Structure-activity relationships of estrogen derivatives as aromatase inhibitors. Effects of heterocyclic substituents

Article abstract of DOI:10.1248/cpb.56.1304

Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic target for the selective lowering estrogen level in patients with estrogen-dependent breast cancer. We prepared and tested series of the pyridine- and other heterocyclic ring-containing derivatives of 2- and 4-aminoestrones, estrone, and estradiol, compounds 5, 10, 12 and 15. The isonicotinyl derivatives of 2- and 4-aminoestrone, compounds 5c and 10c, were fairly potent competitive inhibitors of aromatase (K_I, 2.1±0.14 and 1.53±0.08 μm for 5c and 10c, respectively) and other compounds did not show, to a significant extent, the aromatase inhibitory activity. This result suggests that the isonicotinyl-substituted derivatives 5c and 10c would be accessible to the active site of aromatase.

Full text of DOI:10.1248/cpb.56.1304

1304
Chem. Pharm. Bull. 56(9) 1304—1309 (2008)
Vol. 56, No. 9
Structure–Activity Relationships of Estrogen Derivatives as Aromatase
Inhibitors. Effects of Heterocyclic Substituents
Mitsuteru NUMAZAWA,* Sachiko KOMATSU, Takako TOMINAGA, and Kouwa YAMASHITA
Tohoku Pharmaceutical University; 4–4–1 Komatsushima, Aoba-ku, Sendai 981–8558, Japan.
Received May 13, 2008; accepted June 18, 2008; published online June 27, 2008
Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic tar-
get for the selective lowering estrogen level in patients with estrogen-dependent breast cancer. We prepared and
tested series of the pyridine- and other heterocyclic ring-containing derivatives of 2- and 4-aminoestrones, es-
trone, and estradiol, compounds 5, 10, 12 and 15. The isonicotinyl derivatives of 2- and 4-aminoestrone, com-
pounds 5c and 10c, were fairly potent competitive inhibitors of aromatase (Ki, 2.1ꢀ0.14 and 1.53ꢀ0.08 mM for 5c
and 10c, respectively) and other compounds did not show, to a significant extent, the aromatase inhibitory activ-
ity. This result suggests that the isonicotinyl-substituted derivatives 5c and 10c would be accessible to the active
site of aromatase.
Key words aromatase; inhibition activity; estrone derivative; estradiol derivative; isonicotinyl derivative; competitive inhibitor
Experimental
Aromatase is a cytochrome P-450 enzyme responsible for
catalyzing the conversion of the androgens, androstenedione
Materials and General Methods [1b-3H] Androstenedione (AD)
(27.5 Ci/mmol) (3H distribution: 74—79% at 1b) was purchased from New
England Nuclear Corp. (Boston, MA, U.S.A.), and NADPH from Kohjun
(AD) and testosterone to the estrogens, estrone and estradiol,
respectively.1—3) This process appears to proceed with three
Co., Ltd. (Tokyo, Japan). 2- and 4-Nitroestrone (1, 6) were prepared accord-
oxygenations of the androgens, each of which requires 1 mol ing to known method.20) The structures of all of the known compounds used
in this study were identified by 1H-NMR spectrometric analysis and their pu-
of O2 and 1 mol of reduced nicotinamide adenine dinu-
cleotide phosphate (NADPH). The 19-methyl group, as well
as 1b and 2b-hydrogens, are eliminated in the third oxygena-
rities were confirmed by elemental analysis or high resolution-mass spec-
trometry (HR-MS) as well as thin-layer chromatography (TLC).
Melting points were measured on a Yanagimoto melting point apparatus
tive step, resulting in aromatization of the A-ring of the an-
drogens.4—10) The exact nature of the final step remains un-
certain, however; the A-ring conformation is thought to play
a critical role in the stereospecific removal of the two hydro-
gens.
Aromatase inhibitors are useful in treating estrogen-
dependent breast cancer.11) Therefore, several categories of
steroidal and non-steroidal inhibitors were designed. Osawa
and co-workers reported previously that the natural estrogen,
which was thought to be the final product of aromatase re-
action, served as an inhibitor of aromatase, yielding cate-
chol estrogen, 2-hydroxyestrone, as well as 6a-hydroxy-
estrone.12—14) We previously reported structure–activity rela-
tionships of estrogen analogs as aromatase inhibitors to know
the spatial aspects of the active site of aromatase and to de-
(Kyoto, Japan) and were uncorrected. IR spectra were recorded on a Perkin-
Elmer FT-IR 1725X spectrophotometer, and ultraviolet (UV) spectra were
determined in 95% EtOH on a Hitachi 150-20 UV spectrometer (Tokyo,
1
Japan). H-NMR spectra were obtained in CDCl3 solution with a JEOL GX
400 (400 MHz) or JEOL LA 600 (600 MHz) spectrometer (Tokyo, Japan)
using tetramethylsilane as an internal standard, and MS spectra (EI mode)
with a JEOL JMS-DX 303 spectrometer. TLC was performed on E. Merck
precoated silica gel plates (silica gel 60F-254, Darmstadt, Germany). Col-
umn chromatography was conducted with silica gel 60, 70—230 mesh (E.
Merck).
2- and 4-Nitro-3-benzyloxyestra-1,3,5-triene-17-ones (2, 7) A solu-
tion of 2- or 4-nitroestrone (1, 6) (2.35 g, 7.46 mmol) in CH3CN (85 ml) was
added benzyl bromide (1.075 ml, 7.46 mmol) and anhydrous K2CO3 (6.85 g,
49.7 mmol). The mixture was refluxed with stirring for 1 h. The reaction
mixture was filtered and evaporated which was recrystallized from acetone
to give 2- and 4-nitro-3-benzyloxy steroid 2 or 7 (2.96 g or 2.90 g, 98.0% or
96.0%), respectively.
2: mp 237—240 °C. 1H-NMR d: 0.92 (3H, s, 18-Me), 5.20 (2H, s,
velop a novel series of aromatase inhibitors.15) Consequently, OCH2Ph), 6.83 (1H, s, 4-H), 7.38 (5H, m, OCH2Ph), 7.84 (1H, s, 1-H). IR
2-halogeno-, 2-methyl-, 6a-aryl-, and 6b-methylestrones are
good competitive inhibitors of aromatase in human placental
(KBr) cmꢀ1: 1737, 1517. UV lmax (EtOH) nm (e): 274.0 (2290), 340.0
(1520). MS m/z: 405 (Mꢁ), 299, 91. Anal. Calcd for C25H27NO4: C, 74.05;
H, 6.71; N, 3.45. Found: C, 73.86; H, 6.75; N, 3.31.
microsomes (apparent Ki’s ranging between 100 and 660 nM).
On the other hand, many compounds have been reported as
non-steroidal competitive inhibitors of aromatase, including
flavonoids and their analogs.16—19) They have a heteroatom
(sulfur, oxygen, and nitrogen) that would bind to the heme
iron of the aromatase cytochrome P-450 enzyme. In particu-
lar, flavonoids having a nitrogen-containing heterocylic moi-
ety such as pyridine, pyrimidine, imidazole, and triazole
strongly inhibit aromatase.19)
In this study, we examined the structure–activity relation-
ships of the pyridine and other heterocyclic derivatives of es-
trone and estradiol analogs as aromatase inhibitors in human
placental microsomes. Isonicotinyl-substituted derivatives 5c
and 10c were the most potent inhibitors of aromatase.
7: mp 194—198 °C. 1H-NMR d: 0.91 (3H, s, 18-Me), 5.15 (2H, s,
OCH2Ph), 6.87 (1H, d, Jꢂ8.8 Hz, 2-H), 7.29 (1H, d, Jꢂ8.1 Hz, 1-H), 7.34
(5H, m, OCH2Ph). IR (KBr) cmꢀ1: 1741, 1529. UV lmax (EtOH) nm (e):
275.0 (1590). MS m/z: 405 (Mꢁ), 299, 91. Anal. Calcd for C25H27NO4: C,
74.05; H, 6.71; N, 3.45. Found: C, 74.11; H, 6.76; N, 3.30.
2- and 4-Amino-3-benzyloxyestra-1,3,5-triene-17-ones (3, 8) A solu-
tion of 0.1 mol/l NaOH (68 ml) containing Na2S2O4 (13.3 g, 76.4 mmol) was
added to a solution of steroid 2 or 7 (2.86 g, 7.06 mmol) in acetone (405 ml).
After refluxed for 2 h, the reaction mixture was added dropwise AcOH, fol-
lowed by evaporation and extraction with EtOAc. The organic layer was
washed with sat. NaCl and dried with Na2SO4. Evaporation of the solvent,
purification by column chromatography (hexane–EtOAc, 2 : 1, v/v) and re-
crystallization from EtOAc gave 2- or 4-amino-3-benzyloxy steroid 3 or 8
(2.32 g or 2.19 g, 87.6% or 82.7%), respectively.
3: mp 226—229 °C. 1H-NMR d: 0.91 (3H, s, 18-Me), 3.72 (2H, s, 2-
NH2), 5.05 (2H, s, PhCH2O), 6.61 (1H, s, 4-H), 6.70 (1H, s, 1-H), 7.38 (5H,
m, PhCH2O). IR (KBr) cmꢀ1: 3448, 1735, 1520. UV lmax (EtOH) nm (e):
∗ To whom correspondence should be addressed. e-mail: numazawa@tohoku-pharm.ac.jp
© 2008 Pharmaceutical Society of Japan

September 2008
1305
tially according to the original procedure of Siiteri and Thompson.25) All en-
zymatic studies were carried out in 67 mM phosphate buffer, pH 7.5, at a
final volume of 500 ml under initial velocity conditions. The incubation mix-
ture for the IC50 experiment contained 480 mM of NADPH, 300 nM of [1b-
3H]AD, 20 mg protein of the lyophilized microsomes, various concentrations
of inhibitors, and the entire mixture was incubated at 37 °C for 20 min.26) For
the kinetic assay experiment, the incubation mixture contained 480 mM of
NADPH, 20 mg protein of the microsomes, 300 nM of [1b-3H]AD, various
concentrations of inhibitors. The mixture was incubated at 37 °C for 5 min.
Apparent Ki values were calculated using non-linear regression analysis
with GraFit software.27)
296.0 (4650). MS m/z: 375 (Mꢁ), 284, 122, 91. Anal. Calcd for C25H29NO2:
C, 79.96; H, 7.78; N, 3.73. Found: C, 79.65; H, 7.90; N, 3.53.
8: mp 217—220 °C. 1H-NMR d: 0.90 (3H, s, 18-Me), 3.80 (2H, s, 4-
NH2), 5.08 (2H, s, PhCH2O), 6.71 (1H, d, Jꢂ8.5 Hz, 2-H), 6.76 (1H, d,
Jꢂ8.5 Hz, 1-H), 7.37 (5H, m, PhCH2O). IR (KBr) cmꢀ1: 3460, 1739. UV
lmax (EtOH) nm (e): 288.0 (2940). MS m/z: 375 (Mꢁ), 284, 122, 91. HR-
MS Calcd for C25H29NO2: 375.2198. Found: 375.2199.
2- and 4-Substituted 3-Benzyloxyestra-1,3,5-triene-17-ones (4, 9)
A
solution of 2-amino steroid 3 (260 mg, 0.70 mmol) in anhydrous DMF
(43 ml) was added picolinoyl chloride hydrochloride (249 mg, 1.4 mmol) and
pyridine (55 ml) and refluxed for 2 h. The reaction mixture was evaporated
under the reduced pressure, and the resulting residue was purified by column
chromatography (hexane–EtOAc, 5 : 1, v/v) followed by recrystallization
from acetone to afford 2-picolinylamide steroid 4a (122 mg, 36%). 2-
Nicotinyl, isonicotinyl, and isoxazolecarbonyl substituted compounds 4b,
4c, and 4d and 4-picolinyl, nicotinyl, isonicotinyl, and isoxazolecarbonyl
substituted compounds 9 were also obtained from steroid 3 or 8 in the simi-
lar manner as described above.
2- and 4-Substituted Estra-1,3,5-triene-17-ones (5 or 10) A mixture
of 3-benzyloxy steroid 4a (122 mg, 0.26 mmol), Pd–C (390 mg), and 95%
EtOH (63 ml) was stirred under hydrogen atmosphere for 2.5 h. The reaction
mixture was filterd and evaporated, the residue which was recrystallized
from acetone gave 3-hydroxy-2-picolinyl compound 5a (42.0 mg, 42%).
Other 3-benzyloxy steroids 4b—d and steroids 9 were hydrogenated, simi-
larly as above, to yield 3-hydroxy-2-substituted steroids 5b—d and their 4-
isomers 10.
3-Substituted Estrones (12) Method 1: A mixture of 2-methyl-6-ni-
trobenzoic anhydride (MNBA) (191 mg), 4-dimethylaminopyridine (DMAP)
(46 mg), 2-furancarboxylic acid (62 mg) in THF (1 ml) and triethylamine
(0.1 ml) were added to a solution of estrone (11) (30 mg, 0.11 mmol) in THF
(1 ml).21—23) The resulting mixture was stirred at room temperature for 1 h.
Then the reaction mixture was diluted with EtOAc, washed with 5%
NaHCO3 and water, and dried with Na2SO4. After evaporation of the sol-
vent, the crude obtained was recrystallized from acetone to yield 12a. 3-
Thiophenecarbonyl, thiazolecarbonyl, and pyrrolecarbonyl substituted es-
trones (12b—d) were synthesized similarly as described for the synthesis of
12a.
Results and Discussion
Chemistry Previously, it has been reported that estrone
is more potent inhibitor of human placental aromatase, com-
pared to estradiol, although the estrogens are the aromatase
final products. Then, we first employed estrone as a scaffold
of aromatase inhibitors for the synthesis of the estrogen de-
rivatives. 2- or 4-nitroestrone (1 or 6) was treated with benzyl
bromide in CH3CN in the presence of K2CO3 gave 3-benzyl
ethers 2 or 7 which was reduced with Na2S2O4 to yield 2- or
4-aminoestone 3-benzyl ether (3 or 8) (Fig. 1). Treatment of
2-aminoestone 3-benzyl ether (3) with acid chloride (picoli-
noyl chloride, nicotinoyl chloride, isonicotinoyl chloride, or
5-isoxazolecarbonyl chloride) in pyridine gave 2-amide de-
rivatives 4 having a heterocyclic ring at C-2 in fair yield (Ta-
bles 1, 2). Deprotection of a 3-benzyl function of compounds
4 with H2/C afforded finally 2-heterocyclic-substituted
amides 5. 4-Heterocyclic-substituted aminoestrones (10)
were synthesized similarly as described for the synthesis of
the 2-isomers 5.
Next, we employed estrone for a starting material of syn-
thesis of estrone 3-heterocyclic-substituted derivatives 12
(Fig. 2). Estrone (11) was treated with 2-furancarboxylic
acid, 2-thiophenecarboxylic acid, 4-thiazolecarboxylic acid,
Method 2: 5-Isoxazolecarbonyl chloride (120 mg, 0.91 mmol) in pyridine
(6 ml) were added to a solution of estrone (11) (31 mg, 0.11 mmol) in
CH3CN (6 ml) and the mixture was stirred at 60 °C for 9 h. The reaction
mixture was poured into water and extracted with EtOAc. The organic layer or pyrrole-2-carboxylic acid under the mixed anhydride con-
was washed with sat. NaHCO3 and water, dried with Na2SO4, and purified
by recrystallization from EtOAc to give steroid 12e (29 mg, 70%).
3-tert-Butyldimethylsiloxy-17b-substituted Estradiols (14) Method 1:
A mixture of MNBA (688 mg), DMAP (171 mg), 2-furancarboxylic acid
dition (MNBA in the presence of DMAP and triethylamine)
in THF for 1 h to afford to estrone 3-heterocyclic esters
12a—d whereas the 3-isoxazolecarbonyl ester 12d was ob-
tained with 5-isoxazolecarbonyl chloride in pyridine (Table
3).
(220 mg, 2.0 mmol) in THF (5 ml) and triethylamine (0.5 ml) were added to
a solution of steroid 13 (153 mg, 0.40 mmol) in THF (5 ml), and the mixture
was stirred at room temperature for 1 h. The reaction mixture was poured to
sat. NaHCO3 and extracted with EtOAc. The organic layer was washed with
water, and dried with Na2SO4. Evaporation of the solvent followed by re-
crystallization from acetone gave steroid 14a. 17-Thiophenecarbonyl and
thiazolecarbonyl substituted compounds 14b and 14c were prepared in the
similar manner as the synthesis of 14a.
Method 2: 5-Isoxazolecarbonyl chloride (184 mg, 1.40 mmol) was reacted
with 13 (126 mg, 0.33 mmol) under above condition, a crude product puri-
fied by column chromatography (hexane–EtOAc, 9 : 1, v/v) to give 14d
(54 mg, 35%).
17b-Substituted Estradiols (15) 1 mol/l HCl solution (3.8 ml) was
added to a mixture of steroid 14a (123 mg, 0.26 mmol), isopropanol (2.5 ml),
and THF (6.5 ml). The reaction mixture was allowed to stand at room tem-
perature for over night, and the mixture was diluted with EtOAc. The or-
ganic layer was washed with sat. NaHCO3 and water, and dried with
Na2SO4. Evaporation of the solvent, followed by recrystallization from ace-
tone gave steroid 15a (82 mg, 87%). Other 17-substituted estradiols (15b—
d) were obtained by the deprotection of the 3-silylether 10b—d similarly as
described for the preparation of 15a.
Enzyme Preparation Human placental microsomes (sedimented after
60 min at 105000ꢃg) were obtained as described by Ryan.24) They were
washed once with 0.05 mM dithiothreitol, lyophilized and stored at ꢀ80 °C.
No significant loss of activity occurred during the period (6 months) of this
study. The preparation of human placental microsomes was conducted under
the approval of the ethical review committee of Tohoku Pharmaceutical Uni-
versity in accordance with the standard of the Helsinki Declaration.
Aromatase Assay Procedure Aromatase activity was measured essen-
Fig. 1. Synthesis of 2- and 4-Heterocyclic Amides 5 and 10 of Estrone

1306
Vol. 56, No. 9
Table 2. Elemental Analysis and HR-MS Data of 4, 5, 9 and 10
Compound
Calculation
Observed
2-Substituted series
4a
4b
4c
480.2413
480.2413
480.2413
480.2445
480.2448
480.2443
4d
5a
5b
5c
C, 74.02; H. 6.43; N, 5.95
390.1943
C, 74.10; H, 6.47; N, 5.84
390.1921
390.1943
390.1943
390.1942
390.1924
4-Substituted series
9a
9b
9c
10a
10b
10c
480.2413
480.2413
480.2413
390.1943
390.1943
390.1943
480.2427
480.2449
480.2447
390.1918
390.1916
390.1923
d
Fig. 2. Synthesis of Estrone 3-Heterocyclic Esters 12
Fig. 3. Synthesis of Estradiol 17-Heterocyclic Esters 15
Finally, we prepared estradiol 17-heterocyclic-substituted
derivatives 15 (Fig. 3). 3-tert-Butyldimethylsiloxyestradiol
(13) was converted into 17-heterocyclic esters 14 by treat-
ment with the mixed anhydride method or the acyl chloride
in pyridine as described for the synthesis of compound 12.
Hydrolysis of the 3-silyl ethers 14 with 1 mol/l HCl in
propan-2-ol and THF gave 17-heterocyclic-substituted estra-
diol (15) in good yields (Tables 4, 5).
e
Spectral data for all of the compounds synthesized in this
study were consistent with the assigned structures.
Biological Properties Inhibition of aromatase activity
using [1b-3H]AD as a substrate in human placental micro-
somes by the estrogen derivatives was examined in vitro by
enzyme kinetics. This assay quantitates the production of tri-
tiated water released from [1b-3H]AD by aromatization. The
effects of varying the C-2, C-4, and C-3 substitution of es-
trone and the C-17 substitution of estradiol on the activity of
androstenedione aromatization was determined in relation to

September 2008
1307
d
d
a
b

1308
Vol. 56, No. 9
Table 5. Elemental Analysis and HR-MS Data of 12, 14 and 15
Compound
Calculation
Observed
3-Substituted series
12a
12b
12c
12d
12e
C, 75.80; H. 6.64; N, 0
C, 72.60; H, 6.36; N, 0
381.1399
363.1844
C, 72.31; H, 6.34; 3.83
C, 75.78; H, 6.73; N, 0.01
C, 72.39; H, 6.38; N, 0.05
381.1411
363.1839
C, 71.90; H, 6.41; N, 3.80
17b-Substituted series
14a
14b
14c
14d
15a
15b
15c
15d
C, 72.46; H, 8.39; N, 0
C, 72.44; H, 8.52; N, 0
C, 69.95; H, 8.20; N, 0
497.2420
C, 69.37; H, 8.29; N, 2.75
C, 75.31; H, 7.23; N, 0
C, 72.25; H, 6.92; N, 0
C, 68.95; H, 6.61; N, 3.53
C, 71.87; H, 6.91; N, 3.71
C, 70.11; H, 8.12; N, 0
497.2420
C, 69.82; H, 8.16; N, 2.91
C, 75.38; H, 7.15; N, 0
C, 72.22; H, 6.85; N, 0
C, 68.90; H, 6.57; N, 3.65
C, 71.91; H, 6.86; N, 3.81
Fig. 4. Lineweaver–Burk Plots of Aromatase Inhibition by Isonicotinyl
Derivative 10c
Concentrations of the inhibitor: control (0 mM) (ꢀ); 0.96 mM (ꢁ); 1.92 mM (ꢂ);
3.85 mM (ꢃ).
(Fig. 4). Isonicotinyl analogs 5c and 10c were most potent in-
hibitors and their apparent Ki values were 2.19ꢅ0.14 mM and
1.53ꢅ0.08 mM for 5c and 10c, respectively. Nicotinyl deriva-
tives 5b and 10b and picolinyl derivatives 5a and 10a were
less potent inhibitors. For comparison, the Ki values of es-
trone and estradiol were determined to be 2.50ꢅ0.22 mM and
130ꢅ10 mM, respectively.
Table 6. In Vitro Aromatase Inhibition by Estrone and Estradiol Deriva-
tives
Compound
IC50 (mM)
Apparent Ki (mM)
2-Substituted series
a)
5a
5b
5c
5d
208.83ꢅ23.8
75.39ꢅ4.7
31.16ꢅ1.3
—
—
2.19ꢅ0.14
—
Introduction of fluoro, chloro, bromo, methyl, and formyl
groups at C-2 of estrone gave rise to the markedly increased
affinity for aromatase but that of methoxy, nitro, amino func-
tions at the C-2 position did not affect, to a significant extent,
the affinity.15) On the other hand, the similar substitution at
the 4-position of estrone markedly decreased the affinity. The
present results shows that there is no significant difference
between the 2- and 4-subsituted compounds, demonstrating
that there are the similar accessible volume between C-2 and
C-4 positions. Although there is no precise evidence con-
cerning the relation of aromatase-catalyzed 2-hydroxylation
of estrogen to the aromatase reaction, estrogen formation, the
accessible volume of the C-2 and C-4 position would play as
critical role to the both the hydroxylation and the aromatase
reaction. Among the pyridine substitutions, the fact that the
isonicotinyl derivatives 5c and 10c were most potent ones
suggests that the N-hetero atom, orienting p-position on the
pyridine ring, is most fitted the accessible volume. The coor-
dination of the para N atom of pyridine to the heme-Fe of
aromatase would be involved in the inhibition. Compounds
10% inhibition at 100 mM
4-Substituted series
10a
10b
203.06ꢅ19.38
95.64ꢅ5.2
—
—
10c
10d
28.57ꢅ1.8
10% inhibition at 100 mM
1.53ꢅ0.08
—
3-Substituted series
12a
12b
12c
12d
0% inhibition at 100 mM
0% inhibition at 100 mM
10% inhibition at 100 mM
10% inhibition at 100 mM
243.88ꢅ78.1
—
—
—
—
—
12e
17b-Substituted series
15a
15b
0% inhibition at 100 mM
0% inhibition at 100 mM
0% inhibition at 100 mM
0% inhibition at 100 mM
—
—
—
—
15c
15d
For comparison
Estrone
Estradiol
26ꢅ8
5% inhibition at 50 mM
2.50ꢅ0.22
130ꢅ10
a) Not determined.
the aromatase-catatyzed estrogen hydroxylations, especially having the other heterocyclic ring at the C-3 of estrone and at
the catechol estrogen formation. The results are shown in C-17 of estradiol did not access the binding pocket, namely,
Table 6. IC50 values were initially obtained under initial ve- the hetero atoms of the heterocyclic compounds failed the
locity conditions. 2-Amides 5a—c, picolinyl (5a), nicotinyl coordination to the heme moiety of aromatase.
(5b) and isonicotinyl (5c) amides, as well as 4-amide deriva-
The 2- and 4-isonicotinyl amino derivatives of estrone,
tives 6a—c showed fairly inhibitory activity but 4-isoxazole compounds 5c and 10c, were promising compounds for fur-
derivatives 5d and 10d were poor inhibitors. Moreover, es- ther powerful aromatase inhibitors. On the basis of the re-
trone 3- and estradiol 17-furancarbonyl, thiophenecarbonyl, sults, it is presumed that there would be an accessible volume
thiazolecarbonyl, and isoxazolecarbonyl esters (12, 15) did in the regions of C-2 and C-4 positions.
not show any detectable amounts of the aromatase inhibitory
Acknowledgment This work was supported in part by a High Technol-
ogy Research Center Project from the Ministry of Education, Culture,
Sports, and Technology of Japan. Human term placenta was kindly donated
activity. Among the inhibitors examined, the apparent inhibi-
tion constants (Ki), an index of the ability of the enzyme for
the inhibitor, were obtained for isonicotinyl compounds 5c
and 10c by analysis of a Dixon plot. The two compounds
were evaluated as competitive or non-competitive inhibitors
of aromatase in human placental microsomes. Lineweaver–
Burk plots showed that these were competitive inhibitors
by Dr. Michihiro Yuki of Yuki Lady’s Clinic.
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