Synthesis of 3-spiromorpholinone androsterone derivatives as inhibitors of 17β-hydroxysteroid dehydrogenase type 3

Article abstract of DOI:10.1016/j.bmcl.2013.09.072

Spiromorpholinone derivatives were synthesized from androsterone or cyclohexanone in 6 or 3 steps, respectively, and these scaffolds were used for the introduction of a hydrophobic group via a nucleophilic substitution. Non-steroidal spiromorpholinones are not active as inhibitors of 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3), but steroidal morpholinones are very potent inhibitors. In fact, those with (S) stereochemistry are more active than their (R) homologues, whereas N-benzylated compounds are more active than their non substituted precursors. The target compounds exhibited strong inhibition of 17β-HSD3 in rat testis homogenate (87-92% inhibition at 1 μM).

Full text of DOI:10.1016/j.bmcl.2013.09.072

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6360–6362
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 3-spiromorpholinone androsterone derivatives
as inhibitors of 17b-hydroxysteroid dehydrogenase type 3
Guy Bertrand Djigoué a,b, Lucie Carolle Kenmogne a,b, Jenny Roy a, Donald Poirier a,b,
⇑
a Laboratory of Medicinal Chemistry, CHU de Québec – Research Center (CHUL, T4), Québec, Qc, Canada
b Department of Molecular Medicine, Faculty of Medicine, Laval University, Québec, Qc, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 June 2013
Revised 18 September 2013
Accepted 23 September 2013
Available online 7 October 2013
Spiromorpholinone derivatives were synthesized from androsterone or cyclohexanone in 6 or 3 steps,
respectively, and these scaffolds were used for the introduction of a hydrophobic group via a nucleophilic
substitution. Non-steroidal spiromorpholinones are not active as inhibitors of 17b-hydroxysteroid dehy-
drogenase type 3 (17b-HSD3), but steroidal morpholinones are very potent inhibitors. In fact, those with
(S) stereochemistry are more active than their (R) homologues, whereas N-benzylated compounds are
more active than their non substituted precursors. The target compounds exhibited strong inhibition
Keywords:
Steroids
Spiromorpholinone
Synthesis
of 17b-HSD3 in rat testis homogenate (87–92% inhibition at 1 lM).
Ó 2013 Elsevier Ltd. All rights reserved.
Enzyme inhibitor
17b-Hydroxysteroid dehydrogenase
The role of 17b-hydroxysteroid dehydrogenase type
3
The chemical steps involved in the synthesis of target
compounds 6A, 6B, 8A and 8B are shown in Scheme 1A. Steroidal
oxirane 3 was synthesized following three steps as previously
reported.11,12 Briefly, the C-17 ketone of ADT (1) was protected
as a dioxolane, the C-3 alcohol function of 2 was then oxidized in
the presence of tetrapropylammonium perruthenate (TPAP) and
N-methylmorpholine-N-oxide (NMO), and this ketone reacted
regioselectively with trimethylsulfoxonium iodide to generate
the oxirane 3. This compound was subjected to aminolysis with
(17b-HSD3) in androgen-dependent prostate cancer is well
established.1–3 This enzyme converts 4-androstene-3,17-dione
(D
4-dione) into the androgenic hormone testosterone (T) in the
presence of cofactor NADPH.4–6 Even though 17b-HSD3 is almost
exclusively expressed in testes, it is up-regulated in prostate
tumors.7 In the classic pathway, T is further converted into the
most active androgen dihydrotestosterone (DHT) by 5a-reductase
(Fig. 1). In fact, both T and DHT can activate the androgen receptor
(AR) and, consequently, stimulate the proliferation of prostate
cancer cells. To stop the androgen biosynthesis at the level of
(L) or (D) phenylalanine methyl ester to yield the amino-alcohol
4A or 4B. These two amino-alcohols were subjected to lactoniza-
tion using sodium methoxide in THF at room temperature and
the spiromorpholinones 5A and 5B were obtained. For this step,
we adapted and optimized a method previously reported for posi-
tion C-17 of the steroid.14 In place of sodium hydride, low loading
of sodium methoxide (0.6 equiv) was used in a diluted reaction
mixture to avoid racemization of the alpha-hydrogen of the amino
acid ester as well as the formation of side products. The secondary
amines 5A and 5B were benzylated following a nucleophilic substi-
tution to give 7A and 7B. The hydrolysis of C-17-dioxolanes 5A, 5B,
7A and 7B in dioxane and aqueous 5% sulfuric acid generated the
target products 6A, 6B, 8A and 8B.
In order to verify the importance of the steroid scaffold for the
inhibition of 17b-HSD3, non-steroidal spiromorpholinones 11 and
12 were synthesized starting from cyclohexanone and following
the sequence of reactions reported in Scheme 1B. We selected only
non-steroidal (S) isomers based on our preliminary results show-
ing that the (S) spiromorpholinone isomer of steroidal derivatives
D
4-dione, a steroid inactive on AR,8 an inhibitor of 17b-HSD3 could
be used. Since this membrane enzyme was not yet crystallized,
structure–activity relationships (SAR) must be established for
inhibitor development. From our previous laboratory work, it
was established that the presence of a hydrophobic group at
position C-3 of androsterone (ADT) is a good strategy for designing
potent inhibitors of 17b-HSD3.9–13
In order to develop novel inhibitors of 17b-HSD3 (Scheme 1),
we decided to build a new ring system (cycle E) at position 3 of
the ADT nucleus. In fact, introducing a 3-spiroheterocyclic moiety
is a good strategy for adding rigidity and introducing diversified
hydrophobic groups with several orientations. It is thus expected
that such groups can increase the affinity to 17b-HSD3 hydropho-
bic pocket.
⇑
Corresponding author. Tel.: +1 418 654 2296; fax: +1 418 654 2298.
E-mail address: donald.poirier@crchul.ulaval.ca (D. Poirier).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.072

G. B. Djigoué et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6360–6362
6361
O
17
OH
OH
17β-HSD3
5α-Reductase
3
O
O
O
4-androstene-3,17-dione
Testosterone (T)
Dihydrotestosterone (DHT)
(Δ4-dione)
Inhibitor
action
Androgenic
action
Androgenic
action
Prostate
Figure 1. Blocking the biosynthesis of testosterone and dihydrotestosterone by using an inhibitor of 17b-HSD3.
produced a better 17b-HSD3 inhibitory activity than the (R)
isomer. Compounds 11 and 12 were thus obtained with 76% and
61% global yields, in three and four steps, respectively.
All final steroidal and non-steroidal compounds as well as their
intermediates were fully characterized (IR, 1H NMR, 13C NMR and
LRMS) to confirm their chemical structure. To illustrate this, we
reported the data from the steroidal and non-steroidal spiromorp-
holinones 6A, 8A and 12.15–17
spiromorpholinones 11 and 12 are not active, suggesting that the
steroidal nucleus is essential for inhibitory activity, but steroidal
morpholinones produced a very good inhibition of 17b-HSD3.
Compounds with (S) conformation on the carbon bearing a benzyl
(Bn) showed better inhibition than their (R) homologues. In fact,
compound 6A blocked 48.8% of the transformation of
0.1
(18.5%). Similarly, compound8A (58.2%inhibitionat 0.1
D
4-dione at
lM and consequently is a better inhibitor than compound 6B
l
M) is more
The inhibitory activity of compounds 6A, 6B, 8A, 8B, 11 and 12 on
17b-HSD3 was evaluated in a microsomal fraction of rat testes using
a known procedure.18 These compounds were compared to RM-
532–105, a knowninhibitor of17b-HSD3,12 for theirability to inhibit
potent than its stereoisomer 8B (25.6%). In this screening assay, the
inhibitory activity of compound 8A (58.2%)seems comparable to
that of the reference compound RM-532-105 (47.3% at 0.1 lM).
For comparison purposes, we next tested the two steroidal
(S)-spiromorpholinone derivatives 6A and 8A with the know
the transformation of
D
4-dione into T (Table 1). Non-steroidal
X
A
O
O
H
O
O
ii
iv
H
H
HO
(77 or 87%)
iii
H
HN
O
HO
3
1 (X = O)
O
4A (S) or 4B (R)
i
2
O
(X = OCH2CH2O)
(62 or 65%)
v
18
X
X
12
11
19
13 17
16
D
E
1
9
8
2
14
5A 5B
or
vi (
)
15
10
5
B
C
3
N
HN
7
A
O
6
4
O
O
(70 or 61%)
O
7A (S) or 7B (R) (X = OCH2CH2O)
5A (S) or 5B (R) (X = OCH2CH2O)
vii
vi
8A
8B
(R) (X = O)
(S) or
6A
6B
(R) (X = O)
(S) or
(70 or 81%)
(97 or 56%)
B
NH
HN
v
vi
iv
iii
N
OH
O
O
O
O
O
O
O
O
9
11
(S)
10 (S)
12
(S)
Scheme 1. Synthesis of spiromorpholinone derivatives. Reagents and conditions: (i) HOCH2CH2OH, p-TSA, toluene, reflux; (ii) NMO, molecular sieves, TPAP, DCM, rt, 3 h; (iii)
(CH3)3SOI, NaH, DMSO/THF, rt; (iv) ( ) or ( )-phenylalanine methyl ester, MeOH, 90 °C; (v) CH3ONa, THF, rt; (vi) DIPEA, C6H5CH2Br, DCM, 75 °C, 22 h; (vii) H2O/H2SO4, dioxane, rt.
L
D

6362
G. B. Djigoué et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6360–6362
Table 1
Inhibitory activity toward 17b-HSD3 of the target compounds 6A, 6B, 8A, 8B, 11 and 12
b
Structures
Names
R/S
R1
R2
Inhibition at 0.1
l
Ma (%)
Inhibition at 1
l
Ma (%)
Inhibition IC50 (nM)
6A
6B
S
R
Bn
Bn
H
H
48.8 2.0
18.5 26.9
92.0 1.6
63.2 5.8c
22
—
O
8A
8B
S
R
Bn
Bn
Bn
Bn
58.2 1.7
25.6 5.7
90.4 0.7
87.3 2.8
58
—
R2
N
O
R1
O
O
11
12
S
S
Bn
Bn
H
Bn
6.2 6.3
24.2 3.2
19.5 3.8
24.3 4.2
—
—
R2
R1
N
O
O
CF3
RM-532-105d
—
—
—
47.3 12.4
92.1 0.4
14
N
N
S
O
OH
O
a
Transformation of [14C]-4-androstene-3,17-dione (50 nM) into testosterone by 17b-HSD3 in microsomal fraction of rat testes. Results are expressed as mean SD of
triplicate.
b
IC50 values were determined from the inhibition curves reported in Figure 2 using GraphPad-Prism 6 software (GraphPad Inc Solfware).
This inhibition value was obtained from another experiment performed under the same conditions.
Compound 15b in Ref. 12.
c
d
2. Montgomery, R. B.; Mostaghel, E. A.; Vessella, R.; Hess, D. L.; Kalhorn, T. F.;
RM532-105
6A
Higano, C. S.; True, L. D.; Nelson, P. S. Cancer Res. 2008, 68, 4447.
3. Day, J. M.; Tutill, H. J.; Foster, P. A.; Bailey, H. V.; Heaton, W. B.; Sharland, C. M.;
Vicker, N.; Potter, B. V. L.; Purohit, A.; Reed, M. J. Mol. Cell. Endocrinol. 2009, 301,
251.
100
50
0
8A
4. Penning, T. M. Endocr. Relat. Cancer 1996, 3, 41.
5. Mohler, M. L.; Naranayan, R.; He, Y.; Miller, D. D.; Dalton, T. D. Recent Pat.
Endocr. Metab. Immune Drug Discov. 2007, 1, 103.
6. Poirier, D. Expert Opin. Ther. Pat. 2010, 20, 1123.
7. Koh, E.; Noda, T.; Kanaya, J.; Namiki, M. Prostate 2002, 53, 154.
8. Laplante, Y.; Poirier, D. Steroids 2008, 73, 266.
9. Tchedam Ngatcha, B.; Luu-The, V.; Poirier, D. Bioorg. Med. Chem. Lett. 2000, 10,
2533.
10. Tchedam Ngatcha, B.; Luu-The, V.; Labrie, F.; Poirier, D. J. Med. Chem. 2005, 48,
5257.
-12
-10
-8
-6
-4
Inhibitor concentration (Log M)
11. Maltais, R.; Luu-The, V.; Poirier, D. J. Med. Chem. 2002, 45, 640.
12. Maltais, R.; Fournier, M. A.; Poirier, D. Bioorg. Med. Chem. 2011, 19, 4652.
13. Djigoué, G. B.; Simard, M.; Kenmogne, L. C.; Poirier, D. Acta Cryst. 2012, c68,
c231.
Figure 2. Effect of 6A, 8A and RM-532-105 on the transformation of [14C]
D
4-dione
(50 nM) into [14C]-T by 17b-HSD3. Results are expressed as mean SD of triplicate.
14. Rouillard, F.; Roy, J.; Poirier, D. Eur. J. Org. Chem. 2008, 14, 2446.
15. Data for compound 6A: White foam. IR (film, m
, cmÀ1) 3333 (NH), 3032 (CH, Ph),
17b-HSD3 inhibitor RM-532-105 (Fig. 2). Spiromorpholinones 6A
and 8A (IC50 = 22 and 58 nM, respectively) are about twofold and
fourfold less potent than RM-532-105 (IC50 = 14 nM) (Table 1).
These results confirm the potency of 6A and 8A as new lead
compounds for inhibiting 17b-HSD3. Thus, we are confident that
such 3-spiromorpholinone ADT derivatives can be improved by
judicious diversification of the spirocycle as well as localization
of the hydrophobic group. Further work reporting full details of
the diversification of the 3-spiromorpholinone, including chemis-
try, SAR study and biological activity of the target compounds is
underway and will be presented in a full paper in due course.
1736 (C@O, ketone and lactone). 1H NMR À400 MHz (Acetone-d6, d, ppm) 0.81
(s, CH3-19), 0.84 (s, CH3-18), 0.80–2.00 (unassigned CH and CH2), 2.37 (dd,
J1 = 8.7 Hz and J2 = 18.2 Hz, CH-16b), 2.79 and 2.89 (2d of AB system,
J = 13.3 Hz, CH2N), 3.02 and 3.17 (2 m, CHCH2Ph), 3.73 (dd, J1 = 4.0 Hz and
J2 = 8.1 Hz, NCHCO), 7.25 (m, Ph). 13C NMR À100 MHz (CDCl3, d, ppm) 11.3,
13.8, 20.2, 21.7, 27.8, 30.5, 31.0, 31.5, 32.83, 35.0, 35.8, 36.0, 38.0, 39.2, 47.8,
51.4, 52.6, 53.7, 53.8, 58.7, 82.7, 127.1, 128.8 (2C), 129.5 (2C), 137.2, 170.8,
221.3. LRMS (m/z) calcd. for C29H40NO3 [M+H]+ 450.29, found 450.35.
16. Data for compound 8A: White foam. IR (film, m
, cmÀ1) 3063, 3032 (CH, Ph) and
1728 (C@O, ketone and lactone). 1H NMR À400 MHz (Acetone-d6, d, ppm) 0.65
(s, CH3-19), 0.80 (s, CH3-18), 0.82–2.00 (unassigned CH and CH2), 2.22 and 2.59
(2d of AB system, J = 12.5 Hz, CH2N), 2.36 (dd, J1 = 8.7 Hz and J2 = 18.4 Hz, CH-
16b), 3.27 and 4.40 (2d of AB system, J = 13.9 Hz, NCH2Ph), 3.37 (m, CHCH2Ph),
3.51 (dd, J1 = 2.9 Hz and J2 = 5.1 Hz, NCHCO), 7.31 (m, 2Â Ph). 13C NMR
À100 MHz (Acetone-d6, d, ppm) 10.7, 13.1, 20.0, 21.4, 27.7, 30.6, 30.6, 31.6,
33.0, 34.9, 35.1, 35.2, 35.8, 37.9, 39.6, 47.3, 51.2, 54.1, 57.4, 57.7, 65.9, 80.5,
126.5, 127.1, 127.8 (2C), 128.4 (2C), 128.5 (2C), 130.4 (2C), 138.0, 138.1, 169.5,
218.7. LRMS (m/z) calcd. for C36H46NO3 [M+H]+ 540.34, found 540.40.
Acknowledgment
We would like to thank the Canadian Institutes of Health
Research (CIHR) for an operating grant. Careful reading of this
manuscript by Ms. Micheline Harvey is also greatly appreciated.
17. Data for compound 12: Yellowish oil. IR (film, m
, cmÀ1) 3063, 3032 (CH, Ph) and
1720 (C@O, ketone and lactone). 1H NMR À400 MHz (CDCl3, d, ppm) 0.87–1.74
(unassigned CH2 of cyclohexyl), 2.08 and 2.65 (2d of AB system, J = 12.6 Hz,
CH2N), 3.18 and 4.31 (2d of AB system, J = 13.7 Hz, NCH2Ph), 3.26 and 3.54
(2 m, CHCH2Ph); 3.52 (m, NCHCO), 7.28 (m, 2Â Ph). 13C NMR À100 MHz
(CDCl3, d, ppm) 21.2, 21.4, 25.3, 34.3, 35.7, 35.8, 56.0, 58.1, 66.3, 81.7, 126.7,
127.4, 128.0 (2C), 128.4 (2C), 128.5 (2C), 130.3 (2C), 137.5, 137.5, 171.0. LRMS
(m/z) calcd. for C23H28NO2 [M+H]+ 350.20, found 350.20.
References and notes
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18. Djigoué, G. B.; Ngatcha, B. T.; Roy, J.; Poirier, D. Molecules 2013, 18, 914.
T., Jr., Hellman, S., Rosenberg, S. A., Eds.; Lippincott: Philadelphia, 1985; p 193.