Facile alkylation of 4-nitrobenzotriazole and its platelet aggregation inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2017.07.045

We explored the facile alkylation of 4-nitrobenzotriazole under basic conditions and the synthesized derivatives were tested for their potential ADP induced platelet aggregation inhibition activity in comparison with standard drug ticagrelor (selective P2Y12 inhibitor). The nitro group at 4-position is highly activating toward alkylation reactions (under strong basic conditions) and resulted in formation of degradation product like 3-nitrobenzene-1,2-diamine which make isolation of alkyl products very difficult. We optimized the reaction under mild basic condition (potassium carbonate and DMF) which is devoid of any degradation product. This is perhaps the first report of 4-nitrobenzotriazole derivatives possessing platelet aggregation inhibitory activity. Generally activity increases with increase in length of alkyl chain and 1-alkyl positional isomers were found to be more potent than 2-alkyl isomers. The benzoyl derivative was found to be the most potent [compound 22; (4-Nitro-1H-benzotriazol-1-yl)(phenyl)methanone; IC₅₀ = 0.65 ± 0.10 mM] which may be attributed to electronegative oxygen atom and aromatic ring. Benzyl derivatives [compound 20; 1-Benzyl-4-nitro-1H-benzotriazole; IC₅₀ = 0.81 ± 0.08 mM, compound 21; 2-Benzyl-4-nitro-2H-benzotriazole; IC₅₀ = 0.82 ± 0.19 mM] and sulfonyl derivative [compound 23; 1-[(4-Methylphenyl)sulfonyl]-4-nitro-1H-benzotriazole; IC₅₀ = 0.82 ± 0.19 mM] are also found to be highly active. Furthermore, all compounds possess P2Y12 binding affinity as confirmed by VASP/P2Y12 phosphorylation assay.

Full text of DOI:10.1016/j.bmc.2017.07.045

Accepted Manuscript
Facile Alkylation of 4-Nitrobenzotriazole and its Platelet Aggregation Inhibi-
tory Activity
Dhandeep Singh, Om Silakari
PII:
S0968-0896(17)30416-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.045
BMC 13882
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
28 February 2017
20 May 2017
26 July 2017
Please cite this article as: Singh, D., Silakari, O., Facile Alkylation of 4-Nitrobenzotriazole and its Platelet
Aggregation Inhibitory Activity, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.07.045
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Facile Alkylation of 4-Nitrobenzotriazole and its Platelet Aggregation Inhibitory
Activity
Dhandeep Singh, Om Silakari*
Department of Pharmaceutical Sciences and Drug Research,
Punjabi University, Patiala 147002, Punjab, India
E-mail: omsilakari@gmail.com

Abstract: We explored the facile alkylation of 4-nitrobenzotriazole under basic conditions
and the synthesized derivatives were tested for their potential ADP induced platelet
aggregation inhibition activity in comparison with standard drug ticagrelor (selective P2Y12
inhibitor). The nitro group at 4-position is highly activating towards alkylation reactions
(under strong basic conditions) and resulted in formation of degradation product like 3-
nitrobenzene-1,2-diamine which make isolation of alkyl products very difficult. We
optimized the reaction under mild basic condition (potassium carbonate and DMF) which is
devoid of any degradation product. This is perhaps the first report of 4-nitrobenzotriazole
derivatives possessing platelet aggregation inhibitory activity. Generally activity increases
with increase in length of alkyl chain and 1-alkyl positional isomers were found to be more
potent than 2-alkyl isomers. The benzoyl derivative was found to be the most potent
[compound 22; (4-Nitro-1H-benzotriazol-1-yl)(phenyl)methanone; IC50 = 0.65 ± 0.10 mM]
which may be attributed to electronegative oxygen atom and aromatic ring. Benzyl
derivatives [compound 20; 1-Benzyl-4-nitro-1H-benzotriazole; IC50 = 0.81 ± 0.08 mM,
compound 21; 2-Benzyl-4-nitro-2H-benzotriazole; IC50 = 0.82 ± 0.19 mM) and sulfonyl
derivative [compound 23; 1-[(4-Methylphenyl)sulfonyl]-4-nitro-1H-benzotriazole; IC50 =
0.82 ± 0.19 mM] are also found to be highly active. Furthermore, all compounds possess
P2Y12 binding affinity as confirmed by VASP/P2Y12 phosphorylation assay.
Keywords: Alkylation of nitrobenzotriazole; 4-nitrobenzotriazole; 3-Nitrobenzene-1,2-
diamine; Platelets; Ticagrelor

1. Introduction
The benzotriazole is well explored motif to have versatile medicinal effects [1].
Recently, its sodium hydrogen exchanger inhibitory activity is reported [2]. Moreover,
benzotriazoles are imperative in inter-disciplinary science fields as they possess corrosion
inhibitory property [3, 4], optical properties [5], lubricating [6], and synthetic phyto-
hormonal activity [7]. Katritzky et al well explored the chemistry of benzotriazole moiety for
many decades [8-10], although 4-nitro benzotriazoles is relatively less explored [11, 12].
Platelets play imperative role in hemostasis as they stop bleeding from damaged blood
vessels. However, abnormal hemostasis can cause platelet aggregation which in turn could
lead to the formation of blood clots. Hence, there is unmet need to develop platelet
aggregation inhibitors [13, 14]. Adenosine-5’-diphosphate (ADP) is an important mediator of
platelet activation and aggregation [15]. ADP acts on two receptors of platelets: P2Y1
involved in platelet shape change and activation while P2Y12 involved in aggregation of
platelets [16]. P2Y12R belongs to P2Y purinergic GPCR family stimulated by ADP and this
receptor plays crucial role in platelet activation, aggregation, and thrombus formation [17,
18]. Inhibition of ADP induced platelet aggregation is an indicator of selective inhibition of
ADP binding to P2Y12 [19].
There are number of platelet inhibitory drugs like clopidogrel, prasugrel, but they
have very long half-life and are prodrugs which contribute to their side effects. Then,
nucleoside analogs came into existence like cangrelor, but because of phosphate groups, it
has very short half life and thus, they require intravenous administration. Hence, structural
modifications lead to triazolopyrimidine analogs like ticagrelor with advantage of its
metabolic stability and increase binding affinity [20, 21]. The Platelet Inhibition and Patient
Outcomes (PLATO) trials showed that ticagrelor had antithrombotic effects that were
superior to those of clopidogrel in patients with acute coronary syndromes [22].
Benzotriazole structure is similar to imidazopyrimidine of ADP as well as ticagrelor’s
triazolopyrimidine, therefore we modified benzotriazole ring by introducing nitro group at 4th
position and subsequent addition of alkyl groups at 1st and 2nd position. Herein, we prepared
series of nitro benzotriazole derivatives and tested for its potential platelet aggregation
inhibitory activity. Moreover, all synthesized compounds were evaluated for their P2Y12
binding affinity.

Alkylation of benzotriazole is a well reported reaction involving nucleophilic
substitution of benzotriazole nitrogen on alkyl halides or dimethyl sulphate under basic
conditions [10, 23]. Alkylation of 4-nitrobenzotriazole is relatively less explored in
comparison to alkylation of 5-nitrobenzotriazole [24]. Diehl et al reported the alkylations of
4-nitrobenztriazole, but most derivatives synthesized were cyclic saturated, few N-alkyls like
isopropyl derivatives. Indeed, the reported reactions completed in 30 hours [25]. Few alkyl
reactions may be attributed to the fact that alkylation of 4-nitrobenzotriazole result in
impurities formation and tedious separation of isomers. We primarily explored facile
alkylation of 4-nitrobenzotriazole in potassium carbonate and DMF in only 30 minutes with
absence of impurity. Hence, with incessant growing research in this field, there is imperative
need to explore this reaction.
2. Results and Discussion
2.1. Chemistry
Alkylation of 4-nitrobenzotriazole (10 mmol) was carried out using sodium hydroxide
(40 mmol) and alkyl halides (10 mmol) in N,N-Dimethylformamide (DMF) (8-10 mL)
solution as shown in scheme-1 [26]. Three isomers of 4-nitrobenzotriazole methyl derivative
were reported in DMF solvent and described in Scheme-2 [10].
During this reaction a yellow colored compound was always formed with the desired
products (alkyl isomers) formed by all different alkyl halides. Indeed, in thin layer
chromatography the spot of this yellow compound was spread over the spots of isomers and
present in between the isomers. Thus, it hindered with the isolation of isomer products,
although isomers and impurity (yellow compound) could be isolated by using silica gel of
mesh size 400-700. But it is very tedious procedure. As this impurity was common in all
alkyl halides, it was suspected to be degradation product of 4-nitrobenzotriazole. So, a
reaction of 4-nitrobenzotriazole with sodium hydroxide and DMF-without addition of alkyl
halides, was carried out at room temperature, but did not result in yellow compound
formation. Further on heating this solution above 45oC resulted in degradation of 4-
nitrobenzotriazole and same yellow compound was isolated. Thus, reaction of alkyl halides
with 4-nitrobenzotriazole in sodium hydroxide and DMF was tried under cold conditions, but
yellow impurity was still found, besides the isomer products. Upon NMR analysis impurity
was found to be 3-Nitrobenzene-1, 2-diamine.

Scheme-1: Alkylation under strong basic conditions.
Scheme-2: Methylation under strong basic conditions.
Consequently, alkylation was carried out under milder conditions using potassium
carbonate and dry DMF for 30 minutes stirring at room temperature. Here, alkyl products
from methyl to octyl, chloropropyl, benzyl, benzoyl, and tosyl derivatives were prepared and
isolated as described in Scheme-3.

Scheme-3: Alkylation under mild basic conditions. Reagents and Conditions: (a) Potassium
Carbonate, DMF, Stirred for 30 minutes, RX: C2H5Br (4, 5), C3H7Br (6, 7), C4H9Br (8, 9),
C5H11Br (10, 11), C6H13 Br (12, 13), C7H15Br (14, 15), C8H17 (16, 17), BrCH2CH2 CH2Cl (18,
19), C6H5CH2Cl (20, 21); (b) Pyridine, Reflux, 30 min., ArCl: C6H5COCl (22), C7H7SO2Cl
(23).
2.2. Docking
We used iGEMDOCK for docking studies using 4-NTJ (P2Y12R). The result indicated
that all compounds bind to the same pocket as ticagrelor as indicated in Figure 1. Ligand
map and interaction residues of ticagrelor are indicated in Figure 2. Moreover, they bind to
same critical residues that are observed as hot spots, and are indicated in Table 1 [21]. All the
compounds fit into same binding pocket and their interactions energies are summarized in
Table 2. For each interacting residue, Z score is a measure of interaction conservation
between the interacting groups and screening compounds. Z score and pharmacological
preference, and Pharmacological interaction profile tables are provided in supplementary
information.

Figure 1: Figure showing all compounds bind to same pocket as standard drug ticagrelor.
Figure 2: Figure showing interactions residues and ligand map of ticagrelor. Blue, green, and
red lines indicates hydrogen bonding, electrostatic, and steric interactions respectively with
residue.

Table 1: Table showing Pharmacological interactions, W score, consensus interaction, and
Hot spot (indicating residues critical for interaction).
Predicted
Pharmacological Preference Consensus Ratio Hot Spot
Pharmacological
Interaction
H-S SER156
H-S ASN191
H-S CYS194
V-M VAL102
V-M TYR105
V-S TYR105
V-S TYR109
V-S VAL190
V-S ASN191
0.86
0.34
1.00
0.38
0.70
1.00
0.96
0.57
0.71
0.50
0.34
0.53
0.65
0.73
0.92
0.88
0.65
0.77
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Table 2: Total energy in Kcal/Mol. Van der Waals interactions (VDW, Kcal/Mol), Hydrogen
bonding (HBond, Kcal/Mol), electrostatic interactions (Elec, Kcal/Mol), average conpair, and
rank (Based on total energy). Total energy = VDW + HBond + Elec
#Ligand
4-NO2BTZ
TotalEnergy
-81.052
VDW
-66.684
HBond
-14.665
Elec
0.298
AverConPair
35.333
Rank
25
-75.177
-60.423
-14.754
-21.596
39.778
21.722
BTZ
0
0
26
1
-135.259
-113.664
Ticagrelor
-82.2944
-82.339
-84.049
-90.597
-89.592
-91.688
-94.941
-95.946
-65.2992
-65.326
-71.864
-63.855
-72.630
-75.215
-74.844
-80.232
-16.9952
-17.014
-12.185
-27.505
-16.963
-16.473
-20.097
-15.714
33.3077
34.615
34.846
33.929
35.000
32.800
34.867
31.812
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
0
24
23
22
19
21
18
15
14
0
0
0.763
0
0
0
0

-93.353
-99.0164
-96.288
-97.896
-98.576
-102.074
-103.023
-105.139
-103.548
-93.540
-90.359
-113.147
-107.183
-115.186
-105.663
-80.118
-84.231
-81.660
-77.472
-80.946
-86.944
-96.881
-88.751
-88.953
-80.941
-77.413
-99.847
-94.309
-99.705
-94.990
-13.235
-14.7855
-15.095
-21.138
-17.900
-15.997
-6.449
33.188
31.3529
31.824
29.444
28.000
29.684
31.737
27.600
27.100
33.062
32.312
33.210
31.684
32.100
30.773
Compound 9
Compound 10
Compound 11
Compound 12
Compound 13
Compound 14
Compound 15
Compound 16
Compound 17
Compound 18
Compound 19
Compound 20
Compound 21
Compound 22
Compound 23
0
17
10
13
12
11
9
0
0.467
0.714
0.270
0.866
0.306
1.0463
0.504
0
8
-17.434
-15.098
-12.599
-12.945
-13.300
-13.358
-15.481
-10.673
6
7
16
20
3
0
0
0.484
0
4
2
0
5
2.3. Biological Activity
The platelet aggregation inhibitory activity of all synthesized compounds was determined
using ADP-induced platelet aggregation test in-vitro [27, 28]. Inhibition of ADP-induced
platelet aggregation is directly correlated with inhibition of P2Y12 receptor present on
platelets. The results are expressed as the half-maximal inhibitory concentration (IC50) values
presented in Table 3; the mean values of experiments were performed in triplicate. The
benzotriazole possesses platelet aggregation inhibitory activity and its derivatives could
mimic the imidazopyrimidine of ADP as well as triazolopyrimidine of ticagrelor (selective
P2Y12 inhibitor). The introduction of nitro group increased the activity by more than three
times. The activity increases with increase in alkyl chain length octyl derivatives (1-Octyl-4-
nitro-1H-benzotriazole; IC50 = 0.91 ± 0.43 mM) is more potent than heptyl (1-Heptyl-4-nitro-
1H-benzotriazole; IC50 = 0.93 ± 0.92 mM) ones and 1-isomers were found to be more potent

than 2-isomers except methyl derivative. Introduction of aromatic ring result in marked
increase in potency like benzoyl [(4-Nitro-1H-benzotriazol-1-yl)(phenyl)methanone; IC50 =
0.65 ± 0.10 mM], benzyl (1-Benzyl-4-nitro-1H-benzotriazole; IC50 = 0.81 ± 0.08 mM, 2-
Benzyl-4-nitro-2H-benzotriazole; IC50 = 0.82 ± 0.19 mM) and tosyl derivatives (1-[(4-
Methylphenyl)sulfonyl]-4-nitro-1H-benzotriazole; IC50 = 0.86 ± 0.14 mM). The introduction
of polar chloro group in alkyl chain i.e., 3-chloropropyl (1-(3-Cholropropyl)-4-nitro-1H-
benzotriazole; IC50 = 1.07 ± 0.96 mM) was found to be more active than propyl analogue (1-
Propyl-4-nitro-1H-benzotriazole; IC50 = 1.30 ± 0.35 mM).
Table 3: Platelet aggregation inhibitory activity of compounds (1-23)
a,b
Compounds
IC50
Ticagrelor
0.32 ± 0.02c
7.43 ± 0.23
2.06 ± 0.20
1.84 ± 0.36
1.96 ± 0.27
1.80 ± 0.49
1.50 ± 0.32
1.62 ± 0.54
1.30 ± 0.35
1.32 ± 0.97
1.05 ± 0.97
1.07 ± 0.87
1.07 ± 0.96
1.02 ± 0.81
1.04 ± 0.93
0.94 ± 1.29
0.95 ± 1.33
0.93 ± 0.92
0.94 ± 1.06
0.92 ± 0.81
0.91 ± 0.43
Benzotriazole
4-Nitrobenzotriazole
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
Compound 10
Compound 11
Compound 12
Compound 13
Compound 14
Compound 15
Compound 16
Compound 17
Compound 18

Compound 19
Compound 20
Compound 21
Compound 22
Compound 23
1.26 ± 0.17
0.81 ± 0.08
0.82 ± 0.19
0.65 ± 0.10
0.86 ± 0.14
a IC50 is reported in mM (required to produce 50% inhibition in ADP induced platelet
aggregation); b n=3; c IC50 is reported in µM
2.3.1. P2Y12 Binding Assay:
P2Y12 binding assay was carried out using CY-QUANT VASP/P2Y12 ELISA kit. This test
is highly selective and equivalent to flow cytometric PLT VASP/P2Y12 assay [29-33]. All
compounds were tested at 200 µM concentration and inhibited the effect of ADP on PGE1
induced phosphorylation. The PGE1 induced phosphorylation was set as 100% and ADP
inhibited this phosphorylation completely. All compounds were able to block effect of ADP
on this PGE1 induced phosphorylation (Table 4), indicating their specific binding
(antagonist) to P2Y12. Maximum effect was observed for the benzoyl derivative, followed by
benzyl derivatives, indicating the role of aromatic ring in the binding. No per se effect at 200
µM concentration was observed for all compounds. Hence, indicating that compounds do not
possess P2Y12 agonistic activity.
Table 4: Percent Phosphorylation of compounds (1-23)
Compounds
% Phosphorylationa
Ticagrelor
98.2 ± 7.5
8.1 ± 23.6
19.3 ± 22.4
20.4 ± 8.2
20.8 ± 9.8
21.8 ± 8.7
22.5 ± 9.3
21.6 ± 10.5
26.9 ± 12.4
27.1 ± 11.8
34.3 ± 10.5
33.8 ± 10.7
Benzotriazole
4-Nitrobenzotriazole
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9

Compound 10
Compound 11
Compound 12
Compound 13
Compound 14
Compound 15
Compound 16
Compound 17
Compound 18
Compound 19
Compound 20
Compound 21
Compound 22
Compound 23
a n=3
33.7 ± 10.6
32.7 ± 15.9
33.2 ± 13.6
36.1 ± 17.4
37.4 ± 13.1
40.2 ± 9.7
37.4 ± 18.0
39.4 ± 12.1
37.8 ± 13.4
27.4 ± 11.7
40.3 ± 10.7
41.8 ± 8.5
43.8 ± 9.3
39.4 ± 11.8
3. Conclusions
Consequently, alkylation of 4-nitrobenzotriazole in sodium hydroxide was tedious,
perhaps due to severe conditions in sodium hydroxide result in degradation and it precedes
alkylation. Hence, the reactions were carried out in milder basic conditions; potassium
carbonate in DMF. Alkylation of 5-nitrobenzotriazole with potassium carbonate and DMF
with stirring for overnight to 24 hours is reported recently, while Diehl et al reported reaction
of 4-nitrobenzotriazole with sodium hydroxide and acetonitrile in 30 hours. In 4-
nitrobenzotriazole, the reaction is completed within 30 minutes indicating regioactivation
toward nucleophilic substitution of benzotriazole by 4-nitro group which is more activating
than 5-nitro group. The ADP-induced platelet aggregation inhibitory activity of benzotriazole
derivatives may be due to structural similarity with the imidazopyrimidine of ADP as well as
ticagrelor triazolopyrimidine. The introduction of nitro group increased the activity by more
than three times. Hence, bolstering the hypothesis that the derivatives of 4-nitrobenzotriazole
possess platelet aggregation inhibitory activity. The activity increases with increase in alkyl
chain length: octyl derivatives (Compound 18; IC50 = 0.91 ± 0.43 mM) was more potent than
methyl (Compound 2; IC50 = 1.96 ± 0.27 mM). Amongst the positional isomers, 1-isomers
were found to be more potent than 2-isomers except for the methyl derivative. The

introduction of polar chloro group in alkyl chain i.e., 3-chloropropyl (Compound 10; IC50 =
1.07 ± 0.96 mM) was found to be more active than propyl analogue (Compound 6; IC50 =
1.30 ± 0.35 mM). Introduction of aromatic ring result in marked increase in potency like
benzoyl (Compound 22; IC50 = 0.65 ± 0.10 mM), benzyl (Compound 20; IC50 = 0.81 ± 0.08
mM) and tosyl derivatives (Compound 21; IC50 = 0.82 ± 0.19 mM) and thus indicating the
role of aromatic ring in the P2Y12 binding. These three compounds could be further modified
in order to achieve equipotent compound as ticagrelor.
4. Experimental Section
4.1. Chemistry general
Melting points reported are uncorrected. Synthetic procedures employed were monitored for
completion of product formation by Thin Layer Chromatography (TLC) employing 7 cm x
2.5 cm Silica gel 60 F254 precoated TLC plates by Merck. Nuclear Magnetic Resonance
Spectra were recorded on Bruker Avance DPX-200 (400MHz). In 1H-NMR chemical shifts
(δ) are reported in parts per million (ppm) using tetramethylsilane as internal standard.
Coupling constants (J) are reported in Hz (Hertz). Multiplicities are reported using the
following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd =
double doublet, sx = sextet, qu = quintet. Waters Micromass Q-tof Micromass Spectrometer for
HRMS. Waters HPLC system equipped with a pump, an autosampler, a Dual UV detector set
at 300 nm, and an analytical column (Waters, XTerra, RP18, 4.6 mm × 50 mm, 5 μm). The
mobile phase consisted of varying ratios of HPLC grade acetonitrile and triple distilled water
The flow rate was set at 1 mL/min. IR spectra were measured on a Hitachi 270-30 infrared and
Bruker Vector 22 Spectrophotometers. IR spectra were recorded as KBr pellets.
4.1.1. General Procedure for Synthesis 4-nitrobenzotriazole. To cool solution of 2.65 mL
of nitric acid and 10 mL sulfuric acid was added drop wise in a solution of 5.37 g
benzotriazole in 10 mL sulfuric acid. The reaction was kept at room temperature overnight.
The reaction mixture was then over ice and yellow precipitates were formed which are
filtered, washed with water, and dried. The purification was done by slurrying of
nitrobenzotriazole in hot acetonitrile and product was filtered [25].
4.1.2. General Procedure for Synthesis of Alkyl derivatives of 4-nitrobenzotriazole (1-
21). To the mixture of 4-nitrobenzotriazole 10 mmol and 40 mmol dry potassium carbonate,
8-10 mL dry N,N-dimethylformamide was added. Corresponding 10 mmoles of alkyl halides
were added and stirred for 30 minutes [26]. The mixture was washed with water. 1- and 2-

alkyl substituted derivatives were separated using column chromatography (Silica gel #200-
400, Petroleum ether: Ethyl acetate, 95:05).
4.1.3. General Procedure for Synthesis of Acyl/Sulfonyl derivatives (22, 23). To the
mixture of 1.00 mmol of 4-nitrobenzotriazole in 3 mL dry pyridine, 1.80 mmoles of benzoyl
chloride, tosyl chloride were added in small proportions and resultant mixture was heated to
boiling for 30 minutes. After completion of the reaction, the reaction mixture was acidified
with 2M HCl and poured on crushed ice [34]. Precipitated product was filtered and washed
well with water. The product was recrystallized using ethanol (95 %) to give compounds 22
and 23. The acylation of benzotriazole forms thermodynamically more stable 1-acyl
derivative [35].
4.1.4. 4-Nitro-1H-benzotriazole. Yellow Solid; yield: 5.37 g (73%); m.pt. 228-230 oC; 1H-
NMR (400 MHz, CDCl3): δ 16.62 (broad s, 1H, ArH), 8.57-8.55 (d, J = 8.16 Hz, 1H, Ar H),
8.45-8.43 (d, J = 7.8 Hz, 1H, Ar H), 7.66-7.62 (t, J = 8.04 Hz, 1H, Ar H); 13C-NMR (100
MHz, CDCl3): δ 140.02, 139.63, 125.13, 123.66, 118.65, 108.95; HRMS: 165.1214 [ES+];
Exact mass calcd for C6H4N4O2 164.1215 [m/z]; IR (cm-1) 3418, 3247, 1512, 1479, 1337,
1178, 1024, 926, 851, 684, 542; HPLC Purity 100 % at 300 nm, tR= 2.837 min.
1
4.1.5. 1-Methyl-4-nitro-1H-benzotriazole (1). 58 mg; Creamish Solid; m.pt. 203 oC; H-
NMR (400 MHz, CDCl3): δ 8.31-8.29 (d, J = 7.64 Hz, 1H, ArH), 7.94-7.92 (d, J = 8.4 Hz,
1H, Ar H), 7.70-7.66 (t, J = 8.0 Hz, 1H, Ar H), 4.43 (s, 3H, CH3); 13C-NMR (100 MHz,
CDCl3): δ 138.48, 135.84, 126.66, 121.29, 116.26, 34.94; HRMS: 179.1477 [ES+]; Exact
mass calcd for C7H6N4O2 178.1481 [m/z]; IR (cm-1) 3082, 1965, 1855, 1522, 1451, 1399,
1346, 1288, 1059, 885, 822, 736, 577; HPLC Purity 99.61 % at 300 nm, tR= 2.931 min.
4.1.6. 2-Methyl-4-nitro-2H-benzotriazole (2). 32 mg; Creamish Solid; m.pt. 184 oC; 1H-NMR
(400 MHz, CDCl3): δ 8.43-8.41 (d, J = 8.16 Hz, 1H, ArH), 8.37-8.35 (d, J = 7.88 Hz, 1H, Ar
H), 7.53-7.49 (t, J = 8.04 Hz, 1H, Ar H), 4.61 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3): δ
149.16, 138.43, 126.17, 122.13, 118.59, 39.46; HRMS: 179.1479 [ES+]; Exact mass calcd
for C7H6N4O2 178.1481 [m/z]; IR (cm-1) 3068, 1960, 1837, 1519, 1457, 1392, 1338, 1281,
1055, 883, 819, 731, 568; HPLC Purity 100 % at 300 nm, tR= 2.945 min.
4.1.7. 1-Methyl-7-nitro-1H-benzotriazole (3). 10 mg; Brownish Solid; m.pt. 113 oC; 1H-NMR
(400 MHz, CDCl3): δ 8.43-8.41 (d, J = 7.56 Hz, 1H, ArH), 8.28-8.26 (d, J = 8.84 Hz, 1H, Ar
H), 7.57-7.53 (t, J = 8.16 Hz, 1H, Ar H), 4.66 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3): δ

141.16, 125.02, 124.67, 120.70, 108.41, 31.78; HRMS: 179.1480 [ES+]; Exact mass calcd
for C7H6N4O2 178.1481 [m/z]; IR (cm-1) 3064, 1943, 1851, 1504, 1453, 1397, 1348, 1286,
1054, 885, 814, 734, 572; HPLC Purity 98.51 % at 300 nm, tR= 2.844 min.
4.1.8. 1-Ethyl-4-nitro-1H-benzotriazole (4). 57 mg; Yellow-Creamish Solid; 1H-NMR (400
MHz, CDCl3): δ 8.42-8.40 (d, J = 7.68 Hz, 1H, ArH), 8.29-8.26 (d, J = 8.46 Hz, 1H, Ar H),
7.56-7.52 (t, J = 8.08 Hz, 1H, Ar H), 4.96-4.91 (q, J = 7.36 Hz, 2H, N-CH2), 1.80-1.76 (t, J =
7.4 Hz, 3H, N-CH3); 13C-NMR (100 MHz, CDCl3): δ 142.36, 127.74, 126.46, 121.20, 119.15,
64.83, 29.66, 21.76; HRMS: 193.1746 [ES+]; Exact mass calcd for C8H8N4O2 192.1747
[m/z]; IR (cm-1) 3078, 1961, 1852, 1517, 1446, 1395, 1341, 1281, 1054, 881, 827, 733, 572;
HPLC Purity 98.45 % at 300 nm, tR= 2.18 min.
4.1.9. 2-Ethyl-4-nitro-2H-benzotriazole (5). 33 mg; Yellow-Creamish Solid; 1H-NMR (400
MHz, CDCl3): δ 8.44-8.42 (d, J = 8.24 Hz, 1H, ArH), 8.37-8.35 (d, J = 7.84 Hz, 1H, Ar H),
7.53-7.49 (t, J = 8.04 Hz, 1H, Ar H), 5.11-5.06 (q, J = 7.2 Hz, 2H, N-CH2), 1.57-1.54 (t, J =
7.2 Hz, 3H, N-CH3); 13C-NMR (100 MHz, CDCl3): δ 147.28, 125.81, 124.29, 118.94, 113.62,
65.02, 21.13, 18.41; HRMS: 193.1743 [ES+]; Exact mass calcd for C8H8N4O2 192.1747
[m/z]; IR (cm-1) 3085, 1963, 1852, 1527, 1446, 1395, 1342, 1289, 1054, 887, 829, 732, 574;
HPLC Purity 99.39 % at 300 nm, tR= 2.03 min.
4.1.10. 1-Propyl-4-nitro-1H-benzotriazole (6). 69 mg; Yellow Oil; 1H-NMR (400 MHz,
CDCl3): δ 8.42-8.40 (d, J = 7.62 Hz, 1H, ArH), 8.29-8.26 (d, J = 8.46 Hz, 1H, Ar H), 7.56-
7.52 (t, J = 8.08 Hz, 1H, Ar H), 4.86-4.82 (t, J = 7.24 Hz, 2H, N-CH2), 2.24-2.19 (sx, J =
7.32 Hz, 2H, N-CH2), 1.04-1.00 (t, J = 7.49 Hz, 3H, N-CH3); 13C-NMR (100 MHz, CDCl3): δ
143.46, 131.16, 129.04, 124.72, 114.19, 61.20, 27.96, 18.63; HRMS: 207.2009 [ES+]; Exact
mass calcd for C9H10N4O2 206.2013 [m/z]; IR (cm-1) 3086, 2969, 2878, 1727, 1627, 1528,
1446, 1390, 1346, 1307, 1200, 1152, 1104, 1051, 1022, 987, 882, 821, 738, 595; HPLC
Purity 100 % at 300 nm, tR= 2.941 min.
4.1.11. 2-Propyl-4-nitro-2H-benzotriazole (7). 38 mg; Brownish Oil; 1H-NMR (400 MHz,
CDCl3): δ 8.30-8.28 (d, J = 7.68 Hz, 1H, ArH), 7.92-7.90 (d, J = 8.24 Hz, 1H, Ar H), 7.66-
7.62 (t, J = 5.24 Hz, 1H, Ar H), 4.73-4.69 (t, J = 7.12 Hz, 2H, N-CH2), 2.12-2.06 (sx, J =
7.28 Hz, 2H, N-CH2), 1.02-0.98 (t, J = 7.44 Hz, 3H, N-CH3); 13C-NMR (100 MHz, CDCl3): δ
144.72, 130.42, 127.84, 125.67, 115.36, 63.18, 28.71, 17.29; HRMS: 207.2011 [ES+]; Exact
mass calcd for C9H10N4O2 206.2013 [m/z]; IR (cm-1) 2997, 2875, 1724, 1629, 1527, 1446,
1361, 1148, 1053, 984, 829, 735, 597; HPLC Purity 100 % at 300 nm, tR= 2.861 min.

4.1.12. 1-Butyl-4-nitro-1H-benzotriazole (8). 72 mg; Light-Brown Oil; 1H-NMR (400 MHz,
CDCl3): δ 8.29-8.27 (d, J = 7.68 Hz, 1H, ArH), 7.93-7.91 (d, J = 8.16 Hz, 1H, Ar H), 7.67-
7.63 (t, J = 7.96 Hz, 1H, Ar H), 4.76-4.73 (t, J = 7.12 Hz, 2H, N-CH2), 2.06-2.03 (qu, J =
7.36 Hz, 2H, N-CH2), 1.44-1.38 (t, J = 7.52 Hz, 2H, N-CH2), 1.00-0.96 (t, J = 7.32 Hz, 3H,
N-CH3); 13C-NMR (100 MHz, CDCl3): δ 146.45, 137.22, 127.66, 124.74, 123.03, 57.29,
52.21, 33.07, 19.80, 13.52; HRMS: 221.2277 [ES+]; Exact mass calcd for C10H12N4O2
220.2278 [m/z]; IR (cm-1) 3014, 2877, 1721, 1624, 1529, 1438, 1346, 1126, 1023, 973, 831,
727, 592; HPLC Purity 100 % at 300 nm, tR= 3.064 min.
4.1.13. 2-Butyl-4-nitro-2H-benzotriazole (9). 39 mg; Dark-Brown Oil; 1H-NMR (400 MHz,
CDCl3): δ 8.41-8.39 (d, J = 7.64 Hz, 1H, ArH), 8.27-8.25 (d, J = 8.44 Hz, 1H, Ar H), 7.55-
7.50 (t, J = 8.2 Hz, 1H, Ar H), 4.89-4.85 (t, J = 7.28 Hz, 2H, N-CH2), 2.20-2.16 (qu, J = 7.36
Hz, 2H, N-CH2), 1.45-1.38 (sx, J = 7.52 Hz, 2H, N-CH2), 1.01-0.97 (t, J = 7.36 Hz, 3H, N-
CH3); 13C-NMR (100 MHz, CDCl3): δ 145.34, 136.49, 126.13, 125.39, 124.74, 123.03,
52.21, 32.07, 19.65, 13.482; HRMS: 221.2278 [ES+]; Exact mass calcd for C10H12N4O2
220.2278 [m/z]; IR (cm-1) 3012, 2879, 1723, 1627, 1524, 1435, 1349, 1125, 1026, 976, 834,
724, 596; HPLC Purity 100 % at 300 nm, tR= 2.885 min.
4.1.14. 1-Pentyl-4-nitro-1H-benzotriazole (10). 79 mg; Brownish Oil; 1H-NMR (400 MHz,
CDCl3): δ 8.44-8.42 (d, J = 8.96 Hz, 1H, ArH), 8.29-8.26 (d, J = 8.44 Hz, 1H, Ar H), 7.57-
7.52 (t, J = 8.12 Hz, 1H, Ar H), 4.88-4.85 (t, J = 7.32 Hz, 2H, N-CH2), 2.22-2.14 (qu, J =
7.52 Hz, 2H, N-CH2), 1.42-1.33 (m, 4H, N-CH2CH2), 0.93-0.89 (t, J = 6.88 Hz, 3H, N-CH3);
13C-NMR (100 MHz, CDCl3): δ 146.44, 127.52, 126.16, 125.40, 124.38, 123.04, 57.56,
30.81, 28.63, 23.48, 13.85; HRMS: 235.2543 [ES+]; Exact mass calcd for C11H14N4O2
234.2544 [m/z]; IR (cm-1) 3027, 2972, 1734, 1624, 1537, 1432, 1352, 1129, 1028, 967, 837,
723, 596; HPLC Purity 99.89 % at 300 nm, tR= 2.714 min.
4.1.15. 2-Pentyl-4-nitro-2H-benzotriazole (11). 37 mg; Dark-Brown Oil; 1H-NMR (400
MHz, CDCl3): δ 8.41-8.39 (d, J = 7.68 Hz, 1H, ArH), 8.28-8.26 (d, J = 8.48 Hz, 1H, Ar H),
7.55-7.51 (t, J = 7.96 Hz, 1H, Ar H), 4.88-4.84 (t, J = 7.36 Hz, 2H, N-CH2), 2.22-2.14 (qu, J
= 7.48 Hz, 2H, N-CH2), 1.45-1.30 (m, 4H, N-CH2CH2), 0.93-0.89 (t, J = 6.76 Hz, 3H, N-
CH3); 13C-NMR (100 MHz, CDCl3): δ 145.84, 127.03, 125.61, 124.75, 124.01, 122.97,
52.46, 29.94, 28.52, 18.12, 12.91; HRMS: 235.2541 [ES+]; Exact mass calcd for C11H14N4O2
234.2544 [m/z]; IR (cm-1) 3024, 2975, 1724, 1622, 1526, 1437, 1348, 1124, 1026, 964, 835,
726, 598; HPLC Purity 96.74 % at 300 nm, tR= 2.769 min.

4.1.16. 1-Hexyl-4-nitro-1H-benzotriazole (12). 83 mg; Brownish Oil; 1H-NMR (400 MHz,
CDCl3): δ 8.30-8.28 (d, J = 7.72 Hz, 1H, ArH), 7.92-7.89 (d, J = 8.24 Hz, 1H, Ar H), 7.66-
7.62 (t, J = 8.0 Hz, 1H, Ar H), 4.75-4.71 (t, J = 7.16 Hz, 2H, N-CH2), 2.05-1.85 (qu, J = 7.16
Hz, 2H, N-CH2), 1.42-1.33 (m, 6H, N-CH2CH2CH2), 0.87-0.82 (t, J = 7.12 Hz, 3H, N-CH3);
13C-NMR (100 MHz, CDCl3): δ 146.52, 128.83, 126.40, 121.14, 116.26, 108.39, 67.65,
48.96, 31.11, 29.76, 26.32, 13.91; HRMS: 249.2805 [ES+]; Exact mass calcd for C12H16N4O2
248.2810 [m/z]; IR (cm-1) 2952, 2927, 2868, 1724, 1528, 1459, 1347, 1307, 815, 740; HPLC
Purity 97.43 % at 300 nm, tR= 2.954 min.
4.1.17. 2-Hexyl-4-nitro-2H-benzotriazole (13). 22 mg; Yellowish Oil; 1H-NMR (400 MHz,
CDCl3): δ 8.42-8.39 (d, J = 7.7 Hz, 1H, ArH), 8.28-8.26 (d, J = 8.44 Hz, 1H, Ar H), 7.56-
7.52 (t, J = 8.06 Hz, 1H, Ar H), 4.88-4.84 (t, J = 7.36 Hz, 2H, N-CH2), 2.21-2.14 (qu, J =
7.28 Hz, 2H, N-CH2), 1.69-1.32 (m, 6H, N-CH2CH2CH2), 1.31-1.29 (t, J = 3.36 Hz, 3H, N-
CH3); 13C-NMR (100 MHz, CDCl3): δ 145.59, 127.68, 125.13, 122.65, 116.19, 109.14,
66.93, 47.18, 30.49, 28.81, 26.73, 14.63; HRMS: 249.2808 [ES+]; Exact mass calcd for
C12H16N4O2 248.2810 [m/z]; IR (cm-1) 2955, 2924, 2865, 1526, 1463, 1346, 1302, 813, 746;
HPLC Purity 99.71 % at 300 nm, tR= 2.974 min.
4.1.18. 1-Heptyl-4-nitro-1H-benzotriazole (14). 87 mg; Dark-Brown Oil; 1H-NMR (400
MHz, CDCl3): δ 8.23-8.21 (d, J = 7.6 Hz, 1H, ArH), 7.86-7.83 (d, J = 8.38 Hz, 1H, Ar H),
7.60-7.56 (t, J = 7.88 Hz, 1H, Ar H), 4.99-4.83 (t, J= 7.16 Hz, 2H, N-CH2), 1.54-1.17 (m,
10H, N-CH2CH2CH2CH2CH2), 0.81-0.75 (t, J = 6.8 Hz, 3H, N-CH3); 13C-NMR (100MHz,
CDCl3): δ 146.44, 137.21, 126.17, 125.40, 124.40, 123.04, 57.58, 52.49, 31.58, 31.11, 28.67,
26.36, 14.02; HRMS: 263.3075 [ES+]; Exact mass calcd for C13H18N4O2 262.3076 [m/z]; IR
(cm-1) 3029, 2854, 1713, 1643, 1538, 1431, 1349, 1134, 1025, 964, 837, 714, 583; HPLC
Purity 99.79 % at 300 nm, tR= 3.188 min.
4.1.19. 2-Heptyl-4-nitro-2H-benzotriazole (15). 33 mg; Dark-Brown Oil; 1H-NMR (400
MHz, CDCl3): δ 8.40-8.38 (d, J = 7.74 Hz, 1H, ArH), 8.27-8.25 (d, J = 8.48 Hz, 1H, Ar H),
7.55-7.51 (t, J = 8.1 Hz, 1H, Ar H), 4.68-4.65 (t, J = 7.4 Hz, 2H, N-CH2), 1.40-1.22 (m, 10H,
N-CH2CH2CH2CH2CH2), 0.87-0.80 (t, J = 7.4 Hz, 3H, N-CH3); 13C-NMR (100 MHz,
CDCl3): δ 146.21, 136.80, 127.53, 125.03, 124.76, 122.16, 56.06, 51.70, 31.54, 30.15, 26.49,
22.52, 13.15; HRMS: 264.3073 [ES+]; Exact mass calcd for C13H18N4O2 262.3076 [m/z]; IR
(cm-1) 3024, 2857, 1715, 1641, 1535, 1432, 1346, 1138, 1027, 961, 835, 718, 586; HPLC
Purity 99.53 % at 300 nm, tR= 2.937 min.

4.1.20. 1-Octyl-4-nitro-1H-benzotriazole (16). 93 mg; Dark-Brown Oil; 1H-NMR (400 MHz,
CDCl3): δ 8.29-8.27 (d, J = 7.44 Hz, 1H, ArH), 7.93-7.91 (d, J = 8.16 Hz, 1H, Ar H), 7.67-
7.63 (t, J = 8.04 Hz, 1H, Ar H), 4.75-4.72 (t, J = 7.2 Hz, 2H, N-CH2), 2.05-1.21 (m, 12H, N-
CH2CH2CH2CH2CH2CH2), 0.87-0.83 (t, J = 6.68 Hz, 3H, N-CH3); 13C-NMR (100 MHz,
CDCl3): δ 141.34, 136.94, 128.02, 126.16, 124.75, 113.52, 57.58, 31.69, 30.14, 29.02, 28.95,
26.52, 22.59, 14.06; HRMS: 277.3339 [ES+]; Exact mass calcd for C14H20N4O2 276.3342
[m/z]; IR (cm-1) 3042, 2914, 1723, 1637, 1532, 1427, 1342, 1139, 1022, 963, 834, 723, 597;
HPLC Purity 99.76 % at 300 nm, tR= 3.276 min.
4.1.21. 2-Octyl-4-nitro-2H-benzotriazole (17). 24 mg; Dark-Brown Oil; 1H-NMR (400 MHz,
CDCl3): δ 8.41-8.39 (d, J = 7.64 Hz, 1H, ArH), 8.28-8.25 (d, J = 8.36 Hz, 1H, Ar H), 7.55-
7.51 (t, J = 8.2 Hz, 1H, Ar H), 4.87-4.84 (t, J = 7.36 Hz, 2H, N-CH2), 2.19-1.25 (m, 12H, N-
CH2CH2CH2CH2CH2CH2), 0.88-0.83 (t, J = 6.64 Hz, 3H, N-CH3); 13C-NMR (100 MHz,
CDCl3): δ 142.85, 135.36, 126.42, 121.17, 116.36, 113.52, 48.97, 31.66, 29.79, 29.01, 28.93,
26.66, 22.57, 14.05; HRMS: 277.3337 [ES+]; Exact mass calcd for C14H20N4O2 276.3342
[m/z]; IR (cm-1) 3039, 2917, 1728, 1634, 1537, 1423, 1092, 965, 902, 692, 596; HPLC Purity
99.43 % at 300 nm, tR= 3.059 min.
4.1.22. 1-(3-Cholropropyl)-4-nitro-1H-benzotriazole (18). 53 mg; Light-Brownish Oil; 1H-
NMR (400 MHz, CDCl3): δ 8.31-8.29 (d, J = 7.16 Hz, 1H, ArH), 8.02-8.00 (d, J = 7.84 Hz,
1H, Ar H), 7.71-7.67 (t, J = 8.02 Hz, 1H, Ar H), 4.95-4.92 (t, J = 6.56 Hz, 2H, N-CH2), 3.56-
3.53 (t, J = 5.8 Hz, 2H, N-CH2Cl), 2.60-2.54 (qu, J = 6.48 Hz, 2H, N-CH2); 13C-NMR (100
MHz, CDCl3): δ 146.47, 137.39, 127.79, 125.77, 124.66, 123.33, 54.42, 41.24, 33.55;
HRMS: 241.6458 [ES+]; Exact mass calcd for C9H9ClN4O2 240.6463 [m/z]; IR (cm-1) 3064,
2863, 1928, 1782, 1657, 1583, 1397, 1095, 962, 812, 768, 747, 672, 583; HPLC Purity 98.51
% at 300 nm, tR= 2.844 min.
4.1.23. 2-(3-Cholropropyl)-4-nitro-2H-benzotriazole (19). 47 mg; Dark-Brownish Oil; 1H-
NMR (400 MHz, CDCl3): δ 8.42-8.40 (d, J = 7.88 Hz, 1H, ArH), 8.29-8.27 (d, J = 8.58 Hz,
1H, Ar H), 7.58-7.54 (t, J = 8.04 Hz, 1H, Ar H), 5.08-5.05 (t, J = 6.76 Hz, 2H, N-CH2), 3.67-
3.64 (t, J = 6.16 Hz, 2H, N-CH2Cl), 2.69-2.62 (qu, J = 6.56 Hz, 2H, N-CH2); 13C-NMR (100
MHz, CDCl3): δ 145.26, 128.78, 126.88, 121.39, 116.22, 106.32, 45.32, 41.16, 32.27;
HRMS: 240.6459 [ES+]; Exact mass calcd for C9H9ClN4O2 240.6463 [m/z]; IR (cm-1) 3063,
2879, 1925, 1786, 1652, 1584, 1394, 1092, 965, 817, 763, 741, 676, 587; HPLC Purity 97.09
% at 300 nm, tR= 2.686 min.

4.1.24. 1-Benzyl-4-nitro-1H-benzotriazole (20). 52 mg; Creamish Solid; m.pt. 123-125 oC;
1H-NMR (400 MHz, CDCl3): δ 8.26-8.24 (d, J = 7.7 Hz, 1H, ArH), 7.71-7..69 (d, J = 8.4 Hz,
1H, Ar H), 7.56-7.52 (t, J = 7.84 Hz, 1H, Ar H), 7.37-7.34 (m, 3H-Benzyl), 7.29-7.26 (m,
2H-Benzyl), 5.96 (s, 2H, CH2); 13C-NMR (100 MHz, CDCl3): δ 146.13, 129.81, 129.03,
128.46, 127.93, 127.02, 126.79, 125.61, 124.35, 123.60, 117.72, 58.16; HRMS: 255.2439
[ES+]; Exact mass calcd for C14H20N4O2 254.2441 [m/z]; IR (cm-1) 3084, 2974, 1897, 1532,
1476, 1327, 1126, 1084, 963, 902, 683, 592; HPLC Purity 99.90 % at 300 nm, tR= 2.723 min.
4.1.25. 2-Benzyl-4-nitro-2H-benzotriazole (21). 28 mg; Creamish Solid; m.pt. 93-95 oC; 1H-
NMR (400 MHz, CDCl3): δ 8.44-8.42 (d, J = 8.24 Hz, 1H, ArH), 8.23-8.21 (d, J = 7.76 Hz,
1H, Ar H), 7.54-7.48 (m, 1H-Ar, 1H-Benzyl), 7.38-7.37 (m, 2H-Benzyl), 7.26-7.24 (distorted
t, J = 5.88 Hz, 1H-Benzyl), 7.09-7.07 (distorted t, J = 2.6 Hz, 1H-Benzyl), 6.01 (s, 2H, CH2);
13C-NMR (100 MHz, CDCl3): δ 146.34, 129.94, 129.36, 128.2, 127.98, 127.11, 126.19,
125.43, 124.02, 123.14, 117.09, 57.42; HRMS: 255.2438 [ES+]; Exact mass calcd for
C14H20N4O2 254.2441 [m/z]; IR (cm-1) 3083, 2978, 1895, 1536, 1475, 1329, 1123, 1081, 967,
905, 686, 594; HPLC Purity 99.04 % at 300 nm, tR= 2.729 min.
4.1.26. (4-Nitro-1H-benzotriazol-1-yl)(phenyl)methanone (22). Yellowish Solid; yield: 84%;
1H-NMR (400MHz, CDCl3): δ 8.43-8.41 (d, J = 8.36 Hz, 1H, ArH), 8.25-8.23 (d, J = 8.56
Hz, 1H, Ar H), 7.53-7.48 (m, 2H, Ar H), 7.38-7.37 (d, J = 3.32 Hz, 1H-Ar), 7.26-7.22 (m,
2H-Ar), 7.08-7.06 (distorted t, J = 5.24 Hz, 1H-Ar); 13C-NMR (100MHz, CDCl3): δ 154.36,
147.82, 142.19, 134.61, 131.04, 129.57, 128.66, 127.16, 124.31, 122.96, 121.25, 116.49,
114.26; HRMS: 269.2268 [ES+]; Exact mass calcd for C13H18N4O3 268.2272 [m/z]; IR (cm-1)
3273, 1527, 1481, 1349, 1127, 1093, 960, 905, 687, 598; Purity 98.32 % at 300 nm, tR= 2.837
min.
4.1.27. 1-[(4-Methylphenyl)sulfonyl]-4-nitro-1H-benzotriazole (23). Creamish Solid; yield:
92%; 1H-NMR (400MHz, CDCl3): δ 8.56-8.54 (d, J = 8.44 Hz, 1H, ArH), 8.47-8.45 (d, J =
7.92 Hz, 1H, Ar H), 7.65-7.61 (t, J = 8.04 Hz Ar H), 7.57-7.55 (d, J = 8.04 Hz, 2H-Tosyl),
7.15-7.13 (d, J = 7.96 Hz, 2H- Tosyl), 2.32 (s, 3H, CH3); 13C-NMR (100MHz, CDCl3): δ
145.23, 138.69, 136.41, 132.67, 129.83, 129.04, 128.52, 127.13, 126.49, 123.26, 121.89,
117.23, 26.12; HRMS: 319.3084 [ES+]; Exact mass calcd for C13H10N4O4S 318.3079 [m/z];
IR (cm-1) 3283, 1543, 1481, 1328, 1134, 1093, 963, 914, 676, 574; HPLC Purity 99.71 % at
300 nm, tR= 2.623 min.

4.2.
Platelet aggregation inhibitory activity: Platelet aggregation assay was carried out
using Bruker spectrophotometer. PRP (platelet rich plasma) diluted to give an absorbance
unit of 0.5 apx. at 540 nm were taken in quartz cuvette. The 10 µL of ADP was added to the
sample cuvette and mixed. The standard drug ticagrelor and other compounds in
concentration range of 0.1 M to 0.01 nM were added to PRP before the addition of ADP.
Their absorbance was monitored at every 2 minutes and IC50 was calculated based on
aggregation of platelets after 10 minutes [27].
4.2.1. P2Y12-mediated vasodilator-stimulated phosphoprotein (VASP)
phosphorylation assay: VASP phosphorylation was measured by ELISA method using a
CY-QUANT VASP/P2Y12 kit. The general procedure was followed as manufacturer’s
recommendations. The effect of PGE1 on platelets was observed as maximum
phosphorylation. The ADP was taken as standard inhibitor of phosphorylation as P2Y12
agonist. Ticagrelor was standard drug and compounds were tested at 200 µM concentration
along with ADP in test solution. Furthermore, per se effect at this concentration was checked
using PGE1 only without ADP on platelets.
4.3. Docking Studies: 4.3.1. Preparation of compounds: iGEMDOCK was used as
molecular docking tool for carrying out molecular docking simulations. iGEMDOCK is an
accurate and validated software based on GEMDOCK which uses a genetic evolutionary
method for molecular docking and an empirical scoring function [36]. All compounds were
prepared using ChemSketch software (ACD Labs, Canada). After that structure was cleaned
and explicit hydrogens were added. Finally structure was optimized to 3D so that it can be
used in iGEMDOCK. At the end structure was saved in MDL MolFile [V2000, (*.mol)]
format.
4.3.2. Preparation of protein: The crystal structure of 4-NTJ at 2.62 oA resolution was
retrieved from PDB (www.rscb.org). The structure contains seven transmembrane bundle of
α-helices and a carboxy-terminal helix VIII that is parallel to the membrane bilayer [21]. The
defined binding site was set to ‘By bounded ligand’ and standard parameters were set
automatically by the software. “AZJ” Was Set As Binding Cite Centre. After loading the
ligands, output path was set and then docking started. After completion of docking, ‘view
docked pose and post analysis’ was done. Under interaction profile, energy of each
compound was obtained and under interaction analysis interacting residues were obtained.
‘Molegro Molecular Viewer’ software was used to view all results.

Acknowledgement: We are grateful to S. Avtar Singh for NMR analysis (NMR Lab, SAIF
Panjab University, Chandigarh, India). We would like to thank Dr. VT Punniyakoti for
biological evaluation.
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 Highlights
 Regioactivation by 4-nitro group in benzotriazole n-alkylation
 Alkylation under milder basic conditions
 Degradation of 4-nitrobenzotriazole under strong basic conditions
 Antiplatelet aggregation inhibitory activity
 CY QUANT VASP/P2Y12 phosphorylation assay