Discovery and biological evaluation of benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor

Article abstract of DOI:10.1016/j.bmcl.2008.08.068

A novel series of benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor is reported. Starting from a 3.4 μM high throughput screen hit, members of this series have been identified which are full agonists with functional potency <50 nM and oral bioavailability in mice.

Full text of DOI:10.1016/j.bmcl.2008.08.068

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5255–5258
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and biological evaluation of benzo[a]carbazole-based small
molecule agonists of the thrombopoietin (Tpo) receptor
*
Phil B. Alper, Thomas H. Marsilje , Daniel Mutnick, Wenshuo Lu, Arnab Chatterjee, Michael J. Roberts,
Yun He, Donald S. Karanewsky, Donald Chow, Jianmin Lao, Andrea Gerken, Tove Tuntland, Bo Liu,
Jonathan Chang, Perry Gordon, H. Martin Seidel, Shin-Shay Tian
Genomics Institute of the Novartis Research Foundation (GNF), San Diego, CA 92121, USA
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 benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor
Received 22 June 2008
Revised 15 August 2008
Accepted 18 August 2008
Available online 22 August 2008
is reported. Starting from a 3.4
fied which are full agonists with functional potency <50 nM and oral bioavailability in mice.
Ó 2008 Elsevier Ltd. All rights reserved.
lM high throughput screen hit, members of this series have been identi-
Keywords:
Tpo
Thrombopoietin
TpoR
cMpl
Hematopoietic growth factor
Agonist
The condition of low platelet count is referred to as thrombocy-
topenia. A patient is considered to be at risk of spontaneous hem-
orrhaging if their platelet count falls below 20,000/lL. The causes
closed (Fig. 1). EltrombopagTM (1)4–7 was recently the first small
molecule TpoR agonist to be submitted for regulatory approval.
Several additional reports of small molecule drug discovery efforts
have been published.8–23
We screened for TpoR agonists by measuring activation of a
Stat5 response element-driven reporter gene in a human TpoR
responsive Ba/F3 cell line (Ba/F3-hTpoR RGA).12,16 A benzo[a]car-
bazole (7, Fig. 2) was identified as a novel agonist of hTpoR with
of thrombocytopenia are manifold, including e.g., idiopathic
thrombocytopenic purpura (ITP), chronic liver disease, HCV infec-
tion, certain varieties of cancer and as a side effect of certain forms
of cancer chemotherapy. Thrombopoietin (Tpo) is a hematopoietic
growth factor whose agonism of the Tpo receptor (TpoR, also called
cMpl) has been shown to be the principal regulator of megakaryo-
cyte growth and differentiation into platelets. TpoR is a cell surface
receptor that initiates an intracellular signaling cascade that is
mediated by the JAK2 kinase and the transcription factor Stat5.
The administration of PEG modified, truncated Tpo was tested clin-
ically but this therapeutic strategy has been abandoned due to
immunogenicity issues. Novel methods of agonism of the TpoR
thus represents an alternative strategy to address thrombocytope-
nia in patients, currently an area of unmet medical need.1,2
3.4 lM potency and 100% efficacy relative to 30 ng/mL Tpo. The
structure of 7 is a novel chemotype in comparison to the small
molecule TpoR ligands which had been previously described in
the literature,4–23 (Fig. 1). A search of the literature indicated that
the only previously described biological activity of 7 is weak anti-
bacterial and antifungal activity.24 Based upon the unique chemo-
type, low micromolar potency and full efficacy of 7, a preliminary
SAR analoging effort was undertaken. An initial series of
benzo[a]carbazoles was synthesized using known methodology25
and evaluated in vitro in order to map out the key pharmacophore
features necessary for TpoR activation. The functional activities of
this initial series of compounds (7–14) against TpoR are shown
in Table 1.
Despite the ostensibly difficult task of replicating the effect of a
protein–protein interaction with a small molecule ligand, TpoR
agonism has recently received increasing attention from the phar-
maceutical industry. In addition to the injectable peptidic agent
TM3
Romiplostim
which recently obtained regulatory approval, a
Reduction of the carboxylic acid in 7 generated the benzyl alco-
hol 8 which is inactive. Likewise, methylation of either the carba-
zole nitrogen (9) or the phenol (10) is not tolerated. Converting
the carboxylic acid in 7 to the corresponding methyl ester (11) gen-
number of nonpeptidyl small molecule agonists have been dis-
* Corresponding author. Tel.: +1 858 332 4604; fax: +1 858 812 1648.
erated a compound with retained potency (1.3 lM vs 3.4 lM) but
E-mail address: TMarsilje@gnf.org (T.H. Marsilje).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.068

5256
P. B. Alper et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5255–5258
group in the 9-position of the benzo[a]carbazole ring system. Com-
pound 12 was the first sub-micromolar compound identified in the
scaffold with a potency of 870 nM (76% efficacy). A methylene
homologation of the benzoic acid in 12 to an acetic acid (13)26 re-
sults in a ca. 10ꢀ less potent compound (0.87
lM vs 9.2 lM) but
interestingly, efficacy is retained (70% vs 76%). Encouraged by the
sub-micromolar activity of 12, we replaced the methyl group in
12 with a trifluoromethyl group (14) which significantly increases
both potency and efficacy (143 nM, 128%). This level of potency
and efficacy led us to consider 14 as a lead molecule and a starting
point for the further optimization of the benzo[a]carbazole scaf-
fold. At this point, we desired to modify the 5,6-olefin in this series
of molecules for two reasons: (a) the possibility of oxidation of the
5,6-double bond in vivo potentially generating a reactive species
and (b) the flat and fully aromatic nature of this molecule making
intercalation into DNA an undesired possibility. We decided to at-
tempt to switch from the fully aromatic benzo[a]carbazole to a
partially reduced dihydro-benzo[a]carbazole ring system27 and
the resulting compounds are shown in Table 2.
Compound 15 is the partially reduced direct analog of the lead
compound 14. We were pleased to see that partially reducing the
aromaticity of the lead compound 14 generates a compound which
retains much of the potency (473 nM) and efficacy (96%) of the
fully aromatic system. Compound 15 was thus able to serve as a
lead molecule in the optimization of a partially reduced dihydro-
benzo[a]carbazole scaffold. The CF3 group in 15 was systematically
moved to other regiopositions on the scaffold (16–18) in order to
determine if its effect on potency is primarily due to its electronic
effects on the ring system or if it demonstrates a potency effect in a
regiospecific fashion. Compound 16 incorporates a fluorine in the
R4 position for synthetic reasons but we have demonstrated that
the hydrogen to fluorine switch in position R4 reproducibly causes
only a ca. 1–2ꢀ effect on potency in the dihydro-benzo[a]carbazole
scaffold (data not shown). Placing the CF3 group in the R1 position
(16) or R2 position (17) results in compounds with similar activi-
ties to 15 but it is clear that a CF3 group is not tolerated at the R4
position (18). This indicated to us that the positive effects of the
CF3 group on potency may be due to its electron withdrawing elec-
tronic effect with perhaps an unfavorable steric clash in the R4 po-
sition. In order to test this hypothesis, we incorporated two
alternative electron withdrawing groups: a nitrile at the R2 posi-
tion (19) or a carboxylic acid at the R3 position (20).28 Both com-
pounds have significantly reduced activity, demonstrating that
the CF3 group’s effect on potency is due to factors other than ring
electronics.
Figure 1. Representative structures of reported small molecule TpoR agonists.
Figure 2. HTS hit molecule.
Table 1
In vitro human TpoR functional activity of benzo[a]carbazole derivatives
We next systematically affixed a phenyl group to various regio-
positions on the scaffold in order to gauge the steric limitations
afforded by the binding site of the TpoR. A phenyl ring was affixed
in the R1 (21), R2 (22) or R3 (23) positions. Compound 21 is inactive,
showing potential steric intolerance at the R1 position. The R2 po-
sition (22) partially tolerates the phenyl ring (569 nM, 68%) but the
R3 position (23) shows complete tolerance of a phenyl group as
well as similar potency and efficacy compared to the lead molecule
15. Even though 23 and 15 have similar potencies, we were
encouraged by the fact that a tolerated aromatic ring in the R3 po-
sition would afford a wider range of potential follow-up com-
pounds to synthesize in our attempt to increase potency.
Interestingly, a cyclohexyl group is not tolerated in the R3 position
(24) indicating a possible preference for an aromatic system in the
R3 position. The activity of 23 and synthetic accessibility led us to
focus our efforts on aromatic analogs in the R3 position. Based upon
SAR reported in the literature13 and the possibility of a similar
binding mode in the Tpo receptor, we reasoned that hydrophobic
substitution on the R3 phenyl ring may increase activity and syn-
thesized the derivatives 25–37. In general, the SAR indicates that
increased potency correlates with an increase in lipophilicity in
Compounda
R1
R2
R3
R4
EC50
(l
M)
% Efficacyc
b
7
8
9
10
11
12
13
14
H
H
H
H
H
H
CH3
H
H
H
H
H
OH
OH
OH
OCH3
OH
OH
OH
OH
CO2H
CH2OH
CO2H
CO2H
CO2CH3
CO2H
3.4
100
NA
NA
NA
11
76
70
128
>30
>30
>30
1.3
0.87
9.2
H
CH3
CH3
CF3
CH2CO2H
CO2H
0.143
EltrombopagTM (1)
0.038
127
a
All compounds demonstrated satisfactory LC–MS and 1H NMR characterization.
Ba/F3-hTpoR RGA as previously described.10,14
b
c
Relative to 30 ng/mL Tpo. NA, not applicable.
dramatically reduced efficacy (11% vs 100%). This indicates that the
salicylic acid portion of the molecule is very important for both po-
tency and efficacy. Initial SAR exploration on the opposite side of
the scaffold generated compound 12 which incorporates a methyl

P. B. Alper et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5255–5258
5257
Table 2
In vitro human TpoR functional activity of dihydro-benzo[a]carbazole derivatives
b
Compounda
R1
R2
R3
R4
R5
R6
EC50
(l
M)
% Efficacyc
15
16
17
18
19
20
21
22
23
24
25
26
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
H
CF3
H
H
H
H
H
CF3
H
CN
H
CF3
H
H
H
H
CO2H
H
H
H
F
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
1H-Tetrazole-5-yl
C(O)NH2
C(O)N(CH3)2
2-Imidazoline
0.473
0.799
0.333
7.83
>30
7.79
96
101
115
37
NA
74
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
Phenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>30
NA
68
Phenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.569
0.373
>30
Phenyl
c-Hexyl
136
NA
128
154
30
30-Methyl-phenyl
40-Methyl-phenyl
20,60-Dimethyl-phenyl
30,40-Dimethyl-phenyl
30,50-Dimethyl-phenyl
40-Fluoro-30-methyl-phenyl
30-CF3-phenyl
40-N(CH3)2-phenyl
40-n-Butyl-phenyl
40-Propoxy-phenyl
40-Methoxymethyl-phenyl
30-Propoxy-phenyl
40-n-Butyl-phenyl
40-n-Butyl-phenyl
40-n-Butyl-phenyl
40-n-Butyl-phenyl
40-n-Butyl-phenyl
40-Fluoro-30-methyl-phenyl
0.194
0.109
0.232
0.065
0.067
0.035
0.148
0.284
0.026
0.041
0.452
0.043
0.111
0.055
0.119
0.076
0.127
>30
127
128
126
127
136
143
129
132
147
141
140
128
111
132
NA
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
EltrombopagTM (1)
0.038
127
a
All compounds demonstrated satisfactory LC–MS and 1H NMR characterization.
Ba/F3-hTpoR RGA as previously described.10,14
Relative to 30 ng/mL Tpo. NA, not applicable.
b
c
this region of the ligand although individual heteroatoms are toler-
ated. This indicates that this portion of the scaffold binds to a
highly hydrophobic pocket of the receptor. Tolerance for large sub-
stituents in either the 30 or 40-positons of the phenyl ring at R3 indi-
cates that the binding pocket in this region of the TpoR is sterically
large. The 20,60-dimethyl phenyl analog 28 retains the potency but
not the efficacy of the parent molecule 23, which indicates the po-
tential preference of a flat aromatic ligand geometry in the R3 posi-
tion for receptor activation. The most potent analogs identified
from these efforts (31, 34, 35, and 37) have potencies <50 nM
and efficacies >100% relative to Tpo. Two additional compounds
(29 and 30) are only slightly less potent with potencies of ca.
65 nM. The in vitro Ba/F3-hTpoR RGA potency and efficacy of these
analogs are similar to that seen for the clinically efficacious com-
pound EltrombopagTM (1).
With a potent compound (34) in hand, we next investigated the
possibility of modifying the salicylic moiety. Deleting the phenolic
oxygen of 34 to generate 38 results in a 4ꢀ loss of potency. This
loss in potency is partially addressed by introducing a fluorine in
the R4 position generating compound 39 which has 55 nM potency
with 140% efficacy. Substituting the benzoic acid in 38 with a tet-
razole (40), amide (41) or N,N-dimethylamide (42) generates com-
pounds which all show similar potencies and efficacies as 38,
indicating that a negatively charged moiety is not required for
receptor binding and activation. The 2-imidazolone (43) however
does not retain activity, indicating the intolerance of the receptor
to a positively charged moiety in that portion of the ligand.
The pharmacokinetic profile of compound 30 was determined
in BALB/c mice. It has 10% bioavailability with a Cmax of ca. 2
lM
and a half-life of 4.1 h following 20 mg/kg oral dosing.29 This
was considered a reasonable starting point for the final optimi-
zation of the in vivo properties of this series of compounds.
However, at this time we noted that both in vitro and in vivo,
the dihydro-benzo[a]carbazole series of compounds exemplified
by 30 is chemically unstable and reverts to the fully aromatic
benzo[a]carbazole ring system over time via oxidation of the
5,6-ethylene bridge.
In summary, we have described a structurally novel class of
small molecule agonists of the human Tpo receptor. Beginning
with a 3.4
l
M hit (7), we were able to increase potency 130ꢀ gen-
erating compounds with <50 nM potency and full efficacy in acti-
vating the human Tpo receptor. A representative compound from
this series has oral bioavailability in mice. We were successful in
dearomatizing the original hit molecule but noted that the result-
ing compounds are chemically unstable with rearomatization to
the benzo[a]carbazole ring system being facile. Our efforts to ad-
dress this chemical instability pharmaceutical development issue
are discussed in the following communication in this journal.
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SOCl2 (1.9 equiv), pyridine (0.4 equiv), chloroform; (d) KCN (4 equiv), 160 °C,
10 min (50% over two steps); (e) LiOH, dioxane, H2O (160 °C, 10 min), Na, NH3,
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27. The dihydro-benzo[a]carbazole ring system (15–43) is prepared by reacting a
0.1 M solution of 1 equiv of the appropriate hydrazine hydrochloride, 1 equiv
of the appropriate tetralone, and 2.5 equiv of ZnCl2 in acetic acid at 105 °C
overnight. The final compounds are generated using standard synthetic
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28. Prepared by treatment of 15 with LiOH at 150 °C for 10 min in a mixture of
water and dioxane.
29. Formulated as a 2.5 mg/mL in PEG300/D5W (3:1) solution.