A novel class of inhibitors for steroid 5α-Reductase: Synthesis and evaluation of umbelliferone derivatives

Article abstract of DOI:10.1016/S0960-894X(01)00429-2

A series of umbelliferone derivatives was prepared and their 5α-reductase type 1 inhibitory activities were evaluated in cell culture systems. Our studies have identified a new series of potent 5α-reductase type 1 inhibitors and provided the basis for further development for the treatment of human endocrine disorders associated with overproduction of DHT by 5α-reductase type 1. The preliminary structure-activity relationship was described to elucidate the essential structural requirements.

Full text of DOI:10.1016/S0960-894X(01)00429-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2361–2363
A Novel Class of Inhibitors for Steroid 5ꢀ-Reductase:
Synthesis and Evaluation of Umbelliferone Derivatives
Gao-jun Fan,a,b Woongchon Mar,a Man Ki Park,b Eun Wook Choi,a Kinam Kima
and Sanghee Kima,*
aNatural Products Research Institute, Seoul National University, 28 Yungun, Jongro, Seoul 110-460, South Korea
bCollege of Pharmacy, Seoul National University, San 56-1, Shillim, Kwanak, Seoul 151-742, South Korea
Received 24 April 2001; accepted 21 June 2001
Abstract—Aseries of umbelliferone derivatives was prepared and their 5 a-reductase type 1 inhibitory activities were evaluated in
cell culture systems. Our studies have identified a new series of potent 5a-reductase type 1 inhibitors and provided the basis for
further development for the treatment of human endocrine disorders associated with overproduction of DHT by 5a-reductase type
1. The preliminary structure–activity relationship was described to elucidate the essential structural requirements. # 2001 Elsevier
Science Ltd. All rights reserved.
Introduction
steroidal inhibitors have also been designed based on
the structures of the related steroidal inhibitors. Selected
examples of these nonsteroidal inhibitors are benzophe-
none carboxylic acids,9 indole derivatives,6 benzoqui-
nolizinones10 and hydrophenanthren-2-ones.11 More
recently, the evaluation of 6-substituted 1H-quinolin-2-
ones (Fig. 1) as a new class of nonsteroidal inhibitors of
5aR was reported by Hartmann.12
Steroid 5a-reductase (5aR) is an NADPH-dependent
enzyme that catalyzes the reduction of testosterone (T)
into the more potent androgen, dihydrotestosterone
(DHT) (Fig. 1).1 Two different 5a-reductase isozymes,
type 1 and type 2 (5aR-1 and 5aR-2) have been cloned,
expressed, and characterized.1,2 These two isozymes are
differently distributed in the human tissues. While 5aR-
1 is present mainly in hair follicles, sebaceous glands of
the skin (including the scalp) and liver, 5aR-2 is found
predominantly in the prostate, genital skin, seminal
vesicles, epididymis, and liver.2,3 It was reported that
many human endocrine diseases such as acne, male
pattern baldness, alopecia in men and hirsutism in
women are due to elevated levels of DHT.4
In the course of our study to find a potent 5aR-1 inhi-
bitor, we focused our attention on nonsteroidal inhibi-
tors to avoid some of the well-known side effects of
steroidal drugs. On the basis of the transition state
Anumber of different classes of steroidal compounds
that inhibit 5aR have been designed and synthesized as
therapeutic agents for the treatment of DHT-related
pharmacological disorders.5,6 In designing these inhibi-
tors, mimics of the proposed enolate intermediate of the
natural substrate in the reduction were usually
employed.6,7 This approach led to the introduction of
finasteride8 (Proscar1), a 4-azasteroid inhibiting mainly
5aR-2 (Fig. 1). Recently, a number of simpler, non-
*Corresponding author. Tel.: +82-2-740-8913; fax: +82-2-762-8322;
e-mail: pennkim@snu.ac.kr
Figure 1. Chemical structures of testosterone, dihydrotestosterone
(DHT), finasteride and 6-substituted 1H-quinolin-2-ones.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00429-2

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G. Fan et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2361–2363
paradigm along with Hartmann’s results,12 we opted for
the synthesis and biochemical evaluation of the B-ring
substituted coumarin derivatives. To the best of our
knowledge, no coumarin derivatives have been reported
as inhibitors of 5aR. The coumarin skeleton can be
considered as a mimetic of the proposed transition state
of the natural substrate as well as bioisostere of quino-
lin-2-one. We designed and prepared a series of deriva-
tives of umbelliferone (7-hydroxycoumarin), and
screened their 5aR inhibitory activities. Herein, we
report our preliminary results of these studies.
according to a general procedure15 using 7 and its cor-
responding alkylhalide or benzylhalide together with
potassium carbonate in DMF or acetone. Methylation
of coumarin 7 with dimethyl sulfate in aqueous ethanol
solution resulted in 13. Hydrogenation of 15 was con-
ducted in the same manner for compound 4 to afford
16.
Enzyme Inhibitory Activity and Discussion
LNCaP cells are the androgen-sensitive human prostatic
cancer cell line widely available for use in preclinical
investigations16 and it has been reported that 5aR-1 is
expressed in LNCaP cells but not 5aR-2.17 The exis-
tence of androgen sensitivity as well as 5a-reductase
activity in LNCaP cells offered us the opportunity to
study the effects of inhibitors in the enzymatic and in
vitro functional LNCaP responses to androgens in a
single human cell line.
Synthesis
The synthesis of derivatives of umbelliferone 1 was out-
lined in Scheme 1. Propargyl ether 2 was obtained by
heating the solution of umbelliferone 1 and propargyl
bromide together with potassium carbonate and potas-
sium iodide in DMF. Compound 3 was prepared
according to a method of Pettus et al.13 Reduction of 3
under H2 gas in the presence of Lindlar catalyst and
quinoline in EtOAc provided compound 4.
The compounds were evaluated for their steroid 5aR-1
inhibitory activities in cell culture system using LNCaP
cells according to a documented procedure with some
modification.18 Inhibitory effects were represented with
the concentration (mg/mL) giving 50% inhibition (IC50)
relative to the control (0.5% DMSO). In the case of
highly active compounds, IC50 values are also presented
as mM. The obtained results were summarized in Table 1.
8-Allyl-7-hydroxycoumarin 7 was a key intermediate
which can easily be obtained from umbelliferone 1 in
two steps. Treatment of umbelliferone 1 with allyl bro-
mide in H2O/CH2Cl2 two-phase solution in the presence
of sodium hydroxide and tetrabutylammonium iodide
afforded 5 in 82% yield, which was then heated with p-
xylene in sealed tube at 260 ꢀC to afford mainly the C-8
Claisen rearrangement product 7 together with its C-6
isomer 6 in a ratio of 9:1.14
Umbelliferone 1 and 7-alkoxycoumarins 2, 3, and 5 do
not display any inhibitory activity. However, 10,10-
dimethylallyloxycoumarin 4 shows potent inhibitory
activity (IC50=1.3 mM) for the 5aR-1. The considerably
different activity between 1–3, 5, and 4 is possibly a
result of conformational effects of geminal dimethyl
group (the Thorpe–Ingold effect).19 This observation led
us to investigate the compounds 6 and 7 which are the
Claisen rearrangement products of 5. To our delight, 8-
allyl-7-hydroxycoumarin 7 exhibits a potent inhibitory
activity (IC50=0.99 mM) against 5aR-1. The compound
Compound 7 was condensed with phenylisocyanate in
the presence of pyridine in CH2Cl2 at room temperature
to furnish 8. Stirring a solution of 7, benzoyl chloride
and pyridine in CH2Cl2 at 0 ꢀC gave the corresponding
ester 9. Acetylation of 7 using acetyl chloride and pyri-
dine in THF at room temperature afforded ester 10.
Compounds 11, 12, 14, and 15 were synthesized
Scheme 1. Reagents and conditions: (a) propargyl bromide, K2CO3, KI, DMF, 80 ꢀC, 2 h, 98%; (b) (CF3CO)2O, (CH3)2C(OH)CꢁCH, DBU, then
1, CuCl2.H2O, DBU, rt, 2 h, 61%; (c) H2, quinoline, Lindlar cat., EtOAc, rt, 1 h, 73%; (d) allyl bromide, NaOH, KI, cat. (n-Bu)4NI, H2O, CH2Cl2,
rt, 2 h, 82%; (e) p-xylene, 260 ꢀC, 17 h (90% for 7, 10% for 6); (f8) C6H5N¼C¼O, pyr, CH2Cl2, rt, 2 days, 85%; (f9) C6H5COCl, pyr, CH2Cl2, 0 ꢀC,
5 h, 87%; (f10) CH3COCl, pyr, THF, rt, overnight, 89%; (f11) CH3COCH2Cl, K2CO3, acetone, reflux, 4 h, 84%; (f12) BrCH2CH2OH, K2CO3, ace-
tone, reflux, 1 day, 91%; (f13) (CH3)2SO4, NaOH, C2H5OH, H2O, rt, 5 h, 63%; (f14) BnBr, K2CO3, DMF, 60 ꢀC, 3 h, 98%; (f15) CHCC(CH3)2Cl,
K2CO3, DMF, 90 ꢀC, 26 h, 56%; (g) H2, quinoline, Lindlar cat., EtOAc, rt, 5 h, 90%.

G. Fan et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2361–2363
2363
Table 1. Steroid 5a-reductase type 1 inhibitory activities in cell
culture systems using LNCaP cells
References and Notes
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a
Compound
R1
R2
IC50
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Med. Chem. 1995, 2, 583. (b) Holt, D. A.; Levy, M. A.; Met-
calf, B. W. In Advances in Medicinal Chemistry; Maryanoff, B.
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5. For a review of comparative QSAR analysis, see: Kurup,
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(mg/mL) [mM]
Finasteride
1
2
3
4
5
6
7
8
19.8 [53]
>20
>20
>20
0.3 [1.3]
>20
1.7 [8.4]
0.2 [0.99]
0.2 [0.62]
0.15 [0.49]
0.2 [0.82]
>20
>20
>4
>4
>4
13.6 [50.3]
H
H
H
H
H
H
H
CHꢁCCH2
CHꢁCC(CH3)2
CH2¼CHC(CH3)2
CH2¼CHCH2
(see Scheme 1)
H
C6H5NHCO
C6H5CO
CH3CO
CH2¼CHCH2
CH2¼CHCH2
CH2¼CHCH2
CH2¼CHCH2
CH2¼CHCH2
CH2¼CHCH2
CH2¼CHCH2
CH2¼CHCH2
CH2¼CHCH2
CH2¼CHCH2
9
10
11
12
13
14
15
16
CH3COCH2
HOCH2CH2
CH3
C6H5CH2
HCꢁCC(CH3)2
9. Holt, D. A.; Yamashita, D. S.; Konialian-Beck, A. L.;
Luengo, Y. I.; Abell, A. D.; Bergsma, D. J.; Levy, M. A. J.
Med. Chem. 1995, 38, 13.
H2C¼CHC(CH3)2
aDuplicate samples were treated at nontoxic doses to obtain IC50
values.
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Med. Chem. 2000, 43, 3718.
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13. Pettus, Th. R. R.; Inoue, M.; Chen, X.-T.; Danishefsky,
S. J. J. Am. Chem. Soc. 2000, 122, 6160.
6, 6-allyl-7-hydroxycoumarin (IC50=8.4 mM) is also
more active than finasteride (IC50=53 mM) against 5aR-
1, but less active than the corresponding C-8 isomer 7
by about 10-fold.
14. Cairns, N.; Harwood, L. M.; Astles, D. P.; Orr, A. J.
Chem. Soc., Perkin Trans. 1 1994, 3095.
In the investigation of the substituent effects on the 7-
hydroxyl group of 7, the introduction of carbonyl
groups (such as in compounds 8, 9, and 10) resulted in a
slight enhancement of 5aR-1 inhibitory. However, these
compounds are about 100 times more potent than
finasteride. The introduction of alkyl groups into the 7-
hydroxyl position was not suitable for increasing inhi-
bitory potency (13–16). Replacement of the hydro-
phobic alkyl group with an acetonyl group (11) or
ethanol group (12) also caused a drop in potency. From
these results, the existence of a carbonyl group-contain-
ing side chain at the C-7 hydroxyl position of 8-allyl-
coumarin appears to be desirable for potent inhibitory
activity against 5aR-1.
15. Wulff, H.; Rauer, H.; During, T.; Hanselmann, C.; Ruff,
K.; Wrisch, A.; Grissmer, S.; Hansel, W. J. Med. Chem. 1998,
41, 4542.
16. Horoszewicz, J. S.; Leong, S. S.; Kawinski, E.; Karr, J. P.;
Rosenthal, H.; Chu, M. T.; Mirand, E. A.; Murphy, G. P.
Cancer Res. 1983, 43, 1908.
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Motta, M. Prostate 1998, 34, 283.
18. (a) Reichert, W.; Jose, J.; Hartmann, R. W. Arch. Pharm.
(Weinheim) 2000, 333, 201. (b) 5a-Reductase assay: LNCaP
cells were used for 5aR-1 enzyme assay. In brief, cells were
seeded at a density of 2ꢂ105 cells/mL/well in 24-well plates
and cultured for 24 h in media containing 5% hormone-free
fetal bovine serum. Then cells were treated with [3H]-testos-
terone and test samples (final 0.5% DMSO). After an addi-
tional 18 h of incubation, the analysis of [3H]-T and [3H]-DHT
within the medium was carried out by thin-layer chromato-
graphy (TLC). The incubated medium was collected in a
microcentrifuge tube and [3H]-T and [3H]-DHT in medium
were extracted with equal volume of ethyl acetate via vigorous
vortex. The portion of the ethyl acetate extract was dried in
vacuo and resuspended in a 10 mL ethyl acetate. Then, 10 mL
ethyl acetate was subjected to TLC with silica gel (Silica 60W,
Merck, Darmstadt) using chloroform/methanol (96:4, v/v) as
the solvent system. The measurement of [3H]-T and [3H]-DHT
was performed by using an image plate reader (BAS-1500,
Fuji Film, Tokyo). The conversion from T to DHT was cal-
culated from the ratio of the radioactivity of DHT to the sum
of the radioactivity of T and DHT. Inhibitory effects were
represented with the concentration (mg/mL) giving 50% inhi-
bition (IC50) relative to the control (0.5% DMSO).
19. (a) Keese, R.; Meyer, M. Tetrahedron 1993, 49, 2055. (b)
Kirby, A. J. Adv. Phys. Org. Chem. 1980, 17, 183. (c) Mandolini,
L. Adv. Phys. Org. Chem. 1986, 22, 1.
In conclusion, we have designed and evaluated the B-
ring substituted umbelliferone derivatives as 5aR-1
inhibitors. Several compounds showed potent inhibitory
activity towards 5aR-1 isozyme. This new series of
potent 5aR-1 inhibitors could be leads for the develop-
ment of a drug for the treatment of human endocrine
disorders associated with overproduction of DHT by
5aR-1. Further studies for more potent inhibitors based
on the above findings are in process in our laboratory.
Acknowledgements
This study was supported by a grant of the Korea
Health 21 R&D Project, Ministry of Health & Welfare,
Republic of Korea (HMP-00-CH-0014).