化药合成,抗肿瘤新药

Synthesis,acute toxicities,and antitumor e?ects of novel

9-substituted b -carboline derivatives

Rihui Cao,Qi Chen,Xuerui Hou,Hongsheng Chen,Huaji Guan,Yan Ma,

Wenlie Peng and Anlong Xu *

Department of Biochemistry and Center for Biopharmaceutical Research,College of Life Sciences,Sun Yat-sen (Zhongshan)University,135Xin Gang Xi Road,Guangzhou 510275,PR China

Received 3May 2004;revised 27June 2004;accepted 28June 2004

Abstract—A series of novel 9-substituted b -carboline derivatives was synthesized from harmine and L -tryptophan,respectively.Cy-totoxic activities of these compounds in vitro were investigated.The results showed that most compounds of 9-substituted b -carb-oline derivatives had more remarkable cytotoxic activities in vitro than their corresponding parent compounds.Acute toxicities and antitumor e?ects of the selected b -carboline derivatives in mice were also examined.The results demonstrated that a short alkyl or benzyl substituent at position-9increased the antitumor activities signi?cantly and a ethoxycarbonyl or carboxyl substituent at posi-tion-3reduced the acute toxicity and neurotoxicity of these b -carboline derivatives dramatically.Moreover the compounds both with an alkoxycarbonyl or carboxyl substituent at position-3and a short alkyl or benzyl substituent at positon-9exhibited more signi?cant antitumor activities and lower acute toxicities and neurotoxicities than the other compounds.The compound 8c ,having an n -butyl and a carboxyl substituent at position-9and 3,respectively,was found to have the highest antitumor e?ect and the lowest acute toxicity and neurotoxicity.These data suggested that (1)appropriate substituents at both position-9and 3of b -carboline derivatives might play a crucial role in determining their enhanced antitumor activities and decreased acute toxicities and neurotoxic e?ects;(2)the b -carboline derivatives have the potential to be used as antitumor drug leads.Ó2004Published by Elsevier Ltd.

1.Introduction

b -Carbolines are a large group of naturally occurring and syntheti

c indole alkaloids with di?erent degrees of aromaticity,some of which are widely distribute

d in nat-ure,including various plants,1–3marin

e creatures,4in-sects,5mammalians as well as human tissues and body ?uids.6–9These compounds are o

f great interest due to their various biological activities such as intercalatin

g into DNA,10,11inhibiting CDK,12and Topisom-erase,13,14inhibiting monoamine oxidase 15,16as well as interacting wit

h benzodiazepine receptors 17–19and 5-hydroxy serotonin receptors,20and their broad spectrum pharmacological properties including anxiolytic,hyp-notic,anticonvulsant,21–23parasiticidal,24antiviral 25as well as antimicrobial activities.30Recent work 26–28and

our preliminary investigation results demonstrated that the compounds with b -carboline nucleus have potential antitumor activities.Yet we also found that this class of compounds caused remarkable acute neurotoxicity characterized by tremble,twitch,and jumping in exper-imental mice models.

Many previous reports focused on the e?ects of these compounds on the central nervous system (CNS),such as their a?nity with benzodiazepine receptors,17–195-HT 2A and 5-HT 2C receptors 20and imidazoline recep-tor 32,and so on.However so far there have been few such literatures about their cytotoxic activities of these compounds in vitro,even no reports are available deal-ing with systematic and detailed studies of structure–activity relationships on both antitumor activities and neurotoxic activities in vivo.A probable reason for this is that many of the b -carboline derivatives of interest are not readily available.One goal of the present investiga-tions was to synthesize a series of novel b -carboline derivatives and elucidate their preliminary relationships between structures and antitumor activities as well as

0968-0896/$-see front matter Ó2004Published by Elsevier Ltd.doi:10.1016/j.bmc.2004.06.038

Keywords :b -Carboline;Synthesis;Acute toxicity;Antitumor;Neuro-toxicity.

*Corresponding author.Tel.:+86-20-84113655;fax:+86-20-84038377;e-mail:

ls36@

Bioorganic &Medicinal Chemistry 12(2004)

4613–4623

neurotoxicities,and the other aim of this study was to increase the antitumor activities and decrease the neuro-toxic activities of these compounds.The work would help search for new antitumor leading compounds with signi?cant antitumor activities and lower neurotoxicities by simple chemical structural modi?cations on the basis of b -carboline ring.To the best of our knowledge,all 9-substituted b -carboline derivatives except 2a –c are novel compounds,and this is the ?rst time to report the anti-tumor activities and acute toxicities as well as neurotoxi-cities of this class of compounds in mice.

2.Results and discussion

2.1.Synthetic approach

N-alkylation and N-benzylation derivatives of harmine 1could be easily obtained in a good yield through react-ing with alkyl or benzyl bromide in the presence of so-dium hydride in DMF and THF (see Scheme 1).The starting material L -tryptophan reacted with formalde-hyde via well-known Pictet–Spengler condensation and then the products were esteri?ed with relevant alcohol to produce 1,2,3,4-tetrahydro-b -carboline-3-carboxy-lates,respectively.The 1,2,3,4-tetrahydro-b -carboline-3-carboxylates reacted with SeO 2in acetic acid solution by oxidative decarboxylation to produce b -carboline 3and reacted with sulfur in anhydrous xylene by dehydro-genation to a?ord b -carboline-3-carboxylate 5,6,7,respectively.The N-9position of compound 3,5,6,7was further alkylated or benzylated by the action of so-dium hydride in anhydrous DMF followed by addition of the relevant appropriate alkylating and benzylating agents (see Schemes 2and 3)to produce various 9-sub-

stituted b -carboline-3-carboxylate (5a –d ,6a –e ,7a –d ).The compounds 6and 6a –f were hydrolyzed in sodium hydroxide solution to a?ord the compounds 8and 8a –f (see Scheme 4).In our studies,we found that N-alkyl-ation and N-benzylation of b -carboline-3-carboxylate would be accompanied by ester exchange and ester hydrolysis unexpectedly.As a result,separating and purifying the products would become di?cult.Conse-quently,the key step was to use anhydrous DMF so as to guarantee the dryness of reaction system of alkyla-tion and benzylation of b -carboline-3-carboxylate 5,6,7.The chemical structures of all the synthesized novel compounds were con?rmed by FAB-MS,UV,IR,1H NMR,and elemental analyses data.2.2.Cytotoxicity assays

Thirty-seven b -carboline derivatives were examined for cytotoxic activities against a panel of human tumor cell lines.In order to enhance the solubility in aqueous solu-tion,compounds 8and 8a –f were converted into their water-soluble sodium salts and the other

compounds

4614R.Cao et al./Bioorg.Med.Chem.12(2004)4613–4623

investigated were all prepared in the form of hydrochlo-ride by the usual methods before use.The results were summarized in Table1.

As shown in Table1,most studied compounds showed signi?cant cytotoxic activities against several human tu-mor cell lines.9-Substituted harmines,b-carbolines,b-carboline-3-carboxylates,and b-carboline-3-carboxylic acids(except5d,6f,and7d)displayed more remarkable cytotoxic activities than their parent compounds,respec-tively,indicating that the introduction of an appropriate alkyl or benzyl group into9-position of b-carboline ring system facilitates the increase of their cytotoxic activi-ties.

Among all the compounds investigated,9-substituted harmine series demonstrated the highest cytotoxicities while9-substituted butyl b-carboline-3-carboxylates7a, 7c,7d(except7b),and their corresponding parent com-pound7were found to be less active with IC50values of more than100l M against tumor cell lines,suggesting that the substitution of7-methoxy and1-methyl groups might contribute to their increased cytotoxic activities while a long chain alkoxy at3-position might not be pared with the IC50values of other com-pounds,the IC50values of the compounds2a,2b,2f, and2g was lower than50l M,which implicated that the alkyl and benzyl substituents had almost equal po-tency to enhance their cytotoxic activities in vitro.

As for the harmine series,the compound2b with an ethyl group at9-position exhibited the highest cytotoxic activities(except for Lovo cell line).The compound2a having a methyl at position-9showed selective cytotoxic activities against the screened tumor cell lines with the lowest IC50value(8l M)against Hela.The compounds 2f and2g with a penta?uorobenzyl and a phenylpropyl at position-9,respectively,demonstrated the prominent

Table1.Cytotoxicity of b-carboline derivatives in vitro c(IC50,a l M)

Compounds PLA-801b HepG2b Bel-7402b BGC-823b Hela b Lovo b Harmine454654686066

2a29166352843

2b2214284517170

2c1116258692887

2d783132442042527

2e6585579527052

2f424643222831

2g503644462427 31722837927435335

4a219102302204327220

4b130167311171234159

4c1339916410188134

4d124107777348123 5295241293278100160

5a125119195181181175

5b251171247917107

5c136464433310289166

5d>1000>1000>1000>1000>1000312 6275>1000>1000>1000567>1000

6a17010

826014113669

6b8710910710813777

6c71476784760487173

6d7838453813122

6e471169527837

6f>1000>1000387>1000>1000>1000 7>1000>1000>1000>1000>1000>1000

7a675266313394295104

7b1097074826783

7c>1000>1000>10007295445

7d994>1000>1000>1000>1000>1000 8327354369330262198

8a2671472261268988

8b21217913016318362

8c9273921166011

8d100102112528641

8e863161354413

8f573153281005

a Cytotoxicity as IC

50

for each cell line,is the concentration of compound,which reduced by50%the optical density of treated cells with respect to untreated cells using the MTT assay.

b Cell lines include nonsmall cell lung carcinoma(PLA-801),liver carcinoma(HepG2and Bel-7402),gastri

c carcinoma(BGC-823),cervical carci-noma(Hela),colon carcinoma(Lovo).

c Data represent the mean values of three independent determinations.

R.Cao et al./Bioorg.Med.Chem.12(2004)4613–46234615

and broader spectrum of cytotoxic activities against all six human tumor cell lines with IC50values of all lower than50l M.

In contrast to the harmine series,9-substituted b-carbo-line series failed to exhibit obviously increased cytotoxi-cities.The compound3having no substituent on ring system showed a good cytotoxic activity against PLA-801and Lovo cell pounds4a–d displayed al-most equal cytotoxic activities while the compound4d showed higher and selective cytotoxic activity against Lovo cells with IC50values of48l M.

Of all9-substituted b-carboline-3-carboxylate deriva-tives,the compound6d having a benzyl at position-9 displayed strong cytotoxic activities against HepG2, Bel-7402,BGC-823,and Hela with IC50values of38, 45,38,and13l M,respectively.However compounds 5d and7d,having a benzyl at9-position,had little or even no cytotoxic e?ects on the tested cell lines,indicat-

ing that ethoxycarbonyl substituent was preferable for improving cytotoxic activities of this type of com-pounds.The compounds5b,6b,and7b with an ethyl at position-9,respectively,displayed higher cytotoxic activities against human tumor cell lines tested.Mean-while the compound5b showed highly selective cytotox-ic e?ects against PLA-801and Hela with IC50values25 and17l M,respectively.All these results,together with the cytotoxic activity of the compound2b,con?rmed that the ethyl group at position-9might play a crucial role in eliciting distinct cytotoxic activities of these com-pounds.

For the b-carboline-3-carboxylic acid series(8and8a–e),the cytotoxic activities were also enhanced by intro-duction of short alkyl or benzyl substituent into posi-tion-9of the b-carboline ring.The compounds8c and 8e,having an n-butyl and a benzyl at positon-9,respec-tively,showed remarkable cytotoxic activities against Lovo cell lines with IC50values of11and13l M,respec-tively,furthermore the compound8f exhibited the most signi?cant cytotoxic activity against Lovo cell lines with IC50values5l M.

A total analysis of the cytotoxic activities of b-carboline derivatives in vitro clearly suggested that(1)the b-carb-oline nucleus might be an important basis for the design and synthesis of new antitumor drugs;(2)the cytotoxic potencies of b-carboline derivatives depended upon the presence and location and nature of the subtituents, which were introduced into the b-carboline ring.(3) The cytotoxic activities of b-carboline derivatives were enhanced by the introduction of an appropriate substit-uent into position-9of b-carboline ring;(4)the Lovo cell lines were more sensitive to the tested compounds than other tumor cells.

2.3.Assessment of acute toxicity

The LD50values and scores for neurotoxicity of the se-lected b-carboline derivatives in mice after administra-tion were summarized in Table2.Neurotoxicities were the principal acute toxic e?ects observed in mice after receiving the b-carboline derivatives via intraperitoneal (i.p.)route.All the tested compounds resulted in acute toxic manifestation.Of all the compounds investigated, 1,2b,2c,2e,and5a caused remarkable acute toxic e?ects including tremor,twitch,jumping,tetanus,and supination.Death occurred mostly in high dosage group within1min after administration and then reached a peak1h later.For survived animals,the tremor and jumping lasted for about20min and then relieved grad-ually and returned to normal in the next day.Animals were drowsy and exhibited a decrease in locomotor activity after the administration of the compound8, 6d,8d,and8f.Death only occurred in high dosage group10–20min after administration and reached a peak1h later.However,the compounds8c caused no obvious neurotoxic reaction and no death at tested dos-age.Autopsy of the animals that died in the course of experiment and the necropsy?ndings in surviving ani-mals at the end of experimental period(14days)revealed no apparent changes in any organs.The results of toxi-cities suggested that an alkoxycarbonyl or carboxyl sub-stituent at position-3of the b-carboline ring might play a vital role in determining their decreased acute toxici-ties and neurotoxicities,and perhaps the carboxyl sub-stituent is more favorable.

2.4.Evaluation of antitumor activity

The tumor inhibition rates of the selected b-carboline derivatives in mice bearing Lewis lung cancer and Sar-coma180(S180)were summarized in Table3.Harmine (compound1)only showed a moderate antitumor e?ect (34.1%and15.3%against Lewis lung cancer and S180, respectively),however the selected9-substituted b-carb-oline derivatives all exhibited more potent antitumor pounds2c,2d,2e,6d,and8c displayed remarkable antitumor activities with the tumor inhibi-tion rate of more than40%against mice bearing Lewis lung cancer and S180.Moreover the compounds2e and8c demonstrated the highest antitumor e?ect with the tumor inhibition rate of46.9%against mice bearing Lewis lung cancer among all the tested compounds.The results indicated that short alkyl or benzyl substituent at Table2.Acute toxic e?ects of b-carboline derivatives in mice Compound Acute toxicity

LD50(mg/kg,95%CL)Neurotoxic e?ect 159.00(43.50–110.00)++a

2b24.25(22.24–26.44)++

2c26.45(23.91–29.26)++

2e147.82(132.55–164.85)++

5a70.61(64.17–77.70)+

6d240.38(211.38–273.50)À

8135.22(121.62–150.34)À

8c>500À

8d163.48(141.56–188.76)À

8e219.19(193.25–248.61)À

a Acute neurotoxic manifestation were denoted byÔ+ÕandÔÀÕ.AÔ+Õrepresents toxic reactions including tremble,twitch,jumping,teta-nus,and supination,Ô++Õmeans the same reactions with more severity,whileÔÀÕmeans no such reaction.

4616R.Cao et al./Bioorg.Med.Chem.12(2004)4613–4623

position-9of the b-carboline ring facilitated the increase of their antitumor activities.

3.Conclusions

The present investigation reported for the?rst time that b-carboline derivatives had signi?cant antitumor activi-ties in mice bearing Lewis lung cancer and Sarcoma180 but also exhibited remarkable acute neurotoxicity in experimental mice models.Alkyl or benzyl substituent at position-9of b-carboline ring might facilitate their antitumor activities and minimize their acute toxicities but not neurotoxicities.However alkoxycarbonyl or carboxyl substituent at position-3played a crucial role in determining their decreased acute toxicities and neu-rotoxicities.Furthermore,the compounds with appro-priate substituents both at positon-3and9displayed enhanced antitumor activities and decreased neurotoxi-cities simultaneously.The compound8c,having an n-butyl and a carboxyl at position-9and3,respectively, exhibited the highest antitumor e?ect and the lowest acute neurotoxicity and was found to be the most prom-ising leading compound for further investigation.These data suggested that(1)appropriate substituents at both position-9and3of b-carboline derivatives might play a crucial role in determining their enhanced antitumor activities and decreased acute toxicities and neurotoxic e?ects;(2)the b-carboline derivatives have the potential to be used as antitumor drug leads.

Although the antitumor e?ects of the studied b-carbo-line derivatives presented here remained relatively mod-est,it should be noted that the acute neurotoxicities of some compounds decreased dramatically by introduc-tion of an alkoxycarbonyl or carboxyl substituent into position-3of the b-carboline ring.This investigation and?nding would be helpful to further design and de-velop more potent antitumor agents together with low neurotoxicities.Further investigations are in progress in our laboratory to elucidate the molecular mechanisms involved in antitumor activities and neurotoxicities of b-carboline derivatives.To acquire more information about the structural requirements for enhancing antitu-mor activities and minimizing neurotoxicities,the syn-thesis of more new b-carboline derivatives with di?erent substituents at other positions is needed.

4.Experimental

4.1.Materials

All reagents were purchased from commercial suppliers and were dried and puri?ed when pound 1(Harmine)was extracted from Peganum multisectum Maxim,a plant indigenous to western China,according to the method by Duan et al.2Compounds3and5–8 were synthesized from the starting material L-trypto-phan according to the procedures described by Hagen et al.19and Lippke et al.18with a slight modi?cation, respectively.

Melting points were determined in capillary tubes on an electrothermal PIF YRT-3apparatus and without cor-rection.UV spectra were measured on Shimadzu UV 2501PC Spectrometer.FAB-MS spectra were obtained from VG ZAB-HS spectrometer.FTIR were run on a Bruker Equinox55Fourier Transformation Infarred Spectrometer.1H NMR spectra were recorded on a Var-ian INOVA500NB spectrometer.Elemental analyses were carried out on an Elementar Vario EL CHNS Ele-mental Analyzer.Silica gel F254were used in analytical thin-layer chromatography(TLC)and silica gel were used in column chromatography,respectively.

4.1.1.7-Methoxy-1,9-dimethyl-b-carboline(2a).A mix-ture of Harmine1(2.12g,10mmol),anhydrous DMF (50mL),and anhydrous THF(50mL)was stirred at rt until clear,and then60%NaH(0.6g,15mmol)and iodomethane(2mL,30mmol)were added and stirred at rt ter the mixture was evaporated in re-duced pressure.The resulting solution was poured into H2O(100mL),and extracted with ethyl acetate (3·150mL).The organic phase was washed with water and brine,then dried over anhydrous sodium sulfate,?l-tered,and evaporated.The oil obtained was puri?ed by silica column chromatography with ethyl acetate as the eluent.Upon recrystallization,white crystals of2a were obtained(1.8g,80%),mp121–123°C(from ether); FAB-MS m/e227(M+1);UV k max345,332,302,264, 244,214nm;IR(KBr):3380,2744,1628,1570,1469, 1348,1249,1152,1045,810cmÀ1;1H NMR(500MHz, CDCl3):8.24–8.25(1H,m,H-5),7.93–7.96(1H,m,H-3),7.69–7.71(1H,m,H-4), 6.86–6.89(1H,m,H-8), 6.81–6.83(1H,m,H-6), 4.04–4.07(3H,m,NC H3), 3.94–3.95(3H,m,OC H3), 3.04–3.05(3H,m,C H3). Anal.Calcd for C14H14N2O:C,74.33;H, 6.19;N, 12.39.Found:C,74.21;H,6.37;N,12.31.

4.1.2.7-Methoxy-9-ethyl-1-methyl-b-carboline(2b).A mixture of Harmine1(2.12g,10mmol),anhydrous DMF(50mL),and anhydrous THF(50mL)was stirred at rt until clear,and then60%NaH(0.6g,15mmol)and iodoethane(2.5mL,30mmol)were ter the mixture was treated in a manner similar to that de-scribed for2a to a?ord2b(2.0,83%),mp99–101°C (from ether);FAB-MS m/e241(M+1);UV k max345,

Table 3.Antitumor e?ects of b-carboline derivatives against mice

bearing Sarcoma180and Lewis lung carcinoma

Compound Tumor inhibition rate(%)

Lewis lung carcinoma Sarcoma180

134.115.3

2b42.037.6

2c44.040

.9

2e46.945.2

5a35.031.1

6d43.342.1

833.432.2

8c46.943.1

8d43.234.4

8e39.632.2

CTX88.687.5

R.Cao et al./Bioorg.Med.Chem.12(2004)4613–46234617

332,303,265,244,213nm;IR(KBr):3362,3128,1622, 1565,1451,1346,1217,1136,812cmÀ1;1H NMR (500MHz,CDCl3):8.26–8.27(1H,d,J=5Hz,H-5), 7.96–7.98(1H,d,J=5Hz,H-3),7.74–7.75(1H,d, J=4.5Hz,H-4), 6.86–6.90(2H,m,H-8,H-6), 4.52–4.57(2H,m,C H2CH3),3.95(3H,s,OC H3),3.05(3H, s,C H3),1.43–1.46(3H,m,CH2C H3).Anal.Calcd for C15H16N2O:C,75.00;H, 6.67;N,11.67.Found:C, 74.89;H,6.87;N,11.59.

4.1.3.7-Methoxy-9-butyl-1-methyl-b-carboline(2c).A mixture of Harmine1(2.12g,10mmol),anhydrous DMF(50mL),and anhydrous THF(50mL)was stirred at rt until clear,and then60%NaH(0.6g,15mmol)and n-butyl iodide(6mL,50mmol)were added and re?uxed ter the resulting mixture was treated in a man-ner similar to that described for2a to a?ord2c(2.1, 78%),mp104–105°C(from ether);FAB-MS m/e269 (M+1);UV k max346,334,303,265,244,213nm;IR (KBr):3428,2959,2927,1621,1563,1497,1448,1356, 1243,1197,1137,812cmÀ1;1H NMR(500MHz, CDCl3):8.26–8.28(1H,d,J=

5.5Hz,H-5),7.95–7.97 (1H,d,J=9Hz,H-3),7.71–7.72(1H,d,J=5Hz,H-4),

6.85–6.88(2H,m,H-8,H-6), 4.43–4.46(2H,m, J=8Hz,C H2CH2CH2CH3),3.94(3H,s,OC H3),3.01 (3H,s,C H3), 1.78–1.84(2H,m,CH2C H2CH2CH3), 1.41–1.48(2H,m,CH2CH2C H2CH3),0.97–1.00(3H, m,CH2CH2CH2C H3).Anal.Calcd for C17H20N2O:C, 76.12;H,

7.46;N,10.45.Found:C,75.96;H,7.69;N, 10.53.

4.1.4.7-Methoxy-9-hydroxyethyl-1-methyl-b-carboline (2d).A mixture of Harmine1(1.05g,

5.0mmol)and anhydrous DMF(25mL),and anhydrous THF (25mL)was stirred at rt until clear,and then60% NaH(0.3g,7.5mmol)and2-iodoethanol(3mL, 40mmol)were added and re?uxed for5h.Then the resulting mixture was treated in a manner similar to that described for2a to a?ord2d(0.7g,54%),mp204–206°C (from ether);FAB-MS m/e257(M+1);UV k max328, 304,249,211nm;IR(KBr)3295,2696,1629,1569, 1467,1352,1155,1051,810cmÀ1;1H NMR(500MHz, CDCl3):8.15–8.16(1H,d,J=4Hz,H-5),7.93–7.94 (1H,d,J=3.5Hz,H-3),7.63–7.64(1H,d,J=5.0Hz, H-4),

6.88–6.95(2H,m,H-8,H-6),4.66–4.71(2H,t, J=5.5Hz,NC H2CH2OH), 4.06–4.08(2H,m, NCH2C H2OH), 3.94(3H,s,OCH3), 2.99(3H,s, C H3).Anal.Calcd for C15H16N2O2:C,70.31;H,6.25; N,10.94.Found:C,70.13;H,6.46;N,10.8

7.

4.1.

5.9-Benzyl-7-methoxy-1-methyl-b-carboline(2e).A mixture of Harmine1(2.12g,10mmol),anhydrous DMF(50mL),and anhydrous THF(50mL)was stirred at rt until clear,and then60%NaH(0.6g,15mmol)and benzyl bromide(5mL,40mmol)were ter the mixture was treated in a manner similar to that de-scribed for2a to a?ord2e(2.2,67%),mp131–133°C (from ether);FAB-MS m/e303(M+1);UV k max343, 330,301,244,210nm;IR(KBr):3421,2958,1620, 1565,1498,1447,1361,1256,1197,1172,1044, 825cmÀ1;1H NMR(500MHz,CDCl3):8.29–8.30(1H, d,J=5.5Hz,H-5),8.01–8.02(1H,d,J=8.5Hz,H-3), 7.80–7.81(1H,d,J=5.5Hz,H-4),7.23–7.30(3H,m,H-8,H-6,Ar–H),

6.98–

7.00(1H,d,J=7.0Hz,Ar–H), 6.90–6.92(1H,m,Ar–H), 6.76(1H,s,Ar–H), 5.75 (2H,s,NC H2Ar), 3.85(3H,s,OC H3), 2.88(3H,s, C H3).Anal.Calcd for C20H18N2O:C,79.47;H,5.96; N,9.27.Found:C,79.29;H,6.19;N,9.21.

4.1.6.9-(20,30,40,50,6-Penta?uoro)benzyl-7-methoxy-1-methyl-b-carboline(2f).A mixture of Harmine1 (0.53g,2.5mmol),anhydrous DMF(15mL),and anhy-drous THF(15mL)was stirred at rt until clear,and then 60%NaH(0.15g,3.8mmol)and a-bromo-2,3,4,5,6-pen-ta?uorotoluene(0.7mL,4.5mmol)were ter the mixture was treated in a manner similar to that described for2a to a?ord2f(0.64g,65%),mp173–174°C(from ether);FAB-MS m/e393(M+1);UV k max338,325,300,241,209nm;IR(KBr):2961,1622, 1502,1446,1256,1026,816cmÀ1;1H NMR(500MHz, CDCl3):8.32–8.33(1H,d,J=5Hz,H-5),7.94–7.95 (1H,d,J=7.5Hz,H-3),7.72–7.73(1H,d,J=

5.5Hz, H-4),7.26(1H,s,H-8),

6.87–6.89(1H,m,H-6),5.85 (2H,s,C H2Ar), 3.88(3H,s,OC H3), 3.05(3H,s,

C H3).Anal.Calcd for C20H13F5N2O:C,61.22;H,

3.32;N,7.1

4.Found:C,61.09;H,3.46;N,7.06.

4.1.7.9-Phenylpropyl-7-methoxy-1-methyl-b-carboline (2g).A mixture of Harmine1(1.05g,5mmol),anhy-drous DMF(25mL),and anhydrous THF(25mL)were stirred at rt until clear,and then60%NaH(0.3g, 7.5mmol)and1-bromo-3-phenylpropane(3mL, 20mmol)were added and re?uxed ter the mix-ture was treated in a manner similar to that described for2a to a?ord2g(0.84g,51%),mp117–118°C(from ether);FAB-MS m/e331(M+1);UV k max346,332, 302,265,244,211nm;IR(KBr):2995,2931,1623, 1563,1449,1238,1156,1043,801cmÀ1;1H NMR (500MHz,CDCl3):8.25–8.26(1H,d,J=5Hz,H-5), 7.92–7.94(1H,d,J=8.5Hz,H-3),7.69–7.70(1H,d, J=5Hz,H-4),7.29–7.32(2H,m,H-8,H-6),7.20–7.25 (3H,m,Ar–H),6.84–6.86(1H,m,Ar–H),6.63–6.64 (1H,m,Ar–H),4.42–4.45(2H,m,NC H2CH2CH2Ar), 3.84–3.85(3H,s,OC H3),2.88(3H,s,C H3),2.74–2.77 (2H,m,NCH2C H2CH2Ar), 2.12–2.18(2H,m, NCH2CH2C H2Ar).Anal.Calcd for C22H22N2O:C, 80.00;H,6.67;N,8.48.Found:C,79.87;H,6.82;N, 8.39.

4.1.8.9-Methyl-b-carboline(4a).A mixture of norhar-man3(1.68g,10mmol)and anhydrous DMF(50mL) was stirred at rt till clear.Then60%NaH(0.6g, 15mmol)and iodomethane(2mL,30mmol)were added,and the mixture was stirred at rt -ter the resulting mixture was poured into H2O(150mL), and extracted with ethyl acetate(3·150mL).The organ-ic phase was washed with water and brine,then dried over anhydrous sodium sulfate,?ltered,and evaporated. The oil obtained was puri?ed by silica column chroma-tography with petroleum ether–acetone(2:1)as the elu-ent.Upon recrystallization,white crystals of4a were obtained(1.4g,77%),mp108–109°C(from petroleum ether–acetone);FAB-MS m/e183(M+1);UV k max 360,346,289,236,216nm;IR(KBr):2509,1638, 1504,1335,1259,835cmÀ1;1H NMR(500MHz, CDCl3):8.88(1H,s,H-4),8.46–8.47(1H,d,J=5Hz,

4618R.Cao et al./Bioorg.Med.Chem.12(2004)4613–4623

H-1),8.13–8.14(1H,d,J=6Hz,H-8),7.94–7.95(1H,d, J=5Hz,H-3),7.59–7.62(1H,m,H-5),7.45–7.46(1H,d, J=8.5Hz,H-6),7.25–7.30(1H,m,H-7),3.93(3H,s,

C H3).Anal.Calcd for C12H10N2:C,79.12;H,5.49;N,

15.38.Found:C,78.93;H,5.71;N,15.45.

4.1.9.9-Ethyl-b-carboline(4b).A mixture of norharman 3(1.68g,10mmol)and anhydrous DMF(50mL)was stirred at rt till clear,and then60%NaH(0.6g,15mmol) and iodoethane(2.5mL,30mmol)were ter the mixture was treated following the method described for 4a to a?ord yellow oil4b(1.5g,76%),FAB-MS m/e197 (M+1);UV k max365,342,291,239,218nm;IR(KBr): 2485,2022,1633,1500,1462,1355,1239,831cmÀ1;1H NMR(500MHz,CDCl3):8.84(1H,s,H-4),8.42–8.43 (1H,d,J=5Hz,H-1),8.04–8.05(1H,d,J=8Hz,H-8), 7.85–7.86(1H,d,J=5Hz,H-3),7.50–7.53(1H,m,H-5),7.34–7.36(1H,d,J=8Hz,H-6),7.20–7.23(1H,m, H-7),4.26–4.30(2H,m,C H2CH3),1.35–1.38(3H,m, CH2C H3).Anal.Calcd for C13H12N2:C,79.59;H, 6.12;N,14.29.Found:C,79.32;H,6.43;N,14.13. 4.1.10.9-Butyl-b-carboline(4c).A mixture of norharman 3(1.68g,10mmol)and anhydrous DMF(50mL)was stirred at rt till clear,and then60%NaH(0.6g,15mmol) and n-butyl iodide(6mL,50mmol)were added and ref-luxed ter the mixture was treated following the method described for4a to a?ord white crystals4c (1.6g,71%),mp108–109°C(from light petroleum ether–acetone);FAB-MS m/e225(M+1);UV k max 362,348,290,237,217nm;IR(KBr):2525,2030, 1632,1500,1458,1355,1235,823cmÀ1;1H NMR (500MHz,CDCl3):8.86(1H,s,H-4),8.43–8.44(1H, d,J=

5.5Hz,H-1),8.06–8.08(1H,d,J=8Hz,H-8), 7.88–7.89(1H,d,J=4.5Hz,H-3),7.52–7.55(1H,m, H-5),7.38–7.40(1H,d,J=8.5Hz,H-6),7.21–7.25(1H, m,H-7), 4.25–4.28(2H,m,C H2CH2CH2CH3), 1.79–1.85(2H,m,CH2C H2CH2CH3), 1.30–1.38(2H, m,CH2CH2C H2CH3),0.86–0.91(3H,m,CH2CH2-CH2C H3).Anal.Calcd for C15H16N2:C,80.36;H, 7.14;N,12.50.Found:C,80.15;H,7.35;N,12.39. 4.1.11.9-Benzyl-b-carboline(4d).A mixture of norhar-man3(1.68g,10mmol)and anhydrous DMF(50mL) was stirred at rt till clear,and then60%NaH(0.6g, 15mmol)and benzyl bromide(5mL,40mmol)were added and re?uxed ter the mixture was trea-ted following the method described for4a to a?ord white crystals4d(1.8g,69%),mp118–120°C(from ether);FAB-MS m/e259(M+1);UV k max358,344, 289,237,213nm;IR(KBr):3023,1619,1448,1332, 1258,821cmÀ1;1H NMR(500MHz,CDCl3):8.84 (1H,s,H-4),8.48(1H,s,H-1),8.15–8.16(1H,d, J=8Hz,H-8),7.98(1H,s,H-3),7.53–7.56(1H,m,H-5),7.41–7.43(1H,d,J=8Hz,H-6),7.13–7.31(6H,m, J=8Hz,H-7,Ar–H),5.55(2H,s,C H2Ar).Anal.Calcd for C18H14N2:C,83.72;H,5.43;N,10.85.Found:C, 83.57;H,5.69;N,10.7

6.

4.1.12.Methyl9-methyl-b-carboline-3-carboxylate(5a).A mixture of5(2.26g,10mmol)and anhydrous DMF (60mL)was stirred at rt for10min,then60%NaH (0.6g,2mmol)and iodomethane(2mL,30mmol)were ter the mixture was stirred at rt for30min. The resulting mixture was poured into iced-water (150mL)and extracted with ethyl acetate(3·100mL). The combined organic phase was washed with water and brine.Then dried over anhydrous sodium sulfate,?ltered, and evaporated.The yellow oil obtained was puri?ed by silica column chromatography with ethyl acetate as the eluent.After that the solid was collected,it was recrystal-lized from ethyl ether to give a white crystals(1.8g,75%), mp215–216°C(from ether);FAB-MS m/e241(M+1);UV k max358,343,307,272,236,219nm;IR(KBr):3387,2548, 2056,1730,1631,1334,1282,1207cmÀ1;1H NMR (500MHz,CDCl3):8.94(1H,s,H-4),8.88(1H,s,H-1),

8.20–8.21(1H,d,J=7.5Hz,H-8),7.65–7.68(1H,m,Ar–

H),7.50–7.52(1H,d,J=8Hz,Ar–H),7.36–7.39(1H,m, J=8Hz,Ar–H), 4.06(3H,s,OC H3), 4.00(3H,s, NC H3).Anal.Calcd for C14H12N2O2:C,70.00;H,5.00; N,11.67.Found:C,69.83;H,5.23;N,11.59.

4.1.13.Methyl9-ethyl-b-carboline-3-carboxylate(5b).A mixture of5(2.26g,10mmol)and anhydrous DMF (60mL)was stirred at rt for10min,then60%NaH (0.6g,2mmol)and iodoethane(2.5mL,30mmol)were ter the mixture was treated in a manner similar to that described for5a to a?ord white crystals5b(2.0g, 79%),mp155–156°C(from ether);FAB-MS m/e255 (M+1);UV k max358,345,306,273,236,205nm;IR (KBr):3248,1694,1629,1337,1250cmÀ1;1H NMR (500MHz,CDCl3):8.90–9.00(2H,m,H-4,H-1),8.21–8.23(1H,d,J=8Hz,H-8),7.65–7.68(1H,m,H-5), 7.52–7.54(1H,d,J=8Hz,H-6),7.36–7.39(1H,m,H-7),4.51(2H,s,NC H2CH3),4.07(3H,s,OC H3),1.52 (3H,s,NCH2C H3).Anal.Calcd for C15H14N2O2:C, 70.87;H,

5.51;N,11.02.Found:C,70.65;H,5.68;N, 10.93.

4.1.14.Methyl9-butyl-b-carboline-3-carboxylate(5c).A mixture of5(2.26g,10mmol)and anhydrous DMF (60mL)was stirred at rt for10min,then60%NaH (0.6g,2mmol)and n-butyl iodide(6mL,50mmol)were added and re?uxed ter the mixture was treated in a manner similar to that described for5a to a?ord white crystals5c(2.3g,82%),mp181–183°C(from petroleum ether–ethyl ether1:2);FAB-MS m/e283 (M+1);UV k max358,344,306,273,236,222nm;IR (KBr):2959,1735,1625,1361,1246cmÀ1;1H NMR (500MHz,CDCl3):8.94(1H,s,H-4),8.89(1H,s,H-1), 8.19–8.21(1H,d,J=7Hz,H-8),7.62–7.65(1H,m,H-5),7.50-7.52(1H,d,J=7.5Hz,H-6),7.34–7.37(1H,m, H-7),4.41–4.44(2H,m,C H2CH2CH2CH3),4.06(3H, s,OC H3),1.87–1.93(2H,m,CH2C H2CH2CH3),1.35–1.42(2H,m,CH2CH2C H2CH3),0.93–0.96(3H,m, CH2CH2CH2C H3).Anal.Calcd for C17H18N2O2:C, 72.34;H,6.38;N,9.93.Found:C,72.21;H,6.57;N, 9.86.

4.1.1

5.Methyl9-benzyl-b-carboline-3-carboxylate(5d).A mixture of5(2.26g,10mmol)and anhydrous DMF (60mL)was stirred at rt for10min,then60%NaH (0.6g,2mmol)and benzyl bromide(5mL,40mmol)were added and re?uxed ter the mixture was treated in a manner similar to that described for5a to a?ord white crystals5d(2.3g,73%),mp187–188°C(from petroleum

R.Cao et al./Bioorg.Med.Chem.12(2004)4613–46234619

ethyl ether);FAB-MS m/e317(M+1);UV k max356,341, 304,272,235,205nm;IR(KBr):3026,2945,1731,1622, 1335,1242cmÀ1;1H NMR(500MHz,CDCl3):8.89–8.90(2H,d,J=4Hz,H-4,H-1),8.21–8.22(1H,d, J=8Hz,H-8),7.58–7.61(1H,m,H-5),7.47–7.48(1H,d, J=8.5Hz,H-6),7.35–7.38(1H,m,H-7),7.24–7.28(3H, m,Ar–H),7.13–7.15(2H,m,Ar–H),5.60(2H,s,C H2Ar), 4.05(3H,s,OC H3).Anal.Calcd for C20H16N2O2:C, 75.95;H,5.06;N,8.86.Found:C,75.75;H,5.29;N,8.78.

4.1.16.Ethyl9-methyl-b-carboline-3-carboxylate(6a).A mixture of6(2.4g,10mmol)and anhydrous DMF (60mL)was stirred at rt for10min,then60%NaH (0.6g,15mmol)and iodomethane(2mL,30mmol)were ter the mixture was treated in a manner similar to that described for5a to a?ord white crystals6a(1.9g, 75%),mp139–140°C(from ethyl ether);FAB-MS m/e 255(M+1);UV k max358,342,306,272,236,219nm; IR(KBr):3446,3398,2603,1721,1628,1328, 1280cmÀ1;1H NMR(500MHz,CDCl3):8.94(1H,s, H-4),8.86(1H,s,H-1),8.19–8.20(1H,d,J=7.5Hz,H-8),7.66–7.67(1H,m,H-5),7.49–7.51(1H,d,J=8.5Hz, H-6),7.35–7.37(1H,m,H-7), 4.52–4.56(2H,m, OC H2CH3), 3.98(3H,s,NC H3), 1.48–1.51(3H,s, OCH2C H3).Anal.Calcd for C15H14N2O2:C,70.87;H,

5.51;N,11.02.Found:C,70.73;H,5.73;N,11.09.

4.1.17.Ethyl9-ethyl-b-carboline-3-carboxylate(6b).A mixture of6(2.4g,10mmol)and anhydrous DMF (60mL)was stirred rt for10min,then60%NaH (0.6g,2mmol)and iodoethane(2.5mL,30mmol)were ter the mixture was treated in a manner simi-lar to that described for5a to a?ord white crystals6b (1.8g,67%),mp117–118°C(from ethyl ether);FAB-MS m/e269(M+1);UV k max359,344,306,273,237, 221nm;IR(KBr):3413,2984,1717,1633,1334, 1257cmÀ1;1H NMR(500MHz,CDCl3):8.93(1H,s, H-4),8.86(1H,s,H-1),8.18–8.21(1H,d,J=8Hz,H-8),7.62–7.64(1H,m,H-5),7.48–7.50(1H,d,J=8Hz, H-6),7.33–7.36(1H,m,H-7), 4.42–4.56(4H,m, OC H2CH3,NC H2CH3),1.46–1.52(6H,m,OCH2C H3, NCH2C H3).Anal.Calcd for C16H16N2O2:C,71.64; H,

5.97;N,10.45.Found:C,71.41;H,

6.21;N,10.38.

4.1.18.Ethyl9-butyl-b-carboline-3-carboxylate(6c).A mixture of6(2.4g,10mmol)and anhydrous DMF (60mL)was stirred rt for10min,then60%NaH (0.6g,15mmol)and n-butyl iodide(6mL,50mmol)were added and re?uxed ter the mixture was trea-ted in a manner similar to that described for5a to a?ord white crystals6c(2.3g,78%),mp76–77°C(from petro-leum ether–ethyl ether1:2);FAB-MS m/e297(M+1); UV k max359,343,307,273,235,221nm;IR(KBr): 3438,2956,1731,1623,1333,1242cmÀ1;1H NMR (500MHz,CDCl3):8.98(1H,s,H-4),8.89(1H,s,H-1),8.21–8.23(1H,d,J=8Hz,H-8),7.63–7.66(1H,m, H-5),7.51–7.53(1H,d,J=8.5Hz,H-6),7.36–7.38(1H, m,H-7),4.52–4.56(2H,m,OC H2CH3),4.42–4.44(2H, m,C H2CH2CH2CH3), 1.88–1.94(2H,m,CH2C H2-CH2CH3), 1.49–1.52(3H,m,OCH2C H3), 1.35–1.42 (2H,m,CH2CH2C H2CH3),0.93–0.96(3H,m,CH2CH2-CH2C H3).Anal.Calcd for C18H20N2O2:C,72.97;H, 6.76;N,9.46.Found:C,72.78;H,6.98;N,9.37.4.1.19.Ethyl9-benzyl-b-carboline-3-carboxylate(6d).A mixture of5(2.4g,10mmol)and anhydrous DMF (60mL)was stirred rt for10min,then60%NaH(0.6g, 2mmol)and benzyl bromide(5mL,40mmol)were added and re?uxed ter the mixture was treated in a manner similar to that described for5a to a?ord white crystals6d(2.4g,70%),mp126–127°C(from ethyl ether);FAB-MS m/e331(M+1);UV k max356,342,305, 273,234nm;IR(KBr):3425,3060,3027,2974,1723, 1622,1335,1214cmÀ1;1H NMR(500MHz,CDCl3): 8.91(2H,m,H-4,H-1),8.23–8.25(1H,d,J=8Hz,H-8),7.59–7.62(1H,m,H-5),7.49–7.50(1H,d,J=8Hz, H-6),7.36–7.39(1H,m,H-7),7.25–7.27(3H,s,Ar–H), 7.14–7.16(2H,m,Ar–H),

5.62(2H,s,C H2Ar),4.51–4.55(2H,m,OC H2CH3),1.47–1.50(3H,m,OCH2C H3). Anal.Calcd for C21H18N2O2:C,7

6.36;H,5.45;N,8.48. Found:C,76.19;H,5.67;N,8.38.

4.1.20.Ethyl9-(20,30,40,50,60-penta?uoro)benzyl-b-carbo-line-3-carboxylate(6e).A mixture of5(2.4g,10mmol) and anhydrous DMF(60mL)was stirred at rt for 10min,then60%NaH(0.6g,15mmol)and a-bromo-2,3,4,5,6-penta?uorotoluene(3mL,20mmol)were ter the mixture was treated in a manner simi-lar to that described for5a to a?ord white crystals6d (2.6g,62%),mp153–154°C(from ethyl ether);FAB-MS m/e421(M+1);UV k max350,335,299,271,236, 217nm;IR(KBr):3399,3065,2987,1709,1627,1337, 1245cmÀ1;1H NMR(500MHz,CDCl3):9.07(1H,s, H-4),8.86(1H,s,H-1),8.19–8.21(1H,d,J=8Hz,H-8),7.65–7.68(1H,m,H-5),7.59–7.61(1H,d, J=8.5Hz,H-6),7.38–7.41(1H,m,H-7),

5.67(2H,s, C H2Ar),4.52–4.56(2H,m,OC H2CH3),1.49–1.51(3H, m,OCH2C H3).Anal.Calcd for C21F5H13N2O2:C, 60.00;H,3.10;N,

6.6

7.Found:C,59.87;H,3.29;N, 6.5

8.

4.1.21.Ethyl9-phenylpropyl-b-carboline-3-carboxylate (6f).A mixture of5(2.4g,10mmol)and anhydrous DMF(60mL)was stirred at rt for10min,then60% NaH(0.6g,2mmol)and1-bromo-3-phenylpropane (6mL,40mmol)were added and re?uxed ter the mixture was treated in a manner similar to that de-scribed for5a to a?ord white crystals6e(2.0g,56%), mp140–142°C(from ethyl ether);FAB-MS m/e359 (M+1);UV k max358,343,306,273,236,217nm;IR (KBr):3026,2983,2933,1724,1622,1333,1246cmÀ1; 1H NMR(500MHz,CDCl

3

):8.89(2H,m,H-4,H-1), 8.20–8.22(1H,d,J=7.5Hz,H-8),7.61–7.64(1H,m, H-5),7.41–7.42(1H,m,H-6),7.34–7.37(1H,m,H-7), 7.26–7.30(2H,m,Ar–H),7.19–7.22(1H,m,Ar–H), 7.13–7.15(2H,m,Ar–H), 4.52–4.57(2H,m, NC H2CH2CH2Ar), 4.41–4.44(2H,m,NCH2C H2-CH2Ar),2.70–2.73(2H,m,OC H2CH3),2.24–2.30(2H, m,NCH2CH2C H2Ar),1.49–1.52(3H,m,OCH2C H3). Anal.Calcd for C23H22N2O2:C,77.09;H, 6.15;N, 7.82.Found:C,76.88;H,6.38;N,7.75.

4.1.22.Butyl9-methyl-b-carboline-3-carboxylate(7a).A mixture of7(2.68g,10mmol)and anhydrous DMF (80mL)was stirred at rt for10min,then60%NaH (0.6g,15mmol)and iodomethane(2mL,30mmol)were added and stirred at rt ter the mixture was

4620R.Cao et al./Bioorg.Med.Chem.12(2004)4613–4623

treated in a manner similar to that described for5a to af-ford white crystals7a(2.0g,70%),mp235–238°C(from ethyl ether);FAB-MS m/e283(M+1);UV k max358,343, 305,272,236,219nm;IR(KBr):3402,2956,2869,1726, 1625,1333,1238cmÀ1;1H NMR(500MHz,CDCl3): 8.94(1H,s,H-4),8.83(1H,s,H-1),8.18–8.20(1H,d, J=7.5Hz,H-8),7.63–7.66(1H,m,H-5),7.48–7.50 (1H,d,J=8.5Hz,H-6),7.34–7.37(1H,m,H-7),4.46–4.49(2H,m,OC H2CH2CH2CH3),3.97(3H,s,NC H3), 1.84–1.90(2H,m,J=7.0Hz,OCH2C H2CH2CH3), 1.48–1.56(2H,m,OCH2CH2C H2CH3),0.99–1.02(3H, m,OCH2CH2CH2C H3).Anal.Calcd for C17H18N2O2: C,72.34;H,6.38;N,9.93.Found:C,72.25;H,6.58; N,9.85.

4.1.23.Butyl9-ethyl-b-carboline-3-carboxylate(7b).A mixture of6(2.4g,10mmol)and anhydrous DMF (60mL)was stirred at rt for10min,then60%NaH (0.6g,15mmol)and iodoethane(2.5mL,30mmol)were ter the mixture was treated in a manner simi-lar to that described for5a to a?ord white crystals6b (2.0g,65%),mp76–77°C(from ethyl ether);FAB-MS m/e297(M+1);UV k max360,345,306,273,240, 220nm;IR(KBr):3053,2963,2401,1999,1722,1627, 1337,1269cmÀ1;1H NMR(500MHz,CDCl3):9.02 (1H,s,H-4),8.86(1H,s,H-1),8.20–8.22(1H,d, J=8Hz,H-8),7.63–7.67(1H,m,H-5),7.51–7.53(1H, d,J=7.5Hz,H-6),7.35–7.38(1H,m,H-7),4.47–4.51 (4H,m,OC H2CH2CH2CH3,NC H2CH3), 1.84–1.90 (2H,m,OCH2C H2CH2CH3),1.49–1.56(2H,m,OCH2-CH2C H2CH3),0.99–1.02(3H,m,OCH2CH2CH2C H3). Anal.Calcd for C18H20N2O2:C,72.97;H, 6.76;N, 9.46.Found:C,72.83;H,6.97;N,9.39.

4.1.24.Butyl9-butyl-b-carboline-3-carboxylate(7c).A mixture of6(2.4g,10mmol)and anhydrous DMF (60mL)was stirred at rt for10min,then60%NaH (0.6g,15mmol)and n-butyl iodide(6mL,50mmol)were added and re?uxed ter the mixture was trea-ted in a manner similar to that described for5a to a?ord white crystals6c(2.2g,74%),mp94–95°C(from petro-leum ether–ethyl ether1:2);FAB-MS m/e325(M+1); UV k max359,344,307,274,238,221nm;IR(KBr): 3061,2956,2866,1728,1624,1358,1247cmÀ1;1H NMR(500MHz,CDCl3):8.95(1H,s,H-4),8.85(1H, s,H-1),8.19–8.20(1H,d,J=7Hz,H-8),7.60–7.64 (1H,m,H-5),7.49–7.50(1H,d,J=8.5Hz,H-6),7.32–7.36(1H,m,H-7),4.47–4.49(2H,m,OC H2CH2CH2-CH3), 4.37–4.42(2H,m,NC H2CH2CH2CH3), 1.84–1.92(4H,m,OCH2C H2CH2CH3,NCH2C H2CH2CH3), 1.49–1.56(2H,m,OCH2CH2C H2CH3),1.34–1.40(2H, m,NCH2CH2C H2CH3),0.99–1.02(3H,m,OCH2CH2-CH2C H3),0.92–0.95(3H,m,NCH2CH2CH2C H3). Anal.Calcd for C20H24N2O2:C,74.07;H,7.41;N, 8.64.Found:C,73.88;H,7.65;N,8.57.

4.1.2

5.Butyl9-benzyl-b-carboline-3-carboxylate(7d).A mixture of5(2.4g,10mmol)and anhydrous DMF (60mL)was stirred at rt for10min,then60%NaH (0.6g,15mmol)and benzyl bromide(5mL,40mmol) were added and re?uxed ter the mixture was treated in a manner similar to that described for5a to a?ord white crystals7d(2.4g,67%),mp105–106°C (from ethyl ether);FAB-MS m/e359(M+1);UV k max 356,342,305,273,235,205nm;IR(KBr):3051,2959, 2930,2869,1700,1620,1362,1246cmÀ1;1H NMR (500MHz,CDCl3):8.89(1H,s,H-4),8.85(1H,s,H-1),8.19–8.20(1H,d,J=8Hz,H-8),7.56–7.59(1H,m, H-5),7.45–7.46(1H,d,J=8.5Hz,H-6),7.33–7.36(1H, m,H-7),7.22–7.26(3H,m,ArH),7.11–7.13(2H,m, ArH), 5.56(2H,s,C H2Ar), 4.45–4.48(2H,m, OC H2CH2CH2CH3), 1.82–1.88(2H,m,OCH2-

C H2CH2CH3),1.47–1.54(2H,m,OCH2CH2C H2CH3),

0.98–1.01(3H,m,OCH2CH2CH2C H3).Anal.Calcd for C23H22N2O2:C,77.09;H,6.15;N,7.82.Found:C, 76.89;H,6.35;N,7.75.

4.1.26.General procedure for the preparation of b-carb-oline-3-carboxylic acids8a–e.A mixture of the corre-sponding b-carboline-3-carboxylate(6a–f,10mmol), NaOH(50mmol),ethanol(50mL),and H2O(100mL) was re?uxed for1h,and the ethanol was removed on the rotary evaporator.The mixture was neutralized (pH5)with5M HCl and cooled.The precipitate was collected,washed well with H2O,and dried in vacuo.

4.1.27.9-Methyl-b-carboline-3-carboxylic acid(8a).Yel-low solid was obtained(2.32g,99%).Mp267–269°C; FAB-MS m/e227(M+1);UV k max384,360,273,241, 217nm;IR(KBr):3250–2100,1716,1630,1405, 1200cmÀ1;1H NMR(500MHz,DMSO-d6)d12.15 (1H,s,COO H),9.19(1H,s,H-4),9.12(1H,s,H-1), 8.42–8.44(1H,d,J=8.0Hz,H-8),7.81–7.88(2H,m,H-5,H-6),7.50–7.53(1H,m,H-7),4.16(1H,s,NC H3). Anal.Calcd for C13H10N2O2:C,69.03;H, 4.42;N, 12.39.Found:C,68.96;H,4.57;N,12.31.

4.1.28.9-Ethyl-b-carboline-3-carboxylic acid(8b).Yel-low solid was obtained(2.41g,98%).Mp201–202°C; FAB-MS m/e241(M+1);UV k max387,359,273,239, 220nm;IR(KBr):3250–2250,1713,1630,1407,1336, 1198cmÀ1;1H NMR(500MHz,DMSO-d6)d12.10 (1H,s,COO H),9.16(1H,s,H-4),8.97(1H,s,H-1), 8.31–8.33(1H,d,J=8.0Hz,H-8),7.75–7.81(2H,m,H-5,H-6),7.43–7.46(1H,m,H-7), 4.63–4.67(2H,s, NC H2CH3),1.46–1.52(3H,s,NCH2C H3).Anal.Calcd for C14H12N2O2:C,70.00;H,

5.00;N,11.67.Found:C, 69.89;H,5.19;N,11.59.

4.1.29.9-n-Butyl-b-carboline-3-carboxylic acid(8c).Yel-low solid was obtained(2.71g,99%).Mp182–184°C; FAB-MS m/e269(M+1);UV k max387,359,272,238, 221nm;IR(KBr):3250–2250,1710,1630,1334, 1213cmÀ1;1H NMR(500MHz,DMSO-d6)d12.11 (1H,s,COO H),9.14(1H,s,H-4),8.94(1H,s,H-1), 8.42–8.44(1H,d,J=8.0Hz,H-8),7.77–7.79(1H,d, J=8.5Hz,H-5),7.65–7.68(1H,m,H-6),7.34–7.37(1H, m,H-7),4.56–4.59(2H,m,NC H2CH2CH2CH3),1.79–1.85(2H,m,NCH2C H2CH2CH3), 1.28–1.32(2H,m, NCH2CH2C H2CH3),0.87–0.90(3H,m, NCH2CH2CH2C H3).Anal.Calcd for C16H16N2O2:C, 71.64;H,

5.97;N,10.45.Found:C,71.45;H,

6.19;N, 10.36.

4.1.30.9-Benzyl-b-carboline-3-carboxylic acid(8d).Yel-low solid was obtained(2.96g,98%).Mp261–262°C;

R.Cao et al./Bioorg.Med.Chem.12(2004)4613–46234621

FAB-MS m/e303(M+1);UV k max355,342,267, 239nm;IR(KBr):3457,3409,3250–2250,1683,1624, 1334,1225cmÀ1;1H NMR(500MHz,DMSO-d6)d 12.18(1H,s,COO H),9.15(1H,s,H-4),8.96(1H,s, H-1),8.44–8.45(1H,d,J=8.0Hz,H-8),7.79–7.80(1H, d,J=8.0Hz,H-5),7.63–7.66(1H,m,H-6),7.35–7.38 (1H,m,H-7),7.22–7.31(5H,m,Ar–H),5.85(2H,s, NC H2Ar).Anal.Calcd for C19H14N2O2:C,75.50;H, 4.64;N,9.27.Found:C,75.38;H,4.85;N,9.18.

4.1.31.9-(20,30,40,50,60-Penta?uoro)benzyl-b-carboline-3-carboxylic acid(8e).White solid was obtained(3.84g, 98%).Mp>270°C;FAB-MS m/e393(M+1);UV k max 351,338,261,239,218nm;IR(KBr):3500–2250, 1714,1628,1335cmÀ1;1H NMR(500MHz,DMSO-d6)d12.14(1H,s,COO H),9.10(1H,s,H-4),8.83 (1H,s,H-1),8.45–8.47(1H,d,J=8Hz,H-8),7.65–7.68(1H,m,H-5),7.59–7.61(1H,d,J=8.5Hz,H-6), 7.38–7.41(1H,m,H-7),

5.67(2H,s,C H2Ar).Anal.Cal-cd for C19F5H9N2O2:C,58.16;H,2.30;N,7.14.Found: C,58.03;H,2.39;N,7.18.

4.1.32.9-Phenylpropyl-b-carboline-3-carboxylic acid(8f). Yellow solid was obtained(3.2g,97%).Mp213–215°C; FAB-MS m/e331(M+1);UV k max358,346,268,239, 218nm;IR m max3500–2250,1692,1629,1335, 1215cmÀ1;1H NMR(500MHz,DMSO-d6)d12.09 (1H,s,COO H),9.13(1H,s,H-4),9.00(1H,s,H-1), 8.46–8.48(1H,d,J=7.5Hz,H-8),7.76–7.78(1H,d, J=8.0Hz,H-5),7.69–7.72(1H,m,H-6),7.38–7.41 (1H,m,H-7),7.21–7.26(2H,m,ArH),7.13–7.17(3H, m,Ar–H),4.63–4.66(2H,m,NC H2CH2CH2Ar),2.49–2.51(2H,m,NCH2C H2CH2Ar), 2.14–2.20(2H,m, NCH2CH2C H2Ar).Anal.Calcd for C21H18N2O2:C, 76.36;H,

5.45;N,8.48.Found:C,7

6.22;H,5.69;N, 8.38.

4.2.In vitro cytotoxicity assays

Cytotoxicity assays in vitro were carried out using96 microtiter plate cultures and MTT staining according to the procedures described by Al-Allaf and Rashan26 with a slight modi?cation.Cells were grown in RPMI-1640medium containing10%(v/v)fetal calf serum and100l g/mL penicillin and100l g/mL streptomycin. Cultures were propagated at37°C in a humi?ed atmos-phere containing5%CO2.Cell lines were obtained from Shanghai Cell Institute,Chinese Academy of Science. Drug stock solutions were prepared in DMSO.The?nal concentration of DMSO in the growth medium was2% (v/v)or lower,concentration without e?ects on cell rep-lication.The human tumor cell line panel consisted of nonsmall cell lung carcinoma(PLA-801),liver carci-noma(HepG2and Bel-7402),gastric carcinoma (BGC-823),cervical carcinoma(Hela),colon carcinoma (Lovo).In all of these experiments,three replicate wells were used to determine each point.

4.3.Assay of acute toxicities

Healthy C57BL/6mice(9–12weeks)weighing18–22g were housed in rooms where the temperature was approximately24±2°C,with a relative humidity60–70%,and in12h light-dark cycle.The sterile food and water were provided according to institutional guide-lines.All animals were provided by Shanghai Labora-tory Animal Center of Chinese Academy of Science. All animal procedures were approved by the Animal Ethical Committee of the Sun Yat-sen University.Prior to each experiment,mice were fastened overnight and al-lowed free access to water.Various doses of the b-carb-oline derivatives ranging from10to300mg/kg dissolved in0.5%carboxymethyl cellulose sodium(CMC-Na)salt solution were given via intraperitoneal(i.p.)to di?erent groups of healthy C57BL/6mice,and each group con-tained10mice(?ve males and?ve females).After the administration of the compounds,mice were observed continuously for the?rst2h for any gross behavioral changes and deaths,then intermittently for the next 24h and occasionally thereafter for14days,and for the onset of any delayed e?ects.All animals were sacri-?ced at the14th day after drug administration and checked macroscopically for possible damage to the heart,liver,and kidneys.Mice of immediate death fol-lowing drug administration were also examined for any possible organ damage.LD50values were calculated graphically as described.31

4.4.Assay of antitumor activity

Lewis lung cancer and S180sarcoma cell lines were pro-vided by Shanghai Institute of Pharmaceutical Industry. Tumor cells of Lewis lung cancer and S180sarcoma were inoculated to mice.After7days,tumors were taken out and cells harvested.Viable tumor cells(2·106cells/ mouse)were inoculated to the armpit of mice by subcu-taneous injection.Each compound was injected by intra-peritoneal(i.p.)to di?erent group mice(each group containing10female mice)24h after the inoculation at a dosage of7.5mg/kg once a day for consecutive7days. This dose was the maximum tolerated dose for most compounds based on our preliminary studies.Cyto-phosphane(CTX)at30mg/kg was used as a positive control and vehicle as negative control.The weights of animals were recorded every3days.All animals were sacri?ced at the21st day after tumor inoculation and the tumors were excised and weighed.The inhibition rate was calculated as follows:

ðCÀTÞ=CÂ100

T:average tumor weight of treated group;C:average tumor weight of negative control group.

Acknowledgements

We are thankful for the?nancial support of this work by Xinjiang Huashidan Pharmaceutical Co.Ltd and Guangzhou Commission of Science and Technology (97-Z-12-01).We are also grateful to Prof.Huifang Yan and co-workers for performance of the antitumor and acute toxic assays in mice.

4622R.Cao et al./Bioorg.Med.Chem.12(2004)4613–4623

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