SPERGUALIN-RELATED NITRILE COMPOUNDS CONTAINING A PHENYLENE GROUP AND A PROCESS FOR PRODUCING THE SAME

Abstract: The present application relates to novel Spergualin-re­ lated nitrile compounds containing a phenylene group and salts thereof, which are useful as starting materials for the synthesis of Spergualin-related compounds containing a phenylene group, and to a process for the preparation of such nitrile compounds.

Description: Specification Spergualin-related nitrile compounds containing a phenylene group and a process for producing the same. The present invention relates to novel Spergualin-related nitrile compounds containing a phenylene group and salts thereof which are useful as precursors for the synthesis of Spergualin-related compounds having a phenylene group, as well as to a process for producing such nitrile compounds. Spergualin is a compound which has been isolated from the filtrate of a culture of a Spergualin-producing microorganism of the genus Bacillus. Spergualin has the following structure: 16 14 12 10 8 19 15 13 11 9 7 6 5 4 3 2 1 H2NCNH (CH2 ) 4CHCH2CONHCHC0NHCH2CH2CH2CH2NHCH2CH2CH2NH2 NH OH OH Spergualin has the effect of inhibiting the growth of both Gram-positive and Gram-negative microorganisms. It also exhibits appreciable therapeutic and life-prolonging effects in healing experiments with mouse leukemia, L-1210, mouse leukemia FL-4, Ehrlich cancer and sarcoma 180 (5-180) and has a potential for use as an anti-tumor agent (see Japanese Patent Public Disclosure No. 48957/1982). It has been reported that Spergualin can also be obtained by synthesis techniques tsee The Journal of Antibiotics, 34, 1625 (1981)1. Spergualin having a hemiacetal structure in the molecule is instable in an aqueous solution. Various studies have therefore been made with a view to obtaining Spergualin-related compounds which are stable in aqueous solutions and exhibit high activity. It was later found that Spergualin-related compounds having a phenylene group which are represented by the formula VI: EMI2.1 where R1 is a hydrogen atom or a hydroxylower alkyl group; and n is an integer of 3-5 are stable in aqueous solutions and exhibit high activity (see European Pat. Appl. Publ. No. 0 153 720) Conventionally, these Spergualin-related compounds having a phenylene group are synthesized from 1,5-diprotected 115,l0-triazadecanes of the general formula VII: EMI2.2 where X1 and X2 which are the same or different represent an amino protecting group. However, the compounds of the formula VII are not suitable for use in commercial operations because their synthesis requires the use of several amino protecting groups and involves several reaction steps. Mass production of Spergualin-related compounds having a phenylene group which are represented by the formula VI could be carried out efficiently if a method for selectively acylating the primary amine at N10 of Spermidine (1,5,10-triazadecane) present in formula VI is developed. One objet, therefore, of the present invention is to provide a Spergualin-related nitrile compound having a phenylene group which is a useful precursor for the synthesis on an industrial scale of a Spergualin-related compound having a phenylene group. Another object of the present invention is to provide a process for producing said nitrile compound. Accordingly, the present invention relates to Spergualin-related nitrile compounds containing a phenylene group which is represented by the general formula I: EMI3.1 where R is a hydrogen atom, a hydroxylower alkyl group or a lower alkoxy group, and n is an integer of 3-5 and a salt thereof. The present invention relates further to a process for producing a Spergualin-related nitrile compound having a phenylene group which is represented by the formula I or a salt thereof, said process comprising condensing a compound of the general formula II: EMI3.2 where R and n are the same as defined for the formula I, or a reactive derivative thereof, with N-2-cyanothylbutane-1,4-diamine of the formula III: H2N(CH2)4NH(CH2)2CN (III). In an alternative process a compound of the formula I where R is a hydrogen atom or a hydroxylower alkyl group and n is an integer of 3-5 or a salt thereof, can be prepared by condensing a compound of the general formula IV: EMI4.1 where n is as defined above, or a reactive derivative thereof with a compound of the general formula V: EMI4.2 where R is a hydrogen atom or a lower hydroxyalkyl group. The N-2-cyanoethylbutane-1,4-diamine of the general formula III is readily obtained at low cost by condensing 1,4-diaminebutane and acrylonitrile tsee Journal of Medicinal Chemistry, 2, 710 (1964)j. The nitrile compound of the general formula I can be synthesized from the diamine of formula III by selectively acylating the primary amine and the present inventors have accomplished a route of synthesizing a Spergualin-related compound having a phenlyene group by using said nitrile compound as a precursor. By reducing the -CN in the nitrile compound of formula I to -CH2NH2, a compound represented by the general formula VIII: EMI4.3 where R and n are the same as defined for the formula I, can be obtained. Any known reaction may be employed for reducing the -CN to -CH2NH2 without affecting the acid amide in formula I. Preferable methods include the catalytic reduction using a Raney nickel catalyst and the reduction with sodium borohydride in the presence of a transition metal salt such as cobalt chloride or nickel chloride [see Tetrahedron Letters, 52, 4555 (1969)J. The tcr "hydroxyloweralkyl" as aforementioned denotes a straight or branched alkyl group having from 1 to 6, preferably 1 to 4 carbon atoms, which is substituted, preferably terminally, with a hydroxy group. The term lower alkoxy" as aforementioned denotes a straight or branched alkoxy group having from 1 to 6, preferably from 1 to 4 carbon atoms. If R in the general formula I is a substituent other than a hydrogen atom, the steric configuration of the compounds may be any of the S-, R- and SR-forms. Typical examples of the compounds of the general formula I are listed in Table 1 together with their structures and names. EMI6.1 Table 1 EMI6.2 <tb> Compound <tb> No. <SEP> n <SEP> R <SEP> Name <tb> <SEP> 1 <SEP> 3 <SEP> H <SEP> 8-(N-4-(4-guanidinophenyl)butanoyl]- <SEP> <tb> <SEP> glycyl)-l-cyano-3, <SEP> 8-diazaoctane <SEP> <tb> <SEP> 2 <SEP> 5 <SEP> H <SEP> 8-IN-j6-(4-guanidinophenyl)hexanoyl]- <SEP> <tb> <SEP> glycyl)-l-cyano-3,8-diazaoctane <tb> <SEP> 3 <SEP> 3 <SEP> CH2OH <SEP> 8-iN-[4-(4-guanidinophenyl)butanoyl]- <SEP> <tb> <SEP> L-seryl)-l-cyano-3 <SEP> , <SEP> 8-diazaoctane <SEP> <tb> <SEP> 4 <SEP> 5 <SEP> CH20H <SEP> 8-(N-j6-(4-guanidinophenylhexanoyl]- <SEP> <tb> <SEP> L-seryl)-l-cyano-3,8-diazaoctane <tb> <SEP> 5 <SEP> 3 <SEP> OCH3 <SEP> 8-{N-[4-(4-guanidinophenyl)butanoyl]- <SEP> <tb> <SEP> a-methoxyglycyl)-l-cyano-3,8- <SEP> <tb> <SEP> diazaoctane. <tb> Table 2 EMI7.1 Compound <SEP> Zn <SEP> absorption <SEP> Proton <SEP> NMR <SEP> spectra <SEP> . <SEP> 25 <tb> No. <SEP> | <SEP> spectra, <SEP> cm-1 <SEP> | <SEP> 6 <SEP> value <SEP> | <SEP> @ <tb> <SEP> 3450, <SEP> 2950, <SEP> 2870, <SEP> 1.4 <SEP> s <SEP> 2.2 <SEP> (CH2 <SEP> x <SEP> 3), <tb> <SEP> 2270, <SEP> 1650, <SEP> 1630, <SEP> 2.32 <SEP> (CH2), <SEP> 2.69 <SEP> (CH2) <tb> <SEP> 1580, <SEP> 1560, <SEP> 15820, <SEP> 2.74 <SEP> (CH2), <tb> 1 <tb> <SEP> 1460, <SEP> 1420, <SEP> 1260, <SEP> 2.9 <SEP> # <SEP> 3.4 <SEP> (NCH <SEP> x <SEP> 3),. <tb> <SEP> 1160, <SEP> 1030, <SEP> 830, <SEP> 3.80 <SEP> (ch2), <SEP> 7.20 <SEP> (CH <SEP> x <SEP> 2), <tb> <SEP> 580 <SEP> 7.30 <SEP> (ch <SEP> x <SEP> 2) <tb> <SEP> 3400, <SEP> 2950, <SEP> 2860, <SEP> 1.3 <SEP> # <SEP> 2.0 <SEP> (Ch2 <SEP> x <SEP> 5 <SEP> ), <tb> <SEP> 2810, <SEP> 2275, <SEP> 1670, <SEP> 2.34 <SEP> (CH2), <SEP> 2.68 <SEP> (CH2), <tb> <SEP> 1650, <SEP> 1640, <SEP> 1580, <SEP> 3.08 <SEP> (CH2), <tb> <SEP> 2 <SEP> 1560, <SEP> 15250, <SEP> 1470, <SEP> 2.9 <SEP> # <SEP> 3,5 <SEP> (NCH2 <SEP> x <SEP> 3); ; <tb> <SEP> 1460, <SEP> 1420, <SEP> 1260, <SEP> 3.86 <SEP> (CH2), <SEP> <tb> <SEP> 1060, <SEP> 1030, <SEP> 340, <SEP> 7.1 <SEP> s <SEP> 7.4 <SEP> [CH <SEP> x <SEP> 4) <tb> <SEP> 670, <SEP> 630 <tb> <SEP> 3350, <SEP> 2950, <SEP> 2850, <SEP> | <SEP> 1.4 <SEP> # <SEP> 2.2 <SEP> (CH2 <SEP> x <SEP> 3), <tb> <SEP> 2770, <SEP> 2250, <SEP> 1650, <SEP> 2.39 <SEP> (CH2), <SEP> 2.73(CH2), <SEP> <tb> - <SEP> <tb> <SEP> 1630, <SEP> 1575, <SEP> 1540, <SEP> 3.00 <SEP> (CH2), <SEP> -13.0 <tb> 3 <tb> <SEP> 1505, <SEP> 1450, <SEP> 1400, <SEP> 2.9 <SEP> # <SEP> 3.6 <SEP> (NCH2 <SEP> x <SEP> 3) <SEP> (C <SEP> = <SEP> 1, <SEP> H2O) <tb> <SEP> 1250, <SEP> 1060, <SEP> 820, <SEP> 3.79 <SEP> (CH2), <SEP> 4.37 <SEP> (CH), <tb> <SEP> 560, <SEP> 520 <SEP> 7.20 <SEP> (CH <SEP> x <SEP> 2), <tb> <SEP> 7.30 <SEP> (CH <SEP> x <SEP> 2) <tb> <SEP> 3440, <SEP> 2955, <SEP> 2880, <SEP> 1.2 <SEP> # <SEP> 1.9 <SEP> (CH2 <SEP> x <SEP> %) <SEP> , <tb> <SEP> 2280, <SEP> 1650, <SEP> 1630, <SEP> 2.26 <SEP> (CH2), <SEP> 2.59 <SEP> (CH2), <tb> <SEP> 1580, <SEP> 1560, <SEP> 1520, <SEP> 2.9 <SEP> # <SEP> 3.4 <SEP> (NCH2 <SEP> x <SEP> 3), <SEP> - <SEP> 12.1 <tb> 4 <tb> <SEP> 1480, <SEP> 1460, <SEP> 1350, <SEP> 3.74 <SEP> (CH2), <SEP> 4.30 <SEP> (CH), <SEP> (C <SEP> = <SEP> 1, <SEP> H2O) <tb> <SEP> 1270, <SEP> 1150, <SEP> 1070, <SEP> 7.1 <SEP> # <SEP> 7.4 <SEP> (CH <SEP> x <SEP> 4), <tb> <SEP> 1040, <SEP> 990, <SEP> 960, <SEP> 2.97 <SEP> (CH2) <SEP> <tb> <SEP> 920, <SEP> 620 <tb> <SEP> 3400, <SEP> 2950, <SEP> 2860, <SEP> 1.5 <SEP> # <SEP> 2.2 <SEP> (CH2 <SEP> x <SEP> 3), <tb> <SEP> 2840, <SEP> 2275, <SEP> 1670, <SEP> 2.30 <SEP> (CH2), <SEP> 2.73 <SEP> (CH2), <tb> <SEP> 1610, <SEP> 1580, <SEP> 1540, <SEP> 2.9 <SEP> # <SEP> 3,5 <SEP> (NCH2 <SEP> x <SEP> 3), <tb> <SEP> 1520, <SEP> 1470, <SEP> 1460, <SEP> 3.35 <SEP> (OCH2), <SEP> 5.29 <SEP> (CH), <tb> <SEP> 1260, <SEP> 1200, <SEP> 1150, <SEP> 7.20 <SEP> (CH <SEP> x <SEP> 2), <tb> <SEP> 1100, <SEP> 10200 <SEP> 570 <SEP> 7.30 <SEP> (CH <SEP> x <SEP> 2) <tb> The compounds of the general formula I may form salts with acids. Salt-forming acids may be inorganic or organic. Although there is no particular limitation on the acids that may be used, hydrochloric acid is preferable because of the ease of its handling. The IR absorption spectra (measured as KBr tablet), proton NMR spectra (measured in d-MeOH using TMS as a reference material) and specific rotations of hydrochlorides of typical examples of the compound of formula I are shown in Table 2. The compounds of the general forumula II may be condensed with the compound of formula III by any of the routine methods employed in the formation of peptide bonds. Illustrative methods include the carbodiimide process using dicyclohexyl carbodiimide or l-ethyl-8-(3- dimethylaminopropyl)-carbodiimide; the azide process starting from hydrazide; the mixed acid anhydride process; the active ester process using substituted or unsubstituted phenyl esters or hydroxysuccinyl imide esters; the O-acylhydroxylamine derivative process; and the N-acyl compound process. Condensation solvents may be selected from those which are commonly employed in the formation of peptide bonds. Since the compounds of the general formula II are typically used as salts with acids, amides such as dimethylformamide and dimethyl acetamide are preferably used in consideration of their ability to dissolve such salts. When the N-2-cyanoethylbutane-1,4-diamine of the formula III is condensed with the compound of formula II, an acid, for example, hydrochloric acid may be added in an equimolar amount so that only the secondary amine in formula III is converted to a salt form, and this permits selective acylation of the primary amine. Preferential acylation of the primary amine can also be effected by using N-2 cyanoethylbutane-1,4-diamine in an excess amount (1.2 - 2 mole equivalent). The condensation reaction will proceed by using equimolar amounts of the compounds of the formulas II and III, but in order to attain high yields, the compound of formula III is preferably added in an excess amount. The starting compound of the formula II is novel and may be synthesized by the following procedures. A compound of the general formula IV: EMI10.1 (where n is an integer of 3 - 5) is condensed with a compound such as glycine or serine where a carboxyl group is protected by an appropriate protective group and, subsequently, the carboxyl-protecting group is eliminated by a routine method. The condensation reaction may be carried out by any of the aforementioned methods commonly employed in the formation of peptide bonds. A compound of the general formula II wherein R is a lower alkoxy group, or a compound of the general formula IIa: EMI10.2 where R1 is a lower alkoxy group; and n is an integer of 3 - 5 may be synthesized by the following procedures: the compound of formula IV is esterified and subsequently treated with ammonia; glyoxylic acid is attached to the resulting amide so as to form a compound represented by the formula IX; EMI11.1 where n is an integer of 3 - 5 ; a lower alkanol is reacted on the compound of IX in the presence of an acid catalyst so as to cause both O-alkylation and esterification; and subsequently, the ester is hydrolyzed with an alkali. The compound of formula IV may be condensed with the compound of formula V in the third aspect of the present invention in the same manner as described in connection with the condensation reaction employed in the second aspect of the present invention. Before condensation, the compound of formula V may be treated in such a way that only the secondary amine is converted to a salt form, and this permits selective acylation of the primary amine. Preferential acylation of the primary amine can also be effected by using the compound of formula V in an excess amount (1.2 - 2 mole equivalent). The starting compound of formula V is novel and may be synthesized by the following procedures: a compound such as glycine or serine wherein an amino group is protected by an appropriate protective group is condensed with the N-2 cyanoethyl-butane-l,4-diamine of formula III and, EMI11.2 subsequently, the amino protecting group is eliminated by a routine method. Before condensation, the N-2-cyanoethyl butane-i,4-diamine may be treted insuch a way that only the secondary amine is converted to a salt form, and this s permits selective acylation of the primary amine. Preferential acylation of the primary amine may also be effected by using the N-2-cyanoethylbutane-1,4-diamine in an excess amount (1.2 - 2 mole equivalent). Examples The following examples are given for the purpose of further illustrating the present invention but are in no sense to be taken to limit the invention. Example 1: 8-{N-[4-(4-guanidinophenyl)butanoyl]glycyl}-1- cyano-3,8-diazaoctane dihydrochloride A mixture of 14.73 g (57.2 mmol) of 4-(4guanidinophenyl)butyric acid hydrochloride and 7.90 g (68.6 mmol) of N-hydroxysuccinimide was dissolved in 75 mt of dimethylformamide. To the ice-cooled solution, 14.15 g (68.6 mmol) of dicyclohexyl carbodiimide in 75 mL of dimethylformamide was added dropwise. Thereafter, the mixture was stirred overnight at room temperature and the precipitating dicyclohexyl urea was filtered off. The filtrate was added dropwise to a solution of 12.81 g (64.6 mmol) of 8-(N-glycyl)-l-cyano-3,8-diazaoctane monohydrochloride in 75 mQ of dimethylformamide. The resulting mixture was stirred for 1 hour at room temperature. The solvent was distilled off and the solid residue was dissolved in 1,000 mt of water. The solution was loaded onto a column packed with 300 mt of CM-Sephadex C-25 (Na form) (Pharmacia Fine Chemicals). After washing with 1,000 mt of water, the column was subjected to elution by a gradient of water (1,500 m & and 0.5 M NaCQ (1,500 mt). The active fractions were collected and vacuum-evaporated to dryness. Methanol was added to the solid residue and the insoluble NaCt was filtered off. These procedures were repeated twice. The resulting filtrate was loaded onto a column packed with 1,000 mt of Sephadex LH-20 (Pharmacia Fine Chemicals) and eluted with methanol. The active fractions were collected and concentrated under vacuum to produce 21.78 g of a syrup of 8-lN-[4-(4-guanidinophenyl)- butanoyl]glycyl}-1-cyano-3,8-diazaoctane dihydrochloride in a yield of 80.2%. Example 2: 8-{N-[4-(4-guanidinophenyl)butanoyl]glycyl}-1 cyano-3,8-diazaoctane dihydrochloride A mixture of 25.24 g (80.2 mmol) of [4-(4- guanidinophenyl)butanoyl]glycine hydrochloride and 11.1 g (96.2 mmol) of N-hydroxysuccinimide was dissolved in 100 mt of dimethylformamide. To the ice-cooled solution, a solution of 19.9 g (96.2 mmol) of dicyclohexyl carbodiimide in 100 mt of dimethylformamide was added dropwise. Thereafter, the mixture was stirred overnight at room temperature and the precipitating dicyclohexyl urea was filtered off. The filtrate was added dropwise to a solution of 17.0 g (120.3 nol) of N-2-cyanoethylbutane-l,4-diamine in 80 mt of dimethylformamide, and the resulting mixture was stirred for 3 hours at room temperature. The solvent was distilled off and the solid residue was dissolved in 500 mQ of 0.5 M Nana. The resulting solution was loaded onto a column packed with 2,500 mQ of Diaion HP-200 (Mitsubishi Chemical Industries Limited) adjusted with 0.5 M NaCQ. The column was washed with 8,000 mi of 0.5 M NaCQ, 3,000 mQ of 0.25 M NaCQ and 10 mQ of water, and eluted with 2 x 104 mQ of water-methanol (9:1) and 2 x 104 mQ of water-methanol (8:2). The active fractions were collected and concentrated under vacuum to produce 25.74 g of a syrup of 8-{N-[4-(4guanidinophenyl)butanoyl]glycyl}-1-cyano-3,8-diazaoctane dihydrochloride in a yield of 67.7%. Example 3: 8-{N-[4-(4-guanidinophenyl)butanoyl]glycyl}-1 cyano-3,8-diazaoctane dihydrochloride A mixture of 109 mg (0.38 mmol) of 6-(4guanidinophenyl)caproic acid hydrochloride and 52.5 mg (0.46 mmol) of N-hydroxysuccinimide was dissolved in 1 mQ of dimethylformamide. To the ice-cooled solution, 94.9 mg (0.46 mmol) of dicyclohexyl carbodiimide in 1 mQ of dimethylformamide was added dropwise. Thereafter, the mixture was stirred for 8 hours. The stirred mixture was added dropwise to a solution of 24.7 mg (0.46 mmol) of 8-(N-glycyl)-l-cyano-3,8-diazaoctane dihydrochloride in 1 mQ of dimethylformamide after it was mixed with 0.142 m± (1.01 mmol) of triethylamine. The resulting mixture was stirred overnight at room temperature. The precipitating dicyclohexyl urea was filtered off and 20 mt of water was added to the filtrate. The solution was neutralized with 1 N HCt and diluted with water to make a volume of 300 mt. The diluted solution was loaded onto a column packed with 25 mt of CM-Sephadex C-25 (Na form), washed with 250 mt of water and eluted by a gradient of 250 mQ of water and 250 mt of 0.5 M NaCQ. The active fractions were collected and vacuum-evaporated to dryness and the solid residue was subjected to extraction with methanol. Methanol was distilled off the extract and the the solid residue was dissolved in 0.5 M NaCt. The solution was'loaded onto a column packed with 25 mt of Diaion SP 207 (Mitsubishi Chemical Industries Limited) ajdusted with 0.5 N NaCt. The column was washed with 150 mt of 0.5 M NaCt and eluted with 300 m± of water and 200 mt of methanol. The active fractions were collected and concentrated under vacuum to produce 69.4 mg of a syrup of 8-(N-[6-(4-guanidinophenyl) hexanoyl] glycyl)-l-cyano-3, 8-diazaoctane dihydrochloride in a yield of 36.3%. Example 4: 8-{N-[4-(4-guanidinophenyl)butanoyl]-L-seryl}-1 cyano-3,8-diazaoctane dihydrochloride A mixture of 107.5 g (417 nol) of 4-(4-guanidinophenyl)butyric acid hydrochloride and 57.5 g (500 nol) of N-hydroxysuccinimide was dissolved in 500 mt of dimethylformamide. To the ice-cooled solution, 103.2 g (500 mmol) of dicyclohexyl carbodiimide in 500 mt of dimethylformamide was added dropwise and the mixture was stirred for 5 hours at room temperature. The precipitating dicyclohexyl urea was filtered off and the filtrate was added dropwise to a solution of 150.6 g (500 mmol) of S-(N-L-seryl)-1-cyano-3,8- diazaoctane dihydrochloride in 400 mt of dimethylformamide after it was mixed with 140.5 mQ (1 mol) of triethylamine. The resulting mixture was stirred for overnight at room temperature. The solvent was distilled off and the resulting solid residue was dissolved in 5,000 mQ of water. The solution was loaded onto a column packed with 3,000 mQ of CM-Sephadex C-25 (Na form) and, after washing with water (5,000 mi), the column was eluted with 0.2 - 0.3 M NaCl (1,500 mt). The active fractions were collected and vacuumevaporated to dryness. Methanol was added to the solid residue and the insoluble NaCl was filtered off. These procedures were repeated twice. The resulting filtrate was loaded onto a column packed with 12 x 103 mt of Sephadex LH-20 and eluted with methanol. The active fractions were collected and concentrated under vacuum to produce 185.4 g of a syrup of 8-{N-[4-(4-guanidinophenyl)butanoyl]-L-seryl}- l-cyano-3,8-diazaoctane dihydrochloride in a yield of 88.2%. Example 5: 8-{N-[6- (4-guanidinophenyl)hexanoyl]-L-seryl}-l- cyano-3,8-diazaoctane dihydrochloride A mixture of 114 mg (0.40 mmol) of 6-(4-guanidinophenyl)caproic acid hydrochloride and 55.2 mg (0.48 mmol) of N-hydroxysuccinimide was dissolved in dimethylformamide (1 mt). To the ice-cooled solution, 123.8 mg (0.60 mmol) of dicyclohexyl carbodiimide in 1 mt of dimethylformamide was added dropwise and the mixture was stirred for 7 hours at room temperature. The stirred mixture was added dropwise to a solution of 182.6 mg (0.80 mmol) of 8-(N-L-seryl)-l-cyano3,8-diazaoctane in 1 mt of dimethylformamide and the resulting mixture was stirred for 2 hours at room temperature. The precipitating dicyclohexyl urea was filtered off and the filtrate was mixed with 20 mt of water. The mixture was neutralized with 1 N HC & and diluted with 300 ml of water to make a volume of 300 m±. The dilution was loaded onto a column packed with 25 ml of CM-Sephadex C-25 (Na form) and, after being washed with water (250 mi), the column was eluted by a gradient of water (250 ml) and 0.5 M NaCl (250 mt). The active fractions were collected and vacuum-evaporated to dryness and the solid residue was subjected to extraction with methanol. Methanol was distilled off the extract and the solid residue was dissolved in 0.5 M NaCt and the solution was loaded onto a column packed with 25 mt of Diaion SP 207 adjusted with 0.5 M NaCQ. After being washed with 150 mt of 0.5 M NaCt, the column was eluted with 150 mt of water and 150 mt of methanol. The active fractions were collected and concentrated under vacuum to produce 106.6 mg of a syrup of 8-{N-[6-(4-guanidinophenyl)hexanoyl]-L-eryl}-l-cytno-3,8- diazaoctane dihydrochloride in a yield of 51.5%. Example 6: 8-{N-[4-(4-guanidinophenyl)butanoyl]-&alpha;-methoxy- glycyl) -l-cyano-3, 8-diazaoctane dihydrochloride A mixture of 11.03 g (32.0 mmol) of 4-(4-guanidinophenyl)butanoyl-a-methoxyglycine hydrochloride and 5.50 g (48.0 mmol) of N-hydroxysuccinimide was dissolved in 50 m± of dimethylformamide. To the solution, 10.09 g (48 mmol) of dicyclohexyl carbodiimide in 50 mQ of dimethylformamide was added dropwise and the mixture was stirred overnight at room temperature. The precipitating dicyclohexyl urea was distilled off and the filtrate was added dropwise to a solution of 6.80 g (48.0 mmol) of N-2-cyanoethylbutane-1,4diamine in 50 mQ of dimethylformamide. The mixtue was stirred overnight at room temperature. The solvent was concentrated under vacuum and the resulting solid residue was dissolved in 500 mt of water. The solution was loaded onto a column packed with 500 mQ of CM-Sephadex C-25 (Na form) and, after being washed with water (2,000 mR), the column was eluted by a gradient of 1,500 m± of water and 1,500 mQ of 1 M NaCQ. The active fractions were collected and concentrated under vacuum. The solid residue was subjected to extraction with methanol and the solid residue was dissolved in 20 mQ of water. The solution was loaded onto a column packed with 500 mQ of Diaion SP 207 and, after washing with water (1,500 mt), the column was eluted with water-methanol (9:1). The active fractions were collected and concentrated under vacuum to provide 8.39 g of a syrup of 8-{N-[4- (4-guanidinophenyl) butanoyl] -a-methoxyglycyl)-l- cyano-3,8-diazaoctane dihydrochloride in a yield of 52.0% Reference Example 1: Reduction of 8-{N-[4-(4 guanidinophenyl)butanoyl]glycyl)-l cyano-3,8-diazaoctane dihydrochloride 21.71 g (45.8 mmol) of 8-(N-[4-(4-guanidinophenyl) butanoyl] -glycyl)-l-cyano-3, 8-diazaoctane dihydrochloride was dissolved in 70 mi of methanol and the solution was saturated with ammonia gas. After addition of Raney nickel (30 g), the mixture was stirred for 6 hours in a hydrogen stream (15 kg/cm2) at 450C. The catalyst was filtered off and the solvent was distilled off. The resulting solid residue was dissolved in 500 mt of water. The solution was neutralized with 2 N HCt and diluted with 1,000 m± of water. The dilution was loaded onto a column packed with 300 m± of CM-Sephadex C-25 (Na form) and, after washing with water (1,000 ma,, the column was eluted with 0.2 - 0.3 M NaCQ. The active fractions were collected and vacuum-evaporated to dryness. Methanol was added to the solid residue and the insoluble NaCt was filtered off. These procedures were repeated twice and the resulting filtrate was loaded onto a column packed with 1,000 mt of Sephadex LH-20 and elution was conducted with methanol. The active fractions were collected and concentrated under vacuum. The resulting solid residue (13.55 g) was crystallized from methanolethanol (1:4), thereby providing 10.81 g of 10-(N-[4-(4guanidinophenyl)butanoyl]-glycyl}-1,5,10-triazadecane trihydrochloride. Yield, 45.88; m.p., 158 - 1600C. NMR (CH30H-d4) : 6 = 1.4 X 2.5 (CH2 x 5), 2.66 (CH2), 2.9 X 3.4 (NCH2 x 4), 3.78 (CH2), 7.0 X 7.4 (CH x 4) IR (KBr) : v (cm 1) = 3310, 2950, 1660, 1580, 1550, 1515, 1460, 1410, 1255, 1160, 1110, 1030, 830 Reference Example 2: Reduction of 8-{N-[6-(4-guanidino- phenyl)hexanoyl]glycyl)-l-cyano-3,8 diazaoctane dihydrochloride A mixture of 69.4 mg (0.138 mmol) of 8-{N-[6-(4 guanidinophenyl)hexanoyl3-glycyl}-1-cyano-3,8-diazaOctane dihydrochloride and 49.3 mg (0.207 mmol) of cobalt chloride (CoCl2.6H2O) was dissolved in 3 mQ of methanol. To the ice-cooled solution, 104.4 mg (2.76 mmol) of sodium borohydride was slowly added. The mixture was stirred for 1 hour at room temperature and, after addition of water (10 mt), the solution was rendered acidic with 2 N HCl to dissolve the black precipitate. Thereafter, the pH of the solution was adjusted to 7.0 with 2 N NaOH. The solvent was distilled off and the resulting solid residue was subjected to extraction with methanol. Methanol was distilled off the extract and the solid residue was dissolved in 300 mQ of water. The resulting solution was loaded onto a column packed with 25 mt of CM-Sephadex C-25 (Na form) and, after washing with water (300 m±), the column was eluted by a gradient of water (250 mll) and 1 M NaCQ (250 ml). The active fractions were collected and vacuum-evaporated to dryness. After addition of methanol, the insoluble NaCt was filtered off. These procedures were repeated twice and the resulting filtrate was loaded onto a column packed with 150 mi of Sephadex LH-20 and elutd with methanol. The active fractions were collected and concentrated under vacuum. The resulting solid residue (32.1 mg) was crystallized from ethanol to provide 28.4 mg of 10-{N-[6 (4-guanidinophenyl)butanoyl]glycyl}-1,5,10-triazadecane trihydrochloride. Yield, 37.8%; m.p., 148 - 1510C. NMR (CH3OH-d4) : 6 = 1.3 X 2.0 (CH2 x 5), 2.0 N 2.4 (CH2 x 2), 2.62 (CH2), 2.9 N 3.3 (NCH2 x 4), 3.79 (CH2), 7.0 N 7.4 (CH x 4) IR (KBr) : v (cm-1) ¯ 3430, 2950, 2870, 1680, 1660, 1580, 1560, 1520, 1470, 1420, 1260 Reference Example 3: Reduction of 8-(N-[4-(4-guanidino phenyl)butanoyl]-L-seryl-1-cyano 3,8-diazaoctane dihydrochloride 185.4 g (368 mmol) of 8-{N-[4-(4-guanidinophenyl) butanoyl] -L-seryl) -l-cyano-3, 8-diazaoctane dihydrochloride was dissolved in 400 mt of methanol and the solution was saturated with ammonia gas. After addition of Raney nickel (70 g), the saturated solution was stirred overnight in a hydrogen stream (12 kg/cmê) at 40 C. The catalyst was filtered off and the solvent was distilled off the filtrate. The resulting solid residue was dissolved in 500 mQ of water and the solution was adjusted to a pH of 7.0 with 2 N HCt. The so adjusted solution was diluted with 5,000 ml of water and loaded onto a column packed with 2.5 x 10 mQ of CM-Sephadex C-25 (Na form). After washing with water 5,000 mQ), the column was eluted with 0.3 - 0.4 M NaCQ. The active fractions were collected to make a volume of 35 x 103 mQ and diluted four-fold with water. The diluted solution was loaded onto a column packed with 1,500 mQ of CM-Sephadex C-25 (Na form) and eluted with 0.8 M NaCQ. The active fractions were collected to make a volume of 5,000 mi and vacuum-evaporated to dryness. The resulting solid residue was mixed with methanol and the insoluble NaCl was filtered off. These procedures were repeated twice and the resulting filtrate was loaded onto a column packed with 12 x 103 mt of Sephadex LH-20 and eluted with methanol. The active fractions were collected and concentrated under vacuum. The resulting solid residue was crystallized from methanol-ethanol (1:4) to provide 115.44 g of 10-{N-[4-(4 guanidinophenyl)butanoyl]-L-seryl}-1,5,10-triazadecane trihydrochloride. Yield, 57.6%; m.p., 145 - 1460C. [cr]25 - 13.3 (C = 1, H2O) NMR (CH3OH-d4) : 6 = 1.4 # 2.5 (CH2 x 5), 2.67 (CH2), 2.9 N 3.4 (NCH2 x 4), 3.75 (CH2), 4.33 (CH), 7.0 X 7.5 (CH x 4) IR (KBr) : v (cm 1) - 3380, 2925, 1630, 1570, 1500, 1450, 1390, 1250, 1150, 1060 Reference Example 4: Reduction of 8-(N-[6-(4-guanidino- phenyl)hexanoyl]-l-seryl)-l-cyano- 3,8-diazaoctane dihydrochloride A mixture of 106.6 mg (0.206 mmol) of 8-(N-[6-(4- guanidinophenyl)hexanoyl]-L-seryl-1-cyano-3,8-diazaoctane dihydrochloride and 73.5 mg (0.309 mmol) of cobalt chloride (CoCQ2h6H2O) was dissolved in methanol (3 mt). To the ice-cooled solution, 155.9 mg (4.12 mmol) of sodium borohydride was slowly added. After l-hr stirring at room temperature, 10 mQ of water was added to the mixture, which then was rendered acidic with 2 N HC & so as to dissolve the black precipitate. Thereafter, the solution was adjusted to a pH of 7.0 with 2 N NaOH and the solvent was distilled off. The resulting solid residue was subjected to extraction with methanol. Methanol was distilled off the extract and the solid residue was dissolved in 300 mQ of water. The solution was loaded onto a column packed with 25 m± of CM-Sephadex C-25 (Na form) and, after washing with water (300 mt), the column was eluted by a gradient of water (300 mQ) and 1 M NaCl (300 mt). The active fractions were collected and vacuum-evaporated to dryness. Methanol was added to the residue and the insoluble NaCQ was filtered off. These procedures were repeated twice and the resulting filtrate was loaded onto a column packed with 150 mt of Sephadex LH-20 and eluted with methanol. The active fractions were collected and concentrated under vacuum to yield 42.8 mg of the solid residue. The residue was crystallized from ethanol to provide 40.35 mg of 10-(N-[6 (4-guanidinophenyl)hexanoyl]-L-seryl}-1,5,10-triazadecane trihydrochloride. Yield, 35.18; m.p., 156 - 1600C. 25 25 - 12.3 (C = 1, H20) D NMR (CH3OH-d4) : 6 r 1.3 # 2.0 (CH2 x 5), 2.0 X 2.5 (CH2 x 2), 2.70 (CH2), 2.9 X 3.4 (NCH2 x 4), 3.78 (CH2), 4.36 (CH), 7.1 s7.4 (CH x 4) -l IR (KBr) : v (cm 1) 3460, 2960, 2890, 2710, 1690, 1670, 1595, 1570, 1540, 1470, 1270, 1090 Reference Example 5: Reduction of 8-(N-[4-(4-guanidino phenyl)butanoyl]-a-methoxyglycyl)-l- cyano-3,8-diazaoctane dihydrochloride 508 mg of 8-iN-[4-(4-guanidinophenyl)butanoyl]-a- methoxyglycyl)-l-cyano-3,8-diazaoctane dihydrochloride was saturated with ammonia gas. After addition of Raney nickel (200 mg), the solution was subjected to catalytic reduction overnight in a hydrogen stream (9 kg/cmê) at room temperature. The catalyst was filtered off and the solvent distilled off. Water (10 mL) was added to the solid residue and the pH of the solution was adjusted to 7.0 with 2 N HC±. The so adjusted solution was loaded onto a column packed with 100 mQ of CM-Sephadex C-25 (Na form) and, after washing with water (100 mQ), the column was eluted by a gradient of water (300 mi) and 1 M NaCt (300 mt). The active fractions were collected and concentrated under vacuum. Methanol was added to the solid residue and the insoluble NaCl was filtered off. These procedures were repeated twice and the resulting filtrate was loaded onto a column packed with 200 mt of Sephadex LH-20 and eluted with methanol. The active fractions were collected and concentrated under vacuum to provide 342 mg of 10-(N-[4-(4- guanidinophenyl)butanoyl]-&alpha;-methoxyglycyl}-1,5,10- triazadecane trihydrochloride in a yield of 62.1%. NMR (CH30H-d4) : 6 = 1.5 X 2.2 (CH2 x 3), 2.30 (CH2), 2.73 (CH2), 2.9 X 3.5 (NCH2 x 3, CH2), 3.35 (OCH2), 5.29 (CH), 7.20 (CH x 2), 7.30 (CH x 2) IR (KBr) : v (cm ) 1 3400, 2950, 2860, 2840, 2275, 1670, 1610, 1580, 1540, 1520, 1470, 1460, 1260, 1200, 1150, 1100, 1020, 570 Reference Example 6: Synthesis of 8-(N-glycyl)-l-cyano-3,8 diazaoctane monohydrochloride A mixture of 231.1 g (1.1 mol) of carbobenzoxyglycine and 139.3 g (1.21 mol) of N-hydroxysuccinimide was dissolved in 700 mt of dioxane. To the ice-cooled solution, 249.7 g (1.21 mol) of dicyclohexyl carbodiimide in 400 mt of dioxane was added dropwise. Thereafter, the mixture was stirred overnight at room temperature. The precipitating dicyclohexyl urea was filtered off and the solvent was distilled off the filtrate. The solid residue was dissolved in 500 mQ of ethyl acetate. The solution was added dropwise to 233.0 g (1.65 mol) of N-2-cyanoethylbutane-1,4-diamine in 1,000 mQ of ethyl acetate and the mixture was stirred overnight at room temperature. To the reaction solution, 2,000 mQ of a saturated aqueous solution of NaHCO3 was added and the mixture was subjected to three extractions with ethyl acetate (1,000 mQ). The ethyl acetate layers were combined, washed with saturated aqueous sodium chloride twice and dried over anhydrous Na2SO4. The solvent was distilled off and the resulting solid residue (266.6 g) was dissolved in 500 mQ of ethanol. Concentrated HCQ was added to the ethanol solution until it became acidic. The precipitating crystal was filtered off to provide 195.9 g of 8- (carbobenzoxyglycyl) -l-cyano-3, 8-diazaoctane hydrochloride. Yield, 48.3%; m.p., 151 - 153"C. A portion (22.8 g, 68.6 mmol) of the so obtained 8-(carbobenzoxyglycyl)-l-cyano-3,8-diazaoctane hydrochloride was dissolved in 275 mQ of methanol. After addition of 10% Pd-C (1.5 g), the solution was stirred in a hydrogen stream for 2 days. The catalyst was filtered off and the filtrate was concentrated under vacuum to provide 12.8 g of a syrup of 8-(N-glycyl)-l-cyano-3,8-diazaoctane monohydrochloride. This product can be immediately used as the starting material for a subsequent reaction. Reference Example 6: Synthesis of 8-(N-L-seryl)-l-cyano-3,8- diazaoctane hydrochloride A mixture of 598 g (2.5 mol) of carbobenzoxy-Lserine and 317 g (2.75 mol) of N-hydroxysuccinimide was dissolved in 2,500 mQ of dioxane. To the ice-cooled solution, 576 g (2.75 mol) of dicyclohexyl carbodiimide in 2,000 mQ of dioxane was added dropwise. Thereafter, the mixture was stirred overnight at room temperature and the precipitating dicyclohexyl urea was filtered off. The filtrate was added dropwise to a solution of 530 g (3.75 mol) of N-2-cyanoethylbutane-1,4-diamine in 1,000 mt of dioxane and the mixture was stirred overnight at room temperature. Water (1,500 mt) was added to the reaction mixture and the solution was concentrated under vacuum. To the resulting solid residue, 2,500 mt of a saturated aqueous solution of NaHCO3 was added and the mixture was subjected to six extractions with ethyl acetate (1,500 mll), The ethyl acetate layers were combined, washed with saturated aqueous sodium chloride twice and dried over anhydrous Na2SO4. The solvent was distilled off and the resulting solid residue (ca. 700 g) was dissolved in 2,500 mt of ethanol. To the solution, ethanol saturated with SCt gas was added until the solution turned acidic. The precipitating crystal was filtered off to obtain 693 g of 8-(carbobenzoxy-Lseryl)-l-cyano-3,8-diazaoctane hydrochloride. Yield, 69.5%; m.p., 151 - 1530C. 25 - 4.60 (C = 1, CH3OH). D Three hundred and fifty grams (0.88 mol) of the so obtained 8-(carbobenzoxy-L-seryl)-l-cyano-3,8-diazaoctane hydrochloride was dissolved in 4,000 mt of methanol. After adding 8.75 g of 10% Pd-C, the solution was stirred in a hydrogen stream for 2 days. The catalyst was filtered off and the filtrate was concentrated under vacuum to obtain 233 g of a syrup of 8-(N-L-seryl)-l-cyano-3,8-diazaoctane monohydrochloride. This product can be immediately used as the starting material for a subsequent reaction. Reference Example 8: Synthesis of [4-(4-guanidinophenyl) butanoyl] glycine hydrochloride A mixture of 35.0 g (136 mmol) of 4-(4-guanidinophenyl)butyric acid hydrochloride and 18.8 g (163 mmol) of N-hydroxysuccinimide was dissolved in 150 mQ of dimethylformamide. To the ice-cooled solution, 33.6 g (163 mmol) of dicyclohexyl carbodiimide in 100 mQ of dimethylformamide was added dropwise. Thereafter, the mixture was stirred for 5 hours at room temperature and the precipitating dicyclohexyl urea was filtered off. The filtrate was added dropwise to a solution of 22.8 g (163 mmol) of glycine ethyl ester hydrochloride and 24.8 mQ of triethylamine in 200 mQ of dimethylformamide, and the resulting mixture was stirred overnight at room temperature. The solvent was distilled off and the resulting solid residue was dissolved in 500 mt of water. The solution was loaded onto a column packed with 2,500 m± of Diaion HP-20 and, after washing with water, the column was eluted with 25% aqueous methanol. The active fractions were collected and concentrated under vacuum to obtain 38.0 g of a syrup of [4-(4-guanidinophenyl)butanoyl]- glycine ethyl ester hydrochloride in a yield of 81.58. Thirty-eight grams (110 nol) of the so obtained [4- (4-guanidinophenyl)butanoyl]glycine ethyl ester hydrochloride was dissolved in 400 mi of methanol. To the ice-cooled solution, 133 mt of 1 N NaOH was added and the mixture wss stirred for 1 hour at room temperature. The pH of the mixture was adjusted to 7.0 and methanol was distilled off under vacuum. By recovering teh precipitate on filter, 22.8 g of [4-(4-guanidinophenyl)butanoyl]-glycinc was obtained. Yield, 74%; m.p., 2570C (with decomposition). A portion (22.3 g, 80 mmol) of the 14-(4-guanidinophenyl)- butanoyl]glycine was dissolved in 80 m & of 1 N HCt and the solution was vacuum-evaporated to dryness, thereby providing 25.2 g of [4- (4-guanidinophenyl)butanoyl] glycine hydrochloride. Reference Example 9: Synthesis of N-[4-(4-guanidinophenyl) butanoyl]-a-methoxyglycine hydrochloride 51.6 g (0.2 mol) of 4-(4-guanidinophenyl)butyric acid was dissolved in 300 mt of methanol and, after addition of 2 g of Dowex 50W x 4 (H form) (Dow Chemical Company), the solution was boiled for 5 hours. Activated carbon was added to the reaction solution and the mixture was stirred for 30 minutes. Dowex 50W x 4 and activated carbon were filtered off and the filtrate was concentrated under vacuum to obtain 50.69 g (0.187 mol) of methyl 4-(4-guanidinophenyl)butyrate hydrochloride in a yield of 93.3%. The so obtained methyl 4-(4-guanidinophenyl)butyrate hydrochloride (50.69 g) was dissolved in 500 m± of 28% aqueous ammonia and the solution was stirred overnight at room temperature. The reaction mixture was concentrated under vacuum and the solid residue was dissolved in 200 mQ of water. The solution was loaded onto a column packed with 1,000 mQ of CM-Sephadex C-25 (Na form) and, after washing with water (5,000 mQ), the column was eluted with 0.5 M NaC±. The active fractions were concentrated under vacuum and the solid residue was dissolved in 100 mQ of water. The solution was loaded onto a column packed with 1,000 mQ of Diaion SP 207 and, after washing with water (1,200 mt), the column was eluted with water-methanol (9:1). The active fractions were concentrated under vacuum to obtain 41.94 g 41.94 g (0.154 mol) of 4-(4-guanidinophenyl)butaneamide hydrochloride in a yield of 77.2%. A portion (25.7 g, 0.1 mol) of the so obtained 4- (4-guanidinophenyl) butaneamide was dissolved in 257 mQ of dimethylformamide. To the solution, 10.1 g (0.11 mol) of glyoxylic acid and 257 mQ of acetone were added, and the solution was refluxed on an oil bath (900C) for 2.5 hours. The solvent was distilled off and the resulting solid residue was dissolved in 200 mt of water. The solution was loaded onto a column packed with 1,250 mt of CM-Sephadex C-25 (Na form) and elutd with water. The active fractions were collected and concentrated under vacuum to obtain 22.55 g (0.0766 mol) of N-[4-(4 guanidinophenyl) -butanoyl] -a-hydroxyglycine in a yield of 76.6%. A portion (22.0 g, 0.748 mol) of the so obtained N-[4- (4-guanidinophenyl)butanoyl]-a-hydroxyglycine was dissolved in 580 mt of anhydrous methanol. To the ice-cooled solution, 10 mt of concentrated sulfuric acid was added and the mixture was stirred for 3 hours at room temperature. The reaction solution was added to 1,500 m± of water and the mixture was adjusted to a pH of 11.0 with 2 N NaOH. Two hours later, the mixture was adjusted to a pH of 6.5 and concentrated under vacuum. The resulting residue was loaded onto a column packed with 1,250 mt of CM-Sephadex C-25 (Na form) and eluted with water. The active fractions were collected and concentrated under vacuum to obtain 19.33 g (62.7 mmol) of N-[4-(4 guanidinophenyl) butanoyl] -a-methoxyglycine in a yield of 83.8%. 19.33 g (62.7 mmol) of the so obtained N-14-(4- guanidinophenyl)butanoyl]-&alpha;-methoxyglycine was dissolved in 62.7 mt of 1 N HCt and the solution was concentrated under vacuum to obtain 19.84 g of N-[4-(4-guanidinophenyl)butanoyl]-a-methoxyglycine hydrochloride. Advantages of the Invention In the prior art techdniques for the production of Spergualin-related compounds having a phenylene group, the steps of synthesizing a selective amino protected form of Spermidine and subsequently eliminating the protective group have been necessary. In accordance with the present invention, a Spergualin-related compound having a phenylene group can be synthesized from an inexpensive starting material, N-2-cyanoethylbutane-1,4-diamine, through a shorter route. Therefore, the present invention offers an economical process for producing Spergualin-related compounds having a phenylene group.

Claims: Patent Claims: 1. A compound of the formula I EMI33.1 where R is a hydrogen atom, a hydroxyCl-C6 alkyl group or a C1-C6 alkoxy group, and n is an integer of 3-5 and a salt thereof. 2. A compound as defined in claim 1 wherein R is hydrogen, a Cl-C4-alkyl group terminally substituted with hydroxy, or a C1-C4-alkoxy group and n is the integer 3 or 5. 3. A process for the preparation of a compound as defined in claim 1 which comprises a) condensing a compound of the formula II EMI33.2 where R and n are as defined above, or a reactive derivative thereof, with N-2-cyanoethylbutane-1,4-diamine of the formula III: H2N(CH2)4NH(CH2)2CN (III). to obtain a compound of the formula I wherein R is as defined, or b) condensing a compound of the formula IV EMI33.3 where n is as defined above or a reactive derivative thereof with a compound of the general formula V: EMI34.1 where R is a hydrogen or a hydroxylower alkyl group, to obtain a compound of the formula I where R has the aforementioned meaning.

Inventors: FUJI, AKIO, MORIGUCHI, MAKOTO, NAKAMURA, TERUYA, TAKEUCHI, TOMIO, UMEDA, YOSHIHISA, UMEZAWA, HAMAO, PROF.
Applicants: MICROBIAL CHEMISTRY RESEARCH FOUNDATION
Application year: 1986
Main classification: C07C277
IPC codes: C07C279, C07C67, C07C239, C07C277