Structure Based Library Design (SBLD) for new 1,4-dihydropyrimidine scaffold as simultaneous COX-1/COX-2 and 5-LOX inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.06.008

The various scaffolds containing 1,4-dihydropyrimidine ring were designed by considering the environment of the active site of COX-1/COX-2 and 5-LOX enzymes. The structure-based library design approach, including the focused library design (Virtual Combin

Full text of DOI:10.1016/j.bmc.2015.06.008

Accepted Manuscript
Structure Based Library Design (SBLD) for New 1,4-dihydropyrimidine Scaf-
fold as simultaneous COX-1/COX-2 and 5-LOX Inhibitors
Deepak Lokwani, Rajaram Azad, Aniket Sarkate, Pallu Reddanna, Devanand
Shinde
PII:
S0968-0896(15)00502-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.06.008
BMC 12364
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 March 2015
2 June 2015
3 June 2015
Please cite this article as: Lokwani, D., Azad, R., Sarkate, A., Reddanna, P., Shinde, D., Structure Based Library
Design (SBLD) for New 1,4-dihydropyrimidine Scaffold as simultaneous COX-1/COX-2 and 5-LOX Inhibitors,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.06.008
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Structure Based Library Design (SBLD) for New 1,4-dihydropyrimidine Scaffold as
simultaneous COX-1/COX-2 and 5-LOX Inhibitors
Deepak Lokwania, Rajaram Azadb, Aniket Sarkatea, Pallu Reddannab, Devanand Shindea*
aDepartment of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad
431004, Maharashtra, India bSchool of Life Sciences, University of Hyderabad, Hyderabad, India
*Corresponding Author, Email:dbsdeepak10@rediffmail.com
Abstract:
The various scaffolds containing 1,4-dihydropyrimidine ring were designed by considering the
environment of the active site of COX-1/COX-2 and 5-LOX enzymes. The structure-based
library design approach, including the focused library design (virtual combinatorial library
design) and virtual screening was used to select the 1,4-dihydropyrimidine scaffold for
simultaneous inhibition of both enzyme pathways (COX-1/COX-2 and 5-LOX). The virtual
library on each 1,4-dihydropyrimidine scaffold was enumerated in two alternative ways. In first
way, the chemical reagents at R groups were filtered by docking of scaffold with single position
substitution, i.e. only at R1, or R2, or R3….Rn on COX-2 enzyme using Glide XP docking mode.
The structures that do not dock well were removed and the library was enumerated with filtered
chemical reagents. In second alternative way, the single position docking stage was bypassed,
and the entire library was enumerated using all chemical reagents by docking on the COX-2
enzyme. The entire library of approximately 15,629 compounds obtained from both ways after
screening for drug like properties, were further screened for their binding affinity against COX-1
and 5-LOX enzymes using Virtual Screening Workflow. Finally, 142 hits were obtained and
divided into two groups based on their binding affinity for COX-1/COX-2 and for both enzyme
pathways (COX-1/COX-2 and 5-LOX). The ten molecules were selected, synthesized and
evaluated for their COX-1, COX-2 and 5-LOX inhibiting activity.
Keywords: COX-2, COX-1, 5-LOX, Structure Based Library Design (SBLD), Virtual
Combinatorial Library Design, Virtual Screening, Docking Study, 1,4-dihydropyrimidine

1. Introduction:
Non-steroidal Anti-inflammatory Drugs (NSAID’s) are the most popular and well recognized
drug for the treatment against inflammatory diseases.1 These drugs prevent the metabolism of
arachidonic acid to prostaglandins (PGs) by binding and inhibiting Cyclooxygenase (COX)
enzymes and hence exert their therapeutic effects.2 The two distinct isoforms (COX-1 and COX-
2) of COX enzyme discovered in 1990, which led to the discovery of a new class of NSAID’s,
selective for COX-2, name coxibs.3 COX-1 is constitutive and responsible for the biosynthesis of
PGs, which protects the stomach lining from the secreted acid and maintains blood flow in
gastric mucosa. The other isoform, COX-2 is triggered only in proinflmmatory conditions. Thus
the long term use of traditional NSAID’s cause adverse gastrointestinal effects, which are
overcome by the use of selective COX-2 inhibitors.4 Unfortunately, the withdrawal of rofecoxib
from the market in the fall of the year 2004 after its clinical report of its linkage to increased risk
of cardiovascular toxicity has widely affected the safety of coxib5-6. Studies suggest that selective
COX-2 Inhibitors also cause GI injury like conventional NSAIDs and increased systemic blood
pressure and thus have severe side effects on the cardiovascular system and promote
thrombogenesis7.
Arachidonic acid, but also be metabolized through lipoxygenase (LOX) pathway, leading
to the production of leukotrienes (LTs), which are potent inflammatory mediators. Therefore, it
has been shown that selective COX-2 inhibitors, beside of preventing side effect NSAIDs
associated with inhibition of COX-1, lead to upregulation of arachidonic acid metabolism by the
5-LOX enzyme.8, 9 This result in the increased levels of LTB4 in the gastric mucosa of NSAIDs
and selective COX-2 inhibitors treated patients. The high level of LTs may also contribute to the
development of atherosclerosis, myocardial infarction. It is revealed from literature that

simultaneous inhibition of both enzymatic pathways (COX-1/COX-2 and 5-LOX enzymes)
would possibly exhibit enhanced anti-inflammatory potency along with reduced GI tract damage
and other inflammatory side effects.10-12 Thus licofelone, a competitive inhibitor of COX-1,
COX-2 and 5-LOX, is currently in clinical development for the treatment of osteoarthritis and is
expected to possess clinical advantages over the conventional NSAIDs and selective COX-2
inhibitors with a safer GI profile.13, 14 It is also noted from literature that the inhibition of more
than one enzyme involved in different pathways of arachidonic acid metabolism lead to a
synergistic anti-inflammatory effect with enhanced spectrum of activity.15-17
The virtual combinatorial library is generated routinely for small molecules during the
drug discovery cycle and screened based on their predicted physicochemical properties,
activities, specificities, etc.18-20 The virtual screening is useful in selecting a smaller set of
promising hits from a large set of compound libraries. The one of the major advantages of
generating virtual combinatorial library is that one can utilize cost effective computational
techniques to identify a diverse set of compounds with desired physicochemical properties and
specificities.
Recently, we reported the design and synthesis of COX-2 inhibitors having scaffold
containing dihydropyrimidine nucleus.21-22 With the help of Literature data of COX-1/COX-2
and 5-LOX inhibitors and focused virtual combinatorial library design approach, we herein tried
to design new dual enzyme pathways inhibitors by modifying/changing substitution pattern
around 1,4-dihydropyrimidine scaffold. The Fig. 1 showed the steps which we carried out for
developing the new dual inhibiting compounds. The designed library was primarily screened by
predicting ADME properties and then virtually screened for dual inhibition of COX-1/COX-2

and 5-LOX enzymes. Some of the hits were then selected and experimentally evaluated for
COX-1, COX-2 and 5-LOX enzymes.
2. Result and Discussion:
2.1. Design of Scaffolds for Virtual Combinatorial Library Design:
The search for COX selective inhibitors is somewhat complicated by the close structural
similarities between COX-1 and COX-2. Both COX-1 and COX-2 are homodimers of 70 kDa
subunits composed of N-terminal epidermal growth factor like module, α-helical membrane
binding domain and a large C-terminal globular catalytic domain with active site
(Cyclooxygenase and peroxidase containing heme cofactor)23. COX catalytic domain in COX-2
is 20% larger and differs in the presence of side pocket when compared to COX-1. The main and
important difference in side pocket is the presence of valine in COX-2 and Isoleucine in COX-1
at position 523 which enables interaction of the sulfonamide or a sulfate group of the coxibs with
Arg 513 in the side pocket of COX-2.
The 5-LOX is iron containing non-heme enzymes which catalyze the dioxygenation of
archidonic acid to lipid hydroperoxide, leukotrienes A4 (LTA4). The 5-LOX enzyme resides in
the reduced inactive state (FE2+) and need to be in a oxidized active state (FE3+) for performing
peroxidising activity23. The direct approaches for the inhibition of 5-LOX enzyme involve redox
inhibitor or antioxidant, iron-chelating agents and non-redox competitive inhibitors. We mainly
focused on the class of iron-chelating agents which include the compounds containing
hydroxamic acid, N-hydroxyurea, carboxylate or ester groups, as powerful metal ligating groups.
Therefore, in an attempt to design new, potent and balanced dual inhibitors, we made the
structural modifications to our previously reported selective COX-2 inhibitors,

dihydropyrimidine analogues21-22 by substituting with the hydroxamic acid, N-hydroxyurea,
carboxylate or ester groups. The ten new scaffolds (core structures) were designed and various
reagents/building blocks at predefined connections (R groups) for each core structure were
selected based on synthetic feasibility, strategy of maintaining the tricyclic (butterfly or ‘V’
shaped) pattern of COX-2 inhibitors, while the iron chelating groups were added to the side
chain of compounds for 5-LOX activity (Fig. 2).
2.2. Virtual Combinatorial Library Design (VCLD):
The Virtual Combinatorial Library Design (VCLD) is regarded as a virtual reaction library,
within which reagents (building blocks) undergo specific reaction and product is generated out as
the result of all possible combinations of reagents. The concept of in-silico combinatorial
chemistry of CombiGlide was used here to design focused combinatorial libraries for the
inhibition of COX-2 enzyme. The Virtual combinatorial Screening Workflow of CombiGlide
holds the core structure constant and changing the side chain composition.
The structure of 1,4-dihydropyrimidine template along with the predefined connection
site (R1, R2, R3, R4,…Rn) are presented in Fig. 2. The reagents/fragment structures that used for
the construction of virtual 1,4-dihydropyrimidine library were selected from building block
chemical structures available at commercial vender databases (Table S1, Supplementary
material). Before enumerating the virtual library, all reagents were prepared with respect to their
functional group for the identification of bond, which is then replaced when the reagent is added
to core structure. Likewise, the attachment position was identified for each side chain site
(connection site) in each core structure by identifying the bond that defines, where the

substitution will take place. Therefore the virtual library was enumerated by replacing selected
side chain in the core with the other side chain from the reagents.
Each designed 1,4-dihydropyrimidine scaffold (various core) that is without substitution
at R groups were first docked on the COX-2 enzyme. The resulting poses were clustered, and
poses from each cluster were selected. This ensures the widest diversity of core poses while
limiting their number. The core poses were then used as the initial positions of the substituted
molecules in the subsequent docking stages. The virtual library was then enumerated in two
alternative ways. In the first way, the fragment structures at R groups were filtered by docking of
each core structure with single position substitution, i.e. only at R1, R2, R3, R4…Rn on COX-2
enzyme, using Glide XP docking mode. These structures are generated by adding the reagents,
one at a time, to the core structure for each scaffolds and a “single position library” was
generated for each attachment position with a side chain at that position and the core structures at
the other positions. In this single-position docking, a selection algorithm was used to determine
or predict the fully substituted cores having the highest probability of binding to the receptor.
The selection algorithm takes into consideration the XP GlideScores of the docked poses from
the single-position docking. It also checks that the side chains it has identified for a particular
fully substituted core are not predicted to compete for the same part of the receptor binding site.
The structures that do not docked well were removed from the reagent list for the appropriate
attachment position and the library was enumerated for each core with filtered fragment
structures that are identified by the selection algorithm.
In a second alternative way, the single position docking stage was bypassed, and the
entire library was enumerated using all fragment structures at all R positions of core structures
by docking of each generated on the COX-2 enzyme.

The libraries generated by both ways, before docking studies were pre-filtered for drug
like properties using Lipinski’s rule of five. The tool QikProp v3.9 was used to generate ADME
properties of each compound within each library. The properties like molecular weight (> 500),
logp (>5), hydrogen bond donor (>5), hydrogen bond acceptor (>10) and rotatable bond (>10)
were set to filter the compounds in each library. The Fig.3 shows the number of compounds
generated and screened by ADME and docking by both ways of combinatorial library
enumeration process. The entire library of roughly 15,629 compounds having good binding
affinity for COX-2 enzyme, obtained from both ways of combinatorial library enumeration, was
further screened for specifying their binding affinity against COX-1and 5-LOX enzymes using
Virtual Screening Workflow.
2.3. Virtual Screening Workflow:
Virtual screening is the most efficient way of identifying small-molecule inhibitors from a large
drug-like database. The entire library of compounds obtained from VCLD was first screened by
removing the ligands with reactive functional groups and then for their potential affinity against
COX-1 and 5-LOX enzymes using Virtual Screening Workflow. For the selection of dual
inhibitors, the hit structures were simultaneously docked on both enzymes (COX-1and 5-LOX)
using three different precisions (HTVS, SP and XP) of Glide docking. The retained compounds
after docking through one precision were further docked through another higher precision for
accuracy and further screening of compounds. The retention percentage was set for HTVS, SP
and XP to keep the 50%, 20% and 10 % of compounds after docking. The compounds retained
for COX-1 and 5-LOX after all Glide docking levels were 1614 and 454 (HTVS docking), 322
and 90 (SP docking), 32 and 9 (XP docking) respectively. In last, all the structures retained from

SP and XP docking studies for both COX-1 and 5-LOX enzyme were combined, merged for
duplicates and cross docking was done against all three enzymes (COX-1, COX-2 and 5-LOX)
for selecting compounds with dual binding affinity. Finally 142 hits were obtained and divided
into two groups based on their binding affinity for COX-1/COX-2 and for both enzyme pathways
(COX-1/COX-2 and 5-LOX).
Table 1 and Table 2 shows the structure of compounds selected for experimental studies
and their corresponding docking scores respectively. These 10 compounds were selected based
on their potential binding affinity for both enzyme pathways (COX-1/COX-2 and 5-LOX
enzymes), similar binding pose as that of standard and distance between metal ligating groups of
compounds and iron in the active site of 5-LOX enzyme.
2.4. Synthesis of Compounds:
The synthetic routes of selected, designed, final compounds are outlined in Scheme 1 and
Scheme 2. The final compounds were synthesized in major two or three steps. The compounds
1a-7a were synthesized using one pot Biginelli reaction of substituted acetophenones, substituted
aldehyde and thiourea in the presence of sodium t-butoxide and ethanol as solvent. Condensation
of ethyl 3-oxo-3-phenylpropanoate /ethyl 4,4,4-trifluoro-3-oxobutanoate with thiourea in the
presence of sodium ethoxide in boiling ethanol gave Compounds 8a-10a. The compounds 1b-
10b were then synthesized by the reaction of compounds 1a-10a and ethyl chloroacetate/Ethyl 3-
bromopropionate which undergone S-alkylation in the presence of triethylamine and
dimethylformamide. The compounds 1c, 2c-4c and 7c-c9 were then synthesized by reaction of
compounds 1a, 2b-4 and 7b-9b with hydroxylamine hydrochloride in methanol:dichloromethane
(3:1) in the presence of sodium hydroxide.

2.5. In-vitro COX-2 and 5-LOX inhibiting activity:
All 10 compounds were assayed in-vitro for their inhibitory activity against COX-1, COX-2 and
5-LOX enzymes. This initial in-vitro assay was done with a single concentration (µM)
equivalent to 100 µg/ml, for all 10 compounds. The eight compounds except 4c and 9c, nine
compounds except 9c and nine compounds except 6b showed some inhibitory activity with 3.97-
88.43%, 0.33-35.2% and 0.5-51.84% inhibition range respectively against COX-1, COX-2 and
5-LOX enzymes respectively (Table 3). The compound 2b was found to be potent and inhibited
all three enzymes activity COX-1, COX-2 and 5-LOX with inhibitory activity of 70.08%,
35.12% and 51.84% respectively. This compound was further assayed with varying
concentrations to determine their IC50 against COX-1 and 5-LOX, showing the IC50 of
254.82µM and 19.12µM respectively. The same in-vitro activity protocol for determining IC50
was also performed for compounds showing inhibition of activity of enzymes more than 50%
and we obtained IC50 of 22.57µM, 18.41µM, 259.68µM and 31.54µM against COX-1 enzyme
for compounds 1c, 5b, 8c and 10b respectively. The in-vitro data showed that the predictions
from the virtual screening were not perfect, but it did lead to the identification of the new hits
with inhibitory activity against all three enzymes and prepared a base for design of new
compounds for simultaneous inhibition of both enzyme pathways.
2.6. Analysis of Binding Mode:
Based on the identification of the new hits with respect to in-vitro activity, we further analyzed
detailed binding mode of compound 2b in the active site of all the three enzymes (Fig. 4, Fig. 5
& Fig.6). The binding mode of compound 2b was compared with that of corresponding
reference standards for all three enzymes.

It is seen from Fig. 4 that the compound 2b exhibit the similar binding mode as that of
standard celecoxib and formed ‘V’ shaped binding pose in the active site of the COX-2 enzyme.
The overlapping of compound 2b and celecoxib in the active site of the COX-2 enzyme showed
that –CF3 group and phenyl ring with -CH3 group of celecoxib is overlapped by phenyl rings of
compound 2b. However, phenyl ring with -SOCH3 group of celecoxib is overlapped by aliphatic
chain containing -COOC2H5 group of compound 2b. Hence, it is revealed that two phenyl rings
of compound 2b are positioned at the same hydrophobic region where –CF3 and phenyl ring of
celecoxib bind. Whereas -COOC2H5 group of compound 2b bind at the electrostatic region as
similar to -SOCH3 group of celecoxib and form the H-bond with Arg 513. The middle core ring
1,4-dihydropyrimidine of compound 2b overlapped and form H-bond with Tyr 355 as that by
imidazole of celecoxib.
Similarly, the binding pose of compound 2b is visualized and compared with that of
standard atreleuton and tepoxalin in the active site of 5-LOX enzyme (Fig. 5). It was noted that
both phenyl rings and the middle core ring of all three compounds atreleuton, tepoxalin and
compound 2b orient toward same region and exhibit hydrophobic interaction with surrounding
active site amino acids Phe 177, Trp 147, Leu 141, Ala 410 and Phe 151, in the hydrophobic
region of 5-LOX. However, the side chain containing metal binding group of all three
compounds posed region near to Fe2+ and established interactions with it through either carbonyl
or hydroxamte group.
We focused our interest primarily for the design of dual inhibitors of COX-2 and 5-LOX
enzymes, but as compound 2b showed higher in-vitro inhibiting activity and less virtual binding
affinity toward COX-1 enzyme. Therefore, we analyzed and compared binding pose of
compound 2b with that of standard ibuprofen and compound 10b which showed a higher binding

affinity and comparable in-vitro activity (Fig. 6). It was observed that compound 10b bind and
pose similar to ibuprofen. Both these compounds exhibit strong electrostatic interaction with Arg
120. However, compound 2b bind at a region in the active site of the COX-1 enzyme, slightly
away from the region where ibuprofen bind and fit into the pocket by establishing electrostatic
interaction with Arg 83. Due to this, compound 2b showed less docking score as compared to
that of compound 10b. However, it is seen that the presence of carbonyl or hydroxamte group in
the side chain of all compounds, forming strong electrostatic interaction, may be the reason for
in-vitro COX-1 inhibiting activity.
3. Conclusion:
The structure-based library design approach, including the focused library design (virtual
combinatorial library design) and virtual screening was applied to identify simultaneous dual
inhibitors of COX-1/COX-2 and 5-LOX enzymes. The ten 1,4-dihydropyrimidine scaffold were
designed based on substitution pattern, about 15,629 compounds after the enumeration of
combinatorial library were generated and finally 142 hits with dual inhibiting activity were
obtained. The combined computational and observational studies have led to identification new
of hit, compound 2b with inhibitory activity against all three enzymes. The experimental
outcome and analysis of the binding mode of compounds revealed that for the good dual enzyme
pathways (COX-1/COX-2 and 5-LOX) inhibiting activity, the compound must have ‘V’ shaped
structure to bind perfectly in the active site of the COX-2 enzyme, a metal binding group that
binds to Fe2+ in the active site of 5-LOX enzyme and must have an electronegative group to bind
electropositive amino acid in the active site of the COX-1 enzyme. So the overall studies reveal

the valuable clues for future rational design of new, more potent dual inhibitors of COX-1/COX-
2 and 5-LOX enzymes starting from the identified new hit.
4. Experimental Studies:
4.1. Computational Methods:
The process of virtual combinatorial library design was carried out using CombiGlide v3.2
(Schrödinger, LLC, New York, NY, 2014). The fragment structures used for building library
compounds were extracted from reagents structures, selected from Sigma Aldrich. All structures
of reagents were drawn using drawing tools in Maestro v9.4. The structures of reagents were
prepared by selecting a reagent type (a functional group), identifying the functional group that is
replaced when the reagent is added to the core, and performing 2D-to-3D conversion, structure
variation and clean up. This reagent preparation was carried using LigPrep v 2.9. Likewise, core
containing molecule was drawn in maestro v9. 4 and prepared using LigPrep v2.9. The different
attachment position on the core was defined and reagent files for the attachment positions were
assigned. The pre-generated grid file of COX-2 enzyme was used for the docking and screening
of a library. The coordinate for the COX-2 enzyme (PDB Id: 1CX2) was taken from RCSB
Protein Data Bank and prepared for docking using ‘protein preparation wizard’ in Maestro v9.4.
Water molecules in the structures were removed and termini were capped by adding ACE and
NMA residue. The bond orders and formal charges were added for heterogroups and hydrogens
were added to all atoms in the structure. Side chains that are not close to the binding cavity and
do not participate in salt bridges were neutralized. After preparation, the structure was refined to
optimize the hydrogen bond network using OPLS_2005 force field. This helps in reorientation of
the side chain hydroxyl group. The minimization was terminated when the energy converged or

the RMSD reached a maximum cutoff of 0.30 Å. Grids were then defined around refined
structure by centering on ligand using default box size. First core containing molecule was
docked and then both single-position and combinatorial docking and fully enumerated docking
was done for each system separately. The maximum number of structures to be docked was set
to 3000 using SP precision docking mode of Glide. Similarly, the coordinates for COX-1 enzyme
(PDB Id: 1Q4G) and 5-LOX enzyme (PDB Id: 3V99) were taken from RCSB Protein Data
Bank and prepared using ‘protein preparation wizard’ in Maestro v9.4 for virtual screening of
compounds obtained from virtual combinatorial library design.
4.2. Synthesis:
The purities of all the synthesized compounds have been checked by thin-layer chromatography
(TLC) using various non-aqueous solvents. Melting points were determined on SRS OPTIMELT
1
and were uncorrected. H NMR spectra and 13C NMR were recorded on a Bruker DXM-400
spectrometer (400MHz) with DMSO as a solvent. Mass spectra were recorded on Time of flight
mass spectrometer
4.2.1. General Procedure for synthesis of 4,6-disubstituted-1,4-dihydropyrimidine-2-thiol (1a-7a)
A mixture of acetophenones (5 mmol), thiourea (7.5 mmol), and aldehydes (6 mmol) in ethanol
was heated to a clear solution. To this, t-sodium butoxide (2 mmol) was added slowly and the
reaction mixture was refluxed for around 10-14 h. The completion of the reaction was monitored
on TLC. After the completion of the reaction, the solvent was evaporated under vacuum and ice
cold water was added to the mixture. The residue obtained was filtered, dried and purified either

by recrystallization from ethanol or by column chromatography using Ethyl acetate/n-hexane as
mobile phase to obtain intermediate 1a-7a.
4.2.2. General Procedure for synthesis of 2-mercapto-6-substitutedpyrimidin-4(1H)-one (8a-
10a)24
To the 10 ml absolute ethanol, sodium (6 mmol) was added. When sodium was disappeared,
ethyl acetoacetate derivatives (3 mmol) and thiourea ( 4.5 mmol) were added to the solution. The
mixture was refluxed for 20 h. The solvent was evaporated under vacuum and the residue
obtained was dissolved in water and acetified with glacial acetic acid. The product was then
recrystallized from ethanol.
4.2.3. General Procedure for synthesis of ethyl 2-((4,6-disubstituted-1,4-dihydropyrimidin-2-
yl)thio)acetate/propanoate (1b-10b)25
The Compounds (1a-10a) (2 mmol) and triethylamine (3 mmol) was added to
dimethylformamide and heated to 80-90 °C to a clear solution. The ethyl chloroacetate (3 mmol)
or Ethyl 3-bromopropionate (3 mmol) was then added drop wise to the reaction mixture. After
the completion of the reaction, the reaction mixture was poured to ice-water and residue obtained
was extracted with 3 × 25ml of ethyl acetate. The organic layer was then washed with water and
evaporated to give the product which is finally purified by column chromatography using Ethyl
acetate/n-hexane as mobile phase.

4.2.4. General Procedure for synthesis of Final Compounds (1c, 3c-4c, and 7c-9c)
Compounds (1b, 3b-4b and 7b-9b) (2.4 mmol) were dissolved in 30 ml of methanol:
dichloromethane (3:1). To this mixture, hydroxylamine hydrochloride (9.6 mmol) in 3 ml of
water was added at 0 °C and stirred for 10 min. Sodium hydroxide (19.4 mmol) in 3-4 ml of
water was then added and stirred for 30 min. The reaction mixture was warmed to room
temperature, stirred for 3-6 h and TLC was checked at each interval. After the complete
consumption of starting materials, the solvent was evaporated. Residue obtained was diluted with
20 ml of water and acidified with acetic acid. Finally the product obtained was extracted with
dichloromethane (2 × 20 ml), washed with water, dried and evaporated to give the product and
purified by column chromatography using Ethyl acetate/n-hexane as mobile phase.
4.2.4.1. 2-((4,6-diphenyl-1,4-dihydropyrimidin-2-yl)thio)-N-hydroxyacetamide (1c)
Yield: 69%; 1H NMR (400 MHz, DMSO): δ = 4.05 (s, 2H), 5.26-5.27 (d, 1H), 5.74 (s, 1H), 5.76-
5.77 (d, 1H), 7.06 (s, 1H), 7.32-7.38 (m, 3H), 7.43-7.52 (m, 7H), 9.11 (s, 1H).; 13C NMR
(400MHz, CDCl3): δ = 169.38, 153.72, 144.49, 143.23, 135.57, 134.43, 128.52, 126.70,125.89,
111.94, 73.85, 33.46; AP-MS: m/z =340.1 [M+H]+; Elemental Anal. Calcd for C18H17N3O2S
(339.41): requires (Found): C, 63.70 (63.57); H, 5.05 (5.18); N, 12.38 (12.36).
4.2.4.2. ethyl 3-((4,6-diphenyl-1,4-dihydropyrimidin-2-yl)thio)propanoate (2b)
1
Yield: 56%; H NMR (400 MHz, DMSO): δ = 1.17 (t, 3H), 2.94-2.96(t, 2H), 3.46-3.48 (t, 2H),
4.15-4.18 (q, 2H), 5.20-5.22 (d, 1H), 5.75-5.76 (d, 1H), 7.29-7.33 (m, 3H), 7.45-7.51 (m, 7H),
9.17 (s, 1H).; 13C NMR (400MHz, CDCl3): δ = 171.19, 154.99, 144.49, 143.23, 135.58, 128.52,
128.48, 126.70, 125.89, 111.94, 73.85, 60.81, 33.80, 26.85, 14.21; AP-MS: m/z =367.3 [M+H]+;

Elemental Anal. Calcd for C21H22N2O2S (366.48): requires (Found): C, 68.82 (68.80); H, 6.05
(6.15); N, 7.64 (7.77).
4.2.4.3. 2-((6-(4-fluorophenyl)-4-(pyridin-2-yl)-1,4-dihydropyrimidin-2-yl)thio)-N-
hydroxyacetamide (3c)
Yield (solid): 42%; 1H NMR (400 MHz, DMSO): δ = 4.21 (s, 2H), 5.05-5.06 (d, 1H), 5.52-5.53
(d, 1H), 5.72 (s, 1H), 7.08-7.16 (m, 3H), 7.23 (s, 1H), 7.35-7.42 (m, 3H), 7.65-7.70 (m, 2H), 9.13
(s, 1H).; 13C NMR (400MHz, CDCl3): δ = 169.38, 165.55, 163.53, 161.51, 160.67, 156.17,
147.58, 145.09, 141.98, 138.31, 136.90, 131.89, 127.12, 115.88, 113.76, 111.39, 66.34, 33.46;
AP-MS: m/z =359.1 [M+H]+. Elemental Anal. Calcd for C17H15FN4O2S (358.39): requires
(Found): C, 56.97 (56.85); H, 4.22 (4.25); N, 15.63 (15.78).
4.2.4.4. 2-((4-(4-fluorophenyl)-6-phenyl-1,4-dihydropyrimidin-2-yl)thio)-N-hydroxyacetamide (4c)
Yield (solid): 45%; 1H NMR (400 MHz, DMSO): δ =: 3.96 (s, 2H), 5.13-5.14 (d, 1H), 5.74-5.75
(d, 1H), 5.83 (s, 1H), 6.11 (s, 1H), 7.10-7.13 (m, 2H), 7.42-763 (m, 7H), 9.12 (s, 1H).; 13C NMR
(400MHz, CDCl3): δ = 169.38, 167.02, 163.13, 161.12, 153.72, 143.23, 137.09, 135.57, 133.65,
128.52, 128.08, 125.89, 115.66, 114.10, 111.94, 73.85, 33.46; AP-MS: m/z =358.2 [M+H]+;
Elemental Anal. Calcd for C18H16FN3O2S (357.40): requires (Found): C, 60.49 (60.5); H, 4.51
(4.59); N, 11.76 (11.73).
4.2.4.5. ethyl 3-((6-(4-fluorophenyl)-4-phenyl-1,4-dihydropyrimidin-2-yl)thio)propanoate (5b)
Yield (solid): 59%; 1H NMR (400 MHz, DMSO): δ = 1.18-1.21 (t, 3H), 2.87-2.91 (t, 2H), 3.59-
3.64 (q, 2H), 5.17-5.18 (d, 1H), 5.70-5.77 (d, 1H), 7.05-7.08 (m, 2H), 7.31-7.35 (m, 3H), 7.39-
13
7.44 (m, 4H), 9.17 (s, 1H); C NMR (400MHz, CDCl3): δ = 171.19, 163.53, 161.51, 154.99,
144.48, 143.23, 128.17, 126.70, 115.89, 111.94, 73.85, 60.81, 33.80, 29.76, 26.86, 14.21; AP-

MS: m/z =385.4 [M+H]+; Elemental Anal. Calcd for C21H21FN2O2S (384.47): requires (Found):
C, 65.60 (65.69); H, 5.51 (5.58); N, 7.29 (7.36).
4.2.4.6. ethyl 3-((6-(4-fluorophenyl)-4-(thiophen-2-yl)-1,4-dihydropyrimidin-2-yl)thio)propanoate
(6b)
Yield (solid): 52%; 1H NMR (400 MHz, DMSO): δ = 1.15-1.19 (t, 3H), 3.30-3.32 (t, 2H), 4.06-
4.08 (t, 2H), 4.58-4.62 (q, 2H), 5.30-5.32 (d, 1H), 5.79-5.80 (d, 1H), 6.91-6.99 (m, 4H), 7.20-
13
7.27 (m, 3H), 8.96 (s, 1H).; C NMR (400MHz, CDCl3): δ = 171.19, 163.53, 161.51, 150.75,
146.97, 144.30, 139.12, 128.19, 125.92, 125.59, 125.34, 115.88, 112.00, 77.43, 60.81, 33.80,
26.85, 14.21; AP-MS: m/z =391.5 [M+H]+; Elemental Anal. Calcd for C19H19FN2O2S2 (390.49):
requires (Found): C, 58.44 (58.52); H, 4.90 (4.87); N, 7.17 (7.20).
4.2.4.7. 2-((4-cyclopropyl-6-(p-tolyl)-1,4-dihydropyrimidin-2-yl)thio)-N-hydroxyacetamide (7c)
1
Yield (solid): 38%; H NMR (400 MHz, DMSO): δ =: 0.71-0.80 (m, 5H), 2.30 (s, 3H), 4.33 (s,
2H), 4.61-4.63 (d, 1H), 5.36-5.38 (d, 1H), 5.67 (s, 1H), 7.10-7.14 (m, 2H).; 13C NMR (400MHz,
CDCl3): δ = 169.38, 149.41, 144.04, 137.73, 134.48, 130.06, 129.00, 127.01, 112.11, 67.28,
33.46, 21.41, 19.87, 5.14.; AP-MS: m/z =318.2 [M+H]+; Elemental Anal. Calcd for C16H19N3O2S
(317.41): requires (Found): C, 60.54 (60.52); H, 6.03 (6.01); N, 13.24 (13.31).
4.2.4.8. N-hydroxy-2-((4-oxo-6-phenyl-1,4-dihydropyrimidin-2-yl)thio)acetamide (8c)
1
Yield (solid): 40%; H NMR (400 MHz, DMSO): δ = 3.56 (s, 2H), 5.69 (s, 1H), 7.36 (s, 1H),
7.63-7.72 (m, 5H), 9.25 (s, 1H), 7.12 (s, 1H).; 13C NMR (400MHz, CDCl3): δ = 169.38, 167.22,
165.74, 149.20, 135.19, 128.63, 126.66, 114.57, 33.46; AP-MS: m/z =278.2 [M+H]+; Elemental
Anal. Calcd for C12H11N3O3S (277.30): requires (Found): C, 51.98 (51.91); H, 4.00 (4.05); N,
15.15 (15.20).

4.2.4.9. N-hydroxy-3-((4-oxo-6-phenyl-1,4-dihydropyrimidin-2-yl)thio)propanamide (9c)
1
Yield (solid): 42%; H NMR (400 MHz, DMSO): δ = 2.63-2.66 (t, 2H), 3.05-3.09 (t, 2H), 5.28
(s, 1H), 7.43 (s, 1H), 7.66-7.74 (m, 5H), 7.12 (s, 1H), 8.27 (s, 1H).; 13C NMR (400MHz, CDCl3):
δ = 168.29, 165.74, 164.46, 149.20, 148.07, 135.19, 128.63, 126.65, 114.57, 35.78, 27.33; AP-
MS: m/z =292.2 [M+H]+; Elemental Anal. Calcd for C13H13N3O3S (291.33): requires (Found): C,
53.60 (53.68); H, 4.50 (4.55); N, 14.42 (14.40).
4.2.4.10. 2-((3-oxopentyl)thio)-6-(trifluoromethyl)pyrimidin-4(1H)-one (10b)
Yield (solid): 48%; 1H NMR (400 MHz, DMSO): δ = 1.10-1.14 (t, 3H), 2.47-2.50 (q, 2H), 3.12-
13
3.15 (d, 2H), 3.16-3.19 (d, 2H), 7.06 (s, 1H), 8.20 (s, 1H).; C NMR (400MHz, CDCl3): δ =
211.21, 168.89, 167.05, 135.00, 134.75, 134.24, 124.63, 122.48, 121.90, 120.34, 118.19, 41.26,
36.04, 27.58, 8.59; AP-MS: m/z =281.1 [M+H]+; Elemental Anal. Calcd for C10H11F3N2O2S
(280.27): requires (Found): C, 42.85 (42.80); H, 3.96 (3.88); N, 10.00 (10.03).
4.3. In-vitro COX-2 and 5-LOX inhibiting Assay:
4.3.1. Reagents and chemicals
Commercially acquired reagents: TMPD (N, N, N’, N’‐tetramethyl p‐phenylenediamine),
hematin and Tween 20 were purchased from Sigma, Arachidonic acid purchased from Nu‐check
Prep, Inc (MN, USA) and Dimethyl sulfoxide (DMSO) of HPLC grade. All solutions were
prepared in deionised distilled water. All other reagents were of standard quality commercially
available

4.3.2. Extraction and isolation of cyclooxygenase-1 (COX-1) from Ram seminal vesicles:
We had collected Ram seminal vesicles from the local slaughter house and stored in (−80 °C).
Before starting the experiment, It was then taken out from deep freezer (−80 °C) and kept
overnight at 4°C in refrigerator. Process of Extraction and isolation were carried out below 5°C
in cold room.
4.3.2.1. Preparation of microsomes as a source of COX-1
Preparation of microsome was carried out, according to the method of Hemler et al.26, with some
modifications. Ram seminal vesicles were minced and homogenized in buffer containing 0.05 M
Tris–HCl (pH 8), 5 mM EDTA disodium salt, 5 mM diethyl dithiocarbamate and 0.01% sodium
azide. The homogenate was centrifuged at 10,000 × g for 20 min, at 4°C. The supernatant was
filtered through cheese cloth. The filtered supernatant was further centrifuged at 33,000 RPM for
1 h 30 min, 4°C by using ultracentrifuge (Himac, CP-100-α HITACHI) to obtain microsomal
pellet. This microsomal pellet was solubilised in a solubilising buffer containing 0.05 M Tris–
HCl (pH 8), 0.1 mM EDTA disodium salt, 0.1 mM diethyl dithiocarbamate and 0.01% sodium
azide. This microsome fraction was stored at −80°C, and used as enzyme source.
4.3.3. Preparation of microsomes as a source of cyclooxygenase-2
27
Microsome as a source of COX-2 was carried out according to the method by Reddy et al. ,
with some modifications. Human recombinant COX-2 expressed in Spodoptera frugiperda (Sf9)
cell. Sf9 cells were maintained at 28°C in Grace’s insect culture medium. Inscet Cells at 70%
confluency were infected with recombinant baculovirus containing human COX-2. After 72 h of
infection, the cells were collected by centrifugation at 3000 × g for 5 min at 4°C. The pellet was

suspended in a minimum volume of Tris–HCl buffer (50 mM, pH 8.0) containing 5 mM EDTA,
300 mM sucrose, 5 mM diethyl thiocarbamate, 1 µg/ml pepstatin, 1 mM phenol and sonicated
for 3 min. The cell lysate was centrifuged at 100,000 × g for 1 h 30 min at 4°C by using
ultracentrifuge (Himac, CP-100 α HITACHI), and the microsomal pellet obtained was suspended
in Tris–HCl buffer (2.5 mM, pH 8.0) containing 0.5% glycerol, 0.8% Tween 20 and 1 mM
phenol. This solubilized microsomal fraction was stored at −80°C and used as enzyme source.
4.3.4. Cycloxygenase (COX-1 and COX-2) assays
Enzymatic activities of both, COX-1 and COX-2 were measured according to the method of
Copeland et al.28, with slight modifications using a chromogenic assay based on the oxidation of
N,N,N’,N’-tetramethyl-p-phenylene diamine (TMPD), during the reduction of PGG2 to PGH229,
30. The assay mixture contained Tris–HCl buffer (0.5 M) pH 8.0, hematin (5 mM), EDTA (0.5
M), enzyme (COX-1 or COX-2) and the test compound. The mixture was pre-incubated at 25°C
for 5 min and then the reaction was initiated by the addition of substrate AA and TMPD, in total
volume of 1 ml reaction mixture. The enzyme activity was determined by estimating the rate of
TMPD oxidation for the first 60 s of the reaction by following the increase in absorbance at 610
nm. A low rate of non-enzymatic oxidation, observed in the absence of COX-1 and COX-2, was
subtracted from the experimental value while calculating the percent inhibition.
4.3.5. Purification and assay of 5-lipoxygenase (5-LOX)
5‐Lipoxygenase from potato tubers was purified and assayed as per the method described by
Reddanna et al.31. Enzyme activity was assessed using the polarigraphic method with a Clark’s
oxygen electrode on Strathkelvin Instruments, model 782, RC‐300. Reaction mixture contained

50‐µl of enzyme and 10 µl, 40mM of the substrate, (Arachidonic acid) and final volume made
with 100 mM phosphate buffer pH 6.3 to 3 ml. Since lipoxygenases are oxygen‐consuming
enzymes the concentration of oxygen decrease in the reaction mixture, the rate of decrease in
oxygen was taken as a measure of enzyme activity. The reaction was allowed to proceed at 25°C
and the maximum slope generated was taken for calculating enzyme activity. The activity was
expressed as units/mg protein, where one unit is defined as one micro mole of oxygen consumed
per minute. Assay standardization was done with NDGA (Nordihydrogutaric acid), a selective 5-
LOX inhibitor.
Acknowledgement:
The authors are thankful to Indian Council of Medical Research (ICMR), New Delhi for
providing Senior Research Fellowship to Mr. Deepak K. Lokwani (BIC/11(03)/2012). The
authors also thank to the Head, Department of Chemical Technology, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad 431 004 (MS), India for providing the laboratory facility.

References:
1. Bruno, A.; Tacconelli, S.; Patrignani, P. Basic Clin. Pharmacol. Toxicol. 2014, 114, 56.
2. Seibert, K.; Zhang, Y.; Leahy, K.; Hauser, S.; Masferrer, J.; Perkins, W.; Lee, L.; Isakson, P.
Proc. Natl. Acad. Sci. USA 1994, 91, 12013
3. Tanabe, T.; Tohnai, N. Prostaglandins Other Lipid Mediat., 2002, 68-69, 95.
4. Kurumbail, R. G.; Stevens, A. M.; Gierse, J. K.; McDonald, J. J.; Stegeman, R. A.; Pak, J. Y.;
Gildehaus, D.; Miyashiro, J. M.; Penning, T. D.; Seibert, K.; Isakson, P.C.; Stallings, W.C.
Nature 1996, 384, 644.
5. Salvo, F.; Antoniazzi, S.; Duong, M.; Molimard, M.; Bazin, F.; Fourrier-Reglat, A.; Pariente, A.;
Moore, N. Expert Opin. Drug Saf. 2014, 13, 573.
6. Graham, D. J.; Campen, D.; Hui, R.; Spence, M.; Cheetham, C.; Levy, G.; Shoor, S.; Ray, W. A.,
Lancet 2005, 365, 475.
7. Ulbrich, H.; Fiebich, B.; Dannhardt, G. Eur. J. Med. Chem. 2002, 37, 953.
8. Martel-Pelletier J.; Lajeunesse D.; Reboul P.; Pelletier J-P. Ann. Rheum. Dis. 2003, 62, 501.
9. Caroline, C.; Catherine, M. Eur. J. Med. Chem. 2003, 38, 645.
10. Kirchner, T.; Aparicio, B.; Argentieri, D. C.; Lau, C. Y.; Ritchie, D. M. Prostaglandins Leukot.
Essent. Fatty Acids 1997, 56, 417.
11. Skelly, M. M.; Hawkey, C. J. Int. J. Clin. Pract. 2003, 57, 301.
12. Scholz, M.; Ulbrich, H. K.; Soehnlein, O.; Lindbom, L.; Mattern, A.; Dannhardt G. Bioorg. Med.
Chem. 2009, 17, 558.
13. Kulkarni, S.K.; Singh, V.P. Curr. Top. Med. Chem. 2007, 7, 251.
14. Alvaro-Gracia, J. M. Rheumatology 2004, 43, i21.
15. Jantan, I.; Bukhari, S. N. A.; Adekoya, O. A.; Sylte, I. Drug Des. Devel. Ther. 2014, 8, 1405.

16. Ahmad, W.; Kumolosasi, E.; Jantan, I.; Bukhari, S. N. A.; Jasamai, M. Chem. Biol. Drug Des.
2014, 83, 670.
17. Bukhari, S.N.A., Laurob, G.; Jantana, I.; Bifulcob, G.; Amjad, M. W. Bioorg. Med. Chem. 2014,
22, 4151.
18. Weber, L. QSAR Comb. Sci. 2005, 24, 809.
19. Nandi, S.; Bagchi, M. C. Mol. Simula. 2011, 37, 196.
20. Frecer, V.; Megnassan, E.; Miertus, S. Eur. J. Med. Chem. 2009, 44, 3009.
21. Lokwani, D. K.; Mokale, S. N.; Shinde, D. B. Eur. J. Med. Chem. 2014, 73, 233.
22. Lokwani, D.; Shah, R.; Mokale, S.; Shastry P.; Shinde, D. J. Comput. Aided Mol. Des. 2012, 26,
267
23. Charlier, C.; Michaux, C. Eur. J. Med. Chem. 2003, 38, 645
24. Weaver, D. F.; Guillain, B. M. WO Patent 07, 292, 2006.
25. Mobinikhaledia, A.; Foroughifara, N.; Khanpour, M.; Ebrahimi, S. Eur. J. Med. Chem. 2010, 1,
33.
26. Hemler, M.; Lands, W. E.; J. Biol. Chem. 1976, 251, 5575.
27. Reddy, C.M.; Bhat, V.B.; Kiranmai, G.; Reddy, M.N.; Reddanna, P.; Madyastha, K.M. Biochem.
Biophys. Res. Commun. 2000, 277, 599.
28. Copeland, R.A.; Williams, J.M.; Giannaras, J.; Nurnberg, S.; Covington, M.; Pinto, D.; Pick, S.;
Trzaskos, J.M.; Proc. Natl. Acad. Sci. USA 1994, 91, 11202.
29. Egan, R.W.; Paxton, J.; Kuehl Jr., F.A. J. Biol. Chem. 1976, 251, 7329.
30. Pagels, W.R.; Sachs, R.J.; Marnett, L.J.; Dewitt, D.L.; Day, J.S.; Smith, W.L.; J. Biol. Chem.
1983, 258, 6517.
31. Reddanna, P.; Whelan, J.; Maddipati, K.R.; Reddy, C.C. Methods Enzymol. 1990, 187, 268.

Fig. 1: Flow chart of Virtual Combinatorial Library Design and Virtual Screening shows the
phases involved in the search of new simultaneous COX-1/COX-2 and 5-LOX Inhibitors.
Fig. 2: Designed 1,4dihydropyrimidine scaffold for dual inhibition of COX-1/COX-2 and 5-
LOX enzyme.
Fig. 3: Virtual Combinatorial Library Design and Screening of compounds. (a) Enumeration of
library by docking of each core structure with single position substitution for the filtering of
fragments and (b) Enumeration of entire library by selecting each fragment or substitution,
without performing docking of each core structure with single position substitution.
Fig. 4: Binding pose of compounds (a) Overlap of celecoxib (Orange Color Carbon Atoms) and
compound 2b (Green Color Carbon Atoms), (b) Celecoxib (c) compound 2b in the active site of
COX-2 enzyme
Fig. 5: Binding pose of compounds (a) Overlap of tepoxalin (Orange Color Carbon Atoms) and
compound 2b (Green Color Carbon Atoms), (b) tepoxalin (c) compound 2b in the active site of
5-LOX enzyme
Fig. 6: Binding pose of compounds (a) Overlap of Ibuprofen (Orange Color Carbon Atoms) and
compound 10b (Green Color Carbon Atoms), (b) tepoxalin and compound 2b in the active site of
COX-1 enzyme

Fig. 1

Fig. 2

(a)
(b)
Fig. 3