Discovery of a novel series of N-phenylindoline-5-sulfonamide derivatives as potent, selective, and orally bioavailable Acyl CoA:monoacylglycerol acyltransferase-2 inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b00178

Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has attracted interest as a novel target for the treatment of obesity and metabolic diseases. Starting from N-phenylbenzenesulfonamide derivative 1 with moderate potency for MGAT2 inhibition, we explored

Full text of DOI:10.1021/acs.jmedchem.5b00178

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Article
Discovery of a Novel Series of N-Phenylindoline-5-sulfonamide
Derivatives as Potent, Selective, and Orally Bioavailable
Acyl CoA:Monoacylglycerol Acyltransferase-2 Inhibitors
Kenjiro Sato, Hiroki Takahagi, Takeshi Yoshikawa, Shinji Morimoto, Takafumi Takai, Kousuke
Hidaka, Masahiro Kamaura, Osamu Kubo, Ryutaro Adachi, Tsuyoshi Ishii, Toshiyuki Maki, Taisuke
Mochida, Shiro Takekawa, Masanori Nakakariya, Nobuyuki Amano, and Tomoyuki Kitazaki
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 21 Apr 2015
Downloaded from http://pubs.acs.org on April 21, 2015
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Discovery of a Novel Series of NꢀPhenylindolineꢀ5ꢀ
sulfonamide Derivatives as Potent, Selective, and
Orally Bioavailable Acyl CoA:Monoacylglycerol
Acyltransferaseꢀ2 Inhibitors
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Kenjiro Sato, Hiroki Takahagi, Takeshi Yoshikawa, Shinji Morimoto, Takafumi Takai, Kousuke
Hidaka, Masahiro Kamaura, Osamu Kubo, Ryutaro Adachi, Tsuyoshi Ishii, Toshiyuki Maki,
Taisuke Mochida, Shiro Takekawa, Masanori Nakakariya, Nobuyuki Amano, and Tomoyuki
Kitazaki
Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd., 26ꢀ1, MuraokaꢀHigashi 2ꢀ
Chome, Fujisawa, Kanagawa 251ꢀ8555, Japan
Keywords: MGAT2 inhibitor, acyltransferase, triacylglycerol, obesity, metabolic disease,
indoline, conformation
ABSTRACT. Acyl CoA:monoacylglycerol acyltransferaseꢀ2 (MGAT2) has attracted interest as
a novel target for the treatment of obesity and metabolic diseases. Starting from a Nꢀ
phenylbenzenesulfonamide derivative 1 with moderate potency for MGAT2 inhibition, we
explored an effective location of the hydrophobic group at the 1ꢀposition to enhance MGAT2
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inhibitory activity. Shifting the hydrophobic group to the adjacent position followed by
introduction of a bicyclic central core to restrict the substituent orientation produced a Nꢀ
phenylindolineꢀ5ꢀsulfonamide derivative 10b, which displayed much improved potency, with an
IC value of 1.0 nM. This compound also exhibited excellent selectivity (greater than 30,000ꢀ
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fold) against related acyltransferases (MGAT3, DGAT1, DGAT2, and ACAT1). Subsequent
optimization efforts were directed toward improving pharmacokinetic profiles, which resulted in
the
identification
of
5ꢀ[(2,4ꢀdifluorophenyl)sulfamoyl]ꢀ7ꢀ(2ꢀoxopyrrolidinꢀ1ꢀyl)ꢀNꢀ[4ꢀ
(trifluoromethyl)phenyl]ꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ1ꢀcarboxamide (24d) endowed with potent
MGAT2 inhibitory activity (IC = 3.4 nM) and high oral bioavailability (F = 52%, mouse). In a
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0
mouse oral fat tolerance test, oral administration of this compound effectively suppressed the
elevation of plasma triacylglycerol levels.
INTRODUCTION
Dietary fat mainly consists of triacylglycerol (TG), and it is one of the important sources of
energy for our body. Fat absorption includes multiple steps, namely hydrolysis of TG by gastric
and pancreatic lipases to fatty acids and monoacylglycerol (MG), uptake of the cleaved products
in the enterocytes, resynthesis of TG from MG, incorporation of TG in chylomicrons, and
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entrance of chylomicrons into the systemic circulation.
Acyl CoA:monoacylglycerol
acyltransferases (MGATs) are enzymes that catalyze the synthesis of diacylglycerol (DG) from
MG and fatty acyl CoA, which is a crucial step in TG synthesis by the MGAT pathway. MGATs
belong to the DGAT2 gene family of enzymes, and three isoforms (MGAT1, MGAT2, and
4, 5
MGAT3) have been identified till date. Among them, MGAT2 is highly expressed in the small
intestine, and it is considered to play an important role in dietary fat absorption.
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Recently, independent research groups reported that MGAT2ꢀdeficient mice were protected
from highꢀfat dietꢀinduced obesity and exhibited improved insulin sensitivity and glucose
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tolerance. Interestingly, the MGAT2ꢀdeficient mice displayed increased oxygen consumption
in comparison with the wildꢀtype mice. The antiꢀobesity phenotype of the MGAT2ꢀdeficinet
mice is likely associated with enhanced energy expenditure, although the precise
pharmacological basis remains to be elucidated. Furthermore, in the MGAT2ꢀdeficient mice,
fatty and oily stool was not observed, whereas orlistat, a pancreatic lipase inhibitor, exerted these
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types of gastrointestinal adverse effects. These preclinical data strongly suggested that MGAT2
inhibitors can serve as novel antiꢀobesity and antiꢀdiabetic agents.
Despite the potential pharmacological benefits, only a few reports of MGAT2 inhibitors have
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been reported,
and there is no clinical information regarding MGAT2 inhibitors. We initiated
a program to develop MGAT2 inhibitors as a novel treatment for obesity and metabolic diseases.
Because the substrates of MGAT2, MG and fatty acyl CoA, contain long fatty acyl chains, it is
assumed that the high lipophilicity of compounds may lead to a high affinity for MGAT2.
However, it is of considerable importance to produce a drug candidate molecule with low
lipophilicity from the viewpoint of a better chance of successful drug development, and it is
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proposed that the optimal range of lipophilicity (LogD or LogP) lies between 1 and 3.
Therefore, our goal was to deliver a clinical candidate of MGAT2 inhibitor with desirable
lipophilicity.
Novel series of MGAT2 inhibitors were identified from highꢀthroughput screening of our
chemical library. Among several hit chemotypes, we selected a sulfonamide derivative 1 with an
IC value of 130 nM as a starting point on the basis of its low lipophilicity as well as moderate
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potency (Figure 1). An initial structure–activity relationship (SAR) study revealed that the
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ArNHSO Ar structure was indispensable for MGAT2 inhibitory activity, and even slight
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structural modification on this motif led to an abrupt decline in potency. In contrast, the
modification of the phenoxyacetamide moiety at the 1ꢀposition was tolerated, although
hydrophobic groups such as a phenyl ring were required for MGAT2 inhibition. We
hypothesized that optimal arrangement of the hydrophobic group could lead to enhanced
MGAT2 inhibitory activity. On the basis of this concept, rearrangement of the hydrophobic
group to the adjacent position and introduction of a bicyclic central core to restrict the orientation
of the substituent were implemented, leading to the discovery of an indolineꢀ5ꢀsulfonamide
derivative 10b with potent and highly selective MGAT2 inhibitory activity and desired
lipophilicity as a promising lead compound. Our subsequent optimization efforts were directed to
improve the pharmacokinetic profiles of the lead compound 10b. This resulted in the
identification of a compound 24d, which suppressed the elevation of plasma TG levels in an oral
fat tolerance test (OFTT) in mice after oral administration. Here we report the design, synthesis,
and biological activity of Nꢀphenylindolineꢀ5ꢀsulfonamide derivatives as a novel series of
MGAT2 inhibitors. The relationship between MGAT2 potency and electron density on the
carbonyl group at the indoline 7ꢀposition, one of the important pharmacophoric elements, is also
discussed.
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Figure 1. NꢀPhenylbenzenesulfonamideꢀ and Nꢀphenylindolineꢀ5ꢀsulfonamideꢀbased MGAT2
inhibitors.
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CHEMISTRY
Scheme 1 describes the steps to synthesize indolineꢀ5ꢀsulfonamide derivatives 10a–f. The
synthesis started from protection of the nitrogen atom of 5ꢀbromoꢀ7ꢀnitroindoline (2) with a
benzyloxycarbonyl (Cbz) group, followed by palladiumꢀcatalyzed coupling with benzyl
mercaptan to give benzyl sulfide 4. To prepare sulfonyl chloride 5, oxidative chlorination of 4
using Nꢀchlorosuccinimide (NCS) in aqueous acetic acid was employed according to a method
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reported for aryl methoxymethyl sulfides. Sulfonamidation of 5 with anilines smoothly
proceeded in N,Nꢀdimethylacetamide (DMA) without the addition of a base to furnish 6a and 6b
in good yields. Reduction of the nitro group of 6a or 6b and subsequent Nꢀacetylation of the
resulting aniline afforded 8a or 8b, respectively. After removal of the Cbz groups of 8a and 8b,
variations on the substituent at the indoline 1ꢀposition were achieved by reductive amination
with aldehydes under a sodium triacetoxyborohydride or 2ꢀpicoline borane complex condition to
deliver the final products 10a–f.
a
Scheme 1. Synthesis of indolineꢀ5ꢀsulfonamide derivatives 10a–f
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a
Reagents and conditions: (a) NaH, CbzCl, THF, 0 °C to rt, 84%; (b) BnSH, Pd (dba) ,
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Xantphos, DIEA, toluene, reflux, 73%; (c) NCS, AcOH, H O, 0 °C to rt, 97%; (d) 4ꢀ
methoxyaniline or 2,4ꢀdifluoroaniline, DMA, rt, 86–96%; (e) Fe, CaCl , EtOH, H O, reflux, 66%
for 7a); (f) Zn, AcOH, rt, 94% (for 7b); (g) AcCl, DMA, 0 °C to rt, 89–94%; (h) H (1 atm), Pd
on carbon, THF, MeOH, rt, 92–99%; (i) aldehyde, NaBH(OAc) , AcOH, CH CN, rt, 27–68%
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(for 10a–e); (j) PhCH CH CHO, 2ꢀpicolineꢀborane complex, MeOH, AcOH, rt, 93% (for 10f).
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Further derivatizations from 10b are described in Scheme 2. The indoline ring of 10b was
efficiently oxidized to an indole ring by 2,3ꢀdichloroꢀ5,6ꢀdicyanobenzoquinone (DDQ) to afford
the corresponding indole derivative 11. NꢀDeacetylation of 10b by acidic alcoholysis afforded
aniline 12, which was then subjected to reductive amination with formaldehyde to give a Nꢀ
methyl analog 13.
a
Scheme 2. Synthesis of indolineꢀ5ꢀsulfonamide derivatives 11 and 13
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Reagents and conditions: (a) DDQ, THF, rt, 54%; (b) HCl, MeOH, 80 °C, 58%; (c)
formaldehyde, NaBH(OAc) , AcOH, THF, rt, 29%.
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Scheme 3 outlines the synthesis of an analog bearing no substituent at the indoline 7ꢀposition.
The desired compound 20 was prepared from Bocꢀprotected 5ꢀbromoindoline (15) in a manner
analogous to that described in Scheme 1.
a
Scheme 3. Synthesis of indolineꢀ5ꢀsulfonamide derivative 20
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Reagents and conditions: (a) Boc O, THF, rt, 95%; (b) BnSH, Pd (dba) , Xantphos, DIEA,
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toluene, reflux, 100%; (c) NCS, AcOH, H O, 0 °C to rt, 63%; (d) 2,4ꢀdifluoroaniline, pyridine, 0
C to rt, 87%; (e) HCl, AcOEt, rt, 86%; (f) PhCH CHO, NaBH(OAc) , AcOH, CH CN, rt, 70%.
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2 3 3
Compounds with a heterocycle at the indoline 7ꢀposition were synthesized by the method
presented in Scheme 4. The lactam rings of 22a and 22b and a cyclic carbamate of 22e were
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constructed by intramolecular cyclization of 21a, 21b, and 21e, which were prepared by the
acylation of 7b with appropriate acyl chlorides. In an analogous manner, urea formation of 7b
with isocyanates and subsequent intramolecular cyclization gave cyclic urea derivatives 22c and
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2d in good yields. Removal of the Cbz groups of 22a–c was followed by modification of the
nitrogen atom at the indoline 1ꢀposition. For reductive amination of 23a or 23b with
phenylacetaldehyde, the hydrogenation condition in the presence of platinum oxide was more
effective than that in the presence of sodium triacetoxyborohydride, which afforded the desired
products 24a and 24b. Compounds 24c–f were obtained by urea formation of 23a or 23c with
anilines using triphosgene as a carbonyl source.
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Scheme 4. Synthesis of indolineꢀ5ꢀsulfonamide derivatives 24a–f
a
Reagents and conditions: (a) 4ꢀchlorobutyryl chloride or 5ꢀchlorovaleryl chloride, DMA, 0 °C
to rt; (b) 2ꢀchloroethyl isocyanate or 3ꢀchloropropyl isocyanate, THF, 60–70 °C; (c) 2ꢀ
chloroethyl chloroformate, TEA, THF, 0 °C to rt; (d) NaH, DMF, 0 °C to rt, 59–92% over 2
steps (for 22a and 22b), 64–74% over 2 steps (for 22c and 22d), 11% over 2 steps (for 22e); (e)
H (1 atm), Pd on carbon, THF, MeOH, rt, 94–99%; (f) PhCH CHO, H (1 atm), PtO , THF,
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EtOH, rt, 41ꢀ62% (for 24a and 24b); (g) 4ꢀsubstituted aniline, triphosgene, pyridine, TEA, THF,
°C to rt, 62–92% (for 24c–f).
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A synthetic route to the indazole derivative 32 is depicted in Scheme 5. The requisite 5ꢀ
bromoꢀ7ꢀnitroindazole (26) was synthesized by diazotization of oꢀtoluidine 25 and following
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selfꢀcyclization. Regioselectivity for Nꢀalkylation of 26 with 2ꢀ(4ꢀfluorophenyl)ethyl bromide
was not observed, and the desired compound 27 was obtained in a yield of 42% after separation.
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The structures of 27 and the regioisomer 27′ were determined by NMR experiments.
Compound 27 was converted to the final product 32 through structural modifications at the 5ꢀ
and 7ꢀpositions on the indazole ring by methods similar to those described in Scheme 1.
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Scheme 5. Synthesis of 1Hꢀindazoleꢀ5ꢀsulfonamide derivative 32
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Reagents and conditions: (a) NaNO , AcOH, H O, rt, 56%; (b) 2ꢀ(4ꢀfluorophenyl)ethyl
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bromide, Cs CO , DMF, rt, 42%; (c) BnSH, Pd (dba) , Xantphos, DIEA, toluene, reflux, 92%;
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(d) NCS, AcOH, H O, 0 °C to rt, 92%; (e) 2,4ꢀdifluoroaniline, DMA, rt, 87%; (f) H (1 atm), Pd
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on carbon, THF, MeOH, rt, 95%; (g) AcCl, DMA, rt, 46%.
To access the benzimidazole derivatives 39 and 40, the synthetic procedures described in
Scheme 6 were employed. Direct chlorosulfonylation of 2,6ꢀdinitroaniline (33) with
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chlorosulfonic acid was achieved under a previously reported condition, and the obtained
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sulfonyl chloride was subjected to sulfonamidation with 2,4ꢀdifluoroaniline to yield 34.
Sandmeyer reaction of 34 followed by nucleophilic substitution with 4ꢀfluorophenethylamine
furnished 35, which underwent hydrogenation to provide 3,4,5ꢀtriaminobenzenesulfonamide 36.
Upon treatment of 36 with formic acid, Nꢀformylation of the amino groups was accompanied by
construction of a benzimidazole ring to yield 37. Compound 37 was transformed to the final
product 39 by deformylation and Nꢀacetylation. In contrast, refluxing of compound 36 in acetic
acid afforded 2ꢀmethylbenzimidazole 40 after acetylation of the remaining amino group.
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a
Scheme 6. Synthesis of 1Hꢀbenzimidazoleꢀ5ꢀsulfonamide derivatives 39 and 40
a
Reagents and conditions: (a) (1) ClSO H, 100 °C, (2) 2,4ꢀdifluoroaniline, DMA, rt, 72%; (b)
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rt to 60 °C, 97%; (f) AcCl, DMA, rt, 91%; (g) (1) AcOH, reflux, (2) AcCl, DMA, rt, 70%.
Synthesis of a 1,3ꢀbenzoxazolone derivative 49 was achieved by the route illustrated in
1
7
Scheme 7. Compound 43 was synthesized from 2ꢀaminophenol 41 by a twoꢀstep sequence
consisting of Nꢀacylation and subsequent borane reduction. Reaction of 43 with 1,1′ꢀ
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
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4
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4
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4
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4
5
5
5
5
5
5
5
5
5
5
6
carbonyldiimidazole constructed a 1,3ꢀbenzoxazolone scaffold to yield 44, which was elaborated
to the final compound 49 in a manner similar to the method detailed in Scheme 1.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 7. Synthesis of 2ꢀoxoꢀ2,3ꢀdihydroꢀ1,3ꢀbenzoxazoleꢀ6ꢀsulfonamide derivative 49
a
Reagents and conditions: (a) phenylacetyl chloride, NaH, THF, rt, 42%; (b) boraneꢀTHF
complex, THF, rt to 50 °C, 72%; (c) CDI, THF, rt, 81%; (d) BnSH, Pd (dba) , Xantphos, DIEA,
2
3
toluene, reflux, 91%; (e) NCS, AcOH, H O, 0 °C to rt, 74%; (f) 2,4ꢀdifluoroaniline, DMA, rt,
2
73%; (g) Zn, AcOH, 0 °C to rt; (h) AcCl, DMA, rt, 35% over 2 steps.
Scheme 8 describes the synthetic steps to 4ꢀ(2ꢀphenylethoxy)benzenesulfonamide 54.
Sulfonamidation of a commercially available sulfonyl chloride 50 with pꢀanisidine afforded 51,
which was subjected to Mitsunobu reaction with 2ꢀphenylethanol to give 52. After reduction of
the nitro group of 52, acetylation of the resulting amine produced the final compound 54.
a
Scheme 8. Synthesis of 4ꢀ(2ꢀphenylethoxy)benzenesulfonamide derivative 54
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43
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49
50
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52
53
54
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60
a
Reagents and conditions: (a) 4ꢀmethoxyaniline, DMA, rt, 89%; (b) 2ꢀphenylethanol, DIAD,
PPh , 0 °C, 71%; (c) H (1 atm), Pd on carbon, THF, MeOH, rt, 91%; (d) Ac O, pyridine, rt,
37%.
3
2
2
RESULTS AND DISCUSSION
The newly synthesized compounds were tested for MGAT2 inhibitory activity using
membrane fractions from the MGAT2 expression. A sulfonamide derivative 1 was identified as
an initial lead compound with moderate MGAT2 inhibitory activity (EC = 130 nM) via highꢀ
5
0
throughput screening in our library (Figure 2). The initial SAR study revealed that the Nꢀ
arylbenzenesulfonamide motif was essential for MGAT2 inhibitory activity, and potency for
MGAT2 inhibition was not retained after structural conversion of the sulfonamide linkage. In
contrast, the phenoxyacetyl moiety at the 1ꢀposition was tolerated for structural modification.
Simple amidation reactions with various carboxylic acids allowed us to comprehend SAR at the
1
ꢀposition promptly, and several derivatives containing a hydrophobic group such as a phenyl
ring displayed a similar level of MGAT inhibitory activity to 1, although notable enhancement of
potency was not observed (data not shown).
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Structure of an initial lead compound 1.
Given the tolerability for structural modification of the substituent at the 1ꢀposition, we
attempted to rearrange the hydrophobic group from the 1ꢀposition to the 2ꢀposition in order to
expand the chemical series (Figure 3A, II). Consequently, we identified compound 54, which
displayed comparable potency to 1, with an IC value of 130 nM. We assumed that the benzene
5
0
ring at the 1ꢀposition of 1 or the 2ꢀposition of 54 would occupy the same binding site during its
interaction with MGAT2. Figure 4 shows a result of flexible alignment of compounds 1 and 54
1
8
using Molecular Operating Environment software (MOE). Namely, we hypothesized that
MGAT2 would have a hydrophobic space between the 1ꢀ and 2ꢀpositions of this series of
compounds in interaction (Figure 3B), which could be approached by both series I and II.
Meanwhile, the orientation of the phenethyloxy group of 54 likely prefers conformation IIIa to
IIIb because of steric hindrance with the substituent at the 1ꢀposition (Figure 5A). This concept
was supported by calculation of a stable conformation of 54 using MOE. For the aforementioned
interaction with the putative hydrophobic site, compound 54 needs to adopt an energetically
unfavorable conformation similar to IIIb. Based on this hypothesis, we designed a series of
compounds with a bicyclic core (IV), which enables efficient orientation of a hydrophobic
substituent in the desired position (Figure 5B). We anticipated that this conformational
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restriction could reduce the entropic cost on binding and should lead to the enhancement of
MGAT2 inhibition potency.
10
11
12
13
14
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17
18
19
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22
23
24
25
26
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A)
hydrophobic
group
MeO
O
1
Me
NH
MeO
O
Me
NH
MeO
O
1
O O
O
O
O
O
S
S
S
NH
Me
N
N
N
H
H
H
hydrophobic
group
O
2
2
54
hMGAT2 IC50 = 130 nM
I
II
B)
R
O
X
O
O
S
1 NH
N
hydrophobic
site
H
Y
2
Figure 3. A) Repositioning of a hydrophobic group from the 1ꢀposition to the 2ꢀposition. B)
Presumed hydrophobic space between the 1ꢀ and 2ꢀpositions.
Figure 4. Flexible alignment of compounds 1 (cyan) and 54 (magenta) using MOE.
A)
R1
B)
O
Me
NH
O
1
Me
NH
O
1
Me
O
O
R
R1
NH
R2
S
N
hydrophobic
group
H
N
O
R2
O
2
2
IIIa
IIIb
IV
2
Figure 5. A) Conformational preference of the alkoxy (OR ) moiety. B) Design of orienting a
substituent to a presumed hydrophobic site by introduction of bicyclic core systems.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1 summarizes the MGAT2 inhibitory activity of compounds with various bicyclic core
systems. Gratifyingly, an indoline derivative 10a exhibited a 60ꢀfold improvement in potency in
comparison with the parent 54 despite the lowered lipophilicity. This result highlighted that
conformational constraints on the phenethyl group had a significant impact on potency as
expected, presumably due to efficient interaction with the putative hydrophobic site of MGAT2.
The electron density of the aniline moiety had no effect on MGAT2 inhibitory activity. Indeed,
the 4ꢀmethoxyaniline of 10a could be transformed to 2,4ꢀdifluoroaniline, an electronꢀdeficient
aniline, to yield 10b without compromising potency. Introduction of a fluorine atom on the
benzene ring of the phenethyl group also resulted in potent MGAT2 inhibitory activity (10c).
Although desaturation of the central indoline ring to the corresponding indole ring was tolerated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(11), conversion to an indazole derivative 32 or benzimidazole derivative 39 resulted in a more
than 10ꢀfold decrease in potency. In addition, installation of a methyl group at the 2ꢀposition of
the benzimidazole ring of 39 led to a profound loss of potency (40). These data indicated that the
central bicyclic core of 10a–c or 11 would efficiently occupy a hydrophobic and sizeꢀlimited
pocket of MGAT2 during binding. This speculation was consistent with the fact that the
benzoxazolone derivative 49 displayed only weak MGAT2 inhibitory activity. Through these
explorations, the indoline or indole ring was found to serve as a scaffold to optimally orient the
phenethyl group to the presumed hydrophobic site in MGAT2 and confer greatly enhanced
MGAT2 inhibitory activity. In terms of synthetic accessibility, the indoline core was selected for
further exploration of structural requirements at the 1ꢀ and 7ꢀpositions on the central core.
Table 1. MGAT2 inhibitory activity: SAR of central bicyclic systems
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9
a
10
11
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41
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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59
60
hMGAT2
b
Compound
Ar
R
H
H
F
LogD7.4
2.2
IC (nM)
50
2
.2
10a
(
2.0–2.4)
1
.0
1
0b
2.5
(
0.89–1.2)
1.2
1
0c
2.5
(1.0–1.3)
2
.1
11
H
F
F
2.3
2.2
1.5
(
1.8–2.3)
4
1
3
2
9
(
36–48)
1
70
3
(
130–210)
3
300
40
F
1.6
2.0
(
(
3000–3600)
2
100
4
9
H
1700–2600)
a
Inhibitory activity against human MGAT2. IC values are presented as means of duplicate
experiments with their 95% confidence intervals in parentheses. LogD value at pH 7.4.
5
0
b
19
The results from a representative selection of compounds with various substituents at the
indoline 1ꢀ and 7ꢀpositions are presented in Table 2. Deletion of the phenyl ring of the 1ꢀ
phenethyl group of 10b resulted in more than 100ꢀfold decline in potency (10d), demonstrating
the importance of the terminal hydrophobic group at the 1ꢀposition in driving MGAT2 inhibition.
The observation that the 1ꢀphenylpropyl analog 10f was equipotent to the phenethyl analog 10b
indicated that the presumed hydrophobic site of MGAT2 could accommodate an extended or
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
larger substituent. In contrast, the benzyl analog 10e displayed a 10ꢀfold reduction in potency in
comparison with 10b. This was likely the result of the phenyl group at the 1ꢀposition of 10e
being unable to reach the best hydrophobic site.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. MGAT2 inhibitory activity: SAR at the 1ꢀ and 7ꢀpositions of the indoline ring
a
1
2
hMGAT2
IC (nM)
b
Compound
R
R
LogD7.4
1.4
50
2
20
10d
(
180–260)
2
3
10e
10f
20
2.2
2.8
3.7
3.7
2.9
3.1
(
18–31)
3
.7
(
3.0–4.6)
2
70
(
200–380)
1
30
13
(
99–180)
6
.7
2
4a
(
(
4.9–9.1)
7
.3
2
4b
6.5–8.2)
a
Inhibitory activity against human MGAT2. IC values are presented as means of duplicate
5
0
b
19
experiments with their 95% confidence intervals in parentheses. LogD value at pH 7.4.
We also found that the acetylamino group at the indoline 7ꢀposition had a significant
contribution to MGAT2 inhibitory activity. Removal of the acetylamino or carbonyl group
induced an abrupt loss of potency (20 or 13), despite their increases of lipophilicity in
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comparison with 10b. In contrast, incorporation of a lactam ring resulted in the maintenance of
potent MGAT2 inhibitory activity, as illustrated by 24a and 24b. Based on these results, we
hypothesized that the carbonyl oxygen atom at the 7ꢀposition could have a critical interaction
with MGAT2 as a hydrogen bond acceptor (HBA).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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60
To examine the influence of electron density at the carbonyl oxygen on potency, we prepared
and compared lactam, cyclic urea, and cyclic carbamate derivatives, which have different
electron densities at the carbonyl oxygen with a similar size and shape of molecules (Table 3).
The electron density at the carbonyl oxygen of each simplified version V shown in Table 3 was
20
calculated using the semiempirical PM3 (MOPAC) method in Molecular Operating
1
8
Environment software. The cyclic urea 22c displayed more potent inhibitory activity than the
lactam 22a, whereas the cyclic carbamate 22e had attenuated potency in comparison with 22a.
Interestingly, a good correlation between MGAT2 inhibitory activity and electron density at the
carbonyl oxygen was observed. According to the calculation, the electron density increases in the
order of cyclic urea 22c > lactam 22a > cyclic carbamate 22e. It was reported that cyclic ureas
21
have stronger hydrogen bond basicity than the corresponding lactams, which is consistent with
this calculated result. The lower electron density of the cyclic carbamate was presumably driven
by the electron withdrawing nature of the oxygen atom adjacent to the carbonyl group. Potency
enhancement by conversion of a lactam to the corresponding cyclic urea was also observed for
sixꢀmembered rings, as illustrated by the comparison of 22b and 22d. These results suggested
that the carbonyl oxygen at the 7ꢀposition should participate in an interaction with MGAT2 as
HBA and that MGAT2 inhibitory activity could be enhanced by increasing the electron density.
Table 3. MGAT2 inhibitory activity: SAR at the 7ꢀposition of the indoline ring
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
hMGAT2
IC (nM)
Electron charge on theb
c
Compound
X
n
1
1
1
2
2
LogD7.4
2.8
carbonyl oxygen of V
5
0
1
4
22a
22c
22e
CH
2
−0.353
(
10–19)
3
.4
NH
O
−0.394
−0.334
−0.353
−0.399
2.3
(
2.9–3.9)
4
3
2.3
(
33–56)
1
0
2
2
2b
2d
CH
2
3.2
(
8.3–13)
2.2
NH
2.6
(1.8–2.7)
a
Inhibitory activity against human MGAT2. IC values are presented as means of duplicate
experiments with their 95% confidence intervals in parentheses. Electron density on the
carbonyl oxygen of V was calculated by MOPAC using PM3 method. LogD value at pH 7.4.
5
0
b
c
19
Following this, we examined the selectivity against other related acyltransferases of a
representative compound of this novel series of Nꢀphenylindolineꢀ5ꢀsulfonamide derivatives
(
Table 4). Compound 10b exhibited more than 30,000ꢀfold selectivity over MGAT3, DGAT1,
DGAT2, and ACAT1. In addition to the aforementioned potent MGAT2 inhibitory activity and
tolerability for structural modification at the 1ꢀ and 7ꢀpositions of this series of compounds, the
excellent selectivity of 10b made this compound a promising lead compound of novel MGAT2
inhibitors. In contrast, DMPK assessment revealed that compound 10b was rapidly cleared by
liver microsomes both in humans and mice, which was reflected in its high in vivo clearance and
low plasma concentrations after oral administration in mice (Table 5; CLtotal = 4977 mL/h/kg, F
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=
4.6%). Accordingly, further structural modification focused on the improvement of its
pharmacokinetic profiles.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Selectivity of 10b over other acyltransferases
a
a
a
a
hMGAT3
IC (nM)
hDGAT1
hDGAT2
hACAT1
50
Compound
IC (nM)
50
IC (nM)
IC (nM)
5
0
50
1
0b
> 30000
> 30000
> 30000
35000
a
IC values are presented as means of duplicate experiments.
5
0
Table 5. Microsomal clearances and pharmacokinetic profiles of compound 10b
a
in vitro CLint
b, c
Pharmacokinetic profiles in mice
(
ꢁL/min/mg)
Compound
b
c
c
CLtotal
mL/h/kg)
AUC0–8h
(ng·h/mL)
F
(%)
HLM
MLM
(
1
0b
101
220
4977
9.2
4.6
a
b
c
Human/mouse liver microsomal clearance. 0.1 mg/kg, iv. 1 mg/kg, po.
In the initial optimization step, we explored functionalized benzene rings connected through
an alkyl linkage to the indoline 1ꢀposition, which afforded several analogs with potent MGAT2
inhibitory activity such as a difluorophenyl, (trifluoromethyl)phenyl, or (alkoxycarbonyl)phenyl
derivative; however, no appreciable improvement in microsomal stability was detected in
comparison with 10b (data not shown). From these results, we presumed that the poor metabolic
stability of 10b should be attributed to the vulnerability to oxidative metabolism of the alkylene
spacer at the indoline 1ꢀposition. Based on this assumption, compounds with an Nꢀacylꢀtype
substituent at the 1ꢀposition were designed to improve metabolic stability. In addition, in vitro
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
metabolite analysis of related analogs suggested that the Nꢀacetyl group at the 7ꢀposition of 10b
would be easily hydrolyzed, presumably being associated in part with the high in vivo clearance.
This guided us to employ a lactam or cyclic urea at the 7ꢀposition, which was expected to be
more resistant to hydrolysis, instead of the Nꢀacetyl group.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MGAT2 inhibitory activities, microsomal clearances, and plasma concentration after oral
administration of compounds with structural modifications at the indoline 1ꢀ and 7ꢀpositions are
presented in Table 6. Introduction of a ureaꢀtype linkage at the 1ꢀposition provided compound
24c with an IC50 value of 28 nM, which expectedly displayed a significant beneficial
improvement in microsomal stability in comparison with the phenethyl analog 10b or 24a (CLint
(HLM/MLM) = 112/248 ꢁL/min/mg). Although the potency of 24c was weaker than that of the
2
2
corresponding phenethyl analog 24a, their ligand lipophilicity efficiency (LLE) values were
equivalent (LLE = 5.3 (24a) and 5.4 (24c)). We surmised that increasing lipophilicity of the
substituent at the 1ꢀposition would enhance MGAT2 inhibitory activity because this position is
possibly located in a hydrophobic site of MGAT2 during interaction. Indeed, the addition of a
hydrophobic substituent on the terminal benzene ring resulted in increased potency. The 4ꢀ
(trifluoromethyl)phenyl derivative 24d exhibited potent MGAT2 inhibitory activity with an IC50
value of 3.4 nM and an LLE value of 5.4, which was comparable with that of the phenethyl
derivative 24a, while maintaining improved microsomal stability. Introduction of a tertꢀbutyl
group also enhanced MGAT2 inhibitory activity to a similar level as that of 24d (24e: IC = 3.1
5
0
nM). However, 24e displayed inferior metabolic stability to the trifluoromethyl derivative 24d.
This may be caused by the higher elevation of lipophilicity of 24e than that of 24d (LogD7.4
=
3.1 (24d) and 3.6 (24e)). In terms of lipophilicity efficiency, the trifluoromethyl group could
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more effectively achieve an interaction with the putative hydrophobic site of MGAT2 than the
tertꢀbutyl group.
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Table 6. MGAT2 inhibitory activity, microsomal clearances, and plasma concentration
c
in vitro CLint
a
d
hMGAT2
IC (nM)
b
AUC0–8h
(
ꢁL/min/mg)
Compound
R
X
LogD7.4
2.2
(ng·h/mL)
50
HLM
MLM
2
8
24c
CH
2
30
143
NT
(
26–31)
3
.4
2
4d
CH
CH
2
2
3.1
36
84
842
(
3.0–3.9)
Me
Me
3
.1
O
Me
2
4e
3.6
2.5
66
63
146
82
15
87
(
(
2.7–3.5)
N
H
1
.8
24f
NH
1.3–2.4)
a
Inhibitory activity against human MGAT2. IC values are presented as means of duplicate
5
0
b
19
experiments with their 95% confidence intervals in parentheses. LogD value at pH 7.4.
Human/mouse liver microsomal clearance. Mice. 1 mg/kg, po. NT = not tested.
c
d
To further enhance MGAT2 inhibitory activity, a cyclic urea motif was embedded at the 7ꢀ
position with the aim of increasing electron density at the carbonyl oxygen, as mentioned above.
As anticipated, compound 24f displayed more potent activity and lower lipophilicity than 24d,
resulting in a much higher LLE value (LLE = 6.2). Compound 24f also exhibited improved
microsomal stability to a level similar to that of 24d.
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6
Given several compounds endowed with potent MGAT2 inhibitory activity and improved
metabolic stability such as 24d and 24f, we next investigated pharmacokinetic profiles when
compounds were orally administered to mice at a dose of 1 mg/kg. Pharmacokinetic studies
revealed that compound 24d displayed a high plasma concentration (AUC0–8h = 842 ng·h/mL)
and favorable oral bioavailability (F = 52%), which were probably driven by the improved
stability against oxidative metabolism and hydrolysis. In contrast, the tertꢀbutyl analog 24e did
not display improved bioavailability. We assume that the poor bioavailability of 24e is due to the
insufficient metabolic stability and/or deteriorated aqueous solubility in comparison with
compound 24d (4.8 ꢁg/mL (24e) vs 12 ꢁg/mL (24d) at pH 6.8). Interestingly, it was found that
transformation of the lactam ring to a cyclic urea at the 7ꢀposition negatively affected
pharmacokinetic profiles. The AUC level of compound 24f was approximately oneꢀtenth that of
compound 24d, whereas the two compounds exhibited similar in vitro microsomal stabilities and
volumes of distribution (Vdss = 732 and 872 mL/h/kg for 24d and 24f, respectively). This
unexpected result may be because of deterioration of membrane permeability owing to an
0
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additional hydrogen bond donor (PAMPA permeability : 396 nm/sec (24d) vs 94 nm/sec (24f)
at pH 7.4). Consequently, compound 24d was selected for further in vivo evaluation on the basis
of its potent MGAT2 inhibitory activity and desirable pharmacokinetic profiles.
For evaluating the in vivo efficacy, compound 24d was examined for its effect on
hypertriglyceridemia during OFTT using C57BL/6J mice. To inhibit the hydrolysis of plasma
TG by lipoprotein lipase (LPL), mice were pretreated with an LPL inhibitor, Pluronic F127
2
4
(
Poloxamer 407), permitting measurement of the accumulation of plasma TG following olive
oil administration. Compound 24d and vehicle were administered 6 h before the oral olive oil
load, and plasma chylomicron TG concentrations were monitored for 4 h. As shown in Figure 6,
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compound 24d effectively and doseꢀdependently suppressed plasma TG elevation after olive oil
challenge. The TGꢀlowering effect of 24d was significant (p < 0.025) at doses of 10 and 30
mg/kg. A similar effect of reducing the rate of fat entrance into the circulation was observed in
10
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6
MGAT2 gene knockout mice. Accordingly, this result indicated that compound 24d suppressed
plasma TG elevation via MGAT2 inhibition in vivo.
1
8000
16000
14000
12000
#
1
0000
#
8
6
4
2
000
000
000
000
0
-
3
10
30
Vehicle
24d (mg/kg)
Figure 6. Effect of 24d on plasma TG elevation during oral fat tolerance test in C57BL/6J mice.
4d (3, 10, 30 mg/kg) was orally administered 6 h prior to oil challenge. Figure shows area under
2
the curve (AUC) of plasma chylomicron/TG (CM/TG) levels during 4 h after oil challenge. Data
are the mean + SD (n = 5). #, p ≤ 0.025 versus vehicle by William's test.
CONCLUSION
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6
We identified a novel series of Nꢀphenylꢀindolineꢀ5ꢀsulfonamide derivatives as potent,
selective, and orally bioavailable MGAT2 inhibitors. Starting from the sulfonamide derivative 1,
we explored an optimal arrangement of the hydrophobic group as a key pharmacophoric motif.
Conformational restriction by employing a bicyclic central core was useful to orient the
substituents for effective interaction with MGAT2. Consequently, the indoline derivative 10b
served as an attractive lead compound with potent MGAT2 inhibitory activity and high
selectivity over related acyltransferases. We also demonstrated that the carbonyl group at the
indoline 7ꢀposition was crucial for MGAT2 inhibition and that electron density on the carbonyl
group was correlated with potency. Subsequent optimization efforts were conducted to address
the poor DMPK issues of 10b. Structural modification to the presumed metabolically vulnerable
moieties significantly improved oral bioavailability in mice, resulting in the identification of 24d.
Compound 24d effectively suppressed post olive oilꢀloaded TG excursion after oral
administration. These results indicated that this series of MGAT2 inhibitors could have
therapeutic potential for the treatment of metabolic disorders. Further optimization efforts and
pharmacological effects of this series will be described in due course.
0
1
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0
EXPERIMENTAL SECTION
Chemistry general method. Melting points were determined in open capillary tubes on a
1
13
Büchi melting point apparatus B545 and are uncorrected. H NMR and C NMR spectra were
recorded on Bruker AVANCE III (300 MHz) or Bruker Advance III plus (400 MHz)
spectrometer. Chemical shifts are given in parts per million (ppm) downfield from
tetramethysilane (δ) as the internal standard in deuterated solvent and coupling constants (J) are
in Hertz (Hz). Data are reported as follows: chemical shift, integration, multiplicity (s = singlet, d
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=
doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, dd = doublet of doublets, td =
triplet of doublets, bs = broad signal), and coupling constants. All solvents and reagents were
obtained from commercial suppliers and used without further purification. Thinꢀlayer
chromatography (TLC) was performed on Merck silica gel plates 60F . Silica gel column
10
11
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13
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24
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2
54
chromatography was performed on PurifꢀPack (SI or NH, SHOKO SCIENTIFIC). LC–MS
analysis was performed on a Shimadzu Liquid Chromatography–Mass Spectrometer System,
operating in APCI (+ or −) or ESI (+ or −) ionization mode. Analytes were eluted using a linear
gradient of 0.05% TFA containing water/acetonitrile or 5 mM ammonium acetate containing
water/acetonitrile mobile phase, and detected at 220 nm. The purities of compounds submitted
for biological evaluation were > 95% as determined by elemental analyses within ±0.4% of the
calculated values. The purities of intermediates were determined by LC–MS analysis (detection
at 220 nm). Yields are not optimized.
2
5
Benzyl 5ꢀbromoꢀ7ꢀnitroꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ1ꢀcarboxylate (3). To a mixture of 5ꢀ
bromoꢀ7ꢀnitroꢀ2,3ꢀdihydroꢀ1Hꢀindole (61.7 g, 254 mmol, commercially available (purchased
from Small Molecules, Inc.)) and THF (1.6 L) was added sodium hydride (60% oil dispersion,
15.2 g, 380 mmol) at 0 °C. The mixture was stirred at rt under N atmosphere for 3 h. CbzCl
2
(47.1 mL, 330 mmol) was added to the mixture at 0 °C, and the resulting mixture was stirred at rt
for 3 days. The mixture was quenched with crushed ice at 0 °C and THF was removed under
reduced pressure. To the residue was added a mixed solvent of AcOEt/hexane/water (1:3:3 v/v,
700 mL) under stirring. The precipitated solid was collected by filtration to give the title
1
compound as a yellow solid (80.4 g, 213 mmol, 84%). H NMR (300 MHz, DMSOꢀd ) δ 3.20
6
(2H, t, J = 8.3 Hz), 4.19 (2H, t, J = 8.3 Hz), 5.16 (2H, s), 7.30ꢀ7.44 (5H, m), 7.77ꢀ7.82 (1H, m),
7.83ꢀ7.89 (1H, m). Purity 95.8% (LC–MS).
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2
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5
6
Benzyl 5ꢀ(benzylsulfanyl)ꢀ7ꢀnitroꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ1ꢀcarboxylate (4). A mixture of
compound 3 (20.9 g, 55.4 mmol), phenylmethanethiol (7.14 mL, 61.0 mmol), Pd (dba) (5.07 g,
2
3
5
.54 mmol), Xantphos (6.41 g, 11.1 mmmol), and DIEA (29.0 mL, 166 mmol) in toluene (250
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
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7
8
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0
1
2
3
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9
0
mL) was refluxed under N atmosphere for 5 h. After cooling to rt, AcOEt (250 mL) and H O
2
2
(250 mL) were added to the mixture, and insoluble materials were removed by filtration. The
filtrate was diluted with AcOEt (250 mL) and successively washed water, 0.2 M hydrochloric
acid, aqueous NaHCO solution, and brine. The organic layer was dried over Na SO and
3
2
4
i
concentrated under reduced pressure. The residue was crystallized from AcOEt– Pr O to give the
2
+
title compound as a yellow solid (17.0 g, 40.4 mmol, 73%). MS (ESI/APCI) m/z 421.3 [M + H] .
1
H NMR (300 MHz, DMSOꢀd ) δ 3.14 (2H, t, J = 8.3 Hz), 4.16 (2H, t, J = 8.3 Hz), 4.28 (2H, s),
6
5.14 (2H, s), 7.20ꢀ7.42 (10H, m), 7.55 (2H, dd, J = 8.2, 1.8 Hz). Purity 98.1% (LC–MS).
Benzyl 5ꢀ(chlorosulfonyl)ꢀ7ꢀnitroꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ1ꢀcarboxylate (5). To a mixture
of compound 4 (130 g, 309 mmol), acetic acid (960 mL), and water (320 mL) was added
portionwise Nꢀchlorosuccinimide (165 g, 1.24 mol) at 0 °C. The mixture was stirred at rt for 4 h.
i
Precipitated solid was collected by filtration, successively washed with water and Pr O, and
2
dried to give the title compound as a pale yellow solid (119 g, 300 mmol, 97%). This compound
1
was used for the next step without further purification. H NMR (300 MHz, DMSOꢀd ) δ 3.20
6
(
2H, t, J = 8.3 Hz), 4.20 (2H, t, J = 8.3 Hz), 5.16 (2H, s), 7.33–7.42 (5H, m), 7.73 (1H, d, J = 1.4
Hz), 7.77 (1H, d, J = 1.4 Hz). Purity 92.7% (LC–MS).
Benzyl
5ꢀ[(4ꢀmethoxyphenyl)sulfamoyl]ꢀ7ꢀnitroꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ1ꢀcarboxylate
(
6a). A mixture of compound 5 (2.82 g, 7.11 mmol) and 4ꢀmethoxyaniline (1.05 g, 8.53 mmol)
in DMA (30 mL) was stirred at rt overnight. The mixture was quenched with iced water and
extracted with AcOEt. The organic layer was washed with water and brine, dried over MgSO4,
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and concentrated under reduced pressure. The residue was purified by column chromatography
(hexane/AcOEt = 90/10 to 50/50) to give the title compound as a yellow amorphous solid (3.31
−
1
g, 6.85 mmol, 96%). MS (ESI/APCI) m/z 482.1 [M − H] . H NMR (300 MHz, DMSOꢀd ) δ
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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36
37
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3
.22 (2H, t, J = 8.1 Hz), 3.67 (3H, s), 4.22 (2H, t, J = 8.5 Hz), 5.17 (2H, s), 6.78–6.88 (2H, m),
6.95–7.06 (2H, m), 7.30–7.46 (5H, m), 7.77 (1H, d, J = 1.1 Hz), 7.91 (1H, d, J = 1.5 Hz), 10.04
1H, s). Purity 99.8% (LC–MS).
Benzyl 5ꢀ[(2,4ꢀdifluorophenyl)sulfamoyl]ꢀ7ꢀnitroꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ1ꢀcarboxylate
(6b). Compound 5 (77.5 g, 195 mmol) was added portionwise to a solution of 2,4ꢀdifluoroaniline
23.9 mL, 234 mmol) in DMA (750 mL) at 0 °C. The mixture was stirred at rt overnight. The
(
(
mixture was quenched with water at 0 °C and extracted with AcOEt. The organic layer was
washed with water (3 times) and brine, dried over Na SO and concentrated under reduced
2
4,
i
pressure. The residue was crystallized from AcOEt– Pr O to give the title compound as a pale
2
−
1
yellow solid (82.0 g, 167 mmol, 86%). MS (ESI/APCI) m/z 488.1 [M − H] . H NMR (300 MHz,
DMSOꢀd ) δ 3.24 (2H, t, J = 8.3 Hz), 4.25 (2H, t, J = 8.4 Hz), 5.19 (2H, s), 7.00–7.12 (1H, m),
6
7
.18–7.48 (7H, m), 7.81 (1H, d, J = 1.7 Hz), 7.93 (1H, d, J = 1.8 Hz), 10.30 (1H, s). Purity
94.9% (LC–MS).
Benzyl
7ꢀaminoꢀ5ꢀ[(4ꢀmethoxyphenyl)sulfamoyl]ꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ1ꢀcarboxylate
(7a). A mixture of compound 6a (2.45 g, 5.07 mmol), iron powder (1.42 g, 25.3 mmol), and
calcium chloride (0.562 g, 5.07 mmol) in a mixed solvent of EtOH (20 mL) and water (5 mL)
was refluxed overnight. The insoluble material was removed by filtration, and the filtrate was
concentrated under reduced pressure. The residue was extracted with AcOEt. The organic layer
was separated, washed with brine, dried over MgSO , and concentrated under reduced pressure.
4
i
To the residue was added AcOEt and Pr O. The precipitated solid was collected to give the title
2
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