Design, synthesis and antiproliferative effect of 17β-amide derivatives of 2-methoxyestradiol and their studies on pharmacokinetics

Article abstract of DOI:10.1016/j.steroids.2017.09.013

A series of 17β-amide-2-methoxyestradiol compounds were synthesized with an aim to enhance the antiproliferative effect of 2-methoxyestradiol. The antiproliferative activity of 2-methoxyestradiol analogs against human cancer cells was investigated. 2-methoxy-3-benzyloxy-17β-chloroacetamide-1,3,5(10)-triene (5e) and 2-methoxy-3-hydroxy-17β-butyramide-1,3,5(10)-triene (6c) had comparable or better antitumor activity than 2-methoxyestradiol. The elimination half-life of 6c (t_1/2β = 240.93 min) is ten times longer than 2-ME and the area under the curve was seven times (AUC_0-tmin = 2068.20 ± 315.74 μg mL^?1 min) higher than 2-ME, respectively. Whereas 5e had similar pharmacokinetic behavior with 2-ME (t_1/2β = 22.28 min) with a t_1/2β of 29.5 min. 6c had higher blood concentration, longer actuation duration and better suppression rate against S180 mouse ascites tumor than 2-methoxyestradiol.

Full text of DOI:10.1016/j.steroids.2017.09.013

Accepted Manuscript
Design, synthesis and antiproliferative effect of 17β-amide derivatives of 2-
methoxyestradiol and their studies on pharmacokinetics
Xiufang Shi, Zhihao Wang, Feng Xu, Xiang Lu, Haifeng Yao, Dandan Wu,
Shuaijun Sun, Ruifang Nie, Shuo Gao, Panpan Li, Liwen Xia, Zhenzhong
Zhang, Cong Wang
PII:
S0039-128X(17)30180-0
https://doi.org/10.1016/j.steroids.2017.09.013
STE 8164
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 June 2017
29 August 2017
25 September 2017
Please cite this article as: Shi, X., Wang, Z., Xu, F., Lu, X., Yao, H., Wu, D., Sun, S., Nie, R., Gao, S., Li, P., Xia,
L., Zhang, Z., Wang, C., Design, synthesis and antiproliferative effect of 17β-amide derivatives of 2-
methoxyestradiol and their studies on pharmacokinetics, Steroids (2017), doi: https://doi.org/10.1016/j.steroids.
2017.09.013
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Design, synthesis and antiproliferative effect of 17β-amide derivatives of 2-
methoxyestradiol and their studies on pharmacokinetics
Xiufang Shia, Zhihao Wanga, Feng Xua, Xiang Lua, Haifeng Yaoa,b, Dandan Wua,c,
Shuaijun Suna,d, Ruifang Niea,1, Shuo Gaoa,1, Panpan Lia,1, Liwen Xiaa,1, Zhenzhong
Zhanga*, Cong Wang a*
a Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, China; Key
Laboratory of Technology of Drug Preparation (Zhengzhou University), Ministry of Education of China; Key
Laboratory of Henan Province for Drug Quality and Evaluation, China; Key Laboratory of Targeting Therapy and
Diagnosis for Critical Diseases, Henan Province, China; School of Pharmaceutical Sciences, and Institute of Drug
Discovery & Development, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan 450001, China
b Pharmaceutical Department, The People's Hospital of Chizhou, 3 Baiya road, Chizhou, Anhui 247000, China
c
Pharmaceutical Department, Affiliated Hospital of Binzhou Medical College, 661 Yellow River 2nd Road,
Binzhou, Shandong 256600, China
d Department of Pharmacy, Zhengzhou Central Hospital Affiliated to Zhengzhou University, 195 Tongbai Road,
Zhengzhou 450053, Henan, China
* Corresponding author
E-mail addresses: zhangzz08@126.com (Z. Zhang), wangcong@zzu.edu.cn (C. Wang)
1The undergraduate students of School of Pharmaceutical Sciences, Zhengzhou University, China
Abstract
A series of 17β-amide-2-methoxyestradiol compounds were synthesized with an aim
to enhance the antiproliferative effect of 2-methoxyestradiol. The antiproliferative
activity of 2-methoxyestradiol analogs against human cancer cells was investigated.
2-methoxy-3-benzyloxy-17β-chloroacetamide-1,3,5(10)-triene (5e) and 2-methoxy-3-
hydroxy-17β-butyramide-1,3,5(10)-triene (6c) had comparable or better antitumor
activity than 2-methoxyestradiol. The elimination half-life of 6c (t1/2β = 240.93 min) is
ten times longer than 2-ME and the area under the curve was seven times (AUC0-tmin
=2068.20 ± 315.74 μg.ml-1.min) higher than 2-ME, respectively. Whereas 5e had
1

similar pharmacokinetic behavior with 2-ME (t1/2β = 22.28 min) with a t1/2β of 29.5
min. 6c had higher blood concentration, longer actuation duration and better
suppression rate against S180 mouse ascites tumor than 2-methoxyestradiol.
Keywords: 2-methoxyestradiol, analogs, antiproliferative effect, pharmacokinetics
1. Introduction
2-methoxyestradiol (2-ME), a metabolite of estrogen, is an investigational
anticancer agent toward a wide range of tumor models such as breast cancer, prostate
cancer, ovarian cancer, lung cancer and colon cancer, and inhibits tumor growth at
doses showing no clinical signs of toxicity [1, 2]. It also possesses antiangiogenic
activity through a direct apoptotic effect on endothelial cells [3]. Main anticancer
molecular mechanisms of 2-ME are due to the binding to the colchicine binding site
of tubulin, inhibiting polymerization and causing mitotic arrest in G2/M-phase [4]. 2-
ME also inhibits HIF-1a translation and its nuclear translocation, and inhibits
angiogenesis caused by either bFGF or VEGF [5].
Since the antiproliferative effect and antiangiogenic activity of 2-ME [6-8] were
discovered, the synthetic methodology of preparing 2-ME has been a hot focus, and
several synthetic routes have been improved for higher yields and fewer steps [9, 10].
However, due to its low water solubility, low bioavailability [11] and its extensive
metabolism [12, 13], the clinical application of 2-ME was limited.
Fig. 1 here.
Except for improving formulations [14], the synthesis of active 2-ME analogs or
prodrugs has attracted more attentions [15-18]. There are numerous analogs of 2-ME
2

exhibiting potential anticancer activity even better than 2-ME, and some analogs have
been in preclinical or in Phase I clinical trials such as STX140 and ENMD-1198 (Fig.
1) [19-21]. Structural modification of 2-ME mainly focuses on ring A at C-2 or C-3
position and ring D at C-16 or C-17 position and others [22-25]. Structure-activity
relationships (SAR) of 2-ME analogs have made progress [1, 16, 26].
Fig.2 here.
SAR studies showed the substituent at the C-2 position associated with
antiproliferative activity and antitubulin polymerization. Cushman’s group concluded
that the optimum 2-substituent for cytotoxic activity appeared to be an unbranched
chain containing three atoms such as 2-ethoxyestradiol, or 2-(1’-propynyl) estradiol,
or 2-(1’-propenyl) estradiol exhibiting the best antiproliferative and antitubulin profile
[27]. Potter’s group reported that 3-O-sulfamate-2-ethylestradiol and estrone
exhibited high antiproliferative activity in vitro against a wide range of tumor cells
compared to the 2-ethylestradiol [26]. In addition, when the oxygen atom at C-2
position was replaced by bioisosteres NH or S, they displayed slight reduction in
activity.
Further SAR studies demonstrated the substituents at C-3 and C-17 position were
also associated with bioavailability apart from antitumor activity. Treston’s group
found that when the 3-OH group of 2-ME was replaced by hydrogen donor
substituents, such as 3-NHCOH, 3-NHCN, 3-NHCONH2, it showed much better
antiproliferative activities and increased metabolic stability, but the introduction of 3-
NH2 led to substantial loss of activity [28]. And the result indicated that when the C-
17 position of 2-ME was exocyclic double bond. The antiproliferative activity of 3-
3

CONH2 substituent was greater than 3-NHCOH.
Moreover, a series of 2-ME analogs with polar, ionizable, alkyl, endocyclic, or
exocyclic olefins at the C-17 position were prepared. Treston investigated that when
C-17 position was replaced by amine and carboxamide, it had little effect on
antiproliferative activity compared to 2-ME (Fig 2. A1, A2 and A3) [25]. But it
reduced or prevented metabolism to make 17-OH deoxygen, or induce endocyclic or
exocyclic olefins on the D-ring. ENMD-1198 was one example which has been tested
in Phase I clinical trial [23].
Potter [16] showed that optimal activity resulted from the combined presence of a
C-2 XMe group (X=O, CH2 or S), 3-O-sulfamate, and an H-bond acceptor around the
C-17 position. They demonstrated that the C-17 oxygen was not necessary for the
activity. While the oxygen linker between C-17 and the SO2 group was replaced with
a CH2 group or a NH group, H-bond acceptor group (O) and an electronically neutral
group (CH2) were tolerated, an H-bond donating group (NH) led to a marked decrease
in antiproliferative activity (Fig.2. A4 and A5). However, to the best of our
knowledge, there has been no structure-activity relationships established for 17-
acylamino (-NHCOR) substituted 2-ME analogs. In order to further explore the SAR
of 2-ME and enrich the structure types of the 2-ME analogs, we thus set out to
synthesize C-17-acylamino substituent analogs of 2-ME in the hope of discovering
additional antitumor compounds with improved activity and metabolic stability.
In this work, 2-methoxy-3-O-benzyl-17β-aminoestra-1,3,5(10)-triene was
synthesized in two steps from 2-methoxy-3-O-benzylestrone as described by
Gonschior et al [29], and then reacted with various acyl chlorides, followed by
hydrogenolysis to form 17β-acylamide derivatives of 2-methoxyestradiol. The effect
of different substitutions such as alkyl, aryl, halogen, and sulfonamide at the C-17
4

amide chain was investigated. The analogs were further evaluated in vitro
antiproliferation activity against tumor cells, and synthetic compounds 5e, 5n, and 6c
had comparable or better activity than 2-ME. Analogs with acceptable in vitro profiles
were further assessed for pharmacokinetic (PK) properties in vivo.
2. Experimental
2.1. Chemistry
NMR spectra were recorded on a Bruker Avance DPX-400 MHz spectrometer for
13
1H NMR, and 101 MHz for C NMR. High-resolution mass spectra (HRMS) were
recorded on Esquire 3000 mass spectrometer by electrospray ionization (ESI). X-ray
single crystal diffraction data was collected on an Oxford Xcalibur, Gemini, Eos CCD
diffractometer. Estradiol was purchased from Wuhan Kai Lun Chemical New Material
Company. All solvents were analytical grade unless stated otherwise.
2.1.1. General procedure for the preparation of 2-Methoxy-3-benzyloxy-17-carbonyl-
1,3,5(10)-triene (2).
Compound 1 (1.75 g, 4.5 mmol) was dissolved in toluene (60 mL) in a round-
bottom flask. After 30 min reflux, cyclohexanone (18.7 mL, 22.5 mmol) was added
and the mixture refluxed for 1 h, then aluminum isopropoxide (4.627 g, 22.5 mmol)
was added and heated at 140 oC for 24 h. An aqueous solution of sodium bicarbonate
(100 mL) was added into the reaction mixture when the reaction mixture was cooled
to room temperature. After stirring for 1 h, the reaction mixture was extracted with
CH2Cl2 three times. The combined organic layers were washed with an aqueous
solution of sodium bicarbonate, saturated sodium chloride successively and dried over
(Na2SO4). The organic layer was filtered and concentrated under reduced pressure to a
thick colorless oil, then 250 mL petroleum ether was added for recrystallization to
o
1
afford pure product 2 as a white solid (1.42 g, 82%), mp 155-156 C; H NMR (400
5

MHz, CDCl3) δ 7.47 (d, J = 7.1 Hz, 2H), 7.43 – 7.36 (m, 2H), 7.35 – 7.29 (m, 1H),
6.87 (s, 1H), 6.67 (s, 1H), 5.13 (s, 2H), 3.89 (s, 3H), 2.91 – 2.74 (m, 2H), 2.53 (dd, J =
18.9, 8.5 Hz, 1H), 2.46 – 2.37 (m, 1H), 2.34 – 2.23 (m, 1H), 2.23 – 1.95 (m, 4H), 1.71
13
– 1.44 (m, 6H), 0.94 (s, 3H). C NMR (101 MHz, CDCl3) δ 147.69, 146.45, 137.39,
132.31, 128.60, 128.52, 127.76, 127.30, 114.61, 109.63, 77.39, 77.08, 76.76, 71.10,
56.28, 50.39, 48.04, 44.29, 38.30, 35.91, 31.64, 29.04, 26.64, 26.13, 21.59, 13.91.
2.1.2. General procedure for the preparation of 2-methoxy-3-benzyloxy-17-oxime-
1,3,5(10)-triene (3).
Hydroxylamine hydrochloride (0.178 g, 2.564 mmol) was added into a solution of
compound 2 (0.5 g, 1.282 mmol) and pyridine (5 ml). The reaction mixture was
o
allowed to stir at 60 C for 1 h. TLC showed that the reaction was complete. The
o
reaction mixture was cooled to 0 C and substantial ice water was poured into the
mixture. The reaction mixture was filtered and the filter cake was washed with water,
dried in vacuum to afford the crude product. The crude product was purified by silica
gel chromatography to give product 3 as a white solid (0.487 g, 94%), mp 157-159 oC;
1H NMR (400 MHz, CDCl3) δ 9.28 (s, 1H), 7.49 (d, J = 7.2 Hz, 2H), 7.41 (t, J = 7.3
Hz, 2H), 7.34 (t, J = 7.2 Hz, 1H), 6.89 (s, 1H), 6.68 (s, 1H), 5.15 (s, 2H), 3.91 (s, 3H),
2.91 – 2.75 (m, 2H), 2.70 – 2.58 (m, 2H), 2.45 – 2.37 (m, 1H), 2.33 (dd, J = 14.3, 7.3
Hz, 1H), 2.16 – 2.08 (m, 1H), 2.02 – 1.93 (m, 2H), 1.78 – 1.56 (m, 3H), 1.50 – 1.34
(m, 3H), 1.01 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 171.13, 147.68, 146.44, 137.44,
132.60, 128.68, 128.51, 127.74, 127.31, 114.67, 109.73, 77.38, 77.06, 76.74, 71.13,
56.34, 52.89, 44.33, 44.31, 38.09, 34.11, 29.07, 27.35, 26.37, 25.14, 22.93, 17.26.
2.1.3. General procedure for the preparation of 2-methoxy-3-benzyloxy-17β-amino-
1,3,5(10)-triene (4)
To a solution of compound 3 (0.81 g, 2 mmol) in a mixture of MeOH (20 mL) and
6

THF (5 mL) was added MoO3 (0.63 g, 4.4 mmol) at 0 oC and then NaBH4 was added
portion-wise till the reaction was complete confirmed by TLC. 10% solution of
o
potassium hydroxide (5 mL) was added at 0 C and stirred overnight. The suspension
was filtered through a pad of silica gel and filter cake was washed with MeOH. The
combined filtrates were concentrated and dissolved with CH2Cl2, then washed with
water and saturated sodium chloride successively. The organic layers were dried over
(MgSO4) and concentrated to colorless oil. The crude product was purified by column
chromatography on silica gel to give product 4 as a white solid (0.635 g, 81 %), mp
60-62 oC; 1H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 7.1 Hz, 2H), 7.38 (t, J = 7.3 Hz,
2H), 7.35 – 7.29 (m, 1H), 6.86 (d, J = 12.7 Hz, 1H), 6.69 – 6.60 (m, 1H), 5.11 (d, J =
13.6 Hz, 2H), 3.92 – 3.80 (m, 3H), 3.18 (dd, J = 37.0, 28.3 Hz, 1H), 2.88 – 2.63 (m,
2H), 2.40 – 2.15 (m, 4H), 2.00 – 1.71 (m, 3H), 1.62 – 1.21 (m, 6H), 1.07 – 0.84 (m,
3H). 13C NMR (101 MHz, CDCl3) δ 147.65, 146.47, 137.42, 132.45, 128.69, 128.50,
127.74, 127.31, 114.68, 109.79, 77.37, 77.05, 76.74, 71.13, 61.14, 56.35, 51.69, 43.92,
42.82, 38.52, 36.54, 28.99, 27.46, 26.22, 23.61, 12.21.
2.1.4. General procedure for the preparation of analogs 2-methoxy-3-benzyloxy-17β-
amide-1,3,5(10)-triene series (5a - 5n).
To a solution of compound 4 (1 mmol) in CH2Cl2 (10 mL) was added NEt3 (2
mmol) at 0 oC. Acid chloride (1.3 mmol) was added dropwise at 0 oC. After stirring 30
mins, the reaction was done confirmed by TLC. The reaction mixture was extracted
with water and CH2Cl2. The separated organic layer was washed with water and
saturated sodium chloride successively, and dried over (MgSO4), filtered and
concentrated under reduced pressure. The crude product was recrystallized from
acetone and petroleum ether to give products 5a - 5n.
2.1.4.1. 2-methoxy-3-benzyloxy-17β-acetamide-1,3,5(10)-triene (5a)
7

5a, given as a white solid; Yield: 84%; mp 197-198 oC; 1H NMR (400 MHz, CDCl3)
δ7.47 (d, J = 7.3 Hz, 2H), 7.38 (t, J = 7.3 Hz, 2H), 7.31 (dd, J = 12.3, 5.0 Hz, 1H),
6.86 (s, 1H), 6.64 (s, 1H), 5.35 (t, J = 9.0 Hz, 1H), 5.13 (s, 2H), 4.07 – 3.97 (m, 1H),
3.89 (s, 3H), 2.89 – 2.63 (m, 2H), 2.33 – 2.22 (m, 2H), 2.18 (dd, J = 12.0, 6.3 Hz, 1H),
2.03 (s, 3H), 1.92 – 1.76 (m, 3H), 1.46 – 1.29 (m, 6H), 0.93 – 0.85 (m, 1H), 0.75 (s,
3H).
2.1.4.2. 2-methoxy-3-benzyloxy-17β-propionamide-1,3,5(10)-triene (5b)
1
5b, given as a white solid; Yield: 94%; mp 159-160 oC; H NMR (400 MHz, CDCl3)
δ 7.47 (d, J = 7.3 Hz, 2H), 7.39 (dd, J = 10.0, 4.7 Hz, 2H), 7.32 (dd, J = 8.4, 6.2 Hz,
1H), 6.86 (s, 1H), 6.64 (s, 1H), 5.32 (d, J = 9.0 Hz, 1H), 5.13 (s, 2H), 4.03 (dd, J =
17.7, 8.8 Hz, 1H), 3.89 (s, 3H), 2.88 – 2.67 (m, 2H), 2.33 – 2.16 (m, 5H), 1.92 – 1.82
(m, 2H), 1.56 – 1.26 (m, 8H), 1.20 (t, J = 7.6 Hz, 3H), 0.75 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 173.66, 147.64, 146.38, 137.49, 132.98, 128.81, 128.48, 127.70,
127.30, 114.73, 109.92, 77.36, 77.04, 76.72, 71.16, 58.67, 56.39, 51.60, 44.14, 43.32,
38.93, 37.02, 30.01, 29.16, 28.84, 27.43, 26.44, 23.30, 12.12, 10.10.
2.1.4.3. 2-methoxy-3-benzyloxy-17β-butyramide-1,3,5(10)-triene (5c)
1
5c, given as a white solid; Yield: 75%; mp 136-137 oC; H NMR (400 MHz, CDCl3)
δ 7.47 (d, J = 7.2 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.3 Hz, 1H), 6.86 (s,
1H), 6.64 (s, 1H), 5.32 (d, J = 9.1 Hz, 1H), 5.12 (s, 2H), 4.09 – 3.99 (m, 1H), 3.88 (s,
3H), 2.84 – 2.70 (m, 2H), 2.27 (dd, J = 17.1, 9.5 Hz, 2H), 2.23 – 2.16 (m, 3H), 1.92 –
1.69 (m, 5H), 1.51 (dd, J = 15.5, 11.3 Hz, 2H), 1.43 – 1.30 (m, 5H), 0.99 (t, J = 7.4 Hz,
3H), 0.74 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 172.85, 147.64, 146.38, 137.49,
132.99, 128.81, 128.48, 127.71, 127.31, 114.73, 109.91, 77.36, 77.05, 76.73, 71.16,
58.65, 56.38, 51.58, 44.14, 43.35, 39.06, 38.93, 37.05, 29.15, 28.82, 27.43, 26.45,
23.31, 19.36, 13.77, 12.15.
8

2.1.4.4. 2-methoxy-3-benzyloxy-17β-pivaloylamide-1,3,5(10)-triene (5d)
5d, given as a white solid; Yield: 85%; mp 140-141 oC; 1H NMR (400 MHz, CDCl3)
δ 7.47 (d, J = 7.2 Hz, 2H), 7.39 (dd, J = 10.0, 4.7 Hz, 2H), 7.35 – 7.29 (m, 1H), 6.86
(s, 1H), 6.65 (s, 1H), 5.50 (d, J = 8.8 Hz, 1H), 5.13 (s, 2H), 4.02 (q, J = 8.9 Hz, 1H),
3.89 (s, 3H), 2.85 – 2.67 (m, 2H), 2.33 – 2.12 (m, 3H), 1.85 (ddd, J = 11.5, 9.5, 2.8 Hz,
3H), 1.57 – 1.31 (m, 7H), 1.24 (s, 9H), 0.74 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
178.36, 147.63, 146.38, 137.50, 133.00, 128.82, 128.48, 127.70, 127.30, 114.71,
109.93, 77.36, 77.04, 76.72, 71.15, 58.49, 56.39, 51.60, 44.12, 43.55, 38.94, 38.80,
37.16, 29.16, 28.81, 27.74, 27.44, 26.46, 23.34, 12.08.TOF MS ES(+)(m/z):
calculated for C31H42NO3 ([M +H]+) 476.3165; found 476.3168.
2.1.4.5. 2-methoxy-3-benzyloxy-17β-chloroacetamide-1,3,5(10)-triene (5e)
5e, given as a white solid; Yield: 64%; mp 169-170 oC; 1H NMR (400 MHz, CDCl3)
δ 7.47 (d, J = 7.8 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.35 – 7.29 (m, 1H), 6.86 (s, 1H),
6.65 (s, 1H), 6.53 (d, J = 8.9 Hz, 1H), 5.13 (s, 2H), 4.09 (d, J = 15.5 Hz, 2H), 4.05 –
3.96 (m, 1H), 3.89 (s, 3H), 2.90 – 2.67 (m, 2H), 2.38 – 2.17 (m, 3H), 1.96 – 1.77 (m,
3H), 1.55 – 1.31 (m, 7H), 0.80 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 165.64, 147.60,
146.37, 137.44, 132.73, 128.74, 128.51, 127.73, 127.31, 114.61, 109.77, 77.38, 77.06,
76.75, 71.09, 59.12, 56.36, 51.60, 44.11, 43.55, 42.93, 38.85, 36.88, 29.14, 28.61,
27.43, 26.37, 23.35, 12.06.TOF MS ES(+)(m/z): calculated for C28H35ClNO3 ([M
+H]+) 468.2305; found 468.2335.
2.1.4.6. 2-methoxy-3-benzyloxy-17β-(3’-chloro) propionamide-1,3,5(10)-triene (5f)
o
1
5f, given as a yellow solid; Yield: 80%; mp 103-104 C; H NMR (400 MHz,
CDCl3) δ 7.47 (d, J = 7.4 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H),
6.86 (s, 1H), 6.65 (d, J = 8.6 Hz, 1H), 5.53 (t, J = 11.3 Hz, 1H), 5.14 (d, J = 7.5 Hz,
2H), 4.19 – 4.00 (m, 1H), 3.92 – 3.80 (m, 5H), 2.89 – 2.50 (m, 4H), 2.33 – 2.13 (m,
9

3H), 1.92 – 1.85 (m, 2H), 1.84 – 1.77 (m, 1H), 1.38 (dd, J = 18.6, 15.3 Hz, 5H), 1.02
– 0.82 (m, 2H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 169.39, 147.57, 146.32,
137.45, 132.88, 128.74, 128.50, 127.73, 127.31, 114.59, 109.77, 77.38, 77.06, 76.74,
71.10, 59.06, 56.33, 51.53, 44.11, 43.51, 40.56, 40.02, 38.89, 36.97, 29.15, 28.66,
27.44, 26.43, 23.33, 12.15.TOF MS ES(+)(m/z): calculated for C29H37ClNO3 ([M
+H]+) 482.2462; found 482.2461.
2.1.4.7. 2-methoxy-3-benzyloxy-17β-(3’-fluoro) propionamide-1,3,5(10)-triene (5g)
5g, given as a white solid; Yield: 75%; mp 108-109 oC; 1H NMR (400 MHz, CDCl3)
δ 7.47 (d, J = 7.3 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.3 Hz, 1H), 6.86 (s,
1H), 6.64 (s, 1H), 5.58 (s, 1H), 5.13 (s, 2H), 4.08 (dt, J = 26.7, 9.0 Hz, 1H), 3.95 –
3.75 (m, 5H), 2.88 – 2.56 (m, 4H), 2.33 – 2.14 (m, 3H), 1.88 (dd, J = 7.3, 3.8 Hz, 2H),
1.84 – 1.77 (m, 1H), 1.56 – 1.31 (m, 7H), 0.77 (s, 3H).13C NMR (101 MHz, CDCl3) δ
169.41, 147.57, 146.32, 137.45, 132.88, 128.75, 128.51, 127.74, 127.32, 114.59,
109.78, 77.39, 77.08, 76.76, 71.10, 59.06, 56.34, 51.53, 44.11, 43.51, 40.57, 40.00,
38.89, 36.98, 29.15, 28.64, 27.44, 26.43, 23.33, 12.15.
2.1.4.8. 2-methoxy-3-benzyloxy-17β-benzamide-1,3,5(10)-triene (5h)
5h, given as a white solid; Yield: 95%; mp 152-154 oC; 1H NMR (400 MHz, CDCl3)
δ 7.85 – 7.74 (m, 2H), 7.53 (ddd, J = 6.2, 3.6, 1.3 Hz, 1H), 7.50 – 7.44 (m, 4H), 7.39
(dd, J = 10.0, 4.7 Hz, 2H), 7.35 – 7.29 (m, 1H), 6.97 – 6.77 (m, 1H), 6.66 (s, 1H),
6.06 (d, J = 8.8 Hz, 1H), 5.14 (s, 2H), 4.37 – 4.11 (m, 1H), 3.85 (d, J = 30.6 Hz, 3H),
2.92 – 2.67 (m, 2H), 2.45 – 2.21 (m, 3H), 1.89 (ddd, J = 23.9, 10.9, 3.9 Hz, 3H), 1.67
– 1.22 (m, 8H), 0.88 – 0.82 (m, 3H). 13C NMR (101 MHz, CDCl3) δ 167.47, 147.60,
146.35, 137.47, 135.04, 132.90, 131.38, 128.77, 128.62, 128.51, 127.73, 127.32,
126.83, 114.63, 109.81, 77.39, 77.08, 76.76, 71.11, 59.25, 56.37, 51.66, 44.15, 43.78,
38.95, 37.12, 29.19, 28.93, 27.47, 26.47, 23.43, 12.31.
10

2.1.4.9. 2-methoxy-3-benzyloxy-17β-(4’-fluoro) benzamide-1,3,5(10)-triene (5i)
5i, given as a white solid; Yield: 91%; mp 163-164 oC; 1H NMR (400 MHz, CDCl3)
δ 7.85 – 7.76 (m, 2H), 7.47 (d, J = 7.2 Hz, 2H), 7.42 – 7.36 (m, 2H), 7.35 – 7.29 (m,
1H), 7.18 – 7.10 (m, 2H), 6.87 (s, 1H), 6.65 (s, 1H), 5.95 (d, J = 8.9 Hz, 1H), 5.13 (s,
2H), 4.30 – 4.14 (m, 1H), 3.89 (s, 3H), 2.89 – 2.67 (m, 2H), 2.36 – 2.22 (m, 3H), 1.97
– 1.78 (m, 3H), 1.60 – 1.32 (m, 7H), 0.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
166.42, 147.65, 146.40, 137.47, 132.88, 129.16, 129.07, 128.80, 128.50, 127.73,
127.32, 115.72, 115.50, 114.72, 109.91, 77.37, 77.05, 76.73, 71.15, 59.36, 56.40,
51.66, 44.14, 43.77, 38.95, 37.13, 29.16, 28.93, 27.45, 26.45, 23.42, 12.32.TOF MS
ES(+)(m/z): calculated for C29H37FNO3 ([M +H]+) 514.2757; found 514.2771.
2.1.4.10. 2-methoxy-3-benzyloxy-17β-phenylacetamide-1,3,5(10)-triene (5j)
5j, given as a white solid; Yield: 86%; mp 164-166 oC; 1H NMR (400 MHz, CDCl3)
δ 7.46 (d, J = 7.2 Hz, 2H), 7.43 – 7.29 (m, 8H), 6.84 (s, 1H), 6.63 (s, 1H), 5.24 (d, J =
9.1 Hz, 1H), 5.12 (s, 2H), 3.99 (q, J = 9.0 Hz, 1H), 3.88 (s, 3H), 3.68 – 3.56 (m, 2H),
2.74 (q, J = 10.2 Hz, 2H), 2.26 (dd, J = 13.2, 5.4 Hz, 2H), 2.12 (dt, J = 12.6, 6.7 Hz,
1H), 1.90 – 1.71 (m, 3H), 1.49 – 1.17 (m, 7H), 0.50 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 170.90, 147.64, 146.39, 137.48, 135.22, 132.92, 129.41, 129.07, 128.79,
128.48, 127.71, 127.40, 127.31, 114.72, 109.89, 77.38, 77.06, 76.74, 71.15, 58.75,
56.37, 51.54, 44.10, 43.39, 38.83, 36.94, 29.11, 28.63, 27.41, 26.40, 23.27, 11.76.TOF
MS ES(+)(m/z): calculated for C34H40NO3 ([M +H]+) 510.3008; found 510.3050.
2.1.4.11.
2-methoxy-3-benzyloxy-17β-(1’,2’,3’,4’-tetrafluoro)
phenylacetamide-
1,3,5(10)-triene (5k)
5k, given as a white solid; Yield: 90%; mp 163-165 oC; 1H NMR (400 MHz, CDCl3)
δ 7.85 – 7.72 (m, 1H), 7.47 (d, J = 7.2 Hz, 2H), 7.39 (dd, J = 10.0, 4.6 Hz, 2H), 7.32
(dd, J = 8.5, 6.0 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.65 (s, 1H), 6.55 (dd, J = 11.5, 9.1
11

Hz, 1H), 5.13 (s, 2H), 4.21 (tt, J = 8.9, 4.3 Hz, 1H), 3.89 (s, 3H), 2.88 – 2.70 (m, 2H),
2.34 (dd, J = 15.6, 8.4 Hz, 3H), 1.98 – 1.80 (m, 3H), 1.67 – 1.32 (m, 8H), 0.86 (d, J =
6.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 160.25, 147.61, 146.39, 137.43, 132.69,
128.75, 128.51, 127.74, 127.31, 114.62, 113.07, 112.85, 109.79, 77.37, 77.05, 76.73,
71.10, 59.85, 56.37, 51.60, 44.11, 43.70, 38.89, 37.06, 29.14, 28.93, 27.45, 26.40,
23.45, 12.28.
2.1.4.12. 2-methoxy-3-benzyloxy-17β-methanesulfonamide-1,3,5(10)-triene (5l)
1
5l, given as a white solid; Yield: 98%; mp 200-202 oC; H NMR (400 MHz, CDCl3)
δ 7.47 (d, J = 7.4 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.3 Hz, 1H), 6.86 (s,
1H), 6.65 (s, 1H), 5.13 (s, 2H), 4.42 (d, J = 9.5 Hz, 1H), 3.89 (s, 3H), 3.51 (d, J = 4.9
Hz, 2H), 3.37 (q, J = 9.2 Hz, 1H), 3.01 (s, 3H), 2.78 (dt, J = 17.0, 9.3 Hz, 2H), 2.39 –
2.17 (m, 3H), 2.06 – 1.93 (m, 1H), 1.92 – 1.74 (m, 2H), 1.46 – 1.26 (m, 5H), 0.77 (s,
3H).13C NMR (101 MHz, CDCl3) δ 147.61, 146.39, 137.41, 132.58, 128.72, 128.51,
127.75, 127.31, 114.61, 109.73, 77.38, 77.06, 76.75, 71.09, 63.53, 56.34, 51.21, 50.90,
44.14, 42.88, 41.65, 38.83, 36.66, 30.09, 29.09, 27.33, 26.28, 23.21, 11.90.
2.1.4.13. 2-methoxy-3-benzyloxy-17β-p-toluenesulfonamide-1,3,5(10)-triene (5m)
o
1
5m, given as a white solid; Yield: 93%; mp 147-148 C; H NMR (400 MHz,
CDCl3) δ 7.81 (d, J = 8.2 Hz, 2H), 7.46 (d, J = 7.2 Hz, 2H), 7.38 (t, J = 7.3 Hz, 2H),
7.35 – 7.29 (m, 3H), 6.83 (s, 1H), 6.63 (s, 1H), 5.12 (s, 2H), 4.66 (d, J = 9.2 Hz, 1H),
3.88 (s, 3H), 3.18 (q, J = 8.9 Hz, 1H), 2.83 – 2.61 (m, 2H), 2.47 (s, 3H), 2.33 – 2.11
(m, 2H), 1.96 – 1.76 (m, 3H), 1.66 (s, 1H), 1.52 – 1.11 (m, 7H), 0.74 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 147.59, 146.35, 143.23, 138.18, 137.42, 132.68, 129.64,
128.68, 128.50, 127.73, 127.29, 127.15, 114.58, 109.70, 77.38, 77.07, 76.75, 71.08,
63.35, 56.32, 51.12, 44.12, 42.93, 38.77, 36.38, 29.49, 29.07, 27.32, 26.25, 23.16,
21.60, 11.89.
12

2.1.4.14. 2-methoxy-3-benzyloxy-17β-niacinamide-1,3,5(10)-triene (5n)
5n, given as a white solid; Yield: 88%; mp 169-170 oC; 1H NMR (400 MHz, CDCl3)
δ 8.99 (d, J = 2.0 Hz, 1H), 8.75 (dd, J = 4.8, 1.5 Hz, 1H), 8.14 (dt, J = 7.9, 1.9 Hz, 1H),
7.47 (d, J = 7.3 Hz, 2H), 7.40 (dt, J = 14.5, 6.2 Hz, 3H), 7.31 (t, J = 7.3 Hz, 1H), 6.86
(s, 1H), 6.65 (s, 1H), 6.11 (d, J = 8.8 Hz, 1H), 5.13 (s, 2H), 4.24 (q, J = 9.0 Hz, 1H),
3.88 (d, J = 7.7 Hz, 3H), 2.88 – 2.68 (m, 2H), 2.39 – 2.23 (m, 3H), 1.98 – 1.78 (m,
4H), 1.65 – 1.31 (m, 7H), 0.86 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 165.57, 152.21,
147.70, 147.63, 146.40, 137.45, 135.09, 132.79, 130.66, 128.76, 128.50, 127.73,
127.31, 123.57, 114.68, 109.85, 77.38, 77.06, 76.75, 71.13, 59.44, 56.38, 51.67, 44.13,
43.83, 38.92, 37.13, 29.15, 28.89, 27.45, 26.44, 23.42, 12.35.TOF MS ES(+)(m/z):
calculated for C32H37N2O3 ([M +H]+) 497.2804; found 497.2805.
2.1.5. General procedure for the preparation of analogs 2-methoxy-3-hydroxy-17β-
amide-1,3,5(10)-triene series (6a - 6n).
To a solution of compound 5a - 5n (0.3 g) in EtOH (50 mL) was added Pd/C (10 %,
0.03 g). The suspension was degassed and purged with H2 three times. The mixture
o
was stirred under H2 (30 psi) at 60 C for 4 hours. TLC showed that the reaction was
complete. The suspension was filtered through silica gel and filter cake was washed
with EtOH. The combined filtrate was concentrated to dryness and then recrystallized
from petroleum ether and ethyl acetate to give pure products 6a - 6n.
2.1.5.1. 2-methoxy-3-hydroxy-17β-acetamide-1,3,5(10)-triene (6a)
6a, given as a white solid; Yield: 60%; mp 253-254 oC; 1H NMR (400 MHz, CDCl3)
δ 6.80 (s, 1H), 6.66 (s, 1H), 5.55 (s, 1H), 5.38 (d, J = 9.0 Hz, 1H), 4.01 (d, J = 8.9 Hz,
1H), 3.88 (s, 3H), 2.87 – 2.70 (m, 2H), 2.22 (ddd, J = 17.9, 10.4, 4.5 Hz, 3H), 2.11 –
1.97 (m, 3H), 1.94 – 1.75 (m, 3H), 1.60 – 1.21 (m, 7H), 0.76 (d, J = 9.4 Hz, 3H). 13C
NMR (101 MHz, CDCl3) δ 170.04, 144.63, 143.49, 131.68, 129.42, 114.63, 108.15,
13

77.38, 77.06, 76.75, 58.90, 56.11, 51.56, 44.13, 43.27, 38.96, 36.94, 29.01, 28.76,
27.41, 26.55, 23.65, 23.28, 12.14.
2.1.5.2. 2-methoxy-3-hydroxy-17β-propionamide-1,3,5(10)-triene (6b)
6b, given as a white solid; Yield: 54%; mp 171-172 oC; 1H NMR (400 MHz, CDCl3)
δ 6.80 (s, 1H), 6.66 (s, 1H), 5.61 (d, J = 8.7 Hz, 1H), 5.35 (t, J = 11.1 Hz, 1H), 4.08 –
3.97 (m, 1H), 3.88 (s, 3H), 2.83 – 2.72 (m, 2H), 2.30 – 2.13 (m, 6H), 1.92 – 1.73 (m,
4H), 1.55 – 1.29 (m, 7H), 1.20 (t, J = 7.6 Hz, 3H), 0.74 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 173.76, 144.63, 143.49, 131.71, 129.42, 114.63, 108.15, 77.38, 77.07, 76.75,
58.67, 56.11, 51.57, 44.13, 43.34, 38.97, 37.00, 30.02, 29.02, 28.80, 27.42, 26.56,
23.30, 12.12, 10.14.
2.1.5.3. 2-methoxy-3-hydroxy-17β-butyramide-1,3,5(10)-triene (6c)
6c, given as a white solid; Yield: 75%; mp 174-175 oC; 1H NMR (400 MHz, CDCl3)
δ 6.80 (s, 1H), 6.66 (s, 1H), 5.53 (s, 1H), 5.33 (d, J = 9.0 Hz, 1H), 4.09 – 3.99 (m, 1H),
3.88 (s, 3H), 2.85 – 2.74 (m, 2H), 2.29 – 2.16 (m, 5H), 1.93 – 1.77 (m, 3H), 1.69 (dd,
J= 7.4, 2.0 Hz, 1H), 1.57 – 1.27 (m, 8H), 0.99 (t, J = 7.4 Hz, 3H), 0.74 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 172.91, 144.64, 143.52, 131.73, 129.44, 114.63, 108.18,
77.36, 77.04, 76.73, 58.67, 56.11, 51.59, 44.13, 43.36, 39.05, 38.99, 37.04, 29.00,
28.80, 27.41, 26.58, 23.31, 19.36, 13.76, 12.14.TOF MS ES(+)(m/z): calculated for
C23H34NO3([M +H]+) 372.2539; found 372.2538.
2.1.5.4. 2-methoxy-3-hydroxy-17β-pivaloylamide-1,3,5(10)-triene (6d)
o
1
6d, given as a brown solid; Yield: 60%; mp 127-129 C; H NMR (400 MHz,
CDCl3) δ 6.80 (s, 1H), 6.66 (s, 1H), 5.61 – 5.48 (m, 2H), 4.08 – 3.95 (m, 1H), 3.88 (s,
3H), 2.89 – 2.70 (m, 2H), 2.34 – 2.12 (m, 3H), 1.93 – 1.85 (m, 1H), 1.83 – 1.77 (m,
2H), 1.46 – 1.29 (m, 6H), 1.24 (s, 9H), 1.00 – 0.81 (m, 1H), 0.74 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 178.43, 144.61, 143.49, 131.74, 129.43, 114.63, 108.14, 77.38,
14

77.06, 76.75, 58.49, 56.10, 51.57, 44.11, 43.56, 38.99, 38.81, 37.14, 29.02, 28.79,
27.74, 27.42, 26.59, 23.33, 12.08.
2.1.5.5. 2-methoxy-3-hydroxy-17β-chloroacetamide-1,3,5(10)-triene (6e)
6e, given as a white solid; Yield: 69%; mp 226-228 oC; 1H NMR (400 MHz, CDCl3)
δ 6.81 (d, J = 12.2 Hz, 1H), 6.70 – 6.60 (m, 1H), 5.66 (d, J = 3.0 Hz, 1H), 5.45 (d, J =
8.8 Hz, 1H), 4.07 – 3.95 (m, 1H), 3.87 (d, J = 6.4 Hz, 3H), 2.87 – 2.71 (m, 2H), 2.33 –
2.12 (m, 4H), 2.03 (s, 3H), 1.93 – 1.73 (m, 4H), 1.55 – 1.25 (m, 8H), 0.75 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 170.06, 144.65, 143.51, 131.67, 129.41, 114.65, 108.17,
77.40, 77.08, 76.76, 58.90, 56.11, 51.56, 44.13, 43.27, 38.96, 36.95, 29.01, 28.75,
27.41, 26.55, 23.65, 23.29, 12.15.
2.1.5.6. 2-methoxy-3-hydroxy-17β-(3’-chloro) propionamide-1,3,5(10)-triene (6f)
6f, given as a white solid; Yield: 84%; mp 149-150 oC; 1H NMR (400 MHz, CDCl3)
δ 6.80 (s, 1H), 6.66 (s, 1H), 5.56 (dd, J = 9.3, 2.0 Hz, 2H), 4.06 (q, J = 8.9 Hz, 1H),
3.93 – 3.76 (m, 5H), 2.86 – 2.74 (m, 2H), 2.74 – 2.57 (m, 2H), 2.22 (ddd, J = 27.3,
13.0, 5.8 Hz, 3H), 1.94 – 1.84 (m, 2H), 1.84 – 1.78 (m, 1H), 1.52 – 1.30 (m, 7H), 0.76
(d, J = 9.8 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.39, 144.62, 143.48, 131.70,
129.41, 114.62, 108.13, 77.38, 77.07, 76.75, 59.07, 56.10, 51.54, 44.10, 43.52, 40.57,
40.01, 38.95, 36.97, 28.99, 28.64, 27.42, 26.56, 23.33, 12.14.
2.1.5.7. 2-methoxy-3-hydroxy-17β-(3’-fluoro) propionamide-1,3,5(10)-triene (6g)
6g, given as a yellow solid; Yield: 70%; mp 92-93 oC; 1H NMR (400 MHz, CDCl3)
δ 6.80 (s, 1H), 6.66 (s, 1H), 5.61 (s, 2H), 4.06 (q, J = 8.9 Hz, 1H), 3.95 – 3.77 (m, 5H),
2.86 – 2.74 (m, 2H), 2.74 – 2.58 (m, 2H), 2.32 – 2.14 (m, 3H), 1.86 (dd, J = 18.7, 9.5
Hz, 4H), 1.46 – 1.24 (m, 6H), 0.77 (s, 3H).13C NMR (101 MHz, CDCl3) δ 169.43,
144.63, 143.48, 131.69, 129.40, 114.64, 108.15, 77.40, 77.08, 76.76, 59.07, 56.10,
51.54, 44.10, 43.53, 40.58, 40.00, 38.95, 36.98, 28.99, 28.62, 27.42, 26.56, 23.33,
15

12.14.
2.1.5.8. 2-methoxy-3-hydroxy-17β-benzamide-1,3,5(10)-triene (6h)
6h, given as a white solid; Yield: 89%; mp 125-127 oC; 1H NMR (400 MHz, CDCl3)
δ 7.90 – 7.69 (m, 2H), 7.61 – 7.39 (m, 3H), 6.81 (s, 1H), 6.67 (s, 1H), 6.07 (d, J = 8.9
Hz, 1H), 5.56 (s, 1H), 4.24 (q, J = 9.1 Hz, 1H), 3.88 (s, 3H), 2.81 (dd, J = 8.3, 4.8 Hz,
2H), 2.41 – 2.22 (m, 3H), 2.00 – 1.80 (m, 3H), 1.65 – 1.29 (m, 7H), 0.85 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 167.50, 144.63, 143.50, 135.02, 131.69, 131.39, 129.43,
128.62, 126.83, 114.63, 108.15, 77.38, 77.06, 76.75, 59.26, 56.12, 51.66, 44.15, 43.79,
39.01, 37.11, 29.03, 28.92, 27.44, 26.60, 23.43, 12.30.
2.1.5.9. 2-methoxy-3-hydroxy-17β-(4’-fluoro) benzamide-1,3,5(10)-triene (6i)
6i, given as a white solid; Yield: 56%; mp 140-141 oC; 1H NMR (400 MHz, CDCl3)
δ 7.89 – 7.72 (m, 2H), 7.22 – 7.06 (m, 2H), 6.81 (s, 1H), 6.67 (s, 1H), 5.96 (d, J = 8.8
Hz, 1H), 5.48 (s, 1H), 4.37 – 4.08 (m, 1H), 4.04 – 3.72 (m, 3H), 2.83 (dd, J = 17.9,
11.8 Hz, 2H), 2.50 – 2.15 (m, 3H), 1.98 – 1.78 (m, 3H), 1.62 – 1.27 (m, 7H), 0.85 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 166.45, 165.89, 163.39, 144.63, 143.50, 131.65,
131.17, 129.42, 129.17, 129.08, 115.73, 115.51, 114.63, 108.13, 77.38, 77.06, 76.74,
59.35, 56.12, 51.65, 44.13, 43.78, 39.00, 37.11, 29.02, 28.91, 27.43, 26.58, 23.41,
12.32.
2.1.5.10. 2-methoxy-3-hydroxy-17β-phenylacetamide-1,3,5(10)-triene (6j)
o
1
6j, given as a brown solid; Yield: 74%; mp 138-139 C; H NMR (400 MHz,
CDCl3) δ 7.35 (ddd, J = 16.2, 14.0, 7.5 Hz, 6H), 6.79 (s, 1H), 6.65 (s, 1H), 5.52 (s,
1H), 5.26 (d, J = 9.0 Hz, 1H), 3.99 (dd, J = 18.0, 9.0 Hz, 1H), 3.88 (s, 3H), 3.69 – 3.56
(m, 2H), 2.76 (s, 2H), 2.15 (ddd, J = 26.4, 17.0, 8.1 Hz, 4H), 1.93 – 1.68 (m, 5H),
1.56 – 1.14 (m, 8H), 0.50 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 170.93, 144.63,
143.52, 135.20, 131.66, 129.41, 129.07, 127.40, 114.63, 108.15, 77.37, 77.05, 76.73,
16

58.76, 56.10, 51.54, 44.10, 43.40, 38.89, 36.94, 28.96, 28.62, 27.39, 26.53, 23.26,
11.75.
2.1.5.11.
2-methoxy-3-hydroxy-17β-(1’,2’,3’,4’-tetrafluoro)
phenylacetamide-
1,3,5(10)-triene (6k)
o
1
6k, given as a yellow solid; Yield: 76%; mp 164-165 C; H NMR (400 MHz,
CDCl3) δ 7.78 (dddd, J = 10.7, 8.7, 6.6, 2.4 Hz, 1H), 6.80 (s, 1H), 6.66 (d, J = 7.5 Hz,
1H), 6.61 – 6.49 (m, 1H), 5.50 (s, 1H), 4.21 (qt, J = 13.1, 6.5 Hz, 1H), 3.88 (s, 3H),
2.86 – 2.72 (m, 2H), 2.31 (ddd, J = 16.5, 10.7, 6.3 Hz, 3H), 1.96 – 1.79 (m, 3H), 1.60
– 1.31 (m, 7H), 0.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 160.27, 144.61, 143.52,
131.52, 129.41, 114.62, 113.05, 112.85, 108.07, 99.99, 77.36, 77.05, 76.73, 59.85,
56.10, 51.61, 44.11, 43.70, 38.95, 37.06, 28.98, 28.93, 27.43, 26.53, 23.45, 12.27.
2.1.5.12. 2-methoxy-3-hydroxy-17β-methanesulfonamide-1,3,5(10)-triene (6l)
6l, given as a white solid; Yield: 40%; mp 189-191 oC; 1H NMR (400 MHz, CDCl3)
δ 6.80 (s, 1H), 6.66 (d, J = 8.0 Hz, 1H), 5.51 (s, 1H), 4.46 (t, J = 11.8 Hz, 1H), 3.88 (s,
3H), 3.37 (q, J = 9.2 Hz, 1H), 3.00 (d, J = 6.2 Hz, 3H), 2.89 – 2.69 (m, 2H), 2.36 –
2.18 (m, 3H), 1.99 (dt, J = 12.5, 2.9 Hz, 1H), 1.93 – 1.74 (m, 2H), 1.58 – 1.46 (m, 2H),
1.43 – 1.25 (m, 5H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 144.63, 143.52,
131.43, 129.38, 114.63, 108.05, 77.39, 77.07, 76.75, 63.53, 56.10, 51.20, 44.13, 42.88,
41.63, 38.89, 36.62, 30.03, 28.94, 27.32, 26.40, 23.22, 11.90.
2.1.5.13. 2-methoxy-3-hydroxy-17β-p-toluenesulfonamide-1,3,5(10)-triene (6m)
o
1
6m, given as a yellow solid; Yield: 77%; mp 164-165 C; H NMR (400 MHz,
CDCl3) δ 7.80 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 6.78 (s, 1H), 6.65 (s, 1H),
5.46 (s, 1H), 4.51 (s, 1H), 3.88 (s, 3H), 3.19 (q, J = 8.8 Hz, 1H), 2.85 – 2.68 (m, 2H),
2.46 (s, 3H), 2.29 – 2.11 (m, 2H), 1.84 (t, J = 14.7 Hz, 3H), 1.66 (dd, J = 14.9, 8.0 Hz,
1H), 1.40 – 1.15 (m, 7H), 0.74 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 144.60, 143.51,
17

143.22, 138.21, 131.51, 129.63, 129.37, 127.14, 114.58, 108.04, 77.35, 77.03, 76.72,
63.35, 56.08, 51.13, 44.11, 42.92, 38.83, 36.37, 29.52, 28.90, 27.31, 26.37, 23.15,
21.57, 11.88.TOF MS ES(+)(m/z): calculated for C26H34NO4S([M +H]+) 456.2209;
found 456.2269.
2.1.5.14. 2-methoxy-3-hydroxy-17β-niacinamide-1,3,5(10)-triene (6n)
o
1
6n, given as a white solid; Yield: 86%; mp 152-154 C; H NMR (400 MHz,
DMSO) δ 9.01 (d, J = 1.9 Hz, 1H), 8.70 (dd, J = 4.8, 1.4 Hz, 1H), 8.61 (s, 1H), 8.32 –
8.09 (m, 2H), 7.59 – 7.41 (m, 1H), 6.79 (d, J = 21.9 Hz, 1H), 6.46 (s, 1H), 4.17 – 3.95
(m, 1H), 3.76 – 3.67 (m, 3H), 2.72 – 2.59 (m, 2H), 2.23 (dd, J = 26.4, 23.6 Hz, 2H),
1.94 (dd, J = 11.4, 6.8 Hz, 1H), 1.83 – 1.67 (m, 4H), 1.42 – 1.21 (m, 6H), 0.76 (s, 3H).
13C NMR (101 MHz, DMSO) δ 165.51, 152.04, 148.93, 146.02, 144.82, 135.58,
130.97, 130.82, 128.79, 123.79, 116.08, 110.28, 59.53, 56.30, 51.74, 44.39, 44.17,
37.74, 28.92, 27.63, 27.03, 26.63, 23.50, 12.82.
2.2. Biological methods
2.2.1. Cell lines culture
SKN-SH cells, SGC-7901 cells, MCF-7 cells, PC-3 cells and EC-9706 cells were
obtained from School of Pharmaceutical Sciences of Zhengzhou University. SKN-SH
cells were maintained in Dulbecco's Modified Eagle Medium (DMEM); SGC-7901
cells, MCF-7 cells, PC-3 cells and EC-9706 cells were kept in Roswell Park
o
Memorial Institute 1640 (RPMI 1640). All the cells were maintained at 37 C in 5 %
CO2 humidified atmosphere and supplemented with 10 % fetal bovine serum,
penicillin and streptomycin.
2.2.2. Antiproliferative activity assay
The cancer cells in the exponential growth period were treated with trypsin, and
diluted to 5×104 cells/mL. Then 100 μL of the cell suspension was transferred into 96
18

well microtiter plates. After cultured cells for 24 hours, certain concentration of
derivatives of 5a - 5n and 6a - 6n was added in comparison to the control group. After
treatment for 72 h, the cells were performed in SRB staining experiments to stain the
cancer cells. The absorbance of each hole in the 96 well microtiter plates was
measured by Multiskan Spectrum. Then the growth inhibition ratio was calculated
according to the absorbance of each hole (the growth inhibition ratio= (1 -
Absorbance of drug group/Absorbance of control group) × 100 %). The half
inhibition concentration (IC50) was calculated by SPSS 15. All experiments were
performed six times.
2.2.3. In Vivo Pharmacokinetics Studies
Sprague–Dawley rats (200 ± 20 g) were purchased from Experimental Animal
Center of Henan province, and were randomly divided into three groups (6 per group).
The rats used for this study were housed individually under normal conditions, and
fasted overnight before experiment with free access to water. Drugs were dissolved in
a solution consisting of Cremophor EL-Anhydrous ethanol-normal saline (1:2:7). A
dose of 10 mg/kg of 2-ME, 5e and 6c were injected through tail vein. After the
injection of 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 480, 720 min, 0.5 mL of blood
samples was taken from the orbital venous plexus into micro-tubes containing sodium
heparin, and centrifugated immediately at 4000 rpm for 10 min, and the upper plasma
was taken. Anhydrous sodium sulfate (0.1g) was added to 200 μL plasma to
precipitate protein, and sodium fluoride (0.1g) was also added to the plasma collected
from 2-ME derivatives group to inhibit the activity of esterase. Plasma (200 µL) was
extracted with ethyl acetate (1.4 mL) vortexing for 2 min. Following centrifugation at
3000 rpm for 10 min, the organic phase was decanted to a clean tube and evaporated
to dryness under a stream of air at room temperature. The dried sample was then
19

dissolved in 100 μL mobile phase and 20 μL of the solution was injected into the
column. HPLC analysis was performed using a Phenomenex C18 column (5 µm, 250 ×
4.6 mm) eluted with methanol-water at flow rate of 1.0 mL/min. The excited
wavelength and emission wavelength were 288 and 325 nm, respectively. Column
temperature is 30 oC, and the proportion of methanol-water mobile phase is 75:25 (2-
ME), 90:10 (5e), 80:20 (6c), respectively. Pharmacokinetics parameters were
evaluated using practical pharmacokinetics program version 3P97 (supplied by
Chinese Pharmacological Society).
3. Results and discussion
3.1. Chemistry
There are many methods reported for the synthesis of 17-amine. Jourdan et al [16]
and Gonschior et al [29] depicted a way of reduction of the oxime with MoO3/NaBH4
gave the β-amine. Cushman et al [30] and Shah [25] described that the reaction of 2-
methoxy-3-benzylestrone with benzylamine afforded an intermediate Schiff base
which was reduced to the secondary amine with sodium cyanoborohydride and
followed by hydrogenolysis with palladium hydroxide on charcoal as the catalyst to
afford the corresponding amine. Taylor et al [31] used sodium triacetoxyborohydride
(STAB-H, (CH3COO)3BHNa) as reductive amination agent, then hydrogenolysis by
15 wt% of 10% Pd/C to prepare 17β-amino in high yield.
Base on above methods, the synthesis of target compounds 6a - 6n is described in
Scheme 1. In order to synthesize the 17β-acylamide derivatives, 2-methoxy-3-O-
benzylestradiol 1 was prepared from estradiol following previously published
methods [9].
Scheme 1 here.
20

First, compound 1 was oxidized to ketone 2 by Oppenauer oxidation in a yield of
o
82%. Then the reaction of ketone 2 with hydroxylamine in pyridine at 60 C gave
oxime 3 in a yield of 94%. Reduction amination of the oxime 3 with sodium
borohydride and molybdenum trioxide (MoO3/NaBH4) in 4:1 mixture of absolute
methanol and absolute tetrahydrofuran (MeOH/THF) gave 17β-amine 4 in a yield of
76%. Finally, acyl chlorides were hooked-up at 17β-amine of 4 using triethylamine in
dichloromethane to afford 2-methoxy-3-benzylestra-1,3,5(10)-triene-17β-acylamide
compounds 5a - 5n, followed by debenzylation by Pd-C/H2 in dry ethanol yielded the
corresponding phenol 6a - 6n.
The configuration of the C-17 substituent was on the basis of 1H NMR spectra. The
17α and 17β protons not only exhibited different chemical shifts, but showed different
coupling patterns with the amide N-H and the C-16 protons characteristic of the C-17
isomers. C-17 acetamide isomers showed the 17α-H (quasi-axial) as a quartet (d,d,d),
whereas the 17β-H (quasi-equatorial) was a triplet (d,d) [32] . In the spectrum,
compounds 5c and 6c (Fig. 3, b, c) gave a quartet (d,d,d) centered at 4.04 ppm
corresponding to the 17a-H and the C-18 methyl signal at 0.74 ppm which were
assigned to the 17α-H and the C-18 methyl in 3-acetoxy-17β-acetylamine of Lemini
et al reported [32].
The triplet at 3.14 ppm (Fig. 3a) and the singlet at 0.96 ppm in the spectrum of
compound 4 indicating that the 17-amino was obtained as its β-epimer according on
Bruker Avance 400 spectrometer in CDCl3, which consistent with Szendi et al
reported [33] that the 17α-H (quasi-axial) was found at a higher field as a triplet signal,
whereas the one corresponding to 17β-H (quasi-equatorial) appeared as a doublet.
The configuration of 6c was also confirmed by the single crystal X-ray diffraction
21

(Fig. 4). The single crystal was crystallized from a mixture of acetone and water
(ratios of the volume was 2:1) by the slow evaporation method in NMR tube at room
temperature. It can be seen directly the configuration of 17-NH2 of 6c is β-epimer.
Fig.3. Here.
Fig. 4. Here.
3.2 Antiproliferative assay
Antiproliferative activity was assessed against six tumor cell lines (SKN-SH, SGC-
7901, MCF-7, PC-3 and EC-9706). As listed in Table 1, only three compounds 3-
benzyloxy-17-Chloroacetamide (5e), 3-benzyloxy-17-niacinamide (5n), and 17-
butyramide (6c) had similar or even better effects than 2-ME in cancer cells with a
broad-spectrum inhibitory effect. Compound 5e showed better activities with the IC50
values of 2.27 ± 0.36 μmol/L and 2.85 ± 0.455 μmol/L, respectively against PC-3 and
SK-N-SH than 2-ME. 5n showed better activities with the IC50 values of 3.73 ± 0.57
μmol/L and 3.71 ± 0.57 μmol/L, respectively against SK-N-SH and EC-9706. 6c
showed better activities with an IC50 values of 2.95 ± 0.47 μmol/L against EC-9706
and slightly weaker activity against MCF-7 and SGC-7901 than 2-ME.
As can be seen from the data presented in Table 1, there is a general declining trend
of antiproliferative activity for the 17-substituted acylamide analogs as steric bulk
increases relative to 2-ME. It is consistent with the views that H-bond donating group
leads to a marked decrease in antiproliferative activity, which is similar to 17-amine
and 17-carboxamide 2-ME derivatives [25]. But 17-butyramide (6c) displayed
excellent antiproliferative activities against EC-9706, being slightly less potent than
2-ME against MCF-7 and SGC-7901. However, 17-propionamide (6b), 17-acetamide
22

(6a), 17-pivaloylamide (6d) and 17-halogenated amide (6e, 6f, 6g) had little effect on
antitumor activity. There may be a critical size restriction on the 17-acylamide
substituent in the 2-ME derivatives that modulate the interaction of these substances
with tubulin [27].
In addition, aromatic compounds (6h, 6j, 6n), halogenated aromatic compounds (6i,
6k) and sulfonamide compounds (6l, 6m) had poor activity, which indicated that π -
electron effect at C-17 position did not help to enhance anticancer activity. Whereas
17-phenylacetamide (6j) showed moderate antiproliferative activities against EC-
9706 and SGC-7901, 17-p-toluenesulfonamide (6m), 17-niacinamide (6n) displayed
similar or better antiproliferative activities against SK-N-SH. But 17-
methanesulfonamide (6l) was equipotent with 2-ME in EC-9706, which was very
different with the Potter reported no activity in DU-145 [16].
To our surprise, when the C-3 position was benzyloxy, 17-chloroacetamide
compound (5e) exhibited better or equivalent activity with 2-ME. Deprotected 17-
chloroacetamide (6e) was inactive at all. These differences in activity indicate that in
addition to steric effects, electronic effects are also important. As an electron donor,
benzyloxy at C-3 position may change the electronic distribution of the estrogenic
steroids skeleton and affect the binding ability between the colchicine binding sites of
tubulin with the 2-ME analogs. This can be seen from the comparative data showed in
Table 1. The antiproliferative activities between C-3 protection and deprotection 17-
acylamide substituent compounds were just the opposite. Such as compounds 5d, 5e,
5f, and 5g exhibited similar or slightly less potent activities against cancer cell
proliferation comparing with 2-ME, whereas 6d, 6e, 6f, and 6g were inactive at all.
The opposite effects between 5l and 6l, 5m and 6m can also be seen from the table 1.
In addition, 5n and 6n, 5c and 6c displayed comparable antiproliferative activities.
23

Conversely, 5a and 6a, 5b and 6b were inactive at all.
From the above SAR studies, we found that single electronic effects, steric effects,
or H-bond donor at C-3 or C-17 positions were not sufficient to heighten antitumor
activity with acceptable metabolic stability. Perhaps only with suitable steric effects,
electronic effects, H-bond donor at C-17 position and H-bond acceptor at C-3 position
can help enhance metabolic stability, and increase or maintain the anticancer activity.
Table 1 here.
3.3. In Vivo Pharmacokinetics Studies
Based on their antiproliferative activities profiles in vitro, 5e and 6c displayed
considerably similar or improved antiproliferative activity compared with 2-ME, so
we chose compounds 5e and 6c for metabolic stability studies. Rat intravenous
injection was used as an initial screen for PK evaluation in the experiment (Fig. 5).
The elimination half-life (t1/2), plasma concentration-time curve (AUC), clearance and
volume of distribution of the two analogs was calculated via compartmental analyses
using 3P97 Software (Table 2).
Fig. 5 Here.
Table 2 here.
In vivo pharmacokinetics studies data indicated that 2-ME, 5e, and 6c followed a
two-compartment model with different pharmacokinetics parameters after i.v.
administration of at a dose of 10 mg/kg to rats (Table 2). Fig. 5 indicated that
24

comparation in the curve of plasma concentration to time after injecting 2-ME, 5e,
and 6c. The comparison of other parameters within plasma showed that 2-ME had a
lower distribution half-life t1/2α (2.70 ± 1.31 min) and elimination half-life t1/2β (22.28
± 2.59 min) than the 5e, and 6c. It can be seen directly that 5e had a similar plasma
concentration-time curve compared with 2-ME with elimination half-life t1/2β (29.50 ±
4.46 min) from Fig. 5. In other words, like its progenitor compound 2-ME，5e was
rapidly metabolized in vivo, whereas 6c was still detectable in the plasma after 12 h of
exposure. In Table 2, pharmacokinetics analysis of rats intravenously injected with 6c
demonstrated a t1/2α of 40.19 min, a t1/2β of 240.93 min, the AUC0-tmin (2068.20 ±
315.74) μg·mL-1·min, a clearance of 0.0047 ml/min. The elimination half-life of 6c
was ten times longer than 2-ME and area under the curve was seven times higher than
2-ME, respectively, which meant that 6c had higher bioavailability and longer
metabolism than 2-ME. Taken together, compound 6c had superior bioavailability and
might be useful for further in vivo studies.
4. Conclusion
Twenty-eight derivatives of 5a-5n and 6a-6n were synthesized, and cell activity
tests were carried out. Three compounds 5e, 5n and 6c were found to have similar or
better anticancer activity with 2-ME. But in vivo pharmacokinetics study showed
compound 6c had superior bioavailability and prolonged metabolism against S180
mouse ascites tumor. The elimination half-life of 6c (t1/2β = 240.93 min) is ten times
longer than 2-ME (t1/2β = 22.28 min) and the area under the curve was seven times
(AUC0-tmin = 2068.20 ± 315.74 μg.ml-1.min) higher than 2-ME (AUC0-tmin = 263.57 ±
93.83 μg.ml-1.min), respectively, which meant C17-amide is helpful in prolonging
metabolism and in improving bioavailability and C17-amide in place of C17-OH
afforded us a useful direction to synthesize 2-ME derivatives.
25

Taking into consideration the suitable steric effects, electronic effects, hydrogen
bond donor, and hydrogen bond acceptor, we can provide and design new types of
compounds to improve the antiproliferative activity.
Acknowledgments
This work was supported by the National Natural Foundation of China (Grant No.
81573364), the Science and technology key project of Henan province (No.
172102310171), and College Students Innovation and Entrepreneurship Training
Program (SIETP) (Grant No. 201410459058). The authors thank School of
Pharmaceutical Sciences in Zhengzhou University of China, Collaborative innovation
center of Henan Province and so on.
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