Design, synthesis, antimicrobial evaluation and molecular docking studies of some new 2,3-dihydrothiazoles and 4-thiazolidinones containing sulfisoxazole

Article abstract of DOI:10.3109/14756366.2015.1016514

Microbial resistance to the available drugs poses a serious threat in modern medicine. We report the design, synthesis and in vitro antimicrobial evaluation of new functionalized 2,3-dihydrothiazoles and 4-thiazolidinones tagged with sulfisoxazole moiety. Compound 8d was most active against Bacillis subtilis (MIC, 0.007 g/mL). Moreover, compounds 7c-d and 8c displayed significant activities against B. subtilis and Streptococcus pneumoniae (MIC, 0.03-0.06 g/mL and 0.06-0.12 g/mL versus ampicillin 0.24 g/mL and 0.12 g/mL; respectively). Compounds 7a and 7c-d were highly potent against Escherichia coli (MIC, 0.49-0.98 g/mL versus gentamycin 1.95 g/mL). On the other hand, compounds 7e and 9c were fourfolds more active than amphotericin B against Syncephalastrum racemosum. Molecular docking studies showed that the synthesized compounds could act as inhibitors for the dihydropteroate synthase enzyme (DHPS). This study is a platform for the future design of more potent antimicrobial agents.

Full text of DOI:10.3109/14756366.2015.1016514

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–11
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1016514
!
RESEARCH ARTICLE
Design, synthesis, antimicrobial evaluation and molecular docking
studies of some new 2,3-dihydrothiazoles and 4-thiazolidinones
containing sulfisoxazole
Tamer Nasr1, Samir Bondock2,3, and Sameh Eid4
2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Helwan, Cairo, Egypt, Department of Chemistry, Faculty of
3
Science, Mansoura University, Mansoura, Egypt, Department of Chemistry, Faculty of Science, King Khalid University, Abha, Saudi Arabia, and
4BioMed X Innovation Center, Im Neuenheimer Feld 583, Heidelberg, Germany
Abstract
Keywords
Microbial resistance to the available drugs poses a serious threat in modern medicine.
We report the design, synthesis and in vitro antimicrobial evaluation of new functionalized
2,3-dihydrothiazoles and 4-thiazolidinones tagged with sulfisoxazole moiety. Compound 8d
was most active against Bacillis subtilis (MIC, 0.007 mg/mL). Moreover, compounds 7c–d and 8c
displayed significant activities against B. subtilis and Streptococcus pneumoniae (MIC, 0.03–
0.06 mg/mL and 0.06–0.12 mg/mL versus ampicillin 0.24 mg/mL and 0.12 mg/mL; respectively).
Compounds 7a and 7c–d were highly potent against Escherichia coli (MIC, 0.49–0.98 mg/mL
versus gentamycin 1.95 mg/mL). On the other hand, compounds 7e and 9c were fourfolds more
active than amphotericin B against Syncephalastrum racemosum. Molecular docking studies
showed that the synthesized compounds could act as inhibitors for the dihydropteroate
synthase enzyme (DHPS). This study is a platform for the future design of more potent
antimicrobial agents.
Antimicrobial agents, molecular docking,
sulfonamide, thiazole
History
Received 12 December 2014
Revised 25 January 2015
Accepted 26 January 2015
Published online 27 March 2015
Introduction
were solved and reported in the Protein Data Bank (PDB). The
crystal structure of Bacillus anthracis dihydropteroate synthase
(BaDHPS) bound to sulfathiazole-6-hydroxymethyl-7,8-dihydrop-
terin-pyrophosphate (STZ-DHPP) adduct was reported by
Yun et al18. In this structure, the STZ-DHPP adduct occupies
both the PABA and pterin-binding pockets of DHPS (Figure I,
Supplementary data)18. Trimethoprim, the dihydrofolate reductase
inhibitor, is used in combination with sulfa drugs to increase the
therapeutic efficacy19. On the other hand, 2,3-dihydrothiazoles and
4-thiazolidinones have occupied a unique position in the design
Recently, many drug-resistant human pathogenic microbes have
been reported1. This has been attributed to the widespread use of
antibacterial and antifungal agents as well as the inaccurate
diagnosis2. Methicillin-resistant Staphylococcus aureus3, vanco-
mycin-resistant Enterococci4 and azole-resistant Candida species5
are well-known examples of drug-resistant microbes. It is particu-
larly challenging to treat infections caused by these microbes
especially in the case of immunocompromised patients6.
Therefore, it is crucial to search for new potent antimicrobial
agents7. There are two main strategies for the discovery of new
drugs; either to search for a novel lead compound or to modify the
structure of a known drug8. In the second strategy, two or more
pharmacophores are often combined in one molecule to obtain the
synergistic effect9. Sulfonamide derivatives have numerous bio-
logical activities including antibacterial10, carbonic anhydrase
inhibitor11, insulin release inducer12, antiviral13, antifungal14,
anticancer15 and anti-inflammatory activities16. It is known that
sulfonamides reduce the biosynthesis of dihydrofolic acid through
the competitive inhibition of the dihydropteroate synthase enzyme
(DHPS)17; and sulfisoxazole is an example of sulfa drugs that
inhibits the binding of p-amino benzoic acid (PABA) to DHPS
binding pocket. To the best of our knowledge, only two crystal
structures of a sulfa-related drug bound to the active site of DHPS
and synthesis of biologically active antimicrobial agents20–22
.
In this study, we used the reported STZ-DHPP adduct18 as a
lead compound to design potent antimicrobial agents targeting
both the PABA and pterin-binding pockets of the DHPS enzyme,
thereby inhibiting the biosynthesis of the dihydrofolic acid. The
thiazole, methylene and pteridine moieties were replaced by the
3,4-dimethyl-isoxazole, carbonyl and substituted thiazole rings;
respectively (Figure 1). Sulfisoxazole was used as a starting
material and the target compounds were tested in vitro for their
antibacterial and antifungal activities against human pathogenic
microbes. The observed biological results were rationalized by
molecular docking and lipophilicity studies.
Methods
Chemistry
Melting points were determined on digital Gallen-Kamp MFB-
595 instrument (UK) using open capillary tubes and were
uncorrected. IR spectra were recorded on Schimadzu FTIR 440
Address for correspondence: Tamer Nasr, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Helwan University, 11795 Helwan,
Cairo, Egypt. Tel: +20 110067435. E-mail: tamerhefni@yahoo.com

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T. Nasr et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
Occupies the PABA
pocket of DHPS
Figure 1. Design of thaizole derivatives
bearing sulfisoxazole moiety as DHPS
inhibitors.
Occupies the pterin
pocket of DHPS
H
O
N
S
S
STZ-DHPP adduct obtained from soaking
DHPS with the sulfathiazole inhibitor STZ
and DHPP
O
N
N
H
NH
N
N
O
N
H
NH
2
Modification
Modification
H
O
R
N
4
R
N
N
S
1
3
NC
O
NC
O
O
O
N
R
X=
2
S
S
N
H
X
R
spectrometer (Kyoto, Japan) using KBr pellets. Mass spectra 136 (15.9), 111 (0.5), 96 (12.3), 77 (45.1), 52 (100); Anal. Calcd.
were performed on Shimadzu Qp-2010 plus mass spectrometer at for C21H19N5O4S3 (501.60): C, 50.28; H, 3.82; N, 13.96%, Found:
70 eV (Kyoto, Japan). 1H NMR and 13C NMR spectra were C, 50.31; H, 3.85; N, 13.99%.
recorded on a Bruker model (500 MHz or 400 MHz) Ultra Shield
NMR spectrometer (Billerica, MA) in DMSO-d6 using tetra- General procedure for the synthesis of ketene N,S-acetal
methylsilane (TMS) as an internal standard; chemical shifts are potassium salt derivatives 6a–e
reported as ꢀppm units. The elemental analyses were done
Potassium hydroxide (0.56 g, 0.01 mol) was added to a solution of
at the Microanalytical Center, Cairo University, Cairo, Egypt.
compound 3 (3.34 g, 0.01 mol) in DMF (30 mL). The reaction
Sulfisoxazole 1 was purchased from Sigma-Aldrich (St. Louis,
mixture was stirred for 1 h at room temperature. Substituted
MO). 1-Cyanoacetyl-3,5-dimethylpyrazole 223 and 2-cyano-N-(4-
isothiocyanate derivatives 5a–e (0.01 mol) were then added
{[(3,4-dimethylisoxazole-5-yl)amino]sulfonyl}phenyl)acetamide
dropwise and the mixture was stirred for 24 h at room tempera-
3 (24) were synthesized according to the reported procedures.
ture. Without separation, the obtained products were used for
further reactions.
4-Amino-2,3-dihydro-3-phenyl-2-thioxothiazole-5-N-(4-
{[(3,4-dimethylisoxazole-5-yl)amino]sulfonyl}phenyl)
carboxamide (4)
General procedure for the synthesis of functionalized
4-methylthiazole derivatives 7a–e
Phenylisothiocyanate (1.35 g, 0.01 mol), sulfur (0.32 g, 0.01 mol)
and triethylamine (0.3 mL) were added to a hot solution of
compound 3 (3.34 g, 0.01 mol) in a mixture of absolute ethanol
(20 mL) and dimethylformamide (DMF) (5 mL). The reaction
mixture was heated at 65 ꢀC for 8 h. It was allowed to cool, and
then poured on ice water (200 mL) and the medium was
neutralized by dilute HCl. The obtained solid was filtered off,
washed with water (50 mL), air dried and recrystallized from
ethanol/DMF to afford the target compound.
Chloroacetone (0.79 mL, 0.01 mol) was added dropwise to a well-
stirred solution of the intermediate compounds 6a–e (0.01 mol) in
DMF (30 mL) at 0 ꢀC. After complete addition, the reaction
mixture was stirred at room temperature for 18 h then poured on
ice water (200 mL). The medium was neutralized by dilute HCl
and the obtained solid was filtered off, washed with water
(50 mL), air dried and recrystallized from ethanol to afford the
4-methyl-thiaole derivatives 7a–e.
Pale yellow powder, yield (83%), mp 195–196 ꢀC; IR (KBr)
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(3,4-dimethyl-thiazol-2(3H)-ylidene)-acetamide (7a)
m
max/cmꢁ1: 3409–3351 (NH2), 3279 (NH), 3206 (NH), 3045 (CH–
Ar), 2986 (CH-sp3), 1659 (CO), 1175 (C¼S); 1H NMR
(400 MHz, DMSO-d6): ꢀppm ¼ 1.64 (s, 3H, CH3), 2.09 (s, 3H,
Yellowish brown powder, yield (78%), mp 153–154 ꢀC; IR (KBr)
m
max/cmꢁ1: 3238 (NH), 3101 (NH), 3046 (CH–Ar), 2931
0
CH3), 7.10 (s, 2H, NH2), 7.34 (d, J ¼ 9.0 Hz, 2H, phenyl-H2 ,
(CH-sp3), 2182 (CN), 1656 (CO); H NMR (500 MHz, DMSO-
d6): ꢀppm ¼ 1.71 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.11 (s, 3H,
CH3), 3.25 (s, 3H, NCH3),7.61 (s, 1H, thiazol-H5), 7.71
(d, J ¼ 9.0 Hz, 2H, phenyl-H2, phenyl-H6), 7.81 (d, J ¼ 9.0 Hz,
2H, phenyl-H3, phenyl-H5), 9.89 (s, 1H, NHSO2), 10.91 (s, 1H,
CONH); MS m/z (%): 445 (M+, 0.2), 294 (0.2), 266 (0.4), 194
(0.6), 179 (1.00) , 175 (1.5), 151 (1.5), 113 (2.1), 111 (6.1), 96
(5.8), 64 (100); Anal. Calcd. For C19H19N5O4S2 (445.52): C,
51.22; H, 4.30; N, 15.72%, Found: C, 51.20; H, 4.28; N, 15.76%.
1
0
phenyl-H6 ), 7.42 (d, J ¼ 8.0 Hz, 2H, phenyl-H2, phenyl-H6), 7.63
(d, J ¼ 8.0 Hz, 2H, phenyl-H3, phenyl-H5), 7.88 (m, 3H, phenyl-
0
0
0
H3 , Phenyl-H4 , phenyl-H5 ), 10.74 (s, 1H, NHSO2), 11.14 (s, 1H,
NHCO); 13C NMR (125 MHz, DMSO-d6): ꢀppm ¼ 6.3 (CH3), 10.8
(CH3), 105.3 (thiazole-C5), 119.6, 120.6, 128.1, 128.5, 129.3,
130.6, 133.0, 134.9, 142.0, 143.0 (12C, C6H4, C6H5), 154.0
(isoxazole-C4), 156.2, 161.0, 161.8, 162.0 (4C, isoxazole-C3,
isoxazole-C5, thiazole-C4, CONH), 187.0 (C¼S); MS m/z (%):
501 (M+, 0.1), 293 (0.4), 266 (0.1), 251 (0.1), 235 (0.2), 175 (1.9),

DOI: 10.3109/14756366.2015.1016514
New 2,3-dihydrothiazoles and 4-thiazolidinones containing sulfisoxazole
3
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(3-ethyl-4-methyl-thiazol-2(3H)-ylidene)-acetamide (7b)
Anal. Calcd. for C28H31N5O4S2 (565.71): C, 59.45; H, 5.52; N,
12.38%, Found: C, 59.43; H, 5.53; N, 12.41%.
Yellow powder, yield (74%), mp 162–163 ꢀC; IR (KBr)
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(4-methyl-3-phenyl-thiazol-2(3H)-ylidene)-acetamide (7e)
m
max/cmꢁ1: 3362 (NH), 3283 (NH), 3049 (CH–Ar), 2977 (CH-
sp3), 2178 (CN), 1646 (CO); 1H NMR (500 MHz, DMSO-d6):
ꢀppm ¼ 1.23 (t, J ¼ 7.0 Hz, 3H, CH3), 1.71 (s, 3H, CH3), 2.10 Yellow powder, yield (79%), mp 130–131 ꢀC; IR (KBr)
(s, 3H, CH3), 2.11 (s, 3H, CH3), 3.25 (q, J ¼ 7.0 Hz, 2H, CH2),
m
max/cmꢁ1: 3323 (NH), 3238 (NH), 3048 (CH–Ar), 2961 (CH-
7.63 (s, 1H, thiazol-H5), 7.71 (d, J ¼ 9.0 Hz, 2H, phenyl-H2, sp3), 2239 (CN), 1649 (CO); 1H NMR (500 MHz, DMSO-d6):
phenyl-H6), 7.81 (d, J ¼ 9.0 Hz, 2H, phenyl-H3, phenyl-H5), 9.89 ꢀppm ¼ 1.70 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.15 (s, 3H, CH3),
(s, 1H, NHSO2), 10.93 (s, 1H, CONH); 13C NMR (125 MHz, 7.49–7.79 (m, 10H, C6H5, C6H4, thiazol-H5), 9.21 (s, 1H,
DMSO-d6): ꢀppm ¼ 5.9 (CH3), 10.2 (CH3), 13.8 (CH3), 27.9 NHSO2), 10.92 (s, 1H, CONH); 13C NMR (125 MHz, DMSO-
(CH3), 56.0 (CH2), 94.0 (acetamide-C2), 99.5 (CN), 104.9 d6): ꢀppm ¼ 6.3 (CH3), 10.7 (CH3), 14.7 (CH3), 67.3 (acetamide-
(thiazole-C5), 119.9 (2C, phenyl-C2, phenyl-C6), 127.4 (2C, C2), 96.6 (CN), 105.4 (thiazole-C5), 107.5, 121.0, 124.1, 127.9,
phenyl-C3, phenyl-C5), 133.4, 143.6, 155.4, 155.5, 161.3, 163.3, 128.7, 129.4, 137.0, 138.9, 144.5, 156.0, 161.8, 162.2, 165.7
164.2 (7C, phenyl-C1, phenyl-C4, isoxazole-C3, isoxazole-C4, (17C, C6H5, C6H4, isoxazole-C3, isoxazole-C4, isoxazole-C5,
isoxazole-C5, thiazole-C2, thiazole-C4), 165.9 (CONH); MS m/z thiazole-C2, thiazole-C4), 167.0 (CONH); MS m/z (%): 508
(%): 460 ([M+H]+, 1.3), 459 (M+, 3.6), 208 (2.2), 193 (1.4), 175 ([M+H]+, 18.5), 507 (M+, 21.9), 481 (0.8), 411 (14.6), 396
(0.1), 127 (2.5) , 111 (5.1), 96 (1.9), 92 (100); Anal. Calcd. for (18.5), 332 (4.5), 294 (1.7), 266 (5.6), 256 (12.3), 175 (15.2), 130
C20H21N5O4S2 (459.54): C, 52.27; H, 4.61; N, 15.24%, Found: C, (100), 96 (14.3), 93 (26.2), 77 (2.8); Anal. Calcd. for
52.32; H, 4.65; N, 15.27%.
C24H21N5O4S2 (507.58): C, 56.79; H, 4.17; N, 13.80%, Found:
C, 56.83; H, 4.21; N, 13.84%.
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(3-allyl-4-methyl-thiazol-2(3H)-ylidene)-acetamide (7c)
General procedure for the synthesis of N-phenyl-thiazole
derivatives 9a–e
Yellowish green powder, yield (63%), mp 118–119 ꢀC; IR (KBr)
max/cmꢁ1: 3335 (NH), 3275 (NH), 3051 (CH–Ar), 2928 (CH-
A variety of a-halo carbonyl reagents 8a–e (0.01 mol) were added
dropwise to a well-stirred solution of the non-isolable potassium
salt 6e (0.01 mol) in DMF (30 mL) at 0 ꢀC. The reaction mixture
was stirred at room temperature for 18 h then poured on ice water
(200 mL) and the medium was neutralized by dilute HCl. The
obtained solid was filtered off, washed with water (50 mL), air
dried and recrystallized from ethanol to afford the N-phenyl-
thiazole derivatives 9a–e.
m
sp3), 2189 (CN), 1649 (CO); 1H NMR (500 MHz, DMSO-d6):
ꢀppm ¼ 1.64 (s, 3H, CH3), 1.70 (s, 3H, CH3), 2.10 (s, 3H, CH3),
3.87 (m, 2H, CH2N), 5.22 (dd, J ¼ 1.5 Hz, 17.5 Hz, 1H, CH2¼),
5.30 (dd, J ¼ 1.5 Hz, 17.5 Hz, 1H, CH2¼), 5.89 (m, 1H, CH¼),
7.65 (s, 1H, thiazol-H5), 7.71 (d, J ¼ 9.0 Hz, 2H, phenyl-H2,
phenyl-H6), 7.81 (d, J ¼ 9.0 Hz, 2H, phenyl-H3, phenyl-H5), 9.93
(s, 1H, NHSO2), 10.94 (s, 1H, CONH); 13C NMR (125 MHz,
DMSO-d6): ꢀppm ¼ 5.9 (CH3), 10.2 (CH3), 27.9 (CH3), 48.7
(CH2N), 74.0 (acetamide-C2), 100.1 (CN), 104.8 (thiazole-C5),
117.0 (CH2¼), 119.9 (2C, phenyl-C2, phenyl-C6), 127.4 (2C,
phenyl-C3, phenyl-C5), 128.1, 133.3, 133.5, 143.6, 155.6, 161.3,
163.3, 164.3 (8C, CH¼, phenyl-C1, phenyl-C4, isoxazole-C3,
isoxazole-C4, isoxazole-C5, thiazole-C2, thiazole-C4), 165.6
(CONH); MS m/z (%): 472 ([M+H]+, 9.4), 471 (M+, 5.8), 444
(5.5), 375 (3.5), 266 (2.5), 251 (2.9), 177 (9.5), 175 (7.1), 138
(100), 111 (19.4), 96 (9.9); Anal. Calcd. for C21H21Nz5O4S2
(471.55): C, 53.49; H, 4.49; N, 14.85%, Found: C, 53.53; H, 4.53;
N, 14.87%.
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(2,3-dihydro-5-acetyl-4-methyl-3-phenyl-2-ylidene-thia-
zol)-acetamide (9a)
Brown powder, yield (73%), mp 196–197 ꢀC; IR (KBr)
m
max/cmꢁ1: 3347 (NH), 3232 (NH), 3047 (CH–Ar), 2943 (CH-
sp3), 2201 (CN), 1705 (CO), 1663 (CO); 1H NMR (500 MHz,
DMSO-d6): ꢀppm ¼ 1.69 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.11
0
(s, 3H, CH3), 2.50 (s, 3H, COCH3), 7.13 (m, 1H, phenyl-H4 ),
0
7.35–7.45 (m, 2H, phenyl-H2 and phenyl-H6 ), 7.49–7.55 (m, 2H,
0
0
0
phenyl-H3 and phenyl-H5 ), 7.71 (d, J ¼ 9.0 Hz, 2H, phenyl-H2,
phenyl-H6), 7.84 (d, J ¼ 9.0 Hz, 2H, phenyl-H3, phenyl-H5), 9.81
(s, 1H, NHSO2), 10.95 (s, 1H, CONH); 13C NMR (125 MHz,
DMSO-d6): ꢀppm ¼ 5.9 (CH3), 10.2 (CH3), 18.5 (CH3), 56.0
(CH3CO), 77.5 (acetamide-C2), 104.9 (CN), 106.0, 111.1, 119.8,
120.5, 124.1, 127.4, 129.3, 130.0, 133.7, 141.0 (14C, C6H4, C6H5,
thiazole-C4 and thiazole-C5), 143.4, 155.5, 161.3 (3C, isoxazole-
C3, isoxazole-C4, isoxazole-C5), 162.8 (CONH), 171.9 (thiazole-
C2), 195.0 (CH3CO); MS m/z (%): 549 (M+, 0.9), 534 (12.0), 506
(27.4), 472 (18.0), 266 (39.1), 175 (3.8), 96 (77.4), 77 (51.2), 63
(100); Anal. Calcd. for C26H23N5O5S2 (549.62): C, 56.82; H,
4.22; N, 12.74%, Found: C, 56.86; H, 4.27; N, 12.77%.
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(3-adamantyl-4-methyl-thiazol-2(3H)-ylidene)-acetamide
(7d)
Yellow powder, yield (87%), mp 261–262 ꢀC; IR (KBr)
m
max/cmꢁ1: 3334 (NH), 3267 (NH), 3058 (CH–Ar), 2912 (CH-
sp3), 2263 (CN), 1652 (CO); 1H NMR (500 MHz, DMSO-d6):
ꢀppm ¼ 1.65 (m, 9H, 9 ꢂ adamantyl-H), 1.70 (s, 3H, CH3), 2.01
(m, 3H, 3 ꢂ adamantyl-H), 2.08–2.09 (m, 6H, CH3, 3 ꢂ adaman-
tyl-H), 2.14 (s, 3H, CH3), 7.65 (s, 1H, thiazol-H5), 7.71
(d, J ¼ 9.0 Hz, 2H, phenyl-H2, phenyl-H6), 7.78 (d, J ¼ 9.0 Hz,
2H, phenyl-H3, phenyl-H5), 9.80 (s, 1H, NHSO2), 10.90 (s, 1H,
CONH); 13C NMR (125 MHz, DMSO-d6): ꢀppm ¼ 5.7 (CH3), 10.3
(CH3), 18.5, 28.1, 28.8, 29.0, 35.4, 35.5, 39.7, 39.8, 39.9, 40.1,
40.4 (11C, CH3, 10 ꢂ adamantyl-C), 53.3 (acetamide-C2), 100.8
(CN), 104.8 (thiazole-C5), 120.0 (2C, phenyl-C2, phenyl-C6),
127.4 (2C, phenyl-C3, phenyl-C5), 128.1, 137.5, 143.1, 153.9,
160.7, 161.2, 164.1 (7C, phenyl-C1, phenyl-C4, isoxazole-C3,
isoxazole-C4, isoxazole-C5, thiazole-C2, thiazole-C4), 165.8
(CONH); MS m/z (%): 566.71 ([M+H]+, 13.4), 565 (M+, 4.3),
175 (16.9), 135 (11.1), 111 (18.7), 96 (21.9), 93 (100);
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(2,3-dihydro-5-ethylcarboxylate 4-methyl-3-phenyl-2-yli-
dene-thiazol)-acetamide (9b)
Yellow powder, yield (66%), mp 117–118 ꢀC; IR (KBr)
m
max/cmꢁ1: 3357 (NH), 3231 (NH), 3061 (CH–Ar), 2984 (CH-
sp3), 2191 (CN), 1701 (CO), 1652 (CO); 1H NMR (500 MHz,
DMSO-d6): ꢀppm ¼ 1.32 (s, 3H, CH3), 1.69 (s, 3H, CH3), 1.93
(s, 3H, CH3), 2.10 (s, 3H, CH3), 4.1 (s, 2H, OCH2), 7.10–7.35
(m, 5H, C6H5), 7.73 (d, J ¼ 9.0 Hz, 2H, phenyl-H2, phenyl-H6),

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T. Nasr et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
7.86 (d, J ¼ 9.0 Hz, 2H, phenyl-H3, phenyl-H5), 9.88 (s, 1H, (thiazole-C2); MS m/z (%): 604 (M+, 14.3), 492 (2.4), 352 (13.6),
NHSO2), 10.93 (s, 1H, CONH); MS m/z (%): 581 ([M+2]+, 65.7), 337 (1.9), 309 (3.2), 294 (8.4), 111 (100), 96 (11.8), 77 (49.5);
579 (M+, 61.1), 505 (48.1), 483 (57.4), 405 (61.1), 335 (100), 329 Anal. Calcd. for C29H22ClN5O4S2 (604.10): C, 57.66; H, 3.67; N,
(48.1), 294 (62.9), 252 (29.6), 73 (44.4); Anal. Calcd. for 11.59%, Found: C, 57.70; H, 3.71; N, 11.63%.
C27H25N5O6S2 (579.65): C, 55.95; H, 4.35; N, 12.08%, Found:
C, 55.92; H, 4.37; N, 12.14%.
General procedure for the synthesis of compounds 10a–e
Method (A)
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(2,3-dihydro-4-ethylacetate-3-phenyl-2-ylidene-thiazol)-
acetamide (9c)
Chloroacetylchloride (0.79 mL, 0.01 mol) was added slowly to the
non-isolated potassium salts 6a–e (0.01 mol) at 0 ꢀC. Then
the reaction mixture was stirred at room temperature for 12 h.
The reaction mixture was poured portion wise to ice water
(200 mL) and neutralized by dilute HCl. The obtained solid was
separated by filtration, washed with water (50 mL), air dried and
recrystallized from ethanol to afford the 4-thiazolidinone deriva-
tives 10a–e.
Brown powder, yield (53%), mp 119–120 ꢀC; IR (KBr)
m
max/cmꢁ1: 3390 (NH), 3254 (NH), 3064 (CH–Ar), 2985 (CH-
sp3), 2194 (CN), 1725 (CO), 1653 (CO); 1H NMR (500 MHz,
DMSO-d6): ꢀppm ¼ 1.63–1.70 (m, 6H, CH3 and CH3CH2O), 2.08
(s, 3H, CH3), 2.51 (s, 2H, CH2COO), 4.09 (q, J ¼ 7.5 Hz, 2H,
CH2OOC), 7.28 (s, 1H, thiazole-H5), 7.38–7.50 (m, 5H, phenyl-
0
0
0
0
0
H2 , phenyl-H3 , phenyl-H4 , phenyl-H5 and phenyl-H6 ), 7.72
(d, J ¼ 9.0 Hz, 2H, phenyl-H2, phenyl-H6), 7.84 (d, J ¼ 9.0 Hz,
2H, phenyl-H3, phenyl-H5), 9.89 (s, 1H, NHSO2), 10.95 (s, 1H,
CONH); 13C NMR (125 MHz, DMSO-d6): ꢀppm ¼ 5.8 (CH3), 10.2
(CH3), 14.0 (CH3), 39.0 (CH2COO), 60.4 (CH2OOC), 84.0
(acetamide-C2), 105.0, 112.6 (2C, thiazole-C5 and CN), 119.9,
120.4, 120.6, 120.9, 124.0, 127.4, 128.6, 129.3, 129.8 (13C, C6H4,
C6H5 and thiazole-C4), 143.4, 155.5, 161.3 (3C, isoxazole-C3,
isoxazole-C4, isoxazole-C5), 162.6 (CONH), 167.5 (COO), 171.0
(thiazole-C2); MS m/z (%): 580 ([M+1]+, 5.2), 579 (M+, 7.5), 550
(7.4), 506 (6.6), 328 (1.9), 294 (1.9), 285 (1.4), 266 (4.1), 162
(100), 111 (19.7), 96 (19.6), 87 (4.1), 77 (11.6); Anal. Calcd. for
C27H25N5O6S2 (579.65): C, 55.95; H, 4.35; N, 12.08%, Found: C,
55.97; H, 4.31; N, 12.05%.
Method (B)
Chloroacetonitrile (0.63 mL, 0.01 mol) was added to the non-
isolated potassium salts 6a–e (0.01 mol) at 0 ꢀC. Then the reaction
mixture was stirred at room temperature for 15 h. The reaction
mixture was poured portion wise to ice water (200 mL) and the
medium was neutralized by HCl. The obtained solid was
separated by filtration, washed with water (50 mL), air dried
and recrystallized from ethanol. Compound 11 could not be
obtained and instead the product was identical in all respects (mp.,
m.mp., IR spectra) with 10a isolated from reaction of 6a with
chloroacetyl chloride.
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(3-methyl-4-oxothiazolidin-2-ylidene)-acetamide (10a)
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(2,3-dihydro-3,4-diphenyl-2-ylidene-thiazol)-acetamide
(9d)
Yellow powder, yield (73%), mp 206–207 ꢀC; IR (KBr)
m
max/cmꢁ1: 3343 (NH), 3112 (NH), 3062 (CH–Ar), 2930 (CH-
sp3), 2201 (CN), 1747 (CO), 1662 (CO); 1H NMR (500 MHz,
DMSO-d6): ꢀppm ¼ 1.65 (s, 3H, CH3), 2.10 (s, 3H, CH3), 3.55 (s,
3H, NCH3), 3.91 (s, 2H, CH2), 7.72 (d, J ¼ 9.0 Hz, 2H, phenyl-H2,
phenyl-H6), 7.84 (d, J ¼ 9.0 Hz, 2H, phenyl-H3, phenyl-H5), 10.09
(s, 1H, NHSO2), 10.96 (s, 1H, CONH); 13C NMR (125 MHz,
DMSO-d6): ꢀppm ¼ 5.8 (CH3), 10.2 (CH3), 31.2 (CH3), 32.7
(CH2), 77.0 (acetamide-C2), 105.0 (CN), 116.2 (phenyl-C4), 120.5
(2C, phenyl-C2, phenyl-C6), 127.5 (2C, phenyl-C3, phenyl-C5),
134.3 (phenyl-C1), 142.8, 155.4, 161.3 (3C, isoxazole-C3,
isoxazole-C4, isoxazole-C5), 163.4, 172.1, 173.7 (3C, CONH,
COCH2, thiazole-C2); MS m/z (%): 447 (M+, 13.7), 432 (1.8), 421
(3.6), 351 (4.9), 272 (21.1), 266 (15.7), 181 (47.2), 175 (5.6), 111
(29.1), 96 (1.9), 93 (100); Anal. Calcd. for C18H17N5O5S2
(447.49): C, 48.31; H, 3.83; N, 15.65%, Found: C, 48.36; H, 3.87;
N, 15.67%.
Yellow powder, yield (87%), mp 256–257 ꢀC; IR (KBr)
m
max/cmꢁ1: 3413 (NH), 3288 (NH), 3060 (CH–Ar), 2981 (CH-
sp3), 2196 (CN), 1634 (CO); 1H NMR (500 MHz, DMSO-d6):
ꢀppm ¼ 1.70 (s, 3H, CH3), 2.08 (s, 3H, CH3), 7.0–7.50 (m, 11H,
0
0
00
00
C6 H5 , C6 H5 and thiazole-H5), 7.71 (d, J ¼ 9.0 Hz, 2H, phenyl-
H2, phenyl-H6), 7.89 (d, J ¼ 9.0 Hz, 2H, phenyl-H3, phenyl-H5),
9.90 (s, 1H, NHSO2), 10.91 (s, 1H, CONH); MS m/z (%): 570
([M+1]+, 2.1), 569 (M+, 3.1), 394 (0.7), 303 (3.3), 294 (37.1),
275 (0.3), 251 (0.4), 175 (3.3), 111 (13.9), 96 (7.2), 77 (100);
Anal. Calcd. for C29H23N5O4S2 (569.65): C, 61.14; H, 4.07; N,
12.29%, Found: C, 61.11; H, 4.09; N, 12.33%.
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(2,3-dihydro-4-(4-chloro-phenyl)-3-phenyl-2-ylidene-thia-
zol)-acetamide (9e)
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(3-ethyl-4-oxothiazolidin-2-ylidene)-acetamide (10b)
Yellow powder, yield (79%), mp 209–210 ꢀC; IR (KBr)
m
max/cmꢁ1: 3436 (NH), 3282 (NH), 3061 (CH–Ar), 2927 (CH-
sp3), 2199 (CN), 1640 (CO); 1H NMR (500 MHz, DMSO-d6):
Brown powder, yield (71%), mp 139–140 ꢀC; IR (KBr)
m
max/cmꢁ1: 3369 (NH), 3251 (NH), 3052 (CH–Ar), 2928 (CH-
ꢀppm ¼ 1.70 (s, 3H, CH3), 2.10 (s, 3H, CH3), 7.11–7.16 (m, 1H,
sp3), 2194 (CN), 1732 (CO), 1663 (CO); 1H NMR (500 MHz,
DMSO-d6): ꢀppm ¼ 1.27 (t, J ¼ 7.0 Hz, 3H, CH3), 1.65 (s, 3H,
CH3), 2.10 (s, 3H, CH3), 3.92 (s, 2H, CH2), 4.15 (q, J ¼ 7.0 Hz,
2H, NCH2), 7.72 (d, J ¼ 9.0 Hz, 2H, phenyl-H2, phenyl-H6), 7.82
(d, J ¼ 9.0 Hz, 2H, phenyl-H3, phenyl-H5), 10.13 (s, 1H, NHSO2),
10.96 (s, 1H, CONH); 13C NMR (125 MHz, DMSO-d6): ꢀppm
¼ 5.8 (CH3), 10.2 (CH3), 13.7 (CH3), 31.3 (CH2), 40.1 (CH2),
76.3 (acetamide-C2), 105.0 (CN), 116.0 (phenyl-C4), 120.5 (2C,
phenyl-C2, phenyl-C6), 127.5 (2C, phenyl-C3, phenyl-C5), 134.3
(phenyl-C1), 142.8, 155.4, 161.3 (3C, isoxazole-C3, isoxazole-C4,
isoxazole-C5), 163.4, 170.9, 173.7 (3C, CONH, COCH2,
0
phenyl-H4 ), 7.33–7.38 (m, 4H, phenyl-H2 , phenyl-H3 , phenyl-
0
0
0
0
00
00
H5 , phenyl-H6 ), 7.53 (d, J ¼ 8.5 Hz, 2H, phenyl-H2 , phenyl-H6 ),
00
00
7.62 (d, J ¼ 8.5 Hz, 2H, phenyl-H3 , phenyl-H5 ), 7.72
(d, J ¼ 9.0 Hz, 2H, phenyl-H2, phenyl-H6), 7.86 (d, J ¼ 9.0 Hz,
2H, phenyl-H3, phenyl-H5), 7.96 (s, 1H, thiazole-H5), 9.91 (s, 1H,
NHSO2), 10.94 (s, 1H, CONH); 13C NMR (125 MHz, DMSO-d6):
ꢀppm ¼ 5.9 (CH3), 10.3 (CH3), 81.0 (acetamide-C2), 104.9, 116.0
(2C, thiazole-C5 and CN), 119.9, 121.0, 123.0, 124.5, 127.5,
128.4, 128.8, 129.4, 131.5, 132.4, 134.9, 135.3, 141.6 (19C,
2 ꢂ C6H4, C6H5 and thiazole-C4), 143.4, 155.6, 161.3 (3C,
isoxazole-C3, isoxazole-C4, isoxazole-C5), 168.0 (CONH), 172.1

DOI: 10.3109/14756366.2015.1016514
New 2,3-dihydrothiazoles and 4-thiazolidinones containing sulfisoxazole
5
thiazole-C2); MS m/z (%): 461 (M+, 9.3), 435 (10.0), 432 (1.7), Gram-positive bacteria and Escherichia coli (RCMB 010052),
350 (42.1), 266 (11.1), 210 (29.0), 92 (100); Anal. Calcd. for Proteous vulgaris (RCMB 010085) and Klebsiella pneumonia
C19H19N5O5S2 (461.51): C, 49.45; H, 4.15; N, 15.17%, Found: C, (RCMB 010093) as examples of Gram-negative bacteria. The
49.50; H, 4.17; N, 15.19%.
antifungal activities of the target compounds were evaluated
against Aspergillus fumigatus (RCMB 02568), Syncephalastrum
racemosum (RCMB 05922) and Geotricum candidum (RCMB
05097). The preliminary screening of the antibacterial and
antifungal activities was carried out by the agar-diffusion
method. The results were recorded for each tested compound as
the average diameter of inhibition zones (IZs) of microbial growth
around the disks in millimeters and were attributed to the original
tested concentration (5 mg/mL) as a preliminary test. Whatman
filter paper disks were prepared with standard size (6.0 mm
diameter) and reserved into 1.0 Oz screw capped wide mouthed
containers for sterilization. These containers were kept at a
temperature of 150 ꢀC. Then, the standard sterilized filter paper
disks soaked with a solution of the test compound in DMF
(100 mL, 5 mg/mL) were placed on nutrient agar plate seeded with
the proper test organism in triplicates. Standard concentrations of
106 CFU/mL (Colony Forming Units/mL) and 104 CFU/mL were
used for antimicrobial assays. Pyrex glass Petri dishes (9 cm in
diameter) were used and two disks of filter paper were inoculated
in each plate. Ampicillin and gentamycin were used as standard
antibacterial agents; while amphotericin B was used as standard
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(3-allyl-4-oxothiazolidin-2-ylidene)-acetamide (10c)
Greenish brown powder, yield (64%), mp 129–130 ꢀC; IR (KBr)
m
max/cmꢁ1: 3371 (NH), 3230 (NH), 3061 (CH–Ar), 2929
(CH-sp3), 2193 (CN), 1716 (CO), 1653 (CO); 1H NMR
(500 MHz, DMSO-d6): ꢀppm ¼ 1.65 (s, 3H, CH3), 2.10 (s, 3H,
CH3), 3.86 (m, 2H, CH2N), 3.95 (s, 2H, CH2S), 5.29 (dd,
J ¼ 1.5 Hz, 17.5 Hz, 1H, CH2a¼), 5.36 (dd, J ¼ 1.5 Hz, 17.5 Hz,
1H, CH2b¼), 5.86 (m, 1H, CH¼), 7.71 (d, J ¼ 9.0 Hz, 2H, phenyl-
H2, phenyl-H6), 7.83 (d, J ¼ 9.0 Hz, 2H, phenyl-H3, phenyl-H5),
10.11 (s, 1H, NHSO2), 10.98 (s, 1H, CONH); MS m/z (%): 473
(M+, 0.3), 362 (1.7), 294 (1.5), 207 (3.8), 179 (12.6), 175 (2.2),
111 (21.4), 96 (10.0), 92(100); Anal. Calcd. for C20H19N5O5S2
(473.53): C, 50.73; H, 4.04; N, 14.79%, Found: C, 50.77; H, 4.08;
N, 14.82%.
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(3-adamantyl-4-oxothiazolidin-2-ylidene)-acetamide (10d)
Greenish brown powder, yield (88%), mp 132–133 ꢀC; IR (KBr) antifungal agent. Ampicillin, gentamycin, amphotericin B and
max/cmꢁ1: 3380 (NH), 3233 (NH), 3036 (CH–Ar), 2911 (CH- sulfisoxazole were used as reference drugs. DMF alone was
m
sp3), 2191 (CN), 1717 (CO), 1653 (CO); 1H NMR (500 MHz, used as control at the same aforementioned concentration and,
DMSO-d6): ꢀppm ¼ 1.65 (m, 9H, 9 ꢂ adamantyl-H), 1.69 (s, 3H, therefore, showed no visible change in bacterial growth. The
CH3), 2.02 (m, 3H, 3 ꢂ adamantyl-H), 2.08–2.10 (m, 6H, CH3, plates were incubated at 37 ꢀC for 24 h for bacteria and for 48 h
3 ꢂ adamantyl-H), 3.99 (s, 2H, CH2S), 7.72 (d, J ¼ 9.0 Hz, 2H, at 25 ꢀC for fungi. The mean zone of inhibition is measured
phenyl-H2, phenyl-H6), 7.84 (d, J ¼ 9.0 Hz, 2H, phenyl-H3, in mm standard deviation beyond well diameter (6 mm).
phenyl-H5), 10.13 (s, 1H, NHSO2), 11.01 (s, 1H, CONH); Compounds that showed growth IZs (412 mm) were further
MS m/z (%): 568 ([M+H]+, 21.0), 567 (M+, 8.4), 471 (2.7), evaluated for their MICs using the twofold serial dilution
393 (19.6), 273 (19.3), 252 (26.7), 175 (2.7), 112 (100), 111 (3.7), technique25.
96 (27.4); Anal. Calcd. for C27H29N5O5S2 (567.68): C, 57.13;
H, 5.15; N, 12.34%, Found: C, 57.17; H, 5.18; N, 12.39%.
Minimal inhibitory concentration (MIC) measurement
The microdilution susceptibility test in Mu¨ller-Hinton Broth
(Oxoid) and Subouraud Liquid Medium (Oxoid) was used to
assess the antibacterial and antifungal activities, respectively.
2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phe-
nyl)-2-(4-oxothiazolidin-3-phenyl-2-ylidene)-acetamide (10e)
Yellow powder, yield (79%), mp 145–146 ꢀC; IR (KBr) Stock solutions of the tested compounds, ampicillin, gentamycin,
max/cmꢁ1: 3354 (NH), 3262 (NH), 3056 (CH–Ar), 2939 (CH- amphotericin B and sulfisoxazole were prepared in DMF at a
m
sp3), 2197 (CN), 1729 (CO), 1667 (CO); 1H NMR (400 MHz, concentration of 1000 mg/mL. Each stock solution was diluted
DMSO-d6): ꢀppm ¼ 1.64 (s, 3H, CH3), 2.08 (s, 3H, CH3), 4.04 with standard method broth (Difco) to prepare serial twofold
(s, 2H, CH2), 7.46–7.79 (m, 9H, C6H4 and C6H5), 9.88 (s, 1H, dilutions in the range of 500–0.007 mg/mL. Then, 10 mL of
NHSO2), 10.95 (s, 1H, CONH); 13C NMR (100 MHz, DMSO-d6): the broth containing about 106 CFU/mL of test bacteria or
ꢀppm ¼ 5.7 (CH3), 10.1 (CH3), 32.6 (CH2), 77.1 (acetamide-C2), 104 CFU/mL of the test fungus was added to each well of 96-well
105.0 (CN), 116.1, 120.5, 124.6, 128.4, 127.5, 134.3, 138.4, 141.2 microtiter plate. The sealed microplates were incubated at 37 ꢀC
(12C, C6H4 and C6H5), 143.9, 156.8, 161.9 (3C, isoxazole-C3, for 24 h for antibacterial activity and at 25 ꢀC for 48 h for
isoxazole-C4, isoxazole-C5), 164.5, 169.3, 172.1 (3C, CONH, antifungal activity in a humid chamber. At the end of the
COCH2, thiazole-C2); MS m/z (%): 509 (M+, 0.9), 414 (6.3), 243 incubation period, the MICs values were recorded as the lowest
(6.7), 175 (3.9), 96 (12.3), 92 (10.9), 77 (75.3), 63 (100); Anal. concentrations of the substance that had no visible turbidity.
Calcd. for C23H19N5O5S2 (509.56): C, 54.21; H, 3.76; N, 13.74%, Control experiments with DMF and un-inoculated media were run
Found: C, 54.25; H, 3.81; N, 13.77%.
in parallel to the test compounds under the same conditions.
Molecular modeling
Antimicrobial evaluation
For the purpose of this study, the crystal structure of DHPS from
B. anthracis (BaDHPS) bound to the STZ-DHPP adduct18 was
Antimicrobial screening
The antimicrobial screening and measurement of minimal retrieved from the Protein Data Bank (PDB code: 3TYE) and
inhibitory concentrations (MICs) of the studied compounds processed as described previously24. The studied compounds were
were performed at the Regional Center for Mycology and docked into BaDHPS using the flexible Molecular Operating
Biotechnology, Al-Azhar University, Cairo, Egypt. The newly Environment (MOE) Dock methodology (Montreal, QC,
synthesized compounds were evaluated for their in vitro anti- Canada)26. The three-dimensional docking box was defined
microbial activities against the human pathogens Streptococcus around the co-crystallized ligand in the solvent-exposed binding
pneumoniae (RCMB 010010), Bacillis subtilis (RCMB 010067) pocket of the enzyme. The Alpha Triangle Matcher was used to
and Staphylococcus epidermidis (RCMB 010024) as examples of generate the initial placement poses, which were then rescored

6
T. Nasr et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
and prioritized using the London dG Scoring to maximize the to one NH2 and two NH protons at 7.10, 10.74 and 11.14 ppm,
hydrophobic, ionic and hydrogen-bond contacts to the protein. In respectively. The IR spectrum of compound 4 showed absorp-
a subsequent refinement stage, the generated poses were energy tion bands at 3409–3351, 3279, 3206, 1659, 1175 cmꢁ1 charac-
minimized using the MMFF94x force field. Finally, the optimized teristic for NH2, two NH, C¼O and C¼S functions, respectively.
poses were ranked using the GBVI/WSA DG free-energy The mass spectrum of 4 showed a molecular ion peak at
estimates27. The predicted docking poses were visually inspected m/z ¼ 501, which agrees with its molecular formula
and interactions with binding pocket residues were analyzed using (C21H19N5O4S3).
the ligand-interactions diagrams28.
Next, we described the behavior of N-(2-cyanoacetyl)sulfisox-
azole 3 towards alkyl/phenyl isothiocyanates and a-halo carbonyls
as a convenient route to attain some new functionalized thiazole
derivatives. Thus, the base prompted nucleophilic addition of the
active methylene group of 3 to a series of substituted isothio-
Results and discussion
Chemistry
Scheme 1 outlines the synthetic pathway for the preparation of cyanates 5 in DMF gave the non-isolable adduct 6a–e that reacted
4-amino-3-phenylthiazol-2-(3H)-thione 4, 2,3-dihydrothiazoles in situ with chloroacetone to afford the functionalized 2,3-
7a–e and 9a–e and thiazolidin-4-ones 10a–e. The precursor dihydrothiazoles 7a–e. Structures 7a–e were elucidated on the
N-(2-cyanoacetyl)sulfisoxazole 3 was prepared by cyanoacetyla- basis of elemental analyses and spectral data. IR spectra of 7a–e
tion of sulfisoxazole 1 with 1-cyanoacetyl-3,5-dimethylpyrazole 2 revealed only two NH bands at 3362–3323 and 3283–3238 cmꢁ1
in boiling dioxane as previously described from our laboratory24. in addition to lower conjugated CN and C¼O bands at 2189–2178
The Gewald reaction29 of compound 3, as a nitrile containing and 1656–1646 cmꢁ1 regions. 1H NMR spectra of 7a–e exhibited
active methylene moiety, with sulfur and phenyl isothiocyanate in the H-5 proton of the 2,3-dihydrothiazole ring as a sharp singlet
warming ethanol containing a catalytic amount of triethylamine signal at ꢀ ¼ 7.61–7.65 ppm. The 13C NMR spectrum of 7b, as a
afforded 4-amino-3-phenylthiazol-2(3H)-thione 4 in good yield. representative example of the synthesized analogues, showed five
The chemical structure of 4 was confirmed by 1H NMR spectrum aliphatic carbons at ꢀ 5.9, 10.2, 13.8, 27.9 and 56.0 ppm due to
which lacked a signal assignable for the active methylene four CH3 and CH2N, in addition to the nitrile and carbonyl
group and exhibited signals due to the aromatic ring protons carbons of the amide function at ꢀ 99.5 and 165.9 ppm,
at 7.34–7.88 ppm, and three exchangeable signals corresponding respectively.
CH
3
H
O
N
N
O
S
H C
3
N
2
R
H
N
1
O
S
O
O
Sulfur, PhNCS
EtOH, TEA
CN
R
H N
1
2
HN
O
NH
2
Dioxan
N
NC
Ph
S
N
1
3
4
O
S
HN
O
R NCS
KOH, DMF
2
O
R =
5
1
N
H
N
R
5, 6, 7, 10
O
R
8, 9
R
4
2
3
R
1
H
N
a
b
c
d
e
CH -
3
a
b
c
d
e
CH -
CH -
CH CH OOCCH -
C H -
4-Cl -CH -
CH CO-
CH CH OOC-
H
H
H
3
3
3
5
NC
R
2
3
3
CH CH -
3
2
2
-
+
2
S K
6a-e
CH =CH-CH -
2
2
2
adamantyl-
6
C H -
6
5
6
4
6e
6a
N
O
R
R
3
O
O
X
Cl
R
4
CH
Cl
Cl
Cl
3
X= Cl or Br
8
H
N
H
H
N
H
O
N
O
O
S
N
O
S
R
R
N
1
R
R
2
Ph
N
CH
3
N
R
N
1
1
1
2
HCl
NC
O
NC
NC
NC
R
NH
CH
3
3
S
S
R
4
11
9a-e
10a-e
7a-e
Scheme 1. Synthetic route to functionalized thiazoles tagged with sulfisoxazole.

DOI: 10.3109/14756366.2015.1016514
New 2,3-dihydrothiazoles and 4-thiazolidinones containing sulfisoxazole
7
In a similar manner, heterocyclization of the non-isolable S. pneumoniae (MIC, 0.12 mg/mL) and equipotent to gentamycin
potassium salt 6e with a variety of a-halo carbonyl reagents at against E. coli (MIC, 1.95 mg/mL). Moreover, the derivatives 9c–e
room temperature afforded the diverse substituted 2,3-dihy- were more potent than ampicillin against B. subtilis (MIC, 0.007–
drothiazoles 9a–e. 0.12 mg/mL versus 0.24 mg/mL; respectively). Interestingly, the
On the other hand, in situ heterocyclization of 6a–e with thiazoles 9c and 9d showed promising results against K.
chloroacetyl chloride afforded the 4-thiazolidinone derivatives pneumonia (MIC, 1.95, 0.24 mg/mL; respectively).
10a–e. The structures of 10a–e were established through different
Regarding the 4-thiazolidinones 10a–e, compound 10c was
spectroscopic techniques (IR, 1H NMR, 13C NMR, MS) and the most active against the tested Gram-positive bacteria and
elemental analyses data. IR spectra of 10a–e revealed two NH compounds 10a and 10b had half the potency of gentamycin
bands at 3380–3343 and 3280–3233 cmꢁ1 in addition to a nitrile against E. coli (MIC, 3.9 mg/mL versus 1.95 mg/mL). Moreover,
and two C¼O bands at 2201–2193, 1747–1716 and 1667– 10a,b displayed good activities against P. vulgaris and K.
1653 cmꢁ1 regions, respectively. Their mass spectra showed, in pneumonia.
addition to the molecular ion peak, in each case, a fragment ion
On the other hand, the 4-aminothiazole 4 had weak to
peak at m/z 111 corresponding to 3,4-dimethylisoxazolylamino moderate antibacterial activities against most of the tested
radical cation. 1H NMR spectra of 10a–e exhibited the methylene bacterial strains and it was completely inactive against
group of the 4-thiazolidinone ring as a sharp singlet signal at S. pneumonia.
ꢀ4.04–3.91 ppm. The 13C NMR spectrum of 10a, as a represen-
tative example of the synthesized analogues, showed the methy- Antifungal activity
lene carbon of the thiazolidinone ring at ꢀ32.7 ppm in addition to
The results displayed in Table 2 showed that most of the target
the carbonyl carbons of the amide and 4-thiazolidinone ring
compounds had more potent antifungal activities than sulfisox-
functions at ꢀ163.4 and 172.1 ppm, respectively. Moreover, the
azole and that the latter had weaker antifungal activities than
chemoselectivity of compounds 10a–e was chemically confirmed
amphotericin B against the tested microbes (Table 2).
by the alternate synthesis of 10a as a representative example of
The 4-methylthiazole 7c showed excellent activity against G.
the series. Thus, in situ reaction of the potassium salt 6a, prepared
candidum (MIC, 0.03 mg/mL) (Scheme 1) and was equipotent to
from 3 and methyl isothiocyanate in DMF in the presence of
amphotericin B against A. fumigates and S. racemosum (MIC,
potassium hydroxide, with chloroacetonitrile followed by acid
0.12 mg/mL and 7.81 mg/mL; respectively). Moreover, 4-
hydrolysis gave a product that proved identical in all respects
methylthiazole 7d displayed double the potency of amphotericin
(mp., m.mp., IR spectra) with 10a isolated from reaction of 6a
B against S. racemosum (MIC, 3.9 mg/mL versus 7.81 mg/mL) and
with chloroacetyl chloride (see Methods section). On the basis of
good activity against G. candidum (MIC, 0.98 mg/mL).
this finding, the other isomeric structure, 5-thiazolidinone, was
Interestingly, 4-methylthiazole 7e had four times the potency of
discarded.
amphotericin B against S. racemosum (MIC, 1.95 mg/mL versus
To account for the formation of the products 7, 9 and 10, it is
7.81 mg/mL) and excellent activity against G. candidum (MIC,
assumed, as depicted in Scheme 1, that the reaction of 3 with
0.03 mg/mL). It was observed that the thiazoles 4, 7a and 7b had
alkyl/aryl isothiocyanates starts with nucleophilic addition to give
moderate antifungal activities against the tested fungal strains.
6 as intermediate which then undergoes in situ cyclization
On the other hand, N-phenyl-thiazole 9a showed half the
with a-halo carbonyl reagents via elimination of HCl and water
potency of amphotericin B against A. fumigates (MIC, 0.24 mg/mL
to give 7, 9 and 10 as end products. This sequence is compatible
versus 0.12 mg/mL) and good activity against G. candidum (MIC,
with literature reports on reactions of thiocarbamoyl with a-halo
0.49 mg/mL). While, N-phenyl-thiazoles 9c, 9d and 9e displayed
carbonyl reagents to afford the respective thiazole derivatives30,31
.
half the potency of amphotericin B against S. racemosum (MIC,
15.63 mg/mL versus 7.81 mg/mL; respectively) and excellent
activity against G. candidum (MIC, 0.06, 0.03 and 0.98 mg/mL;
respectively).
Antimicrobial evaluation
Antibacterial activity
Finally, 4-thiazolidinones 10a, 10b and 10d showed moderate
antifungal activities. While, 4-thiazolidinones 10c displayed good
activity against A. fumigates and G. candidum (MIC, 1.95 and
0.24 mg/mL; respectively) and fourfold the potency of amphoteri-
cin B against S. racemosum.
Preliminary antibacterial screening was carried out for the target
compounds and the data are presented in Table 1. Interestingly,
some of the synthesized N-substituted sulfisoxazole derivatives
displayed better antibacterial activities compared to the reference
standards ampicillin and gentamycin and were superior to
sulfisoxazole, which has weaker antibacterial activities than
ampicillin and gentamycin (Table 1).
Molecular modeling
Some of the synthesized 4-methylthiazoles 7a–e showed
excellent activities against B. subtilis, S. pneumoniae, E. coli
MOE docking results
and K. pneumonia. Compounds 7c and 7d were fourfold more The docking results obtained from MOE-Dock showed that the
potent than ampicillin against B. subtilis (MIC, 0.06 mg/mL versus studied compounds can be accommodated in the binding pocket
0.24 mg/mL). Additionally, 7c had twofold the activity of ampi- of BaDHPS with a comparable orientation to the one observed in
cillin against S. pneumonia (MIC, 0.06 mg/mL versus 0.12 mg/mL) the STZ-DHPP covalent adduct in the reported crystal structure18.
and 7d was equipotent to ampicillin against the same microbe. On The top-ranked docking poses reproduce the key interactions
the other hand, 7b showed half the potency of ampicillin against observed in the STZ-DHPP–BaDHPS complex (Figure 2A).
B. subtilis (MIC, 0.49 mg/mL versus 0.24 mg/mL). On the other
Most notably, the benzene-sulfonamide moiety in all predicted
hand, 7a–e showed excellent activities against E. coli (MIC, 0.49– binding modes interacts with Ser221 via H-bonding between its
1.95 mg/mL versus 1.95 mg/mL for gentamycin) while 7d dis- sulfonamide group and the backbone NH group, while the phenyl
played half the potency of gentamycin against K. pneumonia ring packs against the side chains of Lys220 and Pro69. Moreover,
(MIC, 0.06 mg/mL versus 0.03 mg/mL). the central phenyl moiety in the studied compounds makes face-
Additionally, the N-phenyl-thiazoles 9a–e showed good to to-edge interaction with Phe189 (H-to-C distances ranging from
˚
excellent antimicrobial activities and 9c was the most active 3.0 to 3.6 A). As shown in Figure 2, this characteristic orientation
among this series; it was equipotent to ampicillin against of the benzene-sulfonamide is common among all studied

8
T. Nasr et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
Table 1. Antibacterial inhibition zone in mm standard deviation and minimal inhibitory concentrations (MIC, mg/mL, between brackets) of some
newly synthesized compounds.
Gram-positive bacteria
Gram-negative bacteria
Compounds
S. pneumoniae
B. subtilis
S. epidermidis
E. coli
P. vulgaris
K. pneumonia
a
4
7a
7b
7c
7d
7e
9a
9b
9c
9d
9e
10a
10b
10c
10d
10e
–
16.0 0.44 (31.25) 18.3 0.67 (7.81)
17.4 0.25 (15.63) 15.2 0.44 (62.5)
11.2 0.33 (NDb)
21.2 0.58 (0.98)
20.3 0.63 (1.95)
20.1 0.58 (0.98)
22.3 0.33 (0.49)
19.5 0.37 (1.95)
17.1 0.37 (15.63) 18.2 0.50 (7.81)
13.7 0.25 (15.63) 10.8 0.44 (ND)
19.8 0.25 (1.95)
9.2 0.53 (ND)
13.0 0.46 (500)
14.6 0.58 (62.5)
23.4 0.63 (0.24)
17.2 0.58 (15.63) 22.8 0.19 (0.24)
14.0 0.58 (250)
18.8 0.38 (3.9)
20.6 0.34 (0.98)
21.2 0.63 (0.98)
19.3 0.56 (3.9)
18.7 0.68 (3.9)
17.7 0.43 (7.81)
20.3 0.44 (1.95)
21.2 0.43 (0.98)
24.6 0.34 (0.06)
24.2 0.44 (0.12)
22.4 0.29 (0.49)
16.7 0.38 (15.63) 19.1 0.41 (3.9)
–
23.6 0.58 (0.12)
19.5 0.44 (1.95)
19.6 0.44 (1.95)
16.9 0.38 (15.63) 18.1 0.44 (7.81)
16.2 0.15 (31.25) 19.8 0.42 (1.95)
22.3 0.53 (0.49)
24.6 0.25 (0.06)
25.1 0.25 (0.06)
21.3 0.41 (0.98)
19.8 0.42 (1.95) 22.3 0.44 (0.49)
– 24.8 0.63 (0.06)
16.3 0.48 (31.25) 17.9 0.30 (7.81)
16.9 0.31 (15.63)
15.4 0.44 (62.5)
16.3 0.25 (31.25) 20.3 0.44 (1.95)
15.2 0.44 (62.5)
–
21.4 0.31 (0.98)
19.3 0.53 (3.9)
25.6 0.63 (0.03)
29.8 0.58 (0.007) 17.4 0.53 (15.63) 17.6 0.19 (7.81)
23.7 0.63 (0.12)
22.6 0.63 (0.24)
15.6 0.25 (31.25)
19.6 0.50 (1.95)
16.1 0.33 (31.25) 14.4 0.25 (125)
16.1 0.68 (31.25) 18.9 0.37 (3.9)
14.3 0.58 (250)
19.6 0.27 (1.95)
17.2 0.58 (15.63) 13.9 0.46 (250)
19.2 0.63 (3.9)
17.2 0.58 (15.63) 16.1 0.53 (31.25) 18.3 0.58 (7.81)
17.4 0.53 (15.63) 20.3 0.58 (1.95)
20.9 0.44 (0.98)
18.1 0.63 (7.81)
15.0 0.43 (62.5)
20.3 0.58 (1.95)
20.0 0.32 (1.95)
17.4 0.53 (15.63) 13.6 0.44 (250)
20.8 0.63 (0.98)
32.4 0.30 (0.24)
NT
–
–
–
NT
21.2 0.45 (0.98)
14.3 0.44 (250)
18.2 0.58 (7.81)
NT
11.2 0.33 (ND)
15.2 0.25 (62.5)
NTc
Sulfisoxazole 19.6 0.44 (1.95)
23.8 0.20 (0.12)
NT
17.5 0.72 (7.81)
25.4 0.18 (0.06)
NT
Ampicillin
Gentamycin
19.9 0.30 (1.95)
23.4 0.30 (0.24)
26.3 0.15 (0.03)
aNo activity.
bND, not determined.
cNT, not tested.
Table 2. Antifungal inhibition zone in mm standard deviation and minimal inhibitory concentrations (MIC, mg/mL, between brackets) of some newly
synthesized compounds.
Fungi
Fungi
Compounds
A. fumigatus
S. racemosum
G. candidum
Compounds
A. fumigatus
S. racemosum
G. candidum
4
16.3 0.25 (31.25) 15.2 0.58 (62.5) 17.3 0.17 (15.63)
13.6 0.25 (250) 11.7 0.34 (ND*) 16.5 0.58 (31.25)
9c
9d
9e
10a
10b
10c
10d
10e
21.3 0.39 (0.98) 17.2 0.16 (15.63) 24.6 0.58 (0.06)
22.3 0.25 (0.49) 16.5 0.25 (15.63.) 25.8 0.58 (0.03)
20.4 0.39 (1.95) 16.5 0.16 (15.63) 21.4 0.58 (0.98)
7a
7b
7c
7d
7e
9a
9b
15.3 0.55 (62.5) 13.4 0.35 (500) 11.5 0.58 (ND)
24.3 0.63 (0.12) 17.6 0.27 (7.81) 26.9 0.35 (0.03)
13.9 0.29 (250)
12.1 0.18 (500)
14.6 0.35 (62.5)
16.8 0.34 (15.63)
22.6 0.65 (0.24)
17.6 0.58 (7.81)
15.3 0.38 (62.5)
16.8 0.58 (15.63)
17.1 0.53 (15.63) 18.8 0.42 (3.9)
20.9 0.31 (0.98)
15.7 0.33 (31.25) 13.8 0.25 (250)
20.2 0.36 (1.95) 16.9 0.27 (1.95)
16.2 0.36 (31.25) 15.0 0.44 (62.5)
20.3 0.39 (1.95) 20.3 0.16 (1.95) 26.6 0.58 (0.03)
23.0 0.34 (0.24) 14.6 0.52 (125) 21.9 0.53 (0.49)
17.6 0.58 (7.81) 15.4 0.25 (62.5) 12.6 0.38 (500)
11.3 0.34 (ND)
12.1 0.25 (500)
Amphotericin B 23.7 0.1 (0.12)
19.7 0.2 (7.81)
28.7 0.2 (0.007) Sulfisoxazole 15.1 0.39 (62.5) 13.2 0.58 (125)
*ND, not determined.
compounds indicating that all of them can occupy the PABA substituent in compounds 9d and 9e was accommodated in
pocket preventing the key substrate from binding, which is the a hydrophobic pocket lined by Met145, Leu214 and Ile143
basis of the inhibitory action of all sulfa drugs against DHPS32,33
.
(Figure 2F), which is in agreement with the reported flexibility of
In addition to the distinctive interactions in the PABA-binding the binding pocket of BaDHPS18.
pockets, the studied compounds exhibit interactions with resi-
The 4-thiazolidinone derivative 10a presented the same H-
dues in the pterin-binding pocket of DHPS that vary slightly bond to Arg254 and formed an extra H-bond from the
between chemical classes. For instance, the 4-methylthiazole thiazolidinone oxygen to the side chain of Asp184 in the back
derivative 7a exhibits cation–ꢁ interaction between its thiazole of the pterin-binding pocket (Figure 2C). On the other hand, the
ring and the charged side chain of Lys220, as well as a hydrogen thiazolidinone ring of compound 10e exhibited 180ꢀ rotation,
bond between its central amide group and the side chain of thereby forming a hydrogen bond to the acidic side chain of
Arg254, which could further stabilize its interaction with DHPS Asp101 instead (Figure 2D).
(Figure 2B).
The bulky phenyl substituent in the N-phenyl-thiazole series
Lipophilicity study
9a–e enforces a bound conformation where the thiazole ring
points deeper into the pterin-binding pocket making interaction
with Lys220 via cation–ꢁ contact. Compounds 9b and 9c bear an
ester substitution on their terminal thiazole rings. This unique
decoration allows for a water-mediated hydrogen bond to the side
chain of Asp184 deeper in the pterin-binding pocket (Figure 2E),
which utilizes the W286 water molecule observed in the
employed crystal structure18. Conversely, the second phenyl
The lipophilic characters of sulfisoxazole and the target com-
pounds were calculated by the online program OSIRIS Property
Explorer. The results are summarized in Table 3 and revealed
that most of the target compounds were more lipophilic than
sulfisoxazole. Thus, they were expected to have an improved
intracellular penetration and consequently better antimicrobial
activities than sulfisoxazole.

DOI: 10.3109/14756366.2015.1016514
New 2,3-dihydrothiazoles and 4-thiazolidinones containing sulfisoxazole
9
Figure 2. Predicted binding modes of the studied compounds generated by MOE-Dock. (A) Interactions with key residues in the crystal structure of the
STZ-DHPP adduct (orange carbons) with BaDHPS (PDB code 3TYE, STZ: sulfathiazole, DHPP: 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate).
(B–F) Docking poses for compounds 7a, 9b, 9e, 10a and 10e, respectively (green: ligand’s carbon, red: oxygen, blue: nitrogen, yellow: sulfur, white:
hydrogen). Protein is shown as light gray cartoons, hydrogen bonds as dotted red lines, and crystal water W286 as magenta sphere. Colors are available
in the online version of the paper.
Regarding the 4-methylthiazoles 7a–e (Scheme 1), the larger Finally, 4-thiazolidinones 10a–e were more polar than 4-
the N-alkyl substitution the higher the lipophilic character methylthiazoles 7a–e and consequently had lower antimicrobial
(Table 3); and consequently the best antimicrobial activities activities than compounds 7a–e (Table 1). It was obvious that the
were observed in the case of N-allyl and N-adamantyl substitu- 4-thiazolidinone 10a was less polar but more active than
tions. The high lipophilic characters of the N-phenyl-thiazoles sulfisoxazole against the examined Gram-negative bacteria;
9a–e (Scheme 1) were among the factors responsible for thus, there should be other factors than its lipophilicity respon-
their observed good antimicrobial activities (Tables 1 and 2). sible for the observed activity.

10 T. Nasr et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
Table 3. Calculated lipophilic characters of the target compounds.
pterin-binding pockets of DHPS. Additionally, the synergistic
effect of both the sulfonamide and the thiazole ring could
explain the targets’ superior antimicrobial profiles. Particularly,
compounds 7a, 7c–e, 9c–d and 10c displayed promising
antimicrobial activities.
Compounds
C log P*
Compounds
C log P
Sulfisoxazole
4
1.02
1.86
2.25
2.66
2.93
4.33
3.75
3.58
3.74
9c
9d
9e
10a
10b
10c
10d
10e
3.76
5.04
5.65
0.91
1.31
1.58
2.98
2.40
7a
7b
7c
7d
7e
9a
9b
Acknowledgements
The authors are thankful to King Khalid University for providing the
facilities to carry out this research.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
*Calculated by online program OSIRIS Property Explorer.
Structure activity relationship
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The results obtained from this study revealed that most of the
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and pterin-binding pockets of DHPS enzyme (Figure 2) could
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Supplementary material available online
Supplementary Figure I