Synthesis of sterically encumbered 11β-aminoprogesterone derivatives and evaluation as 11β-hydroxysteroid dehydrogenase inhibitors and mineralocorticoid receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2013.08.068

11β-Hydroxyprogesterone is a well-known nonselective inhibitor of 11β-hydroxysteroid dehydrogenase (11βHSD) types 1 and 2. It also activates the mineralocorticoid receptor (MR). Modulation of corticosteroid action by inhibition of 11βHSDs or blocking MR i

Full text of DOI:10.1016/j.bmc.2013.08.068

Bioorganic & Medicinal Chemistry 21 (2013) 6274–6281
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of sterically encumbered 11b-aminoprogesterone
derivatives and evaluation as 11b-hydroxysteroid dehydrogenase
inhibitors and mineralocorticoid receptor antagonists
Keyur Pandya , David Dietrich a, , Julia Seibert b, , John C. Vederas a, , Alex Odermatt b,
a,
⇑
⇑
a
Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, Alberta T6G 2G2, Canada
Swiss Center for Applied Human Toxicology and Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50,
CH-4056 Basel, Switzerland
b
a r t i c l e i n f o
a b s t r a c t
Article history:
11b-Hydroxyprogesterone is a well-known nonselective inhibitor of 11b-hydroxysteroid dehydrogenase
Received 7 August 2013
Accepted 29 August 2013
Available online 7 September 2013
(
11bHSD) types 1 and 2. It also activates the mineralocorticoid receptor (MR). Modulation of corticoste-
roid action by inhibition of 11bHSDs or blocking MR is currently under consideration for treatment of
electrolyte disturbances, metabolic diseases and chronic inflammatory disorders. We established condi-
tions to synthesize sterically demanding 11b-aminoprogesterone, which following subsequent nucleo-
philic or reductive amination, allowed extension of the amino group to prepare amino acid derivatives.
Biological testing revealed that some of the 11b-aminoprogesterone derivatives selectively inhibit
Keywords:
1
1Beta-hydroxysteroid dehydrogenase
Mineralocorticoid receptor
Inhibitor
Antagonist
Amino acid–steroid conjugates
Glucocorticoid
1
1bHSD2. Moreover, two compounds that did not significantly inhibit 11bHSDs had antagonist properties
on MR. The 11b-aminoprogesterone derivatives form a basis for the further development of improved
modulators of corticosteroid action.
Ó 2013 Elsevier Ltd. All rights reserved.
3
1
. Introduction
Steroids play an important role in maintenance and regulation
disturbances observed in metabolic syndrome. Mice overexpress-
ing 11bHSD1 specifically in the liver present with steatosis and
4
rather mild insulin resistance but without obesity. Based on these
and additional animal data and clinical observations, inhibition of
11bHSD1-mediated glucocorticoid activation emerged as a promis-
ing strategy to treat metabolic diseases, including type 2 diabetes,
hyperlipidemia, atherosclerosis and osteoporosis.
Since inhibition of 11bHSD2 in the kidney results in cortisol-in-
duced MR activation with sodium and water retention and hyper-
tension, 11bHSD1 inhibitors used for treatment of metabolic
diseases need to be highly selective. Although inhibition of
11bHSD2 needs to be avoided for these applications, it was re-
cently suggested that 11bHSD2 inhibitors may be used to treat pa-
tients on hemodialysis in order to achieve potassium loss as a
result of cortisol-induced MR activation in the colon,6 and
several glycyrrhetinic acid derived 11bHSD2 inhibitors were
of various physiological functions. Specifically, the corticosteroids
are essentially involved in the regulation of carbohydrate, lipid
and protein metabolism, inflammation, and maintenance of water
and electrolyte balance. Corticosteroids exert their effects mainly
through glucocorticoid receptors (GR) and mineralocorticoid
receptors (MR). A chemical hallmark of the endogenous glucocorti-
coids is the existence of inactive 11-oxosteroids and active 11b-
hydroxysteroids that can be interconverted by 11b-hydroxysteroid
5
1
dehydrogenases (11bHSDs) (Fig. 1). Specifically, there are two iso-
forms, 11bHSD1 and 11bHSD2, that control tissue- and cell-specific
concentrations of active glucocorticoids. 11bHSD1 predominantly
catalyzes the reduction of oxosteroids to alcohols, while 11bHSD2
performs the reverse reaction.
Impaired corticosteroid action has been associated with cardio-
metabolic diseases such as hypertension, atherosclerosis, hyperlip-
idemia and diabetes as well as psychiatric disorders. Transgenic
OH
OH
2
O
O
mice overexpressing 11bHSD1 in adipose tissue develop all typical
O
HO
OH
OH
11βHSD1
H
H
H
H
11βHSD2
H
H
⇑
Corresponding authors. Tel.: +1 780 492 5475; fax: +1 780 492 8231 (J.C.V.);
O
O
tel.: +41 61 267 1530; fax: +41 61 267 1515 (A.O.).
E-mail addresses: john.vederas@ualberta.ca (J.C. Vederas), alex.odermatt@
unibas.ch (A. Odermatt).
cortisone
cortisol
Figure 1. Biological equilibrium between cortisone and cortisol.
These authors contributed equally to this study.
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.08.068

K. Pandya et al. / Bioorg. Med. Chem. 21 (2013) 6274–6281
6275
reported.7–10 Due to frequent dialysis, sodium retention is limited
in these patients.
2, it was found that use of a cobalt–boride reduction system30,31
was optimal. Reduction of the azido species to provide 11b-amino-
Some of the adverse cardio-metabolic effects of corticosteroids
are likely to be caused by increased activation of MR in cardiomyo-
cytes, adipocytes and macrophage. Excessive activation of MR by
progesterone (3) was achieved when 2 was refluxed in a CoCl
2
–
NaBH mixture. It is important to note these conditions did not re-
4
2
duce the ketones at C3 or C20. Confirmation of the 11b-amino sub-
stitution was obtained by X-ray crystallographic analysis
(Supplementary information). It should be noted that this repre-
sents an improved method to obtain this skeleton relative to that
aldosterone or cortisol induces detrimental cardiovascular effects,
and MR antagonists improved the morbidity and mortality of pa-
tients with myocardial infarction or heart failure.1
1–13
Further-
2
5,26
more, MR antagonists showed promising results in the treatment
previously reported.
of chronic kidney disease and diabetic nephropathy.1
4–16
Thus, sev-
To add functionality to the amino steroid, we synthesized a ser-
ies of conjugates, as shown in Scheme 2. The glycine-functional-
ized derivative 5 was prepared by reaction of 3 with t-butyl
bromoacetate, followed by acidic hydrolysis of the t-butyl ester.
Alternatively, when 3 was treated with o-nosyl chloride, sulfony-
lated derivative 6 was obtained. Finally, we prepared the 3-(ethyl
2-hydroximino-propionate) derivative 7 by direct displacement
eral pharmaceutical companies are currently developing novel MR
antagonists.
Progesterone and some of its metabolites have been reported to
bind to MR and inhibit 11bHSDs.1
7–19
In the present project, we
used 11a-hydroxyprogesterone as a starting point for the synthesis
of several 11b-aminoprogesterone derivatives with the goal to
identify potential 11bHSD inhibitors and MR antagonists. The lipo-
philicity of steroids has led chemists to introduce various chemical
modifications that can improve their water solubility and bioavail-
3
2
with the corresponding bromide 13.
We propose that amino acid conjugates of steroids can provide
an important handle that can be further functionalized with a vari-
ety of pharmacophores, or to improve water solubility. While
derivatives 5 and 7 can provide this functionality, we also hoped
to prepare derivatives where the steroid served as a ‘side-chain’
2
0
ability. Aminosteroids and amino acid–steroid conjugates are
commonly prepared to overcome these problems.2
1,22
In an effort
to obtain selective inhibitors of one of the 11bHSD isoforms, we
synthesized a small library of aminosteroid and amino acid–steroid
conjugates with the hopes of preparing inhibitors with improved
solubility and bioavailability. Based on previous inhibition studies
of 11b-hydroxyprogesterone (1a),23 we chose to introduce amino
and amino acid functionalities to the 11b-position of progesterone.
This class of molecules presents a synthetic challenge, as only
limited examples of 11b substituted progesterone derivatives ex-
to an
a-amino acid. We attempted to prepare these derivatives
using protected
a
-amino acids bearing a leaving group at the b-po-
sition. In cases where the electrophile was not activated (b-bromo-
alanine), no product was observed. Also, 11b-aminoprogesterone
did not alkylate activated aziridines, which have been used in the
3
3,34
synthesis of lanthionine derivatives.
These results led us to the conclusion that activated, sterically
unencumbered electrophiles, such as those used in the synthesis
of 4–7, are required to overcome the steric demand that the 11b-
aminoprogesterone skeleton provides.
To avoid the challenges of alkylation of 3, we decided to use a
reductive amination approach to increase the diversity of 11b-
aminoprogesterone (Scheme 3). When 3 was treated with enantio-
merically pure Garner’s aldehyde, in the presence of methanol, ace-
2
4–27
ist.
This is based on the steric demands that are introduced
by the rigid steroid backbone, as well as the angular methyl groups
attached to C10 and C13. Following the successful synthesis of
these 11b-amino and amino acid progesterone derivatives, they
were assessed for their potential to inhibit 11bHSD1 and 11bHSD2
as well as for their ability to modulate MR transcriptional activity.
3
tic acid and NaCNBH , the protected amino–alcohol functionalized
derivatives 8a or 8b were obtained in 60% yield. It was found that
2
2
. Results and discussion
3
5
when this reaction was run for longer times (greater than 3–4 h),
.1. Synthesis
0
epimerization at 2 was observed.
While cooler temperatures and shorter reaction times avoided
this problem the diastereomeric products could be separated fol-
lowing deprotection. The acetonide and Boc protecting groups of
To access the sterically hindered 11b-aminoprogesterone back-
bone, we started from commercially available 11
terone (1b). Attempts to introduce the amino functionality via a
number of S 2-type displacement reactions were unsuccessful.
a-hydroxyproges-
8
a or 8b were hydrolyzed in a warm solution of TFA and water
N
to give amino alcohols 9a and 9b.
To convert these amino alcohols to amino acids, we first re-pro-
tected the amino group with Boc using standard conditions to yield
These include displacement via a Mitsunobu reaction with a
DNs-protected glycine nucleophile and the Mitsunobu reaction
with sodium azide as a nucleophile. In each of these cases the de-
sired 11b-substituted product was not observed, and the major
products isolated were a mixture of elimination products (these
compounds were not fully purified or characterized). Finally, we
1
0a or 10b. Oxidation of these protected amino alcohols was suc-
cessfully achieved using Jones oxidation conditions. At this point,
any diastereomeric products that may have been generated during
the reductive amination could be readily separated by silica gel
chromatography. The final amino acid products, which are in es-
2
8
followed the modified Mitsunobu protocol of Loibner and Zbiral
to synthesize 11b-azidoprogesterone (Scheme 1). Treatment of
PPh , diethyl azodicarboxylate and phenol with freshly prepared
hydrazoic acid and 11
sence
c-N-alkylated 2,3-diaminopropionate (Dap) derivatives,
3
2
9
were obtained by TFA cleavage of the Boc protective group to give
amino acids 12a or 12b.
a
-hydroxyprogesterone (1b) gave azido
product 2 in 42% yield. After several attempts at the reduction of
O
O
O
1
1
N3
H2N
HO
H
a
H
b
H
H
H
H
H
H
H
O
O
O
1
a (11-β)
b (11-α)
2
3
1
Scheme 1. Synthesis of 11b-aminoprogesterone. (a) PPh
3
, diethyl azodicarboxylate, phenol, THF, followed by HN
3
and 1b (42%); (b) CoCl
2
ꢀ6H
2 4 2
O, NaBH , H O (62%).

6
276
K. Pandya et al. / Bioorg. Med. Chem. 21 (2013) 6274–6281
Table 1
O
O
H
N
Inhibition of 11bHSD1 and 11bHSD2 by progesterone derivatives
R
O
O
a
H
Compound IC50 M] (mean ± SD)
[
l
H
H
11bHSD1
11bHSD2
1
1
3
4
5
7
8b
10b
12b
a
b
1.16 ± 0.21
0.63 ± 0.13
n.d.
n.d.
n.d.
0.64 ± 0.21
n.d.
n.d.
0.49 ± 0.06
0.40 ± 0.08
2.20 ± 0.12
2.41 ± 0.45
2.69 ± 0.14
1.05 ± 0.26
5.85 ± 1.32
4.21 ± 1.57
6.76 ± 2.00
4
5
(R=tBu)
b
(R=H)
NO2
O
S
O
O
H N
2
O
N
HN
H
c
n.d.
H
H
H
H
H
O
O
3
at a concentration of 10 lM, and IC50 values were determined for
compounds showing more than 70% inhibition (Fig. 2).
Compound 7 with the 3-(ethyl 2-hydroximino propionate) side
chain was the only potent 11bHSD1 inhibitor with an IC50 of
0.64 ± 0.21 lM. However, 7 was not selective and also potently
6
HO
O
H
d
O
N
HO
H
N
O
O
O
Br
13
H
H
inhibited 11bHSD2 with an IC50 of 1.05 ± 0.26
the effect of the parental compound 1b.
lM, thus resembling
O
In contrast to 11bHSD1, several of the 11b-amino derivatives
preferentially inhibited 11bHSD2, that is 3–5, 8b, 10b and 12b, in
the low micromolar range. Interestingly, the S stereoisomers of
compounds 8, 10 and 12 retained higher inhibitory activity com-
pared with the R forms. In summary, activity against 11bHSD2
can be retained with modifications at position 11 on the steroid
backbone. However, compared to 1a and the parental compound
7
Scheme 2. Synthesis of amine substituted 11b-aminoprogesterone derivatives. (a)
t-Butyl bromoacetate, Bu NI, K CO , MeCN (75%); (b) TFA, CH Cl (89%); (c) o-NsCl,
pyridine, CH Cl (61%); (d) 13, Bu NI, K CO , MeCN (33%).
4
2
3
2
2
2
2
4
2
3
2
.2. Biological evaluation of compounds
1
b these 11b-amino derivatives were less active. Nevertheless,
Previous studies showed that 1a is a potent nonselective inhib-
they provide a basis for the development of more potent
compounds.
itor of both 11bHSD enzymes.2
3,36
Morris et al. reported a more
pronounced inhibition of 11bHSD2 compared with 11bHSD1 dehy-
In a next step, 1a and the 11b-aminosteroid derivatives were
tested for direct effects on MR transactivation activity using HEK-
293 cells expressing human MR and a galactosidase reporter gene
under the control of the MR sensitive MMTV promoter. The refer-
ence compound 1a, which has been reported earlier to activate MR
in transfected COS-7 cells with an EC50 of about 50 nM,18 showed
weak agonist activity in transfected HEK-293 cells with an EC50
of 14 ± 5 lM and, as observed previously in the COS-7 cell system,
did not act as antagonist.
Among the 11b-aminosteroid derivatives three compounds
were active. Compound 12a activated MR with an EC50 of
4.1 ± 1.2 lM but did not act as antagonist, whereas compounds 2
2
3
drogenase activity. Interestingly, using rat Leydig cells they ob-
served no inhibition of 11bHSD1 reductase activity. Galignana
et al. provided evidence that 1a is a substrate for both 11bHSD
3
6
enzymes.
In the present study, we determined the inhibitory activity of 1a
and 1b and of derivatives of the latter against cortisone reduction
by human recombinant 11bHSD1 and cortisol oxidation by human
recombinant 11bHSD2 (Fig. 1, Table 1). Both 1a and 1b potently
inhibited 11bHSD1 reductase activity with IC50 of 1.16 ± 0.21 and
0
.63 ± 0.13
lM, respectively, and 11bHSD2 dehydrogenase activity
with IC50 of 0.49 ± 0.06 and 0.40 ± 0.08
lM (Table 1). The novel
derivatives of 1b were then first screened for 11bHSD inhibition
and 6 inhibited aldosterone-induced MR activation with IC50 of
O
HO
2'
2
'
O
N
Boc
O
H2N
O
O
H2N
HN
HN
a
b
H
H
H
H
H
2
H
H
H
H
O
O
8
8
a (2'R)
b (2'S)
9a (2'R)
9b (2'S)
3
O
O
HO
HN
HO
HO
2'
'
2'
HN
Boc
O
H2N
O
O
Boc
HN
HN
HN
c
d
e
H
H
H
H
H
H
H
H
H
O
O
O
1
0a (2'R)
0b (2'S)
11a (2'R)
11b (2'S)
12a (2'R)
12b (2'S)
1
Scheme 3. Synthesis of
c
-substituted amino acid derivatives of 11b-aminoprogesterone. (a) (R)- or (S)-Garner’s aldehyde, MeOH, AcOH, then NaCNBH
3 2
(60%); (b) TFA, H O
5
0 °C; (c) Boc O, NaHCO
2
3
, MeCN, H O (78%, two steps); (d) Jones oxidant, acetone (39%); (e) TFA, CH Cl (quant).
2
2
2

K. Pandya et al. / Bioorg. Med. Chem. 21 (2013) 6274–6281
6277
tilled over sodium in the presence of benzophenone as indicator;
CH Cl was distilled over CaH . Silica gel was purchased from Sili-
2
2
2
cycle (240–400 mesh). Reactions were monitored on silica-coated
plates with a fluorescence indicator. All NMR spectra were re-
corded on Varian Unity or Inova spectrometers, at the indicated
frequencies at room temperature. High-resolution mass spectrom-
etry was recorded on an Agilent 6220 oaTOF, with electrospray
ionization. Reagents were purchased from Sigma–Aldrich, Alfa–Ae-
sar or 3B Scientific Corporation (11b-hydroxyprogesterone) and
used without further purification.
4
.1.1. 11b-Azidoprogesterone (2)
The title compound is synthesized by an adapted procedure.28
Diethyl azodicarboxylate (0.80 mL, 4.6 mmol) is added drop-wise
to a stirred solution of (oven-dried) triphenylphosphine (1.2 g,
4
.6 mmol) and phenol (0.03 g, 0.3 mmol) in anhydrous THF
(
2.4 mL) under argon at room temperature. Stirring is continued
Figure 2. Preliminary inhibition screen of 11bHSD1 and 11bHSD2 by progesterone
derivatives at 10 M. Lysates of HEK-293 cells expressing recombinant human
1bHSD1 or 11bHSD2 were incubated for 10 min at 37 °C in the presence of 200 nM
cortisone and 500 M NADPH to measure 11bHSD1 reductase activity or 50 nM
cortisol and 500 M NAD to measure 11bHSD2 dehydrogenase activity, followed
37
for 2 min, then hydrazoic acid (3.85 mL of 2.4 M solution in ben-
zene, 9.2 mmol) is added, followed by 11 -hydroxyprogesterone
(1b, 0.5 g, 1.53 mmol), and the reaction mixture is refluxed for
20 min. A color change from pink to dark brown indicates comple-
tion of the reaction. After reduced pressure evaporation of solvent,
the crude product is chromatographed twice through silica gel (1st
l
a
1
l
+
l
by determination of the amount of product formed. Results (mean ± SD) are from
three independent experiments.
2 2
column: CH Cl –ethyl ether, 9.5:0.5 to 9:1; second column: ethyl
ether–hexane 5:5 to 8:2) to yield 11b-azidoprogesterone (2,
3
.5 ± 0.2 and 10.9 ± 0.8 lM, respectively, but did not act as an ago-
1
0
(
2
(
.23 g, 42%) as colorless solid. H NMR (400 MHz, CDCl
d, J = 1.7 Hz, 1H), 4.17–4.15 (m, 1H), 2.51–2.35 (m, 5H), 2.24–
.18 (m, 2H), 2.18–2.09 (m, 4H), 2.05–1.93 (m, 1H), 1.91–1.75
m, 2H), 1.74–1.65 (m, 2H), 1.41 (s, 3H), 1.39–1.23 (m, 2H), 1.17–
3
) d: 5.67
nist. The reason for the differences in the sensitivity of COS-7 and
HEK-293 cells for MR activation remains unknown but may be ex-
plained by differences in the experimental procedure and/or the
ability of progesterone and its derivatives to enter the cell. Never-
theless, MR modulation by the three aminoprogesterone deriva-
tives 2, 6 and 12a was observed at lower concentrations than
that by the parental compound 1a, which has been shown to stim-
1
3
0
1
3
3
.96 (m, 3H), 0.90 (s, 3H). C NMR (100 MHz, CDCl ) d: 208.7,
99.3, 171.4, 122.4, 63.5, 57.6, 57.4, 55.1, 42.5, 42.4, 39.0, 35.0,
3.6, 32.3, 31.7, 31.5, 31.2, 24.1, 22.6, 20.4, 14.4. HRMS (ESI) C21-
+
1
8
H
29
3
N O
2
Na m/z: 378.2152 (expected), 378.2142 (found). FTIR
ulate sodium absorption in renal cortical collecting duct cells.
ꢁ1
(
MeOH thin film) cm : 3244, 3015, 2987, 2091, 1710, 1676,
Thus, future studies are needed to explore the use of these amino-
progesterone derivatives as a basis for the development of suitable
MR blockers and to investigate their biological effects.
1
479. ½
ꢂD: +274 (c 0.87, CH
2 2
a Cl ).
4
.1.2. 11b-Aminoprogesterone (3)
To a suspension of azide 2 (1.9 g, 5.4 mmol) and CoCl
3.8 g, 16.1 mmol) in water (10 mL) at room temperature, a
2
ꢀ6H
2
O
3
. Conclusion
(
solution of NaBH (0.92 g, 24.1 mmol) in H O (25 mL) is added
4
2
Despite the steric limitations that are imposed on the 11b-posi-
tion of progesterone, we were able to develop a concise synthesis
of 11b-aminoprogesterone (3) starting from readily available
11a-hydroxyprogesterone (1b). We demonstrated that alkylation
drop-wise (to subside an excessive foaming) with stirring. The
appearance of a black precipitate indicates the formation of a
cobalt boride species. The mixture is then stirred at reflux for
3
.5 h. Upon completion of the reaction, the solution is allowed to
of the newly introduced amino functionality is challenging, and
only activated unbulky electrophiles were useful. Reductive ami-
nation, however did prove to be amenable to extending the func-
tionality of the amine group, and allowed for the synthesis of
amino acid derivatives 12a and 12b. These amino acid derivatives
provide a convenient handle that can be further elaborated in the
search for second generation 11bHSD2 inhibitors and MR antago-
nists. Our results indicate that some of the substituted 11b-amino-
progesterone derivatives prepared display the ability to
preferentially inhibit 11bHSD2. Furthermore, we identified two
compounds with antagonist properties against the MR (2 and 6).
The biological effect of these compounds needs to be further inves-
tigated in suitable cell and animal models.
cool back to room temperature, which is then diluted with ethyl
acetate (30 mL). The resulting biphasic mixture is filtered through
Celite and the aqueous layer is separated. The aqueous phase is ex-
tracted several times, first with ethyl acetate (3 ꢃ 30 mL) and then
with CH
Na SO
purified on silica gel by flash column chromatography (100% ethyl
acetate followed by CH Cl –MeOH, 9.5:0.5) to give the pure amine
(1.1 g, 62%) as a colorless solid. H NMR (500 MHz, CDCl
d, J = 1.7 Hz, 1H), 3.62 (br s, 1H), 2.51–2.34 (m, 5H), 2.26–2.21 (m,
2
Cl
2
(3 ꢃ 30 mL). The combined organic phases are dried
(
2
4
) and concentrated under reduced pressure, and further
2
2
1
3
3
) d: 5.67
(
1
1
3
1
H), 2.20–2.09 (m, 5H), 2.07–2.00 (m, 1H), 1.97–1.88 (m, 2H),
.79–1.68 (m, 2H), 1.44 (s, 3H), 1.35–1.28 (m, 2H), 1.15–1.03 (m,
1
3
3
H), 0.92 (s, 3H). C NMR (125 MHz, CDCl ) d: 209.0, 199.3,
72.2, 122.2, 64.4, 58.4, 56.2, 48.7, 43.1, 39.2, 35.0, 33.9, 32.9,
4
4
. Experimental details
32.0, 31.4, 31.1, 29.8, 24.4, 22.6, 21.6, 16.6. HRMS (ESI) C21H31NO2-
+
H
m/z: 330.2428 (expected), 330.2426 (found). FTIR (MeOH thin
ꢁ1
.1. Chemistry
film) cm : 3381, 3323, 2928, 2874, 1701, 1667, 1446, 1388 ½
a
ꢂ :
D
+
208 (c 0.93, CH Cl ). MP: 142–145 °C.
2
2
All reactions were performed in oven-dried glassware, under a
stream of argon. Solvents were purchased from Fisher and used di-
rectly, unless otherwise noted. When required, solvents were dried
4.1.3. t-Butyl 11b-aminoprogesteronyl acetate (4)
To a mixture of amine 3 (0.100 g, 0.3 mmol), t-butyl bromoace-
tate (88 L, 0.6 mmol), tetrabutyl ammonium iodide and 4 Å MS
as follows: MeOH was distilled over CaH
2
2
; Et O and THF were dis-
l

6
278
K. Pandya et al. / Bioorg. Med. Chem. 21 (2013) 6274–6281
(
0.222 g, 0.6 mmol) in acetonitrile (5 mL) is added K
2
CO
3
(0.166 g,
3 3
28.5 mmol) in CHCl (85 mL) and CH OH (57 mL), is treated with
1
.2 mmol) under argon at room temperature. This mixture is then
hydroxylamine hydrochloride (2.00 g, 28.5 mmol) at room temper-
ature. The mixture is stirred overnight at room temperature and
stirred at reflux for 9 h. The resulting mixture is filtered through
Celite and the solvent is removed under reduced pressure. The res-
idue is dissolved in CH
with water (2 ꢃ 5 mL) followed by brine (1 ꢃ 5 mL). The organic
phase is dried (Na SO ), concentrated under reduced pressure,
and further purified by silica gel flash column chromatography
then concentrated to dryness. The residue is dissolved in CH
(40 mL), washed with 0.1 N HC1 (20 mL) and brine (20 mL), and
dried over Na SO . Evaporation of the solvent gives crystalline
material, which is recrystallized (CH Cl –hexane) to yield the de-
sired product (3.96 g, 68%) as white needles. H NMR (500 MHz,
CDCl ) d: 9.33 (s, 1H), 4.38 (q, J = 7.2 Hz, 2H), 4.27 (s, 2H), 1.38 (t,
J = 7.2 Hz, 3H). C NMR (125 MHz, CDCl
2 2
Cl
2 2
Cl (10 mL) and extracted several times
2
4
2
4
2
2
1
(
ethyl acetate–hexane, 1:1) to give the pure title compound 4
3
1
13
(
0.101 g, 75%) as an off-white solid. H NMR (600 MHz, CDCl
3
) d:
.67 (s, 1H), 3.42 (d, J = 16.8 Hz, 1H), 3.29–3.10 (m, 2H), 2.52–
.35 (m, 5H), 2.25–2.20 (m, 1H), 2.19–2.15 (m, 1H), 2.15–2.13
3
) d: 161.7, 147.9, 62.5,
5 8 3
30.1, 14.3. HRMS (ESI) C H BrNO Na m/z: 231.9580 (expected),
+
5
2
ꢁ1
231.9579 (found). FTIR (MeOH thin film) cm : 3445 (br), 3275,
(
m, 4H), 2.08–2.03 (m, 1H), 1.99–1.90 (m, 1H), 1.90–1.85 (m, 1H),
3057, 2987, 1724, 1472, 1326, 1225, 1184, 1035. Mp: 77–78 °C.
1
1
3
1
3
.79–1.73 (m, 1H), 1.72–1.66 (m, 1H), 1.56 (s, 3H), 1.48 (s, 9H),
.35–1.28 (m, 1H), 1.18 (dd, J = 11.4, 4.5 Hz, 1H), 1.14–1.02 (m,
4.1.7. 11b-N-3-(O-Ethyl-2-hydroxyimino-propanoyl)-
aminoprogesterone (7)
To a mixture of amine 3 (0.05 g, 0.15 mmol), and oxime 13
(0.065 g, 0.3 mmol), in acetonitrile (1 mL) is added tetrabutyl
1
3
3
H), 0.92 (s, 3H). C NMR (125 MHz, CDCl ) d: 209.1, 199.6,
72.5, 171.8, 121.8, 81.4, 64.2, 58.5, 56.1, 55.0, 50.6, 43.3, 40.7,
9.2, 34.6, 34.0, 33.2, 32.0, 31.7, 31.5, 28.1, 24.4, 22.7, 21.8, 15.2.
+
HRMS (ESI) C27
H41NO
4
H
m/z: 444.3108 (expected), 444.3115
2 3
ammonium iodide (0.110 g, 0.3 mmol), K CO (0.085 g, 0.6 mmol)
ꢁ1
(
found). FTIR (MeOH thin film) cm : 3032, 2934, 1731, 1703,
and 4 Å MS under argon at room temperature. The mixture is stir-
red at reflux for 12 h, and then filtered through Celite. The filtrate is
evaporated under reduced pressure, and the residue is dissolved in
1
671, 1367, 1234, 1158. ½
a
2 2
ꢂD: +137 (c 2.00, CH Cl ).
4
.1.4. 11b-Aminoprogesteronyl acetic acid TFA salt (5)
CH
brine (1 ꢃ 2 mL). The organic phase is dried (Na
under reduced pressure, and further purified by silica gel column
chromatography (ethyl acetate–CH Cl , 1:1) to give pure title com-
pound 7 (0.025 g, 33%) as a light yellow solid. H NMR (500 MHz,
CDCl ) d: 8.97 (s, 1H), 5.66 (d, J = 1.6 Hz, 1H), 4.31 (q, J = 7.1 Hz,
2
Cl
2
(2 mL) and extracted with water (2 ꢃ 2 mL) followed by
A solution of ester 4 (0.100 g, 0.23 mmol) in CH Cl
2
2
(2 mL) is
2
SO ), concentrated
4
treated with excess trifluoroacetic acid (4 mL), and stirred at room
temperature for 6 h. After completion of the reaction, the solvent is
evaporated under reduced pressure and the resulting residue is
dried on high vacuum to yield the title amino acid 5 (0.101 g,
2
2
1
3
8
(
9%) as a fluffy white solid. 1H NMR (500 MHz, CD
3
OD) d: 5.73
d, J = 1.5 Hz, 1H), 4.08–4.01 (m, 3H), 2.64–2.50 (m, 4H), 2.42 (dt,
J = 16.7, 4.8 Hz, 1H), 2.39–2.32 (m, 1H), 2.28 (dt, J = 13.5, 5.0 Hz,
H), 2.16 (s, 3H), 2.16–1.94 (m, 3H), 1.95–1.82 (m, 2H), 1.81 (dd,
J = 15.5, 5.0 Hz, 1H), 1.72 (dd, J = 11.8, 4.2 Hz, 1H), 1.58 (s, 3H),
.43–1. 26 (m, 3H), 1.23–1.14 (m, 1H), 0.89 (s, 3H). 13C NMR
125 MHz, CDCl ) d: 211.3, 200.9, 172.8, 170.0, 123.0, 64.2, 58.4,
7.5, 55.5, 42.3, 39.4, 39.1, 35.2, 34.2, 33.6, 32.4, 32.3, 31.0, 27.7,
2H), 3.81 (d, J = 12.9 Hz, 1H), 3.63 (d, J = 13.0 Hz, 1H), 3.19 (s,
1H), 2.54 (dd, J = 13.8, 2.2 Hz, 1H), 2.50–2.29 (m, 5H), 2.24–2.18
(m, 1H), 2.19 (td, J = 4.6, 3.7, 2.1 Hz, 1H), 2.15 (s, 3H), 2.15–2.05
(m, 1H), 2.06–1.97 (m, 1H), 1.91–1.79 (m, 2H), 1.79–1.61 (m,
2H), 1.42 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H), 1.33–1.22 (m, 2H), 1.15
1
13
1
(
(dd, J = 11.5, 4.7 Hz, 1H), 1.14–1.00 (m, 2H), 0.89 (s, 3H). C NMR
3
3
(125 MHz, CDCl ) d: 209.2, 199.8, 172.6, 163.6, 152.5, 121.9, 64.3,
5
2
62.2, 58.4, 56.1, 55.4, 43.2, 40.9, 39.4, 34.5, 34.1, 33.2, 32.0, 31.8,
+
+
4.8, 23.9, 22.0, 14.6. HRMS (ESI) C23
H33NO
4
H m/z: 388.2482 (ex-
31.5, 29.9, 24.5, 22.6, 21.6, 15.4, 14.4. HRMS (ESI) C26
38 2 5
H N O H
ꢁ1
pected), 388.2483 (found). FTIR (MeOH thin film) cm : 3197,
m/z: 459.2853 (expected), 459.2853 (found). FTIR (MeOH thin
ꢁ1
2
962, 1699, 1678, 1422, 1199. ½
a
ꢂD: +112 (c 1.44, H
2
O).
film) cm : 3300, 3058, 2934, 1703, 1668, 1449, 1357, 1189,
015. ½ ꢂD: +152 (c 0.75, CH Cl ).
1
a
2
2
4
.1.5. 11b-N-ortho-Nitrobenzenesulfonyl-aminoprogesterone
(
6)
4.1.8. 11b-N-(3-(N-Tertbutyloxycarbonyl-N,O-acetonide-1-
hydroxy-2(R)-amino-propyl))-aminoprogesterone (8a)
Amine 3 (0.2 g, 0.6 mmol) is mixed with o-nitrobenzenesulfonyl
chloride (1.33 g, 6 mmol) and pyridine (0.48 mL, 6.0 mmol) in CH2-
Cl (5 mL) and then stirred at reflux for 12 h. The resulting mixture
is diluted further with additional CH Cl (5 mL) and then extracted
with water (2 ꢃ 5 mL) and brine (1 ꢃ 5 mL). The organic phase is
dried (Na SO ), concentrated under reduced pressure, and further
purified on silica gel by flash column chromatography (CH Cl fol-
lowed by ethyl acetate–CH Cl , 2:8) to give pure title compound 6
0.19 g, 61%) as a light yellow solid. H NMR (500 MHz, CDCl
This is prepared by adaptation of a known protocol.35 The fol-
lowing is a representative example; both diastereoisomers 8a
and 8b are generated following the same protocol. To a solution
of 3 (330 mg, 1 mmol) in dry MeOH (11 mL), containing acetic acid
(1% v/v), is added (S)-Garner’s aldehyde (230 mg, 1 mmol), and the
mixture is stirred at room temperature under an atmosphere of ar-
gon for 45 min. The resultant imine is reduced by the addition of
2
2
2
2
4
2
2
2
2
1
(
8
3
) d:
.19–8.13 (m, 1H), 7.93–7.87 (m, 1H), 7.83–7.73 (m, 2H), 5.68 (d,
3
NaCNBH (80 mg, 1.25 mmol) in two portions over 30 min. This
is stirred at room temperature for another 2 h, at which point the
reaction is quenched with water (5 mL). The organic solvent is
evaporated, more water is added (5 mL), and the aqueous layer is
J = 1.6 Hz, 1H), 5.48 (d, J = 10.4 Hz, 1H), 4.46 (dtd, J = 10.0, 4.7,
2
3
3
(
1
4
.3 Hz, 1H), 2.51–2.35 (m, 4H), 2.30–2.20 (m, 2H), 2.09–1.96 (m,
H), 1.96–1.80 (m, 2H), 1.77 (s, 3H), 1.76–1.63 (m, 2H), 1.49 (s,
H), 1.48–1.43 (m, 1H), 1.31–1.19 (m, 2H), 1.15–0.99 (m, 2H), 0.53
extracted with CH
are dried over Na
2
Cl
SO
2
(3 ꢃ 15 mL). The combined organic extracts
2
4
, evaporated and the residue is purified by
1
3
s, 3H). C NMR (125 MHz, CDCl
3
) d: 207.8, 199.2, 170.4, 147.8,
36.2, 133.7, 133.4, 129.6, 125.7, 122.7, 63.8, 57.3, 55.2, 51.4, 44.4,
1.9, 39.1, 34.8, 33.9, 32.7, 31.8, 31.7, 30.7, 24.3, 22.6, 21.0, 16.1.
silica gel chromatography (CH
is obtained as a white foam (325 mg, 60%). H NMR (500 MHz,
CDCl ) d: 5.66 (s, 1H), 4.08–3.92 (m, 2H), 3.91–3.82 (m, 1H),
2 2
Cl –MeOH, 96:4). The title product
1
3
+
HRMS (ESI) C27
(
1
H
34
N
2
O
6
SNa m/z: 537.2030 (expected), 537.2028
3.22–3.11 (m, 1H), 3.09–2.91 (m, 2H), 2.66–2.41 (m, 5H), 2.27–
2.09 (m, 5H), 2.08–2.02 (m, 2H), 1.93–1.80 (m, 2H), 1.79–1.64
(m, 2H), 1.60 (s, 3H), 1.51 (br s, 15H), 1.35–1.24 (m, 2H), 1.19
ꢁ
1
found). FTIR (MeOH thin film) cm : 3388, 3092, 2942, 2881,
700, 1667, 1542, 1450, 1355, 1166. ½
a
ꢂD: +275.6 (c 1.13, CH
2 2
Cl ).
(
dd, J = 11.5, 4.5 Hz, 1H), 1.71–1.01 (m, 2H), 0.84 (s, 3H). 13C NMR
4
.1.6. Ethyl 2-(hydroxyimino)-3-bromopropanoate (13)
3
(125 MHz, CD OD) d: 208.9, 199.5, 172.4, 152.6, 121.8, 93.9, 80.8,
The title compound was synthesized as per the literature proce-
dure. Briefly, a stirred solution of ethyl bromopyruvate (3.6 mL,
66.4, 64.2, 58.1, 57.2, 55.1, 55.0, 49.5, 48.9, 43.2, 39.9, 39.3, 34.5,
33.9, 33.1, 31.8, 31.7, 31.5, 28.3, 24.3, 22.6, 21.6, 15.5. HRMS (ESI)
3
2

K. Pandya et al. / Bioorg. Med. Chem. 21 (2013) 6274–6281
6279
C
32
H
50
N
2
O
5
Na+ m/z: 565.3612 (expected), 565.3605 (found). FTIR
evidenced by TLC analysis (CH
basified with NaOH (three drops of a 3 M solution), and extracted
2
Cl
2
–MeOH, 95:5). The solution is
ꢁ1
(
MeOH thin film) cm : 3053, 2974, 2934, 2878, 1699, 1671,
1
617, 1389, 1174, 1086. ½
a
ꢂD: +138 (c 0.74, CH
2
Cl
2
).
with ethyl acetate (3 ꢃ 30 mL). The combined organic extracts
were dried over Na
2
SO
Cl
4
, concentrated and purified by silica gel
4
.1.9. 11b-N-(3-(N-Tertbutyloxycarbonyl-N,O-acetonide-1-
chromatography (CH
uct (181 mg, 78% over two steps). H NMR (600 MHz, CDCl
2
2
–MeOH, 95:5), to yield the desired prod-
1
hydroxy-2(S)-amino-propyl))-aminoprogesterone (8b)
3
) d:
1
H NMR (500 MHz, CDCl
.91–3.82 (m, 1H), 3.15 (br s, 1H), 3.11–3.02 (m, 2H), 2.58–2.30
m, 5H), 2.30–2.19 (m, 2H), 2.15 (s, 3H), 2.10–1.98 (m, 2H), 1.96–
.80 (m, 4H), 1.75 (s, 3H), 1.60 (s, 6H), 1.50 (br s, 9H), 1.40–1.21
3
) d: 5.67 (s, 1H), 4.01–3.86 (m, 3H),
5.69 (s, 3H), 5.14–5.02 (br s, 1H), 3.78–3.64 (m, 3H), 3.18–3.11
(br s, 1H), 3.00–2.91 (m, 1H), 2.96 (dd, 1H, J = 11.0, 3.7 Hz), 2.75–
2.65 (m, 1H), 2.52–2.43 (m, 3H), 2.40–2.30 (m, 1H), 2.26–2.21
(m, 1H), 2.19–2.12 (m, 4H), 2.12–2.02 (m, 3H), 1.94–1.82 (m,
3H), 1.81–1.69 (m, 2H), 1.61–1.56 (m, 1H), 1.52 (s, 3H), 1.48–
1.43 (m, 9H), 1.36–1.25 (m, 3H), 1.26–1.20 (m, 2H), 0.88 (s, 3H).
3
(
1
1
3
(
m, 2H), 1.20–1.17 (m, 1H), 1.17–1.00 (m, 2H), 0.87 (s, 3H).
C
3
NMR (125 MHz, CD OH) d: 208.8, 199.7, 169.4, 155.9, 122.7, 95.4,
8
3
1
3
3.0, 66.8, 64.1, 63.0, 58.2, 56.7, 55.9, 50.9, 43.1, 41.4, 40.6, 39.4,
4.5, 33.6, 33.2, 31.8, 31.4, 30.9, 28.2, 24.3, 22.6, 21.6, 15.5. HRMS
3
C NMR (125 MHz, CDCl ) d: 209.1, 199.4, 172.1, 156.2, 122.1,
79.9, 65.7, 64.2, 58.3, 55.7, 52.1, 49.9, 43.2, 40.3, 39.3, 34.7, 34.0,
+
(
ESI) C32
H
50
N
2
O
5
Na m/z: 565.3612 (expected), 565.3605 (found).
33.2, 31.9, 31.8, 31.6, 29.9, 28.5, 24.5, 22.8, 22.0, 15.5. HRMS (ESI)
ꢁ
1
+
FTIR (MeOH thin film) cm : 3056, 2972, 2935, 2879, 1699, 1671,
C
29
46
H N
2
O
5
H
m/z: 503.3479 (expected), 503.3470 (found). IR
ꢁ1
1
389, 1365, 1207, 1172. ½
aꢂD: +90 (c 0.6, CH
2
Cl
2
).
(MeOH cast film) cm : 3385.4, 2933.3, 1702.2, 1667.7, 1616.1,
523.0, 1452.8, 1365.1, 1171.4, 1031.8, 700.4. ½ ꢂD: +135.6 (c
0.90, MeOH).
1
a
4
.1.10. 11b-N-(3-(1-Hydroxy-2(R)-amino-propyl))-
aminoprogesterone TFA salt (9a)
The following is a representative example; both diastereoiso-
mers 9a and 9b are generated following the same protocol. The
fully protected amino alcohol appended aminoprogesterone 8a
4.1.13. 11b-N-(3-(1-Hydroxy-2(S)-tertbutyloxycarbonyl-amino-
propyl))-aminoprogesterone (10b)
1
3
H NMR (600 MHz, CDCl ) d: 5.66 (s, 1H), 4.99 (br s, 1H), 3.72
(
250 mg, 0.46 mmol) is stirred in a mixture of trifluoroacetic acid
(dd, 2H, J = 4.1, 1.9 Hz), 3.65–3.49 (m, 1H), 3.17–3.13 (m, 1H),
3.10–3.04 (m, 1H), 2.52–2.39 (m, 4H), 2.38–2.32 (m, 1H), 2.24–
2.19 (m, 1H), 2.16–2.11 (m, 4H), 2.11–2.06 (m, 1H), 2.05–1.99
(m, 1H), 1.91–1.88 (m, 1H), 1.87–1.80 (m, 1H), 1.79–1.65 (m,
2H), 1.59–1.53 (m, 3H), 1.46 (s, 3H), 1.44–1.40 (m, 9H), 1.30–1.23
and water (6 mL, 5:1 mixture) at 50 °C for 16 h. The solvents are
evaporated on a high-vacuum rotary evaporator, and then co-evap-
orated with water (3 ꢃ 10 mL), and dried on high vacuum, to afford
the desired product as a white trifluoroacetate salt. This product
was used without further purification. Analytical samples and
(m, 2H), 1.22–1.18 (m, 1H), 1.11–1.03 (m, 2H), 0.84 (s, 3H). 13
C
1
those used for assay were further purified by HPLC. H NMR
3
NMR (125 MHz, CDCl ) d: 209.0, 199.4, 172.1, 156.5, 122.1, 80.0,
(
1
2
600 MHz, D
2
O) d: 5.83 (s, 1H), 4.22–4.16 (m, 1H), 4.16–4.11 (m,
H), 3.99–3.93 (m, 2H), 3.93–3.88 (m, 1H), 3.64–3.56 (m, 1H),
.73 (t, J = 9.2 Hz, 1H), 2.70–2.64 (m, 1H), 2.61–2.51 (m, 2H), 2.46
64.2, 58.4, 56.0, 55.8, 52.9, 49.7, 43.2, 40.4, 39.3, 34.8, 34.0, 33.2,
31.9, 31.8, 31.6, 29.9, 28.5, 24.4, 22.8, 21.8, 15.5. HRMS (ESI) C29-
+
H
46
2
N O
5
H m/z: 503.3479 (expected), 503.3471 (found) IR (MeOH
ꢁ1
(
(
dt, J = 17.0, 5.0 Hz, 1H), 2.37 (ddd, J = 14.8, 4.8, 2.6 Hz, 1H), 2.24
s, 3H), 2.19–2.09 (m, 2H), 2.09–1.98 (m, 3H), 1.95–1.81 (m, 4H),
cast film) cm : 3375.4, 2931.5, 1701.8, 1668.4, 1616.0, 1522.7,
1452.3, 1390.5, 1364.9, 1171.3, 1030.3, 703.2. ½ ꢂD: +120.9 (c
a
1
.43 (s, 3H), 1.42–1.32 (m, 2H), 1.27–1.16 (m, 1H), 0.89 (s, 3H).
0.86, MeOH).
1
3
C NMR (125 MHz, D
2
O) d: 216.0, 203.4, 175.2, 129.1, 121.2,
6
3
2.8, 61.5, 57.6, 56.8, 53.7, 48.3, 46.8, 41.3, 38.0, 36.9, 33.1, 32.5,
1.8, 31.1, 30.7, 23.2, 22.6, 20.2, 13.3. HRMS (ESI) C24H N O H
38 2 3
4.1.14. 11b-N-(3-(1-Carboxy-2(R)-tertbutyloxycarbonyl-amino-
propyl))-aminoprogesterone (11a)
+
m/z: 403.2955 (expected), 403.2948 (found). FTIR (MeOH thin
The following is a representative example; both diastereoiso-
mers 11a and 11b are generated following the same protocol.
The Jones reagent (2 M final concentration) is prepared freshly by
ꢁ
1
film) cm : 3101, 2949, 2564, 1715, 1426, 1212, 1161. ½
a
ꢂD: +91.5
(
c 1.8, H O).
2
dissolution of CrO
by the addition of concentrated H
3
(100 mg, 1 mmol) in water (0.5 mL) followed
SO (0.085 mL, 1.5 mmol). The
4
.1.11. 11b-N-(3-(1-Hydroxy-2(S)-amino-propyl))-
2
4
aminoprogesterone TFA salt (9b)
Boc-protected amino alcohol 10a (127 mg, 0.25 mmol) is stirred
at room temperature in HPLC-grade acetone (2.5 mL). Oxidation
is achieved by the slow addition of freshly prepared Jones reagent
(0.26 mL of a 2 M solution, 0.53 mmol), over 5 min, followed by
stirring at room temperature for a further 1.5 h. The progress of
the reaction is followed by TLC analysis (CH Cl –MeOH, 8:2). Once
2 2
the reaction is deemed complete excess Jones reagent is quenched
with iPrOH (1 mL), and the entire mixture is evaporated. The resi-
1
2
H NMR (600 MHz, D O) d: 5.83 (d, J = 1.6 Hz, 1H), 4.18–4.15 (m,
2
H), 4.07–4.00 (m, 1H), 3.98 (m, 1H), 3.89 (dd, J = 13.9, 6.2 Hz, 1H),
3
.81–3.75 (m, 1H), 2.77–2.64 (m, 2H), 2.62–2.51 (m, 2H), 2.46 (dt,
J = 17.2, 5.1 Hz, 1H), 2.39–2.34 (m, 1H), 2.24 (s, 3H), 2.22–2.12 (m,
H), 2.10–1.98 (m, 3H), 1.97–1.82 (m, 4H), 1.45 (s, 3H), 1.44–1.31
m, 2H), 1.28–1.16 (m, 1H), 0.88 (s, 3H). 13C NMR (125 MHz, D
O)
d: 216.1, 203.7, 175.4, 129.2, 121.2, 62.9, 62.1, 57.5, 56.7, 53.7, 48.5,
2
(
2
4
1
4
1
6.3, 41.4, 37.9, 36.5, 33.1, 32.5, 31.8, 31.1, 30.7, 23.3, 22.5, 20.3,
due is suspended in water (20 mL) and extracted with CH
(2 ꢃ 15 mL) followed by ethyl acetate (2 ꢃ 15 mL). The combined
organic extracts are dried over Na SO , evaporated and purified
by silica gel chromatography (CH Cl –MeOH, 4:1), to yield the
product as a pure white solid (50 mg, 39%). H NMR (600 MHz,
CD OD) d: 5.64 (s, 1H), 4.19–4.09 (m, 1H), 3.75–3.64 (m, 1H),
2 2
Cl
+
3.1. HRMS (ESI)
03.2948 (found). FTIR (MeOH thin film) cm : 3087, 2960, 2570,
703, 1435, 1210, 1168. ½ ꢂD: +89 (c 0.62, CH Cl ).
C
24
38
H N
2
O
3
H
m/z: 403.2955 (expected),
ꢁ1
2
4
a
2
2
2
2
1
4
.1.12. 11b-N-(3-(1-Hydroxy-2(R)-tertbutyloxycarbonyl-amino-
3
propyl))-aminoprogesterone (10a)
3.49–3.41 (m, 1H), 3.03–2.94 (m, 1H), 2.64 (dd, 1H, J = 15.0,
1.9 Hz), 2.60–2.45 (m, 3H), 2.40–2.24 (m, 3H), 2.17–2.06 (m, 3H),
2.00–1.89 (m, 2H), 1.89–1.76 (m, 2H), 1.63–1.58 (m, 1H), 1.60–
1.49 (m, 4H), 1.49–1.40 (m, 9H), 1.40–1.31 (m, 1H), 1.31–1.22
The following is a representative example; both diastereoiso-
mers 10a and 10b are generated following the same protocol.
The deprotected amino alcohol 9a (0.46 mmol) is dissolved in a
water–acetonitrile mixture (6 mL, 1:2 mixture), to which is added
1
3
(m, 2H), 1.19–1.09 (m, 1H), 0.90 (s, 3H). C NMR (125 MHz, CD3-
NaHCO
.48 mmol). The mixture is stirred at room temperature for 2 h,
at which point the starting material is completely consumed as
3
(116 mg, 1.4 mmol) and di-t-butyl-dicarbonate (105 mg,
OD) d: 210.0, 200.2, 174.2, 172.7, 156.4, 121.2, 79.5, 63.0, 57.3,
0
55.3, 54.6, 48.5, 41.5, 38.4, 37.6, 33.4, 33.0, 32.5, 31.1, 31.0, 29.7,
+
27.3, 23.5, 22.3, 19.8, 13.0. HRMS C29
H
44
N
2
O
6
H
(ESI) m/z:

6
280
K. Pandya et al. / Bioorg. Med. Chem. 21 (2013) 6274–6281
5
3
1
17.3272 (expected), 517.3264 (found) IR (MeOH cast film) cmꢁ1:
390.1, 2936.2, 1705.0, 1671.5, 1484.2, 1454.5, 1365.7, 1167.2,
058.5, 1029.2, 871.6. ½ ꢂD: +80.0 (c 1.10, MeOH).
(Waltham, MA, USA), cell culture media from Invitrogen (Carlsbad,
CA), aldosterone from Steraloids (Wilton, NH, USA) and all other
chemicals from Sigma–Aldrich Chemie GmbH (Buchs, Switzerland)
of the highest grade available.
a
4
.1.15. 11b-N-(3-(1-Carboxy-2(S)-tertbutyloxycarbonyl-amino-
propyl))-aminoprogesterone (11b)
4.2.1. 11bHSD enzyme activity
1
H NMR (600 MHz, CD
.76–3.68 (m, 1H), 3.26 (d, 1H, J = 6.2 Hz), 2.81 (app d, 1H,
J = 13.9 Hz), 2.62 (app t, 1H, J = 9.1 Hz), 2.60–2.41 (m, 2H), 2.39–
3
OD) d: 5.66 (s, 1H), 4.18–4.10 (m, 1H),
Inhibition of 11bHSD enzyme activity was measured as de-
1
0
3
scribed earlier. Briefly, HEK-293 cells stably expressing human
11bHSD1 or 11bHSD2 were harvested by centrifugation and cell
pellets were frozen and stored at ꢁ80 °C. Cell pellets were sus-
pended in TS2 buffer (100 mM NaCl, 1 mM EGTA, 1 mM EDTA,
2
1
3
0
1
3
.23 (m, 1H), 2.19 (s, 3H), 2.15–2.04 (m, 3H), 1.98–1.86 (m, 3H),
.86–1.76 (m, 1H), 1.76–1.64 (m, 1H), 1.65–1.59 (m, 1H), 1.51 (s,
H), 1.45–1.40 (m, 9H), 1.38–1.22 (m, 3H), 1.20–1.08 (m, 1H),
.80 (s, 3H). C NMR (125 MHz, CD OD) d: 210.0, 200.5, 178.1,
3
73.3, 156.6, 121.0, 79.5, 63.3, 57.8, 54.9, 53.8, 48.2, 42.1, 38.6,
1 mM MgCl
and immediately used for activity assay. Cell lysates were incu-
bated for 10 min at 37 °C in a final volume of 22 L, containing
2
, 250 mM sucrose, 20 mM Tris–HCl, pH 7.4), sonicated
1
3
l
7.9, 33.7, 33.1, 33.0, 31.2, 30.1, 29.0, 27.3, 23.6, 21.9, 20.4, 13.4.
either vehicle (0.2% DMSO) or the corresponding inhibitor. Inhibi-
tors were diluted in TS2 buffer from stock solutions (10 mM in
+
HRMS (ESI) C29
H
44
N
2
O
6
H
m/z: 517.3272 (expected), 517.3268
ꢁ1
(
found). IR (MeOH cast film) cm : 3413.3, 2935.2, 1702.6,
DMSO). To measure 11bHSD1 activity, the reaction mixture con-
3
1
671.2, 1482.2, 1392.7, 1366.4, 1244.6, 1165.5, 1029.1, 867.8.
tained 200 nM [1,2- H]cortisone and 500
l
M NADPH. 11bHSD2
½
aꢂD: +131.0 (c 1.10, MeOH).
activity was measured similarly at a final concentration of 50 nM
3
+
[
1,2,6,7- H] cortisol and 500 lM NAD . Reactions were stopped
4
.1.16. 11b-N-(3-(1-Carboxy-2(R)-amino-propyl))-
after 10 min by adding an excess of unlabeled cortisone and corti-
sol (2 mM each, in methanol). Steroids were separated by TLC, fol-
lowed by scintillation counting and calculation of substrate
conversion. Data were obtained from three independent
experiments.
aminoprogesterone TFA salt (12a)
The following is a representative example; both diastereoiso-
mers 12a and 12b are generated following the same protocol.
The Boc-protected amino acid derivative 11a (11 mg, 0.02 mmol)
2 2
is stirred at room temperature in a CH Cl –TFA mixture (0.5 mL,
3
:1 mixture) for 30 min. The solvent is evaporated, followed by
4.2.2. MR transactivation activity
co-evaporation with water (3 ꢃ 1 mL), to yield the amino acid
The effect of compounds on the transcriptional activity of MR
was measured as described earlier. Briefly, 150,000 HEK-293
1
40
1
1
1
1
2a as a TFA salt (quant). H NMR (500 MHz, D
2
O) d: 5.79 (br s,
H), 4.08 (dd, 1H, J = 12.5, 4.0 Hz), 4.05–4.01 (m, 1H), 3.79 (dd,
H, J = 11.7, 4.0 Hz), 3.41 (app t, 1H, J = 12.2 Hz), 2.73–2.66 (m,
H), 2.62–2.49 (m, 3H), 2.43 (dt, 1H, J = 17.3, 5.0 Hz), 2.36–2.30
cells/well of a poly-
for 24 h, followed by transfection using calcium phosphate precip-
itation with pMMTV-lacZ b-galactosidase reporter (0.20 g/well),
pCMV-LUC luciferase transfection control (0.04 g/well) and plas-
mid for human recombinant MR (0.35 g/well). The medium was
L-lysine coated 24-well plate were incubated
l
(
m, 1H), 2.21–2.16 (m, 4H), 2.14–2.09 (m, 1H), 2.05–1.96 (m,
H), 1.91–1.77 (m, 4H), 1.48 (app s, 3H), 1.40–1.30 (m, 2H),
.24–1.13 (m, 1H), 0.85 (app s, 3H). 13C NMR (125 MHz, D
O) d:
l
3
1
2
4
1
4
1
l
2
changed 6 h post-transfection and cells were incubated for another
18 h. Cells were washed twice with charcoal-treated DMEM con-
taining 10% FBS and incubated with various concentrations of com-
pounds in the absence of presence of 10 nM aldosterone and for
16.2, 203.9, 175.7. 171.7, 120.9, 62.9, 56.7, 56.6, 53.3, 47.5, 45.5,
1.4, 38.1, 36.5, 32.8, 32.7, 32.0, 31.1, 30.7, 30.7, 23.3, 22.5, 20.1,
+
3.1. HRMS (ESI)
C
24
36
H N
2
O
4
H
m/z: 417.2748 (expected),
ꢁ1
17.2741 (found) IR (MeOH cast film) cm : 2945.7, 1676.9,
24 h. Cells were washed once with PBS and lysed with 50 lL lysis
422.8, 1362.3, 1294.5, 1202.9, 1134.6, 1027.8, 836.7, 721.4. ½
a
ꢂ :
buffer of the Tropix kit (Applied Biosystems, Foster City, CA) sup-
plemented with 0.5 mM dithiothreitol. Lysed samples were frozen
at ꢁ80 °C prior to analysis of b-galactosidase activity using the Tro-
pix kit and luciferase activity using a luciferine-solution. Data were
normalized to the ratio of b-galactosidase to luciferase activity of
the vehicle control and were obtained from three independent
experiments.
D
+
56.6 (c 0.8, MeOH).
4
.1.17. 11b-N-(3-(1-Carboxy-2(S)-amino-propyl))-
aminoprogesterone TFA salt (12b)
1
H NMR (500 MHz, D
J = 12.1, 4.0 Hz), 4.17 (m, 1H), 3.78 (dd, 1H, J = 12.8. 4.0 Hz), 3.62
app t, 1H, J = 12.5 Hz), 2.69 (app t, 1H, J = 9.0 Hz), 2.67–2.60 (m,
H), 2.58–2.47 (m, 2H), 2.40 (dt, 1H, J = 17.3, 5.0 Hz), 2.35–2.29
m, 1H), 2.30–2.16 (m, 4H), 2.15–2.08 (m, 1H), 2.03–1.91 (m,
H), 1.91–1.77 (m, 4H), 1.45 (app s, 3H), 1.40–1.30 (m, 2H),
.25–1.11 (m, 1H) 0.81 (app s, 3H). 13C NMR (125 MHz, D
O) d:
2
O) d: 5.80 (app s, 1H), 4.18 (dd, 1H,
(
1
(
3
1
2
4
2
4
1
4.2.3. Calculations and statistical analysis
Enzyme kinetics was analyzed by nonlinear regression using
the four parameter logistic curve fitting. All data (mean ± SD) were
obtained from at least three independent experiments and signifi-
cance was assigned using the ratio t-test in the GraphPad Prism 5
software.
2
16.1, 203.9, 175.4, 171.5, 121.2, 62.9, 57.0, 53.9, 53.7, 46.0, 43.7,
1.4, 38.2, 37.0, 33.1, 32.6, 31.8, 31.2, 30.8, 30.6, 23.2, 22.5,
0.4,13.3. HRMS (ESI) C24
17.2741 (found) IR (MeOH cast film) cm : 2957.8, 2881.1,
+
H
36
N
2
O
4
H
m/z:: 417.2748 (expected),
ꢁ1
Acknowledgments
676.5, 1431.0, 1360.6, 1203.7, 1137.5, 835.8, 799.2, 721.8. ½
a
ꢂ :
D
+
114.1 (c 0.80, MeOH).
We thank Mark Miskolzie for assistance with NMR analysis, Dr.
Angie Morales-Izquierdo and Dr. Randy Whittal for help with mass
spectrometry, and Dr. Robert McDonald (University of Alberta) for
crystallographic analysis of compound 3. Funding for K.P., D.D., and
J.C.V. was provided by the Natural Sciences Engineering and Re-
search Council of Canada (NSERC) and by the Canada Research
Chair in Bioorganic and Medicinal Chemistry. A.O. and J.S. were
supported by the Swiss National Science Foundation Grant
31003A_140961. A.O. has a Chair for Molecular and Systems Toxi-
cology by the Novartis Research Foundation.
4
.2. Biology
All experiments were performed with intact HEK-293 cells or
homogenates of cells expressing recombinant human proteins.
Construction of expression plasmids of 11b-HSD1 and 11b-HSD2
3
8,39
and of MR and reporter gene have been described earlier.
3
[
1,2- H]-cortisone was purchased from American Radiolabeled
Chemicals (St. Louis, MO), [1,2,6,7- H]-cortisol from Perkin Elmer
3

K. Pandya et al. / Bioorg. Med. Chem. 21 (2013) 6274–6281
6281
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Supplementary data (Tables of crystallographic experimental
details of 3, along with structural diagrams) associated with this
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