Optimization, pharmacophore modeling and 3D-QSAR studies of sipholanes as breast cancer migration and proliferation inhibitors

European Journal of Medicinal Chemistry 73 (2014) 310e324

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Optimization, pharmacophore modeling and 3D-QSAR studies of sipholanes as breast cancer migration and proliferation inhibitors Ahmed I. Foudah, Asmaa A. Sallam, Mohamed R. Akl, Khalid A. El Sayed* Department of Basic Pharmaceutical Sciences of College of Pharmacy, University of Louisiana at Monroe, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 October 2013 Received in revised form 21 November 2013 Accepted 24 November 2013 Available online 12 December 2013

Sipholenol A, a triterpene isolated from the Red Sea sponge Callyspongia siphonella, was previously shown to reverse multidrug resistance in P-glycoprotein-overexpressing cancer cells. Moreover, sipholanes showed promising in vitro inhibitory effects against the invasion and migration of the metastatic human breast cancer cell line MDA-MB-231. The breast tumor kinase (Brk), a mediator of cancer cell phenotypes important for proliferation, survival, and migration, was proposed as a potential target. This study reports additional semisynthetic optimization of sipholenol A esters to improve the breast cancer antimigratory and antiproliferative activities as well as Brk phosphorylation inhibition. Fifteen new sipholenol A analogs (25e39) were semisynthesized. Sipholenol A 4b-40 ,50 -dichlorobenzoate ester (29) was the most potent, with an IC50 value of 1.3 mM in the migration assay. The level of Brk phosphorylation inhibition of 29 was assessed using the Z0 -LYTEÔ kinase assay and Western blot analysis. Active analogs showed no toxicity on the non-tumorigenic epithelial breast cell line MCF10A at doses equal to their IC50 values or higher in migration and proliferation assays, suggesting their selectivity towards malignant cells. Pharmacophore modeling and 3D-QSAR studies were conducted to identify important pharmacophoric features and correlate 3D-chemical structure with activity. These studies provided the evidence for future design of novel antimigratory compounds based on a simplified sipholane structure possessing rings A and B (perhydrobenzoxepine) connected to substituted aromatic esters, with the elimination of rings C and D ([5,3,0]bicyclodecane system). This will enable the future synthesis of the new active entities feasibly and cost-effectively. These results demonstrate the potential of marine natural products for the discovery of novel scaffolds for the control and management of metastatic breast cancer. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Antimigratory Antiproliferative Breast cancer Brk Pharmacophore modeling 3D-QSAR Sipholane

1. Introduction The marine environment was shown to be a rich source of diverse novel chemical structures with promising anticancer activities [1,2]. Besides the chemical novelty associated with those compounds, some of them possess novel mechanisms of action [3,4]. The Red Sea sponge Callyspongia (Siphonochalina) siphonella is a rich source of triterpenoids. So far, thirty triterpenoids have been isolated from this sponge, possessing four different skeletons, namely, sipholane, siphonellane, neviotane, and dahabane [5e10]. Among these, the sipholane triterpenoids, first reported by Kashman and co-workers, are the major group and include sipholenol A (1) and sipholenone A (2) [5]. The sipholane skeleton is composed of perhydrobenzoxepine (rings A and B) and [5,3,0]bicyclodecane system (rings C and D), linked together through an ethylene bridge [6,7]. Sipholenol A and its analogs were able to reverse P-gp* Corresponding author. Tel.: þ1 318 342 1725; fax: þ1 318 342 1737. E-mail address: [email protected] (K.A. El Sayed). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.11.039

induced multidrug resistance (MDR) in human epidermoid cancer cells [8e10]. Recently, sipholanes were reported as inhibitors of breast cancer cell migration and invasion [11]. Breast cancer is the most common cancer and the secondleading cause of cancer-related death among women in the United States [12,13]. It is estimated that over 230,000 new cases of invasive breast cancer will be diagnosed in 2013, with up to 20% of these patients likely to develop metastatic disease. The development of chemotherapy resistance continues to be the main problem in the treatment of metastatic breast cancer [13]. Consequently, there is an urgent need to discover new entities with new molecular targets and low susceptibility to common drug resistance mechanisms in order to improve therapeutic outcomes [2,13]. The intracellular protein kinase, Brk (known as breast tumor kinase, or protein tyrosine kinase 6, PTK6) has been implicated in the development and progression of a number of different tumor types [14]. Brk is one of the Src family tyrosine kinases, which has been cloned from metastatic breast tumor samples and cultured human melanocytes [15,16]. Brk is overexpressed in up to 86% of

A.I. Foudah et al. / European Journal of Medicinal Chemistry 73 (2014) 310e324

invasive human breast tumors, prostate and colon carcinomas [17]. Brk was detected in approximately two-thirds of breast tumors analyzed, where approximately a third of these showed Brk overexpression by levels ranging from five- to forty three-fold compared to normal tissue [17]. Brk is normally expressed in differentiating epithelial cells of the intestine, skin, prostate, and oral cavity, where it has been shown to promote cellular differentiation, apoptosis, and more recently to mediate migration/woundhealing [15]. However, it is neither detected in normal mammary tissues or fibroadenomas, nor at various stages of mammary development. Overexpression of Brk in tumor samples relative to the restricted levels in normal or differentiating tissues suggest that Brk may have a role in the processes underlying tumorigenesis, such as promotion of cancer cell proliferation, migration, and survival [16]. Recent studies suggest that Brk can promote the migration of breast cancer cells through multiple mechanisms and in response to a number of different ligands. Its expression levels increase in association with the carcinoma content of breast tumors, tumor grade, and invasiveness [15e19]. These observations strongly suggest that high levels of de novo expression of Brk make it an attractive therapeutic target in breast cancer. Furthermore, inhibition of Brk kinase activity may provide a potentially novel approach to sensitize the response of tumor cells to other chemotherapeutics and prevent or inhibit metastasis of cancer with enhanced therapeutic windows. A previous study from our laboratory reported the potent antimigratory and anti-invasive activities of several semisynthetic sipholenol A analogs (1e24) against the metastatic breast cancer cells, MDA-MB-231 [11]. Aromatic sipholenol A esters have shown improved antimigratory activity over the ether and carbamate analogs. Accordingly, the main objective of this study was to further investigate the influence of various aromatic ring substitutions on the antimigratory and antiproliferative activities of sipholenol A aromatic ester analogs in breast cancer cells. In addition, the promising activity of sipholenol A and analogs encouraged the reinvestigation of the source sponge C. siphonella to test additional related analogs to establish a preliminary structureeactivity relationship (SAR). This study reports the antimigratory and antiproliferative activities of twenty known natural terpenoids (40e59), possessing two novel sipholane skeletons. Pharmacophore modeling and predictive quantitative structureeactivity relationship (3D-QSAR) studies were carried out using the most active sipholanes (IC50 > 25 mM) in order to identify important pharmacophoric features of the active analogs and correlate 3Dchemical structure with the antimigratory activity that would be of value in guiding lead sipholane design. 2. Results and discussion 2.1. Chemistry Sipholenol A-4-O-benzoate and related analogs were the most active in breast cancer migration inhibition assays [11]. Therefore,

311

fifteen substituted aromatic and heteroaromatic esters of 1 (25e39) were semisynthesized to explore the substitution effects around the aromatic ring and establish a preliminary SAR profile. These new analogs were prepared using various acid chlorides and N,Ndimethylaminopyridine (DMAP) as a catalyst (Scheme 1) [11,20]. The ESI-MS spectrum of 25 showed an [M  H] peak at m/z 597.40, suggesting the molecular formula C37H54FO5 and possible 4-O-50 -fluorobenzoate ester analog of sipholenol A (1). Esterification at C-4 was evident from 1H and 13C NMR data (Tables 1 and 4). Proton H-4 doublet was downfield shifted to dH 5.19, compared with its parent 1 (DdH þ1.27). This was associated with the downfield shift of carbon C-4 (DdC þ3.66), as well as the upfield shift of carbon C-3 (DdC 1.37). Esterification was further supported by a 3 J-HMBC correlation of H-4 with the ester carbonyl C-10 (dC 164.9). The aromatic methine doublet (dH 8.15, J ¼ 8.2 Hz) was assigned to protons H-30 and H-70 based on the 3J-HMBC correlations with C-10 and C-50 (dC 147.2). Therefore, compound 25 was found to be the 4-O-b-fluorobenzoate ester of 1. Similarly, analysis of the 1H, 13C NMR, and MS data of compounds 26e39 confirmed their identity. 2.2. Biological evaluations and structureeactivity relationship Fifty-nine natural and semisynthetic sipholane analogs (1e59) [8,10,11] were screened in vitro in the MTT wound-healing, and Western blot assays [21e23]. The human mammary carcinoma cell lines MDA-MB-231 and MCF-7 were both used in the MTT assay whereas MDA-MB-231 cell line was used in the wound-healing assay (WHA) and Western blot analysis. The non-tumorigenic breast cell line MCF10A was used to evaluate cytotoxicity in the MTT assay. Additionally, a Z0 -LYTE kinase assay was used to evaluate the ability of the most active analog to inhibit PTK6 phosphorylation. 2.2.1. Antimigratory activity against the highly metastatic MDAMB-231 breast cancer cell line All compounds were evaluated for their antimigratory effect in the WHA using the metastatic breast cancer cell line, MDA-MB-231. The WHA is a simple method to study directional cell migration in vitro [24]. The known antimigratory lead 4-S-ethylphenylmethylene hydantoin (S-Ethyl) was used as a positive control at a 50 mM dose [25]. The concentrations required to inhibit the migration of 50% of cells across the wound, IC50 values, were calculated (Table 8). Fig. 1 shows the effect of the most active analogs 25e27, 29e31 and 39 on cell migration compared with the vehicle and positive controls. The parent sipholenol A (1) and most of the natural sipholanes 44e54 and 57e59 showed limited antimigratory activity, with IC50 values greater than 40 mM. Generally, all sipholenol A semisynthetic analogs showed better antimigratory activity profile than parent compound 1 and natural sipholanes. This highlights the importance of replacing the hydrogen-bond donor of the C-4 secondary alcohol group with various substituents (ester, ether, oxime and carbamate) to the antimigratory activity (Table 8). The activity profile of natural sipholanes also

Scheme 1. General semisynthetic scheme of sipholenol A (1) analogs.

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A.I. Foudah et al. / European Journal of Medicinal Chemistry 73 (2014) 310e324 25

H 8

O A

OR

OR 1 4 30

B

1

10

H

H

C

H

H

OR2

1

15

HO

O

H

D19

20

1 5 6 7 8 9 10 11 18 20 21 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

H a a b c d e f g h i j k l m n o p q r s t u v w x

H H a H H H H H H H H H H H H H H H H H H H H H H H

H

H

Compound

CH3

H a b c d e f g

O

O

OH

4'

1'

8'

2'

N H

O 1' S N H O

h 1'

7'

O

m 1'

7'

O

1'

s

1'

7'

3'

NO2

CF3

7'

l O

1'

7' 5'

5' O N 2

CF3 Cl

o

O

7'

1'

p

q

r

O O

7'

1'

5'

5' F C 3

F

7'

k

NO2

n

g O

3'

Cl

3'

Cl

3'

1'

3'

Cl

OCH3

1'

O

7'

1'

7'

f

F

j

H

1'

3'

CH3

20

H

7'

1'

7'

3'

8'

3'

3'

O

1'

7'

1'

O

7'

O

O

H

10

e

7'

O

4

3'

d

i O

O

1'

N

H

OH

4

H

O

3'

4'

H

2

3'

19

H

7'

1'

c

O

OH

H 1

3'

O

b O

O

H

O

7'

1'

H c g

4

HO

1'

H

22 23 24

a

O

OH

Compound R

1'

2'

H

HO

R

3 12 13 14 15 16 17 19

O

H

H

28

Compound R1 R2

4

O

H

H

HO

H

RO N

4

t

suggests that variations at rings C and D have little or no impact on the antimigratory activity. Ester analogs of 1 were more active than ether, oxime and carbamate analogs. This illustrates the important role of the ester carbonyl group as well as the distance limit between the aromatic side chain and C-4 oxygen for optimal antimigratory activity. Longer distances, as represented by oximes or carbamates, significantly decrease the antimigratory activity. Aromatic esters showed a better activity profile than aliphatic esters; for example, the benzoate 8 was twice as active as the acetate 5. The aromatic ester was tolerant to bioisosteric substitution as evident from the comparable activities of isonicotinate (9) and furoate (38) esters to the benzoate ester 8. In addition to the requirement of an aromatic ester at C-4, ortho-, meta- and para-substitutions (C-30 , C-40 and C-50 , respectively) of the aromatic moiety also appeared to influence the antimigratory activity. An electron-donating group (such as OCH3 and CH3) at the para-position (C-50 ) reduced the antimigratory activity in the following order: 28 < 10 < 8 (Table 8). On the other hand, an electron-withdrawing group (EWG) at the para-position, as in 25, enhanced the antimigratory activity by seven-fold as compared to

O

7'

F

1'

CF3

u

CF3 F

v

O O

3'

w

1'

5'

O

3'

NO2

x

electron-donating group (EDG) as in 28, and three-fold enhancement when compared to the unsubstituted aromatic moiety in 8. However, the influence of the electron-withdrawing group on activity appeared to have a limit. Using Craig’s plot, several EWGs were tried and their effect on the antimigratory profile was evaluated. EWG of moderate hydrophobicity was associated with a better antimigratory activity as can be seen from the activities of 25 > 11 > 27 (Table 8). It was interesting to note that replacing the 50 -F group in 25 with a 50 -NO2 in 26 did not improve the activity as would have been expected. This might be due to a steric factor at the para-position or the involvement of the nitro group in unfavorable interaction(s) with the target receptor. Moreover, an EWG at the meta-position (as in 30 and 31) was also associated with a better antimigratory activity versus the unsubstituted benzoate 8. However, analogs with ortho-substitution like 32 and 34 were less active than the unsubstituted benzoate ester (8). The Topliss Scheme, which designates a special series of substituents for use in sequence to find suitably substituted aromatic compounds with biological properties superior to the parent unsubstituted aromatic compound, was partially applied to the

A.I. Foudah et al. / European Journal of Medicinal Chemistry 73 (2014) 310e324 Table 1 13 C NMR spectroscopic data of compounds 25e30.a

1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 10 20 30 40 50 60 70 80

Table 2 13 C NMR spectroscopic data of compounds 31e36.a

dC

Position

313

dC

Position

25

26

27

28

29

30

42.7, qC 35.1, CH2 23.2, CH2 80.8, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.0, qC 121.4, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 164.9, qC 125.7, qC 131.5, CH 115.4, CH 147.2, qC 115.4, CH 131.5, CH e

42.7, qC 35.1, CH2 23.2, CH2 80.8, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.0, qC 121.4, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 165.9, qC 136.2, qC 130.8, CH 123.8, CH 152.2, qC 123.8, CH 130.8, CH e

42.7, qC 35.1, CH2 23.2, CH2 80.8, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.0, qC 121.4, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 164.5, qC 133.7, qC 129.9, CH 125.7, CH 134.6, qC 125.7, CH 129.9, CH 124.1, qC

42.7, qC 35.1, CH2 23.2, CH2 79.7, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.1, qC 121.3, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 165.5, qC 122.9 qC 131.5, CH 113.8, CH 163.5, qC 113.8, CH 131.5, CH 55.6, CH3

42.7, qC 35.1, CH2 23.2, CH2 80.8, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.0, qC 121.4, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 163.8, qC 137.8 qC 131.4, CH 130.3, qC 133.0, qC 130.9, CH 128.7, CH e

42.7, qC 35.1, CH2 23.2, CH2 80.9, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.0, qC 121.5, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 163.4, qC 132.2, qC 124.2, CH 148.2, qC 127.6, CH 130.1, CH 135.6, CH e

a In CDCl3, J in Hz. 100 MHz for 13C NMR. Carbon multiplicities were determined by APT or PENDANT experiments, qC ¼ quaternary, CH ¼ methine, CH2 ¼ methylene, CH3 ¼ methyl carbons.

design of new sipholenol A analogs in an attempt to improve the antimigratory activity. The scheme starts with evaluating the activity of unsubstituted and para-chloro substituted compounds 8 and 11, respectively. Since 11 was more active than 8, we proceeded to the synthesis of 40 ,50 -dichlorobenzoate analog 29, which proved to be the most active analog and considered a potential hit appropriate for further optimization. 2.2.2. Antiproliferative activity against MCF-7 and the highly metastatic MDA-MB-231 breast cancer cell lines The MTT assay is sensitive in vitro assay that allows the measurement of cell proliferation in a quantitative colorimetric fashion by utilizing the ability of metabolically active (viable) cells to reduce the MTT reagent to insoluble purple formazan crystals [22]. At least four concentrations per compound were tested and used to calculate an IC50 value (Table 8). The parent sipholenol A (1) and most of the natural sipholanes showed moderate to high micromolar antiproliferative activity against both cell lines. In general, all sipholenol A semisynthetic analogs showed antiproliferative activity better than the parent 1 and most of natural sipholanes, indicating the importance of introducing bulkier substitutions at the C-4 secondary alcohol group. Fig. 2 shows the antiproliferative activity of the most active analogs 25, 26, 29 and 39 compared to the DMSO control. The antiproliferative structureeactivity relationship (SAR) observed for

1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 10 20 30 40 50 60 70 80

31

32

33

34

35

36

42.7, qC 35.1, CH2 23.2, CH2 79.7, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.1, qC 121.3, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 163.4, qC 132.2, qC 124.2, CH 134.6, qC 127.6, CH 130.1, CH 135.6, CH 124.1, qC

42.7, qC 35.1, CH2 23.2, CH2 80.8, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.0, qC 121.4, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 164.9, qC 133.3, qC 133.5, qC 129.9, CH 134.4, CH 126.7, CH 131.3, CH e

42.7, qC 35.1, CH2 23.2, CH2 79.7, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.1, qC 121.3, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 163.4, qC 125.9, qC 146.8, qC 123.8, CH 133.9, CH 131.7, CH 127.4, CH e

42.7, qC 35.1, CH2 23.2, CH2 79.7, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.1, qC 121.3, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 163.4, qC 118.6, qC 144.2, qC 115.4, CH 134.6, CH 131.4, CH 133.5, CH e

42.7, qC 35.1, CH2 23.2, CH2 80.8, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.0, qC 121.4, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 164.5, qC 133.3, qC 133.5, qC 129.9, CH 134.4, CH 126.7, CH 131.3, CH 124.1, qC

42.7, qC 35.1, CH2 23.2, CH2 79.7, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.1, qC 121.3, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 165.9, qC 121.9, qC 160.6, qC 112.0, CH 136.9, qC 120.6, CH 131.8, CH 124.1, qC

a In CDCl3, J in Hz. 100 MHz for 13C NMR. Carbon multiplicities were determined by APT or PENDANT experiments, qC ¼ quaternary, CH ¼ methine, CH2 ¼ methylene, CH3 ¼ methyl carbons.

MDA-MB-231 cell line is parallel to the one discussed above for the antimigratory activity. However, there was an exception related to the para-substitution at the aromatic ring with (þs/þp) substituents. The less hydrophobic and more electron withdrawing substituents were associated with better antiproliferative activity as follows: NO2 group (26) > Cl (11) > F (25) > CF3 (27) (Fig. 2). Though aromatic esters maintained their higher activity profile than aliphatic esters as previously discussed, a different pattern was observed for the effect of C-30 , C-40 and C-50 substituents (25e37) on the antiproliferative activity in MCF-7 cells. Introduction of either EDG or EWG in meta-(C-40 ) or para-(C-50 ) position resulted in a marginal decrease in the activity, as observed in analogs 25e28 and 30e31. The 40 ,50 -dichlorobenzoate ester, however, showed a two-fold enhancement in activity as compared to the unsubstituted benzoate ester 8. Insertion of EWG in C-30 significantly enhanced the activity, as can be observed for analogs 32e35. The less hydrophobic and more electron withdrawing substituents were associated with better antiproliferative activity in the following order: 33 > 34 > 32 > 35. The most active of all analogs in this cell line was 33 (Table 8). 2.2.3. Cytotoxic activity against the non-tumorigenic MCF10A epithelial cell line The cytotoxicity of sipholanes was assessed in the MTT assay using the non-tumorigenic human breast cell line MCF10A (Fig. 5).

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Table 3 13 C NMR spectroscopic data of compounds 37e39.a

dC

Position

1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 10 20 30 40 50 60 70 80

Table 4 1 H NMR spectroscopic data of compounds 25e28.a

dH, mult. (J in Hz)

Position

37

38

39

42.7, qC 35.1, CH2 23.2, CH2 80.8, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.0, qC 121.4, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 165.9, qC 135.0, qC 115.0, CH 157.5, qC 123.0, qC 126.6, CH 125.8, CH 123.4, qC

42.7, qC 35.1, CH2 23.2, CH2 80.1, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.2, qC 121.4, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 158.0, qC 145.0, qC 117.6, CH 111.9, CH 146.6, CH e e e

42.7, qC 35.1, CH2 23.2, CH2 79.7, CH 77.8, qC 77.3, CH 26.7, CH2 39.5, CH2 72.4, qC 56.5, CH 26.8, CH2 33.8, CH2 57.4, CH 143.0, qC 121.5, CH 24.8, CH2 48.9, CH 82.2, qC 37.2, CH2 24.9, CH2 52.8, CH 35.4, qC 13.2, CH3 29.1, CH3 21.8, CH3 30.1, CH3 30.1, CH3 25.7, CH3 31.7, CH3 29.5, CH3 156.2, qC 145.1, qC 118.9, CH 111.6, CH 152.0, qC e e e

a In CDCl3, J in Hz. 100 MHz for 13C NMR. Carbon multiplicities were determined by APT or PENDANT experiments, qC ¼ quaternary, CH ¼ methine, CH2 ¼ methylene, CH3 ¼ methyl carbons.

All active analogs were nontoxic at concentrations equal to their IC50 values or higher in both WHA and proliferation assays, suggesting their good selectivity towards malignant cells. 2.2.4. Brk phosphorylation inhibition The effect of compound 29 treatment on Brk phosphorylation was evaluated using cell-free Z0 -LYTEÔ assay and cell-based Western blotting analysis. Generally, the cell-free kinase assays use fluorescence-based detection, which are widely used for HTSbased kinase inhibitors discovery due to their automation friendly, easy to use, relatively low cost, and wide availability [26]. The Z0 -LYTEÔ Kinase Assay-Tyr1 peptide kit (Invitrogen) was selected. In this experiment, Tyr1 peptide is used as a substrate; thus, the changes of Z0 -LYTEÔ Tyr1 peptide phosphorylation can directly reflect the Brk kinase activity. The results showed that analog 29 caused a concentration-dependent suppression of Brk phosphorylation, with an IC50 value of 5.3 mM, compared to an IC50 value of 1.25 mM for the positive control natural product staurosporine (Fig. 3). In Western blot analysis, cells were exposed to different doses of analogs 8 and 29. Results showed a relatively large, dosedependent inhibition of Brk phosphorylation after treatment with 29 for 72 h as compared to the vehicle-treated control group, without affecting total Brk levels (Fig. 4). Moreover, the effect of 8

2 3 4 7 8 9 11 12 13 14 16 17 18 20 21 22 24 25 26 27 28 29 30 31 30 40 60 70 80 a

25

26

27

28

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1) 1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s 8.15, d (8.2) 7.35, d (8.2) 7.35, d (8.2) 8.15, d (8.2) e

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1) 1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s 8.20, d (8.2) 8.33, d (8.2) 8.33, d (8.2) 8.20, d (8.2) e

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1) 1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s 8.15, d (8.2) 7.74, d (8.2) 7.74, d (8.2) 8.15, d (8.2) e

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1) 1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s 7.97, d (8.6) 7.44, d (8.6) 7.44, d (8.6) 7.97, d (8.6) 3.85, 3H, s

In CDCl3, J in Hz. 400 MHz for 1H NMR.

on Brk phosphorylation was only detected at a higher dose (20 mM, Fig. 4). These findings indicate that 29 inhibits Brk phosphorylation and its effect is superior to its parent 8.

2.3. Pharmacophore modeling and 3D-QSAR 2.3.1. Pharmacophore model In order to rationalize chemical structure with the antimigratory activity of sipholane analogs, a number of pharmacophore models were generated using PHASE module of the Schrödinger molecular modeling software [27]. Numerous common pharmacophore hypotheses (models) with different combination of variants (features) were generated [28]. Each of these hypotheses contained a maximum of 6 of the following features/sites: hydrogen bond acceptor (A), hydrogen bond donor (D), hydrophobic group (H) and aromatic ring (R). Hypotheses were evaluated on the basis of survival, survival-inactive and post-hoc scores [27,29]. Ten pharmacophoric hypotheses with three different variant combinations (AAADHR, AAAHHR and AADHHR) were selected (Table 9). These represent top scoring hypotheses showing the best alignment of actives [27,30e32]. In general, a good hypothesis should provide superior alignment with active compounds, discriminate between active and inactive compounds and display the lowest possible relative conformational energy values [27]. Fig. 6 shows the alignment of the most active compound (29) with the top scoring hypothesis from each of the three variants. The generated hypotheses successfully identified the most important structural features implicated in the antimigratory activity of sipholanes: C-10 carbonyl oxygen, aromatic ring and C-10 hydroxyl group. The absence of any of these features compromised the antimigratory activity as previously discussed. For example, the C-4-O-benzoyl ester 8 is about

A.I. Foudah et al. / European Journal of Medicinal Chemistry 73 (2014) 310e324 Table 5 1 H NMR spectroscopic data of compounds 29e32.a

Table 6 1 H NMR spectroscopic data of compounds 33e36.a

dH, mult. (J in Hz)

Position 30

31

32

17 18 20 21 22 24 25 26 27 28 29 30 31 30 40 50

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1) 1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s 8.11, d (2.2) e e

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1) 1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s 8.91, s e 8.45, d (8.2)

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1) 1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s 8.91, s e 8.45, d (8.2)

60

7.56, d (8.2)

70

7.87, dd (8.2, 1.8)

7.71, dd (8.2, 7.8) 8.41, d (7.8)

7.71, dd (8.2, 7.8) 8.41, d (7.8)

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1) 1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s e 7.57, d (8.6) 7.61, dd (8.6, 8.6) 7.63, dd (8.6, 8.6) 8.20, d (8.6)

8 9 11 12 13 14 16

a

dH, mult. (J in Hz)

Position

29 2 3 4 7

315

2 3 4 7 8 9 11 12 13 14 16 17 18 20 21 22 24 25 26 27 28 29 30 31 40 50 60 70 a

33

34

35

36

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1)

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1)

1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s 8.49, d (8.6) 7.89, dd (8.6, 8.6) 7.92, dd (8.6, 8.6) 8.47, d (8.6)

1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s 7.35, d (8.6) 7.95, dd (8.6, 8.6) 7.33, dd (8.6, 8.6) 8.15, d (8.6)

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1) 1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s 7.57, d (8.6) 7.61, dd (8.6, 8.6) 7.63, dd (8.6, 8.6) 8.20, d (8.6)

1.38, 1.56, m 1.94, 2.12, m 5.19, d (6.6) 3.55, dd (11.3, 3.6) 1.45, 1.82, m 1.64, m 0.76, m 1.16, 1.52, m 1.66, 1.95, m 1.57, m 5.44, dd (9.2, 5.1) 1.78, 1.98, m 1.77, m 1.60, m 1.68, 1.87, m 2.41, m 1.04, 3H, s 1.26, 3H, s 1.24, 3H, s 1.12, 3H, s 1.73, 3H, s 1.24, 3H, s 1.04, 3H, s 0.90, 3H, s 7.27, d (8.6) e 7.50, d (8.6) 7.98, d (8.6)

In CDCl3, J in Hz. 400 MHz for 1H NMR.

In CDCl3, J in Hz. 400 MHz for 1H NMR.

twice as active as both the C-4-O-phenyl ether 18 and the C-4-Oacetate ester 5. Moreover, sipholenol A (1) is almost as active as its anhydro analog 3 but is significantly more active than the dianhydro analog 4, indicating the importance of the C-10 hydroxyl, but not the C-19 hydroxyl, to the antimigratory activity of sipholenol A and analogs. 2.3.2. 3D-QSAR In order to statistically establish a relationship between the 3Dspatial arrangement of the pharmacophoric features and the antimigratory activity of sipholanes, a 3D-QSAR model was generated. Pharmacophore hypotheses (Table 9) were used to align compounds for building atom-based 3D-QSAR models by partial least square (PLS) analysis [27,30]. Analogs were divided into training and test sets (see Experimental section 4.4 for details). The test set was used to validate the generated models and was composed of analogs with IC50 values covering the entire range of antimigratory activity [31]. Several models containing up to five PLS factors were generated. The best 3D-QSAR model generated was based on the AADHHR.3334 pharmacophore hypothesis (Fig. 7). Table 10 shows the details of the best 3D-QSAR model, which is a five-PLS factor model with good statistics and predictive ability as reflected from r2 (0.910), q2 (0.853) and Pearson-R (0.943) values. These features together with the large F-value (48.4) and the small variance ratio p (9.229e-012) supported the significance of the selected model. This provided a good correlation between predicted and actual antimigratory activity (Table 11). Scatter plots of actual versus predicted activities showed that pIC50 values are effectively predicted for both training and test set compounds (Fig. 8a and b, respectively). These

Table 7 1 H NMR spectroscopic data of compounds 37e39.a

dH, mult. (J in Hz)

Position 37 2 3 4 7 8 9 11 12 13 14 16 17 18 20 21 22 24 25 26 27 28 29 30 31 30 40 50 60 70 a

1.38, 1.94, 5.19, 3.55, 1.45, 1.64, 0.76, 1.16, 1.66, 1.57, 5.44, 1.78, 1.77, 1.60, 1.68, 2.41, 1.04, 1.26, 1.24, 1.12, 1.73, 1.24, 1.04, 0.90, 8.09, e e 7.86, 8.15,

38 1.56, m 2.12, m d (6.6) dd (11.3, 3.6) 1.82, m m m 1.52, m 1.95, m m dd (9.2, 5.1) 1.98, m m m 1.87, m m 3H, s 3H, s 3H, s 3H, s 3H, s 3H, s 3H, s 3H, s s

d (8.2) d (8.2)

1.38, 1.94, 5.14, 3.55, 1.45, 1.64, 0.76, 1.16, 1.66, 1.57, 5.44, 1.78, 1.77, 1.60, 1.68, 2.41, 1.04, 1.26, 1.24, 1.12, 1.73, 1.24, 1.04, 0.90, 7.15, 6.53, 7.61, e e

In CDCl3, J in Hz. 400 MHz for 1H NMR.

39 1.56, m 2.12, m d (6.8) dd (11.3, 3.6) 1.82, m m m 1.52, m 1.95, m m dd (9.2, 5.1) 1.98, m m m 1.87, m m 3H, s 3H, s 3H, s 3H, s 3H, s 3H, s 3H, s 3H, s d (3.7) dd (3.7, 1.8) d (1.8)

1.38, 1.94, 5.20, 3.55, 1.45, 1.64, 0.76, 1.16, 1.66, 1.57, 5.44, 1.78, 1.77, 1.60, 1.68, 2.41, 1.04, 1.26, 1.24, 1.12, 1.73, 1.24, 1.04, 0.90, 7.32, 7.35, e e e

1.56, m 2.12, m d (6.8) dd (11.3, 3.6) 1.82, m m m 1.52, m 1.95, m m dd (9.2, 5.1) 1.98, m m m 1.87, m m 3H, s 3H, s 3H, s 3H, s 3H, s 3H, s 3H, s 3H, s d (3.6) d (3.6)

316

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Table 8 Antiproliferative and antimigratory activities of compounds 1e59 against human breast cancer cell lines. Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

Antiproliferative activity (IC50, mM)

Antimigratory activity (IC50, mM)

MCF-7

MDA-MB-231

MDA-MB-231

44.5 >50 >50 >50 47.9 39.2 >50 21.0 25.3 >50 24.1 43.5 >50 23.1 >50 >50 22.0 >50 >50 24.5 >50 46.8 25.6 >50 33.5 21.6 23.4 41.7 11.9 36.0 37.9 12.1 5.2 11.3 19.2 >50 >50 25.7 21.3 >50 >50 >50 31.2 >50 39.8 >50 >50 27.4 21.8 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50

>50 >50 >50 >50 38.0 26.2 27.9 16.1 18.0 33.4 10.3 39.2 29.6 18.4 19.1 32.7 12.7 29.1 35.7 40.3 47.1 42.7 27.4 38.2 11.3 8.9 19.5 30.6 8.1 34.7 36.3 24.3 38.7 28.7 31.5 36.3 22.2 14.0 11.1 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50

37.5 28.1 35.3 46.7 16.8 20.3 18.0 7.9 10.6 11.8 5.3 16.0 21.0 7.2 9.5 12.3 5.9 12.2 13.4 13.9 18.0 >50 28.5 31.4 2.4 3.4 7.8 17.9 1.3 5.9 6.1 14.1 18.6 22.7 34.5 26.4 11.9 9.3 3.5 28.0 33.0 38.3 39.0 42.0 45.0 47.5 48.0 >50 >50 >50 >50 >50 >50 >50 >50 >50 42.6 >50 >50

results can be visualized using 3D-plots of crucial volume elements occupied by ligands [27]. In such representations, blue cubes represent characteristics of the ligand structure that have moderate to strong positive effect on calculated activity (positive coefficients). By contrast, red cubes represent moderate to strong negative effects on calculated activity (negative coefficients) [27,33]. Fig. 9 shows the combined effects of all features on the antimigratory activity of sipholanes. The most active analog 29

mainly occupies blue regions (Fig. 9a), whereas the least active analog 54 occupies none of the blue regions observed for 29 (Fig. 9b). 3. Conclusion Substituted aromatic sipholenol A esters showed improved antimigratory activity versus the previously reported unsubstituted aromatic ester 8. The 40 ,50 -dichlorobenzoate ester afforded the most active analog 29. The cell-based in vitro activity of 29 was well-correlated with its cell-free Brk phosphorylation inhibitory activity coupled with the absence of any cytotoxicity to MCF10A non-tumorigenic cells. Pharmacophore model and 3D-QSAR analyses characterized the key structural elements of sipholanes required for efficient migration and Brk phosphorylation inhibition. These studies provide the basis for future design of novel antimigratory entities based on a simplified sipholane structure, possessing rings A and B (perhydrobenzoxepine) connected to substituted aromatic esters with the elimination of rings C and D, the [5,3,0]bicyclodecane system. This will make future synthesis of the new active entity more feasible and cost-effective. Results demonstrate the potential of the marine natural products to inspire the discovery of novel Brk inhibitory scaffolds appropriate for use to control metastatic breast cancer. 4. Materials and methods 4.1. General experimental procedures IR spectra were recorded on a Varian 800 FT-IR spectrophotometer. TLC analysis was carried on precoated Si gel 60 F254 500 mm TLC plates (EMD Chemicals), using n-hexane-EtOAc (7:3) as a developing system. For column chromatography, Si gel 60 (Natland International Corporation, 230e400 mm) was used, and gradient n-hexaneeEtOAc solvent system was used as a mobile phase. 1H and 13C NMR spectra were recorded in CDCl3, using tetramethylsilane (TMS) as an internal standard, on a JEOL Eclipse-ECS NMR spectrometer operating at 400 MHz for 1H NMR and 100 MHz for 13C NMR. The ESIMS experiments were conducted with a 3200 Q-trap LC/MS/MS system (Applied Biosystems, Foster City, CA) using Analyst version 1.4.1 software (MDS Sciex; Toronto, Canada) where analytes were ionized using electrospray ionization (ESI) interface operated in the negative mode. 4.2. Preparation of aromatic ester analogs of sipholenol A Esterification of 1. To a solution of 1 (0.052 mmol) in CH2Cl2 (6.0 mL), N,N-dimethylaminopyridine (2 equivalents) and aryl chloride (2 equiv) were added and refluxed overnight (Scheme 1) [11,20]. Reaction mixture was worked out by dilution with CH2Cl2, followed by the addition of water (10.0 mL). The organic layer was then collected and the aqueous layer further extracted with CH2Cl2. Organic layers were then dried over anhydrous Na2SO4 and the solvent was removed in vacuo. Residue was collected and purified on Si gel 60 using gradient n-hexaneeEtOAc system to afford the product. 4.2.1. 4b-O-50 -Fluorobenzoylsipholenol A (25) Compound 25 was prepared according to the abovementioned procedure to afford 25 mg, white powder (86% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1 H and 13C NMR, see Tables 1 and 4; ESI-MS m/z 597.40 [M  H] (calcd for C37H54FO5, 597.40).

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317

Fig. 1. Dose-response antimigratory activity of 25e27, 29e31, and 39 against the highly metastatic MDA-MB-231 human breast cancer cells in wound-healing assay. Error bars indicate the SEM of n ¼ 3/dose. DMSO was used as a vehicle control, while 4-S-ethylphenylmethylene hydantoin (S-Ethyl) was used as a positive control at 50 mM.

4.2.2. 4b-O-50 -Nitrobenzoylsipholenol A (26) Compound 26 was prepared to afford 20 mg, amorphous solid (45% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 1 and 4; ESI-MS m/z 624.30 [M  H] (calcd for C37H54NO7, 624.30).

4.2.4. 4b-O-50 -Methoxybenzoylsipholenol A (28) Compound 28 was prepared to afford 31 mg, colorless oil (78% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 1 and 4; ESI-MS m/z 609.40 [M  H] (calcd for C38H57O6, 609.40).

4.2.3. 4b-O-50 -(Trifluoromethyl)benzoylsipholenol A (27) Compound 27 was prepared to afford 27 mg, white powder (66% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 1 and 4; ESI-MS m/z 647.40 [M  H] (calcd for C38H54F3O5, 647.40).

4.2.5. 4b-O-40 ,50 -Dichlorobenzoylsipholenol A (29) Compound 29 was prepared to afford 28.5 mg, colorless oil (82% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 1 and 5; ESI-MS m/z 647.30 [M  H] (calcd for C37H53Cl2O5, 647.30).

Fig. 2. Effect of sipholenol A analogs 25, 26, 29, and 39 on the viability of the highly metastatic MDA-MB-231 cells. Error bars indicate the SEM of n ¼ 3/dose.

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1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 2 and 5; ESI-MS m/z 613.30 [M  H] (calcd for C37H54ClO5, 613.30). 4.2.9. 4b-O-30 -Nitrobenzoylsipholenol A (33) Compound 33 was prepared to afford 15 mg, white powder (38% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 2 and 6; ESI-MS m/z 624.30 [M  H] (calcd for C37H54NO7, 624.30). 4.2.10. 4b-O-30 -Fluorobenzoylsipholenol A (34) Compound 34 was prepared to afford 20 mg, white powder (65% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 2 and 6; ESI-MS m/z 597.40 [M  H] (calcd for C37H54FO5, 597.40).

Fig. 3. PTK6 phosphorylation inhibition of the most active analog 29 at various concentrations. Results were obtained using a Z0 -LYTE assay kit; error bars indicate the SEM of n ¼ 3/dose; staurosporine was used as a positive control.

4.2.6. 4b-O-40 -Nitrobenzoylsipholenol A (30) Compound 30 was prepared to afford 22 mg, colorless oil (55% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 1 and 5; ESI-MS m/z 624.30 [M  H] (calcd for C37H54NO7, 624.30). 4.2.7. 4b-O-40 -(Trifluoromethyl)benzoylsipholenol A (31) Compound 31 was prepared to afford 26 mg, white powder (72.5% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 2 and 5; ESI-MS m/z 647.40 [M  H] (calcd for C38H54F3O5, 647.40). 4.2.8. 4b-O-30 -Chlorobenzoylsipholenol A (32) Compound 32 was prepared to afford 21 mg, amorphous solid (67% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713,

4.2.11. 4b-O-30 -(Trifluoromethyl)benzoylsipholenol A (35) Compound 35 was prepared to afford 19 mg, white powder (62% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 2 and 6; ESI-MS m/z 647.40 [M  H] (calcd for C38H54F3O5, 647.40). 4.2.12. 4b-O-30 -Fluoro-50 -(trifluoromethyl)benzoylsipholenol A (36) Compound 36 was prepared to afford 24.6 mg, white powder (56% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 2 and 6; ESI-MS m/z 665.30 [M  H] (calcd for C38H53F4O5, 665.30). 4.2.13. 4b-O-40 -Fluoro-50 -(trifluoromethyl)benzoylsipholenol A (37) Compound 37 was prepared to afford 28 mg, amorphous solid (76% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 3 and 7; ESI-MS m/z 665.30 [M  H] (calcd for C38H53F4O5, 665.30).

Fig. 4. Western blot and densitometric analysis of Brk and p-Brk after exposure of MDA-MD-231 cells to 1, 10, and 20 mM treatments of analogs 8 and 29 for 72 h. b-Tubulin was used as a loading control.

A.I. Foudah et al. / European Journal of Medicinal Chemistry 73 (2014) 310e324 Table 9 Various pharmacophore hypotheses generated by PHASE. Hypothesis

Survival score

Survivalinactive score

Post-hoc score

# Matchesa

Energyb

Activityc

AAADHR.348 AAADHR.389 AAAHHR.7687 AAAHHR.5876 AADHHR.3334 AADHHR.7559 AADHHR.3400 AADHHR.308 AADHHR.290 AADHHR.305

3.500 3.497 3.472 3.470 3.483 3.481 3.475 3.471 3.464 3.460

2.181 1.992 2.095 1.907 2.328 2.214 1.991 2.437 2.337 2.129

6.430 6.428 6.402 6.401 6.413 6.411 6.405 6.101 6.094 6.090

19 19 19 19 19 19 19 19 19 19

0.012 0.012 0.012 0.012 0.012 0.012 0.012 0.313 0.313 0.313

5.886 5.886 5.886 5.886 5.886 5.886 5.886 5.886 5.886 5.886

a b c

The number of active ligands that matched the hypothesis. Energy of the reference conformer relative to the lowest-energy conformer. Reference ligand activity expressed as pIC50.

319

4.2.14. 4b-O-20 -Furoylsipholenol A (38) Compound 38 was prepared to afford 30.5 mg, white powder (80% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 3 and 7; ESI-MS m/z 569.30 [M  H] (calcd for C35H53O6, 569.30). 4.2.15. 4b-O-50 -Nitro-20 -furoylsipholenol A (39) Compound 39 was prepared to afford 27 mg, white powder (70% yield); IR nmax (CH2Cl2) 3454, 2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cm1; 1H and 13C NMR, see Tables 3 and 7; ESI-MS m/z 614.30 [M  H] (calcd for C35H52NO8, 614.30). 4.3. In vitro assays Breast cancer cell lines, MCF-7 and MDA-MB-231, were purchased from ATCC (Manassas, VA). Both cell lines were grown in RPMI 1640 medium (GIBCO-Invitrogen, NY) supplemented with

Fig. 5. Cytotoxic activity of 25e39 against the non-tumorigenic human mammary epithelial cells MCF10A. Error bars indicate the SD of n ¼ 3/dose.

Fig. 6. Most active compound 29 aligned with the top scoring hypothesis of variant (a) AAADHR; (b) AADHHR; and (c) AAAHHR. Red spheres represent hydrogen bond acceptors (A), green spheres represent hydrophobic groups (H), cyan spheres represent hydrogen bond donors (D) and brown ring represents aromatic (R) groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7. Alignment based on the pharmacophore hypothesis AADHHR.3334 generated the best 3D-QSAR model. (a) Common pharmacophoric features identified by this hypothesis. All distances are in  A unit. Alignment of some of the most active (b) and least active (c) sipholane A analogs to this pharmacophore. The most active analog 29 is shown in cyan. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

10% fetal bovine serum (FBS) and glutamine (2 mmol/L), penicillin G (100 U/mL), and streptomycin (100 mg/mL) and incubated at 37  C in a humidified incubator under 5% CO2. A stock solution of each compound was prepared in DMSO at a concentration of 25 mM for all assays. Appropriate media (serum-free, 0.5% FBS or 5% FBS) was used to prepare compounds at their final concentrations for each assay. The vehicle (DMSO) control was prepared by adding the maximum volume of DMSO used in preparing test compounds to the appropriate media type such that the final DMSO concentration never exceeded 0.2%. 4.3.1. Wound-healing assay (WHA) The WHA is a simple method for evaluating directional cell migration in vitro [23,24]. MDA-MB-231 cells were plated in sterile 24-well plates and allowed to form a confluent cell monolayer per well (>95% confluence) overnight. Wounds were then inflicted in each cell monolayer using a sterile 200 mL pipette tip. Media was removed and cells were washed twice with PBS and once with fresh RPMI media. Test compounds at the desired concentrations were prepared in fresh media (0.5% FBS) and were added to wells in triplicate. The incubation was carried out for 24 h, after which media was removed and cells were washed, fixed and stained using Diff-QuickÔ staining (Dade Behring Diagnostics, Aguada, Puerto Rico). Cells which migrated across the inflicted wound were counted under the microscope in at least five randomly selected fields (magnification: 400).

Table 10 Statistical parameters of the best atom-based 3D-QSAR model generated by PHASE. Hypothesis

PLSa SDb

AADHHR.3334 1 2 3 4 5 a

0.302 0.253 0.237 0.211 0.166

r2c

Fd

Pe

RMSEf q2g

Pearson-Rh

0.650 0.763 0.799 0.847 0.910

51.9 43.3 34.5 34.7 48.4

7.711e-008 3.673e-009 3.264e-009 7.219e-010 9.229e-012

0.194 0.135 0.151 0.134 0.128

0.847 0.953 0.940 0.938 0.943

0.664 0.836 0.795 0.840 0.853

Number of factors in the partial least squares (PLS) regression model. Standard deviation (SD) of the regression. Value of r2 for the regression. d Variance ratio. Large values of F indicate a more statistically significant regression. e Significance level of variance ratio. Smaller values indicate a greater degree of confidence. f Root-mean-square error of the test set. g Value of q2 for the predicted activity of the test set. h Value of Pearson-R for the predicted activities of the test set. b c

4.3.2. MTT (proliferation assay) The antiproliferative effect in this study was tested on the human breast cancer cell lines, MCF-7 and MDA-MB-231 following the procedure described previously [22]. Briefly, cells in exponential growth were plated in a 96-well plate at a density of 10  103 cells per well, and allowed to attach overnight at 37  C under 5% CO2 in a humidified incubator. Complete growth medium was then replaced with 100 mL of either RPMI serum-free medium (GIBCO-Invitrogen, NY) for MCF-7 cells or RPMI media supplemented with 5% FBS for MDA-MB-231 cells, containing various doses of the specific test compound and incubation resumed at 37  C under 5% CO2 for 72 h. Control and treatment media were then removed, replaced with fresh media, and 50 mL MTT solution (at 1 mg mL1) were added to each well and plates were re-incubated for 4 h. At the end of the incubation period, the color reaction was stopped by removing the media and adding 100 mL DMSO to dissolve the formazan crystals formed. Incubation at 37  C was resumed for up to 20 min to ensure complete dissolution of crystals. Absorbance was determined at l 570 nm using an ELISA plate reader (BioTek, VT, USA). The number of cells per well was calculated against a standard curve prepared at the start of each experiment by plating various numbers of cells (in the range 1000e60,000 cells per well), as counted by a hemocytometer. The IC50 value for each compound was calculated by nonlinear regression (curve-fit) of log (concentration) versus the number of cells, implemented in GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA) [34]. 4.3.3. MTT (cytotoxicity assay) The cytotoxic effect of the sipholenol A and its analogs was tested in culture on the normal human non-tumorigenic mammary epithelial cell line MCF10A. MCF10A (ATCC cat # CRL-10317) cells were maintained in defined medium consisting of Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 5% horse serum, 1% penicillin/streptomycin, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 mg/mL insulin, and 20 ng/mL epidermal growth factor (rhEGF). For subculturing, cells were rinsed twice with sterile Ca2þ and Mg2þ free phosphate buffered saline (PBS) and incubated in 0.25% trypsin containing 0.025% EDTA in PBS for 10 min at 37  C. The released cells were centrifuged, resuspended in fresh media and counted using hemocytometer. Cells in exponential growth were plated in a 96-well plate at a density of 20  103 cells/well, and allowed to attach for 24 h at 37  C under 5% CO2. Complete growth medium was then replaced with 100 mL of DMEM serumfree medium (GIBCO-Invitrogen, NY) containing various doses of the specific test compound and incubation resumed at 37  C under 5% CO2 for 24 h. The cells were then treated with MTT solution

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Table 11 Experimental and predicted activity data for training and test set compounds obtained from the best generated atom-based 3D-QSAR model (PLS factors ¼ 5). Compound

QSAR set

Experimental activity (pIC50)

Predicted activity (pIC50)

1 2 3 4 6 7 8 9 10 11 12 14 15 16 18 19 20 22 23 24 25 26 27 28 29 30 30 31 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

Training Training Training Training Training Test Training Training Test Test Training Training Training Training Training Training Test Training Test Training Training Training Test Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Training Test Test Test Test Test

4.426 4.551 4.452 4.331 4.693 4.745 5.102 4.975 4.928 5.276 4.796 5.143 5.022 4.910 4.914 4.873 4.857 4.278 4.545 4.503 5.620 5.475 5.108 4.747 5.886 5.229 5.226 5.215 4.730 4.644 4.462 4.578 4.924 5.032 5.458 4.553 4.481 4.417 4.409 4.377 4.347 4.323 4.319 4.268 4.229 4.172 4.109 4.071 4.058 4.032 4.650 4.441 4.371 4.291 4.161

4.257 4.556 4.325 4.454 4.766 4.779 5.086 4.996 5.112 5.197 4.683 4.977 4.953 4.915 4.963 4.768 4.919 4.234 4.554 4.548 5.213 5.564 5.033 4.858 5.445 5.157 5.067 5.082 4.805 4.891 4.456 4.801 5.136 5.147 5.623 4.246 4.395 4.192 4.348 4.479 4.504 4.379 4.256 4.168 4.119 4.131 4.083 4.013 4.001 4.018 4.371 4.339 4.182 4.366 4.139

Pharm set

Inactive Active Active Active Active Active Active Active Active Active Active Active Active Active Inactive

Active Active Active Active Active Active Active Active Active Active

Active Active Active Fig. 8. Scatter plots for the predicted and experimental pIC50 values as calculated by the 3D-QSAR model for the (a) training set, and (b) test set compounds. Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive Active Inactive Inactive Inactive

(50 mL/well) and re-incubated for 4 h. The color reaction was stopped by the addition of solubilization/stop solution (DMSO) (100 mL/well), and incubation at 37  C was continued to ensure complete dissolution of the formazan product. Absorbance of the samples was determined at l 570 nm with an ELISA plate reader (BioTek, VT, USA). 4.3.4. Phosphorylation inhibition assay A Z0 -LYTE kinase assay-Tyr1 peptide kit (Invitrogen) was used to assess the ability of sipholenol A and its analogs to inhibit protein tyrosine kinase 6 (PTK6) phosphorylation. Briefly, 10 mL/ well reactions were set up in 384-well plates containing kinase

buffer, 150 mm ATP, 2 mm Z0 -LYTE-Tyr 1 peptide substrate, 5000 ng/ mL PTK6, and test compound (inhibitor). After 1 h of incubation at RT, 5 mL development solution containing site-specific protease was added to each well. Incubation was continued for 1 h. The reaction was then stopped, and the fluorescent signal ratio of 445 nm (coumarin)/520 nm (fluorescein) was determined on an FLx800 plate reader (BioTek), which reflects the peptide substrate cleavage status and/or the kinase inhibitory activity in the reaction.

4.3.5. Western blot analysis Western blot analysis was performed according to the method described previously [35]. Briefly, MDA-MD-231 cells were initially plated at a density of 1  106 cells/100 mm culture plate, allowed to attach overnight in RPMI-1640 media containing 10% FBS. Cells were then washed with PBS and incubated with vehicle control or treatment in serum-free media for 3 days in culture. At the end of treatment period, cells were lysed in RIPA buffer (Qiagen Sciences Inc., Valencia, CA). Protein concentration wasdetermined by the BCA assay (Bio-Rad Laboratories, Hercules,CA). Equivalent amounts of protein were electrophoresed on SDSepolyacrylamide gels. The gels were then electroblotted onto PVDF membranes. These PVDF membranes were then blocked with 2% BSA in 10 mM TriseHCl containing 50 mM NaCl and 0.1% Tween 20, pH 7.4 (TBST) and then, incubated with specific primary antibodies against Brk (Abnova, CA) and p-Brk (Santa Cruz,

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Fig. 9. 3D-QSAR visualization showing the combined effects of all features on the antimigratory activity for (a) the most active analog 29, and (b) the least active sipholane 54. Blue cubes represent positive coefficients whereas red cubes represent negative ones. Analog 29 mostly occupies positive areas that are not occupied by 54. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

CA) and incubated overnight at 4  C. At the end of incubation period, membranes were washed 5 times with TBST and then incubated with respective horseradish peroxide-conjugated antirabbit or anti-mouse secondary antibodies (PerkinElmer Biosciences, MA) in 2% BSA in TBST for 1-h at room temperature followed by rinsing with TBST 5 times. Blots were then visualized by chemiluminescence according to the manufacturer’s instructions (Pierce, Rockford, IL, USA). Images of protein bands from all treatment groups within a given experiment were acquired using Kodak Gel Logic 1500 Imaging System (Carestream Health Inc, New Haven, CT, USA). The visualization of b-tubulin (Cell Signaling Technology, MA) was used to ensure equal sample loading in each lane. All experiments were repeated at least three times and a representative Western blot image from each experiment is shown in Fig. 4. 4.4. Computational details 4.4.1. Pharmacophore model Pharmacophore modeling and 3D-QSAR generation were carried out using PHASE (version 3.5, 2013) module of the Schrödinger suite implemented in Maestro (Maestro 9.3.5, 2012) molecular modeling package. PHASE identifies the spatial arrangement of functional groups that are common and essential for the biological activity of the compounds [27]. The structures of compounds were built using the structure drawing tool and prepared using LigPrep (version 2.5, 2012). Conformers were generated using ConfGen by applying OPLS-2005 force field method with implicit distance-dependent dielectric solvent model at cutoff root mean square deviation (RMSD) of 1  A. Conformers not within 10 kJ/mol of the global minimum were automatically discarded. A maximum of 250 conformers and 100 minimization steps were set. Compounds were divided into a training set of 48 compounds and a test set of 11 compounds using the leave-n-out method. The test set was used for the validation of the generated models. An arbitrary activity threshold value was then assigned to divide training set compounds into 23 actives (IC50 < 25 mM), 10 intermediates (25 < IC50 < 40 mM) and 13 inactives (IC50 > 40 mM). Compounds 18 and 19 were excluded because they lack the carbonyl group found in all other actives and their inclusion may jeopardize the correct selection of features. Four features/sites were considered in generating pharmacophore variants: hydrogen bond acceptor (A), hydrogen bond donor (D), hydrophobic group (H) and aromatic ring (R). The maximum number of sites was set to 6 and minimum to 4. Common pharmacophores were searched

in at least 19 of the 23 active compounds with a final box size of 1 A and minimum intersite distance of 2  A. Resulting pharmacophore hypotheses were then scored using default weights of scoring parameters for both active (survival score) and inactive ones (survival-inactive scores). To further assist in ranking hypotheses, post-hoc score was generated in which an activity reward was added for hypotheses that utilize the most active compound as the reference and a penalty assigned for hypotheses in which the reference ligand shows a high relative conformational energy. Hypotheses were clustered and only those in which actives showed good fitness scores and reasonable relative conformational energies were selected and considered for 3DQSAR model generation. The quality of each pharmacophore is measured in three ways based on the alignments to the input structures: (1) the alignment score, which is the root-meansquared deviation (RMSD) in the site-point positions; (2) the vector score, which is the average cosine of the angles formed by corresponding pairs of vector features (acceptors, donors, and aromatic rings) in the aligned structures; (3) a volume score which measures how well each ligand overlays with the reference ligand and is based on the overlap of van der Waals models of the nonhydrogen atoms in each pair of structures. 4.4.2. 3D-QSAR data setting A total of 10 pharmacophore models were chosen, all of which mapped well onto the most active compound (29). Molecules were divided into training and test sets as described earlier. However, actives within the training set with fitness scores <2.00 were eliminated. Phase presents two options for aligning the 3D structure of compounds: atom-based alignment and pharmacophorebased alignment [27]. In atom-based QSAR models, a molecule is treated as a set of overlapping van der Waals spheres. Pharmacophore-based models are more useful if the compounds are highly flexible or if they exhibit significant chemical diversity. Atom-based QSAR models were built using a grid spacing of 0.5  A and 5 partial least-square (PLS) factors. It is recommended that the number of PLS factors does not exceed 1/5 of the total number of compounds in the training set [27]. Acknowledgments The Louisiana Board of Regents is acknowledged for supporting the new MS facility (LEQSF(2013e14)-ENH-TR-26). The University of Prince Salman Bin Abdulaziz, Saudi Arabia, is acknowledged for the fellowship support of A.F.

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Appendix A

O

O H

O

H

H

O

O

HO H

H H

HO

OH

H

H

H

O

HOO H

O

43

H H

H

OH

H

H

HO

OOH H

H

H H

OH

47 O

O

H H

OH

HO

OH

O

46

H

H H

HO

OH

H

HO

H

O

OH

H

HO

O

OH

H H

OH

O

H

O

OH

OOH

45 OH

OH

OH

O H

OH

HO

42

H

44 H

OH

OH

O

HO

H H

HO

41

H

HO H

O

OH H

H

H

HO

40

OH

OH

O

O

H H

OH

HO

H

OH

H

HO

HO

48

49

50

OH H

OH

O H

H

H H

HO

H

O

H H

OH

H

HO H

HO

56

H

HO

H O

HO HO

H

H H

OH

H H

H

OH

H

55 OH

OH

O

H H

OH

H

OH

H

OH

54 O

OH

O

H O

HO

53

O

H

H

HO

O

52

H

H

OH

O

OH

H

HO

51 OH

Appendix B. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2013.11.039. References [1] T.L. Simmons, E. Andrianasolo, K. McPhail, P. Flatt, W.H. Gerwick, Mol. Cancer Ther. 4 (2005) 333e342. [2] R. Singh, M. Sharma, P. Joshi, D.S. Rawat, Anti-Cancer Agents Med. Chem. 8 (2008) 603e617. [3] D.J. Newman, G.M. Cragg, J. Nat. Prod. 75 (2012) 311e335. [4] I. Ojima, J. Med. Chem. 51 (2008) 2587e2588. [5] S. Carmely, Y. Kashman, J. Org. Chem. 48 (1983) 3517e3525. [6] S. Carmely, Y. Kashman, J. Org. Chem. 51 (1986) 784e788. [7] Y. Kashman, T. Yosief, S. Carmely, J. Nat. Prod. 64 (2001) 175e180. [8] S. Jain, I. Abraham, P. Carvalho, Y.H. Kuang, D.T. Youssef, M.A. Avery, Z.S. Chen, K.A. El Sayed, J. Nat. Prod. 72 (2009) 1291e1298. [9] I. Abraham, S. Jain, C.P. Wu, Y. Kuanga, Z. Shia, X. Chen, M. Khanfar, L. Fu, S.V. Ambudkar, K.A. El Sayed, Z.S. Chen, Biochem. Pharmacol. 80 (2010) 1497e 1506.

H

58

OH

H

H H

HO

H H

O

H

H H

HO

57

O

O

OH

O

O

59

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