In vitro and in vivo antimalarial activity of peptidomimetic protein farnesyltransferase inhibitors with improved membrane permeability

Article abstract of DOI:10.1016/j.bmc.2004.09.020

A series of ester derivatives of 17 with increased lipophilicity were synthesized and tested against P. falciparum in red blood cells, where the benzyl ester derivative 16 exhibited the best inhibition activity (ED ₅₀ = 150 nM). Compound 16 sho

Full text of DOI:10.1016/j.bmc.2004.09.020

Bioorganic & Medicinal Chemistry 12 (2004) 6517–6526
In vitro and in vivo antimalarial activity of peptidomimetic protein
farnesyltransferase inhibitors with improved
membrane permeability
a
Dora Carrico, Junko Ohkanda, Howard Kendrick, Kohei Yokoyama,
a
b
c
d
Michelle A. Blaskovich, Cynthia J. Bucher, Frederick S. Buckner,
d
e,
*
e
f
Wesley C. Van Voorhis, Debopam Chakrabarti, Simon L. Croft,
b
c,
d
Michael H. Gelb, Sa ¨ı d M. Sebti and Andrew D. Hamilton
a,
*
*
a
Department of Chemistry, Yale University, PO Box 208107, New Haven, CT 06520, USA
b
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street,
London WC1E 7HT, UK
Departments of Chemistry and Biochemistry, Campus Box 351700, University of Washington, Seattle, WA 98195, USA
c
d
Drug Discovery Program, H. Lee Moffitt Cancer Center & Research Institute, Departments of Oncology and
Biochemistry & Molecular Biology, University of South Florida, Tampa, FL 33612, USA
e
Department of Medicine, University of Washington, Campus Box 357185, Seattle, WA 98195, USA
Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, FL 32826, USA
f
Received 23 June 2004; revised 13 September 2004; accepted 14 September 2004
Available online 6 October 2004
Abstract—A series of protein farnesyltransferase inhibitor ester prodrugs of FTI-2148 (17) were synthesized in order to evaluate the
effects of ester structure modification on antimalarial activity and for further development of a farnesyltransferase inhibitor with in
vivo activity. Evaluation against P. falciparum in red blood cells showed that all the investigated esters exhibited significant antima-
larial activity, with the benzyl ester 16 showing the best inhibition (ED50 = 150nM). Additionally, compound 16 displayed in vivo
ꢀ
1
ꢀ1
activity and was found to suppress parasitemia by 46.1% at a dose of 50mgkg day against Plasmodium berghei in mice. The
enhanced inhibition potency of the esters is consistent with improved cell membrane permeability compared to that of the free acid.
The results of this study suggest that protein farnesyltransferase is a valid antimalarial drug target and that the antimalarial activity
of these compounds derives from a balance between the hydrophobic character and the size and conformation of the ester moiety.
ꢀ
2004 Elsevier Ltd. All rights reserved.
1. Introduction
an average of 300–500 million people become infected
1
annually. The disease is caused by protozoan parasites
Malaria is a parasitic disease commonly found in many
tropical and subtropical regions of the world. Present
resurgence of this disease and lack of proper treatment
cause several million deaths per year and present a huge
burden to the economy of these countries. Malaria
causes 0.5–2.5 million deaths per year worldwide and
of the genus Plasmodium. The four Plasmodium species
known to infect humans are P. falciparum, P. vivax, P.
ovale, and P. malariae. Of these, P. falciparum is respon-
sible for the most severe form of malaria infections.
Existing treatments for malaria include a limited num-
ber of clinically effective drugs, however the emergence
of drug-resistant parasite strains indicates an urgent
need for discovering new and effective antimalarial
therapeutics.
Keywords: Plasmodium falciparum; Peptidomimetic; Protein farnesyl-
transferase; Ester prodrug.
*
Corresponding authors. Tel.: +1 206 616 9214; fax: +1 206 685 8861
F.S.B.); tel.: +1 206 543 7142; fax: +1 206 685 8665 (M.H.G.);
tel.: +1 203 432 5570; fax: +1 203 432 3221 (A.D.H.);
e-mail addresses: fbuckner@u.washington.edu; gelb@chem.
washington.edu; andrew.hamilton@yale.edu
New strategies for antimalarial chemotherapy include
inhibition of merozoite surface protein-1 (MSP-1)
processing protease as well as inhibition of P. falciparum
fatty acid biosynthesis, phospholipid metabolism, and
(
0
968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.020

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D. Carrico et al. / Bioorg. Med. Chem. 12 (2004) 6517–6526
2
malarial lactate dehydrogenase. Recent studies have
suggested that protein farnesyltransferase (PFT) is a
promising target for the development of novel antipara-
sitic agents. We have previously demonstrated that pro-
tein prenylation occurs in protozoan parasites T. brucei,
T. cruzi, and Leishmania mexicana, and have success-
fully isolated, cloned, and expressed PFT from T. bru-
activity of our peptidomimetic inhibitors. We initially
showed that peptidomimetics FTI-276 and its methyl
ester FTI-277 (Fig. 1), inhibited T. brucei PFT and the
growth of T. brucei bloodstream form at low nanomolar
3
concentrations. These compounds also potently inhib-
ited the growth of T. cruzi amastigote intracellular
8
form. Additionally, we have recently shown that several
of our PFT inhibitors also exhibit potent antimalarial
3
,4
cei. More recently, inhibition activity against P.
5
falciparum by a PFT inhibitor was also observed.
9
activity against P. falciparum in vitro. In this study
FTI-2148, one of our most potent mammalian PFT
inhibitors (IC =1nM), displayed no inhibition against
PFT belongs to a family of proteins known as protein
prenyltransferases that are responsible for protein
prenylation. Three distinct protein prenyltransferases,
PFT, and protein geranylgeranyltransferase-I and II
5
0
P. falciparum (ED >30lg/mL). However, the methyl
5
0
ester FTI-2153 (Fig. 1) potently inhibited P. falciparum
growth with an ED50 = 2lg/mL. It is likely that this
enhanced inhibition is due to the higher membrane per-
meability of the ester compared to that of the free acid.
Guided by these findings, we decided to prepare a series
of ester prodrugs of FTI-2148 to investigate the effect of
ester modification on antimalarial activity. The ester
prodrug strategy is a well-precedented technique com-
monly used to deliver drugs across cellular membranes
(
PGGT-I and II), have been found in higher eukaryo-
6
tes. The prenyltransferases catalyze the covalent trans-
fer of a farnesyl group (15 carbon by PFT) or of a
geranylgeranyl group (20 carbon by PGGT-I or -II) to
cysteine residues near the C-terminus of protein sub-
strates. Many prenylated proteins are known to play
crucial roles in cellular signaling and other regulatory
events that take place near the cytoplasmic surfaces of
1
0
leading to higher drug efficacy.
Based on this
6
cellular membranes.
approach, a series of FTI-2148 esters were designed.
This paper describes our efforts to develop PFT inhibi-
tor ester derivatives with increased lipophilicity that
exhibit in vivo antimalarial activity.
In past years there has been considerable interest in pro-
tein prenyltransferases because malignant Ras activity,
present in approximately 30% of human cancers, was
found to be dependent on post-translational modifica-
tion by PFT. Therefore, PFT has been considered as a
potential target for anticancer therapeutics. PFT is
known to modify proteins that contain a C-terminal
CAAX tetrapeptide, where C is cysteine, AA are ali-
phatic amino acids and X is usually serine or meth-
ionine. On the basis of the CAAX motif we have
successfully developed potent PFT inhibitors, where
the aliphatic dipeptide AA portion was substituted by
rigid hydrophobic spacers (Fig. 1).
2. Results and discussion
2.1. Chemistry
The FTI-2148 esters were prepared as outlined in
Scheme 1. Oxidation of 2-bromo-4-nitrotoluene with
potassium permanganate followed by esterification with
thionyl chloride in methanol gave 1. The resulting 2-
(bromo)-4-nitrobenzoic acid methyl ester (1) was then
coupled to o-tolylboronic acid via a Suzuki coupling
protocol to afford biaryl 2. Reduction of the nitro group
with tin(II) chloride dihydrate, diazotization, and Sand-
meyer reaction with potassium iodide produced the
iodo ester derivative 3. Copper-mediated iodo-cyano
exchange afforded nitrile 4. The cyano intermediate 4
was then hydrogenated with palladium over activated
charcoal and the resulting amine was Boc-protected in
the same pot to afford intermediate 5. Hydrolysis of 5
with LiOH in THF gave the free acid 6.
Based on previous observations supporting PFT as a
potential target for the development of antiparasitic
3
,7
agents,
we decided to investigate the antiparasitic
HS
HS
O
O
S
H
H
N
O
O
N
H N HN
OH
2
H2N
N
H
OH
HN
O
O
S
The resulting benzoic acid derivative 6 was then coupled
with L-methionine methyl, ethyl, or allyl ester using 1-[3-
CVIM
Cys-4-AMBA-Met
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochlo-
ride (EDCI)/1-hydroxybenzotriazole (HOBt) to afford
the respective amide precursors 7, 8, and 9, respectively.
Deprotection under acidic conditions followed by reduc-
tive amination of the resulting free amine with 4(5)-
HS
H2N
O
O
O
O
HN
OR
OR
imidazolecarboxaldehyde and NaBH gave the methyl
4
HN
NH
HN
(10), ethyl (11), and allyl (12) ester prodrugs of FTI-
2
N
148.
S
N
S
H
Since only two of the desired esters, allyl, and ethyl,
could be prepared from commercially available methio-
nine ester precursors, we turned our attention toward
using a transesterification route to synthesize the
R = H
R = CH3 FTI-277
FTI-276
R = H
FTI-2148
R = CH3 FTI-2153
Figure 1. Evolution of CAAX peptidomimetic PFT inhibitors.

D. Carrico et al. / Bioorg. Med. Chem. 12 (2004) 6517–6526
6519
Br
Br
a, b
c
d, e
O
O2N
O
O
O2N
CH3
OCH3
O2N
I
OCH3
OCH3
1
2
3
f
g
h
O
O
O
NC
OCH3
BocHN
OCH3
BocHN
OH
4
5
6
O
S
i
j, k
O
S
NH
HN
BocHN
HN
N
OR
l
13. R = iPropyl
OR
O
O
N
H
m
n
7
8
9
. R = Methyl
. R = Ethyl
. R = Allyl
10. R = Methyl
11. R = Ethyl
12. R = Allyl
14. R = 2-Ethyl-hexyl
15. R = Cyclohexyl
16. R = Benzyl
o
Scheme 1. Reagents and conditions: (a) KMnO
AcOEt, 93%; (e) NaNO , HCl, KI, 83%; (f) CuCN, N-methyl-2-pyrrolidinone, 68%; (g) H
L-methionine ester, EDCI, HOBt, Et N, CH Cl , 69–88%; (j) TFA, 81–100%; (k) 4(5)-imidazolecarboxaldehyde, NaBH
iPrOH, THF, 4A mol sieves, 70ꢁC, 99%; (m) titanium (IV)-2-ethylhexoxide, 10:1 THF/DMF, 4A mol sieves, 60ꢁC, 99%; (n) tetracyclohexyl titanate,
4
, 62%; (b) SOCl
2
, MeOH, 82%; (c) o-tolylboronic acid, Pd(PPh
/10% Pd–C, Boc O, THF, 92%; (h) LiOH, THF, 99%; (i)
, 32–54%; (l) Ti(OiPr)
3 4 2 3 2 2
) , K CO , 92%; (d) SnCl Æ2H O,
2
2
2
3
2
2
4
4
,
˚
˚
˚
˚
1
0:1 THF/DMF, 4A mol sieves, 60ꢁC, 44%; (o) Ti(OiPr) , benzyl alcohol, THF, 4A mol sieves, 85ꢁC, 98%.
4
remaining derivatives. We anticipated that this latter
approach would also circumvent the low yields observed
in the reductive amination step with the allyl and ethyl
ester intermediates (32% and 36%, respectively). Exten-
sive investigations into the application of transesterifica-
tion reaction conditions using various model systems,
revealed that a titanium-mediated transesterification
could be used to efficiently prepare the desired esters
from the methyl ester FTI-2153 (10) in a single step in
very good yields (Scheme 1).
essed (U) protein. The results of the western blot are
shown in Figure 2. All ester prodrugs were more effec-
tive at inhibiting H-Ras processing than the free carb-
oxylate derivative 17 consistent with a higher cell
permeability of the ester derivatives. In this series, the
most potent compounds disrupted H-Ras farnesylation
with an IC50 value of 4nM. In addition, all compounds
selectively inhibited H-Ras processing over Rap1A
processing.
The inhibitory activity of these PFT inhibitor agents was
then evaluated against P. falciparum. Evaluation against
P. falciparum PFT showed that, as expected, all ester
derivatives of FTI-2148 (17) were less effective at inhibi-
ting the parasitic enzyme than the free acid, due to the
inability of PFT to modify substrates that lack a C-ter-
minal carboxylate. The antimalarial activity against P.
falciparum growth displayed by these compounds was
expected to be related to their hydrophobicity and con-
sequently to their logP values. The octanol/water parti-
tion coefficient (logP) values of these compounds were
predicted computationally using the program Qik-
2
.2. Biological activity
The activity of these PFT inhibitor ester derivatives was
first evaluated against mammalian PFT both in vitro and
in whole cell assays. The in vitro activity and selectivity
of the FTI-2148 ester prodrugs are reported in Table 1.
As expected, all the ester derivatives of FTI-2148 (17)
exhibited higher IC50 values compared to the free acid.
This results from the requirement of a C-terminal carb-
oxylate for PFT substrates. In general, all compounds
were more selective at inhibiting mammalian PFT over
PGGT-I in vitro. The ability of these compounds to inhi-
bit farnesylation and geranylgeranylation in whole cells
was evaluated by determining the level of inhibition of
H-Ras and Rap1A processing, respectively. Oncogenic
H-Ras-transformed NIH 3T3 cells were treated with var-
ious concentrations of inhibitors, cells were harvested
and subjected to Western blot analysis. Inhibition of
farnesylation and geranylgeranylation was demonstrated
by a band shift from processed (P) protein to unproc-
1
1
Prop. The results of this study are shown in Table 1.
In general, the estimated logP values follow an expected
pattern where the least lipophilic compound is predicted
to be FTI-2148 methyl ester and the most lipophilic is
the 2-ethyl-hexyl ester of FTI-2148. A comparison of
the computationally obtained logP values and the P.
falciparum ED50 values shows a correlation between
lipophilicity and antiparasitic activity. This is clearly

6520
D. Carrico et al. / Bioorg. Med. Chem. 12 (2004) 6517–6526
Table 1. Inhibition activity of 10–17 against P. falciparum PFT, P. falciparum infected RBC, mammalian PFT, and mammalian PGGT-I
S
O
NH
HN
N
OR
O
N
H
Compound
R
LogP
P. falciparum
P. falciparum
Mammalian
PFT IC50
(nM)
Mammalian
PGGT-I IC50
(nM)
Murine 3T3
fibroblasts
ED50 (nM)
a
PFT IC50 (nM) infected RBC
ED50 (nM)
1
0
1
CH
C
3
4.44
4.88
>1000
750
3300
1900
34
360
69,000
>10,000
25,000
10,000
1
2
H
5
1
1
2
3
H C
2
5.19
5.19
>1000
>1000
1800
800
150
>10,000
>10,000
1000–10,000
10,000
HC
4000
1
1
1
1
4
5
6
7
7.09
4.94
6.09
1.77
>1000
>1000
1000
15
700
350
3900
3500
530
>10,000
>10,000
>10,000
1700
1000–10,000
1000–10,000
1000–10,000
>25,000
H C
2
HC
H
2
C
150
b
H
>66,000
0.82
a
RBC = red blood cell.
Ref. 9.
b
Figure 2. Mammalian H-Ras and Rap1A processing inhibition by 10–17 in whole cell assays.
seen in the two most lipophilic compounds 16 and 14,
that show much higher inhibition activities compared
to less lipophilic 10 and 11.
result was obtained with the cyclohexyl derivative 15
which, while not being the most hydrophobic com-
pound, showed the second highest antiplasmodial activ-
ity in this series. Even though both 15 and 11 were
predicted to be similarly hydrophobic the cyclohexyl
ester derivative 15 was fivefold more active than 11.
These results indicate that, though a higher hydrophobi-
city leads to higher membrane permeation, other factors
such as size and conformation of the ester side chain
may also affect delivery of the compound into the cell
and thus the inhibition activity. Thus, the activity of
these compounds likely reflects a balance between the
ease of membrane permeability, stability against extra-
Evaluation of the seven different esters for in vitro inhi-
bition of P. falciparum growth in red blood cells (Table
1
) showed that all other esters had improved anti-
plasmodial activity compared to the methyl ester. These
results indicate that the higher potency exhibited by the
more hydrophobic esters appears to be due to their
increased ability to enter parasite cells. In this series,
branched esters 13 and 14 displayed a higher potency
than linear ethyl 11 and allyl 12 derivatives. A surprising

D. Carrico et al. / Bioorg. Med. Chem. 12 (2004) 6517–6526
6521
4. Experimental
4.1. General methods
Melting points were determined with an electrothermal
capillary melting point apparatus and are uncorrected.
H and C NMR spectra were recorded on a Bruker
1
13
AM-500 and 400 spectrometer. Chemical shifts were
reported in d (ppm) relative to tetramethylsilane. All
coupling constants were described inHz. Elemental
analyses were performed by Atlantic Microlab, Inc.,
GA. Flash column chromatography was performed on
silica gel (40–63lm) under a pressure of about 4psi. Sol-
vents were obtained from commercial suppliers and
purified as follows: tetrahydrofuran and ether were dis-
tilled from sodium benzophenone ketyl; dichlorometh-
ane was distilled over calcium hydride. Synthesized
final compounds were checked for purity by analytical
HPLC, which was performed using a Rainin HP con-
troller and a Rainin UV-C detector with a Rainin
Figure 3. Inhibition of P. falciparum growth in red blood cells by
compounds 10–16. Seven different esters of compound 17 (FTI-2148)
were tested at the indicated concentrations for in vitro inhibition
of P. falciparum 3D7 growth in red blood cells. The malaria growth
3
14
was measured by the [ H]hypoxanthine-incorporation assay and
expressed as % incorporation of the no-drug control level.
250mm · 4.6mm, 5lm Microsorb C-18 column, eluted
with gradient 10–90% of CH CN in 0.1% TFA in H O
3
2
in 30min. 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.
Table 2. In vivo study results of 16 against P. berghei
Group
Dose
(mg/kg)
% Parasitemia
% Inhibition
Control
Chloroquine
0
12.42
0.00
6.70
0.0
100
46.1
4.1.1. 2-Bromo-4-nitrobenzoic acid. A solution of 2-bro-
mo-4-nitrotoluene (25.2g, 116mmol) in pyridine
50
50
16
(
100mL) and water (200mL) was warmed to 97ꢁC and
KMnO (101g, 642mmol) was added in portions. The
reaction was refluxed for 5h and an additional amount
of KMnO (27.5g, 174mmol), H O (40mL) and pyri-
4
cellular hydrolytic activity (in serum and red blood cell),
and hydrolysis rate by esterases in the parasite cells.
Optimization of these factors would be expected to
improve antiparasitic inhibition by increasing inhibitor
concentration inside the parasites.
4
2
dine (80mL) were added. The reaction mixture was
allowed to reflux overnight and the hot solution was
then filtered over Celite. The resulting filtrate was acidi-
fied with 12M HCl (110mL) and extracted with AcOEt
(600mL). The organic layer was dried with Na SO and
the solvent was evaporated under vacuum to give the
2 4
In this series, the benzyl ester 16 showed the best inhibi-
tion activity against P. falciparum in red blood cells with
an ED50 value of 150nM (Fig. 3). This promising in
vitro result encouraged us to further evaluate the anti-
malarial activity of benzyl ester 16 in murine malarial
models infected with Plasmodium berghei. Using Petersꢀ
free acid as white crystals (17.6g, 62%). Mp 153–
1
155ꢁC; H NMR (CD OD) d 8.43 (d, J = 2.2Hz, 1H),
3
8.18 (dd, J = 2.2 and 8.5Hz, 1H), 7.86 (d, J = 8.5Hz,
1H); C NMR (CD OD) d 168.47, 150.97, 141.29,
1
3
3
132.98, 130.21, 123.81, 122.46.
1
2
four-day suppressive test, compound 16 was adminis-
tered to P. berghei-infected mice by ip injection at a
4.1.2. 2-Bromo-4-nitrobenzoic acid methyl ester (1).
ꢀ
1
daily dose of 50mgkg and chloroquine was used as a
positive control (Table 2). In this study, the benzyl ester
prodrug was found to suppress parasitemia by 46.1% as
compared to untreated infected controls and promoted
no apparent toxicity in the mice.
Absolute MeOH (50mL) was cooled to 0ꢁC and SOCl
2
(6mL, 82mmol) was added dropwise. A solution of the
free acid (7.5g, 30.5mmol) in MeOH (25mL) was added
and the reaction mixture was then refluxed overnight.
The white solid was filtered, and the filtrate was concen-
trated under reduced pressure. The white precipitates
were combined and recrystallized from hexanes and
3
. Conclusions
ether to afford the product as white crystals (6.53g,
1
8
2%). Mp 82–85ꢁC; H NMR (CDCl ) d 4.02 (s, 3H),
3
In conclusion, we have shown that ester derivatives of 17
with increased lipophilicity show enhanced inhibition
activity against Plasmodium falciparum growth in red
blood cells. Additionally, 16 showed in vivo activity
using murine malaria models infected with Plasmodium
berghei. Further investigations exploring other prodrug
mechanisms in order to generate new antimalarial agents
with potent and long duration of action are ongoing.
7.96 (d, J = 8.4Hz, 1H), 8.24 (dd, J = 2.2 and 8.6Hz,
1H), 8.55 (d, J = 2.2Hz, 1H); C NMR (CDCl ) d
1
3
3
165.61, 149.65, 138.35, 132.17, 129.57, 122.55, 122.40,
53.50.
0
4.1.3.
methyl ester (2). A solution of 1 (0.259g, 1mmol) in
2 -Methyl-5-nitro-biphenyl-2-carboxylic
acid
DMF (1.5mL) was flushed with N2 for 30min.

6522
D. Carrico et al. / Bioorg. Med. Chem. 12 (2004) 6517–6526
0
Pd(PPh ) (0.038g, 33lM) was added and the reaction
4.1.5. 5-Cyano-2 -methyl-biphenyl-2-carboxylic acid methyl
ester (4). A solution of 3 (0.33g, 0.93mmol) and CuCN
(0.187g, 2.1mmol) in N-methyl-2-pyrrolidinone
(4.0mL) was stirred at 200ꢁC for 4h. To the mixture
3
4
was stirred for 10min. To the resulting red reaction mix-
ture, o-tolylboronic acid (0.149g, 1.1mmol) and K CO
2
3
(
0.415g, 3mmol) were added. The mixture was stirred at
0ꢁC for 1h and at 120ꢁC overnight. H O (1.5mL) was
8
added and the mixture was extracted with Et O
was added NaCN solution (0.391g in 8.0mL of H O)
and the mixture was extracted with AcOEt (250mL).
The organic layer was dried with MgSO and the solvent
was evaporated. Purification of the crude product by
SiO2 column chromatography with hexanes/AcOEt =
4:1 afforded the product as a white solid (0.16g, 68%).
Mp 65–68ꢁC; H NMR (CDCl ) d 2.07 (s, 3H), 3.63
(s, 3H), 7.03 (d, J = 6.5Hz, 1H), 7.20–7.33 (m, 3H),
7.56 (d, J = 1.5Hz, 1H), 7.72 (dd, J = 1.7 and 8.1Hz,
2
2
2
(
4 · 3mL). The organic layer was washed with 1M
4
HCl (2 · 1.5mL) and dried over anhydrous MgSO .
Solvent evaporation followed by SiO column chroma-
tography with hexanes/AcOEt = 10:1 afforded the prod-
uct as a pale yellow oil (0.25g, 92%). H NMR
4
2
1
1
3
(
J = 2.2Hz, 1H), 8.07 (d, J = 8.6Hz, 1H), 7.34–7.21 (m,
CDCl ) d 8.26 (dd, J = 2.4 and 8.6Hz, 1H), 8.13 (d,
3
1
3
3
3
1
1
H), 7.08 (d, J = 7.4Hz, 1H), 3.65 (s, 3H), 2.09 (s,
1H), 8.02 (d, J = 8.1Hz, 1H); C NMR (CDCl ) d
3
1
3
H); C NMR (CDCl ) d 166.79, 149.55, 144.68,
166.51, 143.68, 138.91, 135.13, 134.66, 134.48, 130.69,
130.55, 129.83, 128.34, 128.20, 125.60, 117.86, 115.15+,
52.45, 19.97; LRMS (CI) m/z calcd for C H NO H
252, found 252; HRMS (CI) m/z calcd for
C H NO H 252.1025, found 252.1029; Anal. Calcd
3
39.30, 136.72, 135.54, 131.39, 130.30, 128.78, 128.71,
26.20, 126.03, 122.40, 52.89, 20.35; LRMS (CI)
1
6
13
2
+
m/z calcd for C H NO H 272, found 272; HRMS
1
5
13
4
+
CI) m/z calcd for C H NO H 272.0923, found
+
(
1
5
13
4
16 13
2
2
72.0922.
for C H NO : C, 76.48; H, 5.21; N, 5.57. Found: C,
16 13 2
7
6.06; H, 5.12; N, 5.53.
0
4
.1.4. 5-Iodo-2 -methyl-biphenyl-2-carboxylic acid methyl
0
ester (3). To a solution of 2 (4.46g, 16.4mmol) in AcOEt
120mL) was added SnCl Æ2H O (39.6g, 175mmol). The
4.1.6. 5-(tert-Butoxycarbonylamino-methyl)-2 -methyl-
biphenyl-2-carboxylic acid methyl ester (5). A solution
of 10% Pd–C (0.116g) in THF (10mL) was preactivated
(
2
2
reaction was refluxed for 4h and cooled to room temper-
ature. To the mixture AcOEt (370mL) was added
and the resulting mixture was poured into saturated
for 30min under 40psi H atmosphere. To the mixture a
2
solution of 4 (0.551g, 2.20mmol) and Boc O (0.972g,
2
NaHCO (500mL). The organic layer was washed with
3
4.45mmol) in THF (12mL) was added and the solution
was hydrogenated under 60psi H2 atmosphere over-
night. The mixture was filtered over Celite and the fil-
trate was concentrated under reduced pressure.
Purification with SiO2 column chromatography with
hexanes/AcOEt = 4:1 afforded the desired product as a
brine (300mL) and dried over anhydrous MgSO4.
Solvent evaporation afforded the free amine desired
1
product (3.68g, 93%). H NMR (CDCl ) d 1.95 (s,
3
3
H), 3.44 (s, 3H), 3.94 (br s, 2H), 6.26 (d, J = 2.4Hz,
1
J = 7.4Hz, 1H), 7.04–7.13 (m, 3H), 7.74 (d, J = 8.5Hz,
H), 6.45 (dd, J = 2.5 and 8.5Hz, 1H), 6.93 (d,
1
white solid (0.719g, 92%). Mp 68–71ꢁC; H NMR
1
3
1
1
1
H); C NMR (CDCl ) d 167.65, 150.52, 146.09,
(CDCl ) d 1.46 (s, 9H), 2.05 (s, 3H), 3.60 (s, 3H), 4.39
3
3
42.80, 135.66, 133.06, 129.68, 128.49, 127.37, 125.58,
19.17, 114.20, 113.21, 51.82, 20.36; LRMS (CI)
(d, J = 6.0Hz, 2H), 4.91 (br s, 1H), 7.05 (d, J = 7.8Hz,
1H), 7.14 (d, J = 1.3Hz, 1H), 7.17–7.25 (m, 3H), 7.34
+
m/z calcd for C H NO H 242, found 242; HRMS
1
(dd, J = 1.7 and 8.1Hz, 1H), 7.94 (d, J = 8.1Hz, 1H);
C NMR (CDCl ) d 167.49, 155.84, 143.40, 142.89,
5
15
2
+
CI) m/z calcd for C H NO H 242.1181, found
13
(
1
5
15
2
3
2
42.1178.
141.34, 135.26, 130.52, 129.56, 129.45, 129.17, 128.41,
27.31, 125.87, 125.25, 51.84, 44.22, 28.37, 20.01; LRMS
1
+
A solution of the free amine (1.85g, 7.64mmol) in HCl
(ESI) m/z calcd for C H NO H 356, found 356;
21 25 4
+
(
(
(
2.8mL), H O (2.4mL), AcOH (0.8mL) and MeOH
2
2.0mL) was cooled to 0ꢁC. To the mixture NaNO2
0.604g, 8.75mmol) in H O (1.8mL) was added and
HRMS (ESI) m/z calcd for C H NO H 356.1862,
21 25 4
found 356.1872.
2
0
the mixture was stirred at 0ꢁC. A mixture of KI
1.90g, 11.4mmol) in HCl (5mL) was added and the
4.1.7. 5-(tert-Butoxycarbonylamino-methyl)-2 -methyl-
biphenyl-2-carboxylic acid (6). To a solution of 5 (1.2g,
3.38mmol) in THF (12mL) and MeOH (3mL), was
(
reaction was stirred at room temperature for 5min and
then at 60ꢁC for 15min. The mixture was extracted with
AcOEt (300mL) and the organic layer was washed with
5% Na SO (200mL). The organic layer was dried over
2 3
MgSO and the solvent was evaporated. Purification by
added LiOH (0.487g, 11.6mmol) in H O (9.5mL) and
2
the mixture was refluxed overnight. The reaction mix-
ture was neutralized with 10% citric acid (2.5mL) and
the solvent was evaporated. The residue was then acidi-
fied with 10% citric acid (23mL) and extracted with
AcOEt. The organic layer was dried over anhydrous
4
SiO2 column chromatography with hexanes/AcOEt =
8
:1 afforded the product as a yellow oil (2.23g, 83%).
H NMR (CDCl ) d 2.06 (s, 3H), 3.60 (s, 3H), 7.04 (d,
J = 7.3Hz, 1H), 7.16–7.30 (m, 3H), 7.63 (d, J = 1.8Hz,
1
MgSO and concentrated under vacuum to afford the
4
3
1
desired product as a colorless foam (1.14g, 99%). H
1
8
1
1
H), 7.67 (d, J = 8.4Hz, 1H), 7.78 (dd, J = 1.8 and
NMR (CDCl ) d 1.45 (s, 9H), 2.06 (s, 3H), 4.40 (br s,
3
1
3
.3Hz, 1H); C NMR (CDCl ) d 166.16, 143.62,
2H), 4.93 (br s, 1H), 7.07 (d, J = 7.3Hz, 1H), 7.13 (s,
1H), 7.18–7.25 (m, 3H), 7.35 (d, J = 8.4Hz, 1H), 8.02
3
38.80, 135.32, 134.15, 131.10, 130.37, 128.50, 127.54,
27.44, 127.26, 126.63, 124.31, 50.97, 18.94; LRMS
1
3
(d, J = 8.1Hz, 1H); C NMR (CD OD) d 170.05,
3
+
(
CI) m/z calcd for C H IO H 353, found 353; HRMS
1
157.48, 143.58, 143.14, 142.01, 135.41, 130.45, 130.08,
129.53, 129.36, 128.57, 127.02, 125.57, 125.11, 79.24,
43.53, 27.64, 19.19; LRMS (ESI) m/z calcd for
5
13
2
+
(
CI) m/z calcd for C H IO H 353.0039, found
15 13 2
3
53.0037.

D. Carrico et al. / Bioorg. Med. Chem. 12 (2004) 6517–6526
6523
+
C H NO H 342, found 342; HRMS (ESI) m/z calcd
stirred for 30min at room temperature. To the mixture
was added NaBH (0.072g, 1.9mmol) and the reaction
2
0
23
4
+
for C H NO H 342.1705, found 342.1712.
2
0
23
4
4
was stirred for 36h. Evaporation of the solvent afforded
a residue which was extracted with 10% MeOH in
CH Cl (350mL) and saturated NaHCO (80mL). The
0
.1.8. 2-{[5-(tert-Butoxycarbonylamino-methyl)-2 -meth-
yl-biphenyl-2-carbonyl]-amino}-4-methylsulfanyl-butyric
4
2
2
3
acid methyl ester (7). A solution of benzoic acid 6 (1.03g,
organic layer was washed with brine and dried over
anhydrous MgSO . Evaporation of the solvent followed
by purification by SiO column chromatography with
2
3
.01mmol), methionine methyl ester hydrochloride
0.667g, 3.33mmol), HOBt (0.455g, 3.33mmol) and
Et N (0.46mL, 3.33mmol) in CH Cl (15mL) was
4
(
9:1:0.1 = CH Cl /MeOH/NH OH afforded the product
2
3
2
2
2
4
cooled to ꢀ10ꢁC. To the cooled solution was added
EDCI (0.590g, 3.01mmol) and the mixture was stirred
for 36h at room temperature. The reaction mixture
was extracted with CH Cl (130mL) and satd. NaHCO
3
as a white foam (0.48g, 54%). TLC R 0.23 (CH Cl /
f 2 2
1
MeOH/NH OH 9:1:0.1); HPLC t 11.93; H NMR
4
R
(CDCl ) d 1.58–1.63 (m, 1H), 1.85–1.87 (m, 1H), 2.01–
3
2.19 (m, 8H), 3.66 (s, 3H), 3.80 (s, 2H), 3.86 (s, 2H),
4.43–4.52 (m, 1H), 6.03 (d, J = 7.4Hz, 1H), 6.88 (s,
1H), 7.16–7.42 (m, 6H), 7.48 (s, 1H), 7.78 (dd, J = 7.9
2
2
(
55mL). The organic layer was then extracted with 10%
citric acid (50mL) and brine. Anhydrous MgSO was
4
1
3
added and the dried organic layer was concentrated
under vacuum. The crude product was purified by
SiO column chromatography with 2:1 = hexanes/ethyl
and 26.7Hz, 1H); C NMR (CDCl ) d 172.32, 168.57,
3
140.56, 140.29, 139.97, 136.58, 135.64, 130.97, 130.87,
130.55, 130.04, 129.79, 129.63, 129.36, 128.75, 127.80,
2
acetate to afford a white foamy product (1.00g, 69%).
126.56, 52.85, 52.78, 52.24, 45.61, 31.83, 29.71, 20.42+,
1
H NMR (CDCl ) d 1.45 (s, 9H), 1.52–1.65 (m, 1H),
15.56; LRMS (ESI) m/z calcd for C H N O SH
467, found 467; HRMS (ESI) m/z calcd for
C H N O SH 467.2117, found 467.2112.
3
25 30
4
3
1
4
1
.81–1.91 (m, 1H), 1.96–2.20 (m, 8H), 3.66 (s, 3H),
.39 (d, J = 5.2Hz, 2H), 4.57–4.67 (m, 1H), 4.96 (br s,
H), 5.90 (d, J = 7.5Hz, 1H), 7.11 (s, 1H), 7.15–7.41
+
2
5
30
4
3
1
3
(m, 5H), 7.94 (dd, J = 8.0Hz (each), 1H); C NMR
(CDCl ) d 172.15, 167.91, 156.23, 140.33, 136.71,
4.1.12. 2-[(5-{[(1H-Imidazol-4-ylmethyl)-amino]-methyl}-
2 -methyl-biphenyl-2-carbonyl)-amino]-4-methylsulfanyl-
0
3
1
1
3
31.05, 130.95, 130.55, 130.29, 129.63, 129.33, 128.83,
26.88, 126.73, 126.64, 80.15, 52.75, 52.10, 44.58,
2.11, 29.68, 28.76, 20.44, 15.61; LRMS (ESI) m/z calcd
butyric acid ethyl ester (FTI-2390) (11). This compound
was prepared in a manner similar to that described for
FTI-2153 (10) with 8 to give the product as a white foam
(42mg, 36%). TLC R 0.31 (CHCl /MeOH/NH OH
+
for C H N O SH 487, found 487; HRMS (ESI) m/z
5
2
6
34
2
f
3
4
+
calcd for C H N O SH 487.2267, found 487.2270.
1
9:1:0.1); HPLC tR 12.56; H NMR (CDCl ) d 1.23 (t,
2
6
34
2
5
3
J = 7.2Hz, 3H), 1.56–1.65 (m, 1H), 1.80–1.91 (m, 1H),
1.97–2.22 (m, 8H), 3.82 (s, 2H), 3.87 (s, 2H), 4.06–4.17
(m, 2H), 4.53–4.65 (m, 1H), 5.95 (d, J = 7.0Hz, 1H),
6.91 (s, 1H), 7.15–7.44 (m, 6H), 7.56 (s, 1H), 7.90 (dd,
0
.1.9. 2-{[5-(tert-Butoxycarbonylamino-methyl)-2 -meth-
yl-biphenyl-2-carbonyl]-amino}-4-methylsulfanyl-butyric
acid ethyl ester (8). This compound was prepared in a
similar manner to that described for compound 7 with
4
1
3
J = 8.0 and 20.7Hz, 1H); C NMR (CDCl ) d 171.86,
3
6
and methionine ethyl ester hydrochloride to yield a
168.40, 140.69, 139.93, 136.68, 135.54, 131.02, 130.92,
130.60, 130.51, 130.17, 129.90, 129.70, 129.34, 128.80,
127.86, 126.72, 61.92, 52.97, 52.36, 45.65, 32.12, 29.65,
colorless foam. This compound was immediately used
for the next reaction without further purification.
2
0.53, 15.67, 14.53; LRMS (FAB) m/z calcd for
0
+
4
.1.10. 2-{[5-(tert-Butoxycarbonylamino-methyl)-2 -meth-
C H N O SH 481, found 481; HRMS (FAB)
26 32 4 3
m/z calcd for C H N O SH
2
481.2275.
+
yl-biphenyl-2-carbonyl]-amino}-4-methylsulfanyl-butyric
acid allyl ester (9). This compound was prepared in a
similar manner to that described for compound 7 with
481.2273, found
6
32
4
3
6
and methionine allyl ester sulfonate to afford a white
4.1.13. 2-[(5-{[(1H-Imidazol-4-ylmethyl)-amino]-methyl}-
2 -methyl-biphenyl-2-carbonyl)-amino]-4-methylsulfanyl-
butyric acid allyl ester (FTI-2391) (12). This compound
was prepared in a manner similar to that described for
FTI-2153 (10) with 9 to afford the product as a colorless
0
foamy residue. This compound was used for the next
reaction without further purification.
4
2
.1.11. 2-[(5-{[(1H-Imidazol-4-ylmethyl)-amino]-methyl}-
0
-methyl-biphenyl-2-carbonyl)-amino]-4-methylsulfanyl-
foam (60mg, 32%). TLC Rf 0.30 (CHCl /MeOH/
3
1
butyric acid methyl ester (FTI-2153) (10). A solution of 7
(
NH OH 9:1:0.1); HPLC t 13.44; H NMR (CDCl ) d
4
R
3
0.92g, 1.90mmol) in CH Cl (9.2mL) was cooled to
2
1.54–1.67 (m, 1H), 1.79–1.92 (m, 1H), 1.95–2.22 (m,
8H), 3.82 (s, 2H), 3.88 (s, 2H), 4.55 (d, J = 5.7Hz,
2H), 4.57–4.69 (m, 1H), 5.20–5.32 (m, 2H), 5.79–5.91
(m, 1H), 5.94 (d, J = 7.6Hz, 1H), 6.91 (s, 1H), 7.13–
7.46 (m, 6H), 7.56 (s, 1H), 7.91 (dd, J = 8.2 and
2
0
ꢁC. To the cooled solution was added TFA (10mL)
and the reaction was stirred at room temperature for
0min. Evaporation of the solvent was followed by
extraction with CH Cl (150mL) and 5% NH OH
3
2
2
4
1
3
(100mL). The organic layer was washed with brine
and dried over MgSO . Evaporation of the solvent af-
forded the free amine (0.74g). This material was used
for the next reaction without further purifications.
21.7Hz, 1H); C NMR (CDCl ) d 171.32, 168.28,
3
140.87, 139.27, 136.31, 136.06, 135.80, 135.43, 135.15,
133.09, 131.69, 131.09, 130.21, 129.54, 129.09, 126.75,
124.57, 121.56, 119.39, 66.48, 52.40, 51.39, 40.78,
4
3
1.56, 29.74, 20.14, 15.50; LRMS (FAB) m/z calcd
+
To a solution of the free amine (0.74g, 1.91mmol) in
MeOH (7.2mL) was added 4(5)-imidazolecarboxalde-
hyde (0.184g, 1.91mmol) and the reaction mixture was
for C H N O SH 493, found 493; HRMS (FAB)
27 32 4 3
m/z calcd for C H N O SH
4
+
493.2273, found
2
7
32
3
493.2272.

6524
D. Carrico et al. / Bioorg. Med. Chem. 12 (2004) 6517–6526
4
2
.1.14. 2-[(5-{[(1H-Imidazol-4-ylmethyl)-amino]-methyl}-
-methyl-biphenyl-2-carbonyl)-amino]-4-methylsulfanyl-
cyclohexanol was removed by vacuum distillation and
the obtained tetracyclohexyl titanate was immediately
used in the synthesis of ester 15 without further purifica-
0
butyric acid isopropyl ester (FTI-2392) (13). To a
solution of FTI-2153 (10) (23.6mg, 0.051mmol) in
˚
iPrOH (1mL) was added two small 4A molecular sieves.
1
tion. H NMR (CDCl ) d 1.28 (br s, 5H), 1.55 (br s, 1H),
3
1.74 (br s, 2H), 1.90 (br s, 2H), 3.62 (br s, 1H).
The mixture was flushed with N and stirred at room
2
temperature for 15min. To the mixture was added four
drops of Ti(OiPr) and an additional amount of iPrOH
A solution of FTI-2153 (10) (25.5mg, 0.055mmol) in
10:1 THF/DMF (2mL) was added to a round bottom
flask containing freshly prepared tetracyclohexyl titan-
ate (0.16mmol) with a syringe to avoid minimal expo-
sure to atmospheric moisture. To the mixture, was
4
(
N . HPLC analysis was used to determine completion of
1mL). The mixture was stirred at 70ꢁC for 3days under
2
the reaction. Evaporation of the solvent gave a residue
that was dissolved in MeOH (2mL) and H O (0.1mL).
˚
added five small 4A molecular sieves and the reaction
2
The resulting mixture was concentrated and filtered over
SiO2 with 5:1:0.1 = CHCl /MeOH/NH OH. Evapora-
was stirred at 60ꢁC for 9days under N atmosphere.
2
During this period, additional amounts of THF were
added to keep approximately 2mL of solvent in the
reaction flask. The solvent was evaporated and to the
3
4
tion of the solvent gave the desired product, which
was further purified by preparatory HPLC to give a
white foam (25mg, 99%). TLC R 0.34 (CHCl /MeOH/
residue was added MeOH (2mL) and H O (0.1mL).
2
f
3
1
NH OH 9:1:0.1); HPLC tR 13.18; H NMR (CDCl ) d
The reaction mixture was stirred for an additional
5min at room temperature and was concentrated under
vacuum. The residue was filtered over SiO2 with
5:1:0.1 = CHCl /MeOH/NH OH. Evaporation of the
4
3
1
1
4
.17–1.24 (m, 6H), 1.51–1.64 (m, 1H), 1.78–1.89 (m,
H), 1.96–2.23 (m, 8H), 3.86 (s, 2H), 3.91 (s, 2H),
.48–4.63 (m, 1H), 4.94–5.01 (m, 1H), 5.94 (d,
3
4
J = 7.9Hz, 1H), 6.93 (s, 1H), 7.14–7.63 (m, 7H), 7.89
(
solvent gave the crude product, which was further puri-
fied by preparatory HPLC to afford the final product as
1
3
dd, J = 7.9 and 19.0Hz, 1H); C NMR (CDCl ) d
3
1
1
1
1
3
71.06, 168.27, 140.81, 139.31, 136.34, 136.05, 135.68,
35.15, 133.05, 131.04, 129.99, 129.55, 129.29, 129.06,
26.73, 124.62, 121.57, 69.89, 52.58, 51.34, 40.74,
a colorless foam (13mg, 44%). HPLC tR 15.14;
H
NMR (CDCl ) d 1.18–1.44 (br m, 5H), 1.46–1.56 (br
3
m, 1H), 1.56–1.74 (br m, 3H), 1.74–1.89 (br m, 3H),
1.90–2.20 (br m, 8H), 4.22 (s, 2H), 4.33 (s, 2H), 4.47–
4.61 (br m, 1H), 4.68–4.79 (br s, 1H), 6.27 (br d,
J = 16.9Hz, 1H), 6.87–7.67 (m, 7H), 7.80 (br s, 1H),
1.68, 29.65, 22.00, 20.22, 15.53; LRMS (FAB) m/z
+
calcd for C H N O SH 495, found 495; HRMS
2
7
34
4
3
+
(
FAB) m/z calcd for C H N O SH 495.2430, found
27 34 4 3
1
3
4
95.2430.
8.34 (br s, 1H); C NMR (CDCl ) d 172.10, 169.58,
3
1
39.98, 139.90, 139.46, 137.79, 137.48, 131.77, 131.76,
4
2
.1.15. 2-[(5-{[(1H-Imidazol-4-ylmethyl)-amino]-methyl}-
-methyl-biphenyl-2-carbonyl)-amino]-4-methylsulfanyl-
131.73, 129.76, 129.60, 128.82, 128.34, 128.31, 127.75,
125.41, 73.26, 51.03, 50.36, 30.66, 30.34, 28.68, 28.12,
24.13, 22.51, 18.90, 14.18; LRMS (FAB) m/z calcd for
0
butyric acid 2-ethyl-hexyl ester (FTI-2393) (14). This
ester was prepared in a similar manner to that described
for 13 with FTI-2153 (10), titanium (IV)-2-ethylhex-
oxide in 10:1 THF/DMF to yield the product as a color-
less foam (40mg, 99%). TLC R 0.37 (CHCl /MeOH/
+
C H N O SH 535, found 535; HRMS (FAB) m/z
4
3
0
38
3
+
calcd for C H N O SH 535.2743, found 535.2745.
3
0
38
4
3
4.1.17. 2-{[(5-[(1H-Imidazol-4-ylmethyl)-amino]-methyl}-
2 -methyl-biphenyl-2-carbonyl)-amino]-4-methylsulfanyl-
f
3
1
0
NH OH 9:1:0.1); HPLC tR 17.73; H NMR (CDCl ) d
4
3
0
1
2
.73–0.86 (m, 6H), 1.12–1.30 (m, 9H), 1.42–1.57 (m,
H), 1.71–1.85 (m, 1H), 1.88–2.16 (m, 8H), 3.76 (s,
H), 3.82 (s, 2H), 3.90 (d, J = 5.6Hz, 2H), 4.49–4.62
butyric acid benzyl ester (FTI-2628) (16). A solution of
FTI-2153 (10) (146mg, 0.313mmol) in THF (3.1mL)
was flushed with N for 25min. To the solution was
2
(
7
m, 1H), 5.85 (d, J = 8.0Hz, 1H), 6.82–7.57 (m, 8H),
added benzyl alcohol (3.2mL, 31mmol), and activated
˚
4A molecular sieves (0.62g). The reaction was stirred
1
.83 (dd, J = 7.8 and 17.6Hz, 1H); C NMR (CDCl3)
3
d 171.85, 168.27, 140.83, 140.79, 139.38, 136.43,
under N2 for an additional 15min, and Ti(OiPr)4
(21.7lL) was added. The reaction mixture was stirred
1
1
4
2
36.06, 133.30, 132.99, 131.09, 130.35, 130.17, 129.57,
29.33, 129.10, 126.75, 125.26, 68.44, 52.31, 51.33,
1.24, 39.01, 31.91, 30.64, 29.68, 29.23, 24.02, 23.30,
at 85ꢁC for 2days under N atmosphere. The solvent
2
was evaporated and the residue was dissolved in MeOH
(16mL) and H O (0.8mL) and stirred for an additional
0.23, 15.51, 14.39, 11.29; LRMS (FAB) m/z calcd for
2
+
C H N O SH 565, found 565; HRMS (FAB) m/z
3
10min at room temperature. The resulting mixture was
concentrated and filtered over SiO2 with 5:1:0.1 =
CHCl /MeOH/NH OH. Evaporation of the solvent
2
44
4
3
+
calcd for C H N O SH 565.3212, found 565.3210.
3
2
44
4
3
3
4
4
2
.1.16. 2-[(5-{[(1H-Imidazol-4-ylmethyl)-amino]-methyl}-
-methyl-biphenyl-2-carbonyl)-amino]-4-methylsulfanyl-
gave the crude product, which was further purified by
preparatory HPLC to give 16 as a white foam
0
1
butyric acid cyclohexyl ester (FTI-2394) (15). Tetra-
cyclohexyl titanate was prepared by reacting titanium
tetraisopropoxide (0.143g, 0.5mmol) with freshly dis-
tilled cyclohexanol (0.26mL, 2.5mmol) in anhydrous
(164.5mg, 98%). HPLC tR 15.61; H NMR (CDCl ) d
3
1.55–168 (m, 1H), 1.78–1.88 (m, 1H), 1.89–2.10 (m,
8H), 4.19 (s, 2H), 4.30 (s, 2H), 4.52–4.66 (br m, 1H),
5.07 (s, 2H), 6.27 (br d, J = 30Hz, 1H), 6.96–7.56 (m,
1
3
toluene (3.1mL) under N atmosphere and the tolu-
2
12H), 7.78 (s, 1H), 8.30 (s, 1H); C NMR (CDCl ) d
3
ene–isopropanol azeotrope was removed by distillation.
Additional toluene was added periodically with continu-
ous removal of toluene–isopropanol azeotrope in order
to drive the reaction to completion. Excess unreacted
171.32, 168.47, 140.88, 139.13, 136.28, 135.73, 135.45,
135.32, 135.17, 133.07, 130.99, 130.08, 129.60, 129.28,
129.15, 129.05, 128.76, 128.70, 124.44, 121.59, 67.73,
52.33, 51.48, 40.79, 31.46, 29.65, 20.12, 15.49; LRMS

D. Carrico et al. / Bioorg. Med. Chem. 12 (2004) 6517–6526
6525
+
FAB) m/z calcd for C H N O SH 543, found 543;
(
HRMS (FAB) m/z calcd for C H N O SH
16 and the control drug, chloroquine, were adminis-
tered i.p. for four consecutive days in a 0.2mL volume
at, respectively, doses of 50mg/kg and 10mg/kg. On
day 5, blood smears were taken, fixed in methanol
and Giemsa stained. The percentage of suppression
was calculated from the parasitemia in untreated and
treated groups.
3
1
34
4
3
+
3
1
34
4
3
5
43.2430, found 543.2428.
4
2
.1.18. 2-[(5-{[(1H-Imidazol-4-ylmethyl)-amino]-methyl}-
-methyl-biphenyl-2-carbonyl)-amino]-4-methylsulfanyl-
0
butyric acid. 2TFA salt (FTI-2148.2TFA) (17). A solu-
tion of FTI-2153 (10) (1.41g, 3.02mmol) in THF
(
5
21mL) was cooled to 0ꢁC and LiOH (0.251g,
.99mmol) in H O (12.1mL) was added dropwise. The
4.1.22. In vitro mammalian PFT and PGGT-I activity
assay. In vitro inhibition assays of mammalian PFT and
PGGT-I were done by measuring the incorporation of
2
reaction was stirred at room temperature for 30min
and neutralized with 0.5N HCl (15mL). Solvent evapo-
ration gave a white residue which was further purified by
gel permeation on Sephadex LH-20 with MeOH/
CHCl = 1:1 to give FTI-2148. The product was dis-
3
solved in CH Cl (9.5mL) and MeOH (1.1mL) and
2
3
[ H] FPP (Amersham Biosciences, Piscataway, NJ) and
[ H] GGPP (Perkin–Elmer Life and Analytical Sciences,
3
Boston, MA) into wild type H-Ras (PFT) and H-Ras-
CVLL (PGGT-I), respectively, as previously de-
1
5
scribed. Briefly, 75lg of 60,000g post-microsomal
supernatant from Daudi cells was incubated in the pres-
ence of increasing concentration of compound, 10lg H-
Ras or H-Ras-CVLL substrate, and 0.5lCi/sample of
2
the solution was cooled in ice-water bath. TFA
1.3mL) was then added and the solvent was rapidly re-
moved under vacuum to give the product as a cream yel-
(
1
3
3
low TFA salt (1.72g, 84%). HPLC tR 10.46; H NMR
(
(
4
(
1
1
1
1
4
either [ H] FPP or [ H] GGPP. Samples were TCA-pre-
cipitated and then filtered onto glass fiber filters;
CD OD) d 1.65–1.82 (m, 1H), 1.95–2.26 (m, 9H), 4.39
3
3
3
s, 2H), 4.39–4.48 (m, 1H), 4.48 (s, 2H), 7.13–7.33 (m,
H), 7.45 (d, J = 12.0Hz, 1H), 7.61–7.73 (m, 2H), 7.74
unbound [ H] FPP or [ H] GGPP was washed through
the filters. Samples were counted on a scintillation coun-
ter and activity compared to vehicle controls to obtain
IC50 values.
1
3
s, 1H), 8.81 (s, 1H); C NMR (CD OD) d 175.09,
3
71.77, 142.41, 141.05, 139.10, 137.76, 137.18, 134.49,
33.75, 131.75, 131.09, 130.49, 130.21, 129.74, 127.21,
26.12, 122.75, 53.27, 52.07, 41.99, 32.12, 31.25, 20.89,
4.1.23. Mammalian Ras and Rap1A processing assay.
Subconfluent NIH 3T3 mouse fibroblast cells stably
transfected with H-Ras61L (H-Ras/3T3) were treated
for 48h with increasing concentrations of the com-
pounds to determine their inhibitory activity against
the farnesylation of H-Ras protein and geranylgeranyl-
ation of Rap1A protein. Cells were lysed in 30mM
+
5.45; LRMS (ESI) m/z calcd for C H N O SH
2
4
28
4
3
53, found 453; HRMS (ESI) m/z calcd for
+
C H N O SH 453.1960, found 453.1952.
2
4
28
4
3
4.1.19. LogP. Theoretical values of logP were predicted
using QikProp program established at Yale Univer-
1
3
sity. The 3D structures were entered by using XChem-
Edit and were optimized using BOSS program. The
octanol/water partition coefficients were computed with
Hepes, pH7.5, 10mM NaCl, 5mM MgCl , 25mM
2
NaF, 1mM EGTA, 1% Triton-X-100, 10% glycerol,
10lg/mL aprotinin, 10lg/mL soybean trypsin inhibitor,
25lg/mL leupeptin, and 2mM phenylmethylsulfonylfluo-
ride on ice for 30min. Cleared lysates were resuspended
in 2· SDS-PAGE sample buffer and run on 12.5% SDS-
PAGE gels to separate proteins. Proteins were trans-
ferred to nitrocellulose and probed with 25lg/mL
Y13-289 pan-Ras antibody for detection of H-Ras or
with 0.2lg/mL Rap1 antibody (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA). Results were visualized using per-
oxidase-conjugated antirat or antirabbit secondary
antibodies (Jackson ImmunoResearch, West Grove,
PA) and an enhanced chemiluminescence detection sys-
tem (Perkin–Elmer).
1
1
the commercial software QikProp version 1.6.
4
.1.20. Assay for in vitro inhibition of P. falciparum
1
4
growth. P. falciparum 3D7 in human red blood cells
0.4% parasitemia, 2% hematocrit) was cultured with
the compound (added as 1lL DMSO solution) in a
(
2
0
00-lL medium using a 96-well plate. After 48h,
3
.8lCi of [8- H]hypoxanthine (American Radiolabeled
Chemicals) was added to the culture, and the incubation
was continued for an additional 24h. The malaria
growth was quantified by measuring the radioactivity
incorporation into the parasites and expressed as %
incorporation of the no-drug control level. Murine
3
T3 fibroblasts were grown in 96-well plates in the pres-
4.1.24. Assay for in vitro inhibition of P. falciparum PFT.
The PFT assay used to determine the IC s of the com-
ence of serially diluted compounds for 72h. Viability
was quantified using the AlamarBlue reagent (Alamar
Biosciences Inc., Sacramento, Calif.).
5
0
1
6
pounds is based on the glass fiber filter method. Assays
were carried out in 30mM potassium phosphate pH7.7,
with 5mM DTT, 0.5mM MgCl , 20lM ZnCl , 0.3lCi
2
2
3
4.1.21. In vivo antimalarial test. Blood was taken from
donor mice infected with P. berghei (ANKA strain)
and diluted in heat inactivated fetal calf serum to a
parasitemia of 1% (equivalent of 1 · 10 infected erythro-
cytes); 0.2mL of the diluent was used to infect female
BALB/c mice (18–20g). The mice were randomly
sorted into groups of 5 and dosing commenced 2h
post-infection. Compounds were prepared as a stock
solution of 10mg/mL in 10% DMSO/PBS. Compound
(0.75lM) [ H] farnesyl pyrophosphate (15lCi/mmol,
American Radiolabeled Chemicals.), 1lM RAS-CVIM
protein substrate in a total volume of 20lL which
3
7
included 1lL of FTI solution in DMSO and 3lL of par-
1
7
tially purified Pf PFT. Assays in the absence of FTI
and Pf PFT were included as positive and negative con-
trols, respectively. Reaction mixtures were incubated at
30ꢁC for 30min, and the reaction was terminated by
addition of 200lL of 10% HCl/ethanol. After overnight

6526
D. Carrico et al. / Bioorg. Med. Chem. 12 (2004) 6517–6526
incubation at room temperature, the mixtures were fil-
tered onto a Whatman glass fiber filter (VWR, San
Francisco, CA) using a 96-well vacuum manifold. After
washing with 100% ethanol, the filter was cut, and indi-
vidual slices were counted in a beta-scintillation counter.
IC50 values were calculated using linear regression anal-
7. (a) Yokoyama, K.; Trobridge, P.; Buckner, F. S.; Sholten,
J.; Stuart, K. D.; Van Voorhis, W. C.; Gelb, M. H. Mol.
Biochem. Parasitol. 1998, 94, 87–97; (b) Couto, A. S.;
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. Nepomuceno-Silva, J. L.; Yokoyama, K.; de Mello, L. D.
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3
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versus concentration of compounds.
9
. Ohkanda, J.; Lockman, J. W.; Yokoyama, K.; Gelb, M.
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Acknowledgements
We thank the National Institutes of Health CA67771,
AI54384, Medicines for Malaria Venture (Geneva) and
UNDP/World Bank/WHO Special Programme for
Research and Training in Tropical Diseases (TDR) for
support of this work.
1
0. For examples see: (a) Taub, M. E.; Larsen, B. D.;
Steffansen, B.; Frokjaer, S. Int. J. Pharm. 1997, 146,
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