.7; N, 4.6. Found

C, 43.8; H, 4.8; N, 4.6.

tert-Butyl (1S)-1-ethyl-3,3,4,4,4-pentaﬂuoro-2-oxobutyl-

carbamate (6). Compound 6 was prepared according to

the procedure described for 4 using the Weinreb amide⁵²

(2.00 g, 8.13 mmol) prepared from Boc-Abu-OH 3 (data

for the Weinreb amide, tert-butyl (1S)-1-{[methoxy

(methyl)amino]carbonyl}propylcarbamate: ¹H NMR

(400 MHz, CDCl₃) d 5.16 (br d, 1H, NH), 4.63 (m, 1H),

3.77 (s, 3H), 3.21 (s, 3H), 1.77 (m, 1H), 1.67–1.53 (m,

1H), 1.43 (s, 9H), 0.94 (dd, J=7.3, 7.3 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃) d 173.3, 155.7, 79.6, 61.7,

51.7, 32.2, 28.5, 26.2, 10.0. IR (neat) 3350, 1702, 1662

cm^ꢀ1.Anal.calcd for C ₁₁H₂₂N₂O₄: C, 53.6; H, 9.0; N,

11.4. Found: C, 53.9; H, 9.1; N, 11.4), pentaﬂuoroethyl

iodide (19.5 g, 79.3 mmol) and 1.5 M methyl lithium–

lithium bromide complex in ether (20.5 mL, 30.7

mmol).The crude product was puriﬁed by column

chromatography on silica gel (10% EtOAc/isohexane)

to yield 6 (1.63 g, 66%) as a white solid. In this case the

product also sublimed, which aﬀected the yield.An

analytical sample was further puriﬁed by sublimation.

¹H NMR (270 MHz, CDCl₃) d 5.10–4.90 (br d, 1H,

NH), 4.87–4.72 (m, 1H), 1.97–1.88 (m, 1H), 1.72–1.54 (m,

1H), 1.44 (s, 9H), 0.99 (dd, J=7.4, 7.4 Hz, 3H). ¹³C NMR

(68 MHz, CDCl₃) d 194.6 (dd, J=27, 27 Hz), 155.2, 118.0

(qdd, J=286, 33, 33 Hz), 107.4 (ddq, J=267, 267, 38 Hz),

80.9, 57.4, 28.4, 24.3, 9.7. IR (neat) 3364, 1756, 1706

cm^ꢀ1.Anal.calcd for C ₁₁H₁₆F₅NO₃: C, 43.3; H, 5.3; N,

4.6. Found C, 43.1; H, 5.4; N, 4.6.

tert-Butyl 1-(2,2,3,3,3-pentaﬂuoro-1-hydroxypropyl)cyclo-

8 was prepared

propylcarbamate (8). Compound

according to the procedure described for 7 using Boc-

ACPC-C₂F₅5 (1.00 g, 3.30 mmol). The crude product

was puriﬁed by column chromatography on silica gel

(5% EtOAc/pentane) to yield 8 (0.820 g, 81%) as a

white solid.In this case the product also sublimed,

1

which aﬀected the yield. H NMR (270 MHz, CDCl₃) d

6.08 (d, J=7.1 Hz, 1H), 5.09 (br s, 1H), 3.31 (m, 1H),

1.44 (s, 9H), 1.22–1.11 (m, 1H), 1.10–0.91 (m, 3H). ¹³C

NMR (100 MHz, CDCl₃) d 159.5, 119.1 (qdd, J=287,

35, 35 Hz), 114.1 (ddq, J=261, 259, 36 Hz), 81.9, 75.7

(dd, J=24, 20 Hz) 33.5, 28.3, 14.8, 14.1. IR (neat) 3500–

3300, 1661 cm^ꢀ1.Anal.calcd for C ₁₁H₁₆F₅NO₃: C, 43.3;

H, 5.3; N, 4.6. Found C, 43.3; H, 5.4; N, 4.6.

tert-Butyl (1S)-1-ethyl-3,3,4,4,4-pentaﬂuoro-2-hydroxy-

butylcarbamate (9). Compound 9 was prepared accord-

ing to the procedure described for 7 using Boc-Abu-

C₂F₅6 (0.670 g, 2.20 mmol). The crude product [1:4

mixture of diastereomers (A/B)] was puriﬁed by column

chromatography on silica gel (7% EtOAc/pentane) to

give 9 (0.62 g, 91%) as a white solid. Separation of the

two diastereomers (A, B) was achieved.In this case, the

1

product also sublimed. H NMR (400 MHz, CDCl₃) d

(A) 5.03 (br d, 1H), 4.21–3.77 (br m, 1H), 4.03 (m, 1H),

3.60 (m, 1H), 1.93–1.66 (m, 2H), 1.43 (s, 9H), 0.98 (dd,

J=7.3, 7.3 Hz, 3H); (B) 4.79 (br d, 1H), 4.79–4.39 (br

m, 1H) 4.17 (m, 1H), 3.88–3.70 (m, 1H), 1.79–1.54 (m,

2H), 1.44 (s, 9H) 1.00 (dd, J=7.3 7.3 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃) d (A) 157.4, 119.2 (qdd,

tert-Butyl (1S)-3,3,4,4,4-pentaﬂuoro-2-hydroxy-1-propyl-

butylcarbamate (7). Boc-Nva-C₂F₅4 (1.10 g, 3.44

2560

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

J=287, 36, 36 Hz), 114.5 (ddq, J=262, 256, 35 Hz),

80.9, 70.4 (dd, J=24, 21 Hz), 52.5, 28.4, 24.5, 10.7; (B)

157.6, 119.0 (qdd, J=288, 36, 36 Hz), 114.5 (ddq,

J=258, 258, 35 Hz), 81.0, 71.5 (dd, J=26, 20 Hz), 55.3,

28.4, 23.5, 11.2. IR (neat) 3500–3250, 1660 cm^ꢀ1.Anal.

calcd for C₁₁H₁₈F₅NO₃: C, 43.0; H, 5.9; N, 4.6. Found

C, 42.9; H, 6.0; N, 4.5.

mL). KCN (3.40 g, 52.2 mmol) was added and the

reaction mixture was stirred at room temperature for 1

h.The mixture was partitioned between water (100 mL)

and EtOAc (200 mL).The water phase was further

extracted with EtOAc (2Â200 mL).The combined

organic layers were washed with saturated aqueous

NaHCO₃(2Â300 mL) and brine, dried (Na₂SO₄), ﬁl-

tered, and concentrated under reduced pressure to yield

crude cyanohydrin, which was directly protected: The

crude cyanohydrin was dissolved in CH₂Cl₂(100 mL).

Acetic anhydride (2.46 mL, 26.1 mmol) and pyridine

(2.11 mL, 26.1 mmol) were added and the resulting

mixture was stirred at room temperature overnight.The

reaction mixture was diluted with CH₂Cl₂and washed

with aqueous 1 M HCl (2Â200 mL), H₂O (100 mL),

saturated aqueous NaHCO₃(2Â200 mL), and brine

(4S) - 4 - Amino - 1,1,1,2,2 - pentaﬂuoro - 3 - heptanol (10).

Compound 7 (0.420 g, 1.31 mmol, only the major dia-

stereomer) was dissolved in 3.6 M HCl/EtOAc (3.70

mL, 13.3 mmol) and stirred for 3 h. The solvent was

evaporated to give a solid.The residue was dissolved in

water (15 mL) for removal of the hydrochloride salt.pH

was adjusted to 12.5 by addition of 1M aqueous NaOH.

The aqueous layer was extracted with ether (4Â20 mL).

The combined organic layers were dried (MgSO₄), ﬁl-

tered, and concentrated under reduced pressure to give

the free amine 10 (0.200 g, 69%) as a white solid. In this

case, the product also sublimed, which aﬀected the

yield. ¹H NMR (270 MHz, DMSO-d₆) 3.90 (ddd,

J=23.0, 5.3, 5.3 Hz, 1H), 2.94–2.81(m, 1H), 1.62–1.39

(m, 2H), 1.38–1.16 (m, 2H), 0.91–0.79 (m, 3H). ¹³C

NMR (68 MHz, DMSO-d₆) 121.2 (qdd, J=287.3, 37.4,

35.4 Hz), 115.2 (ddq, J=261.2, 255.9, 34.6 Hz), 71.9

(dd, J=24, 20 Hz), 50.5, 33.8, 18.6, 14.0. IR (neat) 3378,

3330, 1613 cm^ꢀ1.Anal.calcd for C ₇H₁₂F₅NO: C, 38.0;

H, 5.5; N, 6.3. Found C, 38.1; H, 5.5; N, 6.2.

(1Â200 mL).The organic layer was dried (MgSO ), ﬁl-

4

tered, and concentrated under reduced pressure.The

crude product was puriﬁed by column chromatography

on silica gel (gradient system: 5% to 20% EtOAc/pen-

tane) to aﬀord 15 (3.28 g, 93%) as an oil in a 2:3 mix-

1

ture of diastereomers. H NMR (400 MHz, CDCl₃) d

5.43 (br d, NH) and 5.38 (d, J=4.6 Hz, NH)(1H), 4.72–

4.55 (m, 1H), 4.14–4.03 (m) and 4.02–3.90 (m)(1H), 2.14

(s) and 2.11 (s) (3H), 1.75–1.30 (m, 4H), 1.43 (s, 9H),

0.94 (dd, J=7.3, 7.3 Hz, 3H). ¹³C NMR (100 MHz,

CDCl₃) d 169.1,168.9, 155.5, 155.2, 115.6, 115.4, 80.7,

80.4, 64.7, 63.6, 51.3, 51.1, 32.5, 32.2, 20.5, 20.4, 19.1,

19.0, 13.8, 13.7. IR (neat) 3351, 1760, 1714, 1694 cm^ꢀ1

.

1-(1-Aminocyclopropyl)-2,2,3,3,3-pentaﬂuoro-1-propanol

(11). Compound 11 was prepared according to the pro-

cedure described for 10, using 8 (0.770 g, 2.52 mmol), to

give the product as an oil (0.490 g, 95%), which crys-

tallized upon storage.In this case the product also sub-

limed. ¹H NMR (400 MHz, CDCl₃) d 3.25 (dd, J=18.7,

6.7 Hz, 1H), 2.96 (br s, 2H), 0.97–0.81 (m, 2H), 0.71–

0.62 (m, 1H), 0.61–0.53 (m, 1H); ¹³C NMR (100 MHz,

CDCl₃) d 119.1 (qdd, J=286, 35, 35 Hz), 114.8 (ddq,

J=260, 258, 36 Hz), 73.4 (dd, J=24, 21 Hz) 34.2, 14.3,

13.0. IR (neat) 3386, 3324, 1611 cm^ꢀ1.Anal.calcd for

C₆H₈F₅NO: C, 35.1; H, 3.9; N, 6.8. Found C, 35.5; H,

4.0; N, 6.5.

Anal.calcd for C ₁₃H₂₂N₂O₄Â1/5H₂O: C, 57.0; H, 8.2;

N, 10.2. Found C, 57.1; H, 8.0; N, 10.0.

{1-[(tert-Butoxycarbonyl)amino]cyclopropyl}(cyano)methyl

acetate (16). Compound 16 was prepared according to

the procedure described for 15 using Boc-ACPC-H^54,55

14 (2.32 g, 12.5 mmol) and KCN (4.23 g, 64.9 mmol), to

1

yield 16 (1.59 g, 50%). H NMR (400 MHz, CDCl₃) d

5.47 (s, 1H), 5.10 (br s, 1H), 2.15 (s, 3H), 1.44 (s, 9H),

1.30–0.79 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) d

169.1, 155.2, 115.7, 80.6, 64.8, 34.0, 20.5, 14.0, 11.9. IR

(neat) 3345, 1752, 1686 cm^ꢀ1.Anal.calcd for

C₁₂H₁₈N₂O₄: C, 56.7; H, 7.1; N, 11.0. Found C, 56.8; H,

7.3; N, 10.7.

(4S) - 4 - Amino - 1,1,1,2,2 - pentaﬂuoro - 3 - hexanol (12).

Compound 12 was prepared according to the procedure

described for 10, using 9 (0.540 g, 1.76 mmol, 1:4 mixture

of diastereomers).Concentration under reduced pressure

was performed carefully and not to complete dryness, to

minimize sublimation, giving 12 as a white solid (0.370 g,

101%) in a 1:4 mixture of diastereomers (A:B). ¹H NMR

(400 MHz, CDCl₃) d (A+B) 3.97 (ddd, J=24.2, 4.4, 4.4

Hz, 0.8H), 3.78–3.67 (ddd, J=23.6, 5.1, 1.1 Hz, 0.2H),

3.24 (m, 0.2H), 2.94 (m, 0.8H), 3.23–2.26 (br m, 2H),

1.88–1.40 (m, 2H), 1.03–0.95 (m, 3H). ¹³C NMR

(100 MHz, CDCl₃) d (A+B) 119 (m), 115 (m), 69.9 (dd,

J=27, 21 Hz, B), 68.7 (dd, J=28, 21 Hz, A), 54.3 (B),

49.7 (A), 27.5 (A), 25.4 (B), 10.9(B), 10.4 (A). IR (neat)

3375, 3298, 1594 cm^ꢀ1.Anal.calcd for C ₆H₁₀F₅NO: C,

34.8; H, 4.9; N, 6.8. Found C, 35.0; H, 5.3; N, 6.4.

(2S)-2-[(tert-Butoxycarbonyl)amino]-1-(1H-tetrazol-5-yl)-

pentyl acetate (17). A mixture of 15 (2.68 g, 9.91 mmol),

triethylamine hydrochloride (2.73 g, 19.8 mmol) (pre-

pared from triethylamine and HCl/ether), and sodium

azide (1.29 g, 19.8 mmol) in toluene (75 mL) was heated

at reﬂux overnight.The reaction mixture was diluted

with toluene and extracted with water (3Â200 mL).The

combined water layers were acidiﬁed to pH 1 with con-

centrated HCl, and extracted with EtOAc (3Â400 mL).

The combined organic phases were washed with brine,

dried (MgSO₄), ﬁltered and concentrated under reduced

pressure to give pure 17 (2.86 g, 92%). The product is a

mixture of diastereomers and a pair of rotamers as

deduced by NMR. ¹H NMR (400 MHz, CDCl₃) d 6.33–

6.05 (m, 1H), 5.7 (br d), 5.46 (br d), 5.41 (bd) and 5.24

(m) (1H), 4.30–4.06 (m, 1H), 2.11 (s) and 2.10 (s) (3H),

1.65–1.20 (m, 4H), 1.36 (s) and 1.35 (s)(9H), 0.95–0.85

(m, 3H). ¹³C NMR (100 MHz, CDCl₃) d 170.4, 170.2,

169.9, 156.7, 156.6, 82.0, 81.1, 80.8, 69.2, 68.5, 68.1,

(2S)-2-[(tert-Butoxycarbonyl)amino]-1-cyanopentyl ace-

tate (15). Boc-Nva-H^52,5313 (2.63 g, 13.1 mmol) was

dissolved in a mixture of THF (70 mL) and water (80

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

2561

54.8, 53.0, 52.5, 33.5, 33.0, 28.4, 28.1, 20.9, 20.7, 19.3,

19.2, 13.9, 13.7. IR (KBr) 3364, 1760, 1694 cm^ꢀ1.Anal.

calcd for C₁₃H₂₃N₅O₄: C, 49.8; H, 7.4; N, 22.4. Found

C, 49.6; H, 7.4; N, 22.3.

¹³C NMR (68 MHz, CD₃OD) 2:3 mixture of diaster-

eomers d 158.8, 158.4, 66.2, 65.4, 56.3, 56.2, 32.6, 30.8,

19.8, 19.6, 14.2. IR (neat) 3600–2400, 1594, 1500, 1065

cm^ꢀ1.Anal.calcd for C ₆H₁₄ClN₅O: C, 34.7; H, 6.8; N,

33.7. Found C, 34.9; H, 6.9; N, 33.9.

{1-[(tert-Butoxycarbonyl)amino]cyclopropyl}(1H-tetrazol-

5-yl)methyl acetate (18). Compound 18 was prepared

according to the procedure described for 17, using 16

(12.5 mmol), to give 18 (1.98 g, 53% over three steps

from compound 14).NMR recorded at ambient tem-

perature showed traces of a rotamer.However, at 55 ^ꢁC

free rotation was achieved. ¹H NMR (400 MHz, CDCl₃,

55 ^ꢁC) d 5.90 (s, 1H), 5.61 (br s, 1H), 2.13 (s, 3H), 1.38

(s, 9H), 1.22–1.2 (m, 4H). ¹³C NMR (100 MHz, CDCl₃,

55 ^ꢁC) d 169.9, 157.2, 155.1, 81.7, 70.1, 34.6, 28.4, 20.8,

15.4, 13.2. IR (KBr) 3366, 1732, 1698 cm^ꢀ1.Anal.calcd

for C₁₂H₁₉N₅O₄: C, 48.5; H, 6.4; N 23.6. Found: C,

48.7; H, 6.6; N 23.3.

(1-Aminocyclopropyl)(1H-tetrazol-5-yl)methanol hydro-

chloride (22). Compound 22 was prepared according to

the procedure described for 21, using 20 (1.28 g, 5.03

1

mmol), to yield 22 in quantitative yield (0.970 g). H

NMR (400 MHz, CD₃OD) d 5.04 (s, 1H), 1.25–1.08 (m,

4H). ¹³C NMR (100 MHz, CD₃OD) d 158.2, 68.2, 39.0,

10.1, 9.0. IR (neat) 3600–2400, 1599, 1523, 1074 cm^ꢀ1

Anal.calcd for C H₁₀ClN₅O: C, 31.3; H, 5.3; N 36.6.

Found: C, 31.2; H, 5.4; N 36.3.

.

5

tert-Butyl 1-{[(2-cyanoethyl)amino] carbonyl}butylcarba-

mate (23). Boc-Nva-OH 1 (3.00 g, 13.8 mmol), 3-ami-

nopropionitrile

(1.02

mL,

13.8

mmol),

tert-Butyl (1S)-1-[hydroxy(1H-tetrazol-5-yl)methyl]butyl-

carbamate (19). Aqueous 1 M K₂CO₃(100 mL) was

added to a solution of 17 (2.61 g, 8.34 mmol) in MeOH

(100 mL), and the resulting mixture was stirred at room

temperature overnight.The reaction mixture was diluted

with water and acidiﬁed to pH 1 with concentrated HCl.

The water phase was extracted with EtOAc (3Â300 mL).

The combined organic layers were washed with brine,

dried (MgSO₄), ﬁltered and concentrated under reduced

pressure to give pure 19 (2.99 g, 99%) as a white solid. ¹H

NMR (400 MHz, CD₃OD) 2:3 mixture of diastereomers

d 5.12 (d, J=3.1 Hz) and 4.93 (d, J=6.3 Hz)(1H), 3.91–

3.78 (m, 1H), 1.70–1.20 (m, 4H), 1.35 (s) and 1.34 (s)

(9H), 1.00–0.87 (m, 3H). ¹³C NMR (100 MHz, CD₃OD)

2:3 mixture of diastereomers d 159.7,159.5, 158.1, 157.9,

80.3, 80.2, 69.5, 68.5, 55.9, 34.0, 33.0, 28.80, 28.76, 20.5,

20.3, 14.29, 14.25, including traces of rotamers. (KBr)

3330, 1671 cm^ꢀ1.Anal.calcd for C ₁₁H₂₁N₅O₃: C, 48.7;

H, 7.8; N, 25.8. Found C, 48.8; H, 8.0; N, 25.6.

dicyclohexylcarbodiimide (DCC) (2.85 g, 13.8 mmol),

N-hydroxybenzotriazole (HOBT) (1.86 g, 13.8 mmol)

were dissolved in DMF (50 mL) and stirred at 0 ^ꢁC for

48 h.The reaction mixture was ﬁltered and the solvent

evaporated.The residue was chromatographed on silica

gel (3:2 ethyl acetate/pentane) to yield 23 (3.10 g, 83%)

as a white solid.[ a]_D=ꢀ31.9^ꢁ(c 1.0, MeOH). ¹H NMR

(270 MHz, CDCl₃) d 6.74 (br t, 1H, NH), 4.91 (br d, 1H,

NH), 4.05 (m, 1H), 3.59–3.44 (m, 2H), 2.62 (dd, J=6.4,

6.4 Hz, 2H), 1.86–1.25 (m, 4H), 1.45 (s, 9H), 0.94 (dd,

J=7.3, 7.3 Hz, 3H). ¹³C NMR (68 MHz, CDCl₃) d

173.4, 156.0, 118.2, 80.4, 54.5, 35.7, 34.7, 28.5, 19.0, 18.4,

13.9. IR (KBr) 2246, 1685, 1662 cm^ꢀ1.Anal.calcd for

C₁₃H₂₃N₃O₃: C, 58.0; H, 8.6; N, 15.6. Found C, 58.1; H,

8.6; N, 15.4.

tert-Butyl 1-{[(2-cyanoethyl)amino] carbonyl}cyclopro-

pylcarbamate (24). Compound 24 was prepared accord-

ing to the procedure described for 23, using Boc-ACPC-

OH 2 (1.02 g, 5.07 mmol), 3-aminopropionitrile (0.373

mL, 5.07 mmol), DCC (1.10 g, 5.32 mmol), HOBT (0.86

g, 5.32 mmol) and DMF (20 mL). The residue was

chromatographed on silica gel (5% MeOH/dichloro-

tert-Butyl 1-[hydroxy(1H-tetrazol-5-yl)methyl]cyclopro-

pylcarbamate (20). Compound 20 was prepared accord-

ing to the procedure described for 19 using 18 (1.87 g,

6.29 mmol), to yield 20 (1.48 g, 92%) as a white solid.

NMR recorded at ambient temperature showed traces

of a rotamer.However, at 55 ^ꢁC free rotation was

achieved: ¹H NMR (400 MHz, CD₃OD, 55 ^ꢁC) d 4.89 (s,

1H), 1.35 (s, 9H), 1.21–1.05 (m, 2H), 0.93–0.75 (m, 2H).

¹³C NMR (100 MHz, CD₃OD, 55 ^ꢁC) d 159.5, 159.1,

80.9, 70.2, 37.5, 28.7, 12.7, 12.3. IR (KBr) 3284, 1665

cm^ꢀ1.Anal.calcd for C ₁₀H₁₇N₅O₃: C, 47.1; H, 6.7; N,

27.4. Found C, 47.4; H, 6.8; N, 27.5.

1

methane) to yield 24 (1.09 g, 85%) as a white solid. H

NMR (400 MHz, CDCl₃) d 7.10–6.90 (br, 1H , NH),

5.18 (br s, 1H, NH), 3.53 (dt, J=6.3, 6.3 Hz, 2H), 2.61

(t, J=6.3 Hz, 2H), 1.56 (m, 2H), 1.46 (s, 9H), 1.04 (m,

2H). ¹³C NMR (100 MHz, CDCl₃) d 173.1, 156.0, 118.4,

81.4, 36.2 (C, NCH₂), 28.5, 18.7, 18.2. IR (neat) 2249,

1694, 1660 cm^ꢀ1.Anal.calcd for C ₁₂H₁₉N₃O₃: C, 56.9;

H, 7.6; N, 16.7. Found C, 57.2; H, 7.7; N, 16.6.

tert-Butyl 1-(1H-tetrazol-5-yl)butylcarbamate (25). Die-

thylazodicarboxylate (DEAD) (2.6 mL, 16.6 mmol) was

added dropwise to a mixture of 23 (1.77 g, 6.59 mmol),

triphenylphosphine (4.33 g, 16.4 mmol), and tri-

methylsilyl azide (2.16 mL, 16.5 mmol) in THF (40 mL)

at 0 ^ꢁC under N₂atmosphere.The mixture was slowly

warmed to room temperature and stirred for 48 h.The

reaction mixture was diluted with water and extracted

with EtOAc (2Â100 mL).The combined organic layers

were washed with saturated aqueous NaHCO₃(2Â100

mL) and brine (100 mL), dried (MgSO₄), ﬁltered, and

concentrated under reduced pressure.The residue was

(2S)-2-Amino-1-(1H-tetrazol-5-yl)-1-pentanol hydrochlor-

ide (21). Compound 19 (1.89 g, 6.97 mmol) were dis-

solved in 3.6 M HCl/EtOAc (20.0 mL, 72.0 mmol) and

stirred for 3 h.The solvent was evaporated and the pre-

cipitated crude product was suspended in CH₂Cl₂, ﬁl-

tered, washed with more CH₂Cl₂, and dried in vacuo

(50 ^ꢁC) to yield pure 21 (1.36 g, 94%) as a white solid.

¹H NMR (270 MHz, CD₃OD) 2:3 mixture of diaster-

eomers d 5.45 (d, J=3.6 Hz) and 5.25 (d, J=5.0

Hz)(1H), 3.78–3.64 (m, 1H), 1.85–1.20 (m, 4H), 0.98

(dd, J=7.3, 7.3 Hz) and 0.91 (dd, J=7.2, 7.2 Hz) (3H).

2562

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

chromatographed on silica gel (35% EtOAc/pentane) to

give the cyanoethyl protected tetrazole (not completely

pure), which was subsequently deprotected: 1 M Aqu-

eous NaOH (9.60 mL, 9.60 mmol) was added to the tet-

razole derivative dissolved in THF (60 mL) and stirred at

room temperature for 5 h.The THF was evaporated and

the residue diluted with saturated aqueous NaHCO₃

(100 mL) and washed with ether (3Â100 mL).The water

phase was acidiﬁed with 1M aqueous HCl and extracted

with EtOAc (3Â300 mL).The combined organic layers

were washed with brine, dried (MgSO₄), ﬁltrered, and

concentrated under reduced pressure to yield pure 25

(0.883 g, 56%) as a white solid. [a]_D=ꢀ46.2^ꢁ(c 1.0,

for C₄H₈ClN₅: C, 29.7; H, 5.0; N, 43.3. Found C, 29.5;

H, 5.0; N, 43.0.

tert-Butyl (1S)-1-{[(phenylsulfonyl)amino]carbonyl}butyl-

carbamate (29). 1,1-carbonyldiimidazole (CDI) (5.78 g,

35.7 mmol) was added to a solution of Boc-Nva-OH 1

(2.50 g, 11.5 mmol) in dry THF (200 mL) and stirred for

30 min. A mixture of DBU (2.58 mL, 17.3 mmol) and

benzenesulfonamide (3.62 g, 23.0 mmol) in dry THF (20

mL) was added and the reaction mixture was stirred for

a further 6 h.The solvent was reduced to half the initial

volume by evaporation and thereafter was diluted in

CH₂Cl₂(300 mL) and washed with 5% aqueous citric

acid (2Â250 mL), water (250 mL) and brine (250 mL).

The organic layer was dried (MgSO₄), ﬁltered, and

concentrated under reduced pressure.The crude was

puriﬁed by column chromatography on silica gel (5%

MeOH/CHCl₃) to yield 29 (2.64 g, 64%) as a white

solid.[ a]_D=ꢀ24.1^ꢁ(c 1.0, MeOH). ¹H NMR (270 MHz,

CDCl₃) d 9.70 (br s, 1H), 8.20–7.85 (m, 2H), 7.67–7.59

(m, 1H), 7.58–7.47 (m, 2H), 5.08–4.85 (m, 1H), 4.07 (m,

1H), 1.83–1.62 (m, 1H), 1.59–1.17 (m, 3H), 1.43 (s, 9H),

0.86 (dd, J=7.3, 7.3 Hz, 3H). ¹³C NMR (68 MHz,

CDCl₃) d 170.4, 156.4, 138.7, 134.1, 129.1, 128.5, 81.3,

54.7, 33.1, 28.4, 18.8, 13.8. IR(neat) 3349, 1717, 1665

cm^ꢀ1.Anal.calcd for C ₁₆H₂₄N₂O₅SÂ2/3H₂O: C, 52.2;

H, 6.9; N, 7.6. Found: C, 52.4; H, 6.9; N, 7.2.

1

MeOH). H NMR (270 MHz, CD₃OD) d 4.95 (m, 1H,

overlap with H₂O signal), 2.01–1.78 (m, 2H), 1.52–1.20

(m, 2H), 1.44 (s, 9H), 0.97 (dd, J=7.3, 7.3 Hz, 3H).

¹³C NMR (100 MHz, relaxation delay=6 s, CD₃OD)

d 160.2, 157.8, 80.9, 47.2, 36.8, 28.8, 20.0, 13.9. IR

(KBr) 3330, 1672, 1520 cm^ꢀ1.Anal.calcd for

C₁₀H₁₉N₅O₂: C, 49.8; H, 7.9; N, 29.0. Found C, 49.8;

H, 7.8; N, 28.6.

tert - Butyl 1 - (1H - tetrazol - 5 - yl)cyclopropylcarbamate

(26). Compound 26 was prepared according to the pro-

cedure described for 25, using 24 (2.83 g, 11.2 mmol),

DEAD (4.39 mL, 27.9 mmol), triphenylphosphine (7.33

g, 27.9 mmol), trimethylsilyl azide (3.65 mL, 27.9 mmol)

and THF (60 mL), with following exceptions: the reac-

tion was stirred for 3 days, the cyanoethyl protected

tetrazole was chromatographed on silica gel (gradient

system: 35–75% EtOAc/pentane) and then treated with

1M aqueous NaOH (10.0 mL, 10.0 mmol) in THF (70

mL) and stirred at room temperature overnight, to yield

tert-Butyl 1-{[(phenylsulfonyl)amino]carbonyl}cyclopro-

pylcarbamate (30). Compound 30 was prepared accord-

ing to the procedure described for 29 using Boc-ACPC-

OH 2 (2.41 g, 12.0 mmol), CDI (6.42 g, 39.6 mmol),

DBU (4.00 mL, 26.8 mmol) and benzenesulfonamide

(4.99 g, 31.8 mmol). The reaction was heated to reﬂux

for 24 h and then an additional 76 h.Puriﬁcation by

column chromatography on silica gel (5% MeOH/

CHCl₃) gave 30 (3.68 g, 90%) as a white solid. ¹H NMR

(270 MHz, CDCl₃) d 9.69 (br s, 1H), 8.12–7.98 (m, 2H),

7.69–7.58 (m, 1H), 7.57–7.47 (m, 2H), 5.81–5.16 (br m,

1H), 1.57–1.46 (m, 2H), 1.42 (s, 9H), 1.12–1.02 (m, 2H).

¹³C NMR (68 MHz, CDCl₃) d 170.7, 156.1, 138.5,

133.8, 128.9, 128.3, 81.8, 36.5, 28.1, 19.1. IR (neat)

3331, 3262, 1710, 1686 cm^ꢀ1.Anal.calcd for

C₁₅H₂₀N₂O₅S: C, 52.9; H, 5.9; N, 8.2. Found: C, 52.6;

H, 6.1; N, 7.9.

1

26 as a slightly yellow solid (0.365 g, 15%). H NMR

(400MHz, CD₃OD) d 1.57–1.51 (m, 2H), 1.46 (s, 9H),

1.44–1.38 (m, 2H). ¹³C NMR (100 MHz, relaxation

delay=4 s, CD₃OD) d 162.0, 158.4, 81.4, 28.8, 20.0. IR

(neat) 3248, 1681, 1537 cm^ꢀ1.Anal.calcd for

C₉H₁₅N₅O₂Â1/10H₂O: C, 47.6; H, 6.8; N, 30.8. Found

C, 47.6; H, 6.6; N, 30.6.

1-(1H-Tetrazol-5-yl)-1-butanamine hydrochloride (27).

Compound 25 (0.766 g, 3.17 mmol) was dissolved in

2.7 M HCl/EtOAc (12.0 mL, 32.4 mmol) and stirred for

3 h.Precipitated product was isolated by ﬁltration,

washed with EtOAc, and dried in vacuo to aﬀord pure

25 (0.479 g, 85%) as a white solid. [a]_D=ꢀ7.0^ꢁ(c 1.0,

MeOH). ¹H NMR (400 MHz, CD₃OD) d 4.81 (dd,

J=7.1, 7.1 Hz, 1H), 2.18–2.05 (m, 2H ), 1.49–1.28 (m,

2H), 0.99 (dd, J=7.3, 7.3 Hz, 3H). ¹³C NMR

(100 MHz, relaxation delay=6 s, CD₃OD) d 159.5, 47.8,

35.9, 19.4, 13.9. IR (KBr) 1548, 1073 cm^ꢀ1.Anal.calcd

for C₅H₁₂ClN₅: C, 33.8; H, 6.8; N, 39.4. Found C, 33.7;

H, 6.8; N, 39.3.

N - [( 2S) - 2 - Aminopentanoyl]benzenesulfonamide hydro-

chloride (31). Compound 29 (2.48 g, 6.96 mmol) was

dissolved in 3.6 M HCl/EtOAc (19.3 mL, 69.6 mmol)

and stirred at room temperature for 3 h.The reaction

was concentrated under reduced pressure to yield pure

31 (1.93 g, 95%) as a beige solid. [a]_D=+58.5^ꢁ(c 1.0,

1

MeOH). H NMR (270 MHz, CD₃OD) d 8.10–7.95 (m,

2H), 7.77–7.66 (m, 1H), 7.65–7.53 (m, 2H), 3.89 (dd,

J=5.6, 6.8 Hz, 1H), 1.86–1.66 (m, 2H), 1.43–1.11 (m,

2H), 0.81 (dd, J=7.3, 7.3 Hz, 3H). ¹³C NMR (68 MHz,

CD₃OD) d 169.6, 140.5, 135.3, 130.3, 129.4, 54.9, 34.1,

18.8, 14.0. IR (neat) 3500–2400, 1724 cm^ꢀ1.Anal.calcd

for C₁₁H₁₇ClN₂O₃S: C, 45.1; H, 5.9; N, 9.6. Found: C,

45.4; H, 6.0; N, 9.3.

1 - ( 1H - tetrazol - 5 - yl)cyclopropanamine hydrochloride

(28). Compound 28 was prepared according to the pro-

cedure described for 27, using 26 (0.336 g, 1.49 mmol)

with the following exceptions: the crystals were washed

with CH₂Cl₂and dried in vacuo (60 ^ꢁC) to aﬀord 28 in

quantitative yield. ¹H NMR (400 MHz, CD₃OD) d

1.75–1.57 (m, 4H). ¹³C NMR (100 MHz, CD₃OD) d

162.4, 31.2, 15.0. IR (neat) 1578, 1042 cm^ꢀ1.Anal.calcd

N - [(1 - Aminocyclopropyl)carbonyl]benzenesulfonamide

hydrochloride (32). Compound 32 was prepared accord-

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

2563

ing to the procedure described for 31, using 30 (3.68 g,

10.8 mmol), to yield the 32 (2.69 g, 90%) as a beige

solid. ¹H NMR (270 MHz, CD₃OD) d 8.10–8.01 (m,

2H), 7.77–7.67 (m, 1H), 7.66–7.55 (m, 2H), 1.73–1.38

(m, 4H). ¹³C NMR (CD₃OD, 68 MHz) d 170.3, 140.4,

135.3, 130.2, 129.6, 37.4, 14.0. IR (neat) 3500–2400,

1686.Anal.calcd for C ₁₀H₁₃ClN₂O₃S: C, H, N.C,

43.40; H, 4.73; N, 10.12. Found: C, 43.42; H, 4.84; N,

9.98.

OSilÂHCl, where AA=Asp(OFm), d-Glu(OFm), Leu,

Ile or Cha)⁴⁵(5 equiv) in CH₂Cl₂(2 mL), a solution of

HATU (4 equiv) in DMF (2 mL) and TMP (9 equiv)

were added, in the written order.The columns were

placed in an overhead mixer or magnetically stirred in

the Quest 210 apparatus for 2 h.The resin was washed

with DMF (8Â5 mL) and CH₂Cl₂(7Â5 mL).Removal

of the Sil group was accomplished by treatment with

5% TFA/CH₂Cl₂(6 mL) for 20 min.Finally, the resin

was washed with CH₂Cl₂(7Â5 mL) and MeOH (7Â5

mL) and dried.The procedure was repeated to yield 37a

and 37b, respectively.The resins were dried in vacuo

and analyzed by amino acid analysis, ¹³C NMR

(DMSO-d₆, not shown), and LC–MS (after cleavage of

a analytical sample). 37a: MS (calcd 804.4) 805.5

(M+H⁺).Amino acids analysis (on-resin) Glu, 11.1;

Leu, 1.03; Ile, 0.98; Cha, 0.88 (corrupted). 37b: MS

(calc. 1097.6) 1098.6 (M+H⁺).Amino acids analysis

(on-resin) Asp, 1.08, Glu, 1.05; Leu, 0.98; Ile, 0.93; Cha,

0.95.

N-[(2S)-2-Aminopentanoyl]benzenesulfonamide (33). The

hydrochloride 31 (1.88 g, 6.43 mmol) was dissolved in

EtOH (50 mL). Propene oxide (4.95 mL, 70.7 mmol)

was added.The resulting mixture was stirred and heated

at 50 ^ꢁC for 3 h.The precipitated product was collected

by ﬁltration and washed with diethyl ether and dried in

vacuo (50 ^ꢁC) to yield the zwitterion 33 (1.10 g, 67%) as

a white solid.[ a]_D=+14.0^ꢁ(c 1.0, DMSO). H NMR

1

(270 MHz, DMSO-d₆) d 7.82–7.73 (m, 2H), 7.75–7.51

(br s, 1H); 7.44–7.33 (m, 3H), 3.55–3.20 (m, 1H, overlap

with H₂O signal), 1.74–1.46 (m, 2H), 1.33–1.08 (m, 2H),

0.81 (dd, J=7.3, 7.3 Hz, 3H). ¹³C NMR (68 MHz,

DMSO-d₆) 172.4, 145.4, 130.2, 127.7, 126.9, 54.7, 31.8,

17.7, 13.8. IR(neat) 3300–2450, 1620 cm^ꢀ1.Anal.calcd

for C₁₁H₁₆N₂O₃S: C, 51.5; H, 6.3; N, 10.9. Found: C,

51.7; H, 6.3; N, 11.0.

General procedure for the preparation of the inhibitors

Suc-^D-Glu-Leu-Ile-Cha-P1 (38–46a) and Suc-Asp-D-Glu-

Leu-Ile-Cha-P1 (38–46b). Aliquots of the resins 37a or

37b (42 mmol) were swollen in DMF (and CH₂Cl₂) (1.7

mL) for 20–30 min in columns equipped with poly-

propylene frits or in Quest 210 columns.The P1 build-

ing block (10–12, 21, 22, 27, 28, 33 and 34) (5 equiv)

dissolved in DMF or CH₂Cl₂(0.5 mL), a solution of

HATU (4 equiv) in DMF (2 mL), and the base (DIEA

or TMP, 4–14 equiv) were added to the resin in the

written order.The ﬁnal solvent composition was 4:1

DMF/CH₂Cl₂(3.5 mL). The columns were placed in an

overhead mixer or magnetically stirred in the Quest 210

apparatus for 2–5 h and then washed with DMF (8Â5

mL).Removal of the ﬂuorenylmethyl ester (Fm group)

was accomplished by stirring the resin for 20 min in

25% piperidine/DMF.Successive washings were done

with DMF (8Â5 mL), CH₂Cl₂(7Â5 mL), 5% TFA/

CH₂Cl₂(3Â5 mL), CH₂Cl₂(7Â5 mL), and ﬁnally

MeOH (7Â5 mL).The resins were dried in vacuo.

Cleavage of the peptides from the resins was performed

with UV irradiation (350 nm).Thus, aliquots of resin

(50 mg) in MeOH (1.5 mL) were irradiated for 2 h

under N₂-atmosphere in quartz tubes equipped with

septa.The solid support was removed by ﬁltration and

the solvent evaporated to yield the crude residues as oils

or solids, which were puriﬁed by preparative RP-HPLC

(according to method A, B, or C).The fractions corre-

sponding to the major peak were combined, lyophilized

and subsequently redissolved in a small volume of H₂O/

MeCN (occasionally supplemented with aqueous

NH₄OAc to assist solubilization) for analysis by MS,

analytical RP-HPLC and amino acid analysis.The

concentration of the solutions was calculated from

amino acid analysis data and aliquots containing 0.5 or

1 mmol were transferred to 2 mL vials and lyophilized.

The purity of the target compounds was >95%

according to the HPLC analyses (UV, 220 nm), unless

otherwise stated.The average total yield based on the

loading of TentaGel-P-linker-2 35 (i.e., over 6–8 steps)

was 10–25%.It should be noted that the photolysis

releases only ca.75% of the products, as determined

N - [(1 - Aminocyclopropyl)carbonyl]benzenesulfonamide

(34). Compound 34 was prepared according to the pro-

cedure described for 33, using the hydrochloride 32

(2.41 g, 8.71 mmol), to yield 34 (1.87 g, 89%) as a beige

solid. ¹H NMR (270 MHz, DMSO-d₆) d 8.09 (br s, 1H),

7.86–7.75 (m, 2H), 7.47–7.33 (m, 3H), 1.27–1.02 (m,

2H) 1.01–0.80 (m, 2H). ¹³C NMR (68 MHz, DMSO-d₆)

d 172.5, 145.5, 130.2, 127.8, 127.1, 36.5, 12.2. IR

(neat) 3300–2800, 1627 cm^ꢀ1.Anal.calcd for

C₁₀H₁₂N₂O₃S: C, 50.0; H, 5.0; N, 11.7 Found: C, 50.3;

H, 5.3; N, 11.6.

TentaGel-P-linker-Suc-OH (36). TentaGel-P-linker-2 35

(2.50 g, 0.418 mmol) was swollen in CH₂Cl₂(25 mL) for

10–20 min in a column equipped with a polypropylene

frit. Succinic anhydride (0.418 g, 4.18 mmol) and DIEA

(0.714 mL, 4.18 mmol) were added. The column was

placed in an overhead mixer for 18 h.The resin was

washed with CH₂Cl₂(5Â20 mL), treated with 5% TFA/

CH₂Cl₂(2Â10 mL) and successively washed with

CH₂Cl₂(5Â20 mL) and MeOH (5Â20 mL).The resin

36 was dried in vacuo and analyzed by ¹³C NMR. ¹³C

NMR (270 MHz, CDCl₃) d 21.8 (CH₃), 28.3 (CH₂), 29.9

(CH₂), 38.7 (CH₂NH), 40.9 (resin), 56.3 (OCH₃), 67.0–

75.5 (CH and PEG), 108.5, 111.2 (CH Ar), 130.0

(broad signal, resin), 135.0, 139.2, 145.5, 154.2 (ipso

Ar), 167.3 (NHC¼O), 171.0 (OC¼O), 174.0

(HOC¼O).

Preparation of TentaGel-P-linker-Suc-^D-Glu(OFm)-Leu-

Ile-Cha-OH (37a) and TentaGel-P-linker-Suc-Asp(OFm)-

D-Glu(OFm)-Leu-Ile-Cha-OH (37b). Aliquots of the

resin 36 (75 mmol) were swollen in DMF (2.3 mL) for

20–30 min in columns equipped with polypropylene frits

or in Quest 210 columns.A freshly prepared solution of

the amino acid Sil ester hydrochloride (H-AA-

2564

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

from amino acid analysis of the resin after cleavage.

Some of the target peptides were oxidized before or

after cleavage as described under the speciﬁc heading

below.

lyophilised.The resulting alcohol was dissolved in DMF/

CH₂Cl₂(4:1, 2.5 mL) and cooled to 0^ꢁC.Dess–Martin

periodinane (approximately 5 equiv) was added and the

resulting reaction mixture was stirred for 7 h.The reaction

was quenched with 10% aqueous Na₂S₂O₃and the sol-

vent evaporated.The product was puriﬁed according to

method B to yield 41b with a ﬁnal purity of 91%.MS

(calcd 892.5) 893.4 (M+H⁺).Amino acids analysis Asp,

1.10; Glu, 1.05; Leu, 1.05; Ile, 0.95; Cha, 0.85.

Suc-^D-Glu-Leu-Ile-Cha-Nva-C₂F₅(38a). The compound

was prepared according to the general method using 10

and TMP (4 equiv), with the exception that oxidation

was performed prior to cleavage from the resin.Thus,

the peptide resin comprising the alcohol 10 was swollen

in CH₂Cl₂(3.5 mL) for 20 min. Dess–Martin period-

inane (5 equiv) was added and the resulting mixture

Suc-^D-Glu-Leu-Ile-Cha-ACPC-CN₄H (42a). The com-

pound was prepared according to the general method

using 22 and DIEA (14 equiv), and oxidized and pur-

iﬁed as described for compound 38a.The desired pro-

duct was not the major component in the crude mixture

after cleavage (before puriﬁcation) as indicated by in the

HPLC chromatogram. MS (calcd 761.4) 762.5 (M +

H⁺). Amino acids analysis Glu, 1.09; Leu, 0.98; Ile,

1.00; Cha, 0.94.

rotated for 18 h.The resin was washed with CH Cl₂

2

(7Â5 mL) and MeOH (7Â5 mL).Final puriﬁcation of

38a was done according to method B.MS (calcd 8274. )

828.2 (M+H⁺), 946.2 (M+H⁺+H₂O).Amino acids

analysis Glu, 0.99; Leu, 1.01; Ile, 0.99; Cha, 1.00.

Suc-Asp-^D-Glu-Leu-Ile-Cha-Nva-C₂F₅(38b). The com-

pound was prepared according to the general method

using 10 and TMP (4 equiv), and oxidized and puriﬁed

as described for compound 38a. MS (calcd 942.4) 943.4

(M+H⁺) 961.4 (M+H⁺+H₂O).Amino acids analysis

Asp, 1.01; Glu, 1.03; Leu, 1.00; Ile, 0.97; Cha, 1.00.

Suc-Asp-^D-Glu-Leu-Ile-Cha-ACPC-CN₄H (42b). The

compound was prepared according to the general

method using 22 and DIEA (14 equiv), and oxidized as

described for compound 38a.Final puriﬁcation of 42b

was carried out according to method C.The desired

product was not the major component in the crude

mixture after cleavage (before puriﬁcation) as indicated

by in the HPLC chromatogram. MS (calcd 876.4) 877.1

(M + H⁺). Amino acids analysis Asp, 1.04; Glu, 1.03;

Leu, 1.00; Ile, 0.96; Cha, 0.96.

Suc-^D-Glu-Leu-Ile-Cha-ACPC-C₂F₅(39a). The com-

pound was prepared according to the general method

using 11 and TMP (4 equiv), and oxidized and puriﬁed

as described for compound 38a. MS (calcd 811.4) 812.2

(M+H⁺) 930.2 (M+H⁺+H₂O).Amino acids analysis

Glu, 1.02; Leu, 0.99; Ile, 1.00; Cha, 0.99.

Suc-^D-Glu-Leu-Ile-Cha-1-(1H-tetrazol-5-yl)-1-butanamide

(43a). The compound was prepared according to the

general method using 27 and DIEA (14 equiv).Final

puriﬁcation of 43a was done according to method B.

MS (calcd 749.4) 750.6 (M+H⁺).Amino acids analysis

Glu, 1.02; Leu, 1.00; Ile, 0.97; Cha, 1.02.

Suc-Asp-^D-Glu-Leu-Ile-Cha-ACPC-C₂F₅(39b). The

compound was prepared according to the general

method using 11 and TMP (4 equiv), and oxidized and

puriﬁed as described for compound 38a.MS (calcd

926.4) 927.4 (M+H⁺) 945.4 (M+H⁺+H₂O).Amino

acids analysis Asp, 1.02; Glu, 1.02; Leu, 1.00; Ile, 0.97;

Cha, 0.99.

Suc-Asp-^D-Glu-Leu-Ile-Cha-1-(1H-tetrazol-5-yl)-1-butan-

amide (43b). The compound was prepared according to

the general method using 27 and DIEA (14 equiv).Final

puriﬁcation of 43b was performed according to method

B. MS (calcd 864.5) 865.7 (M+H⁺).Amino acids ana-

lysis Asp, 1.00; Glu, 1.05; Leu, 1.02; Ile, 0.97; Cha, 0.96.

Suc-^D-Glu-Leu-Ile-Cha-Abu-C₂F₅(40a). The compound

was prepared according to the general method using 12

and TMP (4 equiv), and oxidized as described for com-

pound 38a.Final puriﬁcation of 40a was done using

method C. MS (calcd 813.4) 814.2 (M+H⁺) 832. 2

(M+H⁺+H₂O).Amino acids analysis Glu, 09. 1; Leu,

0.93; Ile, 1.07; Cha, 1.08.

Suc-^D-Glu-Leu-Ile-Cha-1-(1H-tetrazol-5-yl)cyclopropan-

amide (44a). The compound was prepared according to

the general method using 28 and DIEA (14 equiv).Final

puriﬁcation of 44b was performed according to method

B to yield 44a as a (3:5) mixture of d-/l-Cha diaster-

eomers. MS (calcd 733.4) 734.2 (M + H⁺).Amino

acids analysis Glu, 1.01; Leu, 1.04; Ile, 0.99; Cha, 0.95.

Suc-Asp-^D-Glu-Leu-Ile-Cha-Abu-C₂F₅(40b). The com-

pound was prepared according to the general method

using 12 and TMP (4 equiv), and oxidized and puriﬁed

as described for compound 38a. MS (calcd 928.4) 929.4

(M+H⁺) 947.4 (M+H⁺+H₂O).Amino acids analysis

Asp, 1.00; Glu, 1.02; Leu, 1.01; Ile, 0.97; Cha, 1.01.

Suc-Asp-^D-Glu-Leu-Ile-Cha-1-(1H-tetrazol-5-yl)cyclopro-

panamide (44b). The compound was prepared according

to the general method using 28 and DIEA (14 equiv).

Final puriﬁcation of 44b was carried out according to

method B. MS (calcd 848.4) 849.6 (M + H⁺).Amino

acids analysis Asp, 1.00; Glu, 1.01; Leu, 1.02; Ile, 0.97;

Cha, 1.02.

Suc-Asp-^D-Glu-Leu-Ile-Cha-Nva-CN₄H (41b). The com-

pound was prepared according to the general method

using 21 and DIEA (14 equiv), with the exception that

oxidation was performed in solution since the on-resin

procedure produced a complex mixture of compounds.

Thus, the peptidic alcohol was cleaved from the resin

(mixture of compounds, see the chemistry section) and

puriﬁed by preparative HPLC according to method B and

Suc-^D-Glu-Leu-Ile-Cha-Nva-NHSO₂Ph (45a). The com-

pound was prepared according to the general method

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

2565

using 33 and TMP (9 equiv).Final puriﬁcation of 45b

was carried out according to method B.MS (calcd

864.4) 865.6 (M+H⁺).Amino acids analysis Glu, 09. 9;

Leu, 1.02; Ile, 0.96; Cha, 1.02, Nva, 1.02.

aqueous Na₂CO₃(35 mL) and 1,4-dioxane (17 mL) and

cooled to 0 ^ꢁC. Fmoc-Cl (2.00 g, 7.74 mmol) dissolved

in 1,4-dioxane (17 mL) was added dropwise to the stir-

red mixture.Stirring was continued at room tempera-

ture for 45 h and the pH was kept at around 10–11.1 M

aqueous HCl was added for adjustment to pH 8.The

reaction mixture was washed with diethyl ether (4Â90

mL).Ether (100 mL) was added to the aqueous layer,

which was subsequently acidiﬁed to pH 2 with 1M

aqueous HCl under vigorous stirring.The two phases

were separated and the aqueous layer was extracted with

additional ether (2Â100 mL).The combined organic

layers were washed with brine (100 mL), dried (MgSO₄)

and concentrated under reduced pressure to give the

Suc-Asp-^D-Glu-Leu-Ile-Cha-Nva-NHSO₂Ph (45b). The

compound was prepared according to the general

method using 33 and TMP (9 equiv).Final puriﬁcation

of 45b was performed according to method B.MS

(calcd 979.5) 980.4 (M + H⁺).Amino acids analysis

Asp, 1.01; Glu, 1.00; Leu, 1.03; Ile, 0.98; Cha, 0.99,

Nva, 0.99.

Suc-^D-Glu-Leu-Ile-Cha-ACPC-NHSO₂Ph (46a). The

compound was prepared according to the general

method described above using 34 and TMP (9 equiv).

Final puriﬁcation of 46a was performed according

to method C. MS (calcd 848.4) 849.7 (M+H⁺).

Amino acids analysis Glu, 0.99; Leu, 0.98; Ile, 1.04;

Cha, 0.99.

1

product 48 (0.222 g, 33%) as a white solid. H NMR

(270 MHz, DMSO-d₆) d 7.88 (dd, J=1.3, 7.4 Hz, 2H),

7.77 (s, 1H), 7.70 (broad d, J=7.4), 7.41 (ddd, J=1.3,

7.4, 7.4 Hz, 2H), 7.31 (ddd, J=1.2, 7.4, 7.4 Hz, 2H),

4.32–4.11 (m, 3H), 3.96 (s, 1H), 1.01–0.75 (m, 2H), 0.70–

0.50 (m, 2H). ¹³C NMR (68 MHz, DMSO-d₆) d 174.0,

156.4, 143.8, 140.7, 127.6, 127.1, 125.4, 120.1, 71.4, 65.5,

46.6, 35.2, 10.9, 10.1. IR (neat) 3446, 3269, 3500–2400,

1721, 1663 cm^ꢀ1.Anal.calcd for C ₂₀H₁₉NO₅: C, 68.0; H,

5.4; N, 4.0. Found: C, 68.1; H, 5.7; N, 3.8.

Suc-Asp-^D-Glu-Leu-Ile-Cha-ACPC-NHSO₂Ph

(46b).

The compound was prepared according to the general

method described above using 34 and TMP (9 equiv).

Final puriﬁcation of 46b was performed according to

method C. MS (calcd 963.4) 964.3 (M+H⁺).Amino

acids analysis Asp, 1.02; Glu, 1.00; Leu, 0.96; Ile, 1.01;

Cha, 1.00.

General procedure for the SPPS of the inhibitors Suc-^D-

Glu-Leu-Ile-Cha-P1-OH (50–54a) Suc-Asp-D-Glu-Leu-

Ile-Cha-P1-OH (49b, 50–54b). The peptide inhibitors

49b and 50–54 were synthesized by standard Fmoc/t-Bu

solid-phase peptide synthesis techniques using a parallel

approach.H-Cys(Trt)-2-chlorotrityl resin and Fmoc-

Phe-Wang resin were commercial products.The other

starting resins; H-Nva-2-chlorotrityl resin, H-ACPC-2-

chlorotrityl resin, H-allylglycine-2-chlorotrityl resin,

and (1-aminocyclopropyl)(hydroxy)acetic acid-2-chloro-

trityl resin (from 48) were synthesized by loading of the

corresponding Fmoc-protected amino acids using a

slightly modiﬁed literature procedure.⁶⁸In short, 2-

chloro-tritylchloride resin (1.47 mmol/g, 500 mol),

Fmoc-AA-OH (300 mol) and DIEA (1.2 mmol) were

agitated for 60 min inaCH₂Cl₂(5 mL).End-capping was

performed by washing with CH₂Cl₂/MeOH/DIEA

(17:2:1) followed by CH₂Cl₂, DMF, and CH₂Cl₂.Pep-

tide assembly was performed on a 33 mol scale using

Fmoc-AA-OH (0.13 mmol), TBTU (0.13 mmol) and

DIEA (0.26 mmol) in DMF (0.5 mL) with a coupling

time of 60 min.The Fmoc group was removed by 25%

piperidine in DMF for 1+10 min.Terminal acylation

was accomplished by succinic anhydride (0.33 mmol)

and DIEA (0.33 mmol) in DMF (0.5 mL) for 60 min.

The peptides were cleaved from the resin in 0.5 mL

TFA/H₂O/triethylsilane (18:1:1) for 60 min, and the

resin was subsequently ﬁltered oﬀ.The TFA was par-

tially evaporated followed by precipitation of the crude

peptide by addition of diethyl ether.The products were

collected by centrifugation and washed several times

with fresh diethyl ether, air-dried and puriﬁed by pre-

parative RP-HPLC (method A, unless otherwise stated).

The target compounds were lyophilized and subse-

quently redissolved in a small volume of H₂O/MeCN

(occasionally supplemented with aqueous NH₄OAc to

assist solubilization) for analysis by MS, analytical RP-

HPLC and amino acid analysis.The concentration of

(1-Aminocyclopropyl)(hydroxy)acetic acid (47). The

Boc-ACPC-H 14 (1.06 g, 5.73 mmol) was dissolved in a

mixture of THF (60 mL) and water (80 mL).KCN

(1.95 g, 29.9 mmol) was added and the reaction mixture

was stirred at room temperature for 3 h.The mixture

was extracted with EtOAc (2Â150 mL).The combined

organic layers were washed with saturated aqueous

NaHCO₃(2Â150 mL) and brine (150 mL), dried

(Na₂SO₄), ﬁltered, and concentrated under reduced

pressure to yield crude cyanohydrin as an oil, which was

directly hydrolyzed without further puriﬁcation.The

crude cyanohydrin was dissolved in a mixture of 1,4-

dioxane (60 mL) and concentrated HCl (60 mL) and

thereafter gently heated to reﬂux for 20 h.The reaction

mixture was concentrated under reduced pressure to

aﬀord the crude hydrochloride salt of the a-hydroxy

carboxylic acid as a brown paste.Propene oxide (60.

mL, 85.6 mmol) was added to the hydrochloride salt

dissolved in EtOH (70 mL).The reaction mixture was

stirred and heated at 50 ^ꢁC for 3 h, during which time

the zwitterionic product precipitated.The reaction mix-

ture was cooled to room temperature and ﬁltered.The

isolated product was washed with cold diethyl ether and

dried in vacuo (38 ^ꢁC) to yield the zwitterion 47 (0.310 g,

41% ) as a white solid. ¹H NMR (270 MHz, DMSO-d₆)

d 3.60 (s, 1H), 0.83–0.75 (m, 1H), 0.73–0.56 (m, 3H). ¹³C

NMR (68 MHz, DMSO-d₆) d 174.2, 68.1, 36.0, 6.8, 5.5.

IR (neat) 3379, 3500–2250, 1636, 1581, 1542 cm^ꢀ1

.

Anal.calcd for C ₅H₉NO₃: C, 45.8; H, 6.9; N, 10.7

Found: C, 46.0; H, 7.1; N, 10.3.

(1 - {[(9H - Fluoren - 9 - ylmethoxy)carbonyl]amino}cyclo-

propyl)(hydroxy)-acetic acid (48). The zwitterion 47

(0.246 g, 1.88 mmol) was dissolved in a mixture of 10%

2566

A. Johansson et al. / Bioorg. Med. Chem. 11 (2003) 2551–2568

the solutions was calculated from amino acid analysis

data and aliquots containing 1 mmol were transferred to

2-mL vials and lyophilized.The purity of the target

compounds was >95% according to the HPLC ana-

lyses (UV, 220 nm).The average total yields were 35–

45%.Analysis data are described under the speciﬁc

heading below.

Enzyme inhibition

The inhibition of the protease activity of the full-length

HCV NS3 (protease-helicase/NTPase) was measured by

a continuous ﬂuorescent assay, as described pre-

viously.^39,40,69Brieﬂy, 1 nM enzyme was incubated in 50

mM HEPES pH 7.5, 10 mM DTT, 40% glycerol, 0.1%

n-octyl-b-d-glucoside, 3.3% DMSO with 25 mM cofac-

tor (KKGSVVIVGRIVLSGK, peptide 2K-NS4A) and

inhibitor at 30 ^ꢁC for 10 min, the reaction was initiated

by addition of 0.5 mM substrate (Ac-DED(Edans)EEA-

bu [COO]ASK(Dabcyl)–NH₂).

Suc-Asp-^D-Glu-Leu-Ile-Cha-ACPC-COOH (49b). The

compound was prepared according to the general SPPS

method up to the HPLC puriﬁcation, starting with 48 to

yield the corresponding peptidic alcohol, which was

oxidized in solution to yield 49b.Thus, the crude pep-

tide alcohol (38 mg, 44 mmol) was dissolved in DMF (2

mL) and cooled to 0 ^ꢁC.Dess–Martin periodinane (171

mg, 404 mmol) was added and the resulting reaction

mixture was stirred for 4 h.The reaction was quenched

with 10% aqueous Na₂S₂O₃and the solvent evaporated.

The product was puriﬁed by RP-HLPC according to

method C to yield the product as a white solid (15.7 mg,

43%). MS (calcd 852.4) 853.2 (M + H⁺).Amino acids

analysis Asp, 1.00; Glu, 1.00; Leu, 1.01; Ile, 0.99; Cha,

0.99.

Molecular modeling

Molecular modeling studies were performed with

MacroModel (version 7.1) and the new Maestro inter-

face (version 3).⁷⁴All molecular calculations used the

Amber* force ﬁeld with the united atom charge set.^75,76

The eﬀect of water as solvent was modeled by the GB/

SA solvation method.⁷⁷The crystal structure 1CU1

(chain A) was used as starting structure.³²This structure

was ﬁtted to the force ﬁeld and modiﬁed to be suitable

for docking studies as described previously.⁴⁰His 528

was assumed to have the proton in position 4.The

docking studies were performed as reported.Since the

acyl sulfonamides exceed the template residues of the

crystal in the P1⁰region this part was investigated thor-

oughly.Various conformations of the acyl sulfonamides

were considered by rotating (i) the single bond between

the Ca of residue P1 and the carbonyl group and (ii) the

single bond between the amide-nitrogen and the sulfur

in order to ﬁnd possible interaction patterns in the P1⁰

region during docking.

Suc-^D-Glu-Leu-Ile-Cha-Cys-OH (50a). MS (calcd 729.4)

730.1 (M + H⁺).Amino acids analysis Glu, 10. 0; Leu,

1.01; Ile, 1.01; Cha, 1.00; Cys, 0.98.

Suc-Asp-^D-Glu-Leu-Ile-Cha-Cys-OH (50b). MS (calcd

844.4) 845.1 (M + H⁺).Amino acids analysis Asp,

1.01; Glu, 0.99; Leu, 1.01; Ile, 1.00; Cha, 1.00; Cys, 0.99.

Suc-^D-Glu-Leu-Ile-Cha-Nva-OH (51a). MS (calcd 725.4)

726.3 (M+H⁺).Amino acids analysis Glu, 10.0; Leu,

1.00; Ile, 1.00; Cha, 1.01; Nva, 1.00.

Suc-Asp-^D-Glu-Leu-Ile-Cha-Nva-OH (51b). MS (calcd

840.5) 841.2 (M+H⁺).Amino acids analysis Asp, 10. 1;

Glu, 0.99; Leu, 1.00; Ile, 1.00; Cha, 1.00; Nva, 1.01.

Acknowledgements

We gratefully acknowledge support from the Swedish

Foundation for Strategic Research (SSF) and Medivir

AB, Huddinge, Sweden.We also thank Karolina Ers-

mark for assistance with the chemical syntheses and Adolf

Gogoll for valuable comments on NMR assignment of

some of the ﬂuoro-containing compounds.

Suc-^D-Glu-Leu-Ile-Cha-ACPC-OH (52a). MS (calcd

709.4) 710.2 (M+H⁺).Amino acids analysis Glu, 10. 0;

Leu, 1.00; Ile, 1.00; Cha, 1.00.

Suc-Asp-^D-Glu-Leu-Ile-Cha-ACPC-OH

(52b).

MS

(calcd 824.4) 825.2 (M+H⁺).Amino acids analysis

Asp, 1.00; Glu, 0.98; Leu, 0.99; Ile, 1.04; Cha, 0.99.

References and Notes

Suc-^D-Glu-Leu-Ile-Cha-AllylGly-OH (53a). MS (calcd

723.4) 724.2 (M+H⁺).Amino acids analysis Glu, 10. 2;

Leu, 0.99; Ile, 0.99; Cha, 1.00.

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Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-length NS3 (protease-helicase/NTPase): A comparative study of different C-terminals

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