Sulfadiazine guanidyl derivatives: Synthesis, characterization and docking studies for potential antituberculosis agents

Article abstract of DOI:10.14233/ajchem.2018.21487

Due to the wide range of pharmacological and biological activities, guanidine considered as one of the attractive pharmacophores. The presence of -CN3 group in guanidine compounds makes them to have efficient affinity towards various substituents with wid

Full text of DOI:10.14233/ajchem.2018.21487

Asian Journal of Chemistry; Vol. 30, No. 9 (2018), 2141-2144
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2018.21487
Sulfadiazine Guanidyl Derivatives: Synthesis, Characterization
and Docking Studies for Potential Antituberculosis Agents
1,*
1
2
1
MAHESH BHAT , B.C. GANAVI , B.K. SAGAR and E. VIJAY SHEKAR
1P.G. Department of Chemistry, J.S.S. College for Women, Saraswathipuram, Mysore-570 009, India
2Department of Studies in Chemistry, University of Mysore, Manasagangothri, Mysore-570 006, India
*Corresponding author: E-mail: maheshbhat08@gmail.com
Received: 29 May 2018;
Accepted: 11 June 2018;
Published online: 31 July 2018;
AJC-19033
Due to the wide range of pharmacological and biological activities, guanidine considered as one of the attractive pharmacophores. The
presence of -CN3 group in guanidine compounds makes them to have efficient affinity towards various substituents with wide range of
biochemical activities. In the present study, novel sulfadiazine guanidyl derivatives were synthesized and characterized by FT-IR, LC-MS
and NMR spectral studies. The synthesized compounds were screened for antituberculosis activity and molecular docking study with
InhA proteins was carried out. It was found that the compounds with electron donating groups exhibited superior activity.
Keywords: Guanidines, Antituberculosis activity, Docking studies, InhA proteins.
zole)-1,3-diethyl-4-aryl substituent’s and screened for anti-
inflammatory and analgesic activities [7]. In our previous works,
we found that guanidinyl benzothiazole derivatives also act as
antimicrobial, antioxidant, antitubercular agents, etc. [8-10].
Sulfadiazine belongs to sulfonamide groups, used as anti-
bacterial agent for curing the mild-to-moderate infections of
the sensitive organisms [11]. Like other sulfonamides, sulfa-
diazine is used for the treatment for isosyncratic liver injury
[12]. Herein, we reported the synthesis of sulfadiazine deri-
vatives, which posses an interesting profile of antioxidant
activity.
In this investigation, we have synthesized a series of sulfa-
diazine guanidinyl derivative through multi step reaction, initially
by condensation then finally reaction with substituted aniline
with good yields. Further, the synthesized compounds were
characterized by LC-MS, IR and NMR. The newly synthesized
derivatives were evaluated for their antioxidant potential towards
DPPH free radical scavenging activity and also comparative
studies were done.
INTRODUCTION
The compound containing -CN3 group are called as guan-
idine. Guanidine is similar to the structure of imide urea, So
the chemistry and properties of guanidines are resembles with
imide urea [1]. The guanidine group is found in wide variety
of both natural and synthetic compounds. The presence of
guanidine functionality enhances activity and also helpful to
interact with functional groups of enzyme or receptors, because
of these properties, the study of guanidine derivatives becomes
more important [2]. The guanidine sub-unit exhibits hydrogen
bonding, this also responsible for the enhanced activity [3]. In
this way, guanidine compounds are useful pharmacophores in
medicines and wide applications in synthetic chemistry, organic
materials, sensors, advanced synthetic molecular recognition
devices and phase transfer catalyst [4-6]. Guanidines used
for the curing the diabetic syndrome and biguanidine are
characteristics properties towards the type two mellitus diabetic
therapy.
Similar to thiourea derivatives, guanidines exhibited the
potent antimicrobial agents. Kamoda et al. [7] synthesized
mono substituted aryl ester derivatives of guanidine (TG44),
an anti H. pylori agent. Recently benzothiazole guanidines
were synthesized with methyl and 1-[2-(substituted benzothia-
EXPERIMENTAL
Synthesis of benzyolisothiocyanates (Step-1):A solution
of benzoyl chloride (0.014 mol) in acetone (20 mL) was taken
in a 100 mL round bottom flask and to that suspension of
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2142 Bhat et al.
Asian J. Chem.
ammonium thiocyanate (0.018 mol) in acetone (20 mL) was
added portionwise and refluxed for 0.5 h. After yellow solid
appeared, the reaction mixture was cooled to room temperature.
Synthesis of thiourea derivative of sulfadiazine (Step-
2): Sulfadiazine (0.014 mol) in acetone (20 mL) was added
dropwise to Step-1 product and heated to reflux for 3 h. After
completion of the reaction, the mixture was poured into crushed
ice and filtered the solid precipitate and dried. The crude material
was purified by recrystallization from ethanol.
C22H19N7O3S, m.w.: 517.12, Yellow solid, m.p. 187-188 ºC.
Elemental analysis % Calcd. (found): C, 55.70 (56.43); H, 3.70
(3.58); N, 18.95 (18.02). FT-IR (KBr, νmax, cm-1): 3310 (-NH),
3148 (SO-NH), 3052 (Ar-C-H), 1725 (C=O), 1610, 13420 (NO2),
1525 (Ar C=C), 1360, 1318 (C-C), 1335 (C-N), 1350, 1145 (SO-
NH). 1H NMR (400 MHz, DMSO-d6): δ 11.22 (s, 1H), 9.87 (s,
1H), 8.42-8.40 (t, 1H), 7.96-7.94 (d, J = 8.4 Hz, 1H), 7.85-7.81
(m, 3H), 7.82-7.80 (d, J = 7.6 Hz, 2H), 7.65-7.63 (d, J = 8.8 Hz,
2H), 7.59-7.57 (d, J = 8.4 Hz, 2H), 7.46-7.43 (t, 2H), 7.32-6.29
(m, 2H), 4.16 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 174.5,
170.2, 159.40, 157.6, 142.8, 140.3, 138.6, 136.7, 135.2, 132.4,
131.8, 130.1, 129.8, 126.4, 120.3, 117.5, 115.9, 113.4. LCMS
Found: m/z 518.3 for [M+1]+ peak and calculated 517.3.
N-[(phenylamino)(4-(N-pyrimidin-2-ylsulfamoyl)
phenylamino)methylene]benzamide (3):Yeild: 89.6 %, m.f.:
C23H20N6O3S, m.w.: 472, off white solid, m.p. 117-118 ºC.
Elemental analysis % Calcd. (found): C, 61.00 (60.52); H, 4.27
(4.32); N, 17.79 (17.58). FT-IR (KBr, νmax, cm-1): 3270 (-NH),
3125 (SO-NH), 3042 (Ar-C-H), 1718 (C=O), 1608, 1320
(NO2), 1564 (Ar C=C), 1340, 1310 (C-C), 1348 (C-N), 1372,
1160 (SO-NH). 1H NMR (400 MHz, DMSO-d6): δ10.62 (s, 1H),
9.92 (s, 1H), 8.34- 8.32 (t, 1H), 8.10-7.98 (d, J = 8.0 Hz, 1H),
8.96-8.94 (J = 7.2 Hz, 2H), 7.93-7.91 (m, 3H), 7.87-7.85 (d, J
= 9.6 Hz, 2H), 7.72-7.70 (d, J = 7.6 Hz, 2H), 7.64-7.62 (d, J =
8.0 Hz, 2H), 7.43- 7.41 (t, 2H), 7.37-6.35 (m, 2H), 4.12 (s, 1H).
13C NMR (100 MHz, CDCl3): δ172.3, 168.7, 157.5, 152.9, 141.6,
141.2, 139.8, 137.1, 133.5, 132.2, 131.4, 130.0, 128.4, 127.7,
121.5, 116.2, 114.4, 112.3. LC-MS Found: m/z 473.13 for
[M+1]+ peak and calculated 472.15.
N-[(3,4-Dimethylphenylamino)(4-(N-pyrimidin-2-yl-
sulfamoyl) phenylamino)methylene] benzamide (4):Yield:
85.8 %, m.f.: C26H20N6O3S, m.w.: 500, brown solid, m.p. 190-
192 ºC. Elemental analysis % Calcd. (found): C, 62.38 (61.95);
H, 4.83 (4.89); N, 16.79 (16.27). FT-IR (KBr, νmax, cm-1): 3218
(-NH), 3145 (SO-NH), 3110 (Ar-C-H), 2983 (CH3), 1690 (C=O),
1620, 1335 (NO2), 1535 (Ar C=C), 1350, 1310 (C-C), 1343 (C-N),
1340, 1175 (SO-NH). 1H NMR (400 MHz, DMSO- d6): 11.43
(s, 1H), 9.92 (s, 1H), 8.48-8.46 (t, 1H), 7.98-7.93 (s, 1H), 7.92
(s, 3H), 7.66 (s, 1H), 7.59-7.57 (d, J = 8.0 Hz, 2H), 7.51-7.50
(d, J = 5.6 Hz, 1H), 7.43-7.39 (t, 2H), 7.18-6.97 (m, 2H), 4.02
(s, 1H), 3.03 (s, 3H), 2.86 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ 175.5, 171.2, 158.5, 156.3, 144.2, 141.2, 139.4, 136.1, 134.8,
131.9, 131.6, 129.9, 129.4, 125.4, 121.7, 118.2, 115.4, 113.4,
38.27, 20.83. LC-MS Found: m/z 501.01 for [M+1]+ peak and
calculated 500.
Synthesis of sulfadiazine gunidinyl derivatives (Step-3):
All guanidines were synthesized by the reported method [10].
The thiourea of sulfadiazine was mixed with different substituted
anilines in DMF in equimolar ratios, with two equivalents of
triethylamine. The temperature was maintained below 5 ºC using
an ice bath, one equivalent of mercuric chloride was added to
the reaction mixture with vigorous stirring. The ice bath was
removed after 0.5 h and the mixture was stirred overnight.
Progress of the reaction was monitored by TLC until completion.
Chloroform (20 mL) was added to the reaction mixture and the
suspension was filtered through a cillate bed to remove HgS. The
solvent from the filtrate was evaporated under reduced pressure
and the residue was redissolved in CH2Cl2 (20 mL), washed with
water (4 × 50 mL) and the combined organic phase washed with
brine (20 mL), dried over anhydrous Na2SO4. The solvent was
evaporated and the residue was purified by column chromato-
graphy and finally recrystallized in ethanol [11] (Scheme-I).
N-[(2-Nitrophenylamino)(4-(N-pyrimidin-2-ylsulf-
amoyl)phenylamino)methylene]benzamide (1): Yield: 87.7
%, m.f. C23H19N7O3S, m.w.: 517.12, off-white solid, m.p. 175-
176 ºC. Elemental analysis % Calcd. (found): C, 55.70 (56.35);
H, 3.70 (3.65); N, 18.95 (18.46). FT-IR (KBr, νmax, cm-1): 3280
(-NH), 3142 (SO-NH), 3055 (Ar-C-H), 1710 (C=O), 1617, 1340
(NO2), 1540 (Ar C=C), 1347, 1305 (C-C), 1340 (C-N), 1355,
1165 (SO-NH). 1H NMR (400 MHz, DMSO-d6): δ 10.82 (s,
1H), 9.64 (s, 1H), 8.63- 8.61 (d, J = 9.6 Hz, 2H), 8.23-8.21
(m, 3H), 8.17-8.15 (d, J = 7.2 Hz, 2H), 7.96-7.94 (t, J = 7.6
Hz, 14.2 Hz, 2H), 7.82 (s, 1H), 7.64-7.62 (d, J = 8.8 Hz, 2H),
7.53-7.51 (d, J = 8.0 Hz, 1H), 7.46- 7.43 (t, J = 4.8, 13.6 Hz,
2H), 7.23-6.20 (m, 2H), 4.08 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 175.92, 169.3, 158.2, 157.8, 141.6, 139.3, 137.5,
135.4, 134.5, 131.6, 130.2, 129.9, 129.2, 125.9, 119.6, 115.3,
113.1, 112.7. LC-MS Found: m/z 518.23 for [M+1]+ peak and
calculated 517.12.
N-[(4-nitro phenylamino)(4-(N-pyrimidin-2-ylsulfamoyl)-
phenylamino)methylene]benzamide (2):Yield: 84.1 %, m.f.:
R: 2-NO2,
4-NO2,
H,
3,4-Dimethyl
Scheme-I: Synthesis of sulfadiazine gunidinyl derivatives

Vol. 30, No. 9 (2018)
Synthesis and Docking Studies of Sulfadiazine Guanidyl Derivatives 2143
Antituberculosis activity: The tuberculosis disease is
caused by Mycobacterium tuberculosis and it mainly causes
the lungs and synthesized Sulfadiazene derivatives was checked
against M. tuberculosis (H37Rv strain):ATCC No. 27294 by in
vitro micro-plateAlamar Blue assay method [13]. The antimyco-
bacterial activity of compounds was screened by microplate
Alamar Blue assay (MABA) for M. tuberculosis 100 µg/mL
and serial half fold solutions of test compounds were prepared.
In 96 well micro-titre plate serial two-fold dilutions of synthe-
sized compounds were added to each of the wells (100 µg/mL
to 0.2 µg/mL) and to that 100 µL of Middlebrook 7H9 broth
was added. After addition of the test solutions, plates were
covered with parafilm and incubated at 37 ºC for 5 days. After
completion of the incubation, freshly prepared 25 µL of 1:1
mixture Alamar Blue reagent and 10 % Tween 80 was added
to each of the wells and once again incubated for 24 h at 37 ºC.
The plates were cooled to room temperature. Appearance of
the blue color in the wells indicated as no bacterial growth and
compound was sensitive to that concentration. The pink colour
appearance in the wells indicated the resistance of bacteria,
which represented absence of activity. MIC of the compound
was defined as the minimum concentration of drug, which
prevents the colour change from blue to pink.
Antioxidant activity: Free radicles scavenging ability
by DPPH radical assay: DPPH scavenging activity of isolated
compound was determined as previously reported.Various con-
centration of tested sample in aliquot of 100 µL were mixed
with 100 µL of 40 µM methonolic solution of DPPH in 96-
well micro-titer plate the decrease in absorbance at 517 nm
was recorded after the incubation of 15 min at room tempera-
ture. The absorbance of DPPH solution with only methanol
and without the sample used as the control. The BHT was used
as standared to compare the activity. The assay was carried out
in triplicate.The percentage inhibition of the DPPH radical by
the sample was calculated according to the formula [14].
RESULTS AND DISCUSSION
Four molecules of new sulfadiazine gunidinyl derivatives
were synthesized. In first step, benzoyl chloride reacted with
potassium thiocyanate in acetone, on refluxing white precipitate
was formed. By cooling reaction mixture and added solution
of 4-amino-N-pyrimidin-2-ylbenzene sulfonamide in acetone
was added drop-wise. In second step, equimolar ratios of
sulfadiazine thiourea derivative and different amines were taken
in DMF and two equivalents of triethylamine and one equi-
valent of HgCl2 was added and stirred at overnight at room temp-
erature. So in this way, sulfadiazine gunidinyl derivatives were
synthesized by efficient way with benzyol thiourea and substi-
tuted aniline in the presence of HgCl2 and triethylamine. The
synthesized compounds were characterized by 1H NMR and
LC-MS spectral analysis.
Antituberculosis activity:All the tested compounds showed
antituberculosis activity and minimum inhibition concentra-
tions are in between 100 to 12.5 µg/mL. In the series of synthe-
sized compound, 3,4-dimethyl substituted compound exhibited
enhanced activity in the series. The activity is lower for nitro
substitutents, however activity has gradually increase for
without substitution to 3,5 dimethyl substitution. It is confirmed
that the presence of electron donating increases with electron
donating group and decreases with electron withdrawing group.
Molecular docking studies:The molecular docking study
of targeted benzothiazole derivatives with enoyl acyl carrier
reductase (InhA) (PDB ID-1ZID) of M. tuberculosis was carried
out to interpret the molecular interactions with proteins. InhA
is key enzyme, which is involved in the type II fatty acid biosyn-
thesis pathway of M. tubecrulosis. InhA inhibition blocks the
biosynthesis of mycolic acid, which is lipid involved in the
formation of cell wall of Mycobacterium (Fig. 1). Here, com-
pounds showed the remarkable docking score -5.944 to -5.023,
which can be compared with the standard drug pyrazinamide
with -5.549 and ciprofloxacin-5.3 as docking scores.The
docking scores of synthesized compounds with InhA are shown
in Table-1.
AC − AS
Inhibition (%) =
×100
AC
whereAC is the absorbance of control andAS is the absorbance
of sample at 15 min
TABLE-1
ANTI-TB AND DOCKING STUDY OF
THE SYNTHESIZED COMPOUND
Molecular docking: Molecular docking studies were
performed with the help of Schrodinger software suite, maestro
9.35 version (2011). The structure of the molecules was drawn
in Chem ISIS draw and saved as mole file and used for the
determination of ability of the compound to interact with
selected protein. The 3D crystallographic structure of protein
enoyl acyl carrier reductase (InhA) of M. tuberculosis (PDB:ID:
1ZID) was retrieved from protein data bank (www.rch.org/pdb).
The protein structure were pre-processed and refined by protein
preparation wizard. Further, it was minimized by OPLF-2005
(optimized potential for liquid simulations) force field to get
the 0.3 Aoroot mean square deviations (RMSD). Lig prep
program by OPLS-2005 force field was used for the optimiza-
tion of selected ligand for generation of lowest energy state.The
molecule docking studies of ligands and protein were deter-
mined by GLIDE .The activity was ranked on the basis of
docking score. Those docking scores depend on how the ligands
fill the target protein [15].
Compound No.
R
2-NO2
4-NO2
H
MIC (µg/mL)
Docking score
-5.944
1
2
3
4
100
50
25
12.5
3.12
-5.148
-5.032
-5.032
-5.549
3,4-(CH3)2
Pyrazinamide
Antioxidant activity: In guanidine derivatives, guanidine
unit had capacity to form hydrogen bond. In addition to this,
synthesized compounds containing -4NO2, -2NO2, -H, -3,4
dimethyl groups, which are capable of releasing H+ ion and
form anionic moiety, which are responsible for the scavenging
action ROS scavenging ability of synthesized compounds
evaluated by DPPH assay method. Antioxidant activities of
the synthesized compounds were determined for 500 µg/mL to
1.6 µg/mL also IC50 values were determined (Table-2). Compound
4 has remarkable activity (IC50 = 259.65 µg/mL).

2144 Bhat et al.
Asian J. Chem.
Fig. 1. Molecular docking study of compound 4
3. R.G.S. Berlinck, Fortschr. Chem. Org. Naturst., 66, 119 (1995);
TABLE-2
IC50 VALUES OF ANTIOXIDANT ACTIVITY
https://doi.org/10.1007/978-3-7091-9363-1_2.
4. K. Burgess, ed., Solid-Phase Organic Synthesis; John Wiley & Sons:
New York, USA (2000).
5. G.J. Durant, Chem. Soc. Rev., 14, 375 (1985);
https://doi.org/10.1039/cs9851400375.
Compound No.
IC50 values (µg/mL)
425.21
1
2
377.23
3
343.78
6. A. Echavarren, A. Galan, J.M. Lehn and J. De Mendoza, J. Am. Chem.
Soc., 111, 4994 (1989);
4
259.65
https://doi.org/10.1021/ja00195a071.
Ascorbic acid
9.26
7. O. Kamoda, K. Anzai, J. Mizoguchi, M. Shiojiri, T. Yanagi, T. Nishino
and S. Kamiya, Antimicrob. Agents Chemother., 50, 3062 (2006);
https://doi.org/10.1128/AAC.00036-06.
8. M. Bhat and S.L. Belagali, Res. Chem. Intermed., 42, 6195 (2016);
https://doi.org/10.1007/s11164-016-2454-6.
Conclusion
An attempt has been made for the synthesis of sulfadiazine
compounds with guanidinyl functionality. The synthesized
compounds showed excellent antituberculosis activity, whose
MIC values were upto 12.5 µg/mL, which is also supported
by molecular docking study. The resonance stabilization of
guanidinyl group was mainly responsible for the observed
activity and different groups on N-substituted phenyl rings of
guanidinyl influence the activity. Some of the compounds also
exhibited remarkable antioxidant activity.
9. M. Bhat, S.L. Belagali and D.C. Shyamala, The Natural Products J.,
7, 286 (2017);
https://doi.org/10.2174/2210315507666170530091940.
10. M. Bhat and S.L. Belagali, Anti-Infective Agents, 16, 1 (2018);
https://doi.org/10.2174/2211352516666180425151720.
11. M. Varga, Textbook of Rabbit Medicine, Elsevier Health: UK, edn 2,
pp. 137-177 (2014).
12. V.P. Arcangelo andA.M. Peterson, Pharmacotherapeutics forAdvanced
Practice: A Practical Approach, Lippincott Williams & Wilkins, vol.
536, pp 959 (2006).
13. M.C.S. Lourenco, M.V.N. de Souza, A.C. Pinheiro, M. de L. Ferreira,
R.S.B. Gonçalves, T.C.M. Nogueira and M.A. Peralta, ARKIVOC, 181
(2007);
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
https://doi.org/10.3998/ark.5550190.0008.f18.
14. M.K. Raj, C. Balachandran, V. Duraipandiyan, P.Agastian, S. Ignacimuthu
and A. Vijayakumar, Med. Chem. Res., 22, 3823 (2013);
https://doi.org/10.1007/s00044-012-0393-3.
15. M. Fathimunnisa, H. Manikandan, K. Neelakandan, N. Rajendra Prasad,
S. Selvanayagam and B. Sridhar, J. Mol. Struct., 1122, 205 (2016);
https://doi.org/10.1016/j.molstruc.2016.06.012.
REFERENCES
1. Y. Yamamoto and S. Kojima, Synthesis and Chemistry of Guanidine
Derivatives: The Chemistry of Amidines and Imidates, John Wiley &
Sons Ltd., vol. 2 (1991)
2. D. Lednicer and L.A. Mitscher, eds., The Organic Chemistry of Drugs
Synthesis; Wiley: New York, vol. II, (1980).