Efficient synthesis and biological activity of Psammaplin A and its analogues as antitumor agents

European Journal of Medicinal Chemistry 96 (2015) 218e230

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

Original article

Efficient synthesis and biological activity of Psammaplin A and its analogues as antitumor agents Suckchang Hong a, 1, Yoonho Shin b, 1, Myunggi Jung a, Min Woo Ha a, Yohan Park c, Yeon-Ju Lee d, Jongheon Shin b, Ki Bong Oh e, Sang Kook Lee b, *, Hyeung-geun Park a, * a

Research Institute of Pharmaceutical Science and College of Pharmacy, Seoul National University, Seoul 151-742, South Korea Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-742, South Korea College of Pharmacy, Inje University, 607 Obang-dong, Gimhae, Gyeongnam 621-749, South Korea d Korea Institute of Ocean Science and Technology, Global Bioresources Research Center, Ansan 426-744, South Korea e Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 March 2015 Received in revised form 1 April 2015 Accepted 1 April 2015 Available online 6 April 2015

We describe a new concise method for the synthesis of psammaplin A and its analogues, and antitumor activity of psammaplin A analogues. Psammaplin A was obtained with 41% yield in 5 steps from 3bromo-4-hydroxybenzaldahyde and ethyl acetoacetate via Knoevenagel condensation and a-nitrosation as key steps. Twenty eight analogues of psammaplin A were prepared employing the new synthetic approach. Structureeactivity relationship study against cytotoxicity reveal that the free oxime group and disulfide functional group were responsible for high cytotoxicity. Also the bromotyrosine component was relatively tolerable and hydrophobic aromatic groups preserved the cytotoxicity. The cytotoxicity of aromatic group is dependent on the size and spatial geometry. Among them, five compounds showed comparable cytotoxicity to psammaplin A. Compound 30 exhibited potential HDAC inhibitory activity and in vivo antitumor activity. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Psammaplin A Structureeactivity relationship HDAC Histone deacetylase inhibitor

1. Introduction Psammaplin A (PsA) was originally independently isolated by several different research groups from the Psammaplysilla sponge or unidentified sponges in 1987 [1]. Psammaplin A has a unique symmetrical structure of bromotyrosine-derived disulfide dimers [2,3]. Biological activity studies have revealed that psammaplin A has various bioactivities such as antimicrobial activity [4], cytotoxicity against the leukemia cell line P388 [1,5], and inhibition of DNA gyrase [4], DNA topoisomerase [6], farnesyl protein transferase [7], and leucine aminopeptidase [7]. Additionally, psammaplin A

Abbreviations: DNMT, DNA methyltransferase; ERK, extracellular signal-regulated kinases; FBS, fetal bovine serum; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitors; IC50, half maximal inhibitory concentration; HRP, horseradish peroxidase; MAPKs, mitogen-activated protein kinases; PPARr(gamma), peroxisome proliferator-activated receptor r(gamma); PsA, psammaplin A; PVDF, polyvinylidene fluoride; RPMI, Roswell Park Memorial Institute; SAHA, suberoylanilide hydroxamic acid; SDS, sodium dodecyl sulfate; SRB, sulforhodamine B; TBS, tris-buffered saline; TCA, trichloroacetic acid; TSA, trichostatin A. * Corresponding authors. E-mail addresses: [email protected] (S.K. Lee), [email protected] (H.-g. Park). 1 S.H. and Y.S. contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2015.04.001 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

activates PPARg and induces apoptosis in human breast tumor cells [8]. Psammaplin A was also shown to be a potent inhibitor of both DNA methyltransferase (DNMT) and histone deacetylase (HDAC) [9,10]. Histone deacetylase inhibitors (HDACi) are candidates for beneficial cancer therapeutic agents. HDACi suppress the progression of tumorigenesis through epigenetic regulation of target protein acetylation including transcription factors, chaperones, signaling transduction molecules, and DNA repair proteins [11,12]. HDACi also regulate the expression of several genes that were involved in the angiogenesis signaling pathway [13]. Indeed, several HDACi are under clinical trials, and some compounds including vorinostat (SAHA) and romidepsin (FK-228) have already been approved as cancer chemotherapeutic agents for cutaneous Tcell lymphoma therapy by the U.S. FDA [14,15]. Moreover, several potent synthetic HDACi were also recently reported [16,17]. Recently, the Shin group, one of our research collaborators, also isolated psammaplin A from an unidentified sponge collected from the southern region of Korea, and confirmed significant cytotoxicity against the leukemia cell line as reported in previous studies [7]. Although the biological activities of psammaplin A are promising, the abundance of psammaplin A in marine natural products is very

S. Hong et al. / European Journal of Medicinal Chemistry 96 (2015) 218e230

low. Our programs for the development of novel cancer chemotherapeutic agents are ongoing, therefore, we aimed to develop very efficient methods for the synthesis of psammaplin A and its analogues. Synthetic methods of psammaplin A and the structureeactivity relationship along with mechanism study against cytotoxicity have been disclosed by several research groups [18e25]. All of the synthetic methods employed a-keto acid as the main intermediate to introduce a-oxime functionality. The a-keto acid could be prepared via various intermediates; oxazolone [20,26e28] from amino acids, benzylidenehydantoin [18] or benzylidenerhodanin [23] from benzaldehydes, or olefins [22] from a-oxyphosphonates and benzaldhydes. Although their synthetic methods are very efficient in 6e8 steps, the synthesis from amino acids were somewhat limited in diversity due to the limited commercial availability of amino acids, and the other methods involved several intermediates prior to a-oxime acids. To improve such shortcomings, we developed a new efficient and concise synthetic method of psammaplin A. Employing the new efficient synthetic method, a structureeactivity relationship study utilizing the more diverse analogues of psammaplin A was attempted to procure the potentially tumor active compounds. We report herein a new, efficient concise synthetic method of psammaplin A [29] and its analogues, and a structureecytotoxicity relationship (SAR) study of psammaplin A analogues. In addition, a selected compound was evaluated for HDAC inhibitory activity, underlying molecular mechanism of the growtheinhibitory activity of human lung cancer cells, and in vivo antitumor activity in the A549 human lung cancer cell-implanted mouse xenograft models. 2. Result and discussion 2.1. Chemistry As shown in the synthetic strategy depicted in Scheme 1, we planned to introduce the a-oxime moiety directly by a-nitrosation of ester, followed by a-H-transfer. First, we needed to prepare 3 as the substrate of nitrosation (Scheme 2). The alkylation of ethyl acetoacetate with 3-bromo-4-O-tetrahydropyranylbenzyl bromide (2) gave the mono-alkylated product 3 in only 35% yield with the corresponding dialkylated compound (Scheme 2). Because the mono-alkylation of active methylene dicarbonyl compounds with alkyl halides typically accompanies dialkylated compounds, the chemical yield is generally low and the purification process of mono-alkylated compounds from dialkylated ones is sometimes very difficult, depending on the alkyl halides. Thus, we finally chose Knoevenagel condensation, followed by reduction to obtain the mono-alkylated substrate of nitrosation. Initially, Knoevenagel condensation of ethyl acetoacetate with 3-bromo-4hydroxybenzaldehyde (4) using piperidine and acetic acid under benzene solvent successfully gave the condensation product 5. However, the 3-bromo moiety was partially removed during the reduction of the unsaturated double bond by catalytic hydrogenation. Therefore, we changed our synthetic method by introducing the 3-bromo group after the reduction step (Scheme 2). Knovenagel condensation of ethyl acetoacetate with 4-hydroxybenzaldehyde (7) successfully gave a,b-unsaturated ester 8 (95%). Compound 8 was reduced to 6 by the catalytic hydrogenation using Pd/C and H2 in methanol (>99%). Selective bromination could be accomplished

219

by treatment of 6 with KBrO3 and KBr under 0.5 M-HCl in methanol, resulting in 9 (92%) [29]. Although the substrate 9 for nitrosation was successfully prepared, we attempted to improve the reduction conditions to selectively reduce the olefin only in the presence of bromide, which can also enable us to obtain diverse reducable functional group substituted analogues of Psammaplin A under catalytic hydrogenation resulting short steps. After several trials, we finally found tri-n-butyltin hydride as a selective reducing agent. Knovenagel condensation of ethyl acetoacetate with 3bromo-4-hydroxybenzaldehyde (4), followed by selective reduction tri-n-butyltin hydride in toluene afforded compound 9 (95% from 4). Next, direct nitrosation of 9 was performed by modification of Barry's conditions [30,31]. The treatment of n-butylnitrite to 9 in the presence of sodium ethoxide base under EtOH solvent at 0
C generated the corresponding a-NO substituted analogue of 9. Then, the subsequent elimination of ethyl acetate of 9 by addition of ethoxide anion to the acetyl group, followed by rearrangement afforded the key intermediate a-oxime ester 10 in a high chemical yield (72%). The ethyl ester of 10 was hydrolyzed to the corresponding acid 11 with 1 N KOH in ethanol (99%). Activation of 11 by coupling with N-hydroxypthalimide using DCC in 1,4-dioxane, followed by addition of cystamine finally afforded psammaplin A (1) (85%), which is an improved process compared to our preliminary communication report [29] by no protection of 10 and no isolation of intermediate 12. According to the optimized synthetic method (Scheme 3), twenty of psammaplin A analogues were prepared from the corresponding aromatic aldehydes (Scheme 4). The O-methylation of 1, 19 and 25 using methyl iodide in the presence of potassium carbonate under DMF afforded 33e35, respectively (Scheme 5). Disulfide modified analogues (36e40) were prepared as shown in Scheme 6. Activation of 11a by coupling with N-hydroxypthalimide using DCC in 1,4-dioxane, followed by addition of 2-aminoethylsulfide, 1,6-diaminohexane, 2aminoethanthol, 2-aminoethyl-chloride, or 3-aminopropyl chloride afforded 36e40, respectively. 2.2. Cytotoxic activity of Psammaplin A analogues and structureeactivity relationship The cytotoxicity of the prepared psammaplin A (1) and its analogues (13e40) was evaluated against two human cancer cell lines (A549 lung cancer cell line and HCT116 colon cancer cell line). As shown in Table 1, variable cytotoxicity was observed depending on the functional group on the phenyl group. The removal of 3bromide and 4-hydroxy group from 1 decreased cytotoxicity (entry 2, 13). The hydrophobic tert-butyl group (entry 4, 15) showed comparable cytotoxicity with psammaplin A itself. In the case of halides, 4-Cl (17) exhibited the highest activity, with the order as follows; 4-Cl > 4-F > 4-Br (entries 5e7). An even higher cytotoxicity was observed in the 3,4-dichloride analogue (19) compared to 4-Cl (17). However, 3,5-dichloride (20) and 2,3,5-trichloride (21) analogues did not show increased activity. The electron donating alkoxy group showed dramatic activity depending on the O-alkyl group. Among the aliphatic alkoxy derivatives (22e26), 4ethoxyphenyl analogue (23) showed the highest cytotoxicity in the following order; Et > Me ~ cyclopentyl > n-Pr > n-Bu (entries 11e14). In the aromatic alkoxy group, 4-benzyloxy analogue (28)

Scheme 1. Synthetic strategy for the key intermediate of a-oxime acid.

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S. Hong et al. / European Journal of Medicinal Chemistry 96 (2015) 218e230

Scheme 2. Initial trials for the synthesis of ethyl a-(3-bromo-4-hydroxybenzyl) acetoacetate (3) [29].

Scheme 3. Optimized synthetic method of Psammaplin A (1).

had comparable cytotoxicity with psammaplin A. However, a loss of activity was observed in the phenyloxy group (27). Among the prepared analogues, b-naphthyl analogue caused the highest cytotoxicity (entry 19, 30). It is notable that the a-naphthyl

derivative (31) showed complete loss of cytotoxicity. The role of oxime and disulfide in cytotoxicity was examined. The partial or complete loss of cytotoxicity of O-methylated oximes (33e35, Table 2) revealed that the free oxime moiety is very important for

S. Hong et al. / European Journal of Medicinal Chemistry 96 (2015) 218e230

Scheme 4. Preparation of Psammaplin A analogues from various aryl aldehydes (13e32).

Scheme 5. Preparation of O-alkylated analogues of Psammaplin A (33e35).

Scheme 6. Preparation of the disulfide modified analogues of Psammaplin A (36e40).

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Table 1 Cytotoxic activity of Psammaplin A (1) and its analogues (13e32)a.

Entry

R

A549b IC50(mM)

HCT116c IC50(mM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

3-Br-4-OH-Ph (1) Ph (13) 4-Me-Ph (14) 4-t-Bu-Ph (15) 4-F-Ph (16) 4-Cl-Ph (17) 4-Br-Ph (18) 3,4-Cl2-Ph (19) 3,5-Cl2-Ph (20) 2,3,5-Cl3-Ph (21) 4-MeO-Ph (22) 4-EtO-Ph (23) 4-n-PrO-Ph (24) 4-n-BuO-Ph (25) 4-Cyclopenthoxy-Ph (26) 4-PhO-Ph (27) 4-BnO-Ph (28) 4-Nitro-Ph (29) b-Napthyl (30) a-Napthyl (31) 9-Antracenyl (32)

1.18 6.55 6.09 1.78 3.50 2.58 5.89 1.60 2.28 5.09 2.81 1.37 13.48 41.13 1.40 >100 1.12 2.66 1.20 >100 18.47

1.62 6.22 5.63 1.67 3.78 2.03 7.24 1.46 2.10 4.23 4.39 1.61 15.54 23.75 5.34 20.35 1.57 1.80 1.30 >100 28.44

a b c

All values are means of at least three experiments. Human lung cancer cells. Human colon cancer cells.

cytotoxic activity. Additionally, the importance of disulfide was confirmed by the partial or completely loss of activity in sulfide analogue (36) and diamidohexane analogue (37). Monomeric sulfide and electrophilic terminal chlorides also showed partial or complete loss of activity. These cumulative results suggest as follow: 1) the free oxime and disulfide linker is very important for the cytotoxicity against cancer cells, which is in accord with the previous reports [19e22,24,25]. 2) the 3-bromo-4-hydroxybenzyl group is tolerable and the hydrophobic aromatic groups preserved the cytotoxicity. 3) The cytotoxicity is dependent on the size and spatial geometry of aromatic group. 2.3. Mechanism of action studies with compound 30 Based on the SAR study of psammaplin A and its analogues in regard to cytotoxic activity against human cancer cells, compound 30 (entry 19 in Table 1), which showed the highest cytotoxicity among psammaplin A analogues was selected for further detailed mechanism of action studies in A549 human lung cancer cells [19e22,24,25,32]. Compound 30 exhibited a concentrationdependent growth inhibition against A549 cells with an IC50 value of 1.2 mM after a 72 h incubation (Fig. 1A). Morphological features with treatment of 30 (0.2e1.6 mM) for 24 h were also observed under a phase-contrast microscope. The cell shape was gradually changed in a concentration-dependent manner and manifested with a shrunken and sharp shape at the highest concentration (1.6 mM) (Fig. 1B). Similar morphological features were observed with the treatment of trichostatin A (TSA, 0.4 mM), a wellknown HDAC inhibitor, suggesting the possibility that 30 could also act as a HDAC inhibitor. To further determine whether the analogue of psammaplin A also inhibits the HDAC activity, the A549 cells were treated with psammaplin A (PsA, 1.6 mM) or various concentrations of compound 30 (0.2e1.6 mM) for 24 h, and then the cells were collected and total cell lysates (each 50 mg) were analyzed for the HDAC enzymatic activity using a fluorometric HDAC assay.

Compound 30 also exhibited the HDAC enzymatic inhibitory activity in a concentration-dependent manner and the inhibitory activity was comparable to psammaplin A (PsA, Fig. 2A). When the acetylation levels of histone 3 were analyzed by Western blot analysis, the expression of acetylated histone 3 protein was increased with the treatment of compound 30 in a concentration dependent manner, as expected by psammaplin A, confirming that compound 30 also functionally inhibited HDAC activity in the A549 cells (Fig. 2B). We next elucidated how the growtheinhibitory activity of psammaplin A analogue 30 in the A549 cells affects signaling pathways including AKT and ERK activities. Interestingly, although AKT/ERK pathways are crucial for proliferation and survival, we found that the levels of p-AKT and p-ERK were increased in a concentration-dependent manner compared to control cells (Fig. 3). Similar results were also reported by gemcitabine and BKM120 in the A549 (KRAS mutant) and SNU-1 (KRAS mutant) cells, respectively [33,34]. Therefore, this result might be a cell-line specific phenomenon. Further detailed studies in the cells with a diverse genetic background are warranted to better understanding the growtheinhibitory activity of psammaplin A analogues. 2.4. In vivo antitumor activity of compound 30 The antitumor activity of 30 was performed using an in vivo nude mouse xenograft model bearing A549 cells [35]. Since previous study already exhibited the antitumor activity of psammaplin A in a nude mouse xenograft model [10], the antitumor activity of compound 30 was determined and compared to psammaplin A in an A549 cell-implanted xenograft model. When the tumor size reached approximately 50e60 mm3 after injection with the A549 cells, compound 30 (15 or 30 mg/kg) or psammaplin A (PsA, 30 mg/ kg) was intraperitoneally administered to mice three times per week. The tumor volume in the control group was approximately 800 mm3 35 days after the cells were subcutaneously implanted into the right flank of each mouse. Paclitaxel (5 mg/kg) was used as a positive control under the same experimental condition. Compared to the vehicle-treated control groups, compound 30 significantly inhibited the tumor growth, and the inhibition rates were 30.4% and 47.6% at 15 mg/kg and 30 mg/kg, respectively, for compound 30 at the end of the experiments (Fig. 4A). Tumor weights were also significantly reduced by the treatment with compound 30 (Fig. 4B). Similar results were observed in the treatment of psammaplin A (PsA). No body weight changes or over toxicity were observed in the in vivo experiment with compound 30 (data not shown). Immunohistochemical analysis using the Ki-67 antibody also showed that compound 30 inhibited the expression of the proliferation biomarker Ki-67 in both central and edge region of tumor tissues (Fig. 4C). Additionally, hematoxylin and eosin staining data revealed that although there was no significant change in cell number in the central region, the cells in the edge or peripheral region of tumor tissues were significantly reduced, suggesting that the outgrowth of tumor size was inhibited by compound 30 in the in vivo xenograft models (Fig. 4D). 3. Conclusion In the present study, we developed a new and concise method of synthesis to efficiently obtain psammaplin A employing Knoevenagel condensation and a-nitrosation as key steps from 3-bromo-4hydroxy-benzaldehyde and ethyl acetoacetate. We assumed that the synthetic processes are fast and cost-effective, and have a high overall yield compared to previously reported methods. Based on the potential biological activity of psammaplin A, we also synthesized analogues of psammaplin A by applying the new developed

S. Hong et al. / European Journal of Medicinal Chemistry 96 (2015) 218e230 Table 2 Cytotoxic activity of Psammaplin A analogues (33e40)a.

Entry 1

2

Comp. no

A549b IC50 (mM) 9.2

>100

HCT116c IC50 (mM) 10.7

>100

223

synthetic methods. Structureeactivity relationship study with the synthetic psammaplin A analogues in the cytotoxicity against cancer cells was performed, and the functionality of chemical structures were set up to better understand the biological activity of psammaplin A and its analogues. The free oxime group and disulfide functional group were responsible for high cytotoxicity. However, the bromotyrosine component was relatively tolerable and hydrophobic aromatic groups preserved the cytotoxicity. The cytotoxicity of aromatic group is dependent on the size and spatial geometry. One analogue, b-naphthyl derivative of psammaplin A (compound 30) was considered as a promising candidate with high cytotoxic activity. Mechanism of action study of compound 30 also revealed that the compound inhibits HDAC enzymatic activity and in vivo tumor growth in murine xenograft models. The in vivo antitumor activity of psammaplin A analogue is reported for the first time in this study. Taken together, these data suggest that the b-naphthyl analogue of psammaplin A have the potential antitumor activity and, therefore, should be prioritized in the development of cancer chemotherapeutic agents. 4. Experimental section 4.1. Chemistry

3

>100

>100

4

2.5

>100

5

20.1

>100

6

40.0

85.1

All reagents bought from commercial sources were used without further purification. Organic solvents were concentrated under reduced pressure using a Büchi rotary evaporator. TLC analyses were performed using Merck precoated TLC plate (silica gel 60 GF254, 0.25 mm). Flash column chromatography was carried out using E. Merck Kieselgel 60 (230e400 mesh). Infrared (IR) spectra were recorded on a JASCO FT/IR-300E spectrometer. Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were measured on JEOL JNM-LA 300 or Varian Gemini 2000 [300 MHz (1H)] spectrometer, JEOL JNM-GSX 400 [400 MHz (1H), 100 MHz (13C)] spectrometer, Bruker AMX 500 [125 MHz (13C)] spectrometer, and JEOL JNM-ECA600 [150 MHz (13C)] spectrometer. All 1H NMR spectra were assigned in ppm relative to CHCl3 (d 7.24) or CH3OH (d 3.3) or DMSO (d 2.49). All 13C NMR spectra were assigned in ppm relative to the central CDCl3 (d 77) or CD3OD (d 49.8) or DMSO-d6 (d 39.5). Coupling constants (J) in 1H NMR and 13C NMR are in Hz. High-resolution mass spectra (HRMS) were measured on a JEOL JMS 700 or JEOL JMS 600-W spectrometer. Melting points were measured on a Büchi B-540 melting point apparatus and were not corrected. 4.2. General procedure for the synthesis of Psammaplin A (1) and its analogues (13e32)

7

>100

>100

8

>100

>100

4.2.1. Preparation of compound 5 Acetic acid (190 mL, 3.3 mmol) and piperidine (54 mL, 0.55 mmol) were added to solution of ethyl acetoacetate (704 mL, 5.5 mmol) in benzene (10 mL) in two-neck round bottom flask. Equipped with DeaneStark apparatus and reflux condenser, 3-bromo-4hydroxybenzaldehyde (4, 1 g, 5 mmol) was added and the mixture was refluxed for 6 h until no more benzaldehyde 4 was observed by TLC analysis. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (100 mL) and extracted with 0.2 N NaOH (50 mL  3). The aqueous phase was acidified until pH 4 using 1 N HCl in ice-water bath. Then extracted

a b c

All values are means of at least three experiments. Human lung cancer cells. Human colon cancer cells.

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Fig. 1. Anti-proliferative activity of compound 30 in cultured human A549 lung cancer cells (A) and morphological changes were observed (B).*p < 0.01; **p < 0.005, compared with the control.

by ethyl acetate (100 mL  3), washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue 5 (1.48 g, 95% yield) was obtained as a yellow solid. mp ¼ 113
C; 1H NMR(300 MHz, CDCl3) (E/Z form) d ¼ 7.52, 7.44 (d, J ¼ 1.83 Hz. 1H), 7.42, 7.34 (s, 1H), 7.18, 6.88 (m, 2H), 4.28, 4.20 (q, J ¼ 7.14 Hz, 2H), 2.31, 2.30 (s, 3H), 1.24, 1.23 (t, J ¼ 7.14 Hz, 3H) ppm; 13C NMR(100 MHz, CDCl3) d ¼ 203.8, 194.9, 167.9, 164.5, 154.8, 154.5, 139.7, 138.9, 134.1, 133.8, 133.2, 132.6, 130.9, 130.6, 126.6, 126.5, 116.5, 110.6, 61.9, 61.6, 31.2, 26.4, 14.1, 13.9 ppm; FT/IR ¼ 3342, 1719, 1595, 1499, 1380, 1296, 1205, 1043, 822, 759 cm1; HRMS (FAB) calcd for [C13H14BrO4]þ 313.0075, found: 313.0082. In case of other analogues, after completion of the reaction, the reaction mixture was diluted with ethyl acetate (100 mL), washed with water (25 mL) and brine (25 mL) in order, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane:ethyl acetate ¼ 10e5:1) to afford desired products. 4.2.2. Preparation of compound 9 Tri-n-butyl hydride (1.7 mL, 6.4 mmol) was added to solution of 5 (1 g, 3.2 mmol) in toluene (15 mL) in two-neck round bottom flask. Equipped with reflux condenser, the mixture was refluxed for 1 h until no more 5 was observed by TLC analysis. After completion of the reaction, the toluene was evaporated and the residue was diluted with ethyl acetate (100 mL), washed with water (25 mL) and brine (25 mL) in order, dried over anhydrous MgSO4, filtered,

Fig. 2. The effect of compound 30 on HDAC enzymatic activity (A) and protein level of acetyl-H3 (B) in A549 cells.*p < 0.05; **p < 0.01, compared with the control.

and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane:ethyl acetate ¼ 10e5:1) to afford 9 (997 mg, 99% yield) as yellow oil. 1H NMR (300 MHz, CDCl3) d ¼ 7.21 (d, J ¼ 2.19 Hz, 1H), 6.93 (dd, J1 ¼ 8.34 Hz, J2 ¼ 2.19 Hz, 1H), 6.80 (d, J ¼ 8.4 Hz, 1H), 4.08 (q, J ¼ 7.14 Hz, 2H), 3.68 (t, J ¼ 7.68 Hz, 1H), 2.99 (d, J ¼ 7.5 Hz, 2H), 2.14 (s, 3H), 1.14 (t, J ¼ 7.14 Hz, 3H) ppm; 13 C NMR (100 MHz, CDCl3) d ¼ 202.5, 168.9, 151.2, 132.2, 131.3, 129.2, 116.0, 109.8, 61.5, 61.1, 32.4, 29.4, 13.8 ppm; FT/IR ¼ 3428, 2982, 1711, 1497, 1419, 1359, 1290, 1216, 1176, 1042, 820, 778 cm1; HRMS (FAB) calcd for [C13H16BrO4]þ 315.0232, found: 315.0236. 4.2.3. Preparation of compound 10 Sodium ethoxide (313 mg, 4.6 mmol) was added to solution of 9 (630 mg, 2 mmol) in ethanol (10 mL) at 0
C. n-Butylnitrite (257 mL, 2.2 mmol) was added and the mixture was stirred for 3 h at rt. After completion of the reaction, the ethanol was evaporated and the residue was diluted with ethyl acetate (200 mL), washed with 1NHCl (50 mL) and brine (50 mL) in order, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane:ethyl acetate ¼ 6e3:1) to afford 10 (435 mg, 72% yield) as a white solid. m.p. ¼ 87
C; 1H NMR (300 MHz, CD3OD) d ¼ 7.35 (d, J ¼ 2.01 Hz, 1H), 7.05 (dd, J1 ¼ 8.42 Hz, J2 ¼ 2.04 Hz, 1H), 6.77 (d, J ¼ 8.25 Hz, 1H), 4.21 (q, J ¼ 6.96 Hz, 2H), 3.80 (s, 2H), 1.25 (t, J ¼ 7.14 Hz, 3H) ppm; 13 C NMR (100 MHz, CD3OD) d ¼ 166.1, 154.6, 152.7, 135.2, 131.1, 131.0, 117.9, 111.3, 63.4, 30.7, 15.2 ppm; FT/IR ¼ 3347, 1720, 1493, 1420, 1288, 1203, 1023, 858, 802, 759, 725 cm1; HRMS (FAB) calcd

S. Hong et al. / European Journal of Medicinal Chemistry 96 (2015) 218e230

225

Fig. 3. The effects of compound 30 on the expression of signal transduction proteins.

for [C11H13BrNO4]þ 302.0028, found: 302.0035. The regiochemical configuration of oxime (11) could be assigned as E-form by 1He1H NOESY spectroscopic analysis. 4.2.4. Preparation of compound 11 10 (302 mg, 1 mmol) was added to 1 M KOH solution (3 mL) in ethanol. The mixture was stirred for 6 h until no more 10 was observed by TLC analysis. If the reaction was not completed, the mixture was supplement by water (1 mL). After completion of the reaction, the reaction mixture was diluted with ethyl acetate (30 mL) and extracted with 0.2 N NaOH (15 mL  3). The aqueous phase was acidified until pH 4 using 1 N HCl in ice-water bath. Then extracted by ethyl acetate (50 mL  3), washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue 11 (270 mg, 99% yield) was obtained as a pale yellow solid. m.p. ¼ 137
C; 1H NMR (300 MHz, CD3OD) d ¼ 7.36 (d, J ¼ 2.04 Hz, 1H), 7.07 (dd, J1 ¼ 8.24 Hz, J2 ¼ 2.04 Hz, 1H), 6.77 (d, J ¼ 8.43 Hz, 1H), 3.79 (s, 2H) ppm; 1H NMR(300 MHz, DMSO-d6) d ¼ 12.44 (s, 1H), 12.28 (s, 1H), 10.06 (s, 1H), 7.27 (d, J ¼ 2.01 Hz, 1H), 7.00 (dd, J1 ¼ 8.25 Hz, J2 ¼ 2.01 Hz, 1H), 6.84 (d, J ¼ 8.22 Hz, 1H), 3.68 (s, 2H) ppm; 13C NMR (100 MHz, CD3OD) d 167.6, 154.6, 152.8, 135.2, 131.1, 117.8, 111.3, 30.5 ppm; FT/IR ¼ 3340, 1715, 1602, 1495, 1421, 1289, 1217, 1023, 823, 772 cm1; HRMS (FAB) calcd for [C9H9BrNO4]þ 273.9715, found: 273.9709. 4.2.5. Synthesis of Psammaplin A (1) N,N0 -Dicyclohexylcarbodiimide (272 mg, 1.32 mmol) and Nhydroxyphthalimide (215 mg, 1.32 mmol) were added to a solution of 11 (301 mg, 1.1 mmol) in 1,4-dioxane (5 mL). The mixture was stirred for 2 h until no more 11 was observed by TLC analysis. Cystamine dihydrochloride (124 mg, 0.55 mmol) was dissolved in methanol (2.5 mL) with triethylamine (338 mL, 2.42 mmol). This solution was added to reaction mixture and stirred for 4 h. After completion of the reaction, the solvent was evaporated and the residue was diluted with ethyl acetate (100 mL), washed with water (25 mL) and brine (25 mL) in order, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane:ethyl acetate ¼ 2e1:1)

Fig. 4. Antitumor effect of compound 30 in a tumor xenograft model. During the experimental procedure, the volume of tumors (A) and the weight of end-point tumor tissues (B) were measured. The tumor sections were also stained by immunohistochemistry method for Ki-67 (C) and H&E (D).*p < 0.05; **p < 0.01, compared with the control.

to afford psammaplin A (1) (310 mg, 85% yield) as a white solid. m.p. ¼ 144
C; 1H NMR (300 MHz, CD3OD) d 7.36 (d, J ¼ 2.19 Hz, 2H), 7.06 (dd, J1 ¼ 8.22 Hz, J2 ¼ 2.19 Hz, 2H), 6.75 (d, J ¼ 8.22 Hz, 2H), 3.78 (s, 4H), 3.51 (t, J ¼ 6.6 Hz, 4H), 2.79 (t, J ¼ 6.78 Hz, 4H) ppm; 13C NMR (100 MHz, CD3OD) d 166.7, 154.5, 153.9, 135.3, 131.5, 131.4, 117.8, 111.3, 40.4, 39.3, 29.5 ppm; FT/IR ¼ 3371, 1658, 1534, 1493, 1423, 1358, 1285, 1209, 1017, 983, 801, 720 m1; HRMS (FAB) calcd for [C22H25Br2N4O6S2]þ 662.9582, found: 662.9558. 4.2.6. Analogue 13 Following the general procedure of psammaplin A (1) from benzaldehyde, 13 was obtained as a white solid (overall 47% yield).

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m.p. ¼ 137
C; 1H NMR (300 MHz, CD3OD) d 7.26e7.09 (m, 10H), 3.90 (s, 4H), 3.49 (t, J ¼ 6.6 Hz, 4H), 2.77 (t, J ¼ 6.78 Hz, 4H) ppm; 13C NMR (125 MHz, CD3OD) d 66.8, 154.0, 138.9, 130.9, 130.1, 128.0, 40.3, 39.3, 30.7 ppm; FT/IR ¼ 3270, 1655, 1529, 1425, 1200, 1000, 747, 704 cm1; HRMS (FAB) calcd for [C22H27N4O4S2]þ 475.1474, found: 475.1471. 4.2.7. Analogue 14 Following the general procedure of psammaplin A (1) from 4methylbenzaldehyde, 14 was obtained as a white solid (overall 49% yield). m.p. ¼ 146
C; 1H NMR (300 MHz, CD3OD) d 7.12 (d, J ¼ 7.97 Hz, 4H), 7.00 (d, J ¼ 7.97 Hz, 4H), 3.85 (s, 4H), 3.47 (t, J ¼ 6.57 Hz, 4H), 2.76 (t, J ¼ 6.78 Hz, 4H), 2.23 (s, 6H) ppm; 13C NMR (125 MHz, CD3OD): d ¼ 166.8, 154.2, 137.6, 135.7, 130.8, 130.7, 40.3, 39.3, 30.3, 21.9 ppm; FT/IR ¼ 3276, 2923, 1657, 1528, 1427, 1204, 1003, 783, 723 cm1; HRMS (FAB) calcd for [C24H3N4O4S2]þ 503.1787, found: 503.1788. 4.2.8. Analogue 15 Following the general procedure of psammaplin A (1) from 4-tbutylbenzaldehyde, 15 was obtained as a white solid (overall 53% yield). m.p. ¼ 86
C; 1H NMR (300 MHz, CD3OD) d 7.25e7.15 (m, 8H), 3.86 (s, 4H), 3.47 (t, J ¼ 6.6 Hz, 4H), 2.76 (t, J ¼ 6.96 Hz, 4H), 1.25 (s, 18H) ppm; 13C NMR (125 MHz, CD3OD) d 166.8, 154.2, 150.9, 135.7, 130.6, 127.0, 40.3, 39.3, 36.0, 32.6, 30.2 ppm; FT/IR ¼ 3284, 2962, 1660, 1529, 1427, 1363, 1213, 1018, 758 cm1; HRMS (FAB) calcd for [C30H43N4O4S2]þ 587.2726, found: 587.2731. 4.2.9. Analogue 16 Following the general procedure of psammaplin A (1) from 4fluorobenzaldehyde, 16 was obtained as a white solid (overall 46% yield). m.p. ¼ 142
C; 1H NMR (300 MHz, CD3OD) d 7.29e7.22 (m, 4H), 6.96e7.88 (m, 4H), 3.87 (s, 4H), 3.50 (t, J ¼ 6.6 Hz, 4H), 2.79 (t, J ¼ 6.96 Hz, 4H) ppm; 13C NMR(125 MHz, CD3OD) d 166.6, 163.7 (d, J ¼ 241.3), 153.8, 134.9 (d, J ¼ 3.15), 132.6 (d, J ¼ 7.9), 116.6 (d, J ¼ 21.36), 40.3, 39.3, 29.9 ppm; FT/IR ¼ 3284, 2926, 1660, 1529, 1508, 1429, 1221, 1158, 1016, 980, 822, 754 cm1; HRMS (FAB) calcd for [C22H25F2N4O4S2]þ 511.1285, found: 511.1277. 4.2.10. Analogue 17 Following the general procedure of psammaplin A (1) from 4chlorobenzaldehyde, 17 was obtained as a white solid (overall 43% yield). m.p. ¼ 125
C; 1H NMR (300 MHz, DMSO-d6) d 11.94 (s, 2H), 8.12 (t, J ¼ 5.7 Hz, 2H), 7.31 (d, J ¼ 8.42 Hz, 4H), 7.22 (d, J ¼ 8.42 Hz, 4H), 3.80 (s, 4H), 3.43 (q, J ¼ 6.21 Hz, 4H), 2.82 (t, J ¼ 6.93 Hz, 4H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.1, 151.4, 135.8, 130.7, 130.6, 128.2, 38.2, 37.0, 28.4 ppm; FT/IR ¼ 3390, 3266, 1654, 1624, 1530, 1491, 1425, 1221, 1001, 804, 771 cm1; HRMS (FAB) calcd for [C22H25Cl2N4O4S2]þ 543.0694, found: 543.0694. 4.2.11. Analogue 18 Following the general procedure of psammaplin A (1) from 4bromobenzaldehyde, 18 was obtained as a white solid (overall 36% yield). m.p. ¼ 108
C; 1H NMR (300 MHz, CD3OD) d 7.40e7.33 (m, 4H), 7.20e7.12 (m, 4H), 3.86 (s, 4H), 3.50 (t, J ¼ 6.75 Hz, 4H), 2.79 (t, J ¼ 6.96 Hz, 4H) ppm; 13C NMR (125 MHz, CD3OD) d 166.5, 153.4, 138.3, 133.2, 132.9, 121.7, 40.4, 39.3, 30.2 ppm; FT/IR ¼ 3283, 1658, 1530, 1487, 1428, 1208, 1071, 1011, 800, 756 cm1; HRMS (FAB) calcd for [C22H25Br2N4O4S2]þ 630.9684, found: 630.9695. 4.2.12. Analogue 19 Following the general procedure of psammaplin A (1) from 3,4dichlorobenzaldehyde, 19 was obtained as a white solid (overall 40% yield). m.p. ¼ 159
C; 1H NMR (300 MHz, CD3OD) d 7.40 (d, J ¼ 2.01 Hz, 2H), 7.37e7.32 (m, 2H), 7.18 (dd, J1 ¼ 8.25 Hz,

J2 ¼ 2.01 Hz, 2H), 3.87 (s, 4H), 3.51 (t, J ¼ 6.78 Hz, 4H), 2.80 (t, J ¼ 6.75 Hz, 4H) ppm; 13C NMR (150 MHz, CD3OD) d 166.3, 153.0, 139.8, 133.8, 132.9, 132.1, 131.9, 130.9, 40.4, 39.4, 30.0 ppm; FT/ IR ¼ 3272, 1658, 1531, 1470, 1204, 1132, 1031, 758 cm1; HRMS (FAB) calcd for [C22H23Cl4N4O4S2]þ 610.9915, found: 610.9932. 4.2.13. Analogue 20 Following the general procedure of psammaplin A (1) from 3,5dichlorobenzaldehyde, 20 was obtained as a white solid (overall 37% yield). m.p. ¼ 181
C; 1H NMR (300 MHz, CD3OD) d 7.17e7.08 (m, 6H), 3.78 (s, 4H), 3.43 (t, J ¼ 6.78 Hz, 4H), 2.73 (t, J ¼ 6.78 Hz, 4H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.0, 150.6, 141.0, 133.9, 133.8, 127.7, 127.5, 125.9, 38.2, 37.0, 28.6 ppm; FT/IR ¼ 3410, 3221, 1649, 1621, 1566, 1530, 1428, 1021, 984, 673 cm1; HRMS (FAB) calcd for [C22H23Cl4N4O4S2]þ 609.9837, found: 611.1027. 4.2.14. Analogue 21 Following the general procedure of psammaplin A (1) from 2,3,5-trichlorobenzaldehyde, 21 was obtained as a white solid (overall 39% yield). m.p. ¼ 191
C; 1H NMR (400 MHz, CD3OD) d 7.42 (d, J ¼ 2.44 Hz, 2H), 7.05 (d, J ¼ 2.36 Hz, 2H), 4.01 (s, 4H), 3.55 (t, J ¼ 6.64 Hz, 4H), 2.85 (t, J ¼ 6.72 Hz, 4H) ppm; 13C NMR (100 MHz, CD3OD) d 166.3, 152.0, 140.7, 135.5, 134.5, 132.7, 130.0, 129.9, 40.4, 39.4, 30.0 ppm; FT/IR ¼ 3217, 3067, 2371, 2321, 1658, 1530, 1429, 1222, 1051, 1024, 737 cm1; HRMS (FAB) calcd for [C22H21Cl6N4O4S2]þ 680.9107, found: 678.9143. 4.2.15. Analogue 22 Following the general procedure of psammaplin A (1) from 4methoxybenzaldehyde, 22 was obtained as a pale yellow solid (overall 45% yield). m.p. ¼ 132
C; 1H NMR (300 MHz, CD3OD) d 6.96 (dd, J1 ¼ 123.96 Hz, J2 ¼ 8.79, 8H), 3.82 (s, 4H), 3.70 (s, 6H), 3.48 (t, J ¼ 6.75 Hz, 4H), 2.76 (t, J ¼ 6.75 Hz, 4H) ppm; 13C NMR (125 MHz, CD3OD) d 166.8, 160.4, 154.4, 131.9, 130.8, 115.5, 56.4, 40.3, 39.3, 29.8 ppm; FT/IR ¼ 3308, 2949, 1661, 1510, 1441, 1248, 1178, 1035, 978, 817, 753 cm1; HRMS (FAB) calcd for [C24H31N4O6S2]þ 535.1685, found: 535.1688. 4.2.16. Analogue 23 Following the general procedure of psammaplin A (1) from 4ethoxybenzaldehyde, 23 was obtained as a white solid (overall 42% yield). m.p. ¼ 154
C; 1H NMR (300 MHz, DMSO-d6) d 11.79 (s, 2H), 8.04 (t, J ¼ 5.7 Hz, 2H), 7.10 (d, J ¼ 8.55 Hz, 4H), 6.79 (d, J ¼ 8.55 Hz, 4H), 3.94 (q, J ¼ 7.2 Hz, 4H), 3.73 (s, 4H), 3.41 (q, J ¼ 6.6 Hz, 4H), 2.81 (t, J ¼ 7.2 Hz, 4H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.3, 156.8, 152.1, 129.7, 128.5, 114.2, 62.8, 38.1, 37.0, 28.0, 14.6 ppm; FT/IR ¼ 3390, 3271, 2981, 2870, 1653, 1625, 1509, 1250, 1000, 924, 696 cm1; HRMS (FAB) calcd for [C26H35N4O6S2]þ 563.1998, found: 563.2007. 4.2.17. Analogue 24 Following the general procedure of psammaplin A (1) from 4-npropoxybenzaldehyde, 24 was obtained as a white solid (overall 47% yield). m.p. ¼ 168
C; 1H NMR (300 MHz, DMSO-d6) d 11.78 (s, 2H), 8.04 (t, J ¼ 5.7 Hz, 2H), 7.10 (d, J ¼ 8.4 Hz, 4H), 6.79 (d, J ¼ 8.4 Hz, 4H), 3.85 (t, J ¼ 6.6 Hz, 4H), 3.73 (s, 4H), 3.41 (q, J ¼ 6.6 Hz, 4H), 2.81 (t, J ¼ 7.2 Hz, 4H), 1.68 (sextet, J ¼ 6.6 Hz, 4H), 0.94 (t, J ¼ 7.5 Hz, 6H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.3, 157.0, 152.1, 129.7, 128.5, 114.2, 68.8, 38.1, 37.0, 28.0, 22.0, 10.4 ppm; FT/IR ¼ 3390, 3265, 2961, 1652, 1529, 1509, 1475, 1424, 1249, 1000, 696 cm1; HRMS (FAB) calcd for [C28H39N4O6S2]þ 591.2311, found: 591.2315. 4.2.18. Analogue 25 Following the general procedure of psammaplin A (1) from 4-n-

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butoxybenzaldehyde, 25 was obtained as a white solid (overall 42% yield). m.p. ¼ 167
C; 1H NMR (300 MHz, DMSO-d6) d 11.78 (s, 2H), 8.04 (t, J ¼ 5.7 Hz, 2H), 7.10 (d, J ¼ 8.4 Hz, 4H), 6.79 (d, J ¼ 8.4 Hz, 4H), 3.89 (t, J ¼ 6.6 Hz, 4H), 3.73 (s, 4H), 3.41 (q, J ¼ 6.6 Hz, 4H), 2.81 (t, J ¼ 7.2 Hz, 4H), 1.65 (quintet, J ¼ 6.3 Hz, 4H), 1.40 (sextet, J ¼ 7.5 Hz, 4H), 0.91 (t, J ¼ 7.8 Hz, 6H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.3, 157.0, 152.1, 129.7, 128.4, 114.2, 67.0, 38.1, 37.0, 28.0, 18.7, 13.6 ppm; FT/IR ¼ 3390, 3269, 2958, 2872, 1653, 1509, 1248, 1000, 696 cm1; HRMS (FAB) calcd for [C30H43N4O6S2]þ 619.2624, found: 619.2618. 4.2.19. Analogue 26 Following the general procedure of psammaplin A (1) from 4cyclopentoxybenzaldehyde, 26 was obtained as a white solid (overall 37% yield). m.p. ¼ 145
C; 1H NMR (300 MHz, DMSO-d6) d 11.78 (s, 2H), 8.04 (t, J ¼ 5.7, 2H), 7.09 (d, J ¼ 8.55 Hz, 4H), 6.76 (d, J ¼ 8.55 Hz, 4H), 4.72 (t, J ¼ 6.0 Hz, 4H), 3.72 (s, 4H), 3.41 (q, J ¼ 6.6 Hz, 4H), 2.81 (t, J ¼ 7.2 Hz, 4H), 1.88e1.82 (m, 4H), 1.67e1.50 (m, 12H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.3, 155.9, 152.1, 129.7, 128.2, 115.1, 78.4, 38.1, 37.0, 32.2, 28.0, 23.5 ppm; FT/ IR ¼ 3384, 3194, 2954, 2871, 1654, 1631, 1535, 1507, 1451, 1245, 1010, 983, 695 cm1; HRMS (FAB) calcd for [C32H43N4O6S2]þ 643.2624, found: 643.2620. 4.2.20. Analogue 27 Following the general procedure of psammaplin A (1) from 4phenoxybenzaldehyde, 27 was obtained as a white solid (overall 44% yield). m.p. ¼ 144
C; 1H NMR (300 MHz, CD3OD) d 7.31e7.22 (m, 8H), 7.07e7.01 (m, 2H), 6.92e6.88 (m, 4H), 6.84e6.80 (m, 4H), 3.88 (s, 4H), 3.51 (t, J ¼ 6.57 Hz, 4H), 2.80 (t, J ¼ 6.75, Hz 4H) ppm; 13 C NMR (100 MHz, DMSO-d6) d 163.3, 156.9, 154.7, 151.8, 131.9, 130.3, 129.9, 123.2, 118.7, 118.3, 38.2, 37.0, 28.2 ppm; FT/IR ¼ 3283, 3060, 2927, 1659, 1530, 1489, 1428, 1238, 1016, 871, 692 cm1; HRMS (FAB) calcd for [C34H35N4O6S2]þ 659.1998, found: 659.1985. 4.2.21. Analogue 28 Following the general procedure of psammaplin A (1) from 4benzoxybenzaldehyde, 28 was obtained as a white solid (overall 52% yield). m.p. ¼ 176
C; 1H NMR (300 MHz, DMSO-d6) d 11.82 (s, 2H), 8.06 (t, J ¼ 5.67 Hz, 2H), 7.42e7.27 (m, 10H), 7.13 (d, J ¼ 8.52 Hz, 4H), 6.88 (d, J ¼ 8.52 Hz, 4H), 5.03 (s, 4H), 3.75 (s, 4H), 3.44e3.41 (m, 4H), 2.81 (t, J ¼ 6.6 Hz, 4H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.4, 156.7, 152.1, 137.2, 129.8, 128.9, 128.4, 127.7, 127.6, 114.6, 69.2, 38.2, 37.0, 28.1 ppm; FT/IR ¼ 3230, 1654, 1624, 1527, 1509, 1423, 1253, 1000, 725, 692 cm1; HRMS (FAB) calcd for [C36H39N4O6S2]þ 687.2311, found: 687.2307. 4.2.22. Analogue 29 Following the general procedure of psammaplin A (1) from 4nitrobenzaldehyde, 29 was obtained as a pale yellow solid (overall 41% yield). m.p. ¼ 142
C; 1H NMR (300 MHz, CD3OD) d 8.08 (d, J ¼ 8.79 Hz, 4H), 7.47 (d, J ¼ 8.79 Hz, 4H), 4.01 (s, 4H), 3.51 (t, J ¼ 6.60 Hz, 4H), 2.81 (t, J ¼ 6.78 Hz, 4H) ppm; 13C NMR (125 MHz, CD3OD) d 166.3, 152.6, 148.7, 147.0, 131.9, 125.2, 40.4, 39.3, 30.9 ppm; FT/IR ¼ 3233, 2927, 1658, 1518, 1429, 1345, 1208, 1013, 744 cm1; HRMS (FAB) calcd for [C22H25N6O8S2]þ 565.1175, found: 565.1175. 4.2.23. Analogue 30 Following the general procedure of psammaplin A (1) from 2naphthaldehyde, 30 was obtained as a pale yellow solid (overall 45% yield). m.p. ¼ 156
C; 1H NMR (300 MHz, DMSO-d6) d 11.94 (s, 2H), 8.14 (t, J ¼ 5.67 Hz, 2H), 7.85e7.79 (m, 6H), 7.68 (s, 2H), 7.49e7.39 (m, 6H), 4.00 (s, 4H), 3.44 (q, J ¼ 6.42 Hz, 4H), 2.82 (t, J ¼ 6.96 Hz, 4H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.3, 151.7,

227

134.5, 133.0, 131.6, 127.7, 127.5, 127.4, 127.3, 126.7, 126.0, 125.4, 38.1, 37.0, 29.2 ppm; FT/IR ¼ 3222, 3053, 2930, 2519, 1657, 1527, 1462, 1358, 1223, 997, 791, 746 cm1; HRMS (FAB) calcd for [C30H31N4O4S2]þ 575.1787, found: 575.1786. 4.2.24. Analogue 31 Following the general procedure of psammaplin A (1) from 1naphthaldehyde, 31 was obtained as a pale yellow solid (overall 37% yield). m.p. ¼ 92
C; 1H NMR (300 MHz, CD3OD) d 8.08 (d, J ¼ 8.04 Hz, 2H), 7.69 (d, J ¼ 7.68 Hz, 2H), 7.56 (dd, J1 ¼ 7.32 Hz, J2 ¼ 1.83 Hz, 2H), 7.40e7.29 (m, 4H), 7.23e7.15 (m, 4H), 4.24 (s, 4H), 3.30 (t, J ¼ 6.78, 4H), 2.57 (t, J ¼ 6.78, 4H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.5, 152.0, 133.3, 132.5, 131.5, 128.4, 126.7, 126.0, 125.7, 125.6, 125.4, 123.8, 38.2, 36.9, 26.4 ppm; FT/IR ¼ 3225, 3049, 2924, 1658, 1627, 1530, 1017, 985, 792, 735 cm1; HRMS (FAB) calcd for [C30H31N4O4S2]þ 575.1787, found: 575.1782. 4.2.25. Analogue 32 Following the general procedure of psammaplin A (1) from 9anthracenecarboxaldehyde, 32 was obtained as a yellow solid (overall 29% yield). m.p. ¼ 137
C; 1H NMR (300 MHz, pyridine-d5) d 14.38 (s, 2H), 8.96 (d, J ¼ 8.97 Hz, 4H), 8.79 (t, J ¼ 5.49 Hz, 2H), 8.39 (s, 2H), 7.99 (d, J ¼ 8.4 Hz, 4H), 7.55e7.38 (m, 8H), 5.32 (s, 4H), 3.34 (q, J ¼ 6.24 Hz, 4H), 2.47 (t, J ¼ 6.78 Hz, 4H) ppm; 13C NMR (100 MHz, pyridine-d5) d 165.0, 153.8, 132.2, 131.4, 130.7, 129.3, 127.1, 126.4, 125.9, 125.3, 38.8, 37.8, 24.4 ppm; FT/IR ¼ 2925, 2854, 2349, 1670, 1508, 1457, 1251, 803, 680 cm1; HRMS (FAB) calcd for [C38H35N4O4S2]þ 675.2100, found: 675.2123. 4.3. General procedure for the synthesis of Psammaplin A analogues (33e35) 4.3.1. Analogue 33 Potassium carbonate (691 mg, 5 mmol) was added to solution of psammaplin A (1) (332 mg, 0.5 mmol) in acetone (5 mL). Iodomethane (616 mL, 5 mmol) was added and the mixture was stirred for 20 h at room temperature. After completion of the reaction, the acetone was evaporated and the residue was diluted with ethyl acetate (100 mL), washed with water (5 mL) and brine (25 mL) together, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane:ethyl acetate ¼ 2:1) to afford 33 (227 mg, 63% yield) as a yellow solid. m.p. ¼ 81
C; 1H NMR (300 MHz, CDCl3) d 7.43 (d, J ¼ 2.19 Hz, 2H), 7.18 (dd, J1 ¼ 8.43 Hz, J2 ¼ 2.19 Hz, 2H), 7.04 (t, J ¼ 6.03 Hz, 2H), 6.76 (d, J ¼ 8.43 Hz, 2H), 3.98 (s, 6H), 3.82 (s, 6H), 3.79 (s, 4H), 3.60 (q, J ¼ 6.42 Hz, 4H), 2.80 (t, J ¼ 6.42 Hz, 4H) ppm; 13 C NMR (100 MHz, CDCl3) d 162.5, 154.4, 151.5, 133.9, 129.7, 129.4, 111.8, 111.4, 63.1, 56.2, 38.2, 37.6, 28.5 ppm; FT/IR ¼ 3394, 2938, 1671, 1522, 1495, 1440, 1284, 1255, 1048 cm1; HRMS (FAB) calcd for [C26H33Br2N4O6S2]þ 719.0208, found: 719.0220. 4.3.2. Analogue 34 Following the synthetic procedure of 33, potassium carbonate and iodomethane were used only half-amount (2.5 mmol). Using the compound 22 (267 mg 0.5 mmol), 34 was obtained as a white solid (214 mg, 76% yield). m.p. ¼ 67
C; 1H NMR (300 MHz, CDCl3) d 7.19 (d, J ¼ 8.61 Hz, 4H), 7.04 (t, J ¼ 6.06 Hz, 2H), 6.79e6.74 (m, 2H), 6.72e6.65 (m, 2H), 3.96 (s, 6H), 3.82 (s, 4H), 3.73 (s, 6H), 3.58 (q, J ¼ 6.21 Hz, 4H), 2.78 (t, J ¼ 6.42 Hz, 4H) ppm; 13C NMR (100 MHz, CDCl3) d 162.7, 158.1, 152.1, 130.2, 128.2, 113.8, 62.9, 55.2, 38.2, 37.6, 28.8 ppm; FT/IR ¼ 3353, 2936, 2834, 1671, 1613, 1510, 1248, 1178, 1044, 916, 693 cm1; HRMS (FAB) calcd for [C26H35N4O6S2]þ 63.1998, found: 563.1993.

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4.3.3. Analogue 35 Following the synthetic procedure of 33, potassium carbonate and iodomethane were used only half-amount (2.5 mmol). Using the compound 28 (343 mg 0.5 mmol), 35 was obtained as a white solid (239 mg, 67% yield). m.p. ¼ 93
C; 1H NMR (300 MHz, CDCl3) d 7.34e7.19 (m, 10H), 7.16e7.12 (m, 4H), 6.99 (t, J ¼ 6.03 Hz, 2H), 6.79e6.75 (m, 4H), 4.92 (s, 4H), 3.90 (s, 6H), 3.76 (s, 4H), 3.52 (q, J ¼ 6.42 Hz, 4H), 2.71 (t, J ¼ 6.42 Hz, 4H) ppm; 13C NMR (100 MHz, CDCl3) d 162.7, 157.4, 152.1, 137.1, 130.3, 128.5, 127.8, 127.4, 114.7, 69.9, 62.9, 38.2, 37.6, 28.8 ppm; FT/IR ¼ 3402, 2936, 1673, 1509, 1220, 1045, 772 cm1; HRMS (FAB) calcd for [C38H43N4O6S2]þ 715.2624, found: 715.2622. 4.4. General procedure for the synthesis of Psammaplin A analogues (36e40) 4.4.1. Preparation of compound 11a Following the general procedure of 11 from 4benzoxybenzaldehyde, 11a was obtained as a yellow solid (overall 70% yield). m.p. ¼ 123
C; 1H NMR (300 MHz, DMSO-d6) d 12.77 (s, 1H), 12.23 (s, 1H), 7.43e7.28 (m, 5H), 7.11 (d, J ¼ 8.61 Hz, 4H), 6.50 (d, J ¼ 8.61 Hz, 4H), 5.04 (s, 2H), 3.73 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) d 165.2, 156.8, 150.5, 137.2, 129.7, 128.8, 128.4, 127.7, 127.6, 114.7, 69.2, 29.0 ppm; FT/IR ¼ 2349, 2308, 1611, 1511, 1246, 1178, 1025, 811, 741, 697, 644 cm1; HRMS (FAB) calcd for [C16H15NO4]þ 285.1001, foun: 285.0999. 4.4.2. Analogue 36 N,N0 -Dicyclohexylcarbodiimide (248 mg, 1.2 mmol) and Nhydroxyphthalimide (196 mg, 1.2 mmol) were added to solution of 11a (285 mg, 1 mmol) in 1,4-dioxane (4 mL). The mixture was stirred for 2 h until no more 11a was observed by TLC analysis. 2,20 Thiobis(ethylamine) (57 mL, 0.5 mmol) was dissolved in methanol (2 mL) with triethylamine (279 mL, 2 mmol). This solution was added to reaction mixture and stirred for 4 h. After completion of the reaction, the solvent was evaporated and the residue was diluted with ethyl acetate (80 mL), washed with water (20 mL) and brine (20 mL) in order, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane:ethyl acetate ¼ 2e1:1) to afford 36 (272 mg, 83% yield) as a white solid. m.p. ¼ 156
C; 1H NMR (300 MHz, DMSO-d6) d 11.85 (s, 2H), 8.02 (s, 2H), 7.41e7.30 (m, 10H), 7.15 (d, J ¼ 7.89 Hz, 4H), 6.90 (d, J ¼ 7.89 Hz, 4H), 5.03 (s, 4H), 3.77 (s, 4H), 3.32 (broad, 4H), 2.62 (t, 4H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.3, 156.8, 152.2, 137.2, 129.9, 129.0, 128.4, 127.8, 127.6, 114.6, 69.2, 38.5, 30.2, 28.1 ppm; FT/IR ¼ 3275, 2862, 1646, 1620, 1530, 1509, 1453, 1247, 1007, 731 cm1; HRMS (FAB) calcd for [C36H39N4O6S]þ 655.2590, found: 655.2589. 4.4.3. Analogue 37 Following the synthetic procedure of 36, using the 1,6diaminohexane (65 mL, 0.5 mmol), 37 was obtained as a white solid (189 mg, 58% yield). m.p. ¼ 157
C; 1H NMR(300 MHz, DMSOd6) d 11.68 (s, 2H), 7.87 (m, 2H), 7.41e7.29 (m, 10H), 7.09 (d, J ¼ 8.7 Hz, 4H), 6.87 (d, J ¼ 8.7 Hz, 2H), 5.02 (s, 4H), 3.71 (s, 4H), 3.07 (q, J ¼ 6.93 Hz, 4H), 1.37 (m, 4H), 1.17 (m, 4H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.2, 156.7, 152.4, 137.2, 129.8, 128.9, 128.4, 127.7, 127.6, 114.6, 69.1, 30.7, 29.0, 28.1, 26.0 ppm; FT/IR ¼ 3294, 3034, 2925, 1657, 1508, 1454, 1240, 1175, 1014, 737, 694 cm1; HRMS (FAB) calcd for [C38H43N4O6]þ 651.3183, found: 651.3183. 4.4.4. Analogue 38 Following the synthetic procedure of 36, using the cysteamine (77 mg, 1 mmol), 38 was obtained as a white solid (155 mg, 45% yield). m.p. ¼ 145
C; 1H NMR (300 MHz, DMSO-d6) d 11.79 (s, 1H),

8.05 (t, J ¼ 6.03 Hz, 1H), 7.41e7.28 (m, 5H), 7.15 (d, J ¼ 8.61 Hz, 2H), 6.89 (d, J ¼ 8.61 Hz, 2H), 5.04 (s, 2H), 3.74 (s, 2H), 3.32e3.18 (m, 2H), 2.54e2.50 (m, 2H), 2.31 (t, J ¼ 7.86 Hz, 1H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.3, 156.7, 152.1, 137.2, 129.8, 128.9, 128.4, 127.7, 127.6, 114.6, 69.1, 42.1, 38.1, 23.2 ppm; FT/IR ¼ 3392, 3173, 3057, 2923, 2860, 1658, 1628, 1535, 1506, 1464, 1375, 1298, 1240, 1003, 736, 696 cm1; HRMS (FAB) calcd for [C18H21N2O3S]þ 345.1273, found: 345.1266. 4.4.5. Analogue 39 Following the synthetic procedure of 36, using the 2chloroethylamine hydrochloride (116 mg, 1 mmol), 39 was obtained as a white solid (225 mg, 65% yield). m.p. ¼ 152
C; 1H NMR (300 MHz, DMSO-d6) d 11.84 (s, 1H), 8.07 (t, J ¼ 5.88 Hz, 1H), 7.43e7.27 (m, 5H), 7.11 (d, J ¼ 8.61 Hz, 4H), 6.88 (d, J ¼ 8.61 Hz, 4H), 5.03 (s, 2H), 3.73 (s, 2H), 3.62 (t, J ¼ 6.57 Hz, 2H), 3.45 (q, J ¼ 6.06 Hz, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.5, 156.7, 151.9, 137.2, 129.8, 128.8, 128.4, 127.7, 127.6, 114.6, 69.1, 42.9, 40.7, 28.0 ppm; FT/ IR ¼ 3388, 3176, 3033, 2860, 1659, 1628, 1532, 1507, 1374, 1299, 1243, 1000, 759, 698 cm1; HRMS (FAB) calcd for [C18H20ClN2O3]þ 347.1162, found: 347.1159. 4.4.6. Analogue 40 Following the synthetic procedure of 36, using the 3chloropropylamine hydrochloride (130 mg, 1 mmol), 40 was obtained as a white solid (224 mg, 62% yield). m.p. ¼ 148
C; 1H NMR (300 MHz, DMSO-d6) d 11.74 (s, 1H), 8.03 (t, J ¼ 5.85 Hz, 1H), 7.43e7.28 (m, 5H), 7.13 (d, J ¼ 8.5 Hz, 2H), 6.89 (d, J ¼ 8.5 Hz, 2H), 5.04 (s, 2H), 3.75 (s, 2H), 3.57 (t, J ¼ 6.39 Hz, 2H), 3.25 (q, J ¼ 6.57 Hz, 2H), 1.87 (pent, J ¼ 6.57 Hz, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) d 163.5, 156.7, 152.4, 137.2, 129.8, 128.9, 128.4, 127.7, 127.6, 114.6, 69.1, 43.0, 38.9, 36.4, 28.1 ppm; FT/IR ¼ 3393, 3178, 2866, 1659, 1627, 1546, 1508, 1466, 1375, 1236, 1005, 752, 696 cm1; HRMS (FAB) calcd for [C19H22ClN2O3]þ 361.1319, found: 361.1322. 4.5. Cell culture Human lung cancer cells (A549) and colon cancer cells (HCT-116) were provided by the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics-antimycotics (PSF; 100 units/mL penicillin G sodium, 100 ng/mL streptomycin, and 250 ng/mL amphotericin B). The cells were incubated at 37
C and 5% CO2 in a humidified atmosphere. 4.6. Cytotoxicity assay (sulforhodamine B assay) Cells (3.5  104 cells/mL) were treated with various concentrations of compounds (total volume of 200 mL/well) in 96-well culture plates for 72 h. After treatment, the cells were fixed with 10% TCA solution, and cell viability was determined with a sulforhodamine B (SRB) assay [36]. The results were expressed as percentages relative to solvent-treated control incubations, and IC50 values were calculated using non-linear regression analysis using Table curve software. 4.7. Western blot analysis A549 cells were cultured with the various concentrations of compound 30 for 24 h. After washing with PBS, cells were lysed in extraction buffer (250 mM TriseHCl at pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% b-mercapto-ethanol, protease inhibitor cocktail and phostop) at 4
C. The same amount of protein in each lysate was loaded and separated by SDSepolyacrylamide gel electrophoresis and then transferred to a PVDF membrane.

S. Hong et al. / European Journal of Medicinal Chemistry 96 (2015) 218e230

Membranes were blocked with 5% bovine serum albumin in Trisbuffered saline (TBS) containing 0.01% Tween-20 for 2 h at room temperature, before overnight incubation with primary antibody at 4
C. After incubation, membranes were rinsed three times with TBS and were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. After washing, membranes were subjected to WestZol (iNtRON biotechnology, Seongnam, Korea), and were visualized using the LAS-4000 imaging system. 4.8. HDAC activity assay The A549 cell lysate HDAC activity was measured by using the HDAC Fluorescent Activity Assay Kit (BioVision, CA, USA) according to manufacturer's instructions. Briefly, the HDAC fluorometric substrate and assay buffer were added to cell lysates in a 96-well format and incubated at 37
C for 30 min. The reaction was stopped by adding lysine developer, and the mixture was incubated for another 30 min at 37
C. Additional negative controls included incubation without the cell lysate, without the substrate, or without both. TSA served as the positive control. A fluorescence plate reader with excitation at 360 nm and emission at 450 nm was used to quantify HDAC activity. 4.9. In vivo antitumor activity in xenograft model Six week-old male athymic mice (BALB/c nu/nu) were purchased Central Lab. Animal Inc. (Seoul, Korea). A549 cell suspension (1  107 cells in 0.1 mL of RPMI) was injected subcutaneously into the right flank of each mouse on day 0. The mice were treated when their tumor volume reached 50e60 mm3. The animals were randomly divided into five groups (six animals per group). Compound 30 (15 or 30 mg/kg body weight), psammplin A (30 mg/kg body weight) dissolved in 0.05% tween-80 in 0.9% NaCl was administered intraperitoneally three times a week. The positive control group was treated with paclitaxel (5 mg/kg body weight), and the negative control group was treated with an equal volume of the vehicle. The tumor volume was monitored two times per week for 35 days using calipers and estimated using the following formula: tumor volume (mm3) ¼ (width)  (length)  (height)  p/6. The body weight of each mouse was also monitored for toxicity. 4.10. Immunohistochemistry of tumor tissues The immunohistochemical analysis of tumor tissues was conducted to detect cell proliferation biomarkers using the Ki-67. Sections of the tumor tissues were incubated at 4
C overnight with the antibodies for Ki-67, detected using the EnVision Plus/HRP detection system (Dako, Carpinteria, CA, USA), and counterstained with hematoxylin and eosin. The stained section was observed under an inverted phase-contrast microscope and photographed. 4.11. Statistical analysis All of the experiments were repeated at least three times. Data were expressed as the mean ± standard deviation (SD) for the indicated number of independently performed experiments and analyzed using Student's t-test. Values of P < 0.05 were considered statistically significant. Acknowledgment This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (No. 20100020428 and No. 2009-0083533). This work was also supported by BK21 Plus Program in 2014.

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