Synthesis and biological evaluation of new 2-methoxyestradiol derivatives: Potent inhibitors of angiogenesis and tubulin polymerization

Bioorganic Chemistry 113 (2021) 104988

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Synthesis and biological evaluation of new 2-methoxyestradiol derivatives:

Potent inhibitors of angiogenesis and tubulin polymerization

,

,*

Moran Sun^a, Yixin Zhang^a, Jinling Qin^a, Mengyu Ba^a, Yongfang Yao^{a *}, Yongtao Duan^b

,

Hongmin Liu^{a *}, Dequan Yu^c

^aSchool of Pharmaceutical Sciences, and Institute of Drug Discovery & Development, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan 450001, China

^bHenan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases, Children’s Hospital Afﬁliated to Zhengzhou University, Zhengzhou University,

Zhengzhou 450018, China

^cChinese Academy of Medical Sciences, Beijing 100021,China

A R T I C L E I N F O

A B S T R A C T

Keywords:

Here, we report the structural optimization of a hit natural compound, 2-ME₂(2-methoxyestradiol), which

exhibited inhibitory activity but low potency on tubulin polymerization, anti- angiogenesis, MCF-7 proliferation

and metastasis in vitro and in vivo. A novel series of 3,17-modiﬁed and 17-modiﬁed analogs of 2-ME₂were

synthesized and investigated for their antiproliferative activity against MCF-7 and another ﬁve different human

cancer cell lines leading to the discovery of 9i. 9i bind to tubulin colchicine site tightly, inhibited tubulin

polymerization and disrupted cellular microtubule networks. Cellular mechanism studies revealed that 9i could

induce G2/M phase arrest by down-regulated expression of p-Cdc2, P21 and cell apoptosis by regulating

apoptosis-related proteins (Parp, Caspase families) in a dose-dependent manner. Importantly, 9i signiﬁcantly

inhibited HUVEC tube formation, proliferation, migration and invasion. The inhibitory effect against angio-

genesis in vivo was conﬁrmed by zebraﬁsh xenograft. Furthermore, 9i could effectively inhibit the proliferation

and metastasis of MCF-7 cells in vitro and in zebraﬁsh xenograft. The satisfactory physicochemical property and

metabolic stability of 9i further indicated that it can act as a promising and potent anti-angiogenesis, inhibiting

proliferation and metastasis of breast cancer agent via targeting tubulin colchicine binding site.

Tubulin

Angiogenesis

Metastasis

Breast cancer

1. Introduction

in the development, progression, and metastasis of breast cancer, and

aids in supplying nutrients to promote cancer cells growth and metas-

Breast cancer, one of the most popular cancers around the world, has

been the leading cause of tumor-related death for females [1]. Although

some novel therapy strategy has prolonged the survival time of breast

cancer patients, the treatment of breast cancer remains extremely un-

favorable and suppressing breast cancer progression, especial metas-

tasis, is still very imperious. According to the American Cancer Society

in 2018, breast cancer metastasis was regarded as the main cause of

therapeutic failure and responsible for over 80% of patient mortality

[2]. Breast cancer metastasis is a complicated process in which breast

cancer cells disseminate from the primary tumor to distant secondary

organs through the lumina or blood vessels. Recent progression has

focused on the understanding of the molecular signaling pathway that

regulates breast cancer metastasis, which displayed that angiogenesis

may be a potential therapeutic target for breast cancer metastasis pre-

vention and inhibition [3,4]. It is well known that angiogenesis is critical

tasis. Angiogenesis for breast cancer is thought as a trustworthy pre-

dictor of poor prognosis and a key risk factor for breast cancer metastasis

[5,6]. Thus, there is an urgent need to explore novel and strong anti-

angiogenic molecules with the activity of signiﬁcantly inhibiting

breast cancer growth and metastasis.

Tubulin, the necessary protein for the formation of the mitotic

spindle and mitotic division of cell, has been an important target for the

treatment of breast cancer in the past decades [7]. Tubulin inhibitors,

including microtubule stabilizers such as taxanes, and microtubule

destabilizers like vinca alkaloids and colchicine, disrupt microtubule

dynamic and arrest cells in G2/M phase, eventually cancer cells or

human umbilical vein endothelial cells (HUVEC) will undergo apoptosis,

which can lead to tumor growth suppression and angiogenesis inhibition

[8–12]. Also, different research suggested that tubulin could regulate

the expression of angiogenesis related growth factors, such as vascular

* Corresponding authors.

E-mail addresses: yongfangyao@zzu.edu.cn (Y. Yao), duanyongtao860409@163.com (Y. Duan), liuhm@zzu.edu.cn (H. Liu).

https://doi.org/10.1016/j.bioorg.2021.104988

Received 18 March 2021; Received in revised form 8 May 2021; Accepted 11 May 2021

Available online 13 May 2021

M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

endothelial growth factor (VEGF) and Hypoxia-inducible factor 1 (HIF-

1) [13–15]. Considering the vital role of tubulin for breast cancer pro-

gression and metastasis, a number of tubulin inhibitors have been

approved to treat breast cancer in clinic including paclitaxel, epothilone,

vinfulnine and eribulin (Fig. 1) [16]. In general, these inhibitors bind to

the taxane or vinca alkaloid binding sites in tubulin with a large mo-

lecular weight, complicated chemical structures, relative hydrophobic-

ity, poor water solubility and strong neurotoxicity, which has limited

their further use in clinic [17]. Tubulin inhibitors that bind to the

colchicine binding site can largely overcome the above drawbacks and

have therapeutic advantages over taxanes and vinca alkaloids. Unfor-

tunately, there is no FDA approved drugs that speciﬁcally target the

colchicine binding site of tubulin for the cancer treatment till date. And

more, these reported tubulin colchicine binding site inhibitors, such as

LG-25 and G-1 with potent anti-breast cancer growth in vitro and in

vivo, almost never involved in angiogenesis and metastasis [18–20].

Therefore, there is still an urgent need to discover potent tubulin

colchicine binding site inhibitors with novel chemical diversity, strong

anti-angiogenesis, suppressing proliferation and metastasis of breast

cancer.

2. Results and discussion

2.1. Chemistry

To explore the preliminary structure-activity relationships of various

sulfone substituents on the 17β-oxygen atom, we prepared a series of 3-

O-sulfamate-17β-sulfonic acid ester 9a-9h endowed with electron-

donating and withdrawing groups. 2-Methoxyestradiol was reacted

with benzyl chloride under the presence of sodium hydroxide to afford

2-methoxy-3-O-benzyl estradiol 6 (Scheme 1). Sulfonylation of 6 by

reaction with corresponding sulfonyl chloride under dry pyridine pro-

ceeded smoothly to give 7 in excellent yield. This was followed by

deprotection with Pd/C under a hydrogen atmosphere to generate

compound 8. Finally, sulfamoylation of 8 was achieved by reaction with

a solution of sulfamoyl chloride in DMA following the method of Okada

and co-workers [26] to give sulfamate 9a-9h.

We also wished to evaluate the impact of replacement of the 17-O-

sulfamate with different functional groups, such as methyl, acetyl,

nitrogen-containing heterocycles and N, N-dimethylformamide, on

antiproliferative activity. The synthesis of this comprehensive series of

3-O-sulfamate estradiol derivatives are thus illustrated in Scheme 2.

After some investigation, it was determined that compound 7i, 7j,

7o, 7n could be achieved by a nucleophilic substitution reaction of 6

with corresponding halide, in the presence of NaH or pyridine. Subse-

quently, 9i, 9j, 9o and 9n was obtained using the aforementioned

deprotection and sulfamoylation conditions. Attempts to introduce an

unreactive pyrimidine or pyridine bromides using the same approach

and Ullmann coupling reaction [27], however, proved unsuccessful.

Gratifyingly, the expected aryl ether 7k was accessed in 85% yield by

Buchwald-Hartwig cross-coupling reaction conditions [28]. Likewise,

7k and 7m was converted into the corresponding phenols under stan-

dard catalytic hydrogenolytic conditions, and the nitro group of 7l was

simultaneously reduced into amino, which all in turn were sulfamoy-

lated to give 9k, 9l, 9m respectively.

Nature resources such as micro-organisms and plants provide a vast

abundance of metabolites with numerous activities, which make natural

products an ideal starting point for drug screening and the identiﬁcation

of novel chemotherapeutics [21]. Considering the advantages of natural

products in discovering novel leading compounds and our continuous

interest in tubulin [7,22–25], an in-house natural products library with

about 1000 compounds have been screened to identify hits of anti-

tubulin polymerization, anti-angiogenesis, suppressing proliferation

and metastasis of breast cancer for further optimization. From the

screening, 2-ME₂was identiﬁed as a preliminary hit containing the

moderate inhibitory effect on tubulin polymerization and MCF-7 pro-

liferation with the IC₅₀value of 36.824 and 3.901 μM, respectively.

Preliminary biological evaluation showed that this hit could, to some

extent, inhibit angiogenesis and breast cancer migration. As shown in

Fig. 2, these results has encouraged us to further optimize hit (2-ME₂)

structure for improving the potency of inhibiting tubulin polymeriza-

tion, anti-angiogenesis, intervention of breast cancer growth and

metastasis, which has led to the discovery of lead compound (9i).

With above 3-O-sulfamte compounds in hand, we want to better

understand the role played by sulfamate on the key interactions with the

binding site. Thus, carbamate was used as a potential bioisostere to

replace sulfamate and a small series 3-O-carbamate analogs were syn-

thesized from the appropriate aryl ether 8, as shown in Scheme 3.

Compound 8 was reacted with trichloroacetyl isocyanate to afford N-

trichloroacetyl carbamates 10, which were then hydrolyzed with K₂CO₃

in MeOH/THF/H₂O to yield 11a, 11i, 11l, 11n, 11o respectively.

Fig. 1. Representive tubulin inhibitors against breast cancer.

2

M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

Fig. 2. Identiﬁcation of hit compound (2-ME₂) from our natural compound library and further optimizations to discover lead compound (9i).

Scheme 1. Reagents and conditions: a. RSO₂Cl, dry pyridine, rt; b. Pd/C, H₂, MeOH, THF, rt; c. NH₂SO₂Cl, DMA, 0 ^◦C to rt.

2.2. Biology

carcinoma), MGC-803 (human gastric cancer), HepG2 (human liver

cancer) and Hela (human cervix carcinoma) cells using the SRB and

2.2.1. Antiproliferative activity and structure activity relationships

All sulfamates and carbamates synthesized were evaluated, together

with 2-ME₂(1) and 4, for their cytotoxic effects on MCF-7 (human breast

adenocarcinoma) and another ﬁve individual cancer cell lines including

U937 (histiocytic lymphoma cancer), A549 (human non-small-cell lung

CCK-8 assay. The results obtained from this in vitro screening are pre-

sented in Table 1. In the sub-groups of 3-O-sulfamate-17-O-sulfonate

derivatives (9a-9h), 9a displayed the most promising antiproliferative

activity with IC₅₀of from 0.133 μM to 0.633 μM. Introduction of an

ethyl- or CF₃-substituent (9b-9c) on the sulfonyl caused a slight

3

M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

Scheme 2. Reagents and conditions: a. CH₃I, NaH, DMF, rt, overnight; b. 2-bromoquinoline, NaH, TBAI, dry DMF ,100 ^◦C, 6 h; c. Pd (OAc)₂, CsCO₃, BINAP, toluene,

100 ^◦C; d. NMe₂COCl, NaH, MeCN,100 ^◦C, 10 h; e.CH₃COCl, dry pyridine, rt, 30 min; f. Pd/C, H₂, MeOH, THF, rt; g. NH₂SO₂Cl, DMA, 0 ^◦C to rt.

Scheme 3. Reagents and conditions: a. Cl₃CC(O)NCO, dry THF, rt; b. K₂CO_3,H₂O/MeOH/ THF, rt.

reduction in activity. In contrast, the phenyl sulfonate derivatives (9d-

9h), without any exception, was 10- to 40- fold less active than 9a,

suggesting that the increased steric bulk around the hydrogen bond

acceptor sulfonyl reduced its accessibility. Based on the evidence of

above results, the steric hindrance of 17-position appears to be critical

for antiproliferative activity. This could also be explained by another

example of N, N-dimethyl formate 9n, which led to a substantial loss of

activity relative to corresponding carbamate derivative 9p. The most

striking compound is sulfamate 9i, in which a small group methyl ether

was directly attached at the 17-position, affording >50% growth inhi-

bition at concentrations below 150 nM in all cell lines, especially for

MCF-7 with about 56 folds increasing in contrast with 2-ME₂. This

4

M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

–

3-sulfamate group of 9i to form a hydrogen bond (β-tubulin, O⋅⋅⋅H N:

Table 1

Biological evaluation of synthesized compounds and 2ME₂.

3.08 Å). Its interaction is further stabilized by hydrogen bond formed

between 17-methoxy group and the amine group of the side chain Lys

Compound

IC₅₀± SD (µM) ^a

–

254 (β-tubulin, O⋅⋅⋅H N: 3.41 Å), indicating the important role of

U937

A549

MGC-803

MCF7

HepG2

Hela

methoxy group for inhibitory activity. As shown in Fig. 3 (panel D), 9i

efﬁciently bind to the [³H]colchicine binding domain of tubulin

compared with the positive control CA-4, which demonstrated that 9i

directly interacted with tubulin by the pocket of colchicine site. The in

vitro tubulin polymerisation inhibition activity of 9i was evaluated

2ME₂

9a

9b

9c

1.261

0.159

0.409

0.542

3.552

＞5

3.216

0.138

0.663

1.087

＞5

4.123

0.133

0.111

0.216

＞5

3.901

0.165

0.920

0.579

＞5

0.307

0.633

1.217

0.957

＞5

0.616

0.124

0.169

0.511

＞5

9d

9e

9f

＞5

subsequently, with colchicine (4

μ

M) as a positive control group. Puri-

＞5

ﬁed and unpolymerized tubulin was incubated with 9i at different

9g

9h

9i

＞5

concentrations (2 μM, 4 μM, 8 μM). The ﬂuorescence intensity becoming

＞5

stronger with time indicated the occurrence of tubulin polymerization. It

was found that the increased tendency of the ﬂuorescence intensity was

obviously slowed down with higher concentrations of 9i (Fig. 2, panel

0.139

＞5

0.101

＞5

0.141

＞5

0.069

＞5

0.075

＞5

0.063

＞5

9j

9k

9l

＞5

E). The IC₅₀value of compound 9i was 4.875

μ

M against tubulin that

9m

9n

9o

9p

8a

8b

8f

＞5

was more potent than colchicine with IC₅₀of 6.6

μ

M [29]. It revealed

＞5

2.33

0.778

＞5

4.212

2.265

＞5

1.263

0.382

＞5

4.383

1.256

＞5

8.099

6.207

＞5

1.961

0.580

＞5

that 9i was a novel tubulin polymerization inhibitor. As tubulin poly-

merization and microtubule assembly plays a vital role in the mainte-

nance of cellular morphology and basic cellular functions, an

immunoﬂuorescence assay was carried out to reveal whether compound

9i could disrupt the microtubule dynamics in living cells. As shown in

Fig. 3 (panel F), the microtubule network of MCF-7 cells in the control

group was arranged and organized normally, with ﬁlamentous micro-

tubule surrounding the cell nucleus. Compound 9i produced moderate

disrupt of the spindle formation at a concentration of 50 nM. When the

concentration increased (100 and 200 nM), the microtubule spindles

around the cell center were signiﬁcantly reduced, cell shape change

from spindle to round, irregular or multiangular, and some spot-like

disordered structures were observed clearly, similar to CA-4. These

morphological changes of microtubules indicated that 9i dramatically

interfered with mitosis of MCF-7 and inhibited tubulin polymerization,

which possibly eventually lead to tumor cell death.

2.473

＞5

5.926

＞5

2.654

＞5

2.284

＞5

4.392

＞5

2.837

＞5

8i

＞5

8l

＞5

8n

8o

11a

11i

11l

11n

11o

4

＞5

0.333

0.686

0.113

0.228

0.614

0.419

a

Each compound was tested in triplicate; the data are presented as the mean

± SD.

unexpected result might be due to methyl ether which is functioned as a

sterically unhindered bond acceptor group, once again conﬁrming steric

size of the 17-subtituent is important. Unsurprisingly, the corresponding

non-sulfamoylated analogue 11i, was dramatically less potent than 9i in

all cell lines, indicating that 3-O-sulfamate substitution is highly

favored. Actually, most of the phenol series compounds proved to

2.2.3. 9i induces G2/M phase arrest and regulated the expression of G2/M-

related proteins in MCF cells.

It has been reported that most tubulin polymerization inhibitors

disrupt the cell cycle at G2/M phase [30]. Thus, the effect of compound

9i on cell cycle progression was investigated in MCF-7 cells by ﬂow

cytometry analysis, using propidiumiodide (PI) staining. MCF cells were

treated with 9i at different concentrations (0, 50, 100 and 200 nM) for

12 h and 100 nM 9i at different times (0, 6 h, 12 h, 24 h) respectively. As

shown in Fig. 4 (panel A-D), the number of cells arrested at G2/M phase

was increased, depending on concentration, from 16.3% (control) to

38.1% (50 nM), 54.0% (100 nM), and 75.1% (200 nM) accordingly.

Similarly, the accumulation of MCF-7 cells at G2/M phase was markedly

increased with a longer exposure to compound 9i. These results sug-

gested that 9i could induce cell cycle arrest at the G2/M phase in a dose-

and time-dependent manner.

possess modest or no activity (IC₅₀> 5

ethyl sulfonate 8b, displaying fairly good activity with a IC₅₀of 2.473

M against the U937 cell line. These ﬁndings in the 3-O-carbamate series

were similar to those obtained in the phenol series, wherein all de-

rivatives show uniformly IC₅₀of >5 M. Based on this ﬁnding, it

μM), with the exception of the 17-

μ

revealed that replacement of the 3-O-sulfamate with a carbamate group

was deleterious to retain good antiproliferative activity, while 17-O-

carbamate compounds have been reported to deliver maintained or

improved activity in some cases compared to 17-O-sulfamate analogues.

In an attempt to improve water solubility of 2ME₂analogues, several

pyridinyl group was introduced at 17-position, derived from its potential

character to form H-bond with water or binding site. Unfortunately, all

compounds bearing aromatic heterocyclic at 17-position exhibited weak

To acquire the underlying mechanisms of G2/M phase arrest induced

by compound 9i, we detected the protein level of Cyclin B1, Cdc2 and

P21, which effectively control cell cycle progression through increasing

the G2/M fraction [31]. As depicted in Fig. 4 (panel E-F), the results

demonstrated that levels of p-Cdc2 and P21 were down-regulated in a

dose-dependent manner. Meanwhile, compound 9i obviously increased

Cyclin B1 expression with increasing concentrations (0, 50, 100, 200

nM).

activity with IC₅₀values of >5 μM against tested cell lines. Collectively,

the structural optimization and SAR studies led to the discovery of 9i

that exhibited high potency on MCF-7. Further in-depth in vitro and in

vivo activity assays were subsequently performed with compound 9i.

2.2.2. 9i bind to the colchicine site of tubulin and inhibit the microtubule

polymerization

2.2.4. 9i induced caspase-dependent apoptosis

It has been reported that 2ME₂occupies exactly the same binding site

of β-tubulin that is occupied by colchicine [19]. In this paper, molecular

docking was performed to elucidate the binding mode of compound 9i

with tubulin-microtubule system by docking 9i into the active site of

tubulin (PDB code: 1SA0). The docking pose in Fig. 3 (panel A-C) show

that compound 9i could be well-anchored into the hydrophobic pocket

of β-tubulin subunit. While Lys 352 residues are in close interaction with

Mitotic arrest of cancer cells by tubulin inhibitor is generally asso-

ciated with cellular apoptosis [32]. The effects of compound 9i on MCF-

7 cell apoptosis were evaluated in an Annexin V-APC/PI double staining

assay and analyzed by a ﬂow cytometry. The results were illustrated in

Fig. 5 (panel A-B). Compared with the control group (6.65%, early and

late apoptosis), the apoptosis rates increased gradually from 20.90% to

21.31% and 39.58% after being treated with 9i at 50, 100, and 200 nM

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M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

Fig. 3. Compound 9i bind to the colchicine-binding site of tubulin and inhibited microtubule polymerization. (A) Molecular docking results of compound 9i binding

mode within the αβ-tubulin heterodimer. The α- and β- tubulin subunits are represented as green ribbons. (B) 2D binding models and hydrogen-bond interactions. (C)

3D binging models of 9i in different conﬁguration. (D) Effect of compound 9i on tubulin binding of [³H] colchicine. (E) Tubulin polymerization inhibitory activity of

9i. (F) The effects of 9i and CA-4 on the organization of celluar microtubule network of MCF-7 cells observed under a confocal microscope. The photomicrographs

shown are representative of at least three independent experiments performed. (For interpretation of the references to color in this ﬁgure legend, the reader is

referred to the web version of this article.)

for 48 h, respectively. These results conﬁrmed that 9i clearly induced

cell apoptosis in MCF-7 cells in a dose-dependent manner.

2.2.5. 9i inhibited migration, invasion and tube formation of vascular

endothelial cells

Caspases, a family of cysteine-aspartic proteases, are the central

executioners of apoptosis. As a critical step in the process of apoptotic

cellular death, caspase activation is mediated by various inducers [33].

caspase-3 is responsible for chromatin condensation and DNA frag-

mentation, which represents the hallmark of apoptosis. The caspase

cascade usually begins with the activation of casase-8 followed by

caspase-3, which eventually trigger nuclear fragmentation. The cleavage

of poly (ADP-ribose) polymerase (Parp) is an important indicator of

apoptosis and generally considered to be an indicator of caspase-3

activation [34]. In order to explore the intrinsic mechanism of the

apoptosis effects produced by 9i, western blotting assay was conducted

on MCF-7 cells threated with 9i at different concentrations for 48 h.

β-Actin was used as a loading control. We observed that 9i induced

activation of the initiator caspase-9 in a dose-dependent manner (Fig. 5,

panel C-D). Meanwhile, the activation of executioner caspase-3 and

cleavage of its substrate Parp during the apoptotic process were also

investigated. The effect of 9i on caspase-3 and Parp cleavage was

consistent with concentration at which it causes G2/M arrest in MCF-7

cells.

Recently, several clinical trials are ongoing to evaluate the potential

of vascular disrupting agents or antiangiogenic drugs in combination

with conventional therapies to treat breast cancer metastasis [35]. Many

tubulin binding agents, especially CA-4, show vascular-disrupting and

antiangiogenic effects against tumor endothelium [36], and for that

reason we examined effects of 9i on endothelial cells in vitro. Specially,

human umbilical vein endothelial cells (HUVECs) seeded on matrigel

was used as a model to study the antivascular activity of 9i. Endothelial

cell migration to the tumor site is essential for the process of angio-

genesis. Microtubules-targeting agents have also been reported to pre-

vent the formation of new blood vessels or interfere with cell migration

at low doses.

The antivascular activity of 9i was evaluated by a HUVEC tube for-

mation assay. As shown in Fig. 6 (panel A-B), relative to the control, 9i

effectively reduced the width and length of “tubule-like” networks

formed by HUVEC after 6 h incubation at the concentrations of 1 μM and

10 μM. We then perform image analysis to obtain a quantitative deter-

mination of the segment length, the area and the number of meshes, the

percent of area covered by HUVECs, and the number of branching points

after 6 h treatment [35].

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M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

Fig. 4. Compound 9i induced G2/M arrest in MCF-7 cells. (A) Cells were treated with indicated concentrations of 9i for 24 h and proﬁles were obtained by ﬂow

cytometry. (B) The percentage of cell cycle from (A) were illustrated in plots. (C) Cells were treated with 100 nM 9i for 0, 6, 12, 24 h respectively and analyzed by

ﬂow cytometry. (D) The percentage of cell cycle from (C) were illustrated in plots. (E) MCF-7 cells were treated with 9i, then cells were harvested and the protein

expressions of Cyclin B1, p-Cdc2 and P21 were detected by western blotting assay. (F) Quantitative analysis of the expression of Cyclin B1, p-Cdc2 and P21 from

experiments as in (E). Data are presented as mean ± SD of at least three independent experiments.

A wound healing assay was then performed to evaluate the capacity

2.2.7. Antiangiogenic effects in zebraﬁsh embryos

of 9i to prevent cell motility. As shown in Fig. 6 (panel C-D), after

The zebraﬁsh model for antiangiogenesis is a well-studied and

adopted tumor model system used for drug screening purposes. The

advantages of zebraﬁsh xenografts for antiangiogenesis screening in-

cludes low cost, transparent embryos, rapid propagation and the ability

to perform large number of transplants. Moreover, major molecular

pathways regulating angiogenesis in mammalian system are conserved

in zebraﬁsh. Except for angiogenic potential, it also allows evaluation of

metastatic potential of cancer [37]. The effect of compound 9i on em-

bryonic angiogenesis in zebraﬁsh was determined, in which the vascular

endothelial cells were labeled with green ﬂuorescent protein (GFP). Tg

(ﬂk: EGFP) Zebraﬁsh embryos (12hpf) were treated with different doses

exposed to compound 9i at 0.1, 1 and 10μМ for 48 h, 9i obviously

decrease the closure of would scratching in a conﬂuent monolayer of

HUVEC cells, with a dose-response relationship observed. Then,

HUVECs cells were treated with 0.1, 1 and 10

μМ of 9i, and the

migration ability was further detected by transwell assay. It was found

that, with the increase in the concentration of 9i, the number of

migrated endothelial cells gradually decrease (Fig. 6, E-F), and the in-

vasion ability of cells gradually weakened. These results collectively

indicated that 9i has signiﬁcant effect on prohibiting HUVECs tubular

structure formation and migration.

of compound 9i (0.5, 1, 1.5μМ) and 30hpf zebraﬁsh was imaged by

2.2.6. 9i inhibited migration and invasion of MCF-7 cells

ﬂuorescent microscopy to observe the affection of applied agents on

intersegmental vessels (ISVs) development. As shown in Fig. 8, the

normally developing embryos in the control group had 22–24 ISVs,

Metastasis is the leading cause of morbidity and mortality for breast

cancer patients. In this work, we evaluated whether 9i could suppress

migration and invasion of MCF-7 cells by wound healing and transwell

assay respectively. As shown in Fig. 7 (panel A-B), after treatment for 48

h at indicated doses, 9i apparently inhibit the wound healing of MCF-7

cells compared with 0 h. Transwell assays show cell quantity from upper

side of transwell membrane to bottom side were obviously decreased

with the increase in the compound’s concentrations, conﬁrming that 9i

could hinder the MCF-7 cells invasion (Fig. 7, panel C-D).

whereas, for zebraﬁsh embryos treated with 0.5 and 1 μM of 9i, the

number and length of ISVs considerably reduced with concentration

increasing, while preserving ﬂuorescence in the doral aorta and major

cranial vessels. At the doses ≥1.5 μM, ISVs formations were seriously

impaired and developmental defects were observed, indicating a dose-

dependent inhibition pattern. Our results suggest that 9i is a powerful

vasoactive reagent and could greatly blocked the growth of angiogenesis

at low doses.

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M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

Fig. 5. 9i induces MCF-7 cells apoptosis. (A) Apoptosis ratio detection by Annexin/ PI double staining assay through ﬂow cytometry analysis treated with indicated

concentrations of compound 9i for 48 h. The diverse cell stages were given as live (Q4), early apoptotic (Q3), late apoptotic (Q2) and necrotic cell (Q1). (B) The

quantitative analysis of apoptotic rate at early and advanced stages of MCF-7 cells. (C) Western blot analysis of the apoptosis related proteins. (D) The quantitative

analysis of the protein levels. The data was presented as the mean ± SD of three independent tests.

2.2.8. In vivo anti-tumor and -metastasis effects of compound 9i in a

zebraﬁsh xenograft

2.2.9. Physicochemical property and metabolic stability of compound 9i

To evaluate the drug-likeness of compound 9i, various physico-

chemical and metabolic parameters had been tested. As shown in

Table 2, 9i has a polar surface area (PSA) of 87.65 placed between 80

and 90 Å, well within the range considered appropriate for passive ab-

sorption. Distribution coefﬁcients displayed an excellent drug-like

cLogD value which was 3.5. Aqueous solubility ranged from a moder-

ate 45–90 µg/mL at pH 2.0 for 9i to a more acceptable value of up to 210

µg/mL at pH 6.5. On the whole, the lead compound 9i possessed

favorable solubility and permeability properties. Then metabolic lia-

bilities of compound 9i were also assessed with human (HLM) liver

microsomes. The in vitro degradation half-life in HLM was 86 min for 9i,

and there was promising value for intrinsic clearance (Clint) which was

105 µL/min/mg protein.

To evaluate in vivo anti-tumor and -metastasis activity, we investi-

gated the proliferation and metastasis of MCF-7 Cells induced by 9i

using the zebraﬁsh xenograft model, a well-studied and adopted in vivo

platform for drug screening and translational research in human carci-

nomas. Accordingly, Cell-Tracker Red CMTPX labeled MCF-7 cells were

microinjected and developed into the yolk of Tg(ﬂk: EGFP) embryos, and

different concentrations of 9i were added. 72

h after xeno-

transplantation, zebraﬁsh were processed by ﬂuorescence microscopy to

evaluate the effects of 9i on the tumor mass development and cancer

cells metastasis. 2ME₂was used as a positive control. As shown in Fig. 9,

when tumor cells were microinjected into zebraﬁsh embryos, after 72 h

the MCF-7 cells in the control group disseminated and widely migrated

away from the primary area, and some tumor cells even migrated in the

blood vessel of the zebraﬁsh tail. However, treatments with 9i could

markedly blocked MCF-7 cells migration and invasion in zebraﬁsh xe-

nografts. In addition to the anti-metastasis effect, comparison of the

ﬂuorescence intensities and size of MCF-7 cells to untreated xenografts

at 72 hpf indicated that 9i could suppress the growth of tumor in a dose-

dependent manner. The tumor almost completely disappeared upon 1.5

3. Conclusions

In the current investigation, a total of 27 novel 3,17-modiﬁed and 17-

modiﬁed analogs of 2-ME₂were designed, synthesized and evaluated.

Among them, the 3-O-sulfamate-17-methoxy derivative 9i exhibited a

potent inhibitory activity against the proliferation, migration, and in-

vasion of MCF-7 breast cancer cells. Molecular mechanisms illustrated

that compound 9i could disrupt microtubule networks, induce cells

MCF-7 apoptosis and arrest MCF-7 cells at G2/M phase. Scratch and

transwell assays showed that 9i dose-dependently suppress the prolif-

eration, migration and invasion of HUVECs. Tube formation assays

μ

M dosages of 9i, which was further evidenced by the result of reduced

ﬂuorescence intensities and decreased tumor area compared with the

control group and 2-ME₂group. All these research showed the potential

of 9i to be the candidate to inhibit the metastasis and proliferation of

breast cancer.

8

M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

Fig. 6. Compound 9i displayed antivascular activity in vitro. (A) Typical images depicting the vascular structure formation of HUVEC cells by treatments with 9i for

6 h. (B) Quantitative evaluation of indicated concentration 9i on standard parameters of HUVEC tubule formation. Mean ± SEM, three experiments. ** p< 0.01; ***

p < 0.001. (C) Conﬂuent HUVECs in a monolayer were wound, and images were captured at 0 h and 48 h after treatments with 9i. (D) Inhibitory rates of 9i on the

HUVECs migration. (E) The invasion suppressing effects of 9i against HUVECs cells by transwell assay. (F) Quantitative analysis of the migration ability. * p < 0.05,

** p < 0.01; *** p < 0.001, vs. control; n = 3.

revealed that 9i severely impaired the tube forming capacity of HUVECs.

Molecular docking, radioligand binding assay, tubulin polymerization

assay in vitro and immunoﬂuorescence studies in cells suggested 9i

could bind to tubulin colchicine binding site, cause disruption of tubulin

polymerization, and interfered with the normal formation of mitotic

spindles via the depolymerization of microtubules. In addition, the

evaluation of in vivo activity illustrated that 9i obviously inhibit the

proliferation and metastasis of MCF-7 cells, and angiogenesis in trans-

genic zebraﬁsh. Together with the promising physicochemical property

and metabolic stability, 9i may serve as a potential treatment agent as

tubulin inhibitor with the activity of anti-angiogenesis, inhibiting

proliferation and metastasis of breast cancer.

4. Experimental procedures

4.1. Biological section

MCF-7, MGC-803, Hela and HepG-2 cell lines were cultured in a base

medium (90% DMEM and 10% FBS), A549 and U937 were cultured in a

base medium (90% PRMI-1640 and 10% FBS) at 37 ^◦C, 5% CO₂and a

saturated humidity atmosphere. All the mediums were supplemented

with 10% FBS and 1% ampicillin/streptomycin (all cells were purchased

9

M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

Fig. 7. Effects of 9i on MCF-7 cells migration and invasion in vitro. (A) Images of MCF-7 cells migration inhibited by 9i determined in wound-healing assay. All

tested samples were treated with serum-free DMEM. (B) Inhibitory rates of 9i on MCF-7 cells migration. (C) Inhibition of cellular invasion by 9i in transwell assay. (D)

Quantitative analysis of the migration ability of MCF-7 cells after the treatment of 9i. The experiments were repeated three times and results were indicated as means

± SD. * p < 0.05, ** p < 0.01; *** p < 0.001 vs untreated control.

Fig. 8. Anti-angiogenic effects of 9i in zebraﬁsh embryos assay. (A) Inhibitory effects of compound 9i on the angiogenesis of transgenic zebraﬁsh. Representative

images from three independent experiments were shown. The left angiogenic vessels were magniﬁed and shown in the right section. (B) Histogram showed the

numbers of zebraﬁsh ISVs per ﬁeld under confocal microscopy. Results represented the means ± SD. ** p < 0.01; *** p < 0.001 compared to untreated control.

10

M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

Fig. 9. (A) Inhibitory effects of 9i on the proliferation and metastasis of MCF-7 cells in zebraﬁsh xenograft models. CMTPX labeled MCF-7 cells (red) were

microinjected into zebraﬁsh embryos, and indicated concentration of 9i were added. 2ME₂was used as a positive control. (B) Quantiﬁcation of the ﬂuorescent area of

the tumor xenografts, representing total MCF-7 cells in zebraﬁsh. (C) Fluorescence intensity of the tumor xenografts in trunk. (For interpretation of the references to

color in this ﬁgure legend, the reader is referred to the web version of this article.)

acid. Bonded dye was solubilized in 10 mM unbuffered trisbase, and the

Table 2

absorbance was read at a wavelength 560 nm according to color in-

Physicochemical parameters and metabolic stability of compound 9i.

tensity using quant universal microplate spectrophotometer (Biotek,

MW^a

PSA^a

cLogD^a

Solubility^b

mL)

(

μ

g/

Degradation

half-life (min)

in human

In Vitro

Winooski, VT). Cell viability (%) was calculated using the following

equation: cell viability (%) = (a treatment/a control) × 100%. The data

represented the mean of three independent experiments in triplicate and

were expressed as means ± SD. The IC₅₀was calculated by the Logit

method.

Clint (µL/

min/mg

protein) in

human

pH

pH 6.5

2.0

395.18

a

87.65

3.5

45–90

105–210

86

105

Calculated using ChemAxon JChem software;

4.1.2. CCK-8 assay

b

Kinetic solubility determined by nephelometry (SolpH).

U937 Cells were seeded in 96-well plates at a density of 8 × 10³per

well and exposed to different concentrations of compounds for 72 h.

from Shanghai Institutes for Biological Sciences, Chinese Academy of

Sciences, Shanghai, China).

Subsequently, 10 μL CCK-8 solution was added to each well, and co-

incubated for another 3–4 h at 37 ^◦C. The absorbance was determined

at 450 nm. Data are calculated with GraphPad Prism5 software.

4.1.1. Cell lines growth inhibition. SRB procedure

A549, MGC-803, MCF-7, HepG2 and Hela cells in exponential

growth phase were seeded in 96-well plates with appropriate cell line

density (3 × 10³–4 × 10³cells/well). Then cells were incubated in a 5%

CO₂incubator at 37 ^◦C for 24 h. When the cells adhered, different

concentrations of compounds as shown in the Figures and Tables were

added to every well. After incubation for another 72 h, cell proliferation

was quantiﬁed using SRB (Sulforhodamine B) to determine the number

of viable cells. Brieﬂy, cells were ﬁxed by addition of cold trichloroacetic

4.1.3. Immunoﬂuorescent staining

MCF-7 cells (3 × 10⁴cells/well) were plated onto square coverslip (1

cm side), after 12 h of incubation, cells were treated with different

concentration of 9i, CA-4 and the vehicle control 0.1% DMSO for 48 h,

respectively. The cells were rinsed twice with PBS and ﬁxed by 4%

paraformaldehyde for 20 min, permeabilized using 0.5% Triton X-100

for another 5 min and blocked with 5% BSA for 40 min. Subsequently,

microtubule detection was performed using a ﬂuorescent antibody

acid (100

were washed with water and dried at 65 ^◦C. Sulforhodamine B solution

μ

L, 0.4%) to the wells (10% (w/v) for 30 min at 4 ^◦C. Plates

against α-tubulin in 5% BSA and incubated for 5–6 h at room tempera-

ture or overnight at 4 ^◦C. To stain cell nucleus, crystals were incubated

with Hoechst for 10 min. After washing with PBST to remove redundant

stain, the samples were visualized under an Olympus laser scanning

confocal microscope.

(100 μL) at 0.4% (w/v) in 1% acetic acid was added to each well, and the

resulting mixtures were further incubated for 20 min at room temper-

ature. Unbound dye was removed by washing ﬁve times with 1% acetic

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M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

4.1.4. Cell cycle distribution assay

Matrigel gel and incubated for 45 min at 37 ^◦C for gelling. Then, cells

were trypsinized and seeded at 6 × 10⁴per upper chamber, and cultured

in 1% fetal bovine serum medium containing different concentrations of

9i. The chamber was then placed in a 24-well plate to which 10% FBS

MCF-7 cells (15 × 10⁴cells/well) were seeded in 6-well culture plate.

◦

After 24 h incubation at 37 C, exponentially cells were treated with

indicated concentration of compound 9i for 12 h and 24 h respectively.

The cells were harvested and washed with cold PBS, then ﬁxed with 75%

ethanol in PBS at ꢀ 20 ^◦C for overnight. Then the ﬁxed cells were washed

◦

had been added. After 48 h incubation at 37 C, both chambers were

washed with PBS three times, and then ﬁxed with 4% paraformaldehyde

for 15 min, followed by staining with crystal violet. The non-invasive

cells on the upper surface membrane were wiped with cotton swabs.

The image of the cells on the bottom face was recorded and quantiﬁed

under an inverted microscope. The results were the means calculated

from three replicates of each experiment.

twice with PBS buffer and subsequently stained with 500 μL staining

solution containing ﬂuorescein isothiocyanate propidium iodide (PI, 5

mg) for 15 min in darkness. Samples were immediately tested for DNA

content with ﬂow cytometry instrument (Becton, Dickinson and Com-

pany, USA). The percentage of cell cycle phases was analyzed by the

software supplied in the instrument.

4.1.10. Zebraﬁsh angiogenesis assay

4.1.5. Cell apoptosis assay

The transgenic ﬂk: enhanced GFP zebraﬁsh embryos were generated

by pair-wise mating, collected and sorted into the 6-well plate (n = 30

per well) with 1 mL of aquaculture water (0.2 g/L of Instant Ocean Salt

in distilled water) and raised at 28 ^◦C. Then the embryos staged at 12 h

post fertilization (hpf) were treated with the indicated concentrations of

9i, which were added into embryo water. About 30 embryos were

screened for each concentration gradient. At 30hpf, the treated embryos

were anesthetized with 0.02% tricaine in embryo water and photo-

graphed. The numbers of ISVs were recorded under confocal micro-

scopy. Results were obtained from three independent determinations

and presented as mean ± SD.

MCF-7 cells at 8 ×10⁴cells/well were seeded in 12-well cell culture

plates overnight, and then the cultured cells were incubated with a dose

range of compounds 9i for 48 h. The treated or untreated cells were

washed twice with PBS and stained by Annexin V-PE and PI for 15 min

without light exposure according to the manufacturer’s protocol.

Apoptosis was quantiﬁed using a ﬂow cytometer (Becton, Dickinson and

Company, USA).

4.1.6. Western blot assay

Exponentially growing MCF-7 cells were seeded in 10 mm dishes at a

density of 6×10⁵/dish and incubated overnight. The cultured cells were

treated with indicated concentrations of 9i for 48 h. After the cells were

washed with cold PBS, the supernatant was removed. Cells were scraped

off the tissue culture dish, and then lysed by 100 µL ice-cold RIPA lysis

buffer for 20 min, with occasional agitation. The supernatant was

collected by centrifuging at 12,000g for 10 min at 4 ^◦C. The protein

concentration in the supernatant was determined by using a BCA protein

assay kit. After BCA analysis to quantify proteins, samples were prepared

in SDS-PAGE loading buffer, then boiled for 10 min at 100 ^◦C. Western

blot analyses were conducted after separation by SDS-PAGE electro-

phoresis and transfer to nitrocellulose ﬁlter (NC) membranes. Immu-

noblotting was performed according to the antibody manufacturers’

recommendations.

4.1.11. Zebraﬁsh xenografts injection and treatment

MCF-7 cells were labeled with Red CMTPX according to the manu-

facturer’s protocol. Before the microinjections, Tg(ﬂk:EGFP) embryos

were kept at 28 ^◦C and manually dechorionated few hours. At 48 hpf, 4

nL of cells suspension containing 400 labelled cells was microinjected

into the yolk of anesthetized embryos by a pneumatic pico pump

(PV820, World Precision Instruments, USA). After injection, embryos

◦

were incubated to recover for at least 1 h at 28 C, the embryos were

distributed to 6-well plates with 20 embryos placed in each well. The

injected xenografts were exposed to the indicated doses of 9i and

◦

maintained at 35 C. DMSO (0.3%) was used as a vehicle control and

2ME₂as a positive control. The survival and development of the xeno-

grafted embryos was recorded every day until the end of experiment. At

one day post injection (dpi), ﬂuorescent microscopy was used to

examine MCF-7 cells size, death and migration in caudal region of

anesthetized zebraﬁsh.

4.1.7. Tube formation assay

Matrigel matrix (Basement Membrane Matrix, BD Biosciences) was

thawed at 4 ^◦C overnight. At this point, 50

μL of the matrix solution was

added to each well of a 96-well plate. After gelling at 37 ^◦C for 30 min,

the matrix was overlaid with 200

μ

L of medium containing 1 × 10⁵

4.2. General chemistry (the detailed information is in Supplementary

Information)

HUVECs per well, which was incubated for 6 h to allow capillary tubes to

form. Different concentrations of 9i were added in the cultures and

incubated for 6 h to monitor the morphological changes of cells and

tubes. The disappearance of existing vasculature was photographed at

10 × magniﬁcation. Values were expressed as percent change form

control cultures grown with complete medium. Standard dimensional

parameters (percent area covered by HUVECs and total length of the

HUVEC network per ﬁeld) were noted, and standard topological pa-

rameters (number of meshes and branching points per ﬁeld) were

estimated.

All chemicals were purchased from commercial sources and used

without further puriﬁcation unless otherwise noted. Solvents and re-

agents were dried and puriﬁed according to the methods described in

the literature. Column chromatography (CC) was carried out on silica

gel (200–300 mesh, Qingdao Ocean Chemical Company, China). Thin-

layer chromatography (TLC) analyses were carried out on silica gel

GF254 (Qingdao Ocean Chemical Company, China) glass plates.

¹HNMR and ¹³CNMR were respectively recorded on a Bruker AV-400

(400 MHz) nuclear magnetic resonance spectrometer at 400 and 100

MHz as deuterated chloroform (CDCl₃) or deuterated dimethyl sulfoxide

(DMSO‑d₆) solutions. Chemical shifts were reported in parts per million

(ppm) relative to tetramethyl silanes as internal standard. High-

resolution mass spectra (HRMS) were recorded with a Q-TOF micro

mass spectrometer.

4.1.8. Would healing migration assays

MCF-7 cells were seeded in 6-well plates and cultivated for 24 h.

Scratches were made in conﬂuent monolayer using 200 μL pipette tips

and washed with PBS to remove the detached cells. The media con-

taining different concentrations of 9i were added to the scratched

monolayers. The migrated cells were photographed under a light mi-

croscope at indicated time points from the scratch. The migration dis-

tance of cells migrated to the wound area was measured manually and

compared with the initial distance.

4.2.1. General procedure for synthesis of compound 7a-7h.

A solution of compound 6 (200 mg, 0.51 mmol) in pyridine (5 mL)

was stirred and corresponding substituted sulfonyl chloride (3.06 mmol)

was added portionwise. The mixture was stirred for 30 min at room

temperature. Ethyl acetate (3 × 15 mL) and water 15 mL was added and

the organic layer was separated, washed with water then brine, dried

4.1.9. Transwell cell invasion experiment

The upper surface of transwell membrane was precoated with 40 μL

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M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

(MgSO₄) and evaporated. The residue was puriﬁed by ﬂash chroma-

tography using mixtures of petroleum ether and ethyl acetate (typically

between 30:1 and 5:1 by volume) to give 7a-7h.

11.57, 8.06. HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₁H₃₁NO₇S₂,

496.1542, found 496.1546. Purity: 97.62%.

2-Methoxy-3-sulfamoyloxy-17β-O-triﬂuoromethanesulfonylestra-1,3,5

(10)-triene(9c): Compound 6 (267 mg, 0.68 mmol) was reacted with

triﬂuoromethyl sulfonyl chloride (0.2 mL, 1.7 mmol) in the presence of

DMAP (498 mg, 4.08 mL) in anhydrous DCM (12 mL) to give 7c as a

white solid, which continue the hydrogenation and sulfamoyl reaction

according to general procedures. The resultant crude solid was puriﬁed

by ﬂash chromatography (petroleum ether/ethyl acetate 5:1) to give a

white powder (250 mg, 85%); m. p. 156.0–158.1 ^◦C; ¹H NMR (400 MHz,

DMSO) δ 7.83 (s, 2H), 7.00 (s, 2H), 4.27 (d, J = 6.3 Hz, 1H), 3.78 (s, 3H),

2.80–2.68 (m, 2H), 2.39–2.15 (m, 2H), 1.97–1.79 (m, 4H), 1.75–1.62

(m, 2H), 1.54–1.15 (m, 5H), 0.83 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ

149.47, 138.65, 136.79, 128.27, 122.92, 110.58, 71.44, 55.88, 47.25,

45.98, 43.56, 38.35, 33.86, 33.43, 28.25, 27.41, 25.92, 23.68, 17.49,

13.42. HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₀H₂₆F₃NO₇S₂,

536.1103, found 536.1106. Purity: 97.03%.

4.2.2. General procedure for preparation of compound 8

Pd/C (80 mg, 10%) was added to a solution of 7 (0.426 mmol) in

MeOH (15 mL) and THF (15 mL). The mixture was stirred under a H₂

atmosphere for 1 h at room temperature and the catalyst was ﬁltered off.

The solvents were removed under reduced pressure to give the crude

product, which was used in next step without further puriﬁcation.

4.2.3. General procedure for preparation of sulfamate 9a-9h from 8

An ice-cold solution of sulfamoyl chloride (200 mg, 1.732 mmol) in

DMA (1.5 mL) was added to 8 (crude product). After 3 h of stirring at

room temperature, ethyl acetate (3 × 10 mL) and water (10 mL) was

added, the organic layer was separated and washed with water and

brine, dried (MgSO₄), and concentrated. The residue was puriﬁed by

silica gel chromatography using mixtures of petroleum ether and ethyl

acetate (typically between 2:1 and 3:1 by volume) to afford 9a-9h as

white solids with a yield between 85% and 92%.

2-Methoxy-3-sulfamoyloxy-17β-O-benzenesulfonylestra-1,3,5(10)-

triene (9d): m.p. 103.7–104.8 ^◦C; ¹H NMR (400 MHz, DMSO) δ 7.94 (d, J

= 7.8 Hz, 2H), 7.80 (d, J = 12.1 Hz, 3H), 7.69 (t, J = 7.6 Hz, 2H), 6.95

(d, J = 10.6 Hz, 2H), 4.37 (t, J = 8.0 Hz, 1H), 3.75 (s, 3H), 2.70 (d, J =

4.8 Hz, 2H), 2.25 (d, J = 12.2 Hz, 1H), 2.11 (t, J = 10.8 Hz, 1H), 1.89 (d,

J = 6.2 Hz, 1H), 1.76 (d, J = 11.3 Hz, 1H), 1.62 (s, 2H), 1.48 (d, J = 12.2

2-Methoxy-17β-O-methanesulfonylestra-1,3,5(10)-trien-3-ol (8a): m.p.

167.5–168.9 ^◦C; ¹H NMR (400 MHz, CDCl₃) δ 6.80 (s, 1H), 6.67 (s, 1H),

5.48 (s, 1H), 4.60 (dd, J = 9.0, 7.9 Hz, 1H), 3.89 (s, 3H), 3.04 (s, 3H),

2.80 (dd, J = 9.7, 6.7 Hz, 2H), 2.35–2.18 (m, 3H), 2.11–2.03 (m, 1H),

1.93–1.74 (m, 3H), 1.56–1.28 (m, 6H), 0.90 (s, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 144.65, 143.59, 131.23, 129.34, 114.62, 108.06, 89.46, 56.09,

49.08, 43.99, 43.33, 38.53, 38.28, 36.40, 28.87, 27.97, 27.15, 26.31,

23.05, 11.78. Purity: 97.54%.

Hz, 1H), 1.38–1.15 (m, 5H), 1.02 (t, J = 11.5 Hz, 1H), 0.76 (s, 3H). ¹³

C

NMR (101 MHz, DMSO) δ 149.45, 138.41, 136.73, 136.23, 134.16,

129.61, 128.11, 127.57, 122.89, 110.47, 89.63, 55.84, 47.91, 43.56,

42.66, 37.61, 35.25, 28.05, 27.35, 26.39, 25.40, 22.46, 11.51. HRMS

(ESI-TOF) m/z [M + Na]⁺calcd for C₂₅H₃₁NO₇S₂, 544.1542, found

544.1546. Purity: 98.29%.

2-Methoxy-17β-O-ethanesulfonylestra-1,3,5(10)-trien-3-ol (8b): m. p.

154.5–156.2 ^◦C; ¹H NMR (400 MHz, CDCl₃) δ 6.80 (s, 1H), 6.67 (s, 1H),

5.47 (s, 1H), 4.60 (dd, J = 9.0, 8.0 Hz, 1H), 3.89 (s, 3H), 3.16 (q, J = 7.4

Hz, 2H), 2.84–2.75 (m, 2H), 2.34–2.18 (m, 3H), 2.08 (t, J = 6.0 Hz, 1H),

1.92–1.76 (m, 3H), 1.53–1.31 (m, 9H), 0.90 (s, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 144.65, 143.59, 131.28, 129.36, 114.62, 108.07, 88.97, 56.09,

49.13, 45.59, 44.01, 43.37, 38.56, 36.42, 28.89, 28.03, 27.16, 26.34,

23.06, 11.81, 8.35. Purity: 98.27%.

2-Methoxy-3-sulfamoyloxy-17β-O-(4-methylbenzene)sulfonylestra-

1,3,5(10)-triene (9e): m. p. 108.2–110.4 ^◦C ¹H NMR (400 MHz, DMSO) δ

7.81 (s, 4H), 7.48 (d, J = 7.9 Hz, 2H), 6.95 (d, J = 9.1 Hz, 2H), 4.34 (t, J

= 8.1 Hz, 1H), 3.75 (s, 3H), 2.70 (d, J = 4.9 Hz, 2H), 2.43 (s, 3H), 2.25 (t,

J = 15.5 Hz, 1H), 2.12 (t, J = 11.3 Hz, 1H), 1.88 (s, 1H), 1.76 (d, J =

11.4 Hz, 1H), 1.58 (dd, J = 20.7, 9.9 Hz, 3H), 1.37–1.09 (m, 6H), 0.76 (s,

3H). ¹³C NMR (101 MHz, DMSO) δ 149.45, 144.68, 138.44, 136.75,

133.40, 130.01, 128.13, 127.58, 122.89, 110.48, 89.39, 55.85, 47.93,

43.56, 42.66, 37.61, 35.33, 28.05, 27.35, 26.40, 25.41, 22.46, 21.10,

11.52. HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₆H₃₃NO₇S₂,

558.1698, found 558.1699. Purity: 97.33%.

2-Methoxy-17β-O-(4-methoxybenzene sulfonyl) estra-1,3,5(10)-trien-3-

ol (8f): m.p. 84.7–86.5 ^◦C; ¹H NMR (400 MHz, CDCl₃) δ 7.88 (d, J = 8.9

Hz, 2H), 7.03 (d, J = 8.9 Hz, 2H), 6.77 (s, 1H), 6.65 (s, 1H), 4.36 (t, J =

8.4 Hz, 1H), 3.92 (s, 3H), 3.87 (s, 3H), 2.87–2.66 (m, 2H), 2.27–2.10 (m,

2H), 2.04–1.92 (m, 1H), 1.88–1.78 (m, 2H), 1.76–1.65 (m, 2H),

1.49–1.40 (m, 2H), 1.28 (dq, J = 7.1, 4.7 Hz, 2H), 1.20–1.10 (m, 2H),

0.86 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.52, 144.63, 143.55,

131.27, 129.98, 129.30, 128.80, 114.60, 114.25, 108.04, 89.68, 56.06,

55.69, 49.04, 43.96, 43.27, 38.44, 36.16, 28.84, 27.72, 27.13, 26.26,

23.00, 11.77. Purity: 98.14%.

2-Methoxy-3-sulfamoyloxy-17β-O-(4-methoxybenzene)sulfonylestra-

1,3,5(10)-triene (9f): m. p. 100.8–102.1 ^◦C; ¹H NMR (400 MHz, DMSO) δ

7.86 (d, J = 8.7 Hz, 2H), 7.82 (s, 2H), 7.18 (d, J = 8.7 Hz, 2H), 6.96 (d, J

= 8.4 Hz, 2H), 4.30 (t, J = 8.1 Hz, 1H), 3.88 (s, 3H), 3.75 (s, 3H), 2.70 (d,

J = 4.8 Hz, 2H), 2.27 (d, J = 14.1 Hz, 1H), 2.12 (t, J = 11.0 Hz, 1H), 1.88

(s, 1H), 1.76 (d, J = 11.7 Hz, 1H), 1.58 (dd, J = 21.3, 9.8 Hz, 3H), 1.25

(ddd, J = 22.3, 16.8, 8.6 Hz, 5H), 1.05 (t, J = 11.5 Hz, 1H), 0.75 (s, 3H).

¹³C NMR (101 MHz, DMSO) δ 163.34, 149.45, 138.45, 136.74, 129.89,

128.13, 127.66, 122.90, 114.71, 110.48, 89.13, 55.84, 55.82, 47.98,

43.58, 42.62, 37.62, 35.36, 28.06, 27.37, 26.40, 25.42, 22.47, 11.51.

HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₆H₃₃NO₈S₂, 574.1648,

found 574.1646. Purity: 98.05.

2-Methoxy-3-sulfamoyloxy-17β-O-methanesulfonylestra-1,3,5(10)-

triene (9a): This compound was prepared according to abovementioned

general procedures and the resultant crude solid was puriﬁed by ﬂash

chromatography (petroleum ether/ethyl acetate 2:1) to give a white

powder (yield 90%); m.p. 192.2–193.0 ^◦C ¹H NMR (400 MHz, DMSO) δ

7.83 (s, 2H), 6.99 (s, 2H), 4.54 (t, J = 8.2 Hz, 1H), 3.77 (s, 3H), 3.17 (s,

3H), 2.74 (d, J = 5.0 Hz, 2H), 2.39 (d, J = 9.3 Hz, 1H), 2.19 (d, J = 8.6

Hz, 2H), 1.89 (d, J = 9.1 Hz, 1H), 1.85–1.66 (m, 3H), 1.46–1.26 (m, 6H),

0.80 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 149.47, 138.52, 136.78,

128.21, 122.94, 110.50, 88.63, 55.87, 48.16, 43.72, 42.70, 37.73,

37.44, 35.52, 28.10, 27.43, 26.48, 25.52, 22.54, 11.55. HRMS (ESI-TOF)

m/z [M + Na]⁺calcd for C₂₀H₂₉NO₇S₂, 482.1385, found 482.1383.

Purity: 98.28%.

2-Methoxy-3-sulfamoyloxy-17β-O-(4-aminobenzene)sulfonylestra-

1,3,5(10)-triene(9 g): Compound 7 g was prepared using the general

procedure described above by the addition of compound 6(200 mg,

0.51 mmol) and 4-nitrobenzene sulfonyl chloride (678 mg, 3.06 mmol)

in dried pyridine (5 mL). In the hydrogenation step, the nitro group was

simultaneously reduced to amino group according to general procedure.

The crude product from the sulfamoyl rection was puriﬁed by column

chromatography (petroleum ether/ethyl acetate 5:1) to give a white

powder (yield 88%); m. p. 127.8–129.4 ^◦C; ¹H NMR (400 MHz, DMSO) δ

10.37 (s, 1H), 8.59 (s, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.48 (s, 2H), 7.33 (d,

J = 8.6 Hz, 2H), 6.72 (s, 1H), 6.42 (s, 1H), 4.30 (t, J = 8.1 Hz, 1H), 3.70

(s, 3H), 2.60 (d, J = 4.3 Hz, 2H), 2.19 (d, J = 11.9 Hz, 1H), 2.02 (d, J =

10.0 Hz, 1H), 1.86 (s, 1H), 1.72 (d, J = 9.4 Hz, 1H), 1.56 (d, J = 11.9 Hz,

3H), 1.23 (ddd, J = 32.3, 15.7, 7.0 Hz, 6H), 0.75 (s, 3H). ¹³C NMR (101

2-Methoxy-3-sulfamoyloxy-17β-O-ethanesulfonylestra-1,3,5(10)-triene

(9b): m. p. 202.8–205.1 ^◦C; ¹H NMR (400 MHz, DMSO) δ 7.78 (s, 2H),

7.00 (d, J = 2.9 Hz, 2H), 4.53 (t, J = 8.2 Hz, 1H), 3.78 (s, 3H), 3.29 (s,

2H), 2.75 (d, J = 4.5 Hz, 2H), 2.41 (s, 1H), 2.21 (d, J = 6.2 Hz, 2H),

1.93–1.68 (m, 4H), 1.45–1.26 (m, 9H), 0.82 (s, 3H). ¹³C NMR (101 MHz,

DMSO) δ 149.47, 138.51, 128.23, 122.92, 110.54, 88.44, 55.90, 48.17,

44.19, 43.70, 42.76, 37.76, 35.65, 28.10, 27.50, 26.46, 25.54, 22.54,

13

M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

MHz, DMSO) δ 145.55, 144.56, 144.35, 129.87, 129.03, 128.15, 127.98,

116.56, 115.52, 109.73, 89.20, 59.72, 55.76, 47.97, 43.33, 42.71,

38.07, 35.44, 28.24, 27.35, 26.72, 25.68, 22.49, 20.73, 14.05, 11.57.

HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₅H₃₂N₂O₇S₂, 559.1651,

found 559.1649. Purity: 98.72%.

7H), 0.88 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 160.03, 145.56, 144.34,

139.13, 132.85, 130.17, 129.57, 128.28, 115.59, 110.65, 109.76, 83.22,

55.78, 49.14, 43.69, 42.63, 38.31, 36.84, 28.38, 27.67, 26.96, 26.09,

22.87, 12.10. HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₄H₃₁N₃O₅S,

496.1984, found 496.1983. Purity: 99.42%.

2-Methoxy-3-sulfamoyloxy-17β-O-naphthalenesulfonylestra-1,3,5(10)-

triene (9h): m.p. 113.1–114.6 ^◦C; ¹H NMR (400 MHz, DMSO) δ 8.67 (s,

1H), 8.24 (dd, J = 18.6, 8.4 Hz, 2H), 8.12 (d, J = 8.0 Hz, 1H), 7.90 (d, J

= 8.7 Hz, 1H), 7.83–7.68 (m, 4H), 6.95 (s, 1H), 6.90 (s, 1H), 4.42 (t, J =

8.2 Hz, 1H), 3.71 (s, 3H), 2.68 (d, J = 5.4 Hz, 2H), 2.20 (d, J = 11.9 Hz,

1H), 2.08 (t, J = 10.7 Hz, 1H), 1.90 (s, 1H), 1.77–1.51 (m, 4H), 1.31 (dd,

J = 21.9, 11.2 Hz, 3H), 1.17 (d, J = 7.2 Hz, 2H), 0.99 (t, J = 11.5 Hz,

1H), 0.79 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 149.43, 138.40, 136.71,

134.75, 133.14, 131.55, 129.77, 129.55, 129.52, 129.29, 128.09,

127.95, 127.91, 122.87, 122.39, 110.47, 89.72, 55.83, 55.45, 47.86,

43.51, 42.70, 37.58, 35.28, 28.04, 27.40, 26.37, 25.36, 22.47, 11.55.

HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₉H₃₃NO₇S₂, 594.1698,

found 594.1701. Purity: 97.12%.

2-Methoxy-3-sulfamoyloxy-17β-O-(pyridin-2-yl)estra-1,3,5(10)-triene

(9 k): Method as for 7 l using compound 6 (240 mg, 0.61 mmol), Cs₂CO₃

(299 mg, 0.92 mmol), BINAP (76 mg, 0.12 mmol), 2,4-dichloropyridine

(132 μL, 1.22 mmol) and Pd (OAc)₂(14 mg, 0.06 mmol) in toluene (10

mL) at 100 ^◦C for 6 h. Flash column chromatography (petroleum ether/

ethyl acetate 15:1) afforded compound 7 k (262 mg, 85%). Further

hydrogenation and sulfamoyl reaction according to general procedure

1

◦

give 9 k as a white solid. m. p. 186.2–189.0 C; H NMR (400 MHz,

DMSO) δ 8.13 (s, 1H), 7.81 (s, 2H), 7.67 (t, J = 7.2 Hz, 1H), 6.99 (s, 2H),

6.93 (s, 1H), 6.78 (d, J = 7.7 Hz, 1H), 4.94 (s, 1H), 3.76 (s, 3H), 2.75 (s,

2H), 2.40–2.15 (m, 4H), 1.81 (dd, J = 32.6, 22.0 Hz, 4H), 1.48–1.25 (m,

7H), 0.91 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 163.96, 149.96, 147.34,

139.47, 139.24, 137.22, 128.74, 123.44, 117.22, 111.48, 110.96, 83.39,

56.34, 49.61, 44.44, 43.05, 38.36, 37.23, 28.70, 28.12, 27.15, 26.34,

23.36, 12.58. HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₄H₃₀N₂O₅S,

481.1875, found 481.1878. Purity: 98.27%.

2-Methoxy-17β-O-methylestra-1,3,5(10)-trien-3-ol (8i): NaH (113 mg,

4.71 mmol) was added to the solution of compound 6 (308 mg, 0.785

mmol) in DMF (3 mL) and stirring was continued overnight under the

atmosphere of argon. Then water (15 mL) was added carefully and the

suspension was extracted with ethyl acetate (3 × 15 mL). The combined

organic layers were washed with brine, dried (MgSO₄) and concen-

trated. The residue was puriﬁed by ﬂash chromatography (petroleum

ether/ethyl acetate 35:1) to give 7i as a white powder (303 mg, 95%).

The hydrogenation step was conducted according to general procedure

to afford compound 8i. m. p. 229.8–231.1 ^◦C; ¹H NMR (400 MHz,

DMSO) δ 8.62 (s, 1H), 6.76 (s, 1H), 6.44 (s, 1H), 3.71 (s, 3H), 3.32–3.19

(m, 4H), 2.78–2.53 (m, 2H), 2.28 (d, J = 5.1 Hz, 1H), 2.09 (d, J = 8.6 Hz,

1H), 2.02–1.86 (m, 2H), 1.77 (d, J = 9.2 Hz, 1H), 1.62 (d, J = 7.2 Hz,

1H), 1.46–1.10 (m, 7H), 0.72 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ

145.54, 144.31, 130.21, 128.23, 115.55, 109.69, 89.80, 57.05, 55.75,

49.55, 43.68, 42.76, 38.28, 37.45, 28.35, 27.23, 26.93, 26.21, 22.61,

11.56. Purity: 99.17%.

2-Methoxy-3-sulfamoyloxy-17β-O-(6-chloropyrimidin-4-yl)estra-1,3,5

(10)-triene (9 m): white solid. m. p. 219.2–221.8 ^◦C; ¹H NMR (400 MHz,

DMSO) δ 8.65 (s, 1H), 7.81 (s, 2H), 7.16 (s, 1H), 6.99 (s, 2H), 5.01 (t, J =

8.0 Hz, 1H), 3.76 (s, 3H), 2.75 (d, J = 5.0 Hz, 2H), 2.37 (d, J = 12.3 Hz,

2H), 2.23 (d, J = 10.0 Hz, 1H), 1.89–1.79 (m, 2H), 1.74 (s, 1H),

1.62–1.36 (m, 7H), 0.90 (s, 3H). HRMS (ESI-TOF) m/z [M + Na]⁺calcd

for C₂₃H₂₈ClN₃O₅S, 516.1438, found 516.1438. Purity: 97.14%.

2-Methoxy-17β-dimethyl formyl estra-1,3,5(10)-trien-3-ol (8n): To a

solution of compound 6 (396 mmol, 1.0 mmol) in acetonitrile (12 mL),

was added N, N-dimethylcarbamic chloride (158 μL, 1.72 mmol) and

NaH (242 mg, 10.09 mmol). The mixture was stirred at 100 ^◦C for 10 h

before being evaporated to remove acetonitrile. The residue was

extracted with ethyl acetate (3 × 20 mL) and the combined organic

layers were washed with brine, dried (MgSO₄) and concentrated. Flash

chromatography on silica (petroleum ether/ethyl acetate 3:1) afforded

compound 7n (421 mg,90%) as a white powder. Then general hydro-

genation procedure gives compound 8n. ¹H NMR (400 MHz, CDCl₃) δ

6.78 (s, 1H), 6.64 (s, 1H), 5.62 (s, 1H), 4.62 (t, J = 8.4 Hz, 1H), 3.85 (s,

3H), 2.92 (s, 6H), 2.81–2.72 (m, 2H), 2.23 (dd, J = 15.1, 6.7 Hz, 3H),

1.94–1.83 (m, 2H), 1.72 (d, J = 8.9 Hz, 1H), 1.60–1.24 (m, 7H), 0.85 (s,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 156.77, 147.64, 146.40, 137.50,

132.97, 128.89, 128.48, 127.71, 127.32, 114.75, 109.96, 83.49, 71.16,

56.39, 49.69, 44.16, 42.92, 38.59, 37.01, 36.35, 35.85, 29.16, 28.15,

27.32, 26.44, 23.27, 12.29. Purity: 97.41%.

2,17β-dimethoxy-3-sulfamoyloxyestra-1,3,5(10)-triene

(9i):

This

compound was prepared using general sulfamoyl procedure described

above and the resultant crude solid was puriﬁed by ﬂash chromatog-

raphy (petroleum ether/EtOAc 3:1) to give a white powder (yield 86%).

m. p. 181.3–182.8 ^◦C; ¹H NMR (400 MHz, DMSO) δ 7.81 (s, 2H), 6.98 (s,

2H), 3.76 (s, 3H), 3.31–3.23 (m, 4H), 2.72 (d, J = 5.0 Hz, 2H), 2.34 (s,

1H), 2.18 (s, 1H), 2.05–1.89 (m, 2H), 1.81 (d, J = 11.9 Hz, 1H), 1.63 (d,

J = 7.4 Hz, 1H), 1.44–1.17 (m, 7H), 0.72 (s, 3H). ¹³C NMR (101 MHz,

DMSO) δ 149.46, 138.80, 136.72, 128.22, 122.93, 110.41, 89.73, 57.05,

55.83, 49.55, 43.94, 42.69, 37.84, 37.36, 28.17, 27.21, 26.62, 25.96,

22.60, 11.51. HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₀H₂₉NO₅S,

418.1766, found 418.1769. Purity: 98.94%.

2-Methoxy-3-sulfamoyloxy-17β-dimethylcarbamoylestra-1,3,5(10)-

triene(9n): white solid. m. p. 200.1–202.6 ^◦C; ¹H NMR (400 MHz,

DMSO) δ 7.81 (s, 2H), 6.98 (s, 2H), 4.49 (t, J = 8.1 Hz, 1H), 3.76 (s, 3H),

2.83 (d, J = 16.2 Hz, 6H), 2.74 (d, J = 4.9 Hz, 2H), 2.36 (d, J = 10.3 Hz,

1H), 2.21 (s, 1H), 2.11 (d, J = 8.2 Hz, 1H), 1.81 (t, J = 8.7 Hz, 2H), 1.67

(s, 1H), 1.40–1.22 (m, 7H), 0.81 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ

155.61, 149.45, 138.71, 136.74, 128.24, 122.93, 110.48, 82.55, 55.85,

48.95, 43.84, 42.30, 37.84, 36.40, 35.85, 35.37, 28.18, 27.75, 26.59,

25.74, 22.75, 12.02. HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

2-Methoxy-3-sulfamoyloxy-17β-O-(isoquinolin-3-yl)estra-1,3,5(10)-

triene (9j): m.p.118.1–120.0 ^◦C ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J

= 8.8 Hz, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H),

7.66–7.58 (m, 1H), 7.42–7.35 (m, 1H), 7.08 (s, 1H), 6.96 (s, 1H), 6.92

(d, J = 8.8 Hz, 1H), 5.26 (t, J = 8.4 Hz, 1H), 5.04 (s, 2H), 3.89 (s, 3H),

2.85 (dd, J = 8.7, 3.9 Hz, 2H), 2.65–2.47 (m, 1H), 2.31 (t, J = 8.3 Hz,

2H), 2.05–1.92 (m, 2H), 1.91–1.79 (m, 1H), 1.65–1.34 (m, 7H), 1.00 (s,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 162.33, 148.93, 146.68, 140.58,

138.41, 136.79, 130.29, 129.33, 127.36, 127.21, 124.99, 124.10,

123.76, 113.56, 110.58, 83.51, 56.37, 49.96, 44.52, 43.10, 38.18,

37.28, 28.67, 28.09, 27.08, 26.46, 23.42, 12.27. HRMS (ESI-TOF) m/z

[M + Na]⁺calcd for C₂₈H₃₂N₂O₅S, 531.2032, found 531.2035. Purity:

98.46%.

C

₂₂H₃₂N₂O₆S, 475.1981, found 475.1983. Purity: 98.64%.

2-Methoxy-17β-acetoxyestra-1,3,5(10)-trien-3-ol

(8o):

m.

p.

190.6–192.1 ^◦C; ¹H NMR (400 MHz, CDCl₃) δ 6.81 (s, 1H), 6.67 (s, 1H),

5.49 (s, 1H), 4.72 (dd, J = 9.0, 7.9 Hz, 1H), 3.88 (s, 3H), 2.80 (dd, J =

9.7, 6.7 Hz, 2H), 2.33–2.17 (m, 3H), 2.09 (s, 3H), 1.89 (ddd, J = 7.9, 5.6,

3.0 Hz, 2H), 1.81–1.70 (m, 1H), 1.60–1.27 (m, 7H), 0.86 (s, 3H). ¹³C

NMR (101 MHz, CDCl₃) δ 171.25, 144.61, 143.50, 131.61, 129.48,

114.62, 108.11, 82.73, 56.08, 49.81, 44.11, 42.92, 38.56, 36.95, 28.97,

27.60, 27.30, 26.52, 23.24, 21.20, 12.10. Purity: 98.38%.

2-Methoxy-3-sulfamoyloxy-17β-O-(5-aminopyridin-2-yl)estra-1,3,5

1

(10)-triene (9 l): m.p. 121.2–123.4 ^◦C; H NMR (400 MHz, DMSO) δ

9.10 (s, 1H), 8.57 (s, 1H), 7.96 (d, J = 2.7 Hz, 1H), 7.51 (dd, J = 8.8, 2.8

Hz, 1H), 6.98 (s, 2H), 6.79–6.73 (m, 2H), 6.45 (s, 1H), 4.85 (t, J = 8.1

Hz, 1H), 3.70 (s, 3H), 2.72–2.58 (m, 2H), 2.34–2.23 (m, 2H), 2.15 (t, J =

8.2 Hz, 1H), 1.84–1.76 (m, 2H), 1.71 (s, 1H), 1.37 (dd, J = 17.0, 9.0 Hz,

2-Methoxy-3-sulfamoyloxy-17β-acetoxyestra-1,3,5(10)-triene (9o): m.

p. 214.3–215.1 ^◦C; ¹H NMR (400 MHz, DMSO) δ 7.82 (s, 2H), 6.99 (d, J

= 4.2 Hz, 2H), 4.61 (t, J = 8.3 Hz, 1H), 3.77 (s, 3H), 2.74 (d, J = 4.8 Hz,

14

M. Sun et al.

Bioorganic Chemistry 113 (2021) 104988

2H), 2.36 (d, J = 8.8 Hz, 1H), 2.15 (ddd, J = 14.9, 13.6, 7.1 Hz, 2H),

2.02 (s, 3H), 1.80 (dd, J = 20.3, 12.1 Hz, 2H), 1.73–1.62 (m, 1H),

1.47–1.24 (m, 7H), 0.80 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 170.79,

149.96, 139.15, 137.25, 128.71, 123.43, 110.95, 82.34, 56.34, 49.54,

44.29, 42.87, 38.28, 36.89, 28.66, 27.71, 27.10, 26.22, 23.24, 21.34,

12.39. HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₁H₂₉NO₆S,

446.1716, found 446.1715. Purity: 99.04%.

NMR (101 MHz, DMSO) δ 159.05, 156.69, 146.04, 144.80, 136.90,

131.52, 131.08, 130.68, 128.75, 116.06, 110.74, 110.20, 83.52, 56.23,

49.64, 44.19, 43.10, 38.80, 37.35, 28.88, 28.18, 27.47, 26.59, 23.36,

12.59. HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₅H₃₁N₃O₄,

460.2315, found 460.2317. Purity: 98.64%.

2-Methoxy-3-carbamoyl-17β-dimethylcarbamoylestra-1,3,5(10)-triene

(11n): m.p.237.6–238.8 ^◦C; ¹H NMR (400 MHz, DMSO) δ 6.87 (s, 1H),

6.64 (s, 1H), 4.49 (t, J = 8.3 Hz, 1H), 3.70 (s, 3H), 2.84 (d, J = 18.3 Hz,

6H), 2.76–2.65 (m, 2H), 2.40–2.28 (m, 1H), 2.21 (t, J = 8.6 Hz, 1H),

2.16–2.04 (m, 1H), 1.81 (d, J = 11.0 Hz, 2H), 1.71–1.60 (m, 1H),

1.51–1.24 (m, 7H), 0.82 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 156.12,

146.05, 144.94, 130.51, 128.73, 116.08, 110.28, 99.99, 83.10, 79.69,

56.28, 49.46, 44.09, 42.86, 38.77, 36.97, 36.36, 28.87, 28.24, 27.41,

26.47, 23.25, 12.57. HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

2-Methoxy-3-sulfamoyloxy-17β-carbamoylestra-1,3,5(10)-triene (9p):

This compound was prepared using the method of Potter et al [17]. m. p.

184.9–189.2^◦C; ¹H NMR (400 MHz, DMSO) δ 7.82 (s, 2H), 6.98 (s, 2H),

6.41 (d, J = 21.4 Hz, 2H), 4.47 (t, J = 8.3 Hz, 1H), 3.76 (s, 3H), 2.73 (s,

2H), 2.36 (d, J = 8.6 Hz, 1H), 2.20 (s, 1H), 2.07 (d, J = 7.9 Hz, 1H), 1.81

(t, J = 9.4 Hz, 2H), 1.65 (s, 1H), 1.52–1.20 (m, 7H), 0.78 (s, 3H). ¹³

C

NMR (101 MHz, DMSO) δ 156.78, 149.45, 138.75, 136.71, 128.22,

122.92, 110.45, 81.23, 55.84, 49.14, 43.86, 42.24, 37.84, 36.50, 28.17,

27.44, 26.59, 25.77, 22.65, 11.89. HRMS (ESI-TOF) m/z [M + Na]⁺

calcd for C₂₀H₂₈N₂O₆S, 447.1668, found 447.1667. Purity: 98.25%.

C₂₃H₃₂N₂O₅, 439.2311, found 439.2310. Purity: 97.39%.

Declaration of Competing Interest

4.2.4. General procedure for preparation of carbamates (compounds 11).

Trichloroacetyl isocyanate (0.21 mL, 1.7 mmol) was added to a so-

lution of corresponding compound 8 (0.87 mmol) in anhydrous THF.

Stirring was continued for 10 h at room temperature prior to water was

added. The product was extracted with ethyl acetate (3 × 15 mL) and the

combined organic layers were washed with brine, dried (MgSO₄) and

concentrated to give the desired crude product 10, which was used for

the next step directly.

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

Acknowledgements

This work was supported by National Natural Science Foundation of

China (Project No.81903623 and 22007086), Henan science and tech-

nology key project (No. 202102310148) and Henan Medical Science and

Technology Program (No. 2018020601).

A solution of K₂CO₃(516 mg, 3.73 mmol) in water (12 mL) was

added to a solution of above crude product in THF (12 mL) and MeOH

(12 mL). The mixture was stirred for 3 h at room temperature and at this

time a large amount of white solid is precipitated. Filtered and obtained

the target product 11 with the yield greater than 85% over two steps.

2-Methoxy-3-carbamoyl-17β-O-methanesulfonylestra-1,3,5(10)-triene

(11a): m.p.206.2–208.1 ^◦C; ¹H NMR (400 MHz, DMSO) δ 6.89 (s, 1H),

6.66 (s, 1H), 4.55 (t, J = 8.3 Hz, 1H), 3.72 (s, 3H), 3.18 (s, 3H),

2.78–2.65 (m, 2H), 2.39 (s, 1H), 2.27–2.13 (m, 2H), 1.95–1.87 (m, 1H),

1.82–1.70 (m, 3H), 1.43 (p, J = 11.6 Hz, 4H), 1.29 (dd, J = 15.1, 7.5 Hz,

2H), 0.82 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 146.09, 145.04, 130.31,

128.71, 116.09, 110.35, 89.25, 79.66, 56.32, 48.65, 43.97, 43.25,

38.68, 37.92, 36.09, 28.80, 27.94, 27.30, 26.26, 23.05, 12.09. HRMS

(ESI-TOF) m/z [M + Na]⁺calcd for C₂₁H₂₉NO₆S, 446.1716, found

446.1719. Purity: 97.69%.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bioorg.2021.104988.

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