Design, synthesis and evaluation of novel diaryl pyrrolopyrimidine and pyrrolothiazine derivatives as inhibitors of tumor necrosis factor stimulated gene-14 (TSG-14) production

Article abstract of DOI:10.1016/j.ejmech.2014.03.068

A novel series of pyrrolothiazines 2-4 and pyrrolopyrimidines 5-7 have been synthesized. The structures of these compounds were established by spectroscopic and element microanalytical data. The newly synthesized compounds were evaluated as inhibitors of TSG-14. The most effective results were obtained by the S-sec-butyl derivatives 6e (80%) and the N-ethyl derivatives 4e (70%).

Full text of DOI:10.1016/j.ejmech.2014.03.068

European Journal of Medicinal Chemistry 78 (2014) 419e424
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and evaluation of novel diaryl pyrrolopyrimidine
and pyrrolothiazine derivatives as inhibitors of tumor necrosis factor
stimulated gene-14 (TSG-14) production
,
b
c
a
Khalid M.H. Hilmy a , Dalia H. Soliman , Esmat B.A. Shahin , Hala S. El-Deeb ,
*
Salah M. El-Kousya
a Department of Chemistry, Faculty of Science, Monufyia University, Shebin El-Kom, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
c Department of Biochemistry, Faculty of Medicine (Girls), Al-Azhar University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of pyrrolothiazines 2e4 and pyrrolopyrimidines 5e7 have been synthesized. The struc-
tures of these compounds were established by spectroscopic and element microanalytical data. The
newly synthesized compounds were evaluated as inhibitors of TSG-14. The most effective results were
obtained by the S-sec-butyl derivatives 6e (80%) and the N-ethyl derivatives 4e (70%).
Ó 2014 Published by Elsevier Masson SAS.
Received 21 October 2013
Received in revised form
5 March 2014
Accepted 23 March 2014
Available online 24 March 2014
Keywords:
Pyrrolopyrimidines
Pyrrolothiazines
Gene-14 (TSG-14)
TNF-
a
1. Introduction
addition, circulating TSG-14 levels increase in several pathological
conditions, such as autoimmune, infectious, and degenerative dis-
TNF-stimulated gene 14/Pentraxinprotein 3 (TSG-14/PTX3) is a
gene inducible by tumor necrosis factor-alpha (TNF- ), inter-
leukine-1 (IL- ) and lipopolysaccharide (LPS) in fibroblasts, mac-
turbances [9e11]. Interestingly, expression of the TSG-14 gene is
controlled by TNF- [3,12,13] and in a positive feedback, TSG-14
appears to enhance the production of TNF- when exposed to LPS
a
a
b
b
a
rophages and endothelial cells [1e4]. TSG-14 gene encodes a 42-kd
secreted glycoprotein that belongs to long pentraxin family of
proteins 381 amino acids, it also shares homology with the short
pentraxin family of acute-phase proteins such as C-reactive protein
(CRP) and serum amyloid P component (SAP) [1,2]. TSG-14 gene is
[1]. Consequently, TSG-14 could be considered as a potential target
for the development of new anti-inflammatory therapies. The
validation of the use of small molecules as inhibitors of TNF-
a
induced kinases and their role in altering the levels of
inflammatory-related proteins has encouraged us to investigate the
inhibitory effect of orally available small molecules on TSG-14 [14].
This could be considered as a chemicalegenetic interaction that
benefits from the advantages of orally available small molecule
inhibitors, over the currently used biopharmaceuticals [15e18].
Moreover, to date there is not any reported data of small molecule
inhibitors of TSG-14, only the biochemicals, cycloheximide, the
most common laboratory reagent used to inhibit protein synthesis
[19,20] and the antioxidant pyrrolidine dithiocarbamate [19,21].
Here in, we report the synthesis and evaluation of a new series
of pyrrolo[2,3-d]pyrimidine and pyrrolo[2,3-d]thiazine derivatives
as inhibitors of TSG-14. The design of the new compounds was
based on their structural similarity to purines and pyrimidines that
one of the TNF-a stimulated gene (TSGs); such genes are known as
inflammatory genes that stimulate the inflammatory response [5].
This altered cytokine-induced gene expression contributes to the
etiology of inflammatory diseases. Previous studies have suggested
a role for TSG-14 in the inflammatory process, it binds to the C1q
component of the complement cascade [6] high levels of TSG-14
were detected in the serum of LPS-injected humans and mice
[3,7], and in the joint fluid of rheumatoid arthritis patients [8]. In
* Corresponding author. P.O. Box 73, Souhbra Misr, Cairo, Egypt.
E-mail address: hilmykhaled@yahoo.com (K.M.H. Hilmy).
http://dx.doi.org/10.1016/j.ejmech.2014.03.068
0223-5234/Ó 2014 Published by Elsevier Masson SAS.

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K.M.H. Hilmy et al. / European Journal of Medicinal Chemistry 78 (2014) 419e424
Table 1
play key roles in cellular processes. One important class of pyrim-
idine is 2-thiopyrimidine (2-TP) and its derivatives [22], which have
attracted substantial interest of synthetic-biochemists [23,24].
TSG-14 inhibition values for some of the synthesized compounds.
Compd. No
TSG-14 (ng/ml)a
% Inhibition of TSG-14
2a
2c
2d
2e
3a
3c
3d
3e
4a
4c
4d
4e
6a
6c
6d
6e
7a
7c
7d
7e
Control
LPS
0.10 ꢀ 0.011
0.098 ꢀ 0.002
0.10 ꢀ 0.006
0.056 ꢀ 0.006
0.10 ꢀ 0.008
0.099 ꢀ 0.002
0.1 ꢀ 0.01
0
2
0
44
0
1
0
1
50
2
2
70
65
61
60
80
23
0
2. Results and discussion
2.1. Chemistry
A representative procedure for this synthesis is shown in
Scheme 1. Compounds 1aee was considered the starting key ma-
terials using synthetic sequence reported in previous work [25].
The reaction of 1aee with carbon disulfide in pyridine afforded the
corresponding pyrrolothiazine-2-thione derivatives 2aee. Subse-
0.099 ꢀ 0.01
0.05 ꢀ 0.001
0.098 ꢀ 0.012
0.098 ꢀ 0.002
0.03 ꢀ 0.001
0.035 ꢀ 0.006
0.039 ꢀ 0.006
0.040 ꢀ 0.002
0.02 ꢀ 0.001
0.077 ꢀ 0.002
0.1 ꢀ 0.006
quent alkylation of pyrrolothiazin-2-thione 2aee with
a-halo and
b-halo alkyl halide gave the corresponding S-alkylated pyrrolo-
thiazine-4-imine derivatives 3aee and 4aee. Upon refluxing
compounds 2aee with 5% potassium hydroxide solution, 4-
iminopyrrolopyrimidine derivatives 5aee were directly obtained.
Alternatively, 3-ethylpyrrolo- pyrimidines 6aee and 3-amino pyr-
rolopyrimidines 7aee were obtained by stirring of 2aee with
ethylamine or hydrazine hydrate, independently, for seven days.
The synthesized compounds were purified by standard procedures
like column chromatography and crystallization. All the assigned
structures were confirmed by spectroscopic and element micro-
analytical data. (c.f. experimental and Scheme 1).
0.1 ꢀ 0.01
0
33
e
0.067 ꢀ 0.002
0.10 ꢀ 0.006
0.5 ꢀ 0.006
e
a
Conc. of LPS-stimulated TSG-14 production in rat whole blood, expressed as
means ꢀ S.E.M. (n ¼ 3).
whole serum rat was followed where production was determined
by comparison of yield, after LPS injection, with a control to which
no test compound was added [26,27]. One variation from
this protocol is that serum TSG-14/PTX3 levels were determined at
4e6 h after LPS injection, which is a time point of maximal eleva-
tion of serum TSG-14, as described in the literature [22], results
2.2. Elisa measurement of TSG-14/PTX3
The newly synthesized derivatives were evaluated for their
ability to inhibit LPS stimulated TSG-14/PTX3. It is well documented
that TSG-14 become elevated in the serum of mice, rat and human
after injection with bacterial lipopolysaccharide, it is also induced
presented in Table 1. In the course of evaluating the TNF-
a
by TNF-a [22,23]. Thus, the same protocol for TNF-a evaluation in
Scheme 1. Synthesis of pyrrolothiazines 2e4 and pyrrolopyrimidines 5e7 series. Reagents and conditions: (i) CS2, Pyridine, reflux, 3 h; (ii) CH3CH2I, NaH, DMF, stirring, 24 h; (iii) 2-
Bromobutane, NaH, DMF, stirring, 24 h; (iv) 5% KOH, reflux in w.b., 2 h; (v) CH3CH2NH2, stirring, 7 days; (vi) NH2NH2.H2O, stirring, 7 days.

K.M.H. Hilmy et al. / European Journal of Medicinal Chemistry 78 (2014) 419e424
421
inhibitory effect of new small molecules we attempted to explore
sulfur containing derivatives. The prototypical vicinal diaryl was
held constant in all the prepared compounds, while varying sub-
stituents on the pyrimidine or thiazine heterocycle. The most
potent compounds of the synthesized series was the 6-(4-
4.1.1.1. 6,7-Diphenyl-4-iminopyrrolo[2,3-d][1,3]thiazin-2(1H,4H,7H)-
thione (2a). Yield: (2.21 g, 66%); mp: 245e247 ꢁC. IR (KBr) cmꢂ1
:
3277 (NH), 1568 (C]S), 1635 (C]NH); MS (m/z, %): 335.00 (Mþ,
100), 336.00 (Mþ1, 19.70), 337.00 (Mþ2, 12.60). 1H NMR ((DMSO-d6)
d/ppm: 6.72 (s, 1H, Pyrrole-H), 6.95e7.48 (m, 10H, AreH), 9.58 (s,
Bromophenyl)-3-ethyl-4-iminepyrrolo-pyrimidine-2-thione
6e,
1H, NH-, D2O exchangeable), 12.07 (s, 1H,imineeNHe, D2O
exchangeable). Anal. calcd for C18H13N3S2 (335.06): C, 64.45; H,
3.91; N, 12.53; S, 19.12. Found: C, 64.17; H, 3.54; N, 12.75; S, 19.47.
showing an 80% inhibition in the TSG-14 induction, in fact all de-
rivatives of this series were potent inhibitors of the TSG-14. Insights
into the structure activity relationship of the tested compounds
showed that within the same series of compounds the 6-(4-
bromophenyl) derivatives displayed the best inhibitory effect fol-
lowed by the unsubstituted 6-phenyl derivatives. This can be
clearly observed from the results obtained from the 2-sec-
butylthio-4-iminepyrrolothiazines 4a and 4e (50% and 70%,
respectively), the 3-amino-4-imine pyrrolopyrimidine-2-thione
derivatives 7a and 7e (23% and 33%, respectively) and the
4-iminepyrrolothiazine-2-thione 2e (44%). Moreover, it was
observed that these N-alkylated derivatives 6aee were more potent
compared to their S-alkylated analogs 4aee and 3aee. Further-
more, it seemed that increasing the bulk of the S-substituent,
resulted in more effective compounds, as observed in the results
obtained for 4e and 2e (70% and 44%, respectively). It could be
concluded that upon comparing the activity of the S-alkylated de-
rivatives to their N-alkylated counterparts, it was observed that the
N-alkyl derivatives were more potent, especially for the bromo and
unsubstituted derivatives 4a and 4e.
4.1.1.2. 6-(4-Fluorophenyl)-4-imino-7-phenylpyrrolo[2,3-d][1,3]thia-
zin-2 (1H,4H,7H)-thione (2b). Yield: (2.75 g, 78%); mp: 248e250 ꢁC.
IR (KBr) cmꢂ1: 3277 (NH), 1570 (C]S), 1640 (C]N); MS (m/z, %):
353.00 (Mþ, 100), 353.90 (Mþ1, 30.63), 355.00 (Mþ2, 12.18). 1H NMR
(DMSO-d6) d/ppm: 6.76 (s, 1H, Pyrrole-H), 6.90e7.47 (m, 9H, AreH),
9.59 (s, 1H, NHe, D2O exchangeable), 12.07 (s, 1H, imineeNHe, D2O
exchangeable). Anal. calcd for C18H12FN3S2 (353.05): C, 61.17; H,
3.42; F, 5.38; N, 11.89; S, 18.14. Found: C, 61.47; H, 3.12; F, 5.08; N,
11.50; S, 18.38.
4.1.1.3. 6-(4-Chlorophenyl)-4-imino-7-phenylpyrrolo[2,3-d][1,3]
thiazin-2 (1H,4H,7H)-thione (2c). Yield: (2.65 g, 72%); mp: 240e
242 ꢁC. IR (KBr) cmꢂ1: 3271 (NH), 1565 (C]S), 1625 (C]NH); MS
(m/z, %): 369.00 (Mþ, Cl35, 100), 370.00 (Mþ1, 27.66), 371.00 (Mþ2
,
Cl37, 45.89). 1H NMR (DMSO-d6)
d/ppm: 6.77 (s, 1H, Pyrrole-H),
6.88e7.50 (m, 9H, AreH), 9.59 (s, 1H, NH-, D2O exchangeable),
12.07 (s, 1H, imineeNHe, D2O exchangeable). 13C NMR (DMSO-d6)
d
/ppm: 97.88, 101.96, 128.52, 128.71, 129.03, 129.50, 129.67, 132.61,
134.62, 135.66, 153.76 (C]N), 170.61, 180.91(C]S). Anal. Calcd for
18H12ClN3S2 (369.02): C, 58.45; H, 3.27; Cl, 9.58; N, 11.36; S, 17.34.
3. Conclusions
C
In conclusion, we found that nine of the tested compounds were
potent inhibitors of TSG-14. The most potent derivatives were 6e
and 4e with 80% and 70% inhibition, respectively. Both of them
were 6-(4-bromophenyl) derivatives. Compounds 6a and 6c
showed good inhibitory action (65% and 61%, respectively), while
4a displayed moderate activity as it inhibited 50% of the induced
gene. These new series of compounds may serve as valuable new
agents that could play an important role in controlling acute and
chronic inflammatory reactions through novel targets that play a
crucial role in these processes.
Found: C, 58.13; H, 3.57; Cl, 9.23; N, 11.04; S, 17.67.
4.1.1.4. 4-Imino-7-phenyl-6-p-tolylpyrrolo[2,3-d][1,3]thiazin-
2(1H,4H,7H)-thione (2d). Yield: (2.72 g, 78%); mp: 238e240 ꢁC. IR
(KBr) cmꢂ1: 3267 (NH), 1558 (C]S), 1631 (C]NH); MS (m/z, %):
349.00 (Mþ, 100), 350.00 (Mþ1, 27.08), 351.00 (Mþ2, 12.66). 1H NMR
(DMSO-d6) d/ppm: 2.17 (s, 3H, CH3), 6.75 (s, 1H, Pyrrole-H), 6.78e
7.48 (m, 9H, AreH), 9.59 (s, 1H, NH-, D2O exchangeable), 12.08 (s,
1H, imineeNHe, D2O exchangeable). Anal. calcd for C19H15N3S2
(349.07): C, 65.30; H, 4.33; N, 12.02; S, 18.35. Found: C, 65.10; H,
4.66; N, 11.63; S, 18.03.
4. Experimental
4.1.1.5. 6-(4-Bromophenyl)-4-imino-7-phenylpyrrolo[2,3-d][1,3]
thiazin-2 (1H,4H,7H)-thione (2e). Yield: (3.06 g, 74%); mp: 265e
267 ꢁC. IR (KBr) cmꢂ1: 3272 (NH), 1562 (C]S), 1639 (C]NH); MS
4.1. Chemistry
Melting points were determined on a Stuart melting point
apparatus and are uncorrected. IR spectra (KBr) were measured on
Jasco FT/IR 460 plus (Japan), 1H NMR and 13C NMR spectra were
recorded on Gemini 300 MHz in DMSO-d6 as solvent, using
tetramethyl-silane (TMS) as internal reference standard. The
chemical shifts values are expressed in ppm. Elemental analysis (C,
H, and N) was performed by a Vario III CHN analyzer (Germany).
All compounds were within ꢀ0.4% of the theoretical values. Mass
spectra were run on DI analysis Shimadzu QP-2010 plus
mass spectrometer. All spectroscopic analysis were made at the
Microanalytical Unit of Cairo University. The progress of the reac-
tion and the purity of the compounds were monitored by TLC
analytical silica gel plates 60 F254 eluting with CH2Cl2/CH3OH
(39:1). The chemical reagents used in synthesis were purchased
From Fluka, Sigma, and Aldrich.
(m/z, %): 413.00 (Mþ, Br79, 67.2), 414.00 (Mþ1, 24.66), 415.00 (Mþ2
,
Br81, 74.1). 1H NMR (DMSO-d6)
d/ppm: 6.81 (s,1H, Pyrrole-H), 6.88e
7.46 (m, 9H, AreH), 9.62 (s, 1H, NH-, D2O exchangeable), 12.07 (s,
1H, imineeNHe, D2O exchangeable).Anal. calcd for C18H12BrN3S2
(413.97): C, 52.18; H, 2.92; Br, 19.28; N, 10.14; S, 15.48; found: C,
52.54; H, 2.90; Br, 19.08; N, 10.43; S, 15.15.
4.1.2. General methods of preparation of 3aee
A mixture of 2aee (14 mmol), ethyl iodide (3310 mg, 21.2 mmol)
and anhydrous NaH (100 mg, 41 mmol) in DMF (30 ml) was stirred
for 24 h. Then the mixture was poured onto ice. The precipitate was
then filtered and crystallized out from methanol.
4.1.2.1. 2-(Ethylthio)-6,7-diphenylpyrrolo[2,3-d][1,3]thiazin-4(7H)-
imine (3a). Yield: (3.55 g, 70%); mp: 135e137 ꢁC. IR (KBr) cmꢂ1
:
1631 (C]NH), 3293 (NH); MS (m/z, %): 363.00 (Mþ, 17.81), 364.00
4.1.1. General procedure for the synthesis of compounds 2aee
A mixture of 2-amino-3-cyano-1-phenyl-5-arylpyrroles 1aee
[25] (10 mmol) and carbon disulfide (10 ml) was refluxed in pyri-
dine (8 ml) for 3 h. On addition of diethyl ether gave 2aee. The
precipitate was crystallized from ethanol.
(Mþ1, 4.75), 365 (Mþ2, 1.63). 1H NMR (DMSO-d6)
d
/ppm: 1.32 (t, 3H,
CH3), 3.15 (q, 2H, CH2), 6.70 (s, 1H, H-Pyrrole), 7.00e7.46 (m, 10H,
AreH), 13.66 (s, 1H, imineeNHe, D2O exchangeable). Anal. calcd for
C20H17N3S2 (363.08): C, 66.08; H, 4.71; N, 11.56; S, 17.64. Found: C,
65.88; H, 4.40; N, 11.26; S, 17.32.

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K.M.H. Hilmy et al. / European Journal of Medicinal Chemistry 78 (2014) 419e424
4.1.2.2. 2-(Ethylthio)-6-(4-fluorophenyl)-7-phenylpyrrolo[2,3-d][1,3]
thiazin-4(7H)-imine (3b). Yield: (4.53 g, 85%); mp: 140e142 ꢁC. IR
(KBr) cmꢂ1: 1632 (C]NH), 3291 (NH); MS (m/z, %): 381.00 (Mþ,
21.92), 382.00 (Mþ1, 7.65), 383.00 (M þ2, 2.55). 1H NMR (DMSO-d6)
(409.11): C, 64.52; H, 4.92; F, 4.64; N, 10.26; S, 15.66; Found: C,
64.13; H, 4.59; F, 4.33; N, 10.59; S, 15.98.
4.1.3.3. 2-(Sec-butylthio)-6-(4-chlorophenyl)-7-phenylpyrrolo[2,3-d]
[1,3] thiazin-4(7H)-imine (4c). Yield: (5.05 g, 85%); mp: 160e162 ꢁC
.IR (KBr) cmꢂ1: 1643 (C]NH), 3286 (NH); MS (m/z, %): 425.00 (Mþ,
Cl35, 100), 426.00 (Mþ1, 29.79), 427.00 (Mþ2,Cl37, 44.90). 1H NMR
(DMSO-d6) d/ppm: 0.96 (t, 3H, CH3), 1.39 (d, 3H, CH3), 4.13 (m, 2H,
CH2), 3.26 (m, 1H, CH), 6.77 (s, 1H, H-pyrrole), 6.86e7.46 (m, 9H,
AreH), 13.66 (s, 1H, imineeNHe, D2O exchangeable). Anal. calcd for
d/ppm: 1.31 (t, 3H, CH3), 3.11 (q, 2H, CH2), 6.80 (s, 1H, H-Pyrrole),
7.05e7.54 (m, 9H, AreH), 13.69 (s, 1H, imineeNHe, D2O exchan-
geable).Anal. calcd for C20H16FN3S2 (381.08): C, 62.97; H, 4.23; F,
4.98; N, 11.01; S, 16.81. found: C, 62.58; H, 4.55; F, 4.65; N, 11.33; S,
16.45.
4.1.2.3. 6-(4-Chlorophenyl)-2-(ethylthio)-7-phenylpyrrolo [2, 3-d]
[1,3]thiazin-4(7H)-imine (3c). Yield: (4.44 g, 80%); MP: 162e164 ꢁC.
IR (KBr) cmꢂ1: 1628 (C]NH), 3299 (NH); MS (m/z, %): 397.00 (Mþ,
Cl35, 4.82), 398.00 (Mþ1, 4.10), 399.00 (Mþ2, Cl37, 2.06). 1H NMR
C22H20ClN3S2 (426.01): C, 62.03; H, 4.73; Cl, 8.32; N, 9.86; S, 15.05; .
Found: C, 62.36; H, 4.40; Cl, 8.02; N, 9.50; S, 15.35.
4.1.3.4. 2-(Sec-butylthio)-7-phenyl-6-p-tolylpyrrolo[2,3-d][1,3]thia-
zin-4(7H)-imine (4d). Yield: (4.65 g, 82%); mp: 160e162 ꢁC IR (KBr)
cmꢂ1: 1639 (C]NH), 3286 (NH); (m/z, %): 405.00 (Mþ, 66.62),
(DMSO-d6) d/ppm: 1.34 (t, 3H, CH3), 3.13 (q, 2H, CH2), 6.79 (s, 1H, H-
Pyrrole), 7.13e7.53 (m, 9H, AreH), 13.67 (s, 1H, imineeNHe, D2O
exchangeable). Anal. calcd for C20H16ClN3S2 (397.05): C, 60.36; H,
4.05; Cl, 8.91; N, 10.56; S, 16.12. Found: C, 60.03; H, 4.44; Cl, 8.55; N,
10.23; S, 16.47.
406.00 (Mþ1, 20.68), 407.00 (Mþ2, 8.35). 1H NMR (DMSO-d6)
d/ppm:
0.97 (t, 3H, CH3), 1.39 (d, 3H, CH3), 2.32 (s, 3H, CH3), 4.15 (m, 2H,
CH2), 3.20 (m, 1H, CH), 6.78 (s, 1H, H-pyrrole), 6.88e7.49 (m, 9H,
AreH), 13.65 (s, 1H, imineeNHe, D2O exchangeable). Anal. calcd for
4.1.2.4. 2-(Ethylthio)-7-phenyl-6-p-tolylpyrrolo[2,3-d][1,3]thiazin-
4(7H)-imine (3d). Yield: (4.06 g, 77%); mp: 160e162 ꢁC. IR (KBr)
cmꢂ1: 1634 (C]NH), 3287 (NH); MP (m/z, %): 377.00 (Mþ, 31.03),
C23H23N3S2 (405.59): C, 68.11; H, 5.72; N, 10.36; S, 15.81. Found: C,
68.44; H, 5.36; N, 10.06; S, 15.47.
378.00 (Mþ1, 17.04), 379.00 (Mþ2, 6.80). 1H NMR (DMSO-d6)
d
/ppm:
4.1.3.5. 6-(4-Bromophenyl)-7-phenyl-2-(Sec-butylthio)pyrrolo[2,3-d]
[1,3] thiazin- 4(7H)-imine (4e). Yield: (5.58 g, 85%); mp: 155e
157 ꢁC. IR (KBr) (cmꢂ1): 1655 (C]NH), 3302 (NH); MS (m/z, %):
1.36 (t, 3H, CH3), 2.28 (s, 3H, CH3), 3.13 (q, 2H, CH2), 6.73 (s, 1H, H-
Pyrrole), 7.02e7.50 (m, 9H, AreH), 13.66 (s, 1H, imineeNHe, D2O
exchangeable). 13C NMR (DMSO-d6)
d
/ppm: 14.84(CH2CH3), 22.77
469.00 (Mþ, Br79, 15.07), 470.00 (Mþ1, 7.83), 471.00 (Mþ2, Br81
,
(SCH2CH3), 24.74 (CH3), 98.80, 101.96, 127.83, 128.01, 128.12,128.44,
16.51). 1H NMR (DMSO-d6)
d/ppm: 0.94 (t, 3H, CH3), 1.37 (d, 3H,
128.99, 135.68, 137.72, 139.03, 151.52(C]N), 162.69. Anal. calcd for
CH3), 1.51 (m, 2H, CH2), 4.17 (m, 1H, CH), 6.80 (s, 1H, H-pyrrole),
6.91e7.50 (m, 9H, AreH), 12.63 (s, 1H, imineeNHe, D2O
exchangeable). Anal. calcd for C22H20BrN3S2 (469.03) : C, 56.17; H,
4.29; Br, 16.98; N, 8.93; S, 13.63. Found: C, 56.51; H, 4.50; Br, 16.64;
N, 8.60; S, 13.31.
C
21H19N3S2 (377.10): C, 66.81; H, 5.07; N, 11.13; S, 16.99. found: C,
66.46; H, 4.78; N, 10.93; S, 16.66.
4.1.2.5. 6-(4-Bromophenyl)-2-(ethylthio)-7-phenylpyrrolo[2,3-d][1,3]
thiazin-4(7H)-imine (3e). Yield: (4.63 g, 75%); mp: 165e167 ꢁC. IR
(KBr) cmꢂ1: 1634 (C]NH), 3301 (NH); MS (m/z, %): 441.00 (Mþ,
Br79, 42.03), 442.00 (Mþ1, 21.04), 443.00 (Mþ2, Br81, 7.80). 1H NMR
4.1.4. General procedure for the synthesis of compounds 5aee
The compounds 2aee (10 mmol) was refluxed with aqueous 5%
KOH (5 ml) on a steam bath for 2 h, and then cooled to room
temperature; the precipitate was collected and washed with water.
The filtrate and washings were acidified with glacial acetic acid to
precipitate the pyrrolopyrimidine dithione 5aee. The precipitate
was crystallized from ethyl acetate.
(DMSO-d6) d/ppm: 1.32 (t, 3H, CH3), 3.11 (q, 2H, CH2), 6.84 (s, 1H, H-
Pyrrole), 6.91e7.51 (m, 9H, AreH), 13.67 (s, 1H, imineeNHe, D2O
exchangeable). Anal. calcd for C20H16BrN3S2 (442.40): C, 54.30; H,
3.65; Br, 18.06; N, 9.50; S, 14.50. Found: C, 54.01; H, 3.33; Br, 17.71;
N, 9.18; S, 14.15.
4.1.3. General method for preparation of 4aee
4.1.4.1. 6,7-Diphenyl-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-
A mixture of 2aee (14 mmol), 2-Bromobutane (2900 mg,
2.12 mmol) and anhydrous NaH (100 mg, 41 mmol) in DMF (30 ml)
was stirred for 24 h. Then the mixture was poured onto ice. The
precipitate was then filtered and crystallized from ethanol.
dithione (5a). Yield: (2.34 g, 70%); mp: 245e247 ꢁC. IR (KBr) cmꢂ1
:
3382 (NH), 1620 (C]S); MS (m/z, %): 335.00 (Mþ, 100.00), 336.00
(Mþ1, 19.7), 337.00 (Mþ2, 9.60). 1H NMR (DMSO-d6)
d/ppm: 6.79 (s,
1H, pyrrole-H), 6.85e7.46 (m, 10H, AreH), 9.93 (s, 1H, NH1e, D2O
exchangeable), 11.95 (s, 1H, NH3e, D2O exchangeable). Anal. calcd
for C18H13N3S2 (335.45): C, 64.45; H, 3.91; N, 12.53; S, 19.12. Found:
C, 64.09; H, 3.60; N, 12.17; S, 19.45.
4.1.3.1. 2-(Sec-butylthio)-6,7-diphenylpyrrolo[2,3-d][1,3]thiazin-
4(7H)-imine (4a). Yield: (4.38 g, 80%); mp: 150e152 ꢁC. IR (KBr)
cmꢂ1: 1650 (C]NH), 3287 (NH); MS (m/z, %): 391.00 (Mþ, 100),
392.00 (Mþ1, 27.96), 393.00 (Mþ2,12.56). 1H NMR (DMSO-d6)
d
/
4.1.4.2. 6-(4-Fluorophenyl)-7-phenyl-1H-pyrrolo[2,3-d]pyrimidine-
2,4(3H,7H)-dithione (5b). Yield: (2.18 g, 62%); mp: 248e250 ꢁC. IR
(KBr) cmꢂ1: 3381 (NH), 1622 (C]S); MS (m/z, %): 353.00 (Mþ,
88.00), 354.00 (Mþ1, 21.70), 355.00 (Mþ2, 9.60). 1H NMR (DMSO-d6)
ppm: 0.95 (t, 3H, CH3), 1.38 (d, 3H, CH3), 3.23 (m, 1H, CH), 4.14 (m,
2H, CH2), 6.76 (s, 1H, H-pyrrole), 6.87e7.48 (m, 10H, AreH), 13.66 (s,
1H, imineeNHeD2O exchangeable). Anal. calcd for C22H21N3S2
(391.12): C, 67.49; H, 5.41; N, 10.73; S, 16.38. Found: C, 67.28; H,
5.03; N, 10.40; S, 16.08.
d
/ppm: 6.75 (s, 1H, pyrrole-H), 6.79e7.53 (m, 9H, AreH), 9.93 (s, 1H,
NH1, D2O exchangeable), 11.90 (s, 1H, NH3e, D2O exchangeable).
Anal. calcd for C18H12FN3S2 (353.05): C, 61.17; H, 3.42; F, 5.38; N,
11.89; S, 18.14. Found: C, 61.36; H, 3.10; F, 5.18; N, 11.57; S, 18.48.
4.1.3.2. 2-(Sec-butylthio)-6-(4-fluorophenyl)-7-phenylpyrrolo[2,3-d]
[1,3] thiazin-4(7H)-imine (4b). Yield: (4.86 g, 85%); m.p.180e182 ꢁC.
IR (KBr) cmꢂ1: 1648 (C]NH), 3291 (NH). m/z, %: 409.00 (Mþ, 100),
4.1.4.3. 6-(4-Chlorophenyl)-7-phenyl-1H-pyrrolo[2,3-d]pyrimidine-
410.00 (Mþ1, 27.49), 411.00 (Mþ2, 12.45). 1H NMR (DMSO-d6)
d/
2,4(3H,7H)-dithione (5c). Yield: (2.21 g, 60%); m.p. 240e242 ꢁC. IR
ppm: 0.95 (t, 3H, CH3), 1.37 (d, 3H, CH3), 4.12 (m, 2H, CH2), 3.21 (m,
1H, CH), 6.75 (s, 1H, H-pyrrole), 6.85e7.49 (m, 9H, AreH), 13.64 (s,
1H, imineeNHe, D2O exchangeable). Anal. calcd for C22H20FN3S2
(KBr) cmꢂ1, 3378 (NH), 1618 (C]S); MS (m/z, %): 369.00 (Mþ, Cl35
,
100.00), 370.00 (Mþ1, 21.20), 371.00 (Mþ2, Cl37, 41.60). 1H NMR
(DMSO-d6) /ppm: 6.75 (s,1H, H-pyrrole), 6.86e7.51 (m, 9H, AreH),
d

K.M.H. Hilmy et al. / European Journal of Medicinal Chemistry 78 (2014) 419e424
423
9.91 (s, 1H, NH1e), 11.93 (s, 1H, NH3e, D2O exchangeable). 13C NMR
(DMSO-d6) /ppm: 97.88, 101.96, 128.52, 128.71, 129.03, 129.50,
MS (m/z, %): 360.00 (Mþ, 8.19), 361.00 (Mþ1, 9.61), 362.00 (Mþ2
8.68). 1H NMR (DMSO-d6)
/ppm: 1.15 (t, 3H, CH3), 2.24 (s, 3H, CH3),
,
d
d
129.67, 132.61, 134.62, 135.66, 153.67, 170.61, 180.91 (2C]S). Anal.
calcd for C18H12ClN3S2 (369.90): C, 58.45; H, 3.27; Cl, 9.58; N, 11.36;
S, 17.34. Found: C, 58.13; H, 2.96; Cl, 9.42; N, 11.03; S, 17.04.
2.84 (q, 2H, CH2), 6.77 (s, 1H, H-Pyrrole), 6.80e7.44 (m, 9H, AreH),
8.27 (s, 1H, NH-, D2O exchangeable), 10.35 (s, 1H, imineeNHe, D2O
exchangeable). 13C NMR (DMSO-d6)
d/ppm: 12.80 (CH2CH3), 20.61
(CH3), 34.26(NCH2CH3), 96.32, 99.50, 100.57, 127.75, 128.41, 128.47,
128.95, 135.73, 136.04, 136.87, 149.87, 162.18(C]N), 171.78(C]S).
Anal. calcd for C21H20N4S (360.48): C, 69.97; H, 5.59; N, 15.54; S,
8.90. Found: C, 69.62; H, 5.28; N, 15.20; S, 8.55.
4.1.4.4. 7-Phenyl-6-p-tolyl-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-
dithione (5d). Yield: (2.19 g, 63%); mp: 238e240 ꢁC. IR (KBr)
(cmꢂ1): 3378 (NH), 1613 (C]S); MS (m/z, %): 349.00 (Mþ, 100.00),
350.00 (Mþ1, 23.70), 351.00 (Mþ2, 12.60). 1H NMR (DMSO-d6)
d/
ppm: 2.33 (s, 3H, CH3), 6.71 (s, 1H, pyrrole-H), 6.88e7.45 (m, 9H,
AreH), 9.95 (s, 1H, NH1e, D2O exchangeable), 11.90 (s, 1H, NH3-,
D2O exchangeable). Anal. calcd for C19H15N3S2 (349.48): C, 65.30; H,
4.33; N, 12.02; S, 18.35. Found: C, 65.62; H, 4.03; N, 12.35; S, 18.04.
4.1.5.5. 6-(4-Bromophenyl)3-ethyl-3,4-dihydro-4-imino-7-phenyl-
1H-pyrrolo [2,3-d]pyrimidine-2(7H)-thione (6e). Yield: (6.30 g,
75%); mp: 235e237 ꢁC. IR (KBr) cmꢂ1: 3379 (NH), 1586 (C]S),
1630.84 (¼NH); MS (m/z, %): 424.00 (Mþ, Br79, 20.96), 425.00 (Mþ1
,
9.25), 426 (Mþ2, Br81, 10.39). 1H NMR (DMSO-d6)
d/ppm: 1.15 (t, 3H,
4.1.4.5. 6-(4-Bromophenyl)-7-phenyl-1H-pyrrolo[2,3-d]pyrimidine-
2,4(3H,7H)-dithione (5e). Yield: (2.50 g, 71%); mp: 248e250 ꢁC. IR
(KBr) (cmꢂ1): 3387 (NH), 1630 (C]S); MS (m/z, %): 414.00 (Mþ,
CH3), 2.84 (q, 2H, CH2), 6.77 (s, 1H, H-Pyrrole), 6.80e7.43 (m, 9H,
AreH), 8.26 (s, 1H, NH-, D2O exchangeable), 10.35 (s, 1H, iminee
NHe, D2O exchangeable). 13C NMR (DMSO-d6)
d/ppm: 12.74
88.00), 415.00 (Mþ1, 21.70), 416.00 (Mþ2, 9.6). 1H NMR (DMSO-d6)
d/
(CH2CH3), 34.23(NCH2CH3), 96.35, 99.55, 100.59, 127.79, 128.45,
128.49, 128.85, 128.90, 135.70, 136.11, 149.88, 162.17(C]N),
171.79(C]S). Anal. calcd for C20H17BrN4S (425.35): C, 56.48; H,
4.03; Br, 18.79; N, 13.17; S, 7.54. Found: C, 56.28; H, 4.40; Br, 18.46;
N, 13.35; S, 7.20.
ppm: 6.74 (s, 1H, pyrrole-H), 6.79e7.63 (m, 9H, AreH), 9.94 (s, 1H,
NH1e, D2O exchangeable), 11.92 (s, 1H, NH3e, D2O exchangeable).
Anal. calcd for C18H12BrN3S2 (414.35): C, 52.18; H, 2.92; Br, 19.28; N,
10.14; S, 15.48. Found: C, 52.36; H, 3.10; Br, 19.12; N, 10.50; S, 15.15.
4.1.5. General methods of preparation of 6aee
4.1.6. General methods of preparation of 7aee
A mixture of 2aee (20 mmol) and excess of ethylamine, stirring
the mixture for 7 days, and then the solvent was concentrated. The
residue was purified by column chromatography using eluent
CH2Cl2:CH3OH (39:1).
A mixture of 2aee (10 mmol) and hydrazine hydrate (5.006 mg,
10 mmol) was stirred for about 7 days. Then added ice; filtered and
washed with water. The precipitate was crystallized from ethanol.
4.1.6.1. 3-Amino-6,7-diphenyl-4-imino-1,3,4,7-tetrahydro-2H-pyr-
rolo[2,3-d] pyrimidine-2-thione (7a). Yield: (2.33 g, 70%); mp: 210e
212 ꢁC. IR (KBr) cmꢂ1: 3295.75 (NH), 1568 (C]S), 1635.84 (¼NH),
2926 (NH2); MS (m/z,%): 333.00 (Mþ, 42.20), 334.00 (Mþ1, 10.27)
4.1.5.1. 3-Ethyl-3,4-dihydro-4-imino-6,7-diphenyl-1H-pyrrolo[2,3-d]
pyrimidine-2(7H)ethione (6a). Yield: (5.39 g, 78%); mp: 210e
212 ꢁC. IR (KBr) cmꢂ1: 3380 (NH), 1580 (C]S), 1630 (¼NH); MS (m/
z, %): 346.00 (Mþ, 100.00), 347.00 (Mþ1, 27.08), 348.00 (Mþ2, 8.94).
335.00 (Mþ2, 3.43). 1H NMR (DMSO-d6)
d/ppm: 6.37 (s, 2H, NH2,
1H NMR (DMSO-d6)
d/ppm: 1.15 (t, 3H, CH3), 2.81 (q, 2H, CH2), 6.76
D2O exchangeable), 6.91 (s, 1H, H-Pyrrole), 6.961e7.47 (m, 10H, Are
H), 8.30 (s, 1H, NHe, D2O exchangeable), 10.43 (s, 1H, imineeNHe,
D2O exchangeable). Anal. calcd for C18H15N5S (333.42): C, 64.84; H,
4.53; N, 21.01; S, 9.62. Found: C, 64.45; H, 4.31; N, 21.13; S, 9.42.
(s, 1H, H-Pyrrole), 6.77e7.46 (m, 10H, AreH), 8.27 (s, 1H, NH-, D2O
exchangeable), 10.35 (s, 1H, imineeNHe, D2O exchangeable). Anal.
Calcd. for C20H18N4S (346.46): C, 69.34; H, 5.24; N, 16.17; S, 9.25.
found: C, 69.02; H, 5.57; N, 16.47; S, 8.90.
4.1.6.2. 3-Amino-6-(4-fluorophenyl)-4-imino-7-phenyl-1,3,4,7-
4.1.5.2. 3-Ethyl-6-(4-fluorophenyl)3,4-dihydro-4-imino-7-phenyl-
1H-pyrrolo [2,3-d]pyrimidine-2(7H)ethione (6b). Yield: (5.09 g,
70%); mp: 225e227 ꢁC. IR (KBr) cmꢂ1: 3384 (NH), 1583 (C]S),
1630.84 (¼NH); MS (m/z, %): 364.00 (Mþ,10.69), 365.00 (Mþ1, 2.88),
tetrahydro-2H-pyrrolo[2,3-d]pyrimidine-2-thione
(7b). Yield:
(2.73 g, 78%); mp: 222e224 ꢁC. IR (KBr) cmꢂ1: 3299 (NH), 1571 (C]
S), 1635.84 (¼NH), 2929 (NH2); MS (m/z, %):351.00 (Mþ, 86.03),
352.00 (Mþ1, 21.24), 353.00 (Mþ2, 11.11). 1H NMR (DMSO-d6)
d/
366.00 (Mþ2, 2.50). 1H NMR (DMSO-d6)
d/ppm: 1.15 (t, 3H, CH3),
ppm: 6.37 (s, 2H, NH2, D2O exchangeable), 6.92 (s, 1H, H-Pyrrole),
6.88e7.52 (m, 9H, AreH), 8.30 (s, 1H, NH-, D2O exchangeable),10.41
(s, 1H, imineeNHe, D2O exchangeable). Anal. calcd for C18H14FN5S
(351.40): C, 61.52; H, 4.02; F, 5.41; N, 19.93; S, 9.12. Found: C, 61.23;
H, 3.68; F, 5.20; N, 19.60; S, 8.84.
2.83 (q, 2H, CH2), 6.77 (s, 1H, H-Pyrrole), 6.79e7.45 (m, 9H, AreH),
8.26 (s, 1H, NH-, D2O exchangeable), 10.35 (s, 1H, imineeNHe, D2O
exchangeable). Anal. calcd for C20H17FN4S (364.45):C, 65.91; H,
4.70; F, 5.21; N, 15.37; S, 8.80. Found: C, 65.58; H, 4.38; F, 4.90; N,
15.02; S, 9.13.
4.1.6.3. 3-Amino-6-(4-chlorophenyl)-4-imino-7-phenyl-1,3,4,7-
4.1.5.3. 6-(4-Chlorophenyl)3-ethyl-3,4-dihydro-4-imino-7-phenyl-
1H-pyrrolo [2,3-d]pyrimidine-2(7H)ethione (6c). Yield (5.32 g,
70%); mp: 280e282 ꢁC IR (KBr) cmꢂ1: 3382 (NH), 1581 (C]S), 1636
(¼NH); MS (m/z, %): 380.00 (Mþ, Cl35, 15.96), 381.00 (Mþ1, 20.86),
tetrahydro-2H-pyrrolo[2,3-d]pyrimidine-2-thione
(7c). Yield:
(2.49 g, 68%); mp: 200e202 ꢁC. IR (KBr) cmꢂ1: 3301 (NH), 1569 (C]
S), 1635.84 (¼NH), 2899 (NH2); MS (m/z, %): 367.00 (Mþ, Cl35, 5.26),
368.00 (Mþ1,4.68), 369.00 (Mþ2, Cl37, 100). 1H NMR (DMSO-d6)
d/
382.00 (Mþ2, Cl37, 7.96). 1H NMR (DMSO-d6)
d/ppm: 1.16 (t, 3H,
ppm: 6.37 (s, 2H, NH2, D2O exchangeable), 6.85 (s, 1H, H-Pyrrole),
6.90e7.45 (m, 9H, AreH), 8.32 (s,1H, NH-, D2O exchangeable),10.43
(s, 1H, imineeNHe, D2O exchangeable), Anal. calcd for C18H14ClN5S
(367.86): C, 58.77; H, 3.84; Cl, 9.64; N, 19.04; S, 8.72. Found: C,
58.55; H, 3.60; Cl, 9.33; N, 18.69; S, 8.41.
CH3), 2.81 (q, 2H, CH2), 6.76 (s, 1H, H-Pyrrole), 6.79e7.45 (m, 9H,
AreH), 8.26 (s, 1H, NH-, D2O exchangeable), 10.35 (s, 1H, ]NHe,
D2O exchangeable).Anal. calcd for C20H17ClN4S (380.90): C, 63.07;
H, 4.50; Cl, 9.31; N,14.71; S, 8.42.found: C, 63.37; H, 4.15; Cl, 8.98; N,
14.40; S, 8.23.
4.1.6.4. 3-Amino-4-Imino-6-(4-methylphenyl)-7-phenyl-1,3,4,7-
4.1.5.4. 3-Ethyl-3,4-dihydro-4-imino-7-phenyl-6-p-tolyl-1H-pyrrolo
[2,3-d] pyrimidine-2(7H)-thione (6d). Yield: (5.04 g, 70%); mp:
198e200 ꢁC. IR (KBr) cmꢂ1: 3371 (NH), 1579 (C]S), 1630.84 (¼NH);
tetrahydro-2H-pyrrolo[2,3-d]pyrimidine-2-thione
(7d). Yield:
(2.49 g, 72%); mp: 220e222 ꢁC. IR (KBr) cmꢂ1: 3289 (NH), 1558 (C]
S), 1635.84 (¼NH), 2926 (NH2); MS (m/z, %): 347.00 (Mþ, 87.22),

424
K.M.H. Hilmy et al. / European Journal of Medicinal Chemistry 78 (2014) 419e424
348.00 (Mþ1, 3. 11), 349.00 (Mþ2, 11). 1H NMR (DMSO-d6)
d
/ppm:
References
2.20 (s, 3H, CH3), 6.37 (s, 2H, NH2, D2O exchangeable), 6.86 (s,1H, H-
Pyrrole), 7.02e7.46 (m, 9H, AreH), 8.26 (s, 1H, NH-, D2O
exchangeable), 10.42 (s, 1H, imineeNHe, D2O exchangeable). 13C
[1] D.G. Souza, A.C. Soares, V. Pinho, H. Torloni, L.F.L. Reis, M.T. Martins,
A.A.M. Dias, American Journal of Pathology 160 (5) (2002) 1755e1765.
[2] A.A. Dias, A.R. Goodman, J.L. Dos Santos, R.N. Gomes, A. Altmeyer, P.T. Bozza,
M.F. Horta, J. Vilcek, L.F. Reis, Journal of Biology 69 (6) (2001) 928e936.
[3] G.W. Lee, A.R. Goodman, T.H. Lee, J. Vilcek, Journal of Immunology 153 (1994)
3700e3707.
NMR (DMSO-d6) d/ppm: 20.61 (CH3), 96.32, 99.50, 100.57, 127.75,
128.41, 128.47, 128.85, 128.95, 136.04, 136.87, 148.28, 149.87, 162.10
(C]N), 171.78 (C]S). Anal. calcd for C19H17N5S (347.44): C, 65.68;
H, 4.93; N, 20.16; S, 9.23. Found: C, 65.35; H, 4.70; N, 19.80; S, 9.03.
[4] H.G. Wisneiwski, J. Vilcek, Cytokine & Growth Factor Reviews 15 (2e3) (2004)
129e146.
[5] B.B. Aggrawal, A. Samanta, M. Feldman, TNF-alpha, in: J.D. Oppenheim,
M. Feldmann (Eds.), Cytokine Reference, Academic Press, London 2, 2001, pp.
413e434.
4.1.6.5. 3-Amino-6-(4-bromophenyl)-4-imino-7-phenyl-1,3,4,7-
[6] A.J. Nauta, B. Bottazzi, A. Mantovani, G. Salvatori, U. Kishore, W.J. Schwaeble,
A.R. Gingras, S. Tzima, F. Vivanco, J. Egido, O. Tijsma, E.C. Hack, M.R. Daha,
A. Roos, European Journal of Immunology 33 (2) (2003) 465e473.
[7] M. Introna, V.V. Alles, M. Castellano, G. Picardi, L. De Gioia, B. Bottazzai, G. Peri,
F. Breviario, M. Salmona, L. De Gregorio, T.A. Dragani, N. Srinivasan,
T.L. Blundell, T.A. Hamilton, A. Mantovani, Blood 87 (1996) 1862e1872.
[8] M.M. Luchetti, G. Piccinini, A. Mantovani, G. Peri, C. Matteucci, G. Pomponio,
M. Fratini, P. Fraticelli, P. Sambo, C. Di Loreto, A. Doni, M. Introna, A. Gabrielli,
Clinical and Experimental Immunology 119 (2000) 196e202.
[9] M.E. Suliman, A.R. Qureshi, J.J. Carrero, P. Barany, M.L. Yilmaz, S. Snaedal-
Jonsdottir, A. Alvestrand, O.H. Rger, B. Lindholm, P. Stenvinkel, Quarterly
Journal of Medicine 101 (5) (2008) 397e405.
tetrahydro-2H-pyrrolo[2,3-d]pyrimidine-2-thione
(7e). Yield:
(3.08 g, 75%); mp: 280e282 ꢁC. IR (KBr) cmꢂ1: 3311 (NH), 1573 (C]
S), 1635.84 (¼NH), 2946 (NH2); MS (m/z, %): 411.00 (Mþ, Br79
,
98.34), 412.00 (Mþ1, 25.39), 413.00 (Mþ2, Br81, 100.00). 1H NMR
(DMSO-d6) d/ppm): 6.37 (s, 2H, NH2, D2O exchangeable), 6.76 (s,1H,
H-Pyrrole), 6.91e7.48 (m, 9H, AreH), 8.31 (s, 1H, NH, D2O
exchangeable), 10.40 (s, 1H, imineeNHe, D2O exchangeable). Anal.
calcd for C18H14BrN5S (411.01): C, 52.44; H, 3.42; Br, 19.38; N, 16.99;
S, 7.78. Found: C, 52.23; H, 3.11; Br, 19.2; N, 16.66; S, 7.42.
[10] X. He, B. Han, M. Liu, American Journal of Physiology e Lung Cellular and Mole
Physiology 292 (5) (2007) L1039eL1049.
[11] E.P. Diamandis, L. Goodglick, C. Planque, M.D. Thornquist, Clinical Cancer
Research 17 (8) (2011) 2395e2399.
[12] T.H. Lee, G.W. Lee, E.B. Ziff, J. Vilcek, Molecular and Cellular Biology 10 (1990)
1982e1988.
4.2. Elisa measurement of TSG-14
Compounds to be tested were administered orally to the albino
rats (male, weight approx. 200 g) 30 min prior to LPS (Escherichia
coli B055/B5, 0.1 mg/kg ip) challenge. Three rats were dosed orally
(30 mg/kg) with a suspension of compound in carboxy methyl-
cellulose 0.5% as described in the literature [26]. Sera were assayed
in duplicates with Rat Pentraxin 3 (PTX3)/TG-14 ELISA Kit, 4-6 hr
after LPS injection, which is a time point of maximal elevation of
serum TSG-14, as described in the literature [22]. Percent inhibition
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This assay employs the quantitative sandwich enzyme immu-
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wells and any PTX3 present is bound by the immobilized antibody.
4.2.2. Sensitivity of the assay
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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