Design and synthesis of novel 1,3,4-oxadiazole based azaspirocycles catalyzed by NaI under mild condition and evaluated their antidiabetic and antibacterial activities

Article abstract of DOI:10.1002/jhet.4200

A modest, efficient, and mild synthetic procedure has been developed for the synthesis of novel series of 1,3,4-oxadiazole containing azaspirocycles derivatives. The reaction of 1,3,4-oxadiazole derivative with diverse azaspiro compounds under room temperature condition with helps of sodium iodide catalyst and polar aprotic solvent. Numerous compensations of this strategy embrace less time required, yield increment, consumption of all reactants, and mild condition. All synthesized compounds evaluated for in vitro antidiabetic and antibacterial screening. Among them some compounds show significant biological response.

Full text of DOI:10.1002/jhet.4200

1
Design and synthesis of novel 1,3,4-oxadiazole based azaspirocycles
catalyzed by NaI under mild condition and evaluated their antidiabetic and
antibacterial activities
a
a
b
a
A. J. Radia , J. N. Lalpara , I. J. Modasiya ,G. G. Dubal *
a
Department of chemistry, School of science, RK University, Gujarat, India
b
Department of Microbiology, school of science, RK University, Gujarat, India
*
Email- gaurang.dubal@rku.ac.in, (Tel.) +919662051944
Abstract
A modest, efficient, and mild synthetic procedure has been developed for the synthesis of
novel series of 1,3,4-oxadiazole containing azaspirocycles derivatives. The reaction of 1,3,4-
oxadiazole derivative with diverse azaspiro compounds under room temperature condition
with helps of sodium iodide catalyst and polar aprotic solvent. Numerous compensations of
this strategy embrace less time required, yield increment, consumption of all reactants, mild
condition. All synthesized compounds evaluated for in vitro antidiabetic and antibacterial
screening. Among them some compounds show significant biological response.
Keywords: Azaspirocycles, Mild reaction, 1,3,4-oxadiazole, Antidiabetic, Antibacterial
1
. INTRODUCTION
Proficient synthesis of synthetic drug like molecules has been the focal point of the
exploration for chemist and biologist because of they play a significant role in drug discovery
[
1]. Heterocycles containing spiro moieties available in huge number of plants such as
lactones, alkaloids, and terpenoids. The substituted spiro compounds have significant
importance in pharmaceutical field owing to diverse biological response, numerous synthetic
drugs are relying upon spiro nucleus. six-member piperidine fused spiro derivatives used for
decrease hypertension [2]. trospium has combined pyrrolidine spiro motif utilized for
overactive bladder [3], combination of both diazaspiro[4.5]decan derivatives as potential
chitin synthase inhibitors and antifungal agent [4], diazaspiro[5.5]undecane Derivatives use
for pain treatment as dual μ‑Opioid Receptor Agonists and σ Receptor Antagonists and
1
radioligands for sigma-1 receptors [5-6], also use for treatment of diseases including
Huntington’s disease (HD), Parkinson’s disease (PD), Alzheimer’s disease (AD) [7-8]. Some
spiro derivatives shows antibacterial activity [9] and antidiabetic activity [10] identical
virtuous. Several specific syntheses for different member with dissimilar hetero atom
containing spiro scaffolds including bicyclic construction amidines [11], advanced angular
[
12]
spirocycles or liner spirocycles , via enantioselective hydrogenation [13]. a four-member
ring containing spirocycles synthesized by different strategies [14]. Several drugs contain
various spirocycles shows in Fig. 1.
Another motif oxadiazoles profound class of heterocyclic chemistry that stood out because of
various application in medicinal field and synthetic field. oxadiazole based on their isomeric
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2
form (1,2,3-, 1,2,4-, 1,2,5-, 1,3,4-), among these isomers 1,2,3-oxadiazole is unstable [15],
and 1,3,4-oxadiazole derivatives are extremely stable [16]. A plenty of literature, 1,3,4-
oxadiazole derivatives implanted in conceivably vigorous molecules [17] Preparation of
1
,3,4-oxadiazole several methods have been reported in literature. frequently used synthetic
route for preparation of 1,3,4-oxadiazole including reaction between acid hydrazide and acid
chlorides, another one directly cyclization of diacylhydrazines by means of various
dehydrating agents such as phosphorous pentoxide [18], phosphorous oxychloride [19],
thionyl chloride [20], triflic anhydride [21], polyphosphoric acid [22]. reaction of hydrazine
with carboxylic acid have been reported with diverse oxidizing agents such as PEG
(polyethylene glycol) with dichlorophosphate [23], TBTU [24], Burgess Reagent [25],
cyanuric chloride [26], Deoxo-Fluor [27]. 1,3,4-oxadiazole derivatives are reported to shows
diverse activities such as anticancer [28], anti-HIV [29], antibacterial [30], antifungal [31-
3
2], anticonvulsant [33], antiviral [34] and antitumor activities [35]. few marketed drugs are
shown in Fig. 2.
Figure 1. Some marketed drug containing spirocycles
Figure 2. Some marketed drug containing 1,3,4-oxadiazole
2
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3
The appearance of bacterial confrontation from the antibiotics signifies a thoughtful
apprehension for clinical experts during the most recent decade. Some specific drug resistant
including methicilline against Staphylococcus aureus [36], and vancomycin resistant
enterococci gram positive bacteria [37]. in our studies on this article targeted synthesis of
antibacterial and antidiabetic dual nature compounds. on review of literature basis, we choose
two different pharmacophore 1,3,4-oxadiazole and spirocycles that shows significant desired
activities. our designed synthetic tactic we got extremely significant results for antibacterial
and antidiabetic activities.
2
2
. RESULTS AND DISCUSSION
.1 Chemistry
In our preliminary study preparation of 1,3,4-oxadiazole which was prepared by different
methods but we choose phosphorous oxychloride method. first, esterification of benzoic acid
undergoes H SO we got benzoate (2). further it reacts with hydrazine hydride to form
2
4
hydrazide (3) adduct. furthermore, oxidative cyclization of hydrazide by phosphorous
oxychloride and chloroacetic acid under solvent free condition for 4 hr reflux to get desired
adduct (4). Yield of synthesized compounds were obtained between 80-86%. Synthesized
1
compound 2, 3, and 4 were characterized by H NMR spectroscopy and mass spectrometry.
Scheme 1. Preparation of substituted 1,3,4-oxadiazole
0
Reaction condition: (a) H SO , MeOH, reflux, 2h, (b) NH NH , EtOH, 80 C, 2h, (c) POCl ,
Chloroacetic acid, 80 C, reflux 4h.
2
4
2
2
3
0
3
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4
Now, we move towards the final reaction, we choose 2-(chloromethyl)-5-(4-nitrophenyl)-
1
,3,4-oxadiazole (4), and 1-oxa-4-thia-8-azaspiro[4.5]decan-2-one (5a) has chosen as a
model reaction. In the absence of catalyst, we could not obtain desired product in desired
amount even after 10 h of stirring in ethanol solvent. We tried to different solvent condition
like methanol, DMF, and acetonitrile among them we got some yield increment and
satisfactory result in acetonitrile solvent. Furthermore, we choose acetonitrile solvent in all
the reaction but this process takes required more time and some less yield as per our
expectation. So that we were applied different catalyst in reaction with different mole
percentage.
For the preparation of 8-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methyl)-1-oxa-4-thia-8-
0
azaspiro[4.5]decan-2-one (6a) first we tried polar protic solvents like ethanol at 50 C for 10
hr but didn’t get exemplary yield also tried methanol but we show yield decrement. So, that
our focus moved on polar aprotic solvents like DMF and acetonitrile amongst them
acetonitrile gave good yield compare to DMF solvent (Table 1). Synthesized compound
didn’t give desired yield so, we applied catalyst to enhance reaction rate as well yield
increment. Initially we applied 10% mol tetrabutylammonium iodide (TBAI) 46% yield
obtained which was much better than without catalyst. After that we used 10% mol Tetra-n-
butylammonium bromide (TBAB) and p-Toluenesulfonic acid (P-TSA) we got 37% and 21%
yield respectively (Table 1). Finally, we done this reaction with sodium iodide catalyst
because of it shows higher yield respect to all catalyst. Variation in mole % of sodium iodide
like 10%, 15%, 20% and 25% but we got maximum yield with 20% mole of catalyst. All the
entries summarized in Table 1 (entry 1-11). series of azaspirocycles containing 1,3,4-
oxadiazole moiety shows in Scheme 2.
a
Table 1. Model reaction and optimization of reaction conditions
Reaction
condition
b
Entry
Catalyst (mol %)
Time (h)
Yield (%)
0
1
2
3
4
5
6
7
8
9
-
-
-
-
EtOH, 50 C
MeOH, 50 C
10
10
10
10
5
10
10
5
23
19
32
29
46
37
21
49
56
0
0
MeCN, 50 C
0
DMF, 50 C
TBAI (10%)
TBAB (10%)
P-TSA (10%)
NaI (10%)
NaI (15%)
MeCN, rt
MeCN, rt
MeCN, rt
MeCN, rt
MeCN, rt
3
4
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5
1
0
NaI (20%)
MeCN, rt
3
70
1
1
NaI (25%)
MeCN, rt
3
65
a
Reaction conditions: 2-(chloromethyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (1.71 mmol, 4),
-oxa-4-thia-8-azaspiro[4.5]decan-2-one (1.9 mmol, 5a), K CO (5 mmol) and solvents 6 mL
1
2
3
at different temperatures
b
Isolated yield
Bold value shows final optimal condition of reaction
Scheme 2. Preparation of azaspirocycles holding 1,3,4-oxadiazole moiety
Table 2. Substitution of azaspiro compounds
Table 3. Final synthesized adducts containing azaspiro and 1,3,4-oxadiazole pharmacophore
5
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6
2
.2 Biology
Synthesized compounds 6a-j tested minimum inhibitory concentration (MIC) determination
by the agar well diffusion method [38–40] employed in this work, all synthesized compounds
activity checked against Gram-positive and Gram-negative bacteria alike Bacillus
megaterium, Bacillus subtilisis, E. coli, and Klebsiella. yielded larger zone of inhibition in
primary screening then secondary screening done with 10 µg/ml, 25 µg/ml, 50 µg/ml
concentrations. Results against 50 µg/ml concentration shown in table 2. checked their zone
of growth inhibition in millimetre (mm). amongst them compound 6a was extremely active
against gram positive bacteria Bacillus megaterium, Bacillus subtilisis and growth inhibition
value nearer to standard drug erythromycin. compounds 6b and 6e also shows moderate
activity compare to erythromycin. gram-negative bacterial strains in secondary screening
same compounds 6a, 6b and 6e shows moderate active compare to standard drug tetracycline
(Table 4).
a
Table 4. Antibacterial secondary screening of synthesized compounds
b
Zones of growth inhibition
Gram-negative
Comp. Gram-positive bacteria
Entry
bacteria
code
Bacillus
Bacillus
E.
EC50
µg/mL
Klebsiella
c
megaterium subtilisis coli
1
2
3
4
5
6
7
8
9
6a
6b
6c
6d
6e
6f
6g
6h
6i
16
13
6
15
13
4
6
12
9
6
3
5
4
11
7
0
0
11
4
2
2
3
0
10
9
nt
nt
11
8
7
4
6
28.12 ± 0.40
34.61 ± 0.65
75.00 ± 1.50
64.28 ± 1.25
32.14 ± 0.57
45.00 ± 1.35
50.00 ± 1.55
56.25 ± 1.48
45.00 ± 1.37
112.5 ± 2.32
nt
7
14
10
9
8
10
4
1
1
1
0
1
2
6j
T
E
nt
19
nt
nt
18
nt
16
17
nt
25.00 ± 0.50
a
b
c
Sample concentration 50 µg/mL
Zone of growth inhibition measured in millimetre (mm)
EC concentration for gram positive bacteria Bacillus megaterium
50
T - Tetracycline
E - Erythromycin
0
Value shows no inhibition
6
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7
nt- Not tested
Bold value shows highly active compounds from synthesized compounds
Synthesized compounds 6a-j tested for antidiabetic screening by in vitro α-amylase inhibitory
study. α-amylase inhibitory assay was performed by 3,5-dinitrosalicylic acid (DNSA) method
[
41]. all synthesized compounds percentage inhibition checked by different sample
concentration like 50 µg/mL, 75µg/mL, 100 µg/mL 125 µg/mL. For a reference we used
standard drug acarbose, compare the results of both acarbose and synthesized compounds to
calculate their IC50 value in µg/mL. among them are 6a, 6b, and 6c shows significantly
activity and rest are shows good to moderate activity (Table 5). compound 6a highly active
and percent of inhibition very close to standard drug acarbose. All the data mentioned in
Table 5.
Table 5. in vitro antidiabetic screening of synthesized compounds.
%
Inhibition
IC50
µg/mL
Entry Compound
5
0μg/mL 75μg/mL 100μg/mL 125μg/mL
1
2
3
4
5
6
7
8
9
6a
6b
6c
6d
6e
6f
6g
6h
6i
37.91
36.28
34.64
29.19
37.33
27.72
22.51
31.87
32.52
30.78
38.52
48.23
47.07
43.60
39.58
47.97
35.81
31.02
40.77
38.46
36.55
49.44
60.09
57.32
54.37
47.94
58.48
45.69
40.38
49.56
51.12
50.41
61.18
82.21
80.67
76.48
61.29
81.04
60.85
57.36
71.77
73.32
68.78
84.69
78.07
81.76
89.64
103.93
79.80
106.86
114.02
100.72
97.28
99.49
75.90
1
0
1
6j
1
Acarbose
Bold value shows highly active compounds from synthesized compounds
3
. CONCLUSION
In summary, we have designed efficient methodology for the synthesis of bioactive and
potent heterocycles which was the combinations of two different pharmacophore 1,3,4-
oxadiazole and azaspirocycles under the mild reaction condition. synthesized compounds
screened for antidiabetic activity and antibacterial activity to get significant results.
synthesized molecules will find application in versatile are like medicinal research field and
organic chemistry.
4
4
. EXPERIMENTAL
.1 General
All chemicals, solvents and media were purchased from sigma Aldrich, combi-block,
enamine, Himedia, SRL. all purchased chemicals were used without further purification,
reactions were continuous monitored by thin layer chromatography (TLC) on silica gel-(G60
F254 (Merck)) of 0.5 mm thickness, visualizing with ultraviolet light (254 and 365nm), or
with iodine vapor or aq. KMnO4. Melting points were determined using a Buchi B-540
7
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8
capillary apparatus. NMR spectra were recorded on a Bruker Advance 400 MHz
1
13
spectrometer (400 MHz for H NMR and 101 MHz for C NMR) respectively in solvents
like CDCl , DMSO and chemical shifts are referenced to the solvent residual signals with
3
respect to tetramethylsilane. standard abbreviations are used to represent signals multiplicities
1
for H NMR spectrum s - singlet, d - doublet, t - triplet, q - quartet, m - multiplate. Elemental
analysis was carried out on Euro EA 3000 elemental analyser and the results are in agreement
with the structures assigned. The control of reaction temperature was monitored by ruby
thermometer. Mass spectra were recorded on a Shimadzu GC-MS-QP-2010 mass
spectrometer in EI (70eV) model using direct inlet probe technique and m/z is reported in
atomic units per elementary charge.
4
.2 Antibacterial assay
The method is based on the principle that involves the ability of the compound to inhibit the
growth of organisms, as exhibited by a clear zone of inhibition [38-40]. The lowest
concentration inhibiting the growth of the organism is recorded as the MIC. To check
antimicrobial activity, using culture media: nutrient broth, nutrient agar plates. The inoculum
was prepared previously. For inoculum preparation, take 50 ml Nutrient broth in a flask and
inoculate wire loop culture of bacterial strains. for fungi Bacterial culture incubates at 37 ℃
for 24 hours. All synthesized compounds were dissolved in DMSO (Stock solution:2000
µg/ml concentration). For primary screening, prepare three dilutions 1000 µg/ml, 500 µg/ml
and 250 µg/ml from the stock solution of synthesized compounds. To check MIC prepared
nutrient agar plate and allowed to solidify. after solidification, bacterial strains were spread
on to the solidify plates by spread plate technique and make well on the agar plates by using a
7
mm cupbearer. After preparation of well, synthesized compound dilutions (1000 µg/ml, 500
µg/ml and 250 µg/ml) were added 100 µl into the well of plates. These bacterial plates were
0
incubated at 37 C for 24 hours. The diameter of zone of inhibition extending laterally around
the wells were measured. For secondary screening, the synthesized compound found active in
primary screening were similarly diluted to obtain 200 µg/ml 10 µg/ml, 25 µg/ml, 50 µg/ml
concentrations. The diameter of the zone of inhibition was measured in millimetre (mm).
4
.3 In vitro α-Amylase Inhibitory study
The α-Amylase inhibition assay was performed using the 3,5-dinitrosalicylic acid (DNSA)
method. All the compounds were dissolved in 10% DMSO and were further dissolved in
buffer at pH 6.9 to give concentrations ranging from 10-1000 µg/ml. A volume of 200 ml of
α-amylase solution (2 units/ml) was mixed with 200µl of the dissolved compounds and was
incubated for 10 minutes at 30°C.There after 200µl of starch solution (1% in water (w/v))
was added to each tube and incubated for 3 minutes. The reaction was terminated by the
addition of 200µl DNSA reagent (12gm of sodium potassium tartrate tetra hydrate in 8.0ml of
2
M NaOH and 20ml of 96 mM of 3,5-dinitrosalicylic acid solution) and was boiled for 10
minutes in a water bath at 85-90°C. The mixture was cooled to ambient temperature and was
diluted with 5ml of distilled water, and the absorbance was measured at 540 nm using UV-
Visible spectrometer. The blank with 100% enzyme activity was prepared by replacing the
dissolved compounds with 200 µL of buffer. A blank reaction was similarly prepared using
the dissolved compounds at each concentration in the absence of enzyme solution. A positive
8
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9
control was prepared using acarbose (125µg/mL-10µg/mL) and the reaction was performed
similarly to the reaction with dissolved compounds as mentioned above. The α-amylase
inhibitory activity was expressed as percent inhibition and was calculated using the equation
given below: The % of a-Amylase inhibition was plotted against the concentration of
dissolved compounds and calculated their IC50 (µg/mL) value respectively [41].
%
of α amylase inhibition = 100× Abs100% control-Abs sample / Abs100% control
Procedure for the synthesis of methyl 4-nitrobenzoate (2).
To a stir solution of 4-nitro benzoic acid (89.75 mmol) in Methanol (150 ml) was added
0
sulphuric acid and reaction mixture was reflux at 60 C for 2h, Progress of reaction was
monitored by TLC, After completion of reaction, reaction mixture was concentrated and
poured into ice cold water (400 ml), solid material was precipitate out which was filtered out
and dried u/vacuum to get methyl-4-nitrobenzoate (2).
Methyl 4-nitrobenzoate (2) Yield: 85.04% (14.0 g) as an off-white solid material. Mp: 94-
0
1
9
3
7
6 C. H NMR (400 MHz, DMSO) δ 8.36 (d, J=8.4 Hz, 2H), 8.20 (d, J=8.8 Hz, 2H), 3.92 (s,
H), Mass m/z: 181.15. Elemental Analysis: C H NO , Calculated: C, 53.04; H, 3.90; N,
.73; O, 35.33. Found: C, 53.14; H, 3.75; N, 7.53.
7
8
4
Procedure for the synthesis of 4-nitrobenzohydrazide (3).
To a stir solution of methyl-4-nitrobenzoate (71.76 mmol) in Ethanol (130 ml) was added
0
hydrazine hydrate (143.52 mmol) and reaction mixture was reflux at 78 C for 2h, Progress of
reaction was monitored by TLC, After completion of reaction, reaction mixture was
concentrated and poured into ice cold water (200 ml), solid material was precipitate out
which was filtered out washed with water and dried u/vacuum to get 4-nitrobenzo hydrazide
(3).
4
2
-nitrobenzohydrazide (3) Yield: 84.61 % (11.0 g) as a light-yellow solid material. Mp: 212-
14 C. H NMR (400 MHz, DMSO) δ: 10.14 (s, 1H), 8.30 (d, J=8.8 Hz, 2H), 8.04 (d, J=8.4
0
1
Hz, 2H), 4.65 (s, 2H), Mass m/z: 181.15.Elemental Analysis: C H N O , Calculated: C,
7
7
3
3
4
6.41; H, 3.90; N, 23.20; O, 26.50. Found: C, 46.25; H, 3.70; N, 23.26.
Procedure for the synthesis of 2-(chloromethyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (4).
To a stir solution of Phosphoryl chloride (80 ml), 4-nitrobenzo hydrazide (44.16 mmol) and
0
0
chloroacetic acid (44.16 mmol) were added at 0 C, then reaction mixture was reflux at 80 C
for 4h, Progress of reaction was monitored by TLC, After completion of reaction, reaction
mixture was concentrated and poured into ice cold water (200 ml), solid material was
precipitate out which was filtered out washed with water and dried u/vacuum to get 2-
(chloromethyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (4)
2
-(chloromethyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (4) Yield: 86.71 % (6.0 g) as an off-
0
1
white material, Mp: 134-136 C. H NMR (400 MHz, DMSO) δ: 8.45 (d, J=8.4 Hz, 2H), 8.29
d, J=8.8 Hz, 2H), 5.19 (s, 2H), Mass m/z: 239.62, Elemental Analysis: C H ClN O ,
(
9
6
3
3
Calculated: C, 45.11; H, 2.52; Cl, 14.79; N, 17.54; O, 20.03. Found: C, 45.18; H, 2.56; Cl,
4.69; N, 17.34.
1
9
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1
0
General procedure for the synthesis of 1,3,4-oxadiazole containing azaspirocycles (6a-j)
To a stir solution of azaspirocycles derivatives (6a-j) (1.90 mmol) in Acetonitrile (6 ml)
K CO (5.72 mmol) was added and reaction mixture was stirred at rt for 15 min then 2-
2
3
(chloromethyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (1.71 mmol) and NaI (20 mol%) were
added then reaction mixture was stirred at rt for 3h, Progress of reaction was monitored by
TLC, After completion of reaction, reaction mixture was poured into water (10 ml) and
extracted with EtOAc (10-20 ml), combined organic layer was dried over
Na SO ,concentrated u/vacuum to get crude material which was purified by column
2
4
chromatography using 30% Ethyl acetate/n-Hexane as a mobile phase to get pure compounds
6a-j).
(
8
-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methyl)-1-oxa-4-thia-8-azaspiro[4.5]decan-2-one
0
1
(6a). Yield: 70.02% (0.320 g) as a light brown solid material, Mp: 180-182 C. H NMR (400
MHz, CDCl ) δ: 8.43 (d, J=8.8 Hz, 2H), 8.32 (d, J=8.8 Hz, 2H), 3.99 (s, 2H), 3.79 (s, 2H),
3
2
2
8
.87 (d, J=8.0 Hz, 2H), 2.80 (d, J=4.4 Hz, 2H), 2.29 (d, J=13.6 Hz, 2H), 2.16 (d, J=8.4 Hz,
1
3
H), C NMR (101 MHz, CDCl ) δ: 171.74, 164.31, 163.75, 149.70, 129.21, 127.99, 124.40,
3
9.49, 51.61, 50.29, 39.14, 31.82, 29.69, Mass m/z: 376.39, Elemental Analysis:
C H N O S, calculated: C, 51.06; H, 4.28; N, 14.89; O, 21.25; S, 8.52, Found: C, 51.16; H,
16
16
4
5
4
.22; N, 14.80.
6
6
-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methyl)-2-oxa-6-azaspiro[3.3]heptane (6b). Yield:
0
1
5% (0.24 g) as a light yellow solid, Mp: 176-178 C. H NMR (400 MHz, CDCl ) δ: 8.40 (d,
3
13
J=8.8 Hz, 2H), 8.28 (d, J=8.8 Hz, 2H), 4.79 (s, 4H), 3.92 (s, 2H), 3.65 (s, 4H), C NMR
101 MHz, CDCl ) δ: 172.89, 162.13, 147.90, 129.27, 128.09, 125.25, 77.37, 76.74, 63.17,
(
3
6
2.21, 50.16, 32.25. Mass m/z: 302.29, Elemental Analysis: C H N O , calculated: C,
14 14 4 4
5
5.63; H, 4.67; N, 18.53; O, 21.17 Found: C, 55.56; H, 4.62; N, 18.43.
6
7
8
2
1
3
-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methyl)-6-azaspiro[3.4]octan-2-one (6C). Yield:
0
1
6.22% (0.300 g) as a brown solid material. Mp: 198-200 C. H NMR (400 MHz, CDCl ) δ:
3
.43 (d, J=9.2 Hz, 2H), 8.31 (d, J=8.8 Hz, 2H), 4.12 (s, 2H), 3.19 (s, 2H), 3.13 (t, J=3.6 Hz,
13
H), 3.07 (s, 4H), 2.22 (t, J=7.2 Hz, 2H), C NMR (101 MHz, CDCl ) δ: 200.19, 184.34,
3
76.87, 167.49, 145.72, 134.48, 129.62, 126.50, 70.07, 64.52, 56.87, 52.30, 39.85. Mass m/z:
28.33, Elemental Analysis: C H N O , calculated: C, 58.53; H, 4.91; N, 17.06; O, 19.49,
1
6
16
4
4
Found: C, 58.43; H, 4.90; N, 17.26.
7
6
-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methyl)-7-azaspiro[3.5]nonan-2-one (6d). Yield:
0
1
7.74% (0.200 g) as a creamish solid material. Mp:178-180 C. H NMR (400 MHz, CDCl )
3
δ: 8.41 (d, J=8.8 Hz, 2H), 8.30 (d, J=8.8 Hz, 2H), 3.94 (s, 2H), 2.81 (s, 4H), 2.64 (s, 4H),
1
3
1
1
.85 (s, 4H). C NMR (101 MHz, CDCl ) δ: 202.32, 176.02, 166.62, 159.41, 137.15, 132.30,
3
28.91, 127.12, 124. 08, 74.84, 63.69, 57.27, 51.04, 46.34, 37.57, 34.19. Mass m/z: 342.35,
Elemental Analysis: C H N O , calculated: C, 59.64; H, 5.30; N, 16.37; O, 18.69, Found:
1
7
18
4
4
C, 59.54; H, 5.10; N, 16.32.
8
-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methyl)-2,8-diazaspiro[4.5]decan-3-one
(6e).
0
1
Yield: 70.59% (0.180 g) as a creamish solid material. Mp: 166-168 C. H NMR (400 MHz,
CDCl ) δ: 8.41 (d, J=8.8 Hz, 2H), 8.30 (d, J=8.8 Hz, 2H), 3.96 (s, 2H), 3.23 (d, J=1.2 Hz,
3
1
0
This article is protected by copyright. All rights reserved.

1
1
1
3
2
H), 2.71 (m, 2H), 2.60 (m, 2H), 2.25 (s, 2H), 1.81 (t, J=5.6 Hz, 4H), C NMR (101 MHz,
CDCl ) δ: 175.61, 172.79, 165.29, 151.43, 137.34, 133.59, 129.47, 127.35, 57.04, 54.83,
3
5
0.16, 47.30, 37.46, 36.23. Mass m/z: 357.37, Elemental Analysis: C H N O , calculated:
17 19 5 4
C, 57.14; H, 5.36; N, 19.60; O, 17.91, Found: C, 57.24; H, 5.30; N, 19.65.
7
8
-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methyl)-2-oxa-7-azaspiro[3.5]nonane (6f). Yield:
4.67% (0.440 g) as a light yellow solid material. Mp: 208-210 C. H NMR (400 MHz,
0
1
CDCl ) δ: 8.43 (d, J=8.8 Hz, 2H), 8.31 (d, J=8.8 Hz, 2H), 4.45 (s, 4H), 3.92 (s, 2H), 2.57 (m,
3
1
3
4
1
H), 1.98 (t, J=5.2 Hz, 4H), C NMR (101 MHz, CDCl ) δ: 164.57, 163.66, 149.63, 129.28,
3
27.96, 124.39, 81.57, 52.21, 50.45, 38.06, 34.68. Mass m/z: 330.34, Elemental Analysis:
C H N O , calculated: C, 58.17; H, 5.49; N, 16.96; O, 19.37, Found: C, 58.12; H, 5.40; N,
16
18
4
4
1
6.86.
2
7
-((5-azaspiro [2.4] heptan-5-yl)methyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (6g). Yield:
0
1
5.44% (0.350 g) as a creamish solid material. Mp: 194-196 C. H NMR (400 MHz, CDCl )
3
δ: 8.42 (d, J=8.8 Hz, 2H), 8.33 (d, J=8.8 Hz, 2H), 4.11 (s, 2H), 3.03 (t, J=6.8 Hz, 2H), 2.78
1
3
(
s, 2H), 1.95 (t, J=6.8 Hz, 2H), 0.66 (t, J=8.4 Hz, 4H), C NMR (101 MHz, CDCl ) δ:
3
1
3
72.03, 163.84, 148.62, 134.82, 128.82, 126.24, 67.48, 52.44, 39.51, 13.87. Mass m/z:
00.32, Elemental Analysis: C H N O , calculated: C, 59.99; H, 5.37; N, 18.66; O, 15.98,
1
5
16
4
3
Found: C, 59.90; H, 5.32; N, 18.60.
2
7
8
-((2-azaspiro [4.4] nonan-2-yl)methyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (6h). Yield:
0
1
8.44% (0.350 g) as a yellow solid material. Mp: 186-188 C. H NMR (400 MHz, CDCl ) δ:
3
.42 (d, J=8.8 Hz, 2H), 8.32 (d, J=8.8 Hz, 2H), 4.08 (s, 2H), 2.90 (m, 2H), 2.70 (m, 2H), 1.84
1
3
(t, J=7.2 Hz, 2H), 1.65 (m, 8H). C NMR (101 MHz, CDCl ) δ: 173.52, 164.81, 146.86,
3
1
34.26, 126.05, 123.18, 67.48, 52.68, 50.65, 42.09, 26.37. Mass m/z: 328.37, Elemental
Analysis: C H N O , calculated: C, 62.18; H, 6.14; N, 17.06; O, 14.62, Found: C, 62.10; H,
1
7
20
4
3
6
.12; N, 17.16.
8
7
-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methyl)-1-oxa-8-azaspiro[4.5]decane (6i). Yield:
0
1
5.50% (0.350 g) as a creamish solid material. Mp: 178-180 C. H NMR (400 MHz, CDCl )
3
δ: 8.42 (d, J=8.8 Hz, 2H), 8.32 (d, J=8.8 Hz, 2H), 3.98 (s, 2H), 3.87 (t, J=6.8 Hz, 2H), 2.73
13
(
m, 4H), 1.97 (t, J=6.8 Hz, 2H), 1.75 (t, J=6.8 Hz, 6H). C NMR (101 MHz, CDCl ) δ:
3
1
2
1
74.65, 162.87, 150.08, 136.47, 130.48, 127.82, 86.79, 71.04, 54.69, 51.38, 38.38, 32.83,
9.82. Mass m/z: 344.37, Elemental Analysis: C H N O , calculated: C, 59.29; H, 5.85; N,
6.27; O, 18.58, Found: C, 59.20; H, 5.80; N, 16.25.
17
20
4
4
9
-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methyl)-3-oxa-9-azaspiro[5.5]undecane
(6j).
0
1
Yield: 81.18% (0.300 g) as a light yellow solid material. Mp: 188-190 C. H NMR (400
MHz, CDCl ) δ: 8.42 (d, J=8.8 Hz, 2H), 8.32 (d, J=8.8 Hz, 2H), 3.98 (s, 2H), 3.69 (t, J=5.2
3
1
3
Hz, 4H), 2.67 (m, 4H), 1.69 (m, 4H), 1.61 (m, 4H). C NMR (101 MHz, CDCl ) δ: 172.01,
3
1
64.43, 150.87, 136.82, 130.41, 125.18, 68.53, 48.47, 37.41, 32.90. Mass m/z: 358.40,
Elemental Analysis: C H N O , calculated: C, 60.32; H, 6.19; N, 15.63; O, 17.86 Found: C,
1
8
22
4
4
6
0.22; H, 6.13; N, 15.60.
ACKNOWLEDGEMENTS
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1
This article is protected by copyright. All rights reserved.

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The authors are thankful to the Department of Chemistry, School of science, RK University
Rajkot) for providing laboratory facilities and National Facility For Drug Discovery (NFDD)
(
for providing spectral data.
SUPPORTING INFORMATION
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Experimental of synthesis and biological activity, H NMR, C NMR spectra and Mass
spectra for all compounds were provided as Supplementary material.
DISCLOSURE STATEMENT
No potential conflict of interest was reported by the authors.
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