NO831475L - PROCEDURE FOR THE PREPARATION OF N- (N'- (2-CHLORETHYL) -N'-NITROSO-CARBAMOYL) -OOLOPEPTID ESTERS AND AMIDES - Google Patents

Abstract

Compounds having the formula: <IMAGE> wherein: <IMAGE>R4 = -OCH3, -NH2, NH-R4, R4 being an oligopeptidic residue of which the terminal carboxyl group is blocked as a methyl ester or amide group. They are prepared by reacting the corresponding aminoacids or oligopeptides with an ester or an amide of a second aminoacid or of a second oligopeptide, in presence of an appropriate basic peptidic catalyst.

Description

With alkylantienes, a new drug class has been introduced in cancer chemotherapy in recent years, namely nitrosoureas, e.g. 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) and 1-(2-chloro-ethyl)-3- (4-methylcyclohexyl)-1-nitrosourea (MeCCNU), Proceeding of the 7th new drug seminar on the nitrosourea, (Washington, D.C., Dec. 15-16, 1975), Cancer Treat. Rep.,

60, 645-811 (1976)). In order to obtain compounds with a better therapeutic index, a number of new nitrosoureas with different substituents have recently been synthesized and experimentally tested, e.g. sugar derivatives. (Anderson et al., Cancer Res. 35., 761 (1976), Hayat et al., Cancer Chemother. Pharmacol. 3, 217 (1979).

In contrast to the previously synthesized compounds, in the present case it concerns oligopeptide esters which carry an N-(2-chloroethyl)-N-nitroso-carbamoyl grouping on the amino group. As starting material for the synthesis of N-[N<1->(2-chloroethyl)-N'-nitroso]-carbamoyl oligopeptide esters, N-[N'-(2-chloroethyl)-N'-nitroso]-carbamoyl amino acids are used or - oligopeptides which in turn are conveniently obtained by reacting N-(2-chloroethyl)-N-nitrosocarbamoylazide with amino acids or oligopeptides. Under the influence of per se known peptide catalysts such as e.g. dicyclohexylcarbodiimide (DCC) reacts the aforementioned N-IN'-(2-chloroethyl)-N'-nitroso]-carbamoylamino acids or -oligopeptides to amides so that these with the ester or amide of another amino acid or another oligopeptide forms a peptide bond. The reaction can be schematically illustrated as follows:

R2= OH or -NH-R^, in which R^ denotes an oligopeptide residue with a free carboxyl group peptidically bound above the amino end group.

Examples of R^:

-glycyl-glycine,

-glycyl-glycyl-glycine and peptides of other amino acids^such as e.g. tripeptide, as well as mixed oligopeptides such as: glycyl-alanine,

alanyl-leucine,

leucyl-alanine,

glycyl-tyrosine,

leucyl-glycyl-tyrosine,

leucyl-glycyl-proline,

R4=OCH3, NH2, -NHR^, in which R^ denotes alkyl or aryl, resp. an oligopeptide residue peptidically bound above the amino end group whose carboxyl end group in turn exists as a methyl ester or amide.

Examples of R^:

-glycine methyl ester,

-glycyl-glycyl-methyl ester,

-glycyl-glycyl-glycyl methyl ester,

peptides of other amino acids to tripeptides as well as mixed oligopeptides such as:

glycyl alanine methyl ester,

leucyl-glycyl-proline methyl ester,

leucyl-alanine amide,

glycyl-tyrosine-amide.

The reaction of the starting material I with the ester or the amide of a second amino acid or an oligopeptide II, which may be the same or different from the first amino acid or the first oligopeptide, conveniently takes place under cooling (approx. 0°C), but can also be carried out at room temperature without problems. For example, 1 mmol of N-(N"-(2-chloroethyl)-N'-nitrosocarbamoyl)-amino acid and 1 mmol of amino acid methyl ester are dissolved together in 20 ml of dichloromethane. In this solution, 1.1 mmol of

(2 30 mg) of dicyclohexylcarbodiimide, dissolved in 10 ml of dichloromethane. The reaction mixture is then stirred for a further 1 hour and allowed to stand overnight at room temperature. After filtering off N,N'-dicyclohexylurea, the filtrate is evaporated under vacuum. The impure product is obtained which is purified by column chromatography on silica gel with ether/dichloromethane of different mixing ratios. After elution, the main fractions are combined and evaporated under vacuum. The residue is taken up in dichloromethane, n-pentane is added if necessary and placed in a refrigerator for crystallisation.

The following examples illustrate the invention:

Example 1

Preparation of N-[N'-(2-chloroethyl)-N'-nitroso-carbamoyl]-glycyl-L-valine methyl ester (general formula III)

1 mmol of N-[N<1->(2-chloroethyl)-N<*->nitroso-carbamoyl-glycine] and 1 mmol of L-valine methyl ester were dissolved together in 20 ml of dichloromethane. In this solution, 1.1 mmol of dicyclohexylcarbodiimide (DCC) dissolved in 10 ml of dichloromethane was added dropwise while stirring. The reaction mixture was stirred for an additional 1 hour and allowed to stand at room temperature overnight. After filtering off N,N'-dicyclohexylurea, the filtrate was evaporated under vacuum. . The impure product was purified by column chromatography (silica gel, eluted with ether/dichloromethane 1:10). From the main fraction, the pure product was obtained after removal of the solvent in vacuo and by recrystallization from dichloromethane/n-pentane. Light yellow needles, yield: approx. 70%, m.p. 97 to 98°C (dec.);

elementary analysis:

''"H-NMR spectroscopic investigation gave characteristic peaks at & = 0.95 ppm (m, -C(CH3)2); 8= 2.20 ppm (m, -CH=);

= 3.50 ppm (t, C1-CH2-;; £ = 3.75 ppm (s, -OCH3);

= 4.20 ppm (-N-CH2-C0- and -CH2"N-N0); = 4.60 ppm

(m, -N-CH-C-); δ = 6.70 ppm (d, -NH-); S = 7.70 ppm (t, -NH-). The mass spectrum showed no signal for a molecular ion, but characteristic fragments at M-107 and M-108, which are attributed to cleavage of Cl(CH2)2~NN0 respectively. Cl(CH2)2-N=NOH.

Example 2

Preparation of N-[N<1->(2-chloroethyl)-N<1->nitroso-carbamoyl]-L-valyl-L-valine methyl ester (general formula III)

1 mmol N-[N'-(2-chloroethyl)-N'-nitroso-carbamoyl]-L-valine and 1 mmol L-valine methyl ester were treated as described in example 1. The product crystallized from dichloromethane/n-pentane as pale yellow needles, yield 65%, m.p. 125 to 126°C (dec.);

elementary analysis:

"^H-NMR spectroscopic examination gave the following characteristic peaks: £ = 0.9-1.1 ppm (m, -C(CH3)2); = 2.20 ppm (m, -CH=);

£ = 3.50 ppm (t, C1-CH2~); £ = 3.75 ppm (s, -OHCH 3 );

δ = 4.20 ppm (t, -CH2-N-NO); £ = 4.50 ppm (m, -N-CH-CO);

£ - 6.60 ppm (d, -NH-); £ = 7.50 ppm (d, -NH-). The mass spectrum showed a molecular peak at 364 and peaks at m/e = 258 and 257 (M-107 and M-108). The last two peaks arise from cleavage of Cl-CH2-CH2-N-NO or Cl-CH2-CH2N-NOH, either by simple cleavage of the N-C bond or by a McLafferty rearrangement.

Example 3

Preparation of N-[N'-(2-chloroethyl)-N<1->nitroso-carbamoyl]-L-valyl—L-methionine methyl ester (general formula III)

1 mmol N-[N<*->(2-chloroethyl)-N'-nitroso-carbamoyl]-L-valine and 1 mmol L-methionine methyl ester were treated as described in Example 1. Yellowish needles (dichloromethane/n-pentane) ,

yield approx. 70%, m.p. 81 to 82°C (dec.).

Elemental analysis:

'''H-NMR spectroscopic examination gave the following characteristic peaks: = 1.00 ppm (m, -C(CH 3 ) 2 ); £ = 2.10 ppm (-CH2~S-CH3; -CH=); £ = 2.55 ppm (t, -CH2"); £ = 3.50 ppm (t, Cl-CH2-);

= 3.80 ppm (s, -OHCH3); £ = 4.20 ppm (-CH2~N-NO);

£ = 4.40 ppm (m, -N-CH-CO); 6 = 4.80 ppm (m, -N-CH-CO);

£ = 6.80 ppm (d, -NH-); £ = 7.50 ppm (d, -NH-). The mass spectrum showed a molecular peak at 396 and peaks at m/e = 289 and 288 (M-107 and M-108).

Example 4

Preparation of N-[N'-(2-chloroethyl)-N'-nitroso-carbamoyl]-L-isoleucyl-L-leucine methyl ester (general formula III)

1 mmol N-[N<1->(2-chloroethyl)-N'-nitroso-carbamoyl]-L-isoleucine and 1 mmol L-leucinemethyster were treated as described in example 1. Light yellow needles (dichloromethane/n-pentane) , dividend approx. 60%, m.p. 128 to 129°C (dec.) .

Elemental analysis:

^H-NMR spectroscopic examination gave the following peaks:

$ = 1.00 ppm (-CH3); S = 1.25-2.00 ppm (-CH=; -CH2~);

£ = 3.50 ppm (t, C1-CH2~); S = 3.80 ppm (s, -OCH3);

$ = 4.20 ppm (t, CH2-N-N0); 8 = 4.40 ppm (m, -N-CH-CO);

S = 4.70 ppm (m, -N-CH-CO); 8 = 6.30 ppm (d, -NH-);

8 = 7.45 ppm (d, -NH-).

The mass spectrum showed a molecular peak at 392 and peaks at m/e = 286, 285 (M-107, M-108).

Example 5

Preparation of N-[N<1->(2-chloroethyl)-N<1->nitroso-carbamoyl]-L-phenylalanyl-L-valine methyl ester (III, R^ = -CH2~CgH^;

R2= -CH(CH3)2)

1 mmol N-(N<1->(2-chloroethyl)-N'-nitroso)-carbamoyl-L-phenylalanine and 1 mmol L-valine methyl ester were treated as described in Example 1. Yellow needles (dichloromethane/n-pentane) , dividend approx. 60%, m.p. 107 to 108°C.

Elemental analysis:

<1>H-NMR spectroscopic examination gave the following peaks: δ = 0.85 ppm (m, -C(CH 3 ) 2 ); 8 = 2.10 ppm (m, -CH=);

& = 3.15 ppm (d, -CH 2 -pH); & = 3.40 ppm (t, Cl-CH2-);

i = 3.70 ppm (s, -OCH3); & = 4.10 ppm (t, -CH2~N-N0);

S = 4.45 and 4.80 ppm (m, -N-CH-CO); & = 6.35 ppm (d, -NH-); 1= 7.25 ppm (-CgH5) ; 8 = 7.55 ppm (d, -NH-). The mass spectrum showed a molecular peak at 412 and peaks at m/e = 305, 304 (M-107, M-108).

Example 6

Preparation of N-[N'-(2-chloroethyl)-N<1->nitroso-carbamoyl]-L-phenylalanyl-L-leucine methyl ester (general formula III)

1mmol N-[N<1->(2-chloroethyl)-N<1->nitroso-carbamoyl]-L-phenyl-alanine and 1 mmol L-leucine methyl ester were treated as described in Example 1. Light yellow needles (dichloromethane/pentane ), yield approx. 60%, m.p. 96 to 96.5°C.

Elemental analysis:

^"H-NMR spectroscopic examination gave characteristic peaks at: 8 = 0.90 ppm (-C(CH3)2); 8 = 1.50 ppm (m, -CH2CH=); 8= 3.15 ppm (d , -CH2-pH); δ= 3.45 ppm (t, Cl-CH2-);

δ = 3.75 ppm (s, -OHCH3); 8 = 4.10 ppm (t, -CH 2 ~N-N 0 );

i = 4.55 ppm (m, -N-CH-CO); 8 = 4.80 ppm (m, -N-CH-CO);

8= 6.20 ppm (d, -NH-); 8 = 7.30 ppm (-CgH5); 8 = 7.50 ppm

(d, -NH-).

The mass spectrum exhibited molecular peaks at 426 and peaks at

m/e = 319, 318 (M-107, M-108).

Example 7

Preparation of N-[N'-(2-chloroethyl)-N<1->nitroso-carbamoyl]-L-phenylalanyl-L-phenylalanine methyl ester (general formula III)

1 mmol N-[N'-(2-chloroethyl)-N<1->nitroso-carbamoyl]-L-phenylalanine and 1 mmol L-phenylalanine methyl ester were treated as described in Example 1. Light yellow needles (dichloromethane/n-pentane) , dividend approx. 60%, m.p. 116 to 117°C.

Elemental analysis:

^"H-NMR spectroscopic examination gave the following peaks: 8 = 3.10 ppm (CH2-ph); 8= 3.40 ppm (t, C1-CH2~); £ = 3.70 ppm (s, -0CH3 ); & = 4.10 ppm (t, -CH2~N-N0); 8 = 4^75 ppm (-N-CH-CO); 8 = 6.10 ppm (d, -NH-); 8 = 7.20 ppm (-CgH5);

8 = 7.40 ppm (d, -NH-).

The mass spectrum exhibited molecular peaks at 4 60 and peaks at

m/e = 353, 352 (M-107, M-108).

Example 8

Preparation of N-[N<1->(2-chloroethylj-N<*->nitroso-carbamoyl]-L-phenylalanyl-L-methionine methyl ester (general formula III)

1 mmol N-[N'-(2-chloroethyl)-N<1->nitroso-carbamoyl]-L-phenylalanine and 1 mmol L-methionine methyl ester were treated as described in Example 1. Light yellow needles (dichloromethane/n-pentane) , dividend approx. 70%, m.p. 74 to 75°C.

Elemental analysis:

"'"H-NMR spectroscopic examination gave the following characteristic peaks: £ = 2.05 ppm (-CH 2 -S-CH 3 ); S = 2.45 ppm (-CH2~);

S = 3.20 ppm (d, -CH2-ph); £ = 3.45 ppm (t, Cl-CH2-);

S = 3.75 ppm (s, -OCH3); S = 4.10 ppm (t, -CH2-N-NO);

£= 4.65 ppm (m, -N-CH-CO); h= 4.80 ppm (m, -N-CH-CO);

£= 6.50 ppm (d, -NH-); 5 = 7.25 ppm (-CgH5); £ = 7.50 ppm

(d, -NH-).

The mass spectrum showed a molecular peak at 444 and peaks at m/e = 337, 336 (M-107, M-108).

Example 9

Preparation of N-[N<1->(2-chloroethyl)-N<*->nitroso-carbamoyl]-L-methionyl-L-phenylalanine methyl ester (general formula III)

1 mmol N-[N<*->(2-chloroethyl)-N<1->nitroso-carbamoyl]-L-methionine and 1 mmol L-phenylalanine methyl ester were treated as described in Example 1. Light yellow needles (dichloromethane/n- pentane), yield approx. 70%, m.p. 70 to 71°C.

Elemental analysis:

<1>H-NMR spectroscopic examination gave the following peaks: £= 2.05 - 2.20 ppm (-CH 2 -S-CH 3 ); £= 2.65 ppm (t, -CH 2 ~ ); £= 3.10 ppm (d, -CH 2 -ph) ; £ = 3.45 ppm (t, C1-CH2~); S = 3.75 ppm (s, -OCH3); £ = 4.10 ppm (t, -CH2~N-NO); £= 4.70-4.90 ppm (m, -N-CH-CO); £ = 6.80 ppm (d, -NH-); 8 = 7.20 ppm (m, -C 6 H 5 ); 8 = 7.65 ppm (d, -NH-). The mass spectrum showed a molecular peak at 444 and peaks at m/e = 337, 336 (M-107, M-198).

Claims (13)

1. N-[N'-(2-chloroethyl)-N<1-> nitroso-carbamoyl]-oligopeptide esters and amides of general formula in which R. = -OCH^ , -NH2 , NH-R^ where R^ denotes an oligopeptide residue whose carboxyl end group is in turn blocked as a methyl ester or amide group. 2. N-[N'-(2-chloroethyl)-N1-nitroso-carbamoyl]-glycyl-L-valine methyl ester. 3. N-[N'-(2-chloroethyl)-N <1-> nitroso-carbamoyl]-L-valyl-L-valine methyl ester. 4. N-[N'~ (2-chloroethyl)-N'-nitroso-carbamoyl]-L-valyl-L-methionine methyl ester. 5. N-[N1-(2-chloroethyl)-N1-nitroso-carbamoyl]-L-isoleucyl-L-leucine methyl ester. 6. N-[N'~ (2-chloroethyl)-N'-nitroso-carbamoyl]-L-phenyl-alanyl-L-valine methyl ester. 7. N-[N'~ (2-chloroethyl)-N'-nitroso-carbamoyl]-L-phenyl-alany 1-L-leucine methyl ester.' 8. N-[N'-(2-chloroethyl)-N'-nitroso-carbamoyl]-L-phenyl-alanyl-L-phenylalanine methyl ester. 9. N-[N1-(2-chloroethyl)-N'-nitroso-carbamoyl]-L-phenyl-alanyl-L-methionine methyl ester. 10. N-[N'-(2-chloroethyl)-N'-nitroso-carbamoyl]-L-methionyl-L-phenylalanine methyl ester. 11. Process for the production of N-[N1-(2-chloroethyl)-N'-nitroso-carbamoyl]-oligopeptide esters or amides according to claim 1, characterized in that N-[N'-(2-chloroethyl)-N1- nitroso-carbamoyl] amino acids or oligopeptides are reacted with an ester or an amide of a second amino acid or an oligopeptide which may be similar or different from the first, under the influence of a suitable basic peptide catalyst and under cooling, to N-[N < 1-> (2-chloroethyl)-N'-nitroso-carbamoyl]-oligopeptide esters or amides. 12. Method according to claim 11, characterized in that the N-[N*-(2-chloroethyl)-N'-nitroso-carbamoyl]-amino acids or oligopeptides used as starting material are prepared from N-(2-chloroethyl)-N- nitroso-carbamoylazide and amino acid respectively. oligopeptide in a manner known per se in a suitable solvent, in particular dichloromethane, and that this reaction solution without isolation is used for reaction with the amino acid ester or amide or the corresponding oligopeptide. 13. Method according to claim 11 or 12, characterized in that dicyclohexylcarbodiimide is used as basic peptide catalyst.