mitigating CYP3A4 inhibition was the amino group of the
pyridine ring. Substitution off the amino group may hinder the
interaction with CYP enzyme by virtue of steric hindrance, while
electron-withdrawing groups would be expected to further reduce
the propensity to interact with the heme iron center. We have
examined several simple substitutents (e.g. Me and Et) off the
amino group, and found minimal impact on the in vitro/cellular
properties (data not shown), which made this position a
potentially productive area for modulating the drug-like
properties of resulting analogues without compromising potency.
Compound 6s appeared to have the most robust response in
delaying the progression of tumor at the 1 mg/kg dose. It was
further evaluated in a dose response study in the same MV4-11
xenograft model with doses of 0.1, 1, and 10 mg/kg for 2 weeks
(Figure 1). Compound 1, used as a positive control, was dosed at
0.1 and 10 mg/kg. As shown in Figure 1, 6s potently inhibited the
progression of tumor in a dose-dependent fashion, even at an
extremely low dose of 0.1 mpk. At the higher doses, tumors
regressed completely and long-lasting tumor growth suppression
persisted for an extended period after dosing was halted. Minimal
body weight loss (<5%) was observed in animals treated with 6s
at all doses, indicating that it was well tolerated in mice at the
efficacious doses. As a biomarker of FLT3 inhibition, the pFLT3
level in the tumors was also measured in a separate acute study.
Dose-dependent inhibition of FLT3 phosphorylation with 6s (up
to 79% inhibition of pFLT3 versus vehicle control at 10 mpk
dose) was found to be consistent with tumor growth inhibition
occurring via inhibition of the FLT3 signaling pathway.
The more elaborate amino substituents we focused on were
alkylsulfonylethyl groups, which combined both the electron-
withdrawing and metabolic stability properties offered by the
sulfone group. To our delight, both the methanesulfone (6f) and
ethanesulfone (6g) analogues retained excellent in vitro activities,
in addition to a clean CYP3A4 profile (IC50 > 10 M with or
without pre-incubation). The SAR of the alkylsulfonylethyl
groups was then expanded to confirm the broader utility of such
groups.
The synthesis of urea analogues mostly originated from an
initial Suzuki coupling between boronic acid 7 and heteroaryl
bromides 8, followed by acid-mediated removal of the N-Boc
protecting group to give the biaryl intermediate 9 (Scheme 2).
The more reactive anilino amine group of 9 reacted with the
known carbamate 10 to generate urea analogues 2.17
Interestingly,
the
combination
of
3-F
and
methanesulfonylethyl group (6h) resulted in substantial loss of
cell activity. Both the secondary and tertiary sulfonamide
analogues (6l-m) were potent in cells with good kinome
selectivity. Unfortunately, they suffered from poor oral
bioavailability in rats (9% and 7%, respectively). Both the
Figure 1. In vivo efficacy and pFLT3 inhibition of compound
6s in mouse xenograft model.
cyclopropylsulfonylethyl
(6n)
and
the
extended
methanesulfonylpropyl (6o) derivatives worked well. For the
non-sulfone analogues, the N,N-diethylacetamide compound 6p
was slightly less potent in the MV4-11 cell proliferation assay.
The reverse sulfonamide also produced a potent analogue (6g),
albeit with poor rat bioavailability (2%). Interestingly, a tertiary
amyl alcohol group could mimic the methanesulfonylethyl group,
providing compound 6r with a balanced profile of potent cell
activity and clean CYP3A4. Combining the best sulfones off the
amino group with the 2-F substituent in the central phenyl ring
yielded a crop of highly potent compounds with minimal CYP
inhibition in the time-dependent assay. Select examples (6s-u)
are shown in Table 4.
2000
1500
Untreated
Vehicle 1%HPMC
6S 0.1mpk
1000
6S 1mpk
6S 10mpk
1
1
0.1mpk
10mpk
500
0
0
10
20
30
40
50
60
70
80
90
Study Day
Several lead compounds (6f, 6r-t) were selected for a broader
profiling of selectivity and ADMET properties (Table 5). In the
selectivity cell assays versus other members of the PDGFR
family kinases, all four compounds were shown to have 10-20
fold reduced potency in inhibiting the phosphorylation of Kit and
PDGFR, in comparison to the corresponding pFLT3 IC50 values.
These compounds were much more selective over CSF1R in the
phosphorylation assay, demonstrating over 100-fold separation.
These compounds were also clean in a patch clamp hERG
channel inhibition assay with IC50 values greater than 10 M.
Even though these compounds were highly bound to plasma
proteins, their high exposure in mouse after oral dosing in
conjunction with potent cellular activity predicted sufficient
target coverage with once daily dosing.
PO QDx14
The phenylacetamides were prepared according to the
sequence outlined in Scheme 3. Acylation of the anion of
acetonitrile with various esters 11 yielded ketonitriles 12, which
were cyclized with hydroxylamine using a two-step procedure to
provide 3-aminoisoxazoles 13. On the other hand,
bromophenylacetic acids 14 were converted to the corresponding
boronate esters 15 via
a
Suzuki-Mukiyama coupling.
Condensation of 13 with 15 afforded the key intermediates 16.
The aminopyridines 18 were prepared from nucleophilic
substitution of 2-fluoropyridines 17 with various amines. A
second Suzuki-Mukiyama coupling between 16 and 18 gave the
phenylacetamides 19.
Scheme 2.a General synthetic routes to urea analogues.
The antitumor efficacy of compounds 6f and 6r-t was
assessed at 1 mg/kg orally once daily (QD), an efficacious dose
for compound 1 in a subcutaneous flank-tumor xenograft model
in athymic nude mice using the MV4-11cell line. With respect to
tumor growth delay (TGD) and Log cell kill at equivalent dose,
all of these compounds with the exception of 6f exceeded the
performance of 1 (TGD = 44 d and log cell kill = 2.0) (Table 5).16
Additional factors, including tissue distribution and enzyme
binding kinetics, might have affected the performance of the
compounds in the xenograft model, in addition to cell activity
and plasma exposures.
Liu, Gang
