Identification of a pharmacophore for thrombopoietic activity of small, non-peptidyl molecules. 2. Rational design of naphtho[1,2-d]imidazole thrombopoietin mimics

Article abstract of DOI:10.1021/jm0255365

The invention of a new class of naphtho[1,2-d]-imidazole thrombopoietin mimics based on a pharmacophore-hypothesis for small-molecule thrombopoietic agonists is discussed. Parallel array synthesis and purification techniques allowed for the rapid explorat

Full text of DOI:10.1021/jm0255365

3576
J . Med. Chem. 2002, 45, 3576-3578
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. 2. Ra tion a l
Design of Na p h th o[1,2-d ]im id a zole
Th r om bop oietin Mim ics
Kevin J . Duffy,*,† Alan T. Price,† Evelyne Delorme,‡
Susan B. Dillon,† Celine Duquenne,†
Connie Erickson-Miller,† Leslie Giampa,‡
Yifang Huang,† Richard M. Keenan,† Peter Lamb,‡
Nannan Liu,† Stephen G. Miller,‡ J on Rosen,‡
Anthony N. Shaw,† Heather Smith,‡
Kenneth J . Wiggall,† Lihua Zhang,† and
J uan I. Luengo†
F igu r e 1. Comparison of pyrazol-4-ylidenehydrazine TPO
mimic 1 and thiosemicarbazone TPO mimic 2, illustrating the
three key features for potent thrombopoietic activity: central
metal chelate, lipophilic end portion, and a distal acidic
functionality.
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: The invention of a new class of naphtho[1,2-d]-
imidazole thrombopoietin mimics based on a pharmacophore
hypothesis for small-molecule thrombopoietic agonists is dis-
cussed. Parallel array synthesis and purification techniques
allowed for the rapid exploration of structure-activity rela-
tionships within this class and for the improvement in TPO
mimetic potencies and efficacies.
In the preceding paper,1 we disclosed the discovery
of a novel series of salicylaldehyde thiosemicarbazone
thrombopoietin (TPO) mimics and the optimization of
their potency based on structural comparisons with the
known, potent pyrazol-4-ylidenehydrazine class of TPO
mimics.2 The successful use of structure-activity rela-
tionships from the pyrazol-4-ylidenehydrazines to im-
prove potency in the salicylaldehyde thiosemicarbazones
is very strong evidence that these two structurally
diverse series of TPO agonists share similar or identical
pharmacophores. Figure 1 summarizes the three key
structural features previously identified for potent TPO
agonist activity: (1) lipophilic functionality and (2)
acidic functionality on opposite ends of the molecule
separated by (3) a heteroatom metal chelate in the
central portion. Encouraged by this previous successful
implementation of our pharmacophore hypothesis for
small-molecule TPO mimics, we initiated studies toward
the rational design of a further structurally distinct
series of TPO mimics using these three structural
elements (acidic-chelate-lipophilic).
Of the many possible structures considered, the
synthetic accessibility of a naphtho[1,2-d]imidazole
template prompted us to investigate the viability of
2-substituted 9-hydroxynaphtho[1,2-d]imidazoles as po-
tential new templates. Figure 2 shows the important
structural features of this new template in relation to
our pharmacophore hypothesis. The chelation motif is
provided by the imidazole N-1, the 9-hydroxy group, and
an o-hydroxyaryl substituent at C-2. Acidic and lipo-
F igu r e 2. Design of the naphtho[1,2-d]imidazole template.
philic groups can be readily incorporated at distal ends
of the template on the naphthalene ring and on the
2-hydroxyaryl group, respectively. From a synthetic
point of view acidic-substituted naphthalenes are more
readily available than lipophilic naphthalenes, and so
commercially available 4-amino-5-hydroxynaphthalene-
1-sulfonic acid 3a and 5-amino-4-hydroxynaphthalene-
2-sulfonic acid 3b were chosen as initial starting
materials (Scheme 1). To prepare the required inter-
mediate o-diaminonaphthols 4a ,b, naphthalenes 3a ,b
were coupled with diazotized 4-aminobenzenesulfonic
acid to afford the intermediate diazo compounds, which
were then reduced with sodium dithionite to afford the
desired diamines 4a ,b isolated as the dihydrochloride
salts. 2-Hydroxy-1-naphthaldehyde and 1-hydroxy-2-
naphthaldehyde were selected as representative lipo-
philic hydroxyarylaldehydes, and their condensations
with diamines 4a ,b were investigated. Oxidative cyclo-
condensation of the two isomeric aldehydes with di-
amines 4a ,b with DDQ or sodium hydrogen sulfite then
furnished the desired naphtho[1,2-d]imidazoles 5a ,b
and 6a ,b.
From tests in the BAF-3/TPO-Rluc cell line,3-5 lu-
ciferase assay indeed showed that naphtho[1,2-d]imi-
dazoles 5a and 6a were TPO mimics with potencies of
35 and 4.8 µM, respectively and efficacies in the range
of 30% TPOmax. The potencies for the 7-sulfonic acids
5b and 6b were 26 and 9.7 µM, respectively, and are of
similar magnitude compared to the 5-sulfonic acid
isomers. Although the activities for these two com-
pounds were relatively weak when compared to the
other two structural classes of TPO mimics, they did
* 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/jm0255365 CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/19/2002

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3577
Ta ble 1. TPO Mimetic Activity for Naphtho[1,2-d]imidazoles
5a ,b, 6a ,b, and 8 As Shown by the Activation of Luciferase
Expression in the BAF-3/TPO-Rluc Cell Line and in the
Proliferation of the UT7/TPO-R Cell Line
Sch em e 1. Synthesis of Naphtho[1,2-d]imidazoles 5
and 6a
luciferase
efficacy
proliferation
EC50
efficacy
EC50
compound
(% TPO)
(µM)
(% TPO)
(µM)
5a
5b
6a
6b
8
52
11
28
27
36
35
26
4.8
9.7
1.2
<10
<10
<10
<10
70
1
Sch em e 2. Synthesis of Naphtho[1,2-d]imidazole 8a
a
(a) NH2-(p-C6H4)SO3H, NaNO2, aqueous HCl, room temp; (b)
Na2S2O7, aqueous HCl, 50 °C; (c) HO-(naphthyl)-CHO, DDQ,
pyridine, MeOH, reflux; (d) HO-(naphthyl)-CHO, NaHSO3,
EtOH, water, reflux.
a
(a) NaHSO3, EtOH, water, reflux.
agonist activity to effectively support the proliferation
of UT7/TPO cell lines during the longer 3-day prolifera-
tion assay. However, biphenyl analogue 8 was 5- to 30-
fold more potent in the luciferase assay than the
naphthols and consequently showed good activity in the
UT7/TPO proliferation assay with an EC50 of ap-
proximately 1 µM.
To expand upon this potency increase, several mul-
ticomponent arrays comprising over 300 compounds
overall were rapidly synthesized containing different
functionality in the biphenyl portion of the naphtho-
[1,2-d]imidazoles and differing in the nature (sulfonic,
carboxylic, tetrazole, acyl sulfonamide, etc.) and position
(5- and 7-substituted) of the acidic functionality (a full
account is outside the scope of this letter and will be
published shortly6). Illustrative is the synthesis of one
member, the 2-[3-(3,4-dimethylphenyl)-2-hydroxyphe-
nyl]naphtho[1,2-d]imidazole 8 shown in Scheme 2. The
requisite aldehyde 7 was prepared as described previ-
ously1 and oxidatively condensed with the diamine 4b
to provide the biphenylnaphtho[1,2-d]imidazole 8 in
high yield, isolated as the hydrochloride salt (Scheme
2).
Figure 4 illustrates the SAR trends gathered from the
over 300 naphtho[1,2-d]imidazoles wherein one intrigu-
ing divergence from those of the other two series is
conspicuous: the 2-methoxy analogues (Figure 4; X )
OMe) were generally more potent than the 2-hydroxy
analogues (Figure 4; X ) OH), the opposite trend as seen
for the thiosemicarbazones.1 This prompted the synthe-
sis of des-hydroxy analogues (Figure 4; X ) H) to
investigate whether the third chelatable heteroatom
could be completely deleted in the naphtho[1,2-d]imi-
dazole series. The 3-(4-tert-butylphenyl) group was
chosen as one of the optimal lipophilic substituents, and
both 7-sulfonic and 7-carboxylic acid8 analogues were
prepared.
F igu r e 3. Overlay of the metal-chelating heteroatoms of
pyrazol-4-ylidenehydrazine 1 (red) thiosemicarbazone 2 (green)
and the naphtho[1,2-d]imidazole 8 (blue).5
serve to validate our current hypothesis regarding a
shared pharmacophore for small-molecule mimetics of
TPO.
In a strategy similar to that undertaken with the
thiosemicarbazone TPO mimics,1 biphenyl groups were
chosen as isosteric replacements of the N-arylpyrazoli-
none functionality of the potent pyrazol-4-ylidenehy-
drazine class of TPO mimics and were anticipated to
be an improvement over the naphthol functionality
already incorporated in compounds 5 and 6. Modeling
predicts that lipophilic 3-biaryl functionality would be
optimum (Figure 3) as illustrated by the overlay of
pyrazol-4-ylidenehydrazine 1, salicylaldehyde thiosemi-
carbazone 2, and a naphtho[1,2-d]imidazole substituted
at the 2-position with a 3-(3,4-dimethylphenyl)-2-hy-
droxyphenyl group, compound 8.
Indeed, biphenyl 8 proved to be more potent and
efficacious than the naphthol analogues 5 and 6. Table
1 compares the potencies and efficacies of naphtho[1,2-
d]imidazoles 5a ,b, 6a ,b, and 8 in the BAF-3/TPO-Rluc
cell line and toward the proliferation of UT7/TPO cell
lines.7 As can be seen in Table 1, naphthols 5a ,b and
6a ,b all showed activation of luciferase expression in
the BAF-3/TPO-Rluc cell line but lacked sufficient
As can be seen in Table 2, deletion of the lower
heteroatom does result in significant increases in po-

3578 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Letters
F igu r e 5. CD41+ assay for 10. Human bone marrow CD34+
cells (2 × 106 cells/mL) in IMDM containing fetal calf serum
(20% v/v) and recombinant human stem cell factor (100 ng/
mL) were incubated with 10 for 10 days and then stained with
FITC-anti-IgG1 isotype control and FITC-anti-CD41 (Pharm-
ingen clone SZ22) and analyzed on a Becton-Dickinson FAC-
Scan flow cytometer.
F igu r e 4. General SAR trends elucidated from array syn-
theses of over 300 naphtho[1,2-d]imidazoles.
Ta ble 2. TPO Mimetic Activity for Naphtho[1,2-d]imidazoles 9
and 10 As Shown by the Proliferation of the UT7/TPO-R Cell
Line
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for the synthesis and characterization of naphtho[1,2-d]-
imidazole TPO mimics 5a ,b, 6a ,b, and 8-10. This material is
available free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Duffy, K. J .; Shaw, A. N.; Delorme, E.; Dillon, S. B.; Erickson-
Miller, C.; Giampa, L.; Huang, Y.; Keenan, R. M.; Lamb, P.; Liu,
N.; Miller, S. G.; Price, A. T.; Rosen, J .; Smith, H.; Wiggall, K.
J .; Zhang, L.; Luengo, J . I. Identification of a Pharmacophore
for Thrombopoietic Activity of Small, Non-Peptidyl Molecules.
1. Discovery and Optimization of Salicylaldehyde Thiosemicar-
bazone Thrombopoietin Mimics. J . Med. Chem. 2002, 45,
3573-3575.
compound
Y
efficacy (% TPO)
EC50 (µM)
9
10
SO3H
CO2H
100
60
0.08
0.30
(2) Duffy, K. J .; Darcy, M. G.; Delorme, E.; Dillon, S. B.; Eppley, D.
F.; Erickson-Miller, C. L.; Giampa, L.; Hopson, C. B.; Huang,
Y.; Keenan, R. K.; Lamb, P.; Leong, L.; Liu, N.; Miller, S. G.;
Price, A. T.; Rosen, J .; Shah, R.; Shaw, A. N.; Smith, H.; Stark,
K. C.; Tian, S.-S.; Tyree, C.; Wiggall, K. J .; Zhang, L.; Luengo,
J . I. Hydrazinonaphthalene and Azonaphthalene Thrombopoi-
etin Mimics Are Nonpeptidyl Promoters of Megakaryocytopoie-
sis. J . Med. Chem. 2001, 44, 3730-3745.
tency in both the sulfonic acid 9 and carboxylic acid 10
naphtho[1,2-d]imidazoles over the hydroxy-containing
compounds (e.g., compound 8). This leads to an intrigu-
ing conclusion that the pharmacophore requirements
are quite similar but not identical for the naphtho[1,2-
d]imidazole series of TPO mimics compared with the
pyrazol-4-ylidenehydrazine and thiosemicarbazone se-
ries.
(3) Lamb, P.; Rosen, J . Drug discovery using receptors that modu-
late gene expression. J . Recept. Signal Transduction Res. 1997,
17, 531-543.
(4) Takebe, Y. Luciferase assay: A rapid and sensitive method for
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18, 425-432.
(5) MOE 2001.01; Chemical Computing Group Inc.: Quebec, Canada,
1997-2001.
As with these two previously described series of TPO
mimics, the naphtho[1,2-d]imidazoles are completely
selective for TPO-R over other cytokine responsive cell
lines (e.g., EPO, G-CSF, IL-3). They also stimulated a
similar pattern of cell-signaling protein phosphorylation
(STAT-5, MAPK) as TPO itself, and the more potent
analogues are able to promote the growth and dif-
ferentiation of purified CD34+ cells from human bone
marrow into CD41 expressing megakaryocytic precursor
cells as shown for carboxylic acid analogue 10 in Figure
5.
(6) Price, A. T.; et al. Manuscript in preparation.
(7) Komatsu, N.; Kunitama, M.; Yamada, M.; Hagiwara, T.; Kato,
T.; Miyazaki, H.; Eguchi, M.; Yamamoto, M.; Miura, Y. Estab-
lishment and characterization of the thrombopoietin-dependent
megakaryocytic cell line, UT-7/TPO. Blood 1996, 87, 4552-4560.
(8) The requisite 5,6-diamino-4-hydroxynaphthalenecarboxylic acid
was prepared as shown below. Full experimental procedures are
given in the Supporting Information.
(9) Duffy, K. J .; et al. Manuscript in preparation.
In this manuscript, we have provided strong evidence
that a defined pharmacophore exists for activation of
the TPO receptor by small-molecule agents. A full
account of the application and further refinement of this
pharmacophore model toward the optimization of po-
tency and efficacy of the naphtho[1,2-d]imidazole series
of TPO mimics introduced here will be reported shortly.
Further, a full investigation into the metal binding
aspects of the pharmacophore and into the mode of
interaction of the TPO mimics with the TPO receptor
is beyond the scope of this manuscript.9
(a) (EtO2CCH2)2, KOt-Bu, t-BuOH, reflux, 79% (b) NaOAc, Ac2O,
reflux then hydrazine, room temp. 69%; (c) 6N aqu. HCl, reflux
54%; (d) NH2SO2(p-C6H4)-N2+Cl-, aqu. HCl, room temp.; (e)
SnCl2, conc. HCl, 95 °C, 43% over 2 steps.
J M0255365