WO2008091525A2

WO2008091525A2 - Anthelmintic macrolactams from nonmuraea turkmeniaca ma 7364

Info

Publication number: WO2008091525A2
Application number: PCT/US2008/000616
Authority: WO
Inventors: Sheo B. Singh; Sloan Ayers; Kenneth Mohn; Christine M. Brown; Donald Thompson; Olga Genilloud
Original assignee: Merck & Co., Inc.
Priority date: 2007-01-19
Filing date: 2008-01-17
Publication date: 2008-07-31
Also published as: WO2008091525A8; WO2008091525A3

Abstract

Novel macrolactams, 6-desmethyl-N-methylfluvirucin A1 and N-methylfluvirucin A1, isolated from the acetone extract of Nonomuraea turkmeniaca MA7364 using bioassay-guided fractionation, useful as anthelmintics; characterization of structure of 6-desmethyl-N-methylfluvirucin A1 and N-methylfluvirucin A1 via NMR and MS; and characterization of biological activity of 6-desmethyl-N-methylfluvirucin A1 and N-methylfluvirucin A1.

Description

TITLE OF THE INVENTION

ANTHELMINTIC MACROLACTAMS FROM NONMURAEA TURKMENIACA MA7364

BACKGROUND OF THE INVENTION Productivity to the livestock industry is significantly impacted by infections caused by internal parasites (e.g., Haemonchus contortus). A number of new classes of anthelmintic drugs have been approved for clinical practice since 1960 including the current classes of synthetic drugs such as the benzimidazoles and imidazothiazoles (tetramisole/levamisole). However, the discovery of the avermectin class of macrolactones in the early 1980's represented the most significant of discoveries not only as anthelmintic agents for animal health but also for treatment of parasitic infections to humans, particularly onchocerciasis (river blindness). This discovery led to the anthelmintic drugs, ivermectin and doramectin, which have excellent broad-spectrum activity and superior potency. Resistance to all of these classes of drugs has been observed, however, leading to the continuing need of further research to discover new classes of anthelmintics, especially those with novel modes of action.

SUMMARY OF THE INVENTION

Two new macrolactams, 6-desmethyl-iV-methylfIuvirucin A₁ and N-methylfluvirucin A₁, have been isolated from the acetone extract of Nonomuraea turkmeniaca MA7364. These compounds were isolated by bioassay-guided fractionation. The structures of these compounds were elucidated by comparison of their NMR and MS data to those of previously reported fluvirucins, and confirmed by 2D-NMR. The compound 6-desmethyl-iV-methylfluvirucin A₁ exhibited in vitro activity (EC₉₀ 15 μg/mL) against Haemonchus contortus larvae, whereas the compound iV-methylfluvirucin A₁, while a bit less active in vitro (EC₉₀ 29 μg/mL), showed modest in vivo activity against a surrogate organism Heligmosomoides polygyrus at 50 mg/kg.

DETAILED DESCRIPTION OF THE INfVENTION

To discover anthelmintic agents, extracts of bacterial fermentations were screened for in vitro activity against H. contortus, a prevalent parasitic worm species that infects livestock. In vivo activity was evaluated using Heligmosomoides polygyrus infected mice. An acetone extract of Nonomuraea turkmeniaca MA7364 showed in vitro activity and was selected for further study. Bioassay-guided fractionation using the in vitro assay led to the isolation of two new macrolactams of the fluvirucin class, specifically compounds 1 and 2, below:

1 R = H

2 R = CH₃

The producing organism was obtained from a soil sample collected in Mexico and identified as a closely related species to Nonomuraea turkmeniaca.

The fermentation broth generated from the producing organism was extracted with acetone and successively chromatographed on Amberchrome, Sephadex LH-20, and RP-HPLC affording 14.1 mg (4.7 mg/L) and 8.3 (2.8 mg/L) of compounds 1 and 2, respectively, as colorless amorphous powders. HRESIFTMS of compound 1 exhibited the molecular formula C₂₃H₄₄N₂O₅. The UV/VIS spectrum showed only end-absorption. The formula indicated an index of hydrogen deficiency of three. The carbon count was confirmed by the ¹³C-NMR spectrum (CD₃OD), which showed 23 signals. The DEPT spectrum indicated the presence of four methyl groups, ten methylenes, and eight methines. One quaternary signal was present at <5_C 177.18 ppm, indicating a carbonyl group, which would account for one of the indices of hydrogen deficiency. Lack of resonances for additional sp or sp² carbons in the ¹³C- NMR spectrum suggested that compound 1 must contain two rings. Prominent features of the ¹H-NMR spectrum include a methyl triplet at δu 0.88 ppm, methyl doublets at (5_H 1.18 and δ_H 1.25 ppm, and a methyl singlet at <5_H 2.74 ppm. The chemical shift of the latter signal suggested the presence of an N- methyl group. Numerous signals between δ_H 3.4-4.0 ppm as well as a doublet at δ^ 4.90 ppm was consistent with the presence of a sugar residue, which would be a source of one of the two rings present. A multiplet present at δ_H 8.39 ppm that showed no correlation in the HSQC spectrum is consistent with an amide proton, however, this is unusual because an amide proton would be expected to exchange with CD₃OD. By ¹H NMR, the amide proton does exchange, albeit very slowly. The presence of numerous overlapping protons in the <5_H 1.0-1.6 ppm range indicated a high degree of saturation, consistent with the low index of hydrogen deficiency.

Comparison of the data described above with the corresponding literature data resulted in a very close match to a class of macrolactams isolated from the genus Actinomadura. It should be noted that in 1998, numerous species of Actinomadura were reclassified as Nonomuraea, including N. turhneniaca. These compounds contain a branched 14-membered lactam, a mycosamine or 4-ep/-mycosamine moiety, and have exhibited antifungal and antiviral activities. The branching can vary from three methyl groups to three ethyl groups, as well as combinations of methyl, ethyl, and hydroxyethyl groups. Positions C-2, C- 6, and C-IO are always substituted with the three alkyl groups in this class of compounds. The amino sugar moiety is always attached to the macrocycle either at C-3 or at C-9. The C-3 sugar-substituted compounds are the fluvirucin "A" compounds (Ai and A₂). It was apparent from the COSY and the HMBC spectral data (Figure 1 ) that compounds 1 and 2 had a sugar attached at C-3 like the fluvirucin A series. From coupling constant data, the sugar component of 1 and 2 was determined to be A-epi- mycosamine, which is also present in the fluvirucin A compounds. It is also apparent from the HMBC (Figure 1) and the COSY spectral data that compounds 1 and 2 possess methyl substitution at C-2 and ethyl substitution at C-10. The major difference between 1 and 2 to the fluvirucins is the presence of an N-methyl singlet at δ-a 2.74 ppm. This N-methyl group could be attached to the amide nitrogen, or to the sugar amine. The HMBC spectrum clearly shows a correlation from the N-methyl protons to C-3' of the sugar (Figure 1). Another major difference between 1 and the other macrolactams of this class is the absence of an alkyl branch at C-6 in 1.

Compound 2 showed a molecular formula of C₂₄H₄₆N₂O₅ indicating an index of hydrogen deficiency of three. This formula differs from 1 by an additional carbon and two additional hydrogens which was attributed to a methyl group that resonated at δ_H 0.92 ppm which was assigned to the methyl group substituted at C-6. The ¹H-NMR spectrum of 2 was practically identical to that of fluvirucin A], except for the presence of an additional methyl singlet at (5_H 2.75 ppm, corresponding to an N-methyl group. The large coupling constant (10.5 Hz) for H-2 for both compounds indicates an anti- relationship between H-2 and H-3, also consistent with the fluvirucins.

Compounds 1 and 2 were first evaluated in the in vitro H. contortus assay as described earlier. Both compounds 1 and 2 reduced motility of L3 larvae and showed EC₉₀ (effective concentration of compounds that exhibited 90% cessation of motility of larvae) of 15 and 29 μg/mL, respectively. These compounds were further evaluated in an in vivo mouse model infected with H. polygyrus. Compounds were administered subcutaneously at a dose of 50 mg/kg. Three mice were used for evaluation of each compound and data was averaged. Compound 1 showed no reduction in worm counts while 2, though less active in vitro, resulted in an average of 42% reduction in worm counts. Ivermectin was used as a positive control which was significantly more potent (EC₉₀ «2 μg/mL) in the in vitro assay as well as the in vivo assay (98-100% kill of larvae at 5-10 mg/kg dosed subcutaneously).

Example 1 - Reagents and Equipment

All reagents were obtained from Sigma-Aldrich and were used without further purification. NMR spectra were obtained on a Varian Inova 500 MHz spectrometer operating at 500 MHz for ¹H and 125 MHz for ¹³C nuclei in CD₃OD. Optical rotations were obtained on a Perkin-Elmer 241 Polarimeter, and IR spectral data were obtained on a Perkin-Elmer Spectrum One spectrometer. High-resolution mass spectra were obtained on a Thermo Finnigan LTQ-FT with the standard Ion Max API source (without the sweep cone) and ESI probe.

Example 2 - Source of Producing Organism, and Fermentation The organism was obtained from a soil sample collected under banana trees in Las Tuxtlas,

Veracruz, Mexico. The strain was isolated after pretreatment of the soil with benzethonium chloride and plating on NZ-Amine-based medium.

The organism was grown on agar plates at 28°C consisting of yeast extract (3 g/L), malt extract

(10 g/L), dextrose (4 g/L), and agar (20 g/L), adjusted to pH 7. Agar plugs were used to inoculate 250 mL baffled flasks containing 50 mL ATCC-2 medium, which consists of soluble starch (20 g/L), dextrose (10 g/L), NZ Amine EKC (Kerry) (5 g/L), Difco beef extract (3 g/L), Bacto peptone (5 g/L), yeast extract (5 g/L), and CaCO₃ (1 g/L), adjust to pH 7.0 with NaOH before addition Of CaCO₃. After three days incubation (220 rpm, 28°C), these flasks were used to inoculate (2% inoculum) IL production flasks containing 225 mL KHC medium consisting of dextrin (20 g/L), /?-cyclodextrin (10 g/L), primary yeast (10 g/L), tomato paste (20 g/L), CoCl₂ ^»6H₂O (5 mg/L), adjusted to pH 7.2. Production flasks were incubated for 13 days (220 rpm, 28°C). Total cultures were harvested and kept at -20° C until thawed for metabolite extraction. Example 3 - Identification of the Actinomvcete Strain

The actinomycete strain was identified as a new member of the genus Nonomuraea on the basis of its complete 16S rDNA sequence aligned with nucleotide sequences from Genbank. The taxonomic position of the strain was determined by phylogenetic analysis of the aligned 16S rDNA sequences of validated species of the genus Nonomuraea and other members of the family Streptosporangiaceae. From the phylogenetic analysis based on the maximum parsimony method, the strain was closely related to the species N. turkmeniaca, a relationship highly supported by the bootstrap value (93 %).

Example 4 - Extraction and Isolation of 6-Desmethyl-N-methylfluvirucin A₁ and N-methylfluvirucin A₁ Three liters of fermentation broth was mixed with 3 L acetone and shaken for one hour on a platform shaker. The extract was filtered in a sintered-glass funnel using celite as a filter aid. This extract was concentrated by rotary evaporation to aqueous (~2.8L). This concentrate was loaded onto Amberchrome CG 161M (~150mL of resin, column 1 "-diameter) at 15 mL/min. The column was then eluted with a 150 min 5-100% gradient of methanol/water at 10 mL/min followed by an additional 30 minutes with 100% methanol. Fractions were collected every five minutes, totaling 36 fractions. Fractions 30-32 were active in the in vitro assay, and were combined and evaporated to give 1.54 g of a white solid. This active material was dissolved in 5 mL of 1 : 1 CH₂Cl₂ZMeOH and loaded onto a Sephadex LH-20 column using a 1 "-diameter column, and approximately 275 mL of resin. The column was eluted with 1:1 CH₂Cl₂ZMeOH at 2.5 mL/min. Fractions were collected every 2.5 min., 80 fractions total. Fractions 25-31 were active in the in vitro assay. They were combined and concentrated to give 202 mg of active material. This active material was fractionated using five equal injections by preparative RP-HPLC (Waters Symmetry Ci₈, 300xl9mm, 7μm, 50 min gradient of 10-90% aqueous acetonitrile both containing 0.1% TFA, 10 mL/min). Fractions 16-17 (48.1 mg) contained 1, and fractions 18-20 (58.3 mg) contained 2. Purification of these fractions by analytical HPLC using an Agilent Zorbax SB Ci₈ column, 250x4.6mm, 5μm, 1 mL/min, 20 min gradient of 10-90% aqueous acetonitrile + 0.1% TFA followed by lyophilization afforded compound 1 (14.1 mg) and compound 2 (8.3 mg) as colorless amorphous powders.

Example 5 - Characterization of 6-Desmethyl-N-methylfluvirucin A₁ (1)

White solid: [α]_D ²³ +13.0 (c 0.9, methanol); IR (ZnSe film) v_max 3320, 2947, 2917, 2857, 1674, 1625, 1548, 1465, 1436, 1203, 1134 cm^"1; ¹H-NMR (CD₃OD, 500 MHz) δ 8.39 (IH, dd, J= 8.0, 4.0 Hz, H- 14), 4.90 (IH, d, J= 1.5 Hz, H-I '), 4.01 (IH, q, J= 6.5 Hz, H-5'), 3.92 (IH, m, H-2'), 3.79 (IH, m, H- 4'), 3.77 (IH, m, H-3), 3.72 (IH, m, H-13a), 3.41 (IH, t, J= 3.0 Hz, H-3'), 2.74 (3H, s, N-CH₃), 2.58 (IH, m, H-13b), 2.53 (IH, dq, J= 10.5, 7.0 Hz, H-2), 1.60-1.00 (H-4, H-5, H-6, H-7, H-8, H-9, H-I l, H-12), 1.33 (IH, m, H-16a), 1.25 (3H, d, J= 6.5 Hz, H-6'), 1.18 (3H, d, J= 6.5 Hz, H-15), 1.18 (IH, m, H-16b), 0.88 (3H, t, J= 7.0 Hz, H-17); ¹³C-NMR (CD₃OD, 125 MHz) δ 177.18 (C-I), 103.79 (C-I '), 84.53 (C-3), 67.64 (C-5'), 67.42 (C-4'), 66.48 (C-2'), 57.40 (C-3'), 46.30 (C-2), 40.00 (C-13), 39.65 (C-IO), 32.86, 32.36, 30.12 (N-CH₃), 28.31, 28.15 (C-16), 27.94, 26.98, 25.00, 24.64, 20.75, 16.69 (C-6'), 16.59 (C-15), 12.22 (C-17); HRESEFTMS m/z 429.33224 (calcd for C₂₃H₄₄N₂O₅ + H, 429.33305).

Example 6 - Characterization of N-Methylfluvirucin A₁ (2)

White solid: [α]_D ²³ +37.3 (c 0.4, methanol); IR (ZnSe film) V_1113x 3325, 2948, 2856, 1675, 1625, 1548, 1466, 1204, 1136 cm ¹; ¹H-NMR (CD₃OD, 500 MHz) δ 8.41 (IH, dd, J= 8.0, 4.0 Hz, H-14), 4.90 (IH, d, J= 1.5 Hz, H-I'), 4.05 (IH, q, J= 6.5 Hz, H-5'), 3.94 (IH, m, H-2'), 3.82 (IH, m, H-3), 3.80 (IH, m, H-4'), 3.70 (IH, m, H-13a), 3.42 (IH, t, J= 3.0 Hz, H-3'), 2.75 (3H, s, N-CH₃), 2.58 (IH, m, H-13b), 2.55 (IH, dq, J= 10.5, 7.0 Hz, H-2), 1.80-1.00 (H-4-H-5, H-7-H-9, H-I l-H-12), 1.72 (IH, m, H-6), 1.26 (3H, d, J= 7.0 Hz, H-6'), 1.18 (3H, d, J= 7.0 Hz, H-15), 0.92 (3H, d, J= 7.0 Hz, H-18), 0.87 (3H, t, J= 7.0 Hz, H-17); ¹³C-NMR (CD₃OD, 125 MHz) δ 177.18 (C-I), 103.95 (C-I'), 84.47 (C-3), 67.60 (C-5'), 67.46 (C-4'), 66.40 (C-2'), 57.43 (C-3'), 46.39 (C-2), 40.07 (C-13), 39.28 (C-10), 34.88, 33.44, 32.46 (C- 6), 30.12 (N-CH₃), 28.17, 28.1 1, 27.88, 27.54, 24.64, 23.88, 21.08 (C-18), 16.74 (C-6^r), 16.60 (C-15), 12.22 (C-17); HRESIMS m/z 443.34784 (calcd for C₂₄H₄₆N₂O₅ + H, 443.34871).

Example 7 - Biological Assays

The in vitro assay against H. contortus was used as described in Michael, B.; Meinke, P.T.; Shoop, W. J. Parasitol. 2001, 87(3), 692-696. The in vivo mouse assay was modified from Fonseca- Salamanca, F.; Martinez-Grueiro, M.M.; Martinez-Fernandez, A.R. Parasitol. Res. 2003, 91, 321-324, and performed as follows: Swiss Webster mice (Vendor Taconic Labs, approx. 3Og) were inoculated with 200-400 L3 H. polygyrus larvae. The mice were checked for infection around day 12 post inoculation, then dosed subcutaneously with test compound in triplicate. On day three post treatment, mice were euthanized and the intestine was collected (from below stomach to above cecum), opened, and placed in five mLs of sterile water. Mucosa was scraped and rinsed through a 200-mesh screen. The rinseate was then examined for presence of worms and worms were counted. Worm counts for treated mice were then compared to infected, untreated mice as negative controls, and infected mice treated with ivermectin (5- 10 mg/kg) as positive controls. While the invention has been described and illustrated with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additions of procedures and protocols may be made without departing from the spirit and scope of the invention. For example, effective dosages other than the particular dosages as set forth herein above may be applicable as a consequence of variations in the responsiveness of the mammal being treated for any of the indications with the compounds of the invention indicated above. Likewise, the specific pharmacological responses observed may vary according to and depending upon the particular active compounds selected or whether there are present pharmaceutical carriers, as well as the type of formulation and mode of administration employed, and such expected variations or differences in the results are contemplated in accordance with the objects and practices of the present invention. It is intended, therefore, that the invention be defined by the scope of the claims which follow and that such claims be interpreted as broadly as is reasonable.

Claims

WHAT IS CLAIMED IS:

1. A substantially pure form of the compound of the formula:

wherein R is hydrogen or methyl; and pharmaceutically acceptable salts and individual diastereomers thereof.

2. A method for treating a parasitic infection in a mammalian patient, said method comprising administering to said patient a therapeutically effective amount of the compound of Claim 1 , or a pharmaceutically acceptable salt or individual diastereomer thereof.