NOVEL MACROLIDE ANTIBIOTICS
20200231562 · 2020-07-23
Inventors
- Frank Surup (Manden, DE)
- Heinrich Steinmetz (Hildesheim, DE)
- Kathrin Mohr (Braunschweig, DE)
- Konrad Viehrig (Saarbrucken, DE)
- Rolf Müller (Blieskastel, DE)
- Markus Nett (Jena, DE)
- Sebastian Schieferdecker (Jena, DE)
- Hans-Martin Dahse (Jena, DE)
- Michael Wolling (Hannover, DE)
- Andreas Kirschning (Celle, DE)
Cpc classification
C07D313/00
CHEMISTRY; METALLURGY
C07H21/00
CHEMISTRY; METALLURGY
C12P17/08
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C07H17/08
CHEMISTRY; METALLURGY
International classification
C07D313/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a compound according to general formula (I), which exhibits bioactivity; methods for the production of the compound; to pharmaceutical compositions comprising one or more of the compound(s); and to the use of the compound(s) as a medicament.
Claims
1. A compound of the general formula (I): ##STR00040## or a pharmacologically acceptable salt thereof, wherein A represents a group of the formula: ##STR00041## R.sup.1, R.sup.2, R.sup.3 and, if present, R.sup.5 each independently represents a hydrogen atom; a halogen atom; a hydroxyl group; an amino group; a mercapto group; a C.sub.1-C.sub.12 alkyl group; a C.sub.2-C.sub.12 alkenyl group; a C.sub.2-C.sub.12 alkynyl group; a heteroalkyl group containing 1 to 11 carbon atoms; a C.sub.3-C.sub.10 cycloalkyl group, a heterocycloalkyl group containing 3 to 10 ring atoms, a (C.sub.1-C.sub.6)alkyl-(C.sub.3-C.sub.7)cycloalkyl group, a(C.sub.1-C.sub.6)heteroalkyl-(C.sub.3-C.sub.7)cycloalkyl group, aC.sub.6-C.sub.14 aryl group, a heteroaryl group containing 5 to 14 ring atoms, an ar-(C.sub.1-C.sub.6)alkyl group, or a heteroar-(C.sub.1-C.sub.6)alkyl group containing 5 to 10 ring atoms; R.sup.4 represents a hydrogen atom; a halogen atom; a hydroxyl group; an amino group; a C.sub.1-C.sub.12 alkyl group; or a heteroalkyl group containing 1 to 11 carbon atoms; or R.sup.4 and R.sup.3 are taken together to form an oxygen or sulphur atom, or a group NH.
2. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein R.sup.1, R.sup.2 and, if present, R.sup.5 each independently represents a hydrogen atom; a halogen atom; a hydroxyl group; an amino group; a mercapto group; a C.sub.1-C.sub.12 alkyl group; a C.sub.2-C.sub.12 alkenyl group; a C.sub.2-C.sub.12 alkynyl group; a heteroalkyl group containing 1 to 11 carbon atoms; a C.sub.3-C.sub.10 cycloalkyl group, a heterocycloalkyl group containing 3 to 10 ring atoms, a (C.sub.1-C.sub.6)alkyl-(C.sub.3-C.sub.7)cycloalkyl group, a (C.sub.1-C.sub.6)heteroalkyl-(C.sub.3-C.sub.7)cycloalkyl group, a C.sub.6-C.sub.14 aryl group, a heteroaryl group containing 5 to 14 ring atoms, an ar-(C.sub.1-C.sub.6)alkyl group, or a heteroar-(C.sub.1-C.sub.6)alkyl group containing 5 to 10 ring atoms; and R.sup.3 and R.sup.4 are taken together to form an oxygen atom.
3. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein R.sup.1 and R.sup.2 each independently represents a hydrogen atom; a halogen atom; a hydroxyl group; a heteroalkyl group containing 1 to 11 carbon atoms; or a heterocycloalkyl group containing 3 to 10 ring atoms.
4. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein R.sup.1 represents a hydrogen atom; a hydroxyl group or a heteroalkyl group containing 1 to 11 carbon atoms; and R.sup.2 represents a hydroxyl group; or a heterocycloalkyl group containing 3 to 10 ring atoms.
5. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein R.sup.1 represents a heteroalkyl group containing 1 to 11 carbon atoms; and R.sup.2 represents a heterocycloalkyl group containing 3 to 10 ring atoms.
6. The compound according to claim 1, wherein R.sup.5 represents a hydrogen atom; a halogen atom; a hydroxyl group; an amino group; a C.sub.1-C.sub.12 alkyl group; a C.sub.2-C.sub.12 alkenyl group; a C.sub.2-C.sub.12 alkynyl group; or a heteroalkyl group containing 1 to 11 carbon atoms.
7. The compound according to claim 1, wherein the compound is selected from the group consisting of: ##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046##
8. A pharmaceutical composition comprising at least one compound according to claim 1 and, optionally, one or more carrier substance(s), excipient(s) and/or adjuvant(s).
9. A combination preparation containing at least one compound according to claim 1 and at least one further active pharmaceutical ingredient.
10-11. (canceled)
12. A recombinant polyketide synthase (PKS) capable of synthesizing a compound according to general formula (I), wherein the PKS comprises at least one polypeptide, or a functional variant thereof, according to any one of SEQ ID NOs. 11 to 19 or SEQ ID NOs. 34 to 46.
13. An isolated, synthetic or recombinant nucleic acid comprising: (i) a sequence encoding a PKS of the invention, wherein the sequence has a sequence identity to the full-length sequence of SEQ ID NO. 1 or SEQ ID NO. 20 from at least 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% to 100%; (ii) a sequence encoding a portion of a PKS of the invention, wherein the sequence has a sequence identity to the full-length sequence of any of SEQ ID NOs. 2 to 10 or SEQ ID NOs. 21 to 33 from at least 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% to 100%; (iii) a sequence completely complementary to any nucleic acid sequence of (i) or (ii); or (iv) a sequence encoding a polypeptide according to any of SEQ ID NOs. 11 to 19 or SEQ ID NOs. 34 to 46.
14. A vector comprising at least one nucleic acid according to claim 13.
15. A host cell comprising at least one nucleic acid according to claim 13.
16. A method for the preparation of a compound of formula (I), the method comprising the steps of: (a) culturing a host cell of claim 15; and (b) separating and retaining the compound from the culture broth.
17. A method for treating as patient susceptible to or suffering from a bacterial infection, comprising administering to the patient an effective amount of a compound of claim 1.
18. The method of claim 18 wherein the patient is suffering from a Gram-positive bacterial infection.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0105]
[0106]
[0107]
[0108] Since carbon nuclei in furanose sugars are generally less shielded than in related pyranoses and the anomeric configuration can be differentiated by their chemical shifts, the .sup.13C NMR data of 1 are characteristic for an -arabinofuranose configuration (observed .sub.C 110.6; 83.8; 79.1; 86.3; 63.0, methyl furanoside .sub.C 109.3; 81.9; 77.5; 84.9; 62.4)..sup.[1] The absolute configuration of the arabinose moiety was determined to be
[0109] The relative configuration of the aglycon could be derived from vicinal coupling constants and ROESY spectra (
[0110] Having the relative configuration of the aglycon assigned the absolute configuration remained to be established. In this case the usual chemical derivatization was dispensable because the inherent information of the absolute configuration of the d-()-arabinosyl residue could be utilized: As a strong ROESY correlation between 1-H and 7-H confirmed the typical solution conformation of the glycoside. Consequently, a weak but unambiguous correlation between 4-H and methyl 16-H.sub.3 permitted assigning the configuration of the chiral center C-7 (
[0111] 1D and 2D NMR data of disciformycin B (2) showed that the only difference to 1 was the shifting of the .sup.2,3 double bond to position .sup.3,4 with E configuration as indicated by the large coupling constant (J.sub.3,4=15.3 Hz). Coupling constants and ROESY correlations of the C-6 to C-11 part remained largely unchanged compared to 1. Therefore, a (6S,7R,11R) configuration can be concluded for 2 as well. To determine the configuration of C-2 a structure model of compound 2 was calculated by MM+ with HyperChem. Due to the rigid structure parts, i.e. the double bond, the keto and the ,-unsaturated ester, the core part of 2 is locked in a twisted-like configuration, with protons 4-H, 6-H and methyl group 16-H.sub.3 pointing above the main plain. Strong ROESY correlations between 4-H and 2-H on the one and from 3-H to methyl group 15-H.sub.3 on the other hand indicate a 2S configuration.
[0112]
[0113]
[0114] The identified carbonyl signals in the .sup.13C NMR spectrum could be assigned to a ketone (C-5) as well as to two ester functions (C-1, C-23). Aside from the carbonyl groups, four additional carbon atoms (C-8, C-9, C-12, C-13) are sp.sup.2-hybridized according to their chemical shifts (cf. Table 5 in Example 6 below). Thus, 3 must feature two carbon-carbon double bonds and two ring structures in order to comply with the required degrees of unsaturation. The signals in the .sup.1H NMR spectrum were attributed to their directly attached carbon atoms by heteronuclear single-quantum coherence (HSQC). Proton-proton correlation spectroscopy (COSY) revealed six discrete spin systems, all of which could be connected through heteronuclear multiple bond correlation (HMBC) spectroscopy (
[0115] The configuration at C-3 was determined as R by preparation of the diastereomeric -methoxy--trifluoromethylphenylacetic acid (MTPA) ester derivatives, removal of the furanose with In(OTf).sub.3, and calculation of the .sup.RS values. Subsequently, the stereochemistry of the chiral centers C-2, C-6, C-7, and C-11 was concluded from a 2D NOESY experiment. Since no NOE correlation was detected between H-2 and H-3, the two protons must be anti-oriented, i.e. they are placed on opposite sides of the macrolide ring. Together with the preceding Mosher analysis, a 2S configuration could hence be deduced. In the NOESY spectrum, H-3 exhibits crosspeaks with both hydrogen atoms at C-4, suggesting proton-proton torsion angles of about 60 and a staggered conformation, which is also in good agreement with the respective .sup.3J.sub.HH values (Table 5). It is evident that the spatial orientations of H-4a and H-4b cannot be derived from their NOE interactions with H-3. However, H-4b also shows an exclusive NOE correlation with H-7, which is only possible when H-4b occupies an axial position and H-7 is located on the same side of the macrolide ring as H-3. The lack of a NOE correlation between H-6 and H-7 hence established the 6S, 7R configuration. Diagnostic NOEs were observed from H-9 to H-7, H-10b and H-11. The latter proton correlates with H-10b, but not with H-10a, which itself shows crosspeaks with its geminal partner and H.sub.3-16. Taken together, H-10b must have an axial orientation and H-11 should be found on the same side of the molecule as H-3 and H-7, indicating an 11R configuration. The NOE-derived 2S, 3R, 6S, 7R, 11R stereochemistry of the macrolide core was confirmed using a biosynthetic approach that has proven reliable for the configurational assignment of oxygen-bearing stereogenic centers in macrolides and other polyketide natural products.
[0116] To resolve the stereochemistry of the furanose moiety in 3, the sugar was cleaved from the macrolide core by treatment with In(OTf).sub.3, followed by a derivatization with 2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one (PMP). Co-chromatography against the PMP derivatives of commercially available pentoses indicated 3 to contain either arabinose or xylose. Since a chromatographic separation of the corresponding PMP derivatives could not be achieved, the released sugar was independently subjected to reductive amination with anthranilic acid. The product was compared to accordingly prepared standards and, in this way, the sugar residue of 3 was identified as arabinose. A subsequent conversion of the unmodified sugar into a thiacarbamoyl-thiazolidine derivative and chromatographic analysis revealed
[0117] An inspection of the NMR spectra of gulmirecin B (4) revealed the structural relatedness to 3. The missing double bond equivalent (in comparison to 3) could be readily attributed to the absence of the isovalerate moiety at C-6 and the corresponding carbonyl function. Furthermore, the secondary alcohol at C-3 was replaced by a fully reduced methylene function (.sub.H 1.66 ppm; .sub.C 28.0 ppm) in 4. This suggests the dehydratase (DH) and enoyl reductase (ER) domain of GulF to be operational (
[0118]
[0119]
[0120]
[0121]
[0122]
[0123] The present invention is now further illustrated by the following examples from which further features, embodiments and advantages of the present invention may be taken.
EXAMPLES
[0124] The compounds of the present invention can be prepared in a number of ways well known to one skilled in the art of organic synthesis. The compounds of the present invention can be synthesized using methods known in the art of synthetic organic chemistry or by biotechnological approaches such as fermentation as appreciated by those skilled in the art.
[0125] General Experimental Procedures:
[0126] Optical rotations were determined with a Perkin-Elmer 241 or a JASCO P-1020 polarimeter. UV spectra were recorded with a Shimadzu UV-Vis spectrophotometer UV-2450 or a Varian UV/Visible Cary spectrophotometer. IR spectra were recorded on a Bruker FT-IR (IFS 55) spectrometer. NMR spectra were recorded with Bruker AM 300 (.sup.1H 300 MHz, .sup.13C 75 MHz), Avance III 500 (.sup.1H 500 MHz, .sup.13C 125 MHz), ARX 600 (H 600 MHz, .sup.13C 150 MHz) and Bruker Ascend 700 (with a 5 mm TXI cryoprobe (.sup.1H 700 MHz, .sup.13C 175 MHz)) spectrometers. HRESIMS mass spectra were obtained with an Exactive Mass Spectrometer (Thermo-Scientific) or an Agilent 1200 series HPLC-UV system combined with an ESI-TOF-MS (Maxis, Bruker) [column 2.150 mm, 1.7 m, C.sub.18 Acquity UPLC BEH (Waters), solvent A: H.sub.2O+0.1% formic acid; solvent B: AcCN+0.1% formic acid, gradient: 5% B for 0.5 min increasing to 100% B in 19.5 min, maintaining 100% B for 5 min, F.sub.R=0.6 mLmin.sup.1, UV detection 200-600 nm].
Example 1 Biosynthesis and Biophysical Analysis of Disciformycin A and B
[0127] Cultivation A (10 L Scale):
[0128] Angiococcus disciformis AngJ1 was cultivated in 10 L of medium E (per liter: skimmed milk 4 g; soy meal 4 g; yeast extract 2 g; starch 10 g; MgSO.sub.47 H.sub.2O 1 g; Fe-EDTA 8 mg, glycerol 5 g; behenyl alcohol 35 mg) in a biofermenter b10 (Aktiengesellschaft fr Biotechnologische Verfahrenstechnik, Schweiz) at 30 C. for 216 h. The pH was regulated with potassium hydroxide (2.5%) and sulfuric acid between 7.3 and 7.5. The stirrer speed was 100-400 rpm, aerated with 0.05 vvm compressed air. The dissolved oxygen content within the fermentation broth was regulated by the stirrer speed to pO.sub.2 20%. Fermentation was carried out with addition of 1% adsorber resin XAD-16.
[0129] Purification A:
[0130] For isolation of the active metabolites, XAD-16 was harvested by centrifugation; cells were separated from XAD-16 by flotation and discarded. The XAD was eluted with 750 mL acetone and 750 mL methanol, separately. Bioassays against S. aureus showed that the antibacterial activity was concentrated in the acetone extract. The organic solvent of the acetone fraction was evaporated in vacuo until mostly water remained. This was extracted twice with ethylacetate. The ethylacetate was removed in vacuo; the material was dissolved in 85% aqueous methanol and extracted twice with heptane; subsequently the lower methanol phase was adjusted to 70% methanol and extracted twice with dichloromethane. Bioassays revealing the water fraction contained the main antibacterial activity, antibiotic metabolite 1 was isolated in a bioassay guided fractionation strategy. The extract was fractionated by RP MPLC (column 48030 mm, ODS/AQ C18 (Kronlab), gradient 37% to 100% methanol in 60 min, flow 30 mL/min, UV peak detection at 210 nm). Active fractions were combined and further fractionated by preparative RP HPLC column 25021 mm, VP Nucleodur C18 Gravity 5 am, gradient 37% to 100% methanol in 25 min, 50 mM sodium acetate, flow 20 mL/min). A final step of preparative RP HPLC (column 25021 mm, VP Nucleodur C18 Gravity 5 lam, gradient 28% to 55% methanol in 25 min, 0.5% acetic acid, flow 20 mL/min) provided 1.0 mg of disciformycin A (1).
[0131] Cultivation B (70 L Scale):
[0132] A. disciformis AngJ1 was cultivated in 70 L of medium P (peptone [Marcor] 2 g/L, starch 8 g/L, probion single cell protein 4 g/L, yeast extract 2 g/L, CaCl.sub.2 1 g/L, MgSO.sub.4 1 g/L, Fe-EDTA 8 mg/L, pH 7.5) in a biofermenter P150 (Bioengineering AG, Wald CH) at 30 C. for 148 h. The fermentation parameters were as described for the 10 L fermentation.
[0133] Purification B:
[0134] For isolation of disciformycin A (1) and B (2), XAD-16 and wet cell mass were harvested by centrifugation. Combined cells and XAD were washed with 30% methanol (1.5 L), and subsequently extracted with methanol (4 L) and acetone (1 L). Methanol and acetone extracts were combined, evaporated and subjected to a solvent partition. The extract was dissolved in 70% aqueous methanol and extracted twice with dichloromethane. The obtained material (10.0 g) was fractionated on silica gel 100 (0.063-0.200 mm, approximately 1 kg) by a gradient from dichloromethane/methanol 98:2 to 8:2 (98:2, 95:5, 92.5:7.5 90:10, 80:20 500-750 mL each). The bioactive fraction 90:10 (219.2 mg) was subjected to preparative RP HPLC (column 25021 mm, VP Nucleodur C18 Gravity 5 am, gradient 30% to 55% acetonitrile in 25 min, flow 20 mL/min) and afforded 74.3 mg of material containing 1 and 2. A final step of silica gel HPLC (column 25021 mm, Nucleosil 100 7 m, isocratic tert-butylmethylether/heptane 1:3 with methanol 2%, flow 25 mL/min) provided 25.4 mg of 1 and 7.6 mg of 2.
[0135] Disciformycin A (1):
[0136] colorless, amorphous powder, [].sup.20.sub.D51 (c 0.1, MeOH); UV (MeOH) .sub.max 229 (sh); IR (KBr) 3431, 2961, 2929, 2874, 1731, 1647, 1455, 1383, 1254, 1193, 1124, 1076, 1006, 873 cm.sup.1; .sup.1H, .sup.13C, COSY, HMBC and ROESY NMR data see Table 1; HRESIMS m/z 547.2510 [M+Na].sup.+ (calcd for C.sub.27H.sub.40O.sub.10Na, 547.2514).
TABLE-US-00003 TABLE 1 NMR spectroscopic data of disciformycin A (1) in [D.sub.4]methanol (.sup.1H at 600 MHz, .sup.13C at 150 MHz). # .sup.13C, mult. .sup.1H, mult. COSY HMBC ROESY 1 169.1, C 2 134.5, C 3 130.0, CH 5.88, ddq (8.1, 4a, 4b 4, 15 15 > 4b 7.3, 1.5) 4 44.0, CH.sub.2 4.01, dd 3, 4b, 15 2, 3, 5 4b, 16 (18.3, 8.1) 3.58, dd 3, 4a 4a (18.3, 7.3) 5 203.9, C 6 80.8, CH 5.05, d (9.5) 7 5, 7, 8, 1 16, 4a > 4b 7 84.2, CH 4.26, d (9.5) 6 5, 6, 8, 9, 1 9, 1 > 16 8 134.9, C 9 128.8, CH 5.32, brd (11.0) 10a, 10b, 16, 7, 16 7, 10b, 11 10 31.8, CH.sub.2 2.82, ddd (14.7, 9, 10b, 11 8, 9, 11 10b, 16, 17 11.7, 11.0) 9, 10a, 9, 10a, 11 2.13, m 11, 16 11 75.4, CH 5.58, dd (11.7, 10a, 10b 1, 9, 10, 12, 10b, 3.3) 13, 17 14 > 17 12 134.9, C 13 123.6, CH 5.41, qq 14, 17 11, 14, 17 17 (6.6, 1.5) 14 13.1, CH.sub.3 1.74, dq 13, 17 12, 13 11 (6.6, 1.5) 15 21.2, CH.sub.3 1.96, brs 3, 4a 1, 2, 3 3 16 12.3, CH.sub.3 1.91, dd 9, 10b 7, 8, 9 6, 10a (1.5, 1.5) 17 18.3, CH.sub.3 1.65, qd 13, 14 11, 12, 13 13 (1.5, 1.5) 1 174.5, C 2 43.4, CH.sub.2 2.30, d (7.3) 3 1, 3, 4/5 4/5 3 27.0, CH 2.11, m 2, 4/5 1, 2, 4/5 4/5 22.7, CH.sub.3 1.00/1.01, d 3 2, 3, 4/5 2 (6.6) 1 110.6, CH 5.13, brs 2 7, 3, 4 7 2 83.8, CH 4.06, m 1, 3 1, 3, 4 3 79.1, CH 3.88, dd. 2, 4 1, 2, 5 (5.9, 2.9) 4 86.3, CH 3.94, td 3, 5a, 5b 2, 3 (5.7, 3.3) 5 63.0, CH.sub.2 3.73, dd 4, 5b 3, 4 (12.0, 3.3) 3.64, dd 4, 5a 3, 4 (12.0, 5.7)
[0137] Disciformycin B (2):
[0138] colorless, amorphous powder, [].sub.D+64 (c 0.4, MeOH); UV (MeOH) .sub.max 221 (sh), 240 (3.67); IR (KBr) 3429, 2961, 2936, 2875, 1738, 1704, 1627, 1455, 1379, 1299, 1178, 1074, 1031, 856 cm.sup.1; H, .sup.13C, COSY, HMBC and ROESY NMR data see Table 2; HRESIMS m/z 547.2516 [M+Na].sup.+ (calcd for C.sub.27H.sub.40O.sub.10Na, 547.2514).
TABLE-US-00004 TABLE 2 NMR spectroscopic data of disciformycin B (2) in CHCl.sub.3-d (.sup.1H at 700 MHz, .sup.13C at 175 MHz). # .sup.13C, mult. .sup.1H, mult. COSY HMBC ROESY 1 171.5, C 2 43.0, CH 3.35, dqd (9.3, 6.6, 1.1) 3, 15 3, 4, 15 4, 15 3 145.9, CH 6.60, dd (15.3, 9.3) 2, 4 2, 4, 5, 15 15 >> 2 4 129.9, CH 6.37, dd (15.3, 1.1) 3 2, 3, 5 2, 6, 16 5 192.1, C 6 78.3, CH 5.32, d (10.3) 7 5, 7, 8, 1 4, 16 7 81.1, CH 4.09, d (10.3) 6 5, 6, 8, 9, 16, 1 9, 1 8 133.3, C 9 129.4, CH 5.42, m 10a, 10b 8, 11 7, 10b 10 32.1, CH.sub.2 2.87, ddd (14.6, 11.7, 11.0) 9, 10b 8, 9, 11 10b, 16, 17 2.04, m 9, 10a 8, 9 9, 10a 11 72.8, CH 5.33, dd (11.7, 2.8) 10a, 10b 1, 9, 10, 12, 10b, 14 13, 17 12 133.0, C 13 123.4, CH 5.40, m 14 11, 12, 14, 17 14, 17 14 13.0, CH.sub.3 1.71, dq (6.9, 1.3) 13 12, 13 11 15 14.1, CH.sub.3 1.27, d (6.6) 2 1, 2, 3 2, 3 16 12.5, CH.sub.3 1.91, brs 9 7, 8, 9 6, 10a 17 18.0, CH.sub.3 1.69, dq (1.5, 1.3) 11, 12, 13 10a, 13 1 172.5, C 2 42.8, CH.sub.2 2.36, dd (14.8, 7.3) 3 1, 3, 4/5 4/5 2.31, dd (14.8, 7.3) 3 25.8, CH 2.16, tsept. (7.3, 6.8) 2, 4/5 1, 2, 4/5 4/5 4/5 22.4, CH.sub.3 1.02, d (6.8) 3 2, 3, 4/5 2, 3 1 108.2, CH 5.19, m 2 7, 3, 4 7 2 78.2, CH 4.03, m 1, 3 1, 3 3 78.3, CH 4.03, m 2, 4 2, 5 4 88.1, CH 4.13, m 3, 5a, 5b 1, 3 16, 5 5 62.1, CH.sub.2 3.89, dd (11.6, 2.6) 4, 5b 3 3.83, dd (11.6, 1.7) 4, 5a 3, 4
Example 1A Biosynthesis and Biophysical Analysis of Disciformycin C and D
[0139] Cultivation was performed according to cultivation B (70 L scale) in Example 1 above.
[0140] For isolation of the active metabolites, purification B as described in Example 1 above was conducted, except that the final step of silica gel HPLC was replaced by a further RP HPLC step (column 25021 mm, VP Nucleodur C18 Gravity 5 am, gradient 30% to 55% acetonitrile in 25 min, flow 20 mL/min) where the fraction containing 1 and 2 was further fractionized to thereby obtain 1.0 mg of disciformycin C (5) and 7.4 mg disciformycin D (6) in addition to 1 and 2.
Structure Elucidation of Disciformycins C (5) and D (6)
[0141] A peak at m/z 579.2776 in the HRESIMS spectrum of 5 provided the molecular formula C.sub.28H.sub.44O.sub.11, which implies a formal addition of CH.sub.4O in comparison to disciformycins A (1) and B (2). The NMR spectra (Table 1A) were highly similar to those of 1 and 2. However, the key difference was the shortfall of signals for the C2/C3 respectively C3/C4 double bond. Instead, additional signals of two methines (.sub.H, .sub.C 2.51, 47.5; 3.68, 76.7) and one methoxy moiety (.sub.H, .sub.C 3.25, 57.5) were observed. .sup.1H,.sup.13C HMBC correlations from H.sub.3-15 to C-1/C-2/C-3 and the methoxy signal to C-3 established constitution of 5. The stereochemistry of the new stereo center C-3 was assigned by the strong .sup.1H,.sup.1H ROESY correlation from H-3 to H.sub.3-15 together with the absence of a ROESY correlation from H-3 to H-2.
[0142] The molecular formula of disciformycin D (6) was deduced as C.sub.28H.sub.44O.sub.10S from its [M+Na].sup.+ ion peak cluster at m/z 595.2549 in the HRESIMS spectrum. The NMR spectra of 6 were highly similar to those of 5. The key difference was the high field shift of the signals for C-3 (.sub.C 41.8, .sub.H 3.22) and 3SMe (.sub.C 14.4, .sub.H 2.07). Therefore, disciformycin D (6) was deduced as the 3-thioether derivative of 5.
TABLE-US-00005 TABLE 1A NMR data (.sup.1H 700 MHz, .sup.13C 175 MHz) of disciformycin C (5) and D (6) in CH.sub.3OH-d.sub.4 for 5 and CHCl.sub.3-d for 6. 5 6 # .sup.13C. mult. .sup.1H .sup.13C .sup.1H 1 175.1, C 174.4, C 2 47.5, CH 2.51, br s 44.6, CH 2.52, m 3 76.7, CH 3.68, m 41.8, CH 3.22, m 4 47.5, CH.sub.2 3.02, m 41.4, CH.sub.2 3.03, m 5 205.0, C 201.3, C 6 80.6, CH 5.01, m 78.4, CH 4.96, d (9.8) 7 84.1, CH 4.15, d (9.5) 82.3, CH 4.17, d (9.8) 8 135.8, C 132.6, C 9 129.0, CH 5.30, m 129.8, CH 5.45, dd (11.3, 1.8) 10 32.9, CH.sub.2 2.82, dt (14.3, 32.1, CH.sub.2 2.73, ddd (14.7, 11.8) 11.8, 11.3) 1.97, m 2.00, m 11 73.1, CH 5.81, dd 71.0, CH 5.91, dd (11.8, 1.9) (11.8, 2.3) 12 124.5, CH 123.6, CH 13 135.0, C 5.38, m 133.2, C 5.36, br q (7.0) 14 13.3, CH.sub.3 1.67, m 13.0, CH.sub.3 1.66, d6 (7.0, 1.5) 15 15.1, CH.sub.3 1.14, d (6.9) 17.3, CH.sub.3 1.29, d (6.7) 16 12.3, CH.sub.3 1.85, s 11.7, CH.sub.3 1.79, s 17 18.5, CH.sub.3 1.71, br s 18.4, CH.sub.3 1.71, t (1.5) 1 174.5, C 172.7, C 2 43.6, CH.sub.2 2.28, d (7.3) 42.6, CH.sub.2 2.27, t (7.3) 3 27.2, CH 2.09, m 25.8, CH 2.11, m 4/5 22.8, CH.sub.3 0.97, t (6.5) 22.3, CH.sub.3 0.97, d (6.7) 1 110.7, CH 5.09, s 108.3, CH 5.15 br s 2 83.9, CH 4.01, dd (3.0, 1.3) 84.0, CH 4.03, m 3 79.3, CH 3.82, m 78.1, CH 4.03, brs 4 86.3, CH 3.86, td (5.4, 3.2) 88.0, CH 4.14, m 63.1, CH.sub.2 3.68, dd 62.0, CH.sub.2 3.88, dd (11.8, 3.2) (11.8, 2.8) 5 3.59, dd 3.81, (11.8, 2.0) (11.8, 5.4) 3OMe 57.5, CH.sub.3 3.25, s 3SMe 14.4, CH.sub.3 2.07, s
Example 1B Heterologous Expression System and Precursor Compounds
[0143] To corroborate the biosynthesis proposal as depicted in
Cloning of the Dif Cluster by Transformation-Associated Recombination (TAR)
[0144] Transformation-associated recombination (TAR) is a cloning strategy that allows linear-to-linear homologous recombination of two or more DNA molecules with suitable homologous sequence overlaps to be assembled as an extrachromosomal plasmid in yeast cells. To select for successfully assembled clones, a genetically modified baker's yeast strain with a selectable mutation is used, S. cerevisiae ATCC 4004247. The strategy for cluster assembly of the PKS genes difB-difG is shown in
[0145] The PKS genes difB-difG were amplified as PCR products that overlap with the neighboring fragment over 30-150 base pairs according to the assembly strategy depicted in
[0146] An equimolar mixture of all purified single DNA fragment PCR products was then used for transformation of LEU2-deficient S. cerevisiae ATCC4004247. Clones were selected on leucine-free selection medium. The map of the resulting plasmid pCLY10_BG is shown in
Modification of the Plasmid for Expression in M. xanthus
[0147] The obtained S. cerevisiae clones were screened with colony PCR for correct assembly of the plasmid. Positive clones were then cultivated, their plasmids isolated and transformed into E. coli for plasmid propagation and subsequent restriction analysis. Plasmid DNA from one clone that showed the correct restriction fragments was then transformed into E. coli GB-dir for exchange of the plasmid backbone, which is necessary to allow integration and controllable expression in myxobacterium Myxococcus xanthus DK1622. As a linear capture vector, a PCR product amplified from pMyxZeoPvan plasmid (generated previously by overlap PCR from two plasmids: pMyxoZeo [37], which contains the zeocin resistance cassette and the mx9 integrase gene and pMR3679 [38], which contains the vannilate promotor/repressor system. was generated with primers containing a sequence overhang that is homologous to difB and difG sequences on pCLY10_BG. The plasmid map of the resulting integrative expression plasmid pMyxoZeo_dif is shown in
[0148] Exchange of the plasmid backbone was performed with recombination strain E. coli GB-dir according to standard recombineering protocols [39]. GB-dir clones with recombined plasmid pMyxoZeo_dif were selected by zeocin and ampicillin resistance and verified with colony PCR and restriction analysis. Correct pMyxoZeo_dif plasmids pMyxZeoDif16 and pMyxZeoDif20 were obtained. The sequence of plasmid pMyxZeoDif16 was verified by Illumina sequencing to exclude acquired mutations in the biosynthesis genes.
Heterologous Expression of Precursor Compounds P1 and P2 in M. xanthus
[0149] Plasmids pMyxZeoDif16 and pMyxZeoDif20 were transformed into M. xanthus DK1622 following standard protocols. DK1622 clones were selected on CTT Agar with zeocin and genetically verified by PCR. One genetically verified clone of DK1622 attB::dif20 was then grown in triplicates in a 20 ml scale in liquid culture and induced with either 1 mM or 2 mM potassium vanillate (Kvan) when the culture showed visible planctonic growth. For comparison, a non-induced culture and a zeocin-resistant control mutant DK1622 attB::CPN13, carrying another heterologous gene cluster integrated in its genome were cultivated in parallel. Cultivation was continued until the colour of the cultures was turning brown, indicating the dying phase. Cells and growth medium were then extracted twice with ethyl acetate, the extracts dried, redissolved in methanol and subjected to MS-analysis, as shown in
Structure Elucidation of Products P1 and P2
[0150] To obtain further insight into the compound identity, cultivation was scaled up to 400 ml CYE-medium and 800 ml CTT-medium, induced and extracted as before and the putative precursors P1 and P2 were isolated by preparative LC-MS on a Waters Autopurifier (Eschborn, Germany) high pressure gradient system. From the culture in CYE medium, 0.7 mg of each compound P1 and P2 were obtained, whereas from the culture in CTT, 0.3 mg of P1 and 1.0 mg of P2 were obtained. Subsequently, 0.7 mg of each purified compounds P1 and P2 were dissolved in DMSO-d.sub.6 for NMR measurements on a Bruker Ascend 700 spectrometer (data not shown). By comparison of the obtained spectra for P1 and P2 with the previously recorded spectra of disciformycins A and B, the structures of P1 and P2 were elucidated to consist of the disciformycin macrolacton core, lacking the post-PKS modifications and possessing fully saturated C2-C3 and C3-C4-bonds, where the disciformycins A and B have a double bond, respectively. The structure of P1 is identical to P2 except for its side chain, which lacking two carbon atoms C13 and C14. The structures of P1 and P2 are shown in
Example 2 Assessment of Antimicrobial Activity of Disciformycin A and B
[0151] Bacterial strains. Bacterial wildtype strains used in susceptibility assays were either part of HZI's strain collection or purchased from the German Collection of Microorgansims and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ).
[0152] Susceptibility Testing.
[0153] Minimum inhibitory concentration (MIC) was determined by microbroth dilution. In brief, overnight cultures of bacteria (EBS medium) were diluted to OD.sub.600 0.01. Disciformycin A (1) or B (2) dissolved in DMSO was added directly to the cultures in 96-well plates (Sarstedt, flat bottom) in duplicate and the compound was tested in serial dilution. Methanol was tested as negative control and showed no activity against the test organisms. .sup.[a]Oxytetracyclin hydrochloride, .sup.[b]Gentamycin, and .sup.[c]Vancomycin were tested as positive control. After 16 h incubation at 900 rpm and 30 C. on a microplate shaker (Titramax, Heidolph) absorbance at 600 nm was measured using a microplate reader (POLARstar Omega, BMG Labtech). MIC values were determined by sigmoidal curve fitting. The results are shown in Table 3.
TABLE-US-00006 TABLE 3 Antimicrobial activity of disciformycin A (1) and B (2) Bacterium DSM/ATCC 1 2 Ref..sup.[a, b, c] Gram + Bacillus subtilis 10 4.2 0.83 4.2.sup.[a] Paenibacillus polymyxa 36 16.6 16.6 1.7.sup.[a] Staphylococcus aureus 346 16.6 3.3 0.21.sup.[a] Staphylococcus aureus Newman / 8.0 1.2 / Staphylococcus aureus (MRSA) 11822 4.0 0.6 0.83.sup.[c] Staphylococcus aureus N315 / 8.0 1.2 / (MRSA) Staphylococcus aureus Mu50 700699 2.0 0.6 / (MRSA/VRSA) Staphylococcus camosus 20501 7.8 2.4 / Mycobacterium sp. 43270 n.i. n.i. 0.52.sup.[a] Mycobacterium diemhoferi 43524 33.3 33.3 0.052.sup.[a] Micrococcus luteus 20030 67 n.i. 0.42.sup.[a] Nocardioides simplex 20130 33.3 16.6 3.3.sup.[a] Gram Pseudomonas aeruginosa 50071 n.i. n.i. 42.0.sup.[b] Chromobacterium violaceum 30191 n.i. n.i. 0.83.sup.[a] Escherichia coli 1116 n.i. n.i. 0.83.sup.[a] n.i. = no inhibition up to 67 g/ml
[0154] As demonstrated above, disciformycin A (1) and B (2) have an excellent antimicrobial activity against Gram-positive bacteria, and particularly against staphylococci, such as S. carnosus DSM-20501 (7.8/2.4; MIC in g/mL for 1 and 2, respectively) and S. aureus Newman.sup.[5] (8.0/1.2). In addition, both tested strains of methicillin-resistant S. aureus (MRSA) were inhibited: S. aureus DSM-11822 (4.0/0.6) and S. aureus N315.sup.[6] (8.0/1.2). Moreover, these MRSA strains show reduced susceptibility to other antibiotic classes, such as macrolides and quinolones, indicating a putative novel target to be addressed by compounds according to the invention, e.g. 1 and 2, respectively. Notably, the MICs are in the range of reserve antibiotic vancomycin (cf. Table 3). Most importantly, no cross-resistance was observed to classes of antibiotics in use for human therapy, e.g. vancomycin, as demonstrated by the pronounced activity of 1 and 2 against the methicillin- and vancomycin-resistant S. aureus (MRSA/VRSA) Mu50 (ATCC 700699) (2.0/0.6).
Example 3 Assessment of Antiproliferative Activity of Disciformycin A and B
[0155] Cytotoxicity Assay.
[0156] Half maximal inhibitory concentrations (IC.sub.50) in g/ml of disciformycin A (1) and disciformycin B (2) for inhibiting the proliferative activity of human HCT-116 colon carcinoma cells, mouse fibroblast cells L929, and Chinese hamster ovary CHO-K1 cells were determined. Cells were seeded at 610.sup.3 cells per well of 96-well plates (Corning CellBind) in complete medium (180 l) and directly treated with disciformycin dissolved in DMSO in serial dilution. The compound was tested in duplicate, as well as the internal solvent control. After 5 d incubation, 5 mg/ml MTT in PBS (20 L) was added per well and it was further incubated for 2 h at 37 C. The medium was then discarded and cells were washed with PBS (100 l) before adding 2-propanol/10N HCl (250:1, v/v; 100 l) in order to dissolve formazan granules. The absorbance at 570 nm was measured using a microplate reader (EL808, Bio-Tek Instruments Inc.). The amount of formazan, i.e. relative absorption, is thereby proportional to the number of actively proliferating cells. The results are shown in Table 4.
TABLE-US-00007 TABLE 4 Antiproliferative activity of disciformycin A (1) and disciformycin B (2) IC.sub.50 (g/mL) 1 2 HCT-116 colon carcinoma cells (ACC-581) 13.0 >10 Mouse fibroblast cells L929 (ACC-2) 29 >10 Chinese hamster ovary CHO-K1 cells (ACC-110) 16.6 >10
Example 4 Comparison and Prediction of the Substrate Specificity of the Domains in the Disciformycin PKS Modules
[0157] Assembly of the polyketide is expected to start at C14 with incorporation of malonate (mal) and extension by methylmalonate (mmal), mal, mmal, mal, mal, mmal. The polyketide is released as a macrolide, by formation of an ester bond between the carboxyl group at C1 and the hydroxy group at C11. The order of the PKS modules is DifG, DifB, DifC, DifD, DifE, DifF.
[0158] At Domains.
[0159] Prediction of the substrate specificity of the seven AT domains was done by multiple-alignments of their respective amino acid sequences and comparison of key residues to published data.sup.[3]. Specifically, the specificity of the AT domains within the module sequence was predicted based on occupation of critical position (AT fingerprint) according to Mohanty. The ATs of modules DifB, DifF, DifE and AT1 of module DifG have a motif characteristic for malonate specific ATs. The ATs of modules DifC, DifF and AT2 from DifG have a methylmalonate-specific motif. Accordingly, the predicted substrate of the AT-domains from modules DifB, DifD, DifE and DifG1 is malonate, while AT-domains from modules DifC, DifF and DifG2 are predicted to be methylmalonate. This is consistent with the observations in the disciformycin molecule.
[0160] Kr Domains.
[0161] The polyketide core of disciformycin contains three stereocenters and three double bonds, as can be taken from
[0162] The stereoselectivity of the KR-domains can be predicted from their primary sequence by multiple alignments and screening for sequence motifs.sup.[4]. By multiple alignments of the five KR sequences, loop region and catalytic region were investigated for presence of key residues. The KR of DifB, which reduces the keto group at C11 lacks the typical B-type loop motif and is clearly A-type, because the LDD motif is absent and the critical Trp in the catalytic centre is present. KRs of DifC, DifD and DifF are all B-type, with the conserved LDD-motif being replaced by LED in DifD and LQD in DifF. The KR sequence of DifG shows neither the conserved LDD-motif nor the conserved Trp in the catalytic region. A prediction of its stereoselectivity is therefore not possible by this method. Observed and predicted stereochemistry agree for the KRs of DifC and DifD. For the Z .sup.12,13 double bond and the E .sup.8,9 double bond, the KR domain sequences correspond clearly to A-type KR for DifG and a B-type KR for DifC, respectively. The R-configured hydroxyl group at carbon atom C7, to which the arabinose is attached, results from keto-reduction by the KR of DifD. For the DifG KR, no clear prediction is possible, yet the observed cis-configuration of the double bond at this position suggests that the KR must be A-type, producing a hydroxy-group in S-configuration which is converted to a cis-double bond by the DifG DH domain. Reduction by DifB, which is clearly A-type, should lead to an S-configured OH-group at C11, but an R-configured OH-group is observed here. The cis-double bond at C3 (Z .sup.2,3 double bond) is expected to result from DH activity on an SOH group, but the corresponding DifF KR is B-type. In these two cases, prediction and observed stereochemistry disagree. A possible explanation might be an isomerization during macrocyclization. Notably, module DifF contains a domain of unknown function which appears to be an inactive ER domain. Sequence analysis by BLAST conserved domain search revealed a NADPH binding motif as well as a quinone reductase motif. This unusual domain may catalyze the isomerization which leads to the observed stereochemistry.
Example 5 Synthesis of Disciformycin
[0163] A compound of formula (I) according to the present invention can be synthesized using the starting materials and route of synthesis described below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Preferred methods include, but are not limited to, those methods described below or in the respective references cited. Unless otherwise specified all starting materials and reagents are of standard commercial grade, and are used without further purification, or are readily prepared from such materials by routine methods. Those skilled in the art of organic synthesis will recognize that starting materials and reaction conditions may be varied including additional steps employed to produce compounds encompassed by the present invention.
(1) Preparation of (Z)-3-iodo-2-methylprop-2-en-1-ol.SUP.[7]
[0164] ##STR00010##
[0165] To a solution of CuI (12.8 mg, 67 mol, 0.1 eq.) in THF (0.7 ml) at 20 C. was added propargyl alcohol (39.7 l, 672 mol, 1.0 eq.). A 3 M solution of methylmagnesium bromide in Et.sub.2O (0.49 ml, 1.48 mmol, 2.2 eq.) was added slowly, the temperature was raised to 10 C. and the solution stirred for 30 min. A solution of I.sub.2 (85.3 mg, 672 mol, 1.0 eq.) in Et.sub.2O/THF (0.3 ml, 1:1) was added and the mixture was allowed to warm to room temperature. The solution was poured into a mixture of NH.sub.4Cl (0.7 ml) and brine (0.7 ml). The aqueous phase was extracted with Et.sub.2O (30.4 ml), the combined organic phases were dried (MgSO.sub.4) and concentrated. The residue was purified by column chromatography to furnish the product (100 mg, 504 mol, 75%) in good yield.
(2) Preparation of (Z)-1-(((3-iodo-2-methylallyl)oxy)methyl)-4-methoxybenzene.SUP.[8]
[0166] ##STR00011##
[0167] To a suspension of NaH (13.2 mg, 554 mol 1.1 eq.) in DMF at 0 C. was slowly added a solution of (Z)-3-iodo-2-methylprop-2-en-1-ol (100 mg, 504 mol, 1.0 eq.) in THF (0.3 ml). The resulting mixture was stirred for 30 min, prior to addition of PMBCl (68.4 l, 504 mol, 1.0 eq.) in THF (0.1 ml). After stirring for 2 h, Et.sub.2O and H.sub.2O were added. The phases were separated and the organic phase was washed with H.sub.2O and brine. The dried (MgSO.sub.4) organic layer was concentrated and the residue was purified by column chromatography to give the product (130 mg, 408 mol, 81%) in good yield.
Preparation of (S)-4-benzyl-3-(2-(benzyloxy)acetyl)oxazolidin-2-one.SUP.[9]
[0168] ##STR00012##
[0169] To a solution of (S)-4-benzyloxazolidin-2-one (40.0 mg, 224 mol, 1.0 eq.) in THF (1.1 ml) at 78 C. was added 1.57 M nBuli in hexane (0.14 ml, 224 mol, 1.0 eq.), followed by addition of benzyloxyactyl chloride (38.8 l, 246 mol, 1.1 eq.). After stirring for 1 h at 78 C. the solution was allowed to warm to room temperature over 30 min. The reaction was quenched by addition of NH.sub.4Cl (0.2 ml). The mixture was concentrated to a slurry which was extracted with CH.sub.2Cl.sub.2 (20.4 ml). The combined organic phases were washed with 1 M NaOH (0.3 ml) and brine (0.3 ml), dried (Na.sub.2SO.sub.4) and concentrated. The residue was purified by column chromatography to give the product in very good yield (70.6 mg, 217 mol, 97%).
(3) Preparation of (3S,4R,5R)-4-((acetyl-12-chloranyl)oxy)-5-(((acetyl-12-chloranyl)oxy)methyl)-2-bromotetrahydrofuran-3-yl 2-chloroacetate.SUP.[10]
[0170] ##STR00013##
[0171] To a solution of D-Arabinose (32.7 mg, 218 mol, 1.0 eq.) in methanol (0.8 ml) at 0 C. was dropwise added acetlychloride (12.7 l, 178 mol, 0.9 eq.). The reaction was stirred for 1 h at room temperature. After addition of pyridine (164 l, 2.03 mmol, 9.3 eq.) the mixture was concentrated and the residue was coevaporated with CHCl.sub.3.
[0172] The residue was dissolved in DMF (0.3 ml) and the solution cooled to 0 C. Na.sub.2CO.sub.3 (104 mg, 981 mol, 4.5 eq.) was added, followed by chloroacetyl chloride (78.0 l, 981 mol, 4.5 eq.) in DMF (0.1 ml). The reaction was stirred overnight at room temperature before being quenched by addition of H.sub.2O. After stirring for 30 min, the solution was extracted with Et.sub.2O (30.7 ml). The combined organic layers were dried (MgSO.sub.4) and concentrated. The residue was purified by column chromatography to furnish the product (24.2 mg, 61.1 mol, 28%).
[0173] This compound (24.2 mg, 61.1 mol, 1.0 eq.) was dissolved in AcOH (0.6 ml) and 45% HBr in AcOH (0.8 ml) was added. The reaction was stirred for 1 h at room temperature. CH.sub.2Cl.sub.2 (0.8 ml) and ice-cold H.sub.2O (0.7 ml) were added and the organic layer was washed with cold H.sub.2O (0.7 ml) and cold saturated NaHCO.sub.3 (20.3 ml). The dried organic phase was concentrated to give the product (19.3 mg, 43.4 mol, 71%), which was used in the following glycosylation without further purification.
(4) Preparation of (Z)-2-methylbut-2-en-1-ol.SUP.[11]
[0174] ##STR00014##
[0175] To a suspension of LiAlH.sub.4 (96.0 mg, 2.53 mmol, 2.5 eq.) in Et.sub.2O (1.2 ml) at 0 C. was slowly added a solution of angelica acid methyl ester (0.12 ml, 1.01 mmol, 1.0 eq.) in Et.sub.2O (1.9 ml). The mixture was stirred at room temperature for 1 h before being treated with more LiAlH.sub.4 (30.7 mg, 0.81 mmol, 0.8 eq.) in Et.sub.2O (1.3 ml). The reaction was quenched after 30 min by addition of H.sub.2O (0.3 ml), 15% aqueous NaOH (0.3 ml) and more H.sub.2O (1.0 ml). After filtration the solution was dried (MgSO.sub.4) and concentrated. Kugelrohr distillation afforded the product (65.1 mg, 756 mol, 75%) as an oil.
(5) Preparation of (Z)-2-methylbut-2-enal.SUP.[12]
[0176] ##STR00015##
[0177] To a solution of (Z)-2-methylbut-2-en-1-ol (65.1 mg, 756 mol, 1.0 eq.) in CH.sub.2Cl.sub.2 (1.1 ml) was added MnO.sub.2 (1.08 g, 12.5 mmol, 16.5 eq.) and the reaction was stirred for 24 h at room temperature. The solid was filtered off and the solvent removed under reduced pressure. The product (50.9 mg, 605 mol, 80%) was obtained as a colourless liquid via short path distillation.
(6) Preparation of (R,Z)-3-hydroxy-1-((S)-4-isopropyl-2-thioxothiazolidin-3-yl)-4-methylhex-4-en-1-one.SUP.[13]
[0178] ##STR00016##
[0179] To a solution of N-Acetyl thiazolidinthione (209 mg, 1.03 mmol, 1.7 eq.) in CH.sub.2Cl.sub.2 (4.4 ml) at 40 C. was added TiCl.sub.4 (0.12 ml, 1.09 mmol, 1.8 eq.). After 5 min DIPEA (0.19 ml, 1.09 mmol, 1.8 eq.) was added and the deep red mixture was stirred for 2 h at 40 C. before being cooled to 78 C. (Z)-2-methylbut-2-enal (50.9 mg, 605 mol, 1.0 eq.) in CH.sub.2Cl.sub.2 (0.6 ml) was added to the reaction. After 10 min the mixture was poured into pH7 phosphate buffer (22 ml) and the aqueous phase was washed with CH.sub.2Cl.sub.2 (3). The combined organic phases were dried (Na.sub.2SO.sub.4) and concentrated. The product (134 mg, 466 mol, 77%) was obtained via column chromatography of the residue.
(7) Preparation of (R,Z)-3-((tert-butyldimethylsilyl)oxy)-1-((S)-4-isopropyl-2-thioxothiazolidin-3-yl)-4-methylhex-4-en-1-one.SUP.[14]
[0180] ##STR00017##
[0181] To a stirred solution of (R,Z)-3-hydroxy-1-((S)-4-isopropyl-2-thioxothiazolidin-3-yl)-4-methylhex-4-en-1-one (134 mg, 466 mol, 1.0 eq.) in CH.sub.2Cl.sub.2 (1.0 ml) was added 2,6-lutidine (92.0 l, 792 mol, 1.7 eq.). The mixture was cooled to 78 C. and TBSOTf (214 l, 932 mol, 2.0 eq.) was added dropwise. The reaction was stirred at 78 C. for 1 h and at 0 C. for another 1 h. After addition of pH7 phosphate buffer (3.3 ml) and CH.sub.2Cl.sub.2 the phases were separated and the aqueous phase was extracted with CH.sub.2Cl.sub.2 (23.3 ml). The combined organic phases were washed with H.sub.2O (3.3 ml), dried and concentrated. The product (185 mg, 461 mol, 99%) was obtained by column chromatography.
(8) Preparation of (R,Z)-3-((tert-butyldimethylsilyl)oxy)-4-methylhex-4-enal.SUP.[15]
[0182] ##STR00018##
[0183] To a solution of (R,Z)-3-((tert-butyldimethylsilyl)oxy)-1-((S)-4-isopropyl-2-thioxothiazolidin-3-yl)-4-methylhex-4-en-1-one (185 mg, 461 mol 1.0 eq.) in PhMe (1.1 ml) was added 1 M DIBALH (1.15 ml, 1.15 mmol, 2.5 eq.) at 78 C. The reaction was quenched after 1 h by addition of EtOAc (0.9 ml), followed by addition of saturated Rochelle's salt (2.9 ml). After stirring at room temperature, the phases were separated and the aqueous phase was extracted with EtOAc. The combined organic phases were dried (Na.sub.2SO.sub.4) and concentrated. The residue was purified via column chromatography to furnish the product (105 mg, 433 mol, 94%) in very good yield.
(9) Preparation of Ethyl (R,2E,6Z)-5-((tert-butyldimethylsilyl)oxy)-2,6-dimethylocta-2,6-dienoate.SUP.[16]
[0184] ##STR00019##
[0185] To a solution of (R,Z)-3-((tert-butyldimethylsilyl)oxy)-4-methylhex-4-enal (105 mg, 433 mol, 1.0 eq.) in THF (6.4 ml) was added (carbethoxyethylidene)triphenylphosphorane (471 mg, 1.30 mmol, 3.0 eq.). The mixture was stirred for 24 h at reflux. The crude material was purified by column chromatography to give the product (141 mg, 433 mol, 100%).
(10) Preparation of (R,2E,6Z)-5-((tert-butyldimethylsilyl)oxy)-2,6-dimethylocta-2,6-dien-1-ol.SUP.[17]
[0186] ##STR00020##
[0187] A solution of Ethyl (R,2E,6Z)-5-((tert-butyldimethylsilyl)oxy)-2,6-dimethylocta-2,6-dienoate (141 mg, 433 mol, 1.0 eq) in THF (0.33 ml) was added to a suspension of LiAlH.sub.4 (49.3 mg, 1.30 mmol, 3.0 eq.) in THF (0.08 ml) at 0 C. After stirring for 20 min at room temperature H.sub.2O (0.05 ml) was added, followed by 2M NaOH (0.02 ml). The mixture was filtered through celite and was washed with brine, dried (MgSO.sub.4) and concentrated to give the product (118 mg, 416 mol, 96%).
(11) Preparation of (R,2E,6Z)-5-((tert-butyldimethylsilyl)oxy)-2,6-dimethylocta-2,6-dienal.SUP.[18]
[0188] ##STR00021##
[0189] To a solution of (R,2E,6Z)-5-((tert-butyldimethylsilyl)oxy)-2,6-dimethylocta-2,6-dien-1-ol (118 mg, 416 mol, 1.0 eq.) in Et.sub.2O (2.0 ml) was added MnO.sub.2 (542 mg, 6.24 mmol, 15.0 eq.) and the mixture was stirred for 2 h. The reaction was filtered over a silica gel column, eluting with EtOAc (16 ml). The resulting solution was concentrated to give the product (110 mg, 391 mol, 94%).
(12) Preparation of (S)-4-benzyl-3-((2S,3R,4E,7R,8Z)-2-(benzyloxy)-7-((tert-butyl-dimethylsilyl)oxy)-3-hydroxy-4,8-dimethyldeca-4,8-dienoyl)oxazolidin-2-one.SUP.[19]
[0190] ##STR00022##
[0191] To a solution of (S)-4-benzyl-3-(2-(benzyloxy)acetyl)oxazolidin-2-one (70.6 mg, 217 mol, 1.0 eq.) in PhMe (0.3 ml) was added Et.sub.3N (39.1 l, 282 mol, 1.3 eq.). The mixture was cooled to 50 C., followed by careful addition of Bu.sub.2BOTf (77.4 l, 239 mol, 1.1 eq.). The reaction was stirred for 1.5 h at 50 C. A solution of (R,2E,6Z)-5-((tert-butyldimethyl-silyl)oxy)-2,6-dimethylocta-2,6-dienal (110 mg, 391 mol, 1.8 eq.) in PhMe (0.3 ml) was added to the mixture via transfer cannula. The reaction was warmed to 30 C. and stirred for 1.5 h. The reaction was quenched by addition of MeOH (0.2 ml), pH7 buffer (0.2 ml), and H.sub.2O.sub.2 (0.2 ml) and was stirred for 1 h. The organic phase was separated and the aqueous phase was washed with Et.sub.2O (315 ml). The combined organic phases were dried (MgSO.sub.4) and concentrated. The product (102 mg, 167 mol, 77%) was purified using column chromatography.
(13) Preparation of (S)-4-benzyl-3-((2S,3R,4E,7R,8Z)-2-(benzyloxy)-7-((tert-butyl-dimethylsilyl)oxy)-3-((tert-butyldiphenylsilyl)oxy)-4,8-dimethyldeca-4,8-dienoyl) oxazolidin-2-one .SUP.[20]
[0192] ##STR00023##
[0193] To a solution of (S)-4-benzyl-3-((2S,3R,4E,7R,8Z)-2-(benzyloxy)-7-((tert-butyldimethylsilyl)oxy)-3-hydroxy-4,8-dimethyldeca-4,8-dienoyl)oxazolidin-2-one (102 mg, 167 mol, 1.0 eq.) in DMF (1.0 ml) at 0 C. was added imidazole (56.8 mg, 835 mol, 5.0 eq.). A solution of TBDPSCl (0.13 ml, 501 mol, 3.0 eq.) in DMF (0.2 ml) was added dropwise and the reaction was allowed to warm to room temperature overnight before being quenched by slow addition of saturated NH.sub.4Cl. Et.sub.2O was added and the phases were separated. The aqueous layer was extracted with Et.sub.2O (4). The combined organic phases were washed with saturated NH.sub.4Cl (2) and H.sub.2O (2), dried (MgSO.sub.4) and concentrated. The product (139 mg, 164 mol, 98%) was obtained via column chromatography of the residue.
(14) Preparation of (2R,3R,4E,7R,8Z)-2-(benzyloxy)-7-((tert-butyldimethylsilyl) oxy)-3-((tert-butyldiphenylsilyl)oxy)-4,8-dimethyldeca-4,8-dien-1-ol.SUP.[21]
[0194] ##STR00024##
[0195] To a solution of (S)-4-benzyl-3-((2S,3R,4E,7R,8Z)-2-(benzyloxy)-7-((tert-butyldimethylsilyl)oxy)-3-((tert-butyldiphenylsilyl)oxy)-4,8-dimethyldeca-4,8-dienoyl)oxazolidin-2-one (139 mg, 164 mol, 1.0 eq.) in THF (0.8 ml) and H.sub.2O (4.3 l, 238 mol, 1.5 eq.) at 0 C. was added a 0.65 M solution of lithium borohydride in THF (0.33 ml, 213 mol, 1.3 eq.). After 1 h the reaction was quenched by addition of saturated NH.sub.4Cl (0.8 ml). The aqueous phase was extracted with EtOAc (21.0 ml). The combined organic phases were washed with brine (0.6 ml), dried (Na.sub.2SO.sub.4) and concentrated. The residue was purified via column chromatography to give the pure product (92.9 mg, 138 mol, 84%).
(15) (2S,3R,4E,7R,8Z)-2-(benzyloxy)-7-((tert-butyldimethylsilyl)oxy)-3-((tert-butyl diphenylsilyl)oxy)-4,8-dimethyldeca-4,8-dienal.SUP.[22]
[0196] ##STR00025##
[0197] A solution of DMSO (21.6 l, 304 mol, 2.2 eq.) in CH.sub.2Cl.sub.2 (0.1 ml) was added to a solution of oxalyl chloride (13.0 l, 152 mol, 1.1 eq.) in CH.sub.2Cl.sub.2 (0.2 ml) at 60 C. After 5 min (2R,3R,4E,7R,8Z)-2-(benzyloxy)-7-((tert-butyldimethylsilyl)oxy)-3-((tert-butyl-diphenylsilyl)oxy)-4,8-dimethyldeca-4,8-dien-1-ol (92.9 mg, 138 mol, 1.0 eq.) in CH.sub.2Cl.sub.2 (0.2 ml) was added to the mixture and the solution was stirred at 60 C. for 15 min. NEt.sub.3 (95.9 l 692 mol, 5.0 eq.) was added and the mixture was stirred for 5 min at 60 C. before being warmed to room temperature. The reaction was quenched by addition of H.sub.2O (0.3 ml), the organic layer was washed with brine (0.3 ml), dried (MgSO.sub.4) and concentrated to give the product (91.9 mg, 137 mol, 99%).
(16) Preparation of (5R,9R,E)-5-((1R)-(benzyloxy)(oxiran-2-yl)methyl)-9-((Z)-but-2-en-2-yl)-2,2,6,11,11,12,12-heptamethyl-3,3-diphenyl-4,10-dioxa-3,11-disilatridec-6-ene.SUP.[23]
[0198] ##STR00026##
[0199] To a solution of Me.sub.3S.sup.+I.sup. (42.0 mg, 206 mol, 1.5 eq.) in THF (0.5 ml) was added KHMDS (35.7 mg, 179 mol, 1.3 eq.). After 1 h, a solution of (2S,3R,4E,7R,8Z)-2-(benzyloxy)-7-((tert-butyldimethylsilyl)oxy)-3-((tert-butyldiphenylsilyl)oxy)-4,8-dimethyldeca-4,8-dienal (91.9 mg, 137 mol, 1.0 eq.) in THF (0.2 ml) was added via transfer cannula. The reaction was quenched with H.sub.2O (0.04 ml) after 30 min and the solution was concentrated. The obtained residue was redissolved in Et.sub.2O (0.8 ml) and H.sub.2O (0.5 ml) and the aqueous layer was washed with Et.sub.2O (0.3 ml). The combined organic phases were washed with H.sub.2O (0.3 ml) and brine (0.3 ml), dried (MgSO.sub.4) and concentrated. The residue was purified by column chromatography, yielding the product (93.2 mg, 136 mol, 99%) in very good yield.
(17) Preparation of (2Z,6R,7R,8E,11R,12Z)-6-(benzyloxy)-11-((tert-butyldimethyl silyl)oxy)-7-((tert-butyldiphenylsilyl)oxy)-1-((4-methoxybenzyl)oxy)-2,8,12-trimethyltetradeca-2,8,12-trien-5-ol.SUP.[24]
[0200] ##STR00027##
[0201] To a dry flask containing Mg (9.92 mg, 408 mol, 3.0 eq.) in THF (0.1 ml), (Z)-1-(((3-iodo-2-methylallyl)oxy)methyl)-4-methoxybenzene (13.0 mg, 40.8 mol, 0.3 eq.) was added. After 30 s THF (3 ml) was added and the reaction cooled to 0 C. Additional (Z)-1-(((3-iodo-2-methylallyl)oxy)methyl)-4-methoxybenzene (117 mg, 367 mol, 2.7 eq.) was added. The solution was stirred for further 2 h and then stirring of the reaction was stopped. After 3 h the mixture was added via transfer cannula to a 0.1 M solution of LiCuCl.sub.4 (0.10 ml, 10.2 mol, 0.1 eq.) in THF (0.6 ml) at 35 C. The reaction was stirred for 35 min, then (5R,9R,E)-5-((1R)-(benzyloxy)(oxiran-2-yl)methyl)-9-((Z)-but-2-en-2-yl)-2,2,6,11,11,12,12-heptamethyl-3,3-diphenyl-4,10-dioxa-3,11-disilatridec-6-ene (93.2 mg, 136 mol, 1.0 eq.) in THF (0.6 ml) was added via transfer cannula. After 10 min the reaction was quenched by addition of saturated NH.sub.4Cl (0.2 ml), together with addition of Et.sub.2O (0.5 ml). The mixture was warmed to 10 C. under vigorous stirring. Washing of the organic phase with brine multiple times was followed by extraction of the combined aqueous phases with Et.sub.2O (1). The combined organic phases were dried (MgSO.sub.4) and concentrated. The residue was purified by column chromatography to furnish the product (115 mg, 132 mol, 97%) in very good yield.
(18) Preparation of (6R,7R,11R,E)-6-(benzyloxy)-11-((Z)-but-2-en-2-yl)-7-((tert-butyldiphenylsilyl)oxy)-5-((Z)-4-((4-methoxybenzyl)oxy)-3-methylbut-2-en-1-yl)-8,13,13,14,14-pentamethyl-4,12-dioxa-2-thia-13-silapentadec-8-ene.SUP.[25]
[0202] ##STR00028##
[0203] A stirred suspension of NaH (5.7 mg, 238 mol, 1.8 eq.) in THF (0.2 ml) was treated with (2Z,6R,7R,8E,11R,12Z)-6-(benzyloxy)-11-((tert-butyldimethylsilyl)oxy)-7-((tert-butyldiphenylsilyl)oxy)-1-((4-methoxybenzyl)oxy)-2,8,12-trimethyltetradeca-2,8,12-trien-5-ol (115 mg, 132 mol, 1.0 eq.) for 30 min. Chloromethyl methyl sulfide (22.1 l, 264 mol, 2.0 eq.) was added and the mixture was stirred at 0 C. for 3 h. After quench with saturated NH.sub.4Cl, the aqueous layer was extracted multiple times with Et.sub.2O. The combined organic phases were washed with brine, dried (Na.sub.2SO.sub.4) and concentrated. The residue was purified using column chromatography to give the product (121 mg, 129 mol, 98%).
(19) Preparation of (2Z,6R,7R,8E,11R,12Z)-6-(benzyloxy)-11-((tert-butyldimethyl-silyl)oxy)-7-((tert-butyldiphenylsilyl)oxy)-2,8,12-trimethyl-5-((methylthio) methoxy)tetradeca-2,8,12-trien-1-ol.SUP.[26]
[0204] ##STR00029##
[0205] (6R,7R,11R,E)-6-(benzyloxy)-11-((Z)-but-2-en-2-yl)-7-((tert-butyldiphenylsilyl)oxy)-5-((Z)-4-((4-methoxybenzyl)oxy)-3-methylbut-2-en-1-yl)-8,13,13,14,14-pentamethyl-4,12-dioxa-2-thia-13-silapentadec-8-ene (121 mg, 129 mol, 1.0 eq.) was dissolved in CH.sub.2Cl.sub.2 (5.7 ml) and H.sub.2O (0.57 ml). DDQ (29.3 mg, 129 mol, 1.0 eq.) was added at 0 C. and the mixture was stirred for 1 h at 0 C. The reaction was quenched by addition of saturated Na.sub.2SO.sub.3 solution (8.6 ml). After extraction of the aqueous phase with CH.sub.2Cl.sub.2 (330 ml) the combined organic phases were dried (MgSO.sub.4) and concentrated. Purification by flash chromatography yielded the product (95.6 mg, 117 mol, 91%).
(20) Preparation of (2Z,6R,7R,8E,11R,12Z)-6-(benzyloxy)-11-((tert-butyldimethyl silyl)oxy)-7-((tert-butyldiphenylsilyl)oxy)-2,8,12-trimethyl-5-((methylthio) methoxy) tetradeca-2,8,12-trienoic Acid.SUP.[27]
[0206] ##STR00030##
[0207] A) To a solution of (2Z,6R,7R,8E,11R,12Z)-6-(benzyloxy)-11-((tert-butyldimethyl-silyl)oxy)-7-((tert-butyldiphenylsilyl)oxy)-2,8,12-trimethyl-5-((methylthio) methoxy)tetradeca-2,8,12-trien-1-ol (95.6 mg, 117 mol, 1.0 eq.) in CH.sub.2Cl.sub.2 (1.2 ml) was added MnO.sub.2 (101 mg 1.17 mmol, 10 eq.). The reaction was stirred for 2 h at room temperature and filtered through a Celite pad. The product (92.9 mg, 114 mol, 98%) was obtained by concentration of the filtrate and purification via column chromatography.
[0208] B) The obtained aldehyde (92.9 mg, 114 mol, 1.0 eq.) was dissolved in tert-butyl alcohol (1.5 ml). 2-methyl-2-butene (0.85 ml, 7.98 mmol, 70 eq.), NaClO.sub.2 (227 mg, 2.51 mmol, 22 eq.) and sodium dihydrogenphosphate (227 mg, 1.89 mmol, 17 eq.) in H.sub.2O (1.5 ml) were added. The reaction was stirred for 16 h at room temperature. 0.5 M KHSO.sub.4 and EtOAc were added, the phases separated and the aqueous phase was washed with EtOAc. The combined organic phases were washed with 10% NaHSO.sub.3 and brine, dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure. The product (91.0 mg, 110 mol, 96%) was obtained by column chromatography.
(21) Preparation of (2Z,6R,7R,8E,11R,12Z)-6-(benzyloxy)-7-((tert-butyldiphenyl-silyl)oxy)-11-hydroxy-2,8,12-trimethyl-5-((methylthio)methoxy)tetradeca-2,8,12-trienoic Acid.SUP.[28]
[0209] ##STR00031##
[0210] (2Z,6R,7R,8E,11R,12Z)-6-(benzyloxy)-11-((tert-butyldimethylsilyl)oxy)-7-((tert-butyldiphenylsilyl)oxy)-2,8,12-trimethyl-5-((methylthio)methoxy)tetradeca-2,8,12-trienoic acid (91.0 mg, 110 mol, 1.0 eq.) was dissolved in ethanol (0.6 ml). The reaction was stirred at 55 C. for 2 h after addition of PPTS (8.26 mg, 32.9 mol, 0.3 eq.). After removal of the solvent under reduced pressure, the residue was dissolved in EtOAc, washed with brine, H.sub.2O and dried (MgSO.sub.4). The product (64.4 mg, 89.8 mol, 82%) was obtained by concentration of the organic phase, followed by column chromatography.
(22) Preparation of (3Z,7R,8R,9E,12R)-7-(benzyloxy)-12-((Z)-but-2-en-2-yl)-8-((tert-butyldiphenylsilyl)oxy)-3,9-dimethyl-6-((methylthio)methoxy)oxacyclodo-deca-3,9-dien-2-one.SUP.[29]
[0211] ##STR00032##
[0212] A mixture of (2Z,6R,7R,8E,11R,12Z)-6-(benzyloxy)-7-((tert-butyldiphenylsilyl)oxy)-11-hydroxy-2,8,12-trimethyl-5-((methylthio)methoxy)tetradeca-2,8,12-trienoic acid (64.4 mg, 89.8 mol, 1.0 eq.), DIPEA (0.61 ml, 3.59 mmol, 40 eq.) and 2,4,6-trichlorbenzoylchloride (0.28 ml, 1.80 mmol, 20 eq.) in THF (4 ml) was stirred overnight at room temperature. The mixture was diluted with benzene (12 ml) and added to a solution of DMAP (548 mg, 4.49 mmol, 50 eq.) in benzene (55 ml) at 80 C. over a period of 12 h. After stirring for another hour, the reaction was quenched by addition of saturated NaHCO.sub.3. The aqueous phase was extracted with EtOAc (225 ml). The combined organic phases were dried (Na.sub.2SO.sub.4) and concentrated. The product (34.5 mg, 49.4 mol, 55%) was obtained by column chromatography.
(23) Preparation of (3Z,7R,8R,9E,12R)-12-((Z)-but-2-en-2-yl)-8-((tert-butyldiphenylsilyl)oxy)-7-hydroxy-3,9-dimethyl-6-((methylthio)methoxy)oxa-cyclododeca-3,9-dien-2-one.SUP.[30]
[0213] ##STR00033##
[0214] To a solution of di-tert-butylbiphenyl (263 mg, 988 mol, 20 eq.) in THF (4.0 ml) was added activated lithium wire (24.0 mg, 3.46 mmol, 70 eq.) at 0 C. to give a green solution. The mixture was used to titrate a solution of (3Z,7R,8R,9E,12R)-7-(benzyloxy)-12-((Z)-but-2-en-2-yl)-8-((tert-butyldiphenylsilyl)oxy)-3,9-dimethyl-6-((methylthio)methoxy)oxacyclododeca-3,9-dien-2-one (34.5 mg, 49.4 mol, 1.0 eq.) in THF (4.0 ml) at 78 C. until the green color persisted (3 h). The reaction was quenched by addition of saturated NH.sub.4Cl (9.0 ml). Et.sub.2O (9.0 ml) was added and the phases were separated, followed by extraction of the aqueous phase with Et.sub.2O (3). The combined organic phases were dried (MgSO.sub.4) and concentrated. The product (29.7 mg, 46.9 mol, 95%) was obtained by column chromatography of the residue.
(24) Preparation of (3Z,7R,8R,9E,12R)-12-((Z)-but-2-en-2-yl)-8-((tert-butyldiphenylsilyl)oxy)-3,9-dimethyl-6-((methylthio)methoxy)-2-oxooxacyclo-dodeca-3,9-dien-7-yl 3-methylbutanoate.SUP.[31]
[0215] ##STR00034##
[0216] A mixture of (3Z,7R,8R,9E,12R)-12-((Z)-but-2-en-2-yl)-8-((tert-butyldiphenylsilyl)oxy)-7-hydroxy-3,9-dimethyl-6-((methylthio)methoxy)oxacyclododeca-3,9-dien-2-one (29.7 mg, 46.9 mol, 1.0 eq.), pyridine (19.0 l, 235 mol, 5.0 eq.), DMAP (1.15 mg, 9.38 mol, 0.2 eq.) and 3-methylbutanoyl chloride (28.9 l, 235 mol, 5.0 eq.) was stirred overnight at room temperature. The reaction mixture was diluted with CH.sub.2Cl.sub.2, washed with 1 M HCl, saturated NaHCO.sub.3 and H.sub.2O. The combined organic phases were dried (Na.sub.2SO.sub.4) and the solvent was removed under reduced pressure. The residue was purified using column chromatography to give the product (27.9 mg, 44.1 mol, 94%)
(25) Preparation of (3Z,7R,8R,9E,12R)-12-((Z)-but-2-en-2-yl)-8-((tert-butyldiphenylsilyl)oxy)-6-hydroxy-3,9-dimethyl-2-oxooxacyclododeca-3,9-dien-7-yl 3-methylbutanoate.SUP.[32]
[0217] ##STR00035##
[0218] (3Z,7R,8R,9E,12R)-12-((Z)-but-2-en-2-yl)-8-((tert-butyldiphenylsilyl)oxy)-3,9-dimethyl-6-((methylthio)methoxy)-2-oxooxacyclododeca-3,9-dien-7-yl 3-methylbutanoate (27.9 mg, 44.1 mol, 1.0 eq.) was dissolved in a THF/H.sub.2O mixture (4:1). After addition of 2,6-lutidine (15.4 l, 132.3 mol, 3.0 eq.) and AgNO.sub.3 (37.5 mg, 220.5 mol, 5.0 eq.), the mixture was stirred at room temperature for 45 min. After addition of Et.sub.2O the solution was filtered through Celite. The combined organic phases were washed with saturated aqueous CuSO.sub.4 solution (2), H.sub.2O (1) and then dried (K.sub.2CO.sub.3). The residue obtained by removal of the organic solvent was purified using column chromatography, yielding clean product (24.6 mg, 38.8 mol, 88%).
(26) Preparation of (3Z,7S,8R,9E,12R)-12-((Z)-but-2-en-2-yl)-8-((tert-butyldiphenyl-silyl)oxy)-3,9-dimethyl-2,6-dioxooxacyclododeca-3,9-dien-7-yl-3-methylbutanoate.SUP.[33]
[0219] ##STR00036##
[0220] To a solution of oxalyl chloride (51 l, 73.8 mol, 2.0 eq.) at 78 C. in dichloromethane (4.4 l) was added a solution of DMSO (10 l, 147 mol, 4.0 eq.) in dichloromethane (45 l) over 5 min. The reaction mixture was stirred for another 10 min. A solution of (3Z,7R,8R,9E,12R)-12-((Z)-but-2-en-2-yl)-8-((tert-butyldiphenylsilyl)oxy)-6-hydroxy-3,9-di-methyl-2-oxooxacyclododeca-3,9-dien-7-yl 3-methylbutanoate (24.6 mg, 38.8 mol, 1.0 eq.) in dichloromethane (47 l) was added to the mixture over 5 min. After 1 hour at 78 C. triethylamine (32 l, 233 mol, 6.0 eq.) was added and the solution was allowed to warm up to room temperature. Addition of CH.sub.2Cl.sub.2 (233 l) was followed by washing with H.sub.2O. The organic phase was dried (MgSO.sub.4) and concentrated. Column chromatography gave the product (23.3 mg, 36.9 mol, 95%) in good yield.
(27) Preparation of (3Z,7S,8R,9E,12R)-12-((Z)-but-2-en-2-yl)-8-hydroxy-3,9-dimethyl-2,6-dioxooxacyclododeca-3,9-dien-7-yl 3-methylbutanoate.SUP.[20]
[0221] ##STR00037##
[0222] (3Z,7S,8R,9E,12R)-12-((Z)-but-2-en-2-yl)-8-((tert-butyldiphenylsilyl)oxy)-3,9-dimethyl-2,6-dioxooxacyclododeca-3,9-dien-7-yl 3-methylbutanoate (23.3 mg, 36.9 mol, 1.0 eq.) was dissolved in THF (1.0 ml) at 0 C. A 1 M TBAF solution in THF (0.22 ml, 221 mol, 6.0 eq.) was added dropwise. The reaction was allowed to warm to room temperature over 3 h and was stirred for another 4 h. The reaction was quenched by addition of a saturated NaHCO.sub.3. The aqueous solution was extracted with EtOAc (4). The combined organic phases were dried (MgSO.sub.4) and concentrated. The product (14.2 mg, 36.2 mol, 98%) was obtained by column chromatography of the residue.
(28) Preparation of (3Z,7S,8R,9E,12R)-8-(((2S,3S,4R,5R)-3,4-bis((acetyl-12-chloranyl)oxy)-5-(((acetyl-12-chloranyl)oxy)methyl)tetrahydrofuran-2-yl)oxy)-12-((Z)-but-2-en-2-yl)-3,9-dimethyl-2,6-dioxooxacyclododeca-3,9-dien-7-yl 3-methylbutanoate.SUP.[34]
[0223] ##STR00038##
[0224] A mixture of (3Z,7S,8R,9E,12R)-12-((Z)-but-2-en-2-yl)-8-hydroxy-3,9-dimethyl-2,6-dioxooxacyclododeca-3,9-dien-7-yl 3-methylbutanoate (14.2 mg, 36.2 mol, 1.0 eq.) was dissolved in dichloromethane (0.30 ml), together with molecular sieves 4 (30.0 mg), Hg(CN).sub.2 (17.4 mg, 68.8 mol, 1.9 eq.) and HgBr.sub.2 (6.52 mg, 18.1 mol, 0.5 eq.). The mixture was stirred for 3 h at room temperature. A solution of (3S,4R,5R)-4-((acetyl-12-chloranyl)oxy)-5-(((acetyl-12-chloranyl)oxy)methyl)-2-bromotetrahydrofuran-3-yl 2-chloroacetate (19.3 mg, 43.4 mol, 1.2 eq.) in dichloromethane (0.03 ml) was added and subsequently stirred for 4 d at room temperature. The mixture was filtered, washed with aqueous KI solution and H.sub.2O, dried (NaSO.sub.4) and the solvent was removed under reduced pressure. The residue was purified using column chromatography to give the product (17.8 mg, 23.5 mol, 65%).
(29) Preparation of Disciformycin.SUP.[35]
[0225] ##STR00039##
[0226] To a solution of (3Z,7S,8R,9E,12R)-8-(((2S,3S,4R,5R)-3,4-bis((acetyl-12-chloranyl)oxy)-5-(((acetyl-12-chloranyl)oxy)methyl)tetrahydrofuran-2-yl)oxy)-12-((Z)-but-2-en-2-yl)-3,9-di-methyl-2,6-dioxooxacyclododeca-3,9-dien-7-yl 3-methylbutanoate (17.8 mg, 23.5 mol, 1.0 eq.) in THF/H.sub.2O (1.3 ml, 2:1) was added LiOH.H.sub.2O (8.89 mg, 211 mol, 9.0 eq.) at 0 C. After stirring at room temperature for 3 h, THF was removed under reduced pressure at ambient temperature. The residue was extracted with EtOAc multiple times, the combined organic phases were dried (MgSO.sub.4) and concentrated. The product (10 mg, 19.1 mol, 81%) was obtained after column chromatography.
Example 6 Biosynthesis and Biophysical Analysis of Gulmirecin A and B
[0227] Production and Isolation:
[0228] For the production of gulmirecins, P. fallax strain HKI 727 (DSM 28991) was cultured at 30 C. in Erlenmeyer flasks under oxic conditions with gentle shaking (130 rpm). The broth of a seven day old culture (50 l total volume) grown in MD1 medium (casitone 0.3% (w/v), CaCl.sub.22 H.sub.2O 0.07% (w/v), MgSO.sub.47 H.sub.2O 0.2% (w/v), vitamin B.sub.12 0.00005% (w/v), and 1 ml trace elements solution SL-4 consisting of EDTA 0.05% (w/v), FeSO.sub.47 H.sub.2O 0.02% (w/v), ZnSO.sub.47 H.sub.2O 0.001% (w/v), MnCl.sub.24 H.sub.2O 0.0003% (w/v), H.sub.3BO.sub.3 0.003% (w/v), CoCl.sub.26 H.sub.2O 0.020% (w/v), CuCl2H.sub.2O 0.0001% (w/v), NiCl.sub.26 H.sub.2O 0.0002% (w/v), and Na.sub.2MoO.sub.42 H.sub.2O 0.0003% (w/v)) was filtered through a cellulose-based filter paper (retention capacity 12-25 m) in order to remove the cell biomass. The filtrate was extracted three times with equivalent volumes of ethyl acetate. The organic layers were combined and residual water was removed following the addition of anhydrous sodium sulfate (30 g/l) by another filtration step. Subsequently, the organic extract was concentrated under reduced pressure. The residue was dissolved in a small amount of methanol and subjected to flash column chromatography using 45 g of Polygoprep 60-50 C.sub.18 (Macherey-Nagel) as a stationary phase. To this end, the octadecyl phase had been suspended in 20% aqueous methanol and filled into a glass column (303 cm). Elution started with 150 ml of 20% methanol and was continued with the same volume of 40%, 60%, 80%, and 100% methanol. Antimicrobial activity screening against S. aureus indicated the 80% methanol fraction to contain the bioactive compounds. Furthermore, .sup.1H NMR analysis revealed the same fraction to feature unique signals in the olefinic range between 5.00 and 6.00 ppm.
[0229] Isolation of the gulmirecins was accomplished by two consecutive reverse-phase HPLC steps. The initial separation was conducted on a Nucleodur PFP column (25010 mm, 5 m; Macherey-Nagel) using a linear gradient of methanol in water+0.1% trifluoroacetic acid and a flow rate of 2 ml/min: 10% methanol for 3 min, 10%.fwdarw.100% methanol within 30 min, 100% methanol for 10 min. The gulmirecin-containing fraction was collected between 28 and 32 min post injection. Final purification was achieved on a Nucleodur C.sub.18 HTec column (25010 mm, 5 m; Macherey-Nagel) using an isocratic flow (2 ml/min) of 70% methanol in water+0.1% trifluoroacetic acid. Under these conditions, 3 had a retention time of 11.5 min and 4 possessed a retention time of 14.0 min. The elution of gulmirecins was detected by wavelength monitoring at 210 nm using a diode array detector. A total of 8.2 mg of gulmirecin A (3) and 2.1 mg of gulmirecin B (4) were isolated in this way.
[0230] Gulmirecin A (3):
[0231] [].sup.25.sub.D+98.4 (c 1.0, MeOH); UV (MeOH) .sub.max (log ) 203 (4.28); IR (film) .sub.max 3344, 2938, 1733, 1456, 1374, 1251, 1167, 1074, 1022, 863, 792 cm.sup.1; HRESIMS m/z 541.2660 [MH].sup., calcd 541.2654 for C.sub.27H.sub.41O.sub.11.
TABLE-US-00008 TABLE 5 NMR spectroscopic data of gulmirecin A (3) in chloroform-d.sub.1 (.sup.1H at 500 MHz, .sup.13C at 125 MHz). .sub.H, mult. Pos. c (J in Hz) HMBC NOESY 1 174.3 2 45.1 2.76, dq (10.4, 6.7) 1, 3, 15 15 3 68.9 3.84, ddd (10.4, 4.0, 3.2) 2, 5 4a, 4b, 15 4 44.3 a: 3.03, dd (20.2, 3.2) 2, 3, 5 3, 4b b: 2.79, dd (20.2, 4.0) 5 3, 4a, 7, 15 5 203.5 6 80.4 5.01, d (9.2) 5, 7,23 16 7 83.5 4.18, d (9.2) 6, 8, 9, 16, 18 4b, 9, 18 8 133.1 9 129.9 5.46, ddd (11.5, 3.3, 1.5) 7, 16 7, 10b, 11 10 31.9 a: 2.67, dt (14.5, 11.5) 8, 9, 11, 12 10b, 16 b: 1.98, m 8, 9, 12 9, 10a, 11 11 70.9 5.82, dd (11.5, 1.6) 1, 10, 13, 17 9, 10b, 17 12 132.9 13 123.6 5.35, dq (6.9, 1.6) 14, 17 17 14 13.0 1.65, dd (6.9, 1.6) 12, 13 15 15.2 1.23, d (6.7) 1, 2, 3 2, 3, 4b 16 11.4 1.70, t (1.5) 7, 8, 9 6, 10a 17 18.3 1.67, t (1.6) 11, 12, 13 11, 13 18 108.2 5.12, s 7, 20, 21 7, 19 19 78.7 3.97, d (1.0) 18 20 78.0 3.98, dd (2.0, 1.0) 22 21 87.7 4.09, q (2.0) 20, 22 22 22 61.8 3.78, dt (11.7, 2.0) 20, 21 21 23 172.6 24 42.8 2.26, d (7.1) 23, 25, 26, 27 26, 27 25 25.7 2.09, m 23, 24, 26, 27 26, 27 26 22.3 0.96, d (6.6) 24, 25, 27 24, 25 27 22.3 0.96, d (6.6) 24, 25, 26 24, 25
[0232] Gulmirecin B (4):
[0233] [].sup.25D+113.5 (c 0.9, MeOH); UV (MeOH) .sub.max (log ) 202 (4.19); IR (film) .sub.max 3306, 2929, 1717, 1652, 1558, 1539, 1506, 1456, 1376, 1175, 1020, 876 cm.sup.1; .sup.1H NMR (500 Mhz, methanol-d.sub.4) .sub.H [ppm] (J [Hz]) 1.08 (3H, d, J 6.9, H-15), 1.63 (3H, d, J 1.4, H-16), 1.66 (2H, m, H-3), 1.68 (3H, m, H-17), 1.69 (3H, dd, J 7.0, 1.6, H-14), 1.94 (1H, m, H-10b), 2.46 (1H, ddd, J 20.5, 10.8, 4.2, H-4b), 2.66 (1H, dt, J 14.4, 11.8, H-10a), 2.73 (1H, m, H-2), 2.91 (1H, dt, J 20.5, 4.2, H-4a), 3.59 (1H, dd, J 11.8, 5.4, H-22b), 3.67 (1H, dd, J 11.8, 3.4, H-22a), 3.81 (1H, dd, J 5.8, 3.3, H-20), 3.90 (1H, ddd, J 5.8, 5.4, 3.4, H-21), 4.02 (1H, d, J 8.5, H-7),4.06 (1H, dd, J 3.3, 1.3, H-19), 4.07 (1H, d, J 8.5, H-6), 5.04 (1H, d, J 1.3, H-18), 5.37 (1H, dq, J 7.0, 1.6, H-13), 5.38 (1H, m, H-9), 5.87 (1H, dd, J 11.8, 2.8, H-11); .sup.13C NMR (125 MHz, methanol-d.sub.4) .sub.C [ppm] 11.7 (C-16), 13.1 (C-14), 18.4 (C-17), 18.8 (C-15), 28.0 (C-3), 32.6 (C-10), 35.6 (C-4), 39.0 (C-2), 63.0 (C-22), 72.3 (C-11), 78.8 (C-20), 81.7 (C-6), 83.2 (C-19), 86.3 (C-21), 88.7 (C-7), 109.9 (C-18), 123.9 (C-13), 129.3 (C-9), 135.0 (C-12), 136.4 (C-8), 177.8 (C-1), 210.1 (C-5); HRESIMS m/z 465.2098 [M+Na].sup.+, calcd 465.2095 for C.sub.22H.sub.34O.sub.9Na.
Example 7 Assessment of Antimicrobial Activity of Gulmirecin A and B
[0234] Agar Diffusion Assay.
[0235] Antimicrobial activities of the gulmirecins were determined in a primary screen against Bacillus subtilis ATCC 6633, Staphylococcus aureus SG511, Staphylococcus auricularis DSM 20609, Mycobacterium vaccae IMET 10670, Pseudomonas aeruginosa K799/61, Escherichia coli SG458, Sporobolomyces salmonicolor SBUG 549, Candida albicans ATCC14053 and Penicillium notatum JP 36. To this end, holes with 7 mm diameter were aseptically punched in the respective agar medium. Subsequently, the agar plates were inoculated with the test organisms. 0.5 mg of every test compound was dissolved in 1 mL of methanol, and 50 L of this solution was transferred to a single hole. Ciprofloxacin and amphotericin B served as positive controls. After evaporation of the solvent, the agar plates were incubated depending on the growth conditions of the test organisms. A noticeable antimicrobial activity resulted in an inhibition zone of >10 mm. The test results are shown in Table 6 below, wherein the given values represent the diameters of the respective inhibition zone in the agar diffusion assay.
TABLE-US-00009 TABLE 6 Antimicrobial activity of gulmirecin A (3) and B (4) Microorganism 3 4 Ciprofloxacin Amphotericin B Bacillus subtilis 35 n.a. 30 n.a. Staphylococcus aureus 29 17 19 n.a. Staphylococcus auricularis 31 19 20 n.a. Mycobacterium vaccae 17 n.a. 24 n.a. Pseudomonas aeruginosa n.a. n.a. 38 n.a. Escherichia coli n.a. n.a. 34 n.a. Sporobolomyces salmonicolor n.a. n.a. n.a. 19 Candida albicans n.a. n.a. n.a. 22 Penicillium notatum n.a. n.a. n.a. 18 n.a., no activity observed
[0236] As demonstrated above, gulmirecin A (3) and B (4) have an excellent antimicrobial activity against Gram-positive bacteria, and particularly against staphylococci. However, they were inactive against Gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. Fungi, such as Candida albicans, Penicillium notatum, and Sporobolomyces salmonicolor, were also not affected by the gulmirecins.
[0237] Bouillon Dilution Assay.
[0238] To determine the MIC.sub.90 value of 3, the test organism was cultured in 500 l aliquots of trypric soy broth (peptone from casein 1.7% (w/v), peptone from soymeal 0.3% (w/v),
[0239] The MIC.sub.90 values of 3 against methicillin-resistant S. aureus (MRSA) and S. auricularis A were 23.05 M and 22.48 M, respectively. Ciprofloxacin, which served as a reference, exhibited an MIC.sub.90 of 75.4 M against the MRSA strain in the same test series.
Example 8 Assessment of Cytotoxic Effects of Gulmirecin A and B
[0240] The cytotoxic activity of gulmirecin A (3) and gulmirecin B (4) against human cells was evaluated in the well-established MTT assay using primary monocytes from human peripheral blood as well as transformed lung epithelial cell A-549 cell and leukemic Mono Mac 6 cells. Furthermore, 3 and 4 were tested against K-562, HUVEC, and HeLa cells.
[0241] Isolation of Primary Monocytes, Cell Lines and Growth Conditions.
[0242] Human primary monocytes were isolated from leukocyte concentrates, obtained from the Institute of Transfusion Medicine at the University Hospital Jena, Germany. The concentrates were prepared from the blood of healthy adult human donors who had not taken any anti-inflammatory medication for the 10 days prior to blood donation, as described. In brief, freshly withdrawn peripheral blood was pretreated with citrate-phosphate-dextrose solution as anticoagulant and processed with the 2C+ protocol of the Atreus Whole Blood Processing System (Terum BCT). Peripheral blood mononuclear cells (PBMC) were isolated by dextran sedimentation and centrifugation on LSM 1077 lymphocyte separation medium (PAA Laboratories). For isolation of monocytes, the PBMC were washed twice with ice-cold phosphate buffered saline (PBS) and plated (density 210.sup.7 cells/ml) in culture flasks (Greiner Bio-One) containing PBMC medium (RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 g/ml streptomycin and 2 mM L-glutamine) for 1.5 hours at 37 C., 5% CO.sub.2. Non-adherent cells were removed; adherent monocytes were scraped, washed with ice-cold PBS and resuspended in ice-cold PBS with a purity of >85%, defined by forward- and side-light scatter properties and detection of the CD14 surface molecule by flow cytometry (BD FACS Calibur). Monocytes were cultured in monocyte medium (RPMI 1640 supplemented with 2% heat-inactivated fetal calf serum (FCS, 10%, v/v), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 g/ml)).
[0243] The human monocytic cell line Mono Mac 6 was cultured in RPMI 1640 medium supplemented with FCS (10%, v/v), penicillin (100 U/ml), streptomycin (100 g/ml), insulin (10 g/ml), oxaloacetic acid (1 mM), sodium pyruvate (1 mM), and 1 non-essential amino acids at 37 C. and 5% CO.sub.2. A-549 cells (DSM ACC-107) were cultured in DMEM high glucose (4.5 g/l) medium supplemented with FCS (10% v/v), penicillin (100 U/ml) and streptomycin (100 g/ml) at 37 C. in a 5% CO.sub.2 incubator. K-562 (DSM ACC 10) and HeLa cells (DSM ACC 57) were grown in RPMI 1640, whereas cells of HUVEC (ATCC CRL-1730) were cultured in DMEM medium. The respective media were supplemented with ultraglutamine 1 (10 ml/l), gentamicin sulfate (500 l/l), and FCS (10% v/v).
[0244] MTT Assay.
[0245] Cells (310.sup.5 Mono Mac 6, 110.sup.5 A-549 cells, or 210.sup.6 monocytes per well) were seeded in a 96-well plate in the respective medium (100 l/well). Monocytes were allowed to adhere for 1.5 hours (37 C., 5% CO.sub.2) prior to treatment. Test compounds (0.3% DMSO as vehicle) were added to each well and samples were incubated for 48 hrs. Then, 20 l of thiazolyl blue tetrazolium bromide (MTT, 5 mg/ml PBS) were added, and the incubation was continued at 37 C., 5% CO.sub.2 until blue staining of the vehicle control. Formazan formation was stopped by adding 100 l of lysis buffer (SDS, 10%, w/v in 20 mM HCl) and samples were shaken overnight. Absorbance of each well was measured at 570 nm in a Multiskan microplate spectrophotometer (Thermo Scientific).
[0246] Incubation of the cells with either 3 or 4 for 24 or 48 hours caused no reduction in cell viability at the concentrations required for antibacterial activity. In contrast, the reference compound staurosporine was highly cytotoxic for all three cell types.
[0247] Evaluation of Antiproliferative and Cytotoxic Effects.
[0248] The test substances were dissolved in methanol before being diluted in DMEM. The adherent cells were harvested at the logarithmic growth phase after soft trypsinization using 0.25% trypsin in PBS containing 0.02% EDTA. For each experiment, approximately 10,000 cells were seeded with 0.1 ml culture medium per well of the 96-well microplates. HeLa cells were pre-incubated for 48 h prior to the addition of the test compounds, which were carefully diluted on the subconfluent monolayers. Incubation was then conducted in a humidified atmosphere at 37 C. and 5% CO.sub.2. In case of K-562 cells, the number of viable cells in every well was determined using the CellTiter-Blue1 assay. The adherent HUVEC and HeLa cells were fixed by glutaraldehyde and stained with a 0.05% solution of methylene blue for 15 min. After gently washing, the stain was eluted with 0.2 ml of 0.33 N HCl in the wells. The optical densities were measured at 660 nm in a SUNRISE microplate reader (TECAN).
[0249] 3 and 4 did not inhibit the proliferation of K-562, HUVEC, or HeLa cells at a concentration of 100 M.
Example 9 Identification and Analysis of the Gul Gene Cluster
[0250] Retrobiosynthetic analysis of 3 suggests that its polyketide portion is assembled from an acetate starter unit and six polyketide extender molecules, including three malonyl-CoAs and three methylmalonyl-CoAs. Assuming a co-linear biosynthesis, the genome of P. fallax HKI 727 (DSM 28991) was screened for gene loci featuring seven PKS modules. The gulmirecin (gul) gene cluster was identified taking the substrate specificities of the gate-keeping acyl transferase (AT) domains in every PKS module and the reductive domains into consideration. The gul gene cluster includes six PKS genes (gulA-gulF) having the organization shown in
[0251] The oxygen-bearing stereogenic centers of 3 at C-3, C-7 and C-11 are introduced at different extension steps by NADPH-dependent reduction of the respective Claisen products. They are catalyzed by the ketoreductase (KR) domains of the PKS modules, which can be classified into two groups (A-type and B-type) on the basis of their distinct substrate orientation. Since the relative position of the substrate determines the stereochemical outcome of the reduction, the stereospecificity of KR domains can be inferred from their 3D architecture. By using a sequence-based model, a 3R, 7S, 11R configuration for the macrolide that is offloaded from the PKS assembly line was predicted. While the predictions for the chiral centers at C-3 and C-11 already matched the NOE-derived stereochemistry, the discrepancy at C-7 can be rationalized. The 7S configuration was predicted under the assumption that the AT domain of GulD selects malonyl-CoA as an extender unit. This means that the hydroxyl group at C-6 is not introduced during the GulD-catalyzed polyketide chain extension, but results from an independent reaction at a later biosynthetic stage. The cytochrome P450 GulG is a likely candidate for the expected hydroxylation due its sequence homology to macrolide carbon hydroxylases, such as EryK. Once the hydroxyl group is installed, the original PKS-derived 7S configuration will switch to 7R.
[0252] The structure of 3 suggests the skipping of two reductive domains in the PKS GulF during its assembly.
Example 10 Assessment of Resistance Mutations in S. aureus
[0253] The reference strain S. aureus N315 (genome accession number NC_002745) has been sequentially exposed to increasing concentrations of disciformycin A (1). Resistant mutants developed at a frequency of ca. 10.sup.s as determined by colony-forming units (cfu) count of a defined inoculum treated with the 8MIC of disciformycin A (1). Finally, ten independent resistant mutants were obtained that grew in the presence of 50 g/mL disciformycin A (1). These mutants were analyzed by whole-genome sequencing and comparison to the wildtype reference genome. The results are shown in Table 7.
TABLE-US-00010 TABLE 7 Mutations (amino acid changes) found in Disciformycin- resistant S. aureus N315 mutants. Mutant # RpoB ( subunit) RpoC (subunit) Mt50DscA.1 Q575R, C593W, R594C Mt50DscA.2 Y507D, D611N Mt50DscA.3 H785R, T936A Mt50DscA.4 D611N Mt50DscA.5 C593W, R594C D952N Mt50DscA.6 D611N Mt50DscA.8 Q575R, C593W, R594C Mt50DscA.9 R594C I763M Mt50DscA.10 Q575R, C593W, R594C Mt50DscA.11 Y510H, C593W, R594C
[0254] All of the mutations mapped to the 1 and 3 subunit of S. aureus RNA polymerase. Importantly, no cross-resistance with rifampicin was observed. All of the Diciformycin A-resistant mutants were at least 8-fold resistant towards treatment with Disciformycins B-D and Gulmirecins A and B, which most probably exhibit the same mechanism of action. (Table 8).
TABLE-US-00011 TABLE 8 Susceptibility of Disciformycin A-resistant S. aureus N315 mutants towards disciformycin and gulmirecin derivatives and rifampicin. S. aureus wildtype MIC [g/ml] (WT) and mutants DscA (1) DscB (2) DscC (5) DscD (6) GlmA (3) GlmB (4) RIF WT 4 1 4 0.5 4 32 0.003 Mt50DscA.1 >64 >64 >64 32 >64 >64 0.003 Mt50DscA.2 >64 >64 n.d. n.d. >64 >64 0.005 Mt50DscA.3 >64 >64 >64 64 >64 >64 0.003 Mt50DscA.4 >64 >64 n.d. n.d. >64 >64 0.001 Mt50DscA.5 64 16 32 4 64 >64 0.001 Mt50DscA.6 >64 >64 >64 32 >64 >64 0.001 Mt50DscA.8 >64 >64 n.d. n.d. >64 >64 0.001 N315 Mt50DscA.9 >64 >64 n.d. n.d. >64 >64 0.001 Mt50DscA.10 >64 >64 n.d. n.d. >64 >64 0.001 Mt50DscA.11 >64 32 n.d. n.d. >64 >64 0.001 (Dsc: Disciformycin; Glm: Gulmirecin; RIF: rifampicin; n.d. not determined)
Example 11 Assessment of Intra-Macrophage Activity
[0255] The PMA-differentiated human THP-1 cell line (macrophages) was used as cellular host for infection with S. aureus Newman at an MOI (multiplicity of infection) of 10. After 2 h, extracellular S. aureus cells were eliminated by lysostaphin treatment and compounds were added for 18 h at their respective 1, 4 and 8MIC. Intracellular bacteria were recovered and serial dilutions were streaked out on CASO-Agar for cfu counting. The reference drug rifampicin reduced the number of intracellular bacteria by ca. 4 logs (at 8MIC). Likewise, disciformycin A and B reduced the number of intacellular bacteria by 3 logs without causing any apparent cytotoxicity (
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[0295] The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention.