PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Bioorg Med Chem. Author manuscript; available in PMC 2010 May 15.
Published in final edited form as:
PMCID: PMC2756498
NIHMSID: NIHMS109116

Discovery, Synthesis, and Biological Evaluation of Piperidinol analogs With Anti-tuberculosis Activity

Abstract

Direct anti-tuberculosis screening of commercially available compound libraries identified a novel piperidinol with interesting anti-tuberculosis activity and drug like characteristics. To generate a structure activity relationship about this hit a 22 member optimization library was generated using parallel synthesis. Products of this library 1-((R)-3-(4-chlorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl) phenyl)piperidin-4-ol and 1-((S)-3-(4-(trifluoromethyl) phenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl) phenyl) piperidin-4-ol demonstrated good anti-tuberculosis activity. Unfortunately, side effects were observed upon in vivo anti-tuberculosis testing of these compounds precluding their further advancement, which may be in part due to the secondary pharmacology associated with the aryl piperidinol core.

Introduction

Tuberculosis continues to pose a serious world health threat for which there is an urgent need to develop new therapeutic options.1 In our ongoing effort to discover novel anti-tuberculosis therapeutics we have been performing primary anti-tuberculosis screening of commercially available libraries. This effort produced a piperidinol hit (1 in Figure 1). The anti-tuberculosis activity of 1 was confirmed by resynthesis and biological reevaluation showing good anti-tuberculosis activity with an MIC 1.5 μg/mL. Compound 1 was then selected for further investigation because: (i) it is structurally similar to existing orally bioavailable drugs haloperidol and penfluridol2, 3 shown in Figure 1; (ii) there has been reported bioactivity of a number of compounds bearing 4-chloro-3-trifluoromethylphenyl-4-piperidinol scaffold as antivirals4, antibacterials,5 mu receptor agonists6, and nociceptin (N/OFQ) receptor ligands7 suggesting that it is a biologically useful scaffold for a variety of targets; (iii) there are no reports of similar compounds with anti-tuberculosis activity and thus 1 represents a potentially novel scaffold or pharmacophore for anti-tuberculosis activity, which is unlikely to be cross resistant to existing tuberculosis therapies. To this end, we became interested in the synthesis and evaluation of a hit optimization library about compound 1 in an effort to obtain a preliminary structure-activity relationship (SAR). In this manuscript, the design and synthesis of a series of analogs related to 1 is reported and the resulting anti-tuberculosis SAR is discussed.

Figure 1
Chemical structures of piperidinol library hit 1, haloperidol, and penfluridol.

Chemistry

The chemistry to make racemic and chiral aryl piperidinol analogs is shown in Schemes 1 and and2,2, respectively. 8, 9 The synthesis of racemic products 1 and 2 starts from commercially available piperidinol and racemic epoxide starting materials. Epoxide ring opening by piperidine afforded diol product 1 and 2 in 75 and 79% yield, respectively. (Scheme 1) The synthesis of the chiral piperidinol compounds 4a-p was first performed by reacting optically active epoxide intermediate (S)-(+)-epichlorohydrin or (R)-(-)-epichlorohydrin with a substituted phenol or thiophenol in acetonitrile under reflux in the presence of cesium carbonate to afford chiral epoxide derivative 3.8 The yield of this reaction was limited by a competing epoxide ring opening with the phenoxide nucleophile.10, 11 However, the bis-substituted byproducts12 (scheme 2) were most easily removed by column chromatography purification in the next step, therefore the crude product intermediate 3 was used directly without further purification. The crude preparation of 3 was reacted with 4-[4-chloro-3-(trifluoromethyl)-phenyl]-4-piperidinol in ethanol under reflux to afford alcohol diols 4a-p in 19-50% two step overall yield after purification by silica gel column chromatography (Scheme 2).9 Based on the biological data of the first series, a second set of compounds was designed and synthesized to evaluate changes to the 4-Cl, 3-CF3 phenyl piperidinol core of 4b in an effort to gain further understanding of the A and B-ring SAR, compound 4b was selected for further optimization because it is the most active from the first series and has the highest therapeutic index. As outlined in scheme 3, the same synthetic strategy was employed as in the synthesis of 4a-p except that starting materials bearing different piperidine scaffolds were used. Optically active epoxide (S)-(+)-epichlorohydrin was firstly reacted with 4-chlorophenol in the presence of cesium carbonate to give the chiral epoxide derivative, which was subsequently treated with a panel of 4-piperidines in ethanol under reflux to afford alcohol 5a-e, in overall yield 37-57% after purification by silica gel chromatogrpahy. (Scheme 3).

Scheme 1
Synthesis of piperidinol racemate analogs 1 and 2
Scheme 2
Synthesis of optically active piperidinol analogs 4a-p
Scheme 3
Synthesis of optically active piperidinol analogs 5a-e

Biological activity

All derivatives were evaluated for anti-tuberculosis activity and the results are described in Tables 1 & 2. In general, compounds 1, 2, and 4a-p demonstrated anti-tuberculosis activity with minimum inhibitory concentrations (MICs) ranging from 1.4 to 18.8 μg/mL (Table 1). The most active compounds are: 4b (MIC 1.4 μg/mL) with R stereochemistry at the central secondary hydroxyl group and chloro substitution at para position to the aryl C-ring; and 4m (MIC 1.7 μg/mL) with S stereochemistry at the central secondary hydroxyl group and trifluoromethyl group at para position of the aryl C-ring. In addition, among eight pairs of enantiomers synthesized, the R isomers 4b, 4j, and 4l of three pairs of enantiomers showed more potent anti-tuberculosis activity than their corresponding S isomers, whilst in the remaining five pairs of enantiomers, the S isomers demonstrated approximately equal (4c and 4o) or more than one fold active (4e, 4g, and 4m) anti-tuberculosis activity compared with their corresponding R isomers. Based on these data, there appears to be no correlation or preference between stereochemistry at the central secondary hydroxyl group and anti-tuberculosis activity. The SAR with respect to the phenoxy C ring was clearer with 4-chloro (4a,b) and 4-trifluoromethyl (4m,n) analogs being the most active and unsubsituted phenyl (4c, d and 4e, f) and the 2,5 dimethyl substituted (4o,p) analogs being the least active. The anti-tuberculosis activities of the second set of compounds are shown in Table 2. Compared with parent compound 4b, compounds 5a-c had significant decrease of anti-tuberculosis activity. This demonstrated the importance of 4-Chloro and 3-CF3 groups. Removal of either of these groups is detrimental to anti-tuberculosis activity by comparing compound 5b, 5c, and 4b, removal of both groups in compound 5a led to the significant loss of activity. The tertiary hydroxyl group also has a significant effect on activity as well. Replacement of it with a cyano group resulted in a more active compound 5d and replacement with a dehydration product 5e results led to a complete loss of anti-tuberculosis activity (Table 2).

Table 1
In vitro anti-tuberculosis activity, cytotoxic LD50, and therapeutic indices of piperidinol analogs 1, 2, and 4 optimized at R1 position
Table 2
In vitro anti-tuberculosis activity of piperidinol analogs 5 optimized at R4 position

The cytotoxicities of the compounds were next evaluated against eukaryotic Vero cells (Table 1) to determine their suitability for further advancement. In general cytotoxicities were observed producing narrow therapeutic indices that ranged from 1.5 for 4f to 13.3 for 4b. However, the selectivity of 4b and 4m was sufficient to advance for testing in vivo (>10). In subsequent experiments for anti-tuberculosis efficacy of 4b in mice,13 severe adverse effects were observed resulting in death at doses higher than 50-100 mg/kg. These symptoms precluded further testing of these compounds.

Conclusions

In summary, high-throughput screening of a commercial library identified a piperidinol hit with anti-tuberculosis activities and an unknown mechanism of action. Based on this hit compound, two expanded sets of compounds 4a-p and 5a-e were subsequently designed, synthesized, and evaluated toward anti-tuberculosis activity. Two compounds 4b and 4m were identified with good activity (MIC: 1.4-1.7 μg/mL) and acceptable therapeutic indices (>10). We have showed that improvements in the therapeutic index can be made (Table 1), unfortunately these compounds produced significant side effects when tested in vivo for anti-tuberculosis activity in mice likely due to secondary pharmacology associated with 4-[4-chloro-3-(trifluoromethyl)-phenyl]-4-piperidinol core. This study may highlight some of the problems likely to be encountered as more investigators switch to direct antituberculosis screening rather than single enzyme screening to discover novel anti-tuberculosis compounds from compound libraries. As such commercial libraries are rarely truly random and are most often designed around pharmacologically active scaffolds utilized in discovery of human non-infectious diseases, which could compromise their use for infectious disease particularly those requiring long-term treatment such as tuberculosis. As observed in our study, this increases the risk of unwanted side effects from residual secondary pharmacology associated with the chemical scaffold that may preclude further advancement of the series.

Experimental Section

All solvents were purchased from Sigma-Aldrich and Fisher Scientific and used as received, and remaining chemicals were from Sigma-Aldrich. Thin layer chromatography (TLC) analysis was performed on Merck silica gel 60F254 plates and the spots were visualized under a UV lamp. Melting point (m.p.) was determined using OptiMelt Automated Melting Point System (Stanford Research Systems) and is uncorrected. 1H NMR spectra were recorded at 500 MHz on a Varian Inova NMR instrument. Mass spectra were recorded on a Bruker Esquire LC/MS using ESI. Analytical RP-HPLC was conducted on a Shimadzu HPLC system with a Phenomenex C18 column (100Å, 3 μm, 4.6 × 50 mm), flow rate 1.0 mL/min and a gradient of solvent A (water with 0.1% TFA) and solvent B (acetonitrile): 0-2.00 min 100% A; 2.00-7.00 min 0-100% B (linear gradient). UV detection at 254 and 218 nm.

General procedure for the synthesis of the piperidinols 1, 2, 4a-p, and 5a-e

Acetonitrile (15 mL) was added to (S)-(+)-epichlorohydrin or (R)-(-)-epichlorohydrin (0.188 mL, 2.4 mmol, 1.2 eq.), substituted phenol (2 mmol, 1 eq.), followed by the addition of Cesium carbonate (1.3 g, 4 mmol, 2 eq.). The reaction mixture was stirred under reflux overnight. The reaction was complete based on TLC monitoring. The reaction mixture was filtered and washed with acetonitrile. The filtrate was evaporated in vacuo to give crude product 3, which was used directly for the next step without further purification.

The crude product obtained above was dissolved in ethanol (15 mL), followed by the addition of 4-[4-chloro-3-(trifluoromethyl)-phenyl]-4-piperidinol or the other 4-piperidines (2 mmol). The reaction was stirred under reflux overnight and the reaction was complete based on HPLC analysis. The resulting solution was evaporated in vacuo and the residue was purified by column chromatography on silica gel (hexane-50% ethyl acetate in hexane in a volume of 255 mL, followed by 100% ethyl acetate in a linear gradient in a volume of 204 mL). Evaporation of appropriate fractions gave final product as white powder.

1-(3-(4-chlorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 1 (racemate)

0.35 g (1 mmol scale), 75.4% yield. m.p.: 147-149 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.92 (d, J = 2.0 Hz, 1H), 7.76 (dd, J = 1.7 and 8.5 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.32 (dd, J = 3.3 and 8.8 Hz, 2H), 6.98 (dd, J = 3.3 and 8.8 Hz, 2H), 5.15 (s, 1H, OH), 4.85 (d, J = 4.2 Hz, 1H, OH), 4.01 (dd, J = 3.5 and 9.5 Hz, 1H, CHaO), 3.96 (m, 1H, CHOH), 3.88 (dd, J = 5.8 and 9.5 Hz, 1H, CHa′O), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (dd, J = 6.5 and 12.6 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 464.1, (M-H)- 461.9. HPLC purity: 100% (254 nm), tR = 5.52 min.

4-(4-chloro-3-(trifluoromethyl)phenyl)-1-(2-hydroxy-3-phenoxypropyl)piperidin-4-ol, 2 (racemate)

0.68 g (2 mmol scale), 79.1% yield. m.p.: 127-130 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 2.0 Hz, 1H), 7.77 (dd, J = 2.0 and 8.5 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.29 (m, 2H), 6.95 (m, 3H), 5.15 (s, 1H, OH), 4.82 (br s, 1H, OH), 3.99 (m, 2H, CHaO and CHOH), 3.88 (dd, J = 5.4 and 9.0 Hz, 1H, CHa′O), 2.73 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.43 (dd, J = 6.4 and 12.5 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 430.1, (M-H)- 428.0. HPLC purity: 100% (254 nm), tR = 5.38 min.

1-((S)-3-(4-chlorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4a

0.39 g (2 mmol scale), 42.0% 2-step overall yield. m.p.: 146-149 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.92 (d, J = 2.0 Hz, 1H), 7.76 (dd, J = 1.7 and 8.5 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.32 (dd, J = 3.5 and 8.9 Hz, 2H), 6.98 (dd, J = 3.4 and 8.9 Hz, 2H), 5.15 (s, 1H, OH), 4.84 (d, J = 4.6 Hz, 1H, OH), 4.01 (dd, J = 3.5 and 9.5 Hz, 1H, CHaO), 3.96 (m, 1H, CHOH), 3.88 (dd, J = 5.9 and 9.5 Hz, 1H, CHa′O), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.41 (dd, J = 6.5 and 12.6 Hz, 1H, NCHb), 1.95 (m, 2H), 1.56 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 464.1, (M-H)- 461.9. HPLC purity: 99.1% (218 nm), tR = 5.59 min.

1-((R)-3-(4-chlorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4b

0.30 g (2 mmol scale), 32.3% 2-step overall yield. m.p.: 144-146 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 2.0 Hz, 1H), 7.76 (dd, J = 2.0 and 8.5 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.32 (dd, J = 3.5 and 9.0 Hz, 2H), 6.98 (dd, J = 3.4 and 9.0 Hz, 2H), 5.14 (s, 1H, OH), 4.84 (d, J = 4.4 Hz, 1H, OH), 4.01 (dd, J = 3.7 and 9.8 Hz, 1H, CHaO), 3.96 (m, 1H, CHOH), 3.88 (dd, J = 5.9 and 9.5 Hz, 1H, CHa′O), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.41 (dd, J = 6.4 and 12.5 Hz, 1H, NCHb), 1.95 (m, 2H), 1.56 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 464.1, (M-H)- 461.9. HPLC purity: 100% (254 nm), tR = 5.58 min; 98.8% (218 nm), tR = 5.59 min.

4-(4-chloro-3-(trifluoromethyl)phenyl)-1-((S)-2-hydroxy-3-(phenylthio)propyl)piperidin-4-ol, 4c

0.17 g (2 mmol scale), 19.1% 2-step overall yield. m.p.: 117-119 °C. 1H NMR, 500 MHz (CDCl3): δ 7.84 (s, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.28 (t, J = 7.8 Hz, 2H), 7.18 (t, J = 7.3 Hz, 1H), 3.89 (m, 1H, CHOH), 3.58 (br s, 2H, OH and OH), 3.03 (m, 2H, CH2S), 2.81 (d, J = 11.0 Hz, 1H, NCHb′), 2.71 (m, 2H), 2.58 (dd, J = 2.9 and 12.5 Hz, 1H, NCHb), 2.45 (t, J = 10.3 Hz, 2H), 2.09 (m, 1H), 2.01 (m, 2H), 1.70 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 446.1, (M-H)- 443.9. HPLC purity: 99.4% (254 nm), tR = 5.48 min; 99.5% (218 nm), tR = 5.48 min.

4-(4-chloro-3-(trifluoromethyl)phenyl)-1-((R)-2-hydroxy-3-(phenylthio)propyl)piperidin-4-ol, 4d

0.22 g (2 mmol scale), 24.7% 2-step overall yield. m.p.: 116-119 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 2.0 Hz, 1H), 7.77 (dd, J = 2.0 and 8.3 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.37 (d, J = 7.6 Hz, 2H), 7.31 (t, J = 7.8 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 5.14 (s, 1H, OH), 4.88 (d, J = 4.6 Hz, 1H, OH), 3.80 (m, 1H, CHOH), 3.19 (dd, J = 4.6 and 13.2 Hz, 1H, CHaS), 2.96 (dd, J = 6.7 and 13.2 Hz, 1H, CHa′S), 2.65 (m, 2H), 2.46 (m, 3H), 2.40 (dd, J = 6.4 and 12.7 Hz, 1H, NCHb), 1.94 (m, 2H), 1.55 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 446.1, (M-H)- 443.9. HPLC purity: 99.3% (254 nm), tR = 5.48 min; 99.3% (218 nm), tR = 5.48 min.

4-(4-chloro-3-(trifluoromethyl)phenyl)-1-((S)-2-hydroxy-3-phenoxypropyl)piperidin-4-ol, 4e

0.40 g (2 mmol scale), 46.5% 2-step overall yield. m.p.: 124-126 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 1.7 Hz, 1H), 7.77 (dd, J = 1.7 and 8.3 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.29 (m, 2H), 6.94 (m, 3H), 5.15 (s, 1H, OH), 4.81 (d, J = 4.4 Hz, 1H, OH), 3.99 (m, 2H, CHOH and CHaO), 3.89 (dd, J = 5.5 and 9.2 Hz, 1H, CHa′O), 2.74 (m, 2H), 2.51 (m, 3H), 2.43 (dd, J = 6.4 and 12.7 Hz, 1H, NCHb), 1.96 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 430.1, (MNa)+ 452.1, (M-H)- 427.9. HPLC purity: 100% (254 nm), tR = 5.42 min; 100% (218 nm), tR = 5.43 min.

4-(4-chloro-3-(trifluoromethyl)phenyl)-1-((R)-2-hydroxy-3-phenoxypropyl)piperidin-4-ol, 4f

0.43 g (2 mmol scale), 50.0% 2-step overall yield. m.p.: 124-126 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 1.2 Hz, 1H), 7.77 (dd, J = 1.2 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.30 (t, J = 7.6 Hz, 2H), 6.94 (m, 3H), 5.15 (s, 1H, OH), 4.81 (d, J = 4.4 Hz, 1H, OH), 3.99 (m, 2H, CHOH and CHaO), 3.89 (dd, J = 5.6 and 9.3 Hz, 1H, CHa′O), 2.74 (m, 2H), 2.51 (m, 3H), 2.43 (dd, J = 6.4 and 12.5 Hz, 1H, NCHb), 1.96 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 430.1, (MNa)+ 452.1, (M-H)- 427.9. HPLC purity: 100% (254 nm), tR = 5.41 min; 99.4% (218 nm), tR = 5.42 min.

1-((S)-3-(4-fluorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4g

0.35 g (2 mmol scale), 39.1% 2-step overall yield. m.p.: 114-115 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 2.0 Hz, 1H), 7.77 (dd, J = 1.7 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.12 (m, 2H), 6.97 (m, 2H), 5.15 (s, 1H, OH), 4.81 (d, J = 4.2 Hz, 1H, OH), 3.98 (m, 2H, CHOH and CHaO), 3.87 (dd, J = 5.4 and 9.0 Hz, 1H, CHa′O), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (dd, J = 6.4 and 12.7 Hz, 1H, NCHb), 1.95 (m, 2H), 1.56 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 448.1, (MNa)+ 470.1, (M-H)- 445.9. HPLC purity: 99.5% (254 nm), tR = 5.44 min; 99.5% (218 nm), tR = 5.45 min.

1-((R)-3-(4-fluorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4h

0.38 g (2 mmol scale), 42.4% 2-step overall yield. m.p.: 114-116 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 1.7 Hz, 1H), 7.77 (dd, J = 1.7 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.12 (m, 2H), 6.98 (m, 2H), 5.15 (s, 1H, OH), 4.81 (d, J = 4.4 Hz, 1H, OH), 3.98 (m, 2H, CHOH and CHaO), 3.87 (dd, J = 5.4 and 9.0 Hz, 1H, CHa′O), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (dd, J = 6.4 and 12.5 Hz, 1H, NCHb), 1.96 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 448.1, (M-H)- 445.9. HPLC purity: 99.2% (254 nm), tR = 5.43 min; 98.2% (218 nm), tR = 5.44 min.

1-((S)-3-(4-(trifluoromethoxy)phenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4i

0.43 g (2 mmol scale), 41.8% 2-step overall yield. m.p.: 123-126 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 1.7 Hz, 1H), 7.77 (dd, J = 1.7 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.29 (d, J = 9.0 Hz, 2H), 7.06 (d, J = 9.0 Hz, 2H), 5.15 (s, 1H, OH), 4.85 (d, J = 4.6 Hz, 1H, OH), 4.04 (dd, J = 3.4 and 9.5 Hz, 1H, CHaO), 3.98 (m, 1H, CHOH), 3.92 (dd, J = 5.9 and 9.5 Hz, 1H, CHa′O), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (dd, J = 6.4 and 12.7 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 514.2, (M-H)- 511.9. HPLC purity: 99.0% (254 nm), tR = 5.70 min; 98.3% (218 nm), tR = 5.71 min.

1-((R)-3-(4-(trifluoromethoxy)phenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4j

0.43 g (2 mmol scale), 41.8% 2-step overall yield. m.p.: 123-125 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 2.0 Hz, 1H), 7.77 (dd, J = 2.0 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 9.0 Hz, 2H), 5.15 (s, 1H, OH), 4.85 (d, J = 4.6 Hz, 1H, OH), 4.04 (dd, J = 3.4 and 9.8 Hz, 1H, CHaO), 3.98 (m, 1H, CHOH), 3.92 (dd, J = 6.1 and 9.8 Hz, 1H, CHa′O), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (dd, J = 6.4 and 12.7 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 514.2, (M-H)- 511.9. HPLC purity: 98.9% (254 nm), tR = 5.71 min; 97.5% (218 nm), tR = 5.72 min.

1-((S)-3-(3,4-dichlorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4k

0.41 g (2 mmol scale), 41.1% 2-step overall yield. m.p.: 121-124 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 2.0 Hz, 1H), 7.77 (dd, J = 2.0 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.27 (d, J = 2.9 Hz, 1H), 7.00 (dd, J = 2.9 and 9.0 Hz, 1H), 5.15 (s, 1H, OH), 4.87 (d, J = 4.2 Hz, 1H, OH), 4.08 (m, 1H), 3.95 (m, 2H), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.41 (dd, J = 5.9 and 12.5 Hz, 1H, NCHb), 1.96 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 500.0. HPLC purity: 96.1% (254 nm), tR = 5.69 min; 95.8% (215 nm), tR = 5.69 min.

1-((R)-3-(3,4-dichlorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4l

0.41 g (2 mmol scale), 41.1% 2-step overall yield. m.p.: 136-138 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 1.7 Hz, 1H), 7.77 (dd, J = 2.0 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 9.0 Hz, 1H), 7.27 (d, J = 2.9 Hz, 1H), 7.00 (dd, J = 2.9 and 9.0 Hz, 1H), 5.15 (s, 1H, OH), 4.87 (d, J = 3.9 Hz, 1H, OH), 4.08 (m, 1H), 3.95 (m, 2H), 2.71 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.41 (dd, J = 5.9 and 12.5 Hz, 1H, NCHb), 1.96 (m, 2H), 1.56 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 500.0. HPLC purity: 95.8% (254 nm), tR = 5.68 min; 95.2% (218 nm), tR = 5.69 min.

1-((S)-3-(4-(trifluoromethyl)phenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4m

0.35 g (2 mmol scale), 35.2% 2-step overall yield. m.p.: 135-137 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 2.0 Hz, 1H), 7.77 (dd, J = 2.0 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.15 (d, J = 8.8 Hz, 2H), 5.15 (s, 1H, OH), 4.89 (d, J = 4.2 Hz, 1H, OH), 4.12 (m, 1H), 4.00 (m, 2H), 2.73 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.44 (dd, J = 5.9 and 12.5 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 498.1. HPLC purity: 98.4% (218 nm), tR = 5.68 min.

1-((R)-3-(4-(trifluoromethyl)phenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4n

0.21 g (2 mmol scale), 21.1% 2-step overall yield. m.p.: 138-141 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.92 (d, J = 1.7 Hz, 1H), 7.77 (dd, J = 1.5 and 8.3 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H), 5.15 (s, 1H, OH), 4.89 (d, J = 4.2 Hz, 1H, OH), 4.12 (m, 1H), 4.00 (m, 2H), 2.73 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.44 (dd, J = 5.9 and 12.5 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 498.1. HPLC purity: 96.8% (218 nm), tR = 5.69 min.

1-((S)-3-(2,6-dimethylphenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4o

0.37 g (2 mmol scale), 40.4% 2-step overall yield. m.p.: 138-141 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.93 (d, J = 2.0 Hz, 1H), 7.76 (dd, J = 1.7 and 8.3 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 7.3 Hz, 2H), 6.91 (t, J = 7.3 Hz, 1H), 5.16 (s, 1H, OH), 4.80 (d, J = 4.6 Hz, 1H, OH), 3.99 (m, 1H, CHOH), 3.76 (dd, J = 3.7 and 9.3 Hz, 1H, CHaO), 3.70 (dd, J = 5.6 and 9.3 Hz, 1H, CHa′O), 2.75 (m, 2H), 2.57 (dd, J = 5.9 and 12.5 Hz, 1H, NCHb′), 2.50 (m, 2H, overlapped with DMSO peak), 2.44 (dd, J = 6.6 and 12.5 Hz, 1H, NCHb), 2.26 (s, 6H, CH3 and CH3), 1.95 (m, 2H), 1.59 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 458.1, (MNa)+ 480.1, (M-H)- 455.9. HPLC purity: 100% (254 nm), tR = 5.58 min; 100% (218 nm), tR = 5.58 min.

1-((R)-3-(2,6-dimethylphenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4p

0.43 g (2 mmol scale), 47.0% 2-step overall yield. m.p.: 138-141 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.94 (d, J = 1.7 Hz, 1H), 7.76 (dd, J = 1.5 and 8.3 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 7.6 Hz, 2H), 6.91 (t, J = 7.6 Hz, 1H), 5.16 (s, 1H, OH), 4.80 (d, J = 4.4 Hz, 1H, OH), 3.99 (m, 1H, CHOH), 3.76 (dd, J = 3.9 and 9.3 Hz, 1H, CHaO), 3.70 (dd, J = 5.6 and 9.3 Hz, 1H, CHa′O), 2.75 (m, 2H), 2.57 (dd, J = 6.1 and 12.5 Hz, 1H, NCHb′), 2.50 (m, 2H, overlapped with DMSO peak), 2.44 (dd, J = 6.6 and 12.5 Hz, 1H, NCHb), 2.26 (s, 6H, CH3 and CH3), 1.95 (m, 2H), 1.59 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 458.1, (MNa)+ 480.1, (M-H)- 455.9. HPLC purity: 100% (254 nm), tR = 5.58 min; 100% (218 nm), tR = 5.58 min.

1-((R)-3-(4-chlorophenoxy)-2-hydroxypropyl)-4-phenylpiperidin-4-ol, 5a

0.41 g (2 mmol scale), 56.7% 2-step overall yield. m.p.: 108-111 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.48 (d, J = 7.6 Hz, 2H), 7.32 (m, 4H), 7.20 (t, J = 7.3 Hz, 1H), 6.99 (d, J = 9.0 Hz, 2H), 4.84 (d, J = 4.6 Hz, 1H, OH), 4.77 (s, 1H, OH), 4.02 (dd, J = 3.4 and 9.5 Hz, 1H, CHaO), 3.97 (m, 1H, CHOH), 3.88 (dd, J = 6.1 and 9.5 Hz, 1H, CHa′O), 2.69 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.41 (dd, J = 6.6 and 12.7 Hz, 1H, NCHb), 1.93 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 362.3, (MNa)+ 384.1. HPLC purity: 100% (254 nm), tR = 5.16 min; 100% (215 nm), tR = 5.16 min.

1-((R)-3-(4-chlorophenoxy)-2-hydroxypropyl)-4-(4-chlorophenyl)piperidin-4-ol, 5b

0.35 g (2 mmol scale), 44.2% 2-step overall yield. m.p.: 146-148 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.50 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 4.89 (s, 1H, OH), 4.84 (d, J = 4.4 Hz, 1H, OH), 4.01 (dd, J = 3.7 and 9.8 Hz, 1H, CHaO), 3.97 (m, 1H, CHOH), 3.88 (dd, J = 6.1 and 9.8 Hz, 1H, CHa′O), 2.69 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.41 (dd, J = 6.4 and 12.5 Hz, 1H, NCHb), 1.90 (m, 2H), 1.56 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 396.2, (MNa)+ 418.1, (M-H)- 393.9. HPLC purity: 100% (254 nm), tR = 5.33 min; 99.3% (215 nm), tR = 5.33 min.

1-((R)-3-(4-chlorophenoxy)-2-hydroxypropyl)-4-(3-(trifluoromethyl)phenyl)piperidin-4-ol, 5c

0.32 g (2 mmol scale), 37.2% 2-step overall yield. m.p.: 101-104 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.83 (s, 1H), 7.77 (d, J = 7.3 Hz, 1H), 7.57 (m, 2H), 7.33 (dd, J = 3.4 and 8.2 Hz, 2H), 6.99 (dd, J = 3.4 and 8.8 Hz, 2H), 5.06 (s, 1H, OH), 4.85 (d, J = 4.4 Hz, 1H, OH), 4.02 (dd, J = 3.7 and 9.8 Hz, 1H, CHaO), 3.97 (m, 1H, CHOH), 3.89 (dd, J = 5.9 and 9.5 Hz, 1H, CHa′O), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (dd, J = 6.6 and 12.7 Hz, 1H, NCHb), 1.97 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 430.2, (MNa)+ 452.1, (M-H)- 427.9. HPLC purity: 98.9% (254 nm), tR = 5.45 min; 98.7% (215 nm), tR = 5.45 min.

1-((R)-3-(4-chlorophenoxy)-2-hydroxypropyl)-4-phenylpiperidine-4-carbonitrile, 5d

0.30 g (2 mmol scale), 40.4% 2-step overall yield. m.p.: 74-76 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.54 (d, J = 7.6 Hz, 2H), 7.45 (t, J = 7.3 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 7.33 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 4.94 (d, J = 3.4 Hz, 1H, OH), 3.99 (m, 2H, CHaO and CHOH), 3.89 (dd, J = 7.1 and 10.7 Hz, 1H, CHa′O), 3.05 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (m, 1H), 2.04 (m, 4H). Mass spectrum (ESI) m/z (MH)+ 371.2, (MNa)+ 393.1. HPLC purity: 100% (254 nm), tR = 5.38 min; 98.5% (215 nm), tR = 5.38 min.

(R)-1-(4-chlorophenoxy)-3-(4-(4-chlorophenyl)-5,6-dihydropyridin-1(2H)-yl)propan-2-ol, 5e

0.28 g (2 mmol scale), 37.0% 2-step overall yield. m.p.: 124-126 °C. 1H NMR, 500 MHz (DMSO-d6): δ 7.46 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.20 (br t, 1H, = CH), 4.94 (d, J = 4.2 Hz, 1H, OH), 4.00 (m, 2H, CHaO and CHOH), 3.89 (dd, J = 6.8 and 10.7 Hz, 1H, CHa′O), 3.17 (m, 2H), 2.71 (m, 2H), 2.58 (dd, J = 5.6 and 12.5 Hz, 1H, NCHb′), 2.50 (m, 1H, overlapped with DMSO peak, NCHb), 2.46 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 378.1, (MNa)+ 400.1. HPLC purity: 99.7% (254 nm), tR = 5.57 min; 99.7% (215 nm), tR = 5.56 min.

MIC determination

MIC values were determined against M. tuberculosis (H37Rv) by the microbroth dilution method.14 A broth culture of M. tuberculosis was grown in Middlebrook 7H9 medium with 10% OADC supplement to an OD600 of 0.4–0.6 and aliquots were frozen. The CFU/mL of these aliquots were determined. The tested compounds were serially diluted in DMSO and 4 μL added to microtier plate wells containing 100 μL of 7H9 medium. Subsequently, 2×104 TB bacteria in 100 μL of 7H9 medium were added to each well. The plates were incubated at 37 °C for 9-11 days. The MIC90 was determined by visual inspection and defined as the concentration that inhibited 90% of growth. For each experiment, controls of INH were run; the MIC of INH under these conditions was between 0.032 and 0.064 μg/mL.

Cytotoxicity study

The LD50 for each drug was determined using a microplate dilution method.15 African green monkey kidney cells (Vero cells) were grown in L15 medium without phenol (Invitrogen). The media was supplemented with glutamine, 10 mL/L penicillin-streptomycin solution (10,000 IU/10,000 μg/mL), and 10% bovine calf serum at 37°C. The microtiter plates were sealed and testing was conducted for 72 h at 37°C, alamarBlue® was added to each well, and plates were incubated overnight at 37°C. Plates were read at 570 and 600 nm using a spectrophotometric plate reader, and the absorbance readings were used to calculate the 50% lethal concentration.

Supplementary Material

01

Acknowledgments

We thank National Institutes of Health grant AI057836 for financial support.

Footnotes

Supplementary Material: 1H NMR spectra and HPLC profiles of all target molecules.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

1. Sacchettini JC, Rubin EJ, Freundlich JS. Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis. Nat Rev Microbiol. 2008;6(1):41–52. [PubMed]
2. Williams DA, Lemke TL. Foye's Principles of Medicinal Chemistry. Fifth's Edition. Lippincott Williams & Wilkins; 2002.
3. Gallant DM, Mielke DH, Spirtes MA, Swanson WC, Bost R. Penfluridol: an efficacious long-acting oral antipsychotic compound. Am J Psychiatry. 1974;131(6):699–702. [PubMed]
4. Manikowski A, Verri A, Lossani A, Gebhardt BM, Gambino J, Focher F, Spadari S, Wright GE. Inhibition of herpes simplex virus thymidine kinases by 2-phenylamino-6-oxopurines and related compounds: structure-activity relationships and antiherpetic activity in vivo. J Med Chem. 2005;48(11):3919–29. [PMC free article] [PubMed]
5. Zhi C, Long ZY, Gambino J, Xu WC, Brown NC, Barnes M, Butler M, LaMarr W, Wright GE. Synthesis of substituted 6-anilinouracils and their inhibition of DNA polymerase IIIC and Gram-positive bacterial growth. J Med Chem. 2003;46(13):2731–9. [PubMed]
6. Chen Z, Davies E, Miller WS, Shan S, Valenzano KJ, Kyle DJ. Design and synthesis of 4-phenyl piperidine compounds targeting the mu receptor. Bioorg Med Chem Lett. 2004;14(21):5275–9. [PubMed]
7. Chen Z, Miller WS, Shan S, Valenzano KJ. Design and parallel synthesis of piperidine libraries targeting the nociceptin (N/OFQ) receptor. Bioorg Med Chem Lett. 2003;13(19):3247–52. [PubMed]
8. Yang W, Wang Y, Roberge JY, Ma Z, Liu Y, Michael Lawrence R, Rotella DP, Seethala R, Feyen JH, Dickson JK., Jr Discovery and structure-activity relationships of 2-benzylpyrrolidine-substituted aryloxypropanols as calcium-sensing receptor antagonists. Bioorg Med Chem Lett. 2005;15(4):1225–8. [PubMed]
9. Shagufta, Srivastava AK, Sharma R, Mishra R, Balapure AK, Murthy PS, Panda G. Substituted phenanthrenes with basic amino side chains: a new series of anti-breast cancer agents. Bioorg Med Chem. 2006;14(5):1497–505. [PubMed]
10. Surendra K, Krishnaveni NS, Nageswar YV, Rao KR. Highly regioselective ring opening of oxiranes with phenoxides in the presence of beta-cyclodextrin in water. J Org Chem. 2003;68(12):4994–5. [PubMed]
11. Das B, Krishnaiah M, Thirupathi P, Laxminarayana K. An efficient catalyst-free regio- and stereoselective ring-opening of epoxides with phenoxides using polyethylene glycol as the reaction medium. Tetrahedron Letters. 2007;48(24):4263–4265.
12. Representative characterization of Bis-byproduct (R = 4-Cl, X = O):
An external file that holds a picture, illustration, etc.
Object name is nihms109116u1.jpg

1H NMR, 500 MHz (DMSO-d6): 7.33 (d, J = 8.8 Hz, 4H), 6.99 (d, J = 9.0 Hz, 4H), 5.44 (d, J = 4.6 Hz, 1H, OH), 4.14 (m, 1H), 4.07 (dd, J = 4.6 and 9.8 Hz, 2H), 4.01 (dd, J = 5.9 and 10.0 Hz, 2H). Mass spectrum (ESI) m/z (MH)+ 335.1. HPLC purity: 100% (254 nm and 215 nm), tR = 7.00 min.
13. Lenaerts AJM, Veronica G, Brooks Jason V, Orme Ian M. Rapid in vivo screening of experimental drugs for tuberculosis using gamma interferon gene-disrupted mice. Antimicrobial Agents and Chemotherapy. 2003;47(2):783–785. [PMC free article] [PubMed]
14. Hurdle JG, Lee RB, Budha NR, Carson EI, Qi J, Scherman MS, Cho SH, McNeil MR, Lenaerts AJ, Franzblau SG, Meibohm B, Lee RE. A microbiological assessment of novel nitrofuranylamides as anti-tuberculosis agents. J Antimicrob Chemother. 2008;62(5):1037–45. [PubMed]
15. Collins L, Franzblau SG. Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother. 1997;41(5):1004–9. [PMC free article] [PubMed]