Synthesis and biological activity of novel benzoazoles, benzoazines and other analogs functionalized by 2,4-dihydroxyphenyl moiety

Article abstract of DOI:10.1007/s11164-018-3483-0

Novel benzoazoles, benzoazines, benzothiazoles, benzothiazines and other related compounds possessing a 2,4-dihydroxyphenyl moiety were prepared. The compounds were obtained by the reaction of sulfinylbis[(2,4-dihydroxyphenyl)methanethione]s with the appropriate heterocyclic amines or hydrazines. The structures of the compounds were proved by IR, ¹H NMR, and mass spectral data. Human cancer lines, Candida species and phytopathogenic fungi were used for the evaluation of biological potency of compounds. Additionally, drug-like properties were estimated in silico.

Full text of DOI:10.1007/s11164-018-3483-0

Res Chem Intermed
https://doi.org/10.1007/s11164-018-3483-0
Synthesis and biological activity of novel benzoazoles,
benzoazines and other analogs functionalized by
2,4-dihydroxyphenyl moiety
Joanna Matysiak1
Alicja Skrzypek1 Urszula Głaszcz1
•
•
•
Arkadiusz Matwijczuk2 Bogdan Senczyna1 Joanna Wietrzyk3
Elzbieta Krajewska-Kułak Andrzej Niewiadomy1,5
•
•
•
4
•
_
Received: 8 December 2017 / Accepted: 22 May 2018
Ó The Author(s) 2018
Abstract Novel benzoazoles, benzoazines, benzothiazoles, benzothiazines and
other related compounds possessing a 2,4-dihydroxyphenyl moiety were prepared.
The compounds were obtained by the reaction of sulfinylbis[(2,4-dihydrox-
yphenyl)methanethione]s with the appropriate heterocyclic amines or hydrazines.
1
The structures of the compounds were proved by IR, H NMR, and mass spectral
data. Human cancer lines, Candida species and phytopathogenic fungi were used for
the evaluation of biological potency of compounds. Additionally, drug-like prop-
erties were estimated in silico.
Keywords Benzothiazine Á Benzothiazole Á Benzoazine Á Antiproliferative
activity Á Antifungal activity
&
Joanna Matysiak
joanna.matysiak@up.lublin.pl
1
2
3
4
5
Department of Chemistry, University of Life Sciences in Lublin, Akademicka 15,
20-950 Lublin, Poland
Department of Biophysics, University of Life Sciences in Lublin, Akademicka 13,
20-950 Lublin, Poland
Department of Experimental Oncology, Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences, R. Weigla 12, 53-114 Wrocław, Poland
Department of Integrated Medical Care, Medical University, ul. M. Skłodowskiej-Curie 7A,
Białystok, Poland
Institute of Industrial Organic Chemistry, Annopol 6, 03-236 Warsaw, Poland
123

J. Matysiak et al.
Introduction
Nitrogen and sulfur heterocycles have received a great deal of attention in the
literature due to their role as active pharmacophores. Benzothiazines are a group of
bioactive compounds known to have anticancer, diuretic, and anti-hypertensive
activities [1, 2]. The representatives of this heterocyclic class are assumed to possess
biological activities since they might provide the heteroatoms as potential hydrogen
bond acceptors and the fused phenyl ring for possible p–p interactions. In particular,
1,2-benzothiazine and 1,3-benzothiazine derivatives have exhibited promising
pharmacological properties: anti-inflammatory, antitumor and antiproliferative
activity [3, 4], as well as antomicrobial activity [5–8]. 4H-1,4-benzothiazines
exhibited antifungal potency against Aspergillus fumigates and Candida albicans
[9, 10]. The derivatives of 12(H)-quino[3,4-b][1,4]benzothiazine showed antipro-
liferative activity against two cancer cell lines, SNB-19 and C-32 [11], while
6-substituted 9-fluoroquino[3,2-b]benzo[1,4]thiazines exhibited cytotoxicity as well
as antiproliferative actions against human peripheral blood mononuclear cells
stimulated with phytohemagglutinin A (PHA) [12].
Coupling of 2-alkylthiobenzimidazole with the b-lactam ring produced com-
pounds with antibacterial and antifungal activities [13]. Attachment of other
heterocycles like b-lactam, thiadiazole and oxadiazole to the benzimidazole
skeleton resulted in hybrid compounds of potent antibacterial and/or antifungal
properties [14–16]. A pyridobenzimidazole derivative with unique activities, called
D75-4590, is a specific inhibitor of b-1,6-glucan synthesis and exhibits potent
activities against various Candida species [17]. The series of methyl-4H-1-
benzopyran-4-ones carrying mono- or diamidinobenzimidazoles at different posi-
tions have been described for antibacterial and antifungal activities [18]. Methyl-
benzimidazoles are an important class of compounds for cytotoxic activity against
human tumour cell lines [19].
Recently, there have been various approaches to study the role of the
benzothiazole moiety as antimicrobial [20–23], anticancer [24–27] and antifungal
agents [22, 28–30]. It has been shown that some benzothiazole dimers were free
from irritation, teratogenicity and sensitivity properties compared to monomers and
that the increase in the hydrogen donor count is conductive for cytotoxic activity of
derivatives against the HL-60 cell lines [31]. The novel N-alkylbromobenzothia-
zoles have been evaluated for their anticancer potency [32]. The derivatives of
imidazo[2,1-b][1,3]benzothiazole have been reported for their strong inhibitory
activity against bacterial and fungal strains [33].
Modifications on the heterocycle nucleus resulted in a large number of
compounds characterized by diverse pharmacological activities. The biological
profiles of new generations of benzothiazoles, benzimidazoles and benzthiazines
represent much progress as regards the older compounds. Looking into the
medicinal importance of these compounds’ moiety, it was thought worth
synthesizing some newer derivatives and screening them for their biological
activities.
123

Synthesis and biological activity of novel benzoazoles,…
To extend our studies in this area, a set of new heterocyclic analogs with 2,4-
dihydroxyphenyl functionality were designed and obtained. Following from
previous studies, this group of compounds exhibits strong antifungal and
antiproliferative activity [34–39]. This paper presents the in vitro antifungal
potency of compounds against the panel phytopathogenic fungi and Candida strains
as well as cytotoxic potency against human cancer cell lines. Some properties of
compounds in silico were estimated to explain the influence of the structure of the
compounds on their biological properties.
Experimental
Chemistry
General
¨
Melting points (m.p.) were determined using a BUCHI B-540 (Flawil, Switzerland)
melting point apparatus. The elemental analysis (C, H, N) was performed on a
Perkin-Elmer 2400. The IR spectra were measured with a Perkin-Elmer FT-IR
1725X spectrophotometer (in KBr) or a Varian 670-IR FT-IR spectrometer (ATR)
in the range of 600–4000 cm-1. The NMR spectra (1D NMR) were recorded in
DMSO-d6 using a Varian Mercury 400, Bruker DRX 500 (Bruker Daltonics,
Billerica, MA, USA) or Tesla BS 567A (100 MHz). Chemical shifts (d, ppm) have
been described in relation to tetramethylsilane and coupling constants (J) are
expressed in Hz. The MS spectra (EI, 70 eV) were recorded using the apparatus
AMD-604.
The purity of the compounds was examined by HPLC Knauer (Berlin, Germany)
with a dual pump, a 20-lL simple injection valve and a UV–visible detector (275
and 330 nm). The Hypersil Gold C18 (1.9 lm, 100 9 2.1 mm) column was used as
the stationary phase. The mobile phase included different contents of MeOH and the
aqueous phase (acetate buffer, pH 4, 20 nM). The flow rate was 0.4 ml/min at room
temperature. The retention time of an unretained solute (to) was determined using
KCl. The log k values for 80 or 65% of MeOH (v/v) in the mobile phase are
presented. They were calculated as log k = log(tR - to)/to, where tR is the retention
time of a solute, and to the retention time of an unretained solute.
HO
OH
S
S
OH
1a-12a
R
OH
R-fused heterocyclic system
S
MeOH, reflux 2.5-3 h
O
HO
OH
1-12
STB
Fig. 1 Synthesis scheme of compounds 1–12
123

J. Matysiak et al.
General procedure for the synthesis of compounds 1–12
A mixture of the corresponding amine or hydrazine (5 mmol) (1a–12a) and STB
(10 mmol) in MeOH (80 mL) (Fig. 1) was heated to reflux for 2.5–3 h. The hot
mixture was filtered via a Bu¨chner funnel. Next, the formed solid was filtered off (2,
9–11), and either the filtrate was concentrated (compounds 3–8) or water was added
to afford the solid (1, 12) which was filtered. Finally, the product was recrystallized
from MeOH (compounds 1, 2, 5, 9, 10), from MeOH/H2O (3:1) (4, 11) or from
MeOH/H2O (2:1) (3, 6–8, 12), respectively.
2-(2,4-Dihydroxyphenyl)-9b-methoxy-[1,3,4]thiadiazolo[2,3-a]isoindol-5(9bH)-one
1
IR (KBr) mmax: 3460 (OH), 3024 (C(Ar)-H), 2950 (CH), 1702 (C=O), 1661
(C=N), 1623 (C=N), 1604 (C=C), 1576 (C=C), 1521 (C=C), 1494, 1472, 1452,
1435, 1349, 1322, 1302, 1280, 1217 (C–OH), 1181, 1141, 1123, 1096, 1024, 997,
985, 969, 871, 829, 804, 795, 759, 729, 714 cm-1; 1H NMR (500 MHz, DMSO-d6)
d: 11.14 (s, 1 H, HO–C–(2)), 10.08 (s, 1 H, HO–C(4)), 8.08 (d, J = 8.7 Hz, 1 H, H–
C(6)), 7.89 (m, 1 H, H–C(Ar)), 7.80 (m, 1 H, H–C(Ar)), 7.72 (td, J = 7.5 and
1.5 Hz, 1 H, H–C(Ar)), 7.68 (td, J = 7.5 and 1.4 Hz, 1 H, H–C(Ar)), 6,51 (d,
J = 2.3 Hz, 1 H, H–C(3)), 6.46 (dd, J = 8.7 and 2.3 Hz, 1 H, H–C(5)), 3.71 (s, 3 H,
CH3) ppm; 13C NMR (125 MHz, DMSO-d6) d: 167.7 (C=O), 164.2, 163.5, 161.3,
156.4, 131.6, 131.5, 130.8, 130.3, 129.3, 129.0, 128.9, 108.4, 108.2, 102.3, 52.3
(CH3) ppm; EI-MS (m/z, %): 328 (M?, 48), 296 (72), 268 (3), 162 (100), 149 (5),
132 (23), 130 (14), 119 (2), 104 (70), 102 (3), 90 (2), 76 (18), 63 (2), 51 (10), 40 (7).
4-(4-Hydroxy-6-methyl-4-phenyl-4H-benzo[d][1,3]thiazin-2-yl)benzene-1,3-diol
IR (ATR) mmax: 3251 (OH), 2942, 1618 (C=N), 1593 (C=C), 1531, 1490, 1445 (C-
H), 1340, 1257, 1209 (C–OH), 1182, 1128, 1053, 1019, 982, 920, 882, 845, 810, 746,
2
1
697, 654 cm-1; H NMR (500 MHz, DMSO-d6) d: 14.55 (s, 1 H, HO–C(thiazine),
11.82 (s, 1 H, HO–C(3)), 10.90 (s, 1 H, HO–C(1)), 7.85 (d, J = 9.0 Hz, 1 H, H–
C(Ar)), 7.74 (m, 1 H, H–C(Ar)), 7.57 (m, 2 H, H–C(Ar)), 7.49 (d, J = 8.77 Hz, 1 H,
H–C(Ar)), 7.40 (m, 2 H, H–C(Ar)), 7.25 (m, 1 H, H–C(Ar)), 6.45 (d, J = 8.2 Hz, 1 H,
H–C(Ar)), 6.37 (m, 2 H, H–C(Ar)), 1.70 (s, 3 H, CH3) ppm; 13C NMR (125 MHz,
DMSO-d6) d: 207.8, 164.7, 163.0 162.9, 162.6, 140.5, 139.5, 136.3, 129.9, 129.7,
128.6, 128.1, 126.3, 119.6, 114.3, 110.9, 108.7, 107.9, 103.2, 57.9, 19.5 (CH3) ppm;
ESI–MS (m/z): 362.1 [[M-H]-].
4-(Perfluorobenzo[d]thiazol-2-yl)benzene-1,3-diol 3 IR (KBr) mmax: 3445 (OH),
3350 (OH), 1626 (C=N), 1595 (C=N), 1497 (C=C), 1493, 1333, 1291, 1256, 1228
1
(C–OH), 1173, 1139 (C–F), 1090, 1041, 1013, 980, 946, 876, 794, 749 cm-1; H
NMR (100 MHz, DMSO-d6) d: 11.39 (s, 1 H, HO–C(3) (exchangeable in D2O)),
10.31 (s, 1 H, HO–C(1) (exchangeable in D2O)), 8.05 (d, J = 8.7 Hz, 1 H, H–C(5)),
6.51 (d, J = 2.3 Hz, 1 H, H–C(2)), 6.49 (m, 1 H, H–C(6)) ppm; EI-MS (m/z, %):
315 (M?, 100), 259 (7), 258 (32), 226 (4), 144 (6), 69 (3), 40 (5).
4-(6-Amino-5-chlorobenzo[d]thiazol-2-yl)benzene-1,3-diol 4 IR (KBr) mmax: 3443
(OH), 3383 (OH), 2928 (CH), 2850 (CH), 1628 (C=N), 1591 (C=N), 1543 (C=C),
1511 (C=C), 1470, 1384, 1345, 1277, 1230, 1121 (C–OH), 1178 (C–Cl), 991, 871,
123

Synthesis and biological activity of novel benzoazoles,…
1
853, 794, 728 cm-1; H NMR (500 MHz, DMSO-d6) d: 11.86 (s, 1 H, HO–C(3)),
10.37 (s, 1 H, HO–C(1)), 8.30 (s, 1 H, H–C(benzothiazole)), 8.21 (m, 2 H, H–
C(benzothiazole)), H–C(5)), 6.42 (m, 2 H, H–C(2, 6)) ppm; 13C NMR (125 MHz,
DMSO-d6) d: 162.2, 147.2, 135.9, 134.4, 130.1, 128.7, 127.3, 115.8, 112.3 108.1,
106.5, 102.6, 99.5 ppm; EI-MS (m/z, %): 292 (M?, 100), 258 (5), 223 (6), 176 (18),
153 (30), 147 (5), 124 (4), 97 (6), 69 (5), 53 (3).
4-(5-(5-(2,4-Dihydroxyphenyl)-1,3,4-thiadiazol-2-yl)-1H-benzo[d]imidazol-2-yl)ben-
zene-1,3-diol 5 IR (ATR) mmax: 3402 (OH), 3134 (OH), 2940 (OH), 1631 (C=N),
1598 (C=C), 1525 (C=C), 1496, 1469, 1424, 1315, 1301, 1257 (C–OH), 1163, 1139,
1107, 1050, 1018, 981, 964, 846, 800, 752, 691, 659, 633 cm-1. 1H NMR
(400 MHz, DMSO-d6) d: 11.56 (s, 1 H, OH), 11.28 (s, 1 H, OH), 10.82 (s, 1 H,
OH), 10.17 (s, 1 H, OH), 8.39 (d, J = 1.7 Hz, 1 H, H–C(benzoimidazole)(4)), 8.10
(m, 3 H, H–C(Ar)), 7.91 (d, J = 8.4 Hz, 1 H, H–C(6)), 6.75 (d, J = 2.2 Hz, 1 H, H–
C(3)), 6.59 (m, 2 H, H–C(2,6)), 6.48 (dd, J = 8.7 and 2.2 Hz, 1 H, H–C(5)) ppm;
13C NMR (125 MHz, DMSO-d6) d: 164.7, 161.1, 160.3, 157.3, 156.6, 155.6, 132.0,
130.8, 130.6, 129.3, 127.7, 121.2, 120.7, 120.6, 118.9, 112.4, 112.2, 108.6, 108.5,
102.6, 56.1 ppm; ESI–MS (m/z): 419 [M ? H]?.
4-(5,7,8-Trifluoro-1H-benzo[e][1,3,4]thiadiazin-3-yl)benzene-1,3-diol 6 IR (KBr)
mmax: 3223 (OH), 3088 (H–C(Ar)), 1653 (C=N), 1615 (C=C), 1516 (C=C), 1488,
1464, 1416, 1395, 1373, 1315, 1262, 1225 (C–OH), 1180, 1168, 1116, 1076, 985,
1
948, 920, 844, 825, 816, 777, 712 cm-1; H NMR (500 MHz, DMSO-d6) d: 11.93
(s, 1 H, HO–C(3)), 10.18 (s, 1 H, HO–C(1)), 8.92 (s, 1 H, NH), 7.89 (d, J = 9.4 Hz,
1 H, H–C(5)), 7.24 (m, 1 H, H–C(benzothiadiazine)), 6.37 (m, 2 H, H–C(2, 6))
ppm; 13C NMR (125 MHz, DMSO-d6) d: 192.0, 162.1, 157.8, 146.8, 144.9, 138.0,
135.9, 133.3, 127.3 113.9, 107.9, 102.9, 96.5 ppm; EI-MS (m/z, %): 312 (M?, 100),
260 (8), 187 (61), 165 (86), 153 (27), 150 (49), 135 (89), 108 (22), 99 (13), 88 (7),
80 (12), 69 (16), 52 (16), 39 (11).
4-(5,6,7,8-Tetrafluoro-1H-benzo[e][1,3,4]thiadiazin-3-yl)benzene-1,3-diol
(KBr) mmax: 3234 (OH), 1622 (C=N), 1521 (C=C), 1464, 1404, 1347, 1225 (C–
OH), 1173, 1125, 1023, 985, 842, 811, 737, 712 cm-1 1H NMR (500 MHz,
7
IR
;
DMSO-d6) d: 11.96 (s, 1 H, HO-C(3)), 10.11 (s, 1 H, HO-C(1)), 8.83 (s, 1 H, NH),
7.82 (d, J = 9.4 Hz, 1 H, H–C(5)), 6.35 (d, J = 2.3 Hz, 1 H, H–C(2)), 6.33 (m, 1 H,
H–C(6)) ppm; EI-MS (m/z, %): 330 (M?, 100), 318 (6), 312 (7), 243 (3), 196 (52),
183 (54), 168 (54), 155 (24), 153 (24), 135 (100), 124 (13), 108 (29), 99 (10), 80
(14), 77 (6), 69 (23), 63 (9), 52 (19), 44 (11), 39 (14).
4-(6,8-Dichloro-1H-benzo[e][1,3,4]thiadiazin-3-yl)benzene-1,3-diol 8 IR (KBr)
mmax: 3397 (OH), 3214 (OH), 3063 (C(Ar)-H), 1626 (C=N), 1601 (C=C), 1509
(C=C), 1447, 1382, 1352, 1332, 1265, 1231 (C–OH), 1179, 1127, 1106, 1077, 986,
1
944, 887, 861, 851, 824, 799, 774, 725 cm-1; H NMR (100 MHz, DMSO-d6) d:
12.10 (s, 1 H, HO-C(3) (exchangeable in D2O)), 10.21 (s, 1 H, HO-C(1)
(exchangeable in D2O)), 8.05 (d, 1 H, H–C(5)), 7.50 (m, 2 H, H–C(benzothiadi-
azine) (6, 8)), 6.38 (m, 2 H, H–C(2, 6)) ppm; 13C NMR (125 MHz, DMSO-d6) d:
161.7, 157.1, 152.3, 146.3, 138.7, 134.0, 128.7, 126.4, 124.5, 113.2, 109.3, 107.9,
123

J. Matysiak et al.
102.6 ppm; EI-MS (m/z, %): 327 (M?, 84), 294 (3), 210 (4), 195 (37), 167 (9), 158
(9), 153 (100), 136 (21), 124 (7), 108 (5), 97 (8), 69 (5), 63 (4), 52 (5), 39 (4).
2-(2,4-Dihydroxyphenyl)-4H-benzo[d][1, 3]thiazin-4-one 9 (previously the com-
pound was obtained from 2-aminobenzanilide [5])
4-(1H-Perimidin-2-yl)benzene-1,3-diol 10 IR (KBr) mmax: 3412 (OH), 3057
(C(Ar)-H), 2846 (CH), 1622 (C=N), 1533 (C=C), 1482, 1446, 1300, 1229 (C–
OH), 1150, 1032, 891, 843, 815, 791, 717 cm-1; 1H NMR (500 MHz, DMSO-d6) d:
12.60 (s (broad), 2 H, H–C(1, 3), 10.39 (s, 1 H, NH), 7.73 (d, J = 8.7 Hz, 1 H, H–
C(5)), 7.25 (m, 4 H, H–C(Ar)), 6.80 (m, 2 H, H–C(Ar)), 6.47 (d, J = 2.1 Hz, 1 H,
H–C(2)), 6.44 (dd, J = 8.7 and 2.3 Hz, 1 H, H–C(6)) ppm; EI-MS (m/z, %): 276
(M?, 100), 247 (6), 229 (4), 219 (15), 206 (27), 166 (5), 152 (3), 140 (7), 138 (5),
115 (7), 88 (3), 69 (2), 63 (3), 51 (2), 39 (2), 36 (6).
4-(Benzimidazo[1,2-c]chinazolin-6-yl)benzene-1,3-diol 11 IR (KBr): mmax = 3273
(OH), 1640 (C=N), 1620 (C=N), 1600 (C=C), 1521 (C=C), 1449, 1341, 1301, 1230,
1193, 1120, 979, 818, 771 cm-1; 1H NMR (100 MHz, DMSO-d6) d: 10.03 (s, 1 H,
HO–C(3)), 9.93 (s, 1 H, HO–C(1)), 8.65 (dd, J = 7.9 and 1.3 Hz, 1 H, H–C(Ar)),
7.97 (dd, J = 7.9 and 1.1 Hz, 1 H, H–C(Ar)), 7.93 (d, J = 8.1 Hz, 1 H, H–C(5)),
7.89 (td, J = 8.3 and 1.5 Hz, 1 H, H–C(Ar)), 7.77 (td, J = 8,1 and 1.1 Hz, 1 H, H–
C(Ar)), 7.49 (td, J = 7.9 and 1.2 Hz, 1 H, H–C(Ar)), 7.36 (d, J = 8.3 Hz, 1 H, H–
C(Ar)), 7.26 (td, J = 7.9 and 1.1 Hz, 1 H, H–C(Ar)), 6.83 (d, J = 8.4 Hz, 1 H, H–
C(Ar)), 6.59 (d, J = 2.2 Hz, 1 H, H–C(2)), 6.53 (dd, J = 8.2 and 2.2 Hz, 1 H, H–
C(6) ppm; EI-MS (m/z, %): 327 (M?, 92), 326 (100), 310 (13), 298 (6), 281 (5),
280 (5), 270 (5), 256 (5), 163 (8), 141 (3), 135 (4).
4-(3-Chlorodibenzo[b,g][1,4,6]oxathiazocin-6-yl)benzene-1,3-diol 12 IR (KBr):
mmax = 3255 (OH), 3065 (OH), 1678 (C=N), 1619 (C=N), 1594 (C=N), 1500 (C=C),
1472 (C=C), 1452, 1385, 1343, 1308, 1255, 1127, 1099, 1057, 1007, 988, 836, 807,
1
749 cm-1; H NMR (100 MHz, DMSO-d6) d: 11.88 (s, 1 H, HO–C(3)), 10.25 (s,
1 H, HO–C(1)), 8.32–8.09 (m, 1 H, H–C(Ar)), 7.75–7.68 (m, 1 H, H–C(Ar)),
7.48–7.18 (m, 4 H, H–C(Ar)), 6.95–6.70 (m, 2 H, H–C(Ar)), 6.43–6.34 (m, 2 H, H–
C(2, 6)) ppm; EI-MS (m/z, %): 369 (M?, 10), 333 (5), 257 (11), 253 (100), 244
(89), 220 (26), 218 (82), 202 (6), 192 (13), 183 (6), 162 (28), 137 (83), 109 (12), 91
(9), 80 (60), 77 (5), 69 (7), 65 (15) 63 (16) 53 (9), 52 (7), 44 (6), 39 (11), 36 (4).
Computational methods
The Clog P values and molar refractivity MR were calculated using the ChemDraw
Ultra 10.0 according to the fragmentation method introduced by Crippen [40]. The
tPSA was calculated by Virtual Computational Chemistry Laboratory [41]. The
polar surface area (tPSA) was estimated by the atom-based method [42].
123

Synthesis and biological activity of novel benzoazoles,…
Biological assays
Antiproliferative assay
The following human cell lines were applied in the antiproliferative assay in vitro:
T47D (breast cancer), SW707 (rectal adenocarcinoma), and A549 (nonsmall cell
lung carcinoma) from the American Type Culture Collection (Rockville, MD,
USA), and HCV29T (bladder cancer) from the Fibiger Institute, Copenhagen,
Denmark. The SRB test measuring the cell proliferation inhibition in the in vitro
culture was applied [43]. The experiments were repeated at least 3 times. The IC50
values were calculated by Cheburator 0.9.0 software [44]. The details were
described previously [45].
Activity of the compounds against phytopathogenic fungi
In vitro tests estimating the inhibition of mycelium growth in the agar culture
medium caused by the compound under investigation were performed. The five
strains of phytopathogenic fungi: Alternaria alternata, Botrytis cinerea, Rhizoctonia
solani, Fusarium culmorum, and Phytophthora cactorum were used. The solutions
(suspensions) were prepared with concentrations making it possible to obtain 200
and 20 lg/mL of the studied substance after dilution with the agar culture medium
(PDA). Petri scale pans were used into which the agar culture medium and the
studied substance were poured. When the culture medium set, the infectious
material of the tested fungus in the form of agar discs overgrown with mycelium
were placed at three sites on its surface. After 3–5 days (temperature 22 1 °C)
depending on the mycelium culture, the linear growth of the mycelium was
measured. The compound action was determined from the percentage of mycelium
growth inhibition compared with the control using the equation: J = (C - T)/
C 100%, where J is the percentage of mycelium growth inhibition, C the zone of
mycelium growth in the control combination (mm), and T the zone of the mycelium
growth in the combination with the compound (mm). Carbendazim (Sarfun 500 SC;
Organica, Chemical Comp., Nowa Sarzyna, Poland) and procymidone (Sumilex 500
SC; Sumitomo Chemical Comp, Japan) were used as standards. The results are
given in the five-degree scale, determining the percentage of mycelium growth
inhibition compared with the control. Biological studies were carried out in the
Institute of Industrial Organic Chemistry in Warsaw with the SPR/BFF/01/b
procedures (certificate GLP-OECD—1997) [36].
Antifungal activity of the compounds against the fungi Candida
Fifty strains of Candida albicans taken from the mouth cavity of patients suffering
from tumors were used as the selective material. Itraconazole and fluconazole were
administered for prophylaxis or due to the symptoms of candidosis. The isolates
resistant to the antifungal drugs were chosen for the dilution method testing the
compounds. In addition, the strain was used for comparison with the American Type
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J. Matysiak et al.
Culture Collection (University Boulevard, Manassas, VA, USA) C. albicans (ATCC
10231).
Six isolates of non-albicans Candida (two isolates of C. tropicalis, one each of C.
glabrata, C. krusei, C. paratropicalis, and C. tropicalis) (n = 6) were tested. The
drug resistance by FungitestÒ was also determined for them. The MIC values were
determined by the agar dilution procedure according to the National Committee for
Clinical Laboratory Standards reference document M27 [46]. The details were
presented by Matysiak et al. [47]. Itraconazole (Pliva, Krakow, Poland) and
fluconazole (Janssen-Cilag) tested under the same experimental conditions were
used as the references. MIC values were expressed as the average value from 10
measurements for C. albicans and from 6 for non-albicans Candida.
Results and discussion
Chemistry
Using the reaction with sulfinylbis[(2,4-dihydroxyphenyl)methanethione] (STB)
suitable nitrogen bases—amines, hydraznes or hydrazides—new compounds with
an additional heterocyclic ring have been obtained (Fig. 1; Table 1). Initially, the
process was carried out according to the electrophilic substitution to lead to the
corresponding thioamides. The final compounds were afforded by the elimination of
HF molecule (compounds 3, 4, 6, 7) or HCl (8, 12) leading to thiazole or thiazine
rings, respectively. Formation of an azine ring was an effect of H2S elimination
(compounds 5, 10, 11). In the case of the substrates possessing the carbonyl group,
cycloaddition took place and a thiazine or thiazole ring was obtained with the
additional hydroxyl group (1, 2). Formation of the fused 4H-3,1-benzothizin-4-on
system was an effect of the elimination of the corresponding amine (9) [5].
The synthesis was performed in methanol under reflux (2–3 h) with moderate to
good yields (64–88%), as outlined in Fig. 1. The composition and properties of
products 1–12 are summarized in Table 2. Purity of the compounds was monitored
by the reversed-phase HPLC (CP-18, methanol–water mobile phase). The log
k values of the compounds for the selected system are collected in Table 2. The
other analytical data of the compounds confirmed the proposed constructions.
The OH substituents of the compounds were usually registered as broad singlets
1
at ca. 10–12.5 ppm by H NMR spectroscopy. OH protons were exchangeable in
D2O. Similar data for other derivatives with the benzenediol moiety were obtained
[40]. A strong band in the region of about 1630–1600 cm-1 was recorded which
corresponds to the m (C=N) bond in the IR spectra. The bands in the range of ca.
3500–3150 cm-1 confirmed the presence of OH groups. The C=O moiety was
observed for compounds 1 and 10. In the case of compounds 2 and 5, the carbonyl
group of the substrates was not observed. In the EI-MS, the molecular ion peak M?
of varying intensity was registered.
123

Synthesis and biological activity of novel benzoazoles,…
Table 1 Structures of the compounds and substrates applied in the synthesis
Yield
(%)
77
No.
1.
Product
No.
1a
Substrate
O
OH
Me
N
NH2
O
S
OH
N
O
N
O
2.
3.
2a
3a
62
65
HO
S
N
NH2
O
OH
Me
HO
F
F
HO
F
F
F
F
NH2
N
OH
F
S
F
F
4.
5.
4a
5a
65
70
Cl
HO
NH2
Cl
Cl
N
OH
H2N
S
H2N
H2N
HO
N
NH
N
OH
H2N
OH
N
S
O
HO
H2N
F
N
H
6.
7.
6a
7a
73
77
HN NH2
HO
F
HN
N
S
F
F
F
F
OH
OH
F
F
F
F
HN
N
S
F
F
HN NH2
F
F
HO
F
F
123

J. Matysiak et al.
Table 1 continued
Yield
(%)
No.
8.
Product
No.
8a
Substrate
HN NH2
Cl
69
HO
Cl
HN
N
S
Cl
OH
Cl
Cl
9.
9a
79
O
N
H
S
OH
N
NH2
O
OH
10.
11.
10a
11a
80
70
HO
NH2
NH2
N
OH
NH
NH2
N
N
N
N
N
H
OH
HO
12.
12a
80
HO
OH
NH2
Cl
O
N
S
Cl
O
Cl
Biological activity
Cytotoxic activity in vitro of two compounds, 3 and 11, was assessed against four
human cancer cells. Their potency was expressed as IC50 [lM] which is the
concentration of the substance that inhibited the proliferation rate of tumour cells by
50% as compared to the untreated control cells. The results of the tests are presented
in Table 3. They show that both compounds display substantial activity against all
studied cells, similar to that of cisplatin.
Antifungal potency of the selected compounds was assessed in vitro against five
strains of phytopathogenic fungi (Table 4). The research was carried out at two
different concentrations: 20 and 200 lg/mL. They show that the antifungal potency
of compounds is varied, and that the highest activity was found for compound 7
with the 5,6,7,8-tetrafluoro-1H-benzo[e][1,3,4]thiadiazine ring. At the concentra-
tions of 200 and 20 lg/mL the derivative showed fungistatic action against A.
123

Synthesis and biological activity of novel benzoazoles,…
Table 2 Characterization data of compounds 1–12
No.
Formula
M
Elemental analysis
(calcd.)/(found)/ %
M.p. °C
log k
C
H
N
1.
C16H12N2O4S
C21H17NO3S
C13H5F4NO2S
C13H9N2O2SCl
C21H14N4O4S
C13H7F3N2O2S
C13H6F4N2O2S
C13H8N2O2SCl
C17H12N2O2
328.34
363.43
315.24
292.74
418.43
312.27
330.25
327.19
276.29
327.10
369.82
58.53
58.60
69.40
69.51
49.53
49.60
53.34
53.46
60.28
60.21
50.00
49.88
47.28
47.35
47.72
47.80
73.90
74.01
73.38
73.30
61.71
59.70
3.68
3.66
4.71
4.70
1.60
1.59
3.10
3.09
3.37
3.35
2.26
2.27
1.83
1.84
2.46
2.45
4.38
4.40
4.00
4.02
3.27
3.25
8.53
8.56
3.85
3.87
4.44
4.46
9.57
9.60
13.39
13.43
8.97
9.00
8.48
8.44
8.56
8.59
10.14
10.10
12.84
12.80
3.45
3.47
193–195
123–124
197–199
246–248
220–221
146–147
123–125
185–186
210–212
294–296
210–212
0.027b
2.
- 0.277b
0.313b
3.
4.
0.440b
5.
- 0.357a
- 1.107a
- 0.570a
- 0.402a
- 0.175a
- 0.091a
- 0.175a
6.
7.
8.
10.
11.
12.
C20H13N3S
C19H12ClNO3S
aThe log k values for 80% of MeOH (v/v) in the mobile phase
bThe log k values for 65% of MeOH (v/v) in the mobile phase
Table 3 Antiproliferative activity of compounds 3 and 11 against human cancer cell lines
Compound
Cell line/ICa50/(lM)
HCV29T
A 549
T 47D
SW 707
3.
28.30 8.63
7.28 2.04
10.33 3.20
19.38 3.87
5.56 3.73
16.94 5.14
10.73 0.80
21.56 7.57
19.60 4.54
13.85 0.61
34.77 16.53
11.
Cisplatin
22.57 17.89
aIC50 indicates the compound concentration that inhibits the proliferation rate of tumor cells by 50% as
compared to the control untreated cells. The values are the mean SD of nine independent experiments
123

J. Matysiak et al.
Table 4 Antifungal potency of compounds against phytopathogenic fungi
Compound
Estimation of mycelium growth inhibitiona/(lg/mL)
A. alternata B. cinerea R. solani F. culmorum
P. cactorum
200
20
200
20
200
20
200
20
200
20
2.
1
2
0
2
2
0
0
1
0
1
1
0
0
2
0
2
2
0
0
1
0
1
0
0
–
3
0
0
1
2
2
0
3
0
0
0
0
0
0
3
–
0
2
0
2
0
0
3
0
0
0
1
0
0
3
0
2
0
2
1
0
3
0
1
0
0
0
0
3
4.
5.
7.
9.
10.
b
b
b
–
b
Carbendazim
Procymidone
–
–
b
–
b
b
–
b
b
b
2
2
3
–
–
–
aThe results are given in the five-degree scale determining the percentage of mycelium growth inhibition
compared with the control: 0 = 0–20%; 1 = 21–40%; 2 = 41–60%; 3 = 61–80%; 4 = 81–100%
bStudy was not performed
alternate, P. cactorum and B. cinerea at the levels of 41–60% and 21–40%,
respectively. The compounds under consideration show lower potency compared to
the previously described analogs with the 2,4-dihydroxyphenyl moiety [36].
Antifungal activity of the compounds against pathogenic fungi was also
estimated. It was expressed as the average MIC values against several clinical
strains of C. albicans and non-albicans Candida as well as against the reference
strain of C. albicans (Table 5). The results show that the non-albicans Candida
Table 5 Antifungal activity against C. albicans and non-albicans Candida strains expressed in the
average MIC values (lg/mL)a
Compound
C. albicans ATCC 10231b
C. albicansc (n = 10)
Non-albicans Candidad (n = 6)
1.
100.0 0.0
200.0 0.0
100.0 0.0
50.0 0.0
200.0 0.0
100.0 0.0
200.0 0.0
200.0 0.0
95.0 15.8
200.0 0.0
90.0 45.9
50.0 0.0
100.0 0.0
70.0 25.2
47.5 7.9
27.5 7.9
75.0 38.7
83.3 60.6
23.96 15.0
47.9 30.01
100.0 0.0
83.3 60.6
27.5 7.9
4.
6.
7.
8.
12.
Itraconazole
Fluconazole
12.5 0.0
aMIC the minimal inhibitory concentration caused full inhibition of growth in relation to the control (lg/
mL)
bMIC SD (mean standard deviation of three independent experiments)
cMIC SD (MIC values expressed as the average value from 10 isolates standard deviation)
dMIC SD (MIC values expressed as the average value from 6 isolates standard deviation)
123

Synthesis and biological activity of novel benzoazoles,…
Table 6 Molecular descriptors in silico of compounds
2
tPSA (A )
MR (cm3/mol)
˚
Compound
HBD
HBA
C logP
MlogP
NR
RB
1.
2
3
2
4
5
3
3
3
2
3
2
2
6
4
3
4
8
4
4
4
4
4
5
4
2.00
3.39
3.36
3.21
2.81
1.92
1.92
2.99
1.29
4.49
4.12
5.89
1.96
2.91
2.93
1.86
1.33
2.80
2.93
2.93
1.78
3.00
3.58
3.08
4
4
3
3
5
3
3
3
3
4
5
4
2
2
1
1
3
1
1
1
1
1
1
1
82.36
73.05
53.35
79.37
135.38
64.85
64.85
64.85
69.89
64.85
70.65
66.49
84.67
104.09
68.76
76.57
115.72
72.74
73.14
80.73
71.81
80.43
93.19
97.34
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
HBD the number of H-bond donors, HBA the number of H-bond acceptors, Clog P the octanol–water
partition coefficients, MlogP the Moriguchi octanol–water partition coefficient, RB the number of
rotatable bonds, NR the number of rings; tPSA the polar surface area, MR the molar refractivity
clinical isolates are more sensitive to the considered compound than those of C.
albicans. The strongest antifungal activity was observed for compound 7.
To estimate drug-likeness of the compounds, their molecular descriptors were
calculated in silico (Table 6) and Lipinki’s rule of five was applied [48]. The
partition coefficient of compounds was determined in two different ways: log P and
clog P (Table 6). The results show that the compounds with the exception of
compound 12 meet the Lipinki’s criterion and possess drug-like properties.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, dis-
tribution, and reproduction in any medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were
made.
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