A facile greener synthesis, antimicrobial evaluation and molecular modelling of new 4-aryl-2-(3-(2-(trifluoromethyl)phenyl)-1,8-naphthyridin-2-yl)phthalazin-1(2H)-one derivatives

Article abstract of DOI:10.1007/s11164-018-03711-1

Abstract: The synthesis of 4-aryl-2-(3-(2-(trifluoromethyl)phenyl)-1,8-naphthyridin-2-yl)phthalazin-1(2H)-ones was performed by cyclization of 2-hydrazinyl-3-(2-(trifluoromethyl)phenyl)-1,8-naphthyridine with 2-aroylbenzoic acids in ethanol containing a catalytic amount of concentrated sulfuric acid under solid state conditions. All these synthesized compounds (8a–h) were screened for their in vitro antibacterial activity against gram-positive bacteria such as (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli) and also evaluated for their antifungal activity against Aspergillus Niger and Helmenthosphorium oryzae fungal strains. Some of the products demonstrate good antibacterial activity and moderate antifungal activity. In predominantly, 8b, 8d, 8g, and 8h compounds showed good to excellent antibacterial and antifungal activities. The antimicrobial activity of the compound 8 was further investigated with the help of in LibDock score docking study to predict the active sites. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11164-018-03711-1

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-018-03711-1
A facile greener synthesis, antimicrobial
evaluation and molecular modelling
of new 4‑aryl‑2‑(3‑(2‑(trifuoromethyl)
phenyl)‑1,8‑naphthyridin‑2‑yl)phthalazin‑1(2H)‑one
derivatives
Boda Sakram1 · Dharavath Ravi1 · Mutyala Raghupathi2 ·
Boda Sathish Kumar1 · P. V. Anantha Lakshmi1
Received: 22 June 2018 / Accepted: 14 December 2018
© Springer Nature B.V. 2019
Abstract
The synthesis of 4-aryl-2-(3-(2-(trifuoromethyl)phenyl)-1,8-naphthyridin-2-yl)
phthalazin-1(2H)-ones was performed by cyclization of 2-hydrazinyl-3-(2-
(trifuoromethyl)phenyl)-1,8-naphthyridine with 2-aroylbenzoic acids in ethanol
containing a catalytic amount of concentrated sulfuric acid under solid state con-
ditions. All these synthesized compounds (8a–h) were screened for their in vitro
antibacterial activity against gram-positive bacteria such as (Staphylococcus aureus)
and gram-negative bacteria (Escherichia coli) and also evaluated for their antifun-
gal activity against Aspergillus Niger and Helmenthosphorium oryzae fungal strains.
Some of the products demonstrate good antibacterial activity and moderate antifun-
gal activity. In predominantly, 8b, 8d, 8g, and 8h compounds showed good to excel-
lent antibacterial and antifungal activities. The antimicrobial activity of the com-
pound 8 was further investigated with the help of in LibDock score docking study to
predict the active sites.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1116
4-018-03711-1) contains supplementary material, which is available to authorized users.
*
Boda Sakram
bschemou@gmail.com
1
2
Department of Chemistry, Osmania University, Tarnaka, Hyderabad, Telangana 500007, India
Department of Chemistry, CSIR-Indian Institute of Chemical Technology, Tarnaka 500007,
India
Vol.:(0123456789)
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B. Sakram et al.
Graphical abstract
Keywords Greener chemistry · 1,8-Naphthyridines · Biological activity · Molecular
docking
Introduction
1,8-Naphthyridine has produced great interest in organic synthetic and medicinal
felds. These derivatives have an exceptionally broad spectrum of biological activi-
ties [1–4] due to their pronounced antitumor [5], anti-HIV [6], antimalarial [7], oste-
oporosis [8], and antibacterial activities [9]. The 1,8-naphthyridine derivatives are
also inhibitors of acid secretion in the stomach, referred to as gastric antisecretory
properties [10], and inhibitors of platelet aggregation [11, 12]. Nalidixic acid and
gemifoxacin possess strapping antimicrobial activity and are used efectively for the
treatment of urinary tract infections [13, 14]. A number of other remarkable applica-
tions have also been reported in the literature; for example, the compounds have been
patented as herbicides, fungicides, insecticides, anxiolytic, bactericides, and antihy-
pertensives [15–17]. In addition, heterocyclic complexes containing the phthalazine
and phthalazinone skeletons are present in many compounds isolated from natural
substances, with a wide spectrum of biological activities [18–21] due to their excep-
tionally strong efcacy, such as anticancer [22], antimicrobial [23], tuberculostatic
[24], anticonvulsant [25, 26], and antiinfammatory activities [27]. Azelastine is an
antiallergic agent and a relevant member of this family of compounds that shows a
1 3

A facile greener synthesis, antimicrobial evaluation and…
fair bronchodilatory activity, useful for treating asthma [28], and a plasma glucose-
lowering activity [29].
In recent years, many articles have been published in the area of solid-state
organic synthesis [30–33]. Solid-state conditions have many advantages due to high
purity of the products, simpler reactors, dramatically reduced reaction times, ef-
cient workup procedures, easy reusability, and enhanced product purities; this is
achieved by reducing unwanted side reactions observed in conventional synthetic
methods [34, 35]. In view of this and in continuation of our interest in solid-state
organic reactions on 1,8-naphthyridine derivatives [36, 37], we report herein an
operationally simple, greener and fast methodology for the synthesis of 4-aryl-2-(3-
(2-(trifuoromethyl)phenyl)-1,8-naphthyridin-2-yl)phthalazin-1(2H)-ones in solid-
state grinding methods. However, the majority of these reactions were successfully
performed with 2-hydrazinyl-3-(2-(trifuoromethyl)phenyl)-1,8-naphthyridine and
2-aroyl benzoic acids and their further modifcations.
Experimental
Material and apparatus
All the reactants were used as purchased from Aldrich Chemical Company. All the
melting points were resolute in open capillary tubes using a Cintex apparatus and
were uncorrected. The purity of the compounds was checked by thin layer chro-
matography (TLC) was performed on Merck 60 F-254 silica gel plates, using ethyl
acetate:n-hexane (20:80) as an eluent. Infrared (IR) spectra were recorded on a Per-
kin-Elmer spectrum BX series Fourier transform IR (FT-IR) spectrometer (cm−1),
using KBr discs. The proton nuclear magnetic resonance (1H NMR) spectra were
indicated in chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6) on a
Varian Gemini 400 MHz spectrometer (chemical shifts in δ ppm) using tetramethyl-
silane (TMS) as an internal standard, and mass spectra (MS) were measured on a
Jeol JMSD-300 spectrometer. The biological assay of the synthesized compounds
was tested against human pathogenic bacteria (E. coli & S. aureus) and fungal
species (A. niger & H. oryzae) were obtained from the Deutsche Sammlung von
Mikroorganismen und Zellkulturen. For microbiological, Luria-Bertani (LB) broth
(Lennox) powder was purchased from Oxoid. Selective dishes were used to culture
each strain. The dishes were stored in a refrigerator at 37 °C for 24–48 h. With a dif-
fuse light, a clear, well-defned zone of inhibition will appear at diferent concentra-
tions (250 and 500 ppm). Results were recorded using Chi-square tests with Yates’
correction of diferent compounds.
General procedure for the synthesis of 3‑(2‑(trifuoromethyl)
phenyl)‑1,8‑naphthyridin‑2‑amine (3)
A mixture of 2-aminonicotinaldehyde (1, 1 mmol, 122.12 mg) and 2-(2-(trifuoro-
methyl)phenyl)acetonitrile (2, 1 mmol, 185.15 mg) in KOH (10%) was heated under
neat conditions. On completion of the reaction, as monitored by TLC, the reaction
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B. Sakram et al.
mixture was cooled and treated with chilled water. The solid that precipitated was
fltered, washed with water, and recrystallized from ethanol to give compound 3
with good yield.
3‑(2‑(trifuoromethyl)phenyl)‑1,8‑naphthyridin‑2‑amine (3) Pale yellow solid; yield:
91%; melting point (mp): 151–153 °C; FT-IR (KBr, ν, cm−1): 3483 (NH2), 3059
(C–H), 1637 (C–NH2), 1595 (C=N), 1558, 1429 (C=C), 1309, 1165, 1112 (C–F);
1H-NMR (400 MHz, DMSO-d6, δ, ppm): 8.74 (dd, 1H, J=4.5, 2.0 Hz, Ar–H),
8.10 (dd, 1H, J=7.9, 2.0 Hz, Ar–H), 7.89 (t, 1H, J=7.4 Hz, Ar–H), 7.77 (d, 1H,
J=7.5 Hz, Ar–H), 7.66 (s, 1H, Ar–H), 7.61 (t, 2H, J=7.5 Hz, Ar–H), 7.20 (dd,
1H, J=7.8, 4.4 Hz, Ar–H), 6.33 (s, 2H, NH2); MS [electrospray ionization [EI),
m/z (%)]: 290 (M+H) +. Anal. calcd. for C15H10F3N3: C, 62.28; H, 3.48; N, 14.53%.
Found: C, 62.01; H, 3.40; N, 14.65%.
General procedure for the synthesis of 3‑(2‑(trifuoromethyl)
phenyl)‑1,8‑naphthyridin‑2(1H)‑one (4)
To a cold solution of 3 (1 mmol, 289.26 mg) in 2 M HCl (25 mL) was added NaNO2
solution (1 mmol in 25 mL of water) and the reaction mixture was stirred at room
temperature for 1 h and treated with chilled water. The solid thus obtained was fl-
tered, washed with water, and recrystallized from ethanol to aford 4.
3‑(2‑(trifuoromethyl)phenyl)‑1,8‑naphthyridin‑2(1H)‑one (4) Colorless solid; yield:
89%; mp: 186–188 °C; FT-IR (KBr, ν, cm−1): 3238 (NH), 3028 (C–H), 1654
1
(ring C=O), 1625, 1604 (C=N), 1570, 1427 (C=C), 1313, 1107 (C–F); H-NMR
(400 MHz, DMSO-d6, δ, ppm): 12.40 (s, 1H, NH), 8.16 (dt, 1H, J=14.2, 7.1 Hz,
Ar–H), 7.84 (d, 1H, J=7.7 Hz, Ar–H), 7.75 (t, 1H, J=7.5 Hz, Ar–H), 7.69–7.58
(m, 2H, Ar–H), 7.49–7.42 (m, 2H, Ar–H), 7.29 (dd, 1H, J=7.7, 4.8 Hz, Ar–H); MS
[EI, m/z(%)]: 291 (M+H) +. Anal. calcd. for C15H9F3N2O (%): C, 62.07; H, 3.13; N,
9.65. Found (%): C, 62.01; H, 3.19; N, 9.93.
General procedure for the synthesis of 2‑chloro‑3‑(2‑(trifuoromethyl)
phenyl)‑1,8‑naphthyridine (5)
A solution of 4 (1 mmol, 290. 25 mg) in POCl3 (15 mL) and N,N-diethylamine
(5 mL) was heated in an oil bath at 100 °C and stirred for 4 h. After the reaction
mixture was cooled to room temperature, the resulting reaction mixture was dis-
solved in ethyl acetate (50 mL) and washed with H2O (2×75 mL) and saturated
NaHCO3. The organic phase was dried (Na2SO4) and evaporated to dryness. The
crude product was fltered, washed with water, and recrystallized from n-pentane to
furnish 5.
2‑chloro‑3‑(2‑(trifuoromethyl)phenyl)‑1,8‑naphthyridine (5) White solid; yield:
85%; mp: 181–183 °C; FT-IR (KBr, ν, cm−1): 3039 (C–H), 1604 (C=N), 1548
(C=C), 1311, 1170, 1031 (C–F), 769, 704 (C–Cl); 1H-NMR (400 MHz, DMSO-d6,
1 3

A facile greener synthesis, antimicrobial evaluation and…
δ, ppm): 9.18 (dd, 1H, J=4.2, 2.0 Hz, Ar–H), 8.64–8.55 (m, 2H, Ar–H), 7.94 (t,
1H, J=9.0 Hz, Ar–H), 7.85 (t, 1H, J=7.5 Hz, Ar–H), 7.80–7.74 (m, 2H, Ar–H),
+
7.63 (d, 1H, J=7.5 Hz, Ar–H); MS [EI, m/z (%)]: 309 (M+H) . Anal. calcd. for
C15H8ClF3N2 (%): C, 58.36; H, 2.61; N, 9.08. Found (%): C, 58.45; H, 2.75; N, 9.31.
General procedure for the synthesis of 2‑hydrazinyl‑3‑(2‑(trifuoromethyl)
phenyl)‑1,8‑naphthyridine (6)
A mixture of 5 (1 mmol, 308.69 mg) and hydrazine hydrate (1 mol%) in ethanol
(20 mL) was refuxed on a water-bath for 3.5 h and cooled. The solid thus separated
was fltered and recrystallized from ethanol to give 6.
2‑hydrazinyl‑3‑(2‑(trifuoromethyl)phenyl)‑1,8‑naphthyridine (6) Pale yellow solid;
yield: 86%; mp: 221–223 °C; FT-IR (KBr, ν, cm−1): 3327, 3275, 3192 (–NHNH2),
3032 (C–H), 1602 (C–NHNH2), 1552 (C=N), 1514, 1456 (C=C), 1398, 1234, 1047
(C–F); 1H-NMR (400 MHz, DMSO-d6, δ, ppm): 9.19 (dd, 1H, J=4.2, 2.1 Hz, NH),
8.68–8.52 (m, 2H, Ar–H), 7.95 (d, 1H, J=7.8 Hz, Ar–H), 7.84 (t, 1H, J=7.6 Hz,
Ar–H), 7.81–7.72 (m, 3H, Ar–H), 7.64 (d, 1H, J=7.8 Hz, Ar–H) 3.35 (s, 2H, NH2);
MS [EI, m/z(%)]: 305 (M+H) +. Anal. calcd. for C15H11F3N4 (%): C, 59.21; H, 3.64;
N, 18.41. Found (%): C, 59.40; H, 3.54; N, 18.63.
General experimental procedure for the preparation
of 4‑aryl‑2‑(3‑(2‑(trifuoromethyl)phenyl)‑1,8‑naphthyridin‑2‑yl)
phthalazin‑1(2H)‑ones (8a–8h)
The mixture of compounds 7a–h (1 mmol) and 2-hydrazinyl-3-(2-(trifuoromethyl)
phenyl)-1,8-naphthyridine 6 (1 mmol, 304.30 mg) in the presence of sulfuric acid
(15 mL) was ground in a mortar and pestle at 10–20-min intervals for a predeter-
mined time frame (Table 1). After the completion of the reaction as monitored
by TLC, the mixture was poured into water. The solid obtained was collected and
washed with water. It was dried and recrystallized from methanol to give 8a–h.
Characterization data of compounds 8a–8h
4‑phenyl‑2‑(3‑(2‑(trifuoromethyl)phenyl)‑1,8‑naphthyridin‑2‑yl)phthalazin‑1(2H)‑one
(8a) FT-IR (KBr, ν, cm−1): 3071 (C–H), 1701, 1664 (C=O), 1581 (C=N), 1487,
1
1436 (C=C), 1305, 1232, 1122 (C–F); H-NMR (400 MHz, DMSO-d6, δ, ppm):
8.73 (dd, 2H, J=4.3, 1.8 Hz, Ar–H), 8.11 (dd, 2H, J=7.8, 1.7 Hz, Ar–H), 7.89 (s,
2H, Ar–H), 7.61–7.50 (m, 6H, Ar–H), 7.20 (dd, 2H, J=7.8, 4.4 Hz, Ar–H), 6.53
(s, 3H, Ar–H); 13C-NMR (400 MHz, CDCl3-d6, δ, ppm): 166.42, 164.18, 159.89,
157.67, 144.39, 141.32, 136.31, 135.28, 133.54, 132.77, 131.53, 130.82, 129.62,
128.62 (2C), 127.91, 126.51, 125.68, 125.25, 124.22 (2C), 123.45, 121.55 (2C),
119.72, 118.63, 117.70, 117.01, 112.53; MS [EI, m/z (%)]: 495 (M+H)+. Anal.
calcd. for C29H17F3N4O (%): C, 70.44; H, 3.47; N, 11.33. Found (%): C, 70.16; H,
3.35; N, 11.51.
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B. Sakram et al.
Table 1 Reaction of various 2-aroylbenzoic acids with 2-hydrazinyl-3-(2-(trifuoromethyl)phenyl)-
1,8-naphthyridine
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A facile greener synthesis, antimicrobial evaluation and…
4‑(4‑chlorophenyl)‑2‑(3‑(2‑(trifluoromethyl)phenyl)‑1,8‑naphthyridin‑2‑yl)phthala-
zin‑1(2H)‑one (8b) FT-IR (KBr, ν, cm−1): 3059 (C–H), 1714, 1670 (C=O),
1
1581 (C=N), 1489, 1450 (C=C), 1352, 1251, 1116 (C–F), 765,721 (C–Cl); H-
NMR (400 MHz, DMSO-d6, δ, ppm): 8.53 (d, 2H, J=3.2 Hz, Ar–H), 8.25–8.12
(m, 4H, Ar–H), 7.69 (dd, 7H, J=30.2, 8.5 Hz, Ar–H), 7.50–7.41 (m, 1H, Ar–H),
7.28 (dd, 2H, J=7.6, 4.8 Hz, Ar–H); 13C-NMR (400 MHz, CDCl3-d6, δ, ppm):
164.88, 160.26, 157.38, 145.73, 144.68, 142.25, 136.10, 134.79, 133.43, 132.43,
129.66, 128.67, 127.74, 126.72 (2C), 125.85, 124.39, 123.91 (2C), 123.52, 122.33,
121.19, 119.86, 119.25 (2C), 118.36, 116.68, 113.78, 112.25; MS [EI, m/z(%)]: 529
(M+H)+. Anal. calcd. for C29H16ClF3N4O: C, 65.85; H, 3.05; N, 10.59. Found (%):
C, 65.62; H, 3.19; N, 10.52.
4‑(4‑fluorophenyl)‑2‑(3‑(2‑(trifluoromethyl)phenyl)‑1,8‑naphthyridin‑2‑yl)phthala-
zin‑1(2H)‑one (8c) FT-IR (KBr, ν, cm−1): 3061 (C–H), 1714, 1672 (C=O), 1581,
1
1552 (C=N), 1487, 1448 (C=C), 1350, 1251, 1197 (C–F); H-NMR (400 MHz,
DMSO-d6, δ, ppm): 8.52 (d, 2H, J=3.6 Hz, Ar–H), 8.18 (d, 4H, J=8.7 Hz, Ar–H),
7.70 (dd, 7H, J=29.8, 8.2 Hz, Ar–H), 7.45 (s, 1H, Ar–H), 7.33–7.20 (m, 2H,
Ar–H); 13C-NMR (400 MHz, CDCl3-d6, δ, ppm): 163.69, 160.69, 158.45, 157.02,
151.27, 145.93, 144.74, 139.72, 137.54, 135.74, 133.11, 131.57, 129.45 (2C),
128.58, 128.00, 126.90, 125.32, 124.27 (2C), 123.44, 121.95, 120.01, 118.95 (2C),
+
117.63, 116.32, 115.51, 111.00; MS [EI, m/z(%)]: 513 (M+H) . Anal. calcd. for
C29H16F4N4O: C, 67.97; H, 3.15; N, 10.93. Found (%): C, 67.84; H, 3.26; N, 10.90.
4‑(4‑bromophenyl)‑2‑(3‑(2‑(trifluoromethyl)phenyl)‑1,8‑naphthyridin‑2‑yl)phthala-
zin‑1(2H)‑one (8d) FT-IR (KBr, ν, cm−1): 3071 (C–H), 1710, 1667 (C=O),
1
1580 (C=N), 1494, 1450 (C=C), 1350, 1248 (C–F), 719,192 (C–Br); H-NMR
(400 MHz, DMSO-d6, δ, ppm): 8.52 (s, 2H, Ar–H), 8.22–8.04 (m, 3H, Ar–H), 7.74
(d, 4H, J=6.5 Hz, Ar–H), 7.54–7.38 (m, 5H, Ar–H), 7.27 (s, 2H, Ar–H); MS [EI,
m/z(%)]: 574 (M+H)+. Anal. calcd. for C29H16BrF3N4O (%): C, 60.75; H, 2.81; N,
9.77. Found (%): C, 60.69; H, 2.85; N, 9.92.
4‑(3,4‑difuorophenyl)‑2‑(3‑(2‑(trifuoromethyl)phenyl)‑1,8‑naphthyridin‑2‑yl)phthala-
zin‑1(2H)‑one (8e) FT-IR (KBr, ν, cm−1): 3069 (C–H), 1703, 1660 (C=O), 1579
1
(C=N), 1510, 1436 (C=C), 1305, 1123, 1013 (C–F); H-NMR (400 MHz, DMSO-
d6, δ, ppm): 8.57 (d, 1H, J=3.5 Hz, Ar–H), 8.17 (d, 2H, J=7.3 Hz, Ar–H), 7.92 (s,
2H, Ar–H), 7.84 (d, 2H, J=7.7 Hz, Ar–H), 7.75 (t, 2H, J=7.4 Hz, Ar–H), 7.65 (t,
2H, J=7.5 Hz, Ar–H), 7.47 (d, 2H, J=7.5 Hz, Ar–H), 7.30 (dd, 2H, J=7.5, 4.8 Hz,
Ar–H); MS [EI, m/z(%)]: 531 (M+H)+. Anal. calcd. for C29H15F5N4O (%): C, 65.66;
H, 2.85; N, 10.56. Found (%): C, 65.90; H, 2.81; N, 10.64.
4‑([1,1′‑biphenyl]‑4‑yl)‑2‑(3‑(2‑(trifluoromethyl)phenyl)‑1,8‑naphthyridin‑2‑yl)
phthalazin‑1(2H)‑one (8f) FT-IR (KBr, ν, cm−1): 3053 (C–H), 1720, 1680
1
(C=O), 1598, 1554 (C=N), 1450 (C=C), 1359, 1224, 1197, 1022 (C–F); H-
NMR (400 MHz, DMSO-d6, δ, ppm): 8.78–8.66 (m, 2H, Ar–H), 8.60–8.51 (m,
3H, Ar–H), 8.24–8.09 (m, 4H, Ar–H), 7.90 (s, 1H, Ar–H), 7.77 (dd, 4H, J=13.0,
1 3

B. Sakram et al.
8.6 Hz, Ar–H), 7.61–7.53 (m, 2H, Ar–H), 7.44 (dd, 4H, J=20.7, 8.5 Hz, Ar–H),
7.21 (dd, 1H, J=7.8, 4.4 Hz, Ar–H); MS [EI, m/z(%)]: 571 (M+H)+. Anal. calcd.
for C35H21F3N4O (%): C, 73.68; H, 3.71; N, 9.82. Found (%): C, 73.80; H, 3.77; N,
9.59.
4‑(4‑hydroxyphenyl)‑2‑(3‑(2‑(trifuoromethyl)phenyl)‑1,8‑naphthyridin‑2‑yl)phthala-
zin‑1(2H)‑one (8g) FT-IR (KBr, ν, cm−1): 3445, 3321 (C–OH), 3070 (C–H), 1703,
1
1666 (C=O), 1583 (C=N), 1516, 1436 (C=C), 1305, 1120, 1013 (C–F); H-NMR
(400 MHz, CDCl3-d6, δ, ppm): 11.20 (s, 1H, OH), 8.70 (dd, 1H, J=4.8, 1.6 Hz,
Ar–H), 8.60 (d, 1H, J=2.3 Hz, Ar–H), 7.97 (dd, 2H, J=14.7, 5.2 Hz, Ar–H), 7.81
(s, 1H, Ar–H), 7.77–7.69 (m, 5H, Ar–H), 7.25–7.21 (m, 2H, Ar–H), 7.16–7.12 (m,
4H, Ar–H); MS [EI, m/z(%)]: 511 (M+H)+. Anal. calcd. for C29H17F3N4O2 (%): C,
68.23; H, 3.36; N, 10.98. Found (%): C, 68.20; H, 3.45; N, 11.08.
4‑(4‑methoxyphenyl)‑2‑(3‑(2‑(trifuoromethyl)phenyl)‑1,8‑naphthyridin‑2‑yl)phthala-
zin‑1(2H)‑one (8h) FT-IR (KBr, ν, cm−1): 3072 (C–H), 1708, 1672 (C=O), 1587
1
(C=N), 1485, 1438 (C=C), 1280, 1244 (C–F), 1195, 1097, 1010 (C–OCH3); H-
NMR (400 MHz, DMSO-d6, δ, ppm): 8.51 (ddd, 2H, J=8.5, 4.6, 1.5 Hz, Ar–H),
8.21–8.11 (m, 3H, Ar–H), 8.07 (s, 1H, Ar–H), 7.90 (d, 1H, J=2.0 Hz, Ar–H),
7.79–7.68 (m, 3H, Ar–H), 7.26 (dt, 3H, J=12.7, 7.6 Hz, Ar–H), 7.01 (d, 3H,
J=8.7 Hz, Ar–H), 3.82 (s, 3H, OCH3); MS [EI, m/z(%)]: 525 (M+H)+. Anal. calcd.
for C30H19F3N4O2 (%): C, 68.70; H, 3.65; N, 10.68. Found (%): C, 68.92; H, 3.81;
N, 10.98.
Results and discussion
Synthesis of compounds
The synthetic route used to synthesize title compounds (8a–h) is depicted in
Scheme 1. The starting material 2-amino-3-(2-(trifuoromethyl)phenyl)-1,8-naph-
thyridine (3) was synthesized easily from 2-aminonicotinaldehyde and 2-(2-(tri-
fuoromethyl)phenyl)acetonitrile, and on treatment with NaNO2, it furnished
3-(2-(trifuoromethyl)phenyl)-1,8-naphthyridin-2(1H)-one (4). Treatment of com-
pound 4 with POCl3 and N,N-diethyl amine under refux aforded 2-chloro-3-
(2-(trifuoromethyl)phenyl)-1,8-naphthyridine (5), which on hydrazinolysis with
hydrazine hydrate in boiling ethanol furnished 2-hydrazinyl-3-(2-(trifuoromethyl)
phenyl)-1,8-naphthyridine (6), in very good yield. In this research, a pyridine-based
2-hydrazinyl-1,8-naphthyridine (6) was used as a suitable intermediate for synthesis
of target compounds.
The 2-aroylbenzoic acids (7a–h) which are required for the synthesis of
phthalazinones derivatives were obtained from Friedel and Crafts acylation through
a reported method [29], by means of the reaction between isobenzofuran-1,3-dione,
aromatic hydrocarbon, and anhydrous aluminum trichloride, by Friedel and Crafts
and that a good yield depended on the aromatic hydrocarbon. The cyclization to the
1 3

A facile greener synthesis, antimicrobial evaluation and…
F
3C
O
F
3C
H
a
+
b
N
NH2
H
N
N
NH2
2C
CN
1
4
2
3
F
3C
F
3C
c
d
N
N
Cl
N
N
H
O
5
F
3C
O
OH
O
R1
R2
e
+
+
N
N
NH
NH2
R2
O
O
O
R1
6
7(a-h)
aromatic
hydrocarbon
phthalic
anhydride
f
F
7/8a: R
7/8b: R1 H, R
H
2 = Cl
3C
O
1/R2
=
=
=
=
=
=
=
=
R2
R1
7/8c: R1 H, R
F
=
7/8d: R1 H, R2 Br
N
=
=
N
N
N
7/8e: R1 F R 2
F
,
7/8f: R1 H, R2 Ar
=
=
2
7/8g: R1 H, R
OH
7/8h: R1 H, R2
OCH
=
8(a-h)
2
3
Scheme 1 Synthesis of 4-substituted-2-(3-(2-(trifuoromethyl)phenyl)-1,8-naphthyridin-2-yl)phthalazin-
1(2H)-ones (8a–h). Reagents and conditions: (a) 10% KOH; (b) NaNO2, refux for 1 h; (c) POCl3, N,N-
diethylamine, at 100 °C; (d) N2H4.H2O, EtOH; (e) anhydrous AlCl3, room temperature; (f) ethanol, con-
centrated H2SO4, solid state
4-aryl-2-(3-(2-(trifuoromethyl)phenyl)-1,8-naphthyridin-2-yl)phthalazin-1(2H)-one
derivatives (8a–h) bearing a 3-(2-(trifuoromethyl)phenyl)-1,8-naphthyridine moi-
ety was aforded by the successful condensation of the appropriate para- and meta-
substituted 2-aroylbenzoic acids (7a–h) and 2-hydrazinyl-3-(2-(trifuoromethyl)
phenyl)-1,8-naphthyridine in ethanol containing a catalytic amount of sulfuric acid
under grinding conditions. The reactions proceed efciently with excellent yields at
ambient pressure within a short time and the results are shown in Table 1. The pro-
cess is environmentally benign. The reaction is fairly general, facile, and efcient and
structures of the synthesized compounds were established on the basis of elemental
1
analysis and various spectroscopic methods, viz. IR, H NMR and MS. Elemental
analysis (C, H, and N) data were within 0.4% of the theoretical values.
In the IR spectra of compounds 8a–h showed fve bands characteristic of
1,8-naphthyridine containing a phthalazinone moiety out of which one band
1 3

B. Sakram et al.
for an aromatic hydrogen group at 3072–3053 cm−1 and one band for a carbonyl
group (C=O) of phthalazone moiety at 1720–1660 cm−1. Apart from these bands,
a band in the region 1598–1552 cm−1 corresponding to C=N stretching, a band at
1516–1436 cm−1 for the C=C group, and one band for the C–F (1359–1013 cm−1)
group were also observed. For example, the molecular weight for 4-phenyl-2-
(3-(2-(trifuoromethyl)phenyl)-1,8-naphthyridin-2-yl)phthalazin-1(2H)-one
(8a,
+
C29H17F3N4O) was determined as 495 (M+H) by liquid chromatography–tan-
1
dem MS (LC–MS-MS). Its H NMR spectrum appeared in an aromatic region (δ
8.73–6.53).
A probable reaction mechanism for the most interesting synthesis of 4-aryl-
2-(3-(2-(trifuoromethyl)phenyl)-1,8-naphthyridin-2-yl)phthalazin-1(2H)-ones
is
shown in Scheme 2 which follows the condensation between 2-hydrazinyl-3-(2-
(trifuoromethyl)phenyl)-1,8-naphthyridine 6 and 2-aroylbenzoic acids (7a–h)
to give several intermediates which subsequently undergo cyclization to form the
expected products (8a–h) by sulfuric acid and water as a good leaving group.
Biological activity (antibacterial and antifungal activity)
The antimicrobial screening of 8a–h was tested in vitro for their antibacterial activ-
ity against two strains of bacteria [Escherichia coli (gram − ve)] and Staphylococcus
aureus [gram + ve)] at two concentrations (250 and 500 ppm) using the method
of Vincent and Vincent [38]. The same concentrations of 8a–h (250 and 500 ppm)
were screened for their two fungal species (Aspergillus Niger and Helmenthospho-
rium oryzae). Griseofulvin was used as standard according to the Benson technique
[39]. A flter paper disk (5 mm) was transferred into 250-mL fasks containing
20 mL of working volume of tested solution (100 mg/mL). All fasks were auto-
claved for 20 min at 121 °C. LB broth media surfaces were inoculated with two
investigated antibacterial and two antifungal species. The wells were loaded with 50
µL of diferent investigated compounds. DMSO in sterilized distilled water was used
as the control. Plates were incubated at 37 °C and examined for a zone of inhibition
around each disc after 24–48 h where clear. All these compounds showed good inhi-
bition activity. The activity data indicate that all the compounds are active against
both antibacterial and anti-fungal bacteria. The results obtained are presented in
Table 2; it is inferred that the substitution in the aryl ring as well as at the 4-position
of phthalazin-1(2H)-one linked to diferent derivatives exerted signifcant infuence
on the biological screening. It is gratifying to observe that a majority of the com-
pounds (8b, 8d, 8g, and 8h) displayed good biological activity.
Structure–activity relationship (SAR)
The structure–activity relationship (SAR) is a relation between the molecular
structure and the potential antimicrobial efects. The substituents that have thus
far been reported to confer a signifcant amount of specifc antimicrobial activity
on these synthesized compounds structure–activity relationship-1 (SAR-1) and
1 3

A facile greener synthesis, antimicrobial evaluation and…
HO
O
HO
O
O
OH
H
2SO4
R2
-H
R2
SO4
R1
R1
3C
7(a-h)
F
3C
F
HO
O
OH
R2
R1
-H
+
H
N
OH
NH2
N
N
N
N
N
N
H
R2
H
R1
HO2C
6
H
-H
SO4
2SO4
F
3C
F
3C
R2
R1
H
N
N
N
NH
H
2O
N
-H
2O
R1
R2
N
N
N
H
-H
HO2C
H
CO2
H
-H
2SO4
SO4
F
3C
F
3C
R2
R1
R2
R1
N
-H
2O
N
N
N
N
N
N
N
H
O
-H
O
OH2
8(a-h)
7/8a: R
7/8b: R H, R Cl, 7/8c: R H, R F, 7/8d: R H, R Br, 7/8e:
= = = = = =
1 2 1 2
1/R2 H,
=
R1 F, R F, 7/8f: R 1 H, R 2 Ar, 7/8g: R H, R OH, 7/8h: R H, R OCH
=
=
=
=
=
=
=
=
2
1
2
1
2
1
2
3.
Scheme 2 Plausible mechanism for the formation of 4-substituted-2-(3-(2-(trifuoromethyl)phenyl)-
1,8-naphthyridin-2-yl)phthalazin-1(2H)-one derivatives (8a–h) by concentrated sulfuric acid
structure–activity relationship-2 (SAR-2) are highlighted by the colored boxes,
as shown in Fig. 1. 3-(2-(trifuoromethyl)phenyl)-1,8-naphthyridine (SAR-1))
have strong active, compare to SAR-2 devoid of antibacterial activity. SAR-2 was
devoid of antibacterial activity; when SAR-2 was the substituted 4-arylphthala-
zin-1(2H)-one group, the para substitution was found to be superior to meta or
ortho. In the benzene ring, electron-withdrawing groups like 4-Cl (8b) and 4-Br
(8d) exhibited good antibacterial activities. A majority of synthesized compounds
1 3

B. Sakram et al.
Table 2 Antibacterial and antifungal activity of compounds 8a–h
Zone of inhibition (mm)
Compound
Antibacterial activity
Antifungal activity
Escherichia coli
Staphylococcus
aureus
Aspergillus Niger
Helmenthosphorium
oryzae
250 ppm 500 ppm 250 ppm 500 ppm 250 ppm 500 ppm 250 ppm 500 ppm
8a
09
14
08
14
11
10
15
15
20
–
10
15
11
16
15
12
18
17
10
13
12
14
10
11
16
15
24
–
13
18
15
16
13
14
18
19
12
14
13
15
12
14
18
19
–
15
19
16
18
14
17
21
22
15
16
11
14
15
14
16
18
–
18
19
15
16
17
16
22
21
8b
8c
8d
8e
8f
8g
8h
Pencillin
Griseofulvin
34
34
SAR-1
F
3C
O
R2
R1
SAR-2
N
N
N
N
8(a-h)
8a: R
8b: R
R
8c: R
R
8d: R
R
Br, 8e:
=
2
H,
H,
H,
Cl,
H,
H,
F,
H,
1/R2
=
=
=
=
=
=
=
=
=
=
1
1
R1
R
8f: R1
R2 Ar,
R1
R2 OH, 8h:1R
R
H,
=
2
8g:
F,
F,
OCH
=
=
=
2
1
2
2
3.
Fig. 1 Structure–activity relationship for the antimicrobial activity of the synthesized compounds
having a 4-OCH3 (8g) and 4-OH (8h) substitution group the benzene ring showed
more potent antibacterial activity against S. aureus and gram-negative micro-
organisms such as E. coli. The ring containing 4-OCH3 and 4-OH substitution
exhibited better antifungal activity.
Molecular docking
Exploration of molecular interaction of the 4-aryl-2-(3-(2-(trifuoromethyl)phenyl)-
1,8-naphthyridin-2-yl)phthalazin-1(2H)-one compounds was performed through
molecular docking studies using Discovery Studio 2.1. To study the molecu-
lar interaction of compounds, crystallographic data of penicillin-binding pro-
tein 6 (PBP6) from Escherichia coli (PDB: 3ITA) was retrieved from the Protein
1 3

A facile greener synthesis, antimicrobial evaluation and…
Fig. 2 a Structural model of the penicillin-binding protein 6 from E. coli (PDB: 3ITA) binding site
(sphere); b binding site and binding pattern of title compounds along with control
Fig. 3 Receptor-ligand hydrogen bonds (green color) and bumps (pink color) of the 4-aryl-2-(3-(2-
(trifuoromethyl)phenyl)-1,8-naphthyridin-2-yl)phthalazin-1(2H)-one compounds and penicillin with
active site residues of penicillin-binding protein 6 from E. coli (PDB: 3ITA)
Data Bank (https://doi.org/10.2210/pdb3ita/pdb). The retrieved crystal structure
of PBP6 was cleaned and hydrogen atoms were added. All the heteroatoms were
removed before the docking study. The docking study of 2-(3-(2-(trifuoromethyl)
1 3

B. Sakram et al.
Table 3 Details of LibDock score and ligand interaction data revealed through molecular docking of
4-aryl-2-(3-(2-(trifuoromethyl)phenyl)-1,8-naphthyridin-2-yl)phthalazin-1(2H)-one derivatives on peni-
cillin-binding protein 6 (PDB: 3ITA)
Compound
LibDock score
Interacting atoms
H-Bond distance
Donor Acceptor
No. of H-bonds
8a
111.582
A:SER83:HN-8a:F35
A:SER40:HG-8a:N3
A:LYS43:HZ1-8a:N3
A:SER83:HN-8b:F36
A:LYS43:HZ1-8b:N3
A:SER40:HG-8b:N3
A:SER40:HG-8c:N3
A:SER40:HG-8c:N7
A:LYS43:HZ1-8c:N3
A:SER83:HN-8d:F37
A:SER40:HG-8d:N3
A:LYS43:HZ1-8d:N3
A:LYS43:HZ1-8e:N3
A:SER40:HG-8e:N3
A:SER83:HN-8e:F38
A:LYS43:HZ1-8f:N3
A:SER40:HG-8f:N7
A:SER40:HG-8f:N3
A:SER40:HG-8 g:N3
2.263000
1.806000
2.203000
2.240000
2.160000
1.838000
1.913000
2.465000
1.770000
2.271000
1.849000
2.164000
2.168000
1.850000
2.245000
1.741000
2.384000
1.849000
1.951000
HN
HG
HZ1
HN
HZ1
HG
HG
HG
HZ1
HN
HG
HZ1
HZ1
HG
HN
HZ1
HG
HG
HG
HN
HZ1
HG
HZ1
HN
HN
HZ2
F35
N3
N3
F36
N3
N3
N3
N7
N3
F37
N3
N3
N3
N3
F38
N3
N7
N3
N3
F38
N3
N3
N3
F38
O4
O2
3
8b
115.213
109.163
116.099
108.322
114.051
118.413
115.598
121.482
3
3
3
3
3
3
3
2
8c
8d
8e
8f
8g
A:SER83:HN-8 g:F38 2.382000
A:LYS43:HZ1-8 g:N3 2.189000
A:SER40:HG-8 h:N3
A:LYS43:HZ1-8 h:N3 2.146000
A:SER83:HN-8 h:F38 2.386000
A:SER83:HN-pen:O4
8h
1.911000
Pencillin
2.401000
A:LYS43:HZ2-pen:O2 1.964000
phenyl)-1,8-naphthyridin-2-yl)-4-substituted-phthalazin-1(2H)-ones revealed high
docking scores (LibDock) and binding afnities, in the range of 118.483–108.322,
as compared to penicillin (121.482; Figs. 2 and 3 and Table 3). Based on a review of
the literature, the scafolds naphthyridine and 4-substituted phthalazin-1(2H)-ones
showed antimicrobial activity towards bacteria and fungi, but while performing our
in vitro studies, we did not get positive results on fungi [40–42].
Conclusion
We have successfully developed an efcient, greener, and fast methodology for the
synthesis of 4-aryl-2-(3-(2-(trifuoromethyl)phenyl)-1,8-naphthyridin-2-yl)phthala-
zin-1(2H)-ones through the cyclization of 2-hydrazinyl-3-(2-(trifuoromethyl)
phenyl)-1,8-naphthyridine and 2-aroyl benzoic acids in the presence of sulfuric acid
1 3

A facile greener synthesis, antimicrobial evaluation and…
under solid-state grinding methods. This synthetic strategy ofers important advan-
tages such as short reaction time, high to excellent yields, versatile substituents,
easy work-up, mild reaction conditions, and environmental safety. All compounds
were evaluated for their in vitro antimicrobial activity; compounds 8b, 8d, 8g, and
8h exhibited the highest activity. Moreover, in LibDock molecular docking studies,
results have proved the strong binding afnity and more H-bond interaction.
Acknowledgements The authors are thankful to the Head, Department of Chemistry, Osmania Univer-
sity, for providing laboratory facilities and constant encouragement, and are also thankful to the Direc-
tors, IICT, CFRD-OU-Hyderabad, for providing spectral and analytical data of the synthesized com-
pounds. One of the authors (Dharavath Ravi) is thankful to the UGC-SRF New-Delhi, India, for the
fnancial support.
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