Novel pyrazolyl-s-triazine derivatives, molecular structure and antimicrobial activity

Article abstract of DOI:10.1016/j.molstruc.2017.05.040

A new series of pyrazole-containing s-triazine derivatives were synthesized by reaction of the corresponding s-triazinyl hydrazine derivatives with acetylacetone in the presence of HClO₄ or DMF/TEA. The former method allowed the preparation of the target products with higher yields. All compounds were fully characterized. X-ray single crystal diffraction for two representative compounds (4-(4,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)-1,3,5-triazin-2-yl)morpholine and N-benzyl-4-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)-1,3,5-triazin-2-amine) was studied and the molecular structures were optimized using the DFT/B3LYP method. The structures were found to be in agreement with X-ray structures. The antimicrobial and antifungal activity of the prepared compounds were tested against the growth of several microorganisms.

Full text of DOI:10.1016/j.molstruc.2017.05.040

Accepted Manuscript
Novel pyrazolyl-s-triazine derivatives, molecular structure and antimicrobial activity
Anamika Sharma, Hazem Ghabbour, Shams Tabrez Khan, Beatriz G. de la Torre,
Fernando Albericio, Ayman El-Faham
PII:
S0022-2860(17)30625-7
10.1016/j.molstruc.2017.05.040
MOLSTR 23781
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 31 March 2017
Revised Date: 2 May 2017
Accepted Date: 10 May 2017
Please cite this article as: A. Sharma, H. Ghabbour, S.T. Khan, B.G. de la Torre, F. Albericio, A. El-
Faham, Novel pyrazolyl-s-triazine derivatives, molecular structure and antimicrobial activity, Journal of
Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.05.040.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT

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Novel Pyrazolyl-s-Triazine Derivatives, Molecular Structure and
Antimicrobial Activity
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Anamika Sharma,1 Hazem Ghabbour,2 Shams Tabrez Khan,3 Beatriz G. de la Torre,1,4
Fernando Albericio*5,6,7,8 Ayman El-Faham,*5,9
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School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa;
anamika.aug14@gmail.com (AS)
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2Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh
11451, Saudi Arabia; ghabbourh@yahoo.com (HAG)
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3Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh
11451, Saudi Arabia; stkhan@ksu.edu.sa (STK)
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4School of Laboratory of Medicine and Medical Sciences, University of KwaZulu-Natal, Durban
4001, South Africa; Garciadelatorreb@ukzn.ac.za (BGT)
5Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh
11451, Saudi Arabia; aymanel_faham@hotmail.com; aelfaham@ksu.edu.sa (AEF).
6School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001, South Africa;
albericio@ukzn.ac.za (FA).
7Department of Organic Chemistry, University of Barcelona, Barcelona 08028, Spain.
8CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine,
Barcelona Science Park, Barcelona 08028, Spain.
9Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia,
12321 Alexandria, Egypt.
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Abstract
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A new series of pyrazole-containing s-triazine derivatives were synthesized by reaction of the
corresponding s-triazinyl hydrazine derivatives with acetylacetone in the presence of HClO4 or
DMF/TEA. The former method allowed the preparation of the target products with higher yields.
All compounds were fully characterized. X-ray single crystal diffraction for two representative
compounds (4-(4,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)-1,3,5-triazin-2-yl)morpholine and N-
benzyl-4-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)-1,3,5-triazin-2-amine) was studied
and the molecular structures were optimized using the DFT/B3LYP method. The structures were
found to be in agreement with X-ray structures. The antimicrobial and antifungal activity of the
prepared compounds were tested against the growth of several microorganisms.
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Keywords: Pyrazole, hydrazino-s-triazine, cyanuric chloride, X-ray crystallography,
DFT/B3LYP, antibacterial activities.
34 1. Introduction
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1,3,5-Triazine and its derivatives are key moieties in drug discovery due to their wide
range of biological activities, such as antibacterial [1], antiprotozoal [2], fungicidal [3],
anticancer [1], antiviral [4], cytotoxic [5], sedative and anti-inflammatory [6], herbicide [7], and
analgesic [8] effects and hence these moieties have been found to be useful in numerous fields
[9-13]. Pyrazole and its derivatives have also been found in numerous biological and
pharmaceutical applications, such as hypoglycemic activity inhibitors, deactivators of liver
alcohol dehydrogenase and oxidoreductases [14, 15] and antihypertensive agents [16].
Therefore, the synthesis of new s-triazine derivatives attached to pyrazolyl rings could be an
efficient strategy for the discovery of new drugs.
In literatures, s-triazines with a pyrazolyl fragment in their structure can be synthesized
by cyclotrimerization of aromatic nitriles [17] or from cyanuric chloride (2,4,6-trichloro-1,3,5-
triazine) by direct substitution of first or second chlorine atom by an amine-containing pyrazolyl
moiety [18] or by reaction of s-triazine hydrazine derivatives with acetoacetic ester and 3-
iminobutyronitrile [19]. More recently, pyrazolyl-s-triazine derivatives have been synthesized
by means of reactions of hydrazinyl-s-triazine with 1,3-diketone (aliphatic or aromatic) in the
presence of catalytic amount of trimethylamine [20]. These derivatives showed low toxicity
towards growth-stimulating activity, antidote activity and also inhibitors of the oil-oxidizing
processes [21, 22].
In our ongoing medicinal chemistry project based on the use of s-triazine as a scaffold
[23-25], here we report further modifications of amine-containing s-triazine with various
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substituents by incorporating mono and dipyrazole derivatives (Figure 1), along with the X-ray
single crystal diffraction of two representative compounds was studied with further validation of
molecular structures using the DFT/B3LYP method. The antimicrobial activity of the
synthesized compounds was tested in preliminary assays to offer a clear indication regarding
effect of substituents on the bioactivity.
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Figure 1: Structure of mono- and di-pyrazolyl-s-triazine derivatives.
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2. Experimental Section
2.1. Chemistry
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Materials and method: The solvents were of analytical reagent grade and were used
without further purification. NMR (1H and 13C) spectra were recorded on a JEOL 400 MHz
spectrometer at room temperature in CDCl3 using internal standard δ = 0 ppm. Elemental
analysis were performed on Perkin-Elmer 2400 elemental analyzer. Melting points were
determined on a Mel-Temp apparatus in an open capillary tube and are uncorrected. Fourier
transform infrared spectroscopy (FTIR) spectra were recorded on Shimadzu model IRAffinity-1
Spectrometer from KBr discs. The follow-up of the reaction and purity checks were done using
silica gel-coated TLC plates (Type 60 GF254, Merck). A mixture of methanol-chloroform (1:9)
or ethylacetate-hexane (4:6) was used as eluent.
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2.1.1. Synthesis of 2,4-dichloro-6-substituted 1,3,5-triazine derivatives
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2,4-Dichloro-6-substituted 1,3,5-triazine derivatives were prepared following the reported
method [23-25].
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2.1.1.1. 2, 4-dichloro-6-morpholino-1,3,5-triazine (2a)
White solid in 94% yield; mp = 157-158°C (Lit. [23] mp = 152-153°C). 1H NMR (CDCl3): δ 3.69
(t, 4H, J = 5.4 Hz, 2-CH2N), δ 3.73 (t, 4H, J = 5.4 Hz, 2 -CH2O) ppm.
2.1.1.2. 2,4-dichloro-6-(piperidin-1-yl)-1,3,5-triazine (2b)
White solid in 90% yield; mp = 76°C (Lit. [25] mp = 72-74°C); H NMR (CDCl3): δ 1.60-1.65
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(m, 2H, CH2), 1.69-1.72 (m, 4H, 2CH2), 3.80 (t, 4H, J = 6.1 Hz, 2CH2N) ppm.
2.1.1.3. N-benzyl-4,6-dichloro-1,3,5-triazin-2-amine (2c)
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Off white solid in 88% yield; mp = 115-117°C; H NMR (CDCl3): δ 4.65 (d, 2H, J = 6.4 Hz,
phCH2), 6.61 (bs, 1H, NH), 7.24-7.37 (m, 5H, C6H5) ppm; 13C NMR (CDCl3): δ 45.4, 127.7,
128.1, 128.9, 136.2, 165.8, 169.9, 171.1 ppm. Anal. Calc. for C10H8Cl2N4 (255.10): C, 47.08; H,
3.16; N, 21.96. Found: C, 47.19; H, 3.22; N, 22.06.
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2.1.2. General method for the synthesis of 2-chloro-4,6-disubstituted 1,3,5-triazine
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A solution of amine (20 mmol) in 50 mL DCM was added dropwise at room temperature
to a solution of mono substituted-s-triazine (10 mmol) in 50 mL dichloromethane over 10 min.
The reaction mixture was stirred for 6-8 h at room temperature and then DCM (100 mL) was
added, and the combined organic layer was washed with 1N HCl (2 x 20 mL) and brine (20 mL).
The organic layer was dried over anhydrous MgSO4, and after filtration, the solvent was removed
under vacuum. The crude product obtained was recrystallized from DCM-hexane (1:2).
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2.1.2.1. 4-(4-chloro-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)morpholine (5a)
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White solid in 87% yield; mp = 125-126°C; H NMR (CDCl3): δ 1.52-1.63 (m, 6H, 3CH2),
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3.68-3.75 (m, 12H, 6CH2) ppm; C NMR (CDCl3): δ 24.6, 25.6, 25.8, 43.6, 43.8, 44.0, 44.5,
66.6, 66.9, 163.9, 164.5, 169.5 ppm. Anal. Calc. for C12H18ClN5O (283.76): C, 50.79; H,
12.49; N, 24.68. Found: C, 51.01; H, 12.34; N, 24.54.
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2.1.2.2. N-benzyl-4-chloro-6-morpholino-1,3,5-triazin-2-amine (5b)
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White solid in 85% yield; mp = 158-159°C ; H NMR (CDCl3): δ 3.68 (brs, 4H, 2CH2), 3.77
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(brs, 4H, 2CH2), 4.58 (d, 2H, J = 6.0 Hz, PhCH2 ), 6.10 (brs, 1H, NH), 7.26-7.33 (m, 5H,
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C6H5-) ppm; C NMR (CDCl3): δ 43.9, 44.8, 66.5, 127.3, 127.4, 127.8, 128.6, 128.7, 163.5,
165.6 ppm. Anal. Calc. for C14H16ClN5O (305.77): C, 54.99; H, 5.27; N, 22.90. Found: C,
54.76; H, 5.11; N, 23.18.
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2.1.2.3. N-benzyl-4-chloro-6-(piperidin-1-yl)-1,3,5-triazin-2-amine (5c)
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White solid in 85% yield; mp = 151-152°C ; H NMR (CDCl3): δ 1.53 (brs, 2H, CH2), 1.62
(brs, 4H, 2CH2), 3.71 (brs, 4H, 2CH2), 4.58 (d, 2H, J = 6.0 Hz, PhCH2 ), 6.05 (brs, 1H, NH),
7.25-7.30 (m, 5H, C6H5) ppm; 13CNMR (CDCl3): δ 24.4, 25.4, 43.3, 43.4, 126.2, 126.4, 127.1,
127.4, 127.9, 128.2, 140.9, 164.2, 165.6, 168.0 ppm. Anal. Calc. for C15H18ClN5 (303.79): C,
59.30; H, 5.97; N, 23.05. Found: C, 59.11; H, 6.03; N, 23.28.
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2.1.2.4. 4-chloro-6-morpholino-N-phenyl-1,3,5-triazin-2-amine (5d)
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White solid in 85% yield; mp = 125-127°C; H NMR (CDCl3): δ 3.72 (brs, 4H, 2CH2), 3.82
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(brs, 4H, 2CH2), 5.27 (s, 1H, NH), 7.09 ( t, 1H, J= 7.6 Hz, Ar), 7.32 (dd, 2H, J= 12.4, Ar), -
7.48 (d, 2H, J= 7.2, Ar) ppm; 13C NMR (CDCl3): δ 44.1, 66.5, 66.6, 120.5, 124.0, 128.9,
163.8, 164.6 ppm. Anal. Calc. for C13H14ClN5O (291.74): C, 53.52; H, 4.84; N, 24.01. Found:
C, 53.80; H, 5.04; N, 23.83.
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2.1.2.5. 4-chloro-N,N-diethyl-6-morpholino-1,3,5-triazin-2-amine (5e)
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White solid in 85% yield; mp = 108-109°C; H NMR (CDCl3): δ 1.12 (t, 6H, J = 7.6 Hz,
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2CH3), 3.46-3.56 (m, 4H, 2CH2), 3.66 (t, 4H, J = 3.4 Hz , 2CH2), 3.74 ( t, 4H, J = 4.4 Hz,
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2CH2)ppm; C NMR (CDCl3): δ 12.5, 13.2, 41.4, 41.6, 43.7, 66.6, 66.8, 163.9, 164.4, 169.2
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ppm. Anal. Calc. for C11H18ClN5O (271.75): C, 48.62; H, 6.68; N, 25.77. Found: C, 48.88; H,
6.74; N, 25.98.
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2.1.3. General method for the synthesis of hydrazino -1,3,5-triazine derivatives
The synthesis of hydrazine derivatives was performed following the reported method
with slight modifications [26]: Hydrazine hydrate 80% (10 mL) was added dropwise to a
solution of chloro-substituted-1,3,5-triazine (20 mmol) in 50 mL ethanol at room temperature.
The reaction mixture was then sonicated for 60 min at 60°C. As a part of the work up, ethanol
and excess hydrazine were removed under vacuum, followed by addition of excess diethyl
ether to afford the product as a white solid in excellent yield and purities. In the case of the
synthesis of the dihydrazine derivatives, 20 mL of hydrazine was used for the substitution
process. The mono- and di-hydrazino derivatives were used directly in the next step without
further purification.
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2.1.3.1. 4-(4,6-dihydrazinyl-1,3,5-triazin-2-yl)morpholine (3a)
White solid, mp = 215-217 °C in 95 % yield.
2.1.3.2. 2,4-dihydrazinyl-6-(piperidin-1-yl)-1,3,5-triazine (3b)
White solid, mp = 160-162 °C in 93 % yield.
2.1.3.3. N-benzyl-4,6-dihydrazinyl-1,3,5-triazin-2-amine (3c)
White solid; mp = 180-182 °C in 92 % yield
2.1.3.4. 4-(4-hydrazinyl-6-(piperidin-1-yl)-1,3,5-triazin-2- yl)morpholine (6a)
White solid; mp = 140-141 °C in yield 94%.
2.1.3.5. N-benzyl-4-hydrazinyl-6-morpholino-1,3,5-triazin-2-amine (6b)
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White solid, mp = 155-156 °C in 90% yield.
2.1.3.6. N-benzyl-4-hydrazinyl-6-(piperidin-1-yl)-1,3,5-triazin-2-amine (6c)
White solid, mp = 163-5 °C in 96% yield.
2.1.3.7. 4-hydrazinyl-6-morpholino-N-phenyl-1,3,5-triazin-2-amine (6d)
White solid, m.p. 145-6 °C in 90% yield.
2.1.3.8. N,N-diethyl-4-hydrazinyl-6-morpholino-1,3,5-triazin-2-amine (6e)
White solid, mp = 163-165 °C in 96% yield.
2.1.4. Synthesis of dimethyl(1Н-pyrazol-1-yl)-1,3,5-triazine derivatives
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Pyrazole-s-triazine derivatives were prepared following the reported method with slight
modifications [20, 27]. Two methodologies were applied, as explained below.
Method A: [20] 1,3,5-Triazine dihydrazino derivative (10 mmol) was dissolved in 20 mL DMF
and then acetylacetone (25 mmol in the case of dihydrazine and 12 mmol in the case of
monohydrazine derivatives) was added, followed by addition of triethylamine (8 mmol) in 10
mL DMF with stirring at room temperature. The reaction mixture was refluxed for 6-8 h. TLC
using ethylacetate-hexane (4:6) monitored the progress of the reaction. The solution was allowed
to cool to room temperature, and then ice-cold water was added with continuous stirring. The
reaction mixture was kept in an ice bath for 2 h, and the product was collected by filtration,
washed with cold water (3 x 20 mL), and then dried under vacuum. The crude product was
recrystallized from ethanol to afford the products in 60-75% yields.
Method B: [27] Acetylacetone was added dropwise (22 mmol) to a cold solution of 1,3,5-
triazine dihydrazino or mono hydrazine derivatives (10 mmol) in dilute HClO4 (20 mL). The
resulting mixture was stirred at room temperature overnight. The mixture was neutralized with
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dilute K2CO3, and the white precipitate was filtered, washed with water, and crystallized from
aqueous ethanol to afford pure products in 90-95% yields. For the reaction of dihydrazine
derivatives, double amounts of HClO4, and acetylacetone were used.
2.1.4.1. 4-(4,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)-1,3,5-triazin-2-yl)morpholine (4a)
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White crystals, mp = 187-188°C, in yield 92% (B); 70% (A); IR (KBr, cm-1) 1664, 1620 (C=N),
1595 (C=N, C=C); 1H NMR (CDCl3): δ 2.27 (s, 6H, 2CH3),2.632 (s, 6H, 2CH3), 3.56 (t, 4H, J =
2.4 Hz, 2CH2), 3.86 (t, 4H, J = 4.4 Hz, 2CH2), 5.97 (s, 2H, 2CH) ppm; 13C NMR (CDCl3): δ
10.7, 12.9, 41.2, 63.4, 108.1, 140.5, 149.0, 160.5, 162.4 ppm. Anal. Calc. for C17H22N8O
(354.42): C, 57.61; H, 6.26; N, 31.62. Found: C, 57.88; H, 6.35; N, 31.78
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2.1.4.2. 2,4-bis(3,5-dimethyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)-1,3,5-triazine (4b)
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White crystals, mp = 141-142°C, in 96% yield (B); 73% (A); IR (KBr, cm-1) 1674, 1624 (C=N),
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1595 (C=N, C=C); H NMR (CDCl3): δ 1.60 (m, 6H, 3CH2), 2.27 (s, 6H, 2CH3),2.62(s, 6H,
2CH3), 3.78 (t, 4H, , J = 4.4 Hz, 2CH2), 5.97 (s,2H, 2CH) ppm; 13C NMR (CDCl3): δ 13.5, 15.8,
24.3, 25.5, 45.1, 110.8, 143.3, 151.7, 163.5, 164.7 ppm. Anal. Calc. for C18H24N8. (352.45): C,
61.34; H, 6.86; N, 31.79. Found: C, 61.51; H, 6.99; N, 32.00.
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2.1.4.3. N-benzyl-4,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)-1,3,5-triazin-2-amine (4c)
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White crystals, mp = 120-121°C, in 95% yield (B); IR (KBr, cm-1) 3388 (NH), 1634, 1620
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(C=N), 1595, 1541 (C=N, C=C); H NMR (CDCl3): δ 2.54 (s, 6H, 2CH3), 2.68(s, 6H, 2CH3),
4.69 (d, 2H, J = 6.0 Hz, phCH2), 5.98 (s, 2H, 2CH), 6.45 (t, 1H, NH), 7.24-7.30 (m, 5H, C6H5)
ppm; 13C NMR (CDCl3): δ 13.7,15.7, 45.2, 110.0, 127.1, 127.5, 128.7, 137.4, 143.9, 144.3,
152.1, 152.3, 163.5, 163.7, 166.7 ppm. Anal. Calc. for C20H22N8 (374.45): C, 64.15; H, 5.92; N,
29.93. Found: C, 64.30; H, 6.02; N, 30.04.
2.1.4.4.
4-(4-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)morpholine
(7a)
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Off white crystals, mp = 123-124°C, in 96% yield (B); 72% (A); 75% (A); IR (KBr, cm-1) 1622,
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1589 (C=N), 1541 (C=N, C=C); H NMR (CDCl3): δ 1.59-1.66 (m, 6H, 3CH2), 2.32 (s, 3H,
CH3), 2.62 (s, 3H, CH3), 3.72-3.84 (m, 12H , 6CH2), 5.99 (s, H, CH) ppm; 13C NMR (CDCl3): δ
14.0, 16.1, 24.8, 25.9, 43.9, 44.5, 44.7, 66.6, 110.3, 143.4, 150.9, 163.1, 164.9, 165.6 ppm. Anal.
Calc. for C17H25N7O. (343.44): C, 59.45; H, 7.34; N, 28.55. Found: C, 59.67; H, 7.50; N, 28.81.
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2.1.4.5. N-benzyl-4-(3,5-dimethyl-1H-pyrazol-1-yl)-6-morpholino-1,3,5-triazin-2-amine (7b)
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White crystals, mp = 124-125°C, in 94% yield (B), 72% (A); IR (KBr, cm-1) 3269 (NH), 1643,
1622 (C=N), 1595, 1553 (C=N, C=C) ; 1H NMR (CDCl3): δ 2.27 (s, 3H, CH3),2.59(s, 3H, CH3),
3.69 (brs, 4H, 2CH2), 3.87 (brs, 4H, 2CH2), 4.60 (d, 2H, J = 6.0 Hz), 5.96 (s,1H,CH), 6.20 (brs,
1H, NH), 7.22-7.28 (m, 5H, C6H5) ppm; 13C NMR (CDCl3): δ 13.8, 15.9, 43.7, 44.7, 66.5, 116.4,
127.1, 127.2, 128.4, 138.4, 142.9, 165.1, 166.0 ppm. Anal. Calc. for C19H23N7O (365.44): C,
62.45; H, 6.34; N, 26.83. Found: C, 62.67; H, 6.44; N, 27.05.
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2.1.4.6. N-benzyl-4-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)-1,3,5-triazin-2-amine (7c)
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Off white crystals, mp = 142-143°C, in 92% yield (B); IR (KBr, cm-1) 3336 (NH), 1678, 1622
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(C=N), 1577, 1529 (C=N, C=C);; H NMR (CDCl3): δ 1.56-1.65 (m, 6H, 3CH2), 2.28 (s, 3H,
CH3),2.62 (s, 3H, CH3), 3.76 (brs, 4H, CH2), 4.60 (d, 2H, J = 5.6 Hz, phCH2), 5.95 (s,1H, CH),
7.23-7.32 (m,5H, C6H5) ppm; 13C NMR (CDCl3): δ 13.8, 15.9, 24.6, 25.6, 44.7, 44.8, 110.3,
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127.1, 127.3, 127.4, 128.3, 128.4, 138.7, 142.9, 150.4, 162.5, 164.5, 165.9 ppm. Anal. Calc. for
C20H25N7 (363.47): C, 66.09; H, 6.93; N, 26.98. Found: C, 66.24; H, 7.10; N, 27.08.
2.1.4.7. 4-(3,5-dimethyl-1H-pyrazol-1-yl)-6-morpholino-N-phenyl-1,3,5-triazin-2-amine (7d)
219
220
221
222
223
224
225
Off white crystals, mp = 213-214°C, in 95% yield (B), 70% (A); IR (KBr, cm-1) 3281 (NH),
1633, 1614 (C=N), 1585, 1542 (C=N, C=C);1H NMR (CDCl3): δ 2.29 (s, 3H, CH3),2.61(s, 3H,
CH3), 3.74 (brs, 4H, 2CH2), 3.84 (brs, 4H, 2CH2), 5.98 (s,1H,CH), 7.06 (m, 1H, Ar), 7.28-7.32
(m, 3H, Ar), 7.51 (d, 1H, J = 8.0 Hz, Ar) ppm; 13C NMR (CDCl3): δ 13.9, 16.1, 44.1, 66.5,
110.9, 120.2, 123.4, 128.8, 138.2, 143.2, 151.8, 163.1, 165.1 ppm. Anal. Calc. for C18H21N7O
(351.41): C, 61.52; H, 6.02; N, 27.90. Found: C, 61.66; H, 6.20; N, 28.18.
226
2.1.4.8. 4-(3,5-dimethyl-1H-pyrazol-1-yl)-N,N-diethyl-6-morpholino-1,3,5-triazin-2-amine (7e)
227
228
Off white crystals, mp = 94°C, in 96% yield (B) 74% (A); IR (KBr, cm-1) 1643, 1612 (C=N),
1541 (C=N, C=C); 1H NMR (CDCl3): δ 1.16 (t, 6H, J = 7.6 Hz, 2CH3), 2.33 (s, 3H, CH3), 2.62(s,
3H, CH3), 3.50-3.60 (m, 4H , 2CH2), 3.63-3.86 (m, 8H , 4CH2), 5.98 (s, H, CH) ppm; 13C NMR
(CDCl3): δ 12.8, 13.3, 13.8, 15.7, 41.5, 43.8, 66.8, 110.2, 143.4, 150.7, 162.7, 164.5, 165.4 ppm.
229
230
231
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233
Anal. Calc. for C16H25N7O. (331.42): C, 57.77; H, 7.69; N, 29.83. Found: C, 57.98; H, 7.60; N,
29.58.
234
2.2. X-Ray Crystallography
235
236
237
238
239
240
241
242
243
244
The two compounds 4a and 7c were obtained as single crystals by slow evaporation from
ethanol solution of the pure compound at room temperature. Data were collected on a Bruker
APEX-II D8 Venture area diffractometer, equipped with graphite monochromatic Mo Kα
radiation, λ = 0.71073 Å at 296 (2) and 293 (2) K. Cell refinement and data reduction were
carried out by Bruker SAINT. SHELXT [28, 29] was used to solve the structure. The final
refinement was carried out using the full-matrix least-squares techniques with anisotropic
thermal data for non-hydrogen atoms on ̀. CCDC 1509638 and 1509633, respectively, contain
the supplementary crystallographic data for these compounds. This information can be obtained free
of
charge
from
the
Cambridge
Crystallographic
Data
Centre
via
www.ccdc.cam.ac.uk/data_request/cif.
245
2.3. DFT Calculations
246
247
248
249
250
251
252
Starting geometries were taken from X-ray refined data. The density functional theory (DFT)
geometry optimization with the Gaussian09 program package [30] using the B3LYP (Becke
three parameters Lee–Yang–Parr) exchange correlation functional, which combines the hybrid
exchange functional of Becke [31] with the gradient-correlation functional of Lee, Yang and Parr
[32] and the 6-311G++(d,p) basis set, was performed in gas phase. No solvent corrections were
made with these calculations as it was reported that gas phase calculations frequently correspond
well with crystal structures [33].
253
2.4. Antimicrobial Activity
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255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
The antimicrobial activity of synthesized compounds 4a-c and 7a-e against bacteria and
fungi was determined by modified Kirby-Bauer disk diffusion method [34]. Briefly, cultures of
two Gram-negative bacteria (E. coli ATCC 25922, Ps. aeruginosa ATCC 75853), two Gram-
positive bacteria (M. luteus ATCC 10240, Methicillin-resistant Staphylococcus aureus (MRSA)
ATCC 43300) and C. albicans ATCC 10145 (fungi) were grown in Luria broth, nutrient broth,
Müller-Hinton broth, nutrient broth, and potato dextrose broth, respectively. These organisms
were cultured at their optimal growth temperature i.e., E. coli and MRSA at 37°C, and P.
aeruginosa, M. luteus, MRSA, and C. albicans at 30°C on a rotary shaker at 150 rpm. Agar
plates were seeded with a mixture of exponentially growing test organisms containing 105-106
colony-forming units/ml and molten soft agar (0.7% agar). Plates were allowed to cool and
solidify at room temperature for 10-15 min. Wells of 7 mm in diameter were punched into the
soft agar layer in order to test the antimicrobial activity of the compounds. Compounds were
dissolved in DMSO at 10 mg/mL and then 10 µL of the solution was added to the wells. The
plates were incubated overnight at optimal growth temperatures to dry at room temperature
inside a biological safety cabinet. Then incubation zones of inhibition were measured and
recorded. Tobramycin, fusidic acid and canesten were used as controls.
270
271
3. Results and discussion
3.1. Chemistry
272
273
274
275
The triazine scaffold of cyanuric chloride was conveniently manipulated by the facile
displacement of its chlorine atoms by oxygen- or nitrogen-containing nucleophilic species in the
presence of a hydrogen chloride acceptor. These reactions take place with great regioselectivity
as a result of the temperature-controlled process.
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277
278
279
The mono and dipyrazolyl derivatives were first compounds synthesized following the published
method, where the hydrazinyl-s-triazine was reacted with acetylacetone using DMF as a solvent
in the presence of trimethylamine (method A) [20]. Alternatively, the method based on the use of
HClO4 and then neutralization with K2CO3 was also examined (method B) [27].
280
281
Scheme 1. Synthetic route for the synthesis of pyrazol-s-triazine derivatives.
282
283
The initial nucleophilic substitution of the first chlorine atom of cyanuric chloride 1 by
the first amine (morpholine, piperidine or benzylamine) at 0-5°C afforded the products 2a-c (2,4-
15

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284
285
286
287
288
289
290
291
dichloro-6-substituted-s-triazine derivatives) with excellent yield. The second chlorine of 1
replaced by a second amine at room temperature for 24 h gave the products 5a-e (2-chloro-4,6-
disubstitued-s-triazine derivatives). Products 2a-c and 5a-e were treated with hydrazine hydrate
(80%) in ethanol for 1-2 h under ultrasonic irradiation (US) [26] to afford the hydrazine
derivatives 3a-c and 6a-e, respectively. Compounds 3 and 6 were then reacted with acetyl
acetone following methods A or B [20, 27], as shown in Scheme 1. Method B (using the HClO4)
afforded the products in higher yield under moderate conditions (experimental section). The
spectral data for the new products confirmed their structure.
292
3.2. X-Ray Crystallography
293
294
295
296
297
298
299
300
301
302
303
304
305
306
The crystallographic data and refinement information for compounds 4a and 7c are
summarized in Table 1. The selected bond lengths and bond angles are provided in the
Supplementary Material (Table S1 and S3). Analysis of the asymmetric unit of 4a revealed that
the molecule crystallized in the P 21/c space group with two water molecules (Figure 1 and 2).
All the bond lengths and angles were found in normal ranges [35]. Of the four rings present in
the unit of 4a, the morpholine one had a puckered chair conformation. However, no puckered
conformation was observed in any other ring. Weak π-π stacking was observed in the crystal
structure of 4a (Cg4-Cg4: 3.6590(10) Å; -1-x,-1-y,-1-z). H-bonding was found between 4a and
the water molecules.
The asymmetric unit of 7c contained two molecules (Figure 2 and 3). The molecule
crystallized in the P-1 space group. Bond lengths were in the normal range [35]. The piperidine
ring present in the two molecules had a puckered chair conformation (Cg5-Cg6: 3.6136(12) Å;
1-x,1-y,1-z), providing extra stability to the crystal structure. No classical H-bonding was
observed in the crystal.
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307
Table 1. X-ray crystallographic experimental details of 4a and 7c.
Crystal data
4a
7c
Chemical formula
Mr
C17H26N8O3
390.46
C20H25N7
363.47
Crystal system, space group
Temperature (K)
a, b, c (Å)
Monoclinic, P21/c
293
Triclinic, P-1
296
7.7525 (3), 21.5986 (8),
12.3291 (4)
11.1772 (5), 11.9058 (5),
15.3195 (7)
α
,
β
,
γ
(°)
76.367 (2), 79.752 (2), 78.321
(2)
112.970 (2)
V (Å3)
1900.73 (12)
4
1921.93 (15)
4
Z
Radiation type
µ (mm−1)
Mo K
α
Mo Kα
0.10
0.08
Crystal size (mm)
Data collection
Diffractometer
0.57 × 0.15 × 0.11
0.30 × 0.17 × 0.14
Bruker APEX-II D8 venture
diffractometer
Bruker APEX-II D8 venture
diffractometer
Absorption correction
Multi-scan SADABS Bruker
2014
Multi-scan SADABS Bruker
2014
Tmin, Tmax
0.946, 0.989
0.977, 0.989
No. of measured, independent
and observed [I > 2
reflections
Rint
σ
(I)]
29796, 4353, 3067
0.194
41578, 8821, 5114
0.113
Refinement
R[F2 > 2
σ
( F2)], wR( F2), S
0.059, 0.157, 1.02
0.059, 0.131, 1.03
No. of reflections
No. of parameters
No. of restraints
H-atom treatment
4353
257
0
8821
499
0
H atoms treated by a mixture H atoms treated by a mixture
of independent and
constrained refinement
0.35, −0.45
of independent and
constrained refinement
0.26, −0.33
ꢀρmax
CDCC numbers
,
ꢀρmin (e Å−3)
1509638
1509633
308
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7c
4a
309
310
311
312
Figure 2. ORTEP diagram of the titled compounds. Displacement ellipsoids are plotted at the
40% probability level for non-H atoms.
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7c
313
4a
314
315
Figure 3. Molecular packing of titled compounds viewed with hydrogen bonds, which are drawn
as dashed lines.
316
3.3. Theoretical Calculations (DFT) of 4a and 7c.
317
318
319
DFT geometry optimization were made on a crystal unit using Gaussian09 program package.
The optimized geometry results in the free molecule state were compared to those in the
crystalline state (Table S5 and S6, Supplementary Materials). No negative vibrational modes
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320
321
were obtained. The optimized geometry of 4a and 7c revealed P 21/c and P-1 space group,
respectively (Figure 4).
7c
4a
322
Figure 4. Optimized geometry of 7c and 4a using DFT [B3YLP/6-311++(d,p)]
20

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323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
The optimized geometry of the studied compounds was compared with the structural parameters
obtained from the crystallographic information file (CIF). All the bond lengths and angles
calculated were in agreement with crystal structure. Among all the four rings present in 4a,
puckering was observed in morpholine ring which is quite similar to that present in the crystal
structure. H-bond was found between 4a and water molecules. The O(1)-H(50) and O(52)-H(51)
distance was found to be 1.99 Å (exp. 1.99 Å) and 1.96 Å (exp. 1.89 Å) respectively which
indicated the presence of intermolecular H-bond between 4a and water molecules. Torsion angle
between O(1)-C(43)-C(46)-N(9) and O(1)-C(40)-C(37)-N(9) was found to be 54.60° (Expt.
56.15°) and -54.61° (Expt. -56.28°) which showed slight deviation from the experimental data
but maintaining the puckering for the morpholine ring same as that found in crystal unit.
Optimized geometry of 7c clearly indicates absence of classical H-bonding. Torsion angle
between C(29)-C(32)-C(35)-N(6) and C(29)-C(26)-C(23)-N(6) was found to be -53.71° (Expt. -
55.04°) and 53.59° (Expt. 55.57°) respectively, with values slightly deviating from the
experimental data. The pyridine ring was found to have chair conformation similar to that
present in the crystal.
338
3.4. Preliminary Antimicrobial Activity
339
340
341
342
343
344
345
In the present study, synthesized compounds (4a, 4b, 4c, 7a, 7b, 7c, 7d and 7e) were
evaluated for their in vitro antibacterial and antifungal activities against microbial strains such as
Gram-negative bacteria (E. coli ATCC 25922, P. aeruginosa ATCC 75853), Gram-positive
bacteria (M. luteus ATCC 10240, Methicillin-resistant Staphylococcus aureus (MRSA) ATCC
43300), and fungi (C. albicans ATCC 10145) by the agar-well diffusion method. The results of
antimicrobial testing against strains were obtained as zone of inhibition (mm), as tabulated in
Table 2.
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346
347
348
349
350
351
352
353
354
355
Compounds 4b, 4c, 7b, 7c and 7e exhibited antibacterial activity against bacterial strains,
whereas 4a, 7a and 7d did not. However, 7a showed mild activity against fungi C. albicans, but
4a was inactive against all the strains tested. In addition, 4b and 7b also showed weak activity
against C. albicans.
It is evident from the structure that compounds with one pyrazole molecule (7a-e)
showed better performance than analogs with two (4a-c). Furthermore, presence of piperidine
and benzylamine on triazine ring exhibits the highest activity. Comparatively, presence of
morpholine moiety appeared to have detrimental effects on activity (4a, 7a and 7d bear this
heterocycle). Compound 7c which holds pyrazole, piperidine, and benzylamine, exhibited the
highest activity against all bacterial strains.
356
Table 2: Antimicrobial activities of 4a-c and 7a-ea,b
Gram-negative
E. coli P. aeruginosa
Gram-positive
M. luteus MRSA
Fungi
C. albicans
Compoundsc
4a
4b
4c
7a
7b
7c
7d
7e
-
-
-
-
-
-
-
-
-
13
13e
-
17
19
-
-
-
11
7
-
8
11
7
8
-
17
15
-
9
22
-
wdd
-
9
wdd
-
-
8
9
17
357
358
359
360
361
362
363
364
365
366
367
368
a Tobramycin, fusidic acid and canesten were used as controls.
b Zone of inhibition in mm
c Compounds were dissolved in DMSO (25% in water) at 10 mg/mL and then 10 µL of the solution was added
to the wells
cdwd = weak and diffused zones (weak activity and cannot be measured)
e 20 µL of the solution was added to the wells
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369 Conclusions
370
371
372
373
374
375
376
377
378
379
380
381
In summary, a new series of pyrazole-containing s-triazine derivatives were synthesized
by reaction of corresponding s-triazinyl hydrazine derivatives with acetylacetone in presence of
HClO4 or DMF/TEA. The HClO4 method allowed the preparation of the target products in higher
yields when compared to DMF/TEA approach. X-ray single crystal diffraction of 4a and 7c was
studied and their molecular geometries were optimized using the DFT/B3LYP 6-311G++(d,p)
method. The optimized structures were found to be in good agreement with corresponding X-ray
structures. The synthesized compounds were screened for antimicrobial activity. In the view of
the results obtained, compounds with one pyrazole molecule showed better performance when
compared to analogs with two pyrazole. Presence of piperidine with benzylamine over triazine
ring proves to be an optimum factor for enhancing the activity against all bacterial strains. Based
on our findings, we conclude that s-triazine with pyrazole and piperidine is a promising scaffold
for the development of new active compounds.
382 Acknowledgments
383
384
385
386
387
We thank Dr. Ashish Kumar for fruitful discussions regarding the biological activity during the
preparation of the manuscript. The authors thank the International Scientific Partnership Program
(ISPP) at King Saud University (ISPP# 0061) (Saudi Arabia). The work carried out in South
Africa was partially funded by the National Research Foundation and the University of
KwaZulu-Natal.
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Highlights
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Pyrazolyl-s-triazine.
X-ray crystallography.
DFT/B3LYP.
Anti-microbial activity.