Evaluation of A22 Analogs as Gram-negative Antimicrobial Agents

doi:10.21203/rs.3.rs-1862196/v1

S-(3,4-dichlorobenzyl) isothiourea (A22) and mecillinam are the only known antibacterial agents that act on the rod-shape maintenance mechanism of rod-like bacteria. This makes A22 an attractive lead compound for developing antibiotics with novel mechanism of action. In this study we expanded a limited structure activity relation (SAR) study previously reported and determined the effect of fused carbocyclic and heterocyclic analogs of A22 against three Gram-negative bacteria. Also, the efflux liabilities of the analogs generated here were examined in E. coli and P. aeruginosa.

A22

MreB inhibitors

isothiourea

Gram-negative bacteria

antimicrobial agents

Multidrug-resistant bacteria pose a significant emerging worldwide health threat[1]. Infection involving antibiotic-resistant bacteria are responsible for at least 2 million serious morbidities in the US every year[2]. Around 23,000 fatalities have been reported as a result of antibiotic-resistant infections[2]. This situation is further aggravated by the lack of discovery of new antimicrobials. Antibiotics with unique mechanisms of action are urgently needed to replenish the dwindling antibiotic arsenal and to address the grim scenario of resistance to the antibiotics currently in use at the clinic. The fundamental process of bacterial cytokinesis[3], which is central to bacterial propagation, has emerged as a new prospective target for designing novel antimicrobials. Crucial to cytokinesis is the formation of the Z-ring by the structural protein FtsZ, a bacterial homolog of tubulin. FtsZ hydrolyses GTP to form small filaments that make up a septum which serves as a cytoskeletal scaffold for recruitment of other cell division proteins, ultimately leading to the formation of a divisome [4–6]. FtsZ is highly conserved within the bacterial kingdom and required for bacterial cell survival[7]. Moreover, FtsZ is absent in higher eukaryotes. These fundamental features of FtsZ highlight it as a potential novel target that could be utilized for the discovery of antibiotics against multidrug-resistant bacteria[8].

Several synthetic and natural compounds have been identified as inhibitors of FtsZ. These inhibitors target GTP hydrolysis, FtsZ assembly, or the interaction between FtsZ and other proteins[9]. In this context, TAXIS Pharmaceuticals, Inc. has over a decade of experience associated with the development and evaluation of agents that specifically affect FtsZ. This effort has resulted in the successful development of TXA709 (Fig. 1), a FtsZ-targeting antibiotic against methicillin-resistant Staphylococcus aureus (MRSA), currently undergoing Phase 1 clinical trial[10].

While cell division is governed by FtsZ, bacterial cell elongation is controlled by MreB, a prokaryotic homolog of the protein actin [11]. MreB is capable of forming macromolecular structures through polymerization of filaments driven by ATP hydrolysis, which in turn act as a regulatory timing mechanism to induce depolymerization. MreB is a shape-determining factor conserved in rod-shaped bacteria [12]. It primarily coordinates cell wall biogenesis by interacting with the cell wall elongation system via a membrane protein complex that acts together with peptidoglycan enzymes to deliver lateral peptidoglycan, generating a rod-shape cell [13–16]. This interaction is facilitated thanks to a helical filamentous structure that MreB forms in the inner face of the membrane recently revealed by high-resolution microscopy in vivo [17, 18, 13]. MreB function is imperative for appropriate localization of enzymes involved in peptidoglycan and murein synthesis. Not all aspects of MreB structure and function have been completely elucidated. In Gram-negative bacteria, like E. coli, only a single form of this protein has been observed [19]. Driven by its absence in higher eukaryotes, as well as its pivotal role in terms of bacterial cell shape, cell division and chromosome segregation, MreB has been described as a viable target for the development of novel antibiotics.

S-(3,4-dichlorobenzyl) isothiourea, named A22 (Fig. 2), is one of the best known MreB inhibitors and has been widely studied [20]. A preliminary structure activity relationship (SAR) of S-benzylisothiourea derivatives and related compounds revealed that A22 showed antibacterial activity toward Escherichia coli (MG1655), and Salmonella typhimurium (NBRC13245) at a relatively low concentration (3.13 µg/mL) but was less effective against Gram-positive bacteria (100 µg/mL for Bacillus subtilis and more than 100 µg/mL for Staphylococcus aureus (ATCC 29213)[21, 22]. S-(4-Chlorobenzyl)isothiourea, an analog of A22, showed similar antibacterial activity against these bacteria [22]. A cyclic analog of A22, 1,2,3,4-tetrahydro-1,3,5-triazine, showed anti-Pseudomonas activity against wild-type as well as efflux pump deleted strains equally at a same concentration [23]. Both A22 and its triazine analog showed potential antibacterial activity against P. aeruginosa (4 µg/mL) but not Klebsiella pneumoniae, Acinetobacter baumannii, and S. aureus. Also, the activity of A22 and its cyclic analogs were comparable when tested against P. aeruginosa wild-type strain PAO1 and the N150 mutant strain lacking MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM efflux pumps, whereas levofloxacin exhibited 32-fold weaker inhibition against PAO1 than N150 [23]. A22 induces spherical cells and spherical anucleate cells in E. coli. The spherical cells induced by A22 treatment varied in size, and anucleate cells seemed to be more frequent among the smaller cells. These results suggest that loss of the rod shape in E. coli led to asymmetrical cell division resulting in faulty chromosome segregation. How A22 inhibits MreB function is still under investigation. However, a tentative notion argues in favor of A22 blocking the inorganic phosphate release channel of MreB and thus hindering ATP hydrolysis [24, 25]. Nevertheless, A22 remains the only MreB inhibitor possessing interesting antibiotic properties against some important Gram-negative pathogens.

So far the limited SAR studies of A22 derivatives primarily aimed to evaluate the effects of substitutions on the S-benzyl ring. Moreover, these studies have been further limited to monocyclic S-benzyl groups without any investigations into fused carbocycles or heterocyclic counterparts. In this study we investigate the activities of various isothioureas that differ in the substitution patterns within the monocyclic S-benzyl groups in addition to isothioureas fused to carbocycles or heterocycles against the Gram-negative bacteria E. coli, P. aeruginosa and K. pneumoniae. Additionally, we explored the effects of the S-linked sidechains on the antimicrobial properties of these novel analogs.

Chemistry

The synthetic schemes of all isothioureas are depicted in schemes 1-4. Scheme 1 represents the synthesis of substituted S-benzyl isothioureas 3a-3g and 4 from requisite benzyl bromides 2a-g which were either purchased from commercial sources or prepared from the corresponding benzyl alcohol in two ways; (i) by reacting with PBr₃ or (ii) under Appel reaction conditions (PPh₃ and CBr₄). For the final isothiourea salts, benzyl bromides or chlorides and thiourea are refluxed in ethanol for 2-3 hours. Following ethanol removal, the resulting residue were suspended in dichloromethane to produce the final compounds as hydrobromide or hydrochloride salts.

Scheme 2 represents the preparation of fused carbocyclic or heterocyclic analogs following the same method described above for 3a-g. While bromomethyl naphthalene intermediates are commercially available, the quinoline and isoquinoline intermediates 12c-e were synthesized from the corresponding commercially available starting materials. 6-quinolinylmethanol was directly converted to the corresponding bromide under Appel reaction conditions. Similarly, 8-bromomethyl quinoline was prepared from 8-methylquinoline under free radical bromination conditions (NBS, AIBN, CCl₄). On the other hand, the isoquinoline isothioureas 13e was prepared in two steps: reduction of isoquinoline aldehyde to the corresponding methyl alcohol using NaBH₄ and then conversion of the alcohol to the corresponding bromide.

Benzofuran, benzothiophene and indole isothioureas 16a-c (Scheme 3) were prepared from the commercially available 5-methyl intermediates 14a-c in two steps as described in schemes 1 and 2.

The synthesis of specialized isothioureas (19, 22, 23 and 24) modified with respect to the alkyl chains and the isothiourea functionality are depicted in scheme 4. In order to assess the effect of the chain length, an ethyl linked isothiourea analog, 19, was prepared from the commercially available alcohol 17 in two steps as described in schemes 1-3. Similarly, the methyl substituted analog 22 was prepared to assess the effect of α-substitution on the benzyl side chain. The cyclized isothiourea 23 and the methyl substituted isothiourea 24 were prepared as described earlier [23].

Results

First, the isothiourea salts were evaluated for their antimicrobial activities against three Gram-negative bacteria P. aeruginosa ATCC 27853, E. coli ATCC 25922 and K. pneumoniae ATCC 10031 by determining their minimal inhibitory concentration (MIC) in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines for broth microdilution. The results are presented in Table 1. It appears that A22 and all its analogs are ineffective against P. aeruginosa ATCC 27853 strain (MICs > 256 µg/mL) except for compound 16a, the benzofuran analog possessing respectable MIC of 16 µg/mL.

The results against E. coli ATCC 25922 are somewhat similar to the P. aeruginosa activities. Most of the isothioureas including A22 possess very weak potencies against this strain of E. coli. While most of the analogs have MICs > 256 µg/mL, compounds 3f and 3g (3 and 4-biphenyl isothioureas) do show some hint of activitiy with MICs = 64 µg/mL. Similarly, most of the compounds are ineffective against K. pneumoniae ATCC 10031 except for 13b, 16a, 16b and 22 with MICs ranging between 16-32 µg/mL. In comparison, the control antibiotic azithromycin was equally ineffective against P. aeruginosa (MIC = 32 µg/mL) whereas it showed weak activity against E. coli and K. pneumoniae (4 µg/mL).

The results in Table 1 were surprising as it is well documented that A22 is a potent inhibitor of MreB in E. coli albeit against other E. coli strains [22, 21]. Iwai et al. reported an MIC of 3.13 µg/mL against E. coli (MG1655). Similarly, Yamachika et al. has reported an MIC of 4 µg/mL against P. aeruginosa PAO1 strain. We reasoned that the resistant determinants of the strains used in this study might be different from strains used in other studies and hence the difference in activity profile.

One of the known mechanisms of antimicrobial resistance in Gram-negative bacteria are efflux pumps systems which facilitate the extrusion of a wide range of compounds [26-28]. Many of these efflux pumps in Gram-negative bacteria belong to the resistance-nodulation-cell division (RND) family of tripartite efflux pumps [29, 30]. A good example of this is the AcrAB-TolC efflux pump system in E. coli known to be responsible for the extrusion of a broad range of compounds such as lipophilic antimicrobial drugs, antibiotics, various dyes, detergents, organic solvents, and others [31]. Thus, we assessed the antibacterial activity of A22 and its analogs in E. coli strains where the AcrAB-TolC efflux pump is overexpressed or inactivated as well as their isogenic wild-type strain (Table 2). In strains CGSC #11844 and CGSC #11268 the acrR and marR genes have been deleted respectively resulting in AcrAB upregulation. In strain CGSC #11430 the tolC gene has been deleted resulting in inactivation of the AcrAB-TolC efflux pump.

Table 1. MICs (µg/mL) of A22 and analogs against P. aeruginosa ATCC 27853, E. coli ATCC 25922 and K. pneumoniae (ATCC 10031)

Compound	MIC (µg/mL)
Compound	*P. aeruginosa* ATCC 27853	*E. coli* ATCC 25922	*K. pneumoniae* ATCC 10031
3a (A22)	>256	64	4
3b	>256	>256	256
3c	>256	256	>64
3d	>256	>256	>256
3e	>256	>256	>64
3f	>256	64	64
3g	>256	64	64
5	>256	>256	>64
13a	>256	>256	>64
13b	>256	>256	16
13c	>256	>256	>64
13d	>256	>256	>64
13e	>256	>256	>64
16a	16	>256	32
16b	>256	>256	16
16c	>256	>256	>64
19	>256	256	>64
22	>256	256	32
23	>256	>256	ND
24	>256	>256	ND
Azithromycin	32	4	4
ND, not done

The results of this study are presented in Table 2. A22 and several of its analogs show good potencies against wild-type E. coli strain CGSC #7636. Compound 3b, the 2, 4-dichloro analog of A22, shows identical MICs against wild-type strain (2 µg/mL). Notably, 4-bromo benzyl analog (3d) and benzothiophene analog (16b) also show promising activities (8 µg/mL). It appears that 3a, 3b, 3d, 3g, 13b, 16b and 22 are the substrates of the AcrAB-TolC efflux pump. Strikingly, any isothiourea containing a nitrogen head group as in pyridine analog 5, quinolines 13c and 13d, isoquinoline 13e and indole isothiourea 16d are devoid of any antimicrobial activities irrespective of the efflux pumps characteristics of these E. coli strains.

Table 2. MICs (µg/mL) of A22 and analogs against E. coli efflux mutants

Antibiotics	MIC (µg/mL)
Antibiotics	CGSC #7636 (WT)	CGSC #11844 *(∆acrR; ↑AcrAB)*	CGSC #11268 *(∆marR; ↑AcrAB-TolC)*	CGSC #11430 *(∆tolC)*
3a (A22)	2	4	4	1
3b	2	2	2	0.5
3c	>64	>64	>64	>64
3d	8	4	4	2
3e	>64	>64	>64	>64
3f	64	64	64	64
3g	32	64	64	16
5	>64	>64	>64	>64
13a	>64	>64	>64	>64
13b	16	16	16	4
13c	>64	>64	>64	>64
13d	>64	>64	>64	>64
13e	>64	>64	>64	>64
16a	16	32	32	16
16b	8	16	16	4
16c	>64	>64	>64	>64
19	>64	>64	>64	>64
22	32	64	32	8
Azithromycin	4	4	4	1

In order to further validate that A22-related types of compounds are indeed substrates of RND-type efflux pumps like the E. coli AcrAB-TolC system mentioned above or the Mex system in P. aeruginosa, we evaluated the impact of TXA09155, an in-house efflux pump inhibitor (EPI) developmental candidate [32] (Figure 3), on the activity of A22 and its analogs (Table 3). Use of TXA09155 lowered the MICs of A22 and some of its analogs significantly against wild-type P. aeruginosa strain while its effect on E. coli was less pronounced.

Table 3. Screening of A22 and analogs against P. aeruginosa ATCC 27853 and E. coli ATCC 25922 in combination with efflux pump inhibitor TXA09155 (12.5 μg/mL)

Compounds	*P. aeruginosa* ATCC 27853		*E. coli* ATCC 25922
Compounds	MIC alone (μg/mL)	MIC with TXA09155	MIC alone (μg/mL)	MIC with TXA09155
3a (A22)	>256	0.5	64	4
3b	>256	1.0	>256	>256
3c	>256	256	256	256
3d	>256	1.0	>256	>256
3e	>256	>256	>256	>256
3f	>256	>256	64	64
3g	>256	>256	64	64
5	>256	>256	>256	>256
13a	>256	>256	>256	>256
13b	>256	4	>256	16
13c	>256	>256	>256	>256
13d	>256	>256	>256	>256
13e	>256	>256	>256	>256
16a	16	4	>256	>256
16b	>256	4	>256	>256
16c	>256	>256	>256	>256
22	>256	4	256	256
23	>256	16	>256	16
24	>256	4	>256	>256

Although A22 and some of its analogs were reported to have potent activities against certain E. coli and P. aeruginosa strains, unfortunately they were ineffective against the bacteria strains used in this study. After testing 19 varied analogs of A22, none of them improved on the antibacterial potency of A22 substantially. The monocyclic S-benzyl isothioureas remain to be the most active compounds while fused carbocycles or heterocycles did not show improved antibacterial activities. It appears that some of the analogs are substrate of both E. coli and P. aeruginosa efflux pumps. It is concluded that A22 and its analogs described in this study may not be ideal for development of a broad-spectrum MreB targeting antibiotic agent due to their limited antibiotic effects against certain E. coli and P. aeruginosa strains.

Synthesis reagents, Analytical Instruments and reaction conditions

All reactions, unless otherwise stated, were done under nitrogen atmosphere. Reaction monitoring and follow-up were done using aluminum backed Silica G TLC plates (UV254 e Sorbent Technologies), visualizing with ultraviolet light. Flash column chromatography was done on a Combi Flash Rf Teledyne ISCO using hexane, ethyl acetate, dichloromethane, and methanol. The ¹H (300 MHz or 300 MHz) NMR spectra were done in CDCl₃, Methanol-d₄, and DMSO-d₆ and recorded on a Varian Oxford 300 MHz Multinuclear NMR Spectrometer. Data is expressed in parts per million relative to the residual nondeuterated solvent signals, spin multiplicities are given as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), dt (doublet of triplets), q (quartet), m (multiplet), bs (broad singlet), and bt (broad triplet), and coupling constants (J) are reported in Hertz. HRMS was collected on a LTQ Orbitrap Mass Spectrometer from Thermo Electron CorportaionThe final compound tested for biological activity was analyzed for purity using Shimadzu LCMS-2020 system monitoring absorbances at both 254 and 280 nm. All chemical reagents used in this manuscript were purchased either from Sigma-Aldrich (St. Louis, USA) or from Combi-Blocks Inc. (San Diego, USA).

Experimental

General Procedure:

Preparation of benzyl bromides under Appel reaction conditions

Bromophenyl methanol (1.0 mmol), triphenylphosphine (1.5 mmol) and tetrabromomethane (1.5 mmol) were dissolved in dichloromethane (8 mL). The reaction mixture was stirred overnight in the room temperature. The reaction mixture was concentrated under reduced pressure. The residue was purified on an ISCO chromatography (0-10% ethyl acetate/hexane) to give the products.

Preparation of bromides using PBr₃

Bromophenyl phenol (1.0 mmol) and phosphorus tribromide (1.1 mmol) were dissolved in dichloromethane (6.0 mL). The reaction mixture was stirred for overnight at room temperature. The reaction mixture was quenched with ice water. The reaction mixture was diluted with dichloromethane, and it was washed with saturated sodium bicarbonate followed by brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to give the crude products.

1-Bromo-4-(bromomethyl)benzene (2d)

White solid. Yield: 83%. ¹H NMR (300 MHz) (CDCl₃) δ 7.49-7.46 (m, 2H), 7.28-7.25 (m, 2H), 4.44 (s, 2H).

1-Bromo-3-(bromomethyl)benzene (2e)

Colorless oil. Yield: 56%. ¹H NMR (300 MHz) (CDCl₃) δ 7.55 (s, 1H), 7.43 (d, J = 8 Hz, 1H), 7.32 (d, J = 8 Hz, 1H), 7.22 (t, J = 8 Hz, 1H), 4.43 (s, 2H).

4-(Bromomethyl)-4'-fluoro-1,1'-biphenyl (2f)

Colorless solid. Yield: 73%. ¹H NMR (300 MHz) (CDCl₃) δ 7.57-7.45 (m, 6H), 7.16-7.11 (m, 2H), 4.55 (s, 2H).

3-(Bromomethyl)-4'-fluoro-1,1'-biphenyl (2g)

White solid. Yield: 48%. ¹H NMR (300 MHz) (CDCl₃) δ 7.56-7.54 (m, 3H), 7.47 (dd, J = 8 Hz, J = 2 Hz, 1H), 7.46-7.39 (m, 2H), 7.13 (t, J = 8 Hz, 2H), 4.55 (s, 2H).

8-(bromomethyl)quinoline (12c)

White solid. Yield: 82%. ¹H NMR (300 MHz) (CDCl₃) δ 9.03-9.01 (m, 1H), 8.18-8.15 (d, 1H), 7.85-7.79 (m, 2H), 7.54-7.43 (m, 2H). 5.25 (s, 2H).

6-(bromomethyl)quinoline (12d)

Orange solid. Yield: 76%. ¹H NMR (300 MHz) (CDCl₃) δ 8.93-8.91 (m, 1H), 8.16-8.09 (m, 2H), 7.83-7.82 (d, 1H), 7.77-7.73 (dd, 1H), 7.45-7.41 (q, 1H), 4.67 (s, 2H).

5-(bromomethyl)isoquinoline (12e)

Orange solid. Yield: 74%. ¹H NMR (300 MHz) (CDCl₃) δ 9.32 (s, 1H), 8.67-8.65 (d, 1H), 8.01-7.95 (m, 2H), 7.79-7.77 (d, 1H), 7.60-7.55 (m, 1H), 4.92 (s, 2H).

5-(Bromomethyl)benzofuran (15a)

Colorless oil. Yield: 78%. ¹H NMR (300 MHz) (CDCl₃) δ 7.65-7.63 (m, 2H), 7.48 (d, J = 8 Hz, 1H), 7.34 (dd, J = 9 Hz, J = 2 Hz, 1H), 6.76-6.75 (m, 1H), 4.64 (s, 2H).

5-(Bromomethyl)benzo[b]thiophene (15b)

White solid. Yield: 41%. ¹H NMR (300 MHz) (CDCl₃) δ 7.87-7.85 (m, 2H), 7.48 (d, J = 6 Hz, 1H), 7.39 (dd, J = 8 Hz, J = 2 Hz, 1H), 7.32 (dd, J = 6 Hz, 1H), 4.66 (s, 2H).

15c: t-Butyl 5-(bromomethyl)-1H-indole-1-carboxylate (15c)

Colorless oil. Yield: 17%. ¹H NMR (300 MHz) (CDCl₃) δ 8.12 (d, J = Hz, 1H), 7.62 (d, J = 4 Hz, 1H), 7.58 (d, J = 1 Hz, 1H), 7.35 (dd, J = 8 Hz, J = 2 Hz, 1H), 6.55 (d, J = 5 Hz, 1H), 4.64 (s, 2H), 1.68 (s, 9H).

4-(2-Bromoethyl)-1,2-dichlorobenzene (18)

Colorless oil. Yield: 33%. ¹H NMR (300 MHz) (CDCl₃) δ 7.39 (d, J = 8 Hz, 1H), 7.31 (d, J = 2 Hz, 1H), 7.06 (dd, J = 8 Hz, J = 2 Hz, 1H), 3.54 (t, J = 7 Hz, 2H), 3.12 (t, J = 7 Hz, 2H).

4-(1-Bromoethyl)-1,2-dichlorobenzene (21)

White solid. Yield: 77%. ¹H NMR (300 MHz) (CDCl₃) δ7.53 (s, 1H), 7.43-7.40 (d, 1H), 7.29-7.26 (d, 1H), 5.12-5.07 (q, 1H), 2.03-2.00 (s, 3H).

Preparation of Isothioureas

Bromomethyl intermediates (1.0 mmol) and thiourea (0.8 mmol) were dissolved in ethanol (10 mL). The reaction mixture was refluxed for 2 hours. The reaction mixture was cooled, and ethanol was removed under reduced pressure. The resulting residue was suspended in dichloromethane. The suspension was filtered to give products as solids.

3,4-dichlorobenzyl methylcarbamimidothioate hydrobromide (3a)

White solid. Yield: 86%. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.23 (s,1H), 9.04 (s, 1H), 7.72 (s, 1H), 7.72-7.64 (d, 1H), 7.44-7.41 (d, 1H), 4.51 (s, 2H); ¹³C NMR (75 MHz, DMF-d₇) δ 169.99, 135.16, 132.28, 131.89, 130.77, 130.69, 128.73, 33.82. HRMS (ESI) m/z [M + H]⁺ calcd. for C₈H₉Cl₂N₂S⁺ 234.9858, found 234.9861.

2,4-Dichlorobenzyl carbamimidothioate hydrobromide (3b)

White solid. Yield: 81%. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.14 (bs, 4H), 7.72 (d, J = 2 Hz, 1H), 7.58 (d, J = 9 Hz, 1H), 7.48 (dd, J = 8 Hz, J = 2 Hz, 1H), 4.54 (s, 2H); ¹³C NMR (75 MHz, DMF-d₇) δ 169.93, 134.56, 134.05, 132.49, 131.51, 129.15, 127.62, 32.19. HRMS (ESI) m/z [M + H]⁺ calcd. for C₈H₉Cl₂N₂S⁺ 234.9858, found 234.9861.

4-(t-Butyl)benzyl carbamimidothioate hydrobromide (3c)

White solid. Yield: 42%. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.12 (bs, 2H), 8.94 (bs, 2H), 7.38 (dd, J = 9 Hz, J = 2 Hz, 2H), 7.31 (d, J = 8 Hz, 2H), 4.42 (s 2H), 1.25 (s, 9H); ¹³C NMR (75 MHz, DMF-d₇) δ 170.56, 150.75, 131.65, 128.83, 125.57, 34.15, 30.56. HRMS (ESI) m/z [M + H]⁺ calcd. for C₁₂H₁₉N₂S⁺ 223.1264, found 223.1266.

4-Bromobenzyl carbamimidothioate hydrobromide (3d)

White solid. Yield: 97 %. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.21 (s, 2H), 9.01 (s, 2H), 7.59-7.55 (m, 2H), 7.40-7.36 (m, 2H), 4.48 (s, 2H); ¹³C NMR (75 MHz, DMF-d₇) δ 169.95, 134.62, 131.48, 131.03, 121.12, 33.58. HRMS (ESI) m/z [M + H]⁺ calcd. for C₈H₁₀BrN₂S⁺ 244.9743, found 244.9746.

3-Bromobenzyl carbamimidothioate hydrobromide (3e)

White solid. Yield: 99%. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.17 (bs, 2H), 8.99 (bs, 2H), 7.64 (s, 1H), 7.52 (d, J = 8 Hz, 1H), 7.41 (d, J = 8 Hz, 1H), 7.34 (t, J = 8 Hz, 1H), 4.45 (s, 2H); ¹³C NMR (75 MHz, DMF-d₇) δ 169.87, 137.95, 131.65, 130.70, 130.55, 127.92, 121.67, 33.42. HRMS (ESI) m/z [M + H]⁺ calcd. for C₈H₁₀BrN₂S⁺ 244.9743, found 244.9746.

(4'-Fluoro-[1,1'-biphenyl]-4-yl)methyl carbamimidothioate hydrobromide (3f)

White solid. Yield: 95%. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.21 (s,2H), 9.02 (s, 2H), 7.74-7.64 (m, 4H), 7.51-7.48 (d, 2H), 7.32-7.25 (m, 2H), 4.54 (s, 2H); ¹³C NMR (75 MHz, CD₃OD) δ 170.76, 164.26, 160.00, 140.10, 136.44, 132.87, 129.40, 128.49, 128.38, 127.10, 115.40, 115.11, 34.74. HRMS (ESI) m/z [M + H]⁺ calcd. for C₁₄H₁₄FN₂S⁺ 261.0856, found 261.0859.

(4'-Fluoro-[1,1'-biphenyl]-3-yl)methyl carbamimidothioate hydrobromide (3g)

White solid. Yield: 89%. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.04 (bs, 4H), 7.69-7.66 (m, 3H), 7.60 (d, J = 9 Hz, 1H), 7.46 (t, J = 8 Hz, 1H), 7.39 (d, J = 7 Hz, 1H), 7.31 (t, J = 9 Hz, 2H), 4.50 (s, 2H); ¹³C NMR (75 MHz, DMF-d₇) δ 170.31, 139.80, 136.20, 135.62, 129.17, 128.56, 128.47, 127.80, 127.36, 126.09, 115.50, 115.22, 34.33. HRMS (ESI) m/z [M + H]⁺ calcd. for C₁₄H₁₄FN₂S⁺ 261.0856, found 261.0858.

(6-(trifluoromethyl)pyridin-3-yl)methyl carbamimidothioate hydrochloride (5)

White solid. Yield: 87%. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.38 (s, 2H), 9.18 (s, 2H), 8.82 (s, 1H), 8.15-8.12 (d, 1H), 7.96-7.94 (d, 1H), 4.65 (s, 2H); ¹³C NMR (75 MHz, DMF-d₇) δ 170.47, 151.36, 147.59, 247.13, 146.68, 146.27, 139.36, 137.10, 124.32, 121.42, 121.39, 120.69, 31.66. HRMS (ESI) m/z [M + H]⁺ calcd. for C₈H₉F₃N₃S⁺ 236.0464, found 236.0466.

Naphthalen-1-ylmethyl carbamimidothioate hydrobromide (13a)

White solid. Yield: 95%. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.24 (s, 2H), 9.05 (s, 2H), 8.19-8.16 (d, 1H), 7.99-7.92 (dd, 2H), 7.65-7.47 (m, 4H), 4.99 (s, 2H); ¹³C NMR (75 MHz, DMF-d₇) δ 170.66, 133.84, 131.13, 129.85, 129.11, 128.76, 128.31, 126.64, 126.12, 125.44, 123.73, 32.93. HRMS (ESI) m/z [M + H]⁺ calcd. for C₁₂H₁₃N₂S⁺ 217.0794 found 217.0797.

Naphthalen-2-ylmethyl carbamimidothioate hydrobromide (13b)

White solid. Yield: 98%. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.23 (s, 2H), 9.00 (s, 2H), 7.95-7.87 (m, 4H), 7.56-7.50 (m, 3H), 4.66 (s, 2H); ¹³C NMR (75 MHz, DMF-d₇) δ 170.40, 133.07, 132.70, 132.40, 128.51, 127.97, 127.62, 127.54, 126.71, 126.42, 126.31, 34.77. HRMS (ESI) m/z [M + H]⁺ calcd. for C₁₂H₁₃N₂S⁺ 217.0794 found 217.0797.

Quinolin-8-ylmethyl carbamimidothioate hydrobromide (13c)

White solid. Yield: 90 %. ¹H NMR (300 MHz) (DMF-d₇) δ 9.44 (s, 2H), 8.99-8.98 (m, 2H), 8.48-8.45 (d, 1H), 8.02-8.00 (d, 1H), 7.93-7.91 (d, 1H), 7.66-7.59 (m, 2H), 4.98 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 170.67, 150.54, 145.23, 137.65, 133.45, 131.20, 129.30, 128.60, 126.96, 122.43, 31.22. HRMS (ESI) m/z [M + H]⁺ calcd. for C₁₁H₁₂N₃S⁺ 218.0747 found 218.0750.

Quinolin-6-ylmethyl carbamimidothioate hydrobromide (13d)

Brown solid. Yield: 89 %. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.24 (s, 2H), 9.00 (s, 2H), 8.94-8.92 (d, 2H), 8.41-8.39 (d, 1H), 8.06-8.02 (m, 2H), 7.83-7.79 (d, 2H), 7.61-7.57 (dd, 1H), 4.70 (s, 2H). ¹³C NMR (75 MHz, DMF-d₇) δ 169.99, 150.14, 146.22, 136.73, 133.70, 130.68, 128.60, 128.08, 127.76, 121.75, 34.26. HRMS (ESI) m/z [M + H]⁺ calcd. for C₁₁H₁₂N₃S⁺ 218.0747 found 218.0748.

Isoquinolin-5-ylmethyl carbamimidothioate hydrobromide (13e)

Brown solid. Yield: 66 %. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.42 (s,1H), 9.28 (s, 2H), 9.08 (s, 2H), 8.63-8.62 (d, 1H), 8.18-8.15 (m, 2H), 7.93-7.91 (d, 1H), 7.73-7.68 (m, 1H), 5.01 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 169.34, 153.11, 142.79, 134.23, 133.05, 130.46, 129.28, 127.89, 117.73, 32.18. HRMS (ESI) m/z [M + H]⁺ calcd. for C₁₁H₁₂N₃S⁺ 218.0747 found 218.0749.

Benzofuran-5-ylmethyl carbamimidothioate hydrobromide (16a)

White solid. Yield: 100%. ¹H NMR (300 MHz) (DMSO-d₆) δ 9.14 (bs, 2H), 8.94 (bs, 2H), 8.02 (d, J = 2 Hz, 1H), 7.69 (s, 1H), 7.60 (d, J = 8 Hz, 1H), 7.34 (dd, J = 9 Hz, J = 2 Hz, 1H), 6.96 (d, J = 1 Hz, 1H), 4.55 (s, 2H); ¹³C NMR (75 MHz, DMF-d₇) δ 170.40, 154.23, 146.70, 129.30, 127.77, 125.51, 122.01, 111.42, 106.54, 34.72. HRMS (ESI) m/z [M + H]⁺ calcd. for C₁₀H₁₁N₂OS⁺ 207.0587 found 207.0591.

Benzo[b]thiophen-5-ylmethyl carbamimidothioate hydrobromide (16b)

White solid. Yield: 84%; ¹H NMR (300 MHz) (DMSO-d₆) δ 9.16 (bs, 2H), 8.94 (bs, 2H), 8.01 (d, J = 8 Hz, 1H), 7.90 (s, 1H), 7.81 (d, J = 5 Hz, 1H), 7.45 (d, J = 6 Hz, 1H), 7.39 (d, J = 8 Hz, 1H), 4.58 (s, 2H); ¹³C NMR (75 MHz, DMF-d₇) δ 170.26, 139.82, 138.99, 130.78, 127.91, 125.02, 123.91, 123.57, 122.72, 34.63. HRMS (ESI) m/z [M + H]⁺ calcd. for C₁₀H₁₁N₂S₂⁺ 223.0358 found 223.0360.

t-Butyl 5-((carbamimidoylthio)methyl)-1H-indole-1-carboxylate (16c)

Red solid. Yield: 65 %; ¹H NMR (300 MHz) (CD₃OD) δ 8.12 (d, J = 9 Hz, 1H), 7.65 (d, J = 4 Hz, 1H), 7.63 (s, 1H), 7.35 (dd, J = 8 Hz, J = 2 Hz, 1H), 6.61 (d, J = 3 Hz, 1H), 4.53 (s, 2H), 1.67 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.52, 149.40, 134.88, 130.96, 126.73, 125.21, 121.85, 115.69, 107.24, 83.94, 60.37, 36.77, 28.11, 20.98, 14.15. HRMS (ESI) m/z [M + H]⁺ calcd. for C₁₅H₂₀N₃O₂S⁺ 306.1271 found 306.1273.

3,4-Dichlorophenethyl carbamimidothioate (19)

White solid, Yield: 37 %; ¹H NMR (300 MHz) (DMSO-d₆) δ 8.97 (bs, 3H), 7.60-7.57 (m, 2H), 7.28 (d, J = 7 Hz, 1H), 3.41 (t, J = 8 Hz, 2H), 3.12 (t, J = 7 Hz, 2H); ¹³C NMR (75 MHz, DMF-d₇) δ 170.71, 139.98, 131.05, 130.67, 130.18, 129.43, 128.98, 33.14, 30.65. HRMS (ESI) m/z [M + H]⁺ calcd. for C₉H₁₁Cl₂N₂S⁺ 249.0015, found 249.0018.

1-(3,4-Dichlorophenyl)ethyl carbamimidothioate hydrobromide (22)

White solid, Yield: 80%; ¹H NMR (300 MHz) (DMSO-d₆) δ 9.27 (s,2H), 9.04 (s, 2H), 7.76 (s, 1H), 7.68-7.66 (d, 1H), 7.49-7.45 (d, 1H), 5.23-5.21 (q, 1H), 1.62-1.60 (s, 3H); ¹³C NMR (75 MHz, CD₃OD) δ 167.92, 142.24, 131.83, 131.51, 131.27, 129.75, 127.95, 43.81, 21.64. HRMS (ESI) m/z [M + H]⁺ calcd. for C₉H₁₁Cl₂N₂S⁺ 249.0015, found 249.0018.

Isoquinolin-5-ylmethanol (11)

Isoquinoline-5-carbaldehyde (1.00 g, 6.36 mmol) and sodium borohydride (482 mg, 12.8 mmol) were dissolved in methanol (20 mL) at 0 ^oC. The reaction mixture was stirred from 0 ^oC to room temperature for overnight. The resulting mixture was then quenched with ice water. Methanol was removed and the residue was extracted with dichloromethane and washed with water and brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to give the crude product as a brown oil (957 mg, 95%). The crude product was used directly without further purification; ¹H NMR (300 MHz) (CDCl₃) δ 8.89 (s, 1H), 8.21-8.19 (d, 1H), 7.69-7.67 (d, 1H), 7.64-7.60 (m, 2H), 7.39-7.33 (m, 1H), 4.96 (s, 2H).

3-methyl-6-((naphthalen-2-ylmethyl)thio)-1,2,3,4-tetrahydro-1,3,5-triazine (23)

Methyl amine (32 µL, 0.68 mmol) was mixed well with formaldehyde (135 μL of a 37% wt. solution in water, 1.36 mmol) in dioxane (20 mL). To this solution, naphthalen-2-ylmethyl carbamimidothioate hydrobromide (200 mg, 0.68 mmol) was added. The reaction mixture was heated until a solution was obtained and allowed to stir overnight at room temperature. The residue was purified on an ISCO chromatograph (10% methanol/methylene chloride + 1% ammonia) to give the product as a white solid (123 mg, 67%); ¹H NMR (300 MHz) (CDCl₃) δ 7.90 (s, 1H), 7.73-7.70 (m, 4H), 7.44-7.42 (m, 3H), 4.76 (s, 2H), 4.18 (m, 4H), 2.14 (s, 3H). ¹³C NMR (75 MHz, CD₃OD-CDCl₃) δ 170.82, 133.25, 133.02, 131.05, 130.38, 128.96, 128.32, 128.20, 127.68, 126.59, 126.12, 63.62, 38.80, 35.79. MS (ESI) m/z [M + H]⁺ calcd. for C₁₅H₁₈N₃S⁺ 272.1216, found 272.1218.

3,4-dichlorobenzyl methylcarbamimidothioate hydrobromide (24)

4-(bromomethyl)-1,2-dichlorobenzene (500 mg, 2.08 mmol) and N-methylthiourea ( 156 mg, 1.74 mmol) were dissolved in ethanol (20 mL). The reaction mixture was refluxed for 2 hours. The reaction mixture was cooled, and ethanol was removed under reduced pressure. The resulting residue was suspended in dichloromethane. The suspension was filtered to give a product as a white solid (371 mg, 65%); ¹H NMR (300 MHz) (DMSO-d₆) δ 9.77 (s,1H), 9.47 (s, 1H), 9.15(s,1H), 7.71 (s, 1H), 7.63-7.61 (d, 1H), 7.41-7.39 (d, 1H), 4.58 (s, 2H), 2.84 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆andDMF-d₇) δ 165.39, 137.46, 131.53, 131.23, 131.14, 130.96, 129.59, 33.78, 30.91. MS (ESI) m/z [M + H]⁺ calcd. for C₉H₁₁Cl₂N₂S⁺ 249.0015, found 249.0017.

MIC assays

MIC assays were conducted in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines for broth microdilution [28]. Briefly, log-phase bacteria were added to 96-well microtiter plates (at 5 × 10⁵ CFU/mL) containing 2-fold serial dilutions of compound or comparator drug in cation-adjusted Mueller-Hinton (CAMH) broth, with each compound concentration being present in duplicate. The final volume in each well was 0.1 mL, and the microtiter plates were incubated aerobically for 24 h at 37 °C. Bacterial growth was then monitored by measuring the optical density at 600 nm using a VersaMax plate reader (Molecular Devices, Inc., San Jose, CA, USA), with the MIC being defined as the lowest compound concentration that inhibited growth.

Bacterial Strains

E. coli strains CGSC #7636 (WT), CGSC #11844 (∆acrR; ↑AcrAB), CGSC #11268 (∆marR; ↑AcrAB-TolC) and CGSC #11430 (∆tolC) were obtained from the Coli Genetic Stock Center (YALE University, New Haven, CT, USA). E. coli ATCC 25922, K. pneumoniae ATCC 10031 and P. aeruginosa ATCC 27853, were obtained from the American Type Culture Collection (Manassas, VA, USA).

Conflict of Interest

Ajit K. Parhi, Jesus Rosado-Lugo, Pratik Datta, Yongzheng Zhang and Yi Yuan are associated with TAXIS Pharmaceuticals Inc. and thus declare conflicts of interest.

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Scheme 1 to 4 are available in Supplementary Files section.

Scheme1.png
Scheme 1. Preparation of monocyclic aryl methyl isothioureas: (i) PBr₃, DCM, rt, 12h; (ii) CBr₄, PPh₃, DCM; (iii) thiourea, EtOH, reflux, 3h
Scheme2.png
Scheme 2. Synthesis of fused aryl methyl isothioureas. (i) PBr₃, DCM, rt, 12h; (ii) NBS, AIBN, CCl₄, reflux, 3h; (iii) NaBH_4, MeOH, 0 ^oC-rt, 12h, 95%; (iv) thiourea, EtOH, reflux, 3h
Scheme3.png
Scheme 3. Synthesis of five membered fused heterocyclic isothioureas, (i) NBS, AIBN, CCl₄, reflux, 3h; (ii) thiourea, EtOH, reflux, 3h.
Scheme4.png
Scheme 4. Synthesis of specialized isothioureas, (i) PPh₃, CBr₄, DCM, rt, 12h; (ii) thiourea, EtOH, reflux, 3h; (iii) PBr₃, DCM, rt, 12h; (iv) MeNH₂, HCHO, dioxane, heating to rt, 12h; (v) N-methylthiourea, EtOH, reflux, 3h.

Evaluation of A22 Analogs as Gram-negative Antimicrobial Agents

Status:

Version 1

Abstract

Figures

Introduction

Results And Discussion

Conclusions

Materials And Methods

Declarations

References

Scheme

Supplementary Files

Status:

Version 1