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Synthesis and Biochemical Evaluation of Carbamoylalkenylphenyloxy Carboxylic Acid Derivatives as Non-steroidal 5╨Ю┬▒-Reductase Inhibitors.

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239
(Carbamoylalkeny1)phenyloxy Carboxylic Acids
Synthesis and Biochemical Evaluation of (Carbamoylalkeny1)phenyloxy
Carboxylic Acid Derivatives as Non-steroidal Sa-Reductase Inhibitors
Lars Kattner, Sigrid Gohring, and Rolf W. Hartmann*
Fachrichtung 12.1 Pharmazeutische Chemie, Universitit des Saarlandes. D-66041 Saarbriicken, FRG
Received August 22,1994
Synthese uml bioehemisdte Testung von (Cadmmoylalkenylbhenyloxycarbdurederivatetenals nichtstemidale5a-Reduktaseinhibitoren
Several (carbamoylalkenyl) and (carbamoylalkenyl)phenyloxy carboxylic
acids (Table 1) and some of their ethyl esters (Table 2) were synthesized and
evaluated in vitro as inhibitors of steroid 5a-reductase. Inhibitors of this
enzyme may be useful in treating dihydrotestosterone-relateddiseases such
as prostate cancer and benign prostatic hyperplasia Using an enzyme preparation obtained from human prostate carcinoma tissue, the inhibition values
In theI.
series of free
ranged from 0 to 57 % at the given dose of 100 @
acids, surprisingly, the compounds showed only modest inhibitory potency
(&26 46). By contrast, the ethyl esters displayed inhibition values up to 57
%. Structure-activityrelationships are discussed
Verschiedene (Carbamoylalkenyl) und (Carbamoyla1kenyl)phenyloxycarbonsiuren (Tab. 1)und einige ihrer Ethylester (Tab. 2) wurden synthetisiert
und auf inhibitorische Aktivitiit in vitro zur Hemmung der 5a-Reduktase
untersucht. Von Inhibitoren dieses Enzyms verspricht man sich eine
medikamentose Behandlung Dihydrotestosteron-abhangigerErkrankungen,
wie z.B. des Prostatakarzinom bzw. der benignen Hyperplasie der Prostata
Bei Verwendung einer aus menschlichem Prostatakarzinom erhaltenen
Enzymprapamtion wurden bei einer Inhibitorkonzentration von 100 ph4
Hemmwerte von 0 bis zu 57 % ermittelt. Wiihrend die freien Carbonsauren
nur geringe Henunaktivitit (0-26 %) zeigten, wurde das E n y m von den
entspr. Ethylestern m 0-57 % inhibiert.
~
Prostate cancer ( ) is one of the leading causes of death
of the human m a l 3 . Benign prostatic hyperplasia (BHP)
affects more than 50 % of all human males over the age of
502).Both diseases most often require surgical procedures, so
that an alternative medical treatment would be highly desirable. The cell growth in the prostate isregulated, besides other
factors, by the androgen dihydrotestosterone (DHT), which
is formed in androgen responsive tissue by reduction of
testosterone (T), mediated by the enzyme 5a-reductase
(NADPH: A4-3-oxosteroid-5a-oxidoreductase)3).Two distinct isoenzymes of human 5a-reductase (type 1 and 2),
encoded by two different genes, have already been identified
and characterized4). Mechanistically, it has been suggested
that prior to reduction of testosterone, it binds to an electropositive site of the enzyme, generating an enolate-type intermediate. Hydride transfer from NADPH to C-5 and subsequent addition of H+ on C-4 leads to the formation of DHT,
which is finally released by the enzyme NADP' complex5).
Inhibition of the enzyme 5a-reductase by synthetic substrate (T) or product (DHT) analogues causes a reduction of
prostate size by reducing the DHT level, and thus prolifer
tion of tumor cells c n also be suppressed6).Finasteride (1)%
and Epristeride (2) (Scheme 1) are established steroidal
inhibitors, which dramatically reduce DHT formation in v i m
and in viva Finasteride (1)was introduced into theUS market
in 1992 for the treatment of BPH.
However, steroids are expected to cause undesired hormonal side-effects due to their ability to bind to the androgen
receptor. To avoid this problem, non-steroidal inhibitors,
such as 3 9, and 4 lo) (Scheme 2) have recently been developed, exhibiting IC50 values in a nanomolar range.
3
A
4
!Scheme 1
Arch. P h a m (Weinheim) 328,23%245(1995)
Scheme 2
0VCH Valagsgesellschaft mbH, D-69451Weinheim, 1995
0365-6233195l0303-0239S 5.00 + .25&
240
Kattner, Gohring. and Hartmann
In the course of preliminary work in our group concerning
the development of non-steroidal mimics of Finasteride (1)
and related steroidal inhibitors, a series of non-steroidal analogues thereof have been synthesized. Biochemical evaluation of these compounds revealed inhibition values in the
range of o to 99 % in a concentration of 1 0 0 ~ " 1 In
. our
continuing effort to search for new non-steroidal leads, we
would like to report the synthesis of (carbamoylalkeny1)phenyloxy carboxylic acids and some derivatives thereof,
represented by formulas 5 and 6 (Scheme 3) and the biochemical evaluation of these compounds as non-steroidal
inhibitors of 5a-reductase. We assume that a carboxylic acid
chain may be able to mimic the steroid A ring of steroidal
inhibitor 2. Ring B of the steroid skeleton has been replaced
by a benzene nucleus. Ring C is represented by an olefinic
double bond which, in turn, is connected with a bulky amide
function, comparable to the C-17 substituent of the steroidal
inhibitors 1 and 2.
5
Scheme 3
dl
dl
6
-
7 18: R1-
Chemistry
The strategy used for the synthesis of the series of potential
non-steroidal inhibitors of type 5 and 6, namely 11-18, is
outlined in Scheme 4. Etherification of hydroxycarbonyl
compounds 7 and 8 with ethyl bromoacetate afforded the
desired ethers 9 and 10, respectively, in 98 % yield. Subsequent Wiftig reaction with an ylide generated by treatment
of a corresponding phosphonium bromide with BuLi, gave
the olefins 11/12 as E/Z mixtures which in most cases could
easily be separated by column chromatography (@Table 1).
The separated olefins were subjected to a regioselective
saponification, resulting in the potential inhibitors 13/14.
Finally, the C=C bonds of 11-14 were hydrogenated with
W/C as a catalyst to afford l F 1 8 . For the qynthesis of
potential inhibitors containing R = Me and R = NiPr2 or
NHrBu the appropriate ylide was not reactive enough. Consequently, to obtain the desired olefins, the corresponding
phosphonate in a Witrig-Homer reaction was employed for
the C=C connection step, followed by regioselective saponification, chlorination with S K I 2 and treatment of the chloride with the corresponding amine.
Biological Properties
The inhibitory activities of the compounds 11-18 towards
Sa-reductase were determined in vitro, usin an enzyme
preparation of human PC tissue and a [ lp,2p- Hl-testosterone/testosterone mixture, according to the standard enzyme
inhibition assay modified by OUT group"). The % inhibition
values are given in Tables 1 and 2.
Disappointingly, in the series of free carboxylic acids (Table l), potent inhibitors cannot be found. The most active
compounds are the EIZ-isomeric o-substituted diisopropylamides 1% and 13d with inhibition values of 26 and 22 %,
respectively. Neither variation of R3 (NiF'r2, NHfBu) nor the
s
H. El
R2.H.k
R3 =OH. OEl. M u . W r 2
Scheme 4: a) EtOzCCH&/KKCb, acetone, 24 h. room temp., 98%;
b)BrPPhCHKOR3, BuLi, THF, -60 "C, 1 h. 50-83 %; or i)
@0)2POCH2COOEt/NaH. THF. morn temp., 24 h, 40-65 %, ii) NaOH,
EtOH, 24 h, room temp., iii) SOCl2, 2 h, 70 "C, iv) tBuNHz or iPr2NH.
methylene chloride, 3 h, room temp., c) NaOH. EtOH, 1 h, room temp..
8598%; d) HfldC, MeOH. 20 h, mom temp.. 99 %.
position of the side chain (ortho, mefu) nor introduction of a
methyl group as R2 influences the inhibitory potency of the
acids very much. The configuration of the double bond has
no prominent effect on enzyme inhibition (13, 14), whereas
hydrogenation of the double bond seems to decrease inhibition values (17, 18).
The ethyl esters (Table 2 , l l a/b/c, 12a, 15a, 16a).however,
show 2- to 5-fold increased inhibition values as compared
with the corresponding free acids (Table 1,13a/dg, 14a, 17b,
la).It cannot be excluded that the enzyme preparation
contains other enzymes, such as esterases, leading to partial
hydrolysis of the esters. But the fact that the ester 15b is
completely inactive, while the corresponding free acid 17d
shows some activity makes this possibility unlikely.
In the series of carboxylic esters (Table 2). diisopropylamide substitution (llblc, 15a, 16a) is superior to terr-butylamide substitution (lla, 12a). The compounds bearing a
second ethyl ester group (R3=OEt) (lld, 12b, 1%) are nearly
as potent as those containing a diisopropylamide group
(llb/c, 1%. 16a).A free carboxylic acid in this position (R3=
OH) (lle), however, results in a loss of activity. As seen with
the carboxylic acids (Table I), an increase in inhibition cannot be achieved by varying the position of the side chain from
ortho (11, 15) to mera (12, 16). Here again, with H (llb) or
a methyl group as R2 (llc), similar inhibition values are
obtained. Saturation of the double bond leads to a strong
decrease in activity (15).
Arch Phann (Weinheim) 328,239-245 (1995)
24 1
(Carbamoylalkeny1)phenyloxy Carboxylic Acids
Table I: Inhibition of human 5a-reductase by carboxylicacids 13,14,17, and 18 (conc. 100 phf).
no.
13a
13b
13C
13d
13e
13f
1%
13h
17a
17b
17c
17d
RZ
H
H
H
H
Me
Me
Me
Me
H
H
Me
Me
R3
NHtBu
NHtBu
NiRz
NiRz
NHtBu
NHrBu
NiPrz
NlRz
NHtBu
NiR2
NHrBu
NiPrz
olef.
geom.
8
inhib.
no.
(E)
1
0
14a
26
22
14C
14d
14e
(Z)
(E)
(Z)
(E)
6
(UE)
9
11
6
0
9
13
12
(E)
0
14b
14f
14g
14h
18a
18b
1&
18d
RZ
H
H
H
H
Me
Me
Me
Me
H
H
Me
Me
R3
olef.
geom.
NHrBu
NHtBu
NIRZ
NlRz
NHrBu
NHrBu
NiRz
NlR2
NHrBu
NiPr2
NHtBu
NiRz
(E)
R3
olef.
geom.
(Z)
Q
0
(E)
(Z)
(Q
CUE)
%
inhib.
2
25
11
14
3
6
14
7
3
1
0
8
Table 2: Inhibition of human 5a-reductase by carboxylicesters 11,12,15,and 16 (conc. 100 pM).
no.
lla
llb
llc
1ld
lle
15a
15b
15c
R2
H
H
Me
Me
Me
H
Me
Me
R3
NHtBu
NiPrz
NiPrz
OEt
OH
NiRz
NiRz
OEi
olef.
geom.
Q
0
Q
Q
0
8
no.
RZ
inhib.
6
57
55
39
6
29
0
24
1213
12b
16a
H
NHtBu
Me
OEt
Me
N1R2
Finasteride*
Q
(0
%
inhib.
6
40
43
99
* ICsc-value of Finasteride (1) 3nM
The most promising inhibitors of 5a-reductase of the present series, exhibiting more than 50 % inhibition of DHT
formation, are the Econfigurated olefins l l b and l l c bearing
an ethyl ester function as well as a diisopropylamide group.
The optimization of these new lead structures is underway in
our laboratory.
Thanks are due to the h t s c h e Forschungsgemeinschaft (DFG) and to the
Verband der Chemischen Industrie, Fonds der Chemischen Industrie for
financial support.
Experimental Part
Chemistry
GeneralMethods
'H-NMR spectra: Bruker AW 80 (80MHz) or Ah4 400 (400 MHz), CM313,
TMS as internal standard.-IR spectra: Perkin Flmer 398 spectmphdometer.
- Prep. column chromatography: silica gel Merck 60,0.05-0.20 nun. - All
reactions were performed in purified solvents and were monitored on TLC
Arch P h a m ( W e M m ) 328,23%245 (1995)
plates (Macherey Nagel, Alugram S L GIWzs4). -For all potential inhibitors [ll-181 satisfactory elemental analyses (within 0.4 % of the theoretical
values for C. H. N) were obtained.
synthesis of 9 and 10
To a solution of 50 -17
or 8 in acetone (100 ml) were added 13.8 g
(100 mmol) K2CCh and 8.3 g (50 -1)
ethyl bromoacetate. The mixture
was refluxed for20 h, concentrated,diluted with water (100 ml) and extracted
with ether (3 times with 100 ml each). The combined org. layers were dried
(NazSOd), the solvent was removed under reduced pressure and the residue
was distilled in vacuo to afford 9alb and loelb as colorless oils in 98 % yield.
'H-NMR spectra: 9a.6 @pm) = 1.25 (t, 3H, J = 7 Hz, OCHKH3); 4.23 (q.
2H, J = 7 Hz, OCH2CH3): 4.70 (s, 2H, ArOCHz), 6.75-7.95 (m, 4H, arene):
-
10.55(~,1H,CHO).-10a:S@pm)=1.25(t,3H,J=7Hz,OCH2CH3);4.23
(9. 2H, J = 7 Hz, OCffi?cH3), 4.60 (s, 2H, ArOCH2), 7.00-7.75 (m, 4H,
arene), 10.00 (s, lH, CHO) -9b: S (ppm) = 1.45 (t, 3H, J = 7 Hz, OCHzCH3);
2.68 (s, 3H, Me); 4.28 (q,2H, J = 7 Hz. OCH2CH3); 4.72 (s, 2H, ArOCHz);
6.72-7.85 (m4H, arene). -10b: 6 (ppm) = 1.28 (t. 3H. J = 7 Hz, OCHzCH3):
2.56 (s, 3H, Me); 4.24 (q,2H, J = 7 Hz, OCHzCH3): 4.62 (s. 2H. ArOCHz):
7.W7.70 (m4H. arene).
242
Kattner. Gohring, and Hartmann
Table 3 Physical and spectroscopicproperties of compounds 11-18.
no.
mP.
IR (cm-', D r
or film)*
1la
57
3300 (N-H),
1760 (COOEt)
ec)
'H-NMR (80 MHZ,c m 3 , TMS, 6 (ppm))
1.25 (t,3H, J = 7Hz. MKHz0; 1.40 (s, 9H, rBu); 4.20 (q. 2H,
J = 7Hz MeCHzO); 4.60 (s, 2H, ArOCHz); 6.05 (s, 1H. NH);
6.75 and 7.89 (d each, 1H each, J = 16Hz. olefin); 6.60-7.93 (m
4H, arene)
llb
oil
1760 (COOEt).
1640 (NC=O)
1.34 (m,15H, MKHzO, iRMe); 3.70-4.45 (m,4H, iPrCH,
MeCH20); 4.60 (s, 2H, ArOCHz); 6.80 and 7.75 (d each, 1H
each, J = 16Hz, olefin); 6.80-7.65 (m 4H, arene)
1le
oil
1700 (COOEt).
1640 (NC=O)
1.25 (m.15H. MA3H20, tRMe); 2.43 (s, 3H. Me); 3.40 and 4.00
(m each, 1H each, iKH);
4.15 (q, 2H, J = 7Hz MeCHzO); 4.55
(s, 2H, ArocH2); 5.84 (s, lH, olefin); 6.70-7.40 (m,4H, arene)
1Id
oil
1760 and
1710 (C=O),
1630 (C=C)
1.10 (m, 6H. MeCHzO); 2.15 (s, 3H. Me); 3.70-4.35 (m 4H,
MeCH20); 4.55 (s. 2H. ArOCH2); 5.95 (s, lH, olefin); 6 . S 7 . 3 5
(m,4H. arene)
1le
87
1740 and
1710 (C=O),
1640 (C=C)
1.26 (t.3H. MeCH20); 2.50 (s,3H, Me); 4.18 (q, 2H, MeCHz0);
4.65 (s, 2H, ArOCHz); 5.90 (s. lH, olefin); 6.65-7.40 (m. 4H.
arene); 9.55 (s, lH, COOH)
123
67
3310 (N-H),
1760 (COOEt),
1650 (NC=O)
1.24 (t. 3H. 1 = 7Hz, MKH20). 2.40 (s, 9H, tBu); 4.25 (q, 2H. J
= 7Hz MeCHzO); 4.60(s, 2H. ArocHz); 5.50(s, 1H. NH); 6.27
and 7.50 (d each, 2H, J = 16Hz,olefin); 6.75-7.37 (m,4H. arene)
1%
oil
1760 and
1710 (C=O),
1630 (C=C)
1.25 (m, 6H, MeCHzO); 2.50 (s, 3H. Me); 4.20 (m,4H,
MeCH20); 4.60 (s. 2H. ArOCHz); 6.05 (s. lH, olefin); 6.55-7.40
(m,4H, arene)
1L
131
3350 (N-H),
2980 (OH),
1740 (COOH),
1650 (NC=O),
750 (ar)
1.40 (s, 9H, fBu); 4.69 (s,2H, CHI); 5.65 (s, 2H, NWCOOH)
6.55 and 7.87 (d, lH, J = 16Hz. olefin); 6.70-7.60 (m 4H,
arene)
1%
158
3340 (N-H).
2980 (OH).
1740 (COOH),
1650 (NC=O)
13C
oil
2940 (N-H),
1750 (OH),
1640 (NC=O).
1210 (C-0-C)
1.25 (d, 12H. J = Mz.iRMe); 2.254.40 (m, 2H, tPrCH);4.60 (s,
2H, CHz); 6 . a 8 . 0 0 (m 6H, arene, olefin)
13d
oi1
2940 (OH),
1740 (COOH).
1640 (NC=O),
750 (ar)
1.25 (d, 12H. J = 7Hz, iRMe); 3.3M.40 (m,2H, tPrCH);4.62 (s,
2H. CHz); 6.60 (s. 1H. COOH); 6.62-8.00 (m6H. arene, olefin)
1%
139
3405 (N-H),
2960 (OH),
1700 (COOH),
1640/1550(NC=O).
750 (ar)
1.34 (s, 9H. rBu); 2.44 (s,3H, Me); 4.38 (s, 2H, CH2); 5.96 (s.
1H, olefin);6.34 (s. IH, NH); 6.74-7.42 (m,5H, arene, C W H )
13f
oi1
340513370 (N-H),
2970 (OH),
1700 (COOH),
1640/1550(NC=O)
3H, Me); 4.36 (2s, 2H. CH2); 5.94 (s,
1.30 (s, 9H. ~Bu);2.48 (a,
1H. olefin); 6.63-7.40 (m.5H. arene, NH); 8.70 (s. 1H, COOH)
140
2960 (OH),
1600-1 700
(COOH, NC=O.
C=C), 750 (ar)
Arch P h a m (Weinheim) 328,239-245 (1995)
243
(Carbamoylalkeny1)phenyloxy Carboxylic Acids
Table 3:Continued
no.
mP.
IR (cm-'. Wr
'H-NMR (80 MHz, CDCl3, TMS. 8 (ppm))
ec)
or film)*
13h
oil
2%5 (OH),
1710 (COOH),
1610/1560 (NC=O),
1220 (C-O-C)
1.20 (d. 12H,J = 7Hz, iPrMe); 2.10 (s, 3H, Me); 3.40 and 4.00
(m each. 1H each, i W H ; 4.54 (s, 2H, CHz); 5.95 (s. lH, olefin);
6.75-7.40 (m.4H, arene); 9.70 (s. lH, COOH)
1411
oi1
3350 (N-H),
2%5 (OH),
1765 (COOH),
1655 (NC=O)
1.35 (s, 9H, tBu); 4.58 (s, 2H, CH2); 5.90 (s, lH, NH); 6.35 and
7.45 (d each, 1H each, J = l6H2, olefin); 6.6G7.25 (m, 4H.
arene); 7.75 (s. lH, COOH)
14b
142
3250 (N-H),
2%O (OH),
1740 (COOH),
1630 (NC=O),
1255 (C-0-C)
1.20 (s,9H, rBu); 4.60 (s, 2H, CH2); 5.39 (s, IH, NH); 5.96 and
6.64 (d each. IH each, J = 12I-h. olefin); 6.79-7.32 (m. 4H,
arene); 8.35 (s, lH, COOH)
14C
142
2960 (OH),
1660-1560
(NC=O, COOH,
C=C),
1210(c-0-c)
1.30 (d, 12H, J = 7Hz, t M e ) ; 3.70-4.50 (m,2H, iWH)4.60 (s,
2H, CHz); 6.80 (d, lH, J = 16Hz olefin); 6.80-7.65 (m 5H.
arene, olefin)
14d
oi1
2920 (OH),
1740 (COOH),
1640 (NC=O)
1.25 (d. 12H. J = 7Hz c M e ) ;3.20-4.50 (m. 2H. iWH);4.68 (s,
2H. CHz); 6.08 and 6.57 (d each, 1H each, J = 12H2, olefin);
6.70-7.75 (m. 5H, arene, COOH);
14e
159
3320 (N-H),
2975 (OH),
1700 (COOH),
1630 (NC=O),
1185 (C-O-C)
1.30 (s, 9H, tBu); 2.54 (s, 3H. Me); 4.40 (s, 2H, CH2); 5.53 (s,
lH, NH);6.14 (s, lH, olefin); 6.39 (s. lH, COOH); 6.70-7.42 (m,
4H, arene)
14f
oi1
3335 (N-H),
2950 (OH),
1700 (COOH),
1630/1645 (NC=O)
1.36 (s, 9H. tBu); 2.52 (s,3H. Me); 4.38 (s, 2H, CH2); 6.14 (s,
1H. olefin); 6.35 (s, lH, NH), 6.70-7.50 (m. 5H. arene, COOH)
1%
147
2970 (OH),
1700 (COOH),
1620 (NC=O),
1185 (C-O-C)
14h
141
2970 (OH),
1700 (COOH).
1620 (C=C),
1I85 (C-0-C)
1.30 (m 12H, iRMe); 2.10 and 2.50 (s each, 3H. Me); 3.104.35
(m. 2%8 N H ) ; 4.60 (s, 2H, CHz); 6.10 (s, lH, olefin); 6.80-7.40
(m. 4H, arene); 8.00 (s, lH, COOH)
15a
oil
1760 (COOEt),
1650 (NC=O)
1.20 (m 15H, MeCHzO, iRMe); 2.44-4.40 (m,8H, CH2, iPrCH);
4.64 (s.2H. ArOCH2); 6.55-7.25 (m,4H, arene)
15b
oil
1730 (COOEt),
1640 (NC=O)
1.35 (m 18H. MeCH, MeCH20, iPrMe); 2.204.35 (m. 7H,
MeCH20. r X H , MeCH. CH2); 4.60 (s, 2H. ArOCH2);
6.80-7.40 (m, 4H, arene)
15c
oiI
1760 and 1730
(C=O), 1200
(A-0-CHZ)
1.20 (m 9H, Me); 2.62 (m2H, CHz); 3.65 (m, lH, MeCH); 4.15
(m,4H, MeCHz0); 4.60 (s, 2H, ArCCH2); 6.60-7.30 (m,4H.
arene)
16a
oil
1740 (COOEt)
1640 (NC=O)
1.26 (m 18H, MeCH, MeCHzO, iPrMe); 2.45,3.20 and 4.00
(m each, 7H, MeCH20. IRCH MeCH, CH2); 4.60 (s, 2H,
ArOCH2); 6.60-7.30 (m, 4H, arene)
17a
111
3320 (N-H),
2970 (OH).
1710 (COOH)
1.10 (s, 9H, ~Bu),2.25-3.10 (m 4H. CH2); 4.60 (s, 2H, OCHz);
5.95 (s, 1H. NH); 6.58-7.25 (m4H, arene); 8.55 (s. 1H. COOH)
Arch. Pharm (Weinha'm)328,23p-245 (1995)
244
Kattner, Gohring, and Hartmann
Table 3 Continued
1%
97
2970 (OH)
1730 (COOH),
1210 (C-OC),
755 (ar)
1.20 (m, 12H, iRMe); 2.42-3.18 (m, 4H, CHz); 3.42 and 3.98 (m
each, 1H each. IPrCH); 4.60 (s,2H. OCHz): 6.58-7.27 (m,4H,
arene); 8.75 (s, lH, COOH)
17c
oi I
3420 (N-H),
2960 (OH),
1720 (COOH),
1645 (NC=O)
1.30 (d, 3H, MeCH), 1.32 (s, 9H. rBu), 2.45-2.70 (m 2H, CHZ);
3.65 (m, lH, MeCH); 4.35 (s, 2H, OCHz); 6.55 (s, lH, NH); 6.637.30 (m, 4H, arene); 9.75 (s, lH, COOH)
17d
103
2970 (OH),
1700 (COOH).
1640 (NC=O),
1215 (C-0-C)
1.30 (m, 15H, iRMe, MtCH): 2.60 (m, 2H, CH2); 3.10-4.35
(m, 3H, iPrCH. MeCH); 4.62 (s, 2H, OCHz); 6.65-7.30 (m4H,
arene); 8.02 (s, lH, COOH)
18a
127
3340 (N-H),
2950 (OH),
1720 (COOH),
1610 (NC=O)
1.30 (s, 9H, rBu), 2.35 and 2.80 (m each, 2H each, CH2); 4.60
(s, 2H. OCH2); 5.25 (s, lH, NH); 6.67.38 (m 5H. arene,
COOH)
1%
oil
2970 (OH),
1730 (COOH),
1640 (NC=O)
1.20 (m, 12H,rRMe); 2.70 (m 4H, CHz); 3.38 and 3.80 (m
each, 1H each, iPrCH); 4.54 (s, 2H, OCHz); 6.55-7.30 (m,4H,
arene); 8.10 (s, 1H. COOH)
18C
oil
3410 (N-H),
2960 (OH),
1725 (COOH),
1650 (NC=O),
1165 (C-OC)
1.22 (d, 3H, J = 7Hz MeCH); 1.30 (s, 9H, tBu); 2.58 (m, 2H,
CHz). 3.25 (m lH, MeCH); 4.32 (s, 2H, OCH2);6.27 (s, lH,
NH); 6.67.40 (m,4H, arene); 8.20 (s~1H. COOH)
18d
85
2970 (OH),
1720 (COOH),
1625 (NM),
1180 (C-0-C)
1.25 (m. 15H, tRMe, MeCH); 2.55 (m, 2H, CH2); 3.30 and 4.10
(m each. 3H. iPrCH, MeCH); 4.60 (s. 2H, OCHz); 6.67.30 (m
4H. arene): 8.72 (s. lH, COOH)
* Only very strong andlor characteristic wave numbers are given.
Svnthesis of 11/12: A ) 2 = H
Of winig salt BrPh3PCHzC0R3 in THF (lo dl were
with
5 mmol BuLi (3.3 ml of a 1.5 M solution in hexane) at room temp. After 1 h
the SOlUtiOn Was mkd to -60 "cand 3.5 rnmol 98
in THF (3 ml)
were added. The mixture was stirred f a an additional2 h. Water (10 ml) was
added, THF was removed in vacuo and the aqueous residue was extracted
with ether. nK combined ether layers were dried concentrated*and the
remaining oil was chromatographed (hexane, ethyl acetate; 41) to afford
5G83 % of olefins 11/12 as colorless oils in a ratio of approximately EZ =
5:l (Table 3).
was removed in vacuo and the residue was extracted with ether. The aaueous
layer was acidified with dil. HCI and extracted with ether. The combined org.
layem were dried (&SO4), the solvent was removed in yacuo and the
residue was
with o.6 (5.5 mmol) sOClz
for
at 7o Dc. Excess
s m Zwas removed by distillation and the
oil was dissolved in
chloride
ml). mmol r'R2NH or tBuNHz in
chloride dl
were added dropwise. The mixme wBsstirredat room tenp.
for 3 h and worked up by adding
(10ml). neerg. layer wBs
and the aqueous one was extraded with methylene chloride. The combined
org. layers were dried (Nafiod), concentrated,and the residue was chromatographed (hexane, ethyl acetate; 4:l). Physical and spectrosoopicproperties
of compounds 1V12: Table 3.
Synthesis of 11/12: B) R2 = M e
0.29 g (12 mmol) NaH were suspended in THF (100 ml) and 2.9 g (13
mmd)triethyl phosphonoacetatein THF (50 ml) were added dropwise at 0
Synthesis of 13 and 14
"C.Themixturewasstirredfor3hatroomtemp., 2.1 g(10mmol)ofketone
9b or l o b in THF (30 ml) were added and the mixture was stirred for an
additional 20 h. Wata (20 ml) was added, the mixture was concentrated and
extracted with ether. The combined org. layers were dried (NaZs04). the
solvent was removed under reduced pressure and the residue was chromatographed (hexane, ethyl acetate;101) to afford the diestersas colorless oils
in 40 to 65 ?41 yield (ratio of isomers: approximately EZ=lOl). 0.4 g (0.88
mmol) of diester in methanol (30 ml) was then treated with 35 mg (0.90
mmol) NaOH for 20 h at room temp.Water (10 ml) was added, methanol
For selective saponifcation, 1 mmol of ester 11 or 12 in ethanol (30 ml)
was treated with 1 g (25 mmol) NaOH for 1 h at room temp. Dil. HCI was
added until neutralization. Ethanol was removed and the residue was distributed between aq. sat. NaHCa solution and ether. The aqueous layer was
acidified with dil. HCI, extracted with ether and the combined ether phases
were washed with water, dried (MBso4), and the solvent was removed in
vacuo to afford 13 and 14 in 85-98 910 yield; physical and spectroscopic
properties: Table 3.
Arch Phunn (Weinheim) 32823e245 (1995)
245
(Carbamoylalkeny1)phenyloxy Carboxylic Acids
Synthesis of 15-18
To a solution of 0.1 mmol olefin 11-14 in methanol (20ml) were added
catalytic amounts of Pd/C 5%. The mixture was hydrogenated at 1 atm for 3
h. The catalyst was filtered off and the solvent was removed to give 15-18
in 99 % yield (Table 3).
Enzyme Inhibition Tesl
Reagents
[1.2-3H]-testosterone: DuPont, Bad Homburg, Germany. - Quickszint
Flow 302:Zinsser Analyric, Frankfurt, Germany. - 4-MA(Nfl-diethyl-4methyl-3-oxe4-aza-5a-androstane-17~carboxamide)
was a gift from
Merck Sharp & Dohme, Rahway, New Jersey, U.S.A. - All reagents were
of biochemical or analytical grade. - Methanol and water for HPLC were
glass-distilled and degassed prior to use. - HPLC: reversed-phase
125 x 3 m m ID column and a 11 x 3 m m ID precolumn (Nucleosil Cs 3pm
Macherey & Nagel, Diiren, Germany).
Preparation of tissue
Human prostatic tissue from cancer or BPH patients was placed in ice-cold
0.9 % NaCl solution immediately after surgery. The enzyme preparation was
obtained according to Lirang etal.&)All followingoperationswere performed
at 0-4 OC. The prostates were dissected free from fat and connective tissue,
cut into pieces and weighed. Per 1 g of tissue, 3 ml of 20 mM phosphate
buffer pH 6.5containing 0.32 mM sucrose and 1 mM dithiothreitol were
added. The tissue was homogenized by ten 10-sstrokes at 20,500 rpm of an
Ultraturmx (IKA)in 60-s intervals, filtered through cheesecloth and centrifuged for 60 min at 105,000g in a Beckman ultracentrifuge. The pellet was
resuspended in phosphate buffer. The centrifugation was repeated, the final
pellet resuspended in a minimum volume of phosphate buffer and stored in
300 pl portions at -70 "C. The 105,ooOg pellet suspension contains nuclei,
mitochondria, and microsomesand is referred to as enzyme pre ation. The
protein content was determined by the method of Lowry et a l . c n d was in
the range of 15-25 mg/ml.
Incubation procedure
The assay was performed similar to the procedure of Liang et a1.48).All
values were run in duplicate. The incubation was carried out for 30 min at
37 OC in a total volume of 250 pl citrate buffer (40 mM, pH 5.5). The
incubation mixture contained approximately 125 pg human protein, 100pM
NADPH. 0.2
Tincluding 45 nCi [ 1,2-3H]-Tand 2 % dimethyl sulfoxide
with or without test compound (100phQ.The reaction was started by adding
the prostatic enzyme preparation and stopped by addition of 50 pl NaOH
solution (10M). The steroids were extracted using 0.5 ml diethyl ether. After
centrifugation (5 min) the water layer was frozen and the ether layer was
decanted in fresh tubes and evaporated to dryness.
HPLC procedure
Steroid separation was carried out similar to Cook et al.13).The steroids
were dissolvedin 80pl methanol. 35 pl were injected into the computer-controlled HPLC system by a Jasco autosampler.The HPLCsystem was checked
before use with the labelled reference compounds. Radioactivity was measured using a Berthold LB 506C monitor. Using a methanol : water mixture
(1:l.w/w) with a flow of 0.6 ml/min and an additive flow of 1.5 ml of
scintillator, baseline separation was achieved within 24 min.
Calculationprocedure
The amount of DHT formed was calculated (% DHT). The zero value was
subtracted from the control (cv) and inhibition (iv) value (cv-. and iv-.).
) calculated using the following equation:
Inhibition (Iwas
References
1
H. Klosterhalfen, E. Altenihr, H.D. Frank, Das Prosratakacinom,
Thieme Verlag, Stuttgart. 1982.
2 a)P.C. Walsh, Campbell'sUrology,Vol. 1 (Ed.:P.C. Walsh.A.B.Retik
T.A. Stamey, E.D. Vaughan), W.B. Saunders. Philadelphia. 1992, p
1007-1028; b) M. Krieg, K.D. Voigt, J. Steroid Biochem 1976, 7,
1005-1012;~)
R.S. Kirby,T. Christmas, WorldJ. Urol. 1991,9,41-44;
d) P.K. Siiteri. J.D. Wilson, J. Clin. Invest. 1970.49,1737-1745;e) P.C.
Walsh, A.M. Hutchens,L.L. Ewing,J. Clin. Invest. 1983.72.1772-1777.
3 a) J.D. Wilson, Annu. Rev. Physiol. 1978, 40, 279-306; b) N.
Bruchovsky, J.D. Wilson, J. Biol. Chem. 1%8,243,2012-2021.
4 a) T. Liang. A. Cascien, A.H. Cheung, F.G. Reynolds, G.H. Rasmusson,
Endocrinology 1985. 117, 571-579; b) S. Andersson. D.W. Russell,
Proc. Natl. Acad Sci. USA 1990,87,364&3644; c) S . Andersson. D.M.
Berman.E.P. Jenkins.D.W.Russell,Nature1991,354,159-161;d)E.P.
Jenkins, S. Andersson, J. Imperato-McGinley. J.D. Wilson, D.W.
Russell, J. Clin Invest. 1992,89,293-300;
e) E.P. Jenkins, C.-L. Hsieh.
A. Milatovich, K. Normington, D.M. Berman, U. Francke, D.W. Russell.
Genomim 1991,II,1102-11 12;f ) F. Labrie, Y. Sugimoto. V. Luu-The,
J. Simard, Y. Lachance, D. Bachvarov, G. Leblanc, F. Durocher, N.
Paquet, Endocrinology 1992,131, 1571-1573;g) E.P. Jenkins, S. Andersson, J. ImperateMcGinley. J.D. Wilson. D.W. Russell, J. Clin
Invest. 1992, 89,293-300; h) K. Normington, D.W. Russell, J. Biol.
Chem. 1992,267, 19548-19554; i) K. Ichihara, C. Tanaka, J. Steroid
Biochem 1989. 33. 1249-1251; k) B. Houston, G.D. Chisholm. F.K.
Habib, J. SteroidBiochem 1985,22,461467:1) W.J. Watkins, C.E.P.
Goldring, D.B. Gower, J. SteroidBiochem 1988,29,325-331.
5 a)M.A. Levy, M. Brandt. D.A. Holt, B.W. Metcalf, J. SreroidBiochem.
1989,34571-575;b)M.A.Levy,M. Brandt, J.R.Heys,D.A.Holt,B.W.
Metcalf, Biochemistry 1990,29,2815-2824.
6 J.T. I S ~ C DmgS
S,
Today 1993,29,335-342.
7 a) E. Stoner,J. Steroid Biochem Molec. Biol. 1990,37,375-378;b) J.D.
Mdonnell, J.D. Wilson, F.W. George, J. Geller, F. Pappas, E. Stoner,
J. Clin Endocrinol. Merab. 1992,74,505-508; c) P.J. Catafier, Drugs
Future 1991,16.996-999;d) "The Finasteride Group" Prostate 1993,
22,291-299;e) D.H. Peters, E.M. Sorkin, Dmgs 1993,46,177-208;f )
G.H. Rasmusson.G.F. Reynolds, T.Utne, R.B. Jobson, R.L. Primka, C.
Berman, J.R. Brooks, J. Med. Chem. 1984,27, 1690-1701; g) G.H.
Rasmusson, G.F. Reynolds, N.G. Steinberg, E. Walton, G.F. Patel, T.
Liang, M.A. Cascieri, A.H. Cheung, J.R. Brooks, C. Berman, J. Med
Chem. 1986,29,2298-2315:h) J.R. Brooks, C. Berman, R.L. Primka,
G.F. Reynolds, G.H. Rasmusson, Steroids 1986,47,1-19.
8 a) D.A. Holt, M.A. Levy, H.-J. Oh, J.M. Erb, J.I. Haeslip. M. Brandt,
H.-Y. Lan-Hargest, B.W. Metcalf, J. Med. Chem. 1990.33.943-950 b)
P. Audet, H. Nurcombe, Y. Lamb, D. Jorkasky, K. Loyd-Davies, R.
Morris. Clin. Phamacol. %r. 1993,53, 231-231; c) J.C. Lamb, H.
English, P.L. Levandoski, G.R. Rhodes, R.K. Johnson, J.T. Isaars,
Endocrinology 1992,130,685-694;d) Ref. 5.
9 S.Okada, K. Sawada, N. Kayakiri, Y. Sawada. H. Tanaka, M. Hashimoto, WO 930 301 2-A1.1992,Chem Abstr. 1993,119,49227~.
10 Y. Kumazawa, H. Takami, H. Obase, N. Kishibayashi, A. Ishii. EP
0511477-A1.1992,Chem Abstr. 1993,118,1243926.
1 1 R.W. Hartmann, M. Reichert, S . Gtihring, Eur. J. Med. Chem.1994,29,
807-817.
12 O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem.
1951,193.265-275.
13 S.J. Cook,N.C. Rawlings. R.I. Kennedy, Stemids, 1982.40.369-380.
% I = (1 - ivcm./cvcm.) x 100
[Ph288]
Arch. Phann (Weinheim)3281 23%245(1995)
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acid, synthesis, inhibitors, evaluation, carboxylic, non, biochemical, steroidal, derivatives, reductase, carbamoylalkenylphenyloxy
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