Two years ago, we reported the first total synthesis of marinopyrrole A along with “symmetrical” derivatives, (±)-1 and 12 analogs, which bear the same substituents with the same substitution pattern on both phenyl ring A and B attached to the carbonyl groups (Chart 1) [6]. In addition to those symmetrical marinopyrrole derivatives, we envisage that the “asymmetrical” marinopyrrole derivatives shown in Scheme 1 should have different biological activity than their symmetrical counterparts. In particular, the molecules with diverse functional groups decorated on this unique 1,3-bipyrrole system may adopt specific conformations due to the restricted free rotation of the chiral axis. To this end, a series of novel asymmetrical marinopyrrole derivatives were designed and synthesized.

Chemistries to access both symmetrical and asymmetrical marinopyrrole derivatives were reported previously [2,6]. Briefly, 2-ethoxycarbonyl-3-aminopyrrole 2 was obtained using a published procedure [17]. A PPTS-promoted Clauson–Kaas reaction [2,18] between 2 and 2,5-dimethoxy-tetrahydrafuran 3 in dioxane under reflux gave rise to bispyrrole 4 in 53% yields. The mono-addition of lithiated anisole 5 to bispyrrole ester 4 in THF at −78 °C furnished mono acylated bispyrrole 6 in 85% yield [2]. Friedel–Crafts arylation of 6 with the acid chlorides 8, generated in situ from the corresponding carboxylic acids 7 with thionyl chloride, afforded a series of marinopyrrole precursor 9a–9c. A novel series of asymmetrical marinopyrrole derivatives 10a–10c were obtained by tetrachlorination of the corresponding 9a–9c using 4.1 equivalents of sulfuryl chloride (SO₂Cl₂) in DCM at 0 °C. Demethylation of 10a–10c using BBr₃ in DCM furnished 1a–1c in >90% yield (Scheme 1) [2,6].

The anti-MRSA activity of the novel asymmetrical marinopyrroles shown in Scheme 1 was evaluated against a USA300 strain of community-associated MRSA. One of the derivatives, (4-chloro-2-hydroxyphenyl) (4,4′,5,5′-tetrachloro-2′-(2-hydroxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)methanone (1a), exhibited potent antibacterial activity. The 2–4 fold improvement in antibacterial activity from the parent compound 1 was observed for this novel marinopyrrole derivative 1a as shown in Table 1. The corresponding dimethoxy-congener, (4-chloro-2-methoxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-methoxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)methanone, 10a, was found to be inactive against MRSA at >200 μM. This observation is consistent with the result reported previously for compound 1 and its corresponding methoxy analog [2] as compound 1a showed >1000 fold more potency than 10a. This observed SAR was also supported by the current results from compounds 1b and 1c. Compound 1b, with a chloro-substituent ortho to the hydroxyl group, was >128-fold more potent than 10a, whereas derivative 1c with chloro-substituent para to the hydroxyl group showed >64-fold more anti-MRSA activity than 10b. Compound 1a has the chloro-substituent meta to the hydroxyl group exhibiting 8–16-fold and 4–8-fold more anti-MRSA activity than 1c and 1b, respectively. The fact that the free hydroxyl groups are essential for the antibacterial activity suggested that the binding sites may contain both hydrogen bond donors and hydrogen bond receptors. Although the internal hydrogen bonding between the phenyl hydroxyl and adjacent carbonyl group was observed in an X-ray structure of (−)-1 [1] and compounds 1a–1c may behave similarly, this interaction can be weakened or interrupted if appropriate amino acid residues, such as Arg, His, Asn, Asp, Glu or Gln, are close by for interactions. Although thorough investigation is required to understand the observed structure activity relationships generated by compounds 1a–1c, their physicochemical properties on the OR₅ group in B ring due to the difference in pK_a values of their hydroxyl groups might, at least in part, play an essential role in addition to the different substitution pattern.

Most significantly, not only did compound 1a show increased antibacterial activity compared to the parent compound, marinopyrrole A (1), but its activity was also less inhibited upon the addition of 20% human serum (MIC 12.5–25 µM vs. 100–200 µM). In comparison to contemporary MRSA agents, compound 1a is more potent than vancomycin and linezolid against USA300 MRSA strain TCH1516 [16].

In vitro time-kill studies were performed to characterize killing kinetics of derivative 1a at 20×, 10× and 1× MIC and to compare them with that of the parent natural product marinopyrrole A (−)-1 [16]. Derivative 1a displayed strong concentration-dependent MRSA killing that was much more rapid than vancomycin or linezolid and was similar in profile to the parent compound [16]. The potency of derivative 1a was especially evident at 20× the MIC (3.8 μM), yielding greater than a 4-log kill of MRSA at 4 h. This feature parallels the effects seen with the natural product parent compound (−)-1.

Compound (−)-1 was also shown to have a favorable therapeutic index at 24 h in treated mammalian cells [16]. The natural product marinopyrrole A had a low propensity for toxicity at concentrations below 20× MIC. Upon closer examination, derivative 1a displays more potent MRSA killing effects. For example, the tested concentration of 1a (MIC 0.39 μM) was half of the concentration of the parent natural product tested in time-kill analyses (MIC 0.75 μM or 0.375 μg/mL) [16]. Secondly, at 3.9 μM (10× MIC) and 6 h incubation, 1a causes 3-log decrease in bacterial survival (Figure 1). This improved upon the kinetics of the natural product, (−)-1, which generated only a two-log decrease in bacterial survival [16]. Furthermore, the actual tested compound concentration of (−)-1 was 7.5 μM, a fold higher than that of derivative 1a (3.9 μM) (Figure 1). Similar to (−)-1, this activity is much more rapid than vancomycin or linezolid [16]. In summary, we have discovered a novel natural product based derivative that retains the favorable and potent concentration-dependent bacterial killing characteristic of the natural product but at a lower concentration.

All chemicals and solvents were purchased from commercial suppliers and used without further purification. Preparative flash column chromatography was performed on silica gel 60, 0.040–0.063 mm (EMD Chemicals). ¹H NMR (400 MHz) spectra were recorded on a Varian AS400 with a 60-place automated sample changer. High resolution ESI-MS spectra were recorded on an Agilent ESI-TOF LC-MS 6200 system. Preparative HPLC was performed on a Gilson HPLC system with UV detectors and Gilson 215 liquid handler for auto injection and fraction collections (customized by HT Labs, San Diego, CA, USA). Analytical HPLC was performed on an Agilent 1100 series with diode array detectors and auto samplers. The specifications of HPLC analysis are as follows: flow rate, 1 mL/min; column, Intertsil, 2.5 μm, 4.6 × 150 mm; wavelength, 254 and 280 nm; mobile phase, A: H₂O with 0.1% HCO₂H, B: MeOH, gradient of 50–95% B in 25 min. All tested compounds possessed a purity of not less than 95%.

Ethyl 1′H-1,3′-bipyrrole-2′-carboxylate (4). To a stirred solution of 2,5-dimethoxy-tetrahydrofuran 3 (1.6 mL, 12.4 mmol, 1.3 equiv) and PPTS (3.2 g, 12.4 mmol, 1.3 equiv) in 1,4-dioxane (20 mL) at 50 °C, a solution of aminopyrrole 2 (1.47 g, 9.55 mmol, 1.0 eq.) in 1,4-dioxane (6 mL) was added slowly. The resulting mixture was brought to reflux and stirred for 1 h. The reaction mixture was then concentrated, dissolved in EtOAc (50 mL) and dried over anhydrous MgSO₄. The crude product was purified by flash column chromatography (10% EtOAc in hexane) to give 4 as a yellow solid (1.036 g, 53% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.99 (s, 1H), 7.04–7.01 (m, 2H), 6.89 (t, J = 3.1 Hz, 1H), 6.29 (t, J = 2.9 Hz, 1H), 6.27–6.24 (m, 2H), 4.28 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H).

1′H-1,3′-Bipyrrol-2′-yl(2-methoxyphenyl)methanone (6). To a solution of 2-bromoanisole (1.39 g, 7.43 mmol, 5.63 equiv) in anhydrous THF (5 mL) at −78 °C, a 2.5M solution of BuLi in hexane (3 mL, 7.50 mmol, 5.68 eq.) was added dropwise, the resulting mixture was stirred for 1 h. at −78 °C to generate the lithiated anisole. In an oven-dried flask, compound 4 (270 mg, 1.32 mmol, 1 eq.) was dissolved in THF (5 mL) and cooled to −78 °C, the lithiated anisole 5 was transferred into the solution containing 4 dropwise via syringe, and the resulting mixture was stirred for 1 h. at −78 °C. To the reaction mixture, an aqueous solution of NH₄Cl (5 mL) was added, the mixture was extracted with EtOAc, dried over anhydrous Na₂SO₄. The crude product was purified by flash column chromatography (10% EtOAc in hexane) to give 6 as a light brown solid (0.30 g, 85% yield). ¹H NMR (400 MHz, CDCl₃) δ 9.35 (s, 1H), 7.28–7.22 (m, 1H), 7.20 (dd, J = 7.5, 1.7 Hz, 1H), 7.02 (t, J = 3.0 Hz, 1H), 6.80 (td, J = 7.5, 0.9 Hz, 1H), 6.68 (d, J = 8.4 Hz, 1H), 6.48–6.43 (m, 2H), 6.27 (t, J = 2.8 Hz, 1H), 5.88 (m, 2H), 3.63 (s, 3H).

(2-(4-Chloro-2-methoxybenzoyl)-1′H-1,3′-bipyrrol-2′-yl)(2-methoxyphenyl)methanone (9a). Into a solution of 4-chloro-2-methoxybenzoic acid (125 mg, 0.67 mmol, 1.2 eq.) in benzene (1.5 mL), SOCl₂ (1.5 mL) was added at room temperature and the resulting solution was refluxed for 2 h. The reaction mixture was concentrated under vacuum to generate 4-chloro-2-methoxybenzoyl chloride 8a which was used directly in the next step without purification. A solution of 8a in CH₂Cl₂ (DCM) (2 mL) was added to a slurry of AlCl₃ (96 mg, 1.3 eq.) in DCM (2.5 mL) at 0 °C and then a solution of 6 (150 mg, 0.56 mmol, 1.0 equiv) in DCM (1.5 mL) was added dropwise. The resulting solution was allowed to warm to room temperature and stirred overnight. A saturated solution of NaHCO₃ (10 mL) and DCM (10 mL) was then added and the resulting mixture was stirred for 1 h, and then filtered though Celite^®. The mixture was extracted with DCM (2 × 10 mL), the organic layer was dried over anhydrous Na₂SO₄, and purified by flash column chromatography (hexanes:EtOAc) to afford 172 mg of 9a as a white solid, 70% yield. ¹H NMR (400 MHz, CDCl₃) δ 9.83 (s, 1H), 7.22–7.14 (m, 2H), 7.12 (d, J = 7.8 Hz, 1H), 7.04 (t, J = 3.0 Hz, 1H), 6.95–6.88 (m, 2H), 6.73–6.63 (m, 3H), 6.31 (dd, J = 4.0, 1.7 Hz, 1H), 6.27 (t, J = 2.8 Hz, 1H), 5.83 (dd, J = 4.0, 2.6 Hz, 1H), 3.77 (s, 3H), 3.69 (s, 3H).

(2-(5-Chloro-2-methoxybenzoyl)-1′H-1,3′-bipyrrol-2′-yl)(2-methoxyphenyl)methanone (9b). 5-Chloro-2-methoxybenzoyl chloride (8b) was generated in situ from the corresponding acid using the same method as 8a. Compound 9b (88 mg, 67% yield) was obtained using the same method as 9a described above. ¹H NMR (400 MHz, CDCL₃) δ 9.78 (s, 1H), 7.31 (dd, J = 8.8, 2.7 Hz, 1H), 7.25–7.15 (m, 2H), 7.06 (dd, J = 4.3, 1.7 Hz, 2H), 6.85 (d, J = 8.9 Hz, 1H), 6.77–6.66 (m, 3H), 6.32 (dd, J = 4.0, 1.7 Hz, 1H), 6.30 (t, J = 2.8 Hz, 1H), 5.87 (dd, J = 4.0, 2.6 Hz, 1H), 3.75 (s, 3H), 3.69 (s, 3H).

(2-(3-Chloro-2-methoxybenzoyl)-1′H-1,3′-bipyrrol-2′-yl)(2-methoxyphenyl)methanone (9c). 3-Chloro-2-methoxybenzoyl chloride (8c) was generated in situ from the corresponding acid using the same method as 8a. Compound 9c (90 mg, 68% yield) was obtained using the same method as 9a described above. ¹H NMR (400 MHz, CDCL₃) δ 9.81 (s, 1H), 7.52 (dd, J = 7.8, 1.6 Hz, 1H), 7.39 (dd, J = 8.0, 1.6 Hz, 1H), 7.24–7.19 (m, 1H), 7.10 (dt, J = 5.7, 2.2 Hz, 2H), 6.87 (dd, J = 2.6, 1.6 Hz, 1H), 6.79 (d, J = 7.9 Hz, 1H), 6.66–6.59 (m, 3H), 6.31 (t, J = 2.8 Hz, 1H), 6.05 (dd, J = 4.1, 2.6 Hz, 1H), 3.57 (s, 3H), 3.52 (s, 3H).

(4-Chloro-2-methoxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-methoxybenzoyl)-1′H-[1,3′-bipyrrole]-2-yl)methanone (10a). To a solution of compound 9a (82 mg, 0.19 mmol, 1 eq.) in DCM (5 mL) at 0 °C, SO₂Cl₂ (64 μL, 0.78 mmol, 4.1 equiv) was added dropwise, and the solution was allowed to stir at 0 °C for 1 h. Saturated aqueous NaHCO₃ solution (2 mL) was added and the resulting mixture was extracted with DCM (3 × 4 mL). The combined organic layers were dried with anhydrous MgSO₄, filtered, and concentrated. The residue was purified by flash column chromatography (silica gel, hexane:EtOAc 4:1) to afford 10a (92 mg, 85% yield) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.38 (s, 1H), 7.25–7.21 (m, 2H), 7.13 (d, J = 7.9 Hz, 1H), 6.96–6.94 (m, 2H), 6.76 (d, J = 8.3 Hz, 1H), 6.68 (t, J = 7.8 Hz, 1H), 6.31 (s, 1H), 3.80 (s, 3H), 3.72 (s, 3H); HRMS (ESI-TOF) [M + H]⁺ calcd for C₂₄H₁₆Cl₅N₂O₄ 570.9547, found 570.9537; HPLC purity, 95.2%.

(5-Chloro-2-methoxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-methoxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)methanone (10b). The same procedure described for 10a was used to synthesize 10b (87 mg, 80% yield). ¹H NMR (400 MHz, CDCL₃) δ 10.28 (s, 1H), 7.36 (dd, J = 8.9, 2.7 Hz, 1H), 7.31–7.27 (m, 1H), 7.19 (dd, J = 7.5, 1.7 Hz, 1H), 7.07 (d, J = 2.6 Hz, 1H), 6.88 (d, J = 8.9 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.73 (td, J = 7.5, 0.8 Hz, 1H), 6.33 (s, 1H), 3.78 (s, 3H), 3.73 (s, 3H).

(3-Chloro-2-methoxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-methoxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)methanone (10c). The same procedure described for 10a was used to synthesize 10c (80 mg, 74%). ¹H NMR (400 MHz, CDCL₃) δ 10.30 (s, 1H), 7.57 (dd, J = 7.9, 1.5 Hz, 1H), 7.37–7.27 (m, 2H), 7.11 (dd, J = 7.5, 1.7 Hz, 1H), 6.85 (t, J = 7.9 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 6.67 (t, J = 7.5 Hz, 1H), 6.57 (s, 1H), 3.83 (s, 3H), 3.73 (s, 3H).

(4-Chloro-2-hydroxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-hydroxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)methanone (1a). To a solution of 10a (33 mg, 0.058 mmol) in anhydrous DCM (1 mL) was slowly added 1.0 M solution of BBr₃ in DCM (23 μL, 0.23 mmol, 4 eq.) via a syringe under N₂ at −78 °C. After being stirred for 0.5 h, the mixture was quenched by addition of MeOH (0.5 mL) and extracted with DCM (3 × 10 mL). The combined organic layers were dried over anhydrous MgSO₄, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel, hexanes:12% EtOAc) to give 1a (30 mg, 96% yield) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 11.34 (s, 1H), 10.39 (s, 1H), 9.77 (s, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.43 (dd, J = 8.0, 1.7 Hz, 1H), 7.36 (ddd, J = 8.8, 7.4, 1.6 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 6.95–6.90 (m, 1H), 6.87 (dd, J = 8.6, 2.0 Hz, 1H), 6.68 (s, 1H), 6.53 (ddd, J = 8.0, 7.3, 1.1 Hz, 1H); HRMS (ESI-TOF) [M + H]⁺ calcd for C₂₂H₁₂Cl₅N₂O₄ 542.9234, found 542.9237; HPLC purity 96.6%.

(5-Chloro-2-hydroxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-hydroxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)methanone (1b). The same procedure as 1a was followed to obtain 1b (28 mg, 90%) from 10b (31 mg, 0.055 mmol). ¹H NMR (400 MHz, CDCL₃) δ 11.10 (s, 1H), 10.33 (s, 1H), 9.89 (s, 1H), 8.06 (dd, J = 7.9, 1.8 Hz, 1H), 7.95 (d, J = 2.4 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.71–7.64 (m, 1H), 7.49–7.41 (m, 2H), 6.97 (dd, J = 8.9, 2.1 Hz, 1H), 6.87 (s, 1H); HRMS (ESI-TOF) [M + Na]⁺ calcd for C₂₂H₁₁Cl₅N₂O₄Na 564.9054, found 564.9055; HPLC purity, 95.0%.

(3-Chloro-2-hydroxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-hydroxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)methanone (1c). The same procedure as 1a was followed to obtain 1c (26 mg, 91%) from 10c (29 mg, 0.05mmol). ¹H NMR (400 MHz, CDCL₃) δ 11.74 (s, 1H), 10.48 (s, 1H), 9.93 (s, 1H), 8.06 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 6.96 (t, J = 7.9 Hz, 1H), 6.83 (s, 1H); HRMS (ESI-TOF) [M + Na]⁺ calcd for C₂₂H₁₁Cl₅N₂O₄Na 564.9054, found 564.9058; HPLC purity, 97.4%.

TCH1516, a USA300 strain of community-associated MRSA, was obtained from the American Type Culture Collection and used for biological assays. MICs were determined by broth microdilution according to Clinical and Laboratory Standards Institute guidelines except that Todd-Hewitt broth (THB) was used in place of Mueller-Hinton broth. Assays were done in 96-well tissue-culture treated plates. Vancomycin served as a control antibiotic. MIC assays in 20% human serum were assessed by bacterial metabolic activity in resazurin as described [16].

The bactericidal activity of derivative 1a against the CA-MRSA isolate TCH1516 were assessed by time-kill analysis essentially as described previously [16,19,20,21]. Briefly, MRSA was grown overnight in Todd-Hewitt Broth (THB) at 37 °C with shaking. Following overnight growth, MRSA was inoculated in fresh media for growth to mid-logarithmic phase. At the start of the time-kill assay, bacteria (starting inoculum ~5 × 10⁵ cfu/mL) were added to duplicate 5 mL polystyrene round-bottom tubes (Falcon, Bedford, MA, USA) containing 20×, 10×, 1×, none of the MIC of derivative 1a (0.39 μM) or an equivalent amount of DMSO vehicle control (Figure 1, none). These cultures were incubated in a shaking 37 °C incubator for 24 h. To determine the rate of antibiotic killing, small aliquots were removed from tubes at 0, 3, 6 and 24 h and serially diluted for cfu enumeration on THA plates. The limit of detection for the time-kill assay was 1.6 (log₁₀ cfu/mL).