HALOGENATED COMPOUNDS, PROCESS AND USES THEREOF

Abstract

The present disclosure relates to halogenated fatty acid lactylates, in particular to chlorinated fatty acid lactylates.

The halogenated fatty acid lactylates now disclosed have antimicrobial and/or antibiofilm activity towards healthcare associated microbial infections.

Claims

1. A compound of formula I ##STR00006## or a pharmaceutically acceptable salt, ester or solvate thereof, wherein, R, R.sup.1, R.sup.2 are independently selected from each other; R is a C.sub.8-C.sub.16 alkyl chain comprising at least one halogen selected from the group consisting of Cl, Br and I, wherein the at least one halogen is in-located at any position of said chain; R.sup.1 is H, CH.sub.3, CH.sub.2CH.sub.3 or CH(CH.sub.3)COOH; R.sup.2 is H, CH.sub.3, Cl, Br or I.

2. The compound of claim 1, wherein R is a C.sub.10-C.sub.14 alkyl chain comprising at least one halogen selected from the group consisting of Cl, Br and I, wherein the at least one halogen is located at any position of said chain.

3. The compound of claim 1, wherein R is a C.sub.10-C.sub.14 alkyl chain comprising at least one Cl located at any position of said chain.

4. The compound of claim 1, wherein R is a C.sub.11-C.sub.13 alkyl chain comprising at least one halogen selected from the group consisting of Cl, Br and I, located at any position of said chain.

5. The compound of claim 1, wherein the compound is ##STR00007## and wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are independently selected from each other; R.sup.3, R.sup.4, R.sup.5 are each independently H, CH.sub.3, Cl, Br, or I, and at least one of R.sup.3, R.sup.4 or R.sup.5 is Cl, Br, or I.

6. The compound of claim 5, wherein R.sup.3, R.sup.4, R.sup.5 are each independently H, CH.sub.3, or Cl.

7. The compound of claim 5, wherein at least one of R.sup.3, R.sup.4 or R.sup.5 is Cl.

8. The compound of claim 1, wherein R.sup.1 is H.

9. The compound of claim 1, wherein R.sup.2 is CH.sub.3.

10. The compound of claim 5, wherein R.sup.3 is H or Cl, R.sup.4 is H or Cl, and R.sup.5 is Cl or CH.sub.3.

11. The compound of claim 1, wherein the compound is ##STR00008##

12. (canceled)

13. A method for treating or preventing a microbial infection in a subject, the method comprising administering the compound of claim 1 to the subject.

14. The method of claim 19, wherein the bacterial infection is a coagulase-negative staphylococci infection.

15. The method of claim 14, wherein the coagulase-negative staphylococci infection is a Staphylococcus aureus infection.

16. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1 and a pharmaceutically acceptable excipient.

17. A medical device comprising the compound of claim 1 as a biofilm inhibitor.

18. A cyanobacterium strain with a deposit under the number 1471/1 of Aug. 8, 2019 at CCAP.

19. The method of claim 13, wherein the microbial infection is a bacterial infection.

20. The method of claim 19, wherein the bacterial infection is selected from the group consisting of endocarditis, osteomyelitis, sinusitis, urinary tract infection, chronic prostatitis, periodontitis, and chronic lung infection in cystic fibrosis patients.

21. The medical device of claim 17, wherein the medical device is an implant, a catheter, or a combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The following figures provide preferred embodiments for the present disclosure and should not be seen as limiting the scope of the disclosure.

[0044] FIG. 1. Results of the bioassay for antibiotic activity, where the bioactive fractions F32, F34-F28, F40-F41 and F43-48 showed complete inhibition of Staphylococcus aureus (S54F9 strain) growth. These chromatographic fractions were obtained by HPLC separation of the extract of Sphaerospermopsis sp. LEGE 00249.

[0045] FIG. 2. Mass spectrometry data of bioactive fractions F43-F45 that contain compounds 1 and 2. These chromatographic fractions were obtained by HPLC separation of the extract of Sphaerospermopsis sp. LEGE 00249.

[0046] FIG. 3. Mass spectra of bioactive fractions that contain compounds 9 and 16. These chromatographic fractions were obtained by HPLC separation of the extract of Sphaerospermopsis sp. LEGE 00249.

[0047] FIG. 4. Planar structures of the novel chlorinated fatty acid lactylates 1, 2, 9 and 16.

[0048] FIG. 5. (A) Spectrum view of fraction F44 at 4.6 min showing the m/z 305.1499 and m/z 377.1697, and the in-source-formed species at m/z 269.1733 and m/z 341.1938. Mass differences are shown. (B) Proposed mechanism for the generation of the in-source-formed species m/z 269.1733 and m/z 341.1938.

[0049] FIG. 6. (A) Spectrum view of fraction F35 at 3.6 min showing the m/z 339.1101 and m/z 411.1300, and the in-source-formed species at m/z 375.1540 and m/z 303.1337. (B) Spectrum view of fraction F40 at 4.1 min showing the m/z 373.0706 and m/z 445.0956, and the in-source-formed species at m/z 409.1142, m/z 337.0944 and m/z 301.1179.

DETAILED DESCRIPTION

[0050] The present disclosure relates to halogenated fatty acid lactylates compounds, in particular chlorinated fatty acid lactylate compounds, isolated from the cyanobacterial strain Sphaerospermopsis sp. LEGE 00249. This organism was isolated from a Portuguese freshwater system and is maintained at the LEGEcc in CIIMAR, Matosinhos, Portugal (the strains are commercial and can be purchased at http://lege.ciimar.up.pt/ordering-services/).

[0051] The strain was cultured in Z8 medium at 25° C., with a photoperiod of 14 h/10 h light and dark respectively, and at light intensity of 10-30 μmols photons s.sup.−1m.sup.−1. Cultures were grown up to 50 L with constant aeration and at the exponential phase, cells were harvested through centrifugation, frozen and freeze-dried. The biomass of Sphaerospermopsis sp. LEGE 00249 (7.7 g) was sequentially extracted with hexane, ethyl acetate and methanol yielding crude extracts of 66.07 mg, 352.88 mg and 949.65 mg, respectively. The bioassay guided fractionation of the methanolic extract yielded the novel chlorinated fatty acid lactylates.

[0052] The methanolic extract was submitted to solid phase extraction (Waters Sep-Pak® Vac 35 cc 10 g C18 Cartridges) using mixtures of water/methanol (4:1 to 1:4; 4 to 8 column volumes each), yielding an enriched fraction free of chlorophylls and other pigments. The fraction was then chromatographed using semi-preparative HPLC conditions with a gradient of water/acetonitrile 19:1 to 0:1 (both with 0.1% of formic acid) as mobile phase. Fractions were collected every 30 s (total run time of 70 min) and 140 fractions were collected into 96-well deep well plates, and a 25% of a 67 mg 140-well fractionation was tested against Staphylococcus aureus S54F9. In order to carry out this broth microdilution antibiotic susceptibility test, 50 μL of each HPLC fraction resuspended in 14% MeOH in water were mixed with 50 μL of the S. aureus suspension at 10.sup.6 CFU/mL in 2×MHB in a microtiter plate (final desired inoculum=5.Math.10.sup.5 CFU/mL, final concentration of MeOH in bioassay plate=7%, final volume per well=100 μL). The microtiter plate was replicated onto a selective/differential solid medium (Manitol Salt Agar) in order to distinguish between bacteriostatic and bactericidal activities. Growth controls (broth with bacterial inoculum, no bioactive molecule) as well as sterility (broth only) and solvent controls (broth with 7% MeOH in water) were included [7].

[0053] The bioactive fractions (FIG. 1) were analyzed by UPLC-MS using a gradient of water/acetonitrile 2:3 to 0:1 (both with 0.1% of formic acid). The mass spectra were acquired in negative ion mode on ESI-q-TOF Bruker Impact II mass spectrometer. The analysis of the mass data showed typical chlorine isotope patterns, indicating the active fractions to contain compounds bearing chlorine atoms. More specifically, fractions F43-F45 had compounds with one Cl atom, fractions F35-F38 contained compounds with two Cl atoms and fractions F39-F41 had three Cl atoms (FIGS. 2-3). Fractions F37, F40, F43 and F45 were submitted to NMR experiments along with HRMS that unambiguously established the structure of compounds 1, 2, 9 and 16, presented in FIG. 4. In this way, compound 1 was identified as (S)-2-[(12-chlorododecanoyl)oxy]propanoic acid on the basis of its spectrometric and spectroscopic data. The HRMS analysis showed a monoisotopic m/z 305.1504 [M-H].sup.− (calculated m/z=305.1525), consistent with the molecular formula C.sub.15H.sub.27ClO.sub.4. NMR characterization: .sup.1H NMR (CD.sub.3OD, 600.13 MHz): 1.32 (m, 6H, H6′+H7′+H8′), 1.33 (m, 2H, H5′), 1.34 (m, 2H, H9′), 1.35 (m, 2H, H4′), 1.44 (m, 2H, H10′), 1.44 (d, 3H, J=7.1 Hz, H3), 1.62 (m, 2H, H3′), 1.75 (m, 2H, H11′), 2.37 (m, 2H, H2′), 3.55 (t, 2H, J=6.6 Hz, H12′), 4.99 (q, 1H, J=7.1 Hz, H2) ppm. .sup.13C NMR (CD.sub.3OD, 150.9 MHz): 17.74 (C3), 25.89 (C3′), 27.94 (C10′), 29.99 (C9′), 30.15 (C4′), 30.39 (C5′), 30.54 (CH.sub.2), 30.59 (2×CH.sub.2), 33.84 (C11′), 34.92 (C2′), 45.74 (C12′), 71.18 (C2), 175.06 (C1′), 176.54 (C1) ppm.

[0054] Compound 2 showed a pseudomolecular ion at 305.1509 [M-H].sup.− (calculated m/z=305.1525), presenting the same molecular formula as 2 (C.sub.15H.sub.27ClO.sub.4). Through 1D and 2D NMR experiments the chlorine atom was assigned to position 6 of the dodecanoic acid side chain, and therefore the compound was named as (S)-2-[(6-chlorododecanoyl)oxy]propanoic acid. NMR characterization: .sup.1H NMR (CD.sub.3OD, 600.13 MHz): 0.91 (t, 1H, J=7.0 Hz, H12′), 1.30 (m, 2H, H10′), 1.32 (m, 2H, H8′), 1.33 (m, 2H, H11′), 1.42 (m, 1H, H9′), 1.45 (d, 3H, J=7.1 Hz, H3), 1.49 (m, 1H, H4′), 1.53 (m, 1H, H9″), 1.59 (m, 1H, H4″), 1.66 (m, 1H, H7′), 1.64 (m, 1H, H3′), 1.68 (m, 2H, H3″+H5′), 1.76 (m, 1H, H7″), 1.78 (m, 1H, H5″), 2.41 (t, 2H, J=7.1 Hz, H2′), 3.92 (m, 1H, H6′), 4.99 (q, 1H, J=7.1 Hz, H2) ppm. .sup.13C NMR (CD.sub.3OD, 150.9 MHz): 14.38 (C12′), 17.55 (C3), 23.64 (C11′), 25.40 (C3′), 27.02 (C4′), 27.54 (C9′), 29.95 (C8′), 32.91 (C10′), 34.67 (C2′), 39.31 (C5′), 39.67 (C7′), 64.87 (C6′), 70.63 (C2), 174.74 (C1′), 175.57 (C1) ppm.

[0055] The HRMS data of compound 9 (S)-2-[(6,12-dichlorododecanoyl)oxy]propanoic acid indicated the molecular formula of C.sub.15H.sub.26Cl.sub.2O.sub.4 (m/z=339.1117 [M-H].sup.−, calculated m/z=339.1135). NMR characterization: .sup.1H NMR (CD.sub.3OD, 600.13 MHz): 1.35 (m, 1H, H9′), 1.37 (m, 1H, H9″), 1.46 (d, 3H, J=7.2 Hz, H3), 1.46 (m, 2H, H10′), 1.48 (m, 2H, H4′+H8′), 1.56 (m, 1H, H8″), 1.59 (m, 1H, H4″), 1.65 (m, 2H, H3′), 1.69 (m, 2H, H5′+H7′), 1.77 (m, 4H, H5″+H7″+H11′), 2.40 (m, 2H, H2′), 3.56 (t, 2H, J=6.7 Hz, H12′), 3.93 (m, 1H, H6′), 4.99 (d, 1H, J=7.2 Hz, H2), ppm. .sup.13C NMR (CD.sub.3OD, 150.9 MHz): 17.44 (C3), 25.38 (C3′), 26.99 (C4′), 27.41 (C8′), 27.80 (C10′), 29.46 (C9′), 33.72 (C11′), 34.62 (C2′), 39.29 (C5′), 39.5 (C7′), 45.68 (C12′), 64.78 (C6′), 70.31 (C2), 174.68 (C1′), 175.09 (C1) ppm.

[0056] The structure of 16 (S)-2-[(6,12,12-trichlorododecanoyl)oxy]propanoic acid was assigned on the basis of HRMS (C.sub.15H2.sub.5Cl.sub.3O.sub.4; m/z=373.0707 [M-H].sup.−, calculated m/z=373.0746) and NMR spectroscopic data: .sup.1H NMR (CD.sub.3OD, 600.13 MHz): 1.38 (m, 2H, H9′), 1.42 (d, 3H, J=7.1 Hz, H3), 1.45 (m, 2H, H4′+H8′), 1.56 (m, 4H, H4″+H8″+H10′), 1.64 (m, 2H, H3′), 1.69 (in, 2H, H5′+H7′), 1.78 (in, 2H, H5″+H7″), 2.19 (in, 2H, H11′), 2.4 (in, 2H, H2′), 3.94 (in, 1H, H6′), 4.91 (q, 1H, J=7.1 Hz, 1H2), 5.99 (t, 1H, J=6.1 Hz, H12′) ppm. .sup.13C NMR (CD.sub.3OD, 150.9 MHz): 18.23 (C3), 25.4 (C3′), 27.32 (C8′), 27.1 (C4′), 26.93 (C10′), 29.12 (C9′), 34.9 (C2′), 39.32 (C5′/C7′), 39.45 (C5′/C7′), 44.74 (C11′), 64.76 (C6′), 72.71 (C2), 75.02 (C12′), 175.11 (C1′), 178.48 (C1) ppm.

[0057] Furthermore, the HRMS analysis of the minor components of fractions F29-F50 yielded other novel chlorinated compounds. The calculated molecular formulas were consistent with fatty-acid lactylate-like compounds differing in small number of atoms (table 1, FIGS. 5 and 6).

TABLE-US-00001 TABLE 1 Identification of novel halogenated fatty acid lactylate compounds, in particular chlorinated fatty acid lactylate compounds, by LC-HRMS analysis. New Molecule Proposed Predicted (NM)/In-Source- Molecular m/z Detected Analytical Formed Species Compound Formula [M − H].sup.− m/z [M − H].sup.− Error (F) Observations Mono-Chlorinated 1 C.sub.15H.sub.27ClO.sub.4 305.1524 305.1500 0.0024 NM Lauroyl-1- 2 C.sub.15H.sub.27ClO.sub.4 305.1524 305.1495 0.0029 NM lactylates; 3 C.sub.15H.sub.27ClO.sub.4 305.1524 305.1504 0.0020 NM Isomers 4 C.sub.16H.sub.29ClO.sub.4 319.1681 319.1658 0.0023 NM Methyl lactate 5 C.sub.17H.sub.31ClO.sub.4 333.1837 333.1809 0.0028 NM Ethyl lactate 6 C.sub.18H.sub.31ClO.sub.6 377.1736 377.1707 0.0029 NM Lauroyl-2- 7 C.sub.18H.sub.31ClO.sub.6 377.1736 377.1697 0.0039 NM lactylates; 8 C.sub.18H.sub.31ClO.sub.6 377.1736 377.1702 0.0034 NM Isomers Di-Chlorinated 9 C.sub.15H.sub.26Cl.sub.2O.sub.4 339.1135 339.1109 0.0026 NM Lauroyl-1- 10 C.sub.15H.sub.26Cl.sub.2O.sub.4 339.1135 339.1105 0.0030 NM lactylates; 11 C.sub.15H.sub.26Cl.sub.2O.sub.4 339.1135 339.1107 0.0028 NM Isomers 12 C.sub.15H.sub.26Cl.sub.2O.sub.4 339.1135 339.1105 0.0030 NM 13 C.sub.16H.sub.28Cl.sub.2O.sub.4 353.1291 353.1256 0.0035 NM Methyl lactate 14 C.sub.17H.sub.30Cl.sub.2O.sub.4 367.1448 367.1416 0.0032 NM Ethyl lactate 15 C.sub.18H.sub.30Cl.sub.2O.sub.6 411.1346 411.1300 0.0046 NM Lauroyl-2- lactylate Tri- 16 C.sub.15H.sub.25Cl.sub.3O.sub.4 373.0745 373.0700 0.0045 NM Lauroyl-1- 17 C.sub.15H.sub.25Cl.sub.3O.sub.4 373.0745 373.0713 0.0032 NM lactylates; Isomers 18 C.sub.18H.sub.29Cl.sub.3O.sub.6 445.0956 445.0906 0.0050 NM Lauroyl-2- lactylate

[0058] The culture of Sphaerospermopsis sp. LEGE 00249 was scaled-up in order to increase the yield for the isolation of the novel halogenated fatty acid lactylates, in particular chlorinated fatty acid lactylates, from the biomass produced. The strain was cultivated in a modified BG11 medium, a common medium for freshwater strains. The strain was gradually adapted to outdoor conditions in particular with regards to light intensity and photoperiod using as culture vessel a 7-L bubbled tube placed outdoors. A volume containing 15 g of dry biomass was then transferred to a 40-L Green Wall Panel (GWP®-III) photobioreactor in order to start with an initial biomass concentration of 20 g m.sup.−2 of reactor illuminated surface. For the first days the photobioreactor was tilted backward (North facing) to reduce the light intercepted and thus reduce light stress to the culture, then it was tilted (50°) facing South to increase light availability and thus maximize growth and productivity. The culture was kept at a maximum temperature of 28° C. by circulating cold water inside a stainless-steel serpentine placed within the culture chamber and it was bubbled with air at a flow rate of 0.3 L L.sup.−1 min.sup.−1. Pure CO.sub.2 was injected when the pH value exceeded 7.8. The culture was firstly managed in batch and then in semi-continuous with a 30% daily dilution. Average productivity was 7.6 g m.sup.−1 day.sup.−1 with an average irradiance of 29.6 MJ m.sup.−1 day.sup.−1. At the steady-state the culture was harvested by centrifugation, frozen and lyophilized.

[0059] The lyophilized biomass (29.3 g) was sequentially extracted with hexane, ethyl acetate and methanol. The resultant crude extracts were joined yielding 4.4 g that were fractionated by normal-phase VLC (Si gel 60, 0.015-0.040 mm, Merck KGaA) using an increasing polarity grade, of mixtures of n-hexane/EtOAc (9:1 to 0:1), EtOAc/MeOH (7:3) and MeOH, giving a total of 9 fractions. The resulting fractions were then analyzed by LC-MS that revealed the presence of the chlorinated fatty acid lactylates on the last fraction. More specifically, the compounds 1, 2, 9 and 16 were isolated after several chromatographic steps using reverse-phase column chromatography and reverse-phase HPLC using gradients of water/acetonitrile or water/methanol.

[0060] The biofilm forming ability of coagulase-negative staphylococci, when exposed to compounds 1, 2, 9 and 16, was assessed by quantification of total biomass by violet crystal staining [8].

[0061] The novel chlorinated fatty acid lactylates were able to inhibit the biofilm formation of coagulase-negative staphylococci (Table 2). The calculated concentration that inhibits the biofilm formation by 50% was 55.14, 94.60, 20.10 and 43.40 mg/L for compounds 1, 2, 9 and 16, respectively. Compound 9 showed highest antibiofilm activity.

TABLE-US-00002 TABLE 2 Coagulase-negative staphylococci antibiofilm forming inhibition of compounds 9, 16, 1 and 2 Compound 9 Compound 16 Compound 1 Compound 2 mg/L % inhibition mg/L % inhibition mg/L % inhibition mg/L % inhibition 721 88 150 71 140 74 257 90 360 87 75 68 70 0 128.5 89 180 76 37.5 0 — — 64.25 74 90 66 — — — — 32.12 31 35 62 — — — — 16 0 22.5 56 — — — — — — 11.25 22 — — — — — — 5.65 0

[0062] The minimal inhibitory concentrations (MICs) against S. aureus were calculated for compounds 1, 2, 9 and 16 based on the antibiotic activities presented on FIG. 1. Thus, the predicted MICS range from 1790 mg/L to 5790 mg/L.

[0063] In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims.

[0064] The present disclosure should not be seen in anyway restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0065] The above described embodiments are combinable. The following claims further set out particular embodiments of the disclosure.

REFERENCES

[0066] [1] J. L. Del Pozo, Biofilm-related disease, Expert Review of Antiinfective Therapy, 2017. DOI: 10.1080/14787210.2018.1417036 [0067] [2] WHO, 2011. Report on the burden of endemic health care-associated infection worldwide, Fact sheet on HCAI endemic burden worldwide <https://www.who.int/gpsc/country_work/burden_hcai/en/> [0068] [3] B. Allegranzi, S. B. Nejad, C. Combescure, W. Graafmans, H. Attar, L. Donaldson, D. Pittet, Burden of endemic health-care-associated infection in developing countries: Systematic review and meta-analysis, Lancet. 377 (2011) 228-241. doi:10.1016/S0140-6736(10)61458-4. [0069] [4] S. L. Percival, L. Suleman, C. Vuotto, G. Donelli, Healthcare-associated infections, medical devices and biofilms: risk, tolerance and control, J. Med. Microbiol. 64 (2015) 323-34. doi:10.1099/jmm.0.000032. [0070] [5] H.-C. Flemming, J. Wingender, U. Szewzyk, P. Steinberg, S. A. Rice, S. Kjelleberg, Biofilms: an emergent form of bacterial life, Nat. Rev. Microbiol. 14 (2016) 563-575. doi:10.1038/nrmicro.2016.94. [0071] [6] (a) Boutte, T.; Skogerson, L. “Stearoyl-2-lactylates and oleoyl lactylates” in Emulsifiers in Food Technology (2nd Edition), Norn, V. (Ed), Wiley: New York, 2015, Chapter 11, pp 251-270. (b) Wang, F. C.; Marangoni, A. G. J. Colloid Interface Sci. 2016, 483, 394-403. (c) Shah, R.; Kolanos, R.; Di Novi, M. J.; Mattia, A.; Kaneko, K. J. Food Addit. Contam., Part A 2017, 34, 905-917. (d) Draelos, Z. D.; Donald, A. J. Drugs Dermat. 2018, 17, 671-676. [0072] [7] Wiegand, I., Hilpert, K., & Hancock, R. E. W. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature Protocols, 3(2), 163-175. [0073] [8] Danese P N, Pratt L A, Kolte R, Exopolysaccharide production is required for development of Escherichia coli K-12 architecture, J. Bacteriol. 182(2000): 3593-3596.