2
R. Ding et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
H2N
Cl
N
O
OMe
H
N
H
N
N
N
N
O
N
N
N
O2N
OH
Cl
N
N
O
N
1
H
O
HN
HN
N
H
2
3
O
NH2
N
N
NH
O
N
N
NH2
N
HN
N
N
H
4
S
F
O
N
5
HN
N
N
O
N
S
N
S
O
O
N
N
H
6
7
Figure 1. Selected p97 inhibitors.
R1
structure–activity relationship (SAR) studies on the quinazoline
scaffold led to ML240 (3) and ML241, both of which selectively
bind to the D2 domain.14 More recently, an optimized analog
CB-5083 (4) has been advanced into a phase I clinical trial in solid
tumors, further validating inhibition of p97 as a promising anti-
cancer strategy.15,16 p97 inhibitors based on a 2-(cyclohexylmethy-
lamino) pyrimidine core as exemplified by compound 5 have been
reported.17 Photoaffinity experiments revealed labeling of Asp478
located on the D1–D2 linker in proximity to the D2 ATP-binding
site, suggesting that this class of inhibitors including compound
5 target the D2 domain. This proposed binding mode was further
supported by computational modeling.17 In a high-throughput
screening campaign, alkylsulfanyl-1,2,4-triazoles were identified
as moderate and allosteric inhibitors of p97.18 Extensive medicinal
chemistry and SAR studies gave rise to compounds with signifi-
cantly enhanced activity. Photo-affinity labeling and subsequent
studies of residue mutations have revealed that NMS-873 (6), a
representative inhibitor based on the alkylsulfanyl-1,2,4-triazol
core structure, interacts with a tunnel between the D1 and D2
domains.18,19 Very recently, UPCDC30245 (7) has been shown to
occupy a surface at the interface of D1 and D2 domains.20 This
binding blocks cross-talk between domains and prevents confor-
mational changes necessary for p97 function. In addition to those
shown in Figure 1, a growing list of new p97 inhibitors, including
O
A
H
N
O
N
N
N
R2
O2N
OH
O
N
H
O
B
1 (EerI), R1 = Cl, R2 = Cl
N
H
8, R1 = a, R2 = Cl
1
, R = Cl, R2
=
9
10
a
a
=
O
a =
O
1
2
a
, R
, R
=
Figure 2. EerI derivatives designed to improve solubility.
explored compound 10, in which both Cl groups were replaced
with a solubilizing group.
Our synthesis of EerI derivatives started with the preparation of
isocyanate 15 (Scheme 1). Starting material 1-fluoro-4-nitroben-
zene (11) was displaced with 2-methoxyethanol (12) under basic
conditions to afford nitrate 13, which was subsequently reduced
to aniline 14.25 The resulting aniline 14 was treated with triphos-
gene to generate isocyanate 15, which was used in the next step
immediately without any purification. Aldehyde 17,26 which was
needed to introduce the NFC group present in EerI and its deriva-
tives, was prepared through a simple Wittig reaction between
5-nitrofuran-2-carbaldehyde (16) and (triphenylphosphoranyli-
dene)acetaldehyde. To prepare the central urea structure,
isobutyraldehyde (18) was first converted into oxime 19 through
condensation with hydroxylamine (Scheme 2),27 followed by intro-
duction of a silyl protective group to give protected oxime 20.28
Subsequently, bromination of the isopropyl group in 20 was
accomplished through a radical reaction.28 The resulting bromide
21 was displaced with glycine methyl ester to give oxime 2229 with
concomitant loss of the silyl protective group.
withaferin
A
analogs,21 indole amides,22 trifluoromethyl and
pentafluorosulfanyl indoles,23 and chlorinated analogs of dehy-
drocurvularin,24 has also been reported.
As the first reported p97 inhibitor, EerI has been an invaluable
tool in the studies of p97’s biological functions and potential ther-
apeutic applications. It has displayed a promising anti-cancer
activity with a novel mechanism of inhibition in vitro. Neverthe-
less, our preliminary animal studies revealed that EerI is poorly
soluble (vide infra), which limits its in vivo applications. To cir-
cumvent this problem, we wished to identify EerI derivatives that
possess improved aqueous solubility without a significant loss of
cancer killing activity. Our strategy was to replace the Cl groups
in benzene rings A and B with solubilizing groups (Fig. 2). Since
there was no prior SAR information regarding substituents on the
benzene rings, a relatively small and flexible solubilizing group
such as 2-methoxyethanoxy (a) was examined. To investigate
whether such substitution was tolerated on either ring, we
designed compounds 8 and 9, in which the Cl group on rings A
and B was replaced with a solubilizing group, respectively. We also
With these key fragments in hand, we proceeded to prepare our
target products 8–10 in a synthetic sequence analogous to that
reported previously (Scheme 3).29
A condensation reaction
between oxime 22 and one equivalence of isocyanate 15 followed
by another reaction with one equivalence of commercially avail-
able 4-chlorophenyl isocyanate in one pot gave methyl ester 23.
Conversely, condensation reactions between oxime 22 and
4-chlorophenyl isocyanate followed by isocyanate 15 afforded
methyl ester 25. Esters 23 and 25 were then converted into hydra-
zides 24 and 26, respectively. Target compounds 8 and 9, each of
which contained only one solubilizing group, were obtained after
treatment with aldehyde 17 under condensation conditions.