Crystobactamides

Abstract

The present invention provides cystobactamides of formula (I): R.sup.1—Ar.sup.1-L.sup.1-Ar.sup.2-L.sup.2-Ar.sup.3-L.sup.3-Ar.sup.4-L.sup.4-Ar.sup.5—R.sup.2 and the use thereof for the treatment or prophylaxis of bacterial infections.

Claims

1. A method for treatment or prophylaxis of bacterial infections comprising administering a pharmaceutical composition, wherein the pharmaceutical composition comprises a compound of formula (I)
R.sup.1—Ar.sup.1-L.sup.1-Ar.sup.2-L.sup.2-Ar.sup.3-L.sup.3-Ar.sup.4-L.sup.4-Ar.sup.5—R.sup.2  (I) wherein Ar.sup.1 is an optionally substituted 1,4-phenylene group; Ar.sup.2 is an optionally substituted 1,4-phenylene group; Ar.sup.3 is an optionally substituted 1,4-phenylene group; Ar.sup.4 is an optionally substituted 1,4-phenylene group; Ar.sup.5 is an optionally substituted 1,4-phenylene group; L.sup.1 is a NHCO wherein the nitrogen atom is bound to Ar.sup.1; L.sup.2 is NHCO, wherein the nitrogen atom is bound to Ar.sup.2; L.sup.3 is a formula of: ##STR00109## wherein the NH group is bound to Ar.sup.3, and R.sup.30 is a hydrogen atom or a C.sub.1-3 alkyl group; L.sup.4 is absent or NHCO wherein the nitrogen atom is bound to Ar.sup.4; R.sup.1 is —NH.sub.2, —NO.sub.2, COOR.sup.11, or —CONR.sup.12R.sup.13; wherein R.sup.11, R.sup.12 and R.sup.13 are independently a hydrogen atom or a C.sub.1-6 alkyl group; R.sup.2 is —NH.sub.2, —NO.sub.2, COOR.sup.11a, or —CONR.sup.12aR.sup.13a; wherein R.sup.11a, R.sup.12a and R.sup.13a are independently a hydrogen atom or a C.sub.1-6 alkyl group; or a pharmaceutically acceptable salt, or a pharmaceutically acceptable formulation thereof.

2. The method of claim 1 wherein the compound has formula (VII) ##STR00110## wherein R.sup.51 is a hydrogen atom, or a C.sub.1-6 alkyl group; R.sup.53 is a hydrogen atom, F, Cl, a hydroxy group, a C.sub.1-6 alkyl group or a group of formula —O—C.sub.1-6 alkyl; D is CR.sup.56; R.sup.56 is a hydrogen atom, F, Cl, a hydroxy group, a C.sub.1-6 alkyl group or a group of formula —O—C.sub.1-6 alkyl; R.sup.57 is a hydrogen atom, F, Cl, a hydroxy group, a C.sub.1-6 alkyl group or a group of formula —O—C.sub.1-6 alkyl; R.sup.58 is a hydrogen atom, F, Cl, a hydroxy group, a C.sub.1-6 alkyl group or a group of formula —O—C.sub.1-6 alkyl; R.sup.59 is a hydrogen atom, F, Cl, a hydroxy group, a C.sub.1-6 alkyl group or a group of formula —O—C.sub.1-6 alkyl; R.sup.60 is a hydrogen atom, F, Cl, a hydroxy group, a C.sub.1-6 alkyl group or a group of formula —O—C.sub.1-6 alkyl; R.sup.61 is a hydrogen atom, F, Cl, a hydroxy group, a C.sub.1-6 alkyl group or a group of formula —O—C.sub.1-6 alkyl; and R.sup.8 is a group of the following formula: ##STR00111## wherein R.sup.9 is COOH or CONH.sub.2 and R.sup.10 is COOH or CONH.sub.2; or a pharmaceutically acceptable salt, or a pharmaceutically acceptable formulation thereof.

3. The method of claim 1 wherein the compound has formula (IV) ##STR00112## wherein R.sup.5 is a hydrogen atom or a group of formula —O—C.sub.1-6 alkyl; R.sup.6 is a hydroxy group; R.sup.7 is a group of formula —O—C.sub.1-6 alkyl; and R.sup.8 is a group of the following formula: ##STR00113## wherein R.sup.9 is COOH or CONH.sub.2 and R.sup.10 is COOH or CONH.sub.2; or a pharmaceutically acceptable salt, or a pharmaceutically acceptable formulation thereof.

4. The method of claim 1 wherein the compound is selected from: ##STR00114## ##STR00115## ##STR00116## ##STR00117##

5. The method of claim 1 wherein the pharmaceutical composition optionally comprises one or more carrier substances and/or one or more adjuvants.

Description

FIGURES

(1) FIG. 1: Key 2D NMR correlations (700 MHz, DMSO-d.sub.6) for cystobactamide A (1)

(2) FIG. 2: Key 2D NMR correlations (500 MHz, DMSO-d.sub.6) for cystobactamide C (3)

(3) FIG. 3: Key 2D NMR correlations (700 MHz, DMSO-d.sub.6) for cystobactamide D (4)

(4) FIG. 4: Key 2D NMR correlations of cystobactamide D dimethyl ester (4a)

(5) FIG. 5: Key 2D NMR correlations of cystobactamide E (5)

(6) FIG. 6: Key 2D NMR correlations (700 MHz, DMSO-d.sub.6) of cystobactamide F (6)

(7) FIG. 7: Key 2D NMR correlations (700 MHz, MeOH-d.sub.4) of cystobactamide G (7)

(8) FIG. 8: Key 2D NMR correlations (700 MHz, DMSO-d.sub.6) of cystobactamide H (8)

(9) FIG. 9a-9d: FIG. 9a show the results of the gyrase inhibition assay. FIG. 9b shows the result of the topoisomerase IV inhibition assay. FIG. 9c shows the result of the topoisomerase II inhibition assay. FIG. 9d shows the result of the topoisomerase I inhibition assay.

(10) FIG. 10a-10b: FIG. 10a shows the result of DNA/gyrase complex linearization assays. FIG. 10b shows the result of ATP competition assay.

(11) FIG. 11: Shows the results of the DNA/gyrase complex linearization assay.

(12) FIG. 12: Shows the cystobactamide biosynthesis gene cluster.

BIOLOGICAL EVALUATION OF CYSTOBACTAMIDES

(13) As summarized in Tables 10a/b, cystobactamides were evaluated against several microorganisms and cell lines. All derivatives demonstrated a potent inhibitory effect on various E. coli strains, including a nalidixic acid resistant (NAL.sup.R) isolate. Overall potency (average MIC values) of the tested derivatives increased in the following order: CysA1, CysC<CysB<CysA, CysG<CysF. Importantly, the pathogenic Gram-negative strains A. baumannii and P. aeruginosa were also inhibited by the most active derivatives, CysA, CysB, CysG, and CysF, in the low μg/ml range, which is in terms of MIC values only by one order of magnitude higher than for the reference drug ciprofloxacin.

(14) Average MIC values on Gram-positive bacteria, such as E. faecalis, S. aureus, and S. pneumonia were partly in the sub-μg/ml range and the average potency of CysA and CysB exceeded that of ciprofloxacin.

(15) Furthermore, it was shown that cystobactamides do not inhibit the growth of yeast and mammalian cells, respectively. Thus, the cystobactamides did not cause apparent cytotoxicity.

(16) Susceptibility of Mutant E. coli Strains to Cystobactamides

(17) Quinolones are a widely used class of antibiotics that target the type II topoisomerases, DNA gyrase and topoisomerase IV. Resistance to quinolones is thereby often mediated by mutations in chromosomal genes that lead to alterations in the drug targets. In GyrA the quinolone-resistance determining region (QRDR) is located between amino acids 67 and 106, whereas amino acids 83 (Ser) and 87 (Asp) are most often involved..sup.[1,2] In analogous regions of ParC, the secondary target of quinolones, changes of amino acid 80 (Ser) are found to confer quinolone resistance..sup.[3,4]

(18) Cystobactamides were screened using a panel of E. coli strains with typical mutations in gyrA and parC genes (Table 11). With ciprofloxacin the MIC values increase approximately by factor 30 for the single-step gyrA mutations (strain MI and WT-3.2). However, a combination of both gyrA mutations (strain WT-3) results already in nearly clinical resistance (1 mg/L). A parC mutation (strain WT-4 M2.1) leads to a two-fold increase of the MIC of ciprofloxacin. However, MIC values for cystobactamides did not or only marginally increase for gyrA and parC mutant E. coli strains, which suggests that cystobactamides might interfere with amino acids 87 and 83 of GyrA and amino acid 80 of ParC to a lower extent than observed for ciprofloxacin.

(19) High-level quinolone resistance often results from a combination of several target site mutations and altered efflux mechanisms. The in vitro selected mutant WT III (marR Δ74 bp) does not produce functional MarR, which acts as a repressor of marA expression. This, in turn, leads to overproduction of MarA and AcrAB and overexpression of the AcrAB efflux pump is associated with the MAR (multiple antibiotic resistance) phenotype..sup.[5] E. coli strain WT III was less susceptible to ciprofloxacin treatment by a factor of ca. 4 (cp. E. coli WT). In comparison, MIC values of cystobactamides B, F, and G were still in the μg/ml range. Notably, the MIC of CysF on strain E. coli WT III only increased by factor 2 compared to wildtype E. coli DSM-1116, whereas the MIC of ciprofloxacin increased by ca. factor 10.

(20) TABLE-US-00012 TABLE 10a Antimicrobial activity of cystobactamides (Cys). CysA CysA1 CysB CysC Test organism MIC [μg/ml] Acinetobacter baumannii DSM-30008 7.4 58.9 3.7 32.5 Burkholderia cenocepacia DSM-16553 >59 >59 >59 >65 Chromobacterium violaceum DSM-30191 >59 >59 14.7 16.3 Escherichia coli DSM-1116 0.9 14.7 1.8 16.3 Escherichia coli DSM-12242 (NAL.sup.R) 0.9 29.4 3.7 8.1 Escherichia coli DSM-26863 (tolC3) 0.5 7.4 1.8 4.1 Escherichia coli ATCC35218 0.9 14.7 1.8 16.3 Escherichia coli ATCC25922 0.5 7.4 0.9 8.1 Enterobacter aerogenes DSM-30053 >59 >59 >59 >33 Klebsiella pneumoniae DSM-30104 >59 >59 >59 65 Pseudomonas aeruginosa PA14 >59 58.9 14.7 65 Pseudomonas aeruginosa ATCC27853 >59 58.9 14.7 65 Mycobacterium smegmatis mc.sup.2155 ATCC700084 >59 >59 >59 >65 Bacillus subtilis DSM-10 0.12 1.8 0.46 2.0 Enterococcus faecalis ATCC29212 0.06 3.7 0.23 4.1 Micrococcus luteus DSM-1790 0.06 7.4 0.23 4.1 Staphylococcus aureus ATCC29213 0.12 14.7 0.12 8.1 Streptococcus pneumoniae DSM-20566 0.23 14.7 0.46 8.1 Candida albicans DSM-1665 >59 >59 >59 >65 Pichia anomala DSM-6766 >59 >59 >59 >65 Test organism CysF CysG CIP Acinetobacter baumannii DSM-30008 — — 0.2 Burkholderia cenocepacia DSM-16553 — — 6.4 Chomobacterium violaceum DSM-30191 — — 0.006 Escherichia coli DSM-1116 0.4 0.9 0.006 Escherichia coli DSM-12242 (NAL.sup.R) — 0.05 Escherichia coli DSM-26863 (tolC3) 0.4 0.9 ≤0.003 Escherichia coli ATCC35218 — — 0.006 Escherichia coil ATCC25922 — — ≤0.003 Enterobacter aerogenes DSM-30053 — — 0.2 Klebsiella pneumoniae DSM-30104 — — 0.025 Pseudomonas aeruginosa PA14 3.4 7.1 0.1 Pseudomonas aeruginosa ATCC27853 — — 0.1 Mycobacterium smegmatis mc.sup.2155 ATCC700084 — — 0.4 Bacillus subtilis DSM-10 — — 0.1 Enterococcus faecalis ATCC29212 — — 0.8 Micrococcus luteus DSM-1790 — — 1.6 Staphylococcus aureus ATCC29213 — — 0.1 Streptococcus pneumoniae DSM-20566 — — 1.6 Candida albicans DSM-1665 — — >6.4 Pichia anomala DSM-6766 — — >6.4 CIP reference antibiotic ciprofloxacin — not determined

(21) TABLE-US-00013 TABLE 10b Cytotoxicity of cystobactamides (Cys). GI.sub.50 [μM] Cell lines and primary cells CysA CysA1 CysB CHO-K1 (Chinese hamster ovary) 37-111 >111 >111 HCT-116 (human colon carcinoma) — — >50  HUVEC (human umbilical vein endothelial — — >50  cells) GI.sub.50 [μM] Cell lines and primary cells CysC CysF CysG CHO-K1 (Chinese hamster ovary) ca. 111 >111 37-111 HCT-116 (human colon carcinoma) — — — HUVEC (human umbilical vein endothelial — — — cells) —not determined

(22) TABLE-US-00014 TABLE 11 Antimicrobial activity of cystobactamides (Cys) against E. coil mutant strains. CysA CysA1 CysB CysC Test organism [resistance mutations] MIC [μg/ml] Escherichia coli WT 0.5 14.7 1.8 8.1 Escherichia coil MI [gyrA(S83L)] 3.7 29.4 3.7 16.3 Escherichia coli WT-3.2 [gyrA(D87G)] 3.7 29.4 3.7 32.5 Escherichia coli WT-3 [gyrA(S83L, D87G)] 14.7 >59 7.4 >33 Escherichia coli WT-4 M2.1 [parC(S801)] 0.5 14.7 1.8 8.1 Escherichia coli MI-4 [gyrA(S83L), 0.5 14.7 1.8 16.3 parC(S801)] Escherichia coli WTIII [marRΔ74bp] 14.7 58.9 3.7 65 CysF CysG CIP Test organism [resistance mutations] MIC [μg/ml] Escherichia coli WT — — 0.013 Escherichia coli MI [gyrA(S83L)] — — 0.4 Escherichia coli WT-3.2 [gyrA(D87G)] — — 0.4 Escherichia coli WT-3 [gyrA(S83L, D87G)] — — 0.8 Escherichia coli WT-4 M2.1 [parC(S801)] — — 0.025 Escherichia coli M1-4 [gyrA(S83L), — — 0.4 parC(S801)] Escherichia coli WTIII[marRΔ74bp] 0.9 3.6 0.05 CIP reference antibiotic ciprofloxacin — not determined

(23) Experimental Procedures Cell-Based Assays

(24) Cell Lines and Primary Cells.

(25) Human HCT-116 colon carcinoma cells (CCL-247) were obtained from the American Type Culture Collection (ATCC) and Chinese hamster ovary CHO-K1 cells (ACC-110) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Both cell lines were cultured under the conditions recommended by the respective depositor. Primary HUVEC (human umbilical vein endothelial cells; single donor) were purchased from PromoCell (Heidelberg, Germany) and cultured in Endothelial Cell Growth Medium (PromoCell) containing the following supplements: 2% FCS, 0.4% ECGS, 0.1 ng/ml EGF, 1 ng/ml bFGF, 90 μg/ml heparin, 1 μg/ml hydrocortisone.

(26) Bacterial Strains.

(27) Bacterial wildtype strains used in susceptibility assays were either part of our strain collection or purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ) or from the American Type Culture Collection (ATCC). E. coli strain WT.sup.[6] and E. coli mutants were kindly provided by Prof. Dr. P. Heisig, Pharmaceutical Biology and Microbiology, University of Hamburg.

(28) Cytotoxicity Assay.

(29) Cells were seeded at 6×10.sup.3 cells per well of 96-well plates (Corning CellBind®) in complete medium (180 μl) and directly treated with cystobactamides dissolved in methanol in a serial dilution. Compound were tested in duplicate for 5 d, as well as the internal solvent control. After 5 d incubation, 5 mg/ml MTT in PBS (20 μL) was added per well and it was further incubated for 2 h at 37° C.[7] The medium was then discarded and cells were washed with PBS (100 μl) before adding 2-propanol/10N HCl (250:1, v/v; 100 μl) in order to dissolve formazan granules. The absorbance at 570 nm was measured using a microplate reader (EL808, Bio-Tek Instruments Inc.).

(30) Susceptibility Testing.

(31) MIC values were determined in microdilution assays. Overnight cultures were diluted in the appropriate growth medium to achieve an inoculum of 10.sup.4-10.sup.6 cfu/mL. Yeasts were grown in Myc medium (1% phytone peptone, 1% glucose, 50 mM HEPES, pH 7.0), S. pneumonia and E. faecalis in tryptic soy broth (TSB: 1.7% peptone casein, 0.3% peptone soymeal, 0.25% glucose, 0.5% NaCl, 0.25% K.sub.2HPO.sub.4; pH 7.3); M. smegmatis in Middlebrook 7H9 medium supplemented with 10% Middlebrook ADC enrichment and 2 ml/l glycerol). All other listed bacteria were grown in Müller-Hinton broth (0.2% beef infusion solids, 1.75% casein hydrolysate, 0.15% starch, pH 7.4). Cystobactamides and reference drugs were added directly to the cultures in sterile 96-well plates as duplicates and serial dilutions were prepared. Microorganisms were grown on a microplate shaker (750 rpm, 30-37° C., 18-48 h), except S. pneumonia, which was grown at non-shaking conditions (37° C., 5% CO.sub.2, 18 h). Growth inhibition was assessed by visual inspection and the MIC was defined as the lowest concentration of compound that inhibited visible growth.

(32) Target Identification

(33) To test the anti-gyrase activity of cystobactamides, commercial E. coli gyrase supercoiling kits (Inspiralis) were used. Cystobactamide A inhibited the E. coli gyrase (20.5 nM eq. 1 unit) showing an apparent IC.sub.50 of 6 μM. Cystobactamide A1 inhibited the E. coli gyrase (20.5 nM eq. 1 unit) showing an apparent IC.sub.50 of 2.5 μM. Cystobactamide D inhibited the E. coli gyrase (20.5 nM eq. 1 unit) showing an apparent IC.sub.50 of 1 μM. Cystobactamide C inhibited the E. coli gyrase (20.5 nM eq. 1 unit) showing an apparent IC.sub.50 of 7.7 μM. Cystobactamides thus are novel inhibitors of bacterial DNA gyrase.

(34) IC.sub.50 values of cystobactamide A-D in the Gyrase inhibition assay:

(35) TABLE-US-00015 Compound IC.sub.50/μM cystobactamide A  6 +/− 1.4 cystobactamide A1 2.5 +/− 0.8 cystobactamide C  7.2 +/− 0.74 cystobactamide D 0.7 +/− 0.4

(36) FIG. 9a show the results of the Gyrase inhibition assay. The gyrase reactions were titrated with varying concentrations of cystobactamide A, A1, C and D and resolved by agarose gel electrophoresis. For IC.sub.50 determination the band intensity of the supercoiled plasmid was determined using Adobe Photoshop, plotted vs. [cystobactamide] and fitted using Hill's equation.

(37) Prokaryotic DNA gyrase and topoisomerase IV share a high degree of homology and gyrase inhibitors typically show a topoisomerase IV inhibitory activity..sup.8 To test the influence of the cystobactamides on topoisomerase IV a commercial E. coli topoisomerase IV kit (Inspiralis) was used.

(38) Cystobactamide A inhibited the activity of E. coli topo IV only at the highest tested concentration of 815 μM. Cystobactamide A1 inhibited E. coli topo IV showing an IC.sub.50 value of 6.4+/−1.8 μM. Cystobactamide C inhibited the activity of E. coli topo IV only at the highest tested concentration of 300 μM. Cystobactamide D inhibited E. coli topo IV showing an IC.sub.50 value of 10+/−3 μM.

(39) IC.sub.50 values for cystobactamide A-D in the E. coli Topoisomerase IV inhibition assay:

(40) TABLE-US-00016 Compound IC.sub.50/μM cystobactamide A >160 cystobactamide A1 6.4 +/− 1.8 cystobactamide C >60 cystobactamide D 10 +/− 3

(41) FIG. 9b shows the result of the Topoisomerase IV inhibition assay. The topo IV reactions were titrated with varying concentrations of A-D and resolved by agarose gel electrophoresis. For IC.sub.50 determination the band intensity of the supercoiled plasmid was determined using Adobe Photoshop, plotted vs. [cystobactamide] and fitted using Hill's equation.

(42) Prokaryotic DNA topoisomerase IV and eukaryotic topoisomerase II share a high degree of homology (type IIa topoisomerases) and inhibitors of the prokaryotic enzyme often also inhibits the eukaryotic counterpart..sup.8 To test the influence of the cystobactamides on eukaryotic topoisomerase IV a commercial H. sapiens topoisomerase II kit (Inspiralis) was used.

(43) Cystobactamide A inhibited the activity of human topo II only at the highest tested concentration of 815 μM. Cystobactamide A1 inhibited human topo II showing an IC.sub.50 value of 9+/−0.03 μM. Cystobactamide C inhibited the activity of human topo II only at the highest tested concentration of 300 μM. Cystobactamide D inhibited human topo II showing an IC.sub.50 value of 41.2+/−3 μM

(44) IC.sub.50 values for cystobactamide A-D in the H. sapiens Topoisomerase II inhibition assay:

(45) TABLE-US-00017 Compound IC.sub.50/μM cystobactamide A >160 cystobactamide A1 9 +/− 0.03 cystobactamide C >60 cystobactamide D 41.2 +/− 3

(46) FIG. 9c shows the result of the Topoisomerase II inhibition assay. The topo II reactions were titrated with varying concentrations of A-D and resolved by agarose gel electrophoresis. For IC.sub.50 determination the band intensity of the supercoiled plasmid was determined using Adobe Photoshop, plotted vs. [cystobactamide] and fitted using Hill's equation.

(47) Aside the ATP-dependent type IIa topoisomerases like E. coli gyrase, topoIV and human topoII, the activity of cystobactamides on the ATP-independent human topoisomerase I was tested as well.

(48) IC.sub.50 values for cystobactamide A-D in the H. sapiens Topoisomerase I inhibition assay:

(49) TABLE-US-00018 Compound IC.sub.50/μM cystobactamide A ~1.0 cystobactamide A1 ~0.7 cystobactamide C ~6   cystobactamide D ~33.6 

(50) FIG. 9d shows the result of the Topoisomerase I inhibition assay. The topo I reactions were titrated with varying concentrations of A-D and resolved by agarose gel electrophoresis. For IC.sub.50 determination the band intensity of the supercoiled plasmid was determined using Adobe Photoshop, plotted vs. [cystobactamide] and fitted using Hill's equation.

(51) IC.sub.50(gyrase) vs. IC.sub.50 (topoisomerase IV) value comparison of cystobactamide A-D:

(52) TABLE-US-00019 IC.sub.50/μM ratios ratios gyrase Topo IV IC.sub.50(topo IV)/IC.sub.50(gyrase) cystobactamide A 6 ~815  ~136 cystobactamide A1 2.5 6.4 ~2.6 cystobactamide D 0.7 10 ~14   cystobactamide C 7.2 ~300 ~42  

(53) Cystobactamides A and C show a strong preference for gyrase as molecular target (40-100 fold stronger preference for gyrase). A1 and D both target gyrase and topoisomerase IV almost equally well (2.6-10 fold stronger preference for gyrase).

(54) Generally, there are two described inhibition modes/binding sites for gyrase inhibitors:

(55) 1. Compounds like the fluoroquinolones bind to the GyrA DNA complex and avoid the religation of the nicked dsDNA (gyrase poisoning); and

(56) 2. Aminocoumarins on the other hand bind to the ATP binding pocket on GyrB (competitive inhibition)..sup.8

(57) To test if cystobactamides follow any of those two inhibition modes, DNA/gyrase complex linearization assays (A) and ATP competition assays (B) were performed using cystobactamide D. (A) Here, the complex of DNA and gyrase is trapped using SDS and the gyrase is digested using proteinase K. If the gyrase/DNA complex is trapped by a gyrase inhibitor of type 1 this will lead to the formation of linearized plasmid (as the religation is inhibited). Type 2 inhibitor-bound or compound-free samples will not show the formation of linearized plasmids. The results of the assay are shown in FIG. 10a. Ciprofloxacin (a known gyrase/DNA stabilizer) and cystobactamide D show the formation of linearized plasmid after proteinase K treatment. This effect is not seen for the untreated control. Therefore, it appears likely that cystobactamides stabilize the covalent GyrA-DNA complex in a fashion comparable to the fluoroquionolones. (B) Here, standard gyrase reactions were inhibited using a constant amount of cystobactamide D and titrated with increasing amounts of ATP. If ATP and cystobactamide D would compete for binding at the ATP binding pocket on the gyrase GyrB subunit, increasing amounts of ATP would lead to the formation of supercoiled plasmid in the assay. FIG. 10b shows the assay results. Even at the highest ATP concentration of 10 mM (2000 fold cystobactamide concentration) the gyrase activity is not regained, indicating that the ATP binding pocket is not the binding site of the cystobactamides. This result is in line with the linearization assay results.

(58) FIG. 11 shows the results of the DNA/gyrase complex linearization assay.

(59) Experimental Procedures

(60) Gyrase Supercoiling Assay

(61) To test the anti-gyrase activity of cystobactamides, commercial E. coli gyrase supercoiling kits (Inspiralis, Norwich, UK) were used.3 For standard reactions 0.5 μg relaxed plasmid were mixed with 1 unit (˜20.5 nM) E. coli gyrase in 1× reaction buffer (30 μl final volume, see kit manual) and incubated for 30 minutes at 37° C. The reactions were quenched by the addition of DNA gel loading buffer containing 10% (w/v) SDS. The samples were separated on 0.8% (w/v) agarose gels and DNA was visualized using Roti-GelStain (Carl Roth).

(62) All natural products stock solutions and dilutions were prepared in 100% DMSO and added to the supercoiling reactions giving a final DMSO concentration of 5% (v/v). Ciprofloxacin stock solutions and Dilutions were prepared in 10 mM HCl and 50% DMSO and used 1:10 in the final assay.

(63) Following natural product concentrations were used in the assay:

(64) Cystobactamide A: 815.8 μM; 163 μM; 80 μM, 16 μM; 8 μM; 1.6 μM; 0.8 μM; 0.16 μM; 0.08 μM; 0.016 μM

(65) Cystobactamide A1: 543.5 μM; 108.7 μM; 54 μM; 10.8 μM; 5.4 μM; 1.087 μM; 0.54 μM; 0.108 μM; 0.054 μM; 0.0108 μM

(66) Cystobactamide C: 300 μM; 60 μM; 30 μM; 6 μM; 3 μM; 0.6 μM; 0.3 μM; 0.06 μM; 0.03 μM; 0.006 μM

(67) Cystobactamide D: 347 μM; 173.5 μM; 86.75 μM; 43.38 μM; 21.69 μM; 10.84 μM; 5.42 μM; 2.71 μM; 1.36 μM; 0.68 μM; 0.34 μM; 0.17 μM; 0.085 μM; 0.042 μM; 0.021 μM; 0.0106 μM; 0.0053 μM

(68) Control reactions were: no enzyme and a standard reaction in presence of 5% (v/v) DMSO.

(69) All reaction samples were equilibrated for 10 minutes at room-temperature in the absence of DNA. Then the relaxed plasmid was added to start the reaction.

(70) Proteinase K Linearization Assay

(71) To test if cystobactamides stabilize the covalent complex between DNA gyrase and the nicked DNA substrate, proteinase K linearization assay were performed (see a). Standard gyrase supercoiling assays were run in the presence of cystobactamide D (18 μM; 1.8 μM). Control reactions contained no gyrase, no inhibitor or the known gyrase/DNA complex stabilizer ciprofloxacin (1 μM). The reactions were quenched by the addition of 1/10 volume of 10% SDS. To linearize the nicked DNA-gyrase complexes, 50 μg/ml proteinase K were added to the reactions and incubated for 30 minutes at 37° C. The samples were separated on 0.8% (w/v) agarose gels and DNA was visualized using Roti-GelStain (Carl Roth). To detect linearized plasmid bands the relaxed plasmid was digested by the single-cutting restriction enzyme Ndel.

(72) Gyrase Supercoiling Assay with Varying ATP Concentrations

(73) To test if cystobactamides compete with ATP for binding to the ATP binding pocket on GyrB, standard gyrase supercoiling assays (see a) with varying ATP concentrations were performed. Standard reaction mixes (1 mM ATP) were supplemented with ATP (0.5M ATP stock solution, ATP was purchased from Sigma-Aldrich) to final ATP concentrations of 2.5; 5 and 10 mM. All reactions were performed in triplicates.

(74) Topoisomerase IV Relaxation Assay

(75) To test the anti-topoisomerase IV activity of cystobactamides, commercial E. coli topoisomerase IV relaxing kits (Inspiralis, Norwich, UK) were used.4 For standard reactions 0.5 μg supercoiled plasmid were mixed with 1 unit (˜20.5 nM) E. coli topoisomerase IV in 1× reaction buffer (see kit manual) and incubated for 30 minutes at 37° C. The reactions were quenched by the addition of DNA gel loading buffer containing 10% (w/v) SDS. The samples were separated on 0.8% (w/v) agarose gels and DNA was visualized using Roti-GelStain (Carl Roth).

(76) Following natural product concentrations were used in the assay:

(77) Cystobactamide A: 815.8 μM; 163 μM; 80 μM, 16 μM; 8 μM; 1.6 μM; 0.8 μM; 0.16 μM; 0.08 μM; 0.016 μM

(78) Cystobactamide A1: 543.5 μM; 108.7 μM; 54 μM; 10.8 μM; 5.4 μM; 1.087 μM; 0.54 μM; 0.108 μM; 0.054 μM; 0.0108 μM

(79) Cystobactamide C: 300 μM; 60 μM; 30 μM; 6 μM; 3 μM; 0.6 μM; 0.3 μM; 0.06 μM; 0.03 μM; 0.006 μM

(80) Cystobactamide D: 347 μM; 173.5 μM; 86.75 μM; 43.38 μM; 21.69 μM; 10.84 μM; 5.42 μM; 2.71 μM; 1.36 μM; 0.68 μM; 0.34 μM; 0.17 μM; 0.085 μM; 0.042 μM; 0.021 μM; 0.0106 μM; 0.0053 μM

(81) Control reactions were: no enzyme and a standard reaction in presence of 5% (v/v) DMSO. All reaction samples were equilibrated for 10 minutes at room-temperature in the absence of DNA. Then the relaxed plasmid was added to start the reaction.

(82) Topoisomerase II Relaxation Assay

(83) To test the anti-topoisomerase II activity of cystobactamides, commercial human topoisomerase IV relaxing kits (Inspiralis, Norwich, UK) were used.4 For standard reactions 0.5 μg supercoiled plasmid were mixed with 1 unit (˜20.5 nM) E. coli topoisomerase II in 1× reaction buffer (see kit manual) and incubated for 30 minutes at 37° C. The reactions were quenched by the addition of DNA gel loading buffer containing 10% (w/v) SDS. The samples were separated on 0.8% (w/v) agarose gels and DNA was visualized using Roti-GelStain (Carl Roth).

(84) Following natural product concentrations were used in the assay:

(85) Cystobactamide A: 815.8 μM; 163 μM; 80 μM, 16 μM; 8 μM; 1.6 μM; 0.8 μM; 0.16 μM; 0.08 μM; 0.016 μM

(86) Cystobactamide A1: 543.5 μM; 108.7 μM; 54 μM; 10.8 μM; 5.4 μM; 1.087 μM; 0.54 μM; 0.108 μM; 0.054 μM; 0.0108 μM

(87) Cystobactamide C: 300 μM; 60 μM; 30 μM; 6 μM; 3 μM; 0.6 μM; 0.3 μM; 0.06 μM; 0.03 μM; 0.006 μM

(88) Cystobactamide D: 347 μM; 173.5 μM; 86.75 μM; 43.38 μM; 21.69 μM; 10.84 μM; 5.42 μM; 2.71 μM; 1.36 μM; 0.68 μM; 0.34 μM; 0.17 μM; 0.085 μM; 0.042 μM; 0.021 μM; 0.0106 μM; 0.0053 μM

(89) Control reactions were: no enzyme and a standard reaction in presence of 5% (v/v) DMSO. All reaction samples were equilibrated for 10 minutes at room-temperature in the absence of DNA. Then the relaxed plasmid was added to start the reaction.

(90) Topoisomerase I Relaxation Assay

(91) To test the anti-topoisomerase II activity of cystobactamides, commercial H. sapiens topoisomerase I relaxing kits (Inspiralis, Norwich, UK) were used.4 For standard reactions 0.5 μg supercoiled plasmid were mixed with 1 unit (˜20.5 nM) H. sapiens topoisomerase I in 1× reaction buffer (see kit manual) and incubated for 30 minutes at 37° C. The reactions were quenched by the addition of DNA gel loading buffer containing 10% (w/v) SDS. The samples were separated on 0.8% (w/v) agarose gels and DNA was visualized using Roti-GelStain (Carl Roth).

(92) Following natural product concentrations were used in the assay:

(93) Cystobactamide A: 815 μM; 81.5 μM; 8.15 μM

(94) Cystobactamide A1: 543 μM; 54.3 μM; 5.43 μM

(95) Cystobactamide C: 300 μM; 30 μM; 3 μM

(96) Cystobactamide D: 277 μM; 27.2 μM; 2.77 μM

(97) Control reactions were: no enzyme and a standard reaction in presence of 5% (v/v) DMSO. All reaction samples were equilibrated for 10 minutes at room-temperature in the absence of DNA. Then the relaxed plasmid was added to start the reaction

(98) Quantification and Analysis

(99) To determine IC50 values, the formation of supercoiled (gyrase) or relaxed (topoisomerase I, II IV) plasmid was quantified using Adobe Photoshop (Histogram mode). Plotting of these values versus the compound concentration yielded sigmoidal shaped curves, which were fitted using Hill's equation (Origin Pro 8.5). All determined IC50 values are the averages of three independent experiments.

REFERENCES

(100) [1] T. Gruger, J. L. Nitiss, A. Maxwell, E. L. Zechiedrich, P. Heisig, S. Seeber, Y. Pommier, D. Strumberg, Antimicrob. Agents Chemother. 48, 2004, 4495-4504. [2] H. Schedletzky, B. Wiedemann, P. Heisig, J. Antimicrob. Chemother. 43, 1999, 31-37. [3] A. B. Khodursky, E. L. Zechiedrich, N. R. Cozzarelli, Proc. Natl. Acad. Sci. USA 92, 1995, 11801-11805. [4] A. Schulte, P. Heisig, J. Antimicrob. Chemother. 46, 2000, 1037-1046. [5] D. Keeney, A. Ruzin, F. McAleese, E. Murphy, P. A. Bradford, J. Antimicrob. Chemother. 61, 2008, 46-53. [6] P. Heisig, H. Schedletzky, H. Falkenstein-Paul, Antimicrob. Agents Chemother. 37, 1993, 669-701. [7] T. Mosmann, J. Immunol. Meth. 65, 1983, 55-63. [8] Pommier, Y.; Leo, E.; Zhang, H.; Marchand, C. Chemistry & Biology 2010, 17, 421.

Synthesis of Cystobactamide A and C

(101) First, the synthesis of cystobactamide C is described which can further be elaborated to the other cystobactamides.

(102) 1.1. Cystobactamide C

(103) The following Schemes 1 and 2 provide an overview on the synthesis of individual aromatic building blocks followed by assembling these to generate cystobactamide C.

(104) Alternatively, step e) in Scheme 1 can be modified by using another alcohol (R′OH) instead of .sup.iPrOH. If for example EtOH is used, building blocks of cystobactamide H can be prepared. The same applies for step b) in the second reaction sequence given in Scheme 1. Here, also .sup.iPrOH can be exchanged by any other alcohol (R′OH). If for example MeOH is used, building blocks of cystobactamides C, G and H can be prepared. For the preparation of cystobactamide F, p-amino-benzoic acid derivatives such as p-aminobenzoic acid or corresponding N-protected aminobenzoic acid derivatives and p-nitrobenzoic acids are employed instead of building block B.

(105) ##STR00029##

(106) ##STR00030##

(107) 1.2 Cystobactamide A

(108) The more complex cystobactamides consist of the bisamide that represents cystobactamide C, a bisarylamide (fragment C) and a chiral linker element. In this section fragment C and the chiral linker element are reported first which is followed by the assembling of all three elements to provide cystobactamide A.

(109) 1.2.1 Synthesis of Bisarene C.

(110) ##STR00031##

1.2.2 Synthesis of the Chiral Building Block D with Bisarene C Attached

(111) The synthesis starts from methyl cinnamate and chirality is introduced by the Sharpless asymmetric dihydroxylation. The phenyl ring serves as protecting group for the second carboxylate which is oxidatively liberated. Finally, building block C is attached to the free amino group. The corresponding enantiomeric fragment (ent)-D was prepared using AD mix α instead of AD mix β.

(112) ##STR00032##

(113) ##STR00033##

(114) 2. Experimentals

(115) 2.1 General Experimental Information

(116) All reactions were performed in oven dried glassware under an atmosphere of nitrogen gas unless otherwise stated. .sup.1H-NMR spectra were recorded at 400 MHz with a Bruker AVS-400 or at 500 MHz with a Bruker DRX-500. .sup.13C-NMR spectra were recorded at 100 MHz with a Bruker AVS-400 and at 125 MHz with a Bruker DRX-500. Multiplicities are described using the following abbreviations: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, b=broad. Chemical shift values of .sup.1H and .sup.13C NMR spectra are commonly reported as values in ppm relative to residual solvent signal as internal standard. The multiplicities refer to the resonances in the off-resonance decoupled spectra. These were elucidated using the distortionless enhancement by polarization transfer (DEPT) spectral editing technique, with secondary pulses at 90° and 135°. Multiplicities are reported using the following abbreviations: s=singlet (due to quaternary carbon), d=doublet (methine), t=triplet (methylene), q=quartet (methyl). Mass spectra (EI) were obtained at 70 eV with a type VG Autospec spectrometer (Micromass), with a type LCT (ESI) (Micromass) or with a type Q-TOF (Micromass) spectrometer in combination with a Waters Aquity Ultraperformance LC system. Analytical thin-layer chromatography was performed using precoated silica gel 60 F.sub.254 plates (Merck, Darmstadt), and the spots were visualized with UV light at 254 nm or alternatively by staining with potassium permanganate, phosphomolybdic acid, 2,4-dinitrophenol or p-anisaldehyde solutions. Tetrahydrofuran (THF) was distilled under nitrogen from sodium/benzophenone. Dichloromethane (CH.sub.2Cl.sub.2) was dried using a Solvent Purification System (SPS). Commercially available reagents were used as supplied. Preparative high performance liquid chromatography using a Merck Hitachi LaChrom system (pump L-7150, interface D-7000, diode array detector L-7450 (λ=220-400 nm, preferred monitoring at λ=230 nm)) with column (abbreviation referred to in the experimental part given in parentheses): Trentec Reprosil-Pur 120 C18 AQ 5 μm, 250×8 mm, with guard column, 40×8 mm (C18-SP). Flash column chromatography was performed on Merck silica gel 60 (230-400 mesh). Eluents used for flash chromatography were distilled prior to use. Melting points were measured using a SRS OptiMelt apparatus. Optical rotations [α] were measured on a Polarimeter 341 (Perkin Elmer) at a wavelength of 589 nm and are given in 10.sup.−1 deg cm.sup.2 g.sup.−1.

(117) 2.2 Specific Procedures

4-Aminomethylbenzoate

(118) ##STR00034##

(119) MeOH (200 mL) was provided in a flask and acetyl choride (2.6 mL, 36.5 mmol, 1 eq) was slowly added. Then 4-aminobenzoic acid (5.00 g, 36.5 mmol) was added and the solution was stirred 7 days at room temperature. The solvent was removed under reduced pressure and 4-aminomethylbenzoate (5.38 g, 35.59 mmol, quantitative) was obtained as a beige solid.

(120) The titled compound decomposes before reaching its melting point.

(121) ATR-IR (neat): ũ=2828, 2015, 1724, 1612, 1558, 1508, 1430, 1316, 1280, 1181, 1109, 1072, 1022, 984, 959, 853, 786, 757, 686, 653 cm.sup.−1.

(122) .sup.1H-NMR (400 MHz, CD.sub.3OD): δ 8.19-8.13 (m, 2H), 7.53-7.48 (m, 2H), 3.93 (s, 3H) ppm.

(123) .sup.13C-NMR (100 MHz, CD.sub.3OD): δ 167.2137.0, 132.4, 131.7, 124.2, 53.0 ppm HRMS (ESI): Calculated for C.sub.8H.sub.10NO.sub.2 (M+H).sup.+: 152.0712, found: 152.0706.

4-(4-Nitrobenzamido)methyl benzoate

(124) ##STR00035##

(125) A solution of P(OMe).sub.3 (3.5 mL, 29.8 mmol) in CH.sub.2Cl.sub.2 (100 mL) was cooled with an ice bath, then I.sub.2 (7.56 g, 29.8 mmol) was added. After the solid iodine was completely dissolved, p-nitrobenzoic acid (5.52 g, 29.8 mmol) and Et.sub.3N (4.70 mL, 33.7 mmol) were added in sequential order, and the solution was stirred for 10 minutes in a cooling bath. 4-aminomethylbenzoate (3.00 gr, 19.9 mmol) was added and the mixture was stirred for 10 minutes. After removing the cooling bath, the reaction mixture was stirred for 3 days at room temperature, then diluted with saturated aqueous NaHCO.sub.3 and extracted with dichloromethane (3×). The combined, organic layer was sequentially washed with H.sub.2O, 1 M HCl, H.sub.2O, and brine. The combined organic layers were dried with anhydrous MgSO.sub.4 and the solvent concentrated in vacuo, yielding the title compound (4.4 g, 14.65 mmol, 75%) as a beige solid. mp: 245-246° C.

(126) .sup.1H NMR (400 MHz, DMSO) δ 10.87 (s, 1H.sub.NH), 8.39 (d, J=8.8 Hz, 2H), 8.20 (d, J=8.8 Hz, 2H), 7.99 (d, J=8.8 Hz, 2H), 7.95 (d, J=8.8 Hz, 2H), 3.84 (s, 3H.sub.OMe) ppm.

(127) .sup.13C NMR (100 MHz, DMSO) δ 166.2, 164.9, 149.77, 143.6, 140.7, 130.7, 129.8, 125.3, 124.2, 120.2, 52.4 ppm.

(128) HRMS (ESI): Calculated for C.sub.15H.sub.13N.sub.2O.sub.2Na (M+H).sup.+: 301.0824, found: 301.0828.

4-(4-Nitrobenzamido) benzoate

(129) ##STR00036##

(130) 4-(4-Nitrobenzamido)methyl benzoate (4.32 g, 14.38 mmol) was dissolved in a mixture 1/1 of THF/H.sub.2O (77/77 mL). Then, solid LiOH (5.16 g, 215.66 mmol) was added and the system was stirred at room temperature for 17 hours. 1M HCl was added until pH-1 and the resulting solid was filtered and dried in vacuo. The title compound (3.3 g, 11.54 mmol, 80%) was obtained as a pale yellow solid. mp: 322-324° C.

(131) .sup.1H NMR (400 MHz, C.sub.6D.sub.6) δ 10.83 (s, 1H.sub.CO2H), 8.34 (d, J=8.6 Hz, 1H), 8.29 (d, J=8.6 Hz, 1H), 8.13 (d, J=8.6 Hz, 1H), 8.06 (d, J=8.6 Hz, 1H), 7.75 (s, 1.sub.NH) ppm.

(132) .sup.13C NMR (100 MHz, C.sub.6D.sub.6) δ 168.2, 164.6, 162.2, 149.7, 143.9, 141.1, 131.1, 129.8, 123.5, 120.4 ppm.

(133) HRMS (ESI): Calculated for C.sub.14H.sub.9N.sub.2O.sub.5(M−H).sup.−: 285.0511, found: 285.0506.

(Ethyl carbonic) 4-(4-nitrobenzamido)benzoic anhydride

(134) ##STR00037##

(135) To a stirred solution of 4-aminobenzoic acid (1.5 g, 10.9 mmol) and N, N-dimethylaniline (2.0 g, 10.9 mmol) in acetone was added 4-nitrobenzoyl chloride at 0° C. Then, the reaction mixture was allowed to warm to room temperature and stirred for another hour. The resulting solid was filtered and purified by recrystallization in DMF to afford 4-(4-nitro-benzoylamino)-benzoic acid (2.75 g, 88%). 4-(4-Nitro-benzoylamino)-benzoic acid (0.6 g, 2.1 mmol) was dissolved in 14 ml CH.sub.3CN. Then Et.sub.3N (0.31 ml, 2.2 mmol) was added at 0° C. To this resulting solution ethyl chloroformate was added. After stirring for 30 min at 0° C., the white precipitate was filtered and washed with cold CH.sub.3CN, then dried under high vacuum at room temperature to afford the title anhydride 0.5 g, 67%.

(136) .sup.1H-NMR (400 MHz, DMSO, DMSO=2.50 ppm): δ=1.33 (dd, J=7.2 Hz, 3H), 4.37 (q, J=7.2 Hz, 2H), 8.02-8.09 (m, 4H), 8.21 (d, J=8.8 Hz, 2H), 8.40 (d, J=8.8 Hz, 2H), 11.01 (s, 1H).

3-Hydroxy-4-nitromethylbenzoate

(137) ##STR00038##

(138) TMSCHN.sub.2 (2.0 M in Et.sub.2O, 13.20 mL, 26.48 mmol) was added to a solution of 3-hydroxy-2-nitrobenzoic acid (2.50 g, 13.65 mmol) in a mixture of toluene/methanol (81/36 mL) at 0° C. After stirring at 0° C. for 30 minutes, the solvent was evaporated in vacuo to give an oily residue, which was purified by flash chromatography (petroleum ether/ethyl acetate=9:1) to yield the title compound (2.43 g, 12.33 mmol, 90%) as a yellow solid.

(139) mp: 91-92° C.

(140) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 10.49 (s, 1H.sub.-OH), 8.17 (d, J=8.8 Hz, 1H), 7.83 (d, J=1.8 Hz, 1H), 7.61 (dd, J=8.8, 1.8 Hz, 1H), 3.96 (s, 3H) ppm.

(141) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 165.0, 154.8, 138.1, 125.4, 121.8, 120.74, 53.1 ppm. HRMS (ESI): Calculated for C.sub.8H.sub.6NO.sub.5 (M−H).sup.−: 196.0246, found: 196.0249.

3-Isopropoxy-4-nitromethylbenzoate

(142) ##STR00039##

(143) 3-Hydroxy-4-nitromethylbenzoate (2.30 g, 10.89 mmol) was dissolved in THF (100 mL). .sup.iPrOH (1.10 mL, 14.16 mmol) and PPh.sub.3 (3.90 g, 14.70 mmol) were added, and the mixture was stirred until all components were dissolved. DEAD (2.2 M in toluene, 14.16 mmol, 6.50 mL) was added and the mixture was stirred at room temperature 17 hours. The solvent was evaporated in vacuo to give an oily residue, which was purified by flash chromatography (petroleum ether/ethyl acetate=95:5) to yield the title compound (2.61 g, 10.91 mmol, quantitative) as a yellow oil.

(144) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.75 (d, J=8.4 Hz, 2H), 7.64 (dd, J=8.3, 1.6 Hz, 1H), 4.77 (hept, J=6.1 Hz, 1H), 3.95 (s, 3H), 1.41 (s, 3H), 1.40 (s, 3H) ppm.

(145) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 165.5, 150.9, 134.6, 125.2, 121.2, 117.1, 73.2, 52.9, 21.9 ppm.

(146) HRMS (Qtof): Calculated for C.sub.8H.sub.6NO.sub.5 (M+Na).sup.+: 262.0691, found: 262.0700.

3-Isopropoxy-4-aminomethylbenzoate

(147) ##STR00040##

(148) 3-Isopropoxy-4-nitromethylbenzoate (2.60 g, 10.87 mmol) was dissolved in MeOH (91.0 mL) and degassed. Pd/C (10% wt, 0.58 g, 0.54 mmol) was added and vacuum was applied under cooling to remove air. The flask was flushed with H.sub.2 and the suspension was stirred for 17 hours at room temperature. The catalyst was filtered over Celite®, washed with MeOH and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (petroleum ether/EtOAc=7/3). 3-Isopropoxy-4-aminomethylbenzoate was obtained (2.27 g, 10.85 mmol, quantitative) as a light orange solid. mp: 55-57° C.

(149) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.51 (dd, J=8.2, 1.7 Hz, 1H), 7.46 (d, J=1.7 Hz, 1H), 6.66 (dd, J=8.2, 5.1 Hz, 1H), 4.63 (sept, J=5.1 Hz, 1H), 3.85 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H) ppm.

(150) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 167.5, 144.24, 142.3, 124.0, 119.5, 114.1, 113.5, 70.9, 51.8, 22.3 ppm.

(151) HRMS (ESI): Calculated for C.sub.11H.sub.16NO.sub.3 (M+H).sup.+: 210.1130, found: 210.1126.

6-Bromo-2,3-dihydroxybenzaldehyde

(152) ##STR00041##

(153) To a solution of 6-bromo-2-hydroxy-3-methoxybenzaldehyde (25.0 g, 108.2 mmol) in CH.sub.2Cl.sub.2 (270 mL) at −30° C. was slowly added BBr.sub.3 (1 M in CH.sub.2Cl.sub.2, 200.0 mL, 200.0 mmol) via additional funnel over a period of 45 minutes. The solution was allowed to warm to room temperature and stirred 17 hours. H.sub.2O was added and the reaction mixture was stirred for additional 30 minutes. The solution was then extracted with EtOAc (3×) and washed with H.sub.2O. The combined, organic layers were dried over anhydrous MgSO.sub.4, filtered and concentrated in vacuo to give the title compound (22.16 g, 102.11 mmol, 95%) as a yellow solid.

(154) mp: 135-136° C.

(155) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 12.13 (d, J=0.5 Hz, 1H.sub.-OH), 10.27 (s, 1H.sub.-CHO), 7.07 (d, J=8.5 Hz, 1H), 7.02 (dd, J=8.5, 0.5 Hz, 1H), 5.67 (s, 1H.sub.-OH) ppm.

(156) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 198.4, 151.2, 145.0, 124.4, 122.0, 117.5, 116.1 ppm. HRMS (ESI): Calculated for C.sub.7H.sub.4BrO.sub.3 (M−H).sup.−: 214.3943, found: 214.9344.

4-Bromo-3-hydroxymethylbenzene-1,2-diol

(157) ##STR00042##

(158) A solution of 6-bromo-2,3-dihydroxybenzaldehyde (22.16 g, 102.10 mmol) in THF (650 mL) at −40° C. was treated with NaBH.sub.4 (3.86 g, 102.10 mmol) portion wise (3×). The resulting mixture was stirred for 30 minutes at room temperature. A saturated aqueous solution of NH.sub.4Cl was added and the mixture was stirred for another 10 minutes, before being finally treated with 1M HCl. After 10 minutes of additional stirring, the aqueous phase was extracted with EtOAc (3×). The combined, organic extracts were dried over anhydrous MgSO.sub.4 and filtered. The solvent was removed under reduced pressure to yield the title compound (20.27 g, 92.53 mmol, 91%) as a colorless solid.

(159) mp: 90-92° C.

(160) .sup.1H NMR (400 MHz, MeOD) δ 6.88 (d, J=8.5 Hz, 1H), 6.64 (d, J=8.5 Hz, 1H), 4.82 (s, 2H) ppm.

(161) .sup.13C NMR (100 MHz, MeOD) δ 147.1, 146.1, 126.9, 123.9, 116.6, 114.4, 61.1 ppm. HRMS (ESI): Calculated for C.sub.7H.sub.6BrO.sub.3 (M−H).sup.−: 216.9500, found: 216.9505.

5-Bromo-2-phenyl-4H-benzo-[1,3]-dioxin-8-ol

(162) ##STR00043##

(163) A solution of 4-bromo-3-hydroxymethylbenzene-1,2-diol (20.27 g, 92.53 mmol) in THF (550 mL) was treated with PhCH(OMe).sub.2 (20.8 mL, 138.8 mmol) and pTSA.H.sub.2O (0.19 g, 1.02 mmol). The mixture was stirred at room temperature for 5 days. CH.sub.2Cl.sub.2 was added and then washed successively with 5% aqueous NaHCO.sub.3 and brine. The aqueous phase was extracted with EtOAc (3×). The combined, organic extracts were dried over anhydrous MgSO.sub.4, filtered and the solvent was removed under reduced pressure. Purification by flash chromatography (petroleum ether/EtOAc=95/5) afforded 5-bromo-2-phenyl-4H-benzo-[1,3]-dioxin-8-ol (16.02 g, 52.16 mmol, 56%) as a colorless solid.

(164) mp: 89-91° C.

(165) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.62-7.55 (m, 2H), 7.50-7.43 (m, 3H), 7.07 (d, J=8.6 Hz, 1H), 6.78 (d, J=8.6 Hz, 1H), 5.97 (s, 1H), 5.40 (s, 1H.sub.-OH), 4.99 (s, 2H) ppm.

(166) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 144.0, 141.8, 136.1, 130.1, 128.8, 126.7, 124.9, 121.0, 115.0, 109.4, 100.0, 67.8 ppm.

(167) HRMS (ESI): Calculated for C.sub.14H.sub.10BrO.sub.3 (M−H).sup.−: 304.9813, found: 304.9813.

5-Bromo-7-nitro-2-phenyl-4H-benzo-[1,3]-dioxin-8-ol

(168) ##STR00044##

(169) 5-Bromo-2-phenyl-4H-benzo-[1,3]-dioxin-8-ol (6.00 g, 19.54 mmol; max. amount) was dissolved in acetone (250 mL). Then, Ni(NO.sub.3).sub.2.5H.sub.2O (5.68 g, 19.54 mmol) and pTSA.H.sub.2O (3.72 g, 19.54 mmol) were added. The mixture was stirred at room temperature for 2.5 h. The reaction mixture was filtered over Celite®, washed with CH.sub.2Cl.sub.2 and concentrated in vacuo. Purification by flash chromatography (dry load: SiO.sub.2+CH.sub.2Cl.sub.2; petroleum ether/ethyl acetate=9:1) yielded the titel compound (5.08 g, 14.43 mmol, 74%) as a bright yellow solid.

(170) mp: 154-156° C.

(171) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 10.60 (s, 1H.sub.-OH), 7.96 (s, 1H), 7.65-7.57 (m, 2H), 7.48-7.42 (m, 3H), 6.02 (s, 1H), 4.99 (s, 2H) ppm.

(172) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 144.9, 135.5, 133.2, 130.2, 129.0, 128.9, 126.7, 119.2, 109.2, 99.9, 67.4 ppm.

(173) HRMS (ESI): Calculated for C.sub.14H.sub.9BrNO.sub.5 (M−H).sup.−: 359.9664, found: 349.9660.

5-Bromo-8-isopropoxy-7-nitro-2-phenyl-4H-benzo-[1,3]-dioxine

(174) ##STR00045##

(175) 5-Bromo-7-nitro-2-phenyl-4H-benzo-[1,3]-dioxin-8-ol (13.79 g, 39.16 mmol) was dissolved in THF (429 mL). iPrOH (4.00 mL, 50.91 mmol) and PPh.sub.3 (13.87 g, 52.87 mmol) were added, and the mixture was stirred until all components were dissolved. DEAD (2.2 M in toluene, 23.1 mL, 50.91 mmol) was slowly added (via syringe pump) and the mixture was stirred at room temperature 17 hours. The solvent was evaporated in vacuo to give an oily residue, which was purified by flash chromatography (petroleum ether/ethyl acetate=96:4) to yield the title compound (13.08 g, 33.18 mmol, 85%) as a colorless solid.

(176) mp: 87-89° C.

(177) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.59 (s, 1H), 7.59-7.54 (m, 2H), 7.50-7.43 (m, 3H), 5.97 (s, 1H), 5.00 (s, 2H), 4.69 (hept, J=6.2 Hz, 1H), 1.31 (d, J=6.2 Hz, 3H), 1.28 (d, J=6.2 Hz, 3H) ppm.

(178) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 216.8, 149.0, 144.5, 139.9, 135.7, 130.1, 128.8, 126.4, 126.2, 119.8, 112.7, 99.7, 78.1, 67.6, 22.6, 22.4 ppm.

(179) HRMS (Qtof): Calculated for C.sub.14H.sub.9BrNO.sub.5 (M+Na).sup.+: 416.0110, found: 416.0101.

8-Isopropoxy-7-nitro-2-phenyl-4H-benzo-[1,3]-dioxine, 73

(180) ##STR00046##

(181) 5-Bromo-8-isopropoxy-7-nitro-2-phenyl-4H-benzo-[1,3]-dioxine 72 (4.00 g, 10.15 mmol), Pd.sub.2(dba).sub.3 (0.93 g, 1.01 mmol), (PhO).sub.3P (0.53 mL, 2.03 mmol), Cs.sub.2CO.sub.3 (4.30 g, 13.19 mmol) and .sup.iPrOH (4.7 mL, 60.88 mmol) were dissolved in 1,4-dioxane (28 mL). The oil bath was preheated to 60° C. and the mixture was stirred at 80° C. for 1.5 hours. The reaction mixture was filtered through Celite® and washed with EtOAc. The combined, organic extracts were dried over anhydrous MgSO.sub.4 and concentrated in vacuo. The crude material was purified by flash chromatography (petroleum ether/ethyl acetate=96:4) to yield the title compound (2.24 g, 7.10 mmol, 70%) as a pale yellow solid.

(182) mp: 80-82° C.

(183) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.65-7.55 (m, 2H), 7.51-7.41 (m, 3H), 7.37 (d, J=8.5 Hz, 1H), 6.81 (d, J=8.5 Hz, 1H), 6.01 (s, 1H), 5.19 (d, J=15.5 Hz, 1H), 5.03 (d, J=15.5 Hz, 1H), 4.71 (hept, J=6.2 Hz, 1H), 1.32 (d, J=6.2 Hz, 3H), 1.28 (d, J=6.2 Hz, 3H) ppm.

(184) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 147.67, 144.27, 140.55, 136.26, 129.85, 128.72, 126.54, 126.34, 118.82, 116.69, 99.61, 77.71, 66.44, 22.65, 22.41 ppm.

(185) HRMS (QTof): Calculated for C.sub.17H.sub.17NO.sub.5Na (M+Na).sup.+: 338.1004. Found: 338.1003.

6-Hydroxymethyl-2-isopropoxy-3-nitrophenol

(186) ##STR00047##

(187) To a mixture of 8-isopropoxy-7-nitro-2-phenyl-4H-benzo-[1,3]-dioxine (4.24 g, 13.43 mmol) in MeOH (102 mL) and CH.sub.2Cl.sub.2 (42 mL) at 0° C. was added camphor sufonic acid (3.12 g, 13.43 mmol). The mixture was stirred at room temperature for 17 hours. The reaction mixture was quenched with Et.sub.3N until pH-8, concentrated in vacuo and purified by flash chromatography (petroleum ether/ethyl acetate=7:3) to yield the title compound (2.75 g, 12.09 mmol, 90%) as a brownish solid. mp: 39-41° C.

(188) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.46 (d, J=7.4 Hz, 1H), 7.12 (d, J=7.4 Hz, 1H), 6.61 (s, 1H.sub.-OH), 4.81 (d, J=3.5 Hz, 2H), 4.39 (hept, J=7.4 Hz, 1H), 1.36 (s, 3H), 1.35 (s, 3H) ppm.

(189) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 148.9, 138.5, 132.4, 122.1, 116.5, 79.2, 61.3, 22.5 ppm.

(190) HRMS (ESI): Calculated for C.sub.10H.sub.12NO.sub.5(M−H).sup.−: 226.0715, found: 226.0717.

2-Hydroxy-3-isopropoxy-4-nitrobenzaldehyde

(191) ##STR00048##

(192) 6-Hydroxymethyl-2-isopropoxy-3-nitrophenol (2.97 g, 13.05 mmol) was dissolved in CH.sub.2Cl.sub.2 (58 mL). Then MnO.sub.2 (11.35 g, 130.53 mmol) was added and the mixture was stirred at rt 17 h. The mixture was filtered over Celite® and washed with CH.sub.2Cl.sub.2. The solvent was concentrated to give the title compound (2.38 g, 10.57 mmol, 81%) as a brown oil.

(193) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 11.44 (s, 1H.sub.-CHO), 9.97 (s, 1H.sub.-OH), 7.39 (d, J=8.4 Hz, 1H), 7.23 (d, J=8.4 Hz, 1H), 4.88 (hept, J=6.2 Hz, 1H), 1.33 (s, 3H), 1.32 (s, 3H) ppm.

(194) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 196.39, 156.53, 149.36, 139.74, 127.28, 122.57, 114.32, 77.42, 77.16, 22.51. ppm.

(195) HRMS (ESI): Calculated for C.sub.10H.sub.10NO.sub.5 (M−H).sup.−: 224.0559. Found: 224.0535.

2-Hydroxy-3-isopropoxy-4-nitrobenzoic acid

(196) ##STR00049##

(197) 2-Hydroxy-3-isopropoxy-4-nitrobenzaldehyde (2.36 g, 10.49 mmol) was dissolved in tert-buthanol (71 mL). 2-Methyl-2-butene (2M in THF, 36.7 mL, 73.45 mmol) and a solution of NaClO.sub.2 (2.85 g, 31.48 mmol) and NaH.sub.2PO.sub.4 (6.32 g, 47.22 mmol) in H.sub.2O (51 mL) were added in sequential order. The reaction mixture was stirred at room temperature for 17 hours. 6M NaOH was added until ph˜10 and the solvent was concentrated in vacuo. H.sub.2O was added and the organic layer was extracted with petroleum ether (2×). The aqueous layer was acidified with 6M HCl until pH-1 and extracted with ethyl acetate (3×). The organic extracts were combined, dried over MgSO.sub.4 and filtered. The solvent was concentrated in vacuo to yield the title compound (1.90 g, 7.87 mmol, 75%) as a dark wax.

(198) .sup.1H NMR (400 MHz, MeOD) δ 7.72 (d, J=8.7 Hz, 1H), 7.15 (d, J=8.7 Hz, 1H), 4.86-4.82 (m, 1H), 1.28 (s, 3H), 1.26 (s, 3H) ppm.

(199) .sup.13C NMR (100 MHz, MeOD) δ 172.7, 158.0, 140.0, 125.8, 117.4, 113.8, 77.5, 22.6 ppm.

(200) HRMS (ESI): Calculated for C.sub.10H.sub.10NO.sub.6 (M−H).sup.−: 240.0508, found: 240.0510.

2-Hydroxy-3-isopropoxy-4-nitro benzoate

(201) ##STR00050##

(202) TMSCHN.sub.2 (2.0 M in Et.sub.2O, 0.87 mL, 1.75 mmol) was added to a solution of 2-hydroxy-3-isopropoxy-4-nitrobenzoic acid (0.32 g, 1.35 mmol) in a mixture of toluene/methanol (10.4/2 mL) at 0° C. After stirring at 0° C. for 30 minutes, the solvent was evaporated in vacuo to give an oily residue, which was purified by flash chromatography (SiO.sub.2.Et.sub.3N; petroleum ether/ethyl acetate=95:5) to yield the title compound (0.24 g, 0.94 mmol, 57%) as a yellow oil.

(203) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 11.29 (s, 1H.sub.-OH), 7.63 (d, J=8.8 Hz, 1H), 7.12 (d, J=8.8 Hz, 1H), 4.84 (hept, J=6.2 Hz, 1H), 4.00 (s, 3H), 1.32 (s, 3H), 1.31 (s, 3H) ppm.

(204) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 198.2, 188.9, 176.1, 170.0, 157.0, 149.2, 139.8, 123.9, 115.7, 113.4, 77.4, 53.2, 22.5 ppm.

(205) HRMS (ESI): Calculated for C.sub.11H.sub.12NO.sub.6 (M−H).sup.−: 254.0665, found: 254.0666.

2-Benzyloxy-3-isopropoxy-4-nitrobenzoate

(206) ##STR00051##

(207) 2-Hydroxy-3-isopropoxy-4-nitrobenzoate (0.17 g, 0.69 mmol) was dissolved in THF (7.5 mL). BnOH (92.6 μL, 0.89 mmol) and PPh.sub.3 (0.24 g, 0.93 mmol) were added, and the mixture was stirred until all components are dissolved. DEAD (2.2 M in toluene, 0.41 mL, 0.89 mmol) was slowly added (via syringe pump) and the mixture was stirred at room temperature 17 hours. The solvent was evaporated in vacuo to give an oily residue, which was purified by flash chromatography (petroleum ether/ethyl acetate=95:5) to yield the title compound (0.20 g, 0.58 mmol, 85%) as a colorless oil.

(208) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.53 (d, J=8.6 Hz, 1H), 7.50 (d, J=8.6 Hz, 1H), 7.48-7.44 (m, 2H), 7.42-7.35 (m, 3H), 5.14 (s, 2H), 4.74 (hept, J=6.2 Hz, 1H), 3.86 (s, 3H), 1.28 (s, 3H), 1.26 (s, 3H) ppm.

(209) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 165.3, 153.4, 148.4, 145.7, 136.4, 130.9, 128.7, 128.7, 128.7, 125.1, 119.3, 78.2, 76.4, 52.8, 22.5 ppm.

(210) HRMS (QTof): Calculated for C.sub.18H.sub.19NO.sub.6Na (M+Na).sup.+: 368.1110, found: 368.1112.

2-Benzyloxy-3-isopropoxy-4-nitrobenzoic acid

(211) ##STR00052##

(212) 2-Benzyloxy-3-isopropoxy-4-nitrobenzoate (0.23 g, 0.67 mmol) was dissolved in a mixture 1/1 of THF/H.sub.2O (3.5/3.5 mL). Then, solid LiOH (0.16 g, 6.67 mmol) was added and the reaction mixture was stirred at room temperature for 17 hours. The aqueous layer was acidified with 1M HCl until pH-1 and extracted with EtOAc (3×). The organic extracts were combined, dried over anhydrous MgSO.sub.4 and filtered. The solvent was concentrated in vacuo to yield the title compound (0.21 g, 0.63 mmol, 95%) as a yellow wax.

(213) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.91 (d, J=8.7 Hz, 1H), 7.58 (d, J=8.7 Hz, 1H), 7.41 (s, 5H), 5.35 (s, 2H), 4.71-4.62 (m, 1H), 1.36 (s, 3H), 1.35 (s, 3H) ppm.

(214) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 164.3, 152.8, 149.7, 144.7, 134.1, 129.8, 129.4, 129.2, 126.98, 120.0, 79.1, 77.7, 22.5 ppm.

(215) HRMS (ESI): Calculated for C.sub.17H.sub.16NO.sub.6 (M−H).sup.−: 330.0978, found: 330.0976.

4-(2-(Benzyloxy)-3-isopropoxy-4-nitro benzamido)-3-isopropoxybenzoate

(216) ##STR00053##

(217) 2-Benzyloxy-3-isopropoxy-4-nitrobenzoic acid (51.5 mg, 0.16 mmol) was dissolved in CH.sub.2Cl.sub.2 (8 mL) and preactivated with Ghosez's reagent (66.0 μL, 0.50 mmol) for 3 hours at 40° C. 3-Isopropoxy-4-aminomethylbenzoate (0.12 g, 0.55 mmol) was dissolved in CH.sub.2Cl.sub.2 (8 mL) and N,N-diisopropylethylamine (DIPEA) was added (0.20 mL, 1.12 mmol). The solution containing the acid chloride was then added and the reaction mixture stirred for 2 days at 40° C. The solvent was then removed and the crude product was purified by preparative HPLC (RP-18; run time 100 min; H.sub.2O/MeCN=100:0.fwdarw.0:100; tr=80 min) providing the title compound (56.9 mg, 0.11 mmol, 68%) as a light yellow oil.

(218) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 10.33 (s, 1H.sub.-NH), 8.55 (d, J=8.5 Hz, 1H), 7.85 (d, J=8.7 Hz, 1H), 7.70 (dd, J=8.5, 1.7 Hz, 1H), 7.59 (d, J=8.7 Hz, 1H), 7.57 (d, J=1.7 Hz, 1H), 7.25-7.12 (m, 5H), 5.25 (s, 2H), 4.75-4.67 (m, 1H), 4.67-4.59 (m, 1H), 3.93 (s, 3H), 1.40 (d, J=6.2 Hz, 6H), 1.28 (d, J=6.0 Hz, 6H) ppm.

(219) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 167.0, 161.4, 151.1, 147.9, 146.1, 145.2, 134.1, 132.9, 132.9, 130.0, 129.4, 128.7, 125.79, 125.6, 123.3, 120.1, 119.5, 113.3, 78.9, 77.4, 71.7, 52.3, 22.6, 22.1 ppm.

(220) HRMS (ESI): Calculated for C.sub.28H.sub.31N.sub.2O.sub.8 (M+H).sup.+: 523.2080, found: 523.2075.

4-(4-Amino-2-hydroxy-3-isopropoxybenzamido)-3-isopropoxybenzoate

(221) ##STR00054##

(222) 4-[2-(Benzyloxy)-3-isopropoxy-4-nitrobenzamido]-3-isopropoxy-benzoate (7.9 mg, 0.015 mmol) was dissolved in MeOH (0.5 mL) and degassed. Pd/C (10% wt, 2 mg, 0.0014 mmol) was added and vacuum was applied under cooling to remove air. The flask was flushed with H.sub.2 and the suspension was stirred for 3 hours at room temperature. The catalyst was filtered off over Celite®, washed with MeOH and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (petroleum ether/ethyl acetate=7:3) and the title compound was obtained (5.8 g, 0.014 mmol, 96%) as a yellow oil.

(223) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 12.21 (s, 1H.sub.-OH), 8.81 (s, 1H.sub.-NH), 8.49 (d, J=8.5 Hz, 1H), 7.69 (dd, J=8.5, 1.8 Hz, 1H), 7.58 (d, J=1.7 Hz, 1H), 7.07 (d, J=8.8 Hz, 1H), 6.28 (d, J=8.7 Hz, 1H), 4.80-4.72 (m, 1H), 4.72-4.63 (m, 1H), 4.28 (s, 2H.sub.-NH2), 3.91 (s, 3H), 1.44 (d, J=6.1 Hz, 6H), 1.34 (d, J=6.2 Hz, 7H) ppm.

(224) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 168.5, 166.9, 156.4, 146.5, 146.0, 132.7, 132.0, 125.1, 123.40, 121.5, 119.1, 113.4, 106.5, 106.3, 77.4, 74.4, 72.0, 52.3, 22.9, 22.4 ppm.

(225) HRMS (ESI): Calculated for C.sub.21H.sub.25N.sub.2O.sub.6 (M−H).sup.−: 401.1713, found: 401.1716.

4-(tert-butoxycarbonylamino)benzoic acid

(226) ##STR00055##

(227) 4-Aminobenzoic acid (1.00 g, 7.29 mmol) was dissolved in 1,4-dioxane (15 mL) and H.sub.2O (7 mL). Et.sub.3N (2.0 mL, 14.58 mmol) was added to the solution and the reaction mixture was stirred for 5 minutes at room temperature. Di-tert-butyl dicarbonate (3.18 g, 14.58 m mol) was then added to the solution in one portion and the reaction mixture was stirred for 24 hours. Following removal of the solvent in vacuo, 3M HCl was added to the residue yielding a white precipitate. The slurry was then filtered and washed with H.sub.2O before drying in under high vacuum. Recrystallization from hot methanol yielded the titled compound as a colorless solid (1.63 g, 6.85 mmol, 94% yield).

(228) mp: 192-194° C.

(229) .sup.1H NMR (400 MHz, DMSO) δ 9.73 (s, 1H.sub.-CO2H), 7.83 (d, 2H, J=8.9 Hz), 7.55 (d, 2H, J=8.9 Hz), 1.47 (s, 9H) ppm.

(230) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 167.1, 152.6, 143.8, 130.4, 124.0, 117.2, 79.7, 28.1 ppm.

(231) HRMS (ESI): Calculated for C.sub.12H.sub.15NnaO.sub.4 (M+Na).sup.+: 260.0893, found: 260.0897. The spectroscopic data are in accordance with those reported in the literature (J. Am. Chem. Soc. 2012, 134, 7406-7413).

Methyl-4-(4-(4-(tert-butoxycarbonyl)amino)benzamido)-2-hydroxy-3-isopropxybenzamido)-3-isopropoxybenzoate

(232) ##STR00056##

(233) 4-(Tert-butoxycarbonylamino)benzoic acid (40.0 mg, 0.17 mmol) was dissolved in CH.sub.2Cl.sub.2 (8.4 mL) and preactivated with Ghosez's reagent (22.5 μL, 0.17 mmol) for 2 hours at room temperature. 4-(4-Amino-2-hydroxy-3-isopropoxybenzamido)-3-isopropoxybenzoate (68.4 mg, 0.17 mmol) was dissolved in CH.sub.2Cl.sub.2 (8.4 mL) and N,N-diisopropylethylamine (DIPEA) was added (59.2 μL, 0.34 mmol). The solution containing the acid chloride was then added and the reaction mixture stirred for 1 day at room temperature. The solvent was then removed and the crude product was purified by preparative HPLC (RP-18; run time 100 min; H.sub.2O/MeCN=100:0.fwdarw.0:100; tr=70 min) providing the title compound as a light yellow oil (47.3 mg, 0.076 mmol, 72%).

(234) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.98 (d, J=7.5 Hz, 2H), 7.78 (d, J=1.4 Hz, 1H), 7.72 (dd, J=7.5, 1.4 Hz, 1H), 7.69 (s, 1H.sub.-NH), 7.68 (d, J=7.3 Hz, 3H), 7.56 (d, J=7.5 Hz, 1H), 7.17 (d, J=7.5 Hz, 1H), 5.72 (s, 1H.sub.-NH), 5.49 (s, 1H.sub.-NH), 4.02-3.96 (m, 2H), 3.95 (d, J=3.7 Hz, 3H), 1.49 (s, 9H), 1.46 (d, J=5.6 Hz, 6H), 1.41 (d, J=5.5 Hz, 6H) ppm.

(235) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 166.89, 166.67, 166.61, 158.88, 154.93, 146.90, 141.47, 135.07, 134.68, 131.70, 130.38, 130.38, 127.26, 127.17, 123.25, 121.40, 120.63, 120.63, 115.87, 114.85, 113.39, 106.06, 80.65, 75.89, 74.13, 52.08, 28.41, 28.41, 28.41, 21.80, 21.80, 21.80, 21.80 ppm.

(236) HRMS (ESI): Calculated for C.sub.33H.sub.38N.sub.3O.sub.9 (M−H).sup.−: 620.2687, found: 620.2689.

Methyl-4-(4-(4-aminobenzamido)-2-hydroxy-3-isopropxybenzamido)-3-isopropoxybenzoate

(237) ##STR00057##

(238) Methyl-4-(4-(4-(tert-butoxycarbonyl)amino)benzamido)-2-hydroxy-3-isopropxybenzamido)-3-isopropoxybenzoate (40.0 mg, 0.064 mmol) was dissolved in a mixture 10/1 dichloromethane/trifluoroacetic acid (1 mL) and stirred 17 hours at room temperature. The solvent was removed under reduced pressure and the residual acid was removed under high vacuum to give the titled compound (33.4 mg, 0.064 mmol, quantitative) as yellow oil.

(239) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.86 (d, J=1.4 Hz, 1H), 7.83 (s, 1H.sub.-NH), 7.79 (dd, J=7.5, 1.4 Hz, 1H), 7.75 (d, J=7.5 Hz, 1H), 7.70 (d, J=7.5 Hz, 2H), 7.65 (d, J=7.5 Hz, 1H), 7.05 (d, J=7.5 Hz, 1H), 6.94 (s, 1H.sub.-NH), 6.75 (d, J=7.5 Hz, 2H), 6.09 (s, 1H.sub.-OH), 4.02-3.97 (m, 1H), 3.95-3.89 (s, 3H), 3.92 (m, 1H), 3.85 (s, 2H.sub.-NH), 1.47 (d, J=5.7 Hz, 6H), 1.40 (d, J=5.5 Hz, 6H) ppm.

(240) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 166.89, 166.67, 166.61, 158.88, 152.59, 146.90, 135.07, 134.68, 131.70, 130.93, 130.93, 127.17, 123.25, 122.42, 121.40, 115.87, 114.85, 114.35, 114.35, 113.39, 106.06, 75.89, 74.13, 52.08, 21.80, 21.80, 21.80, 21.80 ppm.

(241) HRMS (ESI): Calculated for C.sub.28H.sub.32N.sub.3O.sub.7 (M+H).sup.+: 522.2162, found: 522.2160.

Cystobactamide C

(242) ##STR00058##

(243) Methyl-4-[4-(4-aminobenzamido)-2-hydroxy-3-isopropxybenzamido]-3-isopropoxybenzoate (30.0 mg, 0.058 mmol) was dissolved in a mixture 1/1 of THF/H.sub.2O (0.3/0.3 mL). Then, solid LiOH (13.9 mg, 0.58 mmol) was added and the reaction mixture was stirred at room temperature for 17 hours. The aqueous layer was acidified with 1M HCl until pH-1 and extracted with ethyl acetate (3×). The organic extracts were combined, dried over anhydrous MgSO.sub.4 and filtered. The solvent was concentrated in vacuo to yield the title compound (27.4 mg, 0.054 mmol, 93%) as a yellow oil.

(244) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.91 (d, J=1.4 Hz, 1H), 7.87 (dd, J=7.5, 1.4 Hz, 1H), 7.70 (d, J=7.5 Hz, 2H), 7.65 (d, J=7.5 Hz, 1H), 7.53 (d, J=7.5 Hz, 1H), 7.05 (d, J=7.5 Hz, 1H), 6.95 (s, 1H.sub.-NH), 6.77 (s, 1H.sub.-NH), 6.75 (d, J=7.5 Hz, 2H), 6.12 (s, 1H.sub.-OH), 3.97-3.89 (m, 2H), 3.85 (s, 2H.sub.-NH), 1.40 (d, J=5.5 Hz, 6H), 1.39 (d, J=5.5 Hz, 6H) ppm.

(245) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 167.79, 166.67, 166.61, 158.88, 152.59, 149.81, 136.38, 135.07, 134.68, 130.93, 130.93, 125.08, 123.25, 122.80, 122.42, 120.37, 114.35, 114.35, 113.76, 113.39, 106.06, 75.89, 74.13, 21.80, 21.80, 21.80, 21.80 ppm.

(246) HRMS (ESI): Calculated for C.sub.28H.sub.32N.sub.3O.sub.7 (M+H).sup.+: 508.2006, found: 508.2008.

(2S,3R)-Methyl 2,3-dihydroxy-3-phenylpropanoate

(247) ##STR00059##

(248) AD mix β (20.0 g) was dissolved in a mixture of tBuOH/H.sub.2O (1:1; 142 mL) at 25° C. Afterwards, CH.sub.3SO.sub.2NH.sub.2 (1.36 g, 14.3 mmol, 1.0 eq.) was added and the reaction mixture cooled to 0° C. Then, methylcinnamate (2.31 g, 14.3 mmol, 1.0 eq.) was added and the resulting mixture was vigorously stirred for 16 h at 0° C. Stirring was continued for additional 6 h at 25° C. The reaction mixture was hydrolyzed by addition of an aqueous Na.sub.2SO.sub.3 solution (21.4 g, 170 mmol, 12.0 eq.) and stirring was continued for additional 2.5 h. The reaction mixture was diluted with ethyl acetate and the layers were separated. The aqueous layer was extracted with EtOAc (3×). The combined organic layers were washed with H.sub.2O (1×) and dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Purification by flash chromatography (petroleum ether/ethyl acetate=1:1) afforded the desired diol (2.21 g, 11.3 mmol, 79%) as a colorless solid. The spectroscopic data are in accordance with those reported in the literature.

(249) R.sub.f=0.38 (PE/EtOAc 1:1); m.p.=84-85° C. (lit: m.p.=80-81° C.); [α].sub.D.sup.20=−9.8°(c 1.28, CHCl.sub.3) {lit.: [α].sub.D.sup.26=−9.8° (c 1.07, CHCl.sub.3)};

(250) .sup.1H-NMR (400 MHz, CDCl.sub.3, CHCl.sub.3=7.26 ppm): δ=7.42-7.29 (5H, m, ArH), 5.03 (1H, dd, J=2.7, 7.2 Hz, H-3), 4.38 (1H, dd, J=2.7, 6.0 Hz, H-2), 3.82 (3H, s, H-8), 3.12 (1H, d, J=6.0 Hz, OH-α), 2.76 (1H, d, J=7.2 Hz, OH-β) ppm;

(251) .sup.13C-NMR (100 MHz, CDCl.sub.3, CHCl.sub.3=77.16 ppm): δ=173.3 (q, C-1), 140.1 (q, C-4), 128.6 (2C, t, C-6), 128.3 (t, C-7), 126.3 (2C, t, C-5), 74.8 (t, C-2), 74.6 (t, C-3), 53.1 (p, 0-8) ppm; HRMS (ESI): m/z calculated for C.sub.10H.sub.12O.sub.4Na [M+Na].sup.+: 219.0633; found 219.0633.

(2R,3S)-Methyl 2-acetoxy-3-bromo-3-phenylpropanoate (3)

(252) ##STR00060##

(253) To (2S,3R)-Methyl 2,3-dihydroxy-3-phenylpropanoate (2.15 g, 10.9 mmol, 1.0 eq.) was added HBr/HOAc (33%; 16.9 mL) dropwise at 25° C. The resulting mixture was heated to 45° C. and stirred for 30 min. Then, the reaction mixture was cooled to 25° C. and poured into an ice-cooled NaHCO.sub.3-solution (40 mL). The aqueous layer was extracted with Et.sub.2O (3×). The combined organic layers were washed with H.sub.2O (1×) and with brine. Then, the combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Purification by flash chromatography (petroleum ether/ethyl acetate=12.5:1) gave the title compound (2.32 g, 7.71 mmol, 71%) as a colorless solid. The spectroscopic data are in accordance with those reported in the literature.

(254) R.sub.f=0.79 (PE/EtOAc 1:1); m.p.=78-82° C. (lit: m.p.=78-79° C.); [α].sub.D.sup.20=+89.9° (c 1.74, CHCl.sub.3) {Lit.: [α].sub.D.sup.26=+100.3° (c 1.36, CHCl.sub.3)};

(255) .sup.1H-NMR (400 MHz, CDCl.sub.3, CHCl.sub.3=7.26 ppm): 6=7.46-7.44 (2H, m, H-6), 7.36-7.30 (3H, m, H-5, H-7), 5.65 (1H, d, J=6.3 Hz, H-3), 5.35 (1H, d, J=6.3 Hz, H-2), 3.71 (3H, s, H-9), 2.11 (3H, s, H-10) ppm;

(256) .sup.13C-NMR (100 MHz, CDCl.sub.3, CHCl.sub.3=77.16 ppm): δ=169.7 (q, C-1), 167.5 (q, C-8), 136.8 (q, C-4), 129.3 (t, C-7), 128.7 (4C, t, C-5, C-6), 75.4 (t, C-3), 52.9 (p, C-9), 49.3 (t, C-2), 20.6 (p, C-10) ppm;

(257) HRMS (ESI): m/z calculated for C.sub.12H.sub.13O.sub.4BrNa [M+Na].sup.+: 322.9895; found 322.9891.

(2 S,3R)-Methyl 2-acetoxy-3-azido-3-phenylpropanoate

(258) ##STR00061##

(259) (2S,3R)-Methyl 2-acetoxy-3-azido-3-phenylpropanoate (2.27 g, 7.55 mmol, 1.0 eq.) was dissolved in DMF (27.0 mL) at 25° C. Then, NaN.sub.3 (1.96 g, 30.2 mmol, 4.0 eq.) was added and the resulting mixture was heated up to 40° C. for 3 h. After cooling the reaction mixture was cooled to 25° C. and EtOAc was added. The organic layer was washed with H.sub.2O (2×), followed by brine (1×). The combined, organic phases were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Purification by flash chromatography (petroleum ether/ethyl acetate=10:1) afforded the title compound (1.77 g, 6.71 mmol, 89%) as yellow oil. The spectroscopic data are in accordance with those reported in the literature.

(260) R.sub.f=0.24 (PE/EtOAc=10:1); [α].sub.D.sup.20=−97.8° (c 2.3, CHCl.sub.3); {lit.: [α].sub.D.sup.26=−104.2° (c 2.33, CHCl.sub.3)};

(261) IR: {tilde over (v)}=2955 (w), 2103 (s, azide), 1747 (s, C═O), 1495 (w), 1454 (m), 1437 (m), 1373 (m), 1210 (s), 1099 (m), 1030 (m), 910 (m), 751 (m), 701 (s) cm.sup.−1;

(262) .sup.1H-NMR (400 MHz, CDCl.sub.3, CHCl.sub.3=7.26 ppm): δ=7.42-7.33 (5H, m, ArH), 5.24 (1H, d, J=4.8 Hz, H-2), 5.07 (1H, d, J=4.8 Hz, H-3), 3.69 (3H, s, H-9), 2.14 (3H, s, H-10) ppm;

(263) .sup.13C-NMR (100 MHz, CDCl.sub.3, CHCl.sub.3=77.16 ppm): δ=169.9 (q, C-1), 168.0 (q, C-8), 134.6 (q, C-4), 129.3 (t, C-7), 129.0 (2C, t, C-6), 127.6 (2C, t, C-5), 74.9 (t, C-2), 65.4 (t, C-3), 52.8 (p, C-9), 20.5 (p, C-10) ppm;

(264) HRMS (ESI): m/z calculated for C.sub.12H.sub.13N.sub.3O.sub.4Na [M+Na].sup.+: 286.0804; found 286.0805.

(2S,3R)-Methyl 3-azido-2-methoxy-3-phenylpropanoate

(265) ##STR00062##

(266) (2S,3R)-Methyl 2-acetoxy-3-azido-3-phenylpropanoate (2.5 g, 1.0 eq) was dissolved in 190 ml THF at 0° C. Then a solution of KOH (0.5M, 10.0 eq) was added dropwise and the reaction mixture was stirred at 0° C. for 5 h. Afterwards, aqueous 2N HCl was added to the reaction mixture and the aqueous phase was extracted with ethyl acetate. The organic phases were combined and dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to afford the crude acid which was directly used for the next step without further purification. The crude material (0.5 g, 1.0 eq) was dissolved in 17 ml methyl iodide. Then, CaSO.sub.4 (2.6 g, 8.0 eq) and Ag.sub.2O (1.7 g, 3.0 eq) were added and stirring of the suspension was carried out in the dark at room temperature for 22 h. Then, the crude mixture was filtered and concentrated in vacuum to give the title compound (70% yield) which can be directly used in the next step without further purification.

(267) [α].sub.D.sup.20=−143.7° (c 1.1, CHCl.sub.3);

(268) .sup.1H-NMR (400 MHz, CDCl.sub.3, CHCl.sub.3=7.26 ppm): δ=3.44 (s, 3H), 3.61 (s, 3H), 3.94 (d, J=6.4 Hz, 1H), 4.79 (d, J=6.4 Hz, 1H), 7.35-7.36 (m, 5H);

(269) .sup.13C-NMR (100 MHz, CDCl.sub.3, CHCl.sub.3=77.0 ppm): δ=52.2, 59.1, 66.9, 84.7, 127.7, 128.7, 128.9, 135.1, 170.0;

(270) HRMS (ESI): m/z calculated for C.sub.11H.sub.13N.sub.3O.sub.3Na [M+Na]: 258.0855; found 258.0852.

(2S,3S)-tert-Butyl 3-azido-2-methoxy-3-phenyl propanoate

(271) ##STR00063##

(272) To a stirred solution of (2S,3R)-Methyl 3-azido-2-methoxy-3-phenylpropanoate (1.2 g, 1.0 eq) in 100 ml THF was added an aqueous solution of KOH (0.5 M, 10.0 eq) dropwise. The reaction mixture was stirred for 5 h at rt and hydrolyzed by addition of 2N HCl. The aqueous phase was extracted with ethyl acetate and the combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to give carboxylic acid (1.2 g, 98% yield) which was subjected to the next reaction without further purification. Crude acid (0.3 g, 1.0 eq) and 3.9 ml dimethylformamide di-tert-butyl acetal (3.9 ml, 12 eq) were dissolved in 8 ml toluene at room temperature. The resulting reaction mixture was heated up to 80° C. and stirred for 7 h. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography (petroleum ether/ethyl acetate=30:1) to afford the title compound (0.34 g, 89% yield).

(273) [α].sub.D.sup.20=−113.3° (c 1.0, CHCl.sub.3);

(274) .sup.1H-NMR (400 MHz, CDCl.sub.3, CHCl.sub.3=7.26 ppm): δ=1.26 (s, 9H), 3.45 (s, 3H), 3.85 (d, J=7.2 Hz, 1H), 4.70 (d, J=7.2 Hz, 1H), 7.34-7.35 (m, 5H);

(275) .sup.13C-NMR (100 MHz, CDCl.sub.3, CHCl.sub.3=77.0 ppm): δ=27.7, 58.6, 67.2, 82.3, 85.1, 128.2, 128.6, 128.9, 135.2, 168.5;

(276) HRMS (ESI): m/z calculated for C.sub.14H.sub.19O.sub.3N.sub.3Na [M+Na].sup.+: 300.1324; found 300.1332.

(2S,3S)-4-tert-Butyl 1-methyl 2-azido-3-methoxysuccinate

(277) ##STR00064##

(278) To a stirred solution of (2S,3S)-tert-butyl 3-azido-2-methoxy-3-phenylpropanoate (310 mg, 1.0 eq) in a solvent mixture of 3 ml CHCl.sub.3, 13 ml CH.sub.3CN and 26 ml H.sub.2O NaIO.sub.4 (7.2 g, 30 eq) and RuCl.sub.3 (0.3 eq, 69 mg) were added portionwise at room temperature. The reaction mixture was heated under refluxing conditions for 3 h. A white precipitate formed upon cooling to room temperature. The solid was filtered off and the filtrate was extracted with diethyl ether. The combined organic phases were concentrated under reduced pressure to yield the crude product. This material was dissolved in 9 ml methyl iodide. Then, CaSO.sub.4 (1.2 g, 8.0 eq) and Ag.sub.2O (778 mg, 3.0 eq) were added and the reaction mixture was stirred in the dark at room temperature for 22 h. After filtration the filtrate was concentrated under reduced pressure to afford the title compound in pure form so that it can directly be employed in the next step without further purification.

(279) .sup.1H-NMR (400 MHz, CDCl.sub.3, CHCl.sub.3=7.26 ppm): δ=1.51 (s, 3H), 3.48 (s, 3H), 4.15 (d, J=3.6 Hz, 1H), 4.21 (d, J=4.0 Hz, 1H);

(280) .sup.13C-NMR (100 MHz, CDCl.sub.3, CHCl.sub.3=77.0 ppm): δ=28.1, 53.0, 59.5, 63.4, 81.2, 83.0, 167.7, 168.3.

(2S,3R)-1-tert-Butyl 4-methyl 2-methoxy-3-[4-(4-nitrobenzamido)benzamido]succinate

(281) ##STR00065##

(282) The crude mixture (2S,3S)-4-tert-butyl 1-methyl 2-azido-3-methoxysuccinate was dissolved in 12 ml THF, then 0.5 ml water and PPh.sub.3 (881 mg, 3.0 eq) were added. The resulting reaction mixture was warmed up to 50° C. and stirring was continued for 12 hours. Then, the solvent was removed under reduced pressure to afford the crude product which was pure enough to be used directly in the next step. The crude product was dissolved in 5 ml DMF and (ethyl carbonic) 4-(4-nitrobenzamido)benzoic anhydride (481 mg, 1.2 eq) was added at room temperature. After stirring for 20 h, water was added and the aqueous solution was extracted with ethyl acetate. The combined organic phases were concentrated under reduced pressure. Purification by flash column chromatography (petroleum ether/ethyl acetate=2:1) afforded the title compound (81 mg, 16% over four steps).

(283) [α].sub.D.sup.20=−11.8° (c 1.1, CHCl.sub.3);

(284) .sup.1H-NMR (400 MHz, CDCl.sub.3, CHCl.sub.3=7.26 ppm): δ=1.41 (s, 9H), 3.45 (s, 3H), 3.78 (s, 3H), 4.34 (d, J=2.4 Hz, 1H), 5.29 (dd, J=2.4, 9.6 Hz, 1H), 6.76 (d, J=9.6 Hz, 1H), 7.27-7.35 (m, 4H), 8.07 (d, J=8.8 Hz, 2H), 8.26 (2, J=8.8 Hz, 2H), 8.83 (s, 1H);

(285) .sup.13C-NMR (100 MHz, CDCl.sub.3, CHCl.sub.3=77.0 ppm): δ=27.9, 52.9, 54.8, 59.1, 79.8, 83.2, 120.1, 123.8, 128.3, 128.7, 129.6, 140.3, 141.1, 149.7, 164.1, 166.9, 168.0, 169.7.

(286) HRMS (ESI): m/z calculated for C.sub.24H.sub.27O.sub.9N.sub.3Na [M+Na].sup.+: 524.1645; found 524.1647.

(287) ##STR00066##

(288) To a stirred solution of (2S,3R)-1-tert-Butyl 4-methyl 2-methoxy-3-[4-(4-nitrobenzamido)benzamido]succinate (74.3 mg, 0.15 mmol) in 2.5 ml CH.sub.2Cl.sub.2 was added 1.5 ml TFA at room temperature. After stirring for 5 h, the reaction mixture was added water and extracted with ethyl acetate. The combined organic phases were washed with water (three times), dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to give the title compound in quantitative yield (65.9 mg, quant.).

(289) [α].sub.D.sup.20=−16.4° (c 1.1, EtOAc);

(290) .sup.1H-NMR (400 MHz, DMSO, DMSO=2.50 ppm): δ=3.37 (s, 3H), 3.69 (s, J=3H), 4.34 (d, J=4.4 Hz, 1H), 5.09 (dd, J=4.8, 8.8 Hz, 1H), 7.89-7.90 (m, 4H), 8.21 (dd, J=2, 6.8 Hz, 1H), 8.39 (dd, J=2, 6.8 Hz, 1H), 8.55 (d, J=8.8 Hz, 1H), 10.8 (s, 1H).

(291) .sup.13C-NMR (100 MHz, DMSO, DMSO=40.0 ppm): δ=52.9, 54.8, 58.7, 79.5, 120.0, 124.1, 129.0, 129.2, 129.8, 140.8, 142.2, 149.8, 164.7, 166.6, 170.2, 170.9.

(292) HRMS (ESI): m/z calculated for C.sub.20H.sub.19O.sub.9N.sub.3Na [M+Na].sup.+: 468.1019; found 468.1016.

(293) Optical rotation of other enantiomer:

(294) ##STR00067##

(295) [α].sub.D.sup.20=+13.9° (c 1.1, EtOAc);

Methyl-4-(4-(4-((2S,3S)-2,4-dimethoxy-3-(4-(4-nitrobenzamido)benzamido)-4-oxobutanamido)benzamido)-2-hydroxy-3-isopropxybenzamido)-3-isopropoxybenzoate

(296) ##STR00068##

(297) Methyl-4-[4-(4-aminobenzamido)-2-hydroxy-3-isopropxybenzamido]-3-isopropoxybenzoate (15.3 mg, 0.029 mmol) and (2S,3R)-2,4-dimethoxy-3-[4-(4-nitrobenzamido)benzamido]succinate (14.2 mg, 0.032 mmol) were dissolved in CH.sub.2Cl.sub.2 (3.4 mL) and cooled to 0° C. Then, HOAt (5.9 mg, 0.044 mmol), DIPEA (7.7 μL, 0.044 mmol), and EDC. HCl (6.9 mg, 0.036 mmol) were added. The mixture was stirred from 0° C. to room temperature for 17 hours. The solvent was concentrated in vacuo to give an oily residue, which was purified by flash chromatography (petroleum ether/ethyl acetate=94/6) to yield the title compound (20.1 mg, 0.021 mmol, 73%) as a colourless oil.

(298) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.07 (s, 1H.sub.-OH), 8.37 (d, J=7.5 Hz, 2H), 8.20 (d, J=7.5 Hz, 2H), 8.11 (s, 1H.sub.-NH), 8.02 (s, 1H.sub.-NH), 8.01 (d, J=1.4 Hz, 2H), 7.98 (d, J=7.5 Hz, 2H), 7.90 (d, J=1.3 Hz, 1H), 7.81 (dd, J=7.5, 1.4 Hz, 1H), 7.78 (d, J=7.4 Hz, 1H), 7.69 (d, J=7.5 Hz, 1H), 7.61 (d, J=7.5 Hz, 2H), 7.55 (s, 1H), 7.54 (s, 1H.sub.-NH), 7.53 (s, 1H), 7.41 (d, J=7.5 Hz, 1H), 5.72 (s, 1H.sub.-NH), 5.63 (s, 1H.sub.-NH), 5.10 (d, J=3.8 Hz, 1H), 4.76 (d, J=3.8 Hz, 1H), 4.04-3.98 (m, 2H), 3.97 (s, J=3.1 Hz, 3H), 3.74 (s, 3H), 3.32 (s, 3H), 1.47 (d, J=5.7 Hz, 6H), 1.39 (d, J=5.7 Hz, 6H) ppm.

(299) .sup.13C NMR (100 MHz, CDCl.sub.3) δ 173.30, 168.15, 168.07, 167.77, 166.93, 166.88, 166.82, 158.83, 151.01, 146.97, 140.78, 139.42, 138.71, 134.97, 134.55, 131.57, 130.00, 130.00, 129.41, 129.41, 129.39, 129.39, 128.12, 127.53, 127.24, 124.17, 124.17, 123.28, 122.61, 122.61, 121.78, 121.78, 121.44, 115.94, 114.88, 113.30, 106.09, 78.00, 75.89, 74.13, 58.51, 56.50, 52.17, 52.08, 21.80, 21.80, 21.80, 21.80 ppm.

(300) HRMS (ESI): Calculated for C.sub.48H.sub.47N.sub.6O.sub.15 (M−H).sup.−: 947.3178, found: 947.3175.

(301) Cystobactamide A

(302) ##STR00069##

(303) Methyl-4-4-[4-((2S,3S)-2,4-dimethoxy-3-(4-(4-nitrobenzamido)benzamido)-4-oxobutanamido]benzamido}-2-hydroxy-3-isopropxybenzamido)-3-isopropoxybenzoate (15.2 mg, 0.016 mmol) was dissolved in a mixture 1/1 of THF/H.sub.2O (0.2/0.2 mL). Then, solid LiOH (3.8 mg, 0.16 mmol) was added and the reaction mixture was stirred at room temperature for 17 hours. The aqueous layer was acidified with 1M HCl until pH-1 and extracted with ethyl acetate (3×). The organic extracts were combined, dried over MgSO.sub.4 and filtered. The solvent was concentrated in vacuo to yield the title compound (13.3 mg, 0.014 mmol, 90%) as a yellow wax.

(304) [α].sub.D.sup.20=−19.1° (c 1.1, EtOAc)

(305) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.35 (d, J=7.5 Hz, 2H), 8.15 (d, J=7.5 Hz, 2H), 8.00 (d, J=1.8 Hz, 2H), 7.98 (d, J=1.8 Hz, 2H), 7.90 (d, J=1.8 Hz, 1H), 7.86 (dd, J=7.5, 1.8 Hz, 1H), 7.78 (d, J=7.5 Hz, 1H), 7.65 (s, 1H), 7.63 (d, J=7.5 Hz, 2H), 7.58 (s, 1H.sub.-NH), 7.54 (d, J=7.5 Hz, 2H), 7.51 (s, 1H.sub.-NH), 7.10 (s, 1H.sub.-NH), 7.03 (d, J=7.5 Hz, 1H), 6.35 (s, 1H.sub.-NH), 5.57 (s, 1H.sub.-NH), 5.42 (s, 1H.sub.-OH), 4.93 (s, 1H), 4.70 (s, 1H), 4.01 (hept, J=5.6 Hz, 1H), 3.95 (hept, J=5.6 Hz, 1H), 3.38 (s, 3H), 1.48 (s, 6H), 1.47 (s, 6H) ppm.

(306) .sup.13C NMR (100 MHz CDCl.sub.3) δ 173.30, 169.54, 168.18, 168.07, 167.77, 166.88, 166.82, 158.83, 151.01, 149.88, 140.78, 139.42, 138.71, 136.26, 134.97, 134.55, 130.00, 130.00, 129.41, 129.41, 129.39, 129.39, 128.12, 127.53, 125.15, 124.17, 124.17, 123.28, 122.84, 122.61, 122.61, 121.78, 121.78, 120.41, 113.82, 113.30, 106.09, 77.86, 75.89, 74.13, 58.51, 54.58, 21.80, 21.80, 21.80, 21.80 ppm.

(307) HRMS (ESI): Calculated for C.sub.46H.sub.43N.sub.6O.sub.15 (M−H).sup.−: 920.2865, found: 920.2866.

Synthesis of Cystobactamide C Derivatives

(308) ##STR00070## ##STR00071## ##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078##

(309) 1.1. Synthesis of the Different Used Individual Rings

(310) The preparation of the different individual rings that were used during the synthesis of the cystobactamide C derivatives is described here.

Preparation of Ring C

(311) ##STR00079##

Preparation of Ring B

(312) ##STR00080## ##STR00081##

Preparation of Ring A

(313) ##STR00082##

(314) 1.2. Coupling of Ring B and C to Give the Different Prepared BC Fragments

(315) ##STR00083## ##STR00084## ##STR00085## ##STR00086##

(316) 1.3. Coupling of Ring A with BC Fragments

(317) 1.3.1. Coupling of Ring A with BC Fragments (BC1, BC2, BC3, BC5, BC6, BC7) to Synthesize the Cystobactamide C Derivatives (1a)-(23a)

(318) ##STR00087##

(319) TABLE-US-00020 Compound Scaffold R R.sub.1 R.sub.2 R.sub.3  (1a) I iPr iPr 2-OH H  (2a) I iPr iPr 2-OH 2-OH  (3a) I iPr iPr 2-OH 2-OiPr  (4a) I iPr iPr 2-OH 2-F  (5a) I iPr iPr 3-OiPr 2-OH  (6a) II — iPr 2-OH H  (7a) II — iPr 2-OH 2-OH  (8a) II — iPr 2-OH 2-OiPr  (9a) II — iPr 2-OH 2-OMe (10a) II — iPr 3-OiPr 2-OH (11a) III iPr iPr 2-OH H (12a) III iPr iPr 2-OH 2-OH (13a) III iPr iPr 2-OH 2-OiPr (14a) III iPr iPr 3-OiPr 2-OH (15a) IV — iPr 2-OH H (16a) IV — iPr 2-OH 2-OH (17a) IV — iPr 2-OH 2-OiPr (18a) IV — iPr 3-OiPr H (19a) IV — Me 3-OMe H (20a) II — Me 2-OH, H 3OMe (21a) IV — Me 2-OH, H 3OMe (22a) IV — Me 2-OMe, H 3OH (23a) IV — iPr 2,3-diOMe H

(320) 1.3.2. Coupling of Ring A with BC1 Fragment to Synthesize the Cystobactamide C Derivatives (24a)-(31a)

(321) ##STR00088##

(322) TABLE-US-00021 Compound Scaffold R.sub.3 (24a) V (25a) V 0 (26a) V (27a) V (28a) VI (29a) VI (30a) VI (31a) VI

(323) 1.3.3. Coupling of Ring A with BC4 Fragment to Synthesize the Cystobactamide C derivatives (32a)-(33a)

(324) ##STR00097##
2. Experimental

(325) 2.1. General Experimental Information

(326) Starting materials and solvents were purchased from commercial suppliers, and used without further purification. All chemical yields refer to purified compounds, and not optimized. Reaction progress was monitored using TLC Silica gel 60 F.sub.254 aluminium sheets, and visualization was accomplished by UV at 254 nm. Flash chromatography was performed using silica gel 60 Å (40-63 μm). Preparative RP-HPLC was carried out on a Waters Corporation setup contains a 2767 sample manager, a 2545 binary gradient module, a 2998 PDA detector and a 3100 electron spray mass spectrometer. Purification was performed using a Waters XBridge column (C18, 150×19 mm, 5 μm), a binary solvent system A and B (A=water with 0.1% formic acid; B=MeCN with 0.1% formic acid) as eluent, a flow rate of 20 mL/min and a gradient of 60% to 95% B in 8 min were applied. Melting points were determined on a Stuart Scientific melting point apparatus SMP3 (Bibby Sterilin, UK), and are uncorrected. NMR spectra were recorded either on Bruker DRX-500 (.sup.1H, 500 MHz; .sup.13C, 126 MHz), or Bruker Fourier 300 (.sup.1H, 300 MHz; .sup.13C, 75 MHz) spectrometer at 300 K. Chemical shifts are recorded as δ values in ppm units by reference to the hydrogenated residues of deuterated solvent as internal standard (CDCl.sub.3: δ=7.26, 77.02; DMSO-d.sub.6: δ=2.50, 39.99). Splitting patterns describe apparent multiplicities and are designated as s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublet), t (triplet), q (quartet), m (multiplet). Coupling constants (J) are given in Hertz (Hz). Purity of all compounds used in biological assays was 95% as measured by LC/MS Finnigan Surveyor MSQ Plus (Thermo Fisher Scientific, Dreieich, Germany). The system consists of LC pump, autosampler, PDA detector, and single-quadrupole MS detector, as well as the standard software Xcalibur for operation. RP C18 Nucleodur 100-5 (125×3 mm) column (Macherey-Nagel GmbH, Dühren, Germany) was used as stationary phase, and a binary solvent system A and B (A=water with 0.1% TFA; B=MeCN with 0.1% TFA) was used as mobile phase. In a gradient run the percentage of B was increased from an initial concentration of 0% at 0 min to 100% at 15 min and kept at 100% for 5 min. The injection volume was 10 μL and flow rate was set to 800 μL/min. MS (ESI) analysis was carried out at a spray voltage of 3800 V, a capillary temperature of 350° C. and a source CID of 10 V. Spectra were acquired in positive mode from 100 to 1000 m/z and at 254 nm for UV tracing.

(327) 2.2. LC/MS Data for the Triaryl Derivatives

(328) TABLE-US-00022 Compound LC/MS m/z (ESI+)  (1a) 521.99 [M + H].sup.+  (2a) 537.87 [M + H].sup.+  (3a) 579.90 [M + H].sup.+  (4a) 540.07 [M + H].sup.+  (5a) 580.11 [M + H].sup.+  (6a) 479.98 [M + H].sup.+  (7a) 496.02 [M + H].sup.+  (8a) 537.99 [M + H].sup.+  (9a) 509.98 [M + H].sup.+ (10a) 538.11 [M + H].sup.+ (11a) 492.02 [M + H].sup.+ (12a) 508.01 [M + H].sup.+ (13a) 550.02 [M + H].sup.+ (14a) 550.13 [M + H].sup.+ (15a) 449.87 [M + H].sup.+ (16a) 465.93 [M + H].sup.+ (17a) 508.07 [M + H].sup.+ (18a) 492 [M + H].sup.+ (19a) 435 [M].sup.+ (20a) 482 [M + H].sup.+ (21a) 452 [M + H].sup.+ (22a) 452 [M + H].sup.+ (23a) 494 [M + H].sup.+ (24a) 466.20 [M + H].sup.+ (25a) 478.07 [M + H].sup.+ (26a) 493.17 [M + H].sup.+ (27a) 509.12 [M + H].sup.+ (28a) 423.53 [M +].sup.+ (29a) 436.13 [M + H].sup.+ (30a) 451.10 [M + H].sup.+ (31a) 467.11 [M + H].sup.+ (32a) 535 [M + H].sup.+ (33a) 493 [M + H].sup.+

(329) 2.3 General Synthetic Procedures:

(330) a) A mixture of the acid (25 mmol), isopropyl bromide (52 mmol) and potassium carbonate (52 mmol) in 100 ml DMF were heated overnight at 90° C. Excess DMF was then removed under reduced pressure and the remaining residue was partitioned between water and ethyl acetate. The organic layer was dried over sodium sulphate and the excess solvent was then removed under reduced pressure to give the pure product.

(331) c) To a stirred solution of the nitro derivative (10 mmol) in EtOH (60 mL), iron powder (2.80 g, 50 mmol) was added at 55° C. followed by NH.sub.4Cl (266 mg, 5 mmol) solution in water (30 mL). The reaction was refluxed for 1-2 h, then iron was filtered while hot and the filtrate was concentrated under vacuum till dryness. The residue was diluted with water (30 mL) and basified by NaHCO.sub.3 (saturated aqueous solution) to pH 7-8. The mixture was extracted with EtOAc. The combined organic extract was washed with brine, dried (MgSO.sub.4), and the solvent was removed by vacuum distillation. The obtained crude material was triturated with n-hexane, and collected by filtration.

(332) d) Ester hydrolysis was done according to the following reported procedure.′ The ester (0.1 mmol), sodium hydroxide 1M (3 mL) and anhydrous methanol were heated overnight at 45° C. On cooling, the reaction mixture was acidified to pH 1 (3 mL, hydrochloric acid 1 M) and extracted with dichloromethane (3×150 mL). The organic was dried over sodium sulphate and the solvent removed under reduced pressure to leave give the pure product.

(333) m) Amide formation was done according to the following reported procedure..sup.2 A boiling solution of the acid (1 mmol) and the amine (1 mmol) in xylenes 2.5 ml was treated with a 2M solution of PCl.sub.3 in CH.sub.2Cl.sub.2 (0.4 mmol). After 2 hours the excess solvent was evaporated and the residue was purified using column chromatography.

(334) n) To a stirred solution of the acid (2 mmol), amine (2.4 mmol) in anhydrous CHCl.sub.3 (50 mL) under a nitrogen atmosphere, dichlorotriphenylphosphorane (3.0 g, 9 mmol) was added. The reaction was heated at 80° C. for 5 h. Solvent was removed by vacuum distillation. The residue was then purified using flash chromatography.

(335) 2.4 Specific Synthetic Procedures:

Methyl 3-methoxy-4-nitrobenzoate

(336) ##STR00098##

(337) To a stirred mixture of 3-hydroxy-4-nitrobenzoic acid (9.16 g, 50 mmol) and K.sub.2CO.sub.3 (15.2 g, 110 mmol) in DMF (150 mL), dimethyl sulfate (25.2 g, 200 mmol) was added portion wise then the reaction was stirred at 90° C. overnight. After cooling the mixture was poured on to ice cooled water (400 mL), the precipitate was filtered, washed with cold water then n-hexane.

(338) Yield 95% (pale yellow solid), m/z (ES1+) 212 [M+H].sup.+.

3-Methoxy-4-nitrobenzoic acid

(339) ##STR00099##

(340) To a stirred solution of methyl 3-methoxy-4-nitrobenzoate (2.11 g, 10 mmol) in MeOH (30 mL), KOH (1.68 g, 30 mmol) in water (30 mL) was added. The reaction was refluxed for 2 h then MeOH was evaporated by vacuum distillation. The residue was diluted with water (20 mL). The solution was cooled in an ice bath and acidified by KHSO.sub.4 (saturated aqueous solution) to pH 3-4. The precipitated solid was collected by filtration, washed with cold water then n-hexane.

(341) Yield 96% (off-white solid), m/z (ESI+) 198 [M+H].sup.+.

6-Chloro-2-isopropoxy-3-nitropyridine

(342) ##STR00100##

(343) To a stirred solution of 2,6-dichloro-3-nitropyridine (3.86 g, 20 mmol) in toluene (30 mL), isopropanol (1.44 g, 24 mmol) was added. The mixture was stirred at 0° C. for 15 min. then NaH (50-60% in mineral oil, 1.22 g, 28 mmol) was added portion wise under a nitrogen atmosphere, and the reaction was allowed to stir at room temperature overnight. The reaction was quenched with brine, then diluted with water and extracted with EtOAc. The combined organic extract was washed with brine, dried (MgSO.sub.4), and the solvent was removed by vacuum distillation. The residue was dissolved in toluene and purified using flash chromatography (SiO.sub.2, n-hexane-EtOAc=5:1).

(344) Yield 70% (yellowish white crystals), m/z (ES1+) 217 [M+H].sup.+.

2-isopropoxy-3-nitro-6-vinylpyridine

(345) ##STR00101##

(346) To a stirred solution of 6-chloro-2-isopropoxy-3-nitropyridine (650 mg, 3 mmol), and tributyl(vinyl)tin (1.0 g, 3.15 mmol) in toluene (20 mL) under a nitrogen atmosphere, tetrakis(triphenylphosphine) palladium(0) (180 mg, 5% eq.) was added. The reaction was refluxed overnight. Brine was added, and the reaction was extracted with EtOAc. The combined organic extract was washed with brine, dried (MgSO.sub.4), and the solvent was removed by vacuum distillation. The crude product was used directly in the next step without further purification. Yield 90% (yellow liquid), m/z (ES1+) 208 [M]+.

6-Isopropoxy-5-nitropyridine-2-carboxylic acid

(347) ##STR00102##

(348) To a stirred solution of 2-isopropoxy-3-nitro-6-vinylpyridine (625 mg, 3 mmol) in acetone (10 mL), KMnO.sub.4 (1.9 g, 12 mmol) solution in 50% aq. acetone (50 mL) was added. The reaction was stirred at room temperature for 24 h. NaOH 0.5 M (5 mL) was added, then the mixture was filtered and filtrate was concentrated under vacuum. The residue was cooled in an ice bath and carefully acidified by KHSO.sub.4 (saturated aqueous solution) to pH 4-5, then extracted with EtOAc. The combined organic extract was washed with brine, dried (MgSO.sub.4), and the solvent was removed by vacuum distillation. The obtained crude material was triturated with n-hexane, and collected by filtration.

(349) Yield 75% (beige solid), m/z (ES1+) 227 [M+H].sup.+.

Isopropyl 3-isopropoxy-4-{[(6-isopropoxy-5-nitropyridin-2-yl)carbonyl]amino}benzoate

(350) ##STR00103##

(351) To a stirred solution of 6-isopropoxy-5-nitropyridine-2-carboxylic acid (226 mg, 1 mmol), and isopropyl 4-amino-3-isopropoxybenzoate (237 mg, 1 mmol) in a mixture of anhydrous CHCl.sub.3 (50 mL) and DMF (1 mL) under a nitrogen atmosphere, HOBt (676 mg, 5 mmol) was added at 0° C. followed by EDC.HCl (958 mg, 5 mmol). The reaction was allowed to stir at 0° C. for 2 h. then at room temperature overnight. Solvent was removed by vacuum distillation. The residue was dissolved in toluene and purified using flash chromatography (SiO.sub.2, n-hexane-EtOAc=2:1). Yield 70% (pale yellow solid), m/z (ESI+) 446 [M+H].sup.+.

2-formyl-6-methoxyphenyl acetate

(352) ##STR00104##

(353) To a stirred solution of 3-methoxysalicylaldehyde (4.56 g, 30 mmol), and pyridine (2.43 mL, 30 mmol) in DCM (40 mL), acetyl chloride (2.36 g, 30 mmol) was added drop wise. The reaction was stirred at room temperature overnight then the solvent was removed by vacuum distillation. The residue was triturated in cold dil. HCl and filtered, washed with cold water then n-hexane.

(354) Yield 94% (off-white solid), m/z (ESI+) 195 [M+H].sup.+.

6-formyl-2-methoxy-3-nitrophenyl acetate

(355) ##STR00105##

(356) To a stirred ice-cooled suspension of 2-formyl-6-methoxyphenyl acetate (1.94 g, 10 mmol), and KNO.sub.3 (1.01 g, 10 mmol) in CHCl.sub.3 (15 mL), trifluoroacetic anhydride (12 mL) was added. The reaction was stirred in an ice bath for 2 h. then at room temperature overnight. The reaction was diluted very carefully with water (50 mL) and extracted with CHCl.sub.3. The combined organic extract was dried (MgSO.sub.4), and the solvent was removed by vacuum distillation. The residue was dissolved in toluene and purified using flash chromatography (SiO.sub.2, n-hexane-EtOAc=3:1). Yield 45% (yellow semisolid), m/z (ESI+) 239 [M].sup.+.

2-hydroxy-3-methoxy-4-nitrobenzaldehyde

(357) ##STR00106##

(358) To a stirred suspension of 6-formyl-2-methoxy-3-nitrophenyl acetate (957 mg, 4 mmol) in water (30 mL), NaOH (0.8 g, 20 mmol) was added. The reaction was refluxed for 2 h then allowed to stir at room temperature overnight. The solution was cooled in an ice bath and acidified by HCl 2 M to pH 3-4. The precipitated solid was collected by filtration, washed with cold water then n-hexane.

(359) Yield 90% (yellowish brown solid), m/z (ESI+) 197 [M].sup.+.

2-hydroxy-3-methoxy-4-nitrobenzoic Acid

(360) ##STR00107##

(361) To a stirred solution of 2-hydroxy-3-methoxy-4-nitrobenzaldehyde (788 mg, 4 mmol), and NaOH (0.8 g, 20 mmol) in water (50 mL), AgNO.sub.3 (3.4 g, 20 mmol) was added portion wise. The reaction was refluxed overnight, then allowed to cool and filtered through celite. Filtrate was cooled in an ice bath and acidified with HCl 37% to pH 3-4. The precipitated solid was collected by filtration, washed with cold water then n-hexane. Yield 65% (beige solid), m/z (ESI+) 213 [M].sup.+.

Isopropyl 3-isopropoxy-4-[({6-isopropoxy-5-[(4-nitrobenzoyl)amino]pyridin-2-yl}carbonyl)amino]benzoate

(362) ##STR00108##

(363) To a stirred solution of isopropyl 4-{[(5-amino-6-isopropoxypyridin-2-yl)carbonyl]amino}-3-isopropoxybenzoate (207 mg, 0.5 mmol), and pyridine (0.1 mL) in DCM (20 mL), 4-nitrobenzoyl chloride (185 mg, 1 mmol) was added. The reaction was stirred at room temperature overnight then the HCl 2 M (20 mL) was added. The mixture was extracted with DCM then EtOAc. The combined organic extract was dried (MgSO.sub.4), and the solvent was removed by vacuum distillation. The residue was dissolved in toluene and purified using flash chromatography (SiO.sub.2, n-hexane-EtOAc=1:1). Yield 80% (yellow crystals), m/z (ESI+) 565 [M+H].sup.+.

5. References

(364) 1) Valeria Azzarito, Panchami Prabhakaran, Alice I. Bartlett, Natasha Murphy, Michaele J. Hardie, Colin A. Kilner, Thomas A. Edwards, Stuart L. Warriner, Andrew J. Wilson. 2-O-Alkylated Para-Benzamide α-Helix Mimetics: The Role of Scaffold Curvature. Org. Biomol. Chem., 2012, 10, 6469. 2) Alina Fomovska, Richard D. Wood, Ernest Mui, Jitenter P. Dubey, Leandra R. Ferreira, Mark R. Hickman, Patricia J. Lee, Susan E. Leed, Jennifer M. Auschwitz, William J. Welsh, Caroline Sommerville, Stuart Woods, Craig Roberts, and Rima McLeod. Salicylanilide Inhibitors of Toxoplasma gondii. J. Med. Chem., 2012, 55 (19), pp 8375-8391.

6. Activity of these Compounds

(365) Several of these compounds were tested for their activity against an E. coli strain (ToIC-deficient) according to the procedures described above. Most tested compounds showed an activity (MIC) of from 1 to 320 μM.