Potent nonsteroidal progesterone receptor agonists: Synthesis and SAR study of 6-aryl benzoxazines

Article abstract of DOI:10.1016/S0960-894X(02)00025-2

Novel 6-aryl benzoxazines were prepared and examined as progesterone receptor (PR) modulators. In contrast to the structurally related 6-aryl dihydroquinoline PR antagonists, the 6-aryl benzoxazines were potent PR agonists. Compounds 4e, 5b, and 6a with the 2,4,4-trimethyl-1,4-dihydro-2H-benzo[d][1,3]oxazine core were the most potent PR agonists in the series with sub-nanomolar activities (EC₅₀ 0.20-0.35 nM). Compound 6a was more potent than progesterone (P4) in the in vivo decidualization assay in an ovariectomized female rat model by subcutaneous administration with an ED₅₀ of 1.5 mg/kg (vs 5.62 mg/kg for P4).

Full text of DOI:10.1016/S0960-894X(02)00025-2

Bioorganic& Medicinal Chemistry Letters 12 (2002) 787–790
Potent Nonsteroidal Progesterone Receptor Agonists:
Synthesis and SAR Study of 6-Aryl Benzoxazinesy
Puwen Zhang,a,* Eugene A. Terefenko,a Andrew Fensome,a Zhiming Zhang,b Yuan Zhu,b
Jeffrey Cohen,b Richard Winneker,b Jay Wrobela and John Yardleya
aMedicinal Chemistry I, Chemical Sciences, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
bWomen’s Health Research Institute, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
Received 1 October 2001; accepted 21 December 2001
Abstract—Novel 6-aryl benzoxazines were prepared and examined as progesterone receptor (PR) modulators. In contrast to the
structurally related 6-aryl dihydroquinoline PR antagonists, the 6-aryl benzoxazines were potent PR agonists. Compounds 4e, 5b,
and 6a with the 2,4,4-trimethyl-1,4-dihydro-2H-benzo[d][1,3]oxazine core were the most potent PR agonists in the series with sub-
nanomolar activities (EC50 0.20–0.35 nM). Compound 6a was more potent than progesterone (P4) in the in vivo decidualization
assay in an ovariectomized female rat model by subcutaneous administration with an ED50 of 1.5 mg/kg (vs 5.62 mg/kg for P4).
# 2002 Elsevier Science Ltd. All rights reserved.
The progesterone receptor (PR) is one of the intra-
cellular gene regulators known as ‘ligand dependent
transcription factors’.1 PR agonists play an important
role in female reproduction and have been used exten-
sively in female contraception and hormone replace-
ment therapy in combination with estrogens. A selective
PR antagonist is potentially useful for the treatment of
hormone dependent cancers,2 nonmalignant chronic
conditions such as fibroids,3 and endometriosis.4 How-
ever, many of the steroidal PR modulators (e.g., mif-
epristone) have shown undesirable cross-reactivities
with other steroid receptors such as the glucocorticoid
and androgen receptors promoting a need for newer,
more selective PR modulators.
contrast to the 6-aryl dihydroquinolines, most of our
series were potent PR agonists. The synthesis and SAR
of novel 6-aryl benzoxazines will be discussed in this
report.
Synthesis
A general synthesis of 6-aryl benzoxazines is illustrated
in Scheme 1. Treatment of compounds 1a,b with an
appropriate Grignard reagent formed o-amino carbinols
(2) in good yield. Ring closure of 2 with an aldehyde or
a ketone gave 6-bromo benzoxazines (3). A Suzuki
cross-coupling of compound 3 with an arylboronicacid
generated 6-aryl benzoxazines (4a–6e). In the case when
an arylboronicacid was not available, 3 was converted
to the arylborate following a literature procedure6 and
cross-coupled in situ with a suitable haloarene to gen-
erate compounds 4a–6e.7 Alkylation or acylation of 6c
at position 1 afforded 1-substituted 6-aryl benzoxazines
(6f–h).
Recently, several classes of the nonsteroidal PR mod-
ulators have been reported.5 Among a number of inter-
esting series, 6-aryl dihydroquinolines, such as 3-fluoro-
5-(2,2,4-trimethyl-1,2-dihydro-quinolin-6-yl)-benzonitrile,
have shown good PR antagonist activity.5e We prepared
and evaluated a number of novel 6-aryl-1,4-dihydro-
2H-benzo[d][1,3]oxazines (4a–6h) as potential 6-aryl
dihyroquinoline scaffold replacements. However, in
Results and Discussion
yPresented in preliminary form at Gordon Research Conference on
Medicinal Chemistry, Colby-Sawyer College, New London, NH,
USA, August 5–10, 2001.
*Corresponding author. Tel.: +1-484-865-3856; fax: +1-484-865-
9398; e-mail: zhangp@war.wyeth.com
The novel 6-aryl-1,4-dihydro-2H-benzo[d][1,3]oxazines
were evaluated in the alkaline phosphatase8 and PR
competition binding assays using the human T47D
breast carcinoma cell line.8 The results of their PR
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00025-2

788
P. Zhang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 787–790
agonist, antagonist activities and relative binding affinities
are summarized in Table 1. 6-Aryl benzoxazines with
the 2,4,4-trimethyl-1,4-dihydro-2H-benzo[d][1,3]oxazine
core were the most potent PR agonists (Table 1). For
example, compounds 4e, 5b, and 6a elicited PR agonist
activities in the alkaline phosphatase assay in the sub-
nanomole range (EC50 0.20–0.35 nM) and were more
potent PR agonists than progesterone (EC50 0.92 nM)
and comparable to MPA (EC50 0.12 nM, Table 1).
than 20-fold. Furthermore, compounds 5c and 6b with a
spiro-cyclohexyl group at position 4 were 10 times less
potent than their 4,4-dimethyl congeners 5b and 6a.
At position 2, substitution of methyl group for tri-
fluoromethyl moiety marginally reduced the PR agonist
activities in the alkaline phosphatase assay (Table 1).
For example, 2-trifluromethylated compounds 4g and
6c were less active than their corresponding 2-methyl
substituted analogues 4e and 6a. Introduction of an
additional 2-methyl group to compounds 4b, 4e, and 6a
led to the dimethylated compounds 4d, 4h, and 6d (alk.
phos. EC50 9.2–32.0 nM) which were much less active
than their mono-methylated congeners. More surpris-
ingly, when the size of the 2-substituent was increased
by replacing the methyl group with an isopropyl, t-
butyl, thien-2-yl, phenyl, and ethoxyl carbonyl moieties,
the resulting compounds 4i–m switched from PR ago-
nists to PR antagonists (Table 1).
The nature and position of the substituents on the 6-aryl
moieties in the compounds in Table 1 had a significant
effect on the potency of the compounds. For instance,
the para-fluoro substituent on 4n (EC50 23 nM and
binding IC50 166.0, respectively) significantly reduced
the PR agonist activity and binding affinity compared to
the meta-fluoro regio-isomer 4e (EC50 0.35 nM, binding
IC50 7.3 nM). The extra para-fluoro substitutent in 4o
reduced T47D alkaline phosphatase activity and bind-
ing affinity more than 10-fold compared to 4b. Further-
more, the additional meta-nitrile substituent in 4e
compared to compounds 4a caused about an order of
magnitude increase in the T47D alkaline phosphatase
potency.
The NH moiety at position 1 was necessary for activity.
Replacing the NH hydrogen atom of 6c with an acetyl
(6f) or a carbomethoxy group (6g) led to loss of activity
at the highest concentration tested. Compound 6h
showed similar functional activity and binding affinity
compared to its parent compound (6c). Presumably, 6h
was not stable and converted to compound 6c under the
conditions of assays used in our studies.
Replacing one of the 4-methyl groups of 4e with a
phenyl moiety reduced activity of the resulting com-
pound 4f in the T47D alkaline phosphatase assay more
Scheme 1. Reagents and conditions: (a) R2MgBr, ether, 0 ꢀC; (b) R3R4CO, p-TsOH, toluene, rt; (c) ArB(OH)2, Pd(Ph3P)4, Na2CO3, DME/H2O,
85 ꢀC or bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, DMF, 80 ꢀC; ArBr, Pd(dppf)Cl2, 2 N Na2CO3; (d) R5Cl, NaH, DMF, rt.

P. Zhang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 787–790
789
Table 1. Alkaline phosphatase activities and binding affinities of P4, MPA, and 6-aryl benzoxazines
Compd
R
R1
R2
R3
R4
R5
X
Alk. phos.
EC50 (nM)a
Alk. phos.
IC50 (nM)a
PR binding
IC50 (nM)a
P4
MPA
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
5a
5b
5c
6a
6b
6c
6d
6e
6f
0.92
0.12
2.60
2.40
2.9
32.0
0.35
8.20
3.5
10.8
23.0
9.0
41.3
10.0
7.3
3-F
3-Cl
3-Cl
3-Cl
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
CF3
Me
Me
Me
CF3
Me
H
H
H
Me
H
H
H
Me
H
H
H
3-CN, 5-F
3-CN, 5-F
3-CN, 5-F
3-CN, 5-F
3-CN, 5-F
3-CN, 5-F
3-CN, 5-F
3-CN, 5-F
3-CN, 5-F
3-CN, 4-F
3-Cl, 4-F
H
24.7
7.1
1.7
10.9
42.0
NDb
3042
1682
2060
1793
166.0
212.5
27.0
8.1
CH(CH3)2
C(CH3)3
Thien-2-yl
Ph
COOEt
Me
Me
>10,000
>10,000
>10,000
>10,000
>10,000
23.0
47.0
126.6
51.2
92.6
158.8
H
H
H
H
32.0
1.05
0.20
2.40
0.35
3.50
0.8
Me
Me
Me
Cyclohexyl
Me
Cyclohexyl
Me
Me
2.9
Me
Me
Me
CF3
Me
H
H
H
Me
H
H
H
H
H
H
H
S
S
S
S
O
S
S
S
11.5
12.0
4.9
ND
113.4
2087
ND
7.9
Me
Me
Me
Me
Me
Me
9.2
4.55
>10,000
>10,000
0.9
Me
Me
Me
Me
Me
CF3
CF3
CF3
COMe
COOMe
CH2OCOtBu
6g
6h
7c
H
H
>10,000
103.0
301.0
aExperimental values represent the average of at least duplicate determinations.8 The standard deviations for these assays were typicallyꢂ20% of
mean or less.
bND, not determined.
cCompound 7 was prepared by an acidic hydrolysis of compound 4b.
The hemiaminal ring of our compounds could con-
ceivably hydrolyze to an amino carbinol under appro-
priate conditions. This does not appear to be the case
under the conditions of assays used in our studies for
amino carbinol 7 was a weak PR antagonist when
compared to the corresponding potent hemiaminal-
containing agonists 4b–d.
demonstrated good binding activities in the PR com-
petition binding assay. Compound 6a was also active
SC in the in vivo rat decidualization assay.
The structure–activity relationships of 6-aryl benzox-
azines were examined at positions 1, 2, 4, and 6. While
the nature of the substituent at positions 1, 4, and 6
played an important role in the potency and binding
affinities of these compounds as PR agonists, the size of
substituent at position 2 dictated the functional profile
of the 6-aryl benzoxazines. The large groups at position
2 such as isopropyl or phenyl group switched the
compounds from PR agonists to PR antagonists. This
information was useful for the design of new PR
The in vivo decidualization activities of 6a was eval-
uated and compared to P4 and MPA in an ovari-
ectomized mature female Sprague–Dawley rat model
(ꢁ60-day old and 230 g) by subcutaneous injection
(Table 2).9 As illustrated from Table 2, 6a showed
potent in vivo decidualization activity and was more
potent than P4.
Table 2. Rat decidualization activities of P4, MPA, and 6a
In summary, novel 6-aryl benzoxazines as PR mod-
ulators were synthesized and evaluated in the in vitro
and in vivo PR assays. The most potent compounds 4e,
5b, and 6a in the series showed PR agonist activities
comparable to MPA in the in vitro alkaline phosphatase
assay and represented some of the most potent non-
steroidal PR agonists to date. These compounds also
Compd
P4
MPA
6a
Decidualization ED50 (mg/kg)a
5.62
0.40
1.50
aExperimental values represent the average of at least duplicate deter-
minations.9 The standard deviation for the decidualzation assay was
typically ꢂ15% of mean or less.

790
P. Zhang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 787–790
agonists or antagonists using the 6-aryl benzoxazine
scaffold.
(ESI) m/z 295 [MꢃH]ꢃ. Anal. calcd for C18H17FN2O: C,
72.96; H, 5.78; N, 9.45. Found: C, 72.63; H, 5.82; N, 9.27. 3-
[4,4-Dimethyl-2-(trifluoromethyl)-1,4-dihydro-2H-3,1-benzoxazin-
6-yl]-5-fluorobenzonitrile (4g). A white solid: mp 102–103 ꢀC;
1H NMR (DMSO-d6) d 8.07 (s, 1H), 7.91 (d, 1H, J=10.7 Hz),
7.7 (d, 1H, J=10.7 Hz) 7.62 (d, 1H, J=1.98 Hz), 7.53 (dd, 1H,
J=8.33, 1.98 Hz), 7.05 (s, 1H), 6.87 (d, 1H, J=8.33 Hz), 5.39–
5.37 (m, 1H), 1.61 (s, 3H), 1.59 (s, 3H); MS (ES) m/z 349
[MꢃH]ꢃ. Anal. calcd for C18H14F4N2O: C, 61.72; H, 4.03; N,
8.00. Found: C, 61.66; H, 3.94; N, 7.79. 5-(2,4,4-Trimethyl-1,4-
dihydro-2H-3,1-benzoxazin-6-yl)thiophene-2-carbonitrile (5a).
An off-white solid: mp 172–173 ꢀC; 1H NMR (DMSO-d3) d
7.88 (d, 1H, J=4.0 Hz), 7.47 (d, 1H, J=4.0 Hz), 7.43 (d, 1H,
J=2.0 Hz), 7.32 (dd, 1H, J=8.36, 2.4 Hz), 6.77 (s, 1H), 6.60
(d, 1H, J=8.4 Hz), 4.83 (m, 1H), 1.51 (s, 3H), 1.48 (s, 3H),
1.28 (d, 3H, J=5.6 Hz); MS (ESI) m/z 283 [MꢃH]ꢃ. Anal.
Acknowledgement
We are grateful to the assistance of Department of
Analytical Chemistry of Wyeth-Ayerst Research for
elemental analyses, NMR and mass spectroscopy data.
References and Notes
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Found: C, 66.82; H, 5.61; N, 9.77. 4-Methyl-5-(2,4,4-trimethyl-
1,4-dihydro-2H-3,1-benzoxazin-6-yl)thiophene-2-carbonitrile (5b).
A yellowish solid: mp 145–146 ꢀC; 1H NMR (DMSO-d6) d
7.79 (s, 1H), 7.18 (d, 1H, J=2.0 Hz), 7.13 (dd, 1H, J=8.4,
2.0 Hz), 6.68 (s, 1H), 6.54 (d, 1H, J=8.3 Hz), 4.83 (m, 1H),
2.26 (s, 3H), 1.49 (s, 3H), 1.46 (s, 3H), 1.28 (d, 3H, J=5.5 Hz);
MS (ESI) m/z 299 [M+H]+. Anal. calcd for C17H18N2OS: C,
68.43; H, 6.08; N, 9.39. Found: C, 68.29; H, 6.11; N, 9.31. 4-
(2,4,4-Trimethyl-1,4-dihydro-2H-3,1-benzoxazin-6-yl)thiophene-
2-carbonitrile (6a). An off-white solid: mp 175–176 ꢀC; 1H
NMR (DMSO-d6) d 8.39 (d, 1H, J=1.5 Hz), 8.13 (d, 1H,
J=1.5 Hz), 7.47 (d, 1H, J=1.9 Hz), 7.36 (dd, 1H, J=8.4,
1.9 Hz), 6.59 (d, 1H, J=8.4 Hz), 6.41 (s, 1H), 4.78 (m, 1H),
1.51 (s, 3H), 1.47 (s, 3H), 1.28 (d, 3H, J=5.4 Hz); MS (ESI)
+
.
m/z 285 [M+H] . Anal. calcd for C16H16N2OS 0.2H2O: C,
66.73; H, 5.74; N, 9.73. Found: C, 66.82; H, 5.54; N, 9.87. 4-
[4,4-Dimethyl-2-(trifluoromethyl)-1,4-dihydro-2H-3,1-benzoxazin-
6-yl]thiophene-2-carbonitrile (6c). A yellow solid: 1H NMR
(DMSO-d6) d 8.43 (s, 1H), 8.19 (s, 1H), 7.54 (d, 1H,
J=1.98 Hz), 7.46 (dd, 1H, J=8.33, 1.98 Hz), 6.90 (s, 1H), 6.83
(d, 1H, J=8.33 Hz), 5.35 (d, 1H, J=3.57 Hz), 1.59 (s, 3H),
1.57 (s, 3H); MS (ES) m/z 337 [MꢃH]ꢃ.
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Fluoro-5- (2,4,4-trimethyl-1,4-dihydro-2H-3,1-benzoxazin-6-
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yl)benzonitrile (4e). A white solid: mp 163–164 ꢀC; H NMR
1
(DMSO-d6) d 8.02 (t, 1H, J=1.5 Hz), 7.87 (dt, 1H, J=10.6,
2.2 Hz), 7.65 (m, 1H), 7.55 (d, 1H, J=2.2 Hz), 7.44 (dd, 1H,
J=8.4, 2.2 Hz), 6.63 (d, 1H, J=8.4 Hz), 6.58 (s, 1H), 4.82 (m,
1H), 1.52 (s, 3H), 1.50 (s, 3H), 1.28 (d, 3H, J=5.1 Hz); MS
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