Development of Piperazinediones as dual inhibitor for treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2018.02.078

Novel multifunctional 3,6-Diphenyl-1,4-bis(phenylsulfonyl)piperazine-2,5-dione derivatives were designed and synthesized for the treatment of Alzheimer's disease (AD). The designed scaffold has blood brain barrier penetrating ability, acetylcholinesterase (AChE) and matrix metalloproteinase-2 (MMP-2) inhibition potential. Compounds 52 and 46 showed very significant inhibition against AChE, IC₅₀ = 32.45 ± 0.044, 28.65 ± 0.029, BuChE, IC₅₀ = 157.95 ± 0.264, 160.58 ± 0.082 and MMP-2, IC₅₀ = 36.83 ± 0.015, 19.57 ± 0.005 (nM). In the enzyme kinetics study, lead molecule 46 showed non-competitive inhibition of AChE with K_i = 7 nM and competitive inhibition of MMP-2 with K_i = 20 nM. Compounds 52 and 46 inhibited AChE-induced Aβ aggregation at 20 μM. The compounds also exhibited in-vitro antioxidant potential in DPPH assay. Further, compound 46 was found to be a promising neuroprotective agent in MC65 cells. Lead molecule 46 significantly enhanced working memory in scopolamine induced amnesia animal model at dose of 5 mg/kg dose. The mitochondrial membrane potential was restored in animals when treated with compounds 52 and 46.

Full text of DOI:10.1016/j.ejmech.2018.02.078

Accepted Manuscript
Development of Piperazinediones as dual inhibitor for treatment of Alzheimer's
disease
Devendra Kumar, Sukesh K. Gupta, Ankit Ganeshpurkar, Gopichand Gutti, Sairam
Krishnamurthy, Gyan Modi, Sushil Kumar Singh
PII:
S0223-5234(18)30215-0
10.1016/j.ejmech.2018.02.078
EJMECH 10255
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 December 2017
Revised Date: 11 February 2018
Accepted Date: 24 February 2018
Please cite this article as: D. Kumar, S.K. Gupta, A. Ganeshpurkar, G. Gutti, S. Krishnamurthy, G. Modi,
S.K. Singh, Development of Piperazinediones as dual inhibitor for treatment of Alzheimer's disease,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.078.
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Development of Piperazinediones as Dual Inhibitor for Treatment of Alzheimer’s disease
Devendra Kumar, Sukesh K. Gupta, Ankit Ganeshpurkar, Gopichand Gutti, Sairam Krishnamurthy,
Gyan Modi, Sushil Kumar Singh*
Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (Banaras
Hindu University), Varanasi 221005, India.
*
Corresponding author. Tel.: +91 542 6702736; fax: + 91 542 368428.
E-mail address: sksingh.phe@iitbhu.ac.in
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ABSTRACT
Novel multifunctional 3,6-diphenyl-1,4-bis(phenylsulfonyl)piperazine-2,5-dione derivatives were
designed and synthesized for the treatment of Alzheimer’s disease (AD). The designed scaffold has
blood brain barrier penetrating ability, acetylcholinesterase (AChE) and matrix metalloproteinase-2
(
MMP-2) inhibition potential. Compounds 52 and 46 showed very significant inhibition against AChE,
IC50 = 32.45±0.044, 28.65±0.029, BuChE, IC50 = 157.95±0.264, 160.58±0.082 and MMP-2, IC50=
6.83±0.015, 19.57±0.005 (nM). In the enzyme kinetics study, lead molecule 46 showed non-
competitive inhibition of AChE with K =7 nM and competitive inhibition of MMP-2 with K = 20 nM.
3
i
i
Compounds 52 and 46 inhibited AChE-induced Aβ aggregation at 20 µM. The compounds also
exhibited in-vitro antioxidant potential in DPPH assay. Further, compound 46 was found to be a
promising neuroprotective agent in MC65 cells. Lead molecule 46 significantly enhanced working
memory in scopolamine induced amnesia animal model at dose of 5 mg/kg dose. The mitochondrial
membrane potential was restored in animals when treated with compounds 52 and 46.
KEYWORDS: Alzheimer’s disease, piperazine-2,5-dione, acetylcholine esterase (AChE), matrix
metalloproteinase-2 (MMP-2).
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1
. INTRODUCTION
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder primarily
characterized by the loss of memory associated with other cognitive deficits[1]. Among the
several hypotheses proposed to correlate the onset of disease and its progression, the
cholinergic hypothesis is one of the oldest, robust and clinically validated in mild to severe
AD cases. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) has been
extensively explored in AD tissues since the discovery of cholinergic deficit in the disease[2].
The alterations in AChE activity coupled with variations in its polymorphism in brain, CSF
and blood were reported in AD patients[3]. Its (AChE or Ach) level in hippocampus and
cortex was found to be decreased in AD[4].
The other hypotheses being advocated for the development of AD are based on ‘plaques' and
‘tangles,' derived from proteins which hamper the usual cognitive functions of the brain[5].
AD is one of the major amyloidosis with two types of amyloid depositions in the diseased
brain. First, amyloid beta (Aβ) aggregates, a major constituent of plaques, and second, tau
protein which forms neurofibrillary tangles (NFTs), dystrophic neurites, and neuropil
threads[6].
Altered mitochondrial membrane potential plays a pivotal role in the pathogenesis of
neurodegenerative diseases including AD and Parkinson’s disease (PD)[7]. The deposition of
Aβ aggregates in the mitochondria decreases the activity of respiratory chain complexes
leading to decrease in ATP production and mitochondrial membrane potential. Further
mitochondrial Aβ and extracellular neuritic plaques are also found to be involved in reactive
oxygen species (ROS) generation which leads to enhanced apoptosis and neuronal cell
death[8]. The effectiveness of AD therapy at molecular level needs to be stablished by
evalutating the mitochondrial function. There is an imbalance between ROS production and
antioxidant defense system in AD. Generation of ROS is multifactorial phenomena and
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imbalance of bioactive metals is the major player. The metal ions create oxidative stress
leading to Aβ and tau protein aggregation[9].
In normal human brain, the matrix metalloproteinase (MMPs) levels are quite low. However,
it is reported to be upregulated during neurological disorders including AD and after injuries.
The altered MMPs expression in AD had been reported previously[10-12]. Brkie et al.,
measured the expression of MMP gene in choroid plexus epithelial (CP) and cerebrospinal
fluid (CSF) to establish a correlation between MMPs and Aβ induced blood-CSF barrier
(BCSFB) leakage. An increase in MMPs activity was observed simultaneously in CSF with
Aβ1-42 oligomer injection. Intracerebroventricular co-treatment of Aβ oligomers with
GM6001, a non-selective MMP inhibitor, could reduce the Aβ oligomers mediated BCSFB
disruption[13]. It was found that MMP-2 and 9 were engaged in different pathological
cascade of AD[14]. The role of MMP-9 in Aβ promoted neurotoxicity in AD has not been
understood completely[15].
The current treatment protocol for AD provides only symptomatic relief for initial 1-2 years
without addressing the elementary pathogenetic factors responsible for the disease[16].
Therefore, the disease continues to progress with the existing therapies. The development of
multifunctional drugs with ability to provide symptomatic treatment along with the
modulation of pathogenic factors such as Aβ aggregation and oxidative stress is the one of
the major goals of present study.
The compounds for the study were designed by Hybrid pharmacophore approach and
database mining. The two compounds i.e. Donepezil (FDA approved, centrally acting AChE
inhibitor) and PQ912 (in phase II Clinical trials for AD) were selected and used as template
to identify the essential pharmacophore features. The developed pharmacophores were
subsequently used for database mining of “Zinc15” and “Asinex” databases. The similar
pharmacophoric compounds obtained, were further subjected to various filters i.e drug-
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likeness property, in-silico BBB permeability and toxicity. The selected compounds were
docked to identify the functional groups and fragments to provide key interactions with the
targets (AChE and MMP-2) (Figure 1). The fully optimized molecules thus obtained were
promoted to synthesis, in-vitro enzyme assays and in-vivo studies. The potent molecules
obtained from in-vitro study were further investigated for neuroprotection ability in MC65
cells and antioxidant assay. The two most potent compounds, 52 and 46, were selected for in-
vivo studies in AD animal models to evaluate the working memory and learning response
(
Figure 1).
. RESULTS AND DISCUSSION
.1 Chemistry
2
2
The aromatic and aliphatic piperazine-2,5-diones were synthesized according to scheme I
and II. The sulfonamide derivatives (14-35) were prepared by treating substituted aromatic
sulfonyl chloride (1-13) with aliphatic amino acid (11) or aromatic amino acids (12, 13) in
presence of base. The substituted sulfonamides (14-35) were initially characterized by FTIR
1
followed by HNMR. FTIR showed characteristic secondary amine stretching band in the
-1
-1
region of 3300-3200 cm and the presence of hump in the range of 2900-2800 cm , carbonyl
-1
band appeared in between 1690-1650 cm and sulfur dioxide band in between 1100-1070
-1
cm . The carboxy group was found above 9.0 ppm followed by aromatic, amino and
1
methylene protons at 7-8, 4.5-5.5 and below 4.3 ppm respectively in H-NMR. Piperazine-
2
,5-dione derivatives were synthesized by cyclization with different sulfonamides (14-35)
using ethyleneglycol as a cyclizing agent. Compounds 60, 61 were synthesized by reacting
substituted alphatic sulfonyl chlorides (58, 59) with glycine (11) in presence of
sodiumbicarbonate and acetone-water mixture (Scheme II). The FTIR showed bands in
-1
between 3300-3200, 2800-2500, 1680-1630 and 1110-1030 cm for a secondary amine,
carboxy and sulfur dioxide respectively.
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2
.2 In-vitro Biological Evaluation
2
.2.1 AChE, BuChE and MMP-2 inhibition assay: Considering the fact that AD is
multifactorial in nature, we designed multifunctional drugs to address the basic factors
responsible for the disease. The pharmacophore 1,4-bis(phenylsulfonyl)piperazine-2,5-dione
(36) was the starting point for designing the potent in-vivo compounds. The structural
modifications were centered towards the introduction of various substituents on the
peripheral phenyl and central piperazine rings. Our first goal was to find out the significance
of peripheral phenyl rings in the interaction with the enzymes. Therefore, compound 36 and
its aliphatic analogs 62 and 63 were designed, synthesized and evaluated for AChE and
MMP-2 inhibition[17] [18]. In this assay, compound 36 carrying phenyl ring turned out to be
potent in comparison to 62 and 63 (IC , AChE, 36 = 98.53±0.025 nM, IC 62 and 63 =
5
0
50
>
200.00 nM; IC , BuChE, 36 = >200.00 nM; IC , MMP-2, 36 = 82.17±0.024 nM, IC for
50 50 50
6
2 and 63 was>1000.00 nM, Table 1). This may be due to the aromatic-π interaction of
molecule with the target. We further introduced various electron withdrawing groups (EWG)
and electron donating groups (EDG) to the phenyl ring of our lead molecule 36 to explore the
role of substituents p-Cl, m-Cl, p-Br, p-F and p-CH which led to generation of compounds
3
3
8, 39, 40, 43 and 37. Interestingly, compound 38 bearing chloride group at para position on
the phenyl ring showed maximum inhibition of the enzymes over EDG analog 37 (IC ,
5
0
AChE = 61.62±0.094 nM,117.70±0.0930 nM for 38 and 37, respectively; IC , BuChE =
5
0
1
80.21±0.230 nM, >200 nM for 38 and 37, respectively; IC , MMP-2 = 49.69±0.031nM,
50
and 86.74±0.473nM, for 38 and 37 respectively). It was also observed that strong EWG
(
43) and larger size EWG (40) are not well tolerated on AChE binding site (IC , AChE =
50
7
8.32±0.042 nM, 81.84±0.522 nM for 43 and 40, respectively) while no significant
difference was observed in the inhibition of MMP-2 (IC , MMP-2 = 55.13±0.051nM, and
5
0
5
4.68±0.007 nM, for 43 and 40 respectively). This is also evident from the structure of the
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binding interaction of the molecules, indicating that π-π and π-sulfur are crucial for activity.
The role of hydrogen bonding in the interaction with targets was explored by introducing
hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA) groups at para-position of
the lead molecule 36. The amine containing compound 42 could inhibit MMP-2 at lower
concentration compared to methoxy compound 41 (IC , MMP-2 = 53.14±0.014nM, and
5
0
7
5.25±0.022 nM, for 42 and 41 respectively). It is noteworthy that these analogs were found
to be more active as compared to 36 (Table 1).
After optimizing the suitable substituents on the peripheral phenyl ring, we introduced
aromatic feature into the central piperzaine ring. This modification is based on the
observation of π-π interaction with amino acid residues Trp 86, 238 and Asp74 in AChE and
Ala 80, 88 in MMP-2 with the aromatic ring. The addition of phenyl ring into the piperazine
ring, generated compound 46 (IC , AChE = 28.65±0.029 nM; IC , BuChE = 160.58±0.082
5
0
50
nM; IC , MMP-2 = 19.57±0.005 nM) which was found to be superior in comparison to the
5
0
parent compound 36. Interestingly, AChE inhibitory property of compound 46 was slightly
less than donepezil (IC = 225.06±0.041 nM). Based on these results, we introduced phenyl
5
0
ring into our previously synthesized compounds bearing substitution on the peripheral phenyl
ring. In this series total six compounds were synthesized. They were subjected to enzyme
inhibition studies after characterization. As expected, the phenyl piperazine derivatives 48,
and 50 (IC , AChE = 76.39±0.032 nM, 50.62±0.023 nM for 48 and 50, respectively; IC ,
5
0
50
MMP-2 = 62.87±0.074 nM, and 32.71±0.005nM, for 48 and 50, respectively) inhibited both
the enzymes with lower IC50 values as compared to piperazine analogs, 37 and 40,
respectively (IC , AChE = 117.70±0.030 nM, 81.84±0.522 nM for 37 and 40; IC , MMP-2
5
0
50
=
86.74±0.473 nM, and 54.68±0.007 nM, for 37 and 40 respectively). It is important to
point out that slight difference among the amine containing phenyl piperazine 45 and its
parent compound 42 (IC , AChE = 91.33±0.017nM, 86.35±0.029 nM for 45 and 42,
50
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respectively; IC , MMP-2 = 50.55±0.021nM, and 53.14±0.014 nM, for 45 and 42
5
0
respectively) was noticed which could be due to complex interaction of amine with the target.
In this series, hydroxyl and nitro derivatives, 44 and 47, were also tested and found to be
potent inhibitor of enzymes (IC , AChE = 84.25±0.018 nM, 60.76±0.00 nM for 44 and 47,
5
0
respectively; IC , MMP-2 = 57.24±0.040 nM, and 33.47±0.027 nM, for 44 and 47,
5
0
respectively). However, the chloride bearing 46 remained most potent so far in this series.
Finally, we introduced one carbon linker between phenyl and piperazine to reach the Tyr124,
3
41 in AChE and His120, Leu83, Ala84 in MMP-2. The six molecules synthesized in this
category were less active against MMP-2 in comparison to the molecules without a linker.
The chlorine containing compound 52 lost the activity by 10% compared to 46 (IC , AChE =
5
0
3
2.45±0.044 nM, 28.65±0.029 nM for 52 and 46 respectively). However, the bromo
compounds, 56, 57 and nitro compound 55 retained the activity against AChE (IC ,
5
0
5
6.97±0.021 nM, 76.75±0.041 nM and 57.35±0.024 nM for 56, 57 and 55, respectively)
while a small reduction in MMP-2 activity was observed (IC , 38.68±0.045 nM,
5
0
5
8.08±0.024 nM and 53.82±0.042 nM for 56, 57 and 55, respectively). The para-hydroxyl
compound 53 gained the activity in comparison to its analog 44 against AChE (IC ,
5
0
5
9.45±0.132 nM, 84.25±0.018 nM for 53 and 44, respectively).
The mechanism of AChE inhibition was studied in detail by exploring the enzyme kinetics
parameters of compound 46. Vmax and Vmax/K in the Michaelis-Menten graph was found to
m
decrease by increasing the concentration of inhibitor, while K increased (Figure S1,
m
supplementary information). The pattern of Vmax and K along with Lineweaver-Burk plot
m
suggested that compound 46 is acting on both free enzyme and enzyme-substrate complex
(noncompetitive inhibition). In Lineweaver-Burk plot, the lines intersect at the value of 1/[S]
less than zero and 1/[V] greater than zero thus α > 1 indicate that the inhibitor preferentially
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binds to the free enzyme rather than enzyme-substrate complex (Figure 2). The Dixon plot
showed that the compound 46 has Ki = 7 nM (Figure S2, supplementary information).
The mechanism of MMP-2 inhibition was established by the kinetic study. Compound 46
inhibited the enzyme by competitive mode of action as the Vmax of the enzyme remained
constant throughout the enzyme kinetics at different concentrations of inhibitor. The Vmax/Km
decreased gradually on increasing the inhibitor concentration (Figure 3). The value of
dissociation constant (K ) of the enzyme reaction was determined by the graphical
i
method[19] by plotting K vs[I] (I, inhibitor). The inhibitor was found to bind tightly with the
m
enzyme having Ki value of 20 nM (Table S1, Figure S3, S4 supplementary information).
2
.2.2 Blood-Brain Barrier Permeation Assay (PAMPA): Permeability of the synthesized
compounds for blood-brain barrier (BBB) is a prerequisite to target AD. Therefore, the
compounds were screened by parallel artificial membrane permeation assay (PAMPA)[20].
PAMPA model can successfully determine the passive BBB permeation with high accuracy.
The in-vitro permeability of the synthesized compounds and nine commercially available
drugs was performed through porcine brain lipid (PBL) dissolved in dodecane. A linear
correlation was established by using the experimentally obtained permeability (Pe) and
reference (Pe). The relationship used to determine the cutoff for CNS permeability was
Pe(exp) = 1.308Pe(literature)-0.8394, (R2 = 0.9317). The Pe > 4.3×10-6 cm/s was found for
high CNS permeability (Table S2, Figure S5, Supporting information). The Compounds 37-
4
3 showed appreciable CNS permeability, but compounds 62 and 63 have very low CNS
permeability (Table 2).
.2.3 Neuroprotection studies: Human neuroblastoma MC 65 cell line yields Aβ1-40 by
2
conditional expression of C-terminal APP protein. It mediates cell cytotoxicity by DNA
fragmentation which is induced by ROS[21]. The cell line is accompanying with oxidative
stress and Aβ induced cellular toxicity in tetracycline removal condition (-TC). Therapeutic
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suitability and effectiveness of the molecules to prevent oxidative stress was determined by
MTT assay. Most active compound 46 (70-1µM) was selected for the neuroprotection and
cytotoxicity studies. The neuroprotective ability of compound 46 is comparable to that of
tetracycline (TC+). The percentage cell viability of compound 46 was found to be 99.41 in
relation to +TC (Figure 4).
compensate the oxidative stress generated in the absence of TC.
.2.4 AChE-induced Aβ - aggregation assay Metal ions (Fe, Cu, Zn) and AChE are the
MTT assay ascertained that molecule had the ability to
2
:
1
42
two major factors responsible for Aβ aggregation. Peripheral anionic binding site (PAS)
provides the seeding site for the low molecular weight oligomers[22]. Aβ and AChE form the
complex which promotes the neurite network dystrophia and apoptosis. Furthermore, Aβ-
2
+
AChE complex increases intracellular Ca resulting in an imbalance of mitochondrial
membrane potential[23].
A fixed amount of Aβ (10 µM) and variable concentrations of donepezil, compounds 52 and
4
6 (0.5, 10 and 20 µM) were used in the Thioflavin T (ThT) assay. Aβ and AChE with Aβ
were taken as negative control. Donepezil, compounds 52 and 46 did not show significant
results in the assay at 0.5 µM concentration. Donepezil showed over 60% of Aβ aggregation
at 10 µM concentration while, compounds 52 and 46 showed less than 60% aggregation at
same concentration. Further, the Aβ aggregation was approximately 50% at 20 µM for
donepezil but was less than 50% and 40% in case of compounds 52 and 46, respectively
(Figure 5). ThT assay clearly indicated that compounds 52 and 46 were more potent than
donepezil at 10 and 20 µM concentrations.
It was observed in the kinetic study that the designed compounds showed non-competitive
inhibition and thus were more efficient in inhibition of the seeding of Aβ oligomers. The
compounds also showed metal chelating ability and might inhibit the metal induced Aβ
aggregation. The potent nature of compounds 52 and 46 compared to donepezil in Aβ
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aggregation might be due to their dual mechanism of action. The hexapeptide sequence
responsible for nucleation of Aβ was found to be KLVFFA[24]. Compounds 52 and 46
depicted interaction (A chain) with Aβ protein by amino acids valine 24 (V24), phenylalanine
1
9, 20 (F 19, 20), alanine 21 (A21) and lysine 16(K16), leucine 17 (L 17), valine 18 (V18),
phenylalanine 19, 20 (F 19, 20) respectively (Figure S6, supplementary information).
.2.5 Antioxidant Activity Evaluation: DPPH assay is a reliable method to determine the
2
active oxygen scavenging property of compounds. The synthesized piperazine-2,5-diones
were evaluated for their antioxidant potential by DPPH assay. Compounds 44, 45, 42, 53, and
5
4 displayed good antioxidant property (Table 2). The compounds containing –OH or –NH2
type of donor groups are potent oxygen scavenger over other molecules in this series[25].
The compounds 44, 53, 54 containing a hydroxyl group and compound 45 an amino group
also participated in the active oxygen scavenging. Rest of the molecules showed satisfactory
DPPH reduction potential.
2
.2.6 In-vitro metal chelation assay: Biometals (mainly Cu) are found to be involved in the
Aβ aggregation. The toxic oligomeric state of Aβ is stabilized by Aβ-metal complex[26].
2
+
2+
Cu and Fe bound Aβ produces ROS which induces oxidative stress followed by the
neuronal death[27]. The synthesized compounds were investigated for their metal chelation
property by using CuCl . Compound 46 showed copper complexing property as observed in
2
UV and FTIR spectra. The UV spectra of copper complex had a remarkable shift from the
parent compound (Figure 6). FTIR spectra displayed the change in sulfur dioxide peak
indicating that it was involved in the metal chelation (Figure S7 (A), supplementary
information)[28, 29].
2
.3 Behavioral Studies.
Compounds 52 and 46 showed promising in-vitro activity and were further evaluated in AD
animal models. The in-vivo scopolamine model was used to evaluate the anti-cholinesterase
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and dementia conditions in animals[30]. Antiamnesic effect of compounds 52 and 46 was
assessed by Y-Maze and passive avoidance tests. The dose of the compounds was fixed at
half of the lethal dose (LD ) data (Supplementary, section S5).
5
0
2
.3.1 Y-Maze Test: Y-maze test is used to analyze the hippocampal-dependent spatial
working memory of animals. The working memory is assessed by the spontaneous alteration
score[31]. The spontaneous alternation score (% alternation) was significantly decreased in
scopolamine treated group as compared to control, vehicle and donepezil groups. The animals
treated with compound 52 showed an increase in spontaneous alternation score at dose of 10
mg/kg, while compound 46 treated animals showed approximately same alternation score at
5
mg/kg dose (Figure 7A, B). Neophobia and anxiety behaviors of the animals were
determined by monitoring their novel arm entries. Compound 52 decreased neophobia and
anxiety significantly at 10 and 5 mg/kg dosages as compared to scopolamine. Neophobia and
anxiety behavior was also decreased by the compound 46 at 5 mg/kg dose, but its action
decreased upon increase in the dose (Figure 7C, D). The anxiolytic patterns of compound 52
and 46 were further evaluated by observing the time spent by animals in the novel arm,
known arm and center of Y maze. The animals treated with 20 mg/kg of compound 52 spent
significantly more time in the novel arm as compared to the control and vehicle (Figure 7E).
Moreover, at the same dose, compound 46 showed comparatively same anxiolytic activity
(Figure 7F). Further studies indicated that scopolamine treated animals spent most of their
time in the known arm as compared to the novel arm indicating that scopolamine did not
affect the locomotive behavior of the animals. Compounds 52 and 46 increased the working
memory, decreased the neophobia and anxiety but did not alter the locomotive behavior of
animals (Figure 7G, H).
2
.3.2 Passive avoidance test: The learning response is mainly governed by the hippocampus
and the central cholinergic neurons are involved in the passive avoidance learning[32]. The
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transfer latency time (TLT) was significantly increased in retention trial of control, vehicle
and compounds 52 and 46 at doses of 10 and 5 mg/kg. Intraperitoneal (i.p.) injection of
scopolamine caused cognitive impairment hence there was no significant change in the TLT
of retention trial as compared to acquisition trial. Treatment with compounds 52 and 46
showed significant dose related TLT response (Figure 8 A, B). Compound 52 showed an
increase in TLT that was comparable to control, vehicle and donepezil at 10 mg/kg while
nearly similar response was observed at half (5 mg/kg) dose of compound 46. The increase in
the TLT at lower dose indicated that compound 46 was more potent than compound 52.
2
.3.3 Mitochondrial membrane potential in brain region
Aβ can get transported in mitochondria by translocase of the outer membrane ENREF 34[33]
and produces ROS that eventually cause damage to neurons and disturbs the mitochondrial
membrane potential. The mitochondrial membrane potential was maintained in animals when
treated with compounds 52 and 46 at a dose of 5 mg/kg (Figure S8, supplementary
information).
2
.3.4 Neurochemical level estimation
The neurochemical (AChE) level in the animal brain was analyzed by Ellman’s method to
determine the effect of treatment. Scopolamine treatment increased the level of AChE, but it
was significantly decreased by the treatment of donepezil and compounds 52 (10 mg/kg), 46
at 5 mg/kg. Catalase (CAT, catalyzes H O ) level in the brain of animals was determined
2
2
after the behavioral studies. Compound 52 maintained its normal level at a dose of 10 mg/kg
Figure S9 A, B, supplementary information). Histopathological examination of the brain was
(
carried out to determine the pattern of arrangement in the normal, toxic and treated tissues.
Scopolamine treated animal showed some lesions while the standard pattern of the tissues
was found in control, compounds 52 and 46 treated animals (Figure 7I).
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3
. CONCLUSIONS
Novel 3,6-diphenyl-1,4-bis(phenylsulfonyl)piperazine-2,5-dione derivatives were designed
and synthesized by amide coupling followed by cyclization to piperazine ring. The scaffold
contains the BBB penetrating ability along with AChE and MMP-2 inhibition potentials.
Compounds 38, 39, 46, 53, 54, 55, 47, 56 and 50 showed promising inhibition potential
against AChE while compounds 52, 38, 45, 47, 56 and 50 showed high potency against
MMP-2 inhibition. Further, compounds 52 and 46 showed highest inhibition potential for
AChE with IC50 = 32.45±0.044 nM, 28.65±0.029 nM, BuChE, IC50 = 157.95±0.264,
1
60.58±0.082 and MMP-2 IC50 = 36.83±0.015 nM, 19.57±0.005 nM, respectively. These
derivatives also demonstrated critical interaction with targets in molecular modeling studies.
In enzyme kinetics with AChE, lead compounds 52 and 46 showed noncompetitive inhibition
and inhibitor preferentially bound to the free enzyme (α > 1) while MMP-2 was found to be
inhibited competitively. The compounds 52 and 46 were found to be more potent than
standard donepezil in AChE-induced Aβ aggregation studies at dose of 20 µM.
Neuroprotection assay of compound 46 in MC65 cell line indicated that this compound has
approximately equal potency as TC for the inhibition of ROS induced neurodegeneration.
Metal chelation property found in compound 46 was helpful in MMP-2 inhibition and AChE-
induced Aβ aggregation.
In the working memory assessment compounds 52 and 46 could enhance the spontaneous
alteration. Compound 52 showed a significant increase in spontaneous alteration score at 10
mg/kg while 46 has same response at half doe (5 mg/kg dose) indicating that there is
improvement in the immediate working memory. Compound 52 reduced neophobia and
anxiety at the dose of 10 and 5 mg/kg as compared to scopolamine treated rats. It also
displayed significant anti-anxiolytic activity which was monitored by observing the time
spent by the animals in the novel arm. Compounds 52 and 46 showed significant dose-related
1
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response in passive avoidance test. Compound 52 exhibited significant increase in TLT at 10
mg/kg while the similar response was observed at half (5 mg/kg) dose of compound 46.
Further, no significant difference in the mitochondrial membrane potential of the brain in
treated animals was observed compared with control. In the histopathology studies of the
brain of scopolamine treated animals revealed the complete absence of neurodegenerative
lesson in compound treated and control group.
4
4
. METHOD
.1 Chemistry
All the reagents were purchased from the Sigma-Aldrich, Avera, Spectrochem and Alfa
Aesar. The progress of the reactions was monitored by thin layer chromatography (TLC) on
precoated silica gel 60 F254 plates (Merck KGaA) and ultraviolet light (254 nm) or iodine
vapors were used for visualization of spots. Silica gel (60-120 mesh size) was used as
adsorbent in column chromatography for the purification of the compounds. Melting points
were determined by using automated melting point apparatus (Bamstead Electrothermal,
1
13
UK). The H NMR and C NMR spectra were recorded on Bruker Advance, 500 MHz
spectrometer in DMSO-d and CDCl . Chemical shift was measured in the ppm (δ) and
6
3
coupling constant (J) was measured in Hz. Mass analysis was performed on LC-MS
Spectrometer Model Q-ToF Micro Waters Mass spectra with EI ion source and FTIR spectra
was recorded on Bruker ALPHA-T (Germany) ATR /FT-IR instrument.
Purity of the compounds was determined by HPLC (Shimadzu LC 20 AD). Isocratic mobile
phase was delivered by quaternary pumps with flow rate of 1ml/min. The mobile phase
composition was phase A (acetonitrile) and phase B (methanol) in ratio of 1:9. 20 µL samples
were injected into the HPLC column. SPD10AVP detector was used at 310 nm for the
detection of the compounds. CLC C column (5µ, 25 cm x 4.6mm i.d, with CLC ODS, 4cm
1
8
1
5

ACCEPTED MANUSCRIPT
x 4.6 mm. i.d as guard column) was used for purification. The purity of the compounds was
found to be above 98%.
4
.1.1 General procedure for the synthesis of substituted sulfonamides using different
aminoacids (14-21 and 22-35). L-isomers of different amino acids were used for the
1
synthesis and in H NMR spectra it was found that only one isomer was formed indicating
that there is retention in configuration. The substituted aromatic or aliphatic sulfonylchlorides
(1-13, 58, 59) (0.0143 mol, 1 equivalent) were dissolved in the acetone (2 ml). The mixture
was added to water (2 ml) containing glycine/phenylglycine/phenylalanine (0.0143 mol, 1
equivalent) and sodiumbicarbonate (0.0429 mol, 3 equivalent). The final reaction mixture
was stirred at room temperature (rt) for 24 h. The reaction was monitored by TLC using
ethylacetate and methanol (8:2) as the mobile phase. The acetone was evaporated under
reduced pressure and reaction mixture was neutralized with 1N HCl to get the product. The
product was filtered and recrystallized with methanol to get the pure compound[34].
2
-((4-Hydroxyphenyl)sulfonamido)-2-phenylacetic acid (22): It was synthesized as per
general procedure earlier described section (4.1.1) using 4-hydroxybenzenesulfonyl chloride
-
(9) (2.8 g) to get compound 22 as white powder, yield 80%; mp 134-136 ºC; FTIR (KBr) cm
1
1
:
3480 (phenolic-OH-str.), 3400 (-NH-str.), 2400-2800 (-COOH-str.), 1161 (SO -str.); H
2
NMR (500 MHz, DMSO-d ) δ 9.70 (s, 1H, -COOH), 7.81-7.76 (m, 2H, hydroxyphenyl-C ,
6
2
C ), 7.68-7.62 (m, 2H, hydroxyphenyl-C , C ), 7.40-7.25(m, 4H, phenyl-C , C , C , C ), 7.24-
6
3
5
2
3
5
6
1
3
7
.18(m, 1H, phenyl-C ), 5.22 (s, 1H, -OH), 4.82 (s, 1H, -CH), 4.31 (s, 1H, -NH); C NMR
4
(
125 MHz, DMSO-d ) δ 175.12 (-COOH), 161.42 (hydroxyphenyl-C ), 137.16 (phenyl-C ),
6
4
1
1
32.14 (hydroxyphenyl-C ), 128.41 (hydroxyphenyl-C , C , phenyl-C ), 127.70 (phenyl-C ,
1 2 6 4 2
C ), 127.11 (hydroxyphenyl-C , C ), 118.14 (phenyl-C , C ), 64.41 (-CH); MS (ESI): m/z
6
3
5
3
5
+
found 307.9[M ]calculated for C H NO S 307.0.
1
4
13
5
1
6

ACCEPTED MANUSCRIPT
((3-Hydroxyphenyl)sulfonyl)phenylalanine (30): Synthesized as per general procedure
described earlier section (4.1.1) using 3-hydroxybenzenesulfonyl chloride (11) (4.5 g) to get
-1
compound 30 as white powder, yield 73%; mp 162-163 ºC; FTIR (KBr) cm : 3474
1
(
phenolic-OH-str.), 3409 (-NH-str.), 2432-2842 (-COOH-str.), 1151 (SO -str.); H NMR (500
2
MHz, DMSO-d ) δ 9.51 (s, 1H, COOH), 7.39-7.38 (m, 2H, hydroxyphenyl-C , C ), 7.37 (t, J
6
2
6
=
5.5, 1H, hydroxyphenyl-C ), 7.35-7.33 (m, 1H, hydroxyphenyl-C ), 7.31 (t, J = 6.2, 2H,
5 4
phenyl-C , C ), 7.27 (d, J = 10.0, 2H, phenyl-C , C ), 7.22 (t, J = 3.3, 1H, phenyl-C ), 4.51 (t,
3
5
2
6
4
1
3
J = 4.0, 1H, -CH), 3.8 (s, 1H, -NH), 3.31 (s, 1H, -OH), 2.84 (d, J = 0.9, 2H, -CH ); C NMR
2
(125 MHz, DMSO-d ) δ 174.14 (-COOH), 158.47 (hydroxyphenyl-C ), 139.88
6 1
(
hydroxyphenyl-C ), 136.99 (phenyl-C ), 129.14 (hydroxyphenyl-C ), 128.84 (phenyl-C ,
3
1
5
2
C ), 128.17 (phenyl-C , C ), 127.54 (hydroxyphenyl-C ), 123.49 (phenyl-C ), 118.96
6
3
5
4
4
(
hydroxyphenyl-C ), 116.00 (hydroxyphenyl-C ), 58.14 (-CH), 38.14 (-CH ); MS (ESI): m/z
6 2 2
+
found 321.7[M ]calculated for C H NO S 321.0.
1
5
15
5
((3-Bromophenyl)sulfonyl)phenylalanine (34): Synthesized as per the earlier mentioned
general procedure (section 4.1.1) using 3-bromobenzenesulfonyl chloride (13) (5 g) to get
-1
compound 34 as brownish crystal, yield 82%; mp 151-153 ºC; FTIR (KBr) cm : 3441 (-NH-
1
str.), 2441-2814 (-COOH-str.), 1142 (SO -str.); H NMR (500 MHz, DMSO-d ) δ 9.61 (s,
2
6
1
H, COOH), 8.11 (s, 1H, bromophenyl-C ), 7.76 (d, J = 10.3, 1H, bromophenyl-C ), 7.64 (d,
2 6
J = 2.9, 1H, bromophenyl-C ), 7.20 (t, J = 5.8, 1H, bromophenyl-C ), 7.16 (t, J = 6.0, 2H,
4
5
phenyl-C , C ), 7.14-7.7 (m, 3H, pheny-C , C , C ), 5.41 (s, 1H, -NH), 4.47 (t, J = 4.3, 1H, -
3
5
2
4
6
1
3
CH), 2.86 (d, J = 1.2, 2H, -CH ); C NMR (125 MHz, DMSO-d ) δ 175.19 (bromophenyl-
2
6
C ), 142.14 (bromophenyl-C ), 137.51 (bromophenyl-C ), 135.25 (bromophenyl-C ), 131.15
1
3
2
4
(
phenyl-C ), 130.84 (bromophenyl-C ), 129.39 (phenyl-C , C ), 129.13 (phenyl-C , C ),
1 5 2 6 3 5
1
27.11 (phenyl-C ), 124.25 (bromophenyl-C ), 60.01 (-CH), 36.27 (-CH ); MS (ESI): m/z
4 6 2
+
found 383.3 [M ] calculated for C H BrNO S 382.9.
1
5
14
4
1
7

ACCEPTED MANUSCRIPT
4
.1.2 General procedure for synthesis of 1,4-Bis(substituted-phenylsulfonyl)3, 6
diphenyl/benzyl piperazine-2,5-dione derivatives (37-43 and 62, 63). Compounds 14-35, 60
and 61(1.53 mmol, 1 equivalent) were treated with ethylene glycol (12.24 mmol, 8
equivalent) and heated up to 100 ºC to get the clear solution[17]. The reaction mixture was
refluxed at 120-150 ºC for 2-4 h. The final mixture was cooled and poured on crushed ice to
get the solid product. The product was purified by column chromatography using 30% ethyl
acetate in hexane as solvent over Silica gel (60-120 mesh).
1
,4-Bis(phenylsulfonyl)piperazine-2,5-dione (36): Synthesized as per general procedure
described in section (4.1.2) using (phenylsulfonyl)glycine (20) (0.33 g) to get compound 36
as white powder (reported by Koves G et al.,)[35], yield 40%; mp 293-294 ºC; FTIR (KBr)
-1
1
cm : 3030 (Ar-str.), 1641 (C=O-str.), 1162 (SO -str.); H NMR (500 MHz, DMSO-d ) δ
2
6
7
.71(d, J = 8.5. 4H, phenyl-C , C and C , C ), 7.27-7.26 (m, 6H, phenyl-C , C , C and C ,
2 6 2’ 6’ 3 4 5 3’
13
C4’, C ), 4.24 (s, 4H, piperazine-C , C ); C NMR (125 MHz, DMSO-d ) δ 157.71
5’
3
6
6
(
piperazine-C , C ), 137.52 (phenyl-C and C ), 133.31 (phenyl-C , C and C C ), 128.41
2 5 1 1’ 2 6 2’ 6’
(
phenyl-C , C , C and C , C , C ), 48.14 (piperazine-C , C ); MS (ESI): m/z found 394.26
3 4 5 3’ 4’ 5’ 3 6
+
[
M ]; calculated for C H N O S 394.42.
16 14 2 6 2
1
,4-Ditosylpiperazine-2,5-dione (37): It was synthesized as per general procedure described
earlier in section (4.1.2) using tosylglycine (14) (0.35 g) to get compound 37 as white powder
-1
(
reported by Berse C et al.,)[36], yield 36%; mp 281-283 ºC; FTIR (KBr) cm : 3102 (Ar-
1
str.), 1630 (C=O-str.), 1161 (SO -str.); H NMR (500 MHz, DMSO-d ) δ 7.71-7.69 (m, 4H,
2
6
ditosyl-C -C and C -C ), 7.64-7.61 (m, 4H, ditosyl-C -C and C -C ), 4.40 (s, 4H,
2
6
2’ 6’
3
5
3’ 5’
13
piperazine-C , C ), 2.30 (s, 6H, -CH , -CH ); C NMR (125 MHz, DMSO-d ) δ 159.64
3
6
3
3
6
(
piperazine-C , C ), 143.57(ditosyl-C and C ), 139.67(ditosyl-C , C and C , C ),
2 5 1 1’ 2 6 2’ 6’
1
2
31.41(ditosyl-C , C and C , C ), 127.97(ditosyl-C and C ), 48.47 (piperazine-C , C ),
3 5 3’ 5’ 4 4’ 3 6
+
2.41 (phenyl-CH ); MS (ESI): m/z found 422.14 [M ]; calculated for C H N O S 422.47.
3
18 18
2
6 2
1
8

ACCEPTED MANUSCRIPT
1
,4-Bis((4-chlorophenyl)sulfonyl)piperazine-2,5-dione (38): The compound was synthesized
as per general procedure described in section (4.1.2) using ((4-chlorophenyl)sulfonyl)glycine
15) (0.38 g) to get compound 38 as white crystal, yield 37%; mp 162-165 ºC; FTIR (KBr)
(
-1
1
cm : 2965 (Ar-str.), 1641 (C=O-str.), 1120 (SO -str.); H NMR (500 MHz, DMSO-d ) δ 7.80
2
6
(
d, J = 8.5, 4H, bis-chlorophenyl-C , C and C C ), 7.72 (d, J = 7.7.0, 4H bis-chlorophenyl-
2 6 2’, 6’
1
3
C , C and C C ), 4.05 (s, 4H, piperazine-C , C ); C NMR (125 MHz, DMSO-d ) δ
3
5
3’,
5’
3
6
6
1
58.71(piperazine-C , C ), 142.15 (bis-chlorophenyl-C , and C ), 139.45 (bis-chlorophenyl-
2 5 4 4’
C , and C ), 130.74 (bis-chlorophenyl-C , C and C , C ), 129.44 (bis-chlorophenyl-C , C
5
1
1’
2
6
2’
6’
3
+
+2
and C , C ), 48.10 (piperazine-C , C ); MS (ESI): m/z found 463.4, 465.2, 467.8 [M , M ,
3
’
5’
3
6
+
4
M (9:6:1)]; calculated for C H Cl N O S 463.3.
1
6
12
2
2
6 2
1
,4-Bis((3-chlorophenyl)sulfonyl)piperazine-2,5-dione (39): It was synthesized as per general
procedure described in section (4.1.2) using ((3-chlorophenyl)sulfonyl)glycine (16) (0.38 g)
-1
to get compound 39 as white crystal, yield 35%; mp 141-142 ºC; FTIR (KBr) cm : 2962 (Ar-
1
str.), 1652 (C=O-str.), 1132 (SO -str.); H NMR (500 MHz, DMSO-d ) δ 8.03 (s, 2H, bis-
2
6
chlorophenyl-C C ), 7.89-7.75 (m, 2H, bis-chlorophenyl-C C ), 7.68-7.55 (m, 2H, bis-
2
,
2’
6,
6’
chlorophenyl-C C ), 7.47-7.43 (m, 2H, bis-chlorophenyl-C C ), 4.51 (s, 4H, piperazine-C ,
4
,
4’
5, 5’
3
13
C ); C NMR (125 MHz, DMSO-d ) δ 159.41 (piperazine-C , C ), 139.71 (bis-chlorophenyl-
6
6
2
5
C , and C ), 132.51 (bis-chlorophenyl-C , and C ), 131.95 (bis-chlorophenyl-C and C ),
3
3’
1
1’
2
2’
1
30.15 (bis-chlorophenyl-C , and C ), 129.31 (bis-chlorophenyl-C , and C ), 127.47 (bis-
4 4’ 6 6’
chlorophenyl-C , and C ), 49.15 (piperazine-C , C ); MS (ESI): m/z found 463.2, 465.4,
5
5’
3
6
+
+2
+4
4
67.1 [M , M , M (9:6:1)]; calculated for C H Cl N O S 463.3.
16 12 2 2 6 2
1
,4-Bis((4-bromophenyl)sulfonyl)piperazine-2,5-dione (40): This compound was also
synthesized as per general procedure mentioned in section (4.1.2) using ((4-
bromophenyl)sulfonyl)glycine (17) (0.45 g) to get compound 40 as pale brown crystal, yield
-1
1
3
5%; mp 161-164 ºC; FTIR (KBr) cm : 3014 (Ar-str.), 1641 (C=O-str.), 1142 (SO -str.); H
2
1
9

ACCEPTED MANUSCRIPT
NMR (500 MHz, DMSO-d ) δ 7.74 (d, J = 8.7, 4H, bis-bromophenyl-C , C and C ’, C ’),
6
2
6
2
6
7
.64 (d, J = 8.0, 4H bis-bromophenyl-C , C and C ’, C ’), 4.00 (s, 4H, piperazine-C , C );
3 5 3 5 3 6
1
3
C NMR (125 MHz, DMSO-d ) δ 159.51 (piperazine-C , C ), 138.84 (bis-bromophenyl-C ,
6
2
5
4
and C ), 132.10 (bis-bromophenyl-C , and C ), 130.24 (bis-bromophenyl-C , C and C ,
4
’
1
1’
2
6
2’
C ), 129.61 (bis-bromophenyl-C , C and C , C ), 49.27 (piperazine-C , C ); MS (ESI): m/z
6
’
3
5
3’
5’
3
6
+
+2
+4
found 551.4, 553.25, 555.1 [M , M , M (1:2:1)]; calculated for C H Br N O S 551.8.
1
6
12
2
2
6 2
1
,4-Bis((4-methoxyphenyl)sulfonyl)piperazine-2,5-dione (41): Synthesized as per general
procedure described earlier section (4.1.2) using ((4-methoxyphenyl)sulfonyl)glycine (18)
-1
(
0.37 g) to get compound 41 as white powder, yield 31%; mp 162-165 ºC; FTIR (KBr) cm :
1
3
042 (Ar-str.), 1645 (C=O-str.), 1127 (SO -str.); H NMR (500 MHz, DMSO-d ) δ 7.76-7.74
2
6
(
m, 4H, bis-methoxyphenyl-C -C and C -C ), 7.68-7.65 (m, 4H, bis-methoxyphenyl-C -C
2 6 2’ 6’ 3 5
1
3
and C -C ), 4.42 (s, 4H, piperazine-C , C ), 2.80 (s, 6H, -OCH , -OCH ); C NMR (125
3
’
5’
3
6
3
3
MHz, DMSO-d ) δ 162.84 (piperazine-C , C ), 157.51 (bis-methoxyphenyl-C , and C ),
6
2
5
4
4’
1
30.21 (bis-methoxyphenyl-C , and C ), 129.10 (bis-methoxyphenyl-C , C and C C ),
1 1’ 2 6 2’ 6’
1
12.64 (bis-methoxyphenyl-C , C and C C ), 55.41 (-OCH ), 47.27 (piperazine-C , C );
3
5
3’
5’
3
3
6
+
MS (ESI): m/z found 454.8 [M ]; calculated for C H N O S 454.4.
1
8
18
2
8 2
1
,4-Bis((4-aminophenyl)sulfonyl)piperazine-2,5-dione (42): Synthesized as per general
procedure mentioned in section (4.1.2) using ((4-aminophenyl)sulfonyl)glycine (19) (0.35 g)
-1
to get compound 42 as light brown powder, yield 34%; mp 155-157 ºC; FTIR (KBr) cm :
1
3
331, 3291 (NH -str.), 2957 (Ar-str.), 1651 (C=O-str.), 1131 (SO -str.); H NMR (500 MHz,
2
2
DMSO-d ) δ 7.54 (d, J = 8.5, 4H, bis-aminophenyl-C -C and C -C ), 7.38 (d, J = 7.8, 4H,
6
2
6
2’ 6’
bis-aminophenyl-C -C and C -C ), 4.20 (s, 4H, piperazine-C , C ), 1.50 (s, 4H, -NH , -
3
5
3’ 5’
3
6
2
1
3
NH2); C NMR (125 MHz, DMSO-d ) δ 159.74 (piperazine-C , C ), 154.34 (bis-
6
2
5
aminophenyl-C , and C ), 130.14 (bis-aminophenyl-C , and C ), 126.94 (bis-aminophenyl-
4
4’
1
1’
2
0

ACCEPTED MANUSCRIPT
C , C and C , C ), 115.24 (bis-aminophenyl-C , C and C , C ), 48.41 (piperazine-C , C );
2
6
2’
6’
3
5
3’
5’
3
6
+
MS (ESI): m/z found 424.2 [M ]; calculated for C H N O S 424.4.
1
6
16
4
6 2
1
,4-Bis((4-fluorophenyl)sulfonyl)piperazine-2,5-dione (43): Synthesized as per general
procedure described in section (4.1.2) using ((4-fluorophenyl)sulfonyl)glycine (21) (0.36 g)
-1
to get compound 43 as white crystal, yield 36%; mp 182-184 ºC; FTIR (KBr) cm : 3014 (Ar-
1
str.), 1625 (C=O-str.), 1135 (SO -str.); H NMR (500 MHz, DMSO-d ) δ 8.10 (d, J = 8.3, 4H
2
6
bis-chlorophenyl-C , C and C C ), 7.80 (d, J = 7.3, 4H, bis-fluorophenyl-C , C and C
3’,
2
6
2’, 6’
3
5
13
C ), 4.20 (s, 4H, piperazine-C , C ); C NMR (125 MHz, DMSO-d ) δ 175.21 (piperazine-
5
’
3
6
6
C , C ), 159.61 (fluorophenyl-C and C ), 136.14 (fluorophenyl-C and C ), 132.44
2
5
4
4’
1
1’
(
fluorophenyl-C , C and C , C ), 118.57 (fluorophenyl-C , C and C , C ), 49.41
2 6 2’ 6’ 3 5 3’ 5’
+
(
piperazine-C , C ); MS (ESI): m/z found 430.2 [M ]; calculated for C H F N O S 430.4.
3 6 16 12 2 2 6 2
1
,4-Bis((4-hydroxyphenyl)sulfonyl)-3,6-diphenylpiperazine-2,5-dione (44): It was also
synthesized as per general procedure mentioned in section (4.1.2) using 2-((4-
hydroxyphenyl)sulfonamido)-2-phenylacetic acid (22) (0.50 g) to get compound 44 as white
-1
powder, yield 33%; mp 185-187 ºC; FTIR (KBr) cm : 3379 (-OH-str.), 2895 (Ar-str.), 1662
1
(
C=O-str.), 1169 (SO -str.); H NMR (500 MHz, DMSO-d ) δ 7.79-7.77 (m, 4H, bis-
2
6
hydroxyphenyl-C , C and C C ), 7.72-7.56 (m, 4H, bis-hydroxyphenyl-C , C and C C ),
2
6
2’, 6’
3
5
3’, 5’
1
3
7
.48-7.06 (m, 10H, dipheny), 5.30 (s, 2H, piperazine-C , C ), 4.18 (s, 2H, -OH, -OH); C
3 6
NMR (125 MHz, DMSO-d ) δ 164.71(piperazine-C , C ), 162.51(bis-hydroxyphenyl-C , and
6
2
5
4
C ), 136.19 (bis-hydroxyphenyl-C , and C ), 129.71(bis-hydroxyphenyl-C , and C ),
4
’
1
1’
2
6’
1
28.91(bis- hydroxyphenyl-C , and C ), 128.31(dibenzyl-C and C ), 127.84 (dibenzyl-C ,
3 5’ 1 1’ 2
C and C , C ), 127.51(dibenzyl-C , C , C and C , C ,C ), 117.81(dibenzyl-C and C ),
6
2’
6’
3
4
5
3’
4’ 5’
4
4’
+
6
5
9.10 (piperazine-C , C ); MS (ESI): m/z found 578.5 [M ]; calculated for C H N O S
3
6
28 22
2
8
2
78.6.
2
1

ACCEPTED MANUSCRIPT
1
,4-Bis((4-aminophenyl)sulfonyl)-3,6-diphenylpiperazine-2,5-dione (45): Synthesized as per
general procedure mentioned in section (4.1.2) using 2-((4-aminophenyl)sulfonamido)-2-
phenylacetic acid (27) (0.45 g) to get compound 45 as light brown powder, yield 33%; mp
-1
1
74-175 ºC; FTIR (KBr) cm : 3321, 3284 (NH -str.), 2937 (Ar-str.), 1651 (C=O-str.), 1120
2
1
(
SO -str.); H NMR (500 MHz, DMSO-d ) δ 7.74-7.71 (m, 4H, bis-aminophenyl-C , C and
2
6
2
6
C2’, C ), 7.62-7.45 (m, 4H, bis-aminophenyl-C , C and C C ), 7.39-6.80 (m, 10H,
6’
3
5
3’,
5’
1
3
dipheny), 5.21 (s, 2H, piperazine-C , C ), 4.10 (s, 4H, -NH ); C NMR (125 MHz, DMSO-
3
6
2
d ) δ 164.75 (piperazine-C , C ), 154.47 (bis-aminophenyl-C , and C ), 136.01 (bis-
6
2
5
4
4’
aminophenyl-C , and C ), 131.64 (bis-aminophenyl-C , C and C , C ), 130.84 (bis-
1
1’
2
6
2’
6’
aminophenyl-C , C and C , C ), 129.87 (dibenzyl-C and C ), 128.57 (dibenzyl-C , C and
3
5
3’
5’
1
1’
2
6
C , C ), 126.37 (dibenzyl-C , C and C , C ), 115.14 (dibenzyl-C and C ), 69.47
2
’
6’
3
5
3’
5’
4
4’
+
(
piperazine-C , C ); MS (ESI): m/z found 576.2 [M ]; calculated for C H N O S 576.6.
3
6
28 24
4
6
2
1
,4-Bis((4-chlorophenyl)sulfonyl)-3,6-diphenylpiperazine-2,5-dione
(46):
This
was
synthesized as per general procedure described in section (4.1.2) using 2-((4-
chlorophenyl)sulfonamido)-2-phenylacetic acid (26) (0.50 g) to get compound 46 as white
-1
crystal, yield 32%; mp 181-183 ºC; FTIR (KBr) cm : 3014 (Ar-str.), 1647 (C=O-str.), 1124
1
(
SO -str.); H NMR (500 MHz, DMSO-d ) δ 7.82-7.79 (m, 4H, bis-chlorophenyl-C , C and
2
6
2
6
C ’, C ’), 7.78-7.60 (m, 4H, bis-chlorophenyl-C , C and C , C ), 7.35-7.07 (m, 10H,
2
6
3
5
3’
5’
1
3
dipheny), 5.35 (s, 2H, piperazine-C , C ); C NMR (125 MHz, DMSO-d ) δ 169.21
3
6
6
(
piperazine-C , C ), 139.78 (bis-chlorophenyl-C , and C ), 137.97 (bis-chlorophenyl-C , and
2 5 4 4’ 1
C ), 129.68 (bis-chlorophenyl-C , C and C C ), 128.855 (bis-chlorophenyl-C , C and C
3’
1
’
2
6
2’ 6’
3
5
C ), 119.47 (dibenzyl-C and C ), 118.62 (dibenzyl-C - C and C - C ), 67.34 (piperazine-
5
’
1
1’
2
6
2
6’
+
+2
+4
C , C ); MS (ESI): m/z found 615.1, 617.2, 619.4 [M , M , M (9:6:1)]; calculated for
3
6
C H Cl N O S 615.5.
28
20
2
2
6 2
2
2

ACCEPTED MANUSCRIPT
1
,4-Bis((4-nitrophenyl)sulfonyl)-3,6-diphenylpiperazine-2,5-dione (47): Synthesized as per
general procedure described in section (4.1.2) using 2-((4-nitrophenyl)sulfonamido)-2-
phenylacetic acid (23) (0.51 g) to get compound 47 as pale yellow crystal, yield 39%; mp
-1
1
2
24-227 ºC; FTIR (KBr) cm : 3067 (Ar-str.), 1669 (C=O-str.), 1150 (SO -str.); H NMR
2
(
500 MHz, DMSO-d ) δ 8.43 (d, J = 7.2, 4H, bis-nitrophenyl- C , C and C C ), 8.22 (d, J =
6 3 5 3’, 5’
8
.4, 4H, bis-nitrophenyl-C , C and C C ), 7.52-7.08 (m, 10H, dipheny), 5.52 (s, 2H,
2 6 2’, 6’
13
piperazine-C , C ); C NMR (125 MHz, DMSO-d ) δ 165.97 (piperazine-C , C ), 154.12
3
6
6
2
5
(
bis-nitrophenyl-C and C ), 145.02 (bis-nitrophenyl-C , C and C C ), 137.12 (bis-
4 4’ 3 5 3’ 5’
nitrophenyl-C and C ), 130.86 (bis-nitrophenyl-C , C and C C ), 129.27 (dibenzyl-C and
1
1’
2
6
2’ 6’
1
C ), 127.47 (dibenzyl-C , C and C C ), 124.41 (dibenzyl-C , C , C and C , C , C ),
1
’
2
6
2’
6’
3
4
5
3’
4’
5’
+
6
6
3
8.17 (piperazine-C , C ); MS (ESI): m/z found 636.2 [M ]; calculated for C H N O S
3 6 28 20 4 10 2
36.6.
,6-Diphenyl-1,4-ditosylpiperazine-2,5-dione (48): Synthesized as per general procedure
mentioned in section (4.1.2) using 2-((4-methylphenyl)sulfonamido)-2-phenylacetic acid (28)
-1
(
0.47 g) to get compound 48 as white powder, yield 38%; mp 138-140 ºC; FTIR (KBr) cm :
1
3
142 (Ar-str.), 1652(C=O-str.), 1142 (SO -str.); H NMR (500 MHz, DMSO-d ) δ 7.74 (d, J
2
6
=
8.0, 4H, ditosyl-C , C and C C ), 7.67 (d, J = 7.6, 4H, ditosyl-C , C and C C ), 7.43-
2 6 2’, 6’ 3 5 3’, 5’
1
3
7
.01 (m, 10H, dipheny), 5.25 (s, 2H, piperazine-C , C ), 2.18 (s, 6H, -CH , -CH ); C NMR
3 6 3 3
(
125 MHz, DMSO-d ) δ 162.57 (piperazine-C , C ), 141.84 (ditosyl-C and C ), 137.72
6 2 5 1 1’
(
ditosyl-C , C and C , C ), 134.24 (ditosyl-C , C and C , C ), 129.84 (ditosyl-C and C ),
2 6 2’ 6’ 3 5 3’ 5’ 4 4’
1
29.10 (dibenzyl-C and C ), 128.31 (dibenzyl-C , C and C , C ), 127.17 (dibenzyl-C , C
1 1’ 2 6 2’ 6’ 3 5
and C , C ), 126.4 (dibenzyl-C and C ), 69.10 (piperazine-C , C ), 20.41 (-CH ); MS
3
’
5’
4
4’
3
6
3
+
(
ESI): m/z found 574.1 [M ]; calculated for C H N O S 574.6.
30 26 2 6 2
3
,6-Diphenyl-1,4-bis(m-tolylsulfonyl)piperazine-2,5-dione (49): Synthesized as per general
procedure earlier described in section (4.1.2) using 2-((3-methylphenyl)sulfonamido)-2-
2
3

ACCEPTED MANUSCRIPT
phenylacetic acid (29) (0.47 g) to get compound 49 as white powder, yield 37%; mp 152-154
-1
1
ºC; FTIR (KBr) cm : 2951 (Ar-str.), 1614 (C=O-str.), 1146 (SO -str.); H NMR (500 MHz,
2
DMSO-d ) δ 7.93 (s, 2H, bis-tolyl-C and C ), 7.79 (d, J = 8.3, 2H, bis-tolyl-C and C ),
6
2,
2’
6,
6’
7
.58-7.33 (m, 4H, bis-tolyl-C C and C C ), 7.23 (d, J = 8.0, 4H, dipheny-C , C and C ,
4, 5 4’ 5’ 2 6 2’
C ), 7.20-6.53 (m, 6H, dipheny-C , C , C and C , C , C ), 4.24 (s, 2H, piperazine-C , C ),
6
’
3
4
5
3’
4’
5’
3
6
1
3
2
1
.00 (s, 6H, -CH , -CH ); C NMR (125 MHz, DMSO-d ) δ 161.24 (piperazine-C , C ),
3 3 6 2 5
37.61 (ditosyl-C and C ), 135.41 (ditosyl-C and C ), 133.41 (ditosyl-C and C ), 129.31
1
1’
6
6’
2
2’
(ditosyl-C and C ), 128.71 (ditosyl-C and C ), 127.74 (ditosyl-C and C ), 127.31
5 5’ 4 4’ 3 3’
(
dibenzyl-C and C ), 126.74 (dibenzyl-C , C and C , C ), 125.46 (dibenzyl-C , C and C ,
1
1’
2
6
2’
6’
3
5
3’
C ), 123.71 (dibenzyl-C and C ), 69.17 (piperazine-C , C ), 21.41 (-CH ); MS (ESI): m/z
5
’
4
4’
3
6
3
+
found 574.2 [M ]; calculated for C H N O S 574.6.
3
0
26
2
6 2
1
,4-Bis((4-bromophenyl)sulfonyl)-3,6-diphenylpiperazine-2,5-dione (50): Synthesized as per
general procedure mentioned in section (4.1.2) using 2-((4-bromophenyl)sulfonamido)-2-
phenylacetic acid (24) (0.57 g) to get compound 50 as light brown crystal, yield 31%; mp
-1
1
1
81-184 ºC; FTIR (KBr) cm : 3014 (Ar-str.), 1625 (C=O-str.), 1142 (SO -str.); H NMR
2
(
500 MHz, DMSO-d ) δ 7.79 (d, J = 8.6, 4H, bis-bromophenyl-C , C and C ’, C ’), 7.73 (d,
6 2 6 2 6
J = 7.3, 4H, bis-bromophenyl-C , C and C , C ), 7.47-7.04 (m, 10H, dipheny), 4.75 (s, 2H,
3
5
3’
5’
13
piperazine-C3, C6); C NMR (125 MHz, DMSO-d ) δ 163.66 (piperazine-C , C ), 139.62
6
2
5
(
bromophenyl-C and C ), 135.11 (bromophenyl-C and C ), 132.14 (bromophenyl-C , C
1 1’ 4 4’ 2 6
and C , C ), 131.01 (bromophenyl-C , C and C , C ), 130.77 (dibenzyl-C and C ), 130.42
2
’
6’
3
5
3’
5’
1
1’
(
dibenzyl-C , C and C , C ), 129.35 (dibenzyl-C , C and C , C ), 127.46 (dibenzyl-C and
2 6 2’ 6’ 3 5 3’ 5’ 4
+
+2
C ), 69.17 (piperazine-C , C ); MS (ESI): m/z found 704.1, 706.4, 708.3 [M , M ,
4
’
3
6
+
4
M (1:2:1)]; calculated for C H Br N O S 704.4.
2
8
20
2
2
6 2
1
,4-Bis((3-bromophenyl)sulfonyl)-3,6-diphenylpiperazine-2,5-dione (51): Synthesized as per
general procedure given in section (4.1.2) using 2-((3-bromophenyl)sulfonamido)-2-
2
4

ACCEPTED MANUSCRIPT
phenylacetic acid (25) (0.57 g) to get compound 51 as light brown crystal, yield 30%; mp
-1
1
1
90-192 ºC; FTIR (KBr) cm : 3010 (Ar-str.), 1621 (C=O-str.), 1131 (SO -str.); H NMR
2
(
500 MHz, DMSO-d ) δ 7.96 (s, 2H, bis-bromophenyl-C C ), 7.83 (d, J = 8.1, 2H, bis-
6 2, 2’
bromophenyl-C C ), 7.63 (d, J = 5.2, 2H, bis-bromophenyl-C C ), 7.41 (t, J = 7.5, 2H, bis-
6
,
6’
4, 4’
1
3
bromophenyl-C C ), 7.45-7.01 (m, 10H, dipheny), 4.76 (s, 2H, piperazine-C , C );
C
5
,
5’
3
6
NMR (125 MHz, DMSO-d ) δ 163.47 (piperazine-C , C ), 141.41 (bromophenyl-C and C ),
6
2
5
3
3’
1
36.51 (bromophenyl-C and C ), 135.02 (bromophenyl-C and C ), 132.11(bromophenyl-
1 1’ 2 2’
C and C ), 130.74 (bromophenyl-C and C ), 129.35 (bromophenyl-C and C ), 127.44
6
6’
4
4’
5
5’
(
dibenzyl-C and C ), 125.91 (dibenzyl-C , C and C , C ), 123.87 (dibenzyl-C , C , C and
1 1’ 2 6 2’ 6’ 3 4 5
+
+2
C , C , C ), 69.19 (piperazine-C , C ); MS (ESI): m/z found 704.2, 706.2, 708.7 [M , M ,
3
’
4’
5’
3
6
+
4
M (1:2:1)]; calculated for C H Br N O S 704.4.
2
8
20
2
2
6 2
3
,6-Dibenzyl-1,4-bis((4-chlorophenyl)sulfonyl)piperazine-2,5-dione (52): The compound was
synthesized as per general procedure (mentioned in section 4.1.2) using ((4-
chlorophenyl)sulfonyl)phenylalanine (35) (0.53 g) to get compound 52 as white crystal, yield
-1
1
3
2%; mp 177-180 ºC; FTIR (KBr) cm : 3027 (Ar-str.), 1621 (C=O-str.), 1141 (SO -str.); H
2
NMR (500 MHz, DMSO-d ) δ 7.80-7.78 (m, 4H, bis-chlorophenyl-C , C and C C ), 7.78-
6
2
6
2’, 6’
7
.62 (m, 4H, bis-chlorophenyl-C , C and C C ), 7.45-7.03 (m, 10H, dibenzyl), 4.64-4.63
3 5 3’, 5’
13
(
m, 2H, piperazine-C , C ), 3.00 (dd, J = 6.6, 4H, -CH , -CH ); C NMR (125 MHz, DMSO-
3 6 2 2
d ) δ 164.51 (piperazine-C , C ), 140.75 (bis-chlorophenyl-C , and C ), 138.62 (bis-
6
2
5
4
4’
chlorophenyl-C , and C ), 136.72 (bis-chlorophenyl-C , and C ), 131.64 (bis-chlorophenyl-
1
1’
2
6’
C , and C ), 128.71 (dibenzyl-C , C and C C ), 122.00 (dibenzyl-C and C ), 120.62
3
5’
3
5
3’
5’
1
1’
(
dibenzyl-C -C and C - C ), 61.72 (piperazine-C , C ), 30.05 (benzyl-CH ); MS (ESI): m/z
2 6 2’ 6’ 3 6 2
+
+2
+4
found 643.2, 645.1, 647.4 [M : M : M (9:6:1)]; calculated for C H Cl N O S 643.5.
3
0
24
2
2
6 2
3
,6-Dibenzyl-1,4-bis((4-hydroxyphenyl)sulfonyl)piperazine-2,5-dione (53): Synthesized as
per general procedure mentioned in section (4.1.2) using ((4-
2
5

ACCEPTED MANUSCRIPT
hydroxyphenyl)sulfonyl)phenylalanine (31) (0.49 g) to get compound 53 as white powder,
-1
yield 35%; mp 181-183 ºC; FTIR (KBr) cm : 3450 (-OH-str.), 2981 (Ar-str.), 1651 (C=O-
1
str.), 1142 (SO -str.); H NMR (500 MHz, DMSO-d ) δ 7.76 (d, J = 8.5, 4H, bis-
2
6
hydroxyphenyl-C , C and C C ), 7.70 (d, J = 7.4, 4H, bis-hydroxyphenyl-C , C and C
3’,
2
6
2’, 6’
3
5
C ), 7.38-7.12 (m, 4H, dibenzyl-C , C and C , C ), 7.10-6.80 (m, 6H, dibenzyl-C , C , C
5
5
’
2
6
2’
6’
3
4
and C , C , C ), 4.50 (t, J = 6.3, 2H, piperazine-C , C ), 4.02 (s, 2H, -OH, -OH), 3.08 (d,
3
’
4’
5’
3
6
1
3
4
H, -CH2); C NMR (125 MHz, DMSO-d ) δ 163.51(piperazine-C , C ), 161.79 (bis-
6 2 5
hydroxyphenyl-C , and C ), 137.75 (bis-hydroxyphenyl-C , and C ), 128.47 (bis-
4
4’
1
1’
hydroxyphenyl-C , C and C C ), 129.19 (bis-hydroxyphenyl-C , C and C C ), 128.31
2
6
2’
6’
3
5
3’
5’
(
dibenzyl-C and C ), 127.82 (dibenzyl-C , C and C C ), 127.31 (dibenzyl-C , C and C
1 1’ 2 6 2’ 6’ 3 5 3’
C ), 116.78 (dibenzyl-C and C ), 63.27 (piperazine-C , C ), 37.17 (benzyl-CH ); MS (ESI):
5
’
4
4’
3
6
2
+
m/z found 606.2 [M ]; calculated for C H N O S 606.6.
3
0
26
2
8 2
3
,6-Dibenzyl-1,4-bis((3-hydroxyphenyl)sulfonyl)piperazine-2,5-dione (54): Synthesized as
per general procedure mentioned in section (4.1.2) using ((3-
hydroxyphenyl)sulfonyl)phenylalanine (30) (0.49 g) to get compound 54 as white powder,
-1
yield 31%; mp 175-177 ºC; FTIR (KBr) cm : 3431 (-OH-str.), 3171 (Ar-str.), 1661 (C=O-
1
str.), 1131 (SO -str.); H NMR (500 MHz, DMSO-d ) δ 7.98 (s, 2H, bis-hydroxyphenyl-C
2
6
2,
C ), 7.84 (d, J = 8.0, 2H, bis-hydroxyphenyl-C C ), 7.64 (d, J = 5.5, 2H, bis-
2
’
6,
6’
hydroxyphenyl-C C ), 7.43 (t, J = 7.5, 2H, bis-hydroxyphenyl-C C ), 7.34 (t, J = 7.2, 4H,
4
,
4’
5, 5’
dibenzyl-C , C and C , C ), 7.32-6.76 (m, 6H, dibenzyl-C , C , C and C , C C ), 4.45 (t,
3
5
3’
5’
2
4
6
2’
4’ 6’
13
J = 6.5, 2H, piperazine-C , C ), 3.80 (s, 2H, -OH, -OH), 3.10-3.87 (m, 4H, -CH , -CH ); C
3
6
2
2
NMR (125 MHz, DMSO-d ) δ 162.51 (piperazine-C , C ), 157.71 (bis-hydroxyphenyl-C ,
6
2
5
3
and C ), 137.51 (bis-hydroxyphenyl-C , and C ), 136.54 (bis-hydroxyphenyl-C , and C ),
3
’
1
1’
2
2’
1
29.71(bis-hydroxyphenyl-C , and C ), 128.18 (bis-hydroxyphenyl-C , and C ), 127.21 (bis-
6 6’ 4 4’
hydroxyphenyl-C , and C ), 126.45 (dibenzyl-C and C ), 124.14 (dibenzyl-C , C and C
2’
5
5’
1
1’
2
6
2
6

ACCEPTED MANUSCRIPT
C ), 119.42 (dibenzyl-C , C and C C ), 115.28 (dibenzyl-C and C ), 63.47 (piperazine-
6
’
3
5
3’ 5’
4
4’
+
C , C ), 37.16 (benzyl-CH ); MS (ESI): m/z found 606.1 [M ]; calculated for C H N O S
8 2
3
6
2
30 26
2
6
06.6.
,6-Dibenzyl-1,4-bis((4-nitrophenyl)sulfonyl)piperazine-2,5-dione (55): Synthesized as per
3
general procedure given in section (4.1.2) using ((4-nitrophenyl)sulfonyl)phenylalanine (32)
0.53 g) to get compound 55 as pale yellow crystal, yield 40%; mp 213-214 ºC; FTIR (KBr)
(
-1
1
cm : 3087 (Ar-str.), 1657 (C=O-str.), 1145 (SO -str.); H NMR (500 MHz, DMSO-d ) δ 8.40
2
6
(
d, J = 9.0, 4H, bis-nitrophenyl-C , C and C C ), 8.20 (d, J = 5.1, 4H, bis-nitrophenyl- C ,
2 6 2’, 6’ 3
C and C C ), 7.51 (d, J = 8.4, 4H, dibenzyl-C , C and C , C ), 7.47 (t, J = 7.3, 4H,
5
3’, 5’
2
6
2’
6’
dibenzyl-C , C and C , C ), 7.30 (t, J = 6.8, 2H, dibenzyl-C and C ), 4.68 (t, J = 3.1, 2H,
3
5
3’
5’
4
4’
1
3
piperazine-C , C ), 3.15-3.01 (m, 4H, -CH , -CH ); C NMR (125 MHz, DMSO-d ) δ 165.71
3
6
2
2
6
(
piperazine-C , C ), 154.24 (bis-nitrophenyl-C and C ), 145.15 (bis-nitrophenyl-C , C and
2 5 4 4’ 3 5
C C ), 138.72 (bis-nitrophenyl-C and C ), 129.71 (bis-nitrophenyl-C , C and C C ),
3
’
5’
1
1’
2
6
2’
6’
1
29.08 (dibenzyl-C and C ), 127.72 (dibenzyl-C , C and C C ), 126.17 (dibenzyl-C , C ,
1 1’ 2 6 2’ 6’ 3 4
C and C , C , C ), 63.75 (piperazine-C , C ), 37.14 (benzyl-CH ); MS (ESI): m/z found
5
3’
4’
5’
3
6
2
+
6
64.4 [M ]; calculated for C H N O S 664.6.
30 24 4 10 2
3
,6-Dibenzyl-1,4-bis((4-bromophenyl)sulfonyl)piperazine-2,5-dione (56): Synthesized as per
procedure earlier mentioned in section (4.1.2) using ((4-
bromophenyl)sulfonyl)phenylalanine (33) (0.59 g) to get compound 56 as brownish crystal,
general
-1
yield 32%; mp 167-169 ºC; FTIR (KBr) cm : 3015 (Ar-str.), 1661 (C=O-str.), 1109 (SO -
2
1
str.); H NMR (500 MHz, DMSO-d ) δ 7.79 (d, J = 8.5, 4H, bis-bromophenyl-C , C and C
6
2
6
2’,
C ), 7.78 (d, J = 7.5, 4H, bis-bromophenyl-C , C and C C ), 7.35 (d, J = 8.6, 4H, dibenzyl-
6
’
3
5
3’, 5’
C , C and C , C ), 7.33-7.05 (m, 6H, dibenzyl-C , C , C and C , C , C ), 4.44 (t, J = 3.5,
2
6
2’
6’
3
4
5
2’
4’
5’
1
3
2
H, piperazine-C , C ), 3.01-2.90 (m, 4H, -CH , -CH ); C NMR (125 MHz, DMSO-d ) δ
3 6 2 2 6
1
63.74 (piperazine-C , C ), 139.71 (bis-bromophenyl-C and C ), 137.58 (bis-bromophenyl-
2
5
4
4’
2
7

ACCEPTED MANUSCRIPT
C and C ), 132.21(bis-bromophenyl-C , C and C , C ), 131.10 (bis-bromophenyl-C , C
5
1
1’
2
6
2’
6’
3
and C , C ), 130.37 (dibenzyl-C and C ), 129.75 (dibenzyl-C , C and C C ), 128.97
3
’
5’
1
1’
2
6
2’
6’
(dibenzyl-C , C and C , C ), 127.61 (dibenzyl-C and C ), 62.31 (piperazine-C , C ), 37.47
3 5 3’ 5’ 4 4’ 3 6
+
+2
+4
(
benzyl-CH ); MS (ESI): m/z found 732.3, 734.2, 736.2 [M , M , M (1:2:1)]; calculated
2
for C H Br N O S 732.4.
3
0
24
2
2
6 2
3
,6-Dibenzyl-1,4-bis((3-bromophenyl)sulfonyl)piperazine-2,5-dione (57): Synthesized as per
general procedure mentioned in section (4.1.2) using ((3-
bromophenyl)sulfonyl)phenylalanine (34) (0.59 g) to get compound 57 as brownish crystal,
-1
yield 31%; mp 152-154 ºC; FTIR (KBr) cm : 2984 (Ar-str.), 1645 (C=O-str.), 1114 (SO -
2
1
str.); H NMR (500 MHz, DMSO-d ) δ 7.99 (s, 2H, bis-bromophenyl-C and C ), 7.86 (s,
6
2,
2’
2
2
H, bis-bromophenyl-C C ), 7.66 (d, J = 6.5, 2H, bis-bromophenyl-C C ), 7.45 (t, J = 7.7,
6, 6’ 4, 4’
H, bis-bromophenyl-C C ), 7.36 (d, J = 8.0, 4H, dibenzyl-C , C and C , C ), 7.33-7.30
5
,
5’
2
6
2’
6’
(
m, 6H, dibenzyl-C , C , C and C , C C ), 4.48 (t, J = 6.0, 2H, piperazine-C , C ), 3.10-
3 4 5 3’ 4’ 5’ 3 6
1
3
3
1
.00 (m, 4H, -CH , -CH ); C NMR (125 MHz, DMSO-d ) δ 163.74 (piperazine-C , C ),
2 2 6 2 5
41.51 (bis-bromophenyl-C and C ), 137.74 (bis-bromophenyl-C and C ), 136.61 (bis-
3
3’
1
1’
bromophenyl-C and C ), 132.10 (bis-bromophenyl-C and C ), 130.41(bis-bromophenyl-C
4
2
2’
6
6’
and C ), 129.76 (bis-bromophenyl-C and C ), 129.21 (dibenzyl-C and C ), 127.52
4
’
5
5’
1
1’
(
dibenzyl-C , C and C , C ), 125.14 (dibenzyl-C , C and C , C ), 123.71 (dibenzyl-C and
2 6 2’ 6’ 3 5 3’ 5’ 4
C ), 62.24 (piperazine-C , C ), 37.21 (benzyl-CH ); MS (ESI): m/z found 732.1, 734.2, 736.3
4
’
3
6
2
+
+2
+4
[
M , M , M (1:2:1)]; calculated for C H Br N O S 732.4.
30 24 2 2 6 2
1
,4-Bis(butylsulfonyl)piperazine-2,5-dione (62): Synthesized as per general procedure given
in section (4.1.2) using (butylsulfonyl)glycine (60) (0.30 g) to get compound 62 as brownish
-1
1
powder, yield 40 %; mp 124-126 ºC; FTIR (KBr) cm : 1594 (C=O-str.), 1078 (SO -str.); H
2
NMR (500 MHz, DMSO-d ) δ 4.49 (s, 4H, piperazine-C , C ), 4.41 (t, J = 6.9, 4H, -CH ),
6
3
6
2
13
3
.61 (m, 4H, -CH , -CH ), 3.00 (m, 4H, CH ), 1.01 (t, J = 7.2, 6H, CH ); C NMR (125
2
2
2
3
2
8