New Di-(β-chloroethyl)-α-amides on N-(meta-Acylamino-benzoyl)-D,L-aminoacid Supports with Antitumoral Activity

In order to obtain new compounds with antitumoural action the N-(meta-acylaminobenzoyl)-α-acylaminobenzoyl)-α-aminoacids 4-9 were prepared. These compounds were subsequently converted into the corresponding Δ2-oxazolin-5-ones 10-15, which in turn were submitted to a ring opening reaction with di-(β-chloroethyl)amine to afford the peptide supported N-mustards 16-21, which showed low toxicity and cytostatic activity similar to that of sarcolisine against the Ehrlich ascite and Walker 253 carcinosarcoma.


Introduction
The sensivity of tumours toward chemotherapic drugs varies widely in function of their biochemical characteristics, especially with regards to the metabolism of nucleic acids and the activity of the cell enzymatic equipment [1][2]. Among the chemotherapic agents used in the treatment of some malignant tumours the N-mustards are particularly important [3][4][5][6]. The cytostatic action of these compounds is exerted by the blocking alkylating effect on the components involved in the anarchic growth of the cancerous cells. The great disadvantage of the N-mustards used in treatment of certain cancer types is their rather high toxicity. By grafting the di-(β-chloroethyl)amino group on some compounds characteristic of living organisms, the transport of the cytostatic group to the cell level is facilitated, the toxicity lowered and the cytostatic action of these N-mustards by means of their antagonistic and alkylating effect increased [7][8][9][10].
Aminoacids and their derivatives are known to participate in animal metabolism decreasing the toxicity of some drug substances and facilitating the chemotherapic activity [4,6,9,[11][12][13][14]. Based on these considerations the present study is aimed to synthesize new N-mustards with the di-(β-chloroethyl)amino group supported by acylated derivatives of N-(meta-aminobenzoyl)-D,L-asparagic acid, N-(meta-aminobenzoyl)-D,L-asparagine and N-(meta-aminobenzoyl)-D,L-methionine, in order to follow their antitumour action by antagonist and alkylating effect.

4-9
The cyclization reaction may be considered to take place according to a mechanism similar to that described in literature for other 2,4-disubstituted-Δ 2 -oxazolones [11,15,[17][18][19]. The products 10-15 were obtained in 62-99% yields as crystalline compounds with sharp melting points. The oxazolone structures were elucidated by means of elemental analysis data and IR and 1 H-NMR spectral measurements. The IR absorption bands characteristic of the >C=O group are to be found at 1630-1820 cm -1 . The NH group vibrations give a single wide band within the 3080-3500 cm -1 range. The C=N bond is made evident by an absorption maximum at 1610-1625 cm -1 and the C-S bond at 654-725 cm -1 . The 1 H-NMR spectra are indicative of the cyclization realization by the disappearance of the signal characteristic of the acylated amine group at 5.50-6.50 ppm as well as by the heterocyclic proton descreening in virtue of its neighborhoods.
The high reactivity of Δ 2 -oxazolin-5-ones towards numerous nucleophilic reagents [11,17,18,20] prompted us to perform the reaction between the newly obtained oxazolones 10-15 and di-(βchloroethyl)amine to obtain new N-mustards 16-21 with possible higher activity and lower toxicity; the electron of the nitrogen atom being involved in an amidic conjugation system (Scheme 3).
The N-mustards are solid compounds with fixed melting points and stable to long preservation in a moistureless medium. Their structures were elucidated by means of elemental analyses and spectral measurements (IR, 1 H-NMR). The IR spectra of the compounds 16-21 show the amide I and II bands as well as intense absorption maxima within the 670-800 cm -1 range corresponding to the C-Cl valence bond. The spectra of the compounds 18 and 21 show an additional band at 718-771 cm -1 attributable to the C-S group. The 1 H-NMR spectra also reveal the oxazolone ring opening. Some spectra of the synthesized compounds are presented in Figures 1-4.

Biological activity
The six synthesized N-mustards 16-21 were tested for their toxicity (DL 50 -median lethal dose) and an experimental study on their antitumoural activity was performed. The antitumor activity was studied using two experimental tumours frequently used in experiments with functionalized aminoacids: Ehrlich ascite and Walker 256 carcinosarcoma. The data in Table 1 reveal the inhibitory effect of the synthesized N-mustards on the experimental tumors. At the same time the inhibition values on the liquid tumors were noticed to be higher than on the solid ones, meaning that the Ehrlich ascite is more sensitive to the action of the tested substances. The compounds 16-21 containing a p-acetylamino moiety in their structure show the highest inhibition values, especially on the liquid tumor. The di-(β-chloroethyl)amino group grafted on N-(meta-aminobenzoyl)-aminoacids 1-3 is seen to be much less toxic and the obtained di-(β-chloroethyl)-amides 16-21 are selective enough toward the neoplastic cells. In comparison with sarcolisine, a well-known antitumoural agent, the compounds 16-21 showed a similar inhibition on both Ehrlich ascite and Walker 256 carcinosarcoma. The great advantage of the tested compounds consists in their much lower toxicity (DL 50 values) than that of sarcolisine (Table 1). This could be explained by the presence of the peptide support involved in the cytotoxic group transport to the malignant cell level where it would inhibit the abnormal synthesis of some proteins and nucleoproteids by an alkylating mechanism. The cytostatic action of di-(β-chloroethyl)amines may be assumed to consist in the possibility of an intramolecular cyclization in aqueous solution affording the formation of an immonium cation consisting of a three-membered ethyleneiminic cation ring which is unstable and can be easily broken being thus able to act by means of the β-carbon atom as an alkylating reagent for the substances containing active groups such as -NH, -SH, etc. (Scheme 4).

Scheme 4.
The alkylating mechanism of the di-(β-chloroethyl)-amines. The alkylating property of di-(β-chloroethyl)amines allow us to assume that the N-mustards 16-21 are linked by means of the di-(β-chloroethyl)-amino group to the active groups -NH, -SH etc. of proteins, nucleoproteids or desoxyribonucleic acid molecules which participate to the cell multiplication process, and thus the anarchical division of cancerous cells is blocked.
The structure of the final and intermediate synthesized compounds 4-21 was confirmed by elemental analysis and spectral measurements (IR and 1 H-NMR). The experimental study revealed the six N-mustards 16-21 to show low toxicity and significant antimour activity on the Ehrlich ascite and Walker 256 carcinosarcoma tumours, similarly to sarcolisine.

General
All melting points were determined on a Melt-Temp R apparatus equipped with a digital thermometer and are uncorrected. C,H,N analyses were performed on an Elemental Exeter Analytical CE 440 Apparatus. The IR spectra were recorded with KBr pellets on a Digilab Scimitar Series spectrophotometer. The 1 H-NMR spectra were recorded on Bruker ARX-300 (300 MHz) or ARX-400 (400 MHz) spectrometers. Chemical shifts were recorded as δ values in parts per million (ppm). All chemical reagents were obtained from Aldrich Chemical Company.

General synthesis method of N-meta-(formylaminobenzoyl) aminoacids 4-6
N-(meta-aminobenzoyl)-L-aminoacid 1-3, (0.02 mol) was introduced in a reaction flask provided with a refluxing condenser, then 80% formic acid (10 mL) was added and the mixture heated on an oil bath for 30 minutes, at 115-120ºC. After cooling, anhydrous acetone (20 mL) was added dropwise under stirring, when colorless crystalline products separated. The product was then filtered, dried in a vacuum oven at 45ºC and finally recrystallized from an ethyl alcohol-acetone mixture.  (6

General synthesis method of N-(meta-acetylaminobenzoyl)-aminoacids 7-9
A reaction mixture consisting of N-(meta-aminobenzoyl)-L-amino acid 1-3, (0.02 mol), 96% acetic acid (10 mL) and acetic anhydride (4 mL) was refluxed for 4 hours in a reaction flask provided with a refluxing condenser. The reaction mixture was the passed into a crystallizer and allowed to stand for 24 hours at room temperature, when the crystalline products separated. It was filtered off, washed twothree times with acetone (5 mL each portion), dried in a vacuum oven at 45ºC and finally purified by recrystallization from boiling ethyl alcohol. (7). White solid; yield 69.5 % (398 mg); m.p. 120-122ºC. Anal. Calc. for C 13

Toxicity study
The acute toxicity, the DL 50 values respectively, was estimated by intraperitoneal administration of the compounds as a suspension in 1% methylcellulose to groups of four mice each, weighting 20±2 g, according to the classical laboratory procedure [21]. The methylcellulose was chosen in these experiments since it is chemically inert and inactive on the animal tissue [22]. The tested animals were carefully followed and the death rate registered seven days later.

Antitumour activity
The antitumour activity was estimated by using two experimental tumors: Ehrlich ascite and Walker 256 carcinosarcoma. Mice A 2 G, weighing 25-30 g (± 2g) and Wistar rats, weighing 100-130 g (± 15 g), were used. The Ehrlich ascite tumors were transplanted to mice and the Walker 256 carcinosarcoma to rats. The ascite was transplanted intraperitoneally, from donors with 14 day old while the solid tumors was transplanted subcutaneously from donors with 21 day old tumors. The substances were administrated intraperitoneally, as suspensions in 1% methylcellulose. Single injection was made seven days after the transplantation with ascite and fourteen days after the transplantation with solid tumour. Concentrations of 400 mg, 200 mg and 40 mg/ kg body were taken and groups of 20 animals used with every concentration. The reference group of 20 animals with tumours was taken for every tumoural series. The inhibition induced by the compounds under study was estimated according to the literature method [9,11,23] after seven and fourteen days from their administration for the ascite and for solid tumour respectively.