Indole- and indolizine-glyoxylamides displaying cytotoxicity against multidrug resistant cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2008.02.029

We report herein the SAR studies of a series of indole- and indolizine-glyoxylamides 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 of various origins (e.g., breast, colon, uterine).

Full text of DOI:10.1016/j.bmcl.2008.02.029

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1784–1787
Indole- and indolizine-glyoxylamides displaying cytotoxicity
against multidrug resistant cancer cell lines
David A. James,* Keizo Koya, Hao Li, Guiqing Liang, Zhiqiang Xia, Weiwen Ying,
Yaming Wu and Lijun Sun
Synta Pharmaceuticals, 45 Hartwell Ave, Lexington, MA 02421, USA
Received 10 December 2007; revised 10 February 2008; accepted 13 February 2008
Available online 16 February 2008
Abstract—We report herein the SAR studies of a series of indole- and indolizine-glyoxylamides 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 of various origins (e.g., breast, colon, uterine).
ꢀ 2008 Elsevier Ltd. All rights reserved.
One of the leading causes for the failure of chemothera-
peutic agents is the development of resistance to the
drug as a result of prolonged treatment. This phenome-
non is known as multidrug resistance (MDR).1 Some of
the most successful cancer drugs such as paclitaxel,2 vin-
cristine,3 and doxorubicin4 suffer from MDR rendering
them ineffective against certain cancers. Natural prod-
ucts such as the epothilones5 discodermolide,6 and mod-
ified taxanes7 have been discovered that display potent
activity against MDR resistant cancer cell lines. How-
ever there is a continuing search for effective small-mol-
ecule drugs that show MDR cancer cell cytotoxicity.
destabilization of microtubules in cancer cells but the
tubulin binding site is not the same as the well-known
vincristine and colchicine binding sites. It has also been
reported that Indibulin has activity against MDR cell
lines, oral bioavailability, and no neurotoxicity.9 These
characteristics offer advantages over existing microtu-
bule disrupting drugs. Indibulin is currently undergoing
early stage clinical trials for the treatment of solid tu-
mors.10 Li et al.,11 have synthesized and studied a series
of N-heterocyclic indolyl glyoxylamides and found com-
pounds possessing activity against cancer cell lines
including MDR cell lines. These compounds also
showed significant in vivo activity in cancer models.
These findings support the continued investigation of
this class of compounds as anticancer agents.
From our screening program, we found that indole-gly-
oxylamides 1 displayed in vitro cytotoxicity against a
range of cancer cell lines, particularly MDR cancer cell
lines. Recently, we reported that conjugated indole-
imidazole derivatives 2 showed appreciable cytotoxicity
against MDR cancer cell lines.8 These compounds are
structurally similar to the conjugated indole-glyoxyla-
mide derivatives. Essentially the gloxylamide system is
a biostere of the imidazole ketone system. It should be
noted that others have also reported indole-glyoxyla-
mides as anticancer agents. Bacher et al.,9 first reported
the activity of D-24851 (now known as Indibulin) and
its mechanism of action has been extensively studied.
The mechanism of action was shown to be through the
Herein we describe the synthesis, cytotoxicity against
cancer cells including MDR cancer cell lines of a series
indole- and indolizine-glyoxylamide derivatives. The
biological activity of the indole-glyoxylamides support
the potential of this class of compounds and the indoli-
zine series represent a novel class of anticancer agents.
R2
R3
N
HN
O
O
N
O
R2
N
N
Keywords: Indole; Indolizine; Multidrug; Resistant; Cytotoxic; Cancer;
Glyoxylamide.
R1
R1
*
Corresponding author. Tel.: +1 7815417256; fax: +1 7812748228;
e-mail: djames@syntapharma.com
1
2
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.029

D. A. James et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1784–1787
1785
CH3
O
CONH2
N
N
N
CH3
N
N
O
N
I-
N
N
N
N
4a
4b
4c
4d
4e
4f
4g
4j
4h
4i
CH3
CH3
CH3
t-B u
N
Cl
N
N
N
S
N
N
N
N
N
N
O
N
N
N
O
O
SCH3
CH3
S
S
S
S
S
N
4k
4l
4m
4n
4r
4s
4t
4o
4p
4q
Table 1. Comparison of the in vitro cytotoxicity of indole-glyoxyla-
mides against the Taxol resistant HL60/TX100014 cell line
Previously, we had found with the indole-imidazole ser-
ies that substitution of the indole nitrogen was crucial
for activity and a para-chloro group (1 R1 = p-Cl) in
the phenyl ring was well tolerated.8 Therefore, all the
compounds described herein possess this substitution.
A number of compounds were synthesized for initial
screening using the procedure shown in Scheme 1. In-
dole was benzylated with 4-chlorobenzyl chloride to
generate 3. The benzylated indole 3 was then reacted
with oxalyl chloride, and with various amines to gener-
ate the glyoxylamides 4.12
Compound
Cytotoxicity IC50 (lM)13
Taxol
4a (D-24851)
5
0.05
6
4b
4c
4d
4e
4f
>10
0.05
>10
0.05
0.5
>10
0.05
0.05
>10
0.05
0.05
0.05
5
4g
4h
4i
The potency of a selection of the initial series of com-
pounds is summarized in Table 1.13 Compound 4a has
the same structure as D-24851 and is included as a ref-
erence. In particular we were interested in compounds
that showed significant activity against multidrug resis-
tant cell lines such as HL60/TX1000 that is resistant to
Taxol.14 The cell line HL-60/TX1000 was isolated
in vitro by subculturing HL-60 in progressively higher
concentration of Taxol. HL-60/TX1000 cells over-ex-
press MDR1 mRNA and p-glycoprotein, as determined
by Western blot and immunofluorescence labeling with
anti-Pgp antibodies.
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
0.01
5
0.1
1
>10
is suggested by the complete absence of activity for the
pyridinium salt 4e. A similar suggestion of the impor-
tance of the position and accessibility of a nitrogen atom
was observed for the series of quinoline compounds 4f–h.
The quinoline amide 4f was very active but the introduc-
tion of a methyl group adjacent to the quinoline ring
nitrogen atom as shown in 4g resulted in a 100-fold de-
crease of activity. An isomer of 4f with nitrogen atom in
a different position 4h showed almost no potency against
this cell line.
From these results, it is noted that certain heterocyclic
amides show very strong activity against this cell line.
Clear and interesting, SAR is shown for the pyridine ser-
ies 4a–e. The 4-pyridyl compound 4a is very active
whereas the 2- and 3-pyridyl isomers 4b and 4c have
very weak or almost no potency. Interestingly, when a
carboxamide group is introduced into the 4-position of
the 2-pyridyl derivative as represented by 4d strong cyto-
toxicity was observed.
The importance of the electronic nature of the pyridine
nitrogen atom (or a group to replace the nitrogen atom)
Numerous amides comprising of five-membered ring
heterocycles were also synthesized and screened. A selec-
R
HN
O
O
(ii), (iii)
(i)
N
N
H
N
Cl
Cl
4
3
Scheme 1. Reagents and condition: (i) NaH, 4-Chlorobenzyl chloride, rt, 77%; (ii) (COCl)2, ether, reflux; (iii) R–NH2, THF, TEA.

1786
D. A. James et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1784–1787
Table 3. Comparison of the cytotoxicity of 5b and Taxol against a
range of cancer cell lines
tion of interesting compounds is also shown in Scheme 1.
Isoxazoles 4i–j and 4l–m were active and for both pairs
of isomers simple methyl substitution did not affect cyto-
toxicity. However, when a larger more bulky group is
introduced such as a fused phenyl ring (4k) then virtu-
ally all the biological effect is lost. Thiazole amides are
tolerated (4n) but bulky substitution within the thiazole
ring is detrimental to activity (4o). Isothiazole 4p was
shown to be the most potent derivative within this series
and one of the most potent compounds we had discov-
ered within this program (up to five times more active
than the next most potent compounds). The exact rea-
sons for its outstanding activity are uncertain but it ap-
pears to have the correct ring size and orientation of
heteroatoms to produce the strong cytotoxic effect. It
is quite striking to compare compound 4q with 4p and
to note that the introduction of a chloro-substituent
causes almost complete loss of potency, illustrating the
importance of the positioning of the group in the correct
orientations or the delicate electronic nature of the ac-
tive system.
Cell line
Cytotoxicity, IC50 (lM)13,16
5b
Taxol
MDA435
HL60
P388
0.05
0.05
0.01
0.05
0.01
0.05
0.02
0.005
0.005
0.01
0.005
0.005
5
DU145
MES-SA
MES-SA/DX5
HL60/TX1000
5
suggests that 5b is not a substrate for the efflux pump
transporters.
In summary, this SAR study has shown that the indole-
glyoxylamides possess strong cytotoxicity against a
range of cancer cell lines including cell lines that show
resistance to Taxol. The specific orientation of substitu-
ents around the indole center was shown to be vital for
potency suggesting specific interactions of these groups
with a biological target. We have also shown that the in-
dole center core can be replaced with an indolizine cen-
ter core without loss of potency. These indolizine
compounds represent a novel class of anticancer agents.
Next, we investigated different center cores in an attempt
to replace the indole moiety. After screening numerous
cores (the majority of which were completely inactive
or possessed minimal activity) we found that the indoli-
zine compound 5a15 (see Table 2) which is analogous to
the indole derivative 4p had comparable activity. This
discovery shows that for this series of compounds that
indolizine appears to be a suitable biostere for indole.
References and notes
1. For recent reviews, see: (a) La Porta, C. A. M. Curr. Med.
Chem. 2007, 14, 387; (b) Perez-Tomas, R. Curr. Med.
Chem. 2006, 13, 1859.
We also found that substituents in the para-position of
the benzyl-substituent were well tolerated and a cyano-
group (see 5b within Table 2) provided a more potent
compound. Compound 5b was screened against a range
of cancer cell lines originating from different tissues and
the results are summarized in Table 3.16
2. (a) Lagas, J. S.; Vlaming, M. L.; van Tellingen, O. W.; Els,
J.; Jansen, R. S.; Rosing, H.; Beijnen, J. H.; Schinkel, A.
H. Clin. Cancer Res. 2006, 12, 6125; (b) George, J. A.;
Chen, T.; Taylor, C. C. Cancer Res. 2005, 65, 10381.
3. (a) Baran, Y.; Gunduz, U.; Ural, A. U. Exp. Oncol. 2006,
28, 163; (b) Huang, R.; Murry, D. J.; Kolwankar, D.;
Hall, S. D.; Foster, D. R. Biochem. Pharmacol. 2006, 71,
1695; (c) Yang, Y.-X.; Xiao, Z.-Q.; Chen, Z.-C.; Zhang,
G.-Y.; Yi, H.; Zhang, P.-F.; Ii, J.-L.; Zhu, G. Proteomics
2006, 6, 1615.
4. (a) Brooks, T. A.; O’Loughlin, K. L.; Minderman, H.;
Bundy, B. N.; Ford, L. A.; Vredenburg, M. R.; Bernacki, R.
J.; Priebe, W.; Baer, M. R. Invest. New Drugs 2007, 25, 115;
(b) Lou, P.-J.; Lai, P.-S.; Shieh, M.-J.; MacRobert, A. J.;
Berg, K.; Bown, S. G. Int. J. Cancer 2006, 119, 2692; (c)
Meyer-Losic, F.; Quinonero, J.; Dubois, V.; Alluis, B.;
Dechambre, M.; Michel, M.; Cailler, F.; Fernandez, A.-M.;
Trouet, A.; Kearsey, J. J. Med. Chem. 2006, 49, 6908.
5. For a review on epothiolones in clinical trials, see: Larkin,
J. M. G.; Kaye, S. B. Expert Opin. Investig. Drugs 2006,
15, 691.
As shown in Table 3, 5b was effective against all cell lines
including the multidrug resistant cell lines MES-SA/
DX5 and HL60/TX1000 which were resistant to treat-
ment with Taxol. Both these cell lines possess high levels
of MDR1 mRNA and Pgp and show cross resistance to
a wide range of common chemotherapeutic agents. This
Table 2. Comparison of the in vitro cytotoxicity of compounds against
the Taxol resistant HL60/TX100014 cell line
CH3
N
HN
O
S
6. For a recent review, see: Dubois, J. Expert Opin. Ther.
Pat. 2006, 16, 1481.
O
7. For a review on new tubulin inhibitors in the clinic, see:
Kuppens, I. E. L. M. Curr. Clin. Pharmacol. 2006, 1, 57.
8. James, D. A.; Li; Koya, K.; Li, H.; Chen, S.; Xia, Z.;
Ying, W.; Sun, L. Bioorg. Med. Chem. Lett. 2006, 16,
5164.
X
Y
R
9. (a) Bacher, G.; Beckers, T.; Emig, P.; Klenner, T.;
Kutscher, B.; Nickel, B. Pure Appl. Chem. 2001, 73,
1459; (b) Bacher, G.; Nickel, B.; Emig, P.; Vanhoefer, U.;
Seeber, S.; Shandra, A.; Beckers, T. Cancer Res. 2001, 61,
392.
Compound
X
Y
R
Cytotoxicity IC50 (lM)13
4p
5a
5b
C
N
N
N
C
C
Cl
Cl
0.01
0.06
0.02
CN

D. A. James et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1784–1787
1787
10. Kuppens, I.; Witteveen, P.; Schot, M.; Schuessler, V.;
Daehling, A.; Beijnen, J.; Voest, E.; Schellens, J. Invest.
New Drugs 2007, 25, 227.
11. Li, W.-T.; Hwang, D.-R.; Chen, C.-P.; Shen, C.-W.;
Huang, C.-L.; Chen, T.-W.; Lin, C.-H.; Chang, Y.-L.; Lo,
Y.-K.; Tseng, H.-Y.; Lin, C.-C.; Song, J.-S.; Chen, H.-C.;
Chen, S.-J.; Wu, S.-H.; Chen, C.-T. J. Med. Chem. 2003,
46, 1706.
12. The synthesis of 4p described below is representative of the
chemistry for the synthesis of the compounds described in
this paper. A solution of oxalyl chloride (0.44 mL,
5.1 mmol) in ethyl ether (25 mL) was cooled in an ice
bath. To it was added 1-(40-chlorobenzyl)indole (1.01 g,
4.14 mmol) in ethyl ether. The resulting yellow slurry was
refluxed for 2 h. After removal of the solvent in vacuo, the
residue was dissolved in THF (20 mL) and cooled to 0 ꢁC.
5-Amino-3-methylisothiazole (1.32 g, 9.73 mmol) in THF
(20 mL) was added dropwise. The mixture was then
warmed to room temperature, and stirred overnight.
Solvent was removed in vacuo. Silica gel chromatographic
purification gave the product (1.49 g) in 88% yield. 1H
NMR (DMSO-d6) d 2.37 (s, 3H), 5.67 (s, 2H), 7.07 (s, 1H),
7.36 (m, 6H), 7.61 (d, J = 9 Hz, 1H), 8.32 (d, J = 8.1 Hz,
1 Hz), 9.18 (s, 1H); ESMS Calcd (C21H16ClN3O2S): 409.1,
found: 408.1 (MꢀH)+.
13. 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 various concentrations of compounds for
72 h and the IC50 was determined by MTS (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
assay.
14. The cell line HL60/TX1000 was a gift from Dr. Bhalla of
Emory University School of Medicine.
15. For the synthesis of indolizine compounds such as 5a and
5b, see: Li, H.; Xia, Z.-Q.; Chen, S.; Koya, K.; Ono, M.;
Sun, L. Org. Proc. Res. Dev. 2007, 11, 246.
16. Cell lines (tissue origin in parentheses) were all purchased
from ATCC: MDA435 (breast), HL60 (leukemia), DU145
(prostate), MES-SA (uterine), and MES-SA/DX5 (MDR
uterine). See Ref. 13 for cell culture conditions and assay
and see Ref. 14 for origins of HL60/TX1000.