Marinopyrrole Derivatives as Potential Antibiotic Agents against Methicillin-Resistant Staphylococcus aureus (III)

The marine natural product, marinopyrrole A (1), was previously shown to have significant antibiotic activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Although compound (1) exhibits a significant reduction in MRSA activity in the presence of human serum, we have identified key modifications that partially restore activity. We previously reported our discovery of a chloro-derivative of marinopyrrole A (1a) featuring a 2–4 fold improved minimum inhibitory concentration (MIC) against MRSA, significantly less susceptibility to serum inhibition and rapid and concentration-dependent killing of MRSA. Here, we report a novel fluoro-derivative of marinopyrrole A (1e) showing an improved profile of potency, less susceptibility to serum inhibition, as well as rapid and concentration-dependent killing of MRSA.


Introduction
Since we reported the synthesis of novel non-symmetrical marinopyrrole derivatives retaining their potent activity against methicillin-resistant Staphylococcus aureus (MRSA), yet less susceptible to human serum inhibition [1], several research publications on the topic of marinopyrroles have appeared [2][3][4][5][6][7]. Biosynthesis of marinopyrrole A via an N,C-bipyrrole homocoupling catalyzed by two flavin-dependent halogenases was reported by the Moore group [2], and racemic marinopyrrole B by total synthesis and a review of the marinopyrroles were reported by the Clive group [3,4]. After optimization of the key step to avoid the formation of an oxazepine byproduct [5] that was reported in our first total synthesis of marinopyrroles [8], we published the most potent symmetrical marinopyrrole derivative against MRSA and methicillin-resistant Staphylococcus epidermidis (MRSE) [6]. Recently we reported a series of cyclic marinopyrroles as disruptors of Mcl-1 and Bcl-x L binding to Bim [7] and a series of novel marinopyrroles with potential as anticancer agents [9].
The World Health Organization recognizes antibiotic resistance as a serious threat to human health [10]. The global crisis of antibiotic resistance has spread rapidly over the last several decades, with multidrug-resistant MRSA as a major cause of serious infections in the United States [11][12][13][14][15]. From 1999 to 2005, estimated MRSA hospitalizations in the U.S. more than doubled, increasing from 127,000 to 280,000, and accounted for roughly 94,000 infections and close to 19,000 deaths in 2005 [16]. That same year, more people in the U.S. died from MRSA infections than HIV/AIDS (16,000 people). MRSA infections cost U.S. hospitals between $3.2 and $4.2 billion annually [17]. Recent survey documents have shown that MRSA remains one of the most prevalent multidrug-resistant organisms causing healthcare-associated infections, and the MRSA prevalence in 2010 is higher than that reported in 2006 [18]. The introduction of new MRSA antibiotics to clinical practice has been limited primarily to the oxazolidinone, linezolid [19], in 2000, the lipopeptide, daptomycin [20], in 2003, and ceftaroline [21]. Vancomycin remains the most commonly used first line treatment against MRSA. However, overreliance on this drug has resulted in an increase in MRSA with reduced susceptibility to vancomycin [22,23]. The minimum inhibitory concentration (MIC) shift ("the MIC creep") for vancomycin has been especially noticeable in MRSA compared to other S. aureus [24]. In fact, vancomycin efficacy continues to decline, due to pathogen-developed resistance [22,24]. Instances of daptomycin [25] and linezolid [26,27] resistance have also surfaced. There are now several late-stage products in development, including tedizolid, dalbavancin, oritavancin and ceptobiprole. Although these antibiotics may add to the arsenal for combating MRSA resistance, bacteria inevitably develop resistance to all antibacterial agents that are introduced to the clinic [28]. Novel antibiotic agents of new structural classes and further advances in discovery research are urgently needed to overcome the problem of MRSA resistance.
The relative abandonment of the discovery and development of antibiotics by the pharmaceutical industry has opened opportunities for academic researchers to discover new antibiotics that treat these increasingly problematic infections. Here, we report our design and synthesis of novel marinopyrrole derivatives with excellent antibiotic activity against MRSA, but only limited serum inactivation.

Synthesis and Structural Activity Relationships of Non-Symmetrical Marinopyrrole Derivatives
We classified marinopyrroles as "symmetrical" and "asymmetrical/non-symmetrical" in our previous publication to facilitate structure-activity relationship (SAR) discussions [1]. "Symmetrical" derivatives bear the same substituents and patterns on both phenyl Rings A and B attached to the carbonyl groups, while "non-symmetrical" marinopyrroles are those with different substituents for Rings A and B (Chart 1). As we envisaged that the "non-symmetrical" marinopyrrole derivatives should have different and possibly more favorable biological activity than their symmetrical counterparts, in particular, the molecules with diverse functional groups decorated on this unique 1,3-bispyrrole system may adopt specific conformations, due to the restricted free rotation of the chiral axis. Indeed, a series of novel non-symmetrical marinopyrrole derivatives that we designed and synthesized showed potent anti-MRSA activity with a superior antibiotic profile to the parent marinopyrrole A (1) [1].

Chart 1. Marinopyrroles.
To continue our efforts of structure-activity relationship (SAR) optimization, we designed and synthesized a series of novel non-symmetrical marinopyrroles and evaluated their anti-MRSA activity. As shown in Chart 1 and Scheme 1, while Ring A was kept constant, substitutions with different halogen (F, Cl and Br) in the different position of Ring B were examined. Indeed, the effects of different halogen (F, Cl and Br) in Ring B on SARs, physicochemical properties and pH-dependent microspecies are observed, as detailed in Figures 1 and 2.
To understand the significant effects of the physicochemical properties on antibacterial activity, we calculated the pK a 1, pK a 2 and log p of all marinopyrrole derivatives ( Figure 2). All fluoro-substituted marinopyrroles (1c-1e) have lower Clog p-values than 1, while their chloro-(1a) or bromo-(1f) counterparts are up to half a log unit higher. Although pK a 2 values do not vary much (8.1-8.4), the pK a 1 values change from 7.0 to 7.8, due to the substitution of halogen atoms in different positions of Ring B. Careful analysis of pK a data reveals that there are five microspecies, I-V, present, and their distributions depend on the pH, as shown in Figure 2. Although our MIC assays were performed at pH 7.0, microspecies distributions at pH 7.4 and 8.0 are also tabulated, as the latter conditions are usually used for other assays [7,9]. At pH 7.0, 50%-85% of all marinopyrroles are in the form of Microspecies I, with the parent marinopyrrole, 1, being the most predominant (85%) Microspecies I ( Figure 2); marinopyrroles 1 and 1e have similar distributions of Microspecies II, 7.1% and 6.6%, respectively; the rest are increasing from 17% (1f) to 42% (1d) Microspecies II; the variation of Microspecies III distributions is small from 4.2% (1d) to 12.5% (1e); both Microspecies IV and V are from 0.0% to 3.5% at pH 7 and may be considered negligible. Microspecies I-IV distributions of marinopyrroles vary significantly at pH 7.4 and 8.0 (Figure 2). Microspecies I is found in the free hydroxyl form for both phenol groups, which can serve as both hydrogen bond donors and acceptors. Microspecies II and III can provide one free hydroxyl and one phenoxide group, as shown in Figure 2. The former can provide both a hydrogen bond donor and acceptor in Ring A and only a hydrogen bond acceptor or phenoxide for ionic interactions in Ring B, and vice versa for the latter. Microspecies IV has both phenol groups in phenoxide form, which are only available as hydrogen bond acceptors or for ionic interactions. The microspecies and their distributions at different pH should have a significant impact on their antibacterial activity.

In Vitro Time-Kill of Marinopyrrole Derivative 1e
Our previous data showed that the marinopyrrole derivative, 1a, exhibited rapid killing kinetics, and we investigated whether 1e might also show similar kinetics compared to the parent molecule. Derivative 1e displayed strong concentration-dependent MRSA killing similar in profile to the parent compound [30]. The potency of Derivative 1e was especially evident at 20× the MIC (7.8 μM), yielding greater than a 4-log kill of MRSA at 4 h ( Figure 3B). These killing kinetics parallel the effects previously seen with the natural product parent compound, (−)-1 [30]. Importantly, the tested concentration of 1e (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) [30]. Secondly at 3.9 μM (10× MIC), bacterial counts were reduced by nearly two log units at three hours incubation and on average by three log units at six hours. In comparison, at six hours of incubation, the parent natural product only yielded a two log decrease in bacterial counts at concentrations two fold higher (7.5 μM) than that tested for Derivative 1e (3.9 μM) [30]. Furthermore, the actual tested compound concentration of (−)-1 was 7.5 μM, two fold higher than that of Derivative 1e (3.9 μM) (Figure 3). In summary, we have discovered a second novel derivative of a natural product with favorable bactericidal activity against MRSA, even in the presence of human serum. These results provide additional data showing that the marinopyrrole A scaffold is amenable to modifications to increase its antibacterial activity.

Synthesis of Compounds 5c-6f
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, Billerica, MA, USA). 1 H NMR (400 MHz) spectra were recorded on a Varian AS400 with a 60-place automated sample changer (Thermo, Madison, WI, USA). High resolution ESI-MS spectra were recorded on an Agilent ESI-TOF LC-MS 6200 system (Agilent Technologies, Santa Clara, CA, USA). Preparative HPLC was performed on a Gilson HPLC system with UV detectors and a 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 (Agilent Technologies, Santa Clara, CA, USA). 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: H 2 O with 0.1% HCO 2 H, B: MeOH, gradient of 50%-95% B in 25 min. All tested compounds possessed a purity of not less than 95%.

Conclusions
In our continuation of studies of novel non-symmetrical derivatives of the marine natural product, marinopyrrole A, we identified a derivative, designated as 1e, with favorable bactericidal activity against MRSA (MIC = 0.19-0.78 μM). Furthermore, our time-kill studies indicate potent concentration-dependent killing with 1e that is at least comparable or slightly better than the parent natural product in parallel studies. One of the main drawbacks of the natural product has been its significant reduction in anti-MRSA activity (a 128 to 256 fold increase in MIC) in the presence of human serum. Importantly, 1e is clearly less serum-inhibited with only a 32-64 fold increase in MIC in 20% human serum (Figure 3). Future derivatization and SAR optimization will continue to identify more potent analogs with activity in human serum.