ULTRA-VIOLET ABSORBING COMPOUNDS

Abstract

Novel compounds able to absorb UV radiation and comprise antimicrobial properties as well. The compounds are novel and undisclosed scytonemin analogs, able to absorb up to 90% of UV-A radiation and can cover a broader UV to blue light absorption range from 293 nm to 500 nm. When mixtures of more than one compound are used, their synergetic absorption properties can cover an even broader spectrum within 293 nm to 500 nm. The compounds present faint coloration, and some are even colorless, therefor the present compounds can be easily incorporated in formulations to be used in cosmetic products, sunscreens or lenses for sunglasses.

Claims

1. An ultra-violet (UV) radiation absorbing compounds scytonemin (A), 3′-hydroxyscytonemin (B) and 9,3′-dihydroxyscytonemin (C) of formulas: ##STR00003## and stereoisomers thereof, wherein the compounds have antibacterial activity.

2. (canceled)

3. The ultra-violet (UV) radiation absorbing compounds according to claim 1, wherein the compounds absorb light from 293 nm to 500 nm.

4. The ultra-violet (UV) radiation absorbing compounds according to claim 1, wherein the compounds absorb up to 90% of UV-A radiation.

5. (canceled)

6. The ultra-violet (UV) radiation absorbing compounds according to claim 1, wherein the compounds have antioxidant activity.

7. A cosmetic product comprising the ultra-violet (UV) radiation absorbing compounds of claim 1.

8. A sunscreen product comprising the ultra-violet (UV) radiation absorbing compounds of claim 1.

9. A sunglasses comprising the ultra-violet (UV) radiation absorbing compounds of claim 1.

10. A formulation comprising the compounds of claim 1.

11. The formulation according to claim 10, wherein the formulation comprises one compound.

12. The formulation according to claim 10, wherein the formulation comprises more than one compound.

13. Formulations A cosmetic product comprising the formulation according to claim 10.

14. A sunscreen product comprising the formulation according to claim 10.

15. A lens for sunglasses comprising the formulation according to claim 10.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0032] For easier understanding of this application, figures are attached in the annex that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.

[0033] FIG. 1 shows the .sup.1H NMR spectra of several fractions presenting typical scytonemin-like signals;

[0034] FIG. 2 shows the planar structure of 3′-hydroxyscytonemin (C.sub.36H.sub.24O.sub.5N.sub.2)—m/z [M-H]−: 563.1587;

[0035] FIG. 3 shows the planar structure of 3′-hydroxy-3-methoxyscytonemins (C.sub.37H.sub.28O.sub.6N.sub.2)—m/z [M-H]−: 595.1864;

[0036] FIG. 4 shows the planar structure of 3,3′-dihydroxyscytonemin (C.sub.36H.sub.30O.sub.6N.sub.2)—m/z [M-H]−: 585.2061;

[0037] FIG. 5 shows the .sup.1H NMR spectrum (600 MHz, DMSO-d6) of compound 3′-hydroxyscytonemin;

[0038] FIG. 6 shows the colors of scytonemin molecules at 1 mg/mL (from left to right): scytonemin (previously known), 3′-hydroxyscytonemin, 3′-hydroxy-3-methoxyscytonemin (isomer 1), 3′-hydroxy-3-methoxyscytonemin (isomer 2), scytonemin with the molecular formula C.sub.36H.sub.22O.sub.6N.sub.2 (isomer 1), scytonemin with the molecular formula C.sub.36H.sub.22O.sub.6N.sub.2 (isomer 2), 3,3′-dihydroxyscytonemin;

[0039] FIG. 7 shows the UV-Vis spectra of compounds m/z [M-H]−: 579.1589 (A), 581.1750 (B), 585.2061 (3,3′-dihydroxyscytonemin) (C), 591.1594 (D), 609.1689 (E), 611.1853 (F), 577.1369 (G), 673.1862 (H), 595.1864 (3′-hydroxy-3-methoxyscytonemins) (I), 593.1742 (J), 680.2218 (K), 657.1910 (L) and 563.1587 (3′-hydroxyscytonemin) (M);

[0040] FIG. 8 shows the cytotoxicity assays results in SH-SY5Y cell line for the pure compounds scytonemin C.sub.36H.sub.22O.sub.6N.sub.2 (isomers 1 and 2), 3′-hydroxy-3-methoxyscytonemin (isomers 1 and 2), 3′-hydroxyscytonemin and the original scytonemin. 1% DMSO (v/v) was used as negative control and 20% DMSO (v/v) as a positive control;

[0041] FIG. 9 shows the antibiograms of the novel compound 3′-hydroxyscytonemin (15 μg) against S. aureus ATCC 29213. Kanamycin and ampicillin (15 μg) were used as positive controls.

DESCRIPTION OF THE EMBODIMENTS

[0042] Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application.

[0043] The present application relates to scytonemin analogs (scytoneman-like compounds). The compounds were isolated from cyanobacteria collected from a small saltern pond located just behind the primary dune in Mrizika, Morocco (GPS coordinates: 32.955807, −8.779638). Morphological analysis allowed to classify the dominant cyanobacterium as a member of the Lyngbya genus. To explore the chemodiversity of the mat, the biomass was freeze-dried (650 g, d.w.) and extracted using a dichloromethane/methanol mixture (2:1). The crude extract was then fractionated by vacuum liquid chromatography using a solvent gradient with increasing polarity from 100% hexanes to 100% ethyl acetate and then to 100% MeOH. Fractions containing scytonemin-like signals in 1H NMR analysis were pooled and scytonemin was precipitated as described in the literature [8]. The filtered solution was subfractionated through flash chromatography using a gradient of hexanes, ethyl acetate to methanol. The resulting fractions were then analyzed by LC-HRMS, that revealed the presence of scytonemin and other major compounds whose calculated molecular formulas were consistent with scytoneman-like compounds, by only differing in a small number of atoms. The .sup.1H NMR spectra of these fractions presented the typical aromatic signals between 66.5 and 8 ppm, as well as OH signals between 69 and 10 ppm as have been observed for the previously reported scytonemins—Table 1, FIG. 1.

TABLE-US-00001 TABLE 1 Identification of new scytoneman- like compounds by LC-HRMS analysis Standard Difference Main Chemical deviation/ to reduced absorbance m/z [M − H].sup.− formula ppm scytonemin regions    563.1587 a) C.sub.36H.sub.24O.sub.5N.sub.2 3.55 +H.sub.2O UVA 577.1369 C.sub.36H.sub.22O.sub.6N.sub.2 5.30 +O.sub.2 UVA 579.1589 C.sub.36H.sub.24O.sub.6N.sub.2 5.68 +HO.sub.2 UVA/UVB 581.1750 C.sub.36H.sub.26O.sub.6N.sub.2 6.43 +H.sub.3O.sub.2 Green/Blue region     585.2061 b) C.sub.36H.sub.30O.sub.6N.sub.2 6.05 +H.sub.8O.sub.2 UVB 591.1594 C.sub.37H.sub.24O.sub.6N.sub.2 6.41 +CHO.sub.2 UVA/blue region 593.1742 C.sub.37H.sub.26O.sub.6N.sub.2 4.95 +CH.sub.3O.sub.2 UVA    595.1864 c) C.sub.37H.sub.28O.sub.6N.sub.2 7.37 +CH.sub.6O.sub.2 Green/blue region 609.1689 C.sub.37H.sub.26O.sub.7N.sub.2 4.47 +CH.sub.3O.sub.3 UVA/blue region 611.1853 C.sub.37H.sub.28O.sub.7N.sub.2 5.68 +CH.sub.5O.sub.3 UVB 657.1910 C.sub.38H.sub.30O.sub.9N.sub.2 5.62 +C.sub.2H.sub.8O.sub.5 UVB 680.2218 C.sub.41H.sub.33O.sub.8N.sub.2 8.72 +C.sub.5H.sub.11O.sub.4 UVA 673.1862 C.sub.38H.sub.30O.sub.10N.sub.2 5.94 +C.sub.2H.sub.8O.sub.6 UVB a) Structure of the compound in FIG. 2 b) Structure of the compound in FIG. 4 c) Structure of the compound in FIG. 3

[0044] No bibliographic data has reported scytoneman-family compounds with the identified molecular masses, indicating that these compounds are new and had never been isolated or characterized. Based on this data, the new scytoneman compounds were isolated by reverse-phase HPLC using a gradient of water and acetonitrile, and performed their structural elucidation by NMR, 2D-NMR and HRMS2 techniques.

[0045] The new compounds present the general structure of reduced form of formula A and C, and oxidized formula B.

##STR00002##

wherein,
And R1 and R6 are independently selected from H, OH, ═O, O(CH.sub.2).sub.nCH.sub.3 where n=1-16, Br, Cl, N and S;
R2, R3, R4 and R5 are independently selected from H, OH, O(CH.sub.2).sub.nCH.sub.3 where n=0-16, Br, Cl, N and S.

[0046] In the reduced form C, R3 and R4 do not exist when the carbons, to which R3 and R4 are bond, are connected by a double bond.

[0047] Formula A relates to the reduced form of the molecule, with the indol group in NH form), and formula B relates to the oxidized form with the indol group in N form. The R and S isomers of the disclosed compounds are an object of protection of the present application as well.

[0048] More specifically, the following compounds were isolated: 3′-hydroxyscytonemin, two stereoisomers of 3′-hydroxy-3-methoxyscytonemin and two stereoisomers of 3,3′-dihydroxyscytonemin, as shown in FIGS. 2 to 4.

[0049] FIG. 2 shows the planar structure of 3′-hydroxyscytonemin (C36H24O5N2)—m/z [M-H]−: 563.1587.

[0050] FIG. 3 shows the planar structure of 3′-hydroxy-3-methoxyscytonemins (C37H28O6N2)—m/z [M-H]−: 595.1864.

[0051] FIG. 4 shows the planar structure of 3,3′-dihydroxyscytonemin (C36H30O6N2)—m/z [M-H]−: 585.2061.

Example of the Structural Elucidation for Compound: 3′-Hydroxyscytonemin:

[0052] The 1H NMR spectrum of 3′-hydroxyscytonemin, shown in FIG. 5, showed several characteristic singlet signals for scytonemins, that could be ascribed to two NH protons at 611.43 and 612.31 and two OH protons at 69.01 and 610.09. Furthermore, a proton resonating as singlet at 66.45, which is not found in the 1H NMR data of scytonemin, did not show a cross-peak in the HSQC data. This proton was assigned as belonging to an additional, when compared to scytonemin, hydroxyl group, satisfying the HRMS-derived molecular formula (C.sub.36H.sub.24O.sub.5N.sub.2).

[0053] Although the new compounds present only slight differences in molecular composition when compared to scytonemin, these seem to strongly impact their UV-Vis absorption properties and their colors, as shown in FIG. 6. The previous role of scytonemin as a sunscreen active ingredient has been compromised by its dark color, seen in FIG. 6A, because products comprising the previously known dark scytonemin are not only not appealing to consumers, but also comprise the side effect of dying the fabrics that come in contact with such products. However, the different substitution pattern of these new scytonemins disrupts the conjugation of part of the molecules, leading to faint colored or even colorless compounds, overcoming this major issue that has been putting Cosmetic companies away from using these compounds.

[0054] The UV absorption profiles for these molecules and the maximum absorption wavelength can be seen in FIG. 7 and table 2, respectively. This data provides an evidence of the complementarity action of the compounds, covering a very broad range of radiation from 293 nm to 500 nm. This indicates that when used as a mixture, they can provide an efficient protection against UV-B, UV-A and blue light.

TABLE-US-00002 TABLE 2 Maximum absorption wavelengths of the presently described compounds. Main Chemical absorbance Max wavelength m/z [M − H].sup.− formula regions (nm) 563.1587 C.sub.36H.sub.24O.sub.5N.sub.2 UVA 361, 520 577.1369 C.sub.36H.sub.22O.sub.6N.sub.2 UVA 304, 377, 387 579.1589 C.sub.36H.sub.24O.sub.6N.sub.2 UVA/UVB 294, 367 581.1750 C.sub.36H.sub.26O.sub.6N.sub.2 Green/Blue 549 region 585.2061 C.sub.36H.sub.30O.sub.6N.sub.2 UVB 293 591.1594 C.sub.37H.sub.24O.sub.6N.sub.2 UVA/blue 290, 380, 413 region 593.1742 C.sub.37H.sub.26O.sub.6N.sub.2 UVA 361 595.1864 C.sub.37H.sub.28O.sub.6N.sub.2 Green/blue 500, 552 region 609.1689 C.sub.37H.sub.26O.sub.7N.sub.2 UVA/blue 366, 427 region 611.1853 C.sub.37H.sub.28O.sub.7N.sub.2 UVB 288, 304 657.1910 C.sub.38H.sub.30O.sub.9N.sub.2 UVB 288, 304 680.2218 C.sub.41H.sub.33O.sub.8N.sub.2 UVA/UVB 290, 367 673.1862 C.sub.38H.sub.30O.sub.10N.sub.2 UVB 287, 317

Cytotoxicity Tests

[0055] The pure compounds were also tested in cytotoxicity assays using the MTT assay. Compound 3′-hydroxyscytonemin showed considerable bioactivity, reducing SY-SH5Y (neuroblastoma cell line) viability to 37.8% in 24 h and 18.3% in 48h of exposure, at a final concentration of 10 μg mL.sup.−1, as shown in FIG. 8.

Antimicrobial Activity

[0056] The pure compounds were also tested in antimicrobial assays using the disc diffusion method. Compound 3′-hydroxyscytonemin presented antimicrobial activity against Staphylococcus aureus (ATCC 29213) at a final concentration of 15 μg mL.sup.−1, as shown in FIG. 9. Apart from its sunscreen function, the previously known scytonemins have been associated with anticancer and antioxidative activity, but no antimicrobial activity has ever been reported for any of the scytoneman family of compounds until now.

Synthesis

[0057] The scytoneman basic structure can be easily accessed and modified to match the structures herein disclosed through adaptation of currently known organic synthesis methodologies [2, 3] and because its biosynthetic pathway is known, fermentation in a heterologous host can also be used to produce this compound in vivo [4].

Application

[0058] Due to their extensive UV protection properties, the presently disclosed compounds can be used in formulations, particularly formulations for cosmetic products, sunscreens, lenses for sunglasses, or other applications, and thus complying with the need for UV-B, UV-A and blue light protection.

[0059] The fact that these compounds also present antimicrobial activity, combines yet another useful property with the UV protection.

[0060] In one embodiment the compounds are used in cosmetic products.

[0061] Additionally, in one embodiment these compounds can be used individually. In another embodiment the compounds are used in mixtures of more than one compound to extend the UV range of absorption.

[0062] In one embodiment, the formulations comprise one compound described herein. In another embodiment, the formulations comprise more than one compounds described herein.

[0063] This description is of course not in any way restricted to the forms of implementation presented herein and any person with an average knowledge of the area can provide many possibilities for modification thereof without departing from the general idea as defined by the claims. The preferred forms of implementation described above can obviously be combined with each other. The following claims further define the preferred forms of implementation.

REFERENCES

[0064] 1. D'Orazio, J., et al., UV radiation and the skin. Int J Mol Sci, 2013. 14(6): p. 12222-48. [0065] 2. Kumar, R., G. Deep, and R. Agarwal, An Overview of Ultraviolet B Radiation-Induced Skin Cancer Chemoprevention by Silibinin. Curr Pharmacol Rep, 2015. 1(3): p. 206-215. [0066] 3. https://www.wcrf.org/dietandcancer/cancer-trends/skin-cancer-statistics [0067] 4. Krutmann, J., The role of UVA rays in skin aging. Eur J Dermatol, 2001. 11(2): p. 170-1. [0068] 5. Halliday, G. M., et al., UV-A fingerprint mutations in human skin cancer. Photochem Photobiol, 2005. 81(1): p. 3-8. [0069] 6. Gasparro, F. P., Sunscreens, skin photobiology, and skin cancer: the need for UVA protection and evaluation of efficacy. Environ Health Perspect, 2000. 108 Suppl 1: p. 71-8 [0070] 7. Regazzetti, C., et al., Melanocytes sense blue-light and regulate the pigmentation through the Opsin 3. Journal of Investigative Dermatology, 2017. 137(10): p. S299-S299. [0071] 8. Garciapichel, F. and R. W. Castenholz, Characterization and Biological Implications of Scytonemin, a Cyanobacterial Sheath Pigment. Journal of Phycology, 1991. 27(3): p. 395-409. [0072] 9. Proteau, P. J., et al., The structure of scytonemin, an ultraviolet sunscreen pigment from the sheaths of cyanobacteria. Experientia, 1993. 49(9): p. 825-9. [0073] 10. Bultel-Ponce, V., et al., New pigments from the terrestrial cyanobacterium Scytonema sp. collected on the Mitaraka inselberg, French Guyana. J Nat Prod, 2004. 67(4): p. 678-81. [0074] 11. Grant, C. S. and J. W. Louda, Scytonemin-imine, a mahogany-colored UV/Vis sunscreen of cyanobacteria exposed to intense solar radiation. Organic Geochemistry, 2013. 65: p. 29-36. [0075] 12. Itoh, T., et al., Reduced scytonemin isolated from Nostoc commune suppresses LPS/IFNgamma-induced NO production in murine macrophage RAW264 cells by inducing hemeoxygenase-1 expression via the Nrf2/ARE pathway. Food Chem Toxicol, 2014. 69: p. 330-8. [0076] 13. Stevenson, C. S., et al., The identification and characterization of the marine natural product scytonemin as a novel antiproliferative pharmacophore. Journal of Pharmacology and Experimental Therapeutics, 2002. 303(2): p. 858-866. [0077] 14. Matsui, K., et al., The cyanobacterial UV-absorbing pigment scytonemin displays radical-scavenging activity. Journal of General and Applied Microbiology, 2012. 58(2): p. 137-144. [0078] 15. Ekebergh, A., et al., Oxidative coupling as a biomimetic approach to the synthesis of scytonemin. Org Lett, 2011. 13(16): p. 4458-61. [0079] 16. Ekebergh, A., A. Borje, and J. Martensson, Total synthesis of nostodione A, a cyanobacterial metabolite. Org Lett, 2012. 14(24): p. 6274-7. [0080] 17. Balskus, E. P., R. J. Case, and C. T. Walsh, The biosynthesis of cyanobacterial sunscreen scytonemin in intertidal microbial mat communities. Fems Microbiology Ecology, 2011. 77(2): p. 322-332.