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. 2014 Jul 27;5(10):1162–1166. doi: 10.1021/ml500299q

Synthesis of Deuterated Benzopyran Derivatives as Selective COX-2 Inhibitors with Improved Pharmacokinetic Properties

Yanmei Zhang †,*, Micky D Tortorella †, Yican Wang †, Jianqi Liu †, Zhengchao Tu †, Xiaorong Liu †, Yang Bai †, Dingsheng Wen †, Xin Lu †, Yongzhi Lu †, John J Talley ‡,§,*
PMCID: PMC4190635  PMID: 25313331

Abstract

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We designed a series of specifically deuterated benzopyran analogues as new COX-2 inhibitors with the aim of improving their pharmacokinetic properties. As expected, the deuterated compounds retained potency and selectivity for COX-2. The new molecules possess improved pharmacokinetic profiles in rats compared to their nondeuterated congeners. Most importantly, the new compounds showed pharmacodynamic efficacy in several murine models of inflammation and pain. The benzopyran derivatives were separated into their enantiomers, and the activity was found to reside with the S-isomers. To streamline the synthesis of the desired S-isomers, an organocatalytic asymmetric domino oxa-Michael/aldol condensation reaction was developed for their preparation.

Keywords: COX-2, coxib, deuterium, benzopyran, NSAID

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit both COX-1 and COX-2 enzymes and have been a mainstay in clinical medicine for the treatment of inflammation and pain for many years. COX-2-selective inhibitors (coxibs)1,2 developed in the mid 2000s supplanted traditional NSAIDs by virtue of their comparable efficacy with improved gastrointestinal safety.103 Coxibs were initially thought to be able to avoid renal and cardiovascular side effects; however, this proved not to be entirely accurate.104 In the kidney, COX-2 is expressed constitutively in certain regions (i.e., macula densa) and is highly regulated in response to changes in blood volume. COX-2-mediated metabolites (e.g., PGE2 and PGI2) play a mechanistic role in renin release, sodium excretion, and the maintenance of renal blood flow and glomerular filtration rate.105 Coxibs fell into disfavor as clinical medicine because initial data linked them to cardiovascular risks of stroke and myocardial infarction compared with traditional NSAIDs.106 However, data analysis from two recent clinical trials107,108 clearly demonstrated that chronic and even short-term use of NSAIDs increased renal and cardiovascular side-effect risk no lesser than coxibs, and that the risks appeared to be drug-dependent rather than class-dependent. These data among other studies have inspired renewed interest in selective COX-2 inhibitors, particularly with coxibs that have a unique pharmacological profile, which could address unmet medical conditions.3−6 Such clinical conditions include treating pain and inflammation without further compromising renal function and in treating or preventing certain types of cancer associated with inflammation mediated by COX-2.

Benzopyran compounds as selective COX-2 inhibitors were investigated.7−9 It was reported that 2-trifluoromethyl, 3-carboxy, and 4-H substituents were key elements of the benzopyran pharmacophore. Chiral chromatographic separation of 2-trifluoromethyl enantiomers and evaluation of the individual isomers showed that S-isomers were far more effective in blocking COX-2 activity than the corresponding R-isomers. Substitutions at the 5, 6, 7, and 8 positions were optimized for potency, efficacy, and pharmacokinetic profiles. Several clinical candidates were identified including SC-75416 and advanced into clinical trials (Figure 1).

Figure 1.

Figure 1

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Benzopyran template and SC-75416.

Deuteration of compounds is a strategy for creating new chemical entities that has recently been used in drug discovery to make improved drugs. Carbon–deuterium bonds are stronger than the more common carbon–hydrogen bonds, and the replacement of hydrogen atoms with deuterium at metabolically labile positions can significantly improve the PK and thus efficacy of physiologically active compounds by slowing metabolism due to the kinetic isotope effect. Deuterium substituted compounds retain both biochemical potency and selectivity for their target, suggesting that deuterium atom substitution does not appreciably alter ligand–receptor binding. More importantly, replacing hydrogen with deuterium can enable substantial benefits to the overall pharmacological profile of the compounds.10−12 In an effort to develop improved selective COX-2 inhibitors, we designed and synthesized a series of specifically deuterated analogues of benzopyran coxibs. The initial synthetic efforts focused on deuteration of the methyl group, which is outlined in Scheme 1. Commercially available anisoles 1 were deuterium labeled on the methyl group under strongly basic conditions in DMSO-d6 to give compound 2, which converted methyl or dimethyl into CD3 or di-CD3 derivatives.13 Deuterium substituted phenols 3 were prepared by O-demethylation, followed by chlorination or bromination to provide the desired chlorinated or brominated products. Chlorination or bromination may also be conducted prior to O-demethylation if needed. Substituted salicylaldehydes 4 were synthesized by the Duff reaction.14−16 Key intermediate 4 was then condensed with commercially available ethyl trifluorocrotonate to produce the specifically deuterium substituted benzopyran nucleus 5, in a one-step procedure by heating in K2CO3 and DMF. The resulting esters 5 were hydrolyzed with sodium hydroxide yielding the acids 6a–h; see Scheme 1 (see synthesis and characterization of compounds, Supporting Information).

Scheme 1. Synthesis of Trideuteromethyl Derivatives.

Scheme 1

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R represents substituents on C5, C6, C7, or C8 position; RD represents deuterated substituents. Reagents and conditions: (a) potassium t-butoxide, DMSO-d6, 110 °C 6 h; (b) BBr3, DCM, 0 °C, 2 h; (c) HTMA, TFA, rt, 1 h; (d) ethyl 4,4,4-trifluorocrotonate, K2CO3, DMF, 80–100 °C, 2–4 h; (e) NaOH, THF/CH3OH/H2O.

Compound 6i was prepared according to the procedure outlined in Scheme 2. The benzopyran 7 was prepared by a published procedure17 and then coupled with ethynyltrimethylsilane in the presence of a base and a catalytic amount of cuprous iodide and tetrakis(triphenylphosphine)palladium(0) to afford 8. Removal of the trimethylsilyl group was key to yield the terminal deuterated compound 9 (K2CO3 in D2O), and reduction of 9 (D2, Pd/C in ethyl acetate) and finally saponification of the ester with aqueous sodium hydroxide gave deuterated benzopyran 6i (see Scheme 2 and synthesis and characterization of compounds in Supporting Information). The corresponding nondeuterated benzopyran derivatives 6an–in were synthesized by known methods.17

Scheme 2. Synthesis of Pentadeuterated Analogue 6i.

Scheme 2

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Reagents and conditions: (a) CuI, Pd(PPh3)4, ethynyltrimethylsilane, TEA, MeCN, rt, 24 h; (b) K2CO3, anhydrous ethanol, D2O, rt, 30 min; (c) D2, EtOAc, Pd/C, rt, 1 h; (d) NaOH, THF/CH3OH/H2O.

The analogues 6a–i and 6an–in were tested for their inhibitory activity against recombinant human COX-1 and COX-2 enzymes, the results are shown in Table 1 (see in vitro COX enzyme assay in Supporting Information). There were no significant differences in the inhibitory profile against COX-1 or COX-2 between deuterated versus the corresponding nondeuterated analogues. Most analogues had potency for COX-2 with an IC50 < 100 nM and were relatively inactive against COX-1. Within this series of compounds, the analogues bearing a 6-chloro or 6-bromo substituent were the most potent and selective inhibitors of COX-2.

Table 1. Evaluation of Substituted Benzopyrans against COX-1 and COX-2 Enzymes.

graphic file with name ml-2014-00299q_0008.jpg

          rh IC50 (μM)
compd R1 R2 R3 R4 COX-1 COX-2
6a H CD3 Cl CD3   0.054 ± 0.013
6b H CD3 Br CD3   0.043 ± 0.018
6c Br CD3 Br CD3 >100 0.084 ± 0.020
6d H H CD3 H   >10
6e Cl H CD3 H   0.063 ± 0.015
6f CD3 H CD3 H >100 0.15 ± 0.035
6g CD3 H Cl H   0.059 ± 0.009
6h CD3 H Br H   0.039 ± 0.005
6i CD2CD3 H OCF3 H   0.061 ± 0.009
6an H CH3 Cl CH3 >100 0.069 ± 0.025
6bn H CH3 Br CH3 >100 0.19 ± 0.042
6cn Br CH3 Br CH3 >100 0.069 ± 0.008
6dn H H CH3 H   >10
6en Cl H CH3 H >100 0.078 ± 0.022
6fn CH3 H CH3 H   0.100 ± 0.024
6gn CH3 H Cl H   0.051 ± 0.022
6hn CH3 H Br H >100 0.028 ± 0.001
6in CH2CH3 H OCF3 H >100 0.049 ± 0.007
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The pharmacokinetic properties of analogues 6a–i and 6an–in were studied in male SD rats following i.v. and oral administration, the results are described in Tables 2 and 3 (see rat po PK results of deuterated and nondeuterated analogues (Table 2) and rat iv PK results of deuterated and nondeuterated analogues (Table 3) in Supporting Information). There were significant changes in the overall pharmacokinetic profile between deuterated versus the corresponding nondeuterated analogues. Overall the deuterated analogues demonstrated a better PK profile. Analogue 6h bearing a 6-bromo substituent was shown to have the longest half-life.

Table 2. Rat PK of Deuterated Analogues (PO).

  dose (mg/kg) BA% Tmax (h) Cmax (μg/L) Vd (L/kg) t1/2 (h) AUC(0–∞) (μg/L·h) CLz/F (L/h/kg)
6a 10 92.8 1.167 20900 0.26 2.1 118331.438 0.086
6b 10 111.8 0.5 21300 0.57 3.35 84789.673 0.125
6g 2.5 110.7 0.5 8945 0.316 3.37 38881.983 0.066
6h 10 107.4 0.417 35300 0.53 5.2 142686.966 0.072
6i 30 60.2 1.25 33475 0.491 4.542 402662.508 0.075
6an 10 78.5 0.438 6592 1.25 3.09 12846.927 0.195
6bn 2.5 56.7 0.375 5338 1.13 3.53 12529.512 0.211
6gn 2.5 128.5 0.25 2720 1.37 1.75 4627.624 0.547
6hn 5 46.3 0.438 11523 0.65 1.51 17190.28 0.298
6in 25 78.6 1.625 29525 0.678 9.542 511440.855 0.049
SC-75416 10 67.5 0.75 8152.5 2.314 6.68 42223.264 0.242
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Table 3. Rat PK of Deuterated Analogues (IV).

  dose (mg/kg) Cmax (μg/L) Vd (L/kg) t1/2 (h) AUC(0–∞) (μg/L·h) CLz/F (L/h/kg)
6a 5 38750 0.287 2.525 63759.714 0.079
6b 5 26175 0.529 2.733 37914.156 0.135
6g 1 9068.75 0.404 3.909 14043.753 0.072
6h 5 38975 0.461 4.186 66436.832 0.077
6i 10 51625 0.39 5.946 222923.421 0.047
6an 1 5035 0.45 1.5 4898.988 0.207
6bn 1 16681 0.38 2.30 8843.992 0.119
6gn 1 4131 0.59 0.59 1440.446 0.706
6hn 2.5 38581 0.15 0.58 14843.535 0.172
6in 5 37250 0.301 5.446 130985.986 0.038
SC-75416 5 37187 1.353 5.78 31271.923 0.16
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Moreover, compounds 6a, 6b, 6g, and 6h were efficacious in several key animal models used to assess the potency of NSAIDs including the air pouch model of PGE2 release and inflammation, paw edema, hyperalgesia in rats and the adjuvant induced arthritis model in mice; see Table 4. All compounds displayed potent efficacy and dose-dependent suppression in each of these disease models.

Table 4. In Vivo Murine Models of Inflammation and Pain.

compd air pouch EC50 (mg/kg) edema EC50 (mg/kg) hyperalgesia EC50 (mg/kg) arthritis ED50 (mg/kg)
celecoxib 0.69 ± 0.10 2.81 ± 0.43 5.0 ± 0.64 1.57 ± 0.21
6a 0.47 ± 0.05 5.18 ± 1.01 2.77 ± 0.25  
6b 0.57 ± 0.10 2.26 ± 0.36 3.82 ± 0.75  
6g 0.45 ± 0.03 6.48 ± 1.25 3.33 ± 0.49  
6h 0.27 ± 0.02 3.00 ± 0.31 2.19 ± 0.32 0.42 ± 0.08
6an 0.77 ± 0.10      
6gn 0.61 ± 0.09      
6hn 0.47 ± 0.06 3.47 ± 0.62 3.52 ± 0.77  
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As an example of the dose-dependent efficacy that we observed for these compounds, a full dose response curve for compound 6h, tested in the rat adjuvant arthritis model is shown in Figure 2.

Figure 2.

Figure 2

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Dose-dependent inhibition of arthritis by compound 6h. Indomethacin (2 mg/kg) was used a positive control and PBS was used as the vehicle (Veh).

Normally, separation of the enantiomers can be accomplished by classical diastereomeric salt formation (selective crystallization using chiral amines, such as α-methylbenzylamine) or by chiral chromatography. We discovered that the protected α,α-diphenyl-2-pyrrolidinemethanol18−20 (compound 12 of Scheme 3) was able to catalyze the formation of S-stereoisomer with more than 80% ee, which is a much more efficient method for making the compound especially for large scale synthesis. Iminium activation of α,β-unsaturated aldehydes 11(21) by 12 leads to enantioselective oxa-Michael addition with 4 and generates enamine intermediates that undergo subsequent intramolecular 6-exo-trig aldol condensations to give the corresponding S-6a–i(18) (see Scheme 3 and page 9 of Supporting Information). X-ray analysis of S-6h established that the absolute configuration at C2 was S; see Figure 3 (see crystal structure of S-6h, Supporting Information). As expected, S-stereoisomers were found to be 2–5 times more potent for COX-2 than their corresponding racemic mixtures; see Table 5.

Scheme 3. Enantioselective Synthesis of (S)-2-(Trifluoromethyl)-2H-chromene-3-carboxylic Acids.

Scheme 3

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R represents substituents on C5, C6, C7, or C8 position. Reagents and conditions: (a) 2-nitrobenzoic acid, CH2Cl2, rt, 24 h; (b) OXONE, DMF, rt, 24 h.

Figure 3.

Figure 3

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Ortep picture of S-6h.

Table 5. COX-2 Inhibition of S-Stereoisomers.

graphic file with name ml-2014-00299q_0009.jpg

  ee value (%) rh IC50 (μM) COX-2 rh IC50 (μM) COX-1
S-6a 86.5 0.016 >100
S-6b 80.7 0.009 >100
S-6g 89.7 0.02 >100
S-6h 90.2 0.014 >100
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In conclusion, we synthesized a series of specifically deuterium substituted benzopyran analogues as potent and selective COX-2 inhibitors. Analogues 6a, 6b, 6g, and 6h showed not only excellent selective COX-2 inhibition but also a significantly improved PK profile. These compounds were efficacious in the gold standard rodent models of inflammation and pain. We also developed a new enantioselective synthetic protocol for preparation of S-stereoisomers for this series of molecules. Collectively, these compounds deserve further evaluation as clinical candidates for the treatment of inflammatory mediated diseases.

Supporting Information Available

Complete experimental details along with the characterizations of the synthesized compound. This material is available free of charge via the Internet at http://pubs.acs.org.

Y.Z. is grateful to the Guangzhou Government and Guangzhou Institute of Biomedicine and Health (GIBH), Chinese Academy of Sciences (CAS) for the joint sponsorship of this work under the Drug Discovery Pipeline (DDP) grant.

The authors declare no competing financial interest.

Supplementary Material

ml500299q_si_001.pdf (4MB, pdf)

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Associated Data

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Supplementary Materials

ml500299q_si_001.pdf (4MB, pdf)

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