Disulfide linked pyrazole derivatives inhibit phagocytosis of opsonized blood cells

Article abstract of DOI:10.1016/j.bmcl.2013.02.064

Immune thrombocytopenia (ITP) is caused by production of an autoantibody to autologous platelets. ITP can be treated either by reducing platelet destruction or by increasing platelet production. Fcγ receptor mediated phagocytosis of the opsonized blood ce

Full text of DOI:10.1016/j.bmcl.2013.02.064

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2324–2327
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Disulfide linked pyrazole derivatives inhibit phagocytosis
of opsonized blood cells
Meena K. Purohit a,b, Iain Scovell c, Anton Neschadim c, Yulia Katsman c, Donald R. Branch c,
Lakshmi P. Kotra a,d,e,
⇑
a Center for Molecular Design and Preformulations, Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada M5G 1L7
b Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Rajasthan, India
c Research & Development, Canadian Blood Services, Toronto, Ontario, Canada
d McLaughlin Center for Molecular Medicine, University of Toronto, Toronto, Ontario, Canada
e Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 3M2
a r t i c l e i n f o
a b s t r a c t
Article history:
Immune thrombocytopenia (ITP) is caused by production of an autoantibody to autologous platelets. ITP
Received 14 January 2013
Revised 7 February 2013
Accepted 13 February 2013
Available online 26 February 2013
can be treated either by reducing platelet destruction or by increasing platelet production. Fcc receptor
mediated phagocytosis of the opsonized blood cells is a well-accepted mechanism for the underlying
pathogenesis of ITP and inhibition of this phagocytosis process with small molecules is a potential strat-
egy for the development of drugs against ITP. A broad screen indicated that 4-methyl-1-phenyl-pyrazole
derivative (1) could inhibit the phagocytosis of opsonized blood cells with weak potency. We reveal here
the discovery of the polysulfide products, synthesis of various 1-phenyl-pyrazole derivatives, and the bio-
logical evaluation of pyrazole derivatives as inhibitors of phagocytosis for potential use as therapeutics
for ITP. Substitution at C4 of the pyrazole moiety in the disulfide-bridged dimers influenced the potency
in the increasing order of 10 ffi 11 ffi 16 < 19 < 20. A novel scaffold, 20 with an IC50 of 100 nM inhibiting
opsonized blood cell phagocytosis was identified as a potential candidate for further studies. Confirma-
tion of the disulfide bridge additionally provides clues for the non-thiol or non-disulfide bridge carrying
ligands targeting ITP and other similar disorders.
Keywords:
Immune thrombocytopenia
Phagocytosis
IVIG
Small molecule inhibitors
Macrophages
Ó 2013 Elsevier Ltd. All rights reserved.
Thrombocytes, or platelets, are anucleate circulating blood cells
involved in haemostasis. A normal platelet count for an adult hu-
Immunological destruction of platelets occurs in response to
unknown stimuli, and is called ‘immune thrombocytopenia’ or ‘pri-
mary immune thrombocytopenia’ (ITP) which is caused by produc-
tion of autoantibodies to autologous platelets.5 These antibodies
are generated against platelet membrane glycoproteins such as
GP IIb/IIIa. When the antibodies bind to these glycoproteins they
man is between 150,000 and 450,000 per
thrombocyte count drops below 150,000 per
l
L of blood. When the
L of blood, it is de-
l
fined as thrombocytopenia.1,2 The degree of severity of the throm-
bocytopenia is correlated to the platelet counts—a severe form of
thrombocytopenia manifests at platelet counts between 10,000
and 30,000, associated with bleeding with minor trauma and at
counts less than 10,000, associated with spontaneous bleeding as
well as the risk of internal bleeding. Observed reduced platelet
counts could occur either because of decreased platelet production
such as in bone marrow failure, abnormal platelet distribution
(splenomegaly), or increased platelet destruction. Platelet destruc-
tion could be caused by immune-mediated or non-immune medi-
are marking the platelets for destruction. Fcc receptors (FcRs) ex-
pressed on the macrophages recognize these opsonised platelets
and clear them from circulation (Chart 1A).6,7 ITP can be treated
by reducing platelet destruction or by increasing platelet produc-
tion. Current treatments for ITP include corticosteroids, rituximab,
intravenous immunoglobulins (IVIG and anti-D), administrstion of
thrombopoitin receptor (TPO) agonists and surgical measures such
as splenectomy and there are no treatments that directly target the
ated destructive pathways.1,3,4
A
common pathophysiologic
Fcc receptors, which would directly interfere with the platelet rec-
mechanism leading to thrombocytopenia is the disproportionate
amount of consumption or destruction of platelets in circulation
compared to their rate of production.
ognition.8 These therapies have inherent limitations and chal-
lenges, such as the toxicities associated with immunosuppressant
rituximab, relapse of ITP on discontinuation of the TPO receptor
agonist treatment, and possible exposure to blood-borne infections
from IVIG.
Thrombopoietin (TPO) receptor agonists enhance platelet pro-
duction by stimulating the TPO receptor on pluripotent hemato-
poietic stem cells and in megakaryocytes. For this reason, these
⇑
Corresponding author. Tel.: +1 416 581 7601; fax: +1 416 581 7559.
E-mail address: lkotra@uhnres.utoronto.ca (L.P. Kotra).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.064

M. K. Purohit et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2324–2327
2325
A
B
O
Fcγ receptor
H3C
Antibody
N
SH
N
+
Opsonized blood cell
Macrophage
Phagocytosed blood cell
1
Chart 1. (A) Fcc
receptor-mediated phagocytosis of opsonized blood cells. (B) Structure of 1-phenylpyrazole derivative 1.18,19
agonists have been sought after for the treatment of ITP.9 Although
this approach may increase the production of platelets it does not
interfere with the destruction of the thrombocytes. IVIG is limited
in supply since it is produced from thousands of human plasma do-
nors and is an expensive therapy.10 Molecular mechanisms of ac-
tion of IVIG and anti-D therapies is not yet completely
understood, but a number of theories have been proposed.11–15
One of the most likely mechanisms of action for IVIG and anti-D
R
H3C
H3C
O
N
N
OH
O
N
N
a
b
2 R=phenyl
Edaravone
3 R=5-isoxazolyl
therapy is the competitive blockade of Fc
c receptor on macro-
4
R=2-thiophenyl
phages.16,17 This provides an opportunity for the design of small
molecule therapeutics targeting mononuclear phagocytes as a po-
tential treatment for ITP.
Ph
O
Ph
O
Ph
H3C
O
Earlier, several small molecules carrying sulfhydryl and disul-
fide groups on aromatic and aliphatic compounds were screened
for their ability to inhibit the phagocytosis of opsonized thrombo-
cytes by macrophages.18–23 It was hypothesized that these mole-
cules would interact with the sulfhydryl or disulfide groups on
the cell surface of human mononuclear phagocytes and inhibit
the phagocytosis of opsonized red blood cells (RBC). This is based
on the premise that cell surface proteins of monocyte-macro-
phages carry sulfhydryl and disulfide groups that play an impor-
tant role in endocytic-phagocytic function, and small molecules
carrying such groups would function as antagonists to the
phagocytosis process.24,25 Among these compounds, a pyrazole
derivative 1 (Chart 1B) exhibited weak activity at 1 mM concentra-
tion.18 We further explored this discovery to dissect the chemistry
and structure–activity relationships of this class of molecules.
Here, we reveal the synthesis, unexpected discovery of the polysul-
fide products, their evaluation as inhibitors of phagocytosis of the
opsonized blood cells as therapeutics for ITP.
1-Phenyl-1H-pyrazolyl derivatives were synthesized starting
from edaravone as the key starting material. For the synthesis of
compounds 8–11, appropriate aroyl chlorides and edaravone were
first reacted in the presence of calcium hydroxide to obtain the
respective substituted pyrazoles 2–4 (Scheme 1).26 Compounds
2–4 were then chlorinated using phosphorous oxychloride to ob-
tain compounds 5–7.27 The temperatures and reaction times for
chlorination were dependent on the aromaticity and the acid sta-
bility of the heteroaryl groups at the C4 position of the pyrazole
derivatives 2–4. 4-Acyl-5-chloro-pyrazole derivatives 5–7 were
then treated with sodium sulfide to obtain thiolated pyrazoles
8–11: phenyl derivative 5 yielded the corresponding thiol deriva-
tive 8, which was then refluxed in DMF for 3 h to afford the disul-
fide derivative 10; isoxazole derivative 6 yielded the thiol 9,
whereas compound 7 yielded the disulfide derivative 11. Interest-
ingly, treatment of 5 with sodium sulfide resulted in 8 as well as a
minor byproduct the disulfide 10, which was confirmed by LCMS
and could be separated by chromatography.
H3C
d
CH3
S
S
N
SH
N
N
N
N
N
10
8
R
R
O
H3C
O
H3C
5
, c
N
SH
N
N
Cl
N
6
, c
7, c
9 R=5-isoxazolyl
5 R=phenyl
6 R=5-isoxazolyl
7
R=2-thiophenyl
S
S
O
O
H3C
CH3
S
S
N
N
N
N
11
Scheme 1. Synthesis of compounds 8–11. Reagents and conditions: (a) Edaravone,
Ca(OH)2, R-COCl, anhyd 1,4-dioxane, reflux, 3-4 h; (b) POCl3, reflux for 2 h for
R = phenyl (2), or at 80 °C for 50 min for R = 5-isoxazolyl (3), or at 50 °C for 30 min
for R = 2-thienyl (4); (c) Na2S, anhyd DMF, 60 °C, 2–3 h; (d) DMF, 100 °C, 3 h.
Vilsmeier-Hacck reaction conditions (DMF/POCl3) yielded com-
pound 12 (Scheme 2).28 The resulting aldehyde 12 was then oxi-
dized with potassium permanganate under aqueous conditions to
obtain the carboxylic acid 13.29 Subsequent reaction between the
carboxylic acid 13 and the secondary amine (morpholine or piper-
idine) in the presence of HATU and DIPEA at ambient temperature
yielded compounds 14 and 15 in good yields. C4-Carbamyolated
pyrazole derivatives 14 and 15 were reacted with a mixture of so-
dium sulfide and elemental sulfur to yield compounds 16–17.30,31
Weaker activation of the carbamoyl group at C4 in compounds
14–15 may be responsible for the sluggish nucleophilic displace-
ment of the chloride by the sulfide anion. An important observa-
tion was made that routine TLC and NMR characterizations for
Unlike the aryl acid chlorides, treatment of edaravone with
carbamoyl chlorides under Jensen conditions afforded O5-carba-
mates instead of C4-carbamoylated products. Therefore, an
alternative route was used to synthesize C4-carbamoylated deriv-
atives 16, 17 and 19 (Schemes 2 and 3). Edaravone was first formy-
lated at C4 followed by chlorination in situ at C5 position using

2326
M. K. Purohit et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2324–2327
H3C
CHO
Cl
CH3
H3C
CO2H
H3C
S
N
N
N
N
S
Cl
N
N
a
b
N
N
a
b
c
Edaravone
Edaravone
12
H3C
13
ROC
20
COR
Scheme 4. Synthesis of compound 20. Reagents and conditions: (a) Lawesson’s
reagent, 1,4-dioxane, reflux, 2 h; (b) MeOH, rt, 16 h.
H3C
COR
Cl
CH3
N
N
S
N
N
d
N
N
2-7
compounds were then purified. Interestingly, the thiol derivative
8 with no disulfide impurity exhibited both very weak activity at
1 mM concentration and very weak inhibition of phagocytosis.
16 R=4-morpholinyl
17 R=piperidin-1-yl
14
15
R=4-morpholinyl
R=piperidin-1-yl
Since 8 was active only at a higher concentrations than 5 lM in
our current studies, any observed activity for 8 is thought due to
the presence of disulfide 10 as an impurity. Previously reported
findings from Branch and co-workers in which an in vitro MMA as-
say was applied suggested that disulfides were able to inhibit the
phagocytosis of platelets.18 This supported the idea that the disul-
fide 10 is responsible for the additional activity.
Scheme 2. Synthesis of carbamoylated derivatives 16 and 17. Reagents and
conditions: (a) POCl3/DMF (3:1), W at 100 °C, 4 min; (b) KMnO4, water, reflux,
20 h; (c) HATU, DIPEA, anhyd DMF, rt, 30 min; (d) Na2S, elemental sulfur, anhyd
l
DMF, 120 °C, 3–6 h.
Disulfide derivative 10 was separately synthesized and was
evaluated for its potential to inhibit phagocytosis of anti-D opso-
the identification of the products could mistakenly point to either
sulfhydryl or disulfide products and were insufficient to distin-
guish between the mono- and poly-sulfide derivative. While com-
pound 14 yielded a mixture of polysulfide derivatives, they could
be separated to yield the major product, disulfide derivative 16
(Fig. S-3). A polysulfide mixture of compound 17 was reduced into
the corresponding thiol derivative 18 and the disulfide 19 using so-
dium borohydride in ethanol (Scheme 3), which were then sepa-
rated by semi-preparative HPLC. A dimerized edaravone via a
disulfide linker, 20, was prepared via thionation of edaravone with
Lawesson’s reagent followed by overnight stirring in methanol
(Scheme 4). Compound 20 did not carry any substitution at C4 po-
sition of the pyrazole.
Sulfhydryl and disulfide carrying molecules are prone to oxida-
tion and polymerization as was described above and is a major is-
sue when such compounds are investigated in medicinal
chemistry. Synthesis of pure sulfhydryl and disulfide derivatives
of pyrazoles via careful follow-up of the reactions and the separa-
tion of the polymeric forms provided us with the opportunity to
evaluate the role of sulfhydryl moiety, disulfide and higher order
polysulfides in inhibiting phagocytosis and establish a structure–
activity relationship.
Inhibition of opsonized blood cell phagocytosis for various syn-
thesized compounds was evaluated by MMA assay (Table 1). Com-
pound 8 had a tendency to dimerize, on exposure to air, during
sample preparation for biological evaluations into a mixture of
compounds 8 and 10 (Figs. S-1 and S-2, see Supplementary data).
Several batches of compound 8, either a commercially acquired
batch or the ones synthesized in the laboratory (Figs. S-1 and S2;
see Supplementary data), showed varying proportions of dimer-
ized form, compound 10. For example, an HPLC analysis of 8,
immediately after its synthesis revealed that this compound was
a mixture of thiol 8 and disulfide 10 with a 82:18 ratio, the
nized human RBCs. Compound 10 inhibited Fc
phagocytosis of opsonized RBCs effectively at a 5
(Table 1). Compound 9 carrying an isoxazole moiety at C4 did not
show activity up to a 5 M concentration. Compound 11 with a
2-thiophenyl group at C4, however, was found to inhibit phagocy-
tosis similar to 10 at a 5 M concentration. This further confirmed
c
receptor-mediated
l
M concentration
l
l
the observation that the compounds containing a disulfide func-
tional group are those able to inhibit the phagocytosis event. If
one assumes that the thiol/disulfide exchange between the disul-
fide compounds and the cell surface protein thiol were the mode
of action of the above compounds 10–11, then compounds with
higher order polysulfide links, such as trisulfides, tetrasulfides,
etc., should also be active because the –S–S– bond length in poly-
sulfides such as 16 or 17 (n P1, Scheme 2) is longer and weaker
than that found in disulfides 19. Polysulfides are more reactive to-
wards protein thiol since the dissociation energy of S–S bond in
polysulfide (138–151 kJ molꢀ1 32
is lower than in disulfide (272–
)
293 kJ molꢀ1).32 Therefore, polysulfide derivatives were evaluated
for their potential to inhibit phagocytosis of opsonized RBCs
in vitro. Carbamoylated derivative 16 with a morpholinyl moiety
at C4 was obtained as a mixture of polysulfides (contained 65%
disulfide analog, Table 1), and inhibited phagocytosis at 5 lM up
to 20% in comparison to the control. Compared with 16, however,
the disulfide analog 19 with a piperidine at C4 inhibited up to 34%
of phagocytosis of anti-D opsonized RBCs. One of the reasons for
the low activity of compound 16 could be that the disulfide might
be involved in more than one mechanism along with thiol/disulfide
exchange in inhibiting Fcc receptor-dependent phagocytosis.
Intriguingly, compound 20 devoid of any C4 substitution, was
found to be the most active inhibitor of phagocytosis of human
RBCs, inhibiting 43% of phagocytosis at 5 lM concentration, and
CH3
ROC
COR
H3C
COR
H3C
COR H3C
COR
N
CH3
S
N
a
N
N
N
S
+
SH
N
S
N
N
N
N
2-7
17 R=piperidin-1-yl
18 R=piperidin-1-yl
19 R=piperidin-1-yl
Scheme 3. Reduction of compound 17. Reagents and conditions: (a) Compound 17, NaBH4, EtOH, rt, 1 h.

M. K. Purohit et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2324–2327
2327
Table 1
Supplementary data
Biological activity and cellular toxicity for compounds 8–11, 16, 19 and 20 at 5 lM
concentration, in comparison to the +ve control IVIG
Supplementary data (experimental procedures for the synthesis
of compounds, monocyte monolayer assay (MMA) and cell viability
assay, purity data for compounds 8, 9, 10, 11, 19 and 20, and liquid
chromatography profile for compounds 8 and 10) associated with
this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2013.02.064.
References and notes
1. Gauer, R. L.; Braun, M. M. Am. Fam. Physician 2012, 85, 612.
2. Buckley, M. F.; James, J. W.; Brown, D. E.; Whyte, G. S.; Dean, M. G.; Chesterman,
C. N.; Donald, J. A. Thromb. Haemost. 2000, 83, 480.
3. Veneri, D; Franchini, M; Randon, F; Nichele, I; Pizzolo, G; Ambroseti, A. Blood
Transfus. 2009, 7, 75.
4. Cines, D. B.; Bussel, J. B.; Liebman, H. A.; Luning Prak, E. T. Blood 2009, 113,
6511.
5. Neunert, C.; Lim, W.; Crowther, M.; Cohen, A.; Solberg, L., Jr.; Crowther, M. A.
Blood 2011, 117, 4190.
6. Handin, R. I.; Stossel, T. P. N. Engl. J. Med. 1974, 290, 989.
7. Kuwana, M.; Okazaki, Y.; Ikeda, Y. J. Thromb. Haemost. 2009, 7, 322.
8. George, J. N. Am. J. Hematol. 2012, 87, S12.
9. Kuter, D. J. Eur. J. Haematol. Suppl. 2008, 69, 9.
Compound
% Inhibition of phagocytosis
Apoptotic index
NA
1.29
10. Blackhouse, G.; Xie, F.; Levine, M. A.; Campbell, K.; Assasi, N.; Gaebel, K.;
O’Reilly, D.; Tarride, J.; Goeree, R. J. Popul. Ther. Clin. Pharmacol. 2012, 19,
e166.
IVIG
8
94
0
11. Baerenwaldt, A.; Biburger, M.; Nimmerjahn, F. Expert Rev. Clin. Immunol. 2010,
6(3), 425.
12. Crow, A. R.; Lazarus, A. H. Transfus. Med. Rev. 2008, 22(2), 103.
13. Stangel, M.; Pul, R. J. Neurol. 2006, 253, 18.
14. Hansen, R. J.; Balthasar, J. P. Clin. Lab. 2004, 50, 133–140.
15. Lazarus, A. H. Vox Sang. 2002, 83, 53.
10
11
16
19
20
22
19
20
34
43
1.04
0.69
1.88
1.87
2.78
16. Crow, A. R.; Song, S.; Semple, J. W.; Freedman, J.; Lazarus, A. H. Br. J. Haematol.
2001, 115, 679.
17. Crow, A. R.; Lazarus, A. H. J. Pediatr. Hematol. Oncol. 2003, 25, S14.
18. Rampersad, G. C.; Suck, G; Sakac, D; Fahim, S; Foo, A; Denomme, G. A.; Langler,
R. F.; Branch, D. R. Transfusion 2005, 45, 384.
19. Foo, A. H.; Fletcher, S. P.; Langler, R. F.; Porter, C. H.; Branch, D. R. Transfusion
2007, 47, 290.
20. Branch, D. R.; Gallagher, M. T.; Mison, A. P.; Sy Siok Hian, A. L.; Petz, L. D. Br. J.
Haematol. 1984, 56, 19.
21. Gallagher, M. T.; Branch, D. R.; Mison, A.; Petz, L. D. Exp. Hematol. 1983, 11, 82.
22. Hadley, A. G. Transpl. Immunol. 2002, 10, 191.
23. Arndt, P. A.; Garratty, G. Transfusion 2004, 44, 1273.
24. Carpenter, R. R.; Barsales, P. B. J. J. Immunol. 1967, 98, 844.
25. Sahaf, B.; Heydari, K.; Herzenberg, L. A.; Herzenberg, L. A. Proc. Natl. Acad. Sci.
2003, 100, 4001.
in a separate assay, it was found that its IC50 is approximately
100 nM. Compound 20 did not have any higher order polysulfides.
In summary, we reveal a set of designer pyrazole derivatives as
sulfhydryl, disulfide and polysulfides, the role of the sulfide linker
and the C4 substitution on the pyrazole moiety against phagocyto-
sis of anti-D opsonized human RBCs in vitro. Substitution at C4 of
the pyrazole moiety in the disulfide dimers influenced the potency
in the increasing order of 10 ffi 11 ffi 16 < 19 < 20. A novel scaffold
20 with IC50 of 100 nM inhibiting phagocytosis of opsonized blood
cells was identified as a potential candidate for further study as an
alternative therapeutic for ITP. The degree of polymerization is a
very critical aspect for understanding the biological activity in this
study, and lends itself to designing next generation compounds.
26. Jensen, B. S. Acta Chem. Scand. 1959, 13, 1668.
27. Holzer, W.; Hahn, K. J. Heterocycl. Chem. 2003, 40(2), 303.
28. Barreiro, E. J.; Camara, C. A.; Verli, H.; Brazil-Más, L.; Castro, N. G.; Cintra, W.
M.; Aracava, Y.; Rodrigues, C. R.; Fraga, C. A. J. Med. Chem. 2003, 46(7), 1144.
29. Datterl, B.; Tröstner, N.; Kucharski, D.; Holzer, W. Molecules 2010, 15, 6106.
30. David, A.; Edyta, M.; Brzostowska, A. M.; Alexander, G. J. Org. Chem. 2007, 72,
2951.
31. Ilari, F.; Anderson, G.; Abdul, H.; Lucian, A. L.; Dimitris, S. A. Ind. Eng. Chem. Res.
2006, 45, 7388.
32. Argyropoulos, D. S.; Hou, Y.; Ganesaratnam, R.; Harpp, D. N.; Koda, K.
Holzforschung 2005, 59, 124.
Acknowledgment
This work was supported by a Grant from the Canadian Blood
Services, TO00068.