1. Introduction
With over 9.6 million deaths in 2018, cancer is the second leading cause of death worldwide [
1]. Therefore, there is an urgent demand to develop novel anticancer drugs with high bioactivities and non-conventional modes of action [
2,
3,
4]. Papaverine is an opiate alkaloid isolated from
Papaver somniferum and
Rauwolfia serpentina plants. Chemically, papaverine (1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline) belongs to the benzylisoquinoline-alkaloid class of compounds, with the isoquinoline being substituted at positions 6 and 7 by methoxy groups and at position 1 by a 3,4-dimethoxybenzyl group (
Figure 1). It is a neutral solid with poor solubility in water. Papaverine is an antispasmodic drug which is used for the treatment of impotence and vasospasms (approved by the US Food and Drug Administration and non-FDA-approved) [
5,
6,
7]. As a soft muscle relaxant, vasodilator and narcotic agent, papaverine has a direct relaxant action on the smooth muscle which may be attributed to its ability to inhibit phosphodiesterases and calcium channels [
8,
9,
10]. It relaxes the smooth musculature of the larger blood vessels, particularly coronary, systemic peripheral and pulmonary arteries [
11]. Papaverine increases cerebral blood flow and reduces cerebral vascular resistance through its immediate vasodilating action on cerebral blood vessels and lack of impact on oxygen intake [
5,
6,
7]. Like quinidine, papaverine operates directly on the heart muscle to reduce conduction and extend the refractory period [
12]. Papaverine also showed a potential antiviral activity against respiratory syncytial virus, cytomegalovirus, measles and HIV [
13]. The biological half-life of papaverine hydrochloride given by the oral route is reported to be within the range of 1–2 h. Papaverine hydrochloride is rapidly absorbed orally and undergoes massive initial pass metabolism in the gut wall and liver, and the bioavailability is as low as 30% [
10].
As a potential antitumor drug, papaverine showed selective and potential antitumor activity against several types of cancer cells, including breast carcinoma T47D [
14], MCF-7 and MDA-MB-231 [
15], prostate carcinoma PC-3 [
16], LNCaP [
17,
18], colorectal carcinoma HT29 [
14], hepatocarcinoma HepG2 [
19] and fibrosarcoma HT1080 [
14]. Papaverine was also found to sensitize A549 lung and EO771 breast tumor cells to radiation therapy by inhibiting the mitochondrial complex 1 [
20]. Recently, papaverine has been identified—by in silico screening from the Drug Bank library—as a potential inhibitor for the receptor for advanced glycation end-products (RAGE). In this study, papaverine was found to dramatically inhibit HMGB/RAGE interaction and to suppress the production of inflammatory cytokines (IL-6 and TNF-α) [
21]. In glioblastoma, papaverine significantly inhibited the cell proliferation of human temozolomide-sensitive U87MG and temozolomide-resistant T98G cells with EC
50 values of 29 and 40 μM, respectively, by suppressing the interaction between HMGB1 and RAGE. Moreover, papaverine dramatically reduced tumor volume and delayed tumor growth in a human glioblastoma U87MG xenograft mouse model [
22]. In a further study by the same group, the combination of papaverine with temozolomide significantly and more potently reduced the clonogenicity of T98G cells and delayed the tumor growth in a human glioblastoma U87MG xenograft mouse model [
23]. Given these facts, papaverine has been considered as a potentially druggable scaffold for various tumor types and for RAGE inflammatory and immune disorders.
Metal ions play various crucial roles in human health, e.g., for their functions in drug’s action mechanisms and as indicative agents. In general, metals have several unique features, such as the power of redox activity, flexible coordination approaches and reactivity towards organic substrates [
24]. Due to their reactivity, abnormal metal ion concentrations are related to different pathological disorders, including malignant growth [
25]. Accordingly, metal ion complexes, either as medications or pro-drugs, are appealing for medicinal chemistry. Owning to their versatile reactivity, structure and geometry, metallodrugs provide a unique mode of action. Several studies indicated that the drug (or ligand) bioactivity improves upon binding to metal ions, which might be owed to the release of at least two biologically active species [
26,
27,
28,
29,
30,
31,
32]. To investigate such a concept for a drug, one approach would be to examine the in vitro activity of the metal complexes including the drug as a ligand. The best examples include the conjugate of ferrocene with quinoline, which has completed the phase II clinical trials [
30,
33]; the octahedral polypyridyl meatal-complexes, which enhanced the levels of reactive oxygen species by targeting the mitochondria, and are used photodynamic therapy as photosensitizers [
34,
35,
36,
37,
38,
39,
40]; and the nonsteroidal anti-inflammatory drugs coordinated with metals [
28]. Among different metal complexes, ruthenium, gold and vanadium complexes have attracted particular attention. While quinoline Ru-complexes show potential antimicrobial activities [
27,
29], the
p-cymene Ru-complexes show potential anticancer activities [
41,
42,
43,
44,
45]. The vanadium-based complexes have been extensively investigated as potential anti-diabetic, anti-cancer, antibacterial, antiviral, anti-atherosclerotic and anti-tuberculosis drugs [
46,
47,
48,
49]. After the FDA’s approval of auranofin (tetra-
O-acetylglucose-1-thiolgold(I) triethylphosphine complex) as a therapy for rheumatoid arthritis, the gold-based drugs have attracted special attentioin. The exploitation of gold complexes has led to vast diversity of gold compounds of biological relevance, including anti-cancer, anti-inflammatory and antiparasitic agents [
50,
51,
52,
53,
54,
55,
56,
57,
58].
Although various structural and activity studies have been successfully performed for many drugs, papaverine–metal activity studies are very rare [
59,
60]. Indeed, only one entry of a crystal structure with papaverine as a ligand can be found in the Cambridge structural database [
61]. These facts encouraged us to study the effects of complexing the bioactive benzylisoquinoline moiety of papaverine drug with a set of metals (V
+3, Ru
+3 and Au
+3). Herein, we report the syntheses and structural characterizations of a novel set of papaverine–metal complexes. The biological activities, including the antibacterial and antitumor activities, of the papaverine and its metal derivatives, were evaluated using various microorganisms and human cancer cell lines (MCF-7 and Hep.G2).
2. Materials and Methods
2.1. General Description of Materials
All chemical reagents and solvents were purchased from Merck Co and used without further purification, unless otherwise specified. All the solvents were used after distillation by standard methods.
2.2. Instrumentation
Elemental analyses (carbon, hydrogen and nitrogen content) were verified using a Perkin–Elmer CHN 2400 in the Micro-analytical unit at the Faculty of Science, Cairo University, Egypt. The metal ions were determined gravimetrically by transforming the metals into their corresponding oxides. Molar conductivities of freshly prepared 1.0 mmol/dm−3 solutions in DMSO were assessed using Jenway 4010 conductivity meter. The UV–Vis spectra for papaverine and its metal complexes were determined for a solution of 1.0 mM in DMSO using UV2 Unicam UV/Vis Spectrophotometer with a 1 cm quartz cell. Magnetic measurements were performed on a Sherwood scientific magnetic balance using Guoy’s method and Hg[Co(CNS)4] as calibrants in the micro analytical laboratory, Faculty of Science, Mansoura University, Egypt. The infrared spectra of papaverine ligand and their metal complexes were recorded on Bruker FTIR Spectrophotometer (4000–400 cm−1) in KBr pellets. 1H and 13C-NMR spectra of papaverine ligand and metal complexes were recorded on a Varian Gemini 200 MHz spectrometer using DMSO-d6 as the solvent and TMS as an internal reference. Thermogravimetric analysis (TGA and DTG) was conducted in dynamic nitrogen atmosphere (30 mL/min) with a heating rate of 10 °C/min using a Schimadzu TGA–50H thermal analyzer. The X–ray powder diffraction experiments were carried out using a Rikagu diffractometer. The crystal surface was examined by employing scanning electron microscopy (SEM) using JEOL JSM–840.
2.3. Synthesis of Metal Complexes
A solution of MCl
3 (0.1 mole, MCl
3 = VCl
3 (unhydrated), RuCl
3.3H
2O or AuCl
3.3H
2O) in methanol (0.5 M) was treated under stirring with a methanolic solution of papaverine hydrochloride (0.1 mol, 0.5 M). The pH of resulting mixture was then adjusted to pH ≈ 8 by carefully addition of methanolic ammonia solution (0.1 mol). After the resulting reaction mixture was allowed to stir under the same conditions for additional 3–4 h (during while a solid was formed), the mixture was filtered. The obtained solid product was washed two times with methanol and dried to afford the corresponding papaverine metal complexes in yields of 75–78% (details in
Table 1). The obtained solids were re-crystallized to afford pure products, as indicated by analytical analysis. The metal complexes were used directly without any further purification steps.
Au(III)–papaverine complex: Yield 77%. 1H NMR (DMSO-d6, 400 MHz, ppm): δ 8.25 (d, 1H, arm.3), 7.52 (d, 1H, arm. 4), 7.30 (s, 1H, arm.8), 6.81 (s, 1H, arm. 2′), 6.79 (d, 1H, arm. 5), 6.76 (s, 1H, arm 6′), 6.76 (s, 1H, arm 5′), 4.47 (s, 1H, -CH2-), 3.65 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.93 (s, 3H, OCH3). 13C NMR (DMSO-d6, 120 MHz, ppm): δ 158.3, 152.6, 150.0, 149.0, 147.5, 141.0, 133.3, 132.7, 122.5, 41.2, 118.9, 121.0, 118.9, 112.4, 106.1, 104.7, 56.2, 56.1, 55.9, 55.9.
2.4. Magnetic Susceptibility Measurements
Magnetic susceptibility measurements were carried out on a Sherwood Scientific magnetic balance according to Guoy’s method. The calculation was carried out using following equation:
where X
g is mass susceptibility per gram of sample, C is the calibration constant of the instrument and equal to 1.135, R is the balance reading for the sample and tube, R
0 is the balance reading for the empty tube, M is the weight of the sample in grams and T is the absolute temperature.
2.5. Antibacterial Investigation
The antibacterial activities of papaverine and its metal complexes were tested against the gram-negative bacteria
Klebsiella pneumonia and
Escherichia coli, and the gram-positive bacteria
Staphylococcus epidermidis and
Staphylococcus aureus. The agar hole-well diffusion technique with diameter 4 mm was applied [
62]. The investigated isolates of bacteria were cultivated in tubes and supplemented with nutrient broth. The seeded nutrient broth (1 cm
3) was homogenized in the tube with 9 cm
3 of melted nutrient agar (45 °C). The homogeneous suspension was filled into Petri dishes, and the holes were made in the cool medium. After cooling, 2 × 10
−3 dm
3 of papaverine or one of its metal complexes (at concentration of 1.0 mmol/dm
3) was applied in these holes. The dishes were incubated at 25–27 °C for 24 h, and then the inhibition zone diameters were measured and expressed in mm. The antibacterial activities of examined probes were compared to the activities of augmentin and unasyn at the same concentrations.
2.6. Anticancer Investigation
The two cell lines, MCF-7 and HepG-2, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1%
L-glutamine, HEPES buffer and 50 µg/mL gentamycin. All cell lines were incubated at 37 °C under a humidified atmosphere of 95% air and 5% CO
2 and were sub-cultured two times/week. All stock solutions were prepared in DMSO and the final concentration of DMSO in medium did not exceed 1% (
v/
v), at which cell viability was not inhibited. After the cells were allowed to resume exponential growth for 24 h, they were exposed to drugs at different concentrations in media for 72 h. The antitumor activity levels of papaverine and the corresponding Au complex were evaluated in vitro for comparisons with cisplatin and doxorubicin drugs using the viability assay [
63]. The 50% inhibitory concentration (IC
50), the concentration required to cause toxic effects of 50% in the intact cells, was estimated from graphic plots of the dose–response curves using Graphpad Prism software (San Diego, CA, USA). Evaluation was based on means from at least three independent experiments.
4. Conclusions
In this study, we described the synthesis of novel papaverine-metal (V+3, Ru+3 and Au+3) complexes. The structures of the synthesized complexes were characterized by elemental analysis, molar conductivity, TGA, SEM and several spectroscopic techniques (UV–Vis, XRD, SEM, NMR), which indicated octahedral geometry for these complexes. Biological evaluation of synthesized metal complexes revealed that the papaverine–Au(III) complex, among the complexes we synthesized, possesses potential anticancer activity against both breast cancer MCF-7 cells and human HepG-2 cells. The anticancer activity of Au complex against different cancer cell lines was higher than that of the papaverine ligand alone, which indicates that Au metal complexation improved the anticancer activity of the parent ligand. Interestingly, the Au–complex showed anticancer activity against MCF-7 (IC50 2.87 µg/mL), better than that of cisplatin. Overall, these results indicate that the Au(III)–papaverin complex is a promising antitumor compound that would make it a suitable candidate for further in vivo investigations.