Novel indoline-1- or 3,4-dihydroquinoline-1(2H)-substituted carbothiohydrazides as TPO receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2010.08.028

Synthesis and evaluation of novel series of indoline-1- or 3,4-dihydroquinoline-1(2H)-substituted carbothiohydrazide or carbohydrazide based small molecule compounds as thrombopoietin (TPO) receptor agonists are reported. Members of these compounds have b

Full text of DOI:10.1016/j.bmcl.2010.08.028

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5670–5672
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel indoline-1- or 3,4-dihydroquinoline-1(2H)-substituted
carbothiohydrazides as TPO receptor agonists
*
Peng Cho Tang , He Jun Lu, Yi Qian Chen, Hao Zheng, Peng Song, Li Wang, Qiang Qin, Ai Shen Gong
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:
Synthesis and evaluation of novel series of indoline-1- or 3,4-dihydroquinoline-1(2H)-substituted carbo-
thiohydrazide or carbohydrazide based small molecule compounds as thrombopoietin (TPO) receptor
agonists are reported. Members of these compounds have been identified as full agonists of human c-
mpl in BaF3/TPOR cell line. Indoline-1-carbohydrazide 9b exhibited reasonable pharmacokinetic profile.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 1 June 2010
Revised 22 July 2010
Accepted 5 August 2010
Available online 8 August 2010
Keywords:
Carbothiohydrazide
Carbohydrazide
Thrombopoietin
Agonist
Thrombocytopenia, or low circulating platelet levels, is a fre-
quent finding in a range of medical disorders, such as aplastic
anemia,1 viral infections (HIV,2 hepatics C3), myelodysplasia,4 idio-
pathic thrombocytopenia purpura(ITP),5 and liver dysfunctions.
Thrombopoietin (TPO) is the key cytokine involved in thrombo-
poiesis, and is the endogenous ligand for the thrombopoietin
receptor (TPOR) that is expressed on the surface of megakaryo-
cytes, and megakaryocytic precursors.6 Binding of TPO to its recep-
tor triggers the activation of the JAK-STAT pathway, leading to
changes in gene expression that promote progression along the
megakaryocytic pathway, ultimately leading to the release of
platelets into the peripheral circulation.
Structural elucidation of TPO and its receptor c-mpl resulted in
prompt development of rhTPO and pegylated-rHuMGDF, clinical
trial of which, unfortunately, were stopped for immunogenic
drawbacks.7
Recent research has resulted in development of oral non-peptide
TPO receptor agonists to treat thrombocytopenia. Several non-pep-
tide TPO mimetics (Eltrombopag,8 AKR-501,9 LGD-466510) have en-
tered human clinical trials, and one of them, eltrombopag, has
reached the US market. Non-peptide TPO mimetics are thought to
bear obvious benefits, such as a lower cost of production, an easier
route of administration (orally available), outpatient treatment,
and non-immunogenicity, etc.11 There are still great opportunity
for alternative nonpeptidyl orally administered agents.
further evaluation was reported thereafter.12 We have reported a
series of oxoindolin-3-ylidene ethyl benzothiohydrazides as potent
TPO receptor agonists.13 Herein we report another series of novel
indoline-1- or 3,4-dihydroquinoline-1(2H)-substituted carbothio-
hydrazide or carbohydrazide based compounds as thrombopoietin
receptor agonists.
Commercially available methyl indoline-5-carboxylate (1a) or
methyl 1,2,3,4-tetrahydro-6-quinolinecarboxylate (1b) were trea-
ted with 1,10-thiocarbonyldiimidazole in THF, followed by hydra-
zine hydrate, giving thiosemicarbazides 3a, b in good yields.
Reaction of 3a, b with 3-dimethylamino-ethylidene-oxindoles 4a–
e, followed by a simple sodium hydroxide treatment afforded the
final products 5a–g in moderate to good yields.14 (Scheme 1) 3-
dimethylamino-ethylidene-oxindoles 4a–e were obtained from
commercially available oxindoles through reported procedures.15a,b
Potency of compounds 5a–g as TPO receptor agonists were
tested in a TPO dependent BaF3 cell line by introducing human
TPO receptor into BaF3 cells.16 Structures and bioactivities of 5a–
g were outlined in Table 1.
Compounds 5a, 5b, 5c, and 5g were found to be full agonists of
TPO receptor in the BaF3/TPOR cell line.
Encouraged by the results of 5a–g, their pyrazolone analogues
were also synthesized and tested. 4-[1-(Dimethylamino)ethyli-
dene]-2-aryl-5-methyl-2,4-dihydro-3H-pyrazol-3-ones 6a–b were
reacted with thiosemicarbazides 3a–b, followed by a simple so-
dium hydroxide treatment afforded the final products 7a–d in
moderate to good yields (Scheme 2). 4-[1-(Dimethylamino)ethyli-
dene]-2-aryl-5-methyl-2,4-dihydro-3H-pyrazol-3-ones 6a–b were
obtained from commercially available pyrazolones through re-
ported procedures.17
Some thio-urea compounds with good in vitro thrombopoietic
activities has been disclosed by Nissan researchers, while no
* Corresponding author. Tel.: +86 21 54759101; fax: +86 21 54759072.
E-mail address: tangpc@shhrp.com (P.C. Tang).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.028

P. C. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5670–5672
5671
Table 2
CO2Me
Structures and activities of compounds 7a–d
CO2Me
(CH2)n
N
a
b
a
N
(CH2)n
N
No.
n
Ar
EC50 (nM)
N
H
7a
7b
7c
7d
1
1
2
2
3,4-Dimethylphenyl
5-Indanyl
3,4-Dimethylphenyl
5-Indanyl
6
3
64
35
S
1a, b
2a, b
NMe2
CO2Me
a
Values are means of three experiments. EC50 of Eltrombopag was found to be
(CH2)n
c, d
120 nM under the same condition.
N
+
R
O
NH2NH
N
Ar
S
Table 3
Structures and activities of compounds 8a–c and 9a–b
3a, b
4a-e
CO2H
O
H
N
(CH )n
H 2
N
N
N
R
N
R'
N
Ar
Ar
H
X
N
N
H
S
O
N
O
Ar
a
No.
X
R0
EC50 (nM)
8a
8b
8c
9a
9b
S
S
S
O
O
3,4-Dimethylphenyl
3,4-Dimethylphenyl
3,4-Dimethylphenyl
3,4-Dimethylphenyl
5-Indanyl
–NHMe
22
31
26
42
46
5a-g
Morpholino-
–NHEt
–OH
Scheme 1. Synthesis of carbothiohydrazides 5a–g. Reagents and conditions: (a)
1,10-thiocarbonyldiimidazole, THF, rt, 2 h, 41–55%; (b) NH2NH2ꢀH2O, THF, rt, 1 h,
70–75%; (c) EtOH, AcOH(cat.), 60 °C, 2 h, 60–80%; (d) NaOH, MeOH, rt, >90%.
–OH
a
Values are means of three experiments.
Table 1
Structures and activities of 5a–g
reaction of 7a with various amines gave the corresponding amides
8a–c, and their structures and bioactivities were outlined in Table
3. Sulfur–oxygen exchange in 7a and 7b resulted in carbohydrazide
compounds 9a and 9b respectively, also with good thrombopoietic
activities retained. Synthesis of Compounds 9a and 9b were sum-
marized in Scheme 3.
Selected compounds were converted into their ethanolamine
salts and administered orally at 5 mg/kg dosage respectively to
rats. While most thio-urea compounds, including 7a/7b, were
found only moderately absorbed (data not shown), compounds
9a and 9b did exhibited reasonable pharmacokinetic profiles, as
shown in Table 4.
No.
n
Ar
R
BaF3/TPOR Screeninga
150 nM
300 nM
5a
5b
5c
5d
5e
5f
1
1
1
1
1
2
2
3,4-Dimethylphenyl
4-Methylphenyl
4-Methylphenyl
5-Indanyl
5-Indanyl
3,4-Dimethylphenyl
4-Methylphenyl
H
H
5-F
H
5-F
H
72.4%
47.8%
78.1%
14.3%
3.2%
137.8%
107.3%
122.4%
75.8%
10.6%
30.9%
47.3%
33.4%
101.4%
5g
H
a
The growth rate of BaF3/TPOR in the presence of each compound at 150 nM or
300 nM, expressed by taking the value observed in the presence of 10 ng/ml TPO as
100% standard.
Comparing with 9a, compound 9b was well absorbed orally in
rats. AUC of 9b was four times higher than that of 9a, indicating
a favorable effect of the indane substitution. Clearance half life of
both 9a and 9b were less than 2 h, which may account for the
low plasma exposures of both compounds. Efforts must be taken
CO2H
(CH2)n
N
NMe2
O
H
N
a, b
S
N
H
CO2Me
CO2Me
N
N
Ar
CO2Me
O
N
N
a
b
Ar
N
N
6a-b
7a-d
N
N
O
H
O
NH2NH
N
Scheme 2. Synthesis of carbothiohydrazides 7a–d. Reagents and conditions: (a)
3a,b, EtOH, AcOH(cat.), 60 °C, 1 h, 65–80%; (b) NaOH, MeOH, rt, >90%.
1a
10a
11a
NMe2
O
H
As shown in Table 2, compounds 7a–d were found to be highly
active in the BaF3/TPOR cell line. EC50 of the most potent com-
pound 7b was 3 nM. Ring size of benzene fused heterocycle exhib-
ited clear effect on thrombopoietic activities of the compounds.
Indoline-1-carbothiohydrazides 7a, b are 10 times more potent
than 3,4-dihydroquinoline-1(2H)-carbothiohydrazides 7c, d.
Substitutions on indolino-benzene ring of 7a were varied by
displacement of the carboxylic acid by amides, which were well
tolerated, retaining good TPO receptor agonist activities. Thus,
N
O
N
CO2H
c, d
N
+
H
N
N
N
Ar
N
O
Ar
6a-b
9a-b
Scheme 3. Synthesis of carbohydrazides 9a–b. Reagents and conditions: (a) 1,10-
carbonyldiimidazole, THF, rt, 2 h, 62%; (b) NH2NH2ꢀH2O, THF, rt, 1 h, 81%; (c) EtOH,
AcOH(cat.), 60 °C, 2 h, 63–70%; (d) NaOH, MeOH, rt, >90%.

5672
P. C. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5670–5672
8. Skirvin, J. A. Formulary 2009, 43, 356.
Table 4
9. Fukushima-Shintani, M.; Suzuki, K.; Iwatsuki, Y.; Abe, M.; Sugasawa, K.;
Hirayama, F.; Kawasaki, T.; Nakahata, T. Eur. J. Haematol. 2009, 82, 247.
10. Tomilero, A.; Moral, M. A. Methods Find. Exp. Clin. Pharmacol. 2008, 30, 543.
11. Duffy, K. J.; Darcy, M. G.; Delorme, E.; Dillion, S. B.; Eppley, D. F.; Erickson-
Miller, C.; Giampa, L.; Hopson, C. B.; Huang, Y.; Keenan, R. M.; Lamb, P.; Leong,
L.; Liu, N.; Miller, S. G.; Price, A. T.; Rosen, J.; Shah, R.; Shaw, T. N.; Smith, H.;
Kenneth, C. S.; Tian, S. –S.; Tyree, C.; Wiggall, K. J.; Zhang, L.; Luengo, J. I. J. Med.
Chem. 2001, 44, 3730.
Pharmacokinetic properties of 9a, 9b ethanolamine salts
Compd
Clz/F (L/h/kg)
T1/2 (h)
AUC0–t (ng h/mL)
Cmax (ng/mL)
9a
9b
6.78 3.49
1.23 0.42
1.86 0.27
1.48 0.30
923 504
4460 1640
859 336
3553 884
Clz/F, apparent total plasma clearance; T1/2, half life; AUC, area under the plasma
concentration–time; Cmax, peak plasma concentration.
12. Miyaji, K.; Iwamoto, S.; Shigeta, Y.; Hirokawa, Y.; Owada, S.; Nakamura, T.;
Ishiwata, N. PCT Int. Appl. WO 2007142308 A1, 2007.
13. Tang, P. C.; Lu, H. J.; Chen, Y. Q.; Wang S. L.; Zheng, H.; Wang, L. Bioorg. Med.
Chem. Lett. 2010, in submit.
to improve the metabolic stability of such molecules and reduce
body clearance to obtain better pharmacokinetic profile.
In summary, we have developed indoline-1- and 3,4-dihydro-
quinoline-1(2H)-substituted carbothiohydrazide as well as carbo-
hydrazide based small molecule compounds as TPO mimetics.
Several compounds were found to be full agonists of TPO receptor
in the BaF3/TPOR cell line, although their pharmacokinetic proper-
ties will require further optimization. Efforts to improve PK/PD
profile of such compounds are under way. This may provide an
alternative route to develop novel therapeutic agents to treat
thrombocytopenia.
14. All new compounds were characterized by 1H NMR, 13C NMR, MS and IR. Data
for compound 5a: 1H NMR (400 MHz, DMSO-d6, ppm) d 11.68 (br, 1H), 11.48
(br, 1H), 8.76 (d, 1H, J = 8.8 Hz), 7.88 (s, 1H), 7.82 (d, 1H, J = 8.8 Hz), 7.54 (d, 1H,
J = 5.6 Hz), 7.33 (d, 1H, J = 8.0 Hz), 7.22 (d, 1H, J = 2.0 Hz), 7.15 (dd, 1H,
J1 = 8.0 Hz, J2 = 2.0 Hz), 7.00–7.05 (m, 2H), 6.77–6.82 (m, 1H), 4.39 (t, 2H,
J = 8.4 Hz), 3.23 (t, 2H, J = 8.4 Hz), 2.50 (s, 3H), 2.31 (s, 3H), 2.30 (s, 3H); 13C
NMR (100 MHz, DMSO-d6, ppm) d 167.60, 167.50, 167.36, 147.37, 137.99,
137.87, 136.08, 134.61, 133.03, 132.41, 130.67, 129.11, 128.38, 128.00, 126.49,
126.09, 124.75, 124.42, 123.27, 121.83, 119.23, 116.40, 108.75, 53.07, 26.91,
19.86, 19.55, 15.60; MS (ESI) m/z: 499.6 [M+1]+; IR (KBr): 3434.5, 2922.4,
1680.7, 1607.3, 1505.6, 1449.3, 1370.9, 1279.9, 1255.1, 1198.3, 1107.1, 951.6,
914.6, 874.4, 819.6, 773.0, 695.6, 610.9, 444.1 cmꢁ1. Data for compound 7a: 1H
NMR (400 MHz, DMSO-d6, ppm) d 8.62 (d, 1H, J = 6.8 Hz), 7.79–7.81 (m, 2H),
7.70 (s, 1H), 7.64 (d, 1H, J = 8.0 Hz), 7.19 (d, 1H, J = 8.0 Hz), 4.38 (t, 2H,
J = 8.4 Hz), 3.18 (t, 2H, J = 8.4 Hz), 2.51 (s, 3H), 2.44 (s, 3H), 2.27 (s, 3H), 2.23 (s,
3H); MS (ESI) m/z: 464.6 [M+1]+. Data for compound 9a: 1H NMR (400 MHz,
DMSO-d6, ppm) d 12.31 (br, 1H), 9.70 (br, 1H), 7.90–7.92 (m, 1H), 7.80–7.84 (m,
3H), 7.70–7.74 (m, 1H), 7.23 (d, 1H, J = 8.0 Hz), 4.13 (t, 2H, J = 8.4 Hz), 3.25 (t,
2H, J = 8.4 Hz), 2.52 (s, 3H), 2.50 (s, 3H), 2.44 (s, 3H), 2.36 (s, 3H); MS (ESI) m/z:
448.5 [M+1]+.
Acknowledgements
We thank members of the analytical group of Shanghai Hengrui
Pharmaceutical Ltd for their analytical and spectral determina-
tions, and the Informatics group as well as Dr. Feng Jun for their
useful discussions and enormous support.
15. (a) Sarges, R.; Howard, H. R.; Koe, B. K.; Weissman, A. J. Med. Chem. 1989, 32,
437; (b) 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.
16. 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
References and notes
1. Jenkins, J. M.; Williams, D.; Deng, Y.; Uhl, J.; Kitchen, V.; Collins, D.; Erickson-
Miller, C. L. Blood 2007, 109, 4739.
2. Mannucci, P. M.; Gringeri, A. Ann. Ital. Med. Int. 2000, 15, 20.
3. Espanol, I.; Gallego, A.; Enriquez, J.; Rabella, N.; Lerma, E.; Hernandez, A.; Pujol-
Moix, N. Hepatogastroenterology 2000, 47, 1404.
4. Moody, J. L.; Xu, L.; Helgason, C. D.; Jirik, F. R. Blood 2004, 103, 4503.
5. Andersson, J. Acta Paediatr. Suppl. 1998, 424, 61.
6. Kuter, D. J. Annu. Rev. Med. 2009, 60, 193.
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.
10 ll/well of CCK-8 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.
17. Tang, P. C.; Lu, H.; Chen, Y.; Song, P.; Wang, S. PCT Int. Appl. WO 2009103218
A1, 2009.
7. Kuter, D. J.; Begley, C. G. Blood 2002, 100, 3457.