MaterialsChemicals

Chloroform, Graphite, ethyl acetate H2O2, H2SO4, KMnO4 NaNO3 and NaOH were bought from Sigma (St. Louis, Mo., USA). Fetal Bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine and gentamycin were purchased from Lonza (Belgium).

Radioactive material

Technetium-99 m (99mTc) was eluted as pertechnetate (99mTcO4−) from a 99Mo/99mTc generator provided as a gift from Radio-isotopes Production Facility (RPF), Egyptian Atomic Energy Authority (EAEA), Cairo, Egypt.

Mammalian cell lines

Normal human lung fibroblast cells (MRC-5) were collected from the American Type Culture Collection (ATCC, Rockville, MD).

Docking of the synthesized compounds to the Active site of PARP-1Database preparation

All molecules were build using the builder functionality integrated in MOE2015.10 program. The molecules were subjected to stochastic conformational search and energy minimization using MMFF94X forcefield. All the generated conformation were saved as.mdb file to be used for later docking [29].

Protein targets preparation

PARP-1 crystal structure was downloaded from the protein data bank depository (https://www.rcsb.org/), PDB 4GV7 [30]. For protein preparation, all included water molecules were removed, 3D protonation was performed, and energy minimization and correction of bonding pattern was applied.

Identification of the binding site

The binding site was determined based on the position of the bounded co-crystallized inhibitor using the site finder functionality in MOE.

Docking protocol

Semi-flexible docking was applied using MMFF94x as a force field, triangle matcher as placement method and London dG as scoring function. For validation of the docking protocol, the co-crystallized ligand was included in the docked database and re-docked with the test compounds. Also, the docking protocol was done using Affinity dG as a scoring function to validate the results of the docking [29].

Synthesis of carboxylated nanographene oxide sheets (NGO-COOH)

Graphite oxide suspension was prepared from natural graphite powder using the modified Hummers' process. Exactly 1 g graphite and 1 g NaNO3 were added to 50 mL H2SO4 and the mixture was stirred for 10 min in an ice bath. Following that, the mixture was allowed to warm to room temperature while gradually adding 6 g of KMnO4. The formed suspension was stirred in a water bath (35 ℃), then mixed with one hundred milliliter of deionized water (DI) while keeping the temperature under 60 °C. Finally, 6 mL of hydrogen peroxide (30%) diluted in 200 mL deionized water was added to the suspension to solubilize manganese ions and to prevent the suspension from forming residual permanganate. Centrifugation was performed at 6000 rpm for 10 min then the supernatant solution was extracted and centrifuged several times to remove all the remaining acids and salts. The obtained nanographene oxide (NGO) suspension was sonicated for 30 min to obtain a yellow–brown graphene oxide (GO) suspension. Further centrifugation at 2000 rpm for 15 min was performed to dissolve the remaining unexfoliated graphitic platelets and any formed precipitates were eliminated. For carboxylation of NGO, 10 mL NaOH (12 mg/ mL) was added followed by sonication for 2 h at 800 W to convert OH groups to COOH [31,32,33,34,35,36].

Characterization of NGO-COOH nanosheets

Various techniques were used to characterize NGO-COOH nanosheets to ascertain their form, size, surface area, chemical composition, and dispersion. Transmission electron microscopy (TEM) with an acceleration voltage of 200 kV (Ted Pella, Redding, CA, USA), and dynamic light scattering (DLS) at an acceleration voltage of 200 kV (Ted Pella, Redding, CA, USA) were used for characterization. The XPS peak was deconvoluted using Gaussian components after a Shirley background subtraction. The O/C atomic ratio of the NGO sheets were evaluated using peak area ratio of the XP Score levels and the sensitivity factor of each element in XPS. Raman spectroscopy was carried out at room temperature using a HR-800Jobin-Yvon equipped with a 532 nm Nd-YAG excitation source. UV–Visible spectrophotometry using visible recording spectrophotometer UV-160A, Shimadzu, Japan and Fourier transformer infrared spectroscopy (FT-IR), (Mattson Instruments, Inc., New Mexico, USA was used. Samples were prepared for TEM measurements by placing 5- 20 µL of NGO-COOH dispersed solution on a Cu grid and then dried under an IR lamp while the sample had been diluted by utilizing the same sample quantity of bi-distilled water for DLS measurements.

Synthesis and characterization of 2,4-Dioxo-1,2,3,4-tetrahydroquinazoline-7-sulfonohydrazide Chemistry

The synthetic approach of the target quinazoline-sulfonohydrazide derivative as shown in Scheme 1.

Scheme 1

The synthetic approach of the target quinazoline-sulfonohydrazide derivatives (3 and 4a-c)

Preparation of quinazoline-2,4(1H,3H)-dione (1)

Compound 1 was prepared according to reported method [37]. Yield 72%, m.p. > 250 °C.

Preparation of 2,4-dioxo-1,2,3,4-tetrahydroquinazoline-7-sulfonyl chloride (2)

Compound 2 was prepared according to reported method. Yield 71%, m.p. 310 °C.

2,4-dioxo-1,2,3,4-tetrahydroquinazoline-7-sulfonohydrazide (3)

To a solution of the sulfonyl chloride derivative 2 (2.60 g, 10 mmol) in ethanol (30 mL), hydrazine hydrate (2 mL, 20 mmol) was added and the reaction was continuously stirred at room temperature for 6 h. The formed precipitate was filtered, washed several times with petroleum ether and then crystallized from ethanol to yield the hydrazide derivative 3 as a white powder.

Yield (65%), m.p.268–270 °C, IR (KBr, cm−1): 3344–3320 (4NH); 3132 (CH-aromatic), 2999 (CH-alicyclic); 1750, 1678 (2C = O); 1332, 1138 (SO2). 1HNMR (DMSO-d6, δ ppm): 4.17 (broad s, 2H, NH2, exchangeable with D2O); 7.30, 7.9 (2d, 2H, J = 7.08 Hz, aromatic-H); 8.28 (s, 1H, aromatic-H); 8.24, 11.31, 11.55 (3 s, 3H, 3NH, exchangeable with D2O).13CNMR (DMSO-d6, δ ppm): 114.73, 116.54, 128.06, 132.12, 134.23, 144.24, (aromatic-C); 150.61, 162.54 (2C = O). MS, m/z (%): 257 [M+. + 1] (30.09), 256 [M+] (19.27). Analysis for C8H8N4O4S (256.24), Calcd.: %C, 37.50; H, 3.15; N, 21.87; S, 12.51. Found: %C, 37.78; H, 3.37; N, 22.06; S, 12.38.

Preparation of 2,4-dioxo-N-substituted-1,2,3,4-tetrahydroquinazoline-7-sulfonamide (4a-c)

To a solution of compound 2 (2.60 g, 10 mmol) in ethanol (30 mL), an appropriate amine namely; p-toluidine, 2-aminobenzoic acid and thiazol-2-amine (10 mmol) was added. The reaction mixture was refluxed for 7 h. The formed precipitate was collected by filtration and crystallized from ethanol to give the corresponding derivatives 4a-c.

2,4-Dioxo-N-(p-tolyl)-1,2,3,4-tetrahydroquinazoline-7-sulfonamide (4a)

Yield (74%), m.p. < 300 °C, IR (KBr, cm−1): 3344–3320 (3NH); 3132 (CH-aromatic); 2996 (CH-alicyclic); 1750, 1678 (2C = O); 1332, 1138 (SO2). 1HNMR (DMSO-d6, δ ppm): 2.32 (s, 3H, CH3); 6.95–8.20 (m, 7H, aromatic-H); 11.21, 11.31, 11.50 (3 s, 3H, 3NH, exchangeable with D2O). 13CNMR (DMSO-d6, δ ppm): 20.76 (CH3); 121.31, 123.34, 124.51, 126.85, 129.43, 130.12, 130.67, 138.20, 141.37, 142.84, 144.28, 150.50 (aromatic-C); 150.67, 163.11 (2C = O). MS, m/z (%): 332 [M+. + 1] (45.09), 331 [M+] (41.89). Analysis for C15H13N3O4S (331.35), Calcd.: %C, 54.37; H, 3.95; N, 12.68; S, 9.68. Found: %C, 54.57; H, 3.70; N, 12.93; S, 9.90.

2-(2,4-Dioxo-1,2,3,4-tetrahydroquinazoline-7-sulfonamido) benzoic acid (4b)

Yield (74%), m.p. 280–282 °C, IR (KBr, cm−1): 3350- 3320 (3NH); 3025 (CH-aromatic), 2905 (CH-alicyclic); 1750, 1710, 1680 (3C = O); 1332, 1135 (SO2). 1HNMR (DMSO-d6, δ ppm): 6.71–6.90 (m, 2H, aromatic-H), 7.10 (d, 1H, aromatic-H), 7.14–7.26 (m, 1H, aromatic-H); 7.81, 7.87 (2d, 2H, J = 7.08 Hz, aromatic-H); 8.20 (s, 1H, aromatic-H), 11.08, 11.21, 11.31 (3 s, 3H, 3NH, exchangeable with D2O); 11.55 (s, 1H, OH, exchangeable with D2O). 13CNMR (DMSO-d6, δ ppm): 117.10, 123.61, 128.67, 132.43, 134.70, 130.53, 134.69, 137.90, 141.45, 142.84, 144.67, 150.81 (aromatic-C); 150.61, 162.91, 171.11 (3C = O). MS, m/z (%): 362 [M+. + 1] (36.50), 361 [M+] (28.03), Analysis for C15H11N3O6S (361.33), Calcd.: % C, 49.86; H, 3.07; N, 11.63; S, 8.87. Found: % C, 50.09; H, 3.27; N, 11.85; S, 9.17.

2,4-dioxo-N-(thiazol-2-yl)-1,2,3,4-tetrahydroquinazoline-7-sulfonamide (4c)

Yield (74%), m.p. 265–267 °C, IR (KBr, cm−1): 3348- 3330 (3NH); 3021 (CH-aromatic); 2910 (CH-alicyclic); 1746, 1680 (2C = O); 1332, 1135 (SO2). 1HNMR (DMSO-d6, δ ppm): 6.71, 7.11 (2d, 2H, J = 7.08 Hz, aromatic-H); 7.21- 8.20 (m, 3H, aromatic-H); 11.08, 11.21, 11.50 (3 s, 3H, 3NH, exchangeable with D2O). 13CNMR (DMSO-d6, δ ppm): 124.10, 128.15, 129.63, 131.90, 138.90, 141.45, 142.84, 144.67, (aromatic-C); 154.61, 168.91 (C = O). MS, m/z (%): 324 [M+.] (28.03). Analysis for C11H8N4O4S2 (324.33), Calcd.: % C, 40.74; H, 2.49; N, 17.28; S, 19.77. Found: % C, 40.59; H, 2.27; N, 17.65; S, 19.43.

Evaluation of cytotoxic effectsEvaluation of cytotoxic effects of NGO-COOH nanosheets

The in vitro cytotoxic effect of NGO-COOH was tested using Normal human lung fibroblast cells (MRC-5). The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/mL gentamycin. The cells were maintained at 37 ºC in a humidified atmosphere with 5% CO2 and were sub-cultured 2–3 times per week.

For the cytotoxicity assay, the cell lines were suspended in medium at concentration 5 × 104 cells/ well in Corning® 96-well tissue culture plates, then incubated for 24 h. NGO was then added into 96-well plates (three replicates) to achieve twelve concentrations for it. Six vehicle controls with media were run for each 96 well plate as a control. After incubating for 24 h, the numbers of viable cells were determined by MTT test. The 50% inhibitory concentration (IC50) was estimated using Graphpad Prism software (San Diego, CA. USA) from graphic plots of the dose response curve for each conc [38, 39].

Evaluation of cytotoxic effect of compound 3 and 4a-c

The cytotoxicity assay was performed at Department of therapeutic chemistry/ National Research Center. cytotoxic activity of target compounds 3, 4a-c was determined via three independent experiments by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay against the cell proliferation of human breast cancer MDA-MB-436 cells carrying natural BRCA1 deficient [R].

In vitro PARP inhibition assay

The in vitro inhibition of PARP-1 was measured using an HT F Homogeneous 96-well PARP Inhibition Assay Kit (Trevigen, Ca# 4690-096–K, Gaithersburg, USA), according to the manufacturer’s protocol. The synthesized compounds were dissolved in DMSO and then serially diluted to the required concentrations with distilled water, keeping the final concentration of DMSO lower than 1%. Olaparib was used as positive control. Fluorescence values under the condition of excitation wavelength (544 nm) and emission wavelength (590 nm) were measured using a multi-well spectrophotometer (Molecular Devices SpectraMax M5 microplate reader, Careforde, Chicago, USA). Then, the standard curve was drawn and the inhibition rate of each test compound was calculated. IC50 value of each compound was calculated using GraphPad Prism 6 software [40, 41].

Conjugation of NGO-COOH with compound 3 (NGO-COOH-3)

NGO-COOH nanosheets were condensed with compound 3 (12 mM) in the presence of N,N′-Dicyclohexylcarbodiimide (DCC) (12 mM) in DMF. Dicyclohexylurea formed was removed by filtration and DMF was removed under vacuum. The residue obtained was washed with water to remove excess of amine and traces of DMF. The residue was then purified by column chromatography using chloroform/ethyl acetate, 80:20 as an eluent and then recrystallized from alcohol [42,43,44].

Radiolabeling procedures

The eluted [99mTc]TcO4− was reduced from its hepta-oxidation state to enable the formation of the desired complex [99mTc]TcO4-NGO-COOH using sodium dithionite [45]. Compound 3 and its conjugate with NGO-COOH nanosheets were radiolabeled with 99mTc as follow;

Radiolabeling of compound 3

A volume of 200 μL of freshly eluted 99mTcO4− (20 MBq) was added to appropriate amount sodium dithionite (5- 25 mg) followed by the addition of different amounts of compound 3 (50- 750 mg) dissolved in 5 mL of DMF. The mixture was incubated for 10- 50 min and the pH was adjusted using the appropriate buffer solutions at 4- 8.

Radiolabeling of NGO-COOH-3

A volume of 200 μL of freshly eluted 99mTcO4− (20 MBq) was added to appropriate amount sodium dithionite (5- 25 mg) followed by the addition of different amounts of NGO-COOH (50- 500 mg). The mixture was incubated for 10- 60 min and the pH was adjusted using buffer solutions at 4–8.

Determination of in vitro stability

The in vitro stability of the radiolabeled complexes was studied in saline at 0.5, 2, 4, 6, 8 and 24 h post-incubation. The radiolabeling reactions were kept at 37 °C and a sample from each reaction mixture was withdrawn and the RCY was re-estimated by paper chromatography.

Determination of the radiochemical yield (RCY)

The RCY was determined using ascending paper chromatography. After the designated time interval, samples of each radiolabeling reaction mixture (200 µL, 20 MBq) were applied on strips of Whatman paper no. 3 (13 cm × 1 cm). The applied samples were allowed to air dry. Two different mobile phases were used for development [46,47,48,49,50]. First, chloroform/ethyl acetate mixture (1:2) was used as a mobile phase to check the percentage of free 99mTcO4−. Second saline, was used to determine the percent of reduced hydrolyzed 99mTc-colloid (RH-99mTc). After complete development, each paper strip was allowed to dry and cut into 1 cm pieces and counted in a well-type NaI (Tl) γ-counter (BLC-20, BUCK Scientific). HPLC was used to ensure that the labeled molecule was present as a single species and to ascertain the complexation yield. HPLC analysis of 99mTc were done by injection of 10 µl, after 0.20 µm Millipore filtration, into the column (C-18 reversed phase column) and UV spectrophotometer detector (SPD-6A) adjusted to the 270 nm wavelength. The column was eluted with mobile phase (water (A) and acetonitrile (B) mixed with 0.1% trifluoroacetic acid as the mobile phase. the flow rate was adjusted to 1 ml/min. Fractions of 1 ml were collected separately using a fraction collector up to 30 and counted in a well-type NaI (Tl) detector connected to a single-channel analyzer [51].

The RCY was calculated as follows;

$$Radiolabeled\;complex\;\% = 100-(^Tc\_^}_\%+RH ^ Tc\%).$$

Biodistribution study of radiolabeled complexes

To form a solid tumor, a 0.2 mL of Ehrlich Ascites Carcinoma fluid was administered intramuscularly in the right thigh of female Swiss Albino mice. The animals were well-cared until the tumors became obvious (7- 10 days). The parent tumor line (Ehrlich Ascites Carcinoma) was withdrawn from 7-day-old Swiss Albino donor females and diluted with sterile physiological saline solution to yield 12.5 x 106 cells/mL [52].

The animal study was conducted in accordance with the EAEA Committee on Animal Ethics (EAEA/2020/193) which follows the criteria set upon by the European Community for the use of animals as an experiment.

Biodistribution studies were performed by injecting the solid tumor-bearing mice intravenously with NGO-COOH nanosheets followed by injecting the radiolabeled complexes. The mice were divided into four groups (four mice per group) according to the designated time of dissection. Following the administration of the radiolabeled nanosheets, mice were dissected at 0.5, 1, 2, 4 h post injection (p.i). Blood, solid tumor, and major organs/tissues were collected and wet-weighed. The distribution of the radioactivity in each organ/ fluid was measured ex vivo, the radioactivity in each was detected by a gamma-counter (Perkin Elmer) as presented in Fig. 4. The results were expressed as mean percentage injected dose per gram (%ID/g ± SD) [53,54,55].