Development of (6 R)-2-Nitro-6-[4-(trifluoromethoxy)phenoxy]-6,7-dihydro-5 H-imidazo[2,1- b][1,3]oxazine (DNDI-8219): A New Lead for Visceral Leishmaniasis.

Discovery of the potent antileishmanial effects of antitubercular 6-nitro-2,3-dihydroimidazo[2,1- b][1,3]oxazoles and 7-substituted 2-nitro-5,6-dihydroimidazo[2,1- b][1,3]oxazines stimulated the examination of further scaffolds (e.g., 2-nitro-5,6,7,8-tetrahydroimidazo[2,1- b][1,3]oxazepines), but the results for these seemed less attractive. Following the screening of a 900-compound pretomanid analogue library, several hits with more suitable potency, solubility, and microsomal stability were identified, and the superior efficacy of newly synthesized 6 R enantiomers with phenylpyridine-based side chains was established through head-to-head assessments in a Leishmania donovani mouse model. Two such leads ( R-84 and R-89) displayed promising activity in the more stringent Leishmania infantum hamster model but were unexpectedly found to be potent inhibitors of hERG. An extensive structure-activity relationship investigation pinpointed two compounds ( R-6 and pyridine R-136) with better solubility and pharmacokinetic properties that also provided excellent oral efficacy in the same hamster model (>97% parasite clearance at 25 mg/kg, twice daily) and exhibited minimal hERG inhibition. Additional profiling earmarked R-6 as the favored backup development candidate.

Introduction

n class="Disease">Visceral leishmaniasis (VL) is a class="Chemical">particularly lethal sandfly-borne class="Chemical">pan class="Disease">parasitic disease that is prevalent in more than 60 countries, where it mostly affects underprivileged people in remote rural areas who have limited access to diagnosis and treatment.[1−3] Major outbreaks of VL in East Africa have been attributed to waves of forced migration during periods of conflict, and such epidemics are exacerbated by weak healthcare systems, malnutrition, and HIV/AIDS coinfection.[4,5] Moreover, in this region, the first-line drug combination of paromomycin and sodium stibogluconate was found to be unsuitable for VL patients who were >50 years of age or those with HIV, and no other therapies have shown adequate efficacy.[6,7] Failure of the most recently evaluated new agent, fexinidazole, in a phase II clinical trial for VL in Sudan[8] has now left the clinical pipeline empty, underlining the compelling need to develop more satisfactory medications.[9]

The target product profile (n class="Chemical">TPP) of an optimized new chemical entity for the treatment of VL requires (i) effectiveness against all causative species, in all endemic areas, in both immunocompetent and immunosuppressed individuals, with a clinical efficacy of >95%; (ii) activity against resistant strains; (iii) no adverse safety events requiring monitoring and no contraindications; (iv) no drug–drug interactions (suitable for combination therapy); (v) oral administclass="Chemical">pan class="Species">ration once per day for a maximum of 10 days (or intramuscular dosing three times over 10 days); (vi) stability in relevant climates (3 years); and (vii) affordable cost (<$80, ideally <$10 per course).[10] However, new drug discovery for VL faces formidable challenges, such as inadequate investment, a lack of validated targets, poor translation of in vitro activity into in vivo models, and meager hit rates (<0.1%) for phenotypic screening of compound libraries.[11−13] The latter may be due in part to the concealed location of parasites in acidic parasitophorous vacuoles within macrophages.[14] Furthermore, the unique glycolipid-rich cell surface of the amastigotes presents an additional barrier to chemotherapy.[15] Another issue is that many cellularly active hits may never meet TPP and progression criteria, even after valiant optimization attempts.[13,16] Nevertheless, drug development efforts spearheaded by the Drugs for Neglected Diseases initiative (DNDi) have now shown encouraging progress in several novel classes, including oxaboroles and aminopyrazoles.[12,17,18] Novartis has also disclosed a triazolopyrimidine preclinical lead with utility in vivo against both leishmanial and trypanosomal infections.[19]

The n class="Chemical">2-nitroimidazooxazines are best known for their potent effects against class="Chemical">pan class="Species">Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB).[20] The first drug candidate from this class, pretomanid [PA-824, S-1 (Figure )], has shown excellent safety and bactericidal efficacy in phase II clinical trials for TB,[21] leading to its ongoing combination assessment,[22] while our collaborative work with the TB Alliance on second-generation analogues of S-1 culminated in the advancement of TBA-354 (S-2) into phase I studies.[23] We have recently described the investigation of a novel 7-substituted 2-nitroimidazooxazine class, which in addition to possessing considerable potential against TB has also demonstrated exciting activity against both VL and Chagas disease, resulting in the selection of preclinical VL lead 3.[24] This followed an in-depth analysis of the structurally related 6-nitroimidazooxazole class,[25] where phenotypic screening of some of our initial examples by DNDi had enabled the discovery of previous development nominee 4 (DNDI-VL-2098).[26] The latter was found[27] to be activated by a novel leishmanial nitroreductase (NTR2). In comparison to 4, candidate 3 exhibited an improved safety profile and had similarly notable efficacy in two animal models of VL.[24] Furthermore, while the new TB drug delamanid (5) has also been suggested as a possible VL therapy,[28] it is noteworthy that 3 was substantially more effective than this agent in the highly stringent chronic infection hamster model.[24,25]

Figure 1

Structures of antitubercular or antileishmanial agents.

Structures of antitubercular or antipan class="Disease">leishmanial agents.

As part of our VL lead optimization program with DNDi, it was considered important to develop a few efficacious backup compounds having good physicochemical/pharmacological profiles and better safety, to mitigate development risks. Given the encouraging results with pan class="Chemical">nitroimidazooxazoles and class="Chemical">pan class="Chemical">7-substituted 2-nitroimidazooxazines, we first evaluated various other pretomanid-related scaffolds for VL, including those with a reversed linker at C-6[29,30] and novel nitroimidazooxazepines. We then assessed our larger collection of pretomanid analogues via the medium-throughput screening of ∼900 compounds at the Institut Pasteur Korea (IPK). Finally, a more systematic synthetic approach was employed to redevelop the 6-substituted 2-nitroimidazooxazine class for VL, taking into consideration both enantiomer forms. We now report the findings from these wide-ranging structure–activity relationship (SAR) studies, including the detailed in vitro/in vivo profiling of selected new leads, which resulted in our identification of the title compound as a very promising VL backup candidate.

Chemistry

Scheme outlines the synthetic methods used to prepare eight novel racemic analogues of S-1 featuring changes to the original pan class="Chemical">nitroimidazooxazine core (14, 22, 23, 32, 33, 39, 40, and 47). A common stclass="Chemical">pan class="Species">rategy (based on the original, well-validated route to S-1 and simple derivatives)[31,32] proved to be effective for the first five of these (Scheme A–C), involving the initial reaction of functionalized epoxides (9,[33]17, and 27(34)) with 2,4-dinitroimidazole (8), followed by THP protection of the derived alcohols (10, 18, and 28). In the shorter chain cases (11 and 19), subsequent cleavage of the TBS ether (TBAF) enabled in situ annulation, whereas in the latter instance (29), oxazepine ring formation required additional treatment with a strong base (NaH). Removal of the THP group with methanesulfonic acid and standard alkylation chemistry on alcohols 13, 21, and 31 then gave the aforementioned targets.

Scheme 1

Reagents and conditions: (i) 70–75 or 95 °C, 18–23 h; (ii) 3,4-dihydro-2H-pyran, PPTS, CH2Cl2, 20 °C, 3.5–24 h; (iii) TBAF, THF, 20 °C, 1–4.5 h, or 0–20 °C, 14 h (for 47); (iv) MsOH, MeOH, 20 °C, 1–2 h; (v) 4-OCF3BnBr or 4-BnOBnCl or 4-BnOBnI, NaH, DMF, 20 °C for 3–20 h or 0–20 °C for 0.7–2.2 h; (vi) TBSOTf, Et3N, CH2Cl2, 20 °C, 3 h; (vii) m-CPBA, CH2Cl2, 20 °C, 18 h; (viii) NaH, DMF, 20 °C for 18 h or 0–20 °C for 3.5 h; (ix) 3.3 M HCl, MeOH, 20 °C, 5 h; (x) DIPEA, toluene, 89–105 °C, 67 h.

Reagents and conditions: (i) 70–75 or 95 °C, 18–23 h; (ii) 3,4-dihydro-2H-pyran, PPTS, n class="Chemical">CH2Cl2, 20 °C, 3.5–24 h; (iii) class="Chemical">pan class="Chemical">TBAF, THF, 20 °C, 1–4.5 h, or 0–20 °C, 14 h (for 47); (iv) MsOH, MeOH, 20 °C, 1–2 h; (v) 4-OCF3BnBr or 4-BnOBnCl or 4-BnOBnI, NaH, DMF, 20 °C for 3–20 h or 0–20 °C for 0.7–2.2 h; (vi) TBSOTf, Et3N, CH2Cl2, 20 °C, 3 h; (vii) m-CPBA, CH2Cl2, 20 °C, 18 h; (viii) NaH, DMF, 20 °C for 18 h or 0–20 °C for 3.5 h; (ix) 3.3 M HCl, MeOH, 20 °C, 5 h; (x) DIPEA, toluene, 89–105 °C, 67 h.

For the isomeric n class="Chemical">oxazepines (Scheme D), class="Chemical">pan class="Chemical">THP protection of alcohol 34(24) and desilylation (TBAF) similarly furnished the noncyclized alcohol 36, which was ring-closed (NaH and DMF) and THP-deprotected (HCl) to produce alcohol 38. However, both final step alkylations of 38 unexpectedly cogenerated significant quantities (20–36%) of the isomeric 7-substituted oxazine derivative (41 or 42), together with the desired oxazepine ether (39 or 40). Although this is unconfirmed, it is postulated that this rearrangement may involve an intramolecular SNAr reaction, with attack by the oxazepine alkoxide anion at the imidazole ring junction carbon 9a and subsequent alkylation of the released 2-nitroimidazooxazine 7-alkoxide (but it should be emphasized that no similar rearrangements were detected during the derivatization of 13, 21, 31, or the alcohol precursor to S-1, and that S-1 itself has shown excellent safety and a lengthy 16–18 h half-life in clinical trials for TB,[35] suggesting that 2-nitroimidazooxazine-based VL leads would be unlikely to demonstrate an excessive reactivity toward biological nucleophiles; this is further supported by an observed tolerance of the latter ring system toward several basic nucleophiles in the chemistry reported below). The remaining scaffold, nitrotriazolooxazine 47, was accessed from 5-bromo-3-nitro-1,2,4-triazole (43) and iodide 44,[36] via desilylation/in situ annulation of the major adduct 46, as shown above (Scheme E).

The assembly of new biaryl and heterobiaryl side chain variants of S-1 and its enantiomer, R-1,[37,38] was relatively straightforward (Schemes and 3). Biphenyl analogues (R-58, pan class="Chemical">R-60, and class="Chemical">pan class="Chemical">R-62) were created by Suzuki coupling reactions on halobenzyl ether derivatives of the key 6-R alcohol 56,[32] while combination of 56 with bromide 63(39) afforded the terminal pyridine R-64 (Scheme A). Alternative alkylation of 56 or its 6-S equivalent 65(32) with various isomeric bromomethyl bromopyridines (66, 71, 75, 82,[39] and 97) then set up the Suzuki-based manufacture of novel ortho-linked phenylpyridines [S-69, R-69, S-74, and R-74 (Scheme B,C)] and both meta- and para-linked congeners (Schemes D and 3A,C). This latter work was further expanded to include examples containing a pyridazine, pyrazine, or pyrimidine ring (R-109, R-112, and R-115), using similar chemistry[39] (Scheme D), and to a novel racemic nitrotriazole counterpart of R-84 [88 (Scheme B)], starting from alcohol 86.[40] Lastly, a buffered m-CPBA oxidation of R-84 supplied pyridine N-oxide R-85 (Scheme A).

Scheme 2

Reagents and conditions: (i) NaH, DMF, 0–20 °C, 2.3–3.5 h; (ii) ArB(OH)2, DMF, (toluene, EtOH), 2 M Na2CO3 or 2 M KHCO3, Pd(dppf)Cl2 under N2, 70–88 °C, 2.2–4 h; (iii) NBS, PPh3, CH2Cl2, 20 °C, 3.5 h.

Scheme 3

Reagents and conditions: (i) NaH, DMF, 0–20 °C (or 0–8 °C), 2–2.7 h; (ii) ArB(OH)2, DMF, (toluene, EtOH), 2 M Na2CO3 or 2 M KHCO3, Pd(dppf)Cl2 under N2, 80–89 °C for 2–4 h or 70 °C for 16 h; (iii) m-CPBA, Na2HPO4, CH2Cl2, 20 °C, 16 h.

Reagents and conditions: (i) n class="Chemical">NaH, class="Chemical">pan class="Chemical">DMF, 0–20 °C, 2.3–3.5 h; (ii) ArB(OH)2, DMF, (toluene, EtOH), 2 M Na2CO3 or 2 M KHCO3, Pd(dppf)Cl2 under N2, 70–88 °C, 2.2–4 h; (iii) NBS, PPh3, CH2Cl2, 20 °C, 3.5 h.

Reagents and conditions: (i) n class="Chemical">NaH, class="Chemical">pan class="Chemical">DMF, 0–20 °C (or 0–8 °C), 2–2.7 h; (ii) ArB(OH)2, DMF, (toluene, EtOH), 2 M Na2CO3 or 2 M KHCO3, Pd(dppf)Cl2 under N2, 80–89 °C for 2–4 h or 70 °C for 16 h; (iii) m-CPBA, Na2HPO4, CH2Cl2, 20 °C, 16 h.

Several reference benzyl n class="Chemical">ethers (R-1,[38]R-7,[32]R-118, and R-119) were sourced through direct alkylations of class="Chemical">pan class="Chemical">6-R alcohol 56(32) (Scheme A). Three extended ether targets (R-122, R-123, and R-124) were also formed by Sonogashira reactions on the propargyl ether R-121, derived from the coupling of 56 with bromide 120.[41] Next, the orthogonally diprotected triol 125(42) was employed in complementary syntheses of the novel R and S enantiomers of racemic ether 6(42) (Scheme B,C). Following a Mitsunobu reaction of 125 with 4-(trifluoromethoxy)phenol, selective removal of the PMB group (DDQ), iodination of the resulting alcohol 127, and reaction with 2-bromo-4-nitroimidazole (129) gave silyl ether 130. Treatment of the latter with TBAF and sodium hydride-induced ring closure then produced S-6. Conversely, successive cleavage of the TIPS group from 126, iodination, and then reaction with 129 provided PMB ether 134. Oxidative debenzylation of 134 with DDQ in the absence of water unexpectedly led to partial acetalization of the alcohol product with 4-methoxybenzaldehyde, but this mixture was cleanly converted back to 135 via acid hydrolysis (TsOH/MeOH). Base-assisted annulation of alcohol 135 (NaH) then furnished the second enantiomer, R-6. In subsequent work, 2-pyridinyl ether analogues of both R-6 (R-136, R-137, and R-138) and triazole 47 (139) were accessed from alcohols 56(32) and 86,[40] respectively, via sodium hydride-catalyzed SNAr displacement reactions on halopyridines (Scheme D), while Cu(I)-induced coupling of 56 with aryl isocyanates generated the O-carbamates R-140 and R-141 (Scheme E). Finally, piperazine carbamate R-143 was synthesized by chloroformylation of alcohol 56 and in situ reaction with 1-[4-(trifluoromethoxy)phenyl]piperazine[43] (142) (Scheme F).

Scheme 4

Reagents and conditions: (i) NaH, DMF, 0–20 °C, 0.25–5.5 h; (ii) TBAF, THF, 20 °C, 0.5–18 h; (iii) ArI or ArBr, Et3N, DMF, CuI, Pd(PPh3)2Cl2 under N2, 70 °C for 0.25–1 h or 20 °C for 16 h; (iv) 4-OCF3PhOH, DEAD, PPh3, THF, 0–20 °C, 60 h; (v) DDQ, CH2Cl2, 20 °C, 10–28 h (then TsOH, MeOH, 20 °C, 12 h for 135); (vi) I2, PPh3, imidazole, CH2Cl2, 20 °C, 12–35 h; (vii) 128 or 133, K2CO3, DMF, 85–92 °C, 64–111 h; (viii) CuCl, DMF, 20 °C, 33–43 h; (ix) triphosgene, Et3N, THF, 0–20 °C, 1.7 h, then 142, THF, 20 °C, 3.5 h.

Reagents and conditions: (i) n class="Chemical">NaH, class="Chemical">pan class="Chemical">DMF, 0–20 °C, 0.25–5.5 h; (ii) TBAF, THF, 20 °C, 0.5–18 h; (iii) ArI or ArBr, Et3N, DMF, CuI, Pd(PPh3)2Cl2 under N2, 70 °C for 0.25–1 h or 20 °C for 16 h; (iv) 4-OCF3PhOH, DEAD, PPh3, THF, 0–20 °C, 60 h; (v) DDQ, CH2Cl2, 20 °C, 10–28 h (then TsOH, MeOH, 20 °C, 12 h for 135); (vi) I2, PPh3, imidazole, CH2Cl2, 20 °C, 12–35 h; (vii) 128 or 133, K2CO3, DMF, 85–92 °C, 64–111 h; (viii) CuCl, DMF, 20 °C, 33–43 h; (ix) triphosgene, Et3N, THF, 0–20 °C, 1.7 h, then 142, THF, 20 °C, 3.5 h.

Further linker diversity was accessed through the transformation of n class="Chemical">6-S alcohol 65(32) into the novel class="Chemical">pan class="Chemical">6-R amine hydrochloride, 146 (Scheme A). Following tosylation of 65 and azide displacement, reduction of 6-R azide 145 with propane-1,3-dithiol gave the required amine, which was converted to its hydrochloride salt for improved stability. From this intermediate, reductive alkylation with benzaldehydes (using NaBH3CN), acylation with benzoyl chlorides, or treatment with phenyl isocyanates in the presence of catalytic dibutyltin diacetate yielded the expected benzylamine, benzamide, or phenyl urea derivatives (Scheme A,B). Then, to conclude this study, a variety of shorter O-linked heterobiaryl side chains were constructed using Suzuki couplings on haloheteroaryl ether precursors [156, 159, 167, 170, and 173 (Scheme C–E)]. The latter were obtained directly from alcohol 56, via SNAr reactions on fluoropyridines or chloropyrimidines, or, in the case of 167, from the diprotected triol 125(42) and 6-bromopyridin-3-ol, using the same methodology as described above for R-6.

Scheme 5

Reagents and conditions: (i) TsCl, pyridine, 49 °C, 17 h; (ii) NaN3, DMSO, 64 °C, 3.5 days; (iii) HS(CH2)3SH, Et3N, MeOH, 20 °C, 0.5 h, then HCl, dioxane; (iv) NaBH3CN, AcOH, DMF, 0–20 °C, 7–20 h; (v) DIPEA, DMF, 20 °C, 10–25 h; (vi) NMM or DIPEA, Bu2Sn(OAc)2, DMF, 20 °C, 4–18 h; (vii) NaH, DMF, 0–20 °C, 2.7–3.4 h; (viii) ArB(OH)2, DMF, toluene, EtOH, 2 M Na2CO3, Pd(dppf)Cl2 under N2, 84–89 °C, 1.3–3.5 h; (ix) 6-bromopyridin-3-ol, DEAD, PPh3, THF, 0–20 °C, 89 h; (x) TBAF, THF, 20 °C, 13 h; (xi) I2, PPh3, imidazole, CH2Cl2, 20 °C, 41 h; (xii) 164, K2CO3, DMF, 88 °C, 122 h; (xiii) DDQ, CH2Cl2, 20 °C, 98 h, then TsOH, MeOH, CH2Cl2, 20 °C, 10 h.

Reagents and conditions: (i) TsCl, n class="Chemical">pyridine, 49 °C, 17 h; (ii) NaN3, class="Chemical">pan class="Chemical">DMSO, 64 °C, 3.5 days; (iii) HS(CH2)3SH, Et3N, MeOH, 20 °C, 0.5 h, then HCl, dioxane; (iv) NaBH3CN, AcOH, DMF, 0–20 °C, 7–20 h; (v) DIPEA, DMF, 20 °C, 10–25 h; (vi) NMM or DIPEA, Bu2Sn(OAc)2, DMF, 20 °C, 4–18 h; (vii) NaH, DMF, 0–20 °C, 2.7–3.4 h; (viii) ArB(OH)2, DMF, toluene, EtOH, 2 M Na2CO3, Pd(dppf)Cl2 under N2, 84–89 °C, 1.3–3.5 h; (ix) 6-bromopyridin-3-ol, DEAD, PPh3, THF, 0–20 °C, 89 h; (x) TBAF, THF, 20 °C, 13 h; (xi) I2, PPh3, imidazole, CH2Cl2, 20 °C, 41 h; (xii) 164, K2CO3, DMF, 88 °C, 122 h; (xiii) DDQ, CH2Cl2, 20 °C, 98 h, then TsOH, MeOH, CH2Cl2, 20 °C, 10 h.

Results and Discussion

To establish the SARs against kinetoplastid diseases, 76 new (and several known) pretomanid analogues derived from successive projects with TB Alliance and DNDi were retrospectively tested in replicate assays conducted at the University of Antwerp [LMPH (Tables –4)]. These assays measured activity versus the intracellular amastigote forms of both pan class="Species">Leishmania infantum (class="Chemical">pan class="Species">L. inf) and Trypanosoma cruzi (T. cruzi) and against the bloodstream form of Trypanosoma brucei (T. brucei); cytotoxicity toward human lung fibroblasts (MRC-5 cells, the host for T. cruzi) was also assessed.[44] Much of our VL lead optimization work with DNDi was earlier guided by the findings from single IC50 determinations against Leishmania donovani (L. don) in a mouse macrophage-based luciferase assay[26] performed at the Central Drug Research Institute (CDRI, Pradesh, India), and by follow-up evaluations of in vitro microsomal stability and efficacy in the mouse VL model (Figure A). The best leads were then advanced to further appraisal in the more stringent hamster VL model. Overall, while excellent in vivo efficacy was a key goal for second-generation VL drug candidates, we also aspired (a) to minimize compound lipophilicity (estimated using ACD LogP/LogD software, version 14.04, Advanced Chemistry Development Inc., Toronto, ON) to lessen toxicity risks, (b) to increase aqueous solubility (as judged by kinetic data on dry powder forms of active leads) for optimal oral bioavailability, and (c) to reduce hERG inhibition potential (cf. 4)[24] to improve safety.

Table 1

In Vitro Antiparasitic and Antitubercular Activities for Racemic Nitroheterobicyclic Scaffold Variants

				IC50a,b (μM)					MICb,c (μM)
compd	form	R	CLogP	L. don	L. inf	T. cruzi	T. brucei	MRC-5	MABA	LORA
6d	Aa	OCF3	2.48	0.39	0.95	0.39	>64	>64	2.9	9.6
1e	Ab	OCF3	2.70	0.39	4.0	1.2	>64	>64	1.1	4.4
7e	Ab	OBn	3.32	1.1	5.9	0.38	>64	>64	0.11	2.7
14	Bb	OCF3	3.24	78	>64	50	>64	>64	7.4	55
22	Cb	OCF3	3.74	5.5	2.2	0.53	34	>64	1.2	9.6
23	Cb	OBn	4.36	0.77	2.5	0.27	29	>64	2.2	8.3
24f	Da	OCF3	2.78	0.46	4.0	<0.13	>64	>64	0.63	16
25f	Db	OCF3	2.77	2.6	1.7	<0.13	>64	>64	2.4	7.9
26f	Db	OBn	3.39	2.0	2.6	<0.13	>64	>64	3.1	35
32	Eb	OCF3	2.74	>2	>64	51	>64	>64	>128	>128
33	Eb	OBn	3.36	>2	16	2.3	23	>64	>128	>128
39	Fb	OCF3	2.74	0.63	>64	4.2	>64	58	52	35
40	Fb	OBn	3.36	0.26	>64	2.3	3.2	>64	>128	86
41g	Gb	OCF3	2.88	0.03	0.12	1.2	>64	>64	1.0	7.5
42g	Gb	OBn	3.50	0.05					0.46	3.0
47	Ha	OCF3	2.51		>64	1.3	>64	>64
48e	Hb	OCF3	2.74		>64	0.73	38	>64	>128	>128
49e	Hb	OBn	3.36		>64	0.25	>64	>64	>128	>128
50e	Ib	OCF3	3.26		>64	8.2	46	>64	>128	>128

IC50 values for inhibition of the growth of L. don and L. inf (in mouse macrophages), T. cruzi (on MRC-5 cells), and T. brucei, or for cytotoxicity toward human lung fibroblasts (MRC-5 cells).

Each value (except the single-test L. don data) is the mean of at least two independent determinations. For complete results (mean ± SD), see the Supporting Information.

Minimum inhibitory concentration against M. tb, determined under aerobic (MABA)[59] or hypoxic (LORA)[60] conditions.

TB data from ref (42).

TB data from ref (40).

TB data from ref (30).

Data from ref (24).

Table 4

In Vitro Antiparasitic Activities of 6-O-Linked Biaryl Nitroimidazooxazines

						IC50a,b (μM)
compd	form	link	aza	R	CLogP	L. don	L. inf	T. cruzi	T. brucei	MRC-5
R-157	B	3′	2′	4-OCF3	3.27		0.31	<0.13	7.5	>64
R-158	B	3′	2′	4-F	2.35		0.41	0.30	>64	>64
R-160	B	4′	2′	4-OCF3	3.35		0.64	0.11	9.3	42
R-161	B	4′	2′	4-F	2.43		0.43	<0.13	10	>64
R-168	B	4′	3′	4-OCF3	3.07		0.13	0.15	>64	>64
R-169	B	4′	3′	4-F	2.15		0.20	0.40	>64	>64
S-171c	A	3′	4′,6′	4-OCF3	2.79	0.10	23	2.0	>64	47
R-171	B	3′	4′,6′	4-OCF3	2.79		21	<0.13	>64	>64
R-172	B	3′	4′,6′	4-F	1.87		49	0.68	>64	>64
R-174	B	4′	2′,6′	4-OCF3	2.71		0.65	0.48	>64	>64
R-175	B	4′	2′,6′	4-F	1.80		0.83	0.64	>64	>64

IC50 values for inhibition of the growth of L. don and L. inf (in mouse macrophages), T. cruzi (on MRC-5 cells), and T. brucei, or for cytotoxicity toward human lung fibroblasts (MRC-5 cells).

Each value (except the single-test L. don data) is the mean of at least two independent determinations. For complete results (mean ± SD), see the Supporting Information.

From ref (42).

Figure 2

Schematic diagram of the two lead optimization approaches (A and B) employed.

IC50 values for inhibition of the growth of n class="Species">L. don and class="Chemical">pan class="Species">L. inf (in mouse macrophages), T. cruzi (on MRC-5 cells), and T. brucei, or for cytotoxicity toward human lung fibroblasts (MRC-5 cells).

Each value (except the single-test pan class="Species">L. don data) is the mean of at least two independent determinations. For complete results (mean ± SD), see the Supporting Information.

Minin class="Gene">mum inhibitory concentclass="Chemical">pan class="Species">ration against M. tb, determined under aerobic (MABA)[59] or hypoxic (LORA)[60] conditions.

IC50 values for inhibition of the growth of n class="Species">L. don and class="Chemical">pan class="Species">L. inf (in mouse macrophages), T. cruzi (on MRC-5 cells), and T. brucei, or for cytotoxicity toward human lung fibroblasts (MRC-5 cells).

Each value (except the single-test pan class="Species">L. don data) is the mean of at least two independent determinations. For complete results (mean ± SD), see the Supporting Information.

pan class="Chemical">N-Oxide.

Racemic pan class="Chemical">nitrotriazolooxazine analogue of R-84.

IC50 values for inhibition of the growth of n class="Species">L. don and class="Chemical">pan class="Species">L. inf (in mouse macrophages), T. cruzi (on MRC-5 cells), and T. brucei, or for cytotoxicity toward human lung fibroblasts (MRC-5 cells).

Each value (except the single-test pan class="Species">L. don data) is the mean of at least two independent determinations. For complete results (mean ± SD), see the Supporting Information.

Racemic pan class="Chemical">nitrotriazolooxazine analogue of class="Chemical">pan class="Chemical">R-136.

pan class="Chemical">N-Piperazine.

IC50 values for inhibition of the growth of n class="Species">L. don and class="Chemical">pan class="Species">L. inf (in mouse macrophages), T. cruzi (on MRC-5 cells), and T. brucei, or for cytotoxicity toward human lung fibroblasts (MRC-5 cells).

Each value (except the single-test pan class="Species">L. don data) is the mean of at least two independent determinations. For complete results (mean ± SD), see the Supporting Information.

Scaffold Modification: Initial Hits

As part of our earlier TB studies, we had exn class="Chemical">amined some fundamental changes to the class="Chemical">pan class="Chemical">nitroimidazooxazine “warhead”, including replacement of the nitroimidazole ring by nitropyrazole or nitrotriazole [e.g., 48-50 (Table )] and exchange of the 8-oxygen for sulfur or nitrogen.[40] We had also explored reversal of the C-6 linker (e.g., 24–26)[29,30] and transposition of the side chain to position 7 (e.g., 41 and 42).[24] In a further extension to this work (seeking improved metabolic stability and new active scaffolds), the novel methylated derivatives 14, 22, and 23 were investigated, together with nitroimidazooxazepines 32, 33, 39, and 40. Unfortunately, except for the 7,7-dimethyl derivative 22 [which showed antitubercular potency comparable to that of 1 and excellent stability toward human liver microsomes, HLM, 92% after 1 h (Table )], these compounds proved to be unattractive for TB. Nevertheless, in preliminary antiparasitic screening at the Swiss Tropical Institute, 23, 25, and 26 demonstrated encouraging utility against L. don in a mouse macrophage assay (IC50s of 1.2–1.5 μM),[29] and triazole 49 exhibited striking activity versus Chagas disease (T. cruzi IC50 of 0.084 μM). For greater clarity, we will focus the discussion first on the intended main application (VL) and discuss the other parasite data in a closing section. Follow-up testing of a larger set of compounds at CDRI[26] identified 1, 6, 24, and 40 as being superior for VL [L. don IC50s of 0.26–0.46 μM (Table )], although such hits were still an order of magnitude less potent than 4 and the 7-substituted oxazine 41 (IC50s of 0.03 μM).[24,25]

Table 5

Aqueous Solubility, Microsomal Stability, and in Vivo (mouse) Antileishmanial Efficacy Data for Selected Analogues

	aqueous solubilitya (μg/mL)		microsomal stabilityb [% remaining at 1 (0.5) h]			in vivo efficacy against L. don (% inhibition at dose in mg/kg)c
compd	pH 7	pH 1	H	M	Ham	50	25	12.5	6.25	ED50d
S-1	19		82	94
R-1	18		92	86	31	50
R-6	12		81	79	19	>99	>99	81	42	7.5
22	6.0		92	72
24	3.9		(78)	(88)	(70)		31
S-51	1.6		(78)	(75)	(43)
S-52	2.7		87	67
R-69	0.51	263	35	44	3.0	20
R-74	78	7350	8.0	0.2	0.1
S-77	3.0	211	(75)	(78)	(31)	44
R-77	1.5	167	(68)	(68)	(31)	52
S-81	15	439	(96)	(74)	(15)	35
R-81	6.0	691	(79)	(68)	(0)
R-84	3.0	1040	27 (66)	36 (70)	10 (61)	>99	76	42	36	12
S-89	1.4	479	(88)	(84)	(69)	45
R-89	3.4	503	27 (75)	40 (79)	10 (69)	>99	72	48	17	14
S-91	1.4	384	(73)	(100)	(75)	69
R-91	2.9	12	16 (65)	27 (81)	5.8 (57)	>99	38	6		28
S-92	4.0	1780	(92)	(88)	(59)	37
R-92	5.7	2050	49 (71)	56 (86)	11 (45)	94	54	20		20
R-94	11	4600	58 (85)	66 (77)	10 (37)	83
R-96	40	7140	59	57	5.2	97	48	29	17	28
R-99	2.9	364	37	41	9.2	97	64	31	8	19
R-102	2.1	857	58	56	7.9
R-106	3.9	925	58	67	10	91
116	0.13	32	(88)	(96)	(88)		49
117	0.27	132	(85)	(66)	(64)		23
R-136	110		90	92	48	>99			30
R-147	84	38100	84	61	7.5	12
S-151	132		(100)	(83)	(81)	72
R-151	85		87	86	59	5
S-155	22		(74)	(43)	(64)
R-168	0.36	36	40	35		>99			>99	<3.1
R-169	1.2	325	73	59		>99			72

Kinetic solubility in water (pH 7) or 0.1 M HCl (pH 1) at 20 °C, determined by HPLC (see Method A in Experimental Section).

Pooled human (H), CD-1 mouse (M), or hamster (Ham) liver microsomes; data in parentheses are the percentage parent compound remaining following a 30 min incubation.

Dosing was done orally, once daily for 5 days consecutively; data are the mean percentage reduction of parasite burden in the liver.

Dose in milligrams per kilogram required to achieve a mean 50% reduction in parasite burden.

Kinetic solubility in pan class="Chemical">water (pH 7) or 0.1 M class="Chemical">pan class="Chemical">HCl (pH 1) at 20 °C, determined by HPLC (see Method A in Experimental Section).

Pooled n class="Species">human (H), CD-1 class="Chemical">pan class="Species">mouse (M), or hamster (Ham) liver microsomes; data in parentheses are the percentage parent compound remaining following a 30 min incubation.

Dosing was done orally, once daily for 5 days consecutively; data are the mean percentage reduction of pan class="Chemical">parasite burden in the liver.

Dose in pan class="Chemical">milligrams per kilogram required to achieve a mean 50% reduction in class="Chemical">pan class="Chemical">parasite burden.

These results, togn class="Chemical">ether with evidence of the reduced solubility and more rapid metabolism of analogues with 4-benzyloxybenzyl side chains,[36,42] prompted the further appraisal of 24 in an class="Chemical">pan class="Species">L. don infection VL mouse model. Disappointingly, 24 displayed weak activity [31% inhibition at 25 mg/kg, dosing po daily for 5 days (Table )], despite its reasonable mouse PK profile [50% oral bioavailability, moderate exposure, and 2 h half-life (Table )]. This outcome implied the need to significantly boost in vitro potencies in this class. However, two highly effective phenylpyridine analogues of 24, 116(30) and 117(30) [L. don IC50s of 0.02–0.05 μM (Table )], also failed to deliver useful activity in this in vivo assay under the same dosing regimen (23–49% inhibition). Analysis of their mouse PK data identified low oral bioavailability (11–15%) as a contributing factor here because greater oral exposure led to better efficacy [116 (Table and Figure S1)]. A related concern for both compounds was poor aqueous solubility [0.13–0.27 μg/mL at pH 7 (Table )], while retrospective testing against L. inf later revealed suboptimal potency (IC50s of ∼6 μM). Taken together, these findings reinforced the importance of improving both potency and in vivo PK properties, to achieve suitable efficacy in VL models. To this end, we returned to our sizable pretomanid analogue library, where we had already amassed key solubility and DMPK information from extensive earlier studies with the TB Alliance.

Table 6

Pharmacokinetic Parameters for Selected Compounds in Mice, Rats, and Hamsters

	intravenous (1–2 mg/kg)a					oral (25–50 mg/kg)a
compd	C0 (μg/mL)	CL (mL/min/kg)	Vdss (L/kg)	t1/2 (h)	AUClastb (μg·h/mL)	Cmax (μg/mL)	Tmax (h)	t1/2 (h)	AUClastb (μg·h/mL)	Fc (%)
Mice
R-6						21	5.3	7.8	272
24	0.34	13	2.5	2.0	1.25	1.5	1.0		15.7	50
R-84						16	4.7	9.4	211
R-89						32	3.3	6.1	265
116	0.75	1.2	1.9	20	11.7	0.87	10		32.2	11
117	0.44	5.2	1.8	2.7	3.18	0.84	8.0		12.0	15
R-136						96	3.1	30	1777
Rats
R-6	0.54	11	2.7	3.0	1.51	5.2	6.7	-d	80.6	100
R-136	0.84	6.1	1.5	2.7	2.47	18	2.7	3.5	164	100
Hamsters
R-6	0.66	81	6.5	1.4	0.42	2.1	3.3	2.2	11.2	100
R-84	0.65	23	8.2	6.3	1.30	5.7	3.0	5.8	62.7	100
R-89	0.94	18	4.2	3.9	1.89	4.2	2.0	3.9	26.7	74
R-96	0.93	63	4.0	0.83	0.48	1.9	0.75	0.83	3.95	37
R-99	1.1	8.8	3.2	5.3	3.69	4.7	3.3	5.8	46.5	64
R-106	0.52	59	5.4	1.4	0.71	1.1	1.7	17	3.40	21
R-136						12	3.3	3.1	71.0

The intravenous dose was 1 mg/kg for mice and rats and 2 mg/kg for hamsters. The oral dose was 25 mg/kg for 24, 116, and 117, 50 mg/kg for R-106 and R-136, and 40 mg/kg for the other compounds.

Area under the curve calculated to the last time point (24 or 48 h).

Oral bioavailability, determined using dose-normalized AUClast values.

Not calculable.

Table 2

In Vitro Antiparasitic Activities of 6-OCH2/CH2O-Linked Biaryl Nitroimidazooxazines

						IC50a,b (μM)
compd	form	link	aza	R	CLogP	L. don	L. inf	T. cruzi	T. brucei	MRC-5
S-51c					3.97	0.25	2.3	4.2	>64	>64
S-52c					2.60	0.40	5.2	4.9	>64	>64
S-58d	A	2′		4-OCF3	4.36	0.18	30	0.88	>64	>64
R-58	B	2′		4-OCF3	4.36	0.17	1.1	0.64	>64	>64
R-60	B	3′		4-OCF3	4.36	0.31	2.3	0.43	22	62
R-62	B	4′		4-OCF3	4.36	1.2	>64	0.12	>64	>64
S-64e	A	4′	2	4-CF3, 6-Cl	4.46	0.13	1.5	3.3	54	>64
R-64	B	4′	2	4-CF3, 6-Cl	4.46	0.21	0.87	0.65	43	57
S-69	A	2′	3′	4-OCF3	3.01	28	>64	6.7	>64	>64
R-69	B	2′	3′	4-OCF3	3.01	0.78	2.0	1.9	>64	>64
S-74	A	2′	6′	4-OCF3	3.04	4.8	9.1	6.3	>64	>64
R-74	B	2′	6′	4-OCF3	3.04	0.86	2.0	1.3	18	>64
S-77e	A	3′	2′	4-OCF3	3.01	0.12	1.9	2.0	>64	47
R-77	B	3′	2′	4-OCF3	3.01	0.06	0.63	0.25	46	61
S-79	A	3′	2′	2-F, 4-OCF3	3.57		2.4	3.8	>64	>64
R-79	B	3′	2′	2-F, 4-OCF3	3.57		0.45	0.56	>64	>64
R-80	B	3′	2′	4-OCF2H	2.16		0.51	0.26	>64	>64
S-81e	A	3′	2′	4-F	2.10	0.24	32	3.9	>64	>64
R-81	B	3′	2′	4-F	2.10	0.12	0.73	0.36	>64	>64
S-84e	A	4′	2′	4-OCF3	3.04	0.83	10	1.4	43	>64
R-84	B	4′	2′	4-OCF3	3.04	(0.24)f	0.71	0.043	>64	>64
S-85g	A	4′	2′-Oh	4-OCF3	0.94		9.1	5.4	11	>64
R-85	B	4′	2′-Oh	4-OCF3	0.94		1.6	3.3	>64	>64
88i					3.08		41	0.52	3.5	14
S-89e	A	4′	2′	2-F, 4-OCF3	3.60	0.18	14	1.8	>64	>64
R-89	B	4′	2′	2-F, 4-OCF3	3.60	(0.27)f	0.62	0.078	>64	>64
R-90	B	4′	2′	3-F, 4-OCF3	3.02		3.5	0.025	>64	>64
S-91e	A	4′	2′	2-Cl, 4-OCF3	3.75	0.16	2.4	2.5	35	20
R-91	B	4′	2′	2-Cl, 4-OCF3	3.75	(0.31)f	0.57	0.12	35	41
S-92e	A	4′	2′	4-OCF2H	2.19	0.08	3.4	1.2	46	>64
R-92	B	4′	2′	4-OCF2H	2.19	0.86	0.63	0.078	>64	>64
R-93	B	4′	2′	4-CF3	3.17		6.1	0.072	>64	>64
R-94	B	4′	2′	4-F	2.13	0.18	2.3	0.16	>64	>64
S-96	A	4′	2′	2,4-diF	2.67		1.4	7.8	>64	>64
R-96	B	4′	2′	2,4-diF	2.67	(0.39)f	1.2	0.23	55	>64
R-2	B	4′	3′	4-OCF3	3.01	1.4	4.1	0.27	>64	>64
S-99e	A	4′	3′	2-F, 4-OCF3	3.57	0.33	>64	1.4	>64	>64
R-99	B	4′	3′	2-F, 4-OCF3	3.57	(0.48)f	1.1	0.19	>64	>64
R-100	B	4′	3′	3-F, 4-OCF3	2.99		12	0.24	>64	>64
S-101e	A	4′	3′	2-Cl, 4-OCF3	3.72	0.34	5.2	2.4	23	20
R-101	B	4′	3′	2-Cl, 4-OCF3	3.72		0.61	0.15	34	17
R-102	B	4′	3′	4-OCF2H	2.16		1.9	0.56	>64	>64
R-103	B	4′	3′	4-CF3	3.14		11	0.64	>64	>64
R-104	B	4′	3′	4-F	2.10		1.9	1.7	>64	>64
S-106	A	4′	3′	2,4-diF	2.64		5.3	51	>64	>64
R-106	B	4′	3′	2,4-diF	2.64	(0.64)f	0.85	1.3	>64	>64
R-109	B	4′	2′,3′	4-OCF3	1.52		2.3	0.55	>64	>64
R-112	B	4′	2′,5′	4-OCF3	2.19		2.8	0.27	>64	>64
R-115	B	4′	2′,6′	4-OCF3	2.63		1.4	0.28	>64	>64
116g	C	4′	3′	4-OCF3	3.38	0.05	6.1	<0.13	0.63	>64
117g	C	4′	3′	4-F	2.46	0.02	6.3	<0.13	17	>64

IC50 values for inhibition of the growth of L. don and L. inf (in mouse macrophages), T. cruzi (on MRC-5 cells), and T. brucei, or for cytotoxicity toward human lung fibroblasts (MRC-5 cells).

Each value (except the single-test L. don data) is the mean of at least two independent determinations. For complete results (mean ± SD), see the Supporting Information.

From ref (49).

From ref (47).

From ref (39).

LMPH data (mean of three or four values).

From ref (30).

N-Oxide.

Racemic nitrotriazolooxazine analogue of R-84.

The intravenous dose was 1 mg/kg for n class="Species">mice and class="Chemical">pan class="Species">rats and 2 mg/kg for hamsters. The oral dose was 25 mg/kg for 24, 116, and 117, 50 mg/kg for R-106 and R-136, and 40 mg/kg for the other compounds.

Library Screening and Hit to Lead Assessments for VL

To assist the identification of more active leads from the n class="Chemical">6-substituted class="Chemical">pan class="Chemical">nitroimidazooxazine class, an 898-member library was screened against L. don amastigotes in a seven-point 3-fold dilution macrophage assay at the Institut Pasteur Korea (mid-2010).[45] In total, 248 compounds (28%) showed >50% inhibition at 10 μg/mL, although only 89 (36%) of these displayed >50% inhibition at 3.3 μg/mL, and known actives from the nitroimidazooxazole and 7-substituted oxazine classes were dispersed across both groupings. By eliminating examples from previously inspected classes (including the “reversed C-6 linker” series above), we obtained a starting set of 169 hits. This set was further refined by excluding compounds with a higher propensity for metabolic and/or solubility issues, based on established trends, to give 42 hits, which were retested at CDRI. The most relevant results are summarized in Figure , with almost all of the remaining hits manifesting weaker potencies (L. don IC50s of 0.5–11 μM). We also screened S-1 and S-2, but both had only modest activities (L. don IC50s of 3.9 and 2.6 μM, respectively). Nevertheless, in view of the 10-fold higher potency of racemic 1 [IC50 of 0.39 μM (Table )], this result for S-1 was highly significant as it implied that 6R enantiomers (which have little activity against M. tb(32,37)) may be the more active chiral form for VL. Therefore, we synthesized a trial set of 10 compounds (R-1, R-2, R-7, R-58, R-60, R-62, R-77, R-81, R-84, and R-94) for assessment. In 9 of 10 cases, these 6R enantiomers exhibited 1.1–12-fold superior potencies [L. don IC50s of 0.06–1.4 μM (see Tables S2 and S3)]; hence, the 6R counterparts of selected hits in Figure were also targeted (in line with the approach in Figure A).

Figure 3

Potencies of 17 selected phenotypic screening hits[30,39,42,46−49] against L. don (percent inhibition data from IPK, IC50s from CDRI).

Potencies of 17 selected phenotypic screening hits[30,39,42,46−49] against n class="Species">L. don (percent inhibition data from IPK, ICclass="Chemical">pan class="Species">50s from CDRI).

In an effort to further prioritize the library screening hits for in vivo evaluation, five compounds of high lipophilicity (CLogP > 4 for S-53,[46]S-54,[30]S-55,[47]S-58,[47] and S-64[39]) were omitted from further study and the remaining 12 were assessed for aqueous solubility and microsomal stability (Table and cited references for Figure ). Both the pan class="Chemical">amide S-151[48] and class="Chemical">pan class="Chemical">urea S-155 provided encouraging solubility data (132 and 22 μg/mL, respectively), but the urea unexpectedly showed poor stability toward mouse liver microsomes (MLM, 43% parent after 30 min). Conversely, both the lipophilic arylthiazole S-51[49] (CLogP ∼ 4.0) and the arylpyrimidine S-171[42] were considered of borderline interest because of their modest solubility values (0.9–1.6 μg/mL). While some phenylpyridine hits (e.g., S-77, S-89, S-91, S-92, and S-99)[39] were not substantially more soluble than this at pH 7 (1.4–4.0 μg/mL), these compounds have demonstrated greatly superior results at pH 1 (211–1780 μg/mL). It has been recognized that the low pH of gastric fluid (typically ∼1–2) can enhance the dissolution and oral absorption of such weak bases.[50] Furthermore, close analogue S-2 was advanced to clinical studies for TB partly on the basis of its superior in vivo PK properties in comparison to those of delamanid (5), which are absorption-limited.[23] Concordant with this, the most potent phenylpyridine hits in Figure (S-77, S-81, S-89, S-91, and S-92) also displayed broadly acceptable HLM and MLM stabilities (>70% remaining after 30 min), suggesting their suitability for in vivo studies.

The best six screening hits mentioned above (pan class="Chemical">phenylpyridines S-77, S-81, S-89, S-91, and S-92 and class="Chemical">pan class="Chemical">amide S-151) were then evaluated in the mouse VL model alongside a similar set of 6R enantiomers (R-77, R-84, R-89, R-91, R-92, R-94, and R-151), dosing at 50 mg/kg orally, once daily for 5 days. Results for the meta-linked phenylpyridines R-77, S-77, and S-81 were not particularly impressive [52, 44, and 35% inhibition, respectively (Table )], although the level of activity did track with their respective L. don IC50s (0.06, 0.12, and 0.24 μM, respectively). In contrast, pairwise comparison of para-linked phenylpyridines S-89, S-91, and S-92 with their 6R counterparts unequivocally confirmed the latter as being superior [R-89 (99.5%) vs S-89 (45%), R-91 (99.8%) vs S-91 (69%), and R-92 (94%) vs S-92 (37%)], notwithstanding their slightly higher rates of metabolism (e.g., R-91, 81% parent after 30 min in MLM, vs S-91, 100%). In this series, the 4-fluoro analogue R-94 (L. don IC50 of 0.18 μM) showed reduced utility (83%), whereas the apparently less potent 4-trifluoromethoxy congener R-84 (IC50 of 0.32 μM in the same CDRI assay) provided excellent efficacy (99.4%), despite having lower microsomal stability (36% vs 66% after 1 h in MLM). Overall, the most effective (6S) screening hit was the soluble amide S-151 (72%), but its 6R form (R-151) was unexpectedly poor (5%). However, for this 6-N-linked benzamide class, it was later discovered that the 6S enantiomers had the stronger in vitro potencies (e.g., L. inf IC50s of 5.6 and 12 μM for S-151 and R-151, respectively), suggesting the need for a more systematic investigation of the SAR (Figure B). For better clarity, we will describe this analysis next, before summarizing the results from additional in vivo assessments on all of the most active new VL leads.

SAR of 6-Substituted 2-Nitroimidazooxazines for VL

Following the discovery that many 6R enantiomers had superior in vitro and in vivo activities against VL, a more extensive lead optimization study was initiated to develop additional backup candidates to 4 possessing an advantageous solubility, PK–pan class="Disease">PD, and safety profile. In light of the high potency of ortho-linked biphenyl hit S-58 (class="Chemical">pan class="Species">L. don IC50 0.18 μM), we first sought to establish the optimal linking position for biaryl side chains. Comparison of R-58, R-60, and R-62 in both L. don and L. inf assays (Table ) unexpectedly indicated that ortho linkage was most preferred and that para linkage was least preferred. Therefore, the novel ortho-linked phenylpyridines S-69, R-69, S-74, and R-74 were studied. Here, R-69 and R-74 were equally best, although 1.8-fold less effective than R-58 (L. inf IC50s of 2.0 vs 1.1 μM). Interestingly, these two phenylpyridine isomers showed major differences in both solubility and microsomal stability, with the more soluble R-74 (78 vs 0.51 μg/mL) being metabolized extremely rapidly in all three microsome species [0.1–8% remaining after 1 h (Table )], whereas R-69 was more stable than the para-linked analogue R-84 described above (44% vs 36% after 1 h in MLM).

In the meta-linked n class="Chemical">phenylpyridine series, two new compounds (R-79 and R-80) having terminal ring substituents favored in class="Chemical">pan class="Chemical">para-linked isomers described above (2-F, 4-OCF3, and 4-OCF2H) were marginally more potent than R-77 (4-OCF3) and R-81 (4-F) [L. inf IC50s of 0.45 and 0.51 vs 0.63 and 0.73 μM, respectively (Table )]. Here, replacement of the proximal phenyl ring in R-60 by 2-pyridine (R-77) resulted in a 3.7-fold improvement in activity (L. inf IC50s of 2.3 and 0.63 μM). This enhancement by 2-pyridine was even more pronounced in the para-linked phenylpyridine series (L. inf IC50s of 0.71 and >64 μM for R-84 and R-62, respectively), clarifying that with this heterocycle, ortho linkage was less useful than meta or para linkage. However, in the para-linked set, we considered that the 2′-nitrogen would have less steric protection against oxidation, perhaps accounting for the modest microsomal stabilities of some analogues. Indeed, we could form the N-oxide derivative of R-84 (R-85), which was found to be 2.3-fold less potent (L. inf IC50 of 1.6 μM). Because microsomal stability varied significantly with the substituents in the terminal ring, we evaluated three new congeners (R-90, 3-F, 4-OCF3; R-93, 4-CF3; R-96, 2,4-diF). The most promising of these was R-96 (L. inf IC50 of 1.2 μM, activity ∼2-fold weaker than those of early leads R-84, R-89, R-91, and R-92), which exhibited a better stability profile in MLM and HLM [57–59% vs 16–40% for R-84, R-89, and R-91 (Table )].

As suggested by the screening data (Figure ), replacement of the proximal phenyl ring in pan class="Chemical">R-62 with class="Chemical">pan class="Chemical">3-pyridine (R-2) was less favorable [L. inf IC50 of 4.1 μM vs 0.71 μM for R-84 (Table )]. Nevertheless, the 6R enantiomers of two hits (R-99, 2-F, 4-OCF3; R-101, 2-Cl, 4-OCF3) and the novel 2,4-difluoro analogue R-106 all displayed good potencies (L. inf IC50s of 1.1, 0.61, and 0.85 μM, respectively) and microsomal stabilities at least comparable to those of their 2-pyridine counterparts, although R-101 was cytotoxic (MRC-5 IC50 of 17 μM). By extension, we examined three less lipophilic diaza proximal rings (R-109, R-112, and R-115) that had proven to be very effective in our TB studies,[39] but these turned out to be of less interest (L. inf IC50s of 1.4–2.8 μM). In summary, several new phenylpyridines provided profiles that were attractive for in vivo evaluation, but we had yet to investigate other linker groups. Therefore, we next turned our attention to simpler monoaryl side chains to explore these changes. For this part of the study, we restricted our focus to linkers that had shown particular promise either in the initial screening (e.g., 6-O, 6-NHCO, and 6-NHCONH) or in our earlier TB work.

Commencing with the enantiomer of pretomanid [R-1, pan class="Species">L. inf IC50 of 4.7 μM (Table )], we found variation of the trifluoromethoxy position identified that ortho substitution was best (R-119, class="Chemical">pan class="Species">L. inf IC50 of 1.4 μM), followed by meta substitution (R-118), mimicking findings for biphenyl linkage. Switching to a propargyl ether[42] (R-122) produced much greater activity (L. inf IC50 of 0.33 μM), which was largely retained in trifluoromethylpyridine replacements for the aryl ring[25] (L. inf IC50s of 0.44 and 0.53 μM for R-123 and R-124, respectively). Conversely, removal of the benzylic methylene (R-6) also enabled high potency (L. inf IC50 of 0.53 μM, L. don IC50 of 0.19 μM), while critically allowing the retention of good aqueous solubility (12 μg/mL) and high microsomal stability [79–81% parent after 1 h in MLM and HLM (Table )]. Therefore, we similarly investigated trifluoromethylpyridine analogues of R-6 (R-136, R-137, and R-138) and while there was an ∼2-fold loss of activity, R-136 demonstrated a 9-fold improvement in aqueous solubility (110 μg/mL), together with a slower rate of metabolism (90–92% parent after 1 h in MLM and HLM). However, changing to an O-carbamate linker[48] (R-140, R-141, and R-143) proved to be less satisfactory, with moderate to low potencies observed (L. inf IC50s of 2.1–17 μM).

Table 3

In Vitro Antiparasitic Activities of Variously Linked Monoaryl Nitroimidazooxazines

						IC50a,b (μM)
compd	form	X	aza	R	CLogP	L. don	L. inf	T. cruzi	T. brucei	MRC-5
S-1c	A	OCH2		4-OCF3	2.70	3.9	59	10	>64	>64
R-1d	B	OCH2		4-OCF3	2.70	0.54	4.7	0.40	>64	>64
R-118	B	OCH2		3-OCF3	2.70		2.5	0.31	>64	>64
R-119	B	OCH2		2-OCF3	2.70		1.4	0.20	>64	>64
S-48e	C	OCH2		4-OCF3	2.74		>64	0.74	>64	>64
R-48e	D	OCH2		4-OCF3	2.74		>64	0.54	>64	>64
S-7c	A	OCH2		4-OBn	3.32	1.7	>64	51	>64	>64
R-7c	B	OCH2		4-OBn	3.32	0.14	0.87	0.39	>64	>64
R-122	B	OCH2C≡C		4-OCF3	3.94		0.33	0.11	>64	>64
R-123	B	OCH2C≡C	2	4-CF3	2.47		0.44	1.4	21	28
R-124	B	OCH2C≡C	3	4-CF3	2.47		0.53	1.2	>64	>64
S-6	A	O		4-OCF3	2.48		8.2	7.3	>64	>64
R-6	B	O		4-OCF3	2.48	(0.19)f	0.53	0.15	>64	>64
R-136	B	O	2	4-CF3	2.33	(0.15)f	1.1	0.34	25	>64
R-137	B	O	2	3-CF3	2.13		1.2	0.35	>64	>64
R-138	B	O	2	5-CF3	1.73		0.85	2.4	23	>64
139g					2.37		>64	23	>64	>64
R-24h	A	CH2O		4-OCF3	2.78	0.13	0.86	0.33	57	>64
S-24h	B	CH2O		4-OCF3	2.78	0.11	2.2	<0.13	>64	>64
S-140c	A	OCONH		4-OCF3	2.11	3.3	7.3	6.8	>64	>64
R-140	B	OCONH		4-OCF3	2.11		2.1	3.5	48	>64
R-141	B	OCONH		2-OCF3	2.51		6.4	1.5	>64	>64
S-143i	A	OCOpipj		4-OCF3	1.56	0.88	14	2.1	>64	>64
R-143	B	OCOpipj		4-OCF3	1.56		17	0.27	>64	>64
S-147i	A	NHCH2		4-OCF3	2.26	6.1	>64	11	3.6	>64
R-147	B	NHCH2		4-OCF3	2.26		12	1.8	3.3	>64
R-148	B	NHCH2		3-OCF3	2.26		6.9	<0.13	2.0	>64
R-149	B	NHCH2		2-OCF3	2.26		8.6	0.15	2.1	>64
S-150c	A	NHCO		4-OCF3	1.75		6.0	19	>64	>64
R-150	B	NHCO		4-OCF3	1.75		57	2.5	>64	>64
S-151i	A	NHCO		3-OCF3	1.22	0.25	5.6	25	>64	>64
R-151	B	NHCO		3-OCF3	1.22		12	0.87	>64	>64
S-152i	A	NHCO		2-OCF3	1.40		5.8	53	>64	>64
R-152	B	NHCO		2-OCF3	1.40		18	0.96	>64	>64
S-154c	A	NHCONH		4-OCF3	1.47		10	7.5	22	>64
R-154	B	NHCONH		4-OCF3	1.47		>64	2.3	19	>64
S-155	A	NHCONH		2-OCF3	1.73	0.23	6.8	3.1	4.9	16
R-155	B	NHCONH		2-OCF3	1.73		55	1.3	>64	48

IC50 values for inhibition of the growth of L. don and L. inf (in mouse macrophages), T. cruzi (on MRC-5 cells), and T. brucei, or for cytotoxicity toward human lung fibroblasts (MRC-5 cells).

Each value (except the single-test L. don data) is the mean of at least two independent determinations. For complete results (mean ± SD), see the Supporting Information.

From ref (32).

From ref (38).

From ref (40).

LMPH data (mean of three values).

Racemic nitrotriazolooxazine analogue of R-136.

From ref (30).

From ref (48).

N-Piperazine.

Another option to improve solubility was to replace the pan class="Chemical">ether linkage at C-6 with class="Chemical">pan class="Chemical">nitrogen-based linkers.[48] The 6-amino analogue of R-1 (R-147) had a 5-fold better solubility value at pH 7 (84 μg/mL) and was >2000 times more soluble at pH 1. Nevertheless, this compound was less stable toward microsomes (e.g., 61% vs 86% in MLM) and showed 2.6-fold lower activity (L. inf IC50 of 12 vs 4.7 μM), which was not sufficiently improved by varying the ring substituent position (R-148 and R-149). Alternatively, with a carboxamide or urea linker, the enantiomer preference was reversed, with the original 6S forms (S-150, S-151, S-152, S-154, and S-155) being clearly superior but not particularly potent (L. inf IC50s of ∼6–10 μM). Hence, the only compounds with useful antileishmanial activity were ether-linked at C-6, although it was apparent that the original OCH2 linkage was not optimal and that removal of the benzylic methylene may have metabolic stability and potency advantages. To investigate this further, a small set of O-linked phenylpyridine and phenylpyrimidine derivatives was evaluated (Table ). In the phenylpyridine series (R-157 to R-169), activity against L. inf was similar to or better than that for R-6, with the 3-pyridine isomers preferred (R-168 and R-169, IC50s of 0.13–0.20 μM). These latter compounds exhibited microsomal stabilities comparable to those of the parent linker series above, but their solubility values were inferior (0.36–1.2 μg/mL at pH 7). Finally, contrary to the screening data for S-171, a proximal pyrimidine ring was tolerated only when it was para-linked (R-174 and R-175, L. inf IC50s of 0.65–0.83 μM). Generally, potency against L. inf was more discriminating and tended to better correlate with in vivo outcomes.[24,25]

Throughout the course of these studies, 10 more candidates (R-1, n class="Chemical">R-6, class="Chemical">pan class="Chemical">R-69, R-96, R-99, R-106, R-136, R-147, R-168, and R-169) were screened for activity in the mouse VL model at 50 mg/kg [dosing po daily for 5 d (Table )]. The ortho-linked phenylpyridine R-69 was disappointing (20% inhibition), but the new para-linked analogues R-96, R-99, and R-106 displayed high efficacies (91–97%). More heartening still was the fact that shorter chain phenylpyridines R-168 and R-169 gave essentially complete clearance of the parasite infection (99.9%), as did their monoaryl counterparts, R-6 and R-136. However, both R-1 and its amino-linked equivalent R-147 were unsatisfactory (50 and 12%, respectively), consistent with their weaker in vitro potencies. During the concluding stages of this project, Patterson et al.[38] reported that R-1 was a potential oral treatment for VL on the basis of its in vivo activity in a comparable mouse model at a much larger dose of 100 mg/kg twice daily, but it is clear from these results and other studies[24] that R-1 may not be the optimal development candidate.

Additional Assessments To Determine the Best VL Lead

Dose–response experiments on 7 of the 10 best compounds (pan class="Chemical">R-6, R-84, R-89, R-91, R-92, R-96, and R-99) in the class="Chemical">pan class="Species">mouse VL model yielded ED50 values of 7.5, 12, 14, 28, 20, 28, and 19 mg/kg, respectively (Table and Figure ). The more recent O-linked leads, R-136, R-168, and R-169, were also evaluated at a smaller dose of 6.25 mg/kg and provided parasite burden reductions of 30, 99.7, and 72%, respectively. The most efficacious of these (R-168) produced 84% inhibition at 3.13 mg/kg, representing activity at a level similar to that of the nitroimidazooxazole 4,[25,26] albeit marginal aqueous solubility (0.85 μM at pH 7) deterred its advanced assessment. Instead, we elected to focus initially on the monoaryl ethers R-6 and R-136, together with phenylpyridines R-84 and R-89, which all produced favorable mouse PK data, including excellent oral exposure levels and half-lives of 6–30 h (Table and Figure S1).

Figure 4

Comparative in vivo efficacy in the L. don mouse model: (a) 25 mg/kg and (b) 6.25 mg/kg. All compounds except racemates 24, 116, and 117 are the 6R form.

Compan class="Chemical">parative in vivo efficacy in the L. don class="Chemical">pan class="Species">mouse model: (a) 25 mg/kg and (b) 6.25 mg/kg. All compounds except racemates 24, 116, and 117 are the 6R form.

The selected candidates were further assessed in the chronic pan class="Disease">infection class="Chemical">pan class="Species">hamster model, which is considered the bona fide experimental model for VL because it mimics many features of progressive human disease.[51] Pleasingly, at 50 mg/kg twice daily for 5 days, phenylpyridine R-84 achieved 99.9–100% L. inf clearance in all three target organs, and R-89 was almost as good (Table and Figure ), although both compounds were less inhibitory in bone marrow at 25 mg/kg b.i.d. (81–88%). The monoaryl ethers R-6 and R-136 were even more effective at both dose levels, enabling a 97–99% parasite kill at 25 mg/kg b.i.d. (for comparison, this efficacy level was similar to that observed for 3 at 12.5 mg/kg b.i.d.[24]). Pyridinyl ether R-136 additionally demonstrated fully curative activity (100% parasite clearance in all three organs) at 25 mg/kg b.i.d. in an L. don infection hamster model. Three more phenylpyridines (R-96, R-99, and R-106) were similarly evaluated in the L. inf hamster model, but only R-99 (the 3-pyridyl isomer of R-89) showed any promise (86–98% at 50 mg/kg b.i.d.), paralleling efficacy trends in the mouse model. These results were also broadly in line with the hamster PK data, where R-96 and R-106 displayed inferior oral exposures and oral bioavailabilities (21–37%) much lower than those of the other leads [64–100% (Table and Figure S2)].

Table 7

In Vivo Efficacy Data for Selected Compounds in the Early Curative L. inf Hamster Model

		% inhibition in target organsb
compd	dosea (mg/kg)	liver	spleen	bone marrow
MIL	40	99.0	99.5	96.8
R-6	50	100	100	99.8
	25	98.4	99.2	97.0
	12.5	53.5	47.6	37.3
R-84	50	100	99.9	99.9
	25	98.1	98.4	88.3
	12.5	69.6	55.7	33.6
R-89	50	99.9	99.9	98.9
	25	99.1	93.7	81.3
	12.5	83.5	70.6	50.4
R-96	50	73.0	55.1	55.7
R-99	50	97.7	97.5	86.0
R-106	50	55.7	17.6	45.0
R-136	50	100	100	99.9
	25	99.5	97.3	97.7
	12.5	44.4	43.0	53.0

All test compounds were dosed orally, twice daily for 5 days consecutively; miltefosine (MIL) was dosed once daily for the same period.

Data are the mean percentage reductions in parasite burden in target organs.

Figure 5

Comparative in vivo efficacy in the L. inf hamster model. All compounds are the 6R form.

All test compounds were dosed orally, twn class="Chemical">ice daily for 5 days consecutively; class="Chemical">pan class="Chemical">miltefosine (MIL) was dosed once daily for the same period.

Data are the mean percentage reductions in pan class="Chemical">parasite burden in target organs.

Compan class="Chemical">parative in vivo efficacy in the L. inf class="Chemical">pan class="Species">hamster model. All compounds are the 6R form.

To better discriminate among the four preferred candidates, we next considered key safety features, starting with measuring their interactions with the pan class="Gene">hERG channel. While the class="Chemical">pan class="Chemical">monoaryl ethers R-6 and R-136 posed minimal risk (hERG IC50s of >30 μM), unfortunately, phenylpyridines R-84 and R-89 both caused potent inhibition (IC50s of 0.81 and 0.92 μM, respectively), indicating a strong likelihood of QT prolongation[52] (lead optimization criteria[53,54] mandate an IC50 of >10 μM). This outcome was not anticipated, as 6S counterparts had generated much less concern. The two remaining compounds were then checked for any evidence of mutagenicity in the Ames test. Here, phenyl ether R-6 was negative, but the more soluble pyridinyl ether R-136 unexpectedly yielded a positive result. Although several other nitroimidazole drugs are Ames positive (e.g., metronidazole and fexinidazole),[55] this outcome effectively ruled R-136 out of contention because it would face a more difficult path to achieving regulatory approval.[16,53] Thus, R-6 was identified as the optimal VL lead.

Further Appraisal of VL lead R-6

Additional properties of n class="Chemical">R-6 were measured and weighed against those of the initial preclinical candidate 4 (Table ). The two compounds were comclass="Chemical">pan class="Chemical">parable in terms of molecular weight (345 Da vs 359 Da) and were both highly permeable, but R-6 had a lower measured LogD value (2.59 vs 3.10 for 4 and 2.52 for S-1[48]), superior thermodynamic solubility (23 μM vs 2.8 μM), and a reduced propensity to bind to plasma proteins in various species (82–87% vs 92–96% for 4). This lead also showed only weak CYP3A4 activity (IC50 > 40 μM) and produced a notably favorable rat PK profile, with prolonged exposure and 100% oral bioavailability (Table and Figure S2). These attributes reinforced our conclusion that R-6 (DNDI-8219) was indeed a very promising backup candidate for VL.

Table 8

Additional Comparative Data for Lead Compounds 4 and R-6

property	4a	R-6
molecular weight (Da)	359.3	345.2
LogD (measured)	3.10	2.59
thermodynamic solubility (μM) at pH 7.4	2.8	23
permeability, Papp (×10–6 cm/s) A to B/B to A	22.6/24.7b	32.4/18.9c
plasma protein binding (%)
mouse	96.2	86.7
rat	93.2	82.1
hamster	92.4	87.2
human	93.9	85.2
mutagenic effect (Ames test)	no	nod
hERG IC50 (μM)	10.5	>30
CYP3A4 IC50 (μM)	>25	>40
MABA MIC (μM)	0.046	31e
LORA MIC (μM)	5.9	>128

Most data from refs (24) and (25).

Caco-2 data from ref (61).

MDCK-MDR1 data; no P-gp-mediated efflux.

Not mutagenic in strains TA98 and TA100, in the presence or absence of metabolic activation (S9 fraction).

Single MIC against M. tb, determined under aerobic conditions.

pan class="CellLine">MDCK-MDR1 data; no P-gp-mediated efflux.

Single MIC against pan class="Species">M. tb, determined under aerobic conditions.

A larger scale synthesis of n class="Chemical">R-6 has recently provided a single 170 g batch of high-quality material (HPLC purity of >99.9% and 97.2% ee) in reasonable overall yield (8% over nine linear steps, starting from commercial R-solketal). However, the current synthetic route would still require significant improvement to deliver a scalable, robust, and cost-effective chemical process, in line with the stated class="Chemical">pan class="Chemical">TPP objectives for an affordable drug. Possible alternatives include using the known[56] enantiomer of orthogonally diprotected triol 125 and following the pathway described for S-6 in Scheme B because this enantiomer could be obtained from the cheap, optically pure starting material d-mannitol (via the less expensive S-solketal).[57]

Mechanistic studies of the closely related n class="Chemical">nitroimidazooxazine R-1 demonstclass="Chemical">pan class="Species">rated that it was not activated by the previously identified type I nitroreductase (NTR1) in Leishmania, which mediates the cidal effects of monocyclic nitroheterocyclic drugs such as fexinidazole and nifurtimox.[38,58] Instead, the activity of R-1 was solely triggered by the same novel flavin mononucleotide-dependent NADH oxidoreductase (NTR2) in Leishmania that was employed by nitroimidazooxazoles, such as 4 and 5.[27] This new mode of action was elucidated through a combination of quantitative proteomics and whole genome sequencing of susceptible and drug resistant L. don promastigotes, the latter being generated via culture in the continuous presence of R-1 for 80 days (leading to a reduced level of expression of NTR2).[27] The further observation that R-1 and fexinidazole sulfone displayed additive effects against drug susceptible L. don sparked the suggestion of combination therapy between monocyclic and bicyclic nitro drugs to reduce the likelihood of any future clinical drug resistance.[38] However, we consider that it may be more preferable to look for alternative partner drugs for 3 (or R-6) with greater diversity in their mechanism of action.

One final aspect to consider with pan class="Chemical">R-6 was its efficacy against a wider range of VL and class="Chemical">pan class="Disease">cutaneous leishmaniasis (CL) strains. Overall, R-6 displayed potent broad-spectrum activity against both reference strains and clinical isolates (Table ), comparing favorably with the standard agents sodium stibogluconate, paromomycin, and miltefosine. This lead was also effective against the drug resistant clinical isolates L. don BHU1, L. inf LEM5159, and L. inf MHOM/FR/96/LEM3323 (IC50s of 1.3–5.4 μM), as well as the miltefosine resistant laboratory strain L. inf MHOM/FR/96/LEM3323 C14 MIL4 (IC50 of 0.59 μM). These data confirm that R-6 has excellent potential as a therapy for VL and may have an additional application for the treatment of CL (the more common skin lesion form of leishmaniasis).[1]

Table 9

Inhibitory Activity of R-6 and Clinical VL Drugs against Different Leishmania Strains

	IC50 (μM)a
strain (origin)	R-6	SSGb	Amp Bc	MILd	PMe
VL Strains
L. don MHOM/IN/80/DD8 (India)	0.22	54.3	0.02	2.50	>30
L. don MHOM/ET/67/HU3 (Ethiopia)	0.33	NT	0.05	2.05	NT
L. inf MHOM/MA/67/ITMAP263 (Morocco)	0.51	NT	NT	2.30	136
L. don MHOM/SD/62/1SCL2D (Sudan)	2.16	NT	NT	NT	NT
L. inf MHOM/FR/96/LEM3323 C14 MIL4 (France)f	0.59	NT	NT	>20	78.5
VL Clinical Isolates
L. don BHU1 (India)g	1.34	>150	0.20	3.80	>30
L. don SUKA001 (Sudan)	0.57	29.9	0.05	2.13	>30
L. don GR265 (Ethiopia)	0.19	14.5	0.05	4.60	>30
L. inf LEM5695 (Algeria; dog)	1.77	NT	NT	1.86	165
L. inf MCAN/BR/2002/BH400 (Brazil; dog)	1.23	NT	NT	1.11	64.1
L. inf L3034 (Paraguay; HIV patient)	0.75	NT	NT	1.25	87.7
L. inf LEM5159 (France; HIV patient)f,h	5.41	NT	NT	>20	64.9
L. inf LEM3323 (France; HIV patient)h	2.22	NT	NT	0.74	142
CL Strains
Leishmania aethiopica MHOM/ET/84/KH (Ethiopia)	3.17	NT	0.11	36.1	NT
Leishmania amazonensis MPRO/BR/72/M1841 (Brazil)i	4.68	NT	0.13	15.0	NT
Leishmania major MHOM/SA/85/JISH118 (Saudi Arabia)	2.34	NT	0.05	22.3	NT
Leishmania mexicana MNYC/BZ/62/M379 (Belize)	1.17	NT	0.08	6.55	NT
Leishmania panamensis MHOM/PA/67/Boynton (Panama)	0.34	NT	0.07	21.3	NT
Leishmania tropica Anwari (Syrian clinical isolate)	1.57	>100	0.11	8.22	NT

NT means not tested; some data for clinical VL drugs from refs (26) and (63).

Sodium stibogluconate (IC50 in micrograms per milliliter).

Amphotericin B.

Miltefosine.

Paromomycin.

Resistant to miltefosine.

Resistant to sodium stibogluconate.

Failed amphotericin B treatment.

DsRed2 transgenic strain.

pan class="Chemical">Sodium stibogluconate (IC50 in micrograms per class="Chemical">pan class="Chemical">milliliter).

pan class="Chemical">Amphotericin B.

pan class="Chemical">Miltefosine.

pan class="Chemical">Paromomycin.

Resistant to pan class="Chemical">miltefosine.

Resistant to pan class="Chemical">sodium stibogluconate.

Failed pan class="Chemical">amphotericin B treatment.

SAR of 6-Substituted 2-Nitroimidazo(or 2-Nitrotriazolo)oxazines for Chagas Disease

While the primary objective of this reinvestigation of pretomanid analogues was to develop a backup drug candidate for VL, retrospective screening against the protozoan pan class="Chemical">parasites class="Chemical">pan class="Species">T. cruzi and T. brucei presented an opportunity to assess the possible capacity of these compounds to treat Chagas disease and human African trypanosomiasis (HAT), respectively. A brief inspection of Tables –4 found that only one compound (116) had submicromolar activity against HAT (T. brucei IC50 of 0.63 μM), and this hit could be disregarded on the basis of its less favorable mouse PK profile, inferior aqueous solubility, and poor MDCK-MDR1 cell permeability.[36] Conversely, like 7-substituted 2-nitroimidazooxazines,[24] the majority of compounds displayed interesting potencies against T. cruzi (IC50s of 0.025–1 μM). The selective anti-Chagas activity of nitrotriazolooxazines (e.g., 49; T. cruzi IC50 of 0.25 μM vs >64 μM against L. inf) was particularly striking, being reminiscent of that of a nitrotriazolooxazole analogue of 4.[25] However, further scrutiny revealed that the enantiomers of 48 were roughly equipotent (Table ), while the racemic nitrotriazole counterparts of key VL leads R-6, R-84, and R-136 (47, 88, and 139, respectively) were 9–68-fold less active, suggesting limited utility for this class.

Aside from the less suitable reversed linker congeners (24–26, 116, and 117) and O-linked pan class="Chemical">biaryls (R-157, R-160, R-161, class="Chemical">pan class="Chemical">R-168, and R-171), the most potent 6-substituted 2-nitroimidazooxazine anti-Chagas leads were para-linked phenylpyridines (e.g., R-84, R-89, R-90, R-92, and R-93; IC50s of 0.025–0.078 μM). Here, the 6R enantiomers were clearly superior (by 15- to >400-fold), and the 2-pyridine isomer was preferred, although N-oxidation of the pyridine ring (R-85) was strongly deactivating (77-fold). In the monoaryl subset, arylpropargyl ether R-122 and phenyl ether R-6 seemed highly promising (IC50s of 0.11 and 0.15 μM, respectively), with the latter favored on PK and solubility grounds, whereas similarly active benzylamines R-148 and R-149 were expected to show rapid metabolism.[36] Overall, given the significant hERG liability and higher level of plasma protein binding of the phenylpyridines (e.g., 97.9% for R-84 in mice), R-6 was also regarded as the best new lead for Chagas disease, although its mechanism of action for this application remains to be determined.

Conclusions

In response to a compelling clinical need for more satisfactory VL treatments, recent efforts have been made to reposition leads from other therapeutic areas, seeking to accelepan class="Species">rate new drug development. Promising results with antitubercular class="Chemical">pan class="Chemical">nitroimidazooxazoles and 7-substituted 2-nitroimidazooxazines encouraged us to evaluate additional scaffolds, e.g., nitroimidazooxazepines and methylated or reversed C-6 linker analogues of pretomanid, but these lacked sufficient potency and/or suitable PK and efficacy in the L. don mouse model. However, phenotypic screening of our pretomanid analogue library and follow-up IC50 testing unveiled more active hits spanning a wide lipophilicity range (CLogP values of 1.2–4.5), including several with better solubility and microsomal stability, e.g., phenylpyridines and benzamide S-151. This work also pointed to the generally improved activities of novel 6R enantiomers, which was confirmed for phenylpyridines through comparative appraisal in the mouse VL model. Further studies in this series established that a 4-trifluoromethoxy phenyl substituent, para linkage, and a proximal 2-pyridine ring were preferred for good in vivo PK and efficacy, with two such leads (R-84 and R-89) giving ≥99% parasite clearance in the L. inf hamster model at 50 mg/kg b.i.d. These compounds also showed high potencies against T. cruzi, but unexpectedly high levels of hERG inhibition ultimately terminated their development.

Meanwhile, investigation of the C-6 linker group revealed that the parent n class="Chemical">ether moiety in R-1 (OCH2) was nonoptimal for VL, with shorter and longer chain variants (O and OCH2C≡C, respectively) improving potency against class="Chemical">pan class="Species">L. inf (whereas O-carbamate and N-linked alternatives were poor). Although O-linked phenylpyridine R-168 displayed superb activity in the mouse VL model (99.7% reduction in parasite burden at 6.25 mg/kg, similar to the case for 4), we elected to focus instead on two less lipophilic monoaryl leads (R-6 and pyridine R-136), having superior solubility values (12–110 μg/mL), low hERG risk, and excellent PK profiles in three species (mouse, rat, and hamster). Both compounds delivered high efficacies in the chronic infection hamster model (≥97% inhibition at 25 mg/kg, b.i.d.) and showed weakened binding to plasma proteins, although a positive Ames test for pyridine R-136 dissuaded its further advancement and earmarked R-6 as the favored VL backup candidate to 3. Finally, like the phenylpyridines, R-6 also demonstrated interesting activity against T. cruzi, whereas nitrotriazolooxazine congeners of such leads were less effective. These results provide new insights into the exciting potential of bicyclic nitroimidazoles as novel therapies for the treatment of some challenging neglected diseases.

Experimental Section

Combustion analyses were performed by the Campbell Microanalytical Labopan class="Species">ratory, University of Otago, Dunedin, New Zealand. Melting points were determined using an Electrothermal IA9100 melting point apclass="Chemical">pan class="Chemical">paratus and are as read. NMR spectra were measured on a Bruker Avance 400 spectrometer at 400 MHz for 1H and 100 MHz for 13C and were referenced to Me4Si or solvent resonances. Chemical shifts and coupling constants were recorded in units of parts per million and hertz, respectively. High-resolution electron impact (HREIMS), chemical ionization (HRCIMS), and fast atom bombardment (HRFABMS) mass spectra were recorded on a VG-70SE mass spectrometer at a nominal 5000 resolution. High-resolution electrospray ionization (HRESIMS) mass spectrometry was conducted on a Bruker micrOTOF-Q II mass spectrometer. Low-resolution atmospheric-pressure chemical ionization (APCI) mass spectra were obtained for organic solutions using a ThermoFinnigan Surveyor MSQ mass spectrometer connected to a Gilson autosampler. Optical rotations were measured on a Schmidt + Haensch Polartronic NH8 polarimeter. Column chromatography was performed on silica gel (Merck 230–400 mesh). Chromatographed compounds were typically further purified by crystallization from two solvent combinations, e.g., CH2Cl2 and n-hexane, EtOAc and n-hexane, Et2O and n-pentane, or CH2Cl2 and n-pentane (occasionally, Et2O was added to the latter combination to induce solidification, while some compounds required cooling at −20 °C); more polar compounds were first dissolved in a minimum of 10% MeOH/CH2Cl2 and slowly diluted with n-hexane to give the solid product. Thin-layer chromatography was performed on aluminum-backed silica gel plates (Merck 60 F254), with visualization of components by UV light (254 nm), I2, or KMnO4 staining. Tested compounds (including batches screened in vivo) were all ≥95% pure, as determined by combustion analysis (results within 0.4% of theoretical values) and/or by HPLC conducted on an Agilent 1100 system with diode array detection, using a 150 mm × 3.2 mm Altima 5 μm reversed phase C18 column or a 150 mm × 4.6 mm Zorbax Eclipse XDB 5 μm C8 column and eluting with a gradient (40 to 100%) of 80% CH3CN/water in 45 mM ammonium formate buffer (pH 3.5). Finally, the chiral purity of lead R-6 was assessed by HPLC performed on a Shimadzu 2010 system with diode array detection, employing a 150 mm × 4.6 mm CHIRALPAK AY-H 5 μm analytical column and isocratic elution with 20% EtOH/n-heptane.

Compounds of Table

The following section details the syntheses of compounds 14, 39, and 41 of Table , via representative procedures and key intermediates, as described in Scheme . For the syntheses of the other compounds in Table , see the Supporting Information.

Synthesis of 14 (Scheme A)

Procedure A: 1-[(tert-Butyldimethylsilyl)oxy]-3-(2,4-dinitro-1H-imidazol-1-yl)-2-methylpropan-2-ol (10)

A mixture of 2,4-din class="Chemical">nitro-1H-class="Chemical">pan class="Chemical">imidazole (8) (1.00 g, 6.33 mmol) and tert-butyldimethyl[(2-methyloxiran-2-yl)methoxy]silane[33] (9) (1.81 g, 8.94 mmol) under N2 was stirred at 75 °C for 18 h. The resulting cooled mixture was diluted with EtOAc (250 mL) and washed with aqueous NaHCO3 (150 mL) and brine (125 mL), back-extracting with EtOAc (150 mL). The combined extracts were dried (Na2SO4) and then evaporated to dryness under reduced pressure, and the remaining oil was chromatographed on silica gel. Elution with 10–33% EtOAc/petroleum ether gave 10 (0.92 g, 40%) as a yellow oil: 1H NMR (CDCl3) δ 7.98 (s, 1 H), 4.75 (d, J = 14.0 Hz, 1 H), 4.53 (d, J = 14.0 Hz, 1 H), 3.54 (d, J = 10.2 Hz, 1 H), 3.46 (d, J = 10.1 Hz, 1 H), 2.52 (br s, 1 H), 1.17 (s, 3 H), 0.91 (s, 9 H), 0.09 (2 s, 2 × 3 H); HRCIMS (NH3) calcd for C13H25N4O6Si m/z [M + H]+ 361.1543, found 361.1545.

Procedure B: 1-{3-[(tert-Butyldimethylsilyl)oxy]-2-methyl-2-[(tetrahydro-2H-pyran-2-yl)oxy]propyl}-2,4-dinitro-1H-imidazole (11)

A mixture of n class="Chemical">alcohol 10 (248 mg, 0.688 mmol), 3,4-dihydro-2H-pyran (0.32 mL, 3.51 mmol), and PPTS (95 mg, 0.378 mmol) in anhydrous class="Chemical">pan class="Chemical">CH2Cl2 (10 mL) under N2 was stirred at 20 °C for 5 h. The resulting solution was diluted with CH2Cl2 (100 mL) and washed with aqueous NaHCO3 (2 × 50 mL), water (50 mL), and brine (50 mL), back-extracting with CH2Cl2 (50 mL). The combined extracts were dried (Na2SO4) and then evaporated to dryness under reduced pressure, and the remaining oil was chromatographed on silica gel. Elution with 10% EtOAc/petroleum ether gave 11 (234 mg, 76%) as a yellow oil (a 1:1 mixture of diastereomers): 1H NMR (CDCl3) δ 8.25, 7.99 (2 s, 1 H), 4.88 (d, J = 13.8 Hz, 0.5 H), 4.87 (d, J = 14.0 Hz, 0.5 H), 4.84–4.72 (m, 1 H), 4.62 (d, J = 14.0 Hz, 0.5 H), 4.54 (d, J = 13.8 Hz, 0.5 H), 3.73–3.65 (m, 1 H), 3.60–3.36 (m, 2 H), 3.29 (d, J = 10.6 Hz, 0.5 H), 3.26 (d, J = 10.7 Hz, 0.5 H), 1.95–1.35 (m, 6 H), 1.33, 1.25 (2 s, 3 H), 0.94, 0.87 (2 s, 9 H), 0.10 (2 s, 3 H), 0.04, 0.02 (2 s, 3 H); HRCIMS (NH3) calcd for C18H33N4O7Si m/z [M + H]+ 445.2119, found 445.2125.

Procedure C: 6-Methyl-2-nitro-6-[(tetrahydro-2H-pyran-2-yl)oxy]-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (12)

A solution of n class="Chemical">silyl ether 11 (230 mg, 0.517 mmol) in anhydrous class="Chemical">pan class="Chemical">THF (6 mL) under N2 was treated with TBAF (1.5 mL of a 1 M solution in THF, 1.50 mmol), and the mixture was stirred at 20 °C for 1 h. The resulting solution was concentrated under reduced pressure, then diluted with EtOAc (50 mL), and washed with aqueous NaHCO3 (50 mL) and brine (50 mL), back-extracting with EtOAc (2 × 50 mL). The combined extracts were dried (Na2SO4) and then evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel. Elution with 5% MeOH/CH2Cl2 gave 12 (130 mg, 89%) as a white solid (a 1:1 mixture of diastereomers): mp (CH2Cl2/hexane) 153–156 °C; 1H NMR (CDCl3) δ 7.40, 7.37 (2 s, 1 H), 5.03–4.95 (m, 1 H), 4.54 (dd, J = 11.6, 2.3 Hz, 0.5 H), 4.44 (dd, J = 11.8, 2.7 Hz, 0.5 H), 4.34 (dd, J = 12.7, 2.7 Hz, 0.5 H), 4.18 (d, J = 11.6 Hz, 0.5 H), 4.11 (d, J = 11.7 Hz, 0.5 H), 4.09 (dd, J = 12.5, 2.3 Hz, 0.5 H), 3.89 (d, J = 12.6 Hz, 0.5 H), 3.89–3.81 (m, 1 H), 3.57–3.44 (m, 1 H), 3.42–3.33 (m, 0.5 H), 1.92–1.38 (m, 9 H); APCI MS m/z 284 [M + H]+.

Procedure D: 6-Methyl-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-ol (13)

A solution of n class="Chemical">THP ether 12 (120 mg, 0.424 mmol) in class="Chemical">pan class="Chemical">MeOH (10 mL) was treated with methanesulfonic acid (52.6 mg, 0.547 mmol), and the mixture was stirred at 20 °C for 1 h. The resulting solution was neutralized with aqueous NaHCO3; then the solvents were removed under reduced pressure, and the residue was chromatographed on silica gel. Elution with 5% MeOH/CH2Cl2 gave 13 (80 mg, 95%) as a white solid: mp (MeOH/CH2Cl2/hexane) 185–188 °C; 1H NMR [(CD3)2SO] δ 8.05 (s, 1 H), 5.50 (br s, 1 H), 4.26 (d, J = 11.1 Hz, 1 H), 4.12 (dd, J = 11.1, 2.6 Hz, 1 H), 4.00 (d, J = 12.6 Hz, 1 H), 3.89 (dd, J = 12.6, 2.4 Hz, 1 H), 1.24 (s, 3 H); HREIMS calcd for C7H9N3O4m/z (M+) 199.0593, found 199.0591.

Procedure E: 6-Methyl-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (14)

A solution of n class="Chemical">alcohol 13 (50.7 mg, 0.255 mmol) and class="Chemical">pan class="Chemical">4-(trifluoromethoxy)benzyl bromide (65 μL, 0.406 mmol) in anhydrous DMF (3 mL) under N2 was treated with 60% NaH (20 mg, 0.50 mmol), and the mixture was stirred at 20 °C for 3 h. The resulting mixture was diluted with EtOAc (150 mL) and washed with aqueous NaHCO3 (50 mL), water (2 × 50 mL), and brine (50 mL), back-extracting with EtOAc (100 mL). The combined extracts were dried (Na2SO4) and then evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel. Elution with 1–2% MeOH/CH2Cl2 gave 14 (45 mg, 47%) as a white solid: mp (EtOAc/hexane) 139–141 °C; 1H NMR [(CD3)2SO] δ 7.99 (s, 1 H), 7.31 (br d, J = 9.0 Hz, 2 H), 7.28 (br d, J = 9.0 Hz, 2 H), 4.60 (dd, J = 11.9, 2.8 Hz, 1 H), 4.56 (br s, 2 H), 4.37 (d, J = 11.8 Hz, 1 H), 4.24 (dd, J = 13.2, 2.6 Hz, 1 H), 4.07 (d, J = 13.1 Hz, 1 H), 1.36 (s, 3 H); 13C NMR [(CD3)2SO] δ 147.5, 147.0, 142.2, 137.8, 129.0 (2 C), 120.9 (2 C), 120.0 (q, JC–F = 255.9 Hz), 117.8, 70.6, 69.2, 62.9, 50.7, 17.0. Anal. Calcd for C15H14F3N3O5: C, 48.26; H, 3.78; N, 11.26. Found: C, 48.23; H, 3.98; N, 11.31.

Synthesis of 39 and 41 (Scheme D)

2-Bromo-4-nitro-1-{3-[(tetrahydro-2H-pyran-2-yl)oxy]-4-[(triisopropylsilyl)oxy]butyl}-1H-imidazole (35)

Reaction of 4-(2-bromo-4-n class="Chemical">nitro-1H-imidazol-1-yl)-1-[(triisopropylsilyl)oxy]butan-2-ol[24] (34) with 3,4-dihydro-2H-pyran (4.0 equiv) and PPTS, using procedure B for 1 day (but washing with aqueous class="Chemical">pan class="Chemical">NaHCO3 only and extracting the product four times with CH2Cl2), followed by chromatography of the product on silica gel, eluting with 25–50% CH2Cl2/petroleum ether (forerunners) and then with CH2Cl2, gave 35 (100%) as a colorless oil (a 1:1 mixture of diastereomers): 1H NMR (CDCl3) δ 8.20, 7.82 (2 s, 1 H), 4.69–4.58 (m, 1 H), 4.31–4.10 (m, 2 H), 4.08–3.99 (m, 1 H), 3.92–3.43 (m, 4 H), 2.34–2.23 (m, 0.5 H), 2.12–2.01 (m, 1 H), 1.94–1.41 (m, 6.5 H), 1.18–0.96 (m, 21 H); HRFABMS calcd for C21H39BrN3O5Si m/z [M + H]+ 522.1822, 520.1842, found 522.1838, 520.1826.

4-(2-Bromo-4-nitro-1H-imidazol-1-yl)-2-[(tetrahydro-2H-pyran-2-yl)oxy]butan-1-ol (36)

Reaction of n class="Chemical">silyl ether 35 with class="Chemical">pan class="Chemical">TBAF (1.1 equiv), using procedure C for 3 h (extracting the product five times with EtOAc), followed by chromatography of the product on silica gel, eluting with 33% EtOAc/petroleum ether (forerunners) and then with 33–50% EtOAc/petroleum ether, gave 36 (91%) as a white solid (a 3:1 mixture of diastereomers): mp (Et2O/CH2Cl2/pentane) 89–91 °C; 1H NMR (CDCl3) δ 8.16, 7.79 (2 s, 1 H), 4.65–4.50 (m, 1 H), 4.29–4.12 (m, 2 H), 4.08–3.97 (m, 1 H), 3.85–3.46 (m, 4 H), 2.12–1.73 (m, 5 H), 1.65–1.45 (m, 4 H). Anal. Calcd for C12H18BrN3O5: C, 39.58; H, 4.98; N, 11.54. Found: C, 39.85; H, 5.12; N, 11.57.

Procedure F: 2-Nitro-7-[(tetrahydro-2H-pyran-2-yl)oxy]-5,6,7,8-tetrahydroimidazo[2,1-b][1,3]oxazepine (37)

A solution of n class="Chemical">alcohol 36 (730 mg, 2.00 mmol) in anhydrous class="Chemical">pan class="Chemical">DMF (8 mL) under N2 at 0 °C was treated with 60% NaH (148 mg, 3.70 mmol) and then quickly degassed and resealed under N2. The mixture was stirred at 20 °C for 3.5 h and then cooled to −78 °C (CO2/acetone), the reaction quenched with ice/aqueous NaHCO3 (50 mL), and the mixture extracted with EtOAc (7 × 50 mL). The extracts were washed with brine (50 mL) and then evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–33% EtOAc/petroleum ether first gave forerunners, and then further elution with 50% EtOAc/petroleum ether gave the crude product, which was chromatographed again on silica gel. Elution with 0–2% EtOAc/CH2Cl2 first gave forerunners, and then further elution with 3–4% EtOAc/CH2Cl2 gave 37 (144 mg, 25%) as a white solid: mp (CH2Cl2/hexane) 158–160 °C; 1H NMR (CDCl3) δ 7.48 (s, 1 H), 4.83–4.75 (m, 1 H), 4.39–4.04 (m, 4 H), 3.96–3.82 (m, 2 H), 3.60–3.50 (m, 1 H), 2.25–2.02 (m, 2 H), 1.92–1.71 (m, 2 H), 1.69–1.50 (m, 4 H). Anal. Calcd for C12H17N3O5: C, 50.88; H, 6.05; N, 14.83. Found: C, 51.08; H, 6.08; N, 14.89.

2-Nitro-5,6,7,8-tetrahydroimidazo[2,1-b][1,3]oxazepin-7-ol (38)

A solution of n class="Chemical">THP ether 37 (137 mg, 0.484 mmol) in class="Chemical">pan class="Chemical">MeOH (14 mL) was treated with 3.3 M HCl (0.47 mL, 1.55 mmol). The mixture was stirred at 20 °C for 5 h, cooled to −20 °C, and neutralized with a solution of NH3 in MeOH (0.8 mL of a 7 M solution). The resulting mixture was evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–25% EtOAc/CH2Cl2 first gave forerunners, and then further elution with 25% EtOAc/CH2Cl2 gave 38 (91 mg, 94%) as a white solid: mp (MeOH/CH2Cl2/hexane) 185–187 °C; 1H NMR [(CD3)2SO] δ 8.15 (s, 1 H), 5.28 (br s, 1 H), 4.22 (ddd, J = 14.1, 9.1, 2.0 Hz, 1 H), 4.19–4.09 (m, 1 H), 4.02–3.91 (m, 3 H), 2.09–1.98 (m, 1 H), 1.88–1.76 (m, 1 H). Anal. Calcd for C7H9N3O4: C, 42.21; H, 4.55; N, 21.10. Found: C, 42.41; H, 4.72; N, 21.28.

Procedure G: 2-Nitro-7-{[4-(trifluoromethoxy)benzyl]oxy}-5,6,7,8-tetrahydroimidazo[2,1-b][1,3]oxazepine (39) and 2-Nitro-7-({[4-(trifluoromethoxy)benzyl]oxy}methyl)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (41)

A mixture of n class="Chemical">alcohol 38 (51.0 mg, 0.256 mmol) and class="Chemical">pan class="Chemical">4-(trifluoromethoxy)benzyl bromide (0.205 mL, 1.28 mmol) in anhydrous DMF (1.5 mL) under N2 at 0 °C was treated with 60% NaH (17.5 mg, 0.438 mmol) and then quickly degassed and resealed under N2. The mixture was stirred at 20 °C for 130 min and then cooled to −78 °C (CO2/acetone), the reaction quenched with ice/aqueous NaHCO3 (10 mL), and the mixture added to brine (40 mL) and extracted with CH2Cl2 (5 × 50 mL). The combined extracts were evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–50% EtOAc/petroleum ether first gave forerunners, and then further elution with 50% EtOAc/petroleum ether gave 39 (37 mg, 39%) as a white solid: mp (CH2Cl2/pentane) 119–121 °C; 1H NMR [(CD3)2SO] δ 8.18 (s, 1 H), 7.51 (br d, J = 8.8 Hz, 2 H), 7.36 (br d, J = 7.9 Hz, 2 H), 4.66 (s, 2 H), 4.36 (dd, J = 12.6, 4.6 Hz, 1 H), 4.26–4.13 (m, 2 H), 4.04 (ddd, J = 14.2, 6.4, 3.0 Hz, 1 H), 3.95–3.86 (m, 1 H), 2.19–2.02 (m, 2 H); 13C NMR [(CD3)2SO] δ 151.1, 147.6 (q, JC–F = 1.5 Hz), 140.6, 137.9, 129.3 (2 C), 120.9 (2 C), 120.4, 120.1 (q, JC–F = 256.1 Hz), 74.5, 73.3, 68.7, 42.0, 30.1. Anal. Calcd for C15H14F3N3O5·0.5H2O: C, 47.13; H, 3.96; N, 10.99. Found: C, 46.86; H, 3.63; N, 10.82. HPLC purity of 100%.

Further elution of the column described above with 50–75% n class="Chemical">EtOAc/petroleum class="Chemical">pan class="Chemical">ether gave crude 41, which was chromatographed again on silica gel (as before) to give 41(24) (34 mg, 36%) as a cream solid: mp (EtOAc/hexane) 163–164 °C (lit.[24] mp 158–160 °C); 1H NMR (CDCl3) δ 7.42 (s, 1 H), 7.34 (br d, J = 8.7 Hz, 2 H), 7.20 (br d, J = 8.6 Hz, 2 H), 4.61 (s, 2 H), 4.61–4.54 (m, 1 H), 4.15 (ddd, J = 12.4, 5.7, 3.7 Hz, 1 H), 4.06 (ddd, J = 12.4, 10.0, 5.8 Hz, 1 H), 3.83 (dd, J = 10.6, 4.4 Hz, 1 H), 3.78 (dd, J = 10.7, 4.9 Hz, 1 H), 2.39–2.21 (m, 2 H); APCI MS m/z 374 [M + H]+; HPLC purity of 100%.

The following section details the syntheses of compounds R-58 and pan class="Chemical">R-69 of Table , via representative procedures and key intermediates, as described in Scheme . For the syntheses of the other compounds in Table , see the Supporting Information.

Synthesis of R-58 (Scheme A)

(6R)-6-[(2-Iodobenzyl)oxy]-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (57)

Reaction of (6R)-2-n class="Chemical">nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-class="Chemical">pan class="Chemical">6-ol[32] (56) with 2-iodobenzyl chloride (3.0 equiv) and NaH (1.6 equiv), using procedure G for 3.5 h, followed by chromatography of the product on silica gel, eluting with 0–0.25% MeOH/CH2Cl2 (forerunners) and then with 0.25–0.3% MeOH/CH2Cl2, gave 57 (91%) as a light yellow solid: mp (Et2O) 118–119 °C; 1H NMR (CDCl3) δ 7.85 (br d, J = 7.8 Hz, 1 H), 7.40 (s, 1 H), 7.37–7.33 (m, 2 H), 7.08–7.00 (m, 1 H), 4.72 (d, J = 12.0 Hz, 1 H), 4.72–4.65 (m, 1 H), 4.64 (d, J = 12.0 Hz, 1 H), 4.40 (br dd, J = 12.1, 0.9 Hz, 1 H), 4.25–4.19 (m, 3 H). Anal. Calcd for C13H12IN3O4: C, 38.92; H, 3.02; N, 10.47. Found: C, 39.08; H, 2.98; N, 10.44.

Procedure H: (6R)-2-Nitro-6-{[4′-(trifluoromethoxy)(1,1′-biphenyl)-2-yl]methoxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (R-58)

A mixture of n class="Chemical">iodide 57 (435 mg, 1.08 mmol), class="Chemical">pan class="Chemical">[4-(trifluoromethoxy)phenyl]boronic acid (401 mg, 1.95 mmol), and Pd(dppf)Cl2 (56 mg, 0.077 mmol) in DMF (6 mL) and aqueous KHCO3 (2 mL of a 2 M solution, 4.0 mmol) was degassed, and then N2 was added. The resulting mixture was stirred at 75 °C for 4 h, and then cooled, diluted with brine (100 mL), and extracted with CH2Cl2 (3 × 100 mL). The extracts were evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel. Elution with CH2Cl2 first gave forerunners, and then further elution with 2% MeOH/CH2Cl2 gave R-58 (218 mg, 46%) as a cream solid: mp (CH2Cl2/hexane) 133–135 °C; 1H NMR [(CD3)2SO] δ 8.00 (s, 1 H), 7.50–7.44 (m, 3 H), 7.44–7.35 (m, 4 H), 7.33–7.26 (m, 1 H), 4.57–4.49 (m, 2 H), 4.49 (d, J = 10.9 Hz, 1 H), 4.42 (br d, J = 11.8 Hz, 1 H), 4.22–4.12 (m, 3 H); 13C NMR [(CD3)2SO] δ 147.6 (q, JC–F = 1.5 Hz), 147.1, 142.1, 140.0, 139.3, 134.5, 130.7 (2 C), 130.0, 129.9, 128.3, 127.9, 120.6 (2 C), 120.1 (q, JC–F = 256.4 Hz), 117.9, 68.1, 67.8, 66.8, 46.5. Anal. Calcd for C20H16F3N3O5: C, 55.18; H, 3.70; N, 9.65. Found: C, 55.08; H, 3.66; N, 9.68.

Synthesis of R-69 (Scheme B)

(6R)-6-[(2-Bromopyridin-3-yl)methoxy]-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (68)

Reaction of n class="Chemical">alcohol 56(32) with 2-bromo-3-(bromomethyl)class="Chemical">pan class="Chemical">pyridine (66) (1.6 equiv) and NaH (1.6 equiv), using procedure G at 0 °C for 1 h and then at 20 °C for 80 min, followed by chromatography of the product on silica gel, eluting with 0–0.5% MeOH/CH2Cl2 (forerunners) and then with 0.5–0.67% MeOH/CH2Cl2, gave 68 (83%) as a cream solid: mp (MeOH/CH2Cl2/hexane) 189–190 °C; 1H NMR [(CD3)2SO] δ 8.32 (dd, J = 4.7, 2.0 Hz, 1 H), 8.05 (s, 1 H), 7.80 (dd, J = 7.5, 2.0 Hz, 1 H), 7.46 (dd, J = 7.5, 4.7 Hz, 1 H), 4.73–4.66 (m, 3 H), 4.51 (br d, J = 11.7 Hz, 1 H), 4.39–4.32 (m, 2 H), 4.25 (dd, J = 13.7, 3.4 Hz, 1 H). Anal. Calcd for C12H11BrN4O4: C, 40.58; H, 3.12; N, 15.78. Found: C, 40.81; H, 3.20; N, 15.72.

Procedure I: (6R)-2-Nitro-6-({2-[4-(trifluoromethoxy)phenyl]pyridin-3-yl}methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (R-69)

A stirred mixture of n class="Chemical">bromide 68 (60.3 mg, 0.170 mmol), class="Chemical">pan class="Chemical">[4-(trifluoromethoxy)phenyl]boronic acid (60.5 mg, 0.294 mmol), and Pd(dppf)Cl2 (29.7 mg, 0.041 mmol) in DMF (1.5 mL), toluene (1.0 mL), and EtOH (0.8 mL) was degassed for 7 min (vacuum pump), and then N2 was added. An aqueous solution of Na2CO3 (0.45 mL of a 2 M solution, 0.90 mmol) was added by syringe; the stirred mixture was again degassed for 8 min, and then N2 was added. The resulting mixture was stirred at 87 °C for 190 min and then cooled, diluted with aqueous NaHCO3 (50 mL), and extracted with CH2Cl2 (5 × 50 mL). The combined extracts were evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–0.5% MeOH/CH2Cl2 first gave forerunners, and then further elution with 0.5–0.75% MeOH/CH2Cl2 gave R-69 (62 mg, 84%) as a pale yellow solid: mp (MeOH/CH2Cl2/hexane) 246–248 °C; 1H NMR [(CD3)2SO] δ 8.62 (dd, J = 4.7, 1.7 Hz, 1 H), 8.02 (s, 1 H), 7.89 (dd, J = 7.8, 1.6 Hz, 1 H), 7.67 (br d, J = 8.8 Hz, 2 H), 7.43 (dd, J = 7.7, 4.8 Hz, 1 H), 7.41 (br d, J = 9.0 Hz, 2 H), 4.68–4.57 (m, 3 H), 4.46 (br d, J = 11.9 Hz, 1 H), 4.31–4.17 (m, 3 H); 13C NMR [(CD3)2SO] δ 156.2, 148.9, 148.4, 147.1, 142.1, 138.5, 138.2, 130.8 (2 C), 130.5, 122.8, 120.4 (2 C), 120.1 (q, JC–F = 256.4 Hz), 118.0, 67.8, 67.4, 67.1, 46.6. Anal. Calcd for C19H15F3N4O5: C, 52.30; H, 3.47; N, 12.84. Found: C, 52.02; H, 3.43; N, 12.72.

The following section details the syntheses of compounds n class="Chemical">R-122, class="Chemical">pan class="Chemical">R-136, R-140, R-147, R-151, and R-155 of Table , via representative procedures and key intermediates, as described in Schemes and 5. For the syntheses of the other compounds in Table , see the Supporting Information.

Synthesis of R-122 (Scheme A)

(6R)-2-Nitro-6-[(prop-2-yn-1-yl)oxy]-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (121)

A solution of n class="Chemical">alcohol 56(32) (430 mg, 2.32 mmol) and (class="Chemical">pan class="Chemical">3-bromoprop-1-yn-1-yl)(tert-butyl)dimethylsilane[41] (120) (900 mg, 3.86 mmol) in anhydrous DMF (6 mL) under N2 was treated with 60% NaH (148 mg, 3.70 mmol), and the mixture was stirred at 20 °C for 15 min. The resulting mixture was diluted with water (100 mL), and the precipitate was collected by filtration, washed with water and petroleum ether, and dried. This solid was then redissolved in THF (10 mL); TBAF (6.0 mL of a 1 M solution in THF, 6.0 mmol) was added, and the mixture was stirred at 20 °C for 30 min. The resulting mixture was diluted with EtOAc (100 mL) and washed with water (2 × 100 mL) and brine (100 mL), back-extracting with EtOAc (100 mL). The combined extracts were dried (Na2SO4) and then evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel. Elution with 50% EtOAc/petroleum ether first gave forerunners, and then further elution with EtOAc gave 121 (312 mg, 60%) as a cream solid: mp (Et2O/pentane) 81–83 °C; 1H NMR (CDCl3) δ 7.42 (s, 1 H), 4.63 (ddd, J = 12.5, 3.8, 2.1 Hz, 1 H), 4.42–4.33 (m, 3 H), 4.32 (dd, J = 16.5, 2.5 Hz, 1 H), 4.25 (dd, J = 13.1, 3.6 Hz, 1 H), 4.18 (dt, J = 13.0, 2.5 Hz, 1 H), 2.56 (t, J = 2.4 Hz, 1 H); HRESIMS calcd for C9H9N3NaO4m/z [M + Na]+ 246.0485, found 246.0484.

Procedure J: (6R)-2-Nitro-6-({3-[4-(trifluoromethoxy)phenyl]prop-2-yn-1-yl}oxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (R-122)

A mixture of n class="Chemical">alkyne 121 (108 mg, 0.484 mmol), class="Chemical">pan class="Chemical">1-iodo-4-(trifluoromethoxy)benzene (167 mg, 0.580 mmol), and CuI (2 mg, 0.01 mmol) in triethylamine (5 mL) and DMF (5 mL) was purged with N2. Pd(PPh3)2Cl2 (7 mg, 0.01 mmol) was added, and the mixture was stirred at 70 °C for 15 min under N2 and then cooled, diluted with water (100 mL), and extracted with EtOAc (2 × 100 mL). The extracts were combined and evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel. Elution with 50% EtOAc/petroleum ether first gave forerunners, and then further elution with EtOAc gave R-122 (109 mg, 59%) as a cream solid: mp 150–152 °C; 1H NMR [(CD3)2SO] δ 8.05 (s, 1 H), 7.61 (br d, J = 8.9 Hz, 2 H), 7.39 (br d, J = 8.9 Hz, 2 H), 4.66 (dt, J = 12.1, 2.4 Hz, 1 H), 4.59 (s, 2 H), 4.49 (br d, J = 12.0 Hz, 1 H), 4.41–4.37 (m, 1 H), 4.31 (dt, J = 13.6, 2.0 Hz, 1 H), 4.25 (dd, J = 13.6, 3.2 Hz, 1 H); 13C NMR [(CD3)2SO] δ 148.3 (q, JC–F = 1.5 Hz), 147.0, 142.1, 133.6 (2 C), 121.3 (2 C), 121.0, 119.9 (q, JC–F = 257.1 Hz), 118.0, 86.5, 84.7, 67.8, 66.2, 56.2, 46.6. Anal. Calcd for C16H12F3N3O5: C, 50.14; H, 3.16; N, 10.96. Found: C, 50.19; H, 3.08; N, 10.85.

Synthesis of R-136 (Scheme D)

(6R)-2-Nitro-6-{[5-(trifluoromethyl)pyridin-2-yl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (R-136)

A solution of n class="Chemical">alcohol 56(32) (1.00 g, 5.40 mmol) and class="Chemical">pan class="Chemical">2-fluoro-5-(trifluoromethyl)pyridine (265 mg, 1.61 mmol) in anhydrous DMF (20 mL) under N2 at −10 °C was treated with 60% NaH (275 mg, 6.88 mmol), then quickly degassed, and resealed under N2. Then 2-fluoro-5-(trifluoromethyl)pyridine (1.32 g, 8.00 mmol) was added, and the mixture was stirred at −10 to 0 °C for 1 h. Additional 2-fluoro-5-(trifluoromethyl)pyridine (450 mg, 2.73 mmol) was added, and the mixture was stirred at 20 °C for 130 min. The resulting mixture was cooled to −78 °C (CO2/acetone), the reaction quenched with ice/aqueous NaHCO3 (20 mL), and then the mixture added to brine (100 mL) and extracted with CH2Cl2 (7 × 100 mL). The combined extracts were evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with CH2Cl2 first gave forerunners, and then further elution with 0–2% EtOAc/CH2Cl2 gave the product, which was triturated in Et2O (10 mL) and diluted with pentane (90 mL) to give R-136 (1.66 g, 93%) as a cream solid: mp 140–141 °C; 1H NMR [(CD3)2SO] δ 8.68–8.64 (m, 1 H), 8.13 (br dd, J = 8.7, 2.6 Hz, 1 H), 8.05 (s, 1 H), 7.09 (d, J = 8.7 Hz, 1 H), 5.86–5.79 (m, 1 H), 4.75 (dt, J = 12.3, 2.1 Hz, 1 H), 4.71 (br d, J = 12.2 Hz, 1 H), 4.49 (dd, J = 14.0, 3.4 Hz, 1 H), 4.42 (br d, J = 14.1 Hz, 1 H); 13C NMR [(CD3)2SO] δ 163.9, 146.9, 144.8 (q, JC–F = 4.5 Hz), 142.1, 137.2 (q, JC–F = 3.1 Hz), 124.0 (q, JC–F = 271.5 Hz), 119.7 (q, JC–F = 32.6 Hz), 118.0, 112.1, 68.3, 64.3, 46.6. Anal. Calcd for C12H9F3N4O4: C, 43.65; H, 2.75; N, 16.97. Found: C, 43.80; H, 2.69; N, 17.18.

Synthesis of R-140 (Scheme E)

Procedure K: (6R)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl [4-(Trifluoromethoxy)phenyl]carbamate (R-140)

n class="Chemical">1-Isocyanato-4-(trifluoromethoxy)benzene (70 μL, 0.464 mmol) and class="Chemical">pan class="Chemical">CuCl (3.3 mg, 0.033 mmol) were successively added to a solution of alcohol 56(32) (54.2 mg, 0.293 mmol) in anhydrous DMF (0.7 mL) under N2. The mixture was briefly degassed and resealed under N2 and then stirred at 20 °C for 33 h. The resulting solution was treated with ice/water (5 mL), added to brine (50 mL), and extracted with CH2Cl2 (6 × 50 mL). The combined extracts were evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–3% EtOAc/CH2Cl2 first gave forerunners, and then further elution with 3–4% EtOAc/CH2Cl2 gave R-140 (108 mg, 95%) as a cream solid: mp (CH2Cl2/hexane) 203–205 °C; 1H NMR [(CD3)2SO] δ 10.11 (br s, 1 H), 8.09 (s, 1 H), 7.56 (br d, J = 8.8 Hz, 2 H), 7.30 (br d, J = 8.6 Hz, 2 H), 5.47–5.41 (m, 1 H), 4.69–4.60 (m, 2 H), 4.43 (dd, J = 14.0, 3.4 Hz, 1 H), 4.33 (br dd, J = 14.0, 1.1 Hz, 1 H); 13C NMR [(CD3)2SO] δ 152.2, 146.8, 143.3 (q, JC–F = 1.6 Hz), 142.1, 137.9, 121.7 (2 C), 120.1 (q, JC–F = 255.5 Hz), 119.6 (2 C), 117.9, 68.5, 62.9, 47.0. Anal. Calcd for C14H11F3N4O6: C, 43.31; H, 2.86; N, 14.43. Found: C, 43.43; H, 2.73; N, 14.35.

Syntheses of R-147 and R-151 (Scheme A)

(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl 4-Methylbenzene-1-sulfonate (144)

A mixture of (6S)-2-n class="Chemical">nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-class="Chemical">pan class="Chemical">6-ol[32] (65) (5.00 g, 27.0 mmol) and 4-methylbenzene-1-sulfonyl chloride (10.4 g, 54.6 mmol) in anhydrous pyridine (25 mL) was stirred at 49 °C for 17 h. The cooled solution was added to crushed ice (∼0.7 L), and the resulting precipitate was collected by filtration, washing with water and petroleum ether, to give 144 (8.62 g, 94%) as a cream solid: mp 238–242 °C dec; 1H NMR [(CD3)2SO] δ 8.01 (s, 1 H), 7.85 (br d, J = 8.3 Hz, 2 H), 7.51 (br d, J = 8.0 Hz, 2 H), 5.41–5.34 (m, 1 H), 4.56 (br d, J = 12.3 Hz, 1 H), 4.42 (dt, J = 12.6, 2.6 Hz, 1 H), 4.35 (dd, J = 14.3, 3.2 Hz, 1 H), 4.20 (dt, J = 14.4, 1.9 Hz, 1 H), 2.44 (s, 3 H). Anal. Calcd for C13H13N3O6S: C, 46.02; H, 3.86; N, 12.38. Found: C, 46.25; H, 3.74; N, 12.47.

(6R)-6-Azido-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (145)

A mixture of tosylate 144 (8.61 g, 25.4 mmol) and n class="Chemical">sodium azide (2.48 g, 38.1 mmol) in anhydrous class="Chemical">pan class="Chemical">DMSO (60 mL) was stirred at 64 °C for 84 h. The resulting cooled solution was added to water (250 mL) and extracted with EtOAc (5 × 250 mL); the initially formed emulsion required filtration to remove a dark brown tar. The combined extracts were evaporated to dryness under reduced pressure (at 50 °C), and the residue was triturated in EtOAc (45 mL), diluted with petroleum ether (15 mL), and filtered to give 145 (3.54 g, 66%) as a brown solid. The filtrate was evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel. Elution with 0–0.33% MeOH/CH2Cl2 first gave forerunners, and then further elution with 0.33–0.5% MeOH/CH2Cl2 gave crude 145, which was chromatographed again on silica gel. Elution with Et2O first gave forerunners, and then further elution with 0–0.33% MeOH/CH2Cl2 gave additional 145 (363 mg, 7%) as a cream solid: mp (EtOAc/hexane) 154–156 °C; 1H NMR [(CD3)2SO] δ 8.08 (s, 1 H), 4.67–4.61 (m, 1 H), 4.59 (br dd, J = 11.9, 1.4 Hz, 1 H), 4.55 (ddd, J = 11.9, 2.8, 2.0 Hz, 1 H), 4.32 (dd, J = 13.5, 3.8 Hz, 1 H), 4.17 (br dt, J = 13.5, 1.8 Hz, 1 H); [α]25D 92.6 (c 1.004, DMF). Anal. Calcd for C6H6N6O3: C, 34.29; H, 2.88; N, 39.99. Found: C, 34.57; H, 2.66; N, 40.03.

(6R)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine Hydrochloride (146)

n class="Chemical">Propane-1,3-dithiol (9.2 mL, 91.6 mmol) was added to a mixture of class="Chemical">pan class="Chemical">azide 145 (3.83 g, 18.2 mmol) and triethylamine (12.8 mL, 91.8 mmol) in anhydrous MeOH (75 mL) under N2. After being stirred at 20 °C for 30 min, the mixture was evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–3.5% MeOH/CH2Cl2 first gave forerunners, and then further elution with 5–8% MeOH/CH2Cl2 gave the product free base (2.25 g, 67%) as a yellow solid, which was used directly: 1H NMR [(CD3)2SO] δ 8.06 (s, 1 H), 4.35 (br dd, J = 10.8, 2.1 Hz, 1 H), 4.19–4.10 (m, 2 H), 3.76 (ddd, J = 12.4, 5.5, 1.0 Hz, 1 H), 3.54–3.13 (m, 3 H); APCI MS m/z 185 [M + H]+. This free base was dissolved in MeOH (10 mL) and dioxane (10 mL) and treated with a solution of HCl in dioxane (4.6 mL of a 4 M solution, 18.4 mmol), and then the mixture was diluted with Et2O (100 mL). The resulting oily precipitate was triturated to give 146 (2.29 g, 57%) as a bright yellow powder: mp 208 °C dec; 1H NMR [(CD3)2SO] δ 8.73 (br s, 3 H), 8.18 (s, 1 H), 4.63 (dd, J = 12.2, 2.0 Hz, 1 H), 4.59 (dt, J = 12.1, 2.2 Hz, 1 H), 4.41 (dd, J = 14.0, 5.0 Hz, 1 H), 4.21 (br d, J = 14.1 Hz, 1 H), 4.16–4.07 (m, 1 H); [α]27D 74.7 (c 1.004, H2O); HRESIMS calcd for C6H9N4O3m/z [M – Cl–]+ 185.0669, found 185.0673.

Procedure L: (6R)-2-Nitro-N-[4-(trifluoromethoxy)benzyl]-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (R-147)

n class="Chemical">4-(Trifluoromethoxy)benzaldehyde (130 μL, 0.910 mmol) was added to a mixture of class="Chemical">pan class="Chemical">amine salt 146 (120 mg, 0.544 mmol) and AcOH (65 μL, 1.14 mmol) in anhydrous DMF (5 mL) under N2. The mixture was stirred at 20 °C for 15 min and then cooled to 0 °C. Sodium cyanoborohydride (73 mg, 1.16 mmol) was added, and the mixture was quickly degassed and resealed under N2 and then stirred at 20 °C for 7 h. The resulting mixture was cooled to −78 °C (CO2/acetone), the reaction quenched with ice/aqueous Na2CO3 (10 mL), and the mixture added to brine (40 mL) and extracted with CH2Cl2 (6 × 50 mL). The combined extracts were evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–0.25% MeOH/CH2Cl2 first gave forerunners, and then further elution with 0.33–0.5% MeOH/CH2Cl2 gave R-147 (150 mg, 77%) as a cream solid: mp (Et2O/CH2Cl2/hexane) 120–121 °C; 1H NMR [(CD3)2SO] δ 8.01 (s, 1 H), 7.45 (br d, J = 8.6 Hz, 2 H), 7.30 (br d, J = 8.0 Hz, 2 H), 4.43 (dd, J = 11.2, 2.2 Hz, 1 H), 4.38 (ddd, J = 11.3, 4.3, 1.3 Hz, 1 H), 4.16 (dd, J = 12.7, 4.1 Hz, 1 H), 3.99 (dd, J = 12.7, 2.7 Hz, 1 H), 3.81 (br s, 2 H), 3.29–3.19 (m, 1 H), 2.83 (br s, 1 H); 13C NMR [(CD3)2SO] δ 147.4, 147.1, 142.1, 139.9, 129.6 (2 C), 120.8 (2 C), 120.1 (q, JC–F = 255.6 Hz), 118.1, 68.8, 48.8, 47.2, 46.8. Anal. Calcd for C14H13F3N4O4: C, 46.93; H, 3.66; N, 15.64. Found: C, 47.12; H, 3.54; N, 15.89.

Procedure M: N-[(6R)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl]-3-(trifluoromethoxy)benzamide (R-151)

3-(Trifluoromethoxy)benzoyl chloride (205 mg, 0.913 mmol) was added to a solution of n class="Chemical">amine salt 146 (152 mg, 0.689 mmol) and class="Chemical">pan class="Chemical">DIPEA (0.30 mL, 1.72 mmol) in anhydrous DMF (3 mL) under N2. The mixture was stirred at 20 °C for 10 h and then treated with ice/water (5 mL), added to brine (40 mL), and extracted with CH2Cl2 (4 × 50 mL). The combined extracts were evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–0.5% MeOH/CH2Cl2 first gave forerunners, and then further elution with 0.5–0.67% MeOH/CH2Cl2 gave R-151 (213 mg, 83%) as a cream solid: mp (Et2O/pentane) 98–101 °C dec; 1H NMR [(CD3)2SO] δ 9.01 (br d, J = 6.4 Hz, 1 H), 8.13 (s, 1 H), 7.90 (dt, J = 7.6, 1.3 Hz, 1 H), 7.83–7.78 (m, 1 H), 7.63 (dd, J = 8.0, 7.7 Hz, 1 H), 7.60–7.55 (m, 1 H), 4.69–4.61 (m, 1 H), 4.57 (dd, J = 11.2, 2.3 Hz, 1 H), 4.51 (ddd, J = 11.3, 4.1, 1.4 Hz, 1 H), 4.41 (dd, J = 13.0, 5.1 Hz, 1 H), 4.17 (ddd, J = 13.1, 3.3, 1.2 Hz, 1 H); 13C NMR [(CD3)2SO] δ 165.4, 148.2, 147.2, 142.2, 135.9, 130.5, 126.8, 124.1, 120.1, 120.0 (q, JC–F = 256.8 Hz), 118.4, 68.5, 46.5, 41.6. Anal. Calcd for C14H11F3N4O5: C, 45.17; H, 2.98; N, 15.05. Found: C, 45.37; H, 2.98; N, 15.07.

Synthesis of R-155 (Scheme B)

Procedure N: N-[(6R)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl]-N′-[2-(trifluoromethoxy)phenyl]urea (R-155)

n class="Chemical">1-Isocyanato-2-(trifluoromethoxy)benzene (80 μL, 0.532 mmol) was added to a solution of class="Chemical">pan class="Chemical">amine salt 146 (80.2 mg, 0.364 mmol), DIPEA (0.155 mL, 0.890 mmol), and dibutyltin diacetate (9.3 mg, 0.026 mmol) in anhydrous DMF (2 mL) under N2. The mixture was stirred at 20 °C for 18 h and then treated with ice/water (5 mL), added to brine (40 mL), and extracted with CH2Cl2 (5 × 50 mL). The combined extracts were evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–0.5% MeOH/CH2Cl2 first gave forerunners, and then further elution with 0.75–1% MeOH/CH2Cl2 gave R-155 (118 mg, 84%) as a cream solid: mp (MeOH/CH2Cl2/hexane) 221–225 °C dec; 1H NMR [(CD3)2SO] δ 8.27 (dd, J = 8.5, 1.6 Hz, 1 H), 8.26 (br s, 1 H), 8.11 (s, 1 H), 7.58 (br d, J = 7.0 Hz, 1 H), 7.35–7.25 (m, 2 H), 7.03 (ddd, J = 8.0, 7.7, 1.6 Hz, 1 H), 4.59 (dd, J = 11.3, 1.8 Hz, 1 H), 4.48 (ddd, J = 11.3, 3.0, 2.2 Hz, 1 H), 4.44–4.36 (m, 1 H), 4.31 (dd, J = 12.8, 4.1 Hz, 1 H), 4.12 (dt, J = 12.9, 2.1 Hz, 1 H); 13C NMR [(CD3)2SO] δ 154.2, 147.0, 142.1, 136.7, 132.5, 127.8, 122.1, 121.1, 120.3, 120.2 (q, JC–F = 257.6 Hz), 118.4, 69.7, 47.8, 41.0. Anal. Calcd for C14H12F3N5O5: C, 43.42; H, 3.12; N, 18.08. Found: C, 43.48; H, 2.99; N, 18.03.

The following section details the synthesis of compound pan class="Chemical">R-168 of Table , via representative procedures and key intermediates, as described in Scheme . For the syntheses of the other compounds in Table , see the Supporting Information.

Synthesis of R-168 (Scheme D)

(2R)-2-[(6-Bromopyridin-3-yl)oxy]-3-[(4-methoxybenzyl)oxy]propan-1-ol (163)

Reaction of 2-bromo-5-({(2S)-1-[(4-methoxybenzyl)oxy]-3-[(triisopropylsilyl)oxy]propan-2-yl}oxy)n class="Chemical">pyridine[42] (162) with class="Chemical">pan class="Chemical">TBAF (1.2 equiv), using procedure C for 13 h (extracting the product four times with EtOAc), followed by chromatography of the product on silica gel, eluting with 0–30% Et2O/petroleum ether and CH2Cl2 (forerunners) and then with 2% MeOH/CH2Cl2, gave 163 (94%) as a white solid: mp (CH2Cl2/pentane) 78–80 °C; 1H NMR (CDCl3) δ 8.12 (br d, J = 3.0 Hz, 1 H), 7.35 (br d, J = 8.7 Hz, 1 H), 7.20 (br d, J = 8.8 Hz, 2 H), 7.18 (dd, J = 8.7, 3.1 Hz, 1 H), 6.87 (br d, J = 8.7 Hz, 2 H), 4.51–4.42 (m, 3 H), 3.94–3.81 (m, 2 H), 3.81 (s, 3 H), 3.67 (d, J = 5.1 Hz, 2 H), 1.95 (t, J = 6.4 Hz, 1 H); [α]23D 19.8 (c 3.024, CHCl3). Anal. Calcd for C16H18BrNO4: C, 52.19; H, 4.93; N, 3.80. Found: C, 52.19; H, 4.82; N, 3.84.

Procedure O: 2-Bromo-5-({(2S)-1-iodo-3-[(4-methoxybenzyl)oxy]propan-2-yl}oxy)pyridine (164)

A solution of n class="Chemical">iodine (3.74 g, 14.7 mmol) in anhydrous class="Chemical">pan class="Chemical">CH2Cl2 (9 × 10 mL) was slowly added (dropwise over 1 h) to a stirred mixture of alcohol 163 (3.97 g, 10.8 mmol), imidazole (1.92 g, 28.2 mmol), and triphenylphosphine (3.70 g, 14.1 mmol) in anhydrous CH2Cl2 (50 mL) under N2 at 20 °C (water bath cooling). After being stirred at 20 °C for 41 h, the mixture was concentrated under reduced pressure (at 25 °C), and the residual oil was added to excess petroleum ether (100 mL) at the top of a silica gel column (50 g in petroleum ether), rinsing on with CH2Cl2 (4 × 5 mL). Elution with 0–10% Et2O/petroleum ether first gave forerunners, and then further elution with 20–33% Et2O/petroleum ether gave 164 (5.17 g, 100%) as a colorless oil: 1H NMR (CDCl3) δ 8.11 (d, J = 3.1 Hz, 1 H), 7.36 (d, J = 8.7 Hz, 1 H), 7.22 (br d, J = 8.7 Hz, 2 H), 7.16 (dd, J = 8.7, 3.1 Hz, 1 H), 6.88 (br d, J = 8.7 Hz, 2 H), 4.51 (d, J = 11.6 Hz, 1 H), 4.48 (d, J = 11.6 Hz, 1 H), 4.37–4.30 (m, 1 H), 3.81 (s, 3 H), 3.73 (dd, J = 10.4, 5.2 Hz, 1 H), 3.69 (dd, J = 10.3, 4.9 Hz, 1 H), 3.43 (dd, J = 10.8, 5.5 Hz, 1 H), 3.36 (dd, J = 10.7, 5.4 Hz, 1 H); HRESIMS calcd for C16H18BrINO3m/z [M + H]+ 479.9490, 477.9509, found 479.9489, 477.9508.

2-Bromo-5-({(2R)-1-(2-bromo-4-nitro-1H-imidazol-1-yl)-3-[(4-methoxybenzyl)oxy]propan-2-yl}oxy)pyridine (165)

A mixture of n class="Chemical">iodide 164 (5.17 g, 10.8 mmol), 2-bromo-4-class="Chemical">pan class="Chemical">nitro-1H-imidazole (129) (2.28 g, 11.9 mmol), and powdered K2CO3 (1.79 g, 13.0 mmol) in anhydrous DMF (26 mL) under N2 was stirred at 88 °C for 122 h. The resulting cooled mixture was added to aqueous NaHCO3 (100 mL) and extracted with CH2Cl2 (6 × 100 mL). The combined extracts were evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–30% EtOAc/petroleum ether first gave forerunners, and then further elution with 30–50% EtOAc/petroleum ether gave 165 (4.44 g, 76%) as a pale yellow solid: mp (Et2O/pentane) 86–88 °C; 1H NMR (CDCl3) δ 8.04 (d, J = 3.1 Hz, 1 H), 7.83 (s, 1 H), 7.37 (br d, J = 8.7 Hz, 1 H), 7.24 (br d, J = 8.7 Hz, 2 H), 7.02 (dd, J = 8.7, 3.2 Hz, 1 H), 6.92 (br d, J = 8.7 Hz, 2 H), 4.63–4.56 (m, 1 H), 4.53 (d, J = 11.6 Hz, 1 H), 4.49 (d, J = 11.6 Hz, 1 H), 4.46 (dd, J = 14.8, 3.8 Hz, 1 H), 4.36 (dd, J = 14.7, 7.1 Hz, 1 H), 3.84 (s, 3 H), 3.62 (dd, J = 10.5, 4.1 Hz, 1 H), 3.55 (dd, J = 10.5, 6.1 Hz, 1 H); HRESIMS calcd for C19H18Br2N4NaO5m/z [M + Na]+ 566.9500, 564.9517, 562.9536, found 566.9506, 564.9522, 562.9540.

(2R)-3-(2-Bromo-4-nitro-1H-imidazol-1-yl)-2-[(6-bromopyridin-3-yl)oxy]propan-1-ol (166)

A mixture of n class="Chemical">PMB ether 165 (4.42 g, 8.15 mmol) and class="Chemical">pan class="Chemical">DDQ (1.95 g, 8.59 mmol) in CH2Cl2 (175 mL) was stirred at 20 °C for 48 h. Additional DDQ (202 mg, 0.89 mmol) was added, and stirring was continued at 20 °C for 50 h. The resulting mixture was added to saturated aqueous NaHCO3 (200 mL) and extracted with CH2Cl2 (4 × 150 mL). The extracts were sequentially washed with aqueous NaHCO3 (150 mL); then the combined extracts were evaporated to dryness under reduced pressure (at 30 °C), and the remaining oil was chromatographed on silica gel. Elution with CH2Cl2 first gave forerunners, and then further elution with 3% MeOH/CH2Cl2 gave the crude product mixture (4.09 g) as a pale yellow foam. This material was suspended in 3:1 MeOH/CH2Cl2 (200 mL) and treated with p-toluenesulfonic acid monohydrate (0.79 g, 4.15 mmol), stirring at 20 °C for 10 h. Excess NaHCO3 (0.5 g) was added, and the mixture was concentrated under reduced pressure, then diluted with aqueous NaHCO3 (100 mL), and extracted with CH2Cl2 (5 × 100 mL). The combined extracts were evaporated to dryness under reduced pressure, and the remaining oil was chromatographed on silica gel. Elution with 0–1% MeOH/CH2Cl2 first gave forerunners, and then further elution with 1–2% MeOH/CH2Cl2 gave 166 (3.32 g, 96%) as a cream solid: mp (MeOH/CH2Cl2/hexane) 132–133 °C; 1H NMR [(CD3)2SO] δ 8.58 (s, 1 H), 8.08 (d, J = 3.0 Hz, 1 H), 7.51 (br d, J = 8.7 Hz, 1 H), 7.36 (dd, J = 8.8, 3.2 Hz, 1 H), 5.25 (br t, J = 4.7 Hz, 1 H), 4.85–4.78 (m, 1 H), 4.44 (dd, J = 14.7, 3.9 Hz, 1 H), 4.37 (dd, J = 14.7, 7.8 Hz, 1 H), 3.72–3.57 (m, 2 H); [α]26D 13.3 (c 2.032, DMF). Anal. Calcd for C11H10Br2N4O4: C, 31.31; H, 2.39; N, 13.28. Found: C, 31.55; H, 2.32; N, 13.34.

(6R)-6-[(6-Bromopyridin-3-yl)oxy]-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (167)

A solution of n class="Chemical">alcohol 166 (3.29 g, 7.80 mmol) in anhydrous class="Chemical">pan class="Chemical">DMF (50 mL) under N2 at 0 °C was treated with 60% NaH (419 mg, 10.5 mmol) and then quickly degassed and resealed under N2. The mixture was stirred at 0 °C for 45 min and then at 20 °C for 160 min, then cooled to −78 °C (CO2/acetone), the reaction quenched with ice/aqueous NaHCO3 (20 mL), and the mixture added to brine (200 mL). The resulting mixture was sequentially extracted with CH2Cl2 (150 mL), 10% MeOH/CH2Cl2 (4 × 150 mL), 20% EtOAc/CH2Cl2 (4 × 150 mL), 25% MeOH/CH2Cl2 (4 × 150 mL), CH2Cl2 (2 × 150 mL), and EtOAc (150 mL), and then the combined extracts were evaporated to dryness under reduced pressure (at 30 °C). The crude residue was triturated in water, filtered and dried, then resuspended in warm 5% MeOH/CH2Cl2 (100 mL), cooled, and filtered to give 167 (1.93 g, 73%) as a pale brown solid: mp 244–247 °C; 1H NMR [(CD3)2SO] δ 8.22 (br d, J = 3.0 Hz, 1 H), 8.05 (s, 1 H), 7.61 (br d, J = 8.6 Hz, 1 H), 7.55 (dd, J = 8.8, 3.1 Hz, 1 H), 5.35–5.29 (m, 1 H), 4.69 (dt, J = 12.4, 2.1 Hz, 1 H), 4.64 (br d, J = 12.2 Hz, 1 H), 4.40 (dd, J = 14.0, 3.0 Hz, 1 H), 4.35 (br d, J = 14.1 Hz, 1 H); [α]25D 21.0 (c 1.000, DMF). Anal. Calcd for C11H9BrN4O4: C, 38.73; H, 2.66; N, 16.42. Found: C, 38.67; H, 2.54; N, 16.40.

The filtn class="Species">rate described above was evapoclass="Chemical">pan class="Species">rated to dryness under reduced pressure, and the residue was chromatographed on silica gel. Elution with 0–0.5% MeOH/CH2Cl2 first gave forerunners, and then further elution with 0.5–0.67% MeOH/CH2Cl2 gave the crude product, which was suspended in warm 10% MeOH/CH2Cl2 (15 mL), then diluted with CH2Cl2 (20 mL) and hexane (50 mL), and filtered to give additional 167 (233 mg, 9%).

(6R)-2-Nitro-6-({6-[4-(trifluoromethoxy)phenyl]pyridin-3-yl}oxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (R-168)

Reaction of n class="Chemical">bromide 167 with class="Chemical">pan class="Chemical">Pd(dppf)Cl2 (0.27 equiv) and [4-(trifluoromethoxy)phenyl]boronic acid (1.9 equiv), using procedure I for 210 min, followed by chromatography of the product on silica gel, eluting with 0–0.33% MeOH/CH2Cl2 (forerunners) and then with 0.5% MeOH/CH2Cl2, gave R-168 (86%) as a cream solid: mp (MeOH/CH2Cl2/hexane) 239–241 °C; 1H NMR [(CD3)2SO] δ 8.46 (d, J = 2.9 Hz, 1 H), 8.16 (br d, J = 8.9 Hz, 2 H), 8.08 (s, 1 H), 8.01 (d, J = 8.7 Hz, 1 H), 7.67 (dd, J = 8.8, 3.0 Hz, 1 H), 7.45 (br d, J = 8.1 Hz, 2 H), 5.44–5.36 (m, 1 H), 4.73 (dt, J = 12.4, 2.1 Hz, 1 H), 4.69 (br d, J = 12.0 Hz, 1 H), 4.45 (dd, J = 13.9, 3.0 Hz, 1 H), 4.39 (br d, J = 14.0 Hz, 1 H); 13C NMR [(CD3)2SO] δ 151.9, 148.5 (q, JC–F = 1.4 Hz), 148.3, 146.9, 142.2, 138.8, 137.4, 127.9 (2 C), 123.9, 121.2 (2 C), 121.1, 120.1 (q, JC–F = 256.4 Hz), 118.0, 67.9, 66.0, 46.3; [α]25D 6.98 (c 1.003, DMF). Anal. Calcd for C18H13F3N4O5: C, 51.19; H, 3.10; N, 13.27. Found: C, 51.33; H, 2.94; N, 13.27.

Minimum Inhibitory Concentration Assays (MABA and LORA)

These assays against pan class="Species">M. tb were performed according to the reported procedures.[59,60] Results in Table are the mean of two or three independent determinations (SD data are given in Table S1).

In Vitro Parasite Growth Inhibition Assays

The activity of test compounds against the amastigote stage of the n class="Species">L. don class="Chemical">pan class="Chemical">parasite was measured at CDRI using a mouse macrophage-based luciferase assay, performed according to the published procedures.[26] Replicate assays quantifying the growth inhibitory action of compounds against L. inf, T. cruzi, and T. brucei and assessing any cytotoxic effects on human lung fibroblasts (MRC-5 cells) were conducted at the University of Antwerp (LMPH), as previously described;[44] results in Tables –4 are the mean of at least two (up to 10) independent determinations (SD data are given in Tables S1–S4). Additional assays using a wider range of VL and CL strains and clinical isolates were performed via comparable methods at LMPH[44] or LSHTM[26] (primary peritoneal mouse macrophages infected with cultured promastigotes were incubated at 37 °C for 24 h prior to the addition of test compounds and then further incubated for either 3 or 5 days for CL or VL assays, respectively).

Solubility Determinations

Method A

The solid compound sample was mixed with n class="Chemical">water or 0.1 M class="Chemical">pan class="Chemical">HCl (enough to make a 2 mM solution) in an Eppendorf tube, and the suspension was sonicated for 15 min and then centrifuged at 13000 rpm for 6 min. An aliquot of the clear supernatant was diluted 2-fold with water (or 0.1 M HCl), and then HPLC was performed (as described). The kinetic solubility was calculated by comparing the peak area obtained with that from a standard solution of the compound in DMSO (after allowing for varying dilution factors and injection volumes).

Method B

The thermodynamic solubility of compound pan class="Chemical">R-6 at pH 7.4 was measured by Syngene International Ltd. (Plot No. 2 and 3 Biocon class="Chemical">pan class="Chemical">Park, Jigani Link Road, Bangalore 560099, India). The dry powder was equilibrated with 0.1 M phosphate buffer (pH 7.4) in a glass vial at 25 °C (water bath), shaking for 24 h. After filtration using a 0.45 μm PVDF membrane filter, the concentration of R-6 was determined by HPLC (Waters e2695 system, employing a 150 mm × 4.6 mm XBridge 3.5 μm reversed phase C18 column and isocratic elution with 50% CH3CN in 10 mM ammonium acetate buffer, at 1 mL/min), comparing the peak area obtained with that from a standard solution (0.93 mM) in 1:1:2 EtOH/water/CH3CN.

Microsomal Stability Assays

Compound 22 was tested by n class="Disease">MDS Pharma Servclass="Chemical">pan class="Chemical">ices (22011 30th Dr. SE, Bothell, WA 98021-4444), as described previously.[47] Studies of compounds 24, S-51, S-77, R-77, S-81, R-81, R-84, S-89, R-89, S-91, R-91, S-92, R-92, R-94, 116, 117, S-151, and S-155 (Table ) were run by Advinus Therapeutics Ltd. (21 and 22 Phase II, Peenya Industrial Area, Bangalore 560058, India), using a published procedure[61] in which the compound concentration was 0.5 μM and the incubation time was 30 min. Additional analyses of compounds R-1, R-6, R-69, R-74, R-84, R-89, R-91, R-92, R-94, R-96, R-99, R-102, R-106, R-136, R-147, R-151, R-168, and R-169 were performed by WuXi AppTec (Shanghai) Co., Ltd. (288 FuTe ZhongLu, WaiGaoQiao Free Trade Zone, Shanghai 200131, China) via a reported[25] method; the compound concentration was 1 μM, and the incubation time was 1 h.

Distribution Coefficient

This was measured by WuXi AppTec (Shanghai) Co., Ltd. The LogD value of n class="Chemical">R-6 was found by assessing its distribution between 100 mM class="Chemical">pan class="Chemical">phosphate buffer (pH 7.4) and octanol at room temperature (final matrix contained 1% DMSO), using the shake-flask method and LC–MS/MS analysis.

Permeability Assay

The assay was performed by WuXi AppTec (Shanghai) Co., Ltd. n class="CellLine">MDCK-MDR1 cells were seeded onto class="Chemical">pan class="Chemical">polyethylene membranes in 96-well plates at a density of 2 × 105 cells/cm2, giving confluent cell monolayer formation over 4–7 days. A solution of R-6 (2 μM in 0.4% DMSO/HBSS buffer) was applied to the apical or basolateral side of the cell monolayer. Permeation of the compound in the A to B direction or B to A direction was assessed in triplicate over a 150 min incubation at 37 °C and 5% CO2 (95% humidity). In addition, the efflux ratio of R-6 was also determined. Test and reference compounds were quantified by LC–MS/MS analysis based on the peak area ratio of the analyte/internal standard.

Plasma Protein Binding Assays

The studies of 4 and n class="Chemical">R-6 were performed by WuXi AppTec (Shanghai) Co., Ltd., using equilibrium dialysis across a semipermeable membrane. Briefly, 2 μM compound solutions in plasma were dialyzed against 100 mM class="Chemical">pan class="Chemical">phosphate-buffered saline (pH 7.4) on a rotating plate (150 rpm) incubated at 37 °C for 4 or 6 h. Following precipitation of protein with CH3CN, the amount of compound present in each compartment was quantified by LC–MS/MS; values are the mean of triplicate determinations.

Ames Test

Compounds n class="Chemical">R-6 and class="Chemical">pan class="Chemical">R-136 (at doses of 1.5, 4, 10, 25, 64, 160, 400, and 1000 μg/well) were evaluated in triplicate in the Mini-Ames reverse mutation screen conducted by WuXi AppTec (Suzhou) Co., Ltd. (1318 Wuzhong Ave., Wuzhong District, Suzhou 215104, China). Two strains of Salmonella typhimurium (TA98 and TA100) were employed, in the presence and absence of metabolic activation (rat liver S9). Positive controls (2-aminoanthracene, 2-nitrofluorene, and sodium azide) and a negative (DMSO solvent) control were included.

hERG Assay

The effects of compounds n class="Chemical">R-6, R-84, R-89, and class="Chemical">pan class="Chemical">R-136 on cloned hERG potassium channels expressed in Chinese hamster ovary cells were assessed by WuXi AppTec (Shanghai) Co., Ltd., using the automated patch clamp method. Six concentrations (0.12, 0.37, 1.11, 3.33, 10, and 30 μM) were tested (at room temperature), and at least three replicates were obtained for each.

CYP3A4 Inhibition Assay

This work was performed by WuXi AppTec (Shanghai) Co., Ltd. Compound n class="Chemical">R-6 (at concentclass="Chemical">pan class="Species">rations of 1 and 10 μM) was incubated with NADPH-fortified pooled HLM (0.2 mg/mL) and testosterone (50 μM) in phosphate buffer (100 mM) at 37 °C for 10 min. Following quenching with CH3CN, samples were analyzed for the formation of 6β-hydroxytestosterone by LC–MS/MS, and the percentage inhibition was determined (ketoconazole was the positive control, and tolbutamide was used as an internal standard).

In Vivo Experiments

All animal experiments were performed according to institutional ethical guidelines for animal care. pan class="Species">Mouse model studies (LSHTM) were conducted under lclass="Chemical">pan class="Chemical">icense (PPL X20014A54), according to UK Home Office regulations, Animals (Scientific Procedures) Act 1986, and European Directive 2010/63/EU, and hamster studies (LMPH) were approved by the ethical committee of the University of Antwerp (UA-ECD 2010–17).

Acute VL Infection Assay (mouse model, LSHTM)

Test compounds were orally administered once per day for 5 days consecutively to groups of five female BALB/c pan class="Species">mice infected with 2 × 107class="Chemical">pan class="Species">L. don amastigotes, with treatment commencing 1 week postinfection, as described previously.[26] Miltefosine and AmBisome were positive controls, and parasite burdens were determined from impression smears of liver sections. Efficacy was expressed as the mean percentage reduction in parasite load for treated mice in comparison to untreated (vehicle-only) controls (SD data are provided in Table S5). Derived ED50 values (with 95% confidence limits, as specified in Table S5) were obtained from GraphPad Prism 6 software, using a four-parametric sigmoidal variable slope dose–response curve.

Chronic VL Infection Assay (hamster model, LMPH)

Golden n class="Species">hamsters (weighing 75–80 g) were infected with 2 × 107class="Chemical">pan class="Species">L. inf amastigotes, and 21 days postinfection, treatment groups of six animals each were treated orally twice per day with test compounds (formulated in PEG-400) for 5 days consecutively. Parasite burdens in three target organs (liver, spleen, and bone marrow) were determined by microscopic evaluation of impression smears (stained with Giemsa), and efficacy was expressed as the mean percentage parasite load reduction for treated hamsters in comparison to untreated (vehicle-only) controls (SD data are given in Table S6). Miltefosine was included as a reference drug in all experiments.

Mouse Pharmacokinetics

Testing of compounds 24, 116, and 117 was executed by Advinus Therapeutics Ltd., according to a published protocol.[61] Briefly, compounds were administered to groups of male Swiss albino pan class="Species">mice; intravenous dosing (at 1 mg/kg) employed a solution vehicle comprising 20% class="Chemical">pan class="Chemical">NMP and 40% PEG-400 in 100 mM citrate buffer (pH 3), while oral dosing (at 25 mg/kg) was as a suspension in 0.5% carboxymethylcellulose (CMC) and 0.08% Tween 80 in water. Samples derived from plasma (at 0.083 for iv only, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24, and 48 h) were centrifuged prior to analysis by LC–MS/MS, and the PK parameters were determined using Phoenix WinNonlin software (version 5.2). The remaining compounds (R-6, R-84, R-89, and R-136) were assessed by WuXi AppTec (Shanghai) Co., Ltd.; in this case, oral dosing of female BALB/c mice occurred at 40–50 mg/kg in PEG-400 (sampling at 0.25, 1, 2, 4, 8, and 24 h), and the PK data were obtained using WinNonlin software (version 6.2) following similar LC–MS/MS analysis.

Rat and Hamster Pharmacokinetics

All studies were conducted in fasted animals (either male n class="Species">Sprague-Dawley rats or female golden class="Chemical">pan class="Species">Syrian hamsters) by WuXi AppTec (Shanghai) Co., Ltd. Intravenous dosing (at 1 mg/kg for rats and 2 mg/kg for hamsters) utilized a solution formulation of 20% NMP and 40% PEG-400 in citrate buffer (pH 3). In rats, oral dosing (at 40–50 mg/kg) was as a suspension in 0.08% Tween 80 and 0.5% CMC in water, whereas PEG-400 was the vehicle employed for oral dosing in hamsters (at 40–50 mg/kg). Plasma samples (at 0.083 for iv only, 0.25, 0.5, 1, 2, 4, 8, and 24 h) were analyzed by LC–MS/MS, and the PK parameters were calculated using Phoenix WinNonlin software (version 6.3).