Design, Synthesis and Antibacterial Activities of Triazole-pyrimidine Derivatives as SecA Inhibitors

SecA, a key component of bacterial Sec-dependent secretion pathway, is an attractive target for exploring novel antimicrobials. Along this line, we reported optimization of a hit bistriazole (SCA 21) which has been previously identied as a SecA inhibitor. Herein we describe the synthesis of some novel triazole-pyrimidine derivative by structural modication of SCA 21. Some of them have been evaluated for their antimicrobial activity against against Escherichia coli NR698 (a leaky mutant), Staphylococcus aureus and Bacillus anthracis Sterne.


Introduction
Bacterial pathogens' infectious diseases have become a serious clinical issue in recent years because of the emergence and spread of drug resistance. [1] Thus, the development of novel antibacterial agents, especially those with a new drug target and/or with the capability to overcome drug resistance is an urgent need. [2] In bacterial cells, more than 30% of proteins become functional after being transported outside the cytoplasm. Trans-membrane movement of most of these proteins is via the Sec pathway (i.e. secretion pathway). SecA constitute a key enzyme of the bacterial protein secretion (Sec) pathways. SecA ATPase, one of the essential components of the Sec machinery provides a major pathway to aid translocation of cytosol proteins across or into the cytoplasmic membrane. [3][4][5][6][7][8][9] SecA is considered as an attractive target by researchers in order to nd novel antibacterial drugs because it is a conserved and essential protein in all bacteria and absent in humans. [10,11] Several studies carried out have shown that inhibition of SecA can lead to bacteriostatic and bactericidal effects [12] . From this perspective, we are interested working in targeting SecA, which is a critical protein secretion machinery essential for bacterial survival. [2,11,13] Currently, small organic molecules described in the literature as SecA inhibitors mainly include Rose Bengal, [14] bisthiouracil, [15] bistriazole [16] and their derivatives, [17] thiazolo [4,5-d]pyrimidine derivatives [18] and others. [19][20][21][22] In order to nd new SecA inhibitors, it is worthwhile to explore the chemistry space of known inhibitor to increase structural diversity.
The results and implications in guiding future work in this area are described below.

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The compound in Figure 1 has been identi ed as SecA inhibitor for further optimization in earlier work [32] .
To enhance the potency, we began to simplify the structure by dissecting the lead compound in half and removing part A. Then by changing the methyl to an alkylamine which have been biotinylated, we hypothesize that these compounds, due to the presence of several nitrogen atoms, would have the ability to form more hydrogen bonding interactions with the target protein SecA, and would be bene cial in enhancing antibacterial activity. Also, by changing the methyl to an azidopentyl, we think that could improve the potency because sodium azide is a well-known SecA inhibitor [33] . The general strategy of analogue design is shown in Figure 2.
Compounds synthesis began by reacting commercially available benzoyl chloride with hydrazine carbothioamide, followed by self-condensation of 2 in the presence of 5% sodium hydroxide under re ux conditions to afford 3 [34] (Scheme 1). Compounds 7 and 15 were prepared by following published procedures in Scheme 2 [35] and Scheme 4 [36] . Compounds 8, 9, 17 and 18 were synthesized by reaction of intermediate 7 and 15 with 2,4,6-trichloro pyrimidine at room temperature under weakly basic conditions [37] . Compounds 10 and 19 were obtained by reaction of 3 with respectively 8 and 17 in the presence of potassium carbonate in acetonitrile. Then compounds 10 were deprotonated in tri uoroacetic acid at room temperature to give compounds 11 [38] . The reaction of 11 with 12 gave compounds 13a, b via a nucleophilic attack by unprotonated amine on the ester group, followed by an amide bond formation and release of N-hydroxysuccinimide (Scheme 3) [39] . SCA 107 was synthesized and tested in our earlier work [32] .

Biological evaluation
The newly synthesized compounds 11a (SCA 257), 11b (SCA 255), 13b (SCA 256) and 19 (SCA 259) were rst screened for their in vitro antibacterial activity using a truncated version of E. coli SecA, EcSecAN68, at 25 μM and at 50 μM. There were three compounds (SCA255, SCA256 and SCA259) that showed greater than 50% inhibition at 50 μM, but only one compound (SCA259) showed more than 50% inhibition at 25 Μm, as shown in Figure 2. SCA 259 showed more potent inhibition against EcSecAN68 than SCA107, which was one of the best triazole-primidine inhibitors from our previous study with IC 50 at 30 μM against EcSecAN68 [32] . SCA 259 was then evaluated further at various concentrations to allow the determination of IC 50 value at 5.9 μM. Thus, SCA 259 was more potent than the lead compound SCA21 with IC 50 at 18 μM against EcSecAN68 [32] . These results suggested that the azide pentyl group is bene cial for potent inhibitory activity. The biotinylated compound SCA 256 was more active than compound SCA 255 with the amine group.
Compounds SCA 257 and SCA 259 were also evaluated for their antimicrobial activities against B. anthracis Sterne, S. aureus 6538 and E. coli NR698. The results are shown in Table 1. It showed that SCA 259 has potent inhibitory activity against the three tested bacterial strains, higher than that of the lead compound SCA 21 [32] . Compound SCA 259 showed MIC at 3.1 μM against B. anthracis Sterne, at 1.6 μM against S. aureus 6538 and at 12.5 μM against E. coli NR698, which are comparable to some of our best compounds in this class.

Conclusion
Some novel triazole-pyrimidine derivatives were designed, synthesized and evaluated as SecA inhibitors. Compound SCA 259 with azide pentyl group was the most potent analogue developed from this study.
This compound expressed better inhibitory activity against SecA ATPase than that of the known inhibitor SCA 21.

Experimental
All chemical reagents and solvents used were of reagent grade or puri ed using standard methods. TLC analyses were conducted on silica gel plates (Sorbent Silica G UV254). Column chromatography was carried out on ash silica gel (Sorbent 230-400 mesh). NMR spectra were recorded at 1 H (400 MHz) and 13 C (100 MHz) on a Bruker instrument. Coupling constants (J) and chemical shifts (δ) are given in hertz and ppm respectively, using TMS ( 1 H NMR) and solvents ( 13 C NMR) as internal standards.
General procedure for the synthesis of 8a, b To a solution of 7 (1.04 mmol) in acetonitrile (5 mL) was added K 2 CO 3 (434 mg, 3.14 mmol) and 2,4,6trichloropyrimidine (230 mg, 1.25 mmol) and the mixture was stirred at room temperature. Upon completion, the resulting mixture was diluted with ethyl acetate and then washed with water and brine.
The organic layer was dried over anhydrous Na 2 SO 4 , ltered, and then concentrated in vacuo. Flash chromatography (hexane/AcOEt) of the residue gave 8.
tert-butyl (2-((4,6-dichloropyrimidin-2yl)thio)ethyl)carbamate 8a (287 mg, 85%) General procedure for the synthesis of 11a, b 10 (0.13 mmol) and tri uoroacetic acid tri uoroacetic acid (2.64mg, 0.26 mmol) was stirred at room temperature until no further gas was released. Diethyl ether (5 ml) was added into the clear solution and stirred. After the solid came out, the mixture was suction ltered. The solid was washed by diethyl ether and dried in a vacuum. The solid was dissolved in water and 10 % NaOH was added drop by drop into the solution to get white crystals in an ice bath. After suction ltration, the crystals were washed with water and dried in a vacuum to obtain 11.
Bacteriostatic effect: Bacteriostatic effects were evaluated at 37°C in 96-well microtitier plates as previously described [17] . Minimum inhibitory concentration (MIC) is the lowest concentration of compounds at which bacterial cells were not able to grow at tested condition.