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European Journal of Medicinal Chemistry 45 (2010) 6005e6011

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Semi-synthesis and anti-tumor activity of 5,8-O-dimethyl acylshikonin derivatives

Wen Zhou, Ying Peng, Shao-Shun Li*

School of Pharmacy, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, China

a r t i c l e i n f o

Article history:Received 26 June 2010Received in revised form29 September 2010Accepted 30 September 2010Available online 7 October 2010

Keywords:Shikonin derivativesSemi-synthesisAnti-tumor activityStructureeactivity relationship

* Corresponding author. Tel./fax: þ86 21 34204775E-mail address: ssli@sjtu.edu.cn (S.-S. Li).

0223-5234/$ e see front matter � 2010 Elsevier Masdoi:10.1016/j.ejmech.2010.09.068

a b s t r a c t

A set of twenty-two 5,8-O-dimethyl acylshikonin derivatives were designed and synthesized startingfrom shikonin. The cell-based investigation demonstrated that these dimethylated derivatives were lessactive than or equally effective to shikonin. However, the selective cytotoxicities toward MCF-7 werefound among these derivatives, together with no toxicity in the normal cell. Furthermore, compounds 3f,3p, 3r were subjected to KM mice suffering from S-180 carcinoma subcutaneously, which possessedmore potent than Fluorouracil, a typical anticancer drug used clinically. So we may conclude that themodification to the mother nucleus of shikonin via the methylation is an available approach to acquiringanti-tumor agents with higher selectivity and lower toxicity.

� 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction

Natural products have historically provided new drugs againsta wide variety of diseases, and cancer is certainly no exception.Several plants of Boraginaceae family have been usedmedicinally asanti-inflammatory, antiarthritis, and antimicrobic agents in Europeand East Asia [1,2]. Shikonin and its derivatives, which primarilyoccur in Lithospermun erythrorhizon, have been arousing greatinterest as the hallmark molecules responsible for their significantand fascinating anti-tumor activities by different mechanisms[1,3,4]. Many shikonin derivatives, in which most modificationswere focused on the hydroxyl group of the side chain, have beensynthesized and evaluated as to their anti-tumor effects on variouscancer cell lines. Acetylshikonin, isovalerylshikonin and SH-7exhibited obvious inhibitory actions on topoisomerase I, whichwere stronger than their mother compound shikonin [5e7]. Shi-konin glycosyl derivatives were also reported to show similar orstronger cytotoxicity than shikonin [8]. Therefore, reasonablemodifications to shikonin could be vital for obtaining more prom-ising anti-tumor agents.

However, shikonin derivatives prepared previously, althoughshown to have anti-tumor activities, have not gone into clinicaltrials yet. A lot of shikonin derivatives, showing great potential ascancer inhibitory agents in cell culture studies, were not performed

.

son SAS. All rights reserved.

well in vivo, possibly because of extensively bioreductive alkylationof shikonin derivatives with the nucleophiles such as glutathione,proteins or DNA [1,9,10]. Therefore, only the modification to thealcoholic hydroxyl group of side chainwas insufficient to overcomedefects in the structure of shikonin itself. To our knowledge, therehad been no documents touching upon the methoxylated shikoninderivatives starting from naturally occurring shikonin. To explorethe anti-tumor activities, we masked the phenolic hydroxyl groupsof shikonin with methyl groups to prepare 5,8-O-dimethyl acyl-shikonin derivatives.

Capped shikonin derivatives withmethyl groupsmay contributeto the enhancement of the anti-tumor activity in vivo. It wasreported that the methoxylated flavones have a great advantageover the nonmethyated flavones regarding inhibiting the prolifer-ation of cancer cells at the cancer promotion stage [11]. Althoughseveral literatures demonstrated that the dimethylation of naph-thazarin ring would benefit the cytotoxicity of quinones [12e14],no evidences were provided to support the modifications favorablefor shikonin. Additionally, the discovery of 5,8-O-dimethyl acetyl-shikonin from Onosma argentatum [15], which had the ability tostimulate the growth of normal cells such as human amnionfibroblasts [16], promoted us to gain further insight into themethylated shikonin derivatives. Encouraged by the variousreasons mentioned above, we designed and synthesized a series of5,8-O-dimethyl acylshikonin derivatives starting from shikonin toscreen their cytotoxicities against three cancer cell lines and thenormal cell in vitro, and then further evaluated for the anti-tumoractivities on S-180 xenograft models with the analogues 3f, 3p, 3r.

W. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 6005e60116006

2. Chemistry

Synthesis of 5,8-O-dimethyl acylshikonin derivatives (3aw3v) isdepicted in Scheme 1. Shikonin 1was prepared in bulk according tothe procedure reported by Assimopoulou and Papageorgiou [17].Concretely speaking, all shikoninderivatives contained in theextractfrom Lithospermun erythrorhizon roots were formed into the Cu-complexes with copper acetate, and the chelates were displaced byacids to release the derivatives which were transformed by hydro-lysis into shikonin1.Manyattemptshavebeen continuouslymade todevelop5,8-O-dimethyl shikonin2usingvariousmethyl agentswithshikonin 1 as starting material under different reaction conditions.The standard methylation conditions (Me2SO4, K2CO3) generallyproceeded poorly [18,19], possibly due to its high chemical reactivityand polyoxygenated nature [2,20]. When being treated shikoninwithmethyl iodideand silveroxide, the reactionaffordedonly tracesof the expectedquinone inCHCl3[21]. Nevertheless, the reactionwasperformed smoothly using with methyl iodide in dimethyl form-amide as solvent at the presence of potassium carbonate in 76.6%yield. Next, our attention was turned to the introduction of variouscarboxylic acids to compound 2, which was converted to the targetcompounds 3aw3v with the corresponding organic acids in thepresence of dicyclohexylcarbodiimide (DCC) and dimethylamino-pyridine (DMAP) at room temperature overnight [22].

3. Results and discussion

The in vitro cytotoxic activity of the target derivatives 3aw3vagainst HT-29 (colon cancer), K562 (leukemia), MCF-7 (breastcancer), HSF (Human skin fibroblasts) was evaluated by the cell-based MTT Assay [23] using shikonin as a reference compound.Anti-tumor potency of the tested compounds was displayed by IC50values that were calculated by linear regression analysis of theconcentration-response curves afforded for each compound. Thedata are listed in Table 1. Subsequently, three derivatives (3f, 3p, 3r)were evaluated with an S-180 xenograft model in male KM mice.

The cytotoxic activity of twenty-three compounds tested againstthree kinds of cancer lines and the normal cell revealed that alldimethylated derivatives were less active than, or comparative tothe lead compound shikonin, and that most compounds displayedno cytotoxicity toward the normal cell (IC50 > 100 mM). Thephenomenon was observed for human amnion fibroblasts treatedwith compound 3a (IC50¼ 140 mM) [16]. Although Shikonin showedbetter anti-tumor activity among HT-29, MCF-7, K562(IC50 ¼ 15.8 mM, 1.9 mM, 0.6 mM respectively), no selectivity wasreflected between cancer cells tested and the normal cell(IC50 ¼ 1.3 mM). It could be concluded that the methylation ofthe phenolic hydroxyl group of shikonin drastically enhanced theselectivity in comparison with the positive control, in spite ofdeceasing the cytotoxic activity against cancerous cells with varyingdegrees.

Compared with compound 2, the introduction of saturated alkylgroup at 10-position in the side chain did not significantly affectedanti-tumor potency, irrespective of the number and length of

OOH

OH O OH

OOCH3

OCH3O O

i

21

1 245

8 1'

Scheme 1. Reagents and conditions: (i) CH3I/K2CO3/DMF, 60

branched chains (3aw3d). However, the introduction of a doublebond to the alkyl group produced compound 3e, 3f, whichcontributed to the enhancement of the cytotoxic activity. The datareported in Table 1 demonstrated that the compounds bearing theunsaturated alkyl group have more potent with IC50 values of lessthan 12.2 mM in all cancer lines. For example, compound 3f wasapproximately 5-fold more active against MCF-7 with the IC50values of 3.3 mM than compound 2 (IC50 ¼ 17.2 mM). Again, whetherthe ester contained heteroatom had a profound influence on thecytotoxicity to the target compounds against a panel of cancerlines. A good example was illustrated that compound 3p containinghydroxyl group exhibited the activity comparative to the leadcompound shikonin against MCF-7 (IC50 ¼ 2.2 mM vs.IC50 ¼ 1.9 mM), and it displayed the differential of cytotoxicitiesamong three cancer lines and the normal cell, possibly due tohaving a unique mechanism of action with an ATP-noncompetitiveinhibitor of tyrosine kinases such as v-Src and EGFR [24]. Similarly,Compounds 3r bearing tetrahydrofuryl expressed stronger cyto-toxic activity with the IC50 value of 5.5 mM against MCF-7 thancompound 2 (IC50¼ 17.2 mM). Apparently, the relative positions andtypes of the heteroatom and whether it was saturated seemed tobe critical to cytotoxic activity among compounds 3qw3v. Theresults in vitro indicated that the switch of heteroatom’ positionsand the introduction of double bonds to the ring could sufferedfrom loss of the cytotoxic activity.

Among the prepared compounds, replacing the alkyl groupwiththe aryl moiety (3he3o) caused a sharp drop in cytotoxic activityin all cell lines, suggesting that an increase in steric bulk led toa decrease in anti-tumor effects. The tendency was in accordancewith our previous report [22]. However, the position of methoxysubstituent on aryl moiety had little influence on cytotoxicity.Starting from compound 3l, moving the methoxy group from thepara- to the ortho-positions (3m) had similar activity to that of 3lagainst three cancer cell lines. Replacement of the methoxy groupwith an electron-withdrawing nitro-group resulted in derivative3k, which led to a dramatic drop of anti-tumor potency againstK562. Similarly, except for compound 3n, replacing the phenyl withphenethyl (3i) and cinnamenyl (3j) overall had only minor effectson the cytotoxic activity.

Log P (P ¼ partition coefficient in an octanolewater system) isa standard parameter that is measured for any new compound ofbiological interest in many companies. As shown in Table 1, the logP values of prepared compounds, calculated by ChemDraw, weredistributed in the range from 0.92 to 3.93. Although the obviouscorrelation of log P and anti-tumor activities was not found, thecompounds possessing appropriate log P values would be helpful toenhance the anti-tumor activities. This was supported by theobservation that compounds 3p and 3r, which have the same log Pvalues of 1.8, exhibited more potent than other compoundsprepared. More detailed roles of the log P values in anti-tumoractivities for this class of dimethyl acylshikonin derivatives arefurther to be investigated.

In order to demonstrate if the dimethylated shikonin derivatives3f, 3p, 3r have the ability to inhibit the tumor growth and toxic

H

OOCH3

OCH3O O

O

ii

3a~3v

R

�C, 5 h, N2; (ii) RCOOH, DCC/DMAP/CH2Cl2, rt, overnight.

Table 1Cytotoxicity of shikonin derivatives against HT-29, K562, MCF-7, HSF.

OOCH3

OCH3O O

O3a~3v

R

Compounds R IC50 (mM) Log Pa

HT-29 MCT-7 K562 HSF

3a 20.8 13.4 18.2 87.5 1.67

3b 17.3 15.7 21.1 92.1 2.33

3c 19.6 14.3 13.4 67.6 2.74

3d 28.7 9.8 16.6 78.9 2.89

3e 10.6 7.2 12.2 86.2 2.72

3f 9.7 3.3 11.7 >100 2.90

3g 43.9 11.6 9.5 >100 3.31

3h 60.2 27.3 36.5 >100 3.57

3i 56.3 24.1 22.2 >100 3.93

3j 43.1 17.5 28.5 >100 3.91

3k NO2 78.2 40.3 76.3 >100 2.95

3lOCH3 51.7 35.5 29.2 >100 3.44

3mH3CO

45.6 24.7 33.9 74.5 3.44

3n

OCH3

24.5 16.6 17.8 96.4 3.39

Table 1 (continued)

Compounds RIC50 (mM)

Log PaHT-29 MCT-7 K562 HSF

3o OCH3

OCH3

31.3 20.4 46.7 >100 3.66

3p OH 9.7 2.2 15.3 >100 1.8

3q

O18.9 15.3 14.2 >100 2.19

3r

O11.5 5.7 11.5 >100 1.8

3s

O15.6 10.7 10.9 >100 2.13

3t

O27.9 18.6 12.2 >100 1.68

3u

N23.1 13.5 45.6 >100 2.23

3vN

46.7 37.5 27.6 >100 2.23

2 16.9 17.2 26.4 63.8 1.441 shikonin 15.8 1.9 0.6 1.3 0.92

Mean IC50 values were calculated from at least three independent experiments.a Indicates that the values of log P for all compounds were calculated through

ChemDraw.

W. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 6005e6011 6007

effects in vivo, a study using KM mice given a subcutaneousinjection of sarcoma S-180 was performed. As is shown in Table 2,compound 3f, 3p, 3r inhibited the growth of sarcoma S-180 with53.7%, 59.4% or 66.1% respectively when administered via intra-peritoneal (i.p.) injection at 12 mg/kg once a day for 9 days,compared with the control as physiological saline containing 1%Tween 80 and 0.5% dimethyl-sulfoxide (control tumor weight100%). The inhibitory effect on tumor growth by compound 3f,3p, 3r was exhibited after daily i.p. administration for 9 days.Meanwhile, no significant toxicity was observed in dimethylatedshikonin derivatives-treated mice. The 5-FU, a typical anticancerdrug, used as a positive control, showed 49.2% suppression via i.p..Interestingly, although acetylshikonin was reported to showsignificant suppression of tumor growth [25,26], the shikonin-treated KM mice with 4 mg/kg once a day via i.p. died in four daysin at least three independent experiments, and accompaniedwith apparent adverse effects such as weight reduction, hypo-trichosis and much bloody ascites. When adjusted to 1.5 mg/kg/dayvia the same route of administration, some toxic and side-effectsabovementioned were observed anatomically, and lowered furtherto 1.0 mg/kg/day, the in vivo inhibition effects were also decreasedto 38.9%. The results of the anti-tumor effects in vivo indicated

Table 2Tumor growth inhibition in KM mice implanted with S-180 sarcoma.

Group Dose (mg/kg) Routes Numbers BWC (%) TW(g) mean�SD TWI (%)

D0 D10

Control i.p. 10 10 þ57.8% 1.39 � 0.213f 12 i.p. 10 10 þ38.2% 0.64 � 0.14﹡﹡ 53.7%3p 12 i.p. 10 10 þ44.1% 0.56 � 0.12﹡﹡ 59.4%3r 12 i.p. 10 10 þ48.3% 0.47 � 0.09﹡﹡ 66.1%Shikonin 1.0 i.p. 10 10 þ8.3% 0.85 � 0.13﹡﹡ 38.9%5-FU 25 i.p. 10 10 þ51.2% 0.71 � 0.10﹡﹡ 49.2%

Data were presented as �X � SD, and significance was assessed with Student’s t-test. Differences were considered significant at **p < 0.01, compared with control group; D0

means the day before beginning to dose.; D10 means the 10th day; BWC means average body weight change; TW means average tumor weight; TWI means the averageinhibition rate of tumor weight.

W. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 6005e60116008

dimethylated shikonin derivatives were more potent anti-tumoragents than the positive control, to some extent implying thefact that the modification to hydroxyl groups of parent nucleus ofshikonin via the methylation was available for enhancing the anti-tumor potency of shikonin and lowering its toxicity.

4. Conclusion

We have designed and synthesized twenty-two 5,8-O-dimethylacylshikonin derivatives and one 5,8-O-dimethyl shikonin, andevaluated for their anti-tumor effects using three sorts of cancercells and the normal cell and KM mice implanted S-180 carcinomasubcutaneously. Most of the prepared compounds displayed theselective cytotoxic activities toward MCF-7, together with nocytotoxicity to the normal cell, although they exhibited less activethan or comparative to the positive control shikonin. Moreimportantly, the results in vivo unveiled that the dimethylatedshikonin derivatives showed more potent and selective than shi-konin and 5-Fu. Hopefully, this study may provide valuable infor-mation that the modifications on the mother nucleus of shikoninvia methylation, together with the introduction of a suitableoxygen-containing group at the 10-position in the side chain ofshikonin, are conducive to designing and acquiring prospectiveanti-tumor agents.

5. Experimental protocols

5.1. Chemistry

Reagents and solvents were obtained from commercial supplies.Solvents were dried and purified using standard techniques. Allreactions involving air or moisture sensitive or intermediates werecarried out under nitrogen. Melting points were determined ona SGW X-4 micro-melting point apparatus and are uncorrected.NMR spectra were recorded on Varian Mercury-300 spectrometer(300 MHz for 1H and 75 MHz for 13C), chemical shifts of 1H and 13Cspectra were recorded with tetramethylsilane as internal standard.Mass spectra were recorded on a Shimadzu LCMS-2010EV massspectrometer. Column chromatography was run on silica gel(200e300 mesh) from Qingdao Ocean Chemical Factory.

5.2. Preparation of shikonin (1)

Dried pulverized roots (100 kg) of Lithospermum erythrorhizonSieb. et Zucc, purchased from Liaoning province in China, wereextracted by supercritical CO2 (the device manufactured by Nan-tong Huaan above-critical extraction Co., Ltd. China and equippedwith 48 L of the supercritical CO2 reactor, and its model: HA221-40-48) to obtained dark red viscous residues (2 kg). Methanol(200 mL) was selected to dissolve the resulting extract (30 g) and

filtered to get rid of the insoluble substance. Afterwards the solu-tion of copper acetate (14.0 g) in distilled water (30 mL) was addedto the methanolic solution to chelate naphthoquinone derivatives,and then filtered. Subsequently 10% HCl (150 mL) was applied todisplace copper ions from Cu-complexes, and stirred for 2 h, andthen filtered. Finally 2 L of 1 mol/L NaOH was added to hydrolyzethe filtrate for 12 h at room temperature. The reaction mixture wasadjusted to pH 4 with 10% of HCl and extracted with ethyl acetate(300 mL) in twice. The combined organic layer was washed withbrine (100 mL), and dried by anhydrous Na2SO4 for 1 h, andevaporated in vacuo, and then re-crystallized with n-hexane to giveshikonin 1 (8.5 g) as the purplish red solid. mp: 115w116 �C, 1HNMR (300 MHz, CDCl3): d 12.59 (s, 1H), 12.58 (s, 1H), 7.19 (s, 2H,benzene ring H), 7.16 (s,1H, quinone ring H), 5.20 (t,1H, J¼ 8.10 Hz),4.91 (t, 1H, J ¼ 7.20 Hz), 2.64 (m, 1H), 2.35 (m, 1H), 1.75 (s, 3H), 1.65(s, 3H). 13C NMR (75 MHz, CDCl3): d 180.6, 180.1, 165.7, 165.1, 151.5,137.7, 132.5, 132.3, 132.0, 118.6, 112.3, 111.7, 68.6, 35.1, 25.2, 18.2.ESI-MS: 311.28 (M þ Na)þ.

5.3. 2-(1-Hydroxy-4-methylpent-3-enyl)-5,8-dimethoxynaphthalene-1,4-dione (2)

Shikonin 1 (2.88 g, 0.01 mol), potassium carbonate (13.8 g,0.1 mol) and potassium iodide (100 mg) and dimethyl formamide(20 mL) were stirred for 30 min at 60 �C, and then methyl iodidewas added in batches. The reaction proceeded for 5 h under themonitoring of TLC. Ethyl acetate (30mL) and distilled water (20mL)were poured into the reaction mixture. The organic layer waswashed by distilled water and brine respectively, dried over MgSO4and concentrated in vacuo. The residual oil was purified by flashchromatography on silica gel with ethyl acetate and petroleumether (V:V ¼ 1:4) to afford 2-(1-hydroxy-4-methylpent-3-enyl)-5,8-dimethoxy- naphthalene-1,4-dione 2 (2.24 g, 76.6%) as the redsolid. mp: 56e59 �C, 1H NMR (300 MHz, CDCl3): d 7.32 (s, 2H,benzene ring H), 6.79 (s, 1H, quinone ring H), 5.17 (t, 1H,J ¼ 7.80 Hz), 4.75 (t, 1H, J ¼ 7.20 Hz), 3.98 (s, 6H), 2.45 (m, 2H), 1.72(s, 3H), 1.62 (s, 3H). 13C NMR (75 MHz, CDCl3): d 185.5, 185.1, 154.2,153.8, 150.6, 136.7, 133.9, 120.9, 120.4, 119.1, 69.2, 57.1, 57.0, 35.7,29.4, 26.1, 18.3. ESI-MS: 339.12 (M þ Na)þ.

5.4. General procedure for preparation of1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3-enylcaroxylates (3ae3v)

To 2-(1-hydroxy-4-methylpent-3-enyl)-5,8-dimethoxynaph-thalene-1,4-dione 2 (0.1 mol) and carboxylic acid (0.15 mol) inanhydrous CH2Cl2 were added DCC (0.2 mol) and DMAP (0.05 mol).After stirring overnight at room temperature under nitrogenatmosphere, petroleum ether was added to the reaction mixtureto facilitate precipitates, and then the solution was filtered, and

W. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 6005e6011 6009

concentrated in vacuo. The residual oil was purified by flash chro-matography to give 3ae3v as orange red oil.

5.4.1. 1-(1,4-Dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3-enyl acetate (3a)

Yield 71.9%, orange red oil, 1H NMR (300 MHz, CDCl3): d 7.31(s, 2H, benzene ring H), 6.69 (s, 1H, quinone ring H), 5.90 (m, 1H),5.11 (t, 1H, J ¼ 7.20 Hz), 3.96 (s, 3H), 3.92 (s, 3H), 2.45 (m, 2H), 2.15(s, 3H), 1.68 (s, 3H), 1.58 (s, 3H). 13C NMR (75 MHz, CDCl3): d 184.6,184.1,169.8,155.9,150.5,144.3,138.8,137.7, 135.6,125.1, 120.0,118.0,116.7, 70.4, 61.9, 56.6, 33.9, 25.5, 21.0, 17.7. ESI-MS: 381.14(M þ Na)þ.

5.4.2. 1-(1,4-Dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3-enyl propionate (3b)

Yield 56.2%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.29(s, 2H, benzene ring H), 6.63 (d, 1H, J¼ 1.5 Hz, quinone ring H), 5.91(m,1H), 5.10 (t, 1H, J¼ 7.8 Hz), 3.93 (s, 6H), 2.57 (m, 4H), 1.64 (s, 3H),1.54 (s, 3H), 1.15 (t, 3H, J ¼ 7.2 Hz). ESI-MS: 395.15 (M þ Na)þ.

5.4.3. 1-(1,4-Dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3-enyl butyrate (3c)

Yield 51.8%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.29(s, 2H, benzene ring H), 6.64 (s, 1H, quinone ring H), 5.93 (t, 1H,J¼ 5.1 Hz), 6.12 (t, 1H, J¼ 6.9 Hz), 3.94 (s, 6H), 2.58 (m, 4H), 1.70 (m,5H), 1.49 (s, 3H), 0.96 (t, 3H, J ¼ 7.5 Hz). ESI-MS: 409.15 (M þ Na)þ.

5.4.4. 1-(1,4-Dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3-enyl isobutyrate (3d)

Yield 55.3%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.29(s, 2H, benzene ring H), 6.62 (d, 1H, J¼ 0.9 Hz, quinone ring H), 5.87(m, 1H), 5.10 (t, 1H, J ¼ 5.1 Hz), 3.90 (s, 6H), 2.62 (m, 2H), 2.43 (m,1H), 1.49 (s, 3H), 1.47 (s, 3H), 1.17 (d, 3H, J ¼ 2.4 Hz,), 1.15 (d, 3H,J ¼ 2.4 Hz). ESI-MS: 409.15 (M þ Na)þ.

5.4.5. (E)-1-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl but-2-enoate(3e)

Yield 48.6%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.30(s, 2H, benzene ring H), 7.03 (m, 1H), 6.64 (s, 1H, quinone ring H),5.97 (m, 1H), 5.87 (m, 1H), 5.13 (t, 1H, J ¼ 6.3 Hz), 3.90 (s, 6H), 2.65(m, 2H), 1.92 (m, 3H), 1.72 (s, 3H), 1.54 (s, 3H). ESI-MS: 407.17(M þ Na)þ.

5.4.6. 1-(5,8-Dimethoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-methylbut-2-enoate (3f)

Yield 62.4%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.30(s, 2H, benzene ring H), 6.67 (d, 1H, J ¼ 1.5 Hz, quinone ring H), 5.91(m,1H), 5.88 (s,1H), 5.11 (t, 1H, J¼ 6.3 Hz), 3.94 (s, 6H), 2.70 (m,1H),2.54 (m, 1H), 2.15 (s, 3H), 1.92 (s, 3H), 1.67 (s, 3H), 1.56 (s, 3H). ESI-MS: 421.16 (M þ Na)þ.

5.4.7. 1-(5,8-Dimethoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 2-methylbutanoate (3g)

Yield 45.3%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.27(s, 2H, benzene ring H), 6.62 (m, 1H, quinone ring H), 5.89 (m, 1H),5.10 (t, 1H, J ¼ 5.1 Hz), 3.91 (s, 6H), 2.60 (m, 3H), 1.63 (s, 3H), 1.54 (s,3H), 1.17 (d, 6H, J ¼ 3.0 Hz), 0.94 (m, 2H). ESI-MS: 423.18 (M þ Na)þ.

5.4.8. 1-(1,4-Dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3-enyl benzoate (3h)

Yield 36.7%, orange red oil. 1H NMR (300 MHz, CDCl3): d 8.09(m, 2H, benzene ring H), 7.61 (m, 3H, benzene ring H), 7.33 (s, 2H,benzene ring H), 6.78 (s, 1H, quinone ring H), 6.34 (m, 1H), 5.23 (t,1H, J ¼ 6.9 Hz), 3.98 (s, 3H), 3.88 (s, 3H), 2.66 (m, 2H), 1.68 (s, 3H),1.52 (s, 3H). ESI-MS: 443.15 (M þ Na)þ.

5.4.9. 1-(5,8-Dimethoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-phenylpropanoate(3i)

Yield 46.1%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.31(m, 7H, benzene ring H), 6.56 (d, 1H, J ¼ 1.2 Hz, quinone ring H),5.92 (m, 1H), 5.01 (t, 1H, J ¼ 6.3 Hz), 3.90 (s, 6H), 2.97 (t, 2H,J ¼ 7.8 Hz), 2.71 (t, 2H, J ¼ 7.8 Hz), 2.43 (m, 2H), 1.64 (s, 3H), 1.53(s, 3H). ESI-MS: 471.18 (M þ Na)þ.

5.4.10. 1-(1,4-Dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3-enyl cinnamate (3j)

Yield 41.3%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.72(d, 1H, J ¼ 15.9 Hz), 7.56 (m, 7H, benzene ring H), 6.72 (s, 1H,quinone ring H), 6.51 (d, 1H, J ¼ 15.9 Hz), 5.91 (m, 1H), 5.11 (t, 1H,J ¼ 7.5 Hz), 3.96 (s, 6H), 2.75 (m, 1H), 2.58 (m, 1H), 1.67 (s, 3H), 1.58(s, 3H). ESI-MS: 469.16 (M þ Na)þ.

5.4.11. 1-(1,4-Dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3-enyl 4-nitrobenzoate (3k)

Yield 78.3%, orange red oil. 1H NMR (300MHz, CDCl3): d 8.33 (m,4H, benzene ring H), 7.33 (s, 2H, benzene ring H), 6.79 (d, J¼ 1.2 Hz,quinone ring H), 6.17 (m, 1H), 5.18 (t, 1H, J ¼ 7.2 Hz), 3.95 (s, 3H),3.92 (s, 3H), 2.67 (m, 2H), 1.66 (s, 3H), 1.57 (s, 3H). ESI-MS: 488.13(M þ Na)þ.

5.4.12. 1-(1,4-Dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3-enyl 4-methoxybenzoate (3l)

Yield 33.7%, orange red oil. 1H NMR (300 MHz, CDCl3): d 8.03(d, 1H, J ¼ 7.8 Hz, benzene ring H), 8.02 (d, 1H, J ¼ 7.8 Hz, benzenering H), 7.31 (s, 2H, benzene ring H), 6.95 (d, 1H, J¼ 7.8 Hz, benzenering H), 6.94 (d, 1H, J ¼ 7.8 Hz, benzene ring H), 6.78 (s, 1H, quinonering H), 6.13 (t, 1H, J ¼ 5.7 Hz), 5.23 (t, 1H, J ¼ 6.9 Hz), 4.02 (s, 3H),3.97 (s, 3H), 3.94 (s, 3H), 2.72 (m, 2H), 1.69 (s, 3H), 1.58 (s, 3H).ESI-MS: 473.16 (M þ Na)þ.

5.4.13. 1-(1,4-Dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3–enyl 2-methoxybenzoate (3m)

Yield 31.5%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.83 (m,1H, benzene ring H), 7.51 (m, 1H, benzene ring H), 7.45 (d, 1H,J ¼ 2.4 Hz, benzene ring H), 7.44 (d, 1H, J ¼ 2.4 Hz, benzene ring H),7.02 (m, 2H, benzene ring H), 6.87 (d,1H, J¼ 1.2 Hz, quinone ring H),6.17 (m, 1H), 5.20 (t, 1H, J ¼ 8.1Hz), 3.96 (s, 3H), 3.94 (s, 3H), 3.88(s, 3H), 2.57 (m, 2H), 1.65 (s, 3H), 1.54 (s, 3H). ESI-MS: 473.16(M þ Na)þ.

5.4.14. 1-(5,8-Dimethoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 2-(4-methoxyphenyl)acetate (3n)

Yield 39.2%, orange red oil, 1H NMR (300 MHz, CDCl3): d 7.29(s, 2H, benzene ring H), 7.20 (d, 2H, J¼ 2.7 Hz, benzene ring H), 6.87(d, 2H, J¼ 2.7 Hz, benzene ring H), 6.53 (s, 1H, quinone ring H), 5.92(m,1H), 5.03 (t, 1H, J¼ 7.5 Hz), 3.90 (s, 6H), 3.79 (s, 3H), 3.61 (s, 2H),2.55 (m, 2H), 1.63 (s, 3H), 1.26 (s, 3H). ESI-MS: 487.17 (M þ Na)þ.

5.4.15. (E)-1-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-(3,4-dimethoxyphenyl)acrylate (3o)

Yield 37.4%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.68(s, 1H, J ¼ 15.6 Hz,), 7.31 (m, 5H, benzene ring H), 6.37 (s, 1H,quinone ring H), 6.37 (d, 1H, J ¼ 15.6 Hz), 6.01 (m, 1H), 5.21 (t, 1H,J ¼ 7.5 Hz), 3.96 (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H), 3.83 (s, 3H), 2.35(m, 1H), 2.37 (m, 1H), 1.66 (s, 3H), 1.58 (s, 3H). ESI-MS: 529.18(M þ Na)þ.

5.4.16. 1-(5,8-Dimethoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-hydroxy-3-methylbutanoate (3p)

Yield 37.1%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.31(s, 2H, benzene ring H), 6.67 (s, 1H, quinone ring H), 5.98 (t, 1H,

W. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 6005e60116010

J ¼ 2.7 Hz), 5.10 (t, 1H, J ¼ 8.4 Hz), 3.95 (s, 6H), 2.58 (m, 4H), 1.66(s, 3H), 1.56 (s, 3H), 1.28 (s, 3H), 1.27 (s, 3H). ESI-MS: 439.17(M þ Na)þ.

5.4.17. 1-(5,8-Dimethoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl furan-2-carboxylate (3q)

Yield 65.3%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.61(d, 1H, J ¼ 0.9 Hz, furan ring), 7.32 (s, 2H, benzene ring H), 7.23(m, 1H, furan ring H), 6.75 (d, 1H, J ¼ 0.9 Hz, quinone ring H), 6.67(m, 1H, furan ring H), 6.15 (t, 1H, J ¼ 4.8 Hz), 5.18 (t, 1H, J ¼ 3.6 Hz),3.97 (s, 3H), 3.95 (s, 3H), 2.69 (m, 2H), 1.67 (s, 3H), 1.59 (s, 3H).ESI-MS: 433.13 (M þ Na) þ.

5.4.18. 1-(5,8-Dimethoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl tetrahydro-furan-2-carboxylate (3r)

Yield 60.2%, orange red oil. 1H NMR (300 MHz, CDCl3): d 6.79(s, 2H, benzene ring H), 6.68 (d, 1H, J¼ 0.9 Hz, quinone ring H), 6.00(m,1H), 5.11 (t, 1H, J¼ 6.6 Hz), 4.53 (m,1H, J¼ 4.8 Hz), 4.01 (m, 8H),2.56 (m, 2H), 2.28 (m, 1H), 2.05 (m, 3H), 1.67 (s, 3H), 1.58 (s, 3H).ESI-MS: 437.16 (M þ Na)þ.

5.4.19. 1-(5,8-Dimethoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl furan-3-carboxylate (3s)

Yield 55.8%, orange red oil. 1H NMR (300 MHz, CDCl3, d ppm):d 8.06 (s, 1H, furan ring H), 7.44 (s, 1H, furan ring H), 7.31 (s, 2H,benzene ring H), 6.82 (s, 1H, quinone ring H), 6.80 (s, 1H, furan ringH), 6.09 (t, 1H, J ¼ 6.3 Hz), 5.16 (t, 1H, J ¼ 6.3 Hz), 3.97 (s, 3H), 3.88(s, 3H), 2,68 (m, 2H), 1.66 (s, 3H) 1.58 (s, 3H). ESI-MS: 433.13(M þ Na)þ.

5.4.20. 1-(5,8-Dimethoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl tetrahydro-furan-3-carboxylate (3t)

Yield 50.1%, orange red oil. 1H NMR (300 MHz, CDCl3): d 7.31(s, 2H, benzene ring H), 6.80 (d, 1H, J ¼ 4.2 Hz, quinone ring H), 6.16(t, 1H, J ¼ 5.7 Hz), 5.10 (t, 1H, J ¼ 7.8 Hz), 4.00 (m, 10H), 3.17 (m, 1H),2.60 (m, 2H), 2.21 (m, 2H), 1.67 (s, 3H), 1.57 (s, 3H). ESI-MS: 437.16(M þ Na)þ.

5.4.21. 1-(1,4-Dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3-enyl nicotinate (3u)

Yield 67.9%, orange red oil. 1H NMR (300 MHz, CDCl3): d 9.25(m, 1H, pyridine ring H), 8.81 (m, 1H, pyridine ring H), 8.32 (m, 1H,pyridine ring H), 7.45 (m, 1H, pyridine ring H), 7.32 (s, 2H, benzenering H), 6.71 (s, 1H, quinone ring H), 6.19 (m, 1H), 5.18 (t, 1H,J ¼ 7.2 Hz), 3.97 (s, 3H), 3.94 (s, 3H), 2.76 (m, 2H), 1.66 (s, 3H) 1.59(s, 3H) ESI-MS: 444.15(M þ Na)þ.

5.4.22. 1-(1,4-Dihydro-5,8-dimethoxy-1,4-dioxoaphthalent-2-yl)-4-methypent-3-enyl isonicotinate (3v)

Yield 69.1%, orange red oil. 1H NMR (300 MHz, CDCl3): d 8.88(d, 2H, J ¼ 5.1 Hz, pyridine ring H), 7.88 (d, 2H, J ¼ 5.1 Hz, pyridinering H), 7.28 (s, 2H, benzene ring H), 6.71 (s, 1H, quinone ring H),6.20 (m,1H), 5.19 (m,1H), 3.96 (s, 3H), 3.91 (s, 3H), 2.73 (m, 2H),1.62(s, 3H), 1.56 (s, 3H). ESI-MS: 444.15 (M þ Na)þ.

5.5. Anti-tumor activity in vitro

5.5.1. Cells cultureHT-29 and K562 were grown in RPMI-1640 supplemented with

10% fetal bovine serum (FBS), while MCF-7 and HSF were culturedin Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 10% FBS. Both culture mediums contained 1 mM nonessentialamino acids, 0.1 mM sodium pyruvate, 100 U/mL penicillin, and100 mg/mL streptomycin. The cells were incubated at 37 �C ina humidified atmosphere with 5% CO2.

5.5.2. Assessment of anti-tumor activity by MTT assayThe compounds tested were dissolved in suitable amount of

dimethyl-sulfoxide (DMSO) before the experiment to obtain thesolution having the known concentration, from which a definiteamount was taken, and diluted to the various concentrations withnutrient solution. The concentration of HT-29, K562, MCF-7, andHSF cells was adjusted to 5.0� 104 cells/mL respectively. Cells wereseeded on 96-well plates. After cultured for 24 h in 37 �C humidifiedincubator (5% CO2), cells were incubated in completemediumswiththe absence (negative control) and presence of various concentra-tions of compounds tested, respectively, for 24 h. Each group wasarranged four parallel wells. The supernatant was removed, andthen 20 mL of MTT solution (5 mg/mL) was added to each well. Afterre-incubated for another 4 h,100 mL of DMSOwas added to eachwellfor dissolving the formazan crystals. The percentage of cell viabilitywas determined by measuring the absorbance (Abs) at l ¼ 570 nmusing a Multiskan MK3 microplate reader (Thermo, USA). Survivalpercentage was calculated using the following equation: inhibitoryrate¼ (Abs570control cells � Abs570treated cells)/Abs570control cells� 100%.IC50 values were obtained from linear regression analysis of theconcentration-response curves plotted for each compounds tested.

5.6. Anti-tumor activity in vivo

SarcomaS-180cells (2�106) in0.2mLofphysiological salineweresubcutaneously injected between the femur of the male KM mice(Seven-week-old specific pathogen free (SPF)male KMmicewith theweight of 18e22 g each were obtained from Shanghai LaboratoryAnimal Center, Chinese Academy of Sciences). Tumor-bearing micewere randomly subdivided into 6 groups of 10. After 24 h, miceweretreatedwith compound3f,3p,3rvia an intraperitoneal (i.p.) injectionat a dose of 12mg/kg once a day for 9 days (9 times)while compound1 was administrated with 1 mg/kg. Similarly, 5-fluorouracil (5-FU,25 mg/kg, i.p.) was administered as a positive control. It was notice-able that all compound tested were dissolved in 0.5% DMSO and 1%Tween 80, and diluted to the given concentration by physiologicalsaline. The body weight of mice was observed. Subsequently micewere killed, and the tumors were excised and weighed in day 10.Control mice were injected with 0.5% DMSO and 1% Tween 80in physiological saline (vehicle). Tumor growth inhibition wascalculated as T/C (Treatment/control) by the following formula:T=C ¼ ½ðPC10=10�P

T10=10Þ=ðP

C10=10Þ� � 100%, where C10 isthe tumor weight at day 10 in control group and T10 is the tumorweight at day 10 in treated group.

Acknowledgments

We thank National Natural Science Foundation of China(No.30973604, No.91013012), Shanghai Program on Key BasicResearch (No.08JC1410800)and Comprehensive technology plat-forms for innovative drug R&D (2009ZX09301-007) for financialsupports.

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