S. Sharma et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 1. Previously reported GIRK1/2 activators, 1 and 2, and the new N-(bi-
cyclo[2.2.1]heptan-2-yl)-2-(5-phenyl-2H-tetrazol-2-yl) acetamide,
a starting
lead for GIRK1/2 activator optimization.
Scheme 2. Reagents and conditions: (a) NaN3, Et3N-HCI, toluene, 120 °C; (b)
bromoethyl acetate, NaOEt, EIOH; (c) NaOH, H2O, THF; (d) 2-chloroacethyl
chloride, Et3N, CH2Cl2; (e) NaOEt, EtOH, μW, 90 °C; (f) T3P, Et3N, CH2Cl2, rt.
Scheme 1. Reagents and conditions: (a) CH3CN, n-BuLi, THF, −78 °C to rt; (b)
AcOH, EtOH, reflux; (c) NaCNBH3, AcOH, MeOH, rt; (d) TFA, CH2Cl2, rt.
acidic conditions (AcOH, EtOH, reflux) to yield the desired dis-
ubstituted pyazoloamine, 7. For those substituted hydrazines that were
not commercially available, the synthetic procedure outlined in
Scheme 1B was followed. The Boc-hydrazine, 9, was reacted with the
carbonyl, 8, under reductive amination conditions (NaCNBH3, AcOH,
MeOH, rt) to yield 10, which was subjected to TFA in order to remove
the Boc protecting group and yield the desired hydrazine, 6.18
knowledge from previous work, we attached the 3-methyl-1-cyclohex-
ylpyrazole group to generate 15a, which we found to be a potent and
efficacious GIRK1/2 activator (EC50 = 96 nM; Efficacy = 92%). This
compound was also a GIRK1/4 activator, but it demonstrated an ap-
proximate 3-fold preference for GIRK1/2. Methylation of the amide
nitrogen led to an inactive molecule (data not shown). Moving from the
amide to the thioamide, 15b, produced an active molecule; however,
we observed an approximate 7-fold loss of potency (EC50 = 623 nM).
Further saturated 6-membered analogs were evaluated and the tetra-
hydropyran, 15c, lost activity compared to 15a. However, the 4,4-di-
fluorocyclohexane was equipotent (15d, EC50 = 84 nM), but potency
eroded with substituting dimethyl, 15e, for the difluoro. Further
branched alkyl analogs were less potent (15f–h), and the cyclopropyl
was also inferior, but some activity could be regained with the cyclo-
pentyl group (15j, EC50 = 163 nM). The 5- and 6-membered sulfone
derivatives (15k, l) were also less active; however, these were shown to
have superior in vitro PK properties (vide infra). Branched groups at the
3-position of the pyrazole were not productive analogs (15m–p). The 1-
cyclohexylmethyl group, as we have seen previously, was an active
analog (15q, EC50 = 176 nM); however, this molecule lost selectivity
between GIRK1/2 and GIRK1/4. Other substituents (15r), or 5-mem-
bered pyrazole replacements (15s, t) were not active, nor were pyridine
replacements for the pyrazole (15v).
The synthesis of the final tetrazole or heterocyclic compounds was
completed as outlined in Scheme 2A–C. The tetrazole-containing com-
cyanide with NaN3 (Et3N∙HCl, toluene, 120 °C) to yield 12.19 Next, 12
was reacted with bromoethyl acetate (NaOEt, EtOH)20 and followed by
saponification of the ester (NaOH, H2O, THF) to give the acid coupling
partner, 13. In an analogous fashion to the final targets, the dis-
ubstituted pyrazoloamine, 7, was coupled with 2-chloroacetyl chloride
(Et3N, CH2Cl2) affording the α-chloroamide, 14. This compound could
then be reacted with a heterocyclic partner under basic conditions to
yield the final targets, 15. Also, the acid coupling partner (e.g., 13 or
16) could be reacted with 7 under standard amide coupling procedures
(T3P, Et3N, CH2Cl2) to yield the final targets, 17.21
The initial structure-activity relationship (SAR), which was centered
around the left-hand amide portion, is detailed in Table 1. The initial
HTS hit molecule was resynthesized and tested on HEK293 cells ex-
pressing GIRK channels using thallium flux assays, as previously de-
scribed.8 This molecule demonstrated weak activity against GIRK1/2
(3, EC50 = 1980 nM, Efficacy = 13% of a maximally effective con-
centration of VU0466551), which agreed with previous data showing
non-pyrazole “head groups” to be weak GIRK1/2 activators. Using our
We next set out to evaluate the right-hand phenyl group (Table 2).
Replacement of the phenyl with either 3-pyridyl (16a) or 2-pyridyl
(16b) led to a > 20-fold loss of activity. Addition of halogens (either
mono- or di-halogens) maintained activity. The 3,4-dichloro and 3,4-
difluoro were notable exceptions, with > 10-fold loss of activity. In
2