Synthesis and biological evaluation of N-heterocyclic indolyl glyoxylamides as orally active anticancer agents

Article abstract of DOI:10.1021/jm020471r

A series of N-heterocyclic indolyl glyoxylamides were synthesized and evaluated for in vitro and in vivo anticancer activities. They exhibited a broad spectrum of anticancer activity not only in murine leukemic cancer cells but also in human gastric, breast, and uterus cancer cells as well as their multidrug resistant sublines with a wide range of IC₅₀ values. They also induced apoptosis and caused DNA fragmentation in human gastric cancer cells. Among the compounds studied, 7 showed the most potent activity of growth inhibition (IC₅₀ 17-1711 nM) in several human cancer cells. Given orally, compounds 7 and 13 dose-dependently prolonged the survival of animals inoculated with P388 leukemic cancer cells. N-Heterocyclic indolyl glyoxylamides may be useful as orally active chemotherapeutic agents against cancer and refractory cancerous diseases of multidrug resistance phenotype.

Full text of DOI:10.1021/jm020471r

1706
J . Med. Chem. 2003, 46, 1706-1715
Syn th esis a n d Biologica l Eva lu a tion of N-Heter ocyclic In d olyl Glyoxyla m id es a s
Or a lly Active An tica n cer Agen ts
Wen-Tai Li,† Der-Ren Hwang,† Ching-Ping Chen,† Chien-Wei Shen,† Chen-Long Huang,† Tung-Wei Chen,†
Chi-Hung Lin,‡ Yee-Ling Chang,† Ying-Ying Chang,† Yue-Kan Lo,† Huan-Yi Tseng,† Chu-Chung Lin,†
J eng-Shin Song,† Hua-Chien Chen,† Shu-J en Chen,† Se-Hui Wu,† and Chiung-Tong Chen*,†
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes,
9F, 161, Sec. 6, Min-Chiuan East Road, Nei-Hu, Taipei 114, Taiwan, R.O.C., and
Institute of Microbiology and Immunology, National Yang Ming University,
Shih-Pai, Taipei 112, Taiwan, R.O.C.
Received October 22, 2002
A series of N-heterocyclic indolyl glyoxylamides were synthesized and evaluated for in vitro
and in vivo anticancer activities. They exhibited a broad spectrum of anticancer activity not
only in murine leukemic cancer cells but also in human gastric, breast, and uterus cancer
cells as well as their multidrug resistant sublines with a wide range of IC50 values. They also
induced apoptosis and caused DNA fragmentation in human gastric cancer cells. Among the
compounds studied, 7 showed the most potent activity of growth inhibition (IC50 ) 17-1711
nM) in several human cancer cells. Given orally, compounds 7 and 13 dose-dependently
prolonged the survival of animals inoculated with P388 leukemic cancer cells. N-Heterocyclic
indolyl glyoxylamides may be useful as orally active chemotherapeutic agents against cancer
and refractory cancerous diseases of multidrug resistance phenotype.
Ch a r t 1
In tr od u ction
The indole skeleton often appears in the natural
products with a variety of biological activities.1-5
A
number of clinical therapeutics such as indomethacin,6
indoramin,7 and indorenate8 also consist of an indole
moiety. A newly discovered anticancer agent D-24851
(Chart 1) contains an indole glyoxylamide skeleton and
possesses in vitro/in vivo anticancer activities.9-11 It was
reported that isosteric substitutions with heterocycles
often provides pharmacological and pharmacokinetic
benefits as drug candidates for further development.12-15
Others have shown heterocycle substitutions may cause
changes in the degree of ionization of compounds in
physiological pH resulting in changes of their basicity
and lipophilicity and thus substantial differences in
pharmacokinetic properties.13 Furthermore, heterocycle
substitutions of the phenyl, anilic, or an isosteric
structure were reported of better pharmacological ef-
ficacy14,15 and pharmacokinetic properties,14,15 and less
toxicity.14 A change in the orientation (positions of
substitution) of the isoxazole ring changes the biological
activity.12 It would be interesting to know whether the
replacements of the benzene rings with heterocyclic
rings at 1- and/or 3-positions of the indolyl glyoxylamide
skeleton may enhance their biological activities and
provide benefits and the basis for further development
as drug candidates. We report here the synthesis of a
number of N-heterocyclic indolyl glyoxylamides and the
evaluation of their biological activity of in vitro growth
inhibition of cancer cells and in vivo prolongation of
cancer survival in animals.
Ch em istr y
N-Substituted indolyl glyoxylamides 1-28 were syn-
thesized according to the general synthetic approach
shown in Schemes 1 and 2. According to Route I, the
potassium salt of indole was chosen to react with an
aralkyl halide to give the corresponding N-substituted
product I.16-18 The use of Grignard reagent and sodium
carbonate as bases was restricted, as indole undergoes
preferential alkylation at the 3-position of indole.19,20
The aralkyl halides were prepared either via the halide
substitution of an aralkyl alcohol21-23 or via a 1,3-
dipolar addition reaction to generate the five-membered
heterocyclic ring (5-10).24 Reaction of the N-substituted
indole with oxalyl chloride at nucleophilic 3-position of
indole was facile at 0 °C. Subsequently, on treatment
with a mild base such as triethylamine, the various
N-substituted indoles underwent substitution reaction
to give the desired products.
Alternatively, the indolyl glyoxylamides could also be
synthesized by use of another methodology (Scheme 2,
Route II). The treatment of indole with oxalyl chloride
produced 2-(1H-3-indolyl)-2-oxoethanoyl chloride due to
the high nucleophilic character at 3-position of in-
dole,25,26 and intermediate II was then produced by a
* Author for correspondence. Phone: 886-2-2653-4401 ext. 6587.
Fax: 886-2-2792-9703. E-mail: ctchen@nhri.org.tw.
† National Health Research Institutes.
‡ National Yang Ming University.
10.1021/jm020471r CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/25/2003

N-Heterocyclic Indolyl Glyoxylamides as Anticancer Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1707
Sch em e 1. Synthesis of Indolyl Glyoxylamides: Route Ia
a
Conditions: (i) tBuOK, R1CH2X, THF, 0 °C; (ii) oxalyl chloride, Et2O, 0 °C; (iii) R2NH2, Et3N, THF, 0 °C.
Sch em e 2. Synthesis of Indolyl Glyoxylamides: Route IIa
a
Conditions: (i) oxalyl chloride, Et2O, 0 °C; (ii) R1NH2, Et3N, THF, 0 °C; (iii) tBuOK, R2CH2X, THF, 0 °C.
substitution reaction with an arylamine. Finally, the
N-alkylation of indoles was carried out to give the
desired N-substituted indole glyoxylamides by similar
procedures to those described in Route I.
synthesized indolyl glyoxylamides are listed in Table 1.
In general, N-heterocyclic substitution of indolyl gly-
oxylamide decreased AlogP and thus increased solubility
of the molecules in water. It may be noted, however,
that there was no correlation between the AlogP values
and the in vitro anticancer activity.
Biology
All of the synthesized indolyl glyoxylamides were
screened initially for anticancer activity using the
human gastric NUGC3 cells. The selected active indolyl
glyoxylamides were then further evaluated to obtain
their IC50s, the concentration that causes 50% inhibition
of cancer cell growth, against six human cancer cell
lines, gastric NUGC3, hepatocellular HepG2, breast
MCF7 and its doxorubicin(adriamycin)-resistant MCF7/
ADR subline, uterus MES-SA and its doxorubicin-
resistant MES-SA/Dx5 subline, and one murine leu-
kemic P388 cell line. Anticancer activity was evaluated
with a colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoli-
um (MTS) and phenazine methosulfate (PMS) assay
system27 by measuring the residual cancer cell activity
after treatment with the indolyl glyoxylamides. Apop-
totic changes in morphology, nucleus condensation, and
apoptotic bodies of the cancer cells upon treatment with
indolyl glyoxylamides were visualized with the fluores-
cent Hoechst staining.28 These compounds also exhibited
activity in apoptosis induction as visualized by DNA
fragmentation analysis in the human gastric NUGC3
cancer cells. Compounds 7 and 13 with in vitro anti-
cancer activities were further evaluated for their in vivo
anticancer activity with the leukemic P388 cancer
survival model in young female inbred DBA/2J mice.29
In Vitr o An tica n cer Activity. The IC50 concentra-
tion of compounds 1-4, 6-10, 13-18, 20, 25 and
D-24851 were determined and were shown in Table 2.
These indolyl glyoxylamides with N-heterocyclic rings
at R1- and R2-positions exhibited a broad spectrum of
anticancer activity against the human and murine
cancer cells. Most of these compounds were more active
than D-24851. Nonetheless, it was noted that D-24851
was comparably more potent against P388 than the
other human cancer cells. Selectivity of the activity
among these compounds against various human cancer
cells was observed. Likewise, these indolyl glyoxyl-
amides with diverse chemical structures also exhibited
differential activities in various cancer cell lines. For
example, 23 out of 28 compounds were active against
NUGC3 cells as shown in Table 1, and 15 out of 17
compounds were relatively active in most of the cell lines
tested as indicated in Table 2.
The structures and IC50s in NUGC3 cells of active
compounds 11-13, 21, 22, 28 that consist of a five-
membered heterocycle and D-24851 that contains a six-
membered heterocycle on R1 were listed in Table 1. The
change of a six- to five-membered heterocycle at R1-
position maintains the biological activity. For example,
replacement of the aryl chloride of D-24851 with a furyl
group of compound 23 at R1-position showed similar
activity against NUGC3 cells. On the other hand, with
a five-membered heterocycle on R2-position, the change
from a six- to five-membered heterocycle at R1-position
dramatically increased the activity as noted in com-
pounds 20 and 21. However, the position of the het-
eroatom in the heterocycle is extremely important for
the R1-position as indicated by the IC50 of compounds
Resu lts a n d Discu ssion
P a r tition Coefficien t. The predicted partition coef-
ficient of indolyl glyoxylamides between water and
n-octanol was calculated using the fragmental method.30
The AlogP value is an index of the ability of a molecule
penetrating the cell membrane and tissue. The predicted
AlogP value for D-24851 is 3.30 and those for other

1708 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Li et al.
Ta ble 1. Structure-Activity Relationships of Indolyl Glyoxylamides in Gastric NUGC3 Cells

N-Heterocyclic Indolyl Glyoxylamides as Anticancer Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1709
Ta ble 1. (Continued)
a
b
IC50 values expressed as the mean of triplicate wells of at least three experiments. Not determined because of amorphous solid.
11 and 12. The isosteric isomer pairs, compounds 1 and
2 or 14 and 15, showed comparable anticancer activities.
Compounds 7-9 gradually lose their efficacy as the
size of alkyl side chain of R1 () isoxazolyl) increased. A
similar steric hindrance effect was also observed in the
case of compounds 25 and 27. The absence of aroma-
ticity at R1-position on compound 5 dramatically de-
creased anticancer activity as compared with the aro-
matic compound 7. These observations suggest that the
steric and electronic factors may play crucial roles at
R1-position that influences the binding affinity between
the compound and its target molecule.
The compounds with a variety of heterocyclic amines
at R2-position were synthesized and evaluated for
anticancer activity as listed in Table 1. Replacement of
the six-membered pyridine ring of D-24851 with a five-
membered heteroaromatic or a bulky quinolyl ring on
R2-position did not significantly change the anticancer
activity. As suggested by the data of compounds 2 and
19, isothiazolylamine appears to be an important moiety
at R2-position. Similar observations were also noted that
the potent compounds 6-8 and 11 consist of isothiaz-
olylamine at R2-position.
frequently used doxorubicin was also explored. Most of
these N-heterocyclic indolyl glyoxylamides showed com-
parative or better efficacy in the two doxorubicin-
resistant sublines, breast MCF7/ADR and uterus MES-
SA/Dx5 as compared in the parent breast MCF7 and
uterus MES-SA cancer cells, indicating no cross-
resistance to the multi-drug resistant doxorubicin. The
lack of cross-resistance to multiple drugs may be clini-
cally important. On the other hand, compounds 7-9 lose
their efficacy gradually with increasing size of alkyl side
chain of isoxazolyl R1. Increasing in the size of the alkyl
side chain on R1, somehow, made these molecules better
substrates of the multidrug resistant machinery leading
to a gradual loss in activity as suggested by the data in
Table 2. In agreement with the previously reported
results,9 D-24851 showed its efficacy to a similar extent
toward both MCF-7 and its multidrug-resistant subline
in the present study. The differential IC50 values
observed may be due to the utilization of different assay
methods (MTS vs 2,3-bis(2-methoxy-4-nitro-5-sulfonyl)-
5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide,
XTT, or sulforhodamine B, SRB) and treatment periods.
The results indicated that the incorporation of heteroa-
toms at 1- and 3-positions of indolyl glyoxylamide, in
general, increases the in vitro anticancer activity.
The possibility of cross-resistance of these compounds
with the other anticancer drugs such as the clinically

1710 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Li et al.
Ta ble 2. Anticancer Activity of Indolyl Glyoxylamides in Cancer Cell Linesa
gastric
NUGC3
liver
HepG2
breast
MCF7
breast
MCF-7/ADR
uterus
MES-SA
uterus
MES-SA/DX5
compound
P388
D-24851
1
2
3
4
6
7
8
524 ( 34
524 ( 26
646 ( 31
359 ( 36
676 ( 53
40 ( 6
>10000
1134 ( 430
957 ( 18
958 ( 23
6825 ( 316
3592 ( 193
2608 ( 135
73 ( 2
627 ( 77
828 ( 72
946 ( 71
605 ( 178
1260 ( 116
1197 ( 96
3085 ( 301
736 ( 115
92 ( 9
5416 ( 784
7371 ( 164
652 ( 42
151 ( 5
3018 ( 516
314 ( 17
247 ( 31
439 ( 36
26 ( 6
77 ( 15
10 ( 1
167 ( 22
7 ( 2
5962 ( 116
2537 ( 162
879 ( 30
338 ( 37
1188 ( 130
397 ( 24
2333 ( 154
33 ( 7
>10000
729 ( 9
7216 ( 723
85 ( 11
855 ( 205
19 ( 1
5 ( 1
>10000
240 ( 57
17 ( 7
6 ( 1
18 ( 2
5 ( 1
756 ( 138
54 ( 27
12 ( 3
5 ( 4
62 ( 22
9 ( 1
112 ( 15
3 ( 2
63 ( 10
1711 ( 301
1149 ( 79
42 ( 5
230 ( 57
2131 ( 59
877 ( 46
619 ( 122
497 ( 12
961 ( 55
486 ( 39
438 ( 44
95 ( 4
32 ( 15
673 ( 11
820 ( 7
385 ( 54
349 ( 18
576 ( 8
347 ( 45
378 ( 78
74 ( 20
166 ( 37
2132 ( 187
512 ( 13
310 ( 96
279 ( 7
369 ( 28
280 ( 17
261 ( 27
32 ( 2
9
362 ( 50
431 ( 81
623 ( 126
736 ( 74
603 ( 23
503 ( 55
206 ( 10
262 ( 17
478 ( 23
>10000
>10000
3663 ( 1970
>10000
5668 ( 692
>10000
1889 ( 258
1837 ( 61
>10000
10
13
14
15
16
17
18
20
25
>10000
>10000
>10000
>10000
9390 ( 1057
>10000
9763 ( 409 >1000
>10000 >1000
5044 ( 103 >10000
a
Data were IC50s expressed in nM as mean ( standard deviation of at least three separate experiments.
F igu r e 1. Induction of apoptosis by the treatment with
compound 13 at 100 nM for 16 h in human gastric NUGC3
cancer cells. Apoptotic bodies were evident by Hoechst 33258
staining as indicated in arrows.
F igu r e 2. DNA fragmentation was observed in human gastric
NUGC3 cancer cells treated with several N-heterocyclic indolyl
glyoxylamides.
Ap op tosis In d u ction . Human gastric NUGC3 can-
cer cells were treated with indolyl glyoxylamides for 16
h, and the apoptosis induction was observed by the
morphological change in nucleus condensation, forma-
tion of apoptotic bodies, and as demonstrated by DNA
fragmentation analysis. As shown in Figure 1, apoptotic
bodies of the cancer cells treated with compound 13 at
100 nM were observed after Hoechst staining with a
fluorescent microscope. As shown in Figure 2, several
indolyl glyoxylamides at 100 nM induced DNA frag-
mentation after a 24-h treatment in the cancer cells.
The findings indicate that apoptosis induction may be
a mechanism by which these N-heterocyclic indolyl
glyoxylamides kill the cancer cells. The molecular
target(s) of these new indolyl glyoxylamides is under
investigation although the anticancer activity of D-24851
can be explained by an interaction with the tubulin.9
In Vivo Act ivit y of P r olon ga t ion in Ca n cer
Su r viva l. Female DBA/2J mice of a negative, vehicle
control group consistently survived for 7 to 8 days after
a single intravenous inoculation of one million murine
leukemic P388 cancer cells. In the control experiment,
doxorubicin showed a consistent activity in prolongation
of the survival period in the P388-inoculated DBA/2J
mice. The increase of life span by treatment with
doxorubicin at its maximal tolerated dose was 119 (
18% (mean ( standard deviation) and was used as an
experimental quality control for each experiment. Com-
pounds 7 and 13 exhibited dose-dependent activities in
increasing the life span of cancer-bearing animals as
shown in Figure 3. The in vivo dose-response relation-
ship (p < 0.05, ANOVA) of 7, 13 and D-24851 in
prolonging cancer survival in animals was summarized
in Table 3. An oral dose-dependent in vivo activity
between 7 and D-24851 was observed. Compound 7 and
D-24851 showed equal activity in the prolongation of
cancer survival at oral doses up to 100 mg/kg, whereas
D-24851 was more active at the highest given oral dose,
200 mg/kg. Further prolongation of cancer survival was
observed after a continuation of up to 9 days of daily
oral administrations with 7 and 13 without evident loss
of body weight (data not shown).
As shown in Table 2, the in vitro potency of 7 and 13
was approximately 4- and 6-fold, respectively, higher

N-Heterocyclic Indolyl Glyoxylamides as Anticancer Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1711
Ta ble 3. Prolongation of Cancer Survival in P388-Inoculated
Leukemic Mice by Treatments of 7, 13, and D-24851
dose, mg/kga
7, %
13, %
D-24851, %
25
50
100
200
26 ( 27b
57 ( 20
14 ( 1
27 ( 9
44 ( 22
63 ( 14c
149 ( 28d
27 ( 3
63 ( 17c
100 ( 21d
20 ( 8c
46 ( 24d
a
The doses were administered daily on the next day after P388-
b
inoculation for 4 days. The data in mean ( standard deviation
of three separate experiments were expressed as the percentage
of increase in life span compared in medium survival time.
Medium survival time is the time when 50% of the P388-inoculated
mice in a dosing group are still surviving. c p < 0.05 (ANOVA) for
d
both 7 vs 13 and 13 vs D-24851. p < 0.05 (ANOVA) for 7 vs 13,
7 vs D-24851, and 13 vs D-24851.
further demonstrated an oral anticancer activity of
these N-heterocyclic indolyl glyoxylamides.
It was noted that D-24851 was selectively more potent
against murine P388 than the human tumor cells. The
difference between the IC50 of the human tumor cells
and that of murine P388 cells for the analogues was
generally less than that for D-24851, implying a possible
oral activity for compound 7 against in vivo growth of
the human tumor cells. While orally active against the
leukemic cancer, this observation encourages explora-
tion on the oral activity spectrum against the growth
of human solid tumor xenografts in animals and further
development.
Con clu sion
Cancer is the leading cause of death in the developed
countries. Although chemotherapy is one of the major
treatment options available for cancer, the mechanism
of action of some effective anticancer drugs remains
unclear. Most of the currently used anticancer drugs are
chemicals of small molecule and administered into
patients via a parenteral infusion or bolus injection.
Clinical complications with the parenteral administra-
tions have been documented, and thus extra cares and
cost associated with hospitalization are necessary.
Recent efforts in the discovery of anticancer drugs have
been focused on searching for orally active anticancer
agents. N-Heterocyclic indolyl glyoxylamides induce
apoptosis in cancer cells and demonstrate oral activity
against cancer in animals and may be further explored
as anticancer therapeutics.
F igu r e 3. Prolongation of cancer survival in leukemic DBA/
2J mice orally gavaged with compounds 7 (A) and 13 (B). A
dose-response relationship was observed. A. ]: 5, 0: 25, 9:
50, 4: 100, 2: 200 mg/kg compound 7, O: 0.5% CMC vehicle
control; B. 0: 25, 9: 50, 4: 100, 2: 200 mg/kg compound 13,
[: 0.5% CMC vehicle control.
Exp er im en ta l Section
than that of D-24851 against the growth of P388 cells.
D-24851, however, showed equal or better in vivo oral
activity compared to 7 and 13 in prolonging the survival
of P388-inoculated animals. The oral dose-dependent
difference in in vivo activity between 7 and D-24851
implies a possible involvement of the pharmacokinetic
processes, absorption, distribution, metabolism, and
excretion. Pharmacokinetic characteristics usually ac-
counts for uncorrelated activities between in vitro and
in vivo results. For example, intestinal P-glycoprotein
limits oral bioavailability of paclitaxel and thus its oral
uses.31 Promising P-glycoprotein inhibitors have been
developed to increase the oral bioavailability of drugs.32,33
These observations warrant further detailed pharma-
cokinetic studies for the N-heterocyclic indolyl glyoxyl-
amides. Nonetheless, results of the leukemic P388
cancer survival model showed that the N-heterocyclic
substitution retains the in vivo anticancer activity and
In Vitr o Gr ow th In h ibition Stu d y. A murine leukemic
P388 cell line and a panel of human cancer cell lines, gastric
NUGC3, hepatocellular HepG2, breast MCF7 and its doxoru-
bicin-resistant MCF7/ADR subline, uterus MES-SA and its
doxorubicin-resistant MES-SA/Dx5 subline purchased from
the Food Industry Research and Development Institute (Hsin-
chu, Taiwan), J apanese Collection of Research Bioresources,
National Institute of Health Sciences (Tokyo, J apan) and from
American Type Culture Collection (Manassas, VA), were
seeded at a cell density of 3000 or 4500 for human cancer and
10000 for murine cancer cells/100 µL/well, respectively, in 96-
well flat-bottom plates and incubated for 24 h at 37 °C in a
5% CO2 incubator. Testing compounds were dissolved in
dimethyl sulfoxide (DMSO) purchased from Sigma (St. Louis,
MO) and further diluted into the culture medium for treat-
ments of human cancer cells. The testing sample-containing
media had a final DMSO concentration of e0.3% for in vitro
testing at various concentrations. Culture media with 10 nM
actinomycin D and 0.3% DMSO were used as the positive
reference and vehicle control, respectively. Cancer cells were

1712 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Li et al.
treated in duplicate wells per concentration (200 µL/well) for
72 h at 37 °C, 5% CO2 in an incubator. A colorimetric assay
using MTS/PMS system was used to determine the anticancer
activity of the testing compounds.27 Both MTS and PMS were
purchased from Promega Corp. (Madison, WI). The colorimet-
ric assay measures cell viability based on the cellular activity
in conversion of a tetrazolium salt into a colored soluble
formazan product. The optical density (OD) values were
measured at 490 nm with a 1420 multilabel counter VICTOR
from Wallac (Turku, Finland). The IC50 of an N-heterocyclic
indolyl glyoxylamide that inhibits 50% of the growth activity
of cancer cells was then determined.
Hoech st 33258 Sta in in g. Gastric NUGC3 cancer cells of
5 × 105 seeded in a six-well culture plate were allowed to
attach for 24 h. Cells were then treated with compounds of
various concentrations at 37 °C in a CO2 incubator for another
16 h. The cells were washed with PBS and fixed with 2%
formalin at room temperature for 10 min. After being rinsed
with PBS, the cells were incubated in the staining solution (1
µg/mL Hoechst 33258 and 0.1% Triton X-100 in PBS) at room
temperature for 5 min followed by washing with PBS and
examination with a fluorescent microscope.
DNA F r a gm en ta tion An a lysis. Oligonucleosomal frag-
ments of the genomic DNA induced by treatment with the
indolyl glyoxylamides in gastric NUGC3 cancer cells were
isolated and assessed by agarose gel electrophoresis as previ-
ously described procedures with modification.28 In brief, 2 ×
106 cancer cells were incubated with an appropriate compound
for an optimal period of time and concentration. Both floating
and adherent cells were collected at the end of incubation,
washed once with PBS, and resuspended in the lysis buffer.
After incubation at 50 °C overnight, the samples were treated
with RNase A at 37 °C for 1 h. The DNA was extracted with
a mixture of phenol:chloroform:isoamyl alcohol (25:24:1), and
the supernatant was collected, separated on a 2% agarose gel,
and stained with ethidium bromide. Colchicine was used as a
positive control.
spectral measurements were of spectrograde. Preparative
separations were performed with flash column chromatogra-
phy (Merck silica gel 60, particle size 230-400 mesh) and
analytical TLC was performed on precoated plates (Merck
silica gel 60 F254); compounds were visualized by using UV
light, I2 vapor, or 2.5% phosphomolybdic acid in ethanol with
heating.
Melting points were measured with a Yanaco (MP-500D)
melting point apparatus. Predicted AlogP30 values were cal-
culated using the CeriusII interface from Accelrys Inc. (San
Diego, CA). Infrared (IR) spectra were recorded on a Perkin-
Elmer (Spectrum RX1) spectrophotometer. The proton NMR
spectra were obtained on a Varian Mer-Vx-300 (300 MHz)
Spectrometer. Chloroform-d and dimethyl sulfoxide-d6 were
used as solvent; Me4Si (δ 0.00) was used as an internal
standard. All NMR chemical shifts are reported as δ values,
and coupling constants (J ) are given in hertz (Hz). The
splitting pattern abbreviations are as follows; s, singlet; d,
doublet; t, triplet; q, quartet; br, broad; m, unresolved multiplet
and dd, doublet of doublet. Mass spectra were carried out on
a Hewlett-Packard (1100 MSD) mass spectrometer. Microanal-
yses were performed on a Heraeus CHN-O Rapid microana-
lyzer at the NCTU Instrument Center at National Chiao Tung
University, Hsinchu, Taiwan.
Gen er a l P r oced u r e for th e Syn th esis of 1-4, 11-12,
14, 16-17, 19, 21, 23. N1-(3-Meth yl-5-isoth ia zolyl)-2-[1-(2-
fu r ylm eth yl)-1H-3-in d olyl]-2-oxoa ceta m id e (1). To a mix-
ture of indole (1.17 g, 10 mmol) in tetrahydrofuran (10 mL)
was added a solution of potassium tert-butoxide (1.34 g, 12
mmol) in tetrahydrofuran (10 mL) at 0 °C and stirred at room
temperature for 1 h. 3-(Bromomethyl)furan (1.61 g, 10 mmol)
was added, and the mixture was stirred at room temperature
for 4 h. After the reaction was quenched with the addition of
saturated aqueous NH4Cl solution, the mixture was extracted
with several portions of EtOAc. The combined EtOAc solution
was dried with MgSO4 and concentrated in vacuo. The residue
was purified by silica gel column chromatography (EtOAc/
hexane ) 1:4) to give 1-(3-furylmethyl)-1H-indole (1.73 g, 88%)
as a yellow solid.
In Vivo Ca n cer Su r viva l Stu d y. The in vivo anticancer
activity of the compounds was evaluated by the murine
leukemic P388 model.29 Inbred five-week old female DBA/2J
mice of were purchased from the National Laboratory Animals
Breeding and Research Center (Taipei, Taiwan). Murine
leukemic P388 cells were purchased from the J apanese Col-
lection of Research Bioresources, J apan. P388 cells were
cultured and propagated in RPMI1640 medium supplemented
with the MEM-nonessential amino acids, 50 µM 2-mercapto-
ethanol, and 10% fetal bovine serum. Mice were grouped as
the treatment, negative control and positive control groups at
7 to 8 mice per treatment group. All mice were intravenously
inoculated with one million P388 cells per mouse 1 day before
the treatments initiated. Compounds were dissolved in DMSO
and then diluted with 0.5% carboxymethyl cellulose with the
final concentration of DMSO less than 0.5%. The compounds
were orally gavaged (po) to the mice (0.1 mL per mouse) at
various doses to show a pharmacological dose-response
relationship. Mice of the negative control group were treated
only with the dosing vehicle, 0.5% carboxymethyl cellulose.
Doxorubicin of maximal tolerated dose (10 mg/kg, iv) was used
as a positive experimental reference. The cancer cell-inoculated
animals were monitored twice daily. Survival fractions of the
mice were recorded for each treatment group. A time period
that 50% of the P388-inoculated mice still survive is defined
as the medium survival time and used to calculate a percent-
age (normalized to the medium survival time of the control
group) of increase in life span after treatments. The percentage
of increase in life span is then used as an index for treatment
response. The study was performed three times to obtain the
percentage expressed in mean ( standard deviation for each
dosing group.
To a solution of 1-(3-furylmethyl)-1H-indole (197 mg, 1.0
mmol) in diethyl ether (10 mL) was added oxalyl chloride (254
mg, 2.0 mmol) dropwise at 0 °C. The reaction mixture was
stirred at 0 °C for 3 h, and the solvent was removed under
reduced pressure. The residue was dissolved in tetrahydrofu-
ran (5 mL) and 5-amino-3-methylisothiazole hydrochloride
(151 mg, 1.0 mmol) was added. The mixture was stirred for 3
h and quenched with a saturated aqueous NaHCO3 solution
(4 mL). The organic layer was separated and washed with
brine (20 mL). The aqueous layer was extracted with ethyl
acetate (20 mL × 3). The combined organic layer was dried
with MgSO4 and concentrated in vacuo. The crude mixture
was purified by silica gel column chromatography (EtOAc/
1
hexane ) 1: 2) to give 1 as a yellow solid (259 mg, 71%). H
NMR (CDCl3): δ 10.35 (s, 1H), 9.10 (s, 1H), 8.45 (dd, J ) 6.2,
1.8 Hz, 1H), 7.46-7.36 (m, 5H), 6.8 (s, 1H), 6.32 (d, J ) 1.2
Hz, 1H), 5.26 (s, 2H), 2.46 (s, 3H). ESMS m/z 366.0 (MH+).
N1-(3-Meth yl-5-isoth ia zolyl)-2-oxo-2-[1-(3-th ien ylm eth -
yl)-1H-3-in d olyl]a ceta m id e (2). Yield: 68%. Yellow solid. 1H
NMR (CDCl3): δ 10.42 (s, 1H), 9.05 (s, 1H), 8.37 (d, J ) 7.5
Hz, 1H), 7.31-7.19 (m, 4H), 7.07 (s, 1H), 6.89 (d, J ) 4.8 Hz,
1H), 6.73 (s, 1H), 5.33 (s, 2H), 2.36 (s, 3H). ESMS m/z 382.1
(MH+).
N1-(3-Meth yl-5-isoth ia zolyl)-2-oxo-2-[1-(2-th ien ylm eth -
yl)-1H-3-in d olyl]a ceta m id e (3). Yield: 65%. Yellow solid. 1H
NMR (CDCl3): δ 10.37 (s, 1H), 9.14 (s, 1H), 8.46-8.43 (m, 1H),
7.46-7.26 (m, 5H), 6.92 (d, J ) 3.6 Hz, 1H), 6.81 (s, 1H), 5.47
(s, 2H), 2.46 (s, 3H). ESMS m/z 382.0 (MH+).
N1-(3-Meth yl-5-isoth iazolyl)-2-[1-(2-fu r yl)-1H-3-in dolyl]-
2-oxoa ceta m id e (4). Yield: 69%. Brown solid. 1H NMR
(CDCl3): δ 10.36 (s, 1H), 9.13 (s, 1H), 8.45-8.42 (m, 1H), 7.55-
7.52 (m, 1H), 7.42-7.37 (m, 3H), 6.80 (s, 1H), 6.42-6.34 (m,
2H), 5.36 (s, 2H), 2.46 (s, 3H). ESMS m/z 366.3 (MH+).
N1-(3-Meth yl-5-isoth iazolyl)-2-1-[(3-m eth yl-4,5-dih ydr o-
5-isoxa zolyl)m et h yl]-1H -3-in d olyl-2-oxoa cet a m id e (5).
Syn th esis. All commercially available materials were used
without further purification unless otherwise stated. Tetrahy-
drofuran and diethyl ether were distilled from sodium ben-
zophenone ketyl. CH2Cl2 was distilled from P2O5. Triethyl-
amine was dried over KOH and distilled. Solvents used for

N-Heterocyclic Indolyl Glyoxylamides as Anticancer Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1713
Yield: 68%. Yellow solid. 1H NMR (CDCl3): δ 10.44 (s, 1H),
9.03 (s, 1H), 8.36 (d, J ) 7.2 Hz, 1H), 7.46 (d, J ) 4.5 Hz, 1H),
7.35-7.28 (m, 3H), 5.08-5.02 (m, 1H), 4.35-4.30 (m, 2H), 3.14
(dd, J ) 17.0, 10.5 Hz, 1H), 2.78 (dd, J ) 17.0, 6.9 Hz, 1H),
2.45 (s, 3H), 2.00 (s, 3H). ESMS m/z 383.1 (MH+). Anal.
(C19H18N4O3S) C, H, N.
N1-(3-Meth yl-5-isoth ia zolyl)-2-[1-(5-isoxa zolylm eth yl)-
1H-3-in d olyl]-2-oxoa ceta m id e (6). Yield: 72%. Yellow solid.
1H NMR (CDCl3): δ 12.76 (s, 1H), 9.12 (s, 1H), 8.52 (d, J )
1.5 Hz, 1H), 8.32-8.29 (m, 1H), 7.73-7.69 (m, 1H), 7.38-7.35
(m, 2H), 7.05 (s, 1H), 6.56 (d, J ) 1.5 Hz, 1H), 5.91 (s, 2H),
2.34 (s, 3H). ESMS m/z 367.0 (MH+). Anal. (C18H14N4O3S) C,
H, N.
1H), 8.83 (d, J ) 2.1 Hz, 1H), 8.52-8.48 (m, 2H), 8.19-8.11
(m, 2H), 7.80 (dd, J ) 9.0, 2.4 Hz, 1H), 7.48-7.33 (m, 4H),
7.08 (d, J ) 2.1 Hz, 1H), 5.61 (s, 2H). ESMS m/z 413.1 (MH
+).
N1-(6-Qu in olyl)-2-oxo-2-[1-(3-t h ien ylm et h yl)-1H -3-in -
d olyl]a ceta m id e (17). Yield: 78%. Yellow solid. 1H NMR
(CDCl3): δ 9.66 (s, 1H), 9.17 (s, 1H), 8.88 (dd, J ) 4.2, 1.8 Hz,
1H), 8.51-8.48 (m, 2H), 8.15 (dd, J ) 13.8, 7.5 Hz, 2H), 7.79
(dd, J ) 9.0, 2.4 Hz, 1H), 7.44-7.32 (m, 5H), 7.16 (d, J ) 1.8
Hz, 1H), 6.99 (dd, J ) 4.8, 1.2 Hz, 1H), 5.42 (s, 2H). HRMS
calcd for C24H17N3O3S 411.1041, found 411.1033. Anal.
(C24H17N3O3S) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 5-10, 18, 20,
25. N1-(6-Qu in olyl)-2-1-[(3-m eth yl-5-isoxa zolyl)m eth yl]-
1H-3-in d olyl-2-oxoa ceta m id e (18). To a mixture of propar-
gyl chloride (2.79 g, 37.5 mmol), acetaldoxime (2.23 g, 37.4
mmol), and triethylamine (2 mL) in CH2Cl2 (100 mL) was
added sodium hypochlorite (67.1 mL, 13% active chlorine) at
0 °C. The reaction mixture was allowed to warm to room
temperature, and stirring was continued at room temperature
for 3 h. The mixture was quenched with water, and the
aqueous solution was extracted with CH2Cl2 and dried over
Na2SO4. After removal of the solvent, the residue was chro-
matographed on silica gel (EtOAc/hexane ) 1:4) to give
5-(chloromethyl)-3-methylisoxazole (4.5 g, 46%) as a colorless
liquid.
N1-(3-Meth yl-5-isoth iazolyl)-2-1-[(3-m eth yl-5-isoxazolyl-
)m eth yl]-1H-3-in d olyl-2-oxoa ceta m id e (7). Yield: 77%.
1
Yellow solid. H NMR (CDCl3): δ 10.33 (s, 1H), 9.15 (s, 1H),
8.44 (d, J ) 6.3 Hz, 1H), 7.45-7.38 (m, 3H), 6.82 (s, 1H), 5.96
(s, 1H), 5.48 (s, 2H), 2.49 (s, 3H), 2.52 (s, 3H). HRMS calcd for
C
19H16N4O3S 380.0943, found 380.0940. Anal. (C19H16N4O3S)
C, H, N.
N1-(3-Meth yl-5-isoth ia zolyl)-2-1-[(3-eth yl-5-isoxa zolyl-
)m eth yl]-1H-3-in d olyl-2-oxoa ceta m id e (8). Yield: 68%.
1
Yellow solid. H NMR (CDCl3): δ 10.26 (bs, 1H), 9.14 (s, 1H),
8.68-8.41 (m, 1H), 7.43-7.39 (m, 3H), 6.79 (s, 1H), 5.95 (s,
1H), 5.46 (s, 2H), 2.62 (q, J ) 7.5 Hz, 2H), 2.46 (s, 3H), 1.20 (t,
J ) 7.5 Hz). HRMS calcd for C20H18N4O3S 394.1100, found
394.1104.
To a mixture of indole (1.17 g, 10 mmol) in tetrahydrofuran
(10 mL) was added a solution of potassium tert-butoxide (1.34
g, 12 mmol) in tetrahydrofuran (10 mL) at 0 °C, and the
mixture was stirred at 0 °C for 30 min. This was followed by
the addition of 5-(chloromethyl)-3-methylisoxazole (1.32 g, 10
mmol) and stirring at room temperature for 4 h. The reaction
was quenched at room temperature with a saturated aqueous
NH4Cl solution and extracted with several portions of CH2-
Cl2. The combined organic layer was dried with MgSO4 and
concentrated. The crude product was purified by silica gel flash
column chromatography (EtOAc/hexane ) 1:4) to give 5-(1H-
1-indolylmethyl)-3-methylisoxazole (1.61 g, 76%) as a yellow
solid.
N1-(3-Met h yl-5-isot h ia zolyl)-2-1-[(3-isop r op yl-5-isox-
a zolyl)m et h yl]-1H -3-in d olyl-2-oxoa cet a m id e (9). Yield:
1
67%. Yellow solid. H NMR (CDCl3): δ 10.32 (s, 1H), 9.16 (s,
1H), 8.46-8.43 (m, 1H), 7.48-7.37 (m, 3H), 6.81 (s, 1H), 5.98
(s, 1H), 5.47 (s, 2H), 3.07-2.93 (m, 1H), 2.47 (s, 3H), 1.24 (t, J
) 5.1 Hz, 6H). ESMS m/z 409.1 (MH+).
N1-(3-Met h yl-5-isot h ia zolyl)-2-oxo-2-1-[(3-p h en yl-5-
isoxazolyl)m eth yl]-1H-3-in dolylacetam ide (10). Yield: 63%.
1
Yellow solid. H NMR (CDCl3): δ 10.29 (s, 1H), 9.21 (s, 1H),
8.46 (dd, J ) 5.4, 2.7 Hz, 1H), 7.72 (dd, J ) 6.8, 2.4 Hz, 2H),
7.50-7.37 (m, 6H), 6.81 (s, 1H), 6.42 (s, 1H), 5.56 (s, 2H), 2.47
(s, 3H). ESMS m/z 443.1 (MH+).
To a solution of 5-(1H-1-indolylmethyl)-3-methylisoxazole
(232 mg, 1.0 mmol) in diethyl ether (10 mL) was added oxalyl
chloride (254 mg, 2.0 mmol) dropwise at 0 °C. The reaction
mixture was stirred at 0 °C for 3 h, and the solvent was
concentrated in vacuo to remove the diethyl ether and then
dissolved in tetrahydrofuran (5 mL). To the mixture were
added 6-quinolylamine (216 mg, 1.5 mmol) and triethylamine
(5 mL), and the mixture was stirred for 3 h at room temper-
ature. A saturated aqueous NaHCO3 solution (4 mL) was
added to quench the reaction and the reaction mixture
extracted with ethyl acetate. The combined extracts were dried
with MgSO4 and concentrated in vacuo. The crude compound
was purified by column chromatography (EtOAc/hexane ) 1:1)
N1-(3-Meth yl-5-isoth ia zolyl)-2-oxo-2-[1-(3-p yr id ylm eth -
yl)-1H-3-in d olyl]a ceta m id e (11). Yield: 67%. Yellow solid.
1H NMR (DMSO-d6): δ 12.75 (s, 1H), 9.20 (s, 1H), 8.66 (d, J
) 1.5 Hz, 1H), 8.49 (dd, J ) 4.8, 1.5 Hz, 1H), 8.31 (dd, J )
5.4, 2.7 Hz, 1H), 7.73-7.66 (m, 2H), 7.37-7.31 (m, 3H), 7.05
(s, 1H), 5.70 (s, 2H), 2.35 (s, 3H). ESMS m/z 377.0 (MH+).
N1-(3-Meth yl-5-isoxazolyl)-2-oxo-2-[1-(4-pyr idylm eth yl)-
1
1H-3-in d olyl]a ceta m id e (12). Yield: 73%. Yellow solid. H
NMR (CDCl3): δ 10.10 (s, 1H), 9.05 (s, 1H), 8.60 (d, J ) 5.1
Hz, 2H), 8.48 (d, J ) 7.8 Hz, 1H), 7.44-7.22 (m, 3H), 7.05 (d,
J ) 5.1 Hz, 2H), 6.35 (s, 1H), 5.45 (s, 2H), 2.32 (s, 3H). HRMS
calcd for C20H16N4O3 360.1222, found 360.1228.
N1-(3-Meth yl-5-isoth ia zolyl)-2-[1-(4-cya n oben zyl)-1H-3-
1
to give 10 as a yellow solid (303 mg, 74%). H NMR (CDCl3):
1
in d olyl]-2-oxoa ceta m id e (13). Yield: 71%. Yellow solid. H
δ 9.61 (s, 1H), 9.20 (s, 1H), 8.88 (dd, J ) 4.2, 1.5 Hz, 1H), 8.50
(dd, J ) 9.5, 1.5 Hz, 2H), 8.16 (dd, J ) 14.0, 9.5 Hz, 2H), 7.80
(dd, J ) 3.3, 2.4 Hz, 1H), 7.45-7.38 (m, 4H), 5.95 (s, 1H), 5.48
(s, 2H), 2.25 (s, 3H). ESMS m/z 411.2 (MH+).
NMR (CDCl3): δ 9.39 (s, 1H), 9.15 (s, 1H), 8.46 (d, J ) 9.0 Hz,
1H), 7.65-7.21 (m, 11H), 5.48 (s, 2H). ESMS m/z 401.2 (MH+).
N1-(6-Qu in olyl)-2-[1-(2-fu r ylm et h yl)-1H -3-in d olyl]-2-
oxoa ceta m id e (14). Yield: 84%. Yellow solid. 1H NMR
(CDCl3): δ 9.56 (s, 1H), 9.10 (s, 1H), 8.81 (d, J ) 3.9 Hz, 1H),
8.45-8.40 (m, 2H), 8.12 (d, J ) 8.7 Hz, 1H), 8.06 (d, J ) 8.7
Hz, 1H), 7.73 (dd, J ) 9.0, 2.4 Hz, 1H), 7.48-7.31 (m, 4H),
7.19 (s, 1H), 6.35-6.29 (m, 2H), 5.30 (s, 2H). HRMS calcd for
N1-(3-Meth yl-5-isoxazolyl)-2-oxo-2-[1-(3-th ien ylm eth yl)-
1
1H-3-in d olyl]a ceta m id e (19). Yield: 74%. Yellow solid. H
NMR (CDCl3): δ 10.07 (s, 1H), 9.00 (s, 1H), 8.47-8.43 (m, 1H),
7.43-7.31 (m, 4H), 7.15 (dd, J ) 2.9, 1.2 Hz, 1H), 6.97 (dd, J
) 5.0, 1.2 Hz, 1H), 6.35 (s, 1H), 5.41 (s, 2H), 2.32 (s, 3H).
HRMS calcd for C19H15N3O3S 365.0834, found 365.0829. Anal.
(C19H15N3O3S) C, H, N.
C
24H17N3O3 395.1270, found 395.1270. Anal. (C24H17N3O3) C,
H, N.
N1-(6-Qu in olyl)-2-oxo-2-[1-(2-t h ien ylm et h yl)-1H -3-in -
d olyl]a ceta m id e (15). Yield: 81%. Yellow solid. 1H NMR
(CDCl3): δ 9.64 (s, 1H), 9.18 (s, 1H), 8.88 (dd, J ) 4.2, 1.8 Hz,
1H), 8.51-8.48 (m, 2H), 8.16 (dd, J ) 9.0, 0.9 Hz, 1H), 8.12
(d, J ) 9.0 Hz, 1H), 7.79 (dd, J ) 9.0, 2.4 Hz, 1H), 7.49-7.36
(m, 4H), 7.29-7.26 (m, 1H), 7.07 (dd, J ) 3.6, 1.2 Hz, 1H),
6.98 (dd, J ) 5.1, 3.6 Hz, 1H), 5.56 (s, 2H). ESMS m/z 412.0
(MH+).
N1-(3-Methyl-5-isothiazolyl)-2-1-[(3-methyl-5-isoxazolyl)-
m eth yl]-1H-3-in d olyl-2-oxoa ceta m id e (20). Yield: 71%.
1
Yellow solid. H NMR (CDCl3): δ 10.03 (s, 1H), 9.05 (s, 1H),
8.47-8.44 (m, 1H), 7.46-7.38 (m, 3H), 6.37 (s, 1H), 5.95 (s,
1H), 5.48 (s, 2H), 2.33 (s, 3H), 2.54 (s, 3H). ESMS m/z 364.4
(MH+).
N1-(3-Meth yl-5-isoth ia zolyl)-2-oxo-2-[1-(4-p yr id ylm eth -
yl)-1H-3-in d olyl]a ceta m id e (21). Yield: 69%. Yellow solid.
1H NMR (CDCl3): δ 10.34 (s, 1H), 9.18 (s, 1H), 8.60 (d, J )
6.0 Hz, 2H), 8.48 (d, J ) 7.8 Hz, 1H), 7.45-7.22 (m, 4H), 7.08
N1-(5-Qu in olyl)-2-[1-(4-isoth iazolylm eth yl)-1H-3-in dolyl]-
2-oxoa ceta m id e (16). Yield: 81%. Yellow solid. 1H NMR
(CDCl3): δ 9.64 (s, 1H), 9.25 (s, 1H), 8.87 (dd, J ) 4.2, 1.8 Hz,

1714 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Li et al.
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Capone, F.; Dusonchet, L.; Meli, M.; Raimondi, M. V.; Landino,
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(d, J ) 6.0 Hz, 2H), 6.82 (s, 1H), 5.47 (s, 2H), 2.47 (s, 3H).
ESMS m/z 377.1 (MH+). Anal. (C20H16N4O2S) C, H, N.
N1-(3-Met h yl-5-isoxa zolyl)-2-[1-(4-cya n ob en zyl)-1H -3-
1
in d olyl]-2-oxoa ceta m id e (22). Yield: 69%. Yellow solid. H
NMR (CDCl3): δ 10.07 (s, 1H), 9.04 (s, 1H), 8.48 (d, J ) 8.1
Hz, 1H), 7.65 (d, J ) 8.4 Hz, 2H), 7.44-7.22 (m, 5H), 6.35 (s,
1H), 5.50 (s, 2H), 2.33 (s, 3H). ESMS m/z 385.0 (MH+).
N 1-(4-P yr id yl)-2-[1-(2-fu r ylm e t h yl)-1H -3-in d olyl]-2-
oxoa ceta m id e (23). Yield: 74%. Yellow solid. 1H NMR
(CDCl3): δ 9.44 (s, 1H), 9.00 (s, 1H), 8.46 (dd, J ) 4.7, 1.5 Hz,
2H), 8.39-8.36 (m, 1H), 7.58 (dd, J ) 4.8, 1.5 Hz, 2H), 7.47-
7.28 (m, 4H), 6.35-6.28 (m, 2H), 5.29 (s, 2H). HRMS calcd for
C
20H15N3O3 345.1113, found 345.1115.
N1-(5-Meth yl-3-isoxa zolyl)-2-1-[(1-m eth yl-1H-5-im id a -
zolyl)m et h yl]-1H -3-in d olyl-2-oxoa cet a m id e (24). Yield:
59%. Yellow solid. 1H NMR (CDCl3): δ 11.46 (br, 1H), 8.78 (s,
1H), 8.25-8.22 (m, 1H), 7.71-7.69 (m, 1H), 7.32-7.29 (m, 2H),
7.12 (s, 1H), 6.83 (s, 1H), 6.69 (s, 1H), 5.55 (s, 2H), 3.69 (s,
3H), 2.41 (s, 3H). HRMS calcd for C19H17N5O3 363.1331, found
363.1347.
N1-(4-Met h yl-1,3-t h ia zol-2-yl)-2-1-[(3-m et h yl-5-isox-
a zolyl)m eth yl]-1H-3-in d olyl-2-oxoa ceta m id e (25). Yield:
73%. Yellow solid. 1H NMR (CDCl3): δ 9.10 (s 1H), 8.46-8.43
(m, 1H), 7.41-7.34 (m, 4H), 6.63 (s, 1H), 5.90 (s, 1H), 5.43 (s,
2H), 2.34 (s, 3H), 2.21 (s, 3H). ESMS m/z 381.0 (MH+).
N1-(4-Meth yl-1,3-th ia zol-2-yl)-2-[1-(4-th ia zolylm eth yl)-
1H-3-in d olyl]-2-oxoa ceta m id e (26). Yield: 81%. Yellow
solid. 1H NMR (CDCl3): δ 9.19 (s, 1H), 8.55 (d, J ) 2.1 Hz,
1H), 8.49-8.46 (m, 1H), 7.46-7.31 (m, 4H), 7.06 (s, 1H), 6.65
(s, 1H), 5.60 (s, 2H), 2.41 (s, 3H). HRMS calcd for C18H14N4O2S2
382.0058, found 382.0059.
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N1-(4-Meth yl-1,3-th ia zol-2-yl)-2-1-[(3-isop r op yl-5-isox-
a zolyl)m eth yl]-1H-3-in d olyl-2-oxoa ceta m id e (27). Yield:
1
70%. Yellow solid. H NMR (CDCl3): δ 9.82 (bs, 1H), 9.03 (s,
1H), 8.47-8.43 (1H, m), 7.41-7.36 (m, 4H), 6.76 (s, 1H), 5.92
(s, 1H), 5.44 (s, 2H), 3.00-2.95 (m, 1H), 2.44 (s, 3H), 1.20 (t, J
) 6.9 Hz, 6H). ESMS m/z 409.0 (MH+).
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761.
Ack n ow led gm en t. This study was supported by
grants BP-090-PP01, BP-090-CF03, and BP-091-CF03
of the National Health Research Institutes, Taiwan,
R.O.C. We would like to thank Professor C.-C. Liao of
National Tsing Hua University and Dr. Tony C.-C.
Liang for helpful comments; Drs. Y. S. Chao and C. M.
Chang, and Ms. H. W. Chu and Ms. S. F. J ung, for their
administrative support.
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