Synthesis and structure-activity relationships of a novel and selective bone morphogenetic protein receptor (BMP) inhibitor derived from the pyrazolo[1.5-a]pyrimidine scaffold of Dorsomorphin: The discovery of ML347 as an ALK2 versus ALK3 selective MLPCN probe

Article abstract of DOI:10.1016/j.bmcl.2013.03.113

A structure-activity relationship of the 3- and 6-positions of the pyrazolo[1,5-a]pyrimidine scaffold of the known BMP inhibitors dorsomorphin, 1, LDN-193189, 2, and DMH1, 3, led to the identification of a potent and selective compound for ALK2 versus ALK3. The potency contributions of several 3-position substituents were evaluated with subtle structural changes leading to significant changes in potency. From these studies, a novel 5-quinoline molecule was identified and designated an MLPCN probe molecule, ML347, which shows >300-fold selectivity for ALK2 and presents the community with a selective molecular probe for further biological evaluation.

Full text of DOI:10.1016/j.bmcl.2013.03.113

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3248–3252
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of a novel and
selective bone morphogenetic protein receptor (BMP) inhibitor
derived from the pyrazolo[1.5-a]pyrimidine scaffold of
Dorsomorphin: The discovery of ML347 as an ALK2 versus
ALK3 selective MLPCN probe
Darren W. Engers a,c,d, Audrey Y. Frist e, Craig W. Lindsley a,b,c,d,f, Charles C. Hong a,e,f,g
,
Corey R. Hopkins a,b,c,d,
⇑
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
c Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
d Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN 37232, USA
e Division of Cardiovascular Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA
f Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
g Research Medicine, Veterans Administration TVHS, Nashville, TN 37212, 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 of the 3- and 6-positions of the pyrazolo[1,5-a]pyrimidine scaffold of the
known BMP inhibitors dorsomorphin, 1, LDN-193189, 2, and DMH1, 3, led to the identification of a potent
and selective compound for ALK2 versus ALK3. The potency contributions of several 3-position substitu-
ents were evaluated with subtle structural changes leading to significant changes in potency. From these
studies, a novel 5-quinoline molecule was identified and designated an MLPCN probe molecule, ML347,
which shows >300-fold selectivity for ALK2 and presents the community with a selective molecular
probe for further biological evaluation.
Received 11 February 2013
Revised 22 March 2013
Accepted 27 March 2013
Available online 11 April 2013
Keywords:
ALK2 kinase
Bone morphogenic receptor (BMP)
Pyrazolo[15-a]pyrimidine
Selectivity
Ó 2013 Elsevier Ltd. All rights reserved.
ML347
The bone morphogenetic protein (BMP) signaling pathway
plays critical, diverse roles in embryonic pattern formation, and a
number of disease processes.1 BMP ligands bind and activate the
type-I and type-II BMP receptors, a family of serine–threonine ki-
nases belonging to the TGF-b receptor superfamily, which then
activate downstream mediators Smad1/5/8 by phosphorylation.2
Activated Smad1/5/8 translocate to the nucleus to turn on BMP tar-
get genes. Because there are more than 20 distinct BMP ligands, a
number of extracellular antagonists, three type-II receptors (BMP
type II receptor, BMPRII; Activin type II receptor; ActRIIa and ActI-
Ib), and four BMP type-I receptors (activin-receptor like kinase 1,
ALK1; ALK2, ALK3 and ALK6), the role of targeting an individual
component in various signaling contexts is unclear.
Recently, in a chemical genetic screen for compounds that per-
turb zebrafish embryonic axis, we discovered dorsomorphin (DM),
1, the first small molecule inhibitor of the BMP pathway which
directly targets the type-I receptor.3 DM, 1, and its analog LDN-
193189, 2, have been instrumental in demonstrating the therapeutic
potential of BMP inhibitors for anemia, Duchenne muscular dystro-
phy, atherosclerosis and heterotopic ossification syndromes.1,4
However, the early generation compounds DM, 1, LDN-193189, 2,
and DMH1, 3,5 do not discriminate between ALK1, ALK2, ALK3
and ALK6 (Fig. 1)5, and long-term consequences of pharmacologi-
cal inhibition of all BMP signals are unknown. The issue of subtype
selectivity is particularly germane to fibrodysplasia ossificans pro-
gressiva (FOP), a rare congenital disease of progressive soft tissue
ossification, since it is caused by dysregulated BMP signaling due
to a highly recurrent mutation (R206H) in ALK2.6,7 Although
LDN-193189, 2, could blunt ectopic ossification in a mouse model
expressing a constitutively active form of ALK2 (Q207D),8 inhibi-
tors with greater subtype selectivity might be more desirable as
⇑
Corresponding author at: Department of Pharmacology, Vanderbilt University
Medical Center, Nashville, TN 37232, USA.
E-mail address: corey.r.hopkins@vanderbilt.edu (C.R. Hopkins).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.113

D. W. Engers et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3248–3252
3249
Table 1
HN
SAR of the 3-position of pyrazolo[1,5-a]pyrimidine scaffold (7a–m)
O
N
N
O
N
N
N
N
N
N
N
N
N
R1
N
N
Compd
R1
BMP4 cell IC50 (nM)14,15
1
2
Dorsomorphin (DM),
O
LDN-193189,
N
∗
7a
Inactive
N
N
N
∗
∗
∗
N
7b
7c
7d
Inactive
Inactive
94.1
N
3
DMH1(VU0364852),
N
Figure 1. Structures of previously disclosed BMP inhibitors, Dorsomorphin (DM), 1,
LDN-193189, 2, and DMH1, 3.
NH
lead compounds for FOP. Such inhibitors might also be useful
chemical probes to interrogate the biology of BMP signaling at
the subtype resolution. Moreover, because DM, 1, and LDN-
193189, 2, have important off-target effects, including AMP-acti-
vated kinase (AMPK), platelet-derived growth factor receptor-b
(PDGFRb) and vascular endothelial growth factor type-II receptor
(VEGFR-2/KDR), validity of some of their in vivo effects has been
challenged.5,9 Therefore, we undertook a synthetic effort to devel-
op compounds with greater subtype selectivity, centered around
the central pyrazolo[1.5-a]pyrimidine scaffold which has culmi-
nated in the discovery of an ALK2 selective compound, ML347.
The first round of SAR was designed by keeping the 6-(4-
methoxyphenyl) moiety constant and varying the 3-position (R1).
To that end, compounds 7a–n were synthesized as outlined in
Scheme 1. Starting with the commercially available 2-(4-methoxy-
phenyl)malonaldehyde, 4, and condensing with 1H-pyrazol-5-amine,
5, under acidic conditions afforded the 6-(4-methoxyphenyl)
pyrazolo[1,5-a]pyrimidine in 92% yield,10 which was then
iodinated (NIS, DMF) to give 6.11 The final compounds 7a–n were
synthesized either by converting 6 to the boronate ester (diboronp-
inacol ester, Pd(dppf)Cl2ÁDCM, KOAc, DMF, 100 °C, 16 h) followed
by Suzuki–Miyaura cross-coupling12 with the appropriate aryl
halide (ArX, Pd(dppf)Cl2ÁDCM, K3PO4) or direct Suzuki–Miyaura
cross-coupling with an appropriate aryl boronic acid (R1B(OH)2,
Pd(dppf)Cl2ÁDCM, K3PO4).
N
∗
7e
7f
Inactive
Inactive
N
∗
N
∗
7g
7h
152
N
∗
6732
N
∗
7i
<1
N
∗
N
N
7j
Inactive
Inactive
7k
H
∗
N
7l
Inactive
Inactive
The SAR of the 3-position of the pyrazolo[1,5-a]pyrimidine
scaffold is detailed in Table 1. Based on previous studies from
N
∗
7m
S
∗
O
O
S
N
HN
a,b
N
O
7n
4571
N
+
6
H2N
N
3
N
O
5
4
I
6
O
c,d or e
N
N
our laboratories,5 and others,13 where it was shown that heterocy-
cles with nitrogen in the 4-position were optimal, it was not sur-
prising to see the substituted isoquinoline and 3- or 8-quinoline
compounds inactive (7a–c, e, f). The 4-pyrazole compound (7d)
was active (94.1 nM) as was the 5-quinoline (152 nM), which
was not expected as this is the first compound without a nitrogen
in the 4-position to show such potency. The most potent com-
pound in the BMP4 cell assay was the 4-quinoline (7i, <1 nM),
N
R1
7a-n
Scheme 1. Reactions and conditions: (a) AcOH, EtOH, 170 °C, 10 min,
l
W, 92%; (b)
NIS, DMF; (c) diboronpinacol ester, Pd(dppf)Cl2ÁDCM, KOAc, DMF, 100 °C, 16 h; (d)
R1X, Pd(dppf)Cl2ÁDCM, K3PO4, 1,4-dioxane, H2O, 120 °C, 30 min,
lW, 17%–27% (3
steps); (e) R1B(OH)2, Pd(dppf)Cl2ÁDCM, K3PO4, 1,4-dioxane, H2O, 120 °C, 30 min,
lW, 10%–65% (2 steps).

3250
D. W. Engers et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3248–3252
Table 2
which is consistent with previous findings. Other nitrogen (7j
and 7l) or sulfur compounds (7m) were inactive. However, subtle
changes to the 3-postion substituent led to significant loss of activ-
ity. By changing the 4-quinoline (7i) to the 7-thieno[3,2-b] pyridine
(7n) resulted in nearly complete erosion of activity (4571 nM) even
though these compounds are similar in size and shape.
Having evaluated a number of 3-position substituents, we next
looked at the 4-position of the 6-phenyl substituent. Previous re-
sults from our laboratory5 evaluated substituted alkyl chain sub-
stituents (such as those in 1 and 3) with much success and
additional studies evaluated phenyl replacements (pyridinone,16
unpublished results); however, any phenyl replacements led to
inactive compounds. Thus, the 6-phenyl moiety has remained in-
tact for this study. The synthesis of these analogs follows the out-
lined steps in Scheme 2. Starting with 2-bromomalonaldehyde, 8,
and condensing with 1H-pyrazol-5-amine, 5, under acidic condi-
tions led to 6-bromopyrazolo[1,5-a]pyrimidine, 9.10 Next, an
appropriately substituted 4-phenylboronate ester was reacted
SAR of the 6- and 3-position of the pyrazolo[1,5-a]pyrimidine scaffold (13a–r)
R2
N
N
N
R3
Compd
R2
R3
BMP4 Cell IC50 (nM)14,15
∗
13a
141
NH
N
∗
N
S
13b
Inactive
O
O
∗
∗
with
9
under Suzuki-Miyaura cross-coupling conditions
(Pd(dppf)Cl2ÁDCM, K3PO4) in good yields 56%–73%. The resulting
compound, 11, was then iodinated in the 3-position (NIS, DMF,
73%) and the final compounds (13a–r) were realized after a final
Suzuki–Miyaura cross-coupling12 step with an appropriate boronic
acid (R3B(OH)2, Pd(dppf)Cl2ÁDCM, K3PO4).
13c
13d
529
Cl
O
N
N
∗
<1.0
N
The SAR of these compounds had two points of diversity on the
molecule––with the 6-(4-phenyl)- position having molecules sim-
ilar to that found in LDN-193189, 2; namely six-membered hetero-
cycloalkyl groups (piperazine in LDN-193189). The first R2 group
evaluated was morpholine (Table 2) (13a–g) with the SAR tracking
similarly to that seen in Table 1 and previously. Thus, the most
active compounds were those containing heterocycles in the
4-position of the 3-substituent (4-pyrazole, 13a, 141 nM;
2-chloro-4-pyridine, 13c, 529 nM; 4-quinoline, 13d, <1.0 nM;
3-benzo[b]thiophene, 13g, 418 nM). These same R3 groups were
active when the R3 group was changed to piperazine (13h–n)
and 4-methylpiperazine (13o–r). In each case, the 4-quinoline or
4-pyrazole was the most potent of the R3 groups. Although the
3-benzo[b]thiophene was active in the morpholine groups, the
activity significantly dropped off in the piperazine group (13n,
2636 nM) and was inactive in the 4-methylpiperazine group
(13o). Notably, the 5-quinoline compound (13m) was equipotent
to 7g. This SAR trends mimics that which was seen in our earlier
work on this scaffold.5
∗
∗
∗
13e
13f
13g
Inactive
Inactive
418
N
N
S
∗
13h
13i
<1.0
NH
N
∗
N
N
Inactive
∗
HN
13j
13k
13l
246
N
Cl
Having identified a number of potent inhibitors in the func-
tional BMP4 cell based assay, which with certain structural classes
can be difficult to interpret due to promiscuity, we next sent a
∗
N
Br
Inactive
Inactive
∗
O
Br
∗
∗
N
N
HN
a
N
O
+
Br
13m
13n
13o
13p
117
H2N
N
8
5
N
9
O
O
R2
R2
B
2636
10
c
S
∗
N
N
b
11
N
Inactive
Inactive
R2
R2
S
∗
d
N
I
N
N
N
N
N
12
N
N
R3
∗
13a-r
Scheme 2. Reactions and conditions: (a) AcOH, EtOH, reflux; (b) Pd(dppf)Cl2ÁDCM,
K3PO4, 1,4-dioxane, H2O, 150 °C, 30 min,
lW, 56%–73% (2 steps); (c) NIS, DMF, rt,
73%; (d) R3B(OH)2, Pd(dppf)Cl2ÁDCM, K3PO4, 1,4-dioxane, H2O, 120 °C, 30 min,
lW,
14%–52%.

D. W. Engers et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3248–3252
3251
Table 4
Table 2 (continued)
In vitro pharmacokinetic properties of ML347
Compd
R2
R3
BMP4 Cell IC50 (nM)14,15
O
∗
N
N
13q
523
Cl
N
N
N
∗
N
13r
25.2
ML347
7g
MW
352.4
number of compounds for kinase selectivity to Reaction Biology
Corp. (Malvern, PA). We tested our active compounds against 10
kinases (Table 3) and each of the assays were run in 10-dose IC50
cLogP
TPSA
4.00
52.3
Human
mode with threefold serial dilution starting at 100
lM. The reac-
In vitro PK parameter
Rat
Mouse
tions were carried out at 10 M ATP. The three reference com-
l
CLINT (mL/min/kg)
CLHEP (mL/min/kg)
PPB (% fu)
516
20.2
0.9
148
47.5
1.4
617
78.5
1.4
pounds have been previously run in these selectivity assays and
are shown in Table 3. DM, 1, LDN-193189, 2, and DMH1, 3, are po-
tent against ALK2 (ACVR1) however, all the compounds are equi-
potent or more potent against ALK3 (BMPR1A). In addition, these
compounds have variable selectivity against the other kinases
evaluated; however, DMH1, 3, shows the most selectivity within
these three compounds. Across the board, all compounds tested
were equipotent against ALK1 (ACVRL1) and ALK2 (ACVR1),
however, there were two compounds identified that displayed
selectivity against ALK3 (BMPR1A) and each compound contain a
5-quinoline R3 substituent (7g and 13m). The more potent (and
selective) compound, 7g, has IC50’s of 46 and 32 nM, respectively,
against ALK1 and ALK2; however, the IC50 against ALK3 is
10,800 nM, >300-fold selective over ALK3. In addition, 7g is com-
pletely inactive against all the other kinases tested (with weak
activity against ALK6, 9830 nM and KDR (VEGFR2) 19,700 nM). It
is interesting to note that it appears to be a combination of the
5-quinoline and 4-methoxyphenyl which gives rise to the selectiv-
ity profile, as 13m still retains significant ALK3 activity (539 nM).
Due to the potency of 7g against the BMP4 cell assay, ALK1 and
ALK2 and the significant selectivity against the other kinases, 7g,
has been declared a probe molecule in the MLPCN and redesignat-
ed ML347.17
In order to further the BMP community as to the utility of
ML347, we evaluated this molecule in our Tier 1 in vitro pharma-
cokinetic assays (Table 4). These studies are useful in order to eval-
uate the metabolic stability and predicted clearance in a number of
species in order to inform on possible dosing routes. Utilizing rapid
equilibrium dialysis, the protein binding of ML347 was determined
in human, rat and mouse plasma. The results were similar in all
three species with ML347 displaying high plasma protein binding
(% Fu ꢀ 1.0–1.4). ML347 was also assessed for its intrinsic clearance
in hepatic microsomes. This measure will help predict the in vivo
PBS solubility
4 lM
clearance in the same three species (CLHEP). ML347 was unstable
to oxidative metabolism––possibly due to the labile methoxy
group19––and therefore was predicted to display high clearance
in human and mouse, and moderate-to-high clearance in the rat.
Going forward, the intrinsic clearance is predicting high clearance
after oral dosing, a more appropriate dosing paradigm might be
intraperitoneal dosing for this compound. Further in vivo experi-
ments, including PK, will be reported in due course.
In conclusion, SAR studies of the 3- and 6-positions of the pyr-
azolo[1,5-a]pyrimidine scaffold revealed a potent and selective
inhibitor of ALK2 versus ALK3. These studies further validated that
4-phenyl substituents of the 6-position on the pyrazolo[1,5-
a]pyrimidine scaffold allowed a wide range of substituents, from
ethers to cycloheteroalkyl (morpholine, piperazine, 4-methylpiper-
azine). These studies also revealed that subtle changes of the
3-position substituents can drastically influence the BMP activity
(e.g., 7i vs 7n). These SAR studies culminated in the discovery of
a highly selective ALK2 inhibitor, ML347, which shows >300-fold
selectivity for ALK2 versus ALK3. ML347 is potent in the BMP4 cell
assay (152 nM) as well as the in vitro kinase assay for ALK1
(46 nM) and ALK2 (32 nM) and is devoid of activity in a number
of related kinases. Further studies are planned for this selective
inhibitor in a number of in vivo animal disease models, such as
FOP, and results will be reported in due course.
Acknowledgments
The authors would like to thank Katrina Brewer and Ryan Mor-
rison for technical assistance with the PK experiments. Vanderbilt
Table 3
Kinase selectivity data for selected compounds5,18
Compd
IC50 (nM)
ALK1/ACVRL1 ALK2/ACVR1 ALK3/BMPR1A ALK4/ACVR1B ALK5/TGFBR1 ALK6/BMPR1B BMPR2
TGFBR2 AMPK
KDR/VEGFR2
DM, 1
LDN-193189, 2
DMH1, 3
7d
7g
106.3
13.3
27
52.4
46
26.5
42.1
<5
67.5
40.7
107.9
110
32
33.3
53.4
<5
95
<5
<5
25,740
1825
9622
Inactive
Inactive
1840
5930
326
17,090
565
235
60
74
3845
Inactive
24
Inactive
Inactive
15.9
3640
59
695
2360
102.9
140.4
234.6
1122
21.8
214.7
Inactive
Inactive
Inactive
33,300
10,900
178
3320
3960
75
47.6
102
9830
56.9
60.9
<5
129
92.9
<5
Inactive Inactive Inactive
16 44 13
Inactive Inactive 19,700
236 7260 4300
1.37 39.9 1.68
26
38
327
15
41
10,800
7i
6.78
10.4
<5
55
539
<5
13a
13d
13h
13m
13r
2680
82
2520
960
5330
14
2040
1520
55
14.4
<5
43
26.2
<5
3420
4090
183

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D. W. Engers et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3248–3252
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is a member of the MLPCN and houses the Vanderbilt Specialized
Chemistry Center for Accelerated Probe Development. This work
was generously supported by the NIH/MLPCN Grant U54
MH084659 (C.W.L.), and VA Merit Award (C.C.H.), NIH
R01HL104040 (C.C.H.), and the Developmental Grants from the
Center for Research in Fibrodysplasia Ossificans Progressiva and
Related Disorders (C.R.H, C.C.H).
11. Berdini, V.; Besong, G.E.; Callaghan, O.; Carr, M.G.; Congreve, M.S.; Gill, A.L.;
Griffiths-Jones, C.M.; Madin, A.; Murray, C.W.; Nijjar, R.K.; O’Brien, M.A.; Pike,
A.; Saxty, G.; Taylor, R.D.; Vickerstaffe, E. WO2008/078100, 2008.
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14. BMP-responsive luciferase reporter assays. BMP-responsive C2C12BRA cells,
stably transformed with the Id1 promoter-firefly luciferase reporter; kind gift
of D. Rifkin, NYU Medical Center) were seeded in 96-well plates, and incubated
overnight with the compounds and BMP4 (50 ng/mL). The cells were then
lysed, and cell extracts were then subjected to the firefly luciferase assay using
Steady-Glo luciferase assay kit (Promega). The results were normalized to cell
titers, as measured using Cell Titer-Glo, luminescence assay (Promega).
15. Zilberberg, L.; ten Dijke, P.; Sakai, L. Y.; Rifkin, D. B. Bioorg. Med. Chem. Lett. Cell
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
03.113.
References and notes
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17. VU0469381 (ML347) has been declared a probe via the Molecular Libraries
Probe Production Centers Network (MLPCN) and is available through the
network, see: http://mli.nih.gov.
18. Kinase assay. All kinase assays were conducted by Reaction Biology Corp
(Malvern, PA), as previously reported. In brief, compounds were tested at 10
concentrations by threefold serial dilutions starting at 100 mM. In vitro kinase
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reactions were carried out in the presence of 10 lM (33P)gATP. Eleven human
kinases tested were the BMP type-I receptors ALK-1/ACVRL1, ALK2/ACRV1,
ALK3/BMPR1A and ALK6/BMPR1B, the TGFb type-I receptors ALK4/ACVR1B
and ALK5/TGFbR1, the BMP type-II receptor (BMPR2), the TGFb type-II receptor
(TGFbR2), VEGF type-II receptor (KDR/VEGFR2), AMP-activated protein kinase
(AMPK-A1/B1/G1) and the human platelet-derived growth factor receptor-b
(PDGFRb).
19. LDN-193189, 2, which has replaced the labile methoxy group with piperazine
(similar to 13h–n) was shown to be much more stable in liver microsome
assay (see Ref. 13).
6. Shore, E. M.; Xu, M.; Feldman, G. J.; Fenstermacher, D. A.; Cho, T.-J.; Choi, I. H.;
Connor, J. M.; Delai, P.; Glaser, D. L.; LeMerrer, M.; Morhart, R.; Rogers, J. G.;