Advent of Imidazo[1,2-a]pyridine-3-carboxamides with Potent Multi- and Extended Drug Resistant Antituberculosis Activity

Abstract

A set of nine 2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamides and one 2,6-dimethylimidazo[1,2-a]pyrimidine-3-carboxamide were synthesized. The compounds were evaluated for their in vitro anti-tuberculosis activity versus replicating, non-replicating, multi- and extensive drug resistant Mtb strains. The MIC₉₀ values of seven of these agents were ≤ 1 μM against the various tuberculosis strains tested. A representative compound of this class (1) was screened against seven non-tubercular strains as well as other non-mycobacteria organisms and demonstrated remarkable microbe selectivity. A transcriptional profiling experiment of Mtb treated with compound 1 was performed to give a preliminary indication of the mode of action. Lastly, the in vivo ADME properties of compounds 1, 3, 4, and 6 were assessed. The 2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamides are a drug-like and synthetically accessible class of anti-TB agents that have excellent selective potency against multi- and extensive drug resistant TB and encouraging pharmacokinetics.

Tuberculosis (TB) is a serious global health risk. More than one third of the human population is infected resulting in an estimated 1,700,000 deaths in 2006 (1.5 million in HIV-negative people and 0.2 million in HIV-positive people).¹ Moreover, there were a staggering 14,400,000 cases estimated worldwide in 2006, with 83% of the total cases located in the African, South-East Asia and Western Pacific regions.¹ Mycobacterium tuberculosis, the causative agent of TB, is an airborne pathogen that can be spread from one person to another by close contact. Because it can lie dormant in a latent state for many years, it is a silent killer among the poor, HIV-infected, immune-compromised and the elderly. To make matters worse, multiple-drug resistant TB (MDR-TB, strains that are resistant to first line drugs isoniazid and rifampin) and extensively drug resistant TB (XDR-TB, strains that are resistant to isoniazid and rifampin, as well as any fluoroquinolone and at least one of three injectable second-line drugs, such as amikacin, kanamycin, or capreomycin) are on the rise.² Most alarming is the emergence of extremely drug-resistant tuberculosis “XXDR-TB” (the proposed designation for TB that is resistant to all first- and second-line TB drugs), which is now documented.³ In 2008, there were 12,898 cases of TB provisionally reported in the United States.

A focus of our laboratories is to facilitate the decline of TB by the identification of therapeutically effective anti-TB agents to augment the long dosing regimen of first line drugs.⁵ Herein we call attention to the in vitro potency of the imidazo[1,2-a]pyridine-3-carboxamide scaffold. To our knowledge the anti-TB activity of the 2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamide class is unprecedented. Imidazo[1,2-a]pyridine-3-nitroso derivatives were reported in 2004 to impart notable anti-TB activity (MIC = 3.1 μg/mL vs. H₃₇Rv-TB) concurrent with notable toxicity to VERO cells (IC₅₀ = 3.6 μg/mL).⁶ Also in 2004, rationally designed imidazo[1,2-a]pyridine-3-hydrazones⁷ were reported but were all inactive against H₃₇Rv-TB at 6.25 μg/mL. Most recently, in 2009, functionalized 3-amino-imidazo[1,2-a]pyridines were reported as in vitro Mtb glutamine synthetase inhibitors, but without assessment of the in vitro activity versus H₃₇Rv-TB.⁸ While reports on the syntheses of imidazo[1,2-a]pyridine-3-carboxamides date to 1965,⁹ the 2,7-dimethylimidazo[1,2-a]pyridine architecture is atypical within the cannon of medicinal chemistry literature and is unprecedented within the TB lexicon.

Since 2007, we have had a collaborative agreement with Dow AgroScience to screen their compound inventory for inhibitors of Mtb. This effort, coupled with a program to elaborate novel heterocylic anti-TB agents from fragment-based studies of mycobacterial siderophores,¹⁰ led to the identification of an ethyl 2,7-dimethylimidazo[1,2-a]pyridine-3-carboxylate. This compound had weak activity against H₃₇Rv TB (MIC ~ 65 μM, average) but was nonetheless an attractive heterocyclic scaffold to optimize as we did previously with related heterocyclic classes.¹¹

The simplest synthesis of the imidazo[1,2-a]pyridine-3-carboxylate ring system¹² is the straightforward reaction of 2-amino-4-picoline with ethyl 2-chloroacetoacetate to give the desired heterocyclic scaffold in 78% yield (Scheme 1). Saponification with lithium hydroxide followed by acidic work up gave the free acid which was then easily converted to various amide analogs through classical EDC-mediated couplings in good yields (70% for 1).

Scheme 1.

Synthesis of imidazo[1,2-a]pyridines.

Reagents: (a) Ethyl 2-chloroacetoacetate, DME, reflux, 48 h.; (b) 1. LiOH, EtOH, 2. HCl, 56 h.; (c) EDC, DMAP, R₁, ACN, 16 h.

Our initial structure-activity relationship (SAR) strategy evaluated a representative panel of nine imidazo[1,2-a]pyridine-3-carboxamide analogs. The chosen imidazopyridine analogs included the classical Topliss¹³ set of benzyl, 4-methoxyphenyl, 4-methylphenyl, 4-chlorophenyl and 3,4-dichlorophenyl amides. This set was augmented by ortho- and meta-methoxyphenyl analogs to probe possible steric effects. Next, due to potential metabolic issues associated with a benzylic methylene group, the corresponding aniline was prepared as well as a fluorine replacement for the chlorine. Finally, we explored the influence on potency by changing the imidazo[1,2-a]pyridine to an imidazo[1,2-a]pyrimidine core (as in 10).

Table 1 summarizes the in vitro anti-TB activity of these ten analogs in three different media (GAS¹⁴, GAST¹⁵ and 7H12¹⁴), their potency against non-replicating “latent” TB (LORA¹⁶) and an assessment of their toxicity by the VERO¹⁷ assay. All compounds were potent (MIC <10 μM in the GAS assay media) with the exception of the aniline derived analog (9, MIC >128 μM) suggesting that in the imidazo[1,2-a]pyridine series the benzylic position is important for activity. Additionally, by running the TB assay in three different media (GAS, GAST and 7H12), we eliminated concern that the activity of these compounds might be carbon source dependent, a flaw discovered in the pyrimidine-imidazoles reported by Pethe and colleagues at Novartis¹⁸ as the GAS and GAST assays use glycerol-alanine-salts as the carbon source while the 7H12 media uses palmitic acid.

Table 1.

In vitro evaluation of compounds 1 – 10 against H₃₇Rv-TB in various assays and media (MIC₉₀ in μM) and stability to rat liver (RLM) and human liver (HLM) microsomes.

Compound ID	Mol Wt	Calc. Clog P*	GAS	GAST	7H12	LORA	VERO	RLM % metab. (30 min)	HLM % metab. (30 min)
1	279.34	3.60	0.37	0.69	1.9	53.6	>128	71	59
2	309.36	3.51	2.8	1.9			>128
3	309.36	3.51	1.2	1.0	5.9	31.4	>128	80	47
4	309.36	3.51	0.51	0.50	1.35	10.5	>128	69	30
5	293.36	4.09	0.51	0.80			54.7
6	313.78	4.31	0.50	0.51	1.94	28	>128	79	82
7	348.23	4.90	9.3	14.4
8	297.33	3.74	0.29	0.38	1.98	40.1	>128
9	265.31	3.42	>128	>128
10	310.35	2.51		0.10	0.48	>10	88.7
RMP (Rifampicin)	822.95	6.04	0.8	0.2	0.07	2.6	113
PA-824 (nitroimidazole)	359.26	2.62	0.31	0.21	0.47	4.9	>128

^*Calculated ClogP by ChemDraw version 12.0, GAS = Glycerol-alanine-salts media; GAST = Iron deficient glycerol-alanine-salts with Tween 80 media; 7H12 = 7H9 broth base media with BSA, casein hydrolysate, catalase, palmitic acid; LORA = Low oxygen recover assay; VERO = African green monkey kidney cell line; RLM = Rat liver microsomes, HLM = Human liver microsomes. Values reported are the average of three individual measurements.

SAR analysis based on the whole cell assay readout indicated that 3,4-dichloro analog (7) had diminished activity (MIC’s of 9 – 14 μM) when compared to the 4-chloro (6, 0.5 μM) and 3-fluoro analogs (8, ~0.3 μM). There appeared to be a slight preference for para-substitution in terms of potency (MIC = 2.8 μM for ortho- vs. 1.2 μM for meta- vs. 0.5 μM for para-methoxy analog in the GAS assay media). Comparison of the imidazo[1,2-a]pyrimidine analog (10) to the corresponding imidazo[1,2-a]pyridine analog (3) indicated that the additional nitrogen in the heterocyclic core was well tolerated (sub-micomolar potency) although VERO toxicity (IC₅₀ = 89 μM) was noted. Compounds 1 and 10 were re-screened in the presence of 4% BSA (bovine serum albumin) and 10% FBS (fetal equine serum) and their MIC’s were found to shift less than two fold by ATP and MABA readouts (see supporting information) indicating that protein binding is not a problem.

Encouraged that six of the ten analogs tested had sub-micromolar MIC values against the H₃₇Rv Mtb strain, we next screened compounds 1 and 10 against a panel of single drug resistant strains (Table 2) against controls rifampicin (RIF) and isoniazid (INH), and then three of the more promising compounds (1, 3, 8) against a panel of MDR and XDR clinical strains (Table 2). The difference in potencies of these compounds against the clinical strains may be due to the difference in growth media where growth inhibition for clinical strains was tested in media containing glucose as well as glycerol as carbon source, as well as the fact that many clinical strains exhibit poor growth in vitro since they are not adapted to laboratory conditions and media which may affect their apparent susceptibility to certain inhibitors.

Table 2.

Potency of imidazo[1,2-a]pyridines (1, 3, 8) and imidazo[1,2-a]pyrimidine (10) against single drug resistant strains, MDR-TB and XDR-TB stains (MIC₉₀ in μM).

Strains resistance to drugs:	Control/Compound ID
	RMP	INH	1	3	8	10
Rifampicin	>1	0.23	0.28			1.49
Isoniazid	0.01	>8	0.33			5.84
Kanamycin	0.02	0.43	1.07			1.02
Streptomycin	0.02	0.23	1.02			5.84
MDR-HRESP			2.24	1.01	0.26
MDR-HREZSP			1.12	0.06	0.06
MDR-HCPTh			1.12	0.13	0.26
MDR-HREKP			0.28	0.13	0.26
MDR-HRERb*			0.14	≤0.03	0.13
MDR-HRERb*			0.14	0.03	0.34
MDR-HRERb*			0.28	0.06	0.26
MDR-HREZSKPTh			0.07	≤0.03	0.06
MDR-HREZRbTh			0.14	0.06	0.06
XDR-HRESPOCTh			0.07	0.03	0.07
XDR-HREPKOTh			0.07	0.02	0.03
XDR-HRESPO			0.14	0.02	0.03

Abbreviations: H=Isoniazid, R=Rifampicin, E=Ethambutol, Z=Pyrazinamide, S=Streptomycin, C=Cycloserine, Th=Ethionamide, K=Kanamycin, P=p-aminosalicylic acid, Rb=Rifabutin, Th=Thioacetazone, O=Ofloxacin.

^*Different clinical strains.²⁰ Values reported are the average of three individual measurements.

The excellent activity found when these imidazo[1,2-a]pyridine agents were tested against the drug resistant strains compared favorably to the published MIC values of the nitroimidazole clinical candidate PA-824¹⁹ (MICs against MDR-TB from 0.03 to 0.25 μg/mL or 0.08 to 0.7 μM, comparatively). Furthermore, the improved, and indeed outstanding, potency of these agents against the various drug resistant strains suggests that they inhibit a novel target. Selectivity screening against various non-tubercular mycobacteria revealed that compounds 1 and 10 are also inhibitors of M. avium, M. bovis BCG and M. kansasii but not inhibitors of M. smegmatis, M. abscessus, M. chelonae and M. marinum (Table 3). This unusual selectivity prompted us to further screen compounds 1, 3, 4, 6, 8 and 10 against a panel of representative non-mycobacterial organisms. Compounds 1, 3, 4, 6, 8 and 10 were all found to be inactive against the Gram positive strain of Staphylococcus aureus (MIC >128 μM), the Gram negative strain of Escherichia coli (MIC >128 μM), and the fungus, Candida albicans (MIC >128 μM) further suggesting a mycobacterium specific target of these agents.

Table 3.

Non-tubercular mycobacteria activity and selectivity of imidazo[1,2-a]pyridine (1) and imidazo[1,2-a]pyrimidine (10) (MIC₉₀ in μM).

Compound ID	TB-H37Rv	M. abscessus	M. chelonae	M. marinum	M. avium	M. kansasii	M. bovis BCG	M. smegmatis
1	1.07	> 50	> 50	> 50	1.32	1.32	0.33	> 50
10	5.94	> 50	> 50	> 50	12.00	12.00	2.78	> 50
RMP	0.05	162.3	150.00	< 0.78	< 0.78	< 0.78	< 0.78	162.3

Values reported are the average of three individual measurements.

The in vivo pharmacokinetics (PK) of compounds 1, 3, 4 and 6 were evaluated in Sprague Dawley rats by oral (PO) and intravenous (IV) routes of administration at 10 and 1 mg/kg dosing levels, respectively (Table 4). Compound 4 had moderate in vitro rat microsomal stability (69% metabolized, t_1/2 = 19 min) and also displayed promising PK by having the lowest in vivo clearance (28 mL/min/kg, t_1/2 = 28 min by IV). The aqueous solubility for compounds 1, 3, 4 and 6 were measured at 181, 149, 148, 25 μM, respectively in phosphate buffered saline (PBS) at pH 7.4. Additional in vivo ADME properties including terminal half life (t_1/2β), the area under the curve (AUC), the volume of distribution (V_d) and volume of distribution at steady state (V_dss) for compounds 1, 3, 4, and 6 can be found in the supporting information section. Encouraged by the potency, PK and favorable oral bioavailability of these imdazo[1,2-a]pyridine agents we intend to evaluate various analogs in vivo by the murine Gamma Knock-out (GKO) infection model and the results will be reported in due course.

Table 4.

In vivo PK evaluation of imidazo[1,2-a]pyridines.

Compound ID	PO Cmax (ng/mL)	PO Tmax (hr)	IV t1/2 (hr)	IV clearance (mL/min/kg)	%F
1	3012	0.25	0.35	91	76
3	3140	0.25	0.33	43	43
4	5741	0.25	0.28	28	50
6	1995	0.31	0.4	51	49

Values reported are the average of three individual measurements.

Finally, curious as to the mechanism of action of these agents, we performed transcriptional profiling experiments of M. tuberculosis treated with compound 1 and comparison to the existing database of drug-induced transcriptional profiles indicated that this compound inhibited an aspect of energy generation in the cell (see supporting information). Thus, compound 1 resulted in up-regulation of the cytochrome bd oxidase which is the high oxygen-affinity respiratory enzyme²¹ observed to be up-regulated during oxygen restriction as well as inhibition of respiration by agents such as cyanide, sodium azide, the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and the nitric oxide-releasing pro-drug PA-824.²² In addition, this compound up-regulated the phosphoenolpyruvate carboxykinase which plays an important role in modulating carbon flow during cellular energy restriction²³ and has previously been observed to be up-regulated by stresses such as hypoxia, sodium azide, valinomycin, nigericin, carbonyl cyanide rn-chlorophenylhydrazone, cyanide, PA-824, and the ATP inhibitor dicyclohexylcarboxydiimide, that limit energy generation through respiration.²²

All the data suggest we have discovered a class of compounds with promising attributes of synthetic accessibility, no redox active moieties,¹⁹ impressive potency and selectivity towards replicating, MDR and XDR Mtb strains. This class has good in vivo ADME properties that potentially can be improved through further analog generation. Additionally, compound 1 appears to act by a novel mechanism of action based on transcriptional profiles to known anti-TB agents. With new anti-TB agents desperately needed we offer the imidazo[1,2-a]pyridine class as a potential therapeutic for further development.