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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Med Chem. Author manuscript; available in PMC 2013 August 9.
Published in final edited form as:
PMCID: PMC3430128

6-Trifluoromethyl Pyridoxine: Novel 19F-NMR pH Indicator for In Vivo Detection


pH plays an important role in tumor proliferation, angiogenesis, metabolic control and the efficacy of cytotoxic therapy, and accurate non-invasive assessment of tumor pH promises to provide insight into developmental processes and prognostic information. In this paper, we report the design, synthesis and characterization of two novel pH indicators 6-trifluoromethyl pyridoxine 8 and α4, α5-di-O-[3’-O-(β-D-glucopyranosyl)-propyl]-6-trifluoromethyl pyridoxine 17, and demonstrate 8 as an extracellular 19F-NMR pH probe to assess pHe of various tumors in vivo.

Keywords: pH indicator, synthesis, characterization, 19F-NMR, signal enhancement, tumor in vivo pH detection


The pH gradient between the interstitial and intracellular compartments is involved in many cell regulatory processes, and strongly influences drug uptake.[1] Tumor pH also influences cell thermosensitivity, radiation sensitivity, proliferation and the efficacy of cancer therapy.[2,3] The accurate non-invasive assessment of tumor pH promises to provide insight into the developmental process and prognostic information regarding therapeutic outcome. Previously, we demonstrated that 6-fluoropyridoxine (FPOL, Figure 1) can be used to measure both intra- and extracellular pH simultaneously providing exceptional sensitivity to pH changes in whole blood and the perfused rat heart.[1a,b,46] However, its pKa=8.2 is not ideal for measurements under normal physiological conditions (pH 6.5~7.5). As a continuing work we report herein another strategy through introduction of a trifluoromethyl (CF3−) group instead of a fluorine atom at the 6-position of vitamin B6 with the aims of modifying the pKa to physiological conditions and raising 19F-signal to noise ratio.

Figure 1
The structure of 6-fluoropyridoxine (FPOL).


Design and Synthesis

The introduction of a trifluoromethyl (CF3−) group into an organic compound can bring about remarkable changes in its physical, chemical and biological properties, making them suitable for diverse applications in pharmaceutics and agrochemistry.[7,8] We demonstrated that the introduction of CF3− group in place of fluorine atom in phenols can enhance 19F-NMR signal and modify the pKa value ideal.[9,10]

A wide variety of methods have been developed for introducing a CF3− group into organic compounds,[11] with (trifluoromethyl)trimethylsilane (Me3SiCF3) as a nucleophilic trifluoromethylating reagent becoming the method of choice.[12,13] In this study we demonstrate a strategy that utilizes the iodination derivative 3 of vitamin B6 (1) to react with Me3SiCF3 for synthesis of target compound 6-trifluoromethyl pyridoxine 8 (Figure 2).

Figure 2
Reagents and conditions

A three-step procedure for halogenation of vitamin B6 via 6-aminopyridoxine has been reported for the synthesis of the 19F-NMR pH indicator 6-fluoropyridoxine by the modified Schiemann reaction, resulting in ~28% overall yield.[1,6,14,15] We have now developed a more effective and direct method for obtaining the key intermediate 6-iodopyridoxine (2) in high yield. Reaction of 1 with iodine in 10% aqueous K2CO3 solution and avoiding light afforded 2 in 73% yield. For convenient blocking and de-protection, we initially proposed the acetylation as protecting strategy. Acetylation of 2 as usual work up gave 3, α4, α5-tri-O-acetyl-6-iodopyridoxine (3) in high yield (93%). However, trifluoromethylation of 3 with Me3SiCF3 gave the desired compound 3, α4, α5-tri-O-acetyl-6-trifluoromethyl pyridoxine (4) in only 40% yield. Further purification separated 12% of α4, α5-di-O-acetyl-6-trifluoromethyl pyridoxine (5). Presumably this resulted from the introduction of the highly electron-withdrawing 6-trifluoromethyl group in 4 that makes the para Ac-O3 bond much polarized and activates its C=O group, which competed against the C6-I bond in 3 to consume the trifluoromethylation reagent Me3SiCF3. The proposed mechanism is depicted in Scheme 1.

Scheme 1
The proposed reaction mechanism for the formation of 5.

To improve the process we employed benzyl ether as an alternative protecting group for 3, α4, α5-hydroxyl. Benzylation of 2 with benzyl bromide afforded 3, α4, α5-tri-O-benzyl-6-iodopyridoxine (6) in quantitative yield, which reacted with Me3SiCF3 in a similar procedure for the preparation of 4, giving 3, α4, α5-tri-O-benzyl-6-trifluoromethyl pyridoxine (7) in excellent yield (96%). Overnight hydrogenation of 7 in ethanolic solution with the catalyst of 5% Pd/C provided the partial debenzylation product α4, α5-di-O-benzyl-6-trifluoromethyl pyridoxine (9) in quantitative yield. However, the α4, α5-di-O-benzyl groups could not be removed even with extended reaction times up to 1 week. Testing various acids as co-solvents and catalysts showed that anhydrous AcOH-EtOH (1:10 V/V’) allowed stepwise cleavage of the 3, α5-di-O-benzyl groups in 7 yielding 10 in 1 day, then the α4-O-benzyl group in another day resulting in 8 with total yield of 100%. As expected, 6-trifluoromethyl pyridoxine 8 yielded higher signal to noise than 6-fluoropyridoxine (FPOL) and its derivatives.[1a,b] Importantly, it has an ideal pKa=6.83, but with much less 19F NMR sensitivity to pH (Δδ=1.61ppm) and poorer water solubility (FPOL: 17.8mg/mL, 8: 5.8mg/mL in H2O at room temperature).

We found that modification of 4- and/or 5-methylenehydroxyl moieties of 6-fluoropyridoxine resulted in changes of the pKa and 19F chemical shifts.[1a,b] Previously, we successfully enhanced the solubility of 19F-NMR and 1H-MRI β-galactosidase reporters by conjugating them with carbohydrates.[16] Prompted by these results, we designed another novel molecule 17 with two D-glucoses coupled to the 4- and 5-methylenehydroxyl moieties of 6-trifluoromethyl pyridoxine 8 (Figure 3).

Figure 3
Reagents and conditions

Starting with 2 as the initial molecule, the primary challenge was the regioselective protection among its three hydroxyl groups. However, pKa calculations using the advanced chemistry development software ( indicated the 3-phenolic hydroxyl (pKa=9.27±0.10) is much more acidic than 4- and 5-methylenehydroxyls (pKa=13.31±0.10 and 13.81±0.10, respectively), which suggests that phase-transfer-catalysis at pH=9~10 could provide regioselective protection of the 3-phenolic hydroxyl. To the well-stirred mixture of 2 in bi-phasic CH2Cl2-H2O (pH=9~10) at room temperature which was catalyzed by tetrabutyl-ammonium bromide (TBAB), benzyl bromide (1.1 equiv.) was added dropwise over a period of 4~5h. 3-O-benzyl-6-iodopyridoxine 11 was isolated as a major product in 74% yield. Its stereochemistry was confirmed by its 1H-NMR spectrum where α4, α5-CH2 exhibit doublets with coupling constants of JH-4,HO-4=5.6Hz at 4.87ppm, and JH-5,HO-5=6.4Hz at 4.73ppm. Treatment of 11 with an excess of allyl bromide gave 3-O-benzyl-α4, α5-di-O-allyl-6-iodopyridoxine 12 in quantitative yield. It was then subjected to the procedure described for the preparation of 4 and 7 giving 3-O-benzyl-α4, α5-di-O-allyl-6-trifluoromethyl pyridoxine 13 in an excellent yield (92%). The diallylated derivative 13 proceeded regioselective hydroboration by using 9-borabicyclo-[3.3.1] nonane (9-BBN), and subsequent alkaline oxidation with H2O2 giving 3-O-benzyl-α45-di-O-(3-hydroxypropyl)-6-trifluoromethyl pyridoxine 14 in 86% yield.

Condensation of 14 with 2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl bromide accomplished 3-O-benzyl-α4, α5-di-O-[3’-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl)-propyl]-6-trifluoromethyl pyridoxine 15 in 88% yield. The ESI-MS displayed the expected molecular ion at m/z 1103 and quasimolecular ion at m/z 1104 [M+H], corresponding to the fully adorned derivative with two 2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl units. The identity of 15 was established using 1H and 13C-NMR based on the anomeric protons H-1’ and H-1” of D-glucoses at 4.48 and 4.41ppm, respectively, with two well resolved doublets J1’,2’=J1”,2”=~8.0 Hz and J2’,3’=J2”,3”=~10Hz, which confirmed both D-glucoses in the β-configuration with 4C1 chair conformation, whereas the anomeric carbons C-1’ and C-1” at 101.02 ppm are in accordance.

Compound 15 was deacetylated with NH3/MeOH from 0°C to room temperature giving 3-O-benzyl-α4, α5-di-O-[3’-O-(β-D-glucopyranosyl)-propyl]-6-trifluoromethyl pyridoxine 16 in quantitative yield, and followed by hydrogenation catalyzed with 5% Pd/C overnight affording quantitative yield of target compound α4, α5-di-O-[3’-O-(β-D-glucopyranosyl)-propyl]-6-trifluoromethyl pyridoxine 17 with an overall yield of 52%.

Characterization as 19F-NMR pH Indicator

The 19F chemical shifts upon pH changes (pH electrode) were measured with respect to sodium trifluoroacetate (NaTFA, δF=0ppm). Both 8 and 17 exhibited single narrow 19F-NMR signals in 0.9% saline or PBS essentially invariant (Δδ≤0.03ppm) with temperatures ranging from 25 to 37°C. Figure 4 shows the titration curves of 8 and 17 in saline between pH 2–13 at 25°C. From the titration curves their pKa, δ(acid) and δ(base) were determined from the Henderson-Hasselbach equation[1a,b] (Table 1).

Figure 4
The titration curves of 8 and 17 in saline at 25°C.
Table 1
Acidities and 19F-NMR/pH properties of 8 and 17 at 25°C.

Given that both 8 and 17 show the feasibility as 19F-NMR pH indicators, 8 has a more ideal pKa for sampling biological system in vivo, making it favored for further evaluation.

19F-NMR pH Assessment in Perfused Heart and Whole Blood

The 19F-NMR spectrum (376 MHz) of 8 in Langendorff perfused rat heart exhibited only a single 19F-resonance at δF=16.41ppm corresponding to pH=7.39 (Figure 5a). The intra- and extracellular inorganic phosphates of Langendorff perfused rat heart was sampled by 31P-NMR, which provided the intracellular pHi=7.04 (δPi=4.88ppm) and extracellular pHe=7.42 (δPe=5.32ppm).[1a,b] When the 19F-NMR of 8 is compared to these results it becomes evident that 8 does not enter cells and reports only the extracellular pHe. This was confirmed by the 19F-NMR spectrum of 8 in whole rabbit blood which showed a single 19F peak at δF=16.52ppm (pHe=7.44) (Figure 5b).

Figure 5
19F-NMR spectra (376MHz, 25°C) of 8 in (a) Langendorff perfused rat heart, δF=16.41ppm (pHe=7.39); (b) whole rabbit blood, δF=16.52ppm (pHe=7.44).

In vivo 19F-NMR pH Measurements in Tumors

Extracellular pHe in human tumors has been shown to be associated with tumorigenic transformation, chromosomal rearrangements, induction of growth factors and proteases, extracellular matrix breakdown, and increased migration and invasion.[2c,e] To evaluate the efficacy of this novel 19F-NMR pHe reporter molecule for sampling the tumor microenvironment, it was directly injected into rats bearing pedicle tumors. Firstly, pHe reporter 8 (320mg/kg) and NaTFA (200mg/kg) in DMSO/saline (1:3 V/V’) were injected i.p. into an anesthetized (with isoflurane) Copenhagen rat bearing a Dunning R3327-AT1 prostate tumor (tumor size: 2.4cm×3.1cm×1.8cm). 8 was detected by 19F-NMR in the tumor 30 minutes after injection. The single broad 19F-signal centered at δF=16.28ppm indicated tumor heterogeneity with mean pHe=7.20.[1b]

Similarly, 8 was detected in a 13762NF rat mammary tumor (tumor size: 1.8cm×1.0cm×2.0cm) after 8 minutes following an i.p. injection of the same doses into a Fisher 344 rat. By comparing the 19F-NMR spectral intensities it is estimated that approximately 90% of 8 was in the 13762NF tumor. A broad single 19F-resonance was obtained at δF=16.23ppm (pHe=7.14) with biological clearance over 100 minutes (Figure 6A). This pH value was commensurate with micro electrode measurements in three Fisher 344 rats bearing 13762NF breast tumors various in 3.2, 7.5 and 9.2cm3, showing the most frequent pHe=7.03 (Figure 6B).

Figure 6
(A) In vivo 19F-NMR spectrum (188MHz, 37°C) of 8 (320mg/kg) in Fisher 344 rat bearing pedicle 13762NF breast tumor, i.p. injection, tumor size: 1.8cm×1.0cm×2.0cm, δF=16.23ppm corresponding to pHe=7.14, acquisition time: ...

Encouraged by these in vivo measurements of tumor pHe, we also investigated 6-trifluoromethyl pyridoxine 8 in other animal models. After i.p. injection of 8 (12mg, 54µmol) in DMSO/H2O (1:3 V/V’) into a nude mouse bearing a MatLu rat prostate tumor grown on the thigh (tumor size: 1.6cm×1.4cm×1.0cm), a broad, single 19F peak was observed at δF(8)=15.96 ppm (pHe=6.48) with an acquisition time of 7 minutes (Figure 7).

Figure 7
In vivo 19F-NMR spectrum (188MHz, 37°C) of 8 (12mg) in nude mouse bearing MatLu rat prostate tumor on thigh, i.p. injection, tumor size: 1.6cm×1.4cm×1.0cm, δF(8)=15.96ppm corresponding to pHe=6.48, acquisition time: 7 minutes. ...


Given the relevance of pHi/pHe to tumor development and prognostic outcome, non-invasive techniques to sample cellular pH in vivo have great potential and are increasingly important in therapeutic oncology.[13] As the most electronegative element, fluorine has played a key role in medicinal chemistry; the incorporation of fluorine and/or fluorine-containing groups into an organic molecule often drastically perturbs the properties of the parent compound.[7,8] 19F-MRS has been widely utilized in in vivo studies on drug absorption, distribution, metabolism and excretion due to its favorable MR properties, simplicity, and high sensitivity.[1a,b] In this study, we have successfully synthesized two novel 19F-NMR pH indicators 8 and 17, and identified the following useful characteristics which make them well-suited for the in vivo assessment of pH using 19F-MRS: (a) ideal pKa (6.83~7.84); (b) sensitivity to pH (~0.40 ppm/pH unit); (c) 19F-chemical shift response to pH (ΔδF=1.38~1.61ppm); (d) 19F-signal enhancement, in which 8 was shown to be capable of in vivo sampling pHe in various tumor models. Noting these features of 8 as a 19F-MRS pHe molecular probe, we believe it has promising potential in 19F-MRI investigations of the tumor microenvironment for effective characterization of tumor heterogeneity with spatial and temporal resolution.


General Methods

NMR spectra were recorded on a Varian Inova 400 spectrometer (400 MHz for 1H, 100 MHz for 13C, 376 MHz for 19F, 121 MHz for 31P), 1H and 13C chemical shifts are referenced to TMS as internal standard with CDCl3, or DMSO-d6 as solvents; 19F to a dilute solution of NaTFA in a capillary as external standard, chemical shifts are given in ppm. Mass spectra were obtained by positive and negative ESI-MS using a Micromass Q-TOF hybrid quadrupole/time-of-flight instrument (Micromass UK Ltd). Microanalyses were performed on a Perkin-Elmer 2400CHN microanalyser.

Hg(CN)2 was dried before use at 50°C for 1h, CH2Cl2 was dried over Drierite, acetonitrile was dried on CaH2 and kept over molecular sieves under nitrogen. Solutions in organic solvents were dried with anhydrous sodium sulfate, and concentrated in vacuo below 45°C. 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide was purchased from the Sigma Chemical Company. Column chromatography was performed on silica gel (200~300 mesh), and silica gel GF254 used for TLC was purchased from the Aldrich Chemical Company. Detection was effected by spraying the plates with 5% ethanolic H2SO4 (followed by heating at 110°C for ~10 minutes) or by direct UV illumination of the plate. The purity of the final products was determined by HPLC with ≥ 95%.

6-Iodopyridoxine 2

To a solution of pyridoxine 1 (3.4 g, 20.0 mmol) in 10% K2CO3 aqueous solution (60 mL), iodine (5.04 g, 20.0 mmol) was added. The reaction mixture was vigorously stirred in the dark at room temperature for 2~3 h, after addition of Na2SO3 (320 mg), the reaction was quenched with concentrated HCl up to pH 3, then the precipitate was filtered and dried in vacuo over NaOH to give 2 (4.28 g, 73%) as a yellow powder, 1H-NMR (DMSO-d6), δH: 9.51 (1 H, s, HO-3), 5.82 (1 H, br, α5-OH), 5.15 (1H, br, α4-OH), 4.80 (2 H, d, J = 2.8 Hz, CH2-5), 4.57 (2 H, d, J = 3.2 Hz, CH2-4), 2.31 (3 H, s, CH3-2) ppm; 13C-NMR (DMSO-d6), δC: 150.43 (s, Py-C), 148.25 (s, Py-C), 136.21 (s, Py-C), 134.97 (s, Py-C), 112.11 (s, Py-C), 63.99 (s, CH2-5), 57.05 (s, CH2-4), 18.93 (s, CH3-2) ppm.

Anal. Calcd. for C8H10NO3l (%): C, 32.56, H, 3.42, N, 4.75; Found: C, 32.51, H, 3.39, N, 4.71.

3, α4, α5-Tri-O-acetyl-6-Iodopyridoxine 3

A solution of 2 (0.90 g, 3.0 mmol) in pyridine (20 mL) was treated with acetic anhydride (9 mL). After being stirred from 0°C to r.t. overnight, the mixture was co-evaporated with toluene under reduced pressure and the residue purified by flash silica gel column chromatography (eluent: 2:1 cyclohexane-EtOAc) to afford 3 (1.19 g, 93%) as white crystals, Rf 0.50 (3:2 cyclohexane-EtOAc), 1H-NMR (CDCl3), δH: 5.18 (2 H, s, CH2-5), 5.15 (2 H, s, CH2-4), 2.40 (3 H, s, CH3-2), 2.05, 2.03, 2.01 (9 H, 3s, 3×CH3CO) ppm; 13C-NMR (CDCl3), δC: 173.97, 170.73, 168.43 (3s, 3×CH3CO), 152.83 (s, Py-C), 150.33 (s, Py-C), 132.99 (s, Py-C), 129.83 (s, Py-C), 113.02 (s, Py-C), 66.33 (s, CH2-5), 58.76 (s, CH2-4), 20.94, 20.88, 19.63 (3s, 3×CH3CO), 19.61 (s, CH3-2) ppm.

Anal. Calcd. for C14H16NO6l (%): C, 39.92, H, 3.83, N, 3.33; Found: C, 39.90, H, 3.80, N, 3.30.

3, α4, α5-Tri-O-Acetyl-6-Trifluoromethyl Pyridoxine 4

To a well stirred mixture of 3 (1.0 g, 2.4 mmol), CuI (456 mg, 2.4 mmol, 1.0 equiv.) and KF (168 mg, 2.9 mmol, 1.2 equiv.) in N, N-dimethylformamide (DMF, 5 mL) and N-methylpyrrolidine (NMP, 5 mL), Me3SiCF3 (537 µL, 2.9 mmol, 1.2 equiv.) was added under an argon atmosphere in the dark, then the reaction mixture was stirred at 80°C in a sealed glass pressure tube (15 mL) for 24h. The mixture was diluted with CH2Cl2 (120 mL), filtered through Celite, washed with water, dried (Na2SO4) and concentrated in vacuo. The residue was purified on a silica gel column (2:1 cyclohexane-EtOAc) to yield 4 (0.35 g, 40%) as a syrup, Rf 0.37 (2:1 cyclohexane-EtOAc), 1H-NMR (CDCl3), δH: 5.34 (2 H, s, CH2-5), 5.16 (2 H, s, CH2-4), 2.39 (3 H, s, CH3-2), 2.38, 2.10, 2.02 (9 H, 3s, 3×CH3CO) ppm; 13C-NMR (CDCl3), δC: 170.54, 170.22, 168.41 (3s, 3×CH3CO), 145.04 (s, Py-C2’), 137.81 (s, Py-C3’), 154.80 (s, Py-C4’), 133.90 (q, 3JF-C = 20.6 Hz, Py-C5’), 140.27 (q, 2JF-C = 32.6 Hz, Py-C6’), 121.53 (q, 1JF-C = 272.6 Hz, CF3), 66.27 (s, CH2-5), 57.39 (s, CH2-4), 20.89, 20.72, 20.68 (3s, 3×CH3CO), 19.63 (s, CH3-2) ppm.

Anal. Calcd. for C15H16NO6F3 (%): C, 49.59, H, 4.44, N, 3.86; Found: C, 49.55, H, 4.43, N, 3.84.

α4, α5-Di-O-Acetyl-6-Trifluoromethyl Pyridoxine 5

88 mg, 12% as a syrup, Rf 0.27 (2:1 cyclohexane-EtOAc), 1H-NMR (CDCl3), δH: 5.24 (2 H, s, CH2-5), 5.06 (2 H, s, CH2-4), 2.33 (3 H, s, CH3-2), 2.09, 2.02 (6 H, 2s, 2×CH3CO) ppm; 13C-NMR (CDCl3), δC: 170.12, 168.38 (2s, 2×CH3CO), 144.84 (s, Py-C2’), 137.41 (s, Py-C3’), 154.77 (s, Py-C4’), 133.30 (q, 3JF-C = 18.6 Hz, Py-C5’), 141.67 (q, 2JF-C = 32.8 Hz, Py-C6’), 121.13 (q, 1JF-C = 272.5 Hz, CF3), 66.17 (s, CH2-5), 57.29 (s, CH2-4), 20.68, 20.57 (2s, 2×CH3CO), 19.56 (s, CH3-2) ppm.

Anal. Calcd. for C13H14NO5F3 (%): C, 48.60, H, 4.39, N, 4.36; Found: C, 48.55, H, 4.36, N, 4.32.

3, α4, α5-Tri-O-Benzyl-6-Iodopyridoxine 6

A solution of benzyl bromide (1.43 g, 8.0 mmol) in dry DMF (15 mL) was added dropwise over a period of 1~2h to a well stirred dry DMF (70 mL) solution of 2 (0.69 g, 2.4 mmol) and NaH (341 mg, 8.6 mmol, 60% dispersion in mineral oil), and the stirring continued for additional 4~5h, at the end of time TLC (4:1 cyclohexane-EtOAc) showed reaction complete, then MeOH (15 mL) was added slowly to react with the excess of the NaH. After most of the DMF was removed under reduced pressure at 55°C, the residue was dissolved in CH2Cl2 (125 mL) and washed with water, dried (Na2SO4), filtered and evaporated, the residue was purified by column chromatography on silica gel with 4:1 cyclohexane-EtOAc as the eluent to afford quantitatively 6 (1.32 g) as a syrup, Rf 0.56 (4:1 cyclohexane-EtOAc), 1H-NMR (CDCl3), δH: 7.38 ~ 7.27 (15 H, m, Ph-H), 4.84 (2 H, s, PhCH2-O3), 4.64 (2 H, s, α4-OCH2Ph), 4.56 (2 H, s, CH2-4), 4.54 (2 H, s, CH2-5), 4.41 (2 H, s, α5-OCH2Ph), 2.48 (3 H, s, CH3-2) ppm; 13C-NMR (CDCl3), δC: 155.28 (s, Py-C), 152.76 (s, Py-C), 140.29 (s, Py-C), 136.24 (s, Py-C), 128.66 ~ 128.16 (m, Ph-C), 119.57 (s, Py-C), 76.97 (s, PhCH2-O3), 73.68 (s, α4-OCH2Ph), 73.51 (s, α5-OCH2Ph), 71.87 (s, CH2-4), 63.24 (s, CH2-5), 19.74 (s, CH3-2) ppm.

Anal. Calcd. for C29H28NO3l (%): C, 61.60, H, 4.99, N, 2.48; Found: C, 61.56, H, 4.96, N, 2.45.

3, α4, α5-Tri-O-Benzyl-6-Trifluoromethyl Pyridoxine 7

Trifluoromethylation of 6 (0.89 g, 1.6 mmol) with Me3SiCF3 (356 µL, 1.9 mmol, 1.2 equiv.) in the presence of CuI (304 mg, 1.6 mmol, 1.0 equiv.) and KF (110 mg, 1.9 mmol, 1.2 equiv.) in DMF-NMP (10 mL, 1:1 V/V’) under an argon atmosphere in the dark, according to the procedures described for the preparation of 4, furnished 3, α4, α5-tri-O-benzyl-6-trifluoromethyl pyridoxine 7 (0.78 g, 96%) as a syrup, Rf 0.64 (4:1 cyclohexane-EtOAc), 1H-NMR (CDCl3), δH: 7.40 ~ 7.28 (15 H, m, Ph-H), 4.89 (2 H, s, PhCH2-O3), 4.65 (2 H, s, α4-OCH2Ph), 4.59 (2 H, s, CH2-4), 4.45 (2 H, s, CH2-5), 4.40 (2 H, s, α5-OCH2Ph), 2.54 (3 H, s, CH3-2) ppm; 13C-NMR (CDCl3), δC: 141.44 (s, Py-C2’), 136.50 (s, Py-C3’), 153.44 (s, Py-C4’), 137.60 (q, 3JF-C = 21.4 Hz, Py-C5’), 142.07 (q, 2JF-C = 32.1 Hz, Py-C6’), 122.36 (q, 1JF-C = 273.9 Hz, CF3), 128.92 ~ 128.10 (m, Ph-C), 73.86 (s, PhCH2-O3), 73.70 (s, α4-OCH2Ph), 64.08 (s, α5-OCH2Ph), 64.05 (s, CH2-4), 62.63 (s, CH2-5), 20.01 (s, CH3-2) ppm.

Anal. Calcd. for C30H28NO3F3 (%): C, 70.99, H, 5.56, N, 2.76; Found: C, 70.96, H, 5.54, N, 2.73.

6-Trifluoromethyl Pyridoxine 8

Hydrogenation (H2, 30 psi) of 7 (0.70 g, 1.4 mmol) in anhydrous AcOH-EtOH (70 mL, 1:10 V/V’) catalyzed by Pd/C (5%, 250 mg) for 2 days furnished the target molecule 8 (0.33 g, 100%) as crystals, Rf 0.34 (1:1 cyclohexane-EtOAc), 1H-NMR (DMSO-d6), δH: 4.88 (2 H, s, CH2-5), 4.59 (2 H, s, CH2-4), 4.50 (3 H, br, 3-OH, α4-OH, α5-OH), 2.40 (3 H, s, CH3-2) ppm; 13C-NMR (DMSO-d6), δC: 132.23 (s, Py-C2’), 134.51 (s, Py-C3’), 153.97 (s, Py-C4’), 145.85 (s, Py-C5’), 133.51 (q, 2JF-C = 31.3 Hz, Py-C6’), 122.94 (q, 1JF-C = 272.4 Hz, CF3), 56.55 (s, CH2-4), 55.21 (s, CH2-5), 19.27 (s, CH3-2) ppm.

Anal. Calcd. for C9H10NO3F3 (%): C, 45.58, H, 4.25, N, 5.91; Found: C, 45.54, H, 4.23, N, 5.89.

α4, α5-Di-O-Benzyl-6-Trifluoromethyl Pyridoxine 9

0.58 g, 100%, syrup, Rf 0.52 (4:1 cyclohexane-EtOAc), 1H-NMR (CDCl3), δH: 8.22 (1 H, s, 3-OH), 7.62 ~ 7.55 (10 H, m, Ph-H), 4.91 (2 H, s, α4-OCH2Ph), 4.79 (2 H, s, CH2-4), 4.73 (2 H, s, CH2-5), 4.72 (2 H, s, α5-OCH2Ph), 2.66 (3 H, s, CH3-2) ppm; 13C-NMR (CDCl3), δC: 138.12 (s, Py-C2’), 138.17 (s, Py-C3’), 149.47 (s, Py-C4’), 147.26 (s, Py-C5’), 140.02 (q, 2JF-C = 32.3 Hz, Py-C6’), 121.54 (q, 1JF-C = 273.2 Hz, CF3), 128.50 ~ 127.20 (m, Ph-C), 71.96 (s, α4-OCH2Ph), 71.64 (s, α5-OCH2Ph), 67.09 (s, CH2-4), 62.58 (s, CH2-5), 19.88 (s, CH3-2) ppm.

Anal. Calcd. for C23H22NO3F3 (%): C, 66.18, H, 5.31, N, 3.36; Found: C, 66.13, H, 5.30, N, 3.34.

α4-O-Benzyl-6-Trifluoromethyl Pyridoxine 10

0.46 g, 100%, syrup, Rf 0.34 (4:1 cyclohexane-EtOAc), 1H-NMR (DMSO-d6), δH: 7.38 ~ 7.34 (5 H, m, Ph-H), 5.05 (2 H, s, α4-OCH2Ph), 4.70 (2 H, br, 3-OH, α5-OH), 4.63 (2 H, s, CH2-4), 4.54 (2 H, d, JH-5,HO-5 = 6.2 Hz, CH2-5), 2.52 (3 H, s, CH3-2) ppm; 13C-NMR (DMSO-d6), δC: 132.03 (s, Py-C2’), 137.30 (s, Py-C3’), 153.76 (s, Py-C4’), 147.93 (s, Py-C5’), 136.58 (q, 2JF-C = 32.6 Hz, Py-C6’), 122.54 (q, 1JF-C = 272.8 Hz, CF3), 128.78 ~ 128.27 (m, Ph-C), 73.22 (s, α4-OCH2Ph), 64.40 (s, CH2-4), 61.07 (s, CH2-5), 18.99 (s, CH3-2) ppm.

Anal. Calcd. for C16H16NO3F3 (%): C, 58.71, H, 4.93, N, 4.28; Found: C, 58.68, H, 4.90, N, 4.27.

3-O-Benzyl-6-Iodopyridoxine 11

To a well stirred CH2Cl2-H2O (20 mL, 1:1 V/V’) bi-phasic mixture (pH 9~10) of 2 (1.0 g, 3.4 mmol) and TBAB (0.10 g, 0.31 mmol) as the phase-transfer catalyst, a solution of benzyl bromide (0.65 g, 3.73 mmol, 1.1 equiv.) in CH2Cl2 (10 mL) was added dropwise over a period of 4~5h at room temperature, and the stirring continued for an additional hour. The mixture was extracted with CH2Cl2 (4×20 mL), washed free of alkali, dried (Na2SO4), and concentrated, the residue was purified by column chromatography on silica gel with 3:2 cyclohexane-EtOAc as the eluent to afford major product 11 (1.31 g, 74%) as white crystalline, Rf 0.68 (1:2 cyclohexane-EtOAc), 1H-NMR (CDCl3), δH: 7.41 ~ 7.37 (5 H, m, Ar-H), 4.92 (2 H, s, PhCH2), 4.87 (2 H, d, JH-4,HO-4 = 5.6 Hz, CH2-4), 4.73 (2 H, d, JH-5,HO-5 = 6.4 Hz, CH2-5), 3.73 (2 H, br, α4-OH, α5-OH, exchangeable with D2O), 2.50 (3 H, s, CH3-2) ppm; 13C-NMR (CDCl3), δC: 155.56 (s, Py-C), 152.03 (s, Py-C), 142.98 (s, Py-C), 136.22 (s, Py-C), 129.04 ~ 128.69 (m, Ph-C), 117.97 (s, Py-C), 76.93 (s, PhCH2-O3), 67.03 (s, CH2-4), 57.44 (s, CH2-5), 19.83 (s, CH3-2) ppm.

Anal. Calcd. for C15H16NO3I (%): C, 46.77, H, 4.19, N, 3.64; Found: C, 46.74, H, 4.17, N, 3.62.

3-O-Benzyl-α4, α5-Di-O-Allyl-6-Iodopyridoxine 12

To a well stirred dry DMF (80 mL) solution of 11 (1.20 g, 3.1 mmol) and NaH (0.50 g, 12.5 mmol, 60% dispersion in mineral oil), allyl bromide (1.13 g, 9.33 mmol) in dry DMF (10 mL) was added dropwise over a period of 1~2h, and the stirring continued for additional 4~5h, at the end of time TLC (4:1 cyclohexane-EtOAc) showed reaction complete, then MeOH (15 mL) was added slowly to react with the excess of the NaH. After most DMF was removed under reduced pressure at 55°C, the residue was dissolved in CH2Cl2 (150 mL) and washed with water, dried (Na2SO4), filtered and evaporated, the residue was purified by column chromatography on silica gel with 4:1 cyclohexane-EtOAc as the eluent to afford quantitatively 12 (1.45 g) as a syrup, Rf 0.58 (4:1 cyclohexane-EtOAc), 1H-NMR (CDCl3), δH: 7.43 ~ 7.36 (5 H, m, Ar-H), 5.98 (1 H, dq, 3J1’,2’ = 1.8 Hz, 3J2’,3a’ = 20.0 Hz, 3J2’,3b’ = 9.0 Hz, H-2’), 5.91 (1 H, dq, 3J1”,2” = 1.8 Hz, 3J2”,3a” = 22.4 Hz, 3J2”,3b” = 9.0 Hz, H-2”), 5.35 (1 H, dt, 4J1’,3’ = 1.0 Hz, 2J3a’,3b’ = 2.4 Hz, H-3’), 5.26 (1 H, dt, 4J1’,3’ = 1.0 Hz, 2J3a”,3b” = 2.4 Hz, H-3”), 4.89 (2 H, s, PhCH2), 4.67 (2 H, s, CH2-4), 4.60 (2 H, s, CH2-5), 4.11 (2 H, dt, H-1’), 4.02 (2 H, dt, H-1”), 2.49 (3 H, s, CH3-2) ppm; 13C-NMR (CDCl3), δC: 155.27 (s, Py-C), 152.84 (s, Py-C), 140.40 (s, Py-C), 136.65 (s, Py-C), 134.63 (s, α4-OCH2CH=CH2), 134.26 (s, α5-OCH2CH=CH2), 128.89 ~ 128.14 (m, Ph-C), 119.45 (s, Py-C), 118.25 (s, α4-OCH2CH=CH2), 118.03 (s, α5-OCH2CH=CH2), 76.97 (s, PhCH2-O3), 72.46 (s, α4-OCH2CH=CH2), 72.29 (s, α5-OCH2CH=CH2), 71.96 (s, CH2-4), 63.29 (s, CH2-5), 19.76 (s, CH3-2) ppm.

Anal. Calcd. for C21H24NO3I (%): C, 54.20, H, 5.20, N, 3.01; Found: C, 54.16, H, 5.18, N, 3.00.

3-O-Benzyl-α4, α5-Di-O-Allyl-6-Trifluoromethyl Pyridoxine 13

Trifluoromethylation of 12 (1.10 g, 2.4 mmol) with Me3SiCF3 (537 µL, 2.9 mmol, 1.2 equiv.) in the presence of CuI (456 mg, 2.4 mmol, 1.0 equiv.) and KF (168 mg, 2.9 mmol, 1.2 equiv.) in DMF-NMP (10 mL, 1:1 V/V’) under an argon atmosphere in the dark, according to the procedures described for the preparation of 4 and 7, yielded 13 (0.90 g, 92%) as a syrup, Rf 0.71 (3:1 cyclohexane-EtOAc), 1H-NMR (CDCl3), δH: 7.45 ~ 7.34 (5 H, m, Ar-H), 5.97 (1 H, dq, 3J1’,2’ = 4.8 Hz, 3J2’,3a’ = 20.8 Hz, 3J2’,3b’ = 9.2 Hz, H-2’), 5.91 (1 H, dq, 3J1”,2” = 1.8 Hz, 3J2”,3a” = 22.0 Hz, 3J2”,3b” = 9.0 Hz, H-2”), 5.32 (1 H, dt, 4J1’,3’ = 1.2 Hz, 2J3a’,3b’ = 2.6 Hz, H-3’), 5.23 (1 H, dt, 4J1’,3’ = 0.8 Hz, 2J3a”,3b” = 2.2 Hz, H-3”), 4.96 (2 H, s, PhCH2), 4.71 (2 H, s, CH2-4), 4.61 (2 H, s, CH2-5), 4.10 (2 H, dt, H-1’), 4.05 (2 H, dt, H-1”), 2.58 (3 H, s, CH3-2) ppm; 13C-NMR (CDCl3), δC: 136.50 (s, Py-C2’), 136.12 (s, Py-C3’), 153.32 (s, Py-C4’), 141.73 (q, 3JF-C = 6.8 Hz, Py-C5’), 134.94 (q, 2JF-C = 32.4 Hz, Py-C6’), 122.31 (q, 1JF-C = 273.9 Hz, CF3), 133.38 (s, α4-OCH2CH=CH2), 131.18 (s, α5-OCH2CH=CH2), 129.64 ~ 127.45 (m, Ph-C), 118.24 (s, α4-OCH2CH=CH2), 118.21 (s, α5-OCH2CH=CH2), 78.41 (s, PhCH2-O3), 72.69 (s, α4-OCH2CH=CH2), 72.39 (s, α5-OCH2CH=CH2), 71.21 (s, CH2-4), 64.04 (s, CH2-5), 20.59 (s, CH3-2) ppm.

Anal. Calcd. for C22H24NO3F3 (%): C, 64.86, H, 5.94, N, 3.44; Found: C, 64.82, H, 5.91, N, 3.42.

3-O-Benzyl-α4, α5-Di-O-(3-Hydroxypropyl)-6-Trifluoromethyl Pyridoxine 14

To a solution of 13 (0.41 g, 1.0 mmol) in dry dioxane (10 mL) was added 9-BBN (8 mL, 4 mmol, 0.5 M solution in THF) dropwise at 0°C under argon. The reaction mixture was stirred at r.t. for 24h, cooled to 0°C, aqueous NaOH (3 M, 8 mL) and 30% H2O2 (1.3 mL) were added. The reaction mixture was stirred at r.t. for 2 days, the aqueous phase was extracted with ethyl acetate (4×50 mL). The combined organic phases were washed with saturated NaCl solution, and dried (Na2SO4). The solution was filtered and the filtrate concentrated in vacuo to give an almost colorless syrup, which was purified by column chromatography on silica gel with 1:3 cyclohexane-EtOAc as the eluent to afford 14 (0.38 g, 86%) as a syrup, Rf 0.35 (1:4 cyclohexane-EtOAc), 1H-NMR (CDCl3), δH: 7.42 ~ 7.36 (5 H, m, Ar-H), 4.93 (2 H, s, PhCH2), 4.86 (2 H, s, CH2-4), 4.62 (2 H, s, CH2-5), 3.74 ~ 3.60 (8 H, m, α4-OCH2CH2CH2OH, α5-OCH2CH2CH2OH), 2.55 (3 H, s, CH3-2), 2.92 (2H, br, α4-O(CH2)3OH, α5-O(CH2)3OH), 1.86 ~ 1.76 (4 H, m, α4-OCH2CH2CH2OH, α5-OCH2CH2CH2OH) ppm; 13C-NMR (CDCl3), δC: 135.70 (s, Py-C2’), 130.81 (s, Py-C3’), 153.36 (s, Py-C4’), 136.34 (q, 3JF-C = 8.3 Hz, Py-C5’), 141.59 (q, 2JF-C = 32.0 Hz, Py-C6’), 122.22 (q, 1JF-C = 273.9 Hz, CF3), 129.00 ~ 127.84 (m, Ph-C), 76.85 (s, PhCH2-O3), 72.66 (s, CH2-4), 69.57, 69.56 (2s, α4-OCH2(CH2)2OH, α5- OCH2(CH2)2OH), 63.72 (s, CH2-5), 60.79, 60.63 (2s, α4-O(CH2)2CH2OH, α5-O(CH2)2CH2OH), 32.36, 32.27 (2s, α4-OCH2CH2CH2OH, α5-OCH2CH2CH2OH), 19.78 (s, CH3-2) ppm.

Anal. Calcd. for C22H28NO5F3 (%): C, 59.59, H, 6.36, N, 3.16; Found: C, 59.56, H, 6.34, N, 3.14.

3-O-Benzyl-α4, α5-Di-O-[3’-O-(2, 3, 4, 6-Tetra-O-Acetyl-β-D-Glucopyranosyl)-Propyl]-6-Trifluoromethyl Pyridoxine 15

A solution of 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide (0.75 g, 1.45 mmol, 1.2 equiv.) in anhydrous CH2Cl2 (5 mL) was added dropwise into the solution of 14 (0.34 g, 0.75 mmol) and Hg(CN)2 (0.52 g, 1.21 mmol) as a promoter in dry acetonitrile (10 mL) containing powdered molecular sieves (4 Å, 1.3 g) with vigorous stirring at r.t. under an argon atmosphere in the dark for 12h. The mixture was diluted with CH2Cl2 (60 mL), filtered through Celite, washed with water, dried (Na2SO4) and concentrated in vacuo. The residue was purified on a silica gel column (1:1 cyclohexane-EtOAc) to yield the title compound 15 (0.73 g, 88%) as a syrup, Rf 0.50 (1:1 cyclohexane-EtOAc), 1H-NMR (CDCl3), δH: 7.45 ~ 7.39 (5 H, m, Ar-H), 4.94 (2 H, s, PhCH2), 4.68 (2 H, s, CH2-4), 4.64 (2 H, s, CH2-5), 4.15 ~ 4.09 (4 H, m, α4-OCH2(CH2)2O−, α5-OCH2(CH2)2O−), 3.62 ~ 3.54 (4 H, m, α4-O(CH2)2CH2O−, α5-O(CH2)2CH2O−), 2.57 (3 H, s, CH3-2), 1.94 ~ 1.84 (4 H, m, α4-OCH2CH2CH2O−, α5-OCH2CH2CH2O−), 4.48 (1 H, d, J1’,2’ = 8.0 Hz, H-1’), 4.41 (1 H, d, J1”,2” = 7.6 Hz, H-1”), 5.00 (1 H, dd, J2’,3’ = 10.0 Hz, H-2’), 4.96 (1 H, dd, J2”,3” = 9.8 Hz, H-2”), 5.20 (1 H, dd, J3’,4’ = 3.2 Hz, H-3’), 5.17 (1 H, dd, J3”,4” = 3.6 Hz, H-3”), 5.10 (1 H, dd, J4’,5’ = 5.6 Hz, H-4’), 5.04 (1 H, dd, J4”,5” = 5.4 Hz, H-4”), 3.70 (1 H, m, H-5’), 3.68 (1 H, m, H-5”), 4.26 (1 H, dd, J5’,6a’ = 4.8 Hz, J6a’,6b’ = 13.6 Hz, H-6a’), 4.23 (1 H, dd, J5”,6a” = 4.4 Hz, J6a”,6b” = 12.4 Hz, H-6a”), 3.93 (1 H, dd, J5’,6b’ = 5.6 Hz, H-6b’), 3.90 (1 H, dd, J5”,6b” = 4.8 Hz, H-6b”), 2.06 ~ 1.99 (24 H, 8s, 8×CH3CO) ppm; 13C-NMR (CDCl3), δC: 171.23 ~ 169.37 (8s, 8×CH3CO), 136.37 (s, Py-C2’), 131.00 (s, Py-C3’), 153.41 (s, Py-C4’), 141.18 (q, 3JF-C = 11.5 Hz, Py-C5’), 141.82 (q, 2JF-C = 32.8 Hz, Py-C6’), 122.27 (q, 1JF-C = 274.0 Hz, CF3), 129.00 ~ 127.88 (m, Ph-C), 76.88 (s, PhCH2-O3), 71.49 (s, CH2-4), 68.14 (s, α4-OCH2(CH2)2O−), 68.02 (s, α5-OCH2(CH2)2O−), 67.07 (s, α4-O(CH2)2CH2O−), 67.01 (s, α5-O(CH2)2CH2O−), 63.52 (s, CH2-5), 30.00 (s, α4-OCH2CH2CH2O−), 29.13 (s, α5-OCH2CH2CH2O−), 20.10 (s, CH3-2), 101.02 (s, C-1’, C-1”), 68.58 (s, C-2’), 68.51 (s, C-2”), 71.95 (s, C-3’), 71.87 (s, C-3”), 67.76 (s, C-4’), 67.66 (s, C-4”), 73.01 (s, C-5’), 72.92 (s, C-5”), 61.60 (s, C-6’), 61.50 (s, C-6”), 21.14 ~ 20.63 (8s, 8×CH3CO) ppm. ESIMS: m/z 1103 [M+] (40%), 1104 [M+1] (28%).

Anal. Calcd. for C50H64NO23F3 (%): C, 54.40, H, 5.84, N, 1.27; Found: C, 54.36, H, 5.83, N, 1.25.

3-O-Benzyl-α4, α5-Di-O-[3’-O-(β-D-Glucopyranosyl)-Propyl]-6-Trifluoromethyl Pyridoxine 16

A solution of 15 (0.70 g) in anhydrous MeOH (20 mL) containing 0.5 M NH3 was vigorously stirred from 0°C to rt for 2 days until TLC showed reaction complete, evaporated to dryness in vacuo. Chromatography of the crude syrup on silica gel with EtOAc-MeOH (4:1) afforded 16 (0.49 g) as a syrup in quantitative yield, Rf 0.36 (1:4 MeOH-EtOAc), 1H-NMR (CDCl3), δH: 7.52 ~ 7.39 (5 H, m, Ar-H), 5.00 (2 H, s, PhCH2), 3.60 (2 H, s, CH2-4), 3.49 (2 H, s, CH2-5), 3.69 ~ 3.63 (4 H, m, α4-OCH2(CH2)2O−, α5-OCH2(CH2)2O−), 3.47 ~ 3.37 (4 H, m, α4-O(CH2)2CH2O−, α5-O(CH2)2CH2O−), 2.52 (3 H, s, CH3-2), 1.83 ~ 1.78 (4 H, m, α4-OCH2CH2CH2O−, α5-OCH2CH2CH2O−), 4.29 (2 H, d, JH-2,OH-2 = 7.6 Hz, HO-2’, 2”), 4.61 (1 H, d, JH-3’,OH-3’ = 5.2 Hz, HO-3’), 4.54 (1 H, d, JH-3”,OH-3” = 5.2 Hz, HO-3”), 4.95 (1 H, d, JH-4’,OH-4’ = 4.6 Hz, HO-4’), 4.91 (1 H, d, JH-4”,OH-4” = 4.4 Hz, HO-4”), 4.44 (2 H, br, HO-6’, 6”), 4.11 (1 H, d, J1’,2’ = 8.0 Hz, H-1’), 4.08 (1 H, d, J1”,2” = 7.6 Hz, H-1”), 2.96 (1 H, dd, J2’,3’ = 10.1 Hz, H-2’), 2.91 (1 H, dd, J2”,3” = 9.6 Hz, H-2”), 3.08 (1 H, dd, J3’,4’ = 3.0 Hz, H-3’), 3.05 (1 H, dd, J3”,4” = 3.4 Hz, H-3”), 3.84 (1 H, dd, J4’,5’ = 6.4 Hz, H-4’), 3.80 (1 H, dd, J4”,5” = 6.4 Hz, H-4”), 3.47 (1 H, m, H-5’), 3.43 (1 H, m, H-5”), 3.67 (1 H, dd, J5’,6a’ = 4.6 Hz, J6a’,6b’ = 11.2 Hz, H-6a’), 3.64 (1 H, dd, J5”,6a” = 4.8 Hz, J6a”,6b” = 9.6 Hz, H-6a”), 3.58 (1 H, dd, J5’,6b’ = 5.2 Hz, H-6b’), 3.54 (1 H, dd, J5”,6b” = 4.4 Hz, H-6b”) ppm; 13C-NMR (CDCl3), δC: 136.50 (s, Py-C2’), 131.27 (s, Py-C3’), 153.16 (s, Py-C4’), 141.36 (q, 3JF-C = 16.0 Hz, Py-C5’), 140.25 (q, 2JF-C = 32.1 Hz, Py-C6’), 122.32 (q, 1JF-C = 273.9 Hz, CF3), 128.80 ~ 128.53 (m, Ph-C), 76.88 (s, PhCH2-O3), 72.07 (s, CH2-4), 68.04 (s, α4-OCH2(CH2)2O−), 67.82 (s, α5-OCH2(CH2)2O−), 66.00 (s, α4-O(CH2)2CH2O−), 65.89 (s, α5-O(CH2)2CH2O−), 63.98 (s, CH2-5), 32.66 (s, α4-OCH2CH2CH2O−), 29.76 (s, α5-OCH2CH2CH2O−), 22.57 (s, CH3-2), 103.11 (s, C-1’), 103.04 (s, C-1”), 68.18 (s, C-2’), 67.96 (s, C-2”), 70.38 (s, C-3’), 70.15 (s, C-3”), 65.98 (s, C-4’), 65.89 (s, C-4”), 73.52 (s, C-5’), 73.15 (s, C-5”), 61.30 (s, C-6’), 61.16 (s, C-6”) ppm. ESIMS: m/z 767 [M+] (30%), 768 [M+1] (18%).

Anal. Calcd. for C34H48NO15F3 (%): C, 53.19, H, 6.30, N, 1.82; Found: C, 53.14, H, 6.28, N, 1.80.

α4, α5-Di-O-[3’-O-(β-D-Glucopyranosyl)-Propyl]-6-Trifluoromethyl Pyridoxine 17

Hydrogenation (H2, 30 psi) of 16 (0.45 g) in anhydrous EtOH (30 mL) catalyzed by Pd/C (5%, 125 mg) for 1 day furnished the target molecule 17 (0.40 g, 100%) as a syrup, Rf 0.30 (1:2 MeOH-EtOAc), 1H-NMR (DMSO-d6), δH: 4.78 (2 H, s, CH2-4), 4.54 (2 H, s, CH2-5), 4.94 ~ 4.85 (4 H, m, α4-OCH2(CH2)2O−, α5-OCH2(CH2)2O−), 3.40 ~ 3.15 (4 H, m, α4-O(CH2)2CH2O−, α5-O(CH2)2CH2O−), 2.38 (3 H, s, CH3-2), 1.89 ~ 1.80 (4 H, m, α4-OCH2CH2CH2O−, α5-OCH2CH2CH2O−), 4.40 (1 H, d, J1’,2’ = 7.6 Hz, H-1’), 4.07 (1 H, d, J1”,2” = 8.8 Hz, H-1”), 3.06 (1 H, dd, J2’,3’ = 10.0 Hz, H-2’), 3.02 (1 H, dd, J2”,3” = 9.6 Hz, H-2”), 3.18 (1 H, dd, J3’,4’ = 2.8 Hz, H-3’), 3.10 (1 H, dd, J3”,4” = 3.4 Hz, H-3”), 3.85 (1 H, dd, J4’,5’ = 5.4 Hz, H-4’), 3.81 (1 H, dd, J4”,5” = 5.6 Hz, H-4”), 3.57 (1 H, m, H-5’), 3.54 (1 H, m, H-5”), 3.77 (1 H, dd, J5’,6a’ = 4.6 Hz, J6a’,6b’ = 11.4 Hz, H-6a’), 3.62 (1 H, dd, J5”,6a” = 4.4 Hz, J6a”,6b” = 9.8 Hz, H-6a”), 3.56 (1 H, dd, J5’,6b’ = 5.0 Hz, H-6b’), 3.52 (1 H, dd, J5”,6b” = 4.4 Hz, H-6b”) ppm; 13C-NMR (DMSO-d6), δC: 148.05 (s, Py-C2’), 132.81 (s, Py-C3’), 154.84 (s, Py-C4’), 130.25 (q, 3JF-C = 12.6 Hz, Py-C5’), 135.45 (q, 2JF-C = 32.1 Hz, Py-C6’), 123.71 (q, 1JF-C = 273.6 Hz, CF3), 78.15 (s, CH2-4), 74.37 (s, α4-OCH2(CH2)2O−), 74.14 (s, α5-OCH2(CH2)2O−), 71.13 (s, α4-O(CH2)2CH2O−), 71.08 (s, α5-O(CH2)2CH2O−), 77.27 (s, CH2-5), 30.92 (s, α4-OCH2CH2CH2O−), 29.44 (s, α5-OCH2CH2CH2O−), 21.48 (s, CH3-2), 103.73 (s, C-1’, C-1”), 68.97 (s, C-2’), 68.55 (s, C-2”), 70.98 (s, C-3’), 70.89 (s, C-3”), 67.23 (s, C-4’), 67.18 (s, C-4”), 73.09 (s, C-5’), 72.29 (s, C-5”), 62.17 (s, C-6’), 62.02 (s, C-6”) ppm. ESIMS: m/z 677 [M+] (25%), 678 [M+1] (12%).

Anal. Calcd. for C27H42NO15F3 (%): C, 47.86, H, 6.25, N, 2.07; Found: C, 47.81, H, 6.23, N, 2.04.


The 19F-NMR data versus pH were measured in NMR tubes using a combination pH electrode (Wilmad, Buena, NJ) attached to a pH meter (Corning 220, Sudbury, UK), and for titration curves the pH was altered by addition of NaOH or HCl aq. Solutions. In vivo 19F-NMR data were acquired using a 4.7T horizontal bore magnet with a Varian INOVA Unity system (Palo Alto, CA, USA, 188 MHz 19F).


Fresh whole blood was drawn from the lateral ear of New Zealand white rabbits and stored chilled in the presence of heparin prior to 19F-NMR studies.

Heart Perfusion

Langendorff retrograde perfusion was performed with recycled phosphate-free, modified Kres-Henseleit buffer oxygenated with carbogen at 37°C under a pressure of 100cm H2O, as described in detail previously.[46]

Animal Studies

Animal studies were performed in accordance with protocols approved by the UT Southwestern Institutional Animal Care and Use Committee. Dunning prostate tumor R3327-AT1, rat mammary tumor 13762NF cells were respectively implanted in a skin pedicle on the fore-back of a Copenhagen, Fisher 344 rat, and Dunning prostate tumor Mat-Lu cells were implanted subcutaneously in thighs of nude mice. Anesthesia was induced in an induction chamber with isoflurane and maintained during the surgery with a nose cone at 1.3% isoflurane/air (1.0 dm3/min). Once the tumor had grown to the requisite sizes, animals were anesthetized (isoflurane/air), injected i.p. with a solution of pH indicator 8 and NaTFA, then placed on a platform with a 2 cm diameter home built volume coil around the tumor. The animal-bed was inserted into the bore of the MR scanner and 19F-NMR spectra were acquired immediately after tuning the coil to the 19F resonance frequency. Animal temperature was maintained at 37°C by a warm pad with circulating water during acquisition.

Tumor Micro-Electrode Studies

Measurement of pHe was accomplished with the 20G combination needle electrode (Model 818, Diamond General, Ann Arbor, MI) and digital pH meter (Model 820A, Orion Research). Spatial pHe was mapped in three 13762NF breast tumor bearing female Fisher 344 rats. After anesthetizing the animal, the tumor was gently clamped into a fixed position. The pH electrode was clamped to a micro-caliper insertion device and inserted along the central track of the central plane of the tumor, until the needle reached the opposing side of the tumor wall. The pH needle was withdrawn in 0.5mm steps, and allowed to stabilize for 2 minutes prior to measurement. The pH response was recorded at each step, and the electrode was stepped backwards until withdrawn. This process was repeated along two additional parallel tracks, 0.5 cm anterior and 0.5 cm posterior to the central track.


Supported in part by grants from the DOD Breast Cancer Initiative IDEA award DAMD17-03-1-0343, the DOD Prostate Cancer New Investigator Award W81XWH-05-1-0593, and the Small Animal Imaging Research Program, which is supported in part by NCI U24 CA126608 and P30 CA142543. NMR experiments were conducted at the Advanced Imaging Research Center, an NIH BTRP facility #P41-RR02584. We are also grateful to Dr. Anca Constantinescu and Jennifer McAnally for expert technical assistance and Dr. Pieter Otten for sharing ideas and initial observations.


Sodium trifluoroacetate
Extracellular pH value
Intracellular pH value
Dimethyl sulfoxide
Magnetic resonance spectroscopy
Nuclear magnetic resonance
Magnetic resonance imaging
Thin layer chromatography
N, N-dimethylformamide



The authors declare no competing financial interest.


1. (a) Mason RP. Transmembrane pH Gradients In Vivo : Measurements using fluorinated vitamin B6 derivatives. Curr. Med. Chem. 1999;6:533–551. [PubMed](b) Yu J-X, Kodibagkar VD, Cui W, Mason RP. 19F: A versatile reporter for non-invasive physiology and pharmacology using magnetic resonance. Curr. Med. Chem. 2005;12:819–848. [PubMed](c) Song CW, Griffin R, Park HJ. Influence of tumor pH on therapeutic response. In: Teicher B, editor. Cancer Drug Discovery and Development: Cancer Drug Resistance, Influence of tumor pH on therapeutic response. Totowa, NJ: Humana Press; 2006. pp. 21–42.(d) Hashim AI, Zhang X, Wojtkowiak JW, Martinez GV, Gillies RJ. Imaging pH and metastasis. NMR Biomed. 2011;24:582–591. [PubMed]
2. (a) Tannock IF, Rotin D. Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res. 1989;49:74373–74384. [PubMed](b) Gerweck LE, Seetharaman K. Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer. Cancer Res. 1996;56:1194–1198. [PubMed](c) Gatenby RA, Gawlinski ET. A reaction-diffusion model of cancer invasion. Cancer Res. 1996;56:5745–5753. [PubMed](d) Gerweck LE. Tumor pH: implications for treatment and novel drug design. Seminars in Radiation Oncology. 1998;8:176–182. [PubMed](e) Gerweck LE, Vijayappa S, Kozin S. Tumor pH controls the in vivo efficacy of weak acid and base chemotherapeutics. Mol. Cancer Ther. 2006;5:1275–1279. [PubMed]
3. (a) Guerquin-Kern JL, Leteurtre F, Croisy A, Lhoste JM. pH Dependence of 5-fluorouracil uptake observed by in vivo 31P and 19F nuclear magnetic resonance spectroscopy. Cancer Res. 1991;51:5770–5773. [PubMed](b) Raghunand N, He X, van Sluis R, Mahoney B, Baggett B, Taylor CW, Paine-Murrieta G, Roe D, Bhujwalla ZM, Gillies RJ. Enhancement of chemotherapy by manipulation of tumor pH. Br. J. Cancer. 1999;80:1005–1011. [PubMed](c) Rofstad EK, Mathiesen B, Kindem K, Galappathi K. Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice. Cancer Res. 2006;66:6699–6707. [PubMed](d) Zhang X, Lin Y, Gillies RJ. Tumor pH and its measurement. J. Nucl. Med. 2010;51:1167–1170. [PubMed]
4. Hunjan S, Mason RP, Mehta VD, Kulkarni PV, Aravind S, Arora V, Antich PP. Simultaneous intra- and extra- cellular pH measurement using 19F NMR of 6-fluoropyridoxol. Magn. Reson. Med. 1998;39:551–556. [PubMed]
5. Mehta VD, Kulkarni PV, Mason RP, Constantinescu A, Aravind S, Goomer N, Antich PP. 6-Fluoropyridoxol: a novel probe of cellular pH using 19F NMR spectroscopy. FEBS Lett. 1994;349:234–238. [PubMed]
6. He S, Mason RP, Hunjan S, Mehta VD, Arora V, Katipally R, Kulkarni PV, Antich PP. Development of novel 19F NMR pH indicators: Synthesis and evaluation of a series of fluorinated vitamin B6 analogs. BioOrg. Med. Chem. 1998;6:1631–1639. [PubMed]
7. For the use of organofluorine compounds in medicinal and biomedical chemistry see: Ojima I, McCarthy JR, Welch JT, editors. Biomedical Frontiers of Fluorine Chemistry; ACS Symposium Series 639; American Chemical Society; Washington, DC. 1996. Filler R, Kirk K. Biological properties of fluorinated compounds. In: Hudlicky M, Pavlath AE, editors. Chemistry of Organic Fluorine Compounds II: A Critical Review. Washington, DC: ACS Monograph 187; American Chemical Society; 1995. Elliot AJ. Chemistry of Organic Fluorine Compounds II. Washington, DC: ACS Monograph 187; American Chemical Society; 1995. Fluorinated pharmaceuticals. Sholoshonok VA, editor. Enantiocontrolled Synthesis of Organo-Fluorine Compounds: Stereochemical Challenge and Biomedical Targets. New York: Wiley; 1999.
8. For the use of organofluorine compounds in agrosciences see: Cartwright D. Recent developments in fluorine-containing agrochemicals. In: Banks RE, Smart BE, Tatlow JC, editors. Organofluorine Chemistry: Principles and Commercial Applications. New York: Plenum; 1994. Lang RW. Chemistry of Organic Fluorine Compounds II. Washington, DC: ACS Monograph 187; American Chemical Society; 1995. Fluorinated agrochemicals.
9. Yu J-X, Otten P, Ma Z, Cui W, Liu L, Mason RP. A novel NMR platform for detecting gene transfection: Synthesis and evaluation of fluorinated phenyl β-D-galactosides with potential application for assessing lacZ gene expression. Bioconj. Chem. 2004;15:1334–1341. [PubMed]
10. Yu J-X, Liu L, Kodibagkar VD, Cui W, Mason RP. Synthesis and evaluation of novel enhanced gene reporter molecules: Detection of β-galactosidase activity using 19F NMR of trifluoromethylated aryl β-D-galactopyranosides. Bioorg. Med. Chem. 2006;14:326–333. [PubMed]
11. For general discussion on the synthesis of organofluorine compounds see: Olah GA, Prakash GKS, Chambers RD. Synthetic fluorine chemistry. New York: Wiley; 1992. Furin GG. Synthetic aspects of the fluorination of organic compounds. London: Harward Academic; 1991. McClinton MA, McClinton DA. Trifluoromethylations and related reactions in organic chemistry. Tetrahedron. 1992;48:6555–6666.
12. (a) Prakash GKS, Yudin AK. Perfluoroalkylation with organosilicon reagents. Chem. Rev. 1997;97:757–786. [PubMed](b) Singh RP, Shreeve JM. Nucleophilic trifluoromethylation reactions of organic compounds with (trifluoromethyl)trimethylsilane. Tetrahedron. 2000;56:7613–7632.(c) Roy S, Gregg BT, Gribble GW, Le V-D, Roy S. Trifluoromethylation of aryl and heteroaryl halides. Tetrahedron. 2011;67:2161–2195.
13. (a) Blazejewski JC, Anselmi E, Wilmshurst MP. Extending the scope of ruppert's reagent: trifluoromethylation of imines. Tetrahedron Lett. 1999;40:5475–5478.(b) Urata H, Fuchikami T. A novel and convenient method for trifluoromethylation of organic halides using CF3SiR'3/KF/Cu(I) system. Tetrahedron Lett. 1991;32:91–94.
14. Korytnyk W, Srivastava SC. Chemistry and biology of vitamin B6. 31. Synthesis and physicochemical and biological properties of 6-halogen-substituted vitamin B6 analogs. J. Med. Chem. 1973;16:638–642. [PubMed]
15. Koch V, Schnatterer S. Chemistry of 3-hydroxypyridine Part 1: Bromination and iodination of 3-hydroxypyridine. Synthesis. 1990:497–500.
16. (a) Yu J-X, Mason RP. Synthesis and characterization of novel lacZ gene reporter molecules: Detection of β-galactosidase activity using 19F NMR of polyglycosylated fluorinated vitamin B6. J. Med. Chem. 2006;49:1991–1999. [PubMed](b) Yu J-X, Gulaka PK, Liu L, Kodibagkar VD, Mason RP. Novel Fe3+-based 1H MRI β-galactosidase reporter molecules. ChemPlusChem. 2012;70:370–378. [PMC free article] [PubMed]