Optimized Synthesis of New N-Mustards Based on 2-Mercaptobenzoxazole Derivatives with Antitumor Activity

New di-(β-chloroethyl)-amides of some acids derived from 2-mercaptobenzoxazole were prepared by reaction of the corresponding pivalic mixed anhydrides with di-(β-chloroethyl)-amine. A study regarding the optimization of the chemical reactions was made for the case of di-(β-chloroethyl)-amines. The quantum chemical analysis by Spartan’14 was made in order to establish the most stable configuration of the ground electronic states for the obtained chemical structures and some physico-chemical parameters of N-mustards reported in this paper. Mercaptobenzoxazoles substituted in the side chain with the cytotoxic group show antitumor activity and they inhibit Ehrlich Ascites in an appreciable proportion compared to the drug I.O.B.-82, as our studies evidenced.


Introduction
Cancer can be considered one of the most pressing concerns of research in medicine, chemotherapy, phytochemistry, and biology. Chemotherapy alone or in combination with radio-immunosurgical treatment is the major strategy in the fight against cancer.
However, there are some drawbacks in the administration of various chemicals used as cytostatic drugs. First, their poor selectivity towards cancer cells, they also act on normal cells and are, therefore, toxic for the body. Second, there are concerns about the installation of the resistance of malignant tumors against the used drugs [11]. Nowadays, the most active and widely used class of cytostatics is that of alkylating substances. The cytostatics containing the di-(β-chloroethyl)-amine group in their structure have the widest utility.
When the active group is attached to a radical of an aliphatic hydrocarbon, those N-mustards have a reduced selective action against cancer cells and a high toxicity to the normal cells [12]. N-mustards chemotherapy has taken a step forward in the use of aromatic and heterocyclic di-(β-chloroethyl)-amines. Their lower toxicity is due to the action of the aromatic or heterocyclic support, which causes a decrease in the basicity of the nitrogen atoms and the rate of alkylation, respectively. The main mode of action of di-(β-chloroethyl)-amines consists in the possibility of an intramolecular cyclization with the formation of an immonium cation consisting of an ethylene ring of three atoms, which, being unstable, easily breaks and can react through carbon as an alkylating agent for substances possessing active groups: NH 2 , SH, and so forth.
It is assumed that, through the alkylating group, the N-mustards bind to the active groups NH 2 , SH, and so forth, from two protein macromolecules, nucleoproteins, deoxyribonucleic acids that participate in the multiplication process of the neoplastic cells and, by forming cross-links, forms a bridge between two nucleoprotein macromolecules or protein molecules and enzymes (cross-linking), preventing them from participating in accelerated biosynthesis, stopping the anarchic division of malignant cells [13].
The aim of the present study is to propose new N-mustard compounds for biomedical applications in cancer therapy. As new compounds, they are studied both for the optimization of their obtaining reactions and for establishing some characteristics based on quantum mechanical analysis. Their very low toxicity and the results provided after testing on experimental animals recommend them as potential candidates for antitumor drugs development.

Materials and Methods
The chemical reactants were provided by Merck Company (Darmstadt, Germany) and Fluka (Darmstadt, Germany) and used without any purification.
The purity of the obtained substances was checked by quantitative elemental analysis, as well as Fourier-transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy. Quantitative elemental analysis was performed by using the Exeter Analytical CE 440 (Exeter Analytical UK Ltd., Coventry, UK). BRUKER Tensor-27 FT-IR (ATR) spectrophotometer (Bruker Optik GmbH, Ettlingen, Germany) was used to record the FT-IR spectra. 1 H-NMR spectra (DMSO-d 6 , 400 MHz) were recorded using a BRUKER ARX 400 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with 5 mm QNP 1 H/ 13 C/ 31 P/ 19 F samples and Silicon Graphics INDIGO 2 workstation (Silicon Graphics, Inc., Mountain View, CA, USA).
Statistical models based on the factorial design [31][32][33] were applied to establish the best conditions in which the reaction rates can be maximized. To decide the situations for which the reaction yield does not depend on a given variable, the Student's t-test [32] was applied.
The acute toxicity was established by evaluation of produced mortality. The toxicity degree indicates the maximum bearable dose LD 0 , minimum lethal dose LD 100 , and the killing dose that kills 50% of the experimental animals LD 50 , these representing the best clues in the interpretation of the results. In the determination of the acute toxicity of the compounds, mice weighing 20 ± 2 g were used, the experimental groups consisting of 10 animals of both sexes. The substances were administered intraperitoneally as a suspension in Tween 80 (Sigma-Aldrich, Darmstadt, Germany) and the mortality was recorded at 24 h, 48 h, and 7 days. The general condition, the appearance of the hair, the behavior, the appearance of the tremors, and so forth were observed. The Spearman-Kärber method [36] allowed for the quick determination of LD 50 on a small number of animals, using the next calculation method: where a is the difference between two successive doses of the administrated substance, b is the average number of dead animals in two successive doses, and n is the total number of animals in the group.
To study the cytostatic action, the A 2 G mice weighing 25-30 g (±2 g) were used as experimental animals and Ascita Ehrlich has been used as an experimental tumor. The transplantation was performed intraperitoneally. The substances were administered as suspensions in 1% methylcellulose, by single injections on the 7th day after transplantation. The concentrations of 400, 200, and 40 mg/kg body weight were used. The experimental groups included 20 animals for each concentration, while the control groups had 10 tumored animals. The inhibition was calculated according to the methods from the literature [37], 7 days after the administration of the substances.

Chemical Reactions
The pharmacodynamic action of various chemicals is the result of their interaction with substances from the biological substrate, under the influence of biochemical factors. Consequently, the synthesis and study of a wide range of chemical compounds that contain bioactive groups in the molecule will allow the establishment of a more accurate correlation between structure and biological activity.
Based on the previous research [13][14][15][16][17]38,39], the synthesis of new N mustard derivatives containing the benzoxazole heterocycle and the di-(β-chloroethyl)-amine group in their molecules was made. Thus, it becomes interesting to study the mutual influence of these components that are present in the same molecule, as well as their contribution to the overall biological activity of the substance.
In our research, we aimed to graft the di-(β-chloroethyl)-amine group in different positions on the side chain of the oxazole ring within the 2-mercapto benzoxazole substrate. For this purpose, by the hot dissolution of 2-mercaptobenzoxazole in alcoholic solution containing sodium ethoxide, the sodium salt of 2-mercaptobenzoxazole (I) was obtained, which, treated in water-acetone solution, with chloroformic acid, chloroacetic acid and β-chloropropionic acid, led to benzoxazole-2-yl-mercapto-formic (II), acetic (III), and βpropionic (IV) acids (Scheme 1). animals of both sexes. The substances were administered intraperitoneally as a suspension in Tween 80 (Sigma-Aldrich, Darmstadt, Germany) and the mortality was recorded at 24 h, 48 h, and 7 days. The general condition, the appearance of the hair, the behavior, the appearance of the tremors, and so forth were observed. The Spearman-Kärber method [36] allowed for the quick determination of LD50 on a small number of animals, using the next calculation method: where a is the difference between two successive doses of the administrated substance, b is the average number of dead animals in two successive doses, and n is the total number of animals in the group.
To study the cytostatic action, the A2G mice weighing 25-30 g (±2 g) were used as experimental animals and Ascita Ehrlich has been used as an experimental tumor. The transplantation was performed intraperitoneally. The substances were administered as suspensions in 1% methylcellulose, by single injections on the 7th day after transplantation. The concentrations of 400, 200, and 40 mg/kg body weight were used. The experimental groups included 20 animals for each concentration, while the control groups had 10 tumored animals. The inhibition was calculated according to the methods from the literature [37], 7 days after the administration of the substances.

Chemical Reactions
The pharmacodynamic action of various chemicals is the result of their interaction with substances from the biological substrate, under the influence of biochemical factors. Consequently, the synthesis and study of a wide range of chemical compounds that contain bioactive groups in the molecule will allow the establishment of a more accurate correlation between structure and biological activity.
Based on the previous research [13][14][15][16][17]38,39], the synthesis of new N mustard derivatives containing the benzoxazole heterocycle and the di-(β-chloroethyl)-amine group in their molecules was made. Thus, it becomes interesting to study the mutual influence of these components that are present in the same molecule, as well as their contribution to the overall biological activity of the substance.
In our research, we aimed to graft the di-(β-chloroethyl)-amine group in different positions on the side chain of the oxazole ring within the 2-mercapto benzoxazole substrate. For this purpose, by the hot dissolution of 2-mercaptobenzoxazole in alcoholic solution containing sodium ethoxide, the sodium salt of 2-mercaptobenzoxazole (I) was obtained, which, treated in water-acetone solution, with chloroformic acid, chloroacetic acid and β-chloropropionic acid, led to benzoxazole-2-yl-mercapto-formic (II), acetic (III), and β-propionic (IV) acids (Scheme 1).  The IR spectra of the compound (I) show a characteristic band of the aromatic ring at 3000 cm −1 . The band attributed to the valence vibration of the cyclic C = N group can be noticed at 1481 cm −1 . The C-S group gives absorption at 685 cm −1 . In the 1 H-NMR spectra, the signal of the aromatic protons appears in the form of a doublet in the range 6.94-7.47 ppm.
The IR spectra of the derivatives (II-IV) show a band at 3356-3368 cm −1 corresponding to the valence vibrations of the COOH group and in the immediate proximity, at 2982-3060 cm −1 , an intense absorption band given by the vibrations of the aromatic CH group. The cyclic C = N group is highlighted by the band at 1638-1644 cm −1 . The bands specific to the valence vibration of the C-S connection appear at 720-752 cm −1 . The band at 1076-1080 cm −1 is assigned to the cyclic C-O group. In the 1 H-NMR spectra of the compounds (II-IV), the proton of the COOH group is highlighted at 11.56-11.76 ppm, while signals of the aromatic protons are present at 7.13-7.69 ppm. For compounds (III) and (IV), due to the nucleophilic nature of the COOH group and the two heteroatoms (N and O) of the benzoxazole heterocycle, the peak values for the protons of the CH 2 group are found close to the protons in the benzene nucleus, namely at 7.6-7.11 ppm.
Initially, the sodium salts of the benzoxazole-2-yl-mercapto-formic, acetic, and βpropionic (V-VII) acids were synthesized by refluxing a mixture of acid (II-IV) in alcoholic solution with sodium hydroxide (Scheme 2). The synthesis of the compounds I-IV was confirmed by elemental and spectral analysis (FT-IR, 1 H-NMR).
The IR spectra of the compound (I) show a characteristic band of the aromatic ring at 3000 cm −1 . The band attributed to the valence vibration of the cyclic C = N group can be noticed at 1481 cm −1 . The C-S group gives absorption at 685 cm −1 . In the 1 H-NMR spectra, the signal of the aromatic protons appears in the form of a doublet in the range 6.94-7.47 ppm.
The IR spectra of the derivatives (II-IV) show a band at 3356-3368 cm −1 corresponding to the valence vibrations of the COOH group and in the immediate proximity, at 2982-3060 cm −1 , an intense absorption band given by the vibrations of the aromatic CH group. The cyclic C = N group is highlighted by the band at 1638-1644 cm −1 . The bands specific to the valence vibration of the C-S connection appear at 720-752 cm −1 . The band at 1076-1080 cm −1 is assigned to the cyclic C-O group. In the 1 H-NMR spectra of the compounds (II-IV), the proton of the COOH group is highlighted at 11.56-11.76 ppm, while signals of the aromatic protons are present at 7.13-7.69 ppm. For compounds (III) and (IV), due to the nucleophilic nature of the COOH group and the two heteroatoms (N and O) of the benzoxazole heterocycle, the peak values for the protons of the CH2 group are found close to the protons in the benzene nucleus, namely at 7.6-7.11 ppm.
Initially, the sodium salts of the benzoxazole-2-yl-mercapto-formic, acetic, and β-propionic (V-VII) acids were synthesized by refluxing a mixture of acid (II-IV) in alcoholic solution with sodium hydroxide (Scheme 2). The structure of the compounds V-VII was also investigated by elemental and spectral analysis (FT-IR, 1 H-NMR). The IR spectra for the compounds V-VII show absorption at 1608-1640 cm −1 , specific to the COOgroup. The 1 H-NMR spectra of the compounds V-VII show the aromatic protons as a doublet at 7.12-7.68 ppm and the protons of the CH2 group (in the compounds VI and VII) as a multiplet in the region of 7.05-7.12 ppm.
In order to increase the reactivity of the carboxyl group in sodium salts (V-VII), they were converted by treatment with pivaloyl chloride (trimethylacetic acid chloride) to mixed anhydrides (A-C), capable of reacting rapidly and under mild conditions with di-(β-chloroethyl)-amine. The mixed anhydrides (A-C) are unstable and it is difficult to isolate them, so we chose for their in situ use a dichloromethine solution in which di-(βchloroethyl)-amine is dissolved as a free base. The reaction mixture was stirred for three hours, filtered, and recrystallized from the anhydrous dioxane precipitate to give N-mustards (VIII-X) (Scheme 3). Scheme 2. Synthesis of the sodium salts of benzoxazol-2-yl-mercapto-formic, acetic, and β-propionic acids (V-VII), respectively.
The structure of the compounds V-VII was also investigated by elemental and spectral analysis (FT-IR, 1 H-NMR). The IR spectra for the compounds V-VII show absorption at 1608-1640 cm −1 , specific to the COOgroup. The 1 H-NMR spectra of the compounds V-VII show the aromatic protons as a doublet at 7.12-7.68 ppm and the protons of the CH 2 group (in the compounds VI and VII) as a multiplet in the region of 7.05-7.12 ppm.
In order to increase the reactivity of the carboxyl group in sodium salts (V-VII), they were converted by treatment with pivaloyl chloride (trimethylacetic acid chloride) to mixed anhydrides (A-C), capable of reacting rapidly and under mild conditions with di-(β-chloroethyl)-amine. The mixed anhydrides (A-C) are unstable and it is difficult to isolate them, so we chose for their in situ use a dichloromethine solution in which di-(β-chloroethyl)-amine is dissolved as a free base. The reaction mixture was stirred for three hours, filtered, and recrystallized from the anhydrous dioxane precipitate to give N-mustards (VIII-X) (Scheme 3). ines 2021, 9, x FOR PEER REVIEW 5 of 20 Reactions for obtaining di-(β-chloroethyl)-amides of benzoxazole-2-yl-mercapto-formic (VIII), acetic (IX) and β-propionic (X) acids, respectively.
The structure of the compounds VIII-X was established by elemental and spectral analysis (FT-IR, 1 H-NMR). The IR spectra show characteristic bands of the three N-mustards. In the area 3094-3097 cm −1 , there are the bands corresponding to the vibration of the aromatic CH group. The C = O amide bond produces a well-highlighted band at 1623 cm −1 and 1624 cm −1 . The vibration of the C = N group appears at 1490-1493 cm −1 . In the IR absorption spectra of N-mustards VIII-X, the C-N stretching frequency was identified at 1283-1306 cm −1 , while the C-Cl stretching frequency was identified between 747-789 cm −1 . In the 1 H-NMR spectra of the compounds VIII-X, multiplets between δ = 3.16-3.41 ppm (VIII), δ = 3.55-3.57 ppm (IX) and δ = 2.55-3.39 ppm, respectively, whose integrated surface satisfactorily coincides, in all cases, with the eight existing protons, were obtained for the protons from the N-mustard group.

Sodium Salt of the 2-Mercaptobenzoxazole (I)
First, 0.2 mol 2-mercaptobenzoxazole were treated with 0.2 mol metal sodium dissolved in 120 mL anhydrous ethyl alcohol. The obtained solution was refluxed for 90 min on a water bath, then hot filtered to remove any impurities. Excess of alcohol was removed by distillation under reduced pressure, then, after cooling, the precipitate was separated, filtered in vacuum, and then dried.
Light gray solid (  The structure of the compounds VIII-X was established by elemental and spectral analysis (FT-IR, 1 H-NMR). The IR spectra show characteristic bands of the three N-mustards. In the area 3094-3097 cm −1 , there are the bands corresponding to the vibration of the aromatic CH group. The C = O amide bond produces a well-highlighted band at 1623 cm −1 and 1624 cm −1 . The vibration of the C = N group appears at 1490-1493 cm −1 . In the IR absorption spectra of N-mustards VIII-X, the C-N stretching frequency was identified at 1283-1306 cm −1 , while the C-Cl stretching frequency was identified between 747-789 cm −1 . In the 1 H-NMR spectra of the compounds VIII-X, multiplets between δ = 3.16-3.41 ppm (VIII), δ = 3.55-3.57 ppm (IX) and δ = 2.55-3.39 ppm, respectively, whose integrated surface satisfactorily coincides, in all cases, with the eight existing protons, were obtained for the protons from the N-mustard group. Next, 0.05 mol of sodium salt of 2-mercaptobenzoxazole (I) were dissolved in 60 mL of water-acetone (1:1) to give a clear gray solution. Then, 0.05 mol of acid (chloroformic, chloroacetic and β-chloropropionic) were added in portions, under vigorous stirring. The reaction mixture was heated at 40-45 • C, stirring for 100-120 min. Towards the end, an abundant white-yellow precipitate appeared instantly. It was filtered by vacuum, dried, and purified by recrystallization from boiling water.

Optimization of the Reactions
In the factorial experiments [31][32][33], the reaction yield is considered as being an indicator of the reaction optimization. It is markedly influenced by some conditions of the chemical reactions as temperature, time interval, solvent, solvent mixture, impuri-ties, or pressure. These parameters are considered real variables. Some of these are significant variables.
The yield of the chemical reactions for obtaining the benzoxazoles derivatives VIII-X depends on two significant variables: temperature and time interval of reactions. Let us consider temperature (T, in • C, noted with X 1 ) and interval of time (t, in hours, noted with X 2 ) as being real variables of the experiment. In order to facilitate the calculations, dimensionless variables x 1 and x 2 are introduced instead of the real variables X 1 and X 2 by using the average value of the real variables, noted X i , i = 1, 2, and the half of the variation domain of the variables, noted ∆X i , i = 1, 2, defined by relations (2) and (3): The dimensionless variables depend on the real variables as relation (4) indicates: Let us propose a dependence (5) of the reaction yield [31][32][33], η, on the dimensionless variables, considering the individual influence and the conjugate effects of these variables: The regression coefficients, a i , can be determined using the values of η and of the dimensionless variables obtained in 3 2 experiments organized for the chemical reaction for obtaining benzoxazoles. The results are given in Tables 1-3 and in Figures 1-3 for the molecules VIII, IX and X, respectively. No.
x 1 (X 1 , • C) x 2 (X 2 , h) No.  No.         The absolute values of the coefficients indicate the strength of the dependence of the reaction yield on the corresponding variables. The positive sign of these coefficients indicates the increasing in yield when the corresponding variables increase. The decrease of the reaction yield when the dimensionless variables increase is indicated by the negative sign of the corresponding coefficients.
Using the data from Tables 1-3, the regression coefficients from Table 4 were obtained for the three studied compounds. To decide the situations for which the reaction yield does not depend on a given variable, we must know the t-Student coefficients [32]. For this purpose, supplemental experiments were organized in the vicinity of the middle of the variation domain of the dimensionless variables (see Table 5). The precision of determination of the regression coefficients in relation (5) was estimated by using relation (6): where ( ) In relations (6) and (7) N is the number of the experiments in 3 2 procedure and n is the number of the supplemental experiments organized for precision estimation. To com- The absolute values of the coefficients indicate the strength of the dependence of the reaction yield on the corresponding variables. The positive sign of these coefficients indicates the increasing in yield when the corresponding variables increase. The decrease of the reaction yield when the dimensionless variables increase is indicated by the negative sign of the corresponding coefficients.
Using the data from Tables 1-3, the regression coefficients from Table 4 were obtained for the three studied compounds. To decide the situations for which the reaction yield does not depend on a given variable, we must know the t-Student coefficients [32]. For this purpose, supplemental experiments were organized in the vicinity of the middle of the variation domain of the dimensionless variables (see Table 5). The precision of determination of the regression coefficients in relation (5) was estimated by using relation (6): where In relations (6) and (7) N is the number of the experiments in 3 2 procedure and n is the number of the supplemental experiments organized for precision estimation. To compute the sum of quadratic deviations the data of Table 5 were used. The t-Student coefficients, estimated on the basis of the values of the regression coefficients from Table 4, are given in Table 6. If one supposes that the variables corresponding to the t-Student coefficients are smaller than 3.00, this does not exert a significant influence on the reaction yield, relation (5) can be written for the studied molecules in relations (8)- (10):

Quantum Mechanical Analysis of N-Mustards
Since the N-mustards described above are new compounds, the quantum mechanical characterization is necessary because the provided results can help in obtaining information on the interaction mechanism of these molecules with living cells.
The optimized structures of the three molecules, performed by Spartan'14 software, are given in Figure 4a-c. In Figure 4a-c oxygen atom is red, nitrogen is blue, sulfur is yellow, chlorine is green, carbon is black, and hydrogen is white. Resulting from Figure 4a-c, the reciprocal orientation of the atomic groups in the optimized structure of N-mustards VIII-X and the change in the dipole moment orientation when CH 2 group is introduced in the structures IX and X. The arrow indicates the orientation of the electrical dipole moment of the molecule. pute the sum of quadratic deviations the data of Table 5 were used. The t-Student coefficients, estimated on the basis of the values of the regression coefficients from Table 4, are given in Table 6. If one supposes that the variables corresponding to the t-Student coefficients are smaller than 3.00, this does not exert a significant influence on the reaction yield, relation (5) can be written for the studied molecules in relations (8)

Quantum Mechanical Analysis of N-Mustards
Since the N-mustards described above are new compounds, the quantum mechanical characterization is necessary because the provided results can help in obtaining information on the interaction mechanism of these molecules with living cells.
The optimized structures of the three molecules, performed by Spartan'14 software, are given in Figure 4a-c. In Figure 4a-c oxygen atom is red, nitrogen is blue, sulfur is yellow, chlorine is green, carbon is black, and hydrogen is white. Resulting from Figure  4a-c, the reciprocal orientation of the atomic groups in the optimized structure of N-mustards VIII-X and the change in the dipole moment orientation when CH2 group is introduced in the structures IX and X. The arrow indicates the orientation of the electrical dipole moment of the molecule.   The values of some electro-optical parameters computed using Spartan'14 for the studied molecules are given in Table 7.  The values of some electro-optical parameters computed using Spartan'14 for the studied molecules are given in Table 7. The dipole moment, measuring the separation of the positive and negative electrical charges of the molecule, increases when passing from molecule VIII to molecule X, as results from Table 7. The molecular polarizability, giving information about the induced dipole moment in the molecule by the internal electric field acting in each point in solution, also increases from molecule VIII to molecule X.
The polar surface area (PSA) [47][48][49][50][51] defined as the sum of the polar atoms' surfaces in a molecule is a useful parameter for prediction of drug transport properties. The values of PSA are between 28.490 and 29.781 Å 2 , these molecules being able to cross both the cell membranes and the blood-brain barrier.
The log P [47][48][49][50][51] value is a measure of lipophilicity or hydrophobicity. The chemical structures of the studied molecules are hydrophobic because the values of logP are positive.
The HOMO and LUMO maps of the studied N-mustards are illustrated in Figure 5a-c. HOMO and LUMO orbitals are involved in the chemical reactivity of the molecule, determining the chemical stability, the electrical and optical properties, and the interaction ability of the molecule with other species.
Biomedicines 2021, 9, x FOR PEER REVIEW 13 of 20 The dipole moment, measuring the separation of the positive and negative electrical charges of the molecule, increases when passing from molecule VIII to molecule X, as results from Table 7. The molecular polarizability, giving information about the induced dipole moment in the molecule by the internal electric field acting in each point in solution, also increases from molecule VIII to molecule X.
The polar surface area (PSA) [47][48][49][50][51] defined as the sum of the polar atoms' surfaces in a molecule is a useful parameter for prediction of drug transport properties. The values of PSA are between 28.490 and 29.781 Å 2 , these molecules being able to cross both the cell membranes and the blood-brain barrier.
The log P [47][48][49][50][51] value is a measure of lipophilicity or hydrophobicity. The chemical structures of the studied molecules are hydrophobic because the values of logP are positive.
The HOMO and LUMO maps of the studied N-mustards are illustrated in Figure 5a-c. HOMO and LUMO orbitals are involved in the chemical reactivity of the molecule, determining the chemical stability, the electrical and optical properties, and the interaction ability of the molecule with other species.   Figure 5a-c illustrates the changes in the valence electron cloud distribution by excitation; these changes involve especially the heterocycle region. As results from the data of Table 7 referring to the energy gap |∆E|(eV), the transitions between HOMO and LUMO are made under the UV photons. Figure 5a-c also emphasizes the atomic groups involved in the chemical reactions both in the ground and excited states of N-mustards molecules. Some graphical representations provided by Spartan'14 program [34,35] are illustrated in Figures 6-8. medicines 2021, 9, x FOR PEER REVIEW 14 HOMO map LUMO map (c)        From Figure 9a-c, it results that the highest negative charges are localized near nitrogen atom belonging to the heterocycle and oxygen atom of the C = O group in all studied molecules.
The arrows in Figure 9a-c show the orientation of the electric dipole moment in the ground electronic states of the studied molecules. It is parallel to the CS bond for the mol-  The mice weighing 20 ± 2 g were used and the experimental groups consisted of 10 animals of both sexes. The substances were administered intraperitoneally as a suspension in Twen 80 and the mortality was recorded at 24 h, 48 h, and 7 days. The degree of toxicity (LD 50 ) was determined using the Spearman-Kärber method [36]. The obtained results are listed in Table 8. It is found that the N-mustards (VIII-X) present a very low toxicity compared to that of the di-(β-chloroethyl)-amine free base and a slightly increased toxicity compared to the support intermediates (II-IV). The results confirm the data from the literature according to which the use of heterocyclic structures as transport agents positively influences the decrease of cytotoxic group toxicity [1,[52][53][54].
Thus, in the case of N-mustards, the toxicity is due to the support, which causes a decrease in the basicity of nitrogen atom by involvement in the amide conjugation system of its non-participating electrons.

Antitumor Activity of di-(β-Chloroethyl)-Amides VIII-X
In order to study the cytostatic action, the A 2 G mice weighing 25-30 g (±2 g) as experimental animals were used, and Ascita Ehrlich was used as an experimental tumor. The transplantation was performed intraperitoneally (10 6 cells dose). The substances were administered as suspensions in 1% methylcellulose, by single injections on the 7th day after transplantation. The concentrations of 400, 200, and 40 mg/kg body weight were used. The experimental groups included 20 animals for each concentration and the control groups had 10 tumored animals. The inhibition exerted by N-mustards VIII-X was calculated according to the methods from the literature [37], 7 days after the administration of the substances (Table 9), with an error of 1%. From the experimental data it results: • The tested N mustards presents appreciable antitumor activity; more selective is the N mustard IX: di-(β-chloroethyl)-amide of benzoxazole-2-yl-mercapto-acetic acid which inhibits Ehrlich's Ascites in a proportion close to that of the reference cytostatic, I.O.B.-82; • In addition to the alkylating effect, the synthesized N-mustards may also have a specific antimetabolite effect; • The selective transport of the di-(β-chloroethyl)-amine group performed by the benzoxazole heterocycle and the active group transforms it into a masked group (protected group); • The relationship between the benzoxazole nucleus and the active group does not consist exclusively in transport, but also in their mutual potentiation (convenient or not, direct or indirect).

Conclusions
The paper presents results related to the extension of the grafting reaction of the di-(β-chloroethyl)-amine group on heterocyclic structures.
The technique of obtaining di-(β-chloroethyl)-amides with benzoxazole structure was applied for the first time using the activation of support acids derived from 2mercaptobenzoxazole by transforming them into mixed pivalic anhydrides which, for electronic and steric reasons, can be cleaved by the aminic nitrogen to one of the two carbonyls in the desired direction.
The synthesized products were characterized by elemental and spectral analysis (IR, 1 H-NMR).
The new compounds VIII -X were characterized by Spartan'14 and their electro-optical properties were established.
The importance of the synthesized N mustards among the biologically active products was highlighted by establishing the degree of toxicity and testing their antitumor activity on experimental animals.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.