Design, synthesis, in-vitro, in-vivo and in-silico studies of pyrrolidine-2,5-dione derivatives as multitarget anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2019.111863

In recent years, drug discovery paradigm has been shifted from conventional single target inhibition toward multitarget design concept. In current research, we have reported synthesis, in-vitro, in-vivo and acute toxicity determination of N-substituted py

Full text of DOI:10.1016/j.ejmech.2019.111863

Journal Pre-proof
Design, synthesis, in-vitro, in-vivo and in-silico studies of pyrrolidine-2,5-dione
derivatives as multitarget anti-inflammatory agents
Muhammad Saeed Jan, Sajjad Ahmad, Fida Hussain, Ashfaq Ahmad, Fawad
Mahmood, Umer Rashid, Obaid-ur-Rahman Abid, Farhat Ullah, Muhammad Ayaz,
Abdul Sadiq
PII:
S0223-5234(19)31015-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111863
EJMECH 111863
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 June 2019
Revised Date: 5 November 2019
Accepted Date: 6 November 2019
Please cite this article as: M.S. Jan, S. Ahmad, F. Hussain, A. Ahmad, F. Mahmood, U. Rashid, O.-
u.-R. Abid, F. Ullah, M. Ayaz, A. Sadiq, Design, synthesis, in-vitro, in-vivo and in-silico studies of
pyrrolidine-2,5-dione derivatives as multitarget anti-inflammatory agents, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111863.
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2019 Published by Elsevier Masson SAS.

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Design, Synthesis, In-vitro, In-vivo and In-silico studies of
Pyrrolidine-2,5-dione Derivatives as Multitarget Anti-inflammatory
Agents
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Muhammad Saeed Jan , Sajjad Ahmad , Fida Hussain , Ashfaq Ahmad , Fawad Mahmood ,
,
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Umer Rashid* , Obaid-ur-Rahman Abid , Farhat Ullah , Muhammad Ayaz ,
*
*,1
Abdul Sadiq
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Department of Pharmacy, Faculty of Biological Sciences, University of Malakand, Chakdara,
18000 Dir (L), KP, Pakistan.
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Department of Pharmacy, University of Swabi, Swabi, KP, Pakistan.
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Department of Pharmacy, Sarhad University of Science & Technology, Peshawar, KPK,
Pakistan.
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Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, 22060
Abbottabad, Pakistan.
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Department of Chemistry, Hazara University, Mansehra, Pakistan
Corresponding author (*): Email: umerrashid@cuiatd.edu.pk (Umer Rashid)
Corresponding author (**): Email: sadiquom@yahoo.com, Contact: +92 (0)301-2297 102
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Abstract
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In recent years, drug discovery paradigm has been shifted from conventional single target
inhibition toward multitarget design concept. In current research, we have reported synthesis, in-
vitro, in-vivo and acute toxicity determination of N-substituted pyrrolidine-2,5-dione derivatives
as multitarget anti-inflammatory agents. We synthesized cycloalkyl, alkyl and aryl carbonyl
derivatives by the Michael addition of ketones to N-substituted maleimides using self-assembled
three component system as an organocatalyst. Anti-inflammatory potential of the compounds
was determined by using different in-vitro assays, like cyclooxygenase-1, cyclooxygenase-2 and
5-lipoxygenase, albumin denaturation and anti-protease assays. Amongst the synthesized
compounds, 13a-e series of compounds showed inhibition in low micromolar to submicromolar
ranges. These compounds also demonstrated COX-2 selectivity. Compound 13e with IC value
5
0
0.98 µM and SI of 31.5 emerged as the most potent inhibitor of COX-2. Based on in-vitro
results, in-vivo anti-inflammatory investigations were performed on compounds 3b and 13e via
carrageenan induced paw edema test. The possible mode of action of compounds 3b and 13e
were ascertained with various mediators like histamine, bradykinin, prostaglandin and
leukotriene. In-vivo acute toxicity study showed the safety of synthesized compounds up to 1000
mg/kg dose. The selectivity of the compounds against cyclooxygenase isoforms was supported
by docking simulations. Selective COX-2 inhibitors showed significant interactions with the
amino acid residues present in additional secondary COX-2 enzyme pocket. Furthermore, in-
silico pharmacokinetic predictions confer the drug-like characteristics.
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Keywords: Michael addition; Succinimides; 5-Lipoxygenase; Cyclooxygenase-1/2; Albumin
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7
8
denaturation; Protease inhibition.
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1. Introduction
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Inflammation is a multifactorial disorder frequently associated with pain. It involves raise of
vascular permeability, membrane alteration and protein denaturation [1]. The normal cells or
tissues upon exposure to microbes, chemical or physical agents can get inflamed which may lead
to injury [2]. The intensity of the loss of function depends on the extent and site of the injury.
Inflammation is the body defensive mechanism that is triggered by various stimuli including
radiation, heat, microbial infections and is frequently associated with tissue damages [3-4].
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Arachidonic acid metabolism play a vital role in the mechanism of inflammation [5].
Arachidonic acid metabolized to thromboxane A2 and prostaglandins by the cyclooxygenase-2
(COX-2) cascade, or by the 5-lipoxygenase (5-LOX) pathway upon suitable stimulus of
neutrophils. The arachidonic acid is break down from phospholipids membrane and converted to
prostaglandins and leukotrienes through COX-2 or 5-LOX cascades [6]. Inhibitions of COX-2
and 5-LOX may direct to reduce the production of prostaglandins and leukotrienes. Hence, the
drug having capability to inhibit these enzymes have the potential to give anti-inflammatory and
analgesic effects with a decrease in the gastro-intestinal side effects [7]. Membrane stabilization
is a process of maintaining the integrity of biological membranes [8]. Denaturation of proteins is
a process in which proteins lose their secondary and tertiary structures due to the appliance of the
external stress such as a concentrated inorganic salts, strong acids or bases and organic solvents
or heat [9]. When the biological proteins are denatured, they lose their functions. Proteins
denaturation is a well-known reason of inflammation [10]. Proteases, also called proteinases or
peptidases, are group of enzymes that perform proteolysis, i.e. hydrolysis of peptide bonds which
link the amino acids jointly in the polypeptide chain forming the protein [11]. Proteases
constitute one of the largest functional group of proteins involved in many normal and
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pathological processes. Proteases inhibitions may help in control of several diseases including
inflammation [12-14].
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For the treatment of multifactorial pathologies such as Alzheimer’s disease (AD) and
inflammation, the drug discovery paradigm has been shifted from conventional single target
inhibition towards multitarget design concept. In anti-inflammatory drug discovery, the
development of licofelone as dual cyclooxygenase/lipoxygenase inhibitor is a success of the later
strategy. Aspirin, a nonsteroidal anti-inflammatory drug (NSAID), is the oldest clinically
accepted example of multitarget anti-inflammatory drug [15].
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Organic compounds with cyclic amide group have been reported to possess great
pharmacological importance [16]. Substituted succinimides (pyrrolidine-2,5-diones) are
important compounds in medicinal chemistry [17-18]. Due to the presence of amide group, they
showed excellent in-vivo activity because it can easily cross the biological membrane [19]. A
diversity of biological activities and pharmaceutical uses has been attributed to them [20-31].
Based on the literature evidences, it is obvious that specific functional groups are responsible for
biological activities, and may specifically be effective in the management of inflammation [32-
33]. Some of the ketoester derivatives of succinimides showed strong anti-inflammatory activity
[34].
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The organocatalytic asymmetric Michael addition is a strong reaction for making the C-C bond
formation [35-36]. The asymmetric Michael addition of ketones to maleimides has been explored
by a limited number of researchers. Among them, none has explored the biological importance of
the synthesized compounds. Our synthetic strategy relied on Michael addition of ketones to N-
substituted maleimides using self-assembled three component system as an organocatalyst. We
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have previously synthesized aldehyde and ketoester derivatives of succinimides [37-38]. We
noticed that the synthesis of some of the designed compounds have previously investigated using
different catalysis systems [39-42]. However, to the best of our literature search, we have noticed
that these compounds have not been previously evaluated for any biological activity including
anti-inflammatory activity. Herein, we report synthesis, in-vitro, in-vivo and molecular docking
studies of N-substituted pyrrolidine-2,5-dione derivatives as multitarget anti-inflammatory
agents.
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01 2. Results and discussion
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2.1 Design strategy
General structure of selective COX-2 inhibitor includes two vicinal aryl rings on central 5- or 6-
memebered heterocyclic/acyclic scaffold bearing a small lipophilic moiety or sulfonamide
(responsible for prooxidant activity). COX-2 selectivity of celecoxib is associated with these
structural features (Figure 1a). In the current study, we have constructed pyrrolidine-2,5-dione
based derivatives as shown in Figure 1b. Our design strategy aims to synthesize N-substituted
pyrrolidine-2,5-dione connected at position-3 with cycloalkyl, alkyl and aryl carbonyl
substituents (Figure 1b-c). Cycloalkyl carbonyl compounds are planned to connect with N-
substituted pyrrolidine-2,5-dione ring directly (Figure 1b). While, aryl carbonyl derivatives relate
to a flexible methylene linker (Figure 1c). Considering the importance of rational development of
drug to tame multifactorial inflammation, we decided to design “single-drug-multitarget”
strategy. The investigated multiple targets are represented in Figure 1d.
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Figure 1: (a) General structure of selective COX-2 inhibitor; (b-c) Design strategy of current research;
(d) Multiple anti-inflammatory targets evaluated in current study.
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2.2 Asymmetric ketone derivatives of pyrrolidine-2,5-dione
We synthesized ketone derivatives (3a-l) of pyrrolidine-2,5-dione by adding various cyclic alkyl
/ alkyl ketones to
N-substituted maleimides. Except 3i-j, all the reactions are simple single step
and completed in short time with good isolated yields. We used a tricomponent non-covalent
organocatalyst system for these reactions for the first time as shown in Scheme 1. The synthesis
of 3i-j was carried out in two-step. In first step,
synthesized by the reaction of maleic anhydride with sulfanilamide in diethyl ether to obtain
sufamoyl-phenylmaleanic acid ([(4 sulfamoylphenyl)carbamoyl]prop enoic acid,
N-substituted sulfonamide derivative 7 was
N
-
‐
‐
2
‐
6
)
intermediate. The synthesized intermediate was then cyclized in acetic anhydride in the presence
of sodium acetate to give sulfanilamide derivative 7 (Scheme 2). Cyclohexanone and
cycloheptanone reacted with the
N
-sulfonamide derivative
7
to yield 3i-j.
O
O
O
R1
N
L-isoleucine, thiourea and KOH
20 mol% each)
N R1
O
O
O R
R'
N R1
(
R
O
O
H C
H C
3
3
O
Chloroform (1.0 M), rt
O
R'
1a-e
1f-g
2a-c, 7
3a-j
3k-l
R,R' = H, H (1f
,
3k); CH , CH (1g
,
3l)
3
3
=
O
O
1
S NH2
O
CH3
R =
Br
Cyclic alkyl
1
1
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Scheme 1: Synthesis of the pyrrolidine-2,5-dione derivatives 3a-j.
O
O
O
O
S
o
O
S
O
S
80-100 oC
2
0 C
O
HN
O
+
H N
2
NH2
NH2
N
NH2
O
CHCl3
O
Ac O, NaOAc
O
2
O
HO
O
4
5
6
7
1
1
30
31
Scheme 2: Synthesis of the N-benzene sulfonamide derivative (7) of pyrrolidine-2,5-dione
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1
1
1
1
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The addition of aromatic ketones to maleimide type Michael acceptors is very rare in the
literature. There is only a recent example of aromatic ketones additions to -phenylmaleimide
N
[42]. However, to the best of our literature survey, there is no single example of aromatic or any
other ketones to the maleimide Michael acceptors that we used in our anti-inflammatory study.
In the next Scheme, relevant 4-substituted acetophenones (aryl ketones) were stirred at room
temperature with
7 in the presence of OtBu-L-threonine and 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) in chloroform to obtain 13a-e as outlined in Scheme 3
.
O
O
O
O
OtBu-L-threonine, DBU
CHCl , rt, 51.3-63.2%
S NH2
O
X
N
7
+
CH3
X
O
R
3
R
8-12
13a-e
X = N (13a), X = CH (13b-e
R = -H (13a-b), -OCH3 (13c),
Cl (13d), -CH3 (13e
)
-
)
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Scheme 3: Synthesis of the pyrrolidine-2,5-dione derivatives 13a-e
.
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1
1
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The individual yields and other details of all the compounds are available in experimental section
1
13
and the H and C NMR spectra are provided in the supporting information. Biologically
screened compounds are > 95 % pure as determined by HPLC.
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1
1
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2.3 In-vitro enzyme inhibition assays
2.3.1 Cyclooxygenase (COX-1/COX-2) and 5-LOX inhibition assay
The COX-1 / COX-2 inhibitory activity of the synthesized compounds (3a-j and 13a-c) were
carried out using COX-1 and COX-2 screening assay kits. Celecoxib, indomethacin, diclofenac
sodium and Zileuton was used as corresponding positive controls. The in-vitro enzymes
inhibition results are presented as IC50 values as means of three acquired determinations and
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presented in Table 1. The selectivity index (SI) was calculated as IC50 (COX-1) / IC50 (COX-2)
Table 1). It is evident from the in-vitro results that overall cyclic ketones (3a-h) and alkyl
ketones (3i-j) exhibited poor to moderate inhibition of both COX isoforms. -benzyl derivatives
(
N
3b and 3e showed selectivity towards COX-2. They showed selectivity index of 3.13 and 2.35
respectively. We noticed that presence of bulkier cycloalkyl carbonyl derivatives improve the
COX-2 selectivity. The selectivity index for compounds 3a (cyclohexanone), 3g
(cycloheptanone) and 3h (cyclopentanone) was 1.45, 1.66 and 0.58 respectively. N-sulfonamide-
cyclohexanone/ cycloheptanone derivatives (3i and 3j respectively) showed moderate COX-2
inhibition with selectivity index of 2.04 and 2.36 respectively.
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It is evident from Table 1 that in-vitro results of aryl ketone / N-sulfonamide derivatives (13a-e)
demonstrated marked inhibitions and selectivity against COX-2 isozyme. Compounds of this
series showed IC50 values in submicromolar to low micromolar range. Compound 13e with IC50
value of 0.98
µM and SI 31.5 emerged as the most potent inhibitor of COX-2. The SI for
compounds 13a-d is 4.88, 11.5, 18.7 and 10.9 respectively.
The synthesized compounds were also screened for their 5-LOX inhibitory potentials.
Compounds of series 1 (3a-l) demonstrated moderate to poor 5-LOX inhibition. Sulfonamide-
cycloheptanone derivative 3j exhibited IC value of 16.15 µM. Aryl ketone derivatives 13a-e
5
0
showed good to excellent inhibition of human lipoxygenase (5-LOX). Among all the tested
compounds of this series, 13a and 13e exhibited highest inhibitions attaining IC values of 0.81
5
0
and 0.86 µM respectively, while the standard drug zileuton exhibited IC50 of 0.63 µM (Table 1)
.
Concentrations of the synthesized compounds at which 50% of inhibition is observed (IC50) were
calculated among the inhibition percentages against the tested concentrations using the MS-
Excel program. All the assays were performed in triplicate and values were expressed as means ±
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Standard error means (SEM). Statistical analysis was performed by Two-Way analysis of
difference (ANOVA), followed by Bonferroni tests. The difference was measured to be
statistically significant when the p value
˂ 0.05.
Table 1: In-vitro cyclooxygenase-1 / 2 and 5-LOX inhibition activity of the compounds
IC (µM)
5
0
No.
Structures
COX-1
COX-2
SI
5-LOX
O
O
N
3
3
3
a
65.56 + 1.34
45.08 + 1.29
1.45
78.3 + 1.87
O
O
O
N
b
c
62.57 + 1.12
19.98 + 0.07
3.13
1.64
36.0 + 0.92
O
O
O
N
Br
186.35 + 3.16 113.44 + 2.22
130.5 + 1.64
O
O
O
N
3
3
d
e
53.02 + 1.82
51.52 + 1.31
56.49 + 1.41
21.86 + 0.62
0.93
2.35
69.18 + 2.02
56.2 + 1.00
O
O
O
N
O
O
O
N
O
3
f
48.92 + 1.16
65.64 + 2.38
56.58 + 0.98
39.45 + 1.49
0.86
1.66
26.83 + 0.32
18.44 + 0.09
O
O
O
N
3
g
O
10

O
O
3
3
3
3
3
h
i
N
30.26 + 0.82
43.39 + 1.19
49.10 + 1.81
33.31 + 1.63
39.25 + 1.23
51.35 + 1.55
21.26 + 1.07
20.73 + 0.82
63.88 + 1.93
53.47 + 1.41
0.58
2.04
2.36
0.52
0.73
98.55 + 2.09
21.31 + 1.28
16.15 + 0.09
62.38 + 1.66
53.45 + 1.39
O
O
O
S
O
N
NH
2
O
O
O
O
S
O
N
NH
2
j
O
O
O
O
O
k
l
N
O
O
N
O
O
O
O
N
S
NH2
1
1
3a
3b
43.70 + 2.01
48.66 + 1.89
8.94 + 0.06
4.23 + 0.19
4.88
11.5
0.81 + 0.02
5.29 + 0.26
N
O
O
O
O
O
N
S
NH
2
O
O
O
O
S
O
N
NH2
1
3c
O
39.21 + 1.13
2.10 + 0.03
18.7
4.74 + 0.02
O
O
O
O
S
O
N
NH
2
1
1
3d
3e
68.79 + 2.15
30.87 + 1.53
6.29 + 0.01
0.98 + 0.01
10.9
31.5
12.59 + 0.42
0.86 + 0.01
O
O
Cl
O
O
S
O
N
NH
2
O
O
H C
3
1
77
Celecoxib
72.52 + 1.32
0.28 + 0.02
269
-
11

Diclofenac
0.48 + 0.01
0.25 + 0.01
-
10.05 + 1.02
0.07 + 0.01
-
0.05
3.57
-
-
Indomethacin
-
Zileuton
0.63 + 0.03
1
1
78
79
SI= IC50 (COX-1) / IC50 (COX-2)
1
1
1
1
1
1
1
1
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81
82
83
84
85
86
87
2.3.2. Inhibition of Albumin denaturation and protease
Albumin denaturation and protease inhibitory assays were also performed for the synthesized
compounds as shown in Table 2. Among our tested compounds of series-A, compound 3b
showed good albumin denaturation and protease inhibition potentials with IC values of 24.53
5
0
and 17.52
(Albumin denaturation) and 13.39
inhibitor 13e exhibited IC50 values of 16.89 and 15.29
Table 2: In-vitro albumin denaturation and protease inhibition activities of the compounds.
µM respectively. Compound 13a showed excellent inhibition with IC values of 5.36
50
µM (protease inhibition). Most active COX-2 and 5-LOX
µM respectively.
Compound
Albumin denaturation
Protease inhibition
a
IC (µM)
IC (µM)
50
50
3
a
40.54
24.53
157.04
59.58
53.44
50.57
94.62
97.17
66.34
17.52
99.94
77.10
63.47
117.10
63.08
85.51
ND
3
b
3
c
3
d
3
e
3
f
3
g
3
h
b
3
i
ND
3
j
ND
ND
3
k
77.84
151.35
88.70
13.39
45.65
14.91
3
l
65.56
c
1
3a
3b
3c
5.36
1
16.11
24.85
1
12

1
3d
3e
Diclofenac sodium
19.66
16.89
30.52
21.48
15.29
42.72
1
1
1
1
1
1
1
88
89
90
91
92
93
a
b
c
The calculated IC50 value for was µM. Data is represented as mean ± S.E.M; n = 3; ND = not determined; bold
value = most potent compound
1
1
1
1
1
1
2
2
2
2
2
2
94
95
96
97
98
99
00
01
02
03
04
05
2.4. In-vivo anti-inflammatory activity
Anti-inflammatory activity of the compounds was assessed by carrageenan-induced paw edema
method of the experimental animals. Carrageenan induced paw edema model is a COX-2–
dependent model of inflammation. Therefore, we selected two compounds having high COX-2
inhibitory potentials (high selectivity index) from the synthesized two series of derivatives
(compound 3b from series 1 and 13e from series 2).
2.4.1. Acute toxicity
The dose chosen for in-vivo testing were based upon the results of preliminary range finding
tests from LD to LD and were ranged from 5-2000 mg/kg body weight consisting of 8 doses.
0
100
Three replicates were tested for each of eight doses with positive and negative control. The
detailed dosing regimens and animal specifications for the tested compound such as 3b and 13e
were given in above Table 3
.
2
2
2
2
2
2
2
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07
08
09
10
11
12
In the acute toxicity study of both the tested compounds (3b and 13e), no related mortalities were
recorded in animals treated with a single dose of 1000 mg/kg body weight. Therefore, the
approximate lethal dose (LD ) of both compounds in the experimental mice was 1000 mg/kg.
5
0
There were no clinical signs in the, eyes and mucus membrane (nasal), skin and fur, respiratory
rate, autonomic effects (salivation, piloerection, perspiration, defecation and urinary
incontinence) circulatory signs and central nervous system (ptosis, gait, drowsiness, convulsion
and tremors) among mice administered 1000 mg/kg body weight of both tested compounds (3b
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2
2
2
13
14
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&
13e). According to organization for economic cooperation and development (OECD)
guidelines for acute oral toxicity, an LD dose of > 300 – 2000 is categorized as category 4 and
5
0
hence the drug is found to be safe.
2
2
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Table 3: Animal group specification and quantity of drug administered for acute toxicity
studies with compound 3b and 13e.
Group
Animals
Tested drug 3b + 13e
(
mg/kg b.wt)
Male
Female
1
2
3
8
8
8
8
8
8
5
25
50
4
5
6
7
8
8
8
8
8
8
8
8
100
200
300
400
8
9
8
8
8
8
8
8
500
1000
2000
1
0
2
18
2
2
2
2
2
2
2
2
2
19
20
21
22
23
24
25
26
27
2.4.2 Carrageenan induced paw edema test
In preliminary screening test of compound 3b and 13e for anti-inflammatory activity, it has been
observed from Table 3 that the tested compound 3b and 13e revealed better anti-inflammatory
properties. The anti-inflammatory activity of the tested compound 3b was 34.54 % (P
˂
0.001) at
st
th
1 h and remained significant till 5 h (39.49%, P
˂0.001) at highest dose (100mg/kg)
comparable to that of standard drug aspirin (47.54-57.64%). In addition, the tested compound
13e displayed excellent activity (55.17-68.22%) than that of positive control at 100 mg/kg body
weight.
2.4.3 Anti-inflammatory mechanism of the synthesized compounds
14

2
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2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
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2
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
For the investigation of possible anti-inflammatory mechanism of tested compounds 3b and 13e
we used various mediators in in vivo animal model.
-
2.4.3.1. Effect of compounds on paw edema induced by Histamine
Histamine induced inflammation was significantly changed when treated with the tested dose of
st
positive control i.e. chlorpheniramine maleate at 1mg/kg body weight at 1 h (71.80%) and
th
remained significant till 5 h. Similarly, tested compound 13e demonstrated significant
st
inhibitory potential (32.40%) at 1 h at the dose of 100 mg/kg and was remained significant till
th
5 h. Likewise, the tested compound 3b at dose of 100 mg/kg did not displayed good anti-
th
inflammatory effect (17.10%) till 4 h of the administration of histamine induced inflammation
(Figure 2a)
.
2.4.3.2. Effect of compounds on paw edema induced by Bradykinin
Mean changes in paw edema volume of mouse pretreated with the test compounds at 100 mg/kg
body weight were measured at 1, 2, 3, 4 and 5 h after administration of bradykinin (20 µg/ml).
Both the tested compounds exhibited less activity in bradykinin induced inflammation as
rd
compared to the positive control. The tested compound 3b displayed 12.40% inhibition at 3 h
of bradykinin injection while 13e showed 27.13% inhibition which was not comparable to that of
positive control HOE 140. The results are shown in Figure 2b
.
2.4.3.3. Effect of compounds on paw edema induced by Prostaglandin
Administration of prostaglandin E (0.01 µg/ml) was associated with an increase in paw edema.
2
The prostaglandin E inflammatory changes were significantly modified by treatment with tested
2
compounds 3b, 13e (100 mg/kg) and Celecoxib (50 mg/kg). The tested compound 3b
st
significantly reduced the PGE
2
induced paw edema with 50.50 % at 1 h which reached to
th
th
maximal level at 4 h (64.60%) and remained significant till 5 h. Similarly, 13e displayed
15

th
2
2
2
2
2
2
2
2
2
2
2
2
51
52
53
54
55
56
57
58
59
60
61
62
promising result (61.60-81.80%) till 5 h of the experimental procedure. Celecoxib
comparatively displayed maximum percent inhibition of paw inflammation (65.90-87.10%)
(Figure 2c).
2.4.3.4. Effect of compounds on paw edema induced by leukotriene
In leukotriene induced inflammatory effect, the tested compounds 3b and 13e demonstrated a
concentration dependent anti-inflammatory activity at 100 mg/kg body weight (Figure 2d). The
edema produced by leukotriene injection (10 µg/ml) was inhibited by the tested compound in
nd
dose dependent manner. The compound 3b exhibited maximum inhibition (60.10 %) at 2 h
rd
while 13e displayed marked anti-inflammatory activity with 74.40 % at 3 h after administration
of leukotriene which was comparatively much closer to that of standard drug. Montelukast used
rd
as positive control displayed 77.40% percent inhibition of paw inflammation at 3 h.
2
2
63
64
Table 4: Anti-inflammatory activity of the tested compound using carrageenan induced paw edema in
mice.
Compound
Dose
Percentage inhibition of Edema (Mean + SEM)
1
h
2h
3h
4h
5h
Vehicle
Aspirin
10ml/kg
7.66±3.753
7.39±2.475
11.41±3.069
14.45±1.568
7.31±3.204
*
**
***
***
***
***
100mg/kg 47.54±4.12
25 mg/kg 24.33±3.28
54.63±1.47
53.26±1.36
56.92±2.95
57.64±1.55
***
***
***
***
***
***
*
**
***
***
***
3
b
28.33±1.20
37.43±2.12
49.63±2.15
23.33±1.20
27.81±2.79
23.37±2.16
47.84±2.56
55.07±2.33
***
***
5
0 mg/kg 34.54±4.59
41.14±2.19
44.51±3.43
***
***
1
00mg/kg 44.68±4.79
51.64±1.81
49.68±3.28
***
***
***
***
***
***
***
***
***
***
***
1
3e
25 mg/kg 45.63±1.47
0 mg/kg 51.52±2.82
00mg/kg 55.17±3.09
53.03±1.93
57.33±2.97
63.84±2.94
51.54±4.12
54.17±2.42
56.62±3.24
59.33±2.97
68.22±3.98
***
***
5
54.24±5.62
57.26±1.36
***
***
1
61.65±2.69
66.07±2.33
2
2
65
66
Data expressed as mean percent inhibition ± SEM. Two-way repeated measures ANOVA followed by Bonferroni’s post hoc test. ns; non
significance, *P<0.05, **P<0.01, ***P<0.001, compared to vehicle control. n=8 mice per group.
2
67
16

2
68
69
2
Figure 2: (a) Percent inhibition produced by tested compound 3b and 13e (100 mg/kg) in histamine
induced paw edema model in mice. (b) Percent inhibition produced by tested compound 3b and 13e (100
mg/kg) in bradykinin induced paw edema model in mice; (c) Percent inhibition produced by tested
compound 3b and 13e (100 mg/kg) in prostaglandin E2 induced paw edema model in mice; (d) Percent
inhibition produced by tested compound 3b and 13e (100 mg/kg) in leukotriene induced paw edema
model in mice. Each percent point represents the mean for group of 08 mice. Data was analyzed by two-
way ANOVA post test. *P < 0.05, **P < 0.01, ***P < 0.001, n.s; indicates non-significant;
270
271
272
273
274
275
276 2.5. Docking studies
277
278
279
280
281
282
Docking studies on the targets was carried out using Molecular Operating Environment (MOE
2016.08) software. Crystal structure of COX-2 in complex with SC-558 was retrieved from
Protein Data Bank (PDB code 1CX2). In protein data bank repository, two forms of 5-LOX are
available. Crystal structure human 5-LOX with no co-crystallized ligand was obtained from PDB
(accession No. 3O8Y). While, the crystal structure of another human 5-LOX with co-crystalized
substrate, arachidonic acid, is also available (PDB code = 3V99). We preferred to carry out
17

2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
docking studies on 3O8Y due to mutation (S663D) and absence of many amino acids in 3V99
[43-44].
2.5.1. Docking studies on COX-2 enzyme
Docking studies on the target enzymes were carried out to support our in-vitro experimental
results and to analyze the binding orientation and ligand-enzyme interactions. We have explored
the predictive power of docking simulations and performed docking studies were carried out on
all possible enantiomers. All the synthesized compounds of two series (3a-j and 13a-d) were
docked into the binding pockets of COX-1 and COX-2 isoforms. Although, the active sites of
both isoforms are very similar, however in COX-2, an additional secondary side pocket is
present above the Arg120/Tyr355. This additional pocket is bordered by small Val523
(isoleucine in COX-1). Furthermore, COX-2 also contains conserved Arg513 (replaced with
His513 in COX-1). Other residues important for COX-2 selectivity are His90, Gln192, Leu352
and Ser353. To investigate the selectivity, we only performed docking simulations on COX-2
isozyme. Docking simulations on the reference drugs celecoxib and indomethacin were also
performed. Two-dimensional (2-D) binding interaction pattern of both drugs is shown in
Supporting Information (Figure S-1). Analysis of the 2-D interaction plot of celecoxib, a
selective COX-2 inhibitor, reveals that it interacts amino acid residues (His90, Leu352, Ser353
and Arg513) present in additional pocket COX-2 pocket (Figure S1-b in Supporting
Information). Similarly, indomethacin displayed interactions with Ser353 and Tyr355.
Compounds of series 1 (3a-j) were docked into the binding site of COX-2. It is interesting to
note that (S, S)-isomer, with few exceptions, have more negative binding affinity values than (R,
R)-isomer, (S, R)-isomer and (R, S)-isomer. Interestingly, the most active COX-2 inhibitor 3b has
shown high negative value in (R, R)-form. The computed binding affinity for (R, R)-isomer of
18

306
307
308
309
310
311
most active COX-2 inhibitor 3b is -6.95 kcal/mol. While, for (S, S)- 3b, (S, R)- 3b and (R, S)- 3b,
it was found to be -6.81, -6.71 and -6.60 kcal/mol. Binding interaction pattern shown in Figure
3a revealed that in (S, S)- 3b, cyclohexanone ring oriented towards the secondary pocket and
forms hydrogen bond interactions with His90. While, the (R, R)-3b forms hydrogen bond
interactions with His90 and π-H interactions between Tyr355. Hence, we can predict here that
(R, R)-2 may have the more contribution towards bioactivity against COX-2.
312
3
13
14
Figure 3: Two-dimensional (2-D) binding interaction pattern of; (a) (S, S)-3b; (b) (R, R)-3b in the
3
binding site of 1CX2.
315
316
317
318
319
320
321
Analysis of docked poses of aryl ketone / N-sulfonamide derivatives (13a-e) showed that all the
compounds adopted similar conformations to the native ligand SC-558 (highly selective COX-2
inhibitor). Figure 4a represents the superposed binding poses of R-isomers (13a-e) and native
SC-558. The secondary pocket residues are shown in red spheres. While, the orientations of all
the ligands in S-isomeric form are shown in Figure 4b. The two-dimensional binding
interactions of R-sulfonamides (13a-b and 13d) are shown in Supporting Information (Figure
S-2).
19

322
323
324
325
326
Figure 4: (a) Ribbon model of the superimposed binding poses of compounds 13a-e (R-isomer) into the
binding site of human COX-2 (1CX2). The secondary pocket residues (His90, Gln192, Leu352,
Ser353 Arg513 and Val523 are shown as red spheres. While, some other important residues are shown
as yellow spheres; (b) Binding orientation of all compound 13a-e (S-isomer).
327
3
28
29
Figure 5: Two-dimensional (2-D) binding interaction pattern of; (a) R-13c; (b) R-13e in the binding site
3
of 1CX2.
330
331
332
333
20

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
2.5.2. Docking studies on 5-LOX enzyme
All the synthesized compounds were also docked into the binding site of human 5-LOX (PDB
accession code 3O8Y). Human 5-LOX consist of a hydrophobic cavity having Leu367, Leu362,
Ile406, Ala410, Val604, Leu607. The iron atom, liganded with His367, His372, His550 and
Asn554, is present at the center of the active site. Phe177 is also an important residue and
participates in the complex stabilization. Docking simulations on the reference drug Zileuton was
also performed. Two-dimensional (2-D) binding interaction pattern of Zileuton in the binding
site of 3O8Y is shown in Supporting Information (Figure S-3). Analysis of the 2-D interaction
plot of Zileuton reveals that it forms hydrogen bond interactions with Asn554, Gln607 and Ala
672. While, Leu607 and Asn180 forms arene-H interactions.
We docked our both series (3a-j and 13a-e) into the binding site of 3O8Y. All the possible
isomers were included, and their binding affinities were also analyzed. To explore the binding
mode of most active series of the compounds (13a-e). The 2-D interaction plot of R- and S-13e
into the active site of 5-LOX is shown in Figure 6. S-13e forms hydrogen bond interactions with
Asn180, His550, Pro668 and Ala672. While, R-13e established hydrogen bond interactions with
Asn180, Leu607 and Gln611. Oxygen atom of sulfonamide coordinated with Fe to stabilize the
ligand-enzyme complex. The computed binding affinity for S-13e and R-13e is -8.3684 and -
8.9405 kcal/mol respectively. Complete docking analysis of the most active compound of 3g
from first series of the compounds (3a-j) is presented in Supporting Information (Figure S-4
and S-5).
21

357
3
58
59
Figure 6: Two-dimensional (2-D) binding interaction pattern of; (a) S-13e; (b) R-13e in the binding site
3
of 5-LOX (3O8Y).
360
361
362
363
364
365
366
367
368
369
370
371
372
2.6. Preliminary In-silico pharmacokinetic studies
The aim of this study is to predict the human intestinal absorption, blood brain barrier
penetration and toxicities of the synthesized compounds. These in-silico pharmacokinetic
properties were predicted by using online AdmetSAR (http://lmmd.ecust.edu.cn/admetsar1)
server. These properties with probability output are tabulated in Table 5. All the compounds are
predicted to absorbed in intestine. The blood brain barrier (BBB) penetration was also predicted.
Inflammation has played a critical role in Alzheimer’s disease (AD) and other neurodegenerative
disorders (NDs). Cyclooxygenases (COX-1 and COX-2) and lipoxygenases (LOXs) affects the
progression of neurodegenerative diseases [45]. Lipoxygenase-5 (LOX-5) enzyme is widely
distributed in central nervous system (CNS) and in NDs its expression level increases [46].
Therefore, LOX-5 is considered as important target for neuroinflammation (NI) [47-48].
Similarly, COX-2 inhibition is also important to treat NDs [49]. The BBB penetration data in
Table 5 showed that all compounds are permeable and hence CNS active to treat the NDs like
22

3
3
3
3
73
74
75
76
AD. Moreover, AMES toxicity and carcinogenicity was also computed. All the compounds were
found to be non-AMES toxic and non-carcinogens.
Table 5: In-silico pharmacokinetic descriptors for compounds 3a-j and 13a-e and reference
Drugs.
BBB
Compound
HIA
AMES Toxicity
Carcinogens
penetration
+ (1.000 ) + (0.9964)
+ (1.000) + (0.9968)
+ (0.9973) + (0.9954)
Non-AMES toxic
Non-carcinogens
(0.8819)
a
3
3
3
3
3
3
3
3
3
3
3
3
1
1
1
1
a
(
0.8625)
Non-AMES toxic
0. 0.8142)
Non-AMES toxic
0.8191)
Non-AMES toxic
0.8376)
Non-AMES toxic
0.8245)
Non-AMES toxic
0.7192)
Non-AMES toxic
0.8601)
Non-AMES toxic
0.8720)
Non-AMES toxic
0.7037)
Non-AMES toxic
0.6862)
Non-AMES toxic
0.8768)
Non-AMES toxic
0.8774)
Non-AMES toxic
0.8774)
Non-AMES toxic
0.8774)
Non-AMES toxic
0.8774)
Non-AMES toxic
0.8774)
Non-carcinogens
(0.9068)
b
c
(
Non-carcinogens
(0.8458)
(
Non-carcinogens
(0.8748)
d
e
+ (1.000)
+ (1.000)
+ (0. 9923)
+ (0.9940)
(
Non-carcinogens
(0. 0.8976)
(
Non-carcinogens
(0.9180)
f
+ (0.9944) + (0.9877)
+ (09961) + (0.9963)
+ (0.9973) + (0.9966)
+ (0.9958) + (0.9781)
+ (0.9925) + (0.9798)
(
Non-carcinogens
(0.9003)
g
(
Non-carcinogens
(0.88143)
h
i
(
Non-carcinogens
(0.7634)
(
Non-carcinogens
(0.7984)
j
(
Non-carcinogens
(0.7982)
k
l
+ (1.000)
+ (1.000)
+ (0.9971)
+ (0.9799)
(
Non-carcinogens
(0.7029)
(
Non-carcinogens
3a
3b
3c
3d
+ (0.9912) + (0.9213)
+ (0.9936) + (0.9785)
+ (0.9964) + (0.9674)
+ (0.9950) + (0.9482)
(
0.7029)
Non-carcinogens
0.7029)
Non-carcinogens
0.7029)
Non-carcinogens
0.7029)
(
(
(
(
(
(
(
23

Non-AMES toxic
0.8774)
Non-AMES toxic
0.7185)
Non-AMES toxic
0.9133)
Non-carcinogens
0.7029)
1
3e
+ (0.9963) + (0.9717)
+ (1.000) + (0.9713)
(
(
Non-carcinogens
0.7905)
b
Celecoxib
(
(
Non-carcinogens
0.8728)
b
Indomethacin + (0.9509) + (0.9381)
(
(
a
b
3
3
77
78
Probability output is given in parenthesis; Reference drugs
3
79 3. Conclusions
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
Inflammation has been described and investigated by multiple researchers due to its association
with numerous diseased conditions. The use of various types of drugs for the management of
inflammation has been reported with different molecular targets and peculiar mechanisms. The
exploitation of each target against inflammation has been provided with myriads of applications
and drawbacks. The way a specific drug bind with a receptor, and functional groups of a specific
drug candidate carry a lot of information regarding its fate and association with adverse drug
reactions. Worldwide, various groups of researchers are continuously exploring novel molecular
targets and novel drug candidates for various diseases including inflammation. Our current
investigation describes the synthesis of N-substituted pyrrolidine-2,5-dione connected at
position-3 with cycloalkyl, alkyl and aryl carbonyl substituents. The synthesized compounds
were evaluated for their anti-inflammatory potentials by using different in-vitro assays like
cyclooxygenase-1, cyclooxygenase-2, 5-lipoxygenase, albumin denaturation and anti-protease
assays. It is evident from the in-vitro results that overall cyclic ketones (3a-h) and alkyl ketones
(3i-j) exhibited poor to moderate inhibitions of both COX isoforms. N-benzyl derivatives 3b and
3e showed selectivity towards COX-2. Compound 3b demonstrated IC value of 19.98 µM and
5
0
selectivity index (SI) of 3.13. Amongst the synthesized compounds, 13a-e series of compounds
showed inhibitions in low micromolar to submicromolar ranges. These compounds also
24

3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
demonstrated COX-2 selectivity. Compound 13e with IC50 value 0.98 µM and SI of 31.5
emerged as the most potent inhibitor of COX-2. Compounds 3b and 13e were further
investigated for in-vivo anti-inflammatory activity via carrageenan induced paw edema test. The
possible mode of action of compounds 3b and 13e were ascertained with various mediators like
histamine, bradykinin, prostaglandin and leukotriene. In-vivo acute toxicity study showed the
safety of synthesized compounds up to 1000 mg/kg dose. Extensive docking analysis were
carried out considering all the possible isomers of the compounds. Unfortunately, we were
unable to separate and identify the ratio of isomers in our compounds. However, by exploiting
the predictive power of computational tools, binding affinity data and binding orientation of all
the possible isomers were investigated. The selectivity of the compounds against cyclooxygenase
isoforms was supported by docking simulations. Selective COX-2 inhibitors showed significant
interactions with the amino acid residues His90, Gln192, Leu352 and Ser353 present in
additional secondary COX-2 enzyme pocket. Preliminary in-silico pharmacokinetic studies have
shown that all compounds are CNS active and thus these compounds can also be used to treat
neuroinflammation.
The focus of the current research is to identify ligands that can act on multiple anti-inflammatory
targets. This study represents a unique example of asymmetric N-substituted pyrrolidine-2,5-
dione derivatives targeting anti-inflammatory enzymes. The findings of the research in terms of
asymmetric catalysis, scaffold structure, in-vitro/in-vivo screening results, in-silico docking on
various targets and preliminary pharmacokinetic predictions are encouraging. Further
investigations are required to explore the type and position of substituents. Current study
described substitution at position-3 of N-substituted pyrrolidine-2,5-dione, however, the effect of
substitution at position-4 (vicinal substituents) of N-substituted pyrrolidine-2,5-dione will be
25

4
4
4
20
21
22
taken into consideration. For COX-2 selectivity, cycloalkyl / alkyl groups were not favorable
substituents. In conclusion, pyrrolidine-2,5-dione is still an important scaffold and further
structural modification may lead to design and synthesize of potent anti-inflammatory drugs.
4
4
23 4. Materials and methods
24
4.1. General
4
4
4
4
4
4
4
4
4
4
4
4
4
4
25
26
27
28
29
30
31
32
33
34
35
36
37
38
All the reagents and solvents were purchased from standard commercial vendors and were used
without any further purification. N-substituted pyrrolidine-2,5-dione, sulfanilamide, cycloalkyl,
1
13
alkyl and aryl carbonyl reagents were purchased from Sigma Aldrich. H and C-NMR spectra
were recorded in deuterated solvents on a Bruker spectrometer at 400 and 100 MHz respectively
using tetramethyl silane (TMS) as internal reference. Chemical shifts are given in δ scale (ppm).
The progress of all the reactions was monitored by TLC on 2.0 x 5.0 cm aluminum sheets pre-
coated with silica gel 60F254 with a layer thickness of 0.25 mm (Merck). LC-MS spectra were
obtained using Agilent technologies 1200 series high performance liquid chromatography
comprising of G1315 DAD (diode array detector) and ion trap LCMS G2445D SL. Final
products were analyzed for their purity on Schimadzu system using C18 reversed phase column
and isocratic solvent system of water/methanol (10:90) at room temperature. Biologically
screened compounds are > 95 % pure as determined by HPLC. Elemental analyses were
conducted using Elemental Vario EI III CHN analyzer (for Series 1) and LECO-932 CHNS
Analyzer (LECO Corporation, USA) (for Series 2).
4
4
4
4
39
40
41
42
4.2. General information of the Synthesized compounds (3a-l)
All the compounds were synthesized by the addition of different ketones to maleimides. The
respective ketone (2.0 equiv) was added to maleimide (1.0 equiv). Organocatalyst assembly
consisting of L-isoleucine, thiourea and KOH (0.2 equiv each) were added to chloroform (1M).
26

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
All the reactions were completed at room temperature. Thin layer chromatography (TLC) was
used for monitoring of reactions. The finishing of the starting material on TLC plate was
considered as completion of each reaction. The reaction was diluted with chloroform (15 ml) and
transferred to a separating funnel. Then added 15 ml of water, shake well and allowed to separate
the two layers. The organic layer containing crude compound was separated from the water
layer. Repeat the same extraction three times. Concentrate the crude compound by using rotary
evaporator and absorbed on silica gel surface. The solid form of the crude compound was loaded
to column chromatography for purification. In column chromatography, n-hexane and ethyl
acetate were used as eluting solvents. The yield of the final product was calculated from the
1
13
obtained pure compound. The products' structures were confirmed by H & C NMR and the
data was also compared with the reported literature.
4.2.1. 3-(2-oxocyclohexyl)-1-phenylpyrrolidine-2,5-dione (3a) [39- 41]
1
White solid. Yield = 71%. R = n-hexane-ethyl acetate (3:1) = 0.41. H NMR (400 MHz, CDCl )
f
3
(ppm): 1.51-1.82 (m, 3H), 1.96-2.04 (m, 1H), 2.07-2.22 (m, 2H), 2.31-2.48 (m, 2H), 2.52-2.67
(m, 1H), 2.82-2.90 (m, 1H), 3.02-3.12 (m, 1H), 3.19-3.33 (m, 1H), 7.24-7.33 (m, 2H), 7.35-7.40
1
3
3
(m, 1H), 7.44-7.50 (m, 2H). C NMR (100 MHz, CDCl ) (ppm): 22.1, 25.4, 29.3, 30.1, 31.4,
32.6, 38.5, 40.4, 41.7, 50.1, 52.3, 126.5, 126.5, 128.7, 129.2, 131.9, 175.1, 177.4, 210.2. HPLC
purity = 95.2 %, T = 10.0 min. LC-MS found for C H NO (m/z) = 272.2 [M+H]. Analysis
R
16 17
3
calcd (%): C, 70.83; H, 6.32; N, 5.16. Found (%): C, 71.03; H, 6.29; N, 5.21.
4.2.2. 1-benzyl-3-(2-oxocyclohexyl)pyrrolidine-2,5-dione (3b) [39-41]
Off-white solid. Yield = 69 %. R = n-hexane-ethyl acetate (3:1) = 0.45. The R value in n-
f
f
1
hexane and ethyl acetate (4:1) was calculated as 0.45. H NMR (400 MHz, CDCl
3
) (ppm): 1.49-
1.79 (m, 3H), 1.83-2.01 (m, 2H), 2.12-2.26 (m, 1H), 2.33-2.63 (m, 3H), 2.80-2.99 (m, 2H), 3.02-
27

1
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
3
3.16 (m, 1H), 4.65 (d, J = 7.38 Hz, 2H), 7.26-7.38 (m, 5H). C NMR (100 MHz, CDCl ) (ppm):
24.0, 26.2, 27.9, 32.0, 32.9, 39.5, 40.2, 40.9, 41.8, 49.9, 51.5, 126.9, 127.6, 128.3, 129.1, 129.8,
133.9, 175.2, 178.3, 210.2. HPLC purity = 96.4 %, T = 10.9 min. LC-MS found for C H NO
3
R
17 19
(m/z) = 286.2 [M+H]. Analysis calcd (%): C, 71.56; H, 6.71; N, 4.91; Found (%): C, 71.43; H,
6.73; N, 4.94.
4.2.3. 1-(4-bromophenyl)-3-(2-oxocyclohexyl)pyrrolidine-2,5-dione (3c) [40, 41]
1
Yellow solid. Yield = 75% %. R
CDCl
7.24-7.28 (m, 2H), 7.59-7.64 (m, 2H). C NMR (100 MHz, CDCl
f
= n-hexane-ethyl acetate (4:1) = 0.43. H NMR (400 MHz,
3
) (ppm): 1.64-1.77 (m, 3H), 2.06-2.21 (m, 4H), 2.47-2.52 (m, 3H), 1.96-3.02 (m, 2H),
1
3
3
) (ppm): 23.1, 24.3, 27.4,
28.1, 30.7, 31.5, 32.5, 33.8, 41.8, 42.5, 43.6, 54.1, 54.8, 124.1, 129.2, 131.0, 133.6, 175.6, 177.8,
211.6. HPLC purity = 98.1 %, T = 13.9 min. LC-MS found for C H BrNO (m/z) = 350.1
R
16 16
3
[M+H]. Analysis calcd (%): C, 54.87; H, 4.61; N, 4.00 %. Found (%): C, 54.97; H, 4.60; N, 4.03.
4.2.4. 3-(5-methyl-2-oxocyclohexyl)-1-phenylpyrrolidine-2,5-dione (3d)
1
White solid. Yield = 79 % %. R = n-hexane-ethyl acetate (4:1) = 0.49. H NMR (400 MHz,
f
CDCl
3
) (ppm): 0.95-0.96 (m, 1H), 1.14-1.20 (m, 3H), 1.26-1.44 (m, 1H), 1.61-1.83 (m, 1H),
1.87-2.03 (m, 2H), 2.16-2.36 (m, 2H), 2.43-2.81 (m, 2H) 2.94-2.97 (m, 1H), 3.03-3.15 (m, 1H),
1
3
7.19-7.27 (m, 2H), 7.31-7.33 (m, 1), 7.38-7.41 (m, 2H). C NMR (100 MHz, CDCl ) (ppm): 17.
3
7, 17.7, 21.3, 21.4, 26.9, 26.9, 32.0, 32.4, 33.4, 33.6, 34.9, 35.2, 35.7, 37.1, 37.2, 37.4, 38.2, 41.0,
41.2, 41.3, 46.1, 47.5, 51.3, 126.8, 126.8, 126.9, 128.7, 129.3, 132.2, 132.5, 175.8, 175.9, 178.6,
178.7, 210.4, 210.8. HPLC purity = 96.5 %, T = 11.2 min. LC-MS found for C H NO (m/z)
R
17 19
3
= 286.5 [M+H]. Analysis calcd (%): C, 71.56; H, 6.69; N, 4.91. Found (%): C, 71.76; H, 6.71; N,
4.93.
4.2.5. 1-benzyl-3-(5-methyl-2-oxocyclohexyl)pyrrolidine-2,5-dione (3e)
28