SiO2-H3BO3 promoted solvent-free, green and sustainable synthesis of bioactive 1-substituted-1H-tetrazole analogues

Article abstract of DOI:10.1039/c4nj02079k

In the present study, a focused library of multi functionalized 1-substituted-1H-tetrazole analogues 4(a-o) were synthesized, which were typically accessed via a facile, solvent-free and green synthetic protocol. The reaction involves expeditious reusable catalyst (SiO₂-H₃BO₃) promoted condensation between a variety of heterocyclic/aromatic amines, sodium azide and triethyl ortho-formate, eliminating the use of an environmentally toxic solvent. This new eco-friendly and sustainable protocol resulted in a remarkable enhancement in the synthetic efficiency (90-97%, yield) with high purity. This silica-boric acid catalyst is air and water stable and easy to prepare from cheap silica and boric acid. The present methodology is a green protocol offering several advantages such as an excellent yield of products, a simple operational procedure, minimizing production of chemical wastes, mild reaction conditions, a shorter reaction profile, easy preparation of the catalyst and its recyclability up to five cycles without any appreciable loss in catalytic activity. The optimization conditions achieved in the present study revealed that 2.5 mol% of the SiO₂-H₃BO₃ catalyst under solvent-free conditions at 90 °C are the best suited conditions for the synthesis of tetrazole derivatives in excellent yields. The present protocol is applicable to a broader substrate scope (electron-rich and electron-deficient). Compounds possessing a nicotinic acid nucleus (4e, 4f, 4g) displayed strongest inhibition against AChE with IC₅₀ values of 0.43 μM, 0.21 μM and 0.26 μM, respectively. The results revealed that the electronic effect of substituents inflicts a prominent effect on AChE activity. This journal is

Full text of DOI:10.1039/c4nj02079k

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Page 1 of 39
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SiO2-H3BO3 promoted solvent-free, green and sustainable synthesis of bioactive 1-
substituted-1H-tetrazole analogues
Mehtab Parveen,*a Faheem Ahmad,a Ali Mohammed Malla,a Shaista Azaza
aDivision of Organic Synthesis, Aligarh Muslim University, Aligarh, 202002, India
Abstract
In the present study, a focused library of multi functionalized 1ꢀsubstitutedꢀ1Hꢀtetrazole
analogues 4 (a-o) were synthesized, which were typically accessed via a facile, solventꢀfree
and green synthetic protocol. The reaction involves expeditious reusable catalyst (SiO2ꢀ
H3BO3) promoted condensation between a variety of heterocyclic/aromatic amines, sodium
azide and triethyl orthoꢀformate, eliminating the use of environmentally toxic solvent. This
new ecoꢀfriendly and sustainable protocol resulted in a remarkable enhancement in the
synthetic efficiency (90ꢀ97 %, yield) with high purity. This silicaꢀboric acid catalyst is air and
water stable and easy to prepare from cheap silica and boric acid. The present methodology is
a green protocol offering several advantages such as excellent yield of products, simple
operational procedure, minimizing production of chemical wastes, mild reaction conditions,
shorter reaction profile, easy preparation of catalyst and its recyclability up to five cycles
without any appreciable loss in catalytic activity. The optimization conditions achieved in the
present study, revealed that 2.5 mol% of SiO2ꢀH3BO3 catalyst under solventꢀfree condition at
o
90 C are the best suited conditions for the synthesis of tetrazole derivatives in excellent
yields. The present protocol is applicable to a broader substrate scope (electronꢀrich and
electronꢀdeficient). Compounds possessing nicotinic acid nucleus (4e, 4f, 4g) displayed
strongest inhibition against AChE with IC50 values of of 0.43 ꢁM, 0.21 ꢁM and 0.26 ꢁM,
respectively. The results revealed that, electronic effect of substituents inflict prominent
effect on AChE activity.
Key words: Solventꢀfree, SiO2ꢀH3BO3, Tetrazoles derivatives, Green protocol, AChE, BuChE
*
Corresponding author: Mehtab Parveen
Eꢀmail: mehtab.organic2009@gmail.com; Tel: +91ꢀ9897179498

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1.
Introduction
Tetrazoles belong to a class of 5ꢀmembered nitrogen containing heterocyclic compounds
possessing highest nitrogen content among the known stable heterocycles. The first
compound comprising a tetrazole ring is thought to be 2ꢀphenylꢀ2Hꢀtetrazoleꢀ5ꢀcarbonitrile,
which was synthesized and characterized in 1885.1 In the last few decades, the chemistry of
Nꢀcontaining heterocycles (tetrazoles), have received considerable attention due to their
promising biological properties especially antiviral,2 anticonvulsant,3 antiinflammatory,4
antimicrobial,5 antinociceptive,6 antifungal,7 antiꢀHIV,8 antiulcer,9 antiallergic,10
antiproliferative,11 analgesic12 and anticancer.13 They have also been shown to be potential
P2X7ꢀantagonists,14 TNF alpha inhibitors15 and inhibitors of anandamide cellular uptake.16
The importance of tetrazole and its derivatives in medicinal chemistry has augmented
because of their use as lipophilic spacers and as metabolically stable surrogate for a
carboxylic acid group in drug design.17 A number of drugs approved by the Food and Drug
Administration (FDA) which are medicinally important possess tetrazole moiety in their
structures.18 It has been reported, that the lipophilic nature of tetrazole moiety in a drug
improves its oral bioavailability and cell penetration.19 In this regard, tetrazole chemistry has
gained so much interest due to their potential therapeutic profile. Valsartan, losartan and
irbesartan, are wellꢀknown antihypertensive drugs belonging to the class of nonpeptide
angiotensinꢀII inhibitors and possess biphenyl tetrazolyl moiety in their structure (Fig. 1).
Similarly, TAKꢀ456 a broadꢀspectrum antifungal drug also bears a tetrazole ring in its
structure (Fig. 1).

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Valsartan (Diovan)
Losartan
Irbesartan
TAKꢀ456
Fig. 1 Drugs in the market with a tetrazole moiety
Tetrazoles have also found a wide range of applications in materials as specialty
explosives,20 information recording systems,21 rocket propellants22 and agrichemical
applications.23 They are endowed with befitting coordinating property as nitrogenꢀcontaining
heterocyclic ligands are capable to form stable complexes with several metal ions and behave
as precursors to a variety of nitrogen heterocycles in organic synthesis.24,25 Moreover,
tetrazole ring is a crucial intermediate in the synthesis of other more complex heterocycles,
through various rearrangements.26 It has been recently documented that nitrogen containing
heterocyclic compounds possess inherent acetylcholinesterase inhibition potency and hence,
have emerged as one of the most promising candidates for the treatment of Alzheimer’s
disease (AD).27 As a consequence of such immense synthetic utility and inherent
pharmacological applications of tetrazoles, there is a need for the development of efficient
and elegant protocols for their synthesis.
Among tetrazoles, 1ꢀsubstituted tetrazoles have received much attention because of
their wide applications.28 However, the lack of convenient methods for the synthesis of 1ꢀ

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substituted tetrazoles, strongly restricts their potential application in medical practice.
Although many 5ꢀsubstituted tetrazoles are known, yet only a very few 1ꢀsubstituted
tetrazoles have been described.
Several sophisticated methodologies have been postulated in the literature for the
synthesis of tetrazole nucleus. The most convenient method to achieve 5ꢀsubstituted 1Hꢀ
tetrazoles is [3+2] cycloaddition between organic/inorganic azides or hydrazoic acid and
corresponding nitriles in the presence of varied catalysts viz. Cu2O,29a Pd(OAc)2/ZnBr2,29b
29f
Zn(OTf)3,29c ZrOCl2,29d Zn/Al hydrotalcite,29e Pd(PPh3)4
and ZnS.29g However, 1ꢀ
substituted tetrazole can be synthesized by acidꢀcatalyzed cycloaddition between isocyanides
and hydrazoic acid30 or cyclization between primary amines, triethyl orthoformate, and
sodium azide in presence of acetic acid31 or by using different catalysts such as In(OTf)3,32a
32d
Yb(OTf)3,32b SSA,32c [Hbim]BF4 and recently natrolite zeolite.32e Although, all of these
methods are worthwhile, however a number of them have several shortcomings such as, use
of expensive and hazardous reagents, application of high boiling solvents, prolonged reaction
times, tedious workup procedures, high temperature, toxic waste generation, difficulty in
separation and recovery of the catalyst and the presence of hydrazoic acid which is highly
toxic and explosive. Hence, it is of great practical importance to develop a more efficient and
environmentally benign method that avoids or minimize all of these complications.
In recent years, the use of reusable heterogeneous catalysts has received considerable
attention in organic synthesis as a result of their economic and environmental benefits. Such
reagents not only simplify purification processes but also help in preventing the discharge of
toxic reaction residues into the environment. The use of solid heterogeneous catalysts for the
development of clean processes is a theme of extreme importance in the chemical industry.
Solid support based heterogeneous catalysts are capable of driving several impracticable
organic transformations faster and under mildest possible conditions. These catalysts make

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the synthetic process economically feasible as they can be easily recovered by simple
filtration from the reaction mixture and can be reused several times to achieve very high
turnover numbers and also reduces the risk of discharge of toxic reaction residues into the
environment when compared with the conventional homogeneous catalysts. The development
of selective and reusable solid support catalysts for organic reactions has been a very active
area of research. During the last few decades, silicaꢀsupported catalysts have achieved
considerable reputation because of their many advantageous properties such as higher
mechanical and thermal stabilities, higher efficiency, larger surface area, better selectivity,
cheap, lower toxicity, reusability and easy separation of the catalyst from the reaction
mixture.33,34
Motivated by these findings, and in continuation of our ongoing efforts endowed with
the finding of new synthetic protocols35 under the principles of green chemistry, herein, we
report for the first time silicaꢀsupported boric acid (SiO2ꢀH3BO3) catalyzed synthesis of 1ꢀ
substituted 1Hꢀtetrazoles under thermal solvent free condition. Silicaꢀsupported boric acid
(SiO2ꢀH3BO3) catalyst was prepared by employing standard procedures depicted in the
literature (Scheme 1).36 It has emerged as a promising heterogeneous solid acid catalyst in
many organic transformations36ꢀ38 and possesses environmentally benign properties such as
nonꢀtoxicity, biocompatibility, recyclability, physiological inertness, inexpensiveness and
thermal stability. The structure and morphology of the catalyst was established on the basis of
FTꢀIR, powder Xꢀray diffraction (XRD), scanning electron microscopy (SEM), energyꢀ
dispersive Xꢀray spectroscopy (EDX) and transmission electron microscopy (TEM). The
synthesized compounds were screened for aetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) inhibition studies.
Scheme: 1 Synthetic pathway for the synthesis of catalyst

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2.
Results and discussions
In the present study a library of bioactive 1ꢀsubstitutedꢀ1Hꢀtetrazole derivatives 4(aꢀo) have
been synthesized by using environmentally benign heterogenous catalyst (SiO2ꢀH3BO3) under
solvent free conditions. This protocol offers several advantages over other existing synthetic
methodologies in terms of yield and purity of products, operational procedure, reaction times,
catalyst stability and recyclability. This synthetic scheme possesses diverse applicability and
is compatible to a range of functional groups (electron donating/ electron withdrawing). In
context to the potential applications of nitrogen containing heterocyclic compounds against
Alzheimer’s disease (AD),27 we have tested our synthesized compounds for the possible
AChE and BuChE inhibition studies. The results revealed that, electronic effect of
substituents inflict prominent effect on AChE activity.
2.1.
Characterization of silica-supported boric acid catalyst
The FTꢀIR spectrum of the catalyst (SiO2ꢀH3BO3) is depicted in (Fig. 2) and was recorded
using the KBr disk technique. The FTꢀIR spectrum displayed a stretching band of SiOꢀH at
3434 cmꢀ1, while the stretching band of BOꢀH appeared at 3227 cmꢀ1. The peak at 2260 cmꢀ1
was assigned to the BꢀOH combination and the peak at 1187 cmꢀ1 was attributed to the
bending vibration of BꢀOH. The SiꢀOꢀSi asymmetric stretching vibration band appeared at
1091 cmꢀ1 and the low frequency modes at 800 cmꢀ1 were due to the symmetric SiꢀOꢀSi
stretching vibrations. The peaks at 927 and 649 cmꢀ1 for the stretching and bending vibration
of borosiloxane linkage (SiꢀOꢀB), respectively and a BꢀO stretching band of borosiloxane at
1435 cmꢀ1 observed in the FTꢀIR spectrum of the catalytic system, authenticates the loading
of H3BO3 on the SiO2 surface.

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Fig. 2 FTꢀIR spectrum of catalyst SiO2ꢀH3BO3
Formation of the catalytic system (SiO2ꢀH3BO3) was further confirmed by powder
XRD analysis (Fig. 3). The XRD pattern showed a single broad peak at 2θ = 22o indicating
fine dispersion of boric acid on the silica material and thus confirming the formation of
amorphous SiO2ꢀH3BO3 matrix. The absence of reflection at 2θ = 28o for free boric acid39,40
indicated that boron atoms are mainly engaged in the formation of borosiloxane bridges in the
catalyst.
Fig. 3 Powder XRD analysis of catalyst SiO2ꢀH3BO3

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SEM analysis was employed to study the surface morphology of the catalytic system
(Fig. 4). SEM micrographs of the catalyst showed that H3BO3 particles were of uneven size
and shape, well dispersed and no conglomeration of H3BO3 particles was found on the
surface of the silica gel. The increased reactivity of boric acid supported on silica material is
believed to be due to the catalystꢀsupport interactions and the resultant changes in the surface
properties of the reactive sites. TEM micrograph (Fig. 5) of the catalytic system further
displayed uniform distribution of H3BO3 particles as black dots on the surface of silica,
approving the formation of the expected catalytic system. EDX analysis (Fig. 6) of the
catalyst showed the presence of Si, B and O elements suggesting the formation of SiO2ꢀ
H3BO3 catalytic system.
Fig. 4 SEM image of catalyst SiO2ꢀH3BO3

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Fig. 5 TEM image of catalyst SiO2ꢀH3BO3
Fig. 6 Energy dispersive Xꢀray (EDX) analysis of catalyst SiO2ꢀH3BO3

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2.2.
Chemistry
The synthetic pathway of a series of 1ꢀsubstitutedꢀ1Hꢀtetrazoles 4(aꢀo) is presented in
Scheme 2. Herein, a series of compounds were typically accessed via a solvent free facile
environmentally benign cyclization reaction, involving various heterocyclic/aromatic amines
with sodium azide and triethyl orthoꢀformate to yield target 1ꢀsubstitutedꢀ1Hꢀtetrazoles in
excellent yields (90ꢀ97 %) with high degree of purity.
The structural elucidation of all the synthesized compounds 4 (a-o) has been
1
established on the basis of elemental analysis, IR, H NMR, 13C NMR and mass spectral
studies. The analytical results for C, H and N were found to be within ± 0.3% of the
theoretical values. The spectral analysis has been in good corroboration with the expected
structural framework of the synthesized compounds. All the synthesized compounds showed
the absence of ꢀNH2 peak in the IR spectrum, which confirmed the reaction at the target
amine moiety of the substrates. Moreover, all the compounds displayed a characteristic peak
for C=N and N=N group, resonating at around 1578ꢀ1603 cmꢀ1 and 1503ꢀ1514 cmꢀ1,
respectively, which signifies the formation of tetrazole ring.

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Scheme: 2 Synthetic pathway for the synthesis of tetrazole derivatives 4 (a-o)
Characteristic peaks for the different substituted functional moieties such as methoxy,
1
nitro and carboxylic group’s etc., have been discussed in the experimental section. In H
NMR spectral analysis, each synthesized compound displayed a sharp singlet resonating at
around δ 8.07ꢀ8.95, attributed to the Hꢀ5 (ꢀCH=N) proton of the tetrazole nucleus, this

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prominent downfield shift displayed by Hꢀ5 proton of the tetrazole nucleus is probably due to
its acidic nature imposed by adjacent nitrogen atoms of the tetrazole ring (See supplementary
information). In 13C NMR spectral study, the absorption band resonating at around δ 135ꢀ148
has been assigned to the sp2 hybridized carbon of the tetrazole ring (See supplementary
information). The detailed spectral assignment has been discussed in experimental section.
The mass spectral analysis of all the synthesized tetrazole derivatives has been found to be in
good conformity with the proposed structures.
In our present study, a series of 1ꢀsubstitutedꢀ1Hꢀtetrazole 4 (a-o) were synthesized
from different heterocyclic/aromatic amino compounds with NaN3 and triethyl orthoꢀformate
at reflux temperature in acetic acid in the absence of catalyst. It was observed that the
reaction took prolonged time (4ꢀ6 hrs) for completion with a moderate yield (61ꢀ72 %) of the
products (Table 1).
In order to develop efficient, ecoꢀfriendly and environmentally benevolent approach
for the synthesis of 1ꢀsubstitutedꢀ1Hꢀtetrazole compounds, we explored the efficacy of silicaꢀ
supported boric acid (SiO2ꢀH3BO3) catalyst by carrying out the solvent free reaction of
variety of substituted amino compounds with sodium azide and triethyl orthoꢀformate at 90
oC. The reaction progressed smoothly and resulted in the formation of corresponding
products 4(a-o) in excellent yields (90ꢀ97 %) within (30ꢀ55 mins) as monitored by TLC
(Table 1). The present protocol is applicable to a broader substrate scope (electronꢀrich and
electronꢀdeficient). The plausible mechanistic pathway for the synthesis of target compounds
4 (a-o) is shown in Scheme 3. In the present study, we probed the optimized reaction
conditions including loading of catalyst, effect of temperature and solvents in terms of time
and yield on a model reaction using 5ꢀmethylisoxazolꢀ3ꢀamine (1a) with sodium azide (2)
and triethyl orthoꢀformate (3) to establish the best possible reaction conditions for the
synthesis of tetrazole derivatives.

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Table 1 Silicaꢀsupported boric acid (SiO2ꢀH3BO3) catalyzed synthesis of 1ꢀsubstitutedꢀ1Hꢀ
tetrazole derivatives 4 (aꢀo)
Products
Structure
Reaction in presence Reaction in presence M.P (oC)
of acetic acid of catalyst
Time
(hrs)
Yield
(%)
Time
(min)
Yield
(%)
5
69
72
30
95
93
160
4a
4b
4.5
45
189ꢀ191
4
5
65
68
35
40
96
91
174
196
4c
4d
4.7
63
50
97
231ꢀ233
4e
4.2
5.5
67
70
42
46
95
90
211
203
4f
4g

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6
61
35
96
98
4h
(95ꢀ96)31b
5.5
5.0
64
67
40
55
92
91
116
4i
(114ꢀ115)44
263
4j
4.8
4.2
71
66
48
43
97
93
174
4k
181ꢀ183
4l
5
72
70
68
35
40
42
95
94
96
127
4m
4n
4o
(128)45
5.5
5.3
79
(77) 32c,45
171

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To achieve the optimum concentration of catalyst, the model reaction was
investigated for different concentrations 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mol% (entries 2ꢀ7,
o
Table 2) of catalyst (SiO2ꢀH3BO3) at 90 C under solvent free condition. The model reaction
was primarily tested in the absence of catalyst, it was found that reaction took elongated time
(8 hrs) for completion with poor yield (42 %) of product obtained (entry 1, Table 2). The
model reaction was then tested for 0.5 mol% of catalyst followed by 1.0, 1.5, 2.0, 2.5 and 3.0
mol% and the results were noted in terms of reaction time and yield. The results revealed that
at every subsequent increase in concentration of catalyst by 0.5 %, there has been noteworthy
increase in the yield from 64ꢀ95 %, with drop in reaction time from 8ꢀ0.5 hrs. It can be
inferred from (entry 6, Table 2) that 2.5 mol% of the catalyst is satisfactory to gain the
o
optimum yield in the shortest reaction time under neat conditions at 90 C. Using less than
2.5 mol% of catalyst, moderate yield of the product (64ꢀ83 %) was obtained with higher
reaction time. The use of excess mole% of catalyst (>2.5 mol%) does not further enhance the
yield, possibly due to the saturation of the catalyst surface.
Table 2 Effect of catalyst loading on the yield and reaction time of model reaction (4a)a
Entry
Catalyst (mol%)
Time (hrs)b
Yield (%)c
No catalyst
8.0
4.5
4.0
2.0
1.0
0.5
0.5
42
64
71
75
83
95
95
1
2
3
4
5
6
7
0.5
1.0
1.5
2.0
2.5
3.0
aReaction condition: 5ꢀmethylisoxazolꢀ3ꢀamine (1a, 2 mmol), NaN3 (2, 2 mmol), triethyl
orthoꢀformate (3, 2.4 mmol), solvent free, 90 oC; bReaction Progress monitored by TLC.
cIsolated yield of products.

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To optimize the reaction temperature, we conducted the model reaction at different
temperatures under solvent free condition. It was observed that the increase in temperature
o
o
from 25 C to 90 C, has a prominent effect on the reaction in terms of yield and reaction
o
time. Initially model reaction was allowed to stir at room temperature (25 C), the results
obtained were not satisfactory with prolonged reaction time of 3 hrs and moderate yield of 54
%. The increase in temperature coupled with stirring produced a profound effect on the yield
of the product from 54ꢀ95 % during the course of reaction (entries 1ꢀ5, Table 3). However,
further increase in the temperature up to 110 oC did not show any further increase in the yield
o
of the product. Thus, keeping in view the above optimized reaction conditions, 90 C was
chosen as the optimal temperature for all the reactions.
Table 3 Effect of temperature on model reactiona
Entry
Temperature (oC)
Time (hrs)b
Yield (%)c
25
50
3.0
2.5
2.0
1.0
30d
30d
54
77
82
86
95
95
1
2
3
4
5
6
70
80
90
110
aReaction condition: 5ꢀmethylisoxazolꢀ3ꢀamine (1a, 2 mmol), NaN3 (2, 2 mmol), triethyl
orthoꢀformate (3, 2.4 mmol), catalyst (2.5 mol%), solvent free, refluxing at different
temperatures.
bReaction Progress monitored by TLC (entry 1ꢀ4, hrs).
cIsolated yield of products.
dReaction Progress monitored by TLC (entry 5ꢀ6, min).

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To evaluate the efficiency of the solvent free reaction conditions in comparison with
solvent conditions, the model reaction was examined in different solvent systems in the
presence of 2.5 mol% of SiO2ꢀH3BO3 catalyst. The model reaction was initially performed in
common organic solvents like MeOH and EtOH, a moderate yield of the product (4a) was
obtained after a stretched time period (entries 2ꢀ3, Table 4). This moderate yield can be
attributed to the nucleophilic nature of these solvents, due to presence of lone pair of
electrons on oxygen atom. Thus nucleophilic competition between these solvents (MeOH,
EtOH) and primary amine group of the substrate for electrophilic carbon of triethyl orthoꢀ
formate will eventually resulted in lower yield of desired product. Moreover, this low yield in
these coordinating solvents (MeOH, EtOH) can be ascribed due to the accessibility of the
acidic Lewis sites of B(OH)2 in these solvents. Whereas in acetic acid, the yield of the desired
product increased significantly with reduced reaction time (entry 4, Table 4). This
improvement in yield in CH3COOH solvent is probably due to its ability to facilitate the
elimination of ethoxy group (OEt) from triethyl orthoꢀformate, thus enhances the
electrophilicity of carbon, rendering it more feasible for nucleophilic attack by the amines.
However, when the reaction was carried out in nonꢀcoordinating solvents like, THF and
DMF, no further significant increase in the yield of the product was observed in comparison
to CH3COOH, after a stretched reaction time (entries 5ꢀ6, Table 4). Moreover, when the
reaction was explored under solventꢀfree condition, it was noted that the yield of product
enhanced sharply with significant drop in reaction time. Thus, nature
(nucleophlicity/protocity) of the solvent has a considerable effect on the yield and reaction
time. These results suggest that, solvent free reaction condition coupled with 2.5 mol% of
SiO2ꢀH3BO3 at 90 oC is the best synthetic approach for the synthesis of 1ꢀsubstituted tetrazole
derivatives in terms of reduced reaction times and excellent yields of the products.

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Table 4 Effect of various solvents on model reactiona
Entry
Solvent
Time (hrs)b
Yield (%)c
Solvent free
MeOH
30 min
95
59
54
79
73
76
1
2
3
4
5
6
7
8
5
9
8
EtOH
CH3COOH
THF
DMF
aReaction condition: 5ꢀmethylisoxazolꢀ3ꢀamine (1a, 2 mmol), NaN3 (2, 2 mmol), triethyl
orthoꢀformate (3, 2.4 mmol), catalyst (2.5 mol%), different solvents, refluxing at 90 oC.
bReaction Progress monitored by TLC.
cIsolated yield of products.
A comparative study of a variety of heterogeneous catalysts was conducted to confirm
the superiority of silicaꢀboric acid over other heterogeneous acid catalysts. When the model
reaction was investigated with silicaꢀsupported catalysts such as HClO4ꢀSiO2, NaHSO4ꢀSiO2,
NH4OAcꢀSiO2 and P2O5ꢀSiO2 the reaction took prolonged time period, and the yields were
not satisfactory (entries 2ꢀ5, Table 5). However, relatively good yield of the product 4a (77ꢀ
81 %) was obtained by using Yb(OTf)3 and In(OTf)3 as heterogeneous catalysts but again
reaction took protracted time period for completion (entries 6ꢀ7, Table 5). When the model
reaction was coupled with silicaꢀboric acid catalyst, the yield of the product 4a (95 %)
increased exponentially with prominent drop in reaction time. These results suggest that
silicaꢀboric acid is the catalyst of choice for the present synthetic protocol.

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Table 5 Comparison of catalytic activity of different catalysts on the model reactiona
Entry
Catalyst
SiO2ꢀH3BO3
SiO2ꢀHClO4
SiO2ꢀ NaHSO4
SiO2ꢀ NH4OAc
SiO2ꢀ P2O5
Yb(OTf)3
Time (hrs)b
Yield (%)c
30 min
95
50
55
59
62
77
81
1
2
3
4
5
6
7
5
7
6
6.5
5
In(OTf)3
3
aReaction condition: 5ꢀmethylisoxazolꢀ3ꢀamine (1a, 2 mmol), NaN3 (2, 2 mmol), triethyl
orthoꢀformate (3, 2.4 mmol), different catalyst (2.5 mol%), solvent free, refluxing at 90 oC.
bReaction Progress monitored by TLC.
cIsolated yield of products.
The reusability of the silicaꢀboric acid catalyst was also examined on model reaction
in order to satisfy the green chemistry criteria. The recycling of catalyst is an important step
as it reduces the cost of the process. The catalyst was reused five times and the results
demonstrate that catalyst can be reused as such without a significant loss in yield (Fig. 7).
After completion of the reaction, the catalyst was removed by simple filtration. The
o
recovered catalyst was dried under vacuum at 100 C for 6 hrs and tested up to five more
reaction cycles. Recycling and reuse of the catalyst showed minimal decreases in yields. The
product was obtained in 93%, 91%, 88%, 85%, 80% yields after successive cycles. (Fig. 7),
thus proving the catalyst’s reusability. To ascertain the variation in morphological features of
the recovered catalyst, we carry out its SEMꢀEDX analysis. It was observed that the
composition of the catalytic system was almost consistent with the fresh catalyst (Fig. 8) and
also there was no significant change in the morphology of the catalyst (Fig. 9) as compared to
the fresh catalyst.

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Fig. 7 Recycling data of the catalyst (SiO2ꢀH3BO3) for the model reaction
Fig. 8 Energy dispersive Xꢀray (EDX) analysis of recovered catalyst after five runs

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Fig. 9 SEM image of recovered catalyst after five runs
Under these optimized reaction conditions discussed above, the scope and generality
of the present protocol was further demonstrated by carrying out the reaction of different
heterocyclic/aromatic amines with sodium azide and triethyl orthoꢀformate under solvent free
condition. All the reactions proceeded smoothly to afford the desired products 4(a-o) in
excellent yields (90ꢀ97 %) within 30ꢀ55 min (Table 1).
The probable silicaꢀboric acid catalyzed mechanistic pathway for the synthesis of
tetrazole derivatives is shown in Scheme 3, in which the Lewis acidity of the catalyst
probably has an important role in the promotion of the cyclization process. It is believed that
silicaꢀboric acid activates the ethoxy group of triethyl orthoꢀformate and facilitates its
elimination by cleaving the CꢀO bond of triethyl orthoꢀformate to generate carbenium ion (I),
which is resonance stabilized by oxygen atom of another ethoxy group followed by
nucleophilic attack of amine. The next step involves the expulsion of second ethoxy group
generating carbenium ion (II), which can be stabilized by the neighbouring O or N atom to
form either oxonium (A) or iminium (B) cation, respectively. It is believed that the formation
of iminium cation is preferential over oxonium cation due to the greater basicity of nitrogen
with respect to oxygen. The next step involves the sequential attack of azide anion leading to

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the formation of proposed imidoylazide intermediate (III).41,42 As the three nitrogens bend
away from the linear arrangement and the fourth nitrogen atom comes within the bonding
distance, eventually leads to the formation of cyclized tetrazoles 4 (a-o).
Scheme: 3 Plausible mechanistic catalytic loop for the synthesis of compounds 4 (a-o)

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2.3.
AChE and BuChE inhibition results
All the synthesized tetrazole derivatives 4 (a-o) were analyzed for acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE) inhibition studies. The IC50 values for AChE and
BuChE inhibition are summarized in Table 6. It can be inferred from the data reported in
Table 6, that compounds bearing nicotinic acid nucleus i.e., 4e, 4f and 4g exhibited strongest
inhibition to AChE with an IC50 values of 0.43±0.02 ꢁM, 0.21±0.02 ꢁM and 0.26±0.01 ꢁM,
respectively, followed by compound 4c (IC50 = 1.03±0.01 ꢁM), 4d (IC50 = 1.25±0.01 ꢁM),
4b (IC50 = 1.46±0.01 ꢁM), 4a (IC50 = 1.52±0.02 ꢁM), 4m (IC50 = 2.08±0.02 ꢁM), 4n (IC50 =
2.13±0.02 ꢁM), 4o (IC50 = 2.17±0.01 ꢁM), 4l (IC50 = 2.71±0.01 ꢁM), 4k (IC50 = 2.89±0.01
ꢁM), 4j (IC50 = 3.01±0.02 ꢁM), 4h (IC50 = 3.29±0.02 ꢁM) and 4i (IC50 = 3.32±0.02 ꢁM). Of
these nicotinic acid analogues 4f was found to be most potent AChE inhibitor. Among the
isoxazole nucleated tetrazole analogues 4 (a-d), compound 4c (IC50 = 1.03±0.01 ꢁM) with
methyl and bromine substituted isoxazole ring displayed encouraging activity against AChE,
methoxy substituted isoxazole ring (compound 4d) showed lower activity than 4c by a value
of 0.22 but higher than methyl substituted isoxazole analogues (compound 4a and 4b) by a
value of 0.27 and 0.21, respectively. These results suggest that the nature of substituent inflict
a prominent effect on AChE activity. Compound 4m, 4n and 4o comprising pyridine nucleus
exhibited nearly similar results with IC50 values of 2.08±0.02 ꢁM, 2.13±0.02 ꢁM and
2.17±0.01 ꢁM, respectively. Among benzothiazole nucleated tetrazole derivatives, compound
4l (IC50 = 2.71±0.01 ꢁM) with methoxy substituent attached to the benzothiazole nucleus at 6
position exhibited better AChE inhibition. It was noticed that when the methoxy group at the
6 position in compound 4l is replaced by chlorine (compound 4k), the activity decreased
significantly by 0.18. However, when the same methoxy group was replaced by nitro group
in compound 4j, there was further dip in AChE inhibition by value of 0.30. Thus the nature of
substituent at 6 position in benzothiazole based tetrazole derivatives plays an essential role in

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AChE inhibition activity. It can be seen that compound 4h and 4i exhibited moderate activity
among all the synthesized tetrazole derivatives with IC50 values of 3.29±0.02 ꢁM and
3.32±0.02 ꢁM, respectively. In view of the above results it can be concluded that in general
electron pumping groups enhance AChE activity while as electron withdrawing groups
reduce it. The level of in vitro BuChE inhibition was also examined using the same method.
Most of these compounds exhibited moderate inhibitory potency against BuChE, but there
were no apparent patterns in the levels of inhibition.

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Table 6 In vitro inhibition IC50 (µM) and selectivity of tetrazole derivatives and tacrine for
hAChE and hBuChE
Entry
Product
IC50 (µM)a
AChEb ± SEM BuChEc ± SEM
Selectivity for
h
AChEd
h
h
1.52±0.02
1.46±0.01
1.03±0.01
1.25±0.01
0.43±0.02
0.21±0.02
0.26±0.01
3.29±0.02
3.32±0.02
3.01±0.02
2.89±0.01
2.71±0.01
2.08±0.02
2.13±0.02
2.17±0.01
0.09±0.02
1.05±0.03
1.71±0.01
2.29±0.02
4.58±0.02
2.43±0.01
1.53±0.01
0.85±0.03
2.39±0.02
1.69±0.01
13.64±0.02
19.57±0.02
27.30±0.01
0.39±0.03
0.70±0.01
0.54±0.02
0.14±0.01
0.69
1.17
2.23
3.67
5.64
7.31
3.27
0.72
0.51
4.53
6.77
10.07
0.19
0.33
0.25
0.16
1
2
4a
4b
4c
4d
4e
4f
3
4
5
6
7
4g
4h
4i
8
9
10
11
12
13
14
15
16
4j
4k
4l
4m
4n
4o
Tacrine
(Standard)
aIC50 values represent the concentration of inhibitor required to decrease the enzyme activity
by 50%; SEM = Standard error of the mean
bhAChE = Acetylcholinesterase from human serum
chBuChE = Butyrylcholinesterase from human serum
dSelectivity for hAChE = IC50 (hBuChE)/IC50 (hAChE).

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3.
Conclusions
The present protocol reports the convenient, ecoꢀfriendly and scalable protocol approach for
the synthesis of tetrazole derivatives 4 (a-o) in excellent yields (90ꢀ97 %) by employing
silicaꢀsupported boric acid as a catalyst. This solventꢀfree, green synthetic procedure
eliminates the use of toxic solvents and thus makes it attractive one in organic synthesis. The
noteworthy features of this protocol are shorter reaction time, high purity, mild reaction
conditions, operational simplicity, cleaner reaction profile, enhanced reaction rates and easy
workup. Moreover, SiO2ꢀH3BO3 exhibited stability towards air and water and is easy to
prepare from cheap materials. The present protocol demonstrates that, optimization
o
conditions i.e., 2.5 mol% of catalyst under solventꢀfree condition at 90 C are the best
parameters for the synthesis of tetrazole analogues in excellent yields for scalable purposes.
All the compounds displayed moderate to good AChE inhibition potency. The results validate
that the nature of substituents attached to the heterocyclic moieties is crucial for biological
activity. We believe that this synthetic approach provides a better scope for the synthesis of
tetrazole analogues and will be a more practical alternative to the other existing methods.
4.
Experimental
4.1.
Materials and general methods
All reagents were purchased from SigmaꢀAldrich (India), Alfa Aesar and Merck as ‘synthesis
grade’ and used without further purification. The purity of all the compounds was checked by
thin layer chromatography (TLC) on glass plates coated with silica gel G254 (EꢀMerck).
Human AChE (EC: 3.1.1.7) and BuChE (EC: 3.1.1.8) lyophilized powder were purchased
from SigmaꢀAldrich. Melting points of all synthesized compounds were determined on a
Kofler apparatus and are uncorrected. Fourier transformꢀinfrared (FTꢀIR) spectra were
recorded in the 400ꢀ4000 cmꢀ1 waveꢀnumber range with a PerkinꢀElmer (2000 FTIR)

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1
Spectrometer by KBr pellet method. H NMR and 13C NMR spectra were recorded on a
Bruker AvanceꢀII 400 MHz instrument in pure deuterated DMSOꢀd6 solvent. The chemical
shifts (δ) are reported in ppm relative to the TMS as an internal standard and J values are
reported in Hertz. Mass spectra were recorded on a JEOL Dꢀ300 mass spectrometer.
Elemental analysis (C, H, N) was conducted using Thermo Scientific (FLASH 2000) CHN
Elemental Analyser. Xꢀray diffractograms (XRD) of the catalyst was recorded in the 2θ range
of 20ꢀ80° with scan rate of 4°/ min on a Shimadzuꢀ6100 Xꢀray diffractometer with Niꢀfiltered
Cu Kα radiation at a wavelength of 1.54060 Ao. The scanning electron microscope (SEMꢀ
EDX) analysis was obtained using a JEOL (JSMꢀ6510) equipped with an energy dispersive
Xꢀray spectrometer at different magnification and transmission electron microscope (TEM)
results were recorded using JEOL (JEMꢀ2100F) model.
4.2.
Synthesis of silica-supported boric acid (SiO2-H3BO3) catalyst
Boric acid (3.0 g) was taken in a 250 ml round bottom flask with 60 mL water and heated to
o
60ꢀ80 C. Silica gel (60ꢀ120 mesh, 27.0 g) was added gradually with constant stirring and
refluxed for 5 hrs. Water was evaporated under reduced pressure and the residue was stirred
o
at 100 C for 6ꢀ7 hrs under vacuum to give free flowing white powder of silicaꢀboric acid
(Scheme 1). It was kept under vacuum at room temperature.36 The various catalysts i.e. SiO2ꢀ
43d
HClO4,43a SiO2ꢀNaHSO4,43b SiO2ꢀNH4OAc43c and SiO2ꢀP2O5
used for the comparative
study with respect to silicaꢀboric acid have been synthesized according to the previously
published standard procedures.
4.3.
General procedure for the synthesis of 1-substituted-1H-tetrazoles
To a mixture of amine 1 (2.0 mmol), NaN3 2 (2.0 mmol) and triethyl orthoꢀformate 3 (2.4
mmol), 2.5 mol% of silicaꢀsupported boric acid was added and the reaction mixture was

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o
allowed to stirr at 90 C for 30ꢀ55 min. After completion of the reaction (as monitored by
TLC), the reaction mixture was allowed to cool at room temperature and diluted with cold
water (5 mL). The catalyst was separated by filtration and the resulting solution was extracted
with ethyl acetate (3×10 mL). The combined organic layers were washed with brine, dried
over anhydrous Na2SO4 and evaporated under reduced pressure. The crude product obtained
was crystallized with chloroformꢀmethanol to afford the pure products. The recovered
catalyst was reused for subsequent cycles without a significant loss in yield.
4.4.
Spectral characterization
-tetrazol-1-yl) isoxazole (4a)
4.4.1. 5-methyl-3-(1
H
Compound 4a crystallized from CHCl3ꢀMeOH as yellow crystalline solid. Yield: 95%, m.p.
160 °C; Anal. calc. for C5H5N5O: C, 39.74; H, 3.33; N, 46.34; found: C, 39.75; H, 3.34; N,
46.36. IR ν cmꢀ1: 1065 (CꢀO), 1128 (CꢀN), 1508 (N=N), 1584 (C=N). 1H NMR (400 MHz,
KBr
max
DMSOꢀd6, δ, ppm): 2.24 (s, 3H, CH3), 6.35 (s, 1H,isoxazole ring), 8.07 (s, 1H, ꢀCH=Ntetrazole ring).
13C NMR (100 MHz, DMSOꢀd6, δ, ppm): 15.25 (CH3), 102.44 (Cꢀ4), 135.05 (ꢀCH=Ntetrazole
ring), 148.54 (Cꢀ3), 155.03 (Cꢀ5). MS (ESI) m/z: 151.05 [M+H] +•.
4.4.2. 3-methyl-5-(1H-tetrazol-1-yl) isoxazole (4b)
Compound 4b crystallized from CHCl3ꢀMeOH as brownish solid. Yield: 94%, m.p. 189ꢀ191
°C; Anal. calc. for C5H5N5O: C, 39.74; H, 3.33; N, 46.34; found: C, 39.76; H, 3.32; N, 46.37.
1
cmꢀ1: 1058 (CꢀO), 1135 (CꢀN), 1511 (N=N), 1578 (C=N). H NMR (400 MHz,
KBr
max
IR ν
DMSOꢀd6, δ, ppm): 2.15 (s, 3H, CH3), 6.38 (s, 1H,isoxazole ring), 8.15 (s, 1H, ꢀCH=Ntetrazole ring).
13C NMR (100 MHz, DMSOꢀd6, δ, ppm): 17.24 (CH3), 104.20 (Cꢀ4), 137.18 (ꢀCH=Ntetrazole
ring), 143.25 (Cꢀ5), 152.31 (Cꢀ3). MS (ESI) m/z: 151.05 [M+H] +•.

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4.4.3. 4-bromo-3-methyl-5-(1H-tetrazol-1-yl) isoxazole (4c)
Compound 4c crystallized from CHCl3ꢀMeOH as white solid. Yield: 96%, m.p. 174 °C; Anal.
KBr
max
calc. for C5H4BrN5O: C, 26.11; H, 1.75; N, 30.45; found: C, 26.13; H, 1.77; N, 30.43. IR ν
1
cmꢀ1: 574 (CꢀBr), 1082 (CꢀO), 1142 (CꢀN), 1509 (N=N), 1587 (C=N). H NMR (400 MHz,
13
DMSOꢀd6, δ, ppm): 2.17 (s, 3H, CH3), 8.26 (s, 1H, ꢀCH=Ntetrazole ring). C NMR (100 MHz,
DMSOꢀd6, δ, ppm): 16.32 (CH3), 102.38 (Cꢀ4), 140.25 (ꢀCH=Ntetrazole ring), 147.28 (Cꢀ5),
156.24 (Cꢀ3). MS (ESI) m/z: 228.96 [M+H] +•.
4.4.4. 5-methoxy-3-(1H-tetrazol-1-yl) isoxazole (4d)
Compound 4d crystallized from CHCl3ꢀMeOH as yellowish crystals. Yield: 92%, m.p. 196
°C; Anal. calc. for C5H5N5O2: C, 35.93; H, 3.02; N, 41.90; found: C, 35.94; H, 3.01; N,
41.93. IR ν cmꢀ1: 1078 (CꢀO), 1140 (CꢀN), 1512 (N=N), 1582 (C=N). 1H NMR (400 MHz,
KBr
max
DMSOꢀd6, δ, ppm): 2.94 (s, 3H, OCH3), 5.78 (s, 1H,isoxazole ring), 8.23 (s, 1H, ꢀCH=Ntetrazole
ring). 13C NMR (100 MHz, DMSOꢀd6, δ, ppm): 53.65 (OCH3), 107.28 (Cꢀ4), 142.20 (ꢀ
CH=Ntetrazole ring), 145.27 (Cꢀ3), 157.80 (Cꢀ5). MS (ESI) m/z: 167.04 [M+H] +•.
4.4.5. 2-(1H-tetrazol-1-yl) nicotinic acid (4e)
Compound 4e crystallized from CHCl3ꢀMeOH as white crystalline solid. Yield: 97%, m.p.
231ꢀ233 °C; Anal. calc. for C7H5N5O2: C, 43.98; H, 2.64; N, 36.64; found: C, 43.97; H, 2.66;
cmꢀ1: 1132 (CꢀN), 1510 (N=N), 1592 (C=N), 1712 (C=O), 3212 (ꢀOH). 1H
KBr
max
N, 36.65. IR ν
NMR (400 MHz, DMSOꢀd6, δ, ppm): 7.23 (t, 1H, Hꢀ5), 7.85 (d, 1H, Hꢀ6), 8.02 (d, 1H, Hꢀ4),
8.59 (s, 1H, ꢀCH=Ntetrazole ring), 9.84 (brs, 1H, OH). 13C NMR (100 MHz, DMSOꢀd6, δ, ppm):
123.59 (Cꢀ5), 129.25 (Cꢀ4), 132.18 (Cꢀ3), 142.24 (Cꢀ6), 144.21 (ꢀCH=Ntetrazole ring), 148.13
(Cꢀ2), 171.35 (C=O). MS (ESI) m/z: 191.04 [M+H] +•.
4.4.6. 6-(1H-tetrazol-1-yl) nicotinic acid (4f)
Compound 4f crystallized from CHCl3ꢀMeOH as light yellow solid. Yield: 93%, m.p. 211
°C; Anal. calc. for C7H5N5O2: C, 43.98; H, 2.64; N, 36.64; found: C, 43.99; H, 2.63; N,