molecules Article
Discovery of N-(Naphtho[1,2-b]Furan-5-Yl) Benzenesulfonamides as Novel Selective Inhibitors of Triple-Negative Breast Cancer (TNBC) Ya Chen 1,† , Yong Tang 2,† , Beibei Mao 1 , Wenchao Li 1 , Hongwei Jin 1 , Liangren Zhang 1, * and Zhenming Liu 1, * 1
2
* †
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China;
[email protected] (Y.C.);
[email protected] (B.M.);
[email protected] (W.L.);
[email protected] (H.J.) Beijing Shenogen Biomedical Co., Ltd., Beijing 102206, China;
[email protected] Correspondence:
[email protected] (L.Z.);
[email protected] (Z.L.); Tel.: +86-10-8280-2567 (L.Z.); +86-10-8280-5514 (Z.L.); Fax: +86-10-8280-5063 (L.Z.); Fax: +86-10-82802724 (Z.L.) Those authors contribute equally to this work.
Received: 11 February 2018; Accepted: 16 March 2018; Published: 16 March 2018
Abstract: Any type of breast cancer not expressing genes of the estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2) is referred to as triple-negative breast cancer (TNBC). Accordingly, TNBCs do not respond to hormonal therapies or medicines targeting the ER, PR, or HER2. Systemic chemotherapy is therefore the only treatment option available today and prognoses remain poor. We report the discovery and characterization of N-(naphtho[1,2-b]furan-5-yl)benzenesulfonamides as selective inhibitors of TNBCs. These inhibitors were identified by virtual screening and inhibited different TNBC cell lines with IC50 values of 2–3 µM. The compounds did not inhibit normal (i.e. MCF-7 and MCF-10A) cells in vitro, indicating their selectivity against TNBC cells. Considering the selectivity of these inhibitors for TNBC, these compounds and analogs can serve as a promising starting point for further research on effective TNBC inhibitors. Keywords: triple-negative breast cancer; three-dimensional similarity search; virtual screening; selective inhibitors
1. Introduction Breast cancer is the most common malignancy and second leading cause of cancer death among women in the United States [1]. As in most countries, breast cancer is the most common cancer in Chinese women today. Cases in China account for 12.2% of all newly diagnosed breast cancers and for 9.6% of all deaths from breast cancer worldwide [2]. Based on DNA microarray expression profiling, breast cancers can be classified into six different subtypes [3–8]: luminal A, luminal B, human epidermal growth factor receptor-2 (HER2)-overexpressing, normal breast tissue-like, basal-like, and claudin-low breast cancers. These subtypes respond differently to therapy and are associated with different outcomes, with the shortest survival times seen in patients with basal-like and HER2-overexpressing subtypes [4,5,9]. Triple-negative breast cancer (TNBC) is an aggressive clinical phenotype characterized by the lack of expression (or minimal expression) of the estrogen receptor (ER) and progesterone receptor (PR) as well as the absence of the human epidermal growth factor receptor-2 (HER2). TNBCs comprise a heterogeneous subgroup of tumors, including but not limited to those classified by expression profiling as basal-like and claudin-low subtypes. TNBCs account for about 15% of all breast cancers [7–10]. Unlike patients suffering from ER/PR-positive or HER2-overexpressing cancers, treatment options Molecules 2018, 23, 678; doi:10.3390/molecules23030678
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Moleculescancers 2018, 23, 678 2 of 12 breast [7–10]. Unlike patients suffering from ER/PR-positive or HER2-overexpressing cancers, treatment options for patients with TNBC are currently limited to systemic cytotoxic chemotherapy [11]. The are overall survival ratestoofsystemic TNBC patients lower than those of patients for patients with TNBC currently limited cytotoxicare chemotherapy [11]. The overall suffering from other phenotypes of breast cancer (in both early and advanced stages) [12,13]. These survival rates of TNBC patients are lower than those of patients suffering from other phenotypes of facts highlight the urgent need for effective medicines for the treatment of TNBCs. breast cancer (in both early and advanced stages) [12,13]. These facts highlight the urgent need for Currently, compounds targetingofthe vascular endothelial growth factor (VEGF), poly (ADPeffective medicines for the treatment TNBCs. ribose) polymerase (PARP), HSP90, and aurora kinase are growth under investigation clinical trials as Currently, compounds targeting the vascular endothelial factor (VEGF),inpoly (ADP-ribose) therapeutics (PARP), for metastatic TNBCs Herein, weare report the investigation computer-guided discovery of Npolymerase HSP90, and [14]. aurora kinase under in clinical trials as (naphtho[1,2-b]furan-5-yl)benzenesulfonamides as effective and selective inhibitors of TNBCs. These therapeutics for metastatic TNBCs [14]. Herein, we report the computer-guided discovery of compounds serve as starting points for the development of effective N-(naphtho[1,2-b]furan-5-yl)benzenesulfonamides as effective anddrugs. selective inhibitors of TNBCs.
These compounds serve as starting points for the development of effective drugs. 2. Results and Discussion 2. Results and Discussion 2.1. Three-Dimensional Similarity Search and Bioassays 2.1. Three-Dimensional Similarity Search and Bioassays Estrogens are known to stimulate cell proliferation and increase the risk of the development of Estrogens aretypes known to stimulate cell proliferation anduterus increase the risk of In theorder development of several different of cancers, in particular breast and cancers [15]. to identify severalinhibitors different of types of cancers, particular breast and uterus cancers [15]. In order to identify novel novel breast cancers,in we employed similarity-based computational approaches to search inhibitors breast cancers, we employed similarity-basedaccessed computational approaches to search the the SPECSof compound library (http://www.specs.net/, by May 2014) for candidate SPECS compound library (http://www.specs.net/, byagent May 2014) for candidate compounds. compounds. 17β-estradiol and IC-163 (Figure 1), aaccessed potential for breast cancer identified by 17β-estradiol and IC-163 (Figure 1),[16], a potential agent for identified by Beijing Shenogen Beijing Shenogen Biomedical Co. were chosen asbreast querycancer molecules for 3D similarity search Biomedical (Figure 2). Co. [16], were chosen as query molecules for 3D similarity search (Figure 2).
Figure of IC-163. Figure 1. 1. Chemical Chemical structure structure of IC-163.
In total, approximately 200 k compounds were screened with ROCS, an alignment-based virtual In total, approximately 200 k compounds were screened with ROCS, an alignment-based virtual screening engine quantifying the similarity of pairs of molecules based on their molecular shapes and screening engine quantifying the similarity of pairs of molecules based on their molecular shapes chemical features [17,18]. The most interesting compounds (selected by visual inspection) were reand chemical features [17,18]. The most interesting compounds (selected by visual inspection) were ranked with EON (version 2.2.0, OpenEye Scientific Software Inc., Santa Fe, NM, USA) to evaluate re-ranked with EON (version 2.2.0, OpenEye Scientific Software Inc., Santa Fe, NM, USA) to evaluate compound similarity with regard to electrostatics. EON quantifies the similarity between pairs of compound similarity with regard to electrostatics. EON quantifies the similarity between pairs of molecules based on their electrostatic potential maps. Comparison with EON resulted in rankmolecules based on their electrostatic potential maps. Comparison with EON resulted in rank-ordered ordered list of 435 candidate molecules, which was further reduced by clustering with ECFP_6 and list of 435 candidate molecules, which was further reduced by clustering with ECFP_6 and FCFP_6 fingerprints. In total, 32 candidate compounds (Figure S1) were selected by visual inspection FCFP_6 fingerprints. In total, 32 candidate compounds (Figure S1) were selected by visual inspection (taking into account calculated aqueous solubility) and purchased from SPECS for experimental (taking into account calculated aqueous solubility) and purchased from SPECS for experimental evaluation. evaluation. Twenty-five of the selected compounds originate from 17β-Estradiol as query and seven Twenty-five of the selected compounds originate from 17β-Estradiol as query and seven from IC-163. from IC-163.
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Commercial library from Specs ~200000 compounds
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Commercial library from Specs ~200000 compounds
ROCS 2 Queries (17β-estradiol and IC-163)
ROCS
EON Clustering
Solubilit y_level
2 Queries (17β-estradiol and IC-163)
Selected Hits 32
EON Clustering
Selected Hits 32
Solubilit y_level
435 hits 435 hits
Figure 2. Scheme for three-dimensional virtual screening. Figure 2. Scheme for three-dimensional virtual screening. Figure 2. Scheme for three-dimensional virtual screening.
The inhibition rates of these 32 compounds were measured on the TNBC cell lines MDA-MB-231 The inhibition rates of these 32 compounds were measured on the TNBC cell lines MDA-MBand SUM-159, as well asrates the non-TNBC breast cancer cellmeasured line MCF-7the (Figure The interesting The inhibition of these 32 compounds were TNBCS2). cell linesmost MDA-MB231 and SUM-159, as well as the non-TNBC breast cancer cell on line MCF-7 (Figure S2). The most compounds candidates were B09 cell andline C10. Compound identified 231 andidentified SUM-159, among as well those as the 32 non-TNBC breast cancer MCF-7 (Figure B09, S2). The most by interesting compounds identified among those 32 candidates were B09 and C10. Compound B09, interesting identified among thoseinhibited 32 candidates were B09with and IC C10. Compound B09, had similarity searchcompounds using 17β-estradiol as query, MCF-7 cells = 1.45 µM and 50 identified by similarity search using 17β-estradiol as query, inhibited MCF-7 cells with IC50 = 1.45 μM identified by similarity search using 17β-estradiol as query, inhibited MCF-7 cells with IC 50 = 1.45 μM almost no growth-inhibitory effect on MDA-MB-231 and SUM-159. 17β-Estradiol is an endogenous and had almost no growth-inhibitory effect on MDA-MB-231 andSUM-159. SUM-159. 17β-Estradiol and had almost no growth-inhibitory effect on MDA-MB-231 and 17β-Estradiol is anis an molecule directly interacting with the estrogen receptor. This may explain why B09 only inhibited endogenous molecule directly interacting with the receptor.This This may explain why B09 endogenous directly interacting the estrogen estrogennot receptor. explain B09 only only MCF-7 cells. B09molecule is structurally related towith 17β-estradiol only withmay respect to why its 3D shape but inhibited MCF-7 cells. B09B09 is structurally notonly only with respect to3D itsshape 3D shape inhibited MCF-7 cells. is structurallyrelated relatedto to 17β-estradiol 17β-estradiol not with respect to its also its 2D structure (Figure 3). On the contrary, C10 showed good inhibition of TNBC cell lines but also (Figure 3).3). On showedgood good inhibition of TNBC cell lines but its also2D itsstructure 2D structure (Figure Onthe thecontrary, contrary, C10 C10 showed inhibition of TNBC cell lines (IC50 = 2.32 µM for MDA-MB-231; IC50 = 3.45 µM for SUM-159) but low inhibition of MCF-7 cells 50 = μM 2.32 for μMMDA-MB-231; for MDA-MB-231; 3.45μM μMfor for SUM-159) SUM-159) but inhibition of MCF-7 cellscells (IC50 (IC50 (IC50 =(IC 2.32 ICIC 50 50 = =3.45 butlow low inhibition of MCF-7 (IC50 ==20 20μM; µM; Table 1). Its chemical structure is to similar to that with of IC-163 with respect to the 3D Table 1). chemical Its chemical structure similar that of respect to the 3D molecular = 20 μM; Table 1). Its structure isissimilar to that ofIC-163 IC-163 with respect to the 3D molecular molecular shape and electrostatic properties (ShapeTanimoto coefficient = 0.817; EON_ShapeTanimoto electrostatic properties (ShapeTanimoto coefficient shapeshape and and electrostatic properties (ShapeTanimoto coefficient = =0.817; 0.817;EON_ShapeTanimoto EON_ShapeTanimoto coefficient = 0.784, where values of 1 denote compounds with identical properties) butbut notnot with respect coefficient = 0.784, where values of 1 denote compounds with identical properties) with coefficient = 0.784, where values of 1 denote compounds with identical properties) but not with to the 2D structure to therespect 2D structure (Figure 3). (Figure 3). respect to the 2D structure (Figure 3). Table 1. Activities B09and andC10 C10 measured measured on cell lines. Table 1. Activities ofofB09 ondifferent different cell lines. Table 1. Activities of B09 and C10 measured on different cell lines. IC50 (μM) Compound IC50 (µM) IC (μM) Compound MDA-MB-231 50SUM-159 MCF-7 Compound MDA-MB-231
MDA-MB-231 ~20
B09
B09 C10 B09 C10
~20 2.32 2.32~20
C10
A
2.32
OH
A
O
OH
NH2
O
H
OH
17beta-estradiol
OH HN
O OHOCH3
IC-163
O
B09 OCH3
OH O
20
B
OH S
OH
O
1.45
20 1.45 20
B09
HO
HO
3.45
MCF-7
MCF-7 1.45
S
17beta-estradiol H H
OHHO
SUM-159 >40
>40 >40 3.453.45
B
S
O
H
H HO
NH2
O
H
SUM-159
HN
O
S S
OH O
O
O O S O C10
OH OH O
Figure (A) Structures of 17β-estradiol,B09, B09, IC-163 IC-163 and alignment of B09 withwith Figure 3. (A)3. Structures of 17β-estradiol, and C10. C10;(B) (B)Structure Structure alignment of B09 O 17β-estradiol and of C10 with IC-163. 17β-estradiol IC-163 and of C10 with IC-163. C10
Figure 3. (A) Structures of 17β-estradiol, B09, IC-163 and C10. (B) Structure alignment of B09 with 17β-estradiol and of C10 with IC-163.
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2.2. Hit Follow-up and Expansion 2D similarity search based on ECFP_6 and FCFP_6 was conducted to identify further purchasable analogs of C10 for experimental evaluation. A total of 12 analogs of C10 were purchased from SPECS and tested on MDA-MB-231 and SUM-159 cell lines. The measured inhibition rates are reported in Figure S3. All compounds were initially tested only with two TNBC cell lines for cell viability at 5 µg/mL. Following this test, the inhibitory activity of any compounds with inhibition rates above 30% at 5 µg/mL (eight compounds) were tested on four TNBC cell lines and one non-TNBC cell line MCF-7 (Table 2). All eight compounds inhibited MDA-MB-231 and MDA-MB-453 cells with IC50 values lower than 10 µM. Most compounds inhibited SUM-159 and BT-20 cells with IC50 values greater than 10 µM; seven of these compounds inhibited MCF-7 cells with IC50 values greater than 40 µM. Among them, Compounds 2-5 and 2-8 exhibited the strongest inhibitory effect on all tested TNBC cell lines and had no inhibitory effect on MCF-7. Table 2. Activities of eight analogs of C10 measured on different cell lines. IC50 (µM)
Compound Tamoxifen IC-163 C10 2-1 2-3 2-5 2-6 2-7 2-8 2-9 2-11
MDA-MB-231
MDA-MB-453
SUM-159
BT-20
MCF-7
2.03 >40 5.34 4.36 7.99 3.12 4.97 3.95 2.96 6.22 6.92
3.64 >40 2.30 3.19 8.59 2.95 3.36 3.65 3.09 4.61 5.72
13.48 >40 11.13 30.76 >40 2.91 19.21 >40 2.50 18.06 >40
8.54 >40 10.63 13.34 >40 >40 25.02 >40 10.10 >40 >40
9.08 >40 9.38 13.97 >40 >40 >40 >40 >40 >40 >40
All these compounds are based on a N-(naphthalen-1-yl)benzenesulfonamide scaffold (Figure 4), with different decorations in para position of the benzenesulfonamide and/or the substituent at the naphthalene ring. Compounds C10, 2-1, 2-3, 2-6, and 2-7 are known inhibitors of myeloid cell leukemia 1 (Mcl-1) [19]. Compound 2-11 is an antimalarial heme detoxification protein (HDP) inhibitor [20], and 2-9 is an antitumor agent with inhibition of signal transducer and activator of transcription 3 (STAT3) [21,22]. We did not identify literature on the bioactivity of 2-5 and 2-8. In terms of molecular structure, 2-5 and 2-8 clearly differ from other compounds of that series. They are decorated with a naphtho[1,2-b]furan in para position of the benzenesulfonamide. Compound 2-5 is a methyl carboxylate, while 2-8 is an ethyl carboxylate. Due to structural novelty and good bioactivity, 2-5 and 2-8 were selected for another iteration of hit expansion based on 2D similarity search.
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OH
OH
OH O
O O S
HN
2-6
OH
OH S
S
HN
5 of 12
O
O O S
O O S
HN
2-1
OH
O
2-9
2-11
OH S
O HN O S
O
O
O
O
O O
O O HN O S
O HN O S
F
O
O
2-3
2-5
2-8
O OH
OH S
OH Br
S
S
N N NH
O HN O S
O
O
O
O
Cl
O HN O S
O
2-7
OH Cl
OH S
O O S
HN
HN
O O S
O
O
2-2
2-4
HN
O O S
2-10
HN
O O S
2-12
Figure 4. Structures of 12 compounds resulting from a similarity search based for C10.
Figure 4. Structures of 12 compounds resulting from a similarity search based for C10. A total of 40 analogs of 2-5 and 2-8 were purchased from SPECS and tested on MDA-MB-231, SUM-159, MCF-7 cells (Figure S4). were Twenty-four of these analogs did nottested exhibiton activity (any A total ofand 40 analogs of 2-5 and 2-8 purchased from SPECS and MDA-MB-231, compounds with ancells inhibition rateS4). above 50% were considered to be active). (3-12) (any SUM-159, and MCF-7 (Figure Twenty-four of these analogs did One not compound exhibit activity inhibitedwith all three of the cell lines, 3-9, 3-17, 3-18,considered 3-20, and 3-37 had an effect two TNBC(3-12) compounds an inhibition ratewhile above 50% were toonly be active). One on compound cell lines. Eleven compounds inhibited one TNBC cell line. Compounds with substitutions in para inhibited all three of the cell lines, while 3-9, 3-17, 3-18, 3-20, and 3-37 only had an effect on two position of the benzenesulfonamide tended to be more active (Tables 3–4). All tested compounds TNBC cell lines. Eleven compounds inhibited one TNBC cell line. Compounds with substitutions with R3 = methyl and R1 = ethoxy were active against TNBC, regardless of the length of R2. in para position of the benzenesulfonamide tended to be more active (Tables 3 and 4). All tested Compounds with a large substituent in R2 tended to be less active on SUM-159 cells. Compounds 3 = methyl and R1 = ethoxy were active against TNBC, regardless of the length of R2 . compounds with R with R1 = ethyl were more active on MDA-MB-231 with R2 = methoxy than R2 = ethoxy. Compounds 2 tended to be less active on SUM-159 cells. Compounds3 with Compounds with a large substituent in Rinactive with a methoxy substituent in R2 were on SUM-159. Replacement of the methyl moiety at R 1 R = by ethyl were more active onto MDA-MB-231 R2 (e.g., = methoxy thandid R2 = ethoxy. a phenyl ring (e.g., 3-11 3-22) or other with groups 2-8 to 3-28) not result Compounds in substantial with changes in activity against the three cell lines, indicating that the substituent at the R3 position is likely a methoxy substituent in R2 were inactive on SUM-159. Replacement of the methyl moiety at R3 by a 1 showed less relevant for TNBC inhibition. Compounds with an isopropyl or chlorine substituent in R phenyl ring (e.g., 3-11 to 3-22) or other groups (e.g., 2-8 to 3-28) did not result in substantial changes 2 was low activity on the MDA-MB-231 and noindicating activity onthat SUM-159 cells. When at 1) the R3 was methyl, 2)isRlikely in activity against three cell lines, the substituent R3 position less methoxy or ethoxy and 3) R1 was methyl, ethyl, methoxy, ethoxy, or fluorine, almost all compounds 1 relevant for TNBC inhibition. Compounds with an isopropyl or chlorine substituent in R showed of this combination had different levels of inhibitory activities on both TNBC cell lines. low activity on MDA-MB-231 and no activity on SUM-159 cells. When (1) R3 was methyl, (2) R2 was 1 was methyl, ethyl, methoxy, ethoxy, or fluorine, almost all compounds methoxy or ethoxy (3) Rrates Table 3.and Inhibition of analogs of 2-5 and 2-8 identified by 2D similarity search (Part 1). of this combination had different levels of inhibitory activities on both TNBC cell lines.
Compound
R1
R2
R3
3-17 3-8 3-32 3-1 3-9 3-37
-OCH2CH3 -CH2CH3 -CH(CH3)2 -Cl -OCH2CH3 -OCH2CH3
-CH3 -OH -OH -OH -OCH3 -OCH2CH3
-CH3 -CH3 -CH3 -CH3 -CH3 -CH3
Inhibition% MDA-MB-231 SUM-159 68.567 60.956 −1.131 0.221 24.718 −0.964 31.450 −0.754 63.008 60.778 59.687 66.789
MCF-7 40.549 −6.706 −7.568 −12.178 24.251 42.503
with a methoxy substituent in R were inactive on SUM-159. Replacement of the methyl moiety at R by a phenyl ring (e.g., 3-11 to 3-22) or other groups (e.g., 2-8 to 3-28) did not result in substantial changes in activity against the three cell lines, indicating that the substituent at the R3 position is likely less relevant for TNBC inhibition. Compounds with an isopropyl or chlorine substituent in R1 showed low activity on MDA-MB-231 and no activity on SUM-159 cells. When 1) R3 was methyl, 2) R2 was methoxy or678 ethoxy and 3) R1 was methyl, ethyl, methoxy, ethoxy, or fluorine, almost all compounds6 of 12 Molecules 2018, 23, of this combination had different levels of inhibitory activities on both TNBC cell lines. Table 3. Inhibition rates analogsofof2-5 2-5and and 2-8 identified similarity search (Part 1). 1). Table 3. Inhibition rates of of analogs identifiedbyby2D 2D similarity search (Part
Compound Compound
1 R R1
3-17 3-17 3-8 3-8 3-32 3-32 3-1 3-1 3-9 3-9 3-37 3-37 3-23 3-20 3-11 3-22 3-4 3-31 3-30 3-14 2-5 3-10 3-33 3-29 3-34 3-26 3-36 2-8 3-3 3-25 3-28 3-12 3-39 3-13 3-40
-OCH2CH3 -OCH2 CH3 -CH2CH3 -CH2 CH3 -CH(CH3)2 -CH(CH3 )2 -Cl -Cl -OCH -OCH22CH CH33 -OCH -OCH22CH CH33 -OCH2 CH3 -CH2 CH3 -CH2 CH3 -CH2 CH3 -CH(CH3 )2 -CH(CH3 )2 -H -CH3 -OCH3 -F -Cl -Br -COOH -H -CH3 -OCH3 -OCH3 -OCH3 -OCH3 -OCH3 -F -COOH -COOH
R2R2 -CH3 -CH -OH 3 -OH -OH -OH -OH -OH -OCH 3 -OCH 3 -OCH 2CH 3 -OCH 2 CH 3 -OCH2 CH2 OCH3 -OCH3 -OCH2 CH3 -OCH2 CH3 -OCH3 -OCH2 CH3 -OCH3 -OCH3 -OCH3 -OCH3 -OCH3 -OCH3 -OCH3 -OCH2 Ph -OCH2 CH3 -OCH2 CH3 -OCH2 CH2 CH2 CH3 -OCH2 CH2 CH2 CH2 CH3 -OCH2 CH3 -OCH3 -OCH(CH3 )2 -OCH2 CH3 -OCH2 CH3
Inhibition% Inhibition% MDA-MB-231 MDA-MB-231 SUM-159 SUM-159 -CH3 68.567 60.956 -CH3 68.567 60.956 -CH3 −1.131 0.221 -CH3 −1.131 0.221 -CH3 24.718 −0.964 -CH3 24.718 −0.964 -CH 3 31.450 −0.754 -CH 31.450 −0.754 3 -CH 3 63.008 60.778 -CH 63.008 60.778 3 -CH 3 59.687 66.789 -CH 59.687 66.789 3 -CH3 62.014 49.127 -CH3 53.486 65.439 -CH3 41.115 0.175 -Ph 53.616 −0.556 -CH3 37.770 −0.319 -CH3 32.074 −1.330 -CH3 50.872 0.862 -CH3 62.843 48.214 -CH3 31.005 56.793 -CH3 64.477 35.466 -CH3 32.822 −0.507 -CH3 45.039 1.3243 -CH3 10.991 −0.042 -CH3 51.036 1.581 -CH3 16.752 −0.123 -CH3 62.936 61.771 -CH3 51.145 42.064 -CH3 39.505 61.902 -CH2 CH2 CH3 61.911 46.000 -C(CH3 )3 71.378 90.386 -CH3 42.330 −0.193 -CH3 19.635 −0.635 12.487 0.000 -CH2 CH2 CH3 R3R3
MCF-7 MCF-7 40.549 40.549 −6.706 −6.706 −7.568 −7.568 −12.178 −12.178 24.251 24.251 42.503 42.503 14.707 31.221 13.005 2.238 14.222 2.952 29.311 10.194 34.480 2.819 14.480 2.848 25.556 −4.385 29.745 48.321 33.448 76.319 4.948 5.137 2.525
Compounds with more than one substituent or a fused ring system in the position of the phenyl ring of the benzenesulfonamide tended to have poor bioactivity (Table 4). The compounds available from SPECS and tested within the scope of this study only cover two or three methyl groups at different position of the benzene; other substituent groups like two or three methoxy groups were not measured. In addition, a compound including a tetracycline moiety (3-21) exhibited a low inhibitory activity on TNBC cells (Table S1).
3-12 -OCHmore -OCH -C(CH 71.378system90.386 90.386 76.319 3-12 -OCH 33 -OCH -C(CH 71.378 76.319 Compounds with than one 333substituent or a33))33fused ring in the position of the phenyl 3-12 -OCH -OCH -C(CH 71.378 90.386 76.319 3-39 -F 3 -OCH(CH 3))22 -CH333)3 42.330 −0.193 4.948 3-39 -F -OCH(CH -CH 42.330 −0.193 4.948 3-39 -F -OCH(CH 33) 2 -CH 3 42.330 −0.193 4.948 3-39 -F -OCH(CH 3) 2 -CH 3 42.330 −0.193 4.948 ring of the3-13 benzenesulfonamide tended 4). The compounds available 3-13 -COOH -OCH 2CH CH33 to have poor -CH33 bioactivity 19.635(Table −0.635 −0.635 5.137 3-13 -COOH -OCH -CH 19.635 5.137 -COOH -OCH 22CH 3 -CH 3 19.635 −0.635 5.137 3-13 -COOH 2CH3 3 19.635 −0.635 5.137 3-40 and tested -COOH within -OCH -OCH 2CH CH33 -CH-CH 2CH CH 2CH CH33 only 12.487 0.000 2.525 3-40 -COOH -OCH -CH 12.487 0.000 2.525 from SPECS the of this study cover two or three methyl groups at 3-40 -COOH -OCH 22scope CH 3 -CH 22CH 22CH 3 12.487 0.000 2.525 3-40 -COOH -OCH2CH3 -CH2CH2CH3 12.487 0.000 2.525 different position ofwith themore benzene; other substituent groups likeintwo or three methoxy groups were Compounds with than one one substituent substituent or or a fused fused ring ring system system in the position position of of the the phenyl phenyl Compounds more than than the Compounds with more more one substituent substituent or or aaa fused fused ring ring system system in in the the position of of the the phenyl phenyl Compounds with than one position not measured. In addition, a compound including a tetracycline moiety (3-21) exhibited a low ring of the benzenesulfonamide tended to have poor bioactivity (Table 4). The compounds available ring of the the benzenesulfonamide tended to to have have poor poor bioactivity bioactivity (Table (Table 4). 4). The The compounds compounds available available Molecules 2018, 23,benzenesulfonamide 678 7 of 12 ring of benzenesulfonamide tended ring of the tended to have poor bioactivity (Table 4). The compounds available from SPECS and tested within the scope of this study only cover two or three methyl groups at inhibitory activity on TNBC cells (Table S1). from SPECS SPECS and and tested tested within within the the scope scope of of this this study study only only cover cover two two or or three three methyl methyl groups groups at at from
from SPECS and tested within the scope of this study only cover two or three methyl groups at different position position of of the the benzene; benzene; other other substituent substituent groups groups like like two two or or three three methoxy methoxy groups groups were were different different position of of the the benzene; benzene; other other substituent substituent groups groups like like two two or or three three methoxy methoxy groups groups were were different position not measured. measured. In In addition, addition, aa compound compound including including aa tetracycline tetracycline moiety moiety (3-21) (3-21) exhibited exhibited aa low low not not measured. In addition, a compound including a tetracycline moiety (3-21) exhibited a low Table 4. Inhibition identified by 2D 2D similarity search (Part 2). 2). rates of analogsincluding of 2-5 anda 2-8 identified by similarity search (Part not measured. In addition, a compound tetracycline moiety (3-21) exhibited a low inhibitory activity on TNBC cells (Table S1). inhibitory activity activity on on TNBC TNBC cells cells (Table (Table S1). S1). inhibitory inhibitory activity on TNBC cells (Table S1). Table 4. 4. Inhibition Inhibition rates rates of of analogs analogs of of 2-5 2-5 and and 2-8 2-8 identified identified by by 2D 2D similarity similarity search search (Part (Part 2). 2). Table Table 4. Inhibition Inhibition rates rates of of analogs analogs of of 2-5 2-5 and and 2-8 2-8 identified identified by by 2D 2D similarity similarity search search (Part (Part 2). 2). Table 4.
Compound Compound Compound
Compound Compound Compound 3-27 3-27 3-27 3-27 3-27 3-27
4 4 R 4 RR R 44 R 4 R
R222R R R 2 R
2
R2
Inhibition% Inhibition% Inhibition% Inhibition% Inhibition% Inhibition% MDA-MB-231 MDA-MB-231 SUM-159 MDA-MB-231 SUM-159SUM-159 MCF-7 MDA-MB-231 SUM-159 MCF-7 MDA-MB-231 SUM-159 MCF-7 MDA-MB-231 SUM-159 MCF-7
-OCH 3 3 -OCH -OCH -OCH 33 -OCH 3 -OCH 3
48.15348.153 10.205 10.205 48.153 48.153 10.205 48.153 48.153 10.205
35.546 10.205 35.546 35.546 10.205 35.546
3-7 3-7 3-73-7 3-7
-OCH 3 -OCH -OCH -OCH 33 3 -OCH 3
0.431 0.431 0.431 0.431 0.431
0.085 0.085 0.085 0.085
−4.733 −4.733 0.085 −4.733 −4.733
3-6 3-6 3-6 3-63-6
-OCH22CH CH33 -OCH -OCH 2CH 3 -OCH 2 CH -OCH 2CH 3 3
43.365 43.365 43.365 43.365 43.365
0.292 0.292 0.292 0.292
7.724 7.724 7.724 0.292 7.724
3-7
3-6 3-5 3-5 3-5 3-53-5
Molecules 2018, 23, 678 Molecules 2018, 23, 678 Molecules 2018, 2018, 23, 23, 678 678 Molecules Molecules 2018, 23, 678 Molecules 2018, 23, 678
3-5
-OCH3
0.431
-OCH2CH3 -OCH33 -OCH -OCH 3 -OCH -OCH 3 3
-OCH3
43.365
41.065 41.065 41.065 41.065 41.065
0.757 0.757 0.757 0.757
41.065
0.085
MCF-7 MCF-7
35.546 35.546
−4.733
−4.733 7.724
0.292 21.98 21.98 21.98 0.757 21.98
0.757
7.724 7 77 77 7
21.98
of of of of of of
12 12 12 12 12 12
21.98
3-15 3-15 3-15 3-15 3-15 3-15 3-15
-OCH -OCH 3 3 3 -OCH -OCH33333 -OCH -OCH -OCH 3
45.58445.584 45.584 45.584 45.584 45.584 45.584
0.037 0.037 0.037 0.037 0.037 0.037
0.037 18.546 18.546 18.546 18.546 18.546 18.546
18.546
3-38 3-38 3-38 3-38 3-38 3-38 3-38
-OCH 3 -OCH 3 -OCH -OCH33333 3 -OCH -OCH -OCH 3
50.55850.558 50.558 50.558 50.558 50.558 50.558
1.346 1.346 1.346 1.346 1.346 1.346
14.827 1.346 14.827 14.827 14.827 14.827 14.827
14.827
3-24 3-24 3-24 3-24 3-24 3-24 3-24
-OCH333 -OCH -OCH 3 3 -OCH -OCH 333 -OCH -OCH 3
46.563 46.563 46.56346.563 46.563 46.563 46.563
0.233 0.233 0.233 0.233 0.233 0.233
34.910 34.910 0.233 34.910 34.910 34.910 34.910
34.910
3-16 3-16 3-16 3-16 3-16 3-16 3-16
-OCH 2CH3 -OCH CH333 22CH -OCH 2 -OCH 2 2 CH 3 3 -OCH -OCH CH -OCH222CH CH333
48.171 48.17148.171 48.171 48.171 48.171 48.171
0.732 0.732 0.732 0.732 0.732 0.732
43.439 43.439 43.439 0.732 43.439 43.439 43.439
43.439
3-35 3-35 3-35 3-35 3-35 3-35 3-35
-OCH222CH CH333 -OCH 2 CH 3 -OCH -OCH 2 CH -OCH 222CH 333 3 -OCH CH -OCH 2CH 3
33.853 33.853 33.85333.853 33.853 33.853 33.853
0.352 0.352 0.352 0.352 0.352 0.352
24.499 24.499 0.352 24.499 24.499 24.499 24.499
24.499
3-2 3-2 3-2 3-23-2 3-2 3-2
-OCH CH -OCH2222CH CH3333 -OCH -OCH -OCH 222CH 333 3 2 CH -OCH CH -OCH 2CH 3
24.255 24.255 24.255 24.255 24.25524.255 24.255
−0.884 −0.884 −0.884 −0.884 −0.884 −0.884
17.700 17.700 17.700 −0.884 17.700 17.700 17.700
17.700
3-19 3-19 3-19 3-19 3-19 3-19 3-19
-OH -OH -OH -OH -OH -OH -OH
19.306 19.306 19.306 19.306 19.306 19.30619.306
0.380 0.380 0.380 0.380 0.380 0.380
16.340 16.340 16.340 16.340 16.340 0.380 16.340
16.340
3-18 3-18 3-18 3-18 3-18 3-18 3-18
-OCH -OCH3333 -OCH -OCH 333 -OCH -OCH -OCH 3 3
59.421 59.421 59.421 59.421 59.42159.421 59.421
51.425 51.425 51.425 51.425 51.425 51.425
33.244 33.244 33.244 33.244 33.244 51.425 33.244
33.244
Ten compounds compounds with with good good inhibition inhibition rates rates on on both both TNBC TNBC cell cell lines lines were were selected selected for for the the Ten Ten with good inhibition rates on cell lines were for Ten compounds compounds good inhibition ratesand on both both TNBC TNBC cell linesHowever, were selected selected for the the measurement of IC 50 values on MDA-MB-231 SUM-159 cell lines. the aqueous measurement of IC IC50 50 with values on MDA-MB-231 and SUM-159 cell lines. However, the aqueous 50 measurement of values on MDA-MB-231 and SUM-159 cell lines. However, the aqueous 50 values on MDA-MB-231 and SUM-159 cell lines. However, the aqueous measurement of 50 2 50with Ten compounds good inhibition on both cell lines selected for the measurement of IC IC 50 values on MDA-MB-231 and SUM-159 cellTNBC However, thewere aqueous solubility of these compounds is poor. Hydrolysis of the ester in the R is expected to result 222 position solubility of these these compounds is poor. poor. Hydrolysisrates of the the ester in the the Rlines. position is expected expected to result result solubility of compounds is Hydrolysis of ester in R position is to 222 position solubility of these compounds is poor. Hydrolysis of the ester in the R is expected to result 2 solubility of these compounds isMDA-MB-231 poor. Hydrolysis of the ester in the R position is hydrolyzed expected the to 2-5 result in improved water solubility while maintaining biological activity. Therefore, we by measurement of IC values on and SUM-159 cell lines. However, aqueous solubility in improved water solubility while maintaining biological activity. Therefore, we hydrolyzed 2-5 by 50 solubility in improved improved water water solubility while while maintaining maintaining biological biological activity. activity. Therefore, Therefore, we we hydrolyzed hydrolyzed 2-5 2-5 by by in in improved water whilealkaline maintaining biological Therefore, we hydrolyzed by refluxing it for for twosolubility days under under alkaline condition in the theactivity. presence of an an aqueous aqueous solution2-5 with 2 refluxing it two days condition in presence of solution with of these compounds is poor.under Hydrolysis of the ester the R position is expected to with result in improved refluxing ithydroxide for intoin the presence of refluxing for two two days days under alkaline alkaline condition condition presence of an an aqueous aqueous solution solution with potassium and tetrahydrofuran (Scheme 1) obtain 2-5-COOH. potassiumithydroxide hydroxide and tetrahydrofuran tetrahydrofuran (Scheme 1) 1)into tothe obtain 2-5-COOH. potassium and (Scheme obtain 2-5-COOH. hydroxide and tetrahydrofuran (Scheme 1) to obtain 2-5-COOH. waterpotassium solubility while maintaining biological activity. Therefore, we hydrolyzed 2-5 by refluxing it for potassium hydroxide and tetrahydrofuran (Scheme 1) to obtain 2-5-COOH. Most of the tested compounds inhibited both TNBC cell lines with similar strength (Table 5). Most of of the tested tested compounds inhibited both TNBC TNBC cell lines lines with similar similar strength strength (Table (Table 5). 5). Most the compounds inhibited both cell with Most of the tested compounds inhibited both TNBC cell lines with similar strength (Table 5). two days under alkaline condition in the presence of an aqueous solution with potassium hydroxide Most of the tested compounds inhibited both TNBC cell lines with similar strength (Table 5). Compound 2-5-COOH had an approximately 10-fold reduced inhibitory activity compared to the Compound 2-5-COOH had an approximately 10-fold reduced inhibitory activity compared to the Compound 2-5-COOH had an approximately 10-fold reduced inhibitory activity compared to the Compound 2-5-COOH had an approximately 10-fold reduced inhibitory activity compared to the ester compound (2-5). Its IC 50 values were greater than 20 μM for both cell lines. and tetrahydrofuran (Scheme 1) to obtain 2-5-COOH. ester compound compound (2-5). (2-5). Its IC IC50 50 values were greater greater than 20 20 μM μM for for both both cell cell lines. lines. 50 ester Its values were than 50 values ester were greater than 20 μM for both cell lines. 50 ester compound compound (2-5). (2-5). Its Its IC IC50 50 values were greater than 20 μM for both cell lines.
measurement of IC50 values on MDA-MB-231 and SUM-159 cell lines. However, the aqueous solubility of these compounds is poor. Hydrolysis of the ester in the R2 position is expected to result in improved water solubility while maintaining biological activity. Therefore, we hydrolyzed 2-5 by refluxingMolecules it for2018, two days under alkaline condition in the presence of an aqueous solution with 23, 678 8 of 12 potassium hydroxide and tetrahydrofuran (Scheme 1) to obtain 2-5-COOH. Most ofMost the of tested compounds inhibited TNBCcell cell lines with similar strength (Table 5). the tested compounds inhibitedboth both TNBC lines with similar strength (Table 5). Compound 2-5-COOH had had an approximately reduced inhibitory activity compared Compound 2-5-COOH an approximately 10-fold 10-fold reduced inhibitory activity compared to the to the ester compound were greater than than 20 forfor both cell lines. ester compound (2-5). (2-5). Its ICIts50 IC values were greater 20µM μM both cell lines. 50 values
Scheme 1. Hydrolysis routeofofCompound Compound 2-5 to to Compound 2-5-COOH. Scheme 1. Hydrolysis route 2-5 Compound 2-5-COOH. Table 5. IC
for compounds measured on two TNBC cell lines.
50 Table 5. IC50 for compounds measured on two TNBC cell lines.
Compound
Compound 2-5
2-5 2-8 3-3 2-8 3-9 3-12 3-17 3-18 3-20 3-28 3-37 2-5-COOH
(µM) ICIC5050(μM) MDA-MB-231 SUM-159 MDA-MB-231 SUM-159 3.12 2.91 3.12 2.91 2.96 2.50 3.25 8.66 2.96 2.50 2.68 11.91 1.77 5.20 10.04 >40 5.49 ~24
2.66 11.53 1.94 24.15 16.97 14.41 4.09 24.25
Seven of the most potent compounds were chosen for evaluation of their bioactivity in further cell lines. In addition to three TNBC cell lines (MDA-MB-231, MDA-MB-436, and SUM-159) and the breast cancer cell line MCF-7, we employed the normal mammary epithelial cell line MCF-10A, which was used for testing the compounds for their effects on normal breast cells (Table 6). The results show that most of the compounds exhibited a weak inhibition of MCF-10A cells. Compound 3-17, the most potent TNBC inhibitor identified in this study, also had the strongest inhibition on MCF-10A (IC50 = 0.66 µM). Thus, 3-17 may be less promising for the treatment of TNBC. From a molecular structure point of view, the main differences of these compounds are the substituents of the phenyl ring of the benzenesulfonamide (2-5, 2-8, 3-3, and 3-12 are methoxy; 3-9, 3-17, and 3-37 are ethoxy), and/or naphtho[1,2-b]furan (Figure 5). Six compounds are esters, and Compound 3-17 is a ketone. Calculations suggest that 3-3 and 3-12 are less soluble in water, probably because of their longer carbon alkyl substituents (Table 6).
substituents of the phenyl ring of the benzenesulfonamide (2-5, 2-8, 3-3, and 3-12 are methoxy; 3-9, 317, and 3-37 are ethoxy), and/or naphtho[1,2-b]furan (Figure 5). Six compounds are esters, and Compound 3-17 is a ketone. Calculations suggest that 3-3 and 3-12 are less soluble in water, probably because of their longer carbon alkyl substituents (Table 6). Molecules 2018, 23, 678
9 of 12
Table 6. IC50 of compounds measured on different cell lines and calculated molecular properties. IC50 (μM) Properties Table 6. IC50 of compounds measured on different cell lines and calculatedCalculated molecularMolecular properties.
Compound
Compound Tamoxifen 2-5 Tamoxifen 2-8 2-5 3-3 2-8 3-3 3-9 3-9 3-123-12 3-17 3-173-37 3-37
MDA-MB231 2.03 MDA-MB-231 3.12 2.03 2.96 3.12 2.96 3.25 3.25 2.68 2.68 11.91 11.91 1.77 1.77 5.49 5.49
MDA-MBSUMMolecular ADMET_Solu MCF-7 MCF-10A AlogP 436 IC (µM) 159 _Weight bility_level Calculated Molecular Properties 50 10.02 13.48 9.08 >40 371.5 6.319 1 MDA-MB-436 SUM-159 MCF-7 MCF-10A Molecular_Weight AlogP ADMET_Solubility_level 1.99 2.91 >40 >40 425.5 3.514 2 10.02 13.48 9.08 >40 371.5 6.319 1 2.80 2.50 >40 425.5 439.5 3.863 2 2 1.99 2.91 >40>40 >40 3.514 2.80 2.50 >40 >40 439.5 3.863 2 a 3.36 8.66 ND >40 467.5 4.842 1 a 3.36 8.66 ND >40 467.5 4.842 1 2.08 2.66 >40 439.5 439.5 3.863 2 2 2.08 2.66 NDND >40 3.863 7.13 11.53 ND 5.063 7.13 11.53 ND >40 >40 467.5 467.5 5.063 1 1 ~0.01 1.94 ND 0.66 423.5 3.747 2 ~0.01 1.94 0.66 453.5 423.5 3.747 2 2 1.41 4.09 NDND > 40 4.211 1.41 4.09ND a : not ND 453.5 4.211 2 determined.> 40
ND a: not determined.
Figure 5. Structures of compounds in Table 6.
3. Materials and Methods 3.1. Overall Protocol 3D screening methods taking into account the molecular shape and electrostatic maps of molecules were employed to identify compounds of interest in the SPECS compound library. Compounds were selected for purchase and experimental testing in cell-based assays taking into account their calculated aqueous solubility and structural diversity (assessed by a cluster analysis). An iterative 2D similarity search was conducted to follow up on active compounds and identify (further) derivatives for testing (Figure 6).
3D screening methods taking into account the molecular shape and electrostatic maps of molecules were employed to identify compounds of interest in the SPECS compound library. Compounds were selected for purchase and experimental testing in cell-based assays taking into account their calculated aqueous solubility and structural diversity (assessed by a cluster analysis). Molecules 2018, 23, 10 of 12 An iterative 2D678 similarity search was conducted to follow up on active compounds and identify (further) derivatives for testing (Figure 6). Virtual Screening Queries 17β-estradiol
IC-163
3D Shape Smilarity Search
Specs
Breast Cancer Cell Lines
3D Electrostatic Smilarity Search
MDA-MB-231 MDA-MB-453 SUM-159 BT-20 MCF-7 MCF-10A
Solubility_level Clustering
Biological Assays
Cell Viability Assay Inhibition% → IC50 Inhibition%
Good compound(s)
2D Similarity Search
Good compound(s)
Cell Viability Assay
SAR analysis
IC50
Figure6.6.Workflow Workflowof ofvirtual virtualscreening screening and and bioassays. bioassays. Figure
3.2. Three-Dimensional Three-DimensionalSimilarity Similarity Search Search 3.2. The SPECS SPECS compound compound library (http://www.specs.net/, (http://www.specs.net/,accessed accessedMay May2014) 2014)was wasprepared preparedwith withaa The workflow developed developedwith withPipeline PipelinePilot Pilotv7.5 v7.5(PP (PP7.5, 7.5,Accelrys AccelrysSoftware, Software,Inc., Inc.,San SanDiego, Diego,CA, CA,USA.), USA.), workflow in which components were removed and the structures standardized. The prepared in which minor minorsalt salt components were removed andchemical the chemical structures standardized. The database was filtered with “Blockbuster” filter of FILTER 2.2.1, OpenEye Scientific Software, Inc., prepared database was filtered with “Blockbuster” filter(version of FILTER (version 2.2.1, OpenEye Scientific Santa Fe, NM, USA) Fe, to remove molecules withmolecules undesiredwith physicochemical properties withproperties respect to Software, Inc., Santa NM, USA) to remove undesired physicochemical molecular weight, number ofweight, heavy atoms, andofaqueous Next, the databases wereNext, processed with respect to molecular number heavy solubility. atoms, and aqueous solubility. the with OMEGA (versionwith 2.4.5,OMEGA OpenEye[23] Scientific Software, Inc., Santa Fe, NM,Software, USA) to generate up databases were[23] processed (version 2.4.5, OpenEye Scientific Inc., Santa to 500 for each Fe, NM,conformations USA) to generate up tomolecule. 500 conformations for each molecule. ROCS [17,24] OpenEye Scientific Software Inc., Santa NM,Fe, USA) wasUSA) employed ROCS [17,24] (version (version3.2.0, 3.2.0, OpenEye Scientific Software Inc., Fe, Santa NM, was for 3D shape The lowest energy conformers 17β-estradiol IC-163 generated with employed forcomparison. 3D shape comparison. The lowest energyofconformers of and 17β-estradiol and IC-163 OMEGA served as inputserved for screening with ROCS. EON was employed re-rank the top-ranked generated with OMEGA as input for screening with ROCS. EON wastoemployed to re-rank the molecules obtained with ROCSwith based on the similarity electrostatic properties. properties. top-ranked molecules obtained ROCS based on theofsimilarity of electrostatic 3.3. Water WaterSolubility SolubilityPrediction Prediction and and Structure Structure Cluster Cluster Analysis Analysis 3.3. ◦ C was calculated with the ADMET solubility prediction module of Water solubility solubility at at 25 25 °C Water was calculated with the ADMET solubility prediction module of Discovery Studio 2.5 (Accelrys Software, Inc., Inc., San San Diego, Diego,CA, CA,USA). USA). We We removed removed molecules molecules with with Discovery Studio 2.5 (Accelrys Software, ADMET solubility level in 0 (extremely low) and 1 (very low) to ensure that the chosen compounds ADMET solubility level in 0 (extremely low) and 1 (very low) to ensure that the chosen compounds haveacceptable acceptablesolubility. solubility. The The remaining remaining compounds compoundswere were clustered clustered based based on on ECFP_6 ECFP_6 and and FCFP_6 FCFP_6 have fingerprints to to assist assist the the selection selection of of compounds compounds for for experimental experimental testing. testing. fingerprints
3.4. Two-Dimensional Similarity Search 3.4. Two-Dimensional Similarity Search The most promising compounds, C10, 2-5, and 2-8, served as templates for 2D similarity search based on ECFP_6 or FCFP_6 fingerprints. 3.5. Cell Viability Assays Four human TNBC cell lines (MDA-MB-231, MDA-MB-453, SUM-159, and BT-20), a non-TNBC breast cancer cell line (MCF-7) and a normal mammary epithelial cell line (MCF-10A) were cultured with DMEM (phenol free) supplemented with 2.5% CS-FBS and 1% L-Glu. 1.2 × 103 of cells were seeded into 384-well microplates and maintained for 24 h in an incubator at 37 ◦ C in a 5% CO2 , saturated humidified atmosphere. Different compounds were added into cells with 9 concentrations
Molecules 2018, 23, 678
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from 0.156 to 40 µM for 72 h. Tamoxifen was the positive control. Then CCK-8 solution was added into cells and incubated for another 4 h. Absorbance was measured with a Microplate Reader at 450/600 nm. The IC50 values of the compounds on TNBC cell lines were derived using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). 4. Conclusions 3D and 2D similarity searches led to the identification of N-(naphtho[1,2-b]furan-5-yl)benzenesulfonamides as novel inhibitors of TNBCs. The most potent compounds (2-5 and 2-8) obtained IC50 values of 2–3 µM on different TNBC cell lines and showed no inhibitory activity on normal (i.e. MCF-7 and MCF-10A) cells in vitro, indicating their selectivity against TNBC cells. These compounds and derivatives thereof could serve as starting points for further research and development of selective TNBC inhibitors. Supplementary Materials: The following are available online. Figure S1: Structures of 32 compounds from 3D similarity search; Figure S2: Inhibition rates of 32 compounds at 10 µM from 3D similarity search; Figure S3: Inhibition rates of 12 compounds at 5 µg/mL from C10 2D similarity search; Figure S4: Inhibition rates of 40 compounds at 5 µg/mL from Compounds 2-5 and 2-8 2D similarity search; Table S1: Inhibition rates of analogs of 2-5 and 2-8 identified by 2D similarity search (Part 3). Acknowledgments: This work was supported by the National Natural Science Foundation of China [grant numbers 21772005 and 21572010]. Author Contributions: Z.L., L.Z. Y.C. and Y.T. conceived and designed the research; Y.C. performed the computational experiments, wrote the paper, and prepared the data, figures, and tables. Y.C., Y.T. and H.J. analyzed the data. Y.T. performed the bioassays; B.M. and W.L. synthesized the compounds; Z.L., L.Z. and H.J. commented and revised on the manuscript. All authors approved the final version of the manuscript. Conflicts of Interest: The authors declare that they have no conflict of interests.
Abbreviations TNBC ER PR HER2 VEGF 3D ROCS 2D ECFP_6 FCFP_6
triple-negative breast cancer estrogen receptor progesterone receptor human epidermal growth factor receptor 2 the vascular epidermal growth factor three-dimensional Rapid Overlay of Chemical Structures two-dimensional extended connectivity fingerprints of maximum diameter 6 function class fingerprints of maximum diameter 6
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Sample Availability: Samples of the compounds 2-5, 2-8, 3-1 to 3-40 are available from the authors. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
