CHLOROTONIL DERIVATIVES

Abstract

The present invention relates to novel chlorotonil derivatives of formula (I) and the use thereof for the treatment or prophylaxis of bacterial infections and malaria.

##STR00001##

Claims

1. A compound of general formula (I): ##STR00048## wherein A-E together are a group of formula ##STR00049## G-U together are a group of formula ##STR00050## V-W together are a group of formula ##STR00051## L-Q together are a group of formula ##STR00052## X-Y-Z together are a group of formula —C(═O)—C(Cl).sub.2—C(═O)—, —C(OH)═C(Cl)—C(═O)— or —C(═O)—C(Cl)═C(OH)—; R.sup.1 is a hydrogen atom, a halogen atom, NO.sub.2, ONO.sub.2, N.sub.3, OH, NH.sub.2, SH, CN, or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; and R.sup.2 is a hydrogen atom, a halogen atom, NO.sub.2, ONO.sub.2, N.sub.3, OH, NH.sub.2, SH, CN, or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; or R.sup.1 and R.sup.2 together are a group of formula —O—; with the proviso that all of A-E, G-U, V-W and L-Q do not at the same time possess a double bond; or a pharmaceutically acceptable salt, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

2. A compound according to claim 1 of general formula (II): ##STR00053## wherein A-E together are a group of formula ##STR00054## G-U together are a group of formula ##STR00055## V-W together are a group of formula ##STR00056## X-Y-Z together are a group of formula —C(═O)—C(Cl).sub.2—C(═O)—, —C(OH)═C(Cl)—C(═O)— or —C(═O)—C(Cl)═C(OH)—; R.sup.1 is a hydrogen atom, a halogen atom, NO.sub.2, ONO.sub.2, N.sub.3, OH, NH.sub.2, SH, CN, or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; and R.sup.2 is a hydrogen atom, a halogen atom, NO.sub.2, ONO.sub.2, N.sub.3, OH, NH.sub.2, SH, CN, or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; or R.sup.1 and R.sup.2 together are a group of formula —O—; or a pharmaceutically acceptable salt, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

3. A compound according to claim 1 of general formula (III): ##STR00057## wherein X-Y-Z together are a group of formula —C(═O)—C(Cl).sub.2—C(═O)—, —C(OH)═C(Cl)—C(═O)— or —C(═O)—C(Cl)═C(OH)—; R.sup.1 is a hydrogen atom, a halogen atom, NO.sub.2, ONO.sub.2, N.sub.3, OH, NH.sub.2, SH, CN, or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; and R.sup.2 is a hydrogen atom, a halogen atom, NO.sub.2, ONO.sub.2, N.sub.3, OH, NH.sub.2, SH, CN, or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; or R.sup.1 and R.sup.2 together are a group of formula —O—; or a pharmaceutically acceptable salt, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

4. A compound according to claim 1 of general formula (IV): ##STR00058## wherein X-Y-Z together are a group of formula —C(═O)—C(Cl).sub.2—C(═O)—, —C(OH)═C(Cl)—C(═O)— or —C(═O)—C(Cl)═C(OH)—; R.sup.1 is a hydrogen atom, a halogen atom, NO.sub.2, ONO.sub.2, N.sub.3, OH, NH.sub.2, SH, CN, or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; and R.sup.2 is a hydrogen atom, a halogen atom, NO.sub.2, ONO.sub.2, N.sub.3, OH, NH.sub.2, SH, CN, or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which groups may optionally be substituted; or a pharmaceutically acceptable salt, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

5. A compound according to claim 1, wherein R.sup.1 is a halogen atom, OH, ONO.sub.2 or a group of formula —O—C.sub.1-6 alkyl which group may be substituted by one or two hydroxy groups and/or by an optionally substituted phenyl group; and R.sup.2 is OH.

6. A compound according to claim 1, wherein R.sup.1 is OH; and R.sup.2 is a halogen atom, OH, ONO.sub.2 or a group of formula —O—C.sub.1-6 alkyl which group may be substituted by one or two hydroxy groups and/or by an optionally substituted phenyl group.

7. A compound according to claim 1, wherein R.sup.1 is F, Cl, Br, OH, ONO.sub.2, OMe, OEt, OBu, OBuOH, Oisoamyl, OBn or glycerol; and R.sup.2 is OH.

8. A compound according to claim 1, wherein R.sup.1 is OH and R.sup.2 is Cl, OMe or OEt.

9. A compound according to claim 1 having the following formula (V): ##STR00059## or a pharmaceutically acceptable salt, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

10. A compound according to claim 1 having the following formula (VI): ##STR00060## or a pharmaceutically acceptable salt, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

11. A compound according to claim 1 having the following formula (VII): ##STR00061## or a pharmaceutically acceptable salt, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

12. A compound according to claim 1 which is selected from the following compounds: ##STR00062## ##STR00063## or a pharmaceutically acceptable salt, solvate or hydrate or a pharmaceutically acceptable formulation thereof.

13. Pharmaceutical composition comprising a compound according to claim 1 and optionally one or more carrier substances and/or one or more adjuvants.

14-15. (canceled)

16. A method for treating a subject suffering from or susceptible to a bacterial infection, comprising: administering to the subject an effective amount of a compound of claim 1.

17. The method of claim 1 wherein the subject is identified as suffering from or susceptible to a bacterial infection and the compound is administered to the identified subject.

18. A method for treating a subject suffering from or susceptible to malaria, comprising: administering to the subject an effective amount of a compound of claim 1.

19. The method of claim 1 wherein the subject is identified as suffering from or susceptible to malaria and the compound is administered to the identified subject.

Description

EXAMPLES

[0104] Activity Testing

[0105] Antimicrobial Assay:

[0106] Bacterial strains used in susceptibility assays (minimum inhibitory concentrations, MIC) were either part of our internal strain collection or were purchased form the German Collection of Microorganisms and Cell Cultures (DSMZ). All compounds were prepared as DMSO stocks and MIC values were determined in standard microbroth dilution assays. Overnight cultures of bacteria were diluted in cation-adjusted Muller-Hinton broth (BBL™, BD) and were adjusted to approximately 10.sup.5 cfu/mL. For the E. coli culture, 3 μg/mL of polymyxin B nonapeptide (PMBN) was added to increase permeability of the outer membrane as chlorotonils were previously shown to not penetrate into Gram-negative cells. Bacteria were grown in the presence of the derivatives in serial dilution for 16 h at their optimal growth temperature. MIC values were determined according to CLSI guidelines (antibiotic concentration at which no visible bacterial growth is observed).

[0107] Cytotoxicity Assay

[0108] The murine fibroblast cell line L-929 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) and maintained under conditions recommended by the depositor. Cells were seeded at 6×10.sup.3 cells per well of sterile 96-well plates in 180 μL complete medium and treated with the compounds in serial dilution after 2 h of equilibration. After 5 d, viability was assessed by the MTT method. After 1.5 h incubation with 0.5 mg/mL MTT reagent, cells were washed with PBS and 100 μL of 2-propanol/10 N HCl (250:1) was added. The absorbance at 570 nm was determined on a microplate reader and cell viability was expressed as percentage relative to the respective solvent control. Half-inhibitory concentrations (IC.sub.50) were determined by sigmoidal curve fitting.

[0109] Antiplasmodial Assay

[0110] Parasite Culture:

[0111] Two laboratory strains of P. falciparum, the chloroquine sensitive 3D7 and the multi-resistant Dd2 were kept in continuous culture as previously described (Trager and Jensen 1976). In brief, parasites were kept in complete culture medium (RPMI 1640, 25 mM HEPES, 2 mM L-glutamine, 50 μg/ml gentamicin and 0.5% w/v AlbuMAX) at 37° C., 5% CO.sub.2 and 5% oxygen at 5% hematocrit with daily change of medium. Synchronization was performed with sorbitol twice a week (Lambros and Vanderberg 1979).

[0112] In Vitro Drug Sensitivity Assay

[0113] All compounds were dissolved in DMSO at stock dilutions between 25 and 100 mM; the reference drug chloroquine diphosphate (MW: 515.86) was diluted in distilled water and DMSO, respectively. Further dilutions were prepared in complete culture medium so that final concentrations of solvent did not interfere with parasite growth. Antiplasmodial activity of the different compounds was tested in a drug sensitivity assay against the two laboratory strains using the histidine-rich protein 2 (HRP2) assay as described previously (Noedl 2005). In brief: 96 well plates were pre-coated with the different compounds in a threefold dilution before ring stage parasites were added in complete culture medium at a hematocrit of 1.5% and a parasitemia of 0.05% in a total volume of 225 μl per well. After three days of incubation plates were frozen until analyzed by HRP2-ELISA. All compounds were evaluated in duplicate in at least two independent experiments. The 50% inhibitory concentrations (IC.sub.50) were determined by analysing the nonlinear regression of log concentration—response curves using the drc-package v0.9.0 of R v2.6.1 (Vienna Austria 2008).

[0114] Zebrafish Embryo Toxicity

[0115] The maximum tolerated concentration (MTC) was determined on zebrafish larvae of the AB wildtype line and the TLF wild type line. Larvae were placed into a 96-well plate (one larva per well) and incubated in a solution at different concentrations (100, 50, 25, 10, 1 μM) of ChA, ChA-Epo2, ChB or ChB-Epo at two days post fertilization (dpf) for AB and in a solution at different concentrations of ChA (100, 50 μM) ChA-Epo2 (25, 10, 1 μM), ChB (100, 50 μM) or ChB-Epo (100, 50, 25 μM) at one dpf for TLF. Five zebrafish larvae were used per condition. The incubated embryos were kept in compound solutions at 28° C. until five dpf and monitored daily by microscopy. The final MTC result was recorded at five dpf. Compound solutions were prepared in the larvae medium Danieau's. Additionally a solution of 0.5% DMSO in Danieau's was used as vehicle control and Danieau's alone as a negative control.

REFERENCES

[0116] Lambros, C. and J. P. Vanderberg (1979). “Synchronization of Plasmodium falciparum erythrocytic stages in culture.” J Parasitol 65(3): 418-20. [0117] Trager, W. and J. B. Jensen (1976). “Human malaria parasites in continuous culture.” Science 193(4254): 673-5. [0118] Noedl, H., J. Bronnert, et al. (2005). “Simple histidine-rich protein 2 double-site sandwich enzyme-linked immunosorbent assay for use in malaria drug sensitivity testing.” Antimicrob Agents Chemother 49(8): 3575-7. [0119] Vienna Austria R Foundation for Statistical Computing. 2008. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing 1:26673.

[0120] Determination of Water Solubility

[0121] Method 1:

[0122] 10 mg of compound was suspended in 1 L of tap water and the solution was stirred at room temperature for 18 h. Upon completion, solution was sonicated for another hour, centrifuged and the supernatant was separated from the precipitate. Both aliquots were lyophilized, and weighed. This procedure was performed in triplicates.

[0123] Method 2:

[0124] A standard curve with 10 calibration points of the compound of interest is plotted using HPLC-MS and serial dilution. The dilutions and stock solutions were prepared in THF as a solvent. Following the measurements, a saturated solution in water of the compound in study was also measured. Upon plotting the standard curve using the peak areas of the MS peaks, the water samples were plotted along the curve and the solubility was then calculated.

[0125] Detailed NMR Conditions

[0126] All 1D (.sup.1H and .sup.13C) and 2D (COSY, ROESY, HSQC-DEPT and HMBC) NMR spectra were recorded on a Bruker Ascend 700 spectrometer with a 5 mm TXI cryoprobe (1H at 700 MHz, 13C at 175 MHz). 2D Experiments were recorded using standard pulse programs. The samples were dissolved in CDCl.sub.3 and the chemical shifts of the solvent signals at 7.26 ppm (δH) and 77.16 ppm (δC) were considered as internal standard (reference signal). The observed chemical shift (δ) values are given in ppm and the coupling constants (J) in Hz. For ROESY experiments measurements were carried out with mixing times of 400 ms.

[0127] Detailed LCMS and LCMSMS Conditions

[0128] The measurements to detect all chlorotonil derivatives were performed on a Dionex Ultimate 3000 RSLC system using a BEH C18, 50×2.1 mm, 1.7 μm dp column (Waters, Germany). Separation of 1 μl sample was achieved by a linear gradient from (A) H.sub.2O+0.1% FA to (B) ACN+0.1% FA at a flow rate of 600 μL/min and 45° C. The gradient was initiated by a 0.5 min isocratic step at 5% B, followed by an increase to 95% B in 6 min to end up with a 2 min step at 95 B before re-equilibration under the initial conditions. UV spectra were recorded by a DAD in the range from 200 to 600 nm. The LC flow was split to 75 μL/min before entering the maXis 4G hr-ToF mass spectrometer (Bruker Daltonics, Germany) using the Apollo ESI source. Mass spectra were acquired in centroid mode ranging from 150-2500 m/z at a 2 Hz scan rate. Settings for MS/MS measurements were: minimum precursor intensity is set to 10000. Full scan spectra are acquired at 2 Hz followed by MS/MS spectra acquisition at variable scan speed ranging from 1 to 3 Hz, as a function of precursor intensity. CID energy varies linearly from 30, 35, 45, to 55 eV with respect to the precursor m/z from 300, 600, 1000, to 2000 m/z. The collision cell is set to ramp collision energy (80-120% of the set value with equal weights of both values), collision RF (700 to 1000 Vpp with equal weights of both values) and transfer time (90-110 μs) for every MS/MS scan. The number of precursors was set to 2 and precursors were moved to an exclusion list for 0.2 min after two spectra were measured (typical chromatographic peak width was 0.10-0.15 min). Precursors were reconsidered if their intensity changed fivefold.

[0129] Syntheses

[0130] Chemical structures of natural chlorotonils A and B and the chlorotonil derivatives of the present invention:

##STR00018## ##STR00019## ##STR00020##

[0131] Ch-A-Epo:

##STR00021##

[0132] Chlorotonil A (0.21 mmol, 100 mg) was dissolved in chloroform (10 ml) and meta-Chloroperoxybenzoic acid (m-CPBA) (0.252 mmol, 43.35 mg) in chloroform (5 ml) was added to the solution dropwise over 30 min. The mixture was left stirring at room temperature for 16 hours. When no trace of starting material was observed (TLC:silica, CHCl.sub.3:DCM, 1:1, UV, R.sub.f), the solution was concentrated under reduced pressure and the mixture was purified using flash chromatography (silica, CHCl.sub.3:DCM, 1:1, UV) to yield Ch-A-Epo1 (0.03 mmol, 15.6 mg, 15% yield) and Ch-A-Epo2 (0.17 mmol, 83.2 mg, 80% yield) both as white powder. HRMS (ESI, +ve) C.sub.26H.sub.32Cl.sub.2O.sub.5 [M+H].sup.+ calculated for 495.1700, found 495.1702.

[0133] NMR-Data of Ch-A-Epo 1:

TABLE-US-00001 [00022] H δ.sub.H m J(Hz) COSY ROESY C δ.sub.C HMBC — —  1 168.0  2 4.53 q  7.0 23 —  2 47.1 23, 1, 3 —  3 192.0 —  4 81.4 —  5 196.0  6 3.70 dd 12.2, 6.6 7, 15 15  6 49.3 12, 16, 7, 8, 15, 5, 13, 14  7 2.17 td 10.8, 4.2 6, 8, 8, 24/26  7 31.5 6, 9, 12, 24, 11, 15, 5  8 2.45 13  2.3 9, 7, 9  8 29.0 9, 10, 12, 24/26, 25 24, 16  9 2.83 d  1.7 8, 11α, 8, 25,  9 65.6 10, 24, 8, 6, 25 15 12 — — 10 57.4  11α 1.59 t 13.1 9, 7, 12, 7 11 36.5 9, 10, 7, 12, 12/11β 13  11β 1.93- m — 2.01 12 1.93- m 12 28.6 2.01 13 5.55- m 13 132.2 5.59 14 5.46- m 14 124.2 5.52 15 2.98 m  2.1 16, 6, 13, 6 15 42.7 6, 7, 9, 13, 14, 12/11 14, 16, 17, β 26 16 2.71- m 17, 15, 7 16 33.4 6, 13, 14, 2.77 24/26, 15 15, 19, 18, 26 17 5.28 t  9.1 16, 18, 18, 17 139.3 15, 16, 18, 19 14/20 19, 26, 21 13/21 18 5.86 t 10.9 19, 17 19, 17, 18 125.6 16, 15, 19, 14/20 20, 21 13/21 19 6.03 td 11.5, 1.3 18, 20, 18, 19 124.0 17, 18, 20, 17, 21 14/20, 21, 22 17, 16 20 5.46- m 20 130.4 5.52 21 5.55- m — 21 70.4 5.59 22 1.35 d  6.7 21/14 13/21, 22 21.1 20, 19, 21 14/20 23 1.65 d  7.0 2 2 23 17.1 3, 1, 2 24 0.91- m 24 10.4 0.93 25 1.27 s 9, 25 24.7 9, 10, 11 12/11β, 11α 26 0.91- m 26 15.8 0.93

[0134] NMR-Data of Ch-A-Epo2:

TABLE-US-00002 [00023] H δ.sub.H m J(Hz) COSY ROESY C δ.sub.C HMBC — —  1 168.0  2 4.54 q  7.0 23 —  2 47.2 23, 1, 3 —  3 191.8 —  4 81.4 —  5 197.7  6 3.66 dd 12.0, 6.5 7, 15 15  6 48.7 12, 16, 7, 8, 15, 5, 13, 14  7 1.97 td 11.2, 5.5 6, 8, 8,  7 37.0 6, 9, 12, 24/26 24, 11, 15, 5  8 2.34 6  6.2 9, 7, 9  8 27.8 9, 10, 24/26, 25 12, 24, 16  9 3.06 d  5.6 8 8, 25,  9 64.5 10, 24, 15 8, 6, 12 — — 10 59.2  11α 1.41 t  8.6 9, 7, 12, 7 11 39.1 9, 10, 7, 12/11β 12, 13  11β 2.11 dd 14.1, 3.6 — 12 2.04- m 12 27.9 2.07 13 5.60 App  9.6 13 132.7 d 14 5.47 ddd 4.8, 2.3 14 123.7 15 2.98- m 6 15 42.4 6, 7, 3.01 9, 13, 14, 16, 17, 26 16 2.74- m 17, 15, 7 16 33.3 6, 13, 2.79 24/26, 15 14, 15, 19, 18, 26 17 5.25 t  9.3 16, 18, 18, 17 139.0 15, 16, 19 14/20 18, 19, 13/21 26, 21 18 5.86 t 10.9 19, 17 19, 17, 18 125.6 16, 15, 14/20 19, 20, 13/21 21 19 5.99- m 18, 20, 18, 19 123.7 17, 18, 6.03 17, 21 14/20, 20, 21, 17, 16 22 20 5.51 dd 15.3, 1.9 20 130.4 21 5.60 App  9.6 — 21 70.4 d 22 1.31 d  6.7 21/14 13/21, 22 21.0 20, 19, 14/20 21 23 1.65 d  7.0 2 2 23 17.2 3, 1, 2 24 0.93 App  6.6 24 9.7 d 25 1.35 s 9, 25 23.1 9, 10, 12/11β, 11 11α 26 0.93 App  6.6 26 15.7 d

[0135] Ch-B-Epo:

##STR00024##

[0136] Ch-B1-Epo and Ch-B3-Epo (0.018 mmol, 8.3 mg, 80% yield, 15:85 ratio) as white powder were prepared from ChB3 following the same procedure described above for Ch-A-Epo. (TLC:silica, CHCl.sub.3:DCM, 1:1, UV, R.sub.f). HRMS (ESI, +ve) C.sub.26H.sub.33ClO.sub.5 [M+H].sup.+ calculated for 461.2089, found 461.2087.

[0137] NMR-Data of ChB1-Epo:

TABLE-US-00003 [00025] H δ.sub.H m J(Hz) COSY C δ.sub.C HMBC —  1 168.2 —  2 4.38 q  7.0 23  2 44.8 23, 1, 3 —  3 187.8 — —  4 109.2 — —  5 194.2 —  6 3.34 dd 12.3, 6.5 7, 15  6 45.7 5, 4, 15, 7, 16, 8  7 1.9 4 td 17.4, 5.9 6, 8  7 36.0 24, 8, 11, 12/11α 15, 6, 5  8 2.44-2.52 m 9, 7, 24  8 28.4 24, 7, 9, 12  9 3.03 d  5.4 8  9 64.7 10, 7, 8, 25 — 10 59.3 —  11α 2.05-2.16 m 11β 11 38.7 12, 7, 10, 9, 13  11β 1.38-1.46 m 12/11α 12, 7, 10, 9, 13 12 2.05-2.16 m 11β 12 28.5 7, 10, 13 13 5.62 d 10.0 14, 11β 13 132.7 15, 11a, 7, 12/11α 12, 14 14 5.51 ddd 10.1, 4.6, 2.1 13, 15 14 124.4 13, 12, 15 15 2.73-2.80 m 14, 6, 12 15 42.1 14, 13 16 2.52-2.60 m 17, 26, 16 33.0 15, 26, 17, 18, 15 14, 18 17 5.37 t  9.6 16, 18 17 138.7 15, 16, 19, 26, 21 18 5.88 t 10.8 19, 17 18 126.0 16, 15, 19, 20 19 6.32 t 13.4 18, 20 19 123.3 17, 18, 20, 21 20 5.55 dd 15.2, 2.1 21, 19 20 131.7 21, 18 21 5.43-5.49 m 22, 20 21 70.5 22, 19 22 1.35 d  6.1 21 22 20.8 20, 21 23 1.44 d  6.9 2 23 12.1 3, 1, 2 24 0.85 d  7.1 8 24 10.2 9, 8, 7 25 1.34 s 25 23.0 9, 10, 11 26 0.94 d  6.6 16 26 16.8 16, 15, 17

TABLE-US-00004 [00026] H δ.sub.H m J(Hz) COSY C δ.sub.C HMBC — —  1 175.1 —  2 4.58 q  6.9 23  2 46.1 23, 1, 3 — —  3 180.9 — — —  4 107.7 — — —  5 193.9 —  6 3.29 dd 12.0, 7.0 7, 15  6 47.4 5, 15, 7, 16, 8  7 1.81 td 16.7, 6.1 6, 8,  7 36.5 24, 8, 11, 12/11α 15, 6  8 2.78-2.85 m 9, 7, 24  8 28.8 24, 7, 12  9 2.99 d  5.3 8  9 65.0 10, 7, 8, 25 — — 10 59.4 —  11α 1.99-2.14 m 11β 11 39.0 12, 7, 10, 9, 13  11β 1.38-1.46 m 12/11α 12, 7, 10, 9, 13 12 1.99-2.14 m 11β 12 28.7 7, 10, 13 13 5.53-5.58 m 14, 15, 13 132.8 15, 12, 14 11β 12/11α 14 5.46 ddd 10.0, 4.3, 2.3 13, 15 14 125.0 15 2.87-2.94 m 14, 6 15 41.3 16 2.31-2.41 m 17, 26, 16 33.6 15, 26, 17, 15 14, 18 17 5.32 t 10.0 16, 18 17 139.0 15, 16, 19, 26, 21 18 5.79 t 10.7 19, 17 18 125.7 16, 15, 19, 20, 17, 21 19 6.13 t 13.2 18, 20 19 124.1 17, 18, 20, 21 20 5.43 dd 15.4, 1.8 21, 19 20 131.1 22, 21, 18 21 5.58-5.64 m 22, 20 21 69.6 22, 19 22 1.35 d  6.6 21 22 20.6 20, 21 23 0.98 d  7.0 2 23 14.6 3, 1, 2 24 0.81 d  7.1 8 24 11.0 9, 8, 7 25 1.29 s 25 23.7 9, 10, 11 26 0.73 d  6.5 16 26 16.6 16, 15, 17

[0138] Ch-A-Epo 1-OMe:

##STR00027##

[0139] Ch-A-Epo1 (0.01 mmol, 5 mg) was dissolved in methanol (2 ml) and concentrated sulfuric acid (99.99%, 50 μL) was added. Solution was left stirring at room temperature for 5 h. Upon completion, the reaction mixture was diluted with water (100 ml) and the mixture was frozen and lyophilized. The resultant white powder was then purified using flash chromatography (silica, CHCl.sub.3:DCM, 1:1, UV). The desired product, Ch-A-Epo1-OMe (0.007 mmol, 3.7 mg, 70% yield) was obtained as a white powder. HRMS (ESI, +ve) C.sub.27H.sub.36Cl.sub.2O.sub.6 [M+H].sup.+ calculated for 527.1962, found 527.1963.

TABLE-US-00005 H δ.sub.H m J(Hz) COSY ROESY C δ.sub.C HMBC — — 1 168.0  2 4.54 q 7.0 23  23 2 47.2 23, 1, 3 — 3 192.1 — 4 81.4 — 5 196.7  6 3.83 dd 11.8, 6.8 7, 15 9, 15, 6 49.6 5, 7, 12, 16, 24, 12 15  7 2.34 td 11.1, 3.8 6 6, 8, 7 34.4 8, 12, 24 11α, 26,  8 2.01-2.05 m 9, 7, 24 9, 7, 24 8 37.5 8, 12, 24  9 3.60 bs 8, 11β 6, 8, 24 9 76.14 7, 8, 10, 11, 16, 22, 24 — — 10 77.1  .sup. 11α 1.42 t 13.3 12, 11β 7, 11β 11 38.2 7, 9  11β 1.76 d 13.7 11α, 9, 11α, 12, 12, 7 27 12 2.29-2.29 m 11α, 11β, 6, 24 12 28.8 11, 14, 13 8, 13 5.59-5.64 m 13 133.9 14 5.49-5.52 m 14 124.0 15 3.01-3.05 m 16, 6, 12 6, 16 15 43.1 16 2.74-2.78 m 15, 26, 15, 26 16 33.5 17 5.33 t 9.3 16, 18 18 17 139.3 16, 19, 20, 26, 18 18 5.88 t 10.9 19, 17 17, 18 125.6 16, 19, 20 21/13, 20/14 19 6.05 t 13.3 18, 20/14 16, 15 19 124.0 18, 17, 21 20 5.49-5.52 m 20 130.4 21 5.59-5.64 m 21 70.4 22 1.33 d 6.7 21/13 21/13, 22 22.1 20, 21 20/14 23 1.65 d 7.1 2  2 23 17.1 3, 1, 2 24 1.02 d 7.5 8 6, 8, 9, 24 12.3 8, 7, 9 12, 25 25 1.22 s 9, 11α, 25 21.7 9, 10, 11 24, 27 26 0.98 d 6.4 16  7, 16 26 15.8 17, 15, 16 27 3.18 s 9, 24, 27 48.7 10 25

[0140] Ch-A-Epo1-OEt:

##STR00028##

[0141] Ch-A-Epo1-OEt (65% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-OMe with the use of ethanol instead of methanol. HRMS (ESI, +ve) C.sub.28H.sub.38Cl.sub.2O.sub.6 [M+H].sup.+ calculated for 541.2118, found 541.2117.

TABLE-US-00006 H δ.sub.H m J(Hz) COSY ROESY C δ.sub.C HMBC — — 1 168.0  2 4.54 q 7.0 23 23 2 47.2 23, 1, 3 — 3 192.1 — 4 81.4 — 5 196.7  6 3.83 dd 11.8, 6.8 7, 15 9, 15, 6 49.6 5, 7, 12, 16, 24, 12 15  7 2.34 td 11.1, 3.8  6 6, 8, 7 34.4 8, 12, 24 11α, 26,  8 2.01-2.05 m 9, 7, 24 9, 7, 24 8 37.5 8, 12, 24  9 3.60 bs 8, 11β 6, 8, 24 9 76.14 7, 8, 10, 11, 16, 22, 24 — — 10 77.1  .sup. 11α 1.42 t 13.3 12, 11β 7, 11β 11 38.2 7, 9  11β 1.76 d 13.7 11α, 9, 11α, 12, 12, 7 27 12 2.29-2.29 m 11α, 11β, 6, 24 12 28.8 11, 14, 13 8, 13 5.59-5.64 m 13 133.9 14 5.49-5.52 m 14 124.0 15 3.01-3.05 m 16, 6, 12 6, 16 15 43.1 16 2.74-2.78 m 15, 26, 15, 26 16 33.5 17 5.33 t 9.3 16, 18 18 17 139.3 16, 19, 20, 26, 18 18 5.88 t 10.9 19, 17 17, 18 125.6 16, 19, 20 21/13, 20/14 19 6.05 t 13.3 18, 20/14 16, 15 19 124.0 18, 17, 21 20 5.49-5.52 m 20 130.4 21 5.59-5.64 m 21 70.4 22 1.33 d 6.7 21/13 21/13, 22 22.1 20, 21 20/14 23 1.65 d 7.1  2  2 23 17.1 3, 1, 2 24 1.02 d 7.5  8 6, 8, 9, 24 12.3 8, 7, 9 12, 25 25 1.22 s 9, 11α, 25 21.7 9, 10, 11 24, 27 26 0.98 d 6.4 16 7, 16 26 15.8 17, 15, 16 27 3.36-3-43 m 28 9, 24, 27 56.7 10, 28 25 28 1.13 t 6.9 27 28 16.1 28

[0142] Ch-A-Epo1-Cl:

##STR00029##

[0143] Ch-A-Epo1 (0.01 mmol, 5 mg) was dissolved in CHCl.sub.3 (2 ml) and concentrated hydrochloric acid (12 N, 50 μL) was added. Solution was left stirring at room temperature for 5 h. Upon completion, the reaction mixture was diluted with water (100 ml) and the mixture was frozen and lyophilized. The resultant white powder was then purified using flash chromatography (silica, CHCl.sub.3:DCM, 1:1, UV). The desired product, Ch-A-Epo1-Cl (88% yield) was obtained as a white powder. HRMS (ESI, +ve) C.sub.26H.sub.33Cl.sub.3O.sub.5 [M+H].sup.+ calculated for 531.1466, found 531.1465.

[0144] Ch-A-Epo2-OMe:

##STR00030##

[0145] Ch-A-Epo2-OMe (85% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-OMe. HRMS (ESI, +ve) C.sub.27H.sub.36Cl.sub.2O.sub.6 [M+H].sup.+ calculated for 527.1962, found 527.1963.

TABLE-US-00007 H δ.sub.H M J(Hz) COSY ROESY C δ.sub.C HMBC — — 1 168.0  2 4.54 q 7.0 23 — 2 47.2 23, 1, 3 — 3 192.1 — 4 81.3 — 5 196.9  6 3.81 dd 12.0, 6.3 7, 12, 15 15, 24, 6 49.9 5, 8, 12, 16, 12 15  7 2.23 td 11.75, 3.95 6, 12, 24 7 34.3 6, 8, 9, 11, 12, 15, 24, 25, 13  8 2.32-2.34 m 9, 7, 24 9, 6 8 29.6 9, 10, 7, 24  9 2.86 bs 8, 11α, 8, 25, 9 88.4 10, 24, 8, 25, 24 24 16, 11, 27 — — 10 73.5  .sup. 11α 1.56 ddd 13.7, 3.5, 1.3 9, 12, 25, 13, 11 42.5 9, 10, 7, 8, 11β 7 13  11β 1.47-1.52 m 9, 12, — 11α 12 2.39-2.43 m 7, 11α, 6, 24 12 28.9 11β, 13, 14 13 5.59-5.66 m 14, 15, 7 17, 12, 13 133.9 15, 11α 14 5.46-5.54 m 13, 15, 17, 15 14 123.9 16 15 3.01-3.05 m 16, 6, 12, 6, 16, 15 43.2 13, 14 19, 14, 13 16 2.71-2.79 m 15, 26, 15, 19, 16 33.6 15, 19, 17, 17, 18 18, 17, 26, 14 14 17 5.33 t 9.3 16, 18, 26, 14, 17 139.3 26, 16, 18, 19 13 19, 20, 15 18 5.88 t 11 19, 17 20 18 125.7 16, 15, 19, 20 19 6.01-6.09 m 18, 20, 16, 15, 19 124.0 21, 18, 20, 17 17, 21 17 20 5.46-5.54 m 19, 21 22 20 130.3 21 5.59-5.66 m 22, 20 — 21 70.4 22 1.32 d 6.7 21 20 22 22.1 20, 21, 23 23 1.65 d 7.0  2 — 23 17.1 3, 1, 2 24 1.03 d 7.6 8, 9, 7 6, 9, 12 24 12.4 8, 7, 9, 12 25 1.27 s 9, 11 25 29.3 9, 10, 11 26 0.97 d 6.4 16  7 26 15.7 17, 15, 16, 18 27 3.39 s 8, 9 27 57.3 9

[0146] Ch-A-Epo2-OEt:

##STR00031##

[0147] Ch-A-Epo2-OEt (82% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-OEt. HRMS (ESI, +ve) C.sub.28H.sub.38Cl.sub.2O.sub.6 [M+H].sup.+ calculated for 541.2118, found 541.2117.

TABLE-US-00008 H δ.sub.H M J(Hz) COSY ROESY C δ.sub.C HMBC — — 1 168.0  2 4.54 q 7.0 23 — 2 47.2 23, 1, 3 — 3 192.1 — 4 81.3 — 5 196.9  6 3.81 dd 12.0, 6.3 7, 12, 15 15, 24, 6 49.9 5, 8, 12, 16, 15 12  7 2.23 td 11.7, 3.9 6, 12, 24 7 34.3 6, 8, 9, 11, 12, 15, 24, 25, 13  8 2.32-2.34 m 9, 7, 24 9, 6 8 29.6 9, 10, 7, 24  9 2.86 bs 8, 11α, 25, 8, 25, 9 88.4 10, 24, 8, 16, 11, 24 24 27 — — 10 73.5  .sup. 11α 1.56 ddd 13.7, 3.5, 9, 12, 11β 25, 13, 11 42.5 9, 10, 7, 8, 13 1.3 7  11β 1.47-1.52 m 9, 12, 11α — 12 2.39-2.43 m 7, 11α, 11β, 6, 24 12 28.9 13, 14 13 5.59-5.66 m 14, 15, 7 17, 12, 13 133.9 15, 11α 14 5.46-5.54 m 13, 15, 16 17, 15 14 123.9 15 3.01-3.05 m 16, 6, 12, 13, 6, 16, 15 43.2 14 19, 14, 13 16 2.71-2.79 m 15, 26, 17, 15, 19, 16 33.6 15, 19, 17, 26, 14 18 18, 17, 14 17 5.33 t 9.3 16, 18, 19 26, 14, 17 139.3 26, 16, 18, 19, 20, 13 15 18 5.88 t 11 19, 17 20 18 125.7 16, 15, 19, 20 19 6.01-6.09 m 18, 20, 17 16, 15, 19 124.0 21, 18, 20, 17 17, 21 20 5.46-5.54 m 19, 21 22 20 130.3 21 5.59-5.66 m 22, 20 — 21 70.4 22 1.32 d 6.7 21 20 22 22.1 20, 21, 23 23 1.65 d 7.0  2 — 23 17.1 3, 1, 2 24 1.03 d 7.6 8, 9, 7 6, 9, 12 24 12.4 8, 7, 9, 12 25 1.27 s 9, 11 25 29.3 9, 10, 11 26 0.97 d 6.4 16  7 26 15.7 17, 15, 16, 18  .sup. 27α 3.34-3.38 m 28, 27α 8, 9 27 64.9 9  27β 3.70-3.75 m 28, 27β 28 1.92 t 7.0 27 28 15.7

[0148] Ch-A-Epo2-OBu:

##STR00032##

[0149] Ch-A-Epo2-OBu (90% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-OMe where 1-butanol was used instead of methanol. HRMS (ESI, +ve) C.sub.30H.sub.42Cl.sub.2O.sub.6 [M+H].sup.+ calculated for 569.2431, found 569.2433.

[0150] Ch-A-Epo2-Oisoamyl:

##STR00033##

[0151] Ch-A-Epo2-Oisoamyl (80% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-OMe where isomayl alcohol was used instead of methanol. HRMS (ESI, +ve) C.sub.31H.sub.44Cl.sub.2O.sub.6 [M+H].sup.+ calculated for 583.2588, found 583.2587.

[0152] Ch-A-Epo2-OBuOH:

##STR00034##

[0153] Ch-A-Epo2-OBuOH (65% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-OMe where 1,4-butanediol:THF (1:1) was used instead of methanol. HRMS (ESI, +ve) C.sub.30H.sub.42Cl.sub.2O.sub.7 [M+H].sup.+ calculated for 585.2380, found 585.2381.

[0154] Ch-A-Epo2-Cl:

##STR00035##

[0155] Ch-A-Epo2-Cl (95% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-Cl. HRMS (ESI, +ve) C.sub.26H.sub.33Cl.sub.3O.sub.5 [M+H].sup.+ calculated for 531.1466, found 531.1465.

TABLE-US-00009 H δ.sub.H M J(Hz) COSY ROESY C δ.sub.C HMBC — — 1 168.0  2 4.53 q 7.0 23 — 2 47.3 23, 1, 3 — 3 192.1 — 4 81.3 — 5 196.9  6 3.81 dd 11.7, 6.7 7, 12, 15 15, 24, 6 49.8 5, 8, 12, 16, 15 12  7 2.57 td 11.3, 3.8 6, 12, 24 7 33.9 6, 8, 9, 11, 12, 15, 24, 25, 13  8 2.43-2.49 m 9, 7, 24 9, 6 8 29.6 9, 10, 7, 24  9 3.84 t 1.7 8, 11α, 25, 8, 25, 9 70.1 10, 24, 8, 16, 11, 24 24 27 — — 10 73.5  .sup. 11α 1.68-1.73 m 9, 12, 11β 25, 13, 11 41.0 9, 10, 7, 8, 13 7  11β 1.68-1.73 m 9, 12, 11α — 12 2.43-2.49 m 7, 11α, 11β, 6, 24 12 28.6 13, 14 13 5.60-5.65 m 14, 15, 7 17, 12, 13 133.4 15, 11α 14 5.50-5.54 m 13, 15, 16 17, 15 14 124.5 15 3.01-3.08 m 16, 6, 12, 13, 6, 16, 15 43.3 14 19, 14, 13 16 2.74-2.82 m 15, 26, 17, 15, 19, 16 33.6 15, 19, 17, 26, 14 18 18, 17, 14 17 5.33 t 9.3 16, 18, 19 26, 14, 17 139.3 26, 16, 18, 19, 20, 13 15 18 5.89 t 10.9 19, 17 20 18 125.7 16, 15, 19, 20 19 6.06 t 12.9 18, 20, 17 16, 15, 19 124.0 21, 18, 20, 17 17, 21 20 5.50-5.54 m 19, 21 22 20 130.4 21 5.60-5.65 m 22, 20 — 21 70.3 22 1.33 d 6.7 21 20 22 21.2 20, 21, 23 23 1.65 d 7.0  2 — 23 17.2 3, 1, 2 24 1.13 d 7.7 8, 9, 7 6, 9, 12 24 14.8 8, 7, 9, 12 25 1.40 s 9, 11 25 31.1 9, 10, 11 26 1.01 d 6.5 16  7 26 15.8 17, 15, 16, 18

[0156] Ch-A-Epo2-Br:

##STR00036##

[0157] Ch-A-Epo2-Br (70% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-Cl where HBr (purum ≥62%) was used instead of HCl and THF as a solvent instead of CHCl.sub.3. HRMS (ESI, +ve) C.sub.26H.sub.33BrCl.sub.2O.sub.5 [M+H].sup.+ calculated for 575.0961, found 575.0962.

[0158] Ch-A-Epo2-OH:

##STR00037##

[0159] Ch-A-Epo2-OH (50% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-OMe where a mixture of THF:water (1:1) was used as a solvent instead of methanol. HRMS (ESI, +ve) C.sub.26H.sub.34Cl.sub.2O.sub.6 [M+H].sup.+ calculated for 513.1805, found 513.1805.

TABLE-US-00010 H δ.sub.H M J(Hz) COSY ROESY C δ.sub.C HMBC — — 1 168.0  2 4.55 q 7.0 23 — 2 47.5 23, 1, 3 — 3 192.1 — 4 81.3 — 5 196.9  6 3.84 dd 11.8, 6.8 7, 12, 15 15, 24, 6 49.7 5, 8, 12, 16, 15 12  7 2.35 td 11.3, 3.9 6, 12, 24 7 34.6 6, 8, 9, 11, 12, 15, 24, 25, 13  8 2.41-2.47 m 9, 7, 24 9, 6 8 37.5 9, 10, 7, 24  9 3.47 d 2.0 8, 11α, 25, 8, 25, 9 79.1 10, 24, 8, 16, 11, 24 24 27 — — 10 73.5  .sup. 11α 1.58-1.60 m 9, 12, 11β 25, 13, 11 42.1 9, 10, 7, 8, 13 7  11β 1.58-1.60 m 9, 12, 11α — 12 2.41-2.47 m 7, 11α, 11β, 6, 24 12 29.5 13, 14 13 5.59-5.65 m 14, 15, 7 17, 12, 13 132.9 15, 11α 14 5.48-5.54 m 13, 15, 16 17, 15 14 124.2 15 3.01-3.06 m 16, 6, 12, 13, 6, 16, 15 43.4 14 19, 14, 13 16 2.73-2.79 m 15, 26, 17, 15, 19, 16 33.7 15, 19, 17, 26, 14 18 18, 17, 14 17 5.33 t 9.3 16, 18, 19 26, 14, 17 139.4 26, 16, 18, 19, 20, 13 15 18 5.89 t 10.9  19, 17 20 18 125.8 16, 15, 19, 20 19 6.03-6.08 m 18, 20, 17 16, 15, 19 124.2 21, 18, 20, 17 17, 21 20 5.48-5.54 m 19, 21 22 20 130.7 21 5.59-5.65 m 22, 20 — 21 70.9 22 1.33 d 6.6 21 20 22 22.1 20, 21, 23 23 1.66 d 7.0  2 — 23 17.1 3, 1, 2 24 1.07 d 7.6 8, 9, 7 6, 9, 12 24 12.1 8, 7, 9, 12 25 1.31 m 9, 11 25 28.7 9, 10, 11 26 0.98 d 6.5 16  7 26 15.7 17, 15, 16, 18

[0160] Ch-A-Epo2-ONO.sub.2:

##STR00038##

[0161] Ch-A-Epo2-ONO.sub.2 (80% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-Cl where HNO.sub.3 69% was used instead of HCl and THF as a solvent. HRMS (ESI, +ve) C.sub.26H.sub.33Cl.sub.2NO.sub.8 [M+H].sup.+ calculated for 558.1656, found 558.1654.

TABLE-US-00011 H δ.sub.H M J(Hz) COSY ROESY C δ.sub.C HMBC — — 1 168.0  2 4.52 q 7.0 23 — 2 47.6 23, 1, 3 — 3 192.1 — 4 81.3 — 5 196.9  6 3.79 dd 11.8, 6.8 7, 12, 15 15, 24, 6 50.0 5, 8, 12, 16, 15 12  7 2.20 td 11.3, 4.1 6, 12, 24 7 35.1 6, 8, 9, 11, 12, 15, 24, 25, 13  8 2.29-2.34 m 9, 7, 24 9, 6 8 32.6 9, 10, 7, 24  9 4.81 t 1.7 8, 11α, 25, 8, 25, 9 88.2 10, 24, 8, 16, 11, 24 24 27 — — 10 73.5  .sup. 11α 1.71-1.73 m 9, 12, 11β 25, 13, 11 43.2 9, 10, 7, 8, 13 7  11β 1.47 t 13.4 9, 12, 11α — 12 2.44-2.48 m 7, 11α, 11β, 6, 24 12 28.6 13, 14 13 5.58-5.63 m 14, 15, 7 17, 12, 13 132.9 15, 11α 14 5.50-5.54 m 13, 15, 16 17, 15 14 123.9 15 3.03-3.06 m 16, 6, 12, 13, 6, 16, 15 43.2 14 19, 14, 13 16 2.73-2.77 m 15, 26, 17, 15, 19, 16 33.6 15, 19, 17, 26, 14 18 18, 17, 14 17 5.30-5.33 m 16, 18, 19 26, 14, 17 139.2 26, 16, 18, 19, 20, 13 15 18 5.88 t 10.8 19, 17 20 18 125.8 16, 15, 19, 20 19 6.02-6.07 m 18, 20, 17 16, 15, 19 124.0 21, 18, 20, 17 17, 21 20 5.50-5.54 m 19, 21 22 20 130.7 21 5.58-5.63 m 22, 20 — 21 70.6 22 1.32-1.35 m 21 20 22 21.1 20, 21, 23 23 1.64 d 7.0  2 — 23 17.1 3, 1, 2 24 1.13 d 7.6 8, 9, 7 6, 9, 12 24 12.1 8, 7, 9, 12 25 1.32-1.35 m 9, 11 25 28.7 9, 10, 11 26 0.96 d 6.5 16  7 26 15.7 17, 15, 16, 18

[0162] Ch-A-Epo2-F:

##STR00039##

[0163] Ch-A-Epo2-F (40% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-Cl where HF (48 wt. % in H.sub.2O) was used instead of HCl and THF as a solvent and the reaction was done in a flacon tube. HRMS (ESI, +ve) C.sub.26H.sub.33Cl.sub.2FO.sub.5 [M+H].sup.+ calculated for 515.1762, found 515.1761.

[0164] Ch-A-Epo2-Glycerol:

##STR00040##

[0165] Ch-A-Epo2-Glycerol (45% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-OMe where glycerol:THF (1:1) was used instead of methanol. HRMS (ESI, +ve) C.sub.29H.sub.40Cl.sub.2O.sub.8 [M+H].sup.+ calculated for 587.2173, found 587.2175.

[0166] Ch-A-Epo2-OBn:

##STR00041##

[0167] Ch-A-Epo2-OBn (50% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-OMe where benzyl alcohol:THF (1:1) was used instead of methanol. HRMS (ESI, +ve) C.sub.33H.sub.40Cl.sub.2O.sub.6 [M+H].sup.+ calculated for 603.2275, found 603.2272.

[0168] Ch-B-Epo-Cl:

##STR00042##

[0169] Ch-B-Epo-Cl (80% yield), white powder was prepared from Ch-B-Epo following the same procedure described above for compound Ch-A-Epo1-Cl. HRMS (ESI, +ve) C.sub.26H.sub.34Cl.sub.2O.sub.5 [M+H].sup.+ calculated for 497.1856, found 497.1856.

[0170] Ch-B1-Epo-OMe:

##STR00043##

[0171] Ch-B1-Epo-OMe (53% yield), white powder was prepared from Ch-B1-Epo following the same procedure described above for compound Ch-A-Epo1-OMe. Extraction of the reaction with dichloromethane was accomplished before purification instead of freeze drying. HRMS (ESI, +ve) C.sub.27H.sub.37ClO.sub.6 [M+H].sup.+ calculated for 493.2351, found 493.2346.

TABLE-US-00012 H δ.sub.H m J(Hz) COSY C δ.sub.C HMBC — 1 168.0 —  2 4.40 q 6.9 23 2 44.9 23, 1, 3 — 3 188.6 — — 4 109.4 — — 5 193.4 —  6 3.50 dd 12.3, 6.7 7, 15 6 46.7 5, 4, 15, 7, 12  7 2.21 td 17.5, 4.3 6, 8, 12 7 33.3 24, 8, 12, 11/15, 6  8 2.45-2.49 m 7, 24 8 30.7 24, 7, 12, 9, 10  9 2.83 br s  8 9 88.4 24, 25, 7, 11, 27, 10 — — 10 73.6 —  .sup. 11α 1.51 q 13.0  12 11 42.2 12, 7, 10, 9, 13  11β 1.54 m 12 12, 7, 10, 9, 13 12 2.40-2.45 m 7, 11 12 29.4 13 5.62 d 10.0  14, 15, 12 13 133.9 11, 12, 7, 14 14 5.50 ddd 10.1, 4.2, 2.7 13, 15, 12 14 124.7 13, 12, 15, 6 15 2.76-2.80 m 14, 6 15 42.7 7, 14, 13 16 2.56-2.62 m 17, 26, 15 16 33.2 15, 26, 6, 17, 14, 18 17 5.41 t 9.6 16, 18 17 139.0 16, 19, 26 18 5.88 t 10.8  19, 17 18 125.8 16, 15, 19, 20 19 6.36 t 13.3  18, 20 19 123.4 17, 18, 20, 21 20 5.55 dd 15.3, 2.2 21, 19 20 131.5 22, 21, 18, 19 21 5.45-5.49 m 22, 20 21 70.4 22, 19, 20, 1 22 1.35 d 6.6 21 22 20.7 20, 21 23 1.44 d 7.1  2 23 12.2 3, 1, 2 24 0.97 d 7.4  8 24 13.0 9, 8 25 1.27 s 25 29.2 9, 10, 11 26 0.98 d 6.2 16 26 16.9 16, 15, 17 27 3.36 s 27 57.5 9

[0172] Ch-B1-Epo-OEt:

##STR00044##

[0173] Ch-B1-Epo-OEt (53% yield), white powder was prepared from Ch-B1-Epo following the same procedure described above for compound Ch-B1-Epo-OMe. HRMS (ESI, +ve) C.sub.28H.sub.39ClO.sub.6 [M+H].sup.+ calculated for 507.2508, found 507.2501.

TABLE-US-00013 H δ.sub.H m J(Hz) COSY C δ.sub.C HMBC — —  1 168.1 —  2 4.40 q 6.9 23  2 45.0 23, 1, 3, 4 — —  3 188.8 — — —  4 109.3 — — —  5 193.0 —  6 3.49 dd 12.3, 6.6 7, 15  6 46.8 5, 4, 15, 7, 12  7 2.29 dd 17.4, 3.9 6, 8  7 33.5 24, 8, 12, 11/15, 6  8 2.36-2.47 m 7, 24  8 31.7 24, 7, 9, 10, 12  9 2.92 br s  8  9 86.5 24, 25, 7, 11, 27, 10 — — 10 73.4 — 11 1.52-1.56 m 13.0 12 11 42.3 12, 7, 10, 9, 13 12 2.36-2.47 m 7, 11 12 29.6 13 5.63 d 10.0 14, 15, 12 13 134.1 11, 12, 7, 14 14 5.52-5.48 m 13, 15, 12 14 124.7 13, 12, 15, 6 15 2.77 br s 14, 6 15 43.0  7 16 2.56-2.64 m 17, 26, 15 16 33.3 15, 26, 6, 17, 14, 18 17 5.42 t 9.6 16, 18 17 139.3 16, 19, 26 18 5.88 t 10.8 19, 17 18 126.0 16, 15, 19, 20 19 6.36 t 16.1 18, 20 19 123.6 17, 18, 20, 21 20 5.55 d 15.3 21, 19 20 131.6 22, 21, 18, 19 21 5.45-5.48 m 22, 20 21 70.5 22, 19, 20, 1 22 1.35 d 6.5 21 22 20.9 20, 21 23 1.44 d 6.9  2 23 12.3 3, 1, 2 24 0.95 d 7.7  8 24 13.0 9, 8, 7 25 1.26 s 25 29.3 9, 10, 11 26 0.97 d 6.5 16 26 16.8 16, 15, 17  .sup. 27α 3.65 m 28, 27β  .sup. 27α 65.1 28  27β 3.36 m 28, 27α  27β 28 28 1.18 t 7.0 27α, 27β 28 16.0 27

[0174] Ch-B1-Epo-Cl:

##STR00045##

[0175] Ch-B1-Epo-Cl (75% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-Cl. HRMS (ESI, +ve) C.sub.26H.sub.34Cl.sub.2O.sub.5 [M+H].sup.+ calculated for 497.1856, found 497.1860.

TABLE-US-00014 H δ.sub.H m J(Hz) COSY C δ.sub.C HMBC — — 1 168.2 —  2 4.37 q 6.9 23 2 44.8 23, 1, 3 — — 3 188.3 — — — 4 109.3 — — — 5 193.0 —  6 3.50 dd 11.8, 6.6 7, 15 6 46.5 5, 4, 15, 7  7 2.54 dd 11.7, 4.0 6, 12 7 32.8  6  8 2.56-2.64 m 24 8 38.2  9 3.83 s 5.4 9 70.1 10, 7, 11, 24 — — 10 73.9 —  .sup. 11α 1.72 t 13.2  12/11β 11 40.7 12  11β 1.61 ddd 13.6, 3.0, 1.7  .sup. 11α 7, 10 12 2.45-2.52 m  11α 12 29.6 13 13 5.64 d 10.0  14 13 133.3 15, 7, 12 14 5.53-5.58 m 13, 15 14 125.1 12, 15 15 2.77-2.84 m 14, 6 15 42.7 16 16 2.57-2.64 m 17, 26 16 33.1 17 5.42 t 9.6 16, 18 17 138.8 16, 19, 20 18 5.89 t 10.8  19, 17 18 125.9 16, 19, 20 19 6.36 t 13.0  18, 20 19 123.4 18, 21, 17 20 5.53-5.58 m 15.2, 2.1 21, 19 20 131.6 21, 18 21 5.45-5.50 m 22 21 70.5 22, 19 22 1.35 d 6.7 21 22 20.8 20, 21 23 1.43 d 7.0  2 23 12.1 3, 1, 2 24 1.06 d 7.6  8 24 15.3 9, 8, 7 25 1.40 s 25 31.1 9, 10, 11 26 1.01 d 6.5 16 26 16.7 16, 15, 17

[0176] Ch-B1-Epo-ONO.sub.2:

##STR00046##

[0177] Ch-B1-Epo-ONO.sub.2 (71% yield), white powder was prepared following the same procedure described above for compound Ch-A-Epo1-Cl where HNO.sub.3 69% was used instead of HCl and THF as a solvent. HRMS (ESI, +ve) C.sub.26H.sub.34ClNO.sub.8 [M+H].sup.+ calculated for 524.2046, found 524.2051.

TABLE-US-00015 H δ.sub.H m J(Hz) COSY C δ.sub.C HMBC — 1 167.9 —  2 4.37 q 7.0 23 2 45.0 23, 1, 3, 4 — 3 188.4 — — 4 109.3 — — 5 192.2 —  6 3.49 dd 12.1, 6.7 7, 15 6 46.4 5, 4, 15, 8/16  7 2.18 td 17.2, 4.3 6, 8 7 34.4 24, 11/15  8 2.3-2.48 m 7, 24 8 33.2 24, 9, 10, 12  9 4.79 m 8, 11α 9 88.3 7, 11, 10 — — 10 71.7 —  .sup. 11α 1.71 ddd 13.8, 3.0, 1.5 11β, 12 11 42.8 7, 10, 9  11β 1.46 t 13.5  11α, 12 12 12 2.48 m 7, 11, 13 12 29.3 13 5.63 dt 10.1, 1.5 14, 15, 12 13 133.0 11, 12 14 5.55-5.58 m 13, 15, 12 14 125.5 12, 15 15 2.77-2.84 m 14, 6 15 42.7 16 2.55-2.62 m 17, 26, 15 16 33.2 15, 26, 17, 14 17 5.40 t 9.4 16, 18 17 138.7 16, 19, 26 18 5.89 t 10.8  19, 17 18 126.2 16, 20 19 6.34 t 13.3  18, 20 19 123.6 17, 21 20 5.55-5.58 m 21, 19 20 131.8 22, 21, 18 21 5.45-5.50 m 22, 20 21 70.7 22, 19, 20, 1 22 1.35 d 6.6 21 22 20.9 20, 21 23 1.44 d 6.9  2 23 12.3 3, 1, 2 24 1.06 d 7.7  8 24 12.7 9, 8, 7 25 1.32 s 25 28.8 9, 10, 11 26 0.97 d 6.6 16 26 16.9 16, 15, 17

[0178] Ch-A-Epo2-polyEpo:

##STR00047##

[0179] Ch-A-Epo2 (0.02 mmol, 10 mg) was dissolved in chloroform (2 ml) and meta-Chloroperoxybenzoic acid (m-CPBA) (0.08 mmol, 14 mg) in chloroform (5 ml) was added to the solution dropwise over 30 min. The mixture was left stirring at room temperature for 16 hours. However, an aliquot of 0.5 ml was taken every 3 h, concentrated to dryness.

[0180] When no trace of starting material was observed (TLC:silica, CHCl.sub.3: DCM, 1:1, UV, R.sub.f), the remaining aliquot was joined with the latter ones and was concentrated under reduced pressure and the mixture was purified using flash chromatography (silica, CHCl.sub.3:DCM, 1:1, UV) to yield the following derivatives as white powder.

[0181] Ch-A-tetraEpo-A HRMS (ESI, +ve) C.sub.26H.sub.32Cl.sub.2O.sub.8 [M+H].sup.+ calculated for 543.1547, found 543.1551;

[0182] Ch-A-tetraEpo-B HRMS (ESI, +ve) C.sub.26H.sub.32Cl.sub.2O.sub.8 [M+H].sup.+ calculated for 543.1547, found 543.1552;

[0183] Ch-A-triEpo-A HRMS (ESI, +ve) C.sub.26H.sub.32Cl.sub.2O.sub.7 [M+H].sup.+ calculated for 527.1598, found 527.1607;

[0184] Ch-A-triEpo-B HRMS (ESI, +ve) C.sub.26H.sub.32Cl.sub.2O.sub.7 [M+H].sup.+ calculated for 527.1598, found 527.1606

[0185] Results

TABLE-US-00016 TABLE 1 Water solubility of selected compounds Compound Method 1 Method 2 Chlorotonil A (ChA)  3 μg/mL ± 0.4 1.8 μg/mL ChA-Epo2  14 μg/mL ± 0.3 10.3 μg/mL ChA-Epo2-Cl 280 μg/mL ± 0.4 320 μg/mL ChA-Epo2-ONO.sub.2 310 μg/mL ± 0.5 380 μg/mL

TABLE-US-00017 TABLE 2 MIC values in μg/mL of various chlorotonil derivatives against Gram-positive bacteria and a sensitive E. coli strain (nd: not determined). MIC [μg/ml] B. C. E. subtilis glutamicum S. coli DSM- DSM- aureus TolC Compound 10 20300 Newman (+PMBN) Chlorotonil A (ChA) 0.0125 0.0125 0.0125 0.4 ChA-Epo2 0.025 0.05 0.025 0.1 ChA-Epo1 3.2 >3.2 3.2 >3.2 ChA-Epo2-OBuOH >3.2 3.2 >3.2 1.6 ChA-Epo2-OMe >3.2 >3.2 8-4 3.2 ChA-Epo2-OBu 0.8 >3.2 1.6 >3.2 ChA-Epo2-Oisoamyl 0.8 3.2 1.6 0.8 ChA-Epo2-Br 0.05 0.1 0.05 0.2 ChA-Epo2-Cl 0.1 0.4 0.05 0.8 ChA-Epo2-OEt >3.2 >3.2 4-2 >3.2 ChA-Epo2-OH >6.4 >6.4 8-4 >6.4 ChA-Epo2-ONO.sub.2 0.025 0.1 0.025 0.1 ChA-Epo2-OBn 0.4 3.2 0.4 >3.2 ChA-Epo2-glycerol >3.2 >3.2 4 >3.2 ChA-Epo2-F 0.2 0.4-0.8 0.2 0.8 ChA-Epo1-Cl 3.2 >3.2 3.2 3.2 ChA-Epo1-OMe 6.4 >6.4 1.6 0.8-1.6 ChA-Epo1-OEt >6.4 >6.4 >1.6 1.6 Chlorotonil B (ChB) 0.2 0.1 0.2 >3.2 ChB-Epo nd nd 0.05 nd ChB-Epo-Cl nd nd 0.2 nd

TABLE-US-00018 TABLE 3 IC.sub.50 values in nM of various chlorotonil derivatives against P. falciparum 3D7 (sensitive strain) and P. falciparum Dd2 (resistant strain). Compound P. falciparum 3D7 P. falciparum Dd2 Chloroquine (reference) 7.4 291.5 Chlorotonil A (ChA) 18.7 20.7 ChA-Epo1 96.5 229.4 ChA-Epo2 51.3 87.1 ChA-Epo2-OMe >11 μM >11 μM ChA-Epo2-OEt >11 μM >11 μM ChA-Epo2-OBu >11 μM >11 μM ChA-Epo2-Oisoamyl >11 μM >11 μM ChA-Epo2-Cl 38.7 45.9 ChA-Epo2-Br 25.1 64.4 ChA-Epo1-Cl >2095 >2095 ChA-Epo2-OH >2168 >2168 ChA-Epo2-glycerol >1895 >1895 ChA-Epo2-F 135.6 328.8 ChA-Epo2-ONO.sub.2 0.92 2.2 Chlorotonil B (ChB) 96.6 63.6 ChB-Epo 158.1 182.0

TABLE-US-00019 TABLE 4 IC.sub.50 values in nM of various chlorotonil derivatives against the L-929 cell line. Compound L-929 (murine fibroblasts) Chlorotonil A (ChA) 1100 ChA-Epo1 10300 ChA-Epo2 2100 ChA-Epo2-OMe >100,000 ChA-Epo2-OEt >100,000 ChA-Epo2-OBu 49400 ChA-Epo2-Oisoamyl 44700 ChA-Epo2-Cl 4200 ChA-Epo2-Br 1100

TABLE-US-00020 TABLE 5 MIC values in μg/mL of various chlorotonil derivatives against a range of Gram-positive bacteria ChA- ChB- ChB1- bacterial strain ChA Epo2 ChB Epo Epo S. epidermidis 28765 0.1 0.2 >3.2 0.8 0.05 E. faecalis 20478 0.05 0.8 >3.2 3.2 0.8 E. faecalis 29212 0.05 1.6 >3.2 >3.2 0.8 E. faecium 17050 0.025 0.4 >3.2 3.2 32 (VRE) E. faecium 20477 0.1 3.2 >3.2 >3.2 0.2 M. luteus 0.0125 0.025 0.4 0.1 0.0125 M. smegmatis 0.8 0.003 0.0125 >3.2 16 B. megaterium 0.003 0.00125 0.1 0.05 0.0125 S. pneumoniae 20566 0.006 0.2 1.6 0.8 0.4 S. pneumoniae 11865 0.2 8 >16 >32 — (PRSP)

TABLE-US-00021 TABLE 6 Observations noted on fish larvae of AB line at five dpf (incubation started at two dpf) ChA-Epo2 ChB-Epo 100 μM died died 50 μM died died 25 μM died OK 10 μM died OK 1 μM OK OK MTC 1 μM 25 μM

TABLE-US-00022 TABLE 7 Observations noted on fish larvae of TLF line at five dpf (incubation started at one dpf) ChA-Epo2 ChB-Epo 100 μM — tail malformation 50 μM — slight tail malformation 25 μM 1 died, 4 delayed in OK development 10 μM 4 delayed in development — 1 μM OK — MTC 1 μM 25 μM

[0186] The chlorotonil derivatives of the present invention are superior in their pharmaceutical properties and structurally different to the known naturally produced chlorotonils. The invention demonstrates a very efficient chemistry. The two-step transformations applied are scalable to multi-gram scale and easily handled. The setup does not require any anhydrous systems or any complicated setups. All reactions are done at room temperature and the purification on both steps is done using normal phase chromatography, which facilitates the efficient upscaling of such systems. None of the used reagents is highly priced. All these facts display a system that can be applied in any lab set up and can be utilized for large-scale process development.

[0187] Due to the introduction of an epoxide on one of the double bonds and subsequent addition of a number of different substituents, the aqueous solubility could be increased when compared to the parent compound. A significant improvement in water solubility of chlorotonil from less than 2 μg/mL to over 300 μg/mL for some of the derivatives was observed (Table 1). This has helped in determining the maximum tolerated concentration (MTC) of the epoxide derivatives on two zebrafish lines (Tables 6 and 7), whereas this was not possible on the natural derivatives because of their low solubility. The derivatives were assessed in a number of biological assays and were shown to exhibit a broad antibiotic activity in the low nM range against a range of Gram-positive bacteria (Table 5), where additionally, some of the derivatives were intriguingly active on Plasmodium falciparum without displaying significant toxicity on a model cell line (Tables 2 to 4).