CAMPTOTHECIN DERIVATIVES AND ITS MANUFACTURING METHOD

Abstract: This invention relates to camptothecin derivatives and pharmaceutically acceptable salts thereof, its manufacturing method and antineoplastic agent containing it. The compound of this invention is camptothecin derivatives modified in B-ring and E-ring thus show improved water solubility and enhanced antineoplastic effect.

Description: TITLE OF THE INVENTION Camptothecin derivatives and its manufacturing method TECHNICAL FIELD This invention relates to camptothecin derivatives and pharmaceutically acceptable salts thereof, expressed by the following chemical formula (I) , its manufacturing method and antineoplastic agent containing it as an active ingredient Wherein; R is hydrogen or -(CH^.-N .^, (R, is hydrogen or a general protecting group of amine; R- is a lower alkyl, hydroxyethyl or acetoxyethyl; further, R 2 may form heterocyclic compound by binding with an adjacent nitrogen) ; n is 1 or 2; R- is hydrogen or -OR 4 [R 4 is hydrogen or C ^(R j is methyl or CH 3 OCH 2 -) ; C NH R 6 (R 6 s isopropyl, 0 o phenyl or -CH-CH-C1.); CH~OR 7 (R 7 is methyl, ethyl or CH j OCH-CH--) ] ; Wherein; When n is 2 and R 4 is hydrogen, R is not hydrogen; and when n is 2 and R- is H, R is not hydrogen; also, when R is -CH-CH 2 NHCH 3 , R_ is not hydrogen. BACKGROUND OF ART Since the isolation of camptothecin from the Chinese tree, Camptotheca acuminata. by Wall and co-workers (J. Am. Chem. Soc. , 1966, 88, 3888), there had been many approaches to synthesize camptothecin. However, the development of camptothecin as an effective antineoplastic agent was unsuccessful due to its severe toxicity in the first clinical trial in 1970. Thereafter, Liu et al. reported in 1985 that camptothecin had a specific mode of action to inhibit topoisomerase I. Thus, considerable interest has focused on this compound. In particular, since topoisomerase I inhibitor itself was not used clinically, it is expected that with its actual development, camptothecin may be very efficiently used in combination with other antineoplastic che otherapeutics having different modes of action. Recently, various studies for the development of camptothecin derivatives have been proposed in order to reduce the toxicity of camptothecin and to further enhance its antineoplastic activities. Among these related studies, the clinical trial of CPT-ll(Irinotecan) synthesized by Yakurt-Honsha Co. of Japan in 1986 showed that it exhibited excellent antineoplastic activities with less toxicity (Japanese Patent Laid Open Publication No. 64-61482) and followed by other pharmaceutical companies such as Smithkline Beecham (Topotecan) and Glaxo (MDO-camptothecin and 9-aminocamptothecin) . Among them, CPT-11 is launched. In particular, it is noticeable that said CPT-11 compound has exhibited excellent antineoplastic activities in the treatment of incurable solid tumors such as the lung cancer. Since a majority of camptothecin derivatives developed hitherto were semi-synthetic compounds which were obtained from the chemical modification of camptothecin, there were some difficulties in procuring camptothecin and in developing derivatives with a variety of structures because of restrictions of said chemical modification. In addition, these reported total synthesis are not satisfactory in view of both chemical and optical yields. DISCLOSURE OF INVENTION Accordingly, the inventor et al. have confirmed that through the total synthesis for a series of novel compounds expressed by said general formula (I) and followed by in vitro test, these compounds with different mode of action may be used as effective antineoplastic agents. Thus, the present invention has been completed. Camptothecin is a fused ring system, composed of a quinoline (A and B) , fused to a pyrrolidine ring (C) , fused to an alpha-pyridone ring (D) which in turn is fused to a lactone ring(E). In particular, further careful review has been made on camptothecin's mode of action to inhibit topoisomerase I and various structures of camptothecin derivatives developed hitherto, and attempts have been also made in such a manner to modify the substituents of B-ring or E-ring in camptothecin. In consideration of the fact that the conventionally developed derivatives have suffered from some side effects such as nausea, vomiting and cystitis due to extremely poor water solubility, the introduction of side chain at B-ring of camptothecin was made in such a manner that in order to enhance the solubility. Hence, the side chain including amino groups was introduced at the 7-position of camptothecin. In view of the mode of action suggested by Crow that camptothecin binds covalently with topoisomerase I fJ. Med. Chem. , 1992, 35, 4160-4164], the modification of camptothecin at E-ring was attempted so as to enhance its reactivity. The processes for manufacturing camptothecin derivatives and its intermediates are described in more detail as set forth hereunder, in accordance with the practice of this invention. The compounds based upon EXAMPLES can be prepared in accordance with the practice of this invention as specified in the following Scheme 1. Scheme 1 condensation Wherein ; R, n and R 3 are the same as described in the above and R' is hydrogen or -(CH^.-N - j R-.; R 1 is a general protecting group of amine; R- is the same as described in the above. Friedlander condensation of aminoketone compound(II) with tricyclic Ketone(III) affords the compound expressed by the general formula(I) [Organic reactions, 28, 37-202, Wiley & Sons Inc, New York (1932)]. The condensation of the compounds (II) and (III) is conducted, in general, in the presence of acid at room temperature or under the heating condition. The following inert solvents should be used for the reactions so as not to affect the reactions, for example, aromatic hydrocarbons(toluene, benzene, xylene etc.), hydrocarbon halide(dichloromethane, chloroform, 1,1- dichloroethane, 1,2-dichoroethane etc.), lower alcohols, amides (N,N-dimethylformamide etc.), or acetic acid. The reactions may be conducted using inorganic acid(hydrochloric acid or sulfuric acid) or organic acids (methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, acetic acid, etc.). The reaction time ranges from 1 to 48 hrs, and the reaction temperature is 30 to 150°C. As a typical reaction condition, it is most preferable that reflux is made available in the presence of p-toluenesulfonic acid in toluene. When R is -CH 2 CH 2 NHR 2 (R 1 is hydrogen) in the synthesis of general formula(I), said amino protecting group is removed through the catalytic hydrogenation using platinum or palladium. When R' is hydrogen in the compound (II) , compound(I) is obtained after carbonyl group of the compound(II) is protected by acetalization with ethylene glycol or by formation of shiff base with p-toluidine according to known process. [Chem. Ber. 76, 1099(1943)] When R- is hydroxy group in the compound(III) , thus obtained compound(I) , wherein R- is hydroxy group, is reacted with such reagents as R^Cl R 6 NCO and R-OCH-C1 to 0 produce the compound(I) wherein the hydroxy group of R 3 is thus modified. In particular, when R 6 NC0 is used, the compound expressed by the general formula(I) can be obtained by using the catalytic amounts of tin complex (e.g., di-n-butyltin diacetate, etc.) . The aminoketone(II) can be prepared in accordance with Scheme 2. Scheme 2 O mannich reaction (HNR j R,) OC N _: amino protecuon reduction Wherein ; R' , R_, and R 2 are the same as described in the above. A novel aminoketone compound(II) of this invention can be prepared as follows: 2'-nitroacetophenone reacts with monoalkyla ine (e.g. , ethylamine, propylamine or isopropylamine) , monoarylamine(e.g. , benzylamine) or dialkylamine(e.g. , morpholine, piperidine or diethylamine) in the presence of cone, hydrochloric acid, through Mannich reaction with paraformaldehyde to give the compound(IV) . The reaction is conducted at 30-80°C for 1 to 48 hrs. In case of the compound(IV) having monoalkylamine or monoarylamine, the general protecting group of amine is introduced to give the said compound(IV) . According to this invention, Cbz is a preferable protecting group of amine. The compound(IV) is treated with sodium dithionite in lower alcohol solvent to give the compound(II) . The reaction is conducted at 30 to 100°C for 1 to 10 hrs . As specified in Scheme 3 below, a novel tricyclic ketone(III) of this invention can be prepared from the compound(IX) which is already known. From the compound(III) , tricyclicketone(n is 2 an d R 3 is H) is already known compound prepared in accordance with a convention method(U.S. Pat. 4,894,456(1990)). Scheme 3 . Wherein ; R-, n, R 5 and 6 are the same as described in the above and R- is the general protecting group of hydroxyl group. During α-alkylation reaction where n is 2, the compound(IX) reacts with base such as KOtBu or NaH in inert solvent including N, N-dimethylformamid or 1,2- dimethoxyethane. Then, the reaction mixture is reacted with 2-bromoethanol protected with tetrahydropyranyl, methoxymethyl or methoxyethoxymethyl to give the compound(VIII) . The reaction is preferably conducted at 30 to 60°C for 18 to 48 hrs. Meanwhile, when n is 1, α-hydroxymethylation is conducted using formaldehyde in alcoholic solvent and followed by the protection with the general protecting group of OH to give the compound(VIII) . The preferable protecting group of this invention is methoxymethyl or methoxyethoxymethyl. The compound(VII) was obtained by the reduction of compound(VIII) using Raney-Nickel as a catalyst in a co- solvent of acetic acid and acetic anhydride. The reaction is conducted at 30 to 80°C for 1 to 10 hrs. The rearrangement via nitroso compound is done by reacting the compound(VII) with sodium nitrite in a co-solvent of acetic anhydride and acetic acid at 0 to 50°C for 1 to 24 hrs. Thus, obtained nitroso compound is refluxed in the solvent such as CC1 4 at 60 to 90°C for 5 to 18 hrs to give the compound(VI) . When n is 1 or 2, the reaction route of the diester (VI) becomes slightly different. In case that n is 2, compound(VI) is hydrolyzed in a kali aqueous solution including LiOH, NaOH and KOH in methanol and treated with acidic aqueous solution such as acetic acid or hydrochloric acid to give the compound(Vc) . Said hydrolysis is conducted at 0 to 50°C for 30 mins to 5 hrs. The lactonization in acidic aqueous solution is conducted at 0 to 50°C for 1 to 48 hrs. Compound(Vc) is treated with base such as potassium t- butoxide in the amine solvents such as N,N- dimethylformamide or N,N-dimethylacetamide and oxidized with the bubbling of 0 2 under triethylphosphite to give the compound(V) . The reaction is conducted at -10 to 50°C for 30 ins to 5 hrs. Meanwhile, when n is 1, compound(VI) s treated with organic base such as DBU, DBN or triethylamine in aromatic hydrocarbon solvent such as benzene, toluene or xylene to produce the compound(Va) . The reaction is conducted at 0°C to 100°C for 10 mins to 5 hrs. The compound(Va) is treated with Os0 4 in a pyridine solvent to give the compound(Vb) . This reaction is conducted at 10 to 50°C for 1 to 10 hrs. The lactonization of the compound(Vb) is carried out as the same as described in the above. The compound(V) where n is 2, is treated with acid to produce compound (III) . The reaction is conducted at 10 to 80°C for 30 mins to 10 hrs. In case of the compound(V) wherein n is 1, said deketalization is done after introducing the substituent by treating R- j CCl, R 6 NC0 0 for the preparation of the compound(III) . In case of the compound(I) of this invention where R is CH 2 CH 2 NR.,R 2 , the conversion of secondary amine into the salts of inorganic acids(e.g., hydrochloric acid, sulfuric acid, phosphoric acid, etc.) or organic acids (e.g., acetic acid) makes it possible to prepare physiologically acceptable salts. BRIEF DESCRIPTION OF DRAWINGS Figure 1 illustrates the difference of ILS(%) in various dose between the compound prepared in example 30 and camptothecin. Figure 2 illustrates the inhibitory effects on mice melanorma cells in accordance with the compound prepared in example 30. BEST MODE FOR CARRYING OUT THE INVENTION This invention is explained in more detail by the following examples and experiments, but the claims are not limited to these examples and experiments. EXAMPLES 1 : Preparation of 3-morpholino-l-(2'- nitropheny1)propan-l-one A solution of morpholine(2.27g, 0.026mol) in ethanol (10ml) was treated with c-HCl(3ml) for 10 mins, concentrated under reduced pressure to prepare morpholine HC1 salt. To this mixture, 2 '■-nitroacetophenone (3.3g, 0.02mol), paraformaldehyde(0.8g, 0.029mol), c-HCl(O.lml) and anhydrous ethanol(10ml) were added. The reaction mixture was stirred under reflux for 30 hrs. This reaction mixture was then cooled to room temperature, alkalized with 10% sodium carbonate solution, and the reaction mixture was extracted with dichloromethane(3 x 50ml) . The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product. The crude product was purified by flash column chromatography with 5% ethanol in dichloromethane to give the desired product(1.5g, 35%) as an oil. 1 H-NMR (CDC1 3 ) 6 3.1(m, 3H) , 3.5(m, 4H) , 3.75(m, 3H) , 3.99(m, 2H) , 7.85 (m, 3H) , 8.15(d, IH, J=8Hz) EXAMPLE 2 : Preparation of 3-piperidino-l- (2 -nitrophenyl) propan-l-one In the same procedure as in EXAMPLE 1, the desired product (458mg,29%) was yielded using piperidine (515mg, 6.06mmol) , 2 ' -nitroacetophenone ( lg, 6.06mmol) , paraformaldehyde(260mg, 8.67mmol) , c-HCl (1ml) and anhydrous ethanol. 1.5(m, 6H) , 2.38(t, 4H, J=5Hz) , 2.76(t, 2H, J=6Hz), 3.00(t, 2H, J=6HZ), 3.99 (m, 2H) , 7.40-7.75(m, 3H) , 8.13(d, IH, J=8Hz) EXAMPLE 3 ; Preparation of 3-pyrrolidino-l- (2'-nitrophenyl) propane-1-one In the same procedure as in EXAMPLE 1, the desired product (347mg, 23%) was yielded using pyrrolidine(560mg, 7.88mmol) , 2 ' -nitroacetophenone ( lg, 6.06mmol) , paraformaldehyde (260mg, 8.67mmol), c-HCl (1ml) and anhydrous ethanol (5ml) . EXAMPLE 4 ; Preparation of 3- (N-benzyl-N- carbobenzyloxyamino. -l-(2'-aminophenyl)propane-1-one A solution of benzylamine(2.79g, 0.026mol) in ethanol(10ml) was treated with c-HCl(3ml) for 10 min, concentrated under reduced pressure to prepare benzylamine«HCl salt. To this solution, 2'-nitroacetophenone (3.3g, 0.02mol), paraformaldehyde (800mg, 0.029mol), c-HCl(0.1ml) and anhydrous ethanol(10ml) were added. The reaction mixture was stirred under reflux for 30 hrs. This reaction mixture was then cooled to room temperature, alkalized with 10% sodium carbonate solution, and the reaction mixture was extracted with dichloromethane (3 x 50ml) .The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give the crude product, which was dissolved in dichloromethane and cooled to 0°C. To this mixture, triethylamine (1.2g, 0.012mol) was added and followed by another dropwise addition of benzylchloroformate(2ml) . The reaction mixture was stirred under nitrogen flow at 0°C for 1 hr, heated to room temperature and then stirred for additional 5 hrs. After removing the solvent under reduced pressure, the mixture was dissolved in ethylacetate(100ml) . The organic layer was washed with water (3 x 30ml) and brine (30ml) and dried over anhydrous sodium sulfate. After the filtration, the crude product was obtained by removing the solvent. The residue was purified by flash column chromatography with n-hexane-ethylacetate(l:10) to give the desired product (1.5g, 36%). 1 H-NMR(CDC1 3 ) 6 3.0(m, 2H) , 3.70(t, 2H, J=7Hz) , 4.60(s, 2H) , 5.17(S,2H), 7.10-7.70(m, 13H) , 8.10(d, IH, J=8Hz) In the same procedure as in EXAMPLE 4 , the compounds of EXAMPLE 5-8 were prepared and the results were as follows: EXAMPLE Preparation of 3-(N-2'-acetoxyethyl-N- carbobenzyloxγamino)-!- (2 '-nitrophenyl. ropane- 1-one mixture of 3-[ (N- (2 ' -hydoxyethyl) -N- carbobenzyloxyamino ) ] -1- (2 '-nitrophenyl) propane -l-one(40mg, 0.107mmol) and 4-dimethylamino pyridine (1.2mg, O.Olmmol) was dissolved in anhydrous dichloromethane(2ml) , then added triethylamine (0.02ml, 0.160mmol) and acetic anhydride(0.02ml, 0.214mmol). The reaction mixture was stirred at room temperature for 30 mins and washed with a saturated ammonium chloride aqueous solution, water and brine successively. Then, the organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue, so obtained, was purified by column chromatography with ethylacetate-n-hexane(l:3) to give the desired product(4Omg,90%) as a yellow oil. 1 H-NMR(400MHZ, CDCl 3 ) _ 8.123(d, IH, J=8.4HZ), 7.607-7.261(m, 8H) , 5.136(s, 2H) , 4.194(m, H) , 3.784-3.754(m, 2H) , 3.660-3.646(m, 2H) , 3.172 (m, 2H) , 2.047(s, 3H) EXAMPLE 10 : Preparation of 3-morpholino-l- (2'- a inophenyl)propane-1-one To a solution of 3-morpholino-l-(2'-nitrophenyl) propane-l-one(200mg, 0.758mmol) dissolved in 10ml of 95% ethanol was added sodium dithionite(660mg, 3.79mmol) and the reaction mixture was stirred under reflux for 3 hrs. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography with ethanol-dichloromethane-triethylamine to yield the desired product (174mg, 89%) as an oil. 1 H-NMR (CDC1 3 ) . 3.20(m, 3H) , 3.50(m, 3H) , 3.5(m, 4H) , 3.99(m, 2H) , 7.5-7.8(m, 3H) , 8.08(d, IH, J=8Hz) EXAMPLE 11 : Preparation of 3-piperidino-l- (2 -aminophenyl propane-1-one The same procedure as in EXAMPLE 10 was applied t the compound of EXAMPLE 2(200τng, 0.758mmol) and sodiu dithionite(660mg, 3.79mmol) so that the desired produc (174mg,89%) was yielded. EXAMPLE 12 Preparation of 3-pyrrolidino-l (2'-amino henyl) ropane-1-one The same procedure as in EXAMPLE 10 was applied t the compound of EXAMPLE 3 (400mg) and sodium dithionit (1.052g, 6.05mmol) so that the desired product(235mg) wa yielded. In the same procedure as used in EXAMPLE 10, th compounds of EXAMPLE 13-17 were prepared and the result were as follows: EXAMPLE 18 Preparation of dl-7-r2-(N-benzyl-N- carbobenzyloxyamino)ethyl1camptothecin A mixture of 3-(N-benzyl-N-carbobenzyloxyamino) -l-(2'-aminophenyl)propane-l-one(543mg, 1.4mmol) and 4-ethyl-6-oxo-l , 4 , 7 , 8-tetrahydro-4-hydroxy-pyrano [3,4-f]indolizine-3,10(6H)-dione(263mg, l.Ommol) was dissolved in toluene(50ml) . The reaction mixture was stirred under reflux for 30 mins, and p-toluenesulfonic acid(25mg, 0.13mmol) was added to the mixture. The reaction mixture was stirred in a Dean-Stark trap under reflux for 9 hrs. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography with ethylacetate-dichloromethane (1:1) to give the desired product(507mg, 59%) as a yellow powder. 1 H-NMR (CDC1 3 ) δ 1.04(t, 3H, J=8Hz) , 1.90(m, 2H) , 3.10-3.90(m, 4H) , 4.53(s, 1/2 x 2H) , 4.61(s, 1/2 x 2H) , 4.95(Ξ,l/2 x 2H) , 5.07(s, 1/2 x 2H) , 5.30(s, 2H) , 5.31(d, IH, J=16HZ) , 5.75(d, IH, J=16Hz) , 7.20-8.20(m, 15H) In the same procedure as in EXAMPLE 18, the compounds of EXAMPLE 19 to 24 were prepared and the results were as follows: EXAMPLE 25 : Preparation of dl-7-T2-(N-hvdroxyethyl- N-carbobenzyloxyamino)ethyl * |camptothecin A mixture of dl-7-[2-(N-acetoxyethyl-N- carbobenzyloxyamino) ethyl] camptothecin(28.2mg, 0.05mmol) and potassium carbonate (25mg, O.lSmmol) was dissolved in mixed a solution of methanol and water(1:1, 1.5ml) and the reaction mixture solution was stirred at room temperature for 3 hrs. The reaction mixture was acidified with IN HC1 solution, extracted with dichloromethane and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography with dichloromethane-methanol(20:1) to give the desired product (I7mg, 60%) as a yellow solid. 1 H-NMR (400MHz, CDC1 3 ) δ 8.171-7.762(m, 10H) , 5.710(d, 2H, J=16.8Hz), 5.308 (d, 2H, J=16.8Hz) ,5.301(s, 2H) , 5.207(s, 2H) , 3.868-3.466(m, 8H) , 1.926-1.832(m, 2H) , 1.033(t, 3H, J=7.6HZ) EXAMPLE 26 : Preparation of dl-7-[2-(N-benzylamino, ethyl]camptothecin dl-7-[2-(N-Benzyl-N-carbobenzyloxyamino)ethyl] camptothecin(5Omg, O.Oδmmol) was dissolved in glacial acetic acid(20ml) and treated with 10% Pd/C(25mg) . The reaction mixture was stirred under hydrogen flow of 40 psi for 9 hrs, and the catalyst was removed through cellite pad by filtration under reduced pressure. The filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography with 10% methanol-dichloromethane to give the desired product (24mg, 63%) as a yellow powder. 1 H-NMR (400MHz, CDC1 3 ) . 0.89(t, 3H, J=8Hz) , 1.87(m, 2H) , 2.90(m, 2H) , 3.5(m, 4H) , 3.73(s, IH) , 5.33(s, 2H) , 5.43(s, 2H) , 6.52(s, IH) , 7.25(m, 6H) , 7.70-8.30(m, 4H) EXAMPLE 27 : Preparation of dl-7-f2- (N-acetoxyethylamino) ethyl] camptothecin The same procedure as in EXAMPLE 26, was carried out. 1 H-NMR (400MHz, CDC1 3 +MeOH-d . 8.323(d, IH, J=8.8HZ), 8.138(d, IH, J=8.4Hz) , 7.812 (t, IH, J=6.8Hz), 7.675(t, IH, J=6.8Hz), 7.647(s, IH) , 5.742(d, lH J=16.4Hz) , 5.324-5.284 (m, 2H) , 4.169-4.122(m, 2H) , 3.373(t, 2H, J=7.6Hz), 3.108- 3.040(m, 4H) , 2.090(s, 3H) , 2.024(s, 3H) , 1.955- 1.803(m, 2H) , 1.033(t, 3H, J=7.6Hz) EXAMPLE 28 : Preparation of dl-7-T2- (N-hvdroxyethyl amino) ethyl]camptothecin The same procedure as in EXAMPLE 26, was carried out. 8.284(d, IH, J=8.0HZ), 8.226(d, IH, J=8.8Hz), 7.863(t, IH, J=6.8Hz) , 7.756(t, IH, J=6.8Hz) , 7.730(S, IH) , 5.682(d,lH,J=13.6Hz) , 5.641(d, IH, J=13.6HZ), 5.130(s, 2H) , 3.800-3.190(m, 8H) , 1.962-1.898(m, 2H) , 1.031(t, 3H, J=4.4Hz) , 2.035(S, 3H) EXAMPLE 29 : Preparation of dl-7-T2-(N-ethyla ino)ethyl] camptothecin A mixture of 2'-amino-2-(N-carbobenzyloxy-N -ethyla ino) propiophenone (34mg, O.llmmol) and 4-ethyl-6-oxo-l,4,7,8-tetrahydro-4-hydroxy-pyrano[3, 4-f]indolizine-3, 10(6H)-dione(34mg, O.lmmol) was dissolved in toluene and was refluxed for 30 mins. After addition of p-toluenesulfonic acid (19.2mg, O.lmmol), the reaction mixture was then refluxed in a Dean-Stark trap for 3 hrs. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography with dichloromethane-methanol(10:1) to give the desired product( 40mg,67%) as a solid. ^-NMR (400MHz, DMSO-d 6 ) 6 8.280(d, IH, J=8.6HZ), 8.184(d, IH, J=7.4Hz), 7.873-7.753(m, 2H) , 7.471(d, 2H, J=7.2Hz), 7.111(d, 2H, J=7.2Hz) , 7.356(s, IH) , 6.523(s, IH) , 5.439(s, 2H) , 3.534-2.753(m, 6H) , 2.217(s, 3H) , 1.671-1.643 (m, 2H) , 1.230(t, 3H, J=6.8Hz), 0.743(t, 3H,J=7.2Hz) EXAMPLE 30 ; Preparation of dl-7- \2-(N-2-methyl ethylamino)ethyl]camptothecin The same procedure as in EXAMPLE 29 was carried out. 1 H-NMR (400MHz, DMSO-d 6 ) 6 8.870-8.567(m, 2H) , 8.298-8.155(m, 2H) , 7.867(d,2H, J=7.6Hz), 7.741(S, IH) , 7.500(d, 2H, J=7.6Hz), 5.828(s, 2H) , 5.795(s, 2H) , 3.958-3.820(m, 2H) , 2.666(s, 3H) , 2.320-2.200(m, 2H) , 1.614(d, 6H, J=6.4Hz), 1.533(t, 2H) , 1.267(t, 3H) EXAMPLE 31 : dl-7-[2-(Morpholino)ethyl]camptothecin trifluoro-acetate The same procedure as in EXAMPLE 24 was carried out except that trifluoroacetic acid was used instead of p-toluenesulfonic acid. 1 H-N_m (200MHz, DMSO-d 6 ) δ 0.92(t, J=6Hz, 3H) , 1.94(m, 2H) , 2.90(m, 4H) , 3.30- 3.65(m, 8H) , 5.47(s, 2H) , 5.48(s, 2H) , 6.56(s, IH) , 7.70-8.30(m, 4H) EXAMPLE 32 : Preparation of 6-cvano-l.l-(ethylenedioxy) -7-[!'-(ethoxγcarbonyl)-3 -(methoxymethyloχypropyl]-5-oxo -Δ 6<8 -tetrahvdroindolizine 6-Cyano-l, 1-(ethylenedioxy)-7-[ (ethoxycarbonyl)methyl]-5- oxo-Δ 6(8) -tetrahydroindolizine(608.6mg, 2mmol) was dissolved in anhydrous dimethylformamide(3ml) , cooled to 5°C and after addition of potassium t-butoxide(258.lmg, 2.3mmol), the reaction mixture was stirred at the same temperature for 30 mins. To this mixture, 2-brmoethanolmethoxy-methylether (1.352g, 8mmol) was added and the reaction mixture was stirred at 45-50°C for 20 hrs. The reaction mixture was diluted with water(50ml) , extracted with dichloromethane (100ml) . The organic layer was washed with water(80ml) and brine(80ml) , dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography with ethylacetate -n-hexane( 2:1) to give the desired product(734.8mg, 95%) as a pale yellow solid. IR(neat) 2224, 1734, 1656 cm '1 1 H-NMR (400MHz, CDC1 3 ) δ 1.254(t, 3H, J=6.8Hz), 1.987-2.020(m, IH) , 2.410(t, 2H, J=6.8Hz) , 2.43-2.449(m, IH) , 3.447-3.485 (m, IH) 3.564-3.603 (m, IH) , 4.117-4.201 (m, 7H) , 4.225(q, 2H J=7.6HZ) ,4.584, 4.609(ABq, 2H, J=6.8Hz) , 6.367 (s,lH) MS (El) m/e 392 (M + ), 377(M * -CH 3 ) , 361 (M * -OCH 3 ) , 347 (M + -C 2 H s O) EXAMPLE 33 : Preparation of 6- (acetamidomethyl) -1,1 - (ethylenedioχy)-7-n / - (ethoχycarbonyl)-3°C- (methoxymethyl oxγpropyl) ]-5-oxo-Δ 6(8> -tetrahvdroindolizine 250 Drops of Raney-Nickel (50% aqueous slurry) was washed with water (5ml) and acetic acid (10ml) 5 times respectively and added into a parr bottle. To this solution, a compound(743.8mg, 1.9mmol) , obtained in EXAMPLE 32, acetic anhydride(30ml) and acetic acid(10ml) were added and the reaction mixture was hydrogenated at a pressure of 45 psi and 45-50°C for 3 hrs. The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with dichloromethane -methanol (20: 1) to give the desired product(789.5mg,95%) as a yellow oil. IR(neat) 1733, 1660, 1635 cm "1 1 H-NMR (400MHZ, CDC1-.) δ 1.214(t, 3H, J=6.8HZ), 1.924(s, 3H) , 1.939-1.98 (m, IH) , 2.382(t, 2H, J=6.8H2), 2.399-2.40(m, IH) , 3.343(s, 3H) , 3.44-3.58(m, 2H) , .075-4.131(m, 7H) , 4.145(q, 2H) , 4.34, 4.39(d, d, 2H) , 4.571, 4.592 (ABq, 2H, J=6.8HZ), 6.306(S, IH) , 6.61(brs, IH) MS(El) m/e 438(M * ), 423(M * -CH 3 ), 407 (flT-OCH j ) , 395(M'-COCH j ) , 393 (M * -C 2 H 5 0 ) EXAMPLE 34 : Preparation of 6-( cetoxymethyl)- 1.1- (ethylenedioxγ)-7-[i'-(ethoxycarbonyD-S'-(methoxymet hyloxγpropyl)]-5-oxo-A 6<8 -tetrahvdroindolizine To a compound(789.5mg, 1.8mmol) obtained in EXAMPLE 33, acetic anhydride(15ml) , acetic acid(5ml) and sodium nitrite(621.2mg, 9mmol) were added and the reaction mixture was stirred at 5 to 10°C for 6 hrs. After removing the inorganic material, so formed by filtration, the filtrate was concentrated under reduced pressure to give nitroso compound. With addition of carbon tetrachlonride (50ml) , the mixture was heated under reflux for 18 hrs. The reaction mixture was washed with water(10ml) and brine(10ml), dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography with dichloromethane -methanol( 20:1) to give the desired product(72O g,91%) as a colorless oil. IR(neat) 1740, 1733, 1654 cm "1 1 H-NMR (400MHz, CDC1 3 ) 1.217(t, 3H, J=6.8Hz) , 1.86-1.95(m, IH) , 2.056(s, 3H) , 2.368(t, 2H) , 2.38-2.43(m, IH) , 3.333(s, 3H) , 3.41-3.57(m, 2H) , 4.087-4.119 (m, 9H) , 4.569(s, 2H) , 5.259(S, 2H) , 6.304(S, IH) MS (El) m/e 439 (M * ) , 408 (M + -OCH,) , 396 (M + -C0CH,) EXAMPLE 35 : Preparation of 1,1-(ethylenedioxy)- (5 / -methoxγmethγloxyethγl-2 H, 5 / H, 6 H-6-oxopγrano) f3 ' .4 ' -f1-5-oxo-Δ 6<8) -tetrahvdroindolizine A mixture of the compound (720mg,1.64mmol) obtained in prepared by EXAMPLE 34 and methanol(15ml) was added to LiOH'H-O (172mg, 4.1mmol) dissolved in water(5ml) and stirred at 25 to 30°C for 2 hrs. The reaction mixture was concentrated under reduced pressure to remove methanol and with addition of water(15ml), dichloromethane(50ml) and acetic acid(5ml) , the reaction mixture was stirred at 25 to 30°C for 10 hrs. The organic layer was separated from the reaction mixture, washed with water (10ml) and brine(10ml) and dried over anhydrous magnesium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified by column chromatography with dichloromethane- methanol(30:1) to give the desired product(72O g,91%) as a white solid. IR(neat) 1747, 1662 cm '1 .,H-NMR (400MHz, CDCl 3 ) _ 2.178-2.216(m, 2H) , 2.404(t, 2H, J=6.8Hz), 3.371(s, 3H) , 3.599-3.643(m, 2H) , 3.701(t, IH, J=6.8Hz), 4.109-4.192(m, 6H) , 4.618(s, 2H) , 5.278, 5.43(ABq, 2H, J=16.4HZ), 6.169(s, IH) MS(El) m/e 351(M * ), 320(M + -0CH 3 ) , 307(M * -C0 2 ) EXAMPLE 36 : Preparation of 1,1-(ethγlenedioχy)-(5 / -methoχymethγloxyethyl-5 -hγdroxγ-2 / H. 5'H. 6 / H- 6-oxopyrano) r3'_4'-f1-5-oxo-Δ 6<8) -tetrahvdroindolizine Potassium t-butoxide(273mg, 2.43mmol) was added to a mixture of the compound(570.3mg, 1.63mmol) obtained in EXAMPLE 35 dissolved in anhydrous DMF(20ml) and stirred at 0 to 5°C for 30 mins. The reaction mixture was cooled to -10 to -5°C and with addition of triethyl phosphite (974 μl, 5.68mmol), the mixture was stirred for 2.5 hrs, while bubbling the oxygen. Water(10ml) was added to the mixture and pH was adjusted to 3.5 with IN-HCl. Then, the reaction mixture was extracted with dichloromethane (30ml x 2 times), washed with water(20ml) , and brine(20ml) and dried over anhydrous magnesium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified by column chromatography with dichloromethane -methanol (25:1) to give the desired product (416.4mg, 69.8%) as a white solid. IR(neat) 3503, 1749, 1654 cm "1 1 H-NMR (400MHz, CDC1 3 ) δ 1.919-1.974(m, IH) , 2.010-2.063 (m, IH) , 2.348(t,2H, J=6.8HZ), 3.30(s, 3H) , 3.542-3.597 (m, IH) , 3.639- 3.684(m, IH) , 4.002-4.1541(m, 7H) , 4.516, 4.494 (ABq, 2H, J=6.4HZ) , 5.111, 5.557 (ABq, 2H, J=16Hz) , 6.541(s, IH) MS(El) m/e 367 (M * ) , 336(M + -0CH 3 ) , 323(M + -C0 2 ) EXAMPLE 37 : Preparation of 1,5-dioxo-(5 / -hvdroxyethyl-5 - hvdroxy-2 H, 5'H, 6'H-6-oxopyrano) r3 .4'-fl- Δ 6(8) -tetrahydroindolizine A mixture of THF(200ml) , water(lml) and 6N HC1(2.5ml) was added to the compound(146.lmg, 0.4mmol) obtained in EXAMPLE 36 and stirred at 50-55°C for 1 hr. The reaction mixture was concentrated under reduced pressure. The residue was purified by Dianion" HP-20 (Mitsubishi) with acetonitrile-water(4:1) and concentrated under reduced pressure to give the desired product (93.2mg,84%) as a black resin. IR(KBr) 3470, 1749, 1654 cm "1 'H-NMR (400MHz, CDC1 3 ) . 1.92-2. ll(m, 2H) , 2.962(t, 2H, J=6.0Hz), 3.674-3.86 ( , 2H) , 4.326(t, 2H, J=6.0Hz), 5.259, 5.627(ABq, 2H , J=16 . 8Hz ) , 5 . 287 ( s , IH) , 7 . 284 ( s , IH) MS ( EI ) m/e 279 (M + ) , 261 (M + -H 2 0) , 235 (M * -C 2 H 5 0H) EXAMPLE 38 ; Preparation of dl-18-hydroxγcamptothecin A solution of 2-aminobenzaldehyde ethyleneacetal (75.6mg, 0.46mmol) and toluene (8ml) was added to the compound(85.2mg, 0.31mmol) obtained in EXAMPLE 37 and refluxed in a Dean-Stark apparatus for 1 hr. p-toluene- sulfonic acid (catalytic amount) was added to the reaction mixture and refluxed again for 3 hrs. The solid, so formed, was filtered and purified by column chromatography with dichloromethane-methanol(15:1) to give the desired product(57.8mg,52%) as a pale brown solid. IR(neat) 3400, 1743, 1658 cm "1 1 H-NMR (400MHz, CDCl 3 :DMSO-d 6 = 3:1) . 2.01-2.20(m, 2H) , 3.51-3.86(m, 2H) , 5.315(s, 2H) , 5.34, 5.55(ABq, 2H) , 6.418(s, IH) , 7.557(s, IH) , 7.676(t, IH) , 7.83(t, IH) , 8.03(d, IH) , 8.18(d, IH) , 8.570(s, IH) MS(EI) m/e 364(M * ), 320(M * -C0,) EXAMPLE 39 ; Preparation of dl-18-methoxymethyloxγ Methoxymethyl chloride (6μl, O.Oδmmol) and diisopropylethylamine(12μl, 0.07mmol) was added to a suspension of dl-18-hydroxycamptothecin(20mg, 0.05mmol) in dichloromethane(2ml) and stirred at 25 to 30°C for 48 hrs. With addition of dichloromethane(25ml) , the reaction mixture was washed with a saturated ammonium chloride(10ml) , water(lθ l) , 10% hydrogen sulfate potassiu (10ml) , water(10ml) and brine(10ml) successively. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography with dichloromethane-methanol (30:1) and PTLC with dichloromethane-methanol (20: 1) to give the desired product (5mg,23%) as a yellow solid. IR(KBr) 3566, 1733, 1652 cm '1 1 H-NMR (400MHz, CDC1 3 ) δ 2.15-2.34(m, 2H) , 3.387(s, 3H) , 4.08-4.22(m, 2H) , 4.604(d, d, 2H) , 5.313(s, 2H) , 5.62, 5.78(ABq, 2H) , 6.6(Ξ, IH) , 7.68(t, IH) , 7.726(s, IH) , 7.84(t, IH) , 7.96(d, IH) , 8.24(d, IH) , 8.41(s, IH) MS(El) m/e 408 (M * ) HRMS m/e M + Calcd; 408.1321, Obsd; 408.1336 In the same manner as in EXAMPLE 39, the compounds( R=H and n=2) of EXAMPLE 40 to 46, expressed by the general formula(I) , were prepared. EXAMPLE 47 : Preparation of 6-cvano-l,l-(ethγlenedioxy)-7- [l '-(ethoxγcarbonyl)-2'-hydroxγethγl]-5-oxo-A 6<8) - tetra ydroindolizine A mixture of 35% formaldehyde(30ml) , dioxane(50ml) , water(20ml) and ethanol(20ml) was added to 6-cyano-l,l- (ethylenedioxy)-7-[ (ethoxycarbonyl)methyl]-5-oxo-Δ 6(8 - tetrahydroindolizine(500mg, 1.64mmol) and stirred at 25 to 30°C for 15 hrs. With addition of dichloromethane (120ml), the reaction mixture was washed with water(120ml x 3 times) and brine(120ml) . The separated organic layer was dried over anhydrous magnesum sulfate and concentrated under reduced pressure. The residue was purified by column chromatography with dichloromethane -methanol( 25:1) to give the desired product( 318mg, 58%) as a white solid. IR(KBr) 3310, 2224, 1735, 1647 cm -1 1 H-NMR (400MHz, CDC1 3 ) δ 1.275(t, 3H, J=7.2Hz) , 2.407 (t, 2H, J=6.8Hz) , 4.029-4.221(m, 9H) , 4.239(q, 2H) , 6.383(s, IH) MS (El) m/e 334 (M * ) , 316(M * -H 2 0) , 304 (M * -CH 3 OH) EXAMPLE 48 ; Preparation of 6-cvano-l,l-(ethylenedioxγ) -7-fl / -(ethoxγcarbonyl)-2 -(methoχyethoxγmethyloxγethyl) ] -5-oxo-Δ 6(8 -tetrahγdroindolizine Dichloromethane(0.7ml) was added to the compound(51.6mg, 0.15mmol) obtained in EXAMPLE 47 and cooled in ice bath. Then, with a slow addition of diisopropylethylamine (30μl, 0.17mmol) and MEM-C1 (35μl, 0.31mmol), the resulting solution was stirred at 25 to 30°C for 20 hrs. To the reaction mixture, dichloromethane (15ml) was added and washed with a saturated sodium bicarbonate aqueous solution (10ml x 2 times) , water(10ml) and brine (10ml) . The separated organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography with dichlorometήane-methanol(25:1) to give the desired product(41mg, 62%) as a white solid. IR(neat) 2224, 1734, 1661 cm '1 1 H-NMR (400MHz, CDC1-) δ 1.289(t, 3H, J=6.8Hz), 2.422(t, 2H, J=6.8Hz), 3.410 (s, 3H) , 3.548-3.598(m, 2H) , 3.653-3.680(m, IH) , 3.725-3.748(m, IH) , 3.912 (dd, IH, J=10Hz, J=6.8Hz), 4.098(dd, IH, J=10Hz, J=6.8Hz), 4.129- .216(m, 6H) , 4.236(q, 2H) , 4.336(t, IH, J=6.8Hz), 4.706, 4.742 (ABq, 2H, J=6.8Hz), 6.441(8, IH) MS (El) m/e 423(M * +H) , 422 (M + ) , 392 (M + -OCH 3 ) , 378 (M * -C-H-0) EXAMPLE 49 : Preparation of 6- (acetamidomethyl)- 1,1- (ethylenedioxγ) -7-[l / -(ethoxycarbonγl)-2 / - (methoxyeth oxγmethγloxyethγl) ]-5-oxo-Δ 6<8) -tetrahγdroindolizine With the compound(39.6mg, 0.09mmol) obtained in EXAMPLE 48, the reaction and work-up was carried out in the same procedure as in EXAMPLE 33 and then, the residue was purified by column chromatography with dichloromethane- methanol ( 20 : 1) to give the desired compound(42mg, 96%) as a colorless oil. IR(neat) 1733, 1661, 1656 cm "1 1 H-NMR (400MHz, CDC1 3 ) _ 1.247(t, 3H, J=7.2HZ), 1.943(s, 3H) , 2.393(t, 2H, J=6.8HZ), 3.405(s, 3H) , 3.546-3.569 (m, 2H) , 3.660 -3.686(m, 2H) , 3.809(dd, IH, J=6.4Hz, J=9.2Hz) , 4.093-4.192(m, 7H) , 4.205(g, 2H) , 4.467(dq, 2H, J=6.4Hz), 4.652(t, 2H, J=6.8Hz), 4.719, 4.750(ABq, 2H, J=6.8Hz), 6.349(s, IH) , 6.658(s, IH) MS (El) m/e 468 (M * ), 453(M * -CH 3 ) , 425 (M * -C 2 H 5 0) EXAMPLE 50 ; Preparation of 6- (acetoxymethγl)-l,l- (ethylenedioxγ)-7-fl - (ethoxγcarbonyP-2'-(methoxyethoxym ethyloxyethyl) ]-5-oxo-Δ 6<8> -tetrahydroindolizine With the compound(359mg, 0.76mmol) obtained in EXAMPLE 49, the reaction and work-up was carried out in the same procedure as used in EXAMPLE 34 and then, the residue was purified by column chromatography with dichloromethane : methanol (30:1) to give the desired compound(234mg, 65%) as a colorless oil. IR(neat) 1733, 1740, 1661 cm "1 1 H-NMR (400MHz, CDC1 3 ) δ 1.230(t, 2H, J=7.2HZ) , 2.057(s, 3H) , 2.361(t, 2H, J=6.8Hz) , 3.381(s, 3H) , 3.521-3.542 (m, 2H) , 3.620- 3.653(m, 2H) , 3.723(dd, IH, J=6.0Hz, J=9.6Hz) , 4.082-4.209(m, 8H) , 4.225(5, 2H) , 4 , 716-4.675 (ABq, 2H, J=6.8HZ) , 5.259(5, 2H) , 6.313(s, IH) MS (El) m/e 469 (M*), 426(M + -COCH 3 ) EXAMPLE 51 ; Preparation of 6-(acetoxymethγl)-l,l- (ethγlβnedioxγ)-7-fl'-(ethoxγcarbonγDvinyl]-5-oxo-Δ 6<8) - tetrahγdroindolizine A mixture of dry benzene(8ml) and DBU(416μl, 2.78mmol) was added to the compound(522mg, l.lmmol) obtained in EXAMPLE 50 and stirred at room temperature for 3 hrs. With addition of dichloromethane (20ml) , , the reaction mixture was washed with a saturated ammonium chloride aqueous solution(15ml x 2) and water(15ml) . The separated organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography with dichloromethane- methanol(30:1) to give the desired product(363mg, 90%) as a yellowish white solid. IR(neat) 1729, 1719, 1655 cm '1 1 H-NMR (400MHz, CDC1 3 ) δ 6.551(s, IH) , 6.109, 5.773(s, S, IH X 2) , 5.008(s, 2H) , 4.243(q, 2H, J=7.2Hz), 4.104-4.161(m, 6H) , 2.398(t, 2H, J=6.8HZ), 2.021(s, 3H) , 1.293(t, 3H, J=7.2Hz) MS ( El ) m/e 3 63 (M* ) , 32 0 EXAMPLE 52 : Preparation of 6-(acetoxymethyl) -1,1- (ethylenedioxy)-7-fl'-(ethoxycarbonyl)-1 -(hydroxy)-2 - (hydroxy)ethyl]-5-oxo-Δ 6<8) -tetrahydroindolizine Pyridine(3.9ml) was added to the compound(285mg, 1.79mmol) obtained in EXAMPLE 51, injected with 0s04 (240mg, 0.94mmol) (0.08M toluene, 11.8ml) and stirred at room temperature for 4 hrs in the dark. After the reaction was completed, the reaction mixture was added with a solution of NaHSO 3 (480mg) and water(7ml) and stirred for 1 hr. The reaction mixture was extracted with dichloromethane (30ml x 5) , and the combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified by column chromatography with dichloromethane-methanol(20:1) to give the desired product(280mg,90%) as a white solid. IR(neat) 3307, 1735, 1654 cm '1 1 H-NMR (400MHz, CDC1 3 ) _ 6.455(s, IH) , 5.303(ABq, 2H, J=11.7Hz), 4.611(s,lH) 4.085-4.611(m, 9H) , 3.855, 3.826(dd, IH, J=6.8, 11.2HZ) , 2.641(brt, IH, J=6.8Hz), 2.384(t, 2H, J=6.8HZ) , 2.063(s, 3H) , 1.290(t, 3H, J=6.8Hz) MS (El) m/e 397 (M * ), 354 EXAMPLE 53 : Preparation of l,l-(ethylenedioχy)-(5 -hydroxy methyl-S'-hvdroxy-Z'H. 5'H. 6'H-6-oxopyrano) T3' ,4 -f] -5-OXO-A6(8)-tetrahydroindolizine With the compound(325mg, 0.82mmol) obtained in EXAMPLE 52, the reaction and work-up was carried out in the same procedure as in EXAMPLE 35 and then, the residue was purified by column chromatography with dichloromethane- methanol(20:1) to give the desired compound (211mg, 84%) as a white solid. IR(neat) 3421, 1750, 1654 cm ' , 1 H-NMR (400MHz, CDCl 3 ) δ 6.599(s, IH) , 5.564, 5.255(ABq, 2H, J=16.0Hz), 4.072-4.238(m, 7H) , 3.772, 3.733(dd, 2H, J=11.6Hz), 2.418(t, 2H, J=6.8Hz) MS(El) m/e 309(M * ), 293, 280, 279, 265 HRMS m/e (M * ) Calcd : 309, 0849, Obsd : 309, 0.847 EXAMPLE 54 t Preparation of 1.1-(ethylenedioxy) - (S'-acetoxymethyl-S'-hvdroxy-Z 'H, 5'H. 6Η-6- oxopyrano) T3 7 ,4 -f]-5-oxo-Δ 6<8) - tetrahvdroindolizine The compound(119mg, 0.38mmol) obtained in EXAMPLE 53 was dissolved in anhydrous dichloromethane(10ml) and with addition of pyridine(93μl, 1.15mmol) and acetic anhydride(47μl, 0.50mmol) , the reaction mixture was stirred at room temperature for 20 hrs. The reaction mixture was diluted with dichloromethane(20ml) , washed with 10% potassium bisulfate aqueous solution(20ml) , water(20ml) and brine(20ml), and dried over anhydrous magnesium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified by column chromatography with dichloromethane-methane(20:l) to give the desired compound(127mg,94%) as a white solid. IR(neat) 3437, 1751, 1657 cm '1 1 H-NMR (400MHz, CDCl 3 ) δ 6.586(Ξ, IH) , 5.636, 5.295(ABq, 2H, J=16.4Hz) , 4.302(d, IH, J=11.2HZ) , 4.116-4.215 ( , 7H) , 4.004 (Ξ, IH) , 2.427(t, 2H, J=6.8Hz) , 2.084(s, 3H) In the same procedure as used in EXAMPLE 54 , the compounds of EXAMPLE 55 to 57 were prepared and the results were as follows: EXAMPLE 58 : Preparation of 1,5-dioxo- (S'-acetoxymethγl-S'- hvdroχy-2 'H. 5'H.6 Η-6-oxopyrano) f3 ' .4 ' - t] -Δ 6(8 - tetrahvdroindolizine 80% trifluoroacetic acid(1.3ml) was added to the compound(127mg, 0.36mmol) obtained in EXAMPLE 54 and stirred at room temperature for 2 hrs. The reaction mixture was concentrated under reduced pressure and with addition of brine, the reaction mixture was extracted with dichloromethane(20ml x 3) and dried over anhydrous magnesium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified by column chromatography with dichloromethane- methanol (20:1) to give desired product(theoretical yield) as a yellow solid. IR(KBr) 3412, 1746, 1660 cm '1 1 H-NMR (400MHz, CDC1-) δ 7.318(s, IH) , 5.687, 5.388(ABq, 2H, J=17.1Hz), 4.405(t, 2H) , 4.370(d, IH, J=11.7Hz), 4.155(d, IH, J=11.7HZ), 3.007(t, 2H, J=6.8Hz), 2.080(s, 3H) MS(El) m/e 307(M * ) In the same manner as used in EXAMPLE 58, the compounds(R=H and n=2) of EXAMPLE 59 to 61, expressed by the general formula(II),were prepared. EXAMPLE 62 : Preparation of dl-20-desethyl-20-acetoxymethyl camptothecin With the compound(19mg, 0.06mmol) obtained from EXAMPLE 58 and N-(2-aminobenzylidene) -p-toluidine(20mg, 0.09mmol) , the reaction and work-up was carried out in the same procedure as in EXAMPLE 38 and then, the residue was purified by PTLC with dichloromethane -methanol (20: 1) to give the desired compound(9mg, 36%) as a brown solid. IR(CHC1 3 ) 3400, 1749, 1740, 1654 cm "1 1 H-NMR ( 400MHZ , CDC1 3 + CD 3 OD=4 : 1 ) 8.493(s, IH) , 8.223(d, IH, J=8.4Hz) , 7.988(d, IH, J=8.0HZ), 7.867(t, IH, J=7.2Hz), 7.767(s, IH) , 7.703(t, IH, J=7.2HZ) , 5.697, 5.433(ABq, 2H, J=16.4Hz), 5.334(s, 2H) , 4.460(d, IH, J=11.2Hz), 4.330(d, IH, J=11.2HZ), 2.087(s, 3H) LRMS(EI) m/e 392(M*), 331, 320, 305 HRMS m/e (M * ) Calcd : 392.1008, Obsd : 392.1006 In the same manner as in EXAMPLE 38, the compounds (R=H and n=l) of EXAMPLE 63 to 65, expressed by the general formula(I), were prepared. EXAMPLE 66 Preparation of dl-20-desethyl-20- hydroxymethylcamptothecin A mixture of methanol (6ml) , water(2ml) and LiOH* H 2 0(33mg, 0.78mmol) was added to dl-20-desethyl-20- acetoxymethylcamptothecin (140mg, 0.36mmol) and the reaction mixture was stirred at room temperature for 1.5 hrs. The reaction mixture was concentrated under reduced pressure to remove methanol and stirred in an ice bath for 30 mins until the pH of the solution became controlled 3- 3.5 with IN HCl . The solid, so formed, was filtrated, washed with water, isopropanol and ether successively, and under P 2 0 5 , dried in vacuum for 3 hrs to give the desired product(83mg, 66%) as a yellowish white soild. IR(KBr) 3436, 1743, 1657 cm '1 1 H-NMR (400MHz, CDCl 3 +DMSO-d 6 =l: 1) <5 8.608(s, IH, 8.157(d, IH, J=8.0Hz) , 8.106(s, IH) , 8.050(d, IH, J=8.0Hz) , 7.829(t, IH, J=7.3Hz) , 7.673 (t, IH, J=7.3HZ) , 6.741(s, IH) , 5.441, 5.360(ABq, 2H, J=16.1Hz) , 5.304(s, 2H) , 3.850-3.89 (m, IH) , 3.681-3.723 (m, IH) LRMS(EI) m/e 350(M * ), 320, 306 HRMS m/e (M * ) Calcd : 350.0903, Obsd : 350. 0917 In the same manner as in EXAMPLE 39, the compounds(R=H and n=l) of EXAMPLE 67 to 69, expressed by the general formula(I) , were prepared. EXPERIMENT 1 : Cvtotoxicity of the compounds according to this invention 1) Materials Cancer cell lines In the determination of cytotoxicity, the following cell lines, purchased from ATCC(U.S.A. ) , were used for the experiments as the target organisms: L1210(ATCC CCL 219) , A172(ATCC CRL 1620) , A427(ATCC HTB 53) , A549(ATCC CCL185) , SK-NEP-1(ATCC HTB 48) , CAOV-3 (ATCC HTB 75) , HEC -1-B(ATCC HTB 113) , DLD-1(ATCC CCL 221) and KATO-III (ATCC HTB 103), CAKI-2(ATCC HTB 47) . Preparation of culture medium for cancer cells Distilled water was used for the preparation of culture medium. 1 liter of powdered RPMI 1640 medium was dissolved in distilled water and with addition of NaHC0 3 (2. Og) , the resulting solution was stirred. After the pH of the solution was adjusted to 6.8 to 7.4, the medium was filtered by 0.22μm filter. The fetal bovine serum was thermally treated at 56°C for 30 mins before use. Reagents RPMI 1640 culture medium for cancer cells, fetal bovine serum, sodium bicarbonate and trypsin-EDTA buffer were purchased from Gibco Co. And MTT(3, 4, 5-dimethylthiazol -2-yl) -2 ,5-diphenyltetrazolium bromide) and sulforhodamine B(SRB) reagent were obtained fro Sigma Co. and other general reagents were of G.R. degree. Test materials Camptothecin, purchased from Sigma Co. , was dissolved in dimethylsulfoxide and with addition of Dulbec co's phosphate buffered saline(DPBS) , the concentration of dimethylsulfoxide was adjusted to 10% before use. In case of the synthesized test materials dissolved in dimethylsulfoxide, the concentration of dimethylsulfoxide was also adjusted to 10%, while the other packed with DPBS. The final concentration of dimethyl sulfoxide given to cancer cells was adjusted to less than 1%, which had no influence on the growth of cancer cells. 2. Methods The cancer cells were grown at 37°C, 5% C0 2 in RPMI 1640 medium containing 10%(v/v) fetal serum(FBS) , 50 μg/ml gentamicin and 2 g/L sodium bicarbonate. (In order to detach anchorage-dependent cell lines grown by adhering to a culture flask) the medium was removed, washed with PBS one time and with addition of 2 to 3ml of trypsin-EDTA buffer,the monolayers of cancer cells were wholly covered to stand for the time being. Then, a suspension of the cultured cancer cells was prepared, diluted with a medium and inoculated on 96-well plate so that the number of cells may be the same as described in the following Table 1. 20 μl of sample(PBS solution in a control well) was inoculated to 96-well microplate and cultured for 3 days. After cultivation, the anti-neoplastic activites were measured by SRB assay[Test Scheme 1] . As for anchorage-independent cell such as L1210, cells were inoculated so that the number of cell may also be the same as described in the following Table 1. After being cultured for 2 days, the cytotoxicity against the cells were measured by MTT assay[Test Scheme 2]. The optical density was measured using an automatic microplate reader. The concentration of cell-growth inhibition rate by 50% was calculated by GI 50 (computer PCS version 4.1 by Probit method). As per the experiment one time, the mean of optical density on about 2 to 3 wells was used for the calculation. Table 1 : Number of inoculated cells per Test Scheme 1 : SRB assay 180μl of cell suspension in medium + 20 μl of sample or PBS 4- Cultured at 37°C for 3 days, 5% carbon dioxide. Precipitated with TCA. Left at 4°C for l hr. Washed with distilled water 5 times and dried. Stained with 0.4% SRB solution for 30 mins. Washed with 1% HAc solution 5 times. Dissolved in 10 mM Tris buffer. * Measurement of optical density at 520nm. Calculation of GI 50 Test Scheme 2 : MTT assay 180 μl of cell suspension in medium + 20μl of sample or PBS Cultured at 37°C for 2 days, 5% carbon dioxide. Added with 50μl of MTT solution(2mg/ml in PBS solution). Cultured for 4 hrs. Centrifuged at 2,000 rp for 10 mins. .J. Removal of supernatant. Dissolved formazan crystals in DMSO. Measurement of optical density at 570 nm. Calculation of GI 50 3. Results The cytotoxicity results of each cell line were as table 2 and 3: 66 Table 2 KATO Exa "M A _7 ;.«-: iDLD-1 < mC - ! CA0V ! S -ϊ ' 1210 [MEAN NO. i : ll-B 3 lNEP-1 III 18 0.14 ; 0.73 0.65 1.8 1.21 I 0.0077 8.6 i 0.43 i 1.83 19 0.31 1.32 0.31 i 4.7 0.08 ' 0.013 0.29 0.62 26 0.23 I 0.15 0.17 ; 1.37 ; 0.09 ! 0.0031 0.46 2.18 0.35 29 0.07 0.05 , 0.26 ! 7.6S 0.1. 0.015 0.21 0.38 1.19 30 0.057 ! 0.065 i 0.064 i 1.65 ! 0.025 10.00077 0.024 0.08 0.27 38 0.83 1.63 0.91 - 20.5 2.3 0.17 3.54 2.3 4.27 39 0.45 ' 1.07 0.76 , 1.34 0.23 I 0.018 0.13 ' 0.18 0.57 Table 3 EXPERIMEN T 2 ; Antitumor activities of the—compounds according to this invention The antitumor activities may be usually proven by determining tumor growth inhibitory effects on solid tumor and prolongation effects of life span on artificially tumor-bearing animals. Antitumor activities were measured through the experiments on prolongation effects of life span against L1210-bearing mice and tumor growth inhibitory effects against B16-bearing mice. To evaluate the antitumor activities on L1210 leukemia, a certain number of L1210 cells(in vitro) was intraperitoneally inoculated for propagation and the L1210 cells in ascites were harvested from animals sacrificed by cervical dislocation on 7th day after tumor was implanted. To investigate tumor growth inhibitory effects against B16 melanoma, a certain number of B16 cells(in vitro) was subcutaneously inoculated for propagation and tumor cells were resected from animals scrificed by cervical dislocation on 14th day after tumor was implanted. The following is a detailed description of this EXPERIMENT for antitumor activities. Experimental animals Male BDF 1 mice, weighing about 20 grams, were used. Animal rooms were controlled at 23+2°C and 60+5% relative humidity, under HEPA filter, and maintained in specific pathogen free (SPF) . Experiment 2-1: Prolongation effects of life span against L1210-bearing mice in accordance with the compounds of this invention. 5x10 s of L1210 leukemia cell, obtained in vitro form, were intraperitoneally inoculated in BDF 1 mice for propagation. The L1210 tumor cells in ascites were aseptically harvested from BDF, mice sacrificed by cervical dislocation on 7th day after tumor implantation. The tumor cells were passaged at least three times prior to use in the test. 5 x 10 s cell were intraperitoneally inoculated into BDF, mice on day 0 and the sample (O.lml/lOg) were intraperitoneally administration on days 1, 3 and 5. Six mice were used for each experimental group. Antitumor activities were evaluated by the mean survival time of a group of mice and also expressed by the ILS(%) value and the mean animal weights for day 1 and day 5. Increased life span(ILS) ILS(%) = { (MST of sample treated group /MST of solvent group) -1) x 100 Results The compound of example 18 given 1st, 3rd and 5th days after the day of tumor inocualtion increased the life span(ILS) of L1210-bearing mice by 33% and 64% at total doses of lOmg/kg and 50mg/kg, respectively (Tabel 4). The compound of example 26 given 1st, 3rd and 5th days after the day of tumor inoculation increased the life span(ILS) of L1210-bearing mice by 72% and 75% at total doses of lOmg/kg and 50mg/kg, respectively (Table 5) . Table. 5 Prolongation effects of life against L1210 of the compound prepared in example 26 Dose Survival days 30 days : B.W. Compound (mg/kg) MST ILS(S-) survivor , Change(g) Control 9.125 0/6 + 1.77 Compound of 10 15.67 72 0/6 0.09 example 26 Compound of 50 16.00 75 0/6 - 0.57 example 26 The compound of example 30 given 1st, 3rd and 5th days after the day of tumor inoculation increased the life span(ILS) of L1210-bearing mice by 42% and 119% at total doses of lOmg/kg and 50mg/kg, respectively (Table 6) . Control 9.125 0/6 + 1.77 j Compound of 10 13.00 .9 0/6 * 0.74 i example 30 j Compound of 50 20.00 119 0/6 - 0.57 I example 30 Since the compound of example 30 exhibited better dose dependency and ILS(%) than other compound, the inventor et al. tried to elucidate a higher dose level and amounts showing antitumor activities in experimental animals administered with a higher dose. When the compound of example 30 was given 1st, 3rd and 5th days after the inoculation at total doses of 8.75, 17.5, 35, 70 and 140mg/kg, the ILS(%) showed 58.7, 79.7, 92.4, 143 and 110%, respectively(Table 7) . When the compound of example 30 was given on day 1, 3 and 5 after the tumor inoculation, the maximum ILS of 143% was obtained at total dose of 70mg/kg. The antitumor activity of the compound of example 30 against intraperitoneally-i planted L1210 was evaluated in parallel with that of camptothecin as shown in Figure 1. The compound of example 30 gave higher ILS(%) and wider range where the effect was siginificant. Experiment 2-2: Tumor growth inhibitory effects on B16- bearing mice in accordance with the compounds of this invention 10° of B16 melanoma cell, obtained in vitro form, were subcutaneously in BDF, mice for propagation. The B16 melanomas were transfered on day 14. The fresh tumors except for necrotized center were aseptically resected from tumor-bearing mice sacrificed by cervical dislocation. The tumor cells were passaged at least injected three times prior to use in the test. lg subcutaneously donor tumor to be subcutaneously was homogenized in cold sterilized saline to give a final concentration of lg/lO l. The 1:10 tumor suspension is injected into all BDF, mice subcutaneously using 0.2ml/mouεe. The mice were subcutaneously implanted with such homogenized solution and 7 to 8th day after the tumor implantation, the mice having a certain size of tumors were selected for experiments. Each experimental group were administered with the sample (O.lml/lOg) on 8th, 12th and 16th day after the inoculation of tumor. Eight mice were used for each experimental group. The size of the tumors was calculated by direct measurement of the diameter of the tumors, using the formula. Tumor size = (a 2 b)/2 Where b is the largest diameter and a is the diameter perpendicular to b and growth curves were thus obtained. (Figure 2) The inhibition rate of tumor growth on the basis of tumor size was calculated according to the formula. Wherein; IR(%) = (l-TW c /TW c )xl00 TW, is the mean tumor volume(weight) of sample treated groups. TW c is the mean tumor volume(weight) of contro group. Test results When the compound of example 30 was given 8, 12 an 16 days after the tumor inoculation at total doses of 35 , 70 and 140mg/kg , the IR(%) showed 27, 54.8 and 67.72%, respectively (Table 8) . Table. 8 Tumor growth inhibitory effects against B 1 6 melanoma of the compound prepared i_ evampie 30 12.Compound ofi3.Compo_nd ofl4.Compoun Tumor l.Camptothecin Control I example 30 example 30 [example 30 I weight lOmg/kg J35mg/kg 70mg kg jl40mg kg 'day 20 3.92 + 0.32 1.61 + 0.30 I 2.34 + 0.44 ' 1.71 + 0.18 1.11 + 0 UR(?.) 59?- 40?_ 56?_ Futher, 20th day after the compound of example 30 was administered, the percentage of IR on the weight of the incised tumor showed 40, 56 and 71.6%, respectively. The maximum IR of 67.72% was obtained at total dose of 140mg/kg. The changes in tumor volumes were outlined in Figure 2. Such results clearly imply that the compound of example 30 has not only better antitumor activity on ascites tumor and solid type tumor, but also less toxicity and a wide range of safety margin, compared with camptothecin.

Claims: WHAT IS CLAIMED IS: 1. Camptothecin derivative and pharmaceutically acceptable salts thereof, expressed by the following general formula[ I ] Wherein: R represents hydrogen or -(CH 2 ) 2 -NR,R 2 , (R, is hydrogen or a general protecting group of amine; R 2 is a lower alkyl, hydroxyethyl or acetoxyethyl; Further,R 2 may form heterocyclic compound by binding with an adjacent nitrogen) ; n is 1 or 2 ; R 3 is hydrogen or -OR,, [R A is hydrogen or C R 5 (R 5 is methyl or CH 3 OCH 2 ~) ; C NH R 6 (R 6 is isopropyl , o 0 phenyl or -CH 2 CH 2 C1 . ) ; CH 2 OR 7 (R 7 is methly, ethly or CH 3 OCH 2 CH 2 - . ) ] ; Wherein; When n is 2 and R 4 is hydrogen, R is not hydrogen; And when n is 2 and R- is hydrogen, R is not hydrogen; Also, when R is -CH 2 CH 2 NHCH-, R 3 is not hydrogen. 2. Intermediate of camptothecin synthesis expressed by the following formula[II] [ II ] Wherein; R' is -(CH 2 ) 2 -NR,R 2 , (R, is a general protecting group of amine; R 2 is a lower alkyl, hydroxyethyl, or acetoxyethyl; R 2 may form heterocyclic compound by binding with an adjacent nitrogen) ; 3. Intermediate of camptothecin synthesis expressed by the following formula[III] n is 1 or 2; R 3 is hydrogen or -0R 4 [R 4 is hydrogen or C ^(Iv j is methyl or CH 3 OCH 2 -) ; C NH R 6 (R 6 is isopropyl, 0 o phenyl, and -CH 2 CH 2 C1.); CH 2 OR 7 (R- is methyl, ethyl or -CH 2 OCH 2 CH 3 ) ] ; Wherein; When n is 2 and R 4 is hydrogen, R is not hydrogen; And when n is 2 and R- is hydrogen, R is not hydrogen; Also, when R is -CH-CH 2 NHCH-, R 3 is not hydrogen. 4. Process for manufacturing the compound expressed by the following general formula[I] wherein the compound expressed by the following formul [II] is reacted with the compound expressed by the compound[III] under acidic condition Wherein; R is hydrogen or -(CH 2 ) 2 -NR,R 2 , (R, is hydrogen or a general protecting group of amine; is lower alkyl, hydroxyethyl or acetoxyethyl; further,R 2 may form heterocyclic compound by binding with an adjacent nitrogen) ; R' is hydrogen or -(CH- j -Nl^ -, (R, is a general protecting group of amine; R j is a lower alkyl, hydroxyethyl or acetoxyethyl; further, R 2 may form heterocyclic compound by binding with an adjacent nitrogen; n is 1 or 2 ; R 3 is hydrogen or -0R [R A is hydrogen or C R 5 (R 5 is methyl or CH-OCH--) ; C NH R 6 (R 6 is isopropyl, 0 0 phenyl or -CH 2 CH 2 C1.) ; CH 2 OR 7 (R 7 is methyl, ethyl or CH 3 OCH 2 CH 2 -. ) ] ; Wherein; When n is 2 and R 4 is hydrogen, R is not hydrogen; And when n is 2 and R 3 is hydrogen, R is not hydrogen; Also, when R is -CH-CH 2 NHCH-, R 3 is not hydrogen. 5. Antineoplastic agent containing camptothecin derivatives expressed by the following general formula[I] and pharmaceutically acceptable salt thereof Wherein; R represents hydrogen or -(CH^.-NR- j R-,, (R, is hydrogen or a general protecting group of amine; R 2 is a lower alkyl, hydroxyethyl or acetoxyethyl; Further,R 2 may form heterocyclic compound by binding with an adjacent nitrogen) ; n is 1 or 2 ; R 3 is hydrogen or -0R 4 [ R 4 is hydrogen or C R 5 (R 5 is methyl or CH 3 OCH 2 ~ ) ; C NH R 6 (R 6 is isopropyl , δ o phenyl or -CH 2 CH-C1. ) ; CH 2 OR 7 (R 7 is methyl, ethyl or CH 3 OCH 2 CH 2 -. ) . Wherein; When n is 2 and R 4 is hydrogen, R is not hydrogen; And when n is 2 and R- is hydrogen, R is not hydrogen; Also, when R is -CH 2 CH 2 NHCH 3 , R 3 is not hydrogen.

Inventors: CHA KYEONG HOI, HAH JEONG MI, JEW SANG SUP, KIM HEE JIN, KIM JONG MIN, KIM JOON KYUM, KIM MYOUNG GOO, LEE HEE SOON, LEE KWANG KYUN, OK KWANG DAE, CHA, KYEONG, HOI, HAH, JEONG, MI, JEW, SANG, SUP, KIM, HEE, JIN, KIM, JONG, MIN, KIM, JOON, KYUM, KIM, MYOUNG, GOO, LEE, HEE, SOON, LEE, KWANG, KYUN, OK, KWANG, DAE
Applicants: CHA KYEONG HOI, CHONG KUN DANG CORP, HAH JEONG MI, JEW SANG SUP, KIM HEE JIN, KIM JONG MIN, KIM JOON KYUM, KIM MYOUNG GOO, LEE HEE SOON, LEE KWANG KYUN, OK KWANG DAE, CHA, KYEONG, HOI, CHONG KUN DANG CORPORATION, HAH, JEONG, MI, JEW, SANG, SUP, KIM, HEE, JIN, KIM, JONG, MIN, KIM, JOON, KYUM, KIM, MYOUNG, GOO, LEE, HEE, SOON, LEE, KWANG, KYUN, OK, KWANG, DAE
Application year: 1996
Main classification: C07D491
IPC codes: A61K31, A61P35, C07D491