Pentafluorosulfanyl-Substituted Benzopyran Analogues As New Cyclooxygenase-2 Inhibitors with Excellent Pharmacokinetics and Efficacy in Blocking Inflammation

Article abstract of DOI:10.1021/acs.jmedchem.6b01484

In this report, we disclose the design and synthesis of a series of pentafluorosulfanyl (SF₅) benzopyran derivatives as novel COX-2 inhibitors with improved pharmacokinetic and pharmacodynamic properties. The pentafluorosulfanyl compounds showe

Full text of DOI:10.1021/acs.jmedchem.6b01484

Article
pubs.acs.org/jmc
Pentafluorosulfanyl-Substituted Benzopyran Analogues As New
Cyclooxygenase‑2 Inhibitors with Excellent Pharmacokinetics and
Efficacy in Blocking Inflammation
Yanmei Zhang,*,† Yican Wang,† Chuang He,† Xiaorong Liu,† Yongzhi Lu,† Tingting Chen,† Qiong Pan,†
Jingfang Xiong,† Miaoqin She,† Zhengchao Tu,*,† Xiaochu Qin,† Minke Li,† Micky D. Tortorella,†
and John J. Talley*,‡
†Drug Discovery Pipeline, Guangzhou Institutes of Biomedicine and Health, 190 Kaiyuan Avenue, Science City, Guangzhou 510530,
P.R. China
‡Euclises Pharmaceuticals, St. Louis, Missouri 63108, United States
S
* Supporting Information
ABSTRACT: In this report, we disclose the design and synthesis of a series of pentafluorosulfanyl (SF5) benzopyran derivatives
as novel COX-2 inhibitors with improved pharmacokinetic and pharmacodynamic properties. The pentafluorosulfanyl
compounds showed both potency and selectivity for COX-2 and demonstrated efficacy in several murine models of inflammation
and pain. More interestingly, one of the compounds, R,S-3a, revealed exceptional efficacy in the adjuvant induced arthritis (AIA)
model, achieving an ED50 as low as 0.094 mg/kg. In addition, the pharmacokinetics of compound R,S-3a in rat revealed a half-life
in excess of 12 h and plasma drug concentrations well above its IC90 for up to 40 h. When R,S-3a was dosed just two times a
week in the AIA model, efficacy was still maintained. Overall, drug R,S-3a and other analogues are suitable candidates that merit
further investigation for the treatment of inflammation and pain as well as other diseases where COX-2 and PGE2 play a role in
their etiology.
Various types of prostanoids, such as PGE2, PGD2, PGI2, and
TXA2, metabolized from PGH2, may contribute to human pathol-
ogies if unregulated. For example, PGE2 is closely associated with
inflammation, fever, and pain, whereas PGI2 is linked to inflam-
mation and vasodilation.10−15 Thus, inhibition of cyclooxygenases
can lower the production of PGH2 and its downstream metab-
olites and reduce inflammation.16 Cyclooxygenases consist of
two forms, COX-1 and COX-2. COX-1 is generally consti-
tutively produced in tissues such as the gastrointestinal (GI)
tract and blood with a primary role in homeostasis. COX-2 is
absent or expressed at low levels in most tissues, however, it
becomes significantly upregulated at sites of inflammation.17−20
The anti-inflammatory properties of traditional NSAIDs are
primarily due to inhibition of COX-2, however, nonselective
INTRODUCTION
■
We recently described the use of deuterium to enhance the
drug like properties of benzopyran based COX-2 inhibitors.1 In
this report, the application of a pentafluorosulfanyl moiety is
explored as a chemical strategy for enhancing both the pharma-
cokinetic and pharmacodynamic properties of these compounds.
The pentafluorosulfanyl (SF5) moiety is currently being
deployed to improve the physicochemical properties of drug-
like molecules.2,3 It has been shown that the SF5 moiety
can improve the bioavailability and half-life of some drugs4,5 as
well as enhance the chemical and thermal stability of other
molecules.6,7 In addition, the SF5 moiety has been demon-
strated to endow molecules with fluorous behavior, enhanced
electron-withdrawing characteristics, increased lipophilicity and
larger steric bulk.8
Cyclooxygenases (COX) are essential rate-limiting enzymes
Received: October 7, 2016
for the conversion of arachidonic acid to prostaglandins (PGH2).9
© XXXX American Chemical Society
A
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inhibition of COX-1 may result in gastrointestinal damage,
bleeding, and ulceration in some patients. Thus, selective
COX-2 inhibitors were developed as better NSAIDs and
referred to as coxibs.21−24 However, several of the first- and
second-generation coxibs were associated with increased
incidences of adverse cardiovascular effects and quickly fell
out of favor.25,26 However, recent studies demonstrated that the
risks appear to be drug-dependent rather than class-depen-
dent,27,28 inspiring renewed interest in the development of
what’s referred to as third-generation COX-2 inhibitors, coxibs
that have unique physicochemical properties with improved
safety pharmacological profiles. Benzopyran compounds as
selective COX-2 inhibitors have been investigated29−33 as well
as deuterated benzopyran analogues.1 The deuterated com-
pounds were shown to retain potency and selectivity for
COX-2 but possessed improved pharmacokinetics and pharma-
codynamics in rodent models of inflammation and pain com-
pared to their nondeuterated congeners. It was also revealed
that substitution at the 6-position was optimal for maintaining
high potency against COX-2.34,35
Scheme 1. General Method for the Preparation of
Benzopyran Derivatives 3a and 3b
a
a
Reagents and conditions: (a) (CH2)6N4 (HMTA), CF3CO2H
(TFA), rt, 1 h; (b) (i) ethyl 4,4,4-trifluorocrotonate, KF, DMSO,
130 °C, 12 h, (ii) NaOH/MeOH/H2O, rt, 1−2 h. 1a = 4-(penta-
fluorosulfanyl)phenol; 1b = 3-(pentafluorosulfanyl)phenol.
Bromo-substituted anisole (5) was then converted to the corre-
sponding phenol by O-dealkylation with potassium n-octane-
thiolate and immediately converted to the corresponding
salicylaldehyde 6 by treatment with hexamethylenetetramine
in trifluoroacetic acid; see Scheme 2. Then 6 was condensed
a
Scheme 2. Preparation of Target Compound (7)
The pentafluorosulfanyl group (SF5) is a unique moiety, and
when incorporated into compounds, endow molecules with a
high dipole moment without increasing molecular polarity
resulting in interesting physicochemical properties, including
better thermal stability as well as hydrolytic and chemical
stability, higher density, and lipophilicity that can lead to better
biological activity (Figure 1).36−42 To some extent, it bears
a
Reagents and conditions: (a) MeI, K2CO3, DMF, 12 h; (b) Br2,
HOAc, 80 °C, 12 h; (c) (i) 1-octanethiol, tert-BuOK, DMF, 110 °C,
12 h, (ii) HMTA, TFA, rt, 2 h; (d) (i) ethyl 4,4,4-trifluorocrotonate,
KF, DMSO, 130 °C, 12 h, (ii) NaOH/MeOH/H2O, rt, 1−2 h.
Figure 1. Benzopyran and SF5-substituted derivatives.
similarity to the trifluoromethyl group but is slightly more elec-
tronegative, lipophilic, and appreciably bulkier.43 These rare
properties make the SF5 moiety suitable for applications in drug
development.44−46
with ethyl 4,4,4-trifluorocrotonate to produce the anticipated
benzopyran ester. Finally, the ester was hydrolyzed to give
R,S-7. A separate synthetic scheme was developed for the
preparation of 6-alkyl substituted intermediates 10a−10e
(Scheme 3). Briefly, 2-hydroxy-5-(pentafluorosulfanyl)benzal-
dehyde (2a) was treated with either sodium borohydride (8a)
or with excess Grignard reagent to produce benzyl alcohol
derivatives (8b−8e). The benzylic hydroxyl was then removed
by treatment of 8a−8e with two equivalents of ethyl chlorofor-
mate followed by reduction with excess sodium borohydride
in THF to afford the 2-alkyl derivatives 9a−9e.47 The 2-alkyl
derivatives 9a−9e were then converted to the corresponding
benzaldehydes 10a−10b and thence to benzopyrans (R,S-11a−
11e), see Scheme 3.
RESULTS
■
Chemistry. In an effort to develop different selective COX-2
inhibitors, we introduced a SF5 group on the benzopyran
pharmacophore. Although the availability of SF5 reagents
remains limited, new synthetic methods were developed to
prepare key intermediates allowing for the synthesis of a series
of SF5-substituted benzopyran derivatives.
The initial synthesis focused on introducing the SF5 moiety
into the 6-position or 7-position. According to published pro-
cedures,1,34 substituted salicylaldehydes 2a/2b were prepared
from commercially available meta or para-(pentafluorosulfanyl)-
phenol 1a and 1b and then condensed with ethyl 4,4,4-
trifluorocrotonate to produce the benzopyran ester. Finally, the
ester was hydrolyzed to give R,S-3a, SF5 at the 6-position or
R,S-3b, and SF5 at the 7-position (Scheme 1). It was observed
that R,S-3a was potent in inhibiting COX-2, whereas R,S-3b
was much less active (Table 1).
We next turned our attention to the synthesis of 8-bromo
6-substituted SF5 containing benzopyran derivative 7. Because
of our inability to monobrominate 1a, we prepared the corre-
sponding anisole derivative 4, which was readily monobrominated.
The individual R or S enantiomers were obtained either by
preparative chiral chromatography or by the catalytic asymmetric
synthesis shown in Scheme 4.
Cyclooxygenase Assays. The potency of the compounds
was determined against human COX-1 (hCOX-1) and human
recombinant COX-2 (hCOX-2) enzymes using a substrate-
based assay.34,48−50 Most of the compounds were tested as
racemates as separation of the R/S isomers resulted in lower
yields. The IC50 values showed that many of the SF5-substituted
molecules were poorly active against COX-1 but very active
against COX-2, with the most potent molecules being R,S-3a,
B
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a
binding, over 99.9% indicating these compounds are tightly
bound in the blood and may have diminished off and on target
side effects.
Scheme 3. General Synthesis of Benzopyrans R,S-11a−11e
Next we tested selected compounds (R,S-3a, S-3a, R,S-11a,
R,S-11b, R,S-11c, and R,S-11e) in the dofetilide binding assay,
which is a high-throughput surrogate assay for predicting hERG
activity. hERG is a potassium ion channel in cardiac tissues,
thus hERG inhibition is an important antitarget that must be
avoided during discovery and drug development. All the
compounds tested showed no binding up to 50 μM, indicating
that this series of compounds will not likely interfere with
hERG.
Crystal Structure of R,S-3a and Predicted Interaction
between R-3a or S-3a and COX-2. To better understand the
structural configuration of this class of chemistry, crystal struc-
ture of R,S-3a was determined (Olex251 was used for structure
refinement). The structure has been deposited in the Cam-
bridge Crystallographic Data Centre and assigned to the
deposition number CCDC1505776. The crystal of R,S-3a
belongs to space group P21/c with cell parameters a (9.10556),
b (11.3030), c (12.5197), α (90), β (91.1775), and γ (90). There
was only one R,S-3a molecule present in an asymmetric unit.
The R-3a molecule is related to S-3a through a symmetric center.
To investigate the mechanism of COX-2 inhibited by R,S-3a,
R,S-3a was fitted into the active site of COX-2 (PDB 4PH9)52
using Auto dock Vina53 (Figure 2). In accordance with the
result of enzyme assay (Table 1), the predicted inhibition
activity of S-3a was apparently higher than R-3a. The binding
affinity energy (BAE) was −10.2 and −8.7 kcal/mol for S-3a
and R-3a, respectively. We speculate that the subpocket Psel
(marked by red circle in Figure 2C,D) that accommodates the
−CF3 group is critical for enantioselectivity. Psel prefers to
accommodate the −CF3 group of S-3a, but not that of R-3a,
because it has a deep front side (Figure 2C, marked by a red
circle and named Psel-F) and shallow back side (Figure 2D,
marked by a red circle and named Psel-R). S-3a forms three
H-bonds with COX-2, and the group −CF3 is accommodated
well by Psel-F (Figure 2C). When R-3a docked to COX-2, one
H-bond is lost because its −CF3 group is pushed away by the
shallow Psel-R (Figure 2D).
a
Reagents and conditions: (a) NaBH4, EtOH, reflux, 2 h for R1 = H,
or excess R1MgBr, THF, 0−25 °C, 12 h; (b) (i) EtOCOCl, Et3N,
DCM, 0−25 °C, 2 h, (ii) NaBH4, EtOH, 0−25 °C, 2 h; (c) HMTA,
TFA, 80 °C, 12 h; (d) (i) ethyl 4,4,4-trifluorocrotonate, Et3N, DMF
80−100 °C, 12−18 h, (ii) NaOH/MeOH/H2O, rt, 2 h.
S-7, R,S-11a, and R,S-10c yielding IC50 values of 18, 8 and 26,
and 46 nM, respectively (Table 1). On the basis of the
structure−activity relationship, it was found that the position of
the SF5 moiety on the aromatic ring is critical for potency as
compound R,S-3a was found to be active against COX-2,
whereas R,S-3b, the same molecule with the SF5 moiety shifted
from position 6 to 7, was much less active. Thus, positioning
of the SF5 moiety was found critical for compound potency
against COX-2.
Another interesting observation was that molecule R-7
showed some activity against COX-2, although approximately
100 times less active than S-7. This is an important finding, as it
has previously been shown that the R-isomers of benzopyran
based COX-2 inhibitors were largely inactive whereas the
S-isomers were highly active. This suggests that the SF5 moiety
allows the R-isomeric form of the compound to bind to the
active site of COX-2, albeit weakly.
̀
Serum Protein and the Human Ether-a-go-go-Related
Gene (hERG) Binding of Selective Compounds. As long as
efficacy is not compromised, it has been suggested that high
plasma protein binding may be a desirable property for new
coxibs to possess as a way of limiting their off and on target side
effects. For example, during vascular inflammation, COX-2 is
up-regulated to produce the prostaglandin PGI2, a vascular
vasodilator causing relaxation of veins, large arteries, and arte-
rioles. It is believed that blockade of PGI2 may be responsible
for the increased incidence of heart attack and strokes
associated with NSAIDs. Thus, we tested the SF5 compounds
for human plasma protein binding by equilibrium dialysis
(Table 2). All the compounds tested showed very high plasma
Pharmacokinetic Profile of R,S-3a. It has been reported
that the SF5 moiety can enhance the pharmacokinetics of some
drugs. R,S-3a was tested for PK in rats by dosing the compound
orally and intravenously at 5 mg/kg (Table 3). The compound
demonstrated an excellent PK profile with a half-life of >12 h, a
Cmax of ∼15 mg/L, and 100% bioavailability. Even after 45 h,
the rat plasma drug concentration was above the IC90 required
for COX-2 inhibition as determined in the enzyme assay
(Figure 3). Whether this molecule has a longer duration of
action in other species remains to be determined. To put this
observation into perspective, a structurally similar deuterated
a
Scheme 4. Catalytic Asymmetric Synthesis of Selected Benzopyrans
a
Reagents and conditions: (a) 2-nitrobenzoic acid (20 mol %), (R)-2-(diphenyl((trimethylsilyl)oxy)methyl)pyrrolidine or (S)-2-(diphenyl-
((trimethylsilyl)oxy)methyl)pyrrolidine (20 mol %), (E)-4,4,4-trifluorobut-2-enal, CH2Cl2, rt, 24 h; (b) KHSO5, DMF, rt, 24 h.
C
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Table 1. SF5-Substituted Benzopyrans Tested against Human COX-1 and Human COX-2 Enzymes
a
IC50 values represent the half-maximal (50%) inhibitory concentration, as determined in triplicate.
Air Pouch Model. An air pouch was produced by the
subcutaneous injection of sterile air into the back of rats.55−59
The pouch mimics a synovial cavity providing a localized
environment to study an inflammatory response. The rats were
then administered either control or compound R,S-3a orally at
various doses. Carrageenan was then injected into the air pouch
to produce inflammation. The air pouch was monitored for
PGE2 levels, which is a marker of inflammation, and the efficacy
of R,S-3a was determined. Drug R,S-3a blocked PGE2 levels in
the pouch in a dose-dependent manner, showing an ED50 of
∼0.37 mg/kg, demonstrating that compound R,S-3a gets to the
site of inflammation (Table 4).
Carrageenan Induced Paw Edema. The carrageenan paw
edema model was used to detect local inflammation mediated
by prostaglandin production.60−62 Carrageenan was injected
into right hind paw of rats. One half-hour before the injection,
compound R,S-3a was orally dosed at different concentrations.
Caliper measurements of the paws were taken at baseline (just
prior to carrageenan injection) and again between 1 and 4 h
postcarrageenan injection. Differences between injected versus
noninjected and compound treated versus nontreated paws
were determined. Compound R,S-3a demonstrated concen-
tration-dependent suppression of paw edema, yielding an ED50
of ∼4 mg/kg (Table 4).
Table 2. Human Plasma Protein Binding Was Measured by
Rapid Equilibrium Dialysis
a
conc
time
(h)
mean % nound
compd
(μM)
(n = 3)
SD (%)
0.04
chlorpromazine HCl
(positive control)
10
5
99.82
atropine (negative control)
R,S-3a (SF-1001)
R,S-3b (SF-1002)
S-7 (SF-1003)
10
10
10
10
10
10
10
10
5
5
5
5
5
5
5
5
24.55
99.90
99.94
99.91
99.95
99.96
99.95
99.92
0.69
0.07
0.07
0.01
0.03
0.01
0.01
0.07
R-7 (SF-1004)
S-3a (SF-1005)
R,S-11a (SF-1006)
R,S-11e (SF-1009)
a
Chlorpromazine HCl and atropine was used as controls, high protein
binding versus low protein binding compound, respectively.
benzopyran compound made by our team and published in
20151 had a half-life of 4.2 h. Thus, these data suggest that the
addition of the SF5 group may be beneficial for enhancing PK
for this single compound, although more SF5 compounds will
have to be tested for PK and in different species before we can
make any firm conclusions.
In Vivo Efficacy in Rodent Models of Inflammation
and Pain. On the basis of its excellent pharmacokinetics,
compound R,S-3a was selected for in vivo efficacy evaluation
in rat models of inflammation and pain. As a comparator com-
pound, celecoxib was chosen as it has extensively been studied
in these models.
Thermal Hyperalgesia. Increased sensitivity to pain was
assessed in the carrageenan paw edema model as described
above.63−65 One half-hour after oral administration of R,S-3a at
varying doses, carrageenan was injected into right hind paws
of the rats, and paw withdrawal thermal latency (PWTL) was
D
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Figure 2. Predicted interaction between S-3a or R-3a (Cambridge Crystallographic Data Centre deposition no.: CCDC1505776) and COX-2 (PDB
4PH9). (A,B) Predicted docking pose of S-3a (yellow sticks) and R-3a (blue sticks), respectively. Key residues of binding pocket are shown as sticks,
and the H-bonds are labeled. (C,D) Surface presentation of binding pocket, (D) is generated by rotating (C) 180° around its vertical axis. This
diagram is prepared using Pymol.54
a
Table 3. Rat Pharmacokinetic Profile for Compound R,S-3a
Table 4. In Vivo Summary of Compound R,S-3a versus
Celecoxib in Rat Models of Inflammation and Pain
a
administration
rat no.
PO
♂4
IV
♂4
R,S-3a ED50
n
celecoxib ED50
n
model
air pouch
(mg/kg)
(mg/kg)
dose level mg/kg
AUC(0−∞) μg·h/L
T1/2 (h)
5
5
0.37
4.0
0.69
2.8
414200
12.5
364120
17.3
paw edema
hyperalgesia
2.8
5.0
Tmax (h)
7.75
0.033
31688
0.223
AIA (paw edema)
AIA (arthritis score)
AIA (movement disorder)
0.093
0.094
0.12
1.6
Cmax (μg/L)
Vd
15300
0.341
113.8%
0.71
9.2
F (%)
a
a
Compounds were dosed at various concentrations to determine the
Compound was dosed both orally (PO) and intravenously (IV).
n
ED50 values. ED50 values represent the half-maximal (50%) effective
dose, as determined in triplicate.
Adjuvant-Induced Arthritis Model (AIA). As an animal
model for inflammation and reactive rheumatoid arthritis, AIA
is proven to be a valuable model for testing and discovering
NSAIDs.66−68 Each rat (n = 6/dose group) received a sub-
cutaneous injection of adjuvant into the base of the tail.
Fourteen days after the injection, only rats that showed
significant left hind paw swelling were selected for the study
(>95% responded, typically 6 of 6 animals). The rats were then
divided into several groups including vehicle and different
concentrations of compound R,S-3a, dosed orally from day
15 to 25. At day 25, the rats were evaluated for disease. In the
absence of drug, severe disease progression was observed.
However, drug R,S-3a effectively stopped all disease parameters
including paw edema (Figure 4), arthritis, and movement
disorder with ED50 values of 0.093, 0.094, and 0.12, respectively
(Figure 5 and Table 3). Measurement of paw edema was paw
volume, arthritis score was based on erythema and swelling of
ankles, wrists, and toes (scores ranging from 0 to 16), and
Figure 3. PK profile for compound R,S-3a in rats. Compound shows a
long extended duration in the plasma.
measured before and 3 h post injection using an PL-200 hot
plate device. A significant decrease in PWTL was observed in
the vehicle group 3 h post injection, indicating increased heat
sensitivity, but compound R,S-3a demonstrated a concen-
tration-dependent reversal of the observed thermal hyperalgesia
with an ED50 of ∼5.6 mg/kg (Table 4). Almost complete rever-
sal was observed between 10 and 20 mg/kg doses.
E
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drug candidate for the treatment of inflammation and asso-
ciated pain as it outperformed celecoxib in all parameters.
Future studies will focus on investigational new drug
enabling activities such as large scale manufacturing of com-
pound S-3a and advanced toxicity studies in rodents and dogs.
EXPERIMENTAL SECTION
■
General. All commercial chemicals and solvents were obtained
from commercial sources and were used without further purification.
Solvents were chromatography grade and were used without further
purification. Thin layer chromatography (TLC) analysis was per-
formed using Merck silica gel 60 F-254 thin layer plates. LC-MS
analyses were performed on the Agilent 1200 HPLC/MCD electro-
spray mass spectrometer in positive/negative ion mode. The scan
range was 100−1000d. High resolution mass spectra were obtained in
the ABSciex TOF5600+ MS using electrospray ionization. Preparative
reverse phase HPLC was performed on a Shimadzu LC-20AP
equipped with a C18 column using a methanol/water gradient. The
purity of tested compounds was ≥95% determined by HPLC analysis
conducted on the Agilent 1260 system using a reverse phase C18
column with diode array detector unless stated otherwise. Chiral
GC-column: Chiralpak OD-H, AS-H, OJ-H, 4.6 mm × 250 mm 5 μm.
Specific rotation value were recorded on Autopol IV-T (λ = 589 nm,
Figure 4. Efficacy of drug R,S-3a at 10 mg/kg versus vehicle in
blocking paw edema in the AIA rat model.
1
50 mm cell, 25 °C). H and 13C NMR spectra were recorded on a
Bruker 500 or Bruker 400 (at 500 and 125 MHz or 400 and 100 MHz,
respectively) and are reported to CDCl3 (δ 1H, 7.26, and 13C, 77.16).
19F NMR (at 471 MHz). Coupling constants (J) are given in Hz.
2-Hydroxy-5-(pentafluorosulfanyl)benzaldehyde (2a). To a sol-
ution of 4-(pentafluorosulfanyl)phenol (2.0 g, 9.1 mmol) in tri-
fluoroacetic acid (TFA) (20 mL) was added hexamethylenetetramine,
(HTMA) (1.78 g, 12.7 mmol). The mixture was stirred at 80 °C for
4 h and then cooled to room temperature. To the mixture, H2O
(40 mL) was added and then stirred at room temperature for another
0.5 h. The mixture was then extracted with EtOAc, and the organic
layer was washed with saturated NaHCO3, water, and brine. The
resulting organic phase was dried over Na2SO4, concentrated, and
purified by column chromatography, yielding the compound as a
white solid 1.02 g in 45% yield. 1H NMR (400 MHz, CDCl3) δ 11.31
(s, 1H), 9.94 (s, 1H), 7.99 (d, J = 2.7 Hz, 1H), 7.90 (dd, J = 9.2, 2.7
Hz, 1H), 7.02 (d, J = 9.26 Hz, 1H).
Figure 5. Efficacy of drug R,S-3a at various doses, vehicle, 0.1, 0.3, 1, 3,
and 10 mg/kg (X-axis) in blocking paw edema, arthritis, and
movement index. Arthritis score is depicted by the red squares,
movement disorder index is shown by the blue circles, and paw edema
is illustrated by the green triangles.
movement disorder was based on the standard of movement of
the animals (scores ranging from 0 to 3).
To determine whether compound R,S-3a would be effective
with less frequent dosing due to its long plasma half-life, the
study was repeated, this time dosing at 10 mg/kg every 5 days,
starting from day 15 post adjuvant injection and ending on day
25 (two doses as opposed to ten). As hoped, drug R,S-3a still
demonstrated efficacy, again blocking all three disease param-
eters including paw edema, arthritis, and movement disorder
with inhibition scores of 66.9 6.9, 69.6 30.3, and 79.2
40.9, respectively. These data bring up the possibility that drug
R,S-3a may be able to be dosed less frequently.
2-Hydroxy-4-(pentafluorosulfanyl)benzaldehyde (2b). The title
compound was prepared by the same procedure as 2a as colorless oil
1
in 40% yield. H NMR (500 MHz, CDCl3) δ 11.09 (s, 1H), 10.03
(s, 1H), 7.74 (d, J = 8.5 Hz, 1H), 7.46 (d, J = 6.5 Hz, 1H), 7.46
(s, 1H). MS (MM-ES + APCI) m/z: 247.1 [M − H]−.
R,S-6-(Pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chromene-3-
carboxylic Acid (R,S-3a). Step 1: A mixture of 2-hydroxy-5-
pentafluorosulfanylbenzaldehyde (2a) (0.64 g, 2.6 mmol), anhydrous
KF (0.30 g, 5.2 mmol), and ethyl 4,4,4-trifluorocrotonate (0.87 g,
5.2 mmol) in anhydrous DMSO (30 mL) was stirred at 130 °C for 5 h.
The mixture was cooled to room temperature and extracted with
EtOAc. The organic layer was dried over Na2SO4, concentrated, and
purified by column chromatography to give ethyl 6-(pentafluor-
osulfanyl)-2-(trifluoromethyl)-2H-chromene-3-carboxylate as a yellow
solid 0.3 g in 31% yield. 1H NMR (400 MHz, CDCl3) δ 7.66−7.73 (m,
3H), 7.04 (d, J = 9.2 Hz, 1H), 5.78 (d, J = 6.8 Hz, 1H), 4.34−4.37 (q,
J = 7.2 Hz, 2H), 1.35−1.39 (t, J = 7.2 Hz, 3H). This product was used
directly in the next step without further purification. Step 2: A solution
of ethyl 6-(pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chromene-3-
carboxylate (0.3 g, 0.75 mmol) was dissolved in (20 mL, MeOH/H2O =
10/1) and treated with NaOH (0.30 g, 7.5 mmol). The resulting
solution was stirred at room temperature overnight. The solution was
concentrated to remove the ethanol, acidified to pH = 3, and extracted
with EtOAc. The organic layer was dried over Na2SO4, concentrated,
and purified by column chromatography to give the target compound
as a solid 0.27 g in 100% yield. 1H NMR (500 MHz, CDCl3) δ 7.84 (s,
1H), 7.74 (dd, J = 7.8, 2.6 Hz, 1H), 7.68 (d, J = 2.6 Hz, 1H), 7.06 (t,
J = 7.8 Hz, 1H), 5.76 (q, J = 6.6 Hz, 1H). 13C NMR (126 MHz,
DMSO-d6) δ 164.39 (s), 154.52 (s), 147.35−146.75 (m), 134.76 (s),
DISCUSSION AND CONCLUSIONS
■
A new series of COX-2 inhibitors has been developed by incor-
porating a pentafluorosulfanyl moiety. Most of compounds
were found to be potent and selective for human recombinant
COX-2 over COX-1. Moreover, one of the molecules, R,S-3a,
was found to have excellent pharmacokinetics in rodents with a
half-life of greater than 12 h. In addition, R,S-3a proved to be
efficacious in multiple models of inflammation and pain,
including the rat air pouch, paw edema, hyperalgesia, and
adjuvant induced arthritis models. Pronounced efficacy of
R,S-3a was observed in the AIA model, yielding an especially
low ED50 of 0.094 mg/kg in blocking edema and arthritis
disease. This is particularly important, as the AIA system is an
industry standard model for predicting human clinical success
of new nonsteroidal anti-inflammatory drugs and coxibs.
To our knowledge, no other anti-inflammatory drug, either
small molecule or protein, has displayed such a low ED50.
A representative of this class of SF5 based drugs may be a good
F
DOI: 10.1021/acs.jmedchem.6b01484
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Journal of Medicinal Chemistry
Article
130.29 (s), 127.65 (s), 124.48 (t, J = 287.4 Hz), 119.30 (s), 118.92 (s),
116.23 (s), 70.41 (q, J = 32.4 Hz). 19F NMR (471 MHz, CDCl3)
δ 84.08 (m, 1F), 63.67 (dd, J = 150.5, 22.1 Hz, 4F), −78.48 (d, J =
6.7 Hz, 3F). HRMS (ESI) calcd for C11H5F8O3S [M − H] 368.9837,
found 368.9840. HPLC: 99.438%.
7-(Pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chromene-3-car-
boxylic Acid (R,S-3b). The title compound was prepared by the same
procedure as R,S-3a as a white solid in 95% yield. 1H NMR (500 MHz,
CDCl3) δ 7.82 (s, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.2 Hz,
1H), 5.75 (q, J = 6.6 Hz, 1H). 13C NMR (126 MHz, DMSO-d6)
δ 164.39 (s), 154.80 (t, J = 17.0 Hz), 152.16 (s), 134.24 (s), 130.43
(s), 124.48 (t, J = 287.4 Hz), 122.43 (s), 120.21 (s), 113.24 (s), 70.20
(q, J = 32.2 Hz). 19F NMR (471 MHz, CDCl3) δ 82.73 (m, 1F), 62.27
(dd, J = 150.2, 22.0 Hz, 4F), −78.35 (d, J = 6.7 Hz, 3F). HRMS (ESI)
calcd for C11H5F8O3S [M − H] 368.9837, found 368.9842. HPLC:
99.443%.
(S)-6-(Pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chromene-3-
carboxylic Acid (S-3a). A mixture of S-12a (R2 = H) (100 mg,
0.28 mmol) and Oxone (209 mg, 0.34 mmol) in DMF (10 mL) was
stirred at room temperature overnight. The solution was then diluted
with water and extracted with EtOAc. The organic layer was dried over
Na2SO4, concentrated, and purified by column chromatography to give
the title compound 57 mg in 55% yield. 1H NMR (500 MHz, DMSO-
d6) δ 8.20 (s, 1H), 8.00 (s, 1H), 7.89 (d, J = 9.0, 1H), 7.23 (d, J =
9.0 Hz, 1H), 6.08 (q, J = 7.0 Hz, 1H). 13C NMR (126 MHz, DMSO-
d6) δ 164.32 (s), 154.47 (s), 146.99 (t, J = 17.2 Hz), 134.73 (s), 130.23
(s), 127.60 (s), 123.28 (q, J = 287.5 Hz), 119.24 (s), 118.83 (s),
116.17 (s), 70.36 (q, J = 32.3 Hz). 19F NMR (471 MHz, CDCl3)
δ 85.46−83.19 (m, 1F), 63.67 (dd, J = 150.5, 22.0 Hz, 4F), −78.48 (d,
J = 6.5 Hz, 3F). HRMS (ESI) calcd for C11H6F8O3S [M − H]
368.9837, found 368.9834. HPLC: 98.28%.
(R)-6-(Pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chromene-3-
carboxylic Acid (R-3a). The procedure described for the preparation
of R-3a was used substituting (R)-2-(diphenyl((trimethylsilyl)oxy)-
methyl)pyrrolidine as the chiral catalyst. 1H NMR (400 MHz, CDCl3)
δ 7.85 (s, 1H), 7.76 (dd, J = 9.0, 2.6 Hz, 1H), 7.70 (d, J = 2.6 Hz, 1H),
7.09 (dd, J = 9.0, 2.6 Hz, 1H), 5.80 (q, J = 6.6 Hz, 1H). HRMS (ESI)
calcd for C11H5F8O3S [M − H] 368.9837, found 369.0002. HPLC:
99.56%.
The mixture was stirred at 80 °C for 8 h and then cooled to room
temperature. To the above mixture, H2O (40 mL) was added and the
resulting mixture stirred at room temperature for another 0.5 h. The
mixture was then extracted with EtOAc, and the organic layer was
washed with saturated NaHCO3, water, and brine. The resulting
organic phase was dried over Na2SO4, concentrated, and purified by
column chromatography to give the title compound 0.46 g in 30%
yield. 1H NMR (400 MHz, CDCl3) δ 11.92 (s, 1H), 9.93 (s, 1H), 8.19
(s, 1H), 8.00 (s, 1H).
(R,S)-8-Bromo-6-(pentafluorosulfanyl)-2-(trifluoromethyl)-2H-
chromene-3-carboxylic Acid (R,S-7). The title compound was
1
prepared by the same procedure as R,S-3a in 60% yield. H NMR
(400 MHz, DMSO-d6) δ13.69 (s, 1H), 8.28 (q, J = 2.6 Hz, 2H), 8.02
(s, 1H), 6.30 (q, J = 7.0 Hz, 1H). 13C NMR (126 MHz, DMSO-d6)
δ 164.49 (s), 152.11 (s), 147.13 (t, J = 17.9 Hz), 134.81 (s), 133.06
(s), 127.33 (s), 124.70 (t, J = 287.4 Hz), 120.99 (s), 120.20 (s), 109.56
(s), 71.67−71.42 (m). 19F NMR (471 MHz, DMSO-d6) δ 86.25−
85.60 (s, 1F), 65.40−65.07 (dd, J = 151.9, 21.9 Hz, 4F), −77.56 (d, J =
7.0 Hz, 3F). HRMS (ESI) calcd for C11H5BrF8O3S [M− H] 446.8942,
found 446.9130. HPLC: 93.84%.
(S)-8-Bromo-6-(pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chro-
mene-3-carboxylic Acid (S-7). A mixture of S-12b (R2 = Br) (104 mg,
0.24 mmol) and Oxone (172 mg, 0.28 mmol) in DMF (10 mL) was
stirred at room temperature overnight. The solution was then diluted
with water and extracted with EtOAc. The organic layer was dried over
Na2SO4, concentrated, and purified by column chromatography to give
the title compound 72 mg in 67% yield. 1H NMR (500 MHz, CDCl3)
δ 7.97 (d, J = 2.4 Hz, 1H), 7.81 (s, 1H), 7.63 (d, J = 2.4 Hz, 1H), 5.88
(q, J = 6.4 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 164.10 (s),
151.61 (s), 147.01 (t, J = 17.9 Hz), 133.94 (s), 132.45 (s), 126.77 (s),
124.46 (t, J = 287.4 Hz), 120.62 (s), 120.22 (s), 109.04 (s), 76.92−
67.30 (m). 19F NMR (471 MHz, CDCl3) δ 82.73 (m, 1F), 63.86 (dd,
J = 150.8, 22.1 Hz, 4F), −78.58 (d, J = 6.4 Hz, 3F). HRMS (ESI) calcd
for C11H4BrF8O3S [M − H] 446.8942, found 446.8957. HPLC:
98.29%.
(R)-8-Bromo-6-(pentafluorosulfanyl)-2-(trifluoromethyl)-2H-
chromene-3-carboxylic Acid (R-7). The procedure described for the
preparation of R-7 was used substituting (R)-2-(diphenyl((trimeth-
ylsilyl)oxy)methyl)pyrrolidine as the chiral catalyst. The NMR data
was essentially identical to S-7. HRMS (ESI) calcd for C11H4BrF8O3S
[M − H] 446.8942, found 446.9134. HPLC: 90.91%.
1-Methoxy-4-(pentafluorosulfanyl)benzene (4). To a solution of
4-(pentafluorosulfanyl)phenol (5.0 g, 22.7 mmol) and anhydrous
K2CO3 (9.4 g, 68.1 mmol) in DMF (75 mL) was added iodomethane
(3.2 g, 22.7 mmol) at 0 °C. The resulting mixture was stirred at room
temperature overnight. The mixture was then diluted with water and
extracted with EtOAc. The organic layer was dried over Na2SO4,
concentrated, and purified by column chromatography to give the
2-(Hydroxymethyl)-4-(pentafluorosulfanyl)phenol (8a). To a
solution of 2a (0.8 g, 3.2 mmol) in EtOH (15 mL) was added
NaBH4 (0.17 g, 4.5 mmol), and then the mixture was refluxed for 2 h.
The mixture was then extracted with EtOAc, and the organic layer was
washed with brine. The resulting organic phase was dried over
Na2SO4, concentrated, and purified by column chromatography to give
the title compound 0.65 g in 81% yield. 1H NMR (400 MHz, CDCl3)
δ 7.91 (s, 1H), 7.60 (d, J = 9.0 Hz, 1H), 7.44 (s, 1H), 6.92 (d, J =
9.0 Hz, 1H), 4.94 (s, 2H). MS (MM-ES + APCI) m/z: 249.0 [M − H]−.
2-(1-Hydroxyethyl)-4-(pentafluorosulfanyl)phenol (8b). To a
solution of 2a (0.13 g, 0.5 mmol) in anhydrous THF (6 mL) was
added CH3MgBr (3.0 mL, 3.7 mmol, 1.3 mol/L) dropwise at room
temperature. Then the mixture was stirred at room temperature
overnight; after that, the mixture was acidified to pH = 3 and extracted
with EtOAc. The organic layer was dried over Na2SO4, concentrated,
and purified by column chromatography to give title compound 0.13 g
1
compound 4.5 g in 85% yield. H NMR (400 MHz, CDCl3) δ 7.69−
7.72 (d, J = 7.5 Hz, 2H), 6.92−6.94 (d, J = 7.5 Hz, 2H), 3.87 (s, 3H).
2-Bromo-1-methoxyphenyl(pentafluorosulfanyl)benzene (5). To
a mixture of 1-methoxy-4-(pentafluorosulfanyl)benzene (2.0 g,
8.5 mmol) in AcOH (30 mL) was added Br2 (6.8 g, 42.7 mmol),
and the mixture was stirred at 80 °C overnight. Then solution was
diluted with saturated NaHSO3 solution and extracted with EtOAc.
The organic layer was dried over Na2SO4, concentrated, and purified
by column chromatography to give the title compound 1.76 g in 66%
1
yield. H NMR (400 MHz, CDCl3) δ 7.97 (s, 1H), 7.70−7.73 (d,
J = 7.5 Hz, 1H), 6.92−6.94 (d, J = 7.5 Hz, 1H), 3.97 (s, 3H).
3-Bromo-2-hydroxy-5-(pentafluorosulfanyl)benzaldehyde (6).
Step 1: To a solution of t-BuOK (1.29 g, 8.6 mmol) in DMF
(50 mL) was added 1-octanethiol (1.29 g, 8.6 mmol). The resulting
mixture was stirred at room temperature for 10 min, then 1-methoxy-
4-(pentafluorosulfanyl)benzene (1.76 g, 5.6 mmol) was added. The
resulting mixture was stirred at 110 °C for 1 h. Then the mixture was
diluted with H2O, acidified to pH = 1 and extracted with EtOAc. The
organic layer was dried over Na2SO4, concentrated, and purified by
column chromatography to give 2-bromo-4-(pentafluorosulfanyl)-
1
in 94% yield. H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 7.56 (dd,
J = 8.9, 2.6 Hz, 1H), 7.38 (d, J = 2.5 Hz, 1H), 6.89 (d, J = 9.0 Hz, 1H),
5.12 (q, J = 6.5 Hz, 1H), 1.61 (d, J = 6.6 Hz, 3H). MS (MM-ES +
APCI) m/z: 263.0 [M − H].
2-(1-Hydroxypropyl)-4-(pentafluorosulfanyl)phenol (8c). The title
compound was prepared by the same procedure as 8b, in 50% yield.
1H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 7.56 (dd, J = 9.0, 2.7 Hz,
1H), 7.34 (d, J = 2.7 Hz, 1H), 6.89 (d, J = 9.0 Hz, 1H), 4.82 (t,
J = 13.6 Hz, 1H), 1.89 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H).
2-(Hydroxy(phenyl)methyl)-4-(pentafluorosulfanyl)phenol (8d).
The title compound was prepared by the same procedure as 8b in
1
phenol, 1.41 g in 84% yield. H NMR (400 MHz, CDCl3) δ 7.91
(s, 1H), 7.64−7.66 (d, J = 7.5 Hz, 1H), 7.06−7.08 (d, J = 7.5 Hz, 1H).
Step 2: To a solution of the product of Step 1 (1.4 g, 4.7 mmol) in
TFA (20 mL) was added HTMA (0.9 g, 6.6 mmol) portionwise.
1
40% yield. H NMR (400 MHz, CDCl3) δ 7.66−7.55 (m, 3H), 7.45
G
DOI: 10.1021/acs.jmedchem.6b01484
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Journal of Medicinal Chemistry
Article
(dd, J = 10.4, 4.8 Hz, 3H), 7.35 (t, J = 7.4 Hz, 2H), 1.26 (s, 1H). MS
(MM-ES + APCI) m/z: 325.0 [M − H]−.
3-Benzyl-2-hydroxy-5-(pentafluorosulfanyl)benzaldehyde (10d).
The title compound was prepared by the same procedure as 9a in
20% yield. 1H NMR (400 MHz, CDCl3) δ 11.65 (s, 1H), 9.91 (s, 1H),
7.86 (s, 1H), 7.71 (s, 1H), 7.37−7.28 (m, 2H), 7.24 (m, 3H), 4.05
(s, 2H). MS (MM-ES + APCI) m/z: 337.0 [M − H]−.
3-Butyl-2-hydroxy-5-(pentafluorosulfanyl)benzaldehyde (10e).
The title compound was prepared by the same procedure as 9a in
25% yield. 1H NMR (400 MHz, CDCl3) δ 11.57 (s, 1H), 9.91 (s, 1H),
7.82 (s, 1H), 7.74 (s, 1H), 2.77−2.67 (m, 2H), 1.66−1.56 (m, 2H),
1.40 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H).
2-(1-Hydroxybutyl)-4-(pentafluorosulfanyl)phenol (8e). The title
compound was prepared by the same procedure as 8b in 35% yield. 1H
NMR (500 MHz, CDCl3) δ 8.82 (s, 1H), 7.52 (d, J = 8.8 Hz, 1H),
7.35 (s, 1H), 6.83 (d, J = 8.8 Hz, 1H), 4.84 (s, 1H), 3.81 (s, 1H),
1.93−1.66 (m, 2H), 1.56−1.28 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H). MS
(MM-ES + APCI) m/z: 291.1 [M − H]−.
2-Methyl-4-(pentafluorosulfanyl)phenol (9a). A solution of
2-(hydroxymethyl)-4-(pentafluorosulfanyl)phenol (170 mg, 0.68 mmol)
and trimethylamine (1.50 g, 14.9 mmol) in CH2Cl2 (10 mL) was
treated with ethyl chloroformate (650 mg, 6.0 mmol) at 0 °C and then
allowed to warm to room temperature for 3 h. The solution was
concentrated in vacuo and the residue dissolved in ethanol (3 mL) and
water (20 mL) and treated with NaBH4 (1.80 g, 39.7 mmol) at 0 °C.
The solution was allowed to warm to room temperature overnight and
then acidified to pH = 3 with 3 N HCl and then extracted with
CH2Cl2. The organic extract was dried over Na2SO4, filtered, and
concentrated to give the title compound that was used directly in the
next step without further purification. 1H NMR (400 MHz, CDCl3) δ
7.53 (s, 1H), 7.46 (dd, J = 8.8, 2.7 Hz, 1H), 6.79 (d, J = 8.8 Hz, 1H),
2.28 (s, 3H). MS (MM-ES + APCI) m/z: 233.0 [M − H]−.
2-Ethyl-4-(pentafluorosulfanyl)phenol (9b). To a mixture of 8b
(0.13 g, 0.49 mmol) and trimethylamine (TEA) (1.82 g, 18 mmol) in
CH2Cl2 was added ethyl chloroformate (0.75 g, 6.9 mmol) at 0 °C and
then stirred at room temperature for 3 h. After the reaction completed,
the mixture was concentrated and dissolved in EtOH (3 mL). To
above solution, a solution of NaBH4 (2 g, 55.2 mmol) in H2O
(20 mL) was added at 0 °C. The resulting mixture was then stirred at
room temperature overnight. Then the mixture was acidified to pH = 3
and extracted with CH2Cl2. The organic layer was dried over Na2SO4
and concentrated to give title compound, which was used directly in
the next step without further purification.
8-Methyl-6-(pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chro-
mene-3-carboxylic Acid (R,S-11a). The title compound was prepared
1
by the same procedure as R,S-3a in 95% yield. H NMR (500 MHz,
CDCl3) δ 7.82 (s, 1H), 7.60 (s, 1H), 7.51 (s, 1H), 5.80 (d, J = 6.5 Hz,
1H), 2.31 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.39 (s),
152.50 (s), 146.52 (s), 135.14 (s), 130.80 (s), 126.04 (s), 125.18 (s),
124.47 (t, J = 287.4 Hz), 118.67 (s), 70.37 (d, J = 32.3 Hz), 14.86 (s).
19F NMR (471 MHz, CDCl3) δ 84.74 (m, 1F), 63.72 (dd, J = 150.5,
21.4 Hz, 4F), −78.78 (d, J = 6.3 Hz, 3F). HRMS (ESI): calcd for
C12H7F8O3S [M − H] 382.9994, found 383.0004. HPLC: 95.42%.
8-Ethyl-6-(pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chro-
mene-3-carboxylic Acid (R,S-11b). The title compound was prepared
1
by the same procedure as R,S-3a in 95% yield. H NMR (500 MHz,
CDCl3) δ 7.82 (s, 1H), 7.60 (d, J = 2.5 Hz, 1H), 7.52 (d, J = 2.6 Hz,
1H), 5.80 (q, J = 6.7 Hz, 1H), 2.71 (m, 2H), 1.24 (t, J = 7.6 Hz, 3H).
13C NMR (126 MHz, DMSO-d6) δ 165.20 (s), 152.64 (s), 148.29−
146.87 (m), 135.20 (s), 132.64 (s), 130.10 (s), 125.67 (s), 122.78 (t,
J = 287.4 Hz), 119.95 (s), 119.53 (s), 71.52−70.60 (m), 22.86 (s),
14.30 (s). 19F NMR (471 MHz, CDCl3) δ 84.65 (m, 1F), 63.70 (dd,
J = 150.4, 22.1 Hz, 4F), −78.55 (d, J = 6.7 Hz, 3F). HRMS (ESI): calcd
for C13H9F8O3S [M − H] 397.0150, found 397.0171. HPLC: 98.18%.
6-(Pentafluorosulfanyl)-8-propyl-2-(trifluoromethyl)-2H-chro-
mene-3-carboxylic Acid (R,S-11c). The title compound was prepared
1
4-(Pentafluorosulfanyl)-2-propylphenol (9c). The title compound
by the same procedure as R,S-3a in 90% yield. H NMR (500 MHz,
1
was prepared by the same procedure as 9a in 45% yield. H NMR
CDCl3) δ 7.81 (s, 1H), 7.58 (d, J = 2.5 Hz, 1H), 7.51 (d, J = 2.6 Hz,
1H), 5.79 (q, J = 6.7 Hz, 1H), 2.65 (m, 2H), 1.64 (m, 2H), 0.97 (t, J =
7.4 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.51 (s), 152.33
(s), 146.74 (t, J = 16.6 Hz), 135.19 (s), 130.33 (s), 125.43 (s), 124.57
(t, J = 287.4 Hz), 119.07 (s), 118.79 (s), 70.51 (q, J = 31.8 Hz), 30.75
(s), 22.16 (s), 13.40 (s). 19F NMR (471 MHz, CDCl3) δ 84.65 (m,
1F), 63.70 (dd, J = 150.5, 21.9 Hz, 4F), −78.54 (d, J = 6.7 Hz, 3F).
HRMS (ESI): calcd for C14H11F8O3S [M − H] 411.0307, found
411.0309. HPLC: 99.14%.
(400 MHz, CDCl3) δ 7.52 (s, 1H), 7.45 (dd, J = 8.8, 2.7 Hz, 1H), 6.79
(d, J = 8.8 Hz, 1H), 2.61 (t, J = 21.6, 13.7 Hz, 2H), 1.73−1.57 (m, 2H),
0.98 (t, J = 7.3 Hz, 3H). MS (MM-ES + APCI) m/z: 261.0 [M − H]−.
2-Benzyl-4-(pentafluorosulfanyl)phenol (9d). The title compound
1
was prepared by the same procedure as 9a in 50% yield. H NMR
(400 MHz, CDCl3) δ 7.59−7.49 (m, 2H), 7.32 (t, J = 7.3 Hz, 2H),
7.23 (t, J = 6.7 Hz, 3H), 6.82 (d, J = 8.5 Hz, 1H), 4.02 (s, 2H). MS
(MM-ES + APCI) m/z: 309.0 [M − H]−.
2-Butyl-4-(pentafluorosulfanyl)phenol (9e). The title compound
8-Benzyl-6-(pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chro-
1
was prepared by the same procedure as 7e in 47% yield. H NMR
mene-3-carboxylic Acid (R,S-11d). The title compound was prepared
1
(500 MHz, CDCl3) δ 7.55 (s, 1H), 7.47 (d, J = 8.8 Hz, 1H), 6.81 (d,
J = 8.8 Hz, 1H), 6.71 (s, 1H), 2.70−2.62 (m, 2H), 1.64−1.61 (m, 2H),
1.47−1.36 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H). MS (MM-ES + APCI)
m/z: 275.1 [M − H]−.
2-Hydroxy-3-methyl-5-(pentafluorosulfanyl)benzaldehyde (10a).
The title compound was prepared by the same procedure as 2a in 25%
yield. 1H NMR (400 MHz, CDCl3) δ 11.56 (s, 1H), 9.91 (s, 1H), 7.83
(s, 1H), 7.76 (s, 1H), 2.33 (s, 3H). MS (MM-ES + APCI) m/z: 261.0
[M − H]−.
by the same procedure as R,S-3a in 95% yield. H NMR (500 MHz,
CDCl3) δ 7.82 (s, 1H), 7.55 (s, 2H), 7.31 (t, J = 7.4 Hz, 2H), 7.23 (d,
J = 7.3 Hz, 1H), 7.18 (d, J = 7.2 Hz, 2H), 5.79 (q, J = 6.5 Hz, 1H),
4.08−3.96 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 164.39 (s),
152.20 (s), 146.75 (t, J = 16.9 Hz), 138.90 (s), 134.91 (s), 130.61 (s),
129.24 (s), 128.48 (s), 126.34 (s), 125.80 (s), 124.77 (t, J = 287.4 Hz),
119.28 (s), 118.89 (s), 70.56 (q, J = 32.3 Hz), 34.15 (s). 19F NMR
(471 MHz, CDCl3) δ 84.30 (m, 1F), 63.70 (dd, J = 150.4, 22.2 Hz,
4F), −78.61 (d, J = 6.5 Hz, 3F). HRMS (ESI): calcd for C18H11F8O3S
[M − H] 459.0307, found 459.0308. HPLC: 99.37%.
3-Ethyl-2-hydroxy-5-(pentafluorosulfanyl)benzaldehyde (10b).
To a solution of 9b (0.12 g, 0.49 mmol) in TFA (6 mL) was added
HTMA (0.20 g, 1.4 mmol) portionwise. The mixture was stirred at
80 °C for 12 h and then cooled to room temperature. To the above
mixture was added 3 N HCl (3 mL) and the resulting mixture stirred
at room temperature for another 0.5 h. The mixture was then extracted
with EtOAc, and the organic layer was washed with saturated
NaHCO3, water, and brine. The resulting organic phase was dried over
Na2SO4, concentrated, and purified by column chromatography to give
8-Butyl-6-(pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chro-
mene-3-carboxylic Acid (R,S-11e). The title compound was prepared
1
by the same procedure as R,S-3a in 90% yield. H NMR (500 MHz,
DMSO-d6) δ 13.46 (s, 1H), 8.03 (d, J = 2.5 Hz, 1H), 7.99 (d, J = 11.5
Hz, 1H), 7.78 (d, J = 2.5 Hz, 1H), 6.10 (q, J = 7.0 Hz, 1H), 2.74−2.65
(m, 1H), 2.65−2.55 (m, 1H), 1.58−1.45 (m, 2H), 1.34−1.24 (m, 2H),
0.88 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.36
(s), 152.19 (s), 146.56 (d, J = 17.0 Hz), 135.15 (s), 130.49 (s), 130.19
(s), 125.32 (s), 123.33 (t, J = 287.4 Hz), 118.96 (s), 118.58 (s), 70.36
(q, J = 32.2 Hz), 31.02 (s), 28.49 (s), 21.71 (s), 13.58 (s). 19F NMR
(471 MHz, DMSO-d6) δ 88.58−86.89 (m, 1F), 66.35−64.67 (m, 4F),
−77.53 (d, J = 7.1 Hz, 3F). HRMS (ESI) calcd for C15H13F8O3S
[M − H] 425.0463, found 425.0459. HPLC: 92.88%.
1
the title compound 40 mg (30% yield for two steps). H NMR (400
MHz, CDCl3) δ 11.58 (s, 1H), 9.91 (s, 1H), 7.83 (d, J = 2.7 Hz, 1H),
7.76 (d, J = 2.6 Hz, 1H), 2.75 (q, J = 7.5 Hz, 2H), 1.30−1.22 (m, 3H).
MS (MM-ES + APCI) m/z: 275.0 [M − H]−.
2-Hydroxy-5-(pentafluorosulfanyl)-3-propylbenzaldehyde (10c).
The title compound was prepared by the same procedure as 8b in
25% yield. MS (MM-ES + APCI) m/z: 289.1.0 [M − H]−.
(S)-6-(Pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chromene-3-
carbaldehyde (S-12a), (R2 = H). A mixture of 2a (100 mg, 0.40 mmol),
H
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Journal of Medicinal Chemistry
Article
(E)-4,4,4-trifluorobut-2-enal (74 mg, 0.60 mmol), (S)-2-(diphenyl-
(trimethylsilyloxy)methyl) pyrrolidine (20 mg, 0.06 mmol), and
2-nitrobenzoic acid (10 mg, 0.06 mmol) in EtOAc (10 mL) was
stirred at room temperature overnight. The solution was then concen-
trated and purified by column chromatography to give the title
compound 113 mg in 80% yield. 1H NMR (400 MHz, CDCl3), δ 9.69
(s, 1H), 7.74 (s, 1 H), 7.58 (m, 2 H), 7.08 (d, J = 7.5 Hz, 1H), 5.99 (q,
J = 9 Hz, 1H).
at −20 °C until LC-MS analysis. The rats were humanely euthanized
by carbon dioxide 24 h after experiment without pain.
Plasma Sample Preparation. The plasma samples were prepared
using protein precipitation method. First, 50 μL plasma samples were
added to 1.5 mL tube and vortex for 3 min, then 150 μL of acetonitrile
containing internal standard were added and vortexed for 5 min, and
finally spin tubed in centrifuge at 16000g for 40 min at 4 °C. The
standard curve was prepared as follows: The compound was dissolved
in DMSO at a concentration of 2 mg/mL and diluted with 50% aq
methanol solution to series concentration. Series concentration solu-
tion was prepared as plasma sample was.
Liquid Chromatography and Mass Spectrometry Conditions.
Chromatographic analyses were performed with a HPLC system
consisting of a LC-10ADvp pump (Shimadzu, Japan), MPS3C auto-
matic sampler (Gerstel Auto Sampler, Germany), and API 3000 triple
quadrupole tandem mass spectrometer (AB Co., USA). The column
used for the separation was a Capcell PAK C18 (2.0 mm × 50 mm,
5 μm). The HPLC mobile phases consisted of water (0.1% form acid)
(A) and methanol (0.1% form acid) (B). A gradient program was used
for the HPLC separation with 0.3 mL/min. The initial composition
was 30% B, increased linearly to 100% B in 1.5 min, and maintained at
100% B for 1.5 min, decreased linearly to 30% B in 0.3 min, then
maintained for 0.7 min. The column eluent was directly introduced
into electrospray ionization (ESI) interface.
(S)-8-Bromo-6-(pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chro-
mene-3-carbaldehyde (S-12b), (R2 = Br). A mixture of 6 (R2
=
Br) (100 mg, 0.30 mmol), (E)-4,4,4-trifluorobut-2-enal (74 mg,
0.60 mmol), (S)-2-(diphenyl(trimethylsilyloxy)methyl) pyrrolidine
(20 mg, 0.06 mmol), and 2-nitrobenzoic acid (10 mg, 0.06 mmol)
in EtOAc (10 mL) was stirred at room temperature overnight. The
solution was then concentrated and purified by column chromatog-
1
raphy to give the title compound 106 mg in 80% yield. H NMR
(400 MHz, CDCl3), δ 9.69 (s, 1H), 7.74 (s, 2 H), 7.58 (s, 1 H), 5.99
(m, J = 9 Hz, 1H).
In Vitro COX Enzyme Assay. Compounds were evaluated for
COX inhibitory activity in vitro by using Cayman’s COX Fluorescent
Inhibitor Screening Assay Kit (Cayman Chemical Company, Ann
Arbor, MI, USA; item nos. 701070 and 701080). Experiments were
performed according to the manufacturer’s instruction. Human
COX-1 and human recombinant COX-2 enzymes were preincubated
with serially diluted test compounds for 15 min at rt, the heme and
fluorometric substrates were added and incubated for another 15 min
at rt. The reaction was started by the addition of arachidonic acid and
allowed to proceed for 2 min. The fluorescence was measured at a
530 nm excitation wavelength and a 595 nm emission wavelength
using a microplate reader (Envision, PerkinElmer), and the data were
analyzed using Graphpad Prism 5 (Graphpad Software, Inc.).
Fluorescence Polarization hERG Assay. The Inhibition activity
of compounds on hERG potassium channel was determined using
Predictor hERG Fluorescence Polarization Assay Kit (Life Tech-
nologies, Carlsbad, CA, USA). Experiments were performed according
to the manufacturer’s instructions. In brief, the reactions were carried
out in 384-well plates including 10 μL of Predictor hERG membrane
and 10 μL of Predictor hERG Tracer Red with appropriate amount of
compound or positive control. Reactions were incubated for 2 h at rt
and then read on an EnVision Multilabel Reader (PerkinElmer, Inc.)
using polarized excitation and emission filters. Data were analyzed
using Graphpad Prism 5 (GraphPad Software Inc., San Diego, CA).
Human Plasma Protein Binding. Human plasma protein
(Southern Medical University) binding (PPB) of test compounds
was measured by rapid equilibrium dialysis device using RED Device
Inserts (Thermo Scientific). Reactions were carried out for 5 h, and
then the drug concentrations in the sample chamber and buffer
chamber were determined by LC/MS (API 3000 three triple quad-
rupole tandem mass spectrometer AB company) (LC-10ADvp pump
(Shimadzu). Using these values, PPB of test compounds were defined
using the following equation: PPB rate = [1 − (concentration buffer
chamber/concentration plasma chamber)] × 100%.
In Vivo Pharmacodynamics. The assay listed here was per-
formed in accordance with Public Health Service policies, the Animal
Welfare Act, and the Laboratory Animal Committee (LAC) of GIBH
(Guangzhou Institute of Biomedicine and Health, Chinese Academy
of Sciences) Policy on Humane Care.
Pharmacokinetic Studies. Compounds were dissolved in suitable
solvent. Pharmacokinetic properties of SD rats (male) were deter-
mined following IV and oral administration. Animals were randomly
distributed into two experimental groups (n = 4). The oral groups
were given 5 mg/kg of compound by gastric gavage. The other group
was dosed by injection into the tail vein (1 mg/kg). After single
administration, whole blood samples (100−200 μL) were obtained
from the orbital venous plexus at the following time points after
dosing: 5, 10, and 30 min and 1, 2, 3, 4, 6, 8, 11, and 24 h (PO); 2, 10,
30 min and 1, 2, 3, 4, 6, 8, 11, and 24 h (IV). Whole blood samples
were collected in heparinized tubes. The plasma fraction was imme-
diately separated by centrifugation (8000 rpm, 6 min, 4 °C) and stored
Data Analysis. After centrifugation of plasma samples, 100 μL of
the supernatant was transferred to a 96-well plate and analyzed by
LC-MS/MS for each individual test compound. The pharmacokinetics
parameters were calculated using the pharmacokinetic software
DAS.2.0.
Air-Pouch Model of Inflammation. Air pouches were produced
by a subcutaneous injection of 20 mL of sterile air into the back area
of male Sprague−Dawley rats (Southern Medical University, China)
after anesthetization using carbon dioxide. The compounds were
administrated orally (10 mL/kg) as a suspension in 0.5% carboxy-
methyl cellulose at the dose 0.03, 0.1, 0.3, 1, 3, and 10 mg/kg (n = 6)
and the vehicle as the control. One day post the air pouch production,
6 mL of 1% carrageenan (Sigma, USA) saline solution was injected
into the air cavities to produce an inflammatory reaction 1 h after
administration. Then 3 mL of washing solution (9 g/L NaCl, 2 mg/L
EDTA, 10 mg/L indomethacin) was injected into the air cavity and
shaking. After performing euthanasia to the rats 3 h post the car-
rageenan injection, 1 mL of exudate washing solution in the air cavity
was collected into the EP tube and centrifuged at 8000g for 5 min at
4 °C, the supernatant was collected to analyze the PGE-2 using PGE-2
ELISA kit (R&D Systems, USA). Immediately after collecting the
exudates, the gastric mucosa was taken from the stomach and homo-
genized in washing solution (weight: volume = 1:20), centrifuged at
12000g for 15 min at 4 °C, and the supernatant was collected to
determine the PGE-2 using PGE-2 ELISA kit. The EC50 was analyzed
using the software Grafit 5 (Erithacus Software Ltd., Horley, UK).
Carrageenan Induced Rat Foot Paw Edema. Paw edema was
induced by injecting subcutaneously 0.2 mL of 3% λ-carrageenan
(Sigma, USA) saline solution into the right hind paw of male
Sprague−Dawley rats (Southern Medical University, China) with the
weight from 180 to 220 g. The compounds were administrated orally
as a suspension in 0.5% carboxymethyl cellulose at the dose 1, 3, 10,
30, 100 mg/kg (n = 6) and the vehicle as the control at the time 0.5 h
before immunization. Paw volumes were measured by water dis-
placement with a plethysmometer PV-200 (Chengdu Technology and
Market Co., China); immediately after immunization and 3 h post
immunization, the percentage of inhibition was calculated by comparing
the paw volume increment of treated animals, and the EC50 was analyzed
using the software Grafit 5 (Erithacus Software Ltd., Horley, UK).
Carrageenan Induced Rat Foot Paw Hyperalgesia. Paw
hyperalgesia was induced by injecting subcutaneously 0.2 mL of 3%
λ-carrageenan (Sigma, USA) saline solution into the right hind paw of
male Sprague−Dawley rats (Southern Medical University, China) with
the weight from 180 to 220 g. The compounds were administrated
orally as a suspension in 0.5% carboxymethyl cellulose at the dose of 1,
3, 10, 30, and 100 mg/kg (n = 6) and the vehicle as the control at the
I
DOI: 10.1021/acs.jmedchem.6b01484
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
time 0.5 h before immunization. Hyperalgesia was measured as the
antinociceptive response to thermal stimuli from a radiant heat source
positioned under a Plexiglas floor directly beneath the right hind paw
before immunization and 3 h post immunization using the PL-200
thermal stabbing pain equipment (Chengdu Technology and Market
Co., Ltd., China), the percentage of inhibition was calculated by com-
paring the paw withdrawal latency of treated and controlled animals,
and the EC50 was analyzed using the software Grafit 5 (Erithacus
Software Ltd., Horley, UK).
the project funded by Guangdong Science & Technology Project
(2013B050800009) (Y.Z.), CAS Key Technology Talent Pro-
gram China (2013) (Z.T.), and Guangdong Science & Tech-
nology Project (2013B040200031) (Z.T.).
ABBREVIATIONS USED
■
F, oral bioavailability; PK, pharmacokinetics; PD, pharmacody-
namics; AUC, area under the curve; Tmax, time to maximum
drug concentration; Cmax, maximum concentration of drug;
MMP, matrix metalloprotease; COX-1, cyclooxygenase 1;
COX-2, cyclooxygenase 2
Adjuvant Induced Rat Arthritis. The adjuvant arthritis was
induced by injection of 1 mg of heat-killed Mycobacterium tuberculosis
(Difco, USA) in 0.2 mL of mineral oil (Sigma, USA) into the base of
the tail of the male Lewis rat (Charles River, China) with the weight
from 170 to 200 g. The volume of the left hind paw was measured
using a plethysmometer (Chengdu Technology and Market Co., Ltd.,
China) on day zero and 14th post adjuvant injection, and the rats on
the day 14 having a left hind paw volume of 0.375 mL greater than that
on day zero post adjuvant injection were randomly divided into six
groups with each containing six animals. Treatment was initiated on
day 15 and until day 24 after the immunization. Different dosages of
the compound suspended in 0.5% methylcellulose (0.1, 0.3, 1, 3, and
10 mg/kg and vehicle) were administrated by oral gavage one time
daily. The left hind paw volume and the clinical score of each rat were
examined on day 25 after immunization, and the clinical scoring for
each limb ranged from 0 to 4 (0 = no erythema or swelling, 1 = slight
erythema or swelling of one of the toes or fingers, 2 = erythema and
swelling of more than one toe or finger, 3 = erythema and swelling of
the ankle or wrist, 4 = complete erythema and swelling of toes or
fingers and ankle or wrist, and inability to bend the ankle or wrist).
The percent inhibition was calculated by comparing the paw with-
drawal latency of treated animals, and the EC50 was analyzed using the
software Grafit 5 (Erithacus Software Ltd., Horley, UK).
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.6b01484.
■
S
HPLC traces, HRMS reports, H, 13C, 19F NMRs, and
1
chiral HPLC traces (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*For Y.Z.: phone, 86-20-32015277; E-mail, zhang_yanmei@
gibh.ac.cn.
■
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gibh.ac.cn.
*For J.J.T.: phone, 1-314-650-2151; E-mail, jjtalley@euclises.
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ORCID
Yanmei Zhang: 0000-0002-2300-7390
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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The research work was financially supported by the Guangzhou
Science & Technology Project (2011Y2-00026 and 201508020131)
to support GIBH Drug Discovery Pipeline Development (Y.Z.),
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