A.T. Osuma, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126603
Fig. 2. 2,3 pyrazolopyrimidines as KCNQ2/3 modulators.
Fig. 1. Optimization of HTS hits led to compound 3.
Table 1
Compound 3 compared to Retigabine.
Compounds
KCNQ2/3 EC50 (μM)
3
0.91
0.56
4.38
149
112
99
Retigabine
Flupirtine
a
% efficacy relative to that of 10 µM Retigabine.
activity of 0.91 μM, compared favorably to Retigabine and Flupirtine
(Table 1) and was considered an excellent starting point for further
The general synthesis of compounds 2 is shown in Scheme 1. Con-
enones 5, which were reacted with ethyl 5-amino-1H-pyrazole-4-car-
boxylate under acidic conditions to give the pyrazolopyrimidine esters
6.10 The esters 6 were converted to the acids 7 followed by a Curtius
rearrangement reaction and a deprotection step to afford the amines 8.
The amines 8 were then subjected to amide coupling conditions with
various acids to yield compounds 2.
Scheme 2. Synthesis of compounds 11 i) malonitrile, bis(triphenylphosphine)
palladium(II) chloride, NaH, THF, 0 °C to reflux, ii) hydrazine monohydrate,
BuOH, 125 °C, iii) 15, AcOH, EtOH, 140 °C, iv) 17, pyridine, DCM, 25 °C.
convergent synthesis as shown in Scheme 2. Palladium catalyzed cross-
coupling of aryl iodides 12 with malononitrile gave the corresponding
phenylmalononitriles 13, followed by a reaction with hydrazine to give
the pyrazole-3,5-diamines 14. The pyrazole-3,5-diamines were then
reacted with various substituted enones 15 (installation of R3 sub-
stituent) to afford 16. Numerous compound libraries of compound 11
were generated. Representative SAR for compound 18 (R3 = H, Fig. 3)
Further chemistry efforts focused on the optimization of compound
3 where considerations were given to modifications of the substitution
patterns around the pyrazolopyrimidine core (Fig. 2). The substituents
R1 and the entire amide group bearing R2 were moved around the ring
system as shown in compound 9. This exercise afforded the 2,3-sub-
stitued pyrazolopyrimidines 10 as a group of novel KCNQ2/3 mod-
ulators.
The general SAR on the pyrazolopyrimidines shows that hydro-
phobic groups are well tolerated for R1 and R2. 4-substitued phenyls
consistently gave good potency. The 4-triflouromethyl group gave
consistent and robust activity across a variety of R2 substituents with
the ethyl-cyclopentane compound 32 showing a superior potency of
0.05 μM amongst the initial group of compounds studied at the Hit-to-
Lead stage. The 4-fluoro (26) and 4-trifluromethoxy (29) analogs
showed reasonable KCNQ2/3 activity, at 0.14 μM and 0.31 μM, re-
spectively. Although the unsubstituted phenyl compound 19 was in-
active, the introduction of larger R2 groups such as methyl-cyclohexane
(21) and ethyl-cyclohexane (22), gave good activity of 0.29 μM and
0.13 μM, respectively. In addition, the inactivity of compounds 20 and
24 revealed the need for the R2 substituent to have at least one me-
thylene spacer when compared to compounds 21 and 25.
Structural investigation of scaffold 10 revealed the optimal R2 to be
4-substitued phenyls, with 4-triflouromethyl and 4-fluoro phenyls being
the most active analogs. Additionally, alkyls and cycloalkyls were the
optimal R2 representatives. R3 substituents were introduced to the
scaffold as represented in structure 11. However, no substituents larger
than a methyl group were tolerated.
Compounds 11 were easily assessable via an efficient 5-step
Further investigation to probe the pyrazolopyrimidine SAR by
substituting around the core gave further improvements of KCNQ2/3
activity. As mentioned earlier, substituents larger than a methyl group
in any of the 5, 6 or 7 positions gave significant reduction in potency
For example, compound 33, with a t-butyl substitution at the 7
position, exhibits an EC50 of > 32 μM. Representatives of compound 34
with methyl substitutions at the 5, 6 and 7 positions are shown in
Table 3. In general, methyl substitution at the 6 position or 5,7-di-
methyl substitution appears to significantly boost potency when com-
pared to their corresponding unsubstituted analogs. Compounds 35 and
38 with no 5, 6 or 7 substitutions are generally weaker than their 6-
Scheme 1. Synthesis of compounds 2 i) DMF-DMA, Et3N, 95 °C, ii) AcOH,
100 °C, iii) 1 N NaOH, THF, 65 °C, iv) diphenylphosphoryl azide, Et3N, 110 °C,
v) 4 N HCl in 1,4-dioxane, vi) HATU, DIEA, DCM, 25 °C.
2