Identification of new anti-inflammatory agents based on nitrosporeusine natural products of marine origin

Article abstract of DOI:10.1016/j.ejmech.2017.04.015

Nitrosporeusines A and B are two recently isolated marine natural products with novel skeleton and exceptional biological profile. Interesting antiviral activity of nitrosporeusines and promising potential in curing various diseases, evident from positive data from various animal models, led us to investigate their anti-inflammatory potential. Accordingly, we planned and synthesized nitrosporeusines A and B in racemic as well as enantiopure forms. The natural product synthesis was followed by preparation of several analogues, and all the synthesized compounds were evaluated for in vitro and in vivo anti-inflammatory potential. Among them, compounds 25, 29 and 40 significantly reduced levels of nitric oxide (NO), reactive oxygen species (ROS) and pro-inflammatory cytokines. In addition, these compounds suppressed several pro-inflammatory mediators including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-κB (NF-κB), and thereby can be emerged as potent anti-inflammatory compounds. Furthermore, all possible isomers of lead compound 25 were synthesized, characterized and profiled in same set of assays and found that one of the enantiomer (?)-25a was superior among them.

Full text of DOI:10.1016/j.ejmech.2017.04.015

Accepted Manuscript
Identification of new anti-inflammatory agents based on nitrosporeusine natural
products of marine origin
Satish Chandra Philkhana, Abhishek Kumar Verma, Gorakhnath R. Jachak,
Bibhabasu Hazra, Anirban Basu, D. Srinivasa Reddy
PII:
S0223-5234(17)30270-2
10.1016/j.ejmech.2017.04.015
EJMECH 9362
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 December 2016
Revised Date: 5 April 2017
Accepted Date: 7 April 2017
Please cite this article as: S.C. Philkhana, A.K. Verma, G.R. Jachak, B. Hazra, A. Basu, D.S. Reddy,
Identification of new anti-inflammatory agents based on nitrosporeusine natural products of marine
origin, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.015.
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Graphical abstract

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Identification of new anti-inflammatory agents based on
nitrosporeusine natural products of marine origin
Satish Chandra Philkhana, ab† Abhishek Kumar Verma,c† Gorakhnath R. Jachak, ab Bibhabasu
Hazra,c Anirban Basu*cand D. Srinivasa Reddy*ab
a. CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India
b. Academy of Scientific and Innovative Research (AcSIR), New Delhi, India.
c. National Brain Research Centre, NH-8, Manesar, Gurgaon, Haryana-122051, India
*Corresponding authors: Dr. D. Srinivasa Reddy ds.reddy@ncl.res.in
anirban@nbrc.ac.in
; Dr. Anirban Basu
ABSTRACT
Nitrosporeusines A and B are two recently isolated marine natural products with novel skeleton
and exceptional biological profile. Interesting antiviral activity of nitrosporeusines and promising
potential in curing various diseases, evident from positive data from various animal models, led
us to investigate their anti-inflammatory potential. Accordingly, we planned and synthesized
nitrosporeusines A and B in racemic as well as enantiopure forms. The natural product synthesis
was followed by preparation of several analogues, and all the synthesized compounds were
evaluated for in vitro and in vivo anti-inflammatory potential. Among them, compounds 25, 29
and 40 significantly reduced levels of nitric oxide (NO), reactive oxygen species (ROS) and pro-
inflammatory cytokines. In addition, these compounds suppressed several pro-inflammatory
mediators including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear
factor-κB (NF-κB), and thereby can be emerged as potent anti-inflammatory compounds.
Furthermore, all possible isomers of lead compound 25 were synthesized, characterized and
profiled in same set of assays and found that one of the enantiomer (-)-25a was superior among
them.
Keywords. Inflammation, pro-inflammatory factors, nitrosporeusines, maleimycin, Amano PS
lipase, enzymatic kinetic resolution

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1. Introduction
Inflammation is a biological response to harmful stimuli such as pathogens that cause tissue and
cell damage.1 It is considered as a defensive measure taken by the organism to eliminate noxious
stimuli and to begin the healing process. It is classified as either acute or chronic, depending on
whether it involves a short response or a prolonged one, respectively.2 During an inflammatory
response, mediators, such as pro-inflammatory cytokines (e.g., interleukin-1β (IL-1β), IL-6, IL-
12, and the chemokine IL-8), tumor necrosis factors (e.g., TNF-α and TNF-β), interferons (e.g.,
IFN-γ), eicosanoids (e.g., prostaglandins and leukotrienes) and vasoactive amines (e.g.,
histamine) are released.3 The transcription factor nuclear factor-κB (NF-κB), plays a central role
in the inflammatory response by regulating the expression of various genes encoding pro-
inflammatory cytokines, adhesion molecules, chemokines, growth factors, and inducible
enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS).4 In
spite of the fact that inflammation is primarily a protective response, the chronic and
uncontrolled inflammation becomes detrimental to tissues.5 The inferences of the chronic
inflammation in the pathogenesis of arthritis, cancer, cardiovascular, autoimmune as well as viral
infections have made it a serious medical issue.6 Therefore, research has been directed in recent
years to develop safer and potent anti-inflammatory drugs to attenuate the severity of
inflammation.6,7 As the human immune system is a complex process involving many factors and
can go awry many times, it is very challenging to develop novel efficient chemical entities for
treating inflammation.8
Figure 1: Natural products nitrosporeusines A (1) and B (2)

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As a part of our continuous interest9 in search of biologically active natural molecules with anti-
inflammatory activity, we have come across a novel class of compounds with
benzenecarbothioccyclopenta[c]pyrrole-1,3-dione scaffold - nitrosporeusines. Nitrosporeusines
A (1) and B (2) are two new marine natural products having unique skeleton isolated10 by Lin
and co-workers from Arctic Chucki sea (Figure 1). The structures of both compounds were
established through detailed NMR and single crystal X ray studies. Biological evaluation of these
compounds revealed that both compounds 1 and 2 have promising potential in treating influenza
virus strains A/WSN/33(H1N1).10 Following that, in vivo studies by Chen and co-workers
showed in mouse models that, nitrosporeusine A has exceptional potential in treating wide range
of diseases such as rhinitis, oral ulcer, chronic heart failure, acute renal failure and renal fibrosis
and all of them are claimed in a series of patents11 (see figure 2 for details). These biological
activity results reported for nitrosporeusines, in particular with compound 1 are very impressive,
and attracted our attention where we planned for a complete study over this novel scaffold. Due
to close association of many microbial and viral infections with inflammation,12 we envisioned to
synthesize and study the nitrosporeusines in detail towards its anti-inflammatory potential.
Figure 2: Exceptional activity of nitrosporeusine A (1)

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Accordingly, the first total synthesis of nitrosporeusines A & B using a simple chemistry was
achieved from this group and reported in a preliminary communication.13 The developed route is
flexible and now we extended it to generate a library of analogues around the natural product
skeleton. Full account of design, synthesis and biological evaluation on nitrosporeusine scaffold
is discussed in the present paper.
2. Results and Discussion:
2.1 Chemistry
Nitrosporeusines A and B share a common structural core with difference only in relative
stereochemistry between hydroxy group and ring junction.
Scheme 1: Approach towards nitrosporeusines and their analogues
So, both the natural products are envisioned to be obtained from a common intermediate –
maleimycin, which in turn could be obtained from compound 3. This maleimide moiety (3) could
be prepared from cyclopent-1-ene-1,2-dicarboxylic acid in few synthetic operations. While we
were planning for the synthesis, we chose a strategy which would be amenable for synthesis of
both natural products, as well as analogue synthesis. We planned to exploit the key maleimide
intermediate to make several building blocks for analogues. Our initial aim was to prepare
compound 3 in good quantities, so following literature procedures,14a,b we treated cyclopent-1-
ene-1,2-dicarboxylic acid with acetic anhydride/NH3 which was then subjected to intramolecular
condensation using trifluoroacetic anhydride resulting in desired maleimide compound 3 in good
yields (Scheme 2). After scaling up the reaction and making sufficient quantities, we first
explored the synthesis of nitrosporeusines A and B in racemic forms. Towards that we subjected

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compound 3 to microwave mediated allylic oxidation using selenium dioxide,15 and after few
optimizations we could obtain desired allylic alcohol (4) in 68% yield (brsm).
Scheme 2: Synthesis of racemic nitrosporeusines A and B
Alternately, the same alcohol 4 was obtained by allylic bromination (NBS/hυ/CHCl3) followed
by acetylation and hydrolysis protocol (Scheme 2). Having the required racemic maleimycin in
hand we next prepared required acid fragment for Michael addition. Commercially available 4-
hydroxybenzoic acid along with solid Lawesson’s reagent was dissolved in acetonitrile and
irradiated under microwave conditions (100 ºC/ 15 mins)16 which gave 4-hydroxybenzothioic S-
acid (7). We then subjected 4 to Michael addition with 4-hydroxybenzothioic S-acid (7) using
water as solvent and obtained both nitrosporeusines A and B in 1:3 diastereomeric ratio (Scheme
2). Both the compounds were racemic, but the diastereomers formed were cleanly separated
using silica gel column chromatography, characterized and compared with literature reports.10
Here, the observed selectivity could possibly be due to strong intermolecular H-bonding between
thio-ester carbonyl and hydroxy groups, which directs the incoming nucleophilic addition
towards forming intermediate I as major compound giving nitrosporeusine B (Scheme 3). The

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other possibility of addition could result in formation of intermediate II resulting in
nitrosporeusine A as minor product. Similar H-bonding mediated anti-aza-Michael addition
reactions were reported in literature17 which helped us in proposing this probable mechanism.
The successful racemic synthesis of natural products with a simple and efficient route
encouraged us to synthesize natural products in enantiopure forms.
H
Intermolecular H-Bonding
coordinates and directs the face
addition
O
O
O
SH
NH
HO
O
OH
H
O
OH
NH
O
S
OH
NH
O
S
O
HO
HO
O
I
II
More favorable and major product
observed from experiment
Less favorable minor product observed
from experiment
OH
O
OH
O
O
NH
S
NH
O
S
HO
O
O
nitrosporeusine B
HO
nitrosposreusine A
Observed 3: 1 selectivity
Scheme 3: Probable explanation for observed selectivity
The use of enzymatic kinetic resolution as a tool to resolve alcohols is well documented in
literature.18 We subjected alcohol 4 to different conditions in which one of the alcohols would
selectively get acetylated and give enantiopure compounds (Scheme 4). Thus after a few
attempts with different lipases and conditions (Scheme 4), alcohol 4 was resolved using vinyl
acetate in presence of Amano PS lipase in dry THF to give acetate (-)-5 and corresponding pure
alcohol (+)-4 with > 98%ee. The optical purity of (+)-4 was also confirmed by HPLC and optical
rotation values.19 Here it is interesting to note that (+)-4 is a natural product maleimycin with
good antibacterial and anti-proliferative activities.14c We also synthesized (-)-4 and (+)-5 from (-

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)-5 and (+)-4, respectively, and compared their optical rotation values with enantiomers obtained
in enzymatic resolution previously.
Enzyme
Solvent/time
THF, rt, 24h
THF, rt, 24h
THF, rt, 12h
Observation
Candida antartica lipase B
Candida rugusa lipase
100 wt% Amano PS lipase
10% conversion to (-)-5
17% conversion to (-)-5
30% yield for isolated (-)-4, 87%ee
150 wt% Amano PS lipase
dry THF, rt, 4h
47% yield for isolated (-)-4, 99%ee
Scheme 4: Synthesis of enantiopure alcohols
Having (+)-4 in hand, we then subjected it to Michael addition conditions using 4-
hydroxybenzothioic S-acid (7) to furnish the target compounds nitrosporeusines A and B in
enantiopure forms. To our surprise, the naturally occurring (+)-maleimycin ((+)-4) did not yield
natural nitrosporeusines (+)-1 and (-)-2, instead their enantiomers (-)-1 and (+)-2 were obtained.
So, we treated the unnatural maleimycin ((-)-4) under same conditions to obtain (+)-
nitrosporeusine A and (-)-nitrosporeusine B which are in full agreement with those reported in
literature10 (Scheme 5). Based on above observations, we could probably propose that natural
(+)-maleimycin may not be the biogenetic precursor for the natural nitrosporeusines A and B.
After documenting all the results obtained,13 we next planned to expand the scope of these
interesting natural products by synthesizing a series of analogues around the nitrosporeusine
scaffold and subject them for various anti-inflammatory studies. To obtain close analogues of

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nitrosporeusines we exploited the maleimide intermediate (3) by synthesizing Michael acceptors
3, 4, 5, 6 around it.
Scheme 5: Synthesis of enantiopure nitrosporeusines
Towards the preparation of thio-acids 7-12, we relied on our earlier method where we
used Lawesson’s reagent under microwave conditions (Scheme 6) to convert carboxylic acids to
corresponding thio-acids.16 These thio-carboxylic acids are found to dimerise immediately even
when stored at 4 °C, so we freshly prepared them when required and partially purified them over
short silica column before use.
Scheme 6: Synthesis of thio-acids from corresponding carboxylic acids

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Scheme 7: Synthesis of nitrosporeusine analogues

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As an initial attempt at synthesis of analogues we subjected 3, 4 and 5 to Michael addition with
thioacetic acid, thiobenzoic acid and various thio-acids 7-12 to get corresponding nitrosporeusine
analogues with major being all syn isomer (Scheme 7). The diastereomers obtained from 4 were
cleanly separated using column chromatography. The stereochemistry of these products was
assigned based on the 1H NMR patterns observed in natural products nitrosporeusine A and B. In
the 1H NMR spectrum the splitting pattern of the C6 proton which couples with both the adjacent
protons at C5 and C7, explains the syn or anti relation between hydroxy group and the ring
junction. In both the natural products the extent of coupling between C6 and C5 protons is more
or less the same; but in case of nitrosporeusine A, owing to the syn relationship between the C6
and C7 protons, the C6 proton couples with the C7 proton to a larger extent as compared to
nitrosporeusine B, where the concerned protons (C6 and C7) are anti to each other. As a result,
C6 proton of nitrosporeusine A shows a greater extent of splitting than nitrosporeusine B (figure
3). Apart from this, on comparing both natural products, we can also observe that the pattern of
C4 – C5 protons is different depending on whether hydroxy group is syn or anti to ring junction
(Figure 3).
C6 proton
C4- C5 protons
more splitting
Anti
H
N
1 2 1
2
8
O
O
H
S
OH
4
HO
6
O
5
(+)-nitrosporeusine A
1
C6 proton
less splitting
C4- C5 protons
Syn
2
1 1
H
N
8
O
O
H
S
OH
4
O
HO
6
5
( )-nitrosporeusine B
2
Figure 3: NMR pattern observed for C6, C4, C5 protons in nitrosporeusines

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1
Similar pattern of C6, C4 and C5 protons were observed in H NMR spectra of diastereomers
obtained from 4 and different thio-acids, which enabled us to assign the relative stereochemistry
between hydroxy group and ring junction in them. Here, we synthesized eleven new analogues
13-16, 18-21 and 28, 29, 33 from racemic maleimycin (4).
O
O
O
NH
O
A
Br
O
O
A
SH
NH
O
or
or
NH
NH
THF:H2O, 16 h, rt.
O
O
S
O
S
O
S
O
O
A
6
A
A
S
CH3,
A =
F
H
H
N
H
H
N
O
O
N
O
O
N
O
O
O
O
S
O
S
S
S
F
O
O
O
35
34
36
37
H
H
N
H
N
O
O
O
O
N
O
O
O
O
H
H
S
S
H
S
F
S
S
O
S
O
F
O
O
38
39
40
H
N
H
O
O
N
O
O
H
S
O
S
S
O
S
O
42
41
Scheme 8: Synthesis of di-substituted and unsaturated nitrosporeusine analogues
The Michael addition of thioacetic acid, 2-chlorobenzothioic S-acid, 2-phenylethanethioic S-acid
and 4-fluorobenzothioic S-acid on compound 3 gave corresponding nitrosporeusine analogues 17
and 22-24. These compounds were all racemic and are fully characterised by spectral techniques

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(1H, 13C, HRMS). Similarly the reaction of different thio-acids on compound 5 gave six new
acetate analogues 25-27 and 30-32 and all compounds are fully characterised. However, in spite
of best efforts we could not separate diastereomers formed from 5 but nevertheless
diastereomeric acetates 25-27, 30-32 we screened as such for biological activity (Scheme 7). In
total we made around 21 new analogues using the substrates 3, 4 and 5. The synthesis of
analogues over compound 6 was then explored where Michael addition using thio-acids gave
entirely new and interesting compounds. Instead of simple addition adducts, we observed
Michael addition followed by elimination to give olefin analogues which further underwent
second Michael addition with thio-acids giving rise to di-substituted analogues. Accordingly,
when compound 6 was subjected to Michael addition using 4-fluorobenzothioic S-acid, it
resulted in compound 35 (Scheme 8). Probably, Michael addition followed by
dehydrobromination resulted in compound 35 which is an interesting intermediate for generating
a variety of compounds around this skeleton. For example, the newly generated double bond
potentially can be used for addition of various nucleophiles, dihydroxylation, allylic oxidation
etc. Similar olefin analogues were obtained when compound 6 reacts with thioacetic acid and 2-
phenylethanethioic S-acid. But when the same reaction was performed on 6 using thiobenzoic
acid, it resulted in product 37 in good yields, which is the result of substitution of bromine.
Compound 37 is also an interesting intermediate towards the library creation. Similar product 42
was obtained when Michael addition performed using thiophene-2-carbothioic S-acid. During all
these reactions we also isolated compounds 38 – 41 in minor quantities as a mixture of
diastereomers which were also considered for biological screening. These compounds have been
later prepared exclusively by prolonging the reaction time and addition of more equivalents of
respective thio-acids. Following this approach we could prepare compounds 34 – 37 and 40 – 42.
In total by using different Michael acceptors and thio-acids we prepared 32 new compounds
which were well characterized by spectral techniques. The inflammatory studies on all
compounds were planned in a systematic manner where first cytotoxicity and NO inhibition
studies were to be carried out. Based on the results we thought to select few compounds which
would be analysed for their inflammatory potential with different pro-inflammatory factors.
2.2 Biology
2.2.1 Anti-inflammatory activity

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First, all compounds were evaluated for their inhibition of NO production in LPS stimulated
RAW264.7 cells and nitrite levels, a strong metabolite of NO, were measured in culture media
using Griess reagent.20 During inflammation, large amount of NO are produced and in turn
amplify inflammatory response to multiple fold. The primary results indicated that almost all
compounds evidently decreased NO level in LPS treated cell as shown in Table 1.
Simultaneously, cytotoxicity of all compounds were assessed using MTT assay and IC50 was
calculated (Table 1).
Table 1: Cytotoxicity and LPS induced NO inhibitory activity in RAW 264.7 cells
Cytotoxicity (A)
95%
NO inhibition (B)
95%
Com-
pound
(-)-1
(+)-1
(±)-2
(+)-2
(-)-2
(-)-4
(+)-4
(-)-5
13
Selectivity
index (A/B)
14.66905
14.14485
1.925433
22.98817
9.849822
2.259645
2.631492168
0.147653
3.7762
IC50
(µM)
616.1
1015.6
433.8
2914.9
1383.9
216.7
319.2
128
confidence
IC50
confidence
interval (µM)
interval (µM)
(µM)
42
513.7-802.9
856-1281.4
307.4-592.6
2307-3449.4
1045.3-1536.8
103.8-354.7
284.3-367.8
98.4-137.7
31.2-56.3
60-84.6
71.8
225.3
126.8
140.5
95.9
182.8-289.4
98.3-146.7
118.1-157.3
79.4-116.3
96.3-152.8
682.3-993.5
112.5-186.9
664.7-1004.7
107.7-165.2
121.3
866.9
147.9
832.8
132
558.5
856.7
834.5
472.6-637.2
622.4-983.3
727.4-967.9
1.028698
6.32197
14
15

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773.7
674.4
667.7
717.1
479.4
17063
383.6
834.5
425.6
12135.6
9076.1
1912
682.8-867.8
493.7-819.6
512.3-788.6
604.6-833.6
294.5-601
142.8
59.2
127.3
178.3
72.4
65.2
389.8
59.8
56.9
34.3
101.4
64.3
318
116.4-204.7
46.8-76.9
106.3-146.8
119.7-213.4
49.8-87.4
47.8-78.1
327.5-466.1
43.6-77.5
33.5-83.6
24-43.2
5.418067
11.39189
5.24509
4.021873
6.621547
261.7025
0.984094
13.95485
7.479789
353.8076
89.50789
29.73561
1.489623
297.0519
23.56745
9
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
34
36
38
40
41
42
15948-18666
305.1-459.8
773.8-923.1
301.4-556.2
11853-12738
8113-10263
1628.4-2249.9
355.6-513.9
9332.1-11382
1283.5-1983.5
109.2-228.5
84.7-109.3
74.1-121.8
52.9-93.8
271.2-380
24.5-46.1
44.7-87.6
15.6-26.1
18.9-43.7
83.6-123.6
14.5-25.4
42.8-76.1
48.6-74.8
473.7
10872.1
1607.3
184.5
93.07
210.6
36.6
68.2
20.5
30.6
98.3
19.8
57.5
65
3.041503
2.142421
505.4343
12.36174
0.410769
183.2-237.4
10007.6 9438.3-10837.8
710.8
26.7
525.6-984.4
13.5-42.6

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Figure 4: Selected compounds based on cytotoxicity and no inhibition potential
However, the compounds showing effective NO inhibitory activity with low cytotoxicity were
screened based on the selectivity index (cytotoxicity IC50/NO inhibition IC50). Compounds 25,
29 and 40 were selected for further study (figure 4), since they exhibited superior selectivity
indices (≥300) among all the derivatives. Further, the anti-inflammatory potency of compounds
25, 29 and 40 were determined by measuring the level of intracellular reactive oxygen species
(ROS) in LPS treated RAW 264.7 cells. ROS generation is a key marker for inflammation and is
reported in several cases as a triggering factor for apoptosis.21
Figure 5: Effects of compounds on ROS level in LPS stimulated RAW 264.7 cells. The plot
represents the mean fluorescence intensity (MFI) of ROS generation in presence of three selected
compounds. Data represent mean ± SD of three independent experiments. **p<0.01 and
***p<0.001in comparison to LPS-treated values.

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As depicted in Figure 5, increase of ROS in LPS treatment was reduced significantly in presence
of all these three compounds in a dose dependent manner as analysed by mean fluorescence
intensity (MFI). Inflammation is the body’s first response of the immune system to infection or
irritation. During the inflammatory process, large quantities of the inflammatory mediators are
produced by the inducible isoforms of iNOS and COX-2.22 Thus we checked the levels of iNOS
and COX-2 by immunoblotting in response to drugs treatment in LPS administered cells. We
found that 50 µM of all three selected compounds are efficient in reducing iNOS and COX-2
levels compared to LPS (Figure 6A & 6B).
Figure 6: Protein isolated from RAW 264.7 cells of control, LPS and LPS+drug conditions were
analysed by immunoblot. The graphs represent the densitometric quantification of protein bands-
iNOS (A), COX-2 (B) and phospho-NFκB (C) normalised to β-actin. Values represent mean ±
SD from 3 independent experiments. *p<0.05, **p<0.01 and ***p<0.001 in comparison to LPS
treated values.

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Since NF-κB activation is an important marker of LPS-induced inflammation, we assessed
influence of these compounds on NF-κB activation. NF-κB is present in the cytoplasm as an
inactivated dimer composed of p65 and p50 subunits. In response to inflammatory stimuli, IκB is
phosphorylated and degraded, and NF-κB is released and translocated into the nucleus,23 where it
binds promoter region of many inflammatory genes, including iNOS, COX-2, and TNF-α and
augments their expression.24 Our results showed that 25, 29 and 40 potentially decreased LPS
induced NF-κB activation as analysed phosphorylated NF-κB level (pNF-κB) by western blot
(Figure 6C).
Figure 7: Cytokine Bead assay (CBA) analysis of protein extract isolated from RAW 264.7 cells
treated with LPS along with compounds. Addition of compounds (50 µM) revealed substantial
decrease in the levels of IL-12 (A), IL-6 (B), TNF-α(C),MCP-1 (D) and IFN-γ (E) compared to
LPS treated cells. Data represent mean ± SD of three independent experiments. *p<0.05,**p<0.01
and ***p<0.001in comparison to LPS treated values.
Under the pathogenic attack in our body, activated macrophages release numerous pro-
inflammatory cytokine and inflammatory mediators.25 Hence, the macrophage cell line provides
an excellent model for drug screening and evaluation of potential inhibitors of the inflammatory

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response. LPS treatment can induce inflammation, resulting in the extreme production of
numerous pro-inflammatory mediators including IL-12, TNF-α, IFN-γ, MCP-1 and IL-6.
However, the anti-inflammatory activity of compounds 25, 29 and 40 were evaluated by
measuring the expression of these inflammatory cytokines in LPS and compound treated
RAW264.7 cells by CBA. As evidenced from figure 7, it was found that 50 µM dose of all these
compounds effectively suppressed pro-inflammatory mediators.
Figure 8: Mouse serum samples were used to study anti-inflammatory activity of drugs in vivo.
There were significant reductions in the levels of IL-12 (A), IL-6 (B), TNF-α (C), MCP-1 (D),
and IFN-γ (E) in compounds (10 mg/kg body weight) treated serum samples compared to only
LPS injected mice. Values represent mean ± SD of serum samples collected from five mice.
*p<0.05, **p<0.01 and ***p<0.001 in comparison to LPS treated values.
In vivo testing is often employed over in vitro because it is better suited for observing the overall
effects of an experiment on a living subject. Therefore, we next investigated anti-inflammatory
activity of compounds in vivo using serum samples collected intracardially from mouse. We
measured levels of pro-inflammatory cytokines by CBA and based on the results, compound 25

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was found very promising in in vivo though other two compounds were also effective in reducing
cytokines level when compared with LPS treated group (Figure 8). As evident from all in vitro
and in vivo studies, compound 25 showed the most potent anti-inflammatory activity among
them.
Scheme 9: Synthesis of active enantiopure analogues of nitrosporeusines
So, we planned to prepare four optically pure isomers of 25 to further understand whether the
spatial arrangements contribute to anti-inflammatory activity. Towards this we relied on
enantiopure compounds (-)-4 and (+)-4 to obtain desired alcohols which were then converted to
respective enantiopure acetates. Compound (-)-4, on subjection to previously optimized Michael
addition conditions with 4-fluorobenzothioic S-acid gave desired adducts (-)-28a and (+)-28b in
approximately 1:5 diastereomeric ratio. Both the compounds were then carefully separated using
silica gel chromatography and characterized. The compound (+)-28b was then acetylated using
Ac2O/pyridine conditions to obtain enantiopure acetate (+)-25b. Similarly, (-)-28a was converted
to acetate (-)-25a (Scheme 9). Using similar reactions with (+)-4 as starting material, acetate
compounds (+)-25a and (-)-25b have been obtained in enantiopure forms.

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2.2.2. Anti-inflammatory activity of enantiomers of compound 25
With all the four diastereomers of 25 in hand, we again assessed them through in vitro and in
vivo assays to identify the potent lead compound among them.
Figure 9: Effects of enantiomers on NO and ROS level in LPS stimulated RAW 264.7 cells. The
plot represents the fold change of NO (A) and mean fluorescence intensity (MFI) of ROS
generation (B) in presence of four enantiomers. Data represent mean ± SD of three independent
experiments. *p<0.05 and **p<0.01 in comparison to LPS-treated values.
The anti-inflammatory efficiency of enantiomers was determined by measuring the NO level in
culture supernatants as well as intracellular ROS in LPS treated RAW 264.7 cell. Although
marked reduction in NO and ROS level were observed in all enantiomers treated cells, (-)-25a
showed most potential inhibition among them (Figure 9). Next, we checked the levels of iNOS,
COX-2 and pNF-κB by immunoblotting in response to enantiomers treatment in LPS treated
cells. Among them, enantiomer (-)-25a most significantly attenuated NF-κB activation and a
comparable reductions in iNOS and COX-2 expression were observed (Figure 10).

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Figure 10: Protein isolated from RAW 264.7 cells of control, LPS and LPS + enantiomer (30
µM) conditions were analysed by immunoblot. The graphs represent the densitometric
quantification of protein bands- COX-2 (A), iNOS (B) and phospho-NF-κB (C) normalised to β-
actin. Values represent mean ± SD from 3 independent experiments. *p<0.05 in comparison to
LPS treated values.
Consistently, enantiomer (-)-25a, in comparison to others, significantly reduced the expressions
of pro-inflammatory cytokines in LPS treated cells as assessed through CBA analysis (Figure
11). From all the above biological studies it may be concluded that compound (-)-25a showed
potent anti-inflammatory activity among all the analogues and its structure and stereochemistry
closely relates to nitrosporeusine A. Incidentally, nitrosporeusine A showed significant activity in
various animal models for treatment of different diseases (as shown in figure 2).

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Figure 11: CBA analysis of protein extract isolated from RAW 264.7 cells treated with LPS
along with enantiomers. Addition of compounds (30 µM) showed substantial decrease in the
levels of IL-12 (A), IL-6 (B), TNF-α (C),MCP-1 (D) and IFN-γ (E) compared to LPS treated
cells. Data represent mean ± SD of three independent experiments. *p<0.05,**p<0.01 in
comparison to LPS treated values.
Figure 12: Identification of active nitrosporeusine analogue through synthesis

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3. Conclusion
The intriguing structure and exceptional biological activity reported for nitrosporeusines A and B
has encouraged us to plan and execute a medicinal chemistry program around nitrosporeusine
scaffold in identifying new anti-inflammatory agents. We achieved first synthesis of
nitrosporeusines A and B in both racemic and enantiopure forms using short and simple route
with key steps involving allylic oxidation (SeO2) and Michael addition (in water). We
synthesized four isomers of the natural products in enantiopure forms by employing gram scale
kinetic enzymatic resolution method using Amano PS lipase. Later, we diverted our efforts
towards synthesis of 32 close analogues of nitrosporeusines. All the synthesized compounds
were profiled for their anti-inflammatory potential in different assays. The initial screening with
respect to NO inhibition and cytotoxicity has shown that three of the compounds are promising
which prompted us to profile them in further studies. Accordingly, compounds 25, 29 and 40
have been studied in detail with various inflammatory markers and 25 showed the most
promising anti-inflammatory potential. Four enantiomers of compound 25 were further prepared
and evaluated for their anti-inflammatory activity using same assays, and finally the enantiomer
(-)-25a was recognized as the potential lead from this project.
4. Experimental Section
4.1 Chemistry
All reactions were carried out in oven-dried glassware under a positive pressure of argon or
nitrogen unless otherwise mentioned with magnetic stirring. Air sensitive reagents and solutions
were transferred via syringe or cannula and were introduced to the apparatus via rubber septa.
All reagents, starting materials and solvents were obtained from commercial suppliers and used
as such without further purification. Reactions were monitored by thin layer chromatography
(TLC) with 0.25 mm pre-coated silica gel plates (60 F254). Visualization was accomplished with
either UV light, or by immersion in ethanolic solution of phosphomolybdic acid (PMA), para-
anisaldehyde, 2,4-DNP, KMnO4, Ninhydrin solution or Iodine adsorbed on silica gel followed by
heating with a heat gun for ~15 sec. Column chromatography was performed on silica gel (100-
200 or 230-400 mesh size). Deuterated solvents for NMR spectroscopic analyses were used as
received. All 1H NMR and 13C NMR spectra were obtained using a 200 MHz, 400 MHz, and 500
MHz spectrometer and coupling constants were measured in Hertz. In some of the compounds,

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we observed doubling of peaks due to diastereomers. All chemical shifts were quoted in ppm
relative to TMS, using the residual solvent peak as a reference standard. The following
abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, br = broad. HRMS (ESI) were recorded on ORBITRAP mass analyzer
(Thermo Scientific, Q Exactive). Mass spectra were measured with ESI ionization in MSQ
LCMS mass spectrometer. Infrared (IR) spectra were recorded on a FT-IR spectrometer.
Chemical nomenclature was generated using Chembiodraw ultra 14.0. Melting points of solids
were measured in melting point apparatus. Optical rotation values were recorded on P-2000
polarimeter at 589 nm.
Experimental procedures for the preparation of compounds 1, 2, 3, 4, 5, 6 as well as
HPLC copies of 4, (-)-4, (+)-4 has been reported previously by us.18 Data pertaining to the
characterization and purity of each of the compounds (-)-1, (+)-1, (+)-2, (-)-2, 3, 4, (-)-4, (+)-4, 5,
6 have also been given earlier.18 Here all the synthesized analogues were tested for purity on C18
column and found to be greater than 90% pure. Compounds 35, 37, 39 were not considered for
screening due to lack of HPLC purity.
4.1.1. Procedure A: General procedure for synthesis of analogues through Michael addition
In a round-bottomed flask equipped with a magnetic stirrer, thioacid (1.1 eq.), unsaturated
compound (Michael acceptor) (1 eq.), and THF:water (1:1) were charged. The reaction mixture
was stirred vigorously at room temperature for 10 to 12h, then it was diluted with EtOAc and
extracted twice (2x3 mL). The combined organic layer was washed with saturated aqueous
NaHCO3 solution followed by brine solution and concentrated in vacuo to obtain a crude mixture
which was purified by column chromatography (silica gel; EtOAc/Petroleum ether) to obtain the
desired nitrosporeusine analogues.
#All the products are isolated in 55-71% overall yield.
4.1.2. Procedure B: General procedure for synthesis of thio-acids
To a solution of substituted aromatic acid (1 eq.) in dry acetonitrile was added Lawesson’s
reagent (0.5 eq.) and was subjected to microwave irradiation in closed vessel at 100 °C for 15
minutes (Antonpaar monowave 300 instrument). The reaction mixture was evaporated to dryness
and the crude residue obtained was diluted with EtOAc and washed several times with 1N HCl,

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then with brine solution and dried over anhydrous Na2SO4. The combined organic layers were
concentrated in vacuo and purified by silica gel column chromatography with elution of 20-30%
EtOAc/Petroleum ether.
* All thio-acids are prepared freshly before reaction and used immediately for reactions
4.1.3. Procedure C: General procedure for acetylation
To a solution of alcohol (1 eq.) in pyridine as solvent was added 1.5 eq. of acetic anhydride and
0.1 eq. of dimethylaminopyridine (DMAP) and stirred at room temperature for 6-8 hours. The
reaction was diluted with ethylacetate and slowly added with 1N HCl until pH turns acidic. The
layers were separated and organic layer was washed with water, brine and dried over anhydrous
Na2SO4. The crude reaction reaction mixture obtained upon concentrating in vacuo was purified
over column chromatography (silica gel; 5-10% EtOAc/CH2Cl2) to obtain the desired acetate
compounds.
4.1.4.
S-((3aS*,6S*,6aR*)-6-Hydroxy-1,3-dioxohexahydrocyclopenta[c]pyrrol-3a(1H)-yl)
ethanethioate (13) and S-((3aR*,6S*,6aS*)-6-Hydroxy-1,3-dioxohexahydrocyclopenta [c]pyrrol-
3a(1H)-yl) ethanethioate (14)
Following general procedure A alcohol 4 has been treated with thioacetic acid to get
diastereomers
13
and
14
in
approximately
1:1.2
ratio.
1
Compound 13 (20 mg) obtained as white solid. IRυmax (film) 3809, 1708, 1692, 1515 cm-1; H
NMR (400 MHz, CD3OD) δ 4.60-4.56 (m, 1H, CH2CHOH), 3.34-3.30 (d, 1H,
(OH)CHCHCONH), 2.33 (s, 3H, CH3CO), 2.31-2.28 (m, 1H, (OH)CHCH2CH2C(SCOCH3))
2.02-1.89
(m,
1H,
(OH)CHCH2CH2C(SCOCH3)),
1.88-1.81
(m,
1H,
(OH)CHCH2CH2C(SCOCH3)), 1.79-1.77 (m, 1H, (OH)CHCH2CH2C(SCOCH3)); 13C NMR
(100 MHz, CD3OD) δ 198.0 (S-C=O), 181.0 (SCCONHCOCH), 177.1 (SCCONHCOCH), 73.9
(CH2CHOH), 61.0 ((HO)CHCHCONH), 60.7 (CH3COSCCH), 35.2, 33.4, 29.8 (CH3CO);
HRMS (ESI) m/z calculated for C9H11NO4S [M+Na]+ 252.0301, found 252.0299. RTHPLC 5.538
min, purity >90%, 25:75 H2O/ MeOH. Compound 14 (23 mg) obtained as white solid. Melting
1
Point 120-129 °C; IRυmax (film) 3809, 1708, 1692, 1515 cm-1; H NMR (400 MHz, CD3OD):
δ 4.53-4.51 (m, 1H, CH2CHOH), 3.17-3.15 (d, 1H, (OH)CHCHCONH), 2.31 (s, 3H, CH3CO),
2.23-2.19 (m, 2H, CH2CH2C(SCOCH3)), 1.89-1.88 (m, 1H, (OH)CHCH2CH2C(SCOCH3)),

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1.70-1.65 (m, 1H, (OH)CHCH2CH2C(SCOCH3)); 13C NMR (100 MHz, CD3OD): δ 196.9 (S-
C=O), 179.6 (SCCONHCOCH), 176.7 (SCCONHCOCH), 74.8 (CH2CHOH), 63.6
((HO)CHCHCONH), 58.3 (CH3COSCCH), 32.9, 31.7, 28.0 (CH3CO); HRMS (ESI): m/z
calculated for C9H11NO4S [M+Na]+ 252.0301, found 252.0299. RTHPLC 4.22 min, purity >95%,
10:90 H2O/ MeOH.
4.1.5. S-((3aS*,6S*,6aR*)-6-Hydroxy-1,3-dioxohexahydrocyclopenta[c]pyrrol-3a(1H)-yl) 2-
phenylethanethioate
(15)
&
S-((3aR*,6S*,6aS*)-6-Hydroxy-1,3-dioxohexahydrocyclo
penta[c]pyrrol-3a(1H)-yl) 2-phenylethanethioate (16)
Following general procedure A, alcohol 4 has been treated with freshly prepared 2-
phenylethanethioic S-acid to get diastereomers 15 and 16 in approximately 1:2 ratio. Compound
1
15 (25 mg) obtained as yellow solid. H NMR (400 MHz, CD3OD) δ 7.36 -7.28 (m, 5H,
CH2C6H5), 4.55-4.53 (m, 1H, CH2CHOH), 3.84 (s, 2H, COCH2C6H5), 3.25-3.23 (d, J = 7.6 Hz,
1H, (OH)CHCHCONH), 2.31-2.27 (m, 1H, (OH)CHCH2CH2C(SCOPh)), 1.99-1.87 (m, 1H,
13
(OH)CHCH2CH2C(SCOPh)), 1.85-1.75 (m, 2H, (OH)CHCH2CH2C(SCOPh)); C NMR (125
MHz, CD3OD) δ 199.9 (S-C=O), 181.0 (SCCONHCOCH), 177.1 (SCCONHCOCH), 134.5,
131.0, 129.9, 128.8, 73.9 (CH2CHOH), 61.0 ((HO)CHCHCONH), 50.3 (PhCH2COSCCH), 49.7,
35.2, 33.5; HRMS (ESI) m/z calculated for C15H15NO4S [M+Na]+ 328.0614, found 328.0601.
RTHPLC = 6.10 min, purity >95%, 25:75 H2O/ MeOH. Compound 16 (45 mg) obtained as white
1
solid. H NMR (500 MHz, CD3OD) δ 7.35-7.30 (m, 5H, CH2C6H5), 4.52-4.51 (m, 1H,
CH2CHOH), 3.86 (s, 2H, COCH2C6H5), 3.13 (s, 1H, (OH)CHCHCONH), 2.21-2.18 (m, 2H,
(OH)CHCH2CH2C(SCOPh)), 1.91-1.87 (m, 1H, (OH)CHCH2CH2C(SCOPh)), 1.69-1.66 (m, 1H,
(OH)CHCH2CH2C(SCOPh)); 13C NMR (125 MHz, CD3OD) δ 198.9 (S-C=O), 179.6
(SCCONHCOCH), 176.7 (SCCONHCOCH), 133.0, 129.5, 128.3, 127.2, 74.8 (CH2CHOH),
63.6 ((HO)CHCHCONH), 58.4 (PhCH2COSCCH), 48.6, 33.0, 31.7; HRMS (ESI) m/z
calculated for C15H15NO4S [M+Na]+ 328.0614, found 328.0601. RTHPLC = 5.72 min, purity
>93%, 25:75 H2O/ MeOH.
4.1.6. S-((3aR*,6aS*)-1,3-dioxohexahydrocyclopenta[c]pyrrol-3a(1H)-yl) ethanethioate (17)
Following general procedure A, compound 3 has been treated with commercially available
thioacetic acid to get 17 (71 mg) as white solid. Melting point 120-122 °C. 1H NMR (400 MHz,
CDCl3) δ 8.96 (br s, 1H, NH), 3.27-3.24 (d, J = 8.8 Hz, 1H, CH2CHCONH), 2.34 (s, 3H,

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CH3CO),
2.32-2.24
(m,
2H,
CH2CH2CH2C(SCOCH3)),
1.83-1.54
(m,
4H,
CH2CH2CH2C(SCOCH3)); 13C NMR (100 MHz, CDCl3) δ 196.3 (S-C=O), 178.2
(SCCONHCOCH), 178.2 (SCCONHCOCH), 59.5 (CH3COSCCH), 54.7 (CH2CHCONH), 36.2
(CH3CO), 29.7, 29.6, 24.3; HRMS (ESI) m/z calculated for C9H11NO3S [M+Na]+ 236.0348,
found 236.0352. RTHPLC 4.90 min, purity >90%, 40:60 H2O/ MeOH
4.1.7. S-((3aS*,6S*,6aR*)-6-Hydroxy-1,3-dioxohexahydrocyclopenta[c]pyrrol-3a(1H)-yl) 2-
chlorobenzothioate (18)
&
S-((3aR*,6S*,6aS*)-6-Hydroxy-1,3-dioxohexahydrocyclopenta
[c]pyrrol-3a(1H)-yl) 2-chlorobenzothioate (19)
Following general procedure A, alcohol 4 has been treated with freshly prepared 2-
chlorobenzothioic S-acid to get diastereomers 18 and 19 in approximately 1:3 ratio. Compound
18 (26 mg) obtained as white solid. Melting point 173-174 °C; IRυmax (film) 3743, 2925, 2320,
1
1707, 1515 cm-1; H NMR (400 MHz, CD3OD) δ 7.72-7.69 (m, 1H, Aromatic-CClCH=CH-
CH=CH), 7.55-7.53 (m, 2H, Aromatic-CClCH=CH-CH=CH), 7.45-7.43 (m, 1H, Aromatic-
CClCH=CH-CH=CH), 4.67-4.63 (m, 1H, CH2CHOH), 3.46 (d, J = 7.6 Hz, 1H,
(OH)CHCHCONH), 2.43-2.39 (m, 1H, (OH)CHCH2CH2C(SCOPh)), 2.08-1.99 (m, 2H,
13
(OH)CHCH2CH2C(SCOPh)), 1.86-1.83 (m, 1H, (OH)CHCH2CH2C(SCOPh)); C NMR (100
MHz, CD3OD) δ 196.0 (S-C=O), 183.0 (SCCONHCOCH), 179.3 (SCCONHCOCH), 139.7,
136.9, 134.6, 134.3, 133.0, 130.9, 76.3 (CH2CHOH), 64.0 ((HO)CHCHCONH), 63.3
(PhCOSCCH), 37.7, 35.9; HRMS (ESI) m/z calculated for C14H12NO4ClS [M+Na]+ 348.0068,
found 348.0061. RTHPLC 4.46 min, purity 90%, 40:60 H2O/MeOH. Compound 19 (82 mg)
obtained as white solid. Melting point 154-158 °C; IRυmax (film) 3743, 2925, 2320, 1707, 1515
1
cm-1; H NMR (200 MHz, CD3OD) δ 7.78-7.76 (m, 1H, Aromatic-CClCH=CH-CH=CH), 7.58-
7.50 (m, 3H, Aromatic-CClCH=CH-CH=CH), 4.63-4.60 (td, J = 3.7, 1.2 Hz, 1H, CH2CHOH),
3.34-3.32 (m, 1H, (OH)CHCHCONH), 2.39-2.31 (m, 2H, (OH)CHCH2CH2C(SCOPh)), 1.97-
13
1.95 (m, 1H, (OH)CHCH2CH2C(SCOPh)), 1.82-1.80 (m, 1H, (OH)CHCH2CH2C(SCOPh)); C
NMR (100 MHz, CDCl3) δ 192.6 (S-C=O), 178.3 (SCCONHCOCH), 176.1 (SCCONHCOCH),
135.0, 133.2, 131.3, 131.1, 129.9, 126.9, 75.4 (CH2CHOH), 63.7 ((HO)CHCHCONH), 59.2
(PhCOSCCH), 33.2, 32.5; HRMS (ESI) m/z calculated for C14H12NO4ClS [M+Na]+ 348.0068,
found 348.0061. RTHPLC = 6.70 min, purity >99%, 40:60 H2O/ MeOH.

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4.1.8.
S-((3aS*,6S*,6aR*)-6-Hydroxy-1,3-dioxohexahydrocyclopenta[c]pyrrol-3a(1H)-yl)
benzothioate (20) & S-((3aR*,6S*,6aS*)-6-Hydroxy-1,3-dioxohexahydrocyclopenta[c] pyrrol-
3a(1H)-yl) benzothioate (21)
Following general procedure A, alcohol 4 has been treated with commercially available
Thiobenzoic acid to get diastereomers 20 and 21 in approximately 1:2 ratio. Compound 20 (26
mg) as white solid. Melting point 185-186 ºC; IRυmax (film) 3743, 2927, 2320, 1741, 1706, 1531
1
cm-1; H NMR (400 MHz, CD3OD) δ 7.91-7.89 (dd, J = 8.4, 1.1 Hz, 2H, 2H,2 x Aromatic-
orthoCH), 7.66-7.64 (m, 1H, Aromatic-para CH), 7.53-7.49 (m, 2H, 2 x 2 x Aromatic-meta
CH), 4.62-4.59 (m, 1H, CH2CHOH), 3.42 (d, J = 7.6 Hz, 1H, (OH)CHCHCONH), 2.42-2.39
(dd,
J
=
13.2, 7.1 Hz, 1H, (OH)CHCH2CH2C(SCOPh)), 2.08-2.02 (m, 2H,
(OH)CHCH2CH2C(SCOPh)), 1.84-1.82 (m, 1H, (OH)CHCH2CH2C(SCOPh)); 13C NMR (100
MHz, CD3OD) δ 193.6 (S-C=O), 181.1 (SCCONHCOCH), 177.2 (SCCONHCOCH), 137.4,
135.6, 130.3, 128.4, 73.9 (CH2CHOH), 61.1 ((HO)CHCHCONH), 60.6 (PhCOSCCH), 35.3,
33.7; HRMS (ESI) m/z calculated for C14H13NO4S [M+Na]+ 314.0457, found 314.0456. RTHPLC
= 6.02 min, purity >90%, 25:75 H2O/ MeOH. Compound 21 (47 mg) as white solid. Melting
1
point 180-182 ºC; IRυmax (film) 3743, 2927, 2320, 1741, 1706, 1531 cm-1; H NMR (400 MHz,
CD3OD) δ 7.94-7.91 (m, 2H, 2 x Aromatic-ortho CH), 7.69-7.66 (m, 1H, Aromatic-paraCH),
7.54-7.51 (m, 2H, 2 x Aromatic-meta CH), 4.59-4.58 (m, 1H, CH2CHOH), 3.34-3.42 (m, 1H,
(OH)CHCHCONH), 2.40-2.33 (m, 2H, (OH)CHCH2CH2C(SCOPh)), 1.96-1.95 (m, 1H,
(OH)CHCH2CH2C(SCOPh)), 1.78-1.77 (m, 1H, (OH)CHCH2CH2C(SCOPh)); 13C NMR (100
MHz, CD3OD) δ 191.1 (S-C=O), 178.1 (SCCONHCOCH), 175.27 (SCCONHCOCH), 134.21,
132.54, 127.23, 125.36, 73.36 (CH2CHOH), 62.28 ((HO)CHCHCONH), 56.75 (PhCOSCCH),
31.75, 30.32; HRMS (ESI) m/z calculated for C14H13NO4S [M+Na]+ 314.0457, found 314.0456.
RTHPLC = 10.8 min, purity >88%, 40:60 H2O/ MeOH.
4.1.9. S-((3aR*,6aS*)-1,3-Dioxohexahydrocyclopenta[c]pyrrol-3a(1H)-yl) 4-fluorobenzothioate
(22)
Following general procedure A, compound 3 has been treated with freshly prepared 4-
fluorobenzothioic S-acid to get 22 (40 mg) as white solid. Melting point 186-187 °C; IRυmax
1
(film) 3744, 2922, 1770, 1647 cm-1; H NMR (400 MHz, CDCl3) δ 8.40 (br s, 1H, NH) 7.96-
7.89 (m, 2H, 2 x Aromatic-CCH=CH-CF), 7.18-7.08 (m, 2H, 2 x Aromatic-CCH=CH-CF), 3.39-
3.35 (m, 1H, CH2CHCONH), 2.48-2.28 (m, 2H, CH2CH2CH2C(SCOPh)), 2.09-1.91 (m, 2H,