Synthesis and structure-activity relationships of novel benzofuran farnesyltransferase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.01.074

A series of benzofuran-based farnesyltransferase inhibitors have been designed and synthesized as antitumor agents. Among them, 11f showed the most potent enzyme inhibitory activity (IC₅₀ = 1.1 nM) and antitumor activity in human cancer xenogra

Full text of DOI:10.1016/j.bmcl.2009.01.074

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1753–1757
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of novel benzofuran
farnesyltransferase inhibitors
Kohsuke Asoh, Masami Kohchi, Ikumi Hyoudoh, Tatsuo Ohtsuka, Miyako Masubuchi, Kenichi Kawasaki,
Hirosato Ebiike, Yasuhiko Shiratori, Takaaki A. Fukami, Osamu Kondoh, Toshiyuki Tsukaguchi, Nobuya Ishii,
*
Yuko Aoki, Nobuo Shimma, Masahiro Sakaitani
Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., 200-Kajiwara, Kamakura, Kanagawa 247-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzofuran-based farnesyltransferase inhibitors have been designed and synthesized as anti-
tumor agents. Among them, 11f showed the most potent enzyme inhibitory activity (IC50 = 1.1 nM) and
antitumor activity in human cancer xenografts in mice.
Received 28 July 2008
Revised 21 January 2009
Accepted 23 January 2009
Available online 11 February 2009
Ó 2009 Published by Elsevier Ltd.
Keywords:
Farnesyltransferase inhibitors
Anti cancer
Tipifarnib
Benzofuran
Membrane-bound GTP binding proteins (G-proteins) act as
molecular switches to regulate cell growth by cycling between
the inactive GDP-bound state and the active GTP-bound state. In
tumor cells, the constitutive activation of some G-proteins contrib-
utes to their malignant growth properties. In normal cells, this
switching mechanism is highly regulated and G-proteins are found
predominantly in their inactive GDP-binding state. All of these G-
proteins originally have the CAAX tetrapeptide motif (C: Cys, A:
an aliphatic amino acid, X: Ser, Met, Gln, Ala) at their C-terminal.1
Farnesyltransferase (FTase) enzymes recognize this CAAX tetra-
peptide motif and transfer the farnesyl group to the cysteine thiol.
This farnesylation is critical for membrane binding and the biolog-
ical function of G-proteins.2 In the last decade, many classes of
FTase inhibitors have been reported and discussed as antitumor
agents.3 Tipifarnib (R115777) is one of the most potent FTase
inhibitors that is undergoing clinical trials.4 In order to discover
new potent FTase inhibitors, we searched for a novel core template
in place of the quinolinone moiety of tipifarnib. The first templates,
4-substituted phenyl rings 1 and bicyclic structures 2 such as ben-
zofuran and benzothiophene, displayed moderate FTase inhibition
but no antiproliferative activity against tumor cells. Further modi-
fication studies resulted in the second templates, the biaryl series 3
which showed weak activity against both FTase and tumor cells.
We report herein our design and synthesis of the series of benzo-
furan FTase inhibitors 4 using the X-ray structure of human FTase.
(Fig. 1).
N
NH2
N
N
O
R2
Het
R2
*
*
Cl
1
2
2
Cl
t-
R = CN, CO Bu
2
tipifarnib
N
N
R4
R2
A
Het
O
*
R2
R1
B
R3
4
3
* Corresponding author. Tel.: +81 467 47 6046; fax: +81 467 45 6824.
E-mail address: sakaitanimsh@chugai-pharm.co.jp (M. Sakaitani).
Figure. 1. Modification of tipifarnib from core structures 1 through 3 to give
benzofuran derivative (4).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.01.074

1754
K. Asoh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1753–1757
N
N
N
N
O
O
OH
OH
R2
a
b
c
H
O
R2
O
R2
HO
O
R1
R2
R1
R1
Br
5
Br
Ar
Br
6
7
8
R2 = NO2, CO2Me, CN
R1 = Cl, Br, I, CN, CO2t-Bu
8d: R1 = CO2t-Bu
8e: R1 = CO2H
d
i
8q: R2 = CO Me
2
N
O
8r : R2 = CONH2
N
e
f
N
S
8v: R2 =
S O
NH
N
NH2
O
N
O
j
k
*
N
H
N
O
O
R2
R2
O
R2
R1
R1
R1
8s: R2 = CHO
8t : R2 = CH2OH
8u: R2 = CH2NMe2
Ar
Br
g
h
Br
9
10
11
Scheme 1. Reagents and conditions: (a) 4-substituted phenacyl bromide, K2CO3, CH3CN, reflux, 1 h, 36–80%; (b) 1-methyl-2-TES-imidazole, n-BuLi, THF, À78 °C, 55–81%; (c)
ArB(OH)2 Pd2(dba)3, PPh3, Na2CO3, toluene/MeOH/H2O, 80 °C, 17–97%; (d) TFA/CH2Cl2, rt, 1 h, 73%; (e) i—LiOH THF/H2O, 46%; ii—WSCI, HOBt, DIPEA, appropriate amine, THF,
51% for 8r, 58% for 8v; (f) i—LiOH, THF, rt, ii—HNMeOMe, WSCI, HOBt, DIPEA, DMF, rt, 56%, (iii) LAH, THF, À78 °C, 96%; (g) NaBH4, MeOH, rt, 1.5 h, 90%; (h) dimethyamine,
NaB(OAc)3H, AcOH, THF, rt, 72%; (i) 2-methyl-2-propanesulfinamide, Ti(OEt)4, toluene, 70 °C, 2 h, 52–70%, (j) i—1-methyl-2-TES-imidazole, n-BuLi, THF, rt, 3 h, 56–76%,
ii—chiral resolution by chiral HPLC system; (k) i—2-(substituted phenyl)-[1,3,2]dioxaborinane, Pd(PPh3)4, K3PO4, DMF, 100 °C, 1 h, 81–98%, ii—HCl, THF, rt, 60–93%.
Table 1
Activity of benzofuran (R = OH) series
N
N
OH
A
O
R2
R1
B
R3
Compound
R1
R2
R3
FTase/K-rasa IC50 (nM)
QG56 IC50 (nM)
8a
8b
8c
8d
So
8f
8g
8h
8i
Cl
Br
I
CO2t-Bu
CO2H
NO2
OMe
CN
CN
CN
CN
CN
CN
CN
CN
CN
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
CO2Me
CONH2
CHO
CH2OH
CH2NMe2
H
H
H
H
H
H
H
H
170
360
360
>1000
850
30
250
6.4
3.3
8.5
2.8
11
1885
1477
>10000
6248
>10000
547
4967
1477
89.5
38.8
22.9
23.2
145
2-F
8j
8k
8l
3-OMe
3-CN
3-Me
3-F
4-OMe
4-CN
4-F
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
8m
8n
8o
8p
8q
8r
8s
8t
6.3
4
36
32.6
158
36.3
8.2
15.2
16.8
142
3.4
7.2
3.2
0.9
2
CN
CN
CN
CN
1
11
8u
CN
H
O
O
N
N
N
8v
CN
3-OMe
2.6
ntb
O
H
N
8w
Cl
H
H
6.4
2.4
73.6
14.5
O
8x
CN
CN
a
Human farnesyltransferase.
Not tested.
b

K. Asoh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1753–1757
1755
:
:
:
vehicle
1000
25 mg/kg
50 mg/kg
: 100 mg/kg
: 200 mg/kg
100
0
5
10
10
120
110
100
Figure. 2. Crystal structure of FTase with 8 k (pink) and 8w (cyan). Cyano group on
A-ring of 8 k makes a hydrogen bond (3.1 Å) to Arg202B. There is a bridging water
between the carbonyl oxygen on benzofuran of 8w and amide of Phe360B.
90
80
70
The general synthesis of benzofuran derivatives 4 is outlined in
Scheme 1. Intermediate 6 was provided from substituted benzalde-
hyde 5 by coupling with the appropriate phenacyl bromide under
basic condition.5
0
5
Days after the initial dosing
Figure. 3. Effect of 11f on QG56 tumor growth in mouse xenograft models. Tumor
volume (TV) was measured twice/week (upper graph). Body weight (BW) was
measured 5 times/week and relative BW calculated (lower graph).
Compound 7 was prepared by treatment of 1-methyl-2-TES-
imidazole with n-BuLi followed by the addition of the appropriate
6. A variety of aryl substituents were introduced into 7 by Suzuki
coupling reaction to give biaryl 8 as a racemate (R4 = OH). The t-bu-
tyl group in 8d was hydrolyzed to carboxylic acid (8e). The methyl
ester group in 8q was converted to amides (8r and 8v) and an alde-
hyde (8s) which was further converted to an alcohol (8t) and ami-
no derivative (8u). The synthesis of the other benzofuran series 11
(R4 = NH2) is also described in Scheme 1. Sulfineimine 9 was pre-
pared by tetraethyl orthotitanate mediated condensation of 6 with
sulfineamide.6 Arylation of 9 with 1-methyl-2-TES-imidazole-5-
lithium followed by chiral resolution using an HPLC system with
a chiral column afforded 10.7,8 Suzuki coupling of 10 with 2-
(substituted phenyl)-[1,3,2]dioxaborinane, followed by deprotec-
tion of the t-butylsulfine group under acidic condition gave the de-
sired product 11.
The enzyme inhibitory activity (FTase/K-ras) and antiprolifera-
tive activity against human non-small cell lung carcinoma
(QG56) of compounds (8) were evaluated.9 The results from com-
pound 8 having a hydroxyl group at the chiral center are shown
in Table 1. Our first lead compound (8a) showed weak FTase inhi-
bition (IC50 = 170 nM) and low antiproliferative activity
(IC50 = 1885 nM). Introduction of a cyano group (8 h) or a nitro
group (8f) on the A-ring resulted in significant increase of enzyme
inhibitory activities (6.4 and 30 nM respectively). Replacement of
the chlorine atom with another halogen (8b and 8c), t-butylester
(8d), carboxylic acid (8e), or methoxy (8 g) resulted in reduction
of enzyme inhibitory activity (from 250 to 1000 nM). Further mod-
ification of the substituent R3 in the compound (8 h) was carried
out. The compound having a 3-CN group (8 k) showed potent en-
zyme inhibitory activity (2.8 nM) and antiproliferative activity
(23 nM). The X-ray crystal structure of FTase complexed with 8 k
shown in Figure. 2 suggests that the hydrogen bonding between
the cyano group on the A-ring and Arg202B improves the enzyme
inhibitory activity.10 No obvious interaction of the cyano group on
the B-ring with the enzyme was seen. In fact, all the compounds
having small substituents on the B-ring (8i–p) showed similar
FTase inhibition (2.8–11 nM) compared with a non-substituted
phenyl (8 h, 6.4 nM) but exhibited improved antiproliferative
activity against QG56 cells (23–158 nM). The effects of the substi-
tuent R2 on the benzofuran ring of compound 8 are summarized in
Table 1 (note 8q–v). Interestingly, replacement of the nitro group
Table 2
Activity of benzofuran (R4 = NH2) series
N
N
NH2
*
A
O
R2
R1
B
R3
Compound R1 R2
R3
Chiral FTase/K-rasa IC50 (nM) QG56 IC50 (nM)
Table 3
Pharmacokinetics of 11f in micea
11a
11b
11c
11d
11e
11f
CN NO2
CN NO2
CN NO2 3-OMe
CN CN
CN CN
CN CN
H
H
S
R
S
S
S
S
1.5
49
0.8
1.2
0.7
1.1
5.9
206.1
1.1
1.5
2
Cmax
g/mL)
AUC (0–24 h)
*
CL/F
t1/2
BA (%)
(
l
(
lg h/mL)
(mL/min/kg)
(h)
3-OMe
3-CN
3-F
IV 25 mg/kg
PO 25 mg/kg
PO 100 m g/kg
—
1.57
5.01
15.4
10.2
31.3
27.0
40.8
41.0
1.2
2.4
12.3
—
66
51
2
a
a
Human farnesyltransferase.
Vehicle: 0.06 M HCl, 0.44% CMC distilled water.

1756
K. Asoh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1753–1757
O
O
OHC
OH
Br
O
O
Br
CN
NC
NC
a
b
c
d
OH
F
MeO
F
F
Br
12
F
13
14
15
16
N
O
S
N
S O
N
F
N
N
NH
NH2
e
f
O
g
O
O
CN
CN
CN
NC
NC
NC
F
F
11f (99.5% ee)
17
18 (S : R = 20 : 1)
Scheme 2. Reagents and conditions: (a) i—4-fluoroboronic acid, Pd(OAc)2, PPh3, Na2CO3, MeOH, reflux, 14 h, ii—BBr3, CH2Cl2, 0 °C to rt, 87%; (b) i—paraformaldehyde, MgCl2,
Et3 N, THF, reflux, 2 h, ii—NBS, CHCl3, reflux, 2 h; (c) 4-cyanophenacyl bromide, K2CO3, CH3CN, reflux, 1 h, 78% from 13; (d) Zn(CN)2, Pd(PPh3)4, DMF, 100 °C, 4 h, 90%; (e) (S)-2-
methyl-2-propanesulfinamide, Ti(OEt)4, toluene, 70 °C, 2 h, 90%; (f) 5-bromo-N-methyl-imidazole, EtMgBr, CH2Cl2, THF, rt, 3 h, 80%; (g) i—4 M HCl in THF, rt, 1 h; ii—
crystallization from H2O/MeOH/EtOH, 70%.
with a carbonyl function such as a methylester (8q) amide (8r, 8v),
or CHO (8s), showed 3- to 8-fold higher enzyme inhibitory activity
(0.9–3.2 nM) than 8j (8.5 nM). X-ray crystal structure of FTase with
8w (Fig. 2) revealed a bridging water molecule forming a hydrogen
bonding between the oxygen atom of the carbonyl in the substitu-
ent R2 on the benzofuran and the amide back bone of the
Phe360B.10 Thus, the compounds with a carbonyl moiety have
strong activity against FTase (8a vs. 8w). Compounds 8t and 8x also
showed strong enzyme inhibitory activity (1.0 and 2.4 nM, respec-
tively, with FTase). These results indicate that not only a carbonyl
moiety but also a hydrogen bond accepting (HBA) group is essen-
tial to increase the enzyme inhibitory activity. 8x has strikingly
strong cellular activity (14.5 nM) even though it has no substituent
(R3 = H) on the B-ring.
lective addition of an imidazole moiety into chiral sulfineimine
17 to give 18. Although the addition of 1-methyl-2-TES-imidazole
treated with n-BuLi resulted in low diastereoselectivity (S:R = 2:1),
the Grignard condition, treated by EtMgBr, showed dramatically
improved selectivity (S:R = 20:1). This new synthetic process has
significant advantage for further scale-up synthesis because all
the intermediates can be purified by crystallization without using
column chromatography.12
In summary, we discovered a series of benzofuran compounds
showing potent FTase inhibitory activity. A cyano group at the
para-position of the A-ring (R1 position) showed excellent FTase
inhibition. Substituent of the functional group at the B-ring (R3 po-
sition) increased antiproliferative activity against human cancer
cell lines. Introduction of an HBA group at the R2 position resulted
in improved inhibitory activities both in enzyme and cellular as-
says. X-ray crystal structure of FTase with 8 k and 8w revealed
important hydrogen bonding of (1) the cyano group at the R1 posi-
tion of 8 k with Arg202 and (2) the carbonyl group at the R2 posi-
tion of 8w with a water molecule bound to the protein. Compound
11f, a clinical candidate, showed strong tumor regression in the
QG56 human NSCLC xenograft model with no noticeable body
weight loss. 11f also showed good pharmacokinetics profile in
mice. Furthermore, we established diastereoselective synthesis of
11f applicable to large scale synthesis.
The results from in vitro evaluation of compound 11 (R4 = NH2)
are shown in Table 2. With regard to the stereochemistry of the
chiral amino group, the nitro derivative with an (S)-configuration
(11a) showed 33 times higher enzyme inhibitory activity than
the corresponding (R)-isomer (11b). Furthermore, in the (S)-isomer
series, replacement of the nitro group (R2) with a cyano group and
the introduction of favorable substituents (R3) on the B-ring led us
to identify compounds (11d–f) showing potent inhibitory activities
against both FTase (0.7–1.2 nM) and QG56 cells (1.5–2.0 nM)
(Table 2).
The antitumor activity of compounds 11c–f was evaluated with
a human cancer xenograft model showing potent antitumor activ-
ity in vivo. The in vivo efficacy of 11f in human non-small cell lung
carcinoma (QG56) xenograft in mice is shown in Figure. 3.11 11f
was administered 5 times a week for 2 weeks orally at doses of
25–200 mg/kg per day. 11f showed strong tumor regression with
no noticeable body weight loss at the dose of 200 mg/kg.
After evaluation of several parameters including solubility, bio-
logical stability, and pharmacokinetics we identified 11f as a clin-
ical candidate. The pharmacokinetics data in mice of 11f is
presented in Table 3. 11f was administered intravenously at dose
of 25 mg/kg and orally at doses of 25 and 100 mg/kg. Oral bioavail-
ability of 11f was more than 50% and AUC was increased dose-
dependently. It has been confirmed that there was a good correla-
tion between AUC and tumor growth inhibition in vivo.
Acknowledgments
We are grateful to Mr. Toshihiko Fujii and Mr. Kiyoaki Sakata for
in vitro biological assays. We also thank Dr. Satoshi Sogabe for
analysis of the X-ray crystallographic data and Dr. Kazunao
Masubuchi and Ms. Miyuki Asai for synthesis of the compounds.
We thank Drs. Mamoru Suzuki and Noriyuki Igarashi and Prof. Nor-
iyoshi Sakabe at the Photon Factory for assistance with the beam
line station (BL-6B).
References and notes
1. (a) Clarke, S. Annu. Rev. Biochem. 1992, 61, 355; (b) Cox, A. D.; Der, C. J. Crit. Rev.
Oncog. 1992, 3, 365; (c) Gibbs, J. B. Cell 1991, 65, 1; (d) Newman, C. M. H.;
Magee, A. I. Biochim. Biophys. Acta 1993, 1155, 79; (e) Schafer, W. R.; Rine, J.
Annu. Rev. Genet. 1992, 26, 209.
2. Tamanoi, F.; Kato-Stankiewicz, J.; Jiang, C.; Machado, I.; Thapar, N. J. Cell.
Biochem. Suppl. 2001, 37, 64.
The chiral synthesis of 11f was established by modification of
the synthetic route shown in Scheme 1 and outlined in Scheme
2. The most dramatic improvement was made in the diastereose-

K. Asoh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1753–1757
1757
3. (a) Leonard, D. M. J. Med. Chem. 1997, 40, 2971; (b) Sebti, S. M.; Hamilton, A. D.
Drug Discov. Today 1998, 3, 26.
4. Jabbour, E.; Kantarjian, H.; Cortes, J. Expert Opin. Investig. Drugs 2007, 16, 381.
5. Stille,J.R.;Ward,J.A.;Leffelman,C.;Sullivan,K.A.TetrahedronLett.1996,37,9353.
6. Cogan, D. J.; Liu, G.; Kim, K.; Bradley, B. J.; Backes, J.; Ellman, J. A. J. Am. Chem.
Soc. 1998, 120, 8011.
11. Materials and methods human tumor cell line: The QG56 human non-small
cell lung cancer cell line was cultured in RPMI-1640 medium supplemented
with 10% (v/v) fetal bovine serum. Animals: 5-week-old male athymic nude
mice were subjected to the experiment. Compound: 11f was dissolved in
0.06 M HCl, 0.44% CMC distilled water. Determination of antitumor activities:
The in vivo evaluation procedure for anticancer drugs was based on the
National Cancer Institute (NCI) guidelines. A single cell suspension of QG56
(5.0 Â 106 cells per mouse) was inoculated subcutaneously into the right flank
of each mouse. The tumor volume was estimated from two-dimensional
measurements using the equation ab2/2, where a and b represent tumor length
and width, respectively. Drug administration was initiated on the day of
grouping (day 0). 11f was administered orally once a day from days 0 to 4 and
days 7 to 11.
7. Carpenter, A. J.; Chadwick, D. J. Tetrahedron 1986, 42, 2358.
8. Prepared diastereomers were separated and purified by chiral column
chromatography over DAICEL CHIRACEL OD (eluent: hexane/2-propanol 1:1).
9. Assay methods described in: Sakaitani, M.; Masubuchi, K.; Kohchi, M.;
Hyoudoh, I,; Asoh, K.; Asai, M. WO Patent 04/037816, 2004.
10. Crystals of rat FTase with compounds were obtained within a week under the
crystallization condition of 10% (w/v) PEG 6000, 0.2 M Mg acetate, 0.1 M Na
acetate buffer (pH4.5) at 4 °C. The structures in complex with compounds 8 k
and 8w were solved at 2.6 and 2.4 Å, respectively, and their coordinates have
been deposited in the Protein Data Bank under the access codes 2ZIS and 2ZIR.
12. 11f was analyzed by chiral column chromatography over DAICEL
CHIRALPAKÒ AS (eluent: hexane/ethanol, 4:1) to determine enantiomeric
excess.