Synthesis and in vitro-anticancer and antimicrobial evaluation of some novel quinoxalines derived from 3-phenylquinoxaline-2(1 H)-thione

Article abstract of DOI:10.1002/ardp.200600012

Two novel series derived from 3-phenylquinoxaline-2(1H)-thione 2 and 2-(hydrazinocarbonylmethylthio)-3-phenylquinoxaline 6 have been synthesized. Eight out of twenty six new compounds were selected at the National Cancer Institute for evaluation of their

Full text of DOI:10.1002/ardp.200600012

Arch. Pharm. Chem. Life Sci. 2006, 339, 437–447
S. A. M. El-Hawash et al.
437
Full Paper
Synthesis and in vitro-Anticancer and Antimicrobial
Evaluation of Some Novel Quinoxalines Derived from
3-Phenylquinoxaline-2(1H)-thione
Soad A. M. El-Hawash1, Abeer E. Abdel Wahab2
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
2 Genetic Engineering and Biotechnology Research Institute (GEBRI), Mubarak City for Scientific Research
and Technology Application, Borg El-Arab, Alexandria, Egypt
Two novel series derived from 3-phenylquinoxaline-2(1H)-thione 2 and 2-(hydrazinocarbonylme-
thylthio)-3-phenylquinoxaline 6 have been synthesized. Eight out of twenty six new compounds
were selected at the National Cancer Institute for evaluation of their in vitro-anticancer activity.
Among them, compounds 3b, 3c, 4b, and 4c displayed moderate to strong growth inhibition
activity against most of the tested sub-panel tumor cell lines with GI50 10– 5 to 10– 6 molar concen-
trations. Compound 4b exhibited a significant value of percent tumor growth inhibition against
breast cancer at concentration a10– 8 M. Compound 4c showed moderate selectivity towards leu-
kemia cell lines with GI50 of 1.8 to 3.8 lM (selectivity ratio = 5.7). Preliminary antimicrobial
testing revealed that compounds 7a, 7b, 8a, 11a, and 11b were as active as ampicillin against B.
subtilis (MIC = 12.5 lg/mL). Compounds 7b and 8a were also nearly as active as ampicillin against
E. coli (MIC = 12.5 lg/mL). In addition, compounds 4a, 7b, 10b, and 11a were as active as ampicil-
lin against P. aerugenosa (MIC = 50 lg/mL). However, compounds 7b, 8a, and 10b showed mild
activity against C. albicans (MIC = 50 lg/mL). The values of minimum bactericidal concentrations
indicated that compounds 4a and 7b were bactericidal against B. subtilis and P. aerugenosa,
respectively, while compound 10b was bactericidal against both organisms. However, compound
11a was bactericidal against E. coli, P. aerugenosa, and S. aureus.
Keywords: Antitumor activities, Antimicrobial activities / 2-(Hydrazinocarbonylmethylthio)-3-phenylquinoxaline / N-Ar-
ylchloroacetamides / 3-Phenylquinoxaline-2(1H)-thione /
Received: January 17, 2006; accepted: March 16, 2006
DOI 10.1002/ardp.200600012
topoisomerase II inhibitors, 2-[4-(7-chloroquinoxalin-2-
yl)-phenoxy]propionic acid (XK469) [5] and chloroquinox-
alinesulfonamide (CQS) [6] (Fig. 1), in addition to the non-
nucleoside reverse transcriptase inhibitors, (S)-3-ethyl-6-
fluoro-4-isopropoxy-carbonyl-3,4-dihydroquinoxalin-
2(1H)-one (GW420867) [9] and 4-isopropoxycarbonyl-6-
methoxy-3-(methylthiomethyl)-3,4-dihyroquinoxalin-2-
(1H)-thione (HBY 097) [8] (Fig. 1).
Introduction
Quinoxaline and quinoxalinone derivatives have
received much attention due to their versatile biological
properties, especially antitumor [1–6], anti-HIV [7–9],
and antimicrobial [10–13] activities. Among these deriv-
atives some found their way in clinical application. For
example, the two known antineoplastic quinoxaline
These findings prompted us to investigate some of our
previously reported quinoxalines [14, 15] for their in
vitro-antitumor effect. The results obtained from their
screening have shown interesting tumor growth inhibi-
tion on various cell lines between 10– 6 –10– 5 M [16].
Some of these compounds exhibited significant values of
percent growth inhibition at 10– 7 M. For example: 1-(4-
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
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438
S. A. M. El-Hawash et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 437–447
Figure 1. Chemical structure of antineoplastic quinoxaline
topoisomerase II inhibitors, XK469, CQS, and non-nucleoside
reverse transcriptase inhibitors, GW420867, HBY 097.
Figure 2. Chemical structure of 1-(4-methoxyphenyl)-4-phenyl-
1,2,4-triazolo[4,3-a]quinoxaline (A) and 2-aryl-5-phenyl-1H-
1,2,4-triazino-[4,3-a]quinoxalines (B).
Scheme 1. Synthesis route of 2-(N-
arylcarbamoylmethylthio) and 2-(N-
arylcarbonylmethylsulfonyl)-3-phe-
nylquinoxalines (3a–d and 4a–d)
and 2-[arylidene and (1-substituted-
ethylidene)-hydrazinocarbonyl-
Reagents: i = H2N-CS-NH2 / H2SO4; ii = HSCH2CO2C2H5 / K2CO3 / dry acetone;
iii = ClCH2CO2C2H5 / anhydrous CH3CO2Na / EtOH; iv = 4-RC6H4NHCOCH2Cl / anhydrous CH3CO2Na / EtOH;
v = KMnO4 / gl. AcOH; vi = NH2NH2 N H2O / EtOH; vii = R-CO-R1 / EtOH.
methylthio]-3-phenylquinoxalines
7a–d.
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Arch. Pharm. Chem. Life Sci. 2006, 339, 437–447
Anticancer and Antimicrobial Quinoxalines
439
Scheme 2. Synthesis route of 2-(N-
arylthiocarbamoyl-hydrazinocarbo-
nylmethylthio)-3-phenylquinoxalines
8a, b and derived ring systems
(compounds 9a,b; 10a,b; 11a,b; and
12a,b).
Reagents: i = 4-RC6H4NCS / DMF, EtOH; ii = H2SO4; iii = HgO / dry dioxane; iv = ClCH2CO2C2H5 / abs. EtOH;
v = 4-R1C6H4COCH2Br / dry dioxane.
methoxyphenyl)-4-phenyl-1,2,4-triazolo[4,3-a]quinoxa-
line (A) and 2-aryl-5-phenyl-1H-1,2,4-triazino-[4,3-a]quin- tance of substituted 1,3,4-thiadiazoles [17–19], 1,3,4-oxa-
oxalines (B) (Fig. 2). diazole [20–22], thiazoles and thiazolidinones [23–26]
Moreover, owing to the increasing biological impor-
In view of the above mentioned results and in conti- particularly in the field of chemotherapy, it was planed
nuation of our interest in biologically active quinoxa- to synthesize additional derivatives. These derivatives
lines [14–16], the aim of the present study was to synthe- comprise the quinoxaline backbone linked to the above
size and investigate the in vitro-anticancer and antimicro- mentioned heterocyclic ring systems by various atom
bial activity of some novel quinoxalines which were spacers (compounds 9a, b; 10a, b; 11a, b; and 12a–d,
designed as structural relatives to the anticancer CQS Scheme 2), in order to investigate the effect of these
and the non-nucleoside reverse transcriptase inhibitor structure variants on the anticipated antitumor and/or
HBY 097 (Fig. 1). The new series comprised the com- antimicrobial activity.
pounds, namely: 2-(N-arylcarbamoylmethylthio) and 2-(N-
arylcarbonylmethylsulfonyl)-3-phenylquinoxalines (3a–
d and 4a–d); 2-[arylidene and (1-substituted-ethylidene)-
hydrazinocarbonylmethylthio]-3-phenylquinoxalines
Results and Discussion
(7a–d, Scheme 1) and 2-(N-arylthiocarbamoyl-hydrazino- Chemistry
carbonylmethylthio)-3-phenylquinoxalines
(8a, b; The preparation of target compounds was conducted
Scheme 2). The substitution pattern of these derivatives according to the sequence of reactions depicted in
was selected to confer different electronic environment Schemes 1 and 2. The starting compound 2-chloro-3-phe-
to the molecules.
nylquinoxaline 1 was obtained as previously described
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[27]. 3-Phenyl-quinoxaline-2(1H)-thione 2 was obtained by parameters (GI50, TG1, and LC50) were calculated for each
reacting 1 with thio-urea as reported for the preparation cell line. The GI50 value corresponds for the compounds’
of related compounds [28]. Treatment of 2 with the appro- concentration causing 50% decreases in net cell growth.
priate N-aryl substituted chloroacetamide afforded the The TG1 value is the compounds’ concentration resulting
respective 2-(N-arylcarbamoylmethylthio)-3-phenylqui- in total growth inhibition and the LC50 value is the com-
noxalines 3a–d. The target 2-[N-arylcarbamoylmethylsul- pounds’ concentration causing a net 50% loss of initial
fonyl)-3-phenylquinoxalines 4a–d were obtained by oxi- cells at the end of the incubation period (48 h). Sub-panel
dation of 3a–d with potassium permanganate in glacial and full panel mean-graph midpoint values (MG-MID) for
acetic acid. 2-(Ethoxycarbonymethylthio)-3-phenylqui- certain agents are the average of individual real and
noxaline 5 was obtained either by treatment of chloroqui- default GI50, TGI, or LC50 values of all cell lines in sub-
noxaline 1 with ethyl thioglycolate in refluxing dry acet- panel and the full panel respectively [31].
one containing anhydrous potassium carbonate (method
In this study, the preliminary screening data indicated
A) or by reacting 2 with ethyl chloroacetate in refluxing that four compounds showed antitumor activity namely:
ethanol containing anhydrous sodium acetate (method 2-(N-arylcarbamoylmethylthio)-3-phenylquinoxalines
B). Treatment of 5 with ethanolic hydrazine hydrate at 3b, c) and 2-(N-arylcarbamoylmethylsulfonyl)-3-phenyl-
room
temperature
gave
2-(hydrazinocarbonyl- quinoxalines (4b, c) (Tables 1–4). Compounds 3b and 4b
methylthio)-3-phenylquinoxaline 6. Reacting the latter exhibited broad-spectrum antitumor activity against
with the selected aromatic aldehyde, acetophenone or 4- most of the tested sub-panel tumor cell lines while com-
chloroacetophenone in refluxing ethanol yielded the cor- pounds 3c and 4c were of narrow spectrum of activity
responding 2-(arylidenehydrazinocarbonylmethylthio)-3- (Table 1). With regard to sensitivity against individual
phenylquinoxalines 7a, b and 2-[1-(arylethylidene)-hydra- cell lines, compounds 3b and 4b proved to be sensitive
zinocarbonylmethylthio]-3-phenyquinoxaline derivatives against most of the tested cell lines. Compound 3b
7c, d. Refluxing 6 with the selected arylisothiocyanate in a showed high sensitivity against lung cancer HOP-62 and
mixture of ethanol and DMF resulted in the formation of colon cancer HCT-116 with GI50 of 5.28 and 5.24 lM and
the corresponding 2-(N-arylthiocarbamoylhydrazinocar- TG1 values of 20.4 and 24.0 lM, respectively. However,
bonylmethylthio)-3-phenylquinoxalines (8a, b). Treat- compound 4b exhibited a super sensitivity profile
ment of the latter with cold concentrated sulfuric acid towards breast cancer HS-578T with GI50 value lying in
gave 2-[(5-substituted-1,3,4-thiadiazol-2-yl)methylthio]-3-
the nanomolar range (GI50 a 0.01 lM and TG1 of 6.88 lM).
phenylquinoxalines 9a, b. Cyclodesulfurazation of 8a, b The compound also showed significant activity against
with freshly prepared yellow mercuric oxide in boiling breast cancer B-549 (GI50 = 1.33 and TGI = 53.1 lM). On
dioxane afforded the respective 2-[(5-substituted-1,3,4- the other hand, compounds 3c and 4c showed remark-
oxadiazol-2-yl)methylthio]-3-phenylquinoxalines 10a, b. able activity against some of the tested cell lines. Com-
Cyclocondensation of 8a, b with ethyl chloroacetate in pound 3c exhibited high activity against ovarian cancer
refluxing ethanol yielded 2-[(3-substituted-4-oxo-thiazoli- OVCAR-4 (GI50 = 1.96 and TG = 15.7 lM). In addition, com-
din-2-ylidene)-hydrazinocarbonylmethylthio]-3-phenyl-
pound 4c showed moderate activity against all leukemia
quinoxalines 11a, b. On the other hand, cyclocondensa- cell lines with GI50 values of 1.80 to 3.83 lM and TC1
tion of 8a, b with phenacyl or 4-chlorophenacylbromide values of 10.3 to 18.8 lM.
gave the corresponding 2-[(3,4-disubstituted-2,3-dihy-
drothiazol-2-ylidene)-hydrazinocarbonylmethylthio]-3-
phenylquinoxaline hydrobromides (12a–d).
The LC50 (cytotoxicity) values were A100 lM for most
tested cell lines. The ratio obtained by dividing the com-
pounds' full panel MG-MID (mM) by its individual sub-
panel MG-MID (lM) is considered as a measure of com-
pound selectivity. Ratios between 3 and 6 refer to moder-
ate selectivity, ratios A6 indicate high selectivity towards
Biology
Antitumor activity
Antitumor activity tests were performed at the National the corresponding cell line, while compounds meeting
Cancer Institute (NCI), Bethesda, MD, USA. Eight of the neither of these criteria are rated non-selective [31]. The
synthesized compounds (3b, 3c, 4b, 4c, 7b, 7d, 12b, and tested compounds 3b, 3c, and 4b proved to be non-selec-
12d) were selected at the NCI and subjected to the NCI in tive with a broad spectrum of activity, while compound
vitro-disease-oriented human cells screening panel assay 4c showed moderate selectivity towards leukemia cell
[29–31]. About 60 cell lines of nine tumor sub-panels lines. Its selectivity ratio was 5.72 (Table 4).
were incubated with five concentrations (0.01–100 lM)
The obtained screening results would indicate that the
for each compound and were used to create log-concen- antitumor activity was only associated with 2-(N-arylcar-
tration vs.%-growth inhibition curves. Three response bamoylmethylthio)-3-phenylquinoxalines (3b, c) and 2-(N-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 437–447
Anticancer and Antimicrobial Quinoxalines
441
Table 1. Growth inhibitory action (GI50) of some selected in vitro tumor cell lines (lM)a).
Compd. NCS
No.
Panel
Sub-panel cell lines (cytotoxicity GI50 lM)
3b
3c
4b
734278
Lung Cancer
Colon Cancer
CNS cancer
HOP-62 (5.28), HOP-92 (15.40), NCI-H 226 (16.70), NCI-H460 (11.80), NCI-H 522 (15.30).
HCT-116(5.24), HT29 (25.10).
SF-268 (15.50), SF-295 (17.50), SF-539 (16.30) SNB-75 (18.80), U 251 (23.60)
SK-MEL-5 (19.30), UACC-62 (20.70), M 14 (23.70).
OVCAR-3 (13.10), OVCAR-4 (13.60), OVCAR-8 (25.50).
786-0 (16.00), ACHN (12.30), CAKI-1(12.50), RXF-393 (19.10), SN 12C (22.60).
NCI/ADR-RES (24.90), HS 578T (16.10), T-47D (13.00).
Melanoma
Ovarian Cancer
Renal Cancer
Breast Cancer
735806
734280
Lung Cancer
Colon Cancer
CNS Cancer
HOP-92 (20.30), NCI-H522 (24.20).
HTC-116 (14.10), HT29 (21.50).
SF-539 (18.60), SNB-75 (21.60).
MALME-3M (17.10), SK-MEL-5 (16.50).
OVCAR-4 (1.96)
Melanoma
Ovarian Cancer
Renal Cancer
Breast Cancer
RXF-393 (18.60)
BT-549 (24.60), T-47D (19.20).
Leukemia
CCRF-CEM (23.40), MOLT-4 (24.40), RPMI-8226 (20.60), SR (18.70).
HOP-92 (12.30), NCI-H460 (18.60), NCI-H522 (18.60), HOP-62(23.20).
SF-295 (15.70), SF-539 (14.00), U251 (21.80).
LOX IMVI (17.50), MALME-3M (19.10), SK-MEL-5 (15.20), UACC-62 (16.00).
OVCAR-3 (21.50).
A498 (19.00), ACHN (21.40), CAKI-1 (20.10), SN12C (18.20).
HS 578T (a 0.01), BT-549 (1.33),MDA-MB-231/ATCC (18.50), MDA-MB-435 (18.10).
HCT-116 (16.00), HCT-15 (21.20), KM12 (21.70), COLO 205 (23.40).
Lung Cancer
CNS Cancer
Melanoma
Ovarian Cancer
Renal Cancer
Breast Caner
Colon Cancer
4c
735807
Leukemia
CCRF-CEM (3.15), HL-60 (TB) (1.80), K-562 (3.83).
HOP-92 (22.30)
HCC-2998 (13.30), HCT-116 (25.70)
SF-539 (18.70)
Lung Cancer
Colon Cancer
CNS Cancer
Melanoma
SK-MEL-5 (23.90)
Ovarian Cancer
Renal Cancer
Prostate Caner
Breast Cancer
OVCAR-4 (21.60).
A498 (21.60)
PC-3 (26.00)
HS 578T (29.70), BT-549 (29.10).
a)
Data obtained from NCI in vitro disease-oriented human cell screen.
Table 2. Median growth inhibitory concentration (GI50, lM) of in vitro sub-panel tumor cell lines.
Compd.
No.
Sub-panel tumor cell linesa)
MG-MIDb)
I
II
III
IV
V
VI
VII
VIII
IX
3b
3c
4b
4c
82.40
64.05
21.78
9.47
30.72
51.36
23.78
77.33
50.25
46.14
26.60
36.47
21.27
32.62
35.80
74.57
42.70
60.04
25.90
66.66
46.25
39.56
39.53
70.08
25.65
46.84
23.55
76.83
43.00
44.95
25.90
31.05
27.44
41.90
19.39
44.97
41.08
47.50
26.91
54.16
a)
I: Leukemia; II: non-small cell lung cancer; III: colon cancer; IV: CNS cancer; V: melanoma; VI: ovarian cancer; VII: renal cancer;
VIII: prostate cancer; IX: breast cancer.
GI50 full panel mean-graph midpoint (lM).
b)
arylcarbamoylmethylsulfonyl)-3-phenylquinoxalines
with narrow spectrum of activity (3c and 4c, Table 1). Oxi-
(4b, c). The results also revealed that the substitution at dation of 2-[(N-arylcarbamoyl)methylthio]quinoxalines
position 4 of the phenyl group of the N-arylcarbamoyl 3a–d to the corresponding methylsulfonyl derivatives
moiety with a chlorine atom yielded compounds with a 4a–d increased activity and selectivity against some of
broad-spectrum anticancer activity (3b and 4b), while the tested sub-panel cell lines. For example, compound
substitution with a fluorine atom yielded derivatives 4b exhibited significant activity against breast cancer
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Table 3. Median total growth inhibitory (TGI) concentrations (lM) of the in vitro sub-panel tumor cell lines and TG1 full panel mean-
graph midpoints (MG-MID).
Compd.
No.
Sub-panel tumor cell linesa)
MG-MIDb)
I
II
III
IV
V
VI
VII
VIII
IX
3b
3c
4b
4c
–c)#
–
58.31
36.43
71.79
95.34
72.70
–
89.14
89.31
68.49
89.23
66.68
86.8
83.08
93.37
97.25
88.6
61.83
99.33
90.05
85.95
91.78
91.67
72.43
96.06
65.50
–
–
–
–
–
94.50
89.76
70.53
99.19
86.87
92.45
75.80
89.92
a)
For sub-panel tumor cell lines see foot note (a) of Table 2.
TG1 (lM) full panel mean-graph midpoint (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 for the active compounds towards the nine tumor cell lines.
Compd.
No.
Sub-panel tumor cell linesa)
I
II
III
IV
V
VI
VII
VIII
IX
3b
3c
4b
4c
0.50
0.74
1.24
5.72
1.34
0.93
1.13
0.70
0.82
1.03
1.01
1.49
1.93
1.46
0.75
0.73
0.96
0.79
1.04
0.81
0.89
1.20
0.68
0.77
1.60
1.01
1.14
0.71
0.96
1.06
1.04
1.74
1.50
1.13
1.39
1.20
a)
For sub-panel tumor cell lines see footnote (a) of Table 2.
Table 5. The inhibition zones (IZ) in mm diameter of the most
active compounds.
sub-panel cell lines especially HS 587T (GI50 a 0.01 lM)
and compound 4c showed moderate selectivity towards
all leukemia sub-panel cell lines (GI50 values, 1.8–3.8 lM).
Its selectivity ratio was 5.72 (Table 4).
Compd. S. aureus B. sub-
P. aeru-
genosa
E. coli C. albi-
cans
No.
tilis
3d
4a
7a
7b
8a
10b
11a
11b
12
13
14
14
19
14
15
17
12
14
14
16
14
14
14
18
15
15
18
19
16
14
15
14
13
14
14
14
14
19
23
24
16
14
21
20
14
22
13
14
Antimicrobial activity
All the newly synthesized compounds were preliminary
evaluated for their in vitro-antibacterial activity against S.
aureus and B. subtilis as Gram-positive bacteria; E. coli and
P. aerugenosa as Gram-negative bacteria. The compounds
were also evaluated for their in vitro-antifungal activity
against C. albicans. Their inhibition zones using the cup
diffusion technique [32] were measured, further evalua-
tion was then carried out on compounds showing reason-
able inhibition zones (A13 mm) to determine their mini-
mal inhibitory concentration (MIC) and minimal bacteri-
cidal concentration (MBC) using the two-fold serial dilu-
tion method [33]. Ampicillin was used as standard anti-
bacterial while clotrimazole was used as antifungal refer-
ence. Dimethylsulfoxide (DMSO) was used as a blank and
showed no antimicrobial activity.
As revealed from Tables 5 and 6, eight compounds (3d,
4a, 7a, b, 8a, 10b, and 11a, b) showed promising antimi-
crobial activity. Compounds 7a, 7b, 8a, 11a, and 11b
exhibited significant activity against B. subtilis. They were
as active as ampicillin (MIC = 12.5 lg/mL). Compounds 7b
and 8a also were nearly as active as ampicillin against E.
coli (MIC = 12.5 lg/mL). In addition, 4a, 7b, 10b, and 11a
were also as active as ampicillin against P. aerugenosa
(MIC = 50 lg/mL). On the other hand, compounds 3d, 4a,
and 10b showed moderate activity towards B. subtilis.
They displayed half the activity of ampicillin
(MIC = 25 lg/mL). Compounds 4a, 10b, and 11a also
exhibited half the activity of ampicillin towards E. coli
(MIC = 25 lg/mL). However, the test compounds exhib-
ited mild antimicrobial activity against S. aureus and C.
albicans.
According to the MIC and MBC limits derived from the
latest National Committee on Clinical Laboratory Stan-
dards (NCCLS), we can determine whether the test com-
pound is bactericidal or bacteriostatic to the test organ-
ism. If the MBC = MIC, the test compound is considered a
bactericidal but if MBC > MIC the test compound is con-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 437–447
Anticancer and Antimicrobial Quinoxalines
443
Table 6. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the most active compounds in
lg/mL.
Compd.
No.
S. aureus
MBC
B. subtilis
P. aerugenosa
E. coli
MBC
C. albicans
MIC
MIC
MBC
MIC
MBC
MIC
MIC
MBC
3d
4a
7a
7b
8a
10b
11a
11b
100
100
50
50
50
25
25
100
5
100
200
50
100
50
50
25
100
25
25
50
25
25
25
25
25
25
25
100
50
100
50
100
50
50
100
100
100
50
100
100
50
50
25
50
12.5
12.5
25
25
50
100
50
100
25
25
25
100
100
100
50
50
50
200
100
100
50
100
100
200
200
12.5
12.5
12.5
25
12.5
12.5
12.5
25
100
100
100
100
50
200
Ampicillin
Clotrimazol
10
5
sidered a bacteriostatic. Accordingly, as revealed from Reacting 6 with aryl isothiocyanates yielded active com-
Table 6, compounds 7a, 8a, and 11a were bactericidal pounds 8, of which compound 8a exhibited significant
against S. aureus, compounds 4a and 10b were bacterici- activity. It was as active as ampicillin towards B. subtilis, E.
dal against B. subtilis; 10b and 11a were also bactericidal coli and had half the activity against P. aerugenosa. Cycli-
against E. coli. In addition, compound 7b and 11a were zation of 8a, b to the substituted 1,3,4-thiadiazole deriv-
bactericidal against P. aerugenosa, while compound 7b atives 9a, b abolished the antimicrobial activity. While
was fungicidal against C. albicans. The remaining com- cyclodesulfurization of 8a, b afforded the corresponding
pounds were bacteriostatic against the test organisms.
1,3,4-oxadiazole analogs 10a, b with promising activity.
The above-mentioned results revealed that compounds Compound 10b was the most active. It had the same activ-
7b, 10b and 11a exhibited a broad spectrum of antimicro- ity as ampicillin against P. aerugenosa and half the activ-
bial activity and were devoid of cytotoxic activity (they ity towards B. subtilis and E. coli. Cyclocondensation of
were devoid of antitumor activity).
8a, b with ethyl bromoacetate gave the corresponding
The activity of the test compounds could be correlated substituted 4-oxothiazolidin-2-ylidene derivatives (11a, b)
to the structure variations and modifications. The with increased activity. Compound 11a was as active as
obtained screening results revealed that 2-[(N-arylcarba- ampicillin towards B. subtilis and P. aerugenosa and had
moyl)methylthio]quinoxalines 3 exhibited antimicrobial half the activity against E. coli. It exhibited moderate
activity. Maximum activity was observed when position 4 activity against S .aureus and C. albicans. On the other
of the phenyl group was substituted with methyl group hand, cyclocondensation with phenacyl bromide or 4-
3d. The compound had half the activity of ampicillin chlorophenacylbromide yielded the respective 3,4-diaryl-
against B. subtilis and P. aerugenosa. Oxidation of such substituted-2,3-dihydrothiazol-2-ylidene derivatives (12a-
compounds to the corresponding 2-(N-arylcarbamoyl)- d) which were devoid of activity.
methylsulfonyl analogs 4 increased the activity against
the Gram-negative bacteria. Compound 4a was the most
active member. It had the same activity as ampicillin
towards P. aerugenosa and half the activity against E. coli.
Experimental
2-(Hydrazinocarbonylmethylthio)-3-phenylquinoxaline 6
did not show any antimicrobial activity. Condensation of
6 with aromatic aldehydes afforded the hydrazones 7a, b
with significant activity against B. subtilis, P. aerugenosa,
E. coli and moderate activity towards S. aureus and C. albi-
cans. When position 4 of the phenyl group of hydrazone
was substituted with a chlorine atom 7b, an increase in
the activity was observed. This compound was as active as
ampicillin against B. subtilis, P. aerugenosa, and E. coli. On
the other hand, condensation of 6 with acetophenone or
4-chloroacetophenone gave inactive compounds 7c, d.
Chemistry
All melting points were determined in open-glass capillaries on
a Gallenkamp melting point apparatus (Sanyo) and are uncor-
rected. The IR spectra were recorded using KBr discs on a Perkin-
Elmer 1430 spectrophotometer (Perkin-Elmer, Norwalk, CT,
1
USA). H-NMR (d ppm) spectra were recorded on a Jeol spectro-
meter (JEOL, Tokyo, Japan) at 500 MHz using TMS as an internal
standard and DMSO-d6 as a solvent. The mass spectra (MS) were
run on a Finnigan mass spectrometer (Thermo Electron Corp.)
model SSQ/7000 (70 eV). The microanalyses were performed at
the Microanalytical Laboratory, National Research Center,
Cairo, Egypt and the data were within l0.4% of the theoretical
values. Following up of the reactions and checking the homoge-
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444
S. A. M. El-Hawash et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 437–447
neity 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 few seconds.
Table 7. Physical and analytical data of the synthesized com-
pounds 3–12.
Compd.
No.
R
R1
Mp. (8C)
cryst. solvent (%)
Yield Mol. Formulaa)
Mol. Wt.
2-(N-Arylcarbamoylmethylthio)-3-phenylquino-
3a
3b
3c
H
–
211–212
DMF/EtOH
216–217
DMF/EtOH
202–203
EtOH
202–203
DMF/EtOH
189–190
EtOH
214–215
EtOH
191–192
EtOH
201–202
EtOH
138–139
EtOH
96
92
78
68
67
85
72
67
82
92
72
93
92
72
97
95
96
98
52
97
40
48
82
73
86
60
C22H17N3OS
371.46
C22H16ClN3OS
405.91
C22H16FN3OS
389.45
C23H19N3OS
385.49
C22H17N3O3S
403.46
C22H16ClN3O3S
437.91
C22H16FN3O3S
421.45
C23H19N3O3S
417.49
C18H16N2O2S
324.40
C16H14N4OS
310.38
C23H18N4OS
398.49
C23H17ClN4OS
432.49
C24H20N4OS
412.52
C24H19ClN4OS
446.96
C23H19N5OS2
445.57
C24H21N5OS2
459.60
C23H17N5S2
427.55
C24H19N5S2
441.58
C23H17N5OS
411.49
C24H19N5OS
425.52
C25H19N5O2S2
485.59
C26H21N5O2S2
499.62
C31H23N5OS2 N HBr
626.61
C31H22ClN5OS2 N HBr
661.05
xalines 3a–d
Cl
–
A
mixture of 3-phenylquinoxaline-2(1H)-thione 2 (0.44 g,
2 mmol), the appropriate N-arylchloroacetamide (2 mmol) and
anhydrous sodium acetate (0.49 g, 6 mmol) in absolute ethanol
was refluxed for 1 h. The mixture was cooled, the white product
separated was filtered, washed with water, dried, and crystal-
lized from the appropriate solvent (Table 7). IR (KBr, cm– 1): 3242
(NH), 1691–1690 (C=O), 1648 (C=N), 1610, 1595 (C=C), 1533 d NH,
1245–1244, 1085 (C-S-C). 1H-NMR of 3d (d ppm): 2.2 (s, 3H, CH3),
4.17 (s, 2H, CH2), 7.06, 7.46 (two d, each 2H, J = 8.4 Hz, C6H4-CH3),
7.55–7.56 (m, 3H, C6H5-C3,4,5-H), 7.69 (t, 1H, J = 7.65 Hz, quinox.
C6-H), 7.74–7.77 (m, 3H, C6H5-C2,6-H and quinox. C7-H), 7.88 (d, J =
8.4 Hz, 1H, quinox. C5-H), 8.01 (d, J = 7.65 Hz, 1H, quinox. C8-H),
10.36 (s, 1H, NH, D2O exchangeable).
F
–
3d
4a
CH3
H
–
–
4b
4c
Cl
–
F
–
4d
5
CH3
–
–
–
6
–
–
195–196
EtOH
222–223
DMF-EtOH
2-(N-Arylcarbamoylmethylsulfonyl)-3-phenylquino-
xalines 4a–d
7a
H
C6H5
A solution of 5% potassium permanganate was added dropwise
to a stirred suspension of the appropriate 3a–d (3 mmol) in gla-
cial acetic acid (10 mL) till the pink color persisted. Stirring was
continued at room temperature over night, then poured onto
cooled sodium sulfite solution. The product was filtered, washed
with water, dried, and crystallized from the appropriate solvent
(Table 7). IR (KBr, cm– 1): 3330–3267 (NH), 1673–1659 (C=O),
1603–1601; 1515–1497 (C=C), 1548-1545 (d NH), 1325–1319,
1157–1147 (SO2), 1273–1262, 1096–1093 (C-S-C). 1H-NMR of 4d
(d ppm): 2.18 (s, 3H, CH3), 4.85 (s, 2H, CH2), 7.03, 7.28 (two d, each
2H, J = 8.4 Hz, C6H4-CH3), 7.49–7.53 (m, 3H, C6H5-C3,4,5-H), 7.74 (d,
2H, J = 6.9 Hz, C6H5-C2,6-H), 8.01 (t, 1H, J = 7.65 Hz, quinox. C6-H),
8.07 (t, 1H, J = 7.65 Hz, quinox. C7-H), 8.19 (d, 1H, J = 7.65 Hz, qui-
nox. C5-H), 8.23 (d, 1H, J = 7.65 Hz quinox. C8-H), 10.32 (s, 1H, NH,
D2O exchangeable). MS of 4b m/z (relative abundance%): [M+9] at
437 absent, 375, 373 [M+-SO2] (2.6, 4.7), 330 (28.9), 247 (25.6), 204
(100), 127(39.6), 77 (88.3).
7b
7c
H
4-ClC6H4 236–237
DMF-EtOH
C6H5
CH3
CH3
H
201–202
DMF-EtOH
7d
8a
4-ClC6H4 223–224
DMF-EtOH
–
209–210
DMF/EtOH
208–209
DMF/EtOH
246–247
DMF/H2O
236–237
DMF-H2O
206–208
DMF-EtOH
207–208
DMF-H2O
151–152
EtOH
234–235
EtOH
234–235
EtOH
237–238
EtOH
236–237
DMF-EtOH
244–245
DMF-EtOH
8b
9a
CH3
H
–
–
9b
10a
10b
11a
11b
12a
12b
12c
12d
CH3
H
–
–
CH3
H
–
–
2-Ethoxycarbonylmethylthio-3-phenylquinoxaline 5
Method A
To a stirred mixture 1 (2.4 g, 10 mmol) and anhydrous potas-
sium carbonate (1.4 g, 10 mmol) in dry acetone (40 mL), ethyl
thioglycolate (1.2 g, 10 mmol) was added. The reaction mixture
was refluxed for 6 h, cooled, and poured onto ice-water. The pre-
cipitate formed was filtered, dried, and crystallized from etha-
nol.
CH3
H
–
H
Cl
H
Cl
H
CH3
CH3
C32H25N5OS2 N HBr
640.05
C32H24ClN5OS2 N HBr
675.08
Method B
a)
Analyzed for C, H, N; the results are within l0.4% of the theo-
retical values.
A mixture of 2 (2.38 gm, 10 mmol), ethyl chloroacetate (1.23 g,
10 mmol) and anhydrous sodium acetate (2.87 g, 30 mmol) in
absolute ethanol (50 mL), was heated under reflux for 2 h. The
mixture was cooled and poured onto ice-water. The product was
filtered, washed with water, dried, and crystallized from etha-
nol. IR (KBr, cm– 1): 1739 (C=O), 1642 (C=N), 1607, 1480 (C=C),
1243, 1151, 1030 (C-O-C), 1088 (C-S-C). 1H-NMR (dppm): 1.17 (t, 3H,
J = 7.6 Hz, CHH CH ), 4.08 (s, 2H, CH2CO), 4.12 (q, 2H, J = 7.6 Hz,
nox. C6-H), 7.73–7.75 (m, 2H, C6H5-C2,6-H), 7.78 (t, 1H, J = 8.4 Hz,
quinox. C7-H), 7.84 (d, 1H, J = 8.4 Hz, quinox. C5-H), 8.02 (d, 1H, J =
8.4 Hz, quinox. C8-H).
2
3
CH2CH3), 7.55-7.56 (m, 3H, C6H5-C3,4,5-H), 7.72 (t, 1H, J = 8.4 Hz, qui-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 437–447
Anticancer and Antimicrobial Quinoxalines
445
and crystallized from the proper solvent (Table 7). IR (KBr, cm– 1):
3254–3244, 3194–3188 (NH), 1613–1602, (C=N), 1516–1502
(C=C), 1549–1534 (d NH), 1244–1241, 1087–1085 (C-S-C). 1H-
NMR of 9b (d ppm): 2.19 (s, 3H, CH3), 4.77 (s, 2H, CH2), 7.06, 7.40
(two d, each 2H, J = 8.4 Hz, C6H4-CH3), 7.51–7.79 (m, 5H, C6H5),
7.87 (t, 1H, J = 8.4 Hz, quinox. C6-H), 8.00 (t, 1H, J = 8.4 Hz, quinox.
C7-H), 8.07 (d, 1H, J = 8.4 Hz quinox. C5-H), 8.08 (d, 1H, J = 7.65 Hz,
quinox. C8-H), 10.07 (s, 1H, NH, D2O exchangeable). MS of 9b m/z
(relative abundance%): 441 [M+., (38.6)], 407 (42.0), 333 (29.5), 237
(100), 204 (53.4), 150 (50.0), 91 (100), 77 (94.3).
2-(Hydrazinocarbonylmethylthio)-3-phenylquinoxaline 6
To a suspension of 5 (3.24 g, 10 mmol) in absolute ethanol
(50 mL), hydrazine hydrate (98%) (5 g, 100 mmol) was added and
the mixture was stirred at room temperature for 24 h. The prod-
uct was filtered, washed with water, dried, and crystallized from
ethanol. IR (KBr, cm– 1): 3298, 3257, 3115 (NH), 1639 (C=O, C=N),
1534 (d NH), 1499, 1483 (C=C), 1242, 1085 (C-S-C). 1H-NMR (d
ppm): 3.93 (s, 2H, CH2), 4.25 (s, 2H, NH2, D2O exchangeable),
7.52–7.57 (m, 3H, C6H5-C3,4,5-H), 7.71 (t, 1H, J = 7.65 Hz, quinox.
C6-H), 7.72–7.76 (m, 2H, C6H5-C2,6-H), 7.79 (t, 1H, J = 7.65 Hz, qui-
nox. C7-H), 7.95 (d, 1H, J = 7.65 Hz, quinox. C5-H), 8.04 (d, 1H, J =
7.65 Hz, quinox. (C8-H), 9.34 (s, 1H, NH, D2O exchangeable).
2[-(5-Arylamino-1,3,4-oxadiazol-2-yl)methylthio]-3-
phenylquinoxalines 10a, b
2-(N-Arylidenehydrazinocarbonylmethylthio)-3-
phenylquinoxalines 7a, b and 2-[(1-
arylethylidene)hydrazinocarbonylmethylthio]-3-
A mixture of 8a or 8b (2 mmol) and freshly prepared yellow HgO
(0.42 g, 1 mmol) in dry dioxane (20 mL) was heated under reflux
for 4 h. The mixture was filtered, the filtrate was evaporated
under reduced pressure, and the residue was crystallized from
the proper solvent (Table 7). IR (KBr, cm– 1): 3228, 3178, 3122
(NH), 1642 (C=N), 1575 (d NH), 1517, 1482 (C=C), 1273, 1083 (C-S-
phenylquinoxalines 7c, d
To a suspension of 6 (0.31 g, 1 mmol) in ethanol (10 mL), the
appropriate aldehyde or ketone (1 mmol) was added. The mix-
ture was refluxed for 1 h. then cooled, filtered, dried, and crys-
tallized from the proper solvent (Table 7). IR (KBr, cm– 1): 3183–
3172 (NH), 1675–1670 (C=O), 1616–1606 (C=N), 1568, 1519–
1518, 1490–1485 (C=C), 1535-1534 (d NH), 1224–1221, 1089–
1088 (C-S-C). 1H-NMR of 7b (d ppm): 4.55 (s, 2H, CH2), 7.43 (d, 2H, J
= 8.4 Hz, C6H4-Cl C3,5-H), 7.45–7.79 (m, 7H, C6H5 and quiox. C6,7-H),
7.81 (d, 1H, J = 7.65 Hz, quinox. C5-H), 7.91 (d, 1H, J = 8.4, quinox.
C8-H), 8.02 (d, 2H, J = 8.4 Hz, C6H4-Cl C2,6-H), 8.24 (s, 1H, CH=N),
11.68 (s, 1/2 H, NH, D2O exchangeable), 11.88 (s, 1/2 H, OH, enolic).
1H-NMR of 7d (d ppm): 2.25 (s, 3H, CH3), 4.59 (s, 2H, CH2), 7.39,
7.79 (two d, each 2H, J = 8.4 Hz C6H4-Cl), 7.41–7.81 (m, 7H, C6H5
and quiox. C6,7-H), 7.92 (d, 1H, J = 7.65 Hz, quinox. C5-H), 8.01 (d,
1H, J = 7.65 Hz, quinox. C8-H), 10.81 (s, 1/2 H, NH enolic, D2O
exchangeable), 10.89 (s, 1/2 H, OH, D2O exchangeable). MS of 7d
m/z (relative abundance%): [M+.] at 446 absent, 279 [M+-
C6H11N2OS, (75.4)], 278 (100), 250 (60.1), 204 (24.6), 151 (10.4), 102
(27.2), 77 (49).
1
C), 1247, 1058 (C-O-C). H-NMR of 10b (d ppm): 2.18 (s, 3H, CH3),
4.73 (s, 2H, CH2), 7.03, 7.32 (two d, each 2H, J = 8.4 Hz, C6H4-CH3),
7.54-7.73 (m, 5H, C6H5), 7.74 (t, 1H, J = 8.4 Hz, quinox.C6-H), 7.81
(t, 1H, J = 8.4 Hz, quinox. C7-H), 7.97 (d, 1H, J = 8.4 Hz, quinox. C5-
H), 8.04 (d, 1H, J = 8.4 Hz, quinox. C8-H), 10.28 (s, 1H, NH, D2O
exchangeable). MS of 10b m/z (relative abundance%): 425 [M+.,
(14.0)], 318 (100), 276 (18.7), 236 (70.8), 134 (56.2), 77 (86.2).
2-[(3-Aryl-4-oxothiazolidin-2-
ylidene)hydrazinocarbonylmethylthio]-3-
phenylquinoxalines 11a, b
A mixture of equimolar amounts of 8a or 8b (1 mmol) and ethyl
chloroacetate in absolute ethanol (10 mL) was heated under
reflux for 6 h. The mixture was cooled to room temperature, the
crystalline product was filtered, dried, and recrystallized from
ethanol (Table 7). IR (KBr, cm– 1) of 11a: 3188 (NH), 1755 (C=O),
1661 (C=O), 1644 (C=N), 1608, 1517 (C=C), 1531 (d NH), 1276,
1087 (C-S-C). 1H-NMR of 11a (d ppm): 4.02 (s, 2H-thiazolidinone C5-
H), 4.63 (s, 2H, CH2), 7.22, 7.36 (two t, each 1H, J = 7.65 Hz, quinox.
2-(N-Arylthiocarbamoylhydrazinocarbonylmethyl-
thio)-3-phenylquinoxalines 8a, b
C6,7-H), 7.41, 7.97 (two d, each 1H, J = 7.65 Hz, quinox. C5,8-H),
7.51–7.68 (m, 10H, Ar-H), 10.40 (s, 1H, NH, D2O exchangeable). IR
of 11b (KBr, cm-1): 3168 (NH), 1731 (C=O), 1678 (C=O), 1660 (C=N),
1602, 1513 (C=C), 1535 (d NH), 1242, 1084 (C-S-C). 1H-NMR of 11b
(d ppm): 1.9 (s, 3H, CH3), 4.02 (s, 2H, thiazolidinone C5-H), 4.67 (s,
2H, CH2), 7.01, 7.2 (two d, each 2H, J = 8.00 Hz, C6H4-CH3), 7.5–8.0
(m, 9H, C6H5-H+ quinox. C5,6,7,8-H); 10.41 (s, 1H, NH, D2O exchange-
able).
A mixture of equimolar amounts of 6 (3.1 gm, 10 mmol) and the
appropriate arylisothiocyanate in absolute ethanol and DMF
(3:1, 4 mL) was heated under reflux for 3 h. The reaction mix-
ture cleared, then a yellow crystalline product was separated.
The mixture was cooled, filtered, washed with ethanol, dried,
and recrystallized from the proper solvent (Table 7). IR (KBr,
cm–1): 3316–3312, 3270–3236, 3186–3179 (NH), 1654–1649
(C=O), 1620–1613 (C=N), 1513–1498 (C=C), 1566–1557, 1272–
1268, 1086–1085, 987–986 (N-C=S), 1238–1235, 1046–1030 (C-
S-C). 1H-NMR of 8b (d ppm): 2.24 (s, 3H, CH3), 4.09 (s, 2H, CH2), 7.08
2-[(3,4-Disubstituted-2,3-dihydrothiazol-2-ylidene)-
hydrazinocarbonyl-methylthio]-3-phenylquinoxaline
hydrobromides 12a-d
(d, 2H, J = 8.4 Hz, CH3-C6H4-C3,5-H), 7.18 (dist. d, 2H, CH3C6H4 C2,6
-
H), 7.55-7.56 (m, 3H, C6H5-C3,4,5-H), 7.73 (t, 1H, J = 6.9 Hz, qui-
nox.C6-H), 7.74–7.76 (m, 3H, C6H5, C2,6-H and quinox. C7-H), 8.01,
8.02 (two d, 2H, J = 8.0 Hz, quinox. C5,8-H), 9.47, 9.67, 10.33 (three
s, 3H, 3 NH, D2O exchangeable).
A mixture of equimolar amounts of 8a or 8b (1 mmol) and phen-
acyl bromide or 4-chlorophenacyl bromide in dry dioxane
(10 mL) was heated under reflux for 2 h. The reaction mixture
cleared, then a white fluffy product was formed. The mixture
was then cooled, filtered by suction, dried, and crystallized from
the proper solvent (Table 7). IR (KBr, cm– 1): 3391–3390 (NH),
1723–1706 (C=O), 1643–1642 (C=N), 1612–1597, 1573–1570,
1519–1509 (C=C), 1537–1535 (d NH), 1282–1252, 1086–1084
(C-S-C). 1H-NMR of 12c (d ppm): 2.25 (s, 3H, CH3), 4.04, 4.21 (two d,
each 1H, J = 15.3 Hz, CH2), 6.89, 6.98 (two t, each 1H, J = 7.60 Hz,
2-[(5-Arylamino-1,3,4-thiadiazol-2-yl)methylthio]-3-
phenylquinoxalines 9a, b
A solution of 8a or 8b (1 mmol) in cold conc. H2SO4 (3 mL) was
stirred at room temperature. The mixture was poured onto
crushed ice, the product was filtered, washed with water, dried,
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446
S. A. M. El-Hawash et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 437–447
quinox. C6,7-H), 6.94 (s, 1H, thiazolidin-C5H), 7.28–7.74 (m, 15 H,
two C6H5, p-tolyl and quiox. C5-H), 8.00–8.02 (dist d, 1H, quinox.
C8-H), 11.94 (s, 1H, NH, D2O-exchangeable). 1H-NMR of 12d (d
ppm): 2.33 (s, 3H, CH3), 4.01, 4.25 (two d, each 1H, J = 14.5 Hz,
CH2), 6.87, 7.33 (two d, each 2H, J = 8.4 Hz, p-tolyl), 6.88 (s, 1H,
thiazolidene C5-H), 7.28–7.74 (m, 12H, C6H5, 4-ClC6H4 and qui-
nox. C5,6,7-H), 8.01 (dist d, 1H, quinox. C8-H), 11.97 (s, 1H, NH, D2O
exchangeable). MS of 12d m/z (relative abundance%): 594 [M+,
(2.5)], 593 (4.3), 592 (4.9), 343 (15), 341 (38.9), 299 (100), 238 (13.1),
236 (48.7), 204 (42.1), 167 (22.5), 133 (35.8), 77 (57.5).
incubation, the plates were examined for growth. Again, the
tube containing the lowest concentration of the test compound
that failed to yield growth on sub-culture plates was judged to
contain the MBC of that compound for the respective test organ-
ism (Table 6).
The authors are grateful to the staff of the Department of
Health and Human Services, National Cancer Institute,
Bethesda, Maryland, USA for carrying out the anticancer
screening of the newly synthesized compounds.
Biology
Antitumor activity
References
Eight of the prepared compounds were selected and tested for
their in vitro-antitumor activity against 60 human tumor cell
lines, derived from nine clinically isolated types of cancer (leuke-
mia, lung, brain, melanoma, colon, ovarian, renal, breast, and
prostate) following the National Cancer Institute (NCI) preclini-
cal antitumor drug discovery screen. Each compound was tested
at five, ten-fold dilutions, a 48 h continuous drug exposure pro-
tocol was used and with a sulforodamine B (SKB) protein assay
the cell viability or growth was estimated [29–31]. The results
are presented in Tables 1–4.
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All the synthesized compounds were evaluated by the agar cup
diffusion technique [32] using a 1mg/mL solution in DMSO. The
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