LT4302B - Potassium salts of n-propionyl-4-hydroxy-3-nitro-l-phenylalanine and n-propionyl-4-metoxy-3-nitro-dl-phenylalanine having antitumour activity - Google Patents

Abstract

This invention describes biologically active compounds, such as water-soluble N-propionil-4-hydroxy-3-nitro-L-phenylalanine and N-propionyl-4-methoxy-3-nitro-DL-phenylalanine potassium salts. The synthetic derivatives of amino acids are slightly toxic, but the experiments with mice have shown a high level of effect against cells affected by cancer. They slow down the growth of sarcoma 180, respectively by 94.5 and 89 per cent, and lengthen the time of living by up to 50 per cent.

Description

The invention relates to non-proteinaceous amino acid chemistry and is directed to potassium salts of N-propionyl-4-hydroxy-3-nitro-L-phenylalanine and N-propionyl-4-methoxy-3-nitro-DL-phenylalanine with antitumor activity.

Some N-acylated proteinaceous amino acids containing alkanecarboxylic acid residues are known to exhibit antitumor activity. Of this type of compounds, N-propionyl-L-valine, N-lauroyl-L-leucine and N-myristoyl-L-isoleucine are characterized by carcinolytic activity [USA Pat. 3845097, 1974 and 3919291, 1975], the most active of which was N-propionyl-L-valine (A-195) [K. Fukushima, S. Toyoshima. Cancer Chemother. Rep. Part 1, Vol. 59, no. 2, p. 309-318 (1975)]:

(CHJ-CHCHCOOH

I hncoch ₂ ch ₃

A-195

Compound A-195 has been studied in clinics [Π.B. lop6aHeBa m πρ. ΧμμμοTepannn 3noKaMecTBeHHbix H0B006pa30BaHMfi. Μτογμ HayKM m τθχημκμ. BMHMTM. Οηκοπογμη. 1982, τ. 12, c. 201]. A-195 was chosen as the closest structural analogue to the prototype of our patented compounds.

N-acetyl-L-cysteine, which inhibits carcinogenesis experimentally, e.g. exhibit chemopreventive properties of cancer [S. De Flora et al. In: C. loannides and D.F.V. Lewis (eds), Drugs, Diet and Disease, Vol. 1: Mechanistic Approaches to Cancer, Ellis Horwood, Hemel Hempstead, UK, 1995, p. 151-203].

Thus, the literature cited above demonstrates that the compounds obtained by the modification of amino acids with alkanecarboxylic acids make them suitable for clinical use in the treatment of cancer.

SUMMARY OF THE INVENTION The present invention relates to novel, low-toxic, water-soluble antitumor compounds obtained by introducing a propionic acid residue into non-proteinogenic aromatic amino acid molecules and modifying the carboxylic group of the resulting derivatives by synthesizing potassium salts (1c and 2c):

RO — C _ # - CHjCHCOOK ₀ / HNCOCH ₂ CH ₃ ,

C, 2c where 1c: L, R = H;

2c: DL, R = CH ₃

Synthesis of compounds 1c and 2c was carried out according to the scheme below:

RO— oz

Oh, N

CHoCHCOOH ² I NH,

2) B

RO o ₂ n - CH, CHCOOH - I

HNCOCH, CH,

1a, 2a

1b, 2b

ROΟ, Ν ^

CHjCHCOOK

HNCOCH ₂ CH ₃

1c, 2c wherein 1a to 1c: L, R = H;

2a-2c: DL, R = CH ₃ .

A: NaOH / H ₂ O, CH ₃ CH ₂ COCl / Et ₂ O; B: HCl / H ₂ O; C: KOH / EtOH.

By condensing propionyl chloride with 4-hydroxy-3-nitro-L-phenylalanine 1a (method A) and 4-methoxy-3-nitro-DL-phenylalanine hydrochloride 2a (method B), respectively [IO. flery-TMC, M. OrpayKac. > KypH. o5ies. χμμμμ, vol.32, No 4, c. 1255-1259 (1962)], N-propionyl-4-hydroxy-3-nitro-L-phenylalanine 1b and N-propionyl-4-methoxy-3-nitro-DL-phenylalanine 2b (stages A + B) were synthesized in aqueous sodium hydroxide solution. N-Propionylated amino acid derivatives 1b and

2b, treatment with potassium alcohol alkaline solution yielded water soluble potassium salts of Npropionyl-4-hydroxy-3-nitro-L-phenylalanine and N-propionyl-4-methoxy-3-nitro-DL-phenylalanine (Step C). The gross formulas, melting points, yields and elemental analysis of the synthesized amino acid derivatives 1b, 2b, 1c and 2c are shown in Table 1. The IR spectra of these compounds are recorded on a Specord 71 (Germany) device with KBr tablets. The IR spectra data are shown in Table 2. ¹ H NMR spectra were recorded on a Hitachi R-22 spectrometer with a working frequency of 90 MHz, internal standard is hexamethyldisiloxane, temperature 35 ° C. Data for ¹ H NMR spectra are shown in Table 3.

The following examples illustrate the present invention.

An example. N-Propionyl-4-hydroxy-3-nitro-L-phenylalanine 1b

2.26 g (10 mM) of 4-hydroxy-3-nitro-L-phenylalanine are dissolved in 16 ml (32 mM) of 2N sodium hydroxide aqueous solution, 15-20 g of ice, 10 ml of ethyl ether are added and 1 ml is added with vigorous stirring. ml (12 mM) of propionyl chloride. After that, stir for 1.5 hours. at a temperature of not more than 2 ° C, ensuring that the reaction contents are always slightly alkaline. The ethereal layer is separated and the aqueous layer is acidified with dilute hydrochloric acid to pH3 with ice-water. The precipitated product is recrystallized from a mixture of alcohol and ethyl acetate. The resulting material 1b is in the form of yellowish crystals.

An example. N-Propionyl-4-methoxy-3-nitro-DL-phenylalanine 2b.

Under similar conditions to Example 1, compound 2b was synthesized from 2.77 g (10 mM) of 4-methoxy-3-nitro-DL-phenylalanine hydrochloride, 16 ml (32 mM) of 2N aqueous sodium hydroxide and 1 ml (12 mM) of propionyl chloride. Substance 2b, recrystallized from a mixture of alcohol and water, is in the form of white crystals. ³

An example. Potassium salt of N-Propionyl-4-hydroxy-3-nitro-L-phenylalanine 1c.

Dissolve 2.82 g (10 mM) of 1b in 20 ml of absolute ethanol and add 5 ml (10 mM) of 2N potassium alcohol solution. Then ml of ether is added to the reaction solution. The separated precipitate is recrystallized from a mixture of ethanol and ether. The resulting material 1c is in the form of red crystals, soluble in water, in hot spirit. Rf = 0.62 (Silufol UV254, eluent: n-butanol saturated with water).

An example. Potassium salt of N-Propionyl-4-methoxy-3-nitro-DL-phenylalanine 2c.

Under similar conditions as in Example 3, 2.96 g (10 mM) of 2b and 5 ml (10 mM) of 2N alcoholic potassium alkali gave 2c. Material 2c, recrystallized from a mixture of alcohol and ether, is a white crystalline solid, soluble in water, in hot ethanol. Rf = 0.44 (Silufol UV254, eluent: n-butanol saturated with water).

An example. Biological evaluation of compounds 1c and 2c.

The acute toxicity of Compounds 1c and 2c was determined in non-linear white mice by intraperitoneal injection of test preparations and observation of test animals for 30 days.

Antineoplastic activity was assayed using three models of grafted tumors, namely, sarcoma 180 (S-180), Erlich ascitic carcinoma (EAC), and L 1210 (L-1210). Tumors in mice were grafted using conventional techniques. The experiments were performed on non-linear and hybrid BDFf mice weighing 18-22 g.

The structural analog N-propionyl-t-valine (A-195) was used for comparison.

Test compounds were dissolved in saline by daily injection into the peritoneal cavity in mice for 2-5 days. The antitumor activity of the compounds was evaluated by determining the percent inhibition of S-180 tumor and EAC and L-1210 ascitic fluid development as compared to controls. Treatment was initiated one day after EAC and L-1210 and three days after S-180 grafting. For evaluation of tumor S-180 growth inhibition, animals were decapitated 5 days after the last injection. The amount of ascitic fluid was determined on day 10 after EAC and on day 8 after L-1210 grafting.

Bioassay data were processed by Lichfield and Wilcoxon's method of variational statistics [J.T. Litchfield, F. Wilcoxon. J. Pharmacol. Exp. Therap., Vol. 96, p. 99-113 (1949)].

The bioassay data for the tested compounds are shown in Table 4.

As shown in Table 4, compounds 1c and 2c are of low toxicity with an acute toxicity 2.5-fold lower than that of A-195. Compounds 1c and 2c inhibit sarcomas 180 growth by 94.5% and 89.1%, respectively, whereas prototype A-195 inhibits this tumor model by 74.9%. 1c and 2c strongly or completely inhibit the development of ascites fluid. Furthermore, at the dose shown in Table 2c 2c, 57.3% prolonged the survival of mice grafted with EAC. Compounds 1c and 2c slightly altered body weight in grafted tumors - only 1c reduced 13.1% in mice with EAC and 2c increased 16.1% in mice with sarcoma 180.

Comparison of the results of biological studies shows that 1c and 2c outperform the structural analog A-195, respectively.

The widely used drug ftorafur inhibits tumor growth of 66% [BKcnepMMeHTanbHan oøeHKa npoTMBOonyxoneBbix npenapaTOB b CCCP m CIUA in sarcoma 180. ΠΟΑ pep. 3.Π. CocpbMHoii, A.B. CbipKMHa (CCCP) m A. ΓοπflMHa, A. KnafiHa (CIUA). M. MepHaiHHa, 1980, c. 250], and its LD50 = 640 mg / kg (female mice) and its LD50 = 750 mg / kg (male mice) when administered intraperitoneally [M.M. ΚραΒΜβΗκο n pp. B kh .: SKcnepuMeHTanbHaji m κπμημμθοKaa cpapMaKonea. Para, 3nHaTHe, 1977, Bbin. 7, c. 100-115].

Thus, the water-soluble potassium salts of N-propionyl-4-hydroxy-3-nitro-L-phenylalanine and N-propionyl-4-methoxy-3-nitro-DL-phenylalanine (1c and 2c) have low toxicity and exhibit significant antitumor activity. can be promising compounds in experimental biology and medicine.

Table. Gross formula, melting point, yield, and elemental analysis of compounds 1b, 2b, 1c and 2c.

Jung. The gross formula Melting point (° C) Yield (%) C H N K UH C12H14N2O6 159-161 78 51.24 ^ 5l; 07 ^b) 5.20 5.00 9.94 9.93 2b C13H16N2O6 151-152 92 52.89 52.70 5.33 5.44 9.35 9.45 1c * ⁵ C ₁₂ H ₁₃ KN ₂ O ₆ 258-263 (See) 90 44.70 44.99 3.98 4.09 8.58 8.74 12.91 12.21 2c C13H15KN2O6 138-142 84 46.82 46.70 4.50 4.52 8.44 8.38 11.90 11.67

^(a) upper value: found; ^(b) Lower value: calculated

Table. IR spectra of compounds 1b, 2b, 1c and 2c (v cm ^-1 )

Jung. C = 0 for carboxyl C = 0 amide NO ₂ NH 1b 1717 1613 1533, 1323 3390 2b 1700 1640 1527, 1353 3310 1c 1623 1595 1520, 1333 3300 2c 1630 1587 1520, 1345 3320

Table. ¹ H NMR Spectra of Compounds 1b, 2b, 1c and 2c (scale 5, chemical shifts md, multiplicity)

Jung. Solvent CH ₃ O β-Phenyl 6-H ch ₂ CHCOO ch ₃ CH ₂ CO 2-H 5-H 1b (CD ₃ ) ₂ CO - 7.92d 7.04d 7.53dd 3.05m 4.69m 0.94h 2.03k 2b (CD ₃ ) ₂ CO 3.85s 7.65d 7.16d 7.48dd 3.04m 4.69m 0.94h 2.11k 1c DMSO ₆ - 7.46d 6.77d 7.07dd 2.86m 4.03m 0.95t 2.05k 2c DMSO-dg 3.82s 7.50d 7.13d 7.35dd 2.93m 3.85m 0.88h 2.00k

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Claims (3)

DEFINITION 1. Compounds of the general structural formulas of the potassium salt of N-propionyl-4-hydroxy-3-nitro-L-phenylalanine and N-propionyl-4-methoxy-3-nitro-DL-phenylalanine: RO-CH ₂ ęHCOOK / o ₂ n ₂ hncoch ck ₃ H wherein R represents the residue of L-phenylalanine, and R represents CH _3, while the residue is DL-phenylalanine. Compounds according to claim 1, characterized in that they exhibit antitumor activity by inhibiting the growth of sarcoma 180 by 94.5% and 89.1%, respectively. 3. The compound N-propionyl-4-methoxy-3-nitro-DL-phenylalanine potassium salt of claim 1, wherein it prolonged the survival of mice grafted with EAC by 57.3%.