Development of a tripeptide mimetic strategy for the inhibition of protein farnesyltransferase

Article abstract of DOI:10.1016/S0040-4020(00)00890-5

This paper describes the development of a novel terphenyl-based tripeptide mimetic of the CAAX carboxy terminal sequence of Ras. We employ a concise synthesis to form a series of differently functionalized terphenyl inhibitors of protein farnesyltransfera

Full text of DOI:10.1016/S0040-4020(00)00890-5

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 9833±9841
Development of a Tripeptide Mimetic Strategy for the Inhibition
of Protein Farnesyltransferase
a
Mohit A. Kothare, Junko Ohkanda, Jeffrey W. Lockman, Yimin Qian,
a
a
a
Â
Michelle A. Blaskovich,b Said M. Sebtib, and Andrew D. Hamilton
*
a,
*
aDepartment of Chemistry, Yale University, P. O. Box 208107, New Haven, CT 06520, USA
bDrug Discovery Program, H. Lee Mof®tt Cancer Center and Research Institute, Department of Biochemistry and Molecular Biology,
University of South Florida, Tampa, FL 33612, USA
Received 14 June 2000; accepted 17 August 2000
AbstractÐThis paper describes the development of a novel terphenyl-based tripeptide mimetic of the CAAX carboxy terminal sequence of
Ras. We employ a concise synthesis to form a series of differently functionalized terphenyl inhibitors of protein farnesyltransferase (PFTase),
exempli®ed by 5, 6 and 7. The key reaction in the synthesis of the terphenyl methyl ester 13, and therefore 6 and 7, was the Pd-catalyzed
chemoselective Suzuki cross-coupling of 3-bromo-4-chloronitrobenzene 16 with an appropriate boronic acid derivative utilizing a commer-
cially available, electron rich phosphine ligand. We further show that one member of this series is a potent inhibitor of PFTase. q 2000
Elsevier Science Ltd. All rights reserved.
Introduction
residue is cysteine, the third and the second residues are
aliphatic (valine or isoleucine) and X is any amino acid
but usually methionine or serine. This modi®cation which
is required for Ras activity is catalyzed by the enzyme
protein farnesyltransferase (PFTase).8 The CAAX sequence
is the minimum recognition element for PFTase. Subse-
quent post-translational modi®cations of the Ras protein
include the proteolysis of the AAX residues and methylation
of the resulting cysteine carboxylic acid. These steps are not
obligatory for membrane association or malignant trans-
forming activity.6 Thus, inhibition of PFTase provides an
attractive target for blocking Ras function and therefore as
an anti-cancer therapy.
The mammalian ras genes (H, K and N) encode a family of
21 kDa GTP binding proteins that are intimately involved in
the regulation of mitogen-stimulated cell division.1±3 The
proteins cycle between their GTP (active) and GDP (inac-
tive) bound states, to regulate the transduction of biological
information from the plasma membrane to the nucleus.
Since only the GTP±Ras complex can trigger mitogen-
activated protein (MAP) kinase cascades (which eventually
lead to cell division), the hydrolysis of GTP by Ras is vital
to avoiding uncontrolled cell growth. Single amino acid
mutations of Ras at positions 12, 13, or 61 lead to
GTPase-de®cient Ras, resulting in uncontrolled cell
growth.3 These mutations in the Ras proteins are common
in human cancers and therefore blocking oncogenic Ras can
provide a new avenue in cancer chemotherapy.4
PFTase is a heterodimer composed of a 48 kDa a-subunit
and a 46 kDa b-subunit, and also requires Zn21 and Mg21
ions for activity.8 The PFTase active site is at the interface
of the two subunits and forms a ternary complex with both
farnesyl diphosphate (FPP) and the CAAX box of the Ras
protein.9 Crystallographic studies using a-hydroxy-
farnesylphosphonic acid and a CVIM tetrapeptide as FPP
and Ras analogues, respectively, suggest that both substrates
bind to PFTase in extended conformations.10 The tetra-
peptide is bound within the active site through hydrophobic
interactions in the region of the Ile-Met peptide bond and
also utilizes hydrogen bonding to the Ile backbone carbonyl.
Additionally, the cysteine sulfur is weakly bound to the
active-site zinc.
Ras proteins must be membrane-bound to participate in the
gene expression process through activation of the MAP
kinase cascades.5±7 Ras proteins are made in the cytosol,
however, a series of post-translational modi®cations cause
their translocation to the plasma membrane. The ®rst modi-
®cation of Ras is farnesylation of the cysteine residue of its
carboxyl terminal sequence CAAX,5 where the fourth
Keywords: protein farnesyltransferase (PFTase); inhibitor; chemoselective
Suzuki cross-coupling; aryl chloride; boronic acid; phosphine ligand;
terphenyl scaffold.
* Corresponding authors. Tel.: 11-203-432-5570; fax: 11-203-432-3221;
e-mail: andrew.hamilton@yale.edu; Said M. Sebti e-mail:
sebti@mof®tt.usf.edu; tel.: 1813-979-6734; fax: 1813-979-6748.
Similar in function to PFTase are the two classes of protein
geranylgeranyltransferases: PGGTase-I and PGGTase-II.11
Both enzymes attach geranylgeranyl groups, 20-carbon
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00890-5

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M. A. Kothare et al. / Tetrahedron 56 (2000) 9833±9841
9834
Figure 1. Previously reported PFTase inhibitors.
isoprenoid units, to the cysteine residues near their
substrates' carboxy terminus. Like PFTase, PGGTase-I
recognizes a CAAX tetrapeptide, but one with a terminal
leucine instead of other amino acids. PGGTase-II recog-
nizes proteins ending in Cys-Cys or Cys-X-Cys motifs.
Since protein geranylgeranylation is more prevalent than
farnesylation, it is imperative to design inhibitors that are
selective for PFTase over PGGTase-I.
spacer.13 This provided a rigid and hydrophobic link
between the cysteine and methionine residues. For example,
analogue (2) inhibited PFTase potently (IC50ˆ150 nM),
con®rming that parts of the tetrapeptide sequence could
be replaced with a structurally de®ned non-amino acid
fragment.
In an endeavor to further reduce the peptidic character of 2,
the Val-Ile-Met residues of CVIM (1) were replaced with
the more conformationally restricted 4-amino-30-carboxy-
biphenyl moiety, resulting in analogue 3. Since the para-
substituted benzene of 2 was topographically a small mimic
of the aliphatic Val-Ile moiety, further modi®cations were
also investigated. It was envisioned that introduction of an
additional phenyl ring onto the aromatic spacer of 2 would
result in a more hydrophobic molecule, that could interact
effectively with the hydrophobic binding pocket of PFTase.
This resulted in the synthesis of FTI-276 (4), an extremely
potent inhibitor of PFTase (IC50ˆ0.61 nM).14 The success
of scaffolds 3 and 4 suggested that combining their essential
features (phenyl substitution and a C-terminal benzoate)
could lead to an improved non-peptide inhibitor of PFTase.
Consequently, we designed a potential mimetic of the AAX
tripeptide based on a terphenyl derivative, exempli®ed by 5
(Fig. 2).
The CAAX tetrapeptides are potent inhibitors of PFTase.12a
However, due to their peptide character, these inhibitors are
metabolically unstable and are poorly taken up by the cells.
In an endeavor to circumvent these problems, we12b and
others12c±e have reported peptidomimetic inhibitors of
PFTase. Herein, we report the design, synthesis and bio-
logical evaluation of novel, terphenyl-based tripeptide
mimetics of CAAX, that are non-peptidic and cell perme-
able and are highly potent and selective inhibitors of
PFTase.
Design of tripeptide mimics as inhibitors of PFTase
Tetrapeptide CVIM (Fig. 1) inhibited PFTase in vitro with
an IC50 of 340 nM but is inactive at inhibiting Ras proces-
sing in whole cells. In an attempt to circumvent low cellular
uptake and metabolic instability problems associated with
peptide inhibitors, we had previously synthesized a series of
peptidomimetics wherein the central hydrophobic dipeptide
core (Val-Ile) was replaced by a 4-aminobenzoic acid
The advantage of this design is that it combines a rigidly
separated N- and C-terminus with a phenyl group that can
mimic the hydrophobic side chains of the AAX tripeptide. To
Figure 2. Designed PFTase inhibitors 5±7.

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M. A. Kothare et al. / Tetrahedron 56 (2000) 9833±9841
9835
Compound 5 superimposes well onto the extended confor-
mation of Ac-CVIM10 that is found in the X-ray crystal
structure bound within the PFTase active site. The distance
between the N-terminal cysteine sulfur and C-terminal
Ê
carboxylate carbon on terphenyl inhibitor 5 (13.2 A for 5)
Ê
is similar to that found in the substrate tetrapeptide (12.0 A
for Ac-CVIM). Superimposition of the N- and C-termini of
the two molecules (Fig. 3) places the central phenyl ring of 5
near the substrate's valine residue and projects another
phenyl ring in the position corresponding to the isoleucine
side chain of the tetrapeptide. These results con®rmed the
potential mimicry of the AAX tripeptide by our terphenyl
derivatives and encouraged us to seek an effective route to
their synthesis.
Figure 3. Overlaid structures of terphenyl inhibitor 5 and Ac-CVIM.
investigate the structural similarities of our terphenyl peptido-
mimetics with the Ras CAAX sequence, molecular modeling
was employed (Fig. 3). Terphenyl compound 5 was
modeled in the absence of solvent with the OPLS-AA force
®eld, using the boss4.1 software package.15 Although
compound 5 is certain to exist in aqueous solution as its
zwitterion, the neutral complex was modeled for computa-
tional simplicity.
Results and Discussion
Chemistry
Inhibitors exempli®ed by 3 and 4 were previously synthe-
sized in this laboratory as peptidomimetics of the CAAX
Scheme 1. Synthesis of analog 5.

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M. A. Kothare et al. / Tetrahedron 56 (2000) 9833±9841
9836
sequence (1) of protein farnesyltransferase (PFTase).16±18
We were interested in pursuing the synthesis of compounds
5±7, because of the above modeling studies and the novelty
of these structures as PFTase inhibitors. In our initial
strategy (Scheme 1), commercially available nitroaniline 8
was brominated and the resulting aryl bromide 9 was
subjected to a Suzuki cross-coupling reaction with phenyl-
boronic acid to afford biphenylaniline 10, which was trans-
formed under Sandmeyer conditions to aryl iodide 11.
Coupling of 11 with 3-methylphenylboronic acid under
Suzuki cross-coupling conditions, afforded terphenyl 12.
The methyl moiety of 12 was oxidized to afford the corre-
sponding terphenyl-carboxylic acid, which was then ester-
i®ed to its terphenyl±methyl ester 13. This ester was later
hydrolyzed and re-esteri®ed to the t-butyl ester 14. Catalytic
reduction of 14, followed by reductive amination of the
resultant amine with an appropriately protected cysteinal
afforded 15. Deprotection of 15 gave the desired terphenyl
inhibitor 5. While giving us access to the terphenyl pepti-
domimetics, this route was long and laborious. We therefore
wanted to explore alternative routes to modi®ed inhibitors
such as 6 and 7.
A facile entry into these terphenyl analogues would involve,
the chemoselective C±C bond formation of a differentially
functionalized aryl dihalide such as 16 with an appropriate
arylboronic acid derivative (Scheme 2). Until the recent
work of Buchwald et al.19 and Fu et al.,20 it was thought
that aryl chlorides (particularly electron rich/neutral or
sterically congested), were poor substrates for palladium-
catalyzed Suzuki cross-coupling reactions. However,
with the advent of electron rich ligands such as 2-(di-t-
butylphosphino)biphenyl (a) and 2-(dicyclohexylphos-
phino)biphenyl (b) (Fig. 4), it is now possible to perform
these reactions in an ef®cient manner.
We decided to apply this modi®cation to the synthesis of the
terphenyl mimetics, 6 and 7 (Schemes 2 and 3). Commer-
cially available nitrobenzene 16, was chosen as a retron for
the synthesis of scaffold 13. We envisioned a chemoselective
Scheme 2. Synthesis of the terphenyl scaffold 13.

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M. A. Kothare et al. / Tetrahedron 56 (2000) 9833±9841
9837
To test our synthetic strategy (chemoselective Suzuki), we
attempted coupling of 17, with commercially available
3-formylphenylboronic acid. Utilizing conditions outlined
in Scheme 2, we obtained the formyl±terphenyl analog 18
in a moderate yield (45%), due presumably to the hydrolytic
deboronation/instability of the formyl boronic acid under
the reaction conditions.21 To overcome this we synthesized
terphenyl±methylester 13, via 3-(methoxycarbonyl)phenyl-
boronic acid22 in a 86% yield (Table 1, entry 9).
Figure 4. Electron rich phosphine ligands (Strem Chemicals).
Suzuki cross-coupling based on the differential reactivity of
the bromine and chlorine substituents of 16. Treatment of 16
with phenylboronic acid (1.0 equiv.) and tetrakis(triphenyl-
phosphine)-palladium(0) afforded biphenyl chloride 17 in
excellent yield. If excess boronic acid (1.3±1.5 equiv.),
was present in the reaction mixture, substitution of the
chloro resulted.
In general, the choice of the palladium catalyst, base,
substrate, and solvents affects the outcome of a Suzuki
cross-coupling reaction.19,20 As indicated in Table 1 (entry
9), the use of 10 mol% Pd(OAc)2, 2-(dicyclohexylphos-
phino)biphenyl and K3PO4 was critical to the success of
the chloride-coupling reaction. Ligands such as 2-(di-t-
butylphosphino)biphenyl or t-tributylphosphine (which have
Table 1. Optimization of the chemoselective Suzuki cross-coupling of biphenyl chloride (17) with 3-(methoxycarbonyl)phenylboronic acid
Entry
mmol (17)
Catalyst (mol%)a
Ligand (4L/Pd)b
ArB(OH)2 (equiv.)c
Base (equiv.)
Conditionsd
Yield (%)
1
2
3
4
5
6
7
8
9
0.25
0.25
0.25
0.10
0.10
0.10
1.0
5
5
5
5
5
10
10
10
10
P(t-Bu)3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.5
Cs2CO3 (3)
KF (3)
608C/24 h
RT to 608C
608C/30 h
608C/overnight
608C/overnight
708C/1.5 h
70±758C/1.5 h
85±908C/3 h
85±908C/1 h
19
a
a
a
b
b
b
b
b
,10e
none
trace
trace
.90e
69
Cs2CO3 (3)
K3PO4 (3)
K3PO4 (2)
K3PO4 (4)
K3PO4 (4)
K3PO4 (4)
K3PO4 (4)
1.5
0.7
55
86
a Catalyst: Pd(OAc)2.
b aˆ2-(di-t-butylphosphino)biphenyl, bˆ2-(dicyclohexylphosphino)biphenyl. Refer, Fig. 4.
c ArB(OH)2: 3-(methoxycarbonyl)phenylboronic acid.
d Solvent: toluene.
e Yield was estimated by 1H NMR integration.
Scheme 3. Synthesis of analogues 6 and 7.

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M. A. Kothare et al. / Tetrahedron 56 (2000) 9833±9841
9838
been used in some aryl chloride Suzuki cross-couplings),
either failed or gave the desired product in a poor yield.
Finally, the use of the pinacol ester of 3-(methoxycarbonyl)-
phenylboronic acid under the aforementioned reaction
conditions did not afford the desired product. This under-
scores the importance of the reactivities of all the reagents,
particularly due to the recalcitrant nature of the Ar±Cl bond
to undergo oxidative addition.
by 8-fold as seen with 5. The phenyl substituent presumably
mimics the iso-butyl side chain of the Ile residue in CVIM
(as in Fig. 3) and binds into the large and complementary
hydrophobic pocket in PFTase. Replacement of the cysteine
residue of 5 with an arylamido or an alkylamido thiol group
(6 and 7) did not improve PFTase or PGGTase-I inhibition
activity. These attempts to rigidify the spacer between the
key thiol group and the terphenyl carboxylate were unsuc-
cessful presumably due to the strict steric requirements of
the PFTase active site. The ¯exible thioalkylamine 5 may be
able to access an optimal conformation that permits binding
to the zinc ion. In contrast, thioarylamide 6 and thioalky-
lamide 7 are less ¯exible and probably constrained in a less
optimal conformation.
Terphenyl ester 13 was then saponi®ed to the corresponding
acid 19 and was further reduced to the terphenyl amino acid
20. Scaffold 20 was then utilized for synthesizing the
PFTase inhibitors 6 and 7 (Scheme 3). Thus, mercapto-
benzoic acid 21 and mercaptopropanoic acid 22 were each
treated with isobutyl choloformate to give the correspond-
ing mixed anhydrides, which upon treatment with terphenyl
amino acid 20 afforded Boc-protected 6 and 7. Removal of
the protecting group under acidic conditions provided the
target compounds, 6 and 7. While this work was in progress,
Fu et al.23 also reported chemoselective Suzuki couplings of
aryl dihalides with an appropriate boronic acid, to prepare
substituted biaryls. However, to the best of our knowledge,
our work is the ®rst report of a chemoselective Suzuki cross-
coupling involving a 1,2-dihalo arene, such as 16, as a
substrate. Furthermore, our method affords the desired
peptidomimetic PFTase inhibitors in an ef®cient manner.
Conclusion
We have successfully demonstrated a chemoselective
Suzuki cross-coupling of an 1,2-dihalo arene such as 16,
with an appropriate arylboronic acid derivative. We have
also designed concise syntheses of PFTase inhibitors 5, 6
and 7. Incorporating a phenyl substituent into the inhibitors
was clearly shown to be an advantage with the superior
inhibition potency of 5 compared to 3. However, further
changes of the reduced cysteine N-terminus, as in 6 and 7,
gave a loss of activity.
Biological results
Experimental
Inhibitors 5±7 were evaluated against PFTase and
PGGTase-I from human Burkitt lymphoma (Daudi) cells
(Table 2).24 The reaction mixture for the assay also
contained [3H]FPP and recombinant H-Ras-CVLS
(PFTase), or [3H]GGPP and H-Ras-CVLL (PGGTase-I)
and different concentrations of inhibitors. The mixtures
were incubated for 30 min at 378C, ®ltered on glass ®ber
®lters, and processed for scintillation counting as described
previously.24
General
All solvents and reagents were purchased from Aldrich, WI
and used directly without puri®cation unless indicated
otherwise. 3-Bromo-4-chloronitrobenzene was purchased
from TCI, OR. 2-(Dicyclohexylphosphino)biphenyl was
purchased from Strem Chemicals, MA. CH2Cl2 was distilled
under nitrogen from calcium hydride. THF was distilled
under argon from sodium benzophenone ketyl. All appara-
tus was oven-dried and cooled in a desiccator, prior to use.
1H and 13C NMR were procured on a Bruker-400 instru-
The IC50 values for inhibition of PFTase are collected in
Table 2 and con®rm the basis of our peptidomimetic strat-
egy. The tetrapeptide CVIM (1) inhibited PFTase with an
IC50 of 340 nM.25 Compound 3, bearing the comformation-
ally restricted biphenyl scaffold (PFTase IC50 114 nM),
showed greater potency than 1. This clearly con®rms that
an appropriately functionalized biphenyl group can act as an
effective mimic of the extended backbone conformation of
the last three amino acids of the Ras protein. Moreover,
introducing an additional phenyl substituent onto the
biphenyl system improved the PFTase inhibition activity
1
ment, with CDCl3 as the internal reference for H (d 7.26)
and 13C (d 77.06). Mass spectral analyses were performed at
University of Illinois at Urbana Champaign, IL. HRMS
(FAB and EI) were procured on Micro-mass 70-4F and
Micro-mass VSE instruments respectively. Reactions were
monitored on silica gel TLC plates, (Aldrich) and visualized
under a uv-lamp (254 nm) or staining with I2, KMnO4,
Vanillin, Indophenol and/or Ninhydrin, where appropriate.
Chromatographic separations were performed on silica gel
columns (2.5£15 cm, Merck grade silica, 240±400 mesh,
Table 2. Inhibition of PFTase and PGGTase-I by CAAX peptidomimetics
Ê
60 A) employing the gravity or ¯ash technique. Fractions
Compound
IC50 (nM) PFTase
IC50 (nM) PGGTase-I
containing 20 mL of the eluent were collected and then
combined, unless otherwise noted. Melting points were
obtained on a Electrochem melting point apparatus and
are uncorrected.
CVIM, (1)a
340
114
0.61
15
.10 000
3800
±
3b
10 000
50
640
.10 000
.10 000
FTI-276, (4)c
5
6
7
2-Phenyl-4-nitro-30-methylbiphenyl (12). 4-Nitroaniline 8
(13.8 g, 0.10 mol) was suspended in 125 mL of acetic acid.
To this solution was added a solution of bromine (15.92 g,
0.10 mol) in 75 mL of acetic acid through a dropping funnel
over a period of 1 h. The mixture was stirred at room
a Ref. 25.
b Ref. 14.
c Ref. 17.

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M. A. Kothare et al. / Tetrahedron 56 (2000) 9833±9841
9839
temperature for 1 h and then warmed to 608C. The resulting
solution was poured into 300 mL of ice-water and the pre-
cipitate was ®ltered. The solid was dissolved in ether and
washed with NaHCO3. After evaporating solvents, the crude
solid was recrystallized from methanol±water to give pale
yellow crystals of 9 (16.5 g, 76%). mp 99±1008C; 1H NMR
(CDCl3, 400 MHz): d 8.38 (s, 1H, ArH), 8.04 (d, Jˆ8.7 Hz,
1H, ArH), 6.74 (d, Jˆ8.7 Hz, 1H, ArH), 4.83 (br s, 2H,
NH2).
(d 6.44, singlet).The mixed brominated compounds
(3.42 g) were dissolved in 50 mL of hot methanol and
then AgNO3 (3.63 g, 21.3 mmol) in 70 mL of methanol
and 5 mL of water was added. The mixture was re¯uxed
for 30 min and then ®ltered. The ®ltrate was washed and
dried. 1H NMR showed 30% of aldehyde, 48% of dimethyl
acetal and 24% of methyl ether. This mixture was treated
with 10 mL of TFA and 2 drops of water to convert the
acetal to the aldehyde. After workup and evaporating
solvents, the residue was treated with tetrabutylammonium
permanganate (2.51 g, 6.95 mmol) in 20 mL of pyridine and
was stirred for 10 h at room temperature. The mixture was
poured into a solution of sodium sul®te (6 g) in 100 mL of
5N HCl and then extracted with ethyl acetate. After evapor-
ating solvents, the residue was extracted with 1N NaOH and
then acidi®ed with 3N HCl. After ethyl acetate extraction
and evaporating solvents, a solid (1.6 g) was obtained. This
solid was mixed with 100 mL of methanol and 3.0 mL of
thionyl chloride and re¯uxed for 5 h. After ¯ash column
2-Bromo-4-nitroaniline 9 (10.85 g, 50.0 mmol) was coupled
with phenylboronic acid (6.4 g, 52.5 mmol) in the presence
of Pd(OAc)2 (575 mg, 5% equiv.) in aqueous acetone
solution. The product was puri®ed by ¯ash column chroma-
tography (ethyl acetate/hexaneˆ2.5:1) to give 2-phenyl-4-
nitroaniline 10 as pale yellow crystals (8.2 g, 77%). mp
1
124±1258C; H NMR (CDCl3, 400 MHz): d 8.09 (d, Jˆ
8.5 Hz, 1H, ArH), 8.07 (s, 1H, ArH), 7.50 (d, Jˆ8.1 Hz,
2H, ArH), 7.43 (m, 3H, ArH), 6.71 (d, Jˆ8.5 Hz, 1H,
ArH), 4.50 (br s, 2H, NH2).
chromatography,
biphenyl 13 was obtained as an oil which solidi®ed on
standing (1.20 g, 46% for four steps).
2-phenyl-4-nitro-30-methoxycarbonyl-
The diazonium salt of 10 was synthesized according to a
known procedure.26 Sodium nitrite (2.51 g, 36.4 mmol) was
added in several portions to 19.4 mL of concentrated
sulfuric acid in a water bath. To this solution was added
23 mL of glacial acetic acid dropwise at 108C. The mixture
was stirred for 20 min at this temperature and then 2-phenyl-
4-nitroaniline (7.10 g, 33.0 mmol) was added in several
portions within 30 min. The brown suspension was stirred
at 108C for 1 h (6 mL of water was added to obtain a clear
solution) and at room temperature for 15 min. To this solu-
tion was added KI (8.71 g, 52.4 mmol) in 25 mL of 2N HCl.
The brown mixture was stirred at room temperature for
20 min and then heated to 708C. Sodium sul®te was added
to the cold mixture to remove iodine. The solid was ®ltered
and recrystallized from methanol to afford 11 as crystals
(8.42 g, 77%). mp 112±1148C; 1H NMR (CDCl3,
400 MHz): d 8.34 (d, Jˆ8.6 Hz, 1H, ArH), 8.09 (s, 1H,
ArH), 7.96 (d, Jˆ8.5 Hz, 1H, ArH), 7.46±7.52 (m, 3H,
ArH), 7.41±7.44 (m, 2H, ArH).
The above methyl ester 13 (1.10 g, 3.3 mmol) was hydro-
lyzed with NaOH (1 equiv.) in methanol. The resulting
carboxylic acid (1.05 g, 3.3 mmol) was treated with
0.43 mL of oxalyl chloride (4.93 mmol, 1.5 equiv.) and
the acid chloride was reacted with KOBut (0.55 g,
4.94 mmol) to give the tert-butyl ester (14). Compound 14
was puri®ed by ¯ash column chromatography (670 mg,
54.2%). mp 49±508C; 1H NMR (CDCl3, 400 MHz): d 8.31
(s, 1H, ArH), 8.27 (d, Jˆ8.4 Hz, 1H, ArH), 7.91 (d,
Jˆ7.2 Hz, 1H, ArH), 7.86 (s, 1H, ArH), 7.62 (d,
Jˆ8.4 Hz, 1H, ArH), 7.21±7.29 (m, 5H, ArH), 7.12±7.15
(m, 2H, ArH), 1.56 (s, 9H, C(CH3)3); 13C NMR (CDCl3,
400 MHz) d 165.1, 147.2, 145.9, 142.0, 139.2, 138.9,
133.5, 132.1, 131.4, 130.5, 129.6, 128.7, 128.4, 128.0,
127.6, 125.5, 122.3 (ArC), 81.2 C(CH3)3, C(CH3)3 28.0;
HRMS (EI, m/z) calcd for C23H21NO4 375.1470, obsd
375.1470.
Coupling of 1-iodo-2-phenyl-4-nitrobenzene 11 (3.25 g,
10.0 mmol) with 3-methylphenylboronic acid (1.36 g,
10.0 mmol) in the presence of palladium acetate (112 mg,
(Method B: Scheme 2, Compound 13). 3-(Methoxycarbonyl)-
phenylboronic acid (0.81 g, 4.5 mmol),22 palladium acetate
(0.07 g, 0.30 mmol, 10 mol%), 2-(dicyclohexylphosphino)-
biphenyl (0.42 g, 1.2 mmol, 4L/Pd) and K3PO4 (2.55 g,
12.0 mmol) were placed in a Schlenk ¯ask under nitrogen.
To this was added 5 mL of dry toluene and the mixture was
stirred at room temperature for 5 min. 2-Chloro-5-nitro-
biphenyl 17 (0.7 g, 3.0 mmol) was then added and the
mixture was heated at 85±908C for 1 h. At the end of this
period, the mixture was ®ltered and the ®ltrate was washed
with 10% citric acid (2£20 mL), followed by 1 M NaHCO3
(2£20 mL) and brine (2£20 mL). The organic layers were
combined and concentrated to afford a brown oil, which was
chromatographed on a silica gel column, using a gradient of
hexanes:EtOAc (100:0!90:10) to afford 13 as a yellow
solid (0.84 g, 86%). mp 1128C; TLC: Rf: 0.31 (silica gel,
hexanes:EtOAc 3:1); 1H NMR (CDCl3, 400 MHz): d 8.46±
8.19 (m, 2H, ArH), 7.89 (t, 2H, ArH), 7.54 (d, 1H, ArH),
7.20±7.05 (m, 7H, ArH), 3.83 (s, 3H, COOCH3); 13C NMR
(CDCl3, 400 MHz): d 166.37 (COOCH3), 147.18, 145.61,
141.91, 139.47, 138.63, 133.87, 131.35, 131.23, 130.29,
129.45, 128.72, 128.22, 128.00, 127.55, 125.32, 122.15
1
5% equiv.) gave compound 12 as an oil (2.75 g, 95%). H
NMR (CDCl3, 400 MHz): d 8.30 (s, 1H, ArH), 8.25
(d, Jˆ8.4 Hz, 1H, ArH), 7.58 (d, Jˆ8.5 Hz, 1H, ArH),
7.26 (m, 3H, ArH), 7.09±7.16 (m, 4H, ArH), 6.99 (s, 1H,
ArH), 6.88 (d, Jˆ7.0 Hz, 1H, ArH), 2.27 (s, 3H, CH3); 13
C
NMR (CDCl3, 400 MHz): d 147.1, 146.9, 141.8, 139.3,
139.2, 137.8, 131.4, 130.1, 129.5, 128.4, 128.1, 127.9,
127.4, 126.7, 125.4, 122.1 (ArC), 21.3 (ArCH3); LRMS
(EI) 289.
2-Phenyl-4-nitro-30-methoxycarbonylbiphenyl (13) and
2-phenyl-4-nitro-30-tert-butoxycarbonylbiphenyl (14).
(Method A: Scheme 1, Compound 13). Compound 12
(2.25 g, 7.78 mmol) was dissolved in 45 mL of carbon tetra-
chloride and then NBS (2.91 g, 16.3 mmol, 2.10 equiv.) was
added. After the addition of dibenzoyl peroxide (38 mg,
2% equiv.), the mixture was re¯uxed until all precipitate
1
disappeared. H NMR showed 25% of mono-brominated
(d 4.40, singlet) and 75% of di-brominated product

Â
M. A. Kothare et al. / Tetrahedron 56 (2000) 9833±9841
9840
(ArC), 52.06 (COOCH3); HRMS (FAB, m/z) calcd for
C20H16NO4 (M1H)1 334.1080, found 334.1078.
2-Phenyl-4-nitro-30-carboxybiphenyl (19). 2-Phenyl-4-
nitro-30-methoxycarbonylbiphenyl 13 (0.44 g, 1.33 mmol)
and NaOH (0.27 g, 6.0 mmol) were placed in a ¯ask. To
this was added 10 mL (1:5)H2O/THF. The mixture was
stirred at room temperature for 24 h. The mixture was
washed with Et2O (1£20 mL) to remove any starting
material. The aqueous layer was acidi®ed to pH,3 followed
by extraction with EtOAc (3£30 mL). The organic layers
were combined and concentrated to afford 19 as an off-white
solid (0.39 g, 93%). mp 2458C (dec.); TLC: Rf: 0.22 (silica
2-Phenyl-4-N-[2(R)-N-tert-butoxycarbonylamino-3-(tri-
phenylmethyl)thiopropyl]amino-30-tert-butoxycarbonyl-
biphenyl (15). Compound 14 (660 mg, 1.76 mmol) was
hydrogenated and then reacted with N-Boc-S-trityl-l-cys-
teinal (1.0 equiv.) in the presence of NaCNBH3 to give the
reductive amination product. After ¯ash column chroma-
tography (hexane/THF 4:1), compound 15 was isolated
(650 mg, 47%). mp 85±868C (dec.); 1H NMR (CDCl3,
400 MHz): d 7.76 (m, 2H, ArH), 7.41±7.44 (m, 6H,
ArH), 7.17±7.30 (m, 13H, ArH), 7.09±7.14 (m, 4H, ArH),
6.61 (d, Jˆ8.3 Hz, 1H, ArH), 6.55 (s, 1H, ArH), 4.57
(br, 1H, Boc amide), 3.87 (m, 2H, Cys a-H, NH), 3.14
(br t, 2H, CH2N), 2.48 (m, 2H, CH2S), 1.57 (s, 9H, But),
1.43 (s, 9H, Boc); Anal calcd for C50H52O4N2S: C 77.32, H
6.70, N 3.61; Found C 77.08, H 6.56, N 3.55.
1
gel, CHCl3:Me2CO:EtOH 100:40:8); H NMR (CDCl3 and
1 drop of CD3OD, 400 MHz): d 8.28±8.31 (m, 2H, Ar), 7.97
(m, 2H, ArH), 7.63 (m, 1H, ArH), 7.15 (m, 7H, ArH); 13C
NMR (CDCl3 and one drop of CD3OD, 400 MHz): d 167.98
(CvO), 146.99, 145.77, 141.86, 138.56, 133.73, 131.24,
131.19, 130.57, 130.39, 129.94, 129.31, 128.81, 128.05,
127.83, 122.01, 124.12 (ArC); HRMS (FAB, m/z) calcd
for C19H14NO4 (M1H)1 320.0924, found 320.0922.
2-Chloro-5-nitrobiphenyl (17). Phenylboronic acid (1.51 g,
10.05 mmol), tetrakis(triphenylphosphine)palladium(0)
(0.35 g, 0.30 mmol, 3 mol%) and K2CO3 (2.76 g,
20.0 mmol) were placed in a Schlenk ¯ask under nitrogen.
To this was added 5 mL (4:1) dry toluene/EtOH and the
mixture was stirred at room temperature for 5 min.
3-Bromo-4-chloronitrobenzene 16 (2.37 g, 10.0 mmol)
was added and the mixture was re¯uxed for 24 h. At the
end of this period, the mixture was ®ltered and the ®ltrate
washed with 10% citric acid (2£20 mL), followed by 1 M
NaHCO3 (2£20 mL) and brine (2£20 mL). The organic layers
were combined and concentrated to afford a brown oil, which
was chromatographed on a silica gel column, using a gradient
of hexanes:Et2O (100:0!80:10) to afford 17 as a colourless
oil, which solidi®ed to a white residue on standing (1.56 g,
88%). mp 908C; TLC: Rf: 0.75 (silica gel, hexanes:EtOAc
2-Phenyl-4-amino-30-carboxybiphenyl (20). 2-Phenyl-4-
nitro-30-carboxybiphenyl 19 (0.39 g, 1.23 mmol), SnCl2´2H2O
(1.40 g, 6.15 mmol) were placed in a ¯ask. To this was
added 20 mL (1:1) EtOAc/EtOH. The mixture was stirred
at re¯ux for 2 h and then cooled to room temperature. The
mixture was then slowly poured into an ice-cold solution of
saturated NaHCO3 (100 mL). The pH of the solution was
adjusted to pH,7, followed by extraction with EtOAc
(3£30 mL). The organic layers were combined and concen-
trated to afford 20 as an off-white solid (0.22 g, 62%). mp
1758C; TLC: Rf: 0.18 (silica gel, CHCl3:Me2CO:EtOH
1
100:40:8); H NMR (CDCl3, 400 MHz): d 8.31±7.79 (m,
1H, ArH), 7.20±7.04 (m, 10H, ArH), 6.72±6.70 (m, 1H,
ArH); 13C NMR (10% CD3OD in CDCl3, 400 MHz):
d 169.64 (CvO), 147.01, 142.68, 135.20, 131.98, 131.95,
131.46, 130.73, 130.53 130.25, 128.33, 128.10, 127.60
126.98, 118.02, 115.33 (ArC); HRMS (FAB, m/z) calcd
for C19H16NO2 (M1H)1 290.1182, found 290.1180.
1
3:1); H NMR (CDCl3, 400 MHz): d 8.16±8.05 (m, 2H,
ArH), 7.57±7.36 (m, 6H, ArH); 13C NMR (CDCl3,
400 MHz): d 146.27, 141.63, 139.33, 136.92, 130.61,
128.97, 128.45, 128.23, 125.89, 122.91 (ArC); HRMS (EI,
m/z): calcd for C12H8NClO2 233.0244 (M)1, found 233.0243.
2-Phenyl-4-N-[2(R)-amino-3-mercaptopropyl]amino-30-
carboxybiphenyl tri¯uoroacetate (5). Compound 15
(440 mg) was deprotected by TFA in the presence of
triethylsilane. The crude product (90% purity) was puri®ed
by preparative HPLC to give compound 5 (145 mg, 52%).
mp 89±908C (dec); [a]D25ˆ112.1 (cˆ0.3, MeOH); 1H NMR
2-Phenyl-4-nitro-30-formylbiphenyl (18). 3-Formylphenyl-
boronic acid (0.02 g, 0.15 mmol), palladium acetate
(0.002 g, 0.01 mmol, 10 mol%), 2-(dicyclohexylphos-
phino)biphenyl (0.01 g, 0.04 mmol, 4L/Pd) and K3PO4
(0.06 g, 0.30 mmol) were placed in a Schlenk ¯ask under
nitrogen. To this was added 5 mL (4:1) of dry toluene/EtOH
and the mixture was stirred at room temperature for 5 min.
This was followed by the addition of 2-chloro-5-nitro-
biphenyl 17 (0.02 g, 0.10 mmol) and the mixture was
re¯uxed for 24 h. At the end of this period, the mixture
was ®ltered and the ®ltrate washed with 10% citric acid
(2£20 mL), followed by 1 M NaHCO3 (2£20 mL) and
brine (2£20 mL). The organic layers were combined and
concentrated to afford a brown oil, which was chromato-
graphed on a silica gel column, using a gradient of hexane-
s:EtOAc (100:0!90:10) to afford 18 as a yellow oil
(0.014 g, 45%). TLC: Rf: 0.40 (silica gel, hexanes:EtOAc
3:1); 1H NMR (CDCl3, 400 MHz): d 10.00 (s, 1H, ArCHO),
8.46 (s, 1H, ArH), 8.21 (d, 1H, ArH), 7.92 (d, 1H, ArH),
7.64±7.60 (m 4H, ArH), 7.52±7.44 (m 4H, ArH), 7.26
(s, 1H, ArH); HRMS (EI, m/z): calcd for C19H13NO3 (M)1
303.0895, found 303.0898.
(CD3OD, 400 MHz):
d
7.77 (m, 2H, ArH), 7.28
(d, Jˆ8.3 Hz, 1H, ArH), 7.18±7.24 (m, 5H, ArH), 7.08±
7.11 (m, 2H, ArH), 6.93 (d, Jˆ8.3 Hz, 1H, ArH), 6.87
(s, 1H, ArH), 3.56±3.65 (m, 2H, Cys a H, CH2N), 3.44±
3.53 (m, 1H, CH2N), 3.00 (dd, Jˆ4.7, 14.6 Hz, 1H, CH2SH),
2.88 (dd, Jˆ5.5, 14.6 Hz, 1H, CH2SH); 13C NMR (CD3OD,
400 MHz); d 169.9 (CvO), 148.9, 143.5, 143.1, 143.0,
135.6, 132.5, 132.0, 131.5, 130.9, 130.7, 129.0, 128.8,
128.0, 127.7, 116.1, 113.2 (ArC), 53.9, 45.3, 25.3; Anal
calcd for C22H22O2N2S´CF3COOH´1.2H2O: C 56.07, H
4.94, N 5.45; Found C 55.89, H 4.68, N 5.37.
2-Phenyl-[4-(3-mercaptobenzoyl)amino]-30-carboxyl-
biphenyl (6). To a solution of 21 (24 mg, 0.10 mmol) and
Et3N (15 mL, 0.10 mmol) in THF (0.5 mL) was added
isobutyl chloroformate (13.5 mL, 0.10 mmol) in THF
(0.5 mL) by syringe at 2208C. After stirring for 15 min at
2158C, a solution of 20 (13 mg, 0.04 mmol) in THF
(0.25 mL) was added by syringe at 2158C, and the mixture

Â
M. A. Kothare et al. / Tetrahedron 56 (2000) 9833±9841
9841
was stirred overnight at room temperature. After evapora-
References
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by ¯ash column chromatography with CHCl3:acetone:
EtOH 100:40:8 to give 2-phenyl-4-[3-S-(t-butoxy-lcarbonyl)-
mercaptobenzoyl] amino-30-carboxylbiphenyl as a yellow oil
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(24 mg, 44%). H NMR (CDCl3, 400 MHz): d 8.25 (s, 1H,
ArH), 8.10 (d, Jˆ7.0 Hz, 1H, ArH), 7.99 (d, Jˆ12.5 Hz, 1H,
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biphenyl (7). To a solution of 22 (24 mg, 0.10 mmol) and
Et3N (15 mL, 0.10 mmol) in dry THF (0.5 mL) was added
isobutyl chloroformate (13.5 mL, 0.10 mmol) in THF
(0.5 mL) by syringe at 2158C. After stirring for 15 min at
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(23 mg, 46%). 1H NMR (CDCl3, 400 MHz): d 7.96 (s, 1H,
ArH), 7.90±7.93 (m, 1H, ArH), 7.82 (br s, 1H, ArH), 7.64
(dd, Jˆ2.0, 8.0 Hz, 1H, ArH), 7.59 (d, Jˆ2.0 Hz, 1H, ArH),
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1
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(t, Jˆ8.4 Hz, 1H, SH), 7.91±7.94 (m, 2H, ArH), 7.80 (s, 1H,
NH), 7.63 (dd, Jˆ2.0, 8.4 Hz, 1H, ArH), 7.53 (d, Jˆ2.0 Hz,
1H, ArH), 7.41 (d, Jˆ8.4 Hz, 1H, ArH), 7.08±7.26 (m, 7H,
ArH), 2.90±2.95 (m, 2H, CH2S), 2.77 (t, Jˆ6.4 Hz, 2H,
CH2CO), 1.72 (t, Jˆ8.4 Hz, 1H, SH); HRMS (EI, m/z)
calcd for C22H19NO3S (M)1 378.1162, found 378.1164.
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Acknowledgements
We thank the National Institutes of Health (CA 67771) for
®nancial support of this work.