Synthesis, Spectroscopic Characterization, and Biological Assessment of Novel Benzothiazole Derivatives Bound to Transition Metal Complexes

Introduction

Since their first identification by Schiff in 1864, the products of condensation between carbonyl compounds and primary amines are known as Schiff bases [1-3]. Schiff bases have found several applications in various fields, including analytical chemistry, catalysis, the food industry, fungicidal agents, agrochemicals, and biological activity [4]. The need for innovative, highly effective antimicrobials with few unwanted side effects is rising in response to the rising incidence of deep mycosis [5].

The benzothiazole ring system is a unique type of bicyclic ring with high significance in the pharmaceutical industry due to its potent biological actions. Compounds containing a simple benzothiazole nucleus are often explored to discover and evaluate products with promising biological activity. The 2-substituted benzothiazole structure, in particular, has gained importance in various therapeutic applications. Changes in the substituent group at the C-2 position can lead to alterations in bioactivity, as evidenced by structure-activity relationship studies [5].

First synthesized by A. W. Hofmann in 1887, substituted benzothiazole has been the subject of various synthetic methods due to its diverse action and facile cyclization mechanism [6]. Benzothiazoles, with their diverse applications and biological activity, especially anti-tumor, anti-inflammatory, antimalarial, antifungal, anticandidal, and other central nervous system (CNS) actions, have garnered significant attention [7]. Novel benzothiazole derivatives including the azo moiety and associated binuclear metal complexes are the focus of our research. We aim to synthesize, characterize, and evaluate their biological activity. This work aligns with our interest in the synthesis of benzothiazole derivatives.

Materials and methods

Merck, BLD Pharma, and Sigma-Aldrich supplied the solvents and reagents. The HBTADH and its metal complexes have their hydrogen, carbon, nitrogen, and sulfur percentages determined using the Perkin Elmer 240C elemental analyzer. The Bruker IMPACT HD mass spectrometer was used for the mass spectrometry analysis. A nuclear magnetic resonance (NMR) spectrometer from Bruker, calibrated with trimethylsilyl, was used to record the spectra of the HBTADH ligand. With the help of a JASCO V650 UV-visible spectrophotometer, electronic absorption spectra were captured in DMF. An FT-IR spectrometer from Bruker was used to acquire KBr pellet FT-IR spectra. We used a Gouy balance to quantify the complexes’ magnetic moments with Hg[Co(NCS)₄] as a standard, and we used Pascal’s constants to establish diamagnetic corrections. Using a Q band ESR Spectrometer, ESR spectra were acquired at room temperature. The instrument in question is the JES-FA200. An examination using powder XRD was carried out using BRUKER D8 VENTURE equipment that utilized Cu-Kα radiation.

Synthesis of HBTADH ligand

Scheme 1 shows the process for developing the HBTADH ligand. The 6-methoxy-2-aminobenzothiazole solution, which was 10 mM ethanolic, was stirred using a magnetic stirring rod in a round-bottom flask. Afterwards, a 1:1 ratio of 2,4-dihydroxybenzaldehyde ethanolic solution (10 mM) was added dropwise. Using TLC, the RM was agitated continuously at 50-60 °C for 2 hours. It was then necessary to cool the mixture to room temperature before isolating and purifying the solid result, which had a yellow hue, using cold ethanol and petroleum ether washes. Vacuum drying using desiccators containing anhydrous CaCl₂ was applied to the Schiff bases.

Scheme 1: Preparation of HBTADH. Click here to View Figure

Synthesis of metal (II) complexes:

While stirring a 20 mM hot ethanolic solution of HBTADH ligand, a hot ethanolic solution of metal sulfates (10 mM) was gradually added drop by drop, resulting in the development of color in the solution. The pH level was adjusted to 7 using a dilute alkali. The solid obtained was filtered and underwent successive washes with hot ethanol to eliminate any impurities. The metal complexes were then dried in vacuum desiccators with anhydrous CaCl₂ added.

Antimicrobial activity

The antibacterial efficacy of the HBTADH ligand and its complexes was assessed through the disc diffusion technique on a nutrient agar medium [21-22]. Antibacterial activity was evaluated in vitro for bacteria. The medication solution was left to soak into the plate for one hour to improve diffusion. After a 24-hour incubation period at 37°C for bacteria and 48 hours for fungus, the plates were examined for the zone of inhibition in mm. The molecule that inhibited bacterial growth was determined after 24 hours of incubation at 37°C in terms of concentration (µg/mL). It’s noteworthy that none of the microorganisms tested were influenced by variations in DMSO content in the medium.

In vitro cytotoxicity

Investigated for cytotoxicity through a brine shrimp bioassay, the synthesized HBTADH ligand, and complexes were examined [23]. With 38 grams of sodium chloride to 1000 milliliters of tap water, half of a tank was previously filled with salt water, and prawn eggs were added. The eggs hatched into nauplii within 48 hours, and the newborn shrimp were collected for the bioassay. Test tubes containing different amounts of dried complexes (2.5, 7.5, 10, and 12.5 mg/10 mL) were prepared, and their cytotoxic potential was assessed by dissolving DMSO in them.

To ensure accurate findings for the cytotoxic activity of the drug, every test tube was supplied with 10 live prawns through a Pasteur pipette. To ensure the reliability of the testing method, a control group was incorporated. After one day, the tubes were microscopically examined to record any pertinent observations and count the number of nauplii that survived. Each experiment consisted of three sets of five replicates. From the collected data, the LC50, LC90, 95% confidence limit, and chi-square were computed. Abbott’s method [24] was employed to adjust the data, accounting for control fatalities with the formula % deaths = [(test-control)/control] x 100.

Results and Discussion

The HBTADH ligand and its metal complexes exhibit color and stability when stored at room temperature. A wide range of organic solvents, including chloroform, methanol, acetonitrile, DMSO, DMF, and DCM, are compatible with HBTADH ligands. The complexes exhibit solubility in dimethyl sulfoxide (DMSO), and nitrobenzene (DMF), and not aqueous whatsoever. Analytical data for all synthesized complexes align well with the anticipated values for 1:2 metal-to-ligand stoichiometric ratios. The prepared complexes measured molar conductance’s at room temperature in nitrobenzene (10^-3 M solutions). Table 1’s results point to the fact that they are not electrolytic [25].

The HBTADH ligand and its metal complexes exhibit color and stability at room temperature. Various organic solvents, including methanol, acetonitrile, chloroform, DMSO, DMF, and DCM, are compatible with HBTADH ligands. The compounds are insoluble in water but dissolve in dimethyl sulfoxide, nitrobenzene, and dry mass solid solvents. Analytical data for all synthesized complexes align well with the anticipated values for 1:2 metal-to-ligand stoichiometric ratios. Table 1 shows that the complexes were not electrolytic when their molar conductance was measured at room temperature in nitrobenzene (10^-3 M solutions) [25].

Table 1: Analytical and physical data of prepared compounds

Comp	Color	MW	% Yield	MP/DP	Element Content						Cond	MM
					M	C	H	N	O	S
HBTADH	Yellow	300.33	86.45	183	–	59.99	4.03	9.33	15.98	10.68	–	–
Fe(BTADH)2	Blue	654.55	93.15	201	8.54	55.05	3.36	8.56	14.68	9.79	1.87	5.57
Co(BTADH)2	Brown	659.65	80.30	203	8.94	54.57	3.34	8.49	14.60	9.70	1.59	4.96
Ni(BTADH)2	Orange	659.35	79.88	202	8.90	54.60	3.34	8.49	14.60	9.71	0.72	3.00
Pd(BTADH)2	Red	706.66	84.98	208	15.00	50.94	3.11	7.93	14.60	9.06	0.22	–
Cu(BTADH)2	Green	664.21	74.10	205	9.57	54.20	3.31	8.43	14.50	9.64	2.28	1.91
Zn(BTADH)2	Yellow	666.05	82.28	208	9.82	54.05	3.30	8.41	14.40	9.61	0.89	–
Cd(BTADH)2	Yellow	713.07	82.13	211	15.77	50.49	3.09	7.85	13.50	8.98	1.39	–
Hg(BTADH)2	Red	800.66	80.86	207	25.05	44.96	2.75	6.99	11.99	7.99	4.25	–
Mn(BTADH)2	Brown	653.59	71.63	201	8.41	55.08	3.37	8.58	14.70	9.79	0.38	5.38

 

FT(IR) spectroscopy

The FTIR data that correspond to the metal complexes and ligands can be found in Table 2. In the infrared spectra of the HBTADH ligand, the phenolic hydroxyl group is represented by a band at 3068 cm-1. Phenol hydroxyl group deprotonation, as indicated by its absence in the complex spectra, implies that it participates in bond formation with the metal ions. Because the azomethine group contains u(>C=N-), the free ligand shows broadband at 1630 cm^-1 in its infrared spectra. Within complexes, this band moves to 1537–1588 cm^-1 as the ligand coordinates with the central divalent metal ions.

The prominent band at 1252 cm^-1 in the free HBTADH ligand spectrum indicates aromatic C-S stretching vibrations, and this vibration shifts to a lower frequency of 1206–1226 cm^-1 upon complexation, signifying bonding of the sulfur (aromatic) group of the HBTADH ligand to the metal ions. Additionally, newly observed bands between 537-635 cm^-1 and 535-599 cm^-1 are attributed to N-N and S-M, while those between 510 and 555 cm^-1 are attributed to O-M. Imino nitrogen, aromatic sulfur, and phenolic oxygen atoms are confirmed to be coordinated with metal ions by the FTIR spectra.

Table 2: Spectral data from FT(IR) analysis of the HBTADH ligand and its metal complexes

Comp	-OH (C4)	-OH (C2)	-OCH3	-C-H=	>C=N-	C-O	Ar -OH	C-S	N®M	S®M	O-M
HBTADH	3349	3068	2943	2830	1630	1328	1265	1252	–	–	–
Fe(BTADH)2	3350	–	2920	2848	1571	1362	1265	1212	635	538	517
Co(BTADH)2	3562	–	2924	2835	1537	1390	1279	1222	629	599	555
Ni(BTADH)2	3350	–	2921	2850	1578	1309	1266	1215	590	571	532
Pd(BTADH)2	3350	–	2922	2850	1565	1312	1264	1215	582	–	530
Cu(BTADH)2	3391	–	2921	2850	1580	1308	1263	1214	567	535	516
Zn(BTADH)2	3381	–	3187	2796	1588	1316	1226	1226	544	–	518
Cd(BTADH)2	3195	–	2981	2836	1583	1324	1252	1215	602	–	551
Hg(BTADH)2	3399	–	2920	2850	1566	1314	1263	1222	537	–	510
Mn(BTADH)2	3352	–	2920	2849	1568	1338	1274	1206	611	552	541

 

Figure 1: IR spectrum of the HBTADH ligand. Click here to View Figure

Figure 2: IR spectrum of the [Fe(BTADH)2] complex Click here to View Figure

Figure 3: IR spectrum of the [Co(BTADH)2] complex. Click here to View Figure

Figure 4: IR spectrum of the [Ni(BTADH)2] complex Click here to View Figure

Figure 5: IR spectrum of the [Pd(BTADH)2] complex Click here to View Figure

Figure 6: IR spectrum of the [Cu(BTADH)2] complex Click here to View Figure

Figure 7: IR spectrum of the [Zn(BTADH)2] complex Click here to View Figure

Figure 8: IR spectrum of the [Cd(BTADH)2] complex Click here to View Figure

Figure 9: IR spectrum of the [Hg(BTADH)2] complex Click here to View Figure

Figure 10: IR spectrum of the [Mn(BTADH)2] complex Click here to View Figure

ESR spectra

The ESR spectrum of the [Cu(BTADH)₂] complex offers essential insights into the degree of electron delocalization and the character of the metal-ligand interaction. Values of g_║, 2.08, and 1.51 were found via spectral analysis, in that order. A deformed octahedral geometry is shown by the greater g_║ value relative to g_⊥ in the [Cu(BTADH)₂] complex. In addition, the complex is in a ²B_1g ground state because an unpaired electron is located in the d_{x2− y2} molecular orbital. The designation “G” signifies significant trade contacts across copper centers [34].

The g-value, obtained from references [35, 36], is below 2.3, indicating a covalent connection between the metal and ligand. This information contributes to a comprehensive understanding of the electronic structure and coordination environment of the [Cu(BTADH)₂] complex.

UV-visible spectra and Magnetic moments

Electronic spectra measured in DMF solutions are summarized in Table 4. There was an increase in energy absorption between 445 and 285 nm, known as a charge transfer band, in every single compound.

An octahedral structure containing five unpaired electrons gives the Fe(II) complex an effective magnetic moment of 5.57 BM [57]. The existence of absorption bands at 585 nm in the electronic spectra, which correspond to the ⁵T_2g→⁵E_g electronic transition, allowed for the confirmation of an octahedral structure for the iron(II) complex [51].

According to the ⁴T_1g(F) → ⁴T_1g(P) u₃,⁴T_1g(F) → ⁴A_2g(F) u₂, and ⁴T_1g(F) → ⁴T_2g (F) u₁, transitions, the cobalt(II) complex exhibited d-d bands at 435, 635, and 900 nm in its electronic spectra, respectively. The complexes’ octahedral shape is supported by these transitions. Three d-d transition bands were visible in the absorption spectra of the nickel(II) complex, at 970, 620, and 529 nm. The presence of an octahedral structure with D₄h symmetry is supported by these bands, which correspond to the ³A_2g(F) → ³T_1g(P) ν₃,³A_2g (F) → ³T_1g(F) ν₂, and ³A_2g (F) → ³T_2g(F) ν₁,transitions, respectively.

In our analysis of the Ni(II), and Co(II) complexes, we considered several factors, including the ligand field stabilization energy (LFSE), covalency, B, and 10 Dq. The B and Dq values of Co(II) complexes were calculated by taking into account the complexes’ covalent nature and applying the u₃/u₁ ratio determined from the transition energy ratio diagram.

According to [50], absorptions at 490 and 686 nm, which are associated with the transitions ²B_1g→²B_2g,and ²B_1g→²E_g, respectively, in the Cu(II) complex, suggested a deformed octahedral geometry enclosing copper(II). Electronic spectra and magnetic moments provided evidence that the Mn(II) complex was structured octahedrally. According to [55], the electronic spectra showed bands at 442 and 375 nm, which were associated with ⁶A_1g→³T_1g(P) and ⁶A_1g→³T_1g (F) electronic transitions, respectively. The complex’s octahedral shape was confirmed by its effective magnetic moment measurement of 5.38 BM [56].

The spectra of the complex showed three bands, one for each of the three lower d orbitals and one for the unoccupied d_x2-y2 orbital, due to three d-d spin-allowed transitions. In the electronic spectra, distinct d-d transition bands were observed at wavelengths of 392 nm, 319 nm, and 283 nm. These transitions were identified as originating from the shifts between ¹A_1g→¹A_2g, ¹A_1g→¹B_1g, and ¹A_1g→¹E_1g energy levels, respectively. These compounds are square and planar, according to the electronic spectra [20].

Table 3: The HBTADH ligand and its metal complexes’ electronic spectrum.

Compound	lnm	Transition
HBTADH	265	p®p*
	283	p®p*
Fe(BTADH)2	442	6A1g → 4T1g(4P)
	375	6A1g → 4Eg(4D)
Fe(BTADH)2	585	5T2g→5Eg
	442, 368, 273	LMCT
Co(BTADH)2	~900	4T1g(F) →4T2g(F) (n1)
	635	4T1g(F) →4T2g(P) (n2)
Ni(BTADH)2	970	3A2g(F)→ 3T2g(F) (n1)
	620	3A2g(F) → 3T1g(F) (n2)
Pd(BTADH)2	392, 319, 283	LMCT
Cu(BTADH)2	686	2B1g → 2A1g (n1)
Zn(BTADH)2	392, 307, 384	LMCT
Cd(BTADH)2	325, 298	LMCT
Hg(BTADH)2	373, 330, 285	LMCT

 

Figure 11: UV spectrum of ligand. Click here to View Figure

NMR spectra

Table 4 presents the 1H NMR spectra (measured in ppm) of both the HBTADH ligand and its corresponding metal complexes dissolved in CDCl₃. The addition of D₂O serves to neutralize the acidic phenolic -OH protons at the C2 position (δ12.07 ppm) in the HBTADH ligand, indicating the involvement of the hydroxyl group in metal ion interaction.

One peak at δ3.91 ppm (s, 3H, H3C-O) is shown in the ¹H-NMR spectra of the ligand HBTADH, originating from the three protons of the methoxy group, one singlet at δ10.74 ppm (s, 1H, Ar-OH, C4), and one singlet at δ9.24 ppm (s, 1H, -CH=) attributable to a methyl group attached to the azomethine group. Additionally, three multiplets are observed at δ7.69-7.86 ppm.

Table 4: Measurements obtained with ¹H NMR spectra

Comp	-OH (C2)	-OH (C4)	-CH=	-OCH3	Aromatic Protons
HBTADH	12.07	10.74	9.24	3.91	6.54-7.86
Pd(BTADH)2	–	9.15	8.19	3.98	6.88-7.58
Zn(BTADH)2	–	9.73	8.12	3.70	6.21-7.89
Cd(BTADH)2	–	9.84	9.08	3.73	6.83-7.80
Hg(BTADH)2	–	9.88	8.71	3.73	6.12-7.91

 

Figure 12: The PMR spectrum of HBTADH ligand Click here to View Figure

Figure 13: PMR spectrum of [Pd(BTADH)2] complex Click here to View Figure

Figure 14: PMR spectrum of [Zn(BTADH)2] complex Click here to View Figure

Figure 15: PMR spectrum of [Cd(BTADH)2] complex Click here to View Figure

Figure 16: PMR spectrum of [Hg(BTADH)2] complex Click here to View Figure

Biological activity

A total of four types of bacteria were tested to determine the antimicrobial activities of the produced HBTADH ligand and its metal complexes. The bacterial samples comprised two strains of Gram-positive bacteria, two strains of Gram-negative bacteria, and two fungal strains.

Table 5 compiles the microbiological data, comparing them to the gold standard in pharmaceuticals. As illustrated in Figure 1, the metal complexes demonstrate increased antibacterial efficacy compared to the free HBTADH ligand, albeit exhibiting lower activity than the standard drugs. The enhanced biological activity of copper complexes might be linked to the atomic radius of the Cu(II) ion [48]. Metal complexes, characterized by high lipophilicity, contribute to their enhanced antibacterial action [49]. Overtone’s theory of cell permeability underscores the importance of lipophilicity as the master regulator of antimicrobial activity. The lipid membrane surrounding an organism’s cell selectively allows passage only to lipid-soluble molecules, making the lipophilic characteristic pivotal in antimicrobial activity. Chelation makes complexes more lipophilic by reducing the metal ion’s polarity, which in turn promotes electron delocalization across the chelate ring. Many aspects contribute to the efficiency of the complexes, including chelation, concentration, shape, stereochemistry, solubility, hydrophobicity, and coordination sites, among others.

Table 5: A study on the antimicrobial activities

Compound	Antibacterial Activity (zone of inhibition)
	S. aureus	B. subtilis	E. coli	P. aeruginosa
HBTADH	8	8	12	0
Fe(BTADH)2	14	13	18	9
Co(BTADH)2	15	9	24	10
Ni(BTADH)2	25	21	20	25
Pd(BTADH)2	8	14	0	9
Cu(BTADH)2	9	8	0	6
Zn(BTADH)2	14	8	0	0
Cd(BTADH)2	8	12	25	7
Hg(BTADH)2	14	8	23	12
Mn(BTADH)2	12	19	0	0
Streptomycin	10	7	13	8

Conclusion

Applying a variety of spectroscopic and analytical methods, mononuclear binary metal(II) complexes were synthesised and characterised from HBTADH. The results indicate that apart from the complex containing Pd(II), all the others have a high spin octahedral structure, tetrahedral geometry for Hg(II), Cd(II), and Zn(II), and square planar geometry for Pd(II). When an azomethine and an oxygen atom from a hydroxyl group join with a metal in a 1:2 ligand stoichiometry, a coordination complex develops.

Against every single bacterium that was examined, the metal complexes exhibited greater antibacterial activity than the free ligand. Compared to the Co(II) and Ni(II) complexes, the Cu(II) complex exhibited a far stronger antibacterial action. The following are the complicated structures that spectral studies have determined:

Scheme 2 Click here to View Scheme

Acknowledgment

The Authors are thankful to Riva Industries, 289/Kirwali, Raigad, Maharashtra, India-410201 for providing spectral data and other activity.

Conflict of Interest

The authors assert that there are no conflicts of interest.

References

1. Wang, H.; Dai, X. J.; & Li, C. J. Nature Chemistry, 2017, 9(4), 374-378.
   CrossRef
2. Wady, A. F.; Hussein, M. B.;  Mohammed, M. M.  Sch. Int. J. Chem. Mater. Sci, 2021, 4, 46-53.
3. Mary, Y. S.; Mary, Y. S.; Krátký, M.; Vinsova, J.; Baraldi, C.; Gamberini, M. C. Journal of Molecular Structure, 2021, 1228, 129680.
   CrossRef
4. Valentová, J.; Varényi, S.; Herich, P.; Baran, P.; Bilková, A.; Kožíšek, J.;  Habala, L. Inorganica Chimica Acta, 2018, 480, 16-26.
   CrossRef
5. Rahim, N. A. H. A.; Abbas, S. S.; Aljuboori, S. B.; Mahmood, A. A. R. Research Journal of Pharmacy and Technology, 2021,14(8), 4129-4136.
6. Qadir, T.; Amin, A.; Salhotra, A.; Sharma, P. K.; Jeelani, I.; Abe, H. Current Organic Chemistry, 2022, 26(2), 189-214.
   CrossRef
7. Shirvani, P.; Fassihi, A.; Saghaie, L.; Van Belle, S.; Debyser, Z.; Christ, F.  Journal of Molecular Structure, 2020, 1202, 127344.
   CrossRef
8. Pathak, N.; Rathi, E.; Kumar, N.; Kini, S. G.; Rao, C. M. Mini reviews in medicinal chemistry, 20(1), 2020, 12-23.
   CrossRef
9. Kumar, G.; Singh, N. P. Bioorganic Chemistry, 2021, 107, 104608.
   CrossRef
10. Spassov, D. S.; Atanasova, M.; Doytchinova, I. Frontiers in Molecular Biosciences, 2023, 9, 1405.
    CrossRef
11. Min, L. J.; Zhai, Z. W.; Shi, Y. X.; Han, L.; Tan, C. X.; Weng, J. Q.; Liu, X. H. Phosphorus, Sulfur, and Silicon and the Related Elements, 2020,195(1), 22-28.
    CrossRef
12. Silva, F. T.; Franco, C. H.; Favaro, D. C.; Freitas-Junior, L. H.; Moraes, C. B.; Ferreira, E. I. European Journal of Medicinal Chemistry, 2016, 121, 553-560.
    CrossRef
13. Shaikh, F. M.; Patel, N. B.; Sanna, G.; Busonera, B.; La Colla, P.;  Rajani, D. P. Medicinal Chemistry Research, 2015, 24, 3129-3142.
    CrossRef
14. Bauer, A.W.; Kirby, W.W.M.; Sherris J.C.; Turck. M. Am. J. Clin. Pathol., 1966, 45, 493.
    CrossRef
15. Gross, D.C.; De Vay, S.E. Physiol. Plant Pathol., 1977, 11, 13 
    CrossRef
16. Piro, O. E.; Echeverría, G. A.; Navaza, A.; Güida, J. A. Inorganica Chimica Acta, 2020, 511, 119831.
    CrossRef
17. Şakıyan, İ.; Özdemir, R.; Öğütcü, H. Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 2014, 44(3), 417-423.
    CrossRef
18. Özkan, E. H.; Kizil, H. E.; Şakiyan, İ.; Nartop, D.; Nurşen, S. A. R. I.; Güleray, A. Ğ. A. R. Gazi University Journal of Science, 2018, 31(2), 408-414.
19. Diaddario, L. L.; Robinson, W. R.; Margerum, D. W. Inorganic Chemistry, 1983, 22(7), 1021-1025.
    CrossRef
20. Heinert, D.; Martell, A. E. Journal of the American Chemical Society, 1963, 85(2), 188-193.
    CrossRef
21. Aly, G. Y.; Rabia, M. K.;  Al‐Mohanna, M. A. Synthesis and Reactivity in Inorganic and Metal-Organic Chemistry, 2004, 34(1), 45-66.
    CrossRef
22. Singh, G.S.; Pheko, T. Spectrochim. Acta, Part A. 2008, 70, 595–600.
    CrossRef
23. Wehrli, F.W.; Marchand, A.P.; Wehrli, S. Interpretation of Carbon-13 NMR Spectra; Wiley: New York, USA, 1988.

---

This work is licensed under a Creative Commons Attribution 4.0 International License.