Probing the hydrophobic pocket of farnesyltransferase: Aromatic substitution of CAAX peptidomimetics leads to highly potent inhibitors

Article abstract of DOI:10.1016/S0968-0896(99)00252-7

Cysteine farnesylation at the carboxylate terminal tetrapeptide CAAX of Ras protein is catalyzed by farnesyltransferase. This lipid modification is necessary for regulatory function of both normal and oncogenic Ras. The high frequency of Ras mutation in human cancers has prompted an intensive study on finding ways of controlling oncogenic Ras function. Inhibition of farnesyltransferase is among the most sought after targets for cancer chemotherapy. We report here the design, synthesis and biological characterization of a series of peptidomimetics as farnesyltransferase inhibitors. These compounds are extremely potent towards farnesyltransferase with IC₅₀ values ranging from subnanomolar to low nanomolar concentrations. They have a high selectivity for farnesyltransferase over the closely related geranylgeranyltransferase-I. Structure-activity relationship studies demonstrated that a properly positioned hydrophobic group significantly enhanced inhibition potency, reflecting an improved complementarity to the large hydrophobic pocket in the CAAX binding site. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00252-7

Bioorganic & Medicinal Chemistry 7 (1999) 3011±3024
Probing the Hydrophobic Pocket of Farnesyltransferase:
Aromatic Substitution of CAAX Peptidomimetics Leads to
Highly Potent Inhibitors
Yimin Qian, a Juan Jose Marugan, c Renae D. Fossum, c Andreas Vogt, d
Said M. Sebti b,* and Andrew D. Hamilton a,*
aDepartment of Chemistry, Yale University, Post Oce Box 208107, New Haven, CT 06511, USA
bDrug Discovery Program, H. Lee Mott Cancer Center and Research Institute, Department of Biochemistry and Molecular Biology,
University of South Florida, Tampa, FL 33612, USA
cDepartment of Chemistry, School of Arts and Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA
dDepartment of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15260, USA
Received 21 June 1999; accepted 23 August 1999
AbstractÐCysteine farnesylation at the carboxylate terminal tetrapeptide CAAX of Ras protein is catalyzed by farnesyltransferase.
This lipid modi®cation is necessary for regulatory function of both normal and oncogenic Ras. The high frequency of Ras mutation
in human cancers has prompted an intensive study on ®nding ways of controlling oncogenic Ras function. Inhibition of farnesyl-
transferase is among the most sought after targets for cancer chemotherapy. We report here the design, synthesis and biological
characterization of a series of peptidomimetics as farnesyltransferase inhibitors. These compounds are extremely potent towards
farnesyltransferase with IC50 values ranging from subnanomolar to low nanomolar concentrations. They have a high selectivity for
farnesyltransferase over the closely related geranylgeranyltransferase-I. Structure±activity relationship studies demonstrated that a
properly positioned hydrophobic group signi®cantly enhanced inhibition potency, re¯ecting an improved complementarity to the
large hydrophobic pocket in the CAAX binding site. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
restricted to leucine, isoleucine, or, to a less extent,
phenylalanine.6 Unlike FTase and GGTase-I, GGTase-
II only modi®es proteins with carboxyl terminal
sequences CXC, CCXX, or XXCC and requires a Rab
escort protein to function.7,8
Protein isoprenylation plays a key role in protein sub-
cellular localization, protein±protein interaction, and
signal transduction. In this process C15 farnesyl and C20
geranylgeranyl groups are covalently linked to the
cysteine residue near the carboxyl terminus through a
thiol alkylation reaction that is catalyzed by the iso-
prenyltransferase enzymes.1 Three di€erent iso-
prenyltransferases have been identi®ed, farnesyltrans-
ferase (FTase), type-I geranylgeranyltransferase
(GGTase-I), and type-II geranylgeranyltransferase
(GGTase-II).2±4 FTase catalyzes farnesylation of pro-
teins with a carboxyl terminal sequence CAAX, where
C is cysteine, A is an aliphatic amino acid, X is methio-
nine, serine, alanine, glutamine, or cysteine.5 GGTase-I
catalyzes geranylgeranylation of proteins with the same
carboxyl terminal sequence, except that the X residue is
Among the three isoprenyltransferases, FTase has been
most intensively studied, owing to its involvement in the
post-translational modi®cation of the low molecular
weight GTPase, Ras.9 Ras cycles between GDP (inac-
tive) and GTP (active) states to transduce growth factor
signals from cell surface receptor tyrosine kinases to the
nucleus.10 Single point mutations of Ras abolish its
GTPase activity and lead to uncontrolled signaling.11
These mutations to Ras are found in over 30% of
all human tumors, particularly pancreatic and colon
cancers.12,13
The regulatory function of Ras is dependent on its
association with the plasma membrane.14 It is now
recognized that Ras undergoes a three step post-
translational modi®cation to convert its cytosolic form
to a membrane bound form. The ®rst, obligatory and
Key words: Farnesyltransferase inhibitors; peptidomimetics; hydro-
phobic pocket; anticancer targets.
* Corresponding authors. Tel.: +1-203-432-5570; fax: +1-203-432-
3221.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00252-7

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Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
sucient step in these modi®cations is the attachment of
a farnesyl group to the cysteine residue of the CAAX
sequence at the carboxyl terminal.15 After farnesylation,
the tripeptide AAX is cleaved by a peptidase and the
resulting carboxylic acid is methylated by a methyl-
transferase.16 Site directed mutagenesis has shown that
farnesylation alone is necessary and sucient for Ras
transforming activity.17 Thus, the inhibition of FTase
provides a new target for potential anticancer drug
design.18±20
Design and Synthesis
We initiated this project with the goal of identifying
peptidomimetics and nonpeptides as FTase inhibitors.
We ®rst replaced the central dipeptide in CAAX with a
variety of spacers including 3-aminomethylbenzoic acid
which could replace the Val-Ile dipeptide in CVIM
without signi®cant loss of inhibition activity.38,39 Fur-
ther rigidi®cation was achieved by using a 4-amino-
benzoic acid spacer between the methionine and
cysteine derivatives. The synthesis of this parent pepti-
domimetic included coupling (l)-methionine methyl
ester with N-Boc-aminobenzoic acid, deprotection and
amino group coupling with N-Boc-S-trityl-(l)-cysteine
by using EDCI or isobutylchloroformate. A further
modi®cation was the replacement of the cysteine amide
bond with a methyleneamino group using a reductive
amination of the corresponding amine with N-Boc-S-
trityl-(l)-cysteinal.31 Scheme 1 describes the synthesis of
compounds 1 and 2.
FTase is a heterodimeric metalloenzyme composed of a
and b subunits with molecular weights of 49 and
46 kDa, respectively.21,22 Both farnesylpyrophosphate
(FPP) and Ras bind to the b subunit.23,24 However,
FTase does not require the full length of Ras for the
farnesylation reaction. Small peptides, as short as four
amino acids with the consensus CAAX sequence can be
farnesylated with kinetic parameters similar to that of
Ras protein.5,25 A recent X-ray structure of FTase with
tetrapeptide Ac-CVIM(Se) and an FPP analogue bound
into the active site shows that the active site is at the
interface of the two subunits.26,27 The tetrapeptide is in
an extended conformation with the Cys-SH bound to
the catalytically important zinc ion (Fig. 1). Impor-
tantly for the present paper, a single hydrogen bond is
formed to the C-terminal amide and a large pocket
formed by two Trp and one Tyr residues provides a
binding cavity for the aliphatic side chain of the penul-
timate residue.
While the rigidity of the 4-aminobenzoic acid spacer
signi®cantly reduces the possible conformations of the
corresponding Val-Ile dipeptide, the simple aromatic
ring does not reach the volume of the side chains of the
Val-Ile dipeptide. In order to probe the size and com-
plementarity of the hydrophobic pocket in FTase, we
have attached di€erent groups to the aromatic ring.
Figure 2 shows the structures of inhibitors with di€er-
ently designed hydrophobic substituents.
The discovery that CAAX tetrapeptides can competi-
tively inhibit FTase has played a key role in the design
of peptidomimetics as FTase inhibitors. So far, several
di€erent approaches have been taken in the search for
in vitro active inhibitors of FTase. These include the
screening of natural products and synthetic libraries, the
design of FPP analogues, bisubstrate transition state
analogues, and most importantly, CAAX peptidomi-
metics.28,29 We and others have previously reported
peptidomimetics and pseudopeptides as FTase inhibi-
tors.30±41 In this paper, we report that systematic
hydrophobic functionalization of our peptidomimetics
to ®t the hydrophobic pocket in the FTase active site
has led to the discovery of FTase inhibitors with sub-
nanomolar inhibition potency.
The synthesis of compounds 3 and 4 was similar to that
of 1 except 4-amino-3-methylbenzoic acid and 4-amino-
3-methoxybenzoic acid, respectively, were used. Com-
pound 7 was prepared from 4-amino-1-naphthoic acid,
itself prepared from the hydrolysis of 4-amino-1-naph-
thalenecarbonitrile. The spacers in compounds 5 and 6
were prepared from the coupling of 4-nitro-2-bromoto-
luene with phenyl or xylylboronic acid under Suzuki
conditions.42 Oxidation of 4-nitro-2-phenyltoluene with
potassium permanganate provided 4-nitro-2-phenyl-
benzoic acid. The carboxylic acid was coupled with (l)-
methionine methyl ester and the nitro group was
reduced with stannous chloride.43 The resulting amine
was coupled with N-Boc-S-trityl-(l)-cysteine to give a
fully protected precursor which was deprotected and
puri®ed to give the ®nal product.
The structure±activity relationship of compounds 3±7
revealed that a phenyl substitution at the 2-position
signi®cantly enhanced potency for FTase inhibition
(Table 1). We next investigated di€erent aromatic sub-
stitutions at the 2-position of the 4-aminobenzoic acid
spacer. This substitution is expected not only to increase
the hydrophobicity of the molecule, but also to restrict
rotation about the phenyl±CO bond. Figure 3 shows the
structures of compounds 8±13, which also include
pseudopeptide modi®cations of the cysteine residue.
The synthesis of compound 8 (FTI-276) and its methyl
ester (FTI-277) is described in Scheme 2. Compounds
8±13 were puri®ed by reverse phase preparative HPLC
(C18 column, linear gradient from 5% acetonitrile to
Figure 1. Active site contacts to the CAAX substrate for FTase.26,27

Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
3013
Scheme 1. Replacement of dipeptide in CVIM with 4-aminobenzoic acid. Reagents: (a) (1) (Boc)2O, NaOH, (2) (l)-methionine methyl ester; (b) (1)
gaseous HCl in methanol, (2) N-Boc-S-trityl-(l)-cysteine, isobutyl chloroformate, Et3N; (c) (1) gaseous HCl in methanol, (2) N-Boc-S-trityl-(l)-
cysteinal, NaB(CN)H3; (d) (1) aqueous LiOH, THF, (2) gaseous HCl in ether; (e) (1) aqueous LiOH, THF, (2) TFA, Et3SiH.
60% acetonitrile in 0.1% TFA aqueous solution over
30 min). The methyl ester of compound 8 (compound
14, FTI-277) was prepared from deprotection of the
fully protected precursor using mercuric chloride and
hydrogen sul®de. FTI-277 was isolated as a hydrochlo-
ride salt without the requirement of HPLC puri®cation.
Raney nickel and hydrazine in methanol.45 The result-
ing amine was coupled with N-Boc-S-trityl-(l)-cysteinal
to give the fully protected precursor, which was depro-
tected and puri®ed to give the pyridine substituted
inhibitors.
The spacer in 9 and 10 was prepared by oxidation of
4-nitro-2-bromotoluene followed by methylation to form
methyl 4-nitro-2-bromobenzoate. The required 2-thi-
enyl- and 1-naphthylboronic acids were prepared from
the corresponding aryl bromides. Scheme 3 shows the
synthesis of peptidomimetics 9 and 10. The H NMR
spectra of 10 showed two isomers due to slow aryl±aryl
bond rotation on the NMR time scale.
Results and Discussion
The ability of the peptidomimetics to inhibit FTase and
GGTase-I in vitro was investigated by using partially
puri®ed FTase and GGTase-I from human Burkitt
lymphoma (Daudi) cells.40 Enzyme preparations were
incubated with [3H]FPP and recombinant H-Ras-CVLS
(FTase) or [3H]GGPP and H-Ras-CVLL (GGTase-I) in
the presence of di€erent concentrations of inhibitors.
After incubation for 30 min at 37ꢀC, the reaction was
stopped and ®ltered on glass ®ber ®lters to separate free
from incorporated label, as described earlier.40 The
1
Incorporation of a pyridine ring into the aromatic
spacer was accomplished through the coupling of
methyl 4-nitro-2-bromobenzoate with trimethyl-
stannylpyridine (Scheme 4).44 The methyl ester was
hydrolyzed with aqueous lithium hydroxide in methanol
and the corresponding carboxylic acid was coupled with
(l)-methionine methyl ester. The nitro group was
reduced using stannous chloride with the 4-pyridyl
derivative, but the same conditions caused amide bond
cleavage in the 2-pyridyl and 3-pyridyl analogues. For
these compounds reduction was best achieved with
Table 1. Inhibition of FTase and GGTase-I by CAAX peptidomi-
meticsa
Peptidomimetics
IC50 (nM)
‹SEM
FTase
IC50 (nM)
‹SEM
GGTase-I
Selectivity
FTase/GGTase-I
1
2
3
4
5
6
213‹71 (4)
267‹104 (6)
825‹225 (2)
2550‹1250 (2)
4.5‹2.9 (5)
405‹105 (2)
143‹26 (3)
0.61‹0.16 (17)
1.3‹0.2 (2)
1.9‹0.3 (2)
3.0 (1)
1200‹216 (3)
3675‹653 (4)
9000 (1)
50000 (1)
267‹94 (3)
400 (1)
3150‹150 (2)
50‹20 (13)
40 (1)
7.0 (1)
400 (1)
680 (1)
310 (1)
1800‹200 (2)
6
14
11
20
59
1
22
82
31
4
7
8 (FTI-276)
9
10
11
12
133
124
59
28
5.5 (1)
5.3‹2.1 (2)
63‹21 (5)
13
14 (FTI-277)
a
Figure 2. Structures of peptidomimetics with improved hydrophobic
spacer.
Numbers in parenthesis indicate the times of independent enzyme
inhibition assay.

3014
Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
gives a sixfold selectivity for FTase over GGTase-I.46
The methyl ester of 1 inhibited H-Ras processing in
NIH 3T3 cells at 200 mM without a€ecting the
geranylgeranylation of Rap1A at concentrations as
high as 400 mM. Although the potency of 1 is in the
submicromolar range, it became a key starting point for
the design of subnanomolar FTase inhibitors.
Reduction of the cysteine amide to its methyleneamino
isostere has been shown to be tolerated in CAAX
pseudopeptides.31 In our series of compounds reduction
of cysteine amide did not a€ect inhibition (2,
IC50=267 nM) but gave improved selectivity for FTase
over GGTase-I (14-fold for 2). Furthermore, the methyl
ester of 2 inhibited H-Ras farnesylation in NIH 3T3
cells at 25 mM, a 10-fold improvement over the methyl
ester of 1. This result clearly con®rmed that one advan-
tage a€orded by the pseudopeptide modi®cation is to
reduce peptidase cleavage.
Figure 3. Peptidomimetics with aromatic substitution at the 2-position
of 4-aminobenzoic acid.
activity of the inhibitors is reported in Table 1 as IC50
values, the concentration at which FTase or GGTase-I
activity was inhibited by 50%.
Tetrapeptide CVIM from the KB-Ras terminal sequence
can inhibit FTase in vitro with an IC50 value around
165 nM.31 In our FTase inhibition studies, CVIM was
used as a control with a measured IC50 value of 150±
340 nM.39 Our initial proposal was that the
central dipeptide of CAAX might simply play a hydro-
phobic role in binding to the FTase active site. This
postulation was consistent with the observation that in
the CAAX sequence the cysteine residue is absolutely
required, the methionine residue is favored and the
central two residues can tolerate a variety of aliphatic or
aromatic amino acids. The recent X-ray structure of
FTase bound to a tetrapeptide26,27 shows no signi®cant
interaction to the amide bond between the central Val and
Ile residues (Fig. 1). To test this hypothesis, we replaced
the central two amino acids in CVIM with a hydrophobic
4-aminobenzoic acid spacer. This replacement maintains
the FTase inhibition activity (IC50=213nM, Table 1) and
Although 1 and 2 are as potent inhibitors as CVIM, it is
clear that the 4-aminobenzoic acid spacer is a poor
mimic of the large hydrophobic side chains in valine and
isoleucine. Therefore, increasing the size and hydro-
phobicity of the 4-aminobenzoic acid spacer should
allow better occupation of the FTase substrate binding
pocket. Attachment of methyl and methoxy substituents
at the 3-position of the 4-aminobenzoic acid spacer led
to a decrease in inhibition potency (IC50=825 and
2250 nM for 3 and 4, respectively). In contrast, sub-
stitution at the 2-position gave a startling improvement
in activities with phenyl-substituted 5 giving an IC50 of
4.5 nM. This corresponds to an increase in FTase inhi-
bition potency of 50-fold when compared to 1. Com-
pound 5 is sevenfold more potent than CIFM, the most
potent FTase inhibitor in CAAX series. The increased
Scheme 2. Synthesis of peptidomimetic FTI-276 and FTI-277. Reagents: (a) (1) phenylboric acid, Pd(OAC)2, aqueous acetone, (2) KMnO4, aqueous
pyridine; (b) (l)-methionine methyl ester, EDCI, HOBT; (c) (1) SnCl2 hydrate, (2) N-Boc-S-trityl-(l)-cysteinal NaB(CN)H3; (d) (1) LiOH, THF, (2)
TFA, Et3SiH; (e) (1) HgCl2, methanol, (2) H2S, methanol.

Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
3015
Scheme 3. Preparation of peptidomimetic 9 and 10. Reagents: (a) (1) KMnO4, aqueous pyridine, (2) methanol, SOCl2; (b) (1) 2-thienylboric acid or
1-naphthylboric acid, Pd(PPh3)4, DMF; (c) (1) aqueous LiOH, MeOH, THF, (2) (l)-methionine methyl ester, EDCI, HOBT; (d) (1) SnCl2 hydrate,
(2) N-Boc-S-trityl-(l)-cysteinal, NaB(CN)H3, methanol; (e) (1) aqueous LiOH, THF; (2) TFA, Et3SiH.
Scheme 4. Preparation of pyridine containing peptidomimetics 11±13. Reagents; (a) Pd(PPh3)2Cl2, CuI, LiCl, toluene; (b) (1) aqueous LiOH,
methanol, (2) (l)-methionine methyl ester, EDCI, HOBT; (c) SnCl2 hydrate; (d) Raney Ni, hydrazine, methanol; (e) N-Boc-S-trityl-(l)-cysteinal,
NaB(CN)H3, methanol; (f) (1) aqueous LiOH, THF, (2) TFA, Et3SiH.
potency of 5 clearly suggested a favorable hydrophobic
interaction of FTase active site with the phenyl ring.
When the phenyl group was replaced by a xylyl ring,
inhibition activity decreased 100-fold. The potency of 6
is reduced compared to 1, possibly due to unfavorable
steric e€ects from the methyl groups. It is interesting to
note that when 4-amino-1-naphthoic acid was used as
the spacer, inhibition potency was not improved (com-
pare 7 and 1 in Table 1).
0.74 nM. Methylation of the carboxylic acid in 8
decreased inhibition potency by 100-fold (IC50=63 nM,
14 in Table 1). However, the methyl ester showed sig-
ni®cant potency in inhibiting Ras farnesylation in whole
cells. Compound 14 (FTI-277) inhibited H-Ras proces-
sing with an IC50 of 100 nM and retained selectivity in
whole cells, inhibiting the processing of Rap1A (a ger-
anylgeranylated protein) with an IC50 of 50 mM.41
Results from Table 1 also indicate that substitution at
the 2-position of the aminobenzoic acid spacer is not
restricted to phenyl groups since both 2-thienyl and
1-naphthyl attachments also lead to potent inhibitors
with IC50 values in the 1±2 nM range. Interestingly, the
selectivity for FTase over GGTase-I is decreased from
82-fold in 5 to only fourfold in 10. This indicates that
large hydrophobic substituents such as naphthalene
are more optimal for strong binding to GGTase-I.
As described for compound 2, the pseudopeptide strat-
egy was also applied to modify the cysteine amide bond
in 5. Compound 8 (FTI-276) showed subnanomolar
inhibition activity with an average IC50 of 0.61 nM (IC50
ranged from 0.33 to 0.90 nM in 17 independent assays)
and a selectivity for FTase over GGTase-I of 82-fold.
Lineweaver±Burk analysis con®rmed that 8 (FTI-276) is
competitive to H-Ras for binding to FTase with a Ki of

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Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
Comparison of the active site residues in the two
enzymes26,27 suggests that the hydrophobic pocket is
larger in GGTase-I, formed by smaller Thr, Phe and
Leu residues in place of Trp102, Trp106 and Tyr361
seen in the crystal structure of FTase (Fig. 1). This dif-
ference has recently been exploited in the development
of a family of inhibitors for GGTase-I.46
Concentrations are expressed in g/100 mL. Melting
points (mp) were determined using an Electrothermal
capillary melting point apparatus and are uncorrected.
All synthesized ®nal compounds (amine hydrochloride
or amine tri¯uoroacetate salts) were checked for purity
by analytical high pressure liquid chromatography
which was performed using a Rainin HP controller and
a Rainin UV-C detector with a Rainin 250Â4.6 mm,
5 mm Microsorb C18 column. Binary solvents consisting
of 10±90% acetonitrile and 0.1% TFA in water were used.
Flow rate was set as 1 mL/min and separation was pro-
grammed for 30 -min running. Preparative HPLC was
performed on a Waters 600E controller and a Waters
490E multi-wavelength UV detector with a 25Â10 cm
Delta-Pak C-18 300 A cartridge column inside a Waters
25Â10 cm Radial Compression Module. Solvents con-
sisting of 20±80% acetonitrile and 0.1% TFA in water
were used with a ¯ow rate of 15 mL/min in 40 min.
Finally, we linked a pyridine ring to the 2-position of
the 4-aminobenzoic acid spacer in an attempt to probe
the presence of hydrogen bond donors in the enzyme
binding region. The recent crystal structure of FTase
(Fig. 1) shows three potential acidic centers in the
hydrophobic pocket for the penultimate Leu side
chain, a Tyr-OH and two Trp-NH groups.26,27 We
used 2-, 3-, and 4-pyridyl groups (11, 12 and 13,
respectively) in an attempt to position the hydrogen
bond accepting pyridine-N at di€erent locations in the
pocket. Table 1 shows that the three pyridine derived
inhibitors share similar inhibition activity for FTase
(IC50 values 3±6 nM) with good selectivity for FTase
over GGTase-I. These three compounds are ®ve- to 10-
fold less active as FTase inhibitors than FTI-276 (com-
pare 8 with 11, 12 and 13 in Table 1), suggesting that no
additional interactions have been exploited. In fact, the
drop in activity presumably re¯ects the less hydrophobic
and more strongly solvated character of pyridyl com-
pared to phenyl. A computational study of the binding
and solvation consequences of a phenyl to pyridyl
modi®cation in FK506 analogues has recently been
published by Jorgensen.47
Farnesyltransferase inhibition assay was determined by
measuring the amount of [3H]-farnesyl group trans-
ferred from [3H]-FPP to recombinant H-Ras-CVLS
protein according to literature procedure.40 Each assay
solution contained the following concentrations of
components in a ®nal volume of 50 mL: 50 mM Tris±
chloride (pH 7.5), 50 mM ZnCl2, 20 mM KCl,
3 mM mgCl2, 500 nM [3H]-FPP (speci®c activity 20 Ci/
mmol), and 15 mM H-Ras. For kinetic assay, the di€er-
ent concentrations of inhibitors and certain amount of
partially puri®ed farnesyltransferase from human
Burkitt lymphoma (Daudi) cells were added to the
above solution at 0ꢀC in 12Â75 mm borosilicate tubes.
After incubation at 37ꢀC for 15 min, the reaction was
stopped by addition of 0.5 mL of 4% SDS and 0.5 mL
of 30% trichloroacetic acid (TCA) solution. The mix-
ture was left on ice for 45 min and then 2 mL of 2%
SDS and 6% TCA was added. The solution was ®ltered
on glass ®ber ®lters and each ®lter was washed ®ve times
with 2 mL of 6% TCA solution. The ®lters were dried
and counted in a scintillation counter. For GGTase-I
assays, the reaction mixture was similar except that
the substrates used were H-Ras-CVLL and [3H]-GGPP
(speci®c activity 20 Ci/mmol). GGTase-I was also par-
tially puri®ed from Daudi cells.40
The structure±activity relationships of our peptidomi-
metics in Table 1 clearly indicates that the strategy of
hydrophobic substitution can lead to highly potent
inhibitors of FTase. These inhibitors share some struc-
tural similarity with the CVIM tetrapeptide including
the methionine residue, the methionine amide bond, the
cysteine amino group and the cysteine side chain.
Recent work by us48 and by Augeri et al.49 has shown
that the N-terminal cysteine group in these inhibitors
can be replaced by nitrogenous base derivatives and that
an extended hydrophobic spacer can be used in place of
the C-terminal methionine.50 In the present paper we
have investigated the third region of the molecules and
identi®ed an aryl-substituted hydrophobic spacer that
gives both potent inhibition of FTase and high selectiv-
ity over GGTase-I.
N-(4-N-Boc-Aminobenzoyl)-methionine methyl ester (15).
To a mixture of 4-aminobenzoic acid (13.7 g, 100 mmol)
in 100 mL of 1 N NaOH and 100 mL of dioxane was
added di-tert-butyl dicarbonate (24.0 g, 110 mmol) at
0ꢀC. The mixture was stirred at rt overnight and then
acidi®ed with 3 N HCl. After extraction with ethyl ace-
tate and evaporation of solvents, a crude solid product
was obtained. This solid was recrystallized from metha-
nol and water to give N-Boc-4-aminobenzoic acid as
Experimental
Nuclear magnetic resonance spectra were acquired
using Bruker AM-300 series spectrometers (300 MHz
for 1H, 75 MHz for 13C). Chemical shifts were expressed
as parts per million and referenced to internal tetra-
methylsilane (TMS), CDCl3 or CD3OD. Electron
impact (EI) and fast atom bombardment (FAB) mass
spectra were obtained on a Varian MAT CH-5 (high
resolution) or VG 7070 (low resolution) mass spectro-
meter. Elemental analysis were performed by Atlantic
Microlabs, Inc., Norcross, GA. Optical rotations [a]d25
were measured using a Perkin±Elmer 241 polarimeter.
white crystals (15.2 g, 65%). Mp 190±191ꢀC. H NMR
1
(CD3OD) d 7.92 (d, J=8.5 Hz, 2H), 7.50 (d, J=8.5 Hz,
2H), 1.51 (s, 9H); 13C NMR (CD3OD) d 169.8, 155.0,
145.8, 131.8, 125.3, 118.5, 81.2, 28.6; HRMS (EI) calcd
for C12H15O4N 237.0961, obsd 237.0956. This acid
(3.55 g, 15 mmol) was dissolved in 150 mL of methylene
chloride. To this solution was added l-methionine methyl
ester hydrochloride (3.0 g, 15mmol) and triethylamine
(2.48 mL). This mixture was cooled in an ice bath and

Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
3017
then EDCI (2.87 g, 15 mmol) was added. The clear
solution was stirred at rt overnight and then extracted
with methylene chloride and 1 N HCl. The organic
phase was washed with concentrated sodium bicarbo-
nate and dried over sodium sulfate. After evaporating
the solvents, a crude solid was obtained which was
recrystallized from ethyl acetate and hexane to give a
acetate and concentrated sodium bicarbonate. The
organic layer was dried and solvents were evaporated. The
residue was puri®ed by ¯ash column chromatography
(ethyl acetate:hexane, 1:1) to give the desired product
(2.52 g, 65%). Mp 85ꢀC (decomp). H NMR (CDCl3) d
1
7.63 (d, J=8.6 Hz, 2H, aromatic), 7.43 (m, 6H, trityl),
7.21±7.32 (m, 9H, trityl), 6.73 (d, J=7.6 Hz, 1H, Met
amide), 6.50 (d, J=8.6 Hz, 2H, aromatic), 4.91 (ddd,
J=5.5, 7.6 Hz, 1H, Met a H), 4.59 (d, J=7.3 Hz, 1H,
Boc amide), 4.25±4.40 (br, 1H, NHPh), 3.80 (m, 1H,
Cys a H), 3.78 (s, 3H, OCH3), 3.09 (d, J=6.3 Hz, 2H,
CH2N), 2.55±2.60 (m, 2H, CH2 SCPh3), 2.46 (d,
J=5.0 Hz, 2H, CH2SCH3), 2.23±2.28 (m, 1H, Met
CH2), 2.07±2.12 (m, 1H, Met CH2), 2.09 (s, 3H, SCH3),
1.43 (s, 9H, Boc); 13C NMR (CDCl3) d 172.8, 166.8,
155.7, 150.8, 144.3, 129.4, 128.8, 127.9, 126.8, 121.5,
115.2, 79.7, 67.0, 52.4, 51.8, 49.2, 46.8, 34.2, 31.6, 30.0,
pure product (4.92 g, 86%). Mp 184±185ꢀC. H NMR
1
(CDCl3) d 7.78 (d, J=8.6 Hz, 2H, aromatic), 7.47 (d,
J=8.6 Hz, 2H, aromatic), 7.01 (d, J=7.0 Hz, 1H,
amide), 6.91 (s, 1H, amide), 4.92 (q, J=7.0 Hz, 1H, Met
a H), 3.79 (s, 3H, OCH3), 2.59 (t, J=5.9 Hz, 2H,
CH2S), 2.18 (m, 1H, Met CH2), 2.11 (m, 4H, Met CH2,
SCH3), 1.53 (s, 9H, But); 13C NMR (CDCl3) d 172.8,
166.6, 152.3, 141.9, 128.3, 127.8, 117.9, 81.2, 52.7, 52.1,
31.6, 30.2, 28.3, 15.6. Anal. calcd for C18H26N2O5S: C
56.53, H 6.85, N 7.29; found C 56.46, H 6.83, N 7.30.
.
28.2, 15.4; Anal. calcd for C40H47O5 N3S2 0.4CH2Cl2: C
64.89, H 6.39, N 5.62, S 8.56; found C 64.66, H 6.43, N
5.69, S 8.50.
N-Boc-S-trityl-cysteinyl-4-aminobenzoyl-methionine methyl
ester (16). Compound 15 (298 mg, 0.78 mmol) was dis-
solved in a mixture of 2 mL of methylene chloride and
2 mL of 3 N HCl in methanol. After 1.5 h, TLC showed
disappearance of starting material. This clear solution
was evaporated and the residue was dried under
vacuum. Amide coupling was carried out through a
mixed anhydride which was prepared by stirring a mix-
ture of N-Boc-S-trityl-l-cysteine (370 mg, 0.80 mmol),
isobutyl chloroformate (0.105 mL, 0.80 mmol) and trie-
thylamine (0.10 mL, 0.80 mmol) in 5 mL of THF at
15ꢀC for 30 min. The hydrochloride salt from Boc
deprotection was then suspended in 3 mL of methylene
chloride and neutralized with 0.15mL of triethylamine.
This solution was added to the mixed anhydride solution
prepared above and the mixture was stirred at rt for 12 h.
After work up and column chromatography puri®cation,
a pure product was obtained (450mg, 79.5%). Mp 98±
99ꢀC. [a]2d5 3.1 (c 1.4, EtOAc). 1H NMR (CDCl3) d 8.52
(br s, 1H, amide), 7.72 (d, J=8.5 Hz, 2H, aromatic), 7.50
(m, 8H, aromatic), 7.29 (m, 9H, aromatic), 7.03 (d,
J=6.8Hz, 1H, amide), 4.93 (q, J=7.4 Hz, Met a H), 4.85
(d, J=7.2Hz, 1H, amide), 3.98 (m, 1H, Cys a H), 3.79 (s,
3H, OCH3), 2.75 (dd, J=13.0, 7.3 Hz, 1H, CH2SCPh3),
2.63 (dd, J=13.0, 5.3 Hz, 1H, CH2SCPh3), 2.59 (t,
J=7.3Hz, 2H, CH2SCH3), 2.29 (m, 1H, Met CH2), 2.12
(m, 4H, Met CH2, SCH3), 1.44 (s, 9H, But); 13C NMR
(CDCl3) d 173.9, 169.7, 166.7, 158.0, 144.2, 140.8, 129.3,
128.3, 128.0, 127.8, 126.7, 110.6, 80.6, 67.1, 54.9, 52.8,
51.9, 33.3, 30.9, 30.4, 28.1, 15.3. Anal. calcd for
C40H45O6N3S2: C 65.99, H 6.24, N 5.77; found C 65.94, H
6.28, N 5.71.
Cysteinyl-4-aminobenzoyl-methionine hydrochloride (1).
The fully protected compound 16 (400 mg, 0.55 mmol)
was hydrolyzed with 1.0 equiv of 0.5 N LiOH in 3 mL of
THF at 0ꢀC. The solvents were evaporated and the
residue was ®rst acidi®ed with 1 N HCl and then
extracted with methylene chloride. After evaporation of
solvents, the carboxylic acid was dissolved in 3 mL of
methylene chloride, 1 mL of THF and 3 mL of 3 N HCl
in dry ether. The solution was maintained at rt for
25 min. The white precipitate was collected and recrys-
tallized twice from ethanol and ether to give 1 shown to
be pure by HPLC (108 mg, 48%). Mp 157ꢀC (decomp).
[a]2d5 22.5 (c 0.5, MeOH). H NMR (CD3OD) d 7.87
1
(d, J=8.7 Hz, 2H, aromatic), 7.73 (d, J=8.7 Hz, 2H,
aromatic), 4.75 (dd, J=4.6, 6.5 Hz, 1H, Met a H), 4.16
(dd, J=5.1, 7.0 Hz, 1H, Cys a H), 3.18 (dd, J=14.7,
5.0 Hz, 1H, CH2SH), 3.04 (dd, J=14.7, 7.3 Hz, 1H,
CH2SH), 2.62 (m, 2H, CH2SCH3), 2.20 (m, 1H, Met
CH2), 2.11 (m, 4H, SCH3, Met CH2); 13C NMR
(CD3OD) d 175.5, 169.8, 166.9, 142.5, 131.0, 129.6,
120.5, 56.7, 53.2, 31.8, 31.6, 26.3, 15.2. Anal. calcd for
.
C15H21O4N3S2 HCl: C 44.16, H 5.44, N 10.30; found C
44.40, H 5.87, N 10.01.
N-[4-N-[2(R)-Amino-3-mercaptopropyl]aminobenzoyl]-
methionine hydrochloride (2). The fully protected com-
pound 17 (567 mg, 0.79 mmol) was dissolved in 3.0 mL
of 0.5 N lithium hydroxide and 3.0 mL of tetra-
hydrofuran. The mixture was stirred at 0ꢀC for 1 h.
After evaporating solvents, the residue was dissolved in
water and extracted with methylene chloride and 1 N
hydrochloric acid. The organic layer was dried and the
solvents were evaporated. The residue was dissolved in a
mixture of 1 mL of methylene chloride and 2 mL of tri-
¯uoroacetic acid. Triethylsilane was added dropwise
until the deep brown color disappeared. The mixture
was maintained at rt for 1 h. The solvents were evapo-
rated and the residue was dried. This residue was dis-
solved in 2 mL of 1.7 N HCl in acetic acid and then
5 mL of 3 N HCl in ether was added followed by further
addition of 20 mL of ether. The white precipitate was
®ltered and dried to give a hydrochloride salt (159 mg,
N-[4-[N-[2(R)-(tert-Butoxycarbonyl)amino-3-(triphenyl-
methyl)thio]propyl]-aminobenzoyl]-methionine methyl ester
(17). To a solution of N-(4-aminobenzoyl)-methionine
methyl ester hydrochloride (1.78 g, 5.6 mmol, prepared
from the deprotection of compound 15) in 60 mL of
methanol and 4 mL of glacial acetic acid was added one
1
equivalent of N-Boc-S-tritylcysteinal (according to H
NMR determination of aldehyde percentage) in 10 mL
of methanol. Sodium cyanoborohydride (528 mg,
8.40 mmol) was added to this deep colored solution at
0ꢀC. The mixture was stirred at rt for 15 h. After eva-
porating solvents, the residue was extracted with ethyl

3018
Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
46%). Analytical HPLC showed purity over 98%. Mp
164±165ꢀC. [a]2d5 11.9 (c 0.15, MeOH). 1H NMR
(CD3OD) d 7.74 (d, J=8.7 Hz, 2H, aromatic), 6.75 (d,
J=8.7 Hz, 2H, aromatic), 4.73 (dd, J=4.6, 9.3 Hz, 1H,
Met a H), 3.45±3.58 (m, 3H, CH2NH and Cys a H),
2.93 (dd, J=4.5, 14.6 Hz, 1 H, CH2SH), 2.80 (dd,
J=5.3, 14.6 Hz, 1H, CH2SH), 2.53±2.64 (m, 2H,
CH2SCH3), 2.15±2.23 (m, 1H, Met CH2), 2.07±2.13 (m,
1H, Met CH2), 2.10 (s, 3H, SCH3); 13C NMR (CD3OD)
d 175.6, 170.3, 152.5, 130.4, 123.4, 113.0, 53.7, 53.1,
44.7, 32.0, 31.6, 25.3, 15.2; LRMS for C15H23O3N3S2
357 (M+, 10), 282 (60), 133 (100).
amine was coupled with N-Boc-S-trityl-(l)-cysteine
using a mixed anhydride method. Mp 100±103ꢀC. H
1
NMR (CDCl3) d 8.69 (1H, s), 7.58 (1H, d, J=8.2 Hz),
7.50 (1H, s), 7.37±7.44 (11H, m), 7.18±7.30 (10H, m),
6.10 (1H, d, J=6.6 Hz), 4.90 (1H, d, J=7.4 Hz), 4.58±
4.63 (1H, m), 4.02 (1H, br s), 3.64 (3H, s), 2.72 (1H, dd,
J=12.7 and 5.5 Hz), 2.63 (1H, dd, J=12.7 and 6.9 Hz),
2.05 (2H, t, J=7.6 Hz), 2.00 (3H, s), 1.84 1.95 (1H, m),
1.61 1.78 (1H, m), 1.41 (9H, s); 13C NMR (CDCl3) d
171.8, 169.1, 168.5, 156.0, 130.1, 129.8, 129.4, 128.7,
128.6, 128.0, 127.9, 126.9, 120.9, 118.2, 80.8, 67.3, 54.2,
52.3, 51.8, 33.0, 31.2, 29.4, 28.2, 15.2. This fully pro-
tected compound was deprotected using the same
method described for compound 2. The ®nal product
was puri®ed by reverse phase preparative HPLC. Mp
150±154ꢀC (decomp). [a]d25 +21.5 (c 0.7, H2O/CH3OH).
1H NMR (CD3OD) d 7.65±7.67 (2H, m), 7.52±7.55 (1H,
m), 7.37±7.41 (5H, m), 4.46 (1H, br s), 3.15 (1H, dd,
J=14.7 and 4.8 Hz), 3.03 (1H, dd, J=14.7 and 7.3 Hz),
2.16±2.18 (1H, m), 2.00±2.10 (5H, m), 1.62±1.79 (1H,
m); 13C NMR (CD3OD) d 172.5, 167.8, 167.0, 142.5,
141.4, 140.5, 133.5, 130.1, 129.7, 129.6, 129.0, 122.3,
119.4, 57.0, 32.8, 31.8, 26.3, 15.0. Anal. calcd for C23H26
F3N3O6S2: C 49.20, H 4.67, N 7.48; found C 49.14, H
4.71, N 7.42.
Cysteinyl-4-amino-3-methylbenzoyl methionine tri¯uoro-
acetate (3). 4-Amino-3-methylbenzoic acid was pro-
tected to give N-Boc-4-amino-3-methylbenzoic acid
(60%). This acid was coupled with (l)-methionine
methyl ester as described in compound 15 to give N-
Boc-4-amino-3-methylbenzoyl methionine methyl ester
(86%). The Boc group was deprotected and the result-
ing amine was coupled with N-Boc-S-trityl-(l)-cysteine
using a mixed anhydride method. The fully protected
precursor was ®rst treated with aqueous LiOH in THF
and then deprotected with TFA and 1.0 equiv of tri-
ethylsilane. Compound 3 was puri®ed by reverse phase
preparative HPLC. Mp 158±163ꢀC. [a]d25 +21.0 (c 1.0,
H2O). 1H NMR (CD3OD) d 7.80 (1H, s), 7.69±7.73 (1H,
m), 7.64 (1H, d, J=8.4 Hz), 4.70±4.75 (1H, m), 4.25
(1H, dd, J=6.9 and 5.2 Hz), 3.19 (1H, dd, J=14.6 and
5.2 Hz), 3.08 (1H, dd, J=14.6 and 6.9 Hz), 2.57±2.65
(2H, m), 2.36 (3H, s), 2.19±2.30 (1H, m), 2.09±2.17 (4H,
m); 13C NMR (CD3OD) d 175.6, 169.7, 167.5, 139.3,
133.2, 131.0, 126.7, 125.8, 125.5, 56.7, 53.5, 32.1, 31.6,
26.5, 18.3, 15.2.
Cysteinyl-4-amino-2-(3,5-dimethylphenyl)benzoyl methio-
nine tri¯uoroacetate (6). This compound was prepared
in exactly the same way described for the preparation of
compound 5 except 4-nitro-2-(3,5-dimethylphenyl)-ben-
zoic acid was used. Final product was puri®ed by reverse
phase preparative HPLC. Mp 135±138ꢀC. 1H NMR
(CD3OD) d 7.66 (2H, m), 7.53 (1H, d, J=8.0 Hz), 7.02
(3H, s), 4.45±4.48 (1H, m), 4.15 (1H, dd, J=7.3 and
4.9 Hz), 3.17 (1H, dd, J=14.6 and 4.9 Hz), 3.05 (1H, dd,
J=14.6 and 7.3 Hz), 2.33 (6H, s), 2.00±2.17 (6H, m),
1.76±1.84 (1H, m); 13C NMR (CD3OD) d 174.8, 172.6,
166.9, 142.9, 141.3, 140.4, 139.3, 133.4, 130.6, 130.1,
127.5, 122.3, 119.3, 57.0, 53.0, 31.7, 31.0, 26.3, 21.5, 15.0.
Cysteinyl-4-amino-3-methoxybenzoyl methionine tri¯uoro-
acetate (4). This compound was prepared with the same
method as described in compound 3. 4-Amino-3-meth-
oxybenzoic acid was protected to give N-Boc-4-amino-
3-methoxybenzoic acid (93%). This acid was coupled
with (l)-methionine methyl ester and the Boc group was
then deprotected. The free amine was coupled with N-
Boc-S-trityl-(l)-cysteine to provide a fully protected
precursor. After deprotection, the crude product was
puri®ed by reverse phase preparative HPLC. Mp 191±
193ꢀC (decom). [a]d25 +19.0 (c 1.0, H2O); 1H NMR
(CD3OD) d 8.23 (1H, d, J=8.4 Hz), 7.56 (1H, s), 7.50
(1H, d, J=8.4 Hz), 4.77 (1H, dd, J=7.5 and 4.8 Hz),
3.92 (3H, s), 3.15 (1H, dd, J=14.6 and 4.8 Hz), 3.00
(1H, dd, J=14.6 and 7.5 Hz), 2.53±2.71 (2H, m), 2.20±
2.32 (1H, m), 2.12±2.18 (1H, m), 2.10 (3H, s); 13C NMR
(CD3OD) d 175.4, 169.6, 167.2, 150.9, 131.9, 130.9,
122.1, 121.1, 111.0, 56.8, 56.6, 53.3, 31.8, 31.6, 26.7,
15.2; m/z (FAB) for the free amine 402 (M+1).
Cysteinyl-4-amino-1-naphthoyl methionine tri¯uoroace-
tate (7). 4-Amino-1-naphthalenecarbonitrile (1.50 g,
8.91 mmol) was dissolved into a 50% KOH solution
(18 mL). The heterogeneous solution was heated at
re¯ux for 2±3 days. Once the solution became homo-
geneous and TLC showed no more starting material, the
deep red solution was cooled and acidi®ed with con-
centrated HCl. The solid was ®ltered and treated with
activated carbon to obtain 1.51 g of 4-amino-1-naph-
thoic acid (91%). Mp 169±171ꢀC. 1H NMR (CD3OD) d
9.09 (1H, d, J=8.5 Hz), 8.13 (1H, d, J=8.2 Hz), 8.03
(1H, d, J=8.5 Hz), 7.48±7.54 (1H, m), 7.38±7.43 (1H,
m), 6.69 (1H, d, J=8.2 Hz); 13C NMR (CD3OD) d
171.4, 151.3, 135.0, 128.5, 127.4, 125.2, 123.9, 123.0,
114.6, 107.4; HRMS calcd for C11H7NO2 187.0633;
found 187.0642. The amino group of this product was
protected with Boc anhydride and the carboxylic acid
was coupled with (l)-methionine methyl ester as descri-
bed before. The Boc group was deprotected and the free
amine was coupled with N-Boc-S-trityl-(l)-cysteine to
give a fully protected compound. This compound was
deprotected as described before to give the ®nal product.
Mp 121±125ꢀC. [a]d25 +2.4 (c 0.8, H2O). 1H NMR
Cysteinyl-4-amino-2-phenylbenzoyl methionine tri¯uoro-
acetate (5). 4-Nitro-2-phenylbenzoic acid (0.30 g,
1.23 mmol) was coupled with (l)-methionine methyl
ester (0.27 g, 1.35 mmol) in the presence of 1.0 equiv of
EDCI and HOBT. The crude product was recrystallized
from ethyl acetate and hexane (86%). The nitro group
was reduced with SnCl2 dihydrate (5.0 equiv) in ethyl
acetate to give the free amine as a yellow solid. This

Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
3019
(CD3OD) d 8.98 (1H, d, J=7.7 Hz), 8.29±8.32 (1H, m),
8.08±8.11 (1H, m), 7.70 (1H, d, J=7.7 Hz), 7.64 (1H, d,
J=7.7 Hz), 7.57±7.61 (1H, m), 4.84±4.89 (1H, m), 4.42
(1H, m), 3.16±3.33 (2H, m), 2.59±2.74 (2H, m), 2.22±
2.36 (1H, m), 2.03±2.13 (4H, m); 13C NMR (CD3OD) d
175.2, 172.4, 168.1, 135.1, 134.1, 132.3, 129.5, 128.3,
128.0, 127.0, 126.1, 126.4, 123.4, 122.5, 56.7, 53.2, 31.6,
31.5, 26.4, 15.2. Anal. calcd for C21H23F3N3O6S2: C
47.19, H 4.34, N 7.86; found C 47.53, H 4.56, N 7.59.
N-[4-[N-[2(R)-(tert-Butoxycarbonyl)amino-3-(triphenyl-
methyl)thio]propyl]amino-2-phenylbenzoyl]-methionine
methyl ester (20). Compound 19 (3.04 g, 7.83mmol)
was dissolved in 100 mL of ethyl acetate. Stannous chlo-
ride hydrate (8.84 g, 39mmol) was added and the mixture
was re¯uxed for 2 h. This solution was poured into
200 mL of concentrated sodium bicarbonate and extrac-
ted with ethyl acetate. After evaporating solvents, the
residue was dissolved in 10mL of methylene chloride and
15mL of 3 N HCl in ether was added. Solvents were eva-
porated and a hydrochloride salt was obtained (2.97 g,
4-Nitro-2-phenylbenzoic acid (18). 4-Nitro-2-bromoto-
luene (6.48 g, 30 mmol) and phenyl boronic acid (3.84 g,
31.5 mmol) was dissolved in 70 mL of acetone. To this
mixture was added 85 mL of water, potassium carbon-
ate hydrate (12.4 g, 75 mmol, 2.5 equiv) and Pd(OAc)2
(0.33 g, 5% equiv). The mixture was re¯uxed for 10 h
and then cooled. The deep black solution was extracted
with ether and 3 N HCl. The ether fraction was passed
through a layer of Celite. After evaporating solvents,
the residue was dried and then recrystallized from
methanol to give ¯ake crystals (4.60 g, 88%). Mp 77±
78ꢀC. 1H NMR (CDCl3) d 8.09±8.11 (m, 2H, aryl),
7.40±7.49 (m, 4H, aryl), 7.30±7.33 (m, 2H, aryl), 2.37 (s,
3H); 13C NMR (CDCl3) d 146.1, 143.5, 143.0, 139.4,
131.1, 128.8, 128.4, 127.8, 124.5, 122.0, 20.7. Anal. calcd
for C13H11NO2: C 73.24, H 5.16, N 6.57; found C 73.11,
H 5.15, N 6.53; LRMS (EI) 213. This compound (3.20
g, 15 mmol) was dissolved in 15 mL of pyridine and
30 mL of water. The mixture was heated to 90ꢀC and
then KMnO4 (14.2 g, 90 mmol) was added in portions.
The mixture was re¯uxed for 5 h. The black solid was
®ltered o€ and the ®ltrate was acidi®ed with 6 N HCl.
The mixture was cooled in an ice bath and the white
precipitate was collected (3.1 g, 85%). Mp 175±176ꢀC.
1H NMR (CDCl3) d 8.25±8.33 (m, 2H, aryl), 8.08 (d,
J=8.9 Hz, 1H, aryl), 7.41±7.51 (m, 3H, aryl), 7.31±7.39
(m, 2H, aryl); 13C NMR (CD3OD) d 170.6, 150.2, 144.4,
140.4, 139.3, 131.6, 129.5, 129.4, 129.3, 126.1, 123.0;
LRMS (EI) 243. Anal. calcd for C13H9O4N: C 64.19, H
3.70, N 5.76; found C 64.23, H 3.74, N 5.70.
96%). Mp 229±230ꢀC. H NMR (CD3OD) d 7.65 (d,
1
J=8.1Hz, 1H, aryl), 7.39±7.46 (m, 7H, aryl), 4.53 (dd,
J=4.3, 9.5 Hz, 1H, Met a H), 3.69 (s, 3H, OCH3), 2.15±
2.23 (m, 1H, Met CH2), 2.00 (s, 3H, SCH3), 1.93±2.11 (m,
2H, CH2S), 1.74±1.83 (m, 1H, Met CH2); 13C NMR
(CD3OD) d 173.4, 171.7, 143.4, 140.1, 137.4, 134.0, 130.9,
129.7, 129.4, 125.5, 122.7, 53.0, 52.9, 31.3, 30.9, 15.1
(expect 10 aromatic C, observed 9).
The above hydrochloride salt (1.27 g, 3.22 mmol) was
dissolved in 10 mL of methanol and then N-Boc-S-tri-
tylcysteinal (1.0equiv, according to 1H NMR determina-
tion of aldehyde percentage) was added. After stirring for
10min, sodium cyanoborohydride (305mg, 1.5 equiv) was
added and the mixture was stirred for 12h at rt. After
evaporating solvents, the residue was extracted with ethyl
acetate and concentrated sodium bicarbonate. Solvents
were evaporated and the residue was puri®ed by ¯ash
column chromatography (1:1=hexane:ethyl acetate,
silica) to give 20 as a ¯u€y foam (1.84 g, 74%). Mp 82±
83ꢀC (decomp). [a]2d5 0.3 (c 1.6, EtOAc). 1H NMR
(CDCl3) d 7.65 (d, J=8.6 Hz, 1H, aryl), 7.34±7.42 (m,
11H, aryl), 7.18±7.29 (m, 9H, aryl), 6.52 (dd, J=2.3,
8.1 Hz, 1H, aryl), 6.34 (d, J=2.3Hz, 1H, aryl), 5.65 (d,
J=7.7Hz, 1H, amide), 4.64 (ddd, J=4.5, 9.3 Hz, 1H, Met
a H), 4.55 (d, J=8.1Hz, 1H, Boc amide), 4.19 (br t, 1H,
NH), 3.78 (br m, 1H, Cys a H), 3.64 (s, 3H, OCH3), 3.09
(t, J=6.1 Hz, 2H, CH2N), 2.44 (m, 2H, CH2SCPh3) 2.04±
2.10 (m, 2H, CH2SCH3), 2.00 (s, 3H, SCH3), 1.81±1.90
(m, 1H, Met CH2), 1.60±1.70 (m, 1H, Met CH2), 1.41 (s,
9H, Boc); 13C NMR (CDCl3) d 172.0, 168.3, 155.7, 149.4,
144.3, 141.6, 141.1, 131.3, 129.5, 128.7, 128.5, 127.9, 127.7,
126.8, 122.6, 113.6, 111.3, 79.8, 67.1, 52.2, 51.7, 49.5, 47.2,
34.3, 31.6, 29.4, 28.2, 15.2. Anal. calcd for
N-[4-Nitro-2-phenylbenzoyl]-methionine methyl ester
(19). Compound 18 (2.43 g, 10 mmol) was suspended in
50 mL of methylene chloride. To this solution in an ice
bath was added (l)-methionine methyl ester hydrochlo-
ride (2.0 g, 10 mmol), triethylamine (1.38 mL, 10 mmol),
EDCI (2.01 g, 10.5 mmol) and HOBT (1.35 g, 10 mmol).
The mixture was stirred at rt for 10 h and then extracted
with methylene chloride and 1 N HCl. The organic layer
was washed with concentrated sodium bicarbonate and
dried. After evaporating solvents, the residue was
recrystallized from ethyl acetate and hexane to give 19
(3.22 g, 83%). Mp 104±105ꢀC. [a]d25 ±5.2 (c 0.9, EtOAc).
1H NMR (CDCl3) d 8.24±8.28 (m, 2H, aryl), 7.85 (d,
J=8.9 Hz, 1H), 7.43±7.52 (m, 5H), 6.01 (d, J=7.5 Hz,
1H, amide), 4.69 (ddd, J=4.5, 7.4 Hz, 1H, Met a H),
3.68 (s, 3H, OCH3), 2.05 (t, J=7.2 Hz, 2H, CH2S), 1.98
(s, 3H, SCH3), 1.88±1.96 (m, 1H, Met CH2), 1.72±1.81
(m, 1H, Met CH2); 13C NMR (CDCl3) d 171.2, 167.3,
148.1, 141.0, 140.6, 137.5, 129.5, 128.7, 128.6, 128.3,
124.7, 121.9, 52.3, 51.5, 30.7, 29.2, 15.0. Anal. calcd for
C19H20O5N2S: C 58.76, H 5.15, N 7.21, S 8.24; found C
58.79, H 5.22, N 7.26, S 8.42.
.
C46H51O5N3S2 0.4CH2Cl2: C 67.65, H 6.29, N 5.10, S
7.77; found C 67.68, H 6.40, N 5.18, S 7.69.
N-[4-[2(R)-Amino-3-mercaptopropyl]amino-2-phenylbenz-
oyl]-methionine tri¯uoroacetate (8, FTI-276). The N-
Boc-S-trityl protected peptide methyl ester 20 (500 mg,
0.63 mmol) was hydrolyzed with 2.0 equiv of 0.5 N
lithium hydroxide at 0ꢀC for 1 h. The obtained free
carboxylic acid was treated with tri¯uoroacetic acid
(2 mL) in methylene chloride (1 mL). Triethylsilane was
added dropwise until the deep yellow color disappeared.
The mixture was maintained at rt for 1 h. After eva-
porating solvents, the residue was dried and washed
with dry ether. The solid was puri®ed by reverse phase
preparative HPLC (Waters 25Â10 cm, C-18 column,
220 nm UV detector, ¯ow rate 15 mL/min, linear gra-
dient from 5% acetonitrile and 95% water with 0.1%
TFA to 60% acetonitrile in 40 min) to give FTI-276 as a

3020
Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
TFA salt (270 mg, 78%). Mp 90ꢀC (decomp). [a]d25
13.6 (c 0.5, MeOH). H NMR (CD3OD) d 7.44 (d,
reagent of 2-thienyl bromide with trimethyl borate. The
crude product was recrystallized from water toꢀgive a
NMR (acetone-d6) d 7.67 (m, 2H), 7.31 (br s, 2H), 7.15
(t, J=3.7 Hz, 1H).
1
1
J=8.4 Hz, 1H, aryl), 7.30±7.39 (m, 5H, aryl), 6.75 (d,
J=8.4 Hz, 1H, aryl), 6.67 (s, 1H, aryl), 4.45 (dd, J=4.2,
5.1 Hz, 1H, Met a H), 3.42±3.58 (m, 3H, CH2N, Cys a
H), 2.90 (dd, J=4.3, 14.5 Hz, 1H, CH2SH), 2.81 (dd,
J=5.5, 14.5 Hz, 1H, CH2SH), 2.17±2.23 (m, 1H,
CH2SCH3), 2.09±2.15 (m, 1H, CH2SCH3), 2.00 (s, 3H,
SCH3), 1.90±1.99 (m, 1H, Met CH2), 1.71±1.81 (m, 1H,
Met CH2); 13C NMR (CD3OD) d 176.4, 173.5, 150.4,
143.0, 141.5, 131.0, 129.7, 129.4, 128.9, 124.6, 115.0,
112.3, 53.3, 49.6, 44.4, 30.8, 30.1, 24.9, 14.8. Anal. calcd
pure 2-thienylboronic acid (65%). Mp 146±147 C. H
Compound 21 (1.30 g, 5.0 mmol) and 2-thienylboronic
acid (1.27 g, 10 mmol) were dissolved in 30 mL of dry
DMF. Trisodium phosphate (3.0 equiv) and Pd(PPh3)4
(300 mg, 5% equiv) were added and the mixture was
heated at 100ꢀC for 12 h. After work up with ether and
2 N HCl, the ether layer was dried and evaporated. The
residue was recrystallized from methanol to give 22 as
pale-brown crystals (0.75 g, 57%). Mp 97±98ꢀC. 1H
NMR (CDCl3) d 8.35 (s, 1H), 8.22 (d, J=8.4 Hz, 1H),
7.83 (d, J=8.6 Hz, 1H), 7.45 (d, J=5.0 Hz, 1H), 7.09±
7.14 (m, 2H), 3.80 (s, 3H); 13C NMR (CDCl3) d 169.6,
150.8, 141.2, 139.1, 137.6, 132.3, 129.7, 129.4, 127.5,
124.1, 54.8 (expect 10 aromatic C, obsd 9).
.
.
for C21H27O3N3S2 CF3COOH H2O: C 48.85, H 5.31, N
7.43, S 11.32; found C 48.90, H 5.35, N 7.41, S 11.38.
N-[4-[2(R)-Amino-3-mercaptopropyl]amino-2-phenylbenz-
oyl]-methionine methyl ester hydrochloride (14, FTI-
277). The above N-Boc-S-trityl protected peptide methyl
ester 20 (900 mg, 1.14 mmol) was dissolved in 5 mL
of methanol. To this mixture was added a solution of
mercuric chloride (774 mg, 2.85 mmol) in 5 mL of
methanol. The mixture was re¯uxed for 20 min. The
clear solution was decanted and the precipitate was col-
lected and dried. This solid was suspended in 10 mL of
methanol and treated with gaseous hydrogen sul®de.
After the removal of black solid, the clear solution was
evaporated to dryness. The residue was then dissolved
in 2 mL of methylene chloride and 5 mL of 3 N hydro-
gen chloride in ether was added. The white solid was
collected and dried to give the pure product FTI-277
(476 mg, 81%). Mp 125ꢀC (foaming). [a]2d5 12.0 (c 0.6,
4-Nitro-2-(1-naphthyl)benzoic acid methyl ester (23). 1-
Naphthylboronic acid was prepared with the same
method described in 22. The crude boronic acid was
used directly without puri®cation. This boronic acid
(2.40 g, 13.95 mmol) was coupled with 4-nitro-2-bromo-
benzoic acid methyl ester (1.95 g, 7.5 mmol) in 50 mL of
DMF at 100ꢀC in the presence of Na3PO4 (22.4 mmol,
3.0 equiv) and Pd(PPh3)4 (430 mg, 5% equiv). The crude
product was puri®ed by ¯ash column chromatography
(hexane:ethyl acetate, 5:1) to give 23 as an oil (1.69 g,
73%). 1H NMR (CDCl3) d 8.34 (d, J=8.5 Hz, 1H), 8.28
(s, 1H), 8.14 (d, J=8.5 Hz, 1H), 7.92 (m, 2H), 7.47±7.56
(m, 2H), 7.41 (d, J=3.8 Hz, 2H), 7.34 (d, J=6.8 Hz,
1H), 3.40 (s, 3H); 13C NMR (CDCl3) d 166.4, 149.2,
142.7, 137.2, 136.8, 133.2, 131.3, 131.0, 128.6, 128.4,
126.6, 126.2, 126.0, 125.0, 124.7, 122.3, 52.3 (expect 16
aromatic C, observed 15). Anal. calcd for C18H13O4N:
C 70.35, H 4.23, N 4.56; found, C 70.21, H 4.14, N 4.37.
1
MeOH). H NMR (CD3OD) d 7.42 (d, J=8.4 Hz, 1H,
aryl), 7.30±7.38 (m, 5H, aryl), 6.77 (d, J=8.4 Hz, 1H,
aryl), 6.71 (s, 1H, aryl), 4.48 (dd, 4.2 and 5.1 Hz, 1H,
Met a H)), 3.68 (s, 3H, OCH3), 3.44±3.58 (m, 3H,
CH2N, Cys a H), 2.90±2.95 (dd, J=4.1, 14.5 Hz, 1H,
CH2SH), 2.79±2.85 (dd, J=4.7, 14.5 Hz, 1H, CH2SH),
2.18±2.22 (m, 1H, CH2SCH3), 2.03±2.16 (m, 1H,
CH2SCH3), 2.00 (s, 3H, SCH3), 1.91±1.97 (m, 1H, Met
CH2), 1.73±1.82 (m, 1H, Met CH2); 13C NMR
(CD3OD) d 173.7, 173.4, 150.7, 143.5, 142.3, 131.2,
129.8, 129.5, 128.6, 125.6, 115.6, 112.2, 53.7, 53.2, 52.8,
45.0, 31.4, 30.9, 25.3, 15.0; LRMS (EI) for C22H29O3
N3S2 447 (M+, 15), 372 (25), 285 (35), 209 (100).
N-[4-Nitro-2-(2-thienyl)benzoyl]-methionine methyl ester
(24). Compound 22 was suspended in a mixture of 1 N
NaOH (2.0 equiv) in methanol and the solution was
re¯uxed for 2 h. After acidi®cation with 2 N HCl, a
carboxylic acid derivative was obtained (100%). This
carboxylic acid was coupled with l-methionine methyl
ester using EDCI and HOBT as coupling reagents as
described before. After ¯ash column chromatography
.
Anal. calcd for C22H29O3N3S2 1.6HCl: C 52.32, H
6.05, N 8.31, S 12.63; found C 52.08, H 6.25, N 8.11, S
12.30.
puri®cation, 24 was obtained (90%). Mp 94±95ꢀC. H
1
Methyl 4-nitro-2-bromobenzoate (21). 4-Nitro-2-bromo-
toluene was oxidized by KMnO4 using the same method
as for the preparation of compound 18 to give 4-nitro-2-
bromobenzoic acid (75%). Mp 175±176ꢀC. This car-
boxylic acid (4.92 g, 20 mmol) was mixed with 50 mL of
methanol and 4 mL of thionyl chloride (54 mmol) and
the mixture was re¯uxed for 3 h. After cooling in an ice
bath, white crystals were ®ltered and washed with 5 mL
of cold methanol to give 4-nitro-2-bromobenzoic acid
methyl ester (4.67 g, 90%). Mp 81±82ꢀC. 1H NMR
(CDCl3) d 8.52 (s, 1H), 8.23 (d, J=8.6 Hz, 1H), 7.92 (d,
J=8.6 Hz, 1H), 3.99 (s, 3H).
NMR (CDCl3) d 8.29 (s, 1H, aryl), 8.16 (d, J=8.4 Hz,
1H, aryl), 7.70 (d, J=8.4 Hz, 1H, aryl), 7.45 (d,
J=5.3 Hz, 1H, thienyl), 7.24 (d, J=3.7 Hz, 1H, thienyl),
7.11 (t, J=3.7 Hz, 1H, thienyl), 6.59 (d, J=7.8 Hz, 1H,
amide), 4.76 (ddd, J=5.2, 7.8 Hz, 1H, Met a H), 3.72 (s,
3H, OCH3), 2.25 (t, J=7.5 Hz, 2H, CH2S), 2.00±2.11
(m, 1H, Met CH2), 2.04 (s, 3H, SCH3), 1.82±1.94 (m,
1H, Met CH2); 13C NMR (CDCl3) d 171.4, 167.2, 148.1,
140.8, 138.2, 133.6, 129.5, 128.1, 128.0, 127.7, 125.1,
122.3, 52.5, 51.8, 30.8, 29.4, 15.2. Anal. calcd for
C17H18O5N2S2: C 51.77, H 4.56, N 7.10; found C 51.84,
H 4.61, N 7.08.
4-Nitro-2-(2-thienyl)benzoic acid methyl ester (22). 2-
Thienylboronic acid was prepared by reacting Grignard
N-[4-Nitro-2-(1-naphthyl)benzoyl]-methionine methyl ester
(25). Compound 23 was ®rst hydrolyzed as described

Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
3021
for compound 24 to give a free carboxylic acid (98%).
Mp 197±198ꢀC. 1H NMR (CDCl3) d 8.33 (d, J=8.6 Hz,
1H), 8.23 (s, 1H), 8.17 (d, J=8.6 Hz, 1H), 7.88 (d,
J=8.2 Hz, 2H), 7.46±7.52 (m, 2H), 7.38±7.42 (m, 2H),
7.33 (d, J=7.0 Hz, 1H); 13C NMR (CD3COCD3) d
167.2, 150.1, 143.1, 139.0, 138.2, 134.4, 132.5, 132.1,
129.2, 127.3, 126.7, 125.8, 125.9, 123.3 (expect 16 aro-
matic C, obsd 13); HRMS calcd for C17H11O4N
293.0688, obsd 293.0686.
N-[4-[N-[2(R)-(tert-Butoxycarbonyl)amino-3-(triphenyl-
methyl)thio]propyl]amino-2-(1-naphthyl)benzoyl]-methionine
methyl ester (27). Compound 25 was reduced to the
corresponding amine by stannous chloride as described
for compound 20. This amine was coupled with N-Boc-
S-trityl-l-cysteinal under reductive amination condi-
tions. After column chromatography (ethyl acetate:hex-
ane, 1:1), compound 27 was obtained (40%, two steps).
1
TLC showed a single spot, but H NMR showed dia-
stereomers caused by the restricted rotation of C±C
1
The coupling of 4-nitro-2-(1-naphthyl)benzoic acid with
l-methionine methyl ester in the presence of EDCI and
HOBT provided the desired product (yield 95%). TLC
of the product showed a single spot, but 1H NMR
showed the presence of diastereomers caused by the
restricted C±C bond rotation. Variable temperature
1H NMR spectrum indicated that peaks at 8.14, 8.00,
3.56, 3.51, 1.83 and 1.74 became broad as temperature
was increased and coalescence temperatures were
ꢁ373 K for methionine side chain methyl group, 383
K for methyl ester group and 393 K for aromatic
hydrogen. Mp 114±115ꢀC. [a]2d5 +37.4 (c 0.7, EtOAc).
1H NMR (CDCl3) d 8.33±8.38 (m, 1H), 8.26 (m, 1H),
8.14 (d, J=8.5 Hz, 0.5H), 8.00 (d, J=8.5 Hz, 0.5H),
7.94±7.98 (m, 2H), 7.42±7.65 (m, 5H), 5.98 (t, 1H,
amide), 4.42 (m, 1H, Met a H), 3.56 (s, 1.5H, OCH3),
3.51 (s, 1.5H, OCH3), 1.83 (s, 1.5H, SCH3), 1.74 (s,
1.5H, SCH3), 1.56±1.64 (m, 1H, Met CH2), 1.33±1.45
(m, 2H, CH2S), 1.09±1.14 (m, 1H, Met CH2); 13C NMR
(CDCl3) d 171.4, 171.1, 166.2, 165.5, 148.8, 148.3, 141.5,
140.6, 139.4, 135.8, 135.1, 133.5, 131.3, 130.9, 130.4,
129.6, 129,4, 128.8, 128.6, 127.4, 127.3, 126.7, 126.6,
126.3, 125.5, 124.7, 124.4, 122.9, 52.4, 51.5, 31.0, 28.8,
28.6, 15.0. Anal. calcd for C23H22O5N2S: C 63.01, H
5.02, N 6.39, S 7.31; found C 62.91, H 5.12, N 6.40, S
7.24.
bond. Mp 91±92ꢀC. H NMR (CDCl3) d 7.84±7.96 (m,
3H, aryl), 7.49±7.66 (m, 4H, aryl), 7.37±7.43 (m, 7H,
aryl), 7.14±7.27 (m, 9H, aryl), 6.60±6.63 (d, J=8.6 Hz,
1H, aryl), 6.33 (m, 1H, aryl), 5.67 (d, J=7.8 Hz, 0.6H,
amide), 5.60 (d, J=7.8 Hz, 0.4H, amide), 4.56 (br d,
J=6.2 Hz, 1H, Boc amide), 4.35±4.44 (m, 1H, Met a
H), 4.30 (br, 1H, NH), 3.78 (br m, 1H, Cys a H), 3.55 (s,
1.9H, OCH3), 3.38 (s, 1.1H, OCH3), 3.06 (t, J=5.8 Hz,
2H, CH2N), 2.44 (m, 2H, CH2SCPh3), 1.90 (s, 1H,
SCH3), 1.79 (s, 2H, SCH3), 1.57±1.68 (m, 0.5H, Met
CH2), 1.36±1.45 (m, 10H, Boc, Met CH2), 1.23±1.32 (m,
2H, CH2SCH3), 0.94±0.98 (m, 0.5H, Met CH2). Anal.
.
calcd for C50H53O5N3S2 0.5CH3COOC2H5: C 70.67, H
6.45, N 4.75; found C 70.63, H 6.47, N 4.86.
N-[4-[2(R)-Amino-3-mercaptopropyl]amino-2-(2-thienyl)-
benzoyl]-methionine hydrochloride (9). The fully pro-
tected compound 26 was ®rst hydrolyzed by 1 equiv of
0.5 N LiOH and then deprotected by TFA in the pre-
sence of triethylsilane. The TFA salt was dried and
washed with ether. The crude product (80% purity
according HPLC analysis) was puri®ed by preparative
HPLC as described for the preparation of FTI-276
(60%). The highly hydroscopic TFA salt was dissolved
in 1 N HCl and the aqueous solution was lyophilized to
give compound 9 as a hydrochloride salt. Mp 110ꢀC
(foaming). [a]2d5
15.5 (c 0.2, MeOH). 1H NMR
N[4-[N-[2(R)-(tert-Butoxycarbonyl)amino-3-(triphenyl-
methyl)thio]propyl]amino-2-(2-thienyl)benzoyl]-methionine
methyl ester (26). Compound 24 was ®rst reduced to its
corresponding amine and this amine was coupled with
N-Boc-S-trityl-l-cysteinal using the same method as for
the preparation of compound 20. After puri®cation by
¯ash column chromatography (ethyl acetate:hexane,
1:1), pure 26 was obtained (46%, two steps). Mp 79±
(CD3OD) d 7.40 (m, 2H, phenyl and thienyl), 7.15 (d,
J=3.3 Hz, 1H, thienyl), 7.03 (t, J=4.2 Hz, 1H, thienyl),
6.77 (s, 1H, phenyl), 6.71 (d, J=8.4 Hz, 1H, phenyl),
4.55 (dd, J=4.3, 4.9 Hz, 1H, Met a H) 3.39±3.57 (m,
3H, CH2N, Cys a H), 2.92 (dd, J=4.5, 14.6 Hz, 1H,
CH2SH), 2.80 (dd, J=5.7, 14.6 Hz, 1H, CH2SH), 2.20±
2.38 (m, 2H, CH2SCH3), 2.03±2.10 (m, 1H, Met CH2),
2.00 (s, 3H, SCH3), 1.81±1.92 (m, 1H, Met CH2); 13C
NMR (CD3OD) d 175.0, 173.0, 150.7, 143.2, 135.2,
131.1, 128.6, 127.7, 127.0, 126.3, 115.4, 112.5, 53.8, 53.1,
44.8, 31.7, 31.1, 25.3, 15.1; LRMS (EI) for C19H25O3
N3S3 439 (M+, 95), 202 (100); HRMS (EI) calcd
439.1058, obsd 439.1063.
80ꢀC (decomp). [a]2d5 +0.9 (c 0.85, EtOAc). H NMR
1
(CDCl3) d 7.60 (d, J=8.5 Hz, 1H, aryl), 7.39±7.42 (m,
6H, trityl), 7.30 (d, J=4.8 Hz, 1H, thienyl), 7.17±7.29
(m, 9H, trityl), 7.07 (m, 2H, thienyl), 6.50 (d, J=8.5 Hz,
1H, aryl), 6.43 (s, 1H, aryl), 6.04 (d, J=5.8 Hz, 1H,
amide), 4.70 (ddd, J=5.4, 5.8 Hz, 1H, Met a h), 4.63 (br
d, J=7.6 Hz, Boc amide), 4.22 (br t, 1H, NH), 3.75 (br,
1H, Cys a h), 3.66 (s, 3H, OCH3), 3.06 (t, J=5.7 Hz,
2H, CH2N), 2.45 (br d, 2H, CH2SCPh3), 2.26 (t,
J=7.6 Hz, 2H, CH2SCH3), 2.00 (s, 3H, SCH3), 1.96
2.02 (m, 1H, Met CH2), 1.73±1.83 (m, 1H, Met CH2),
1.41 (s, 9H, Boc); 13C NMR (CDCl3) d 172.0, 168.2,
155.6, 149.2, 144.3, 141.9, 133.5, 131.1, 129.4, 127.9,
127.4, 126.9, 128.8, 126.1, 123.4, 114.3, 111.7, 79.7, 67.0,
52.3, 51.7, 49.4, 47.0, 34.2, 31.6, 29.5, 28.2, 15.2;
N-[4-[2(R)-Amino-3-mercaptopropyl]amino-2-(1-naphthyl)-
benzoyl]-methionine tri¯uoroacetate (10). The fully pro-
tected compound 27 was deprotected using the same
method as for the preparation of compound 9. Final
product was isolated from preparative HPLC as descri-
bed before (51%). The purity of the ®nal product was
over 99% as indicated from HPLC analysis. Mp 89±
90ꢀC (decomp). [a]2d5 41.2 (c 0.4, MeOH). H NMR
1
showed complicated diastereomers caused by the
restricted rotation of aryl±aryl bond. 1H NMR
(CD3OD) d 7.86±7.94 (m, 2H, naphthyl), 7.73 (d,
J=8.6 Hz, 0.6H, phenyl), 7.35±7.67 (m, 5.4H, naphthyl,
. .
Anal. calcd for C44H49O5N3S3 0 4C6H14: C 67.13, H
6.58, N 5.06, S 11.57; Found C 67.00, H 6.76, N 5.06, S
11.47.

3022
Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
1
phenyl), 6.83±6.88 (m, 1H, phenyl), 6.63±6.67 (m, 1H,
phenyl), 4.17±4.23 (m, 1H, Met a H), 3.41±3.58 (m, 3H,
CH2N, Cys a H), 2.91 (dd, J=4.2, 14.5Hz, 1H, CH2SH),
2.80 (dd, J=5.3, 14.5Hz, 1H, CH2SH), 1.82 (s, 1.4H,
SCH3), 1.80 (s, 1.6H, SCH3), 1.65±1.77 (m, 1H, Met CH2),
1.41±1.52 (m, 2H, CH2SCH3), 1.09±1.32 (m, 1H, Met
method described for compound 31 (92%). H NMR
(Cl3CD) d 1.84±1.97 (m, AB, 2H), 1.96 (s, 3H), 2.18 (t,
7.3 Hz, 2H), 3.64 (s, 3H), 4.62 (m, 1H), 7.19 (d,
J=7.7 Hz, 1H), 7.30 (m, 1H), 7.73 (m, 2H), 8.14 (d,
J=2.2 Hz, 1H), 8.19 (dd, J=2.2, 8.5 Hz, 1H), 8.44 (d,
J=2.0 Hz, 1H), 8.48 (dd, J=1.4, 8.5 Hz, 1H); 13C NMR
(Cl3CD) d 15.21, 29.60, 30.68, 51.80, 52.59, 122.89,
123.41, 124.97, 129.60, 133.61, 136.09, 137.71, 141.27,
148.37, 148.70, 149.44, 167.18, 171.48.
.
.
CH2) Anal. calcd for C25H31O3N3S2 CF3 COOH H2O: C
52.68, H 5.20, N 6.83; found C 52.73, H 5.19, N 6.78.
Methyl 4-nitro-2-(2-pyridyl)benzoate (28). Methyl-2-bromo-
4-nitro benzoate (3 g, 12.41 mmol), (Ph3P)2PdCl2
(435 mg, 0.62 mmol) and CuI (236 mg, 1.24 mmol) were
dissolved in anhydrous toluene (10 mL) and LiCl (2.63 g,
62.04 mmol) and 2-trimethylstannylpyridine in anhydrous
toluene (5 mL) were added. The mixture was re¯uxed for
13 h and then cooled and ®ltered. Evaporation in
vacuum and ¯ash chromatography (from hexane:ether,
7:3 to ether) a€orded 2 g (62% yield) of pure compound
N-[4-Nitro-2-(4-pyridyl)benzoyl]-methionine methyl ester
(33). This compound was prepared with the same
method described for compound 31 (86%). H NMR
(Cl3CD) d 1.89±2.04 (m, AB, 2H), 2.02 (s, 3H), 2.25 (t,
6.9 Hz, 2H), 3.69 (s, 3H), 4.70 (m, 1H), 6.76 (d, J=7.4 Hz,
1H), 7.36 (d, J=4.6 Hz, 2H), 7.78 (d, J=8.4 Hz, 1H),
8.22 (d, J=2.2 Hz, 1H), 8.27 (dd, J=2.2, 8.4 Hz, 1H),
8.64 (d, J=4.6 Hz, 2H); 13C NMR (Cl3CD) d 15.43,
29.73, 30.83, 52.01, 52.76, 123.25, 123.48, 124.84, 129.76,
138.79, 140.97, 145.54, 148.60, 150.17, 166.92, 171.55.
1
1
28. H NMR (Cl3CD) d 3.69 (s, 3H), 7.29 (dd, J=4.6,
7.2 Hz, 1H), 7.56 (d, J=7.8 Hz, 1H), 7.79 (m, 1H), 7.85
(d, J=8.4 Hz, 1H), 8.24 (dd, J=2.2, 8.4 Hz, 1H), 8.40
(d, J=2.2 Hz, 1H), 8.62 (d, J=4.6 Hz, 1H); 13C NMR
(Cl3CD) d 52.49, 122.42, 122.99, 123.06, 124.27, 130.56,
136.84, 137.42, 141.73, 148.82, 149.30, 155.60, 167.90.
N-[4-[N-[2(R)-(tert-Butoxycarbonyl)amino-3-(triphenyl-
methyl)thio]propyl]amino-2-(2-pyridyl)benzoyl]-methionine
methyl ester (34). Compound 31 (370 mg, 0.95 mmol)
and Raney Ni (1 mg, catalyst) were dissolved in MeOH
(15 mL). The mixture was re¯uxed and then hydrazine
(92 mg, 2.87 mmol) was added. After 10 min of re¯ux-
ing, Raney Ni was removed. After evaporation of the
solvent, the residue was puri®ed by ¯ash column chroma-
tography to provide an amino derivative (97% yield).
Longer time of re¯uxing caused ester cleavage by the
Methyl 4-nitro-2-(3-pyridyl)benzoate (29). This com-
pound was prepared with the same method described
1
for compound 28 (53%). H NMR (Cl3CD) d 3.69 (s,
3H), 7.38 (dd, J=4.9, 7.8 Hz, 1H), 7.67 (d, J=7.8 Hz,
1H), 8.05 (d, J=8.4 Hz, 1H), 8.19 (d, J=2.0 Hz, 1H),
8.26 (dd, J=2.0, 8.4 Hz, 1H), 8.54 (m, 1H), 8.64 (m,
1H); 13C NMR (Cl3CD) d 52.71, 122.75, 123.12, 125.60,
131.56, 134.87, 135.62 135.98, 140.46, 148.36, 149.16,
149.73, 166.42.
1
hydrazine. H NMR (Cl3CD) d 1.94 (m, 2H), 2.13 (s,
3H), 2.34 (m, 2H), 3.72 (s, 3H), 4.59 (dd, J=4.4, 8.9 Hz,
1H), 6.75 (dd, J=1.6, 8.2 Hz, 1H), 6.81 (br s, 1H), 7.35
(m, 1H), 7.44 (d, J=8.2 Hz, 1H), 7.54 (m, 1H), 7.81 (m,
1H), 8.55 (d, J=4.6 Hz, 1H); 13C NMR (CD3OD) d
15.16, 31.03, 31.67, 52.81, 53.01, 114.71, 116.82, 123.59,
124.92, 125.32, 131.04, 138.16, 141.91, 149.51, 151.62,
160.09, 172.60, 173.86. This amino derivative was cou-
pled with N-Boc-S-trityl-(l)-cysteinal under the reduc-
tive amination conditions described before. After the
evaporation of methanol, the residue was puri®ed by
¯ash column chromatography (hexane:ethyl acetate 7:3)
Methyl 4-nitro-2-(4-pyridyl)benzoate (30). This com-
pound was prepared with the same method described
1
for compound 28 (45%). H NMR (Cl3CD) d 3.58 (s,
3H), 7.15 (d, J=5.5 Hz, 2H), 7.92 (d, J=8.5 Hz, 1H),
8.07 (d, J= 2.2 Hz, 1H), 8.16 (dd, J=2.2, 8.5 Hz, 1H),
8.54 (d, J= 5.5 Hz, 2H); 13C NMR (Cl3CD) d 52.41,
122.69, 122.82, 124.80, 131.20, 135.58, 140.91 146.52,
148.87, 149.50, 166.14.
1
to provide compound 34 (53%). H NMR (Cl3CD) d
N-[4-Nitro-2-(2-pyridyl)benzoyl]-methionine methyl ester
(31). The methyl benzoate of compound 28 was hydro-
lyzed with 2.0 equiv of lithium hydroxide (0.5 N) in
methanol at rt for 2 h. The mixture was acidi®ed with 1
N HCl and the aqueous solution was lyophilized. The
solid was coupled with (l)-methionine methyl ester
using coupling reagent EDCI (1.0 equiv) and HOBT
(1.0 equiv) and triethylamine. Solvents were evaporated
and the residue was puri®ed, without extraction,
through ¯ash column chromatography (hexane:ethyl
acetate, 1:1) to give pure 31 (83%). 1H NMR (Cl3CD) d
1.88±2.09 (m, AB, 2H), 2.01 (s, 3H), 2.35 (t, 4.18 Hz,
2H), 3.69 (s, 3H), 4.74 (m, 1H), 7.15 (d, J=7.8 Hz, 1H),
7.33 (dd, J=4.6, 7.5 Hz, 1H), 7.57 (d, J=7.8 Hz, 1H),
7.79 (m, 2H), 8.23 (dd, J=2.2, 8.5 Hz, 1H), 8.37 (d,
J=2.2 Hz, 1H), 8.62 (d, J=4.6 Hz, 1H).
1.31 (s, 9H), 1.73±1.95 (m, AB, 2H), 1.93 (s, 3H), 2.17 (t,
J=7.6 Hz, 2H), 2.35 (br s, 2H), 2.98 (br s, 2H), 3.55 (s,
3H), 3.57 (br s, 1H), 4.22 (br s, 1H), 4.61 (m, 2H), 6.41
(s, 1H), 6.44 (m, 1H), 6.90 (d, J=7.7 Hz, 1H), 7.19 (m,
10H), 7.31 (m, 6H), 7.47 (d, J=8.1 Hz, 1H), 7.61 (m,
1H), 8.53 (d, J=4.6 Hz, 1H); 13C NMR (Cl3CD) d
15.26, 28.26, 29.64, 31.65, 34.30, 46.95, 49.46, 51.86,
52.22, 67.00, 79.64, 111.87, 113.65, 122.26, 123.33,
124.12, 126.78, 127.93, 129.44, 130.85, 136.51, 140.40,
144.34, 148.68, 149.39, 155.68, 159.26, 168.88, 172.13.
N-[4-[N-[2(R)-(tert-Butoxycarbonyl)amino-3-(triphenyl-
methyl)thio]propyl]amino-2-(3-pyridyl)benzoyl]-methionine
methyl ester (35). Compound 32 was reduced with Raney
Ni as described for compound 34 to give the correspond-
ing amine (45%). 1H NMR (Cl3CD) d 1.97 (m, 2H), 1.99
(s, 3H), 2.23 (t, 7.4 Hz, 2H), 3.64 (s, 3H), 4.17 (br s, 1H),
4.61 (m, 1H), 6.29 (d, J=7.0 Hz, 1H), 6.47 (d,
J=2.2 Hz, 1H), 6.61 (dd, J=2.2, 8.2 Hz, 1H), 7.26 (m,
N-[4-Nitro-2-(3-pyridyl)benzoyl]-methionine methyl ester
(32). This compound was prepared with the same

Y. Qian et al. / Bioorg. Med. Chem. 7 (1999) 3011±3024
3023
1H), 7.46 (d, J=8.2 Hz, 1H), 7.64 (d, J=7.7 Hz, 1H), 8.50
(m, 2H); 13C NMR (Cl3CD) d 15.32, 29.70, 31.32, 51.83,
52.45, 113.82, 116.20, 123.08, 124.44, 130.57, 136.07,
136.46, 138.20, 148.53, 148.83, 148.96, 168.74, 172.13.
This amine was coupled with N-Boc-S-trityl-(l)-cysteinal
as described before to give 35 (46%). 1H NMR (Cl3CD) d
1.39 (s, 9H), 1.83±1.97 (m, AB, 2H), 2.00 (s, 3H), 2.27 (t,
J=7.4 Hz, 2H), 2.44 (br s, 2H), 3.07 (m, 2H), 3.66 (s, 3H),
3.78 (br s, 1H), 4.63 (m, 1H), 4.78 (br s, 1H), 6.16 (m, 1H),
6.34 (d, J=2.0 Hz, 1H), 6.50 (dd, J=2.0, 8.2 Hz, 1H),
7.28 (m, 10H), 7.43 (m, 6H), 7.51 (d, J=8.2 Hz, 1H), 7.71
(d, J=7.8 Hz, 1H), 8.57 (br s, 2H); 13C NMR (Cl3CD) d
15.33, 28.27, 29.70, 31.34, 34.25, 46.90 48.97, 51.86, 52.44,
67.11, 79.50, 111.46, 113.99, 123.22, 126.85, 127.97,
129.47, 130.64, 132.07, 136.58, 136.98, 138.14, 144.32,
148.00, 148.56, 149.69, 159.73, 168.58, 172.15.
was prepared from the deprotection of 35 as described
in the preparation of 11. 1H NMR (D2O) d 2.09 (s, 3H),
2.10 (m, 2H), 2.53 (m, 2H), 2.78 (m, 2H), 3.53 (m, 4H),
4.44 (m, 1H), 6.72 (d, J=2.0 Hz, 1H), 6.82 (dd, J=2.1,
8.5 Hz, 1H), 7.52 (d, J=8.5 Hz, 1H), 8.02 (m, 1H), 8.50
(d, J=8.1 Hz, 1H), 8.70 (br s, 2H); 13C NMR (CD3OD)
d 15.23, 25.05, 30.67, 30.74, 44.38, 53.14 (2C), 113.94,
115.77, 123.68, 128.11, 131.81, 136.24, 140.85, 141.15,
141.63, 147.69, 151.24, 172.39, 176.24.
N-[4-[2(R)-Amino-3-mercaptopropyl]amino-2-(4-pyridyl)-
benzoyl]-methionine tri¯uoroacetate (13). This com-
pound was prepared from the deprotection of 36 as
1
described in the preparation of 11. H NMR (CD3CN/
D2O) d 2.00±2.17 (m, AB, 2H) 2.13 (s, 3H), 2.45±2.60
(m, 2H), 2.99±2.93 (m, 2H), 3.63±3.48 (m, 4H), 4.49 (m,
1H), 6.80 (d, J=2.0 Hz, 1H), 6.88 (dd, J=2.0, 8.5 Hz,
1H), 7.56 (d, J 8.5 Hz, 1H), 7.99 (d, J=6.6 Hz, 2H), 8.73
(d, J=6.6 Hz, 1H); 13C NMR (CD3CN/D2O) d 15.08,
24.81, 30.52, 30.60, 44.05, 53.02 (2C), 114.20, 115.23,
123.52, 127.81, 131.70, 138.26, 141.53, 150.97, 160.64,
171.49, 175.93; ESMS 435.0 (calcd molecular weight
N-[4-[N-[2(R)-(tert-Butoxycarbonyl)amino-3-(triphenyl-
methyl)thio]propyl]amino-2-(4-pyridyl)benzoyl]-methionine
methyl ester (36). Compound 33 was reduced with
SnCl2 dihydrate as described before to give the corre-
1
sponding amine (60%). H NMR (Cl3CD) d 2.00±2.20
. .
(m, AB, 2H), 2.04 (s, 3H), 2.37 (m, 2H), 3.69 (s, 3H),
4.50 (m, 1H), 6.67 (d, J=2.2 Hz, 1H), 6.73 (dd, J=2.2,
8.3 Hz, 2H), 7.36 (d, J=8.3 Hz, 1H), 7.43 (d, J=5.5 Hz,
2H), 8.48 (d, J=5.5 Hz, 2H); 13C NMR (Cl3CD) d
15.13, 31.15, 31.43, 52.81, 53.13, 114.81, 116.53, 124.92,
125.45, 131.25, 140.45, 149.69, 153.07, 159.21, 172.90,
173.87. This amine was coupled with N-Boc-S-trityl-(l)-
cysteinal under reductive amination conditions to give
434.57). Anal. calcd for C20H26O3N4S2 2CF3COOH
4H2O:C 42.42, H 4.30, N 8.24, S 9.43; found C 42.20, H
4.28, N 8.16, S 9.26.
Acknowledgment
We thank the National Institutes of Health (CA 67771)
for ®nancial support of this work.
1
36 (74%). H NMR (Cl3CD) d 1.20 ( br s, 9H), 1.96±
1.99 (m, AB, 2H), 1.98 (s, 3H), 2.26 (t, J=7.0 Hz, 2H),
2.42 (br s, 2H), 3.04 (br s, 2H), 3.62 (s, 3H), 3.76 (br s,
1H), 4.60 (m, 2H), 4.93 (d, J=7.4 Hz, 1H), 6.35 (br s,
2H), 6.48 (d, J=8.4 Hz, 1H), 7.18 (m, 11H), 7.23 (m,
6H), 7.36 (d, J=7.4 Hz, 1H), 8.50 (br s, 2H); 13C NMR
(Cl3CD) d 15.20, 28.14, 29.65, 31.09, 34.10, 46.80 49.28,
51.79, 52.29, 66.91, 79.51, 111.68, 113.26, 122.55,
123.62, 126.71, 127.84, 129.34, 130.42, 139.04, 144.24,
148.98, 149.39, 149.60, 155.61, 168.55, 172.00.
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