6-trifluoromethylpyridoxine: Novel 19F NMR pH indicator for in vivo detection

Article abstract of DOI:10.1021/jm300520q

pH plays an important role in tumor proliferation, angiogenesis, metabolic control, and the efficacy of cytotoxic therapy, and accurate noninvasive assessment of tumor pH promises to provide insight into developmental processes and prognostic information.

Full text of DOI:10.1021/jm300520q

Article
pubs.acs.org/jmc
19
6‑Trifluoromethylpyridoxine: Novel F NMR pH Indicator for in Vivo
Detection
Jian-Xin Yu,* Weina Cui,† Vincent A. Bourke, and Ralph P. Mason
Advanced Radiological Sciences, Department of Radiology, The University of Texas Southwestern Medical Center at Dallas, 5323
Harry Hines Boulevard, Dallas, Texas 75390-9058, United States
ABSTRACT: pH plays an important role in tumor pro-
liferation, angiogenesis, metabolic control, and the efficacy of
cytotoxic therapy, and accurate noninvasive 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-trifluoromethylpyridoxine 8 and α4,α5-di-O-[3′-O-
(β-D-glucopyranosyl)propyl]-6-trifluoromethylpyridoxine 17
and demonstrate 8 as an extracellular 19F NMR pH probe to
assess pHe of various tumors in vivo.
in pharmaceutics and agrochemistry.7,8 We demonstrated that
INTRODUCTION
The pH gradient between the interstitial and intracellular
■
the introduction of a CF3 group in place of fluorine atom in
phenols can enhance the 19F NMR signal and modify the pKa
compartments is involved in many cell regulatory processes and
value.9,10
strongly influences drug uptake.1 Tumor pH also influences cell
thermosensitivity, radiation sensitivity, proliferation, and the
efficacy of cancer therapy.2,3 The accurate noninvasive
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
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).
A three-step procedure for halogenation of vitamin B6 via 6-
aminopyridoxine has been reported for the synthesis of the 19
F
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 deprotection, we initially proposed the
acetylation as protecting strategy. Acetylation of 2 as the usual
workup 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-trifluorome-
thylpyridoxine (4) in only 40% yield. Further purification
separated 12% of α4,α5-di-O-acetyl-6-trifluoromethylpyridoxine
(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.
Figure 1. Structure of 6-fluoropyridoxine (FPOL).
both intra- and extracellular pH simultaneously providing
exceptional sensitivity to pH changes in whole blood and the
perfused rat heart.1a,b,4−6 However, its pKa = 8.2 is not ideal for
measurements under normal physiological conditions (pH 6.5−
7.5). For the 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 aim of modifying the pKa to physiological conditions and
raising the 19F signal-to-noise ratio.
RESULTS AND DISCUSSION
■
Design and Synthesis. The introduction of a trifluor-
omethyl (CF3) group into an organic compound can bring
about remarkable changes in its physical, chemical, and
biological properties, making it suitable for diverse applications
Received: April 11, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm300520q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. Reagents and conditions: (a) I2, 10% K2CO3, rt 2−3 h, in the dark; Na2SO3; HCl, 73%; (b) Ac2O−pyridine, 0 °C → rt, 24 h, 93% (→3);
(c) NaH, benzyl bromide (3.3 equiv), DMF, rt, 5−7 h, 100% (→6); (d) Me3SiCF3 (1.2 equiv), CuI (1.0 equiv), KF (1.2 equiv), Ar, DMF−NMP
(1:1 v/v′), 80 °C, 24 h, 40% (→4) or 96% (→7); (e) NH3−MeOH, 0 °C → rt, 24 h, quantitative yield (→8); (f) H2 (30 psi), AcOH/EtOH (1:10
v/v′), Pd/C (5% w/w′), rt, 2 days, quantitative yield (→8).
Scheme 1. Proposed Reaction Mechanism for the Formation of 5
Figure 3. Reagents and conditions: (a) benzyl bromide (1.1 equiv), CH2Cl2−H2O, pH 9−10, rt, TBAB, 4−5 h, 74%; (b) NaH, allyl bromide (1.5
equiv), DMF, rt, 4 h, 100%; (c) Me3SiCF3 (1.2 equiv), CuI (1.0 equiv), KF (1.2 equiv), Ar, DMF-NMP (1:1 v/v′), 80 °C, 24 h, 92%; (d) 9-BBN
(2.0 equiv), Ar, dioxane, 0 °C, 24 h; NaOH, H2O2, rt, 48 h, 86%; (e) 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (1.2 equiv), Hg(CN)2 (1.5
equiv), 4 Å molecular sieves, CH2Cl2, rt, 12 h, 88%; (f) NH3−MeOH, 0 °C → rt, 48 h, quantitative yield; (g) 30 psi of H2, EtOH, Pd/C (5% w/w′),
rt, 12 h, quantitative yield.
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-iodopyr-
idoxine (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-trifluoromethylpyridoxine (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-trifluoromethylpyridoxine (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 cosolvents 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 and then the α4-O-benzyl group in another day, resulting in
8 with total yield of 100%. As expected, 6-trifluoromethylpyr-
idoxine 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 =
1.61 ppm) and poorer water solubility ((FPOL) 17.8 mg/mL
and (8) 5.8 mg/mL in H2O at room temperature).
We found that modification of 4- and/or 5-methylenehy-
droxyl 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 carbohy-
drates.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-trifluoromethylpyridoxine 8
(Figure 3).
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 (www.acdlabs.com) indicated
B
dx.doi.org/10.1021/jm300520q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
that 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 biphasic CH2Cl2−H2O (pH 9−10) at room
temperature, which was catalyzed by tetrabutylammonium
bromide (TBAB), was added benzyl bromide (1.1 equiv)
dropwise over a period of 4−5 h. 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 exhibits doublets with coupling constants of
JH‑4,HO‑4 = 5.6 Hz at 4.87 ppm and JH‑5,HO‑5 = 6.4 Hz at 4.73
ppm. 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-
trifluoromethylpyridoxine 13 in excellent yield (92%). The
diallylated derivative 13 underwent with regioselective hydro-
boration by using 9-borabicyclo-[3.3.1]nonane (9-BBN) and
subsequent alkaline oxidation with H2O2, giving 3-O-benzyl-
α4,α5-di-O-(3-hydroxypropyl)-6-trifluoromethylpyridoxine 14
in 86% yield.
Figure 4. 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
19F NMR
δ(acid)
ppm
,
δ(base)
ppm
,
Δδ,
ppm/pH
unit
compd pKa
ppm
pH range
8
6.83
7.84
15.15
15.28
16.76
16.66
1.61
1.38
0.40
0.43
5.40−9.40
6.75−9.93
17
Condensation of 14 with 2,3,4,6-tetra-O-acetyl-β-D-glucopyr-
anosyl bromide gave 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 iden-
tity 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 4.41
ppm, respectively, with two well resolved doublets J1′,2′ = J1″,2″
≈ 8.0 Hz and J2′,3′ = J2″,3″ ≈ 10 Hz, which confirmed both D-
which provided the intracellular pHi 7.04 (δPi = 4.88 ppm) and
extracellular pHe 7.42 (δPe = 5.32 ppm).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 = 16.52 ppm (pHe = 7.44)
(Figure 5b).
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. First, pHe
reporter 8 (320 mg/kg) and NaTFA (200 mg/kg) in DMSO/
saline (1:3 v/v′) were injected ip into an anesthetized (with
isoflurane) Copenhagen rat bearing a Dunning R3327-AT1
prostate tumor (tumor size, 2.4 cm × 3.1 cm × 1.8 cm). 8 was
detected by 19F NMR in the tumor 30 min after injection. The
single broad 19F signal centered at δF = 16.28 ppm indicated
tumor heterogeneity with mean pHe of 7.20.1b
4
glucoses in the β-configuration with C1 chair conformation,
and 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-trifluoromethylpyridoxine 16 in
quantitative yield, 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-tri-
fluoromethylpyridoxine 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
= 0 ppm). Both 8 and 17 exhibited single narrow 19F NMR
signals in 0.9% saline or PBS essentially invariant (Δδ ≤ 0.03
ppm) with temperatures ranging from 25 to 37 °C. Figure 4
shows the titration curves of 8 and 17 in saline between pH 2
Similarly, 8 was detected in a 13762NF rat mammary tumor
(tumor size, 1.8 cm × 1.0 cm × 2.0 cm) after 8 min following
an ip injection of the same doses into a Fisher 344 rat. A broad
single 19F resonance was obtained at δF = 16.23 ppm (pHe
7.14) with biological clearance over 100 min (Figure 6A). This
pH was commensurate with microelectrode measurements in
three Fisher 344 rats bearing 13762NF breast tumors of 3.2,
7.5, and 9.2 cm3, showing the most frequent pHe of 7.03
(Figure 6B).
Encouraged by these in vivo measurements of tumor pHe, we
also investigated 6-trifluoromethylpyridoxine 8 in other animal
models. After ip injection of 8 (12 mg, 54 μmol) in DMSO/
H2O (1:3 v/v′) into a nude mouse bearing a MatLu rat prostate
tumor grown on the thigh (tumor size, 1.6 cm × 1.4 cm × 1.0
cm), a broad, single 19F peak was observed at δF(8) = 15.96 ppm
(pHe 6.48) with an acquisition time of 7 min (Figure 7).
and pH 13 at 25 °C. From the titration curves their pKa, δ(acid)
,
and δ(base) were determined from the Henderson−Hasselbach
equation1a,b (Table 1).
Given that both 8 and 17 show 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 = 16.41 ppm corresponding to pH 7.39 (Figure
5a). The intra- and extracellular inorganic phosphates of
Langendorff perfused rat heart was sampled by 31P NMR,
C
dx.doi.org/10.1021/jm300520q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. 19F NMR spectra (376 MHz, 25 °C) of 8 in (a) Langendorff perfused rat heart, δF = 16.41 ppm (pHe 7.39) and (b) whole rabbit blood, δF
= 16.52 ppm (pHe 7.44).
Figure 6. (A) In vivo 19F NMR spectrum (188 MHz, 37 °C) of 8 (320 mg/kg) in Fisher 344 rat bearing pedicle 13762NF breast tumor: ip injection,
tumor size of 1.8 cm × 1.0 cm × 2.0 cm, δF = 16.23 ppm corresponding to pHe 7.14, acquisition time of 8 min. (B) Distribution of pHe values
measured in a group of three Fisher 344 rats bearing 13762NF breast tumors using needle electrode: mean pH of 7.03 (arrow).
indicators 8 and 17 and identified the following useful
characteristics that 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 = 1.38−1.61 ppm); (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.
EXPERIMENTAL SECTION
■
General Methods. NMR spectra were recorded on a Varian Inova
Figure 7. In vivo 19F NMR spectrum (188 MHz, 37 °C) of 8 (12 mg)
in nude mouse bearing MatLu rat prostate tumor on thigh: ip
injection, tumor size of 1.6 cm × 1.4 cm × 1.0 cm, δF(8) = 15.96 ppm
1
400 spectrometer (400 MHz for H, 100 MHz for 13C, 376 MHz for
1
19F, 121 MHz for 31P). H and 13C chemical shifts are referenced to
TMS as internal standard with CDCl3 or DMSO-d6 as solvents. 19F
corresponding to pHe 6.48, acquisition time of 7 min.
shifts are referenced 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 2400 CHN
microanalyser.
Hg(CN)2 was dried before use at 50 °C for 1 h. CH2Cl2 was dried
over Drierite, and 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 pur-
chased from the Sigma Chemical Co. Column chromatography was
performed on silica gel (200−300 mesh), and silica gel GF254 used for
TLC was purchased from the Aldrich Chemical Co. Detection was
effected by spraying the plates with 5% ethanolic H2SO4 (followed by
heating at 110 °C for ∼10 min) or by direct UV illumination of the
plate. The purity of the final products was determined by HPLC with
≥95%.
CONCLUSION
■
Given the relevance of pHi/pHe to tumor development and
prognostic outcome, noninvasive techniques to sample cellular
pH in vivo have great potential and are increasingly important
in therapeutic oncology.1−3 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 because of its favorable
MR properties, simplicity, and high sensitivity.1a,b In this study,
we have successfully synthesized two novel 19F NMR pH
D
dx.doi.org/10.1021/jm300520q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
6-Iodopyridoxine 2. To a solution of pyridoxine 1 (3.4 g, 20.0
mmol) in 10% K2CO3 aqueous solution (60 mL) was added iodine
(5.04 g, 20.0 mmol). 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
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 C29H28NO3I
(%): 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-trifluoromethylpyridoxine 7. Trifluor-
omethylation 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-trifluoromethylpyridoxine 7 (0.78 g, 96%) as a syrup. Rf =
1
give 2 (4.28 g, 73%) as a yellow powder. H 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 C8H10NO3I (%): 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 room temperature overnight, the
mixture was coevaporated 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
1
0.64 (4:1 cyclohexane−EtOAc). H 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,
1
= 0.50 (3:2 cyclohexane−EtOAc). H NMR (CDCl3), δH: 5.18 (2 H,
2
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 C14H16NO6I (%): C, 39.92; H, 3.83; N, 3.33.
Found: C, 39.90; H, 3.80; N, 3.30.
3JF−C = 21.4 Hz, Py-C5′), 142.07 (q, JF−C = 32.1 Hz, Py-C6′), 122.36
1
(q, JF−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-Trifluoromethylpyridoxine 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.
3,α4,α5-Tri-O-acetyl-6-trifluoromethylpyridoxine 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) was added Me3SiCF3 (537 μL, 2.9 mmol, 1.2 equiv) 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 24 h. 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
1
α4,α5-Di-O-benzyl-6-trifluoromethylpyridoxine 9. Yield: 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
4 (0.35 g, 40%) as a syrup. Rf = 0.37 (2:1 cyclohexane−EtOAc). H
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
3
(s, Py-C2′), 137.81 (s, Py-C3′), 154.80 (s, Py-C4′), 133.90 (q, JF−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.
2
1
(s, Py-C5′), 140.02 (q, JF−C = 32.3 Hz, Py-C6′), 121.54 (q, JF−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,α5-Di-O-acetyl-6-trifluoromethylpyridoxine 5. Yield: 88
1
α4-O-Benzyl-6-trifluoromethylpyridoxine 10. Yield: 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
mg, 12% as a syrup. Rf = 0.27 (2:1 cyclohexane−EtOAc). H 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.
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′) biphasic 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
was added a solution of benzyl bromide (0.65 g, 3.73 mmol, 1.1 equiv)
in CH2Cl2 (10 mL) dropwise over a period of 4−5 h 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 a white
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 1−2 h 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 an additional 4−5 h. At the
end of the time, TLC (4:1 cyclohexane−EtOAc) showed the reaction
to be 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
E
dx.doi.org/10.1021/jm300520q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
crystalline solid. 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) was added allyl
bromide (1.13 g, 9.33 mmol) in dry DMF (10 mL) dropwise over a
period of 1−2 h, and the stirring continued for an additional 4−5 h. At
the end of the time, TLC (4:1 cyclohexane−EtOAc) showed the
reaction to be 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),
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 )2 OH, 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-trifluoromethylpyridoxine 15. A sol-
ution 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 a 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 room temperature under an argon atmosphere in the dark
for 12 h. 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
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′
=
=
=
4
2
2.4 Hz, H-3′), 5.26 (1 H, dt, J1′,3′ = 1.0 Hz, J3a″,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.
1
a syrup. Rf = 0.50 (1:1 cyclohexane−EtOAc). H 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-O-Benzyl-α4,α5-di-O-allyl-6-trifluoromethylpyridoxine 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
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
1
g, 92%) as a syrup. Rf = 0.71 (3:1 cyclohexane−EtOAc). H 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
2
1
(q, JF−C = 32.8 Hz, Py-C6′), 122.27 (q, JF−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.
1
= 32.4 Hz, Py-C6′), 122.31 (q, JF−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′-O-(β-D-glucopyranosyl)propyl]-6-
trifluoromethylpyridoxine 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 the reaction to be
complete. The mixture was then 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
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
room temperature for 24 h and cooled to 0 °C. Aqueous NaOH (3 M,
8 mL) and 30% H2O2 (1.3 mL) were added. The reaction mixture was
stirred at room temperature 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
1
(1:4 MeOH−EtOAc). H 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,
F
dx.doi.org/10.1021/jm300520q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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,
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 ip
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 Microelectrode 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 microcaliper 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 0.5 mm steps and allowed to stabilize for 2 min prior
to measurement. The pH response was recorded at each step, and the
electrode was stepped backward until withdrawn. This process was
repeated along two additional parallel tracks, 0.5 cm anterior and 0.5
cm posterior to the central track.
J
J
1′,2′ = 8.0 Hz, H-1′), 4.08 (1 H, d, J1″,2″ = 7.6 Hz, H-1″), 2.96 (1 H, dd,
2′,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,
J
6a′,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-trifluorome-
thylpyridoxine 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
AUTHOR INFORMATION
Corresponding Author
*Phone: 214-648-2716. Fax: 214-648-4538. E-mail: Jian-Xin.
Yu@UTSouthwestern.edu.
■
1
(1:2 MeOH−EtOAc). H 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
Present Address
†Center for Magnetic Resonance Research, University of
Minnesota, 2021 Sixth Street SE, Minneapolis, Minnesota
55455, United States.
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.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by grants from the DOD
Breast Cancer Initiative IDEA Award DAMD17-99-1-9381, 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 Grants U24 CA126608 and
P30 CA142543. NMR experiments were conducted at the
Advanced Imaging Research Center, an NIH BTRP facility, No.
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.
19F NMR. 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, U.K.), and for titration
curves the pH was altered by addition of NaOH or HCl aqueous
solutions. In vivo 19F NMR data were acquired using a 4.7 T
horizontal bore magnet with a Varian INOVA Unity system (Palo
Alto, CA, U.S., 188 MHz 19F).
Blood. 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 oxy-
genated with carbogen at 37 °C under a pressure of 100 cmH2O, as
described in detail previously.4−6
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 and
rat mammary tumor 13762NF were 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
ABBREVIATIONS USED
■
FPOL, 6-fluoropyridoxine; CF3, trifluoromethyl; Me3SiCF3,
(trifluoromethyl)trimethylsilane; 9-BBN, 9-borabicyclo[3.3.1]-
nonane; NaTFA, sodium trifluoroacetate; pHe, extracellular pH
value; pHi, intracellular pH value; DMSO, dimethyl sulfoxide;
MRS, magnetic resonance spectroscopy; NMR, nuclear
magnetic resonance; MRI, magnetic resonance imaging; TLC,
thin layer chromatography; DMF, N,N-dimethylformamide;
NMP, N-methylpyrrolidine
REFERENCES
■
(1) (a) Mason, R. P. Transmembrane pH gradients in vivo:
measurements using fluorinated vitamin B6 derivatives. Curr. Med.
Chem. 1999, 6, 533−551. (b) Yu, J.-X.; Kodibagkar, V. D.; Cui, W.;
Mason, R. P. 19F: a versatile reporter for non-invasive physiology and
pharmacology using magnetic resonance. Curr. Med. Chem. 2005, 12,
819−848. (c) Song, C. W.; Griffin, R.; Park, H. J. Influence of Tumor
G
dx.doi.org/10.1021/jm300520q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
pH on Therapeutic Response. In Cancer Drug Discovery and
Development: Cancer Drug Resistance, Influence of Tumor pH on
Therapeutic Response; Teicher, B., Ed.; Humana Press: Totowa, NJ,
2006, pp 21−42. (d) Hashim, A. I.; Zhang, X.; Wojtkowiak, J. W.;
Martinez, G. V.; Gillies, R. J. Imaging pH and metastasis. NMR Biomed.
2011, 24, 582−591.
(2) (a) Tannock, I. F.; Rotin, D. Acid pH in tumors and its potential
for therapeutic exploitation. Cancer Res. 1989, 49, 4373−74384.
(b) Gerweck, L. E.; Seetharaman, K. Cellular pH gradient in tumor
versus normal tissue: potential exploitation for the treatment of cancer.
Cancer Res. 1996, 56, 1194−1198. (c) Gatenby, R. A.; Gawlinski, E. T.
A reaction-diffusion model of cancer invasion. Cancer Res. 1996, 56,
5745−5753. (d) Gerweck, L. E. Tumor pH: implications for treatment
and novel drug design. Semin. Radiat. Oncol. 1998, 8, 176−182.
(e) Gerweck, L. E.; Vijayappa, S.; Kozin, S. Tumor pH controls the in
vivo efficacy of weak acid and base chemotherapeutics. Mol. Cancer
Ther. 2006, 5, 1275−1279.
(3) (a) Guerquin-Kern, J. L.; Leteurtre, F.; Croisy, A.; Lhoste, J. M.
pH dependence of 5-fluorouracil uptake observed by in vivo 31P and
19F nuclear magnetic resonance spectroscopy. Cancer Res. 1991, 51,
5770−5773. (b) Raghunand, N.; He, X.; van Sluis, R.; Mahoney, B.;
Baggett, B.; Taylor, C. W.; Paine-Murrieta, G.; Roe, D.; Bhujwalla, Z.
M.; Gillies, R. J. Enhancement of chemotherapy by manipulation of
tumor pH. Br. J. Cancer 1999, 80, 1005−1011. (c) Rofstad, E. K.;
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. (d) Zhang,
X.; Lin, Y.; Gillies, R. J. Tumor pH and its measurement. J. Nucl. Med.
2010, 51, 1167−1170.
lated aryl β-D-galactopyranosides. Bioorg. Med. Chem. 2006, 14, 326−
333.
(11) For general discussion on the synthesis of organofluorine
compounds, see the following: (a) Olah, G. A.; Prakash, G. K. S.;
Chambers, R. D. Synthetic Fluorine Chemistry; Wiley: New York, 1992.
(b) Furin, G. G. Synthetic Aspects of the Fluorination of Organic
Compounds; Harward Academic: London, 1991. (c) McClinton, M. A.;
McClinton, D. A. Trifluoromethylations and related reactions in
organic chemistry. Tetrahedron 1992, 48, 6555−6666.
(12) (a) Prakash, G. K. S.; Yudin, A. K. Perfluoroalkylation with
organosilicon reagents. Chem. Rev. 1997, 97, 757−786. (b) Singh, R.
P.; Shreeve, J. M. Nucleophilic trifluoromethylation reactions of
organic compounds with (trifluoromethyl)trimethylsilane. Tetrahedron
2000, 56, 7613−7632. (c) Roy, S.; Gregg, B. T.; Gribble, G. W.; Le,
V.-D.; Roy, S. Trifluoromethylation of aryl and heteroaryl halides.
Tetrahedron 2011, 67, 2161−2195.
(13) (a) Blazejewski, J. C.; Anselmi, E.; Wilmshurst, M. P. 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, S. C. 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.
(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, R. P. 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. (b) Yu, J.-X.; Gulaka, P. K.; Liu, L.;
Kodibagkar, V. D.; Mason, R. P. Novel Fe3+-based 1H MRI β-
galactosidase reporter molecules. ChemPlusChem 2012, 70, 370−378.
(4) Hunjan, S.; Mason, R. P.; Mehta, V. D.; Kulkarni, P. V.; Aravind,
S.; Arora, V.; Antich, P. P. Simultaneous intra- and extra- cellular pH
measurement using 19F NMR of 6-fluoropyridoxol. Magn. Reson. Med.
1998, 39, 551−556.
(5) Mehta, V. D.; Kulkarni, P. V.; Mason, R. P.; Constantinescu, A.;
Aravind, S.; Goomer, N.; Antich, P. P. 6-Fluoropyridoxol: a novel
probe of cellular pH using 19F NMR spectroscopy. FEBS Lett. 1994,
349, 234−238.
(6) He, S.; Mason, R. P.; Hunjan, S.; Mehta, V. D.; Arora, V.;
Katipally, R.; Kulkarni, P. V.; Antich, P. P. 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.
(7) For the use of organofluorine compounds in medicinal and
biomedical chemistry, see the following: (a) Biomedical Frontiers of
Fluorine Chemistry; Ojima, I., McCarthy, J. R., Welch, J. T., Eds.; ACS
Symposium Series 639; American Chemical Society: Washington, DC,
1996. (b) Filler, R.; Kirk, K. Biological Properties of Fluorinated
Compounds. In Chemistry of Organic Fluorine Compounds II: A Critical
Review; Hudlicky, M., Pavlath, A. E., Eds.; ACS Monograph 187;
American Chemical Society: Washington, DC, 1995. (c) Elliot, A. J.
Fluorinated Pharmaceuticals. In Chemistry of Organic Fluorine
Compounds II; ACS Monograph 187; American Chemical Society:
Washington, DC, 1995. (d) Sholoshonok, V. A., Ed. Enantiocontrolled
Synthesis of Organo-fluorine Compounds: Stereochemical Challenge and
Biomedical Targets; Wiley: New York, 1999.
(8) For the use of organofluorine compounds in agrosciences, see the
following: (a) Cartwright, D. Recent Developments in Fluorine-
Containing Agrochemicals. In Organofluorine Chemistry: Principles and
Commercial Applications; Banks, R. E., Smart, B. E., Tatlow, J. C., Eds.;
Plenum: New York, 1994. (b) Lang, R. W. Fluorinated Agrochemicals.
In Chemistry of Organic Fluorine Compounds II; ACS Monograph 187;
American Chemical Society: Washington, DC, 1995.
(9) Yu, J.-X.; Otten, P.; Ma, Z.; Cui, W.; Liu, L.; Mason, R. P. 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. Bioconjugate Chem.
2004, 15, 1334−1341.
(10) Yu, J.-X.; Liu, L.; Kodibagkar, V. D.; Cui, W.; Mason, R. P.
Synthesis and evaluation of novel enhanced gene reporter molecules:
detection of β-galactosidase activity using 19F NMR of trifluoromethy-
H
dx.doi.org/10.1021/jm300520q | J. Med. Chem. XXXX, XXX, XXX−XXX