Title of Invention

NEW CAMPTOTHECIN DERIVATIVES ,PREPARATIONS THEREOF AND ANTITUMOR AGENTS

Abstract

New camptothecin derivatives of the general formula (1), preparations thereof and antitumor agents comprising the same, wherein R1 represents a hydrogen atom or a C1 - C6 alkyl group, R2 represents a hydrogen or a C1-C6 alkoxy group, R3 represants a hydrogen or halogen atom or a C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, C2-C6 acyloxy or methoxyethoxymelhoxy group, R4 represents a hydrogen or halogen atom, and R5 represents a C1-C6 alkyl, C3-C6 unsaturated alkyl, alkylthioalkyl, alkoxyalkyl, pyridyl or substituted phenyl group, with the proviso that all of the R2, R3 and R4 substituents should not be a hydrogen atom.

Full Text

The present invention relates to new water-soluble camptothecin derlvatlves , a proceess of the preparation thereof and antitumor agents containing the same as an active ingredient. The present inventors have studled and provided a number of new camptothecin (hereinafter referred to CPT) derlvatives with excellent antitumor activities, and found that totally synthetic CPT derivatives carrying a lower alkyl group in 7-position on the B-ring and also hereto and/or alkyl group in 9-, lO- and 11-position on the A-ring among others showed strong antitumor activity (see JP, A, H1-l86892) . The present inventors also made extensive researches to solve a problem of making the derivatives water soluble in case of administration. Especially, CPT derivatives synthesized by subjecting 7-ethylcpt to open the E-lactone ring by the diamines followed by acylation of the hydroxy-methyl group showed excellent water solubility without decrease of antitumor activity contrasting to the known E-ring opened water soluble OPT derivatives (JP,A,H1-131179), - 2 - In addition to further research to obtain other new CPT derivsitives with more excellent antitumor activity and with useful water solubllity in ease of administration, there is also a great demand in the art for creeting new CPT derivatives which solve both toxicity and usage problems. Accordingly, an object of this invention is to provide water soluble new CPT derivatives poaoesaing excellent antitumor activity. It is another object to provide new CPT derivatives which solve both toxicity and usage problem. In order to achieve those objects we provided a process for the preipairation of new water-soluble CPT derivatives prepared from CPT derivetivea carrying a lower aikyl group in 7-position and also herero and/or alkyl group in- 9-, 10- and 11-posltion on the A-ring by subjecting them to open the E-lactone ring by the diamines followed by acyletlon of the hydroxymethyl group. As a result of our extensive safety and galenical researches made for developing new water soluble CPT derivatives, it has been realised to achieve the above mentioned objects, and to provide excellent new CPT derivatives use for antitumor activity. - 3 - In accordance wich the present, invention, there are provided new CPT derivatives represented by the general formula: wherein R1 represents a hydrogen aLom or a C1-C6 alkyl group, R2 represents a hydrogen or a C1-C6 alkoxy group, R3 represents a hydrogen or halogen atom or a C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, C2-C6 acyloxy or methoxyethoxymethoxy group, R4 represents a hydrogen or halogen atom, and R5 represents a C1-C6 alkyl, C3-C6 unsaturated alkyl, alkylthioalkyl, a]koxyalkyl, pyridyl or substituted phenyl group, with the proviso that all of the R2, R3 and R4 substituents should not be a hydrogen atom, and a process for preparing new camptothecin derivatives. In further accordance with the the present invention, there are provided antitumor agents s containing now CPT derivatives of general formula (1) as an active ingredient. The new CPT derivatives of the invention are prepared from CPT derivatives carrying a hydrogen atom or a lower alkyl group in 7-position and also hetoro and/or alkyl group in , 10 and 11-position by N, N-dimavthy1ethyl cnodiamine without 4- -solvent follov;ed by acylation of 17-hydroxyTnethyl group with appropriate acylating agents. The starting CPT derivatives carrying a hydrogen atom or a lower alkyl group in 7-position and also hetero and/or .alkyl group in 9-, lO-and 11-position are the known CPT derivatives prepared from a natural substances (9-methoxyCPT, 10-hydroxyCPT, lO-methoxyCPT, 11-hydroxyCPT, 11-methoxyCPT etc.) or are obtained semisynthetically or synthetically by the known procedures (see JP, A, S58-39684; JP, A, S58-134095; JP, A, S59-51287; JP, A, S59-51289; JP, A, Hl-279891; JP,..A, Hl-185892; JP, A, H4-503505; JP, A, H5-502017; WO-91/04260; WO-92/11263; USP, 5122606 and others) . Although a reaction condition disclosed by in JP, A, Hl-13117 for the E-ring opening by N,N-dimethylethylenediamine followed by acylation of 17-hydroxy 1 group with appropriate acylating agents can be applied, it has been found that aimed compounds were not necessarily prepared in satisfactory yields according to this procedure. We have examined reaction conditions of this procedure and found that in the first step of the E-lactone ring opening reaction by N,N-dimethylethylenediamine, using an excess amount of N,N-dimethylethylenediamine alone without solvent gave the E-ring opened intermediate followed by acylation of 17-hydroxyl group with appropriate acylating agents to afford the aimed compounds in very good yields . As the acylating agent used for the acylation chere is no specific agent, but corresponding acid anhydrides, acid halogenides, for example, acid chlorides, acid bromides and other equivalent acylating agents can be used. A reaction - 5 - mixture of a corresponding carboxylic acid treated with a condensation agent, for example, dicyclohexylcarbodiimide can be also used for the said acylation. Illustrative of the corresponding carboxylic acids used as said acylating agent are, for example, saturated aliphatic acids with 2-2 0 carbon atoms, unsaturated aliphatic acids with 3-2 0 carbon atoms, aliphatic acids with a cycloalkyl group, or aliphatic acids with, for example, a halogen atom or alkylthio, amino, acylamino, hydroxyl, alkoxy, or alkoxycarbonyl group, aromatic acids wi^th 6-20 carbon atoms or aromatic acids with, for example, a halogen atom or hydroxyl, alkoxy or lower alkyl group, heteroaromatic acids or amino acids. Examples of the acylating agent include acecyl chloride, benzoyl chloride, propionyl chloride, butyryl chloride, methoxybenzoyl chloride, fluorobenzcyl chloride, brcmobenzoyl chloride, chlorobenzoyl chloride, nitrobenzoyl chloride, trifluoromethylbenzoyl chloride, naphthoyl chloride, cyclopropanecarbonyl chloride, thenoyl chloride,' crotonyl chloride, cinnamoyl chloride, phenylacetyl chloride, phenylbenzoyl chloride, cyclohexanecarbonyl chloride, stearoyl chloride, oleoyl chloride, metboxycarbonylbezoyl chloride, ethyl succinyl chloride, linoleyl chloride, chlorobutyryl chloride, ethylbenzoyl chloride, methylthiopropionyl chloride, pivaloyl chloride, nicotinoyl chloride, isonicotinoyl chloride and picolinoyl chloride. In the said acylation N,N-dimethylaminopyridine or the like can be present in the reaction as a catalyst. In adding, to keep carefully an anhydrous condition not only in the ring-opening process but in the acylation process - 6- and further, for example, in the pulverization, the purification and the crystalization process can increase a yield of an aimed compound. The new CPT derivatives of this invention show excellent water solubility by being converted to acid addition salts thereof with proper acids such as hydrochloric acid. The compounds of this invention show excellent results in safety and in antitumor activity and therefore can be provided as nev/ antitumor agents. The present invention will now be illustrated in more detail by way of examples. [Examples] [Examples of synthesis] Example 1 Preparation of ring-opened compounds (B1-B13} As a starting material CPT derivatives (A1-A12, for each substituent refer Table 1) with each substituent in 7-position and in 9-, 10- and 11-position on the A-ring were prepared according to the above mentioned literature. 9-Methoxy CPT(A13} separated from natural substances was employed in this exairiple. And for the compounds carrying hydroxyl group the A-ring, the compound (A10) 0-methoxyethoxymethylated by the usual method was used. To 3.0 g, for example, of the starting CPT derivative (A1-A13) v;as added excess anhydrous W, N-dimethylethylenediamine (5-l00 eq. for example 15 ml) . The reaction mixture was stirred under nitrogen at 50°C for 1.5 hr. After reacted the reaction mixture Was evaporated to dryness under reduced pressure. The residue was dissolved in dry methylene chloride (for example 15 ml), and the solution was poured into large - 7 - amount of dry n-hexane (for example 500 ml). The precipitated crystals were filtered, washed v;ith dry n-hexane and dried to give the hydroxyamide (B1-B13, E-lactone-ring opened compounds) in an almost quantitative yield. The yields and each spectral data are shown in the following Table 2. Example -2 Acylation of the 17-hydroxy1 group -To a solution of the above obtained hydroxyamide (for example 1.0 g) in dry methylene chloride (for example 20 ml) was added dropwise an acylating agent (i.2 eq.)in presence of dimethylaminopyridine (DMPA, for example 100 mg) under ice cooling. The reaction mixture was stirred at room temperature overnight and washed with 7% aqueous sodium hydrogen bicarbonate and saturated aqueous sodium chloride solution. The methylene chloride layer was dried over anhydrous scdium sulfate, followed by removal of insoluble materials, and evaporated to dryness under reduced pressure. The residue was subjected to silica gel column chromatography (10% MeOH-CHCl3) and crystallized from chioroform-n-hexane to give the 17-0-acyl-21-N, N-dimethylaminoethylamide derivative (C1-C-c48) related to the invention. As for an 0-methoxyethoxymethyl derivative a solution of the compound in 10% trifluoroacetic acid-methylene chloride was stirred overnight. After stirring triethylamine (eq. mol) was added dropwise under ice cooling to the reaction mixture. The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in methylene chloride, washed with 7% aqueous sodium hydrogen bicarbonate and saturated aqueous sodium chloride solution . The organic layer - 8 - was dried over anhydrous sodium sulfate, followed by removal of insoluble materials, and evaporated to dryness under reduced pressure. The residue was subj ected to silica gel column chromatography (10% MeOH-CHCl3) and crystallized from acetone-CHCl3 to give the 17-0-acyl-21-N,N- dimethylaininoethylamide derivative (C28, C30, C32, C34, C36, C38) related to the invention. The yields of the synthesized derivatives and their spectral data are shown in Table 1 and Table 3. The water solubility data of;- the obtained new cainptothecin derivatives related to the invention are shovm in Table 4 . [Antitumor Effect] For the obtained new camptotheein derivatives of the invention, their results of antitumor activity, toxicology test,- usage-dose, and galenical preparation are described below,. [Antitumor Activity] It is well accepted that antitumor effect for a rodent leads- to a reliable result for antitumor effect in a warmblooded animal. The present inventors investigated antitumor effect for mice as a model. [Antitumor activity for L1210] 5x105 cells of mouse leukemia L1210 were transplanted intraperitoneally to a group of six female CDFl mice (7 v;eeks old, body weight 17-19 g) . The test compound was administered intraperitoneally on day 1, 5 and 3 and i cs life prolonging effect was observed. - 9 - In a case of the administration of a test compound as an acid addition salt the test compound was dissolved in water. The total administration amount was 1.56 mg/kg-400 mg/kg. The antitlumor activity was expressed by the value (T/C%) wherein T denotes the mean survival days in the drug administered group and C denotes those in the non administered group. In case of equal to or more than 125% the drug is considered to be effective. A therapeutic index was calculated by examining the least effective dose and the maximum tolerance dose. [Experimental Result] The antitumor experimental results of the compounds described in the previous example are shovjn in Table 5. As shown in Table 5, the new camptothecin derivatives in the present invention showed about 6 fold more favorable therapeutic index than camptothecin itself. At an optimum dose there was the case that 5 mice amoung 6 mice in the group survived. The results also show their effectiveness at lower doses, remarkable increase of antitumor activity and enlargement of a therapeutic margin. [Toxicity Experiment] Acute toxicity test was conducted by intrapericoneal administration using a group of twenty ICR male mice (4 weeks old, body weight around 20 g) . The results are shouTi* in Table 6. LD50 value was calculated by the Richfield-Willcokson method .from a lethal ratio observing_ the fate of mice during 1 week after administration of a test compound. From the above experimental results, - it is understood that the new camptothecin derivatives have better antitumor - 10 - activity and can be used as less toxic drugs than their mother compound, camptothecin, for a treatment of cancer. The antitumor agents of the present invention can be administered by a injection such as intravenous, intradermal and intramuscular injection, and by oral administration. Especially preferable examples are to administer the compounds" as their acid-addition salts appropriate as m.edicaments by intravenous or oral administration. In an intravenous administration, the dose of each compound above mentioned depends upon the aim of a therapeutic treatment, and is in range 5-4 00 mg/body per day, preferably 20-200 mg/body for adults. In an oral administration a range 50-2000 mg/body per day, preferably 100-1000 mg/body for adults. As a preparation method of the antitumor agents of the present invention, a usual method for each preparation can be selected according to a formulation. As a formulation suitable for absorption through stomach intestine, the antitumor agents of the invention may be prepared in, for example, tablet, powder, granule, capsule or soft capsule, and examiples of the oral liquid preparation include water or oil suspension, solution, syrup and elixir etc. A preparation for injection may be. stored in an ample or a large container. In these formulation an excipient such an antiseptic or dissolvent can be used. A formulation of a liquid preparation may be suspension, solution and emulsion with cily or aqueous vehicle, and may include an excipient such as emulsifier. Corresponding to a 11 - preparation of the antitumor agents of the invention, a content of the active drug is 0,. 1% or more, preferably 1-50%. The preparation examples of the antitumor agents of the invention are further illustrated in, though not limited by, the following examples. Preparation 1 Injection After dissolving the compound C7 (R1 = C2H5, R2=H, R3=CH3, R4=H, R5=C2H4SCH3) in 0 . IN HCl containing equivalent molar HCl, the solution was filtered, and lyophilized to give 5 0 mg of the HCI salt of the compound C7. The salt was sealed into an ample in a germfree condition and stored under cooling in the dark Preparation 2 Tablet The above ingredients were mixed and formed directly into tablets (160 mg/tablet) using a tablet machine. - 12 - Table 1 (Yeilds of each compounds) - 13 - Table 1 (continuous) 14 Table 1 (continuous) - 15 - Table 1 (Appendix) - l6- Tabie 2 (Spector Data of Hydroxy Amide Compounds) - 17 - Table 2 (continuous) - 18 - Table 2 (continuous 2) - 19 - Table 2 (continuous 3) - 20 - Table 2 {continuous h) - 21 - - 22 Table 3 (Spectral Data of 17-0-Acyl-21-amide Compounds) Table 3 -(continuous) 23 Table 3 (continuous 2) mp iD^-lGo-C, Pale yellou powder (from n-Hexane-clilorofona), - 24 - Tabie 3 (continuous 3) 2 5 — Table-3 '(continuous h) - 2 6 - Table 3 (continuous 5) - 27 Table 3 (continuous 6) 28 - Table 3 (ccntinuous 7) - 29 - Table 3 (continuous 8) 30 - ¦Table 3 (co^*.inuous 9) mp 107-]2rC, Yellow needles (from n-hexane-clUoroforai) - 31 - Table 3 (continuous 10) - 32 - Table 3 (continuous H) - 33 - Table 3 {continuous 12) 34 - Table 3 (continuous 13) - 35 Table 3 (continuous 14) mp 102-]05°C. Veiiow prisms(from n-Hexane-chloroform) CsTHiaN.OflF, MS [M+H]^ =69], IRt/'-'^'K^^(cm-') : 1710, 1650, 1620, 1600, 1505. 'H-NMR ((5ppm) in CDClj : l.lJ|(3H, t, J=7Hz), 1.27{3H, t, J^SHZ), 2.16(6H, -s), 2.22-2.i|2(3H, m), 2.il9-2.62{lH, m). 2.82-3.18{3H. m), 3.18-3.^2C1H, m), 3.39(3H, s), 3.50-3.6^(2H. m], 3.8073.93(2H, m),^J.gJi(lH, d, J=19Hz), 5.08(1H, d. J--19Hz),.5.35(lH, d, J=7Hz), 5.40(1H. d, J=7Hz), 5.55-5.77(lH, br), 5.78( 2H, s), 7.01(2H, dd. J=g, 9Hz), 7.22{1H, d, J=3Hz}. 7.27{1H. s), 7.35(1H, dd, J-3, 9Hz), 7.48(lH,br-t. J=6Hz), 7.50{1H, s), 7.90(1H, d, 'J=9Hz), 8.02(2H, dd. J=6, 9Hz). mp 23l7232°C. Colorless powder{from n-Hexane-chlorofonn) [a]^V,)=-6.5(CH30H, c=0.2), C33H35N.O6F, MS [M+i]]^ =603, IRy^^VKD.Ccm"') : 1710, I6i45. 1620, 1595. 1520. 'H-NMH {(5pptn) in DM.S0-d6 : 0.gi(3H. t, J=7Hz), 1.30(3H. t, J=8Hz), 2.11- 2.30(2H, m), 2.53(6H, s), 2.&2-2.90(2H. m), 2.99-3.^0(nH, m). 5.29(2H. s), 5.57(1H, d, J^llHz), 5.62(1H, d. J=llHz}, 6.36(1H. s), 7.33{1H, dd, J=9, 9Hz}, 7.3S-7-48(3H. m). 7.97(211, dd, J=6, 9Hz), 8.0i|(lH, d, J-9Hz)_, 8.9JJ-10.27(1H, br), 10.3^4{1H, s). mp i05-109°C, Veiiow prisms(from n-Hexane-chloroform) [a]'VD=+&2.0{CHCi3, c=0.2), C37H^3N^0aF, MS [H^H]^ =691, IRl.'"*VKBr(cm"') : 1720, 1650, 1620. 1590, 1510. 'H-NMR (oppm) in CDCI3 : 1.1M3H, t, J=7Hz-}. 1.27(311. t, J-8Hz), 2.16(6H. - 36 - - 37 - Tabie 3 (continuous 16) 38 - Table 3 (continuous 17) • - 39 Table 3 (continuous l8) Colorless powderifrom n-Hexane-chloroforin) CnHaJUOt, MS [H^H]^ =523. Ct 6 Yellow fXjwder(from n-Hexane-chloroform) CjoHssN^Ce. MS [M+H]^ =551, C..7 '" Yellow powder(from n-Hexane-chloroform) C31H.0Ni.O7, MS [M^H]* =581, i Ciie mp 179-l82°C, Colorless powder(from n-Hexane-chloroform} C3sH3,rUOTF. MS [M+H]* =645, - 4 0 - Table 4 (Water solubility) Compd. No. solubility(mg/ml) Compd. No. solubility(mg/ml) - 41 - Table 5 (Antitumor activity of camptothecin derivatives; - 42 - Table 5 (continuous) 43 - Table 6 (Acute Toxicity of camptotecin derivatives) 44 - Compd. No. LD50 vaiue{mg/kg) Compd. No. LD50 value(nig/kg) We Claim: 1. New camptothecin derivatives of the general formula (1) wherein R1 represents a hydrogen atom or a C1 - C6 alkyl group, R2 represents a hydrogen or a C1-C6 alcoxy group, R3 represents a hydrogen or halogen atom or a C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, C2-C6 acyloxy or methoxyethoxymethoxy group, R4 represents a hydrogen or halogen atom, and R5 represents a C1-C6 alkyl, C3-C6 unsaturated alkyl, alkyhhioalkyl, alkoxyalkyl, pyridyl or substituted phenyl group, with the proviso that all of the R2, R3 and R4 substituents should not be a hydrogen atom. 45 2. A process for the preparation of new camptothecin derivatives of the general fonnula (1) as claimed in claim 1, wherein camptothecin doivatives of the general formula (2) wherein R1, R2, R3 and R4 have the same meanings as given above, are subjected to the reaction with N, N-dimethylethylenediamine without solvent to open the E-lactone ring followed by the acylation of 17 -hydroxyl group with corresponding acylating agents. 3. Camptothecin derivatives as claimed in claim 1 for use in an anti tumor agent as an active ingredient Dated this 14th day of August 1995 46 New camptothecin derivatives of the general formula (1), preparations thereof and antitumor agents comprising the same, wherein R1 represents a hydrogen atom or a C1 - C6 alkyl group, R2 represents a hydrogen or a C1-C6 alkoxy group, R3 represants a hydrogen or halogen atom or a C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, C2-C6 acyloxy or methoxyethoxymelhoxy group, R4 represents a hydrogen or halogen atom, and R5 represents a C1-C6 alkyl, C3-C6 unsaturated alkyl, alkylthioalkyl, alkoxyalkyl, pyridyl or substituted phenyl group, with the proviso that all of the R2, R3 and R4 substituents should not be a hydrogen atom.

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00948-cal-1995 abstract.pdf

00948-cal-1995 claims.pdf

00948-cal-1995 correspondence.pdf

00948-cal-1995 description(complete).pdf

00948-cal-1995 form-1.pdf

00948-cal-1995 form-18.pdf

00948-cal-1995 form-2.pdf

00948-cal-1995 form-26.pdf

00948-cal-1995 form-3.pdf

00948-cal-1995 letters patent.pdf

00948-cal-1995 reply f.e.r.pdf