Synthesis and antibacterial evaluation of novel heterocyclic compounds containing a sulfonamido moiety

Article abstract of DOI:10.3390/molecules18010832

Aiming for the synthesis of new heterocyclic compounds containing a sulfonamido moiety suitable for use as antibacterial agents, the precursor ethyl {[4-N-(4,6- dimethylpyrimidin-2-yl)sulfamoyl]phenylazo}cyanoacetate was reacted with a variety of active methylene compounds producing pyran, pyridine and pyridazine derivatives. Also, the reactivity of the precursor hydrazone towards hydrazine derivatives to give pyrazole and oxazole derivatives was studied. On the other hand, treatment of the same precursor with urea, thiourea and/or guanidine hydrochloride furnished pyrimidine and thiazine derivatives, respectively. The newly synthesized compounds were tested for antibacterial activity, whereby eight compounds were found to have high activities.

Full text of DOI:10.3390/molecules18010832

Molecules 2013, 18, 832-844; doi:10.3390/molecules18010832
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antibacterial Evaluation of Novel Heterocyclic
Compounds Containing a Sulfonamido Moiety
Mohammad Emad Azab 1, Mohamed M. Youssef 2,†,* and Eman A. El-Bordany 1
1
Organic Synthetic Lab, Chemistry Department, Faculty of Science, Ain Shams University,
Cairo 11566, Egypt
2
Department of Chemistry, Faculty of Science, Taif University, Taif 21974, Saudi Arabia
†
Present address: Chemistry Department, Faculty of Science, Cairo University, Cairo 12613, Egypt.
* Author to whom correspondence should be addressed; E-Mail: mmmyousef@yahoo.com;
Tel.: +966-59-607-2593 or +2-012-2245-1999.
Received: 31 October 2012; in revised form: 4 January 2013 / Accepted: 5 January 2013 /
Published: 11 January 2013
Abstract: Aiming for the synthesis of new heterocyclic compounds containing a
sulfonamido moiety suitable for use as antibacterial agents, the precursor ethyl {[4-N-(4,6-
dimethylpyrimidin-2-yl)sulfamoyl]phenylazo}cyanoacetate was reacted with a variety of
active methylene compounds producing pyran, pyridine and pyridazine derivatives. Also,
the reactivity of the precursor hydrazone towards hydrazine derivatives to give pyrazole
and oxazole derivatives was studied. On the other hand, treatment of the same precursor
with urea, thiourea and/or guanidine hydrochloride furnished pyrimidine and thiazine
derivatives, respectively. The newly synthesized compounds were tested for antibacterial
activity, whereby eight compounds were found to have high activities.
Keywords: cyanoacetylhydrazide; sulfonamide; pyridazines; pyrazoles; oxazole;
pyrimidines; antibacterial activity
1. Introduction
Simple nitrogen-containing heterocycles attached to sulfonamido moieties have received a large
amount of attention in the literature, as a consequence of their exciting biological properties and their
role as pharmacophores of considerable historical importance. Heterocyclic sulfonamides are used as

Molecules 2013, 18
833
carbonic anhydrase inhibitors [1–3], antibacterial agents [4], anticancer, anti-inflammatory and analgesic
agents [5], β3-adrenergic receptor agonists [6], PC-1 inhibitors [7], antifungal agents [8] and antiviral
agents [9]. For these vast biological activities and in continuation of our work [10–18] on the synthesis
of novel heterocyclic systems exhibiting biological activity, we undertook the synthesis of a new series
of compounds incorporating the abovementioned biologically active moieties in one molecule.
2. Results and Discussion
2.1. Chemistry
Synthesis of the precursor hydrazone 3 was achieved by diazotization of sulfamethazine,
[N1-(4,6-dimethyl-2-pyrimidinyl)sulfanilamide, 1] followed by coupling with ethyl cyanoacetate in the
presence of sodium acetate at room temperature [19] (Scheme 1). The spectral data revealed that this
1
compound exists in the hydrazone form (b), as the H-NMR spectrum showed two (exchangeable)
signals at 6.91 and 9.12 corresponding to two NH groups and the MS indicated the molecular ion peak
at 402, which is in accordance with the molecular formula.
Scheme 1. Diazotization and coupling of sulfamethazine; formation of 3.
CH3
CH3
N
N
NaNO2/HCl
SO2
SO2
NH2
N2Cl
H3C
N
N
H
H3C
N
N
H
2
1
CN
CO2Et
CH3
CH3
N
N
CN
N N CH
CO2Et
CN
CO2Et
SO2
SO2
N
H
N
H3C
H3C
N
N
H
N
N
H
3
(a)
(b)
The reactivity of compound 3 towards active methylene reagents was investigated. Firstly, reaction
of hydrazone 3 with dicarbonyl compounds was studied. Thus, when compound 3 reacted with
acetylacetone in refluxing dioxane in the presence of catalytic amounts of triethylamine, the pyranone
derivative 5 was obtained. Formation of 5 is believed to be formed via the intermediate 4 followed by
the intramolecular cyclization with loss of an ethanol molecule. The structure of 5 was confirmed by
the analytical and spectral data. Similarly, reaction of 3 with acetoacetanilide under the same reaction
conditions afforded the pyridinone derivative 7 which is formed through the intermediate 6 followed
by loss of an ethanol molecule (Scheme 2).
Secondly, the behavior of 3 with other active methylene compounds, such as ethyl acetoacetate,
ethyl benzoylacetate, ethyl cyanoacetate and malononitrile, was investigated. This investigation
resulted in the synthesis of polyfunctional substituted pyridazine derivatives 9a–c, through the
intermediate 8 and 11 through the intermediate 10, respectively (Scheme 2). The structures of the
synthesized compounds were elucidated based on their spectral data.

Molecules 2013, 18
Scheme 2. Cyclization of the arylazo cyanoacatate derivative 3 with active methylene
834
compounds and β-dinitriles; formation of substituted pyrans, pyridines and pyridazines.
O
R
N
CO2Et
O
R
N
N
N
O
O
N
O
(a)
-EtOH
H2N
H2N
O
5
4
O
R
N
CO2Et
R
N
Ph
N
N
O
(b)
(c)
-EtOH
H2N
OH
H2N
NHPh
R
N
CO2Et
O
N
7
6
CN
3
R
N
CO2Et
NH2
R
N
CO2Et
NH2
N
NH
EtO2C
O
X
X
a, X = COCH3
b, X = COPh
c, X = CN
9
8
R
N
CO2Et
NH2
R
N
CO2Et
(d)
NH
N
NC
CH3
HN
NH2
N
CN
10
CN
R =
SO2
H3C
N
N
H
11
Reaction conditions: (a) acetylacetone/dioxane/Et3N/∆; (b) acetoacetanilde/dioxane/Et3N/∆; (c) ethyl
acetoacetate, ethyl benzoylacetate, and/or ethyl caynoacetate/dioxane/Et3N/∆; (d) malononitrile/dioxane/Et3N/∆.
The behaviour of 3 towards hydrazine derivatives was examined in order to prepare pyrazoles.
Thus, treatment of 3 with hydrazine hydrate [20] (98%), phenylhydrazine, benzoylhydrazine or
thiosemicarbazide [21] furnished the aminopyrazole derivatives 12a–d, respectively. On the other
hand, reaction of 3 with hydroxylamine hydrochloride in the presence of sodium acetate produced the
isoxazole derivative 13 (Scheme 3). The structure of the prepared compounds were in accordance with
their spectral data.
Pyrazolopyrimidine [22] derivative 16 was obtained when the hydrazone 3 was treated with
cyanoacetohydrazide in refluxing dioxane in the presence of triethylamine. As a speculative
mechanism for the formation of compound 16, the intermediate 14 is firstly formed followed by an
internal nucleophilic attack by the NH group on the cyano group. Then a migration of the two NH
protons to the negatively charged nitrogen atom takes place to form the second intermediate 15.
Finally, 15 cyclized via nucleophilic attack by the NH2 group on the cyano group to produce the
pyrazolopyrimidine derivative 16 (Scheme 3).

Molecules 2013, 18
Scheme 3. Cyclization of the arylazo cyanoacatate derivative 3 with amino compounds;
835
formation of substituted pyrazoles, isoxazoles, pyrazolopyrimidines, pyrimidines and
1,3-thiazines.
R
N
O
NH2
X
N
(a)
(b)
a, X = H
b, X = Ph
c, X = COPh
d, X = CSNH2
N
N
H
12
R
N
O
NH2
H
N
N
O
13
O
R
N
OH
R
N
N
OH
N
R
N
N
N
NH
(c)
N
O
N
H2N
NC
N
CN HN
O
N
H2N
OH
NC
14
3
15
16
NH2
R
R
N
N
N
NH
(d)
(e)
CH3
O
N
H
O
N
R =
SO2
H3C
N
N
H
17
NH2
N
O
a, X = S
b, X = NH
X
N
NH2
18
Reaction conditions: (a) hydrazine hydrate, Phenyl hydrazine, benzoyl hydrazine or
thiosimicarbazide/dioxane/∆; (b) hydroxylamine hydrochloride/dioxane/AcONa/∆; (c) cyanoacetohydrazide/
dioxane/Et3N/∆; (d) urea/EtOH/EtONa/∆; (e) thiourea or guanidine. HCl/EtOH/EtONa/∆.
Treatment of compound 3 with urea, thiourea and guanidine in the presence of ethanolic sodium
ethoxide [23] produces the pyrimidine and thiazine derivatives 17, 18a,b (Scheme 3). The formation of
these compounds is assumed to be occur via the addition of the NH2 or SH groups to the cyano group
followed by cyclization with elimination of an ethanol molecule.
2.2. Antibacterial Activity Evaluation
2.2.1. Agar Diffusion Method
The obtained new compounds were screened in vitro for their antibacterial activities against Gram
positive bacteria [Staphylococcus aureus (ATCC 25923) and Bacillus cereus (ATCC 10987)], Gram

Molecules 2013, 18
836
negative bacteria [Serratia marcesens (ATCC 274) and Proteus mirabilis (SM514)], using the agar
diffusion technique. The results of the antibacterial activity tests are shown in Table 1.
Table 1. Antibacterial activity of the synthesized compounds: Agar diffusion method.
Compound
No.
Gram positive
Gram negative
Proteus mirabilis
Staphylococcus
Bacillus cereus
Serratia
aureus
++
-
marcesens
3
5
7
++
-
++
+
++
+
++
+
++
+
+
+
9a
+
+
9b
+
-
+
+
9c
+
+
-
-
11
+
+
+
+
12a
12b
12c
12d
13
+++
++
++
+++
+
+++
+++
++
++
+
+++
++
++
+
+++
++
++
++
+
+
16
++
++
+
++
+++
+
+++
+++
+++
+++
+++
++
+
17
18a
18b
++
++
+++
The width of the zone of inhibition indicates the potency of antibacterial activity; (-) no antibacterial activity;
(+) mild activity with the diameter of the zones equal to 0.5–0.8 cm, (++) moderate activity with the diameter
of the zones equal to 1.1–1.2 cm; (+++) marked high activity with the diameter of the zones equal to 1.8–2.0 cm.
Most of the synthesized compounds were found to possess some antibacterial activity towards all
the microorganisms used. Compounds 3, 12a, 12b, 12c, 12d, 16, 17, 18b possess the highest
antibacterial activities.
2.2.2. Filter Paper Disc-Diffusion Method
The newly synthesized heterocyclic compounds listed in Table 2 were tested for their antibacterial
activity against Gram positive bacteria [Staphylococcus aureus (ATCC 25923) and Bacillus cereus
(ATCC 10987)], Gram negative bacteria [Serratia marcesens (ATCC 274) and Proteus mirabilis
(SM514)]. The preliminary screening of the investigated compounds was performed using the filter
paper disc-diffusion method. The most active compounds were 12a, 12b, 12d, 16, 17, 18a and 18b,
which were strongly inhibitory to all or some of the tested bacteria. Compounds 3, 7 and 12c showed
moderate activities against the tested bacteria. The rest of compounds showed low or no sensitivity at
all to the bacteria under investigation, and the results are summarized in Table 2.

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Table 2. Antibacterial activity of the synthesized compounds.
Inhibition zone (mm)
Comp. No.
Gram-positive
Gram-negative
Staphylococcus aureus Bacillus cereus Serratia marcesens Proteus mirabilis
3
5
12
0
10
0
11
5
8
7
7
9a
13
3
11
5
6
3
6
4
9b
6
7
2
6
9c
6
6
8
5
11
5
7
7
8
12a
12b
12c
12d
17
15
12
16
7
16
12
11
12
7
16
13
10
7
18
11
10
11
8
13
5
16
17
18a
18b
13
17
9
12
18
19
11
12
8
13
19
22
16
18
15
17
22
24
17
14
7
15
21
20
Chloram-phenicol®
Ampicilin®
The sensitivity of microorganisms to the tested compounds is identified in the following manner:
Highly sensitive = Inhibition zone 15–20 mm; Moderately sensitive = Inhibition zone: 10–15 mm;
Slightly sensitive = Inhibition zone: 5–10 mm; Not sensitive = Inhibition zone: 0–5 mm; Each result
represents the average of triplicate readings.
3. Experimental
3.1. General
All melting points reported are uncorrected and were determined on a Stuart electric melting point
apparatus. The microanalysis were within ±0.4% of theoretical values and were determined at the
Microanalytical Unit of the Faculty of Science, Cairo University. The IR spectra were measured on a
Perkin-Elmer 1600 FT-IR using the KBr wafer technique. The mass spectra were recorded on a
1
Shimadzu GCMS-QP-1000EX mass spectrometer at 70 e.v. The H-NMR spectra were recorded in
CDCl3 or DMSO-d6 solutions on a Bruker 200 MHz instrument using TMS as internal standard with
chemical shifts expressed in ppm. 13C-NMR spectra were recorded on a Varian Mercury 300 MHz
spectrometer using TMS as an internal standard and DMSO-d6 as solvent. TLC was performed on
ready-to-use Merck 60 silica gel plates to monitor the reactions and test the purity of the new
synthesized compounds.
3.1.1. Ethyl {[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenylazo}cyanoacetate (3)
This compound was prepared according to a previously reported method [19]. Yellow solid (from
ethanol), yield 81%, m.p. 228–230 °C. IR (/cm−1): 3232, 3192 (NH), 3053 (CHar), 2989 (CHal), 2221

Molecules 2013, 18
838
(C≡N), 1723 (C=O), 1610 (C=N), 1563 (N=N), 1327, 1150 cm−1 (SO2). 1H-NMR (DMSO-d6): 1.22 (t,
3H, OCH2CH3, J = 4.4 Hz), 2.21 (s, 6H, 2 CH3), 4.27 (q, 2H, OCH2CH3, J = 4.4 Hz), 6.91 (s, 1H, NH,
D2O exchangeable), 7.57–8.15 (m, 5H, Ar-H + pyrimidine-H), 9.12 (s, 1H, NH, D2O exchangeable).
MS (%): molecular ion peak at 402 (3.5) and M+1 at 403 (4.5). C17H18N6O4S (402); Calcd: C, 50.74;
H, 4.51; N, 20.88; S, 7.97; Found: C, 51.00; H, 4.70; N, 21.10; S, 7.80.
3.1.2. Reaction of 3 with Acetylacetone or Acetoacetanilide; Formation of 5 and 7
A mixture of 3 (5 mmol, 2.06 g) and acetylacetone or acetoacetanilide (5 mmol) was dissolved in
dioxane (40 mL) containing triethylamine (5 drops). The reaction mixture was heated under reflux for
6–8 h and concentrated. The solid product that precipitated after cooling was filtered of and
crystallized from the proper solvent to compound 5 and 7, respectively.
5-Acetyl-4-amino-3-{[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenylazo}-6-methyl-2H-pyran-2-
one (5): Pale yellow solid (from dioxane), yield 68% (1.55 g), m.p. 263–265 °C. IR (/cm−1): 3433,
1
3364, 3224, 3171 (NH2, NH), 2996, 2943 (CH), 1689, 1661 (C=O) cm−1. H-NMR (DMSO-d6): 2.21
(s, 6H, 2 CH3), 2.47 (s, 3H, CH3), 2.68 (s, 3H, COCH3), 5.66(s, 2H, NH2, D2O exchangeable), 6.95 (s, 1H,
13
NH, D2O exchangeable), 7.51–8.22 (m, 5H, Ar-H + pyrimidine-H). C-NMR (DMSO-d6): 20.3 (CH3),
25.1 (2CH3), 35.6 (CH3CO), 113.4, 118.5, 119.3, 121.7, 122.3, 124.8, 125.5, 128.4, 131.6, 133.9, 138.4
(Ar-C), 166.4, 181.5 (CO). MS (%): molecular ion peak at 456 (3.5) and M+1 at 457 (4.5). C20H20N6O5S
(456); Calcd: C, 52.62; H, 4.42; N, 18.41; S, 7.02; Found: C, 52.40; H, 4.70; N, 18.20; S, 7.30.
5-Acetyl-4-amino-3-{[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]-phenylazo}-6-hydroxy-1-phenylpyridin-
2(1H)-one (7): Yellow solid (from dioxane), yield 60% (1.60 g), m.p. 271–274 °C. IR (/cm−1): 3502,
1
3435, 3364, 3228, 3176 (OH, NH2, NH), 2996, 2943 (CH), 1691, 1660 (C=O) cm−1. H-NMR
(DMSO-d6): 2.23 (s, 6H, 2 CH3), 2.64 (s, 3H, COCH3), 5.78(s, 2H, NH2, D2O exchangeable), 6.94 (s,
1H, NH, D2O exchangeable), 7.23–8.12 (m, 10H, Ar-H + pyrimidine-H), 10.34 (s, 1H, OH, D2O
exchangeable). 13C-NMR (DMSO-d6): 25.1 (2CH3), 35.5 (CH3CO), 114.1, 116.8, 119.2, 120.0, 120.7,
122.3, 124.5, 125.6, 128.3, 129.7, 132.1, 134.3, 140.6, 141.4, 143.1 (Ar-C), 161.7, 184.1 (CO). MS
(%): molecular ion peak at 533 (2.7). C25H23N7O5S (533); Calcd: C, 56.28; H, 4.34; N, 18.38; S, 6.01;
Found: C, 56.00; H, 4.60; N, 15.20; S, 6.10.
3.1.3. Reaction of 3 with β-ketoesters, β-cyanoesters and β-dicarbonitriles; Formation of 9a–c and 11
Equimolar amounts of 3 (5 mmol, 2.06 g) and an active methylene compound, namely ethyl
acetoacetate, ethyl benzoylacetate, ethyl cyanoacetate or malononitrile (5 mmol) in dioxane (40 mL)
containing triethylamine (5 drops), were heated under reflux for 8–10 h. The solid product so obtained
on cooling was collected by filtration and crystallized from the appropriate solvent to give compounds
9a–c and 11, respectively.
Ethyl 5-acetyl-4-amino-1-[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenyl-1,6-dihydro-6-oxopyrid-
azine-3-carboxylate (9a): Yellow solid (from dioxane), yield 51% (1.24 g), m.p. 260–263 °C. IR
1
(/cm−1): 3443, 3356, 3229, 3169 (NH2, NH), 1728, 1686, 1666 (C=O) cm−1. H-NMR (DMSO-d6):
1.29 (t, 3H, OCH2CH3, J = 4.3 Hz), 2.24 (s, 6H, 2 CH3), 2.68 (s, 3H, COCH3), 4.25 (q, 2H, OCH2CH3,

Molecules 2013, 18
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J = 4.3 Hz), 5.76(s, 2H, NH2, D2O exchangeable), 6.89 (s, 1H, NH, D2O exchangeable), 7.49–8.14 (m,
5H, Ar-H + pyrimidine-H). 13C-NMR (DMSO-d6): 15.2 (CH3), 25.1 (2CH3), 35.5 (CH3CO), 63.2
(CH2), 110.1, 117.3, 119.4, 120.3, 126.5, 129.7, 132.4, 134.1, 139.1, 142.7 (Ar-C), 161.5, 168.4, 183.9
(CO). MS (%): molecular ion peak at 486 (22.4). C21H22N6O6S (486); Calcd: C, 51.84; H, 4.56; N,
17.27; S, 6.59; Found: C, 52.00; H, 4.70; N, 16.90; S, 6.80.
Ethyl 4-amino-5-benzoyl-1-[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenyl-1,6-dihydro-6-oxo-
pyridazine-3-carboxylate (9b): Orange solid (from dioxane-water), yield 52% (1.43 g), m.p. 272–275 °C.
IR (/cm−1): 3435, 3345, 3230, 3173 (NH2, NH), 2989, 2941 (CH3), 1730, 1688, 1662 (C=O) cm−1.
1H-NMR (DMSO-d6): 1.30 (t, 3H, OCH2CH3, J = 4.4 Hz), 2.22 (s, 6H, 2 CH3), 4.23 (q, 2H,
OCH2CH3, J = 4.4 Hz), 5.81 (s, 2H, NH2, D2O exchangeable), 6.92 (s, 1H, NH, D2O exchangeable),
7.33–8.17 (m, 10H, Ar-H + pyrimidine-H). MS (%): molecular ion peak at 548 (13.1). C26H24N6O6S
(548); Calcd: C, 56.93; H, 4.41; N, 15.32; S, 5.85; Found: C, 57.10; H, 4.70; N, 15.10; S, 5.60.
Ethyl 4-amino-5-cyano-1-[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenyl-1,6-dihydro-6-oxopyrid-
azine-3-carboxylate (9c): Yellow solid (from dioxane), yield 59% (1.38 g), m.p. 281–284 °C. IR
1
(/cm−1): 3445, 3349, 3240, 3179 (NH2, NH), 2218 (C≡N), 1728, 1690 (C=O) cm−1. H-NMR
(DMSO-d6): 1.28 (t, 3H, OCH2CH3, J = 4.4 Hz), 2.26 (s, 6H, 2 CH3), 4.28 (q, 2H, OCH2CH3, J = 4.4 Hz),
5.78 (s, 2H, NH2, D2O exchangeable), 6.94 (s, 1H, NH, D2O exchangeable), 7.52–8.11 (m, 5H, Ar-H +
pyrimidine-H). MS (%): molecular ion peak at 469 (18.1). C20H19N7O5S (469): Calcd; C, 51.17; H,
4.08; N, 20.88; S, 6.83; Found: C, 51.30; H, 3.80; N, 21.10; S, 7.00.
Ethyl 4-amino-5-cyano-1-[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenyl-1,6-dihydro-6-iminopy-
ridazine-3-carboxylate (11): Light brown solid (from acetic acid), yield 71% (1.38 g), m.p. 293–296 °C.
IR (/cm−1): 3430, 3339, 3235, 3176 (NH2, NH), 2220 (C≡N), 1726 (C=O) cm−1. 1H-NMR (DMSO-d6):
1.30 (t, 3H, OCH2CH3, J = 4.4 Hz), 2.27 (s, 6H, 2 CH3), 4.22 (q, 2H, OCH2CH3, J = 4.4 Hz), 5.67 (s,
2H, NH2, D2O exchangeable), 6.88 (s, 1H, NH, D2O exchangeable), 7.42–8.16 (m, 5H, Ar-H +
pyrimidine-H), 10.88 (br s, 1 H, =NH, D2O exchangeable). MS (%): molecular ion peak at 468 (24.8).
13C-NMR (DMSO-d6): 15.3 (CH3), 25.1 (2CH3), 63.4 (CH2), 115.3 (CN), 118.1, 119.3, 120.8, 121.4, 125.3,
127.1, 129.7, 132.4, 135.1, 139.7, 153.3 (Ar-C), 164.3 (CO). MS (%): molecular ion peak at 468 (19.5).
C20H20N8O4S (468); Calcd: C, 51.27; H, 4.30; N, 23.92; S, 6.84; Found: C, 51.50; H, 4.10; N, 24.10; S, 7.10.
3.1.4. Reaction of 3 with Diamino Compounds; Formation of 12a–d
To a solution of 3 (5 mmol, 2.06 g) in dioxane (40 mL), hydrazine hydrate (98%), phenylhydrazine,
benzoylhydrazine or thiosemicarbazide (5 mmol, 0.25 mL) was added and the reaction mixture was
refluxed for 2–6 h. The solid product which formed on heating was collected and crystallized from the
proper solvent to afford compounds 12a–d, respectively.
5-Amino-4-{[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenylazo}-pyrazol-5(1H,2H)-one (12a):
Brown solid, (from acetic acid), yield 79% (1.53 g), m.p. 261–263 °C. IR (/cm−1): 3444, 3351, 3239,
1
3171 (NH2, NH), 1677 (C=O), 1626 (C=N) cm−1. H-NMR (DMSO-d6): 2.25 (s, 6H, 2 CH3), 5.85 (s,
2H, NH2, D2O exchangeable), 6.87 (s, 1H, NH, D2O exchangeable), 7.42–8.19 (m, 5H, Ar-H +
pyrimidine-H), 8.99, 10.63 (br s, 2 H, 2NH, D2O exchangeable). 13C-NMR (DMSO-d6): 24.1 (2CH3),

Molecules 2013, 18
840
101.1, 110.3, 117.2, 119.8, 121.4, 123.6, 128.1, 133.5, 140.3 (Ar-C), 164.9 (CO). MS (%): molecular
ion peak at 388 (36.8). C15H16N8O3S (388); Calcd: C, 46.38; H, 4.15; N, 28.85; S, 8.26; Found: C,
46.60; H, 3.90; N, 29.10; S, 8.10.
5-Amino-4-{[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenylazo}-1-phenylpyrazol-5(2H)-one
(12b): Light brown solid, (from acetic acid), yield 63% (1.46 g), m.p. 254–256 °C. IR (/cm−1): 3435,
1
3346, 3231, 3167 (NH2, NH), 1679 (C=O), 1619 (C=N) cm−1. H-NMR (DMSO-d6): 2.24 (s, 6H, 2
CH3), 5.90 (s, 2H, NH2, D2O exchangeable), 6.86 (s, 1H, NH, D2O exchangeable), 7.32–8.13 (m, 10H,
Ar-H + pyrimidine-H), 10.61 (s, 1H, NH, D2O exchangeable). C21H20N8O3S (464); Calcd: C, 54.30; H,
4.34; N, 24.12; S, 6.90; Found: C, 54.50; H, 4.10; N, 24.30; S, 7.10.
5-Amino-1-benzoyl-4-{[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]-phenylazo}pyrazol-5(2H)-one
(12c): Brown solid, (from acetic acid), yield 56% (1.38 g), m.p. 248–250 °C. IR (/cm−1): 3439, 3342,
1
3221, 3174 (NH2, NH), 1680, 1652 (C=O), 1617 (C=N) cm−1. H-NMR (DMSO-d6): 2.25 (s, 6H,
2 CH3), 5.93 (s, 2H, NH2, D2O exchangeable), 6.89 (s, 1H, NH, D2O exchangeable), 7.39–8.13 (m,
10H, Ar-H + pyrimidine-H), 10.66 (s, 1H, NH, D2O exchangeable). C22H20N8O4S (492); Calcd: C,
53.65; H, 4.09; N, 22.75; S, 6.51; Found: C, 53.80; H, 3.90; N, 22.50; S, 6.30.
5-Amino-1-carbothioamido-4-{[4-N-(4,6-dimethyl-pyrimidin-2-yl)sulfamoyl]phenylazo}-pyrazol-
5(2H)-one (12d): Brown solid, (from acetic acid), yield 66% (1.47 g), m.p. 277–280 °C. IR (/cm−1):
1
3438, 3337, 3212, 3162 (NH2, NH), 1678 (C=O), 1615 (C=N) cm−1. H-NMR (DMSO-d6): 2.21 (s,
6H, 2 CH3), 5.82 (s, 2H, NH2, D2O exchangeable), 6.26 (s, 2H, NH2, D2O exchangeable), 6.96 (s, 1H,
NH, D2O exchangeable), 7.48–8.19 (m, 5 H, Ar-H + pyrimidine-H), 10.65 (s, 1H, NH, D2O
exchangeable). C16H17N9O3S2 (447); Calcd: C, 42.94; H, 3.83; N, 28.17; S, 14.33; Found: C, 43.20; H,
4.00; N, 27.90; S, 14.10.
3.1.5. 3-Amino-4-{[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenylazo}-isoxazol-5(2H)one (13)
To dioxane (40 mL) containing sodium acetate (0.5 g), the hydrazone 3 (5 mmol, 2.06 g) and
hydroxylamine hydrochloride (5 mmol, 0.35 g) were added. The mixture was refluxed for 8 h., left to
cool then poured onto ice/water. The solid product so formed was filtered off, dried and crystallized
from dioxane-water as pale brown solid, yield 61% (1.21 g), m.p. 267–269 °C. IR (/cm−1): 3434,
1
3339, 3218, 3164 (NH2, NH), 1679 (C=O), 1616 (C=N) cm−1. H-NMR (DMSO-d6): 2.22 (s, 6H, 2
CH3), 5.88 (s, 2H, NH2, D2O exchangeable), 6.94 (s, 1H, NH, D2O exchangeable), 7.52–8.21 (m, 5 H,
13
Ar-H + pyrimidine-H), 10.77 (s, 1H, NH, D2O exchangeable). C-NMR (DMSO-d6): 24.1 (2CH3),
98.3, 110.1, 117.2, 119.3, 121.4, 123.6, 128.1, 138.5, 145.6 (Ar-C), 173.0 (CO). MS (%): molecular
ion peak at 389 (21.3). C15H15N7O4S (389); Calcd: C, 46.27; H, 3.88; N, 25.18; S, 8.23; Found: C,
46.10; H, 4.10; N, 24.90; S, 8.10.
3.1.6. 5-Amino-3-{[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenylazo}-2,7-dihydroxypyrazolo-
[1,5-a]pyrimidine (16)
Compound 3 (5 mmol, 2.06 g) and cyanoacetohydrazide (5 mmol, 0.45 g) were dissolved in
dioxane (40 mL) containing a few drops of triethylamine. The mixture was refluxed for 19 h., left to

Molecules 2013, 18
841
cool then poured onto dil.HCl/ice. The solid product precipitated was filtered off, dried and
crystallized from dioxane as orange solid, yield 55% (1.25 g), m.p. 291–293 °C. IR (/cm−1): 3503,
3423, 3331, 3221, 3169 (OH, NH2, NH), 1619 (C=N) cm−1. 1H-NMR (DMSO-d6): 2.21 (s, 6H, 2 CH3),
5.86 (s, 2H, NH2, D2O exchangeable), 6.92 (s, 1H, NH, D2O exchangeable), 7.42–8.27 (m, 6H, Ar-H +
2pyrimidine-H), 10.88 (s, 1H, OH, D2O exchangeable), 11.43 (s, 1H, OH, D2O exchangeable)
13C-NMR (DMSO-d6): 24.1 (2CH3), 103.4, 111.7, 117.3, 120.1, 122.3, 123.7, 128.0, 130.4, 137.2,
139.3, 141.4, 145.1 (Ar-C). MS (%): molecular ion peak at 455 (13.9). C18H17N9O4S (455); Calcd: C,
47.47; H, 3.76; N, 27.68; S, 7.04; Found: C, 47.20; H, 4.00; N, 27.90; S, 6.90.
3.1.7. Reaction of 3 with Urea, Thiourea & Guanidine; Formation of 17, 18a,b
To a mixture of 3 (5 mmol, 1.42 g) and the proper amino compound (6 mmol) (urea thiourea or
guanidine) in absolute ethanol (40 mL), sodium ethoxide (0.23 g of Na in 10 mL ethanol) was added.
The reaction mixture was refluxed for 6–8 h, concentrated and cooled. The separated solid was filtered
off, washed with water several times and crystallized from the proper solvent to afford compounds
17,18a,b, respectively.
6-Amino-5-{[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenylazo}-pyrimidine-2,4-(1H,3H)-dione (17):
Yellow solid (from dioxane), yield 77% (1.47 g), m.p. 270–272 °C. IR (/cm−1): 3448, 3347, 3212,
3162 (NH2, NH), 1670 (C=O) cm−1. 1H-NMR (DMSO-d6): 2.27 (s, 6H, 2 CH3), 5.62 (s, 2H, NH2, D2O
exchangeable), 6.87 (s, 1H, NH, D2O exchangeable), 7.56–8.18 (m, 5H, Ar-H + pyrimidine-H), 9.41–9.89
(br, 2H, 2NH, D2O exchangeable). 13C-NMR (DMSO-d6): 24.1 (2CH3), 95.3, 110.1, 118.7, 120.4, 123.1,
126.3, 131.6, 135.2, 142.9 (Ar-C), 159.3, 162.7 (CO). MS (%): molecular ion peak at 416 (11.2).
C16H16N8O4S (416); Calcd: C, 46.15; H, 3.87; N, 26.91; S, 7.70; Found: C, 46.40; H, 3.70; N, 27.10; S, 7.90.
2,6-Diamino-5-{[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenyl-azo}-4H-1,3-thiazin-4-one (18a):
Orange solid (from dioxane), yield 69% (1.47 g), m.p. 296–299 °C. IR (/cm−1): 3441, 3355, 3225,
3176 (NH2, NH), 1667 (C=O) cm−1. 1H-NMR (DMSO-d6): 2.25 (s, 6H, 2CH3), 5.33 (s, 2H, NH2, D2O
exchangeable), 5.65 (s, 2H, NH2, D2O exchangeable), 6.84 (s, 1H, NH, D2O exchangeable), 7.51–8.23
(m, 5H, Ar-H + pyrimidine-H). 13C-NMR (DMSO-d6): 24.1 (2CH3), 107.9, 110.1, 119.3, 120.8, 123.2, 125.0,
130.1, 136.0, 143.9, 151.2 (Ar-C), 167.4 (CO). MS (%): molecular ion peak at 432 (7.7). C16H16N8O3S2
(432); Calcd: C, 44.43; H, 3.73; N, 25.91; S, 14.83; Found: C, 44.20; H, 3.80; N, 26.10; S, 15.00.
2,6-Diamino-5-{[4-N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenylazo}-pyrimidin-4(1H)-one (18b):
Orange solid (from dioxane), yield 61% (1.27 g), m.p. 261–263 °C. IR: 3433, 3364, 3189 (NH2, NH),
1
1669 (C=O) cm−1. H-NMR (DMSO-d6): 2.29 (s, 6H, 2 CH3), 5.38 (s, 2H, NH2, D2O exchangeable),
5.67 (s, 2H, NH2, D2O exchangeable), 6.89 (s, 1H, NH, D2O exchangeable), 7.43–8.24 (m, 5H, Ar-H +
13
pyrimidine-H), 9.83 (br., 1H, NH, D2O exchangeable). C-NMR (DMSO-d6): 24.1 (2CH3), 92.9,
110.1, 118.5, 120.1, 121.7, 125.4, 130.1, 135.5, 141.3, 153.0 (Ar-C), 162.1 (CO). MS (%): molecular
ion peak at 415 (14.5). C16H17N9O3S (415); Calcd: C, 46.26; H, 4.12; N, 30.34; S, 7.72; Found: C,
46.10; H, 3.50; N, 30.10; S, 7.60.

Molecules 2013, 18
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3.2. Antibacterial Screening
The newly synthesized heterocyclic compounds were tested for their antimicrobial activity against
Gram positive bacteria (Staphylococcus aureus and Bacillus cereus) and Gram negative bacteria
(Serratia marcesens and Proteus mirabilis).
Medium: For all bacteria (Nutrient Medium), consisting of (g/L distilled water): peptone, 5 and
meat extract, 3. pH was adjusted to 7.0. For solid media, 2% agar was added. All media were sterilized
at 121 °C for 20 min.
3.2.1. Agar Diffusion Method [24]
One mg of each of the newly synthesized compounds was dissolved in dimethyl sulphoxide
(DMSO, 1 mL) then made up to 10 mL with sterile water to give a concentration of 100 g/mL. A
solution of the tested compounds was placed separately in the agar medium. The inhibition zones were
measured after 24 h incubation.
3.2.2. Filter Paper Disc-Diffusion Method [25]
Proper concentrations of microbial suspensions were prepared from one-day-old liquid stock
cultures incubated on a rotary shaker (100 rpm). The mycelia were then subdivided by mechanical
stirring at speed No. 1 for 30 min. Turbidity of bacteria was adjusted with a spectrophotometer at 350 nm
to give an optical density of 1.0. Appropriate agar plates were aseptically surface inoculated uniformly
by a standard volume (ca. 1 mL) of the microbial broth culture of the tested bacteria.Whatman No. 3
filter paper discs of 10 mm diameter were sterilized by autoclaving for 15 min at 121 °C. Test
compounds were dissolved in 80% ethyl alcohol to give final concentration of 5 μg/mL. The sterile
discs were impregnated with the test compounds (5 μg/disc). After the impregnated discs have been air
dried, they were placed on the agar surface previously seeded with the organism to be tested. Discs
were gently pressed with forceps to insure thorough contact with the media. Each test compound was
conducted in triplicate. Plates were kept in the refrigerator at 5 °C for 1 h to permit good diffusion
before transferring them to an incubator at 37 °C for 24 h.
4. Conclusion
Several new pyridines, pyrans, pyridazines, pyrazoles, isoxazoles and thiazines that contain a
sulfonamido moiety were prepared using simple methods, their structures were proven by spectral
methods and they were tested for their antibacterial activities. Most of these compounds showed
promising activities against both Gram-positive and Gram-negative bacteria. These results are
encouraging for synthesis of similar compounds in the near future.
Acknowledgements
The authors are greatly appreciative to Fatma Farag, Biochemistry Department, Faculty of Science,
Ain Shams University, for helping in carrying out the antibacterial activity tests for the newly
synthesized compounds.

Molecules 2013, 18
843
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available from the authors.
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