H. Yamamoto et al.
Bioorganic & Medicinal Chemistry Letters 38 (2021) 127858
Scheme 1. Reagents and conditions: (a) SOCl
2
, ethanol, quant; (b) NaOH, tetrahydrofuran, 90%; (c) SOCl2, DMF, 88%; (d) (Diphenylmethylene)hydrazine, pyridine,
CH
DMF, 77%; (j) 2-Chloro-4-(tributylstannyl)pyrimidine, Pd(PPh
-methylpyrimidine, lithium hexamethyldisilazide, 73%; (m) 1-Boc-4-(4-aminophenyl)piperazine, 43%; (n) 20, pyridine, ethanol, 74%; (o) NaH, DMF, 43%; (p)
Trifluoroacetic acid, CH Cl , 80%; (q) 1-bromo-2-methoxyethane, N,N-diisopropylethylamine, acetonitrile, 72%.
2
Cl
2
, 99%; (e) NaH, tetrahydrofuran, 83%; (f) HClaq, 88%; (g) 20, pyidine, ethanol, 59%; (h) Sodium ethoxide, toluene, 74%; (i) N-iodosuccinimide, NaHCO
3
,
3 4
)
, CuI, DMF, 59%; (k) 4-(4-Boc-piperazin-1-yl)aniline, Methanol, tetrahydrofuran, 56%; (l) 2-chloro-
4
2
2
9
–11 showed significant decreases in enzyme inhibitory activity, sug-
were evaluated in ALK2 (R206H) – expressing C2C12 cells to determine
5,16
gesting that the 3-pyridyl group is essential for interacting with the
enzyme. Finally, the fused bicyclic pyrazole moiety of 8 was optimized.
The results showed that the IC50 value of alicyclic type 12 and 13 were
lower than that of 8 (Table 3). X-ray crystallographic analyses were
conducted to examine the SAR in a structural context. It was expected
that soaking 8 would be difficult because the N-methoxyethyl group of 8
caused a steric clash with a neighboring protomer in the crystal. Thus, 7
possessing the N-methyl group was selected for crystal structure anal-
ysis. A previous study revealed that 7 directly binds to the ALK2
their ability to inhibit BMP4-induced ALP activity.1
biochemical marker of osteoblastic differentiation of C2C12 cells.
ALP is a useful
1
3
Compound 8 showed higher potency than RK59638, which was
consistent with the biochemical ALK2 (R206H) assay (Table 4). More-
over, 8 showed improvements in human and rat microsomal stability
and reduced the human ether-a-go-go related gene (hERG) inhibition
compared with RK-59638 (Table 4).
The synthesis of 8 was achieved via two divergent routes: route A
included the Stille cross-coupling reaction and route B included a
nucleophilic substitution reaction using lithium hexamethyldisilazide
(Scheme 1).
(
R206H) ATP-binding pocket in the same manner as RK-59638 (PDB
code 6ACR) (Fig. 2A).
Compound 7 formed two hydrogen bonds between the amino-
pyrimidine moiety and the main chain amine and carbonyl of the His286
in the hinge region of the enzyme. Three hydrogen bonds were formed
between the nitrogen of the 3-pyridyl group of 7, the carboxyl group of
First, key intermediate 20 was synthesized. Commercially obtained
p-dioxanone 14 was treated with thionyl chloride in ethanol to give the
corresponding ethyl ester 15.17 Carboxylic acid 16 was formed by base
hydrolysis of 15 and then treated with thionyl chloride in N,N-dime-
1
7
Glu248 from the
αC helix, and the amino group of Lys235 from the β3
thylformamide (DMF) to give the corresponding acid chloride 17.
strand via conserved water molecules. A hydrogen bond was formed
between the nitrogen of the pyrazole ring of 7 and the amino group of
Lys235 via a conserved water molecule. Another weak hydrogen bond
might have formed between the oxygen atom of the morpholinopyrazole
moiety of 7 and Asp293 and Ser290 via a conserved water molecule. The
oxygen atom and the water molecule were 3.4 Å apart. Moreover, the
morpholine moiety was located near Tyr219. The methylene carbon
atoms at the morpholine moiety and the aromatic ring of Tyr219 were
Compound 17 was coupled using benzophenone hydrazine with pyri-
dine to give the amide product 18.18 Compound 19 was obtained by
intramolecular dehydrohalogenation of 18 with sodium hydride in
tetrahydrofuran, followed by deprotection using aqueous hydrochloric
acid to give the intermediate 20.18 Next, as shown in Scheme 1, for route
A, commercially obtained 3-oxo-3-(pyridin-3-yl)propionic acid ethyl
ester 21 was condensed with the hydrazide 20 in pyridine to give the
corresponding acyl hydrazone 22.19 Compound 22 was cyclized and
hydrolyzed using sodium ethoxide to give carboxylic acid 23. Acid 23
4
.0 Å apart, and they formed a CH-
π
interaction. Structural super-
◦
position of RK-59638 and 7 showed that the fused morpholinopyrazole
motif of 7 induced a significant positional change in Tyr219 on the P
was treated with N-iodosuccinimide in DMF at 25 C to give the corre-
sponding iodide 24.18 The Stille reaction of iodide 24 with 2-Chloro-4-
loop (Fig. 2B), suggesting that the motif is important for CH-
π
interac-
(tributylstannyl)pyrimidine produced chloride 25. Displacement of the
1
1
tion with Tyr219 in ALK2 (R206H).
chloride 25 with 1-Boc-4-(4-aminophenyl)piperazine gave 26. As
In addition to the biochemical ALK2 (R206H) assay, RK-59638 and 8
shown in Scheme 1, for route B, commercially available methyl
4