Journal of Medicinal Chemistry
Article
(Cb) were determined by subtraction; accordingly, Kf was computed.
The p-FP was used at a concentration of around 2.5 mM, and
different excess of base stoichiometry were used (from 3 up to 20) to
ensure a 1:1 complexation. Bruker Curve Fit software was used to
mathematically resolve overlapping bands into their Gausso−
Lorentzian components. Five measurements were performed for
each substance, and accordingly the average and standard deviations
were calculated. Consequently, after five runs, the ε’s were averaged
and used. In addition, ε was measured at the same day of analysis to
ensure accuracy.
(g) In terms of bioisosterism, when considering CF2H as a
lipophilic hydrogen bond donor bioisostere of NH2 (or
CH3), placed α to H-bond-accepting functional groups,
a pronounced H-bond-donating ability is presented,
making this moiety an appealing bioisostere in drug
design. Interestingly, even the monofluoromethyl
(CFH2) exhibits in such cases some H-bond-donating
ability. Importantly, it should be taken into account that
the H-bond basicity of such functional groups as well as
the lipophilicity is expected to be significantly altered.
Infrared Wavenumber Shifts. Δν(OH) was calculated as 3614
− ν(OH•••X) [cm−1] and is equal to the shift of the OH vibration of
p-FP when the hydrogen bond complex is formed.
EXPERIMENTAL SECTION
Determination of pKHB Values (NMR Measurements). NMR
experiments for the determination of pKHB were carried out in CCl4 at
25 °C. The 19F and 1H spectra were acquired with proton and fluorine
decoupling, respectively. The pKHB values of the ketones were
extracted from the 19F NMR chemical shifts of complexed p-FP
compared to that of the internal reference p-F-anisole (p-FA), which
are dependent on the Δδmax of each complex, according to the
procedure reported by Taft and co-workers47 and Dalvit et al.33,35
(see SI eq 1). The calculation of Kf where A0 and B0 are the initial
concentrations of p-FC6H4OH (p-FP) and the base, respectively. In
addition, δ is the relative 19F NMR chemical shift (ppm) for the
equilibrium mixture to that for 0.01 M (p-FP) to 0.01 M p-
FC6H4OCH3 in CCl4, and Δmax is the maximum 19F NMR chemical
shift (ppm) of the formed complex relative to uncomplexed p-FP.
Determination of Octanol−Water Partition Coefficients
(log P): “Shake-Flask” Method (UV−Vis Determination). The
partition coefficients were calculated as the logarithm of the ratio of
the compound concentration in the octanol phase to its concentration
in the aqueous phase. The shake-flask method was used for the
determination of log Po/w values.75 Both octanol and water were
presaturated with each other for at least 24 h prior to the experiment.
The different compounds were dissolved in water-saturated octanol to
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General Methods. All reagents were obtained from commercial
suppliers and were dried using standard methods when necessary. 4-
Fluorophenol (p-FP) was sublimed under vacuum and dried in an
oven-dried Schlenk tube. CCl4 was dried before use on activated
molecular sieves (4 Å). 1H, 13C, and 19F NMR spectra were obtained
using either an 11.7 T spectrometer (500, 125, and 471 MHz,
respectively) or a 7.0 T spectrometer (300, 75, and 282 MHz,
respectively). Chemical shifts are reported in parts per million (δ,
ppm). 1H NMR chemical shifts were referenced to the residual CDCl3
(δ = 7.26 ppm) and DMSO-d6 (δ = 2.50 ppm). 19F NMR chemical
shifts are reported downfield from external trifluoroacetic acid in D2O.
In 13C NMR measurements, the signal of CDCl3 (δ = 77 ppm) was
used as reference. Column chromatography was performed with silica
gel 60 (230−400 mesh). UV absorbance for log Po/w calculations was
recorded on an UV−vis spectrophotometer from Amersham
Biosciences, Ultrospec 2100-pro model. Solutions of 10 mg/mL
were prepared in CDCl3 and DMSO-d6 for the determination of H-
bond acidity (A values)72 with standard deviations in the calculation
of A from repeated measurements less than 1%. Fourier transform
infrared spectroscopy (FT-IR) analyses were performed using the
Bruker Equinox 55 FT-IR instrument. The spectra were recorded at a
resolution of 1 cm−1. A 10 mm path length, quartz Infrasil cell was
used.
obtain a concentration of 10 mM. The maximum wavelength (λmax
)
for each compound was determined, and the absorbance was recorded
using UV−vis spectroscopy. Measurements of the concentrations of
compounds were performed on dilute solutions giving absorbance in
the range of 0.2−1. Following preliminary experiments, for identifying
an optimal water/octanol ratio,76 to a volume (usually 45 mL) of
octanol-saturated water, a volume (usually 0.3 mL) of the water-
saturated octanol solution with the dissolved compound was added.
In certain cases, other water/octanol ratios were needed. The mixture
was gently shaken for 5 min. The solutions were then centrifuged at
3000 rpm for 5 min. An aliquot of the octanolic phase was diluted,
and absorbance was measured. The experiment was repeated at least
thrice for each sample under identical conditions, and each result is
presented as an average with the standard deviation of these
measurements. The extraction ratio was obtained by difference, and
log Po/w was calculated taking into account the volume ratio between
water and octanol.
Materials. Difluoromethylated compounds 1a-d-F2 and 2c-F2,
depicted in Table 1, were synthesized according to procedures
previously reported by us using O-diethyl(bromodifluoromethyl)
phosphonate as a difluorocarbene precursor.14,18,44,45 All compounds
used in this study were of high degree of purity (>95%, by 1H NMR).
2-(Difluoromethoxy)pyridine (4-F2) and 1-(Difluoromethyl)-
pyridin-2(1H)-one (5-F2). 2-Hydroxy pyridine (1.9 g, 20 mmol)
was dissolved in 100 mL of acetonitrile in a round-bottom flask, which
was placed in an ice bath. A solution of 4 M KOH (100 mL, 400
mmol) was then added to the mixture, which was stirred vigorously
for a few minutes. The phosphonate (7.1 mL, 40 mmol) was then
added dropwise to the reaction mixture, which was kept in the ice
bath for an additional ∼30 min. After that time, the ice bath was
removed and the reaction mixture was stirred at RT for an additional
∼2.5 h. The organic layer was separated, and the aqueous phase was
extracted with ether (∼100 mL). The combined organic layers were
dried over Na2SO4 and evaporated with no heat to provide a crude
mixture that holds products 4-F2 and 5-F2. Purification of this
mixture on silica using a gradient of 10−15% ethyl acetate in hexane
provided the desired products in a pure form as observed from their
1H, 13C, and 19F NMR spectra, which fitted their previously reported
19F NMR-Based log Po/w Determination. The partition
coefficients were calculated using the 19F NMR spectroscopy-based
method according to Linclau’s protocol.61 Briefly, octanol (2 mL);
2b-F (5−10 mg), 2b-F2 (10 mg), or 2b-F3 (16 mg); 2,2,2-
trifluoroethanol (1−10 mg); and water (2 mL) were added to a 10
mL flask. The mixture was stirred at room temperature for 2 h and
allowed to stand at the same temperature overnight to allow complete
phase separation. Samples (0.5−0.7 mL) were gently taken from each
layer in such a way so as to obtain a pure single solvent. Each sample
was transferred into an NMR tube, and acetone-d6 was added (0.1
mL) as a locking deuterated solvent for 19F NMR spectroscopy. D1’s
of 30 and 60 s were used for the octanol and water samples,
respectively. The numbers of transients (NSs) were 64 and 768-5900
for the octanol and water samples, respectively, and manual
integration was performed. For the separation examination, this
process was repeated thrice (for compounds 2b-F2 and 2b-F3, three
different samples were taken from each layer and were analyzed). In
data in the literature.73,74 Product 4-F2 was obtained at 16.7% yield
and product 5-F2 at 7.7% yield.
Determination of pKHB Values (IR Measurements). All of the
procedures, including the preparation of solutions and filling the IR
cell, were conducted in a dry atmosphere of a glove box. The
equilibrium constant, for the formation of a 1:1 hydrogen-bonded
complex between the acid, p-FC6H4OH (p-FP), and the general base
(B), Kf, was calculated using the IR-based method reported by
Ouvrard et al.46 The free OH band absorbance at 3614 cm−1 in the IR
spectra enabled the calculation of the acid concentration (Ca). The
equilibrium concentrations (presented in mM) of the acid (p-FP, Ca)
were measured, and the concentrations of complex (Cc) and base
4527
J. Med. Chem. 2021, 64, 4516−4531
Zafrani, Yossi
