Butyl methyl imidazolium silica sulfate (BMIm)SS: A novel hybrid nano‐catalyst for highly efficient synthesis of new 1,2-diol monoesters of ibuprofen as the novel prodrugs of ibuprofen having potent analgesic property

Article abstract of DOI:10.1016/j.bioorg.2020.104570

The fabrication, characterization of butyl methyl imidazolium silica sulfate [BMIm]SS as a novel nano hybrid catalyst and its application in synthesis of new ibuprofen (IBP) 1,2-diol mono esters were described. [BMIm]SS catalyzed the reaction of IBP with epoxides to afford the new IBP 1,2-diol mono esters in good to excellent yields. The products were tested in vivo for the analgesic properties on female mice using formalin test. The test results revealed that most compounds, in particular compounds 1h, 1k and 1o displayed potent analgesic activity compare to IBP as a reference drug. No mortality was observed due to the toxicity of the synthesized compounds. The docking analysis was conducted that confirmed the strong binding affinity of active compounds to active site of murine cyclooxygenase-2 (COX-2) enzyme compare to IBP. The in silico pharmacokinetic profile, drug likeness and toxicity predictions were carried out for all compounds which determined that 1h can be suggested as an appropriate future drug candidate.

Full text of DOI:10.1016/j.bioorg.2020.104570

Journal Pre-proofs
Butyl methyl imidazolium silica sulfate (BMIm)SS: A novel hybrid nano‐cat-
alyst for highly efficient synthesis of new 1,2-diol monoesters of ibuprofen as
the novel prodrugs of ibuprofen having potent analgesic property
Mohammad Navid Soltani Rad, Somayeh Behrouz, Esmaeil Atashbasteh,
Seyedeh-Sara Hashemi
PII:
S0045-2068(20)31868-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.104570
YBIOO 104570
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
9 October 2020
4 December 2020
16 December 2020
Please cite this article as: M. Navid Soltani Rad, S. Behrouz, E. Atashbasteh, S-S. Hashemi, Butyl methyl
imidazolium silica sulfate (BMIm)SS: A novel hybrid nano‐catalyst for highly efficient synthesis of new 1,2-diol
monoesters of ibuprofen as the novel prodrugs of ibuprofen having potent analgesic property, Bioorganic
Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104570
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Butyl methyl imidazolium silica sulfate (BMIm)SS: A novel hybrid nano-catalyst for
highly efficient synthesis of new 1,2-diol monoesters of ibuprofen as the novel
prodrugs of ibuprofen having potent analgesic property
Mohammad Navid Soltani Rad, a* Somayeh Behrouz, a* Esmaeil Atashbasteh a Seyedeh-Sara Hashemi b
a
Medicinal Chemistry Research Laboratory, Department of Chemistry, Shiraz University of Technology,
Shiraz 71555-313, Iran
b
Burn and Wound Healing Research Center, Division of Food and Nutrition, Shiraz University of Medical
Sciences, Shiraz, Iran
Abbreviations: IBP, ibuprofen; [BMIm]SS, butyl methyl imidazolium silica sulfate; NSAIDs, nonsteroidal anti-
inflammatory drugs; COX, cyclooxygenase; SSA, silica sulfuric acid; OIHCs, organic-inorganic hybrid catalysts; PGs,
prostaglandins; GI, gastrointestinal; ILs, ionic liquids; SILs, supported ionic liquids; [BMIm]Br, butyl methyl
imidazolium bromide; [BMIm]OH, butyl methyl imidazolium hydroxide; XRD, X-ray diffraction; SEM, scanning
electron microscopy; EDS, Energy-dispersive X-ray spectroscopy; TGA, thermogravimetric analysis, MVD, Molegro
Virtual Docker; RMSD, root-mean-square deviation; Lipinski’s RO5, Lipinski’s rule of 5; nHba, number of hydrogen
bond acceptors; nHbd, number of hydrogen bond donors; nrotb, number of rotatable bonds; TPSA, total polar
surface area
*Corresponding authors. Tel.: +98 71 3735 4500; Fax: +98 71 3735 4520; E-mail address: soltani@sutech.ac.ir
(M.N. Soltani Rad); behrouz@sutech.ac.ir (S. Behrouz)
1

Abstract:
The fabrication, characterization of butyl methyl imidazolium silica sulfate [BMIm]SS as a novel nano hybrid
catalyst and its application in synthesis of new ibuprofen (IBP) 1,2-diol mono esters were described. [BMIm]SS
catalyzed the reaction of IBP with epoxides to afford the new IBP 1,2-diol mono esters in good to excellent yields.
The products were tested in vivo for the analgesic properties on female mice using formalin test. The test results
revealed that most compounds, in particular compounds 1h, 1k and 1o displayed potent analgesic activity
compare to IBP as a reference drug. No mortality was observed due to the toxicity of the synthesized compounds.
The docking analysis was conducted that confirmed the strong binding affinity of active compounds to active site
of murine cyclooxygenase-2 (COX-2) enzyme compare to IBP. The in silico pharmacokinetic profile, drug likeness
and toxicity predictions were carried out for all compounds which determined that 1h can be suggested as an
appropriate future drug candidate.
Keywords: Ibuprofen, 1,2-diol monoesters, [BMIm]SS, analgesic, formalin test, NSAIDs, cyclooxygenase.
2

1. Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are a broad heterogeneous group of compounds that
have been immensely used for decades as highly efficient analgesic [1]. NSAIDs are among the most
frequently used therapeutic agents not only by issue but also many of them are present over-the counter
allowing for self-medication [2]. They are medicated for numerous diseases, including arthritis,
osteoarthritis and musculoskeletal disorders [3]. While most of the biological activities of NSAIDs are
associated with COX-dependent mechanisms; however, several studies have evidenced that NSAIDs
interact with membrane phospholipids in a COX-independent route that could be involved in their
biological activity [4-13]. Structurally, NSAIDs are categorized to salicylates, sulfonanilides, coxibs,
propionic, acetic, enolic and anthranilic acids derivatives [14]. The structures of some popular NSAIDs
are shown in Fig. 1[15].
Cl
NH
Cl
NH
O
H
N
O
OH
OH
O
O
O
Cl
OH
O
Cl
O
HO
OH
O
O
Fenoprofen
Acetaminophen
Ketoprofen
Diclofenac
Aceclofenac
O
OH
O
OH
OH
H
OH
O
H
N
OH
F3C
N
O
S
F
O
N
N
NH2
O
F
O
F3C
O
Mefnamic acid
Diflunisal
Naproxen
Flufenamic acid
Celecoxib
OH
NO2
O
O
OH
OH
OH
O
O
O
O
N
O
O
O
HN
HO
S
O
Indomethacin
Aspirin
Loxoprofen
Nimesulide
Ibuprofen
Cl
O
O
O
O
O
O
N S
N
Cl
N
N
N
N
O
OH
O
NH
HO
O
HN
OH
O
N
OH
Piroxicam
Oxaprozin
Clonixin
Tolmetin
Phenylbutazone
Fig.1. Structure of some NSAIDs from diverse categories.
Ibuprofen (IBP), (R, S) [2-(4-isobutylphenyl) propionic acid] was the first NSAID belongs to category of
propionic acid derivatives that universally used for the therapy of pain and inflammation [16]. It is one of
the strongest orally and highly active NSAID that widely used in the treatment of osteoarthritis,
rheumatoid arthritis, muscle pain and recent surgery [17, 18]. Like most NSAIDs, IBP blocks
cyclooxygenase (COX) enzyme and inhibits prostaglandins (PGs) biosynthesis [19]. In spite of its
remarkable pharmaceutical benefits; however, IBP involves unwanted side effects in particular on
3

human digestion system. Progress in the gastrointestinal (GI) side effects especially stomach ulceration,
bleeding and perforation are the main limitations in its oral administration which is especially due to
local action exerted by direct contact of drug with gastric mucosa [20]. In this context, the free carboxylic
acid moiety in IBP structure plays an important role in maintaining and magnifying the disorders caused
by stomach ulcers [21]. Thus, it is critically essential to convert IBP into relevant prodrugs. Up to date,
diverse drugs, molecules and carriers were used to conjugate with IBP for different purposes like
preparation of mutual prodrugs of IBP, variation in blood-brain barrier and alteration in
pharmacokinetic profiles of IBP [22]. For instance, IBP has been conjugated with sulfa drugs (I) [23],
eugenol (II) [24], menthol (III) [25], thymol (IV) [25], chlorzoxazone (V) [26], acetaminophen (VI) [27],
medoxomil (VII) [28], guaiacol (VIII) [29], N-hydroxy-methylphthalimide (IX) [30], hydroxy
ethylsulfohydroxamic acid (IX) [31], hydroxy ethylnicotinic acid ester (X) [32] and amino acids (XI) [33]
(Fig. 2).
O
S
NHR
O
O
O
O
O
O
O
O
O
O
OMe
,
R=H, COCH3,
N
N
O
(I)
O
(II)
(III)
(IV)
Cl
H
N
O
O
O
O
N
O
O
O
O
O
O
O
O
O
N
OMe
R
(V)
(VI)
(VII)
(VIII)
O
O
O
O
O
O
SO2NHOH
O
O
N
O
O
N
CO2H
H
O
R=H,alkyl, aryl,...
(XII)
(IX)
(X)
(XI)
Fig. 2. Structure of some conjugated drugs and molecules with IBP.
Additionally, IBP was converted to related amides [34, 35] and esters [36] through the reaction with
simple or functionalized amines and alcohols. It is also worthy to mention that IBP has been covalently
tethered to diverse polymers in order to carry the drug and slow release of IBP in controlled release
systems [37-39].
In recent decades, the heterogeneous catalysts have received increasing applications in organic
transformations owing to their environmentally benign nature and plenty of remarkable advantages
such as the ease of handling and separation, inexpensiveness, high selectivity, chemical and thermal
4

constancies [40]. Among the heterogeneous catalysts, the organic-inorganic hybrid catalysts (OIHCs)
have found particular attention [41]. Silica-based OIHCs are fascinating since silica is plentifully available
and displays high durability [42]. Silica sulfuric acid (SSA) is an environmentally benign silica-based
heterogeneous catalyst that has been used in many organic reactions [43]. Additionally, the butyl methyl
imidazolium (BMIm) salts have been extensively applied in many organic transformations either as ionic
liquids or catalysts [44]. In current decades, there has been an enormous interest in immobilization of ILs
on various inorganic and polymeric supports. The supported ILs or SILs are significant owing to their ILs
concomitant, high catalytic activity, ease of separation and recoverability which largely increase their
applicability in industrially important catalytic processes [45-47].
As explained earlier, the application of IBP in its parental shape often provides several side effects and
disorders in patients. To reduce the side effects, the strategy of conversion of IBP into related prodrugs is
traditionally applied. One way for preparation of IBP prodrug is conversion of IBP to related 1,2-diol
monoester which through the best of our knowledge has not yet reported so far. Additionally, since the
remarkable advantageous of OIHCs as a heterogeneous nano catalyst, hereby we would like to report the
synthesis and characterization of butyl methyl imidazolium silica sulfate [BMIm]SS as a novel nano
hybrid catalyst and its application in synthesis of new IBP 1,2-diol monoesters (1a-1o) as potent
analgesic and anti-inflammatory agents (Scheme 1).
O
O
[BMIm]SS (cat.)
O
+
R
OH
MeCN, reflux, 12-36h
O
R
OH
1a-1o
(50-92%)
R= alkyl,alkyloxy,aryloxy,...
O
S
N
[BMIm]SS:
SiO2
O
O
O
N
Scheme 1. Synthesis of new IBP 1,2-diol monoester catalyzed by [BMIm]SS.
5

2. Results and Discussion
2.1. Preparation of [BMIm]SS
The process for the preparation of [BMIm]SS is shown in Scheme 2. Due to Scheme 2, primarily, the
butyl methyl imidazolium bromide [BMIm]Br was prepared via the reaction of N-methyl imidazole and
butyl bromide in anhydrous acetonitrile as a solvent at reflux condition. Afterward of synthesis and
purification of [BMIm]Br, it was diluted in anhydrous DCM in the presence of solid KOH at room
temperature which was vigorously stirred to afford butyl methyl imidazolium hydroxide [BMIm]OH as a
brown oil [48]. In another separate process, the traditional SiO2 was first activated by a flow of 8% O2 in
an argon atmosphere at 400 °C in a furnace for an hour to acquire activated SiO2. The use of O2 flow at
high temperature, produces the extra hydroxyl moieties at the surface of silica structure which is
critically essential for tethering the active species. Afterward, the chlorosulfonic acid was added to
activated-SiO2 at ambient temperature to obtain the fresh silica sulfuric acid (SSA) due to literature [43].
Finally in an acid-base reaction, the SSA powder was suspended in deionized water and titrated with
[BMIm]OH to reach a neutral pH (for control the pH, the pH-meter was used through the course of
reaction). The reaction content was filtered to obtain a brown solid that washed several times with
ethanol, dried in vacuo and stored in a vacuum desiccator at room temperature. The changes in color
from SiO2 to desired catalyst are shown in Fig. 3.
Br
N
N
OH
N
MeCN
KOH
+
Br
reflux, 4h
N
DCM, 24h, rt
N
N
[BMIm]OH
[BMIm]Br
NMI
O
O S O
O
-H2O
r.t.
N
SiO2
N
[BMIm]SS
O
HSO3Cl
r.t.
O
2 (8%) in Ar
SiO2
SiO2
OH
SiO2
O S OH
O

400 C, 1h
SSA
Scheme 2. Procedure for preparation of butyl methyl imidazolium silica sulfate [BMIm]SS as a catalyst.
6

Fig. 3. The changes in color from silica gel to [BMIm]SS.
2.2. Characterization of catalyst
The powder X-ray diffraction pattern of the [BMIm]SS was obtained by the precipitation from freshly
prepared aqueous [BMIm]SS suspension at 10–90 °C (Fig. 4). Based on the XRD pattern, the sample was
an amorphous solid without regular crystalline lattice.
Fig. 4. XRD Pattern of the [BMIm]SS.
The scanning electron microscopy (SEM) image was applied to evaluate the size and morphology of the
[BMIm]SS (Fig. 5). As can be seen in SEM images, the catalyst powders were produced in nano scales, and
these particles were nearly spherical in morphology. Regarding to SEM analysis, using the
microstructural image processing software (MIP software), the acquired histogram determined the
particle size distribution around 20–25 nm (Fig. 6). The chemical composition of nanoparticles was also
determined using Energy-dispersive X-ray spectroscopy (EDS) (Fig. 7). The EDS analysis has indicated
the presence of elements like C, Si, N, O and S which is a good evidence for synthesis of [BMIm]SS.
7

Fig. 5. Scanning electron microscopy (SEM) image of [BMIm]SS.
Fig. 6. Histogram representing the average diameter of [BMIm]SS.
Fig.7. EDS Spectrum of [BMIm]SS.
8

The FT-IR spectrum of the [BMIm]SS is shown in Fig. 8. Since [BMIm]SS is a solid, the IR spectrum was
recorded by the KBr disk technique. FT-IR spectra of silica gel, SSA and [BMIm]SS are shown and
compared as spectra (A), (B) and (C), respectively (Fig. 8). In spectrum (A), the broad bending at 3600
cm-1 is related to the stretching frequency of the OH group on the silica gel substrate, and the broad
bending at 1007-1200 cm-1 is related to the symmetric and anti-symmetric stretching frequency of the
Si-O-Si bond which also are observed for both SSA and [BMIm]SS. A peak pertaining to S=O at 1200 cm-1
which overlaps with Si-O-Si peak is a good criterion for grafting of sulphonic acid on silica gel surface (B,
C). Finally, in spectrum (C), an absorption band at 2850-2950 cm-1 is assigned to CH which is a good
evidence for loading the organic residue into the inorganic part.
Fig. 8. FT-IR Spectrum of the silica gel (A), SSA (B) and [BMIm]SS (C).
The thermogravimetric (TGA) analysis for [BMIm]SS performed at weight of sample in heating rate of
10 °C min-1 in N2 atmosphere within temperature range 26–900 °C. The thermogram of [BMIm]SS is
shown in Fig. 9. The thermogram of the catalyst indicated a thermal stability of [BMIm]SS around 200°C
with mass loss (≈ 2%) below 100 °C, due to removal of physically adsorbed water from the sample. As
shown by TGA analysis, the thermal degradation of [BMIm]SS began at temperatures above 200 °C,
which was attributed to the loss of organic residue loaded onto the silica surface and continues to 400 °C.
9

Subsequent weight loss that occurred at temperatures above 600 ° C was related to the inorganic part of
the catalyst. In general, the [BMIm]SS nearly lost 15% of its overall weight at 900 °C which is a good
criterion for grafting of organic residue on the surface of silica.
102
100
98
96
94
92
90
Figure 2: Thermogram of [BMIm]SS
88
86
84
0
200
400
600
800
1000
Temperature (°C)
Fig.9. Thermogram of [BMIm]SS.
2.3. Synthesis of new 1,2-diol monoesters of IBP derivatives
The synthetic route to access the title compounds was performed due to general pathway illustrated in
Scheme 1. In order to optimize the reaction conditions, we initially selected the ring opening of
cyclohexene oxide with IBP as a model reaction. Firstly, it was attempted to optimize the factors
influences in progress of reaction comprising solvent type, catalyst amount and temperature (Table 1).
As shown in Table 1, the influence of diverse traditional solvents on sample reaction was explored (Table
1, entries 1-10) and it was indicated that among solvents acetonitrile provided the best result.
Additionally, the use of acetone acquired a reasonable yield for 1a; however, the other solvents were not
appropriate enough to be considered as a reaction media. Next, to determine the optimized amount of
[BMIm]SS, the different amounts of catalyst were applied on sample reaction (Table 1, entries 3 and 11-
16). Prior to use of the catalyst, it was indicated by a simple titration that each 0.5 g of catalyst contains
1.22×10-3 mol% of [BMIm] species. Thus, the different amounts of [BMIm]SS were loaded to find out the
10

extent of reaction progress. As can be seen in Table 1, without the catalyst, the reaction was not
conducted at all. In addition, the best result was obtained when 4.88×10-4 mol% (0.2 g) catalyst was
employed. The use of larger amounts of catalyst did not have a tangible effect on progress of reaction.
The effect of temperature was also assessed and revealed that the reaction was poorly achieved at
ambient temperature and the maximum yield was obtained when the reaction was set to reflux
condition.
a
Table 1.The influence of solvent type, catalyst amount and temperature on sample reaction.
O
solvent, [BMIm]SS (xmol%)
O
O
+
T C
OH
O
OH
Yield (%)b
1a
Entry
Solvent
[BMIm]SS (mol %)
Time (h)
T C
1
2
DMF
4.88×10-4
4.88×10-4
4.88×10-4
4.88×10-4
4.88×10-4
4.88×10-4
4.88×10-4
4.88×10-4
4.88×10-4
4.88×10-4
0
1.22×10-4
2.44×10-4
3.66×10-4
6.10×10-4
7.32×10-4
4.88×10-4
4.88×10-4
4.88×10-4
4.88×10-4
100
36
48
24
48
36
48
48
24
48
72
72
24
24
24
24
24
36
36
36
24
46
32
90
36
41
51
57
69
38
NR c
NR
33
50
67
90
90
22
24
63
72
DMSO
MeCN
HMPT
NMP
100
3
reflux
100
4
5
100
6
THF
reflux
reflux
reflux
100
7
Toluene
Acetone
PEG 200
H2O
8
9
10
11
12
13
14
15
16
17
18
19
20
reflux
reflux
reflux
reflux
reflux
reflux
reflux
15
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
20
50
75
a Reaction conditions: IBP(10 mmol), cyclohexen oxide (12 mmol), [BMIm]SS (xmol %) and solvent (30 mL).
b Isolated yield.
c No reaction.
With the optimal reaction conditions in hand, we screened the versatility and the scope of this protocol
using [BMIm]SS as a catalyst. In this regard, the optimized reaction conditions were extended to the
reaction of a variety of structurally diverse epoxides with IBP. Good to excellent yields of the
11

corresponding IBP 1,2-diol monoesters were obtained through the reaction of IBP with cyclohexene
oxide and some terminal epoxides bearing diverse functionalities. Also, good regioselectivity was
observed for the reaction of IBP with terminal epoxides using [BMIm]SS as a catalyst. In this regard, the
ring opening of terminal epoxides was chiefly happened from their less hindered sides (>90%) as
indicated by GC analysis. The structure of synthesized compounds 1a-1o is shown in Fig.10.
O
O
O
O
O
O
O
OH
OH
OH
1a
1b
1c
24h, Yield (%): 90
14h, Yield (%): 88
12h, Yield (%): 92
O
O
O
O
O
O
O
O
O
O
OH
OH
OH
1d
1e
1f
36h, Yield (%): 50
12h, Yield (%): 90
17h, Yield (%): 80
OMe
O
O
O
O
O
O
O
O
O
OH
OH
OH
1g
1h
1i
12h, Yield (%): 88
22h, Yield (%): 85
20h, Yield (%):90
O
O
O
O
O
O
O
O
O
OH
O
OH
1j
OH
Cl
1k
1l
16h, Yield (%): 90
15h, Yield (%): 88
18h, Yield (%): 85
O
O
O
O
O
O
O
O
O
OH
OH
O
OH
O
1m
1n
1o
18h, Yield (%): 80
12h, Yield (%): 92
14h, Yield (%): 88
Fig. 10. Structure of novel 1,2-diol monoester of IBP.
As shown before, without the catalyst, the reaction was not conducted at all even in trace amount,
hence the catalyst has undeniable role in progress of reaction. As it is well indicated, for the ring opening
of epoxides, the presence of nucleophilic, basic or acidic conditions are usually essential. However
neither MeCN nor IBP are known as strong nucleophile and acid, respectively. For example, IBP is a weak
acid with pKa=5.3 [49] which does not seem to be able to catalyze the reaction alone. Previously, we
reported that the [BMIm]Br as an efficient base-free media for ring opening of epoxide via carboxylic
acids [50]. In the same circumstances, we proposed a plausible mechanism for the ring opening of
epoxides via IBP that catalyzed by [BMIm]SS ( Scheme 3).
12

O
O
S
H-cation exchange
N
O
SiO2
O
O
S O
O
(1)
(2)
SiO2
O
OH
+
+
N
O
N
O
OH
N
(i)
O
H
O
protonation
O
O
SiO2
O
S
O
+
SiO2
O
S OH
O
R
R
O
(ii)
H
O
O
O
O
O
anion exchange
N
H
+
SiO2
N
O
S
O
SiO2
(3)
(4)
O
S
O
+
O
O
R
O
O
O
N
R
N
(iii)
O
O
H
internal collapse
O
O
O
R
R
OH
Scheme 3. A plausible mechanism for [BMIm]SS catalyzed the reaction of IBP and epoxides.
As can be seen in Scheme 3, in an initial step, because of ionic nature of immobilized ionic liquid on
surface of [BMIm]SS, the strong electrostatic interaction between IBP and [BMIm]SS causes the
dissociation of IBP followed by hydrogen-cation exchange which acquires SSA and [BMIm]ibuprofenate
(i). Next (Step 2), the in situ generated SSA transfers the proton to epoxide to provide SSA-epoxide
conjugate (ii). Then, through an anion exchange between (i) and (ii), an adduct of ibuprofanate-epoxide
(iii) is generated followed by regeneration of [BMIm]SS (Step 3). Finally, the irreversible internal
collapse of (iii) results in 1,2-diol mono-esters of IBP.
2.4. The reusability of [BMIm]SS
To assess the reusability of [BMIm]SS, this catalyst was applied for synthesis of 1a as a sample reaction
after several consecutive runs. To this end, after each run, the reaction mixture was filtered in vacuo
through a sintered-glass funnel, and the residual solid was washed consecutively with ethanol and dried
in a vacuum oven at 90 °C for 1h. The catalyst was then reused promptly without further purification,
and no fresh catalyst was charged in latter runs. The catalyst was examined in seven consecutive runs
which the results are depicted in Table 2. Due to obtained results (Table 2), the [BMIm]SS can be
recycled and reused for successive trials without remarkable loss of its activity.
13

Table 2. The reusability of [BMIm]SS in successive trails for synthesis of 1a. a
[BMIm]SS
O
O
O
+
OH
O
MeCN, reflux, 24h
OH
1a
Run
1
2
3
4
5
6
7
Time (h)
24
Yield (%) b
90 c
90
89
87
84
81
79
24
24
24
24
24
24
a Reagent and conditions: IBP (10 mmol), cyclohexene oxide (12 mmol), [BMIm]SS (4.88×10-4 mol%), MeCN (30 mL), 24 h
b Isolated Yield
c Fresh catalyst
2.5. Biological assessment
2.5.1. Formalin test
Formalin test is one of the standard tests for measuring pain response. In this study, animals' response
to pain caused by formalin was measured [51].
Experiments were performed on female mice (25–30g, n=54) purchased and kept in Comparative and
Experimental Medicine Center of Shiraz University of Medical Sciences, Shiraz, Iran. Animal selection,
care and sacrifice protocols were adhered to the Animal Care Committee of Iran Veterinary Organization
guidelines. The mice were kept under a standard 12 h light/dark cycle at 21±2 C with ad libitum access
to food and water. Animals were randomly divided into 3 equal groups of control, positive control and
treatment. Before the inception of the experiment, mice were moved to a testing lab for at least 60 min
and put in the formalin testing boxes for habituation for at least 30 min. A mirror was placed underneath
at a 45° angle to allow a clear view of the paws. The synthesized compounds 1a-1o or IBP were dissolved
in olive oil and were fed orally to mice 10 min before pain tests. The dose of 1 μL per gram of mouse was
prepared for synthesized compounds and each animal was then gavaged using a special needle. Formalin
(1%, 20 μL; SC) was subcutaneously injected into right hind foot. These behaviors were scored and
recording in several times (5, 10, 15, 20, 25, and 30 min) after formalin injection. The total time taken (in
seconds) to hold, lick, and bite the foot to which formalin was injected was measured at intervals of the
14

first 5 min for acute pain and up to 30 min as chronic pain. Any 1a-1o that can reduce this time has a
better analgesic effect. The selection of these time intervals as an indicator of acute and chronic pain was
based on previous studies [52]. These studies have shown that the first 5 min after formalin injection,
when the animal shows severe painful behaviors, indicates acute pain, and then the second phase, pain
(chronic pain) begins that does not show severe painful behaviors. Motor response to pain was recorded
in the form of numbers zero, 1, 2 and 3 due to Dubuisson and Dennis method [53, 54]. The zero number
indicates the status that animal was in perfect balance while walking and its weight was distributed on
both legs. The number 1 indicates the status that animal could not bear the weight of the body on the foot
that was injected with formalin or had difficulty in walking. The number 2 indicates that the animal was
lifting the paw to which formalin was injected and had no contact with the floor of the chamber and the
number 3 indicates the status that animal licked or shook the formalin-injected paw. Pain score during
60 min was calculated as 12 blocks of 5 min, the average pain score in each block was calculated
according to the following equation:
푇0 + 푇1 + 푇2 + 푇3
Pain Score =
20
In this equation, T0, T1, T2 and T3, indicate the number of 15 seconds that the animal exhibited zero, 1, 2,
and 3 behaviors over a 5-minute period, respectively.
The analgesic effect curves of synthetic compound, IBP, control and blank group were plotted. The pain
scores versus time for the most potent compounds were shown in Fig. 11. As can be seen in Fig. 11, the
synthetic compounds 1h, 1o, 1k, 1e, 1l and 1c showed the best analgesic activity in comparison with IBP,
blank and control. The majority of tested compounds demonstrated the good activity at early stage of
oral administration at 5 min. Among tested compounds, 1h showed very strong activity compare to IBP
and others with pain score of 0.66 at 5 min. The second potent compound was 1o which displayed
slightly weaker pain score at 5 min but exhibited equal activities with 1h at other time intervals. It is
worthy to mention that, when compounds 1h, 1o and 1k were applied, the animals merely felt pain at 5
min while at other times they didn’t feel pain at all. The analgesic effect curves pertaining to 1e, 1l and 1c
also proved the remarkable analgesic activity compare to parental IBP. The pain scores for 1a, 1b, 1d, 1f,
15

1g, 1i, 1j, 1m, and 1n which are not shown here also demonstrated stronger or equal activities in
comparison with IBP at different time intervals (see the Electronic Supplementary Information).
Fig.11. Analgesic effect curves of the most potent synthetic compounds 1h, 1o, 1k, 1e, 1l and1c.
2.5.2. The acute pain and chronic pain
In Fig. 12, the comparison of the mean pain scores in the acute and chronic stages of the formalin test
between compounds, IBP, blank, and control groups are given. Long-term pain is called chronic or
persistent pain and the pain which is quickly relieved is called acute pain. For this purpose, we
considered mice’s pain for the first 5 min of acute pain and averaged pain for 30 min for chronic pain. As
can be seen in Fig. 12, the most potent compounds 1h, 1o, 1k, 1e, 1l and1c had better acute and chronic
pain scores than IBP. Also 1f and 1g demonstrated lower acute and chronic pain scores than IBP,
whereas 1d, 1i and 1m displayed poor performance from both acute and chronic pain aspects (see the
16

Electronic Supplementary Information). Furthermore, 1a showed better performance for chronic pain
but exhibited higher pain score for acute pain. However, 1b displayed the opposite effect of 1a against
acute and chronic pains. In general, the animals felt less pain if the synthesized compounds were used
either after 5 minutes or more. Thus, it can be concluded that IBP 1,2-diol monoesters not only could be
used to treat the acute pain but also the chronic pain.
Fig. 12. The comparison of the mean pain scores in the acute and the chronic pain for the most potent compounds 1h, 1o, 1k,
1e, 1l and1c.
17

2.5.3. Toxicity studies
The animals were examined for one month after administration of 1a-1o and the mortality rate was
recorded. No mortality was observed due to the toxicity of the synthesized compounds.
2.6. Molecular docking study
Nowadays, molecular docking study is well-established as a powerful tool for prediction of probable
interactions between drugs/drug candidates and target receptor/enzyme binding sites. Since some of
our studied compounds exhibited good to excellent analgesic activity, we were encouraged to investigate
their binding mode in the active site of target protein. In this context, the docking studies of compounds
1e, 1h, 1k, and 1o as the most potent compounds were assessed using Molegro Virtual Docker (MVD 6.0)
software with its default settings [55]. Compounds 1i and 1m are the structural isomers of 1h and 1o,
respectively which showed weak analgesic activity. The question that comes to mind, why the difference
in position of naphthyl moieties (α in 1h and β in 1i) caused the remarkable difference in activity
between 1h and 1i? In addition, why the change in position of a double bond from allyl in 1o to vinyl in
1m also resulted in activity gap? To answer these questions, the docking studies for 1i and 1m were
achieved to gain a correlation between their activity and interaction with an active site of enzyme.
Previously, it was reported that the anti-inflammatory and analgesic effects of IBP are due to the
inhibition of COX-2 enzyme [56]. Therefore, the crystal structure of murine cyclooxygenase-2 (COX-2) in
complex with IBP was selected as a template defined by PDB code: 4PH9. Initially, the ligand-bound
crystallographic structure of COX-2 (4PH9) with 1.81 Å resolution was retrieved from the Protein Data
Bank (http://www.rcsb.org). To modify the structure of selected enzyme, the assignment of hydrogen
positions was achieved based on the default rules and all missing hydrogens were compensated. Then,
the water molecules and the present ligands which are not involved in the binding of COX-2 enzyme and
new ligand were removed. Afterward, the correct atom types and correct bond types were defined. The
docking study was performed and reported based on 40 independent runs. The dock poses of selected
compounds were analyzed to identify their interactions with COX-2 enzyme. Moreover, the definition of
the active site of COX-2 enzyme was achieved by the amino acid residues within a 7Å radius around the
ligands including IBP, 1e, 1h, 1i, 1k, 1m, and 1o. The geometry optimization of selected compounds and
IBP was attained via DFT method at the B3LYP/6-311+G** level of theory using Gaussian09 program
18

package [57]. The validation of docking setup was performed through redocking of the co-crystallized
IBP at the active site of COX-2. Previously, it was reported that RMSD value less than 2Å is adequate to
validate the docking setup [58]. The results indicated that the root-mean-square deviation (RMSD) value
between the docked and co-crystallized IBP was 0.65 Å. Therefore, the validated docking protocol was
applied to investigate the interaction and binding mode of compounds 1e, 1h, 1i, 1k, 1m, and 1o with the
active site of COX-2 enzyme. An overlay view of docked conformations of 1e, 1h, 1i, 1k, 1m, 1o and IBP
at the binding site of COX-2 enzyme is illustrated in Fig. 13. Interestingly, similar to IBP, 1e, 1h, 1i, 1k,
1m, 1o were located at the same binding site. However, due to their larger molecular size, a part of
molecules were placed outside of the active site and interacts with amino acids near the active site.
Fig. 13. Overlay view of docked conformations of 1e (orange), 1h (light blue), 1i (green), 1k (dark blue), 1m (violet), 1o
(pink) and IBP (yellow) in binding site of COX-2 enzyme.
IBP was located near the apex of COX-2 active site during its binding to COX-2 enzyme [59]. Among the
different amino acids near the entrance of the active site of COX-2 enzyme, Arg121 and Tyr356 play an
important role for desirable interaction of enzyme with IBP [60]. Apparently, guanidinium moiety of
Arg121 and the hydroxyl group of Tyr356 provided double and single H-bonds with the carboxylate
group of IBP, respectively. In addition, the hydrophobic interactions between lipophilic parts of IBP and
amino acids presented in the substrate channel, stabilized this compound in the active site of enzyme.
Thus, Val350, Leu353, Val524, and Ala528 interacted with the benzyl moiety of IBP. Val350 and Leu360
made the hydrophobic interactions with α-Me group of IBP. Also, several hydrophobic interactions were
detected between the isobutyl moiety of IBP and Leu353, Leu385, Trp388, Phe519, Met523, Val524,
19

Gly527, Ala528, and Ser531. The docked conformation and H-bonds of IBP in the active site of COX-2
enzyme are shown in Fig. 14.
Fig. 14. Docked conformation and H-bonds (light blue) of IBP in active site of COX-2 enzyme.
Owing to apparent differences in activity of 1h and 1i, molecular docking study was achieved using
MVD software to better understanding the correlation between the activity and the binding mode of
compounds. Fig. 15 shows the docked conformation and H-bond of 1h and 1i in the active site of COX-2
enzyme. By default, MVD software identified five binding sites. As expected, 1h was located in the active
site where it also occupied by IBP. Due to the larger size of 1h in comparison with IBP and small size of
active site, a part of 1h was located outside of the active site cavity and provided extra binding like
hydrophobic interactions with other amino acids near the active site. The carboxylate group of 1h
participated in double H-bonds with Arg121 and single H-bond with Tyr356 which stabilized 1h in the
active site of enzyme (Fig. 15a). Also, the hydroxyl group of 1h afforded additional H-bond with Tyr356.
The α-naphthyl moiety of 1h prepared the hydrophobic contacts with Tyr349, Val350, Leu353, Tyr386,
Trp388, Met523, Val524, Gly527, Ala528, and Ser531. The rest of molecule involved some hydrophobic
interactions with Pro86, Leu93, Val89, Tyr116, Val117, and Ser120 which are near the active site of
enzyme.
Compound 1i was twisted to some extent in the active site of enzyme and in contrary to 1h, β-naphthyl
of 1i located outside of the active site and stabilized through non-bonded interactions with His90,
Tyr349, Leu353, Trp388, Arg514, Ala517, Ile518, Phe519, and Val524 (Fig. 15b). The carboxylate group
of 1i contributed in H-bonds with Tyr349 and Tyr386. In addition, one H-bond interaction was detected
between hydroxyl moiety of 1i and Met523. The rest of molecule was placed in a pocket and interacted
with Val345, Tyr349, Val350, Val524, Gly527, Ala528, and Ser531. The calculated ∆G values for IBP, 1h
20

and 1i were –97.73, –164.22 and –167.87 (kcal/mol), respectively. While ∆G value for 1i was more
negative than ∆G value for IBP and 1h; however, the analgesic activity of 1i was less than those of IBP
and 1h. It seems that the H-bonding interaction of a ligand with both Arg121 and Tyr356 is essential for
its analgesic activity. This hypothesis obviously can be confirmed by obtained results from docking and
experimental studies where the lack of these H-bond interactions regarding 1i caused the weak analgesic
activity.
Fig. 15. Docked conformation and H-bonds (light blue) of 1h (a) and 1i (b) in active site of COX-2 enzyme.
(a)
(b)
The docked conformation and H-bonds of 1m are shown in Fig. 16a. As shown in Fig. 16a, the eugenolyl
moiety of 1m was placed outside of an active site and contributed in hydrophobic interactions with
Val89, Val117, and Tyr116. Also, the isobutyl group of IBP core in 1m located outward of the active site
and interacted with Leu385, Tyr386, and Met 523. Thus, the isobutyl group lost numerous hydrophobic
interactions with amino acids present in the active site. The benzyl and α-Me groups of IBP core
interacted with a hydrophobic pocket consists of Val350, Leu353, Ser354, Phe519, Met523, Val524,
Gly527, Ala528. The carboxylate, hydroxyl, phenoxy, and methoxy groups of 1m participated in four H-
bond interactions with Tyr356. Additionally, the triple H-bond interactions of Arg121 with hydroxyl and
phenoxy groups were detected.
The docking analysis of 1o revealed the contribution of carboxylate group in H-bond interaction with
Tyr356. Also, the hydroxyl residue of 1o afforded additional H-bond interactions with Arg121 and
Tyr356 (Fig.16b). Due to larger size of 1o compare to IBP, the eugenolyl moiety of 1o located outside of
the active site and participated in hydrophobic interactions with Pro86, Val89, Leu93, Val117, Ser120,
and Ser472. The IBP core of 1o was completely located in an active site and interacted with a
21

hydrophobic pocket formed by Val350, Leu353, Ser354, Leu360, Phe382, Leu385, Trp388, Phe519,
Met523, Val524, Gly527, Ala528, Ser531, and Leu532.
(a)
(b
Fig. 16. Docked conformation and H-bonds (light blue) of 1m (a) and 1o (b) in active site of COX-2 enzyme.
The calculated ∆G values were –172.99 and –176.17 (kcal/mol) for 1m and 1o, respectively. Due to
result, despite the energy gap for binding of 1m and 1o to the active site of COX-2 enzyme was negligible;
however, their analgesic activities were not comparable. Obviously, the lack of proper hydrophobic
interactions of the isobutyl group of IBP core in 1m can be compensated by seven strong H-bond
interactions to afford a ∆G value similar to that of 1o. Considering the obtained results, it can be
concluded that beside the H-bond interaction of a ligand with Arg121 and Tyr356 present in an active
site of COX-2 enzyme, the proper hydrophobic interactions between a ligand and the active site of COX-2
enzyme played a vital role to determine the analgesic activity.
The docked conformation and H-bonds of compound 1e are shown in Fig. 17a. The IBP core of 1e was
sited in the active site of COX-2 enzyme and interacted with a hydrophobic pocket formed by Val350,
Leu353, Ser354, Leu360, Trp388, Phe519, Met523, Val524, Gly527, Ala528, Ser351, and Leu532. The
carboxylate group afforded three H-bond interactions with Arg121 and one H-bond interaction with
Tyr356. The hydroxyl group of 1e participated in two H-bond interactions with Arg121 and Tyr356. The
aliphatic chain of molecule oriented outward of the active site and interacted with Val89, Leu93, Ile113,
Val117, Tyr116, and Ser120. In the case of 1k, the IBP core of 1k was placed in the active site and
interacted with Val350, Leu353, Ser354, Leu385, Tyr386, Trp388, Phe519, Met523, Val524, Gly527,
Ala528, and Ser531 (Fig. 17b). The carboxylate and hydroxyl groups of 1k participated in four H-bond
interactions with Arg121 and Tyr356. The rest of molecule was placed out of the active site and provided
22

non-bonded hydrophobic interactions with Val89, Leu93, Ile113, Val117, Phe358, and Leu360. The
calculated ∆G values for 1e and 1k were –135.30 and –145.52 (kcal/mol), respectively. Indeed, the
obtained results from the docking study have confirmed the superior analgesic activity of 1e and 1k in
comparison with IBP which is in a good agreement with the obtained experimental results.
(b)
Fig. 17. Docked conformation and H-bonds (light blue) of 1e (a) and 1k (b) in active site of COX-2 enzyme.
(a)
2.7. Quantum study
The energy of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular
orbital (LUMO) were calculated at the B3LYP/6–31G** level of theory for the optimized structure of 1e,
1h, 1i, 1k, 1m, 1o and IBP to rationalize the binding affinity of these compounds (Table 3). The
HOMO/LUMO views for the optimized geometry of the most potent compounds 1h, 1o, 1k and IBP were
shown in Fig. 18. The HOMO–LUMO energy gaps for 1e, 1h, 1i, 1k, 1m, 1o and IBP as well as the
hardness and softness values were depicted in Table 3.
As depicted in Table 3, except that of 1e which has a close softness with IBP, all other active compounds
were softer than IBP and thus had more polarizability than IBP.
Table 3. Calculated HOMO, LUMO, hardness, and softness for 1e, 1h, 1i, 1k, 1m, 1o and IBP using B3LYP/6–31G** level of
theory
Ligand
1e
HOMO a
-6.670
-5.700
-5.733
-5.731
-5.427
-5.630
-6.645
LUMO a
-0.760
-1.134
-1.297
-0.798
-0.820
-0.794
-0.766
Softness a
0.169
0.219
0.225
0.203
0.217
0.207
0.170
Hardness b
5.909
1h
4.567
1i
4.437
1k
4.933
1m
1o
4.607
4.836
IBP
5.879
a Unit in electron volt (ev)
b Unit in ev-1
23

Despite of higher calculated polarizabilities compare to IBP and hence expecting more antagonistic
property, nevertheless 1i and 1m displayed weaker analgesic profiles which attributed to unsuitable
steric conditions for strong binding at active site of enzyme.
LUMO (1h)
LUMO (1o)
HOMO (1h)
HOMO (1o)
LUMO (IBP)
LUMO (1k)
HOMO (1k)
HOMO (IBP)
Fig. 18. HOMO and LUMO views for the optimized geometry of the most potent compounds 1h, 1o, 1k and IBP.
2.8. In silico pharmacokinetic profile
Recently, the expanded approaches on the basis of in silico prognostication have found a powerful tool
for methodical assessments of physiochemical properties [61]. In this regard, different physiochemical
parameters and molecular descriptors were developed to estimate toxicity risks, drug likeness and drug
scores in drug candidate molecules [62, 63]. The concept developed by Lipinski, so called Lipinski’s rule
of five (RO5), transpired as a simple way to predict the drug likelihood for candidate drugs. Due to RO5, a
drug candidate should (i) have a molecular weight less than 500 Dalton, (ii) avoid more than 10
hydrogen bond acceptors (sum of N and O), (iii) exhibit 10 or fewer rotatable bonds, (iv) have an
octanol–water partition coefficient (log P) value not more than 5; and (v) include the hydrogen bond
donors (sum of NH and OH) less than 5. A drug candidate molecule that does not conform to these rules
likely shows inferior pharmacokinetic profile for oral administration.
To study the in silico pharmacokinetic profile for synthesized compounds, we used a well-known and
fully established OSIRIS DataWarrior V4.7.1 freeware [64]. This software encompasses the database for
commercial none drug molecules or traded drugs. The risks of side effects, such as mutagenic,
tumorigenic, irritant and reproductive effects, can be assessed using this software. The predicted toxicity
risks by OSIRIS DataWarrior for all synthetic compounds and IBP were recorded in Table 4.
24

Table 4. Toxicity risks predicted by OSIRIS DataWarrior for all synthesized compounds and IBP.
Compound
Mutagenic
None
None
None
High
Tumorigenic
None
None
None
None
None
None
None
None
None
None
None
None
None
None
High
Reproductive Effective
Irritant
None
Low
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
IBP
None
High
None
High
None
High
None
None
None
None
None
None
None
None
None
High
Low
High
None
High
None
None
None
None
None
Low
None
High
High
None
None
None
None
None
None
None
None
None
None
None
None
High
None
As indicated in Table 4, except that of 1d and IBP as a reference drug, all synthesized compounds were
predicted to exhibit none risk of mutagenic. Also except that of 1o, other synthesized compounds and IBP
were predicted to implicate none risk of tumorigenic. Furthermore, except that of 1b, 1d, 1f and IBP that
showed the reproductive effective, other studied compounds were anticipated to be harmless from
reproductive effective point of view. In addition, IBP, 1a, 1e, 1g-1k and 1m were predicted to
accommodate with no risk of irritant properties, whereas 1b, 1c and 1l were predicted to involve the low
risk. It is also worth mentioning that 1d, 1f, 1n and 1o were at high risk of irritant properties.
In addition to the toxicity risks prediction by OSIRIS DataWarrior, this software is capable to define
some substantial descriptors or physiochemical parameters. To this end, we computed the Lipinski’s
parameters to show whether compounds are obeyed from Lipinski’s RO5 or not. As shown in Table 5, all
computed molecular weights for compounds were ranged in 278.39–426.55 Dalton (<500 g/mol).
Additionally, the number of hydrogen bond acceptors (nHba) for all compounds were 3–5 which conform
the RO5. The number of hydrogen bond donors (nHbd) for all compounds were equal to 1 which obeys
from the Lipinski’s RO5. Apart from 1d, 1f, 1g, 1k, 1m and 1o, the other compounds involved the
number of rotatable bonds (nrotb) between 6–10 which almost were similar to commercial drugs that lie
within the range of RO5 (Table 5).
One of the most important parameters in Lipinski’s RO5 is lipophilicity factor which traditionally is
represented as an octanol–water partition coefficient (cLog P). Most known drugs were reported to have
25

the cLog P values <5. The cLog P values for all synthesized compounds were computed in range of 3.70–
5.48. Except that of 1h, 1i, 1m and 1o, all others exhibited cLog P below to the defined limit by RO5 (<5).
The aqueous solubility of a molecule (cLog S) is an essential parameter that remarkably affects
absorption and distribution specifications. Most common drugs have a cLog S value>−4. As can be seen in
Table 5, except that of 1b, 1d, 1e and 1f, all compounds had the values less than indicated threshold
value (<−4). Total polar surface area (TPSA) is an effective parameter for prediction of drug transport
properties. TPSA is specified as a sum of surfaces of polar atoms (usually oxygen, nitrogen, and attached
hydrogen) in a molecule. This parameter correlates very well with the human intestinal absorption and
blood–brain barrier penetration. For most usual drugs, TPSA content are known to be less than 140 Å2.
As shown in Table 5, all synthesized compounds involved TPSA values less than 140 Å2.
Table 5. Physicochemical properties predicted by OSIRIS DataWarrior software.
Compound mw
nHba nHbd nrotb
cLogP
4.14
3.72
4.29
3.70
4.53
4.09
4.22
5.48
5.48
4.98
4.22
4.90
5.31
4.63
5.24
3.00
cLogS TPSA Drug likeness
Drug score
0.35
0.24
0.45
0.08
0.66
0.13
0.55
0.34
0.34
0.44
0.55
0.36
0.28
0.30
0.10
0.24
1a
1b
1c
1d
1e
1f
304.43
3
3
4
5
4
4
5
4
4
4
5
4
5
4
5
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
8
-4.20 46.53
-3.46 46.53
-4.14 55.76
-3.43 72.83
-3.49 55.76
-3.65 55.76
-4.16 64.99
-5.75 55.76
-5.75 55.76
-4.83 55.76
-4.16 64.99
-4.88 55.76
-5.07 64.99
-4.49 55.76
-4.87 64.99
-2.90 37.30
-3.69
-0.86
0.82
-23.48
0.82
-7.26
0.97
0.82
0.82
0.82
0.93
0.92
-1.20
0.82
-1.71
0.08
278.39
356.46
348.44
322.44
336.47
386.49
406.52
406.52
384.51
386.49
390.91
426.55
370.49
426.55
206.28
10
11
10
12
11
10
10
10
11
10
12
10
13
4
1g
1h
1i
1j
1k
1l
1m
1n
1o
IBP
Drug likeness is another parameter that predicted by current software. Among the compounds, 1a, 1b,
1d, 1f, 1m and 1o displayed negative drug likeness while the other compounds were found to have the
positive values.
In addition, the software uses the above parameters to give the drug score to indicate whether it can be
considered as a drug candidate or not. According to Table 5, 1e and 1d were indicated to have maximum
and minimum drug score values, respectively. Regarding to all parameters like drug likeness, drug score
and remarkable analgesic activity, 1h can be suggested as an appropriate future drug candidate.
26

3. CONCLUSION
In summary, the fabrication, characterization and application of butyl methyl imidazolium silica sulfate
[BMIm]SS as a novel nano hybrid catalyst and its application in synthesis of new IBP 1,2-diol mono esters
were described. [BMIm]SS was proved to be an efficient nano-hybrid catalyst with thermal and chemical
stability. This catalyst was a low cost and an environmentally benign that simply prepared and reused for
many reaction runs without a significant decline in its reactivity. The synthesized compounds were
tested in vivo for the analgesic properties on female mice using formalin test. The test results revealed
that most compounds, especially 1c, 1e, 1h, 1k, 1l and 1o displayed potent analgesic activity compare to
IBP as a reference drug. The docking analysis was conducted to gain a correlation between compound’s
activity and interaction with the active site of murine cyclooxygenase-2 (COX-2). The results confirmed
stronger binding affinity of active compounds to active site of enzyme compare to IBP. The in silico
pharmacokinetic profile, drug likeness and toxicity predictions were carried out for all compounds which
determined 1h as potent analgesic drug candidate for further investigations.
4. EXPERIMENTAL
4.1. General
All chemicals were purchased from Merck or Sigma-Aldrich. Solvents were purified by standard
procedures, and stored over 3Å molecular sieves. Reactions were followed by TLC using SILG/UV 254
silica-gel plates. Column chromatography was performed on silica gel 60 (0.063–0.200 mm, 70–230
mesh; ASTM). IR spectra were obtained using a Shimadzu FT-IR-8300 spectrophotometer. GC/MS was
performed on a Shimadzu GC/MS-QP1000-EX apparatus (m/z; rel. %). Elemental analyses were
1
performed on a Perkin–Elmer 240-B micro-analyzer. H- and 13C-NMR spectrum were recorded on
Brüker Avance-DPX-250/400 spectrometer operating at 300/75 MHz, respectively. Chemical shifts are
given in δ relative to tetramethylsilane (TMS) as an internal standard, coupling constants J are given in
Hz. The scanning electron micrograph was achieved using SEM (VEGA//TESCAN-LMU) instrument. The
patterned X-ray diffraction (XRD) was obtained using X'PERT PRO MPD Panalytical. The TGA test was
1
13
performed using METTLER TOLEDO instrument. Data involving InChI (Key), HNMR and CNMR for all
products can be found in Electronic Supplementary Information.
27

4.2. General procedure for synthesis of [BMIm]SS
In a round-bottomed flask, butyl bromide (15 mmol) was added to the solution of N-methyl imidazole
(10 mmol) in anhydrous acetonitrile (50 mL). The reaction mixture was then placed on magnetic stirrer
for 4h under reflux condition to obtain butyl methyl imidazolium bromide [BMIm]Br. Afterward, to
solution of [BMIm]Br (4 mmol) in 100 mL anhydrous DCM, it was added solid KOH (12 mmol) and
stirred on magnetic stirrer for 24h at room temperature. Then, the reaction content was filtered by a
sintered glass and the filtrate was evaporated to obtained almost pure [BMIm]OH as a brown oil [48].
Next, the SSA powder [43] was suspended in 100 mL deionized water and [BMIm]OH was added
dropwise to the reaction vessel to finally reach a neutral pH (for control the pH, the pH-meter was used
through the course of reaction). The obtained [BMIm]SS was filtered and washed several times with
ethanol (3×10 mL). The [BMIm]SS was kept in a vacuum oven to dry completely and stored in a
desiccator at room temperature.
4.3. General procedure for synthesis of new 1,2-diol monoester of IBP derivatives (1a-1o)
In a round-bottomed flask, it was added IBP (10 mmol), epoxide (12 mmol) and 0.2 g [BMIm]SS in 30 mL
anhydrous acetonitrile. The reaction mixture was placed on magnetic stirrer under reflux conditions for
12-36h (Fig. 10). After completion of the reaction (TLC check), the catalyst was separated by a sintered
glass, and the solvent was evaporated in vacuo (rotary evaporator). Then, the residue was diluted in
CHCl3 (150 mL) and washed with H2O (3 × 150 mL). Afterward, the product was purified by a short
column chromatography eluted by solvents described below.
4.4. Data for synthesized compounds
4.4.1. 2-hydroxycyclohexyl 2-(4-isobutylphenyl)propanoate (1a)
This compound was obtained as a yellow oil using column chromatography on silica gel (EtOAc:n-hexane,
1:20); Yield: 2.73 g, 90%; Rf = 0.46 (EtOAc:n-hexane, 1:3); IR (liquid film) νmax cm-1 = 3500 (O‒H, Stretch.),
3100 (sp2 C‒H, Stretch.), 2967 (sp3 C‒H, Stretch.), 1740 (C=O, Stretch.), 1481 (aromatic C=C, Stretch.),
1410 (sp3 C‒H, Bend.), 1150 (C‒O, Stretch.), 890 (sp2 C‒H, Bend.). 1H NMR (300 MHz, CDCl3) δppm = 0.90
(d, J = 6.6 Hz, 6H, (CH3)2CHCH2), 1.06–1.35 (complex, 6H, 3CH2), 1.47 (d, J = 7.2 Hz, 3H, CH3CHCO2), 1.57–
1.67 (complex, 3H, CH2, (CH3)2CHCH2), 1.79–1.88 (complex, 2H, CHCO2, CHOH), 2.42 (d, J = 7.2 Hz, 2H,
PhCH2), 2.71 (s, 1H, OH), 3.64–3.76 (m, 1H, CO2CH), 7.06 (d, J = 8.1 Hz, 2H, aryl), 7.18 (d, J = 8.1 Hz, 2H,
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aryl). 13C NMR (75 MHz, CDCl3) δppm = 16.87 (C-12), 22.28 (C-20), 22.81 (C-21), 23.41 (C-9, C-10), 28.02
(C-22), 30.46 (C-8), 31.30 (C-18), 39.71 (C-11), 46.48 (C-7), 75.04 (C-17), 82.93 (C-16), 128.06 (C-1, C-5),
129.10 (C-2, C-4), 133.42 (C-6), 140.33 (C-3), 174.10 (C-13). MS (EI): m/z (%) = 304.20 (19.5) [M+]. Anal.
Calcd for C19H28O3: C, 74.96; H, 9.27. Found: C, 75.09; H, 9.38.
4.4.2. 2-hydroxybutyl 2-(4-isobutylphenyl)propanoate (1b)
This compound was obtained as a yellow oil using column chromatography on silica gel (EtOAc:n-hexane,
1:20); Yield: 2.45 g, 88%; Rf = 0.66 (EtOAc:n-hexane, 1:3); IR (liquid film) νmax cm-1 = 3500 (O‒H, Stretch.),
3050 (sp2 C‒H, Stretch.), 2948 (sp3 C‒H, Stretch.), 1746 (C=O, Stretch.), 1438 (aromatic C=C, Stretch.),
1407 (sp3 C‒H, Bend.), 1167 (C‒O, Stretch.), 860 (sp2 C‒H, Bend.). 1H NMR (300 MHz, CDCl3) δppm = 0.90–
0.92 (complex, 9H, CH3CH2, (CH3)2CHCH2), 1.42–1.63 (complex, 5H, CH3CHCO2, CH3CH2), 1.79–1.92 (m,
1H, (CH3)2CHCH2), 2.18 (s, 1H, OH), 2.45 (d, J = 7.2 Hz, 2H, PhCH2), 3.66–3.79 (complex, 2H, CHOH,
CHCO2), 3.93–4.01 (m, 1H, OCHAHB), 4.11–4.18 (m, 1H, OCHAHB), 7.10 (d, J = 7.5 Hz, 2H, aryl), 7.20 (d, J =
7.8 Hz, 2H, aryl). 13C NMR (75 MHz, CDCl3) δppm = 8.19 (C-20), 17.11 (C-12), 23.72 (C-9, C-10), 25.81 (C-
18), 29.49 (C-8), 43.94 (C-11), 46.59 (C-7), 70.76 (C-16), 73.41 (C-17), 127.56 (C-1, C-5), 128.35 (C-2, C-
4), 132.81 (C-6), 139.40 (C-3), 172.25 (C-13). MS (EI): m/z (%) = 278.19 (20.7) [M+]. Anal. Calcd for
C17H26O3: C, 73.34; H, 9.41. Found: C, 73.23; H, 9.28.
4.4.3. 2-hydroxy-3-phenoxypropyl 2-(4-isobutylphenyl)propanoate (1c)
This compound was obtained as a yellow oil using column chromatography on silica gel (EtOAc:n-hexane,
1:20); Yield: 3.27 g, 92%; Rf = 0.60 (EtOAc:n-hexane, 1:3); IR (liquid film) νmax cm-1 = 3400 (O‒H, Stretch.),
3062 (sp2 C‒H, Stretch.), 2971 (sp3 C‒H, Stretch.), 1738 (C=O, Stretch.), 1463 (aromatic C=C, Stretch.),
1403 (sp3 C‒H, Bend.), 1280 (Ar‒O, Stretch.), 1164 (C‒O, Stretch.), 897 (sp2 C‒H, Bend.). 1H NMR (300
MHz, CDCl3) δppm = 0.93 (d, J = 6.6 Hz, 6H, (CH3)2CHCH2), 1.54 (d, J = 7.2 Hz, 3H, CH3CHCO2), 1.82–1.93 (m,
1H, (CH3)2CHCH2), 2.49 (d, J = 7.2 Hz, 2H, PhCH2), 3.12 (br s, 1H, OH), 3.76–3.91 (complex, 3H,
CHCO2CH2), 4.17–4.37 (complex, 3H, CHOH, CH2OPh), 6.87–7.33 (complex, 9H, aryl). 13C NMR (75 MHz,
CDCl3) δppm = 15.96 (C-12), 22.77 (C-9, C-10), 29.56 (C-8), 40.98 (C-11), 46.35 (C-7), 67.44 (C-16), 69.24
(C-17), 70.66 (C-18), 115.36 (C-22, C-26), 121.79 (C-24), 128.23 (C-1, C-5), 128.94 (C-2, C-4), 129.68 (C-
23, C-25), 133.94 (C-6), 141.45 (C-3), 158.96 (C-21), 175.08 (C-13). MS (EI): m/z (%) = 356.20 (22.8)
[M+]. Anal. Calcd for C22H28O4: C, 74.13; H, 7.92. Found: C, 74.01; H, 7.78.
4.4.4. 2-hydroxy-3-(2-(4-isobutylphenyl)propanoyloxy)propyl methacrylate (1d)
This compound was obtained as a yellow oil using column chromatography on silica gel (EtOAc:n-hexane,
1:10); Yield: 1.74 g, 50%; Rf = 0.49 (EtOAc:n-hexane, 1:3); IR (liquid film) νmax cm-1 = 3500 (O‒H, Stretch.),
3037 (sp2 C‒H, Stretch.), 2960 (sp3 C‒H, Stretch.), 1750 (C=O, Stretch.), 1702 (conjugated C=O, Stretch.),
1469 (aromatic C=C, Stretch.), 1402 (sp3 C‒H, Bend.), 1160 (C‒O, Stretch.), 865 (sp2 C‒H, Bend.). 1H NMR
(300 MHz, CDCl3) δppm = 0.89 (br s, 6H, (CH3)2CHCH2), 1.50 (br s, 3H, CH3CHCO2), 1.83–1.95 (complex, 3H,
29