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.


Introduction
The growing problem of antibiotic resistance is prominent in medical reports and the scientific literature, which highlight the emergence of multidrug resistant bacteria [1,2]. For example, Bacillus anthracis continues to be one of the most fatal pathogens to humans and has become a major concern due to its potential use as a bioterrorism weapon [3]. The threat of bioterrorism arises from dormant spores of B. anthracis, which can readily germinate into an infectious form upon inhalation [4]. Like other Gram-positive bacteria, resistance of B. anthracis to traditional antimicrobials can complicate treatment regimens. New drugs are essential to address these resistant strains, particularly in situations requiring urgent treatment without knowledge of the resistance profile as in a bioterror event [5,6].
Inhibition of the critical metabolic enzyme dihydrofolate reductase (DHFR) is an actively pursued area in antibacterial research, and its value as a target has been validated by the success of the antibiotic trimethoprim (TMP) [7]. New compounds with pharmacokinetics differing from those of TMP are sought to address different sites of infection and then, indirectly, the problem of bacterial resistance. In addition, some bacteria, including B. anthracis, encode a chromosomal DHFR that is resistant to TMP but can be targeted by other antifolates, as we have demonstrated previously [8][9][10]. We now have an expanded library of dihydrophthalazine appended 2,4-diaminopyrimidines with demonstrated potency against the DHFR [6] found in B. anthracis and other Gram-positive bacteria [11][12][13][14][15][16]. In particular, alteration of the substituent at the C1 stereocenter of the dihydrophthalazine has been demonstrated to modulate interactions at the interface of the protein surface and the surrounding solvent. In our effort to develop a more active drug for B. anthracis, our current library presents a refinement of the group at this position to optimize potency against this organism.

Chemistry
In an effort to develop more active compounds against B. anthracis and other Gram-positive bacteria, an earlier synthetic strategy to prepare related structures was modified [14,15]. In this project, we synthesized a series of racemic targets as shown in Schemes 1 and 2. Starting with commercially available phthalazine (1), treatment with an organolithium or organomagnesium reagent (compounds 2a-h) in THF under anhydrous conditions furnished racemic adducts 3a-h. These substrates were further subjected to N-acylation using acryloyl chloride and triethylamine to obtain the 1-(phthalazin-2(1H)-yl)prop-2-en-1-one derivatives 4a-h. Acrylamides 4a-h were then linked to the known 2,4-diaminopyrimidine intermediate 5 [15] via a Heck coupling in the presence of Pd(OAc) 2 and N-ethylpiperidine to afford targets 6a-h in yields of 40%-87% (Scheme 1) [16,17].
In addition, we have also developed a synthetic route for the preparation of several ester-containing drug candidates (Scheme 2). These targets were assembled by addition of t-butyl lithioacetate to 1 to give ester 8, followed by N-acylation with acryloyl chloride to give racemic t-butyl 2-(phthalazin-2(1H)-yl)acetate (9a) in 87% yield. Mild hydrolysis of 9a using catalytic Bi(OTf) 3 led to acid 10, which was re-esterified using this same catalyst in the presence of ethanol or methanol [18] to give 9b and 9c, respectively, in 95% yields. Finally, Heck coupling of 9a-c furnished the desired ester-substituted derivatives 11a-c in 74%-78% yields. Scheme 1. Synthesis of drug candidates 6a-h. Scheme 2. Synthesis of drug candidates 11a-c.

Biological Potency
The potency of our synthesized compounds was evaluated in a whole cell model using bacterial cultures, and for activity against the purified DHFR protein target. The ability to halt the growth of standardized cultures gives insight into the utility of the compound as a potential therapeutic, but it does not inform on the cellular target. In the case of whole cells, the lowest concentration of compound needed to inhibit all visible bacterial growth was assessed as in previous studies [9,10,12,15,16] and followed the Clinical Laboratory Standards Institute guidelines [19]. These values are reported in Table 1 as the minimum inhibitory concentration (MIC) in μg/mL. The activity of each compound was evaluated by its ability to halt the enzymatic reaction carried out by the purified DHFR protein in a 94% standardized assay. The results are reported as the compound concentration, in nM, required to inhibit the enzyme activity to one-half the uninhibited rate. This concentration was then used in combination with the substrate affinity of the DHFR enzyme, in this case the K M for dihydrofolate, to derive the inhibition constant K i as reported in Table 1. The combination of the MIC and the K i allowed unbiased assessment of compound potency between bacterial species. These studies build upon previous results [9,10,12,15,16] and highlight a clear preference for small or planar groups at the C1 dihydrophthalazine stereocenter. Compounds 6a-d are derivatives bearing alkyl substituents at this site, similar to RAB1 (R = n-Pr), but with variable lengths. Of these modified derivatives, 6b (R = Et) showed the greatest activity, while 6a (R = Me) was intermediate. Derivatives 6c (R = n-Bu) and 6d (R = s-Bu) proved the least efficacious within this series. Placement of heteroaromatic groups and acetic ester moieties at C1 of the dihydrophthalazine, as in 6f-h and 11a-c, respectively, yielded moderately active structures, but these possessed the lowest activities in the current screening. While compounds 11a-c did not demonstrate exceptional potency, the intent was to utilize the ester-bearing modifications as pro-drugs. Within the body, numerous esterase enzymes would carry out cleavage of these esters to generate the acid [21]. It was anticipated that this form would be more soluble in aqueous medium and would be more potent than the parent compound. This, however, was apparently not the case. Furthermore, while we have prepared the acid, we have been unable to purify it to an acceptable level for screening. Finally, the installation of a cyclopropyl group at C1 gave structure 6e, which is the most potent compound generated to date. Based on available crystallographic studies of RAB1, the cyclopropyl moiety likely forms favorable stacking interactions with an arginine residue at position 53 within the B. anthracis DHFR binding site ( Figure 1) [9,10]. Interactions between the DHFR protein and the RAB1 (R = n-Pr) inhibitor. This structure illustrates the position of substituents R at the C1 stereocenter of the dihydrophthalazine with a black oval; selected residues are labeled. It is hypothesized that the superior potency of compound 6e (R = cyclopropyl) results from stacking interactions with the guanidinium group of Arg 53.

General Information
Commercial anhydrous N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) were stored under dry N 2 and transferred by syringe into reactions when required. Tetrahydrofuran (THF) was dried over KOH pellets and distilled from LiAlH 4 prior to use. K 2 CO 3 was dried at 120 °C under high vacuum for a period of 16 h before use. All other commercial reagents were used as received. Unless otherwise specified, all reactions were run under dry N 2 in oven-dried glassware. The saturated NaCl and NH 4 Cl used in workup procedures were aqueous solutions. Reactions were monitored by thin layer chromatography (TLC) on silica gel GF plates (Analtech, 21521). Preparative separations were performed by chromatography on silica gel (Davisil ® , grade 62, 60-200 mesh) mixed with UV-active phosphor (Sorbent Technologies, UV-05). Band elution for all chromatographic separations was monitored using a hand-held UV lamp. Melting points were uncorrected. FT-IR spectra were run as thin films on NaCl disks. 1 H-and 13 C-NMR spectra were measured at 300 MHz or 400 MHz ( 1 H) and 75 or 100 MHz ( 13 C) in the indicated solvent. Chemical shifts (δ) are referenced to internal (CH 3 ) 4 Si and coupling constants (J) are given in Hz. Elemental analyses were ±0.4% from Atlantic Microlab, Inc. (Norcross, GA, USA).

Synthesis of 1-(Phthalazin-2(1H)-yl)prop-2-en-1-ones 4a-h
. A stirred solution of phthalazine (1) (2.00 g, 15.4 mmol) in dry THF (50 mL) was treated dropwise with a solution of methyllithium (2a, 1.5 M in ether, 11.3 mL, 16.9 mmol) over a period of 15 min at -20 °C. The reaction was stirred at this temperature for 45 min and was then poured into saturated NH 4 Cl (50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic extracts were washed with saturated NaCl (100 mL), dried (MgSO 4 ), filtered, and concentrated under vacuum to afford 3a as a dark brown liquid. The crude product 3a was dissolved in dichloromethane (DCM, 50 mL), and triethylamine (1.86 g, 2.56 mL, 18.4 mmol) was added, followed by dropwise addition of acryloyl chloride (1.39 g, 1.25 mL, 15.4 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h. The reaction was then quenched with saturated NaCl (100 mL), the organic layer was separated, and the aqueous layer was extracted with DCM (2 × 50 mL). The combined organic extracts were washed with saturated NaCl (50 mL), dried (MgSO 4 ), filtered, and concentrated to afford the crude product. The crude product was purified on a silica gel column eluted with hexanes:EtOAc (4:1) to afford 4a as a pale yellow liquid (2.60 g, 84%).
To a stirred solution of 1 (2.00 g, 15.4 mmol) in dry THF (50 mL) was added dropwise cyclopropylmagnesium chloride (0.5 M in THF, 33.8 mL, 16.9 mmol) over a period of 10 min at 0 °C. The reaction was stirred at 0 °C for 2 h and was then quenched with saturated NH 4 Cl (50 mL) and extracted with ethyl acetate (2 × 50 mL). The combined extracts were washed with saturated NaCl, dried (MgSO 4 ), filtered, and concentrated to give 3e as a light brown oil. The crude product 3e was acylated as described for compound 4a using triethylamine (1. The solution was warmed to -25 °C, and stirring was continued at this temperature for 30 min. The reaction mixture was cooled back to -78 °C, and a solution of 1 (2.00 g, 15.3 mmol) in dry THF (20 mL) was added dropwise over 30 min. The reaction mixture was stirred at this temperature for 2 h. The mixture was poured into saturated NH 4 Cl (100 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic extracts were then washed with saturated NaCl (50 mL), dried (MgSO 4 ), filtered, and concentrated under vacuum to afford 3f as a light brown oil. The crude product 3f was dissolved in DCM (30 mL), and triethylamine (2.37 g, 3.26 mL, 23.4 mmol) was added, followed by dropwise addition of acryloyl chloride (1.59 g, 1.43 mL, 17.6 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h. The reaction was then quenched with saturated NaCl (25 mL), and the organic layer was separated. The aqueous layer was extracted with DCM (2 × 30 mL), and the combined organic extracts were washed with saturated NaCl (50 mL), dried (MgSO 4 ), filtered, and concentrated to afford the crude product. The product was purified on a silica gel column eluted with hexanes-EtOAc