Introduction

In reports from the World Health Organization, tuberculosis (TB) is listed as one of the ten leading causes of death worldwide in 2016 [1]. There are an estimated 10.4 million new incidents of TB annually, and the number of deaths is around 1.8 million. TB is one of the deadliest diseases caused by a single infectious agent, and is the single largest cause of death for individuals infected with human immunodeficiency virus. The number of new multidrug-resistant (MDR)-TB patients in 2016 was estimated to be 490,000, of whom about 6.2% are infected with extensively drug-resistant (XDR)-TB, against which existing anti-TB drugs are ineffective [1]. There is a clear and urgent need to develop new drugs for the treatment of this disease, in order to reduce the worldwide spread of TB infections.

To address this problem, we screened for novel antimycobacterial substances having an effective spectrum of activity with a new mode of action, from microbial products. As a part of this program, we discovered a structurally analogous mixture of caprazamycins (CPZs), a group of novel lipo-nucleoside antibiotics, in a culture broth of Actinomycete strain Streptomyces sp. MK 730-62F2 [2, 3]. Its major component, caprazamycin B (CPZ-B), showed excellent antimycobacterial activity in vitro against drug-susceptible and multidrug-resistant Mycobacterium tuberculosis strains, although the compounds do have some shortcomings, including its onerous separation from a complex mixture by using HPLC and its extremely poor water-solubility profile [4].

In the process of structural analysis of CPZs, two compounds with the core structure, caprazene and caprazol, were obtained very efficiently from the mixture, as shown in Scheme 1 [3]. By using advanced medicinal chemistry with core compounds, we aimed to create new derivatives that overcome the disadvantages of CPZs. As a result, CPZEN-45, which is expected to be a promising new anti-TB drug, was discovered [4].

Scheme 1
scheme 1

Preparation of caprazene and caprazol from a mixture of caprazamycins A−G

CPZEN-45, a derivative of caprazene, showed excellent antibacterial activity against M. tuberculosis and MDR-TB strains, and showed excellent therapeutic efficacy in a murine TB model infected with an XDR-TB strain resistant to ten drugs [5]. Currently, CPZEN-45 is under development as an inhaled MDR/XDR-TB drug [6, 7].

In previous research, we showed that CPZEN-45 exhibits selective anti-TB activity by specifically inhibiting the phospho-N-acetyl glucosaminyl transferase, WecA [8] involved in arabinogalactan biosynthesis (Fig. 1) [9]. This mechanism is different from that of the parent compounds, CPZs, which acts on bacterial translocase I (also known as MraY in various bacteria, or MurX in M. tuberculosis), a paralog of WecA, which is involved in peptidoglycan biosynthesis (Fig. 1) [10]. WecA is a novel target as a drug for tuberculosis. It is very interesting that CPZEN-45, the 1‴-anilide derivative of caprazene, acquired WecA inhibitory activity, although the starting material, caprazene, has no enzyme inhibitory activity.

Fig. 1
figure 1

Biosynthetic pathway of arabinogalactan in M. tuberculosis (a), teichoic acid in B. subtilis 168 (b), and bacterial peptidoglycan (c). Reactions ‘a’ and ‘b’ surrounded by dashed lines indicate the reactions observed in this report. P phosphate, GlcNAc N-acetyl-d-glucosamine, Rha rhamnose, Galf galactofuranoside, Araf arabinofuranoside, ManNAc N-acetyl-d-mannosamine, Gro glycerol, MurNAcX5 N-acetyl-d-muramate pentapeptide, PBPs penicillin binding proteins. Since radiolabeled UDP-GlcNAc was used as the substrate for the enzyme assays in this report, MarY activity was evaluated with the subsequent reaction by MurG

Because of its attractive biological activity and interest in the synthetic chemistry, total synthesis of CPZEN-45 using pure chemical methods has been reported [11, 12], but there has not yet been any reports on the structure−activity relationships (SAR) of CPZEN-45. Herein, we describe the SAR of five analogs, 15, of CPZEN-45 (Fig. 2), which were prepared by focusing on the seven-membered ring and an anilide moiety of CPZEN-45.

Fig. 2
figure 2

Structures of CPZEN-45 and its analogs

Results

Synthesis

As outlined in Scheme 2, compounds 2 and 3 were prepared according to the synthetic method [4] of CPZEN-45. The condensation reaction of N-Boc caprazene with 4-propylaniline or 2-butylaniline was effectively attained using 4-(4, 6-dimethoxy-1, 3, 5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) to produce amides 6 and 8. Subsequent removal of the protecting group (Boc) from the primary amino group by trifluoroacetic acid efficiently produced analogs 2 and 3 of CPZEN-45.

Scheme 2
scheme 2

Synthesis of 24 from caprazene. Reagents and conditions: a RNH2, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 95% aqueous 2-propanol, room temperature; b CF3COOH, MeOH, room temperature; c NaBH4, MeOH, room temperature

On the other hand, treatment of 5″-N-Boc CPZEN-45 (7) [4] with sodium borohydride induced the reduction of the double bond on the seven-membered diazepinone ring, resulting in saturated compound 9 stereoselectively, with high yield. The S configuration at C-2‴ position of 9 was determined using X-ray crystallography (see Fig. S1 in Supplemental). The expression of this stereoselectivity is presumed to be due to the steric influence of uracil and N-protected 5-amino-5-deoxy-d-ribose moiety located in the beta face. Similar deprotection of the amino group as described above proceeded smoothly to produce 4.

Compound 5 was prepared using caprazol, which is one of the core components obtained from a mixture of caprazamycins. Compound 5 corresponds to the 3‴-hydroxyl derivative of 4. With respect to the seven-membered ring moiety, 5 has a structure closer to the parent natural product caprazamycins than CPZEN-45. As shown in Scheme 3, 5 was readily synthesized via 5″-N-t-butoxycarbonylation of caprazol, introduction of 4-butylaniline through an amide-linkage, and deprotection of the amino group.

Scheme 3
scheme 3

Synthesis of 5 from caprazol. Reagents and conditions: a Boc2O, Et3N, aqueous 1,4-dioxane, room temperature; b 4-butylaniline, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 95% aqueous 2-propanol, room temperature; c CF3COOH, MeOH, room temperature

Effects of the analogs of CPZEN-45 on enzyme activity

To investigate the SAR of CPZEN-45 analogs, we used an enzyme assay system we previously established, which uses cell lysates of Bacillus subtilis [9]. In addition to MraY, which is widely distributed among bacteria, B. subtilis also has a WecA ortholog, TagO, as the enzyme that catalyzes the first step of the biosynthesis of teichoic acid (Fig. 1) [13].

The effects of the analogs of CPZEN-45 on the activity of TagO were evaluated using sonicated cell lysate of BKKZ2202 as the enzyme. The IC50 value of CPZEN-45 against TagO was 55 nM, the strongest activity among the compounds tested. Compounds 1 and 2 were also effective, but their activity was inferior to that of CPZEN-45. Compound 3 showed moderate inhibition, but 4 and 5 were not effective (Fig. 3 and Table 1). No compounds tested inhibited MraY or MurG effectively, whereas caprazamycin B inhibits MraY and/or MurG at an IC50 of less than 500 nM. One thing to be noted is that MraY activity was evaluated by the subsequent reactions with MurG [14, 15] (see Materials and Methods and Fig. 1c), because radiolabeled UDP-GlcNAc was used as the substrate of this enzyme assay.

Fig. 3
figure 3

Effect of the analogs of CPZEN-45 on the TagO activity of cell lysates of B. subtilis BKKZ2202 strain. a Autoradiographic image of TLC, developed and subjected to radioautography as described in Materials and methods, showing the effects of the analogs of CPZEN-45 on the synthesis of the product of TagO. b TagO activities calculated from the autoradiographic intensity of the TagO products shown in (a). Circle, CPZEN-45; triangle, 1; square, 2; diamond, 3; cross, 4; plus, 5

Table 1 Antibacterial activities against B. subtilis strains and enzyme inhibition activities of CPZEN-45 and its analogs

Effects of the analogs of CPZEN-45 on the viability of B. subtilis 168-derived strains

To evaluate the contribution of tagO and mraY to the resistance of the analogs of CPZEN-45, the MIC of these compounds against B. subtilis strains were determined (Table 1). CPZEN-45 showed the highest antibacterial activity among all of the compounds tested against the control strain BKZ2201, with an MIC of 8 µg/mL. The activities of the analogs of CPZEN-45 were 2−32 times lower than CPZEN-45. The tagO-overexpressing strain BKKZ2202 was at least four times as resistant, compared with BKKZ2201, as any of the analogs tested, except compound 5. All of the analogs showed similar antibacterial activity against the mraY-overexpressing strain BKKZ2203 and BKKZ2392 strain which overexpresses mraY and murG, as BKKZ2201. The MICs of caprazamycin B against BKKZ2203 and BKKZ2392 were quite different from those against BKKZ2201.

Effects of the analogs of CPZEN-45 on the viability of mycobacterial strains

The antimycobacterial activity of CPZEN-45 and its analogs is shown is Table 2. In slowly growing mycobacteria, 1, 2 and 3 showed similar or weaker activity at MIC values of 1−16 times higher, compared with CPZEN-45. 4 and 5 were not effective at an MIC of ≥4 µg/mL. In rapidly growing bacteria, all of the compounds tested were hardly effective, although CPZEN-45 had better activity than the other analogs.

Table 2 Antibacterial activities of CPZEN-45 and its analogs against Mycobacterium strains

Discussion

Although caprazene was inactive, antibacterial activity was restored for its 1‴-anilide derivatives, as exemplified by CPZEN-45, which contains a 4-butylaniline. It has previously been shown that, in the process of discovery of CPZEN-45, increasing the alkyl chain at the para-position of aniline slightly improves the antimicrobial activity, but at the same time enhances the hemolytic side effects [4]. Therefore, analogs 1 and 2 of CPZEN-45 with a shortened alkyl chain were synthesized, and the effect of the small structural change to CPZEN-45 on the antimycobacterial activity and inhibition of TagO activity was evaluated in sonicated cell lysates of B. subtilis. As the result, it is revealed that as the carbon chain become shorter, from butyl to propyl to ethyl, inhibitory activity against TagO and antimycobacterial activity decreased.

Because 4-butylanilide was shown to have more effective inhibitory activity, we next tested the effect of the position of the butyl group of the anilide moiety using data from CPZEN-45 and 3. The inhibitory activity of 3 against TagO and its antibacterial activity against Mycobacterium strains were significantly reduced compared to that of CPZEN-45, indicating that the para substitution of the butyl group is superior to the ortho substitution.

We also checked the importance of the double bond on the seven-membered ring. Compounds 4 and 5, with saturated ring systems whose stereochemistry is the same as that of caprazamycins, were prepared for the investigation of MraY and TagO inhibitory activity. The inhibitory activities of these compounds against TagO were notably lower than that of CPZEN-45, suggesting that the double bond on the seven-membered ring system is structurally important for binding to TagO. This observation also suggests that the synthesis of inhibitors of TagO or WecA utilizing caprazol, one of the core compounds of CPZs, is less promising than anticipated.

With respect to MraY/MurG subsequent reactions, none of the analogs tested inhibited these reactions effectively, whereas caprazamycin B, the parent compound of CPZEN-45, showed inhibitory effects against the subsequent reactions. Data from the enzyme assay described above correspond reasonably well with antibacterial activity against B. subtilis strains.

These results indicate that the presence of double bond at C-2‴ and C-3‴ on the seven-membered ring is crucial for inhibitory activity against TagO, and the position and chain length of the alkyl group attached to the anilide moiety is also important. This observation suggests that there is a strict structural limitation on the degree of inhibitory activity of CPZEN-45 for TagO.

This finding appeared to be mostly applicable to WecA, the ortholog of TagO, of slowly growing mycobacteria, according to data on the antibacterial activity of the analogs of CPZEN-45. In rapidly growing mycobacteria the analogs of CPZEN-45 were hardly effective. This lack may be because these compounds had lower affinity to WecA, or to lower permeability of the cell membrane, or because they were well recognized by an efflux pump in rapidly growing mycobacteria. Even though CPZEN-45 was not so effective, it showed better inhibitory activity against M. smegmatis and M. vaccae than the other analogs, supporting the superiority of this compound.

Although these observations should also be discussed from the viewpoint of structural biology, the crystal structure of TagO/WecA orthologs has not been reported in any species to date. Even for protein expression and purification, there is only one report using the thermophilic bacterium Thermotoga maritima [16]. We are now trying to overexpress WecA of M. tuberculosis using E. coli and M. smegmatis, in order to perform subsequent structural analysis. If this trial successful, we will be able to confirm the findings in this report, and elucidate the mode of mechanism of CPZEN-45 against bacterial WecA, using the structural information.

Materials and methods

General information

Melting point was determined on a Kofler block and is uncorrected. NMR spectra were recorded on an AVANCE III 600 spectrometer (1H at 600 and 13C at 150.9 MHz) (Bruker, Billerica, MA, USA) at 300 K, unless stated otherwise. The chemical shifts (δ) of 1H and 13C spectra were measured downfield from internal Me4Si, and were confirmed, when necessary, by shift-correlated 2D spectra. The mass spectra were recorded using a Thermo Scientific LTQ Orbitrap XL mass spectrometer (HRESI-MS) or Thermo Scientific LTQ XL (ESI) spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Thin layer chromatography (TLC) was performed on a Kieselgel 60 F254 (Merck, Darmstadt, Germany), and column chromatography was carried out on a Wakogel C-200 (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Elemental analysis was performed using a Perkin Elmer PE 2400 II analyzer (PerkinElmer Inc., Waltham, MA, USA). Optical rotations were measured on a P-1030 polarimeter (JASCO Corporation, Tokyo, Japan).

Preparation of caprazene-1‴-anilides

5″-N-t-Butoxycarbonylcaprazene 4-propylanilide (6)

To a solution of N-Boc caprazene [4] (an addition salt of triethylamine, 602 mg, 0.793 mmol) in 2-propanol−water (19:1, 12 mL) were added 4-propylaniline (323 mg, 2.39 mmol) and 4-(4, 6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (329 mg, 1.19 mmol) and the mixture was stirred for 3 h at room temperature. Concentration gave a residue, which was dissolved in 5% aq potassium hydrogen sulfate (35 mL). The aqueous solution was washed with EtOAc and neutralized with sodium hydrogen carbonate (1.3 g). The precipitate obtained was extracted with EtOAc. The organic solution was washed with saturated aq sodium chloride, dried (Na2SO4), and concentrated. The residue was purified by silica gel column chromatography (7:1 CHCl3−MeOH) to give 6 (516 mg, 84%) as a colorless solid. ESI-MS: m/z calcd for C36H50N6NaO13 (M+Na)+ 797.3, found 797.3; 1H NMR (600 MHz, DMSO-d6) δ 0.87 [3H, t, J= 7 Hz, CH3(CH2)2C6H4NH], 1.31 [9 H, s, (CH3)3C−O], 1.56 (2H, m, CH3CH2CH2−), 2.35 (3H, s, CH3N-5‴), 2.94 (3H, s, CH3N-8‴), 5.02 (1H, s, H-1″), 5.59 (1H, d, J = 8 Hz, H-5), 5.63 (1H, slightly br d, J= ~2 Hz, H-1′), 6.39 (1H, t, J= 6 Hz, H-3‴), 7.13 and 7.52 [each 2H, d, J= 8.5 Hz, CH3(CH2)2C6H4NH], 7.30 (1H, br s, HN-5″), 7.79 (1H, d, J = 8 Hz, H-6), 10.14 (1H, s, HN-1‴), 11.31 (1H, s, HN-3).

5″-N-t-Butoxycarbonylcaprazene 2-butylanilide (8)

To a solution of N-Boc caprazene (an addition salt of triethylamine, 600 mg, 0.791 mmol) in 2-propanol−water (19:1, 12 mL) were added 2-butylaniline (594 mg, 3.98 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (330 mg, 1.19 mmol) and the mixture was stirred for 2 h at room temperature. Concentration gave a residue, which was dissolved in 5% aq potassium hydrogen sulfate (35 mL). The aqueous solution was washed with EtOAc and neutralized with sodium hydrogen carbonate (1.3 g). The precipitate obtained was extracted with EtOAc. The organic solution was washed with saturated aq sodium chloride, dried (Na2SO4), and concentrated. The residue was purified by silica gel column chromatography (7:1 CHCl3−MeOH) to give 8 (359 mg, 58%) as a colorless solid. ESI-MS: m/z calcd for C37H52N6NaO13 (M+Na)+ 811.3, found 811.3; 1H NMR (600 MHz, DMSO-d6) δ 0.80 [3H, t, J= 7 Hz, CH3(CH2)3C6H4NH], 1.19 (2H, m, CH3CH2CH2CH2−), 1.36 [9H, s, (CH3)3C–O], 1.41 (2H, m, CH3CH2CH2CH2−), 2.36 (3H, s, CH3N-5‴), 3.00 (3H, s, CH3N-8‴), 5.03 (1H, s, H-1″), 5.60 (1H, d, J = 8 Hz, H-5), 5.61 (1H, slightly br d, J= ~2 Hz, H-1′), 6.47 (1H, t, J= 6 Hz, H-3‴), 7.37 (1H, br s, HN-5″), 7.82 (1H, d, J = 8 Hz, H-6), 9.72 (1H, s, HN-1‴), 11.33 (1H, s, HN-3).

Caprazene 4-propylanilide (2)

A solution of 6 (196 mg, 0.253 mmol) in trifluoroacetic acid−methanol (4:1, 6 mL) was kept for 2 h at room temperature. The reaction solution was concentrated, and to the resulting syrup was added diethyl ether. The precipitate obtained was thoroughly washed with diethyl ether to give 2 bis-trifluoroacetate (218 mg, 94%) as a colorless solid, [α]D20 + 83° (c 1, MeOH); HRESI-MS: m/z calcd for C31H43N6O11 (M + H)+ 675.2984, found 675.2987; 1H NMR (600 MHz, DMSO-d6) δ 0.87 (3H, t, J= 7 Hz, CH3CH2CH2−), 1.56 (2H, m, CH3CH2CH2−), 2.39 (3H, br s, CH3N-5‴), 2.96 (3H, s, CH3N-8‴), 5.12 (1H, slightly br s, H-1″), 5.59 (1H, d, J= 2 Hz, H-1′), 5.61 (1H, slightly br d, J= 8 Hz, H-5), 6.40 (1H, br s, H-3‴), 7.14 and 7.52 [each 2H, d, J= 8.5 Hz, CH3(CH2)2C6H4NH], 7.67 (1H, slightly br d, J= 8 Hz, H-6), 8.11 (3H, br s, NH3+), 10.17 (1H, s, HN-1‴), 11.33 (1H, s, HN-3). Anal Calc for C31H42N6O11 2CF3COOH H2O: C 45.66, H 5.04, N 9.13. Found: C 45.39, H 5.23, N 9.02.

Caprazene 2-butylanilide (3)

A solution of 8 (226 mg, 0.286 mmol) in trifluoroacetic acid−methanol (4:1, 6.8 mL) was kept for 2 h at room temperature. The reaction solution was concentrated, and to the resulting syrup was added diethyl ether. The precipitate obtained was thoroughly washed with diethyl ether to give 3 bis-trifluoroacetate (253 mg, 94%) as a colorless solid, [α]D20 + 76° (c 1, MeOH); HRESI-MS: m/z calcd for C32H45N6O11 (M + H)+ 689.3141, found 689.3146; 1H NMR (600 MHz, DMSO-d6) δ 0.81 (3H, t, J= 7 Hz, CH3CH2CH2CH2−), 1.20 (2H, m, CH3CH2CH2CH2−), 1.42 (2H, m, CH3CH2CH2CH2−), 2.39 (3H, br s, CH3N -5‴), 3.03 (3H, s, CH3N-8‴), 5.13 (1H, slightly br s, H-1″), 5.59 (1H, d, J= 2 Hz, H-1′), 5.66 (1H, slightly br d, J= 7 Hz, H-5), 6.47 (1H, br s, H-3‴), 7.70 (1H, slightly br d, J= 7 Hz, H-6), 8.14 (3H, br s, NH3+), 9.72 (1H, slightly br s, HN-1‴), 11.35 (1H, s, HN-3). Anal Calc for C32H44N6O11 2CF3COOH H2O: C 46.26, H 5.18, N 8.99. Found: C 45.95, H 5.56, N 8.99.

5″-N-t-Butoxycarbonyl-3‴-deoxycaprazol 4-butylanilide (9)

To an ice-cooled solution of 7 (986 mg, 1.25 mmol) in methanol (20 mL) was added sodium borohydride (711 mg, 18.7 mmol). After the mixture was stirred for 3 h at room temperature, the reaction was quenched by the addition of dry-ice. Concentration gave a residue, which was extracted with 1-butanol. The organic solution was washed with water and concentrated. The resulting solid was purified by column chromatography (upper layer of 5:1:1 EtOAc−1-BuOH−20% aq AcOH) to give 9 (831 mg, 81%) as a colorless solid. An analytical sample was prepared by crystallization from methanol−water, m.p. 163~164 °C; [α]D20 − 9° (c 1, MeOH); ESI-MS: m/z calcd for C37H54N6NaO13 (M+Na)+ 813.4, found 813.4; 1H NMR (600 MHz, DMSO-d6) δ 0.88 (3H, t, J= 7 Hz, CH3CH2CH2CH2−), 1.27 (2H, m, CH3CH2CH2CH2−), 1.34 [9H, s, (CH3)3C-O], 1.47 (2H, m, CH3CH2CH2CH2−), 1.95−2.07 (2H, m, H-3‴), 2.12 (3H, s, CH3N-5‴), 2.44 (2H, m, CH3CH2CH2CH2−), 2.88 (1H, slightly br d, J= 14.5 Hz, H-4‴a), 3.00 (1H, slightly br d, J= 14 Hz, H-5″a), 3.06 (3H, s, CH3N-8‴), ~3.35 (1H, H-5″b), 3.40 (1H, slightly br t, J= 12 Hz, H-4‴b), 3.71 (1H, d, J= 10 Hz, H-6‴), 3.73−3.77 (2H, H-2′ and H-2″), 3.78−3.84 (2H, H-3′ and H-3″), 3.88 (1H, br, H-4″), 4.07 (1H, d, J= 10 Hz, H-5′), 4.16 (1H, d, J= 8 Hz, H-4′), 4.33 (1H, t, J= 4 Hz, H-2‴), 4.91 (1H, d, J= 5.5 Hz, HO-2″), 4.92 (1H, d, J= 6 Hz, HO-3″), 4.96 (1H, s, H-1″), 5.03 (1H, s, H-1′), 5.07 (1H, d, J= 6 Hz, HO-3′), 5.38 (1H, d, J= 8 Hz, H-5), 5.43 (1H, d, J= 4 Hz, HO-2′), 6.87 and 7.36 [each 2H, d, J= 8.5 Hz, CH3(CH2)3C6H4NH], 7.02 (1H, slightly br d, J = 8.5 Hz, HN-5″), 7.61 (1H, d, J= 8 Hz, H-6), 9.39 (1H, s, HN-1‴), 11.16 (1H, s, HN-3); 13C NMR (150.9 MHz, DMSO-d6) δ 13.7 (CH3CH2CH2CH2−), 21.3 (C-3‴), 21.6 (CH3CH2CH2CH2−), 28.1 [(CH3)3C−O], 32.8 (CH3CH2CH2CH2−), 33.6 (CH3N-5‴), 34.1 (CH3CH2CH2CH2−), 36.6 (CH3N-8‴), 41.8 (C-5″), 53.5 (C-4‴), 61.6 (C-2‴), 63.7 (C-6‴), 68.7 (C-3′), 71.1 (C-3″), 74.2 (C-5′), 74.6 (C-2′), 75.4 (C-2″), 77.5 [(CH3)3C-O], 82.1 (C-4′), 83.1 (C-4″), 89.0 (C-1′), 100.1 (C-5), 109.9 (C-1″), 120.5, 127.3, 135.8, and 137.4 (aromatic), 139.1 (C-6), 149.6 (C-2), 155.8 (COHN-5″), 163.1 (C-4), 167.4 (C-1‴), 171.0 (C-7‴). Anal Calc for C37H54N6O13 1.5H2O: C 54.34, H 7.02, N 10.28. Found: C 54.22, H 7.23, N 10.19.

3‴-Deoxycaprazol 4-butylanilide (4)

A solution of 9 (223 mg, 0.273 mmol) in trifluoroacetic acid−methanol (4:1, 6.7 mL) was kept for 2 h at room temperature. The reaction solution was concentrated, and to the resulting syrup was added diethyl ether. The precipitate obtained was thoroughly washed with diethyl ether to give 4 bis-trifluoroacetate (250 mg, 98%) as a colorless solid, [α]D20 + 22° (c 1, MeOH); HRESI-MS: m/z calcd for C32H47N6O11 (M + H)+ 691.3297, found 691.3313; 1H NMR (600 MHz, DMSO-d6) δ 0.88 (3H, t, J= 7 Hz, CH3CH2CH2CH2−), 1.27 (2H, m, CH3CH2CH2CH2−), 1.47 (2H, m, CH3CH2CH2CH2−), 2.14 (3H, slightly br s, CH3N-5‴), 2.44 (2H, m, CH3CH2CH2CH2−), 3.08 (3H, s, CH3N-8‴), 4.40 (1H, s, H-2‴), 4.93 (1H, s, H-1′), 4.99 (1H, s, H-1″), 5.46 (1H, slightly br d, J= 7.5 Hz, H-5), 7.45 (1H, slightly br d, J= 7.5 Hz, H-6), 8.03 (3H, br s, NH3+), 9.47 (1H, s, HN-1‴), 11.18 (1H, s, HN-3). Anal Calc for C32H46N6O11 2CF3COOH H2O: C 46.16, H 5.38, N 8.97. Found: C 46.29, H 5.49, N 8.90.

Preparation of caprazol-1‴-anilide

5″-N-t-Butoxycarbonylcaprazol (10)

To a suspension of caprazol [4] (tetrahydrate, 346 mg, 0.534 mmol) in water−1, 4-dioxane (2:1, 10 mL) were added triethylamine (145 mg, 1.43 mmol) and di-t-butyl dicarbonate (167 mg, 0.765 mmol) and the mixture was stirred for 1 h at room temperature. To the resulting solution was added 28% aq ammonia (0.1 mL) and the solution was concentrated to give a colorless solid of 10 (421 mg, 97%) as an addition salt of triethylamine. This solid was used in the next reaction without further purification.

Compound 10 as an addition salt of triethylamine; ESI-MS m/z calcd for C27H41N5NaO15 (M+Na)+ 698.3, found 698.3; 1H NMR (600 MHz, DMSO-d6) δ 1.01 [9H, t, J = 7 Hz, (CH3CH2)3N], 1.34 [9H, s, (CH3)3C–O], 2.31 (3H, s, CH3N-5‴), 2.89 (1H, d, J = 15 Hz, H-4‴a), 2.98 (3H, s, CH3N-8‴), 3.25 (1H, d, J = 15 Hz, H-4‴b), 3.66 (1H, d, J = 10 Hz, H-6‴), 4.94 (1H, s, H-1″), 5.55 (1H, d, J = 8 Hz, H-5), 5.59 (1H, d, J = 2.5 Hz, H-1′), 7.02 (1H, d, J = 8.5 Hz, HN-5″), 7.92 (1H, d, J= 8 Hz, H-6), 11.32 (1H, br s, HN-3). Anal Calc for C27H41N5O15 Et3N 2H2O: C 48.76, H 7.44, N 10.34. Found: C 48.55, H 7.58, N 10.05.

5″-N-t-Butoxycarbonylcaprazol 4-butylanilide (11)

To a solution of 10 (an addition salt of triethylamine, 2.28 g, 2.80 mmol) in 2-propanol−water (10:1, 44 mL) were added 4-butylaniline (480 mg, 3.22 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (1.06 g, 3.83 mmol) and the mixture was stirred at room temperature. The same amounts of 4-butylaniline and DMT-MM were added after 5 and 15 h, and the mixture was further stirred for 2 days. Concentration gave a syrup, which was extracted with 5% aq potassium hydrogen sulfate (150 mL) and EtOAc (50 mL). The aqueous solution was washed with EtOAc and neutralized with sodium hydrogen carbonate (8.18 g). The precipitate obtained was extracted with EtOAc and the organic solution was washed with saturated aq sodium chloride, dried (Na2SO4), and concentrated. The residue was purified by silica gel column chromatography (6:1 CHCl3−MeOH) to give 11 (1.03 g, 44%) as a colorless solid; [α]D20 − 11° (c 0.5, MeOH); ESI-MS: m/z calcd for C37H54N6NaO14 (M+Na)+ 829.4, found 829.3; 1H NMR (600 MHz, DMSO-d6) δ 0.88 [3H, t, J= 7 Hz, CH3(CH2)3C6H4NH], 1.27 (2H, m, CH3CH2CH2CH2−), 1.37 [9H, s, (CH3)3C–O], 1.46 (2H, m, CH3CH2CH2CH2−), 2.37 (3H, s, CH3N-5‴), 2.97 (1H, d, J = 15 Hz, H-4‴a), 3.13 (3H, s, CH3N-8‴), 3.43 (1H, d, J = 15 Hz, H-4‴b), 3.65 (1H, d, J = 10 Hz, H-6‴), 4.08 (1H, d, J = 10 Hz, H-5′), 4.18 (1H, d, J = 8 Hz, H-4′), 4.37 (2H, s, H-2‴ and H-3‴), 4.87 (1H, s, H-1′), 4.94 (1H, s, H-1″), 5.35 (1H, d, J = 8 Hz, H-5), 6.85 and 7.35 [each 2H, d, J= 8.5 Hz, CH3(CH2)3C6H4NH], 6.92 (1H, slightly br d, J = 8.5 Hz, HN-5″), 7.58 (1H, d, J= 8 Hz, H-6), 9.38 (1H, s, HN-1‴), 11.14 (1H, s, HN-3). Anal Calc for C37H54N6O14 2H2O: C 52.72, H 6.94, N 9.97. Found: C 52.95, H 7.24, N 9.99.

Caprazol 4-butylanilide (5)

A solution of 11 (187 mg, 0.222 mmol) in trifluoroacetic acid−methanol (4:1, 5 mL) was kept for 2 h at room temperature. The reaction solution was concentrated, and to the resulting syrup was added diethyl ether. The precipitate obtained was thoroughly washed with diethyl ether to give 5 bis-trifluoroacetate (209 mg) as a colorless solid. The solid was dissolved in 1-BuOH and the solution was washed successively with 5% aq sodium hydrogen carbonate and water. Concentration gave a solid, which was chromatographed (upper layer of 2:1:1 EtOAc−1-PrOH−H2O) to give 5 trifluoroacetate (114 mg, 59%) as a colorless solid; [α]D20 + 17° (c 0.5, MeOH); HRESI-MS: m/z calcd for C32H47N6O12 (M+H)+ 707.3246, found 707.3249; ESI-MS: m/z 819.3 (M+CF3COOH−H); 1H NMR (600 MHz, DMSO-d6) δ 0.88 (3H, t, J= 7 Hz, CH3CH2CH2CH2−), 1.26 (2H, m, CH3CH2CH2CH2−), 1.46 (2H, m, CH3CH2CH2CH2−), 2.38 (3H, s, CH3N-5‴), 2.43 (2H, m, CH3CH2CH2CH2−), 3.00 (1H, slightly br dd, J= ~2 and 14 Hz, H-4‴a), 3.05 (1H, slightly br dd, J= ~2 and ~13 Hz, H-5″a), 3.14 (1H, slightly br d, J= ~13 Hz, H-5″b), 3.15 (3H, s, CH3N-8‴), 3.22 (1H, slightly br d, J= 14 Hz, H-4‴b), 3.58 (1H, d, J= 10 Hz, H-6‴), 3.66 (1H, ddd, J= 4.5, 6.5 and 9 Hz, H-3′), 3.73 (1H, t, J= 4.5 Hz, H-2′), 3.76 (1H, t, J= 4 Hz, H-2″), 3.98−4.04 (2H, m, H-3″ and H-4″), 4.09 (1H, d, J= 10 Hz, H-5′), 4.19 (1H, d, J= 9 Hz, H-4′), 4.37 (1H, m, H-3‴), 4.42 (1H, d, J= 5 Hz, H-2‴), 4.83 (1H, s, H-1′), 4.96 (1H, s, H-1″), 5.15 (1H, d, J= 6.5 Hz, HO-3′), 5.22 (1H, d, J= 5.5 Hz, HO-3″), 5.24 (2H, d, J= 4 Hz, HO-2″ and HO-3‴), 5.44 (1H, d, J= 8 Hz, H-5), 5.52 (1H, d, J= 4 Hz, HO-2′), 6.85 and 7.38 [each 2H, d, J= 8.5 Hz, CH3(CH2)3C6H4NH], 7.42 (1H, d, J= 8 Hz, H-6), 8.02 (3H, br s, NH3+), 9.48 (1H, s, HN-1‴), 11.16 (1H, s, HN-3); 13C NMR (150.9 MHz, DMSO-d6) δ 13.7 (CH3CH2CH2CH2−), 21.7 (CH3CH2CH2CH2−), 32.7 (CH3CH2CH2CH2−), 34.0 (CH3CH2CH2CH2−), 36.3 (CH3N-5‴), 38.8 (CH3N-8‴), 39.6 (C-5″), 58.2 (C-4‴), 63.0 (C-6‴), 66.5 (C-2‴), 67.3 (C-3‴), 68.4 (C-3′), 69.6 (C-3″), 74.2 (C-2″), 74.4 (C-2′), 74.8 (C-5′), 78.0 (C-4″), 81.4 (C-4′), 89.6 (C-1′), 99.9 (C-5), 109.6 (C-1″), 117.3 (CF3, q, JC, F = 302 Hz), 120.4, 127.2, 135.6, and 137.6 (aromatic), 138.9 (C-6), 149.5 (C-2), 157.6 (CF3COOH, q, JC, F = 30 Hz), 163.2 (C-4), 167.2 (C-1‴), 169.8 (C-7‴). Anal Calc for C32H46N6O12 CF3COOH 3H2O: C 46.68, H 6.11, N 9.61. Found: C 46.56, H 6.07, N 9.39.

Construction of B. subtilis 168 derivatives overexpressing tagO, mraY and murG

The Bacillus subtilis-Escherichia coli shuttle vector pHYermA was constructed using pHY300PLK (Takara bio Inc. Shiga, Japan) [17], as a source of replication origins, and erythromycin-resistant ermA gene from a Staphylococcus aureus strain. Fragments of tagO and mraY genes of B. subtilis were cloned into pHYermA, and the resulting plasmids were designated as pHYermA-tagO and pHYermA-mraY, respectively. For the construction of plasmid pHYermA-mraYmurG, a DNA fragment containing mraY-murD-spoVE-murG genes (part of an operon consisting of ten genes) was amplified by PCR, inserted into pHYermA, and the murD-spoVE region was cut out using the restriction enzyme HindIII. E. coli HST08 strain (Takara Bio Inc., Shiga, Japan) was used for plasmid construction. Transformation of B. subtilis 168 strains using the constructed plasmids was performed as previously described [9]. Erythromycin at concentrations of 500 and 5 µg/mL was used for the selection of transformants of E. coli and B. subtilis, respectively. The resulting strains (Table 3) were used for MIC evaluation of CPZEN-45 analogs by microbroth dilution in LB medium, Miller (Thermo Fisher Scientific Kabushiki Kaisha, Kanagawa, Japan). The strains were incubated for 16 h at 37 °C, and the MIC values were evaluated.

Table 3 Bacillus subtilis strains and plasmids used in this study

Evaluation of enzyme activity of MraY and TagO of B. subtilis

The enzyme activity of TagO was measured as previously described, with some modifications [9]. Log phase cell culture (OD600 ≈ 0.4) of B. subtilis BKKZ2202 was collected, washed twice with ice-cold buffer A (50 mM MOPS-KOH (pH 7.9), 10% Glycerol, 5 mM MgCl2, 5 mM 3-mercapto-1,2-propanediol) and resuspended in 1/40 volume of buffer A. The cell suspensions were probe-sonicated five times for 60 s each with 60 s breaks on ice, and centrifuged at 3000 rpm to remove unbroken cells. The supernatant, which was used as the enzyme source, was stored at –80 °C until use. For the evaluation of enzyme activity, the reaction mixture, contained 250 ng of total protein/µL, 125 µM undecaprenyl phosphate (Larodan AB, Solna, Sweden), 4 µCi/mL uridine diphosphate N-Acetyl-[1-14C] d-Glucosamine (American Radiolabeled Chemicals Inc., St. Louis, MO), and 0.1% CHAPS in a total volume of 0.05 mL of buffer A, followed by incubation for 2 h at 30 °C. The reaction was stopped by adding 0.25 mL of chloroform/methanol (2:1), mixed vigorously, centrifuged (3000 rpm, 15 min), and the resulting lower phase containing the radiolabeled product was washed with 50 µL of water and subsequent vigorous mixing. The resulting mixture was centrifuged, and the lower phase was chromatographed on a silica gel Kieselgel 60 F254 (Merck) by developing with CHCl3 /MeOH/H2O, ≈14 M NH4OH (56:42:6.8:2.7) and subsequent autoradiography (Typhoon 9400, GE Healthcare). The intensity of the autoradiographic image was quantified using ImageJ software (National Institutes of Health).

The enzyme activity of MraY was evaluated by the subsequential reactions with another transglycosylase MurG, which catalyzes the addition of N-acetyl-d-glucosamine to the MraY product (Fig. 1). In this reaction, the same procedure as used in the TagO reaction was applied, but cell lysates of the BKKZ2392 strain, instead of the BKKZ2203 strain, were used, and 100 µM UDP-MurNAc-pentapeptide (UK Bacterial Cell Wall Biosynthesis Network, University of Warwick, Coventry, UK) was added. In this experiment, caprazamycin B was used as a control.

Antimycobacterial activity

The MICs against bacteria of CPZEN-45 and its analogs (Table 2) were measured using a serial agar dilution method using Middlebrook 7H10 agar (Thermo Fisher Scientific Kabushiki Kaisha) with glycerol and OADC enrichment (Thermo Fisher Scientific Kabushiki Kaisha). MIC values were measured after 2−14 days at 37 °C.