Synthesis and biological evaluation of oxoindolin-3-ylidene ethyl benzothiohydrazides as non-peptide TPO mimics

Article abstract of DOI:10.1016/j.bmcl.2010.07.040

A novel series of oxoindolin-3-ylidene ethyl benzohydrazides were designed, synthesized, and identified as small molecule agonists of thrombopoietin (TPO) receptor c-mpl. Sulfur-oxygen exchange in oxoindolin-3-ylidene ethyl benzohydrazides was found to im

Full text of DOI:10.1016/j.bmcl.2010.07.040

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5062–5064
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of oxoindolin-3-ylidene ethyl
benzothiohydrazides as non-peptide TPO mimics
*
Peng Cho Tang , He Jun Lu, Yi Qian Chen, Sheng Lan Wang, Hao Zheng, Li Wang
Shanghai Hengrui Pharmaceuticals Co., Ltd, 279 Wenjing Rd., Shanghai 200245, China
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 oxoindolin-3-ylidene ethyl benzohydrazides were designed, synthesized, and identified
as small molecule agonists of thrombopoietin (TPO) receptor c-mpl. Sulfur–oxygen exchange in oxoindo-
lin-3-ylidene ethyl benzohydrazides was found to improve their agonistic activities. Several oxoindolin-
3-ylidene ethyl benzothiohydrazides have been identified as full agonists of c-mpl.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 12 April 2010
Revised 8 July 2010
Accepted 9 July 2010
Available online 15 July 2010
Keywords:
TPO
c-mpl
Non-peptide
Thrombocytopenia
Platelets are essential in the process of blood clotting and repair
of damaged blood vessels. Thrombocytopenia, or low circulating
platelet levels, can be a debilitating condition associated with a
range of medical disorders, including platelets immunogenicity
(ITP),1 liver dysfunctions, drug treatments (IFN,2 heparin3), and vir-
al infections (HIV,4 hepatitis C5), and is also a common side effect in
cancer patients receiving intensive chemotherapy treatments. Cur-
rent methods to manage thrombocytopenia includes corticoste-
roids, intravenous immunoglobulin, anti-D immunoglobulin,
rituximab, vinca alkaloids, azathioprine, cyclophosph-amide,
mycophenolate mofetil, cyclosporine, as well as splenectomy,6 all
of which suffer from serious side effects or low efficacy.
Thrombopoietin (TPO), a cytokine produced primarily by the li-
ver and kidney, regulates platelet production by stimulating prolif-
eration and differentiation of hematopoietic stem cells,
megakaryocytic progenitor cells, and megakaryocytes via activa-
tion of its receptor, c-mpl.7–11 Continuing understanding of the
structure and function of TPO led to the development of the first
generation thrombopoietic growth factors, including rhTPO and
PEG-rHuMGDF. Clinical study of both of them, unfortunately, were
halted because of the risk of antigenicity.12
trials, and Eltrombopag and AMG-531 have reached the US market
in 2008.
AMG-531, a TPO mimetic peptide, is a 60 kDa molecule that
consists of disulfide-bonded human IgG1 heavy chain constant re-
gions with two identical peptide sequences linked covalently
through a polyglycine bridge. AMG-531 was administered intrave-
nously or subcutaneously and showed remarkable ability to pro-
mote platelet counts in clinical trials.13 Eltrombopag, however, is
a non-peptide hydrazone small molecule, with a molecular weight
of 564 kDa.13 Eltrombopag is the first and so far the only one oral
treatment for thrombocytopenia.
According to Duffy et al., a heteroatom metal chelate in the cen-
tral portion of the molecule is indispensable for its TPO receptor
agonist activity.14 Nissan researchers have reported a novel thio-
phene carbohydrazone compound NIP-004 (Fig. 1) as human TPO
receptor (c-mpl) activator.15
Ar2
CO2H
H
N
H
N
S
N
H
Despite the problem of autoantibody formation, the results of
the clinical trials with the first generation TPO molecules provided
a stimulus for continuing the search and development of non-
immunogenic TPO molecules, namely, the second generation
thrombopoietic agonists. Several agents have progressed to clinical
S
O
O
NH
O
H
M
O
M
N
Ar1
Cl
Cl
NIP-004
Designed Molecules
* Corresponding author.
Figure 1. NIP-004 and designed oxoindolin-3-ylidene ethyl benzohydrazides as
E-mail address: tangpc@shhrp.com (P.C. Tang).
metal chelators.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.040

P. C. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5062–5064
5063
Table 1
Chemical structures and bioactivities of 5aa–la
a
Compound
Ar1
R
Ar2
BaF3/TPOR (%)
150 nM
300 nM
5aa
5ab
5ac
5ba
5bb
5ca
5cb
5cc
5da
5db
5ea
5eb
5ec
5fa
5fb
5ga
5gb
5ha
5hb
5ia
4-Me-Ph
4-Me-Ph
4-Me-Ph
4-Me-Ph
4-Me-Ph
3-Me-Ph
3-Me-Ph
3-Me-Ph
4-Et-Ph
H
H
H
5-F
5-F
H
H
H
H
H
H
H
H
H
H
H
4-CO2H-Ph
3-CO2H-Ph
2-CO2H-Thiophene-5-yl
4-CO2H-Ph
3-CO2H-Ph
4-CO2H-Ph
3-CO2H-Ph
2-CO2H-Thiophene-5-yl
4-CO2H-Ph
3-CO2H-Ph
4-CO2H-Ph
3-CO2H-Ph
2-CO2H-Thiophene-5-yl
4-CO2H-Ph
3-CO2H-Ph
4-CO2H-Ph
3-CO2H-Ph
4-CO2H-Ph
3-CO2H-Ph
11.5
6.1
57.6
45.2
39.6
147.1
60.2
90.2
47.5
56.8
70.2
45.3
82.3
61.5
55.9
39.5
12.1
68.4
33.9
96.3
29.5
31.0
28.2
19.6
13.8
81.3
100.2
10.5
38.8
23.3
20.9
10.3
8.2
13.6
5.8
16.2
7.2
21.3
5.5
4.7
19.3
8.6
21.5
10.2
12.5
16.8
8.1
8.2
26.9
35.5
4-Et-Ph
3,4-DiMe-Ph
3,4-DiMe-Ph
3,4-DiMe-Ph
Ph
Ph
5-Indanyl
5-Indanyl
5-Indanyl
5-Indanyl
4-Me-Ph
4-Me-Ph
4-Me-Ph
4-Me-Ph
4-Me-Ph
4-Me-Ph
H
5-F
5-F
5-NHAc
5-NHAc
5-Pyrrol-1-yl
5-Pyrrol-1-yl
4-F
4-CO2H-Ph
3-CO2H-Ph
4-CO2H-Ph
3-CO2H-Ph
4-CO2H-Ph
4-CO2H-Ph
5ib
5ja
5jb
5ka
5la
6-F
a
The growth rate of BaF3/TPOR in the presence of each compound at 150 or 300 nM, expressed by taking the value observed in the presence of 10 ng/ml TPO as 100%
standard.
Oxoindolin-3-ylidenes were also reported as good metal chela-
Potency of compounds 5aa–la as TPO receptor agonists were
tested in a TPO dependent BaF3/TPOR cell line, which was estab-
lished by introducing human TPO receptor into BaF3 cells.19 The
structures of 5aa–la were outlined in Table 1.
tors.16 Replacement of the 2-(3,4-dichlorophenyl)-thiophene-3-ol
group in NIP-004 with 1-aryl-indolin-2-ones results in a new
group of fine metal chelators (Fig. 1), with the potential to be
TPO mimicking compounds of considerable activity. Herein, we re-
port our efforts on synthesis and evaluation of oxoindolin-3-yli-
dene ethyl benzothiohydrazides as TPO receptor agonists. Several
compounds were found to be full agonists of TPOR in our in vitro
model.
Most of the above compounds were found to be only weakly ac-
tive or inactive at all in the BaF3/TPOR cell line, except for com-
pounds 5ba, 5ha, and 5la, which appeared to be full agonist of
TPOR at 300 nM in our assay. Compounds 5ba, 5ha, and 5la are
all benzoic acids, while all thiophene-2-carboxylic acid compounds
(5ac, 5cc, and 5ec) exhibited weak TPO activity. Lack of TPO mim-
icking potency of compound 5bb indicates the positive effect of the
4-carboxylic acid substitution on the right phenyl ring. The 5-F
substitution seems to be optimal, considering the less potent com-
pounds 5aa, 5ka, and 5la. Bulky substituents on the oxoindoline
group were not tolerated (5ia–jb).
Ethoxycarbonyl substituted benzoic acids or thiophene-2-car-
boxylic acids were reacted with tert-butyl N-aminocarbamate, fol-
lowed by de-protection of the Boc group with TFA to give the
corresponding benzohydrazides, which were reacted with 3-
dimethylamino-ethylidene-oxindoles 4a–l to afford the coupling
products. A simple sodium hydroxide treatment afforded the cor-
responding carboxylic acid products 5aa–la.17 3-Dimethylamino-
ethylidene-oxindoles 4a–l were obtained from commercially avail-
able oxindoles through reported procedures.18
Shifting of the 4-methylphenyl group in 5ba to 7-position of the
oxindole group was proved to be futile. Compound 10 (Scheme 2)
was synthesized and found to be inactive in the BaF3/TPOR assay,
F
F
F
a
c
O
b
O
N
N
H
H
Br
N
H
O
6
7
8
F
F
CO2H
H
N
d, e
N
H
O
N
O
H
N
NMe2
H
O
9
10
Scheme 2. Synthesis of 7-substituted oxindole compound 10. Reagents and conditions: (a) NBS, CH3CN, rt, overnight, 44%; (b) p-tolylboronic acid, Pd(PPh3)4, NaHCO3, DME/
H2O (1:1), reflux, 8 h, 62%; (c) MeC(OMe)2NMe2, CHCl3, reflux, 8 h, 95%; (d) 3a, EtOH, AcOH (cat.), reflux, 6 h, 90%; (e) NaOH, MeOH, H2O, reflux, 3.5 h, 95%.

5064
P. C. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5062–5064
O
Ar
NHNH2
Among the active compounds, compound 11c with 4-methyl phe-
nyl and a 5-fluoro substitution on the oxindole structure was again
the most potent compound, in line with what was seen in the oxo-
series compounds.
b
a
Ar CO2H
Ar
NHNHBoc
2a, b, c
O
1a, b, c
3a, b, c
In summary, sulfur–oxygen exchange in oxoindolin-3-ylidene
ethyl benzohydrazides resulted in dramatic improvement in their
thrombopoietic activities. Members of these compounds were
found to be full agonists of human c-mpl in our in vitro assay. In
vitro and in vivo pharmacokinetic and efficacy studies of selected
compounds are under way. This may provide an alternative route
to develop novel therapeutic agents to treat thrombocytopenia.
R
N
H
N
Ar2
c, d
N
+
O
H
O
N
Ar1
O
N
Ar1
R
4a-l
5aa-la
Acknowledgments
Scheme 1. Synthesis of substituted benzohydrazides 5aa–la. Reagents and condi-
tions: (a) DCC, NEt3, DMAP, rt, 2 h, 67–90%; (b) TFA, CH2Cl2, rt, 2 h, 75–92%; (c)
EtOH, AcOH (cat.), reflux, overnight, 55–85%; (d) NaOH, MeOH, rt, >90%.
The authors thank members of the analytical group of Shanghai
Hengrui Pharmaceuticals Ltd for their analytical and spectral
determinations, as well as the Informatics group and Dr. Feng Jun
for their useful discussions and enormous support.
R1
CO2H
H
N
N
References and notes
H
S
N
O
1. Anderson, J. Acta Paediatr. Suppl. 1998, 424, 61.
2. Garcia-Suarez, J.; Burgaleta, C.; Hernanz, N.; Albarran, F.; Tobaruela, P.; Alvarez-
Mon, M. J. Haematol. 2000, 110, 98.
Ar
Figure 2. Chemical structures of compounds 11a–e.
3. Depasse, F.; Samama, M. M. Ann. Biol. Clin. (Paris) 2000, 58, 317.
4. Mannucci, P. M.; Gringeri, A. Ann. Ital. Med. Int. 2000, 15, 20.
5. Espanol, I.; Gallego, A.; Enriquez, J.; Rabella, N.; Lerma, E.; Hernandez, A.; Pujol-
Moix, N. Hepatogastroenterology 2000, 47, 1404.
6. Kobos, R.; Bussel, J. B. Clin. Lymphoma Myeloma 2008, 8, 33.
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8. Lok, S.; Kaushansky, K.; Holly, R. D. Nature 1994, 369, 565.
9. Kaushansky, K.; Lok, S.; Holly, R. D. Nature 1994, 369, 568.
10. Wending, F.; Maraskovsky, E.; Debili, N. Nature 1994, 369, 571.
11. Bartley, T. D.; Bogenberger, J.; Hunt, P. Cell 1994, 77, 1117.
12. Kuter, D. J. Eur. J. Haematol. 2007, 80, 9.
Table 2
TPO receptor agonist activity of benzothiohydrazides 11a–e
a
Compound
Ar
R1
EC50 (lM)
11a
11b
11c
11d
11e
3,4-DiMe-Ph
5-Indanyl
4-Me-Ph
3,4-DiMe-Ph
5-Indanyl
H
H
5-F
5-F
5-F
0.082
>0.2
0.044
0.103
>0.2
13. Kuter, D. J. Clin. Lymphoma Myeloma 2009, 9, S347.
14. Duffy, K. J.; Price, A. T.; Delorme, E.; Dillon, S. B.; Duquenne, C.; Erickson-Miller,
C.; Giampa, L.; Huang, Y.; Keenan, R. M.; Lamb, P.; Liu, N.; Miller, S. G.; Rosen, J.;
Shaw, A. N.; Smith, H.; Wiggal, K. J.; Zhang, L.; Luengo, J. I. J. Med. Chem. 2002,
45, 3576.
a
Average results of three experiments. EC50 of Eltrombopag was found to be
120 nM under the same condition.
15. Nakamura, T.; Miyakawa, Y.; Miyamura, A.; Yamane, A.; Suzuki, H.; Ito, M.;
Ohnishi, Y.; Ishiwata, N.; Ikeda, Y.; Tsuruzoe, N. Blood 2006, 107, 4300.
16. Hassaan, A. M. A. Synth. React. Inorg. Met.-Org. Chem. 1997, 27, 835.
17. All new compounds were characterized by 1H NMR and MS. Data for
compound 5aa: 1H NMR (400 MHz, DMSO-d6) d 11.50 (br, 1H), 11.18 (br,
1H), 8.00–8.14 (m, 4H), 7.54 (t, J = 8.1 Hz, 1H), 7.32–7.41 (m, 4H), 7.02–7.07 (m,
2H), 6.81 (dd, J1 = 8.1 Hz, J2 = 2.2 Hz, 1H), 2.51 (s, 3H), 2.41 (s, 3H); MS (ESI) m/
z: 428.5 [M+1]+. Data for compound 11c: 1H NMR (400 MHz, DMSO-d6) d 8.10–
7.96 (3H, m), 7.38–7.25 (4H, m), 7.03 (1H, t, J = 8.0 Hz), 6.84–6.77 (2H, m), 6.65
(1H, dd, J1 = 8.4 Hz, J2 = 4.0 Hz) 2.81 (3H, s), 2.38 (3H, s); MS (ESI) m/z: 462.4
[M+1]+.
18. Philips, D. P.; Hudson, A. R.; Nguyen, B.; Lau, T. L.; McNeill, M. H.; Dalgard, J. E.;
Chen, J. H.; Penuliar, R. J.; Miller, T. A.; Lin, Z. Tetrahedron Lett. 2006, 47, 7137.
19. Wild-type BaF3 cell line was transfected with an EX-B0010-M02 plasmid
containing TPO receptor gene and neomycin, screened by G418 (Gibco, US) to
get the stable monoclonal BaF3-TPOR cell line. In a 96-well plate, to each well
indicating suitable aryl substitution on the oxoindoline ring to be
vital for TPO receptor activating potency.
Sulfur is generally accepted as a stronger chelator than oxy-
gen.20 Considering the importance of chelation in thrombopoietic
activities, sulfur–oxygen exchange in compound 5ba may result
in stronger chelation and thus higher TPOR agonist potency. Boc
protected benzohydrazides 2a,b were treated with Lawensson’s re-
agents to give the corresponding benzothiohydrazides, which
underwent similar reaction sequence as 2a,b in Scheme 1, provid-
ing oxoindolin-3-ylidene benzothiohydrazides 11a–e (Fig. 2) in
good overall yields.17 Structures and thrombopoietic activities of
11a–e were outlined in Table 2.
was added 100 ll of the cell suspension, the blank control, negative control,
rhTPO and the test compound of different concentrations. All measurements
were made in triplicate. The plate was incubated for 24 h at 37 °C in 5% CO2.
CCK-8 (10 ll/well) was added and the plate was cultured for another 4 h. OD450
value was recorded with a VICTOR3 (Perkin-Elmer 1420) instrument, and EC50
was calculated with Origin 7.0.
Compounds 11a, 11c, and 11d were found to be full agonists of
human TPO receptor c-mpl, among which 11c was the most potent
compound. Compounds 11b and 11e surprisingly exhibited only
weak thrombopoietic activities, without any obvious explanation.
20. Peters, R. W. J. Hazard. Mater. 1999, 66, 151.