Benzoxazinones as PPARγ agonists. Part 1: SAR of three aromatic regions

Article abstract of DOI:10.1016/S0960-894X(03)00401-3

A series of benzoxazinones was synthesized as PPARγ agonists. The compounds were obtained in seven steps, and SAR was developed by variations to the core shown below. The compounds were tested as functional agonists in the induction of the aP2 gene in preadipocytes, and the most potent compound in the series has an EC₅₀=0.51 μM. The potency was further confirmed through a PPAR-Gal4 construct. Efficacy has been demonstrated in the db/db mouse model of hyperglycemia.

Full text of DOI:10.1016/S0960-894X(03)00401-3

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2359–2362
Benzoxazinones as PPARꢀ Agonists.
Part 1: SAR of Three Aromatic Regions
Philip J. Rybczynski,* Roxanne E. Zeck, Donald W. Combs,y Ignatius Turchi,
Thomas P. Burris,{ Jun Z. Xu, Maria Yang and Keith T. Demarest
Johnson and Johnson Pharmaceutical Research and Development, LLC, Raritan, NJ 08869, USA
Received 28 February 2003; revised 15 April 2003; accepted 16 April 2003
Abstract—A series of benzoxazinones was synthesized as PPARg agonists. The compounds were obtained in seven steps, and SAR
was developed by variations to the core shown below. The compounds were tested as functional agonists in the induction of the aP2
gene in preadipocytes, and the most potent compound in the series has an EC50=0.51 mM. The potency was further confirmed
through a PPAR-Gal4 construct. Efficacy has been demonstrated in the db/db mouse model of hyperglycemia.
# 2003 Elsevier Science Ltd. All rights reserved.
PPARg is a member of the peroxisome proliferator-
activated receptor family and has been the subject of
extensive research for mechanistic importance in glucose
and lipid homeostasis.1 The receptor is widely dis-
tributed in the spleen, the colon, adipose tissue and
macrophages, and found to a lesser extent in the liver,
the pancreas and skeletal muscle.1a Target genes that
are upregulated or downregulated have been identified
from white and brown adipose tissue, skeletal muscle
and the liver.1c The details of how receptor activation
leads to glucose homeostasis are not fully understood.
Studies suggest that adipogenesis provides increased
lipid metabolism and free fatty acid uptake in adipose
tissue, leading to increased insulin sensitivity and glucose
metabolism in muscle and liver.1b,d,g Recent evidence
supporting this mechanism is that a PPARg agonist
induces glycerol kinase gene expression in adipocytes, thus
promoting triglyceride formation in that tissue, and redu-
cing circulating free fatty acids.1h PPARg is also a target
protein for a growing number of agonists useful in the
treatment of Type 2 diabetes.2 These include Rosiglita-
zone1 and Pioglitazone1, both marketed for this utility.
seen in the literature. Select compounds were screened
at 1 mM for aP2 gene induction in pooled human pre-
adipocytes. The aP2 gene is essential for the maturation
of preadipocytes adipocytes, and offered a large window
of activation for primary screening and EC50 deter-
minations.3 The series of racemic benzoxazinones4 in
Table 1 emerged from this assay. Results indicated that
the preferred substitution pattern on the aryl ether is
1,2-, while PPARg agonists in the literature typically
show a 1,4- substitution pattern (Rosiglitazone1,5 Pio-
glitazone1,6 Farglitazar1,7 JTT-5018 and others). This
difference provided impetus to develop SAR in the ser-
ies with respect to the position and spacing of the acidic
moiety, amide substitution, and substitution on the
aromatic portion of the benzoxazinone. Also, in the
early stages of the program, after identifcation of the
Table 1. PPARg aP2 Induction screening of library compounds9
A PPARg discovery program was initiated with a search
of the corporate compound library. In the search para-
digm, a phenyl substituted with carboxylic acid was
used in place of the TZD-substituted phenyl typically
Compd
COOH pos(n)
Functional assaya (Fold induction)
1
2
3
4
5
2(1)
3(1)
3(2)
4(1)
4(2)
6.2
5.5
5.6
3.2
3.6
*Corresponding author. Tel. +1-908-704-4511; fax: +1-908-203-
8109; e-mail: prybczyn@prdus.jnj.com
yCurrent address: Bristol-Myers Squibb Co., Princeton, NJ 08543,
USA.
aValues are the mean of two experiments for activation of aP2 gene in
pooled human preadipocytes.
{Current address: Eli Lilly and Co., Indianapolis, IN 46285, USA.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00401-3

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P. J. Rybczynski et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2359–2362
Scheme 1. (a) K2CO3, DMF, 0 ꢁC to rt overnight, 70%. (b) (i) H2, Pd/C, EtOH. (ii) TBSCl, imidazole, DMF, 60% for 2 steps. (c) (i) NaH, benzyl or
phenethyl halide, DMF. (ii) CH3SO3H, MeOH, H2O, 60–80% for 2 steps. (d) Bu3P, ADDP, PhH, 60–95%. (e) NaOH, MeOH, H2O, 80–95%.
benzoxazinone series, the TZD-substituted naphthyl
compound MCC-555 was licensed from Mitsubishi
Chemical as an early development candidate. Clinical
data was not yet available on MCC-555, so there was no
data indicating incorporation or avoidance of a parti-
cular structural feature in the lead compound. In the
interest of maintaining a diverse set of clinical com-
pounds, the benzoxazinone series was explored as a
backup series.
Functional potency was determined in pooled human
preadipocytes by measurement of aP2 gene induction.
Induction was quantified with a branched DNA tech-
nique previously described.3 A PPARg-Gal4Luciferase
cotransfection assay11 confirmed that the compounds
exert their agonist activity through PPARg. The com-
pounds showed weak PPARa activity in a screening assay
(data not shown) and were not tested for PPARd activity.
The data for a representative number of compounds tes-
ted in this manner are shown in Table 2. Compounds 1, 2,
10–13 explored the position and spacing of the carboxylic
acid with both 3- or 4-chlorobenzyl amides. 1,2- disposal
on a phenylacetic acid provided the best results. The
amide substituent was varied as the 3,4-dichlorobenzyl-,
3,4-dichlorophenethyl- and 3,4-dichlorophenylamides
(14, 15, 16). Extending the linkage by one atom as in 15
provided no advantage. Direct linkage as in 16 improved
the binding value but decreased the potency in the func-
tional assay. In compound 17 the heterocycle was con-
verted to the corresponding benzoxazine and suffered
tremendously in both assays (compare to 14). Turning to
fluorinated derivatives (18–20), increased potency was
observed relative to the chloro and dichloro analogues
while trifluoromethyl analogue had the highest potency in
both the binding and functional assays. Emphasis was
next placed on electron rich or neutral benzyls (21–24).
Binding and functional data was modest in all cases.
The compounds were synthesized as outlined in
Schemes 1 and 2. 2-Nitrophenol was alkylated with
a-bromo-g-butyrolactone to provide 6, followed by
nitro reduction, cyclization to the benzoxazinone and
protection of the primary alcohol (7). Alkylation of the
amide with either a benzyl or phenethyl halide and
deprotection provided Mitsunobu substrate 8. Forma-
tion of the phenyl ether (9) and saponification provided
target compounds 10–15, 17–30. The route was altered
to obtain N-aryl amides, shown in Scheme 2. Palladium
mediated coupling of the aryl bromide and aniline pro-
vided 31.10 Deprotection and cyclization to 32 yielded a
product that was elaborated to target compound 16 in a
manner identical to the conversion of 8–10.
Finally the role of substitution on the benzoxazinone
aromatic was explored, and data for a representative set
of compounds is provided (25–30). The 4-chlorobenzyl
amide and 2-phenylacetic acid were held constant for
this portion of the study. Overall, incorporation of sub-
stituents at the 6- or 7-positions did not improve the
potency of the target compounds. Additional substitu-
tion patterns had the same effect (data not shown).
In vivo efficacy with the series was required to demon-
strate the potential for further exploration, although
none of these analogues had the potency needed for
preclinical evaluation. Compound 11 was chosen for
this study early in the SAR work, and a 27% decrease in
plasma glucose was observed after 5 days of oral dosing
at 30 mg/kg in db/db mice. This compound also had
satisfactory metabolic stability in both human liver
microsomes and hepatic S9 fraction (t1/2>50 min in
each), and good oral bioavailability in rats (30 mg/kg,
AUC0ꢀ24 h=308 mM-h, t1/2=20 h). A more robust
effect would be anticipated from compound 20, but the
Scheme 2. (a) Pd(dba)2, BINAP, PhCH3, 66%. (b) (i) BBr3, xylene,
CH2Cl2. (ii) NaH, a-bromo-g-butyrolactone, DMF, 55% for 2 steps.

P. J. Rybczynski et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2359–2362
PPARg aP2 Gene functional and PPARg-Gal4 luciferase data
2361
9
Table 2.
Compd
R1
R2
COOH pos(n)
aP2a(mM)
PPARg-Gal4b (mM)
1
2
3-ClBn
3-ClBn
4-ClBn
4-ClBn
4-ClBn
4-ClBn
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
7-F
2(1)
3(1)
2(0)
2(1)
2(2)
3(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2(1)
2.4
>10
>10
2.1
>10
>10
2.6
>10
5.9
>10
3.2
1.3
0.51
4.6
5.0
5.4
4.4
>10
4.2
6.2
>10
6.0
>10
0.12
1.9
>5
>5
1.3
>5
>5
0.96
>5
0.57
>5
1.1
0.59
0.50
>5
2.0
0.52
1.1
>5
2.1
1.8
>5
1.8
>5
0.19
10
11
12
13
14
15
16
17c
18
19
20
21
22
23
24
25
26
27
28
29
30
3,4-Cl2Bn
3,4-Cl2Ph(CH2)2
3,4-Cl2Ph
3,4-Cl2Bn
4-FBn
3,4-F2Bn
4-CF3Bn
Bn
4-CH3Bn
4-CH3OBn
3,4-OCH2OBn
4-ClBn
4-ClBn
4-ClBn
4-ClBn
4-ClBn
4-ClBn
7-CH3
7-CH3C(O)
6-COOH
6-CH3O
6,7-CHCHCHCH
Rosiglitazone
aValues are the mean of two experiments for activation of aP2 gene in pooled human preadipocytes.
bValues are the mean of two experiments with a PPARg-Gal4Luc construct.
cBenzoxazine in place of benzoxazinone.
4. (a) Frechette, R.; Wells, M. U.S. Patent 5, 854,242, 1998;
Chem. Abstr 1997, 127, 50652. (b) Burris, T. P.; Rybczynski,
P. J. WO01/87860, 2001; Chem. Abstr. 2001, 135, 371750. (c)
Burris, T. P.; Demarest, K. T.; Combs, D. W.; Rybczynski,
P. J.; Turchi, I. J. WO01/87861; Chem. Abstr. 2001, 135,
371751. (d) Burris, T. P.; Combs, D. W.; Rybczynski, P. J.
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purpose of this study was to prove that the series can
potentially provide a backup. These results have
encouraged the continued synthesis and evaluation of
compounds in the chemical series with the goal of
increased in vitro potency, while maintaining acceptable
parameters of pharmaceutical suitability.
5. Wagstaff, A. J.; Goa, K. L. Drugs 2002, 62, 1805.
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11. Transfection assay materials and method for PPARg:

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P. J. Rybczynski et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2359–2362
HEK293 cells were grown in DMEM/F-12 Media supple-
overnight. Cells were challenged by test compounds and incu-
bated for 24 h at 37 ꢁC in 5%CO2 incubator. Luciferase activity
was assayed using the Steady-Glo Luciferase Assay Kit from
Promega. DMRIE-C Reagent was purchased from GIBCO Cat.
No.10459-014. OPTI-MEM I Reduced Serum Medium was
purchased from GIBCO Cat. No. 31985. Steady-Glo Luciferase
Assay Kit were from Promega Part# E254B.
mented with 10%FBS and glutamine (GIBCOBRL). The cells
were co-transfected with DNA for PPARg-Gal4 receptor and
Gal4-Luciferase Reporter using the DMRIE-C Reagent. On the
following day, the DNA-containing medium were replaced with
5% Charcoal treated FBS growth medium. After 6 h, cells were
seeded in 96-well plate and incubated at 37ꢁC in CO2 incubator