Design and biological activity of (S)-4-(5-{[1-(3-Chlorobenzyl)-2-oxopyrrolidin-3-ylamino]methyl}imidazol-1- ylmethyl)benzonitrile, a 3-aminopyrrolidinone farnesyltransferase inhibitor with excellent cell potency

J . Med. Chem. 2001, 44, 2933-2949

2933

Design a n d Biologica l Activity of (S)-4-(5-{[1-(3-Ch lor oben zyl)-2-

oxop yr r olid in -3-yla m in o]m eth yl}im id a zol-1-ylm eth yl)ben zon itr ile, a

3-Am in op yr r olid in on e F a r n esyltr a n sfer a se In h ibitor w ith Excellen t Cell

P oten cy

Ian M. Bell,* Steven N. Gallicchio, Marc Abrams,^†Douglas C. Beshore, Carolyn A. Buser,^†

J . Christopher Culberson,^‡J oseph Davide,^†Michelle Ellis-Hutchings,^†Christine Fernandes,^†J ackson B. Gibbs,^†

Samuel L. Graham, George D. Hartman, David C. Heimbrook,^†Carl F. Homnick, J oel R. Huff,

Kelem Kassahun,^§Kenneth S. Koblan,^†Nancy E. Kohl,^†Robert B. Lobell,^†J oseph J . Lynch, J r,^|

Patricia A. Miller,^†Charles A. Omer,^†,∧A. David Rodrigues,^§Eileen S. Walsh,^†and Theresa M. Williams

Departments of Medicinal Chemistry, Cancer Research, Molecular Systems, Drug Metabolism, and Pharmacology, Merck

Research Laboratories, West Point, Pennsylvania 19486

Received April 5, 2001

The synthesis, structure-activity relationships, and biological properties of a novel series of

imidazole-containing inhibitors of farnesyltransferase are described. Starting from a 3-amino-

pyrrolidinone core, a systematic series of modifications provided 5h , a non-thiol, non-peptide

farnesyltransferase inhibitor with excellent bioavailability in dogs. Compound 5h was found

to have an unusually favorable ratio of cell potency to intrinsic potency, compared with other

known FTIs. It exhibited excellent potency against a range of tumor cell lines in vitro and

showed full efficacy in the K-rasB transgenic mouse model.

In tr od u ction

series of modifications to a tetrapeptide lead.¹¹The

central piperazinone moiety in these structures repre-

sents a constraint of the original tetrapeptide backbone,

and we decided to evaluate other peptidomimetic cyclic

constraints. We initially investigated the 3-aminopyr-

rolidinone template, which has found widespread use

in biologically active molecules.^12,13We were interested

in the inherent â-turn bias of this cyclic constraint,

which may mimic the bioactive conformation of some

peptide-like inhibitors.¹⁴The exocyclic amino group in

3-aminopyrrolidinones also offered a convenient divalent

synthetic handle to explore substitution patterns that

are unavailable to other FTIs such as arylpiperazinones.

This report describes our initial investigations on such

pyrrolidine-based inhibitors of farnesyltransferase and

their evolution to give highly potent antitumor agents.

Mutation of the GTP-binding protein Ras, a central

player in cell signaling pathways that govern cell

growth, can lead to cellular transformation and uncon-

trolled proliferation.¹The observation that mutant ras

genes are often found in human tumors suggests that

inhibition of Ras function might provide an effective

anticancer therapy.^2,3Extensive studies on Ras have

revealed that a series of posttranslational modifications

are required for its biological function, the first step of

which is alkylation of a cysteine residue near its

C-terminus with a farnesyl group.^4,5Thus, the enzyme

farnesyltransferase (FTase), which catalyses this step,

was identified as a potential target for chemotherapeutic

agents. Although subsequent studies have undermined

the concept of farnesyltransferase inhibitors (FTIs) as

ras-directed agents, such inhibitors have shown efficacy

in a variety of preclinical cancer models and, more

recently, in the clinic.⁶Our objective was to discover a

potent, selective FTI suitable for oral dosing.

Ch em ica l Meth od s

The basic strategy for the synthesis of 3-aminopyrroli-

dinone farnesyltransferase inhibitors is illustrated in

Scheme 1, using the (S)-enantiomers as examples.

Commercially available amines 1 were coupled with (S)-

BOC methionine under standard EDC conditions to

afford the amides 2. Unfortunately, for the less nucleo-

philic anilines 1a and 1d -f, these conditions caused loss

of stereochemical integrity in the resulting products. For

most of the aniline substrates we have investigated, use

of PyBOP as the coupling reagent mitigated epimeriza-

tion and efficiently generated the amide linkage. For

2-chloroaniline (1d ) and 4-chloroaniline (1f), however,

even the standard PyBOP conditions gave some epimer-

ization, necessitating a further adaptation of the reac-

tion conditions. Thus, when 5 equiv of 2-chloroaniline

was used and the reaction was only allowed to proceed

for 30 min at ambient temperature, the desired anilide

2d was afforded in 86% yield and > 99% ee.

A variety of FTIs have been identified, including

peptidomimetics derived from the C-terminal CaaX

amino acid sequence common to FTase substrates,⁷

natural products,⁸mimics of farnesyl pyrophosphate

(FPP),⁹and non-peptide non-thiol inhibitors.¹⁰An ex-

ample of the latter class of compounds is the N-

arylpiperazinone template developed at Merck via a

* To whom correspondence should be addressed at the Department

of Medicinal Chemistry, Merck Research Laboratories, WP14-2, P.O.

Box 4, West Point, PA 19486. Tel: (215) 652-6455. Fax: (215) 652-

7310. E-mail: ian_bell@merck.com.

^†Department of Cancer Research.

^‡Department of Molecular Systems.

^§Department of Drug Metabolism.

^|Department of Pharmacology.

^∧Current address: Pfizer Inc., 2800 Plymouth Rd., Ann Arbor, MI

48105.

Published on Web 07/31/2001

2934 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18

Bell et al.

Sch em e 1^a

a

Reagents: (a) either (S)-BOC methionine, EDC, HOBT, DIEA, and DMF or (S)-BOC methionine, PyBOP, DIEA, and CH₂Cl₂; (b)

MeI; (c) [(Me)₃Si]₂NLi, THF; (d) HCl, EtOAc; (e) 1-(4-cyanobenzyl)-5-imidazole carboxaldehyde, DIEA, NaCNBH₃, MeOH.

Sch em e 2^a

a

Reagents: (a) HCl, TMOF, MeOH; (b) Ph₃CBr, Et₃N, DMF; (c) 4-cyanobenzyl bromide, CH₃CN; (d) MeOH; (e) NaBH₄, MeOH; (f)

ClSO₂Me, DIEA, CH₂Cl₂; (g) 4a -c, DIEA, NaI, DMF.

Sch em e 3^a

Construction of the pyrrolidinone ring system was

initially accomplished by employing conditions similar

to those reported by Freidinger and co-workers.¹⁵Fol-

lowing treatment of amides 2 with excess iodomethane,

the cyclization of the resulting sulfonium salts was

effected with sodium hydride in DMF-CH₂Cl₂at 0 °C.

However, the isolated BOC-protected aminopyrrolidi-

nones 3 were found to be approximately 15% epimer-

ized. A modified procedure, in which the sulfonium salts

were treated with lithium hexamethyldisilylamide in

THF at 0 °C, provided the pyrrolidinones 3 in enantio-

merically pure form and in good chemical yield (60-

95%). Removal of the protecting group with HCl in

EtOAc and reductive alkylation of the resulting amine

hydrochlorides 4 with 1-(4-cyanobenzyl)-5-imidazole-

carboxaldehyde¹¹using sodium cyanoborohydride in

MeOH afforded the desired analogues 5. Use of alterna-

tive conditions that employed sodium triacetoxyboro-

hydride in DCE was complicated by formation of borane

complexes with the product, which resulted in difficult

purifications and suboptimal yields.

Scheme 2 details the preparation of homologues 11.

In analogy with previous work,¹⁶imidazole-4-acetic acid

(7) was esterified and regioselectively protected with

triphenylmethyl bromide to give the imidazole 8. Alkyl-

ation of 8 with 4-cyanobenzyl bromide¹⁶in acetonitrile

at 50 °C afforded the imidazolium salt that, following

methanolic removal of the trityl group, led to the

hydrogen bromide salt of 9. Reduction of 9 with sodium

borohydride in MeOH gave the alcohol cleanly and

a

Reagents: (a) 2-aminopyridine, AlMe₃, CH₂Cl₂; (b) n-Bu₃P,

DBAD, THF.

conversion to the mesylate 10 proceeded as expected.

Finally, alkylation of amines 4a -c with freshly pre-

pared mesylate 10 in DMF, in the presence of DIEA and

sodium iodide, afforded the desired analogues 11 in

moderate yield (20-40%).

A small number of analogues containing heterocyclic

rings could not be prepared by the previously described

sulfonium salt route due to incompatibility with io-

domethane. Therefore, an alternative design strategy

was developed in our laboratory, and an example is

shown in Scheme 3.¹⁷Treatment of 2-aminopyridine

with trimethylaluminum in CH₂Cl₂and then reaction

of the resulting aluminum amide with (S)-BOC ho-

moserine lactone provided amide 14. The intermediate

14 was cyclized under Mitsunobu conditions¹⁸using tri-

n-butyl phosphine and di-tert-butyl azodicarboxylate in

THF to afford the heterocyclic pyrrolidinone 15 in good

A 3-Aminopyrrolidinone Farnesyltransferase Inhibitor

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2935

Sch em e 4^a

a

Reagents: (a) (BOC)₂O, DIEA, DMF; (b) NaOH, THF, H₂O; (c) 1-(4-cyanobenzyl)-5-imidazole carboxaldehyde, HOAc, NaCNBH₃, MeOH;

(d) HCl, EtOAc; (e) RCO₂H, HOBT, EDC, DMF.

Sch em e 5^a

yield and high enantiomeric excess (95% yield, >99%

ee). The 1-heterocyclic pyrrolidinones generated in this

fashion were converted to the desired compounds, in

analogy with the route described in Scheme 1.

To rapidly explore SAR of the substituent on the

pyrrolidine nitrogen, a related series of acyl pyrrolidines

was investigated (Scheme 4). Ample quantities of pyr-

rolidine 23 were prepared using the straightforward

route shown, and treatment of 23 with a variety of

carboxylic acids under EDC-mediated coupling condi-

tions afforded the analogues 24-37. This strategy was

also applied to the synthesis of a small library, in which

a simple aqueous workup procedure afforded the final

products in good to excellent purity (70-99%).

Transformation of the 3-aminopyrrolidinone second-

ary amino group into the corresponding tertiary amine

was accomplished by reductive alkylation of amines 5b

or 5h with various aldehydes to give compounds 38-

49. The synthetic details of two related tertiary amine

analogues, 52 and 54, are shown in Scheme 5. Incor-

poration of the acetaldehyde subunit in structure 51 was

accomplished by reductively alkylating amine 5h with

2-(tert-butyldimethylsilyloxy)acetaldehyde,¹⁹followed by

removal of the silyl group and subjection of the resulting

alcohol to classical Swern conditions.²⁰Reductive ami-

nation of aldehyde 51 with either morpholine or BOC-

piperazine led to compounds 52 and, after deprotection,

54.

Biologica l Meth od s

Compounds were tested as inhibitors of FTase in vitro

using purified recombinant human enzyme²¹to catalyze

the reaction between [1-³H]FPP and a recombinant

protein substrate containing the K-Ras C-terminus (PD

CVIM²²). Incorporation of the labeled farnesyl group

into the protein substrate was quantitated by precipita-

tion of total protein with acid and scintillation counting.^9a

The inhibitory activity of the compounds is reported as

an IC₅₀value, the concentration of inhibitor required

to reduce radiolabel incorporation by 50% compared

with an uninhibited control experiment. The final

a

Reagents: (a) 2-(tert-butyldimethylsilyloxy)acetaldehyde, NaC-

concentration of organic solvent (either methanol or

NBH₃, HOAc, MeOH; (b) TBAF, THF; (c) oxalyl chloride, DMSO,

TEA, CH₂Cl₂; (d) R₂NH, HOAc, NaCNBH₃, MeOH; (e) HCl, EtOAc.

DMSO) in the assay incubations was 0.5 vol %. Inhibi-

tion of geranylgeranyl-protein transferase type I (GG-

Tase-I) was evaluated in a scintillation proximity assay

using enzyme prepared as described previously.²³The

enzymatic reaction between [1-³H]GGPP and a biotiny-

lated peptide that represents the C-terminus of the

K4B-Ras protein (biotinyl-GKKKKKKSKTKCVIM) was

carried out in the presence of phosphates (5 mM ATP)

2936 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18

Bell et al.

and varying concentrations of inhibitor.²⁴Reaction was

terminated by addition of streptavidin scintillation

proximity assay beads (Amersham), and the mixtures

were subsequently analyzed by scintillation counting,

reporting IC₅₀values as described above. For the FTase

and GGTase inhibition assays, most determinations

were carried out at least twice and the repeats were

generally within 2-fold of the original value. Standard

deviations from the mean value are shown for IC₅₀

values determined three or more times.

required to inhibit cell growth 90% under the standard

assay conditions²⁹is reported as an IC₉₀value. The

cytotoxic endpoint for compounds was evaluated for

normal cultured Rat1 cells by measuring the highest

concentration of drug compatible with 80% cell survival,

as determined by MTT staining.³⁰

In vivo antitumor efficacy was evaluated in the mouse

mammary tumor virus-K-rasB transgenic mouse model,

with the compound being administered by subcutaneous

infusion from an Alzet osmotic minipump. A full de-

scription of this efficacy model and experimental condi-

tions has been published elsewhere.³¹Pharmacokinetic

studies of compound 5h in dogs were performed accord-

ing to standard protocols. The compound was dosed to

three dogs either intravenously (1 mg/kg in DMSO) or

orally (3 mg/kg in 0.5% methocellulose suspension), and

the resulting plasma levels were quantitated by LCMS.

Class III electrophysiologic activity was assessed in

anesthetized dogs with the compound of interest being

administered by intravenous infusion in an escalating-

dose protocol. Plasma levels of compound and electro-

cardiogram data were determined before and during the

infusions. The effects on the QT_cinterval (the duration

from the start of the QRS complex to the end of the T

wave, corrected for heart rate) were correlated with drug

concentrations in plasma to yield an EC₁₀value, the

concentration of compound which prolonged the QT_c

interval by 10%.

The affinity of compounds for the I_Krchannel was

evaluated in radioligand competition experiments analo-

gous to [³H]dofetilide binding assays.²⁵The human ERG

potassium channel was stably expressed in 293 embry-

onic kidney cells,²⁶and plasma membrane fractions

prepared from these cells were used for competition

experiments with [³⁵S]-MK499 (L-706,000).²⁷Results

are reported as inflection points (IP) for the test

compounds to inhibit binding of the radioligand. Most

determinations were carried out at least twice and the

repeats were generally within 2-fold of the original

value. Standard deviations from the mean value are

shown for IP values determined three or more times.

Inhibition of FTase in cells was evaluated by a cell-

based radiotracer assay for farnesyltransferase inhibi-

tion (CRAFTI), in which the concentration of compound

required to displace 50% of a potent, radiolabeled FTI

from FTase in cultured H-ras-transformed Rat1 cells

was determined. This radiotracer, [¹²⁵I]-4-{[5-({(2S)-4-

(3-iodophenyl)-2-[2-(methylsulfonyl)ethyl]-5-oxopiperazin-

1-yl}methyl)-1H-imidazol-1-yl]methyl}benzonitrile, has

∼50 000 high-affinity binding sites (apparent K_d∼ 1

nM) in the Rat1 cell line. The nonspecific binding signal,

determined by the addition of a 1000-fold excess of

unlabeled competitor FTI, is typically 5-fold lower than

the specific binding signal. The assay provides compa-

rable results using a variety of cell lines and is per-

formed as follows. Cells are seeded at 200 000 cells per

well in 24-well tissue culture plates and cultured for

16 h. The radiotracer (∼300-1000 Ci/mmol) is diluted

into culture media to a concentration of 1 nM, along

with the desired concentration of test compound, and

then added to the cell monolayers. The test compound

was diluted in DMSO and the final concentration of

DMSO in the assay was 0.1 vol %. After a 4 h incubation

at 37 °C, the cells are briefly rinsed with phosphate-

buffered saline, removed from the culture plate by

trypsinization, and then subjected to γ-counting using

a CobraII γ-counter (Packard Instrument Co.). Dose-

inhibition curves and IC₅₀values are derived from a

four-parameter curve-fitting equation using SigmaPlot

Software. In cases where determinations were carried

out at multiple times, the repeats were generally within

2-fold of the original value. Standard deviations from

the mean value are shown for IC₅₀values determined

three or more times.

Resu lts a n d Discu ssion

Our initial conception of 3-aminopyrrolidinone-based

FTIs consisted of this central heterocyclic template

positioned between the previously identified cyano-

benzylimidazole¹⁶moiety and a hydrophobic aryl ring,

in analogy with piperazinone-based inhibitors of the

enzyme.¹¹We thought that investigation of such a

system would allow an evaluation of the potency and

other properties of 3-aminopyrrolidinone FTIs with

respect to their piperazinone counterparts. A systematic

study to vary the length of the linkers between these

two end groups and the pyrrolidinone unit, as well as

to explore any stereochemical preference of FTase for

such structures, was undertaken, and the results are

summarized in Table 1.

As shown, the only compounds that exhibited IC₅₀

values against FTase below 100 nM were those in which

a methylene linker connected the imidazole to a 1-

benzyl- or 1-phenyl-substituted aminopyrrolidinone (com-

pounds 5a , 5b, and 6b). Among the inhibitors with this

substitution pattern, FTase exhibited a modest prefer-

ence for the (S)-enantiomer (compare, for example,

compounds 5b and 6b). This stereochemical preference

proved to be quite general for the more potent pyrroli-

dine-containing inhibitors of this structural class (data

not shown), and the remaining tables only detail the

(S)-enantiomers. The set of backbone variants described

in Table 1 was counterscreened against the related

enzyme GGTase-I. While all the compounds were selec-

tive for FTase vs GGTase-I, the most potent inhibitors

of this latter enzyme contained an ethylene linker in

the backbone between the imidazole and amino groups

and exhibited a preference for a longer linker to the

phenyl probe (see compounds 11a -11c).

The inhibition of anchorage-independent cell growth

on poly(HEMA)-coated microtiter plates for H-ras-

transformed Rat1 cells and a variety of human tumor

cell lines was assayed using conditions analogous to

those described in the literature.²⁸Anchorage-indepen-

dent growth inhibition was also evaluated for Rat1 cells

transformed by oncogenic H-ras, K-ras, or H-ras-CVLL,

suspended in soft agar. The concentration of FTI

Evaluation of the most potent FTIs (compounds 5a ,

5b, and 6b) in the cell-based assay for binding to FTase

A 3-Aminopyrrolidinone Farnesyltransferase Inhibitor

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2937

Ta ble 1. 3-Aminopyrrolidinone Inhibitors of FTase: Backbone Modifications

a

b

Stereochemistry at C-3 of pyrrolidinone. Number of replicates used to determine IC₅₀values shown in parentheses.

(CRAFTI) provided a surprise: the IC₅₀values were

2-5-fold lower in the cell-based assay than in the in

vitro enzyme assay. This observation contrasted with

most compounds tested in these assays, such as the

N-arylpiperazinones,³²for which the inhibitory activity

in the cell assay was at best equivalent to the intrinsic

activity against the enzyme, and it suggests that these

aminopyrrolidinones are unusually effective in terms of

cell activity. While this result was unexpected, it should

be noted that the assays are measuring different end-

points under different conditions. The FTase inhibition

assay measures incorporation of a labeled farnesyl

group into a CaaX-containing protein, while in the cell-

based assay the displacement of a potent radioligand

from the FTase active site is analyzed. Moreover, the

CRAFTI assay superimposes a number of other factors

onto the simple competition experiment, such as the

activity of the P-glycoprotein pump (vide infra). The

observation of lower IC₅₀values in the cell-based assay

compared to the intrinsic enzyme assay simply indicates

that the compounds are, for a given level of intrinsic

inhibitory activity, unusually potent in cells.

The most potent analogue (5b) had an intrinsic IC₅₀

value of 29 nM against FTase but was 5 times more

potent in the cell-based assay (CRAFTI IC₅₀) 5.7 nM).

The ability of 5b to inhibit binding of the radioligand

to FTase in cells correlated well with its effects on the

growth of H-ras-transformed Rat1 cells on poly(HEMA)-

coated microtiter plates.²⁸Specifically, the growth of

such cells was inhibited by 5b with an IC₅₀value of 33

nM. While this IC₅₀for growth inhibition is not remark-

able in itself, it is noteworthy that it is essentially

equivalent to the intrinsic IC₅₀of compound 5b against

FTase, again demonstrating an unusually favorable

2938 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18

Ta ble 2. 3-Aminopyrrolidinone Inhibitors of FTase

Bell et al.

a

b

Number of replicates used for determination shown in parentheses. IC₅₀determined with preincubation of inhibitor and enzyme.

ratio of cell potency to intrinsic potency. This interesting

property encouraged us to pursue analogues of com-

pound 5b and the corresponding anilide 5a .

The first analogues of 5a and 5b to be examined

explored the effects of simple chloro substitution around

the phenyl hydrophobe, and the results of this SAR

study are detailed in Table 2. For the anilides (5d -f)

intrinsic potency against the enzyme decreased in the

order 2-Cl > 3-Cl > 4-Cl. Thus, the 2-chloroanilide 5d

was found to be the best inhibitor of FTase both

A 3-Aminopyrrolidinone Farnesyltransferase Inhibitor

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2939

intrinsically (IC₅₀) 1.5 nM) and in the cell-based

binding assay (IC₅₀) 14 nM), although the addition of

this substituent reduced the compound’s ability to

function in the cell assay. Attenuated performance in

cells is often attributed to increased protein binding or

poorer cell penetration properties, although the ad-

ditional chlorine atom in 5d seemed to have little effect

on the compound’s lipophilicity (for 5d , log P ) 1.75,

compared with the des-chloro analogue 5a , log P ) 1.87)

or human serum protein binding (for 5d , free fraction

) 25%, compared with 5a , free fraction ) 22%). These

observations indicate that the differences in relative cell

potency were not simply due to changes in physical

properties or protein binding but probably involved

other factors (vide infra).

A parallel series of substituted 1-benzylpyrrolidinones

was also examined (compounds 5g-i in Table 2). In

terms of intrinsic potency against FTase, the 4-chloro

analogue 5i was equipotent with the unsubstituted

version 5b, while the 2-chloro analogue 5g was about

6-fold more potent and the 3-chloro compound 5h 15-

fold more potent than 5b. The chloro-substituted ana-

logues in Table 2 were counterscreened against GG-

Tase-I and showed uniformly good selectivity against

this related enzyme. The relative levels of intrinsic and

cellular inhibition of FTase varied somewhat for the

three chlorobenzyl compounds, but gratifyingly, the

most potent analogue, 5h , had a 4-fold lower IC₅₀in the

cell-based assay, consistent with the results observed

for the compounds in Table 1. Many factors control the

ability of a compound to function in a cell-based assay,

such as penetration of the cell membrane, protein

binding, chemical stability, and maintenance of suitable

concentrations in the relevant cellular compartments,

so it is difficult to explain such phenomena simply. A

well-known factor in cell potency is the P-glycoprotein

pump (PGP), which can expel a variety of compounds

from the cytosol and render them less functional in

cells.³³Indeed, the radioligand used for the CRAFTI

assay is known to be a substrate for PGP.³⁴When the

CRAFTI assay was performed in the presence of 20 µM

verapamil, which effectively blockades the P-glycopro-

tein pump,³³the IC₅₀value of 5h shifted 5-fold to 2.5

nM. This observation is consistent with the idea that

5h is not a good substrate for PGP relative to the

radioligand, and this may explain why 3-aminopyrro-

lidinones such as 5h seem to be particularly cell potent.

Since PGP activity is a common cause of resistance to

chemotherapeutics in cancer cells, we were encouraged

by the fact that compound 5h seemed to be a relatively

poor substrate for this pump.

F igu r e 1. Docked structure of compound 5h in the FTase

active site. The R-subunit has been removed for clarity. The

farnesyl group of FPP is shown in magenta. The side chains

that ligate the zinc (Asp297, Cys299, and His362) are colored

pale cyan, and a hydrophobic pocket defined by the Trp102,

Trp106, and Tyr361 side chains (light gray) is indicated.

Conformers of the FTI were generated using J G⁴³within the

FTase active site as determined by Beese et al.⁴⁴The confor-

mations were subsequently minimized within the rigid active

site with the MMFF force field using Macromodel.⁴⁵During

the energy minimization, the imidazole nitrogen which is

coordinated to the zinc was held fixed, and a distance-

dependent dielectric constant of 2.0 was employed. The result-

ing conformations were ordered by energy, and a representa-

tive structure is shown.

QT_cinterval prolongation, often associated with block-

ade of cardiac ion channels.³⁵Indeed, prolongation of

the QT_cinterval has been observed with Merck’s clinical

candidate FTI during Phase I studies.⁶These concerns

led us to counterscreen compounds against the human

I

_Krpotassium channel, which has been implicated in QT_c

effects,^35,36and 5h was found to have moderate binding

affinity at this channel (hERG IP ) 440 nM). Further-

more, analysis of compound 5h in anesthetized dogs

revealed that it caused 10% prolongation of the QT_c

interval at a plasma level of 2.5 µM. Studies on the

parent compound 5b showed that it had a lower affinity

for the ion channel (hERG IP ) 7200 nM; see Table 1)

but that it nonetheless caused a 10% increase in the

QT_cinterval in dogs at a 2-fold lower plasma level (1.2

µM). The lack of absolute correlation between the hERG

channel binding affinity of these two compounds and

their class III activity in dogs may be due to species

variations between the human and canine ion channels,

activity of the compounds at other ion channels, or

possibly to differential protein binding of 5b and 5h .

Whatever the explanation for these discrepancies, it was

clear that even micromolar affinity for this ion channel

might correlate with undesirable cardiac effects, and we

sought modifications that would alleviate such activity.

Molecular modeling studies provided a structure of

5h docked in the FTase active site, using the protein

structure determined by X-ray crystallographic studies

on a ternary enzyme complex (see Figure 1). As shown,

the key interactions are believed to be ligation of the

catalytic zinc by the imidazole moiety, interaction of the

cyanophenyl group with the farnesyl chain, and binding

of the chlorophenyl ring in a hydrophobic pocket defined

by three aromatic residues. The pyrrolidinone ring does

not appear to be making any important interactions

with the protein and is oriented toward the solvent.

It seemed possible that incorporation of polar func-

tionality at key positions in these FTIs might reduce

their affinity for the I_Krchannel while having little effect

on their potency against FTase, so a number of basic

heterocycles were investigated as alternatives to the

One issue that has gained considerable attention in

the field of drug discovery recently is the potential for

2940 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18

Bell et al.

phenyl groups in compounds such as 5a . Table 2

illustrates a number of these analogues (compounds

16-19), and it was apparent that such modifications

could significantly reduce affinity for FTase (compare

16 with 5a ). Compound 19 illustrated, however, that

this strategy may have some merit, since it combined

good potency against the enzyme with good cell pen-

etration and attenuated ion channel binding. We de-

cided that it may be possible to discover heterocyclic

endgroups that were consistent with good enzyme

inhibition in the absence of significant binding in the

hERG assay by using a different template that facili-

tated rapid exploration of such substituents.

nicotinoyl 2-position on activity against FTase. The

unsubstituted analogue 29 was a relatively poor inhibi-

tor (IC₅₀) 2000 nM), but addition of a methyl group

improved the IC₅₀value by 20-fold. Addition of polar

groups at this position had little effect compared with

29 (see structures 31, 32, and 34), but the addition of a

thioethyl side chain to give 36 provided more than a

400-fold improvement in potency. This profound effect

strongly suggested that the thioethyl side chain was

making a specific interaction with some hydrophobic

pocket on the enzyme, perhaps mimicking one of the

amino acid side chains of the CaaX substrate. Modeling

studies supported this idea, revealing low-energy con-

formations of 36 docked in the FTase active site in which

the thioethyl group projected into the same pocket

occupied by the chlorophenyl ring in 5h (compare

Figures 1 and 2). Moreover, when this conformation of

36 was overlaid with the crystallographically deter-

mined bound structure of the peptidomimetic FTI

L-739,750,^7athe thioethyl moiety occupied the same

space as the benzyl side chain in the peptidomimetic

(Figure 2).

A related series of compounds, in which the pyrroli-

dinone carbonyl group is migrated out of the ring to give

1-acylpyrrolidine-based structures, offered one way to

approach this problem. A number of these pyrrolidine-

based FTIs were synthesized and they possessed mod-

erate activity against FTase (see Table 3). Thus, the

pyrrolidine analogue 24 is a simple isomer of structure

5b (Table 1) and this structural change causes a loss of

approximately 4-fold in intrinsic potency against the

enzyme. Replacement of the benzoyl moiety in 24 with

naphthalene-1-carbonyl gave compound 25, which

showed good inhibitory activity against FTase, in anal-

ogy with related piperazine-based FTIs.³⁷The benzoyl

compound 24 was also compared to its simple chloro-

substituted analogues (see structures 26-28 in Table

3) and, in analogy with the chlorobenzyl derivatives in

Table 2, the 3-chloro isomer 27 was found to be the most

potent inhibitor of FTase, with an IC₅₀value of 27 nM.

It seemed likely that the isomeric 1-acylpyrrolidine

series bound to FTase in an analogous fashion to the

pyrrolidinone series but offered an advantage in terms

of ease of synthesis. It was possible to rapidly screen

different acyl substituents, and we hoped that, by

focusing on polar heterocycles as replacements for the

chlorophenyl moieties in compounds such as 27, it would

be possible to identify pyrrolidine-based FTIs that had

reduced affinity for the I_Krchannel.

Compound 36 exhibited good cell potency (CRAFTI

IC₅₀) 6.2 nM) and excellent selectivity against GG-

Tase-I (IC₅₀) 5100 nM). Compared to the 3-chloro-

benzoyl analogue 27, the thioether 36 offered a signifi-

cant improvement in potency against FTase. However,

neither 36 nor any of the other acylpyrrolidines syn-

thesized exhibited the excellent cell potency of com-

pounds such as 5h . We therefore sought other strategies

to reduce the affinity of these aminopyrrolidine-based

FTIs for the potassium channel.

One of the initially attractive features of the ami-

nopyrrolidinone moiety in the context of FTIs was that

the central backbone amino group may be regarded as

a trivalent handle, allowing the exploration of SAR that

is quite distinct from related inhibitors. This central

nitrogen atom may be attached to a cyanobenzylimida-

zole group, which presumably interacts with the active

site zinc ion, and to a potency-enhancing hydrophobe,

such as the chlorophenyl in compound 5h , via the

pyrrolidine ring. This arrangement still leaves one

position vacant on the central amine and, as such,

permits facile exploration from this handle. Results from

an investigation of such SAR in the context of the lead

5b are shown in Table 4 (compounds 38-46).

A subset of the compounds synthesized in this effort

are illustrated in Table 3 (compounds 29-37). A variety

of heterocyclic acyl substituents was investigated, but

the series of nicotinoyl derivatives in Table 3 serves to

illustrate the general trends observed. As was expected,

the more polar derivatives did possess reduced affinity

for the ion channel. Compounds 31 and 32, for example,

had no measurable activity in the hERG assay, whereas

the 3-chlorobenzoyl derivative 27 had a hERG inflection

point of 5800 nM. Unfortunately, those compounds with

the lowest affinity for the ion channel also exhibited

unacceptably weak inhibition of FTase. For example,

while the 2-methoxynicotinoyl derivative 33 showed

very modest binding to the I_Krchannel (hERG IP )

21 000 nM), it was a poor enzyme inhibitor (FTase IC₅₀

) 150 nM). The related sulfide analogue 35 possessed

slightly better activity against FTase (IC₅₀) 83 nM),

but homologation of this structure provided the ethyl

sulfide 36, which displayed an unexpected improvement

in terms of inhibition of FTase (IC₅₀) 4.8 nM).

Substitution at other positions in this nicotinoyl system,

to afford compounds such as the bromide 37, failed to

provide more potent FTIs than 36. Indeed, Table 3

illustrates the profound effects of substitution at this

Initially, the amino group was derivatized with

aliphatic groups such as n-butyl, which had only modest

effects on the intrinsic enzyme potency (FTase IC₅₀

)

54 nM for 39) but led to significant drops in the cell-

based assay (CRAFTI IC₅₀) 42 nM for 39 compared

with 5.7 nM for 5b). The availability of hydrophobic

binding was probed by placing a phenyl group at various

linker lengths from the amine (see compounds 40-43).

The optimal example appeared to be the phenylpropyl

derivative 42, but this possessed only a 2-fold enhanced

IC₅₀value vs FTase intrinsically and had markedly

reduced cell potency (CRAFTI IC₅₀) 74 nM). Reasoning

that the increased lipophilicity of these structures

relative to compound 5b may be partly responsible for

their poor performance in the cell-based assay, we

investigated a number of relatively polar substituents

to give compounds such as the amine 45 and the

pyridine 46. As Table 4 shows, these compounds fared

A 3-Aminopyrrolidinone Farnesyltransferase Inhibitor

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2941

Ta ble 3. 3-Aminopyrrolidine Inhibitors of FTase

a

Number of replicates used to determine IC₅₀values shown in parentheses.

no better in terms of inhibitory potency either intrinsi-

cally or in cells. Indeed, the cell-based potency of amine

45 was particularly poor, which may be attributable to

the basic character of the substituent.

At this point, we concluded that these substituents

had little effect on inhibition of FTase: the intrinsic IC₅₀

values are all within 2-fold of the parent compound 5b.

One explanation for this behavior is that the additional

side chains are not making significant contacts with the

enzyme active site but are oriented toward the solvent.

Indeed, this explanation seems to be consistent with the

modeled structure of the ternary complex of 5h bound

2942 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18

Bell et al.

none of these analogues offered a significant advantage

over the parent compound 5h in terms of hERG activity

(see Table 4).

Evaluation of the pharmacokinetic properties of 5h

in dogs revealed that the compound exhibited low

plasma clearance (CL_p) 5.84 ( 0.12 mL/min/kg) but a

half-life of only 1.07 ( 0.20 h due to a relatively low

volume of distribution (Vd_ss) 0.45 ( 0.07 L/kg). Despite

excellent bioavailability (F ) 81 ( 16%), this profile was

probably not suitable for oral dosing because, if such a

short half-life were observed in humans, it would lead

to significant peak to trough variations and possible

prolongation of the QT_cinterval at the peak plasma

concentrations of drug. In contrast, the low rate of

clearance would facilitate an intravenous dosing pro-

tocol. Therefore, a more accurate examination of the

potency of 5h seemed appropriate in order to establish

if there was a suitable window between therapeutic

plasma levels and potential proarrhythmic effects in

vivo.

As described above, compound 5h exhibited excellent

potency in the cell-based radioligand displacement

assay, with a CRAFTI IC₅₀value that was some 4-fold

lower than its intrinsic IC₅₀for inhibition of FTase in

vitro (see Table 2). The high cell potency of 5h in

CRAFTI translated from the radioligand binding assay

into cell growth assays, and representative results are

detailed in Table 5. As shown, compound 5h inhibited

the anchorage-independent growth of cultured H-ras-

transformed Rat1 cells on poly(HEMA)-coated microtiter

plates with an IC₅₀value of 4.1 nM. This low nanomolar

activity contrasted with the cytotoxic endpoint of the

compound for Rat1 cells, which was determined to be

greater than 100 µM by MTT staining. These data

suggest that the FTI-mediated growth inhibition of the

H-ras-transformed cells was at least partly due to

inactivation of the mutant H-Ras protein. The inhibition

of H- and K-ras-transformed Rat1 cell growth in soft

agar by FTI 5h was also studied. The compound proved

to be extremely potent in this assay, inhibiting 90% of

colony formation at 3-10 nM for the H-ras cell line and

at 30 nM for the K-ras cell line, again showing activity

at concentrations well below those that were observed

to cause cytotoxicity (see Table 5). In contrast to the

observations with H-ras transformed fibroblasts, the

growth of H-ras-CVLL-transformed Rat1 cells, in which

transformation is effected by an engineered Ras protein

that is a selective substrate for GGTase-I, was much

less sensitive to the FTI 5h (IC₉₀) 100-300 nM),

consistent with the fact that the compound is a highly

selective inhibitor of FTase. The fact that growth of this

cell line was inhibited by 5h at submicromolar concen-

trations indicates that a Ras-independent antiprolif-

erative mechanism is operational in such fibroblasts.

F igu r e 2. Docked structure of compound 36 (green) in the

FTase active site overlaid with the bound conformation of the

peptidomimetic FTI L-739,750^7a(white) as determined by

X-ray crystallography.⁴⁴Other details of the docking experi-

ment and graphical representation are described in the Figure

1 legend.

to FTase, in which the amino group is oriented toward

bulk solvent (see Figure 1). The analogues in Table 4

demonstrated generally good selectivity against GG-

Tase-I and the data indicate that this enzyme is

somewhat more sensitive to such substitutions than

FTase. Compared with the parent structure 5b, the

substituents in some cases gave rise to more than 10-

fold enhancements in the inhibitory activity against

GGTase-I, and in other cases abolished all activity. Of

greater concern was the observation that most of these

tertiary amine analogues did not exhibit the same ratio

of cell-based potency to intrinsic potency seen for

compound 5b, suggesting that this favorable behavior

is related to the presence of the secondary amine

functionality in compounds such as 5b and 5h . None-

theless, the discovery of binding to the I_Krchannel by

compounds such as 5h prompted us to reexamine this

kind of substitution pattern. We reasoned that an

additional substituent on the amino group of 5h should

have a minor effect on its binding to FTase and that

some moderately polar substituent might simulta-

neously maintain good cell penetration and also attenu-

ate the affinity for the hERG channel.

For comparison purposes, the phenylpropyl analogue

of 5h was synthesized (compound 47) and, as expected,

it was comparable to 5h in terms of ability to inhibit

FTase but was less effective in cells and possessed very

high affinity for the I_Krchannel (hERG IP ) 24 nM). A

number of polar side chains were added to structure 5h

(compounds 48-54), and it was found that while they

caused minimal changes to the compound’s intrinsic

potency against FTase, the activity in cells ranged from

excellent (CRAFTI IC₅₀) 1.1 nM for compound 48) to

poor (CRAFTI IC₅₀) 300 nM for compound 54). It

appeared that the best ratios of cell-based to intrinsic

potency were obtained with moderately polar side

chains such as the carbamate in 48. Disappointingly,

The picture was more complex when a panel of human

tumor cell lines was examined. Anchorage-independent

growth was inhibited by 5h for a variety of tumor types

but, as has been observed for other FTIs,³⁸there was

no dependence on the mutational state of the ras gene.

Thus, the growth of A549 cells, which contain a mutant

K-ras gene, was inhibited to an almost identical extent

to that of Colo-205 cells, which possess wild-type ras

[poly(HEMA) IC₅₀) 320 nM for A549 and 340 nM for

Colo-205]. Similar results were observed for the human

A 3-Aminopyrrolidinone Farnesyltransferase Inhibitor

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2943

Ta ble 4. 3-Aminopyrrolidinone Inhibitors of FTase

a

Number of replicates used to determine IC₅₀values shown in parentheses.

tumor cell lines LS180 (mutant K-ras) and MDA-MB-

468 (wild-type ras) (see Table 5). In these human cell

lines, the growth inhibition apparently did not result

from inhibition of the prenylation of mutant Ras but

from lack of farnesylation of some other protein(s). This

result was not surprising, since it was known that in

cells K-Ras is geranylgeranylated by GGTase-I in the

presence of a potent inhibitor of farnesyltransferase and

that inhibition of K-Ras processing does not correlate

with inhibition of cell growth.³⁹Compound 5h , a highly

selective FTI, could inhibit farnesylation of K-Ras, but

the protein would still become prenylated and, presum-

2944 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18

Bell et al.

Ta ble 5. Anchorage-Independent Cell Growth Inhibition by

Compound 5h

died during the experiment. The lower dose of 5h was

significantly less toxic and resulted in no lethality.

These experiments demonstrate that 5h acts as an

effective antitumor agent in a K-ras mouse model of

cancer, despite the fact that the mutant K-Ras protein

is presumably prenylated under the experimental con-

ditions.

poly(HEMA)

cell line

IC₅₀(nM)^a

soft agar

cell line

IC₉₀(nM)^a

H-ras

4.1 (2)

3-10 (2)

30 (2)

A549

Colo-205

LS180

KB-31

MDA-MB-468

DLD1

320 ( 88

K-ras

340 ( 300 (4)

530 ( 250 (8)

530 ( 63 (4)

750 ( 490 (4)

1600 ( 300 (3)

>100000 (2)

H-ras-CVLL

100-300 (2)

Con clu sion

A series of 3-aminopyrrolidinone-based inhibitors of

farnesyltransferase has been developed. Systematic

modification of these structures led to the identification

of 5h , a highly potent, low molecular weight, non-thiol,

non-peptide FTI that was efficacious both in cell culture

and in animals. The compound was extremely potent

in cell-based experiments, inhibiting the growth of a

variety of transformed cells with submicromolar IC₅₀

values. While compound 5h exhibited low plasma clear-

ance and excellent oral bioavailability, its relatively

short plasma half-life in conjunction with its effects on

the QT_cinterval in dogs made it unacceptable as a

candidate for oral dosing. Although the window between

plasma levels that showed full efficacy in the K-rasB

transgenic mouse (0.67 µM) and those that caused 10%

prolongation of the QT_cinterval in dogs (2.5 µM) would

potentially allow a successful intravenous dosing pro-

tocol, a variety of modifications have been explored in

an attempt to improve this window to a degree that

would permit oral dosing in a clinical setting. While

binding to the I_Krion channel was significantly reduced

in some cases, the modifications required were not

consistent with maintaining the high levels of potency

against FTase seen with compound 5h . Nonetheless, the

3-aminopyrrolidinone core appeared to confer a very

favorable ratio of cell-based potency to intrinsic potency,

and this was due in part to the fact that compounds

such as 5h appeared to be relatively poor substrates for

the P-glycoprotein pump. This property may help pre-

vent chemoresistance in treated tumors and facilitate

the effort to identify clinically useful chemotherapy

agents.

DuPro-1

a

Number of replicates used to determine IC₅₀values shown in

parentheses. Standard deviations from the mean are shown for

IC₅₀values determined three or more times.

Ta ble 6. Effects of Compound 5h in the K-RasB Transgenic

Mouse Model

MGR^c(mm³/day)

%

dose^a

(mg/kg/day)

plasma level^bprimary

(µM) tumor

unprocessed

total

mDJ 2^d

vehicle

40

120

17.3 ( 3.5 19.9 ( 3.7

0.67 ( 0.10 -3.1 ( 1.7 -3.9 ( 1.8

1.8 ( 0.3 -1.2 ( 2.0 -1.0 ( 2.1

13 ( 2

47 ( 9

79 ( 1

a

Compound dosed for 28 days subcutaneously in 50% aqueous

DMSO using osmotic minipumps, 10 animals per dose group.

b

Plasma level of compound ((SEM) determined at termination

of study using HPLC analysis. ^cMean growth rate of tumor volume

((SEM) measured for both the primary tumor and also for the

d

total tumor volume in the animal. Percentage ((SEM) of mDJ 2

protein in the mouse spleen determined to be unfamesylated by a

standard Western blot assay at the termination of the experiment.

ably, functional. The identity of the real target or targets

of FTIs has been the subject of much research and

discussion.³A number of proteins, including RhoB⁴⁰and

CENP-E,⁴¹have been postulated to be the relevant

FTase substrate(s), but more work remains to be done.

It was clear, in any case, that compound 5h could inhibit

the growth of a variety of artificially transformed cells

and human tumor cell lines and a key question was

whether this effect could be reproduced in vivo.

As an in vivo model of cancer, the K-rasB transgenic

mouse model of cancer has a number of attractive

features, notably that the mice are not immune-

compromised and that the tumors occur and grow

spontaneously in the animals, in contrast to the trans-

planted foreign tumor cells used in nude mouse experi-

ments.³¹We were interested in establishing what

steady-state plasma levels would be required for full

efficacy, so we examined the effects of 5h in K-rasB

transgenic mice with the compound being administered

by subcutaneous infusion for 28 days. Table 6 sum-

marizes the results of these experiments. As can be

seen, at doses of 120 and 40 mg/kg/day, regression of

both primary tumor and total tumor volume was noted.

The regressions seen in these studies represented

maximal responses for both doses, so it may be assumed

that the 120 mg/kg/day regimen constitutes an excessive

dose. The percentage of unprocessed mDJ 2, a protein

substrate for FTase, was quantitated using spleen tissue

from the mice to give a measure of in vivo FTase

inhibition, and the results confirmed the very high level

of FTase inhibition in the 120 mg/kg/day dose group

(79% unprocessed mDJ 2) compared with the 40 mg/kg/

day group (47% unprocessed mDJ 2). The overdosing of

FTI at the highest dose was also manifested in terms

of gross toxicity in the mice in that 20% of the animals

Exp er im en ta l Section

Gen er a l Meth od s. Proton NMR spectra were run at 300

MHz on a Varian VXR-300 or at 400 MHz on a Varian Unity

400 or VXR-400 spectrometer, and chemical shifts are reported

in parts per million (δ) downfield from tetramethylsilane as

internal standard. Fast atom bombardment mass spectra were

recorded on a VG-ZAB-HF spectrometer using glycerol as

matrix. Electrospray mass spectra were recorded on a Micro-

mass ZMD spectrometer. Elemental analyses were performed

using a Perkin-Elmer 2400 model II elemental analyzer. Silica

gel 60 (230-400 mesh) from EM Science was used for column

chromatography, and analytical or preparative thin-layer

chromatography was conducted using EM Science Kieselgel

60 F₂₅₄plates. Thermoseparation HPLC equipment with a

Vydac C18 reversed phase column was used for analytical or

preparative HPLC. The elution used either a gradient of 95/5

to 0/100 A/B; A ) H₂O-0.1% TFA, B ) CH₃CN-0.1% TFA

(method A), or a gradient of 95/5 to 5/95 A/B; A ) H₂O-0.1%

H₃PO₄, B ) CH₃CN (method B). The enantiomeric purity of

the final compounds was established by HPLC using either a

Chiralpak AD or a Chiralcel OD column or by Mosher amide

analysis. For reactions performed under anhydrous conditions,

glassware was either oven- or flame-dried and the reaction

was run under a positive pressure of argon. Tetrahydrofuran

was freshly distilled from sodium/benzophenone; all other

anhydrous solvents were used as purchased from Aldrich.

A 3-Aminopyrrolidinone Farnesyltransferase Inhibitor

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2945

Except where noted, reagents were purchased from Aldrich

and used without further purification. The reported yields are

the actual isolated yields of purified material and are not

optimized.

quenched with saturated aqueous NaHCO₃(2 mL) and most

of the MeOH was removed under reduced pressure. The

residual solution was partitioned between saturated aqueous

NaHCO₃(3 mL) and CH₂Cl₂(5 mL). The aqueous layer was

extracted further with CH₂Cl₂(2 × 5 mL). The combined

organic extracts were dried over MgSO₄, filtered, and concen-

trated in vacuo. The crude product was purified by flash

column chromatography on silica, eluting with CH₂Cl₂-2%

MeOH-0.2% NH₄OH, to yield the desired product, which was

treated with aqueous HCl in acetonitrile to afford the hydro-

chloride salt as a white solid (215 mg, 85%): ¹H NMR (CD₃-

OD) δ 9.15 (1H, s), 7.95 (1H, d, J ) 1.1 Hz), 7.82 (2H, d, J )

8.6 Hz), 7.67 (2H, d, J ) 8.8 Hz), 7.57 (2H, d, J ) 8.6 Hz),

7.43 (2H, dd, J ) 8.6, 7.5 Hz), 7.26 (1H, t, J ) 7.5 Hz), 5.83

(2H, s), 4.81 (1H, d, J ) 15.0 Hz), 4.53 (1H, d, J ) 15.0 Hz),

4.44 (1H, dd, J ) 10.7, 8.7 Hz), 4.00-3.95 (2H, m), 2.75 (1H,

m), 2.31 (1H, m); MS (FAB) m/z ) 372 (M⁺+ H); HPLC purity

) 97.3% (method A, 215 nm); ee ) 93.9%. Anal. (C₂₂H₂₁N₅O‚

1.7HCl‚1.7H₂O‚0.2EtOAc) C, H, N.

(R)-4-{5-[2-(1-Ben zyl-2-oxop yr r olid in -3-yla m in o)eth yl]-

im id a zol-1-ylm eth yl}ben zon itr ile Hyd r och lor id e (12b).

4-[5-(2-H y d r o x y e t h y l)im id a zo l-1-y lm e t h y l]b e n zo n i-

tr ile. To a stirred solution of methyl [1-(4-cyanobenzyl)-1H-

imidazol-5-yl]acetate¹⁶(1.09 g, 4.28 mmol) in methanol at 0

°C was added sodium borohydride (0.96 g, 25.4 mmol) in one

portion. After 3 h, saturated aqueous NH₄Cl (20 mL) was

added, followed by saturated aqueous NaHCO₃(20 mL), and

the mixture was extracted with EtOAc (3 × 75 mL). The

combined organic extracts were dried over MgSO₄, filtered, and

concentrated in vacuo. The crude product was purified by flash

column chromatography on silica, eluting with CH₂Cl_2-5%

MeOH, to yield the desired product as a white solid (490 mg,

50%): ¹H NMR (CDCl₃) δ 7.64 (2H, d, J ) 8.4 Hz), 7.50 (1H,

s), 7.12 (2H, d, J ) 8.6 Hz), 6.95 (1H, d, J ) 0.7 Hz), 5.23 (2H,

s), 3.79 (2H, t, J ) 6.4 Hz), 2.66 (2H, td, J ) 6.4, 0.9 Hz), 1.94

(1H, br s); MS (FAB) m/z ) 228 (M⁺+ H).

(S)-4-{5-[(2-Oxo-1-p h en ylp yr r olid in -3-yla m in o)m eth yl]-

im id a zol-1-ylm eth yl}ben zon itr ile Hyd r och lor id e (5a ).

(S)-2-(ter t-Bu t oxyca r b on yla m in o)-4-(m et h ylm er ca p t o)-

N-p h en ylbu tyr a m id e (2a ). To (S)-N-(tert-butoxycarbonyl)-

methionine (589 mg, 2.36 mmol) in dry CH₂Cl₂(5 mL) under

argon were added PYBOP (1.23 g, 2.36 mmol), aniline (196

µL, 2.14 mmol), and N,N-diisopropylethylamine (655 µL, 3.76

mmol). The reaction mixture was stirred for 1 h and then

quenched with 10% citric acid (20 mL) and extracted with CH₂-

Cl₂(2 × 20 mL). The combined organic extracts were dried

over MgSO₄, filtered, and concentrated in vacuo. The crude

product was purified by flash column chromatography on silica,

eluting with hexane-15% ethyl acetate to yield the product

as a white solid (690 mg, 99%): ¹H NMR (CDCl₃) δ 8.27 (1H,

br s), 7.51 (2H, d, J ) 8.4 Hz), 7.32 (2H, t, J ) 7.8 Hz), 7.12

(1H, t, J ) 7.1 Hz), 5.20 (1H, br d, J ) 7.1 Hz), 4.40 (1H, m),

2.70-2.56 (2H, m), 2.20 (1H, m), 2.14 (3H, s), 2.02 (1H, m),

1.47 (9H, s); MS (FAB) m/z ) 325 (M⁺+ H); HPLC purity )

99.1% (method A, 215 nm).

(S)-2-(ter t-Bu t oxyca r b on yla m in o)-4-(d im et h ylsu lfo-

n iu m )-N-p h en ylbu tyr a m id e Iod id e. (S)-2-(tert-Butoxycar-

bonylamino)-4-(methylmercapto)-N-phenylbutyramide (2a) (680

mg, 2.10 mmol) was dissolved in iodomethane (10 mL, 160

mmol) and the solution was stirred under argon for 48 h. The

iodomethane was removed by distillation under reduced pres-

sure to give the sulfonium salt as a white solid of sufficient

purity for use in the next step: ¹H NMR (CDCl₃) δ 9.77 (1H,

br s), 7.81 (2H, d, J ) 7.9 Hz), 7.29 (2H, t, J ) 7.9 Hz), 7.10

(1H, t, J ) 7.0 Hz), 6.11 (1H, d, J ) 7.0 Hz), 4.79 (1H, m),

3.72 (1H, m), 3.49 (1H, m), 3.28 (3H, s), 3.04 (3H, s), 2.87 (1H,

m), 2.13 (1H, m), 1.44 (9H, s); MS (FAB) m/z ) 339 (M⁺).

(S)-3-(ter t-Bu toxyca r bon yla m in o)-2-oxo-1-p h en ylp yr -

r olid in e (3a ). (S)-2-(tert-Butoxycarbonylamino)-4-(dimethyl-

sulfonium)-N-phenylbutyramide iodide (970 mg, 2.1 mmol) was

stirred in dry THF (40 mL), under argon, at 0 °C, and lithium

bis(trimethylsilyl)amide (1.0 M in THF, 2.1 mL, 2.1 mmol) was

added dropwise. The reaction mixture was stirred at 0 °C for

2 h and then quenched with saturated aqueous NH₄Cl (5 mL),

and most of the THF was removed under reduced pressure.

The residual solution was partitioned between saturated

aqueous NaHCO₃(10 mL) and CH₂Cl₂(20 mL). The aqueous

layer was extracted further with CH₂Cl₂(2 × 20 mL), and the

combined organic extracts were dried over MgSO₄, filtered, and

concentrated in vacuo. The crude product was purified by flash

column chromatography on silica, eluting with hexane-20%

ethyl acetate to yield the pyrrolidinone as a white solid (560

mg, 97%): ¹H NMR (CDCl₃) δ 7.64 (2H, dd, J ) 8.8, 1.0 Hz),

7.39 (2H, dd, J ) 8.5, 7.6 Hz), 7.18 (1H, tt, J ) 7.3, 1.0 Hz),

5.23 (1H, br s), 4.35 (1H, m), 3.87-3.75 (2H, m), 2.80 (1H, m),

2.00 (1H, qn, J ) 10.7 Hz), 1.48 (9H, s); MS (FAB) m/z ) 277

(M⁺+ H); HPLC purity ) 98.5% (method A, 215 nm); ee )

96.4%.

(S)-3-Am in o-2-oxo-1-p h en ylp yr r olid in e Hyd r och lor id e

(4a ). A solution of (S)-3-(tert-butoxycarbonylamino)-2-oxo-1-

phenylpyrrolidine (3a ) (490 mg, 1.8 mmol) in EtOAc (40 mL)

at 0 °C was saturated with HCl(g). After 15 min, the mixture

was concentrated in vacuo to yield the amine hydrochloride

as a pale solid (380 mg, 99%): ¹H NMR (CD₃OD) δ 7.67 (2H,

dd, J ) 8.8, 0.9 Hz), 7.41 (2H, dd, J ) 8.4, 7.7 Hz), 7.23 (1H,

t, J ) 7.4 Hz), 4.26 (1H, dd, J ) 10.9, 8.7 Hz), 3.99 (1H, td, J

) 9.9, 6.2 Hz), 3.96 (1H, m), 2.69 (1H, m), 2.16 (1H, m); MS

(FAB) m/z ) 177 (M⁺+ H); HPLC purity ) 100.0% (method

A, 215 nm).

Meth a n esu lfon ic Acid 2-[1-(4-cya n oben zyl)-1H-im id a -

zol-5-yl]eth yl Ester (10). A solution of 4-[5-(2-hydroxyethyl)-

imidazol-1-ylmethyl]-benzonitrile (250 mg, 1.10 mmol) in dry

CH₂Cl₂(35 mL) at 0 °C, under argon, was treated with N,N-

diisopropylethylamine (233 µL, 1.34 mmol), followed by meth-

anesulfonyl chloride (103 µL, 1.33 mmol). The reaction mixture

was stirred at 0 °C for 3 h and then quenched with saturated

aqueous NaHCO₃(25 mL) and extracted with CH₂Cl₂(3 × 15

mL). The combined organic extracts were dried over MgSO₄,

filtered, and concentrated in vacuo to give the mesylate as a

thick yellow oil, which was used crude for the next reaction:

¹H NMR (CDCl₃) δ 7.66 (2H, d, J ) 8.4 Hz), 7.56 (1H, s), 7.14

(2H, d, J ) 8.4 Hz), 7.01 (1H, d, J ) 0.5 Hz), 5.21 (2H, s), 4.31

(2H, t, J ) 6.7 Hz), 2.95 (3H, s), 2.87 (2H, td, J ) 6.7, 0.7 Hz).

(R)-4-{5-[2-(1-Ben zyl-2-oxop yr r olid in -3-yla m in o)eth yl]-

im id a zol-1-ylm eth yl}ben zon itr ile Hyd r och lor id e (12b).

Methanesulfonic acid 2-[1-(4-cyanobenzyl)-1H-imidazol-5-yl]-

ethyl ester (10) (173 mg, 0.57 mmol), (R)-3-amino-1-benzyl-2-

oxopyrrolidine (synthesized in analogy with compound 4a ) (80

mg, 0.42 mmol), sodium iodide (126 mg, 0.84 mmol), and N,N-

diisopropylethylamine (110 µL, 0.63 mmol) were combined in

dry, degassed DMF (2 mL) and heated to 50 °C, under argon,

for 18 h. The reaction was quenched with saturated aqueous

NaHCO₃(2 mL) and then concentrated under reduced pres-

sure. The residue was partitioned between saturated aqueous

NaHCO₃(5 mL) and CH₂Cl₂(5 mL). The aqueous layer was

extracted further with CH₂Cl₂(3 × 5 mL). The combined

organic extracts were dried over MgSO₄, filtered, and concen-

trated in vacuo. The crude product was partially purified by

flash column chromatography on silica, eluting with a gradient

of CHCl_3-3% to 4% MeOH-0.3% to 0.4% NH₄OH and then

further purified by preparative TLC, eluting with CHCl_3-6%

MeOH-0.6% NH₄OH, to yield the desired product, which was

converted to the hydrochloride salt by treatment with aqueous

HCl in acetonitrile (43 mg, 22%): ¹H NMR (CD₃OD) δ 9.10

(1H, d, J ) 1.5 Hz), 7.83 (2H, d, J ) 8.6 Hz), 7.68 (1H, d, J )

1.3 Hz), 7.51 (2H, d, J ) 8.4 Hz), 7.38-7.28 (5H, m), 5.66 (2H,

s), 4.53 (1H, d, J ) 14.8 Hz), 4.48 (1H, d, J ) 14.6 Hz), 4.24

(S)-4-{5-[(2-Oxo-1-p h en ylp yr r olid in -3-yla m in o)m eth yl]-

im id a zol-1-ylm eth yl}ben zon itr ile Hyd r och lor id e (5a ).

(S)-3-Amino-2-oxo-1-phenylpyrrolidine (4a) (100 mg, 0.57 mmol),

1-(4-cyanobenzyl)-5-imidazolecarboxaldehyde¹¹(132 mg, 0.62

mmol), and acetic acid (98 µL, 1.71 mmol) were stirred in

MeOH (2 mL) for 1 h, then NaCNBH₃(47 mg, 0.74 mmol) was

added. Stirring was continued for 1 h, then the reaction was

2946 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18

Bell et al.

(1H, dd, J ) 10.2, 8.9 Hz), 3.65 (1H, m), 3.47-3.35 (3H, m),

3.09 (2H, dd, J ) 8.4, 7.3 Hz), 2.55 (1H, m), 2.09 (1H, m); MS

(FAB) m/z ) 400 (M⁺+ H); HPLC purity ) 90.2% (method A,

215 nm); ee ) 90.4%. Anal. (C₂₄H₂₅N₅O‚2.5HCl‚0.65CH₃CN‚

0.85C₆H₅CH₃) C, H, N.

to give the titled compound, which was of sufficient purity for

use in the next step: ¹H NMR (CD₃OD) δ 3.76 (1H, m), 3.62

(1H, m), 3.55-3.38 (2H, m), 3.29 (1H, m), 2.24 (1H, m), 1.93

(1H, m), 1.46 (9H, s).

(S )-3-{[1-(4-Cya n ob e n zyl)-1H -im id a zol-5-ylm e t h yl]-

a m in o}p yr r olid in e-1-ca r b oxylic Acid t er t -Bu t yl E st er

(22). (S)-3-Amino-1-(tert-butoxycarbonyl)pyrrolidine (1.18 g,

6.34 mmol) (21), 1-(4-cyanobenzyl)-5-imidazolecarboxalde-

hyde¹¹(1.41 g, 6.68 mmol), and acetic acid (0.363 mL, 6.34

mmol) were stirred in MeOH (35 mL) for 30 min, then

NaCNBH₃(0.44 g, 7.00 mmol) was added. Stirring was

continued for 18 h, then the reaction was quenched with 10%

aqueous citric acid (5 mL), followed by saturated aqueous Na₂-

CO₃(50 mL), and the mixture was extracted with CH₂Cl₂(2

× 100 mL). The combined organic extracts were dried over

Na₂SO₄, filtered, and concentrated in vacuo. The crude product

was purified by flash column chromatography on silica, eluting

with a gradient of CH₂Cl_2-1% to 7% MeOH-0.5% NH₄OH, to

yield the desired product as a white solid (1.96 g, 81% over 3

steps): ¹H NMR (CDCl₃) δ 7.64 (2H, d, J ) 8.4 Hz), 7.52 (1H,

s), 7.16 (2H, d, J ) 7.9 Hz), 6.99 (1H, s), 5.34 (2H, m), 3.63-

2.94 (7H, m), 1.97 (1H, m), 1.59 (1H, m), 1.46 (9H, s).

(S )-3-{[1-(4-Cya n ob e n zyl)-1H -im id a zol-5-ylm e t h yl]-

a m in o}p yr r olid in e Hyd r och lor id e (23). A solution of (S)-

3-{[1-(4-cyanobenzyl)-1H-imidazol-5-ylmethyl]amino}pyrrolidine-

1-carboxylic acid tert-butyl ester (22) (1.92 g, 5.03 mmol) in

EtOAc (100 mL) at 0 °C was saturated with HCl(g). After 15

min, the mixture was concentrated in vacuo to yield the amine

hydrochloride as a white solid (1.97 g, 100%): ¹H NMR (CD₃-

OD) δ 9.02 (1H, d, J ) 1.3 Hz), 8.01 (1H, s), 7.84 (2H, d, J )

8.6 Hz), 7.58 (2H, d, J ) 8.2 Hz), 5.83 (2H, s), 4.42 (2H, s),

4.22 (1H, m), 3.76-3.65 (3H, m), 3.42 (1H, dt, J ) 11.8, 7.7

Hz), 2.55 (1H, m), 2.41 (1H, m).

(S)-4-(5-{[1-(Na p h t h a len e-1-ca r bon yl)p yr r olid in -3-yl-

a m in o]m eth yl}im id a zol-1-ylm eth yl)ben zon itr ile Hyd r o-

ch lor id e (25). (S)-3-{[1-(4-Cyanobenzyl)-1H-imidazol-5-ylm-

ethyl]-amino}pyrrolidine (23) (40 mg, 0.143 mmol), 1-naphthoic

acid (27 mg, 0.157 mmol), EDC (30 mg, 0.157 mmol), 1-hy-

droxybenzotriazole hydrate (24 mg, 0.157 mmol), and N,N-

diisopropylethylamine (27 µL, 0.157 mmol) were combined in

DMF (0.5 mL), and the mixture was stirred at ambient

temperature for 18 h. The solvent was removed under reduced

pressure and the residue was partitioned between 10% aque-

ous citric acid (1 mL) and CHCl₃(2 mL). The organic layer

was discarded, and the aqueous layer was basified by addition

of saturated aqueous Na₂CO₃(1.4 mL) and then extracted with

CHCl₃(2 × 2 mL). The combined organic extracts were dried

over MgSO₄, filtered, and concentrated in vacuo. The crude

product was purified by flash column chromatography on silica,

eluting with CH₂Cl_2-3% MeOH-0.3% NH₄OH to yield the

titled product, which was converted to the hydrochloride salt

by treatment with aqueous HCl in acetonitrile: ¹H NMR (CD₃-

OD) δ 9.06 (1H, rotamer A, d, J ) 1.3 Hz), 9.00 (1H, rotamer

B, d, J ) 1.3 Hz), 8.02-7.85 (4H, rotamers A and B, m), 7.85

(2H, rotamer A, d, J ) 8.4 Hz), 7.73 (2H, rotamer B, d, J )

8.2 Hz), 7.62-7.54 (6H, rotamer A and 4H, rotamer B, m), 7.42

(2H, rotamer B, d, J ) 8.0 Hz), 5.83 (2H, rotamer A, s), 5.67

(2H, rotamer B, s), 4.37-4.00 (4H, rotamers A and B, m),

3.91-3.84 (1H, rotamer A, m), 3.69-3.65 (1H, rotamer B, m),

3.56-3.27 (2H, rotamers A and B, m), 2.61-2.25 (2H, rotamers

A and B, m). The free base was treated with HCl in EtOAc to

afford the hydrochloride salt as a pale solid: MS (electrospray)

m/z ) 436 (M⁺+ H); HPLC purity ) 99.5% (method B, 215

nm). Anal. (C₂₇H₂₅N₅O‚2.4HCl‚2.3H₂O) C, H, N.

(S)-4-(5-{[2-Oxo-1-(2-p yr id yl)p yr r olid in -3-yla m in o]-

m eth yl)im id a zol-1-ylm eth yl}ben zon itr ile Hyd r och lor id e

(16). (S)-3-Am in o-2-oxo-1-(2-p yr id yl)p yr r olid in e Dih y-

d r och lor id e. A solution of (S)-3-(tert-butoxycarbonylamino)-

2-oxo-1-(2-pyridyl)pyrrolidine¹⁷(65 mg, 0.23 mmol) in EtOAc

(5 mL) at 0 °C was saturated with HCl(g). After 15 min, the

mixture was concentrated in vacuo to yield the amine hydro-

chloride as a white solid (59 mg, 100%): ¹H NMR (CD₃OD) δ

8.43 (1H, ddd, J ) 5.1, 1.9, 0.8 Hz), 8.29 (1H, dt, J ) 8.6, 0.9

Hz), 7.96 (1H, ddd, J ) 8.5, 7.4, 1.8 Hz), 7.29 (1H, ddd, J )

7.3, 5.1, 1.1 Hz), 4.37 (1H, dd, J ) 11.0, 8.8 Hz), 4.33 (1H,

ddd, J ) 10.5, 9.2, 1.1 Hz), 3.82 (1H, td, J ) 10.5, 6.7 Hz),

2.70 (1H, m), 2.16 (1H, m); ee > 99%, as determined by

synthesis of the corresponding Mosher amide diastereomers

and ¹H NMR analysis.¹⁷

(S)-4-(5-{[2-Oxo-1-(2-p yr id yl)p yr r olid in -3-yla m in o]-

m eth yl)im id a zol-1-ylm eth yl}ben zon itr ile Hyd r och lor id e

(16). (S)-3-Amino-2-oxo-1-(2-pyridyl)pyrrolidine dihydrochlo-

ride (58 mg, 0.23 mmol) and 1-(4-cyanobenzyl)-5-imidazole-

carboxaldehyde¹¹(56 mg, 0.27 mmol) were combined in MeOH

(1 mL), and the solution was adjusted to ca. pH ≈ 5 (as judged

by wetted pH indicator paper) by addition of N,N-diisopropy-

lethylamine and then stirred at ambient temperature for 30

min. NaCNBH₃(17 mg, 0.27 mmol) was added and the solution

was adjusted to pH ≈ 5 by addition of acetic acid. Stirring was

continued for 18 h, then the reaction was quenched with a few

drops of 10% aqueous citric acid, and the mixture was stirred

for 10 min. Saturated aqueous Na₂CO₃(5 mL) was added and

the mixture was extracted with CH₂Cl₂(4 × 20 mL) and then

EtOAc (2 × 20 mL). The combined organic extracts were dried

over Na₂SO₄, filtered, and concentrated in vacuo. The crude

material was purified by flash column chromatography on

silica, eluting with a gradient of CH₂Cl_2-1% to 6% MeOH-

0.25% NH₄OH, to yield the desired product: ¹H NMR (CDCl₃)

δ 8.38-8.34 (2H, m), 7.71 (1H, ddd, J ) 8.6, 7.3, 1.8 Hz), 7.62

(2H, d, J ) 8.2 Hz), 7.54 (1H, d, J ) 0.9 Hz), 7.19 (2H, d, J )

8.6 Hz), 7.09-7.04 (2H, m), 5.44 (1H, d, J ) 16.5 Hz), 5.39

(1H, d, J ) 16.5 Hz), 4.16 (1H, ddd, J ) 11.2, 9.0, 1.9 Hz),

3.86 (1H, d, J ) 13.7 Hz), 3.78 (1H, ddd, J ) 11.2, 9.9, 6.8

Hz), 3.72 (1H, d, J ) 13.7 Hz), 3.58 (1H, dd, J ) 10.2, 8.0 Hz),

2.27 (1H, m), 1.70 (1H, m). The free base was treated with

HCl in EtOAc to afford the hydrochloride salt as a white solid

(95 mg, 92%): MS (FAB) m/z ) 373 (M⁺+ H); HPLC purity )

98.0% (method B, 215 nm). Anal. (C₂₁H₂₀N₆O‚2.5HCl‚0.4H₂O‚

0.1EtOAc) C, H, N.

(S)-4-(5-{[1-(Na p h t h a len e-1-ca r b on yl)p yr r olid in -3-yl-

a m in o]m eth yl}im id a zol-1-ylm eth yl)ben zon itr ile Hyd r o-

ch lor id e (25). (S)-1-(ter t-Bu toxyca r bon yl)-3-(tr iflu or o-

a cet a m id o)p yr r olid in e. To a stirred solution of (S)-3-

(trifluoroacetamido)pyrrolidine hydrochloride (20) (TCI) (2.08

g, 9.5 mmol) and N,N-diisopropylethylamine (1.82 mL, 10.5

mmol) in CH₂Cl₂(25 mL) was added di-tert-butyl dicarbonate

(2.08 g, 9.5 mmol) in CH₂Cl₂(25 mL). The reaction mixture

was stirred at ambient temperature for 2 h and then parti-

tioned between saturated aqueous Na₂CO₃(30 mL) and CH₂-

Cl₂(50 mL). The aqueous layer was extracted further with

CH₂Cl₂(50 mL). The combined organic extracts were dried over

Na₂SO₄, filtered, and concentrated in vacuo to give a crude

product that was sufficiently pure for use in the next step:

¹H NMR (CDCl₃) δ 6.46 (1H, br d), 4.50 (1H, m), 3.67 (1H, dd,

J ) 11.7, 6.2 Hz), 3.56-3.22 (3H, m), 2.23 (1H, m), 1.95 (1H,

br s), 1.47 (9H, s).

(S)-3-Am in o-1-(ter t-bu toxyca r bon yl)p yr r olid in e (21).

To a stirred solution of (S)-1-(tert-butoxycarbonyl)-3-(trifluo-

roacetamido)pyrrolidine (2.80 g, 9.5 mmol) in THF (80 mL)

and H₂O (10 mL) was added 1.0 N aqueous lithium hydroxide

(10.5 mL, 10.5 mmol), and the resulting mixture was stirred

at ambient temperature for 18 h and then adjusted to pH 7

with 1.0 N aqueous HCl and concentrated to dryness in vacuo

(S )-4-(5-{[(1-B e n zy l-2-o x o p y r r o lid in -3-y l)(3-{t er t -

bu toxyca r bon yla m in o}p r op yl)a m in o]m eth yl}im id a zol-

1-ylm eth yl)ben zon itr ile (44). (S)-4-{5-[(1-Benzyl-2-oxopyr-

rolidin-3-ylamino)methyl]imidazol-1-ylmethyl}benz onitrile (5b)

(90 mg, 0.23 mmol), 3-(tert-butoxycarbonylamino)propional-

dehyde⁴²(81 mg, 0.47 mmol), and acetic acid (27 µL, 0.47

mmol) were stirred in MeOH (1 mL) for 1 h, then NaCNBH₃

(18 mg, 0.29 mmol) was added. Stirring was continued for 18

h, then most of the MeOH was removed under reduced

pressure. The residue was partitioned between saturated

A 3-Aminopyrrolidinone Farnesyltransferase Inhibitor

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2947

aqueous NaHCO₃(3 mL) and CH₂Cl₂(5 mL). The aqueous

layer was extracted further with CH₂Cl₂(2 × 5 mL). The

combined organic extracts were dried over MgSO₄, filtered, and

concentrated in vacuo. The crude product was purified by flash

column chromatography on silica, eluting with CH₂Cl_2-2%

MeOH-0.2% NH₄OH, to yield the desired product as a white

solid (92 mg, 73%): ¹H NMR (CD₃OD) δ 7.76 (1H, d, J ) 0.9

Hz), 7.69 (2H, d, J ) 8.4 Hz), 7.42-7.34 (5H, m), 7.17 (2H, d,

J ) 8.2 Hz), 7.00 (1H, s), 6.50 (1H, br m), 5.71 (1H, d, J )

16.7 Hz), 5.55 (1H, d, J ) 16.7 Hz), 4.50 (1H, d, J ) 14.8 Hz),

4.31 (1H, d, J ) 14.7 Hz), 3.72 (1H, t, J ) 9.2 Hz), 3.63 (1H,

d, J ) 14.1 Hz), 3.58 (2H, t, J ) 6.4 Hz), 3.54 (1H, d, J ) 14.1

Hz), 3.22-3.15 (2H, m), 2.54 (1H, m), 2.43 (1H, m), 2.04 (1H,

m), 1.92 (1H, m), 1.67 (2H, m), 1.43 (9H, s). The free base was

treated with AcOH to afford the acetate salt as a gummy

solid: MS (FAB) m/z ) 543 (M⁺+ H); HPLC purity ) 98.3%

(method B, 215 nm). Anal. (C₃₁H₃₈N₆O₃‚0.95AcOH‚1.2H₂O) C,

H, N.

(1H, d, J ) 13.9 Hz), 3.61 (1H, dd, J ) 8.4, 1.7 Hz), 3.56 (1H,

d, J ) 13.7 Hz), 3.42 (1H, m), 3.36 (1H, ddd, J ) 11.5, 8.5, 3.1

Hz), 3.24-3.11 (2H, m), 2.66 (1H, m), 2.55 (1H, dt, J ) 14.1,

3.8 Hz), 2.10 (1H, m), 1.84 (1H, m).

(S)-2-{[1-(3-Ch lor ob en zyl)-2-oxop yr r olid in -3-yl][3-(4-

cya n oben zyl)im id a zol-4-ylm eth yl]a m in o}eth a n a l (51). To

a solution of oxalyl chloride (35 µL, 0.39 mmol) in anhydrous

CH₂Cl₂(2 mL) at -78 °C was added dimethyl sulfoxide (35

µL, 0.78 mmol) over 5 min. After stirring for 10 min, a solution

of alcohol 50 (140 mg, 0.303 mmol) in CH₂Cl₂(2 mL) was added

over 2 min. The resulting mixture was stirred at -78 °C for

15 min, then triethylamine (210 µL, 1.51 mmol) was added,

and the reaction mixture was allowed to warm to ambient

temperature over 1 h, after which saturated aqueous NH₄Cl

(5 mL) was added. The mixture was poured into saturated

aqueous NaHCO₃(20 mL) and extracted with CH₂Cl₂(3 × 50

mL). The combined organic extracts were dried over Na₂SO₄,

filtered, and concentrated in vacuo to provide the crude product

in sufficient purity for use in the next step: ¹H NMR (CDCl₃)

δ 9.40 (1H, s), 7.61 (2H, d, J ) 8.4 Hz), 7.56 (1H, s), 7.28-

7.26 (4H, m), 7.24 (1H, s), 7.05 (1H, m), 7.03 (1H, s), 5.68 (1H,

d, J ) 16.7 Hz), 5.58 (1H, d, J ) 16.7 Hz), 4.41 (1H, d, J )

14.7 Hz), 4.36 (1H, d, J ) 14.8 Hz), 3.79 (1H, d, J ) 13.7 Hz),

3.73 (1H, d, J ) 13.7 Hz), 3.67 (1H, t, J ) 9.4 Hz), 3.49 (1H,

d, J ) 18.5 Hz), 3.29 (1H, dd, J ) 18.4, 0.8 Hz), 3.20-3.08

(2H, m), 2.06 (1H, m), 1.81 (1H, m).

(S)-4-(5-{[(3-Aminopropyl)(1-benzyl-2-oxopyrrolidin-3-yl)-

a m in o]m eth yl}im id a zol-1-ylm eth yl)ben zon itr ile Hyd r o-

chloride (45). A solution (S)-4-(5-{[(1-benzyl-2-oxopyrrolidin-3-yl)-

(3-{tert-butoxycarbonylamino}propyl)amino]methyl}imidazol-1-

ylmethyl)benzonitrile (44) (625 mg, 1.15 mmol) in EtOAc (25

mL) at 0 °C was saturated with HCl(g). After 15 min, the

mixture was concentrated in vacuo to yield the amine hydro-

chloride as a white solid (642 mg, 100%): ¹H NMR (CD₃OD) δ

9.05 (1H, d, J ) 1.5 Hz), 7.79 (2H, d, J ) 8.4 Hz), 7.75 (1H, s),

7.49 (2H, d, J ) 8.4 Hz), 7.36-7.27 (3H, m), 7.21 (2H, d, J )

8.1 Hz), 5.82 (1H, d, J ) 16.3 Hz), 5.74 (1H, d, J ) 16.1 Hz),

4.49 (1H, d, J ) 14.8 Hz), 4.43 (1H, d, J ) 14.7 Hz), 3.95-

3.78 (3H, m), 3.23 (2H, m), 2.97 (2H, t, J ) 6.4 Hz), 2.77-2.60

(2H, m), 2.13-1.75 (4H, m); MS (FAB) m/z ) 443 (M⁺+ H);

HPLC purity ) 100% (method B, 215 nm). Anal. (C₂₆H₃₀N₆O‚

2.5HCl‚1.45H₂O) C, H, N.

(S)-4-[5-({[1-(3-Ch lor ob en zyl)-2-oxop yr r olid in -3-yl]-

(2-morpholin-4-ylethyl)amino} methyl)imidazol-1-ylmethyl]-

ben zon itr ile Hyd r och lor id e (52). (S)-4-(5-{[(2-ter t-Bu tyl-

dimethylsilyloxyethyl)[1-(3-chlorobenzyl)-2-oxopyrrolidin-3-yl]-

a m in o]m eth yl}im id a zol-1-ylm eth yl)ben zon itr ile. A solu-

tion of amine 5h (203 mg, 0.48 mmol), 2-tert-butyldimethyl-

silyloxyethanal¹⁹(126 mg, 0.73 mmol), and AcOH (83 µL, 1.45

mmol) in MeOH (2 mL) was stirred at ambient temperature

for 30 min. NaCNBH₃(61 mg, 0.0967 mmol) was added in one

portion. After 15 h, the reaction was partitioned between

saturated aqueous NaHCO₃and CH₂Cl₂and extracted with

CH₂Cl₂(3 × 50 mL). The organic extracts were combined, dried

with Na₂SO₄, filtered, and concentrated in vacuo. The crude

product was purified by flash column chromatography on silica,

eluting with a gradient of CH₂Cl_2-1% to 3% MeOH-0.1% to

0.3% NH₄OH, to yield the titled product (213 mg, 76%): ¹H

NMR (CDCl₃) δ 7.62 (2H, d, J ) 8.2 Hz), 7.55 (1H, s), 7.29-

7.23 (4H, m), 7.17 (1H, s), 7.06 (1H, m), 7.03 (1H, s), 5.65 (1H,

d, J ) 16.5 Hz), 5.57 (1H, d, J ) 16.7 Hz), 4.44 (1H, d, J )

14.8 Hz), 4.35 (1H, d, J ) 14.7 Hz), 3.79 (1H, m), 3.78 (1H, d,

J ) 9.2 Hz), 3.66 (1H, d, J ) 13.7 Hz), 3.47 (2H, m), 3.14 (2H,

m), 2.68 (1H, dt, J ) 13.7, 5.5 Hz), 2.52 (1H, dt, J ) 13.7, 6.6

Hz), 2.07 (1H, m), 1.91 (1H, m), 1.27 (9H, s), 0.86 (6H, s).

(S)-4-[5-({[1-(3-Ch lor oben zyl)-2-oxop yr r olid in -3-yl](2-

h ydr oxyeth yl)a m in o}m eth yl)im id a zol-1-ylm eth yl]ben zo-

n itr ile (50). To a stirred solution of (S)-4-(5-{[(2-tert-butyldi-

methylsilyloxyethyl)[1-(3-chlorobenzyl)-2-oxopyrrolidin-3-

yl]amino]methyl}imidazol-1-ylmethyl)benzonitrile (175 mg,

0.303 mmol) in THF (5 mL) was added TBAF (0.46 mL of a 1

M solution in THF, 0.46 mmol). After 10 min, the reaction was

partitioned between H₂O and CH₂Cl₂and extracted with CH₂-

Cl₂(3 × 30 mL). The organic extracts were combined, dried

with Na₂SO₄, filtered, and concentrated in vacuo. The crude

product was purified by flash column chromatography on silica,

eluting with a gradient of CH₂Cl_2-1% to 6% MeOH-0.1% to

0.6% NH₄OH, to yield the titled product (140 mg, 100%): ¹H

NMR (CDCl₃) δ 7.63 (2H, d, J ) 8.4 Hz), 7.58 (1H, s), 7.29-25

(3H, m), 7.25 (1H, s), 7.18 (1H, s), 7.07 (1H, m), 7.02 (1H, s),

5.70 (1H, d, J ) 16.8 Hz), 5.43 (1H, d, J ) 16.7 Hz), 4.44 (1H,

d, J ) 14.8 Hz), 4.86 (1H, d, J ) 14.7 Hz), 3.98 (1H, br s), 3.63

(S)-4-(5-{[[1-(3-Ch lor ob en zyl)-2-oxop yr r olid in -3-yl]-

(2-morpholin-4-ylethyl)amino]methyl}imidazol-1-ylmethyl)-

ben zon itr ile Hyd r och lor id e (52). A solution of aldehyde 51

(46 mg, 0.10 mmol), morpholine (10 µL, 0.12 mmol), and AcOH

(17 µL, 0.30 mmol) in MeOH (2 mL) was stirred at ambient

temperature for 30 min. NaCNBH3 (13 mg, 0.20 mmol) was

added and the reaction mixture was stirred for 48 h and then

partitioned between saturated aqueous NaHCO3 (10 mL) and

CH2Cl2 (20 mL). The aqueous layer was extracted further with

CH2Cl2 (2 × 20 mL), and the organic extracts were combined,

dried with Na2SO4, filtered, and concentrated in vacuo. The

crude product was purified via preparative HPLC (method A),

and the product-containing fractions were lyophilized. The

resulting trifluoroacetate salt was free-based and treated with

HCl in EtOAc to afford the titled product (13 mg, 25%): 1H

NMR (CD3OD) δ 9.04 (1H, s), 7.81 (2H, d, J ) 8.1 Hz), 7.74

(1H, s), 7.49 (2H, d, J ) 8.2 Hz), 7.38-7.30 (3H, m), 7.27 (1H,

s), 5.73 (1H, d, J ) 16.5 Hz), 5.68 (1H, d, J ) 16.1 Hz), 4.57

(1H, d, J ) 14.8 Hz), 4.37 (1H, d, J ) 14.8 Hz), 4.05-3.75 (8H,

m), 3.45-3.15 (7H, m), 3.15-2.90 (2H, m), 2.18 (1H, m), 1.95

(1H, m); MS (FAB) m/z ) 533 (M+ + H); HPLC purity ) 98.7%

(method B, 215 nm). Anal. (C29H33ClN6O2‚3HCl‚0.1CH2Cl2‚

0.3 EtOAc) C, H, N.

(S)-4-(5-{[[1-(3-Ch lor oben zyl)-2-oxop yr r olid in -3-yl](2-

p ip er a zin -1-yleth yl)a m in o]m eth yl}im id a zol-1-ylm eth yl)-

ben zon itr ile Hyd r och lor id e (54). A solution of aldehyde 51

(46 mg, 0.10 mmol), N-tert-butoxycarbonylpiperazine (22 mg,

0.12 mmol), and AcOH (17 µL, 0.30 mmol) in MeOH (2 mL)

was stirred at ambient temperature for 30 min. NaCNBH₃(13

mg, 0.20 mmol) was added and the reaction mixture was

stirred for 48 h and then partitioned between saturated

aqueous NaHCO₃(10 mL) and CH₂Cl₂(20 mL). The aqueous

layer was extracted further with CH₂Cl₂(2 × 20 mL), and the

organic extracts were combined, dried with Na₂SO₄, filtered,

and concentrated in vacuo. The crude product was purified via

preparative HPLC (method A) and the product-containing

fractions were lyophilized. The resulting trifluoroacetate salt

was free-based and treated with HCl in EtOAc to afford the

titled product (20 mg, 30%): ¹H NMR (CD₃OD) δ 9.15 (1H, s),

7.81 (2H, d, J ) 8.4 Hz), 7.75 (1H,s), 7.51 (2H, d, J ) 8.6 Hz),

7.37-7.30 (2H, m), 7.33 (1H, m), 7.17 (1H, m), 5.77 (1H, d, J

) 16.1 Hz), 5.71 (1H, d, J ) 15.9 Hz), 4.58 (1H, d, J ) 15.0

Hz), 4.37 (1H, d, J ) 14.7 Hz), 3.97 (1H, d, J ) 15.6 Hz), 3.95

(1H, m), 3.86 (1H, d, J ) 15.4 Hz), 3.65-3.45 (8H, m), 3.40-

2.90 (6H, m), 2.15 (1H, m), 1.95 (1H, m); MS (FAB) m/z ) 532

(M⁺+ H); HPLC purity ) 95.0% (method B, 215 nm). Anal.

(C₂₉H₃₄ClN₇O‚4HCl‚0.95H₂O) C, H, N.

2948 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18

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J M010156P

Article Doi

Design and biological activity of (S)-4-(5-{[1-(3-Chlorobenzyl)-2-oxopyrrolidin-3-ylamino]methyl}imidazol-1- ylmethyl)benzonitrile, a 3-aminopyrrolidinone farnesyltransferase inhibitor with excellent cell potency

DOI: 10.1021/jm010156p

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