G. D. Ho et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4110–4113
4111
O
S
O
R2
H
N
N
R2
H
N
N
H
N
N
O
O
R1
R1
N
N
8
N
9
1
b
a
NaV1.7 IC50
NaV1.5 IC50
rat-AUC6h
:
:
160 nM
8092 nM
788 nM.h
5
:
c
d
Figure 1. Initial HTS lead compound 1, and its biological data.
O
R2
O
H
N
N
N
H
H
N
N
R2
plasma exposure in rat based on area under curve (AUC) data
(AUC6h: 788 nM h).
R1
R1
N
10
N
Our strategy was to quickly identify Nav1.7 blockers which
exhibited in vivo efficacy for proof of concept. Therefore achieving
high oral plasma exposure was considered as an important goal.
Rat pharmacokinetic (PK) AUC values of the active compounds
were obtained to guide the compound selection for further
in vivo study. In this communication, we describe syntheses and
structure activity relationships (SAR) of the pyrrolo-benzo-1,4-dia-
zine series versus Nav1.5, Nav1.7, and plasma exposure. The PK and
in vivo data of selected compounds are described.
The synthetic route to the key intermediate 5 is summarized in
Scheme 1. The reaction of nitro-aniline 2 with 2,5-dimethoxytetra-
hydrofuran (2,5-DMTHF) in acetic acid, followed by reduction of
the nitro group using BiCl3–NaBH4 yielded pyrrolo-aniline inter-
mediates 3.8 The pyrrole intermediates (3) were then cyclized with
piperidin-4-one or N-substituted piperidin-4-one derivatives to
form 50H-spiro[piperidine-4,40-pyrrolo[1,2-a]quinoxaline] cores
(4–7). Intermediates 5 were the major intermediates for further
diverse modification. Cyclization of pyrrolo-anilines 3 and N-
substituted piperidin-4-one derivatives allowed us to directly
obtain certain desired targets, such as carbamates (4), amides (7),
and amines (6). However this strategy showed restriction due to
limited commercially available substituted piperidin-4-ones. The
late stage intermediates 5 were the subjects for versatile and fea-
sible structure modification. In general the yields of the cyclization
(reaction c in Scheme 1) to form 4–7 were excellent (>80%).
Conversion of intermediates 5 to sulfonamides, N-substituted
amines, amides, and ureas is shown in Scheme 2. Standard proce-
dures were used to prepare these intermediates. The commercially
7
Scheme 2. Reagents and conditions: (a) R2-Ph-SO2Cl, Et3N, CH2Cl2, rt (ꢀ30–50%);
(b) R2-benzyl bromide, DIPEA, DMF (ꢀ60–70%); (c) R2-PhNCO, Et3N, CH3CN, 0 °C to
rt (ꢀ50–60%); (d) R2CO2H, EDC, HOAt, DMAP, DIPEA, CH2Cl2 (ꢀ50–85%).
available sulfonyl chlorides, isocyanates, and substituted benzyl
bromides were reacted with 5 under basic conditions to form sul-
fonamides (8), ureas (10), and benzyl amines (9), respectively.
Substituted amides were prepared under EDC–HOAt coupling con-
dition. The amides shown in Tables 1, 2, 4 and 5 were prepared
from intermediates 5 according to this condition.
Target compounds were tested for their inhibitory activities at
the cloned human VGSCs (Nav1.7 or Nav1.5) expressed in HEK
293 cells by measuring fluorescence from a membrane potential
dye. After 60 min of compound and dye loading, cell depolarization
was evoked on the FLIPR by the addition of veratridine. Maximum
veratridine-induced increase in fluorescence intensity from base-
line was used to plot concentration-effect curves and generate
IC50 values. The rat PK assay was carried out with oral dosing at
10 mpk, and the AUC values were measured over a 0–6 h duration.
The initial SAR study focused on analogs without substitution on
the left-hand-side (LHS) phenyl ring and with varying substitution
on the right-hand-side (RHS) phenyl ring (Table 1). Compounds 11
and 12 with no or cyano substitution, respectively, on the RHS
phenyl ring did not show activity up to 3 lM. Compound 13 with
3-N,N-dimethyl substitution displayed reasonable Nav1.7 inhibi-
tory activity (IC50 381 nM), and poor selectivity against Nav1.5
(ꢀ11-fold). The 3,5-di-methyl substitution (14) however greatly
improved Nav1.7 inhibitory activity (IC50 47 nM) and selectivity
over Nav1.5 (379-fold). Unfortunately 14 did not show detectable
plasma exposure in the rat PK study (AUC6h 0 nM h). Although
H
N
NCbz
R1
N
4-cyano analog 12 was inactive up to 3 lM in the Nav1.7 FLIPR
O
NCbz
assay, it showed some plasma exposure (AUC6h 1011 nM h) in the
rat PK study. Hence additional analogs with 4-cyano-phenyl moiety
were prepared for further SAR study.
4
c
d
HCl
NH
H
O
NH
NO2
NH2
NH2
N
N
a, b
R1
R1
Table 1
R1
SAR of the analogs with the RHS substitution
c
N
O
2
3
5
O
R3
H
N
N
N
O
R
R2
4
N
3
c
N
c
O
R3
O
N
R2
H
N
a
a
R
Nav1.7 IC50 (nM) Nav1.5 IC50 (nM) AUC6h (nM h)
H
N
N
R1
11
H
>3000
>3000
381
ND
ND
4384
17810
ND
1011
633
0
R1
N
12 4-CN
13 3-N(CH3)2
14 3,5-Di-(CH3)2 47
N
6
7
Scheme 1. Reagents and conditions: (a) 2,5-DMTHF, AcOH, reflux (ꢀ70–90%); (b)
BiCl3, NaBH4, EtOH (ꢀ50–80%); (c) cat. maleic acid, EtOH, 80 °C (>80%); (d) H2
(1 atm), Pd/C, EtOH (>90%).
a
IC50 represent single determinations performed with four replicates per con-
centration point.