Christina R. Bourne

Crystal Structure of Bacillus anthracis Dihydrofolate Reductase with the Dihydrophthalazine-Based Trimethoprim Derivative RAB1 Provides a Structural Explanation of Potency and Selectivity

ABSTRACT Bacillus anthracis possesses an innate resistance to the antibiotic trimethoprim due to poor binding to dihydrofolate reductase (DHFR); currently, there are no commercial antibacterials that target this enzyme in B. anthracis. We have previously reported a series of dihydrophthalazine-based trimethoprim derivatives that are inhibitors for this target. In the present work, we have synthesized one compound (RAB1) displaying favorable 50% inhibitory concentration (54 nM) and MIC (≤12.8 μg/ml) values. RAB1 was cocrystallized with the B. anthracis DHFR in the space group P212121, and X-ray diffraction data were collected to a 2.3-Å resolution. Binding of RAB1 causes a conformational change of the side chain of Arg58 and Met37 to accommodate the dihydrophthalazine moiety. Unlike the natural substrate or trimethoprim, the dihydrophthalazine group provides a large hydrophobic anchor that embeds within the DHFR active site and accounts for its selective inhibitory activity against B. anthracis.

Inhibition of Antibiotic-Resistant Staphylococcus aureus by the Broad-Spectrum Dihydrofolate Reductase Inhibitor RAB1

ABSTRACT The bacterial burden on human health is quickly outweighing available therapeutics. Our long-term goal is the development of antimicrobials with the potential for broad-spectrum activity. We previously reported phthalazine-based inhibitors of dihydrofolate reductase (DHFR) with potent activity against Bacillus anthracis, a major component of Project BioShield. The most active molecule, named RAB1, performs well in vitro and, in a cocrystal structure, was found deep within the active site of B. anthracis DHFR. We have now examined the activity of RAB1 against a panel of bacteria relevant to human health and found broad-spectrum applicability, particularly with regard to Gram-positive organisms. RAB1 was most effective against Staphylococcus aureus, including methicillin- and vancomycin-resistant (MRSA/VRSA) strains. We have determined the cocrystal structure of the wild-type and trimethoprim-resistant (Phe 98 Tyr) DHFR enzyme from S. aureus with RAB1, and we found that rotational freedom of the acryloyl linker region allows the phthalazine moiety to occupy two conformations. This freedom in placement also allows either enantiomer of RAB1 to bind to S. aureus, in contrast to the specificity of B. anthracis for the S-enantiomer. Additionally, one of the conformations of RAB1 defines a unique surface cavity that increases the strength of interaction with S. aureus. These observations provide insights into the binding capacity of S. aureus DHFR and highlight atypical features critical for future exploitation in drug development.

Synthesis and biological activity of substituted 2,4-diaminopyrimidines that inhibit Bacillus anthracis

A series of substituted 2,4-diaminopyrimidines 1 has been prepared and evaluated for activity against Bacillus anthracis using previously reported (±)-3-{5-[(2,4-diamino-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one (1a), with a minimum inhibitory concentration (MIC) value of 1-3 μg/mL, as the standard. In the current work, the corresponding isobutenyl (1e) and phenyl (1h) derivatives displayed the most significant activity in terms of the lowest MICs with values of 0.5 μg/mL and 0.375-1.5 μg/mL, respectively. It is likely that the S isomers of 1 will bind the substrate-binding pocket of dihydrofolate reductase (DHFR) as in B. anthracis was found for (S)-1a. The final step in the convergent synthesis of target systems 1 from (±)-1-(1-substituted-2(1H)-phthalazinyl)-2-propen-1-ones 6 with 2,4-diamino-5-(5-iodo-3,4-dimethoxybenzyl)pyrimidine (13) was accomplished via a novel Heck coupling reaction under sealed-tube conditions.

Structure-activity relationship for enantiomers of potent inhibitors of B. anthracis dihydrofolate reductase.

BACKGROUND Bacterial resistance to antibiotic therapies is increasing and new treatment options are badly needed. There is an overlap between these resistant bacteria and organisms classified as likely bioterror weapons. For example, Bacillus anthracis is innately resistant to the anti-folate trimethoprim due to sequence changes found in the dihydrofolate reductase enzyme. Development of new inhibitors provides an opportunity to enhance the current arsenal of anti-folate antibiotics while also expanding the coverage of the anti-folate class. METHODS We have characterized inhibitors of B. anthracis dihydrofolate reductase by measuring the K(i) and MIC values and calculating the energetics of binding. This series contains a core diaminopyrimidine ring, a central dimethoxybenzyl ring, and a dihydrophthalazine moiety. We have altered the chemical groups extended from a chiral center on the dihydropyridazine ring of the phthalazine moiety. The interactions for the most potent compounds were visualized by X-ray structure determination. RESULTS We find that the potency of individual enantiomers is divergent with clear preference for the S-enantiomer, while maintaining a high conservation of contacts within the binding site. The preference for enantiomers seems to be predicated largely by differential interactions with protein residues Leu29, Gln30 and Arg53. CONCLUSIONS These studies have clarified the activity of modifications and of individual enantiomers, and highlighted the role of the less-active R-enantiomer in effectively diluting the more active S-enantiomer in racemic solutions. This directly contributes to the development of new antimicrobials, combating trimethoprim resistance, and treatment options for potential bioterrorism agents.

Identification of novel potential antibiotics against Staphylococcus using structure-based drug screening targeting dihydrofolate reductase.

The emergence of multidrug-resistant Staphylococcus aureus (S. aureus) makes the treatment of infectious diseases in hospitals more difficult and increases the mortality of the patients. In this study, we attempted to identify novel potent antibiotic candidate compounds against S. aureus dihydrofolate reductase (saDHFR). We performed three-step in silico structure-based drug screening (SBDS) based on the crystal structure of saDHFR using a 154,118 chemical compound library. We subsequently evaluated whether candidate chemical compounds exhibited inhibitory effects on the growth of the model bacterium: Staphylococcus epidermidis (S. epidermidis). The compound KB1 showed a strong inhibitory effect on the growth of S. epidermidis. Moreover, we rescreened chemical structures similar to KB1 from a 461,397 chemical compound library. Three of the four KB1 analogs (KBS1, KBS3, and KBS4) showed inhibitory effects on the growth of S. epidermidis and enzyme inhibitory effects on saDHFR. We performed structure–activity relationship (SAR) analysis of active chemical compounds and observed a correlative relationship among the IC50 values, interaction residues, and structure scaffolds. In addition, the active chemical compounds (KB1, KBS3, and KBS4) had no inhibitory effects on the growth of model enterobacteria (E. coli BL21 and JM109 strains) and no toxic effects on cultured mammalian cells (MDCK cells). Results obtained from Protein Ligand Interaction Fingerprint (PLIF) and Ligand Interaction (LI) analyses suggested that all of the active compounds exhibited potential inhibitory effects on mutated saDHFR of the drug-resistant strains. The structural and experimental information concerning these novel chemical compounds will likely contribute to the development of new antibiotics for both wild-type and drug-resistant S. aureus.

Inhibition of Bacterial Dihydrofolate Reductase by 6-Alkyl-2,4-diaminopyrimidines

(±)‐6‐Alkyl‐2,4‐diaminopyrimidine‐based inhibitors of bacterial dihydrofolate reductase (DHFR) have been prepared and evaluated for biological potency against Bacillus anthracis and Staphylococcus aureus. Biological studies revealed attenuated activity relative to earlier structures lacking substitution at C6 of the diaminopyrimidine moiety, though minimum inhibitory concentration (MIC) values are in the 0.125–8 μg mL−1 range for both organisms. This effect was rationalized from three‐ dimensional X‐ray structure studies that indicate the presence of a side pocket containing two water molecules adjacent to the main binding pocket. Because of the hydrophobic nature of the substitutions at C6, the main interactions are with protein residues Leu 20 and Leu 28. These interactions lead to a minor conformational change in the protein, which opens the pocket containing these water molecules such that it becomes continuous with the main binding pocket. These water molecules are reported to play a critical role in the catalytic reaction, highlighting a new area for inhibitor expansion within the limited architectural variation at the catalytic site of bacterial DHFR.

Modified 2,4-diaminopyrimidine-based dihydrofolate reductase inhibitors as potential drug scaffolds against Bacillus anthracis

The current Letter describes the synthesis and biological evaluation of dihydrophthalazine-appended 2,4-diaminopyrimidine (DAP) inhibitors (1) oxidized at the methylene bridge linking the DAP ring to the central aromatic ring and (2) modified at the central ring ether groups. Structures 4a-b incorporating an oxidized methylene bridge showed a decrease in activity, while slightly larger alkyl groups (CH2CH3 vs CH3) on the central ring oxygen atoms (R(2) and R(3)) had a minimal impact on the inhibition. Comparison of the potency data for previously reported RAB1 and BN-53 with the most potent of the new derivatives (19 b and 20a-b) showed similar values for inhibition of cellular growth and direct enzymatic inhibition (MICs 0.5-2 μg/mL). Compounds 29-34 with larger ester and ether groups containing substituted aromatic rings at R(3) exhibited slightly reduced activity (MICs 2-16 μg/mL). One explanation for this attenuated activity could be encroachment of the extended R(3) into the neighboring NADPH co-factor. These results indicate that modest additions to the central ring oxygen atoms are well tolerated, while larger modifications have the potential to act as dual-site inhibitors of dihydrofolate reductase (DHFR).

Synthesis and biological evaluation of 2,4-diaminopyrimidine-based antifolate drugs against Bacillus anthracis.

Due to the innate ability of bacteria to develop resistance to available antibiotics, there is a critical need to develop new agents to treat more resilient strains. As a continuation of our research in this area, we have synthesized a series of racemic 2,4-diaminopyrimidine-based drug candidates, and evaluated them against Bacillus anthracis. The structures are comprised of a 2,4-diaminopyrimidine ring, a 3,4-dimethoxybenzyl ring, and an N-acryloyl-substituted 1,2-dihydrophthalazine ring. Various changes were made at the C1 stereocenter of the dihydrophthalazine moiety in the structure, and the biological activity was assessed by measurement of the MIC and Ki values to identify the most potent drug candidate.

Comparative Study of the Frech Catalyst with Two Conventional Catalysts in the Heck Synthesis of 2,4-Diaminopyrimidine-based Antibiotics

Nano palladium coordination complexes, incorporating pincer ligands are reported to be highly efficient catalysts for C–C coupling reactions, giving excellent yields with low catalyst loading.1–8 A relatively new amino pincer palladium complex has been reported by Frech for C–C bond formation via the Heck, Sonogashira, Stille, Hiyama and Suzuki-Miyaura reactions.3–7 A myriad of palladium complexes exist to promote these C–C bond forming processes.9 However, a serious limitation to the use of these reactions for the synthesis of bioactive molecules stems from the lack of thermal stability and functional group tolerance of many palladium complexes as well as the requirement for relatively high catalyst loadings.4 Decomposition of catalysts at normal reaction temperatures (140–150°C) can result in highly contaminated products that require extensive purification, which markedly decreases the yields.8,10 Conventional catalysts also give modest results with heterocyclic substrates.11,12 In contrast, the Frech catalyst exhibits robust thermal stability due, in large part, to the P-Pd-P (PCP) moiety of the pincer ligand.5,13 This thermal stability, together with its inertness to oxygen and water, are unique qualities of the Frech catalyst and allow it to maintain high activity under a variety of reaction conditions.13 In the present work, we have evaluated the Frech catalyst and compared it with two conventional palladium catalysts for coupling highly-substituted, heterocyclic substrates in the final step of a synthesis of 2,4-diaminopyrimidine-based antibiotics, which have demonstrated activity against inhalation anthrax14–16 and multi-drug resistant staph.17 We now report results which validate the potential of this new catalyst in reactions involving multi-functional heterocyclic substrates. Initially, an evaluation was made of the catalyst, base, and solvent required for the Heck reaction of 2,4-diamino-5-(5-iodo-3,4-dimethoxybenzyl)pyrimidine (1) with (±)-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one (2a) to generate 3a (Scheme 1).14,15,18 Two conventional catalysts, (Ph3P)2PdCl2 and Pd(OAc)2,18 as well as the Frech complex, were examined and compared. The use of (Ph3P)2PdCl2 under standard conditions (round-bottomed flask, 1.25 mol% catalyst relative to substrates 1 and 2a, 1.10 equivalents of N-ethylpiperidine, DMF, argon atmosphere, 140–150°C, 18 h) gave a low yield (37%) of the coupled product 3a with a significant number of impurities. An improved return (42%) was realized in a sealed tube under the same conditions, but impurities still persisted. The use of Pd(OAc)2 (1.25 mol%) provided the products in similar yields (50–52%) under both standard and sealed tube conditions, but with only a slightly improved impurity profile. By comparison, the Frech catalyst afforded consistently high yields (80%) with far fewer contaminants at a loading of just 0.12 mol%. Moreover, the Frech catalyst allowed the reaction to be performed on a larger scale (see below). Scheme 1 Preparation of 3a by Heck Coupling. Heck reactions using pincer catalysts are often critically influenced by the solvent and base employed for the coupling process.19,20 To determine the optimum protocol, several solvents, including DMF, THF, and PhCH3, were studied. For the current application, DMF afforded the best results due to its superior solvating properties for the substrates and high boiling point. A series of bases, which included K2CO3, Cs2CO3, Et3N, DBU and N-ethylpiperidine, was also evaluated. In DMF, N-ethylpiperidine provided the highest yields of coupled products. Reaction temperatures were also varied to optimize the conditions. Using DMF and N-ethylpiperidine, maximum conversions were realized at 140–150°C. Reactions at lower temperatures (110–120°C) were slow and gave low yields even after prolonged heating (36 h). At more elevated temperatures (≥160°C), complex mixtures were formed which hindered purification of the desired products. For catalyst comparison studies, reactions using (Ph3P)2PdCl2, Pd(OAc)2, and the Frech complex were run at 140–150°C for 16–20 h, although 3h–3j required only 8–12 h. Without exception, the Frech catalyst gave higher yields and cleaner products that were more easily purified. Finally, catalytic loading for each catalyst was investigated. Our optimization studies indicated that 1.25 mol% of (Ph3P)2PdCl2 and Pd(OAc)2 was required to achieve complete conversions. Greater amounts of catalyst slightly decreased the yields and increased the number of impurities, while less catalyst resulted in incomplete reactions. In sharp contrast, the Frech complex afforded essentially complete conversions to products at a catalytic loading of only 0.12 mol%. For the class of compounds examined, isolated yields of products were highly reproducible with this quantity of catalyst. Under optimized conventional conditions, the reactions of 1.30 mmol each of 1 with 2a–j were carried out using 1.42 mmol of N-ethylpiperidine and 1.55 × 10−3 mmol (0.12 mol%) of the Frech catalyst in 4 mL of DMF under argon at 140–150°C for 16–20 h. The R groups [propyl (3a), isobutyl (3b), isobutenyl (3c), cyclohexyl (3d), phenyl (3e), 4-methylphenyl (3f), 4-fluorophenyl (3g), benzyl (3h), 4-methylbenzyl (3i) and 4-trifluoromethoxybenzyl (3j)] were carefully chosen to provide a range of agents with potential activity as antibiotics and also to ascertain the structural diversity tolerated by the catalyst. The results are summarized in Table 1. Products 3a–j were highly polar and retained water (from chromatography) or methanol (from recrystallizations) despite extensive efforts to remove them.21 Finally, though our study compared reactions run on a 1.30-mmol scale, the Frech catalyst (at a loading of 0.17 mol%) allowed us to run 20.0-mmol preparative scale reactions to generate lead compounds 3a and 3c in essentially undiminished yields of 78% and 74%, respectively. Table 1 Yields of 3a–j using Three Catalysts Coupling of substrates incorporating such wide functional diversity–a diaminopyrimidine ring, two ethers, a tertiary amide, an imine and (in some cases) fluorine–is rare. The closest analogy to our work involved the use of the Frech catalyst to couple a variety of aryl halides to N,N-dimethylacrylamide.13 In this investigation, the reported transformations were assessed to be nearly quantitative by GC/MS analysis. Table 1 reports of products isolated in our current study. Although our optimized catalyst loading was 0.12 mol%, compared to 0.01 mol% for the acrylamide,13 this parameter would be expected to vary for different compounds. Nevertheless, the marked flexibility of the Frech catalyst to operate effectively on systems bearing such a large range of functional groups is remarkable and of great significance in organic synthesis. In summary, we have used the Frech pincer catalyst to efficiently prepare a series of highly functionalized 2,4-diaminopyrimidine-based antibacterials for biological evaluation. The Frech catalyst proved superior to conventional palladium-based Heck catalysts, giving the desired products in higher yields and with fewer contaminants. The Frech catalyst also exhibited superior activity and thermal stability and reduced the required catalytic loading by a factor greater than ten compared to the other catalysts examined. Such remarkable utility, broad scope of action, and multi-functional group tolerance by the Frech catalyst mandates further exploration in organic synthesis.

Microwave-assisted Heck Synthesis of Substituted 2,4-Diaminopyrimidine-based Antibiotics.

Microwave-assisted organic synthesis is an area of increasing interest for promoting clean, reproducible, high-yielding reactions under mild conditions.1,2 This is evident from the large number of papers and reviews that have appeared on this topic in the recent literature.3–8 In the current work, microwave irradiation was employed to facilitate the intermolecular formation of C–C bonds using a palladium-catalyzed Heck coupling reaction. Microwave conditions have previously been used to accelerate this type of reaction9,10 as well as other metal-catalyzed processes such as the Suzuki, Sonogashira and Negishi couplings.11,12 However, the use of microwave irradiation to induce reactions of highly functionalized molecules has not been studied in detail. We, therefore, wish to report our work on a microwave-assisted Heck reaction to prepare a series of antibacterials bearing a variety of functional groups. The goal of the current project is the synthesis 2,4-diaminopyrimidine antibiotics 3a–h, which have potential for the treatment of inhalation anthrax, a bioterror threat. The most important aspect of these drugs is that they selectively inhibit the activity of Bacillus anthracis dihydrofolate reductase (DHFR) but not human DHFR.13,14 DHFR plays a critical role in folate metabolism and is a good target for antibiotic drug candidates. Furthermore, since our compounds incorporate several structural units common to drugs that inhibit DHFR, it is less likely that bacteria exposed to these agents will readily develop a resistance to them. The synthesis involves a Heck reaction of 2,4-diamino-5-(5-iodo-3,4-dimethoxy-benzyl)pyrimidine (1) with a series of (±)-1-(1-substituted-1H-phthalazin-2-yl)prop-2-en-1-ones 2, both of which are available by known methods.13,15 Earlier syntheses of certain examples of 3 by conventional Heck procedures13,15 gave yields of 10%–37%. The products obtained by this method, however, were difficult to purify from the reaction mixture due to extensive side-product formation. We have successfully improved the synthesis by employing microwave irradiation to assist the Heck coupling process. The conventional reactions were carried out using 2.07 mmol each of 1 and 2, 2.27 mmol of N-ethylpiperidine and 0.026 mmol (1.24 mol % relative to substrates 1 and 2) of bis(triphenylphosphine)palladium(II) dichloride catalyst in 8 mL of DMF under argon at 140°C–150°C for 18 h.13,15 This procedure generally afforded the coupled product in low yield (10%–37%) with substantial side-product formation. In an effort to improve this outcome, substrate concentrations and temperatures were varied, but neither of these changes resulted in significant improvement. Attempts to adjust the catalyst loading for this transformation were also examined. An increase in catalyst loading to 2.00 mol % decreased the yield of the product and added to the impurity profile, making isolation of the product more difficult. A decrease in catalyst loading to 0.96 mol % led to incomplete reaction and recovery of starting material along with the desired product. Thus, a catalyst loading of 1.24 mol % proved optimum for the complete conversion to the product with a minimum of side reactions. Finally, the use of other catalysts (e.g. Pd(OAc)2, PdCl2, (Ph3P)4Pd, Pd/C and CuI)13 and bases (e.g. Et3N, DBU, K2CO3 and Cs2CO3)13 failed to improve the conversion to product. Reactions were also carried out on the same molar scale in sealed tubes. This resulted in slightly improved yields (42%–65%), but side-reactions were still problematic. It is conceivable that prolonged heating under conventional and sealed tube conditions caused degradation of the substrates and the catalyst, leading to a more complex product mixture. Thus, a method was sought to decrease the reaction time, which led us to the use of microwave irradiation. Microwave-assisted reactions were run on the same scale as above at 400 W and 150°C under argon for 60–80 min and gave superior conversions to products with far fewer impurities. A comparison of yields obtained using conventional, sealed tube and microwave conditions is shown in Table 1. Microwave irradiation as an alternative source of heating expedited the reaction, decreased the required catalyst loading by 20% (to 0.96 mol %) and reduced the amount of solvent needed by 25%. A series of reactions was carried out with R = propyl, isobutyl, isobutenyl, phenyl, 4-fluorophenyl, benzyl, 4-methylbenzyl and 4-trifluoromethoxybenzyl to establish the generality of the method. Products prepared in this manner were conveniently purified by placing the crude reaction mixture directly onto a silica gel column and eluting with increasing concentrations of methanol in dichloromethane. The target molecules were isolated as hydrates or solvates16 and were characterized by elemental and spectral analysis. Table 1 Yields of 3 Using Conventional, Sealed Tube and Microwave Conditions 1+2a-h→N-ethylpiperidineDMF,Δ(Ph3P)2PdCl23a-h We have successfully developed a synthesis of 2,4-diaminopyrimidine-based antibiotics that utilizes a microwave-assisted Heck reaction on highly functionalized substrates in the final step. The reaction is superior to reactions performed under conventional or sealed tube conditions, requiring less solvent and catalyst. More importantly, the use of microwave conditions reduced reaction times and provided higher coupling yields with fewer side products.