Synthesis and antibacterial evaluation of new, unsymmetrical triaryl bisamidine compounds

Abstract

Herein we describe the synthesis and antibacterial evaluation of a new, unsymmetrical triaryl bisamidine compound series, [Am]-[indole]-[linker]-[HetAr/Ar]-[Am], in which [Am] is an amidine or amino group, [linker] is a benzene, thiophene or pyridine ring, and [HetAr/Ar] is a benzimidazole, imidazopyridine, benzofuran, benzothiophene, pyrimidine or benzene ring. When the [HetAr/Ar] unit is a 5,6-bicyclic heterocycle, it is oriented such that the 5-membered ring portion is connected to the [linker] unit and the 6-membered ring portion is connected to the [Am] unit. Among the 34 compounds in this series, compounds with benzofuran as the [HetAr/Ar] unit showed the highest potencies. Introduction of a fluorine atom or a methyl group to the triaryl core led to the more potent analogs. Bisamidines are more active toward bacteria while the monoamidines are more active toward mammalian cells (as indicated by low CC₅₀ values). Importantly, we identified compound P12a (MBX 1887) with a relatively narrow spectrum against bacteria and a very high CC₅₀ value. Compound P12a has been scaled up and is currently undergoing further evaluations for therapeutic applications.

New therapies are urgently needed for treatment of Gram-negative bacterial infections. Due to the emergence of bacterial strains resistant to all classes of β-lactam antibiotics (penicillins, cephalosporins, and carbapenems), aminoglycosides, and quinolones we have reached a crisis in the availability of effective therapy.¹ Among the five novel antibiotics introduced since 2000 (linezolid, daptomycin, retapamulin, fidaxomicin and bedaquiline), none are effective against Gram-negative infections.² All of the newly approved antibiotics for Gram-negative infections are analogs of known drugs, e.g., β-lactams, fluoroquinolones, tetracyclines and macrolides.² To manage infections complicated by the continued development of resistance, new therapeutic approaches and rapid development of novel antibiotics are essential.

In collaboration with the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), we have developed a series of bisamidine compounds that show potent activities against prevalent Gram-negative and Gram-positive pathogens.³ Details on the synthesis and structure-activity relationship studies of compounds in the symmetrical head-to-head bisindole series (Figure 1) have been published elsewhere.⁴ In this report we describe the synthesis and biological evaluation of a new, unsymmetrical series, in which one of the indole rings in the triaryl core has been replaced with another aromatic unit (benzimidazole, imidazopyridine, benzofuran, benzothiophene, pyrimidine or benzene) and the central linker unit contains benzene, thiophene, or pyridine rings.

Figure 1.

Unsymmetrical and symmetrical scaffolds

Our initial synthetic efforts focused on the synthesis of unsymmetrical compounds containing an indole, a phenyl linker, and a non-indole 5:6 heterocyclic system. These compounds were prepared from the following three indole derivatives: N-Boc-2-(4-bromophenyl)indole-6-carbonitrile (5), (4-(NBoc-6-cyanoindol-2-yl)phenyl)boronic acid pinacol ester (6), and (N-Boc-6-cyanoindol-2-yl)boronic acid (7) following path A, B or C as illustrated in Scheme 1. Piperidine-catalyzed condensation of 4-methyl-3-nitrobenzonitrile and 4-bromobenzaldehyde provided a mixture of cis- and trans-stilbenes 3. Treatment of 3 with triethylphosphite led to reduction of the nitro group and formation of the indole 4.⁵ Protection of the indole nitrogen with a Boc group provided the intermediate 5, which could then be directly converted to the intermediate nitrile 9 by conducting a Suzuki-Miyaura cross-coupling⁶ with an aryl pinacolboronate (path A). Alternatively, borylation of the bromide 5 with bis(pinacolato)diboron in the presence of a palladium catalyst gave intermediate boronic acid ester 6⁷ which could also be converted to 9 using palladium-catalyzed coupling methodology (path B). Intermediate 9 could also be synthesized by condensation of commercially available boronic acid 7 with aryl bromide 8 (path C). Subsequent treatment of compounds 9 with diamines in the presence of phosphorous pentasulfide provided the desired amidine products 10.⁸

Scheme 1.

General methods for compound preparation. Reagents and conditions: a) piperidine, 125 °C; b) P(OEt)₃, reflux; c) Boc₂O, DMAP, THF; d) PdCl₂(dppf), B₂pin₂, NaOAc, dioxane, 80 °C;e) P₂S₅, diamine, 120-130 °C.

Preparation of benzimidazole and imidazopyridine containing analogs followed path C (Scheme 2). Condensation of 3,4-diaminobenzonitrile (11) and 4-bromobenzaldehyde (2) at reflux in the presence of sodium metabisulfite provided benzimidazole 12.⁹ Treatment of 2-amino-5-cyanopyridine (13) and 2-amino-4-cyanopyridine (14) with 2,4′-dibromoacetophenone (15) provided the corresponding imidazopyridines 16 and 17, respectively.¹⁰ Coupling of the diarylbromides 12, 16 and 17 with (N-Boc-6-cyanoindol-2-yl)boronic acid (7), followed by amidine formation provided products P1a to P3b.

Scheme 2.

Preparation of benzimidazole and imidazopyridine containing analogs. Reagents and conditions: a) Na₂S₂O₅, EtOH, DMSO, H₂O, 100 °C; b) 7, Pd(Ph₃P)₄ or Pd(OAc)₂/2-(Biphenyl)t-Bu₂P, K₂CO₃, EtOH, toluene, 70 °C; c) CF₃CO₂H, MeOH, CH₂Cl₂; d) P₂S₅, NH₂CH₂(CH₂)_nNH₂, 120 °C; e) EtOH, reflux.

Preparation of benzofuran- and benzothiophene-containing analogs followed path A (Scheme 3). The preparation of (6-cyanobenzofuran-2-yl)boronic acid pinacol ester (24) has been optimized and scaled up to hundreds of grams and involves no chromatographic purifications.¹¹ The synthesis began acid (18) to provide the iodide 19 in 51% yield.¹² Conversion of the carboxylic group of 19 to the corresponding nitrile was accomplished in 3 steps, and involved chlorination, amination and dehydration to provide the nitrile 20 in 51% overall yield.¹³ Sonogashira coupling of 20 with TMS-acetylene gave arylacetylene 21 in 89% yield.¹¹ Treatment of 21 with CuI and triethylamine provided benzofuran-6-carbonitrile (22) in 68% yield.¹¹ Iridium-catalyzed borylation of 22 and the commercially available benzofuran-5-carbonitrile (23) occurred selectively at the 2-position to provide (6-cyanobenzofuran-2-yl)boronic acid pinacol ester (24) and (5-cyanobenzofuran-2-yl)boronic acid pinacol ester (25) in 74% and 83% yields, respectively.¹¹ Cross-coupling of 24, 25 and the commercially available (6-cyanobenzothiophen-2-yl)boronic acid (26) with the intermediate 5, followed by amidine formation as described above, provided products P4 to P6f.

Scheme 3.

Preparation of benzofuran and benzothiophene containing compounds. Reagents and conditions: a) ICl, AcOH, 45 °C; b) EtCO₂Cl, Et₃N, THF; c) NH₄OH; d) (CF₃CO)₂O, pyridine, CH₂Cl₂; e) TMS-acetylene, Pd(PPh₃)₂Cl₂, CuI, Et₃N, THF, 40 °C; f) CuI, Et₃N, EtOH, reflux; g) B₂pin₂, [Ir(OMe)COD]₂, hexanes; h) 5, Pd(PPh₃)₄, Na₂CO₃, EtOH, toluene; i) P₂S₅, NH₂CHR(CH₂)_nNH₂, 120 °C; j) P₂S₅, NH₂CH₂CHR’CH₂NH₂, 120 °C.

Minimum inhibitory concentration (MIC) values for compounds P1a-P6f against a range of clinically important Gram-positive and Gram-negative pathogens, including those proficient and deficient in efflux mechanisms, as well as cytotoxic concentration (CC₅₀) values against human HeLa cells, are shown in Table 1. Compounds containing benzimidazole and imidazopyridine heterocyclic systems are more susceptible to efflux by E. coli than those containing benzothiophene and benzofuran systems, as the MIC values against the efflux deficient (TolC−) strains are substantially lower than those against the wild-type (TolC+) strains. Against the efflux-deficient (TolC−) strain, all compounds are very active (MIC <1 μg/mL). The MIC values against K. pneumonia closely resemble those against the E. coli wild type strain (differences within 2-fold). In contrast, their MIC values against P. aeruginosa varied widely, from <1 μg/mL (P6c) to >80 μg/mL (P2a, P6e). These values remained essentially the same against the efflux deficient strain PAO1 Δ(mexAB–oprM), indicating that the reduced potency against Pseudomonas for some compounds was not due to efflux by the mexAB-oprM pump. Most of the compounds are broadly active against Gram-positive pathogens with the exception of compounds P2a and P3a. Notably, the tetrahydropyrimidine amidine homologs P2b and P3b are still very potent against MRSA, VRE and B. anthracis, following a trend that we found for the bisindoles series.⁴ The differences in MIC values caused by a simple change from a 5-membered ring to 6-membered ring amidine in these analogs are striking. Effects of the amidine group on activity were further examined in the indole-benzofuran containing scaffold. Again, the tetrahydropyrimidine amidine imparts the greatest activity; methylimidazoline, hydroxyl- or methoxytetrahydropyrimidine amidines all exhibit significantly lower potency against P. aeruginosa. The fluorotetrahydropyrimidine amidine makes compound P6f nearly as active as the corresponding norfluoro analog P6c, but less cytotoxic. Lastly, we noted that the position of the amidine on the imidazopyridine does not affect the activity but on the benzofuran it has a moderate effect, with the 6-position being generally more preferred. An exception is that P5 is more potent than P6c against B. anthracis.

Table 1.

MIC and CC₅₀ values for compounds P1a – P6f

Cpd #	Structures		MIC (μg/mL)a,b,c									CC50d
	5,6-HetAr	Am	E.col+	E.col−	P.aer	P.ae-	K.pne	B.sub	MRSA	VRE	B.anth	HeLa
1066			1.25	0.16	7.5	2.5	0.24	0.12	0.16	0.16	0.31	32.5
P1a			10	0.31	20	40	20	0.16	1.25	0.63	5	8.6
P1b	”		2.5	0.31	2.5	2.5	5.0	0.08	0.31	0.31	0.31	27.6
P2a			40	0.63	>80	>80	80	0.31	80	20	40	-
P2b	”		5.0	0.31	5.0	5.0	5.0	0.08	0.63	0.63	1.25	19.7
P3a			2.5	0.31	40	>80	5.0	2.5	>80	>80	>80	2.5
P3b	”		2.5	0.31	40	>80	5.0	0.16	2.5	1.25	1.25	20.9
P4			0.63	0.31	10	10	1.25	0.10	0.31	0.31	0.16	5.9
P5			0.63	0.39	2.5	2.5	1.25	0.08	0.31	0.08	0.08	8.5
P6a			0.47	0.24	10.0	15.0	0.94	0.16	0.16	0.16	0.31	9.7
P6c	”		0.63	0.16	0.63	0.47	0.55	0.14	0.10	0.39	5.0	26
P6b	”		1.09	0.63	80.0	80.0	5.0	0.35	0.55	0.63	0.63	7.6
P6b- iso	”		0.63	0.63	60.0	80.0	2.5	0.20	0.31	0.39	0.31	5.7
P6d	”		2.5	0.16	6.3	15.0	1.09	0.08	0.31	0.31	0.55	11.9
P6e	”		2.5	0.24	>80	>80	1.25	0.08	0.31	0.63	0.31	6.0
P6f	”		1.25	0.27	5.0	2.5	0.63	0.16	0.31	0.31	0.31	47.5

^aMIC values obtained by the broth dilution method;¹⁶

^bbacterial strains: Ecol+ = Escherichia. coli 700 TolC+, Ecol− = E. coli TolC -, P.aer = Pseudomonas aeruginosa PAO 1, P.ae- = P. aeruginosa PAO 1 Δ(mexAB–oprM), K. pne = Klebsiella pneumonia 13882, B. subs = Bacillus subtilis BD54, MRSA = methicillin-resistant Staphylococus aureus 1094, VRE = vacomycin-resistant Enterococcus faecalis ATCC 51575, B. anth = Bacillus. anthracis Sterne;

^cMIC ≤1: green; 1< MIC ≤5: blue; 5< MIC ≤20: yellow; MIC >20: pink;

^dcytotoxicity determined by using the vital stain MTS,¹⁷ units in μg/mL.

To determine whether both amidine groups are required for antibacterial activity, we prepared a number of analogs that have different amine-containing groups. Preparation of these analogs followed paths A and B as shown in Scheme 4. Coupling of intermediate 6 with commercially available building blocks 30, 31 and intermediate 5 with (commercially available) 34 followed by amidine formation provided products P7a to P9b.

Scheme 4.

Preparation of monoamidine compounds. Reagents and conditions: a) 6, Pd(PPh₃)₄, Na₂CO₃, DME, H₂O, 75 °C; b) P₂S₅, NH₂-R’-CH₂NH₂, 120 °C; c) 5, Pd(PPh₃)₄, Na₂CO₃, EtOH, toluene, 75 °C; d) P₂S₅, NH₂CH₂(CH₂)_nNH₂, 120 °C.

MIC and CC₅₀ values for P7a to P9b are shown in Table 2. Overall, these compounds possess significantly lower antibacterial activities compared to the bisamidine compounds, and higher cytotoxicity against HeLa cells. Notably, P7a and P8a are potent against the efflux deficient strain of E. coli but are not active against the wild type strain, suggesting that they are strongly susceptible to efflux. Compounds P7a, P8a, P9a and P9b are active against Gram-positive pathogens. These activities sharply decline in the methylimidazoline isomers, P7b and P8b. Lastly, compounds P9a and P9b are highly cytotoxic. Thus, the data suggests that the bisamidine is important for activity against bacteria, especially Gram-negative bacteria, while monoamidines have more inhibitory effects on the growth of mammalian cells.

Table 2.

MIC and CC₅₀ values for compounds P7a – P9b^a

Cpd #	Structures	MIC (μg/mL)								CC50
		E.col+	E.col−	P.aer	K.pne	B. sub	MRSA	VRE	B. anth	HeLa
P7a		35	0.63	>80	70	0.78	7.5	3.75	0.55	2.0
P7b		>80	60.0	>80	>80	10.0	>80	>80	22.5	1.3
P8a		>80	0.63	>80	>80	0.63	2.5	5.0	0.63	5.7
P8b		>80	40.0	>80	>80	40.0	40.0	>80	40.0	1.1
P9a		>80	>80	>80	>80	0.63	1.04	3.3	2.1	≤0.63
P9b		>80	>80	>80	>80	0.63	1.09	3.1	0.47	≤0.63

^abacterial strains: Ecol+ = E. coli 700 TolC+, Ecol− = E. coli TolC -, P.aer = P. aeruginosa PAO 1, K. pne = K. pneumonia 13882, B. subs = B. subtilis BD54, MRSA = methicillin-resistant S. aureus 1094, VRE = vacomycin-resistant E.faecalis ATCC 51575, B. anth = B. anthracis Sterne; MIC ≤1: green; 1< MIC ≤5: blue; 5< MIC ≤20: yellow; MIC >20: pink; CC₅₀ units in μg/mL

Because the benzofuran-containing analog P6c has the most potent antibacterial activity of this series and an acceptable level of cytotoxicity, we decided to explore the indole-aryl-benzofuran scaffold further by varying the central linker unit and the amidine substitutions. Preparation of these compounds followed a route similar to path A, as shown in Scheme 5. Syntheses of N-Boc-2-(4-bromo-3-fluorophenyl)indole-6-carbonitrile (37) and N-Boc-2-(5-bromothiophen-2-yl)indole-6-carbonitrile (40) were based on the route used previously for the preparation of 5. Thus, condensation of 4-bromo-3-fluorobenzaldehyde (35) and 5-bromothiophene-2-carboxaldehyde with 4-methyl-3-nitrobenzonitrile (1), followed by treatment with triethylphosphite, and subsequent protection of the indole nitrogen with a Boc group, provided 6-cyanoindoles 37 and 40, respectively. Coupling of 37, 40 and 42 ¹⁴ with (6-cyanobenzofuran-2-yl)boronic acid pinacol ester (24) followed by amidine formation provided products P10 to P13d.

Scheme 5.

Preparation of compounds with the indole-aryl-benzofuran scaffold. Reagents and conditions: a) piperidine, sulfolane, 145 °C; a’) piperidine, 110 °C; b) P(OEt)₃, 130 °C; c) Boc₂O, DMAP, THF; d) 24,Pd(PPh₃)₄, Na₂CO₃, EtOH, toluene, 70 °C; e) P₂S₅, NH₂(CH₂)₃NH₂, 120 °C; f) 24, Pd(dppf)Cl₂, Na₂CO₃, MeCN, 70 °C; g) P₂S₅, NH₂CHR²CH₂NH₂, 120 °C; h) P₂S₅, NH₂CH₂CHR³CH₂NH₂, 120 °C

Compounds with a 3-methyl substituent on the indole were prepared as shown in Scheme 6, using the Fischer indole synthesis. Condensation of 3-cyanophenylhydrazine (43)¹⁵ with 4′-bromopropiophenone (44) provided a mixture of regioisomers 45 and 46, which were carried on to the next step. Protection of the indole nitrogen with a Boc group followed by coupling of the product mixture with (6-cyanobenzofuran-2-yl)boronic acid pinacol ester (24) provided triaryl compounds 49 and 50, which were separated by silica gel chromatography. Amidination of 49 provided the bisamidine P14 as expected. Under the same conditions, only one nitrile group in 50 was converted to the corresponding amidine, leading to isolation of the monoamidine product P15. We speculate that the 4-cyanoindole functionality in bis-nitrile 50 is hindered with respect to the 6-cyanoindole functionality in bis-nitrile 49, which is the reason for the production of monoamidated compound P15.

Scheme 6.

Synthesis of 3-methylindole containing compounds. Reagents and conditions: a) p-TsOH•H₂O, toluene, reflux; b) Boc₂O, DMAP, DMF; c) Pd(PPh₃)₄, Na₂CO₃, DME, H₂O, 85 °C; d) P₂S₅, NH₂(CH₂)₃NH₂, 120 °C.

MIC and CC₅₀ values for the indole-benzofuran analogs with various linkers are summarized in Table 3. Introduction of methyl and fluoro groups to the core provided potent compounds (P14, P10), while the more polar diaminopropane functionality was less effective (P11). This pattern is consistent with what we observed in the first series (Table 1): addition of nitrogen atoms to the core produced compounds with higher levels of efflux. Replacing the phenyl linker with pyridine or thiophene led to compounds that were more susceptible to efflux and less active against Gram-negative bacteria. Notably, thiophene-linked compound P12a showed very low cytotoxicity. This unique feature suggests that P12a could be useful for narrow-spectrum treatment of select pathogens. For example, we found that P12a is very active against Francisella tularensis (MIC 0.063-2.5 μg/mL across 13 strains tested). We have subsequently optimized the synthesis of P12a to the hundred-gram scale. Further evaluation of this compound is pending and will be reported in the future.

Table 3.

MIC and CC₅₀ values for compounds P10 – P15^a

Cpd #	Structures		MIC (μg/mL)								CC50
	linker	Am	E.col+	E.col−	P.aer	K.pne	B.sub	MRSA	VRE	B. anth	HeLa
P10			0.63	0.31	2.5	0.63	0.08	0.16	0.16	0.08	14.3
P11			2.5	0.63	2.5	1.25	0.24	0.63	1.25	0.63	4.6
P12a			2.80	0.27	66.7	60.0	0.16	0.94	1.56	1.09	182.2
P12b	”		1.25	0.63	26.7	20.0	0.31	1.04	0.63	0.52	14.5
P12c	”		0.63	0.31	10.0	2.5	0.08	0.31	0.31	0.16	8.7
P12d	”		5.0	0.47	40.0	15.0	0.43	1.88	2.5	1.25	12.7
P12e	”		1.25	0.31	53.3	2.5	0.16	0.63	0.63	0.31	22.5
P13a			1.25	0.16	1.25	1.56	0.08	0.31	0.39	0.16	15.6
P13b	”		1.88	0.27	5.0	2.5	0.08	0.31	0.63	0.63	2.2
P13c	”		80	0.63	25	8.8	3.75	2.5	5.0	1.09	16.7
P13d	”		8.3	0.31	8.3	2.5	0.16	0.83	0.63	0.63	19.6
P14	See scheme 6		0.31	0.31	1.25	0.31	0.08	0.16	0.16	0.08	1.5
P15	See scheme 6		20.0	10.0	40.0	20.0	2.5	5.0	5.0	2.5	-

^abacterial strains: Ecol+ = E. coli 700 TolC+, Ecol− = E. coli TolC -, P.aer = P. aeruginosa PAO 1, K. pne = K. pneumonia 13882, B. subs = B. subtilis BD54, MRSA = methicillin-resistant S. aureus 1094, VRE = vacomycin-resistant E. faecalis ATCC 51575, B. anth = B. anthracis Sterne; MIC ≤1: green; 1< MIC ≤5: blue; 5< MIC ≤20: yellow; MIC >20: pink; CC50 units in μg/mL

In summary, we have prepared 34 new unsymmetrical triaryl bisamidine and monoamidine compounds, featuring 15 new triaryl core structures, and evaluated them for antibacterial activities and cytotoxicity. We found that benzofuran can replace one indole ring from the original hit compound (MBX 1066) to deliver broad-spectrum compounds with improved potency such as P5, P6c and P6f. Introduction of a fluorine atom or a methyl group to the triaryl core led to the more potent analogs P10 and P14, respectively. Bisamidines are more active toward bacteria while the monoamidines are more active toward mammalian cells (as indicated by low CC₅₀ values). Importantly, we have identified compound P12a with a relatively narrow spectrum of activity against bacteria and a very high CC₅₀ value. The synthesis of compound P12a has been optimized and scaled up, and the compound is currently under further evaluations for therapeutic applications.