The hit-to-lead optimization of 1,2,3,4,4a,9a-hexahydro-1H-xanthenes as glucocorticoid receptor antagonists

Article abstract of DOI:10.1016/j.cclet.2014.03.017

The structure-activity relationship (SAR) study of a 1,2,3,4,4a,9a- hexahydro-1H-xanthene series of selective, human glucocorticoid receptor α (hGRα) antagonists is reported. Compounds were screened using hydroxyapatite-based GR binding and MMTV-Luc co-transfection reporter gene assays. Four different regions of the scaffold were modified to assess the effects on hGRα antagonism and related potency. Compound 8d exhibits an 8-fold better bioactivity than the original hit 1a, as well as an improved chemical stability, which make it a promising lead for the subsequent optimization.

Full text of DOI:10.1016/j.cclet.2014.03.017

Chinese Chemical Letters 25 (2014) 693–698
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
The hit-to-lead optimization of 1,2,3,4,4a,9a-hexahydro-1H-
xanthenes as glucocorticoid receptor antagonists
Yan-Hui Zhu a, Meng Zhang a, Qun-Yi Li a, Qing Liu a,b, Jie Zhang a, Yun-Yun Yuan a,
Fa-Jun Nan a,b, Ming-Wei Wang a,b,
*
a The National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
b The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
A R T I C L E I N F O
A B S T R A C T
Article history:
The structure–activity relationship (SAR) study of a 1,2,3,4,4a,9a-hexahydro-1H-xanthene series of
Received 6 November 2013
Received in revised form 22 January 2014
Accepted 20 February 2014
Available online 15 March 2014
selective, human glucocorticoid receptor
using hydroxyapatite-based GR binding and MMTV-Luc co-transfection reporter gene assays. Four
different regions of the scaffold were modified to assess the effects on hGR antagonism and related
a (hGRa) antagonists is reported. Compounds were screened
a
potency. Compound 8d exhibits an 8-fold better bioactivity than the original hit 1a, as well as an
improved chemical stability, which make it a promising lead for the subsequent optimization.
ß 2014 Ming-Wei Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.
Keywords:
Glucocorticoid receptor
Antagonist
1,2,3,4,4a,9a-Hexahydroxanthene
Structure–activity relationship
Lead optimization
1. Introduction
We have previously reported the discovery and characteriza-
tion of a small, non-steroidal molecule GR antagonist 1a (Fig. 1,
Glucocorticoids (GCs) exert a wide spectrum of metabolic and
immunological effects via their cognate receptors (glucocorticoid
receptors, GRs) which regulate a variety of important target genes
[1]. Hypercortisolemia seen in Cushing’s disease or long-term
therapy with GCs leads to a number of disease states, such as
impaired glucose tolerance, hypertension, depression and central
obesity [2]. Therefore, selective GR antagonists are useful in
mitigating such conditions.
IC50 = 165.7 nmol/L) identified during a high-throughput screening
(HTS) campaign [8]. Compound 1a had a 20-fold selectivity on hGR
(human glucocorticoid receptor) over hPR (human progesterone
receptor), nearly 28-fold selectivity on hGR over hMR (human
mineralocorticoid receptor), and no activity on hAR (human
androgen receptor) and hER (human estrogen receptor). As a
follow-up study, we describe here the hit-to-lead optimization of
1a and its allyl derivative 8d, as a novel GR antagonist.
A well-known GR antagonist, mifepristone (RU486), binds to
GR, PR (progesterone receptor) and AR (androgen receptor) with
high affinities [3]. Treatment of diabetic mice with RU486 lowered
the hepatic glucose level by reducing the expression of two key
enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glu-
cose 6-phosphatase (G6Pase) involved in gluconeogenesis [4,5].
Data from Abbott Laboratories demonstrated that liver-selective
GR antagonist (e.g., the cholate conjugate of RU486) could
significantly decrease blood glucose levels in ob/ob mice indicating
that GR modulation may have value in the control of metabolic
disorders [6,7].
2. Experimental
Chemistry: Reagents were commercial grade and were used as
received, unless otherwise noted. NMR spectra were recorded on
Varian Mercury 300 or Varian Inova 600 spectrometers. Chemical
shifts were reported in parts per million (ppm), with the solvent
resonance as the internal standard (CDCl3 7.26 ppm, CD3OD
3.31 ppm, DMSO-d6 2.50 ppm for 1H NMR; DMSO-d6 39.52 ppm
for 13C NMR). Silica gel (200–300 mesh, Shanghai Chemical
Reagents Co., Ltd.) was used for column chromatography. Silica gel
60 F254 plates (Qingdao Ocean Chemical Industry Co., Ltd.) were
used for TLC analysis. Ozone generator BGF-YQ (Beijing Guoke
Ozone Application Technology Co., Ltd.) was used for ozonization.
Bioassay: Dexamethasone (Dex) and RU486 were purchased
from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA) and tritiated
*
Corresponding author at: The National Center for Drug Screening, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
E-mail address: mwwang@simm.ac.cn (M.-W. Wang).
1001-8417/$ – see front matter ß 2014 Ming-Wei Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2014.03.017

694
Y.-H. Zhu et al. / Chinese Chemical Letters 25 (2014) 693–698
O
N
(5a), benzyl bromide (5b) and methyl iodide (5c), respectively.
Efforts were also made to replace 2-methylindole with other
groups, such as phenylethynyl, substituted phenyl, quinolin-4-yl,
or pyrrol-3-yl [11,12], which resulted in the total loss of activity
(data not shown). Then, modification of cyclohexane (6a–h) was
5
4
O
3
2
6
4a
9a
implemented by a similar method (Scheme 2), in which
cyclohexanyl was converted into phenyl[1,2]cyclohexanyl (6a),
2-methylcyclohexanyl (6b), cyclopentanyl (6c and 6d) or cyclo-
heptanyl (6e–h) using the corresponding enamines. Finally, effects
of the 4a-morpholinyl group was examined by the introduction of
other N-containing heterocyclic groups, such as piperidinyl (6i),
piperazinyl (6j), pyrrolidinyl (6k), or alkylated amino groups, such
as N,N-dipropylamino (6l) (Scheme 2). Hydrolyzation of 4a-
morpholinyl led to hemiketal 7a, which was subsequently refluxed
with pyridinium 4-toluenesulfonate (PPTS) in the mixture of
benzene and methanol (2:1, v/v) to obtain the 4a-OMe substituted
compound 8a. Other substitutions, such as hydrogen (8b-c), allyl
(8d and 8e) and CN (8g), were introduced by the reaction of
hemiketal 7a or 7b with substituted silico reagents (e.g.,
triethylsilane [13], trimethylallylsilane [14] or trimethylcyanosi-
lane [15]). Hydrogenation of 8e afforded the 4a-propyl derivative
8f. After that, compound 8d was used as the intermediate for the
subsequent modification by ozonization [16] to obtain the
acetaldehyde 9, which allowed a further SAR exploration of
10a–d by the Wittig reaction. Reduction of 9 with sodium
borohydride afforded the alcohol 11a, which was followed by
acetylation in acetic anhydride to yield 11b. Iodization of 11a
followed by elimination in DBU gave the 4a-vinyl substituted
compound 13 (Scheme 3).
O2N
7
1
9
8
5'
3'
2'
N
H
7'
Fig. 1. Structure of 1a.
Dexamethasone ([3H]Dex) from GE Healthcare Life Sciences
(Chalfont St. Giles, Bucks., UK). Fetal bovine serum (FBS) and
charcoal/dextran-treated FBS (CDT-FBS) were bought from
Hyclone (Logan, UT, USA). Steady-Glo Luciferase Assay System
was obtained from Promega Corporation (Madison, WI, USA) and
AlamarBlueTM from US Biological (Salem, MA, USA). FlexStation
384II was the product of Molecular Devices (Sunnyvale, CA, USA).
Experimental procedures and 1H NMR and 13C NMR of all the
compounds can be found in Supporting information.
3. Results and discussion
All these compounds were examined for their binding affinities
to hGR
Our initial strategy was to investigate the impact of substitu-
tions on the phenyl ring of the 1,2,3,4,4a,9a-hexahydro-1H-
xanthene skeleton. The key intermediates 4a–j, prepared from
bimorpholine aminals of the substituted salicylicaldehydes,
underwent [4 + 2] cycloaddition with 1-morpholinocyclohexene
to yield the titled compounds 1a–j according to the procedures
described by Ukhin et al. (Scheme 1) [9,10]. Compound 1a was then
hydrogenated to give 1k, which was then acetylated to afford the
amide analog 1l. Next, we evaluated the influence of the active
hydrogen on the 2-methylindole group. Compounds 5a–c were
synthesized by direct alkylation of 1a or 1b with propyl bromide
a
using [3H]Dex as the probe and their effects on the
transcriptional activity mediated by GR were assessed with a
luciferase reporter gene co-transfection assay in CV-1 cells. The
results showed that modification of the phenyl ring of
1,2,3,4,4a,9a-hexahydro-1H-xanthene with 7-nitro improved the
binding affinity and cellular activity by 2-fold and 5-fold,
respectively (1a vs. 1b). Other electron-withdrawing groups, such
as chloro or bromo substitution, had limited impacts on the assay
(1c and 1e vs. 1b) with the 7-chloro derivative (1c) exhibiting
nearly equal binding affinity to GR and 40% improved cellular
Scheme 1. The synthetic routes for compounds 1a–j and 5a–c. Reagents and conditions: (i) morpholine, i-PrOH, reflux, 77%; (ii) 5-substituted 2-methylindole, 130 8C 59%; (iii)
4-(cyclohex-1-en-1-yl) morpholine, 165 8C, 17–77%; (iv) COONH4, MeOH, Pd/C, r.t., 74%; (v) Ac2O, H2O r.t., 96%; (vi) PrBr (5a) or BnBr (5b) or MeI (5c), NaH, THF, r.t., 20–47%.

Y.-H. Zhu et al. / Chinese Chemical Letters 25 (2014) 693–698
695
Scheme 2. The synthetic routes for compounds 6a–l. Reagents and conditions: (i) substituted enamine, 165 8C, 12–74%.
activity compared with the 7-unsubstituted compound (1b). On
the contrary, 7-substituents of electron-donating groups displayed
an opposite picture. Activities of compounds substituted by 7-OCF3
(1g), 7-OCH3 (1h) or 7-NH2 (1k) lost their activities significantly in
both assays, especially 7-OCF3 (1g), whose IC50 was only
2344 ꢀ 87.4 nmol/L, indicating that electron-donating groups with
modest steric hinderance at this position were unfavorable for the
receptor binding. Substitution on position 5 did not render any
advantages for both GR binding and reporter gene activities,
regardless of electron-withdrawing (1d and 1f), or donating groups
(1i). As for modification of the indole group, it was determined that
alkylation of the nitrogen caused a dramatic decrease in potency (5a
and 5b vs. 1b and 5c vs. 1a), and substitution of 50-methoxy on the 50-
position markedly reduced the potency by 5-fold (1j vs. 1c) (Table 1).
The influence of the size of the cycloalkane was also evaluated by
changing from cyclopentyl to cycloheptyl (6d, 6e vs. 1c). Compared
with cyclohexyl (1c), substitutions of cyclopentyl (6d) and cyclo-
heptyl (6e) improved the binding affinity by nearly 50%, while the
cellular activity was either decreased (6d vs. 1c), or remained
unchanged (6e vs. 1c). Similar results were also observed in 7-nitro
O
8a
ii for
iii for 8b, 8c
8d 8e
N
OH
R6
iv for
v for 8g
,
8a R1 = NO2, R6 = OMe
8b R1 = NO2, R6 = H
O
O
O
i
8c
8d
8e
R1 = Cl, R6 = H
R1 = NO2, R6 = allyl
R1
R1
R1
R1 = Cl, R6 = allyl
vi
8f R1 = Cl, R6 = propyl
8g R1 = NO2, R6 = CN
N
H
N
H
N
H
1a R1 = NO2
7a R1 = NO2
7b
1c
R1 = Cl
R1 = Cl
R7
R8
O
O
O
10a-c
viii for
ix for 10d
vii
10a
R7 = Me, R8 = H
8d
O2N
10b R7 = propyl, R8 = H
10c R7 = isopropyl, R8 = H
10d R7 = CH3, R8 = COOEt
O2N
x
10e
R7 = CH3, R8 = COOH
N
H
N
H
9
xi
I
OR
O
O
O
xiv
xiii
O2N
O2N
O2N
N
H
N
H
N
H
13
11a
12
R = H
xii
11b R = Ac
Scheme 3. The synthetic routes for compounds 8a–g, 9, 10a–e, 11a–b, 12 and 13. Reagents and conditions: (i) AcOH, H2O, reflux, 70–74%; (ii) 7a, MeOH, PPTS, benzene/
methanol (2:1), reflux, 48%; (iii) 7a or 7b, Et3SiH, BF3ꢁEt2O, DCM, ꢂ20 8C, then aq NaHCO3, 85–89%; (iv) 7a or 7b, trimethylallylsilane, TiCl4, DCM, ꢂ78 8C, then aq NH4Cl, 23–
25%; (v) 7a, TMSCN, TMSOTf, ꢂ78 8C to reflux, then aq NaHCO3, 5%; (vi) H2, 10% Pd/C, THF, 64%; (vii) (1) Boc2O, 4-DMAP, DCM; (2) O3, ꢂ78 8C, then Ph3P, ꢂ78 8C to r.t.; (3) TFA,
DCM, 0 8C to r.t., 35% for 3 steps in total; (viii) (1) EtBr (10a) or BuBr (10b) or iBuBr (10c), PPh3; (2) BuLi, THF, ꢂ78 8C to 0 8C; (3) ꢂ78 8C, THF, then aq NH4Cl, 26–35%; (ix) ethyl
2-(triphenylphosphoranylidene)propanoate, toluene, reflux, 71%; (x) NaOH, THF/MeOH/H2O (4:2:1), reflux, 61%; (xi) NaBH4, THF/MeOH (10:1), r.t., 85%; (xii) Ac2O, H2O, r.t.;
(xiii) Ph3P, 1-H-imidazole, I2, toluene, reflux, 83%; (xiv) DBU, benzene, reflux, 61%.

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Y.-H. Zhu et al. / Chinese Chemical Letters 25 (2014) 693–698
Table 1
Results of binding and reporter gene assays for the analogs 1, 5–8, 10–11 and 13.
Compound
hGR
a
binding assaya
Reporter gene assayb
IC50 (nmol/L)
Compound
hGR
a
binding assaya
Reporter gene assayb
IC50 (nmol/L)
IC50 (nmol/L)
IC50 (nmol/L)
Dexamethasone
5.1 ꢀ 0.7
165.7 ꢀ 31.1
273.1 ꢀ 26.4
245.6 ꢀ 18.9
687.6 ꢀ 53.2
327.6 ꢀ 22.3
838.6 ꢀ 43.5
2344 ꢀ 87.4
589.1 ꢀ 29.5
290.6 ꢀ 29.8
1066 ꢀ 112.4
603.3 ꢀ 43.6
10390c
NAd
RU486
6h
0.45 ꢀ 0.3
184.2 ꢀ 22.3
143.8 ꢀ 11.2
352.0 ꢀ 31.4
689.1 ꢀ 68.4
292.9 ꢀ 29.9
61.2 ꢀ 4.9
0.71 ꢀ 0.1
4607 ꢀ 413.5
1956 ꢀ 489.1
3157 ꢀ 292.3
16416 ꢀ 843.5
1634 ꢀ 172.4
644.0 ꢀ 54.2
2162 ꢀ 324.1
702.0 ꢀ 48.1
3210 ꢀ 278.4
8333 ꢀ 677.3
86.12 ꢀ 7.3
1a (NC3327)
825.3 ꢀ 78.4
4485 ꢀ 323.4
2542 ꢀ 431.9
4570 ꢀ 378.5
5275 ꢀ 438.3
9884 ꢀ 727.4
12675 ꢀ 892.7
8912 ꢀ 672.9
6782 ꢀ 69.2
6229 ꢀ 563.5
NDe
1b
1c
1d
1e
1f
6i
6j
6k
6l
7a
1g
1h
1i
7b
111.4 ꢀ 17.5
66.8 ꢀ 7.2
8a
8b
111.2 ꢀ 9.6
350.0 ꢀ 23.7
20.2 ꢀ 2.8
1j
8c
1k
1l
8d
NDe
8e
23.1 ꢀ 3.2
344.0 ꢀ 34.5
706.0 ꢀ 54.2
812.9 ꢀ 76.4
1195 ꢀ 98.4
8065 ꢀ 732.4
3455 ꢀ 134.3
9884 ꢀ 1032.7
6311 ꢀ 98.3
2211 ꢀ 198.4
1168 ꢀ 34.1
702.0 ꢀ 48.1
5a
5b
5c
6a
6b
6c
6d
6e
6f
2565 ꢀ 241
30252 ꢀ 432.4
1600 ꢀ 30.5
385.6 ꢀ 30.2
349.4 ꢀ 20.1
683.9 ꢀ 43.7
103.7 ꢀ 9.4
138.2 ꢀ 14.5
87.5 ꢀ 17.2
302.0 ꢀ 42.1
NDe
8f
63.9 ꢀ 7.7
NDe
8g
64.2 ꢀ 5.4
NDe
10a
10b
10c
10d
10e
11a
11b
13
84.4 ꢀ 6.4
NDe
274.4 ꢀ 27.6
198.2 ꢀ 17.7
524.2 ꢀ 43.8
291.0 ꢀ 20.5
209.3 ꢀ 17.5
139.1 ꢀ 18.4
66.8 ꢀ 7.2
1974 ꢀ 231.2
27026 ꢀ 4324.7
4817 ꢀ 339.3
2143 ꢀ 245.1
2063 ꢀ 101.4
5725 ꢀ 432.9
6g
Each value represents mean ꢀ SEM of three independent experiments.
a
The compounds were evaluated for their binding abilities to hGR
a
in competition with [3H]Dex.
b
Antagonist activities were assessed in CV1 cells transiently co-transfected with pSG5-hGR
The value was obtained from a single experiment.
NA: not active (antagonist mode).
a and reporter plasmids.
c
d
e
ND: not determined.
substituted compounds (6f vs. 1a). Finally, the influence of 4a-
observation. Notably, substitution of 4a-allyl resulted in an 8.2-fold
increase in binding potency accompanied by a 9.6-fold better cellular
activity (8d vs. 1a), whereas shortening of the chain length led to a
reduction of binding by 3-fold and of luciferase activity by 8-fold,
respectively (13 vs. 8d). The same phenomenon was seen in
compounds 8e and 1c. Hydrogenation of the allyl group resulted in
decreases of GR binding and cellular activities (8f vs. 8e). Terminal
alkylation of the allyl group caused a significant reduction of
bioactivities in both assays (10a–c vs. 8d) especially for the propyl
(10b) or isopropyl (10c) substitution, indicating that introduction of
sterically large groups at the terminal olefinic link was inappropriate
for binding to GR.
morpholine group was assessed by replacing it with several other
groups. Compounds 6i–k were synthesized by replacement of 4a-
morpholine with 1-piperidinyl (6i), 1-piperazinyl (6j) or 1-pyrroli-
dinyl (6k), respectively. Binding assays illustrated a profile indicating
substitutions of saturated heterocycles were more potent than
aromatic ones, which is consistent with the results in the reporter
gene assay (6i–k vs. 1c). It appears that the 4a-morpholine group was
not an essential group, as its removal did not alter bioactivity (8b vs.
1a and 8c vs. 1c). Enhanced activities were recorded in compounds
with 4a-hydroxyl (7a vs. 1a and 7b vs. 1c), or 4a-methoxy (8a vs. 1a)
suggesting that reduced hindrance may be responsible for this
Fig. 2. (A) Glucocorticoid receptor binding characteristics of 1a and 8d. (B) Glucocorticoid receptor mediated transcriptional activities of 1a and 8d.

Y.-H. Zhu et al. / Chinese Chemical Letters 25 (2014) 693–698
697
Fig. 3. Effects of 1a and 8d on glucocorticoid receptor (GR)-mediated gluconeogenesis. HepG2 cells were grown in 6-well plates and incubated with various compounds for
24 h. Total RNA was isolated with TRIzol reagent. (A) Phosphoenolpyruvate carboxykinase (PEPCK) and (B) glucose-6-phosphatase (G6Pase) mRNA levels in HepG2 cells were
quantified by real-time RT-PCR. GAPDH was used as an internal control. Dexamethasone (Dex) at 0.01
m
mol/L could increase the expression of PEPCK and G6Pase, both were
, 1a and 8d were able to reduce Dex-induced transcription, and the effect of 3 mol/L 8d was
mol/L Dex plus 3 mol/L
rate-controlling enzymes of gluconeogenesis. As antagonists of human GR
a
m
statistically better than that of 1a at the same concentration. ##P < 0.01 compared with 0.1
1a. Data (means ꢀ SEM) are representative of three independent experiments.
mmol/L Dex-treated cells; *P < 0.05 compared with 0.1
m
m
Fig. 4. Effects of 1a and 8d on differentiation of 3T3-L1 preadipocytes. 3T3-L1 cells were grown in 6-well plates and allowed to reach confluence. Various concentrations of 1a
and 8d were added into the medium and incubated for 1 h. Differentiation was induced by changing medium to the inducing medium containing 10% FBS, 0.5 mmol/L IBMX,
1
mg/mL insulin, 0.3 mmol/L dexamethasone (Dex) and human GRa antagonists (1a or 8d). After two days, the inducing medium were changed to insulin medium and
incubated for two more days. At last, cells were cultured in DMEM containing 10% FBS for four days. Following differentiation, (A) cells were stained with Oil Red O and
representative images were obtained at 200ꢃ original magnification, and (B) triglyceride produced by 3T3-L1 cells was measured, which was normalized by protein content
in the cells. *P < 0.05 and **P < 0.01 compared with cells cultured in inducing medium. Data (means ꢀ SEM) are representative of three independent experiments.
4. Conclusion
chemical stability make this scaffold a promising starting point
for subsequent optimization, including further studies on 4a-
substitution and effects of specific enantiomers.
Optimization of the initial hit compound 1a guided by the SAR
analysis identified an improved GR antagonist 8d (Fig. 2A) that did
not possess any agonist profile in the cell-based reporter gene assay
(data not shown). In the antagonist mode, both compounds 1a and
8d suppressed dexamethasone-mediated cellular activities with 8d
displaying a higher potency (Fig. 2B). Like 1a, compound 8d was
capable of (1) reducing dexamethasone-induced transcription of
two key enzymes in gluconeogenesis, PEPCK and G6Pase, in HepG2
cells (Fig. 3) and (2) reducing dexamethasone-induced 3T3-L1
preadipocyte differentiation (Fig. 4), both are indicative of GR
antagonism. Although 8e was less active in cell-based reporter gene
assay, it might represent a more promising lead due to the existence
of the nitro group on 8d. Furthermore, 4a-alkyl substitution led to an
improved chemical stability compared with the original structure of
the O, N-ketal, which could easily be hydrolyzed in acid solution
under high temperature. Improved antagonistic activity and
Acknowledgments
We are indebted to Dale E. Mais for critical review of this
manuscript. The study was supported in part by grants from the
Ministry of Health of China (Nos. 2012ZX09304-011,
2013ZX09401003-005, 2013ZX09507001 and 2013ZX09507002),
Shanghai Science and Technology Development Fund (No.
13DZ2290300) and Thousand Talents Program in China.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2014.03.017.

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Y.-H. Zhu et al. / Chinese Chemical Letters 25 (2014) 693–698
[9] L.Y. Ukhin, L. Belousova, V. Khrustalev, Reactions of 2-methylindole with
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