2-(4-Methoxyphenoxy)-5-nitro-N-(4-sulfamoylphenyl)benzamide activates Kir6.2/SUR1 KATP channels

Article abstract of DOI:10.1016/j.bmcl.2004.09.057

2-(4-Methoxyphenoxy)-5-nitro-N-(4-sulfamoylphenyl)benzamide and close analogues inhibit glucose stimulated insulin release through activation of Kir6.2/SUR1 K_ATP channels of beta cells.

Full text of DOI:10.1016/j.bmcl.2004.09.057

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5727–5730
2-(4-Methoxyphenoxy)-5-nitro-N-(4-sulfamoylphenyl)benzamide
activates Kir6.2/SUR1 KATP channels
Flemming E. Nielsen, Palle Jacobsen, Anne Worsaae, Per O. G. Arkhammar,
Philip Wahl and John Bondo Hansen*
˚
Discovery, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maløv, Denmark
Received 6 July 2004; revised 21 September 2004; accepted 21 September 2004
Available online 6 October 2004
Abstract—2-(4-Methoxyphenoxy)-5-nitro-N-(4-sulfamoylphenyl)benzamide and close analogues inhibit glucose stimulated insulin
release through activation of Kir6.2/SUR1 KATP channels of beta cells.
Ó 2004 Elsevier Ltd. All rights reserved.
Only a few different structural types of activators of
Kir6.2/SUR1 potassium channels have been described.
These include the 1,2,4-thiadiazine 1,1-dioxide
derivatives like diazoxide, BPDZ 62, BPDZ 73 and
NN4141–4 and certain cyanoguanidine derivatives, for
example, compound 1,5,6 which all fit into the same
pharmacophore model.1 Recently a nitropyrazole (2)
isation of a small series of analogues in order to describe
the structure activity relationship of this compound.
KATP channels are found in different tissues, such as the
heart, vascular smooth muscle, central neurons and pan-
creatic beta cells.8–10 Activators of KATP channels of
smooth muscle (e.g., diazoxide and pinacidil) have been
explored as drugs for treatment of cardiovascular dis-
eases. Recently it has been suggested that activators of
beta cell KATP channels can be used in the treatment
of metabolic diseases through an inhibition of insulin re-
lease to induce beta cell rest.11–13 It has been found that
has been described as
a KATP channel opener
(KATPCO)7 and we now wish to present a novel activa-
tor (2-(4-methoxyphenoxy)-5-nitro-N-(4-sulfamoyl-
phenyl)benzamide) of the KATP channels of the
Kir6.2/SUR1 type as well as the synthesis and character-
H
H
N
H
N
H
N
H
N
H
N
H
N
N
Cl
N
N
N
N
Cl
S
O
N
S
S
O
S
O
Cl
S
O
O
O
O
O
Diazoxide
NN414
BPDZ 62
BPDZ 73
O2N
H
H
N
H
N
H
N
N
F3C
NO2
CF3
N
N
N
NC
O2N
NC
N
CF3
Pinacidil
N
H
1
2
Keywords: Diazoxide; KATP channels; Beta cell; NN414; Insulin release.
*
Corresponding author. Tel.: +45 44434857; fax: +45 44434547; e-mail: jbha@novonordisk.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.057

5728
F. E. Nielsen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5727–5730
R1
R1
MeO
OMe
X
O
X
O
O
O
c
a-b
R3
OH
N
N
H
R4
R2
R2
R2
3a: R1= OCH3, R2= NO2, X= O
3b: R1= H, R2= H, X= O
3c: R1= OCH3, R2= CN, X= O
4-12, 14: X= O
16: X= NH
13: R2= NH2
14: R2= CN
d
15: R1= OCH3, R2= NO2, X= NH
Scheme 1. Synthesis of benzamides 4–14 and 16. Reagents and conditions: (a) SOCl2, 25°C; (b) aniline derivative, TEA, CH2Cl2, 25°C; (c) 5: H2,
Raney-Ni, DMF, EtOH; (d) HNO2, K2Ni(CN)4, water.
KATPCOÕs (e.g., NN414) are able to preserve beta cell
function in presence of high glucose14,15 and inhibit
apoptosis of human beta cells induced by glucose and
IL-1b.16
give the benzamides 4–12. Reduction of the nitro group
of compound 5 gave the amino derivative 13, which
could be converted to the corresponding cyano deriva-
tive 14 using standard Sandmeyer conditions but was
preferably made from the benzoic acid 3c through reac-
tion with 4-aminobenzonitrile. Compound 3c was pre-
pared from 3-bromo-4-hydroxybenzonitrile through
arylation with 4-methoxyphenylboronic acid followed
by carboxylation with n-BuLi/CO2. Reaction of 2-[(4-
methoxyphenyl)amino]-5-nitrobenzoic acid 15, prepared
from 2-fluoro-5-nitrobenzoic acid and p-anisidine, using
an analogous procedure as for the phenoxy derivatives
gave compound 16. Compounds 4–14 and 16 were char-
The KATP channels are constructed as a 4 + 4 hetero-oc-
tamer made from the regulatory sulfonylurea receptor
SUR and the inward rectifier Kir6.2 or Kir6.1. Different
forms of SUR have been cloned and characterised.17,18
In beta cells and neurons SUR1 combine with Kir6.2
to form the KATP channels, whereas SUR2B in combi-
nation with Kir6.1 and Kir6.2 form the channels of vas-
cular smooth muscle and SUR2A in combination with
Kir6.2 forms the channels of the heart.
1
acterised by H NMR and LC–MS.
N-Aryl-2-phenoxybenzamides were prepared as outlined
in Scheme 1 starting from 2-phenoxybenzoic acid deriv-
atives (3a and 3b).19 Thus treatment of 3a,b with excess
thionyl chloride at room temperature followed by evap-
oration in vacuo gave the corresponding benzoyl chlo-
rides, which were reacted with equimolar amounts of
substituted anilines in the presence of triethylamine to
Compound 4 (NNC 55-0557) was initially identified by
screening compound libraries in an assay aiming to find
compounds, which were able to repolarise membrane
potential of bTC3 beta cells depolarised by 10lM glu-
cose. In this assay3–6,20 compound 4 was nearly 10 times
more potent than diazoxide and only about 3 times less
potent than NN414 (Table 1) (IC50: 1.8, 13.7 and
Table 1. Effects of test compounds on beta cell membrane potential and insulin release
Compound
R1, R2, R3, R4
Membrane potentiala
Inhibition of insulin releaseb
bTC3 cells IC50 (lM)
bTC6 cells
IC50 (lM)
Efficacy (%)
4
OCH3, NO2, 4-SO2NH2, H
OCH3, NO2, 4-OCH3, H
OCH3, NO2, 4-F, H
1.8 0.5
3.4 1.5
7.2 3.0
8.1 3.7
9.9 3.1
NS
0.82 0.16
1.6 0.20
5.67 3.67
1.12 0.21
20.53 5.35
10.37 10.27
25.74 13.52
10.4 1.83
NS
79 0.91
73 3.95
52 4.58
75 1.63
31 4.94
33 7.23
33 6.74
56 3.25
—
5
6
7
OCH3, NO2, 4-COCH3, H
OCH3, NO2, 3-SO2NH2, H
OCH3, NO2, 2-SO2NH2, H
OCH3, NO2, 4-Cl, CH3
OCH3, NO2, 4-Cl, CH2CH3
H, H, 4-OCH3, H
8
9
10
11
12
13
14
16
10.1 1.2*
8.3 0.05*
NS
OCH3, NH2, 4-OCH3, H
OCH3, CN, 4-OCH3, H
OCH3, NO2, 4-SO2NH2, H
NS*
2.5 2.4
NS
NS
48.54 45.55
26.62 7.36
—
17 17
1
4.2
Diazoxide
BPDZ 73
NN414d
13.7 0.25c
0.25 0.02
0.6 0.1
22.98 2.83c
0.46 0.11
0.25 0.05
25 3.70c
75 1.1
73 3.0
a Repolarisation of beta cell membrane potential in presence of 10mM glucose. Values are mean SD of at least three experiments, unless otherwise
noted (*: n = 2).
b Inhibition of glucose stimulated insulin release from bTC6 cells. Values are mean SEM of at least three experiments.
c Data from Tagmose et al.6
d Data from Nielsen et al.4

F. E. Nielsen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5727–5730
5729
0.6lM, respectively). The compound was subsequently
found to inhibit glucose stimulated insulin release4 from
bTC6 beta cells (Table 1) (IC50 = 0.8lM) and freshly
isolated rat islets (IC50 = 0.73 0.39lM (n = 6); 80%
max efficacy), which is approximately 30 times more po-
tent than diazoxide (bTC6: IC50 = 22.98lM and islets:
IC50 = 20.28 8.82 (n = 7), 36% max efficacy) but less
potent than NN414 (bTC6: IC50 = 0.25 0.05lM and
islets: IC50 = 0.18 0.07lM (n = 8), 72% max efficacy).
the amide NH to NCH3 (10) or NCH2CH3 (11) also
gave significantly active but less potent compounds
compared to 4.
Reduction of the nitro group of compound 5 gave the
inactive aniline 13. Transforming 13 to the cyano deriv-
ative (14) regained some of the beta cell activity. The
efficacy of 14 in the membrane assay was low (49%)
compared to that of 4 (100%, data not shown), high-
lighting the importance of the electron withdrawing
nitro group. This is substantiated by the complete lack
of activity of 2-phenoxy-N-(4-methoxyphenyl)benza-
mide (12). The 4-methoxyphenylaminobenzamide deriv-
ative 16 was inactive.
The ability of compound 4 to hyperpolarise the beta cell
membrane and inhibit glucose stimulated insulin release
strongly suggests that it activates KATP channels
although inhibition of insulin release can be mediated
through other mechanisms.21 To substantiate a direct
effect on the channel compound 4 was examined for
effects on HEK 293 cells expressing human Kir6.2 and
human SUR1. It was found that 4 potently repolarised
the cell membrane that was depolarised with the KATP
Conclusion
2-(4-Methoxyphenoxy)-5-nitro-N-(4-sulfamoylphenyl)-
benzamide has been identified as a new activator of
Kir6.2/SUR1 KATP channels. Although the compound
or its close analogues are not drug candidates due to,
for example, low solubility, multiple hydrogen bond
acceptors and high polar surface area, they do provide
leads for further optimisations.
channel blocker tolbutamide. The potency (IC50
=
1.3 0.7lM, (n = 4), 100% max efficacy), which is sim-
ilar to the effects observed on beta cell membrane poten-
tial and insulin release, is considerably improved to that
of diazoxide (33 11lM) and similar to that of BPDZ
73 (0.8 0.01lM).22
To further explore the mechanism of action of 4, the ef-
fects of the compound were examined using the patch
clamp technique in the whole cell configuration (Fig.
1).4 It was found that 4 (0.1, 1 and 10lM) potently
increases the ion current through the Kir6.2/SUR1
channel with an efficacy at 10lM similar to that of diaz-
oxide (300lM). Together these studies strongly suggest
that 4 inhibits glucose activated insulin release through
an activation of Kir6.2/SUR1 channels on the beta cell.
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