Synthesis and anti-tumor activity of a fluorinated analog of medroxyprogesterone acetate (MPA), 9α-fluoromedroxyprogesterone acetate (FMPA)

Article abstract of DOI:10.1248/cpb.54.1567

We synthesized 9α-fluoromedroxyprogesterone acetate (FMPA) in order to test whether it is a more potent anti-angiogenic agent than medroxyprogesterone acetate (MPA), which has been widely used as a therapeutic agent for breast and endometrium cancers. FMPA was previously synthesized in 10 steps (total yield: 1%). An efficient synthesis of FMPA has been achieved in 6 steps (total yield: 12%). We examined the anti-tumor effect of FMPA, complexed with dimethyl-β-cyclodextrin (DM-β-CyD), on rat mammary carcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA). FMPA showed great anti-tumor effect on DMBA-induced rat mammary carcinomas.

Full text of DOI:10.1248/cpb.54.1567

November 2006
Chem. Pharm. Bull. 54(11) 1567—1570 (2006)
1567
Synthesis and Anti-tumor Activity of a Fluorinated Analog of
Medroxyprogesterone Acetate (MPA), 9a-Fluoromedroxyprogesterone
Acetate (FMPA)
,
a
a
a
b
b
Natsuko MURATA,* Shiho FUJIMORI, Yoshitatsu ICHIHARA, Yoshio SATO, Taketo YAMAJI,
b
b
a
a
c
Hiroshi TSUBOI, Masayuki UCHIDA, Hiroto SUZUKI, Masashi YAMADA, Tsutomu OIKAWA,
Hideo NEMOTO, Junko NOBUHIRO, Tominari CHOSHI, and Satoshi HIBINO
d
e
e
e
a
Pharmaceuticals Development Department, Meiji Dairies Corporation; 1–2–10 Shinsuna, Koto-ku, Tokyo 136–8908,
b
Japan: Division of Research and Development, Meiji Dairies Corporation; 540 Naruda, Odawara, Kanagawa 250–0862,
Japan: Faculty of Health and Social Work, Kanagawa University of Human Services; 1–10–1 Heiseicho, Yokosuka,
Kanagawa 238–8522, Japan: Faculty of Pharmaceutical Sciences, University of Toyama; 2630 Sugitani, Toyama
9
c
d
e
30–0194, Japan: and Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University; 1 Sanzo, Gakuen-cho,
Fukuyama, Hiroshima 729–0292, Japan.
Received June 30, 2006; accepted August 8, 2006; published online August 18, 2006
We synthesized 9a-fluoromedroxyprogesterone acetate (FMPA) in order to test whether it is a more potent
anti-angiogenic agent than medroxyprogesterone acetate (MPA), which has been widely used as a therapeutic
agent for breast and endometrium cancers. FMPA was previously synthesized in 10 steps (total yield: 1%). An ef-
ficient synthesis of FMPA has been achieved in 6 steps (total yield: 12%). We examined the anti-tumor effect of
FMPA, complexed with dimethyl-b-cyclodextrin (DM-b-CyD), on rat mammary carcinomas induced by 7,12-di-
methylbenz[a]anthracene (DMBA). FMPA showed great anti-tumor effect on DMBA-induced rat mammary car-
cinomas.
Key words 9a-fluoromedroxyprogesterone acetate; synthesis; medroxyprogesterone acetate; anti-angiogenic agent; dimethyl-
benz[a]anthracene; mammary carcinoma
5)
Neovascularization is involved in tumor growth as well as system. In addition, FMPA (1), complexed with g-cyclo-
rheumatoid arthritis, diabetic retinopathy and age-related dextrin (g-CyD), has shown anti-tumor effect on rat mam-
1,2)
macular degeneration (AMD). Tumor growth is often ac- mary carcinomas induced by 7,12-dimethylbenz[a]anthra-
companied by angiogenesis, the resulting blood vessels sup- cene (DMBA) which is greater than the effect of the parent
5)
plying nutrients necessary to tumor cells. The development compound, MPA (2). FMPA (1) inhibits the activity of plas-
of new blood vessels is also a crucial step in the invasion and minogen activator (PA), an important protease playing an es-
metastases of cancer cells of breast cancer, melanoma, lung sential role in the process of angiogenesis, in bovine endothe-
3)
6)
cancer, prostate cancer and other cancers. Thus, discovery lial cells. However, further biological evaluations such as in
of drugs which suppress angiogenesis has become one of the vitro anti-tumor tests have been difficult, because the first
major focuses in cancer research. Angiostatin and endostatin, synthesis method of FMPA (1) developed had low overall
which are recombinant proteins, could be such drugs, but re- yield (1%) and required many steps (10 steps). To overcome
search on low-molecular inhibitors is still being vigorously these problems, a practical synthetic route to FMPA (1)
studied in pharmaceutical industry.
which involves six-steps and has 12% overall yield has been
As part of a research program to find novel anti-angio- newly established. We here describe the full details of both
genic drug candidates, we synthesized 9a-fluoromedroxy- the synthesis of FMPA (1) and its anti-tumor effect on rat
progesterone acetate (FMPA, 17a-acetoxy-9a-fluoro-6a- mammary carcinoma when complexed with dimethyl-b-
4)
methylprogesterone) (1) (Fig. 1). FMPA (1) is a fluorinated cyclodextrin (DM-b-CyD).
analog of medroxyprogesterone acetate (MPA) (2) (Fig. 1),
which is widely used as a therapeutic agent for breast and en- Results and Discussion
dometrium cancer and possesses anti-angiogenic activity.
For the synthesis of 9a-fluoro-MPA, FMPA (1), we uti-
FMPA (1) exhibited an in vitro anti-angiogenic activity lized the 11b-hydroxy group of the steroid nucleus to intro-
1
00 times as strong as MPA (2) in a chorioallantoic mem- duce a fluorine atom at the 9a-position on both routes
4)
brane (CAM) assay system and exhibited a 7-fold stronger (Charts 1, 2). The initial route to 1 as shown in Chart 1 uses
anti-angiogenic effect than MPA in a rabbit corneal assay the commercially available 11b-17a-dihydroxy-4-pregnen-
3
1
,10-dione (3) as a starting material. Here, acetylation of the
1b-hydroxy group of 3 (100%) gave the compound 4. Se-
quential, acetalization and treatment with m-chloroperben-
zoic acid (mCPBA) afforded the epoxide 6, which was re-
acted with CH MgBr to yield the 5-hydroxy-6-methylpreg-
3
nene 7. Treatment of 7 with 5% KHSO followed by treat-
4
ment with 0.05 N NaOH afforded the 6-methylpregnene 9
(
23% yield from 4). The fluorination of the 9a-position of 9
7)
Fig. 1. The Chemical Structure of FMPA and MPA
was carried out by 70% HF in pyridine to produce the 9a-
∗
To whom correspondence should be addressed. e-mail: natsuko_murata@meiji-milk.com
© 2006 Pharmaceutical Society of Japan

1
568
Vol. 54, No. 11
Chart 1
Chart 2
fluoroprogesterone 10 (23%). The 6-methylprogesterone 10 Thus, we succeeded in an efficient and practical synthesis of
was converted into 6a-methylprogesterone 12 by acetaliza- FMPA (1) with 12% overall yield in 6 steps.
8)
tion followed by deacetalization in the usual manner (58%
FMPA (1) showed significant inhibitory effects on the
yield from 10). Finally, acetylation of 12 by the mixed anhy- growth of DMBA-induced rat mammary carcinomas at doses
9)
dride method provided 9a-fluoromedroxyprogesterone ac- of 30 and 120 mg/kg as compared with a control group dur-
etate (1) (FMPA) (36%). The overall yield of the initial syn- ing the treatment and withdrawal periods (Fig. 2). The num-
thetic route was a quite low (1%) in a ten-step sequence.
ber of tumors which appeared after treatment was also signif-
On the other hand, our improved synthesis of FMPA (1) icantly inhibited by FMPA (1) doses of 30 and 120 mg/kg as
Chart 2) was carried out starting from 6a-methylpre- compared with the control group (Fig. 3). It has been re-
(
donisolone 13, which is a cheaper starting material than ported that FMPA (1) complexed with g-CyD showed anti-
1
1b,17a-dihydroxy-4-pregnen-3,10-dione 3. Specifically, tumor effect on DMBA-induced rat mammary carcinomas,
acetylation of 13 was performed with trimethylorthoacetate, and its effect is greater than that of the parent compound,
5)
and then the resulting ortho ester was hydrolyzed to give 6a- MPA (2). The bioavailability of FMPA (1) complexed with
methylpredonisolone 17a-O-acetate 14 (89%). The hydroxyl g-CyD was found to be about 6 times higher than that of
group at the C-21 position of 14 was converted into the me- FMPA (1) suspended in carboxymethyl cellulose sodium salt
sylate, followed by treatment with sodium iodide to yield and orally administered to rats. In a preliminary study of
pregnadiene-3,20-dione 15 (59%). Reduction of 15 with hy- pharmacokinetics of FMPA (1) in rats, DM-b-CyD complex
drogen atmosphere in the presence of chlorotris(triphenyl- exhibited bioavailability about 2 times higher than g-CyD
11)
10)
phosphine)rhodium(I)
,20-dione 16 (90%). Fluorination of 16 was performed plexed with DM-b-CyD in this study. As a result, FMPA (1)
using hydrogen fluoride in pyridine to produce FMPA (1) exhibited strong anti-tumor activity on DMBA-induced rat
26%). The structure of 1 was confirmed by spectroscopy. mammary carcinomas. Therefore, it is suggested that oral ad-
provided 11b-hydroxy-pregnene- complex (unpublished data), so we used FMPA (1) com-
3
(

November 2006
1569
termined by a Shimadzu QP5050 mass spectrometer. Elemental analysis was
done by Yanaco CHN CORDER MT-6.
1
1b-Acetoxy-17a-hydroxy-4-pregnene-3,20-dione (4) A mixture of 4-
pregnene-11b,17a-diol-3,20-dione 3 (2 g, 5.77 mmol), Ac O (10 ml), pyri-
2
dine (20 ml) and 4-dimethylaminopyridine (DMAP) (20 mg, 0.16 mmol) was
stirred for 6 h at room temperature. The reaction mixture was diluted with
water (50 ml) and extracted with CHCl . The CHCl layer was sequentially
3
3
washed with 10% HCl, a 5% aqueous NaHCO3 solution, and brine, dried
over Na SO , and concentrated under reduced pressure. The residue was
2
4
subjected to column chromatography (silica gel, 150 g) using 70%
CHCl /hexane as eluant to give the acetylated derivative 4 (2.24 g, 100%),
3
1
mp 159.5—160 °C (EtOAc/hexane). H-NMR (CDCl ): d 0.85 (3H, s), 2.01
3H, s), 2.21 (3H, s), 3.04 (2H, m), 5.34—5.56 (1H, m), 5.65 (1H, s). MS
m/z: 388 (M ). Anal. Calcd for C H O : C, 71.10; H 8.30. Found: C,
3
(
ꢀ
2
3
32
5
7
1.08; H 8.50.
6
-Methyl-4-pregnene-11b,17a-diol-3,20-dione (9) 11-Acetate 4 (2 g,
5
.15 mmol), ethylene glycol (1.1 g, 17.7 mmol), and p-TsOH (40 mg,
0
.21 mmol) were refluxed in benzene (100 ml) for 7 h while reducing humid-
Fig. 2. Effect of FMPA on DMBA-Induced Mammary Tumor Size in Rats
ity (by the use of a reflux condenser equipped with a moisture separator).
After the reaction temperature lowered to room temperature, the mixture was
washed with brine, dried over Na SO , and concentrated. The residue was
Values represents the meanꢃS.D. of seven to 14 tumors per group. Each group con-
sisted of seven to eight rats. (ꢀ) Distilled Water, (ꢁ) Control (DM-b-CyD), (ꢂ) FMPA
2
4
3
0 mg/kg, (ꢃ) FMPA 120 mg/kg. ** Significant difference from control (pꢄ0.01, Dun-
used directly in the next step without further purification. 70% mCPBA (2 g,
.11 mmol) was added to the residue 5 (2 g, 4.20 mmol) in CHCl (150 ml),
nett multiple comparison test).
,
# ## Significant difference from control (pꢄ0.05,
8
3
pꢄ0.01, nonparametric Dunnett multiple comparison test).
and the mixture was stirred for 12 h at room temperature. The reaction was
sequentially washed with an aqueous Na CO solution and brine, and then
2
3
concentrated. The resultant residue 6 was thoroughly dried, and then dis-
solved in anhyd. THF (30 ml). While vigorously stirring the mixture,
CH MgBr (1.02 M THF, 30 ml, 30.6 mmol) was added. The reaction mixture
3
was refluxed for 18 h, the temperature was lowered to room temperature, and
the mixture was poured into ice-water containing NH Cl. The mixture was
4
extracted with CHCl , and the organic layer was washed with saturated
3
brine, dried over Na SO , and concentrated under reduced pressure. The
2
4
residue 7 was refluxed at 70 °C for 2 h in a mixture of acetone (80 ml) and an
aqueous 5% KHSO solution (40 ml). The organic layer was evaporated, and
4
the residue was reextracted with CHCl . The CHCl layer was washed with
3
3
saturated brine, dried over Na SO , and concentrated under reduced pres-
2
4
sure. Subsequently, the residue 8 was stirred for 2 h in a mixture of 0.05 N
NaOH (100 ml) and MeOH (100 ml) at room temperature. Structures of
crude compounds 5—8 were supported by the detection of main signals in
1
H-NMR spectra, respectively. AcOH (1 ml) was added to the reaction mix-
ture, and the mixture was concentrated under reduced pressure to ca. half
volume. Extraction was performed with CHCl . The CHCl layer was
3
3
washed with brine, dried over Na SO , and concentrated under reduced pres-
sure. The residue was subjected to column chromatography (silica gel, 70 g)
2
4
Fig. 3. Effect of FMPA on the Number of Tumors (Length ꢅ5 mm) Which
Appeared after Treatment of Rats with DMBA-Induced Mammary Cancer
using 30% EtOAc/hexane as eluant to give the title compound 9 (23% from
Values represents the meanꢃS.D. of seven to eight rats per group. (ꢀ) Distilled
1
compound 4, mp 221—225 °C (EtOAc). H-NMR (CDCl ): d 1.03 (3H, s),
3
Water, (ꢁ) Control (DM-b-CyD), (ꢂ) FMPA 30 mg/kg, (ꢃ) FMPA 120 mg/kg. ,
## Sig-
#
1
.05 (3H, s), 1.06 (3H, d, Jꢁ7.33 Hz), 1.27 (3H, d, Jꢁ7.57 Hz), 1.42 (3H, s),
nificant difference from control (pꢄ0.05, pꢄ0.01, nonparametric Dunnett multiple
1
.50 (3H, s), 2.29 (3H, s), 2.30 (3H, s), 2.68—2.71 (2H, m), 3.48 (1H, q,
comparison test).
ꢀ
Jꢁ7.01 Hz), 4.47 (1H, d, Jꢁ2.43 Hz), 5.73 (1H, s). MS: m/z: 360 (M ).
Anal. Calcd for C H O : C, 73.30; H, 8.95. Found: C, 73.40; H, 8.99.
2
2
32
4
ministration of FMPA (1) may be useful for treatment of
mammary cancer.
9
a-Fluoro-17a-hydroxy-6-methyl-4-pregnene-3,20-dione (10) Hy-
drogen fluoride–pyridine [4 ml, HF : pyridineꢁ7 : 3 (w/w), 0.14 mol] was
placed in a Teflon container. While maintaining the exterior temperature at
ꢂ15 °C, under nitrogen, 11b-hydroxy-4-pergnane-3,20-dione 9 (200 mg,
Conclusions
0.55 mmol) was added. The mixture was stirred for 60 h at the same temper-
4)
In a preliminary work, we proposed the anti-angiogenic
ature, and the mixture was sequentially washed with 3% HCl, 5% NaHCO3,
and brine, dried over Na SO , and concentrated under reduced pressure. The
effect of MPA (2) might be increased by giving it a fluorine
2
4
atom at the C-9a position, a novel design. Based on this hy- residue was subjected to column chromatography (silica gel, 30 g) using
3
0% EtOAc/hexane as eluant to give the fluorinated compound 10 (47 mg,
pothesis, two synthetic routes of FMPA (1) were found. The
newer synthetic route is a practical process using a cheaper
starting material. Subsequently, potent anti-angiogenic ef-
1
2
3.3%), mp 232—235 °C (EtOAc). H-NMR (CDCl ): d 0.75 (3H, s), 0.78
3
(
3H, s), 1.11 (3H, d, Jꢁ6.35 Hz), 1.20 (3H, d, Jꢁ4.96), 1.30 (3H, s), 1.37
(
3H, s), 2.27 (3H, s), 2.28 (3H, s), 2.69—2.75 (2H, m), 2.99—3.07 (1H, m),
fects have been verified for the FMPA (1) in several bioassay 3.82 (1H, d, Jꢁ1.84 Hz), 5.85 (1H, d, Jꢁ3.09 Hz), 5.90 (1H, d, Jꢁ2.00 Hz).
4
—6)
ꢀ
systems.
This study also indicates that FMPA (1) has MS m/z: 225 (M ). Anal. Calcd for C22
H O F: C, 72.89; H, 8.62. Found: C,
31 3
7
2.95; H, 8.75.
strong anti-tumor activity, so that FMPA (1) may well be a
useful oral drug for the treatment of human breast and en-
dometrium cancer.
9
a-Fluoro-17a-hydroxy-6a-methyl-4-pregnene-3,20-dione (12) The
-methylpregnene 10 (40 mg, 0.11 mmol), ethylene glycol (0.014 ml,
.25 mmol), and p-TsOH (2 mg, 0.01 mmol) were added to benzene (20 ml),
6
0
and refluxed with heat for 3 h using a reflux condenser equipped with a
moisture separator. The solvent was concentrated under reduced pressure,
Experimental
General Melting points were measured on a Yanagimoto micro-melting and the residue was combined with an aqueous 5% KHSO solution (5 ml)
4
point apparatus MP-500D and were uncorrected. Proton nuclear magnetic
resonance spectra ( H-NMR) were taken with a Varian Unity-500 instrument mixture was concentrated under reduced pressure, and then the residue was
using tetramethylsilane as an internal standard. Mass spectra (MS) were de- extracted with CHCl . The CHCl layer was washed with saturated brine,
and acetone (5 ml). The mixture was refluxed for 2 h at 70 °C. The reaction
1
3
3

1
570
Vol. 54, No. 11
dried over Na SO , and concentrated under reduced pressure. The residue
was subjected to column chromatography (silica gel, 20 g) using 30%
17a-Acetoxy-11b-hydroxy-6a-methyl-4-pregnene-3,20-dione
(16)
2
4
Chlorotris(triphenylphosphine)rhodium(I) (8.51 mg, 9.20 mmol) was added
EtOAc/hexane as eluant to give the methylated derivative 12 (23 mg, 57.5% to a solution of 17a-acetoxy-11b-hydroxy-6a-methyl-1,4-pregnadiene-3,20-
2
7
from compound 10), mp 237—239 °C (EtOAc). [a] ꢀ23.76° (cꢁ0.202,
dione 15 (316 mg, 0.79 mmol) in CH Cl –EtOH (1 : 1; 30 ml). The mixture
D
2 2
1
MeOH). H-NMR (CDCl ): d 0.86 (3H, s), 1.10 (3H, d, Jꢁ6.45 Hz), 1.30
was stirred for 27 h at room temperature (20—25 °C) in a stream of hydro-
3
2
(
3H, s), 2.69—2.79 (2H, m), 2.99—3.03 (1H, m), 3.84 (1H, d, Jꢁ1.80 Hz),
.89 (1H, d, Jꢁ1.80 Hz). MS m/z: 362 (M ). Anal. Calcd for C H O F: C,
2.89; H, 8.62. Found: C, 72.90; H, 8.78.
gen gas (1.5 kg/cm ). After completion of reaction, the reaction mixture was
ꢀ
5
7
concentrated under reduced pressure. The residue was subjected to silica gel
column chromatography (silica gel, 10 g) using 50% EtOAc/hexane as elu-
2
2
31
3
1
7a-Acetoxy-9a-fluoro-6a-methylprogesterone
(1) 9a-Fluorome- ant to give 17a-acetoxy-11b-hydroxy-6a-methyl-4-pregnene-3,20-dione 16
20
droxyprogesterone 12 (20 mg, 0.055 mmol) was stirred for 5 h at ꢂ10 °C in
(285 mg, 90%), mp 193—195 °C (EtOAc). [a] ꢀ55.4° (cꢁ0.773, CHCl ).
D 3
1
the presence of p-TsOH (20 mg, 0.11 mmol), Ac O (0.4 ml, 4.23 mmol), and
H-NMR (500 MHz, CDCl ) d: 0.93 (3H, s), 1.06 (3H, d, Jꢁ6.5 Hz), 1.43
2
3
CH Cl (1 ml) under nitrogen. Water (10 ml) was added to the reaction mix- (3H, s), 2.06 (3H, s), 2.07 (3H, s), 2.92—2.98 (1H, m), 5.73 (1H, d,
ture, and extraction was performed with CHCl . The CHCl layer was Jꢁ1.5 Hz). MS m/z: 402 (M ). Anal. Calcd for C H O : C, 71.61; H, 8.51.
2
2
ꢀ
3
3
24 34
5
washed with an aqueous 5% NaHCO3 solution and saturated brine, dried
over Na SO , and concentrated under reduced pressure. The residue was
Found: C, 71.75; H, 8.53.
17a-Acetoxy-9a-fluoro-6a-methylprogesterone (1) Hydrogen fluo-
2
4
subjected to column chromatography (silica gel, 20 g) using 30% ride–pyridine [4 ml, HF : pyridineꢁ7 : 3 (w/w), 0.14 mol] was placed in a
EtOAc/hexane as eluant to give the 17a-acetoxy-9a-fluoro-6a-methylprog- Teflon container. While maintaining the exterior temperature at ꢂ15 °C,
2
0
esterone (1) (8 mg, 36%), mp 208—210 °C (EtOAc). [a] ꢀ36.0° (c=0.210,
under nitrogen, 17a-acetoxy-11b-hydroxy-6a-methyl-4-pregnene-3,20-
D
ꢂ1
1
CHCl ). IR (KBr) cm : 1742, 1709, 1674. H-NMR (500 MHz, CDCl ): d dione 16 (145 mg, 0.36 mmol) was added. The solution was stirred for 65 h
.68 (3H, s), 1.12 (3H, d, Jꢁ6.5 Hz), 1.31 (3H, s), 2.05 (3H, s), 2.10 (3H, s),
.96 (1H, ddd, Jꢁ2.5, 11.3, 14.3 Hz), 5.90 (1H, d, Jꢁ1.5 Hz). C-NMR
125 MHz, CDCl ) d: 13.3, 17.9, 21.1, 21.3, 23.6, 25.0, 26.2, 26.8, 29.1,
0.3, 33.1, 33.6, 34.4, 37.6, 43.3, 44.6, 46.1, 96.2, 99.4, 123.9, 169.8, 170.6,
98.7, 203.6. MS m/z: 404 (M ). HR-MS m/z: 404.2380 (Calcd for
3
3
0
2
at the same external temperature of ꢂ15 °C. When the reaction was com-
pleted, ice water was added to the reaction mixture, which was then ex-
tracted with EtOAc (50 ml, 30 ml). The organic layer was sequentially
1
3
(
3
3
1
washed with 10% HCl, saturated NaHCO , and brine, and then dried over
3
ꢀ
Na SO . After the solvent was evaporated under reduced pressure, the
2
4
C H O F: 404.2361).
residue was subjected to column chromatography (silica gel, 6 g) using 40%
24
33
4
1
7a-Acetoxy-11b,21-dihydroxy-6a-methyl-1,4-pregnadiene-3,20- EtOAc/hexane as eluant to give 17a-acetoxy-9a-fluoro-6a-methylproges-
dione (14) Pyridinium p-toluenesulfonate (28.0 mg, 0.11 mmol) and
trimethyl orthoacetate (1.01 ml, 7.94 mmol) were added to a solution (60 ml)
of 6a-methylpredonisolone (6a-methyl-11b,17a,21-trihydroxy-1,4-pregna-
terone (1) (37.9 mg, 26%), which was identical with the FMPA synthesized
previously in all respects.
Anti-tumor Effect on Rat Mammary Carcinoma The following ani-
diene-3,20-dione) 13 (2.13 g, 5.69 mmol) in CH Cl . The mixture was mal studies were performed in accordance with the Guiding Principles for
2
2
stirred at room temperature (20—25 °C) for 5 h. When the reaction was the Care and Use of Laboratory Animals approved by the Japanese Pharma-
completed, the reaction mixture was concentrated under reduced pressure. cological Society. DMBA was administered orally at a single dose of
The residue was dissolved in acetone (30 ml). While cooling the mixture on 20 mg/body to female Sprague-Dawley rats about 50 days old. When the di-
ice, an aqueous solution (30 ml) of p-TsOH (1.19 g, 6.26 mmol) was added ameter of the first developed mammary tumor became 0.5—1.5 cm 8—12
thereto. The mixture was stirred for 30 min on ice. Upon completion of reac-
weeks after DMBA administration, the animal was incorporated into the
tion, a saturated aqueous NaHCO3 solution was added and extraction was study. FMPA (1), complexed with DM-b-CyD, was administered to the ani-
performed with EtOAc. The EtOAc layer was washed with brine, dried over mals orally once a day at doses of 30 and 120 mg/kg/day for 3 weeks. Dis-
Na SO , and concentrated under reduced pressure. The residue was purified
by column chromatography (silica gel, 40 g) using 50% EtOAc/hexane as orally once a day at a volume of 6 ml/kg/day for 3 weeks. The two largest
tilled water and DM-b-CyD (498 mg/ml) were administered to the animals
2
4
eluant to give 17a-acetoxy-11b,21-dihydroxy-6a-methyl-1,4-pregnadiene- perpendicular diameters of each tumor were measured on the first day of
2
0
3
(
,20-dione 14 (2.11 g, 89%), mp 159—161 °C (EtOAc). [a] ꢀ18.1° treatment and thereafter once a week for 6 weeks. The multiplied product of
D
1
cꢁ0.490, CHCl ). H-NMR (500 MHz, CDCl ) d: 0.97 (3H, s), 1.13 (3H, d, these diameters was expressed as tumor size. Number of tumors per rat ap-
Jꢁ6.0 Hz), 1.46 (3H, s), 2.02 (3H, s), 2.74—2.82 (1H, m), 4.24 (1H, dd, peared after treatment was also counted.
3
3
Jꢁ4.0, 18.0 Hz), 4.33 (1H, dd, Jꢁ3.5, 18.0 Hz), 6.04 (1H, s), 6.28 (1H, d,
ꢀ
Jꢁ10.0 Hz), 7.25 (1H, d, Jꢁ10.0 Hz). MS m/z: 416 (M ). Anal. Calcd for
Acknowledgments We would like to thank Professor Kazuhiko Saigo
for helpful discussions.
C H O : C, 69.21; H, 7.74. Found: C, 69.08; H, 7.80.
24
32
6
1
7a-Acetoxy-11b-hydroxy-6a-methyl-1,4-pregnadiene-3,20-dione (15)
Methanesulfonyl chloride (MsCl) (0.15 ml, 1.94 mmol) was added to a solu- References
tion of 17a-acetoxy-11b,21-dihydroxy-6a-methyl-1,4-pregnadiene-3,20-
dione 14 (557 mg, 1.34 mmol) in pyridine (10 ml). The mixture was stirred
at between 0 and 5 °C (external temperature) for 3 h. After completion of re-
action, iced water and ethyl acetate were added to the reaction mixture. The
resultant mixture was sequentially washed with 10% HCl, a saturated aque-
ous NaHCO3 solution, and brine, and then dried over Na SO . After the
1) Battegay E. J., J. Mol. Med., 73, 333—346 (1995).
2) Votruba M., Gregor Z., Eye, 15, 424—429 (2001).
3) Folkman J., Shing Y., J. Biol. Chem., 267, 10931—10934 (1992).
4) Sugino E., Fujimori S., Hibino S., Choshi T., Ichihara Y., Sato Y., Ya-
maji T., Tsuboi H., Murata N., Uchida M., Shimamura M., Oikawa T.,
Chem. Pharm. Bull., 45, 421—423 (1997).
2
4
sol0vent was evaporated under reduced pressure, the residue was dissolved
in pyridine (10 ml). Sodium iodide (447 mg, 2.98 mmol) was added to the
solution, and the mixture was refluxed for 50 min. Upon completion of the
reaction, a saturated aqueous Na SO solution was added to the reaction
5) Uchida M., Tsuboi H., Yamaji T., Murata N., Kohno T., Sugino E., Hi-
bino S., Shimamura M., Oikawa T., Cancer Lett., 154, 63—69 (2000).
6) Yamaji T., Tsuboi H., Murata N., Uchida M., Kohno T., Sugino E.,
Hibino S., Shimamura M., Oikawa T., Cancer Lett., 145, 107—114
(1999).
2
3
mixture, and then extraction was performed with EtOAc. The EtOAc layer
was washed with 10% HCl, a saturated aqueous NaHCO solution, saturated
7) Bergstrom C. G., Dodson R. M., J. Am. Chem. Soc., 82, 3479—3480
(1960).
3
brine, and dried over Na SO . The organic layer was evaporated under re-
2
4
duced pressure. The residue was subjected to silica gel column chromatogra-
phy (silica gel, 16 g) using 50% EtOAc/hexane as eluant to give 17a-
acetoxy-11b-hydroxy-6a-methyl-1,4-pregnadiene-3,20-dione 15 (316 mg,
8) Fried J. H., Arth G. E., Sarett L. H., J. Am. Chem. Soc., 81, 1235—
1239 (1959).
9) Carruthers N. I., Garshasb S., J. Org. Chem., 57, 961—965 (1992).
2
0
1
5
9%), mp 138—140 °C (EtOAc). [a]D ꢀ13.2° (cꢁ0.430, CHCl ). H-NMR 10) Djerassi C., Gutzwiller J., J. Am. Chem. Soc., 88, 4537—4538 (1966).
3
(
2
(
500 MHz, CDCl ) d: 0.95 (3H, s), 1.13 (3H, d, Jꢁ6.5 Hz), 1.46 (3H, s),
11) Kozutsumi D., Kawashima A., Sugimoto T., Kotohda Y., Fujimori S.,
Takami M., Kohno T., Oikawa T., Sugino E., Choshi T., Hibino S.,
Biopharm. Drug Dispos., 20, 277—284 (1999).
3
.06 (3H, s), 2.05 (3H, s), 2.90—2.98 (1H, m), 6.05 (1H, t, Jꢁ2.0 Hz), 6.29
ꢀ
1H, dd, Jꢁ2.0, 10.0 Hz), 7.25 (1H, d, Jꢁ10.0 Hz). MS m/z: 400 (M ). Anal.
Calcd for C H O : C, 71.97; H, 8.05. Found: C, 72.04; H, 8.11.
24
32
5