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K. R. Hornberger et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4511–4516
labile sites identified that would alone explain its high clearance.
We also conducted an in vitro metabolite identification study on
1a in mouse and human liver microsomes in the presence of both
NADPH and UDPGA cofactors. Although several minor oxidative
metabolites were detected, the common major metabolite was
an M+176 metabolite corresponding to a single glucuronidation.
Suspecting this glucuronidation to be occurring on the furopyri-
dine 7-amino substituent, we also ran metabolite identification
on the 7-des-amino counterpart of 1a and observed that the glucu-
ronide metabolite had completely disappeared. A glucuronide
metabolite also aligned with the apparent >100% bioavailability
of 1e; depot of glucuronides in bile followed by cleavage and recir-
culation of the parent compound in the intestine is a known mech-
anism for enterohepatic recycling.9
Although we now had a clear sense of the major site of metab-
olism within the chemical series, this was a particularly vexing
medicinal chemistry problem due to the essential function of the
7-amino group in anchoring binding to the hinge of TAK1. Accept-
ing that we could not remove the 7-amino group altogether, we in-
stead wondered whether it would be possible to modulate its
nucleophilicity, for example by attachment of an electron-with-
drawing group to an open site on the furopyridine core. These
hypotheses around metabolism were coming to the fore just as
the kinase selectivity exploration around the pyrazole 4-substitu-
ent was concluding and we were concomitantly seeking alternate
ways to achieve Aurora B and KDR selectivity. We realized that
the furopyridine 3-position offered an opportunity to solve both
the metabolism and kinase selectivity problems in a single stroke.
In addition to providing a venue for anchoring an electron-
withdrawing group for metabolic stability, the 3-position also
directly faces the ribose binding pocket of TAK1. Such a 3-substitu-
tion could thus provide the desired steric clash with E161 of Aurora
B and introduce torsion to the pyrazole 4-substituent for KDR
selectivity. The only remaining obstacle in reduction of this strat-
egy to practice was the synthesis of the 3-substituted compounds.
At this point we also realized that the means to achieve such a
synthesis were already within reach (Scheme 2). Previously1 we re-
ported the synthesis of compound 8 as a by-product of the desired
C2 mono-chlorination of the furopyridine core en route to com-
pound family 1 and 6. Ironically, this by-product, initially per-
and then transformed to 11 and 12 by a series of sequential Suzuki
couplings analogous to that used for compound series 1. Global re-
moval of all protecting groups afforded the 3-Cl substituted com-
pound 13a, and coupling of this chloride with zinc cyanide in
turn gave 13b. For the preparation of all other compounds, it was
necessary to Boc protect the 7-amino group to give 14. Various het-
erocyclic substituents (13c, 13d) could be accessed by Suzuki cou-
pling, although substantial optimization of conditions was needed
to affect coupling of this sterically congested aryl chloride.12 Some
fluorinated analogs (13e, 13f) were accessed by Suzuki coupling of
a vinyl group, followed by ozonolysis with either PPh3 or NaBH4
work-up, and then subsequent fluorination with DeoxoFluor.
The SAR of the furopyridine 3-substituted compounds 13 is de-
scribed in Table 4. In general, most compounds demonstrated good
maintenance of TAK1 biochemical and cellular potency, although a
larger 6-membered heterocyclic substituent (13c) displayed some
loss of activity. All of the 3-substituted compounds traded back
some lipophilic ligand efficiency, but the loss was deemed neces-
sary to address the other deficits of the chemotype. Gratifyingly,
most compounds demonstrated enhanced cellular selectivity for
Aurora B and especially KDR compared to 1e. In particular, com-
pound 13a showed an excellent balance of TAK1 activity and ki-
nase cellular selectivity (>300ꢀ for Aurora B and >1000ꢀ for
KDR). We also evaluated compound 13f against the broader bio-
chemical panel of 192 kinases and confirmed that as selectivity
for Aurora B and KDR improved, so too did the overall selectivity
profile. At ꢁ50ꢀ TAK1 Ki, 13f showed >50% inhibition for only 4/
192 kinases in the panel: FLT3D835Y, HGK, MAP4K5, and MINK1.
We further obtained an X-ray crystal structure of 13a bound to
TAK1–TAB1 fusion protein (Fig. 3) and found that it possessed ex-
actly the features hypothesized to enhance selectivity. The single
largest change in this structure compared to the earlier structure
of 1c was the increased dihedral angle between the furopyridine
core and the 4-pyrazole substituent (ꢁ25° vs ꢁ4° in 1c), bringing
the pyrazole C5 ꢁ1 Å closer to the selectivity residue V42 (L838
in KDR).
Equally gratifying was that the majority of the 3-substituted
analogs had somewhat reduced extraction ratios in mouse and hu-
man liver microsomes compared to 1e. However, our microsomal
preparations were not run in the presence of the cofactors needed
for Phase II metabolism, for example UDPGA, and thus may not
fully capture the impact of the 3-substitutions on glucuronidation.
Indeed, the mouse pharmacokinetics of 13a and 13f (Table 5) were
dramatically improved compared to 1e. Both compounds had
ceived to be
a
synthetic nuisance requiring painstaking
chromatographic separation, now became the linchpin of the syn-
thetic campaign for the core 3-substitution.11 Compound 8 was
deprotected under acidic conditions to 9, iodinated (10) with NIS,
Cl
Cl
Cl
R2
Cl
Cl
Cl
Cl
b
d
c
R4
R4
O
O
O
O
I
R2N
N
H2N
N
H2N
N
H2N
N
8: R = Boc
a
10
11
12
g
9: R = H
R2
e
R2 =
N
Cl
h,i
13c-d
R4
f
O
j, k, l
j, m, l
N
13b
13a
S
13e
13f
R4 =
N
Boc2N
N
N
OTBS
14
Scheme 2. Preparation of 3-substituted analogs 13. Reagents and conditions: (a) 4 N HCl in 1,4-dioxane, 55 °C (68%); (b) NIS, MeCN, 60 °C (41%); (c) R4-B(pin), PdCl2dppf,
K2CO3, 1,4-dioxane/H2O, 75 °C (51%); (d) R2-B(pin), Pd(PPh3)4, Na2CO3, 1,4-dioxane/H2O, 120 °C,
W; (e) concd HCl, 40 °C (50%, 2 steps); (f) Zn(CN)2, Pd(PPh3)4, 1,4-dioxane/
DMF, 140 °C, W (12%); (g) Boc2O, DMAP, DCM, rt (79%); (h) Ar-B(pin), Pd2dba3, XPhos, K3PO4, 1,4-dioxane/H2O, 100 °C, W; (i) 4 N HCl in 1,4-dioxane, MeOH, rt; (j) vinyl-
W (69%); (k) (i) O3, DCM, (ii) NaBH4 (50%); (l) (i) DeoxoFluor, DCM, 35 °C, (ii) 4 N HCl in 1,4-dioxane, 70 °C; (m) (i) O3,
l
l
l
B(pin), Pd2dba3, XPhos, K3PO4, 1,4-dioxane/H2O, 100 °C,
l
DCM, (ii) PPh3 (69%).