The sulfamide moiety affords higher inhibitory activity and oral bioavailability to a series of coumarin dual selective RAF/MEK inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.10.001

Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth inhibition (IC ₅₀ = 8 nM) and good PK prof

Full text of DOI:10.1016/j.bmcl.2013.10.001

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6223–6227
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The sulfamide moiety affords higher inhibitory activity and oral
bioavailability to a series of coumarin dual selective RAF/MEK
inhibitors
Toshihiro Aoki a, Ikumi Hyohdoh a, Noriyuki Furuichi a, Sawako Ozawa a, Fumio Watanabe a,
Masayuki Matsushita a, Masahiro Sakaitani a, Kazutomo Ori a, Kenji Takanashi a, Naoki Harada a,
Yasushi Tomii a, Mitsuyasu Tabo b, Kiyoshi Yoshinari a, Yuko Aoki a, Nobuo Shimma a, Hitoshi Iikura a,b,
⇑
a Research Division, Chugai Pharmaceutical Co., Ltd, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
b Research Division, Chugai Pharmaceutical Co., Ltd, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activ-
ity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth
inhibition (IC50 = 8 nM) and good PK profile (bioavailability of 51% in mouse), resulting in high in vivo
antitumor efficacy in the HCT116 xenograft (ED50 = 4.8 mg/kg). We confirmed the sulfamide moiety
showed no negative impact on tests run on the compound to evaluate DMPK (PK profiles in three animal
species, CYP inhibition and CYP induction) and the safety profile (hERG and AMES tests). Sulfamide 17
had favorable properties that warranted further preclinical assessment
Received 5 August 2013
Revised 27 September 2013
Accepted 1 October 2013
Available online 9 October 2013
Keywords:
MEK
Raf
Ó 2013 Elsevier Ltd. All rights reserved.
Kinase inhibitor
Sulfamide
Anticancer
The Ras/Raf/MEK/ERK pathway is one of the most important
signal transduction pathways in human cancer.1–3 Oncogenic
mutations of Ras or Raf contribute to the constitutive activation
of this pathway. For example, Ras mutations are found in colon
cancer (32–57%), lung cancer (15–50%) and pancreatic cancer
(72–90%) while B-Raf has a high rate of mutation in certain cancers
including melanoma (66%), papillary thyroid (35–70%) and ovarian
(30%).
As drugs for blocking this pathway, MEK or Raf inhibitors have
been tested in clinical trials for cancer (Chart 1).4 The first selective
MEK inhibitor in a clinical trial was CI-1040 (1a),5,6 followed by its
derivatives, including PD0325901(1b)6 and CH4987655(1c).7
GSK1120212 (1d), the most advanced MEK inhibitor, received
FDA approval most recently in V600E or V600K BRAF-mutated
melanoma patients.8,9 These compounds are noncompetitive and
exquisitely selective to MEK. Raf inhibitors have also entered into
clinical trials. Of them, vemurafenib (2) (marketed as Zelboraf,
PLX4032) received FDA approval for the treatment of BRAFV600E
mutation-positive metastatic melanoma in 2011.10
We previously reported that compound CH5126766
(RO5126766) showed both Raf and MEK inhibitory activity of the
Ras/Raf/MEK/ERK pathway.11 The dual, tandem and exquisitely
selective inhibition of a single signal pathway by a single com-
pound was remarkable: MEK was inhibited by the direct binding
to MEK enzyme, and Raf activity was derived, not from direct bind-
ing to Raf, but from Raf/MEK/drug complex stabilization after MEK/
drug complex formation. Characterization of this compound
showed superior antitumor effect compared with that of a pure
MEK inhibitor in a mouse xenograft.11
CH5126766, bearing a sulfamide moiety, was identified12 as a
clinical compound in the hit-to-lead processing of our HTS hit com-
pound 3 (Chart 1).13,14 Coumarin derivative 3 showed highly selec-
tive RAF/MEK inhibition, but its potency was modest in vitro and
in vivo. Moreover, an aniline moiety of the hit compound 3 could
have caused potential issues when developing the drug, because
anilines are known to be AMES-positive,15 be easily metabolized,
and have lower oral bioavailability. We found that incorporating
a sulfamide moiety enhanced inhibitory activity and also afforded
satisfactory PK profiles, resulting in high in vivo efficacy. Sulfamide
Abbreviations: ERK, extracellular signal-regulated kinase; hERG, human ether-a-
go-go related gene; HPCD, 2-hydroxypropyl-b-cyclodextrin; LYSA, lyophilized
solubility assay; MEK, mitogen-activated protein kinase; PAMPA, parallel artificial
membrane permeability assay; PEG, polyethylene glycol; TGI, tumor growth
inhibition.
⇑
Corresponding author. Tel.: +81 550 87 8597; fax: +81 550 87 5326.
E-mail address: iikurahts@chugai-pharm.co.jp (H. Iikura).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.001

6224
T. Aoki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6223–6227
H
N
H
N
After carbamoylation of the phenoic hydroxyl group by
N,N-dimethylcarbamoyl chloride, reduction of a nitro group by SnCl2
afforded aniline derivative 8. Sulfamide 4 was obtained by the
reaction of 8 with sulfamoyl chloride 920,21 or sulfamoyl oxazolizi-
none 10.22 Amide (11), urea (12) and sulfonamide (13) derivatives
were prepared by the coupling of aniline 8 by the usual procedure.
Our SAR study to modify aniline moiety of hit compound 3
showed that compound 4b with a sulfamide group afforded the
strongest inhibition of Raf and MEK (Table 1). A set of compounds
with a functional group at R4 position were evaluated, because
moving the amino group to R4 (amine 8) gave better MEK
inhibition (IC50 = 56 nM) and HCT116 cell growth inhibition
(IC50 = 290 nM) than the original hit (3, IC50’s of 210 and
1200 nM, respectively). Introduction of an amide (11) or urea
(12) did not enhance the Raf or MEK inhibitory activity of amine
8. On the other hand, introduction of a sulfonamide (13a, b) or a
sulfamide (4aÀc) afforded stronger inhibitory activity on Raf/
MEK enzyme and cell growth of HCT116 and HT29. Sulfamide 4a,
which is a derivative after nitrogen substitution of sulfonamide
13a, showed similar bioactivity to sulfonamide 13a with an IC50
value around 30 nM. Sulfamide 4b, which is a derivative after
nitrogen substitution of sulfonamide 13b, demonstrated about
10-fold superior cell growth inhibition over 13b. Sulfamide 4c,
which has two methyl groups at terminal nitrogen also has strong
bioactivity with an IC50 value around 10 nM. Compound 4b has
exquisite selectivity to Raf and MEK inhibitions. No kinases with
O
O
Cl
HO
O
O
F
H
N
H
N
OH
I
I
F
F
F
F
Cl-1040 (1a)
PD0325901 (1b)
H
HO
N
O
O
F
O
N
O
N
H
N
F
H
N
H
N
O
N
N
I
F
O
I
O
F
O
CH4987655 (1c)
GSK1120212 (1d)
O
S
O
H
N
F
Cl
O
F
N
H
N
(2)
vemurafenib
O
O
N
O
N
O
O
O
N
O
O
O
N
S
Cl
N
N
H
H
NH2
F
IC50 < 5 lM in our panel (29 kinases) were observed except Raf and
3
CH5126766 (RO5126766)
MEK (see Supplementary data). We achieved a reduced risk of muta-
genicity (see below) by modifying the aniline moiety, and to increase
the HCT116 activity up to 300-fold by introducing sulfamide.
Evaluation of sulfamide compounds 4aÀc as oral drugs for
water solubility, metabolic stability and membrane permeability
showed comparable or superior values to sulfonamide compounds
13a or 13b (Table 1). Solubility was evaluated by the LYSA
Chart 1. Structures of representative MEK inhibitors (1), a RAF inhibitor (2), HTS hit
compound 3, and CH5126766 (RO5126766).
moieties have attracted attention for their unique electronic and
steric properties.16,17 However, only a small number of reports
have focused on the potential of sulfamide moieties for clinical
usage, and the distinguishing advantages of sulfamide over isoster-
es, such as urea or sulfonamide.18,19 Here we report a SAR study of
the Raf/MEK inhibitory activity of coumarins with a sulfamide and
its isosteres. We also report a physicochemical and a preliminary
safety study of sulfamide compounds as oral drugs.
The synthesis of sulfamide compound 4 is outlined in Scheme 1.
The benzylation of ethyl acetoacetate afforded derivative 6, which
was then converted to coumarin 7 via Pechmann reaction with 4-
chlororesorcinol 5 in a manner similar to reported procedures.13
method,23 and solubility of sulfamide 4a (19
than that of the corresponding sulfonamide 13a (8
l
g/ml) was higher
l
g/ml).
Sulfamide 4b also has better solubility than sulfonamide 13b. This
increase in solubility could be explained by the effect of
introducing a hetero atom (nitrogen). Sulfamide 4c has lower
solubility than sulfamide 4a or 4b, which is a reflection of higher
lipophilicity. Metabolic stability was evaluated using human and
mouse liver microsomes, and metabolic stability of sulfamide 4a
showed better values (CL: 7
l
L/min/mg in human, 8
lL/min/mg
in mouse) than sulfonamide 13a (CL: 11
lL/min/mg in human,
HO
Cl
O
O
O
O
O
HO
Cl
OH
a
b, c
+
NO2
NO2
7
5
6
O
O
S
R1
Cl
N
N
N
O
O
N
O
O
O
O
O
R2
O O
d or d, e
R1
9
R1
O
O
S
Cl
NH2
Cl
N
H
N
O
O O
R2
S
N
N
or
8
4
O
H
f
10
g
O
O
O
R3
Cl
N
H
11-13
Scheme 1. Synthesis of compounds 4, 11–13. Reagents and conditions: (a) 70% H2SO4 aq, rt, 12 h; (b) N,N-dimethylcarbamoyl chloride (1.5 equiv), NaH (1.1 equiv), DMF, rt,
2 h; (c) SnCl2Á2H2O (4.0 equiv), DMF, rt, 12 h; (d) compound 9, pyridine or Et3N, CH2Cl2; (e) 50% HCl aq, THF, 40 °C; (f) compound 10 (3.0 equiv), Et3N (3.0 equiv), CH3CN,
reflux; (g) R3X, Et3N, CH2Cl2.

T. Aoki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6223–6227
6225
Table 1
Enzymatic and cellular activities and pharmaceutical properties of coumarin derivatives
N
O
O
O
O
Cl
R4
b
Compd
R4
IC50 (nM)
MEK1 HCT116
Solubility (
lg/mL)
CL (
l
L/min/mg)
PAMPA (10À6 cm/s)
AUCpo
(lM h)
TGIf (%)
C-Raf
100
HT-29
250
Human
29
Mouse
49
3
8
210
56
1200
290
ND
ND
NDa
6
ND
29
34
38
*
*
NH2
120
370
27
59
O
O
S
4a
4b
4c
11
26
8
28
7
39
4
15
1
19
13
6
7
8
5
70
107
105g
78
N
H
NH2
O
O
23
32
ND
21
52
ND
4
51c,e
S
*
*
N
H
N
H
O
O
3d
S
5
11
96
12
9
4
N
H
N
O
*
170
360
75
ND
ND
ND
33
N
H
O
12
*
*
*
570
56
500
29
4600
30
480
26
ND
8
ND
11
ND
41
ND
3
ND
41c
30c
ND
64
N
H
O
N
O
S
13a
13b
N
H
O
O
NDa
NDa
3
ND
S
50
21
36
33
9
N
H
a
Data not obtainable due to low solubility.
Compounds were evaluated in 24-h exposure studies in mice at 200 mg/kg and formulated as solutions of 5% DMSO, 5% Cremophor EL, 15% PEG400, 15% HPCD, and 60%
b
water.
c
At 100 mg/kg.
At 50 mg/kg.
Sodium salt of 4b was used.
d
e
f
Tumor growth inhibition in the HCT116 human colon cancer xenograft model at 200 mg/kg/day for 11 days.
At 100 mg/kg/day for 11 days.
g
Permeability was evaluated by PAMPA, and was maintained or
even increased in the sulfamide compounds in spite of a higher
hydrophilicity compared with sulfonamide compounds.
A brief study on drug formulation and mouse PK confirmed that
drugs with a sulfamide group could form salts, as does a sulfon-
amide, and therefore higher bioavailability (BA), AUC and dose
could possibly be achieved by forming the salt rather than the free
form. A Na salt of 4b was prepared by reacting with NaOH. The BA
of mouse at 100 mg/kg administered as a Na salt was 31% (AUC:
N
O
O
O
N
O
O
O
O
O
O
O
O
S
O
S
Cl
N
NH2
Cl
N
N
OH
H
H
H
4a
14
16
HO
N
O
O
O
HO
Cl
O
O
O
O
S
O
O
O
S
Cl
N
N
H
N
N
H
H
H
15
51 lM h), which is an improvement over the 10% (AUC: 17 lM h)
of the free form. The major metabolite 4a (de-methylation) was de-
Chart 2. Metabolites of 4b using human liver microsome.
tected 1/10 in AUC to the parent compound.
Oral administrations of sulfamides 4a and 4b showed high
in vivo antitumor effects on xenograft HCT-116 at a dose of 200
and 100 mg/kg once daily, respectively (the experimental proce-
dure was similar to that explained in Fig. 1). The TGI of sulfamides
4a (107%) and 4b (105%) was superior to that of sulfonamide 13a
(64%), aniline 8 (38%) and amide 11 (33%), and was attributed to
their higher cell growth inhibition and/or better physicochemical
properties. A TGI of 78% for di-substituted sulfamide 4c is reflected
in its AUC, which was lower than those of compounds 4a and 4b.
Modifications of the sulfamide at the terminal nitrogen afforded
a set of potent Raf/MEK inhibitors (Table 2). Sulfamide 4e or 4g
with an electron withdrawing group, sulfamide 4h with a hydroxyl
group, and sulfamide 4i with an ether group have potent Raf/MEK
inhibition and HCT116 cell growth inhibition. Sulfamide 4d with
an oxygen located next to the terminal nitrogen, sulfamide 4f with
41 lL/min/mg in mouse). Though no comparison between sulfam-
ide 4b and sulfonamide 13b was available (solubility of 13b is too
low to evaluate stability), the metabolic stability of sulfamide 4b
itself was in the acceptable ranges. Metabolites analysis of terminal
mono-substituted sulfamide 4b using human liver microsomes
yielded 4a (de-methylation of the sulfamide terminal nitrogen),
in addition to compounds 14–16 (Chart 2). This result suggested
that the reduced stability of the sulfamides with a modified termi-
nal nitrogen atom was due to metabolism from de-methylation.
There could be a difference in the metabolic stability of the N-aryl
and N-alkyl sulfamides. In addition, no hydrolysis of sulfamide
moiety (N–S dissociation) was observed. Because aniline was not
detected after microsomes treatment, the risk for AMES positive
by aniline metabolites could be considered low (see below).

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T. Aoki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6223–6227
Table 3
600
400
200
0
Vehicle
In vitro profile of sulfamide 17
Sulfamide 17 12.5 mg/kg
Sulfamide 17 25 mg/kg
Sulfamide 17 50 mg/kg
N
O
F
O
O
O
O
O
S
N
NH2
H
F
17
a
IC50 (nM)
MEK1
9
CYP inhibition: IC50 (lM)
C-Raf
5
HCT116
2C9 (À/+)
12/12
3A4 (À/+)
14/18
8
a
IC50 (À) and IC50 (+) values were determined after a 30-min pre-incubation
without and with NADPH, respectively.
0
5
10
Days after first treatment
4a was detected at 14
lM h, which was higher than that of the par-
Figure 1. In vivo efficacy of sodium salt of 17 in the HCT116 human colon cancer
xenograft model. HCT116 cells were inoculated subcutaneously into the right flank
of BALB-nu/nu mice. Tumors were allowed to establish growth after implantation
before start of treatment. Sulfamide 17 was administered orally once daily for
11 days, from day 0 to 10. Tumor size was measured twice per week. Values are
mean SD, n = 4.
ent 4i (9 M h)). Because modification of the terminal nitrogen
l
with various groups was acceptable, such modification could be
used to adjust the physicochemical properties, such as solubility.
While optimization of the carbamate part (points for metabo-
lism, Chart 2) could be a choice, changing carbamate to another
group resulted in decreasing the bioactivity by one order or worse
in our preliminary SAR. To elucidate the potential of this lead and
sulfamide group as a clinical candidate earlier, we utilized a result
of fluorine scan24 to afford sulfamide 17 with keeping carbamoyl
group. Compound 17 demonstrated favorable properties that war-
rant further preclinical assessment (Tables 3 and 4). Sulfamide 17
has potent Raf/MEK inhibitory activity, HCT116 cell growth inhib-
itory activity, and showed relatively weak CYP inhibitory activity
(IC50’s of CYP/HCT116 >1000) and weak CYP induction activity
(see Supplementary data) and thus small or negligible drug–drug
another nitrogen attached to the terminal nitrogen via ethylene
linker, and sulfamide 4j of dialkyl version also showed potent
Raf/MEK inhibition, but poorer HCT116 cell growth inhibitory
activity, mainly because of poorer permeability. The AUC’s of sulfa-
mides 4d and 4e showed comparable or superior values to that of
sulfamide 4b, while the AUC’s of sulfamide 4h and 4i showed smal-
ler values than that of sulfamide 4b. De-alkylated metabolism
could be a reason for smaller AUC (in PK study of 4i, metabolite
Table 2
SAR of sulfamides with modified terminal nitrogen
N
O
O
O
O
Cl
R4
a
Compd
R4
IC50 (nM)
MEK1
Solubility (
lg/mL)
CL (
l
L/min/mg)
PAMPA (10À6 cm/s)
AUCpo
(l
M h)
C-Raf
37
HCT1169
170
Human
Mouse
O
O
4d
4e
58
65
11
15
17
34
3
3
62b
26b
S
O
*
N
N
H
H
O
O
S
*
*
45
40
68
93
ND
4
ND
N
H
N
H
CF
3
O
O
S
NH2
HCl
4f
5
290
2
0.2
0.2
N
H
N
H
O
O
N
S
*
*
*
*
4g
4h
4i
4
6
4
36
46
47
23
42
36
10
8
40
32
51
31
22
51
5
5
4
25
14
9
N
H
N
H
O
O
S
S
OH
O
N
H
O
N
H
O
9
N
H
O
N
H
O
S
4j
16
35
160
50
101
63
2
ND
N
H
N
N
a
Compounds were evaluated in 24-h exposure studies in mice at 200 mg/kg and formulated as solutions of 5% DMSO, 5% Cremophor EL, 15% PEG400, 15% HPCD, and 60%
water and administered as a free form (not sodium salt).
b
At 100 mg/kg.

T. Aoki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6223–6227
6227
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Shimma, N. Bioorg. Med. Chem. Lett. 2011, 21, 1795.
PK profile of sodium salt of 17 in mouse, rat and monkeya
Parameter
Mouse
Rat
Monkey
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J.; Matsumoto, N.; Miura, T.; Matsumura, K.; Seki, N.; Inaba, T.; Kawasaki, H.;
Yamaguchi, T.; Kakefuda, R.; Nanayama, T.; Kurachi, H.; Hori, Y.; Yoshida, T.;
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iv/po dose (mg/kg)
AUCinf (lM h), po
10/20
26
0.9
14
5.0/10
6.0
0.7
33
62
2.5/5.0
2.2
16
25
30
t1/2 (h), iv
Cl (mL minÀ1 kgÀ1
Bioavailability (%)
)
51
a
Vehicle: 5% DMSO/5% Cremophor EL/15% PEG400/15% HPCD/60% water.
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interactions. The hERG inhibition (24% at 10 lM) was also weak
and an AMES test was negative (see above). The compound showed
satisfactory PK data for mouse, rat and monkey (Table 4) with BA
values of 51%, 62% and 30%, respectively.25 High antitumor effect
was observed in the HCT116 xenograft model: ED50 of 4.8 mg/kg
and TGI of >100% at 50 mg/kg (Fig. 1). It showed no serious effects
on body weight or clinical signs. On the other hand, sulfamides
with a mono-substituted terminal nitrogen (like 4b) showed BA
of lower than 5% in monkey (several compounds tested, data not
shown) even though good mouse BA was observed in mouse. The
idea that species difference could be an issue is supported by the
detection of de-methylated compounds (like 4a) from monkey
blood. Species difference in the metabolism of alkylated sulfamides
might need to be considered when developing oral drugs.
In summary, compounds with a sulfamide group showed stron-
ger inhibitory activity on the Raf/MEK enzyme and in a mouse
xenograft than those with a sulfonamide. In our compounds, the
sulfamide group itself had no negative impact on the compound
as an oral drug, as shown by the PK of several species, CYP inhibition
and induction activities, hERG inhibition activities and an AMES
test. Physicochemical properties such as solubility and metabolic
stability were comparable and/or superior to sulfonamides, which
are often used as oral drugs. As a result, a highly potent and selec-
tive Raf/MEK inhibitor with a unique scaffold, 17, was identified.
Acknowledgements
We thank Y. Tachibana-Kondoh, K. Sakata, and T. Fujii for bio-
logical assays, H. Suda for mass spectrometry measurement, and
Chugai Editing Services for proofreading the manuscript.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl. 2013.
10.001.
20. Timberlake, J. W.; Ray, W. J., Jr.; Stevens, E. D.; Klein, C. L. J. Org. Chem. 1989, 54,
5824.
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