CA2159660A1 - Phenylalanine-glycine derivatives, process for preparation thereof, and pharmaceutical composition containing said derivatives - Google Patents

Abstract

A phenylalanine-glycine derivative of the general formula (I):

(See fig I) wherein R represents a residue of an antitumor substance, or a salt or ester thereof, a process for preparation thereof, and a pharmaceutical composition containing the same are described.
The novel conjugate of the phenylalanine-glycine derivative and the antitumor substance exhibits superior antitumor activity in comparison with the case wherein an antitumor substance is administered singly or as a mixture with phenylalanine.

Description

PHENYLALANINE-GLYCINE DERIVATIVES PROCESS
FOR PREPARATION THEREOF AND PHARMACEUTICAL
COMPOSITION CONTAINING SAID DERIVATIVES
BACKGROUND OF THE I~v~NllON
This application is a divisional application of copending Canadian Application Serial No. 2,091,316, filed March 9, 1993.
1. Field of the Invention The present invention relates to a novel conjugate of a phenylalanine derivative with an antitumor substance, a process for preparation thereof, and a pharmaceutical composition, in particular, an antitumor agent, containing it. More particu-larly, the present invention relates to a conjugate of L-phenylalanine-glycine with an antitumor substance, a process for preparation thereof, and a pharmaceutical composition containing it.

2. Description of the Related Art Hitherto, chemotherapeutic agents have proved efficacy in treating tumors, but many problems still remain. For example, chemotherapeutic agents not only have effects on tumor cells, but also affect the host cells and exhibit cell toxicity. Therefore, the agents cannot be administered for a long time to physically weakened patients and thus it was difficult to secure a suffi-cient therapeutic effect. The mechanism of action of chemothera-peutic agents is based on the inhibition of biosynthesis (in particular, that of nucleic acids) in the cells and inhibition of the metabolism necessary to maintain cell life. Strictly speaking, it means that the chemotherapeutic agents were not based on specificity to the tumor. Namely, these agents had toxicity to the normal cells of the host suffering from the tumor as well. It was desired to develop antitumor agents with an improved selectivity to the tumor cells and an improved method for concentrating conventional antitumor agents into the tumor cells.
In the meanwhile, 5-fluorouracil (5-FU) as it is does not - la -exhibit antitumor activity, but exhibits antitumor activity when bonded with the pentose phosphate in the cells to form fluoro-deoxyuridine-5'-monophosphate (FdUMP), fluorouridine-5'-triphosphate (FUTP) or the like. Namely, FdUMP inhibits the thymidylate synthetase activity to inhibit the synthesis of DNA.
FUTP is taken up in an RNA and causes critical damage to the RNA, thereby inhibiting the production of cell proteins. Therefore, if 21ss66a the pentose phosphate in the tumor cells could be increased selectively, selective chemotherapy to tumor cells would become possible by 5-FU
Further, pyruvate kinase is the rate-determining enzyme in the anaerobic glycolysis which relates to the production of pentose phosphate in the cells, and includes L-type, M1-type, and M2-type isoenzymes. Tumors contain almost only M2-type isoenzyme. It was known that the M2-type isoenzyme is selectively inhibited by a low concentration of L-phenylalanine. Therefore, it was expected that L-phenylalanine could cause inhibition specific to the pyruvate kinase activity in the tumor cells and enhance the production of pentose phosphate only in the tumor cells. Thus, Lee disclosed and ascertained a method of enhancing the activity of 5-FU to inhibit the tumor in combination with L-phenylalanine [Med. J. Kagoshima Univ., Vol.37, No. 3-4, 285-308, 1985].
In the method of Lee, however, L-phenylalanine was mixed in a laboratory chew and ingested. 5-FU was separately administered.
Therefore, L-phenylalanine and 5-FU were conveyed separately to the lesion. The present inventors engaged in various studies with the object of conveying L-phenylalanine and 5-FU to the target lesion in a bonded form and separating them quickly at the target site.
As a result, the inventors found that the above object can be achieved by bonding 5-FU with L-phenylalanine-1-acetoxyglycine, and that there is a similar effect in antitumor substances other than 5-FU. The present invention is based on these findings.

SUMMARY OF THE INVENTION
Accordingly, the present invention relates to a phenylalanine-glycine derivative of the general formula (I) fH2 1 (I) NH2cHcoNHcH - COOH

wherein R represents a residue of an antitumor substance, a salt thereof or an ester thereof (hereinafter optionally referred to as the "the present conjugate"), a process for preparation of the present conjugate, and a pharmaceutical composition containing the present conjugate.
In accordance with an embodiment of the present invention there is provided a carbobenzoxyphenylalanine amide of the formula (IV):

c~l ~IV) .

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The glycine moiety contained in the present conjugate may be any residue of the L- or D-glycine, or a mixture of LD-glycine. Further, the phenylalanine moiety is preferably residue of L-phenylalanine, but may also be residue of D-phenylalanine or a mixture of LD-phenylalanine. The present conjugate may be non-toxic salts or esters which the amino acids in the glycine and/or phenylalanine residues can form.
The non-toxic salts may be acid-addition salts or metal complexes. The metal complexes are complexes with, for example, zinc, iron, calcium, magnesium, or aluminum. As the acid-addition salts, there may be mentioned hydrochloride, hydro-bromide, sulfate, phosphate, tannate, oxalate, fumarate,glauconite, alginate, maleate, acetate, trifluoroacetate, citrate, benzoate, succinate, malate, ascorbate, tartrate, or the like. Further, the salts may be carbonates, for example, salts with alkali metals tsodium, potassium salts, etc.), salts with alkaline earth metals (calcium, magnesium salts, etc.), or 2I5966~

- 3a -ammonium salts.
The esters may be any esters conventionally used for amino acids, such as aryl or alkyl esters. In particular, there may be mentioned straight-chain or branched alkyl esters having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, or isobutyl ester.
The antitumor substance residue R in the present conjugate may be a residual group of an alkylating antitumor substance, an antimetabolic antitumor substance, an antibiotic antitumor sub-stance, or the like. As the antitumor substances, there may be mentioned, for example, 5-fluorouracil, 5-amino-7-hydroxy-lH-v-triazolo(4,5-d)pyrimidine, 4-amino-N1~-methylpteroyl-glutamic acid, 4-aminopteroyl-glutamic acid, 6-mercaptopurine, 5-[bis(2-chloroethyl)amino]-uracil, mitomycin C, bleomycin, daunorubicin, doxorubicin, p-[bis(2-chloroethyl)amino]-L-phenylalanine or ester 21~966D

thereof, N,N-bis(2-chloroethyl)-N1, O-propylene-phosphate ester diamine, 4-[p-(bis~2-chloroethyl)amino)phenyl]-butylic acid or es~er thereof.
The present conjugate may be prepared by the steps (a) and (b) as follows:
(a) The step comprising introducing an antitumor substance residue R into a compound of the general formula (IIIb):

CHz OCOCH3 (IIIb) ~ CH20CONHCHCONHCHCOR

wherein R2 represents a benzyloxy group or an alkoxy group with 1 to 4 carbon atoms, to obtain a compound of the general formula (II):

CH2 R (II) ~ CH20CONHCHCONHCHCOR2 wherein R and R2 have the same meanings as above.
(b) The step comprising removing group(s) for protecting amino group(s) from the compound of the general formula (II) to obtain the compound of the general formula (I) or ester thereof.
In the step (a), the compound of the general formula (IIIb) and the antitumor substance are reacted in the presence of an organic solvent (for example, dimethylformamide) and preferably a base at -10 to 50CC, preferably a~ 10 to 30~C, for 10 to 120 minutes, preferably for 15 to 60 minutes, to thereby obtain the compound of the general formula (II). As the base, triethylamine, pyridine or the like may be used. After the reaction is completed, the reaction product is used in the next step without or with purification by recrystallization, distillation, extraction, precipitation, washing, column separation, concentration, lyophilization, or the like. In the step (b), a solution of the compound of the general formula (II) in an organic solvent is added to an alcohol solution of palladium carbon and treated at -10 to 120~C, preferably at -5 to 100~C, for 10 to 300 minutes, preferably for 15 to 240 minutes. After cooling, the filtrate is concentrated to obtain the crystal. Further, the crystal was recrystallized or washed with a solvent to obtain a compound of the general formula (I) with a high purity. If necessary, the D-form and L-form of the compound of the general formula (I) or ester thereof are resolved from each other by, for example, chromatography. The compound of the general formula (I) may be converted to the corresponding salt or ester before used as an antitumor agent.
Examples of the present conjugate will be shown hereinafter:
(1) L-phenylalanyl-2-(5-fluorouracil-1-yl)-D, L-glycine (2) L-phenylalanyl-2-(5-fluorouracil-1-yl)-D, L-glycineamide (3) L-phenylalanyl-2-(5-fluorouracil-1-yl)-D, L-glycinemethylester (4) L-phenylalanyl-2-(5-fluorouracil-1-yl)-D, L-glycineethylester (5) L-phenylalanyl-2-(5-fluorouracil-1-yl)-D, L-glycinepropylester (6) L-phenylalanyl-2-[5-(bis(2-chloroethyl)-amino)uracil-1-yl]-D,L-glycine ,-(7) L-phenylalanyl-2-[5-(bis(2-chloroethyl)amino)-uracil-1-yl]-D,L-glycinemethylester (8) L-phenylalanyl-2-[5-(bis(2-chloroethyl)amino)-uracil-1-yl]-D,L-glycineethylester (9) L-phenylalanyl-2-[p-(bis(2-chloroethyl)amino)-L-phenylalaninemethylester-2-yl]-D,L-glycineethylester (10) L-phenylalanyl-2-(5-fluorouracil-1-yl)-L-glycine (11) L-phenylalanyl-2-(5-fluorouracil-1-yl)-D-glycine The compound of the general formula (IIIb) may be prepared, for example, by the following steps (c) to (e):

21~9660 (c) The step comprising reacting a salt or a compound of the fonmula (v):
[~
(v) fH2 with carbobenzoxychloride to obtain a compound of the formula (IV):

CH2 (Iv) ~ CH20CONHCHCONH~

More particularly, L-phenylalanine amide hydrochloride and sodium hydrogencarbonate are qissolved in water and then carbobenzoxychloride and an organic solvent are added at 0 to 30~C.
The mixture is agitated and reacted, for 1 to 24 hours, preferably for 2 to 8 hours. Further carbobenzoxychloride and sodium hydrogencarbonate may be added thereto. After the reaction is completed, a white crystal is taken out and recrystallized from ethyl acetate to obtain the compound of the formula (IV).
(d) The step comprising reacting the compound of formula (IV) with a glyoxylic acid compound of the general formula (VI):

O O
ll ll (VI) 2ls966~

wherein R2 has the same meaning as above, to obtain a compound of ~he general formula (IIIa):

c~, OH (lIIa) ~ CH OCONHCHCONHCHCOR

wherein R2 has the same meaning as above.
More particularly, the compound of the formula (IV) is dissolved or suspended in an organic solvent. The glyoxylic acid of the general formula (VI) is added to the mixture while agitating at 0 to 30~C and reacted for 50 to 240 hours. The product is concentrated under reduced pressure to obtain a white crystal of the general formula (IIIa).
(e) The step comprising reacting the compound of the general formula (IIIa) with acetic anhydride to thereby obtain the compound of the general formula (IIIb).
More particularly, the compound of the general formula (IIIa) is agitated for 1 to 48 hours at 0 to 30~C in acetic anhydride and pyridine and then the reaction mixture is extracted with ethyl acetate. The resulting solution is washed with hydrochloric acid, water, sodium hydrogencarbonate aqueous solution, distilled water, saturated saline solution, or the like. The organic layer is then concentrated to obtain an oily substance. The oily substance is purified by silica gel chromatography, and recrystallized to obtain the compound of the general formula (IIIb) as a white crystal.
In comparison with the case that an antitumor agent is used singly, or the case that an antitumor agent is used in the form of a mixture with phenylalanine, the antitumor activity is enhanced by the present conjugate containing said antitumor agent. Further, in comparison with the case that the antitumor agent is used singly, the acute toxicity of the present conjugate containing said antitumor agent is reduced.

The present conjugate can be formulated to, for example, syrups, injections, ointments, tablets, or the like. The present conjugate may be contained in the formulation in an amount of 0.1 to 99.5 % by weight, preferably in an amount of 1 to 90 % by weight. The formulation of the present conjugate may be administered orally or parenterally. A dose varies with the method of administration, the extent of the treatment, and also the kind of the antitumor substance contained therein. Generally speaking, however, the dose of the present conjugate is in the range of 100 to 1000 mg/kg/day orally, while in the range of 5 to 500 mg/kg/day parenterally, which is divided into 1 to 4 dosages in a day.
As above, the novel present conjugate of the phenylalanine-glycine derivative and the antitumor substance exhibits a superior antitumor activity compared with the case that an antitumor substance is administered singly, or the case that the antitumor substance is administered in the form of a mixture with phenylalanine.

Examples The present invention now will be further illustrated by, but is by no means limited to, the following examples. The physicochemical data described in the following Examples were obtained by the following methods:
(1) Elemental Analysis A Yanagimoto MT3-type automatic elemental analyzer was used and the decomposition gas was detected with a thermal conductivity type detector (TCD).
(2) Optical Rotation A Nihon Bunko automatic polarimeter DIP-360 was used to measure the optical rotation for a solution of the present conjugate to determine the [~]D-(3) NMR
A Nihon Denshi JNM-GSX500 was used.
(4) Infrared Absorption Spectrum A Nihon Bunko A-202 apparatus was used to measure the infrared absorption spectrum by the KBr tablet method and to determine ~max-(5) Thin Layer Chromatography The Rf value was determined by a hexane-ethyl acetate system, 2t59660 g butanol-acetic acid-pyridine-distilled water system, or 5 %
methanol-dichloro~ethane system.
(6) Melting Point The melting point was measured by a Yanagimoto micro melting point detector (DSC).
Exam~le 1 (1) Pre~aration of carbobenzoxv-L-~henvlalanineamide (IV) ~J ~3 CH20COCl HClH7NCHCONH2 NaHCO3 ~ CH20CONHCHCONH2 (v) (IV) I~phenylalaI~ine amide hydrochloride (V) (1.00 g, 5.0 mmmol) and 1.09 9 of sodium hydrogencarbonate (13.0 mmol) were dissolved in 40 ml of distilled water. The solution was agitated at room temperature while adding 0.72 9 of carbobenzoxychloride (4.2 mmol) and 25 ml of dichloromethane. After 2 hours, a further 0.72 9 of carbobenzoxychloride (4.2 mmol~ and 0.55 9 of sodium hydrogencarbonate (6.5 mmol) were added. When agitation was continued, a white crystal was precipitated in the dichloromethane layer. To the crystal layer, dichloromethane was further added to completely dissolve them, and the dichloromethane was washed with a saturated sodium hydrogencarbonate solution. The dichloromethane layer was dried over anhydrous sodium sulfate, then concentrated under reduced pressure, and recrystallized with dichloromethane-hexane, whereby 1.46 9 of a white compound (IV) was obtained. The physicochemical data of the product was as follows:
Yield: 99.0 %, Melting point: 160.1 to 162.1~C
Elemental analysis (%) Found: C, 68.32; H, 5.93; N, 9.38 1~

Calculated (for C17H1gN2O3): C, 68.44; H, 6.08; N, 9.39 ~a]D: -5.55~ (c 1.0, CH30H) H-NMR (CDCl3): ~3.05 (dd, lH, J=13.8Hz, 7.3Hz) phenylalanyl CH2, ~3.13 (dd, lH, J=13.8Hz, 7.3Hz) phenylalanyl CH2, ~4.30 (m, lH) NCHCO, ~5.09 (s, 2H) Ph-CH2O, ~5.35 (br, 2H) CON_2, ~5.66 (br, lH) CON_2 IR(KBr)~max: 3425m, 3210s, 1690m, 1660s (amide group) Rf: 0.42 5 % methanol-dichloromethane (phosphomolybdic acid reagent W +).
(2) Pre~aration of N-carbobenzoxv-L-~henvlalanvl-D,L-2-hvdroxyalvcinebenzvlester (IIIa-1) CHOCOOCH, ~ ¢~
( IV) CH2 OH

CH20CONI{C~ICONHCHCOOCH

( I I I a - 1 ) To a suspension of 2.81 9 of carbobenzoxyphenylalanine amide (IV) -(9.4 mmol) in dichloromethane, 1.93 9 of benzyl glyoxylic acid (11.8 mmol) was added while agitating at room temperature and reacted for about 160 hours. The resulting suspension was concentrated under reduced pressure to obtain a crystal. The product was filtered with suction while washed with dichloromethane-hexane and then dried to give 3.78 9 of the compound (IIIa-1).
Yield: 86.8 %, Melting point: 120.6 to 127.7~C
Elemental analysis (~) Found: C, 67.00; H, 5.66; N, 6.77 Calculated (for C26H26N2O6): C, 67.52; H, 5.67; N, 6.06 [a]D: -13.0~ (c 1.0, CH30H) H-NMR (CDCl3): ~3.06 (m, 2H) phenylalanyl CH2, ~3.86 (d, 0.5H) OH, ~3.97 (d, 0.5H) OH, ~4.43 (m, lH) Ph-CH2-CH, ~5.06 (m, 2H) Z group CH2, ~5.20 (m, 2H) benzylester CH2, ~5.49 (t, 2H) glycyl CH, ~7.1 to 7.4 (m, 15H) aromatic ring IR(KBr)vmax: 3420m (NH), 3310s (NH), 1750m (COO), 1695m, 1660s (CONH) Rf: 0.45 5 % methanol-dichloromethane (phosphomolybdic acid reagent W+).
(3) PreDaration of N-carbobenzoxv-L-Dhenvlalanvl-D,L-2-acetoxvalvcinebenzYlester (IIIb-1) ~ ~2 OCOCH
(IIIa-1) , ~ CH2OCONHCHCONHCHCOOCH
(CH3CO)2O
(IIIb-1) N-carbobenzoxy-L-phenylalanine-D,L-2-hydroxyglycinebenzylester (IIIa-1) (4.82 9, 10.4 mmol) was dissolved at room temperature in 50.0 ml of acetic a~hydride and 36.5 ml of pyridine and agitated for about 24 hours. After the end of the reaction had been confirmed by thin layer chromatography, the reaction mixture was extracted with 100 ml of ethyl acetate and the organic layer was washed three times with 100 ml of distilled water, then twice with 100 ml of saturated saline solution. The washed organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 5.64 9 of a yellow oily substance. The resulting oily substance was purified by silica gel chromatography using n-hexane/ethyl acetate (2/1) and recrystallized from ethyl acetate-n-hexane to obtain 1.31 9 of the compound (IIIb-1).
Yield: 34.0 ~, Melting point: 112.5 to 114.0~C
Elemental analysis (%) Found: C, 66.63; H, 5.52; N, 5.68 Calculated (for C2gH2gN2O7): C, 66.66; H, 5.59; N, 5.55 [~]D: -7-0~ (c 1.0, CH3OH) 1H-NMR (CDCl3): ~2.05 (s, 3H) acetyl CH3, 83.07 (m, 2H) phenylalanyl C_2, 84.46 (m, lH) phenylalanyl N-CHCO, ~5.06 (m, 2H) Z group CH2, ~5.19 (m, 2H) benzylester CH2, ~6.37 (d, lH) glycyl CH, ~7.1 to 7.4 (m, 15H) aromatic ring IR(~CBr)~max: 3300s (NH), 3060w, 3030w, 2973w, 1770s, 1740s, 1695s, 1665s Rf: 0.42 Hexane: Ethyl acetate = 2:1 (phosphomolybdic acid reagent W+).
(4) Pre~aration of N-carbobenzoxv-L-~henvlalanvl-2-(5-fluorouracil-1-vl-D,L-qlvcinebenzvlester (II-1) H N
O N

(IIIb-1) ~ ~ O f (II-1) HN I F

N-carbobenzoxy-L-phenylalanyl-D,L-2-acetoxyglycinebenzylester (IIIb-1) (0.25 9, 0.5 mmol) and 73.5 mg of 5-fluorouracil (0.57 mmol) were dissolved in 1.0 ml of dimethylformamide at room temperature and 0.51 9 of triethylamine (5.0 mmol) was added and the mixture was agitated for about 20 minutes. After the end of the reaction had been confirmed by thin layer chromatography, the reaction mixture was concentrated under reduced pressure and extracted with 50 ml of ethyl acetate. The organic layer was washed with 20 ml of distilled water and 40 ml of saturated saline solution. The washed layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and recrystallized from ethyl acetate-n-hexane to obtain 0.16 9 of the compound tII-1) .
Yield: 53.1 %, Melting point: 170.3 to 175.0~C
Elemental analysis (%) 215966~ .

Found: C, 62.68; H, 4.68; N, 9.76 Calculated (for C30H27N~C7F): C, 62.71; H, 4.74; N, S.75 [a]D: -7.0~ (c 1.0, CH3OH) 1H-NMR (CDCl3): ~2.8 to 3.0 (m, 1.5H) diastereomeric phenylalanyl CH2, ~3.12 (dd, 0.5H, J=13.8, 5.9Hz), ~4.67 (d, 0.5H, J=7.3Hz) phenylalanyl CHCO, ~4.73 (d, 0.5H, J=6.4Hz) phenylalanyl CHCO, ~4.9 to 5.2 (m, 4H) benzyl CH2, ~5.51 (d, 0.5H), ~5.57 (d, 0.5H), ~5.80 (d, 0.5H) glycyl CH, ~5.84 (d, 0.5H) glycyl CH, ~7.0 to 7.3 (m, 15H) aromatic ring, ~7.50 (d, 0.5H, J=5.5Hz) uracil 6H, ~7.60 (d, 0.5H, J=5.5Hz) uracil 6H, ~9.19 (br, 0.5H) uracil 3NH, ~9.66 (br, 0.5H) uracil 3NH
IR (KBr)~max: 3405m, 3300s, 3160m, 3140m, 1760s, 1700s, 1660s.
(5) Preparation of L-PhenvlalanYl-2-(5-fluorouracil-1-vl)-D,L-qlvcine (I-1~ -Pd/C fH2 (II-1) ~ H2O-L-NH2CHCONHCIHCOOH (I-1) ~ ~I
HN ~ F

Methanol was added dropwise to 246.5 mg of 10 % palladium carbon at 0~C. Further, there was added thereto dropwise at 0~C a solution of 0.29 9 of N-carbobenzoxy-L-phenylalanyl-2-(5-fluorouracil-1-yl)-D,L-glycinebenzylester (II-1) (0.5 mmol) in methanol and cyclohexene added. The solution was heated under reflux at 70 to 80~C for about 20 minutes. After the end of the reaction had been confirmed by thin layer chromatography, the reaction liquid was diluted with methanol and filtered through a fluted filter paper. The filtrate was concentrated under reduced pressure to obtain a white crystal. The crystal was washed with ~ 2159660 ethyl acetate to obtain 0.16 9 of the compound (I-1).
Y:eld: 89.6 %, Melting point: 197.3 to 201.3~C
Elemental analysis Found: C, 48.8; H, 4.5; N, 14.8 Calculated (for C1sH1sOsN4F-H2O): C, 48.9; H, 4.7; N, 15.2 [~]D: 11-9~ (c 2.0, CH3OH) 1H-NMR (D2O): ~2.93 to 2.97 (dd, 2H) diastereomeric phenylalanine CH2, ~3.20 to 3.29 (dd, 2H) diastereomeric phenylalanine CH2, ~4.26 to 4.33 (m, 2H), ~5.79 (s, lH) glycyl C_, ~5.90 (s, lH) glycyl CH, ~7.0 to 7.3 (m, 10H) aromatic ring, ~7.55 (d, lH, J=6Hz) uracil 6_, ~7.85 (d, lH, J=6Hz) uracil 6H
IR (KBr)~max: 3400m, 3170m, 3030m, 2800m, 1690s, 1500m, 1370s, 1240s Rf: 0.62, 0.57 Butanol: Acetic acid: Pyridine: Distilled water = 4:1:1:2 (ninhydrin reagent W+).
(6) The procedures of Examples 1 (4) and (5) were repeated, except that 5-[bis(2-chloroethyl)amino]uracil was used instead of 5-fluorouracil, to obtain L-phenylalanyl-2-[5-(bis(2-chloroethyl)amino)uracil-1-yl]-D,L-glycine at a yield of 45 %. The results of the elemental analysis were as follows:
Found: C, 48.01; H, 4.73; N, 15.02 Calculated (for C19H23OsNsCl2): C, 48.32; H, 4.91; N, 14.83.
Exam~le 2 (1) Preparation of N-carbobenzoxy-L-phenylalanyl-D,L-2-hydroxyglycineethylester (IIIa-2) (IV) CH2C12 fH2 OH
+ ~' ~ CH20CONHCHCONHCHCOOCH2CH3 (IIIa-2) , 2159660 To a suspension of 0.94 9 of carbobenzoxyphenylalanine~ amide (IV) (3.1 mmcl) prepared from Ex~mpie 1(1) in dichloromethane, 0.40 g of ethyl glyoxylate (3.9 mmol) was added while agitating at room temperature, and then the suspension was agitated for about 160 hours. The suspension was concentrated under reduced pressure to obtain a white crystal. The crystal was filtered with suction while washing with dichloromethane-hexane and then dried to obtain 3.78 9 of the compound (IIIa-2).
Yield: 77.6 %, Melting point: 143.7 to 148.7~C
Elemental analysis (%) Found: C, 61.60; H, 5.72; N, 7.02 Calculated (for C21H24N2O6): C, 62.99; H, 6.04; N, 7.00 [~]D: 6.85~ (c 1, DMSO) 1H-MMR (d6-DMSO): ~1.19 (t, 3H, J=6.91) ethylester CH3, ~2.74 (m, lH) phenylalanyl CH2, ~2.98 (m, lH) phenylalanyl CH2, ~4.13 (m, 2H) ethylester CH2, ~4.31 (m, lH) phenylalanyl CH, ~4.93 (d, 2H, J=3.33) Z group CH2, ~5.49 (t, 2H) glycyl CH, ~6.60 (d, 0.5H, J=6.67) O_, ~6.68 (d, 0.5H, J=6.67) OH, ~7.19 to 7.34 (m, 10H) aromatic ring IR(KBr)umax: 3300s, 3060m, 3030m, 2950w, 1747s, 1685s, 1658s, 1530s Rf: 0.32 5 % methanol-dichloromethane (phosphomolybdic acid reagent W+).
(2) PreParation of N-carbobenzoxv-L-Phenvlalanvl-D,L-2-acetoxvql~cineethvlester (IIIb-2) ( CH3C0 ) 20 ~
(IIIa-2) > ~ CH2OCONHCHCONHCHCOOCH2CH3 ~ (IIIb-2) N

N-carbobenzoxy-L-phenylalanine-D,L-2-hydroxyglycineethylester (IIIa-2) (2.65 9, 6.6 mmol) was dissolved in 31.8 ml of acetic anhydride and 23.2 ml of pyridine at room temperature and agitated for about 3 hours. After the end of the reaction had been corfirmed by thin layer chromatsgraphy, the reac~ on mixture was extracted with 70 ml of ethyl acetate and the organic layer was washed three times with 70 ml of distilled water and then twice with 70 ml of saturated saline solution. The washed organic layer was dried over anhydrous sodium sulfate, then concentrated under reduced pressure to obtain 2.31 9 of a yellow oily substance. The oily substance was purified by silica gel chromatography (n-hexane/ethyl acetate = 2/1), and recrystallized (ethyl acetate-hexane) to obtain 1.18 9 of the compound (IIIb-2).
Yield: 40.3 %, Melting point: 141.5 to 147.0~C
Elemental analysis (%) Found: C, 62.13; H, 5.65; N, 6.32 Calculated (for C23H26N2O7): C, 62.43; H, 5.92; N, 6.33 [~]D: 23-55~ (c 1, CHCl3) lH-MMR (CDCl3): ~1.27 (t, 3H, J=7.10) ethylester CH3, ~2.08 (s, lH) acetyl CH, ~3.11 (m, 2H) phenylalanyl CH2, ~4.22 (m, 2H) ethylester CH2, ~4.49 (br, lH) phenylalanyl CHCO, ~5.09 (s, 2H) Z group CH2, ~5.23 (br, lH) phenylalanyl CON , ~6.32 (d, lH, J=9.16) glycyl CH, ~7.16 to 7.37 (m, 10H) aromatic ring IR (~3r)Umax: 3300s, 3050w, 3030w, 2960m, 1735s, 1690m, 1660s, 1540m Rf: 0.54 Hexane: Ethyl acetate = 1:1 (phosphomolybdic acid reagent W+).
(3) PreParation of N-carbobenzoxv-L-phenYlalanvl-2-(5-fluorouracil-1-Y1) -D,L-alvcineethYlester (II-2) HN
O N

(IIIb-2J ~ ~ CH2OCONHCHCONHfHCOOCHzCH3 N ~
(II-2) HN ~ F
o ~ 17 2 1 5 9 6 6 ~

N-carbobenzoxy-L-phenylalanyl-D,L-2-acetoxyglycineethylester (IIIb-2) (0.47 9, 1.1 mmol) and 0.15 9 of 5-fluorouracil (1.20 mmol) were dissolved in 2.2 ml of dimethylformamide at room temperature and then 1.06 9 of triethylamine (10.5 mmol) was added thereto. The mixture was agitated at room temperature for about 20 minutes. After the end of the reaction had been confirmed by thin layer chromatography, the reaction mixture was concentrated under reduced pressure and the residue was extracted with 100 ml of ethyl acetate. The extracted organic layer was washed with 40 ml of distilled water and 80 ml of saturated saline solution. The washed organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and recrystallized (ethyl acetate-hexane) to obtain 0.54 9 of the compound (II-2).
Yield: 90.7 %, Melting point: 98.0 to 102.3~C
Elemental analysis (%) Found: C, 57.98; H, 4.94; N, 10.56 Calculated (for C2sH2sN4O7F): C, 58.59; H, 4.92; N, 10.93 [~]D: -10.i20 (c 2, CHOH) 1H-NMR (CDCl3): ~1.19 (t, 1.5H, J=7.11) ethylester CH3, ~1.25 (t, 1.5H, J=7.11) ethylester CH3, ~3.01 (m, 1.5H) phenylalanyl CH2, ~3.15 (dd, 0.5H, J=13.75, 5.96Hz) phenylalanyl CH2, ~4.24 (m, 2H) ethylester CH2, ~4.67 (d, 0.5H, J=7.3Hz) phenylalanyl C_CO, ~4.73 (d, 0.SH, J=6.4Hz) phenylalanyl C_CO, ~5.05 (m, 2H) Z group CH2, ~5.63 (d, 0.5H, J=8.25Hz) phenylalanyl CON_, ~5.70 (d, 0.5H, J=8.25Hz) phenylalanyl CONH, ~5.78 (d, 0.SH, J=7.33Hz) glycyl CH, ~5.83 (d, 0.5H, J=7.33 Hz) glycyl C_, ~7.05 to 7.31 (m, 10H) aromatic ring, ~7.56 (d, 0.5H, J=5.04Hz) uracil CH, ~7.65 (d, 0.5H, J=5.04Hz) uracil C_, ~9.70 (br, 0.5H) uracil NH, ~10.0 (br, 0.5H) uracil NH
IR (KBr)vmax: 3300s, 3050m, 3020m, 1750s, 1700s, 1660s, 1510m Rf: 0.26 Hexane; Ethyl acetate = 1:1 (phosphomolybdic acid reagent W+).
(4) Preparation of L-Phenvlalanvl-2-(5-fluorouracil-1-vl)-D,L-qlvcineethvlester (I-2) 215966~

Pd/C
,. 2 HC 1 /NH2CHCONHCHCOOCH2CH3 (II 2) H2 0 (I-2) HN ~

A O . lN hydrochloric acid-methanol solution was added dropwise to 98.6 mg of 10 % palladium carbon at 0~C. Further, a O.lN
hydrochloric acid-methanol solution (total volume of 4.7 ml) of 0.10 9 (0.2 mmol) of N-carbobenzoxy-L-phenylalanyl-2-(5-fluorouracil-1-yl)-D,L-glycineethylester (II-2) was added thereto dropwise and then the mixture was agitated under an H2 gas stream at room temperature for 3 hours. After the end of the reaction had been conflrmed by thin layer chromatography, the reaction mixture was diluted with methanol and filtered through a fluted filter paper. The filtrate was concentrated under reduced pressure to obtain a white crystal. The crystal was washed with diethylether and filtered to obtain 65.4 9 of the compound (I-2).
Yield: 72.4 %, Melting point: 174.0 to 185.3~C
Elemental analysis (%) Found: C, 45.72; H, 4.66; N, 12.41 Calculated (for C17H1gOsN4F-2HCl): C, 4S.25; H, 4.69; N, 12.41 [a]D: 27.03~ ~c 1, CH3OH) 1H-NMR (D2O): ~1.31 (t, 1.5H, J=7.10Hz) ethylester CH3, ~1.36 (t, 1.5H, J=7.10Hz) ethylester CH3, ~3.12 (dd, 0.5H, J=13.29, 10.54Hz) phenylalanyl CH2, ~3.38 tm, lH) phenylalanyl CH2, ~3.47 ldd, 0.5H, J=13.29, S.SOHz) phenylalanyl CH2, ~4.37 (m, 2H) ethylester CH2, ~4.47 (m, lH) phenylalanyl CHCO, ~6 36 ~d, lH, J=7.33Hz) glycyl CH, ~7.26 to 7.54 (m, SH) aromatic ring, ~7.90 (d, O.SH, J=S.SHz) uracil CH, ~8.05 (d, 0.5H, J=5.5Hz) uracil CH
IR(KBr)umax: 3420m, 3180m, 3020s, 1705s, 1530m, 1495m, 1470m Rf: 0.24 5 % methanol-dichloromethane (phosphomolybdic acid reagent W+3.
Ex~m~le 3 (1~ Pre~aration of N-carbobenzoxv-L-~henvlalanvl- 2-~-(bis( 2-chloroeth~l)amino)-L-~henvlalaninemethvlester-2-vll-D,L-alvcineethvlester (II-3) ClCH2CH2 > N ~ CH2CHCOOCH3 C

(IIIb-2) CH2 NH

ClCH2CH2 > N ~ CH2CHCOOCH3 (II-3) The compound (IIIb-2) obtained from Example 2(2) was reacted with melphalan methylester [p-(bis(2-chloroethylamino)-L-phenylalaninemethylester] in the manner same as that in Example 2(3) to obtain the compound (II-3). The melphalan methylester was treated with a sodium hydrogen carbonate solution (2. 2 equivalents) to remove the hydrochloride and extracted with dichloromethane.
Yield: 84.3 ~, Melting point: 112.3 to 115. 3~C
Elemental analysis (%) Found: C, 60.04; H, 6.00; N, 7.93 Calculated (for C3sH42O7N4Cl2): C, S9.91; H, 6.03; N, 7.98 [~]D: 19.1~ (c 1, CHCl3) lH-MMR (CDCl3): ~1.26 (t, 3H, J=7.10Hz) ethylester CH3, ~2.73 (dd, 0.5H, J=13.75, 7.79Hz) phenylalanine CH2, ~2.91 (dd, O.5H, J=13.98, 5.73Hz) phenylalanine CH2, ~3.05 (m, 2H)- melphalan Ph-CH2, ~3.57 to 3.62 (m, 4H) melphalan CH2-CH2, ~3.65 to 3.70 (m, 4H) melphalan Cl-~15966a -CH2, ~3.69 (s, 3H) melphalan CH3, ~4.14 (m, 2H) ethylester CH2, ~4.36 (m, lH) melphalan CH-CO, ~5.07 (m, 2H) Z group CH2, ~5.18 (m, lH) melphalan NH, ~6.59 (d, lH, J=8.71Hz) glycyl CH, ~6.96 to 7.31 (m, 14H) aromatic ring IR(KBr)umax: 3300s, 3070m, 3040m, 2950m, 1730s, 1645s, 1610m, 1520s Rf: 0.50, 0.56 Hexane: Ethyl acetate = 1:1 (phosphomolybdic acid reagent W+).
(2) Pre~aration of L-~henvlalanvl-2-~-(bis(2-chloroethvl)amino)-L-~henvlalaninemethvlester-2-vll-D,L-alvcineethvlester (I-3) H2 Cti (II-3) ~ 3HC1/NH2CHCONHCHCOOCH2CH3 Pd/C
NH

ClCH2CH ~ N C ~ CH2CHCOOCH3 (I-3) The compound (II-3) obtained from Example 3(1) was reacted with palladium carbon under an H2 stream in the manner same as that in Example 2(4), to obtain the compound (I-3).
Yield: 89.7 ~, Melting point: 102.3 to 108.0~C
Elemental analysis (%) Found: C, 47.33; H, 5.97; N, 8.16 Calculated (for C27H36OsN4C1-3HC1): C, 47.90; H, 5.80; N, 8.28 ~~]D: 36-5~ (c 1, CH3OH) 1H-NMR (D2O): ~1.25 (t, 3H, J=7.10Hz) ethylester CH3, ~3.17 (m, 2H) phenylalanyl CH2, ~3.27 (m, 2H) melphalan Ph-CH2, ~3.70 to 3.76 (m, 4H) melphalan CH2-C~2-, ~3.86 to 3.90 (m, 4H) melphalan Cl-CH2, ~3.89 (s, 3H) melphalan CH3, ~4.37 (m, 2H) ethyl ester C~ 4.55 (m, lH) melphalan CH-CO, ~7.35 to 7.71 (m, 9H) aromatic ring IR(KBr)umax: 3410s, 3150m, 2950s, 1740s, 1705s, 1610m Rf: 0.38, 0.44 5 ~ methanol-dichloromethane (phosphomolybdic acid reagent WTj.
Exam~le 4: Acute Toxicitv (1) The conditions in the feeding chamber were arranged to a temperature of 23+1~C, a relative humidity of 55+5 %, a ventilation frequency of 20 times per hour, and a lighting term of 12 hours.
Wistar rats (male; five-week old; 120 to 141 9) were used. The rats were placed in metallic cages having wire walls in the front and bottom thereof, at the rate of 5 rats per one cage. They were allowed food (MF: Oriental Yeast) and water ad libitum.
(2) Method of Administration The conjugate (I-1) of the present invention obtained from Example 1(5) was suspended in a 1.5 % methylcellulose aqueous solution. The suspension was a~mi n; stered by force. The amount of the administration was 1 ml per 100 9 body weight of ~n;m~ls forced to fast for 18 hours, that is, 250 mg/kg was administered.
Symptoms of addiction and survival were observed at every one-hour up to 8 hours after the administration, then twice a day until 14 days after administration. No death was observed.
ExamPle 5: Antitumor Activitv The concentration of the solid tumor cells (Sarcoma-180) was aseptically adjusted to 1 X 106/0.2 ml with a medium [prepared by filtering 10 % bovine fetal serum-added MEM (Eagles' Minimum Essential Medium) for sterilization, and stored at 4~C] and subcutaneously implanted at the axillas of ICR mice (5 weeks old;
females; one group consisting of 10 mice). From the next day, the samples (0.15 % physiological saline solution) shown in Table 1 were administered intraperitoneally 10 times every other day. On the 22nd day, the tumors were excised and the inhibition rate (IR) was obtained from the weight of the tumors. The results are shown in Table 1. The inhibition rate (IR) (~) was calculated by the following formula:
IR (%) = ~ - (T/C~ X 100 wherein T is the average tumor weight of the treated group, whereas C is the average tumor weight of the control group.

Table 1 Group Amount Average tumor IR (~) (mg/kg) weight+SD
Control - 3.438+1.481 S-Fu 15 1.467+1.437 57.3 MP 10 1.358+1.369 60.5 5-CAU 10 1.574+1.285 54.2 Phe~5-Fu 15+15 1.385+1.297 59.7 Phe+MP 15+10 1.407+1.295 59.1 Phe+5-CAU 15+10 1.550+1.375 54.9 Phe-5-Fu 30 0.770+0.397 77.6 Phe'-5-Fu 30 0 808+0.389 76.5 Phe-MP 20 0.943+0.587 72.6 Phe-5-CAU 20 0.854+0.664 75.2 5-Fu: 5-Fu alone MP: *Melphalan alone 5-CAU: 5-[bis(2-chloroethyl)amino]uracil alone Phe+5-Fu: Mixture of L-phenylalanine and 5-Fu Phe+MP: Mixture of L-phenylalanine and MP
Phe+5-CAU: Mixture of L-phenylalanine and 5-CAU
Phe-5-Fu: Present Conjugate (I-l) prepared from Example 1 Phe'-5-Fu: Present Conjugate (I-2) prepared from Example 2 Phe-MP: Present Conjugate (I-3) prepared from Example 3 Phe-5-CAU: Present Conjugate prepared from Example 1(6).

Example 6 Pre~aration of Iniection A 500 mg amount of the compound (I-l) of the present invention obtained from Example 1(5) was dissolved in 50 ml of ethanol to prepare an injection.

Although the present invention has been described with reference to specific embodiments, various changes and modifications obvious to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention.

*Trade Mark

Claims (2)

1. A carbobenzoxyphenylalanine amide of the formula (IV):

2. Use of the compound of claim 1 as a pharmaceutical compound for the treatment of a tumor.