Structure-based design of imidazole-containing peptidomimetic inhibitors of protein farnesyltransferase

Article abstract of DOI:10.1039/b508184j

A series of imidazole-containing peptidomimetic PFTase inhibitors and their co-crystal structures bound to PFTase and FPP are reported. The structures reveal that the peptidomimetics adopt a similar conformation to that of the extended CVIM tetrapeptide,

Full text of DOI:10.1039/b508184j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Structure-based design of imidazole-containing peptidomimetic inhibitors of
protein farnesyltransferase
Junko Ohkanda,a Corey L. Strickland,b Michelle A. Blaskovich,c Dora Carrico,a Jeffrey W. Lockman,a
Andreas Vogt,e Cynthia J. Bucher,c Jiazhi Sun,c Yimin Qian,a David Knowles,d Erin E. Pusateri,a
Sa¨ıd M. Sebtic and Andrew D. Hamilton*a
Received 10th June 2005, Accepted 4th November 2005
First published as an Advance Article on the web 9th January 2006
DOI: 10.1039/b508184j
A series of imidazole-containing peptidomimetic PFTase inhibitors and their co-crystal structures
bound to PFTase and FPP are reported. The structures reveal that the peptidomimetics adopt a similar
conformation to that of the extended CVIM tetrapeptide, with the imidazole group coordinating to the
catalytic zinc ion. Both mono- and bis-imidazole-containing derivatives, 13 and 16, showed remarkably
high enzyme inhibition activity against PFTase in vitro with IC50 values of 0.86 and 1.7 nM,
respectively. The peptidomimetics were also highly selective for PFTase over PGGTase-I both in vitro
and in intact cells. In addition, peptidomimetics 13 and 16 were found to suppress tumor growth in
nude mouse xenograft models with no gross toxicity at a daily dose of 25 mg kg−1.
The crystal structure of PFTase bound to a CVIM tetrapeptide
(the CaaX tetrapeptide terminal sequence of K-Ras) and a
Introduction
farnesylpyrophosphate (FPP) analog has been solved.5–7 The
structure reveals that the CVIM binds to a large hydrophobic
pocket in the enzyme in an extended conformation forming two
hydrogen bonds with Gln 167a and Arg 202b, while the Cys
thiol group coordinates to the zinc ion. In earlier generations
of our peptidomimetic PFTase inhibitors, the central aliphatic
dipeptide in CaaX was replaced by a 4-amino-2-phenylbenzamide
spacer to afford the lead inhibitor, FTI-2768,9 (Fig. 1). FTI-276
was found to be a remarkably potent inhibitor in vitro against
PFTase with an IC50 value of 0.5 nM, however, the metabolically
unstable thiol group hampered further development. We10 and
others11 have demonstrated that replacement of the thiol group by
an imidazole maintains enzyme inhibition activity and that non-
thiol-containing peptidomimetics inhibit human tumor growth in
animal models in a more selective and potent manner than the
corresponding thiol contaning inhibitors.10b In this study, we have
replaced the free thiol group with differently lengthed imidazole
During the last decade protein prenylation has become a major
focus in anti-cancer research due to its critical role in the Ras- and
Rho-mediated signaling pathways that are aberrantly activated in
many human cancers.1–3 Protein prenylation is a post-translational
modification that involves the attachment of a C-15 farnesyl or a C-
20 geranylgeranyl group through a thioether linkage to a cysteine
residue near the carboxyl terminus of a target substrate protein.4
Three enzymes comprise the protein prenyltransferase family that
is responsible for catalyzing the prenylation of substrate proteins
in mammalian cells: protein farnesyltransferase (PFTase), type I
protein geranylgeranyltransferase (PGGTase-I), and type II pro-
tein geranylgeranyltransferase (PGGTase-II). PFTase transfers a
C15 farnesyl residue to a cysteine within the CaaX tetrapeptide
sequence found at the carboxyl terminus of prenylated proteins.
The central “aa” residues are usually aliphatic amino acids
while X is commonly methionine or serine. The related protein
PGGTase-I attaches a C20 geranylgeranyl lipid to the cysteine
of the CaaX sequence of target substrates, where the X residue
is predominantly leucine, isoleucine or phenylalanine. Thus, the
prenylation state of a protein, farnesylated or geranylgeranylated,
is dictated by the nature of the terminal X specificity residue. A
third prenyltransferase, PGGTase-II, transfers two geranylgeranyl
residues to protein trafficking Rab proteins containing Cys–Cys
or Cys–Ala–Cys sequences at the C-terminus.
aDepartment of Chemistry, Yale University, PO Box 208107, New Haven,
CT, 06520, USA. E-mail: andrew.hamilton@yale.edu
bStructural Chemistry and Tumor Biology Departments, Schering-Plough
Research Institute, 2015 Galloping Hill Road, K15-3-3855, Kenilworth, New
Jersey, 07033-0539, USA
cDrug Discovery Program, H. Lee Moffitt Cancer Center & Research
Institute, Departments of Oncology and Biochemistry & Molecular Biology,
University of South Florida, Tampa, FL33612, USA
dDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA, 15260,
USA
eDepartment of Pharmacology, University of Pittsburgh, Pittsburgh, PA,
15260, USA
Fig. 1 Design of imidazole-containing peptidomimetics.
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groups and investigated the scope of this change by introducing
various substituents on the N-1 of the imidazole and the ortho
position of the 2-phenyl group in the scaffold (Fig. 1). In addition,
we have solved the crystal structures of several ternary complexes
of PFTase bound to imidazole-containing peptidomimetics and
have assessed their antitumor potency in nude mouse xenograft
models of cancer.
Crystal structures
The crystal structure of the ternary complex of PFTase: FPP: FTI-
2148 (13) was solved and a view of the inhibitor bound into the
active site with the corresponding position of the CVIM tetrapep-
tide superimposed, is shown in Fig. 2. The structure confirmed our
peptidomimetic design strategy, since FTI-2148 adopts a similar
conformation to that of the extended conformation of CVIM
in the active site of PFTase. The two hydrogen bonds between
Gln167a/Met carboxylate, and Arg202b/amido carbonyl group
of the benzoate scaffold are well conserved. The N-1 position
of the imidazole group coordinates to the catalytic zinc ion, and
the hydrophobic biphenyl spacer binds to a hydrophobic pocket
created by aromatic amino acid residues Trp102b, Trp106b, and
Tyr361b.5 The crystal structure of PFTase bound to bis-imidazole
FTI-2287 (16) was also solved (Fig. 3). In this structure the N-1
of the first imidazole group coordinates to the zinc ion, and the
biphenyl scaffold and Met residue of the inhibitor adopt a similar
orientation to that observed in the mono-imidazole derivative FTI-
2148 (13), with the two key hydrogen bonds conserved. The second
imidazole group in 16 does not coordinate the zinc but projects
towards the a-subunit as shown in Fig. 3. The 2-H of this imidazole
Results and discussion
Chemistry
The synthetic approach to compounds 1–17 is shown in Scheme 1.
Compounds 1–14 were synthesized by the previously reported
procedures.9,12 Briefly, Suzuki coupling of 2-bromo-4-nitrobenzoic
acid methyl ester, which was derived from commercially avail-
able 2-bromo-4-nitrotoluene, afforded ortho-methyl substituted
biphenyl scaffold 18. The conversion of the nitro group by
reduction and Sandmeyer reaction to the corresponding iodide,
followed by palladium catalyzed reaction with zinc cyanide and
Pd(PPh3)4 gave 19. Hydrogenation in the presence of Boc2O
afforded the Boc-protected benzylamine derivative, which was
followed by hydrolysis of the methyl ester, and subsequent coupling
with L-methionine methyl ester to give 20. Reductive amination
of free benzylamine 21 with the respective imidazolecarboxyalde-
hydes proceeded efficiently to give the methyl ester derivatives,
and subsequent hydrolysis of the methyl esters afforded 13 and
15. The bis-imidazole-containing compounds were prepared by
alkylation of 21 with N-Boc-4-chloromethylimidazole13 followed
by deprotection, and hydrolysis to give 16 and 17, respectively.
˚
is located within van der Waals contact distance (ca. 4.5 A) with the
phenol ring system in Tyr166a suggesting that a CH–p interaction
may contribute to stabilizing the complex.
Fig. 2 View of the active site of PFTase ternary complex with FTI-2148
(13) (green) and FPP (gray with blue surface). The zinc ion is shown in
orange. The structure of CVIM tetrapeptide (gray) bound to the active site
of PFTase is superimposed.
In vitro and in vivo inhibition activity
Scheme 1 Reagents and conditions: (a) KMnO4, 75%; (b) SOCl2,
MeOH, 78%; (c) o-tolylB(OH)2, Pd(PPh3)4, K2CO3, 83%; (d) SnCl2, 100%;
(e) NaNO2, KI, 81%; (f) Zn(CN)2, Pd(PPh3)4, 91%; (g) H2, 60 psi, 10%
Pd–C, Boc2O, 99%; (h) LiOH, 100%; (i) H–Met–OCH3, EDCI, HOBt,
92%; (j) 50% TFA in CH2Cl2, 100%; (k) imidazolecarboxaldehyde, NaBH4,
86%; (l) 1-t-butoxycarbonyl-4-chloromethylimidazole, NaHCO3, DMF,
88%; (m) 50% TFA in CH2Cl2, 74%; (n) LiOH in THF, 77–100%.
The ability of these compounds to inhibit PFTase and PGGTase-I
in vitro was investigated by using partially purified PFTase and
PGGTase-I from human Burkitt lymphoma (Daudi) cells.14 En-
zyme preparations were incubated with [3H]FPP and recombinant
H-Ras-CVLS (PFTase) or [3H]GGPP and recombinant H-Ras-
CVLL (PGGTase-I) in the presence of different concentrations
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Modification of the second phenyl group in the scaffold led to
compounds 10–12 (Table 2). Interestingly, introducing a methyl
group at the ortho position of 1 led to compound 10, which
showed 10-fold higher potency against PFTase and improved the
selectivity over PGGTase-I (PGGTase-I/PFTase = 40 for 1; 100
for 10). Compound 11, in which the imidazole ring system was
attached to the scaffold at C-2, was less active, indicating that the
position of the imidazole nitrogen is critical for binding to the
active site. Replacement of the ortho-methyl group by a methoxy
group to give 12 did not lead to an improvement in inhibition
activity.
Replacement of the aniline-type scaffold in 10 by a benzylamine-
based system led to a highly potent PFTase inhibitor, 13 (Table 3,
FTI-2148) which showed an IC50 value of 0.86 nM. The corre-
sponding methyl ester prodrug 14 (FTI-2153) potently inhibited
H-Ras processing in whole cell-based assays (IC50 = 0.02 lM)
(Table 3). A related compound 15, which possesses a methyl group
at the C-5 position of the imidazole group, showed reduced activity
compared to 13. Furthermore, installing a second imidazole group
onto the benzyl amine nitrogen as in 16, gave remarkably high
inhibition potency in whole cells with single digit nanomolar IC50
value of 0.005 lM (Fig. 4 and Table 3). Somewhat surprisingly,
the methyl ester prodrug 17 (FTI-2295), which might have been
expected to have improved membrane penetration was found to
inhibit farnesylation in whole cells with a correspondingly higher
IC50 value of 0.02 lM. As shown in Fig. 4, 16 inhibits H-Ras
processing at 5 nM (lanes 5 and 6) without any disruption of
Rap1A processing, whereas 17 inhibits 50% processing only at
20 nM (lanes 13 and 14), suggesting the methyl ester prodrug did
not improve inhibition activity in whole cells. A comparison of
the whole cell activity of 13 (IC50 = 0.2 lM) and that displayed
by 16, reveals that installing a second imidazole group leads to
an improvement in inhibition by 40-fold. This result suggests
that the second imidazole group may contribute to an increase
in membrane permeability of the molecule, less protein binding,
better stability, or higher intracellular concentration.16 We further
investigated the in vivo activity of 13 and 16 using xenograft
nude mice models of human lung carcinomas. Fig. 5 shows the
tumor growth suppression after treatment with each compound
at a 25 mg kg−1 d−1 dose administered subcutaneously. After four
weeks, tumor growth was suppressed by 59 and 47% for 13 and
16, respectively. The tumor growth inhibition activity of 16 was
less marked than 13 in contrast to the high inhibition potency in
whole cells. Preliminary pharmacokinetic results (data not shown)
Fig. 3 View of the active site of PFTase ternary complex with FTI-2287
(16) (green) and FPP (gray with blue surface).
◦
of inhibitors. After incubation for 30 min at 37 C, the reaction
was stopped and filtered onto glass fiber filters to separate free
[3H]FPP from [3H]H-Ras. The IC50 values for the inhibition of
PFTase of a series of compounds 1–9 based on the aniline type
scaffold are reported in Table 1. The unsubstituted imidazole
derivative 1 showed good inhibition activity against PFTase
(IC50 = 28 nM). Introducing a benzyl group onto the N-1 position
of the imidazole group as in 2b retained the activity against
PFTase but gave higher selectivity over PGGTase-I compared
to 1 (IC50 PGGTase-I/PFTase: 41 for 1; 105 for 2b), whereas
the corresponding regioisomer, 2a, in which the imidazole ring is
linked to the aniline scaffold at its 4-position (series a), showed
lower activity. All compounds containing a bulky aromatic group
at N-1 (such as 3–5) showed lower activity against PFTase
compared to 2. The alkyl group containing compounds 6–9 were
also effective inhibitors. Again, the compounds in series b show
higher inhibition potency than in series a, and cyclohexylmethyl
substituted 9b showed higher selectivity, consistent with the fact
that the binding environment around the zinc metal in the active
site of PFTase is considerably different to that of PGGTase-I.15
Fig. 4 Effect of bis imidazole-containing compounds on processing of H-Ras and Rap1A in NIH 3T3 cells. Western blot analysis demonstrates the
inhibition of farnesylated H-Ras or geranylgeranylated Rap1A as seen by the band shift from processed (P) to unprocessed (U) protein. Lane 1: vehicle
control; lane 2: 5 lM FTI-277; lane 3: 5 lM GGTI-298; lanes 4 to 10: 0.001, 0.003, 0.01, 0.03, 0.1, 0.3 and 1 lM 16 (FTI-2287); lanes 11 to 17: 0.001,
0.003, 0.01, 0.03, 0.1, 0.3, and 1 lM 17 (FTI-2295).
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Table 1 Inhibition activity of 1–9 against mammalian PFTase and PGGTase-I
a
b
IC50/nM
IC50/nM
PFTase
Compound
R
H
PFTase
PGGTase-I
1150 (n = 2)
3900 700
PGGTase-I
—
1 (FTI-2118)
28 (n = 2)
603 201
—
2a (FTI-2208)
2b (FTI-2209)
46 28
4833 1305
3a (FTI-2218)
4200 (n = 2)
1400 (n = 2)
2600 (n = 2)
6950 (n = 2)
—
—
4a (FTI-2219)
4b (FTI-2221)
130 (n = 2)
435 (n = 2)
4550 (n = 2)
5a (FTI-2217)
5b (FTI-2220)
5600 (n = 2)
3500 (n = 2)
4550 (n = 2)
6b (FTI-2239)
—
—
36
8
>10 000 (n = 3)
7a (FTI-2236)
8a (FTI-2240)
1230 (n = 2)
1980 (n = 2)
>10 000 (n = 2)
5950 (n = 2)
—
22
—
6900
8b (FTI-2241)
9a (FTI-2237)
9b (FTI-2238)
1060 (n = 2)
6250 (n = 2)
41 18
>10 000 (n = 2)
observed during the course of these experiments, as documented
by no change in the weight, appetite and activity of the mice.
Conclusions
A series of imidazole-containing peptidomimetics was prepared,
and the crystal structures of their complexes with PFTase bound to
FPP showed that the peptidomimetics adopt a similar conforma-
tion to that of the extended CVIM tetrapeptide. In addition, two
distinct hydrogen bonds seen between the CVIM tetrapeptide and
PFTase active site were well conserved, and the N-1 position of the
imidazole group coordinates to the zinc ion. However, in the case
of bis-imidazole derivatives, the second imidazole group projects
away from the active site and is located near Tyr166awithin van der
Waals contact distance, suggesting a possible CH–p interaction.
Both mono- and bis-imidazole-containing derivatives, 13 and 16
showed remarkably high enzyme inhibition activity (IC50 = 0.86
and 1.7 nM, respectively), and suppressed tumor growth in nude
mouse xenograft models with no gross toxicity. Furthermore,
compound 16 showed higher inhibition activity in whole cells
than 13, suggesting that the role of the second imidazole group
Fig. 5 In vivo activity of 13 and 16 using xenograft nude mice models of
human lung carcinomas: 13, 25 mg−1 kg−1 d−1 (ꢀ); 16, 25 mg−1 kg−1 d−1
(ꢁ); control, (᭹).
suggest that 16 is less stable in serum than 13, presumably as
a result of its rapid metabolic degradation due to the second
imidazole group. It is worth noting that there was no gross toxicity
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Table 2 Inhibition activity of 10–12 against mammalian PFTase and
PGGTase-I
Experimental
General procedures
Melting points were determined with an electrothermal capillary
1
melting point apparatus and are uncorrected. H and 13C NMR
spectra were recorded on a Bruker AM-500, 400, and 300
spectrometer. Chemical shifts were reported in d (ppm) relative
to tetramethylsilane. All coupling constants were described in Hz.
Elemental analyses were performed by Atlantic Microlab, Inc.,
GA. Flash column chromatography was performed on silica gel
(40–63 lm) under a pressure of about 4 psi. Solvents were obtained
from commercial suppliers and purified as follows: tetrahydrofu-
ran and ether were distilled from sodium benzophenone ketyl;
dichloromethane was distilled over calcium hydride. Synthesized
final compounds were checked for purity by analytical HPLC,
which was performed using a Rainin HP controller and a Rainin
UV-C detector with a Rainin 250 × 4.6 mm, 5 lm Microsorb C-18
column, eluted with gradient 10 to 90% of CH3CN in 0.1% TFA
in H2O in 30 min. High-resolution mass spectra (HRMS) and
low-resolution mass spectra (LRMS) were performed on a Varian
MAT-CH-5 (HRMS) or VG 707 (LRMS) mass spectrometer.
Compounds 5, 8–9, 11, 13–149,12,17 were prepared by the previously
reported methods.
IC50/nM
Compound
R1
R2
PFTase
PGGTase-I
260 130
10 (FTI-2128)
CH3
2.8 2.0
11 (FTI-2130)
12 (FTI-2134)
CH3
155 (n = 2)
9.5 (n = 2)
388 (n = 2)
OCH3
3710 (n = 2)
may not only be to stabilize the complex but also to increase its
cell permeability. Additional SAR studies on the second imidazole
group of 16 are currently underway.
4-[N-(1H-Imidazol-4-yl)methylamino]-2-phenylbenzoylmethionine
(1, FTI-2118). 4-Hydroxymethylimidazole hydrochloride salt
Table 3 Inhibition activity of 13–17 against mammalian PFTase and PGGTase-I
IC50/nM
Processing IC50/lM
Compound
R1
R2
H
PFTase
PGGTase-I
1700 840
H-Ras
0.2
Rap1A
13 (FTI-2148)
0.86 0.43
>100
>30
>10
>30
14 (FTI-2153)
15 (FTI-2288)
16 (FTI-2287)
CH3
H
60 (n = 2)
4.0 2.3
1.7 0.5
>10 000 (n = 2)
660 330
0.02
>10
0.005
H
400 130
17 (FTI-2295)
CH3
46 (n = 2)
2700 (n = 2)
0.02
>10
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(1.0 g, 7.4 mmol) and p-toluenesulfonyl chloride were suspended
in 1 cm3 of distilled water and 3 cm3 of THF. Sodium hydroxide
(1 N) was added and the pH maintained at approximately 9 over
a period of 3 h. The reaction mixture was extracted with diethyl
ether (3 × 50 cm3). The extracts were combined, dried over
magnesium sulfate and concentrated. The residue was purified
by flash column chromatography (100% ethyl acetate) to give
4-hydroxymethyl-1-p-toluenesulfonylimidazole as a white solid
(1.1 g, 58%). Mp 109–112 C. H NMR (CDCl3) d 7.98 (s, 1H,
imidazole), 7.82 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 7.22
(s, 1H, imid), 4.51 (s, 2H, CH2OH), 2.42 (s, 3H, CH3). 13C NMR
(CDCl3) d 146.68, 144.68, 136.82, 134.72, 130.58, 127.54, 114.28,
57.66, 21.82. HRMS: m/e calcd for C11H12N2O3S, 252.0569,
found 252.0576.
This compound (0.7 g, 2.8 mmol) was dissolved into 7 cm3
of methylene chloride and manganese(IV) oxide (2.0 g, 23 mmol)
was added. The reaction mixture was stirred at room temperature
under a nitrogen atmosphere for 20 h. The solvent, methylene
chloride was filtered through a celite pad, and the combined
filtrates were evaporated. The residue was purified by flash
column chromatography (2 : 3 ethyl acetate–hexanes) to give
(1-p-toluenesulfonylimid◦azol-4-yl)carboxaldehyde as a white solid
(0.45 g, 64%). Mp 94–97 C. 1H NMR (CDCl3) d 9.79 (s, 1H), 8.00
(s, 1H), 7.87 (s, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.0 Hz,
2H), 2.38 (s, 3H). HRMS(FAB) m/e calcd for C11H10N2O3S:
250.0412, found 250.0419.
2.27–2.14 (m, 1H), 1.98 (s, 3H), 1.86–1.75 (m, 1H). FAB [M +
H]+ = 425+.
4-[N-(1-Benzyl-1H-imidazol-4-yl)methylamino]-2-phenylbenzoyl-
methionine (2a, FTI-2208). 4(5)-Hydroxy-methylimidazole (5.0g,
37 mmol) was dissolved in 20 cm3 of dry DMF containing
triethylamine (15.0 g, 148 mmol). Benzyl chloride (9.5 g,
55.5 mmol) was added to the reaction mixture via an addition
funnel over a 5 min period and refluxed for 3 h. Additional
benzyl chloride (3.2 g, 18.7 mmol) and triethylamine (2.6 g,
25.7 mmol) were added and the reaction mixture refluxed an
additional 1 h. The solvents were concentrated and the residue
taken up into chloroform. The solvent was dried over magnesium
sulfate and concentrated. The oily material was crystallized
from acetone to give◦1-benzyl-5-hydroxymethylimidazole (0.4 g,
◦
1
◦
1
5.7%). Mp 137–139 C (Lit.18 139–140 C). H NMR (CDCl3)
d 7.50 (s, 1H, imid), 7.31–7.35 (m, 3H, aromatic), 7.12–7.15
(m, 2H, aromatic), 6.99 (s, 1H, imid), 5.24 (s, 1H, CH2C6H5),
4.51 (s, 2H, CH2OH), 2.16 (br s, 1H, OH). An additional 0.3 g
of 1-benzyl-5-hydroxymethylimidazole was recovered from the
column and combined to give a total of 0.7 g (10%).
The acetone soluble material was further purified by a silica
gel column using 100% acetone as the eluent to give 1-benzyl-4-
hydroxymethylimidazole as a white solid (1.4 g, 20%). Mp 75–
77 ◦C, (Lit.18 79.5–80 ◦C). 1H NMR (CDCl3) d 7.52 (s, 1H, imid),
7.34–7.36 (m, 3H, aromatic), 7.12–7.18 (m, 2H, aromatic), 5.07 (s,
2H, CH2C6H5), 4.58 (s, 2H, CH2OH), 3.51 (br s, 1H, OH).
The above carboxaldehyde (0.10 g, 0.4 mmol) and N-(4-
amino-2-phenylbenzoyl)-methionine methyl ester hydrochloride
salt (0.16 g, 0.4 mmol) were dissolved in 10 cm3 of 95% methanol
and 5% acetic acid and stirred for 15 min before adding sodium
cyanoborohydride (0.05 g, 0.8 mmol). The reaction was stirred
for 1/2 h and twice an additional amount of the carboxaldehyde
(0.165 g, 0.66 mmol) and sodium cyanoborohydride (0.83 g,
1.3 mmol) were added. The reaction was stirred at room tem-
perature under a nitrogen atmosphere for 16 h. The reaction
mixture was concentrated, the residue taken up in ethyl acetate
and washed with a saturated solution of sodium bicarbonate. The
organic phase was dried over magnesium sulfate and concentrated.
The residue was purified by flash column chromatography (3 : 2
ethyl acetate–hexanes) to give 4-[N-(1-p-toluenesulfonylimidazol-
4-yl)methylamino]-2-phenylbenzoylmethionine methyl ester as a
white solid (0.075 g, 7.5%). 1H NMR (CDCl3) d 7.97 (s, 1H,
imid), 7.79 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 8.5 Hz, 1H),
7.41–7.34 (m, 8H), 7.18 (s, 1H, imid), 6.60 (d, J = 6.7 Hz, 1H),
6.46 (s, 1H,), 6.46 (s, 1H), 5.71 (d, 1H), 4.62 (dd, J = 6.10,
6.21 Hz, 1H), 4.27 (s, 2H), 3.64 (s, 3H), 2.44 (s, 3H), 2.10 (t,
J = 7.56 Hz, 2H), 2.00 (s, 3H), 1.93–1.82 (m, 1H), 1.69–1.60
(m, 1H).
1-Benzyl-4-hydroxymethylimidazole (0.6 g, 3◦.2 mmol) was
dissolved into 15 cm3 of dioxane and heated to 80 C. Manganese
dioxide (2.23 g, 25.6 mmol) was added at once and heating
continued for 6 h. The reaction was monitored by TLC using 100%
acetone as the eluent. After cooling slightly, the reaction mixture
was filtered through a celite plug followed by several washings
of methylene chloride. The filtrate was dried over magnesium
sulfate, concentrated, and the residue was purified by flash column
chromatography using 1 : 1 ethyl acetate–hexanes as the eluent to
give 1-benzyl-4-imidazolecarboxaldehyde as an oil (0.55 g, 93%).
The above carboxaldehyde (0.21 g, 1.1 mmol) and N-(4-amino-
2-phenylbenzoyl)-methionine methyl ester hydrochloride9 (0.4 g,
1.0 mmol) were dissolved in 10 cm3 of methanol and stirred for
15 min before adding dropwise 2 cm3 of a methanol solution
containing sodium cyanoborohydride (0.034 g, 1.1 mmol). The
reaction was stirred at room temperature for 4 h. The reaction
mixture was concentrated and the residue taken up in ethyl acetate
and washed with a saturated solution of sodium bicarbonate
followed by a saturated solution of sodium chloride. The organic
phase was dried over magnesium sulfate and concentrated. The
residue was purified by flash column chromatography using a
gradient of 3 : 2 ethyl acetate–hexanes to 100% ethyl acetate as
the eluent. The product was crystallized from ethyl acetate and
hexanes to give 4-[N-(1-benzyl-1H-imidazol-4-yl)methylamino]-
2-phenylbenzoylmethionine methyl ester as a white solid (0.38 g,
The above protected sulfonamide (0.07◦5 g, 0.13 mmol) was
dissolved into 2 cm3 of THF and cooled to 0 C. Lithium hydroxide
(2 cm3, 0.5 M) was slowly added to the reaction mixture and stirred
for 4 h. The pH was adjusted to 4 with 1 N HCl and excess THF
was removed under vacuum. The aqueous layer was lyophilized
and the resulting solid was purified by reverse phase preparative
HPLC. All fractions containing not less than 98.8% were pooled
◦
1
72%). Mp 105–107 C. H NMR (CDCl3) d 7.67 (s, 1H), 7.66
(d, J = 8.6 Hz, 1H, aromatic), 7.35–7.45 (m, 9H, aromatic), 7.18
(m, 1H, aromatic), 6.86 (s, 1H), 6.66 (dd, J = 8.6, 2.4 Hz, 1H,
aromatic), 6.51 (d, J = 2.4 Hz, 1H, aromatic), 5.67 (d, J = 7.7 Hz,
1H), 5.10 (s, 2H, CH2C6H5), 4.61 (m, 1H, a met-H), 4.33 (s, 2H,
CH2NH), 3.64 (s, 3H, OCH3), 2.09 (t, J = 7.7 Hz, 2H, CH2SCH3),
2.00 (s, 3H, SCH3), 1.87 (m, 1H, CH2CH2SCH3), 1.65 (m, 1H,
1
and lyophilized to give 1 as a TFA salt (0.03 g, 52%). H NMR
(DMSO-d6) d 14.44 (s, 2H), 12.60 (s, 1H), 9.01 (s, 1H), 8.07 (d, J =
7.8 Hz, 1H), 7.56 (s, 1H), 7.30–7.24 (m, 8H), 6.65 (d, J = 1.5 Hz,
1H), 6.59 (s, 1H), 4.41 (s, 2H), 4.21 (dd, J = 4.5, 4.2 Hz, 1H),
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CH2CH2SCH3). 13C NMR (CDCl3) d 172.3, 168.7, 149.8, 141.7,
141.4, 140.0, 137.4, 136.1, 131.4, 129.2, 128.9, 128.7, 128.5, 127.9,
127.5, 123.0, 116.9, 114.3, 111.8, 52.5, 51.9, 51.1, 41.8, 31.8, 29.6,
15.4.
This compound (0.10 g, 0.19 mmol) was dissolved into 5 cm3 of
THF and cooled to 0 ◦C. Lithium hydroxide (0.017 g, 0.4 mmol)
dissolved into 5 cm3 of distilled water was slowly added to
the reaction mixture and stirred for 2 h. The pH was adjusted
using 0.5 M HCl and the reaction mixture was lyophilized. The
lyophilized mixture was purified by reverse phase preparative
HPLC and fractions having assays greater than 99% were pooled
and lyop◦hilized to give 2b as the TFA salt (0.058 g, 60%). Mp
120–122 C. 1H NMR (CD3OD) d 8.99 (d, J = 1.2 Hz, 1H, imid),
7.49 (s, 1H, imid), 7.28–7.40 (m, 11H, aromatic), 6.57 (dd, J =
8.7, 2.4 Hz, 1H, aromatic), 6.45 (d, J = 2.4 Hz, 1H), 5.53 (s, 2H,
CH2C6H5), 4.44 (dd, J = 9.3, 4.2 Hz, 1H, a met-H), 4.38 (s, 2H,
CH2NH), 2.12–2.22 (m, 1H, CH2CH2SCH3), 2.01 (s, 3H, SCH3),
1.93–2.09 (m, 2H, CH2SCH3, 1.74–1.79 (m, 1H, CH2CH2SCH3).
13C NMR (CD3OD) d 175.1, 173.2, 150.3, 143.4, 142.5, 137.7,
135.0, 134.6, 131.2, 130.7, 130.4, 129.9, 129.6, 129.0, 128.8, 126.2,
120.0, 115.4, 112.2, 53.2, 52.0, 38.2, 31.9, 31.1, 15.2.
This compound (0.10 g, 0.19 mmol) was dissolved into 5 cm3 of
THF and cooled to 0 ◦C. Lithium hydroxide (0.017 g, 0.4 mmol)
was dissolved into 5 cm3 of distilled water and was slowly added
to the reaction mixture and stirred for 2 h. The pH was adjusted
using 0.5 M HCl and extracted with ethyl acetate. The extracts
were combined, washed first with a saturated solution of sodium
chloride, dried over magnesium sulfate and concentrated to give
◦
1
2a as a white solid (0.058 g, 60%). Mp 143–146 C. H NMR
(CD3OD) d 8.99 (s, 1H, imid), 7.51 (s, 1H, imid), 7.29–7.45 (m,
9H, aromatic), 6.65 (dd, J = 8.3, 2.2 Hz, 1H, aromatic), 6.58 (d,
J = 2.3 Hz, 1H, aromatic), 5.38 (s, 2H, CH2C6H5), 4.41–4.47 (m,
3H, CH2NH; a met-H), 2.01–2.12 (m, 2H, CH2SCH3), 2.00 (s,
3H, SCH3), 1.90–1.96 (m, 1H, CH2CH2SCH3), 1.73–1.78 (m, 1H,
CH2CH2SCH3). 13C NMR (CD3OD) d 175.1, 173.1, 150.5, 143.5,
142.5, 136.5, 135.6, 135.0, 131.2, 130.6, 130.5, 129.8, 129.6, 129.6,
128.7, 126.2, 120.8, 115.5, 112.4, 54.1, 53.2, 39.0, 31.9, 31.1, 15.2.
4-[N-(1-(1-Naphthyl)methyl-1H-imidazol-4-yl)methylamino]-2-
phenylbenzoylmethionine (3a, FTI-2218). 1-(1-Naphthylmethyl)-4-
(hydroxymethyl)imidazole was prepared from 4-(hydroxymethyl)-
imidazole hydrochloride (2.5 g, 18.6 mmol), 1-(chloromethyl)-
naphthalene (4.0 g, 20.4 mmol), and Na2CO3 (3.94 g, 37.2 mmol)
in DMF (30 cm3) by stirring at 100 ◦C for 10 h. After removal
of DMF by distillation under reduced pressure, the residue was
dissolved in acetone (30 cm3). The resulting white precipitates
were collected by filtration, and recrystallized from acetone to give
1-naphthylmethyl-4-(hydroxymethyl)imidazole as a sole product
(1.05 g, 24%). Mp 175–176 ◦C; 1H NMR (CD3OD) d 8.04 (d, J =
8 Hz, 1H, aryl H), 7.92 (d, J = 8 Hz, 1H, aryl H), 7.89 (d, J =
8 Hz, 1H, aryl H), 7.71 (s, 1H, imid-2H), 7.55 (m, 2H, aryl H),
7.47 (t, J = 7.0 Hz, 1H, aryl H), 7.35 (d, J = 7.0 Hz, 1H, aryl
H), 7.02 (s, 1H, imid-5H), 5.68 (s, 2H, CH2O), 4.46 (s, 2H, CH2).
Anal. calcd for C15H14N2O1: C, 75.61; H, 5.92; N, 11.76. Found:
C, 75.52; H, 5.96; N, 11.61. The characterization of the isomer
was based on the result of the NOE measurement in DMSO-d6
at room temperature. NOE between both imidazole-ring protons
and naphthylmethyl protons were 6%, respectively.
To a solution of 1-naphthylmethyl-4-hydroxymethylimidazole
(239 mg, 1 mmol) in dioxane (5 cm3) was added MnO2 (869 mg,
10 mmol) at 80 ◦C. After stirring for 1 h at 80 ◦C, the reaction
mixture was filtered through celite washing the solid with CH2Cl2
(100 cm3), and then the filtrate was concentrated to give the
corresponding aldehyde as colorless oil (253 mg, 100%). 1H NMR
(CDCl3) d 9.82 (s, 1H, CHO), 7.89–7.92 (m, 2H, aryl H), 7.79 (m,
1H), 7.64 (s, 1H, imid-2H), 7.57 (s, 1H, imid-5H), 7.52–7.55 (2H,
m, aryl H), 7.46 (t, J = 8.4 Hz, 1H, aryl H), 7.28 (d, J = 7.4 Hz,
1H, aryl H), 5.59 (s, 2H, NCH2).
4-[N-(1-Benzyl-1H-imidazol-5-yl)methylamino]-2-phenylbenzoyl-
methionine (2b, FTI-2209). 1-Benzyl-5-hydroxymethylimidazole
(0.6 g, 3.2 mmol) was dissolved into 15 cm3 of dioxane and heated
to 85 ◦C. Manganese dioxide (2.22 g, 26.0 mmol) was added at
once and heated for 6 h. After cooling, the reaction mixture was
filtered through a celite plug. The celite was washed several times
with methylene chloride. The filtrate was concentrated and the
oily residue was crystallized from hexanes to give 1-benzyl-5-
imidaz◦olecarboxaldehyde19 as a white solid (0.46 g, 78%). Mp
1
52–54 C; H NMR (CDCl3) d 9.77 (s, 1H, C(O)H), 7.85 (s, 1H,
imid), 7.75 (s, 1H, imid), 7.34–7.36 (m, 3H, aromatic), 7.20–7.24
(m, 2H, aromatic), 5.53 (s, 2H, CH2C6H5).
This carboxaldehyde (0.21 g, 1.1 mmol) and N-(4-amino-2-
phenylbenzoyl)-methionine methyl ester hydrochloride (0.4 g,
1.0 mmol) were dissolved in 10 cm3 of methanol and stirred
for 15 min before adding dropwise a methanol (2 cm3) solution
containing sodium cyanoborohydride (0.034 g, 1.1 mmol). The
reaction was stirred at room temperature for 6 h. The reaction
mixture was concentrated and the residue was taken up in ethyl
acetate and washed with a saturated solution of sodium bicar-
bonate followed by a saturated solution of sodium chloride. The
organic phase was dried over magnesium sulfate and concentrated.
The residue was purified by flash column chromatography using
100% ethyl acetate as the eluent. The product was crystallized from
ethyl acetate and hexanes to give 4-[N-(1-benzyl-1H-imidazol-
5-yl)methylamino]-2-phenylbenzoylmethionine methyl ester as a
◦
1
white solid (0.38 g, 72%). Mp 182–183 C; H NMR (CDCl3)
d 7.68 (s, 1H, imid), 7.30–7.46 (m, 9H, aromatic), 7.10 (s, 1H,
imid), 7.04–7.07 (m, 2H, aromatic), 6.49 (dd, J = 8.5, 2.2 Hz, 1H,
aromatic), 6.35 (d, J = 2.3 Hz, 1H, aromatic), 5.70 (d, J = 7.7 Hz,
1H, C(O)NH), 5.19 (s, 2H, CH2C6H5), 4.62 (dd, J = 7.3 Hz, 2H,
a met-H), 4.17 (d, J = 5.0 Hz, 2H, CH2NH), 3.94 (br t, 1H),
3.64 (s, 3H, OCH3), 2.09 (t, J = 7.7 Hz, 2H, CH2SCH3), 1.88
(m, 1H, CH2CH2SCH3), 1.66 (m, 1H, CH2CH2SCH3). 13C NMR
(CD3OD) d 172.2, 168.6, 149.0, 141.8, 141.1, 136.0, 131.3, 129.3,
128.8, 128.5, 128.0, 126.9, 123.8, 114.2, 111.9, 52.5, 51.9, 49.2,
37.9, 31.7, 29.6, 15.4.
A solution of 1-(1-naphthylmethyl)-4-imidazolecarboaldehyde
(236 mg, 1 mmol) and N-(4-amino-2-phenylbenzoyl)methionine
methyl ester hydrochloride (395 mg, 1 mmol) in MeOH (10 cm3)
was stirred for 15 min at room temperature. To the solution was
added NaBCNH3 (69 mg, 1.1 mmol) in MeOH (2 cm3). After
stirring for 4 h, the solvent was evaporated and the residue was
dissolved in CH2Cl2 (100 cm3), and then the organic layer was
washed with sat. NaHCO3 (30 cm3 × 2), brine, and dried (Na2SO4).
The product was purified by flash column chromatography (100 :
10 : 2 CHCl3–acetone–EtOH) to afford the product as a foam;
1
311 mg (54%) H NMR (CDCl3) d 7.84–7.96 (m, 3H), 7.67 (d,
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J = 8.5 Hz, 1H), 7.59 (s, 1H), 7.53–7.55 (m, 2H), 7.36–7.49 (m,
6H), 7.22 (d, J = 7.0 Hz, 1H), 6.86 (s, 1H), 6.65 (dd, J = 7.7 and
1.8 Hz, 1H), 6.50 (d, J = 1.8 Hz, 1H), 5.67 (d, J = 7.7 Hz, 1H), 5.55
(s, 2H, NCH), 4.68 (br s, 1H, NH), 4.61 (q, J = 7.4 Hz, 1H, aCH),
4.30 (s, 2H, NCH), 3.65 (s, 3H, OCH), 2.12 (t, J = 7.7 Hz, 2H,
CH S), 2.01 (s, 3H, SCH3), 1.84–1.98 (m, 1H, CHHCH2), 1.84–
1.98 (m, 1H, CHHCH2). HRMS (ESI) calcd for C34H34N4O3SH+
579.2430. Found 579.2422.
4-[N-(1-(2-Naphthyl)methyl-1H-imidazol-5-yl)methylamino]-2-
phenylbenzoylmethionine (4b, FTI-2221). 1-(2-Naphthylmethyl)-5-
(hydroxymethyl)imidazole was prepared using 4-(hydroxymethyl)-
imidazole hydrochloride and 2-(bromomethyl)naphthalene by
a similar procedure to that described for 1-benzyl-4 or 5-
hydroxymethylimidazole (8%). Mp 150–151 ◦C. 1H NMR
(CDCl3) d 7.83–7.84 (m, 2H, aryl H), 7.76–7.80 (m, 1H, aryl H),
7.58 (s, 1H, aryl H), 7.55 (s, 1H, imid-2H), 7.49–7.52 (m, 2H, aryl
H), 7.26 (dd, J = 8.5 and 1.8 Hz, aryl H), 7.04 (s, 1H, imid-4H),
5.41 (s, 2H, NCH2), 4.54 (s, 2H, CH2OH), 2.05 (br s, 1H, OH).
This compound was converted to 1-(2-naphthylmethyl)-
4-imidazolecarboxyaldehyde by a similar procedure to that
described for 1-benzyl-4- or 5-imidazolecarboxyaldehyde (100%).
Without purification this aldehyde was reacted with N-(4-
amino-2-phenylbenzoyl)methionine methyl ester hydrochloride
by a similar procedure to that described for 3a to give
4-[N-(1-(2-naphthyl)methyl-1H-imidazol-5-yl)methylamino]-2-
phenylbenzoylmethionine methyl ester as an oil (100%). 1H NMR
(CDCl3) d 7.15–7.80 (m, 14H, aryl H and imid-2H), 7.07 (s, 1H,
imid-4H), 6.43 (d, J = 8.0 Hz, 1H, aryl H), 6.24 (s, 1H, aryl
H), 5.72 (d, J = 7.5 Hz, 1H, CONH), 5.31 (s, 2H, NCH2), 4.60
(q, J = 7 Hz, 1H, aCH), 4.15 (d, J = 4 Hz, 2H, CH2NH), 3.99
(br s, 1H, NH), 3.64 (s, 3H, CO2CH3), 2.08 (t, J = 7.5 Hz, 2H,
CH2S), 2.00 (s, 3H, SCH3), 1.86 (m, 1H, CHHCH2), 1.66 (m, 1H,
CHHCH2). HRMS (ESI) calcd for C34H34N4O3SH+ 579.2430.
Found 579.2432.
To
a
solution of 4-[N-((1-naphthylmethyl-1H-imidazol-
4-yl)methyl)amino]-2-phenylbenzoylmethionine methyl ester
(150 mg, 0.26 mmol) in THF (5 cm3) was added 0.1 N LiOH
(5.2 cm3) at 0 ◦C, and then the mixture was stirred at room
temperature for 1 h. The mixture was concentrated and acidified
with 10% citiric acid. The product was extracted with CH2Cl2
(200 cm3), brine, and dried (Na2SO4). Evaporation of the solvent
1
gave the product 3a as an amorphous (115 mg, 78%). H NMR
(CD3OD) d 7.90–8.03 (m, 3H), 7.89 (s, 1H), 7.27–7.56 (m, 11H),
7.01 (s, 1H), 6.61 (d, J = 8.8 Hz, 1H), 6.52 (s, 1H), 5.65 (s,
2H), 4.36 (m, 1H), 4.25 (s, 2H), 2.15 (m, 1H), 2.05 (m, 1H),
2.01 (s, 3H), 1.90 (m, 1H), 1.73 (m, 1H). HRMS (ESI) calcd for
C33H32N4O3SH+ 565.2273. Found 565.2277.
4-[N-(1-(2-Naphthyl)methyl-1H-imidazol-4-yl)methylamino]-2-
phenylbenzoylmethionine(4a, FTI-2219). 1-(2-Naphthyl-methyl)-4-
(hydroxymethyl)imidazole was prepared using 4-(hydroxymethyl)-
imidazole hydrochloride and 2-(bromomethyl)naphthalene by
a similar procedure to that described for 1-benzyl-4 or 5-
hydroxymethylimidazole, white powder (16%). Mp 109–110 ◦C.
1H NMR (CDCl3) d 7.81–7.86 (m, 3H, aryl H), 7.64 (s, 1H,
imid-2H), 7.60 (s, 1H, aryl H), 7.50–7.54 (m, 2H, aryl H), 7.29
(dd, J = 8.4 and 1.7 Hz, 1H, aryl H), 6.90 (s, 1H, imid-5H), 5.25
(s, 2H, NCH2), 4.61 (s, 2H, CH2OH), 2.18 (br s, 1H, OH).
This compound was converted to 1-(2-naphthylmethyl)-
4-imidazolecarboxyaldehyde by a similar procedure to that
described for 1-benzyl-4 or 5-imidazolecarboxyaldehyde.
Without purification this aldehyde was reacted with N-(4-
amino-2-phenylbenzoyl)methionine methyl ester hydrochloride
by a similar procedure to that described for 3a to give
4-[N-(1-(2-naphthyl)methyl-1H-imidazol-4-yl)methylamino]-2-
phenylbenzoylmethionine methyl ester as an oil, 241 mg (93%).
1H NMR (CDCl3) d 1.63–1.68 (m, 1H, CHHCH2), 1.85–1.90
(m, 1H, CHHCH2), 2.00 (s, 3H, SCH3), 2.10 (t, J = 7.5 Hz, 2H,
CH2S), 3.64 (s, 3H, CO2CH3), 4.31 (br s, 2H, CH2NH), 4.60–4.64
(m, 2H, aCH and NH), 5.23 (s, 2H, NCH2), 5.68 (d, J = 7.5 Hz,
1H, CONH), 6.50 (d, J = 2.5 Hz, 1H, aryl H), 6.66 (dd, J = 8.0
and 2.5 Hz, 1H, aryl H), 6.85 (s, 1H, imid-5H), 7.25–7.86 (m, 14H,
aryl H and imid-2H). HRMS (ESI) calcd for C34H34N4O3SH+
579.2430. Found 579.2422.
The methyl ester was hydrolyzed in a similar manner to that
described for 3a to give 4b as white solid (74%). Mp 180–181 ◦C.
1H NMR (CD3OD) d 7.98 (s, 1H), 7.79 (m, 2H, aryl H), 7.51 (s,
1H, imid-2H), 7.43 (m, 2H, aryl H), 7.29 (m, 4H, aryl H), 7.22 (m,
3H, aryl H), 7.06 (s, 1H, imid-4H), 6.48 (d, J = 7 Hz, 1H, aryl
H), 6.31 (d, J = 1.8 Hz, 1H, aryl H), 5.47 (s, 2H, CH2N), 4.43
(m, 1H, aCH), 4.22 (s, 2H, CH2N), 2.15 (m, 1H, CHHS), 2.04 (m,
1H, CHHS), 1.99 (s, 3H, SCH3), 1.91 (m, 1H, CHHCH2), 1.72
(m, 1H, CHHCH2). HRMS (FAB) m/z calcd for C33H32N4O3S
H+ 565.2273. Found 565.2271.
4-[N -(1-(Isobutyl-1H -imidazol-5-yl)methylamino]-2-phenyl-
benzoylmethionine (6b, FTI-2239). 1-Isobutyl-5-(hydroxymethyl)-
imidazole was prepared using 4-(hydroxymethyl)imidazole
hydrochloride and 1-bromo-2-methylpropane by a similar pro-
cedure to that described for 1-benzyl-4 or 5-hydroxymethyl-
imidazole (12%). 1H NMR (CDCl3) d 7.42 (s, 1H, imid-2H), 6.91
(s, 1H, imid-4H), 4.62 (s, 2H, CH2O), 3.81 (d, J = 7.5 Hz, 2H,
NCH2), 2.13 (m, 1H, CH), 0.92 (d, J = 6.5 Hz, 6H, CH3 × 2).
The corresponding aldehyde was derived from the alcohol by a
similar procedure to that described for 3a, and used for the next
reaction without further purification to give 4-[N-(1-(isobutyl-1H-
imidazol-5-yl)methylamino]-2-phenylbenzoylmethionine methyl
The methyl ester was hydrolyzed in a similar manner to that
described for 3a to give 4a as an amorphous solid (82%). 1H NMR
(CD3OD) d 7.96 (s, 1H), 7.79–7.83 (m, 3H), 7.71 (s, 1H, imid-
2H), 7.47–7.50 (m, 2H, aryl H), 7.38 (d, J = 8 Hz, 1H), 7.29 (m,
6H, aryl H), 7.11 (s, 1H, imid-5H), 6.63 (d, J = 8 Hz, 1H, aryl
H), 6.53 (s, 1H, aryl H), 5.33 (s, 2H, CH2N), 4.39–4.41 (m, 1H,
aCH), 4.29 (br s, 2H, CH2N), 2.14–2.17 (m, 1H, CHHS), 2.05–
2.07 (m, 1H, CHHS), 2.00 (s, 3H, SCH3), 1.73–1.74 (m, 2H, CH2).
HRMS (FAB) m/z calcd for C33H32N4O3S H+ 565.2273. Found
565.2271.
1
ester as an oil (100%). H NMR (CDCl3) d 7.70 (d, J = 8.5 Hz,
1H), 7.37–7.45 (m, 6H, aryl H and imid-2H), 7.00 (s, 1H, imid-
4H), 6.67 (dd, J = 2.2 and 8.5 Hz, 1H, aryl H), 5.75 (d, J = 7.7 Hz,
1H, NH), 4.60–4.64 (m, 1H, aCH), 4.28 (d, J = 4.8 Hz, 2H, CH2),
4.15 (br s, 1H, NH), 3.71 (d, J = 7.7 Hz, 2H, CH2), 3.64 (s, 3H,
CO2CH3), 2.07 (t, J = 7.4 Hz, 2H, SCH2), 2.01–2.04 (m, 1H, CH),
2.00 (s, 3H, SCH3), 1.85–1.90 (m, 1H, CHH), 1.85–1.90 (m, 1H,
CHH), 0.90 (d, J = 6.5 Hz, 6H, 2 × CH3). HRMS (ESI) calcd for
C27H34N4O3SH+ 495.2430. Found 495.2414.
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The methyl ester was hydrolyzed in a similar manner to that
described for 3a to give 6b as an amorphous solid (51%). 1H NMR
(CD3OD) d 8.16 (s, 1H, imid-2H), 7.44 (d, J = 8.5 Hz, 1H, aryl
H), 7.32–7.31 (m, 5H, aryl H), 7.16 (s, 1H, imid-4H), 6.71 (dd,
J = 3.0 and 8.5 Hz, 1H, aryl H), 6.63 (d, J = 3.0 Hz, 1H, aryl
H), 4.39 (dd, J = 4.5 and 8.3 Hz, 1H, aCH), 4.44 (s, 2H, CH2),
3.95 (d, J = 7.5 Hz, 2H, CH2), 2.14–2.23 (m, 2H, SCH and CH),
2.07–2.09 (M, 1H, SCH), 2.01 (s, 3H, SCH3), 1.93–1.95 (m, 1H,
CHH), 1.75–1.77 (m, 1H, CHH), 0.94 (t, J = 6.5 Hz, 6H, CH3 ×
2). HRMS (FAB) m/z calcd for C26H32N4O3S H+ 481.2273. Found
481.2272.
(1 : 1 ethyl acetate–hexanes) to give 4-[N-(1-H-imidazol-4-
yl)methylamino]-2-(2-methylphenyl)benzoylmethionine methyl
1
ester as a white foam (0.36 g, 74%). H NMR (CDCl3) d 7.45 (s,
1H), 7.25–7.35 (m, 13H), 7.09–7.13 (m, 6H), 6.74 (s, 1H), 6.65
(dd, J = 9.3, 1.5 Hz, 1H), 6.34 (d, J = 2.5 Hz, 1H), 5.69 (t, J =
7.35 Hz, 1H), 4.56–4.61 (m, 1H), 4.27 (d, J = 4.8 Hz, 2H), 3.64
(s, 3H), 2.00–2.15 (m, 8H), 1.79–1.86 (m, 1H), 1.48–1.56 (m, 8H);
13C NMR (CDCl3) d 172.4, 167.6, 167.2, 150.3, 142.4, 141.4,
141.2, 139.1, 138.2, 136.6, 132.3, 132.0, 130.8, 130.6, 129.9, 129.2,
128.9, 128.3, 126.5, 126.4, 121.9, 121.4, 119.3, 114.2, 112.0, 75.6,
52.4, 51.7, 41.8, 32.0, 29.6, 20.1, 15.4; FAB [M + H]+ = 695+.
The above compound (0.15 g,◦0.22 mmol) was dissolved into
3.5 cm3 of THF and cooled to 0 C. Lithium hydroxide (18.1 mg
dissolved in 3.5 cm3 of water) was slowly added to the reaction
mixture and stirred for 1 h. The pH was adjusted using 1 N HCl
and placed under vacuum to remove excess THF. The residue was
taken up in ethyl acetate and dried over magnesium sulfate and
concentrated under vacuum. The residue was taken up in 4 cm3
of methylene chloride and 8 cm3 of trifluoroacetic acid and the
reaction mixture was immediately quenched with triethylsilane
until colorless. The reaction was stirred for an addition 2 h and
concentrated to an oil. The residue was taken up in methylene
chloride and 3 N HCl added. The solid was vacuum filtered,
collected and dried under vacuum to give 10 as a HCl salt (0.050 g,
4-[N -(1-(n-Hexyl-1H-imidazol-4-yl)methylamino]-2-phenyl-
benzoylmethionine
(7a,
FTI-2236). 1-n-Hexyl-4-(hydroxy-
methyl)imidazole was prepared using 4-(hydroxymethyl)imidazole
hydrochloride and 1-bromo-hexane by a similar procedure to that
described for 1-benzyl-4 or 5-hydroxymethylimidazole, colorless
oil (25%). 1H NMR (CDCl3) d 7.34 (s, 1H, imid-2H), 6.84 (s, 1H,
imid-5H), 4.55 (s, 2H, CH2), 3.86 (t, J = 7.4 Hz, 2H, CH2), 1.73
(m, 2H, CH2), 1.27 (s, 1H), 1.24 (m, 6H, (CH2)3), 0.86 (t, J =
7.0 Hz, 3H).
The corresponding aldehyde was derived from the alcohol by a
similar procedure to that described for 3a, and used for the next
reaction without further purification to give 4-[N-(1-(n-hexyl-1H-
imidazol-4-yl)methylamino]-2-phenylbenzoyl-methionine methyl
◦
1
50%). Mp 112–115 C. H NMR (300 MHz, CD3OD) d 8.65 (s,
1H), 7.58–7.66 (m, 1H), 7.49 (s, 1H), 7.11–7.25 (m, 4H), 6.80 (dd,
J = 8.6, 2.4 Hz, 1H), 6.50 (d, J = 1.9 Hz, 1H), 4.53 (s, 2H), 4.39–
4.40 (m, 1H), 2.04–2.12 (m, 5H), 1.98 (s, 3H), 1.81–1.90 (m, 1H),
1.46–1.64 (m, 1H); FAB [M + H]+ = 440+.
1
ester as an oil (85%). H NMR (CDCl3) d 7.68 (d, J = 8.6 Hz,
1H, aryl H), 7.36–7.43 (m, 6H, aryl H and imid-2H), 6.81 (s, 1H,
imid-5H), 6.66 (dd, J = 2.6 Hz, 1H, aryl H), 6.50 (d, J = 2.6 Hz,
1H, aryl H), 5.70 (d, J = 7.7 Hz, amido NH), 4.62 (m, 2H, aCH
and NH), 4.28 (s, 2H, CH2), 3.87 (t, J = 7.4 Hz, 2H, CH2), 3.64 (s,
3H, CO2CH3), 2.10 (t, J = 7.7 Hz, CH2), 2.00 (s, 3H, SCH3), 1.88
(m, 1H, CHH), 1.75 (m, 2H, CHH), 1.66 (m, 1H, CHH), 1.29 (m,
6H, (CH2)3), 0.88 (t, J = 6.6 Hz, 3H, CH3). HRMS (ESI) calcd
for C29H38N4O3SH+ 523.2743. Found 523.2725.
4-Nitro-2-(2-methoxyphenyl)benzoic acid. 2-Bromo-4-nitro-
toluene (2.6 g, 11.9 mmol) and 2-methoxyboronic acid (2.4 g,
12 mmol) were dissolved into 35 cm3 of acetone and 40 cm3
of water. Potassium carbonate (4.2 g, 30 mmol) and palladium
acetate (0.15 g, 0.67 mmol) were added and refluxed for 20 h.
The reaction mixture was cooled, extracted with diethyl ether,
filtered over a celite plug and evaporated to give 4-nitro-2-(2-
methoxyphenyl)toluene (1.3 g, 45%) as yellow solid. Mp 76–77◦
C. 1H NMR (CDCl3) d 8.11 (dd, J = 8.3, 2.4 Hz, 1H, aromatic),
8.07 (d, J = 2.2 Hz, 1H, aromatic), 7.40 (d, J = 8.1 Hz, 2H), 7.15
(dd, J = 7.3, 1.5 Hz, 1H), 7.06 (d, J = 7.3 Hz, 1H, aromatic),
7.00 (d, J = 8.3 Hz, 1H, aromatic), 3.78 (s, 3H, OCH3), 2.24 (s,
3H, CH3); 13C NMR (CDCl3) d 156.5, 146.2, 145.4, 140.2, 130.8,
130.5, 129.9, 128.6, 125.3, 122.4, 120.9, 111.0, 55.6, 20.4; HRMS:
calcd for C14H13NO3: m/e 243.0895, found 243.0900.
The methyl ester was hydrolyzed in a similar manner to that
described for 3a to give 7a as an amorphous solid (77%). 1H NMR
(CD3OD) d 7.89 (s, 1H, imid-2H), 7.40 (d, J = 8.6 Hz, 1H, aryl H),
7.35 (m, 4H, aryl H), 7.31 (m, 1H, aryl H), 7.13 (s, 1H, imid-2H),
6.65 (dd, J = 1.8 and 8.6 Hz, 1H, aryl H), 6.58 (d, J = 1.8 Hz, 1H,
aryl H), 4.37 (m, 1H, aCH), 4.31 (s, 2H, CH2), 3.99 (t, J = 7.0 Hz,
2H, CH2), 2.06 (m, 1H, CHH), 2.05 (m, 1H, CHH), 2.00 (s, 3H,
SCH3), 1.91 (m, CHH), 1.76 (m, 3H, CH2 and CHH), 1.29 (m,
6H, (CH2)3), 0.88 (t, J = 4.4 Hz, 3H, CH3). HRMS (FAB) m/z
calcd for C28H36N4O3S H+ 509.2586. Found 509.2584.
4-[N -(1-H-Imidazol-4-yl)methylamino]-2-(2-methylphenyl)-
benzoylmethionine (10, FTI-2128). 1-Triphenylmethyl-imidazole-
4-carboxaldehyde (0.25 g, 0.7 mmol) and N-(4-amino-2-(2-
methylphenylbenzoyl)methionine methyl ester hydrochloride
(0.3 g, 0.7 mmol) were dissolved in 10 cm3 of a 95% methanol–5%
acetic acid solution and stirred for 30 min before adding 1
equivalent of sodium cyanoborohydride (0.044 g, 0.7 mmol). The
reaction was stirred over a period of 3 h while additional aldehyde
was added until all of the amine hydrochloride had disappeared.
The reaction mixture was concentrated, taken up in ethyl acetate
and washed with a saturated solution of sodium bicarbonate.
The organic phase was dried over magnesium sulfate and
concentrated. The residue was purified by flash chromatography
The above compound (1.3 g, 5.7 mmol) was suspended in 10 cm3
of pyridine and 20 cm3 of water and heated to reflux. Potassium
permanganate was added in portions and the reaction was heated
for 16 h. The reaction mixture was filtered through a celite plug and
the filtrate carefully acidified with concentrated HCl. The filtrate
was extracted with diethyl ether, dried over magnesium sulfate and
concentrated to give an oil which was recrystallized from ethyl
acetate and hexanes to give 4-ni◦tro-2-(2-methoxyphenyl)benzoic
acid (0.65 g, 45%). Mp 171–175 C. 1H NMR (CDCl3) d 8.25 (d,
J = 2.2 Hz, 1H, aromatic), 8.22 (s, 1H, aromatic), 8.06 (d, J =
8.6 Hz, 1H, aromatic), 7.40 (t, J = 7.6 Hz, 1H, aromatic), 7.31 (dd,
J = 7.5, 1.5 Hz, 1H, aromatic), 7.09 (t, J = 7.3 Hz, 1H), 6.91 (d,
J = 8.2 Hz, 1H), 3.71 (s, 3H, OCH3). 13C NMR (CDCl3) d 172.3,
490 | Org. Biomol. Chem., 2006, 4, 482–492
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155.9, 150.0, 141.0, 136.2, 131.0, 130.5, 129.9, 127.9, 126.6, 122.0,
121.5, 110.8, 55.1.
washed with H2O (200 cm3 × 3) and brine, and dried over Na2SO4.
The crude product was purified by SiO2 column chromatography
with CHCl3–acetone–EtOH = 100 : 40 : 8 to afford the product
1
4-[N-(1-H-Imidazol-4-yl)methylamino]-2-(2-methoxyphenyl)-
as colorless oil (834 mg, 88%). H NMR (CDCl3) d 8.02 (s, 2H,
benzoylmethionine
phenyl)benzoic acid (0.55 g,
(12,
FTI-2134). 4-Nitro-2-(2-methoxy-
mmol) was coupled with
imid-2H × 2), 7.93 and 7.90 (d each, J = 8.4 Hz each, 1H, aryl
H), 7.53 (d, J = 8.4 Hz, 1H, aryl H), 7.32–7.18 (m, 7H, aryl H
and imid-5H × 2), 5.90 (t, J = 7.6 Hz, 1H, amido NH), 4.60 (m,
1H, aCH), 3.78 (s, 2H, NCH2), 3.68 (s, 4H, CH2N × 2), 3.65 (s,
3H, CO2CH3), 2.17–1.83 (m, 9H, CHH, CH2S, SCH3, tolyl-CH3),
1.62 (s, 18H, Boc × 2).
2
L-methionine methyl ester hydrochloride in the manner described
above to give 4-nitro-2-(2-methoxyphenyl)benzoyl-methionine
methyl ester (0.8 g, 93%) as an oil after purification by flash
1
column chromatography (3 : 2 ethyl acetate–hexanes, silica). H
NMR (CDCl3) d 8.25 (dd, J = 8.5, 2.3 Hz, 1H, aromatic), 8.14
(d, J = 2.2 Hz, 1H, aromatic), 7.90 (d, J = 8.5 Hz, 1H, aromatic),
7.45 (app t, 1H, aromatic), 7.25 (d, J = 6.5 Hz, 1H), 7.08 (app t,
1H, aromatic), 7.01 (d, J = 8.3 Hz, 1H), 6.5 (br s, 1H), 4.66 (m,
1H, a Met-H), 3.81 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 1.87–2.00
(m, 5H), 1.66–1.74 (m, 2H); 13C NMR (CDCl3) d 171.9, 166.9,
156.2, 148.7, 141.5, 137.9, 130.9, 130.6, 130.1, 127.2, 126.3, 122.7,
121.4, 111.1, 55.7, 52.7, 52.0, 31.8, 29.3, 15.4.
To a solution of the Boc-protected bis-imidazole compound
(834 mg, 1.12 mmol) in CH2Cl2 (5 cm3) was added TFA (5 cm3)
dropwise at 0 ◦C, and the mixture was stirred at room temperature
for 15 min. Evaporation of the solvent, followed by purification
of the crude oily product by SiO2 column chromatography with
CHCl3–MeOH–28% NH4OH = 5 : 1 : 0.1 afforded the product
as an amorphous solid (450 mg, 74%). 1H NMR (DMSO-d6)
d 1.86–1.94 (m, 2H, CH2), 2.07–2.33 (m, 8H, CH3, SCH3, and
CH2), 3.67 (s, 3H, CO2CH3), 3.79 (s, 2H, NCH2), 3.84 (s, 4H,
N(CH2)2), 4.40 (br s, 1H, aCH),7.26–7.37 (m, 6H, aryl H), 7.58
(s, 2H, imid-5H), 7.76 (s, 2H, imid-2H), 1.92 (s, 1H, aryl H).
13C NMR (DMSO-d6) d 14.4, 19.8, 29.3, 30.1, 46.8, 51.0, 51.8,
56.3, 72.2, 117.6, 118.0, 125.0, 127.2, 127.6, 129.2, 129.5, 129.8,
130.2, 134.4, 135.3, 139.0, 139.3, 158.0, 168.3, 171.8. LRMS (FAB)
m/z calcd for C29H34N6O3S H+ 547. Found 547. Anal. calcd
for C29H34N6O3S·3CF3CO2H·0.5H2O: C,46.82; H, 4.23; N, 9.36.
Found: C, 46.84; H, 4.22; N, 9.19.
The above compound (0.7 g, 1.7 mmol) was reduced in a
similar manner to that previously reported9 to give 4-amino-2-
(2-methoxyphenyl)benzoylmethionine methyl ester hydrochloride
(0.65 g, 92%). 13C NMR (CD3OD) d 173.4, 171.2, 157.5, 140.3,
138.4, 133.2, 131.8, 131.3, 130.7, 128.7, 127.0, 123.0, 121.7, 112.1,
55.9, 53.0, 31.4, 30.7, 15.0; FAB [M + H]+ = 389+.
This compound (0.3 g, 0.7 mmol) was coupled with triphenylim-
idazole carboxyaldehyde (0.29 g, 0.85 mmol) in a similar manner
described above to give the fully protected compound as a white
foam (0.26 g, 52%), which was deprotected by the same method
described above to give 12 as the hydrochloride salt (0.09 g, 93%).
The HPLC showed 91% purity.
4-[N, N -Bis((1H -imidazol-4-yl)methyl)aminomethyl]-2-(1-
(o-tolyl))benzoylmethionine trifluoroacetate (16, FTI-2287). To a
solution of 17 (450 mg, 0.82 mmol) in THF (20 cm3) was added
0.1 N LiOH (16 cm3) at 0 ◦C and the mixture was stirred at
room temperature for 30 min. After neutralization with 0.5 M
HCl, the solution was concentrated to almost dryness. The residue
was purified by gel permiation on Sephadex LH-20 with MeOH–
CHCl3 = 1 : 1, and the product was dissolved in TFA at 0 ◦C. The
solution was concentrated and the resulting residue was dried in
vacuo overnight. To the yellow residue was added dry Et2O to
precipitate the compound as the TFA salt. The white powdery
product was collected by centrifuge and dried in vacuo (549 mg,
2-[(2ꢀ -Methyl-5-{[(5-methyl-1H-imidazol-4-ylmethyl)amino]-
methyl}biphenyl-2-carbonyl)amino]-4-methylsulfanylbutyric acid
(15, FTI-2288). To a solution of the 2-[(5-aminomethyl-2ꢀ-
methylbiphenyl-2-carbonyl)amino]-4-methylsulfanylbutyric acid
methyl ester (21)12 (33 mg, 0.06 mmol) in MeOH (0.5 cm3) was
added 5-methyl-4-imidazole carboxyaldehyde (6 mg, 0.06 mmol).
After stirring at room temperature for 1 h, NaBH4 (2.1 mg,
0.05 mmol) was added and stirred overnight at room temperature.
Evaporation of the solvent, followed by extraction with 10%
MeOH in CH2Cl2 (40 cm3) from saturated NaHCO3 (20 cm3)
afforded the crude product. The compound was purified by gel
chromatography on Sephadex LH-20 with MeOH–CHCl3 = 1 :
1 to give an amorphous solid (15 mg, 58%). 1H NMR (CD3OD)
d 1.70 (m, 1H, CHHCH2S), 1.95–2.17 (m, 12H, 2 × CH3, SCH3,
CH2S, CHHCH2), 3.67 (s, 3H, CO2CH3), 3.71 (s, 2H, NCH2),
3.84 (s, 2H, NCH2), 7.14–7.53 (m, 8H, aryl H).
1
77%). H NMR (DMSO-d6) d 9.06 (s, 2H), 8.32–8.05 (m, 2H),
7.61 (s, 2H), 7.45 (s, 2H), 7.23–7.11 (m, 6H), 4.22 (br s, 1H, aCH),
3.87 (s, 4H, CH2N × 2), 3.70 (s, 2H, NCH2), 2.20–1.71 (m, 10H,
SCH3, CH3, CH2CH2). HRMS (FAB) m/z calcd for C28H32N6O3S
H+ 533.2335. Found 533.2334.
Crystal structures
Compound 15 was prepared by a similar procedure to that
described for 16 (100%). 1H NMR (CD3OD) d 7.42–7.13 (m, 8H),
4.20 (br s, 1H, aCH), 4H from CH2NHCH2 were overlapped under
the D2O peak at 3.90, 2.23–1.86 (m, 12 H), 1.60 (br s, 1H).
Crystals of the FPT–13 and FPT–16 complexes were prepared by
soaking the respective inhibitors into preformed crystals using
methods previously described.20 X-Ray diffraction data were
collected at the IMCA-CAT 17-BM beamline equipped with a
Mar 165 CCD detector. With the detector set at 130 mm, data
4-[N, N -Bis((1H -imidazol-4-yl)methyl)aminomethyl]-2-(1-
(o-tolyl))benzoylmethionine methyl ester (17, FTI-2295). A so-
lution of 2112 (490 mg, 1.27 mmol), 1-t-butoxycarbonyl-4-
chloromethylimidazole (578 mg, 2.67 mmol), and NaHCO3
(225 mg, 2.67 mmol) in DMF (10 cm3) was stirred at 65 ◦C
overnight. After distillation of DMF, the residue was dissolved
in AcOEt (350 cm3) and H2O (200 cm3), and the organic layer was
◦
˚
were collected in 240 contiguous 0.30 oscillation images at 1 A
˚
wavelength. The data for compound 13 extends to 2 A resolution,
has an Rmerge of 5.3% and 4.5-fold multiplicity. The data for
˚
compound 16 extends to 1.9 A resolution, has an Rmerge of 3.6%
and 4.6-fold multiplicity. The structures were refined using CNX
(Accelrys Inc.) to an Rfactor of approximately 19%.
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In vitro assay and inhibition of protein prenylation in whole cells
6 S. B. Long, P. J. Casey and L. S. Beese, Structure, 2000, 8, 209–222.
7 S. B. Long, P. J. Hancock, A. M. Kral, H. W. Hellinga and L. S. Beese,
Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 12948–12953.
In vitro inhibition assays for PFTase and PGGTase-I were carried
out by measuring the incorporation of [3H]FPP and [3H]GGPP
into recombinant H-Ras-CVLS and H-Ras-CVLL, respectively,
as previously described.14 The in vivo inhibition of farnesylation
and geranylgeranylation was determined based on the level of
inhibition by peptidomimetic compounds of H-Ras and Rap1 pro-
cessing, respectively.21 Briefly, oncogenic H-Ras-transformed NIH
3T3 cells were treated with various concentrations of inhibitors,
and the cell lysates were isolated and proteins separated on a
12.5% SDS-PAGE gel. The separated proteins were transferred
to nitrocellulose and immunoblotted using an anti-Ras antibody
(Y13-258) or an anti-Rap1A antibody (Santa Cruz Biotechnology,
Santa Cruz, CA). Antibody reactions were visualized using either
peroxidase-conjugated goat anti-rat IgG or goat anti-rabbit IgG
(Jackson ImmunoResearch, West Grove, PA) and an enhanced
chemiluminescence detection system.
8 Y. Qian, M. A. Blaskovich, M. Saleem, C. M. Seong, S. P. Wathen,
A. D. Hamilton and S. M. Sebti, J. Biol. Chem., 1994, 269, 12410–
12413.
9 Y. Qian, J. J. Marugan, R. D. Fossum, A. Vogt, S. M. Sebti and A. D.
Hamilton, Bioorg. Med. Chem., 1999, 7, 3011–3024.
10 (a) A. Vasudevan, Y. Qian, A. Vogt, M. A. Blaskovich, J. Ohkanda,
S. M. Sebti and A. D. Hamilton, J. Med. Chem., 1999, 42, 1333–1340;
(b) J. Sun, M. A. Blaskovich, D. Knowles, Y. Qian, J. Ohkanda, R. D.
Bailey, A. D. Hamilton and S. M. Sebti, Cancer Res., 1999, 59, 4919–
4926; (c) J. Ohkanda, J. W. Lockman, M. A. Kothare, Y. Qian, M. A.
Blaskovich, S. M. Sebti and A. D. Hamilton, J. Med. Chem., 2002, 45,
177–188.
11 (a) J. T. Hunt, V. G. Lee, K. Leftheris, B. Seizinger, J. Carboni, J. Mabus,
C. Ricca, N. Yan and V. Manne, J. Med. Chem., 1996, 39, 353–358;
(b) N. J. Anthony, R. P. Gomez, M. D. Schaber, S. D. Mosser, K. A.
Hamilton, T. J. O’Neil, K. S. Koblan, S. L. Graham, G. D. Hartman, D.
Shah, E. Rands, N. E. Kohl, J. B. Gibbs and A. I. Oliff, J. Med. Chem.,
1999, 42, 3356–3368; (c) C. Z. Ding, R. Batorsky, R. Bhide, H. J. Chao,
Y. Cho, S. Chong, J. Gullo-Brown, P. Guo, S. H. Kim, F. Lee, K.
Leftheris, A. Miller, T. Mitt, M. Patel, B. A. Penhallow, C. Ricca, W. C.
Rose, R. Schmidt, W. A. Slusarchyk, G. Vite, N. Yan, V. Manne and
J. T. Hunt, J. Med. Chem., 1999, 42, 5241–5253; (d) T. M. Ciccarone,
S. C. MacTough, T. M. Williams, C. J. Dinsmore, T. J. O’Neill, D. Shah,
J. C. Culberson, K. S. Koblan, N. E. Kohl, J. B. Gibbs, A. I. Oliff, S. L.
Graham and G. D. Hartman, Bioorg. Med. Chem. Lett., 1999, 9, 1991–
1996; (e) C. J. Dinsmore, T. M. Williams, T. J. O’Neill, D. Liu, E. Rands,
J. C. Culberson, R. B. Lobell, K. S. Koblan, N. E. Kohl, J. B. Gibbs,
A. I. Oliff, S. L. Graham and G. D. Hartman, Bioorg. Med. Chem.
Lett., 1999, 9, 3301–3306; (f) C. Z. Ding, J. T. Hunt, C. Ricca and V.
Manne, Bioorg. Med. Chem. Lett., 2000, 10, 273–275.
12 D. Carrico, J. Ohkanda, H. Kendrick, K. Yokoyama, M. A. Blaskovich,
C. J. Bucher, F. S. Buckner, W. C. Van Voorhis, D. Chakrabarti, S. L.
Croft, M. H. Gelb, S. M. Sebti and A. D. Hamilton, Bioorg. Med.
Chem., 2004, 12, 6517–6526.
13 T. Matsui, T. Sugiura, H. Nakai, S. Iguchi, S. Shigeoka, H. Takeda, Y.
Odagaki, Y. Nagao, Y. Ushio, K. Ohmoto, H. Iwamura, S. Yamazaki,
Y. Arai and M. Kawamura, J. Med. Chem., 1992, 35, 3307–3319.
14 A. Vogt, Y. Qian, T. F. McGuire, A. D. Hamilton and S. M. Sebti,
Oncogene, 1996, 13, 1991–1999.
Antitumor activity in the nude mouse tumor xenograft model
Nude mice (Charles River, Wilmington, MA) were maintained
in accordance with the Institutional Animal Care and Use
Committee (IACUC) procedures and guidelines. A549 cells were
harvested, resuspended in PBS and 1 × 107 cells injected s.c. onto
both the right and left flanks of eight week old female nude mice
as reported previously.22 When tumors reached about 150 mm3,
animals were randomized and dosed s.c. with a mini-pump as
described previously22 with 25 mg−1 kg−1 d−1 of either FTI-2148,
FTI-2287, or with an equal volume of vehicle control. The tumor
volumes were determined by measuring the length (l) and the
width (w) and calculating the volume (V = lw2/2) as described
previously.23 Statistical significance between control and treated
animals were evaluated by using Student’s t-test.
15 J. S. Taylor, T. S. Reid, K. L. Terry, P. J. Casey and L. S. Beese, EMBO J.,
2003, 22, 5963–5974.
Acknowledgements
16 Celluar uptake enhancement of polycationic pepitdes have been de-
scribed. For example: (a) P. A. Wender, D. J. Mitchell, K. Pattabiraman,
E. T. Pelkey, L. Steinman and J. B. Rothbard, Proc. Natl. Acad. Sci.
U. S. A., 2000, 97, 13003–13008; (b) C. Pichon, C. Goncalves and P.
Midoux, Adv. Drug Delivery Rev., 2001, 53, 75–94.
This work was supported by a grant from the National Institutes
of Health (CA67771).
Use of the IMCA-CAT beamline 17-BM at the Advanced
Photon Source was supported by the companies of the Industrial
Macromolecular Crystallography Association through a contract
with Illinois Institute of Technology. Use of the Advanced Photon
Source was supported by the U. S. Department of Energy, Office
of Science, Office of Basic Energy Sciences, under Contract No.
W-31-109-Eng-38.
17 J. Ohkanda, F. S. Buckner, J. W. Lockman, K. Yokoyama, D. Carrico,
R. Eastman, K. deLuca-Fradley, W. Davies, S. L. Croft, W. C. Van
Voorhis, M. H. Gelb, S. M. Sebti and A. D. Hamilton, J. Med. Chem.,
2004, 47, 432–445.
18 J. M. Kokosa, R. A. Szafasz and E. J. Tagupa, J. Org. Chem., 1983, 48,
3605–3697.
19 R. Millet, J. Domarkas, R. Houssin, P. Gilleron, J.-F. Goossens, P.
Chavatte, C. Loge´, N. Pommery, J. Pommery and J.-P. He´nichart,
J. Med. Chem., 2004, 47, 6812–6820.
20 C. L. Strickland, P. C. Weber, W. T. Windsor, Z. Wu, H. V. Le, M. M.
Albanese, C. S. Alvarez, D. Cesarz, J. D. Rosario, J. Deskus, A. K.
Mallams, F. G. Njoroge, J. J. Piwinski, S. Remiszewski, R. R. Rossman,
A. G. Taveras, B. Vibulbhan, R. J. Doll, V. M. Girijavallabhan and
A. K. Ganguly, J. Med. Chem., 1999, 42, 2125–2135.
21 J. Sun, Y. Qian, A. D. Hamilton and S. M. Sebti, Oncogene, 1998, 16,
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22 M. A. Blaskovich, J. Sun, A. Cantor, J. Turkson, R. Jove and S. M.
Sebti, Cancer Res., 2003, 63, 1270–1279.
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