New di-(β-chloroethyl)-α-amides on N-(meta-Acylaminobenzoyl)-D, L-aminoacid supports with antitumoral activity

Article abstract of DOI:10.3390/molecules13010177

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 tu

Full text of DOI:10.3390/molecules13010177

Molecules 2008, 13, 177-189
molecules
ISSN 1420-3049
© 2007 by MDPI
www.mdpi.org/molecules
Full Research Paper
New Di-(β-chloroethyl)-α-amides on N-(meta-Acylamino-
benzoyl)-D,L-aminoacid Supports with Antitumoral Activity
Valeriu Sunel 1, Marcel Popa 2, Jacques Desbrières 3, Lenuta Profire 4,*, Pintilie Otilia 1 and
Lionte Catalina 5
1
Department of Organic Chemistry and Biochemistry, Faculty of Chemistry, Al. I. Cuza University,
Iasi, 11 B-dul Carol I, 700506, Romania; e-mails: vsunel@uaic.ro (for Sunel),
povpintilie@yahoo.com (for Otilia)
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; e-mail:
marpopa@ch.tuiasi.ro
3
Institut Pluridisciplinaire de Recherche sur l’Environnement et les Matériaux – Equipe de Physique
et Chimie des Polymèrs (IPREM/EPCP), UMR 5254 CNRS – Pau et Pays de l’Adour University,
Hélioparc Pau Pyrénées 2, av. President Angout, 64053 Pau Cedex 09, 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; E-mail: nprofire@yahoo.com; Phone:
(+40)232 267801; Fax: (+40) 232 211802.
Received: 30 December 2007; in revised form: 23 January 2008 / Accepted: 23 January 2008 /
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.

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Keywords: N-(acylaminobenzoyl)-α-aminoacids; Δ2-oxazolin-5-ones; anticancer agents;
alkylating activity; screening.
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 [3-6]. 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 [7-10].
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, 11-14]. 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.
R
CH COOH
NH
R
CH COOH
NH
HCOOH/ (CH3CO)2O
(CH3COOH)
C
O
C
O
C6H4 NH2 (meta)
C6H4 NH
C
O
R1 (meta)
1-3
4-9
4: R= -CH2-COOH R1= -H; 7: R = -CH2-COOH R1= -CH3
5: R= -CH2-CO-NH2 R1= -H; 8: R = -CH2-CO-NH2 R1= -CH3
6: R= -(CH2)2-S-CH3 R1= -H; 9: R = -(CH2)2-S-CH3 R1= -CH3
1: R= -CH2-COOH
2: R= -CH2-CO-NH2
3: R= -(CH2)2-S-CH3

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179
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
1
were ascertained by means of elemental analyses and IR and H-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.
O
R
CH COOH
NH
R
CH
N
C
O
(CH3CO)2O
- 2 CH3COOH
C
C
O
C6H4 NH
C
O
R1 (meta)
C6H4 NH
C
O
R1 (meta)
10-15
4-9
10: R= -CH2-COOH
R1= -H; 13: R = -CH2-COOH; R1 = -CH3
11: R = -CH2-CO-NH2 R1= -H; 14: R = -CH2-CO-NH2; R1 = -CH3
12: R= -(CH2)2-S-CH3 R1= -H; 15: R = -(CH2)2-S-CH3; R1 = -CH3
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, 17-19]. The products 10-15
were obtained in 62-99% yields as crystalline compounds with sharp melting points. The oxazolone
1
structures were elucidated by means of elemental analysis data and IR and 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
1
cm-1. The 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 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

Molecules 2008, 13
180
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
1
the C-S group. The H-NMR spectra also reveal the oxazolone ring opening. Some spectra of the
synthesized compounds are presented in Figures 1-4.
Scheme 3. Synthesis of the N-mustards 16-21.
O
O
R
CH
C
N(CH2CH2Cl)2
R
CH
N
C
NH(CH2CH2Cl)2
O
NH
C
C
O
C6H4 NH
C
R1 (meta)
C6H4
NH
C
O
R1 (meta)
O
10-15
16-21
16: R= -CH2-COOH
R1= -H; 19: R = -CH2-COOH; R1 = -CH3
17: R = -CH2-CO-NH2 R1= -H; 20: R = -CH2-CO-NH2; R1 = -CH3
18: R= -(CH2)2-S-CH3 R1= -H; 21: R = -(CH2)2-S-CH3; R1 = -CH3
Figure 1. IR spectrum of the N-(meta-formylaminobenzoyl)-L-methionine (6)
Figure 2. IR spectrum of 2-(meta-formylaminophenyl)-4-(β-methylthio-
ethyl)-Δ2-oxazolin-5-one (12).

Molecules 2008, 13
Figure 3. IR spectrum of the α-[N-di-(β-chloroethyl)-amide] of N'-(meta-
181
formylaminobenzoyl)-D,L-methionine (18).
1
Figure 4. H-NMR spectrum of the α-[N-di-(β-chloroethyl)-amide] of N'-
(meta-acetylaminobenzoyl)-D,L-methionine (21).
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.

Molecules 2008, 13
Table 1. DL50 values and inhibition (%) induced by the N-mustards 16-21 on some
182
experimental tumors.
Inhibition (%)
Walker 256
carcinosarcoma
DL50
Compound
mg/Kg body
Ehrlich ascite
mg/Kg body
-
400
35
42
46
56
54
58
56
200
32
38
41
49
48
52
52
40
400
200
21
30
36
42
46
47
48
40
16
770
810
898
986
1020
1270
223
28
32
35
42
41
47
48
27
36
40
48
51
53
50
18
23
31
35
40
41
40
17
18
19
20
21
Sarcolisine
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.
+
CH2
CH2
NH2 R'
- H+
Cl CH2 CH2 N CH2 CH2 Cl
R
Cl CH2 CH2 N
-Cl-
R
+
H2C
N CH2 CH2 NH R'
N CH2 CH2 NH R'
R
Cl CH2 CH2
-Cl-
H2C
R
NH2 R'
- H+
N CH2 CH2 NH R'
R
R' HN CH2 CH2
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.

Molecules 2008, 13
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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 115-120ºC. 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.
N-(meta-Formylaminobenzoyl)-L-asparagic acid (4).White solid; yield 88 % (491 mg); m.p. 76-78ºC.
Anal. Calc. for C12H12N2O6: 51.42 % C, 4.32 % H, 10.00 % N; Found: 51.88 % C, 4.56 % H, 10.33 %
1
N; IR (ν, cm-1): 2500, 3340 (NH); 1525, 1670 (CO); 685 (CH); H-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. 156-158ºC.
Anal. Calc. for C12H13N3O5: 51.60 % C, 4.69 % H, 15.05 % N; Found: 51.38 % C, 5.06 % H, 15.33 %
1
N; IR (ν, cm-1): 2500, 3200 (NH); 1535, 1710 (CO); 685 (CH); H-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).

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184
N-(meta-Formylaminobenzoyl)-L-methionine (6). White solid; yield 72 % (426 mg); m.p. 195-196ºC.
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 45ºC 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-
122ºC. Anal. Calc. for C13H14N2O6: 53.05 % C, 4.80 % H, 9.52 % N; Found: 53.54 % C, 5.03 % H,
1
9.86 % N; IR (ν, cm-1): 2500, 3300 (NH); 1530, 1700 (CO); 690 (CH); H-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. 175-177ºC.
Anal. Calc. for C13H15N3O5: 53.22 % C, 5.16 % H, 14.33 % N; Found: 53.44 % C, 5.63 % H, 14.68 %
1
N; IR (ν, cm-1): 2600, 3300 (NH); 1520, 1680 (CO); 695 (CH); H-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. 272-273ºC.
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
ºC. 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

Molecules 2008, 13
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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 40-45ºC. 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. 180-182ºC. 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);
1
1620 (C=N); 710 (CH); H-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. 198-200ºC. 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. 182-183ºC. 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);
1
1610 (C=N); 1155 (C-O-C); 835 (CH); 654 (C-S); H-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. 149-150ºC. 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. 168-170ºC. 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
1
(C=N); 705 (CH); H-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. 142-143ºC. 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);
1
1078 (C-O-C); 1615 (C=N); 820 (CH); 725 (C-S); H-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).

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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 50-60ºC for one hour and the resulting triethylamine hydrochloride
removed by filtration. The dioxane was then removed by low pressure distillation at a temperature of
40-50ºC 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. 193-194ºC. 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. 198-200ºC. 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,
1
3240 (NH); 1530, 1620 (CO); 745 (C-Cl); 710 (CH); H-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. 177-178ºC. 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. 98-100ºC. 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);
1
1640, 1660 (CO); 750 (C-Cl); 698 (CH); H-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. 168-169ºC. Anal. calc. for C17H22Cl2N4O4: 48.93 % C, 5.31 % H,

Molecules 2008, 13
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13.43 % N, 16.99 % Cl; Found: 49.10 % C, 5.88 % H, 13.70 % N, 17.40 % Cl; IR (ν, cm-1): 3125,
1
3300 (NH); 1535, 1630 (CO); 740 (C-Cl); 698 (CH); H-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. 183-184ºC. 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.
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Sample Availability: Samples of the compounds 16-21 are available from the authors.
© 2008 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.