3,5-Disubstituted thiadiazine-2-thiones: New cell-cycle inhibitors

Article abstract of DOI:10.1007/s12272-012-0104-0

Two series, a and b, of 3-cyclopentyl or (3-cyclohexyl)-5-substituted-3,4, 5,6-tetrahydro-2H-1,3,5-thiadiazine-2-thiones (THTT) 2a-9a and 3b, 4b, 6b-9b, were synthesized to develop new cell cycle inhibitors. Variable and promising in vitro antiproliferati

Full text of DOI:10.1007/s12272-012-0104-0

Arch Pharm Res Vol 35, No 1, 35-49, 2012
DOI 10.1007/s12272-012-0104-0
3,5-Disubstituted Thiadiazine-2-thiones: New Cell-Cycle Inhibitors
Awwad A. Radwan1,5, Abdullah Al-Dhfyan2, Mohammed K. Abdel-Hamid3, Abdullah A. Al-Badr4, and
Tarek Aboul-Fadl3,4
2
1Pharmaceutical Technology Center, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia, Stem Cell
3
Therapy Program, King Faisal Specialized Hospital and Research Center, Riyadh 11211, Saudi Arabia, Department of
4
Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt, and Department of Pharmaceutical
5
Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia, and Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt
(Received May 17, 2011/Revised July 25, 2011/Accepted July 26, 2011)
Two series, a and b, of 3-cyclopentyl or (3-cyclohexyl)-5-substituted-3,4,5,6-tetrahydro-2H-
1,3,5-thiadiazine-2-thiones (THTT) 2a-9a and 3b, 4b, 6b-9b, were synthesized to develop new
cell cycle inhibitors. Variable and promising in vitro antiproliferative activities were shown
with the synthesized THTT derivatives. Compound 5a with a 5-cyclopentyl group on position-
3 and a glutamine residue on position-5 of the THTT moiety showed maximum activity (IC50
= 8.98 µM). Compound 5a possessed notable cell cycle disrupting and apoptotic activities with
enhanced selectivity against cancer cells, suggesting the potential for the development of new
selective cell cycle inhibitors. There is no evident relationship between the cytotoxic activity of
the tested compounds and their lipophilicity. In addition, a pharmacophore based study was
performed to explain the biological activity on structural bases. A successful model was gener-
ated with a good correlation with the observed activity.
Key words: Tetrahydro-2
H-1,3,5-thiadiazine-2-thiones, Cell-cycle inhibitors, Antiproliferative,
Apoptosis, Pharmacophore, Molecular modeling
factors for oncogenesis and mutagens, such as viruses
and inherited predisposing factors, impair the G1
INTRODUCTION
Unlimited and uncontrolled cell proliferation is a checkpoint function. Consequently, more than half of
characteristic of tumor cells (Lopez-Saez et al., 1998; all human cancer cells with impaired G1 checkpoint
Rew and Wilson, 2000). Interruption of cell cycle has a function rely on the G2 checkpoint to survive against
crucial role in cancer progression (Hartwell and Kastan, the DNA damage caused by most cytotoxic cancer
1994). As a result, carcinogenesis can be controlled by treatments. The G2 cell cycle checkpoint is rarely used
agents that can affect cell proliferation.
by normal cells, which makes a cell cycle G2 checkpoint
Various natural and synthetic agents are gaining abrogation strategy attractive against cancer (Dixon
widespread attention due to their cell cycle regulation and Norbury, 2002; Kawabe, 2004). From a medicinal
and modulation activities (Agarwal, 2000; Agarwal et chemistry point of view, synthetic small molecule
al., 2000; Weinstein, 2000; Chinni et al., 2001; Moragoda modulators of the G2/M checkpoint are of particular
et al., 2001; Donato et al., 2002; Katdare et al., 2002; interest. Several clinically important anticancer com-
Lin, 2002; Lin et al., 2002; Park et al., 2002; Singh et pounds, such as vinca alkaloids and taxanes, are effec-
al., 2002; Sun et al., 2002; Tyagi et al., 2002; Chinami tive in controlling some cancer types through their
et al., 2005). In fact, all the suspected contributory prominent inhibition of G2/M transition. Nonetheless,
all these compounds can disrupt tubulin directly; they
have complex chemical structures that restrict possible
Correspondence to: Tarek Aboul-Fadl, Department of Pharma-
chemical modifications. In addition, several prominent
ceutical Chemistry, College of Pharmacy, King Saud University,
disrupters of tubulin, such as nocodazole and colchicine,
Riyadh 11451, Saudi Arabia
lack antitumor efficacy. Therefore, novel chemical struc-
tures that block G2/M phase transition are valuable
Tel: 966-1-467-7341, Fax: 966-1-467-6220
E-mail: fadl@ksu.edu.sa
35

36
A. A. Radwan et al.
as pharmacological probes and as lead structures for compounds were tested for their cell cycle inhibitory
future therapeutic agents (Lazo and Tamura, 2001).
On the other hand, the tetrahydro-(2
activity and the obtained results were explained based
)-1,3,5-thia- on a molecular modeling study.
H
diazine-2-thione (THTT) nucleus displays different
biological activities (Ertan et al., 1992) including anti-
tubercular, antibacterial, antiviral, antifungal, anthel-
menthic and anticancer activities. It has been postu-
lated that the biological activity of these molecules is
MATERIALS AND METHODS
General
The starting cycloalkylamines and aminocarboxylic
mainly dependent on isothiocyanates and dithiocar- acids were purchased from Sigma-Aldrich. All other
bamic acid species that are generated in the biosystem chemicals were of commercially available research-
upon hydrolysis (Goksoyr, 1964; Schade and Rieche, grade. Melting points were determined on an electro-
1966; Pérez et al., 2000). Dithiocarbamates themse- thermal melting point apparatus (Stuart Scientific,
lves are associated with antifungal, antiviral, immu- Stone), and were uncorrected. Precoated silica gel
nomodulatory, cell cycle disruption and many other bio- plates (kiesel gel 0.25 mm, 60G F254, Merck) were
activities (Sunderman and Sunderman, 1958; Heikkila used for thin layer chromatography (TLC). The devel-
et al., 1976; Sauer et al., 1984; Furuta et al., 2002; oping solvent system was chloroform/methanol (10:3)
Deng et al., 2003; Zhang et al., 2006), which could be and the spots were detected by ultraviolet light. In-
an added advantage in developing new drugs against frared (IR) spectra (KBr disc) were recorded on a FT-
cancer. Furthermore, isothiocyanates isolated from IR spectrophotometer (Perkin Elmer) at the research
cruciferous vegetables have been identified as potent center, College of Pharmacy, King Saud University,
1
anticancer agents in animal and human epidemiologi- Saudi Arabia. H-nuclear magnetic resonance (NMR)
cal studies. They induce cell cycle arrest, which impli- spectra were scanned in dimethylsulfoxide (DMSO)-d6
cates them as chemotherapeutic agents in cells with on a NMR spectrophotometer (Bruker AXS Inc.) oper-
1
multidrug resistance phenotypes (Jakubikova et al., ating at 500 MHz for H and 125.76 MHz for 13C at
2005; Mi et al., 2009). These observations combined the aforementioned research center. Chemical shifts
with our data from studies on the synthesis of dithio- are expressed in d-values (ppm) relative to tetrame-
carbamates and THTT (Aboul-Fadl and El-Shorbagi, thylsilane (TMS) as an internal standard. Exchangeable
1996; Aboul-Fadl and Hassanin, 1999; Abd-Elrahman protons were confirmed by addition of drop of D2O.
et al., 2002; Aboul-Fadl and Khallil, 2002; Aboul-Fadl Mass spectra were taken on a model 320 MS spectro-
and Osman, 2002; Aboul-Fadl et al., 2002; Hussein et meter (Varian) at the aforementioned research center.
al., 2004; Radwan and Hussein, 2006; Hyallah and Mass spectral data are given as m/z (Intensity %).
Radwan, 2007), and as a part of studies in progress in Elemental analyses were done on a model 2400 CHNSO
our laboratories to develop various small molecule analyzer (Perkin Elmer). Cytotoxicity and cell cycle
modulators of the G2/M checkpoint (Sarhan et al., inhibitory activity were determined at the Stem Cell
2010), prompted us to develop thiadiazine-2-thiones as Therapy Program, King Faisal Specialized Hospital
a new class of cell cycle disrupting agents. Moreover, and Research Center.
findings suggesting that amino acid derivatives have
a cell cycle arrest activity (Rose et al., 2005; Lu et al.,
2007; Karunakaran et al., 2008) were considered in
Chemistry
General procedure for synthesis of 3,5-disub-
designing the compounds. The driving idea was that stituted-3,4,5,6-tetrahydro-2
H-1,3,5-thiadiazine-2-
the coupling of amino acids or their isosteres with ap- thiones (2a-9a and 3b, 4b, 6b-9b)
propriate thiadiazine-2-thione skeleton would produce
Carbon disulfide (60 mmol, 3.6 mL) was added por-
a new class of molecules with active cell cycle inhib- tion-wise to a stirred mixture of the cyclopentyl- or
ition activity.
cyclohexyl-amine 1a or 1b (10 mmol) and aqueous po-
In the current work, two series of thiadiazine-2- tassium hydroxide (40%, 20 mmol, 1.4 mL). The reac-
thiones with amino acid components at position 5 tion mixture was stirred for 3 h at ambient temper-
were synthesized. The significance of the nature of ature. Formaldehyde solution (35%, 22 mmol, 1.63 mL)
substituents at position 3 and the toxicity of substitu- was added to the mixture and stirring was continued
ents at position 5 of the THTT ring has been reported for further 1 h. The resulting clear solution was added
(Reiche et al., 1960; Zsolnai, 1971). Optimum activities portion-wise over 15 min to a stirred solution of the
were obtained in compounds bearing lipophilic groups appropriate amino carboxylic acid (10 mmol) in 20 mL
at the third position and hydrophilic groups at the of phosphate buffer (pH 7.8). After stirring for 6 h at
fifth position (Weuffen et al., 1963). The synthesized ambient temperature, the reaction mixture was acidi-

Thiadiazine-2-thiones: New Cell-Cycle Inhibitors
37
fied with dilute hydrochloric acid (5%, 15-18 mL) to 2-(3-Cyclopentyl-2-thio-3,4,5,6-tetrahydro-2
H-1,3,
pH 2 and the stirring was continued for further 30 5-thiadiazin-5-yl)-1,5-pentanedioic acid mono-
min. The precipitate was collected by filtration, wash- amide (5a)
ed with 0.5% hydrochloric acid and dried. The crude 1H-NMR (DMSO-d6): 1.39-1.61 (m, 4H, c-pentyl 2CH2 at
solid was crystallized from methanol to yield the C3 and C4), 1.61-1.87 (m, 4H, c-pentyl 2CH2 at C2 and
targeted 2a-9a and 3b, 4b, 6b-9b. Yields, melting C5), 2.25 (m, 1H, c-pentyl C1), 2.51 (t, 2H, HOOCCHCH2-
points and physicochemical data are given in Table I.
CH2CONH2), 2.63 (t, 2H, HOOCCHCH2CH2CONH2),
3.36 (t, 1H, HOOCCHCH2CH2CONH2), 4.42 (s, 2H, N-
CH2-N), 4.47 (s, 2H, N-CH2-S), 6.08 (s, exchangeable
2H, NH2), 12.01 (br s, exchangeable 1H, CO2H); 13C-
3-(3-Cyclopentyl-2-thio-3,4,5,6-tetrahydro-2
H-1,3,
5-thiadiazin-5-yl)propanoic acid (2a)
1H-NMR (DMSO-d6): 1.17-1.33 (m, 4H, 2CH2, cyclopentyl NMR (DMSO-d6): 21.88 (pentanedioic acid monamide
C2 and C4), 1.43-1.69 (m, 4H, 2CH2, cyclopentyl C2 C3), 23.5 (c-pentyl C3 and C4), 27.01 (c-pentyl C2 and
and C5), 2.2 (t, 2H, -CH2CH2COOH), 2.3 (m, 1H, CH, C5), 33.26 (pentanedioic acid monamide C4), 56.3 (c-
cyclopentyl C1), 2.75 (t, 2H, -CH2CH2COOH), 4.01 (s, pentyl C1), 58.8 (C4), 62.93 (pentanedioic acid mon-
2H, N-CH2-N), 4.06 (s, 2H, N-CH2-S); 13C-NMR (DMSO- amide C2), 64.4 (C6), 174.9 (pentanedioic acid mono-
d6): 23.75 (c-pentyl C3 and C4), 27.66 (c-pentyl C2 and amide C1 and C5), 190.5 (C2); Mass m/z [%]: 331 [M+,
C5), 33.12 (propionic C2), 45.66 (propionic C3), 57.17 9.6], 282 [11.3], 208 [12.4], 147 [16.0], 81 [13.6], 58
(c-pentyl C1), 59.7 (C4), 65.02 (C6), 173.5 (C1), 191.7 [12.1], 56 [28.7], 46 [59.8], 47 [100].
(C2); Mass m/z [%]: 274 [M+, 1.1], 235 [3.6], 163 [11.5],
134 [9.1], 116 [7.7], 110 [9.5], 94 [14.5], 78 [44.2], 61 4-[(3-Cyclopentyl-2-thio-3,4,5,6-tetrahydro-2H-1,3,
[10.2], 58 [12.7], 47 [28.4], 43 [100].
5-thiadiazin-5-yl)methyl]benzoic acid (6a)
1H-NMR (DMSO-d6): 1.04-1.41 (m, 4H, c-pentyl 2CH2
at C3 and C4), 1.55-1.8 (m, 5H, c-pentyl 2CH2 at C2
and C5 and 1CH), 3.79 (s, 2H, N-CH2-Ar-), 3.93 (s, 2H,
4-(3-Cyclopentyl-2-thio-3,4,5,6-tetrahydro-2
5-thiadiazin-5-yl)butanoic acid (3a)
H-1,3,
1H-NMR (DMSO-d6): 1.1-1.28 (m, 4H, c-pentyl 2CH2 N-CH2-N), 4.13 (s, 2H, N-CH2-S), 7.25 (d, 2H, 2CH of
at C3 and C4), 1.29-1.69 (m, 6H, c-pentyl 2CH2 at C2 benzene ring C3 and C5), 7.91 (d, 2H, 2CH of benzene
and C5 and CH2CH2CH2COOH), 2.04 (t, 2H, CH2- ring C2 and C6); 13C-NMR (DMSO-d6): 23.5 (c-pentyl
CH2CH2COOH), 2.05 (m, 1H, c-pentyl C1), 2.48 (t, 2H, C3 and C4), 27.64 (c-pentyl C2 and C5), 52.05 (c-
CH2CH2CH2COOH), 4.01 (s, 2H, N-CH2-N), 4.07 (s, pentyl C1), 56.08 (C-benzylic), 59.61 (C4), 64.31 (C6),
2H, N-CH2-S); 13C-NMR (DMSO-d6): 22.69 (butanoic 128.2 (ar C3 and C5), 130.66 (ar C2 and C6), 141.0 (ar
C3), 23.5 (c-pentyl C3 and C4), 27.68 (c-pentyl C2 and C1 and C4), 167.92 (C carboxylic), 191.42 (C2); Mass
C5), 33.47 (butanoic C2), 48.97 (butanoic C4), 57.71 (c- m/z [%]: 336 [M+, 2.4], 334 [1.3], 325 [1.2], 293 [1.1],
pentyl C1), 59.31 (C4), 64.74 (C6), 175.17 (C1), 191.73 282 [4.8], 160 [5.8], 78 [11.8], 58 [9.6], 47 [21.9], 40
(C2); Mass m/z [%]: 288 [M+, 8.2], 267 [4.2], 265 [6.0], [50.4], 44 [74.1], 45 [100], 43 [64.9].
135 [8.7], 77 [43.7], 61 [11.7], 58 [2.2], 47 [24.5], 43
[47.3], 45 [100].
4-[2-(3-Cyclopentyl-2-thio-3,4,5,6-tetrahydro-2
H-
1,3,5-thiadiazin-5-yl)ethyl]benzoic acid (7a)
5-(3-Cyclopentyl-2-thio-3,4,5,6-tetrahydro-2
H-1,3,
1H-NMR (DMSO-d6): 1.52-1.70 (m, 4H, c-pentyl 2CH2
5-thiadiazin-5-yl)pentanoic acid (4a)
at C3 and C4), 1.70-1.88 (m, 4H, c-pentyl 2CH2 at C2
1H-NMR (DMSO-d6): 1.38-1.49 (m, 4H, c-pentyl 2CH2 and C5), 2.79-3.13 (m, 5H, c-pentyl C1 and -CH2CH2-),
at C3 and C4), 1.5-1.61 (m, 8H, c-pentyl 2CH2 at C2 4.32 (s, 2H, N-CH2-N), 4.43 (s, 2H, N-CH2-S), 7.39 (d,
and C5 and CH2CH2CH2CH2COOH), 1.94 (m, 1H, c- Ar-Hs at C2 and C6), 7.88 (d, 2H, Ar-Hs at C3 and
pentyl C1), 2.25 (t, 2H, CH2CH2CH2CH2COOH), 2.66 C5), 12.0 (br s, 1H, COOH); 13C-NMR (DMSO-d6): 23.4
(t, 2H, CH2CH2CH2CH2COOH), 4.23 (s, 2H, N-CH2- (c-pentyl C3 and C4), 26.9 (c-pentyl C2 and C5), 33.0
N), 4.26 (s, 2H, N-CH2-S); 13C-NMR (DMSO-d6): 22.15 (N-CH2CH2), 49.9 (N-CH2CH2), 56.2 (c-pentyl C1),
(pentanoic C3), 23.69 (c-pentyl C3 and C4), 26.18 pen- 58.8 (C4), 64.6 (C6), 128.8 (Ar-C2 and Ar-C6), 129.5
tanoic C4), 27.71 (c-pentyl C2 and C5), 33.62 (pentanoic (Ar-C3 and Ar-C5), 144.7 (Ar-C1), 167.2 (CO2H), 190.5
C2), 49.26 (pentanoic C5), 56.83 (c-pentyl C1), 59.3 (C2); Mass m/z [%]: 341 [12.6], 283 [1.9], 281 [3.7],
(C4), 64.82 (C6), 175.6 (C1), 191.76 (C2); Mass m/z 209 [16.7], 207 [100], 193 [19.5], 147 [14.9], 135 [14.8],
[%]: 302 [M+, 2.5], 292 [11.2], 281 [14.8], 276 [7.0], 250 117 [26.3], 91 [38.2], 73 [56.1], 63 [33.6], 58 [2.9], 52
[4.7], 248 [14.6], 223 [4.8], 165 [6.8], 146 [12.2], 107 [7.5], 51 [4.0].
[13.1], 78 [40.3], 54 [20.4], 46 [27.8], 44 [41.8], 43
[54.7], 40 [100].

38
A. A. Radwan et al.
4-(3-Cyclopentyl-2-thio-3,4,5,6-tetrahydro-2
5-thiadiazin-5-yl)cyclohexanecabroxylic acid (8a)
H
-1,3,
C3-C5 and CH2CH2CH2CH2COOH), 1.71-1.84 (m, 4H,
c-hexyl), 1.85 (m, 1H, c-hexyl C1), 2.21 (t, 2H, CH2 at
1H-NMR (DMSO-d6): 1.24-1.60 (m, 4H, c-pentyl 2CH2 CH2CH2CH2CH2COOH), 2.51 (t, 4H, CH2CH2CH2CH2-
at C3 and C4), 1.67-1.89 (m, 14H, c-pentyl Hs at C2 COOH), 4.38 (s, 2H, N-CH2-N), 4.47 (s, 2H, N-CH2-S);
and C5 and c-hexyl Hs at C1-C6), 2.72-2.85 (m, 1H, c- 13C-NMR (DMSO-d6): 22.76 (c-hexyl C4), 23.08 (pen-
pentyl C1), 4.50 (s, 2H, N-CH2-N), 4.51 (s, 2H, N-CH2- tanoic C3), 23.61 (pentanoic C4), 27.07 (c-hexyl C3
S), 12.05 (br s, 1H, CO2H); 13C-NMR (DMSO-d6): 23.2 and C5), 31.26 (c-hexyl C2 and C6), 32.27 (pentanoic
(c-pentyl C3 and C4), 24.3 (c-hexane C3, C5), 26.8 (c- C2), 55.67 (pentanoic C5), 57.44 (c-hexyl C1), 59.14
hexane C2, C6), 26.9 (c-pentyl C2 and C5), 42.6 (c- (C4), 63.7 (C6), 181.11 (pentanoic C1), 191.14 (C2);
hexane C4), 54.1 (c-pentyl C1), 57.4 (C1), 59.1 (C4), Mass m/z [%]: 316 [M+, 10.2], 281 [12.9], 224 [9.9],
62.1 (C6), 176.1 (CO2H), 191.2 (C2); Mass m/z [%]: 207 [15.3], 178 [15.3], 154 [12.3], 122 [9.9], 101 [14.3],
231 [1.7], 191 [2.4], 189 [25.7], 156 [2.2], 143 [24.2], 78 [10.2], 75 [21.4], 61 [34.2],58 [7.1], 47 [69.1], 43
112 [2.4], 111 [37.7], 110 [100], 96 [22.4], 82 [52.6], 78 [14.9], 40 [100].
[31.9], 76 [48.6], 63 [32.0], 58 [2.3].
4-[(3-Cyclohexyl-2-thio-3,4,5,6-tetrahydro-2
H-1,3,
4-[(3-Cyclopentyl-2-thio-3,4,5,6-tetrahydro-2H-1,3,
5-thiadiazin-5-yl)methyl]cyclohexanecabroxylic
5-thiadiazin-5-yl)methyl]benzoic acid (6b)
1H-NMR (DMSO-d6): 0.98-1.43 (m, 6H, c-hexyl 3CH2
acid (9a)
at C3-C5), 1.58-1.79 (m, 4H, c-hexyl 2CH2 at C2 and
1H-NMR (DMSO-d6): 0.90-1.34 (m, 4H, c-hexane C3 C6), 2.05 (m, 1H, c-hexyl C1), 3.63 (s, 2H, benzyl CH2),
and C5), 1.52-1.60 (m, 4H, c-pentyl 2CH2 at C3 and 4.06 (s, 2H, N-CH2-N), 4.36 (s, 2H, N-CH2-S), 7.32 (d,
C4), 1.71-1.9 (m, 9H, 2CH2 at C2 and C5 and c-hexane 2H, 2CH of Ar C3 and C5), 7.94 (d, 2H, 2CH of Ar C2
C2, C4, C6), 2.11-2.16 (m, 1H, c-hexane C1), 2.46 (d, and C6); 13C-NMR (DMSO-d6): 23.15 (c-hexyl C4), 24.96
2H, N-CH2-), 2.51-2.70 (m, 1H, c-pentyl C1), 4.41 (s, (c-hexyl C3 and C5), 30.9 (c-hexyl C2 and C6), 56.27
2H, N-CH2-N), 4.46 (s, 2H, N-CH2-S), 11.99 (br s, 1H, (N-C-Ar), 57.85 (c-hexyl C1), 58.2 (C4), 62.1 (C6),
CO2H); 13C-NMR (DMSO-d6): 23.4 (c-pentyl C3 and 128.91 (Ar C3 and C5), 130.0 (Ar C2 and C6), 141.03
C4), 26.9 (c-pentyl C2 and C5), 28.2 (c-hexane C3,C5), (Ar C1), 143.48 (Ar C4), 168.68 (CO2H), 191.9 (C2);
29.7 (c-hexane C2, C6), 34.5 (N-CH2), 42.6 (c-hexane Mass m/z [%]: 350 [M+, 7.7], 281 [6.5], 223 [6.8], 191
C4), 55.3 (c-pentyl C1), 57.2 (c-hexane C1), 58.7 (C4), [8.0], 149 [12.9], 112 [6.9], 73 [13.5], 64 [46], 62 [75.8],
65.0 (C6), 176.7 (CO2H), 190.6 (C2); Mass m/z [%]: 58 [10.7], 48 [25.5], 43 [100].
341 [M+-1, 1.1], 288 [1.4], 281 [5.2], 267 [1.8], 221 [3.0],
209 [2.1], 193 [2.1], 189 [22.0], 143 [16.8], 124 [4.4], 4-[2-(3-Cyclohexyl-2-thio-3,4,5,6-tetrahydro-2
H-1,
110 [91.7], 96 [16.2], 78 [80.2], 68 [28.5], 63 [100], 58 3,5-thiadiazin-5-yl)ethyl]benzoic acid (7b)
[1.1].
1H-NMR (DMSO-d6); 1.12-1.26 (m, 6H, c-hexyl C3-
C5), 1.50-1.72 (m, 4H, c-hexyl C2 and C6), 1.75-1.77
-1,3,5- (m, 1H, c-hexyl C1), 2.91 (m, 4H, N-CH2CH2-Ar), 4.45
(s, 4H, N-CH2-N and N-CH2-S), 7.37 (d, 2H, Ar C2 and
4-(3-Cyclohexyl-2-thio-3,4,5,6-tetrahydro-2
thiadiazin-5-yl)butanoic acid (3b)
H
1H-NMR (DMSO-d6): 1.1-1.4 (m, 6H, c-hexyl 3CH2 at C5), 7.87 (d, 2H, Ar C4 and C6); 13C-NMR (DMSO-d6):
C3-C5), 1.5-1.8 (m, 6H, c-hexyl 2CH2 at C2 and C6 24.5 (c-hexyl C4), 25.2 (c-hexyl C3 and C5), 27.8 (c-
and CH2CH2 CH2COOH), 2.0 (m, 1H, CH), 2.28 (t, 2H, hexyl C2 and C6), 33.0 (N-CH2CH2), 50.1 (N-CH2CH2),
CH2CH2CH2COOH), 2.71 (t, 2H, CH2CH2CH2COOH), 56.1 (c-hexyl C1), 57.5 (C4), 64.5 (C6), 128.8 (Ar-C2
4.22 (s, 2H, N-CH2-N), 4.32 (s, 2H, N-CH2-S); 13C-NMR and Ar-C6), 129.3 (Ar-C3 and Ar-C5), 144.6 (Ar-C1),
(DMSO-d6): 22.68 (c-hexyl C4), 24.73 (butanoic C3), 167.3 (CO2H), 189.7 (C2); Mass m/z [%]: 141 [50.7], 98
25.5 (c-hexyl C3 and C5), 28.89 (c-hexyl C2 and C6), [3.6], 83 [47.1], 82 [19.5], 67 [18.0], 63 [14.1], 58 [3.2],
31.68 (butanoic C2), 49.1 (butanoic C4), 58.16 (c-hexyl 55 [100].
C1), 58.18 (C4), 64.7 (C6), 175.7 (COOH), 191.03 (C2);
Mass m/z [%]: 302 [M+, 5.7], 297 [4.5], 293 [4.9], 291 4-(3-Cyclohexyl-2-thio-3,4,5,6-tetrahydro-2
H-1,3,5-
[1.7], 285 [2.5], 282 [5.8], 277 [3.1], 267 [12.7], 194 thiadiazin-5-yl)cycloexane-1-carboxylic acid (8b)
[15.2], 158 [21.4], 135 [14.4], 96 [20.5], 72 [31.8], 63 1H-NMR (DMSO-d6): 1.15-1.27 (m, 4H, N5-c-hexyl C2
[61.5], 58 [11.2], 43 [25.5], 44 [100].
and C6), 1.55-1.94 (m, 14H, N3-c-hexyl C2-C6 and N5-
chexyl C3 and C5), 2.40-2.51 (m, 2H, N5-chexyl C1
-1,3,5- and C4), 2.73-2.84 (m, 1H, N4-c-hexyl C1), 4.63 (s, 2H,
N-CH2-N), 4.67 (s, 2H, N-CH2-S), 12.08 (br s, 1H,
5-(3-Cyclohexyl-2-thio-3,4,5,6-tetrahydro-2
H
thiadiazin-5-yl)pentanoic acid (4b)
1H-NMR (DMSO-d6): 1.35-1.68 (m, 10H, 5CH2 at c-hexyl CO2H); 13C-NMR (DMSO-d6): 24.2 (N3-c-hexane C4),

Thiadiazine-2-thiones: New Cell-Cycle Inhibitors
39
24.5 (N5-c-hexane C3 and C5), 25.3 (N3-c-hexane C3 pression, PTEN loss and constitutive activation of the
and C5), 26.8 (N5-c-hexane C2 and C6), 28.0 (N3-c- mitogen-activated protein kinase (MAPK)/extracel-
hexane C2 and C6), 53.8 (N5-c-hexane C1), 54.9 (N3-c- lular signal-regulated kinase (ERK) kinase/ERK path-
hexane C1), 57.8 (C4), 62.0 (C6), 176.1 (C2), 190.4 way (Oliveras-Ferraros et al., 2008). MCF-12 cells were
(CO2H); Mass m/z [%]: 343 [M++1, 0.1], 143 [1.0], 127 cultured in DMEM/F-12 (GIBCO) supplemented with
[9.1], 112 [1.2], 111 [3.2], 110 [23.2], 83 [15.4], 81 10
[70.9], 78 [59.9], 69 [11.0], 68 [100], 58 [1.9].
µg/mL insulin (Sigma-Aldrich), 500 ng/mL hydro-
cortisone (Sigma-Aldrich), 10% fetal bovine serum
(FBS; Lonza), 10 ng/mL EGF (BD Biosciences) and 1%
ABM (GIBCO). MCF-7 and MDA-MB-468 cells were
4-[(3-Cyclohexyl-2-thio-3,4,5,6-tetrahydro-2H-1,3,5-
thiadiazin-5-yl)methyl]cycloexane-1-carboxylic cultured in DMEM medium (Sigma-Aldrich) supple-
acid (9b)
mented with 10% FBS (Lonza) and 1% ABM (GIBCO).
0.85-0.96 (m, 1H, N5-c-hexyl C4), 1.13-1.35 (m, 4H, For treatment, 2 × 105 cells were cultured in 6-well
N5-c-hexyl C3 and C5), 1.50-1.88 (m, 14H, N3-c-hexyl plates overnight and fresh medium containing the
C2-C6 and N5-c-hexyl C2 and C6), 2.11-2.16 (m, 1H), desired concentration of the test compound was
2.44 (d, 2H, N-CH2-), 2.51-2.91 (m, 1H, N3-c-hexyl C1), added.
4.40 (s, 2H, N-CH2-N), 4.93 (s, 2H, N-CH2-S), 12.00 (br
s, 1H, CO2H); 13C-NMR (DMSO-d6): 24.5 (N3-c-hexyl
WST-1 cell proliferation assay
C4), 25.2 (N3-c-hexyl C3 and C5), 27.7 (N5-c-hexyl C3,
K562 chronic myelogenous leukemia cells were
C5), 28.2 (N3-c-hexyl C2 and C6), 29.7 (N5-c-hexyl C2, purchased from the American Type Culture Collec-
C6), 34.4 (N-CH2), 42.6 (N5-c-hexyl C4), 55.6 (N3-c- tion. Cells were maintained in RPMI 1640 (Sigma-
hexyl C1), 57.0 (N3-c-hexyl C1), 57.4 (C4), 65.0 (C6), Aldrich), supplemented with 10% FBS (Lonza), 100
176.7 (CO2H), 189.9 (C2); Mass m/z [%]: 356 [M+, 0.3], IU/mL penicillin, 100 mg/mL streptomycin and 2 mmol/
341 [1.5], 324 [1.6], 299 [1.4], 279 [2.3], 266 [2.8], 207 L L-glutamine (Sigma-Aldrich). Cells were seeded into
[15.6], 203 [22.8], 147 [10.1], 133 [3.0], 124 [65.8], 110 96-well plates at 4 × 103/well and incubated overnight.
[13.4], 96 [12.3], 91 [15.0], 82 [49.6], 78 [81.2], 67 The medium was replaced with fresh medium con-
[14.7], 63 [100], 58 [19.4].
taining the desired concentrations of the compounds.
After 48 h, 10 L of the WST-1 reagent was added to
each well and the plates were reincubated for 4 h at
µ
Calculation of log P values
The log P values of the synthesized derivatives were 37oC. The amount of formazan was quantified using
computed with a routine method called calculated log an ELISA reader at 450 nm.
P (Clog P) contained in a PC-software package (Mac-
LogP 2.0, BioByte). A representation of the molecular
Flow cytometry analysis of cellular DNA con-
structure where hydrogens are omitted, or ‘suppressed’ tent
(SMILES notation), is entered into the program, which
Cells 2 × 106 cells were fixed in 1 mL ethanol (70%)
computes the log P based on the fragment method for 60 min at room temperature. Harvested cells were
developed by Leo (1993). Data are given in Table I.
resuspended in 1 mL Na citrate (50 mM) containing
250
g RNase A and incubated at 50oC for 60 min.
Next, cells were resuspended in the same buffer
containing 4 g propidium iodine (PI) and incubated
µ
Biological investigations
Cell line
µ
MCF-12A cell line is a non-tumorigenic epithelial for 30 min before being analyzed by flow cytometry
cell line established from tissue taken at reduction (Becton Dickinson). The percentage of cells in various
mammoplasty from a nulliparous patient with fibro- cell cycle phases was determined by using Cell Quest
cystic breast disease, which contains focal areas of Pro software (Becton Dickinson).
intraductal hyperplasia (Paine et al., 1992). MCF7
cells are mammary adenocarcinoma cells that express
Measurement of Annexin V binding by flow
very high levels of estrogen receptor (ER), are nega- cytometry
tive for HER2/neu, and do not have strong anchorage-
Loss of phospholipid asymmetry of the plasma mem-
independent properties (Brooks et al., 1973). MDA-MB- brane is an early event of apoptosis (Fadok et al.,
468 cells were derived from a triple-negative breast 1992; Koopman et al., 1994). Annexin V binds to nega-
carcinoma that shows many of the recurrent basal- tively charged phospholipids such as phosphatidyl-
like molecular abnormalities including ER-progester- serine. During apoptosis, cells react to Annexin V once
one receptor (PR)-HER2–negative status, p53 defici- the chromatin condenses, but before the plasma mem-
ency, epidermal growth factor (EGF) receptor overex- brane loses its ability to exclude propidium iodide (PI).

40
A. A. Radwan et al.
Hence, by staining cells with a combination of fluo- et al., 1995a, 1995b, 1995c).
rescein isothiocyanate (FITC) Annexin V and PI, it is
Molecular flexibility was taken into account by con-
possible to detect non-apoptotic live cells, early apopto- sidering each compound as a collection of conformers
tic cells and late apoptotic or necrotic cells (Vermes et representing a different area of conformational space
al., 1995; Del Bino et al., 1999). Annexin V staining accessible to the molecule within a given energy range.
was performed using a Vybrant Apoptosis Assay Kit DS provides two types of conformational analysis: fast
#2 (Molecular Probes) following the manufacturer’s and best quality. The best option was used, specifying
recommendations. Annexin V stained cells were ana- 250 as the maximum number of conformers. The mole-
lyzed by flow cytometry, measuring the fluorescence cules associated with their conformational models
emission at 530 and less than 575 nm.
were submitted to HipHop hypothesis generation.
Hypotheses approximating the pharmacophore were
described as a set of features distributed within a 3D
Molecular modeling
All experiments were conducted on an Intel Pen- space. This process only considered surface accessible
tium dual-core processor workstation with 2 MB cache functions such as hydrogen-bond acceptor (HBA),
memory and 4 GB RAM. Hypotheses generation was hydrogen-bond acceptor lipid (HBAl), hydrogen-bond
applied against previously described data sets; their donor (HBD), hydrophobic (Hp), hydrophobic aromatic
functionality is available as part of Accelrys Discovery (HpAr), hydrophobic aliphatic (HpAl), negative ion-
Studio 2.5 (DS) software (Accelrys). Molecules were izable (Ni), and positive ionizable (Pi). Misses (the
edited using DS 3D visualization, where conformational number of molecules that do not have to map to all
models for each compound were generated using the features in generated hypotheses), Feature Misses
Poling Algorithm (Smellie et al., 1995a, 1995b, 1995c). (the number of maximal molecules that do not have to
The number of conformations needed to produce a map to each feature in generated hypotheses), and
good representation of a compound's conformational Complete Misses (the number of molecules that do not
space depends on the molecule. Conformation generat- have to map to any feature in a given hypothesis)
ing algorithms were adjusted to produce a diverse set were set as 3, 2, and 2, respectively. A “Principal”
of conformations, avoiding repetitious groups of all value of 2 and MaxOmit features of 0 were assigned to
conformations representing local minima. The confor- all training set molecules with an inter-feature value
mations generated were used to align common mole- of not less than 3.0 Å.
cular features and generate pharmacophoric hypoth-
Mathematical model generation was done using
eses. HipHop used conformations generated to align Microsoft Excel 2007 software in Microsoft Office 2007
chemically important functional groups common to (Microsoft) and SPSS software version 10.0 (SPSS).
the molecules in the study set. A pharmacophoric hypo-
thesis then was generated from these aligned struc-
tures. The models emphasized conformational diversity
under the constraint of 20 kcal/mol energy threshold
above the estimated global minimum based on use of
RESULTS
Chemistry
The target compounds, 2-9a and 3b, 4b, 6b-9b,
the CHARMm force field (Brooks et al., 1983; Smellie were synthesized by reaction of cyclopentylamine, 1a
,
Scheme 1. Synthesis of 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazine-2-thione derivatives, 2a-9a and 3b, 4b, 6b-9b

Thiadiazine-2-thiones: New Cell-Cycle Inhibitors
41
Table I. Physicochemical constants of 5-(Cyclopentyl)-3-substituted-tetrahydro-2H-1,3,5-thiadiazine-2-thione derivati-
ves, 2a-9a, and 5-(Cyclohexyl)-3-substituted-tetrahydro-2H-1,3,5-thiadiazine-2-thione derivatives, 3b 4b 6b 9b
,
,
-
Moleculara
formula (M. Wt)
Yield
(%)b
M.P.
(oC)
Compd. No.
R
R’
Clog P
0.18
2a
Cyclo-pentyl
-CH2CH2CO2H
C11H18N2O2S2
(274.40)
C12H20N2O2S2
(288.43)
C13H22N2O2S2
(302.46)
C13H21N3O3S2
(331.45)
C16H20N2O2S2
(336.47)
C17H22N2O2S2
(350.50)
C15H24N2O2S2
(328.49)
C16H26N2O2S2
(342.52)
67
70
58
63
72
68
56
67
133-4
123-3
128-9
117-8
130-1
148-9
141-3
167-8
3a
4a
5a
6a
7a
8a
9a
-CH2CH2 CH2CO2H
-CH2CH2 CH2CH2CO2H
-CH(CO2H)CH2 CH2CONH2
0.09
0.12
1.01
1.42
1.79
1.02
0.63
3b
4b
6b
7b
8b
Cyclo-hexyl
-CH2CH2 CH2CO2H
C13H22N2O2S2
(302.46)
C14H24N2O2S2
(316.48)
C17H22N2O2S2
(350.50)
C18H24N2O2S2
(364.53)
66
60
81
61
73
132-3
181-3
144-5
176-7
173-4
0.64
0.68
1.97
2.35
0.46
-CH2CH2 CH2CH2CO2H
C16H26N2O2S2
(342.52)
9b
C17H28N2O2S2
(356.55)
50
191-2
0.07
aElemental analyses were satisfactory within ± 0.5% of the calculated values; bCrude product.
or cyclohexylamine, 1b, with carbon disulfide and cell growth inhibition assay. The general in vitro anti-
potassium hydroxide to produce the corresponding proliferative evaluation of the synthesized compounds
dithiocarbamate potassium salts, which were not was conducted by the use of WST-1 reagent against
isolated. This was followed by cyclocondensation reac- K562 chronic myelogenous leukemia cells for deter-
tion with formaline and appropriate amino acid to pro- mination of IC50 (Table II).
vide the nitrogen atom at position 5 of the thiazidine
ring (Scheme 1 and Table I). Structures of the syn-
thesized compounds were verified on the bases of
Selective apoptotic effects of 5a
Treatment of MCF-7 and MDA-MB-468 breast cancer
spectral and elemental methods of analyses, and were cells with 5a (10 µM) for 48 h increased apoptosis by
consistent with the proposed structures.
33.37% and 30.17%, respectively (Fig. 1). The effect
was not observed when normal cells were treated with
5a under the same conditions and concentration using
Annexin V staining. Of note, the cytotoxicity of 5a was
Investigation of biological activity
In Vitro antiproliferative activity
Antiproliferative activity was measured in vitro by a not associated with any induction of necrosis com-

42
A. A. Radwan et al.
Fig. 1. Apoptotic effects of 5a on normal breast cell line MCF-12 and cancer breast cell line MDA-MB-468. (A) MCF-12 and
MDA-MB-468 cells were harvested after treatment by 5a for 48 h and incubated with annexin V-FITC and PI. Ten thousand
cells were analyzed per determination. Dot plots show Annexin V-FITC binding on the X axis and PI staining on the Y axis.
Dots represent cells as follows: lower left quadrant, normal cells (FITC/PI); lower right quadrant, apoptotic cells (FITC+/PI);
upper left quadrant, necrotic cells (FITC/PI+). (B) Selective apoptotic effects of 5a/10 µM treatment for 48 h. Apoptosis was
increased by 45% and 37% in MCF-7 and MDA-MB-468 breast cancer cells, respectively, while the compound was well
tolerated by MCF-12 normal breast cells.
pared to untreated cells.
Cell cycle blocking activity of 5a
To gain insight into the mechanism by which anti-
proliferation was achieved, the effect on cell cycle dis-
tribution was investigated by fluorescence-activated
cell sorting (FACS) analysis. Compound 5a showed
selectivity to block cancer cells in S phase. Treatment
of MDA-MB-468 cells increased the number of cells in
S phase by 1.5-fold compared with untreated cells.
Fig. 2. Cell cycle-specific blocking activity of 5a on MDA-
MB-468. MDA-MB-468 cells were treated with (10 µM) or
Increased proportion of the cells in S phase was not
associated with compensation (decrease) in G0/G1
phase, which indicates a novel mechanism of action
rather than binding to DNA. Further studies will be
required to clarify this observation. No increase in
sub-G0/G1 phase or M phase was detected, which
showed the specificity in blocking activity of 5a toward
S phase (Fig. 2).
without compound and analyzed at 0 h and 48 h by DNA
flow cytometry. Histograms show the number of cells per
channel (vertical axis) vs DNA content (horizontal axis). The
values indicate the percentage of cells in the relevant
phases of the cell cycle. The analysis show arrest of cells in
S phase was increased by 1.5-fold compared with untreated
cells.
Lipophilicity of the synthesized compounds
The antiproliferative activity in the in vitro assays
prompted us to investigate the lipophilicity of the
DISCUSSION
The target compounds, 2-9a and 3b, 4b, 6b-9b, were
synthesized compounds. Lipophilicity of the synthesiz- synthesized and their structures were confirmed on
ed compounds expressed in the term of their Clog P the bases of spectral and elemental methods of analy-
values (Table I). Computation of the log P was based sis. All spectral data were in accordance with the as-
on the fragment method developed by Leo (1993) con- sumed structures. The most differentiating stretching
tained in a PC-software package.
bands in the IR spectra of the target compounds, 2-9a

Thiadiazine-2-thiones: New Cell-Cycle Inhibitors
43
Table II. Cell growth inhibitory activity of the synthe-
sized THTT derivatives
obtained biological activity was subjected to a mole-
cular modeling study for further investigation. The
highest antiproliferative activity was observed with
the derivative 5a, a result that nominates this mole-
cule for further biological evaluations including its
apoptotic and cell cycle blocking activities in addition
to its selectivity towards cancer cells.
For an anticancer drug, it is critical to kill those
cells with highest tumorigenic potential, where it can
be tolerated very well by surrounding normal tissues.
Many of the side effects of chemotherapeutic agents
result in their lack of selectivity toward cancer cells
rather than normal cells, especially those cells with
high proliferative nature. Therefore, developing novel
small molecules targeting only cancer cells for a given
organ is valuable as pharmacological probes and re-
presents possible lead structures for future thera-
peutic agents. Based on the phenotypic characters of
breast cancer cells used in this study, 5a induced
apoptosis in ER positive breast cancer cells (MCF-7)
as well as triple negative breast cancer cells (MDA-
MB-468). Furthermore, compound 5a showed selecti-
vity to block cancer cells in S phase. Therefore, these
results indicate the need to develope THTT deriva-
a
Compound
IC50
2a
3a
4a
5a
6a
7a
8a
9a
3b
4b
6b
7b
8b
9b
NAb
19.72 ± 0.011
10.30 ± 0.041
18.98 ± 0.011
10.03 ± 0.041
16.85 ± 0.111
11.12 ± 0.061
11.56 ± 0.069
19.81 ± 0.007
NAb
18.14 ± 0.111
13.21 ± 0.011
10.60 ± 0.009
15.92 ± 0.051
aIC50: concentration of the compound (
µM) producing 50%
cell growth inhibition after 48 h of compound exposure, as
determined by the WST-1 assay. Each experiment was run
at least two times, and the results are presented as
average values ± S.D.; bNA: Compounds having IC50 value
> 100 µM.
and 3b
,
4b
,
6b-9b, were stretching absorption for the tives that may be used for treatment of breast cancer,
1
carboxylic acid group at the range of 1695-1722 cm−
which is clinically characterized as more aggressive
and 3490-3525 cm− for the carbonyl and hydroxyl and less responsive to standard treatment.
1
functionalities respectively. Furthermore, aliphatic C-
H stretching around 2895-2995 cm−1, aromatic C-H
Relationship between lipophilicity and anti-
proliferative activity
Lipophilicity is a well-known physico-chemical factor
stretching around 3030-3135 cm−1 and C=S stretching
around 1453-1500 cm−1 were observed. In the NMR
spectra, with the exception of the N3-substituents of affecting biological activities, characterizes the distri-
the THTT moiety, the resonance of the remaining sets bution process of compound in humans and is a key
of the protons of the synthesized derivatives were factor of both pharmacokinetic and pharmacodynamic
almost superimposable in each of the two series, 2-9a properties of drug molecules (plasma protein binding,
and 3b, 4b, 6b-9b. The protons of the C-4 and the C- blood–brain barrier (BBB) penetration, and penetra-
6 methylenes of the THTT ring in 7b appeared as one tion through cell membranes) (Pliska et al., 1996; van
singlet integrating four protons. However, they were De Waterbeemd et al., 2001; Lesyk et al., 2006). Pres-
separated as two singlets each of two protons in the ently, there was no evident relationship between the
remaining derivatives. In general the 1H- and 13C-NMR cytotoxic activity of the tested compounds and their
signals of each THTT derivatives in both series,
a
and lipophilicity. Clearly, lipophilicity has an influence on
b, showed a similar trend in the chemical shift of the activity, but it does not solely determine the cytotoxic
common part of the molecular backbone.
activity of these compounds.
Molecular ion peaks of the synthesized THTT deri-
vatives were detected, except for derivatives 7a 8a
,
,
Molecular modeling studies
7b. However, they revealed the same fragmentation
pattern shown with the other derivatives.
The aim of this part of the project is to drive a 3D
pharmacophore model based on the active molecules
The synthesized compounds were evaluated for their under study. This should allow us to determine what
antiproliferative activity and showed variable activity. molecular components were required for the observed
It was difficult to find a constant activity pattern that cytotoxic activity. In addition, such model can be a
distinguished between the two synthesized series
and . In addition, the effect of amino acid side chain pounds. Acceylrs Discovery Studio software package
variations on activity was not clear. As a result, the (DS) was applied for the automated ligand based
a
useful tool for further optimization of the tested com-
b

44
A. A. Radwan et al.
Table III. Summary of HipHop generated common fea-
ture hypotheses
pharmacophore generation. Two modules can be used
within DS for pharmacophore generation; namely, the
Hypogen and HipHop modules. The first module is an
activity-based alignment derived from a collection of
conformational models of compounds spanning activi-
ties of 4-5 orders of magnitude (the minimum number
of molecules to ensure statistical significance of phar-
macophores computed in the Hypogen algorithm is
16) (Purushottamachar et al., 2007). The second algo-
rithm is a common feature-based alignment of potent
compounds. The activity of the molecules is not taken
into consideration using this model generation mode.
HipHop hypotheses are produced by comparing a set
of conformational models and a number of 3D configur-
ations of chemical features shared among the training
set molecules. Compounds of the training set may or
may not fit all features of the resulting hypothesis, de-
pending on the settings for the parameters Maximum
Omitted Features, Misses, and Complete Misses. The
retrieved pharmacophore models are expected to dis-
criminate between active and inactive compounds
(Kovat and Langer, 2003). In our case “HipHop” was
Hypo-
thesis
Rank
score
Direct
hitb
Partial
hitc
Featuresa
1
2
3
4
5
6
7
8
9
PHAA
PHAA
PNHA
PHAA
PHAA
PHAA
PHAA
PHAA
PHAA
138.448 11011111111 00100000000
133.239 11011111111 00100000000
133.057 11011111111 00100000000
124.575 11011111111 00100000000
124.575 11011111111 00100000000
122.526 11011111111 00100000000
120.173 11011111111 00100000000
119.143 11011111111 00100000000
115.310 11011111111 00100000000
10
NHHA 113.293 11011111111 00100000000
aP, positive ionizable; N, negative ionizable; H, hydrophobic;
b
A, hydrogen bond acceptor; Direct hit indicates whether
(1) or not (0) a molecule in the training set mapped to every
feature in the hypothesis; cPartial hit indicates whether (1) or
not (0) a molecule in the training set mapped to all but one
feature in the hypothesis.
The successful HipHop run resulted in the genera-
the module of choice where the training set molecules tion of 10 hypotheses (Hypo1-10), each composed of
were structurally close and covered a narrow activity four features (Table III). With the exception of Hypo-3
range.
and Hypo-10, all other hypotheses composed of two
HBA, one Pi and one HY features. Based on its highest
rank score and its mapping into all training set mole-
cules, Hypo-1 was considered to be the best hypothesis
Generation of common feature pharmacoph-
ores
In order to generate the best model, several HipHop statistically. The hypothesis was selected for further
runs were carried out by varying the control parame- investigation and analysis.
ters. The tested compounds were divided according to
their relative activities into highly active (3a
6a 8a 9a 3b 8b), moderately active (6b 7a
and inactive (
group was chosen to constitute the test set compounds tures (HBA-1 and HBA-2) at distances of 5.30 Å and
2a 3b 9a). The remaining eleven molecules were 6.97 Å respectively. The fourth feature is HY, laying
considered as the training set to be submitted to Hip- proximal to HBA-1 at a distance of 4.34 Å.
Hop for pharmacophore generation. All synthesized compounds were mapped onto Hypo-
,
4a
,
5a
,
Analysis of Hypo-1 pharmacophore
,
,
,
,
,
,
7b
,
9b),
Hypo-1 is a four-featured hypothesis (Fig. 3) show-
2, 4b) groups. One molecule from each ing a central Pi feature surrounded by two HBA fea-
(
,
,
For the optimized HipHop run, the highest weight 1 using the “best fit” option where HipHop scores the
was assigned to the highly active molecules of the orientation of a mapped compound within the hypo-
training set. This was achieved by assigning a value of thesis features using a “fit value” score. As a quick
2 (which ensures that all of the chemical features in and primary validation of Hypo-1, mapping of the test
the compound will be considered in building hypo- set compounds found to show a good agreement
thesis space) and 0 (which forces mapping of all fea- between the fit value and the biological activity (Table
tures of the compound) in the principal and maximum IV). Of note, derivative 2a, which displayed almost no
omitting features columns, respectively, for each of activity, showed the lowest fit value among all com-
these molecules. A value of 1 for the principal column pounds under study.
ensures that at least one mapping for each of gener-
Initial assessment of the results shown in Table IV
ated hypotheses will be found, and a value of 1 for the revealed a moderate correlation between the fit value
maximum omitting features column ensures that all and the biological activity of each of the tested com-
but one feature must map for all other training set pounds. The highly active compounds showed an
compounds. The features selected for this run were average fit value of 3.65 where the moderately active
HBD, HBA, Pi, Ni, and hydrophopic (HY) features.
derivatives showed a lower fit value average of 3.32.

Thiadiazine-2-thiones: New Cell-Cycle Inhibitors
45
Fig. 3. Top scoring HipHop pharmacophore Hypo-1 showing intra feature distances (Å). Features are color coded as:
Hydrogen bond acceptor, green; hydrophobic, cyan; positive ionizable, red.
On the other hand, derivatives 2a and 4b showed fit for activity. The Pi feature mapped well onto the basic
values of 2.40 and 3.09, respectively. However, a better N5 nitrogen of the thiadiazine nucleus. The calculated
correlation was observed when taking the relative pKb value (The University of Massachusetts, Boston)
energy of the fitted conformer into consideration. This for this nitrogen ranges between 5.04 and 5.97 (data
initial correlation encouraged us to generate a linear not shown) that suggests its protonation within the
model based on “fit value” and “relative energy” to pre- acidic tumor environment.
dict the biological activity of similar compounds. The
Such a feature is thought to be critical for activity,
generated model (Eq. (1)) showed very good statistics where the slight displacement of N5 away from Pi
and was used successfully to calculate the activity of center is evident, as observed in 2a and 4b (Fig. 5),
the tested compounds (Table IV).
can partially explain their lack of activity. The second
feature in common for all compounds is the HBA-1
IC50 = 33.37
n = 12; Se = 1.37;
−
6.48 fit value + 0.36
= 0.91
E
(kcal/mol) (1) feature. All compounds (including the inactive ones)
mapped their thione group onto HBA-1 perfectly.
R
Further structural modifications need to be tried at
Where
n: number of compounds; Se: standard error of the thione moiety before any conclusions can be drawn
estimation;
R: multiple correlation coefficient. of the importance of such a feature for activity. Hydro-
A closer look at the mapped structures (Fig. 4) phobic groups connected to the N3 atom are found to
revealed the importance of certain structural features be oriented into the HY feature. Although the position
of the hydrophobic group is critical for activity, there
Table IV. Output for Hypo-1 mapping and predictive
model
is no evidence that the size of such group has a de-
terment effect on the cytotoxic activity. The second
HBA feature (HBA-2) lies on the opposite side of other
features at a distance of 11.23 Å from HBA-1. Mapping
of a suitable structural moiety onto HBA-2 seems to
play a very important role for activity. It was inter-
esting to find that either oxygen atoms of the terminal
carboxylic group could place itself well at the center of
HBA-2. Failure to do so, as observed for 2a (Fig. 5a),
led to a complete loss of activity. This comes with the
finding that the ability of a compound to map onto
HBA-2 depended on the length of the side chain linker
separating the terminal carboxylic acid from the thia-
diazine ring. As a general observation, a linker of 3-5
carbon atoms was found to be essential for activity. In
case of derivatives containing aromatic linkers, ac-
tivity can be retained for linkers of up to 6 carbon atoms
length. However, the observation that these aromatic
derivatives were less active highlights the role of the
linker flexibility for optimum activity. Further studies
IC50
M)
Fit
E
Pred IC50
(µM)
Compound
Residual
(
µ
value (kcal/mol)a
2a
3a
4a
5a
6a
7a
8a
9a
3b
4b
6b
7b
8b
9b
NA
2.405 5.41942
20.06
19.83
10.18
10.54
19.74
13.87
19.70
12.79
11.09
15.97
18.73
13.16
10.34
11.09
NA
0.11
0.12
1.56
0.29
2.98
1.42
1.23
1.29
NA
19.72 3.757 1.36706
10.30 3.799 3.09675
18.98 3.604 0.59368
10.03 3.768 1.31440
16.85 3.375 5.66810
11.12 3.866 2.93480
11.56 3.714 8.74698
19.81 3.552 1.21528
NA
3.094 6.50734
18.14 3.276 17.2509
13.21 3.499 5.91861
10.60 3.799 3.51856
15.92 3.407 3.05694
0.59
0.05
0.26
1.29
aEnergy of the fitted conformer relative to its global mini-
mum energy

46
A. A. Radwan et al.
Fig. 4. Active compounds (stick) mapped onto Hypo-1. Hydrogen atoms are hidden for simplicity.
the bases of the obtained results, a current investi-
gation is in progress to improve the cell cycle inhibi-
tory activity/selectivity profile of this type of com-
pounds THTT.
ACKNOWLEDGEMENTS
This work was supported from Research Center of
College of Pharmacy (grant No. C.P.R.C.247). The
authors are grateful to Mr. Manograran Pulicat and
Mr. Amer Al-Mazrou for their help in running and
analyzing Flowcytometry and to Dr. Chaker Adra the
director of Stem Cell Therapy Program, King Faisal
Specialist Hospital and Research Centre, for his tech-
nical support in carrying the biological investigations.
Fig. 5. Inactive molecules mapped onto Hypo-1, (
A) 2a; (B)
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