Conjugated indole-imidazole derivatives displaying cytotoxicity against multidrug resistant cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2006.07.020

We report herein the SAR studies of a series of indole-imidazole compounds. that demonstrate substantial in vitro anti-proliferative activities against cancer cell lines, including multidrug resistance (MDR) phenotypes. The in vitro cytotoxic effects have

Full text of DOI:10.1016/j.bmcl.2006.07.020

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5164–5168
Conjugated indole-imidazole derivatives displaying
cytotoxicity against multidrug resistant cancer cell lines
David A. James, Keizo Koya, Hao Li, Shoujun Chen, Zhiqiang Xia,
Weiwen Ying, Yaming Wu and Lijun Sun*
Synta Pharmaceuticals, 45 Hartwell Ave, Lexington, MA 02421, USA
Received 24 April 2006; revised 29 June 2006; accepted 6 July 2006
Available online 25 July 2006
Abstract—We report herein the SAR studies of a series of indole-imidazole compounds. that demonstrate substantial in vitro anti-
proliferative activities against cancer cell lines, including multidrug resistance (MDR) phenotypes. The in vitro cytotoxic effects have
been demonstrated across a wide array of tumor types, including hematologic and solid tumor cell lines of various origins (e.g.,
leukemia, breast, colon, and uterine).
ꢀ 2006 Elsevier Ltd. All rights reserved.
The development of resistance to chemotherapy with
existing anti-cancer drugs has challenged the pharma-
ceutical industry to rapidly identify and develop new
chemical entities able to counteract this unmet medical
need. Prolonged treatment of cancer cells with certain
drugs can result in an acquired resistance of these cells
toward multiple drugs. This phenomenon is known as
multidrug resistance (MDR).1 While the concise mecha-
nism of MDR is not completely understood it is known
that MDR is often associated with an overexpression of
ATP-binding cassette (ABC) transporters.2 The two
best-known ABC transporters are P-glycoprotein
(P-gp) and multidrug resistance protein 1 (MDR1) that
effectively pump out the anti-cancer drug from MDR-
cancer cells. Other mechanisms believed to be associated
with MDR in cancer cells include: increased expression
of anti-apoptotic genes and decreased expression of
pro-apoptotic genes,3 overexpression of specific tubulin
isotypes,4 decreased expression of topoisomerases,5
and overexpression of major vault protein.6
epothilones,8 discodermolide,9 and modified taxanes10
display potent activity against MDR resistant cancer cell
lines. However, there is a continuing search for effective
small-molecule drugs that show MDR cancer cell
cytotoxicity.
As part of our program to develop anti-cancer deriva-
tives we found that conjugated indole-imidazole deriva-
tives 1 display in vitro cytotoxicity against a range of
cancer cell lines, even MDR cancer cell lines.11 These
compounds are structurally related to the bisindole alka-
loids which include the topsentins 2.12 The topsentins
display a range of biological activities including anti-tu-
mour activity.13 Herein we describe the synthesis, cyto-
toxicity against cancel cell lines, and SAR of these
indole-imidazole derivatives.
H
N
R2
R3
N
O
N
R3
N
R2
O
N
H
N
R1
Various strategies have been employed to overcome
MDR, the most common being inhibition of P-gp and
related proteins to effectively block the efflux of the
drug.7 Numerous MDR-reversal agents have been
reported but most have undesirable side effects such as
toxicity. Other complex natural products such as the
R1
N
H
2
1
R1 ,R2 ,R3 = H or Br or OH
The synthesis of the initial series is shown in Scheme 1.
Early in the program we discovered that when the indole
nitrogen is protected with a benzyl-group activity was
dramatically improved therefore all compounds
Keywords: Indole; Imidazole; Multidrug resistant cancer.
*
Corresponding author. E-mail: lsun@syntapharma.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.020

D. A. James et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5164–5168
5165
HO
COR3
O
R3
N
N
O
N
O
O
N
SEM
N
N
(vi)
(iv),(iii)
R2
H
N
N
N
Cl
Cl
Cl
11
12 R2 =SEM R3 =E t
13
R3 =Et
16 R3 =3-pyridyl
15
(v), (iii)
R2 =SEM R3 =3-pyridyl
R2 =HR3 =3-pyridyl
(iv)
14
R1
R1
R1
N
N
O
N
O
CHO
N
SEM
CHO
N
H
N
SEM
4
(i)
(iv)
(ii),(iii)
Cl
N
N
N
N
H
Cl
Cl
3
R1 =H
8
9
R1 =H
5
6
R1 =2-pyridyl
R1 =2-pyridyl
O
7 R1 =CH2OTBS
10 R1 =CH2OH
N
(vii), (iii)
N
N
SEM
17
O
CO2H
N
COR4
N
O
N
N
O
O
N
N
SEM
(iv)
N
H
(viii) or (ix)
H
N
N
N
Cl
Cl
Cl
19
18
20 R4 =OCH3
4
21
R =NHEt
4
22
R =N(CH3)2
Scheme 1. Reagents and conditions: (i) NaH, 4-chlorobenzyl chloride, rt, 76%; (ii) LDA, 4, 32–50%; (iii) MnO2, rt, 80–90%; (iv) HCl/EtOH, reflux,
32–85%; (v) TBAF, rt, 81; (vi) R3MgX, rt, 41–43%; (vii) LDA, 17, 40; (viii) concd H2SO4, MeOH, reflux, 89%; (ix) EDC, DMAP, NH2R4, rt,
60–70%.
discussed here possess this substitution. Commercially
available indole-3-carboxaldehyde was readily benzylat-
ed with 4-chlorobenzyl chloride to generate 3. The key
reaction for the synthesis of this series was the coupling
between the indole aldehyde 3 and various protected
imidazoles. This reaction was based on a similar proce-
dure previously described for the synthesis of
topsentins.14
by refluxing with HCl in ethanol to furnish the final
compounds 8–10.16
The TBS-protected intermediate 7 was a key intermedi-
ate from which numerous derivatives were synthesized
as shown in Scheme 1. The TBS group was selectively
removed with TBAF at low temperature followed by
oxidation of the hydroxyl group to the aldehyde 11.
Reaction of 11 with ethyl magnesium bromide produced
the intermediate secondary alcohol 12 which is oxidized
with manganese dioxide followed by deprotection with
ethanolic HCl to provide ketone 15. Similarly, reaction
of 11 with the Grignard reagent derived from 3-bromo-
pyridine produced 13 which was oxidized and deprotec-
ted as described above to produce 16. The secondary
alcohol 14 was also isolated to provide a direct compar-
ison with 16 which should determine the effect of the
conjugated linkage between the imidazole and pyridine
rings.
The SEM-protected imidazoles 4 were synthesized from
the corresponding imidazoles by reaction with NaH/
SEM-Cl. Reduction of 2-[(5-toluene-4-sulfonyl)-1H-imi-
dazol-4-yl]pyridine15 with Na/naphthalene formed
4-pyridineimidazole, protection as described provided
the appropriate imidazole 4 for the synthesis of 6. The
primary hydroxyl group and the imidazole-NH of com-
mercially available (1H-imidazol-4-yl)methanol were
protected sequentially as a TBS-ether using TBS-Cl,
imidazole followed by treatment with NaH/SEM-Cl.
This produced the required doubly protected imidazole
for the synthesis of the key intermediate 7. Coupling
of the aldehyde 3 with the appropriate SEM-protected
imidazoles 4 produced the secondary alcohols which
were readily oxidized to the conjugated ketones by man-
ganese dioxide to produce the protected intermediates
5–7. Removal of the SEM group of 5–7 was achieved
Carboxylic acid derivatives were synthesized via the pro-
tected imidazole derivative 17 as shown in Scheme 1.
The synthesis of 17 was based on a similar procedure
reported for the synthesis of 2-(4,4-dimethyl-4,5-dihy-
drooxazol-2-yl)-benzo[d]imidazo[2,1-b]thiazole.17 Cou-
pling of 17 with the indole aldeyde 3 provides the

5166
D. A. James et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5164–5168
intermediate 18. Treatment with ethanolic HCl removes
both the SEM group and liberates the dimethyloxazole
producing the carboxylic acid 19. From 19, the methyl
ester 20 and amides 21 and 22 were synthesized using
standard esterification or amide coupling chemistry.
cytotoxicity. However, both the secondary amide 21
and the tertiary amide 22 showed weak potency. The dif-
ferences in activity between these derivatives could possi-
bly be explained by poor cell permeation from the
carboxylic acid and amides rather than a substituent
effect.
Derivatives 23–25 (see Table 2 for structures) all possess
an imidazopyridine linked to the indole ring via a car-
bonyl group. These compounds were synthesized by
the coupling reaction between the indole aldehyde 3
and the appropriate protected imidazopyridine, oxida-
tion with manganese oxide, and deprotection of the
SEM group as previously described.
From this SAR study it appeared that compounds with a
general structure 26 were active against all cell lines
including the MDR cell lines. In general, compounds that
possessed a hydrogen bond acceptor conjugated to the
imidazole ring showed submicromolar activity. Multiple
conformations resulting from rotation around the bond
connecting the imidazole ring to the hydrogen bond
acceptor exist as represented by 26. We hypothesized that
cyclization producing fused imidazole derivatives 27
could stabilize a conformation where the hydrogen bond
acceptor is fixed in a particular conformation.
The cytotoxicity of the initial series of conjugated in-
dole-imidazole compounds is summarized in Table 1.18
In particular we were interested in compounds that
showed significant activity against multidrug resistant
cell lines such as HL60/TX1000 that are resistant to
Taxol as shown in Table 1.19
X
N
X
O
N
O
N
N
H
H
N
The first observation to note is that the unsubstituted
imidazole derivative 8 shows minimal activity but substi-
tution with certain groups produces significant activity.
Susbtitution with a 2-pyridyl group 9 produces potent
activity but a hydroxy(methyl) group 10 causes signifi-
cant loss of activity. When a conjugated ketone group
is introduced activity is maintained as shown by com-
pound 15. The importance of a conjugated group is
clearly demonstrated by a direct comparison of com-
pounds 14 and 16. Compound 16 possesses a pyridyl
group directly conjugated via a carbonyl to the imidaz-
ole ring and shows potent activity. When the conjuga-
tion is disrupted between the imidazole ring and the
pyridine, as in the secondary alcohol 14, a serious
decrease in activity is observed.
N
Cl
Cl
26
27
The cytotoxic activities of the imidazopyridine series of
compounds against the Taxol-resistant cell line HL60/
TX100018 are shown in Table 2.18 Our restricted confor-
mation hypothesis gained support when it was found
that the indole-pyridoimidazole compound 23 showed
a 10-fold increase in potency compared to any of the in-
dole-imidazole derivatives shown in Table 1. Also of
note is serious loss of activity for the isomer 24 where
the nitrogen atom is moved adjacent to the imidazole
ring. Additionally, if the NH of the fused imidazole ring
is methylated as in 25 significant loss of activity was
observed. This series of compounds illustrates that the
The carboxylic acid derivatives provided further SAR in-
sight. While the parent carboxylic acid 19 showed mini-
mal activity the methyl ester 20 displayed strong
Table 1. Comparison of the in vitro cytotoxicity of indole-imidazole
compounds against the Taxol-resistant HL60/TX100019 cell line
Table 2. Comparison of the in vitro cytotoxicity of indole-pyridoim-
idazole compounds against the Taxol resistant HL60/TX100019 cell
line
R
N
O
O
R
N
H
N
N
Cl
Cl
Compound
R
Cytotoxicity
18
IC50 (lM)
Compound
R
Cytotoxicity
18
IC50 (lM)
Taxol
8
—
H
5
10
0.5
5
Taxol
—
5
N
N
9
2-Pyridyl
CH2OH
CH(OH)-3-pyridyl
COEt
23
0.05
N
10
14
15
16
19
20
21
22
H
10
0.5
0.5
10
0.5
5
N
N
24
25
10
10
CO-3-pyridyl
COOH
N
H
N
N
COOCH3
CONHEt
CON(CH3)2
N
H3C
5

D. A. James et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5164–5168
5167
Table 3. Comparison of the cytotoxicity of 23 and Taxol against a range of cancer cell lines
18,20
Compound
Cytotoxicity IC50
(lM)
MDA435
HL60
P388
DU145
MES-SA
MES-SA/DX5
HL60/TX1000
23
Taxol
0.05
0.005
0.05
0.005
0.05
0.01
0.05
0.005
0.01
0.005
0.05
5
0.02
5
9. Kowalski, R.; Giannakakou, P.; Gunasekera, S.; Longley,
R.; Day, B.; Hamel, E. Mol. Pharmacol. 1997, 52, 613.
10. Ojima, I.; Wang, T.; Miller, M.; Micheal, L.; Lin, S.;
Borella, C.; Geng, Z.; Pera, P.; Bernacki, R. Bioorg. Med.
Chem. Lett. 1999, 9, 3423.
position of the nitrogen atom in the fused pyridine ring
(hydrogen bond acceptor) and the presence of the imid-
azole NH are important for activity.
Compound 23 was screened against a range of cancer
cell lines originating from different tissues and the results
are summarized in Table 3.20 As shown in Table 3, 23
was effective against all cell lines including the multidrug
resistant cell lines MES-SA/DX5 and HL60/TX1000
which were resistant to treatment with Taxol. Both these
cell lines posses high levels of MDR1 mRNA and P-gps
and show cross resistance to a wide range of common
chemotherapeutic agents. The success of 23 suggests
that it is not a substrate for the efflux pump transporters
and its mechanism of action is quite effective against
these cell lines. Additionally 23 is approximately 10
times more active than structurally related topsentin
derivatives against the P388 cell line.21.
11. Koya, K.; Sun, L.; Ono, M.; James, D.; Ying, W.; Chen,
S. U.S. Patent US6,743,919 B2, 2004.
12. (a) Yang, C.-G.; Huang, H.; Jiang, B. Curr. Org. Chem.
2004, 8, 1691; (b) Bartik, K.; Breakman, J.-C.; Daloze, D.;
Stoller, C.; Huysecom, J.; Vandevyver, G.; Ottinger, R.
Can. J. Chem. 1987, 65, 2118; (c) Tsujii, S.; Rinehart, K. J.
Org. Chem. 1988, 5446.
13. Burres, N.; Barber, D.; Gunasekera, S.; Shen, L.; Clement,
J. Biochem. Pharmacol. 1991, 745.
14. (a) Achab, S. Tetrahedron Lett. 1996, 37, 5503; (b)
Kawasaki, I.; Katsuma, H.; Nakayama, Y.; Yamashita,
M.; Ohta, S. Heterocycles 1998, 48, 1887.
15. Van Leusen, A. M.; Schut, J. Tetrahedron Lett. 1976, 4,
285.
16. (a) The synthesis of 23 described below is representative of
the chemistry for the synthesis of the compounds
described in this paper: [1-(4-chlorobenzyl)-1H-indol-
3-yl]-[1-(2-trimethyl-silanylethoxymethyl)-1H-imidazo[4,5-
c]pyridin-2-yl]-methanone. A solution of 1-(2-trimethyl-
silanylethoxymethyl)-1H-imidazo[4,5-c]pyridine (250 mg,
1.0 mmol) in dry THF (50 mL) was cooled to À78 ꢁC in
dry-ice acetone bath. To this solution was added lithium
diisopropylamide (0.6 mL, 2 M solution in heptane/ethyl-
benzene/THF, 1.2 mmol) and the reaction mixture was
stirred at À78 ꢁC for 30 min. To this solution, 1-(4-
In summary, our SAR study of the indole-imidazole
derivatives provided us with a lead towards developing
a pharmacophore for in vitro activity against a range
of cancer lines including MDR cell lines. Using this pre-
liminary design we improved the activity 100-fold
through a possible stabilization of a conformation re-
quired for activity. Further studies are being conducted
to determine the mode of action of these compounds to
provide an insight into the design of future drugs active
against MDR-carcinoma cells.
chlorobenzyl)-1H-indole-3-carboxaldehyde
(390 mg,
1.21 mmol) dissolved in THF (20 mL) was added drop-
wise. The reaction mixture was stirred at À78 ꢁC for
60 min then quenched with saturated NaHCO3 and
allowed to warm to room temperature. The resultant
solution was extracted with ethyl acetate (3· 50 mL), the
combined extracts were washed with water, dried over
MgSO4, and the solution was filtered. Solvent was
removed under reduced pressure to produce a brown oil.
This product was dissolved in CH2Cl2 (50 mL) and MnO2
(500 mg) was added. The resultant suspension was stirred
at room temperature overnight then filtered through a
plug of Celite. Solvent was removed under reduced
pressure and the crude product was purified by silica gel
column chromatography eluting with a gradient of ethyl
acetate/hexane (1:1, v/v) to ethyl acetate. The desired
product was isolated as a yellow oil which was a mixture
of isomers (yield 408 mg, 74%); (b) [1-(4-Chlorobenzyl)-
1H-indol-3-yl]-(1H-imidazo[4,5-c]pyridin-2-yl)-methanone
23: a solution of [1-(4-chlorobenzyl)-1H-indol-3-yl]-[1-(2-
trimethyl-silanylethoxymethyl)-1H-imidazo[4,5-c]pyridin-
2-yl]-methanone (300 mg, 0.54 mmol) in ethanol (50 mL)
and 2 N HCl (20 mL) was heated to reflux for 2 h. After
allowing to cool to room temperature, the solution was
neutralized with 2 N NaOH and ethanol was removed
under reduced pressure. The resultant suspension was
extracted with ethyl acetate (3· 50 mL), the combined
extracts were washed with water, dried over MgSO4, and
the solution was filtered. Solvent was removed under
reduced pressure to produce the pure desired product as a
References and notes
1. (a) Ling, V. Cancer Chemother. Pharmacol. 1997, 40
(suppl), S3; (b) Kaye, S. Curr. Opin. Oncol. 1998, 10 (suppl
1), S15.
2. For recent reviews, see: (a) Tian, Q.; Zhang, J.; Chan, E.;
Duan, W.; Zhou, S. Drug Dev. Res. 2005, 64, 1; (b) Leslie,
E.; Deeley, R.; Cole, S. Toxicol. Appl. Pharmacol. 2005,
204, 216; (c) Polgar, O.; Bates, S. Biochem. Soc. Trans.
2005, 33, 241.
3. Kim, C.; Gollapudi, S.; Lee, T.; Gupta, S. Int. J. Oncol.
1997, 11, 945.
4. Ferlini, C.; Raspaglio, G.; Mozzeti, S.; Cicchillitti, L.;
Fillippetti, F.; Gallo, D.; Fattorusso, C.; Campiani, G.;
Scambia, G. Cancer Res. 2005, 2397.
5. Wessel, I.; Jensen, P.; Falck, J.; Mirski, S.; Cole, S.;
Sehested, M. Cancer Res. 1997, 57, 4451.
6. (a) Hu, Y.; Stephen, A.; Cao, J.; Tanzer, L.; Slapak, C.;
Harrison, S.; Devanarayan, V.; Dantzig, A.; Starling, J.;
Rome, L.; Moore, R. Int. J. Cancer 2002, 149; (b)
Ferguson, R.; Roisean, E.; Jackson, S.; Stanley, A.; Joyce,
A.; Harnden, P.; Morrison, E.; Patel, P.; Phillips, R.;
Selby, P.; Banks, R. Int. J. Cancer 2005, 115, 155.
7. Robert, J.; Jarry, C. J. Med. Chem. 2003, 46, 4805.
8. For recent reviews, see Altmann, K. Curr. Pharm. Des.
2005, 11, 1595.

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1
white solid (yield 257 mg, 92%). H NMR (DMSO-d6) d
72 h and the IC50 was determined by MTS (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
assay.
5.83 (s, 2H), 7.36 (m, 2H), 7.62 (m, 4H), 7.81 (s, 1H), 8.43
(m, 2H), 9.14 (s, 1H), 9.53 (s, 1H); ESMS calcd
(C22H15ClN4O): 420.12, found: 421.1 (M+H)+.
19. The cell line HL60/TX1000 was a gift from Dr. Bhalla of
Emory University School of Medicine. HL-60/TX1000
was isolated in vitro by subculturing HL-60 in progres-
sively higher concentration of Taxol. HL-60/TX1000 cells
overexpress MDR1 mRNA and P-glycoprotein, as deter-
mined by Western blot and immunoflourescence labeling
with anti-P-gp antibodies.
20. Cell lines (tissue origin in parentheses) were all purchased
from ATCC: MDA435 (breast), HL60 (leukemia), P388
(murine leukemia), DU145 (prostate), MES-SA (uterine)
and MES-SA/DX5 (MDR uterine). See Ref. 15 for cell
culture conditions and assay, and see Ref. 16 for origins of
HL60/TX1000.
17. Clements-Jewery, S.; Danswan, G.; Gardner, C.;
Matharu, S.; Murdoch, R.; Tully, R.; Westwood, R. J.
Med. Chem. 1988, 31, 1220.
18. Cell culture. Cell lines were maintained in
RPMI1640(GIBCO) supplemented with 10% FC, 100 U/
mL penicillin, 100 lg/mL streptomycin, and 2 nM L-
glutamine. The cells were split every third day and diluted
to a concentration of 2· 105 cells/mL one day before the
experiment. All experiments were performed on exponen-
tially growing cell culture. Drug treatment and MTS assay.
A stock solution of the drug was prepared by dissolving
the compound at a concentration of 1 mM in DMSO.
Final concentrations were obtained by diluting the stock
solution directly into the tissue medium. Cells were
incubated with varying concentrations of compounds for
21. IC50 for the most potent topsentin derivative against P388
cell line was reported as 0.6 lM. Sakemi, S.; Sun, H. J.
Org. Chem. 1991, 56, 4304.