Synthesis, characterization, bioactivity, and POM analyses of isothiochromeno[3,4-e][1,2]oxazines

Article abstract of DOI:10.1007/s00044-012-0392-4

A series of 18 new 3,4-disubstituted-isothiochromeno[3,4-e][1,2]oxazines 28-45 has been obtained from the 3′,4′-di-substituted-4′H- spiro[isothiochromene-3,5′-isoxazol]-4(1H)-ones 10-27 in refluxing HCl acid/ethanol. A series of 15/18 compounds 28-45 was selected by the National Cancer Institute (NCI, Bethesda, USA) and were evaluated against a full panel of 60 primary human tumor cell lines derived from nine human cancer types, all of which showed antiproliferative activity in the micromolar range. The most active compound number 37 (S722910) showed high potency against all the tested cell lines with a GI₅₀ mean value in the range of 30-80 μM; TGI and LC₅₀ values were 12-16 μM having positive response on 98 and 63 % of the tested cell lines (Breast-MCF7 and NCS-SF-268) respectively. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-012-0392-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4798–4809
DOI 10.1007/s00044-012-0392-4
ORIGINAL RESEARCH
Synthesis, characterization, bioactivity, and POM analyses
of isothiochromeno[3,4-e][1,2]oxazines
•
Brahim Bennani Abdelali Kerbal Bouchra F. Baba
•
•
•
Maria Daoudi Ismail Warad Mohamad Aljofan
•
•
•
Ahmed M. Alafeefy Vijay Masand Taibi B. Hadda
•
Received: 2 August 2012 / Accepted: 4 December 2012 / Published online: 26 January 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of 18 new 3,4-disubstituted-iso-
thiochromeno[3,4-e][1,2]oxazines 28–45 has been obtained
from the 30,40-di-substituted-40H-spiro[isothiochromene-
3,50-isoxazol]-4(1H)-ones 10–27 in refluxing HCl acid/
ethanol. A series of 15/18 compounds 28–45 was selected
by the National Cancer Institute (NCI, Bethesda, USA) and
were evaluated against a full panel of 60 primary human
tumor cell lines derived from nine human cancer types, all
of which showed antiproliferative activity in the micro-
molar range. The most active compound number 37
(S722910) showed high potency against all the tested cell
lines with a GI50 mean value in the range of 30–80 lM;
TGI and LC50 values were 12–16 lM having positive
response on 98 and 63 % of the tested cell lines (Breast-
MCF7 and NCS-SF-268) respectively.
Keywords Leukemia ꢀ Breast cancer inhibition ꢀ
Spiro-isothiochromeno[3,4-e][1,2]oxazines ꢀ
POM analyses
Abbreviation
GI50 Growthinhibition; the drugconcentration resultingin
a50 % reductioninthenetproteinincreasecompared
with control cells during the drug incubation
TGI
Cytostatic activity; the drug concentration resulting
in total growth inhibition
LC50 \The concentration of drug resulting in a 50 %
reduction in the measured protein at the end of the
drug treatment compared with that at the beginning,
thus indicating a net loss of cells following treatment
POM Petra/Osiris/Molinspiration
B. Bennani ꢀ A. Kerbal (&) ꢀ B. F. Baba ꢀ M. Daoudi
M. Aljofan
College of Pharmacy, Qassim University, Buraidah,
Saudi Arabia
´
Laboratoire de Chimie Organique, Faculte desz Sciences
Dhar El-Mehraz, Universite Sidi Mohammed Ben Abdellah,
´
30000 Fez, Morocco
e-mail: kerbal20@yahoo.fr
A. M. Alafeefy
Department of Pharmaceutical Chemistry, College of Pharmacy,
Salman Bin Abdulaziz University, P.O. Box 173,
Alkharj 11942, Saudi Arabia
T. B. Hadda (&)
Laboratoire Chimie des Materiaux, Faculte des Sciences
´
60000 Oujda, Morocco
e-mail: taibi.ben.hadda@gmail.com
´
d’Oujda, Universite Mohammed Premier,
´
V. Masand
Department of Chemistry, Vidya Bharati College,
Camp, Amravati, Maharashtra, India
I. Warad
Department of Chemistry, Science College, King Saud
University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
M. Aljofan
Department of Microbiology, School of Medicine, Nursing
and Health Sciences, Monash University, Caulfield East,
VIC, Australia
123

Med Chem Res (2013) 22:4798–4809
4799
Introduction
isoxazolidine compounds 10–27 and their new aromatic
planar derivatives 28–45 (Scheme 1).
Isoxazoline derivatives have been shown to be efficient
precursors for many synthetic intermediates including
c-amino alcohols and b-hydroxy ketones (Kozikowski,
1984; Kanemasa et al., 1990). Spiro-isoxazolines display
interesting biologic properties such as herbicidal, plant-
growth regulatory, and antitumor (Howe and Shelton,
1990; Smietana et al., 1999). Many 4-chromanone
derivatives are versatile intermediates for the synthesis of
natural products such as brazillin, hematoxylin, ripario-
chromene, clausenin, calonlide A, and inophyllum (Ellis
et al., 1997; Chenera et al., 1993). Chromanone hetero-
cycles have attracted much attention owing to their
important pharmacological properties (Ellis et al., 1997).
Their high synthetic utility and pharmacological impor-
tance have prompted us to synthesize and study the
antitumor activity of some previously prepared spiro-
Currently, we are working on the synthesis and
pharmacomodulation of spiro-heterocyclic compounds,
which have dual antibacterial/antiviral pharmacophores,
such as spiro-isoxazolines (Bennani et al., 2007a; 2004;
Al Houari et al., 2008), spiro-isoquinolines (Hadda
et al., 2007, 2008; Akkurt et al., 2006, 2010), and
spiro-isoxazolidin (Bennani et al., 2007b). This will
allow us to gain insight into the therapeutic behavior of
larger heterocyclic systems containing both isothiochr-
omane and isoxazol rings as potential therapeutic
agents, and delocalised p-conjugated rigid pharmaco-
phore site(s). The biologic activities of these com-
pounds and their derivatives were determined by High
Screening Techniques (Gray and Wickstrom, 1996) to
investigate their suitability as a new class of antitumor
agents.
O
O
N
O
N
O
+
R2
S
S
10-27
R2
H+
R1
R1
1-5
6-9
R2
H2O
1: R2 = H
6: R1 = H
2
N
3
4
1
2: R2 = CH3
3: R2 = OCH3
4: R2 = NO2
5: R2 = Cl
O
7: R1 = CH3
8: R1 = OCH3
9: R1 = NO2
10
7
9
S
5
R1
8
6
28-45
16, 34: (R1, R2) = (CH3, CH3)
10, 28: (R1, R2) = (H, H)
11, 29: (R1, R2) = (H, CH3)
12, 30: (R1, R2) = (H, OCH3)
13, 31: (R1, R2) = (H, NO2)
14, 32: (R1, R2) = (H, Cl)
15, 33: (R1, R2) = (CH3, H)
22, 40: (R1, R2) = (OCH3, OCH3)
23, 41: (R1, R2) = (OCH3, NO2)
24, 42: (R1, R2) = (OCH3, Cl)
25, 43: (R1, R2) = (NO2, CH3)
26, 44: (R1, R2) = (NO2, OCH3)
27, 45: (R1, R2) = (NO2, Cl)
17, 35: (R1, R2) = (CH3, OCH3)
18, 36: (R1, R2) = (CH3, NO2)
19, 37: (R1, R2) = (CH3, Cl)
20, 38: (R1, R2) = (OCH3, H)
21, 39: (R1, R2) = (OCH3, CH3)
Scheme 1 Synthesis of new compounds 28–45 from precursors 10–27
123

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Med Chem Res (2013) 22:4798–4809
Table 1 1H NMR data (d in ppm; J in Hz) of selected compounds of series 28–45
Compound
(R1, R2)
(H, H)
dR1
dR2
dH6
dH9
dH (Arom)
28
–
–
7.70
8.30
7.10–7.60
(s, 1H)
7.70
(dd, Jo = 9; Jm = 2)
(m, 13H)
7.15–7.60
(m, 12H)
7.10–7.60
(m, 11H)
6.80–7.60
(m, 11H)
7.15–8.20
(m, 11H)
7.15–8.30
(m, 11H)
33
34
35
36
45
(CH3, H)
2.50
–
8.35
(s, 3H)
2.40
(s, 1H)
7.70
(dd, Jo = 9.0; Jm = 1.0)
(CH3, CH3)
(CH3, OCH3)
(CH3, NO2)
(NO2, Cl)
2.50
8.30
(s, 3H)
2.45
(s, 3H)
3.80
(s, 1H)
7.7
(dd, Jo = 9.0; Jm = 1.0)
8.30
(s, 3H)
2.50
(s, 3H)
–
(s, 1H)
7.75
(dd, Jo = 9.0, Jm = 1.0))
8.30
(s, 3H)
–
(s, 1H)
7.80
(dd, Jo = 9.0; Jm = 1.0)
8.10
–
(s, 1H)
(dd, Jo = 9.0; Jm = 1.0)
Table 2 13C NMR data (d in ppm) of selected compounds of series 28–45
Compounds (R1,R2) d R1 d R2 dC6 dC3 dC10b d C–H aromat.
d C aromat.
28
33
34
35
36
45
(H, H)
–
–
120.80 161.50 163.00 120.10; 122.20; 124.30; 125.95; 128.30;
128.50; 128.90; 129.30; 129.50; 131.00
114.80; 118.40; 128.70; 130.10;
136.70; 138.60
(CH3,
H)
21.50 –
120.80 161.60 163.00 122.20; 122.50; 124.50; 126.00; 128.40;
128.50; 129.50; 130.10; 130.90
115.00; 118.70; 127.00; 129.05;
136.70; 138.60; 140.80
(CH3,
CH3)
21.40 21.50 120.60 161.50 162.80 122.10; 122.40; 124.30; 125.90; 128.20;
129.25; 130.05; 130.90
114.80; 118.80; 126.10; 127.15;
136.70; 138.55; 138.60; 139.50
(CH3,
OCH3)
21.50 55.20 120.60 161.10 162.80 114.00; 122.12; 122.40; 124.30; 125.90;
128.70 130.20; 131.00
114.70; 118.80; 121.40; 127.25;
136.70; 138.60 160.60
(CH3,
NO2)
21.50 –
121.45 159.70 163.90 122.15; 122.20; 123.70; 124.50; 126.30;
129.10; 130.50; 130.70
114.55; 117.95; 126.15;135.40;
136.90; 138.60; 139.30; 148.30
(NO2,
Cl)
–
–
121.80 160.60 163.60 121.90; 122.40; 124.50; 124.70; 126.70;
129.20; 129.70; 131.90
112.80; 116.85; 124.50; 136.25;
137.00; 137.20; 138.90; 147.90
Evaluation of in vitro antitumoral activity of 28–45
Results and discussion
A series of disubstituted isothiochromeno[3,4-e][1,2]oxa-
zines 28–45 were selected by the National Cancer Institute
for evaluation of their in vitro anticancer activity (Table 3).
As shown in Table 3, compound 37 is the only derivative
that shows cytotoxic activity in this series. Lipophilicity
properties can be evaluated by experimental (shake-flask
method) or fragmentation methods. However, neither the
degree of lipophilicity (LogP) nor the molecular polar sur-
face area (TSPA) correlates positively with the cytotoxic
activity. Compounds 34 and 37 were further tested for their
in vitro activity against 60 primary human cancer cell lines
(Tables 4, 5, 6, 7).
Synthesis
The starting materials, 30,40-di-substituted-40H-spiro[isothio-
chromene-3,50-isoxazol]-4(1H)-ones 10–27, were obtained in
good yield from the corresponding p-substituted benzaldoxi-
mes 1–5 by 1,3-dipolar cycloaddition with appropriate (3Z)-3-
(4-methylbenzylidene)-1H-isothiochromen-4(3H)-ones 6–9 by
our previously reported procedure (Bennani et al., 2007a;
2004; Al Houari et al., 2008). The new compounds 28–45
were prepared in good yields by acidic hydrolysis of precur-
sors 10–27 with one equivalent of HCl in reluxing ethanol
(Scheme 1).
From the data in Table 3, it appeared that only the
isothiochromeno-oxazines 34 and 37 showed marked
cytotoxic activity compared to the rest of series 28–45. In
this case, the degree of lipophilicity of the aryls substituent
does not correlate positively with the cytotoxic activity. A
direct influence of the lipophilic group from the
Compounds 10–27 were previously prepared and char-
acterized in our laboratory (Bennani et al., 2007a; 2004; Al
Houari et al., 2008). The new compounds 28–45 were
stable at ambient temperature. Their structures were
determined by IR, MS, and NMR spectroscopy. The
selected NMR data are regrouped in Tables 1 and 2.
123

Med Chem Res (2013) 22:4798–4809
4801
Table 3 In vitro cytotoxic
activity of selected compounds
against three cell lines
Compounds
(NCI-Ref.)
(R1, R2)
Growth percentages
Activity
(Lung)
NCI-H460
(Breast)
MCF7
(CNS)
SF-268
28
29
30
31
32
33
34
35
36
37
39
40
42
43
45
(S722904)
(S722914)
(S722917)
(S722918)
(S722915)
(S722916)
(S722913)
(S722907)
(S722908)
(S722910)
(S722905)
(S722909)
(S722906)
(S722912)
(S722911)
(H, H)
94
92
104
51
97
62
95
41
36
86
97
2
97
66
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
(H, Me)
(H, OMe)
(H, NO2)
(H, Cl)
101
100
92
106
101
95
(Me, H)
94
69
(Me, Me)
(Me, OMe)
(Me, NO2)
(Me, Cl)
93
91
96
97
Inactive
Inactive
Active
99
83
91
37
(OMe, Me)
(OMe, OMe)
(OMe, Cl)
(NO2, Me)
(NO2, Cl)
84
98
116
51
99
85
103
105
102
111
104
Inactive
Inactive
Inactive
Inactive
Inactive
102
85
97
97
Table 4 Comparative study of
in vitro antitumor activity of hits
34 and 37 against leukemia and
non-small cell lung cancer cell
lines
Panel
Cell line
GI50 (lM) for 34 & 37
34
37
Leukemia
HL-60(TB)
K-562
1.00 9 10-4
2.29 9 10-5
3.71 9 10-5
6.92 9 10-5
4.20 9 10-5
[10-4
1.82 9 10-5
2.06 9 10-5
1.25 9 10-5
1.18 9 10-5
3.65 9 10-5
[10-4
MOLT-4
RPMI-8226
SR
Non-small cell lung
A549/ATCC
EKVX
[10-4
[10-4
[10-4
[10-4
HOP-62
HOP-92
6.81 9 10-5
[10-4
2.65 9 10-5
[10-4
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
[10-4
1.66 9 10-5
[10-4
[10-4
[10-4
[10-4
1.51 9 10-5
5.38 9 10-5
isothiochromeno nucleus appeared to be more important
for activity. The in vitro antitumor activity of compounds
34 and 37 were further investigated (Tables 4, 5, 6).
A comparison of antitumor screening results of com-
pounds 34 and 37 with regard to their in vitro activity
against 60 cell lines reveals mechanistic specificity notably
in the leukemia and breast cancer cell lines (Tables 5, 6).
Compound 37 has micromolar activity against all the leu-
kemia cell lines, while its other analogs have no activity
against one of the same strains. It appears that in this
limited series, the presence of a more polar substituent on
the oxazole ring enhances the activity. Further enhance-
ment of activity was attempted, but not achieved by the
substitution of the second aryl group for compound 40 on
the para position (compound 40 vs. 37). There appears to
be no gain in activity with the disubstituted-analogs com-
pared to the corresponding mono-substituted compound 37
(data not shown).
123

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Med Chem Res (2013) 22:4798–4809
Table 5 Comparative in vitro
antitumor activity of
Panel
Cell line
GI50 (lM) for 34 & 37
diastereoisomers 34 and 37
against CNS, melanoma,
ovarian, and renal cancer cell
lines
34
37
CNS cancer
SF-268
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
9.48 9 10-5
[10-4
[10-4
4 9 10-5
[10-4
[10-4
[10-4
[10-4
[10-4
2 9 10-5
[10-4
[10-4
1.21 9 10-5
[10-4
[10-4
[10-4
2 9 10-5
5 9 10-5
2.21 9 10-5
[10-4
[10-4
[10-4
SF-295
SF-539
SNB-19
U251
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
786-0
5.31 9 10-5
2.56 9 10-5
[10-4
Ovarian cancer
10-5
[ 9 10-4
[10-4
[10-4
1.16 9 10-5
2.12 9 10-5
[10-4
Renal cancer
A498
ACHIN
[10-4
CAKI-1
RXF 393
SN12C
[10-4
[10-4
1.13 9 10-5
[10-4
TK-10
UO-31
[10-4
Table 6 Comparative in vitro
antitumor activity of hits 34 and
37 against prostate, breast, and
colon cancer cell lines
Panel
Cell line
GI50 (lM) for 34 & 37
34
37
PC-3
[10-4
2.16 9 10-5
[10-4
Prostate cancer
Breast cancer
DU-145
[10-4
MCF7
[10-4
3.12 9 10-5
[10-4
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-549
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
4.42 9 10-5
4.12 9 10-5
3.14 9 10-5
4.62 9 10-5
[10-4
[10-4
T-47D
2.54 9 10-5
[10-4
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
[10-4
5.75 9 10-5
[10-4
6.27 9 10-5
8.12 9 10-5
[10-4
[10-4
5.56 9 10-5
HT29
[10-4
KM12
[10-4
SW-620
[10-4
123

Med Chem Res (2013) 22:4798–4809
4803
D-Sa
Table 7 Osiris calculations of compounds 28–45
Compd.
MW
Toxicity risksa
Osiris calculationsb
MUT
TUMO
IRRI
REP
CLP
Sa
DLa
28
353
367
383
398
387
367
381
397
412
401
383
397
413
428
417
412
428
432
350
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
?
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
?
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
?
?
?
?
?
–
6.13
6.45
6.03
6.00
6.75
6.45
6.76
6.34
6.32
7.06
6.03
6.34
5.92
5.90
6.64
6.32
5.90
6.62
1.18
-5.62
-5.97
-5.54
-6.08
-6.36
-5.97
-5.31
-5.98
-6.43
-6.70
-5.54
-5.98
-5.56
-5.10
-6.38
-5.43
-6.10
-6.82
-3.04
-0.09
2.00
0.23
0.26
0.30
0.14
0.25
0.33
0.29
0.32
0.16
0.29
0.38
0.32
0.37
0.17
0.30
0.16
0.17
0.14
0.25
29
30
3.54
31
-6.71
4.66
32
33
2.46
34
–
1.88
35
–
2.15
36
–
-8.10
3.27
37
–
38
–
3.74
39
–
1.89
40
–
3.16
41
–
-5.81
4.54
42
–
43
–
-8.36
-5.81
-5.68
5.24
44
–
45
Camptoc
–
?
Active risk = -, non-active risk = ?
a
MUT mutagenic, TUMO tumorogenic, IRRI irritant, REP reproductive effective
CLP cLogP, S solubility, DL druglikness, D-S drug-score
Campto camptothecin
b
c
POM molecular virtual screening
Molinspiration calculations
Osiris calculations
Drug likeness is calculated by the methodology developed
by Molinspiration (Table 8). The method is very robust and
is able to process practically all organic and most organo-
metallic molecules. Molecular Polar Surface Area TPSA is
calculated based on the methodology published (Ertl et al.,
2000) as a sum of fragment contributions. O– and N– cen-
tered polar fragments are considered. PSA has been shown
to be a very good descriptor characterizing drug absorption,
including intestinal absorption, bioavailability, Caco-2
permeability, and blood–brain barrier penetration. Predic-
tion results of compounds 28–45 molecular properties
(TPSA, GPCR ligand, and ICM) are valued (Tables 7, 8, 9).
Structure-based design is now fairly routine, but many
potential drugs fail to reach the clinic because of ADME-
Tox liabilities. One very important class of enzymes
responsible for many ADMET problems is the cytochromes
P450 (Hokuldsson, 1988). Inhibition of these or the pro-
duction of unwanted metabolites can result in many adverse
drug reactions. Of the most important program, Osiris is
already available online and it may be used in success with
complementary Molinspiration and Petra software (POM)
as we have previously demonstrated (Sheikh et al., 2011;
Chohan et al., 2010). Prediction results of compounds
28–45 toxicity risks (MUT, TUMO, IRRI, and REP) are
valued (Table 7). It appears that the new series 28–45 is less
toxic than clinical drug, the camptothecin (campto). This is
a great advantage in favor of the titled candidates 28–45. On
other hand, CLP constitutes a serious problem (CLP should
be inferior to 5). This means there are more phenyl than it
should be. The D-S of compounds 28–45 is encouraging (D-
S = 0.14–0.38); it is similar or more important than D-S of
Compto (DS = 0.25).
Pi-charge calculations
PETRA is a software package comprising various empirical
methods for the calculation of physicochemical properties in
organic molecules. All 18 methods are empirical in nature.
The following chemical effects can be quantified: heats of
formation, bond dissociation energies, sigma charge distri-
bution, p-charge distribution, inductive effect, resonance
effect and delocalization energies, and polarizability effect.
123

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Med Chem Res (2013) 22:4798–4809
Table 8 Molinspiration calculations of compounds 28–45
Compd
Molinspiration calculationsa
Drug-likenessb
GPCRL
TPSA
NONI
NV
VOL
ICM
KI
0.08
NRL
0.06
PI
EI
0.05
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
a
26
26
35
72
26
26
22
35
72
26
35
35
45
72
35
72
81
72
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
310
327
336
334
324
333
350
352
350
340
336
352
361
350
349
350
359
347
-0.02
-0.06
-0.07
-0.16
-0.02
0.13
-0.14
-0.20
-0.20
-0.18
-0.14
0.03
-0.17
-0.22
-0.22
-0.29
-0.21
-0.01
-0.21
-0.26
-0.33
-0.26
-0.22
-0.26
-0.20
-0.33
-0.25
-0.33
-0.32
-0.32
0.03
0.04
0.03
0.04
-0.02
-0.01
-0.07
0.01
-0.06
0.06
-0.04
0.04
0.11
0.18
0.14
-0.24
-0.10
-0.19
-0.06
-0.07
-0.10
-0.06
-0.19
-0.07
-0.19
-0.19
-0.16
-0.39
-0.26
-0.24
-0.20
-0.20
-0.26
-0.19
-0.24
-0.20
-0.24
-0.23
-0.18
-0.36
-0.01
-0.10
0.01
-0.09
0.01
-0.15
-0.06
-0.12
-0.05
-0.01
-0.06
-0.01
-0.12
-0.04
-0.12
-0.11
-0.10
-0.07
0.01
0.04
0.04
-0.01
0.03
0.01
0.04
-0.10
0.02
-0.07
0.02
-0.10
-0.09
-0.07
-0.07
-0.06
-0.06
NONI number OH–NH interaction, NV Number of violation, VOL volume
b
GPCR GPCR ligand, ICM ion channel modulator, KI kinas inhibitor, PI protease inhibitor, EI enzyme inhibitor
Table 9 Selected p-charge of
heteroatoms of compounds
28–45
Compound
Substituents
R1
Partial p-charge of heteroatom (in e)
R2
O1
N2
S5
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
H
H
-0.09
-0.09
-0.07
-0.09
-0.09
-0.09
-0.09
-0.07
-0.09
-0.09
-0.05
-0.05
-0.04
-0.06
-0.05
-0.04
-0.03
-0.04
-0.00
-0.00
-0.01
-0.01
-0.00
-0.00
-0.00
-0.01
-0.01
-0.00
-0.01
-0.01
-0.02
-0.02
-0.01
-0.02
-0.02
-0.02
-0.15
-0.15
-0.12
-0.10
-0.15
-0.15
-0.15
-0.12
-0.10
-0.15
-0.17
-0.11
-0.09
-0.08
-0.11
-0.13
-0.12
-0.13
H
CH3
OCH3
NO2
Cl
H
H
H
CH3
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
NO2
NO2
NO2
H
CH3
OCH3
NO2
Cl
H
CH3
OCH3
NO2
Cl
CH3
OCH3
Cl
The series 28–45 of oxazolidines have been subjected to
delocalized-charge calculations by the PETRA method of
the nonhydrogen common atoms, obtained from the partial
p-charge of the heteroatoms, have been used to model the
bioactivity against cancer. We give here, as example, the
compounds 36 and 37. It is found that the negative charges
123

Med Chem Res (2013) 22:4798–4809
4805
of the oxygen and nitrogen atoms of oxazolidine group and
the partial p positive charges of sulfur and supplementary
aryls contribute positively in favor of an antitumoral
activity, and this is in good agreement with the mode of
would be prudent first to seek new derivatives having GI50
values around 1 micromolar or less so that the assay can
be conducted more efficiently, though the interest in
compounds 34 and 37 due to their particular stereo-
chemistry remains undiminished. A further consideration
relates to the poor solubility of the aromatic derivatives in
water; for the cell line assays to work, it is important to
prepare derivatives that are more soluble. Substituting the
two aryls on C2 and C3 of oxazine ring with some other
groups such as alkyloxy or amino is possible and might
produce the desired effect.
antitumoral action of the compounds bearing (Xd-
–
Spacer–Yd?) pharmacophore(s) site. It was hypothesized
that difference in charges between two heteroatoms of the
same pharmacophore site (Xd- spacer Yd?) may facilitate
the intercalation in DNA of cancerous strains. It is further
found that the activity increases with increase in negative
charge of one heteroatom of the common pharmacophore
fragment of the compounds (36 and 37). This possible
synergistic and streamlined working procedure may lead to
highly active isomeric/tautomeric DNA receptor ligands. In
fact, the dissymmetric isoxazoline 37, which can appar-
ently recognize specific DNA sequences, could have
potential for gene targeting which merits investigation of
its capacity to inhibit access of activators or repressors to
regulatory sites in DNA controlling gene expression
(Waring et al., 2002).
Experimental protocols
General procedure
NMR spectra (1H, l3C) were recorded on a Bruker DPX
1
200 (operating at 200.12 MHz for H, 50.32 MHz for l3C)
1
or on a Bruker AM 300 (operating at 300.13 MHz for H,
at 75.47 MHz for l3C) spectrometer. NMR data are listed
in ppm and are reported relative to tetramethylsilane
(1H, 13C), residual solvent peaks being used as internal
standard with external calibration. Complete assignments
of the 13C spectra required non-decoupled 13C NMR
spectra with selective. Infra-red spectra were recorded in
KBr pellets using a Brucker IFS28 FTIR spectrometer.
Mass spectrometer (70 eV) and elemental analysis were
Conclusion
Hydrolysis of spiro-compounds 10–27 with hydrochloric
acid in the refluxing ethanol has been demonstrated to be a
powerful and optimal method for isothiochromane-oxazole
aromatic cyclization in the preparation of a new hetero-
cyclic family, the 3,4-di-substituted-isothiochromeno[3,4-
e][1,2]oxazines 28–45. This work provides for the first time
a simple one-pot synthetic methodology for the preparation
of a wide range of 28–45 compounds which have analog or
hybrid O,S-pharmacophore site of O,O-pharmacophore site
of the natural alkaloid Camptothecin (Campto). The cyto-
toxic activity of this new family suggests a promising novel
approach to the design of prospective compounds for the
treatment of Leukemia and breast cancer.
´
performed by the Service du Centre Universitaire Regional
d’Interface CURI (University Sidi Mohamed Ben Abdellah,
`
Fes, Morocco).
Biologic evaluation
In vitro anticancer activity was measured by the National
Cancer Institute (NCI/USA) against three cell lines, NCI
H460 (Lung), MCF7 (Breast), and SF-268 (CNS) as a
preliminary step. Then, the obtained hits are tested against
60 tumor types. In the current protocol, each cell line was
inoculated and preincubated on a microtiter plate. Test
agents were then added at a single concentration and the
culture incubated for 48 h. End-point determinations were
made using Alamar blue (Gray and Wickstrom, 1996).
Results for each test agent were reported as the percent
growth of the treated cells compared to the untreated
controls. Compounds which reduce the growth of any one
of the cell lines to approximately 32 % or less (negative
numbers indicate cell death) were passed on for evalua-
tion in the full panel of 60 cell lines over a 5-log dose
range. All compounds were tested at a final concentration
of 10-4 M.
As a guide for future work, the data reported here
indicate that the 28–45 compounds have a definite
potential efficacy that merits development through modi-
fication to both the isothiochromeno and the substituents
on C-2 and C-3 of oxazine ring. We note, however, that
although the compounds are not nominally camptothecin
analogs, there is no necessary presumption that they do in
fact resemble camptothecin and thereby target topoiso-
merase-I. It might be possible to address this question by
testing their effect(s) on two cell lines, one of which has
normal topoisomerase I and the other has a mutant
(camptothecin-resistant) enzyme. If there was a difference
in GI50 value, this would indicate that topoisomerase I is a
critical target for the drugs. Given the rather low GI50
values of the present compounds, around 200 lM, it
123

4806
Med Chem Res (2013) 22:4798–4809
139.60). MS (EI, 70 eV): [M]?ꢀ = m/z : M = 367
[C24H17NOS, (19 %)], 234.20 (100 %). Elemental analysis
for C24H17NOS: Cal. (found): C 78.45 (78.37); H 4.66
(4.58); N 3.81 (3.78).
General synthesis
Synthesis of 30,40-diaryl-40H-spiro[isothiochromene-3,50-
isoxazol]-4(1H)-ones: 10–27
In an erlenmeyer equipped with a bulb for addition, a
mixture of 10 mmol of the 3-arylideneisothiochroman-4-
one 1–5 and 12 mmol of oxime 6–9 in 20 mL of chloro-
form was placed in an ice-salt bath under magnetic stirring.
Then, 10 mL of a sodium hypochlorite solution (NaOCl,
18°) was added and stirring was maintained for one more
hour. The organic phase was separated, washed with water,
and dried on anhydrous sodium sulfate. The obtained res-
idue after evaporation of the solvent was recrystallized
from ethanol. This series was previously well characterized
3-(4-methoxyphenyl)-4-phenylisothiochromeno[3,4-e][1,2]
oxazine: 30 White powder. Yield = 61 %. M.p. = 148–
150 °C. IR (KBr, t cm-1): 3128 (=CH), 2985 (CH), 1542
(C=C), 1483 (–C=N), 1H NMR (250 MHz, CDCl3) d ppm:
8.25 (dd, 1H, H9, Jo = 8.5; Jm = 2), 7.65 (s, 1H, H6),
6.80–7.06 (m, 13H, Haromat), 3.70 (s, 3H, O–CH3). 13C
NMR (250 MHz, CDCl3) d ppm: Methoxy (55.30), C6
(120.80), C3 (161.20), C10b (162.90), C–H aromat. (114.10;
122.20; 122.40; 124.40; 126.00; 128.75; 129.40; 129.80;
131.20), C aromat. (114.80; 130.50; 131.20; 136.75;
138.70; 160.65). MS (EI, 70 eV): [M]?ꢀ = m/z : M = 383
[C24H17NO2S, (71 %)]; 234.15 (100 %). Elemental anal-
ysis for C24H17NOS: Cal. (found): C 75.17 (74.93); H 4.47
(4.42); N 3.65 (3.57).
1
by H, 13C NMR, MS, and elemental analyses (Bennani
et al., 2007a; 2004; Al Houari et al., 2008).
Synthesis of 3,4-di-substituted-isothiochromeno[3,4-
e][1,2]oxazines: 28–45
3-(4-nitrophenyl)-4-phenylisothiochromeno[3,4-e][1,2]
oxazine: 31 White powder. Yield = 50 %. M.p. = 202–
204 °C. IR (KBr, t cm-1): 3139 (=CH), 2991 (CH), 1543
(C=C), 1484 (–C=N). 1H NMR (250 MHz, CDCl3) d ppm:
8.30 (dd, 1H, H9, Jo = 8.0; Jm = 1), 7.75 (s, 1H, H6),
7.10–8.16 (m, 12H, Haromat). 13C NMR (250 MHz,
CDCl3) d ppm: C6 (121.65), C3 (159.70), C10b (164.00), C–
H aromat. (122.10; 122.30; 123.80; 124.60; 126.40;
129.20; 129.40; 129.75; 131.00), C aromat. (114.60;
117.85; 129.30; 135.30; 137.00; 138.70; 148.40). MS (EI,
70 eV): [M]?ꢀ = m/z : M = 398 [C23H14N2O3S, (74 %)];
89.15 (100 %). Elemental analysis for C23H14N2O3S: Cal.
(found): C 69.33 (69.27); H 3.54 (3.47); N 7.03 (6.96).
A volume of 10 mL of concentrated hydrochloric acid was
carefully added, drop by drop, to a solution of 2 mmol of
compounds 10–27 in 15 mL of EtOH. Agitation at
refluxing solvent was maintained for 10 h after the addi-
tion. The organic phase was concentrated to half initial
volume. White solide is obtained and recuperated by fil-
tration, washed several times with water, and was recrys-
tallized from ethanol.
3,4-diphenylisothiochromeno[3,4-e][1,2]oxazine: 28 White
powder. Yield = 52 %. M.p. = 156–157 °C. IR (KBr, t
cm-1): 3140 (=CH), 2990 (CH), 1540 (C=C), 1480 (–C=N).
1H NMR (250 MHz, CDCl3) d ppm: 8.30 (dd, 1H, H9,
Jo = 9; Jm = 2), 7.70 (s, 1H, H6), 7.10–7.60 (m, 13H,
Haromat). 13C NMR (250 MHz, CDCl3) d ppm: C10b
(163.00), C3 (161.50), C6 (120.80), C–H aromat. (120.10;
122.20; 124.30; 125.95; 128.30; 128.50; 128.90; 129.30;
129.50; 131.00), C aromat. (114.80; 118.40; 128.70;
130.10; 136.70; 138.60). MS (EI, 70 eV): [M]?ꢀ = 353
[C23H15NOS, (100 %)]. Elemental analysis for C23H15NOS:
Cal. (found): C 78.16 (77.98); H 4.28 (4.23); N 3.96 (4.17).
3-(4-chlorophenyl)-4-phenylisothiochromeno[3,4-e][1,2]
oxazine: 32 White powder. Yield = 55 %. M.p. = 168–
170 °C. IR (KBr, t cm-1): 3141 (=CH), 2988 (CH), 1537
1
(C=C), 1481 (–C=N). H NMR (250 MHz, CDCl3) d ppm:
8.25 (dd, 1H, H9, Jo = 8.5; Jm = 1), 7.65 (s, 1H, H6),
7.10–7.70 (m, 12H, Haromat). 13C NMR (250 MHz, CDCl3)
d ppm: C6 (121.10), C3 (160.60), C10b (163.30), C–H aromat.
(121.20; 122.25; 122.30; 124.50; 126.20; 127.50; 127.65;
127.85; 128.70; 128.85), C aromat. (114.60; 118.30; 126.15;
128.75; 135.70; 136.85; 136.70). MS (EI, 70 eV):
[M]?ꢀ = m/z : M = 387 [C23H14ClNOS, (74 %)], 89.15
(100 %). Elemental analysis for C23H14ClNOS: Cal. (found):
C 71.22 (70.97); H 3.64 (3.59); N 3.61 (3.57).
3-(4-methylphenyl)-4-phenylisothiochromeno[3,4-e][1,2]oxa-
zine: 29 White powder. Yield = 55 %. M.p. = 163–
165 °C. IR (KBr, t cm-1): 3139 (=CH), 2991 (CH), 1541
(C=C), 1479 (–C=N). 1H NMR (250 MHz, CDCl3) d ppm:
8.30 (dd, 1H, H9, Jo = 9; Jm = 1), 7.70 (s, 1H, H6),
7.10–7.70 (m, 12H, Haromat), 2.40 (s, 3H, CH3). 13C NMR
(250 MHz, CDCl3) d ppm: Methyl (21.40), C6 (120.80), C3
(161.50), C10b (162.90), C–H aromat. (122.15; 122.30;
124.40; 126.00; 128.30; 128.70; 129.30; 129.35; 131,00), C
aromat. (114.80; 118.60; 126.00; 130.30; 136.70; 138.60;
4-(4-methylphenyl)-3-phenylisothiochromeno[3,4-e][1,2]
oxazine: 33 White powder. Yield = 60 %. M.p. = 138–
141 °C. IR (KBr, t cm-1): 3141 (=CH), 2991 (CH), 1542
(C=C), 1481 (–C=N). 1H NMR (250 MHz, CDCl3) d ppm:
123

Med Chem Res (2013) 22:4798–4809
4807
8.35 (dd, 1H, H9, Jo = 9; Jm = 1), 7.70 (s, 1H, H6),
7.15–7.60 (m, 12H, Haromat), 2.50 (s, 3H, CH3). 13C NMR
(250 MHz, CDCl3) d ppm: Methyl (21.50), C6 (120,80), C3
(161.60), C10b (163.00), C–H aromat. (122.20; 122.50;
124.50; 126.00; 128.40; 128.50; 129.50; 130.10; 130.90), C
aromat. (115.00; 118.70; 127.00; 129.05; 136.70; 138.60;
140.80). MS (EI, 70 eV): [M]?ꢀ = m/z : M = 367
[C24H17NOS, (31 %)], 236.10 (100 %). Elemental analysis
for C24H17NOS: Cal. (found): C 78.45 (78.12); H 4.66
(4.53); N 3.81 (3.76).
[C24H16N2O3S, (49 %)], 221 (100 %). Elemental analysis
for C24H16N2O3S: Cal. (found): C 69.89 (69.75); H 3.91
(3.87); 6.79 (6.73).
3-(4-chlorophenyl)-4-(4-methylphenyl)isothiochromeno
[3,4-e][1,2]oxazine: 37 White powder. Yield = 50 %.
M.p. = 133–135 °C. IR (KBr, t cm-1): 3141 (=CH), 2992
(CH), 1541 (C=C), 1479 (–C=N). 1H NMR (250 MHz,
CDCl3) d ppm: 8.30 (dd, 1H, H9, Jo = 9; Jm = 1), 7.70 (s,
1H, H6), 7.10–7.60 (m, 11H, Haromat), 2.50 (s, 3H,
CH3). 13C NMR (250 MHz, CDCl3) d ppm: CH3 (21.50),
C6 (120.95), C3 (160.60), C10b (163.25), C–H aromat.
(122,20; 122,30; 124,40; 126,10; 128,80; 129,60; 130,25;
130,80), C aromat. (114.60; 118.45; 126,70; 27,50; 135.60;
136.75; 138.60; 138.85). MS (EI, 70 eV): [M]?ꢀ = m/z :
M = 401 [C24H16ClNOS, (3 %)], 221 (100 %). Elemental
analysis for C24H16ClNOS: Cal. (found): C 71.72 (71.68);
H 4.01 (3.97); N 3.49 (3.45).
3,4-bis(4-methylphenyl)isothiochromeno[3,4-e][1,2]oxazine:
34 White powder. Yield = 70 %. M.p. = 163–165 °C.
IR (KBr, t cm-1): 3137 (=CH), 2991 (CH), 1542 (C=C),
1481 (–C=N). 1H NMR (250 MHz, CDCl3) d ppm: 8.30 (dd,
1H, H9, Jo = 9; Jm = 1), 7.70 (s, 1H, H6), 7.10–7.60 (m,
11H, Haromat), 2.50 (s, 3H, CH3), 2.40 (s, 3H, CH3). 13C
NMR (250 MHz, CDCl3) d ppm: 2CH3 (21.40, 21.50), C6
(120.60), C3 (161.50), C10b (162.80), C–H aromat. (122.10;
122.40; 124.30; 125.90; 128.20; 129.25; 130.05; 130.90), C
aromat. (114.80; 118.80; 126.10; 127.15; 136.70; 138.55;
138.60; 139.50). MS (EI, 70 eV): [M]?ꢀ = m/z : M = 381
[C25H19NOS, (33 %)], 250.10 (100 %). Elemental analysis
for C25H19NOS: Cal. (found): C 78.71 (78.65); H 5.02
(4.97); N 3.67 (3.63).
4-(4-methoxyphenyl)-3-phenylisothiochromeno[3,4-e][1,2]
oxazine: 38 White powder. Yield = 60 %. M.p. = 138–
140 °C. IR (KBr, t cm-1): 3139 (=CH), 2991 (CH), 1541
(C=C), 1482 (–C=N). 1H NMR (250 MHz, CDCl3) d ppm:
8.35 (dd, 1H, H9, Jo = 9; Jm = 1), 7.70 (s, 1H, H6),
7.00–7.70 (m, 13H, Haromat), 3.90 (s, 3H, O–CH3). 13C
NMR (250 MHz, CDCl3) d ppm: OCH3 (55.30), C6
(120.70), C3 (161.50), C10b (163.10), C–H aromat. (114.80;
120.70; 122.10; 122.40; 124.35; 125.95; 128.32; 128.50;
129.45), C aromat. (114.45; 118.70; 122.05; 129.00;
136.65; 138.55; 159.95). MS (EI, 70 eV): [M]?ꢀ = m/z :
M = 383 [C24H17NO2S, (7 %)]; 222 (100 %). Elemental
analysis for C24H17NO2S: Cal. (found): C 75.17 (74.95); H
4.47 (4.42); N 3.65 (3.61).
3-(4-methoxyphenyl)-4-(4-methylphenyl)isothiochromeno
[3,4-e][1,2]oxazine: 35 White powder. Yield = 65 %.
M.p. = 181–183 °C. IR (KBr, t cm-1): 31437 (=CH),
2989 (CH), 1538 (C=C), 1481 (–C=N). 1H NMR
(250 MHz, CDCl3) d ppm: 8.30 (dd, 1H, H9, Jo = 9;
Jm = 1), 7.70 (s, 1H, H6), 6.80–7.60 (m, 11H, Haromat),
3.80 (s, 3H, O–CH3), 2.45 (s, 3H, CH3). 13C NMR
(250 MHz, CDCl3) d ppm: CH3 and OCH3 (21.50, 55.20),
C6 (120.60), C3 (161.10), C10b (162.80), C–H aromat.
(114.00; 122.12; 122.40; 124.30; 125.90; 128.70 130.20;
131.00), C aromat. (114.70; 118.80; 121.40; 127.25;
136.70; 138.60 160.60). MS (EI, 70 eV): [M]?ꢀ = m/z :
M = 397 [C25H19NO2S, (74 %)], 248.25 (100 %). Ele-
mental analysis for C25H19NO2S: Cal. (found): C 75.54
(75.46); H 4.82 (4.78); N 3.52 (3.48).
4-(4-methoxyphenyl)-3-(4-methylphenyl)isothiochromeno
[3,4-e][1,2]oxazine: 39 White powder. Yield = 65 %.
M.p. = 182–184 °C. IR (KBr, t cm-1): 3138 (=CH), 2991
(CH), 1541 (C=C), 1482 (–C=N). 1H NMR (250 MHz,
CDCl3) d ppm: 8.30 (dd, 1H, H9, Jo = 9; Jm = 1), 7.65 (s,
1H, H6), 6.85–7.55 (m, 11H, Haromat), 3.80 (s, 3H, O–
CH3), 2.45 (s, 3H, CH3). 13C NMR (250 MHz, CDCl3) d
ppm: (OCH3 (55.25), CH3 (21.50), C6 (120.60), C3
(161.15), C10b (162.80), C–H aromat. (114.00; 122.15;
122.40; 124.35; 125.90; 129.90; 130.15; 131.00), C aromat.
(114.70; 118.80; 121.45; 127.25; 138.10; 138.60; 160.55).
MS (EI, 70 eV): [M]?ꢀ = m/z : M = 397 [C25H19NO2S,
(39 %)], 248 (100 %). Elemental analysis for C25H19NO2S:
Cal. (found): C 75.54 (75.46); H 4.82 (4.78); N 3.52 (3.47).
4-(4-methylphenyl)-3-(4-nitrophenyl)isothiochromeno[3,4-e]
[1,2]oxazine: 36 White powder. Yield = 56 %. M.p. =
219–223 °C. IR (KBr, t cm-1): 3141 (=CH), 2991 (CH),
1539 (C=C), 1481 (–C=N). 1H NMR (250 MHz, CDCl3) d
ppm: 8.30 (dd, 1H, H9, Jo = 9; Jm = 1), 7.75 (s, 1H, H6),
7.15–8.20 (m, 11H, Haromat), 2.50 (s, 3H, CH3). 13C NMR
(250 MHz, CDCl3) d ppm: CH3 (21.50), C6 (121.45), C3
(159.70), C10b (163.90), C–H aromat. (122.15; 122.20;
123.70; 124.50; 126.30; 129.10; 130.50; 130.70), C aromat.
(114.55; 117.95; 126.15;135.40; 136.90; 138.60; 139.30;
148.30). MS (EI, 70 eV): [M]?ꢀ = m/z : M = 412
3,4-bis(4-methoxyphenyl)isothiochromeno[3,4-e][1,2]oxa-
zine: 40 White powder. Yield = 60 %. M.p. = 133–
135 °C. IR (KBr, t cm-1): 3139 (=CH), 2989 (CH), 1541
(C=C), 1482 (–C=N). 1H NMR (250 MHz, CDCl3) d ppm:
123

4808
Med Chem Res (2013) 22:4798–4809
8.35 (dd, 1H, H9, Jo = 9; Jm = 1), 7.70 (s, 1H, H6),
6.60–7.55 (m, 11H, Haromat), 3.90 (s, 3H, O–CH3), 3.80
(s, 3H, O–CH3). 13C NMR (250 MHz, CDCl3) d ppm:
2OCH3 (55.25, 55.30), C6 (120.55), C3 (161.10), C10b
(162.90), C–H aromat. (113.95; 114.85; 122.00; 122.40;
124.35; 125.90; 129.65; 132.30), C aromat. (114.25;
118.90; 121.40; 122.25; 136.60; 138.55; 159.90; 160.50).
MS (EI, 70 eV): [M]?ꢀ = m/z : M = 413 [C25H19NO3S,
(28 %)]; 266.20 (100 %).
C24H16N2O3S: Cal. (found): C 69.89 (69.81); H 3.91
(3.89); N 6.79 (6.75).
3-(4-methoxyphenyl)-4-(4-nitrophenyl)isothiochromeno
[3,4-e][1,2]oxazine: 44 White powder. Yield = 60 %.
M.p. = 196–198 °C. IR (KBr, t cm-1): 3140 (=CH), 2991
(CH), 1540 (C=C), 1481 (–C=N). 1H NMR (250 MHz,
CDCl3) d ppm: 8.50 (dd, 1H, H9, Jo = 9; Jm = 1), 7.80 (s,
1H, H6), 6.90–8.30 (m, 11H, Haromat), 3.85 (s, 3H, O–
CH3). 13C NMR (250 MHz, CDCl3) d ppm: OCH3 (55.35),
C6 (121.60), C3 (161.20), C10b (163.10), C–H aromat.
(122.30; 122.40; 124.50; 124.70; 126.70; 129.20; 129.70;
131.90), C aromat. (112.85; 117.20; 120.30; 137.00;
Elemental analysis for C25H19NO3S: Cal. (found): C
72.62 (72.55); H 4.63 (4.57); N 3.39 (3.34).
4-(4-methoxyphenyl)-3-(4-nitrophenyl)isothiochromeno
[3,4-e][1,2]oxazine: 41 White powder. Yield = 50 %.
M.p. = 191–193 °C. IR (KBr, t cm-1): 3140 (=CH), 2991
(CH), 1540 (C=C), 1481 (–C=N). 1H NMR (250 MHz,
CDCl3) d ppm: 8.30 (dd, 1H, H9, Jo = 9; Jm = 1), 7.75
(s, 1H, H6), 7.00–8.20 (m, 11H, Haromat), 3.90 (s, 3H,
O–CH3). 13C NMR (250 MHz, CDCl3) d ppm: OCH3
(55.35), C6 (121.45), C3 (160.30), C10b (164.10), C–H
aromat. (115.20; 122.15; 122.23; 123.75; 124.50; 126.35;
129.10; 132.15), C aromat. (114.20; 115.20 118.05;
121.50; 135.40; 136.80; 138.60; 148.25; 159.70). MS (EI,
70 eV): [M]?ꢀ = m/z : M = 428 [C24H16N2O4S, (13 %)],
221 (100 %). Elemental analysis for C24H16N2O4S: Cal.
(found): C 67.28 (67.17); H 3.76 (3.65); N 6.54 (6.49).
137.60; 138.80; 147.75; 160.85). MS (EI, 70 eV): [M]?ꢀ
=
m/z : M = 428 [C24H16N2O4S (8 %)]. Elemental analysis
for C24H16N2O4S: Cal. (found): C 67.28 (67.17); H 3.76
(3.69); N 6.54 (6.51).
3-(4-chlorophenyl)-4-(4-nitrophenyl)isothiochromeno[3,4-e]
[1,2]oxazine: 45 White powder. Yield = 50 %. M.p. =
176–178 °C. IR (KBr, t cm-1): 3139 (=CH), 2990 (CH),
1540 (C=C), 1478 (–C=N). 1H NMR (250 MHz, CDCl3) d
ppm: 8.50 (dd, 1H, H9, Jo = 9; Jm = 1), 7.80 (s, 1H, H6),
7.15–8.30 (m, 11H, Haromat). 13CNMR (250 MHz, CDCl3)
d ppm: C6 (121.80), C3 (160.60), C10b (163.60), C–H aromat.
(121.90; 122.40; 124.50; 124.70; 126.70; 129.20; 129.70;
131.90), C aromat. (112.80; 116.85; 124.50; 136.25; 137.00;
137.20; 138.90; 147.90). MS (EI, 70 eV): [M]?ꢀ = m/z :
M = 432 [C23H13ClN2O3S] (3 %), 118 (100 %). Elemental
analysis for C23H13ClN2O3S: Cal. (found): C 63.82 (63.76);
H 3.03 (3.01); N 6.47 (6.45).
3-(4-chlorophenyl)-4-(4-methoxyphenyl)isothiochromeno
[3,4-e][1,2]oxazine: 42 White powder. Yield = 57 %.
M.p. = 162–164 °C. IR (KBr, t cm-1): 3141 (=CH), 2990
(CH), 1542 (C=C), 1480 (–C=N). 1H NMR (250 MHz,
CDCl3) d ppm: 8.30 (dd, 1H, H9, Jo = 9; Jm = 1), 7.70
(s, 1H, H6), 7.00–7.60 (m, 11H, Haromat), 3.90 (s, 3H,
O–CH3). 13C NMR (250 MHz, CDCl3) d ppm: OCH3
(55.55), C6 (121.81), C3 (161.10), C10b (163.12), C–H
aromat. (116.13; 121.93; 123.03; 123.95; 124.55;5; 148.28;
159.79). MS (EI, 70 eV): [M]?ꢀ = m/z : M = 417
[C24H16ClNO2S, (39 %)], 248.15 (100 %). Elemental
analysis for C24H16ClNO2S: Cal. (found): C 68.98 (68.87);
H 3.86 (4.78); N 3.35 (3.32).
Acknowledgments This work was supported by the grants from the
Ministry of Education of Morocco (PGR-UMP-BH-2005 and PRO-
TARS I). We are indebted to the National Cancer Institute (NCI) of
the United States for biologic tests.
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