Synthesis and bioactivity of new Finasteride conjugate

Article abstract of DOI:10.1016/j.bmcl.2011.03.102

Finasteride is a synthetic 4-azasteroid compound that acts by inhibiting type II 5α-reductase, the enzyme that converts the androgen testosterone to 5α-dihydrotestosterone. It was approved by the US FDA for the treatment of benign prostatic hyperplasia and male pattern baldness. Here the acylation product of Finasteride C-18 amide N-polimod was synthesized by employing acylation reaction with polimod amide as a pivotal intermediate. The structure of the key intermediate and target molecule was confirmed by infrared spectrum, ¹H NMR and ¹³C NMR spectra and mass spectrum, and the inhibition of the steroid 5α-reductase and the rats' benign prostatic hyperplasia by the new Finasteride conjugate and Finasteride was also determined. The inhibition of the Finasteride conjugate on 5α-reductase was stronger than that of Finasteride. Prostate hyperplasia of rats was reduced by Finasteride conjugate treatment similar to the Finasteride treatment. However, the Finasteride conjugate treated animals showed better viable condition than the Finasteride treated ones, suggesting the new compound may have improved toxicity profile than Finasteride.

Full text of DOI:10.1016/j.bmcl.2011.03.102

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3439–3442
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and bioactivity of new Finasteride conjugate
Zhao Shuang a, , Wu Jiazhen b,d, , Yang Lijuan c, Li Zhuo a, Yu Dahai b, Li Jinfeng e, Yu Jing c,
b,
Liang Yongtao a, Wang En-si a,c, , Fang Xuexun
⇑
⇑
a College of Life Science, Jilin University, Changchun 130021, PR China
b Key Laboratory of Molecular Enzymology and Enzyme Engineering of the Ministry of Education, Jilin University, Changchun 130012, PR China
c College of Pharmacy, Jilin University, Changchun 130021, PR China
d State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China
e Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Finasteride is a synthetic 4-azasteroid compound that acts by inhibiting type II 5a-reductase, the enzyme
that converts the androgen testosterone to 5a-dihydrotestosterone. It was approved by the US FDA for
the treatment of benign prostatic hyperplasia and male pattern baldness. Here the acylation product of
Finasteride C-18 amide N-polimod was synthesized by employing acylation reaction with polimod amide
as a pivotal intermediate. The structure of the key intermediate and target molecule was confirmed by
infrared spectrum, 1H NMR and 13C NMR spectra and mass spectrum, and the inhibition of the steroid
Received 22 October 2010
Revised 9 March 2011
Accepted 29 March 2011
Available online 5 April 2011
Keywords:
Synthesis
Finasteride
Conjugates
5a-Reductase
Polimod
5a
-reductase and the rats’ benign prostatic hyperplasia by the new Finasteride conjugate and Finasteride
was also determined. The inhibition of the Finasteride conjugate on 5 -reductase was stronger than that
a
of Finasteride. Prostate hyperplasia of rats was reduced by Finasteride conjugate treatment similar to the
Finasteride treatment. However, the Finasteride conjugate treated animals showed better viable condi-
tion than the Finasteride treated ones, suggesting the new compound may have improved toxicity profile
than Finasteride.
Ó 2011 Elsevier Ltd. All rights reserved.
Finasteride1 a synthesized steroid chemical compound, is an
idiosyncratic inhibitor of intracellular enzymes type II 5a-reduc-
The acylation reaction of Finasteride and acetyl chloride was
examined first in order to realize the peptide reaction between Fin-
asteride and polimod. However, the result demonstrated that Fin-
asteride and polimod could not produce compound 8 as expected
under such a condition because of the inability of polimod to pro-
duce acyl-chlorine effectively. Therefore, a new synthetic route
was designed, that polimod acyl-amine was produced first, which
reacted with Finasteride. In this way, compound 8 was obtained.
(Fig. 1)
tase produced in the process of androgen testosterone metaboliz-
ing into dihydrotestosterone (DHT)2 which is applied to the
treatment of benign prostatic hyperplasia (BPH).3–8 Since struc-
tural modifications of steroid drugs by glycosidation and peptida-
tion have been shown to have a dramatic effect on their physical,
chemical, and biological properties biological activities and conse-
quently benefit in their therapeutic applications9,10, the synthesis
of glycosidation and peptidation of steroidal conjugates are of sig-
nificance for research and development of new steroid-based
drugs.9–15 Polimod (Pidotimod), a synthetic dipeptide, is an orally
bioactive immune enhancer,16 which promotes not only non-idio-
syncratic immune reactions,17 but also idiosyncratic immune reac-
tions.18 Under the inspiration of the glycosidation and peptidation
of steroid, we examined the possibility of Finasteride and polimod
could be joined into a new conjugate molecule, and studied the ac-
When Testosterone (T) was used as substrate, the Km value of
5
a
-reductase was 4.087 0.102
minÀ1. The results showed that Finasteride inhibited 5
effectively. It’s IC50 was 147.49 2.84 nM (Fig. 2). The inhibition of
the Finasteride and polimod conjugate (compound 8) on 5 -reduc-
l
M, Vmax was 0.651 0.010
lM
a-reductase
a
tase was stronger than that of Finasteride. It’s IC50 was
87.98 1.76 nM (Fig. 3).
We next examined the therapeutic effect of compound 8 on rat
model of BPH and compared its effect with Finasteride treatment.
A significant difference of the weight of prostates (Fig. 4) and pros-
tate index was observed (data not shown, no significant differences
in body weight were observed among difference groups of the ani-
mals) between the testosterone propionate (TP) control group and
the normal control group (P <0.05). The increase of the size of pros-
tate and an obvious hyperplasia exist as well through observation
(data not shown). These results proved that the hypodermic injec-
tive suppression of the steroid 5a-reductase by the new conjugate.
We also observed whether the new conjugates could possess the
pharmacological effect of Finasteride, and reduce its side effects.
⇑
Corresponding authors.
E-mail address: dahaiyu.lipase@yahoo.com.cn (F. Xuexun).
Joint first authors.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.102

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Z. Shuang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3439–3442
S
S
O
N
(CH3)3CNH2
O
N
N
H
N
H
DCC
O
CONH
O
CO2H
C(CH3
)
3
1
2
O
O
OH
O
OH
OH
KMnO4
NaIO4
NH3
H
H
H
H
H
H
H
H
H
O
O
HO
O
N
H
O
3
4
5
O
O
OH
C(CH3
)
OH
3
S
O
N
CO
H
N
H
NaBH3CN
2
DDQ/BSTFA
H
H
O
H
NH
H
H
O
N
H
O
N
H
H
H
O
O
N
H
6
7
8
Figure 1. The synthetic route of Finasteride conjugates (compound 8).
12
14
12
10
8
10
8
6
6
4
4
2
2
0
0
.4
-.2
0.0
.2
.4
.6
.4
-.2
0.0
.2
.4
.6
[T(µmol)]-1
[T(µmol)]-1
Figure 2. Lineweaver–Burk plot of Finasteride inhibition of 5a-reductase.
Figure 3. Lineweaver–Burk plot of Finasteride-polimod inhibition of 5
a
-reductase.
tions of testosterone propionate promoted the prostate hyperpla-
sia of rats successfully.
appeared single-layered, and a few basal cells and basement mem-
brane were observed. There was little luminal gland secretion, and
nor expansion in the gland cavity. No hyperplasia was found in
interstitial tissues (Fig. 5A).
Compared with the castrated negative control group, the
growth of prostate and seminal vesicle had been greatly stimulated
in TP control group (Fig. 4). After administration of treatment with
Finasteride conjugate with increased dose (2, 10, 50 mg kgÀ1 dÀ1
,
The prostate gland of the rats in the TP control group arranged
relatively tight, and the size of the gland cavity significantly de-
creased. The columnar gland epithelium was single-layered, how-
ever, most of which was pseudostratified, with its thickness
increased, and arranged disorderly. Some of the epithelium
stretched out papillarily or serratedly towards lumen, and the
fibrosis of the interstitial tissues was observed (Fig. 5B).
In the Finasteride group, the prostate gland of the rats arranged
relatively in order, and the gland cavity recovered to the normal
size. The pseudostratified epithelia reduced, and the stretched
gland epithelial reduced and the hyperplasia of the interstitial tis-
sues decreased (Fig. 5C).
14 d), the prostate and seminal vesicle’s weight in the treatment
group declined significantly, showing the inhibitory effect on be-
nign prostatic hyperplasia in Finasteride conjugate group was sim-
ilar to Finasteride group (Fig. 4A), while no statistical differences
were found between Finasteride conjugate groups and Finasteride
group although the absolute weight value appear higher in the Fin-
asteride conjugate group of the same dosage (Fig. 4A).
We next performed histochemistry of the prostates of the ani-
mals for a closer look of the effects of the Finasteride and Finaste-
ride conjugate treatment. The prostate gland of the rats in the
normal control group arranged orderly. The columnar epithelium

Z. Shuang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3439–3442
3441
Figure 4. The inhibition of BPH in castrated rats by Finasteride and Finasteride-polimod conjugate. (A) The weight of prostate in each group. (B) The weight of seminal
vesicles in each group. Group 1: normal control, Group 2: negative control, Group 3: TP control, Group 4: Finasteride (10 mg kgÀ1 dÀ1) treated, Group 5–7: Finasteride
conjugate (2, 10, 50 mg kgÀ1 dÀ1) treated. TP (5 mg kgÀ1 dÀ1) was injected everyday except the normal group. The value of each group was shown as mean SE (n = 8),
⁄P <0.05, ⁄⁄P <0.01 versus the control group.
Figure 5. The histochemistry of the rats’ prostate slices in each group. (A) normal control group (HE Â 40). (B) TP control group (HE Â 40). (C) Finasteride treated group
(HE Â 40). (D) Finasteride conjugate treated group (HE Â 40). TP (5 mg kgÀ1 dÀ1) was injected everyday except the normal group. The dose used in Finasteride and the
Finasteride conjugate treatment group was 10 mg kgÀ1 dÀ1
.
When used at same dose as the Finasteride group, the Finaste-
ride conjugates treatment group significantly inhibited prostato-
plasia. The prostate gland arranged orderly similar to the animals
in the normal control group. The prostate gland arranged orderly,
and the pseudostratified epithelia and the epithelial protrusions
disappeared. The hyperplasia of the interstitial tissues were also
decreased (Fig. 5D). Taken together, these histochemistry results
for the Finasteride conjugates showed slightly improvement com-
pared to the Finasteride group.
We also observed that the rats in the Finasteride conjugate
group were more active than those in the Finasteride group. The
rats in the castrated control group and Finasteride group were less
active, their skin elasticity reduced, and their fur color was dark
and gloomy. However, the rats in the treatment group had almost
no such appearance (Table 1).
common prostate disease.19 It leads to symptoms of urinary hesi-
tancy, frequent urination, dysuria, increased risk of urinary tract
infections, and urinary retention. A safe, effective and relatively
inexpensive treatment for BPH is in urgent need in clinical research.
In vivo, 5a-reductase is an NADPH-dependent enzyme that
catalysis the reduction of testosterone to the more biologically ac-
tive dihydrotestosterone (DHT), and the accumulation of DHT in
prostate tissue is main cause for BPH. The inhibition of 5
tase activity can reduce the DHT levels in vivo, so as to achieve
the effect of inhibition of BPH.20–23 Thus 5
-reductase inhibitors
can be used as an effective drug for the treatment of BPH.24,25
Finasteride is a selecting type II 5 -reductase inhibitor which
a-reduc-
a
a
has been approved for clinical use. Polimod (Pidotimod) is a syn-
thetic dipeptide molecule with biological and immunological
activity on both the adaptive and the innate immune responses.
It may thus reduce the toxic effect in where the original drug (Fin-
asteride) effects. The results show that comparing with Finasteride,
Prostate disease is becoming the most prominent health issue in
the elderly men. Benign prostatic hyperplasia (BPH) is the most

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Z. Shuang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3439–3442
Table 1
Vital signs in each group of animals
Group
Activity (number of animals)
Normal Reduced Significantly
Hair lose, skin elasticity and fur color (number of animals)
Normal Spotted hair lose, skin elasticity Patched hair lose, severe skin elasticity
reduction, normal fur color reduction, dark fur color
activity
reduced activity
Normal
Negative
Positive
Finasteride
8
1
3
0
1
0
4
4
2
3
0
3
1
6
4
8
2
4
0
0
0
0
1
1
5
4
5
3
3
4
Finasteride conjugate
(2 mgÀ1 kgÀ1 dayÀ1
Finasteride conjugate
)
3
1
4
5
1
2
3
3
3
4
2
1
(10 mgÀ1 kgÀ1 dayÀ1
Finasteride conjugate
(20 mgÀ1 kgÀ1 dayÀ1
)
)
Note: The total number is animals in each group is 8.
3. McConnell, J. D.; Bruskewitz, R.; Walsh, P.; Andriole, G.; Lieber, M.; Holtgrewe,
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tase in vitro and showed improved outcome in vivo, in the rat
model with BPH (Fig. 4). The tissue-specific expression patterns
a
-reduc-
of the 5a-reductase isozymes have been reported previously. The
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inal vesicle, prostate, and liver. The expression level of this isozyme
in the seminal vesicle is lower than that in the prostate26 which
may account for the fact that Finasteride and Finasteride conjugate
were more effective in reducing the organ weight of the prostate
than that of the seminal vesicles in the TP treated rats in our test
(Fig. 4). The rats in the Finasteride conjugate group also showed
better vital signs such as activity, skin elasticity, and fur color than
those in the Finasteride group (Table 1). These suggest that the Fin-
asteride conjugate may have other function on the animals than
that of Finasteride, possibly with improved immune response or
reduced toxicity. It would be of interest to further investigate the
immune modulation activity of the Finasteride conjugate and com-
pare it with Polimod. In addition, it is also important to study the
pharmaco kinetics and metabolism of this compound and see if the
Polimod moiety is cleaved in vivo. Our results warrant further re-
search of this new inhibitor of 5
nign prostate hyperplasia.
a-reductase in the treatment of be-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.102.
27. Hirosumi, J.; Nakayama, O.; Fagan, T.; Sawada, K.; Chida, N.; Inami, M.;
Takahashi, S.; Kojo, H.; Notsu, Y.; Okuhara, M. J. Steroid Biochem. Mol. Biol. 1995,
52, 357.
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