7-Substituted 2-Nitro-5,6-dihydroimidazo[2,1-b][1,3]oxazines: Novel Antitubercular Agents Lead to a New Preclinical Candidate for Visceral Leishmaniasis.

Within a backup program for the clinical investigational agent pretomanid (PA-824), scaffold hopping from delamanid inspired the discovery of a novel class of potent antitubercular agents that unexpectedly possessed notable utility against the kinetoplastid disease visceral leishmaniasis (VL). Following the identification of delamanid analogue DNDI-VL-2098 as a VL preclinical candidate, this structurally related 7-substituted 2-nitro-5,6-dihydroimidazo[2,1-b][1,3]oxazine class was further explored, seeking efficacious backup compounds with improved solubility and safety. Commencing with a biphenyl lead, bioisosteres formed by replacing one phenyl by pyridine or pyrimidine showed improved solubility and potency, whereas more hydrophilic side chains reduced VL activity. In a Leishmania donovani mouse model, two racemic phenylpyridines (71 and 93) were superior, with the former providing >99% inhibition at 12.5 mg/kg (b.i.d., orally) in the Leishmania infantum hamster model. Overall, the 7R enantiomer of 71 (79) displayed more optimal efficacy, pharmacokinetics, and safety, leading to its selection as the preferred development candidate.

■ INTRODUCTION

The neglecten class="Chemical">d tropical disease pan class="Disease">visceral leishmaniasis (lass="Chemical">pan class="Gene">VL) is the second deadliest parasitic disorder (after malaria), being most prevalent in Brazil, Sudan, Ethiopia, and the Indian subcontinent, with an estimated 350 million people at risk of infection.[1] Transmitted by sand flies, the disease first manifests as an irregular fever, anemia, leukopenia, and hepatosplenomegaly and is usually fatal within two years if left untreated.[2] About 300000 new cases arise annually, almost half in children, and at least 35 countries have reported the occurrence of HIV coinfection (with up to 34% incidence), which gives a significantly higher mortality rate.[3,4] Unfortunately, none of the existing VL drugs (antimonials, paromomycin, liposomal amphotericin B, or miltefosine 1; see Figure 1) is universally effective nor free from further drawbacks such as parenteral administration (for all except 1), toxicity, high cost, and emerging resistance.[5] Furthermore, there is no available vaccine despite renewed efforts.[6] Clinical investigation of the orally active aminoquinoline sitamaquine (2) has been abandoned due to its toxicity and less satisfactory efficacy,[7] and new phase II trials of the repositioned oral agent fexinidazole (3)[8] for VL have also been interrupted due to patient relapses.[9] With no other candidates under clinical evaluation at present, there is a desperate need for the development of more effective, safe, and affordable oral remedies for VL.

Figure 1

Structures of antitubercular or antileishmanial agents.

Structures of antitubercular or antin class="Chemical">pan class="Disease">leishmanial agents.

We have recently reporten class="Chemical">d that phenotypic screening by the Drugs for Neglected Diseases initiative (DNDi) of some pan class="Chemical">nitroimidazole derivatives arising from our early studies with the lass="Chemical">pan class="Chemical">TB Alliance unexpectedly led to the identification of DNDI-VL-2098 (4) as a preclinical candidate for VL.[10,11] Our opening assignment with TB Alliance had been to prepare and evaluate novel nitroheterobicyclic analogues of the tuberculosis (TB) drugs delamanid (5) and pretomanid (PA-824, 6),[12,13] seeking a possible third active scaffold for the construction of a backup series. However, among the fused 5/6 ring systems examined, only the metabolically labile 2-nitroimidazothiazines retained significant antitubercular potency,[14] returning our attention to the original oxazine class where we uncovered heterobiaryl derivatives of 6 with better efficacy (e.g., TBA-354, 7).[15,16] One important consideration in the design of a superior second-generation TB candidate was the potential for cleavage of the aromatic side chain via oxidative metabolism of the 6-oxymethylene linker; therefore, several alternative linker and steric protection strategies were explored, albeit with limited success.[17-19] A final, more innovative way to address this issue was to invoke a scaffold hopping approach[20] by relocating aromatic side chains from the 6-position to the 7-position of the 2-nitroimidazooxazine core, with attachment via the same inverted linker (CH2OR) that was present in 6-nitroimidazooxazole 5. This was equivalent to a one carbon expansion of the oxazole ring between C-2 and C-3 (Figure 2). The rationale for this design concept stemmed from initial evidence[21] that delamanid (5) was highly stable toward metabolism as well as from a report[22] that 7-methyl derivatives of 6 retained excellent antitubercular potency, suggesting that such an approach merited investigation.

Figure 2

Scaffold hopping to 7-substituted 2-nitroimidazooxazines.

Scaffoln class="Chemical">d hopping to pan class="Chemical">7-substituted 2-nitroimidazooxazines.

Serendipitously, we soon discovered[23] that this novel “pan class="Chemical">7-substituted oxazine” class not only showed considerable promise for lass="Chemical">pan class="Chemical">TB (as later confirmed by others[24,25]), it also displayed potent antileishmanial activity comparable to the 6-nitroimidazooxazoles in early screening assays. Therefore, following the success with 4, this new series was similarly repositioned for VL as part of an extensive backup program run in collaboration with DNDi. In this paper, we first highlight some critical VL hit to lead assessments on the original subset of compounds that had been prepared for TB. We then detail the findings of our lead optimization study directed at developing backups to 4 having an improved physicochemical/pharmacological profile and better safety, which culminated in the selection of a new preclinical candidate for VL. Finally, in light of these encouraging results and the excellent activities of this novel 7-substituted 2-nitroimidazooxazine class against both TB and Chagas disease, we point to related analogues that might be worthy of further assessment for the latter applications.

■ CHEMISTRY

To rapidn class="Gene">ly access some initial examples, the racemic n class="Chemical">7-H and lass="Chemical">pan class="Chemical">7-methyl alcohol intermediates, 13 and 20, were first sought (Scheme 1A). These could be obtained in very good overall yield (62–79%) via similar five-step reaction sequences, starting with base catalyzed alkylation of 2-bromo-4-nitroimidazole (8) using 4-bromobut-1-ene or 4-iodo-2-methylbut-1-ene[26] (15). Dihydroxylation of the resulting alkene (OsO4/NMO), selective TIPS protection of the primary hydroxyl group, sodium hydride-induced ring closure, and acid-catalyzed desilylation[27] completed the synthesis of both alcohols, although in the case of 20, the final two steps required gentle warming. The benzyl ether targets 14, 21–23, 25, and 27–29 were then formed by standard alkylation and Suzuki coupling methodology (Scheme 1A,B). Next, the two enantiomers of early TB lead 29 (34 and 38) were also generated via preparative chiral HPLC separation of the 7R and 7S forms of acetate derivative 30, followed by hydrolysis to the chiral alcohols (32 and 36) and elaboration as before (Scheme 1C). Here, the absolute configurations of 34 and 38 were subsequently established through an independent chiral synthesis of the 7R enantiomer (see the Supporting Information), involving alkylation of 8 with the iodide derived from 2-[(2R)-2-methyl-1,4-dioxaspiro[4.5]decan-2-yl]ethan-1-ol.[28]

Scheme 1

aReagents and conditions: (i) Br(CH2)2CH=CH2 or 15, K2CO3, DMF, 60–73 °C, 4.5–11 h; (ii) OsO4, NMO, CH2Cl2,20 °C, 4 h; (iii) TIPSCl, imidazole, DMF, 20 °C, 2–3d;(iv)NaH, DMF, 0–20 °C, 3.4 h (for 12), or 0–20 °C, 2.5 h then 46 °C,3h(for 19); (v) 1% HCl in 95% EtOH, 20 or 44 °C, 1.5–3d;(vi)ArCH2Br or 4-BnOBnCl, NaH, DMF, 0–20 °C, 2.5–7 h; (vii) ArB(OH)2, toluene, EtOH, 2 M Na2CO3, Pd(dppf)Cl2 under N2, 86–90 °C, 20–75 min; (viii) Ac2O, pyridine, 20 °C, 38 h; (ix) preparative chiral HPLC (see text); (x) K2CO3, aq MeOH, 20 °C, 4 h.

aReagents and conn class="Chemical">ditions: (i) Br(CH2)2CH=CH2 or 15, n class="Chemical">K2CO3, lass="Chemical">pan class="Chemical">DMF, 60–73 °C, 4.5–11 h; (ii) OsO4, NMO, CH2Cl2,20 °C, 4 h; (iii) TIPSCl, imidazole, DMF, 20 °C, 2–3d;(iv)NaH, DMF, 0–20 °C, 3.4 h (for 12), or 0–20 °C, 2.5 h then 46 °C,3h(for 19); (v) 1% HCl in 95% EtOH, 20 or 44 °C, 1.5–3d;(vi)ArCH2Br or 4-BnOBnCl, NaH, DMF, 0–20 °C, 2.5–7 h; (vii) ArB(OH)2, toluene, EtOH, 2 M Na2CO3, Pd(dppf)Cl2 under N2, 86–90 °C, 20–75 min; (viii) Ac2O, pyridine, 20 °C, 38 h; (ix) preparative chiral HPLC (see text); (x) K2CO3, aq MeOH, 20 °C, 4 h.

pan cn class="Gene">lass="Chemical">Mitsunobu coupling of lass="Chemical">pan class="Chemical">alcohol 13 with appropriate phenols (Scheme 2A) successfully led to the 7-H phenyl ethers 39 and 45, together with the 4-iodo analogue 48; the latter enabled biphenyl derivatives 49 and 53, following Suzuki couplings. However, because Mitsunobu reactions were expected to be more problematic for the sterically hindered 7-methyl alcohol 20,[29] a different approach was employed to prepare compounds 44 and 47 (Scheme 2B). Commencing with 2-chloro-4-nitroimidazole (40), alkylation with iodide 15[26] and buffered reaction of alkene 41 with m-CPBA provided epoxide 42 in high yield (80%). Ring opening of 42 with phenols (K2CO3, MEK, 82 °C) then gave alcohol intermediates that could be ring closed to 7-substituted oxazines, as above. An attempt to combine the last two steps in one pot[30] (by exposing 42 to 1.2 equiv of NaH and 4-(trifluoromethoxy)phenol in DMF at 75–86 °C) led to markedly inferior results (27% 44, with 30% 43); equally, ring opening of 42 with 4-iodophenol in DMF (K2CO3,83 °C, 8 h) also gave a lower yield of 50 (60%) due to partial displacement of the 2-chlorine. Suzuki couplings on the ring-closed iodide 51 readily furnished biphenyl derivatives 52 and 54; terminal fluoropyridines 55 and 56 were similarly obtained from 48 and 51 through the use of a weaker base (KHCO3).

Scheme 2

aReagents and conditions: (i) ArOH, DEAD, PPh3, THF, 0–20 °C, 15–51 h; (ii) ArB(OH)2, DMF, (toluene, EtOH), 2 M Na2CO3 or 2 M KHCO3, Pd(dppf)Cl2 under N2, 70–91 °C, 1.5–5 h; (iii) 15, K2CO3, DMF, 73 °C, 14 h; (iv) m-CPBA, Na2HPO4, CH2Cl2, 0–20 °C, 18 h; (v) ArOH, K2CO3, MEK, 82–83 °C, 8–10 h; (vi) NaH, DMF, 0–20 °C, 2–2.5 h; (vii) 57 or 97, NaH, DMF, 0–20 °C, 2.3–3 h; (viii) bis(pinacolato)diboron, KOAc, DMF, Pd(dppf)Cl2 under N2,84–90 °C, 3.5 h, then 104,2MNa2CO3, Pd(dppf)Cl2 under N2,85–90 °C, 2.5–3.5 h.

aReagents and conn class="Chemical">ditions: (i) ArOH, DEAD, n class="Chemical">PPh3, lass="Chemical">pan class="Gene">THF, 0–20 °C, 15–51 h; (ii) ArB(OH)2, DMF, (toluene, EtOH), 2 M Na2CO3 or 2 M KHCO3, Pd(dppf)Cl2 under N2, 70–91 °C, 1.5–5 h; (iii) 15, K2CO3, DMF, 73 °C, 14 h; (iv) m-CPBA, Na2HPO4, CH2Cl2, 0–20 °C, 18 h; (v) ArOH, K2CO3, MEK, 82–83 °C, 8–10 h; (vi) NaH, DMF, 0–20 °C, 2–2.5 h; (vii) 57 or 97, NaH, DMF, 0–20 °C, 2.3–3 h; (viii) bis(pinacolato)diboron, KOAc, DMF, Pd(dppf)Cl2 under N2,84–90 °C, 3.5 h, then 104,2MNa2CO3, Pd(dppf)Cl2 under N2,85–90 °C, 2.5–3.5 h.

The assembn class="Gene">ly of various n class="Chemical">biaryl side chains featuring a proximal lass="Chemical">pan class="Chemical">2-pyridine ring was typically quite straightforward (Scheme 2C). Bromo-2-pyridinyl ethers (58, 60, 98, and 100) were easily formed[17] via sodium hydride-catalyzed SNAr reactions of alcohols 13 and 20 with the fluoropyridines 57 and 97, and Suzuki couplings then supplied the phenylpyridine or bipyridine targets in generally high yields (62–98%). Nevertheless, it proved very challenging to prepare analogues 105 and 106 having a 2-pyridine terminal ring. One-pot treatment of bromides 58 and 60 with bis(pinacolato)diboron (to generate the boronate ester), followed by in situ Suzuki coupling[31] with 2-bromo-5-fluoropyridine, gave 105 and 106 in poor yields (8–15%). However, a copper(I)-facilitated Suzuki approach,[32] designed to mitigate facile protodeboronation of the required 2-pyridyl boronate, was not any better (15% yield of 106).

For more efficient synthesis of n cn class="Gene">lass="Chemical">7-H lass="Chemical">pan class="Chemical">biaryl analogues having a proximal 3-pyridine ring, an epoxide-opening strategy (Scheme 3A) was preferred over the Mitsunobu route described above. Epoxide 67 was obtained in 72% optimized yield from 2-chloro-4-nitroimidazole (40), via alkene 66; in this case, the slow epoxidation step was best achieved under nonbuffered conditions at higher concentration (with initial cooling). Ring opening of 67 with 6-bromopyridin-3-ol (68) (K2CO3, MEK, 81–82 °C) gave mainly alcohol 69 (51% using 2 equiv for 35 h, or 57% from 4 equiv and 14 h), together with small amounts of the oxazine 70 (6–12%). Ring closure of purified 69 (NaH, DMF, 0–20 °C) then gave additional 70 in excellent yield (91%). Comparable results were obtained for scale-up of 39 from 67 (62%) as well as for reaction of epoxide 42 with pyridinol 68 and ring closure, leading to oxazine 89 (Scheme 3D). As expected, bromides 70 and 89 both proved to be excellent substrates for Suzuki couplings to access the remaining racemic phenylpyridine and bipyridine targets.

Scheme 3

aReagents and conditions: (i) Br(CH2)2CH=CH2 or 72 or 80, K2CO3, DMF, 70–74 °C, 19–72 h; (ii) m-CPBA, CH2Cl2,0–20 °C, 34 h; (iii) 68, K2CO3, MEK, 81–84 °C, 19–42 h; (iv) NaH, DMF, 0–20 °C, 2.5–3.5 h; (v) ArB(OH)2, DMF, (toluene, EtOH), 2 M Na2CO3 or 2 M KHCO3, Pd(dppf)Cl2 under N2,70–90 °C, 2–4h; (vi) 1 MHCl, MeOH, 0–20 °C, 6 h; (vii) TsCl, pyridine, –10 to 20 °C, 13–15 h; (viii) DBU, CH2Cl2, 0–20 °C, 8–9 h.

aReagents and conn class="Chemical">ditions: (i) Br(CH2)2CH=CH2 or 72 or 80, n class="Chemical">K2CO3, lass="Chemical">pan class="Chemical">DMF, 70–74 °C, 19–72 h; (ii) m-CPBA, CH2Cl2,0–20 °C, 34 h; (iii) 68, K2CO3, MEK, 81–84 °C, 19–42 h; (iv) NaH, DMF, 0–20 °C, 2.5–3.5 h; (v) ArB(OH)2, DMF, (toluene, EtOH), 2 M Na2CO3 or 2 M KHCO3, Pd(dppf)Cl2 under N2,70–90 °C, 2–4h; (vi) 1 MHCl, MeOH, 0–20 °C, 6 h; (vii) TsCl, pyridine, –10 to 20 °C, 13–15 h; (viii) DBU, CH2Cl2, 0–20 °C, 8–9 h.

By pan cn class="Gene">lass="Chemical">alkylating 2-chloro-4-nitroimidazole (40) with lass="Chemical">pan class="Chemical">(4R)-4-(2-iodoethyl)-2,2-dimethyl-1,3-dioxolane[33] (72) (or its optical isomer, 80[33]), it was possible to transform the above racemic route into a viable chiral synthesis for delivery of both enantiomers of two advanced leads, 71 and 93 (Scheme 3B,C). The two chiral acetal products 73 and 81 were readily converted into the R and S enantiomers of epoxide 67 (76 and 84) by successive hydrolysis (to diols 74 and 82), tosylation at the primary hydroxyl, and internal substitution to form the oxirane ring (DBU). These chiral epoxides were then elaborated to the final products by reaction with 68, ring closure, and Suzuki coupling, as previously described.

The prepan class="Chemical">pan class="Species">ration of lass="Chemical">pan class="Chemical">biaryl congeners 123, 126, 129, 131–133, and 139 in which the first ring was pyridazine, pyrazine, or pyrimidine followed similar procedures to those developed for the pyridine analogues. Thus, sodium hydride-induced SNAr reactions of alcohols 13 and 20 with haloheterocycles 121, 124, and 127 readily provided the bromoheteroaryl ether intermediates needed for final step Suzuki couplings (Scheme 4A). However, the remaining arylpyrimidine target (139) required prior assembly of the biaryl side chain. Initial protection of 2-chloropyrimidin-5-ol (134) as an ethoxymethyl ether derivative (135), followed by Suzuki coupling and acidic deprotection, supplied arylpyrimidinol 137 in excellent yield (83% from 134; Scheme 4B). Reaction of 137 with epoxide 67 produced a 5:2 mixture of the alcohol 138 and the ring closed oxazine (139); treatment of 138 with sodium hydride then completed this synthesis.

Scheme 4

aReagents and conditions: (i) 121 or 124 or 127, NaH, DMF, 0–20 °C, 2.5–3 h; (ii) ArB(OH)2, DMF, toluene, EtOH, 2 M Na2CO3 or 2 M KHCO3, Pd(dppf)Cl2 under N2,80–90 °C, 2.3–3.5 h; (iii) EtOCH2Cl, K2CO3, DMF, 20 °C, 16 h; (iv) 1.25 M HCl in MeOH, 53 °C, 4 h; (v) 137, K2CO3, MEK, 83 °C, 24 h; (vi) NaH, DMF, 0–20 °C, 3 h.

aReagents and conn class="Chemical">ditions: (i) 121 or 124 or 127, n class="Chemical">NaH, lass="Chemical">pan class="Chemical">DMF, 0–20 °C, 2.5–3 h; (ii) ArB(OH)2, DMF, toluene, EtOH, 2 M Na2CO3 or 2 M KHCO3, Pd(dppf)Cl2 under N2,80–90 °C, 2.3–3.5 h; (iii) EtOCH2Cl, K2CO3, DMF, 20 °C, 16 h; (iv) 1.25 M HCl in MeOH, 53 °C, 4 h; (v) 137, K2CO3, MEK, 83 °C, 24 h; (vi) NaH, DMF, 0–20 °C, 3 h.

Scheme 5 outlines the methods used to obtain compounds 142, 144, 147, 149, 152, 154, 157, 159–161, 170, and 178, whose side chains contained either pan class="Chemical">piperazine or lass="Chemical">pan class="Chemical">piperidine linked to an aryl group. Ring opening of epoxides 67 and 42 with the known or commercial amines 140, 145, 150,[34] and 155 easily generated the expected β-amino alcohols in high yield (Scheme 5A,B). These alcohols could be ring closed to the final products with sodium hydride upon mild heating, albeit yields for the 7-methyl analogues were generally significantly lower, in part due to greater purification difficulties. Chloroformylation of alcohols 13 and 20 (triphosgene/Et3N) and in situ reaction with arylpiperazine 140 also led to the O-carbamates 160 and 161 in only modest yield (33–35%; Scheme 5C) on account of similar purification issues; the isolation of alkyl chloride and diethyl carbamate derivatives under the same reaction conditions has been reported recently.[35] Lastly, synthesis of the two O-linked arylpiperidines, 170 and 178, was eventually achieved in each case via a lengthy seven-step route (Scheme 5D), after the failure of a more direct plan (ring opening of epoxide 67 with piperidinol 163[36] in the presence of erbium triflate[37]). Here, piperidinol 163[36] was first sourced in three steps by Buchwald amination of 1-bromo-4-fluorobenzene with 1,4-dioxa-8-azaspiro[4.5]decane,[38] ketal hydrolysis, and reduction (NaBH4). Reaction of epoxides 162[39] and 171[39] with 163 (NaH, DMF, 70 °C) and TBS protection of the liberated hydroxyls provided the desired ethers 165 and 173 in good yield (47–68% overall). Successive benzyl removal (via hydrogenolysis), iodination, and alkylation of 2-chloro-4-nitroimidazole (40) then gave the TBS-protected adducts, 168 and 176, which were readily desilylated (TBAF) and ring closed to furnish the final targets.

Scheme 5

aReagents and conditions: (i) amine (140, 145, 150, or 155), MEK (or DME), 70–85 °C, 16–84 h; (ii) NaH, DMF, 40–60 °C or 0–20 °C (for 170)or20–39 °C(for 178), 1.5–5 h; (iii) triphosgene, Et3N, THF, 20 °C, 30 min, then 140, THF, 20 °C, 2 h; (iv) 163, NaH, DMF, 70 °C, 14–28 h; (v) TBSOTf, 2,6-lutidine, CH2Cl2, 0–20 °C, 1–2.5 d; (vi) H2 (60 psi), 10% Pd–C, EtOH, EtOAc, 20 °C, 45–51 h; (vii) I2, PPh3, imidazole, CH2Cl2, 20 °C, 17–19 h; (viii) 2-chloro-4-nitroimidazole, K2CO3, DMF, 63–75 °C, 70–72 h; (ix) TBAF, THF, 20 °C, 5–25 h.

aReagents and conditions: (i) amine (140, 145, 150, or 155), pan class="Gene">MEK (or DME), 70–85 °C, 16–84 h; (ii) lass="Chemical">pan class="Chemical">NaH, DMF, 40–60 °C or 0–20 °C (for 170)or20–39 °C(for 178), 1.5–5 h; (iii) triphosgene, Et3N, THF, 20 °C, 30 min, then 140, THF, 20 °C, 2 h; (iv) 163, NaH, DMF, 70 °C, 14–28 h; (v) TBSOTf, 2,6-lutidine, CH2Cl2, 0–20 °C, 1–2.5 d; (vi) H2 (60 psi), 10% Pd–C, EtOH, EtOAc, 20 °C, 45–51 h; (vii) I2, PPh3, imidazole, CH2Cl2, 20 °C, 17–19 h; (viii) 2-chloro-4-nitroimidazole, K2CO3, DMF, 63–75 °C, 70–72 h; (ix) TBAF, THF, 20 °C, 5–25 h.

■ RESULTS AND DISCUSSION

The structures and in vitro antiparasitic and antitubercular potencies of 75 novel pan class="Chemical">7-substituted 2-nitroimidazooxazine derivatives prelass="Chemical">pared in two collabolass="Chemical">pan class="Species">rative projects are provided in Tables 1 and 2. While compounds 14, 21–23, 25, 27–29, 34, 38, 39, 44, 45, 47, 49, and 52–54 were initially designed and evaluated for TB, for clarity purposes, we will focus the discussion first on the more recent VL work with DNDi. Here, new synthesis was directed at the optimization of solubility, efficacy, and safety, primarily through the incorporation of heterocycles to reduce compound lipophilicity[11] (estimated by CLogP data derived from ACD LogP/LogD software, version 12.0; Advanced Chemistry Development Inc., Toronto, Canada). Kinetic aqueous solubility measurements were conducted on dry powder forms of particular examples that were being considered for further evaluation. Target compounds were initially screened only once against Leishmania donovani (L. don) using a mouse macrophage-based luciferase assay conducted at the Central Drug Research Institute (CDRI, India).[10] Nevertheless, to gain a clearer understanding of the SAR (in view of some unexpected in vivo outcomes), the entire set was finally re-evaluated at the University of Antwerp (LMPH) in replicate assays against three protozoan parasites: Leishmania infantum (L. inf), Trypanosoma cruzi, and Trypanosoma brucei.[40] Assessments of cytotoxicity were concurrently conducted on both human lung fibroblasts (MRC-5 cells; the host for T. cruzi) and primary peritoneal mouse macrophages (the host for L. inf), which revealed that the compounds were generally nontoxic (MRC-5 IC50s > 55 μM except for 117: IC50 35 μM), as confirmed for TB (VERO assay[41] IC50s > 128 μM for 71 of 72 compounds).

Table 1

In Vitro Antiparasitic and Antitubercular Activities and Calculated Lipophilicities of 7-Substituted 2-Nitroimidazooxazines

compd	form	X	aza	R	CLogP	IC50a,b (μM)				MICc,b (μM)
						L. don	L. inf	T. cruzi	MRC-5	MABA	LORA
4d					3.47	0.03	0.17	2.6	>64	0.046	5.9
14	A	H		4-OCF3	3.30	0.03	0.12	1.2	>64	1.0	7.5
21	A	Me		4-OCF3	3.68	0.31	0.30	0.75	>64	0.55	3.3
22	A	H		4-OBn	3.55	0.05				0.46	3.0
23	A	Me		4-OBn	3.93	0.28				0.20	1.4
25	B	H		4-F	4.21	0.02	0.17	0.53	>64	0.08	1.3
27	B	Me		4-F	4.59	0.22	1.8	0.84	>64	0.085	0.61
28	B	H		4-OCF3	5.14	0.19	0.40	2.1	>64	0.055	1.5
29	B	Me		4-OCF3	5.52	1.1	1.1	0.54	>64	0.093	1.4
34	Be	Me		4-OCF3	5.52	0.24	1.3	0.57	>64	0.063	1.1
38	Bf	Me		4-OCF3	5.52	1.3	1.3	0.77	>64	0.74	6.8
39	C	H		4-OCF3	3.37	0.04	0.047	0.061	>64	5.2	4.7
44	C	Me		4-OCF3	3.75	0.10	0.13	0.14	>64	0.94	6.8
45	C	H		4-OBn	3.62	0.02				>128	>128
47	C	Me		4-OBn	4.00	0.12				0.44	>128
49	D	H		4-F	4.11	0.01	0.47	0.063	>64	0.18	>128
52	D	Me		4-F	4.49	0.20	0.34	0.35	>64	0.085	1.4
53	D	H		4-OCF3	5.03	0.06	0.28	0.24	>64	0.08	0.73
54	D	Me		4-OCF3	5.41	0.52	0.36	0.72	>64	0.11	1.3
Phenylpyridines
55	D	H	3	4-F	2.94	>10	0.083	0.027	>64	0.24	3.5
56	D	Me	3	4-F	3.32	0.12	0.16	0.30	>64	0.29	2.7
59	D	H	2′	4-F	3.66	0.01	0.050	0.027	>64	0.025	<0.25
61	D	Me	2′	4-F	4.04	0.09	0.097	0.23	>64	0.17	1.0
62	D	H	2′	2,4-diF	3.65	0.07	0.037	0.030	>64	0.10	2.4
63	D	Me	2′	2,4-diF	4.03		0.11	0.21	>64	0.089	2.5
64	D	H	2′	4-OCF3	4.58	0.05	0.35	0.12	>64	0.027	0.47
65	D	Me	2′	4-OCF3	4.96	0.26	3.8	1.0	>64	0.13	5.3
71	D	H	3′	4-F	3.03	0.06	0.093	0.27	>64	0.23	2.4
79	De	H	3′	4-F	3.03	(0.03)g	0.080	0.35	>64	0.11	3.2
87	Df	H	3′	4-F	3.03	(0.08)g	0.22	0.29	>64	1.1	3.9
90	D	Me	3′	4-F	3.41	0.65	0.59	0.26	>64	0.35	3.9
91	D	H	3′	2,4-diF	3.03	0.02	0.030	0.13	>64	0.36	8.9
92	D	Me	3′	2,4-diF	3.41	0.31	0.17	0.27	>64	0.40	4.9
93	D	H	3′	4-OCF3	3.96	0.05	0.12	0.17	>64	0.032	0.86
94	De	H	3′	4-OCF3	3.96		0.11	0.26	>64	0.024	1.5
95	Df	H	3′	4-OCF3	3.96		0.13	0.13	>64	0.34	1.6
96	D	Me	3′	4-OCF3	4.34	0.65	4.0	0.25	>64	0.05	0.88
99	E	H	2′	4-F	3.55	0.31	0.14	0.16	59	1.7	3.0
101	E	Me	2′	4-F	3.93		0.18	0.35	>64	0.94	5.0
102	E	H	2′	2,4-diF	3.55	0.25	4.0	0.23	>64	>128	>128
103	E	Me	2′	2,4-diF	3.93		0.24	0.22	>64	0.69	4.8
Bipyridines
105	D	H	2′,2	4-F	2.55	0.06	50	0.34	>64	0.15	11
106	D	Me	2′,2	4-F	2.93	0.15	0.34	0.25	>64	0.40	9.5
107	D	H	2′,3	4-F	2.49	0.27	47	0.54	>64	0.074	15
108	D	Me	2′,3	4-F	2.87	0.22	0.40	0.82	>64	1.9	6.2
109	D	H	2′,3	2,4-diF	2.60	>10	0.19	0.29	>64	0.25	4.2
Bipyridines
110	D	Me	2′,3	2,4-diF	2.98		0.20	0.43	>64	1.0	6.6
111	D	H	2′,3	4-CF3	2.89	0.13	2.5	0.68	>64	0.09	2.5
112	D	H	3′,3	4-F	1.87	0.09	>64	0.55	>64	2.3	21
113	D	Me	3′,3	4-F	2.25	0.09	0.67	0.57	>64	2.7	43
114	D	H	3′,3	2,4-diF	1.98	0.08	52	0.35	>64	1.2	41
115	D	Me	3′,3	2,4-diF	2.36	0.11	0.28	0.38	>64	0.94	18
116	D	H	3′,3	4-CF3	2.61	0.07	7.3	0.46	>64	0.12	58
117	E	H	2′,3	4-F	2.38	>10	44	0.13	35	>128	>128
118	E	Me	2′,3	4-F	2.76		1.1	0.52	>64	11	7.8
119	E	H	2′,3	2,4-diF	2.50	0.25	6.7	0.13	>64	4.3	10
120	E	Me	2′,3	2,4-diF	2.88		0.59	0.49	>64	9.3	7.9
Other Heterobiaryls
123	D	H	2′,3′	4-F	2.73	0.07	8.4	0.67	>64	0.35	23
126	D	H	2′,5′	4-F	3.09	0.15	0.71	4.7	>64	0.15	>128
129	D	H	2′,6′	4-F	2.58	0.10	0.29	0.17	>64	0.04	1.7
131	D	Me	2′,6′	4-F	2.96	0.32	0.29	0.43	57	0.44	6.3
132	D	H	2′,6′,3	4-F	1.41	0.53	>64	3.3	>64	0.24	21
133	D	Me	2′,6′,3	4-F	1.79	1.3	6.1	2.9	>64	23	54
139	D	H	3′,5′	4-F	2.79	0.07	0.21	0.26	62	0.27	20

ICso values for inhibition of the growth of Leishmania donovani and Leishmania infantum (in mouse macrophages) and Trypanosoma cruzi (on MRC-5 cells), 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) please refer to the Supporting Information.

Minimum inhibitory concentration against M. tb, determined under aerobic (MABA)[41] or hypoxic (LORA)[56] conditions.

Data from ref 11.

(R)-Enantiomer.

(S)-Enantiomer.

LMPH data (mean of 2 values).

Table 2

In Vitro Antiparasitic and Antitubercular Activities and Calculated Lipophilicities of Cyclic Amine-Based Analogues

compd	form	X	CLogP	IC50a,b (μM)				MICc,b (μM)
				L. don	L. inf	T. cruzi	MRC-5	MABA	LORA
142	A	H	2.16	0.19	2.3	1.7	>64	1.8	9.9
144	A	Me	2.54	0.70	0.73	1.3	>64	0.34	6.8
147	B	H	3.36	0.88	0.87	2.9	>64	2.1	11
149	B	Me	3.74	1.0	0.32	0.87	>64	0.22	8.3
152	C	H	2.84	0.45	0.84	2.4	>64	2.0	22
154	C	Me	3.22	0.22	0.32	0.59	>64	0.23	48
157	D	H	1.17	4.8	45	11	>64	46	>128
159	D	Me	1.55	2.8	6.5	2.8	>64	93	>128
160	E	H	2.42	>100	>64	1.5	>64	2.5	22
161	E	Me	2.80	0.29	0.72	3.9	>64	3.4	8.4
170	F	H	3.50		0.24	0.22	>64	0.78	20
178	F	Me	3.88		0.35	0.51	>64	0.37	19

IC50 values for inhibition of the growth of Leishmania donovani and Leishmania infantum (in mouse macrophages) and Trypanosoma cruzi (on MRC-5 cells), 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) please refer to the Supporting Information.

Minimum inhibitory concentration against M. tb, determined under aerobic (MABA)[41] or hypoxic (LORA)[56] conditions.

In Vitro Antiparn class="Chemical">asitic and Antitubercular Activities and Calculated Lipophilicities of pan class="Chemical">7-Substituted 2-Nitroimidazooxazines

ICso values for inhibition of the growth of n class="Chemical">pan class="Species">Leishmania donovani and lass="Chemical">pan class="Species">Leishmania infantum (in mouse macrophages) and Trypanosoma cruzi (on MRC-5 cells), or for cytotoxicity toward human lung fibroblasts (MRC-5 cells).

Each value (except the singn class="Gene">le test pan class="Species">L. don data) is the mean of at least two independent determinations. For complete results (mean ± SD) please refer to the Supporting Information.

Minimum inhibitory concentn cn class="Gene">lass="Species">ration against lass="Chemical">pan class="Species">M. tb, determined under aerobic (MABA)[41] or hypoxic (LORA)[56] conditions.

LMPH n class="Chemical">data (mean of 2 values).

In Vitro Antiparn class="Chemical">asitic and Antitubercular Activities and Calculated Lipophilicities of pan class="Chemical">Cyclic Amine-Based Analogues

IC50 values for inhibition of the growth of n class="Chemical">pan class="Species">Leishmania donovani and lass="Chemical">pan class="Species">Leishmania infantum (in mouse macrophages) and Trypanosoma cruzi (on MRC-5 cells), or for cytotoxicity toward human lung fibroblasts (MRC-5 cells).

Each value (except the singn class="Gene">le test pan class="Species">L. don data) is the mean of at least two independent determinations. For complete results (mean ± SD) please refer to the Supporting Information.

Minimum inhibitory concentn cn class="Gene">lass="Species">ration against lass="Chemical">pan class="Species">M. tb, determined under aerobic (MABA)[41] or hypoxic (LORA)[56] conditions.

Early Hit to Lead Assessments for pan class="Gene">VL. Through an agreement between lass="Chemical">pan class="Chemical">TB Alliance and DNDi, 58 nitroimidazole derivatives were screened against L. don at the Swiss Tropical Institute. All five 7-substituted oxazines (including 22, 23, and 28) demonstrated excellent potencies in the in vitro mouse macrophage assay (IC50s 0.065–0.17 μM, similar to racemic 4), prompting the inclusion of 28 alongside rac-4 (and another oxazole[11]) in a proof-of-concept in vivo assessment at the London School of Hygiene and Tropical Medicine (LSHTM). However, the level of activity observed for 28 in this L. don mouse model (49% inhibition at 50 mg/kg, dosing po daily for 5 d; Table 3) was not notable in comparison to the results for rac-4 (99% at 6.25 mg/kg),[11] suggesting that further optimization of the side chain would be necessary. Indeed, while 28 showed good stability on exposure to mouse liver microsomes (MLM: 75% remaining after 1 h, Table 3) and gave a mouse pharmacokinetic (PK) profile comparable to rac-4 (Table 4 and Supporting Information, Figures S1 and S2), it was very hydrophobic (CLogP 5.14) and displayed poor solubility (∼58 ng/mL at pH 7, Table 3; 62-fold lower than for rac-4[11]). Such compounds typically exhibit high levels of plasma protein binding (PPB), which can limit efficacy.[42] We have also observed that increased linker flexibility can be detrimental to in vivo activity.[17,43]

Table 3

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

compd	aq solubilitya (μg/mL)		microsomal stabilityb [% remaining at 1 (0.5) h]			in vivo efficacy against L. don (mouse) (% inhibition at dose in mg/kg)c
	pH 7	pH 1	H	M	Ham	50	25	12.5	6.25	3.13	1.56
4d	2.4		(92)	(89)	18 (54)			>99	>99	83	49
14	4.8		57	10
28	0.058		73	75	46	49
29	0.39		85	77
34			86	79
38			86	59
39	4.0		85 (96)	57 (70)	(23)		87
44	2.3		58 (86)	50 (61)	2.1 (16)		100	100	83	25
49	0.055		(88)	(75)	(45)		>99
53	0.13		(97)	(94)	(94)		65
54	2.6		(89)	(85)	(81)		30
55	0.36
59	0.13	2.8	41	43 (60)	12 (16)	67
61	0.69	13		(33)	(2.6)
62	0.13	1.5	25	19	2.7
71	0.32	164	44	34 (63)	14 (52)	100	98	76	59
79	0.45	237	58	69	34				93
87	0.47	234	63	41	5				85
90	0.19	74	50	28	32	41
91	0.72	221	45	31	8.6	91
93	0.15	92	53	41	37	>99			>99	97	50
94	0.37	110	50	53						>99	84
95	0.40	141	52	46						52	57
99	0.027	0.56
107	0.87			(81)	(51)
108	4.5			(68)	(39)
111	0.36			(72)	(70)
112	3.9		(93)	(87)	(70)	44
113	2.3			(82)	(43)
116	0.30			(82)	(81)
129	1.8		61	56	13	85
139	0.59
142	10	14900	85	77	15	55
152	49	21100	57	58	0
170	6.1	34300	33	17	0.7	45
178	2.2	9970	11	1.2	0.2

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

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

Data from ref 11

Table 4

Pharmacokinetic Parameters for Selected Compounds in Various Species

compd	intravenous (1–2 mg/kg)a						oral (5–40 mg/kg)a
	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 (μ·h/mL)	Fc(%)
					Mice
rac-4d	0.88	9.5	1.7	2.2	1.69	4.1	4.0		33.5	79
28						3.3	8.0	5.2	47.1e
34						2.0	6.0	8.1	31.9e
39	0.36	48	3.2	1.1	0.341	1.3	0.5		3.86	45
44	0.79	12	2.5	2.8	1.31	1.4	3.0		11.5	35
49	2.9	0.52	0.40	6.7	31.6	4.2	6.0		84.7	11
53	0.66	1.3	1.9	17	11.2	14	8.0		376	100
54	0.43	2.3	5.0	27	5.26	0.79	10		22.7	17
71						13	4.0	3.4	112
112	14	0.70	0.12	2.1	24.7	14	2.0		95.3	31
					Rats
71	1.3	3.9	0.94	3.0	4.36	0.49	3.3	6.7	4.27	22
79	1.5	6.3	0.86	1.7	2.65	0.79	3.3	3.1	4.08	34
87	1.1	5.5	1.4	3.2	3.14	0.71	3.3	3.7	5.64	41
					Hamsters
71	2.5	11	1.0	1.2	3.12	0.94	2.7	4.7	4.83	26
79						1.4	2.0	4.2	6.82
87						0.73	2.7	10	3.68

The intravenous dose was 1 mg/kg for mice and rats and 2 mg/kg for hamsters. The oral dose in mice was 25 mg/kg, except for 28 and 34 (40 mg/kg) and 112 (12.5 mg/kg); in other species, the oral doses were 5 mg/kg (rats) or 12.5 mg/kg (hamsters).

Area under the curve calculated to the last time point (10, 24, or 48 h).

Oral bioavailability, determined using dose normalized AUClast values.

Data for racemic 4 from ref 11.

Extrapolated AUCinf value.

Aqueous Solubin class="Gene">lity, Microsomal Stability, and in Vivo Antipan class="Disease">leishmanial Efficacy Data for Selected Analogues

Kinetic solubin class="Gene">lity (μg/mL) in pan class="Chemical">water (pH 7) or 0.1 M lass="Chemical">pan class="Chemical">HCl (pH 1) at 20 °C, determined by HPLC (see the Experimental Section, Method A).

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

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

Pharmacokinetic Parameters for Sen class="Gene">lected Compounds in Various Species

The intravenous dose wn class="Chemical">as 1 mg/kg for n class="Species">mice and lass="Chemical">pan class="Species">rats and 2 mg/kg for hamsters. The oral dose in mice was 25 mg/kg, except for 28 and 34 (40 mg/kg) and 112 (12.5 mg/kg); in other species, the oral doses were 5 mg/kg (rats) or 12.5 mg/kg (hamsters).

Area under the curve can class="Gene">lculated to the last time point (10, 24, or 48 h).

Oral bioavain class="Gene">lability, determined using dose normalized AUClast values.

Extrapolaten class="Chemical">d AUCinf value.

While these n class="Chemical">pan class="Species">mouse studies were being conducted, a further 30 lass="Chemical">pan class="Chemical">7-substituted oxazine derivatives were screened against L. don in the luciferase assay at CDRI.[10] On the basis of the single IC50 data obtained for 14, 21–23, 25, 27–29, 34, 38, 39, 44, 45, 47, 49, and 52–54 (Table 1), several preliminary SAR conclusions were drawn: (1) the 7-H series was generally 5–10-fold more potent than the 7-methyl series; (2) 4-trifluoromethoxy and 4-benzyloxy substituents (forms A and C) provided equivalent potency; (3) for biaryl analogues (forms B and D), 4-fluoro was preferred over 4-trifluoromethoxy as the final ring substituent (as observed[11] in the 6-nitroimidazooxazole series); (4) a shorter linker (forms C and D) was preferred in the majority of cases. Thus, the most active analogues appeared to be 14, 22, 25, 39, 45, 49, and 53 (IC50s 0.01–0.06 μM, similar to 4). However, benzyl ether 14 did not display suitable metabolic stability (10% parent left after 1 h with MLM; Table 3), while evidence from the 6-substituted oxazine series[17] for the more rapid metabolism of benzyloxybenzyl analogues dissuaded further testing of 22 and 45. Moreover, following the disappointing results with 28, we were not optimistic of good in vivo efficacy with close analogue 25 despite its improved potency. Therefore, we elected to initially investigate 39, 49, and 53 as potential leads, together with two counterparts from the 7-methyl series, 44 and 54, to enable a head-to-head comparison.

The selecten class="Chemical">d compounds were advanced to parallel pan class="Species">mouse PK profiling and efficacy studies in the lass="Chemical">pan class="Species">mouse VL model. Encouragingly, both phenyl ether 44 (the direct analogue of rac-4) and biphenyl congener 49 showed excellent efficacy at 25 mg/kg (99.9–100% inhibition; Table 3 and Figure 3b). Surprisingly, the more potent 7-H counterpart of 44 (39) was slightly less active in this assay (87% inhibition), mimicking findings for the 2-H equivalent of rac-4.[11] Moreover, the biphenyl derivatives of 39 and 44 (53 and 54) were also less impressive than 49 (65% and 30% inhibition, respectively). However, while these latter results appeared to track well with the single determination L. don data, they did not seem to line up with the almost equivalent mean potencies vs L. inf (Table 1). The findings also appeared to conflict with the kinetic solubility and microsomal stability data (Table 3), where 49 was as poorly soluble as 28 (55 vs 58 ng/mL), but the more stable analogue 54 (85 vs 75% in MLM) was 45-fold more soluble than 28 (2.6 μg/mL). Solubility is discussed further in the next section.

Figure 3

Comparative in vivo efficacy against L. don in the mouse model: (a) 50 mg/kg, (b) 25 mg/kg, and (c) 6.25 mg/kg.

Compan class="Chemical">pan class="Species">rative in vivo efficacy against lass="Chemical">pan class="Species">L. don in the mouse model: (a) 50 mg/kg, (b) 25 mg/kg, and (c) 6.25 mg/kg.

Analysis of the n class="Chemical">pan class="Species">mouse PK data (Table 4) provided greater insight, revealing that 39 had a 4-fold higher lass="Chemical">pan class="Species">rate of clearance than its 7-methyl derivative 44 (48 vs 12 mL/min/kg), resulting in a short half-life (1.1 h vs 2.8 h for 44) and quite poor oral exposure (see the Supporting Information, Figure S1). Interestingly, with iv administration, the PK profiles for 44 and rac-4 were fairly similar, but 44 did not perform as well under oral dosing, with rather modest absorption (Cmax 1.4 μg/mL, 3-fold less than for rac-4) contributing to reduced exposure and moderate oral bioavailability (35% vs 79%). The oral parameters for compound 54 were also mediocre (poor Cmax of 0.79 μg/mL and low oral bioavailability of 17% offsetting its lengthy 27 h half-life), potentially explaining its inferior efficacy in the mouse VL model. However, the findings for 49 and 53 were more puzzling, with the less efficacious 53 demonstrating greater oral exposure (see the Supporting Information, Figure S1), superior oral bioavailability (100% vs 11% for 49), and an extended half-life (17 h vs 6.7 h for 49). Nevertheless, like 28, 53 was particularly hydrophobic (CLogP 5.03), so high PPB may be a major issue limiting its efficacy.[42] We have previously observed that PK data is not always correctly predictive of in vivo efficacy ranking.[43]

These promising results prompten class="Chemical">d further appraisal of the most active compounds, 44 and 49. In the pan class="Species">mouse lass="Chemical">pan class="Gene">VL model, 44 provided robust dose–response data (Table 3), giving an ED50 value of 4.2 mg/kg (cf. 3.0 mg/kg for rac-4[11]). Unfortunately, additional studies of 49 in this model (using material prepared elsewhere) were unable to replicate the original result; we postulate that this discrepancy may be due in part to the extremely poor aqueous solubility and inadequate oral bioavailability of this compound, rendering oral suspension formulations particularly sensitive to particle size. Nevertheless, the optimal in vivo assay for assessing the efficacy of test compounds against VL is the chronic infection hamster model, which better reproduces the clinical pathology of human disease.[44] In the L. don hamster model at CDRI, leads 44 and 49 were almost equally effective at 50 mg/kg, with 5 days of oral dosing leading to 53% and 51% inhibition of parasite infection in the spleen, whereas rac-4 gave 86% inhibition under the same dose regimen.[11]

A significant factor in the suboptimal activity of 44 in the n class="Chemical">pan class="Species">hamster model was thought to be its exceptionally rapid metabolism in this species, as revealed by the lass="Chemical">pan class="Species">hamster microsomal stability data (only 16% remaining after 0.5 h vs 49% for rac-4[11]). Therefore, 44 was later reassessed in the L. inf early curative hamster model at LMPH, comparing a twice-daily dose regimen (25 mg/kg b.i.d.) with a once daily dose of 50 mg/kg. The results (Table 5) slightly favored the twice daily regimen for all three target organs; hence, this protocol became standard for most test compounds. However, unlike 4, 44 was not curative at this dose level. Another liability with 44 was its greater inhibition of the hERG channel (IC50 3.8 vs 10.5 μM for 4), with IC50 values in excess of 10 μM required to minimize QT prolongation risk.[45] Hence, as lead compounds for VL, 44 and 49 fulfilled many suggested criteria[46] but still had key deficiencies, reflecting their origin as screening hits in a scarcely studied new class.

Table 5

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

compd	dose regimena (mg/kg)	% inhibition in target organsb liver spleen bone marrow
		1	40, qd	92.6	99.5	89.0
4c	25, qd	100	99.9	99.7
	12.5, qd	99.0	98.7	94.0
44	25, b.i.d.	92.2	91.1	82.5
	50, qd	88.6	89.5	73.0
71	25, b.i.d.	99.9	99.4	99.6
	12.5, b.i.d.	99.9	99.5	99.4
	6.25, b.i.d.	98.0	95.7	96.3
	3.13, b.i.d.	68.3	69.8	64.5
	12.5, qd	95.2	87.5	92.8
79	12.5, b.i.d.	99.5	99.4	96.8
	6.25, b.i.d.	91.0	91.6	73.3
87	12.5, b.i.d.	88.2	80.8	82.3
	6.25, b.i.d.	53.1	46.7	35.0

All compounds were dosed orally, once or twice daily for five days consecutively.

Data are the mean percentage reduction of parasite burden in target organs.

Data from ref 11.

In Vivo Efficacy Data for Sen class="Gene">lected Lead Compounds in the pan class="Species">L. inf lass="Chemical">pan class="Species">Hamster Model

Aln class="Gene">l compounds were dosed orally, once or twice daily for five days consecutively.

Data are the mean percentage ren class="Chemical">duction of parasite burden in target organs.

n cn class="Gene">lass="Species">SAR of lass="Chemical">pan class="Chemical">7-Substituted 2-Nitroimidazooxazines for VL. Following the identification of 4 as a preferred drug candidate and the discovery of 44 and 49 as unoptimized new leads, a backup program was launched to develop second-generation agents for VL having better solubility, PK–PD, and safety profile.[11] Because of the inferior profile of 44 in comparison to 4 in several key areas, we elected to center our synthetic strategy mainly on bicyclic side chains, employing heterocycles to modulate lipophilicity and solubility. Six-membered ring nitrogen-containing variants were preferred due to their greater metabolic stability;[47] ortho-substitution of aryl groups and meta-linkage of rings were also investigated as additional options to increase solubility.[48] Recognizing that few orally active registered drugs have solubility values below 1 μM at pH 7.4 (the pH of blood),[49] we aspired to achieve at least 10-fold higher than this for the best compounds.[46] We also aimed to exploit the low pH of gastric fluid (∼1–2) to improve dissolution and oral absorption of analogues containing pyridine and other bases.[50] Hence, we set a minimum solubility requirement for the preferred final candidate of being noninferior to delamanid (5) (0.31 μg/mL at pH 7 and 116 μg/mL at pH 1),[11] an approved TB drug in Europe and Japan.[12]

On the basis of the wider in vitro screening results, it was apparent that the pan class="Chemical">7-substituted oxazines could not be used for African lass="Chemical">pan class="Disease">trypanosomiasis (T. brucei IC50s mostly >64 μM, none <1 μM; see Supporting Information, Tables S1 and S2). However, unlike the 6-nitroimidazooxazoles, this new oxazine class generally showed interesting potencies against T. cruzi (IC50s 0.03–1 μM), suggesting the possibility of dual utility to treat both VL and Chagas disease. Further analysis of data for the 65 racemic compounds tested indicated a modest trend for the best VL leads to have high potencies against T. cruzi (see Supporting Information, Figure S4). Hence, for simplicity, we will focus this part of the SAR discussion entirely on the intended primary application (VL), emphasizing the key L. inf results.

To begin with, a reanalysis of the initian class="Gene">l data set (up to and including 54; Table 1) confirmed weak trends on pan class="Species">L. inf for the lass="Chemical">pan class="Chemical">7-H analogues to be more potent and a shorter linker length to be preferred (e.g., 39: IC50 0.047 μM), but there was no consistent preference for 4-fluoro as the terminal ring substituent. Nevertheless, in view of the better in vivo efficacy of 49 and similarly substituted nitroimidazooxazoles,[11] we retained this latter design element in the majority of cases. Thus, compounds 55 and 56 first investigated the effect of replacing the second phenyl ring of 49 by pyridine (ΔCLogP–1.2 units). Pleasingly, this led to a 2–6-fold potency increase, with 55 (IC50 0.083 μM) also being 6.5-fold more soluble than 49 (0.36 vs 0.055 μg/mL, Table 3). Exchange of the first phenyl ring by 2-pyridine (59 and 61; ΔCLogP –0.5 units) resulted in even better activity (59:IC50 0.050 μM), and in this case solubility values were ∼20-fold higher at low pH (2.8–13 μg/mL; calcd pKa 2.83) although still rather modest. Therefore, we examined the addition of an ortho fluorine in the phenyl ring (62 and 63) in an attempt to break up the planarity.[48] However, while this change was well tolerated, there was no improvement in solubility and microsomal stability was reduced (19% vs 43% in MLM for 62 vs 59, Table 3). In an alternative approach, we tried meta-linkage of the rings (99 and 101–103), but although the activity was generally acceptable, this led to inferior solubility (99: 27 ng/mL).

Turning instead to n class="Chemical">pan class="Chemical">3-pyridine as a first ring, potency was maximized by lass="Chemical">pan class="Chemical">2,4-difluorophenyl substitution (91: IC50 0.030 μM) although the 4-fluoro and 4-trifluoromethoxy (7-H) analogues (71 and 93) were also useful (IC50s 0.093 and 0.12 μM, respectively). Importantly, aqueous solubility values were up to 13-fold better than for 49 at neutral pH (91: 0.72 μg/mL) and 3 orders of magnitude better at low pH (91: 221 μg/mL). This was consistent with a greater lipophilicity reduction for the 3-pyridine (ΔCLogP –1.1 units) and a slightly higher basicity (e.g., 71: calcd pKa 3.76). Final assessment of the enantiomers of two examples, 71 and 93, identified the R forms (79 and 94) as slightly preferred for both potency and microsomal stability (particularly in the case of 79).

In view of the promising results with n class="Chemical">pan class="Chemical">phenylpyridines, we elected to investigate the more hydrophilic lass="Chemical">pan class="Chemical">bipyridines (105–120). Most of these showed interesting potencies in the initial L. don screen and further assessments had identified 108, 112, and 113 as being of potential interest based on their improved solubilities in comparison to 49 (2.3–4.5 vs 0.055 μg/mL). However, on retesting, almost all of the 7-H compounds displayed markedly inferior utility against L. inf (IC50s 2.5 to >64 μM) while the 7-methyl bipyridines retained moderate potencies (IC50s0.20–1.1 μM). It is intriguing to speculate that this might indicate a “minimum lipophilicity” requirement for activity (e.g., CLogP ∼ 2.5) because a similar pattern was noted for all of the more hydrophilic analogues (see analysis of racemic 7-H data set, Figure 4). Another strategy for heterobiaryl analogues of 49 was to exchange the first phenyl ring with pyridazine, pyrazine, or pyrimidine (123, 126, 129, 131–133, and 139). Of these, pyrimidine (129, 131, and 139) provided the best activity (IC50s 0.21–0.29 μM), although combining this with a pyridine ring (132, 133) led to a dramatic loss of potency (21- to >220-fold). Overall, pyrimidine 129 had the best aqueous solubility (1.8 μg/mL; 33-fold better than 49) along with acceptable metabolic stability.

Figure 4

Effect of lipophilicity on potency against L. inf for 35 racemic 7-H analogues.

Effect of lipophin class="Gene">licity on potency against pan class="Species">L. inf for 35 racemic lass="Chemical">pan class="Chemical">7-H analogues.

More structurally diverse targets (142, 144, 147, 149, 152, 154, 157, 159–161, 170, and 178; Table 2) were designed on the premise that arylated pan class="Chemical">cyclic amines can be effective bioisosteres for biphenyls, thus facilitating substantial boosts in solubility.[51,52] Several side chains of this type have previously shown promise for lass="Chemical">pan class="Chemical">TB and/or VL,[11,18] including in the recent development of antileishmanial aminopyrazole ureas.[53] It was initially encouraging to see four examples (arylpiperazine 142, aryloxypiperidines 152 and 154, and arylpiperazine carbamate 161) exhibiting reasonable potencies in the L. don screen (IC50s 0.19–0.45 μM), with 142 and 152 displaying a markedly better solubility profile than 49 (10–49 μg/mL at pH 7, 15–21 mg/mL at low pH). However, the L. inf data did not fully match the L. don results; instead, the 7-methyl analogues were clearly favored over the 7-H compounds (by 3–7-fold), and the more lipophilic piperidines 149 and 154 were superior (IC50s 0.32 μM). The hydrophilic benzoylpiperazines 157 and 159 were particularly poor in both assays, as was the 7-H arylpiperazine carbamate 160. In view of these SAR findings, two O-linked phenylpiperidines (170 and 178) were subsequently designed as structurally closer mimetics for the O-linked biphenyl 49. Gratifyingly, 170 demonstrated both good potency (IC50 0.24 μM) and much better solubility than 49 (6.1 μg/mL at pH 7, 34 mg/mL at pH 1), albeit the microsomal stability of this compound (17–33% in MLM and HLM) was regarded as quite marginal.

Integn cn class="Gene">lass="Species">ration of the initial lass="Chemical">pan class="Species">L. don data with the kinetic solubility and microsomal stability results led to the selection of nine new racemic analogues of 49 for testing in the L. don mouse model (dosing at 50 mg/kg for 5 d; Table 3 and Figure 3a). Encouragingly, a first experiment on 3-pyridine derivative 71 (4-FPh) yielded a 100% parasite clearance from the liver in all mice. Following this, 4-trifluoromethoxy congener 93 was found to be equally efficacious (99.5%), whereas the 2,4-difluoro example 91 was slightly less effective (91% inhibition). However, the less potent 7-methyl derivative of 71 (90) and the more potent 2-pyridine analogue 59 were only moderately active (41% and 67%, respectively); it is possible that the higher crystallinity (larger particle size) of 59 may have contributed to poor oral bioavailability.[42,48] Two more soluble heterobiaryl analogues, bipyridine 112 and phenylpyrimidine 129, also displayed lower efficacy (44% and 85% inhibition); oral PK data on 112 (Table 4 and Supporting Information, Figure S2) were comparable to those of 71, so this may be a potency issue (as suggested by the disparate L. inf and L. don IC50s of >64 vs 0.09 μM). Finally, the inferior in vivo outcomes for two potential bioisosteres of 49, phenylpiperazine 142 (55%) and O-linked phenylpiperidine 170 (45%), may be attributed to either weaker in vitro activity on retesting (for 142: L. inf IC50 2.3 μM) or more rapid metabolism (for 170), as indicated above. No adverse effects were noted in any of the in vivo experiments and the percentage weight changes for the mice were well within normal thresholds (see the Supporting Information, Table S4).

Dose–response appraisan class="Gene">l of 71 in this n class="Species">mouse model provided an ED50 value of 5.1 mg/kg (cf. 4.2 mg/kg for 44), whereas the trifluoromethoxy analogue 93 was unexpectedly ∼3-fold better (50% at 1.56 mg/kg; Table 3). Therefore, the enantiomers of both 71 and 93 were assessed and in each case the R form (79 and 94) gave higher efficacy, with 94 (84% at 1.56 mg/kg) outperforming the preclinical candidate 4 (49%). Meanwhile, 71 was further evaluated in the lass="Chemical">pan class="Species">L. inf hamster model at LMPH. A dose regimen of 25 or 12.5 mg/kg b.i.d. for 5 days enabled parasite burden reductions exceeding 99% for all three target organs (Table 5 and Figure 5), similar to 4 at 25 mg/kg once daily (qd). However, 71 was slightly less effective than 4 when given via the 12.5 mg/kg qd schedule. A final head-to-head comparison of the enantiomers of 71 confirmed 79 as the preferred stereoisomer based on its superior efficacy at two dose levels. This result was also supported by favorable PK data, e.g., a higher exposure than 87 in hamsters, with an acceptable half-life (3.1 h) and good oral bioavailability (34%) in the rat (Table 4 and Supporting Information, Figure S3). Although 94 was not tested in the hamster model, it is thought that 79 may still offer some advantages as a lead candidate, e.g., lower lipophilicity (by ∼1 log unit) and reduced molecular weight (this could lessen PPB and improve safety),[42] slightly better solubility (a calculated pKa value of 3.76 vs 3.42 for 94), and a physical form more suitable for oral administration.

Figure 5

Comparative in vivo efficacy against L. inf in the hamster model (LMPH).

Compan class="Chemical">pan class="Species">rative in vivo efficacy against lass="Chemical">pan class="Species">L. inf in the hamster model (LMPH).

In line with our initial objective to develop improved drug candidates as backups to 4, it was pertinent to examine some additional properties of 79 (Table 6). Compared to 4, 79 had a very similar molecular weight (370 vs 359 Da) and provided thermodynamic solubility values that were clearly superior to 4 as the pH approached the measured pKa value of 3.95. It also had a lower experimental Log D value (2.45 vs 3.10), close to that of pan class="Chemical">pretomanid (6).[18] Furthermore, like 4,[54] 79 displayed high permeability (without being a substlass="Chemical">pan class="Species">rate for P-gp mediated efflux), although it did show a slightly greater binding to human plasma proteins (96.5 vs 93.9%). In terms of safety, 79 gave a low inhibition of hERG (IC50 > 30 μM), did not inhibit CYP3A4 (IC50 > 100 μM), and was not mutagenic (Ames test). These characteristics broadly match the suggested criteria for clinical development of a new entity for VL,[55] so following a belated concern with 4, 79 has now been selected as a new preclinical candidate.

Table 6

Additional Comparative Data for Lead Compounds 4 and 79

property	4	79
molecular weight (Da)	359.3	370.3
Log D (measured)	3.10	2.45a
pKa (measured)		3.95a
thermodynamic solubility (μM):
pH 7.4	2.8	5.4b
pH 6.5/5.0		3.1/18
permeability:
Papp (× 10–6 cm/s) A to B/B to A	22.6/24.7c	29.2/26.2d
human plasma protein binding (%)	93.9	96.5
mutagenic effect (Ames test)	no	noa,e
hERG IC50 (μM)	10.5	>30
CYP3A4 IC50 (μM)	>25	>100

For racemate (71).

Preclinical batch.

Caco-2 data from ref 54.

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

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

Adn class="Chemical">ditional Compapan class="Species">rative Data for Lead Compounds 4 and 79

Preclinican class="Gene">l batch.

pan cn class="Gene">lass="Gene">MDCK-MDR1 data; no lass="Chemical">pan class="Gene">P-gp mediated efflux.

Not mutagenic in strains TA98 and TA100, either in the presence or absence of metabon class="Gene">lic activation (S9 fraction).

n cn class="Gene">lass="Species">SAR of lass="Chemical">pan class="Chemical">7-Substituted 2-Nitroimidazooxazines for TB. Although the primary goal of our work with DNDi was a new drug for VL, the series was originally designed and exemplified for TB, seeking a novel second-generation backup to 6 (now in phase II/III clinical trials[13]). Hence, the antitubercular activities of the 7-substituted oxazine derivatives have remained an aspect of significant ongoing interest. The work began with the preparation of an exploratory set of four compounds (14, 22, 39, and 45; Table 1). Growth inhibitory effects against Mycobacterium tuberculosis (M. tb, strain H37Rv) were studied under both aerobic (replicating) and hypoxic (nonreplicating) conditions (MABA[41] and LORA[56] assays, respectively), in recognition of the varying modes of action of 6 under each state[57] and the suggestion that optimizing for hypoxic activity may lead to agents with better sterilizing ability against persistent bacteria;[56] recorded MIC data (for at least 90% inhibition) represent the mean of 2–5 independent measurements. Compared with racemic 6 (MICs of 1.1 and 4.4 μM in MABA and LORA, respectively),[14] compounds 14 and 22 showed potencies of similar magnitude, stimulating further interest and the synthesis of more than 30 new analogues, including 21, 23, 25, 27–29, 44, 47, 49, and 52–54 (Table 1). These featured two design elements that had proven most advantageous for enhancing in vivo efficacy in early studies of 4 and 6, namely biaryl extension, and methylation adjacent to the ring oxygen.[11,30,58]

From this larger data set, it was observed that 7-methyl congeners (e.g., 21, 23, 44, and 47) were generally slightly more effective than pan class="Chemical">7-H counterlass="Chemical">parts and that biphenyl side chains (e.g., 25, 27–29, 49, and 52–54) provided roughly an order of magnitude further improvement in MABA MIC values (whereas LORA data were less responsive to these changes). The lass="Chemical">pan class="Chemical">phenylbenzyl derivative 29 was earmarked as a potential early lead based on its better MIC profile (0.093 and 1.4 μM in MABA and LORA) and good stability toward MLM and HLM (77–85% parent remaining after a 1 h exposure, Table 3). Thus, for preliminary proof of principle, the enantiomers of 29 (34 and 38) were prepared and assessed in the acute TB infection mouse model alongside 6, dosing orally at 100 mg/kg daily (5 days/week) for three weeks. In this experiment, the R enantiomer 34 displayed equivalent efficacy to 6, but the S form 38 was 5-fold less active (Figure 6), in accordance with its weaker potency and MLM stability data. Nevertheless, the very high lipophilicity of 34 (CLogP 5.52) and its inferior PK profile in comparison to the shorter linked analogue 53 (Table 4) imply that far better in vivo effects might be achievable with optimized compounds (as shown for VL; cf. 94 vs 28).

Figure 6

Comparison of 6, 34, and 38 in the acute TB infection mouse model.

Comparison of 6, 34, ann class="Chemical">d 38 in the pan class="Disease">acute TB infection lass="Chemical">pan class="Species">mouse model.

The early preference for 7-methyn class="Gene">l substitution was not a consistent pattern across pan class="Chemical">heterobiaryl derivatives, where the most potent examples, notably lass="Chemical">pan class="Chemical">phenylpyridines 59, 64, 93 and 94, as well as phenylpyrimidine 129 (MABA MICs 0.02–0.04 μM), were 7-H compounds. As found for the 6-substituted series,[16] bipyridine and other heterobiaryl analogues were generally less impressive (except the 4-CF3 congener 111), particularly when the rings were meta-linked (corresponding phenylpyridines 99 and 101–103 also displayed markedly reduced activity). Finally, arylated cyclic amine bioisosteres (Table 2) showed moderate to weak potencies overall, with the hydrophilic benzoylpiperazines 157 and 159 being especially poor. For side chains A–C, 7-methyl compounds exhibited an order of magnitude better aerobic activity than their 7-H counterparts, although LORA results were disappointing for these and the related O-linked phenylpiperidines (170 and 178). Nevertheless, it was recognized that most of these new 7-substituted oxazines possessed a 4-fluoro substituted terminal ring, whereas more lipophilic 4-trifluoromethoxyphenyl (or 4-CF3pyridine) termini were favored for TB[16-18,43,58] (as seen for phenylpyridines 93–95 vs 71, 79, and 87). Therefore, a few additional examples of the latter type (189–194 and 198; Supporting Information, Table S3) were also made and evaluated. Here, 191 and 198 (4-OCF3) were 10- to 16-fold more effective than 123 and 139 (4-F) in both TB assays (192–194 were also 2–8-fold better than 126, 129, and 131 in MABA), confirming this same SAR pattern. Overall, taking into account potency[46] (needing to be superior to 5[11]), solubility, and metabolism effects, it is considered that phenylpyridine 94 is the most promising lead for TB.

■ CONCLUSIONS

Through a scaffoln class="Chemical">d hopping design stn class="Species">rategy, lass="Chemical">pan class="Chemical">7-substituted 2-nitroimidazooxazines were identified as a third, highly active nitroimidazole-based class of antitubercular agents, having a remarkable similarity in properties to 2-substituted 6-nitroimidazooxazoles. Phenotypic screening of some unoptimized early examples against kinetoplastid diseases led to the detection of two compounds (44 and 49) having significant efficacy against VL in mouse and hamster models, although these proved to be inferior to preclinical lead 4 as potential drug candidates. On the basis of our experiences in the original two classes (with 4 and 6), we then sought to develop more suitable second-generation agents for VL by systematically exploring heterocyclic side chain variants of biphenyl lead 49. Replacement of one or both phenyl rings by pyridine (or pyrimidine etc.) enabled large modulations in lipophilicity (ΔCLogP –0.5 to –2.7 units), with concomitant improvements in aqueous solubility (2–71-fold at pH 7 and ∼4000-fold at pH 1 for phenyl-3-pyridines). In a complementary bioisostere approach, the incorporation of piperazine or piperidine for the first ring produced even greater solubility enhancements (e.g., 170: 34 mg/mL at low pH). However, more subtle strategies (viz. ortho-substitution of aryl groups and meta-linkage of aryl rings) proved less beneficial overall.

Interestingly, potency against n class="Chemical">pan class="Species">L. inf appeared to show some dependence on lipophilicity, with the most effective lass="Chemical">pan class="Chemical">7-H compounds falling in a CLogP range of 2.9–4.0, and compounds of CLogP < 2.5 having weak or negligible activity. This was aptly demonstrated by the improved potency of fluorinated phenylpyridines (5–16-fold over 49), in which the pyridine could be either terminal or proximal to the linker, whereas the combination of two pyridine rings was strongly deactivating except in the presence of a 7-methyl substituent. Phenylpyrimidine and phenylpiperidine were the only other side chains to provide substantial activity in this assay. It has recently been shown[59] that a novel nitroreductase (NTR2) in Leishmania is responsible for the activation of nitroimidazooxazoles such as 4; therefore, differential nitroreductase binding may be a major factor behind the in vitro SARs for both VL and TB.[57] Evaluation of a representative set of nine racemic compounds in the VL mouse model pinpointed phenylpyridines 71 and 93 as the most efficacious, with 93 being as impressive as 4 (50% inhibition at 1.56 mg/kg). In the chronic infection L. inf hamster model, 71 (at 12.5 mg/kg b.i.d.) achieved >99% reductions in parasite burden for all three target organs. Subsequent synthesis and assessment of the enantiomers of both leads identified the R forms (79 and 94) as superior, and in the case of 79 this outcome was reinforced by excellent results in the L. inf hamster model and favorable PK data in the hamster and rat. Importantly, 79 (DNDI-0690) also provided a better safety profile than 4 and has now been selected as a new preclinical candidate for VL.

Finaln class="Gene">ly, as found for the n class="Chemical">nitroimidazooxazole series,[11] it was intriguing to note that some of the best lass="Chemical">pan class="Gene">VL leads (e.g., 59, 79, 93, 94, and 129) showed highly potent in vitro effects against TB, with both R enantiomers and 4-trifluoromethoxy analogues most preferred, pointing to 94 (MABA MIC 0.024 μM) as the favored TB candidate for further evaluation. The S form of 93 (95) also displayed interesting activity against T. cruzi (IC50 0.13 μM), indicating a possible application for treating Chagas disease. This investigation has therefore revealed that the 7-substituted 2-nitroimidazooxazine class has exciting potential to treat up to three neglected diseases and can deliver drug candidates that are worthy of examination in ongoing studies.

■ EXPERIMENTAL SECTION

Combustion analyses were performen class="Chemical">d by the Campbell Microanalytical Labopan class="Species">ratory, University of Otago, Dunedin, New Zealand. Melting points were determined using an Electrothermal IA9100 melting point aplass="Chemical">palass="Chemical">pan class="Species">ratus 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 ppm and hertz, respectively. High-resolution fast atom bombardment (HRFABMS) mass spectra were determined on a VG-70SE mass spectrometer at nominal 5000 resolution. High-resolution electrospray ionization (HRESIMS) mass spectra were determined 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). Thin-layer chromatography was carried out 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 ≥95% pure, as determined by combustion analysis (results within 0.4% of theoretical values) and/or by HPLC conducted on an Agilent 1100 system, using a 150 mm × 3.2 mm Altima 5 μm reversed phase C18 column with diode array detection. Preparative reversed phase HPLC was performed using a Gilson Unipoint system (322-H pump, 156 UV/vis detector) with 250 mm × 21 mm Synergi Max-RP 4 μm C12 or Zorbax 7 μm SB-C18 columns. Finally, preparative chiral HPLC was carried out on similar equipment by employing a 250 mm × 20 mm CHIRALPAK IA 5 μm semipreparative column, while chiral purity was assessed using 250 mm × 4.6 mm CHIRALPAK IA or CHIRALPAK AS-H 5 μm analytical columns.

Compounds of . The fon class="Gene">llowing section details the syntheses of compounds 14, 25, 34, 44, 55, 59, and 79 of Table 1, via representative procedures and key intermediates, as described in Schemes 1–3. For the syntheses of all of the other compounds in Table 1, please refer to the Supporting Information.

Synthesis of . A mixture of 2-bromo-4-n cn class="Gene">lass="Chemical">nitro-lass="Chemical">pan class="Chemical">1H-imidazole (8) (2.50 g, 13.0 mmol), 4-bromobut-1-ene (2.00 mL, 19.7 mmol), and powdered K2CO3 (5.39 g, 39.0 mmol) in anhydrous DMF (25 mL) under N2 was stirred at 73 °C for 4.5 h. The resulting cooled mixture was added to ice/aqueous NaHCO3 (200 mL) and extracted with EtOAc (4 × 200 mL). The extracts were washed with water (200 mL) and then evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–10% EtOAc/petroleum ether first gave foreruns, and then further elution with 20% EtOAc/petroleum ether gave 9 (2.96 g, 92%) as a pale-yellow oil that solidified on cooling: mp 28–30 °C. 1H NMR (CDCl3) δ 7.77 (s, 1 H), 5.75 (ddt, J = 17.1, 10.2, 6.9 Hz, 1 H), 5.18 (dq, J = 10.2, 1.1 Hz, 1 H), 5.12 (dq, J = 17.1, 1.4 Hz, 1 H), 4.09 (t, J = 7.0 Hz, 2 H), 2.58 (qt, J = 6.9, 1.2 Hz, 2 H). HRFABMS calcd for C7H9BrN3O2 m/z [M + H]+ 247.9858, 245.9878, found 247.9860, 245.9882.

Procedure B: n class="Chemical">pan class="Chemical">4-(2-Bromo-4-nitro-1H-imidazol-1-yl)butane-1,2-diol (. lass="Chemical">pan class="Chemical">Osmium tetroxide (3.20 mL of a 4% aqueous solution, 0.524 mmol) was added to a solution of alkene 9 (2.56 g, 10.4 mmol) and 4-methylmorpholine 4-oxide (1.83 g, 15.6 mmol) in CH2Cl2 (65 mL). The mixture was stirred at 20 °C for 4 h, and the resulting precipitate was collected by filtration, washing with CH2Cl2 and water, to give 10 (2.29 g, 79%) as a cream solid: mp (THF/Et2O/pentane) 99–101 °C. 1H NMR [(CD3)2SO] δ 8.55 (s, 1 H), 4.77 (br d, J = 5.0 Hz, 1 H), 4.58 (br t, J = 5.6 Hz, 1 H), 4.20–4.07 (m, 2 H), 3.47–3.37 (m, 1 H), 3.34 (dt, J = 10.7, 5.4 Hz, 1 H), 3.24 (dt, J = 10.7, 5.9 Hz, 1 H), 2.03–1.92 (m, 1 H), 1.76–1.63 (m, 1 H). Anal. (C7H10BrN3O4) C, H, N.

The remaining filtn class="Chemical">pan class="Species">rate above was added to an ice-cold aqueous solution of lass="Chemical">pan class="Chemical">sodium sulphite (100 mL), and the aqueous portion was saturated with salt and extracted with EtOAc (10 × 100 mL). The combined organic portions 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 foreruns, and then further elution with EtOAc gave additional 10 (572 mg, 20%).

Procedure C: n class="Chemical">pan class="Chemical">4-(2-Bromo-4-nitro-1H-imidazol-1-yl)-1-[(triisopropylsilyl)oxy]butan-2-ol (. lass="Chemical">pan class="Chemical">Chlorotriisopropylsilane (2.35 mL, 11.0 mmol) was slowly added to a solution of diol 10 (2.86 g, 10.2 mmol) and imidazole (1.54 g, 22.6 mmol) in anhydrous DMF (25 mL) under N2, and then the mixture was stirred at 20 °C for 2 d. The resulting mixture was added to ice–water (150 mL) and extracted with 50% EtOAc/petroleum ether (4 × 100 mL). The extracts were washed with water (100 mL) and then concentrated under reduced pressure (at 30 °C), and the remaining oil was chromatographed on silica gel. Elution with 0–20% EtOAc/petroleum ether first gave foreruns, and then further elution with 33% EtOAc/petroleum ether gave 11 (4.19 g, 94%) as a white solid: mp (CH2Cl2/pentane) 90–91 °C. 1H NMR (CDCl3) δ 7.89 (s, 1 H), 4.24 (dd, J = 7.7, 6.2 Hz, 2 H), 3.74 (dd, J = 9.6, 3.5 Hz, 1 H), 3.67–3.58 (m, 1 H), 3.53 (dd, J = 9.6, 6.8 Hz, 1 H), 2.59 (d, J = 3.8 Hz, 1 H), 1.95–1.82 (m, 2 H), 1.18–1.02 (m, 21 H). Anal. (C16H30BrN3O4Si) C, H, N.

Procedure n class="Chemical">D: pan class="Chemical">2-Nitro-7-{[(triisopropylsilyl)oxy]methyl}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (. A solution of lass="Chemical">pan class="Chemical">alcohol 11 (1.89 g, 4.33 mmol) in anhydrous DMF (35 mL) under N2 at 0 °C was treated with 60% NaH (262 mg, 6.55 mmol) and then quickly degassed and resealed under N2. The mixture was stirred at 0 °C for 25 min and at 20 °C for 3 h, then cooled (CO2/acetone), quenched with ice/aqueous NaHCO3 (10 mL), added to brine (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 0–20% EtOAc/petroleum ether first gave foreruns, and then further elution with 0–4% EtOAc/CH2Cl2 gave 12 (1.48 g, 96%) as a pale-yellow solid: mp (CH2Cl2/pentane) 121–123 °C. 1H NMR (CDCl3) δ 7.42 (s, 1 H), 4.49–4.40 (m, 1 H), 4.17 (ddd, J = 12.3, 5.8, 3.7 Hz, 1 H), 4.06 (ddd, J = 12.3, 10.3, 5.4 Hz, 1 H), 4.03 (dd, J = 10.7, 4.1 Hz, 1 H), 3.95 (dd, J = 10.7, 5.8 Hz, 1 H), 2.42–2.33 (m, 1 H), 2.33–2.20 (m, 1 H), 1.18–1.03 (m, 21 H). Anal. (C16H29N3O4Si) C, H, N.

Procedure E: n class="Chemical">pan class="Chemical">(2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-7-yl)methanol (. lass="Chemical">pan class="Chemical">Silyl ether 12 (1.48 g, 4.16 mmol) was treated with a solution of 1% HCl in 95% EtOH[27] (63 mL, 15.1 mmol). The mixture was stirred at 20 °C for 36 h and then cooled (CO2/acetone) and neutralized with a solution of NH3 in MeOH (8.0 mL of 2 M). The resulting mixture was evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–2% MeOH/CH2Cl2 first gave foreruns, and then further elution with 2–4% MeOH/CH2Cl2 gave 13 (804 mg, 97%) as a light-yellow solid: mp (THF/MeOH/CH2Cl2/hexane) 179–181 °C. 1H NMR [(CD3)2SO] δ 8.04 (s, 1 H), 5.12 (t, J = 5.8 Hz, 1 H), 4.53–4.43 (m, 1 H), 4.13 (ddd, J = 12.5, 5.8, 3.0 Hz, 1 H), 4.04 (ddd, J = 12.4, 11.0, 5.1 Hz, 1 H), 3.70–3.59 (m, 2 H), 2.23–2.13 (m, 1 H), 2.10–1.96 (m, 1 H). Anal. (C7H9N3O4) C, H, N.

Procedure F: n class="Chemical">pan class="Chemical">2-Nitro-7-({[4-(trifluoromethoxy)benzyl]oxy}-methyl)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (. A solution of lass="Chemical">pan class="Chemical">alcohol 13 (40.2 mg, 0.202 mmol) in anhydrous DMF (2 mL) under N2 at 0 °C was treated with 60% NaH (13.7 mg, 0.343 mmol) and then quickly degassed and resealed under N2. 4-(Trifluoromethoxy)benzyl bromide (60 μL, 0.375 mmol) was added, and the mixture was stirred at 20 °C for 165 min, then cooled (CO2/acetone), quenched with ice/aqueous NaHCO3 (10 mL), 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 0–0.5% MeOH/CH2Cl2 first gave foreruns, and then further elution with 0.5% MeOH/CH2Cl2 gave 14 (52 mg, 69%) as a cream solid: mp (CH2Cl2/hexane) 158–160 °C. 1H NMR [(CD3)2SO] δ 8.06 (s, 1 H), 7.47 (br d, J = 8.7 Hz, 2 H), 7.35 (br d, J = 7.9 Hz, 2 H), 4.77–4.69 (m, 1 H), 4.60 (s, 2 H), 4.13 (ddd, J = 12.5, 5.8, 3.0 Hz, 1 H), 4.05 (ddd, J = 12.5, 10.8, 5.2 Hz, 1 H), 3.76 (dd, J = 11.1, 3.9 Hz, 1 H), 3.73 (dd, J = 11.1, 5.1 Hz, 1 H), 2.27–2.17 (m, 1 H), 2.17–2.03 (m, 1 H). 13C NMR [(CD3)2SO] δ 147.9, 147.6 (q, J = 1.4 Hz), 142.0, 137.6, 129.2 (2 C), 120.9 (2 C), 120.1 (q, J = 256.0 Hz), 117.7, 76.7, 71.4, 70.9, 41.8, 22.6. Anal. (C15H14F3N3O5) C, H, N.

Synthesis of . A mixture of n cn class="Gene">lass="Chemical">alcohol 13 (130 mg, 0.653 mmol) and lass="Chemical">pan class="Chemical">4-iodobenzyl bromide (262 mg, 0.882 mmol) in anhydrous DMF (5 mL) under N2 at 0 °C was treated with 60% NaH (40 mg, 1.00 mmol) and then quickly degassed and resealed under N2. The mixture was stirred at 20 °C for 2.5 h, then cooled (CO2/acetone), quenched with ice/aqueous NaHCO3 (10 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 CH2Cl2 first gave foreruns, and then further elution with 1–1.5% EtOAc/CH2Cl2 gave 24 (165 mg, 61%) as a cream solid: mp (CH2Cl2/hexane) 169–171 °C. 1H NMR (CDCl3) δ 7.68 (br d, J = 8.3 Hz, 2 H), 7.41 (s, 1 H), 7.05 (br d, J = 8.2 Hz, 2 H), 4.59–4.52 (m, 3 H), 4.14 (ddd, J = 12.3, 5.7, 3.8 Hz, 1 H), 4.05 (ddd, J = 12.3, 10.0, 5.6 Hz, 1 H), 3.80 (dd, J = 10.6, 4.3 Hz, 1 H), 3.75 (dd, J = 10.6, 5.0 Hz, 1 H), 2.37–2.20 (m, 2 H). HRFABMS calcd for C14H15IN3O4 m/z [M + H]+ 416.0107, found 416.0105.

Procedure H: 7-{[(4′-Fn class="Gene">luoro[1,1′-biphenyl]-4-yl)methoxy]methyl}-2-n class="Chemical">nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]lass="Chemical">pan class="Chemical">oxazine (. A stirred mixture of iodide 24 (35 mg, 0.084 mmol), 4-fluorophenylboronic acid (15.8 mg, 0.113 mmol), and Pd(dppf)Cl2 (2.1 mg, 0.003 mmol) in toluene (1.8 mL) and EtOH (0.7 mL) was degassed for 5 min (vacuum pump), and then N2 was added. An aqueous solution of Na2CO3 (0.35 mL of 2 M, 0.70 mmol) was added by syringe, and the mixture was stirred at 90 °C for 20 min, and then cooled, diluted with aqueous NaHCO3 (50 mL), and extracted with CH2Cl2 (4 × 50 mL). The extracts were evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–1% EtOAc/CH2Cl2 first gave foreruns, and then further elution with 1–1.5% EtOAc/CH2Cl2 gave 25 (30.5 mg, 94%) as a cream solid: mp (CH2Cl2/pentane) 147–149 °C. 1H NMR [(CD3)2SO] δ 8.07 (s, 1 H), 7.71 (br dd, J = 8.9, 5.4 Hz, 2 H), 7.64 (br d, J =8.2 Hz, 2 H), 7.43 (br d, J = 8.3 Hz, 2 H), 7.29 (br t, J = 8.9 Hz, 2 H), 4.77–4.69 (m, 1 H), 4.61 (s, 2 H), 4.13 (ddd, J = 12.5, 5.8, 3.0 Hz, 1 H), 4.05 (ddd, J = 12.4, 10.9, 5.2 Hz, 1 H), 3.77 (dd, J = 11.0, 3.9 Hz, 1 H), 3.74 (dd, J = 11.1, 5.1 Hz, 1 H), 2.28–2.17 (m, 1 H), 2.17–2.03 (m, 1 H). 13C NMR [(CD3)2SO] δ 161.9 (d, J = 244.4 Hz), 148.0, 142.0, 138.4, 137.2, 136.3 (d, J = 3.0 Hz), 128.6 (d, J = 8.1 Hz, 2 C), 128.1 (2 C), 126.6 (2 C), 117.7, 115.7 (d, J = 21.2 Hz, 2 C), 76.8, 72.0, 70.8, 41.8, 22.6. Anal. (C20H18FN3O4) C, H, N.

Synthesis of . n cn class="Gene">lass="Chemical">Acetic anhydride (3.60 mL, 38.1 mmol) was added to a suspension of lass="Chemical">pan class="Chemical">alcohol 20 (see Supporting Information) (807 mg, 3.79 mmol) in anhydrous pyridine (7.0 mL). The mixture was stirred at 20 °C for 38 h and then added to ice–water (150 mL) and extracted with CH2Cl2 (5 × 100 mL). The extracts were evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with CH2Cl2 first gave foreruns, and then further elution with 1–6% EtOAc/CH2Cl2 gave 30 (962 mg, 100%) as a cream solid: mp (CH2Cl2/pentane) 145–147 °C. 1H NMR (CDCl3) δ 7.44 (s, 1 H), 4.27 (d, J = 11.9 Hz, 1 H), 4.20 (d, J = 11.9 Hz, 1 H), 4.14 (dt, J = 12.7, 5.9 Hz, 1 H), 4.08 (ddd, J = 12.7, 8.3, 5.6 Hz, 1 H), 2.32 (ddd, J = 14.5, 8.3, 6.1 Hz, 1 H), 2.10 (dt, J = 14.5, 5.7 Hz, 1 H), 2.09 (s, 3 H), 1.50 (s, 3 H). HRFABMS calcd for C10H14N3O5 m/z [M + H]+ 256.0934, found 256.0941.

pan cn class="Gene">lass="Chemical">[(7R)-7-Methyl-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-oxazin-7-yl]methyl acetate (. Racemic lass="Chemical">pan class="Chemical">acetate 30 (990 mg) was separated into pure enantiomers by preparative chiral HPLC, using a CHIRALPAK IA column and an isocratic solvent system of 40% EtOH in hexane at a flow rate of 6 mL/min, to first give 35 (427 mg, 43%) as a cream solid, having identical 1H NMR data to 30 that was used directly in the next step; [α][26]D –6.0 (c 1.00, CHCl3).

Further elution of the HPLC column gave 31 (428 mg, 43%) as a cream solid that was used directly in the next step. pan class="Chemical">1H NMR (CDCl3) δ 7.44 (s, 1 H), 4.27 (d, J = 11.9 Hz, 1 H), 4.20 (d, J = 11.8 Hz, 1 H), 4.14 (dt, J = 12.7, 5.9 Hz, 1 H), 4.08 (ddd, J = 12.7, 8.3, 5.6 Hz, 1 H), 2.32 (ddd, J = 14.5, 8.3, 6.1 Hz, 1 H), 2.10 (dt, J = 14.5, 5.7 Hz, 1 H), 2.09 (s, 3 H), 1.50 (s, 3 H). [α][26]D 6.0 (c 1.00, lass="Chemical">pan class="Chemical">CHCl3). Chiral HPLC (using a CHIRALPAK IA analytical column and eluting with 40% EtOH in hexane at 0.5 mL/min) determined that the ee of each enantiomer was 100%.

Procedure I: n class="Chemical">pan class="Chemical">[(7R)-7-Methyl-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-7-yl]methanol (. A stirred solution of lass="Chemical">pan class="Chemical">ester 31 (427 mg, 1.67 mmol) in MeOH (36 mL) was treated with K2CO3 (256 mg, 1.85 mmol), and then water (4 mL) was added dropwise. The mixture was stirred at 20 °C for 4 h and then cooled in ice and neutralized with 0.1 M HCl (37 mL). The resulting mixture was evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–1% MeOH/CH2Cl2 first gave foreruns, and then further elution with 1–2.5% MeOH/CH2Cl2 gave 32 (343 mg, 96%) as a light-yellow solid that was used directly in the next step. 1H NMR [(CD3)2SO] δ 8.03 (s, 1 H), 5.23 (br t, J =5.4Hz, 1 H), 4.13 (dt, J = 13.0, 6.0 Hz, 1 H), 4.05 (ddd, J = 12.9, 8.1, 5.6 Hz, 1 H), 3.54 (dd, J = 11.6, 4.9 Hz, 1 H), 3.48 (dd, J = 11.6, 5.2 Hz, 1 H), 2.21 (ddd, J = 14.4, 8.1, 5.9 Hz, 1 H), 2.00 (dt, J = 14.4, 5.8 Hz, 1 H), 1.32 (s, 3 H). [α][27]D –18.0 (c 1.00, DMF).

pan cn class="Gene">lass="Chemical">(7R)-7-{[(4-Bromobenzyl)oxy]methyl}-7-methyl-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (. Reaction of lass="Chemical">pan class="Chemical">alcohol 32 with 4-bromobenzyl bromide (1.3 equiv) and NaH, using procedure G for 3 h, followed by chromatography of the product on silica gel, eluting with CH2Cl2 (foreruns) and then with 1% EtOAc/CH2Cl2, gave 33 (57%) as a white solid: mp (CH2Cl2/hexane) 157–159 °C. 1H NMR (CDCl3) δ 7.46 (br d, J = 8.3 Hz, 2 H), 7.39 (s, 1 H), 7.12 (br d, J = 8.3 Hz, 2 H), 4.50 (s, 2 H), 4.09 (ddd, J = 12.5, 6.9, 6.0 Hz, 1 H), 4.01 (ddd, J = 12.5, 7.0, 6.0 Hz, 1 H), 3.62 (d, J = 10.2 Hz, 1 H), 3.58 (d, J = 10.2 Hz, 1 H), 2.37 (ddd, J = 14.4, 7.0, 6.0 Hz, 1 H), 2.10 (ddd, J = 14.4, 6.9, 6.1 Hz, 1 H), 1.46 (s, 3 H). [α][27]D 31.0 (c 1.00, CHCl3). HRFABMS calcd for C15H17BrN3O4 m/z [M + H]+ 384.0382, 382.0402, found 384.0385, 382.0398.

(7R)-7-Methyl-2-n class="Chemical">pan class="Chemical">nitro-7-({[4′-(trifluoromethoxy)[1,1′-biphenyl]-4-yl]methoxy}methyl)-6,7-dihydro-5H-imidazo[2,1-b][1,3]lass="Chemical">pan class="Chemical">oxazine(. Reaction of bromide 33 with 4-(trifluoromethoxy)phenylboronic acid (1.5 equiv) and Pd(dppf)Cl2 (0.15 equiv), using procedure H at 88 °C for 75 min, followed by chromatography of the product on silica gel, eluting with 0–0.5% EtOAc/CH2Cl2 (foreruns) and then with 0.5–1.5% EtOAc/CH2Cl2, gave 34 (90%) as a cream solid: mp (CH2Cl2/hexane) 165–167 °C. 1H NMR (CDCl3) δ 7.58 (br d, J = 8.7 Hz, 2 H), 7.52 (br d, J = 8.2 Hz, 2 H), 7.38 (s, 1 H), 7.32 (br d, J = 8.1 Hz, 2 H), 7.28 (br d, J = 8.1 Hz, 2 H), 4.61 (d, J = 12.1 Hz, 1 H), 4.58 (d, J = 12.1 Hz, 1 H), 4.11 (ddd, J = 12.4, 7.2, 5.8 Hz, 1 H), 4.01 (ddd, J = 12.6, 6.5, 6.1 Hz, 1 H), 3.67 (d, J = 10.2 Hz, 1 H), 3.63 (d, J = 10.2 Hz, 1 H), 2.40 (ddd, J = 14.4, 6.6, 6.1 Hz, 1 H), 2.13 (ddd, J = 14.5, 7.3, 6.0 Hz, 1 H), 1.48 (s, 3 H). [α][27]D 37.0 (c 1.00, CHCl3). Anal. (C22H20F3N3O5) C, H, N.

Synthesis of . Reaction of 2-chloro-4-n class="Chemical">pan class="Chemical">nitro-lass="Chemical">pan class="Chemical">1H-imidazole (40) with 4-iodo-2-methylbut-1-ene[26] (15) (1.1 equiv) and powdered K2CO3 (2.0 equiv), using procedure A for 14 h, gave 41 (84%) as a white solid: mp (CH2Cl2/petroleum ether) 70–72 °C. 1H NMR (CDCl3) δ 7.72 (s, 1 H), 4.93–4.87 (m, 1 H), 4.72–4.66 (m, 1 H), 4.13 (t, J = 7.1 Hz, 2 H), 2.52 (br t, J = 7.0 Hz, 2 H), 1.80 (br s, 3 H). Anal. (C8H10ClN3O2) C, H, N.

n cn class="Gene">lass="Chemical">2-Chloro-1-[2-(2-methyloxiran-2-yl)ethyl]-4-nitro-1H-imidazole(. lass="Chemical">pan class="Chemical">3-Chloroperoxybenzoic acid (14.4 g of 70%, 58.4 mmol) was added to a mixture of alkene 41 (10.4 g, 48.2 mmol) and disodium hydrogen phosphate (10.4 g, 73.3 mmol) in CH2Cl2 (300 mL) at 0 °C. The mixture was stirred at 20 °C for 4 h, and then additional m-CPBA (2.40 g, 9.74 mmol) and CH2Cl2 (50 mL) were added. The resulting mixture was stirred at 20 °C for a further 14 h and then cooled to –20 °C and washed with an ice-cold aqueous solution of sodium sulphite (200 mL of 10%), back-extracting with CH2Cl2 (2 × 200 mL). The organic portions were sequentially washed with aqueous NaHCO3 (200 mL) and brine (100 mL) and then combined and concentrated under reduced pressure, and the remaining oil was chromatographed on silica gel. Elution with 3:1 CH2Cl2/petroleum ether first gave foreruns, and then further elution with 3:1 CH2Cl2/petroleum ether and 0–2.5% EtOAc/CH2Cl2 gave 42 (10.6 g, 95%) as a cream solid: mp (CH2Cl2/petroleum ether) 87–89 °C. 1H NMR (CDCl3) δ 7.79 (s, 1 H), 4.13 (t, J = 7.6 Hz, 2 H), 2.67 (br d, J = 4.4 Hz, 1 H), 2.62 (br d, J = 4.3 Hz, 1 H), 2.19 (dt, J = 14.3, 7.7 Hz, 1 H), 2.04 (dt, J = 14.3, 7.4 Hz, 1 H), 1.40 (s, 3 H). Anal. (C8H10ClN3O3)C, H, N.

Procedure J: n class="Chemical">pan class="Chemical">4-(2-Chloro-4-nitro-1H-imidazol-1-yl)-2-methyl-1-[4-(trifluoromethoxy)phenoxy]butan-2-ol (. lass="Chemical">pan class="Chemical">4-(Trifluoromethoxy)phenol (0.280 mL, 2.16 mmol) was added to a mixture of epoxide 42 (200 mg, 0.863 mmol) and powdered K2CO3 (422 mg, 3.05 mmol) in anhydrous MEK (2.0 mL) under N2, and then the mixture was stirred at 82 °C for 10 h. The resulting cooled mixture was diluted with water (50 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 CH2Cl2 first gave foreruns, and then further elution with 0–2% EtOAc/CH2Cl2 gave 43 (272 mg, 77%) as a pale-yellow oil. 1H NMR (CDCl3) δ 7.81 (s, 1 H), 7.17 (br d, J =9.1 Hz,2 H), 6.90 (br d, J = 9.2 Hz, 2 H), 4.33–4.20 (m, 2 H), 3.85 (d, J = 9.0 Hz, 1 H), 3.82 (d, J = 9.0 Hz, 1 H), 2.23 (ddd, J = 13.8, 9.3, 6.5 Hz, 1 H), 2.21 (s, 1 H), 2.04 (ddd, J = 13.8, 9.6, 6.6 Hz, 1 H), 1.40 (s, 3 H). HRESIMS calcd for C15H16ClF3N3O5 m/z [M + H]+ 412.0697, 410.0725, found 412.0700, 410.0722.

pan cn class="Gene">lass="Chemical">7-Methyl-2-nitro-7-{[4-(trifluoromethoxy)phenoxy]methyl}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (. Reaction of lass="Chemical">pan class="Chemical">alcohol 43 with NaH (1.7 equiv), using procedure D for 2 h, followed by chromatography of the product on silica gel, eluting with CH2Cl2, gave 44 (61%) as a cream solid: mp (CH2Cl2/pentane) 134–136 °C. 1H NMR [(CD3)2SO] δ 8.10 (s, 1 H), 7.31 (br d, J = 9.0 Hz, 2 H), 7.07 (br d, J = 9.2 Hz, 2 H), 4.20 (s, 2 H), 4.19 (dt, J = 13.3, 6.1 Hz, 1 H), 4.13 (ddd, J = 13.2, 8.1, 5.6 Hz, 1 H), 2.38 (ddd, J = 14.4, 7.9, 6.2 Hz, 1 H), 2.18 (dt, J = 14.4, 5.8 Hz, 1 H), 1.49 (s, 3 H). [13]C NMR [(CD3)2SO] δ 157.0, 147.2, 142.2, 142.1 (q, J = 1.6 Hz), 122.5 (2 C), 120.1 (q, J = 255.2 Hz), 117.7, 115.9 (2 C), 80.4, 72.4, 39.5, 27.0, 21.3. Anal. (C15H14F3N3O5) C, H, N.

Synthesis of . DEAn class="Chemical">D (0.270 mL, 1.74 mmol) was added dropwise to a stirred solution of pan class="Chemical">alcohol 13 (251 mg, 1.26 mmol), lass="Chemical">pan class="Chemical">4-iodophenol (377 mg, 1.71 mmol), and PPh3 (448 mg, 1.71 mmol) in anhydrous THF (3 mL) under N2 at 0 °C. After being stirred at 20 °C for 32 h, the mixture was concentrated under reduced pressure to give an oil, which was chromatographed on silica gel. Elution with CH2Cl2 first gave foreruns, and then further elution with 0–2% EtOAc/CH2Cl2 gave the crude product, which was further purified by chromatography on silica gel. Elution with 33–50% EtOAc/petroleum ether first gave foreruns, and then further elution with 10% MeOH/CH2Cl2 gave 48 (433 mg, 86%) as a cream solid: mp (MeOH/CH2Cl2/hexane) 224–227 °C. 1H NMR [(CD3)2SO] δ 8.08 (s, 1 H), 7.62 (br d, J = 9.0 Hz, 2 H), 6.86 (br d, J = 9.0 Hz, 2 H), 4.94–4.85 (m, 1 H), 4.31 (dd, J = 11.1, 3.4 Hz, 1 H), 4.25 (dd, J = 11.1, 5.8 Hz, 1 H), 4.18 (ddd, J = 12.6, 5.8, 3.0 Hz, 1 H), 4.09 (ddd, J = 12.5, 10.8, 5.2 Hz, 1 H), 2.35–2.26 (m, 1 H), 2.25–2.12 (m, 1 H). Anal. (C13H12IN3O4) C, H, N.

Procedure n class="Gene">L: pan class="Chemical">7-{[4-(6-Fluoropyridin-3-yl)phenoxy]methyl}-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (. A stirred mixture of lass="Chemical">pan class="Chemical">iodide 48 (70.3 mg, 0.175 mmol), (6-fluoropyridin-3-yl)boronic acid (42.3 mg, 0.300 mmol), and Pd(dppf)Cl2 (19.5 mg, 0.0266 mmol) in DMF (2.3 mL), toluene (1.5 mL), and EtOH (1.0 mL) was degassed for 8 min (vacuum pump), and then N2 was added. An aqueous solution of KHCO3 (0.40 mL of 2 M, 0.80 mmol) was added by syringe, and the stirred mixture was again degassed for 9 min and then N2 was added. The resulting mixture was stirred at 85 °Cfor 2 h, and then cooled, diluted with aqueous NaHCO3 (50 mL), and extracted with CH2Cl2 (6 × 50 mL). The extracts were evaporated to dryness under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–4% EtOAc/CH2Cl2 first gave foreruns, and then further elution with 4–7% EtOAc/CH2Cl2 gave 55 (61 mg, 94%) as a cream solid: mp (MeOH/CH2Cl2/hexane) 197–198 °C. 1H NMR [(CD3)2SO] δ 8.51 (br d, J = 2.6 Hz, 1 H), 8.24 (td, J = 8.2, 2.6 Hz, 1 H), 8.10 (s, 1 H), 7.69 (br d, J = 8.8 Hz, 2 H), 7.24 (dd, J = 8.6, 2.6 Hz, 1 H), 7.13 (br d, J = 8.8 Hz, 2 H), 4.99–4.88 (m, 1 H), 4.39 (dd, J = 11.1, 3.3 Hz, 1 H), 4.33 (dd, J = 11.1, 5.8 Hz, 1 H), 4.20 (ddd, J = 12.5, 5.7, 2.9 Hz, 1 H), 4.11 (ddd, J = 12.4, 10.9, 5.2 Hz, 1 H), 2.39–2.29 (m, 1 H), 2.29–2.15 (m, 1 H). 13C NMR [(CD3)2SO] δ 162.2 (d, J = 235.1 Hz), 158.2, 147.8, 144.8 (d, J = 15.1 Hz), 142.1, 139.8 (d, J = 8.0 Hz), 133.7 (d, J = 4.6 Hz), 128.7, 128.1 (2 C), 117.8, 115.3 (2 C), 109.5 (d, J = 37.7 Hz), 76.0, 68.8, 41.7, 22.4. Anal. (C18H15FN4O4) C, H, N.

Synthesis of . A mixture of n cn class="Gene">lass="Chemical">alcohol 13 (500 mg, 2.51 mmol) and lass="Chemical">pan class="Chemical">5-bromo-2-fluoropyridine (57) (0.52 mL, 5.05 mmol) in anhydrous DMF (10 mL) under N2 at 0 °C was treated with 60% NaH (151 mg, 3.78 mmol) and then quickly degassed and resealed under N2. Further 57 (0.52 mL, 5.05 mmol) was added, and the mixture was stirred at 20 °C for 2.5 h. The resulting mixture was cooled (CO2/acetone), quenched with ice/aqueous NaHCO3 (20 mL), and then added to brine (100 mL) and extracted with CH2Cl2 (8 × 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 0–1% EtOAc/CH2Cl2 first gave foreruns, and then further elution with 2–4% EtOAc/CH2Cl2 gave 58 (778 mg, 87%) as a white solid: mp (MeOH/CH2Cl2/hexane) 182–184 °C. 1H NMR [(CD3)2SO] δ 8.30 (br d, J = 2.6 Hz, 1 H), 8.07 (s, 1 H), 7.94 (dd, J = 8.8, 2.6 Hz, 1 H), 6.91 (br d, J = 8.8 Hz, 1 H), 4.95–4.86 (m, 1 H), 4.58 (dd, J = 12.0, 3.3 Hz, 1 H), 4.52 (dd, J = 12.0, 6.0 Hz, 1 H), 4.17 (ddd, J = 12.6, 5.8, 2.8 Hz, 1 H), 4.09 (ddd, J = 12.5, 11.0, 5.2 Hz, 1 H), 2.35–2.25 (m, 1 H), 2.24–2.10 (m, 1 H). Anal. (C12H11BrN4O4) C, H, N.

Procedure N: n class="Chemical">pan class="Chemical">7-({[5-(4-Fluorophenyl)pyridin-2-yl]oxy}methyl)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (. A stirred mixture of lass="Chemical">pan class="Chemical">bromide 58 (150 mg, 0.422 mmol), 4-fluorophenylboronic acid (117 mg, 0.836 mmol), and Pd(dppf)Cl2 (83.1 mg, 0.114 mmol) in DMF (4.5 mL), toluene (3 mL), and EtOH (2 mL) was degassed for 10 min (vacuum pump), and then N2 was added. An aqueous solution of Na2CO3 (1.05 mL of 2 M, 2.1 mmol) was added by syringe, the stirred mixture was again degassed for 10 min, and then N2 was added. The resulting mixture was stirred at 89 °C for 2.5 h and then cooled, diluted with aqueous NaHCO3 (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 foreruns, and then further elution with 3% EtOAc/CH2Cl2 gave 59 (143 mg, 91%) as a cream solid: mp (MeOH/CH2Cl2/hexane) 180–181 °C. 1H NMR [(CD3)2SO] δ 8.47 (br d, J = 2.6 Hz, 1 H), 8.09 (s, 1 H), 8.05 (dd, J = 8.6, 2.6 Hz, 1 H), 7.71 (br dd, J =8.9, 5.4 Hz, 2 H), 7.30 (br t, J = 8.9 Hz, 2 H), 6.98 (br d, J = 8.6 Hz, 1 H), 4.99–4.90 (m, 1 H), 4.64 (dd, J = 12.0, 3.4 Hz, 1 H), 4.58 (dd, J = 12.0, 6.0 Hz, 1 H), 4.19 (ddd, J = 12.5, 5.8, 2.7 Hz, 1 H), 4.10 (ddd, J = 12.5, 11.1, 5.1 Hz, 1 H), 2.38–2.28 (m, 1 H), 2.27–2.13 (m, 1 H). 13C NMR [(CD3)2SO] δ 162.1, 161.9 (d, J = 244.4 Hz), 147.8, 144.5, 142.0, 137.9, 133.3 (d, J = 3.1 Hz), 128.9, 128.4 (d, J = 8.1 Hz, 2 C), 117.8, 115.8 (d, J = 21.5 Hz, 2 C), 110.8, 76.0, 66.3, 41.7, 22.5. Anal. (C18H15FN4O4) C, H, N.

Synthesis of . Reaction of 2-chloro-4-n class="Chemical">pan class="Chemical">nitro-lass="Chemical">pan class="Chemical">1H-imidazole (40) with (4R)-4-(2-iodoethyl)-2,2-dimethyl-1,3-dioxolane[33] (72) (0.96 equiv) and powdered K2CO3 (1.03 equiv), using procedure A for 3 d, followed by chromatography of the product on silica gel, eluting with 0–33% Et2O/petroleum ether (foreruns) and then with 33–50% Et2O/petroleum ether, gave 73 (74%) as a light-yellow solid: mp (Et2O/pentane) 73–75 °C. 1H NMR (CDCl3) δ 7.81 (s, 1 H), 4.23 (ddd, J = 14.2, 7.7, 5.3 Hz, 1 H), 4.18 (ddd, J = 14.2, 8.0, 7.1 Hz, 1 H), 4.10 (dd, J = 7.9, 6.1 Hz, 1 H), 4.09–4.01 (m, 1 H), 3.60 (dd, J = 7.8, 5.7 Hz, 1 H), 2.12–2.01 (m, 1 H), 2.01–1.90 (m, 1 H), 1.43 (s, 3 H), 1.36 (s, 3 H). [α][26]D 39.2 (c 1.020, CHCl3). Anal. (C10H14ClN3O4) C, H, N.

Procedure O: n class="Chemical">pan class="Chemical">(2R)-4-(2-Chloro-4-nitro-1H-imidazol-1-yl)butane-1,2-diol (. Dilute lass="Chemical">pan class="Chemical">HCl (13 mL of a 1 M solution, 13.0 mmol) was added dropwise to a stirred solution of acetonide 73 (2.86 g, 10.4 mmol) in MeOH (39 mL) at 0 °C. The mixture was stirred at 20 °C for 6 h and then cooled in ice, treated with K2CO3 (0.90 g, 6.51 mmol), and stirred until the neutralization was complete. Following filtration to remove inorganic material (washing with MeOH), the solvents were removed under reduced pressure (at 30 °C), and the residue was chromatographed on silica gel. Elution with 0–67% EtOAc/petroleum ether first gave foreruns, and then further elution with EtOAc gave 74 (2.39 g, 98%) as a cream solid: mp (MeOH/CH2Cl2/hexane) 115–117 °C. 1H NMR [(CD3)2SO] δ 8.55 (s, 1 H), 4.79 (d, J = 5.0 Hz, 1 H), 4.60 (t, J = 5.6 Hz, 1 H), 4.21–4.08 (m, 2 H), 3.45–3.36 (m, 1 H), 3.36–3.29 (m, 1 H), 3.22 (dt, J = 10.7, 5.9 Hz, 1 H), 2.03–1.91 (m, 1 H), 1.75–1.62 (m, 1 H). [α][24]D 29.4 (c 2.008, DMF). Anal. (C7H10ClN3O4) C, H, N.

Procedure P: pan class="Chemical">(2R)-4-(2-Chloro-4-nitro-1H-imidazol-1-yl)-2-hydroxybutyl 4-methylbenzenesulfonate (. A solution of tosyl chloride (2.28 g, 12.0 mmol) in anhydrous lass="Chemical">pan class="Chemical">pyridine (3 mL, then 2 × 1.5 mL to rinse) was added dropwise to a stirred solution of diol 74 (2.35 g, 9.97 mmol) in anhydrous pyridine (5 mL) under N2 at –10 °C. The mixture was stirred at –10 to 0 °C for 2 h and then at 20 °C for 13 h. The resulting solution was cooled in ice and then added to ice–water (100 mL) and extracted with CH2Cl2 (4 × 100 mL). The combined extracts were concentrated to dryness under reduced pressure (at 30 °C), and the remaining oil was chromatographed on silica gel. Elution with 0–2% EtOAc/CH2Cl2 first gave foreruns, and then further elution with 2–50% EtOAc/CH2Cl2 gave 75 (3.38 g, 87%) as a cream foam that was used directly in the next step. 1HNMR (CDCl3) δ 7.78 (s, 1 H), 7.78 (br d, J = 8.3 Hz, 2 H), 7.37 (br d, J = 8.0 Hz, 2 H), 4.22 (dd, J = 7.7, 6.0 Hz, 2 H), 4.04 (dd, J = 10.5, 3.4 Hz, 1 H), 3.95 (dd, J = 10.5, 6.6 Hz, 1 H), 3.87–3.78 (m, 1 H), 2.62 (br d, J = 4.3 Hz, 1 H), 2.47 (s, 3 H), 1.99–1.83 (m, 2 H). APCI MS m/z 392, 390 [M + H]+.

Procedure Q: n class="Chemical">pan class="Chemical">2-Chloro-4-nitro-1-{2-[(2R)-oxiran-2-yl]ethyl}-1H-imidazole (. 1,8-Dilass="Chemical">pan class="Chemical">azabicyclo[5.4.0]undec-7-ene (1.45 mL, 9.70 mmol) was added dropwise to a stirred solution of tosylate 75 (3.38 g, 8.67 mmol) in anhydrous CH2Cl2 (32 mL) under N2 at 0 °C. The mixture was stirred at 0 °C for 3h, at 0–20 °C for 2 h, and then at 20 °C for 3 h. The resulting solution was added to a mixture of ice and brine (100 mL) and extracted with CH2Cl2 (4 × 100 mL). The combined extracts were concentrated to dryness under reduced pressure (at 30 °C), and the remaining oil was chromatographed on silica gel. Elution with CH2Cl2 first gave foreruns, and then further elution with CH2Cl2 gave 76 (1.78 g, 94%) as a cream solid (after freezing): mp (CH2Cl2/pentane) 59–61 °C. 1H NMR (CDCl3) δ 7.81 (s, 1 H), 4.29–4.15 (m, 2 H), 2.99–2.91 (m, 1 H), 2.86 (dd, J = 4.7, 4.0 Hz, 1 H), 2.54 (dd, J = 4.8, 2.6 Hz, 1 H), 2.36–2.25 (m, 1 H), 1.87–1.76 (m, 1 H). [α][25]D 43.6 (c 1.009, CHCl3). Anal. (C7H8ClN3O3) C, H, N.

Procedure R: n class="Chemical">pan class="Chemical">(2R)-1-[(6-Bromopyridin-3-yl)oxy]-4-(2-chloro-4-nitro-1H-imidazol-1-yl)butan-2-ol (. A mixture of lass="Chemical">pan class="Chemical">epoxide 76 (1.76 g, 8.07 mmol), 6-bromopyridin-3-ol (68) (2.82 g, 16.2 mmol), and powdered K2CO3 (2.23 g, 16.1 mmol) in anhydrous MEK (21 mL) under N2 was stirred at 80–82 °C for 42 h. The resulting cooled mixture was added to water (100 mL), washing in residues with MeOH/CH2Cl2, and then extracted with 10% MeOH/CH2Cl2 (3 × 100 mL) and 25% EtOAc/CH2Cl2 (3 × 100 mL). The combined extracts were concentrated to dryness under reduced pressure, and the remaining oil was chromatographed on silica gel. Elution with 0–40% EtOAc/petroleum ether first gave foreruns, and then further elution with 50% EtOAc/petroleum ether gave 77 (1.71 g, 54%) as a cream solid: mp (MeOH/CH2Cl2/hexane) 134–135 °C. 1H NMR [(CD3)2SO] δ 8.58 (s, 1 H), 8.12 (d, J = 3.1 Hz, 1 H), 7.54 (d, J = 8.7 Hz, 1 H), 7.39 (dd, J = 8.8, 3.2 Hz, 1 H), 5.30 (d, J = 4.9 Hz, 1 H), 4.27–4.14 (m, 2 H), 3.99 (dd, J = 10.0, 4.8 Hz, 1 H), 3.95 (dd, J = 10.0, 5.5 Hz, 1 H), 3.86–3.77 (m, 1 H), 2.11–2.00 (m, 1 H), 1.95–1.83 (m, 1 H). [α][24]D 7.95 (c 1.006, DMF). Anal. (C12H12BrClN4O4) C, H, N.

Further elution of the above con class="Gene">lumn with 4:1 n class="Chemical">EtOAc/lass="Chemical">pan class="Chemical">petroleum ether gave impurities, and then elution with EtOAc gave crude oxazine 78 (0.46 g), which was chromatographed again on silica gel. Elution with 0–0.4% MeOH/CH2Cl2 first gave foreruns, and then elution with 0.5% MeOH/CH2Cl2 gave purified 78 (305 mg, 11%) as a cream solid (see data below).

pan cn class="Gene">lass="Chemical">(7R)-7-{[(6-Bromopyridin-3-yl)oxy]methyl}-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (. Reaction of lass="Chemical">pan class="Chemical">alcohol 77 with NaH (1.4 equiv), using procedure D (but extracting the product four times with 10% MeOH/CH2Cl2 and then four times with CH2Cl2), followed by chromatography of the product on silica gel, eluting with 0–0.5% MeOH/CH2Cl2 (foreruns) and then with additional 0.5% MeOH/CH2Cl2, gave 78 (94%) as a cream solid: mp (MeOH/CH2Cl2/hexane) 211–212 °C. 1H NMR [(CD3)2SO] δ 8.19 (d, J = 3.1 Hz, 1 H), 8.10 (s, 1 H), 7.59 (br d, J = 8.7 Hz, 1 H), 7.47 (dd, J = 8.8, 3.2 Hz, 1 H), 4.96–4.89 (m, 1 H), 4.43 (dd, J = 11.2, 3.2 Hz, 1 H), 4.37 (dd, J = 11.2, 5.8 Hz, 1 H), 4.18 (ddd, J = 12.5, 5.8, 2.9 Hz, 1 H), 4.09 (ddd, J = 12.5, 10.9, 5.2 Hz, 1 H), 2.35–2.26 (m, 1 H), 2.25–2.13 (m, 1 H). [α][24]D –61.9 (c 1.002, DMF). Anal. (C12H11BrN4O4) C, H, N.

(7R)-7-({[6-(4-Fluorophenyn class="Gene">l)pyridin-3-yl]oxy}methyl)-2-n class="Chemical">nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]lass="Chemical">pan class="Chemical">oxazine (. Reaction of bromide 78 with 4-fluorophenylboronic acid (1.9 equiv) and Pd(dppf)Cl2 (0.25 equiv), using procedure N at 87 °C for 200 min (but extracting the product three times with 10% MeOH/CH2Cl2 and then three times with CH2Cl2), followed by chromatography of the product on silica gel, eluting with 0–0.5% MeOH/CH2Cl2 (foreruns) and then with 0.5–0.67% MeOH/CH2Cl2, gave 79 (87%) as a cream solid: mp (MeOH/CH2Cl2/hexane) 205–208 °C. 1H NMR [(CD3)2SO] δ 8.43 (d, J = 2.9 Hz, 1 H), 8.11 (s, 1 H), 8.07 (br dd, J = 8.9, 5.5 Hz, 2 H), 7.94 (d, J = 8.8 Hz, 1 H), 7.56 (dd, J = 8.8, 3.0 Hz, 1 H), 7.28 (br t, J = 8.9 Hz, 2 H), 5.00–4.91 (m, 1 H), 4.47 (dd, J = 11.2, 3.2 Hz, 1 H), 4.41 (dd, J = 11.2, 5.8 Hz, 1 H), 4.20 (ddd, J = 12.5, 5.7, 2.9 Hz, 1 H), 4.11 (ddd, J = 12.4, 11.0, 5.2 Hz, 1 H), 2.39–2.29 (m, 1 H), 2.29–2.16 (m, 1 H). [α][23]D –62.6 (c 1.006, DMF). Anal. (C18H15FN4O4) C, H, N.

Compounds of . The fon class="Gene">llowing section details the syntheses of compounds 142 and 160 of Table 2, via representative procedures and key intermediates, as described in Scheme 5. For the syntheses of all of the other compounds in Table 2, please refer to the Supporting Information.

Synthesis of . A mixture of n cn class="Gene">lass="Chemical">epoxide 67 (see the Supporting Information) (150 mg, 0.689 mmol) and lass="Chemical">pan class="Chemical">1-(4-fluorophenyl)piperazine (140) (186 mg, 1.03 mmol) in MEK (3 mL) in a sealed vial was stirred at 70 °Cfor51 h. The resulting cooled mixture was transferred to a flask (in CH2Cl2) and evaporated to dryness under reduced pressure (at 30 °C), and then the residue was chromatographed on silica gel. Elution with 0–0.3% MeOH/CH2Cl2 first gave foreruns, and then further elution with 1–2% MeOH/CH2Cl2 gave 141 (225 mg, 82%) as a pale-yellow oil. 1H NMR (CDCl3) δ 7.87 (s, 1 H), 6.97 (br dd, J = 9.2, 8.3 Hz, 2 H), 6.87 (br dd, J = 9.2, 4.6 Hz, 2 H), 4.27 (dd, J = 8.2, 5.7 Hz, 2 H), 3.67–3.58 (m, 1 H), 3.57 (v br s, 1 H), 3.19–3.07 (m, 4 H), 2.85–2.77 (m, 2 H), 2.59–2.51 (m, 2 H), 2.41 (dd, J = 12.3, 4.0 Hz, 1 H), 2.37 (dd, J = 12.3, 9.5 Hz, 1 H), 1.98–1.87 (m, 1 H), 1.82–1.70 (m, 1 H). HRESIMS calcd for C17H22ClFN5O3 m/z [M + H]+ 400.1366, 398.1390, found 400.1370, 398.1397.

pan cn class="Gene">lass="Chemical">7-{[4-(4-Fluorophenyl)piperazin-1-yl]methyl}-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (. Reaction of lass="Chemical">pan class="Chemical">alcohol 141 with NaH, using procedure D at 40 °C for 2 h, followed by chromatography of the product on silica gel, eluting with 0–0.5% MeOH/CH2Cl2 (foreruns) and then with 1% MeOH/CH2Cl2, gave 142 (67%) as a pale-yellow solid: mp (CH2Cl2/hexane) 213–215 °C. 1H NMR [(CD3)2SO] δ 8.07 (s, 1 H), 7.04 (br dd, J = 9.2, 8.6 Hz, 2 H), 6.94 (br dd, J = 9.3, 4.7 Hz, 2 H), 4.79–4.68 (m, 1 H), 4.13 (ddd, J = 12.6, 5.9, 2.9 Hz, 1 H), 4.05 (ddd, J = 12.5, 10.8, 5.1 Hz, 1 H), 3.13–3.03 (m, 4 H), 2.73 (dd, J = 13.5, 6.6 Hz, 1 H), 2.70–2.60 (m, 5 H), 2.29–2.19 (m, 1 H), 2.09–1.95 (m, 1 H). 13C NMR [(CD3)2SO] δ 156.0 (d, J = 235.6 Hz), 148.0, 147.9 (d, J = 1.6 Hz), 142.1, 117.7, 117.1 (d, J = 7.6 Hz, 2 C), 115.2 (d, J = 21.7 Hz, 2 C), 76.0, 60.5, 53.2 (2 C), 49.0 (2 C), 41.8, 24.3. Anal. (C17H20FN5O3)C,H,N.

Synthesis of . n cn class="Gene">lass="Chemical">Triphosgene (145 mg, 0.489 mmol) was added to a mixture of lass="Chemical">pan class="Chemical">alcohol 13 (192 mg, 0.964 mmol) and triethylamine (0.40 mL, 2.87 mmol) in anhydrous THF (15 mL). The mixture was stirred at 20 °C for 30 min, and then a solution of 1-(4-fluorophenyl)piperazine (140) (347 mg, 1.93 mmol) in anhydrous THF (5 mL) was added. The resulting mixture was stirred at 20 °Cfor 2 h and then quenched with saturated NH4Cl (100 mL) and extracted with EtOAc (2 × 100 mL). The combined extracts were evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel. Elution with 0–2% MeOH/CH2Cl2 gave crude material, which was successively recrystallized from CH2Cl2/hexane and EtOAc/hexane, to give 160 (130 mg, 33%) as a cream solid: mp 177–180 °C. 1H NMR [(CD3)2SO] δ 8.08 (s, 1 H), 7.06 (br dd, J = 9.1, 8.7 Hz, 2 H), 6.97 (br dd, J = 9.2, 4.7 Hz, 2 H), 4.84–4.73 (m, 1 H), 4.37 (dd, J = 12.2, 3.2 Hz, 1 H), 4.29 (dd, J = 12.2, 5.8 Hz, 1 H), 4.15 (ddd, J = 12.5, 5.8, 2.7 Hz, 1 H), 4.06 (ddd, J = 12.4, 11.3, 5.1 Hz, 1 H), 3.61–3.45 (m, 4 H), 3.14–2.98 (m, 4 H), 2.30–2.20 (m, 1 H), 2.17–2.03 (m, 1 H). 13C NMR [(CD3)2SO] δ 156.3 (d, J = 236.3 Hz), 154.1, 147.8, 147.7 (d, J = 1.6 Hz), 142.0, 117.9 (d, J =7.5 Hz, 2 C), 117.7, 115.3 (d, J = 22.0 Hz, 2 C), 75.8, 65.5, 49.1 (2 C), 43.4 (2 C), 41.7, 22.3. Anal. (C18H20FN5O5) C, H, N.

Minimum Inhibitory Concentpan cn class="Gene">lass="Species">ration Assays (MABA and LORA). These were carried out according to the published protocols.[41,56]

In Vitro Parn class="Chemical">asite Growth Inhibition Assays. The activity of test compounds against the amastigote stage of the pan class="Species">L. don lass="Chemical">parasite was assessed at CDRI using a lass="Chemical">pan class="Species">mouse macrophage-based luciferase assay, performed according to the reported procedures.[10] Further assays measuring the growth inhibitory action of compounds against L. inf, T. cruzi, and T. brucei, and determining any cytotoxic effects on human lung fibroblasts (MRC-5 cells), were conducted at the University of Antwerp (LMPH), as detailed in a recent article.[40]

Solubility Determinations. Method A. The solid compound sample was mixed with pan class="Chemical">water or 0.1 M lass="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 conducted. 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 4 at pH 7.4 was measured by Drugabilis, 5 rue Jean-Baptiste Clément, 92290 Châtenay-Malabry, France. The dry powder was stirred with 0.12 M pan class="Chemical">phosphate buffer (pH 7.4) at 20 °C for 24 h. After filtlass="Chemical">pan class="Species">ration using a 0.22 μm PVDF membrane filter, the concentration of 4 was determined by HPLC with reference to a standard solution; the final value is the mean from two independent assays.

Method C. The thermodynamic solubility of compound 79 at pH 6.5 and 5.0 was measured by WuXi AppTec (Shanghai) Co., Ltd., 288 FuTe ZhongLu, WaiGaoQiao Free Trade Zone, Shanghai 200131, China. Aliquots of the compound pan class="Chemical">DMSO stock (10 mM) were transferred to fasted state simulated intestinal fluid buffer (pH 6.5) or fed state simulated intestinal fluid buffer (pH 5.0), and the mixtures were shaken for 24 h at room tempelass="Chemical">pan class="Species">rature. Following sampling by a Whatman filter device, the compound concentrations were determined by UV spectroscopy with reference to three calibration standards (2, 100, and 200 μM).

Method D. The thermodynamic solubility of compound 79 at pH 7.4 was measured by Syngene International Ltd., Plot No. 2 and 3 Biocon Park, Jigani Link Rd, Bangalore 560099, India. The dry powder was equilibpan class="Species">rated with 0.1 M lass="Chemical">pan class="Chemical">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 79 was determined by HPLC, comparing the peak area obtained with that from a standard solution (0.86 μM) in 1:1:2 EtOH/water/CH3CN.

Microsomal Stability Assays. Tests on initial compounds 14, 29, 34, 38, and 39 (Table 3) were run by MDS Pharma Services, 22011 30th Drive SE, Bothell, WA 98021-4444, as previously described.[58] Compounds 39, 44, 49, 53, 54, 59, 61, 71, 107, 108, 111–113, and 116 were evaluated by Advinus Therapeutics Ltd., 21 and 22 Phase II, Peenya Industrial Area, Bangalore 560058, India, using a published procedure[54] in which the compound concentpan class="Species">ration was 0.5 μM and the incubation time was 30 min. Additional analyses on compounds 28, 44, 59, 62, 71, 79, 87, 90, 91, 93–95, 129, 142, 152, 170, and 178 were performed by WuXi AppTec (Shanghai) Co., Ltd., 288 FuTe ZhongLu, WaiGaoQiao Free Trade Zone, Shanghai 200131, China, via a reported method.[11]

Distribution Coefficient ann class="Chemical">d p. The n class="Chemical">octanol–lass="Chemical">pan class="Chemical">water partition coefficient (Log P) of 4 at 20 °C was measured in duplicate by Advinus Therapeutics Ltd., Bangalore, India, using the shake-flask method with HPLC analysis. Log D and pK data for 79 were measured by WuXi AppTec (Shanghai) Co., Ltd. The Log D value was found by assessing the distribution of 79 between 100 mM phosphate buffer of pH 7.4 and octanol at room temperature (final matrix contained 1% DMSO), using the shake-flask method and LC-MS/MS analysis. The pK value was obtained by UV spectroscopy, employing 80% MeOH as the initial cosolvent.

Pln class="Chemical">asma Protein Binding Assay. Studies of 4 and 79 were conducted by WuXi AppTec (Shanghai) Co., Ltd., using equilibrium dialysis across a semipermeable membrane. Briefly, a 2 μM compound solution in plasma (0.5% pan class="Chemical">DMSO) was dialyzed against 100 mM lass="Chemical">pan class="Chemical">phosphate buffered saline (pH 7.4) on a rotating plate incubated for 4 or 6 h at 37 °C. 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.

Permeability n class="Chemical">Assay. This was performed by WuXi AppTec (Shanghai) Co., Ltd. n class="Gene">MDCK-MDR1 cells were seeded onto lass="Chemical">pan class="Chemical">polyethylene membranes in 96-well plates at 2 × 105 cells/cm2, giving confluent cell monolayer formation over 4–7 d. A solution of 79 (2 μM in 0.4% DMSO/HBSS buffer) was applied to the apical or basolateral side of the cell monolayer. Permeation of the compound from A to B direction or B to A direction was determined in triplicate over a 150 min incubation at 37 °C and 5% CO2 (95% humidity). In addition, the efflux ratio of 79 was also determined. Test and reference compounds were quantified by LC-MS/MS analysis based on the peak area ratio of analyte/internal standard.

Ames Test. Compound 71 (at doses of 1.5, 4, 10, 25, 64, 160, 400, and 1000 μg/well) was evaluated in the Mini-Ames reverse mutation screen conducted by WuXi AppTec (Suzhou) Co., Ltd., 1318 Wuzhong Avenue, Wuzhong District, Suzhou 215104, China. Two Salmonella strains (TA98 and TA100) were employed, both in the presence and absence of metabolic activation (pan class="Species">rat liver S9). Positive controls (lass="Chemical">pan class="Chemical">2-aminoanthracene, 2-nitrofluorene, and sodium azide) and a negative (DMSO solvent) control were included.

n cn class="Gene">lass="Gene">hERG Assay. The effects of compounds 44 and 79 on cloned lass="Chemical">pan class="Gene">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. The study was performed by WuXi AppTec (Shanghai) Co., Ltd. Compound 79 (at concentpan class="Species">rations of 1 and 10 μM) was incubated with lass="Chemical">pan class="Gene">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. Aln class="Gene">l animal experiments were performed according to institutional ethical guidelines for animal care. Antitubercular efficacy studies in pan class="Species">mice were approved by the UIC IACUC (UIC AWA no. A3460-01; ACC application no. 12-183). For lass="Chemical">pan class="Gene">VL, mouse model studies (LSHTM) were conducted under license from the UK Home Office (license no. PIL 70/6997), hamster studies at CDRI were approved by the CSIR-CDRI animal ethics committee (license no. 19/2009/PARA/IAEC), and hamster studies at LMPH were approved by the ethical committee of the University of Antwerp (UA-ECD 2010-17).

n cn class="Gene">lass="Disease">Acute TB Infection Assay. Each compound (including 6, which was employed as an internal reference standard) was administered orally to a group of 7 lass="Chemical">pan class="Species">M. tb-infected BALB/c mice at 100 mg/kg daily for 5 days a week for three weeks, beginning on day 11 postinfection, in accordance with published protocols.[41,58] The results were typically recorded as the ratio of the average reduction in colony forming units (CFUs) in the compound-treated mice/the average CFU reduction in the mice treated with 6.

n cn class="Gene">lass="Disease">Acute VL Infection Assay (lass="Chemical">pan class="Species">Mouse Model, LSHTM). Test compounds were orally dosed once per day for 5 days consecutively to groups of five female BALB/c mice infected with 2 × 107 L. donovani amastigotes, with treatment commencing 1 week postinfection, as described.[10] Miltefosine (1) 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.

n cn class="Gene">lass="Disease">Chronic VL Infection Assay 1 (lass="Chemical">pan class="Species">Hamster Model, CDRI). Golden hamsters (weighing 40–45 g) were infected intracardially with 1 × 107 L. donovani amastigotes, and then, 15 days later, all animals were subjected to splenic biopsy to assess the level of infection. Groups of hamsters having an appropriate infection grading (5–15 amastigotes/100 spleen cell nuclei) were treated with test compounds, starting on day 17 and dosing orally once per day for 5 days, according to the usual procedure.[10] Post-treatment splenic biopsies taken 12 days after the first dose were employed to determine the intensity of infection, as previously reported.[10]

pan cn class="Gene">lass="Disease">Chronic VL Infection Assay 2 (lass="Chemical">pan class="Species">Hamster Model, LMPH). Golden hamsters (weighing 75–80 g) were infected with 2 × 107 L. infantum amastigotes, and 21 days postinfection, treatment groups of 6 animals each were dosed orally once or 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 load reduction for treated hamsters in comparison to untreated (vehicle-only) controls. Miltefosine (1) was included as a reference drug in all experiments.

n cn class="Gene">lass="Species">Mouse Pharmacokinetics. Compound 28 was evaluated by UNT Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699 (using a method approved by the UNTHSC IACUC; AWA no. A3711–01). Following oral administlass="Chemical">pan class="Species">ration to female BALB/c mice at 40 mg/kg as a suspension in 0.5% carboxymethylcellulose/water, blood samples were collected (at time intervals of 0.5, 1, 1.5, 2, 4, 6, 8, and 24 h), centrifuged, and analyzed by LC-MS/MS to generate the required PK parameters. Compound 34 was assessed by MDS Pharma Services, 22002 26th Avenue SE, Suite 104, Bothell, WA 98021-4444, via a similar procedure (but employing mixed gender CD-1 mice and an oral formulation of 0.5% carboxymethylcellulose and 0.08% Tween 80 in water). Studies of compounds 39, 44, 49, 53, 54, and 112 were conducted by Advinus Therapeutics Ltd., 21 and 22 Phase II, Peenya Industrial Area, Bangalore 560058, India, according to a published protocol.[54] Briefly, compounds were administered to groups of male Swiss Albino mice; intravenous dosing (at 1 mg/kg) employed a solution vehicle comprising 20% 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 and 0.08% Tween 80 in water (except for 112, where the iv solution was 10% NMP, 10% cremophor EL and 10% propylene glycol in saline, and oral dosing at 12.5 mg/kg was as a suspension in 7% Tween 80 and 3% EtOH 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 WinNonlin software (version 5.2). Finally, 71 was examined by WuXi AppTec (Shanghai) Co., Ltd.; in this case, oral dosing of female BALB/c mice was at 25 mg/kg in PEG-400 (sampling at 0.25, 1, 2, 4, 8, and 24 h), and the PK data were derived following similar LC-MS/MS analysis.

n cn class="Gene">lass="Species">Rat and lass="Chemical">pan class="Species">Hamster Pharmacokinetics. Compounds 71, 79, and 87 were assessed in male Sprague–Dawley rats and female golden 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 5 mg/kg) was as a suspension in 0.08% Tween 80 and 0.5% carboxymethylcellulose in water, whereas PEG-400 was the vehicle employed for oral dosing in hamsters (at 12.5 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 WinNonlin software (version 6.3).

■ ASSOCIATED CONTENT

Supporting Information

The Supporting Information is availabn class="Gene">le free of charge on the pan class="Gene">ACS Publications website at DOI: 10.1021/lass="Chemical">pan class="Gene">acs.jmedchem.7b00034.

Additional biological assay data, synthetic schemes, graphs of PK and assay data, experimental procedures and characterizations for compounds, combustion analytical data, and representative NMR spectra (pan class="Disease">PDF) Molecular formula strings spreadsheet (CSV)

■ AUTHOR INFORMATION

ORCID

Andrew M. Thompson: 0000-0003-2593-8559

Notes

The authors decn class="Gene">lare no competing financial interest.