Synthesis of some new quinoxalines and 1,2,4-triazolo[4,3-a]-quinoxalines for evaluation of in vitro antitumor and antimicrobial activities

Article abstract of DOI:10.1002/ardp.200600061

Two novel series of quinoxalines derived from 3-phenylquinoxalin-2(1H)-one and 2-hydrazino-3-phenylquinoxaline, namely 1-substituted-3-phenylquinoxaline- 2(1H)-ones, 2a-c, 3a-d, and 4; 2-(3-oxo-3,3a,4,5,6,7-hexahydroindazol-2-yl)-3- phenylquinoxaline 6; N

Full text of DOI:10.1002/ardp.200600061

564
Arch. Pharm. Chem. Life Sci. 2006, 339, 564–571
Full Paper
Synthesis of Some New Quinoxalines and 1,2,4-Triazolo[4,3-a]-
quinoxalines for Evaluation of in vitro Antitumor and
Antimicrobial Activities
Soad A. M. El-Hawash1,, Nargues S. Habib1, and Mervat A. Kassem2
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
2 Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Alexandria, Alexandria,
Egypt
Two novel series of quinoxalines derived from 3-phenylquinoxalin-2(1H)-one and 2-hydrazino-3-
phenylquinoxaline, namely 1-substituted-3-phenylquinoxaline-2(1H)-ones, 2a–c, 3a–d, and 4; 2-
(3-oxo-3,3a,4,5,6,7-hexahydroindazol-2-yl)-3-phenylquinoxaline 6; N- cyclopentylidene or benzyli-
dene-N9-(3-phenylquinoxaline-2-yl)hydrazines, 7 and 18; 1-substituted-4-phenyl-1,2,4-triazolo[4,3-
a]quinoxalines, 9, 10, 12, 13, 14, and 16 have been synthesized in order to evaluate their antitu-
mor and antimicrobial activities. Preliminary screening at NCI showed that compounds 2b, 2c,
3b, 3c, and 9 exhibited a moderate to strong growth inhibition activity on various tumor panel
cell lines between 10– 6 to 10– 5 molar concentrations. Compound 3b was the most active with a
broad spectrum of activity. Compound 3c showed selectivity towards CNS-cancer SF-639, leuke-
mia CCRF-CEM, and melanoma SK-MEL-5 (GI50 = 4.03, 6.46, and 4.17 lM, respectively). On the
other hand, the in vitro microbiological data revealed that the prepared compounds showed
mild antimicrobial activity.
Keywords: Anticancer / Antimicrobial activity / 2-Hydrazino-3-phenylquinoxaline / 1-Substituted-3-phenylquinoxalin-
2(1H)-ones / 1-Substituted-4-phenyl-1,2,4-triazolo[4,3-a]quinoxalines /
Received: March 28, 2006; accepted: May 16, 2006
DOI 10.1002/ardp.200600061
ies: 1-(4-methoxyphenyl)-4-phenyl-1,2,4-triazolo[4,3-a]qui-
noxaline (A), 2-(4-substituted phenyl)-5-phenyl-1H-1,2,4-
triazino[4,3-a]quinoxalines (B), 2-(N-arylcarbamoyl-
Introduction
As a part of our ongoing research program on quinoxa-
line derivatives [1–4], which may serve as leads for
designing novel chemotherapeutic agents, we have
recently reported the synthesis, anticancer and antimi-
crobial evaluation of various series of quinoxalines [3, 4].
The results obtained from their in vitro screening have
shown interesting percent tumor-growth inhibition on
various cell lines. Some of these compounds exhibited
significant values of percent growth-inhibition at 10– 8 to
10– 6 molar concentrations. In particular from these ser-
methylthio)-3-phenylquinoxalines (C), and 2-(N-arylcarba-
moylmethylsulfonyl)-3-phenylquinoxalines (D) (Fig. 1).
Moreover, some of our previously reported 1,2,4-tria-
zolo[4,3-a]quinoxalines [2] and some derivatives from 3-
phenylquinoxalin-2(1H)-thione [4] exhibited promising
antifungal and antibacterial activities. Some of these
compounds were as active as the reference antibiotics
nystatin and ampicillin.
The above mentioned results prompted us to continue
our investigation on quinoxalines and quinoxalinones in
order to achieve new lead compounds for future develop-
ment as antitumor and/or antimicrobial agents. In this
context, a new series of 1-substituted-3-phenylquinoxa-
lin-2(1H)-ones (2a–c, 3a–d, and 4) have been synthesized
to further explore their antitumor and antimicrobial
Correspondence: Soad A. M. El-Hawash, Department of Pharmaceuti-
cal Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria,
Egypt.
E-mail: soadhawash@yahoo.com
Fax: +20 348 73-273
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2006, 339, 564–571
Antitumor and Antimicrobial Quinoxalines
565
Figure 1. The structure of biologically active quinoxalines A–D.
activities. The type of substituents to derivatize these
compounds was selected to determine their effect with
regard to activity.
Reagents:
i
=
4-R–C6H4COCH2Br or 3-pyridyl-COCH2Br/K2CO3 /DMF; ii = 4-
RC6H4NH-COCH2Cl/K2CO3 /DMF; iii = ClCH2-CO2C2H5 /K2CO3 /DMF; iv = POCl3; v =
NH2NH2 N H2O/abs. EtOH; vi = ethyl cyclohexanone-2-carboxylate/dry xylene; vii =
ethyl cyclopentanone-2-carboxylate/dry xylene.
Motivated by the significant antifungal and antitumor
activity recorded for the triazolo[4,3-a]-quinoxalines [2,
3], here, we report the synthesis of some new 1,2,4-tria-
zolo[4,3-a]quinoxalines (9, 10, 12, 13, and 14) in order to
achieve further knowledge of the structure-activity rela-
tionship. Moreover, some derivatives obtained from the
reaction of 2-hydrazino-3-phenylquinoxaline with some
b-ketoesters and a,b-unsaturated esters have been also
prepared (compounds 6, 7, and 16) to investigate the
effect of such structural modification on the anticipated
biological effect.
Scheme 1. Synthetic pathway of compounds 2–8.
Results and discussion
Chemistry
The synthetic procedures adopted to obtain the newly
synthesized compounds are depicted in Schemes 1, 2,
and 3. The starting compound 3-phenylquinoxalin-2(1H)-
one 1 was prepared according to previously reported pro-
cedure [5]. Reacting 1 with a variety of a-halocarbonyl
compounds following the reaction conditions adopted
for the synthesis of related compounds [6] afforded a ser-
ies of 1-substituted-3-phenylquinoxalin-2-(1H)-ones (2a–c,
3a–d, and 4). Thus, refluxing a mixture of 1 and phenacyl
bromide, p-chlorophenacyl bromide, or a-bromo-3-acetyl-
pyridine in dimethyl formamide containing anhydrous
potassium carbonate gave the respective 1-(arylcarbonyl-
methyl)-3-phenylquinoxalin-2(1H)-ones, 2a–c. Similarly,
Reagents: i = HCHO/ethylene glycol; ii = 2-acetylbuterolactone/dry xylene; iii = diethyl
oxalate/dry xylene; iv = succinic anhydride/glacial AcOH; v = phthalic anhydride / glacial
AcOH.
Scheme 2. Synthetic pathway of compounds 9–14.
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S. A. M. El-Hawash et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 564–571
with formalin following the reaction condition reported
for the preparation of related compounds [9]. Treatment
of 5 with 2-acetylbuterolactone did not yield the corre-
sponding 2-(pyrazol-1-yl) derivative 11 as reported for the
preparation of related compounds [10]. However, unex-
pectedly 1-methyl-4-phenyl-triazolo[4,3-a]quinoxaline 10
was obtained. The structure of 10 was confirmed by IR
1
and H-NMR spectra (Experimental section) and chemi-
cally by non equivocal synthesis by refluxing 5 in acetic
anhydride [2]. Reacting 5 with diethyloxalate in boiling
dry xylene afforded 1-(ethoxycarbonyl)-4-phenyl-1,2,4-
triazolo[4,3-a]quinoxaline 12. In addition, refluxing 5
with succinic anhydride or phthalic anhydride in glacial
acetic acid resulted in the formation of the respective 1-
substituted-4-phenyl-1,2,4-triazolo[4,3-a]quinoxalines, 13
and 14, as described for the synthesis of analogous com-
pounds [11].
Reagents: i = C2H5OCH=C(X)CO2C2H5; ii = C6H5CH=C(CN)CO2C2H5
Scheme 3. Synthetic pathway of compounds 16 and 18.
Treatment of 5 with diethyl ethoxymethylenemalo-
nate or ethyl (ethoxymethylene)cyanoacetate in presence
1-(N-arylcarbamoylmethyl)-3-phenylquinoxaline-2(1H)-
ones 3a–d were obtained by the reaction of 1 with N-aryl- of anhydrous potassium carbonate failed to produce the
chloroacetamides. Furthermore, 1-(ethoxycarbonyl- expected pyrazolone derivatives 15a,b as reported for the
methyl)-3-phenylquinoxaline 4 was also obtained by the synthesis of analogous compounds [12] and instead, 4-
treatment of 1 with ethyl chloroacetates.
phenyl-1,2,4-triazolo[4,3-a]quinoxalin 16 was obtained.
The present work was extended to study the reaction The structure of this compound was also confirmed by IR
1
of 2-hydrazino-3-phenylquinoxaline 5 with a variety of and H-NMR spectra and chemically by non equivocal
dicarbonyl compounds and a,b-unsaturated carbonyl synthesis by refluxing 5 in formic acid or triethyl ortho-
compounds. Compound 5 was prepared as previously formate [5]. The IR spectrum of compound 16 lacked
described [7]. Reacting 5 with ethyl cyclohexanone-2-car- absorption bands corresponding to CN or C=O of the
ester group which would be present in the expected pyra-
for the preparation of analogous compounds [8] yielded zoles 15a,b. Also, the 1H-NMR spectrum of 16 lacked the
2-(3-oxo-3,3a,4,5,6,7-hexahydroindazol-2-yl)-3-phenylqui-
triplet and quartet of the ethyl ester and the NH proton
noxaline. The product tautomerized directly to 2-(3- of pyrazole 15a. In addition, m.p., mixed m.p., TLC, IR,
hydroxy-4,5,6,7-tetrahydroindazol-2-yl)-3-phenylquinoxa-
and 1H-NMR of 16 were superimposed with that obtained
boxylate by adapting the reaction conditions reported
line 6 as indicated from the IR and1H-NMR spectra. The IR for the compound prepared unequivocally.
spectrum showed the OH absorption band at 3431 cm– 1
Furthermore, reacting 5 with ethyl benzylidenecya-
and lacked the absorption band characteristic for C=O. noacetate in presence of piperidine yielded unexpectedly
1H-NMR revealed
a
D2O-exchangeable signal at
d
2-(benzylidenehydrazino)-3-phenylquinozaline 18 in-
10.91 ppm and lacked the signal corresponding to the stead of the target 2-(pyrazol-1-yl)-3-phenylquinoxaline
proton at C3a. On the other hand, treatment of 5 with derivative 17. This compound has the same TLC, m.p., IR,
ethyl cyclopentanone-2-carboxylate under different reac- and 1H-NMR spectra as the Schiff's base obtained by react-
tion conditions (reflux in dry xylene, fusion in oil-bath at ing 5 with benzaldehyde [5].
160–1708C or reflux in glacial acetic acid) failed to pro-
duce the corresponding 2-(3-oxo-3a,4,5,6-tetrahydro-3H- Biology
Antitumor activity
cyclopentapyrazol-2-yl)-3-phenylquinoxaline 8. Instead,
2-[(1-ethoxycarbonylcyclopentan-2-ylidene)hydrazino]-3-
Antitumor activity was performed at National Cancer
phenylquinoxaline 7 was the product. The IR spectrum of Institute (NCI), Bethesda, Maryland, USA. Nine of the
7 was characterized by the presence of NH and ester car- synthesized compounds 2b,c; 3b,c; 9; 12; 13; 14, and 16
bonyl absorption bands at 3216 and 1732 cm– 1, respec- were selected by the NCI and subjected to the NCI in vitro
tively. The 1H-NMR spectrum showed triplet and quartet disease-oriented human cells screening panel assay [13,
signals at d 1.14 and 4.04 ppm corresponding to ethyl 14]. About 60 cell lines of nine tumor subpanels were
protons of the ester group. 4-Phenyl-1,2-dihydrotria- incubated with five concentrations (0.01–100 lM) for
zolo[4,3-a]quinoxaline 9 was prepared by treatment of 5 each compound and were used to create log concentra-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 564–571
Antitumor and Antimicrobial Quinoxalines
567
Table 1. Growth inhibitory action (GI50) of some selected in vitro tumor cell lines (lM)a).
Cpd.
No.
NSC
Panel
Sub-panel cell lines
(cytotoxicity GI50 lM)a)
2b
734784
leukemia
SR (37.00)
lung cancer
ovarian cancer
renal cancer
HOP-62 (33.40), HOP-92 (27.20)
OVCAR-4 (33.40)
786-D (27.00), TK-10 (25.70)
2c
734785
734279
lung cancer
HOP-92 (27.70)
3b
leukemia
SR (6.54), MOLT-4 (12.60)
lung cancer
HOP-62 (11.90), HOP-92 (8.77), NCI-H226 (10.20), NCI-H 460 (9.79); NCI-H 522
(3.50)
colon cancer
CNS cancer
melanoma
ovarian cancer
renal cancer
prostate cancer
breast cancer
HCT-116 (6.23); HT 29 (11.14) KM 12 (8.30)
SF-295 (5.09), U 251 (13.20)
M 14 (11.40), SK-MEL-5 (4.17); UACC-62 (12.90)
OVCAR-3 (5.56), OVCAR-5 (11.10); OVCAR-8 (11.30)
CAKI-1 (9.04)
PC-3 (9.99)
NCI/ADR-RES (4.46)
3c
735805
734790
leukemia
CCRF-CEM (6.46), HL-60 (Tb) (14.10)
HOP-92 (22.50)
HCT-116 (24.00
SF-639 (4.03)
SR-MEL-5 (7.38)
lung cancer
colon cancer
CNS cancer
melanoma
renal cancer
breast cancer
A 498 (13.70)
MDA-MB-231/ATC (29.90)
9
leukemia
lung cancer
CNS cancer
MOLT-4 (24.20), SR (25.00)
HOP-92 (15.00)
SNB-75 (25.80)
a)
Data obtained from NCI in vitro disease-oriented human cell screen.
tion versus% growth inhibition curves. Three response significant activity against leukemia CCRF-CEM, CNS can-
parameters (GI50, TG1, and GI50) were calculated for each cer SF-639, and melanoma SK-MEL-5, CI50 values (6.46,
cell line. The GI50 value corresponds to the compound’s 4.03, 7.38 lM). On the other hand, compound 2b showed
concentration causing 50% decreases in net cell growth. notable activity against lung cancer HOP-92, renal cancer
The TG1 value is the compound's concentration resulting 786-D, and TK-10 (GI50 values, 27.20, 27.00, 25.70 lM).
in total growth inhibition and the LC50 is the compound's Compound 9 showed moderate activity against leukemia
concentration causing a net 50% loss of initial cells at the MOLT-4, lung cancer HOP-92, and CNS cancer SNB-75 GI50
end of the incubation period (48 h). Sub-panel and full (24.70, 15.00, 25.80 lM) while compound 2c showed mar-
panel mean-graph midpoint values (MG-MID) for certain ginal activity against lung cancer HOP-92 (IG50
=
agents are the average of individual real and default GI50, 27.00 lM). Tables 2 and 3 indicate the GI50, TGI sub-panel
TGI, or LC50 values of all cell lines in sub-panel and full and full panel mean-graph midpoint (MG-MID) values for
panel, respectively.
the active compounds respectively. In addition, the active
In this study, the preliminary screening data of NCI compounds showed LC50 A 100 lM except compound 3b
indicated that five compounds, 2b,c; 3b,c, and 9, showed against melanoma SK-MEL-5 and 3c against CNS Cancer
considerable antitumor activity (Table 1). Compound 3b SF-539; their LC50 values were 68.90 and 87.40 lM, respec-
was the most active with a broad spectrum of activity. It tively.
exhibited activity against all of the tested cell lines. It
The ratio obtained by dividing the compound full
showed significant activity against lung cancer NCI- panel MG-MID (lM) by its individual sub-panel MG-MID
H522, colon cancer HCT-116, CNS caner SF-295, mela- (lM) is considered as a measure of selectivity [15]. Ratios
noma SK-MEL-5, ovarian cancer OVCAR-3, and breast can- A6 indicate selectivity toward the corresponding sub-
cer NCI/ADR-RES (GI50 values 3.50, 6.23, 5.09, 4.17, 5.56, panel cell line, while ratios between 3 and 6 refer to mod-
4.46 lM, respectively). In addition, compound 3c showed erate selectivity. Accordingly, the tested compounds
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568
S. A. M. El-Hawash et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 564–571
Table 2. Median growth inhibitory concentrations (GI50, lM) of in vitro sub-panel tumor cell lines.
Cpd.
No.
Sub-panel tumor cell linesa)
MG-MIDb)
I
II
III
IV
V
VI
VII
VIII
IX
2b
2c
3b
3c
9
78.90
–
14.90
23.40
54.20
66.20
9.20
18.40
79.80
84.80
9.30
–
14.00
84.40
91.80
53.90
–
16.80
55.40
85.20
87.20
–
18.60
72.30
95.40
56.90
91.00
19.60
73.60
97.40
48.00
–
18.10
66.20
96.60
43.40
–
32.50
41.70
91.00
67.30
–
24.30
73.60
82.90
65.97
98.10
19.70
63.40
86.60
c)
a)
I: Leukemia; II: non-small cell lung cancer; III: colon cancer; IV: ovarian cancer; V: melanoma; VI: ovarian cancer; VII: renal can-
cer; VIII: prostate cancer; IX: breast cancer.
GI50 full panel mean-graph mid-point (lM).
b)
c)
Median growth inhibitory concentration A100 lM.
Table 3. Median total growth inhibitory (TGI) concentrations (lM) of the in vitro sub-panel tumor cell lines.
Cpd.
No.
Subpanel tumor cell linesa)
MG-MIDb)
I
II
III
IV
V
VI
VII
VIII
IX
c)
2b
2c
3b
3c
9
–
–
–
96.11
–
70.27
98.47
94.61
–
–
–
–
–
–
–
–
97.77
–
80.86
98.49
–
–
–
–
–
–
–
–
99.32
–
83.84
96.72
99.10
88.69
–
–
71.81
88.12
97.32
67.70
–
–
81.05
–
–
94.17
98.40
–
87.00
–
a)
For sub-panel tumor cell lines see footnote (a) of Table 2.
TG1 (lM) full panel mean-graph mid-point (MG-MID) = the average sensitivity of all cell lines towards the test agent.
Sub-panel TG1 value A100 lM.
b)
c)
Table 4. Selectivity ratios of the active compounds towards the
nine tumor cell lines.
proved to be non-selective except compound 3c which
showed only mild selectivity toward leukemia with ratio
of 2.7 (Table 4).
Cpd.
No.
Subpanel tumor cell linesa)
III IV VI VII
0.72 1.25 0.76 1.16 1.37 1.52 0.98
From the above mentioned results it could be con-
cluded that the antitumor activity was associated with 1-
substituted-3-phenylquinoxalines (2b, 2c, 3b, and 3c) and
4-phenyl-1,2-dihydro-1,2,4-triazolo[4,3-a]quinoxaline 9.
The 1-(4-chlorophenylcarbamoylmethyl)-3-phenylquin-
oxaline 3b was the most active with a broad spectrum of
activity. Replacement of Cl by F produced compound 3c
with narrow spectrum of activity. Removal of NH group
between the aryl moiety and the carbonyl group in com-
pounds 3b and 3c resulted in a decrease of activity (com-
pounds 2b and 2c). Replacement of p-substituted phenyl
group by a pyridyl moiety resulted in a further decrease
of activity 2c.
I
II
V
VIII IX
2b
2c
3b
3c
9
0.84 1.0
0.98 1.07 0.98 0.98 0.98 1.08 0.98 0.98 0.98
1.32 1.07 1.41 1.17 1.06 1.01 1.09 0.61 0.81
0.79 0.75 1.14 0.88 0.86 0.96 1.52 0.86
1.60 1.02 0.94 1.02 0.91 0.89 0.90 0.95 1.05
2.7
a)
For sub-panel tumor cell lines see footnote (a) of Table 2.
measured. Cefotaxim was used as antibacterial reference,
while nystatin was used as antifungal reference. As
revealed from Table 5, compounds 2a and 2b showed con-
siderable antimicrobial activity against S. aureus (IZ = 28,
29 mm), while compounds 3b and 7 showed weak activ-
ity (IZ = 21, 22 mm). On the other hand, compounds 6
and 16 exhibited weak activity against E. coli (IZ =
17 mm). Compounds 4, 7, and 9 showed mild activity
against C. albicans (IZ = 18–19 mm). However, the tested
compounds were less active then the reference antibio-
tics used.
Antimicrobial activity
All the synthesized compounds were preliminary evalu-
ated for their in vitro antibacterial activity against two
representative types of bacteria, Staphylococcus aureus as
Gram-positive bacteria and Escherichia coli as Gram-nega-
tive bacteria. They were also evaluated for their in vitro
antifungal activity against C. albicans. Their inhibition
zones using the agar cup diffusion technique [16] were
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Arch. Pharm. Chem. Life Sci. 2006, 339, 564–571
Antitumor and Antimicrobial Quinoxalines
569
Table 5. Inhibition zones of the active compound (in mm).
Table 6. Physical and analytical data of the synthesized com-
pounds.
Compound No.
S. aureus
E. coli
C. albicans
Cpd.
No.
X
R
M.p. [oC]
cryst. solvent [%]
Yield
Mol. formulaa)
(mol. weight)
2a
2b
3b
4
6
7
9
16
28
29
21
16
16
16
16
17
16
16
17
16
23
–
16
16
16
18
16
19
19
16
16
–
2a
2b
2c
3a
3b
3c
3d
4
CH
CH
N
–
H
Cl
H
H
Cl
F
191–192
DMF-EtOH
206–207
DMF-H2O
238–240
DMF-H2O
258–259
DMF-H2O
231–232
DMF-H2O
242–243
EtOH
89.5
C22H16N2O2
(340.39)
C22H15ClN2O2
(374.83)
C21H15N3O2
(341.37)
C22H17N3O2
(355.40)
C22H16ClN3O2
(389.84)
C22H16FN3O2
(373.39)
C23H19N3O2
(369.43)
C18H16N2O3
(308.34)
C21H18N4O
(342.40)
C22H22N4O2
(374.45)
C15H12N4
(248.29)
C16H12N4
(260.30)
C18H14N4O2
(318.34)
C18H14N4O2
(318.34)
C22H14N4O2
(366.38)
C15H10N4
(246.27)
a)
–
–
22
–
–
–
97
96
DMF
Cefotaxim
Nystatin
98
32
–
22
–
95
a)
–
94
Inactive inhibition zone a16 mm.
–
CH3
–
254–255
DMF-EtOH
113–114
EtOH
254–255
EtOH
96
The data recorded in Table 5 revealed that the 1-substi-
tuted 3-phenylquinoxalin-2(1H)-ones 2a, 2b, and 3b were
the most active compounds. Substitution at position 4 of
the phenyl group with chlorine atom increased the activ-
ity 2b and 3b, while substitution with fluorine or a
methyl group decreased the activity 3c and 3d. Replace-
ment of the phenyl group by pyridyl ring greatly
decreased the activity 2c. Insertion of NH between the
aryl moiety and the carbonyl function also decreased the
activity 3a–c. On the other hand, 1-ethoxycarbonyl-
methy derivative 4 was devoid of activity. This revealed
that the aryl moiety in such a series of compounds was
essential for activity. The results also revealed that the
hydrazone derivative 7 showed antimicrobial activity.
However, insertion of hydrazone function in a ring struc-
ture (6) reduced the activity. Out of the tested 1,2,4-tria-
zolo[3,4-a]quinoxalines only the unsubstituted tria-
zolo[3,4-a]quinoxalines 16 and the 1,2-dihydro derivative
9 showed some antimicrobial activity. On the other
hand, all 1-substituted-tiazoloquinoxalines 10, 12, 13,
and 14 were devoid of activity.
–
81.5
78
6
–
–
7
–
–
112–113
EtOH
75
9
–
–
124–125
EtOH
46
10
12
13
14
16
18
–
–
220–222
EtOH
119–120
EtOH
71.4
62
–
–
244–245
DMF-EtOH
202–203
DMF-EtOH
192–193
EtOH
80.7
64.5
98
118–120
EtOH
90.9
C21H16N4
(324.39)
a)
Analyzed for C, H, N, the results are within l0.4% of the theo-
retical values.
The authors are grateful to the staff of the Department of
Health and Human Services, National Cancer Institute,
Bethesda, Maryland, U.S.A. for carrying out the anticancer
screening of the newly synthesized compounds.
standard and DMSO-d6 as a solvent. The microanalyses were per-
formed at the Microanalytical Laboratory, National Research
Center, Cairo, Egypt and the data were within l0,4% of the theo-
retical values. Following up of the reactions and checking the
homogeneity of the compounds were made by TLC on silica gel-
protected aluminium sheets (Type 60 F254, Merck, Darmstadt,
Germany) and the spots were detected by exposure to UV-lamp
at k 254 nm for a few seconds.
Experimental
Chemistry
1-(Arylcarbonylmethyl)-3-phenylquinoxaline-2(1H)-ones
2a–c
A mixture of 3-phenylquinoxaline-2(1H)-one 1 (0.44 g, 2 mmol)
and phenacylbromide or 4-chlorophenacylbromide or 3-(bro-
moacetyl)pyridine (4 mmol) and anhydrous potassium carbo-
nate (0.55 g, 2.5 mmol) in dimethylformamide (10 mL) was
heated under reflux for 5 h. The reaction mixture was cooled,
All melting points were determined in open glass capillaries on
a Gallenkamp melting point apparatus and are uncorrected. The
IR spectra were recorded using KBr discs on a Perkin-Elmer 1430
spectrophotometer (Perkin- Elmer).1H-NMR (d ppm) spectra were
recorded on a JNM-LA 400 FT NMR system (400 MHz) and on a
Jeol (500 MHz) spectrometer (both JEOL, Tokyo, Japan). 13C-NMR
spectra were run on Jeol spectrometer using TMS as internal
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570
S. A. M. El-Hawash et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 564–571
poured onto ice-cold water. The obtained product was filtered,
dried, and crystallized from aqueous dimethylformamide (Table
6).
2-[(1-Ethoxycarbonylcyclopentan-2-yliden)hydrazino]-3-
phenylquinoxaline 7
This compound has been prepared as described under 6 from 5
(0.47 g, 2 mmol) and ethyl cyclopentanone-2-carboxylate (0.34 g,
2.2 mmol).
IR (KBr, cm– 1): 1694–1691 (C=O ketonic); 1662–1642 (C=O qui-
nox.); 1599–1595, 1585-1552, 1490–1486 (C=N, C=C). 1H-NMR
for 2b (500 MHz, d ppm): 5.95 (s, 2H, CO-CH2); 7.4 (t, 1H, J = 7.6 Hz,
quinox C6-H); 7.45–7.49 (m, 3H, C6H5-C3,4,5-H); 7.52 (d, 2H, J = 8.0
Hz, phenyl C2,6-H), 7.56 (t, 1H, J = 7.6 Hz, quinox. C7-H); 7.86, 8.14
(two d, each 2H, J = 8.4 Hz, p-Cl-C6H4), 7.92 (d, 1H, J = 7.6 Hz, qui-
nox. C5-H); 8.22 (d, 1H, J = 7.6 Hz, quinox. C8-H).
IR (KBr, cm– 1): 3216, (NH); 1732 (C=O ester); 1659, 1643 (C=N);
1605, 1513, 1479 (C=C); 1534 (d NH), 1264, 1033 (C-O-C). 1H-NMR
(400 MHz, d ppm): 1.14 (t, 3H, J = 7.5 Hz, CO2CH2CH3); 1.77 (quin,
2H, J = 7.5 Hz, cyclopentyl-C4-H); 1.98 (q, 2H, J = 7.5 Hz, cyclope-
nyl-C5-H); 2.40 (t, 2H, J = 7.5 Hz, cyclopentyl-C3-H);3.51(t, 1H, J =
7.5 Hz, cyclopentyl-C1-H), 4.04(q, 2H, J = 7.5 Hz, CO2CH2CH3); 7.59-
7.64 (m, 3H, C6H5-C3,4,5-H); 7.68–7.79 (m, 2H, quinox. C6,7-H); 8.13
(dd, 1H, J = 7.65, 1.5 Hz, quinox. C5-H); 8.31 (dd, 1H, J = 7.2, 1.2 Hz,
quinox. C8-H); 8.73–8.76 (m, 2H, C6H5-C2,6-H).
1-(N-Arylcarbamoylmethyl)-3-phenylquinoxaline-2(1H)-
ones 3a–d
The title compounds were prepared as described under 2a–c
from 1 and the appropriate N-substituted chloroacetamides
(Table 6).
4-Phenyl-1,2-dihydro-1,2,4-triazolo[4,3-a]quinoxaline 9
Formalin solution (40%, 0.2 mL) was added to a suspension of 5
(0.47 g, 2 mmol) in ethylene glycol (5 mL). The reaction mixture
was refluxed for 15 min, then cooled to room temperature and
the formed crystalline product was filtered, dried, and recrystal-
lized from ethanol. IR (KBr, cm– 1): 3191 (NH); 1663 (C=N); 1608,
1515 (C=C); 1534 (d NH).1H-NMR (500 MHz, d ppm): 5.23 (d, 2H, J =
6.0 Hz, CH2); 7.54 (t, 1H, J = 8.4 Hz, C6H5-C4-H); 7.73-7.92 (m, 4H,
C6H5C2,3,5,6-H); 8.06 (t, 1H, J = 7.65 Hz, triazoloquinox. C7-H); 8.14
(t, 1H, J = 7.65 Hz, triazoloquinox. C8-H); 8.13 (d, 1H, J = 7.65 Hz,
triazoloquinox. C6-H); 8.27 (dd, 1H, J = 7.65, 1.6 Hz, triazoloqui-
nox. C9-H); 8.74 (t, 1H, J = 6 Hz, NH, D2O exchangeable).
IR (KBr, cm– 1): 3292–3269 (NH); 1660–1651 (C=O); 1599–
1597, 1579–1577, 1517–1499 (C=N, C=C); 1537–1535 (d NH). 1H-
NMR for 3d (500 MHz, d ppm): 2.21 (s, 3H, CH3); 5.16 (s, 2H,
COCH2-N); 7.08 (d, J = 8.4 Hz, 2H, p-tolyl C3,5-H); 7.39 (t, 1H, J = 7.65
Hz, quinox. C6-H); 7.45 (d, J = 8.4 Hz, 2H, p-tolyl-C2,6-H); 7.47–7.55
(m, 5H, C6H5); 7.60 (t, J = 7.65, quinox. C7-H); 7.90 (dd, J = 7.65, 1.55
Hz, 1H, quinox. C5-H); 8.25 (dd, J = 7.65, 2.3 Hz, 1H, quinox. C8-H);
10.39 (s, 1H, NH, D2O-exchangeable). 13C-NMR for 3d (125 MHz, d
ppm): 20.98 (CH3); 46 (N-CH2); 115.3, 128.48, 133.09, 133.72 (p-
tolyl C2,3,4,1, respectively); 119.74, 124.28, 129.75, 130.3, 136.21,
136.66, 153.41, 165.15 (quinox. C8,6,7,5,4a,8a,3,2, respectively); 154.5
(NHCO).
1-Methyl-4-phenyl-1,2,4-triazolo[4,3-a]quinoxaline 10
A mixture of 5 (0.47 g, 2 mmol) and 2-acetylbuterolactone
(0.28 g, 2.2 mmol) in dry xylene (5 mL) was refluxed for 3 h. The
reaction mixture was cooled, the obtained crystalline product
was filtered, dried, and recrystallized from ethanol. IR (KBr,
cm–1): 1660 (C=N); 1606, 1515 (C=C). 1H-NMR (500 MHz, d ppm):
2.47 (s, 3H, CH3); 7.57-7.59 (m, 3H, C6H5-C3,4,5-H); 7.65 (t, 1H, J =
7.65 Hz, triazoloquinox. C7-H); 7.70 (t, 1H, J = 7.65 Hz, triazoloqui-
nox. C8-H); 8.06 (dd, 1H, J = 7.65, 1.6 Hz, triazoloquinox. C6-H);
8.30 (dd, 1H, J = 7.65, 1.6, triazoloquinox. C9-H); 8.69-8.72 (m, 2H,
C6H5-C2,6-H).
1-(Ethoxycarbonylmethyl)-3-phenylquinoxaline-2(1H)-
one 4
The title compound was prepared as described under 2a–c and
3a–d from 1 and ethyl chloroacetate. IR (KBr, cm– 1): 1742 (C=O
ester); 1646 (N-C=O) 1602, 1579 (C=N, C=C); 1226, 1182, 1078 (C-
O-C). 1H-NMR (400 MHz, d ppm): 1.21 (t, 3H, J = 7.0 Hz, CH2CH3);
4.16 (q, 2H, J = 7.0 Hz, CH2CH3), 5.15 (s, 2H, N-CH2-CO); 7.43 (t, 1H,
J = 7.5 Hz quinox. C6-H); 7.49-7.54 (m, 3H-C6H5C3,4,5-H); 7.55 (d, 2H,
J = 7.5 Hz, C6H5 C2,6-H); 7.63 (t, 1H, J = 7.5, quinox. C7-H); 7.92 (d,
1H, J= 8 Hz, quinox. C5-H); 8.21(d, 1H, J = 8.0 Hz, quinox.C8-H).
1-Ethoxycarbonyl-4-phenyl-1,2,4-triazolo[4,3-
a]quinoxaline 12
This compound was prepared in analogy to 10 from 5 (0.47 g, 2
mmol) and diethyl oxalate (0.32 g, 2.2 mmol). IR (KBr, cm– 1):
1733 (C=O ester); 1644 (C=N); 1607, 1501 (C=C); 1281, 1248, 1194,
1041 (C-O-C). 1H-NMR (500 MHz, d ppm): 1.42 (t, 3H, J = 7.0 Hz,
CH2CH3); 4.58 (q, 2H, J = 7.0 Hz, CH2CH3); 7.59–7.63 (m, 3H, C6H5-
C3,4,5-H); 7.74 (t, 1H, J = 7.65 Hz, triazoloquinox. C7-H); 7.76 (t, 1H, J
= 7.65 Hz, triazoloquinox. C8-H); 8.15 (dd, J = 7.65, 3 Hz, triazolo-
quinox. C6-H); 8.62–8.68 (m, 3H, C6H5-C2,6-H and triazoloquinox.
C9-H).
2-(3-Hydroxy-4,5,6,7-tetrahydroindazol-2-yl)-3-
phenylquinoxaline 6
A
mixture of 2-hydrazino-3-phenylquinoxaline 5 (0.47 g,
2 mmol) and ethyl cyclohexanone-2-carboxylate (0.38 g,
2.2 mmol) in dry xylene (5 mL) was heated under reflux for 3 h.
The reaction mixture was cooled and the obtained yellow crys-
talline product was filtered, dried, and recrystallized from etha-
nol. IR (KBr, cm– 1): 3431 (enolic OH); 1608 (C=N), 1551, 1483
(C=C). 1H-NMR (500 MHz, d ppm): 1.61, 1.69 (two m, 4H, indazole
C5,6-H); 2.2 (dist. t, 2H, indazole C7-H); 2.16–2.26 (m, 2H, indazole
C4-H); 7.01–7.47 (m, 4H, Ar-H); 7.59 (t, 1H, J = 7.65 Hz, quinox. C6-
H); 7.85 (t, 1H, J = 7.65 Hz, quinox. C7-H); 7.90 (t, 1H, J = 7.5 Hz,
C6H5-C4-H); 8.03 (dd, 1H, J = 7.65, 1.5 Hz, quinox. C5-H); 8.12 (dd,
1H, H, J = 7.65, 1,5 Hz, quinox. C8-H); 10.9 (s, 1H, enolic OH, D2O
exchangeable).
3-(4-Phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-
yl)propanoic acid 13
A mixture of 5 (0.47 g, 2 mmol) and succinic anhydride (0.18 g,
2 mmol) in glacial acetic acid (5 mL) was heated under reflux for
5 h. The reaction mixture was cooled to ambient temperature.
The obtained crystalline product was filtered, dried, and recrys-
tallized from DMF-EtOH (Table 6). IR (KBr, cm– 1): 3474–3251 (br
OH); 1741 (C=O); 1644 (C=N); 1607, 1515 (C=C).1H-NMR (500 MHz,
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Arch. Pharm. Chem. Life Sci. 2006, 339, 564–571
Antitumor and Antimicrobial Quinoxalines
571
d ppm): 3.03 (t, 2H, J = 6.9 Hz, CH2-C=N); 3.68 (t, 2H, J = 6.9 Hz,
CH2COOH); 7.57–7.59 (m, 3H, C6H5-C3,4,5,-H); 7.60 (t, 1H, J = 7.65
Hz, triazoloquinox. C7-H); 7.66 (t, 1H, J = 7.65 Hz, triazoloquinox.
C8-H); 8.08 (dd, 1H, J = 7.0, 1.5 Hz, triazoloquinox. C6-H); 8.28 (dd,
1H, J = 8.4 Hz, 1.5 Hz, triazoloquinox. C9-H); 8.69-8.71 (m, 2H,
C6H5-C2,6-H), 12.2 (s, 1H, OH, D2O exchangeable). Mass spectrum
m/z (%) : 318 [M+9] (31); 319 [M+ + 1] (10); 273 [M+-CO2H] (100), 219
(35); 218 (18); 192 (6); 115 (7); 103 (6); 102 (11); 92 (5); 91 (19); 77
(26); 76 (14); 75 (10); 64 (25); 63 (23); 62 (12).
protein assay was used to estimate cell viability or growth [13–
15]. The results are presented in Tables 1–4.
Antimicrobial activity
All the synthesized compounds were evaluated by the agar cup
diffusion technique [16] using a 1mg/mL solution in DMF. The
test organisms were Staphylococcus aureus (ATCC 29213) as Gram-
positive bacteria; Escherichia coli (ATCC 25922) as Gram-negative
bacteria. Candida albicans (ATCC 10231) was also used as a repre-
sentative for fungi. Each 100 mL of sterile molten agar (at 458C)
received 1 mL of 24 h broth culture, and then the seeded agar
was poured into sterile petri dishes. Cups (8 mm in diameter)
were cut in the agar. Each cup received 0.1 mL of the 1 mg/mL
solution of the test compounds. The plates were then incubated
at 378C for 24 h or for 48 h for C. albicans, respectively. A control
using DMF without the test compound was included for each
organism. Cefotaxim was used as standard antibacterial, while
nystatin was used as antifungal reference. The resulting inhibi-
tion zones are recorded in Table 5.
2-(4-Phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-yl)benzoic
acid 14
This compound was prepared similarly to 13 from equimolar
amounts of 5 and phthalic anhydride in glacial acetic acid. IR
(KBr, cm– 1): 3414–3256 (OH), 1727 (C=O); 1660 (C=N), 1608,
1515, 1484 (C=C). Mass spectrum m/z (%): 367 [M+9 + 1] (13); 366
[M+9] (56); 322 [M+9–CO2] (25); 321 [M+9–CO2H] (100); 219 (72); 218
(16); 105 (11); 104 (39); 103 (7); 102 (18); 92 (10); 91 (10); 90 (39);
89 (8); 64 (17); 63 (18).
4-Phenyl-1,2,4-triazolo[4,3-a]quinoxaline 16
A mixture of 5 (0.47 g, 2 mmol), diethyl ethoxymethylenemalo-
nate or ethyl (ethoxymethylene)cyanoacetate (2 mmol), and
anhydrous potassium carbonate (0.14 g, 2 mmol) in absolute
ethanol (10 mL) was heated under reflux for 3 h. The reaction
mixture was cooled to room temperature, the obtained white
crystalline needles were filtered, dried, and recrystallized from
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1
ethanol. IR (KBr, cm– 1): 1662, 1645 (C=N); 1610, 1509 (C=C). H-
NMR (400 MHz, d ppm): 7.63–7.64 (m, 3H, C6H5-C3,4,5-H); 7.72 (t,
1H, J = 7.56 triazoloquinox. C7-H); 7.60 (t, 1H, J = 7.65, triazoloqui-
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2-Benzylidenhydrazino-3-phenylquinoxaline 18
To a solution of 5 (0.47 g, 2 mmol) and few drops of piperidine in
ethanol (10 mL) ethyl benzylidenecyanoacetate (0.4 g, 2 mmol)
was added. The reaction mixture was heated under reflux for
3 h. After cooling, the obtained crystalline product was filtered,
dried, and recrystallized from ethanol. IR (KBr, cm– 1): 3339 (NH),
1646 (C=N), 1607, 1523 (C=C), 1560 (d NH). 1H-NMR (400 MHz, d
ppm): 7.15 (t, J = 7.65 Hz, 1H, quinox. C6-H), 7.36–7.55 (m, 10 H,
two C6H5); 7.63 (t, J = 7.65, 1H, quinox. C7-H); 7.76 (dd, J = 7.65, 2.4
Hz, 1H quinox. C5-H); 8.06 (dd, J = 7.6, 2.3 Hz, 1H, quinox. C8-H);
8.48 (s, 1H, CH=N-); 11.11 (s, 1H, NH, D2O exchangeable).
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Antitumor activity
The prepared compounds were tested for their in vitro antitumor
activity against 60 human tumor cell lines, derived from nine
clinically isolated types of cancer (leukemia, lung, colon, brain,
melanoma, ovarian, renal, prostate, and breast) following the
NCI preclinical antitumor drug discovery screen. Each com-
pound was tested at five-, ten-fold dilutions; a 48 h continuous
drug exposure protocol was used and a sulforodamine B (SRB)
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i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com