Design, synthesis and biological evaluation of novel hydroxy-phenyl-1H-benzimidazoles as radical scavengers and UV-protective agents

Figures & data

Scheme 1. Synthesis of compounds 2–15.

Table 1. Results of antioxidant assays. Each value was obtained from three experiments (Mean ± SE).

Product	DPPH (μmolTrolox/mmol)(p ≤ 0.05)	FRAP (μmolTrolox/mmol)(p ≤ 0.05)	PCL (μmolTrolox/mmol)(p ≤ 0.05)
PBSA	< LOQ^a	0.79 ± 0.06	< LOQ^a
2	2.04 ± 0.15	12.23 ± 0.25	11.36 ± 0.08
3	2145.31 ± 45.8	2707.29 ± 29.52	22,838 ± 836,26
4	2.03 ± 0.09	16.22 ± 0.89	2.06 ± 0.05
5	21.41 ± 043	19.87 ± 0.22	159.1 ± 4.3
6	1362.12 ± 133.96	2713.3 ± 45.17	1924.35 ± 101.82
7	1.67 ± 0.08	21.94 ± 0.12	9.66 ± 0,04
8	1974.58 ± 16.89	2663.21 ± 32.53	19190.6 ± 443.18
9	16.47 ± 0.54	37.3 ± 0.35	198.53 ± 2.65
10	0.87 ± 0.05	23.04 ± 0.09	10.93 ± 0.05
11	1811.02 ± 61.7	2723.19 ± 35.74	1614.675 ± 19.95
12	1771.82 ± 84.75	2433.28 ± 21.74	13174.31 ± 240.68
13	32.48 ± 2.38	31.63 ± 1.96	160.26 ± 4.09
14	1.185 ± 0.02	2.62 ± 0.06	6.98 ± 0.27
15	1241.03 ± 9.43	2355.15 ± 52.55	1515.65 ± 75.56

LOQ^a limit of quantification.

Figure 1. Comparison between spectra of PBSA and molecules with sulfonic acid moiety on benzimidazole ring: 2, 3, 4, 5, 6.

Figure 2. Comparison between spectra of PBSA and molecules without substituents on benzimidazole ring: 7, 8, 9, 10, 11.

Figure 3. Comparison between spectra of PBSA and molecules with carboxylic acid moiety on benzimidazole ring: 12, 13, 14, 15.

Figure 4. Stability studies in formulation (formulations were submitted to accelerated aging at 40 °C for 63 days).

Table 2. Values of λ max and ɛ.

	λ max (nm)	ɛ
PBSA	302	25 000
2	308	25 000
3	313	23 000
4	315	21 000
5	318	23 000
6	328	22 200
7	306	24 000
8	311	16 000
9	315	14 000
10	315	17 000
11	325	12 000
12	317	21 000
13	321	20 000
14	312	24 000
15	332	17 000

Table 3 . Protecting efficacy^a.

Formulatedcompounds	SPF (p ≤ 0.05)	UVA/UVB (p ≤ 0.05)	UVAPF (p ≤ 0.05)	λc^b(nm)
PBSA	4.56 ± 1.09	0.26 ± 0.05	1.04 ± 0.06	333
2	4.74 ± 0.15	0.51 ± 0.01	1.36 ± 0.07	346
3	9.83 ± 0.64	0.74 ± 0.04	3.63 ± 0.23	358
4	2.22 ± 0.13	0.96 ± 0.01	1.06 ± 0.07	355
5	2.18 ± 0.14	1.13 ± 0.01	1.21 ± 0.04	370
6	5.99 ± 0.12	0.89 ± 0.03	2.72 ± 0.08	383
8	5.20 ± 0.20	0.78 ± 0.02	1.90 ± 0.09	368
9	3.57 ± 0.23	1.10 ± 0.05	1.89 ± 0.05	380
10	1.96 ± 0.14	1.01 ± 0.02	1.06 ± 0.08	378
11	2.85 ± 0.22	0.90 ± 0.01	1.35 ± 0.1	381
12	2.21 ± 0.17	1.00 ± 0.04	1.88 ± 0.12	370
13	2.85 ± 0.17	1.27 ± 0.02	1.58 ± 0.10	368
14	6.01 ± 0.31	0.70 ± 0.05	2.04 ± 0.11	350
15	3.69 ± 0.24	0.96 ± 0.08	1.87 ± 0.08	382

^a Values are the mean of three independent determinations, each of which at least five readings.

^b Wavelength at which the integral of the spectral absorbance curve reaches 90% of the area under the curve from 290 to 400 nm.

Table 4. Antioxidant activity of finished formulations^a.

Formulation	PCL μmol Trolox/g formulation(p ≤ 0.05)
PBSA	<LOQ^b
2	0.33 ± 0.03
3	2212.09 ± 68.35
4	0.06 ± 0.02
5	0.95 ± 0.02
6	101.98 ± 1.08
8	2972.107 ± 145.63
9	8.66 ± 0.04
10	0.5 ± 0.02
11	153.35 ± 8.14
12	1373.39 ± 19.43
13	4.97 ± 0.17
14	0.16 ± 0.02
15	86.99 ± 6.95

^a Values are the mean of three independent determinations.

^b Limit of quantification.

Table 5. Cytotoxicity results expressed as IC₅₀ values on NCTC-2544 cells.

Compounds	IC50 (μM)
PBSA	>50
2	26.4 ± 2.7
3	>50
4	>50
5	>50
6	23.1 ± 2.9
7	26.1 ± 2.4
8	>50
9	>50
10	>50
11	>50
12	>50
13	>50
14	>50
15	>50

Table 6. Phototoxicity test data.

Compound	Concentration (μM)	% Cell survival
		UVA 20 J/cm^2	UVB 0.5 J/cm^2	UVB 1 J/cm^2
		IC		69.1 ± 3.2%	63.1 ± 3.2%	33.8 ± 3.1%
PBSA	50	74.5 ± 1.4%	75.2 ± 4.5%	64.2 ± 3.1%
2	20	77.3 ± 2.8%	78.0 ± 3.5%	46.7 ± 3.4%
3	50	77.4 ± 4.0%	71.5 ± 2.5%	58.1 ± 2.6%
4	50	76.9 ± 2.0%	74.3 ± 3.2%	37.7 ± 1.7%
5	50	79.0 ± 6.0%	70.8 ± 3.9%	38.6 ± 3.7%
6	20	18.4 ± 2.3%	59.3 ± 3.4%	29.7 ± 2.1%
7	20	72.8 ± 2.9%	74.2 ± 1.3%	44.0 ± 4.1%
8	50	62.9 ± 4.3%	72.8 ± 1.0%	43.9 ± 3.4%
9	50	3.7 ± 0.5%	1.1 ± 0.3%	0.5 ± 0.1%
10	50	4.8 ± 0.6%	19.8 ± 2.4%	3.5 ± 0.3%
11	50	1.2 ± 0.2%	45.6 ± 2.6%	27.0 ± 2.4%
12	50	74.3 ± 2.0%	76.3 ± 3.6%	64.5 ± 3.5%
13	50	80.7 ± 1.3%	69.0 ± 0.2%	57.5 ± 2.4%
14	50	76.7 ± 3.4%	74.2 ± 3.4%	58.4 ± 2.5%
15	50	12.2 ± 1.3%	60.1 ± 2.3%	25.6 ± 2.7%

Table 7. Evaluation of photostability^a.

Product	Residual product (p ≤ 0.05)
PBSA	96.7% ± 1.8%
3	94.9% ± 0.8%
12	98.4% ± 0.9%

^a Values are the mean of four independent experiments.

Figure 5. SPF values (reported in dark grey) and UVA-PF values (reported in light grey) of the tested formulations.

Table 8. Mixed commercial filters used in the formulations.

UV filter composition	SPF 15	SPF 30
INCI name	Amount	Amount
Butyl methoxydibenzoylmethane	2.3%	4.7%
Ethylhexyl methoxycinnamate	4.0%	8.0%
Octocrylene	4.0%	8.0%

Table 9. In vivo evaluation of selected formulations.

Emulsion	In vivo SPF
Oxisol (12) 3%	2.68
Mixed filters SPF 15	15.78
Oxisol (12) 3%+Mixed Filters SPF15	21.74