- Email: [email protected]
Design, synthesis and characterization of potent microtubule inhibitors with dual anti-proliferative and anti-angiogenic activities
Accepted Manuscript Design, synthesis and characterization of potent microtubule inhibitors with dual antiproliferative and anti-angiogenic activities Huijun Zhang, Xiong Fang, Qian Meng, Yujia Mao, Yan Xu, Tingting Fan, Jing An, Ziwei Huang PII:
S0223-5234(18)30599-3
DOI:
10.1016/j.ejmech.2018.07.043
Reference:
EJMECH 10578
To appear in:
European Journal of Medicinal Chemistry
Received Date: 16 April 2018 Revised Date:
30 June 2018
Accepted Date: 16 July 2018
Please cite this article as: H. Zhang, X. Fang, Q. Meng, Y. Mao, Y. Xu, T. Fan, J. An, Z. Huang, Design, synthesis and characterization of potent microtubule inhibitors with dual anti-proliferative and anti-angiogenic activities, European Journal of Medicinal Chemistry (2018), doi: 10.1016/ j.ejmech.2018.07.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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microtubule inhibitors with dual
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Design, synthesis and characterization of potent
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anti-proliferative and anti-angiogenic activities Huijun Zhang1, Xiong Fang1,2, Qian Meng1, Yujia Mao1, Yan Xu1, Tingting Fan1,
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Jing An3,4,*, Ziwei Huang1,3,* 1
School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China
2
Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084, P.
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R. China
Department of Medicine, University of California San Diego, La Jolla, CA 92093
4
Nobel Institute of Biomedicine, Zhuhai, 519080, P. R. China
Corresponding authors:
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*
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Dr. Ziwei Huang. Email: [email protected]. Phone: 0086-10-6279-6890.
Dr. Jing An. Email: [email protected]. Phone: 858-822-0333.
Keywords: Fused 4-aryl-4H-chromenes; microtubule inhibitors; anti-proliferation; anti-angiogenesis; drug development 1
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ABSTRACT Microtubule has been an important target for anticancer drug development. Here we
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report the discovery and characterization of a series of fused 4-aryl-4H-chromene-based derivatives as highly potent microtubule inhibitors. Among a total of 37 derivatives synthesized, 23 exhibited strong in vitro anti-proliferative activities against A375 human
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melanoma cells. The relationship between the biological activities of these microtubule inhibitors and their chemical structure variations was analyzed. Studies of compounds
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27a, 19a and 9a in parallel with colchicine as the positive control compound in a panel of biological assays revealed that these compounds blocked cell cycle progression, increased apoptosis, and inhibited HUVEC capillary tube formation at low nanomolar concentrations. The most potent compound 27a was also tested in eight additional cancer
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cell lines besides A375 cells and two non-cancer cells and showed potent and selective activity on these cancer cells. To understand the molecular and structure mechanism of action of these compounds, tubulin polymerization and molecular docking studies were
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carried out for 27a as the representative. The results were consistent with the mechanism by which 27a interacts with the colchicine binding site on tubulin and disrupts tubulin
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polymerization. With potent dual actions of microtubule destabilization and vascular disruption described above, this small molecule can serve as a valuable research probe of the function and role of microtubules in human diseases and promising lead for developing new therapeutic agents.
1. INTRODUCTION
Cancer, one of the major causes of death worldwide [1], is characterized by abnormal 2
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cell proliferation and inappropriate cell survival. One approach to prevent this proliferation to promote cancer cell death is to use chemotherapeutic drugs that target microtubules [2, 3]. The well-organized function and regulated dynamics of microtubules
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contribute to the structure of the cytoskeleton, mediation of intracellular transport [4, 5], cell division, signaling networks, and many other biological processes [6, 7], making microtubules successful and efficacious cellular targets for the development of traditional
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chemotherapeutic agents [8-11]. For example, taxanes [12, 13] and vinca alkaloids [14, 15] have been used for the treatment of leukemia and solid tumors, and additional
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microtubule-targeting agents are still being pursued intensively [16-19].
Colchicine, extracted from the meadow saffron colchicium autumnale, is one of the best known microtubule-destabilizing agents [20]. Colchicine binding-site inhibitors (CBSI) are also potent destroyers of tumor vasculature [6, 20-27]. They bind to tubulins
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in endothelial cells of tumor vessels, disrupt cytoskeleton, cell shape, and cellular connections and eventually interrupt of the blood flow to the tumor [28]. Also they can selectively affect tumor neovasculature, rather than normal tissues [28-32]. Several small
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molecule vascular disrupting agents (VDAs), including CA-4P, Oxi4503, AVE8062, and EPC2407, are currently undergoing clinical trials [32, 33]. Additional extensive efforts on
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the design and discovery of novel VDAs would further expedite the development of these drugs that target microtubule and show special advantages over other cancer therapies [34-39].
One of the drug development approaches has been based on the use of 4-aryl-4H-chromenes, which was emerged from the studies of natural products and synthetic small molecules. Their broad pharmacological properties have made them 3
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medically privileged scaffolds [40-43]. Extensive research indicates that they exhibit strong antitumor activities through multiple pathways, including the inhibition of
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microtubule polymerization, disruption of tumor vasculature, and antagonism of the Wnt pathway. One representative compound, EPC2407, which has entered into Phase II clinical trials as a vascular-disrupting agent, has also been identified as a tubulin inhibitor
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binding to the colchicine-binding site [44-46]. Similarly, the 4H-chromene analog 1,
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featuring a sesamol-derived ring and resembling closely the structure of podophyllotoxin (PT), exhibited strong antimitotic microtubule destabilizing activity (Figure 1) [47]. Subsequent studies reported that substitution of 7-methoxy, 2-NH2, and 3-CN groups for the methylenedioxy ring A and lactone D, respectively, in oxa-PTs yielded
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7-methoxy-4-aryl-4H-chromene 2 that showed markedly improved cytotoxicity against 60 human cancer cell lines [48]. Similarly, 5-pyrido 4H-chromene analog 3 displayed Wnt pathway inhibitory activity (IC50 = 6 nM) and potent anti-proliferative activity
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against HCT116 human colon carcinoma cells (IC50 = 15 nM) [49, 50]. Furthermore,
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virtual screening and SAR-guided modification has identified 4-heteroaryl-4H-chromene (HFI437) as a first generation small molecule inhibitor of insulin-regulated amino-peptidase (IRAP) [51]. Overall, these highly functionalized 4H-chromenes have shown diverse bioactivities. Most of the 4-aryl-4H-chromene-based derivatives have great advantages for clinical applications as they possess dual biological activities (i.e., tubulin inhibition and vascular disruption). Furthermore, these molecules can overcome
4
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drug resistance by binding directly to the colchicine binding site on tubulin and selectively target established tumor vasculature [35, 37, 41].
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Our laboratory has previously developed mHA11, a 4-aryl-4H-chromene derivative that inhibits tubulin by binding to the colchicine-binding site [52]. mHA11 was derived
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by modification of HA14-1, a small molecule originally reported by us as an inhibitor of both tubulin and Bcl-2 [52, 53]. The reported potent bioactivities of 4-aryl-4H-chromenes
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with dual actions as tubulin-binding VDAs prompted us to investigate whether novel and potent chromene-based VDAs could be developed by modifying the structure of mHA11. To our knowledge, a number of structure-activity relationship (SAR) studies have established that the polyalkoxybenzene moiety of the 4H-chromenes is a crucial
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pharmacophore for mimicking colchicine and podophyllotoxin, and that small hydrophobic groups, such as N(CH3)2, -OCH3, and -NH2 at the 7- or 8-positions or 2-NH2 and 3-CN, generate compounds that inhibit cancer cell growth. In addition, fusion of the
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pyrrole ring at the 7,8-position also increases the potency [44]. Fusion of rings at the
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6,7-position has not yet been explored in the design of new classes of fused 4-aryl-4H-chromene-based derivatives. Thus, we designed different five-member or six-member rings fused at the 6,7- or 7,8-positions of mHA11 to investigate their activities (Figure 2). We fused a 1,3-dioxolane at the 6,7-position, following the introduction of an -OH group at the C-8 position. We also prolonged the fused
5
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4H-chromenes by designing two classes of linear and angular 4-aryl-4H-chromene
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derivatives by incorporating some different groups into the fused imidazole ring.
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Figure 1. 4-aryl-4H-chromene-based derivatives and colchicine binding site inhibitors
Figure 2. Structural modifications of the lead compound mHA11 2. RESULTS
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2.1
Chemistry
Fused 4-aryl-4H-chromene derivatives were prepared by a one-pot three-component
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cascade reaction of starting materials 4 (different substituted benzaldehydes), ethyl cyanoacetate or malononitrile, and synthesized intermediates 5 (different fused phenol
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analogs). The synthetic route was described in Scheme 1. The three-component reaction was carried out by three steps in the presence of piperidine at room temperature (rt) or 80
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℃. The Knoevenagel condensation of aldehydes and active methylene compounds (ethyl cyanoacetate or malononitrile) gave the first intermediate (benzylidenemalononitrile). The ortho-position of the phenols with electron-rich density then underwent Michael addition to form the next intermediate. Subsequently, the phenolic hydroxyl group
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participated in an intramolecular attack at the nitrile group and underwent tautomerization (namely, hetero-Thorpe-Ziegler reaction) [47].
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Scheme 1. Synthesis of 6,7-fused or 7,8-fused-4-aryl-4H-chromene derivatives via a
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three-component cascade reaction.
R1
R3
+
NC
O or
CHO 4
O
CN
CN
+
B n A
o OH piperidine, EtOH, N2, rt or 80 C
R4 or
5
R3
OH n
R3
R1
B 6 n A 7
R1
X
X 7 8 R4
C
R2
R2
1. Knoevenagel reaction 2. Michael reaction 3. hetero-Thorpe-Ziegler reaction
R2
O
NH2
C
8 D n X = -COOCH2CH3, -CN
O
NH2
6,7-fused or 7,8-fused-4-aryl-4H-chromene derivatives
D
The synthetic approaches of different fused phenol analogs were summarized in Scheme 2. Indolin-6-ol 9 was prepared according to a modified Leimgruber-Batcho indole 7
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synthesis [54]. Commercially available 4-methyl-3-nitrophenol was protected with a benzyl group to provide the intermediate 7, followed by condensation reaction of 7 with
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DMF-DMA and pyrrolidine, reductive cyclization and deprotection to obtain 6-hydroxyindole 8 in one step using 10% Pd-C and H2, and then reduction of indole ring in the presence of NaBH(CN)3 and AcOH to yield the desired 9 (Scheme 2a).
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Simultaneously, following the same reductive condition, the indolin-4-ol 11 was obtained
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by using the purchased 10 as the starting material (Scheme 2b). Amide reduction of 12 in the presence of LiAlH4 gave 1,2,3,4-tetrahydroquinolin-7-ol 13 (Scheme 2c), and 5-hydroxylquinoline
14
was
hydrogenated
with
PtO2
to
yield
1,2,3,4-tetrahydroquinolin-5-ol 15 under acidic conditions (Scheme 2d).
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Next, sesamol (16) was treated with MOMCl to give the MOM-protected intermediate 17, followed by the regioselective lithiation of 17 with n-BuLi, boration with B(OMe)3, and oxidation with H2O2 in a one-pot synthesis to afford the intermediate 18 according to
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the reported method [55]. The protection of the MOM group afforded the sesamol analog
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19 under acidic condition (Scheme 2e). Another sesamol analog 22 was prepared by treating the 3,4-dihydroxybenzaldehyde 20 with 1,2-dibromoethane in the presence of K2CO3 to yield the intermediate 21 which was further oxidized with mCPBA (Scheme 2f). Replacement of one oxygen from the dioxane ring of 22 with nitrogen gave the fused phenol analog 27, which was synthesized with the route described in Scheme 2g. According to the reference method, the starting material was 4-methoxy-2-nitrophenol 8
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23, which was reduced to 2-amino-4-methoxyphenol 24 using Fe powder as the reductant, followed by ring closure of 24 with 2-chloroacetyl chloride to yield 25 under
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alkaline conditions [56]. Reduction of 25 then afforded the intermediate 26, which was further treated with BBr3 to remove the methyl group to obtain 27 in the final step.
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Eventually, as shown in Scheme 1, these fused phenol analogs (9, 11, 13, 15, 19, 22, 27) were converted to the corresponding desired compounds 9a-i, 11a-c, 13a-c, 15a-b,
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19a-b, 22a, and 27a-b, respectively, following the general procedure for the one-pot three-component cascade reaction. These synthesized compounds are summarized in
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Table 1.
9
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2.
Synthetic
route
for
fused
phenol
analogs.
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Scheme
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Reagents and Conditions: (a) BnBr, K2CO3, DMF, rt, 16 h; (b) (i) DMF-DMA, pyrrolidine, 120℃, 3 h; (ii) 10% Pd-C, H2, THF; (c) NaBH(CN)3, AcOH, 0℃-rt, 3 h; (d)
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LiAlH4, THF, 65℃, 16 h; (e) PtO2, H2, conc. HCl, EtOH/MeOH, rt, 26 h; (f) MOMCl, NaH, THF, 0℃-rt, 16 h; (g) n-BuLi, B(OMe)3, THF, 0℃-rt, 0.5 h-1 h, and then AcOH, H2O2, rt, 5 h; (h) 2 N HCl, MeOH, rt, 12 h; (i) BrCH2CH2Br, K2CO3, acetone, 60℃, 36 h; (j) (i) mCPBA, CH2Cl2, 40℃, 12 h; (ii) NaOH, MeOH, rt, 15 min; (k) Fe powder, NH4Cl, MeOH/THF/H2O(2:1:1), 80℃, 4 h; (l) 2-chloroacetyl chloride, K2CO3, ACN, 80℃, 2 h; (m) LiAlH4, THF, rt, 16 h; (n) BBr3, CH2Cl2, -78℃-rt, 2 h. 10
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The preparation of linearly and angularly fused 4-aryl-4H-chromene derivatives presented
in
Scheme
3
began
with
4-amino-3-nitrophenol
(28)
and
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2-amino-3-nitrophenol (32) as the starting materials, respectively. These were reduced by Raney-Ni to yield the intermediates 29 (or 33), which were further transformed to the compounds 30a-30b (or 34a-34b) through the above mentioned three-component cascade
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reaction. 30a was treated with triethyl orthoformate or triethyl orthoacetate to afford the
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desired compounds 31a-31b. When 30a (or 30b, 34a) reacted with BrCN to obtain compounds 31c (or 31d, 35a), subsequent acylation of 31c (or 35a) provided compounds 31e (or 35b) in the presence of cyclopropanecarbonyl chloride. 30a (or 34b) was then reacted with different substituted benzaldehydes to afford compounds 31f-31h (or 35c)
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utilizing Na2S2O5 as catalytic reagent.
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Scheme 3. Synthetic route for linearly and angularly fused 4-aryl-4H-chromene
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derivatives.
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Reagents and Conditions: (a) Raney-Ni, H2, THF, rt, 5 h; (b) Piperidine, EtOH, N2, 80℃, 16 h; (c) 31a or 31b: triethyl orthoformate or triethyl orthoacetate, ZrCl4, MeOH, rt, 3 h; 31c
and
31d:
BrCN,
MeOH/THF,
rt,
16
h;
31f-31h
and
35c:
5-methylthiophene-2-carboxaldehyde/3,4,5-trimethoxybenzaldehyde/3-bromo-4,5-dimeth oxybenzaldehyde/3-fluorobenzaldehyde, Na2S2O5, DMF, 100℃, 6 h; (e) 31e and 35b: Cyclopropanecarbonyl chloride, DMAP, pyridine, rt, 16 h. 12
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1.
Chemical
structures
of
synthesized
compounds.
R2
R3
H
H
Cl
9b
H
H
Cl
9c
H
H
9d
H
H
9e
H
9g
9h
B-n-A
C-n-D
(6,7-)
(7,8-)
H
CO2C2H5
C-1-NH
-
H
CN
C-1-NH
-
Br
H
CN
C-1-NH
-
F
H
CN
C-1-NH
-
OCH3
H
CN
C-1-NH
-
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9f
X
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9a
R4
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Compd R1
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Table
OCH3
OCH3
Br
H
CN
C-1-NH
-
OCH3
OCH3
Br
H
CO2C2H5
C-1-NH
-
OCH3
OCH3
OCH3
H
CN
C-1-NH
-
13
F
F
F
H
CN
C-1-NH
-
11a
H
H
Cl
-
CO2C2H5
-
NH-1-C
11b
OCH3
OCH3
Br
-
CO2C2H5
-
11c
OCH3
OCH3
Br
-
CN
-
13a
H
H
Cl
H
CO2C2H5
C-2-NH
-
13b
OCH3
OCH3
Br
H
CO2C2H5
C-2-NH
-
13c
OCH3
OCH3
Br
H
CN
C-2-NH
-
15a
H
H
Cl
-
CO2C2H5
-
NH-2-C
15b
OCH3
OCH3
Br
-
CN
-
NH-2-C
16a
H
H
Cl
H
CO2C2H5
O-1-O
-
16b
OCH3
OCH3
H
CO2C2H5
O-1-O
-
NH-1-C
NH-1-C
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OCH3
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9i
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16c
OCH3
OCH3
OCH3
H
CN
O-1-O
-
16d
OCH3
OCH3
Br
H
CN
O-1-O
-
19a
OCH3
OCH3
Br
OH
CN
O-1-O
-
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OCH3
OCH3
OCH3
OH
CN
O-1-O
-
22a
OCH3
OCH3
Br
H
CN
O-2-O
-
27a
OCH3
OCH3
Br
H
CN
O-2-NH
-
27b
OCH3
OCH3
OCH3
H
CN
O-2-NH
-
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2.2 Biological evaluations
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19b
2.2.1 In vitro anti-proliferative activities and SAR analysis
All synthesized compounds, including the positive control compounds mHA11,
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EPC2407, colchicine, and paclitaxel, were evaluated for their anti-proliferative activities against human melanoma A375 cells, after the incubation with gradient concentrations of
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compounds for 72 h. Cell viability was measured with the CellTiter 96® AQueous One Solution Cell Proliferation Assay（MTS）kit (Promega) according to the manufacturer’s
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instructions. The results presented in Table 2 indicated that 27a, 27b and 19a displayed stronger anti-proliferative activities in A375 cells than colchicine (or paclitaxel), with IC50 values of 16 pM and 369 pM for 27a and 27b respectively. Comparison with mHA11 (IC50 value of 902.3 nM) revealed that the 6,7-fused 4-aryl-4H-chromene derivatives (9a, 9g, 13a, 16a) had approximately 2-3-fold higher anti-proliferative activities except for 13b. On the contrary, 7,8-fused 4-aryl-4H-chromene derivatives 15
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(11a-b, 15a) lost their anti-proliferative activities (IC50＞10 µM). Likewise, replacement of the 4-(3-chlorophenyl) group of 16a with a 3,4,5-trimethoxyphenyl group led to the
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loss of activity of derivative 16b. The most potent compound 27a as described above was further evaluated on eight additional cancer cell lines (CHL-1, B16-F10, HeLa，A549, HT-29, HCT116, SW480, Jurkat) and two non-cancer cells (HUVECs and PBMCs). As a
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positive control, colchicine was included in these assays. As shown in Table 3, compound
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27a exhibited potent biological activities on these cancer cell lines. In contrast, compound 27a displayed no cytotoxic effects on normal, non-cancer HUVECs and PBMCs, suggesting the specificity of the effects of compound 27a for cancer cells.
Fusion of a five-member dihydropyrrole at the C6,7-positions and replacement of
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C3-carboxylate with carbonitrile group, or transformation of chlorine on the 4-aryl to bromine yielded three derivatives 9b-c that showed anti-proliferative activities similar to 9a. The 9d with 3-fluorophenyl was about 10-fold better than 9b, and subsequent
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introduction of polymethoxy substituents onto the 4-aryl ring (compounds 9f and 9h)
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gave a further slight improvement. Fusion of a five-member dihydropyrrole fused at the C7,8-positions, resulting in 11c, reduced the activities by more than 3-fold when compared to 9f. Ring expansion of dihydropyrrole to form tetrahydropyridine (15b) also decreased the activity by 2-fold compared with 11c. Taking together, the data indicated that the scaffolds containing the 6,7-fused 4-aryl-4H-chromene were superior to the 7,8-fused 4-aryl-4H-chromene scaffolds. 16
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We then explored the effect of different 6,7-fused rings on anti-proliferative activity. Given that 1,3-dioxolane is a commonly fused ring found in natural products, such as
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podophyllotoxin derivatives, we also synthesized the two compounds 16c-d with fused 1,3-dioxolane and found their activities were similar to that of 9f or 9h. Nevertheless, introduction of the –OH group at the C-8 position gave compound 19a with a
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3-bromo-4,5-dimethoxyphenyl group that was more active than 19b and exhibited a
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nearly 8-fold increase compared with 16d. Therefore, the introduction of small polar groups (-OH, -NH2) was favorable for increasing the potency of 6,7-fused 4-aryl-4H-chromenes.
We next made a comparison between the fusion of five-member versus six-member
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rings at the 6,7-position. 16d with a fused 1,3-dioxolane was more potent than 22a with 1,4-dioxane. Conversely, 9f with fused tetrahydropyridine was 2-fold less potent than 13c with fused dihydropyrrole. Most interestingly, when the morpholine ring fused at the
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6,7-position, compound 27a was 750-fold more active than 13c and 18-fold more active
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than 27b. Introduction of O,N-containing hetero ring fused at the 6,7-position therefore greatly increased the potency.
Finally, we tested the linearly and angularly fused 4-aryl-4H-chromene derivatives (31a-h and 35a-c). Fusion of a 1H-imidazole ring at the 6,7-position of compound 31a resulted in a 18-fold reduction in anti-proliferative activity compared with 9h. Introduction of different sized groups at the 2-position of the 1H-imidazole ring, such as 17
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methyl (31b), amino (31c-d and 35a), cyclopropanecarboxamide (31e and 35b), 5-methylthiophen-2-yl (31f), or substituted phenyl groups (31g-h and 35c), revealed that
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three compounds (31b, 31d and 35a-b) with small substituents retained a certain degree of activity (IC50<1 µM). The rest of the compounds with large substituents completely lost their anti-proliferative activities. These results suggested that fusion of unsaturated
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heterocycle at the 6,7-position or 7,8-position might be detrimental for activity, and an
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increased steric bulkiness at these two positions results in a great decrease in potency. Table 2. In vitro anti-proliferative activitya of the synthesized compounds against human A375 melanoma cells.
b
(nM)
Compd
IC50±
b
(nM)
IC50±
9a
268.3±18.01
16c
32.7±13.20
9b
332.0±91.95
16d
23.3±11.55
19a
3.0
34.5±7.48
19b
16.7±5.77
ND
22a
38.6±19.63
28.0±22.91
27a
0.016±0.005
9d
9e
9f
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233.7±178.27
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9c
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Compd
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546.7±213.62
27b
0.369±0.301
9h
22.0±12.29
31a
382.0±92.26
9i
ND
31b
11a
＞10000
31c
11b
＞10000
31d
11c
70.0±40.00
31e
13a
441.0±431.57
13b
787.7±349.54
13c
12.3±14.57
31h
＞10000
15a
＞10000
35a
103.3±47.26
35b
110.0±75.50
1345.3±1104.92
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＞10000
676.7±265.02
＞10000
31f
＞10000
31g
＞10000
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EP
150.0±77.95
AC C
15b
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9g
16a
330.0±50.21
35c
5113.3±2172.15
16b
＞10000
mHA11
902.3±230.73
colchicine
9.7±2.89
EPC2407(racemate) 27.7
19
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paclitaxel a
47.8±0.06
Anti-proliferative activity was determined using a MTS assay. bIC50 values are presented
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as compound concentration required to inhibit A375 cells proliferation by 50%. Data were analyzed in Microsoft Excel and plotted in GraphPad Prism 5, and expressed as
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means with standard deviations from at least three independent experiments. ND, not detected.
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Table 3. In vitro anti-proliferative activity of compound 27a and colchicine against eight cancer cell lines (CHL-1, B16-F10, HeLa, A549, HT-29, HCT116, SW480, Jurkat) and two non-cancer cells (HUVECs and PBMCs).
IC50±SD (nM)
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Cell lines
Colchicine
3.98±0.60
3.60±0.95
around 32
around 32
8.6±1.23
9.5±1.63
A549
31.5±13.80
62.7±14.55
HT-29
11.6±0.44
11.5±1.52
HCT116
11.7±0.65
13.9±0.83
SW480
15.3±1.43
14.9±2.37
Jurkat
10.7±3.73
19.8±13.60
CHL-1 B16-F10
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HeLa
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27a
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HUVEC
>10000
>10000
PBMCs
>10000
>10000
Induction of G2-M arrest and cell apoptosis of A375 cells
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2.2.2
Cell cycle assays were conducted to verify whether the newly synthesized compounds
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could arrest mitosis of A375 cells. Cell cycle progression was followed by PI staining and flow cytometry analysis. A375 cells were incubated with different concentrations of the
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selected compounds for 24 h. The untreated cells showed a normal cell cycle pattern while all the compound treated groups showed a dose-dependent inhibition of cell cycle similar to that of colchicine control (Figure 3). Compounds 27a, 19a, and 9f all caused
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obvious G2-M arrest when supplied at 50 nM.
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Figure 3. Analysis of cell cycle. (A) Representative results of effects of compounds 27a, 19a and 9f on cell cycle progression of A375 cells, measured by flow cytometry analysis. A375 cells were treated with different concentrations of compounds 27a, 19a and 9f for 24 h. The percentage of cells in each cycle phase is indicated. (B) Graph summarizing the cell cycle distribution affected by compounds 27a, 19a, and 9f of at least three independent experiments. 22
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We then tested whether these active compounds could induce apoptosis in cancer cells by exposing A375 cells to 10, 100 and 1000 nM of 27a, 19a, and 9f for 24 h and 48 h. All samples were stained with annexin V-FITC and PI for analysis. This method divided cells
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into four groups: live populations (annexin V-/PI-), early apoptotic populations (annexin V+/PI-), late apoptotic populations (annexin V+/PI+), and necrotic populations (annexin V+/PI+). The early apoptotic and late apoptotic cells were regarded as the apoptotic
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population. Results in Figure 4 indicated that apoptosis was increased after treatment with 27a, 19a, and 9f but not with the vehicle control, and the extent of apoptosis was
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concentration dependent. At 24 h, a concentration of 1000 nM of 27a induced apoptosis in 19.8% of the cells, whereas at 48 h it induced apoptosis in 32.8% of the cells.
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Treatment with 19a and 9f also gave similar results in A375 cells.
23
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Figure 4. Analysis of cell apoptosis. A375 cells (106/well) were seeded in a six-well plate and incubated overnight. Different concentrations (10, 100, 1000 nM) of 27a, 19a, and 9f and colchicine were added and the cells were incubated for 24 h and 48 h. Samples of 1×106 cells were then collected by centrifugation, resuspended in 500 µL of 1×binding 24
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buffer, stained with annexin V-FITC and PI for 5 min protecting from light, and analyzed by flow cytometry. (A) 24 h. (B) 48 h. The apoptotic group was indicated in the A+/PIand A+/PI+ regions. The percentage of cell populations was calculated from at least two
Immunofluorescence staining of interfered microtubule
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2.2.3
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independent experiments.
We examined whether our compounds could directly disrupt the distribution of
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microtubules by immunofluorescence staining of tubulin in A375 cells. After treatment with different concentrations of 27a, 19a, and 9f for 24 hours, the cells changed from a long-spindle shape to a shrunken round or multi angular shape. Staining results (Figure 5) showed that untreated cells had a normal microtubule distribution whereas the candidates
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treated cells displayed a disorganized distribution of tubulin and had a coenocytic appearance, which may be arisen from incomplete cell division. These observations
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confirmed that 27a, 19a, and 9f could interfere with microtubule organization and retard
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mitosis in A375 cells.
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Figure 5. Effects of 27a, 19a and 9f on cellular microtubule distribution. Green represents tubulins and blue represents nuclei. Images (magnification: 630X) were obtained using Olympus cellSens microscope. Anti-colonogenic activities
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2.2.4
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We further conducted colony formation assays to determine if our candidate compounds could inhibit the clone-forming ability of tumor cells. Figure 6 indicated that 27a, 19a, and 9f at a concentration of 10 nM could inhibit colony formation by A375 cells.
Three
compounds
displayed
26
similar
inhibitory
effects.
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Figure 6. Inhibition effects on A375 cells colony formation. A375 cells were seeded in a 60 mm culture dish and left for several days to form colonies. Cultures were photographed after staining with 0.1% crystal violet. (A) Untreated control. (B) Representative results of colony formation. (C) Statistic summary of three independent
2.2.5
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experiments. Colony numbers were counted by ImageJ. Anti-vascular activities
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Numerous studies indicate that many microtubule-binding agents can disrupt vascular activities [21]. These include flavonoids, chalcones, and many other tubulin inhibitors
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that inhibit angiogenesis through different mechanisms [22]. We tested whether our synthesized compounds could also inhibit vascular formation by seeding human umbilical vein endothelial cells (HUVECs) on solidified matrigel and allowing the cells form tube structures for 6-8 h. The control groups showed an integral net of tubes whereas the compounds treated cells failed to develop whole tubes when the compounds were given at low nanomolar concentration (Figure 7). These responses were also 27
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dose-dependent. These results suggest that mHA series compounds have potent anti-angiogenic effects. To exclude cytotoxic effects of the compounds on HUVECs, cell viability at 24 h was
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determined with the CellTiter 96® AQueous One Solution Cell Proliferation Assay（MTS） kit (Promega). At concentration below 100 nM, none of them showed noticeable toxicity,
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27a had the weakest toxic among our three compounds (Figure 7D).
Figure 7. Inhibition of in vitro vascular tube formation. HUVECs (104/well) were seeded on matrigel with 1, 10 or 100 nM 27a, 19a and 9f (B) or without drugs (vehicle control) (A) in a 96-well plate. Images were captured after 6-8 h at 37°C (magnification: 100X). (C) Measurements of total lengths of tubular structures using ImageJ from three 28
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independent experiments.
(D) Cytotoxic evaluations. Results are presented as mean ±
SD of three experiments.
Inhibition of tubulin assembly
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2.2.6
The tubulin assembly assay is a good indicator of direct interruption with tubulin. In
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order to investigate the inhibitory effects of 27a on tubulin polymerization, >97% pure tubulin was treated with 27a and control compounds for 2h at 37°C (Figure 8). The
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contents of polymerized microtubules were monitored by measuring the absorbance at 340 nm every 60 sec for 2h. Compound 27a at 10 µM exhibited significant inhibitory effects on microtubule polymerization, as did colchicine, which was consistent with other
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published reports [57-59].
Figure 8.
Effects of 27a on tubulin polymerization. Tubulin was incubated alone
(control) and with each of the following compounds: 10 µM paclitaxel , 10 µM
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colchicine, and 10 µM 27a in fresh general tubulin buffer. Polymerization was measured by absorbance at 340 nm in kinetic mode (Enspire Microplate Reader). Each point
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represents the mean of three independent experiments. 2.3 Molecular docking studies
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To understand the structural basis for the mechanism of action of the above described compounds, molecular docking simulations were performed to predict the binding modes
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of two representative compounds 27a and 19a. As shown in Figure 9A, the re-docking validation step indicated that the co-crystallized ligand DAMA-colchicine was redocked into the colchicine-binding site with small RMSD of 0.4482 Å (energy score (S)= -9.7569 kcal/mol). Taking into consideration that compounds 19a and 27a possess R- and
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S-isomers, we carried out molecular docking studies of (R)-19a and (S)-19a, and (R)-27a and (S)-27a, respectively. The docking studies exhibited that (R)-19a and (R)-27a had similar binding mode with DAMA-colchicine in the colchicine-binding site, and
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superimpose well with (R)-EPC2407 (Figure 9B-C). The 3-bromo-4,5-dimethoxyphenyl
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rings of (R)-19a and (R)-27a can suitably occupy the hydrophobic pocket formed by β-tubulin Cys241, Leu248, Ala250, Leu255, Ala316, Ala317, Val318, and Ala354. Meanwhile, the fused 4H-chromene scaffolds of (R)-19a and (R)-27a lie in the docked pocket, with O-containing and N-containing rings at the 6,7-positions deeply embedded into the small hydrophobic subpocket surrounded by α-tubulin Ala180, Val181, and β-tubulin Met259, Asn258, Val315, Val351, and Lys352. It is noteworthy that a hydroxyl 30
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group or an amino group at the 8-position can favor the formation of a hydrogen bond with the surrounding residues (Ala180, Lys352, and Asn258). In addition, C3-CN group
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of (R)-27a may be more suitable than that of (R)-19a and (R)-EPC2407 in forming two hydrogen bonds with residue Ala250, which may explain the higher biological potency of
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27a.
Figure 9. The proposed binding modes of (R)-27a and (R)-19a in the crystal structure of the complex of tubulin with DAMA-colchicine (PDB 1SA0) [60]. (A) Overview of the docking pocket of the co-crystallized DAMA-colchicine (gray) and its docked pose (green) in the colchicine site. (B) Superimposed binding mode of (R)-27a (green) and 31
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DAMA-colchicine (gray) and surrounding. (C) Superimposed binding mode of (R)-19a (green) and DAMA-colchicine (gray). (D) Superimposition of the docked poses of
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(R)-19a (blue), (R)-27a (green) and (R)-EPC2407 (gray). 3. DISCUSSIONS
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A new group of fused 4-aryl-4H-chromene-based derivatives was synthesized using a one pot three-component cascade reaction of substituted benzaldehydes, ethyl
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cyanoacetate or malononitrile, along with purchased or synthesized phenol analogs. The diamino group of 4-aryl-4H-chromenes (30a-b and 34a-b) can be transformed into linear and angular 4-aryl-4H-chromene derivatives by forming an imidazole ring. Compound 27a showed an increased anti-proliferative activity in A375 cells by 600-fold, 1700-fold,
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and 2900-fold, when compared to colchicine, EPC2407 and paclitaxel, respectively.
The SAR analysis of these derivatives suggests that 6,7-fused chromenes have better
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activity when compared to 7,8-fused chromenes. Among the saturated rings fused at
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6,7-positions, six-member O,N-containing rings had a higher potency than that of rings containing only one nitrogen or two oxygens, while five-member O-containing rings showed similar potency to that of five-member N-containing rings. When saturated rings are fused at the 6,7-position of the chromenes, the introduction of –OH or –NH2 improves the anti-proliferative activities. Introduction of a 3-bromo-4,5-dimethoxyphenyl ring at the C-4 position and a nitrile group at C-3 position is the optimal combination for
32
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maintaining high activity of 4-aryl-4H-chromene-based derivatives. When unsaturated heterocycles, including 1H-imidazole, 2-amino-1H-imidazole, 2-methyl-1H-imidazole,
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2-(5-methylthiophen-2-yl)-1H-imidazole, or 2-phenyl-1H-imidazole rings, are fused at 6,7-position or 7,8-position, these different sized rings decrease the activity due to the increase in steric hindrance.
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Three representative compounds, 27a, 19a, and 9f, differed in their functional groups but showed effective biological activities. Probative flow cytometry analysis of cell cycle
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progression and the induction of apoptosis demonstrated that these newly synthesized compounds also caused a similar G2-M phase blockade and an equivalent increase in cell apoptosis to that achieved by the positive control colchicine. Direct immunofluorescence staining of tubulin and cell-free, purified protein based evaluation of tubulin
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polymerization also indicated a disruption of microtubule dynamics, further confirming these agents as tubulin polymerization inhibitors that bind at the colchicine binding site. Interestingly, evidence is accumulating that most microtubule binding drugs (MBDs) also
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have anti-angiogenic activities [6, 21, 22]. Indeed, we found that our compounds 27a, 19a, and 9f also inhibited capillary-like tube formation of HUVEC cells embedded in
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matrigel. All three agents exhibited strong disruption activities when compared with colchicine. The concentration required to elicit an anti-vascular effect was lower than that required to inhibit mitosis, block cell cycle, or induce apoptosis, which is consistent with the findings of many other in vitro studies [22],[61]. One explanation might be the greater sensitivity of endothelial cells than tumor cells to these drugs in some instances. A further notable observation was the inhibitory effects on tumor neovascularization that occur at 33
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concentrations as much as 10-fold lower than those that induce cell toxicity [62]. Many similar drugs retain much of their antitumor activities against cancer in drug-resistant mouse models [63],[64], which may be due to their anti-angiogenic actions. These
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findings indicated that the anti-vascular actions of the MBDs are therapeutically important and may represent a promising area of research in drug administration. We further investigated the structural mechanism of action for the most potent compound 27a
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through molecular docking simulations. The binding mode predicted by docking calculations explains the better activity of 27a over 19a. The availability of a highly
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potent microtubule inhibitor such as 27a will enable further mechanistic studies and will yield information important for guiding the development of more effective and specific microtubule targeting agents. 4. CONCLUSIONS
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In summary, our results demonstrated that 27a, among all the compounds studied and reported here, is a potent anti-tumor agent with dual actions of microtubule destabilization and vascular disruption. It shows very strong biological activity in human
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A375 melanoma cells as well as eight other cancer cell lines. This highly potent small
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molecule can serve as a tool to further delineate the detailed mechanism of its dual actions and develop new therapeutics for clinical applications. 5. EXPERIMENTAL SECTION
5.1
Chemistry
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Unless otherwise stated, all commercial chemicals and reagents were used directly without further purification, anhydrous solvents were commercially available. All
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reactions were monitored by analytical thin layer chromatography (TLC) using silica gel GF 254 thin-layer plates (Qingdao Marine Chemical Factory, China). TLC was visualized by UV light (254 nM) or stained with alkaline KMnO4 aqueous solution. Flash
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column chromatography (FC) was performed on 100−200 mesh (or 200-300 mesh) silica 13
C NMR spectra were
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gel (Qingdao Marine Chemical Factory, China). 1H NMR and
obtained on a Bruker AV 400 instrument with as tetramethylsilane (TMS δ 0.00) as internal standard. Dimethyl sulfoxide-d6 (DMSO-d6) or Chloroform-d (CDCl3) was used as the solvent. High-resolution mass spectrometry (HR-MS) data were measured on a
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Waters Xevo G2 QTof instrument (Drug Facility of Tsinghua University). All target compounds exhibited a purity of at least 95%.
5.1.1． ． Ethyl2-amino-4-(3-chlorophenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-car
of
intermediate
9
was
presented
in
supporting
information),
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synthesis
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boxylate (9a). To a mixture of 9 (100 mg, 0.74 mmol) (the general procedure for the
3-chlorobenzaldehyde (104 mg, 0.74 mmol) and ethyl 2-cyanoacetate (83 µL, 0.78 mmol) in anhydrous EtOH (5 mL) was added piperdine (136 µL, 1.48 mmol). The reaction mixture was stirred for 16 h at 80℃ under N2 atmosphere. The resulting reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography eluting with CH2Cl2/MeOH (200:1-100:1, v/v) 35
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to afford the title compound (9a) as a slight yellow solid (210 mg, 77% yield). 1H NMR (400 MHz, CDCl3) δ 7.20 (s, 1H), 7.17–7.03 (m, 3H), 6.73 (s, 1H), 6.30 (br., 2H), 6.25
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(s, 1H), 4.78 (s, 1H), 4.14–3.98 (m, 2H), 3.55 – 3.47 (m, 2H), 2.98 – 2.79 (m, 2H), 1.16 (q, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.49 (s), 160.56 (s), 151.23 (s), 150.89 (s), 148.33 (s), 133.82 (s), 129.46 (s), 128.00 (s), 126.33 (s), 126.04 (s), 126.01
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(s), 124.81 (s), 114.82 (s), 96.87 (s), 78.76 (s), 59.49 (s), 47.82 (s), 40.22 (s), 29.14 (s),
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14.44 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C20H19N2O3Cl, 371.1084; found, 371.1161.
5.1.2.
2-amino-4-(3-chlorophenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbonitrile
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(9b). According to the synthetic procedure of 9a, compound 9b was obtained by using 3-chlorobenzaldehyde, malononitrile and 9 as an off-white solid (82% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.34 (t, J = 7.7 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.18 (s, 1H),
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7.14 (d, J = 7.6 Hz, 1H), 6.84 (s, 2H), 6.61 (s, 1H), 6.09 (s, 1H), 5.70 (s, 1H), 4.58 (s,
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1H), 3.38 (br., 2H), 2.76 (br., 2H).13C NMR (400 MHz, DMSO-d6) δ 160.56 (s), 152.59 (s), 149.43 (s), 147.76 (s), 133.09 (s), 130.53 (s), 127.04 (s), 126.54 (s), 126.09 (d, J = 11.0 Hz), 123.85 (s), 120.72 (s), 109.75 (s), 95.09 (s), 55.75 (s), 46.75 (s), 28.29 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C18H14ClN3O, 324.0825; found, 324.0898.
5.1.3. 2-amino-4-(3-bromophenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbonitrile 36
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(9c). According to the synthetic procedure of 9a, compound 9c was obtained by using 3-bromobenzaldehyde, malononitrile and 9 as a slight yellow solid (86% yield). 1H NMR
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(400 MHz, DMSO-d6) δ 7.40 (d, J = 7.8 Hz, 1H), 7.32 (s, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 6.85 (s, 2H), 6.61 (s, 1H), 6.09 (s, 1H), 5.70 (s, 1H), 4.57 (s, 1H), 3.38 (br., 2H), 2.77 (br., 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.02 (s), 153.06
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(s), 150.16 (s), 148.21 (s), 131.32 (s), 130.37 (s), 129.91 (s), 127.01 (s), 124.33 (s), 122.27 (s), 110.22 (s), 95.55 (s), 56.23 (s), 47.21 (s), 28.75 (s). HRMS (ESI, m/z): [M +
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H]+ calcd. for C18H14BrN3O, 368.0320; found, 368.0385.
5.1.4.
2-Amino-4-(3-fluorophenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbonitrile
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(9d). According to the synthetic procedure of 9a, compound 9d was obtained by using 3-fluorobenzaldehyde, malononitrile and 9, and was isolated as a slight yellow solid (25% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.34 (dd, J = 14.2, 7.4 Hz, 1H), 7.02 (t, J
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= 7.7 Hz, 2H), 6.96 (d, J = 10.0 Hz, 1H), 6.83 (s, 2H), 6.62 (s, 1H), 6.10 (s, 1H), 5.69 (s, 13
C NMR (101 MHz, DMSO-d6) δ
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1H), 4.59 (s, 1H), 3.37 (br., 2H), 2.77 (br., 2H).
163.48 (s), 161.05 (s), 160.60 (s), 152.58 (s), 149.87 (d, J = 5.9 Hz), 147.78 (s), 130.54 (d, J = 8.2 Hz), 126.00 (s), 123.87 (s), 123.45 (s), 120.77 (s), 114.05 (s), 113.84 (s), 113.44 (s), 113.23 (s), 109.88 (s), 95.12 (s), 55.80 (s), 46.78 (s), 28.32 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C18H14FN3O, 308.1121; found, 308.1190.
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5.1.5. 2-Amino-4-(3-methoxyphenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbonitrile
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(9e). According to the synthetic procedure of 9a, compound 9e was obtained by using 3-methoxybenzaldehyde, malononitrile and 9 as an off-white solid (80% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.21 (t, J = 8.0 Hz, 1H), 6.77 (br., 1H), 6.75 (br., 2H), 6.71 (br.,
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2H), 6.62 (s, 1H), 6.08 (s, 1H), 5.66 (s, 1H), 4.49 (s, 1H), 3.72 (s, 3H), 3.37 (br., 2H),
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2.76 (br., 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.49 (s), 159.29 (s), 152.38 (s), 148.50 (s), 147.71 (s), 129.63 (s), 125.80 (s), 123.88 (s), 119.58 (s), 113.48 (s), 111.28 (s), 110.46 (s), 95.09 (s), 56.20 (s), 54.94 (s), 46.77 (s), 28.33 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C19H17N3O2, 320.1321; found, 320.1395.
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5.1.6.
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3carbonitrile (9f). According to the synthetic procedure of 9a, compound 9f was obtained
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by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 9, and was isolated as
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a slight yellow solid (82% yield). 1H NMR (400 MHz, CDCl3) δ 6.89 (d, J = 1.9 Hz, 1H), 6.70 (d, J = 1.9 Hz, 1H), 6.61 (s, 1H), 6.21 (s, 1H), 4.64 (s, 2H), 4.51 (s, 1H), 3.83 (s, 3H), 3.82 (s, 3H), 3.54 (t, J = 8.3 Hz, 2H), 2.89 (t, J = 8.3 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 159.61 (s), 153.82 (s), 151.87 (s), 148.12 (s), 145.42 (s), 142.76 (s), 126.98 (s), 124.68 (s), 124.01 (s), 120.25 (s), 117.94 (s), 111.50 (s), 110.98 (s), 96.72 (s), 60.64 (s),
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60.42 (s), 56.24 (s), 47.77 (s), 40.75 (s), 29.03 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C20H18N3O3Br, 428.0532; found, 428.0612.
Ethyl
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5.1.7.
2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-
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carboxylate (9g). According to the synthetic procedure of 9a, compound 9g was obtained by using 3-bromo-4,5-dimethoxybenzaldehyde, ethyl 2-cyanoacetate and 9, and
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was isolated as a slight yellow solid (45% yield). 1H NMR (400 MHz, CDCl3) δ 6.96 (d, J = 1.8 Hz, 1H), 6.74 (s, 1H), 6.67 (d, J = 1.7 Hz, 1H), 6.33 (s, 2H), 6.23 (s, 1H), 4.72 (s, 1H), 4.07 (q, J = 17.8, 10.8, 7.1, 3.6 Hz, 2H), 3.78 (d, J = 2.2 Hz, 6H), 3.51-3.48 (m, 2H), 2.95 – 2.84 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.47 (s),
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160.56 (s), 153.32 (s), 151.25 (s), 148.29 (s), 146.01 (s), 144.42 (s), 126.30 (s), 124.65 (s), 123.83 (s), 117.08 (s), 114.74 (s), 111.25 (s), 96.81 (s), 60.56 (s), 59.46 (s), 56.06 (s), 47.76 (s), 40.06 (s), 29.10 (s), 14.48 (s). HRMS (ESI, m/z): [M + H]+ calcd. for
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5.1.8.
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C22H23BrN2O5, 475.0790; found, 475.0869.
2-Amino-4-(3,4,5-trimethoxyphenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbon itrile (9h). According to the synthetic procedure of 9a, compound 9h was obtained by using 3,4,5-trimethoxybenzaldehyde, malononitrile and 9, and was isolated as a slight yellow solid (42% yield). 1H NMR (400 MHz, CDCl3) δ 6.65 (s, 1H), 6.38 (s, 2H), 6.21 (s, 1H), 4.62 (s, 2H), 4.52 (s, 1H), 3.81 (s, 9H), 3.54 (t, J = 8.4 Hz, 2H), 2.89 (t, J = 7.8 39
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Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 159.50 (s), 153.40 (s), 151.69 (s), 148.13 (s), 141.30 (s), 136.97 (s), 126.82 (s), 124.73 (s), 120.44 (s), 111.60 (s), 105.00 (s), 96.69 (s),
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60.90 (s), 56.21 (s), 47.79 (s), 41.37 (s), 29.06 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C21H21N3O4, 380.1532; found, 380.1607.
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5.1.9.
2-Amino-4-(3,4,5-trifluorophenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbonitr
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ile (9i). The synthesis procedure of 9a was used to obtain compound 9i using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 9 as a slight yellow solid ( 51 % yield). 1H NMR (400 MHz, DMSO-d6) δ 7.18 – 7.01 (m, 2H), 6.91 (s, 2H), 6.64 (s, 1H), 6.09 (s, 1H), 5.73 (s, 1H), 4.64 (s, 1H), 3.39 (br., 2H), 2.77 (br., 2H).
13
C NMR (101
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MHz, DMSO-d6) δ 160.70 (s), 152.78 (s), 147.72 (s), 126.14 (s), 123.77 (s), 111.58 (s), 108.85 (s), 95.09 (s), 46.74 (s), 28.26 (s). HRMS (ESI, m/z): [M + H]+ calcd. for
5.1.10.
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C18H12F3N3O, 344.0932; found, 344.1008.
Ethyl
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2-amino-4-(3-chlorophenyl)-4,7,8,9-tetrahydropyrano[2,3-e]indole-3-carboxylate (11a). According to the synthetic procedure of 9a, compound 11a was prepared by using 3-chlorobenzaldehyde, ethyl 2-cyanoacetate and 11 (the preparation of intermediate 11 was presented in supporting information), and was isolated as a slight yellow solid (16% yield). 1H NMR (400 MHz, CDCl3) δ 7.19 (s, 1H), 7.15 – 7.03 (m, 3H), 6.72 (d, J = 8.0 Hz, 1H), 6.34 (d, J = 8.0 Hz, 1H), 6.34 (br., 2H), 4.83 (d, 1H), 4.08-4.02 (m, 2H), 3.62 (t, 40
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J = 8.5 Hz, 2H), 3.15-3.00 (m, 2H), 1.19-1.10 (m, 3H).
13
C NMR (101 MHz, CDCl3) δ
169.52 (s), 160.35 (s), 152.25 (s), 150.90 (s), 145.73 (s), 133.84 (s), 129.46 (s), 128.70
(s), 47.83 (s), 39.84 (s), 26.85 (s), 14.43 (s).
RI PT
(s), 127.89 (s), 126.01 (s), 125.92 (s), 115.97 (s), 115.05 (s), 106.40 (s), 78.93 (s), 59.56 HRMS (ESI, m/z): [M + H]+ calcd. for
SC
C20H19N2O3Cl, 371.1084; found, 371.1155.
5.1.11.
Ethyl
M AN U
2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-4,7,8,9-tetrahydropyrano[2,3-e]indole-3carboxylate (11b). According to the synthetic procedure of 9a, compound 11b was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, ethyl 2-cyanoacetate and 11 (the preparation of intermediate 11 was presented in supporting information), and was
TE D
isolated as a slight yellow solid (8.5 % yield). 1H NMR (400 MHz, CDCl3) δ 6.95 (s, 1H), 6.75 (d, J = 7.9 Hz, 1H), 6.68 (s, 1H), 6.38 (d, J = 7.9 Hz, 1H), 6.38 (s, 2H), 4.78 (s, 1H), 4.14-4.03 (m, 2H), 3.80 (s, 3H), 3.78 (s, 3H), 3.63 (t, J = 8.4 Hz, 2H), 3.14-3.04 (m, J =
EP
15.9, 7.5 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.53 (s),
AC C
160.35 (s), 153.38 (s), 151.69 (s), 145.98 (s), 145.75 (s), 144.58 (s), 128.66 (s), 123.83 (s), 117.26 (s), 116.42 (s), 115.34 (s), 111.35 (s), 106.75 (s), 78.98 (s), 60.63 (s), 59.59 (s), 56.20 (s), 47.78 (s), 39.76 (s), 29.84 (s), 26.87 (s), 14.53 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C22H23N2O5Br, 475.0790; found, 475.0858.
5.1.12. 2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-4,7,8,9-tetrahydropyrano[2,3-e]indole-3 41
ACCEPTED MANUSCRIPT
-carbonitrile (11c). According to the synthetic procedure of 9a, compound 11c was obtained by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 11 (the
RI PT
preparation of intermediate 11 was presented in supporting information), and was isolated as a slight yellow solid (45% yield). 1H NMR (400 MHz, CDCl3) δ 6.88 (d, J = 1.9 Hz, 1H), 6.71 (d, J = 1.8 Hz, 1H), 6.60 (d, J = 8.0 Hz, 1H), 6.35 (d, J = 8.0 Hz, 1H), 4.67 (s,
13
C NMR (101 MHz, CDCl3) δ 159.38 (s), 153.77 (s), 152.90 (s),
M AN U
8.3, 4.5 Hz, 2H).
SC
2H), 4.56 (s, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.63 (t, J = 8.5 Hz, 2H), 3.09-3.03 (m, J =
145.42 (d, J = 6.4 Hz), 142.75 (s), 128.91 (s), 123.99 (s), 120.17 (s), 117.95 (s), 115.14 (s), 112.15 (s), 111.50 (s), 106.69 (s), 60.67 (d, J = 5.7 Hz), 56.25 (s), 47.80 (s), 40.35 (s), 26.76 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C20H18BrN3O3, 428.0532; found,
5.1.13.
TE D
428.0604.
Ethyl
2-amino-4-(3-chlorophenyl)-6,7,8,9-tetrahydro-4H-pyrano[3,2-g]quinoline-3-carbox
EP
ylate (13a). According to the synthetic procedure of 9a, compound 13a was prepared by
AC C
using 3-chlorobenzaldehyde, ethyl 2-cyanoacetate and 13 (the preparation of intermediate 13 was presented in supporting information), and was isolated as a light red solid (35% yield). 1H NMR (400 MHz, CDCl3) δ 7.19 (s, 1H), 7.15-7.7.06 (m, 3H), 6.58 (s, 1H), 6.30 (br., 2H), 6.11 (s, 1H), 4.75 (s, 1H), 4.11-3.99 (m, 2H), 3.24 (d, J = 3.6 Hz, 2H), 2.68-2.50 (m, 2H), 1.85 (dd, J = 11.3, 5.9 Hz, 2H), 1.15 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.56 (s), 160.61 (s), 150.97 (s), 147.69 (s), 144.15 (s), 133.79 (s), 42
ACCEPTED MANUSCRIPT
129.80 (s), 129.45 (s), 128.00 (s), 126.03 (s), 125.97 (s), 118.61 (s), 113.70 (s), 100.35 (s), 78.94 (s), 59.48 (s), 41.88 (s), 39.73 (s), 26.54 (s), 22.12 (s), 14.44 (s). HRMS (ESI,
RI PT
m/z): [M + H]+ calcd. for C21H21N2O3Cl, 385.1241; found, 385.1320.
5.1.14.
Ethyl
SC
2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-6,7,8,9-tetrahydro-4H-pyrano[3,2-g]qui noline-3-carboxylate (13b). According to the synthetic procedure of 9a, compound 13b
M AN U
was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, ethyl 2-cyanoacetate and 13 (the preparation of intermediate 13 was presented in supporting information), and was isolated as a red-brown solid (25 % yield). 1H NMR (400 MHz, CDCl3) δ 6.96 (d, J = 1.8 Hz, 1H), 6.66 (d, J = 1.7 Hz, 1H), 6.60 (s, 1H), 6.29 (br., 2H), 6.10 (s, 1H), 4.69 (s, 1H),
TE D
4.14-4.01 (m, 2H), 3.25 (t, J = 4.9 Hz, 2H), 2.64-2.61 (m, J = 6.3, 4.0 Hz, 2H), 1.93-1.80 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.56 (s), 160.64 (s), 153.36 (s), 147.71 (s), 146.09 (s), 144.47 (s), 144.23 (s), 129.67 (s), 123.90 (s), 118.58
EP
(s), 117.11 (s), 113.66 (s), 111.35 (s), 100.30 (s), 79.04 (s), 60.61 (s), 59.48 (s), 56.14 (s),
AC C
41.87 (s), 39.61 (s), 26.56 (s), 22.12 (s), 14.52 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C23H25N2O5Br, 489.0947; found, 489.0958.
5.1.15.
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-6,7,8,9-tetrahydro-4H-pyrano[3,2-g]qui noline-3-carbonitrile (13c). According to the synthetic procedure of 9a, compound 13c was obtained by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 13 (the 43
ACCEPTED MANUSCRIPT
preparation of intermediate 13 was presented in supporting information), and was isolated as an off-white solid (69% yield). 1H NMR (400 MHz, DMSO-d6) δ 6.92 (d, J = 1.4 Hz,
RI PT
1H), 6.82 (d, J = 1.5 Hz, 1H), 6.79 (br., 2H), 6.50 (s, 1H), 6.08 (s, 1H), 5.89 (s, 1H), 4.50 (s, 1H), 3.80 (s, 3H), 3.70 (s, 3H), 3.12 (br., 2H), 1.79-1.61 (m, 2H).
13
C NMR (101
MHz, DMSO-d6) δ 161.07 (s), 153.75 (s), 147.49 (s), 145.68 (s), 145.00 (s), 144.59 (s),
SC
129.09 (s), 122.91 (s), 121.28 (s), 117.64 (s), 117.16 (s), 112.22 (s), 109.07 (s), 99.32 (s),
M AN U
60.44 (s), 56.54 (s), 56.39 (s), 26.57 (s), 21.74 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C21H20N3O3Br, 442.0688; found, 442.0760.
5.1.16.
Ethyl
2-amino-4-(3-chlorophenyl)-7,8,9,10-tetrahydro-4H-pyrano[2,3-f]quinoline-3-carbox
TE D
ylate (15a). According to the synthetic procedure of 9a, compound 15a was prepared by using 3-chlorobenzaldehyde, ethyl 2-cyanoacetate and 15 (the preparation of intermediate 15 was presented in supporting information), and was isolated as a light red solid (33%
EP
yield). 1H NMR (400 MHz, CDCl3) δ 7.19 (d, J = 1.1 Hz, 1H), 7.16-7.03 (m, 3H), 6.65
AC C
(d, J = 8.3 Hz, 1H), 6.33 (br., 2H), 6.20 (d, J = 8.2 Hz, 1H), 4.79 (s, 1H), 4.14-3.98 (m, 2H), 3.25 (t, J = 5.4 Hz, 2H), 2.77 (t, J = 6.5 Hz, 2H), 1.99-1.91 (m, 2H), 1.15 (t, J = 6.0, 3H). 13C NMR (101 MHz, CDCl3) δ 169.56 (s), 160.44 (s), 150.88 (s), 147.00 (s), 144.39 (s), 133.81 (s), 129.43 (s), 127.92 (s), 126.92 (s), 125.98 (s), 113.70 (s), 111.07 (s), 108.60 (s), 78.89 (s), 59.51 (s), 41.51 (s), 40.01 (s), 21.62 (s), 20.63 (s), 14.43 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C21H21N2O3Cl, 385.1241; found, 385.1319. 44
ACCEPTED MANUSCRIPT
5.1.17. 2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-7,8,9,10-tetrahydro-4H-pyrano[2,3-f]qu
RI PT
inoline-3-carbonitrile (15b). According to the synthetic procedure of 9a, compound 15b was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 15 (the preparation of intermediate 15 was presented in supporting information), and was isolated
SC
as an off-white solid (83% yield). 1H NMR (400 MHz, CDCl3) δ 6.89 (d, J = 1.9 Hz, 1H),
M AN U
6.72 (d, J = 1.8 Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H), 6.20 (d, J = 8.4 Hz, 1H), 4.64 (s, 2H), 4.53 (s, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.26 (t, J = 5.4 Hz, 2H), 2.73 (t, J = 6.5 Hz, 2H), 2.03 – 1.85 (m, 2H).
13
C NMR (101 MHz, CDCl3) δ 159.48 (s), 153.77 (s), 146.74 (s),
145.41 (s), 145.05 (s), 142.71 (s), 126.95 (s), 124.05 (s), 117.95 (s), 111.61 (s), 111.38
TE D
(s), 109.83 (s), 108.43 (s), 60.64 (s), 56.28 (s), 41.37 (s), 40.53 (s), 21.43 (s), 20.57 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C21H20N3O3Br, 442.0688; found, 442.0745.
5.1.18.
EP
6-amino-8-(3-chlorophenyl)-8H-[1,3]dioxolo[4,5-g]chromene-7-carboxylate
Ethyl (16a).
AC C
According to the synthetic procedure of 9a, compound 16a was prepared by using 3-chlorobenzaldehyde, ethyl 2-cyanoacetate and 16 (the preparation of intermediate 16 was presented in supporting information), and was isolated as an off-white solid (62% yield). 1H NMR (400 MHz, CDCl3) δ 7.18 (s, 1H), 7.16 – 7.03 (m, 3H), 6.52 (s, 1H), 6.45 (s, 1H), 6.30 (br., 2H), 5.91 (s, 1H), 5.87 (s, 1H), 4.80 (s, 1H), 4.17 – 3.97 (m, 2H), 1.16 (t, J = 7.1 Hz, 3H).
13
C NMR (101 MHz, CDCl3) δ 169.27 (s), 160.32 (s), 149.99 (s), 45
ACCEPTED MANUSCRIPT
146.96 (s), 144.57 (s), 143.28 (s), 134.03 (s), 129.59 (s), 127.96 (s), 126.41 (s), 125.96 (s), 117.60 (s), 107.87 (s), 101.62 (s), 98.00 (s), 78.09 (s), 59.64 (s), 40.62 (s), 14.43 (s).
RI PT
HRMS (ESI, m/z): [M + Na]+ calcd. for C19H16NO5Cl, 396.0717; found, 396.0609.
5.1.19.
SC
Ethyl-6-amino-8-(3,4,5-trimethoxyphenyl)-8H-[1,3]dioxolo[4,5-g]chromene-7-carbox ylate (16b). According to the synthetic procedure of 9a, compound 16b was prepared by
M AN U
using 3,4,5-trimethoxybenzaldehyde, ethyl 2-cyanoacetate and 16 (the preparation of intermediate 16 was presented in supporting information), and was isolated as a slight yellow solid (24% yield). 1H NMR (400 MHz, CDCl3) δ 6.52 (s, 2H), 6.40 (s, 2H), 6.27 (br., 2H), 5.92 (s, 1H), 5.88 (s, 1H), 4.75 (s, 1H), 4.10-4.06 (m,, 2H), 3.80 (s, 6H), 3.78 13
C NMR (101 MHz, CDCl3) δ 169.45 (s), 160.37 (s),
TE D
(s, 3H), 1.18 (t, J = 7.1 Hz, 3H).
153.07 (s), 146.76 (s), 144.45 (s), 143.80 (s), 143.25 (s), 136.50 (s), 118.38 (s), 107.80 (s), 104.66 (s), 101.56 (s), 97.87 (s), 78.55 (s), 60.88 (s), 59.52 (s), 56.15 (s), 41.10 (s),
AC C
452.1326.
EP
14.55 (s). HRMS (ESI, m/z): [M + Na]+ calcd. for C22H23NO8, 452.1424; found,
5.1.20.
6-amino-8-(3,4,5-trimethoxyphenyl)-8H-[1,3]dioxolo[4,5-g]chromene-7-carbonitrile (16c). According to the synthetic procedure of 9a, compound 16c was prepared by using 3,4,5-trimethoxybenzaldehyde, malononitrile and 16 (the preparation of intermediate 16 was presented in supporting information), and was isolated as an off-white solid. 1H 46
ACCEPTED MANUSCRIPT
NMR (400 MHz, DMSO-d6) δ 6.88 (s, 2H), 6.67 (s, 1H), 6.64 (s, 1H), 6.50 (s, 2H), 6.00 (s, 1H), 5.96 (s, 1H), 4.57 (s, 1H), 3.73 (s, 6H), 3.63 (s, 3H). 13C NMR (101 MHz,
RI PT
DMSO-d6) δ 160.51 (s), 152.97 (s), 146.73 (s), 143.98 (s), 142.51 (s), 141.63 (s), 136.34 (s), 120.59 (s), 115.42 (s), 107.29 (s), 104.51 (s), 101.64 (s), 97.71 (s), 59.93 (s), 55.85 (s), 40.96 (s), 18.88 (s). HRMS (ESI, m/z): [M + Na]+ calcd. for C20H18N2O6, 405.1165;
SC
found, 405.1062.
M AN U
5.1.21.
6-amino-8-(3-bromo-4,5-dimethoxyphenyl)-8H-[1,3]dioxolo[4,5-g]chromene-7-carbo nitrile (16d). According to the synthetic procedure of 9a, compound 16d was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 16 (the preparation of
TE D
intermediate 16 was presented in supporting information), and was isolated as an off-white solid (74% yield). 1H NMR (400 MHz, DMSO-d6) δ 6.98 (d, J = 1.8 Hz, 1H), 6.96(s, 2H), 6.87 (d, J = 1.8 Hz, 1H), 6.68 (s, 1H), 6.65 (s, 1H), 6.02 (s, 1H), 5.97 (s, 1H),
EP
4.63 (s, 1H), 3.81 (s, 3H), 3.70 (s, 3H).
13
C NMR (101 MHz, DMSO-d6) δ 160.56 (s),
AC C
153.39 (s), 146.93 (s), 144.40 (s), 144.12 (s), 143.50 (s), 142.55 (s), 122.36 (s), 120.42 (s), 116.83 (s), 114.87 (s), 111.71 (s), 107.22 (s), 101.72 (s), 97.81 (s), 59.99 (s), 56.07 (s), 55.00 (s). HRMS (ESI, m/z): [M + Na]+ calcd. for C19H15N2O5Br, 431.0164; found, 431.0085.
5.1.22. 6-Amino-8-(3-bromo-4,5-dimethoxyphenyl)-4-hydroxy-8H-[1,3]dioxolo[4,5-g]chrom 47
ACCEPTED MANUSCRIPT
ene-7-carbonitrile (19a). According to the synthetic procedure of 9a, compound 19a was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 19 (the
RI PT
preparation of intermediate 19 was presented in supporting information), and was isolated as an off-white solid (50% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H), 6.97 (d, J = 1.8 Hz, 1H), 6.85 (d, J = 1.9 Hz, 1H), 6.82 (br., 2H), 6.19 (s, 1H), 5.95 (s, 1H), 5.91
SC
(s, 1H), 4.60 (s, 1H), 3.81 (s, 3H), 3.70 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.67
M AN U
(s), 153.39 (s), 144.37 (s), 144.27 (s),143.61 (s), 134.19 (s), 133.99 (s), 129.61 (s), 122.25 (s), 120.50 (s), 116.81 (s), 115.98 (s), 111.63 (s), 101.33 (s), 98.03 (s), 60.02 (s), 56.10 (s), 55.28 (s), 30.72 (s), 18.58 (s). HRMS (ESI, m/z): [M + 3H]+ calcd. for C19H15BrN2O6, 449.0113; found, 449.0165.
TE D
5.1.23.
6-Amino-4-hydroxy-8-(3,4,5-trimethoxyphenyl)-8H-[1,3]dioxolo[4,5-g]chromene-7-c arbonitrile (19b). According to the synthetic procedure of 9a, compound 19a was
EP
prepared by using 3,4,5-trimethoxybenzaldehyde, malononitrile and 19 (the preparation
AC C
of intermediate 19 was presented in supporting information), and was isolated as an off-white solid (20% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.79 (s, 1H), 6.73 (s, 2H), 6.49 (s, 2H), 6.17 (s, 1H), 5.94 (s, 1H), 5.90 (s, 1H), 4.55 (s, 1H), 3.73 (s, 6H), 3.62 (s, 3H).
13
C NMR (101 MHz, DMSO-d6) δ 160.57 (s), 152.94 (s), 144.10 (s), 141.65 (s),
136.33 (s), 133.95 (s), 129.50 (s), 120.64 (s), 116.44 (s), 104.47 (s), 101.23 (s), 98.08 (s),
48
ACCEPTED MANUSCRIPT
59.94 (s), 55.86 (s), 55.68 (s), 41.32 (s), 18.88 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C20H18N2O7, 399.1114; found, 399.1185.
RI PT
5.1.24.
7-amino-9-(3-bromo-4,5-dimethoxyphenyl)-3,9-dihydro-2H-[1,4]dioxino[2,3-g]chro
SC
mene-8-carbonitrile (22a). According to the synthetic procedure of 9a, compound 22a was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 22 (the
M AN U
preparation of intermediate 22 (the preparation of intermediate 22 was presented in supporting information) was presented in supporting information), and was isolated as an off-white solid (4% yield). 1H NMR (400 MHz, DMSO-d6) δ 6.99 (d, J = 1.8 Hz, 1H), 6.95 (s, 2H), 6.87 (d, J = 1.8 Hz, 1H), 6.59 (s, 1H), 6.55 (s, 1H), 4.64 (s., 1H), 4.20 (br.,
TE D
2H), 4.17 (br., 2H), 3.81 (s, 3H), 3.70 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 161.13 (s), 153.85 (s), 144.06 (s), 143.34 (s), 142.40 (s), 142.40 (s), 122.84 (s), 120.97 (s), 117.31 (s), 117.28 (s), 116.55 (s), 115.97 (s), 112.21 (s), 104.64 (s), 100.00 (s), 64.67 (s),
EP
64.34 (s), 60.46 (s), 56.56 (s), 55.40 (s), 17.34 (s). HRMS (ESI, m/z): [M + H]+ calcd. for
AC C
C20H17BrN2O5, 445.0321; found, 445.0387.
5.1.25.
7-Amino-9-(3-bromo-4,5-dimethoxyphenyl)-2,3,4,9-tetrahydrochromeno[6,7-b][1,4] oxazine-8-carbonitrile (27a). According to the synthetic procedure of 9a, compound 27a was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 27 (the preparation of intermediate 27 was presented in supporting information), and was isolated 49
ACCEPTED MANUSCRIPT
as a slight yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 6.95 (d, J = 1.7 Hz, 1H), 6.84 (d, J = 1.8 Hz, 1H), 6.82 (br., 2H), 6.31 (s, 1H), 6.22 (s, 1H), 6.02 (s, 1H), 4.52 (s, 1H),
RI PT
4.02 (t, J = 4.0 Hz, 2H), 3.80 (s, 3H), 3.70 (s, 3H), 3.22 (br., 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.83 (s), 153.34 (s), 144.21 (s), 142.36 (s), 139.87 (s), 134.92 (s), 122.40 (s), 120.87 (s), 116.78 (s), 114.87 (s), 111.72 (s), 109.80 (s), 100.66 (s), 79.19 (s), 64.44
SC
(s), 60.03 (s), 56.09 (s), 55.19 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C20H18BrN3O4,
M AN U
444.0481; found, 444.0548.
5.1.26.
7-Amino-9-(3,4,5-trimethoxyphenyl)-2,3,4,9-tetrahydrochromeno[6,7-b][1,4]oxazine -8-carbonitrile (27b). According to the synthetic procedure of 9a, compound 27b was
TE D
prepared by using 3,4,5-trimethoxybenzaldehyde, malononitrile and 27 (the preparation of intermediate 27 was presented in supporting information), and isolated as a slight yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 6.74 (s, 2H), 6.48 (s, 2H), 6.31 (s, 1H),
13
C NMR (101 MHz, DMSO-d6) δ 160.78 (s), 152.91 (s), 142.33 (s),
AC C
3.22 (br., 2H).
EP
6.21 (s, 1H), 5.97 (s, 1H), 4.47 (s, 1H), 4.02 (t, J = 3.9 Hz, 2H), 3.72 (s, 6H), 3.63 (s, 3H),
142.25 (s), 139.79 (s), 136.19 (s), 134.69 (s), 121.04 (s), 114.93 (s), 110.40 (s), 104.53 (s), 100.65 (s), 79.19 (s), 64.45 (s), 59.96 (s), 55.86 (s), 55.56 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C21H21N3O5, 396.1481; found, 396.1558.
General synthetic procedure for intermediates 30a-b, 34a-b, and compounds 31a-h, 35a-c were descripted in supporting information. 50
ACCEPTED MANUSCRIPT
5.2
Cell culture
A375, CHL-1, HeLa, HT-29, HCT116 and SW480 cells were cultured in complete
RI PT
growth medium (DMEM high glucose medium, PH7.4, supplemented with 10% fetal bovine serum, 100 mg/mL streptomycin, and 100 units/mL penicillin). B16-F10, A549 and Jurkat cells were growing in RPMI-1640 medium supplemented with 10% fetal
SC
bovine serum, 100 mg/mL streptomycin, 100 units/mL penicillin and 2mM L-glutamine. HUVECs were kindly provided by Prof. Qin Shen’s lab at Tsinghua University and
M AN U
cultured in endothelial growth medium purchased from ScienCell. PBMCs were kindly provided by Dr. Xingquan Zhang from University of California, San Diego, and cultured in RPMI-1640 medium (with 10% fetal bovine serum, 100 mg/mL streptomycin, 100 units/mL penicillin and 5 U/mL IL-2). All cells were incubated in incubator at 37°C in 5%
5.3
TE D
CO2 and 90% relative humidity.
Measurement of cell viability
EP
The anti-proliferative effects of the compounds on tumor cells were measured by examining cell survival after exposure to compounds for 72 h. Briefly, 1000-5000 cells
AC C
(A375, CHL-1, B16-F10, HeLa, HT-29, HCT116, SW480, MCF-7 and HUVECs) or 100000 cells (Jurkat and PBMCs) per well were seeded in a 96-well cell culture plate overnight at 37ºC in an incubator in 5% CO2 and humidified atmosphere and then treated with different concentrations of 27a, 19a, 9f, and colchicine in fresh medium for 72 h. Cell viability was determined by adding 20 µL/well of the CellTiter reagent (96® Aqueous One Solution Cell Proliferation Assay kit, Promega) and incubating for an additional 2h. The absorbance at 490nm was then recorded using a microplate reader 51
ACCEPTED MANUSCRIPT
(Envision).
5.4
Cell cycle analysis
RI PT
A375 (1×106/well) were plated in a 6-well plate and cultured overnight. The cells were then treated with DMSO (vehicle control) or various doses of 27a, 19a, or 9f for 24 h. After incubation with 0.25% trypsin, two washes with PBS, the cells were resuspended in
SC
100 µL of PBS plus 1 mL 75% cold ethanol and fixed at -20°C overnight. The cells were then centrifuged at 1500 rpm for 5 min and washed twice with 1X buffer A, and
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incubated with 0.25 mg/mL RNAse at 37°C for 30 minutes. The cells were stained with 5 µL propidium iodide (PI) (KeyGEN BioTECH) for another 30 min in dark, according to the manufacturer’ manual, and examined using FACScalibur system (Becton Dickinson).
5.5
Apoptosis assay
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The data were analyzed using FlowJo7.5 analysis software.
Apoptosis was determined with an Annexin V-FITC/PI apoptosis detection kit
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(Bioworld). A375 cells (1×106/well) were seeded in a 6-well plate and incubated
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overnight. Different concentrations (10, 100, 1000 nM) of 27a, 19a, 9f, and colchicine as positive control were added to the cultured cells and incubated for 24 h and 48 h. The cells were trypsinized, washed twice in cold PBS, and centrifuged at 2000 rpm for 5 min. The pellet was resuspended in 400 µL staining solution and stained with 5 µL Annexin V-FITC and 10 µL PI for 15 min protecting from light at room temperature. Samples were examined using an LSRFortessa flow cytometer (BD) and four distinct populations of cells were detected. The normal healthy, early apoptosis, late apoptosis, and necrotic 52
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cells were respectively represented by annexin V-/PI-, annexin V+/PI-, annexin V+/PI+ and annexin V-/PI+ populations. Analyses were performed using the software supplied
5.6
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with the instrument.
Immunofluorescence staining of tubulin
A375 cells were seeded on the coverslips in a 6-well plate overnight. Cells were then
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incubated with 27a, 19a, 9f, or vehicle control separately for 24 h. Thereafter, 4% paraformaldehyde were used to fix cells for 30 min at 4°C and followed by incubation
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with 0.1% Triton X-100 permeabilizing buffer for 15 min at room temperature. After washing with PBS and blocking with 2% BSA in PBS for 1 h at room temperature, cells were incubated with 1:2000 monoclonal anti-α-tubulin antibody in PBS at 4°C overnight. Then cells were washed with PBS and incubated, while protecting from light, with 1:200
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fluorescein isothiocyanate (FITC)–labeled anti-mouse IgG antibody (Sigma) at room temperature. After staining with 4,6-diamidino-2-phenylindole (DAPI) to label the nuclei, the cells were washed and finally immobilized with mounting medium on glass slides.
Colony formation assay
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5.7
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Cell images were visualized using a laser scanning confocal microscope (Zeiss).
1.0 × 104 A375 cells/well were seeded in 60 mm culture dishes and incubated overnight. Different concentrations of compounds in fresh medium containing 10% FBS were then added. The cells were left for several days to form colonies before they were subjected to microscopic examination. Clones containing over 50 cells were regarded as one colony [65]. Colony numbers were quantified by ImageJ. 53
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5.8
Tube formation assay
Matrigel (BD) was dissolved overnight at 4°C, and 96-well plates and tips were
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pre-chilled before adding matrigel. After adding 50 µL of matrigel to each well and leaving it to solidify for 45 min at 37°C, endothelia cell medium (ScienCell) containing DMSO (control) or different concentrations of 27a, 19a, or 9f were added. After 6-8 h,
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fluorescence microscopy images were captured (Olympus). Total length of the formed
5.9
Tubulin polymerization assay
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tube were measured by ImageJ.
HTS-tubulin polymerization assay biochem kit (Cat. #BK004P) was purchased from Cytoskeleton. Tubulin polymerization assay was conducted following the instructions.
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Briefly, freshly prepared 4 mg/mL tubulin (>97% pure, plus 2% microtubule associated proteins) in cold G-PEM buffer was added into each well of a half-area 96-well plate in the presence of test compounds (10× in Genral Tubulin Buffer). Absorbance at 340nm
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(Enspire).
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was immediately recorded at 37oC for 2 h using the kinetic settings of spectrophotometer
5.10 Molecular docking modeling
The complex crystal structure of α/β–tubulin heterodimers with DAMA-colchicine was downloaded from the PDB database (PDB 1SA0). The molecular docking modeling was performed using the Molecular Operating Environment (MOE2015.10) software. Receptors and ligands were prepared according to the MOE standard tutorial. Docking 54
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program was first validated by re-docking of the co-crystallized DAMA-colchicine in the colchicine-binding pocket. All parameters for docking modeling were set to default
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values in the run program. AUTHOR INFORMATION
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Author Contributions
Huijun Zhang designed and performed the biological experiments, analyzed the data and
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wrote the manuscript. Xiong Fang designed and synthesized the compounds, analyzed the SAR data and molecular modeling results and helped write the manuscript. Yujia Mao and Yan Xu helped the design of the modeling and biological studies and contributed to the analyses of the results. Qian Meng performed flow-apoptosis and tubulin
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polymerization assays. Tingting Fan conducted cell proliferation assays on HUVECs. Ziwei Huang and Jing An oversaw the entire project and revised the manuscript. All
Notes
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authors have reviewed the results and approved the final version of the manuscript.
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Declaration of interest: none.
ACKNOWLEDGEMENTS
This work was supported by the grant from the Tsinghua-Peking Joint Center for Life Sciences (to Z. H.), Tsinghua University (to Z.H.), the National Institutes of Health (GM 57761, to Z.H and J.A.), and the California Institute for Regenerative Medicine (to Z.H.). We also thank Prof. Qin Shen of Tsinghua University for the gift of HUVECs and Dr. 55
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Xingquan Zhang from UCSD for the gift of PBMCs.
ABBREVIATIONS USED:
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SAR, structure-activity relationship; TBMs, Tubulin-binding molecules; CBSTs, colchicine binding site agents; MTAs, microtubule targeting agents; HUVECs, human
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umbilical vein endothelia cells; BMSCs, bone marrow stromal cells.
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A series of fused 4-aryl-4H-chromene-based derivatives were designed, and synthesized
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Biological assays were conducted to assess their anti-proliferation and anti-angiogenesis activities
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Molecular docking studies showed that fused 4-aryl-4H-chromene scaffold can mimic DAMA-colchicine in the colchicine-binding site
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Compound 27a exhibited potent anti-proliferation and anti-angiogenesis activities
S0223-5234(18)30599-3
DOI:
10.1016/j.ejmech.2018.07.043
Reference:
EJMECH 10578
To appear in:
European Journal of Medicinal Chemistry
Received Date: 16 April 2018 Revised Date:
30 June 2018
Accepted Date: 16 July 2018
Please cite this article as: H. Zhang, X. Fang, Q. Meng, Y. Mao, Y. Xu, T. Fan, J. An, Z. Huang, Design, synthesis and characterization of potent microtubule inhibitors with dual anti-proliferative and anti-angiogenic activities, European Journal of Medicinal Chemistry (2018), doi: 10.1016/ j.ejmech.2018.07.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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microtubule inhibitors with dual
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Design, synthesis and characterization of potent
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anti-proliferative and anti-angiogenic activities Huijun Zhang1, Xiong Fang1,2, Qian Meng1, Yujia Mao1, Yan Xu1, Tingting Fan1,
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Jing An3,4,*, Ziwei Huang1,3,* 1
School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China
2
Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084, P.
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R. China
Department of Medicine, University of California San Diego, La Jolla, CA 92093
4
Nobel Institute of Biomedicine, Zhuhai, 519080, P. R. China
Corresponding authors:
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*
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Dr. Ziwei Huang. Email: [email protected]. Phone: 0086-10-6279-6890.
Dr. Jing An. Email: [email protected]. Phone: 858-822-0333.
Keywords: Fused 4-aryl-4H-chromenes; microtubule inhibitors; anti-proliferation; anti-angiogenesis; drug development 1
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ABSTRACT Microtubule has been an important target for anticancer drug development. Here we
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report the discovery and characterization of a series of fused 4-aryl-4H-chromene-based derivatives as highly potent microtubule inhibitors. Among a total of 37 derivatives synthesized, 23 exhibited strong in vitro anti-proliferative activities against A375 human
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melanoma cells. The relationship between the biological activities of these microtubule inhibitors and their chemical structure variations was analyzed. Studies of compounds
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27a, 19a and 9a in parallel with colchicine as the positive control compound in a panel of biological assays revealed that these compounds blocked cell cycle progression, increased apoptosis, and inhibited HUVEC capillary tube formation at low nanomolar concentrations. The most potent compound 27a was also tested in eight additional cancer
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cell lines besides A375 cells and two non-cancer cells and showed potent and selective activity on these cancer cells. To understand the molecular and structure mechanism of action of these compounds, tubulin polymerization and molecular docking studies were
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carried out for 27a as the representative. The results were consistent with the mechanism by which 27a interacts with the colchicine binding site on tubulin and disrupts tubulin
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polymerization. With potent dual actions of microtubule destabilization and vascular disruption described above, this small molecule can serve as a valuable research probe of the function and role of microtubules in human diseases and promising lead for developing new therapeutic agents.
1. INTRODUCTION
Cancer, one of the major causes of death worldwide [1], is characterized by abnormal 2
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cell proliferation and inappropriate cell survival. One approach to prevent this proliferation to promote cancer cell death is to use chemotherapeutic drugs that target microtubules [2, 3]. The well-organized function and regulated dynamics of microtubules
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contribute to the structure of the cytoskeleton, mediation of intracellular transport [4, 5], cell division, signaling networks, and many other biological processes [6, 7], making microtubules successful and efficacious cellular targets for the development of traditional
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chemotherapeutic agents [8-11]. For example, taxanes [12, 13] and vinca alkaloids [14, 15] have been used for the treatment of leukemia and solid tumors, and additional
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microtubule-targeting agents are still being pursued intensively [16-19].
Colchicine, extracted from the meadow saffron colchicium autumnale, is one of the best known microtubule-destabilizing agents [20]. Colchicine binding-site inhibitors (CBSI) are also potent destroyers of tumor vasculature [6, 20-27]. They bind to tubulins
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in endothelial cells of tumor vessels, disrupt cytoskeleton, cell shape, and cellular connections and eventually interrupt of the blood flow to the tumor [28]. Also they can selectively affect tumor neovasculature, rather than normal tissues [28-32]. Several small
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molecule vascular disrupting agents (VDAs), including CA-4P, Oxi4503, AVE8062, and EPC2407, are currently undergoing clinical trials [32, 33]. Additional extensive efforts on
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the design and discovery of novel VDAs would further expedite the development of these drugs that target microtubule and show special advantages over other cancer therapies [34-39].
One of the drug development approaches has been based on the use of 4-aryl-4H-chromenes, which was emerged from the studies of natural products and synthetic small molecules. Their broad pharmacological properties have made them 3
ACCEPTED MANUSCRIPT
medically privileged scaffolds [40-43]. Extensive research indicates that they exhibit strong antitumor activities through multiple pathways, including the inhibition of
RI PT
microtubule polymerization, disruption of tumor vasculature, and antagonism of the Wnt pathway. One representative compound, EPC2407, which has entered into Phase II clinical trials as a vascular-disrupting agent, has also been identified as a tubulin inhibitor
SC
binding to the colchicine-binding site [44-46]. Similarly, the 4H-chromene analog 1,
M AN U
featuring a sesamol-derived ring and resembling closely the structure of podophyllotoxin (PT), exhibited strong antimitotic microtubule destabilizing activity (Figure 1) [47]. Subsequent studies reported that substitution of 7-methoxy, 2-NH2, and 3-CN groups for the methylenedioxy ring A and lactone D, respectively, in oxa-PTs yielded
TE D
7-methoxy-4-aryl-4H-chromene 2 that showed markedly improved cytotoxicity against 60 human cancer cell lines [48]. Similarly, 5-pyrido 4H-chromene analog 3 displayed Wnt pathway inhibitory activity (IC50 = 6 nM) and potent anti-proliferative activity
EP
against HCT116 human colon carcinoma cells (IC50 = 15 nM) [49, 50]. Furthermore,
AC C
virtual screening and SAR-guided modification has identified 4-heteroaryl-4H-chromene (HFI437) as a first generation small molecule inhibitor of insulin-regulated amino-peptidase (IRAP) [51]. Overall, these highly functionalized 4H-chromenes have shown diverse bioactivities. Most of the 4-aryl-4H-chromene-based derivatives have great advantages for clinical applications as they possess dual biological activities (i.e., tubulin inhibition and vascular disruption). Furthermore, these molecules can overcome
4
ACCEPTED MANUSCRIPT
drug resistance by binding directly to the colchicine binding site on tubulin and selectively target established tumor vasculature [35, 37, 41].
RI PT
Our laboratory has previously developed mHA11, a 4-aryl-4H-chromene derivative that inhibits tubulin by binding to the colchicine-binding site [52]. mHA11 was derived
SC
by modification of HA14-1, a small molecule originally reported by us as an inhibitor of both tubulin and Bcl-2 [52, 53]. The reported potent bioactivities of 4-aryl-4H-chromenes
M AN U
with dual actions as tubulin-binding VDAs prompted us to investigate whether novel and potent chromene-based VDAs could be developed by modifying the structure of mHA11. To our knowledge, a number of structure-activity relationship (SAR) studies have established that the polyalkoxybenzene moiety of the 4H-chromenes is a crucial
TE D
pharmacophore for mimicking colchicine and podophyllotoxin, and that small hydrophobic groups, such as N(CH3)2, -OCH3, and -NH2 at the 7- or 8-positions or 2-NH2 and 3-CN, generate compounds that inhibit cancer cell growth. In addition, fusion of the
EP
pyrrole ring at the 7,8-position also increases the potency [44]. Fusion of rings at the
AC C
6,7-position has not yet been explored in the design of new classes of fused 4-aryl-4H-chromene-based derivatives. Thus, we designed different five-member or six-member rings fused at the 6,7- or 7,8-positions of mHA11 to investigate their activities (Figure 2). We fused a 1,3-dioxolane at the 6,7-position, following the introduction of an -OH group at the C-8 position. We also prolonged the fused
5
ACCEPTED MANUSCRIPT
4H-chromenes by designing two classes of linear and angular 4-aryl-4H-chromene
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SC
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derivatives by incorporating some different groups into the fused imidazole ring.
AC C
EP
TE D
Figure 1. 4-aryl-4H-chromene-based derivatives and colchicine binding site inhibitors
Figure 2. Structural modifications of the lead compound mHA11 2. RESULTS
6
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2.1
Chemistry
Fused 4-aryl-4H-chromene derivatives were prepared by a one-pot three-component
RI PT
cascade reaction of starting materials 4 (different substituted benzaldehydes), ethyl cyanoacetate or malononitrile, and synthesized intermediates 5 (different fused phenol
SC
analogs). The synthetic route was described in Scheme 1. The three-component reaction was carried out by three steps in the presence of piperidine at room temperature (rt) or 80
M AN U
℃. The Knoevenagel condensation of aldehydes and active methylene compounds (ethyl cyanoacetate or malononitrile) gave the first intermediate (benzylidenemalononitrile). The ortho-position of the phenols with electron-rich density then underwent Michael addition to form the next intermediate. Subsequently, the phenolic hydroxyl group
TE D
participated in an intramolecular attack at the nitrile group and underwent tautomerization (namely, hetero-Thorpe-Ziegler reaction) [47].
EP
Scheme 1. Synthesis of 6,7-fused or 7,8-fused-4-aryl-4H-chromene derivatives via a
AC C
three-component cascade reaction.
R1
R3
+
NC
O or
CHO 4
O
CN
CN
+
B n A
o OH piperidine, EtOH, N2, rt or 80 C
R4 or
5
R3
OH n
R3
R1
B 6 n A 7
R1
X
X 7 8 R4
C
R2
R2
1. Knoevenagel reaction 2. Michael reaction 3. hetero-Thorpe-Ziegler reaction
R2
O
NH2
C
8 D n X = -COOCH2CH3, -CN
O
NH2
6,7-fused or 7,8-fused-4-aryl-4H-chromene derivatives
D
The synthetic approaches of different fused phenol analogs were summarized in Scheme 2. Indolin-6-ol 9 was prepared according to a modified Leimgruber-Batcho indole 7
ACCEPTED MANUSCRIPT
synthesis [54]. Commercially available 4-methyl-3-nitrophenol was protected with a benzyl group to provide the intermediate 7, followed by condensation reaction of 7 with
RI PT
DMF-DMA and pyrrolidine, reductive cyclization and deprotection to obtain 6-hydroxyindole 8 in one step using 10% Pd-C and H2, and then reduction of indole ring in the presence of NaBH(CN)3 and AcOH to yield the desired 9 (Scheme 2a).
SC
Simultaneously, following the same reductive condition, the indolin-4-ol 11 was obtained
M AN U
by using the purchased 10 as the starting material (Scheme 2b). Amide reduction of 12 in the presence of LiAlH4 gave 1,2,3,4-tetrahydroquinolin-7-ol 13 (Scheme 2c), and 5-hydroxylquinoline
14
was
hydrogenated
with
PtO2
to
yield
1,2,3,4-tetrahydroquinolin-5-ol 15 under acidic conditions (Scheme 2d).
TE D
Next, sesamol (16) was treated with MOMCl to give the MOM-protected intermediate 17, followed by the regioselective lithiation of 17 with n-BuLi, boration with B(OMe)3, and oxidation with H2O2 in a one-pot synthesis to afford the intermediate 18 according to
EP
the reported method [55]. The protection of the MOM group afforded the sesamol analog
AC C
19 under acidic condition (Scheme 2e). Another sesamol analog 22 was prepared by treating the 3,4-dihydroxybenzaldehyde 20 with 1,2-dibromoethane in the presence of K2CO3 to yield the intermediate 21 which was further oxidized with mCPBA (Scheme 2f). Replacement of one oxygen from the dioxane ring of 22 with nitrogen gave the fused phenol analog 27, which was synthesized with the route described in Scheme 2g. According to the reference method, the starting material was 4-methoxy-2-nitrophenol 8
ACCEPTED MANUSCRIPT
23, which was reduced to 2-amino-4-methoxyphenol 24 using Fe powder as the reductant, followed by ring closure of 24 with 2-chloroacetyl chloride to yield 25 under
RI PT
alkaline conditions [56]. Reduction of 25 then afforded the intermediate 26, which was further treated with BBr3 to remove the methyl group to obtain 27 in the final step.
SC
Eventually, as shown in Scheme 1, these fused phenol analogs (9, 11, 13, 15, 19, 22, 27) were converted to the corresponding desired compounds 9a-i, 11a-c, 13a-c, 15a-b,
M AN U
19a-b, 22a, and 27a-b, respectively, following the general procedure for the one-pot three-component cascade reaction. These synthesized compounds are summarized in
AC C
EP
TE D
Table 1.
9
ACCEPTED MANUSCRIPT
2.
Synthetic
route
for
fused
phenol
analogs.
TE D
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SC
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Scheme
EP
Reagents and Conditions: (a) BnBr, K2CO3, DMF, rt, 16 h; (b) (i) DMF-DMA, pyrrolidine, 120℃, 3 h; (ii) 10% Pd-C, H2, THF; (c) NaBH(CN)3, AcOH, 0℃-rt, 3 h; (d)
AC C
LiAlH4, THF, 65℃, 16 h; (e) PtO2, H2, conc. HCl, EtOH/MeOH, rt, 26 h; (f) MOMCl, NaH, THF, 0℃-rt, 16 h; (g) n-BuLi, B(OMe)3, THF, 0℃-rt, 0.5 h-1 h, and then AcOH, H2O2, rt, 5 h; (h) 2 N HCl, MeOH, rt, 12 h; (i) BrCH2CH2Br, K2CO3, acetone, 60℃, 36 h; (j) (i) mCPBA, CH2Cl2, 40℃, 12 h; (ii) NaOH, MeOH, rt, 15 min; (k) Fe powder, NH4Cl, MeOH/THF/H2O(2:1:1), 80℃, 4 h; (l) 2-chloroacetyl chloride, K2CO3, ACN, 80℃, 2 h; (m) LiAlH4, THF, rt, 16 h; (n) BBr3, CH2Cl2, -78℃-rt, 2 h. 10
ACCEPTED MANUSCRIPT
The preparation of linearly and angularly fused 4-aryl-4H-chromene derivatives presented
in
Scheme
3
began
with
4-amino-3-nitrophenol
(28)
and
RI PT
2-amino-3-nitrophenol (32) as the starting materials, respectively. These were reduced by Raney-Ni to yield the intermediates 29 (or 33), which were further transformed to the compounds 30a-30b (or 34a-34b) through the above mentioned three-component cascade
SC
reaction. 30a was treated with triethyl orthoformate or triethyl orthoacetate to afford the
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desired compounds 31a-31b. When 30a (or 30b, 34a) reacted with BrCN to obtain compounds 31c (or 31d, 35a), subsequent acylation of 31c (or 35a) provided compounds 31e (or 35b) in the presence of cyclopropanecarbonyl chloride. 30a (or 34b) was then reacted with different substituted benzaldehydes to afford compounds 31f-31h (or 35c)
AC C
EP
TE D
utilizing Na2S2O5 as catalytic reagent.
11
ACCEPTED MANUSCRIPT
Scheme 3. Synthetic route for linearly and angularly fused 4-aryl-4H-chromene
EP
TE D
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SC
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derivatives.
AC C
Reagents and Conditions: (a) Raney-Ni, H2, THF, rt, 5 h; (b) Piperidine, EtOH, N2, 80℃, 16 h; (c) 31a or 31b: triethyl orthoformate or triethyl orthoacetate, ZrCl4, MeOH, rt, 3 h; 31c
and
31d:
BrCN,
MeOH/THF,
rt,
16
h;
31f-31h
and
35c:
5-methylthiophene-2-carboxaldehyde/3,4,5-trimethoxybenzaldehyde/3-bromo-4,5-dimeth oxybenzaldehyde/3-fluorobenzaldehyde, Na2S2O5, DMF, 100℃, 6 h; (e) 31e and 35b: Cyclopropanecarbonyl chloride, DMAP, pyridine, rt, 16 h. 12
ACCEPTED MANUSCRIPT
1.
Chemical
structures
of
synthesized
compounds.
R2
R3
H
H
Cl
9b
H
H
Cl
9c
H
H
9d
H
H
9e
H
9g
9h
B-n-A
C-n-D
(6,7-)
(7,8-)
H
CO2C2H5
C-1-NH
-
H
CN
C-1-NH
-
Br
H
CN
C-1-NH
-
F
H
CN
C-1-NH
-
OCH3
H
CN
C-1-NH
-
EP H
AC C
9f
X
TE D
9a
R4
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Compd R1
SC
RI PT
Table
OCH3
OCH3
Br
H
CN
C-1-NH
-
OCH3
OCH3
Br
H
CO2C2H5
C-1-NH
-
OCH3
OCH3
OCH3
H
CN
C-1-NH
-
13
F
F
F
H
CN
C-1-NH
-
11a
H
H
Cl
-
CO2C2H5
-
NH-1-C
11b
OCH3
OCH3
Br
-
CO2C2H5
-
11c
OCH3
OCH3
Br
-
CN
-
13a
H
H
Cl
H
CO2C2H5
C-2-NH
-
13b
OCH3
OCH3
Br
H
CO2C2H5
C-2-NH
-
13c
OCH3
OCH3
Br
H
CN
C-2-NH
-
15a
H
H
Cl
-
CO2C2H5
-
NH-2-C
15b
OCH3
OCH3
Br
-
CN
-
NH-2-C
16a
H
H
Cl
H
CO2C2H5
O-1-O
-
16b
OCH3
OCH3
H
CO2C2H5
O-1-O
-
NH-1-C
NH-1-C
SC
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TE D
AC C
OCH3
RI PT
9i
EP
ACCEPTED MANUSCRIPT
16c
OCH3
OCH3
OCH3
H
CN
O-1-O
-
16d
OCH3
OCH3
Br
H
CN
O-1-O
-
19a
OCH3
OCH3
Br
OH
CN
O-1-O
-
14
ACCEPTED MANUSCRIPT
OCH3
OCH3
OCH3
OH
CN
O-1-O
-
22a
OCH3
OCH3
Br
H
CN
O-2-O
-
27a
OCH3
OCH3
Br
H
CN
O-2-NH
-
27b
OCH3
OCH3
OCH3
H
CN
O-2-NH
-
SC M AN U
2.2 Biological evaluations
RI PT
19b
2.2.1 In vitro anti-proliferative activities and SAR analysis
All synthesized compounds, including the positive control compounds mHA11,
TE D
EPC2407, colchicine, and paclitaxel, were evaluated for their anti-proliferative activities against human melanoma A375 cells, after the incubation with gradient concentrations of
EP
compounds for 72 h. Cell viability was measured with the CellTiter 96® AQueous One Solution Cell Proliferation Assay（MTS）kit (Promega) according to the manufacturer’s
AC C
instructions. The results presented in Table 2 indicated that 27a, 27b and 19a displayed stronger anti-proliferative activities in A375 cells than colchicine (or paclitaxel), with IC50 values of 16 pM and 369 pM for 27a and 27b respectively. Comparison with mHA11 (IC50 value of 902.3 nM) revealed that the 6,7-fused 4-aryl-4H-chromene derivatives (9a, 9g, 13a, 16a) had approximately 2-3-fold higher anti-proliferative activities except for 13b. On the contrary, 7,8-fused 4-aryl-4H-chromene derivatives 15
ACCEPTED MANUSCRIPT
(11a-b, 15a) lost their anti-proliferative activities (IC50＞10 µM). Likewise, replacement of the 4-(3-chlorophenyl) group of 16a with a 3,4,5-trimethoxyphenyl group led to the
RI PT
loss of activity of derivative 16b. The most potent compound 27a as described above was further evaluated on eight additional cancer cell lines (CHL-1, B16-F10, HeLa，A549, HT-29, HCT116, SW480, Jurkat) and two non-cancer cells (HUVECs and PBMCs). As a
SC
positive control, colchicine was included in these assays. As shown in Table 3, compound
M AN U
27a exhibited potent biological activities on these cancer cell lines. In contrast, compound 27a displayed no cytotoxic effects on normal, non-cancer HUVECs and PBMCs, suggesting the specificity of the effects of compound 27a for cancer cells.
Fusion of a five-member dihydropyrrole at the C6,7-positions and replacement of
TE D
C3-carboxylate with carbonitrile group, or transformation of chlorine on the 4-aryl to bromine yielded three derivatives 9b-c that showed anti-proliferative activities similar to 9a. The 9d with 3-fluorophenyl was about 10-fold better than 9b, and subsequent
EP
introduction of polymethoxy substituents onto the 4-aryl ring (compounds 9f and 9h)
AC C
gave a further slight improvement. Fusion of a five-member dihydropyrrole fused at the C7,8-positions, resulting in 11c, reduced the activities by more than 3-fold when compared to 9f. Ring expansion of dihydropyrrole to form tetrahydropyridine (15b) also decreased the activity by 2-fold compared with 11c. Taking together, the data indicated that the scaffolds containing the 6,7-fused 4-aryl-4H-chromene were superior to the 7,8-fused 4-aryl-4H-chromene scaffolds. 16
ACCEPTED MANUSCRIPT
We then explored the effect of different 6,7-fused rings on anti-proliferative activity. Given that 1,3-dioxolane is a commonly fused ring found in natural products, such as
RI PT
podophyllotoxin derivatives, we also synthesized the two compounds 16c-d with fused 1,3-dioxolane and found their activities were similar to that of 9f or 9h. Nevertheless, introduction of the –OH group at the C-8 position gave compound 19a with a
SC
3-bromo-4,5-dimethoxyphenyl group that was more active than 19b and exhibited a
M AN U
nearly 8-fold increase compared with 16d. Therefore, the introduction of small polar groups (-OH, -NH2) was favorable for increasing the potency of 6,7-fused 4-aryl-4H-chromenes.
We next made a comparison between the fusion of five-member versus six-member
TE D
rings at the 6,7-position. 16d with a fused 1,3-dioxolane was more potent than 22a with 1,4-dioxane. Conversely, 9f with fused tetrahydropyridine was 2-fold less potent than 13c with fused dihydropyrrole. Most interestingly, when the morpholine ring fused at the
EP
6,7-position, compound 27a was 750-fold more active than 13c and 18-fold more active
AC C
than 27b. Introduction of O,N-containing hetero ring fused at the 6,7-position therefore greatly increased the potency.
Finally, we tested the linearly and angularly fused 4-aryl-4H-chromene derivatives (31a-h and 35a-c). Fusion of a 1H-imidazole ring at the 6,7-position of compound 31a resulted in a 18-fold reduction in anti-proliferative activity compared with 9h. Introduction of different sized groups at the 2-position of the 1H-imidazole ring, such as 17
ACCEPTED MANUSCRIPT
methyl (31b), amino (31c-d and 35a), cyclopropanecarboxamide (31e and 35b), 5-methylthiophen-2-yl (31f), or substituted phenyl groups (31g-h and 35c), revealed that
RI PT
three compounds (31b, 31d and 35a-b) with small substituents retained a certain degree of activity (IC50<1 µM). The rest of the compounds with large substituents completely lost their anti-proliferative activities. These results suggested that fusion of unsaturated
SC
heterocycle at the 6,7-position or 7,8-position might be detrimental for activity, and an
M AN U
increased steric bulkiness at these two positions results in a great decrease in potency. Table 2. In vitro anti-proliferative activitya of the synthesized compounds against human A375 melanoma cells.
b
(nM)
Compd
IC50±
b
(nM)
IC50±
9a
268.3±18.01
16c
32.7±13.20
9b
332.0±91.95
16d
23.3±11.55
19a
3.0
34.5±7.48
19b
16.7±5.77
ND
22a
38.6±19.63
28.0±22.91
27a
0.016±0.005
9d
9e
9f
EP
233.7±178.27
AC C
9c
TE D
Compd
18
ACCEPTED MANUSCRIPT
546.7±213.62
27b
0.369±0.301
9h
22.0±12.29
31a
382.0±92.26
9i
ND
31b
11a
＞10000
31c
11b
＞10000
31d
11c
70.0±40.00
31e
13a
441.0±431.57
13b
787.7±349.54
13c
12.3±14.57
31h
＞10000
15a
＞10000
35a
103.3±47.26
35b
110.0±75.50
1345.3±1104.92
M AN U
SC
＞10000
676.7±265.02
＞10000
31f
＞10000
31g
＞10000
TE D
EP
150.0±77.95
AC C
15b
RI PT
9g
16a
330.0±50.21
35c
5113.3±2172.15
16b
＞10000
mHA11
902.3±230.73
colchicine
9.7±2.89
EPC2407(racemate) 27.7
19
ACCEPTED MANUSCRIPT
paclitaxel a
47.8±0.06
Anti-proliferative activity was determined using a MTS assay. bIC50 values are presented
RI PT
as compound concentration required to inhibit A375 cells proliferation by 50%. Data were analyzed in Microsoft Excel and plotted in GraphPad Prism 5, and expressed as
SC
means with standard deviations from at least three independent experiments. ND, not detected.
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Table 3. In vitro anti-proliferative activity of compound 27a and colchicine against eight cancer cell lines (CHL-1, B16-F10, HeLa, A549, HT-29, HCT116, SW480, Jurkat) and two non-cancer cells (HUVECs and PBMCs).
IC50±SD (nM)
TE D
Cell lines
Colchicine
3.98±0.60
3.60±0.95
around 32
around 32
8.6±1.23
9.5±1.63
A549
31.5±13.80
62.7±14.55
HT-29
11.6±0.44
11.5±1.52
HCT116
11.7±0.65
13.9±0.83
SW480
15.3±1.43
14.9±2.37
Jurkat
10.7±3.73
19.8±13.60
CHL-1 B16-F10
AC C
HeLa
EP
27a
20
ACCEPTED MANUSCRIPT
HUVEC
>10000
>10000
PBMCs
>10000
>10000
Induction of G2-M arrest and cell apoptosis of A375 cells
RI PT
2.2.2
Cell cycle assays were conducted to verify whether the newly synthesized compounds
SC
could arrest mitosis of A375 cells. Cell cycle progression was followed by PI staining and flow cytometry analysis. A375 cells were incubated with different concentrations of the
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selected compounds for 24 h. The untreated cells showed a normal cell cycle pattern while all the compound treated groups showed a dose-dependent inhibition of cell cycle similar to that of colchicine control (Figure 3). Compounds 27a, 19a, and 9f all caused
AC C
EP
TE D
obvious G2-M arrest when supplied at 50 nM.
21
EP
TE D
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SC
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ACCEPTED MANUSCRIPT
AC C
Figure 3. Analysis of cell cycle. (A) Representative results of effects of compounds 27a, 19a and 9f on cell cycle progression of A375 cells, measured by flow cytometry analysis. A375 cells were treated with different concentrations of compounds 27a, 19a and 9f for 24 h. The percentage of cells in each cycle phase is indicated. (B) Graph summarizing the cell cycle distribution affected by compounds 27a, 19a, and 9f of at least three independent experiments. 22
ACCEPTED MANUSCRIPT
We then tested whether these active compounds could induce apoptosis in cancer cells by exposing A375 cells to 10, 100 and 1000 nM of 27a, 19a, and 9f for 24 h and 48 h. All samples were stained with annexin V-FITC and PI for analysis. This method divided cells
RI PT
into four groups: live populations (annexin V-/PI-), early apoptotic populations (annexin V+/PI-), late apoptotic populations (annexin V+/PI+), and necrotic populations (annexin V+/PI+). The early apoptotic and late apoptotic cells were regarded as the apoptotic
SC
population. Results in Figure 4 indicated that apoptosis was increased after treatment with 27a, 19a, and 9f but not with the vehicle control, and the extent of apoptosis was
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concentration dependent. At 24 h, a concentration of 1000 nM of 27a induced apoptosis in 19.8% of the cells, whereas at 48 h it induced apoptosis in 32.8% of the cells.
AC C
EP
TE D
Treatment with 19a and 9f also gave similar results in A375 cells.
23
AC C
EP
TE D
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SC
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ACCEPTED MANUSCRIPT
Figure 4. Analysis of cell apoptosis. A375 cells (106/well) were seeded in a six-well plate and incubated overnight. Different concentrations (10, 100, 1000 nM) of 27a, 19a, and 9f and colchicine were added and the cells were incubated for 24 h and 48 h. Samples of 1×106 cells were then collected by centrifugation, resuspended in 500 µL of 1×binding 24
ACCEPTED MANUSCRIPT
buffer, stained with annexin V-FITC and PI for 5 min protecting from light, and analyzed by flow cytometry. (A) 24 h. (B) 48 h. The apoptotic group was indicated in the A+/PIand A+/PI+ regions. The percentage of cell populations was calculated from at least two
Immunofluorescence staining of interfered microtubule
SC
2.2.3
RI PT
independent experiments.
We examined whether our compounds could directly disrupt the distribution of
M AN U
microtubules by immunofluorescence staining of tubulin in A375 cells. After treatment with different concentrations of 27a, 19a, and 9f for 24 hours, the cells changed from a long-spindle shape to a shrunken round or multi angular shape. Staining results (Figure 5) showed that untreated cells had a normal microtubule distribution whereas the candidates
TE D
treated cells displayed a disorganized distribution of tubulin and had a coenocytic appearance, which may be arisen from incomplete cell division. These observations
EP
confirmed that 27a, 19a, and 9f could interfere with microtubule organization and retard
AC C
mitosis in A375 cells.
25
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SC
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ACCEPTED MANUSCRIPT
TE D
Figure 5. Effects of 27a, 19a and 9f on cellular microtubule distribution. Green represents tubulins and blue represents nuclei. Images (magnification: 630X) were obtained using Olympus cellSens microscope. Anti-colonogenic activities
EP
2.2.4
AC C
We further conducted colony formation assays to determine if our candidate compounds could inhibit the clone-forming ability of tumor cells. Figure 6 indicated that 27a, 19a, and 9f at a concentration of 10 nM could inhibit colony formation by A375 cells.
Three
compounds
displayed
26
similar
inhibitory
effects.
SC
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ACCEPTED MANUSCRIPT
M AN U
Figure 6. Inhibition effects on A375 cells colony formation. A375 cells were seeded in a 60 mm culture dish and left for several days to form colonies. Cultures were photographed after staining with 0.1% crystal violet. (A) Untreated control. (B) Representative results of colony formation. (C) Statistic summary of three independent
2.2.5
TE D
experiments. Colony numbers were counted by ImageJ. Anti-vascular activities
EP
Numerous studies indicate that many microtubule-binding agents can disrupt vascular activities [21]. These include flavonoids, chalcones, and many other tubulin inhibitors
AC C
that inhibit angiogenesis through different mechanisms [22]. We tested whether our synthesized compounds could also inhibit vascular formation by seeding human umbilical vein endothelial cells (HUVECs) on solidified matrigel and allowing the cells form tube structures for 6-8 h. The control groups showed an integral net of tubes whereas the compounds treated cells failed to develop whole tubes when the compounds were given at low nanomolar concentration (Figure 7). These responses were also 27
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dose-dependent. These results suggest that mHA series compounds have potent anti-angiogenic effects. To exclude cytotoxic effects of the compounds on HUVECs, cell viability at 24 h was
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determined with the CellTiter 96® AQueous One Solution Cell Proliferation Assay（MTS） kit (Promega). At concentration below 100 nM, none of them showed noticeable toxicity,
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27a had the weakest toxic among our three compounds (Figure 7D).
Figure 7. Inhibition of in vitro vascular tube formation. HUVECs (104/well) were seeded on matrigel with 1, 10 or 100 nM 27a, 19a and 9f (B) or without drugs (vehicle control) (A) in a 96-well plate. Images were captured after 6-8 h at 37°C (magnification: 100X). (C) Measurements of total lengths of tubular structures using ImageJ from three 28
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independent experiments.
(D) Cytotoxic evaluations. Results are presented as mean ±
SD of three experiments.
Inhibition of tubulin assembly
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2.2.6
The tubulin assembly assay is a good indicator of direct interruption with tubulin. In
SC
order to investigate the inhibitory effects of 27a on tubulin polymerization, >97% pure tubulin was treated with 27a and control compounds for 2h at 37°C (Figure 8). The
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contents of polymerized microtubules were monitored by measuring the absorbance at 340 nm every 60 sec for 2h. Compound 27a at 10 µM exhibited significant inhibitory effects on microtubule polymerization, as did colchicine, which was consistent with other
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published reports [57-59].
Figure 8.
Effects of 27a on tubulin polymerization. Tubulin was incubated alone
(control) and with each of the following compounds: 10 µM paclitaxel , 10 µM
29
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colchicine, and 10 µM 27a in fresh general tubulin buffer. Polymerization was measured by absorbance at 340 nm in kinetic mode (Enspire Microplate Reader). Each point
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represents the mean of three independent experiments. 2.3 Molecular docking studies
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To understand the structural basis for the mechanism of action of the above described compounds, molecular docking simulations were performed to predict the binding modes
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of two representative compounds 27a and 19a. As shown in Figure 9A, the re-docking validation step indicated that the co-crystallized ligand DAMA-colchicine was redocked into the colchicine-binding site with small RMSD of 0.4482 Å (energy score (S)= -9.7569 kcal/mol). Taking into consideration that compounds 19a and 27a possess R- and
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S-isomers, we carried out molecular docking studies of (R)-19a and (S)-19a, and (R)-27a and (S)-27a, respectively. The docking studies exhibited that (R)-19a and (R)-27a had similar binding mode with DAMA-colchicine in the colchicine-binding site, and
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superimpose well with (R)-EPC2407 (Figure 9B-C). The 3-bromo-4,5-dimethoxyphenyl
AC C
rings of (R)-19a and (R)-27a can suitably occupy the hydrophobic pocket formed by β-tubulin Cys241, Leu248, Ala250, Leu255, Ala316, Ala317, Val318, and Ala354. Meanwhile, the fused 4H-chromene scaffolds of (R)-19a and (R)-27a lie in the docked pocket, with O-containing and N-containing rings at the 6,7-positions deeply embedded into the small hydrophobic subpocket surrounded by α-tubulin Ala180, Val181, and β-tubulin Met259, Asn258, Val315, Val351, and Lys352. It is noteworthy that a hydroxyl 30
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group or an amino group at the 8-position can favor the formation of a hydrogen bond with the surrounding residues (Ala180, Lys352, and Asn258). In addition, C3-CN group
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of (R)-27a may be more suitable than that of (R)-19a and (R)-EPC2407 in forming two hydrogen bonds with residue Ala250, which may explain the higher biological potency of
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27a.
Figure 9. The proposed binding modes of (R)-27a and (R)-19a in the crystal structure of the complex of tubulin with DAMA-colchicine (PDB 1SA0) [60]. (A) Overview of the docking pocket of the co-crystallized DAMA-colchicine (gray) and its docked pose (green) in the colchicine site. (B) Superimposed binding mode of (R)-27a (green) and 31
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DAMA-colchicine (gray) and surrounding. (C) Superimposed binding mode of (R)-19a (green) and DAMA-colchicine (gray). (D) Superimposition of the docked poses of
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(R)-19a (blue), (R)-27a (green) and (R)-EPC2407 (gray). 3. DISCUSSIONS
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A new group of fused 4-aryl-4H-chromene-based derivatives was synthesized using a one pot three-component cascade reaction of substituted benzaldehydes, ethyl
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cyanoacetate or malononitrile, along with purchased or synthesized phenol analogs. The diamino group of 4-aryl-4H-chromenes (30a-b and 34a-b) can be transformed into linear and angular 4-aryl-4H-chromene derivatives by forming an imidazole ring. Compound 27a showed an increased anti-proliferative activity in A375 cells by 600-fold, 1700-fold,
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and 2900-fold, when compared to colchicine, EPC2407 and paclitaxel, respectively.
The SAR analysis of these derivatives suggests that 6,7-fused chromenes have better
EP
activity when compared to 7,8-fused chromenes. Among the saturated rings fused at
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6,7-positions, six-member O,N-containing rings had a higher potency than that of rings containing only one nitrogen or two oxygens, while five-member O-containing rings showed similar potency to that of five-member N-containing rings. When saturated rings are fused at the 6,7-position of the chromenes, the introduction of –OH or –NH2 improves the anti-proliferative activities. Introduction of a 3-bromo-4,5-dimethoxyphenyl ring at the C-4 position and a nitrile group at C-3 position is the optimal combination for
32
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maintaining high activity of 4-aryl-4H-chromene-based derivatives. When unsaturated heterocycles, including 1H-imidazole, 2-amino-1H-imidazole, 2-methyl-1H-imidazole,
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2-(5-methylthiophen-2-yl)-1H-imidazole, or 2-phenyl-1H-imidazole rings, are fused at 6,7-position or 7,8-position, these different sized rings decrease the activity due to the increase in steric hindrance.
SC
Three representative compounds, 27a, 19a, and 9f, differed in their functional groups but showed effective biological activities. Probative flow cytometry analysis of cell cycle
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progression and the induction of apoptosis demonstrated that these newly synthesized compounds also caused a similar G2-M phase blockade and an equivalent increase in cell apoptosis to that achieved by the positive control colchicine. Direct immunofluorescence staining of tubulin and cell-free, purified protein based evaluation of tubulin
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polymerization also indicated a disruption of microtubule dynamics, further confirming these agents as tubulin polymerization inhibitors that bind at the colchicine binding site. Interestingly, evidence is accumulating that most microtubule binding drugs (MBDs) also
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have anti-angiogenic activities [6, 21, 22]. Indeed, we found that our compounds 27a, 19a, and 9f also inhibited capillary-like tube formation of HUVEC cells embedded in
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matrigel. All three agents exhibited strong disruption activities when compared with colchicine. The concentration required to elicit an anti-vascular effect was lower than that required to inhibit mitosis, block cell cycle, or induce apoptosis, which is consistent with the findings of many other in vitro studies [22],[61]. One explanation might be the greater sensitivity of endothelial cells than tumor cells to these drugs in some instances. A further notable observation was the inhibitory effects on tumor neovascularization that occur at 33
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concentrations as much as 10-fold lower than those that induce cell toxicity [62]. Many similar drugs retain much of their antitumor activities against cancer in drug-resistant mouse models [63],[64], which may be due to their anti-angiogenic actions. These
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findings indicated that the anti-vascular actions of the MBDs are therapeutically important and may represent a promising area of research in drug administration. We further investigated the structural mechanism of action for the most potent compound 27a
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through molecular docking simulations. The binding mode predicted by docking calculations explains the better activity of 27a over 19a. The availability of a highly
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potent microtubule inhibitor such as 27a will enable further mechanistic studies and will yield information important for guiding the development of more effective and specific microtubule targeting agents. 4. CONCLUSIONS
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In summary, our results demonstrated that 27a, among all the compounds studied and reported here, is a potent anti-tumor agent with dual actions of microtubule destabilization and vascular disruption. It shows very strong biological activity in human
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A375 melanoma cells as well as eight other cancer cell lines. This highly potent small
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molecule can serve as a tool to further delineate the detailed mechanism of its dual actions and develop new therapeutics for clinical applications. 5. EXPERIMENTAL SECTION
5.1
Chemistry
34
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Unless otherwise stated, all commercial chemicals and reagents were used directly without further purification, anhydrous solvents were commercially available. All
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reactions were monitored by analytical thin layer chromatography (TLC) using silica gel GF 254 thin-layer plates (Qingdao Marine Chemical Factory, China). TLC was visualized by UV light (254 nM) or stained with alkaline KMnO4 aqueous solution. Flash
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column chromatography (FC) was performed on 100−200 mesh (or 200-300 mesh) silica 13
C NMR spectra were
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gel (Qingdao Marine Chemical Factory, China). 1H NMR and
obtained on a Bruker AV 400 instrument with as tetramethylsilane (TMS δ 0.00) as internal standard. Dimethyl sulfoxide-d6 (DMSO-d6) or Chloroform-d (CDCl3) was used as the solvent. High-resolution mass spectrometry (HR-MS) data were measured on a
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Waters Xevo G2 QTof instrument (Drug Facility of Tsinghua University). All target compounds exhibited a purity of at least 95%.
5.1.1． ． Ethyl2-amino-4-(3-chlorophenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-car
of
intermediate
9
was
presented
in
supporting
information),
AC C
synthesis
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boxylate (9a). To a mixture of 9 (100 mg, 0.74 mmol) (the general procedure for the
3-chlorobenzaldehyde (104 mg, 0.74 mmol) and ethyl 2-cyanoacetate (83 µL, 0.78 mmol) in anhydrous EtOH (5 mL) was added piperdine (136 µL, 1.48 mmol). The reaction mixture was stirred for 16 h at 80℃ under N2 atmosphere. The resulting reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography eluting with CH2Cl2/MeOH (200:1-100:1, v/v) 35
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to afford the title compound (9a) as a slight yellow solid (210 mg, 77% yield). 1H NMR (400 MHz, CDCl3) δ 7.20 (s, 1H), 7.17–7.03 (m, 3H), 6.73 (s, 1H), 6.30 (br., 2H), 6.25
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(s, 1H), 4.78 (s, 1H), 4.14–3.98 (m, 2H), 3.55 – 3.47 (m, 2H), 2.98 – 2.79 (m, 2H), 1.16 (q, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.49 (s), 160.56 (s), 151.23 (s), 150.89 (s), 148.33 (s), 133.82 (s), 129.46 (s), 128.00 (s), 126.33 (s), 126.04 (s), 126.01
SC
(s), 124.81 (s), 114.82 (s), 96.87 (s), 78.76 (s), 59.49 (s), 47.82 (s), 40.22 (s), 29.14 (s),
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14.44 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C20H19N2O3Cl, 371.1084; found, 371.1161.
5.1.2.
2-amino-4-(3-chlorophenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbonitrile
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(9b). According to the synthetic procedure of 9a, compound 9b was obtained by using 3-chlorobenzaldehyde, malononitrile and 9 as an off-white solid (82% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.34 (t, J = 7.7 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.18 (s, 1H),
EP
7.14 (d, J = 7.6 Hz, 1H), 6.84 (s, 2H), 6.61 (s, 1H), 6.09 (s, 1H), 5.70 (s, 1H), 4.58 (s,
AC C
1H), 3.38 (br., 2H), 2.76 (br., 2H).13C NMR (400 MHz, DMSO-d6) δ 160.56 (s), 152.59 (s), 149.43 (s), 147.76 (s), 133.09 (s), 130.53 (s), 127.04 (s), 126.54 (s), 126.09 (d, J = 11.0 Hz), 123.85 (s), 120.72 (s), 109.75 (s), 95.09 (s), 55.75 (s), 46.75 (s), 28.29 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C18H14ClN3O, 324.0825; found, 324.0898.
5.1.3. 2-amino-4-(3-bromophenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbonitrile 36
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(9c). According to the synthetic procedure of 9a, compound 9c was obtained by using 3-bromobenzaldehyde, malononitrile and 9 as a slight yellow solid (86% yield). 1H NMR
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(400 MHz, DMSO-d6) δ 7.40 (d, J = 7.8 Hz, 1H), 7.32 (s, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 6.85 (s, 2H), 6.61 (s, 1H), 6.09 (s, 1H), 5.70 (s, 1H), 4.57 (s, 1H), 3.38 (br., 2H), 2.77 (br., 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.02 (s), 153.06
SC
(s), 150.16 (s), 148.21 (s), 131.32 (s), 130.37 (s), 129.91 (s), 127.01 (s), 124.33 (s), 122.27 (s), 110.22 (s), 95.55 (s), 56.23 (s), 47.21 (s), 28.75 (s). HRMS (ESI, m/z): [M +
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H]+ calcd. for C18H14BrN3O, 368.0320; found, 368.0385.
5.1.4.
2-Amino-4-(3-fluorophenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbonitrile
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(9d). According to the synthetic procedure of 9a, compound 9d was obtained by using 3-fluorobenzaldehyde, malononitrile and 9, and was isolated as a slight yellow solid (25% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.34 (dd, J = 14.2, 7.4 Hz, 1H), 7.02 (t, J
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= 7.7 Hz, 2H), 6.96 (d, J = 10.0 Hz, 1H), 6.83 (s, 2H), 6.62 (s, 1H), 6.10 (s, 1H), 5.69 (s, 13
C NMR (101 MHz, DMSO-d6) δ
AC C
1H), 4.59 (s, 1H), 3.37 (br., 2H), 2.77 (br., 2H).
163.48 (s), 161.05 (s), 160.60 (s), 152.58 (s), 149.87 (d, J = 5.9 Hz), 147.78 (s), 130.54 (d, J = 8.2 Hz), 126.00 (s), 123.87 (s), 123.45 (s), 120.77 (s), 114.05 (s), 113.84 (s), 113.44 (s), 113.23 (s), 109.88 (s), 95.12 (s), 55.80 (s), 46.78 (s), 28.32 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C18H14FN3O, 308.1121; found, 308.1190.
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5.1.5. 2-Amino-4-(3-methoxyphenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbonitrile
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(9e). According to the synthetic procedure of 9a, compound 9e was obtained by using 3-methoxybenzaldehyde, malononitrile and 9 as an off-white solid (80% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.21 (t, J = 8.0 Hz, 1H), 6.77 (br., 1H), 6.75 (br., 2H), 6.71 (br.,
SC
2H), 6.62 (s, 1H), 6.08 (s, 1H), 5.66 (s, 1H), 4.49 (s, 1H), 3.72 (s, 3H), 3.37 (br., 2H),
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2.76 (br., 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.49 (s), 159.29 (s), 152.38 (s), 148.50 (s), 147.71 (s), 129.63 (s), 125.80 (s), 123.88 (s), 119.58 (s), 113.48 (s), 111.28 (s), 110.46 (s), 95.09 (s), 56.20 (s), 54.94 (s), 46.77 (s), 28.33 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C19H17N3O2, 320.1321; found, 320.1395.
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5.1.6.
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3carbonitrile (9f). According to the synthetic procedure of 9a, compound 9f was obtained
EP
by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 9, and was isolated as
AC C
a slight yellow solid (82% yield). 1H NMR (400 MHz, CDCl3) δ 6.89 (d, J = 1.9 Hz, 1H), 6.70 (d, J = 1.9 Hz, 1H), 6.61 (s, 1H), 6.21 (s, 1H), 4.64 (s, 2H), 4.51 (s, 1H), 3.83 (s, 3H), 3.82 (s, 3H), 3.54 (t, J = 8.3 Hz, 2H), 2.89 (t, J = 8.3 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 159.61 (s), 153.82 (s), 151.87 (s), 148.12 (s), 145.42 (s), 142.76 (s), 126.98 (s), 124.68 (s), 124.01 (s), 120.25 (s), 117.94 (s), 111.50 (s), 110.98 (s), 96.72 (s), 60.64 (s),
38
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60.42 (s), 56.24 (s), 47.77 (s), 40.75 (s), 29.03 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C20H18N3O3Br, 428.0532; found, 428.0612.
Ethyl
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5.1.7.
2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-
SC
carboxylate (9g). According to the synthetic procedure of 9a, compound 9g was obtained by using 3-bromo-4,5-dimethoxybenzaldehyde, ethyl 2-cyanoacetate and 9, and
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was isolated as a slight yellow solid (45% yield). 1H NMR (400 MHz, CDCl3) δ 6.96 (d, J = 1.8 Hz, 1H), 6.74 (s, 1H), 6.67 (d, J = 1.7 Hz, 1H), 6.33 (s, 2H), 6.23 (s, 1H), 4.72 (s, 1H), 4.07 (q, J = 17.8, 10.8, 7.1, 3.6 Hz, 2H), 3.78 (d, J = 2.2 Hz, 6H), 3.51-3.48 (m, 2H), 2.95 – 2.84 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.47 (s),
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160.56 (s), 153.32 (s), 151.25 (s), 148.29 (s), 146.01 (s), 144.42 (s), 126.30 (s), 124.65 (s), 123.83 (s), 117.08 (s), 114.74 (s), 111.25 (s), 96.81 (s), 60.56 (s), 59.46 (s), 56.06 (s), 47.76 (s), 40.06 (s), 29.10 (s), 14.48 (s). HRMS (ESI, m/z): [M + H]+ calcd. for
AC C
5.1.8.
EP
C22H23BrN2O5, 475.0790; found, 475.0869.
2-Amino-4-(3,4,5-trimethoxyphenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbon itrile (9h). According to the synthetic procedure of 9a, compound 9h was obtained by using 3,4,5-trimethoxybenzaldehyde, malononitrile and 9, and was isolated as a slight yellow solid (42% yield). 1H NMR (400 MHz, CDCl3) δ 6.65 (s, 1H), 6.38 (s, 2H), 6.21 (s, 1H), 4.62 (s, 2H), 4.52 (s, 1H), 3.81 (s, 9H), 3.54 (t, J = 8.4 Hz, 2H), 2.89 (t, J = 7.8 39
ACCEPTED MANUSCRIPT
Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 159.50 (s), 153.40 (s), 151.69 (s), 148.13 (s), 141.30 (s), 136.97 (s), 126.82 (s), 124.73 (s), 120.44 (s), 111.60 (s), 105.00 (s), 96.69 (s),
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60.90 (s), 56.21 (s), 47.79 (s), 41.37 (s), 29.06 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C21H21N3O4, 380.1532; found, 380.1607.
SC
5.1.9.
2-Amino-4-(3,4,5-trifluorophenyl)-4,6,7,8-tetrahydropyrano[3,2-f]indole-3-carbonitr
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ile (9i). The synthesis procedure of 9a was used to obtain compound 9i using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 9 as a slight yellow solid ( 51 % yield). 1H NMR (400 MHz, DMSO-d6) δ 7.18 – 7.01 (m, 2H), 6.91 (s, 2H), 6.64 (s, 1H), 6.09 (s, 1H), 5.73 (s, 1H), 4.64 (s, 1H), 3.39 (br., 2H), 2.77 (br., 2H).
13
C NMR (101
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MHz, DMSO-d6) δ 160.70 (s), 152.78 (s), 147.72 (s), 126.14 (s), 123.77 (s), 111.58 (s), 108.85 (s), 95.09 (s), 46.74 (s), 28.26 (s). HRMS (ESI, m/z): [M + H]+ calcd. for
5.1.10.
EP
C18H12F3N3O, 344.0932; found, 344.1008.
Ethyl
AC C
2-amino-4-(3-chlorophenyl)-4,7,8,9-tetrahydropyrano[2,3-e]indole-3-carboxylate (11a). According to the synthetic procedure of 9a, compound 11a was prepared by using 3-chlorobenzaldehyde, ethyl 2-cyanoacetate and 11 (the preparation of intermediate 11 was presented in supporting information), and was isolated as a slight yellow solid (16% yield). 1H NMR (400 MHz, CDCl3) δ 7.19 (s, 1H), 7.15 – 7.03 (m, 3H), 6.72 (d, J = 8.0 Hz, 1H), 6.34 (d, J = 8.0 Hz, 1H), 6.34 (br., 2H), 4.83 (d, 1H), 4.08-4.02 (m, 2H), 3.62 (t, 40
ACCEPTED MANUSCRIPT
J = 8.5 Hz, 2H), 3.15-3.00 (m, 2H), 1.19-1.10 (m, 3H).
13
C NMR (101 MHz, CDCl3) δ
169.52 (s), 160.35 (s), 152.25 (s), 150.90 (s), 145.73 (s), 133.84 (s), 129.46 (s), 128.70
(s), 47.83 (s), 39.84 (s), 26.85 (s), 14.43 (s).
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(s), 127.89 (s), 126.01 (s), 125.92 (s), 115.97 (s), 115.05 (s), 106.40 (s), 78.93 (s), 59.56 HRMS (ESI, m/z): [M + H]+ calcd. for
SC
C20H19N2O3Cl, 371.1084; found, 371.1155.
5.1.11.
Ethyl
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2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-4,7,8,9-tetrahydropyrano[2,3-e]indole-3carboxylate (11b). According to the synthetic procedure of 9a, compound 11b was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, ethyl 2-cyanoacetate and 11 (the preparation of intermediate 11 was presented in supporting information), and was
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isolated as a slight yellow solid (8.5 % yield). 1H NMR (400 MHz, CDCl3) δ 6.95 (s, 1H), 6.75 (d, J = 7.9 Hz, 1H), 6.68 (s, 1H), 6.38 (d, J = 7.9 Hz, 1H), 6.38 (s, 2H), 4.78 (s, 1H), 4.14-4.03 (m, 2H), 3.80 (s, 3H), 3.78 (s, 3H), 3.63 (t, J = 8.4 Hz, 2H), 3.14-3.04 (m, J =
EP
15.9, 7.5 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.53 (s),
AC C
160.35 (s), 153.38 (s), 151.69 (s), 145.98 (s), 145.75 (s), 144.58 (s), 128.66 (s), 123.83 (s), 117.26 (s), 116.42 (s), 115.34 (s), 111.35 (s), 106.75 (s), 78.98 (s), 60.63 (s), 59.59 (s), 56.20 (s), 47.78 (s), 39.76 (s), 29.84 (s), 26.87 (s), 14.53 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C22H23N2O5Br, 475.0790; found, 475.0858.
5.1.12. 2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-4,7,8,9-tetrahydropyrano[2,3-e]indole-3 41
ACCEPTED MANUSCRIPT
-carbonitrile (11c). According to the synthetic procedure of 9a, compound 11c was obtained by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 11 (the
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preparation of intermediate 11 was presented in supporting information), and was isolated as a slight yellow solid (45% yield). 1H NMR (400 MHz, CDCl3) δ 6.88 (d, J = 1.9 Hz, 1H), 6.71 (d, J = 1.8 Hz, 1H), 6.60 (d, J = 8.0 Hz, 1H), 6.35 (d, J = 8.0 Hz, 1H), 4.67 (s,
13
C NMR (101 MHz, CDCl3) δ 159.38 (s), 153.77 (s), 152.90 (s),
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8.3, 4.5 Hz, 2H).
SC
2H), 4.56 (s, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.63 (t, J = 8.5 Hz, 2H), 3.09-3.03 (m, J =
145.42 (d, J = 6.4 Hz), 142.75 (s), 128.91 (s), 123.99 (s), 120.17 (s), 117.95 (s), 115.14 (s), 112.15 (s), 111.50 (s), 106.69 (s), 60.67 (d, J = 5.7 Hz), 56.25 (s), 47.80 (s), 40.35 (s), 26.76 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C20H18BrN3O3, 428.0532; found,
5.1.13.
TE D
428.0604.
Ethyl
2-amino-4-(3-chlorophenyl)-6,7,8,9-tetrahydro-4H-pyrano[3,2-g]quinoline-3-carbox
EP
ylate (13a). According to the synthetic procedure of 9a, compound 13a was prepared by
AC C
using 3-chlorobenzaldehyde, ethyl 2-cyanoacetate and 13 (the preparation of intermediate 13 was presented in supporting information), and was isolated as a light red solid (35% yield). 1H NMR (400 MHz, CDCl3) δ 7.19 (s, 1H), 7.15-7.7.06 (m, 3H), 6.58 (s, 1H), 6.30 (br., 2H), 6.11 (s, 1H), 4.75 (s, 1H), 4.11-3.99 (m, 2H), 3.24 (d, J = 3.6 Hz, 2H), 2.68-2.50 (m, 2H), 1.85 (dd, J = 11.3, 5.9 Hz, 2H), 1.15 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.56 (s), 160.61 (s), 150.97 (s), 147.69 (s), 144.15 (s), 133.79 (s), 42
ACCEPTED MANUSCRIPT
129.80 (s), 129.45 (s), 128.00 (s), 126.03 (s), 125.97 (s), 118.61 (s), 113.70 (s), 100.35 (s), 78.94 (s), 59.48 (s), 41.88 (s), 39.73 (s), 26.54 (s), 22.12 (s), 14.44 (s). HRMS (ESI,
RI PT
m/z): [M + H]+ calcd. for C21H21N2O3Cl, 385.1241; found, 385.1320.
5.1.14.
Ethyl
SC
2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-6,7,8,9-tetrahydro-4H-pyrano[3,2-g]qui noline-3-carboxylate (13b). According to the synthetic procedure of 9a, compound 13b
M AN U
was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, ethyl 2-cyanoacetate and 13 (the preparation of intermediate 13 was presented in supporting information), and was isolated as a red-brown solid (25 % yield). 1H NMR (400 MHz, CDCl3) δ 6.96 (d, J = 1.8 Hz, 1H), 6.66 (d, J = 1.7 Hz, 1H), 6.60 (s, 1H), 6.29 (br., 2H), 6.10 (s, 1H), 4.69 (s, 1H),
TE D
4.14-4.01 (m, 2H), 3.25 (t, J = 4.9 Hz, 2H), 2.64-2.61 (m, J = 6.3, 4.0 Hz, 2H), 1.93-1.80 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.56 (s), 160.64 (s), 153.36 (s), 147.71 (s), 146.09 (s), 144.47 (s), 144.23 (s), 129.67 (s), 123.90 (s), 118.58
EP
(s), 117.11 (s), 113.66 (s), 111.35 (s), 100.30 (s), 79.04 (s), 60.61 (s), 59.48 (s), 56.14 (s),
AC C
41.87 (s), 39.61 (s), 26.56 (s), 22.12 (s), 14.52 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C23H25N2O5Br, 489.0947; found, 489.0958.
5.1.15.
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-6,7,8,9-tetrahydro-4H-pyrano[3,2-g]qui noline-3-carbonitrile (13c). According to the synthetic procedure of 9a, compound 13c was obtained by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 13 (the 43
ACCEPTED MANUSCRIPT
preparation of intermediate 13 was presented in supporting information), and was isolated as an off-white solid (69% yield). 1H NMR (400 MHz, DMSO-d6) δ 6.92 (d, J = 1.4 Hz,
RI PT
1H), 6.82 (d, J = 1.5 Hz, 1H), 6.79 (br., 2H), 6.50 (s, 1H), 6.08 (s, 1H), 5.89 (s, 1H), 4.50 (s, 1H), 3.80 (s, 3H), 3.70 (s, 3H), 3.12 (br., 2H), 1.79-1.61 (m, 2H).
13
C NMR (101
MHz, DMSO-d6) δ 161.07 (s), 153.75 (s), 147.49 (s), 145.68 (s), 145.00 (s), 144.59 (s),
SC
129.09 (s), 122.91 (s), 121.28 (s), 117.64 (s), 117.16 (s), 112.22 (s), 109.07 (s), 99.32 (s),
M AN U
60.44 (s), 56.54 (s), 56.39 (s), 26.57 (s), 21.74 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C21H20N3O3Br, 442.0688; found, 442.0760.
5.1.16.
Ethyl
2-amino-4-(3-chlorophenyl)-7,8,9,10-tetrahydro-4H-pyrano[2,3-f]quinoline-3-carbox
TE D
ylate (15a). According to the synthetic procedure of 9a, compound 15a was prepared by using 3-chlorobenzaldehyde, ethyl 2-cyanoacetate and 15 (the preparation of intermediate 15 was presented in supporting information), and was isolated as a light red solid (33%
EP
yield). 1H NMR (400 MHz, CDCl3) δ 7.19 (d, J = 1.1 Hz, 1H), 7.16-7.03 (m, 3H), 6.65
AC C
(d, J = 8.3 Hz, 1H), 6.33 (br., 2H), 6.20 (d, J = 8.2 Hz, 1H), 4.79 (s, 1H), 4.14-3.98 (m, 2H), 3.25 (t, J = 5.4 Hz, 2H), 2.77 (t, J = 6.5 Hz, 2H), 1.99-1.91 (m, 2H), 1.15 (t, J = 6.0, 3H). 13C NMR (101 MHz, CDCl3) δ 169.56 (s), 160.44 (s), 150.88 (s), 147.00 (s), 144.39 (s), 133.81 (s), 129.43 (s), 127.92 (s), 126.92 (s), 125.98 (s), 113.70 (s), 111.07 (s), 108.60 (s), 78.89 (s), 59.51 (s), 41.51 (s), 40.01 (s), 21.62 (s), 20.63 (s), 14.43 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C21H21N2O3Cl, 385.1241; found, 385.1319. 44
ACCEPTED MANUSCRIPT
5.1.17. 2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-7,8,9,10-tetrahydro-4H-pyrano[2,3-f]qu
RI PT
inoline-3-carbonitrile (15b). According to the synthetic procedure of 9a, compound 15b was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 15 (the preparation of intermediate 15 was presented in supporting information), and was isolated
SC
as an off-white solid (83% yield). 1H NMR (400 MHz, CDCl3) δ 6.89 (d, J = 1.9 Hz, 1H),
M AN U
6.72 (d, J = 1.8 Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H), 6.20 (d, J = 8.4 Hz, 1H), 4.64 (s, 2H), 4.53 (s, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.26 (t, J = 5.4 Hz, 2H), 2.73 (t, J = 6.5 Hz, 2H), 2.03 – 1.85 (m, 2H).
13
C NMR (101 MHz, CDCl3) δ 159.48 (s), 153.77 (s), 146.74 (s),
145.41 (s), 145.05 (s), 142.71 (s), 126.95 (s), 124.05 (s), 117.95 (s), 111.61 (s), 111.38
TE D
(s), 109.83 (s), 108.43 (s), 60.64 (s), 56.28 (s), 41.37 (s), 40.53 (s), 21.43 (s), 20.57 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C21H20N3O3Br, 442.0688; found, 442.0745.
5.1.18.
EP
6-amino-8-(3-chlorophenyl)-8H-[1,3]dioxolo[4,5-g]chromene-7-carboxylate
Ethyl (16a).
AC C
According to the synthetic procedure of 9a, compound 16a was prepared by using 3-chlorobenzaldehyde, ethyl 2-cyanoacetate and 16 (the preparation of intermediate 16 was presented in supporting information), and was isolated as an off-white solid (62% yield). 1H NMR (400 MHz, CDCl3) δ 7.18 (s, 1H), 7.16 – 7.03 (m, 3H), 6.52 (s, 1H), 6.45 (s, 1H), 6.30 (br., 2H), 5.91 (s, 1H), 5.87 (s, 1H), 4.80 (s, 1H), 4.17 – 3.97 (m, 2H), 1.16 (t, J = 7.1 Hz, 3H).
13
C NMR (101 MHz, CDCl3) δ 169.27 (s), 160.32 (s), 149.99 (s), 45
ACCEPTED MANUSCRIPT
146.96 (s), 144.57 (s), 143.28 (s), 134.03 (s), 129.59 (s), 127.96 (s), 126.41 (s), 125.96 (s), 117.60 (s), 107.87 (s), 101.62 (s), 98.00 (s), 78.09 (s), 59.64 (s), 40.62 (s), 14.43 (s).
RI PT
HRMS (ESI, m/z): [M + Na]+ calcd. for C19H16NO5Cl, 396.0717; found, 396.0609.
5.1.19.
SC
Ethyl-6-amino-8-(3,4,5-trimethoxyphenyl)-8H-[1,3]dioxolo[4,5-g]chromene-7-carbox ylate (16b). According to the synthetic procedure of 9a, compound 16b was prepared by
M AN U
using 3,4,5-trimethoxybenzaldehyde, ethyl 2-cyanoacetate and 16 (the preparation of intermediate 16 was presented in supporting information), and was isolated as a slight yellow solid (24% yield). 1H NMR (400 MHz, CDCl3) δ 6.52 (s, 2H), 6.40 (s, 2H), 6.27 (br., 2H), 5.92 (s, 1H), 5.88 (s, 1H), 4.75 (s, 1H), 4.10-4.06 (m,, 2H), 3.80 (s, 6H), 3.78 13
C NMR (101 MHz, CDCl3) δ 169.45 (s), 160.37 (s),
TE D
(s, 3H), 1.18 (t, J = 7.1 Hz, 3H).
153.07 (s), 146.76 (s), 144.45 (s), 143.80 (s), 143.25 (s), 136.50 (s), 118.38 (s), 107.80 (s), 104.66 (s), 101.56 (s), 97.87 (s), 78.55 (s), 60.88 (s), 59.52 (s), 56.15 (s), 41.10 (s),
AC C
452.1326.
EP
14.55 (s). HRMS (ESI, m/z): [M + Na]+ calcd. for C22H23NO8, 452.1424; found,
5.1.20.
6-amino-8-(3,4,5-trimethoxyphenyl)-8H-[1,3]dioxolo[4,5-g]chromene-7-carbonitrile (16c). According to the synthetic procedure of 9a, compound 16c was prepared by using 3,4,5-trimethoxybenzaldehyde, malononitrile and 16 (the preparation of intermediate 16 was presented in supporting information), and was isolated as an off-white solid. 1H 46
ACCEPTED MANUSCRIPT
NMR (400 MHz, DMSO-d6) δ 6.88 (s, 2H), 6.67 (s, 1H), 6.64 (s, 1H), 6.50 (s, 2H), 6.00 (s, 1H), 5.96 (s, 1H), 4.57 (s, 1H), 3.73 (s, 6H), 3.63 (s, 3H). 13C NMR (101 MHz,
RI PT
DMSO-d6) δ 160.51 (s), 152.97 (s), 146.73 (s), 143.98 (s), 142.51 (s), 141.63 (s), 136.34 (s), 120.59 (s), 115.42 (s), 107.29 (s), 104.51 (s), 101.64 (s), 97.71 (s), 59.93 (s), 55.85 (s), 40.96 (s), 18.88 (s). HRMS (ESI, m/z): [M + Na]+ calcd. for C20H18N2O6, 405.1165;
SC
found, 405.1062.
M AN U
5.1.21.
6-amino-8-(3-bromo-4,5-dimethoxyphenyl)-8H-[1,3]dioxolo[4,5-g]chromene-7-carbo nitrile (16d). According to the synthetic procedure of 9a, compound 16d was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 16 (the preparation of
TE D
intermediate 16 was presented in supporting information), and was isolated as an off-white solid (74% yield). 1H NMR (400 MHz, DMSO-d6) δ 6.98 (d, J = 1.8 Hz, 1H), 6.96(s, 2H), 6.87 (d, J = 1.8 Hz, 1H), 6.68 (s, 1H), 6.65 (s, 1H), 6.02 (s, 1H), 5.97 (s, 1H),
EP
4.63 (s, 1H), 3.81 (s, 3H), 3.70 (s, 3H).
13
C NMR (101 MHz, DMSO-d6) δ 160.56 (s),
AC C
153.39 (s), 146.93 (s), 144.40 (s), 144.12 (s), 143.50 (s), 142.55 (s), 122.36 (s), 120.42 (s), 116.83 (s), 114.87 (s), 111.71 (s), 107.22 (s), 101.72 (s), 97.81 (s), 59.99 (s), 56.07 (s), 55.00 (s). HRMS (ESI, m/z): [M + Na]+ calcd. for C19H15N2O5Br, 431.0164; found, 431.0085.
5.1.22. 6-Amino-8-(3-bromo-4,5-dimethoxyphenyl)-4-hydroxy-8H-[1,3]dioxolo[4,5-g]chrom 47
ACCEPTED MANUSCRIPT
ene-7-carbonitrile (19a). According to the synthetic procedure of 9a, compound 19a was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 19 (the
RI PT
preparation of intermediate 19 was presented in supporting information), and was isolated as an off-white solid (50% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H), 6.97 (d, J = 1.8 Hz, 1H), 6.85 (d, J = 1.9 Hz, 1H), 6.82 (br., 2H), 6.19 (s, 1H), 5.95 (s, 1H), 5.91
SC
(s, 1H), 4.60 (s, 1H), 3.81 (s, 3H), 3.70 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.67
M AN U
(s), 153.39 (s), 144.37 (s), 144.27 (s),143.61 (s), 134.19 (s), 133.99 (s), 129.61 (s), 122.25 (s), 120.50 (s), 116.81 (s), 115.98 (s), 111.63 (s), 101.33 (s), 98.03 (s), 60.02 (s), 56.10 (s), 55.28 (s), 30.72 (s), 18.58 (s). HRMS (ESI, m/z): [M + 3H]+ calcd. for C19H15BrN2O6, 449.0113; found, 449.0165.
TE D
5.1.23.
6-Amino-4-hydroxy-8-(3,4,5-trimethoxyphenyl)-8H-[1,3]dioxolo[4,5-g]chromene-7-c arbonitrile (19b). According to the synthetic procedure of 9a, compound 19a was
EP
prepared by using 3,4,5-trimethoxybenzaldehyde, malononitrile and 19 (the preparation
AC C
of intermediate 19 was presented in supporting information), and was isolated as an off-white solid (20% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.79 (s, 1H), 6.73 (s, 2H), 6.49 (s, 2H), 6.17 (s, 1H), 5.94 (s, 1H), 5.90 (s, 1H), 4.55 (s, 1H), 3.73 (s, 6H), 3.62 (s, 3H).
13
C NMR (101 MHz, DMSO-d6) δ 160.57 (s), 152.94 (s), 144.10 (s), 141.65 (s),
136.33 (s), 133.95 (s), 129.50 (s), 120.64 (s), 116.44 (s), 104.47 (s), 101.23 (s), 98.08 (s),
48
ACCEPTED MANUSCRIPT
59.94 (s), 55.86 (s), 55.68 (s), 41.32 (s), 18.88 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C20H18N2O7, 399.1114; found, 399.1185.
RI PT
5.1.24.
7-amino-9-(3-bromo-4,5-dimethoxyphenyl)-3,9-dihydro-2H-[1,4]dioxino[2,3-g]chro
SC
mene-8-carbonitrile (22a). According to the synthetic procedure of 9a, compound 22a was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 22 (the
M AN U
preparation of intermediate 22 (the preparation of intermediate 22 was presented in supporting information) was presented in supporting information), and was isolated as an off-white solid (4% yield). 1H NMR (400 MHz, DMSO-d6) δ 6.99 (d, J = 1.8 Hz, 1H), 6.95 (s, 2H), 6.87 (d, J = 1.8 Hz, 1H), 6.59 (s, 1H), 6.55 (s, 1H), 4.64 (s., 1H), 4.20 (br.,
TE D
2H), 4.17 (br., 2H), 3.81 (s, 3H), 3.70 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 161.13 (s), 153.85 (s), 144.06 (s), 143.34 (s), 142.40 (s), 142.40 (s), 122.84 (s), 120.97 (s), 117.31 (s), 117.28 (s), 116.55 (s), 115.97 (s), 112.21 (s), 104.64 (s), 100.00 (s), 64.67 (s),
EP
64.34 (s), 60.46 (s), 56.56 (s), 55.40 (s), 17.34 (s). HRMS (ESI, m/z): [M + H]+ calcd. for
AC C
C20H17BrN2O5, 445.0321; found, 445.0387.
5.1.25.
7-Amino-9-(3-bromo-4,5-dimethoxyphenyl)-2,3,4,9-tetrahydrochromeno[6,7-b][1,4] oxazine-8-carbonitrile (27a). According to the synthetic procedure of 9a, compound 27a was prepared by using 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile and 27 (the preparation of intermediate 27 was presented in supporting information), and was isolated 49
ACCEPTED MANUSCRIPT
as a slight yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 6.95 (d, J = 1.7 Hz, 1H), 6.84 (d, J = 1.8 Hz, 1H), 6.82 (br., 2H), 6.31 (s, 1H), 6.22 (s, 1H), 6.02 (s, 1H), 4.52 (s, 1H),
RI PT
4.02 (t, J = 4.0 Hz, 2H), 3.80 (s, 3H), 3.70 (s, 3H), 3.22 (br., 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.83 (s), 153.34 (s), 144.21 (s), 142.36 (s), 139.87 (s), 134.92 (s), 122.40 (s), 120.87 (s), 116.78 (s), 114.87 (s), 111.72 (s), 109.80 (s), 100.66 (s), 79.19 (s), 64.44
SC
(s), 60.03 (s), 56.09 (s), 55.19 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C20H18BrN3O4,
M AN U
444.0481; found, 444.0548.
5.1.26.
7-Amino-9-(3,4,5-trimethoxyphenyl)-2,3,4,9-tetrahydrochromeno[6,7-b][1,4]oxazine -8-carbonitrile (27b). According to the synthetic procedure of 9a, compound 27b was
TE D
prepared by using 3,4,5-trimethoxybenzaldehyde, malononitrile and 27 (the preparation of intermediate 27 was presented in supporting information), and isolated as a slight yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 6.74 (s, 2H), 6.48 (s, 2H), 6.31 (s, 1H),
13
C NMR (101 MHz, DMSO-d6) δ 160.78 (s), 152.91 (s), 142.33 (s),
AC C
3.22 (br., 2H).
EP
6.21 (s, 1H), 5.97 (s, 1H), 4.47 (s, 1H), 4.02 (t, J = 3.9 Hz, 2H), 3.72 (s, 6H), 3.63 (s, 3H),
142.25 (s), 139.79 (s), 136.19 (s), 134.69 (s), 121.04 (s), 114.93 (s), 110.40 (s), 104.53 (s), 100.65 (s), 79.19 (s), 64.45 (s), 59.96 (s), 55.86 (s), 55.56 (s). HRMS (ESI, m/z): [M + H]+ calcd. for C21H21N3O5, 396.1481; found, 396.1558.
General synthetic procedure for intermediates 30a-b, 34a-b, and compounds 31a-h, 35a-c were descripted in supporting information. 50
ACCEPTED MANUSCRIPT
5.2
Cell culture
A375, CHL-1, HeLa, HT-29, HCT116 and SW480 cells were cultured in complete
RI PT
growth medium (DMEM high glucose medium, PH7.4, supplemented with 10% fetal bovine serum, 100 mg/mL streptomycin, and 100 units/mL penicillin). B16-F10, A549 and Jurkat cells were growing in RPMI-1640 medium supplemented with 10% fetal
SC
bovine serum, 100 mg/mL streptomycin, 100 units/mL penicillin and 2mM L-glutamine. HUVECs were kindly provided by Prof. Qin Shen’s lab at Tsinghua University and
M AN U
cultured in endothelial growth medium purchased from ScienCell. PBMCs were kindly provided by Dr. Xingquan Zhang from University of California, San Diego, and cultured in RPMI-1640 medium (with 10% fetal bovine serum, 100 mg/mL streptomycin, 100 units/mL penicillin and 5 U/mL IL-2). All cells were incubated in incubator at 37°C in 5%
5.3
TE D
CO2 and 90% relative humidity.
Measurement of cell viability
EP
The anti-proliferative effects of the compounds on tumor cells were measured by examining cell survival after exposure to compounds for 72 h. Briefly, 1000-5000 cells
AC C
(A375, CHL-1, B16-F10, HeLa, HT-29, HCT116, SW480, MCF-7 and HUVECs) or 100000 cells (Jurkat and PBMCs) per well were seeded in a 96-well cell culture plate overnight at 37ºC in an incubator in 5% CO2 and humidified atmosphere and then treated with different concentrations of 27a, 19a, 9f, and colchicine in fresh medium for 72 h. Cell viability was determined by adding 20 µL/well of the CellTiter reagent (96® Aqueous One Solution Cell Proliferation Assay kit, Promega) and incubating for an additional 2h. The absorbance at 490nm was then recorded using a microplate reader 51
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(Envision).
5.4
Cell cycle analysis
RI PT
A375 (1×106/well) were plated in a 6-well plate and cultured overnight. The cells were then treated with DMSO (vehicle control) or various doses of 27a, 19a, or 9f for 24 h. After incubation with 0.25% trypsin, two washes with PBS, the cells were resuspended in
SC
100 µL of PBS plus 1 mL 75% cold ethanol and fixed at -20°C overnight. The cells were then centrifuged at 1500 rpm for 5 min and washed twice with 1X buffer A, and
M AN U
incubated with 0.25 mg/mL RNAse at 37°C for 30 minutes. The cells were stained with 5 µL propidium iodide (PI) (KeyGEN BioTECH) for another 30 min in dark, according to the manufacturer’ manual, and examined using FACScalibur system (Becton Dickinson).
5.5
Apoptosis assay
TE D
The data were analyzed using FlowJo7.5 analysis software.
Apoptosis was determined with an Annexin V-FITC/PI apoptosis detection kit
EP
(Bioworld). A375 cells (1×106/well) were seeded in a 6-well plate and incubated
AC C
overnight. Different concentrations (10, 100, 1000 nM) of 27a, 19a, 9f, and colchicine as positive control were added to the cultured cells and incubated for 24 h and 48 h. The cells were trypsinized, washed twice in cold PBS, and centrifuged at 2000 rpm for 5 min. The pellet was resuspended in 400 µL staining solution and stained with 5 µL Annexin V-FITC and 10 µL PI for 15 min protecting from light at room temperature. Samples were examined using an LSRFortessa flow cytometer (BD) and four distinct populations of cells were detected. The normal healthy, early apoptosis, late apoptosis, and necrotic 52
ACCEPTED MANUSCRIPT
cells were respectively represented by annexin V-/PI-, annexin V+/PI-, annexin V+/PI+ and annexin V-/PI+ populations. Analyses were performed using the software supplied
5.6
RI PT
with the instrument.
Immunofluorescence staining of tubulin
A375 cells were seeded on the coverslips in a 6-well plate overnight. Cells were then
SC
incubated with 27a, 19a, 9f, or vehicle control separately for 24 h. Thereafter, 4% paraformaldehyde were used to fix cells for 30 min at 4°C and followed by incubation
M AN U
with 0.1% Triton X-100 permeabilizing buffer for 15 min at room temperature. After washing with PBS and blocking with 2% BSA in PBS for 1 h at room temperature, cells were incubated with 1:2000 monoclonal anti-α-tubulin antibody in PBS at 4°C overnight. Then cells were washed with PBS and incubated, while protecting from light, with 1:200
TE D
fluorescein isothiocyanate (FITC)–labeled anti-mouse IgG antibody (Sigma) at room temperature. After staining with 4,6-diamidino-2-phenylindole (DAPI) to label the nuclei, the cells were washed and finally immobilized with mounting medium on glass slides.
Colony formation assay
AC C
5.7
EP
Cell images were visualized using a laser scanning confocal microscope (Zeiss).
1.0 × 104 A375 cells/well were seeded in 60 mm culture dishes and incubated overnight. Different concentrations of compounds in fresh medium containing 10% FBS were then added. The cells were left for several days to form colonies before they were subjected to microscopic examination. Clones containing over 50 cells were regarded as one colony [65]. Colony numbers were quantified by ImageJ. 53
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5.8
Tube formation assay
Matrigel (BD) was dissolved overnight at 4°C, and 96-well plates and tips were
RI PT
pre-chilled before adding matrigel. After adding 50 µL of matrigel to each well and leaving it to solidify for 45 min at 37°C, endothelia cell medium (ScienCell) containing DMSO (control) or different concentrations of 27a, 19a, or 9f were added. After 6-8 h,
SC
fluorescence microscopy images were captured (Olympus). Total length of the formed
5.9
Tubulin polymerization assay
M AN U
tube were measured by ImageJ.
HTS-tubulin polymerization assay biochem kit (Cat. #BK004P) was purchased from Cytoskeleton. Tubulin polymerization assay was conducted following the instructions.
TE D
Briefly, freshly prepared 4 mg/mL tubulin (>97% pure, plus 2% microtubule associated proteins) in cold G-PEM buffer was added into each well of a half-area 96-well plate in the presence of test compounds (10× in Genral Tubulin Buffer). Absorbance at 340nm
AC C
(Enspire).
EP
was immediately recorded at 37oC for 2 h using the kinetic settings of spectrophotometer
5.10 Molecular docking modeling
The complex crystal structure of α/β–tubulin heterodimers with DAMA-colchicine was downloaded from the PDB database (PDB 1SA0). The molecular docking modeling was performed using the Molecular Operating Environment (MOE2015.10) software. Receptors and ligands were prepared according to the MOE standard tutorial. Docking 54
ACCEPTED MANUSCRIPT
program was first validated by re-docking of the co-crystallized DAMA-colchicine in the colchicine-binding pocket. All parameters for docking modeling were set to default
RI PT
values in the run program. AUTHOR INFORMATION
SC
Author Contributions
Huijun Zhang designed and performed the biological experiments, analyzed the data and
M AN U
wrote the manuscript. Xiong Fang designed and synthesized the compounds, analyzed the SAR data and molecular modeling results and helped write the manuscript. Yujia Mao and Yan Xu helped the design of the modeling and biological studies and contributed to the analyses of the results. Qian Meng performed flow-apoptosis and tubulin
TE D
polymerization assays. Tingting Fan conducted cell proliferation assays on HUVECs. Ziwei Huang and Jing An oversaw the entire project and revised the manuscript. All
Notes
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authors have reviewed the results and approved the final version of the manuscript.
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Declaration of interest: none.
ACKNOWLEDGEMENTS
This work was supported by the grant from the Tsinghua-Peking Joint Center for Life Sciences (to Z. H.), Tsinghua University (to Z.H.), the National Institutes of Health (GM 57761, to Z.H and J.A.), and the California Institute for Regenerative Medicine (to Z.H.). We also thank Prof. Qin Shen of Tsinghua University for the gift of HUVECs and Dr. 55
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Xingquan Zhang from UCSD for the gift of PBMCs.
ABBREVIATIONS USED:
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SAR, structure-activity relationship; TBMs, Tubulin-binding molecules; CBSTs, colchicine binding site agents; MTAs, microtubule targeting agents; HUVECs, human
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umbilical vein endothelia cells; BMSCs, bone marrow stromal cells.
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A series of fused 4-aryl-4H-chromene-based derivatives were designed, and synthesized
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Biological assays were conducted to assess their anti-proliferation and anti-angiogenesis activities
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Molecular docking studies showed that fused 4-aryl-4H-chromene scaffold can mimic DAMA-colchicine in the colchicine-binding site
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Compound 27a exhibited potent anti-proliferation and anti-angiogenesis activities