Synthesis and Biological Activity of New 7-Amino-oxazolo[5,4-d]Pyrimidine Derivatives

The synthesis of a series of novel 7-aminooxazolo[5,4-d]pyrimidines 5, transformations during their synthesis and their physicochemical characteristics have been described. Complete detailed spectral analysis of the intermediates 2–4, the N′-cyanooxazolylacetamidine by-products 7 and final compounds 5 has been carried out using MS, IR, 1D and 2D NMR spectroscopy. Theoretical research was carried out to explain the privileged formation of 7-aminooxazolo[5,4-d]pyrimidines in relation to the possibility of their isomer formation and the related thermodynamic aspects. Additionally, the single-crystal X-ray diffraction analysis for 5h was reported. Ten 7-aminooxazolo[5,4-d]pyrimidines 5 (SCM1–10) were biologically tested in vitro to preliminarily evaluate their immunological, antiviral and anticancer activity. Compounds SCM5 and SCM9 showed the best immunoregulatory profile. The compounds displayed low-toxicity and strongly inhibited phytohemagglutinin A-induced proliferation of human peripheral blood lymphocytes and lipopolysaccharide-induced proliferation of mouse splenocytes. Compound SCM9 caused also a moderate suppression of tumor necrosis factor α (TNF-α) production in a human whole blood culture. Of note, the compounds also inhibited the growth of selected tumor cell lines and inhibited replication of human herpes virus type-1 (HHV-1) virus in A-549 cell line. Molecular investigations showed that the compounds exerted differential changes in expression of signaling proteins in Jurkat and WEHI-231 cell lines. The activity of SCM5 is likely associated with elicitation of cell signaling pathways leading to cell apoptosis. The compounds may be of interest in terms of therapeutic utility as inhibitors of autoimmune disorders, virus replication and antitumor agents.

The aim of this work was the synthesis of 7-aminooxazolo [5,4-d]pyrimidines series with 5amino-3-methyl-isoxazol-4-yl substituent at position 2 of their structure to find novel derivatives exhibiting potential immunosuppressive activity. It is worth noting that oxazolo [5,4-d]pyrimidine system is structurally similar to purine analogs of naturally occurring nucleic acid bases. Such a structure may, in theory, condition antitumor and/or antiviral activity, so for this purpose, several in vitro immunological and antitumor assays have been designed and carried out. Finally, on the basis of the obtained results, an attempt to establish a plausible mechanism of the biological action of these compounds has been made. Since that time, many derivatives of oxazolo [5,4-d]pyrimidine system have been synthesized and extensively researched for their potential biological activity. Some oxazolo [5,4-d]pyrimidines are well known as adenosine receptor antagonists (Structure II in Figure 1) [2], inhibitors of receptor tyrosine kinase (Structure III in Figure 1) [3], inhibitors of adenosine kinase (Structure IV in Figure 1) [4], effective inhibitors of ricin [5] and Shiga toxin inhibitors (Structure V in Figure 1) [6]. The antiviral (Structure VI in Figure 1) [7], anti-angiogenic (Structure VII in Figure 1) [8] and immunosuppressive (Structure VIII in Figure 1) [9] activity of some of them has been also revealed. Besides, some oxazolo [5,4-d]pyrimidines can also activate the caspase cascade (Structure IX in Figure 1) [10] and inhibit ubiquitin-activating enzymes (E1 enzymes), particularly NEDD8-activating enzyme (NAE) (Structure X in Figure 1) [11], which may result in their antineoplastic activity.
The aim of this work was the synthesis of 7-aminooxazolo [5,4-d]pyrimidines series with 5-amino-3-methyl-isoxazol-4-yl substituent at position 2 of their structure to find novel derivatives exhibiting potential immunosuppressive activity. It is worth noting that oxazolo [5,4-d]pyrimidine system is structurally similar to purine analogs of naturally occurring nucleic acid bases. Such a structure may, in theory, condition antitumor and/or antiviral activity, so for this purpose, several in vitro immunological and antitumor assays have been designed and carried out. Finally, on the basis of the obtained results, an attempt to establish a plausible mechanism of the biological action of these compounds has been made.

Synthesis and Some Physicochemical Properties of New 7-Aminooxazolo[5,4-d]Pyrimidines 5
In general, there are two known main methods of synthesis of oxazolo [5,4-d]pyrimidines, namely, through cyclization of the pyrimidine ring on existing, appropriately substituted, oxazole derivative or through cyclization of the oxazole ring on pyrimidine derivative [26]. For the first time, this system was synthesized just by using the second method [1], where 2-mercapto-5-benzoylamino-4-hydroxypyrimidine was cyclized with phosphoryl trichloride (POCl 3 ). Herein, the described series of new oxazolo [5,4-d]pyrimidines 5 is synthesized according to the synthetic pathway outlined in Scheme 1.
Molecules 2020, 25 In general, there are two known main methods of synthesis of oxazolo [5,4-d]pyrimidines, namely, through cyclization of the pyrimidine ring on existing, appropriately substituted, oxazole derivative or through cyclization of the oxazole ring on pyrimidine derivative [26]. For the first time, this system was synthesized just by using the second method [1], where 2-mercapto-5-benzoylamino-4-hydroxypyrimidine was cyclized with phosphoryl trichloride (POCl3). Herein, the described series of new oxazolo [5,4- The following reagents and substrates have been used in the synthesis of the considered compounds: starting materials, such as esters of 2-cyano-3-alkoxy-2-butenoate acids, which were used to synthesize known 5-amino-3-methyl-4-isoxazolecarboxylic acid [27], were prepared according to the highly efficient methods precisely described in [28]. 5-Amino-3-methylisoxazole-4carbonyl chloride (1, Scheme 1) was also prepared using the method described earlier [12]. Similarly to the method described by Freeman and Kim [29], 5-amino-2-(5-amino-3-methylisoxazol-4-yl)oxazole-4-carbonitrile (2) was obtained in the reaction, which was conducted in 1-methyl-2pyrrolidinone (NMP) at room temperature, between the commercially available aminomalononitrile tosylate (AMNT) and compound 1. Then, ethyl N-{4-cyano-2- [5-(1-ethoxyethylidene)amino-3methylisoxazol-4-yl]-oxazol-5-yl}ethanimidate (4) was obtained through the intermediate imidate, i.e., ethyl N- [2-(5-amino-3-methylisoxazol-4-yl)-4-cyanooxazol-5-yl]ethanimidate (3), by heating of oxazole 2 in an excess of triethyl orthoacetate under reflux. It is necessary to emphasize that the compound 3 has one and the product 4 has two centers of Z-E isomerism, respectively, so there is a The following reagents and substrates have been used in the synthesis of the considered compounds: starting materials, such as esters of 2-cyano-3-alkoxy-2-butenoate acids, which were used to synthesize known 5-amino-3-methyl-4-isoxazolecarboxylic acid [27], were prepared according to the highly efficient methods precisely described in [28]. 5-Amino-3-methylisoxazole-4-carbonyl chloride (1, Scheme 1) was also prepared using the method described earlier [12]. Similarly to the method described by Freeman and Kim [29], 5-amino-2-(5-amino-3-methylisoxazol-4-yl)-oxazole-4 -carbonitrile (2) was obtained in the reaction, which was conducted in 1-methyl-2-pyrrolidinone (NMP) at room temperature, between the commercially available aminomalononitrile tosylate (AMNT) and compound 1. Then, ethyl N-{4-cyano-2-[5-(1-ethoxyethylidene)amino-3-methylisoxazol-4-yl] -oxazol-5-yl}ethanimidate (4) was obtained through the intermediate imidate, i.e., ethyl N-[2-(5-amino-3-methylisoxazol-4-yl)-4-cyanooxazol-5-yl]ethanimidate (3), by heating of oxazole 2 in an excess of triethyl orthoacetate under reflux. It is necessary to emphasize that the compound 3 has one and the product 4 has two centers of Z-E isomerism, respectively, so there is a possibility that, theoretically, two in the case of 3 or four single isomers in the case of 4 or their mixtures can be created, respectively. Anyway, a spectral analysis revealed that only one isomer was formed in both cases for the compounds 3 and 4. We have not determined which one, as this fact seems to be of little importance for a course of the next stage of the synthesis. The reactions of the imidate 4 with a 30% ethanolic solution of the appropriate aliphatic amines, conducted at room temperature, resulted in a series of final oxazolo [5,4-d]pyrimidines 5. It should be noted that the molecule of compound 4 has three reactive centers with lowered electron density that are susceptible to attack by nucleophilic agents, the nitrile carbon and the carbons of both imidate moieties. Therefore, three possible competitive reactions can occur and result in various products. Our research indicates that the desired compounds 5 form via the labile intermediate amidines 3-IP that are formed by the nucleophilic addition of the corresponding amine to the nitrile group of the compound 4 or 3. In this stage, an excess of the primary aliphatic amine as a strong nucleophile agent causes the removal of the ethoxyethylidene moiety (in the case of the compound 4) from the amino group of the isoxazole ring with simultaneous cyclization to the pyrimidine ring, while acetamidines 6 (only in the case of primary amines) are produced as by-products. However, secondary aliphatic amines (such as N,N-diethylamine, R, R 1 = Et) did not give the expected products 5 under these conditions but N -cyanooxazolylacetamidine 7 (7m, R,R 1 = Et for N,N-diethylamine) in low yield (≈14%) and the mixture of product of the decomposition of the compound 4, i.e., the compounds 2 and 3. It is worth mentioning that the 1 H-NMR spectrum (recorded at 297.8 K ≈ 25 • C) of N -cyanooxazolylacetamidine 7m (for a detailed description of its spectrum, see Section 3.1.2 of "Preparation and experimental properties of compounds 2-5, 7" and for the spectrum, see Figure S76 in the Supplementary Material) shows that the corresponding protons of the N-ethyl groups are not magnetically equivalent. This suggests that inhibition of rotation around the bond between the quaternary acetamidine carbon atom and the nitrogen atom occur. This supposition has been supported with the theoretically predicted rotational barrier that has been estimated to be about 23 kcal·mol −1 (see Section 3.1.4. Computational details). A similar energy barrier is needed for free rotation of the N,N-dimethyl group in a known useful aprotic polar solvent, N,N-dimethylformamide (DMF), for which the experimentally estimated energy barrier is about 21 kcal·mol −1 [30]. In DMF, this inhibition of rotation is caused by fact that bond between carbonyl carbon and nitrogen has partial double bond character, which is connected with a significant contribution of the imidic acid tautomer to the resonance structure. In the case of N -cyanooxazolylacetamidine 7m, the reason could be similar.
Primary aliphatic amines with rigid bulky steric hindrance (with a secondary aliphatic radical like isopropyl or cyclohexyl) usually gave the mixture of products (like 2, 5 and 6) with very low abundance of the oxazolo [5,4-d]pyrimidines 5. In the cases of isopropylamine and cyclohexylamine, the yield of appropriate pure oxazolo [5,4-d]pyrimidines 5 was about 9% (5k) and 22% (5l), respectively. However, in the case of t-butylamine, only the products of decomposition of substrate 4 were isolated. Similarly, in the case of aromatic amines (such as aniline, R = Ph, R 1 = H and p-toluidine, R = p-MePh, R 1 = H), neither oxazolo [5,4-d]pyrimidines 5 nor N -cyanooxazolyl-acetamidines 7 were observed to be formed. In these cases, the imidate 4 underwent only decomposition to the mixture of the compounds 3 and 2 under these conditions, so only the mixture of all of them was isolated. This is probably caused by the much less nucleophilicity of the aromatic amine nitrogen, making aromatic amines behave inertly under these reaction conditions.
It is necessary to underline that during the course of all the reactions of the imidate 4 with amines, the creation of some quantity of the substrate 2 and the intermediate imidate 3 was observed as a result of decomposition of the compound 4. Besides, it appears that compound 4 is unstable in ethanolic solution and slowly decomposes through the intermediate imidate 3 to the substrate 2. The isolated, from reaction mixture, intermediate imidate 3 has been also undergone the reactions with the appropriate aliphatic amines (like in the case of the imidate 4) giving the same final oxazolo [5,4-d]pyrimidines 5 as in the case of the imidate 4, which, in addition to spectral analysis, supports and chemically proves the molecular structure of the compound 3 (for example, preparation of the compound 5i, see Method C in Section 3.1.2 "Preparation and experimental properties of compounds 2-5, 7").
Theoretically predicted heats (enthalpies ∆ f H 298 ) of formation, opposite to binding energies, of N -cyanooxazolylacetamidines 7 (see calculation details in 3.1.4. Computational details), are greater than the ones of structurally corresponding isomeric oxazolo [5,4-d]pyrimidines 5, which means that oxazolo [5,4-d]pyrimidines 5 should be more thermodynamically stable than N -cyanooxazolyl-acetamidines 7. This points out that the observed cases of formation of N -cyanooxazolylacetamidines 7 are rather kinetically and/or sterically controlled, especially taking into account that N -cyanooxazolylacetamidines 7 were formed only in the case of reaction with secondary amines, like N,N-diethylamine, for example. Besides, the formation of N -cyanooxazolylacetamidines 7 suggests that final 7-aminooxazolo [5,4-d]pyrimidines 5 can be produced through only unstable intermediate amidines 3-IP that is formed as a result of the nucleophilic addition of appropriately active amine to cyano group of the imidate 4 or the intermediate imidate 3 because only these amidines 3-IP can cyclize into oxazolo [5,4-d]pyrimidines 5 contrary to N -cyanooxazolylacetamidines 7. Theoretically, N -cyanooxazolylacetamidines 7 could cyclize into N-6-substituted-oxazolo [5,4-d]pyrimidin-7-imines 8 but only in the cases where R 1 = H (see Scheme 1). Formation of such similar imines 8 have been previously described by Ohtsuka [31]. However, imines 8 should be rather unstable, especially, in the presence of water (moisture) which can lead them to decompose into N-6-substituted-oxazolo [5,4-d]pyrimidin-7-ones 9. Actually, the formation of imines 8 and/or oxazolo [5,4-d]pyrimidin-7-ones 9 was not observed under the applied reaction conditions during the conducted research. Besides, this fact is also supported on the basis of the theoretical research on the example of the compounds 5a and 5c, for a simulation carried out for single isolated molecule in a vacuum environment. The calculated difference of energy (∆E 298 ) between thermal-corrected energies of the considered compounds (5a and 5c) and their respective isoforms (8a and 8c) points out that the compounds 5a and 5c should be more thermodynamically stable by about 13 kcal·mol −1 (E stab = −∆E 298 = 13.06 and 13.23 kcal·mol −1 ) than the theoretical compounds 8a and 8c, respectively (see calculation details in 3.1.4. Computational details). The calculations for simulated ethanol solutions of considered molecules conducted with the Conductor-like Polarizable Continuum Model (CPCM) [32] give similar results, i.e., E stab = 12.43 and = 12.83 kcal·mol −1 for the compounds 5a and 5c, respectively. It can be seen that the electrostatic field generated by ethanol molecules has very little effect on the stabilization of the imino isomers 8 compared to vacuum. Finally, the 7-aminooxazolo [5,4-d]pyrimidine structure of the final products 5 were unquestionably proven by X-ray crystallography on the basis of single-crystal X-ray diffraction measurement of the compound 5h.
It should be also noted that compounds 5 may exhibit amine-imine tautomerism (see Scheme 1), similar to purines and pyrimidine bases (e.g., adenine and cytosine), due to the mobility of the proton of the amino group at the seventh position of the oxazolopyrimidine ring. Therefore, there is probability that this proton may easily jump to nitrogen at the sixth position of the pyrimidine ring of the oxazolopyrimidine system due to the fact that the nitrogen atom of amino group at the seventh position, the nitrogen atom of the pyrimidine ring at the sixth position and the carbon atom at seventh position form a semi-cyclic amidine moiety. Theoretical estimation (on the example of the compound 5h) of free enthalpy (∆G trans(298) = 10.748 kcal·mol −1 ) and the equilibrium constant (K trans = [imine]/[amine] ≈ 1.323 × 10 −8 proton transition as a result of amine-imine tautomerism suggests that the amine tautomer of the compound 5h should dominate with a huge advantage in the state of thermodynamic equilibrium, which could have been expected because of partial loss of aromaticity by oxazolopyrimidine ring system of imine tautomer. It can be figured out that one molecule of imine tautomer of the compound 5h occurs with over 75.5 million molecules of its amine tautomer in the equilibrium (see calculation details in 3.1.4. Computational details). For ethanol environment simulation, the results (∆G trans(298) = 10.752 kcal·mol −1 and K trans ≈ 1.313 × 10 −8 ) are very similar (insignificant difference) to those of a vacuum one what would suggests that that electric field generating by the solvent does not influence on the equilibrium in this case. Unfortunately, the CPCM calculations do not take into account the possibility that hydrogen bonds may form between the protic solvent, which is ethanol, and the molecules of the considered compounds, which is a disadvantage of this method.
Taking into account some chemical instability of the compound 4 and the formation of by-products (2, 6 and 7) during the reaction, the yields (calculated for purified products) of expected 7-aminooxazolo [5,4-d]pyrimidines 5 have ranged from about 9% (for isopropyl 5k derivatives) to 66% (for 3-(N,N-dimethylamino)propyl derivative 5i) depending on the primary aliphatic amine used. Since the compound 4 slowly decomposes in ethanol solution, reactions with some amines in aprotic carbon tetrachloride (CCl 4 ) were also carried out (see Method B for the compound 5c and 5i in Section 3.1.2 "Preparation and experimental properties of compounds 2-5, 7"). However, it appeared that the yields of final products 5 are much lower in this case. For example, for the crude compound 5i (R = 3-(N,N-dimethylamino)propyl) and 5c (R = propyl), the yield was only 31% and 13%, respectively, which indicates that CCl 4 has proven to be a worse solvent than ethanol to carry out for these reactions. Therefore, research on the selection of optimal conditions for these reactions will be continued in order to obtain better yields. The structures of all the new compounds were proven by ESI-MS, IR, 1 H, 13 C-NMR and 2D NMR COSY spectroscopy, with single-crystal X-ray diffraction measurement (on the example of the compound 5h) and, additionally, through appropriate chemical transformations. 2D NMR spectra were particularly useful in the unambiguous assignment of chemical shifts to the corresponding protons and to some quaternary carbon atoms. The investigated compounds 5 are highly melting (with decomposition) solids, insoluble in chloroform and water, slightly soluble in methanol and ethanol but well soluble in DMSO (at room temperature).
The packing of 5h is mainly dictated by N-H···O and N-H···N hydrogen bonds (Table S2). The nitrogen atom (N27) from the amine group acts as hydrogen-bond donor via H atoms to the oxygen atom (O11) from the morpholine ring of adjacent molecules, forming the N27-H27···O11 i hydrogen bond of 2.889(2) Å [symmetry code: (i) −x, −y + 1, −z], which leads to a dimer formation. On the other hand, molecules are linked through N7-H7···N22 ii hydrogen bond of 2.959(3) Å [symmetry code: (ii) x + 1, y − 1, z] between the second amine group as a donor and the nitrogen atom of the 1,2-oxazole ring as an acceptor. The combination of these interactions results a ladder-like chain structure ( Figure 3).

Estimation of Cytotoxicity of Tested Compounds on A-549 Reference Cell Line
Selection of therapeutically useful compounds, in order that they could be potential drugs, depends not only on their significant biological actions but also on bioavailability and possibly lowest cytotoxic properties. The compounds were therefore evaluated for their cytotoxic action by application of a reference A-549 cell line at a concentration range of 62.5-15.6 µM using respective dilutions of the solvent (DMSO) in the culture medium as control cultures (Table 1).  Table 8).

Influence of the Tested Compounds on Lymphocyte Proliferation
The ability to suppress mitogen-induced lymphocyte proliferation by the compounds SCM1-10 was evaluated in the model of PHA-induced proliferation of human PBMC, in which the compounds exhibited differentiated suppressive activity with best results for SCM5 and SCM9 (Table 2). At concentration of 50 µM, the suppressive acitivity of SCM5, SCM9 and teriflunomide were 69.8%, 60.7% and 37.9%, respectively. Therefore, these two compounds with strongest suppressive effects with regard to PHA-induced PBMC proliferation at a lower concentration range were selected for the subsequent more advanced experiments. The tested compounds SCM5 and SCM9 appeared to be even more efficient in inhibition of LPS-stimulated mouse splenocytes ( Figure 4) which reflects B cell proliferation [33]. This suppressive effect was more profound in comparison with that one of teriflunomide.  The tested compounds SCM5 and SCM9 appeared to be even more efficient in inhibition of LPSstimulated mouse splenocytes ( Figure 4) which reflects B cell proliferation [33]. This suppressive effect was more profound as in comparison with that one of teriflunomide.

Influence of the Tested Compounds on Growth of Tumor Cell Lines
To evaluate potential antitumor activity of the compounds, A-431 epidermal, HT-29 colon and L-1210 leukemia cell lines (Table 3) were used. Cisplatin was the reference anticancer drug in the study. The results showed that the compounds displayed differential ability to inhibit growth of these

Influence of the Tested Compounds on Growth of Tumor Cell Lines
To evaluate potential antitumor activity of the compounds, A-431 epidermal, HT-29 colon and L-1210 leukemia cell lines (Table 3) were used. Cisplatin was the reference anticancer drug in the study. The results showed that the compounds displayed differential ability to inhibit growth of these cell lines. The best results were observed for HT-29 cell line where SCM9 caused (still significant) 30% growth inhibition at a concentration of 12.5 µM, albeit the activity was weaker than the one of reference cisplatin. Testing the ability of the compounds to inhibit LPS-induced TNF α production in the human whole blood culture (Table 4) revealed that SCM5 was inactive whereas SCM9 demonstrated dose-dependent suppression with a significant inhibition only at 25 µM concentration.

Antiviral Properties of the Compounds
The next aim of this study was to examine the antiviral activity of SCM5 and SCM9 in vitro against HHV-1. The treatment of A-549 cells infected with HHV-1 with SCM compounds resulted in significant reductions in virus yields, reaching up to a 3.5-log 10 decrease for SCM5 and a 2.2-log 10 decrease for SCM9 in comparison with appropriate DMSO control as it is shown in Figure 5. A 4-log 10 reduction of virus titer demonstrates the ability of the tested compounds to inactivation of viruses to a level acceptable by the European Standards. In addition, SCM9, and to a lesser degree SCM5, significantly enhanced the viability of A-549 cells infected with the virus (data are not shown).
Molecules 2020, 25, x FOR PEER REVIEW 11 of 29 The next aim of this study was to examine the antiviral activity of SCM5 and SCM9 in vitro against HHV-1. The treatment of A-549 cells infected with HHV-1 with SCM compounds resulted in significant reductions in virus yields, reaching up to a 3.5-log10 decrease for SCM5 and a 2.2-log10 decrease for SCM9 in comparison with appropriate DMSO control as it is shown in Figure 5. A 4-log10 reduction of virus titer demonstrates the ability of the tested compounds to inactivation of viruses to a level acceptable by the European Standards. In addition, SCM9, and to a lesser degree SCM5, significantly enhanced the viability of A-549 cells infected with the virus (data are not shown). A-549 cells were infected with HHV-1 and after 1h of virus absorption at 37 °C, inoculum of virus was removed and the infected cells were incubated for 48 h with SCM5, SCM9 compounds (31.2 µM) and DMSO as the solvent control. Acyclovir (50 µM) was used as a reference drug. The viral titer was expressed with reference to the TCID50 value, which is based on the cytopathic effect caused by this virus in approximately 50% of infected cells. The results, which were obtained from quadruplicate determinations (mean ±SE), are presented as the log10 TDIC50/mL values in relation with appropriate DMSO control. The level of virus was defined according to the requirements presented in PN-EN 14476:2013+A1:2015.

Influence of the Compounds on Expression of Signaling Molecules in Cell Lines
The researched compounds were tested for their influence on expression of signaling molecules playing roles in apoptosis in Jurkat T cell model. The expression of the following signaling proteins was analyzed: caspases3, 7, 8, 9, Bcl-2, Fas, NFκB and p53. Of interest, the compounds did not alter expression of these proteins except one instance, namely, SCM5 caused a complete block in Bcl-2 expression (data are not shown). Since the expression of MAP kinases may also predict a fate of cells (activation, differentiation or apoptosis) the expression of ERK, JNK and p38 in Jurkat cells have been we additionally measured. The results (Table 5) show that the compounds have differently affected expression of signaling molecules. SCM5 strongly suppressed the expression of ERK1, ERK2, JNK and to a lesser degree p38 subunits. On the other hand, the compound SCM9 did not significantly affect the expression of ERK, but elevated the expression of JNK, p38α and p38β. A-549 cells were infected with HHV-1 and after 1h of virus absorption at 37 • C, inoculum of virus was removed and the infected cells were incubated for 48 h with SCM5, SCM9 compounds (31.2 µM) and DMSO as the solvent control. Acyclovir (50 µM) was used as a reference drug. The viral titer was expressed with reference to the TCID 50 value, which is based on the cytopathic effect caused by this virus in approximately 50% of infected cells. The results, which were obtained from quadruplicate determinations (mean ±SE), are presented as the log 10 TDIC50/mL values in relation with appropriate DMSO control. The level of virus was defined according to the requirements presented in PN-EN 14476:2013+A1:2015.

Influence of the Compounds on Expression of Signaling Molecules in Cell Lines
The researched compounds were tested for their influence on expression of signaling molecules playing roles in apoptosis in Jurkat T cell model. The expression of the following signaling proteins was analyzed: caspases3, 7, 8, 9, Bcl-2, Fas, NFκB and p53. Of interest, the compounds did not alter expression of these proteins except one instance, namely, SCM5 caused a complete block in Bcl-2 expression (data are not shown). Since the expression of MAP kinases may also predict a fate of cells (activation, differentiation or apoptosis) the expression of ERK, JNK and p38 in Jurkat cells have been we additionally measured. The results (Table 5) show that the compounds have differently affected expression of signaling molecules. SCM5 strongly suppressed the expression of ERK1, ERK2, JNK and to a lesser degree p38 subunits. On the other hand, the compound SCM9 did not significantly affect the expression of ERK, but elevated the expression of JNK, p38α and p38β. The determination of MAP kinase expression in WEHI-231 cells, which were exposed to the compounds, revealed no significant changes in the case of SCM9 with except 2-fold increase in the level of p38δ (Table 6). On the other hand, SCM5 caused a strong decrease in expression of ERK-1, ERK-2 and to a lesser degree of JNK and p38 subunits. No significant changes were also observed with regard to MAP kinase expression in WEHI 213 cells in the case of SCM9 apart from 2× increase in p38δ (Table 6). More marked changes were noted in the case of SCM5 which increased the expression levels of p38γ (2.7×) and p38δ (3.7). In contrast, more significant changes in expression of signaling molecules, associated with apoptosis, were found in WEHI-231 cells incubated with SCM5. The changes ( Table 7) included strong elevations in expression of caspase 8 (8×), caspase 9 (23×), NFκB (17.9×), Bcl-2 (10×) and Fas (8.8×). SCM9, on the other hand, did not significantly change the expression of these molecules. The results, presented above, revealed in vitro immunosuppressive activity of the selected compounds (SCM5 and SCM9), which was, amongst the other, manifested by inhibition of cell proliferation and tumor cell growth. Both compounds exhibited the stronger antiproliferative effect on B cells in the mouse model than on T cells in the human peripheral blood cell population. Interestingly, only the compound SCM9 inhibited LPS-induced TNF α production which suggests its anti-inflammatory character. The changes in the expression of signaling molecules by SCM9 in Jurkat cells, such as 2× increase in JNK and an increase in p38α may indicate a proapoptotic action of the compound towards T cells associated with changes in MAP kinase expression [34,35]. However, it is intriguing that the signaling pathways induced by the compound did not involve caspase, NFκB, BcL-2 and Fas pathway despite very strong immunosuppressive properties of SCM9 towards WEHI-231. It is possible that the suppressive and anti-inflammatory action of SCM9 may engage still another signaling pathway, which could not be identified by the limited application of signaling molecules used in this study. In the case of SCM5, its immunosuppressive mechanism of action is more much easier to explain taking into consideration elicitation of signaling pathways by the compound. Firstly, the compound abrogated cell activation as evidenced by a strong, although differential decrease of expression of all MAP kinases, such as: ERK1 and ERK2, JNK and p38 subunits, which concerted involvement in cell differentiation and activation is essential [36]. More importantly, in the model of WEHI-231 cells, the compound induced strong amplification in expression of all molecules involved in apoptosis such as: caspase 8 [37], caspase 9 [38], NFκB [37,38], Bcl-2 [39] and Fas [40]. The level of cytotoxicity against A-549 cells at concentration of 62.5 µM is in accordance with our results on cell signaling showing that the proapoptotic action of SCM5 is more evident than that of SCM9. Of interest, the antiproliferative activity of both compounds on B cells, was stronger than this one observed with PBMC (predominantly T cells), since it was also high at lower concentration. For example, SCM5 at 25 µM/mL concentration, inhibited PBMC and splenocyte proliferation by 28% and 81.5%, respectively. This phenomenon may be of importance when potential therapeutic applications of the compounds are considered, for example in autoimmune disorders, particularly involving B cells and in neoplastic diseases. The ability of the compounds to interfere with the virus replication would require further research in terms of mechanism of action. Interestingly, stimulation of expression of p38 γ and p38 δ in WEHI 231 cells by the compounds may have significance in inhibition of virus replication [41]. Such properties, comparable to that of Acyclovir ® , may enable application possibility of SCM9 as a potential antiviral drug on condition further advanced and positive pharmacological research.
The preliminary analysis of structure/activity relationship of the considered compounds 5a-5j (SCM1-10) led us to the following general conclusions. Changes of substituents at position 7 in the structure of 7-aminooxazolo [5,4-d]pyrimidines 5 have resulted in varying degrees of biological activity. Amongst all the compounds that were biologically tested, the most active compounds, 5e (SCM5) and 5i (SCM9), have lipophilic chains with five linearly arranged non-hydrogen atoms, i.e., 7-N-pentyl and 7-(N,N-dimethylamino)propyl, respectively. In the opposition, there are the compounds with more hydrophilic substituents, such as 2-(morpholin-4-yl)ethyl in a molecule of the compound 5h (SCM8) or hydroxylalkyl ones in a molecule of the compounds 5f (SCM6) and 5g (SCM7), that showed no significant biological activity in the conducted tests.

Materials and Methods
Commercially available reagents, i.e., aminomalononitrile tosylate (AMNT), triethyl orthoacetate and aliphatic amines were purchased from following suppliers such as TCI (Tokyo, Japan) and Sigma-Aldrich (Merck Group, Darmstadt, Germany), and were used without further purification. Thin layer chromatography (TLC) analyses were conducted using Alugram SIL G/UV 254nm plates (Macherey-Nagel, Düren, Germany) and visualized by ultraviolet (UV) light at 254 nm (UV A. KRÜSS Optronic GmbH, Hamburg, Germany). Column chromatography was carried out using Silicagel 60 and the solvent system of eluent was composed of chloroform and ethyl acetate (9:1 or 1:1) or chloroform and methanol (9:1). Melting points were measured on uniMELT 2 apparatus (LLG, Meckenheim, Germany) and were uncorrected. Electrospray Ionization Mass Spectroscopy (ESI-MS) spectra were recorded with a compact TM Electrospray Ionisation-Quadrupole-Time of Flight (ESI-Q-TOF) apparatus (Bruker Daltonik GmbH, Bremen, Germany). All compounds for ESI-MS experiments were dissolved in methanol and detected as positive ions. Theoretical monoisotopic masses of ions were calculated (calc.) using Bruker Compass Data Analysis 4.2 software (Bruker Daltonik GmbH, Bremen, Germany). The Attenuated Total Reflectance IR (ATR-FT-IR) spectra (4000-450 cm −1 ) were recorded on a Nicolet iS50 FT-IR spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). using clean solid forms of the compounds (on the diamond crystal surface, 32 scans, resolution: 1 cm −1 , measurement temperature: 20-25 • C). IR Spectra were recorded with ATR intensity correction. It is known, that the relative intensity of bands in an ATR spectrum increases with wavelength causing the distortion of relative peak intensities in comparison with the classical transmission experiment [42].
Omnic Specta software (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for IR spectra analysis. Baselines of all IR spectra were corrected with autocorrection (fit order 2) and then all the spectra were normalized. 1 H-NMR (300.15 MHz), 13 (2) The reaction was carried out according to the protocol described by Freeman and Kim with slight modifications [29].

Method A (in ethanol)
To compound 4 (0.2 g, 0.58 mmol), a 30% solution of methylamine in ethanol (1 mL) was added in one portion. The reaction mixture was cooled to 5-10 • C and stirred for 0.5 h. When the reaction was complete, as shown by TLC, the precipitate formed was filtered off, dried and crystallized from ethanol. The compound 5a (0.062 g, 41 Figure S31 in the Supplementary Material); 13 C-NMR (DMSO-d 6 , 75.5 MHz): δ (ppm) 11.3 (carbon of methyl group of the isoxazole moiety), 23.4 (carbon of methyl group at position 5 of the oxazolopyrimidine system), 31.9 (carbon of N-methyl group at position 7 of the oxazolopyrimidine system), 81.6 (quaternary carbon C4 of the isoxazole ring), 116.7 (quaternary bridgehead carbon C7a of the oxazolopyrimidine system), 153.6, 154.4, 154.9, 157.0 (quaternary carbon C5 of the oxazolopyrimidine system), 157.2 (quaternary carbon C3 of the isoxazole ring), 169.2 (quaternary carbon C5 of the isoxazole ring) (see Figure S32 in the Supplementary Material). 1 H-13 C 2D COSY NMR spectrum of 5a (see Figure S33 in the Supplementary Material).

Method A (in ethanol)
To compound 4 (0.2 g, 0.58 mmol), a 30% solution of ethylamine in ethanol (1 mL) was added in one portion. The reaction mixture was cooled to 5-10 • C and stirred for 0.5 h. When the reaction was complete, as shown by TLC, the formed precipitate was filtered off, dried and crystallized from ethanol. The compound 5b (0.063 g, 39

Method B (in tetrachloromethane)
To compound 4 (0.14 g, 0.40 mmol), a 30% solution of propylamine in CCl 4 (1 mL) was added in one portion. The reaction mixture was cooled to 5-10 • C and stirred for 1 h. Then, the mixture was warmed to room temperature and maintained with stirring for 47 h. When the reaction was complete, as shown by TLC, the formed precipitate was filtered off. The solution after filtration was evaporated in a vacuum and the residue was dilution with ethanol. The formed precipitate was filtered off and dried to give the compound 5c as a white solid (0.015 g, 12 Figure S7 in the Supplementary Material). Other spectral analyzes (IR, NMR) confirm that the product is identical to that obtained by Method A.

Method A (in ethanol)
To compound 4 (0.2 g, 0.58 mmol), a 30% solution of ethanolamine in ethanol (1 mL) was added in one portion. The reaction mixture was cooled to 5-10 • C and stirred for 1 h. Then, the mixture was warmed to room temperature and maintained with stirring for 1.5 h. When the reaction was complete, as shown by TLC, the formed precipitate was filtered off, dried and crystallized from ethanol. The compound 5f (0.083 g, 49 Figure S51 in the Supplementary Material).

Method A (in ethanol)
To compound 4 (0.2 g, 0.58 mmol), a 30% solution of 3-aminopropan-1-ol in ethanol (1 mL) was added in one portion. The reaction mixture was cooled to 5-10 • C and stirred for 1 h. Then, the mixture was warmed to room temperature and maintained with stirring for 1.5 h. When the reaction was complete, as shown by TLC, the formed precipitate was filtered off, dried and crystallized from ethanol. The compound 5g (0.103 g, 58.5% Figure S54 in the Supplementary Material).

Method A (in ethanol)
To compound 4 (0.2 g, 0.58 mmol), a 30% solution of 2-(morpholin-4-yl)ethanamine in ethanol (1 mL) was added in one portion. The reaction mixture was cooled to 5-10 • C and stirred for 1 h. Then, the mixture was warmed to room temperature and maintained with stirring for 3.5 h. When the reaction was complete, as shown by TLC, the formed precipitate was filtered off, dried and crystallized from ethanol. The compound 5h (0.087 g, 41 Figure S58 in the Supplementary Material); 1 H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 2.33 (s, 3H, methyl group of the isoxazole moiety), 2.46 (t, 3 J ≈ 4.5 Hz, 4H, two morpholine ring methylene group connected directly with the morpholine ring nitrogen), 2.59 (s, 3H, methyl group at position 5 of the oxazolopyrimidine system), 2.61 (t, 3 J ≈ 6.8 Hz, 2H, methylene group of ethyl chain directly connected with the morpholine ring nitrogen), 3.56 (t, 3 J ≈ 4.5 Hz, 4H, two morpholine ring methylene group connected directly with the morpholine ring oxygen), 4.23 (t, 3 J ≈ 6.5 Hz, 2H, methylene group of the ethyl chain directly connected with the amine nitrogen at position 7 of the oxazolopyrimidine system), 7.26 (brs, 1H, -NH), 7.87 (brs, 2H, -NH 2 ) (see Figure S56 in the Supplementary Material); 13 C-NMR (DMSO-d 6 , 75.5 MHz): δ (ppm) 11.4 (carbon of the methyl group of the isoxazole moiety), 23.2 (carbon of the methyl group at position 5 of the oxazolopyrimidine system), 42.5 (carbon of the ethyl chain methylene group directly connected with the amine nitrogen at position 7 of the oxazolopyrimidine system), 53.6 (carbons of two morpholine ring methylene group connected directly with the morpholine ring nitrogen), 54.9 (carbon of the ethyl chain methylene group directly connected with the morpholine ring nitrogen), 66.2 (carbons of two morpholine ring methylene group connected directly with the morpholine ring oxygen), 81.6 (quaternary carbon C4 of the isoxazole ring), 117.0 (quaternary bridgehead carbon C7a of the oxazolopyrimidine system), 153.7 (quaternary bridgehead carbon C3a of the oxazolopyrimidine system, uncertain assignment), 153.8 (quaternary carbon C2 of the oxazolopyrimidine system), 154.8 (quaternary carbon C7 of the oxazolopyrimidine system, uncertain assignment), 156.8 (quaternary carbon C5 of the oxazolopyrimidine system), 157.2 (quaternary carbon C3 of the isoxazole ring), 169.3 (quaternary carbon C5 of the isoxazole ring) (see Figure S57 in the Supplementary Material).

Method A (in ethanol)
To compound 4 (0.2 g, 0.58 mmol), a 30% solution of N,N-dimethylpropane-1,3-diamine in ethanol (1 mL) was added in one portion. The reaction mixture was cooled to 5-10 • C and stirred for 1 h. Then, the mixture was warmed to room temperature and maintained with stirring for 4 h. When the reaction was complete, as shown by TLC, the formed precipitate was filtered off, dried and crystallized from ethanol. The compound 5i (0.127 g, 66.1%

Method B (in tetrachloromethane)
To compound 4 (0.2 g, 0.58 mmol), 30% solution of N,N-dimethylpropane-1,3-diamine in CCl 4 (1 mL) was added in one portion. The reaction mixture was cooled to 5-10 • C and stirred for 1 h. Then, the mixture was warmed to room temperature and maintained with stirring for 47 h. When the reaction was complete, as shown by TLC, the reaction mixture was evaporated in a vacuum and purified by chromatography on silica gel (CHCl 3 : AcOEt, 9:1). The compound 5i (0.06 g, 30.6%) was obtained as a beige solid: m.p. 202-204 • C (dec.); HR-ESI-MS: m/z [M + H] + calc. for C 15 H 22 N 7 O 2 332.1829, found 332.1824 (see Figure S14 in the Supplementary Material). Other spectral analyses (IR, NMR) confirmed that the product is identical to that obtained by Method A.

Method C
To compound 3 (0.05 g, 0.18 mmol), a 30% solution of N,N-dimethylpropane-1,3-diamine in ethanol (0.25 mL) was added in one portion. The reaction mixture was cooled to 5-10 • C and stirred for 1 h. Then, the mixture was warmed to room temperature and maintained with stirring for 4 h. When the reaction was complete, as shown by TLC, the formed precipitate was filtered off, dried and crystallized from ethanol. The compound 5i (0.007 g, 11.6%) was obtained as a white solid: m.p.  Figure S15 in the Supplementary Material). Other spectral analyses (IR, NMR) confirmed that the product is identical to that obtained by Methods A and B.   To compound 4 (0.3 g, 0.87 mmol), a 30% solution of cyclohexylamine in ethanol (1 mL) was added in one portion. The reaction mixture was cooled to 5-10 • C and stirred for 1 h. Then, the mixture was warmed to room temperature and maintained with stirring for 24 h. When the reaction was complete, as shown by TLC, the formed precipitate was filtered off and washed with chloroform. CHCl 3 -insoluble solid was filtered off. The filtrate was evaporated in a vacuum and purified by chromatography on silica gel (CHCl 3 :MeOH, 9:1). Compound 5l (0. group of the cyclohexyl ring), 7.80 (low intensity, 1H, -NH), 7.96 (brs, 2H, -NH 2 ) (see Figure S73 in the Supplementary Material); 13 C-NMR (DMSO-d 6 , 75.5 MHz): δ (ppm) 11.4 (carbon of methyl group of the isoxazole moiety), 24.5 (carbon of methyl group at position 5 of the oxazolopyrimidine system), 25.9, 26.5, 29.0 (carbons of cyclohexyl methylene groups -CH 2 -), 62.1 (methanetriyl (methine) carbon (-CH-N) of the N-cyclohexyl group at position 7 of the oxazolopyrimidine system), 81.5 (quaternary carbon C4 of the isoxazole ring), 118.3 (quaternary bridgehead carbon C7a of the oxazolopyrimidine system), 154.0, 154.3, 154.9 (three quaternary carbons C2, C3a and C7 of the oxazolopyrimidine system), 156.8 (quaternary carbon C5 of the oxazolopyrimidine system), 157.2 (quaternary carbon C3 of the isoxazole ring), 169.6 (quaternary carbon C5 of the isoxazole ring) (see Figure S74 in the Supplementary Material).

Single Crystal X-ray Diffraction Measurement of the Compound 5h
The single crystals of the oxazolo [5,4-d]pyrimidine derivative 5h suitable for X-ray diffraction analysis were obtained as follows: a small amount of the compound 5h was dissolved in methanol and the solution was stirred for about 10 min at 40 • C to ensure homogeneity and then cooled. After dissolving, the solution sample was sealed with a perforated film and placed under ambient conditions for slow evaporation. During the recrystallization, 3-4 drops of 2-propanol and methanol were added several times. Colorless needle-shaped crystals, suitable for X-ray diffraction experiments, were obtained after several weeks.
The X-ray diffraction data for 5h were obtained with an Xcalibur automated four-circle diffractometer with Ruby CCD camera using Enhance Source Cu-Kα radiation (λ = 1.5418 Å). The data were collected at 200(2) K by using an Oxford-Cryosystems cooler. Data collection, cell refinement, data reduction and analysis were carried out with CrysAlisPro [43]. Diffraction data have been corrected for absorption effects by multiple scans [43]. The crystal structure was solved using SHELXS [44]. Refinements were performed using SHELXL-2014/7 [45] based on F 2 through the full-matrix least-squares method. All H atoms were found in difference Fourier maps, and in the final refinement cycle they were repositioned in their calculated positions and refined using a riding model, with the length of C-H = 0.98-0.99 Å and with U iso (H) = 1.2 U eq (C) for CH 2 and 1.5 U eq (C) for CH 3 ; and N-H = 0.88 Å and with U iso (H) = 1.2 U eq (N) for NH and NH 2 . All non-hydrogen atoms were refined with anisotropic atomic displacement parameters. The structure plots for 5h were prepared with DIAMOND [46]. CCDC No. 2,005,200 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
Crystal data for 5h

Computational Details
A full geometry optimization and the thermochemistry of the following compounds has been performed on the basis of ab initio quantum mechanical DFT (density functional theory) method using the B3LYP [47,48] hybrid density functional with the extended 6-311++G(df,pd) basis set: The molecules of the compounds 5a, 5c, 8a, 8c, 5h, 7m and their theoretical isomers (tautomers) were treated as single isolated molecules in vacuum environment. Additionally, in the case of the compounds 5a, 5c, 8a, 8c, 5h and its imine tautomer, the Conductor-like Polarizable Continuum Model (CPCM) [32] was used for simulation of ethanol environment with Gaussian 16 default set of parameters (Scaling factor for van der Waals radius for all atoms, i.e., alpha = 1.1). All the calculations were computed using the Gaussian 2016 revision C.01 software [49]. To possibly get most accurate approximation the following keywords, i.e., Fopt = (Tight,CalcAll), SCF = (Direct,VeryTight) and Integral(Grid = SuperFineGrid) were used in the calculations. The confirmation that the final structure and presented results correspond to a minimum on the potential energy surface is the absence of imaginary wavenumbers in the results of frequency calculations. The thermochemistry calculations were carried out for standard conditions of temperature and pressure (i.e., T = 298.15 K and p = 1 atm). Stabilization energy (presented here as E stab = −∆E = 13.06 kcal mol −1 for vacuum conditions) of the compound 5a relative to its isomeric 8a was calculated as the difference (∆E 298 = −13.06 kcal·mol −1 ) between thermal-corrected energies (E = E elec. + ZPE + E trans. + E rot. + E vib. ) of the compound 5a and its isoform 8a. Taking into account the ethanol environment, the appropriate values of E stab are 12.43 kcal·mol −1 for the compounds (5a versus 8a). For the compound 5c and its isomeric 8c, ∆E 298 = −13.23 kcal·mol −1 , which gives E stab = 13.23 kcal·mol −1 for vacuum and ∆E 298 = −12.83 kcal·mol −1 , which gives E stab = 12.83 kcal·mol −1 for ethanol environment.
Free enthalpy (∆G trans(298) = 10.748 kcal·mol −1 ) of transition of proton between amine-imine tautomers of the compound 5h was calculated as difference between free enthalpy (∆ f G imine ) of formation of its imine tautomer and free enthalpy (∆ f G amine ) of formation of its amine tautomer. Because an equilibrium constant K is related to the standard free enthalpy change ∆G • through the following Potential Energy Surface (PES) was scanned through the change of dihedral angle CH 3 -C-N-CH 2 CH 3 of acetamidine moiety of N -cyanooxazolylacetamidine 7m in the full range of −180-180 • as a result of rotation about C-N bond to find highest energy conformers, which were needed to estimate the rotational energy barrier. As a result, two maxima of energy were found, which are connected with two conformers, for which appropriate dihedral angle CH 3 -C-N-CH 2 CH 3 was as follows −80.839 • and 105.162 • , respectively. Predicted energy (E rot = 23.4 kcal·mol −1 ) needed to overcome a rotational barrier to internal free rotation around bond CH 3 C-N(Et) 2 of acetamidine moiety at fifth position of oxazole ring of N -cyanooxazolylacetamidine 7m has been calculated as difference between free enthalpy of most energetically unstable (where appropriate dihedral angle CH 3 -C-N-CH 2 CH 3 is −80.839 • ) and most energetically stable conformer (where appropriate dihedral angle is −3.838 • ). Details on how to calculate enthalpies and Gibbs free energy of formation with using Gaussian can be found in the paper "Thermochemistry in Gaussian" [50].

Mice
Two month-old, specific pathogen free BALB/c male mice were provided by the Center of Experimental Medicine, Medical University (Białystok, Poland), The mice were kept in 12/12 h light/dark cycles with a free access to filtered tap water and commercial food. The animals were used only as organ donors what does not require approval of ethics committee according to Directive 2010/63/EU of the European Union Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.
Preparation of the Oxazolo [5,4-d]Pyrimidine SCM Derivatives for In Vitro Experiments The compounds SCM1-10 were initially dissolved in DMSO (6.25 mM). Then, the solutions were further diluted in the culture medium. In parallel, respective DMSO solutions were also prepared.

Statistics
The results were subjected to statistical analysis using analysis of variance (one-way ANOVA) in STATISTICA 7 for Windows (TIBCO Software Inc., Palo Alto, CA, USA). Brown-Forsyth s test was used to determine the homogeneity of variance between groups. When the variance was homogenous, analysis of variance (one-way ANOVA) was applied, followed by post hoc comparisons with the Tukey's test to estimate the significance of the difference between groups. Nonparametric data were evaluated with the Kruskal-Wallis analysis of variance. Significance was determined at p < 0.05. The results are presented as mean values ±SE. Two/three independent assays were conducted and the results are presented as mean values from quadruplicate determinations ±SE, taking into account only one representative assay.

Biological Tests
Determination of the Toxicity of the SCM Compounds against the A-549 Cell Line The cytotoxicity of the compounds was determined by measuring growth of human lung epithelial A-549 cells (Criteria of toxicity effect see Table 8). The cells were suspended in a density of 2 × 10 5 /mL in a culture medium, consisting of RPMI-1640 with the addition of 2% FCS, 100 U/mL of penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine (referred to below as "the culture medium"). The compounds were tested at a concentration range of 62.5-15.6 µM. The test was performed in 96-flat bottom plates containing 2 × 10 4 /100 µL /well cells incubated initially for 24 h in a cell culture incubator. Then, the supernatants were removed and appropriate dilutions of the compounds (0.2 mL) were added to the cells in a cell culture medium. The cultures were incubated for 72 h followed by determination of cell viability by a colorimetric MTT method measuring optical density (OD). In parallel, control cultures containing appropriate dilutions of the solvent (DMSO) were also incubated. The results are given in percentage inhibition in relation with appropriate DMSO controls (see Table 1). Data were shown as mean ±SE.

Isolation of the Peripheral Blood Mononuclear Cells (PBMCs)
Venous blood from a single donor was withdrawn into heparinized syringes and diluted twice with phosphate buffered saline (PBS). PBMCs were isolated by centrifugation on a Ficoll-Uropoline gradient (density 1.077 g/mL) at 400× g for 20 min at 4 • C. The interphase cells were then washed three times with Hanks' medium and re-suspended in the culture medium at a density of 2 × 10 6 cells/mL. PBMC were distributed into 96-well flat-bottom plates in 100 µL aliquots (2 × 10 5 cells/well). PHA was added at a concentration of 5 µg/mL. The compounds were tested at the following concentrations of: 50, 25 and 12.5 µM. DMSO at appropriate dilutions served as the control. After three days of incubation in a cell culture incubator, the proliferative response of the cells was determined by colorimetric MTT [51]. The results were obtained as optical density (OD) at 550/630 nm values and presented as mean values from quadruplicate determinations ±SE.

LPS-Induced Proliferation of Mouse Splenocytes
The spleens were pressed against a plastic screen into 0.83% NH 4 Cl solution to lyze erythrocytes (5 min incubation at room temperature). The cells were then washed twice with Hanks' medium, passed through a glass wool column to remove debris, and re-suspended in the culture medium. The cells were then distributed into 96-well flat-bottom tissue culture plates at a density of 2 × 10 5 /100 µL/well. 50 µg/mL of LPS was applied to induce cell proliferation. The compounds were added to the cultures at concentrations of: 25, 12.5, 6.25 and 3.125 µM. After a 3-day incubation, the cell proliferation was determined using a colorimetric MTT colorimetric assay. The results are presented as the mean OD (Optical density) at 550/630 nm from quadruplicate determinations ±SE.

LPS-Induced TNF-α Production in Whole Blood Cell Cultures
Human whole blood was diluted 10 times with RPMI-1640 medium and distributed to 24-well culture plates in 1 mL aliquots. The cultures were stimulated with LPS (100 ng/mL), and the studied compounds were added at concentrations of: 25, 10 and 1 µM. The control cultures contained DMSO in appropriate concentrations. After an overnight incubation, the supernatants were harvested and frozen at −80 • C until cytokine determination. TNF α levels in the supernatants were determined by ELISA kit and the results are shown as percentage of inhibition versus DMSO control (mean values ±SE).

Growth Inhibition of Tumor Cell Lines
The cells were re-suspended in the culture medium and distributed into 96-well flat-bottom plates. L-1210 was present at 1.5 × 10 4 cells/well while HT-29 and A-431 at 2.5 × 10 4 cells/well. The preparations were added to the wells at the concentration range of 50-12.5 µM. Cisplatin was used as a reference drug in the same concentrations. After 3-day incubation in a cell culture incubator, the proliferation was determined using MTT colorimetric method. The data are presented as a mean OD (Optical density) values from quadruplicate wells ±SE. 53 • C for 30 s and elongation in 72 • C for 15 s. GAPDH was used as a housekeeping gene for arbitrary unit calculation for every tested gene. The sequences of primers are enclosed in the Supplementary Material (Table S27).