Identification of a pharmacophore for thrombopoietic activity of small, non-peptidyl molecules. 1. Discovery and optimization of salicylaldehyde thiosemicarbazone thrombopoietin mimics

Article abstract of DOI:10.1021/jm025535c

High-throughput screening has resulted in the discovery of thiosemicarbazone thrombopoietin mimics. A shared pharmacophore hypothesis between this series and a previously identified class, the pyrazol-4-ylidenehydrazines, led to the rapid optimization of

Full text of DOI:10.1021/jm025535c

J . Med. Chem. 2002, 45, 3573-3575
3573
Id en tifica tion of a P h a r m a cop h or e for
Th r om bop oietic Activity of Sm a ll,
Non -P ep tid yl Molecu les. 1. Discover y a n d
Op tim iza tion of Sa licyla ld eh yd e
Th iosem ica r ba zon e Th r om bop oietin
Mim ics
Kevin J . Duffy,*,† Anthony N. Shaw,†
Evelyne Delorme,‡ Susan B. Dillon,†
F igu r e 1. Comparison of salicylaldehyde thiosemicarbazone
screening-hit 2 and potent pyrazol-4-ylidenehydrazine TPO
agonist 1. Activity is measured by expression of luciferase in
the BAF3/TPO-Rluc cell line.15
Connie Erickson-Miller,† Leslie Giampa,‡
Yifang Huang,† Richard M. Keenan,† Peter Lamb,‡
Nannan Liu,† Stephen G. Miller,‡ Alan T. Price,†
J on Rosen,‡ Heather Smith,‡ Kenneth J . Wiggall,†
Lihua Zhang,† and J uan I. Luengo†
bone marrow cultures with full TPO efficacy at EC50
values as low as 1-100 nM. In this report, we describe
the discovery of a second class of small-molecule TPO
mimics, the salicylaldehyde thiosemicarbazones.
High-throughput screening for the induction of a
luciferase reporter gene in a TPO-responsive cell line13,14
resulted in the identification of salicylaldehyde thi-
osemicarbazone 2 (Figure 1) as a weak TPO agonist.
We recognized that this compound shared some struc-
tural similarities with the pyrazol-4-ylidenehydrazine
class of TPO mimics; both the pyrazol-4-ylidenehydra-
zine 1 and the salicylaldehyde thiosemicarbazone 2
contain an arrangement of heteroatoms that can poten-
tially engage a metal ion in a bicyclic, tridentate chelate.
At the time, SAR trends for the pyrazol-4-ylidene-
hydrazines had already been elucidated, with acidic
functionality being optimal on the naphthalene ring and
lipophilic aryl groups being preferred as N substituents
on the pyrazole ring. Because we were intrigued by the
possibility that pyrazol-4-ylidenehydrazines and sali-
cylaldehyde thiosemicarbazones may share the same
pharmacophore for thrombopoietic activity, studies to
improve the potency in the luciferase assay of salicyl-
aldehyde thiosemicarbazone screening hit 2 were initi-
ated.
Incorporation of acidic functionality was found to be
important for potent TPO agonist activity in the pyrazol-
4-ylidenehydrazine series.13 As an initial test of our
common pharmacophore hypothesis, both the sulfonic
and carboxylic acid analogues 5a and 5b were prepared
as shown in Scheme 1. This did indeed result in an over
10-fold improvement in potency for both 5a and 5b;
however, maximum efficacies remained around 30% of
that induced by saturating concentrations of TPO.
To further improve the potency and efficacy of the
salicylaldehyde thiosemicarbazone TPO mimics, a mo-
lecular model was constructed in which the tridentate
chelation atoms of salicylaldehyde thiosemicarbazone
5b and pyrazol-4-ylidenehydrazine 1 were overlayed
(Figure 2). As can be seen, these heteroatoms superim-
pose very well. This model also suggests that the
incorporation of bulky aryl substituents at the 3-position
of the hydroxyphenyl ring will allow access of the
putative lipophilic pocket previously recognized to greatly
enhance potency in the pyrazol-4-ylidenehydrazine se-
ries.13 To test this hypothesis, a series of nine o-methoxy
biaryl aldehydes 7 and the corresponding nine o-hydroxy
GlaxoSmithKline, 1250 South Collegeville Road,
Collegeville, Pennsylvania 19426, and
Ligand Pharmaceuticals, 10275 Science Center Drive,
San Diego, California 92121
Received May 6, 2002
Abstr a ct: High-throughput screening has resulted in the
discovery of thiosemicarbazone thrombopoietin mimics. A
shared pharmacophore hypothesis between this series and a
previously identified class, the pyrazol-4-ylidenehydrazines,
led to the rapid optimization of both potency and efficacy of
the thiosemicarbazones. The application of high-throughput
chemistry and purification techniques allowed for the rapid
elucidation of structure-activity relationships.
The 332 amino acid cytokine thrombopoietin (TPO)
plays a critical role in the regulation of megakaryocy-
topoiesis and is the primary regulator of platelet pro-
duction.1 TPO elicits its biological effect through binding
and activation of the cell-surface receptor, c-mpl (the
TPO receptor). Binding of TPO to c-mpl initiates a
cascade involving several cell-signaling pathways lead-
ing to the irrevocable commitment of the progenitor cell
along the megakaryocytic lineage.2-5 Mature mega-
karyocytes then undergo cell fragmentation to produce
the platelet bodies essential for the eventual formation
of a protective thrombus in compromised blood vessels.
Severely low platelet counts, thrombocytopenia, is a
serious complication in patients receiving intensive
chemotherapy. Currently, management of thrombocy-
topenia is primarily based on platelet transfusion, which
is becoming increasingly costly.6-9 The only approved
platelet growth factor available at this time is the
protein therapeutic rhuIL-11 (Neumega).10 rhuTPO11
and pegylated megakaryocyte differentiation factor12
(peg-MGDF, a pegylated, truncated N-terminal region
of TPO) are two protein agents currently undergoing
clinical trials for the treatment of thrombocytopenia.
Recently we have reported the discovery of the pyrazol-
4-ylidenehydrazines, represented by compound 1, as
small-molecule TPO mimetic agents.13 These com-
pounds were shown to stimulate the proliferation and
differentiation of TPO-responsive cell lines and human
* To whom correspondence should be addressed. Phone: (610) 917-
6770. Fax: (610) 917-4157. E-mail: kevin.j.duffy@gsk.com.
† GlaxoSmithKline.
‡ Ligand Pharmaceuticals.
10.1021/jm025535c CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/19/2002

3574 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Letters
Sch em e 1a
Sch em e 2a
a
(a) NH2NH2, THF; (b) 3,5-dichlorosalicylaldehyde, DMF.
F igu r e 2. Model16 of salicylaldehyde thiosemicarbazone 5b
(green) and pyrazol-4-ylidenehydrazine 1 (red) with the tri-
dentate heteroatom motif overlayed.
biaryl aldehydes 8 were prepared as shown in Scheme
2.17 These 18 aldehydes were condensed with both the
3-carboxythiosemicarbazide A and the 4-carboxythio-
semicarbazide B to afford 36 thiosemicarbazones, 9a -
i, 10a -i, 11a -i, and 12a -i.
a
(a) R-B(OH)2, Pd(PPh3)4, 1,4-dioxane, aqueous Na2CO3; (b)
BBr3, CH2Cl2; (c) a or b, DMF, 4 Å molecular sieves.
The TPO mimetic activity of the 36 salicylaldehyde
thiosemicarbazones was tested by their ability to induce
the proliferation of the TPO-responsive UT7/TPO-R cell
lines in vitro.18 Efficacies at the concentration of maxi-
mal effect19 for all compounds are illustrated in Figure
3 as a percentage relative to the maximal effect of
saturating TPO. As was predicted from the model in
Figure 2, introduction of bulky, lipophilic aryl groups
did indeed result in substantial increases in both
potency and efficacy. The o-hydroxyl group was critical
for high activity, as attested by the weak or lack of
activity for the 18 compounds 9 and 10 containing a
methoxy group in this position. 3-Carboxy and 4-carboxy
analogues were roughly equally efficacious, suggesting
a less stringent requirement for the optimal position of
the acidic functionality.
For ease of representation, the array data in Figure
3 relates only to the maximal efficacy of the salicyl-
aldehyde thiosemicarbazone TPO mimics. Potencies also
improved by incorporating the lipophilic biaryl func-
tionality as shown for selected agonists in Table 1. The
most potent analogues 11b and 12c have potencies of
20 and 30 nM, respectively, with efficacies similar to
those induced by maximal TPO concentrations.
F igu r e 3. Maximal proliferative effect in UT7/TPO-R cell
lines for salicylaldehydes 9a -i, 10a -i, 11a -i, and 12a -i.
Efficacies are expressed as a percentage of the maximal
proliferation induced by saturating TPO concentrations.
It is important to note that all active salicylaldehyde
thiosemicarbazones showed complete selectivity for
TPO-responsive cell lines (UT7/TPO-R, BAF3/TPO-R)
versus other cytokine-responsive cell lines tested [e.g.,
BAF-3 parental (IL-3), UT7/EPO-R, Hep-G2/G-CSF-R].
Further, the agonists induced a pattern of kinase
activation and protein phosphorylation similar to that
induced by TPO itself (data not shown).
In conclusion, high-throughput screening has resulted
in the discovery of a new series of TPO mimics, the
salicylaldehyde thiosemicarbazones. A shared pharma-

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3575
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cophore hypothesis between this series and a previously
identified class, the pyrazol-4-ylidenehydrazines, in
conjunction with molecular modeling led to the rapid
optimization of both potency and efficacy of the salicy-
laldehyde thiosemicarbazones. The application of high-
throughput chemistry and purification techniques fa-
cilitated the simultaneous synthesis of arrays of salicyl-
aldehyde thiosemicarbazones and allowed for the rapid
elucidation of structure-activity relationships. This
pharmacophore hypothesis also made possible the in-
vention of a completely novel class of TPO mimics.20
(14) Lamb, P.; Tapley, P.; Rosen, J . Biochemical approaches to
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(15) Luciferase assay: BAF3/TPO-Rluc cells [(1 × 105)/mL] (starved
of IL-3 overnight) in growth medium containing fetal bovine
serum (FBS) (0.5% v/v) and ZnCl2 (30 µM) were incubated with
compounds (0.32% DMSO final concentration) or rhuTPO at 37
°C (5% CO2, 95% relative humidity) for 3 h. Luciferase activity
was recorded using a Dynatech model 1000 luminometer. Each
data point is the average of triplicate assays.
(16) MOE, version 2001.01; Chemical Computing Group Inc.: Que-
bec, Canada, 1997-2001.
(17) Bromo-2-methoxybenzaldehyde was prepared from 2-bromophe-
nol as show below. Full experimental details are given in the
Supporting Information.
Su p p or tin g In for m a tion Ava ila ble: UT7TPO-R prolif-
eration assay potencies and efficacies for all thiosemicarba-
zones 9-12, experimental details for the synthesis, and
characterization of representative salicylaldehyde thiosemi-
carbazone TPO mimics. This material is available free of
charge via the Internet at http://pubs.acs.org.
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