Design, crystal structures and sustainable synthesis of family of antipyrine derivatives: Abolish to bacterial and parasitic infection

Article abstract of DOI:10.1016/j.molstruc.2019.127010

This work explores the facile synthesis of a series of antipyrine acetylinic framework (3a-3h). The newly synthesized compounds are recognized by IR, NMR (¹H, ¹³C), X-ray diffraction. This collection played indispensable role in the

Full text of DOI:10.1016/j.molstruc.2019.127010

Journal Pre-proof
Design, crystal structures and sustainable synthesis of family of antipyrine derivatives:
Abolish to bacterial and parasitic infection
Gurjaspreet Singh, Pinky Satija, Baljinder Singh, Shweta Sinha, Rakesh Sehgal,
Subash Chandra Sahoo
PII:
S0022-2860(19)31110-X
DOI:
https://doi.org/10.1016/j.molstruc.2019.127010
MOLSTR 127010
Reference:
To appear in: Journal of Molecular Structure
Received Date: 18 July 2019
Revised Date: 30 August 2019
Accepted Date: 31 August 2019
Please cite this article as: G. Singh, P. Satija, B. Singh, S. Sinha, R. Sehgal, S.C. Sahoo, Design, crystal
structures and sustainable synthesis of family of antipyrine derivatives: Abolish to bacterial and parasitic
infection, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.127010.
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Design, Crystal Structures and Sustainable Synthesis of family of antipyrine
derivatives: Abolish to bacterial and parasitic infection.
Gurjaspreet Singha*, Pinky Satijaa, Baljinder Singhb, Shweta Sinhac, Rakesh Sehgalc, Subash
Chandra Sahooa
a Department of Chemistry, Panjab University, Chandigarh 160014, India.
b Department of Biotechnology, Panjab University Chandigarh, 160014, India
c Department of Medical Parasitology, PGIMER, Chandigarh-160012, India.
*Corresponding Author
Dr. Gurjaspreet Singh
Professor,
Department of Chemistry,
P.U., Chandigarh
India
+91-0172-2534428,
Email: gjpsingh@pu.ac.in
1

Graphical Abstract
ABOLIS H
TO
Bacterial
&
Parasitic infection
2

Abstract
This work explores the facile synthesis of a series of antipyrine acetylinic framework (3a-3h).
13
The newly synthesized compounds are recognized by IR, NMR (1H, C), X-ray diffraction.
This collection played indispensable role in the development of biologically active agents.
Our goal is to enable adoption of green chemistry within the pharmaceutical industry for the
benefit of society, industry and the environment. This work articulates a new possible scaffold
for drug discovery against protozoal pathogens (Entamoeba histolytica and Giardia lamblia)
and bacterial strain (Staphylococcus aureus, Vibrio cholera, E.coli, Bacillus subtillus). The
purpose of the present undertaking is to put low price anti parasitic drug using green
technology. It is envisaged that the alliance of anti pyrine derivative and acetylene framework
into a single scaffold boosts the effectiveness of anti bacterial as well as anti-parasitic
treatment and may be vital for meeting future challenges in medicinal world.
Key words: Anti pyrine, Entamoeba histolytica, Giardia lamblia, Microwave methodology,
Acetylene framework
Introduction
The microwave irradiation technique, has been triumphantly making inroads in miscellaneous
chemistry laboratories such as solid- state chemistry, nanomaterial synthesis, and organic
synthesis [1]. The advantage of this fascinating technology is safe, increased reaction kinetics,
energy efficiency, purity and high product yields due to minimized the formation of undesired
side- products during the reactions[2].
Antipyrine (AP) was first synthesized by Knorr in 1883 [3]. It has a biological versatile
structure due to the presence of tribioactive centers like methyl, N and O groups, which
generates interest in the studies of antipyrine derivatives (APDs). 4-Aminoantipyrine (AAP) is
a metabolite of aminophenazone and its derivatives exhibit a fascinating array of
pharmacological activity imputed to their ability to have non covalent interactions with
3

various active site in organism [4].This fragment exhibit a resemblance with metamizole, a
recognized antipyretic agent, analgesic and anti-inflammatory whose potency is
unimpeachable[5,6].
Although there were some recognized reactions grabbing the acetylene framework such as
Huisgen 1, 3-dipolar cycloaddition, Sonogashira, the Pauson-Khand and Nicholas reactions,
the Bergman, Moore and Myers cyclizations yet pharmacological activity of this framework is
not reported much[7].
Bacterial fouling and parasitic infections are devastating diseases impacting the human society
worldwide and global economy significantly. A frightening statistic shows that these
infectious diseases kill more than nine million people every year which is more than any other
diseases like Parkinson’s disease, HIV/AIDS[8,9]. E. histolytica and G. lamblia, protozoan
infection that cause amoebic dysentery and giardiasis, respectively[10-12].This usually shows
broad range of symptoms such as dysentery, bloating, stomachache, cramps[13, 14].
Bacteria can be categorized into two type: Gram positive and Gram negative that depend on
their cell wall structure. The infections due to bacteria may cause severe diseases such as
osteomyelitis, endocarditis, and toxic shock syndrome. The problem of antimicrobial
resistance is due to insufficiency of investment of the pharmaceutical industry[15] and
advanced therapeutic agents.
Nitroimidazole and its derivatives (Fig 1) are the most effective antiamoebic medication, but
have several adverse effects including gastrointestinal disturbance, especially nausea,
vomiting [16]. Hence, in severe infection it carried out with antimicrobial drugs.
4

Benznidazole
Tinidazole
Ornidazole
Delamanid
TBA 354
Pimonidazole
Fexinidazole
1950-
1970
1970- 1990- 2010-
1990
2010
2018
Metronidazole
Azomycin
Pretomanid
Fig 1: Timeline for the discovery of nitro-imidazole anti-infective agents
The general mechanism of action of nitro imidazole derivatives (Fig 1), involves the
formation of reactive nitro radical anion that can lead to DNA degradation and cell death.
Therefore there is an urgent need of an ideal approach to combat them with a novel action
mechanism drug. The proximity of antipyrine derivatives and acetylene framework, a
felicitous choice used to develop new antimicrobial agents.
2. Experimental Protocol
2.1. Materials and measurements.
All commercially available chemicals were used as received without further purification and
organic solvents were used after distillation. 4 amino antipyrine(Avra) Propargyl bromide
(Aldrich), Potassium carbonate(Thomas), 2-hydroxy benzaldehyde (Avra), 3-hydroxy
benzaldehyde(Avra), 4-hydroxy benzaldehyde(Avra), 3-Ethoxy-4-hydroxy benzaldehyde), 2-
hydroxy-3 methoxy benzaldehyde(Avra), 4-hydroxy 3 methoxy benzaldehyde(Alfa Aesar), 2-
hydroxy naphthaldehyde(Alfa Aesar), 2,4 Dihydroxy benzaldehyde(Avra) were used.
2.2. Physical measurements
5

The infrared spectra were recorded on a Thermo Scientific NICOLET IS50
spectrophotometer. The 1H and 13C NMR spectra were carried out on a JEOL (AL 400 MHz)
spectrometer. Microwave synthesis was conducted in Anton Paar Microwave PRO reaction
system. Melting points were measured in a Mel Temp II device using sealed capillaries.
2.3 X-ray Structure determination
Crystals were mounted on Hampton cryoloops. All geometric and intensity data for the
crystals were collected using a Super-Nova (Mo) X-ray diffractometer equipped with a micro-
focus sealed X-ray tube Mo-Kα (λ = 0.71073 Å) X-ray source, and HyPix3000 (CCD plate)
detector of with increasing ω (width of 0.3_ per frame) at a scan speed of either 5 or 10
s/frame. The CrysAlisPro software was used for data acquisition, and data extraction. Using
Olex2[17], the structure was solved with the SIR2004[18] structure solution program using
Direct Methods and refined with the ShelXL[19] refinement package using Least Squares
minimisation. All non-hydrogen atoms were refined with anisotropic thermal parameters.
Detail crystallographic data and structural refinement parameters are summarized in Table 5,
6 and 7.
2.4 Statistical analysis
2.4.1. Antiparasitic activity
Drug sensitivity was conducted by using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, a tetrazole) assay for all Compounds. Highest drug
concentration in each rows was 100ꢀg/ml and then doubling dilutions of the drug were
performed. The plates were incubated at 37°C for 24 hrs and 48hrs. Following incubation at
37°C for 24hr and 48hr, the optical density (OD) was recorded in an ELISA recorder at 570
nm. Each experiment is executed in triplicate sets. IC50 value was measured using Graph Pad
Prism version 6.
2.4.2. Antibacterial activity
In the broth dilution MIC method, various concentrations of the compounds were inoculated
with a standard suspension of test bacteria. Following an overnight incubation at 37 °C, the
MIC was determined by observing the lowest concentration of the compounds that would
inhibit visible growth of the test bacteria. Growth was calculated photometrically by
measuring the optical density (OD) at 600 nm[20].
6

2.5 General details
Synthesis and characterization
2.5.1. Synthesis
2.5.1.1 Representative procedure for the synthesis of anti pyrine Schiff’s base
Antipyrine functionalized Schiff bases (2a-2h) were prepared as condensation of 4 amino
antipyrine and various substituted aromatic aldehyde. Yellow crystalline product is obtained
after irradiating for five minutes as shown in scheme 1. The product was dried at room
temperature.
NH2
O
N
O
R
O
(i)
N
+
(ii)
N
N
R
N
(a-h)
1
2a-2h
OH
R=
O
OH
(a)
OH
OH
(b)
(c)
(d)
OH
OH
OH
OH
O
O
OH
(e)
(f)
(h)
(g)
Scheme 1: Synthesis of anti pyrine functionalized Schiff’s base (2a-2h). Reagent and
Conditions: (i) MW, 5 min, (ii) Ethanol.
7

Methodology for the synthesis of anti pyrine acetylinic framework
Antipyrine functionalized Schiff bases (2a-2h) were converted into terminal alkynes (3a–3h)
(Scheme 2) by using propargyl bromide facilitated by base K2CO3 in DMF and the mixture
was allowed to age for 16hrs. When the reaction was over, the residue was distilled to recover
DMF for the next reaction. After distillation, the residue was poured into cold water, yellow
crystalline solid is obtained.
K2CO3,DMF
R
O
Br
R
OH
+
16hr, Stirring
2a-2h
3a-3h
O
N
N
O
N
O
O
N
N
N
3b(99%)
3a(99%)
O
O
N
N
O
O
O
N
N
N
N
3d(97%)
3c(94%)
N
O
N
O
O
O
O
N
N
N
N
O
3f(98%)
3e(98%)
N
O
O
O
N
O
O
N
N
N
N
3h(95%)
3g(96%)
Scheme 2: Synthesis of Anti pyrine functionalized alkynes (3a-3h) using (2a-2h) with%
yield.
8

2.5.2. Characterization:
Synthesis of (3a) 2a (0.32 mmol), propargyl bromide (0.40 mmol).Mustard Crystalline
Solid.M.p.:158oC-160oC. IR (cm-1): 3293 (C≡C-H), 3113(N=CH), 2116(C≡C), 1617(C=O),
1603 (C=N), 1413 (CH3-C), 1270 (CH2-N), 1168 (O-CH2), 939 (C-C). 1H NMR (25̊ C,
CDCl3): δ(400 MHz) = 9.73 (s, 1H, CH=N), 7.02–7.85 (m, 9H, Ar), 4.75 (d, 2H,-OCH2-),
3.15(s, 3H, N-CH3), 2.57 (t, 1H, -C≡CH), 2.49(s, 3H, C-CH3).
13C NMR (25oC, CDCl3): δ(101MHz)= 161.0, 159.2, 156.6, 151.7, 134.8, 131.7, 129.2,
129.1, 126.8, 124.2, 118.9, 114.8, 78.2, 75.7, 69.8, 55.8, 35.9, 30.8, 18.8, 10.1.
Synthesis of (3b) 2b (0.32mmol), propargyl bromide (0.40mmol). Yellow Crystalline Solid.
M.p.:160oC-162oC. IR (cm-1): 3186 (C≡C-H), 2108(C≡C), 1635 (C=O), 1572(C=N), 1450
(CH3-C), 1392 (CH2-N), 1140 (O-CH2), 959 (C-C).
1H NMR (25oC, CDCl3,): δ(400 MHz)= 9.74 (s, 1H, CH=N), 7.02–8.03 (m, 9H, Ar), 4.76 (d,
2H,-OCH2-), 3.18(s, 3H, N-CH3), 2.56 (t, 1H, -C≡CH), 2.51(s, 3H, C-CH3).
13C NMR (25oC, CDCl3,): δ (101 MHz) = 160.8, 157.9, 157.8, 156.6, 152.1, 139.5, 134.7,
129.5, 129.2, 126.9, 124.4, 121.8, 118.4, 117.3, 112.7, 78.5, 75.6, 55.9, 35.8, 10.1.
Synthesis of (3c) 2c (0.32 mmol), propargyl bromide (0. 40 mmol). Yellow Crystalline Solid.
M.p.:170oC-172oC. IR (cm-1): 3288 (C≡C-H), 2108(C≡C), 1625 (C=N), 1454 (CH3-C), 1384
(CH2-N), 1172 (O-CH2), 967 (C-C). 1H NMR (25oC, CDCl3): δ(400 MHz) = 10.10 (s, 1H,
CH=N), 7.05–8.18 (m, 9H, Ar), 4.79 (d, 2H,-OCH2-), 3.16(s, 3H, N-CH3), 2.51 (t, 1H, -
C≡CH), 2.48(s, 3H, C-CH3).
13C NMR (25oC, CDCl3,): δ(101 MHz)= 160.9, 157.1, 153.2, 152.0, 134.9, 131.1, 129.1,
127.2, 126.7, 126.2, 124.2, 121.4, 119.5, 113.0, 78.6, 75.6, 56.1, 36.0, 10.1.
Synthesis of (3d) 2d (2.84 mmol), propargyl bromide (4.23 mmol).Lemon Coloured Solid( in
the form of Fibres).M.p.:174oC-176oC. IR (cm_1): 3240 (C≡C-H), 3182(N=CH), 2116(C≡C),
1635(C=O), 1597 (C=N), 1428 (CH3-C), 1303 (CH2-N), 1175, 1215 (O-CH2), 920(C-C). 1H
NMR (25oC,CDCl3): δ(400 MHz)= 9.7 (s, 1H, CH=N), 7.05–7.55 (m, 8H, Ar), 4.83 (d, 2H,-
9

OCH2-), 4.21(q, 2H, OCH2CH3), 3.16(s, 3H, N-CH3), 2.55 (t, 1H, -C≡CH), 2.51(s, 3H, C-
CH3), 1.52(t, 3H, OCH2CH3).
13C NMR (25oC, CDCl3): δ(101MHz) =161.0, 157.0, 151.7, 149.1, 149.0, 134.8, 132.3,
129.1, 126.8, 124.2, 122.1, 118.9, 114.0, 110.6, 78.4, 75.9, 64.3, 56.7, 36.0, 10.2.
Synthesis of (3e) 2e (2.90 mmol), propargyl bromide (3.70 mmol).Yellow crystalline solid.
M.p.: 170oC-172oC. IR (cm-1): 3284 (C≡C-H), 2165(C≡C), 1648(C=O), 1571 (C=N), 1470
(CH3-C), 1372 (CH2-N), 920 (C-C), 1135, 1268 (O-CH2).
1H NMR (25oC, CDCl3): δ(400 MHz) = 10.07 (s, 1H, CH=N), 6.69–7.79 (m, 8H, Ar), 4.83
(d, 2H,-OCH2-), 3.90(s, 3H, -OCH3), 3.90(s, 3H, N-CH3), 2.51 (t, 1H, -C≡CH), 2.43(s, 3H, C-
CH3).
13C NMR (25oC, CDCl3): δ(101 MHz) = 160.7, 153.7, 152.9, 152.1, 146.8, 134.9, 132.7,
129.1, 126.7, 124.4, 119.4, 117.8, 113.6, 79.0, 75.9, 60.6, 55.8, 35.9, 10.2.
Synthesis of (3f) 2f (2.90 mmol), propargyl bromide (3.70mmol).Yellow crystalline solid.
M.p.: 166oC-168oC. IR (Neat, cm-1): 3287 (C≡C-H), 2136(C≡C), 1648(C=O), 1575 (C=N),
1448 (CH3-C), 1397 (CH2-N), 1138, 1258 (O-CH2), 959 (C-C). 1H NMR (25oC, CDCl3): δ
(400 MHz) =9.71 (s, 1H, CH=N), 6.65–7.70 (m, 8H, Ar), 4.86 (d, 2H,-OCH2-), 3.94(s, 3H, -
OCH3), 3.16(s, 3H, N-CH3), 2.56 (t, 1H, -C≡CH), 2.51(s, 3H, C-CH3).
13C NMR (CDCl3, 25oC): δ (101 MHz) = 161.0, 156.7, 151.7, 151.5, 139.5, 134.8, 131.1,
129.1, 126.2, 124.2, 123.5, 118.9, 111.1, 78.3, 75.9, 56.7, 55.9, 36.0, 10.1.
Synthesis of (3g) 2g (3.09 mmol), propargyl bromide (4.80 mmol). Mustard crystalline solid.
M.p.: 172oC-174oC. IR (cm-1): 3240 (C≡C-H), 3182(N=CH), 2120(C≡C), 1635(C=O), 1597
(C=N), 1496 (CH3-C), 1303 (CH2-N), 1175, 1215 (O-CH2), 920 (C-C).
1H NMR (25oC, CDCl3): δ(400 MHz) =10.45 (s,1H, -CH=N), 9.49(d, 1H, Naph), 7.28-7.90
(m, 10H, Ar), 4.92 (s, 2H,-OCH2-), 3.20(s, 3H, -NCH3), 2.50 (t, 1H, -C≡CH), 2.49(s, 3H, C-
CH3).
13C NMR (CDCl3, 25oC): δ(101 MHz) = 160.8, 156.6, 156.3, 135.0, 131.9, 129.9, 129.1,
128.2, 127.5, 126.7, 126.2, 124.3, 124.2, 120.0, 115.0, 78.8, 75.9, 57.5, 35.9, 10.4.
10

Synthesis of (3h) 2h( 0.390 mmol), propargyl bromide(0. 480 mmol). Pale Yellow Crystalline
Solid. M.p.: 170oC-172oC. IR (cm-1): 3265 (C≡C-H), 2116(C≡C), 1628(C=O), 1592 (C=N),
1454 (CH3-C), 1305 (CH2-N), 1171, 1257 (O-CH2), 972 (C-C).
1H NMR (25oC, CDCl3): δ (400 MHz) = 10.00 (s, 1H, -CH=N), 6.68-8.14 (m, 7H, Ar), 4.76
(d, 4H,-OCH2-), 3.14(s, 3H,-NCH3), 2.57 (t, 2H, -C≡CH), 2.49(s, 3H, -C-CH3).
13C NMR (CDCl3, 25oC): δ (101 MHz) = 161.0, 160.3, 158.3, 151.6, 135.0, 129.0, 127.4,
126.6, 124.1, 121.2, 119.8, 107.4, 100.6, 78.3, 75.9, 56.2, 36.0, 10.1.
3. Calculated biological properties:
The introduction of single new drug into the market desires nearly 12ꢁyears and costs about
billion dollars for execute this process. High cost, long time duration, high level of risk, highly
complex procedures, uncertainty in the results, are the major threats in the growth of new
drug. To defeat these difficulties, it is required to recruit cost effective drug designing method.
Computer aided drug design are appraised to be substantial equipment for exploration
of pharmacokinetic and pharmacodynamic properties. Proper execution of these methods
could lead to a minimization in cost of drug designing and development. Molecular
hybridization is an attractive strategy which based on the customizing of two different
pharmacophores to fabricate hybrid molecules that can act as “double-edged swords”. It is
considered that these molecules will impart marvellous activity with dual action that can
control resistance[20, 21].
To maintain a pharmaceutically friendly profile, there is a rule called ‘Lipinski rule of five’;
according to this rule, molecules must have molecular weights<500, clog P<5, H bond Donor
<5 and H-bond acceptor <10. For a molecule to have good oral bioavailability, the number of
rotatable bonds should be less than or equal to 10 and TPSAꢁ≤ꢁ140ꢁÅ, or sum of hydrogen
bond donor and acceptor (HBDꢁ+ꢁHBA) should be ≤12. as assessed by Veber rule[22]. The
synthesized compounds 3a-3h was examined for their physicochemical properties using
Molinspiration software. Luckily in our case, all the synthesized compounds obey lipinski rule
as well as Veber rule as summarized in table 1.
11

Table 1: In silico physicochemical properties of Antipyrine functionalized alkynes (3a-
3h)
Comp. ID
M.W
cLog P
TPSA
nON
Nrotb
NViolations
≤500
203
345
345
<5
<140
52.96
48.54
48.54
-
<15
1
<1
0
Lipinski rule
0.81
3.03
3.05
4
5
5
4-AAP
3a
5
0
5
0
3b
345
389
375
375
395
399
171
3.08
3.04
2.84
2.67
4.19
3.22
-0.47
48.54
57.77
57.77
57.77
48.54
57.77
83.88
5
6
6
6
5
6
6
5
7
6
6
5
7
3
0
0
0
0
0
0
0
3c
3d
3e
3f
3g
3h
Metronidazole
The activity score of kinase inhibitor, nuclear receptor ligand, protease inhibitor and enzyme
inhibitor is calculated for synthesised compounds and shown in Table 2.
Table2: Comparative bioactivity of compounds (3a-3h) and reference drug
(metronidazole)
Comp. id
3a
GPCR
ICM
KI
NI
PI
EI
-0.68
-0.7
-1.15
-1.09
-1.08
-1.02
-1.12
-1.06
-0.94
-1.01
-0.87
-0.57
-0.58
-0.56
-0.56
-0.57
-0.54
-0.44
-0.51
-0.59
-0.77
-0.76
-0.76
-0.75
-0.81
-0.78
-0.66
-0.64
-1.74
-0.82
-0.86
-0.85
-0.84
-0.84
-0.86
-0.68
-0.72
-1.68
-0.44
-0.43
-0.43
-0.44
-0.46
-0.43
-0.33
-0.39
-0.32
3b
3c
-0.7
3d
-0.69
-0.68
-0.69
-0.54
-0.6
3e
3f
3g
3h
Met.
-1.09
Pharmacokinetic Profile
12

PK is well described by ADMET[23-25], open source database, represents a number of stages
occur when the drug come in contact with the organism.
All the synthesized acetylinic antipyrine derivatives were investigated through ADMET tool
and it was revealed that all the compounds impart good pharmacokinetic profile. HIA values
of the investigated compounds fall in the range of 97.39-99.84 thus displaying their excellent
absorption behaviour. Caco-2 model revealed a similar value for bis acetylinic antipyrine (3h)
as metronidazole however all other compounds shows good Caco-2 values falling in the range
39.39-57.65.MDCK cell model reveals a little lower value for 3f (0.316). As 3e has very low
value of BBB which is almost similar to the reference drug while for other compounds it
ranges between 0.205 - 0.628.Moreover 3b has excellent value than reference drug. All the
compounds depict excellent binding to the plasma proteins. Skin permeability (log Kp) varies
from-2.19 to -3.44. 3h exhibit eminent value for skin permeability among the synthesized
compounds.
Table 3: Pharmacokinetic performance of anti pyrine functionalized compounds (3a-3h)
and reference drug using ADMET web server
Com. BBB Buffe Caco2 HIA MDCK Plasma
Pure
Skin per- SKLog D SKL
ID
r
protein
binding
Water
solubility
meability
og P
solub
ility
0.182 0.166 39.39
0.628 0.177 58.23
0.061 0.297 41.89
0.069 0.083 56.18
0.449 0.103 57.17
0.205 0.167 57.65
0.187 0.010 47.23
0.140 0.044 21.73
0.462 2.293 18.71
8e+0
97.54 115.14 89.85
10.72
8.09
7.68
3.37
9.60
5.440
0.216
2.05
-
-2.44
-2.41
-2.41
-2.43
-2.52
-2.54
-2.19
-3.44
-2.65
2.66
2.71
2.67
3.04
2.58
2.67
3.8
2.66
2.71
2.67
3.04
2.58
2.67
3.8
3a
3b
3c
97.40 42.33
97.40 42.33
97.39 1.76
97.42 80.57
97.42 0.316
97.81 12.64
99.84 1.27
92.07
93.62
92.21
89.58
92.27
90.83
89.05
3d
3e
3f
3g
3h
Met.
2.98
-2.20
2.98
-
60.06 199.99 90.57
0.64
06
13

3.2Biological Assay:
In vitro anti parasitic effect
Acetylinic antipyrine 3a-3h was assessed for their anti parasitic activities and results against
parasitic strains are epitomized in Fig 2 and Fig 3. Various substituted aldehydes have been
engaged to give a family of Schiff base linked antipyrine derivatives and reconnaissance their
possibilities to curb the parasitic and bacterial infection. The screening results disclosed that
the antipyrine acetylinic derivatives display majestic response on the anti parasitic potency.
Against parasitic strains, antipyrine derivatives manifested curtailed activities compared to
their activity against bacterial strains. The significant activity is shown by compound 3g with
enhanced aromatic region against E. histolytica IC50 value of 10.65 µM at 24 hrs. while 3f (
IC50=1.40 µM) is also found to show marvelous activity against the same strain at 48 hrs as
shown in Fig2
300
E.histolytica
250
200
IC50(µM) 24hrs
IC50(µM) 48hrs
150
100
50
0
3a
3b
3c
3d
3e
3f
3g
3h
met
Fig 2: Anti parasitic activity of compounds (3a-3h) against E. histolytica at 24hrs. and 48
hrs.
Compounds 3b and 3g was displayed superior activity against G.lambia with IC50 values of
44.81, 16.89µM respectively. Moreover 3h manifested magnificent activity (IC50= 4.20µM)
14

as shown in Fig 3 which illustrate the presence of acetylinic framework. Drug efficacy is
strongly dependent on time. The efficiency of all the synthesized compounds increased at
48hrs incubation as shown in Fig 3.
120
G.lambia
100
80
60
G. lamblia IC50(µM) 24hrs
G. lamblia IC50(µM) 48hrs
40
20
0
3a
3b
3c
3d
3e
3f
3g
3h
Met
Comp. ID
Fig 3: Anti parasitic activity of compounds(3a-3h) against G. lambia at 24hrs. and 48
hrs.
Antimicrobial activity
The antibacterial activity was categorized as highly active (>14 mm), moderately active (10-
14 mm) and slightly active (6-10 mm) and less than 5 mm was taken as inactive[26]. It is
revealed from table that 3b, 3c, 3d, 3e, 3h antipyrine acetylinic derivatives exhibit moderate
activity as shown in table 4.
Table 4: Zone of inhibition (mm) of compounds (3a-3h ) against various bacterial strain
Sr.no.
3a
3b
3c
3d
Staphylococcus aureus Bacillus subtillus Vibrio cholera E.coli
-
-
-
-
-
9
-
-
-
9
11
-
-
9
-
-
9
-
9
9
9
-
3e
3f
-
15

3g
3h
-
-
-
-
-
-
9
9
compounds 3c with acetylinic group at para position are found to be the most potent against S.
aureus. Bis Acetylinic antipyrine 3h is the most effective ones against E. coli bacterial strain
with IC50 value of 0.29 mM. The significant activity is shown by compound 3h which is a
bis-acetylinic compound with IC50 value of 0.32 mM against Bacillus subtillus strain and 0.3
mM for vibrio cholera. Acetylinic antipyrine 3e with methoxy group displayed dignified
potency with IC50 value of 0.33 mM against Bacillus subtillus and 0.31mM against vibrio
cholera as shown in Fig 4.
0.35
0.3
0.25
0.2
Staphylococcus aureus
Bacillus subtillus
Vibrio cholera
E.coli
0.15
0.1
0.05
0
3a
3b
3c
3d
3e
3f
3g
3h
Comp. ID
Fig 4: In vitro antibacterial activity of compounds (3a-3h) against bacterial strains of
using microbroth dilution assay (MDA)
4. Result and Discussion:
4.1 Spectroscopic analyses
16

IR spectra
IR spectra of compounds displayed a stretching band at 1628–1648 cm-1 that assigned to
antipyrine ν (C=O) stretching vibrations of carbonyl bond. Since frequency of carbonyl group
appears at 1700 cm-1 around but due to conjugation in the ring, this appears in the region
-1
1628-1648 cm . The band around 2100 cm-1and 3300 cm-1 in antipyrine functionalized
alkyne 3a-3h confirms the formation of C≡C and C≡CH respectively. Moreover, stretching
vibrations of –C=N appear in the range of 1570-1600 cm-1 .
NMR spectra
In the 1H spectra of all compounds, a singlet in the region δ = 9.73–10.45 ppm assigned to
imine proton. Alkynyl proton (C≡CH) for 3a–3h appears around δ = 2.40–2.60 ppm, OCH2
proton which linked to alkynyl group appear as doublet at 4.75-4.92 ppm. The aromatic ring
protons were appeared as multiplets in the region δ~ 8.87–6.74 ppm. Moreover, in case of 3e
and 3f, the –OCH3 protons displayed as singlet in the region δ~3.90 and 3.94 ppm
respectively.
In 13C NMR spectra, for carbonyl carbon appears in the region δ ~ 161.3 ppm due conjugation
within the pyrazole ring and spectra of all other compounds, the aromatic carbon displayed in
the region δ ~ 158.2-120.0 ppm. Two carbons of alkynyl moiety 3a-3h observed in the range
of δ ~ 72.1-77.2ppm. Two methyl carbon allied to pyrazole ring appear at δ ~ 10.0 and 36.0
(attached to nitrogen).
4.2. X-ray structure determination
Sr.no Crystal Structure
Lattice arrangement
17

3a
3b
3c
3e
18

3f
3g
3h
Fig 5: Ball and stick diagram of molecule 3a, 3b, 3c, 3e, 3f, 3g, 3h with colour code as
grey: C, red: oxygen, Blue: Nitrogen, and sky blue: hydrogen and their lattice
arrangement.
The molecule 3a crystalizes in triclinic P-1 space group. The asymmetric unit contain one
molecule per unit cell. The central pyrazolidin moiety along with aromatic group’s forms
planar arrangement with a perpendicular position to the acetylene ether group. Two types of
weak interactions of C ̶H···O and π···π interactions facilities the formation a small cavity like
arrangement favouringπ···π interactions between two aromatic groups along bc-plane. The
molecule 3b has almost similar framework structureand only differs by the position of
acetylene ether group at meta position as compared to ortho position for 3a. The molecule 3b
crystalizes in P 21/n space group of monoclinic system. The asymmetric unit contain one
19

molecule per unit cell. The central pyrazolidin moiety along with aromatic group’s forms
planar arrangement with a perpendicular position to the acetylene ether group. Two weak
interactions of C ̶ H···O type between carbonyl group (O) of pyrazolidin and H-atoms of
arromatic and CH2 group helps a head-tail alignment along ac-plane and with an overall layer
like arrangement. The molecule 3c crystalizes in triclinic P-1 space group. The asymmetric
unit contain one molecule per unit cell. The central pyrazolidin moiety along with aromatic
group’s forms planar arrangement with a perpendicular position to the acetylene ether group.
In the lattice arrangement there is no strong H-bonds however, the weak interactions of C
H···O type between carbonyl group (O) of pyrazolidin and H-atoms of arromatic and CH2
̶
group favours a head-tail alignment along bc-plane and with an overall zig-zag arrangement.
The molecule 3e has an additional methyl ether group attached ortho position to the acetylene
ether group as compared to 3a and 3b. The molecule 3e crystalizes in P 21/c space group of
monoclinic system. The asymmetric unit contain one molecule per unit cell. Weak
interactions of C ̶ H···O, C ̶ H···π type favours layer like alignment along ac-plane where all
acetylene ether group part of one layer and aromatic units of other layer. The molecule 3f has
almost similar structure with that of 3e and only differ by the position of ether groups i.e at
meta and para positions as compared to 3e. The molecule 3f crystalizes in P 21/c space group
of monoclinic system. The asymmetric unit contain two molecules per unit cell. Weak
interactions of C ̶ H···O, C ̶H···π type favours layer like alignment along bc-plane where two
molecules are cluster together either in their un-substituted aromatic (head) end or ether
substituted end (tail) position. The molecule 3g has a naphthalene group attached with an
acetylene ether moiety at ortho position as compared to 3a and 3b. The molecule 3g
crystalizes in P -1 space group of triclinic system. The asymmetric unit contain one molecule
per unit cell. Only one weak interactions of C ̶ H···π type favours layer ribbon like alignment
along ac-plane. The molecule 3h has a similar structural framework as 3e where the two
substituents are acetylene ether type moiety at meta position to each other. The molecule 3h
crystalizes in I2/a space group of monoclinic system. The asymmetric unit contain one
molecule per unit cell. One of the acetylene group anchoring perpendicular to aromatic ring to
form a weak interactions of C ̶ H···π (3.665 (3) Å and forms discreet 0D lattice structure
along ac-plane.
Table 5: Crystallographic Data and Refinement Parameters for the molecules (3a,3b,3c)
20

Complexes
CCDC
3a
1860156
3b
1860155
3c
1860153
Empirical
formula
C21 H19 N3 O2
C21 H19 N3 O2
C21 H19 N3 O2
Mr [g mol-1]
T (K)
345.40
293(2)
0.71073
Triclinic
P-1
7.1765(9)
11.1338(9)
11.9194(15)
77.363(9)
78.259(11)
77.683(9)
895.55(18)
2
345.40
293(2)
0.71073
Monoclinic
P121/n1
11.1873(8)
.2144(4)
23.3211(13)
90
98.036(6)
90
1863.8(2)
4
345.40
293(2)
0.71073
Triclinic
P-1
6.9967(1)
9.7597(2)
14.0933(3)
74.729(2)
82.583(2)
82.153(2)
915.32(3)
2
wavelength (A° )
crystal system
space group
a [Å]
b [Å]
c [Å]
α
β
γ
[deg]
[deg]
[deg]
V [Å3]
Z
ρcalc[g cm-3]
ꢀ
1.280
0.084
1.230
0.081
1.253
0.082
[mm-1]
F (000)
364.2
0.33 × 0.22 × 0.2
9657
728.3
364.2
crystal size [mm]3
reflection collected
independent
reflections
0.31 × 0.26 × 0.19 0.30 × 0.23 × 0.15
13198
3872
12358
3878
3340
GOF on F2
final R
indices(R1/wR2) [I
> 2σ(I)]
1.0565
0.0473/
0.1157
1.11
0.0692/
0.2298
1.0188
0.0476/
0.1233
R indices (R1/wR2)
all data
0.0655/
0.1287
0.1128/
0.3118
0.0653/
0.1358
Table 6: Crystallographic Data and Refinement Parameters for the molecules (3e, 3f, 3g,
3h).
Complexes
CCDC
3e
1860154
3f
3g
3h
1860159
1860157
1860158
Empirical
formula
C22 H21 N3 O3
C44 H37.5 N6 O6 C25 H21 N3 O2
C24 H21 N3 O3
Mr [g mol-1]
T (K)
375.43
293(2)
0.71073
Monoclinic
P121/c1
746.32
99(3)
395.46
99(3)
399.45
99(3)
1.54184
Monoclinic
I12/a1
wavelength (A° )
crystal system
space group
a [Å]
1.54184
Monoclinic
P121/c1
1.54184
Triclinic
P-1
7.5275(3)
9.4115(2)
22.6184(10)
7.4281(3)
15.7933(7)
7.4190(3)
b [Å]
18.6777(12)
10.1999(2)
21

c [Å]
27.8013(11)
90
95.385(4)
90
3891.5(3)
4
1.2738
0.087
11.5282(3)
69.240(2)
84.099(2)
83.363(2)
1025.60(4)
2
27.2477(12)
90
113.106(5)
90
4210.7(4)
8
1.2601
0.085
17.0451(6)
90
106.103(4)
90
1918.82(14)
4
1.299
α
β
γ
[deg]
[deg]
[deg]
V [Å3]
Z
ρcalc[g cm-3]
1.2805
0.083
ꢀ
[mm-1]
0.088
F (000)
792.4
1566.8
416.2
1566.8
crystal size [mm]3
0.25 × 0.15 × 0.15 0.28 × 0.16 × 0.14 0.27 × 0.20 × 0.18
reflection collected
independent
11735
5097
14001
4354
16900
4461
15442
4083
reflections
GOF on F2
1.0010
0.0548/
0.1320
1.0429
0.0683/
0.1796
1.0424
0.0447/
0.1115
1.0166
0.0556/
0.1522
final R
indices(R1/wR2) [I >
2σ(I)]
R indices (R1/wR2) all
data
0.0814/
0.1490
0.0997/
0.2036
0.0673/
0.1246
0.0861/
0.1765
Table 7: Selected Bond Lengths (Å) and bond angles (˚) for the molecules (3a, 3b, 3c, 3e,
3f, 3g, & 3h)
Compound
3a
Length/Å
Length/Å
Length/Å
O23 C6
O23 C24
O20 C10
N8 C11
N8 C7
N9 N13
N9 C14
N9 C10
C24 O23 C6
1.3684(19)
1.432(2)
1.2411(18)
1.398(2)
1.280(2)
1.3966(18)
1.424(2)
C12 C11
C12 C22
C11 C10
C14 C15
O23 C6
O23 C24
O20 C10
N8 C11
1.366(2)
1.485(2)
1.437(2)
1.376(3)
1.3684(19)
1.432(2)
1.2411(18)
1.398(2)
118.04(15)
N9 N13
N9 C14
N9 C10
N13 C12
N13 C21
N13 C21
N8 C7
1.3966(18)
1.424(2)
1.385(2)
1.3498(19)
1.446(2)
1.446(2)
1.280(2)
1.3498(19)
1.385(2)
118.63(13)
N13 C12
C2 C1 C6
Angle/ꢀ
Angle/ꢀ
Angle/ꢀ
C11 C10 N9
C16 C15 C14
C7 N8 C11
120.14(13)
120.28(14)
109.66(12)
125.89(13)
107.32(13)
120.47(13)
C1 C7 N8
120.70(15)
115.44(14)
124.02(16)
120.54(16)
123.65(14)
104.53(14)
119.20(18)
C14 N9 N13
C10 N9 N13
C10 N9 C14
C12 N13 N9
C21 N13 N9
C1 C6 O23
C5 C6 O23
C5 C6 C1
C21 N13 C12 127.15(13)
C11 C12 N13 109.73(13)
C11 C10 O20
N9C10 O20
131.82(16)
22

i-X,-X+Y,-Z
Compound
3b
Length/Å
Length/Å
Length/Å
O20 C13
1.239(3)
1.379(4
1.415(4)
1.381(3
1.279(3)
N9 C14
1.420(3)
1.352(3)
1.443(3)
1.445(4)
1.373(4)
C14 C19
C11 C22
C14 C15
N9 N10
N9 C13
1.376(4)
1.487(4)
1.387(4)
1.410(3)
1.393(3)
O23 C3
O23 C24
N8 C12
N8 C7
N10 C11
N10 C21
C13 C12
C12 C11
Angle/ꢀ
Angle/ꢀ
Angle/ꢀ
C24 O23 C3
C7 N8 C12
C13 N9 N10
C14 N9 N10
C14 N9 C13
118.9(3)
122.1(2)
109.1(2)
120.1(2)
123.5(2)
C11 C12 N8
122.3(2)
C12 C13 O20 131.6(2)
C11 C12 C13 107.4(2)
C12 C11 N10 110.6(2)
C22 C11 N10 121.8(2)
C12 C13 N9
C13 C12 N8
C11 N10 N9
C21 N10 N9
105.2(2)
130.2(2)
107.1(2)
118.7(2)
N9 C13 O20
123.2(2)
C21 N10 C11 126.2(2)
Compound
3c
Length/Å
Length/Å
Length/Å
O20 C10
1.2401(16)
1.3749(18)
1.4256(19)
1.3938(18)
1.2763(18)
1.3916(16)
1.3914(18)
1.4173(19)
1.3471(19)
1.4507(19)
C1 C6
1.386(2)
1.4857(19)
1.368(2)
1.379(2)
1.385(2)
1.373(2)
1.433(2)
1.394(2)
1.458(2)
C19 C18
C15 C16
C18 C17
C17 C16
C14 C15
C6 C5
1.379(3)
1.367(3)
1.374(3)
1.360(3)
1.378(2)
1.381(2)
1.446(3)
1.157(3)
1.37(2)
O23C4
C12 C22
C2 C3
O23 C24
N8 C11
N8 C7
C4 C5
C4 C3
N9 N13
N9 C10
N9 C14
N13 C12
N13 C21
C14 C19
C11 C10
C1 C2
C24 C25
C25 C26
C11 C12
C1 C7
Angle/ꢀ
Angle/ꢀ
Angle/ꢀ
120.90(15)
120.68(16)
122.18(14)
119.00(14)
120.17(14)
112.18(14)
177.4(2)
C24 O23 C4
C7 N8 C11
118.25(12)
120.19(13)
109.47(12)
122.09(12)
124.13(12)
107.61(11)
121.04(13)
128.37(13)
122.33(13)
129.87(12)
C11 C10 N9
C3 C2 C1
104.89(11)
121.30(14)
122.15(14)
125.00(14)
114.96(13)
119.99(14)
118.39(15)
121.68(13)
128.36(14)
122.65(13)
C15 C14 N9
C15 C14 C19
C5 C6 C1
C10 N9 N13
C14 N9 N13
C14 N9 C10
C12 N13 N9
C21 N13 N9
C21 N13 C12
C12 C11 N8
C10 C11 N8
C1 C7 N8
C5 C4 O23
C3 C4 O23
C3 C4 C5
C6 C5 C4
C4 C3 C2
C25 C24 O23
C26 C25 C24
C18 C19 C14
C16 C15 C14
C17 C18 C19
C19 C14 N9
C22 C12 N13
C22 C12 C11
N9 C10 O20
119.27(19)
119.2(2)
119.8(2)
Compound
3e
Length/Å
Length/Å
Length/
Å
O23 C6
1.381(2)
C11 C12
1.364(2)
C6 C1
1.391(2)
23

O23 C24
O20 C10
O27 C5
O27 C28
N9 N13
N9 C10
N9 C14
1.440(2)
1.230(2)
1.359(2)
1.426(2)
1.404(2)
1.404(2)
1.418(2)
C14 C15
C14 C19
C5 C4
1.377(2)
1.385(3)
1.382(3)
0.9300
1.376(3)
1.396(2)
1.278(2)
C6 C5
1.404(2)
1.463(2)
1.398(3)
1.435(2
0.9300
C1 C7
C1 C2
C15 H15
C15 C16
N8 C11
N8 C7
C10 C11
C7 H7
N13 C12
N13 C21
1.366(2)
1.462(2)
Angle/ꢀ
Angle/ꢀ
Angle/ꢀ
C24 O23 C6
C28 O27 C5
C10 N9 N13
C14 N9 N13
C14 N9 C10
C11 N8 C7
C12 N13 N9
Compound
3g
117.43(14)
117.24(17)
109.08(14)
120.06(14)
122.65(15)
120.87(16)
107.09(13)
N9 C10 O20
C11 C10 O20
C11 C10 N9
C1 C7 N8
123.17(16)
132.03(16)
104.76(15)
120.55(17)
120.75(16)
120.79(16)
C21 N13 N9
C21 N13 C12
C1 C6 O23
C7 C1 C6
117.83(14)
123.80(16)
118.21(15)
120.03(16)
118.83(16)
121.14(16)
C5 C6 O23
C5 C6 C1
C2 C1 C6
C2 C1 C7
Length/Å
Length/Å
Length/Å
O24 C14
O27 C2
1.2351(16)
1.3751(17)
1.4279(18)
1.4013(16)
1.4027(18
1.4315(18)
1.4006(17)
1.2789(17)
O24 C14
O27 C2
1.2351(16)
1.3751(17)
1.4279(18)
1.4013(16)
1.4027(18)
1.4315(18)
1.4006(17)
1.384(2)
N17 C16
N17 C25
C15 C16
C15 C14
C16 C26
C1 C11
1.3579(18)
1.4514(19)
1.3669(19)
1.4378(19)
1.482(2)
O27 C28
O27 C28
N13 N17
N13 C14
N13 C18
N12 C15
C1 C2
N13 N17
N13 C14
N13 C18
1.4654(19)
1.4266(19)
N12 C15
C1 C10
N12 C11
Angle/ꢀ
Angle/ꢀ
Angle/ꢀ
C28 O27 C2
C14 N13 N17
C18 N13 N17
C18 N13 C14
C11 N12 C15
C10 C1 C11
C2 C1 C11
Compound
3h
119.40(12)
108.59(11)
119.12(11)
120.95(12)
118.31(12)
124.98(13)
116.12(12)
C16 C15 N12
C14 C15 N12
C14 C15 C16
C15 C16 N17
C26 C16 N17
C19 C18 C23
C23 C18 N13
123.29(13)
128.79(12)
107.66(12)
110.31(12)
121.34(13)
120.77(15)
120.62(14)
C16 N17 N13
C25 N17 N13
C25 N17 C16
C26 C16 C15
C2 C1 C10
107.38(11)
117.96(12)
125.60(13)
128.34(14)
118.88(13)
124.04(13)
118.51(14)
C1 C11 N12
C19 C18 N13
Length/Å
Length/Å
Length/Å
O001 C007
O001 C00B
O002 C00E
O003 C00F
O003 C00O
N004 N006
1.3683(19)
1.4222(19)
1.239(2)
N006 C009
N006 C00E
C007 C00G
C007 C00H
C008 C00D
C009 C00L
1.420(2)
1.383(2)
1.389(2)
1.397(2)
1.368(2)
1.378(2)
N004 C008
N004 C00K
N005 C00C
N005 C00D
C008 C00N
1.349(2)
1.456(2)
1.279(2)
1.393(2)
1.482(2)
1.365(2)
1.428(2)
1.3975(19)
Angle/ꢀ
118.90(13)
117.99(14)
106.99(13)
Angle/ꢀ
120.45(16)
119.61(16)
119.93(18)
Angle/ꢀ
109.99(15)
122.09(16)
127.80(17)
C0B O01 C07
C0O O03 C0F
C08 N04 N06
C0L C09 N06
C0M C09 N06
C0M C09 C0L
C0D C08 N04
C0N C08 N04
C0N C08 C0D
24

C0K N04 N06
C0K N04 C08
C0D N05 C0C
C09 N06 N04
118.75(14)
124.93(14)
120.84(15)
120.48(13)
C0B O01 C07
C0O O03 C0F
C0E N06 N04
C0E N06 C09
118.90(13)
117.99(14)
110.11(14)
127.33(15)
C0G C07 O01
C0H C07 O01
C0H C07 C0G
123.12(15)
115.48(14)
121.40(16)
5. Conclusion and Future Prospects:
In Summary, We have outline a unified strategy for fabricate tangible green drug. These drug
candidates were selected by primary screenings and further subjected to in vitro anti-bacterial
as well as anti parasitic activities comparable or even superior to those of approved drugs like
metronidazole. This study sheds light on biological activity of anti pyrine derivatives and
promotes further research for bio-medical applications. In the long term, such materials will
endorse further research in developing new channels for the medication of infectious diseases.
Acknowledgements
We thankfully acknowledge receipt of financial support from the Department of Science and
Technology, New Delhi, India through the scheme DST SERB, DST-PURSE II , CSIR and
DST FIST (X-ray Crystallography).One of the author Pinky Satija would like to thank DST
for Inspire Fellowship in the form of SRF.
Conflict of Interest
There are no conflicts to declare.
Supplementary appendix
CCDC- 1860156 (3a), 1860155 (3b), 1860153 (3c), 1860154 (3e), 1860159 (3f), 1860157
(3g), 1860158 (3h) contain the supplementary crystallographic data for this paper. These data
can be obtained free of charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk.
List of abbreviation :
AAP - Amino antipyrine
Met
-
Metronidazole
25

MW - Microwave
G.lambia - Giardia lambia
E.histolytica - Entamoeba histolytica
Naph- Naphthalene
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Research highlights:
ꢀ By using microwave methodology, a family of Anti pyrine derivatives were synthesized.
This is an amazing quote of stitching anti pyrine nucleus to acetylene moiety.
ꢀ The protocol afforded the excellent yield in short span of time under eco friendly
conditions.
ꢀ All the synthesized compounds are characterized by IR, NMR (1H, 13C), and Single X-ray
diffraction.
ꢀ The hybrid molecules screened for their physicochemical properties using mol inspiration
software and ADMET web server.
ꢀ This work articulates a new possible scaffold for drug discovery against protozoal pathogens
(Entamoeba histolytica and Giardia lamblia) and bacterial strain (Staphylococcus aureus,
Vibrio cholera E.coli, Bacillus subtillus) and this low price, versatile drug is hopefully
leads to eventual elimination of parasitic disease from human population.