Synthesis, Crystal Structure and Evaluations of Its Cytotoxicity, Anti-microbial and Anti-hydroxyl Radical Activities of a New Co-crystal Compound (C6H6Cl2N2O2S)·(Phen)·H2O①

HUANG Ln-Zhen WANG Y-Nn CAI Zhuo QIU Xiu-Ying

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Synthesis, Crystal Structure and Evaluations of Its Cytotoxicity, Anti-microbial and Anti-hydroxyl Radical Activities of a New Co-crystal Compound (C6H6Cl2N2O2S)·(Phen)·H2O①

HUANG Lan-Zhena, bWANG Ya-NanaCAI ZhuocQIU Xiu-Yinga②

(ab541004)c(530004)

Co-crystal is a very potential kind of drug solid forms, and has a far-reaching influence on designing and preparing drugs. A new 1:1:1 co-crystal compound consisting of 4-amino-3,5-dichloro-benzenesulfonamide, 1,10-phenanthroline and water was synthesized, and its crystal structure was characterized by X-ray diffraction method. The compositions of the co-crystal are self-assembled into a three-dimensional network structure via intermolecular interactions including hydrogen bonds,-stacking, Cl×××Cl interactions and van der Waals’ forces. According to the evaluations of cytotoxicity assays, anti-microbial and anti-hydroxyl radicals, this co-crystal is a potential drug.

4-amino-3,5-dichloro-benzenesulfonamide, co-crystal, cytotoxicity, anti-microbial, anti-hydroxyl radical;

1 INTRODUCTION

Weak intermolecular interactions such as hydro- gen bonds play an important role in molecule-based structural and functional chemistry and biology[1,2]. A co-crystal is a structurally homogeneous crys- talline material that contains two or more neutral building blocks which are present in definite stoichiometric amounts[3]and assembledtogether by weak intermolecular interactions, such as hydrogen bonds,-or C–H×××stacking, van der Waals forces,. The physical and chemical propertiesofco-crystal compoundaresuper tothose ofsingle com- ponents[4], so it plays avery important roleinthesolidchemistryand pharmaceuticalchemistry[5-24].

In the context of pharmaceuticals, crystal engi- neering is an important process and intellectual pro- perty implications related to the control and repro- ducibility of composition and polymorphism[7]. Pharmaceutical co-crystal has become clear that a wide array of multiple component pharmaceutical phases can be rationally designed using crystal engi-neering, and the strategy afforded new intellectual property and enhanced properties for pharma- ceutical substances[4,7, 22]. Some co-crystal com- pounds formed by rac-ibuprofen, rac-flurbiprofen or aspirin with 4,4-bipyridine[22], and some pharma- ceutical molecules by forming novel compositions of ibuprofen, flurbiprofen, and aspirin have been reported[7]. Mino R. Caira[25]reported molecular complexes of sulfonamides and its 1:1 complex with acetylsalicylic acid.

However, the co-crystal compounds are very inadequate and about more than two thousands are recorded in Cambridge Structural Database (CSD), far less than the number of other solid forms. Using active pharmaceutical ingredient (API) and cocrystal former (CCF) to form co-crystal compounds through hydrogen bonds or other non covalent bonds will improve the physical and chemical properties of drugs. This is a good idea in new drug design. In the structure of 4-amino-3,5-dichloro-benzenesulfona- mide, there are sulfamide, amino groups, and chlo- ride substituent, which can form weak intermole- cular interactions with CCF. The polypyridines were often designed in the new chemistry and biology compounds[26-29], exhibiting better biological activi- ties. We herein report the synthesis, crystal structure and evaluations of anticancer, antimicrobial and anti-hydroxyl radical activities of the new 1:1:1 co- crystal compound consisting of 4-amino-3,5-dichlo- ro-benzenesulfonamide, 1,10-phenanthroline and water molecules.

2 EXPERIMENTAL

2. 1 Procurement of the materials

Solvents and chemicals obtained from commercial sources were of reagent grade and used without further purification. 4-amino-3,5-dichloro-benzene- sulfonamide can be synthesized according to the references[30, 31]. IR spectra were taken on a Pekin- Elmer spectrum One FT-IR spectrometer with KBr pallets in the range of 4000～400 cm-1. The elemental analyses for C, H, N and S were per- formed on a Perkin-Elmer 2400II elemental analyzer. The crystal structure was determined by a Bruker FRAMBO CCD area detector[32]. Cytotoxicity analysis was performed using the MTT (3-(4,5-di- methyl-2-thiazolyl)-2,5-diphenyl tetrazolium bro- mide) method, antimicrobial activities were obtained by the serial dilution method, and anti-hydroxyl radical activities were determined on the flow injection chemiluminescence (FI-CL) analysis system according to the reference[33]. Strains and cell lines were obtained from commercial sources.

2. 2 Synthesis of the title co-crystal compound (1)

A mixed solution containing salicylaldehyde (0.02442 g, 0.2 mmol) and 4-amino-3,5-dichloro- benzenesulfonamide (0.04822 g, 0.2 mmol) was stirred and refluxed at 55 ℃ for 1 h in ethanol, and a small amount of formic acid was added to the mixed solution as a catalyst for the synthesis of Schiff base. After 6 h reaction, 1,10-phenanthroline (0.0400 g, 0.22 mmol) and ammonium cerium (IV) sulfate tetrahydrate (0.2007 g, 0.3 mmol) in ethanol (10 mL, 95%) were also added to the aforemen- tioned solution. The mixture was stirred and refluxed at 55 ℃ for 12 h, and then was cooled to room temperature to afford the bright yellow precipitate which was removed by filtration. The filtrate was left at room temperature. Some yellow crystals were obtained after some days, giving yellow needle- shaped single crystals suitable for X-ray diffraction. For C18H16Cl2N4O3S anal. calcd. (%): C, 49.21; H, 3.67; N, 12.75; S, 7.29. Found (%): C, 49.22; H, 3.69; N, 12.74; S, 7.32. IR (KBr,, cm-1): 3489(s), 3386(s), 3305(s), 3024(m), 1678(m), 1613(s), 1554(m), 1494(m), 1460(m), 1409(m), 1332(s), 1261(m), 1219(m), 1162(s), 1128(m), 1051(w), 963(m), 868(m), 842(m), 756(s), 729(s), 626(m), 592(s).For O–H of water: 3489 cm-1; and for -NH2: 3386, 3305 and 1678 cm-1; and for C–H of benzene ring: 3024 cm-1; and for C=C and C=N of Phen: 1613, 1554, and 1494 cm-1; and for -SO2-: 1162, 1128, and 1051 cm-1; and for two C–Cl: 756 and 729 cm-1. Crystal reproducibility is very good, and the production rate is 63.4% (based on 4-amino-3,5- dichloro-benzenesulfonamide).

2. 3 Structure determination and refinement

A yellow single crystal with dimensions of 0.36mm × 0.20mm × 0.18mm was selected for the measurement. The data were collected on a Bruker FRAMBO CCD detector equipped with a graphite- monochromatized Moradiation (= 0.71073 ?) at 153(2) K using an-scan mode, and reduced with the Bruker SAINT. Absolute structure was determined with a Flack parameter= 0.00(1) (Abso- lute structure: Flack H.D. (1983), Acta Cryst. A39, 876～881). In the range of 3.01≤≤25.13° (–8≤≤8, –16≤≤17, –21≤≤21), a total of 13849 reflections were collected, of which 3447 were unique (int= 0.079) and 2402 were observed (> 2()). The structure was solved by direct methods using SHELXS-97(Sheldrick, 2008) and refined by full-matrix least-squares on2using the SHELXL- 97(Sheldrick, 2008)[34]program. The non-hydrogen atoms were assigned by anisotropic displacement parameters in the refinement. Hydrogen atoms cal- culated geometrically were included in the refine- ment by the riding method, with C–H = 0.9300 ? for aryl and N–H = 0.8999～0.9001 ? (iso(H) = 1.2eq(C),iso(H) = 1.2eq(N)), and O–H = 0.8474～0.8541 ? for water (iso(H) = 1.5eq(O)). The crystal of the complex belongs to the orthor- hombicsystem, space group212121, with= 7.4187(18),= 14.602(4),= 17.849(4) ?, C18H16Cl2N4O3S,M= 439.32,= 1933.5(8) ?3,= 2,D= 1.509 g/cm3,= 0.472 mm?1,(000) = 904. 3447 reflections were used in the succeeding refinement.The final cycle of refinement including 253 variable parameters was converged to(2> 2(2)) = 0.0636,(2) = 0.1461 (= 1/[2(F2) + (0.0759)2], where= (F2+ 2F2)/3),= 1.00, (Δ/)max= 0.001, (Δ)max= 0.319, (Δ)min= –0.351 e·??3, completeness to theta = 0.995.

Hydrogen bonds are listed in Table 1. The mole- cular structure of 1 with atomic numbering scheme is illustrated in Fig. 1, and a 2-D sheet structure of 1 in thebplane is illustrated in Fig. 2(A), a 2-D sheet structure of 1 in theplane in Fig. 2(C), and thestacking interaction of 1 in Fig. 2(B).

Table 1. Hydrogen Bonds for 1 (? and °)

Symmetry codes: (i) ?+1/2, ?,?1/2; (ii),?1,; (iii),?1,?1; (iv)+1,,?1

Fig. 1. Crystal structure of co-crystal compound. Displacement ellipsoids are drawn at the 50% probability level

Fig. 2. (A) Crystal packing diagram of co-crystal compoundin theplane, and the distance between Cl(1) and Cl (2) is 3.500 ? (symmetry code:+ 1,– 1/2, –+ 3/2). (B)stacking interaction of co-crystal compound, and some hydrogen atoms are omitted for clarity. (C) Crystal packing diagram of co-crystal compoundin theplane. The two dimension net structures are formed by intermolecular hydrogen bonds,-stacking, Cl×××Cl interactions and van der

Waals’ forces. The dotted lines in the figure are weak intermolecular interactions

2. 4 In vitro cytotoxicity

Cell culture: Cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum, 100 μg·mL-1penicillin and 100 μg· mL-1streptomycin. Cells were maintained at 37 ℃ in a 5% CO2incubator, and the media were changed every three days. MTT assay: Cell viability was determined by measuring the ability of cells to transform MTT to a purple formazan dye. We desig- ned compound sample grows (co-crystal compound, 4-amino-3,5-dichloro-benzenesulfonamide and Phen) and negative control group (physiological saline). Tumor cell lines (DLD-1, HepG2, MGC803, HeLa, HCT116) and normal cell line (HL-7702) were grown in a RPMI 1640 medium supplemented with 10% fetal calf serum, 100 μg·mL-1penicillin and 100 μg·mL-1streptomycin. They were incubated at 37 ℃ in a humidified incubator with 5% CO2and 95% air. Cells at the exponential growth stage were diluted to 3 × 104cells·mL-1with RPMI 1640, and then seeded in 96-well culture clusters (Costar) at a volume of 180 μL per cell, and incubated for 24 h at 37 ℃ in 5% CO2. Then the cells were treated at a volume of 20 μL per cell with various concentrations of complexes. The negative control group was set at the same time, and 5-fluorouracil is a positive control. After incubation of cells for up to 48 h, 20 μL of MTT (5 mg·mL-1) solution was added in each cell. After a further period of incubation (4 h at 37 ℃ in 5% CO2), each cell was added in 100 μL cell lysate (including 10% SDS (sodium dodecyl sulfate) – 5% isobutanol – 0.012 mL·L-1HCl (w/v/v)). After 12 h at 37 ℃,the values of OD were analyzed by a Microplate Reader at a wavelength of 490 nm. The percentage growth inhibitory rate of the treated cells was calculated by (OD negative control – OD compound sample)/OD negative control × 100%. The IC50values were determined by plotting the percentage viability versus the concentration on a logarithmic graph and reading off the con- centration at which 50% cells were viable relative to the control.

2. 5 Anti-microbial activity

The co-crystal compound was prepared into a series of concentrations of 10, 5, 2.5, 1.25 and 0.625 μmol·mL-1using sterilized distilled water. 1 mL of the solution was taken out from various concentra- tions of co-crystal compound, then added into the solution of hydrolysation casein agar of 9 mL at 50～55 ℃, with the final concentration to be 1.0, 0.5, 0.25, 0.125 and 0.0625 μmol·mL-1, respectively. These solutions were quickly spilled into the sterile flat, and then were coagulated. The control sample was set at the same time. Various experimental bacteria were diluted appropriately, and then seeded in the flat plates containing co-crystal compound and control sample with about 105CFU/point (colony-forming unit, the colony forming units CFU), and incubated at 37 ℃for 24 h. Finally, the minimum inhibitory concentration (MIC) values were observed and write-downed. Minimum concentration of the macroscopic observation to inhibit the growth of experimental fungus for the drug is MIC.

2. 6 Anti-hydroxyl radical activity

According to the literature[33], hydroxyl radical scavenging rate was tested by the FI-CL method. The mixed solution containing Fe2+ion, methylene blue, H2O2and water was the input analysis system through the corresponding line, and the resulting light signal was tested by photomultiplier tube and recorded chemical luminescence intensity as the value I0which is the negative control. Using Vit C solution instead of water in the aforementioned mixed solution and the same operating way, the value of chemical luminescence intensity is recorded as Is(Vit C) which is the positive control value. Using a sample solution rather than water in the aforementioned mixed solution and the same operating way, the value of chemical luminescence intensity is recorded as Is(sample). The D-value (I0– Is) is used as clear ·OH quantitative measure, and the hydroxyl radical scavenging rate, namely S, is calculated by the formula S = ((I0– Is)/I0) × 100%.

3 RESULTS AND DISCUSSION

X-ray crystallography reveals that 1 is a co- crystal compound consisting of one 4-amino-3,5- dichloro-benzenesulfonamide, one 1,10-phenanthro- line, and one crystal water molecule, namely (C6H6Cl2N2O2S)·(C12H8N2)·H2O, where C6H6Cl2N2O2S = 4-amino-3,5-dichloro-benzenesul- fonamide and C12H8N2= 1,10-phenanthroline (Fig. 1). In the structure of 1, all the bond lengths and bond angles fall in the normal ranges, and the co-crystal components are assembled together by weak intermolecular interactions containing hydrogen bonds,-stacking, Cl···Cl interactions, and van der Waals’ forces (Fig. 2(A, C)). As shown in Fig. 2(A), a two-dimensional structure is formed by hydrogen bonds (N(3)–H(3B)···O(1), N(4)–H(4A)···N(2), O(1W)–H(1WB)···O(1), and O(1W)–H(1WA)···N(2) (See: Table 1)) and Cl(1)···Cl(2) interactions in theplane, and-stacking is observed along theaxis to further form a three-dimensional structure. The distance between Cl(1) and Cl(2) is 3.500 ? (symmetry code:+ 1 ,– 1/2, –+ 3/2). The short Cl···Cl interaction with the distance of 3.500 ? is weaker than that of 4-amino-3,5-dichloro-benzene-sulfonamide with the distance to be 3.318 ?[31], whichshows that the co-crystal compound is slightly different from the monomer one. As shown in Fig. 2(B), X(1A) is the centre of benzene ring C(1)～C(6) of component 4-amino-3,5-dichloro- benzenesulfonamide, and X(1B) is the centre of benzene ring C(10)C(11)C(12)C(13)C(17)C(18) of component 1,10-phenanthroline (symmetry code:–1,–1,), and X(1C) is the centre of heterocyclic ring C(13)C(14)C(15)C(16)N(2)C(17) of com- ponent 1,10-phenanthroline (symmetry code:,–1,). The distance between X(1A) and X(1B) is 3.639 ?, and that between X(1A) and X(1C) is 3.571 ?, indicating-stacking between the benzene ring of 4-amino-3,5-dichloro-benzenesulfonamide and the benzene and heterocyclic rings of 1,10-phenan- throline, respectively. Moreover, in the 4-amino-3,5- dichloro-benzenesulfonamide molecule fragment S(1)–C(1)–C(2)–C(3)–Cl(1)–C(4)–N(3)–C(5)–Cl(2)–C(6)is planar (maximal deviation from the plane is –0.0583 ?, and mean deviation from the plane is –0.0302 ?; 7.052+ 3.939– 2.741= 0.2782). The intersection anglesare 54.3°, 106.6° and 90.7° between planes S(1)C(1)C(2)C(3)Cl(1)C(4)N(3)-C(5)Cl(2)C(6) and O(1)S(1)O(2), between amino-group planesH(4A)N(4)H(4B) and S(1)C(1)C(2)C(3)Cl(1)C(4)N(3)C(5)Cl(2)C(6), and between amino-group plane H(4A)N(4)H(4B) and plane O(1)S(1)O(2), respectively. This shows that amino-group is perpendicular to the plane O(1)S(1)O(2)anddeviates from the plane S(1)C(1)C(2)C(3)Cl(1)C(4)N(3)C(5)Cl(2)C(6). In the Phen molecule the fragment C(7)–C(16)–N(1)–C(17)–C(18)–N(2) is planar (maximal deviation from the plane is –0.0595 ?, and mean deviation from the plane is –0.0206 ?; 6.991+ 4.600– 2.012= 8.9639).

The cytotoxic potentialities ofI are analyzed in vitro by MTT assay on five different cancer cell lines and one normal live cell line.As shown in Fig. 3, the cell survival inhibition rate increases with the increase of concentration in the range of 8～200 μM, indicating that 1 exhibits significant cytotoxicity in a dose dependent manner. At the concentration of 1000 μM, the cytotoxicity for normal cell line was greater than those of the examined cancer cell lines, indicating that 1 was unsuitable for the anti-tumor drug at such a high concentration. The IC50values are shown in Table 2. The value of IC50for the HCT116 (13.55 ± 1.09) μM is the smallest among the cell lines, and the value forMGC803 is (16.30 ± 2.14) μM, which means that the abilities of inhibition proliferation of 1 for HCT116 and MGC803 cell lines are stronger than those of other examined cell lines. The ability of inhibition proliferation of 1 for HepG2 (41.98 ± 2.83) μM is weaker than that of the normal liver cell line HL-7702 (32.83 ± 7.80) μM, which means that1 exhibitssome harmfulness for the normal liver cells when 1inhibitsthe proliferation of HepG2. Moreover, the inhibition effects for DLD-1 and HeLa are poorer, and the IC50values are more than 200 μM, showing an unremarkable inhibitory effect. In addition, 1 exhibits more significant cytotoxicity than 5-fluorouracil against the examined cell lines. It's worth noting that theabilities of inhibition proliferation of1 are stronger than those of its eutral building block 3,5-dichlorosalfanilamide and 1,10-phenanthroline, which fully embodies the superiority of the co-crystal drug in pharmaceutical chemistry, because co-crystal is a new compound formed by the weak intermolecular interactions, and its physical and chemical properties do not result from the addition of the properties of each building block, but superior to each building block.

Table 2. IC50Values of the Tested Compounds towards Different Cell Lines

IC50values are given in μM. The values are expressed as the mean ± standard deviation (triplicates). DLD-1: human knot rectal cancer cell line; HepG2: human hepatocellular liver carcinoma cell line; MGC803: human gastric cancer cell line; HeLa: human cervical carcinoma cell line; HCT116: human colon cancer cell line; HL-7702: human normal liver cell line. 5-Fluorouracil is a positive control

Fig. 3. Cell inhibition rates assays of HCT116, MCG803, HepG2, DLD-1, HeLa, and HL-7702cell lines treated with various concentrations of 1 for 48h using a MTT method, respectively

Antimicrobial activity experimental results showed that1 can inhibit the bacterial colony grow, and the MIC valuesare 0.25, 0.25 and 1.0 μmol.mL-1for staphylococcus aureus (S. aureus),escherichia coli (E. coli) and pseudomonas aeruginosa (P. aeruginosa), respectively (Table 3). The antimicrobial activities for S. aureus and E. coli are better than that of P. aeruginosa, showing that 1 has certain reference value in the microbial immunology field.

Table 3. Co-crystal Compound Antibacterial Activities for S. aureus, E. coli and P. aeruginosa

Concentration (i): co-crystal compound concentration;Concentration (ii): eventual co-crystal compoundconcentrationin agar. (-): bacterial colony don’t grow; (+): bacterial colony grow.

Free radicals are related with aging, tumor, radiation damage, cytophagy,. The toxicity of hydroxyl radicals (·OH) is the strongest in biology active oxygen. It is of very practical significance to look for ·OH clearing agent and its applications in medicine, food, cosmetics, and so on. The ratios of the elimination of hydroxyl radicals were determi- ned by FI-CL method. It is well known that vitamin C is quite significant in resisting oxidation. Fixed the concentration of 10 ug·mL-1or 10 μmol·L-1, the clear ratio of 1 for hydroxyl radicals is 22.10% and 23.09% bigger than that of vitamin C, respectively (Table 4). At the concentration of 10 μmol·L-1, the clear ratio of 1 for hydroxyl radicals is four times that of vitamin C. 1 is a potential agent on the clearing hydroxyl radicals.

Table 4. Action of Antihydroxyl Radical Activities of 1

4 CONCLUSION

In conclusion, we successfully synthesized a new co-crystal compound (C6H6Cl2N2O2S)·(Phen)·(H2O). It’s structure was characterized, and cytotoxicity test, anti-bacterial activities and the abilities of resisting hydroxyl radicals were studied. It selectively inhibits the proliferation of tumor cells, and the inhibition effects for the HCT116 and MGC803 cell lines are superior to that of HepG2 cell lines. It exhibits obvious antibacterial activities for S. aureus, E. coli, and P. aeruginosa. Moreover, its anti-hydroxyl radical activity is superior to vitamin C. The results show the superiority of co-crystal compound in the design of drug molecules. In fact, pharmaceutical co-crystal used by crystal engineering has a far-reaching influence not only at the interface of chemistry and biology, but also on the advances in drug design and development, and it will be a good mainstream in the new compound drug design.

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26 February 2018;

11 May 2018 (CCDC 884597)

Guangxi Natural Science Foundation (No. 2016GXNSFAA380292), and National Natural Science Foundation of China (No. 21661011)

. Dr, associate professor, female, 44 years old, majoring in coordination chemistry, biochemistry and molecular biology. E-mail: xyqin6688@163.com

10.14102/j.cnki.0254-5861.2011-1985