Synthesis of 6-formyl-pyridine-2-carboxylate derivatives and their telomerase inhibitory activities

Article abstract of DOI:10.1016/S0960-894X(02)01041-7

Twenty-one pyridine-2-carboxylate derivatives were prepared by the coupling of 6-formyl-2-carboxylic acid with the corresponding phenol, thiophenol, and aniline, substituted with various functional groups. Among them, the 3,4-dichlorothiophenol ester (9p) showed the highest in vitro telomerase inhibitory activity and quite significant in vivo tumor suppression activity.

Full text of DOI:10.1016/S0960-894X(02)01041-7

Bioorganic & MedicinalChemistry Letters 13 (2003) 609–612
Synthesis of 6-Formyl-pyridine-2-carboxylate Derivatives
and Their Telomerase Inhibitory Activities
Sang-sup Jew,a,* Boon-saeng Park,a Doo-yeon Lim,a Myoung Goo Kim,a
c
In Kwon Chung,b Joo Hee Kim,b Chung IlHong, Joon-Kyum Kim,c Hong-Jun Park,c
Jun-Hee Leec and Hyeung-geun Parka
aResearch Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University,
Seoul 151-742, South Korea
bDepartment of Biology, College of Science, Yonsei University, Seoul 120-749, South Korea
cChong Kun Dang Pharmaceutical Co., Seoul 152-070, South Korea
Received 15 October 2002; accepted 2 December 2002
Abstract—Twenty-one pyridine-2-carboxylate derivatives were prepared by the coupling of 6-formyl-2-carboxylic acid with the
corresponding phenol, thiophenol, and aniline, substituted with various functional groups. Among them, the 3,4-dichloro-
thiophenolester ( 9p) showed the highest in vitro telomerase inhibitory activity and quite significant in vivo tumor suppression
activity.
# 2003 Elsevier Science Ltd. All rights reserved.
Telomeres are DNA-protein complexes at the ends of
chromosomes, which play an essential protective role
against DNA degradation and aberrant recombination
during cell divisions.1 They consist of tandem guano-
sine-rich nucleotide repeats, such as 50-TTAGGG in
humans.2 They also act as a kind of marker for the
number of cell divisions, which can control the cell
number within the body. In normal somatic cells, the
length of the telomere decreases by 50–200 nucleotides
during each cell division.3 After ca. 20 rounds of cell
division, the telomere achieves a critical level, generat-
ing cellular senescence. However, male germ cells and
80–90% of tumor cells have infinite division capacities
through the extension of telomeres by telomerase, which
is one of the ribonucleoprotein reverse transcriptases.4
Since telomerase is essential for the immortalization of
tumor cells, it has become a new target for anticancer
agents.5
type compounds,6 porphyrins,7 and cyclic polyoxazole8
show potent telomerase inhibitory activities but their
development as clinical drugs may be hindered by their
high molecular weights. Smaller molecules, such as tri-
cyclic amidoanthracene, acridine, and amidofluorenone
derivatives,9 pentacyclic acridinium salts,10 and bisin-
dole derivatives,11 were introduced as moderate inhibi-
tors. As part of our program to develop new antitumor
drugs as small molecules, 1 (IC50=1.0 mM) was chosen
as a lead compound developed by Geron. Co. Ltd. as a
telomerase inhibitor.12 However, because the imine
group in 1 is generally unstable against hydrolysis and
the corresponding aldehyde, 2 (IC50=7.0 mM) itself still
preserves relatively high telomerase inhibitory activity, 2
was modified to develop more potent inhibitors. In this
communication, we report the structure–activity rela-
tionship studies of 6-formyl-pyridine-2-carboxylate
derivatives and their potential value as a new scaffold
via an in vivo antitumor test.
Severalteolmerase inhibitors have been reported as
candidates for new antitumor drugs. Oligonucleotide-
First, we converted the thiobenzylether group of 2 to a
carboxylate group, as 3. The enhanced binding process
between the carbonyland the DNA nucleotide or amino
residues in telomerase might increase the potency of the
inhibitory activity. In addition, the modeling study
*Corresponding author. Tel.: +82-2-880-7872; fax: +82-2-872-9129;
e-mail: ssjew@plaza.snu.ac.kr
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01041-7

610
S. Jew et al. / Bioorg. Med. Chem. Lett. 13 (2003) 609–612
indicated that the conformations of 2 and 3 are quite
similar (data not shown). Twenty-one 6-formyl-pyri-
dine-2-carboxylate derivatives13 were prepared from
pyridine-2,6-dicarboxylic acid 4 in five steps (Scheme 1).
The di-methylester formation of pyridine-2,6-dicar-
boxylic acid, followed by the mono-ester reduction with
NaBH4, gave the mono-alcohol 6. The oxidation of 6
with MnO2 and the following hydrolysis afforded the
acid, 8. Finally, the thioester, ester, and amide deriva-
tives 9a–9u were obtained by the coupling of 8 with the
corresponding thiols (9a–9s), alcohol (9t), and amine
(9u), respectively.
(9m, IC50=79 mM). The p-Cl( 9h, IC50=33 mM) is a
better substituent than p-F (9g, IC50=64 mM) and p-Br
(9i, IC50=56 mM). This tendency is consistent with that
of the di-halide cases (9p, IC50=24 mM; 9s,
IC50=69 mM). The number of chlorides on the aromatic
ring is not directly related to the activity. The best result
was obtained with the 3,4-dichloro derivative 9p
(IC50=24 mM). The conversion of the thioester to the
ester (9t, IC50 >100 mM) and the amide (9u, IC50
>100 mM) abolished the activity, implying that the
more hydrogen bonding abilities of O and NH, rela-
tively to S or the more tautomerism of ester and amide,
relatively to thioester led to unfavorable binding with
the enzyme.
The telomerase inhibitory activities14 of the prepared
derivatives, 9a–9u, along with that of 2, are shown in
Table 1. Most of the thioester derivatives showed com-
parable or higher activities than the reference com-
pound, 2. The meta-CH3 and meta-CH3O derivatives
showed slightly higher activities than the ortho- and
para-derivatives, respectively. Depending on the posi-
tion substituted with the halide, quite wide variations in
the activity were observed. The mono-chloro derivative
series, p-Cl( 9h, IC50=33 mM), shows 2-fold higher
activity than both the o-Cl( 9l, IC50=84 mM) and m-Cl
Before the in vivo antitumor activity test with the best
derivative, 9p, we needed to confirm that 9p is not
associated with cytotoxicity, which is a typicalmechan-
ism of most anticancer agents, such as camptothecin.
The in vitro cytotoxicity15 of 9p was evaluated against
several cancer cell lines. As shown in Table 2, neither of
9p nor 2 showed significant cytotoxicity relative to
camptothecin, suggesting that their antitumor mechan-
ism would be different from cytotoxicity. An in vivo
Scheme 1. Reagents and conditions: (i) SOCl2, CH3OH, reflux, 3 h, 100%; (ii) NaBH4, CH3OH, rt, 4 h, 85%; (iii) MnO2, acetone, 74%; (iv) KOH,
THF/H2O=1:1, 72%; (v) HOBT, EDC, Et3N, the corresponding thiophenol, phenol or aniline derivatives, CH2Cl2, 50–80%.

S. Jew et al. / Bioorg. Med. Chem. Lett. 13 (2003) 609–612
Table 1. Telomerase inhibitory activities of 2-pyridine-carboxylate derivatives
611
Compd
R
X
Telomerase activitya
Compd
R
X
Telomerase activitya
(IC50, mM)
(IC50, mM)
9a
9b
9c
9d
9e
9f
9g
9h
9i
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
4-F
4-ClS
4-Br
4-NO2
4-NHAc
S
S
S
S
S
S
S
100<
76
100<
100<
76
9l
9m
9n
9o
9p
9q
9r
9s
9t
2-ClS
3-ClS
84
79
2,5-Cl2
2,6-Cl2
3,4-Cl2
3,5-Cl2
2,4,6-Cl3
3,4-F2
3,4-Cl2
3,4-Cl2
—
S
S
S
S
S
68
79
24
64
50
69
100<
100<
77b
91
64
33
S
S
S
S
56
75
100<
O
N
—
9j
9k
9u
2
aThe telomeric repeat amplification protocol (TRAP) assay was performed by a modification of the published method.14 The telomerase products
were resolved by 10% nondenaturing polyacrylamide gel electrophoresis, and were visualized by staining with SYBR Green (Molecular Probes,
USA). Signal intensity was quantified with a LAS-1000 Plus Image analyzer (Fuji Film, Japan).
bThe 10-fold lower potency in the IC50 value, as compared to Geron’s data (7.0 mM), might be due to the less sensitive detection method for the
telomerase product in the TRAP assay.
Table 2. In vitro cytotoxicity of 9p using human cancer cell lines
Compd
Cytotoxicitya (IC50, mM)
HT-29
Caki-2
A549
HEC-1-B
HL-60
Camptothecin
2
9p
1.40
113.28
33.09
3.96
300<
300<
2.53
300<
56.93
8.64
175.09
56.44
0.066
26.73
12.97
aIn vitro antiproliferative activities of the analogues against five tumor
cell lines (HT-29, human colon cancer; Caki-2, human renal cancer;
A549, human lung cancer; HEC-1-B, human endometrial cancer; HL-
60, human leukemia cancer) were measured by an SRB assay15 after 3
days of incubation, and are expressed as the doses required to inhibit
the growth of 50% of the cells cultivated (IC50, mM).
antitumor activity assay16 in CX-1 human clone-bearing
athymic nude mice after the subcutaneous administra-
tion of 9p was performed (Fig. 1). A clear tumor-sup-
pressing effect, by 30% in tumor volume at the dose of
20 mg/kg/day, was observed after 37 days. Notably,
there was no significant loss of body weight, which is
normally observed with cytotoxic antitumor agents.
These results suggest that the tumor suppression effect
is associated with telomerase inhibitory activity, not
cytotoxicity.
In conclusion, twenty-one pyridine-2-carboxylate deri-
vatives were prepared. Among them, the 3,4-dichloro-
thiophenolester ( 9p) showed the highest in vitro
telomerase inhibitory activity and quite significant in
vivo tumor suppression activity. These cumulative find-
ings show that the 6-formyl-pyridine-2-carboxylic
thioester would be a useful scaffold for the development
of new anticancer agents, and prove that telomerase
inhibitors can be noveland efficient antitumor drugs
without the side effects emanating from the non-selec-
Figure 1. Antitumor activity of 9p in CX-1 human clone-bearing
athymic nude mice: (A) tumor volume change; (B) body weight
change.
tivity between the normal somatic cells and cancer cells.
Further analyses of the structure–activity relationship
are now underway, replacing the pyridine ring with
another heterocyclic ring system.

612
S. Jew et al. / Bioorg. Med. Chem. Lett. 13 (2003) 609–612
Neidle, S. Bioorg. Med. Chem. Lett. 1999, 9, 2463. (c) Perry,
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