Steroids 95 (2015) 7–16
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First synthesis and characterization for the stereoisomers of Ulipristal acetate Yi Zhao a, Xiaolong Li a, Hong Liu b, Yongguo Yu a, Li Hai a, Li Guo a,⇑, Yong Wu a,⇑ a b
Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, PR China West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
a r t i c l e
i n f o
Article history: Received 15 June 2014 Received in revised form 29 October 2014 Accepted 12 December 2014 Available online 30 December 2014 Keywords: Ulipristal acetate Stereoisomer Synthesis X-ray structure Impurity
a b s t r a c t The three stereoisomers, 11a,17a-isomer I, 11a,17b-isomer II and 11b,17b-isomer III are related substances of the selective progesterone receptor modulator Ulipristal acetate. Herein, we presented an efficient and practical synthesis approach to deliver these three stereoisomers for the first time, and also confirmed the structure of the key intermediate 5a by single-crystal X-ray analysis. Our research will be of immense help for organic chemists to study the impurity profile of Ulipristal acetate. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction The presence of impurities, especially chiral impurities in an active pharmaceutical ingredient (API) has a significant impact on the quality and safety of the drug products. Therefore, it is necessary to study the impurity profiles of any API and try to control it during the manufacturing of a drug product. As per the International Conference on Harmonization (ICH) guidelines any impurities, which are forming at a level of P0.10% with respect to the API, should be identified, synthesized and characterized thoroughly [1,2]. 17a-Acetoxy-11b-(4-N,N-dimethylaminophenyl)-19-norpregna4,9-diene-3,20-dione (Ulipristal, EllaÒ, VA-2914 or CDB-2914, Fig. 1) is a well-known, more specific 19-norprogesterone and a selective progesterone receptor modulator (SPRM) which efficiently binds and inhibits progesterone receptor (PR) in progesterone target tissues [3]. Ulipristal acetate is well characterized for its anti-fertility potency in several of in vivo and in vitro assays. Different from the active metabolites, Ulipristal acetate expresses less antiglucocorticoid activity compared with the most well-known SPRM and mifepristone Besides, it also exhibits benefits for endometriosis treatment, as well as ovarian and breast cancer therapies [4].
⇑ Corresponding authors. Tel.: +86 028 85503235 (Y. Wu), +86 028 85503777 (L. Guo). E-mail addresses: [email protected] (L. Guo), [email protected] (Y. Wu). http://dx.doi.org/10.1016/j.steroids.2014.12.009 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.
Therefore, several methods have been developed so far for the synthesis of Ulipristal acetate [5–9]. However, two chiral centers in the Ulipristal acetate molecule from the starting materials can give rise to four stereoisomers including Ulipristal acetate (Fig. 2) [5,8,9]. Thus, the synthesis of the final product with the required stereochemistry is a significant challenge. Furthermore, these stereoisomers play essential roles for the preparation of Ulipristal acetate and also for the quality control of bulk drugs and drug formulations. In this report, we have designed and synthesized the stereoisomers of Ulipristal acetate. More importantly, the configuration of each isomer was also confirmed.
2. Experimental All reactions were carried out under an argon atmosphere. Most chemicals and solvents were analytical grade and used without further purification. TLC was performed using precoated silica gel GF254 (0.2 mm), while column chromatography was performed using silica gel (100–200 mesh). The melting point was measured on an YRT-3 melting point apparatus (Shantou Keyi instrument & Equipment Co. Ltd., Shantou, China). IR spectra were obtained on a Perkin Elmer 983 (Perkin Elmer, Norwalk, CT, USA). 1H NMR spectra were taken on a Varian INOVA 400 (Varian, Palo Alto, CA, USA) using CDCl3, d-DMSO and D2O as solvent. Chemical shifts are expressed in d (ppm), with tetramethylsilane (TMS) functioning as the internal reference, and coupling constants (J) were expressed in Hz. Mass
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Y. Zhao et al. / Steroids 95 (2015) 7–16
spectra were recorded on an Agilent 1946B ESI-MS instrument (Agilent, Palo Alto, CA, USA). (1) Synthesis of the key intermediate 5: 2.1. 3,3-(Ethylene-dioxy)-5b,10b-epoxyestr-9(11)-ene-17-one(2b) Hexachloroacetone (14.5 mL, 0.0954 mol), 30% aqueous hydrogen peroxide (14.1 mL, 0.477 mol) and sodium phosphate dibasic dodecahydrate (34.5 g, 0.0964 mol) were added to methylene chloride (300 mL) at 0 °C. The mixture was stirred for 1 h at the same temperature. The material 1 (30 g, 0.0954 mol) was added to above mixture. The reaction mixture was stirred for another 18 h at the same temperature. Then, it was poured into a mixture of methylene chloride (200 mL) and ice (160 g). A solution of sodium thiosulfate (79 g, 0.5 mol) in water (300 mL) was added dropwise to the mixture to destroy the excess of hydrogen peroxide. After separation, the organic fraction was washed with water (2 100 mL) and dried on sodium sulfate. The solvent was removed in vacuo to give 32 g (100%) of product, which was a 65:35 mixture of the 5a,10a- and 5b,10b-epoxides showed by HPLC. The obtained crude mixture of epoxides was recrystallized with petroleum ether/ethyl acetate (5:1) to get 12.8 g (40%) white solid 2a. The filtrate was concentrated in vacuo to yield 19.2 g (60%) of a 42:58 mixture of the 5a,10a- and 5b,10b-epoxides. Then the mixture was purified via column chromatography (petroleum ether/ethyl acetate 10:1) to give 6.91 g (36%) white solid 2b. mp: 158–160 °C, MS (m/z): 331.42 [M + H]+, Analysis calculated for C20H26O4: C 72.70, H 7.93; found: C 72.80, H 7.89. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.861 (s, 3H, CH3), 1.201–2.511 (m, 18H), 3.796–3.961 (m, 4H, OACH2), 5.851 (d, 1H, J = 2.8, @CH). 2.2. 3,3-(Ethylene-dioxy)-17a-ethynyl-17b-hydroxy-5b,10bepoxyestr-9(11)-ene (3b) Under argon, 2b (6.8 g, 0.02 mol) was dissolved in dry tetrahydrofuran (70 mL) at 0 °C, and sodium acetylide (1.9 g, 0.04 mol) was added. The mixture was stirred for 1 h. Saturated ammonium chloride solution (50 mL) and water (50 mL) were added, then, the reaction mixture was stirred for 10 min. Then the reaction mixture was concentrated to a volume of 80 mL. The residue was stirred for 3 h at 0 °C. The precipitated crystals were filtered off and dried at 50 °C to yield 7.3 g (100%) of the title compound 3b, mp: 154–156 °C, MS (m/z): 379.48 [M + Na]+, Analysis calculated for C22H28O4: C 74.13, H 7.92; found: C 74.21, H 7.89. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.825 (s, 3H, CH3), 1.081– 2.627 (m, 18H), 2.557 (s, 1H, acetylenic hydrogen), 3.867– 3.946(m, 4H, OACH2), 5.890 (t, 1H, J = 4.4, @CH). 2.3. 3,3-(Ethylene-dioxy)-5b,17b-dihydroxy-11a-(4-N,Ndimethylaminophenyl)-19-nor-17a-pregn-9-ene-21-ethyne(4b) Under anhydrous conditions, a portion (2 mL) of a solution of 4-bromo-N,N-dimethylaniline (12 g, 0.06 mol) in dry tetrahydrofuran (50 mL) and one crystal of iodine was added to the mixture of magnesium (1.7 g, 0.07 mol) in dry tetrahydrofuran (5 mL) at 50 °C. After evidence of reaction was observed, the entire amount
N
O OAc
O Fig. 1. The structures of Ulipristal acetate.
of the reagent was added dropwise. The reaction mixture was stirred for an additional 2 h while it was cooling to room temperature. The mixture was then added dropwise to a suspension of 3b (7.0 g, 0.02 mol) and copper (I) chloride (0.6 g, 0.002 mol) in dry tetrahydrofuran (70 mL) at 0 °C. The reaction mixture was stirred for 1 h, then, it was poured into 10% ammonium chloride (70 mL) solution and extracted with methylene chloride (3 50 mL). The combined organic fractions were washed with water (4 50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give a black oil (21.1 g). The oil was purified via column chromatography (petroleum ether–ethyl acetate 10:1) to yield 5.23 g (55%) light blue solid. mp: 162–164 °C, MS (m/z): 478.62 [M + H]+, Analysis calculated for C30H39NO4: C 75.44, H 8.23, N 2.93; found: C 75.55, H 8.28, N 2.75. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.970 (s, 3H, CH3), 0.842–2.332 (m, 18H), 2.474 (s, 1H, acetylenic hydrogen), 2.923 (s, 6H, NACH3), 3.716 (t, 1H, J = 6.4, CH), 3.820– 4.022(m, 4H, OACH2), 6.680(d, 2H, J = 8.4, ArAH), 7.048 (d, 2H, J = 8.4, ArAH). 2.4. 11a-(4-N,N-dimethylaminophenyl)-17a-ethynyl-17bhydroxyestr-4,9-diene-3-one(5) To a solution of potassium bisulfate (3.7 g, 0.027 mol) in water (50 mL) was added compound 4b (5 g, 0.01 mol) at 0 °C. The mixture was stirred for 5 h and almost became clear. Then, saturated sodium bicarbonate was added to the above mixture to adjust pH to 7–8. The precipitated crystals were filtered off and dried at 50 °C to yield 2.7 g (65%) of the title compound 5. mp: 134– 136 °C, MS (m/z): 416.58 [M + H]+, Analysis calculated for C28H33NO2: C 80.93, H 8.00, N 3.37; found: C 80.85, H 8.18, N3.28. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 1.005 (s, 3H, CH3), 1.396–2.628 (m, 16H), 2.476 (s, 1H, acetylenic hydrogen), 2.920 (s, 6H, NACH3), 3.899 (t, 1H, J = 8.8, CH), 5.664 (s, 1H, @CH), 6.680 (d, 2H, J = 8.4, ArAH), 6.952 (d, 2H, J = 8.4, ArAH). (2) Synthesis of 11a,17a-isomer I: 2.5. 11a-(4-N,N-dimethylaminophenyl)-21-(phenyl-sulfinyl)-19norpregna-4,9,17(20),20-tetraene-3-one (6) To a suspension of compound 5 (1.5 g, 0.0036 mol), triethylamine (2.2 mL, 0.0152 mol) in dry tetrahydrofuran (20 mL), a solution of phenylsulfenyl chloride (0.9 g, 0.0062 mol) in dry tetrahydrofuran (15 mL) was added dropwise while keeping the temperature between 70 and 78 °C. The reaction mixture was stirred for 4 h at 78 °C, then water (20 mL) and methanol (20 mL) was added. The reaction mixture was stirred for 10 min and extracted with methylene chloride (3 40 mL). The combined organic fractions were washed with water (4 20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to get 2.5 g of a reddish brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 5:1) to yield 1.18 g (62.5%) of white solid. mp: 151–155 °C. MS (m/z): 546.78 [M + Na]+, Analysis calculated for C34H37NO2S: C77.97, H 7.12, N 2.67, S 6.12; found: C 77.84, H 7.21, N 2.75, S 6.18. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 1.042 (s, 3H, CH3), 1.213–2.767 (m, 16H), 2.900 (s, 6H, NACH3), 3.967 (t, 1H, J = 8.8, CH), 5.681 (s, 1H, @CH), 6.075 (s, 1H, @CH), 6.634 (d, 2H, J = 8.4, ArAH), 6.888 (d, 2H, J = 8.4, ArAH), 7.460–7.539 (m, 3H, ArAH), 7.626 (d, 2H, J = 7.2, ArAH). 2.6. 11a-(4-N,N-dimethylaminophenyl)-17a-hydroxy-19-norpregna4,9-diene-3,20-dione(7) Compound 6 (1.1 g, 0.0021 mol) was added to a solution of sodium methoxide (0.11 g, 0.0021 mol) in methanol (20 mL). The reaction mixture was stirred at 50 °C for 1 h, then trimethyl
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Y. Zhao et al. / Steroids 95 (2015) 7–16
N
N
O OAc
O
N
O OAc
O
O OAc
O II
I
III
Fig. 2. The stereoisomers of Ulipristal acetate.
O
H 2O2, (Cl 3C)2CO
O
1
+
O
O
Na2HPO 4/CH 2Cl2 rt, 12h, 100%
O
O
O
O
O
2a
1) recrystallization 2) Column chromatography
O
O
2b
21.6%
5α,10α : 5β,10β 65 : 35 N HO
N
1)
HO
MgBr CuCl, THF, 0 oC, 5h
sodium acetylide
2b THF, 0oC, 4h, 100% O
O
2) NH 4Cl, CH2Cl2/H2O, rt, 55%
3b
O
O O
OH 4b
N HO KHSO4 /H2 O,0o C, 4h 65% O 5 Scheme 1. Synthesis of the key intermediate 5 [5,9].
O HC S C
N SCl
1)P(OCH 3)3 /NaOCH 3/CH 3OH
5 triethylamine/THF/-78 oC 4h, 62.5%
2)1.0 M HCl/CH3OH, 0.5h, 54.8% 6
O
N
O
N
HO
O OAc
Ac2O/HClO 4
O
CH 2Cl2, -20 oC, 3h, 86% O
7
I
Scheme 2. Synthesis of 11a,17a-isomer I [9].
phosphite (0.4 g, 0.0032 mol) was added and stirring was continued at 70 °C for 2 h. The reaction mixture was cooled to 20 °C and poured into water (25 mL), then extracted with methylene chloride (3 30 mL). The combined organic fractions were dried over sodium sulfate, filtered, and concentrated in vacuo to give 2.12 g of reddish brown oil. The oil was dissolved in a mixture of 1 N hydrochloric acid (1.3 mL) and methanol (20 mL), then stirred at room temperature for 30 min. Ice water (200 mL) was added to the mixture. After neutralizing with saturated sodium bicarbonate,
the mixture was extracted with methylene chloride (3 20 mL). The combined organic fractions were washed with water (2 20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to get 1.6 g of reddish brown oil. The obtained crude product was purified via column chromatography (petroleum ether– ethyl acetate 10:1) to yield 0.5 g (54.8%) off-white syrupy. MS (m/z): 434.58 [M + H]+, Analysis calculated for C28H35NO3: C 77.56, H 8.14, N 3.23; found: C 77.62, H 8.21, N 3.28. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.881(s, 3H, CH3), 1.232–2.787
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Y. Zhao et al. / Steroids 95 (2015) 7–16
2.9. 11a,17b-Isomer II (Scheme 3)
(m, 16H), 2.258 (s, 3H, COCH3), 2.914 (s, 6H, NACH3), 3.867 (t, 1H, J = 9.2, CH), 5.663 (s, 1H, @CH), 6.673 (d, 2H, J = 8.4, ArAH), 6.934 (d, 2H, J = 8.4, ArAH).
Perchloric acid (0.4 mL, 0.0044 mol) was added to acetic anhydride (2 mL, 0.0212 mol) by dropwise at 10 °C. The obtained solution was stirred for an additional 1 h, then cooled to 30 °C and the solution of compound 8 (0.8 g, 0.002 mol) in dry dichloromethane (20 mL) was added at such rate to keep the temperature between 10 °C and 20 °C, then the reaction mixture was stirred at this temperature for 3 h. The mixture was diluted with dichloromethane (20 mL) and poured into water (20 mL) containing sodium acetate (5.6 g, 0.068 mol). The organic layer was separated, washed with water (3 10 mL), dried over sodium sulfate and concentrated under reduced pressure to give 1.12 g of slightly yellow syrupy. The syrupy was purified via column chromatography (petroleum ether–ethyl acetate 15:1) to yield 0.84 g (86%) of slightly yellow syrupy. FTIR (KBr, diffuse reflectance): cmax 2933; 1731 and 1722(AC@O); 1669 and 1658 (conjugated AC@O); 1568; 1531; 1455; 1371; 1332; 1262; 1222; 1171; HRMS: calculated for C30H37NO4 475.6191, found: 475.6178. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.979 (s, 3H, CH3), 1.193– 2.683 (m, 16H), 1.609 (s, 3H, COCH3), 2.072 (s, 3H, COCH3), 2.917 (s, 6H, NACH3), 3.850 (t, 1H, J = 8.0), 5.687 (s, 1H, CH), 6.666 (d, 2H, J = 8.4, ArAH), 6.989 (d, 2H, J = 8.4, ArAH); 13C NMR {400 MHz, CDCl3 (TMS), d (ppm)}:21.195; 24.087; 25.684; 26.754; 27.789; 30.168; 30.998; 36.676; 36.789; 38.257; 39.268; 40.485; 46.981; 50.848; 66.986; 96.120; 112.689; 122.845; 127.228; 129.187; 131.420; 145.548; 148.528; 156.496; 170.541; 199.431; 203.722.
2.7. 11a,17a-Isomer I Perchloric acid (0.1 mL, 0.0011 mol) was added to acetic anhydride (0.5 mL, 0.0053 mol) by dropwise at 10 °C. The obtained solution was stirred for an additional 1 h, then, cooled to 20 °C and the solution of compound 7 (0.2 g, 0.0005 mol) in dry dichloromethane (10 mL) was added at such rate to keep the temperature between 10 and 20 °C, then the reaction mixture was stirred at this temperature for 3 h. The mixture was diluted with dichloromethane (10 mL) and poured into water (10 mL) containing sodium acetate (1.4 g, 0.017 mol). The organic layer was separated, washed with water (3 10 mL), dried over sodium sulfate and concentrated under reduced pressure to give 0.22 g of slightly yellow syrupy. The syrupy was purified via column chromatography (petroleum ether–ethyl acetate 15:1) to yield 0.19 g (86%) of slightly yellow syrupy. FTIR (KBr, diffuse reflectance): cmax 2923; 1721 and 1718 (AC@O); 1673 and 1662 (conjugated AC@O); 1575; 1528; 1448; 1362; 1313; 1259; 1212; 1178; HRMS: calculated for C30H37NO4 475.6191, found: 475.6165. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.797 (s, 3H, CH3), 1.256– 2.658 (m, 16H), 2.024 (s, 6H, COCH3), 2.945 (s, 6H, NACH3), 3.905 (t, 1H, J = 9.2, CH), 5.686 (s, 1H, @CH), 6.710 (d, 2H, J = 8.4, ArAH), 6.982 (d, 2H, J = 8.4, ArAH); 13C NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 21.181; 24.075; 25.668; 26.761; 27.780; 30.165; 30.951; 36.688; 36.748; 38.268; 39.281; 40.479; 46.951; 50.839; 66.989; 96.110; 112.678; 122.851; 127.214; 129.195; 131.398; 145.525; 148.534; 156.481; 170.517; 199.425; 203.732.
(4) Synthesis of 11b,17b-isomer III: 2.10. 3,3-(Ethylene-dioxy)-17a-ethynyl-17b-hydroxyestr-5(10),9(11)diene (9)
(3) Synthesis of 11a,17b-isomer II: Compound 1 (20 g, 0.0636 mol) was dissolved in dry tetrahydrofuran (200 mL) at 0 °C. The mixture was stirred for 0.5 h, and sodium acetylide (6.0 g, 0.1272 mol) was added. After that, the reaction mixture was stirred for another 1 h. Saturated ammonium chloride solution (200 mL) and water (200 mL) were added and the mixture was stirred for 10 min. Then the reaction mixture was concentrated to a volume of 350 mL. The residue was stirred for 3 h at 0 °C. The precipitated crystals were filtered off and dried at 50 °C to yield 20.6 g (95%) of the title compound 9. mp: 145– 148 °C, MS (m/z): 340.21 [M + H]+, Analysis calculated for C22H28O3: C 77.61, H 8.29; found: C 77.68, H 8.35. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.891(s, 3H, CH3), 1.134–2.662 (m, 18H), 2.579 (s, 1H, acetylenic hydrogen), 3.881–3.998 (m, 4H, OACH2), 5.678 (t, 1H, J = 5.6, @CH).
2.8. 11a-(4-N,N-dimethylaminophenyl)-17b-hydroxy-19-norpregna4,9-diene-3,20-dione(8) Compound 5 (1.2 g, 0.0029 mol) was dissolved in a mixture of acetic acid-H2O-30% sulfuric acid (40:8:1, 10 mL), then, mercuric sulfate (0.085 g, 0.0003 mol) was added to above mixture. The reaction mixture was stirred for 5 h at 60 °C. After neutralizing with saturated sodium bicarbonate, the mixture was extracted with methylene chloride (3 20 mL). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure to give 1.0 g of brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 15:1) to yield 0.82 g (65.2%) of a slightly yellow syrupy. MS (m/z): 434.58 [M + H]+, Analysis calculated for C28H35NO3: C 77.56, H 8.14, N 3.23; found: C 77.48, H 8.01, N 3.18. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.901 (s, 3H, CH3), 1.079 (s, 3H, COCH3), 1.242–2.689 (m, 16H), 2.926 (s, 6H, NACH3), 3.811 (t, 1H, J = 7.2, CH), 5.704 (s, 1H, @CH), 6.676 (d, 2H, J = 8.4, ArAH), 7.016 (d, 2H, J = 8.4, ArAH).
2.11. 17a-Ethynyl-17b-hydroxyestra-4,9-diene-3-one(10) To a solution of compound 9 (20 g, 0.059 mol) in acetic acid (200 mL), perchloric acid (10.7 mL, 0.118 mol) was added dropwise. The reaction mixture was stirred for 0.5 h at room
N HO
N
O
O OAc
5
Ac2O/HClO4
HgSO4/H 2SO 4 CH 3COOH/H 2O, 60oC 5h, 65.2%
CH 2Cl2, -20oC 3h, 88% O
O 8
Scheme 3. Synthesis of 11a,17b-isomer II [9,10].
II
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Y. Zhao et al. / Steroids 95 (2015) 7–16
O
O O
1
OH CH3COOH
sodium acetylide THF,0o C,1h O 95% O
O HO ethylene glycol, p-toluenesulfonic acid triethyl orthoformate CH2Cl2 ,rt, 3h 85.6%
O HO
O
O
CH2Cl2 /Na2 HPO4,0o C,18h 100%
12
MgBr /
O
O
O
+
o
3) ethanol/ 8.5%H2 SO4,0 C, 2h,64%
O
5α,10α : 5β,10β 75 : 25 N
CH2 Cl 2, -20oC, 4h, O 86.9%
O 14
O
O
O
13b
O
Ac 2O/HClO 4
2) Saturated NH4Cl/CH2Cl2 ,0oC
O HO
O
HO CuCl/THF,0oC,3h
11
O 13a
N 1) N
THF/H2O, 60o C, 5h O 79%
10
(CF3 )2 CO/H2 O2
O
HO
HgSO4 /H2 SO4
HClO4 ,rt, 0.5h 87% O
9
O
OH
OAc
III
Scheme 4. Synthesis of 11b,17b-isomer III [5,9,10].
temperature, then, poured into water (500 mL). The precipitated crystals were filtered off and dried at 50 °C to give 15.21 g (87%) off-white solid. mp: 181–185 °C, MS (m/z): 297.18 [M + H]+, Analysis calculated for C20H24O2: C 81.04, H 8.16; found: C 80.95, H 8.11; 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.879 (s, 3H, CH3), 1.202–2.658 (m, 18H), 2.587 (s, 1H, acetylenic hydrogen), 5.682 (s, 1H, @CH). 2.12. 17b-Hydroxy-19-norpregna-4,9-diene-3,20-dione (11) Compound 10 (15.0 g, 0.0506 mol) was dissolved in tetrahydrofuran (150 mL), then, 30% sulfuric acid (50 mL), mercuric sulfate (1.44 g, 0.0051 mol) was added to the above mixture. The reaction mixture was stirred for 5 h at 60 °C. After neutralizing with saturated sodium bicarbonate, the mixture was extracted with methylene chloride (3 50 mL). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure to give brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 10:1) to yield 12.57 g (79%) white solid. mp: 195–196 °C, MS (m/z): 315.19 [M + H]+, Analysis calculated for C20H26O3: C 76.40, H 8.33; found: C 76.45, H 8.41; 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.877(s, 3H, CH3), 1.239 (s, 3H, CH3), 1.363–2.945 (m, 18H), 5.688 (s, 1H, @CH). 2.13. 3,3,20,20-Bis(ethylene-dioxy)-17b-hydroxy-19-norpregna5(10),9(11)-diene (12) To a solution of compound 11 (12 g, 0.0382 mol) in methylene chloride (120 mL), ethylene glycol (21.3 mL, 0.382 mol), trimethyl orthoformate (41.7 mL, 0.382 mol) and p-toluenesulfonic acid (3.3 g, 0.0191 mol) were added. The reaction mixture was stirred at room temperature for 3 h, then saturated sodium bicarbonate was added to adjust pH to 7–8. The organic layer was separated, and the water-course was extracted with methylene chloride (3 50 mL). The combined organic fractions were dried over sodium sulfate and concentrated under reduced pressure to give slightly brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 20:1) to yield 13.1 g (85.6%) white
solid. mp: 185–190 °C, MS (m/z): 403.24 [M + H]+; Analysis calculated for C24H34O5: C 71.61, H 8.51; found: C 71.55, H 8.59; 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.905 (s, 3H, CH3), 1.090 (s, 3H, CH3), 1.173–2.665 (m, 18H), 2.261 (s, 1H, OH), 3.746– 3.819 (m, 1H, AOCH2), 3.933–4.013 (m, 6H, AOCH2), 4.067–4.119 (m, 1H, AOCH2), 5.588 (t, 1H, J = 5.6, @CH). 2.14. 3,3,20,20-Bis(ethylene-dioxy)-17b-hydroxy-5a,10a-epoxy-19norpregna-9(11)-ene (13a) and 3,3,20,20-bis(ethylene-dioxy)-17bhydroxy-5b,10b-epoxy-19-norpregna-9(11)-ene (13b) Under nitrogen, 30% aqueous hydrogen peroxide (4.4 mL, 0.15 mol) and sodium phosphate dibasic dodecahydrate (11.7 g, 0.033 mol) were added to a solution of hexafluoroacetone (3.75 mL, 0.03 mol) in methylene chloride (120 mL). The mixture was stirred for 1 h at 0 °C. Compound 12 (12 g, 0.03 mol) was added to above mixture. The reaction mixture was stirred for another 18 h at the same temperature. Then, it was poured into a mixture of methylene chloride (100 mL) and ice (6 g). A solution of sodium thiosulphate (23.7 g, 0.15 mol) in water (150 mL) was added dropwise to the mixture to destroy the excess of hydrogen peroxide. After separation, the organic fraction was washed with water (2 50 mL) and dried over sodium sulfate. The solvent was removed in vacuo to give 12.6 g (100%) of product, which was a 75:25 mixture of the 5a,10a- and 5b,10b-epoxides showed by HPLC. The obtained crude mixture of epoxides was used in the next step without further purification. MS (m/z): 419.24 [M + H]+; Analysis calculated for C24H34O6: C 68.87, H 8.19; found: C 68.95, H 8.31; 1 H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.885 (s, 3H, CH3), 1.042 (s, 3H, CH3), 1.073–2.641 (m, 18H), 3.751–3.811 (m, 1H, AOCH2), 3.852–4.003(m, 6H, AOCH2), 4.060–4.112 (m, 1H, AOCH2), 5a,10a: 5.830 (t, 1H, J = 6.0, @CH), 5b,10b: (t, 1H, J = 6.0, @CH). 2.15. 11b-(4-N,N-dimethylaminophenyl)-17b-hydroxy-19-norpregna4, 9-diene-3, 20-dione(14)
of
Under anhydrous conditions, a portion (4 mL) of a solution 4-bromo-N,N-dimethylaniline (21 g, 0.105 mol) in dry
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tetrahydrofuran (100 mL) and one crystal of iodine was added to the mixture of magnesium (2.92 g, 0.12 mol) in dry tetrahydrofuran (10 mL) at 50 °C. After evidence of reaction was observed, the entire amount of the reagent was added dropwise. The reaction mixture was stirred for an additional 2 h while it was cooling to room temperature. The mixture was then added dropwise to a suspension of 13a and 13b (12.6 g, 0.03 mol) and copper (I) chloride (0.9 g, 0.003 mol) in dry tetrahydrofuran (130 mL) at 0 °C. The reaction mixture was stirred for 3 h, then, poured into 10% ammonium chloride (70 mL) solution and extracted with methylene chloride (3 100 mL). The combined organic fractions were washed with water (4 50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give black oil (28.5 g). The obtained oil was dissolved in ethanol (200 mL) under a nitrogen atmosphere, and 8.5% aqueous sulfuric acid (8.9 mL) was added. The reaction mixture was stirred for 2 h at 70 °C. After neutralizing with saturated sodium bicarbonate, the mixture was diluted with water (300 mL), and extracted with methylene chloride (3 100 mL). The combined organic fractions were dried over sodium sulfate and concentrated under reduced pressure to give brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 15:1) to yield 8.3 g (64%) of a light blue syrupy. MS (m/z): 434.26 [M + H]+, Analysis calculated for C28H35NO3: C 77.56, H 8.14, N 3.23; found: C 77.48, H 8.24, N 3.17; 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.394 (s, 3H, CH3), 0.858–3.042 (m, 16H,), 1.241 (s, 3H, COCH3), 3.916(s, 6H, NACH3), 4.364 (d, 1H, J = 5.2, CH), 5.767 (s, 1H, @CH), 6.667 (d, 2H, J = 8.0, ArAH), 7.020 (d, 2H, J = 8.0, ArAH).
between 10 °C and 20 °C, then the reaction mixture was stirred at this temperature for 4 h. The mixture was diluted with dichloromethane (50 mL) and poured into water (50 mL) containing sodium acetate (35 g, 0.425 mol). The organic layer was separated, washed with water (3 10 mL), dried over sodium sulfate and concentrated under reduced pressure to give 6.1 g of brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 15:1) to yield 5.17 g (86.9%) of slightly yellow syrupy. FTIR (KBr, diffuse reflectance): cmax 2938; 1742 and 1725 (AC@O); 1662 and 1654 (conjugated AC@O); 1571; 1538; 1449; 1379; 1342; 1269; 1242; 1178; HRMS: calculated for C30H37NO4 475.6191, found: 475.6181. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.702 (s, 3H, CH3), 0.856–2.813 (m, 16H), 2.090 (s, 3H, COCH3), 2.129 (s, 3H, COCH3), 2.935(s, 6H, NACH3), 4.278 (t, 1H, J = 6.4, CH), 5.747 (s, 1H, @CH), 6.683 (d, 2H, J = 8.4, ArAH), 7.021 (d, 2H, J = 8.4, ArAH); 13C NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 21.184; 24.079; 25.688; 26.765; 27.791; 30.178; 30.988; 36.687; 36.791; 38.264; 39.271; 40.487; 46.957; 50.854; 66.979; 96.134; 112.695; 122.851; 127.234; 129.179; 131.442; 145.551; 148.537; 156.499; 170.554; 199.445; 203.731.
3. Discussion 3.1. The formation of the 11a,17a-isomer I The synthesis of the drug substance was based on the very efficient 1,4-addition (Scheme 5). The 5a,10a-epoxide was converted into Ulipristal acetate smoothly. But there always had the 5b, 10bisomer in the starting material. After a series of reactions, the 5b,10b-isomer was transformed into the 11a,17a-isomer I (Scheme 6). Especially, the contents of 15b was up to 45% in the route reported by Dancsi et al. [8]. In this route, the ratio of 15a and 15b was about 55:45, which was used directly in the next step without further separation. And there were only three steps to
2.16. 11b,17b-Isomer III (Scheme 4) Perchloric acid (2.3 mL, 0.025 mol) was added to acetic anhydride (11.8 mL, 0.125 mol) by dropwise at 10 °C. The obtained solution was stirred for an additional 1 h, then, cooled to 30 °C and the solution of compound 14 (5 g, 0.0125 mol) in dry dichloromethane (50 mL) was added at such rate to keep the temperature
N
R
R
Grignard addition O
O O
Ulipristal acetate
O O
5α,10α
OH
4a R= 17β− hydroxy, 17α- ethynyl
3a R= 17β−hydroxy, 17α-ethynyl
O
O 15a R= 17 α−hydroxy, 17β -
16a R= 17α− hydroxy, 17β -
O CH 3
O CH 3
Scheme 5. Synthesis of Ulipristal acetate.
N
R
N
R
O OAc
Grignard addition O
O O
O O
5β,10β
3b R= 17β− hydroxy, 17α - ethynyl
OH
4b R= 17 β− hydroxy, 17 α-ethynyl O
O 15b R= 17α− hydroxy, 17β-
O
O CH3
16b R= 17α−hydroxy, 17β-
O CH 3
Scheme 6. Formation of 11a,17a-isomer I.
11α,, 17α− −isomer Ι
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Y. Zhao et al. / Steroids 95 (2015) 7–16
O HC S C
1) CH3ONa/P(OCH 3)3 2)1.0 M HCl O
O O
O OH
O OH +
O O
O
17
19
18 5 steps
5 steps
Ulipristal acetate and 11β,, 17β-isomer III
11α,, 17β- isomer II and 11β,, 17β-isomer III
Scheme 7. Formation of compound 19.
O HC S C
N
N
N
O
O HO
HO 1)P(OCH3) 3 /CH3ONa
+
2)1.0 M HCl O
O
O
14
21
20 Ac 2O
Ac 2O 11β,, 17β-isomer III
Ulipristal acetate Scheme 8. Formation of compound 14.
O HC S C
N
N
N
O
O HO
HO 1)P(OCH3) 3 /CH3ONa
+
2)1.0 M HCl O
O
O
8
7
6
Ac2O
Ac2O
11α,, 17β -isomer II
11α,, 17α -isomer I Scheme 9. Formation of compound 7 and 8.
obtain Ulipristal acetate. Thus, it was highly possible that the 11a,17a-isomer I was present in the final product. According to HPLC-MS, we found two peaks with the same m/z value as Ulipristal acetate. So, it was necessary to confirm the two peaks. At the beginning, we tried to separate the 11a, 17a-isomer I from the final product. However, the content of the title compound was too low to fit the large scale. Then we attempted to directly get the compound 15b or 16b. Unfortunately, 15a and 15b, or 16a and 16b had the same Rf according to TLC. There was no way to obtain 15b or 16b at a slightly large scale by the means of Dancsi’s route [8]. Previously, we had published our work describing a synthetic route to Ulipristal acetate [9]. Fortunately, in this route, it was found the compound 2a and 2b could be separated on TLC easily, where the Rf value of 2b was slightly larger than 2a. Thus, we designed the above route to synthesize the 11a,17a-isomer I (Scheme 1 and 2). In this route, the contents of 2b was about 35% using hexachloroacetone as the catalyst, mCPBA as epoxidation reagents [9]. In order to improve the contents of 2b, the mixture of 2a and 2b was recrystallized firstly. Then, the filtrate was
Fig. 3. ORTEP view of the key intermediate 5.
concentrated in vacuo to yield a 42:58 mixture of the 2a and 2b. The mixture was separated via column chromatography to give the 5b,10b-isomer 2b.
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Y. Zhao et al. / Steroids 95 (2015) 7–16
Fig. 4. (a) The 1H NMR {400 MHz, CDCl3 (TMS)} of compound 7. (b) The NOEDS {400 MHz, CDCl3 (TMS)} of compound 7 at 0.89 ppm (arrow).
Fig. 5. (c) The 1H NMR {400 MHz, CDCl3 (TMS)} of compound 8. (d) The NOEDS {400 MHz, CDCl3 (TMS)} of compound 8 at 0.90 ppm (arrow).
Fig. 6. (e) The 1H NMR {400 MHz, CDCl3 (TMS)} of compound 14. (f) The NOEDS {400 MHz, CDCl3 (TMS)} of compound 14 at 0.39 ppm (arrow).
3.2. The formation of the 11a,17b-isomer II and 11b,17b-isomer III Dancsi et al. used compound 1 as their starting material to synthesize Ulipristal acetate [8]. The phenyl-sulfinyl compound 17 was obtained after two steps, then, reacted with sodium
methoxide and trimethyl phosphite to get 17a-hydroxy compound 18 (Scheme 7). Our previous work also used compound 1 as the starting material to synthesize Ulipristal acetate [9]. After 5 steps, the phenyl-sulfinyl compound 20 was afforded, then reacted with sodium methoxide and trimethyl phosphite to yield 17a-hydroxy
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Y. Zhao et al. / Steroids 95 (2015) 7–16 Table 1 The differences in 11a,17a-isomer I, 11a,17b-isomer II and 11b,17b-isomer III and Ulipristal acetate. Compound
[a]20 D (c 1.0,CHCl3)
11-CH (ppm)
17-CH3 (ppm)
17-COCH3 (ppm)
17-OCOCH3 (ppm)
Ulipristal acetate 11a,17b-Isomer I 11a,17a-Isomer II 11b,17a-Isomer III
+174° 82° 135° +124°
4.365 3.905 3.850 4.278
0.355 0.797 0.979 0.702
2.137 2.024 1.609 2.129
2.099 2.024 2.072 2.090
Fig. 7. HPLC chromatogram of the crude Ulipristal acetate which was synthesized by Dancsi’s route [8].
Fig. 8. HPLC chromatogram of the crude Ulipristal acetate which was synthesized by Yu’s route [9].
compound 21 (Scheme 8). Thus, most of the product was the 17ahydroxy compound 18 and 21. However, the 17b-hydroxy compound 19 and 14 can also be generated according to collision theory. Especially, when there was 11a-[4-(dimetnylamino)phenyl] in the molecule of compound 6, the generation of 17b-hydroxy compound 8 was easier than the 17b-hydroxy compound 19 and 14 (Scheme 9). From above discussion, we can see that 11a,17a-isomer I, 11a,17b-isomer II and 11b,17b-isomer III might be all present in the final product of Ulipristal acetate. Therefore, further study of the isomers is highly desirable. 3.3. Structure confirmation We tried to cultivate the single-crystal to research their structure. Fortunately the single-crystal of the key intermediate 5 was
cultivated by slow evaporation at room temperature successfully. Its structure had been confirmed by the use of single-crystal X-ray analysis (Fig. 3). It was clear that 4-N,N-dimethylaminophenyl in the molecule of compound 5 was found out at the a-configuration which was not changed in the next reactions. The d (ppm) of 11b-hydrogen was about 3.6–4.0, while 11a-hydrogen was about 4.2–4.4 according to 1H NMR. Thus, the configurations of 4-N,N-dimethylaminophenyl in the molecule of compound I, II and III were easily confirmed. In order to confirm the configurations of 17-acetyl in the molecule of compound I and II and III, we had tried to make NOEDS tests of compound I, II and III. Unfortunately, it was found the 17-acetyl and 17-acetoxy affected each other. Then, we transferred to make NOEDS tests of compound 7 and 8 and 14. According to the 1H NMR and NOEDS of compound 7 and 8 and 14 (Figs. 4–6), the configuration of each compound can been easily
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Y. Zhao et al. / Steroids 95 (2015) 7–16
confirmed. The position of each arrow is just the chemical shift of 13b-methyl. On the basis of the intensity of the signal of 17-acetyl, it can be easily confirmed that the 17-acetyl is at the same side of 13b-methyl. Clearly, the 17-acetyl of compound 7 is at the b-configuration, the 17-acetyl of compound 8 is at the a-configuration, and the 17-acetyl of compound 14 is at the a-configuration. Because the next step is acetylation, the configuration of 17-acetyl was not changed. Thus, that is to say, the configuration of compound I, II and III are the title compounds. The differences of these three isomers are listed in Table 1.
4. Results HPLC Analysis was carried out on a Agilent eclipse XDB-C18 (5 lm, 4.6 150 mm) eluded with 70% CH3OH in water at a flow rate of 1 mL/min and at k = 260 nm. At last, it was found that 0.2% of 11a, 17a-isomer I was present in the Ulipristal acetate synthesized by Dancsi’s route (Fig. 7), while 0.14% of 11b,17b-isomer III was present in the Ulipristal acetate synthesized by our route (Fig. 8). The 11a,17b-isomer II was not detected in any Ulipristal acetate synthesized by above two routes.
5. Conclusion We have developed an efficient and practical synthesis approach to deliver 11a,17a-isomer I, 11a,17b-isomer II and 11b,17b-isomer III for the first time. It is essential and important for comprehending the complete stereoisomers profile of Ulipristal acetate and meeting the stringent regulatory requirements of ICH. This study will provide an access to the reference standard of these three stereoisomers and also will be of immense help for organic chemists to obtain the pure Ulipristal acetate.
Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 81072532 & No. 81102324). References [1] International conference on harmonization (ICH) Guidelines, Q3A (R) Impurities in New Drug Substances, February 2002 (this guideline provides guidance for registration application on the content and qualification of impurities in new drug substances produced by the chemical synthesis). [2] International conference on harmonization (ICH) guidelines, Q3B (R) Impurities in New Drug Substances February 2002 (Guidance for registration or marketing application on the content and qualification of impurities in new drug product). [3] Cook CE, Wani MC, Lee YW, et al. 11b-Substituted progesterone analogs: WO, 8912448 [P]; 1989-12-28. [4] Glasier AF, Cameron ST, Fine PM, Logan SJ, Casale W, Van Horn J, Sogor L, Blithe DL, Scherrer B, Mathe H, Jaspart A, Ulmann A, Gainer E. Ulipristal acetate versus levonorgestrel for emergency contraception: a randomised noninferiority trial and meta-analysis. Lancet 2010;375(9714):555–62. [5] Rao PN, Wang Z, Cessac JW, Rosenberg RS, Jenkins DJA, Diamandis EP. New 11b-aryl substituted steroids exhibit both progestational and antiprogestational activity. Steroids 1998;63:523–30. [6] Kim HK, Rao PN, Burdett JE Jr, Acosta CK. Method for preparing 17a-acetoxy11b-(4-N,N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione, intermediates useful in the method, and methods for the preparation of such intermediates: WO, 9630390 [P]; 1996-10-03. [7] Kim HK, Rao PN, Simmons AM. Method for preparing 17a-acetoxy-11b-(4N,N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione, intermediates thereof, and methods for the preparation of such intermediates: WO, 2004078709 [P]; 2004-09-16. [8] Dancsi L, Visky G, Tuba Z, Csörgei J, Molnár C, Magyari E. Industrial process for the synthesis of 17a-acetoxy-11b-(4-N,N-dimethylaminophenyl)-19norpregna-4,9-diene-3,20-dione and new intermediates of the process: WO, 2007144674 [P]; 2007-12-21. [9] Yongguo Yu, He Yun, Zhao Yi, Hai Li, Yong Wu. A simple and convenient synthetic route to Ulipristal acetate. Steroids 2013;78:1293–7. [10] Palomo C, Oiarbide M, Landa A, et al. Design and synthesis of a novel class of sugar-peptide hybrids: C-linked glycol b-amino acids through a stereoselective ‘‘acetate’’ Mannich reaction as the key strategic element. J Am Chem Soc 2002;124:8637–43.
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First synthesis and characterization for the stereoisomers of Ulipristal acetate Yi Zhao a, Xiaolong Li a, Hong Liu b, Yongguo Yu a, Li Hai a, Li Guo a,⇑, Yong Wu a,⇑ a b
Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, PR China West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
a r t i c l e
i n f o
Article history: Received 15 June 2014 Received in revised form 29 October 2014 Accepted 12 December 2014 Available online 30 December 2014 Keywords: Ulipristal acetate Stereoisomer Synthesis X-ray structure Impurity
a b s t r a c t The three stereoisomers, 11a,17a-isomer I, 11a,17b-isomer II and 11b,17b-isomer III are related substances of the selective progesterone receptor modulator Ulipristal acetate. Herein, we presented an efficient and practical synthesis approach to deliver these three stereoisomers for the first time, and also confirmed the structure of the key intermediate 5a by single-crystal X-ray analysis. Our research will be of immense help for organic chemists to study the impurity profile of Ulipristal acetate. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction The presence of impurities, especially chiral impurities in an active pharmaceutical ingredient (API) has a significant impact on the quality and safety of the drug products. Therefore, it is necessary to study the impurity profiles of any API and try to control it during the manufacturing of a drug product. As per the International Conference on Harmonization (ICH) guidelines any impurities, which are forming at a level of P0.10% with respect to the API, should be identified, synthesized and characterized thoroughly [1,2]. 17a-Acetoxy-11b-(4-N,N-dimethylaminophenyl)-19-norpregna4,9-diene-3,20-dione (Ulipristal, EllaÒ, VA-2914 or CDB-2914, Fig. 1) is a well-known, more specific 19-norprogesterone and a selective progesterone receptor modulator (SPRM) which efficiently binds and inhibits progesterone receptor (PR) in progesterone target tissues [3]. Ulipristal acetate is well characterized for its anti-fertility potency in several of in vivo and in vitro assays. Different from the active metabolites, Ulipristal acetate expresses less antiglucocorticoid activity compared with the most well-known SPRM and mifepristone Besides, it also exhibits benefits for endometriosis treatment, as well as ovarian and breast cancer therapies [4].
⇑ Corresponding authors. Tel.: +86 028 85503235 (Y. Wu), +86 028 85503777 (L. Guo). E-mail addresses: [email protected] (L. Guo), [email protected] (Y. Wu). http://dx.doi.org/10.1016/j.steroids.2014.12.009 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.
Therefore, several methods have been developed so far for the synthesis of Ulipristal acetate [5–9]. However, two chiral centers in the Ulipristal acetate molecule from the starting materials can give rise to four stereoisomers including Ulipristal acetate (Fig. 2) [5,8,9]. Thus, the synthesis of the final product with the required stereochemistry is a significant challenge. Furthermore, these stereoisomers play essential roles for the preparation of Ulipristal acetate and also for the quality control of bulk drugs and drug formulations. In this report, we have designed and synthesized the stereoisomers of Ulipristal acetate. More importantly, the configuration of each isomer was also confirmed.
2. Experimental All reactions were carried out under an argon atmosphere. Most chemicals and solvents were analytical grade and used without further purification. TLC was performed using precoated silica gel GF254 (0.2 mm), while column chromatography was performed using silica gel (100–200 mesh). The melting point was measured on an YRT-3 melting point apparatus (Shantou Keyi instrument & Equipment Co. Ltd., Shantou, China). IR spectra were obtained on a Perkin Elmer 983 (Perkin Elmer, Norwalk, CT, USA). 1H NMR spectra were taken on a Varian INOVA 400 (Varian, Palo Alto, CA, USA) using CDCl3, d-DMSO and D2O as solvent. Chemical shifts are expressed in d (ppm), with tetramethylsilane (TMS) functioning as the internal reference, and coupling constants (J) were expressed in Hz. Mass
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Y. Zhao et al. / Steroids 95 (2015) 7–16
spectra were recorded on an Agilent 1946B ESI-MS instrument (Agilent, Palo Alto, CA, USA). (1) Synthesis of the key intermediate 5: 2.1. 3,3-(Ethylene-dioxy)-5b,10b-epoxyestr-9(11)-ene-17-one(2b) Hexachloroacetone (14.5 mL, 0.0954 mol), 30% aqueous hydrogen peroxide (14.1 mL, 0.477 mol) and sodium phosphate dibasic dodecahydrate (34.5 g, 0.0964 mol) were added to methylene chloride (300 mL) at 0 °C. The mixture was stirred for 1 h at the same temperature. The material 1 (30 g, 0.0954 mol) was added to above mixture. The reaction mixture was stirred for another 18 h at the same temperature. Then, it was poured into a mixture of methylene chloride (200 mL) and ice (160 g). A solution of sodium thiosulfate (79 g, 0.5 mol) in water (300 mL) was added dropwise to the mixture to destroy the excess of hydrogen peroxide. After separation, the organic fraction was washed with water (2 100 mL) and dried on sodium sulfate. The solvent was removed in vacuo to give 32 g (100%) of product, which was a 65:35 mixture of the 5a,10a- and 5b,10b-epoxides showed by HPLC. The obtained crude mixture of epoxides was recrystallized with petroleum ether/ethyl acetate (5:1) to get 12.8 g (40%) white solid 2a. The filtrate was concentrated in vacuo to yield 19.2 g (60%) of a 42:58 mixture of the 5a,10a- and 5b,10b-epoxides. Then the mixture was purified via column chromatography (petroleum ether/ethyl acetate 10:1) to give 6.91 g (36%) white solid 2b. mp: 158–160 °C, MS (m/z): 331.42 [M + H]+, Analysis calculated for C20H26O4: C 72.70, H 7.93; found: C 72.80, H 7.89. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.861 (s, 3H, CH3), 1.201–2.511 (m, 18H), 3.796–3.961 (m, 4H, OACH2), 5.851 (d, 1H, J = 2.8, @CH). 2.2. 3,3-(Ethylene-dioxy)-17a-ethynyl-17b-hydroxy-5b,10bepoxyestr-9(11)-ene (3b) Under argon, 2b (6.8 g, 0.02 mol) was dissolved in dry tetrahydrofuran (70 mL) at 0 °C, and sodium acetylide (1.9 g, 0.04 mol) was added. The mixture was stirred for 1 h. Saturated ammonium chloride solution (50 mL) and water (50 mL) were added, then, the reaction mixture was stirred for 10 min. Then the reaction mixture was concentrated to a volume of 80 mL. The residue was stirred for 3 h at 0 °C. The precipitated crystals were filtered off and dried at 50 °C to yield 7.3 g (100%) of the title compound 3b, mp: 154–156 °C, MS (m/z): 379.48 [M + Na]+, Analysis calculated for C22H28O4: C 74.13, H 7.92; found: C 74.21, H 7.89. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.825 (s, 3H, CH3), 1.081– 2.627 (m, 18H), 2.557 (s, 1H, acetylenic hydrogen), 3.867– 3.946(m, 4H, OACH2), 5.890 (t, 1H, J = 4.4, @CH). 2.3. 3,3-(Ethylene-dioxy)-5b,17b-dihydroxy-11a-(4-N,Ndimethylaminophenyl)-19-nor-17a-pregn-9-ene-21-ethyne(4b) Under anhydrous conditions, a portion (2 mL) of a solution of 4-bromo-N,N-dimethylaniline (12 g, 0.06 mol) in dry tetrahydrofuran (50 mL) and one crystal of iodine was added to the mixture of magnesium (1.7 g, 0.07 mol) in dry tetrahydrofuran (5 mL) at 50 °C. After evidence of reaction was observed, the entire amount
N
O OAc
O Fig. 1. The structures of Ulipristal acetate.
of the reagent was added dropwise. The reaction mixture was stirred for an additional 2 h while it was cooling to room temperature. The mixture was then added dropwise to a suspension of 3b (7.0 g, 0.02 mol) and copper (I) chloride (0.6 g, 0.002 mol) in dry tetrahydrofuran (70 mL) at 0 °C. The reaction mixture was stirred for 1 h, then, it was poured into 10% ammonium chloride (70 mL) solution and extracted with methylene chloride (3 50 mL). The combined organic fractions were washed with water (4 50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give a black oil (21.1 g). The oil was purified via column chromatography (petroleum ether–ethyl acetate 10:1) to yield 5.23 g (55%) light blue solid. mp: 162–164 °C, MS (m/z): 478.62 [M + H]+, Analysis calculated for C30H39NO4: C 75.44, H 8.23, N 2.93; found: C 75.55, H 8.28, N 2.75. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.970 (s, 3H, CH3), 0.842–2.332 (m, 18H), 2.474 (s, 1H, acetylenic hydrogen), 2.923 (s, 6H, NACH3), 3.716 (t, 1H, J = 6.4, CH), 3.820– 4.022(m, 4H, OACH2), 6.680(d, 2H, J = 8.4, ArAH), 7.048 (d, 2H, J = 8.4, ArAH). 2.4. 11a-(4-N,N-dimethylaminophenyl)-17a-ethynyl-17bhydroxyestr-4,9-diene-3-one(5) To a solution of potassium bisulfate (3.7 g, 0.027 mol) in water (50 mL) was added compound 4b (5 g, 0.01 mol) at 0 °C. The mixture was stirred for 5 h and almost became clear. Then, saturated sodium bicarbonate was added to the above mixture to adjust pH to 7–8. The precipitated crystals were filtered off and dried at 50 °C to yield 2.7 g (65%) of the title compound 5. mp: 134– 136 °C, MS (m/z): 416.58 [M + H]+, Analysis calculated for C28H33NO2: C 80.93, H 8.00, N 3.37; found: C 80.85, H 8.18, N3.28. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 1.005 (s, 3H, CH3), 1.396–2.628 (m, 16H), 2.476 (s, 1H, acetylenic hydrogen), 2.920 (s, 6H, NACH3), 3.899 (t, 1H, J = 8.8, CH), 5.664 (s, 1H, @CH), 6.680 (d, 2H, J = 8.4, ArAH), 6.952 (d, 2H, J = 8.4, ArAH). (2) Synthesis of 11a,17a-isomer I: 2.5. 11a-(4-N,N-dimethylaminophenyl)-21-(phenyl-sulfinyl)-19norpregna-4,9,17(20),20-tetraene-3-one (6) To a suspension of compound 5 (1.5 g, 0.0036 mol), triethylamine (2.2 mL, 0.0152 mol) in dry tetrahydrofuran (20 mL), a solution of phenylsulfenyl chloride (0.9 g, 0.0062 mol) in dry tetrahydrofuran (15 mL) was added dropwise while keeping the temperature between 70 and 78 °C. The reaction mixture was stirred for 4 h at 78 °C, then water (20 mL) and methanol (20 mL) was added. The reaction mixture was stirred for 10 min and extracted with methylene chloride (3 40 mL). The combined organic fractions were washed with water (4 20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to get 2.5 g of a reddish brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 5:1) to yield 1.18 g (62.5%) of white solid. mp: 151–155 °C. MS (m/z): 546.78 [M + Na]+, Analysis calculated for C34H37NO2S: C77.97, H 7.12, N 2.67, S 6.12; found: C 77.84, H 7.21, N 2.75, S 6.18. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 1.042 (s, 3H, CH3), 1.213–2.767 (m, 16H), 2.900 (s, 6H, NACH3), 3.967 (t, 1H, J = 8.8, CH), 5.681 (s, 1H, @CH), 6.075 (s, 1H, @CH), 6.634 (d, 2H, J = 8.4, ArAH), 6.888 (d, 2H, J = 8.4, ArAH), 7.460–7.539 (m, 3H, ArAH), 7.626 (d, 2H, J = 7.2, ArAH). 2.6. 11a-(4-N,N-dimethylaminophenyl)-17a-hydroxy-19-norpregna4,9-diene-3,20-dione(7) Compound 6 (1.1 g, 0.0021 mol) was added to a solution of sodium methoxide (0.11 g, 0.0021 mol) in methanol (20 mL). The reaction mixture was stirred at 50 °C for 1 h, then trimethyl
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Y. Zhao et al. / Steroids 95 (2015) 7–16
N
N
O OAc
O
N
O OAc
O
O OAc
O II
I
III
Fig. 2. The stereoisomers of Ulipristal acetate.
O
H 2O2, (Cl 3C)2CO
O
1
+
O
O
Na2HPO 4/CH 2Cl2 rt, 12h, 100%
O
O
O
O
O
2a
1) recrystallization 2) Column chromatography
O
O
2b
21.6%
5α,10α : 5β,10β 65 : 35 N HO
N
1)
HO
MgBr CuCl, THF, 0 oC, 5h
sodium acetylide
2b THF, 0oC, 4h, 100% O
O
2) NH 4Cl, CH2Cl2/H2O, rt, 55%
3b
O
O O
OH 4b
N HO KHSO4 /H2 O,0o C, 4h 65% O 5 Scheme 1. Synthesis of the key intermediate 5 [5,9].
O HC S C
N SCl
1)P(OCH 3)3 /NaOCH 3/CH 3OH
5 triethylamine/THF/-78 oC 4h, 62.5%
2)1.0 M HCl/CH3OH, 0.5h, 54.8% 6
O
N
O
N
HO
O OAc
Ac2O/HClO 4
O
CH 2Cl2, -20 oC, 3h, 86% O
7
I
Scheme 2. Synthesis of 11a,17a-isomer I [9].
phosphite (0.4 g, 0.0032 mol) was added and stirring was continued at 70 °C for 2 h. The reaction mixture was cooled to 20 °C and poured into water (25 mL), then extracted with methylene chloride (3 30 mL). The combined organic fractions were dried over sodium sulfate, filtered, and concentrated in vacuo to give 2.12 g of reddish brown oil. The oil was dissolved in a mixture of 1 N hydrochloric acid (1.3 mL) and methanol (20 mL), then stirred at room temperature for 30 min. Ice water (200 mL) was added to the mixture. After neutralizing with saturated sodium bicarbonate,
the mixture was extracted with methylene chloride (3 20 mL). The combined organic fractions were washed with water (2 20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to get 1.6 g of reddish brown oil. The obtained crude product was purified via column chromatography (petroleum ether– ethyl acetate 10:1) to yield 0.5 g (54.8%) off-white syrupy. MS (m/z): 434.58 [M + H]+, Analysis calculated for C28H35NO3: C 77.56, H 8.14, N 3.23; found: C 77.62, H 8.21, N 3.28. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.881(s, 3H, CH3), 1.232–2.787
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Y. Zhao et al. / Steroids 95 (2015) 7–16
2.9. 11a,17b-Isomer II (Scheme 3)
(m, 16H), 2.258 (s, 3H, COCH3), 2.914 (s, 6H, NACH3), 3.867 (t, 1H, J = 9.2, CH), 5.663 (s, 1H, @CH), 6.673 (d, 2H, J = 8.4, ArAH), 6.934 (d, 2H, J = 8.4, ArAH).
Perchloric acid (0.4 mL, 0.0044 mol) was added to acetic anhydride (2 mL, 0.0212 mol) by dropwise at 10 °C. The obtained solution was stirred for an additional 1 h, then cooled to 30 °C and the solution of compound 8 (0.8 g, 0.002 mol) in dry dichloromethane (20 mL) was added at such rate to keep the temperature between 10 °C and 20 °C, then the reaction mixture was stirred at this temperature for 3 h. The mixture was diluted with dichloromethane (20 mL) and poured into water (20 mL) containing sodium acetate (5.6 g, 0.068 mol). The organic layer was separated, washed with water (3 10 mL), dried over sodium sulfate and concentrated under reduced pressure to give 1.12 g of slightly yellow syrupy. The syrupy was purified via column chromatography (petroleum ether–ethyl acetate 15:1) to yield 0.84 g (86%) of slightly yellow syrupy. FTIR (KBr, diffuse reflectance): cmax 2933; 1731 and 1722(AC@O); 1669 and 1658 (conjugated AC@O); 1568; 1531; 1455; 1371; 1332; 1262; 1222; 1171; HRMS: calculated for C30H37NO4 475.6191, found: 475.6178. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.979 (s, 3H, CH3), 1.193– 2.683 (m, 16H), 1.609 (s, 3H, COCH3), 2.072 (s, 3H, COCH3), 2.917 (s, 6H, NACH3), 3.850 (t, 1H, J = 8.0), 5.687 (s, 1H, CH), 6.666 (d, 2H, J = 8.4, ArAH), 6.989 (d, 2H, J = 8.4, ArAH); 13C NMR {400 MHz, CDCl3 (TMS), d (ppm)}:21.195; 24.087; 25.684; 26.754; 27.789; 30.168; 30.998; 36.676; 36.789; 38.257; 39.268; 40.485; 46.981; 50.848; 66.986; 96.120; 112.689; 122.845; 127.228; 129.187; 131.420; 145.548; 148.528; 156.496; 170.541; 199.431; 203.722.
2.7. 11a,17a-Isomer I Perchloric acid (0.1 mL, 0.0011 mol) was added to acetic anhydride (0.5 mL, 0.0053 mol) by dropwise at 10 °C. The obtained solution was stirred for an additional 1 h, then, cooled to 20 °C and the solution of compound 7 (0.2 g, 0.0005 mol) in dry dichloromethane (10 mL) was added at such rate to keep the temperature between 10 and 20 °C, then the reaction mixture was stirred at this temperature for 3 h. The mixture was diluted with dichloromethane (10 mL) and poured into water (10 mL) containing sodium acetate (1.4 g, 0.017 mol). The organic layer was separated, washed with water (3 10 mL), dried over sodium sulfate and concentrated under reduced pressure to give 0.22 g of slightly yellow syrupy. The syrupy was purified via column chromatography (petroleum ether–ethyl acetate 15:1) to yield 0.19 g (86%) of slightly yellow syrupy. FTIR (KBr, diffuse reflectance): cmax 2923; 1721 and 1718 (AC@O); 1673 and 1662 (conjugated AC@O); 1575; 1528; 1448; 1362; 1313; 1259; 1212; 1178; HRMS: calculated for C30H37NO4 475.6191, found: 475.6165. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.797 (s, 3H, CH3), 1.256– 2.658 (m, 16H), 2.024 (s, 6H, COCH3), 2.945 (s, 6H, NACH3), 3.905 (t, 1H, J = 9.2, CH), 5.686 (s, 1H, @CH), 6.710 (d, 2H, J = 8.4, ArAH), 6.982 (d, 2H, J = 8.4, ArAH); 13C NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 21.181; 24.075; 25.668; 26.761; 27.780; 30.165; 30.951; 36.688; 36.748; 38.268; 39.281; 40.479; 46.951; 50.839; 66.989; 96.110; 112.678; 122.851; 127.214; 129.195; 131.398; 145.525; 148.534; 156.481; 170.517; 199.425; 203.732.
(4) Synthesis of 11b,17b-isomer III: 2.10. 3,3-(Ethylene-dioxy)-17a-ethynyl-17b-hydroxyestr-5(10),9(11)diene (9)
(3) Synthesis of 11a,17b-isomer II: Compound 1 (20 g, 0.0636 mol) was dissolved in dry tetrahydrofuran (200 mL) at 0 °C. The mixture was stirred for 0.5 h, and sodium acetylide (6.0 g, 0.1272 mol) was added. After that, the reaction mixture was stirred for another 1 h. Saturated ammonium chloride solution (200 mL) and water (200 mL) were added and the mixture was stirred for 10 min. Then the reaction mixture was concentrated to a volume of 350 mL. The residue was stirred for 3 h at 0 °C. The precipitated crystals were filtered off and dried at 50 °C to yield 20.6 g (95%) of the title compound 9. mp: 145– 148 °C, MS (m/z): 340.21 [M + H]+, Analysis calculated for C22H28O3: C 77.61, H 8.29; found: C 77.68, H 8.35. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.891(s, 3H, CH3), 1.134–2.662 (m, 18H), 2.579 (s, 1H, acetylenic hydrogen), 3.881–3.998 (m, 4H, OACH2), 5.678 (t, 1H, J = 5.6, @CH).
2.8. 11a-(4-N,N-dimethylaminophenyl)-17b-hydroxy-19-norpregna4,9-diene-3,20-dione(8) Compound 5 (1.2 g, 0.0029 mol) was dissolved in a mixture of acetic acid-H2O-30% sulfuric acid (40:8:1, 10 mL), then, mercuric sulfate (0.085 g, 0.0003 mol) was added to above mixture. The reaction mixture was stirred for 5 h at 60 °C. After neutralizing with saturated sodium bicarbonate, the mixture was extracted with methylene chloride (3 20 mL). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure to give 1.0 g of brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 15:1) to yield 0.82 g (65.2%) of a slightly yellow syrupy. MS (m/z): 434.58 [M + H]+, Analysis calculated for C28H35NO3: C 77.56, H 8.14, N 3.23; found: C 77.48, H 8.01, N 3.18. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.901 (s, 3H, CH3), 1.079 (s, 3H, COCH3), 1.242–2.689 (m, 16H), 2.926 (s, 6H, NACH3), 3.811 (t, 1H, J = 7.2, CH), 5.704 (s, 1H, @CH), 6.676 (d, 2H, J = 8.4, ArAH), 7.016 (d, 2H, J = 8.4, ArAH).
2.11. 17a-Ethynyl-17b-hydroxyestra-4,9-diene-3-one(10) To a solution of compound 9 (20 g, 0.059 mol) in acetic acid (200 mL), perchloric acid (10.7 mL, 0.118 mol) was added dropwise. The reaction mixture was stirred for 0.5 h at room
N HO
N
O
O OAc
5
Ac2O/HClO4
HgSO4/H 2SO 4 CH 3COOH/H 2O, 60oC 5h, 65.2%
CH 2Cl2, -20oC 3h, 88% O
O 8
Scheme 3. Synthesis of 11a,17b-isomer II [9,10].
II
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O
O O
1
OH CH3COOH
sodium acetylide THF,0o C,1h O 95% O
O HO ethylene glycol, p-toluenesulfonic acid triethyl orthoformate CH2Cl2 ,rt, 3h 85.6%
O HO
O
O
CH2Cl2 /Na2 HPO4,0o C,18h 100%
12
MgBr /
O
O
O
+
o
3) ethanol/ 8.5%H2 SO4,0 C, 2h,64%
O
5α,10α : 5β,10β 75 : 25 N
CH2 Cl 2, -20oC, 4h, O 86.9%
O 14
O
O
O
13b
O
Ac 2O/HClO 4
2) Saturated NH4Cl/CH2Cl2 ,0oC
O HO
O
HO CuCl/THF,0oC,3h
11
O 13a
N 1) N
THF/H2O, 60o C, 5h O 79%
10
(CF3 )2 CO/H2 O2
O
HO
HgSO4 /H2 SO4
HClO4 ,rt, 0.5h 87% O
9
O
OH
OAc
III
Scheme 4. Synthesis of 11b,17b-isomer III [5,9,10].
temperature, then, poured into water (500 mL). The precipitated crystals were filtered off and dried at 50 °C to give 15.21 g (87%) off-white solid. mp: 181–185 °C, MS (m/z): 297.18 [M + H]+, Analysis calculated for C20H24O2: C 81.04, H 8.16; found: C 80.95, H 8.11; 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.879 (s, 3H, CH3), 1.202–2.658 (m, 18H), 2.587 (s, 1H, acetylenic hydrogen), 5.682 (s, 1H, @CH). 2.12. 17b-Hydroxy-19-norpregna-4,9-diene-3,20-dione (11) Compound 10 (15.0 g, 0.0506 mol) was dissolved in tetrahydrofuran (150 mL), then, 30% sulfuric acid (50 mL), mercuric sulfate (1.44 g, 0.0051 mol) was added to the above mixture. The reaction mixture was stirred for 5 h at 60 °C. After neutralizing with saturated sodium bicarbonate, the mixture was extracted with methylene chloride (3 50 mL). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure to give brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 10:1) to yield 12.57 g (79%) white solid. mp: 195–196 °C, MS (m/z): 315.19 [M + H]+, Analysis calculated for C20H26O3: C 76.40, H 8.33; found: C 76.45, H 8.41; 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.877(s, 3H, CH3), 1.239 (s, 3H, CH3), 1.363–2.945 (m, 18H), 5.688 (s, 1H, @CH). 2.13. 3,3,20,20-Bis(ethylene-dioxy)-17b-hydroxy-19-norpregna5(10),9(11)-diene (12) To a solution of compound 11 (12 g, 0.0382 mol) in methylene chloride (120 mL), ethylene glycol (21.3 mL, 0.382 mol), trimethyl orthoformate (41.7 mL, 0.382 mol) and p-toluenesulfonic acid (3.3 g, 0.0191 mol) were added. The reaction mixture was stirred at room temperature for 3 h, then saturated sodium bicarbonate was added to adjust pH to 7–8. The organic layer was separated, and the water-course was extracted with methylene chloride (3 50 mL). The combined organic fractions were dried over sodium sulfate and concentrated under reduced pressure to give slightly brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 20:1) to yield 13.1 g (85.6%) white
solid. mp: 185–190 °C, MS (m/z): 403.24 [M + H]+; Analysis calculated for C24H34O5: C 71.61, H 8.51; found: C 71.55, H 8.59; 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.905 (s, 3H, CH3), 1.090 (s, 3H, CH3), 1.173–2.665 (m, 18H), 2.261 (s, 1H, OH), 3.746– 3.819 (m, 1H, AOCH2), 3.933–4.013 (m, 6H, AOCH2), 4.067–4.119 (m, 1H, AOCH2), 5.588 (t, 1H, J = 5.6, @CH). 2.14. 3,3,20,20-Bis(ethylene-dioxy)-17b-hydroxy-5a,10a-epoxy-19norpregna-9(11)-ene (13a) and 3,3,20,20-bis(ethylene-dioxy)-17bhydroxy-5b,10b-epoxy-19-norpregna-9(11)-ene (13b) Under nitrogen, 30% aqueous hydrogen peroxide (4.4 mL, 0.15 mol) and sodium phosphate dibasic dodecahydrate (11.7 g, 0.033 mol) were added to a solution of hexafluoroacetone (3.75 mL, 0.03 mol) in methylene chloride (120 mL). The mixture was stirred for 1 h at 0 °C. Compound 12 (12 g, 0.03 mol) was added to above mixture. The reaction mixture was stirred for another 18 h at the same temperature. Then, it was poured into a mixture of methylene chloride (100 mL) and ice (6 g). A solution of sodium thiosulphate (23.7 g, 0.15 mol) in water (150 mL) was added dropwise to the mixture to destroy the excess of hydrogen peroxide. After separation, the organic fraction was washed with water (2 50 mL) and dried over sodium sulfate. The solvent was removed in vacuo to give 12.6 g (100%) of product, which was a 75:25 mixture of the 5a,10a- and 5b,10b-epoxides showed by HPLC. The obtained crude mixture of epoxides was used in the next step without further purification. MS (m/z): 419.24 [M + H]+; Analysis calculated for C24H34O6: C 68.87, H 8.19; found: C 68.95, H 8.31; 1 H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.885 (s, 3H, CH3), 1.042 (s, 3H, CH3), 1.073–2.641 (m, 18H), 3.751–3.811 (m, 1H, AOCH2), 3.852–4.003(m, 6H, AOCH2), 4.060–4.112 (m, 1H, AOCH2), 5a,10a: 5.830 (t, 1H, J = 6.0, @CH), 5b,10b: (t, 1H, J = 6.0, @CH). 2.15. 11b-(4-N,N-dimethylaminophenyl)-17b-hydroxy-19-norpregna4, 9-diene-3, 20-dione(14)
of
Under anhydrous conditions, a portion (4 mL) of a solution 4-bromo-N,N-dimethylaniline (21 g, 0.105 mol) in dry
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tetrahydrofuran (100 mL) and one crystal of iodine was added to the mixture of magnesium (2.92 g, 0.12 mol) in dry tetrahydrofuran (10 mL) at 50 °C. After evidence of reaction was observed, the entire amount of the reagent was added dropwise. The reaction mixture was stirred for an additional 2 h while it was cooling to room temperature. The mixture was then added dropwise to a suspension of 13a and 13b (12.6 g, 0.03 mol) and copper (I) chloride (0.9 g, 0.003 mol) in dry tetrahydrofuran (130 mL) at 0 °C. The reaction mixture was stirred for 3 h, then, poured into 10% ammonium chloride (70 mL) solution and extracted with methylene chloride (3 100 mL). The combined organic fractions were washed with water (4 50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give black oil (28.5 g). The obtained oil was dissolved in ethanol (200 mL) under a nitrogen atmosphere, and 8.5% aqueous sulfuric acid (8.9 mL) was added. The reaction mixture was stirred for 2 h at 70 °C. After neutralizing with saturated sodium bicarbonate, the mixture was diluted with water (300 mL), and extracted with methylene chloride (3 100 mL). The combined organic fractions were dried over sodium sulfate and concentrated under reduced pressure to give brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 15:1) to yield 8.3 g (64%) of a light blue syrupy. MS (m/z): 434.26 [M + H]+, Analysis calculated for C28H35NO3: C 77.56, H 8.14, N 3.23; found: C 77.48, H 8.24, N 3.17; 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.394 (s, 3H, CH3), 0.858–3.042 (m, 16H,), 1.241 (s, 3H, COCH3), 3.916(s, 6H, NACH3), 4.364 (d, 1H, J = 5.2, CH), 5.767 (s, 1H, @CH), 6.667 (d, 2H, J = 8.0, ArAH), 7.020 (d, 2H, J = 8.0, ArAH).
between 10 °C and 20 °C, then the reaction mixture was stirred at this temperature for 4 h. The mixture was diluted with dichloromethane (50 mL) and poured into water (50 mL) containing sodium acetate (35 g, 0.425 mol). The organic layer was separated, washed with water (3 10 mL), dried over sodium sulfate and concentrated under reduced pressure to give 6.1 g of brown oil. The oil was purified via column chromatography (petroleum ether–ethyl acetate 15:1) to yield 5.17 g (86.9%) of slightly yellow syrupy. FTIR (KBr, diffuse reflectance): cmax 2938; 1742 and 1725 (AC@O); 1662 and 1654 (conjugated AC@O); 1571; 1538; 1449; 1379; 1342; 1269; 1242; 1178; HRMS: calculated for C30H37NO4 475.6191, found: 475.6181. 1H NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 0.702 (s, 3H, CH3), 0.856–2.813 (m, 16H), 2.090 (s, 3H, COCH3), 2.129 (s, 3H, COCH3), 2.935(s, 6H, NACH3), 4.278 (t, 1H, J = 6.4, CH), 5.747 (s, 1H, @CH), 6.683 (d, 2H, J = 8.4, ArAH), 7.021 (d, 2H, J = 8.4, ArAH); 13C NMR {400 MHz, CDCl3 (TMS), d (ppm)}: 21.184; 24.079; 25.688; 26.765; 27.791; 30.178; 30.988; 36.687; 36.791; 38.264; 39.271; 40.487; 46.957; 50.854; 66.979; 96.134; 112.695; 122.851; 127.234; 129.179; 131.442; 145.551; 148.537; 156.499; 170.554; 199.445; 203.731.
3. Discussion 3.1. The formation of the 11a,17a-isomer I The synthesis of the drug substance was based on the very efficient 1,4-addition (Scheme 5). The 5a,10a-epoxide was converted into Ulipristal acetate smoothly. But there always had the 5b, 10bisomer in the starting material. After a series of reactions, the 5b,10b-isomer was transformed into the 11a,17a-isomer I (Scheme 6). Especially, the contents of 15b was up to 45% in the route reported by Dancsi et al. [8]. In this route, the ratio of 15a and 15b was about 55:45, which was used directly in the next step without further separation. And there were only three steps to
2.16. 11b,17b-Isomer III (Scheme 4) Perchloric acid (2.3 mL, 0.025 mol) was added to acetic anhydride (11.8 mL, 0.125 mol) by dropwise at 10 °C. The obtained solution was stirred for an additional 1 h, then, cooled to 30 °C and the solution of compound 14 (5 g, 0.0125 mol) in dry dichloromethane (50 mL) was added at such rate to keep the temperature
N
R
R
Grignard addition O
O O
Ulipristal acetate
O O
5α,10α
OH
4a R= 17β− hydroxy, 17α- ethynyl
3a R= 17β−hydroxy, 17α-ethynyl
O
O 15a R= 17 α−hydroxy, 17β -
16a R= 17α− hydroxy, 17β -
O CH 3
O CH 3
Scheme 5. Synthesis of Ulipristal acetate.
N
R
N
R
O OAc
Grignard addition O
O O
O O
5β,10β
3b R= 17β− hydroxy, 17α - ethynyl
OH
4b R= 17 β− hydroxy, 17 α-ethynyl O
O 15b R= 17α− hydroxy, 17β-
O
O CH3
16b R= 17α−hydroxy, 17β-
O CH 3
Scheme 6. Formation of 11a,17a-isomer I.
11α,, 17α− −isomer Ι
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Y. Zhao et al. / Steroids 95 (2015) 7–16
O HC S C
1) CH3ONa/P(OCH 3)3 2)1.0 M HCl O
O O
O OH
O OH +
O O
O
17
19
18 5 steps
5 steps
Ulipristal acetate and 11β,, 17β-isomer III
11α,, 17β- isomer II and 11β,, 17β-isomer III
Scheme 7. Formation of compound 19.
O HC S C
N
N
N
O
O HO
HO 1)P(OCH3) 3 /CH3ONa
+
2)1.0 M HCl O
O
O
14
21
20 Ac 2O
Ac 2O 11β,, 17β-isomer III
Ulipristal acetate Scheme 8. Formation of compound 14.
O HC S C
N
N
N
O
O HO
HO 1)P(OCH3) 3 /CH3ONa
+
2)1.0 M HCl O
O
O
8
7
6
Ac2O
Ac2O
11α,, 17β -isomer II
11α,, 17α -isomer I Scheme 9. Formation of compound 7 and 8.
obtain Ulipristal acetate. Thus, it was highly possible that the 11a,17a-isomer I was present in the final product. According to HPLC-MS, we found two peaks with the same m/z value as Ulipristal acetate. So, it was necessary to confirm the two peaks. At the beginning, we tried to separate the 11a, 17a-isomer I from the final product. However, the content of the title compound was too low to fit the large scale. Then we attempted to directly get the compound 15b or 16b. Unfortunately, 15a and 15b, or 16a and 16b had the same Rf according to TLC. There was no way to obtain 15b or 16b at a slightly large scale by the means of Dancsi’s route [8]. Previously, we had published our work describing a synthetic route to Ulipristal acetate [9]. Fortunately, in this route, it was found the compound 2a and 2b could be separated on TLC easily, where the Rf value of 2b was slightly larger than 2a. Thus, we designed the above route to synthesize the 11a,17a-isomer I (Scheme 1 and 2). In this route, the contents of 2b was about 35% using hexachloroacetone as the catalyst, mCPBA as epoxidation reagents [9]. In order to improve the contents of 2b, the mixture of 2a and 2b was recrystallized firstly. Then, the filtrate was
Fig. 3. ORTEP view of the key intermediate 5.
concentrated in vacuo to yield a 42:58 mixture of the 2a and 2b. The mixture was separated via column chromatography to give the 5b,10b-isomer 2b.
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Fig. 4. (a) The 1H NMR {400 MHz, CDCl3 (TMS)} of compound 7. (b) The NOEDS {400 MHz, CDCl3 (TMS)} of compound 7 at 0.89 ppm (arrow).
Fig. 5. (c) The 1H NMR {400 MHz, CDCl3 (TMS)} of compound 8. (d) The NOEDS {400 MHz, CDCl3 (TMS)} of compound 8 at 0.90 ppm (arrow).
Fig. 6. (e) The 1H NMR {400 MHz, CDCl3 (TMS)} of compound 14. (f) The NOEDS {400 MHz, CDCl3 (TMS)} of compound 14 at 0.39 ppm (arrow).
3.2. The formation of the 11a,17b-isomer II and 11b,17b-isomer III Dancsi et al. used compound 1 as their starting material to synthesize Ulipristal acetate [8]. The phenyl-sulfinyl compound 17 was obtained after two steps, then, reacted with sodium
methoxide and trimethyl phosphite to get 17a-hydroxy compound 18 (Scheme 7). Our previous work also used compound 1 as the starting material to synthesize Ulipristal acetate [9]. After 5 steps, the phenyl-sulfinyl compound 20 was afforded, then reacted with sodium methoxide and trimethyl phosphite to yield 17a-hydroxy
15
Y. Zhao et al. / Steroids 95 (2015) 7–16 Table 1 The differences in 11a,17a-isomer I, 11a,17b-isomer II and 11b,17b-isomer III and Ulipristal acetate. Compound
[a]20 D (c 1.0,CHCl3)
11-CH (ppm)
17-CH3 (ppm)
17-COCH3 (ppm)
17-OCOCH3 (ppm)
Ulipristal acetate 11a,17b-Isomer I 11a,17a-Isomer II 11b,17a-Isomer III
+174° 82° 135° +124°
4.365 3.905 3.850 4.278
0.355 0.797 0.979 0.702
2.137 2.024 1.609 2.129
2.099 2.024 2.072 2.090
Fig. 7. HPLC chromatogram of the crude Ulipristal acetate which was synthesized by Dancsi’s route [8].
Fig. 8. HPLC chromatogram of the crude Ulipristal acetate which was synthesized by Yu’s route [9].
compound 21 (Scheme 8). Thus, most of the product was the 17ahydroxy compound 18 and 21. However, the 17b-hydroxy compound 19 and 14 can also be generated according to collision theory. Especially, when there was 11a-[4-(dimetnylamino)phenyl] in the molecule of compound 6, the generation of 17b-hydroxy compound 8 was easier than the 17b-hydroxy compound 19 and 14 (Scheme 9). From above discussion, we can see that 11a,17a-isomer I, 11a,17b-isomer II and 11b,17b-isomer III might be all present in the final product of Ulipristal acetate. Therefore, further study of the isomers is highly desirable. 3.3. Structure confirmation We tried to cultivate the single-crystal to research their structure. Fortunately the single-crystal of the key intermediate 5 was
cultivated by slow evaporation at room temperature successfully. Its structure had been confirmed by the use of single-crystal X-ray analysis (Fig. 3). It was clear that 4-N,N-dimethylaminophenyl in the molecule of compound 5 was found out at the a-configuration which was not changed in the next reactions. The d (ppm) of 11b-hydrogen was about 3.6–4.0, while 11a-hydrogen was about 4.2–4.4 according to 1H NMR. Thus, the configurations of 4-N,N-dimethylaminophenyl in the molecule of compound I, II and III were easily confirmed. In order to confirm the configurations of 17-acetyl in the molecule of compound I and II and III, we had tried to make NOEDS tests of compound I, II and III. Unfortunately, it was found the 17-acetyl and 17-acetoxy affected each other. Then, we transferred to make NOEDS tests of compound 7 and 8 and 14. According to the 1H NMR and NOEDS of compound 7 and 8 and 14 (Figs. 4–6), the configuration of each compound can been easily
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Y. Zhao et al. / Steroids 95 (2015) 7–16
confirmed. The position of each arrow is just the chemical shift of 13b-methyl. On the basis of the intensity of the signal of 17-acetyl, it can be easily confirmed that the 17-acetyl is at the same side of 13b-methyl. Clearly, the 17-acetyl of compound 7 is at the b-configuration, the 17-acetyl of compound 8 is at the a-configuration, and the 17-acetyl of compound 14 is at the a-configuration. Because the next step is acetylation, the configuration of 17-acetyl was not changed. Thus, that is to say, the configuration of compound I, II and III are the title compounds. The differences of these three isomers are listed in Table 1.
4. Results HPLC Analysis was carried out on a Agilent eclipse XDB-C18 (5 lm, 4.6 150 mm) eluded with 70% CH3OH in water at a flow rate of 1 mL/min and at k = 260 nm. At last, it was found that 0.2% of 11a, 17a-isomer I was present in the Ulipristal acetate synthesized by Dancsi’s route (Fig. 7), while 0.14% of 11b,17b-isomer III was present in the Ulipristal acetate synthesized by our route (Fig. 8). The 11a,17b-isomer II was not detected in any Ulipristal acetate synthesized by above two routes.
5. Conclusion We have developed an efficient and practical synthesis approach to deliver 11a,17a-isomer I, 11a,17b-isomer II and 11b,17b-isomer III for the first time. It is essential and important for comprehending the complete stereoisomers profile of Ulipristal acetate and meeting the stringent regulatory requirements of ICH. This study will provide an access to the reference standard of these three stereoisomers and also will be of immense help for organic chemists to obtain the pure Ulipristal acetate.
Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 81072532 & No. 81102324). References [1] International conference on harmonization (ICH) Guidelines, Q3A (R) Impurities in New Drug Substances, February 2002 (this guideline provides guidance for registration application on the content and qualification of impurities in new drug substances produced by the chemical synthesis). [2] International conference on harmonization (ICH) guidelines, Q3B (R) Impurities in New Drug Substances February 2002 (Guidance for registration or marketing application on the content and qualification of impurities in new drug product). [3] Cook CE, Wani MC, Lee YW, et al. 11b-Substituted progesterone analogs: WO, 8912448 [P]; 1989-12-28. [4] Glasier AF, Cameron ST, Fine PM, Logan SJ, Casale W, Van Horn J, Sogor L, Blithe DL, Scherrer B, Mathe H, Jaspart A, Ulmann A, Gainer E. Ulipristal acetate versus levonorgestrel for emergency contraception: a randomised noninferiority trial and meta-analysis. Lancet 2010;375(9714):555–62. [5] Rao PN, Wang Z, Cessac JW, Rosenberg RS, Jenkins DJA, Diamandis EP. New 11b-aryl substituted steroids exhibit both progestational and antiprogestational activity. Steroids 1998;63:523–30. [6] Kim HK, Rao PN, Burdett JE Jr, Acosta CK. Method for preparing 17a-acetoxy11b-(4-N,N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione, intermediates useful in the method, and methods for the preparation of such intermediates: WO, 9630390 [P]; 1996-10-03. [7] Kim HK, Rao PN, Simmons AM. Method for preparing 17a-acetoxy-11b-(4N,N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione, intermediates thereof, and methods for the preparation of such intermediates: WO, 2004078709 [P]; 2004-09-16. [8] Dancsi L, Visky G, Tuba Z, Csörgei J, Molnár C, Magyari E. Industrial process for the synthesis of 17a-acetoxy-11b-(4-N,N-dimethylaminophenyl)-19norpregna-4,9-diene-3,20-dione and new intermediates of the process: WO, 2007144674 [P]; 2007-12-21. [9] Yongguo Yu, He Yun, Zhao Yi, Hai Li, Yong Wu. A simple and convenient synthetic route to Ulipristal acetate. Steroids 2013;78:1293–7. [10] Palomo C, Oiarbide M, Landa A, et al. Design and synthesis of a novel class of sugar-peptide hybrids: C-linked glycol b-amino acids through a stereoselective ‘‘acetate’’ Mannich reaction as the key strategic element. J Am Chem Soc 2002;124:8637–43.
