Structure−Activity Relationships of Antitubercular Nitroimidazoles. 1

J. Med. Chem. 2009, 52, 1317–1328

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Structure-Activity Relationships of Antitubercular Nitroimidazoles. 1. Structural Features Associated with Aerobic and Anaerobic Activities of 4- and 5-Nitroimidazoles Pilho Kim,†,§ Liang Zhang,†,# Ujjini H. Manjunatha,†,⊥ Ramandeep Singh,† Sejal Patel,‡,| Jan Jiricek,‡ Thomas H. Keller,‡ Helena I. Boshoff,† Clifton E. Barry, III,† and Cynthia S. Dowd*,†,∞ Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, and NoVartis Institute for Tropical Diseases, 138670, Singapore ReceiVed October 1, 2008

The 4-nitroimidazole PA-824 is active against aerobic and anaerobic Mycobacterium tuberculosis (Mtb) while 5-nitroimidazoles like metronidazole are active against only anaerobic Mtb. We have synthesized analogues of both 4- and 5-nitroimidazoles and explored their antitubercular activities. The nitro group is required for both activities in all compounds. The key determinants of aerobic activity in the 4-nitroimidazoles include the bicyclic oxazine, the lipophilic tail, and the 2-position oxygen. For the 5-nitroimidazoles, neither the corresponding bicyclic analogue nor addition of a lipophilic tail conveyed aerobic activity. Incorporation of a 2-position oxygen atom into a rigid 5-nitroimidazooxazine provided the first 5-nitroimidazole with aerobic activity. Across both series, anaerobic and aerobic activities were not correlated and Mtb mutants lacking the deazaflavin-dependent nitroreductase (Ddn) retained anaerobic sensitivity to some compounds. Aerobic activity appears to be correlated with efficiency as a substrate for Ddn, suggesting a means of structure-based optimization of improved nitroimidazoles. Introduction a

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb ), continues to be a major threat to global public health, killing more than 1.5 million people in 2005.1 One major contributing factor toward this mortality is the extended duration of chemotherapy (6-9 months) required to completely cure disease.2 Tuberculous lesions are hypoxic, with a pO2 less than 10 mmHg.3 Hypoxic Mtb does not actively replicate and is significantly less sensitive to the standard antituberculosis drugs that interfere with processes essential for rapid aerobic growth.4 Thus, hypoxia is thought to contribute to the extended duration of therapy and new agents active against these metabolically altered bacteria are urgently needed. The nitroimidazoles, such as (S)-2-nitro-6-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (PA824, 1),5 are a promising new class of compounds that have shown antitubercular activity under hypoxic conditions and are currently in clinical evaluation. Metronidazole (Mtz, 2-(2methyl-5-nitro-1H-imidazol-1-yl)ethanol, 2), a 5-nitroimidazole, is widely used for the treatment of diseases caused by anaerobic bacteria.6 It has been known for almost 15 years that under low * To whom correspondence should be addressed. Phone: 202-994-8405. Fax: 202-994-5873. E-mail: [email protected]. † National Institute of Allergy and Infectious Diseases. § Present address: Korea Research Institute of Chemical Technology, Daejeon, Korea. # Present address: Walter Reed Army Medical Hospital, Washington, DC. ⊥ Present address: Novartis Institute for Tropical Diseases, Singapore. ‡ Novartis Institute for Tropical Diseases. | Present address: Novartis Institute for Biomedical Research, Cambridge, MA. ∞ Present address: Department of Chemistry, George Washington University, Washington, DC 20052. a Abbreviations: FGD1, F420-dependent glucose-6-phosphate dehydrogenase; MAC, minimum anaerobicidal concentration; MIC, minimum inhibitory concentration; MBP, maltose-binding protein; Mtb, Mycobacterium tuberculosis; Mtz, metronidazole; SAR, structure-activity relationship; TB, tuberculosis; TBS or TBDMS, tert-butyldimethylsilyl; THP, tetrahydro2H-pyran; TBAF, tetrabutylammonium fluoride; TBAI, tetrabutylammonium iodide; 2D NOESY, two-dimensional nuclear Overhauser effect spectroscopy.

oxygen conditions Mtb cultures become sensitive to Mtz treatment.7,8 However, Mtz has no activity against aerobic populations of Mtb and is not used in the treatment of human tuberculosis. 4-Nitroimidazoles based on Mtz were originally synthesized as radiosensitizing agents9 and later found to have significant activity against Mtb.10,11 From a large number of 2,3-dihydro-6-nitroimidazo[2,1-b]oxazole derivatives, compound 3 (2-ethyl-6-nitro-2,3-dihydroimidazo[2,1-b]oxazole, CGI17341) emerged as an interesting tool compound with potent in vitro and in vivo activity against Mtb.10 Further work on this class of compounds led to the discovery of PA-824, a 4-nitroimidazooxazine.5 PA-824 and its analogues have shown potent activity against aerobic and anaerobic Mtb, no cross resistance to current TB drugs, and efficacy in the mouse model of TB infection.5,12-14 PA-824 is currently in clinical development by the Global Alliance for TB Drug Development.15 More recently, a structurally related 4-nitroimidazooxazole, compound 4 ((R)-2-methyl-6-nitro-2-((4-(4-(4-(trifluoromethoxy)phenoxy)piperidin-1-yl)phenoxy)methyl)-2,3-dihydroimidazo[2,1-b]oxazole, OPC-67683), has demonstrated significant antitubercular activity and is also being evaluated in human clinical trials.16 A series of antitubercular and structurally similar nitrofurans have also been reported.17 These compounds appear to be reduced using FGD1 and F420, but through a nitroreductase different from deazaflavin-dependent nitroreductase (Ddn) (Rv3547) used by PA-824.18

10.1021/jm801246z CCC: $40.75  2009 American Chemical Society Published on Web 02/11/2009

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Scheme 1. Synthesis of 5-NO2-824 and des-NO2-824a

a Reagents and conditions: (a) 6, K2CO3, EtOH, sealed tube, 70 °C, 20 h; (b) DHP, PPTS, CH2Cl2, room temp, 2 d; (c) TBAF, THF, room temp, 18 h; (d) AcOH, THF, H2O, 60 °C, 18 h; (e) NaH, TBAI, 4-trifluoromethoxybenzyl bromide, DMF, -78 °C to room temp, 18 h. Compounds 10a and 12 were prepared following the same scheme.

Our objective in the current study was to determine the fundamental structure-activity relationship (SAR) of nitroimidazoles as antitubercular compounds, with special emphasis on the structural requirements for aerobic versus anaerobic activities. A better understanding of the SAR would help us to determine if these diverse groups of nitroimidazole compounds have the same cellular mode of action. In particular, we were interested in the differences or similarities in SAR between the 4-nitroimidazooxazines (typified by PA-824) and the 5-nitroimidazoles (such as Mtz) and whether the SAR for the aerobic activity would mirror that of the anaerobic activity. To simplify the discussion, we will use the numbering for the imidazole ring throughout the paper. Thus, Mtz has a 5-nitro substituent, while PA-824 and compound 3 are referred to as 4-nitro derivatives.

Scheme 2. Synthesis of Analogues To Examine the Importance of the Side Chain of Nitroimidazolesa

Chemistry Simple comparison of the structures of Mtz and PA-824 revealed four key differences: (i) the location of the nitro group; (ii) the presence or absence of the lipophilic side chain; (iii) the electronic nature of the atom at the 2-position of the nitroimidazole; (iv) a monocyclic versus a bicyclic ring structure. We have synthesized a number of compounds that probe the structural space defined by these parameters and then examined their activity against Mtb. Nitro isomers of PA-824 and Mtz. We initially prepared the 5-nitro isomer of PA-824 (11 in Scheme 1). The original synthesis of PA-824 and related compounds was published in two U.S. patents several years ago.12,13 We have improved upon this synthesis and have used this as the basis for much of our synthetic work. The patented synthesis began with 2,4-dinitroimidazole, an explosive. Instead, we used 2-chloro-4-ni-

a Reagents and conditions: (a) NaH, CH3I, DMF, -78 °C to room temp, 18 h; (b) NaH, TBAI, 4-trifluoromethoxybenzyl bromide, DMF, -78 °C to room temp, 18 h; (c) 2-bromoethanol, K2CO3, TBAI, CH3CN, sealed tube, 75 °C, 2 h.

4- and 5-Nitroimidazoles

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Scheme 3. Synthesis of Ring-Opened Analogues of PA-824a

a Reagents and conditions: (a) BrCH2CH2OTBS, K2CO3, MeOH, sealed tube, 75 °C, 18 h; (b) NaOMe, MeOH, room temp, 24 h; (c) NaH, TBAI, 4-trifluoromethoxybenzyl bromide, DMF, -78 °C to room temp, 14 h.

Scheme 4. Synthesis of “Bicyclic” Mtza

a Reagents and conditions: (a) (i) 22, K2CO3, MeOH, sealed tube, 70 °C, 14 h; (ii) TBSCl, imidazole, CH2Cl2, room temp, 1 h; (b) tributyl(vinyl)stannane, Pd(PPh3)4, LiCl, DMF, microwave, 120 °C, 10 min; (c) Hoveyda-Grubbs’ II catalyst, CH2Cl2, sealed tube, 60 °C, 2.5 h; (d) Wilkinson’s catalyst, H2(g), t-BuOH, THF, room temp, 14 h; (e) TBAF, THF, room temp, 10 min. 27b was prepared following the same scheme using (S)-vinyloxirane.

Scheme 5. Synthesis of 2-Methoxy-Mtza

a Reagents and conditions: (a) BrCH2CH2OTBS, K2CO3, MeOH, sealed tube, 75 °C, 18 h; (b) NaOMe, MeOH, room temp, 15 h; (c) TBAF, THF, room temp, 10 min.

troimidazole (5). Condensation of 5 with epoxide 6 gave an isomeric mixture of products. After careful chromatographic separation, alcohols 7a and 7b were isolated in a ratio of 3:1, with a total yield of 86%.19 THP protection of 5-nitroimidazole 7b furnished protected diol 8 in 70% yield. Cyclization of 8 in the presence of TBAF yielded bicyclic compound 9, which was then deprotected with AcOH to give alcohol 10b. The 4-nitro alcohol 10a was obtained following the same procedures from 7a (not shown). Alkylation of 10b using 4-trifluoromethoxybenzyl bromide with NaH and TBAI gave 5-nitro-824 (11). Interestingly, movement of the nitro group from the 4-position to the 5-position of the imidazole ring dramatically changes the chemical reactivity of these compounds. The yields for each of the reactions leading to compound 11 are significantly lower than those using the corresponding 4-nitro isomers. This is typified by the yield of the final alkylation reaction where PA824 was obtained in 70% yield,13 while compound 11 was obtained in 13%. The desnitro analogue (12) was prepared using the same methodology with 2-nitroimidazole as the starting material (see Supporting Information).

To examine the importance of the location of the nitro group in Mtz (2), we also prepared the 4-nitro isomer of this compound (Scheme 2). 2-Methyl-4-nitroimidazole (13) was reacted with 2-bromoethanol and K2CO3 in the presence of TBAI to give 4-nitro-Mtz (14).20 Lipophilic Side Chain Analogues of PA-824 and Mtz. The importance of the lipophilic side chain of PA-824 on aerobic and anaerobic activity was addressed by the compounds shown in Scheme 2. Methylation of alcohol 10a with CH3I and NaH gave the analogue (15) with the 4-trifluoromethoxyphenyl side chain removed in 57% yield.21 Simple alkylation of either Mtz (2) or 4-nitro-Mtz (14)22 with 4-trifluoromethoxybenzyl bromide in the presence of NaH and TBAI gave compounds 16 (33%) and 17 (39%), respectively. Monocyclic Analogue of PA-824 and Bicyclic Analogues of Mtz. Compound 20, a ring-opened analogue of PA-824 (Scheme 3), was prepared as follows: 2-chloro-4-nitroimidazole (5) was alkylated using TBS-protected bromoethanol to give compound 18 (25%) in addition to the corresponding 5-nitro isomer (28 (2.3%) in Scheme 5) as a minor product. Substitution

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Scheme 6. Synthesis of 2-C-824a

a Reagents and conditions: (a) 22, K2CO3, MeOH, sealed tube, 85 °C, 20 h; (b) NaH, TBAI, 4-trifluoromethoxybenzyl bromide, DMF, -78 °C to room temp, 14 h; (c) tributyl(vinyl)stannane, Pd(PPh3)4, LiCl, DMF, microwave, 120 °C, 15 min; (d) Grubbs’ II catalyst, CH2Cl2, sealed tube, 55 °C, 15 h; (e) Wilkinson’s catalyst, H2(g), t-BuOH, THF, room temp, 16 h.

Figure 1. Compounds examining the antitubercular SAR of 4-nitroimidazoles.

of the chlorine followed by TBS deprotection in the presence of NaOMe afforded 19 in 61% yield.23 Alkylation of the hydroxyl group in 19 gave rise to a ring-opened version of PA-824 (20) in 58% yield. To give “bicyclic” analogues of Mtz, we envisioned ring-closing metathesis as a key step (Scheme 4).24 Considering the possible conformations of the bicyclic product, both stereoisomers were prepared. Starting from 2-bromo-4-nitroimidazole (21), an epoxide-opening reaction using (R)-vinyloxirane (22) gave an allylic alcohol which was TBS-protected to afford compound 23a (16% in

two steps). This procedure also produced the corresponding 4-isomer (e.g., 31 (47%) in Scheme 6). Stille coupling of 23a using tributyl(vinyl)stannane furnished the required diene (24a, 67%).25 Ring-closing metathesis using the second generation Hoveyda-Grubbs’ catalyst26 provided the unsaturated bicyclic compound 25a (72%). TBS protection was required to avoid undesired elimination during the synthesis of 25a. Selective reduction of 25a with Wilkinson’s catalyst gave compound 26a in 65% yield.27 TBS deprotection of 26a with TBAF afforded bicyclic Mtz 27a (69%). The same

4- and 5-Nitroimidazoles

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Figure 2. Compounds examining the antitubercular SAR of 5-nitroimidazoles.

procedure was followed using (S)-vinyloxirane to obtain the enantiomer 27b. 2-Position Substitutions in Each Series. To understand the importance of the 2-position substituent on activity, both the 2-methoxy Mtz derivative (30) and the carba analogue (35) of PA-824 were made. In Scheme 5, reaction of 2-chloro-4nitroimidazole (5) with TBS-protected bromoethanol gave the 4- and 5-nitro alkylation products in an 11:1 ratio. Interestingly, preparation of the 5-nitro isomer required use of a protected bromoethanol. The 4- and 5-nitro isomers were separated by careful column chromatography, and their structures were confirmed by 2D NOESY experiments and UV λmax differences.28 The 5-nitro isomer (28) was reacted with sodium methoxide to give 29 (58%) in addition to a small amount of 30 (15%). Subsequent TBS deprotection of 29 with TBAF afforded desired compound 30 in 69% yield. The synthesis of analogue 35 is shown in Scheme 6 and follows the synthetic methodology used to prepare compounds 27a and 27b. Alkylation of 2-bromo-4-nitroimidazole (21) furnished allylic alcohol 31. Compound 31 was then alkylated with 4-trifluoromethoxybenzyl bromide to afford 32 in 62% yield to install the side chain of PA-824. 32 underwent Stille coupling, ring-closing metathesis, and selective reduction reactions, furnishing compounds 33, 34, and 35, respectively. Results and Discussion A Nitro Group is Essential for Both Aerobic and Anaerobic Activities. To assess the importance of the presence of the nitro group, we examined the aerobic MIC (minimal inhibitory concentration, defined as the concentration that prevents 99% of bacterial growth) as well as the anaerobic MAC (minimum anaerobicidal concentration, defined as the drug concentration that causes a 90% reduction in bacterial numbers following 7 days of anaerobic drug exposure) of desnitro-824 (12). Desnitro-824 (12) retained neither the aerobic nor anaerobic activity of the parent compound, indicating that the nitro group is indeed essential for the dual activities of PA-824 (Table 1). SAR of the 4-Nitroimidazole Series Related to PA-824. We approached the SAR of the 4-nitroimidazoles derived from PA-824 systematically as shown in Figure 1. In order to understand the importance of the trifluoromethoxybenzyl ether

side chain on the activity of the 4-nitroimidazoles, we prepared compounds 10a and 15. Removal of the side chain to give the simple “4-nitro alcohol” (10a) resulted in a dramatic and complete loss of both aerobic and anaerobic activities. To rule out that this was simply the very different lipophilicities of an ether compared to an alcohol, we also explored 15 (the simple methyl ether). Compound 15 likewise showed a dramatic loss of aerobic and anaerobic activities when compared to PA-824. These results are in line with the results from Baker et al.12 who showed that 6-(benzyloxy)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (i.e., PA-824 without the OCF3) was inactive against M. boVis BCG while compounds retaining the lipophilic substituent in the para position of the aromatic ring retained aerobic activity. Compound 20 represents an open ring analogue of PA-824 with simple scission of the C-6/C-7 carbon bond of the oxazine ring. As expected, this conservative change retained some of the aerobic activity of the parent compound, PA-824, albeit at a level reduced nearly 10-fold. The anaerobic activity was even more significantly decreased. The response of both activities suggests that the increase in rotational freedom accompanying the loss of the oxazine ring results in a smaller population of active conformers required for recognition by the activating enzyme. Removal of the 2-position oxygen atom from 20 yields compound 17. Interestingly, this change in the electron-donating potential of the 2-substituent leads to a loss in aerobic activity of more than 20-fold, arguing for a very important role of the 2-postion oxygen in determining aerobic activity of the 4-nitroimidazoles. The loss of anaerobic activity of 17 relative to that of Mtz further supports that the 2-oxy substituent is a critical determinant of both aerobic and anaerobic activities in the 4-nitroimidazoles but not 5-nitroimidazoles. Removal of the side chain from compound 17 yields a more Mtz-like compound, 14. The sole difference between 14 and Mtz lies in the position of the nitro group. Compound 14 did not display either aerobic or anaerobic activity. This result confirms the importance of the position of the nitro group in Mtz’s anaerobic activity. The lack of activity of 14 compared to 17 corroborates the observation that the side chain alone cannot confer aerobic or anaerobic activity in the 4-nitro series. To probe the importance of the 2-position of the imidazole further, we returned to the bicyclic core structure of PA-824

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Table 1. Aerobic, Anaerobic, and Enzymatic Activities of Nitroimidazolesa

a

NS ) not a substrate.

and replaced the oxazine oxygen with a methylene unit to give compound 35. This modification restores the rotational constraints of the parent compound, PA-824, while eliminating the electron-donating component at the 2-position. As expected, the aerobic activity of compound 35 was reduced more than 30fold compared to PA-824 and this compound showed negligible anaerobic activity. Interestingly, the olefinic precursor to 35, compound 34, generated as an intermediate following the metathesis reaction, showed comparably low aerobic activity.

While the aerobic activity was comparable, the anaerobic activity of 34 was at least 4-fold better than its saturated analogue. Together, these results indicate that the electronic nature of the 2-position substituent has profound importance for both activities of the 4-nitroimidazoles. Combined with the increase in anaerobic activity seen with the unsaturated carba analogue of PA824 (34), these results suggest that the SAR associated with aerobic activity and the SAR associated with anaerobic activity are distinct within the 4-nitroimidazoles.

4- and 5-Nitroimidazoles

SAR of the 5-Nitroimidazoles Related to Mtz. We employed a similar feature comparison in attempting to understand the SAR of the antitubercular activities of the 5-nitroimidazoles (Figure 2). Beginning with the lipophilic side chain, we found that compound 16, containing the benzyl ether of PA-824 appended to the Mtz core structure, failed to display any aerobic activity against Mtb and indeed showed a significant decrease in anaerobic activity relative to that of Mtz. This observation marks a clear distinction between the 4- and 5-nitroimidazole series. The lipophilic side chain is required for both aerobic and anaerobic activity of the 4-nitroimidazoles. Conversely, the presence of the lipophilic side chain did not confer aerobic activity and decreased anaerobic activity in the 5-nitro series. Building structural elements of PA-824 into Mtz, compound 30 has a 2-position oxygen atom. While this feature is essential for high aerobic and anaerobic activities in the 4-nitro series, this modification alone is not sufficient to convey either activity in the 5-nitro series. Indeed, 30 lost anaerobic activity relative to the parent, Mtz. We were intrigued to note the biological activity data from compound 10b, the “bicyclic” analogue of Mtz containing the 2-position oxygen atom. Compound 10b unexpectedly showed aerobic activity and weak anaerobic activity. The rigid bicyclic structure imparts both aerobic and anaerobic activity compared with compound 30. Further, this result is contrary to that found for the 4-nitro isomer 10a, which was inactive (Table 1). The addition of the side chain from PA-824 to compound 10b yields compound 11. This change improves both the aerobic and anaerobic activity of 10b. Compound 11 can also be viewed as the 5-nitro isomer of PA-824 and is only slightly less active compared to that parent. To understand the influence of the 2-position oxygen atom alone, we prepared compounds 27a/b, conformationally rigid, bicyclic forms of Mtz. This change gave one of the most striking results in this study. Comparing compounds 10b and 27a/b, we find that replacing only the 2-position oxygen atom from compound 10b with a methylene results in the loss of both aerobic and anaerobic activities. This result indicates that for 5-nitroimidazooxazines the 2-position oxygen atom is an essential requirement for both activities. As seen above, this was not found to be the case with 4-nitroimidazooxazines. Compound 35, with a 2-position methylene, retained both aerobic and anaerobic activities. Relationship between Aerobic and Anaerobic SAR. Considering all the compounds together, we can deduce that the structural requirements for aerobic and anaerobic activity for the 4- and 5-nitroimidazoles are fundamentally different. To explore this further we performed MIC and MAC determinations against both wild type Mtb and defined Mtb mutants that were selected for resistance to PA-824. PA-824 is activated by the nitroreductase Ddn (Rv3547) at the expense of cofactor F420. Mutants resistant to this drug fall into three classes. Class A mutants have lost the F420-dependent glucose 6-phosphate dehydrogenase FGD1.29 Class B mutants have disruptions in the biosynthetic genes for cofactor F420. Class C mutants have lesions in Ddn (Rv3547), the protein that reductively activates these prodrugs at the expense of reduced F420.30 Aerobically, all classes of mutants show complete cross-resistance to all of the compounds with aerobic activity described in this study, suggesting that the aerobic SAR was the result of reduction of these compounds only by Ddn (Table 1). Anaerobically, however, compound 11 (5-nitro-824) and compound 34 (the olefin precursor to the 2C analogue of PA-824) both showed significant killing of class C mutants. This result suggests that

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the anaerobic activity of these two compounds was unrelated to activation by this particular nitroreductase. Kinetics of Activation of Nitroimidazoles by Ddn. In order to better differentiate the activities of these compounds, we also directly examined the efficiency of these compounds as substrates of the F420-dependent nitroreductase Ddn (Rv3547). Table 1 shows the kcat/Km values of these compounds, including that for PA-824. These data clearly show that PA-824 is the best substrate for Ddn while Mtz is not a substrate at all. In general, the activity of these compounds as substrates of Ddn is mostly proportional to their observed aerobic killing activity. To a limited extent, both 4- and 5-nitroimidazoles are substrates for Ddn, although bicyclic 4-nitroimidazoles appear to be superior. The SAR associated with aerobic activity in these nitroimidazoles is likely to reflect their interaction with Ddn, suggesting that these compounds could be further optimized, informed by the structure of their interaction with that receptor. Anaerobic activity, however, appears to be more complicated than simple efficiency as a substrate for Ddn.28 Conclusion This study suggests that, even though the 4-nitroimidazoles represented by PA-824 are chemically related to 5-nitroimidazoles such as Mtz, the mechanism of cellular toxicity of these molecules differs substantially. By exploring the common elements of both series of molecules, we were able to both gain aerobic activity (10b) in the 5-nitroimidazoles and lose aerobic activity in the 4-nitro series (34 and 35). This work has elucidated some key structural features that are important for aerobic activity: the preference for the 4-nitroimidazole; the nature of the substituent at the 2-position of the imidazole ring; a hydrophobic substituent at the 6-position of the oxazine ring; and the conformationally rigid bicyclic system. By correlating these structural changes with whole cell activity against wild type and defined mutants resistant to PA-824, we were able to establish which structures made genuine contributions to understanding SAR. By assessing the efficiency of these compounds as substrates for Ddn, the nitroreductase that activates these prodrugs, we were able to further delineate ontarget from off-target compounds. As antibacterial development programs gradually shift toward more whole-cell based screening and lead optimization programs, these results illustrate the ease with which multiple target effects can confuse and mislead medicinal chemists. The improved understanding of the aerobic SAR resulting from these studies should facilitate the rational design and synthesis of even more potent analogues of PA824. Experimental Section Anhydrous solvents and reagents were purchased from SigmaAldrich and used as received unless otherwise stated. Melting points were obtained on an Electrothermal 9100 apparatus and are uncorrected. LC-MS analysis was conducted on an Agilent 1100 series HPLC with attached Agilent quadrupole mass analyzer model G1946 D SL with electrospray ionization in positive ion mode. LC chromatography used a Phenomenex Luna C18(2) column (2 mm × 50 mm, 3 µm) with a water/acetonitrile (each with 0.1% (v/v) formic acid) gradient using a flow rate of 0.3 mL/min. UV detection was with an Agilent diode array detector model G1315A spectrometer at 270 and 310 nm. Proton (1H) and carbon (13C) NMR spectra were recorded at 300 and 75.5 MHz, respectively, on a Varian Gemini spectrometer, using TMS or the solvent peak as an internal standard. Column chromatography was conducted using either silica gel (Geduran, 60, mesh 40-63 µm) or prepacked RediSep columns (Teledyne Isco, Inc., Lincoln, NE) on an Isco CombiFlash Optix10 instrument. Elemental analyses were performed by Atlantic Microlab, Inc. (Norcross, GA).

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HRMS analyses were performed at the W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University, New Haven, CT) or at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH. Isoniazid, rifampicin, Mtz, and methylene blue were obtained from Sigma-Aldrich. PA-824 was prepared as described in U.S. Patent 5,668,127. All stocks were made in 20 mM DMSO. Compound 5 was purchased from TimTec (Newark, DE). (S)-1-(tert-Butyldimethylsilyloxy)-3-(2-chloro-4-nitro-1H-imidazol-1-yl)propan-2-ol (7a) and (S)-1-(tert-Butyldimethylsilyloxy)-3-(2-chloro-5-nitro-1H-imidazol-1-yl)propan-2-ol (7b). A mixture of 5 (2.4 g, 16.2 mmol), 6 (4.6 g, 24.4 mmol), and anhydrous K2CO3 (0.34 g, 2.4 mmol) in absolute EtOH (16 mL) was heated in a sealed tube at 70 °C for 20 h. The solvent and excess 6 were then removed under reduced pressure. CH2Cl2 (50 mL) was added to the residue, and filtered through a bed of Celite (2 cm thick). The filtrate was then concentrated in vacuo to give a brown oil, which was purified via column chromatography (silica gel, 25-50% EtOAc in hexanes) to give 7a as a white crystalline solid (3.9 g, 66%) and 7b as a colorless oil (1.1 g, 20%). 7a: Mp ) 116.5-118.5 °C; 1H NMR (CDCl3) δ 0.10 (s, 6 H), 0.90 (s, 9 H), 2.74 (d, br, J ) 5.0 Hz, 1 H, alcohol-H), 3.61 (dd, J ) 13.5, 3.7 Hz, 1 H), 3.71 (dd, J ) 13.5, 4.2 Hz, 1 H); 13C NMR (CDCl3) δ -5.7, 17.9, 25.6, 50.6, 63.9, 69.8, 122.6, 132.5, 144.8; LC-MS m/z 336.4 [M + H+]. 7b: 1H NMR (CDCl3) δ -0.13 (s, 3 H), 0.04 (s, 3 H), 0.87 (s, 9 H), 3.60-3.64 (m, 2 H), 3.99-4.25 (m, 3 H), 7.80 (s, 1 H); 13C NMR (CDCl3) δ -4.9, 18.0, 25.8, 50.8, 63.8, 70.7, 122.4, 132.9, 145.6; LC-MS m/z 336.4 [M + H+]. 1-((2S)-3-(tert-Butyldimethylsilyloxy)-2-(tetrahydro-2H-pyran2-yloxy)propyl)-2-chloro-5-nitro-1H-imidazole (8). A mixture of 7b (0.78 g, 2.3 mmol), DHP (0.43 mL, 4.6 mmol), and PPTS (0.88 g, 3.5 mmol) in CH2Cl2 (20 mL) was stirred under inert atmosphere at room temperature for 2 days. Saturated NaHCO3 aqueous solution (20 mL) was added to the reaction mixture. The CH2Cl2 layer was separated, and the aqueous layer was extracted with CH2Cl2 (2 × 15 mL). The organic extracts and the original CH2Cl2 layer were combined, washed with brine (30 mL), and dried over MgSO4. The solvent was then removed under reduced pressure to give a palebrown oil, which was purified via column chromatography (silica gel, EtOAc/hexanes ) 3/1) to give 8 (a mixture of two diastereomers) as a colorless oil (0.68 g, 70%). 1H NMR (CDCl3) δ -0.12 (d, J ) 4.5 Hz, 3 H), -0.04 (d, J ) 1.8 Hz, 3 H), 0.85 (s, 4.5 H), 0.86 (s, 4.5 H), 1.50-1.83 (m), 3.25-3.31 (m, 1 H), 3.44 (dd, J ) 12.3, 2.1 Hz, 1 H), 3.49-3.56 (m, 1 H), 3.63 (dd, J ) 10.0, 7.0 Hz, 1 H), 3.73-3.88 (m, 2 H), 3.99-4.15 (m, 3 H), 4.20-4.32 (m, 1 H), 4.54-4.59 (m, 1 H), 7.84 (s, 0.5 H), 7.90 (s, 0.5 H); LC-MS m/z 419.5 [M + H+]. (S)-6,7-Dihydro-3-nitro-6-(tetrahydro-2H-pyran-2-yloxy)-5Himidazo[2,1-b][1,3]oxazine (9). TBAF (1.0 M in THF, 3.0 mL, 3.0 mmol) was added dropwise to a stirred solution of 8 (0.70 g, 1.6 mmol) in anhydrous THF (10 mL) at room temperature. The reaction mixture was further stirred at room temperature under inert atmosphere for 18 h. The solvent was removed under reduced pressure. Aqueous NaHCO3 saturated solution (10 mL) was added to the brown syrupy residue, and the mixture was extracted with CH2Cl2 (3 × 10 mL). The organic extracts were combined, washed with brine, dried over MgSO4, and the solvent was removed under reduced pressure to give a pale-brown syrup, which was purified via column chromatography (silica gel, EtOAc/hexanes ) 3:1 to straight EtOAc) to give 9 as a white crystalline solid (0.10 g, 23%). Mp ) 118.0-119.1 °C; 1H NMR (CDCl3) δ showed a mixture of diastereomers; LC-MS m/z 270.0 [M + H+]. (S)-6,7-Dihydro-3-nitro-5H-imidazo[2,1-b][1,3]oxazin-6-ol (10b). A mixture of 9 (0.22 g, 0.81 mmol), glacial acetic acid (4.0 mL, 69 mmol), tetrahydrofuran (2.0 mL), and H2O (1.0 mL) was heated at 60 °C for 18 h. The solvent and extra reagents were removed under reduced pressure. The residue was triturated with CH2Cl2 to give 10b as a yellow crystalline solid (0.12 g, 75%). Mp ) 174.0-175.2 °C; 1H NMR (CD3OD) δ 3.76 (dd, J ) 12.9, 3.6 Hz, 1 H), 3.98 (dd, J ) 12.9, 3.0 Hz, 1 H), 4.24 (dd, J ) 10.2, 6.6 Hz, 1 H), 4.40 (dd, J ) 10.2, 9.0 Hz, 1 H), 5.44 (dddd, J ) 12.6, 9.6,

Kim et al.

6.6, 3.3 Hz, 1 H), 7.85 (s, 1 H); 13C (CD3OD) δ 46.2, 62.9, 89.8, 115.4; LC-MS m/z 185.9 [M + H+]. (S)-6-(4-(Trifluoromethoxy)benzyloxy)-6,7-dihydro-3-nitro5H-imidazo[2,1-b][1,3]oxazine (11). At -78 °C under argon atmosphere, NaH (60% dispersion in mineral oil, 9.5 mg, 0.24 mmol) was added to a stirred solution of 10b (40.0 mg, 0.21 mmol), 4-trifluromethoxybenzyl bromide (66.0 mg, 0.26 mmol), and tetrabutylammonium iodide (3.7 mg, 0.01 mmol) in anhydrous DMF (5 mL). The mixture was then warmed to room temperature and further stirred at room temperature for 18 h. The reaction was quenched with methanol (0.5 mL). The solvent was removed under reduced pressure. CH2Cl2 (10 mL) was added to the residue and filtered to remove the inorganic salts. The CH2Cl2 solution was then concentrated in vacuo. The yellow gum residue was purified via column chromatography (0-2% MeOH in CH2Cl2) to give a yellow solid, which was further purified via preparative TLC (eluted with 5% MeOH in CH2Cl2) to give 11 as a yellow crystalline solid (10 mg, 13%). [R]D20 +7.4 (c 0.37, MeOH); 1H NMR (CDCl3) δ 3.78 (dd, J ) 11.1, 3.3 Hz, 1 H), 3.89 (dd, J ) 11.1, 3.9 Hz, 1 H), 4.27 (dd, J ) 9.9, 6.6 Hz, 1 H), 4.34 (dd, J ) 9.9, 8.7 Hz, 1 H), 4.60 (s, 2 H), 5.39-5.47 (m, 1 H), 7.18-7.31 (m, 1,4-disubstituted pattern, 4 H), 7.54 (s, 1 H); 13C (CDCl3) δ 45.5, 69.4, 73.2, 85.3, 112.7, 121.3, 129.3, 135.8, 147.5, 149.2, 156.7; LC-MS m/z 360.1 [M + H+]. 2-(2-Methyl-4-nitro-1H-imidazol-1-yl)ethanol (14). A mixture of 2-methyl-4-nitroimidazole (13, 254 mg, 2.0 mmol), 2-bromoethanol (170 µL, 2.4 mmol), potassium carbonate (0.55 g, 4.0 mmol), tetrabutylammonium iodide (15 mg, 0.04 mmol), and acetonitrile (2 mL) was stirred at 75 °C for 2 h. Silica gel (2 g) was added, and the solvent was removed under reduced pressure. The residue was purified twice via column chromatography (0-5% MeOH in CH2Cl2) to give a white solid (76 mg, 22% yield). Mp ) 126.7-127.8 °C (lit.22 130-131 °C); 1H NMR (CD3OD) δ 2.44 (s, 3 H), 3.84 (t, J ) 5.1 Hz, 2 H), 4.12 (t, J ) 5.1 Hz, 2 H), 8.07 (s, 1 H). (S)-6,7-Dihydro-6-methoxy-2-nitro-5H-imidazo[2,1-b][1,3]oxazine (15). NaH (60% dispersion in mineral oil, 24 mg, 0.60 mmol) was added to a stirred solution of 10a (93.0 mg, 0.50 mmol) and CH3I (40.5 µL, 0.65 mmol) in anhydrous DMF (2 mL) at -78 °C. The reaction mixture was then warmed to room temperature and stirred under an inert atmosphere for 18 h. The reaction was quenched with methanol (1 mL). Silica gel was added to the mixture, and the solvent was removed under reduced pressure. The residue was purified via column chromatography (silica gel, 2-5% MeOH in CHCl3) to give 15 as a yellow solid (57 mg, 57%). Mp ) 80.0-80.9 °C; 1H NMR (CDCl3) δ 3.48 (s, 3 H), 3.91-3.96 (m, 1 H), 4.14 (dt, J ) 12.9, 2.4 Hz, 1 H), 4.21 (dd, J ) 12.9, 3.9 Hz, 1 H), 4.34 (d, br, J ) 12.0 Hz, 1 H), 4.63 (ddd, J ) 12.0, 3.6, 2.1 Hz, 1 H), 7.42 (s, 1 H); 13C NMR (CDCl3) δ 47.6, 56.7, 68.3, 69.7, 117.4, 148.3; LC-MS m/z 200.1 [M + H+]. 1-(2-(4-(Trifluoromethoxy)benzyloxy)ethyl)-2-methyl-5-nitro1H-imidazole (16). NaH (60% dispersion in mineral oil, 44 mg, 1.1 mmol) was added to a stirred mixture of 1 (Mtz, 171 mg, 1.0 mmol), 4-trifluoromethoxybenzyl bromide (306 mg, 1.2 mmol), and TBAI (18 mg, 0.05 mmol) in anhydrous DMF (5 mL) at -78 °C. The reaction mixture was then warmed to room temperature and stirred at room temperature under Ar for 18 h. The reaction was quenched with MeOH (10 drops). The solvent was removed under reduced pressure. H2O was added to the residue. The mixture was extracted with CH2Cl2 (3 × 30 mL). The organic extracts were combined, washed with H2O, and dried over MgSO4. The solvent was then removed under reduced pressure to give a brown oil, which was purified via preparative TLC (eluted with 5% MeOH in CH2Cl2) to give 16 as a yellow solid (113 mg, 33%). Mp ) 64.5-65.5 °C; 1 H NMR (CDCl3) δ 2.50 (s, 3H), 3.79 (t, J ) 4.8 Hz, 2H), 4.42 (s, 2H), 4.51 (t, J ) 4.8 Hz, 2H), 7.13-7.19 (m, 4H), 7.94 (s, 1H); 13 C NMR (CDCl3) δ 14.8, 46.7, 68.9, 72.6, 121.2, 128.9, 133.3, 136.3, 149.0, 151.9; LC-MS m/z 346.1 [M + H+]. 1-(2-(4-(Trifluoromethoxy)benzyloxy)ethyl)-2-methyl-4-nitro1H-imidazole (17). NaH (60% dispersion in mineral oil, 40 mg, 1.0 mmol) was added to a stirred mixture of 14 (140 mg, 0.82

4- and 5-Nitroimidazoles

mmol), 4-trifluoromethoxybenzyl bromide (255 mg, 1.0 mmol), and TBAI (18 mg, 0.05 mmol) in anhydrous DMF (5 mL) at -78 °C. The reaction mixture was then warmed to room temperature and stirred under Ar for 18 h. The reaction was quenched with MeOH (2 drops). The solvent was removed under reduced pressure. H2O (30 mL) was added to the residue. The mixture was extracted with CH2Cl2 (3 × 25 mL). The organic extracts were combined, washed with H2O (2 × 100 mL), and dried over MgSO4. The solvent was then removed under reduced pressure to give a brown solid, which was purified via preparative TLC (eluted with EtOAc/hexanes ) 3/1) to give 17 as a yellow solid (111 mg, 39%). Mp ) 86.9-88.5 °C; 1H NMR (CDCl3) δ 1.93 (s, 3 H), 3.24 (t, J ) 5.0 Hz, 2 H), 3.62 (t, J ) 5.0 Hz, 2 H), 4.00 (s, 2 H), 6.66-6.77 (m, 1,4-disubstituted benzene pattern); 13C NMR (CDCl3) δ 13.2, 47.2, 68.5, 72.5, 120.5, 121.1, 129.0, 136.0, 145.4, 146.4, 148.8; LC-MS m/z 346.1 [M + H+]. 1-(2-(tert-Butyldimethylsilyloxy)ethyl)-2-chloro-4-nitro-1Himidazole (18) and 1-(2-(tert-Butyldimethylsilyloxy)ethyl)-2chloro-5-nitro-1H-imidazole (28). A solution of 5 (255 mg, 1.73 mmol), BrCH2CH2OTBS (605 mg, 2.53 mmol), and K2CO3 (99 mg, 0.72 mmol) in MeOH (5 mL) was heated in a sealed tube at 75 °C for 18 h. The reaction mixture was quenched with H2O and extracted with EtOAc (2×). The combined organic layers were dried with MgSO4, filtered, and concentrated. Chromatography on silica gel (EtOAc/hexanes ) 1/1) afforded 18 as a white solid (130 mg, 0.424 mmol, 25%) and 28 as a white solid (19 mg, 0.061 mmol, 3.5%). 18: Mp ) 87.5-88.6 °C; 1H NMR (CDCl3) δ -0.07 (s, 6H), 0.78 (s, 9H), 3.86 (t, J ) 4.8 Hz, 2H), 4.13 (t, J ) 4.8 Hz, 2H), 7.83 (s, 1H); 13C NMR (CDCl3) δ -5.9, 17.9, 25.5, 50.0, 60.8, 121.9, 132.2, 145.2; HRMS (ESMS) calcd for C11H21ClN3O3Si [M + H+] 306.1041, found 306.1035; presence of 2D-NOESY correlationships, aromatic H with CH2, λmax ) 300 nm. 28: Mp ) 47.0-47.8 °C; 1H NMR (CDCl3) δ -0.07 (s, 6H), 0.80 (s, 9H), 3.89 (t, J ) 5.3 Hz, 2H), 4.60 (t, J ) 5.3 Hz, 2H), 7.91 (s, 1H); 13 C NMR (CDCl3) δ -5.8, 18.0, 25.6, 49.2, 61.1, 132.1, 138.8, 139.1; HRMS (ESMS) calcd for C11H21ClN3O3Si [M + H+] 306.1041, found 306.1035; absence of 2D-NOESY correlationships, aromatic H with 2 CH2, λmax ) 310 nm. 2-(2-Methoxy-4-nitro-1H-imidazol-1-yl)ethanol (19). To a solution of 18 (438 mg, 1.43 mmol) in MeOH (10 mL) was added NaOMe (619 mg, 11.5 mmol) at room temperature. The reaction mixture was stirred at room temperature for 24 h and concentrated. Chromatography on silica gel (MeOH/CH2Cl2 ) 1/20) afforded 19 as a white solid (164 mg, 0.874 mmol, 61%). Mp ) 132-133 °C; 1 H NMR (MeOH-d4) δ 3.79 (t, J ) 5.3 Hz, 2H), 3.97 (t, J ) 5.3 Hz, 2H), 4.07 (s, 3H), 7.85 (s, 1H); 13C NMR (MeOH-d4) δ 48.7, 58.6, 61.0, 120.0, 143.5, 152.9; HRMS (ESMS) calcd for C6H10N3O4 [M + H+] 188.0671, found 188.0688. 1-(2-(4-(Trifluoromethoxy)benzyloxy)ethyl)-2-methoxy-4-nitro-1H-imidazole (20). A mixture of 19 (19.6 mg, 0.105 mmol), 4-trifluoromethoxybenzyl bromide (40.1 mg, 0.157 mmol), and n-Bu4NI (4.6 mg, 0.012 mmol) in DMF (1 mL) was prepared under argon atmosphere. To this mixture 60% NaH (5.6 mg, 0.14 mmol) was added at -78 °C. The reaction mixture was stirred at -78 °C for 30 min and then at room temperature for 14 h, quenched with H2O, and extracted with EtOAc (2×). The combined organic layers were dried with MgSO4, filtered, and concentrated. Chromatography on silica gel (EtOAc/hexanes ) 2/1) afforded 20 as a slightly yellow oil (22 mg, 0.061 mmol, 58%). 1H NMR (CDCl3) δ 3.67 (t, J ) 5.0 Hz, 2H), 3.99 (t, J ) 5.0 Hz, 2H), 4.04 (s, 3H), 4.48 (s, 2H), 7.14 (d, J ) 8.4 Hz, 2H), 7.23 (d, J ) 8.4 Hz, 2H), 7.57 (s, 1H); 13 C NMR (CDCl3) δ 44.8, 57.7, 67.5, 72.2, 117.9, 118.6, 120.9, 122.0, 128.8, 135.9, 142.5, 148.6, 150.7; HRMS (ESMS) calcd for C14H15F3N3O5 [M + H+] 362.0964, found 362.0972. (R)-2-Bromo-1-(2-(tert-butyldimethylsilyloxy)but-3-enyl)-5-nitro-1H-imidazole (23a) and (R)-1-(2-Bromo-4-nitro-1H-imidazol-1-yl)but-3-en-2-ol (31). A mixture of 21 (2.0 g, 10.4 mmol), (R)-vinyloxirane (22, 1.00 g, 13.5 mmol), and K2CO3 (560 mg, 4.05 mmol) in MeOH (10 mL) was heated in a sealed tube at 70 °C for 14 h. The reaction mixture was quenched with H2O and extracted with EtOAc (3×). The combined organic layers were dried

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1325

with anhydrous MgSO4, filtered, and concentrated. To the crude organic mixture (2.55 g) in CH2Cl2 (50 mL) was added TBSCl (2.93 g, 19.5 mmol) and imidazole (1.33 g, 19.5 mol) at room temperature. The reaction mixture was stirred at room temperature for 1 h, quenched with H2O, and extracted with CH2Cl2 (2×). The combined organic layers were dried with anhydrous MgSO4, filtered, and concentrated. Chromatography on silica gel (EtOAc/hexanes ) 1/2 to 1/1) afforded (R)-23a (611 mg, 1.62 mmol, 16%) as a slightly yellow oil in addition to the 4-NO2 desTBS product (31, 1.25 g, 4.76 mmol, 48%) as a viscous yellow oil. (R)-23a: [R]D20 -26.5 (c 1.89, CHCl3); 1H NMR (CDCl3) δ 0.01 (s, 6H), 0.83 (s, 9H), 3.90-4.00 (m, 2H), 4.93-4.98 (m, 1H), 5.26 (dd, J ) 17.3, 1.4 Hz, 1H), 5.44 (dd, J ) 10.4, 1.4 Hz, 1H), 5.98 (ddd, J ) 17.3, 10.4, 6.0 Hz, 1H), 7.92 (s, 1H); 13C NMR (CHCl3) δ -5.75, -5.67, 18.0, 25.6, 62.5, 64.2, 119.5, 120.4, 120.7, 131.4, 147.3; HRMS (ESMS) calcd for C13H23BrN3O3Si [M + H+] 376.0692, found 376.0638. 31: [R]D20 -6.86 (c 1.69, CHCl3); 1H NMR (CDCl3) δ 3.95-4.03 (m, 1H), 4.16 (dd, J ) 14.4, 3.3 Hz, 1H), 4.49-4.57 (m, 1H), 5.27 (d, J ) 10.5 Hz, 1H), 5.39 (d, J ) 17.1 Hz, 1H), 5.86 (ddd, J ) 16.8, 10.8, 6.0 Hz, 1H), 7.98 (s, 1H); 13C NMR (CDCl3) δ 53.8, 71.2, 118.9, 120.7, 123.3, 135.9, 146.6; HRMS (ESMS) calcd for C7H9N3O3Br [M + H+] 261.9827, found 261.9829. (S)-2-Bromo-1-(2-(tert-butyldimethylsilyloxy)but-3-enyl)-5-nitro-1H-imidazole (23b). This compound was prepared in the same manner as 23a, using (S)-vinyloxirane. Yield: 23b (15%) and the 4-NO2-desTBS product (45%). 23b: [R]D20 +23.7 (c 0.58, CHCl3); 1 H NMR (CDCl3) δ -0.04 (s, 6H), 0.79 (s, 9H), 3.88-3.98 (m, 2H), 4.90-4.98 (m, 1H), 5.23 (d, J ) 17.3, 1H), 5.40 (d, J ) 10.4, 1H), 5.94 (ddd, J ) 17.3, 10.4, 6.0 Hz, 1H), 7.92 (s, 1H); 13C NMR (CDCl3) δ -5.9, -5.9, 17.9, 25.5, 62.4, 64.1, 119.5, 120.4, 120.6, 131.3, 147.2; HRMS (ESMS) calcd for C13H22BrN3O3Si [M + H+] 376.0692, found 376.0704. (R)-1-(2-(tert-Butyldimethylsilyloxy)but-3-enyl)-5-nitro-2-vinyl1H-imidazole (24a). A mixture of (R)-23a (300 mg, 0.797 mmol), tributylvinylstannane (0.255 mL, 0.874 mmol), Pd(PPh3)4 (37 mg, 0.032 mmol), and LiCl (33 mg, 0.80 mmol) in DMF (2 mL) was heated under microwave at 120 °C for 10 min. The reaction mixture was concentrated under vacuum. Chromatography on silica gel (EtOAc/hexanes ) 1/2) afforded (R)-24a (144 mg, 0.446 mmol, 1 56%) as a slightly yellow oil. [R]20 D -13.7 (c 0.37, CHCl3); H NMR (CDCl3) δ -0.06 (s, 3H), -0.05 (s, 3H), 0.78 (s, 9H), 3.87 (dd, J ) 10.8, 6.6 Hz, 1H), 3.97 (dd, J ) 10.8, 4.1 H, 1H), 4.84-4.90 (m, 1H), 5.18 (dd, J ) 17.4, 1.5 Hz, 1H), 5.39 (dd, J ) 10.4, 1.5 Hz, 1H), 5.56 (dd, J ) 10.8, 1.5 Hz, 1H), 5.95 (ddd, J ) 17.4, 10.8, 5.7 Hz, 1H), 6.36 (dd, J ) 17.1, 1.5 Hz, 1H), 6.55 (dd, J ) 17.1, 10.8 Hz, 1H), 7.85 (s, 1H); 13C NMR (CHCl3) δ -5.84, -5.81, 17.9, 25.5, 60.3, 64.7, 118.6, 120.1, 121.3, 123.2, 132.0, 144.9, 147.1; HRMS (ESMS) calcd for C15H26N3O3Si [M + H+] 324.1743, found 324.1745. (S)-1-(2-(tert-Butyldimethylsilyloxy)but-3-enyl)-5-nitro-2-vinyl1H-imidazole (24b). This compound was prepared in the same manner as (R)-24a. Yield, (S)-24b (53%) as a slightly yellow oil; [R]D20 +14.6 (c 2.23, CHCl3); 1H NMR (CDCl3) δ -0.04 (s, 3H), -0.03 (s, 3H), 0.80 (s, 9H), 3.85-3.91 (m, 2H), 4.82-4.92 (m, 1H), 5.20 (dd, J ) 17.4, 1.5 Hz, 1H), 5.41 (dd, J ) 10.5, 1.5 Hz, 1H), 5.59 (dd, J ) 10.5, 1.5 Hz, 1H), 5.91-6.02 (m, 1H), 6.39 (dd, J ) 17.1, 1.5 Hz, 1H), 6.52-6.61 (m, 1H), 7.86 (s, 1H); 13C NMR (CHCl3) δ -5.79, -5.75, 18.0, 25.5, 60.3, 64.7, 118.6, 120.2, 121.3, 123.3, 132.0, 144.9, 147.2; HRMS (ESMS) calcd for C15H26N3O3Si [M + H+] 324.1743, found 324.1749. (R)-6-(tert-Butyldimethylsilyloxy)-3-nitro-5,6-dihydroimidazo[1,2-a]pyridine (25a). To a solution of (R)-24a (81 mg, 0.25 mmol) in CH2Cl2 (10 mL) was added Hoveyda-Grubbs second generation catalyst (22 mg, 0.035 mmol). The reaction mixture was stirred at 60 °C for 2.5 h. Chromatography on preparative TLC (EtOAc/hexanes ) 1/1) afforded (R)-25a (54 mg, 0.18 mmol, 72%) as a slightly brown solid. [R]D20 -17.8 (c 0.48, CHCl3); mp ) 106-108 °C; 1H NMR (CDCl3) δ 0.09 (d, J ) 0.9 Hz, 6H), 0.92 (s, 9H), 3.50 (dd, J ) 10.5, 9.0 Hz, 1H), 4.11 (dd, J ) 10.5, 4.2 Hz, 1H), 4.82-4.87 (m, 1H), 6.76 (s, 2H), 8.06 (s, 1H); 13C NMR

1326 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

(CHCl3) δ -5.3, -5.2, 18.4, 26.0, 63.9, 66.4, 118.0, 122.9, 132.3, 138.9, 153.7; HRMS (ESMS) calcd for C13H22N3O3Si [M + H+] 296.1430, found 296.1423. (S)-6-(tert-Butyldimethylsilyloxy)-3-nitro-5,6-dihydroimidazo[1,2-a]pyridine (25b). This compound was prepared in the same manner as (R)-25a. Yield: (S)-25b (72%) as a slightly brown solid. 1 [R]20 D +19.9 (c 1.36, CHCl3); mp ) 108-109 °C; H NMR (CDCl3) δ 0.09 (d, J ) 0.9 Hz, 6H), 0.92 (s, 9H), 3.50 (dd, J ) 10.5, 9.0 Hz, 1H), 4.11 (dd, J ) 10.5, 4.2 Hz, 1H), 4.82-4.87 (m, 1H), 6.76 (s, 2H), 8.06 (s, 1H); 13C NMR (CHCl3) δ -5.6, -5.5, 18.1, 25.7, 63.3, 66.2, 117.8, 122.5, 138.8, 150.3, 153.7; HRMS (ESMS) calcd for C13H22N3O3Si [M + H+] 296.1430, found 296.1429. (R)-6-(tert-Butyldimethylsilyloxy)-3-nitro-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine (26a). A solution of (R)-25a(58 mg, 0.20 mmol) in a t-BuOH (1.5 mL) and THF (0.5 mL) was prepared and degassed by freezing and thawing (3×). To the mixture was added Wilkinson’s catalyst (17 mg, 0.018 mmol) and degassed by freezing and thawing (1×). Under a hydrogen balloon atmosphere the reaction mixture was stirred at room temperature for 14 h. Chromatography on preparative TLC (EtOAc/hexanes ) 1/1) afforded (R)-26a (34 mg, 0.11 mmol, 57%) as a slightly yellow solid in addition to the starting material ((R)-25a, 22 mg, 0.074 mmol, 37%). [R]D20 0.00 (c 0.31, CHCl3); mp ) 68.0-68.9 °C; 1H NMR (CDCl3) δ 0.04 (s, 3H), 0.06 (s, 3H), 0.88 (s, 9H), 2.23-2.34 (m, 1H), 2.64-2.76 (m, 1H), 2.82-3.06 (m, 2H), 3.66 (dd, J ) 10.8, 7.5 Hz, 1H), 3.89 (dd, J ) 10.8, 3.6 Hz, 1H), 4.39-4.47 (m, 1H), 7.81 (s, 1H); 13C NMR (CHCl3) δ -5.6, -5.5, 18.4, 23.1, 25.7, 27.9, 60.1, 65.4, 115.7, 128.6, 132.0, 152.9; HRMS (ESMS) calcd for C13H24N3O3Si [M + H+] 298.1587, found 298.1594. (S)-6-(tert-Butyldimethylsilyloxy)-3-nitro-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine (26b). This compound was prepared in the same manner as (R)-26a. Yield: (S)-26b (55%) as a slightly yellow solid in addition to the starting material ((S)-25b, 12 mg, 0.039 mmol, 42%). [R]D20 +0.01 (c 0.75, CHCl3); mp ) 67.2-68.0 °C; 1 H NMR (CDCl3) δ 0.03 (s, 3H), 0.05 (s, 3H), 0.88 (s, 9H), 2.23-2.34 (m, 1H), 2.63-2.75 (m, 1H), 2.84-3.02 (m, 2H), 3.66 (dd, J ) 10.8, 7.5 Hz, 1H), 3.89 (dd, J ) 10.8, 3.6 Hz, 1H), 4.39-4.47 (m, 1H), 7.81 (s, 1H); 13C NMR (CHCl3) δ -5.61, -5.56, 18.1, 23.0, 25.7, 27.9, 60.0, 65.4, 115.7, 150.5, 152.8; HRMS (ESMS) calcd for C13H24N3O3Si [M + H+] 298.1587, found 298.1595. (R)-5,6,7,8-Tetrahydro-3-nitroimidazo[1,2-a]pyridin-6-ol (27a). To a solution of (R)-26a (22 mg, 0.074 mmol) in THF (1 mL) was added 1.0 M TBAF in THF (0.10 mL, 0.10 mmol) at room temperature. The reaction mixture was stirred at room temperature for 10 min. Chromatography on preparative TLC (MeOH/EtOAc ) 1/9) afforded (R)-27a (9.4 mg, 0.051 mmol, 69%) as a white solid. [R]D23 -10.7 (c 0.43, MeOH); mp ) 163.5-164.1 °C; 1H NMR (MeOH-d4) δ 2.34-2.45 (m, 1H), 2.66-2.78 (m, 1H), 2.83-3.02 (m, 2H), 3.66 (dd, J ) 12.0, 6.3 Hz, 1H), 3.92 (dd, J ) 11.6, 3.5 Hz, 1H), 4.47-4.54 (m, 1H), 8.09 (s, 1H); 13C NMR (MeOH-d4) δ 23.9, 29.1, 61.9, 64.6, 117.8, 151.2, 155.2; HRMS (ESMS) calcd for C7H10N3O3 [M + H+] 184.0722, found 184.0724. (S)-5,6,7,8-Tetrahydro-3-nitroimidazo[1,2-a]pyridin-6-ol (27b). This compound was prepared in the same manner as 27a. Yield: (S)-27b (91%) as a white solid. [R]D20 +8.3 (c 0.29, MeOH); mp ) 159.8-161.0 °C; 1H NMR (MeOH-d4) δ 2.34-2.46 (m, 1H), 2.63-2.80 (m, 1H), 2.82-3.02 (m, 2H), 3.66 (dd, J ) 12.0, 6.3 Hz, 1H), 3.92 (dd, J ) 11.7, 3.3 Hz, 1H), 4,47-4.53 (m, 1H), 8.09 (s, 1H); 13C NMR (MeOH-d4) δ 24.0, 29.1, 61.9, 64.9, 117.8, 151.2, 155.2; HRMS (ESMS) calcd for C7H10N3O3 [M + H+] 184.0722, found 184.0723. 1-(2-(tert-Butyldimethylsilyloxy)ethyl)-2-methoxy-5-nitro-1Himidazole (29). To a solution of 28 (29 mg, 0.095 mmol) was added NaOMe (50 mg, 0.93 mmol) at room temperature. The reaction mixture was stirred at room temperature for 15 h and concentrated. Chromatography on silica gel (EtOAc/hexanes ) 1/1) afforded 29 as a slightly yellow oil (17 mg, 0.055 mmol, 58%) in addition to 30 (2.7 mg, 0.014 mmol, 15%). 1H NMR (CDCl3) δ -0.07 (s, 6H), 0.80 (s, 9H), 3.89 (t, J ) 5.3 Hz, 2H), 4.60 (t, J ) 5.3 Hz, 2H), 7.91 (s, 1H); 13C NMR (CDCl3) δ -5.8, 18.0, 25.6, 46.2, 57.3,

Kim et al.

61.1, 131.0, 135.3, 155.4; HRMS (ESMS) calcd for C12H24N3O4Si [M + H+] 302.1536, found 302.1538. 2-(2-Methoxy-5-nitro-1H-imidazol-1-yl)ethanol (30). To a solution of 29 (17 mg, 0.055 mmol) in THF (1 mL) was added 1.0 M TBAF in THF (0.070 mL, 0.070 mmol) at room temperature. The reaction mixture was stirred at room temperature for 10 min and concentrated. Chromatography with preparative TLC gave 30 as a white solid (7.1 mg, 0.038 mmol, 69%). Mp ) 99.0-99.8 °C; 1 H NMR (CDCl3) δ 3.91 (t, J ) 5.6 Hz, 2H), 4.15 (s, 3H), 4.40 (t, J ) 5.6 Hz, 2H), 7.78 (s, 1H); 13C NMR (CDCl3) δ 46.3, 57.9, 61.3, 131.5, 135.5, 155.4; LC-MS m/z 188.1 [M + H+]. 1-((R)-2-(4-(Trifluoromethoxy)benzyloxy)but-3-enyl)-2-bromo4-nitro-1H-imidazole (32). A mixture of 31 (166 mg, 0.633 mmol), 4-trifluoromethoxybenzyl bromide (250 mg, 0.95 mmol), and n-Bu4NI (23 mg, 0.063 mmol) in DMF (5 mL) was prepared under argon atmosphere. To this mixture 60% NaH (30 mg, 0.76 mmol) was added at -78 °C. The reaction mixture was stirred at 0 °C for 30 min and then at room temperature for 14 h, quenched with H2O, and extracted with EtOAc (2×). The combined organic layers were dried with MgSO4, filtered, and concentrated. Chromatography on silica gel (EtOAc/hexanes ) 3/4) afforded 32 as a slightly yellow oil (171 mg, 0.392 mmol, 62%). [R]D20 -18.9 (c 1.95, CHCl3); 1H NMR (CDCl3) δ 4.26-4.40 (m, 3H), 4.48 (d, J ) 12.0 Hz, 1H), 4.78 (d, J ) 12.0 Hz, 1H), 5.58-5.63 (m, 2H), 5.87-5.98 (m, 1H), 7.31 (d, J ) 8.7 Hz, 2H), 7.38 (d, J ) 8.7 Hz, 2H), 8.09 (s, 1H); 13C NMR (CDCl3) δ 52.3, 69.4, 77.6, 118.4, 120.4, 120.7, 121.1, 121.9, 122.9, 128.8, 133.0, 135.8, 146.6, 148.46, 148.48; HRMS (ESMS) calcd for C15H14BrF3N3O4 [M + H+] 436.0120, found 436.0132. 1-((R)-2-(4-(Trifluoromethoxy)benzyloxy)but-3-enyl)-4-nitro2-vinyl-1H-imidazole (33). A mixture of 32 (741 mg, 1.70 mmol), tributyl(vinyl)stannane (0.55 mL, 1.9 mmol), Pd(PPh3)4 (79 mg, 0.068 mmol), and LiCl (72 mg, 1.70 mmol) in DMF (5 mL) was prepared. The reaction mixture was heated under microwave at 120 °C for 15 min. The reaction mixture was concentrated under vacuum. Chromatography on silica gel (EtOAc/hexanes ) 1/3-1/1) afforded 33 as a slightly yellow oil (590 mg, 1.54 mmol, 91%). [R]D20 -13.0 (c 0.59, CHCl3); 1H NMR (CDCl3) δ 3.96-4.13 (m, 3H), 4.25 (d, J ) 12.0 Hz, 1H), 4.57 (d, J ) 12.3 Hz, 1H), 5.37-5.46 (m, 2H), 5.56 (d, J ) 10.8 Hz, 1H), 5.65-5.76 (d, J ) 10.8 Hz, 1H), 6.37 (dd, J ) 17.1, 1.5 Hz, 1H), 6.53 (dd, J ) 17.1, 11.1 Hz, 1H), 7.08-7.17 (m, 4H), 7.74 (s, 1H); 13C NMR (CDCl3) δ 50.8, 69.6, 78.5, 118.6, 120.9, 121.0, 121.2, 121.4, 122.0, 123.2, 129.0, 133.4, 135.7, 144.8, 146.9, 148.76, 148.78; HRMS (ESMS) calcd for C17H17F3N3O4 [M + H+] 384.1171, found 384.1167. (R)-6-(4-(Trifluoromethoxy)benzyloxy)-5,6-dihydro-2-nitroimidazo[1,2-a]pyridine (34). A mixture of 33 (560 mg, 1.54 mmol) and Grubbs’ second generation catalyst (130 mg, 0.15 mmol) in CH2Cl2 (5 mL) was heated in a sealed tube at 55 °C for 15 h. The reaction mixture was concentrated under vacuum. Chromatography on silica gel (EtOAc/hexanes ) 1/1) afforded 34 as a gray-white solid (320 mg, 0.901 mmol, 59%). [R]D20 -189.6 (c 0.51, CHCl3); mp ) 101.7-102.8 °C; 1H NMR (CDCl3) δ 4.14-4.22 (m, 1H), 4.34-4.42 (m, 2H), 4.59 (s, 2H), 6.43-6.48 (m, 1H), 6.65 (d, J ) 10.2 Hz, 1H), 7.14 (d, J ) 8.4 Hz, 2H), 7.30 (d, J ) 8.7 Hz, 2H), 7.77 (s, 1H); 13C NMR (CDCl3) δ 48.3, 67.6, 69.7, 118.5, 120.2, 120.7, 120.9, 121.9, 129.0, 130.2, 135.8, 141.0, 147.3, 148.7; HRMS (ESMS) calcd for C15H13F3N3O4 [M + H+] 356.0858, found 356.0851. (R)-6-(4-(Trifluoromethoxy)benzyloxy)-5,6,7,8-tetrahydro-2nitroimidazo[1,2-a]pyridine (35). A solution of 34 (144 mg, 0.405 mmol) in THF/t-BuOH (2 mL:6 mL) was degassed by freezing and thawing (3×). To this mixture was added Wilkinson’s catalyst (72 mg, 0.078 mmol) at room temperature. The reaction mixture was degassed by freezing and thawing (1×). The reaction mixture was stirred at room temperature under a H2(g) balloon for 16 h and concentrated under vacuum. Chromatography on silica gel (EtOAc/hexanes ) 1/1) afforded 35 as a yellow oil (73 mg, 0.20 mmol, 50%). [R]D20 -13.2 (c 1.8, CHCl3); 1H NMR (CDCl3) δ 1.95-2.06 (m, 1H), 2.27-2.36 (m, 1H), 2.84-3.08 (m, 2H),

4- and 5-Nitroimidazoles

4.07-4.19 (m, 3H), 4.55 (d, J ) 12.0 Hz, 1H), 4.63 (d, J ) 11.7 Hz, 1H), 7.16 (d, J ) 8.7 Hz, 2H), 7.31 (d, J ) 8.7 Hz, 2H), 7.61 (s, 1H); 13C NMR (CDCl3) δ 19.7, 24.2, 49.6, 69.6, 69.8, 118.6, 118.7, 121.0, 122.0, 128.8, 136.0, 144.1, 147.2, 148.7; HRMS (ESMS) calcd for C16H15F3N3O6 [M + HCO2-] 402.0913, found 402.0929. Mtb MIC Assay. A stock culture of Mtb H37Rv (ATCC 27294) was grown to OD 0.5 in Middlebrook 7H9 broth (Difco) supplemented with 0.05% Tween-80, 0.2% glycerol, and albumin/NaCl/ glucose (ADC) complex. The culture was diluted 1:1000 in 7H9based medium before aliquoting 50 µL into each well of a 96-well plate. The inhibitors were dissolved in DMSO to make stock solutions of 50 µmol/mL. Inhibitors were added to the first row of wells of the 96-well plate with 100 µL of 7H9-based medium. After pipet mixing and use of a multichannel pipet, 50 µL was removed from each well in the first row and added to the second row. 2-Fold dilution in this manner was carried out to give eight dilutions of each inhibitor (1000-7.8 µM). The plates were incubated for 2 weeks at 37 °C, and the MIC99 values were read macroscopically using an inverted plate reader. Each measurement was made three independent times. Minimum Anaerobicidal Concentration Estimation Experiment. For oxygen depletion assays, an early log phase Mtb culture in glycerol free Dubos broth was diluted 100-fold and 20 mL was transferred to tubes (Pyrex 16 mm × 125 mm culture tubes) to maintain a head space ratio of 0.5 as described previously.31 The tubes were sealed with paraplast and incubated for 20 days under uniform stirring at 180 rpm using magnetic stirring bars. 1.5 µg/ mL of methylene blue was added to a reference tube to visualize oxygen depletion. 100 µL of Mtb NRP-2 stage cells was exposed to various concentrations of drugs in a 96-well microplate. 2-Fold drug dilutions in DMSO (1.95-500 µM) and a DMSO control were used. Handling of NRP-2 cells was done in a vinyl anaerobic chamber (Coy Laboratories, MI) fitted with a Coy model 10 gas analyzer and a vacuum air lock chamber. The anaerobic chamber was maintained under 90% nitrogen and 10% hydrogen. 96-Well plates were placed in a type A biobag anaerobic chamber (Becton Dickinson, MD) along with an oxygen indicator strip and incubated at 37 °C for 7 days. After drug exposure, the cells were washed three times with fresh Dubos broth and serially diluted by 2-fold dilutions. The minimal anaerobicidal concentration (MAC) against Mtb was estimated by measuring the number of viable bacilli in each dilution after drug treatment as follows: 5 µL of cell suspension from each well was spotted in a single well of a 7H11 agarcontaining 96-well plate. After 3 weeks of incubation at 37 °C, the MAC was read as the concentration of drug causing a 90% reduction in visible growth. Expression and Purification of Mtb-Rv3547 in E. coli. The Mtb-Rv3547 genomic PCR fragment was amplified using XbaIFP (5′-cga ccc ttg gtc tct aga atc agc gtc atg ccg-3′) and D3-RP (5′-tgg ctt cac gtt cgg caa gct tgg gaa cgg tca-3′). The PCR fragment was digested with XbaI and HindIII and was cloned in a pMALc2x vector digested with the same restriction enzymes for expression of Mtb-Rv3547 with the N-terminal MBP tag. Rv3547 DNA sequencing in pMAL-c2x-Rv3547 was performed using malE-FP (NEB no. S12375) and M13 seq (NEB no. S12115) primers. MBPtagged Rv3547 was reproducibly expressed in E. coli turbo cells (NEB). Protein induction at 37 °C with 300 µM IPTG yielded Rv3547-MBP protein in the cytosol. The fusion protein was purified to homogeneity using amylose resin affinity column chromatography (NEB no. E8021) per the manufacturer’s instructions. Nitroreductase Assay. F420 purified from Methanobacterium thermoautotrophicum Marburg was a kind gift from Lacy Daniels, Texas A&M University, Kingsville, TX. Reduced F420 (F420H2) was prepared using a recombinant Mtb glucose 6-phosphate dehydrogenase as described,29 followed by heat inactivation of FGD1 at 55 °C water bath for 15 min and ultrafiltration (Centricon 10 kDa). Reduction of 1 or other nitroimidazoles by Rv3547 was monitored by oxidation of F420H2 at 400 nm using a Cary UV spectrophotometer at 25 °C. Initial rates were calculated on the basis of the gain of absorbance at 400 nm due to oxidation of F420 (ε400 ) 25.71

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1327

mM-1 cm-1). Reactions were performed in phosphate buffer, pH 7.4, 50 µM F420, 0.6 µM Rv3457, and varying amounts of PA-824 and analogues. Apparent steady-state kinetic parameters, Km and Vmax, were obtained by fitting initial rate data to the MichaelisMenten equation.

Acknowledgment. This work was funded (in part) by the intramural research program of National Institute of Allergy and Infectious Diseases, NIH, and (in part) by a grant from the Bill and Melinda Gates Foundation and the Wellcome Trust through the Grand Challenges in Global Health Initiative. We thank Dr. Lacy Daniels (Texas A&M University Health Science Center) for F420 and Michael Goodwin (TRS, NIAID, NIH), John Lloyd (NIDDK, NIH), and Wesley White (NIDDK, NIH) with assistance in obtaining analytical data. Supporting Information Available: Experimental procedures for the preparation of desnitro-824 (12) and purity information for target compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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