Discovery of betamethasone 17α-carbamates as dissociated glucocorticoid receptor modulators in the rat

Article abstract of DOI:10.1016/j.bmc.2008.07.037

A series of betamethasone 17α-carbamates were designed, synthesized, and evaluated for their ability to dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepression, in rat cell lines. A number of alkyl substituted betamethasone 17α-carbamates were identified with excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC₅₀ 5.1 nM) and indicated dissociated profiles in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase, GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these compounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose, insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocorticoid receptor modulators can be identified in rodents.

Full text of DOI:10.1016/j.bmc.2008.07.037

Bioorganic & Medicinal Chemistry 16 (2008) 7535–7542
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of betamethasone 17a-carbamates as dissociated glucocorticoid
receptor modulators in the rat
a
a
a
a
a
Amjad Ali a, , James M. Balkovec , Mark Greenlee , Milton L. Hammond , Greg Rouen , Gayle Taylor ,
Monica Einstein b, Lan Ge b, Georgianna Harris b, Terri M. Kelly b, Paul Mazur b, Shilpa Pandit b,
Joseph Santoro b, Ayesha Sitlani b, Chuanlin Wang b, Joann Williamson c, Michael J. Forrest c,
Ester Carballo-Jane c, Silvi Luell d, Karen Lowitz d, Denise Visco d
*
a Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, United States
b Department of Metabolic Disorders, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, United States
c Department of Pharmacology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, United States
d Department of Laboratory Animal Resources, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 April 2008
Revised 3 July 2008
Accepted 16 July 2008
Available online 20 July 2008
A series of betamethasone 17a-carbamates were designed, synthesized, and evaluated for their ability to
dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepres-
sion, in rat cell lines. A number of alkyl substituted betamethasone 17 -carbamates were identified with
a
excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC50 5.1 nM) and indicated dissociated profiles
in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase,
GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these com-
pounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose,
insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocor-
ticoid receptor modulators can be identified in rodents.
Keywords:
Dissociated glucocorticoid receptor
modulators
17 Alpha betamethasone carbamates
Transactivation
Ó 2008 Published by Elsevier Ltd.
Transrepression
Anti-inflammatory activity
Impaired effects on glucose, insulin,
triglycerides and body weights
1. Introduction
of GCs in target tissues are mediated by GR, the expression of
which is ubiquitous. GR is a member of a superfamily of nuclear
Glucocorticoids (GCs), for example, betamethasone 1 and pred-
nisolone 2, are among the most effective therapies available for the
treatment of allergic and chronic inflammatory diseases.1–3 How-
ever, the long-term use of GCs is limited by their severe, and some-
times irreversible, side effects such as diabetes, osteoporosis,
glaucoma, skin atrophy, and muscle wasting. An unmet medical
need, therefore, is the development of agents with improved ther-
apeutic ratios compared to currently available steroids, particu-
larly upon systemic administration. The last decade has provided
important advances in our understanding of the molecular basis
of GC-mediated actions or gene expression, and this work may lead
to the development of improved GCs and more specific therapies in
the future.4,5 Indeed, efforts to identify a new generation of com-
pounds with improved therapeutic profiles are currently under-
way, and recent investigations have focused on the molecular
mechanisms of glucocorticoid receptor (GR) action.6–9 The effects
receptors, and it modulates gene expression by two different regu-
latory mechanisms, namely transactivation (TA) and transrepres-
sion (TR).10,11 Transactivation (TA) depends on the binding of GR
to cis-acting glucocorticoid response elements (GREs) in the pro-
moter region of the glucocorticoid target genes leading to an in-
crease in the transcription rate of the respective genes. In
contrast, TR is mediated via protein–protein interactions with
other transcription factors such as AP-112 and NF- B,13 thereby
j
suppressing the transcriptional activities of these factors. The dis-
section of these two mechanisms has been achieved by a single
amino acid substitution in the GR, such as A458T, Y735F, and
I747T, which results in reduced transactivational activity.14–16 In
addition, studies of genetically engineered GRdim mice that carry
the substitution A458T in the dimerization domain, which impairs
the ability of the GR to bind to DNA, revealed that thymocyte apop-
tosis and erythroblast proliferation require the transactivating
function of GR, whereas the anti-inflammatory activity does
not.17 These findings therefore suggest that GCs lacking transacti-
vation activity, so-called dissociated GCs, would quite likely sepa-
* Corresponding author. Tel.: +1 732 594 8549; fax: +1 732 594 2210.
E-mail address: amjad_ali@merck.com (A. Ali).
0968-0896/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.07.037

7536
A. Ali et al. / Bioorg. Med. Chem. 16 (2008) 7535–7542
rate the therapeutic effects from the major side effects. In a pro-
gram aimed at discovering such novel GC ligands while retaining
a major portion of the steroid nucleus for glucocorticoid-like activ-
OAc
OAc
O
O
HO
TMSO
OH
OTMS
ity, we have synthesized a series of betamethasone derived 17
carbamates represented by the general structure 3. The advantage
of these 17 -carbamate structures is the possible dissociation of
a-
a
b
H
H
F
H
F
H
a
O
O
4
5
anti-inflammatory activity and the associated side effects when
compared to other compounds containing the intact steroidal skel-
eton. Identification of such an anti-inflammatory steroid would
OH
O
OAc
H
N
O
represent
a significant structure–activity relationship break-
TMSO
O
TMSO
through. Our initial interest was peaked by recent reports in the lit-
erature indicating that certain betamethasone derived steroidal
GCs displayed intriguing in-vivo characteristics. For example, it
has been reported that betamethasone valerate exerts much less
thymolytic activity in rats than in mice, although it exhibits strong
therapeutic activity in both species.18 In addition, species specific
differences in TA function were observed for a series of betameth-
asone esters.19 Also, maternal administration of betamethasone es-
ters induced adrenal hypertrophy in rat fetuses,20 but adrenal
atrophy in mice fetuses,21 whereas betamethasone, dexametha-
sone, and esterified dexamethasone induced adrenal atrophy in
both species.22 These observations raise the possibility that beta-
methasone derivatives can act as dissociated GCs. In the present
study, we have characterized the TA and TR function of a series
OH
c
H
H
O
F
H
F
H
O
O
6
OH
O
H
N
HO
O
d
H
O
F
H
O
7
Scheme 1. Reagents and conditions: (a) TMS–Cl, imidazole, DMF, rt, 16 h; (b) AcOH,
1.0 M TBAF in THF, 0 °C, 4 h; (c) ethyl isocyanate, toluene, rt, 48 h; (d) MeOH/CHCl3/
6 N HCl (10:2:1), rt, 21 h.
of 17a-carbamates derived from betamethasone, both in-vitro
and in-vivo. We demonstrate that these dissociated GCs have po-
tent anti-inflammatory activity in the rat with impaired effects
on glucose, insulin, triglyceride, and body weight.
OAc
OAc
O
O
H
N
TMSO
TMSO
O
OH
OH
OH
a
H
H
O
O
O
F
H
F
H
HO
HO
OH
OH
O
O
H
H
6
24
F
H
H
H
OH
O
O
O
O
H
N
b
prednisolone 2
betamethasone 1
HO
O
H
O
OH
F
H
O
H
25
N
R
HO
O
H
Scheme 2. Reagents and conditions: (a) CuCl DMF, (R)-1-phenethyl isocyanate, rt,
O
4.5 h; (b) MeOH/CHCl3/6 N HCl (10:2:1), rt, 24 h.
F
H
O
the corresponding amine and 1,8-bis(dimethylamino)naphthalene
3 R= alkyl, alkenyl, benzyl
with trichloromethyl chloroformate. 23 The target 17
a-carbamates
7–25 were prepared in good overall yields, and their structures
were confirmed on the basis of 1H NMR, LCMS, high-resolution
mass spectra and C, H, N analyses.
2. Chemistry
3. Biological evaluation
The target compounds were synthesized by the routes as shown
in Schemes 1 and 2. The key step involved reaction of the appropri-
ately substituted isocyanates with the monosilyl-protected beta-
methasone derivative 6. The preparation of 6 began by bis-silyl
protection of betamethasone acetate 4 to afford 5, which was
selectively deprotected using 1.0 M TBAF in acetic acid. Treatment
of 6 with a variety of commercially available isocyanates yielded
the desired carbamates, and clean deprotection of both the silyl
and acetyl groups was achieved under acidic conditions (Scheme
1). Hindered carbamates, for example, 25 were synthesized by
treatment of 6 with excess isocyanate in the presence of a catalytic
amount of copper (I) chloride (Scheme 2). Non-commercially avail-
able isocyanates of interest were prepared by treating a mixture of
The results of the biological evaluation of the compounds are
presented in Tables 1–3. Each compound was first evaluated for
its ability to specifically bind to the a-isoform of GR in a competi-
tion binding assay, which constitutes the primary event in the ac-
tion of a GR modulator. Counter screen binding assays were
routinely performed on a subset of compounds against PR to mon-
itor steroid receptor selectivity and many of the newly synthesized
analogs displayed good selectivity toward GR over PR (Table 3).
The ability of the compounds to repress transcription was evalu-
ated by glucocorticoid mediated inhibition of MMP-1 expression
as determined by ELISA measurement of secreted MMP-1 protein
levels from PMA stimulated A549 cells. The transactivation poten-
tial of each compound was evaluated by its ability to induce tyro-

A. Ali et al. / Bioorg. Med. Chem. 16 (2008) 7535–7542
7537
Table 1
Evaluation of betamethasone 17
a carbamate glucocorticoid modulators
OH
O
H
N
HO
R
O
H
O
F
H
O
Compound
R
GRa
TATb,c
GSd,c
MMP-1e,c
EC50 (nM)
b
IC50 (nM)
EC50 (nM)
% dex
EC50 (nM)
% dex
% dex
7
8
9
10
11
12
13
Ethyl
Propyl
Butyl
Pentyl
iso-propyl
Allyl
5.1
3.6
2.8
n.r.f
n.r.f
n.r.f
117
n.r.f
n.r.f
5.3
10.2
10.3
6.1
37.3
8.6
n.r.f
n.r.f
n.r.f
0.4
23.4
34.7
31.3
60.3
26
15.9
18.7
22
18
1.9
39
104
103
98
97.5
105
106
94
2.1
21.5
17.4
2.4
n.r.f
n.r.f
0.37
10.9
70.1
16
64
tert-Butyl
1.6
O
O
14
15
16
17
18
28.6
0.5
211
n.r.f
705
n.r.f
646
156
99.9
15.8
40.7
30.6
54.6
63.2
47
103
54
83
107
0.3
n.r.f
—
0.8
53.3
0.1
21
97.5
65.9
44.7
56.3
80.3
55.1
352
12.3
5.7
35
—
576
223
61.8
50
19
23.2
a
Data reported were derived from duplicate wells and three independent experiments. Mean IC50 values were determined from 10-point, one-third log concentration
response curves and standard errors were 620%.
b
Rat tyrosine amino transferase (TAT) assay in rat H4 cells.
Experiments were run in duplicate. EC50 values were determined from eight-point, one-third log concentration response curves and standard errors were 620%.
Rat glutamine synthetase (GS) assay in skeletal muscle cells.
Human matrix metalloproteinase I (MMP-1) assay in A549 cells.
n.r.: not reported; EC50 values not reported in cases where % dex 635%.
c
d
e
f
sine aminotransferase (TAT) and glutamine synthetase (GS) activ-
ity in rat H4 and skeletal muscle cells, respectively. TAT is a liver
specific, GR responsive gene, the product of which initiates tyro-
sine catabolism.24 GS catalyzes the conversion of glutamate to glu-
tamine, an important regulator of skeletal muscle.25–27 These TAT
and GS functional assays were used as markers for hepatic- and
muscle-specific GR transactivation. In-vivo activity readouts in-
cluded effects on glucose, insulin, triglyceride, depression of body
weight gain without a change in food consumption, and anti-
inflammatory activity, which constitute significant aspects of the
in-vivo profile of a glucocorticoid in the rat. The hGR, transactiva-
tion (rTAT and rGS), and transrepression (MMP-1) activities for a
selection of compounds are provided in Tables 1 and 2.
Most of the compounds presented in Table 1 exhibited signifi-
cant affinity and selectivity toward the glucocorticoid receptor
and many bound with approximately the same or better affinity
than prednisolone (IC50 = 13.8 nM). Small unbranched alkyl deriv-
atives, for example, R = Et 7, bound with exceptional affinity and
selectivity for GR versus PR (IC50 5.1 = nM vs >1000 nM) (Table
3). The N-Et derivative 7 was evaluated further in functional assays
of TA and TR and, quite gratifyingly, afforded a completely dissoci-
ated profile in rat. Interestingly, however, 7 was found to be a po-
tent inducer of GS activity in mouse and human skeletal muscle
cells.28 This apparent species specific difference in TA activity is
not well understood, but is in line with literature reports for a ser-
ies of structurally related betamethasone esters.19 In rat, however,
as shown in Table 1, 7 exhibited a weak agonist profile in func-
tional assays of TA, yet displayed characteristics of a full agonist
in functional assays of TR. Larger alkyl substituents (e.g., 16, 17)
generally led to reduced affinity and selectivity for GR. The intro-
duction of larger substituents also led to partial agonist profiles
and produced analogs that no longer appeared to be dissociated.
Indeed, an n-butyl substituent (i.e., 9) appeared to be the longest
linear group that could be tolerated while maintaining a fully dis-
sociated profile. Both branched alkyl substituents (e.g., 13, 16) and
the introduction of polarity (e.g., 14) led to decreases in affinity and
to less interesting partial agonist profiles in our cellular based as-
says of TA and TR. In fact, our SAR studies have shown that among
those variations examined any departure from small straight-chain
alkyl substituents such as N-Et of 7, resulted in either loss of affin-
ity for GR or to a partial agonist profile. As shown in Table 2, steri-
cally bulkier substituents, for example, N-cyclohexyl 21, were

7538
A. Ali et al. / Bioorg. Med. Chem. 16 (2008) 7535–7542
Table 2
Sterically hindered 17a carbamates
OH
O
H
N
HO
R
O
H
O
F
H
O
Compound
R
GRa
TATb,c
GSd,c
MMP-1e,c
EC50 (nM)
b
IC50 (nM)
EC50 (nM)
% dex
EC50 (nM)
% dex
% dex
20
21
22
Cyclopentyl
Cyclohexyl
Adamantyl
3.8
3.5
5.8
n.r.f
2.0
9.8
6.0
42.9
80.8
n.r.f
n.r.f
—
23
24.7
—
2.8
1.3
3.4
76.1
82
84.1
25
5.2
1.3
70.3
37.3
66.1
71.7
1.6
0.6
92
49
21.5
4.9
87.5
93
26
a
Data reported were derived from duplicate wells and three independent experiments. Mean IC50 values were determined from 10-point, one-third log concentration
response curves and standard errors were 620%.
b
Rat tyrosine amino transferase (TAT) assay in rat H4 cells.
Experiments were run in duplicate. EC50 values were determined from eight-point, one-third log concentration response curves and standard errors were 620%.
Rat glutamine synthetase (GS) assay in skeletal muscle cells.
Human matrix metalloproteinase I (MMP-1) assay in A549 cells.
n.r.: not reported; EC50 values not reported in cases where % dex 635%.
c
d
e
f
Table 3
Binding affinities of 17
receptors
derivatives N-Et 7 (Clp 26 mL/min/kg, t1/2 2.2 h, Vdss 3.22 L/kg, %F
a
carbamates against human GR versus other steroidal
43) and N-Pr 8. On the basis of their dissociated profiles in our
in-vitro assays and promising pharmacokinetic data above, 7 and
8 were selected for further in-vivo evaluation. We initially exam-
ined 7 under acute inflammatory TR conditions using the murine
LPS challenge model of inflammation.29 Thus, upon oral adminis-
OH
O
H
N
HO
R
O
H
O
tration of 7, a dose-dependent inhibition of TNF-
a was observed,
F
H
similar for that of prednisolone (ED50 = 0.1 mg/kg for 7 vs 0.5 mg/
kg for prednisolone). Figure 1 depicts the dose–response curve
for prednisolone versus 7. The anti-inflammatory efficacy of 7
and 8 was also determined in a chronic 7 day rat cotton pellet
granuloma assay30 at total daily doses of 5, 10, and 19 mg/kg given
p.o. b.i.d from day 1. As shown in Table 4, compounds 7 and 8 pro-
duced dose-dependent inhibition of the cotton pellet granuloma in
female Lewis rats on days 4 and 7. At 19 mg/kg, compounds 7 and 8
inhibited the cotton pellet granuloma by 45% and 51%, respec-
tively, and this compared to prednisolone at 2.5 mg/kg which
showed 49% inhibition at this dose. We next turned our attention
toward examining the in-vivo side effect profile of 7 and 8.
O
Compound
R
Binding IC50 (nM)a
hPR
hGR
7
8
12
Ethyl
Propyl
Allyl
5.1
3.6
17.4
>1000
394
>1000
19
21
5.7
3.5
>1000
120
Cyclohexyl
ED50
(mg/kg, p.o.)
26
5.2
176
150
Prednisolone
7
0.5
0.1
130
110
90
a
Data reported were derived from duplicate wells and three independent
experiments. Mean IC50 values were determined from 10-point, one-third log
concentration response curves and standard errors were 620%.
70
50
tolerated but these derivatives generally exhibited reduced selec-
tivity for GR versus the other steroidal receptors (Table 3) and
many had partially dissociated profiles in functional assays of TA
and TR.
30
10
-10
-30
0.001
0.01
0.1
1
10
100
3.1. In-vivo evaluation
Dose (mg/kg, p.o.)
A number of the N-alkyl 17-
a carbamates displayed good levels
Figure 1. Prednisolone and 7 dose dependently inhibit mouse LPS-induced TNF
a
of oral bioavailability in the rat including the fully dissociated
production.

A. Ali et al. / Bioorg. Med. Chem. 16 (2008) 7535–7542
7539
Table 4
The effects of prednisolone, compounds 7 and 8 on change in body weight, spleen, thymus, and adrenal weights and inhibition of granuloma formation in female Lewis rats with
implanted cotton pellets
Treatment (mg/kg)
Change in body
weight on Day 4 (g)
Change in body
weight on Day 7 (g)
Spleen weight
(mg)
Thymus weight
(mg)
Adrenal weight
(mg)
% Inhibition
granuloma
Vehicle
7.0 3.0
14.4 4.3
438.3 878.7
401.3 76.8
41.3 7.6
Prednisolone
1.0
2.5
6.0
1.5 2.1**
À1.0 2.1**
À3.6 2.5**
3.0 2.3**
À0.4 2.7**
À4.1 3.4**
334.7 16.2**
266.8 11.2**
260.0 22.1**
219.5 39.6**
117.8 38.0**
104.0 26.8**
38.7 5.3
26.9 4.7**
27.9 6.3**
37.6 15.9
49.3 6.0
56.2 6.8
Compound 7
5.0
10
19
5.9 4.4
7.3 3.7
5.6 3.7
11.0 4.2
10.0 3.9
7.5 4.6**
330.9 17.5**
311.2 18.4**
305.3 15.4**
170.7 29.1**
144.9 23.2**
119.9 36.7**
39.0 3.4
36.s8 7.4
30.8 6.6**
35.6 18.3
33.5 14.5
44.6 8.6
Compound 8
5.0
10
19
1.1 4.5**
3.9 2.1
3.9 3.6
5.0 6.6**
7.1 3.5**
6.6 3.4**
296.1 11.7**
274.3 15.2**
258.0 13.1**
105.5 15.7**
91.4 21.6**
82.9 19.3**
34.7 5.7
34.6 10.3
25.6 2.8**
32.7 12.8
40.0 15.7
51.0 8.6
Significant difference from vehicle at p < 0.05 (*) or p < 0.01(**) with Dunnett’s t-test.
Increases in glucose, insulin, and triglyceride levels and decreases
in body weight gain constitute significant undesired activity of cur-
rently available GCs. Samples collected from the rat cotton pellet
granuloma study allowed measurement of all of the above param-
eters and indicated an improved side effect profile for 7 and 8 con-
2.5
2.0
1.5
1.0
0.5
0.0
sistent with
a
dissociated GR modulator. Thus, whereas
administration of prednisolone at 2.5 mg/kg led to significant de-
creases in body weight on day 7 (À0.4 g 2.7 g) versus vehicle
(+14.4 g 4.3 g), weight loss with compound 7 at 19 mg/kg on
day 7 was less pronounced (+7.5 g 4.6 g). The 7 day organ
weights for the prednisolone treated groups (spleen, thymus, and
adrenal) indicated that these lymphoid organs were subject to
the catabolic actions of the steroid. Interestingly, compounds 7
and 8 had similar effects on organ weights compared to the pred-
nisolone treated groups. Decreases in plasma insulin and triglycer-
ide levels were also evident (Fig. 2) and in the case of plasma
triglyceride levels these differences were not statistically signifi-
cant compared to vehicle. On day 7, the rats were administered
an oral glucose test (OGTT) with dextrose dosed at 3 mg/kg
(Fig. 3). Rats dosed with 7 exhibited a glucose excursion similar
to vehicle 2 h post dose, while the prednisolone dosed animals pos-
sessed a strong spike consistent with conventional steroid therapy.
Rats dosed with 8 exhibited a glucose excursion at the higher doses
of 10 and 19 mg/kg, similar to that observed with prednisolone at
2.5 and 6.0 mg/kg (Fig. 3). To further profile 7, effects on plasma
glucose, insulin, triglyceride, and % body weight change were as-
sessed in the Zucker Rat chronic dosing model.31 In this five day
study, 7 was dosed at 30 mg/kg alongside dexamethasone at
0.3 mg/kg and the GR antagonist RU486 at 30 mpk. As shown in
Figure 4, dexamethasone exhibited a profound effect on all four
parameters at low doses. In comparison, the N-Et derivative 7
showed little effect on body weight, glucose, insulin, and triglycer-
ide levels and the differences were not statistically significant com-
pared to vehicle or the GR antagonist RU 486. Interestingly, co-
administration of compound 7 (30 mg/kg) with dexamethasone
(0.3 mg/kg) led to normalization of glucose, insulin, and body
weight change parameters indicating that 7 was able to antagonize
the TA activity of dexamethasone. A similar effect was observed by
co-administration of the GR antagonist RU 486.
90
75
60
45
30
15
0
Figure 2. The effects of prednisolone, vehicle, and compounds 7 and 8 on plasma
insulin and triglyceride levels in female Lewis rats with implanted cotton pellets.
of these compounds exhibit a fully dissociated profile in functional
assays of TA and TR in the rat. In-vivo administration of 7 caused a
significant anti-inflammatory response yet showed little effects on
glucose, insulin, and triglyceride levels. A reduced catabolic effect
was also observed. Taken together, these results indicate that dis-
sociated GR modulators can be identified in the rat starting from
traditional steroidal platforms and that these compounds may of-
fer therapeutic advantages in the treatment of diseases in which
GR plays a critical role, including inflammatory and immunological
disorders.
4. Conclusion
In conclusion, we have demonstrated that a series of 17-a car-
bamates derived from betamethasone retain significant activity
and selectivity for the glucocorticoid receptor. In addition, many

7540
A. Ali et al. / Bioorg. Med. Chem. 16 (2008) 7535–7542
LCMS analyses were performed on a Waters Micromass ZQ single
125
120
115
110
105
100
quadrupole mass spectrometer. Elemental analyses were per-
formed by Robertson Microlit Laboratories, Inc., Madison, NJ, and
are within 0.4% of the calculated values unless otherwise stated.
Reagents were purchased commercially and used without further
purification unless otherwise stated.
5.1.1. (11b,16b)-9-Fluoro-16-methyl-3,20-dioxo-11,17-bis
[(trimethylsilyl)oxy]pregna-1,4-dien-21-yl acetate (5)
To
a
solution of (11b,16b)-9-fluoro-11,17-dihydroxy-16-
acetate (18.79 g,
methyl-3,20-dioxopregna-1,4-dien-21-yl
43.25 mmol) and imidazole (58.89 g, 864.9 mmol) in 200 mL of
dry N,N-dimethylformamide was added neat trimethylsilyl chlo-
ride (55.0 mL, 433 mmol) dropwise during 15 min. The resulting
pale yellow solution was stirred at room temperature for 16 h.
The solution was diluted with ethyl acetate and washed succes-
sively with water, satd NH4Cl, water, satd NaHCO3, water, and
brine. The organic phase was dried over Na2SO4, filtered, and evap-
orated in vacuo to afford 24.8 g (99%) of 5 as a pale yellow oil. 1H
NMR (500 MHz, CDCl3): d 0.25 (s, 9H), 0.26 (s, 9H), 0.94 (s, 3H),
1.15–1.25 (m, 1H), 1.21 (d, J = 7.3 Hz, 3H), 1.49 (s, 3H), 1.50–1.65
(m, 1H), 1.61 (d, J = 13.7 Hz, 1H), 1.85–2.0 (m, 3H), 2.18 (s, 3H),
2.15–2.25 (m, 1H), 2.25–2.50 (m, 3H), 2.55–2.65 (m, 1H), 4.35–
4.40 (m, 1H), 4.75 (d, J = 17.5 Hz, 1H), 4.84 (d, J = 17.5 Hz, 1H),
6.13 (s, 1H), 6.36 (dd, J = 10.1, 1.8 Hz, 1H), 7.06 (d, J = 10.1 Hz,
1H). MS (ESI): m/z = 579.3 (MH+).
Figure 3. The effects of prednisolone, vehicle, and compounds 7 and 8 on plasma
glucose levels following an oral glucose tolerance test (OGTT) in female Lewis rats
with implanted cotton pellets.
5. Experimental
5.1. Chemistry
1H NMR spectra were recorded on a Varian InNova 500 MHz
instrument in CDCl3 or DMSO-d6 solutions, unless otherwise sta-
ted. Analytical thin-layer chromatography (TLC) was carried out
using Merck silica gel 60 F254 plates. All compounds were detected
as single spots on TLC plates and visualized using UV light and
phosphomolybdic acid stain. Low-resolution mass spectra (MS)
were determined on a Micromass Platform LC by electrospray po-
sitive ionization. High-resolution mass spectra (HRMS) were deter-
mined on a Thermo Electron LTQ FT Ultra using ESI ionization.
5.1.2. (11b,16b)-9-Fluoro-17-hydroxy-16-methyl-3,20-dioxo-11-
[(trimethylsilyl)oxy]-pregna-1,4-dien-21-yl acetate (6)
A solution of (11b,16b)-9-fluoro-16-methyl-3,20-dioxo-11,17-
bis[(trimethylsilyl)oxy]pregna-1,4-dien-21-yl
acetate
(26 g,
ꢀ43 mmol) in 430 mL of tetrahydrofuran (THF) was cooled to
0 °C and neat acetic acid (4.95 mL, 86.5 mmol) was added followed
70
60
50
40
30
20
10
0
700
600
500
400
300
200
100
0
A Vehicle
B Dex 0.3 mpk
C RU486 30 mpk
D Compound 7 30 mpk
E Dex + RU486
F Dex + Compound 7
A
B
C
D
E
F
A
B
C
D
E
F
1500
1250
1000
750
15
10
5
0
-5
500
-10
-15
250
A
B
C
D
E
F
A
B
C
D
E
F
Figure 4. The effects of dexamethasone, vehicle, compound 7, and RU486 on plasma glucose, insulin, triglyceride, and % body weight change in Zucker fatty rat chronic dosing
model.

A. Ali et al. / Bioorg. Med. Chem. 16 (2008) 7535–7542
7541
by dropwise addition of a 1.0 M solution of tetra-n-butylammo-
nium fluoride in THF (43.3 mL, 43.3 mmol). After stirring at 0 °C
for 4 h, the ice bath was removed and the reaction was allowed
to warm to room temperature and stirred for an additional 16 h.
Most of the THF was removed by rotary evaporation in vacuo
and the residue was partitioned between ethyl acetate and water.
The organic layer was washed successively with water, satd NH4Cl,
water, satd NaHCO3, water, and brine. The organic layer was dried
over Na2SO4, filtered, and evaporated under vacuum to give an off-
white solid. Purification by flash chromatography through a 1.6 kg
column of silica gel eluting with 4% MeOH in dichloromethane pro-
vided 16.1 g (74%) of 6 as a white solid. 1H NMR (500 MHz, CDCl3):
d 0.25 (s, 9H), 1.06 (s, 3H), 1.15–1.25 (m, 1H), 1.17 (d, J = 7.1 Hz,
3H), 1.49 (s, 3H), 1.45–1.60 (m, 1H), 1.64 (d, J = 14 Hz, 1H), 1.85–
2.2 (m, 5H), 2.18 (s, 3H), 2.35–2.55 (m, 3H), 2.55–2.65 (m, 1H),
4.35–4.40 (m, 1H), 4.94 (AB, 2H), 6.11 (s, 1H), 6.35 (dd, J = 10.3,
1.8 Hz, 1H), 7.05 (d, J = 10 Hz, 1H). MS (ESI): m/z = 507.2 (MH+).
5.1.6. (11b,16b)-9-Fluoro-11,21-dihydroxy-16-methyl-3,20-dio-
xopregna-1,4-dien-17-yl butylcarbamate (9)
From 6 and n-butyl isocyanate. MS (ESI): m/z = 492.3 (MH+). C,
H, N (C27H38FNO6) calcd %C 65.97, %H 7.79, %N 2.85; found %C
65.86, %H 7.93, %N 2.69.
5.1.7. (11b,16b)-9-Fluoro-11,21-dihydroxy-16-methyl-3,20-dio-
xopregna-1,4-dien-17-yl isopropylcarbamate (11)
From 6 and isopropyl isocyanate. MS (ESI): m/z = 478.3 (MH+).
C, H, N (C26H36FNO6) calcd %C 65.39, %H 7.60, %N 2.93; found %C
65.23, %H 7.64, %N 2.97.
5.1.8. (11b,16b)-9-Fluoro-11,21-dihydroxy-16-methyl-3,20-dio-
xopregna-1,4-dien-17-yl allylcarbamate (12)
From 6 and allyl isocyanate. MS (ESI): m/z = 476.3 (MH+). C, H, N
(C26H34FNO6) calcd %C 65.67, %H 7.21, %N 2.95; found %C 65.08 ,%H
7.30, %N 3.15.
5.1.3. (11b,16b)-21-(Acetyloxy)-9-fluoro-16-methyl-3,20-dioxo-
11-[(trimethylsilyl)-oxy]pregna-1,4-dien-17-yl ethylcarbamate
5.1.9. (11b,16b)-9-Fluoro-11,21-dihydroxy-16-methyl-3,20-dio-
xopregna-1,4-dien-17-yl tert-butylcarbamate (13)
From 6 and tert-butyl isocyanate. MS (ESI): m/z = 492.3 (MH+).
C, H, N (C27H38FNO6) calcd %C 65.97, %H 7.79, %N 2.85; found %C
65.83, %H 8.07, %N 2.76.
To
a mixture of (11b,16b)-9-fluoro-17-hydroxy-16-methyl-
3,20-dioxo-11-[(trimethyl-silyl)oxy]-pregna-1,4-dien-21-yl ace-
tate (3.11 g, 6.15 mmol) in 41 mL of toluene was added neat ethyl
isocyanate (20.5 mL, 259 mmol), and the mixture was refluxed for
48 h. The resulting solution was cooled to room temperature and
evaporated under vacuum to leave a slightly gummy solid. Flash
chromatography through a 1 kg column of silica gel eluting with
tert-butyl methyl ether/hexane/chloroform (1:1:1) gave 2.22 g
(67%) of the title compound as a white solid. 1H NMR (500 MHz,
CDCl3): d 0.26 (s, 9H), 0.94 (s, 3H), 1.1–1.3 (m, 4H), 1.34 (d,
J = 6.7 Hz, 3H), 1.48 (s, 3H), 1.45–1.60 (m, 1H), 1.78 (d,
J = 13.5 Hz, 1H), 1.8–2.1 (m, 3H), 2.16 (s, 3H), 2.15–2.55 (m, 4H),
2.55–2.65 (m, 1H), 3.1–3.3 (m, 2H), 4.35–4.45 (m, 1H), 4.54 (d,
J = 16 Hz, 1H), 4.82 (d, J = 16 Hz, 1H), 5.0–5.1 (m, 1H), 6.11 (s,
1H), 6.34 (d, J = 10 Hz, 1H), 7.04 (d, J = 10 Hz, 1H). MS (ESI): m/
z = 578.3 (MH+).
5.1.10. (11b,16b)-9-Fluoro-11,21-dihydroxy-16-methyl-3,20-dio-
xopregna-1,4-dien-17-yl cyclopentylcarbamate (20)
From 6 and cyclopentyl isocyanate. MS (ESI): m/z = 504.2 (MH+).
C, H, N (C28H38FNO6) calcd %C 66.78, %H 7.61, %N 2.78; found %C
66.62, %H 7.75, %N 2.65.
5.1.11. (11b,16b)-21-(Acetyloxy)-9-fluoro-11-hydroxy-16-
methyl-3,20-dioxopregna-1,4-dien-17-yl (1R)-1-
phenylethylcarbamate (24)
To
a mixture of (11b,16b)-9-fluoro-17-hydroxy-16-methyl-
3,20-dioxo-11-[(trimethyl-silyl)oxy]-pregna-1,4-dien-21-yl ace-
tate (100 mg, 0.197 mmol) and copper (I) chloride (0.5 mg,
0.005 mmol) in 1 mL of N,N-dimethylformamide was added neat
(R)-1-phenylethyl isocyanate (0.085 mL, 0.60 mmol). The pale
green reaction mixture was stirred at room temperature in the
dark for 4.5 h before being partitioned between ethyl acetate and
water. The organic layer was washed successively with water,
sat. NH4Cl, water, and brine and then dried over Na2SO4. Filtration
and evaporation in vacuo gave a foam which was purified by flash
chromatography through a 40 g column of silica gel, eluting with
hexane/tert-butyl methyl ether/chloroform (1:1:1), to provide
67 mg of the title compound as a colorless oil. MS (ESI): m/
z = 654.4 (MH+).
5.1.4. (11b,16b)-9-Fluoro-11,21-dihydroxy-16-methyl-3,20-dio-
xopregna-1,4-dien-17-yl ethylcarbamate (7)
Solid (11b,16b)-21-(acetyloxy)-9-fluoro-16-methyl-3,20-dioxo-
11-[(trimethylsilyl)oxy]-pregna-1,4-dien-17-yl
ethylcarbamate
(2.22 g, 3.84 mmol) was dissolved in 128 mL of a pre-mixed solu-
tion of methanol/chloroform/6 N HCl (10:2:1) and stirred at room
temperature. After 16 h, an additional 2 mL of 6 N HCl was added.
After an additional 5 h, the solution was partitioned between ethyl
acetate and water. The organic phase was washed successively
with satd NaHCO3, water, and brine. The organic layer was dried
over Na2SO4, filtered, and evaporated in vacuo to give a pale yellow
oil. Purification by flash chromatography through a column of
500 g of silica gel, eluting with hexane/ethyl acetate/dichlorometh-
ane/methanol (3:3:3:1) yielded 0.890 g (50%) of 7 as a white pow-
der. 1H NMR (500 MHz, CDCl3): d 0.96 (s, 3H), 1.15 (t, J = 7 Hz, 3H),
1.15–1.30 (m, 1H), 1.41 (d, J = 7.3 Hz, 3H), 1.47 (d, J = 13.9 Hz, 1H),
1.56 (s, 3H), 1.50–1.65 (m, 1H), 1.9–2.1 (m, 3H), 2.2–2.7 (m, 7H),
3.1–3.3 (m, 2H), 4.03 (d, J = 18 Hz, 1H), 4.26 (d, J = 18 Hz, 1H),
4.30–4.45 (m, 1H), 5.2–5.3 (m, 1H), 6.14 (s, 1H), 6.34 (dd, J = 10,
1.6 Hz, 1H), 7.21 (d, J = 10 Hz, 1H). MS (ESI): m/z = 464.2 (MH+).
C, H, N (C25H34FNO6) calcd %C 64.78, %H 7.39, %N 3.02; found %C
64.78, %H 7.66, %N 3.02.
5.1.12. (11b,16b)-9-Fluoro-11,21-dihydroxy-16-methyl-3,20-
dioxopregna-1,4-dien-17-yl (1R)-1-phenylethylcarbamate (25)
(11b,16b)-21-(Acetyloxy)-9-fluoro-11-hydroxy-16-methyl-
3,20-dioxopregna-1,4-dien-17-yl
(1R)-1-phenylethylcarbamate
24 (42 mg, 0.064 mmol) was dissolved in 1.25 mL of a pre-
mixed solution of methanol/chloroform/6 N HCl (10:2:1) and
stirred at room temperature for 24 h. The solution was then
partitioned between ethyl acetate and water, and the organic
phase was washed with water (twice) and brine and dried over
Na2SO4. Filtration and evaporation under vacuum gave
a
white film which was purified by preparative thin-layer chro-
matography on silica gel, eluting with ethyl acetate/hexane/
dichloromethane/methanol (95:95:95:15), to yield 20 mg of
(11b,16b)-9-fluoro-11,21-dihydroxy-16-methyl-3,20-dioxopreg-
na-1,4-dien-17-yl (1R)-1-phenylethylcarbamate as a white solid.
MS (ESI): m/z = 540.3 (MH+). HRMS for C31H39FNO6 (M+1)+:
calcd 540.2757; found 540.2756.
5.1.5. (11b,16b)-9-Fluoro-11,21-dihydroxy-16-methyl-3,20-dio-
xopregna-1,4-dien-17-yl propylcarbamate (8)
From 6 and propyl isocyanate. MS (ESI): m/z = 478.3 (MH+). C, H,
N (C26H36FNO6) calcd %C 65.39, %H 7.60, %N 2.93; found %C 65.40,
%H 7.85, %N 2.85.

7542
A. Ali et al. / Bioorg. Med. Chem. 16 (2008) 7535–7542
Mazur, P.; Pandit, S.; Santoro, J.; Sitlani, A.; Wang, C.; Williamson, J.; Miller, D.
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P.; Thompson, C. M.; Wang, C. F.; Williamson, J. M.; Miller, D. M.; Pandit, S.;
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5.1.13. (11b,16b)-9-Fluoro-11,21-dihydroxy-16-methyl-3,20-
dioxopregna-1,4-dien-17-yl (1S)-1-phenylethylcarbamate (26)
From 6 and (1S)-1-phenyl isocyanate. MS (ESI): m/z = 540.3
(MH+). HRMS for C31H39FNO6 (M+1)+: calcd 540.2757; found
540.2756.
5.1.14. (11b,16b)-9-Fluoro-11,21-dihydroxy-16-methyl-3,20-
dioxopregna-1,4-dien-17-yl (1S)-1-(methoxycarbonyl)-
ethylcarbamate (14)
From 6 and (S)-2-isocyanatopropionic acid methyl ester. MS
(ESI): m/z = 522.2 (MH+). HRMS for C27H37FNO8 (M+1)+: calcd
522.2497; found 522.2498.
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Acknowledgment
The authors wish to thank Debbie Zink for providing high-reso-
lution mass spectral analyses.
Supplementary data
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Cell-based assay procedures for hGRa, hPR, glutamine synthe-
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can be found in the online version. Procedures are also available for
the in-vivo evaluation in the mouse LPS challenge assay, rat cotton
pellet granuloma assay and Zucker fatty rat model. Supplementary
data associated with this article can be found, in the online version,
at doi:10.1016/j.bmc.2008.07.037.
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