Structure-activity relationship study of bone morphogenetic protein (BMP) signaling inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.06.052

A structure-activity relationship study of dorsomorphin, a previously identified inhibitor of SMAD 1/5/8 phosphorylation by bone morphogenetic protein (BMP) type 1 receptors ALK2, 3, and 6, revealed that increased inhibitory activity could be accomplished by replacing the pendent 4-pyridine ring with 4-quinoline. The activity contributions of various nitrogen atoms in the core pyrazolo[1,5-a]pyrimidine ring were also examined by preparing and evaluating pyrrolo[1,2-a]pyrimidine and pyrazolo[1,5-a]pyridine derivatives. In addition, increased mouse liver microsome stability was achieved by replacing the ether substituent on the pendent phenyl ring with piperazine. Finally, an optimized compound 13 (LDN-193189 or DM-3189) demonstrated moderate pharmacokinetic characteristics (e.g., plasma t_1/2 = 1.6 h) following intraperitoneal administration in mice. These studies provide useful molecular probes for examining the in vivo pharmacology of BMP signaling inhibition.

Full text of DOI:10.1016/j.bmcl.2008.06.052

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4388–4392
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study of bone morphogenetic protein (BMP)
signaling inhibitors
b
a
a
c
b
b
Gregory D. Cuny a, , Paul B. Yu , Joydev K. Laha , Xuechao Xing , Ji-Feng Liu , Carol S. Lai , Donna Y. Deng ,
*
Chetana Sachidanandan d, Kenneth D. Bloch b, Randall T. Peterson d
a Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham & Women’s Hospital and Harvard Medical School, 65 Landsdowne Street,
Cambridge, MA 02139, USA
b Division of Cardiology, Department of Medicine and the Anesthesia Center for Critical Care Research, Massachusetts General Hospital and Harvard Medical School, Thier 505,
50 Blossom Street, Boston, MA 02114, USA
c Aberjona Laboratories, Inc., 100 Cummings Center, Suite 242-F, Beverly, MA 01915, USA
d Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A structure–activity relationship study of dorsomorphin, a previously identified inhibitor of SMAD 1/5/8
phosphorylation by bone morphogenetic protein (BMP) type 1 receptors ALK2, 3, and 6, revealed that
increased inhibitory activity could be accomplished by replacing the pendent 4-pyridine ring with 4-
quinoline. The activity contributions of various nitrogen atoms in the core pyrazolo[1,5-a]pyrimidine ring
were also examined by preparing and evaluating pyrrolo[1,2-a]pyrimidine and pyrazolo[1,5-a]pyridine
derivatives. In addition, increased mouse liver microsome stability was achieved by replacing the ether
substituent on the pendent phenyl ring with piperazine. Finally, an optimized compound 13 (LDN-
193189 or DM-3189) demonstrated moderate pharmacokinetic characteristics (e.g., plasma t1/2 = 1.6 h)
following intraperitoneal administration in mice. These studies provide useful molecular probes for
examining the in vivo pharmacology of BMP signaling inhibition.
Received 29 May 2008
Revised 16 June 2008
Accepted 17 June 2008
Available online 27 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Bone morphogenetic proteins (BMPs) are a group of >25 protein
ligands that comprise a subset of the transforming growth factor b
(TGF-b) family. BMPs modulate a multitude of biological processes,
including bone and cartilage formation during embryogenesis.1a
However, they are also intimately involved with numerous non-
osteogenic developmental and physiological processes throughout
adulthood as well as several pathological conditions.
BMPs bind to two classes of cell surface bone morphogenetic
protein receptors (BMPR-I and BMPR-II).1a The BMPR-I receptor
class consists of three receptor types, activin receptor-like ki-
nase-2 (ALK-2 or ActR-IA), ALK-3 (BMPR-IA) and ALK-6 (BMPR-
IB). The BMPR-II receptor class is comprised of three receptor
types, BMPR-II, ActR-IIA and ActR-IIB. Binding of BMPs results in
the formation of heterotetrameric complexes containing two type
I and two type II receptors. In addition to an extracellular binding
domain, each BMP receptor contains an intracellular serine/threo-
nine kinase domain. Following binding of BMPs, constitutively ac-
tive type II receptor kinases phosphorylate type I receptor kinase
domains that in turn phosphorylate BMP-responsive SMADs 1, 5,
and 8, which can enter the cell nucleus and function as transcrip-
tion factors.1b Phosphorylation of these specific SMADs results in
various cellular effects, including growth regulation and differenti-
ation. Signaling via BMP receptors may also activate other path-
ways, including mitogen activated protein kinase (MAPK).1c
Several diseases are known to arise from inborn defects in the
BMP signaling pathway, including idiopathic pulmonary arterial
hypertension,2 hereditary hemorrhagic telangiectasia syndrome
and juvenile familial polyposis syndrome,3 all of which involve
loss-of-function mutations in BMP receptors or co-receptors. Ac-
quired defects in the BMP signaling pathway are thought to con-
tribute to metastasis of prostate carcinoma4 and renal cell
carcinoma.5 Other disorders, such as fibrodysplasia ossificans pro-
gressiva (FOP)6 and anemia of chronic disease7 may result from in-
creased BMP signaling. For conditions where increased BMP
signaling contributes to disease pathogenesis, inhibitors may offer
therapeutic benefit.
Inhibition of BMP signal transduction could be envisioned to oc-
cur through various mechanisms, including antagonizing BMP
binding to BMPRs or inhibition of the intracellular BMP receptor ki-
nase activity.8 Numerous endogenous protein antagonists that
sequester BMP ligands preventing engagement with BMP receptors
are known, including noggin, follistatin, chordin and gremlin. Small
molecule antagonists of the BMP ligand-receptor interaction have
not been identified, possibly due to difficulties antagonizing this
protein–protein interaction.9 In addition, the structural diversity
of BMP receptors and ligands, and functional redundancy of both
systems might pose a challenge for effective blockade of extracel-
* Corresponding author. Tel.: +1 617 768 8640; fax: +1 617 768 8606.
E-mail address: gcuny@rics.bwh.harvard.edu (G.D. Cuny).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.052

G. D. Cuny et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4388–4392
4389
lular domains. However, inhibition of SMAD phosphorylation by
NMe2
CN
N
CN
BMPR-I intracellular kinase domains with small molecules may
provide more efficient signal transduction pathway inhibition. This
latter approach has been used to identify inhibitors (i.e., SB-
431542) of the TGF-b1 receptor kinase ALK5.10
HN
H2N
a
b
c
N
N
N
Recently, dorsomorphin, 1,7a,11,12 was discovered as an inhibitor
of SMAD 1/5/8 phosphorylation by BMP type 1 receptors (ALK2, 3,
and 6) utilizing a phenotypic screen to identify compounds that
perturb embryonic dorsoventral axis formation (see Fig. 1). Fur-
thermore, this inhibition was shown to decrease BMP-regulated
hepatic hepcidin gene transcription, leading to increased iron lev-
els in vivo.7a However, 1 only demonstrated moderate inhibition of
9
10
8
R
N
Br
N
N
N
d
N
N
N
N
SMAD 1/5/8 phosphorylation by BMPR-I with an IC50 ꢀ 0.5
lM and
11
12, R = Cbz
13, R = H
lacks metabolic stability. Herein, we describe the results of a struc-
ture–activity relationship (SAR) study to optimize BMP signaling
inhibition of SMAD 1/5/8 phosphorylation. In addition, we ad-
dressed the metabolic stability of this compound series and report
a pharmacokinetic study for an optimized derivative.
N
e
N
Scheme 2. Method B. Reagents and conditions: (a) (MeO)2CHNMe2, 110 °C, 16 h,
100%; (b) NH2NH2ꢁHBr, EtOH/H2O, 110 °C, 4 h, 80%; (c) 4-BrPhCH(CHO)2, AcOH,
EtOH, MW, 170 °C, 5 min, 54%; (d) N-Cbz-piperazine, Pd2(dba)3, (2-biphenyl)di-
tert-butylphosphine, KOBu-t, DME, 100 °C, 20 h, 20–30%; (e) H2 (1 atm), 5% Pd/C
(57% H2O), MeOH/CH2Cl2, rt, 4 h, 86%.
The synthesis of substituted pyrazolo[1,5-a]pyrimidine deriva-
tives was initially accomplished according to Scheme 1 (Method
A). Arylacetonitriles, 2, were allowed to react with dimethyforma-
mide dimethylacetal (DMF-DMA) to give 3. In the case of pyridine
or quinoline acetonitrile salts, an equivalent of triethylamine was
also added. Cyclization of 3 in the presence of hydrazine hydrobro-
mide gave 2-amino-1H-pyrazoles 4a. Condensation of 4a–c with
various malondialdehydes in acetic acid and ethanol either under
conventional or microwave (MW)13 heating yielded pyrazolo[1,5-
a]pyrimidine derivatives 5a–c. In the case of 5c, palladium-medi-
ated cross-coupling with arylboronic acids also gave 5a. This reac-
tion was useful for preparing derivatives where the corresponding
arylacetonitriles were not readily available. Dealkylation of the 3-
or 4-methoxy ethers on the pendent phenyl rings was accom-
plished with hydrobromic acid in acetic acid with microwave heat-
ing to give 6. Finally, alkylation in one step with R2N(CH2)nCl or in
two steps with Cl(CH2)nCl followed by amine addition gave 7.
Two other routes were subsequently developed for the synthe-
sis of pyrazolo[1,5-a]pyrimidine derivative 1314 and other analogs
that contained an amine on the 3- or 4-position of the pendent
phenyl ring. The first alternate route, depicted in Scheme 2 (Meth-
od B), began in a similar manner as previously described starting
with 8,15 except that 2-(4-bromophenyl)malondialdehyde was
used to generate 11. Next, a palladium-mediated cross coupling
with N-Cbz-piperazine yielded 12. Removal of the benzyl carba-
mate by hydrogenation (1 atm) in the presence of 5% Pd/C gave 13.
The second alternate route to 13, depicted in Scheme 3 (Method
C), began with 2-amino-1H-pyrazole, 4b, which was allowed to re-
act with 2-bromomalondialdehyde to give 6-bromopyrazolo[1,5-
a]pyrimidine, 15a. A palladium-mediated cross-coupling with 4-
4-(tert-butoxycarbonyl) piperazin-1-ylphenylboronic acid pinacol
ester yielded 16. Next, a regioselective bromination of the C-3 car-
bon with N-bromosuccinimide (NBS) in dichloromethane at room
temperature gave 17a in 79% yield. Palladium-mediated cross-cou-
pling of this aryl bromide with quinoline-4-boronic acid produced
18a in a moderate 46% yield. Finally, deprotection of the tert-butyl
carbamate with 4N HCl in a mixture of 1,4-dioxane and methanol
gave 13 as the hydrochloride salt. This method was also used to
prepare several other derivatives, including 18c that contains a
C-2 substituent.
O
N
N
N
N
1
N
Figure 1. BMP signaling inhibitor of SMAD 1/5/8 phosphorylation.
N
NMe2
CN
CN
a
b
HN
H2N
c
Ar
R1
Ar
4a, R1 = Ar
4b, R1 = H
4c, R1 = Br
2
3
R2
R2
N
N
e
N
N
The synthesis of pyrrolo[1,2-a]pyrimidine derivatives is illus-
trated in Scheme 4 (Method D). 2-Trichloromethylketopyrrole,
19, was regioselectively brominated affording 20.16 Next, regiose-
lective nitration with concentrated nitric acid gave 21.17 This com-
pound was allowed to react with sodium methoxide in methanol
producing the methyl ester 22. Palladium-mediated cross-coupling
of this pyrrole bromide with quinoline-4-boronic acid generated
23 in 60% yield.12a Reduction of the nitro group with hydrogen (1
atm) in the presence of 10% Pd/C gave 24, which was used imme-
diately without purification. Condensation with 2-(4-methoxy-
phenyl)malondialdehyde in acetic acid and ethanol yielded
pyrrolo[1,2-a]pyrimidine derivative 25. Heating this material at
110 °C in aqueous sulfuric acid for 2 h gave 26 via ester hydrolysis
and subsequent decarboxylation.18 Prolonged heating of 25 for 2
days resulted in ether hydrolysis producing 27. Finally, alkylation
of the phenol afforded 28.
N
N
R1
R1
5a, R1 = Ar,
2 = 3- or 4-OMe-Ph
5b, R1 = H,
2 = 4-OMe-Ph
5c, R1 = Br,
R2 = H, Ph or 4-OMe-Ph
6, R1 H or Ar,
R
R2 = 3- or 4-OH-Ph
f
d
7, R1 H or Ar,
R
R
2 = 3- or 4-O[(CH2)nNR2]-Ph
Scheme 1. Method A. Reagents and conditions: (a) (MeO)2CHNMe2, Et3N (for
pyridine and quinoline salts), DMF, 110 °C, 4–6 h, 100%; (b) NH2NH2ꢁHBr, EtOH/
H2O, 110 °C, 6 h, 45–80%; (c) ArCH(CHO)2, AcOH, EtOH, 110 °C, 6 h (or MW, 170 °C,
5 min); (d) ArB(OH)2, Pd2(dba)3, 2-dicyclohexylphosphino-20,40,60-triisopropylbi-
phenyl, K3PO4, n-BuOH, MW, 150 °C, 8 min, 84–90%; (e) HBr/HOAc, MW, 130 °C,
8 min, 65–86%; (f) R2N(CH2)nClꢁHCl, Cs2CO3, NaI (cat), DMF, 60 °C, 3 h, (or MW,
140 °C, 6 min), 30–75% or Cl(CH2)nCl, K2CO3, DMF, MW, 140 °C, 6 min, then R2NH,
NaI (cat), DMF, MW, 150 °C, 10 min, 30–60%.

4390
G. D. Cuny et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4388–4392
R
R1
N
R2
CO2Me
R1
N
HN
H2N
a
O
b
N
R1
N
HN
O2N
d
e
N
N
HN
R3
4b R1 = H
15a R1 = H, R2 = Br
N
R2
14 R1 = Me
15b R1 = Me, R2 = 4-OMe-Ph
N
N Boc
16 R =
N
19 R1 = CCl3, R2 = R3 = H
R2
a
23
20 R1 = CCl3, R2 = Br, R3 = H
21 R1 = CCl3, R2 = Br, R3 = NO2
22 R1 = OMe, R2 = Br, R3 = NO2
b
c
N
N
d
R1
c
N
N
R5O
Br
R4
17a R1 = H, R2
=
N Boc
CO2Me
N
N
17b R1 = Me, R2 = 4-OMe-Ph
f
HN
H2N
R2
N
N
R1
N
N
25 R4 = CO2Me, R5 = Me
26 R4 = H, R5 = Me
27 R4 = H, R5 = H
N
R3
g
24
h
18a R1 = H, R2
=
, R3 = 4-quinolyl
N Boc
N
18b R1 = Me, R2 = OMe, R3 = 4-Py
e
13 R1 = H, R2
=
, R3 = 4-quinolyl
NH
O
N
f, g
N
18c R1 = Me, R2 = O(CH2)2-N-Me-piperizine, R3 = 4-Py
N
N
i
Scheme 3. Method C. Reagents and conditions: (a) BrCH(CHO)2 (or 4-OMe-
PhCH(CHO)2 for 15b) AcOH, EtOH, 80 °C, 7 h, 49%; (b) B(O[C(CH3)2]2O)-4-Ph-N-
Boc-piperazine, Pd(PPh3)4, K2CO3, dioxane/H2O, MW, 150 °C, 8 min, 90% (or 110 °C,
3 h, 86%); (c) NBS, CH2Cl2, rt, 5 h, 79%; (d) quinoline-4-boronic acid, Pd2(dba)3, 2-
dicyclohexylphosphino-20,40,60-triisopropylbiphenyl, K3PO4, n-BuOH, MW, 150 °C,
15 min, 46%; (e) 4 N HCl in 1,4-dioxane, MeOH, rt, 24 h, 95%; (f) HBr/HOAc, MW,
130 °C, 8 min, 81%; (g) Cl(CH2)2Cl, K2CO3, DMF, MW, 140 °C, 6 min, then N-Me-
piperizine, NaI (cat), DMF, MW 150 °C, 10 min, 57%.
28
N
Scheme 4. Method D. Reagents and conditions: (a) Br2, CHCl3, 0 °C, 57%; (b) HNO3
(70%), Ac2O, ꢂ40 °C to rt, 40%; (c) NaOMe, MeOH, rt, 99%; (d) quinoline-4-boronic
acid, Pd(PPh3)4, Na2CO3, 1,4-dioxane, reflux, 16 h, 60%; (e) H2 (1 atm), 10% Pd/C,
MeOH, rt, 0.5 h; (f) 4-MeOPhCH(CHO)2, AcOH, EtOH, reflux, 16 h, 73%; (g) 40%
aqueous H2SO4, 110 °C, 2 h, 91%; (h) 40% aqueous H2SO4, 110 °C, 2 days, 71%; (i)
piperidyl-N-CH2CH2ClꢁHCl, 60% NaH, DMF, rt, 24 h, 80%.
The synthesis of pyrazolo[1,5-a]pyridine derivatives is outlined
in Scheme 5 (Method E). A palladium-mediated cross-coupling of
3-bromopyridine, 29, with 4-methoxyphenylboronic acid pro-
duced 30 in 58% yield.19 This pyridine derivative was converted
to the 1-aminopyridinium salt 31a utilizing O-(2,4-dinitro-
phenyl)hydroxylamine.20 Cyclization of 31a upon treatment with
methyl propiolate gave regioisomers 32a and 32b in a 1:2 ratio
and a combined yield of 33% over two steps.21 In a similar man-
ner, 29 was converted to 33a and 33b (1:2 ratio) in 37% yield, via
intermediate 31b. Compound 33a was further converted to 34 via
a palladium-mediated coupling. Then, 32a and 34 were hydro-
lyzed with aqueous sodium hydroxide and the resulting carbox-
ylic acids were subjected to metal-mediated decarboxylative
coupling22 with 4-bromoquinoline in the presence of Pd(acac)2
and CuI producing 35 and 36, respectively, albeit in low yields
(10–22%). Finally, exposure of 36 to 4 N HCl in 1,4-dioxane re-
sulted in removal of the tert-butyl carbamate yielding 37 as the
hydrochloride salt.
Evaluation of BMP4-induced phosphorylation of SMAD 1/5/8
was performed using a sensitive cytoblot cellular ELISA assay in
the presence of varying concentrations of test compounds.23a,b
Functional IC50 values were calculated for the inhibitory effects
of test compounds on phosphorylation of SMAD 1/5/8 and are
shown in Tables 1–3.23c
Introduction of an aminoether at the 4-position of the pendent
phenyl ring on the pyridine derivatives (e.g., 1 and 39–41 vs 38)
increased activity three to fifteen fold in addition to improving
aqueous solubility (Table 1). Introduction of a substituent on the
2-position of the pyrazolo[1,5-a]pyrimidine ring (43) abolished
activity. In addition, activity was dramatically affected by the nat-
ure of the substituent on the 3-position of the pyrazolo[1,5-
a]pyrimidine ring. Removal (44) or replacement of the 4-pyridyl
in 1 with 3-pyridyl (45) or phenyl (46) resulted in complete loss
of activity. Replacement of the pyridine ring with 3-fluoro-4-pyri-
dyl (47) likewise resulted in reduced activity.
MeO
R1
NH2
+
Br
c
a
b
N
N
N
_
2,4-di-NO2Ph-O
29
31a R1 = 4-OMe-Ph
31b R1 = Br
30
R1
R1
N
N
N
N
e, f
R2
CO2Me
N
32a R1 = 4-OMe-Ph, R2 = H
32b R1 = H, R2 = 4-OMe-Ph
33a R1 = Br, R2 = H
35 R1 = OMe
36 R1
37 R1
=
=
N
N
N Boc
NH
33b R1 = H, R2 = Br
g
d
34 R1
=
, R2 = H
N
N Boc
Scheme 5. Method E. Reagents and conditions: (a) 4-MeOPhB(OH)2, Pd(PPh3)4,
K3PO4, 1,4-dioxane, 100 °C, 18 h, 58%; (b) 2,4-di-NO2PhONH2, CH3CN, 40 °C, 20 h;
(c) HC„CCO2Me, K2CO3, DMF, rt, 33–37% over two steps (32a:32b and
33a:33b ꢀ 1:2); (d) B(O[C(CH3)2]2O)-4-Ph-N-Boc-piperazine, Pd(PPh3)4, K2CO3,
1,4-dioxane/H2O, 110 °C, 5 h, 73%; (e) NaOH, EtOH/H2O (6:1),
D, 3 h; (f) 4-
bromoquinoline, Pd(acac)2, CuI, K2CO3, 1,10-phenanthroline, 4 Å MS, NMP, 165 °C,
24 h, 10–22% (over two steps); (g) 4 N HCl in 1,4-dioxane, MeOH, rt, 24 h.

G. D. Cuny et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4388–4392
4391
Table 1
Due to these significant substituent effects on the 3-position of
the pyrazolo[1,5-a]pyrimidine ring and the demonstrated influ-
ence of heterocyclic substituents on other TGF-b receptor type
inhibitors,24 quinolines attached through various positions were
examined (Table 2). In compound 52, where the pyrazolo[1,5-
a]pyrimidine ring is connected to the 4-position of the quinoline,
a significant increase in activity was observed. Introduction of an
aminoether to the 4-position of the pendent phenyl ring (53) in-
creased potency as was previously observed for the pyridine deriv-
atives (vida supra). Replacing the aminoether with piperazine (13)
was also well tolerated. However, transposing this substituent to
the 3-position of the pendent phenyl (58) resulted in significant
loss of activity. Likewise, introduction of a chloride to the 7-posi-
tion of the quinoline ring (59 vs 52 and 60 vs 13) resulted in de-
creased activity.
Next, the contributions of the nitrogen atoms in the 1- and 4-
positions of the pyrazolo[1,5-a]pyrimidine ring were examined
(Table 3). The importance of the N-1, but not the N-4, nitrogen
atoms was previously demonstrated for KDR kinase inhibition by
pyrazolo[1,5-a]pyrimidine derivatives.25 Similarly, in the present
series of compounds the N-1 (28 vs 54) appears necessary for po-
tent inhibition of BMP4-induced phosphorylation of SMAD 1/5/8,
whereas the N-4 was not essential (37 vs 13).
IC50 determinations for inhibition BMP4-induced phosphorylation of SMAD 1/5/8
R1
N
N
R3
N
R2
Compound
R1
R2
R3
Method
IC50 (lM)
1
38
39
40
41
42
43
44
45
46
47
Pip-CH2CH2O-4-Ph
4-MeO-Ph
4-Py
4-Py
4-Py
4-Py
4-Py
4-Py
4-Py
H
H
H
H
H
H
H
Me
H
H
H
H
—
A
A
A
A
A
C
A
A
A
C
0.43
6.5
2.0
0.50
0.45
4.5
Morph-CH2CH2O-4-Ph
Et2N-CH2CH2O-4-Ph
NMP-CH2CH2O-4-Ph
NMP-CH2CH2O-3-Ph
NMP-CH2CH2O-4-Ph
Pip-CH2CH2O-4-Ph
Pip-CH2CH2O-4-Ph
Pip-CH2CH2O-4-Ph
NMP-CH2CH2O-4-Ph
>20
>20
>20
>20
3-Py
Ph
3-F-4-Py
3.3
Pip, piperidinyl; Morph, morpholinyl; NMP, N-methylpiperazinyl; Py, pyridyl.
Table 2
IC50 determinations for inhibition BMP4-induced phosphorylation of SMAD 1/5/8
Both the original lead compound 1 and a more potent derivative
53 demonstrated poor metabolic stability in mouse liver micro-
R1
N
N
somes (1: half-life (t1/2) of 10.4 min and intrinsic clearance (CLint
of 133 6.6 L/min/mg protein; 53: t1/2 of 13.3 min and CLint of
104 3.4
L/min/mg protein).23b,26 However, replacement of the
)
l
N
5
l
2
ether on the pendent phenyl ring with piperazine resulted in a sig-
nificant increase in mouse liver microsome stability. For example,
13 demonstrated a t1/2 of 82 min and CLint of 16.9 5.6 lL/min/
N
R2
mg protein. Based on the potency and metabolic stability of 13, it
was selected for in vivo pharmacokinetic analysis following a sin-
gle bolus intraperitoneal (ip) administration of 3 mg/kg in male
and female C57B16 mice.23b The results of this study are shown
in Table 4. The pharmacokinetics of 13 was similar in both male
and female mice. The average maximal plasma concentrations
Compound
R1
Position
R2
Method
IC50 (lM)
48
49
50
51
52
53
54
55
56
57
13
58
59
60
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
6
8
5
3
4
4
4
4
4
4
4
4
4
4
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
C
C
C
C
C
A
A
A
A
A
A,B,C
C
C
>20
>20
3.0
>20
0.055
0.010
0.090
5.0
0.75
0.25
0.0049
20
4-MeO-Ph
NMP-CH2CH2O-4-Ph
Pip-CH2CH2O-4-Ph
H
Ph
HO-4-Ph
Piperazinyl-4-Ph
Piperazinyl-3-Ph
4-MeO-Ph
were slightly higher in males (1.54
lM) than in females
(1.29 M) and were reached quickly (<5 min) following adminis-
l
tration. The plasma half-life (1.6 h) and the average AUC values
1
(994 and 1030 ng h/mL) were similar in male and female mice.
In conclusion, a structure–activity relationship study of dorso-
morphin, 1, a previously identified inhibitor of SMAD 1/5/8 phos-
phorylation by BMP type 1 receptors ALK2, 3, and 6, revealed that
increased inhibitory activity could be accomplished by replacing
the pendent 4-pyridine ring with 4-quinoline. The nitrogen atom
in the 1-position of the pyrazolo[1,5-a]pyrimidine ring was deter-
mined to be necessary for inhibitory activity. However, the nitro-
gen atom in the 4-position was not vital. In addition, increased
mouse liver microsome stability was achieved by replacing the
ether substituent on the pendent phenyl ring with piperazine. Fi-
nally, an optimized compound 13 (LDN-193189 or DM-3189)
demonstrated moderate pharmacokinetic characteristics (e.g.,
plasma t1/2 = 1.6 h) following intraperitoneal administration in
mice. Evaluation of this inhibitor in various animal disease mod-
els in which BMP signaling has been hypothesized to play a role,
such as FOP and anemia of chronic disease, are currently on-
going.
0.50
0.35
Piperazinyl-4-Ph
C
Pip, piperidinyl; NMP, N-methylpiperazinyl.
Table 3
IC50 determinations for inhibition BMP4-induced phosphorylation of SMAD 1/5/8
R1
Y
N
X
N
Compound
R1
X
Y
Method
IC50 (lM)
Table 4
Pharmacokinetic analysis of 13 in plasma following bolus intraperitoneal adminis-
tration in mice (N = 3/sex)
25
26
28
35
37
MeO
MeO
Pip-CH2CH2O
MeO
Piperazinyl
N
N
N
CH
CH
CCO2Me
D
D
D
E
>20
>20
2.6
0.15
0.005
CH
CH
N
Sex
Dose (mg/kg)
Cmax
(l
M)
tmax (min)
t1/2 (h)
AUC1 ng h/mL
N
E
Male
Female
3.0
3.0
1.54
1.29
<5
<5
1.6
1.6
994
1030
Pip, piperidinyl.

4392
G. D. Cuny et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4388–4392
11. (a) Also know in the literature as ‘‘compound C”.; This compound was initially
Acknowledgments
reported as an AMP-activated protein kinase (AMPK) inhibitor, see: (b) Zhou,
G.; Myers, R.; Li, Y.; Chen, Y.; Shen, X.; Fenyk-Melody, J.; Wu, M.; Ventre, J.;
Doebber, T.; Fujii, N.; Musi, N.; Hirshman, M. F.; Goodyear, L. J.; Moller, D. E. J.
Clin. Invest. 2001, 108, 1167.
The authors thank Partners Healthcare for financial support. This
work was also supported in part by NIH Grants HL079943 (P.B.Y.),
HL074352 (K.D.B.), and HL079267 (R.T.P.), the Harvard NeuroDis-
covery Center (G.D.C., X.X. and J.K.L.), a Pulmonary Hypertension
Association Mentored Clinical Scientist Award (P.B.Y.) and a grant
from the GlaxoSmithKline Research & Education Foundation for Car-
diovascular Disease (P.B.Y.). The pharmacokinetic study was per-
formed by Absorption Systems (Exton, PA) and the microsome
stability experiments were performed by Cyprotex (Macclesfield,
UK).
12. For similar compounds reported as KDR kinase inhibitors see: (a) Fraley,
M. E.; Hoffman, W. F.; Rubino, R. S.; Hungate, R. W.; Tebben, A. J.;
Rutledge, R. Z.; McFall, R. C.; Huckle, W. R.; Kendall, R. L.; Coll, K. E.;
Thomas, K. A. Bioorg. Med. Chem. Lett. 2002, 12, 2767; (b) Fraley, M. E.;
Rubino, R. S.; Hoffman, W. F.; Hambaugh, S. R.; Arrington, K. L.; Hungate,
R. W.; Bilodeau, M. T.; Tebben, A. J.; Rutledge, R. Z.; Kendall, R. L.; McFall,
R. C.; Huckle, W. R.; Coll, K. E.; Thomas, K. A. Bioorg. Med. Chem. Lett.
2002, 12, 3537.
13. Daniels, R. N.; Kim, K.; Lebois, E. P.; Muchalski, H.; Hughes, M.; Lindsley, C. W.
Tetrahedron Lett. 2008, 49, 305.
14. 13: 1H NMR (DMSO-d6)d 9.75 (d, J = 2.2 Hz, 1H), 9.40 (br s, 1H), 9.29 (d, J = 5.9 Hz,
1H), 9.28 (d, J = 2.2 Hz, 1H), 9.07 (s, 1H), 8.70 (d, J = 8.4 Hz, 1H), 8.51 (d, J = 5.9 Hz,
1H), 8.47 (d, J = 8.4 Hz, 1H), 8.21 (t, J = 7.6 Hz, 1H), 7.99 (t, J = 7.6 Hz, 1H), 7.88 (d,
J = 8.8 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H), 3.51–3.58 (m, 4H), 3.20–3.30 (m, 4H);
HRMS m/z 407.1979 (calcd for C25H23N6, MH+, 407.1979).
Supplementary data
15. (a) Prepared from quinoline-4-carboxaldehyde in three steps: (i) reduction
(NaBH4, MeOH, 0 °C to rt); (ii) conversion of the alcohol to a chloride (2 equiv
SOCl2, DCM, 0 °C to rt); (iii) conversion of the chloride to a nitrile (NaCN, DMF,
rt).; For an alternate procedure see: (b) Engler, T. A.; Furness, K.; Malhotra, S.;
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Supplementary data associated with this article can be found, in
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