Next Article in Journal
Chemical Composition of Ground Pearl (Eurhizococcus colombianus) Cysts
Previous Article in Journal
Reaction of Nitrilimines with 2-Substituted Aza-heterocycles. Synthesis of Pyrrolo[1,2-a]pyridine and Pyrimido[2,1-d]1,2,3,5- tetrazine
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

1
Department of Organic Chemistry and Biochemistry, Faculty of Chemistry, Al. I. Cuza University, Iasi, 11 B-dul Carol I, 700506, Romania
2
Department of Natural and Synthetic Polymers, Faculty of Chemical Engineering and Environmental Protection, Gh. Asachi Technical University, 71A B-dul Mangeron, Iasi, 700050, Romania
3
IPREM/EPCP, UMR 5254 CNRS, University of Pau et Pays de l’Adour, Hélioparc Pau Pyrénées 2, av. President Angout, 64053 Pau, France
4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gr. T. Popa Medicine and Pharmacy University, 16 Universitatii Street, 700115, Romania
5
Medical Clinic, Emergency Clinic Hospital, Faculty of Medicine, Gr. T. Popa Medicine and Pharmacy University, 16 Universitatii Street, 700115, Romania
*
Author to whom correspondence should be addressed.
Molecules 2008, 13(1), 177-189; https://doi.org/10.3390/molecules13010177
Submission received: 30 December 2007 / Revised: 23 January 2007 / Accepted: 23 January 2007 / Published: 28 January 2008

Abstract

:
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-chloro-ethyl)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.

Results and Discussion

The synthesis of the new di-(β-chloroethyl)amides with their active group supported by N-(meta-acylaminobenzoyl)-D,L-aminoacids was performed in several steps. First the N-(meta-acylamino-benzoyl)-L-aminoacids 4-9 were obtained by reaction of the previously obtained N-(meta-aminobenzoyl)-L-aminoacids 1-3 [15, 16] with formic acid when N-(meta-formylaminobenzoyl)-L-aminoacids 4-6 compounds have been obtained or acetic anhydride in acetic acid medium when N-(meta-acetylaminobenzoyl)-L-aminoacids 7-9 compounds have been obtained (Scheme 1).
Scheme 1. Synthesis of the N-(meta-acylaminobenzoyl)-L-aminoacids 4-9.
Scheme 1. Synthesis of the N-(meta-acylaminobenzoyl)-L-aminoacids 4-9.
Molecules 13 00177 g005
The six N-acyl derivatives which had not been previously decribed in the literature were obtained as crystalline products melting within a narrow temperature range. The structures of these compounds were ascertained by means of elemental analyses and IR and 1H-NMR spectral measurements. Absorption bands characteristic of these structures were noticed in their IR spectra, namely the amide I and amide II bands within 1635-1710 cm-1 and 1520-1583 cm-1, respectively. Every compound showed an absorption band characteristic of the meta-substituted benzene ring within the 685-830 cm-1 range. In addition to that, the compound 6 and 9 also presented bands characteristic of the C-S valence bands at 728-754 cm-1. The signal at 5.50 ppm in the 1H-NMR of the compounds 1-3, was also presented in the spectra of the reaction products 4-9 between 5.70-6.30 ppm due to the conjugation effects with the >C=O group.
By heating the N-(meta-acylaminobenzoyl)-L-aminoacids 4-9 with acetic anhydride at 100°C the ring closure at the α-carboxyl group takes place, which results in the formation of Δ2-oxazolin-5-ones 10-15 (Scheme 2).
Scheme 2. Synthesis of the Δ2-oxazolin-5-ones 10-15.
Scheme 2. Synthesis of the Δ2-oxazolin-5-ones 10-15.
Molecules 13 00177 g006
The cyclization reaction may be considered to take place according to a mechanism similar to that described in literature for other 2,4-disubstituted-Δ2-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 1H-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 1H-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 Δ2-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-(β-chloroethyl)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, 1H-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 1H-NMR spectra also reveal the oxazolone ring opening. Some spectra of the synthesized compounds are presented in Figure 1, Figure 2, Figure 3 and Figure 4.
Scheme 3. Synthesis of the N-mustards 16-21.
Scheme 3. Synthesis of the N-mustards 16-21.
Molecules 13 00177 g007
Figure 1. IR spectrum of the N-(meta-formylaminobenzoyl)-L-methionine (6)
Figure 1. IR spectrum of the N-(meta-formylaminobenzoyl)-L-methionine (6)
Molecules 13 00177 g001
Figure 2. IR spectrum of 2-(meta-formylaminophenyl)-4-(β-methylthio-ethyl)-Δ2-oxazolin-5-one (12).
Figure 2. IR spectrum of 2-(meta-formylaminophenyl)-4-(β-methylthio-ethyl)-Δ2-oxazolin-5-one (12).
Molecules 13 00177 g002
Figure 3. IR spectrum of the α-[N-di-(β-chloroethyl)-amide] of N'-(meta-formylaminobenzoyl)-D,L-methionine (18).
Figure 3. IR spectrum of the α-[N-di-(β-chloroethyl)-amide] of N'-(meta-formylaminobenzoyl)-D,L-methionine (18).
Molecules 13 00177 g003
Figure 4. 1H-NMR spectrum of the α-[N-di-(β-chloroethyl)-amide] of N'-(meta-acetylaminobenzoyl)-D,L-methionine (21).
Figure 4. 1H-NMR spectrum of the α-[N-di-(β-chloroethyl)-amide] of N'-(meta-acetylaminobenzoyl)-D,L-methionine (21).
Molecules 13 00177 g004

Biological activity

The six synthesized N-mustards 16-21 were tested for their toxicity (DL50 – 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.
Table 1. DL50 values and inhibition (%) induced by the N-mustards 16-21 on some experimental tumors.
Table 1. DL50 values and inhibition (%) induced by the N-mustards 16-21 on some experimental tumors.
CompoundDL50 mg/Kg bodyInhibition (%)
Ehrlich asciteWalker 256 carcinosarcoma
mg/Kg body-4002004040020040
16770353228272118
17810423832363023
18898464135403631
19986564942484235
201020544841514640
211270585247534741
Sarcolisine223565248504840
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 (DL50 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.
Scheme 4. The alkylating mechanism of the di-(β-chloroethyl)-amines.
Molecules 13 00177 g008
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.

Conclusions

Six new di-(β-chloroethyl)-α-amides 16-21 were prepared by the ring opening reaction of the synthesized oxazolones 10-15 using di-(β-chloroethyl)-amine. The new Δ2-oxazolin-5-ones 10-15 in turn were obtained by the action of acetic anhydride on the N-(meta-acylaminobenzoyl)- α-aminoacids 4-9, which were prepared by the reaction of formic acid or acetic anhydride with the N-(m-aminobenzoyl)-α-aminoacids 1-3.
The structure of the final and intermediate synthesized compounds 4-21 was confirmed by elemental analysis and spectral measurements (IR and 1H-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.

Experimental Section

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 1H-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 115-120°C. 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 45°C and finally recrystallized from an ethyl alcohol–acetone mixture.
N-(meta-Formylaminobenzoyl)-L-asparagic acid (4).White solid; yield 88 % (491 mg); m.p. 76-78°C. Anal. Calc. for C12H12N2O6: 51.42 % C, 4.32 % H, 10.00 % N; Found: 51.88 % C, 4.56 % H, 10.33 % N; IR (ν, cm-1): 2500, 3340 (NH); 1525, 1670 (CO); 685 (CH); 1H-NMR δ (CD3CN): 2.30 (d, 2H, CH2); 4.95 (t, 1H, CH); 6.20 (s, 1H, NH); 7.65 (t, 1H, Ar); 7.80 (d, 1H, Ar); 8.05 (d, 1H, Ar); 8.10 (s, 1H, Ar).
N-(meta-Formylaminobenzoyl)-L-asparagine (5). White solid; yield 78 % (434 mg); m.p. 156-158°C. Anal. Calc. for C12H13N3O5: 51.60 % C, 4.69 % H, 15.05 % N; Found: 51.38 % C, 5.06 % H, 15.33 % N; IR (ν, cm-1): 2500, 3200 (NH); 1535, 1710 (CO); 685 (CH); 1H-NMR δ (CF3COOD, 300 MHz): 2.25 (d, 2H, CH2); 4.90 (t, 1H, CH); 6.00 (s, 1H, NH); 7.60 (t, 1H, Ar); 7.85 (d, 1H, Ar); 8.0 (d, 1H, Ar); 8.15 (s, 1H, Ar).
N-(meta-Formylaminobenzoyl)-L-methionine (6). White solid; yield 72 % (426 mg); m.p. 195-196°C. Anal. Calc. for C13H16N2O4S: 52.70 % C, 5.40 % H, 9.45 % N, 10.81 % S; Found: 52.68 % C, 5.23 % H, 9.31 % N, 10.58 % S; IR (ν, cm-1): 3339, 3136 (NH); 1706, 1635 (CO); 828 (CH); 754 (C-S); 1H-NMR δ (DMSO-d6, 400 MHz): 2.08 (s, 3H, CH3); 2.50 (m, 4H, CH2); 4.57 (t, 1H, CH); 6.26 (d, 1H, NH); 7.80 (t, 1H, Ar); 8.34 (d, 1H, Ar); 8.74 (s, 1H, Ar); 9.06 (d, 1H, NH); 9.80 (d, 1H, CH).

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 two-three times with acetone (5 mL each portion), dried in a vacuum oven at 45°C and finally purified by recrystallization from boiling ethyl alcohol.
N-(meta-Acetylaminobenzoyl)-L-asparagic acid (7). White solid; yield 69.5 % (398 mg); m.p. 120-122°C. Anal. Calc. for C13H14N2O6: 53.05 % C, 4.80 % H, 9.52 % N; Found: 53.54 % C, 5.03 % H, 9.86 % N; IR (ν, cm-1): 2500, 3300 (NH); 1530, 1700 (CO); 690 (CH); 1H-NMR δ (CD3COCD3, 300 MHz): 2.25 (d, 2H, CH2); 4.50 (s, 3H, CH3); 5.05 (t, 1H, CH); 5.70 (s, 1H, NH); 7.70 (t, 1H, Ar); 7.85 (d, 1H, Ar); 8.00 (d, 1H, Ar).
N-(meta-Acetylaminobenzoyl)-L-asparagine (8). White solid; yield 73.8 % (432 mg); m.p. 175-177°C. Anal. Calc. for C13H15N3O5: 53.22 % C, 5.16 % H, 14.33 % N; Found: 53.44 % C, 5.63 % H, 14.68 % N; IR (ν, cm-1): 2600, 3300 (NH); 1520, 1680 (CO); 695 (CH); 1H-NMR δ (CD3COCD3, 300 MHz): 2.30 (d, 2H, CH2); 4.50 (s, 3H, CH3); 5.00 (t, 1H, CH); 5.80 (s, 1H, NH); 7.75 (t, 1H, Ar); 7.90 (d, 1H, Ar); 8.05 (d, 1H, Ar); 8.25 (s, 1H, Ar).
N-(meta-Acetylaminobenzoyl)-L-methionine (9). Grey solid; yield 70.32 % (432 mg); m.p. 272-273°C. Anal. Calc. for C14H18N2O4S: 54.19 % C, 5.80 % H, 9.03 % N, 10.32 % S; Found: 53.98 % C, 5.63 % H, 9.07 % N, 10.18 % S; IR (ν, cm-1): 2800, 3304 (NH); 1583, 1684 (CO); 830 (CH); 728 (C-S); 1H-NMR δ (DMSO-d6, 400 MHz): 1.90 (s, 3H, CH3); 2.06 (s, 3H, CH3); 2.12-2.60 (m, 4H, CH2); 4.55 (t, 1H, CH); 7.38 (t, 1H, Ar); 7.66 (d, 1H, Ar); 7.80 (d, 1H, Ar); 8.35 (s, 1H, Ar); 9.05 (d, 1H, NH); 9.15 (s, 1H, NH).

General synthesis method of 2-(meta-acylaminophenyl)-4-(β-carboxymethyl-, β-amidomethyl-, β-methyl-thioethyl)-Δ2-oxazolin-5-ones 10-15

N-(meta-acylaminobenzoyl)-L-amino acid 4-9 (0.02 mol) and acetic anhydride (14 mL) were introduced in a flask provided with a reflux condenser and heated on an oil bath for 60 minutes at 140 °C. After cooling, a thin stream of the resulting solution was poured under stirring into a mixture of anhydrous ethyl ether (40 mL) and anhydrous petroleum ether (50 mL). The solvent mixture was then decanted and the remaining viscous mass washed repeatedly with anhydrous ethyl ether till it turned into a fine powder. It was filtered off, repeatedly washed on the filter with anhydrous ethyl ether and allowed to dry in a vacuum oven for 8 hours at 40-45°C. Finally it was purified by recrystallization from anhydrous dioxane.
2-(meta-Formylaminophenyl)-4-(β-carboxymethyl)-Δ2-oxazolin-5-one (10). Yellow solid; yield 67 % (351 mg); m.p. 180-182°C. Anal. Calc. for C12H10N2O5: 54.97 % C, 3.84 % H, 10.68 % N; Found: 54.08 % C, 4.24 % H, 10.12 % N; IR (ν, cm-1): 3320, 3520 (NH); 1775, 1800 (CO); 1080 (C-O-C); 1620 (C=N); 710 (CH); 1H-NMR δ (CD3CN, 300 MHz): 2.90-3.00 (m, 2H, CH2); 4.95 (t, 1H, CH); 6.60 (s, 1H, NH); 7.65 (t, 1H, Ar); 7.95 (d, 2H, Ar); 8.20 (s, 1H, Ar).
2-(meta-Formylaminophenyl)-4-(β-amidomethyl)-Δ2-oxazolin-5-one (11). Yellow solid; yield 69 % (360 mg); m.p. 198-200°C. Anal. Calc. for C12H10N3O4: 55.17 % C, 4.24 % H, 16.09 % N; Found: 55.41 % C, 4.54 % H, 16.61 % N; IR (ν, cm-1): 3325, 3500 (NH); 1760, 1795 (CO); 1088 (C-O-C); 1625 (C=N); 715 (CH); 1H-NMR δ (DMSO-d6, 300 MHz): 2.90-3.00 (m, 2H, CH2); 5.00 (t, 1H, CH); 6.60 (s, 2H, NH2); 7.70 (t, 1H, Ar); 7.95 (d, 2H, Ar); 8.20 (s, 1H, Ar).
2-(meta-Formylaminophenyl)-4-(β-methylthioethyl)-Δ2-oxazolin-5-one (12). Yellow solid; yield 62.2 % (345 mg); m.p. 182-183°C. Anal. Calc. for C13H14N2O3S: 56.11 % C, 5.03 % H, 10.07 % N, 11.51 % S; Found: 55.83 % C, 4.87 % H, 10.01 % N, 11.32 % S; IR (ν, cm-1): 3429 (NH); 1751, 1631 (CO); 1610 (C=N); 1155 (C-O-C); 835 (CH); 654 (C-S); 1H-NMR δ (DMSO-d6, 300 MHz): 2.08 (s, 3H, CH3); 2.50 (m, 4H, CH2); 4.57 (t, 1H, CH); 6.22 (d, 1H, NH); 7.80 (t, 1H, Ar); 8.32 (d, 2H, Ar); 8.39 (d, 1H, Ar); 8.74 (s, 1H, Ar); 9.70 (d, 1H, CH).
2-(meta-Acetylaminophenyl)-4-(β-carboxymethyl)-Δ2-oxazolin-5-one (13). Yellow solid; yield 63.8 % (352 mg); m.p. 149-150°C. Anal. Calc. for C13H12N2O5: 56.52 % C, 4.38 % H, 10.14 % N; Found: 56.98 % C, 4.74 % H, 10.62 % N; IR (ν, cm-1): 3084, 3210 (NH); 1730, 1770 (CO); 1084 (C-O-C); 1620 (C=N); 728 (CH); 1H-NMR δ (CD3CN, 300 MHz): 2.95 (d, 2H, CH2); 4.50 (s, 3H, CH3); 5.20 (t, 1H, CH); 6.50 (s, 2H, NH); 7.70 (t, 1H, Ar); 8.00 (d, 2H, Ar); 8.25 (s, 1H, Ar).
2-(meta-Acetylaminophenyl)-4-(β-amidoethyl)-Δ2-oxazolin-5-one (14). White solid; yield 61.5 % (337 mg); m.p. 168-170°C. Anal. Calc. for C13H13N3O4: 56.72 % C, 4.76 % H, 15.27 % N; Found: 56.91 % C, 4.95 % H, 15.43 % N; IR (ν, cm-1): 3100, 3320 (NH); 1770, 1800 (CO); 1084 (C-O-C); 1620 (C=N); 705 (CH); 1H-NMR δ (DMSO-d6, 300 MHz): 2.80 (d, 2H, CH2); 4.50 (s, 3H, CH3); 5.05 (t, 1H, CH); 5.70 (s, 1H, NH); 6.60 (s, 2H, NH2); 7.70 (t, 1H, Ar); 7.90 (d, 2H, Ar); 8.20 (s, 1H, Ar).
2-(meta-Acetylaminophenyl)-4-(β-methylthioethyl)-Δ2-oxazolin-5-one (15). Brown solid; yield 58.7 % (342 mg); m.p. 142-143°C. Anal. Calc. for C14H16N2O3S: 57.53 % C, 5.47 % H, 9.58 % N, 10.95 % S; Found: 57.18 % C, 5.21 % H, 9.24 % N, 10.81 % S; IR (ν, cm-1): 3080, 3240 (NH); 1680, 1815 (CO); 1078 (C-O-C); 1615 (C=N); 820 (CH); 725 (C-S); 1H-NMR δ (DMSO-d6, 400 MHz): 2.07 (s, 3H, CH3); 2.26 (s, 3H, CH3); 2.57 (m, 4H, CH2); 4.57 (m, 1H, CH); 5.73 (s, 1H, NH); 7.80 (t, 1H, Ar); 8.33 (d, 1H, Ar); 8.39 (d, 1H, Ar); 8.74 (s, 1H, Ar).

General synthesis of α-[N-di-(β-chloroethyl)amides] of the N’-(meta-acylaminobenzoyl)-D,L-amino-acids 16-21

Δ2-Oxazolin-5-ones 10-15 (0.02 mol), di-(β-chloroethyl)amine hydrochloride (0.004 mol), anhydrous dioxane (20 mL) and triethylamine (0.556 mL) were introduced in a flask provided with a refluxing condenser, heated at 50-60°C for one hour and the resulting triethylamine hydrochloride removed by filtration. The dioxane was then removed by low pressure distillation at a temperature of 40-50°C and the viscous product remaining in the flask solved in absolute methyl alcohol (25 mL). After the vacuum distillation of the methyl alcohol, a sticky product resulted which turned into a solid product, after repeated washings with anhydrous ethyl ether. It was finally purified by recrystallization from boiling anhydrous methyl alcohol.
α-[N-di-(β-Chloroethyl)amide] of N’-(meta-formylaminobenzoyl)-D,L-asparagic acid (16). Light-yellow solid; yield 51.4 % (39 mg); m.p. 193-194°C. Anal. Calc. for C16H19Cl2N3O5: 47.54 % C, 4.74 % H, 10.39 % N, 17.54 % Cl; Found: 47.22 % C, 4.50 % H, 10.24 % N, 17.68 % Cl; IR (ν, cm-1): 3350, 3600 (NH); 1640, 1680 (CO); 770 (C-Cl); 705 (CH); 1H-NMR δ (CD3COCD3, 300 MHz): 2.60 (d, 2H, CH2); 4.20 (t, 2H, CH2); 4.80 (t, 1H, CH); 5.10 (t, 2H, CH2); 7.70 (t, 1H, Ar); 7.85 (d, 1H, Ar); 7.95 (d, 1H, Ar); 8.10 (s, 1H, Ar).
α-[N-di-(β-Chloroethyl)diamide] of N’-(meta-formylaminobenzoyl)-D,L-asparagic acid (17). Yellow solid; yield 50.5 % (40.5 mg); m.p. 198-200°C. Anal. calc. for C16H20Cl2N4O4: 47.65 % C, 5.00 % H, 13.89 % N, 17.58 % Cl; Found: 47.85 % C, 5.15 % H, 14.11 % N, 17.92 % Cl; IR (ν, cm-1): 3090, 3240 (NH); 1530, 1620 (CO); 745 (C-Cl); 710 (CH); 1H-NMR δ (CD3CN, 300 MHz): 2.90 (d, 2H, CH2); 4.40 (t, 2H, CH2); 4.95 (t, 1H, CH); 5.35 (t, 2H, CH2); 6.60 (s, 2H, NH2); 7.60 (t, 1H, Ar); 7.80 (d, 2H, Ar); 8.20 (s, 1H, Ar).
α-[N-di-(β-Chloroethyl)amide] of N’-(meta-formylaminobenzoyl)-D,L-methionine (18). Light brown solid; yield 56.38 % (47.30 mg); m.p. 177-178°C. Anal. calc. for C17H23Cl2N3O3S: 48.57 % C, 5.47 % H, 10.00 % N, 16.90 % Cl, 7.61 % S; Found: 48.31 % C, 5.40 % H, 9.85 % N, 16.83 % Cl, 7.41 % S; IR (ν, cm-1): 3065, 3472 (NH); 1700 (CO); 771 (C-S); 801 (C-Cl); 848 (CH); 1H-NMR δ (DMSO-d6, 400 MHz): 2.10 (s, 3H, CH3); 2.56 (m, 4H, CH2); 3.50 (t, 4H, CH2); 3.82 (t, 4H, CH2); 4.56 (t, 1H, CH); 6.20 (d, 1H, NH); 7.80 (t, 1H, Ar); 8.43 (d, 1H, Ar); 8.48 (d, 1H, Ar); 8.73 (s, 1H, Ar); 9.07 (d, 1H, NH); 9.68 (d, 1H, CH).
α-[N-di-(β-Chloroethyl)amide] of N’-(meta-acetylaminobenzoyl)-D,L-asparagic acid (19). Grey solid; yield 52.8 % (44 mg); m.p. 98-100°C. Anal. calc. for C17H21Cl2N3O5: 48.82 % C, 5.06 % H, 10.05 % N, 16.95 % Cl; Found: 48.35 % C, 5.32 % H, 10.23 % N, 17.12 % Cl; IR (ν, cm-1): 3340, 3500 (NH); 1640, 1660 (CO); 750 (C-Cl); 698 (CH); 1H-NMR δ (CD3CN, 300 MHz): 2.90-3.00 (m, 2H, CH2); 4.95 (t, 1H, CH); 5.80 (s, 1H, NH); 7.65 (t, 1H, Ar); 7.95 (d, 2H, Ar); 8.20 (s, 1H, Ar).
α-[N-di-(β-Chloroethyl)diamide] of N’-(meta-acetylaminobenzoyl)-D,L-asparagic acid (20). Yellow solid; yield 53.8 % (44.8 mg); m.p. 168-169°C. Anal. calc. for C17H22Cl2N4O4: 48.93 % C, 5.31 % H, 13.43 % N, 16.99 % Cl; Found: 49.10 % C, 5.88 % H, 13.70 % N, 17.40 % Cl; IR (ν, cm-1): 3125, 3300 (NH); 1535, 1630 (CO); 740 (C-Cl); 698 (CH); 1H-NMR δ (CD3CN, 300 MHz): 2.75 (d, 2H, CH2); 4.50 (t, 2H, CH2); 4.90 (t, 1H, CH); 5.30 (t, 2H, CH2); 6.60 (s, 2H, NH2); 7.70 (t, 1H, Ar); 7.90 (d, 2H, Ar); 8.20 (s, 1H, Ar).
α-[N-di-(β-Chloroethyl)amide] of N’-(meta-acetylaminobenzoyl)-D,L-methionine (21). Light brown solid; yield 58.3 % (50.6 mg); m.p. 183-184°C. Anal. calc. for C18H25Cl2N3O3S: 49.76 % C, 5.76 % H, 9.67 % N, 16.35 % Cl, 7.37 % S; Found: 49.58 % C, 5.62 % H, 9.46 % N, 16.17 % Cl, 7.28 % S; IR (ν, cm-1): 3010, 3328 (NH); 1685 (CO); 718 (C-S); 670 (C-Cl); 855 (CH); 1H-NMR δ (DMSO-d6, 400 MHz): 2.11 (s, 3H, CH3); 2.30 (s, 3H, CH3); 2.56 (m, 4H, CH2); 3.28 (t, 4H, CH2); 3.85 (t, 4H, CH2); 4.56 (t, 1H, CH); 6.02 (s, 1H, NH); 7.80 (t, 1H, Ar); 8.32 (d, 1H, Ar); 8.42 (d, 1H, Ar); 8.73 (s, 1H, Ar); 9.08 (d, 1H, NH).

Toxicity study

The acute toxicity, the DL50 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 A2G, 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.

References

  1. Capasso, S.; Sica, F.; Mazarella, L.; Balboni, G.; Guerrini, R.; Salvadori, S. Acid catalysis in the formation of dioxopiperazines from peptides containing tetrahydroisoquinoline-3-carboxylic acid at position 2. Int. J. Pept. Protein Res. 1995, 45, 567–573. [Google Scholar]
  2. Paci, A.; Rieutord, A.; Brion, F.; Prognon, P. Separation methods for alkylating antineoplastic compounds. J. Chromatogr. B. Biomed. Sci. Appl. 2001, 764, 255–287. [Google Scholar] [CrossRef]
  3. Friedlos, F.; Denny, A.W.; Palmer, B.D.; Springer, C.J. Mustard prodrugs for activation by Escherichia coli nitroreductase in gene-directed enzyme prodrug therapy. J. Med. Chem. 1997, 40, 1270–1275. [Google Scholar]
  4. Sosnovsky, G.; Baysal, M.; Erciyas, E. In the search for new anticancer drugs. 28. Synthesis and evaluation of highly active aminoxyl labeled amino acid derivatives containing the [N'-(2-chloroethyl)-N'-nitrosoamino]carbonyl group. J. Pharmaceut. Sci. 1994, 83, 999–1005. [Google Scholar] [CrossRef]
  5. Zheleva, A.; Raikov, Z.; Ilarionova, M.; Todorov, D. Spin labeled amino acid nitrosourea derivatives-synthesis and antitumour activity. Pharmazie 1995, 50, 25–26. [Google Scholar]
  6. Zheleva, A.; Raikov, Z.; Ilarionova, M.; Carpenter, B.; Todorov, D.; Armstrong, N. Potential antimelanomic drugs. I. Synthesis and antimelanomic effect of a spin labelled D,L-amino acid containing a 2-chloroethylnitrosocarbamoyl group. Pharmazie 1996, 51, 602–604. [Google Scholar]
  7. Sosnovsky, G.; Prakash, I.; Rao, N.U. In the search for new anticancer drugs. XXIV: Synthesis and anticancer activity of amino acids and dipeptides containing the 2-chloroethyl- and [N'-(2-chloroethyl)-N'-nitroso]-aminocarbonyl groups. J. Pharmaceut. Sci. 1993, 82, 1–10. [Google Scholar] [CrossRef]
  8. Sunel, V.; Popa, M.; Popa, A.; Soldea, C. Cellulose derivatives of the N-mustard type with cytostatic activity. Cellulose Chem. Technol. 2000, 34, 269–274. [Google Scholar]
  9. Popa, M.; Balaita, L.; Sunel, V. Immobilization of alpha-[di-beta-chloroethyl)-amide]-N-(m-nitrobenzoyl)-D,L-asparagic acid on xanthan. J. Biomed. Appl. 2003, 18, 83–94. [Google Scholar]
  10. Sunel, V.; Basu, C. Synthesis of antitumoural substances. Synthesis of 2-[m-(N-carbobenzoxy)-methylaminophenyl]-4-(beta-carboxymethyl)-Δ2-oxazolinone-5. An. St. Univ. "Al. I. Cuza" Iasi s.Ic. 1999, 7, 111–116. [Google Scholar]
  11. Sunel, V.; Lionte, C.; Basu, C. New antitumour alkylating compounds with N-[m-(arylthio-carbamoyl)-aminobenzoyl]-asparagic acids as support. Chem. Indian J. 2005, 2, 1–6. [Google Scholar]
  12. Sunel, V.; Basu, C.; Drochioiu, G.; Filimon, M. New di-(beta-chloroethyl)-alpha-amides of N-(m-nitrobenzoyl)-L-asparagic acid and N-(m-nitrobenzoyl)-L-asparagine with cytostatic action. Rev. Med. Chir. Soc. Med. Nat. Iasi 2000, 106, 505–510. [Google Scholar]
  13. Soldea, C.; Sunel, V. Synthesis and study on toxicity of some di-(beta-chloroethyl)-amines with p-N-(carboxyethyl)-methylamino benzoic acid as support. Comp. Renol. Acad. Bulg. Sci. 1995, 48, 67–70. [Google Scholar]
  14. Cecal, A.; Sunel, V.; Ghimiciu, L. Kinetic and analytical study of precipitation reactions with 110AgNO3 of some di-(beta-chloroethyl)-amino derivatives and hydrochlorides with esters of N-(p-aminobenzoyl)-L-aspartic acids as carriers from dimethylformamide-water solution. J. Radioanal. Chem. 1983, 78, 247–253. [Google Scholar]
  15. Sunel, V.; Basu, C.; Soldea, C.; Sirbu, D. New di-(beta-chloroethyl)-amides with potential antitumoural activity derived from m-nitro- and m-amino-benzoyl-D,L-asparagic acids. An. St. Univ. "Al. I. Cuza" Iasi 1998, s.Ic. 6, 35–44. [Google Scholar]
  16. Sunel, V.; Lionte, C.; Popa, M.; Pintilie, O.; Mungiu, O. C.; Teleman, S. Synthesis of new methionine derivatives for the treatment of paracetamol-induced hepatic injury. Eurasian Chem. Tech. J. 2002, 4, 201–222. [Google Scholar]
  17. Pintilie, O.; Moise, M.; Profire, L.; Sunel, V. Synthesis and biological activities of some beta-lactamic derivatives. Farmacia 2006, 14, 61–65. [Google Scholar]
  18. Pintilie, O.; Profire, L.; Sunel, V.; Popa, M.; Pui, A. Synthesis and antimicrobial activity of some new 1,3,4-thiadiazole and 1,2,4-triazole compounds having a D,L-methionine moiety. Molecules 2007, 12, 103–113. [Google Scholar]
  19. Sunel, V.; Ciugureanu, C.; Budeanu, C.; Antohe, N.; Popa, M. Synthesis of antitumoural substances. Azotiperites with esters of p-nitro- and p-amino-benzoyl-D,L-asparagic acids as support. Rev. Chim. 1988, 39, 577–581. [Google Scholar]
  20. Sunel, V.; Ciobanu, A.; Profire, L.; Basu, C. Synthesis and study on toxicity of some new (sulphon-amidophenyl)-amides of N-(m-nitrobenzoyl)-D,L-asparagine. Rev. Med. Chir. Soc. Med. Nat. Iasi 2005, 109, 671–676. [Google Scholar]
  21. Pavelescu, M.; Hriscu, A. Laboratory manual of pharmacology; "Gr. T. Popa" Medicine and Pharmacy University Press: Iasi, Romania, 1983. [Google Scholar]
  22. Stanescu, V. Pharmaceutical Practice.; Academy Press: Bucharest, 1988. [Google Scholar]
  23. Graff, A.; Bielka, H. Aspects of Experimental Oncology; Academy Press: Bucharest, 1962. [Google Scholar]
  • Sample Availability: Samples of the compounds 16-21 are available from the authors.

Share and Cite

MDPI and ACS Style

Sunel, V.; Popa, M.; Desbrières, J.; Profire, L.; Otilia, P.; Catalina, L. New Di-(β-chloroethyl)-α-amides on N-(meta-Acylaminobenzoyl)- D,L-aminoacid Supports with Antitumoral Activity. Molecules 2008, 13, 177-189. https://doi.org/10.3390/molecules13010177

AMA Style

Sunel V, Popa M, Desbrières J, Profire L, Otilia P, Catalina L. New Di-(β-chloroethyl)-α-amides on N-(meta-Acylaminobenzoyl)- D,L-aminoacid Supports with Antitumoral Activity. Molecules. 2008; 13(1):177-189. https://doi.org/10.3390/molecules13010177

Chicago/Turabian Style

Sunel, Valeriu, Marcel Popa, Jacques Desbrières, Lenuta Profire, Pintilie Otilia, and Lionte Catalina. 2008. "New Di-(β-chloroethyl)-α-amides on N-(meta-Acylaminobenzoyl)- D,L-aminoacid Supports with Antitumoral Activity" Molecules 13, no. 1: 177-189. https://doi.org/10.3390/molecules13010177

Article Metrics

Back to TopTop