Pyrazolylthiazole as Δf508-cystic fibrosis transmembrane conductance regulator correctors with improved hydrophilicity compared to bithiazoles

Article abstract of DOI:10.1021/jm100235h

Deletion of phenylalanine residue 508 (ΔF508) in the cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR) is a major cause of CF. Small molecule correctors of defective ΔF508-CFTR cellular processing hold promise for CF therapy. We previously identified and characterized bithiazole CF corrector 1 and s-cis-locked bithiazole 2. Herewe report the regiodivergent synthesis of Nγ and Nβ isomers of thiazole-tethered pyrazoles with improved hydrophilicity compared to bithiazoles. We synthesized a focused library of 54 pyrazolylthiazoles 3, which included examples of both regioisomers 4 and 5. The thiazole-tethered pyrazoles allowed incorporation of property-modulating functionality on the pyrazole ring (ester, acid, and amide) while retaining ΔF508-CFTR corrector activity (EC₅₀) of under 1 μM. The most active pyrazolylthiazole (14h) has an experimentally determined log P of 4.1, which is 1.2 log units lower than bithiazole CF corrector 1.

Full text of DOI:10.1021/jm100235h

3772 J. Med. Chem. 2010, 53, 3772–3781
DOI: 10.1021/jm100235h
Pyrazolylthiazole as ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator Correctors
with Improved Hydrophilicity Compared to Bithiazoles
Long Ye,† John M. Knapp,† Panjamaporn Sangwung,‡ James C. Fettinger,† A. S. Verkman,‡ and Mark J. Kurth*,†
†Department of Chemistry, University of California, Davis, Davis, California 95616, and ‡Department of Medicine and Physiology,
University of California, San Francisco, California, 94143-0521
Received February 23, 2010
Deletion of phenylalanine residue 508 (ΔF508) in the cystic fibrosis (CF) transmembrane conductance
regulator protein (CFTR) is a major cause of CF. Small molecule “correctors” of defective ΔF508-
CFTR cellular processing hold promise for CF therapy. We previously identified and characterized
bithiazole CF corrector 1 and s-cis-locked bithiazole 2. Herein, we report the regiodivergent synthesis of
Nγ and Nβ isomers of thiazole-tethered pyrazoles with improved hydrophilicity compared to
bithiazoles. We synthesized a focused library of 54 pyrazolylthiazoles 3, which included examples of
both regioisomers 4 and 5. The thiazole-tethered pyrazoles allowed incorporation of property-
modulating functionality on the pyrazole ring (ester, acid, and amide) while retaining ΔF508-CFTR
corrector activity (EC50) of under 1 μM. The most active pyrazolylthiazole (14h) has an experimentally
determined log P of 4.1, which is 1.2 log units lower than bithiazole CF corrector 1.
Introduction
original bithiazole scaffold, that would allow for incorpora-
tion of property-modulating functionality while retaining
corrector activity. Because of the lack of useful crystal struc-
ture data for ΔF508-CFTR, we embarked on a ligand-based
compound design strategy wherein many of the core structural
features of our previously identified bithiazole CF correctors
were retained while simultaneously incorporating property-
modulating modifications with the aim of improving hydro-
philicity relative to 1. Target compounds were synthesized
and screened for corrector activity. This effort led us to a
pyrazolylthiazole core (3) and the production of a focused
library of pyrazolylthiazoles, including regioisomers 4and 5.As
Figure 1 illustrates, pyrazole 3 retains many aspects of the core
framework of 1 but replaces the right-hand thiazole with a
pyrazole ring. Compared to the thiazole of 1, the pyrazole ring
accommodates the attachment of extra functionality, such as a
C5 (see R3 in 4) or C3 (see R3 in 5) amide, to modulate
properties such as log P.
One vexing drawback with the bithiazole core of 1 is that it
essentially precludes modification. For example, consider the
right-hand thiazole ring of 1 (red substructure): positions S1,
C2, N3, and C4 are fixed and addition of substituents at C5
forces the two thiazole rings to adopt an orthogonal posture
that is quite different from the conformation of s-cis-locked 2,
our best bithiazole corrector. In contrast, pyrazoles, which are
considered a privileged heterocyclic structure with many
reports of biological activity,9 allow for modification at C5
(see 3), Nβ (see 4), and Nγ (see5). Asillustrated in Figure1, we
reasoned that, as a starting point, retaining the N-(4-methyl-
thiazol-2-yl)pivalamide on the left-hand thiazole ring and the
5-chloro-2-methoxyaryl on the right-hand thiazole ring (see
bithiazole 1) might impart corrector activity to pyrazolyl-
thiazole 3 while allowing for the introduction of property-
modulating functionality “X” at C5 (see 3) of the pyrazole
ring. However, there are two important differences caused by
Cystic fibrosis (CFa), a serious disease afflicting ∼1 in 2500
in the Caucasian population,1 is caused by inherited muta-
tions in the CF transmembrane conductance regulator
(CFTR) gene.2 The most common disease-causing mutation
of CFTR is ΔF508, a deletion of phenylalanine at position
508, which accounts for ∼70% of all CF alleles such that
∼90% of CF patients carry at least one copy of ΔF508-CFTR
allele.3 The ΔF508 mutation alters CFTR biosynthesis, re-
sulting in a misfolded, rapidly degraded protein that is poorly
trafficked out of the endoplasmic reticulum to the cell sur-
face.4 Without physiological CFTR chloride channel func-
tioning at epithelia cell membranes in lung, pancreas, and
intestine, water and salt secretion are compromised, causing
chronic respiratory infections, airway obstruction by viscous
mucous, pancreatic exocrine insufficiency, etc.5
Small-molecule therapy for ΔF508-caused CF is thought to
have considerable promise.6 In our previous work, bithiazole
lead compound 1 (Figure 1) was identified as a “corrector”
capableof partiallyrestoringthe targeting of ΔF508-CFTR to
the cell membrane in transfected cells and primary cultures of
bronchial epithelial cells from ΔF508 patients.7 In follow-up
work, nanomolar corrector potency of the bithiazole class was
obtained by locking the thiazole core of 1 into an s-cis
conformation (see 2 in Figure 1).8 Although s-cis-locked
bithiazole corrector 2 affords improved potency compared
to the original corrector 1, its high hydrophobicity (clogP
6.65) is a concern for further development. Therefore, our
focus has been to identify a new chemotype, based on the
*To whom correspondence should be addressed. Phone: (530) 554-
2145. Fax: (530) 752-8995. E-mail: mjkurth@ucdavis.edu.
a Abbreviations: CFTR, cystic fibrosis transmembrane conductance
regulator; SAR, structure-activity relationship; CF, cystic fibrosis; ER
endoplasmic reticulum.
r
pubs.acs.org/jmc
Published on Web 04/07/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3773
Scheme 1. Synthesis of Nγ-Substituted Pyrazolylthiazolesa
Figure 1. SAR around early bithiazole lead 1 led to s-cis-locked
bithiazole 2, but these compounds have poor hydrophilicity. The
structural features of these bithiazoles inspired the design of
pyrazolylthiazole libraries based on differentially Nβ- and Nγ-
substituted analogues 4 and 5, respectively.
a Reagents and conditions: (a) R1CO2H, CDI, DMF, 85 °C; (b)
the thiazole f pyrazole modification. First, the aniline moiety
of 1 is replaced with a benzylicmoietyin 3; this might causethe
loss of a possible hydrogen bonding interaction with the target
protein. Second, the thiazole “S” is replaced with a pyrazole
“C;X” (X = carboxylic acid derivative in this work); this
mightcausenonproductiveinteraction with the target protein.
On the other hand, the X-moiety could be exploited to
improved drug properties, such as log P in this work, and
might also lead to alternative productive interactions with the
target protein. These two thiazole f pyrazole implications are
highlighted by the ellipses in 1 versus 3 as well as the red
substructures in 4 and 5 (Figure 1). To investigate the con-
diethyl oxalate, LHMDS, THF, -78 °C; (c) R2NHNH2, EtOH; (d)
0
R3R3 NH, AlMe3, DCM, 0 °C; (e) NaOH, THF/H2O; (f) ethanol amine,
MW, 160 °C, 30 min.
7 was then subjected to Claisen reaction with diethyl oxalate
in the presence of 2.1 equiv of LHMDS, giving 1,3-diketone 8
in excellent yield (>90%). With the 2,4-dioxo-4-(thiazol-5-yl)-
butanoate 8 in hand, we turned to pyrazole formation and
expected both pyrazole regioisomers, 9 and 10, in the reaction
with monosubstituted hydrazines. In fact, the condensation
reaction between 8 and substituted hydrazines proceeds with
excellent selectivity to deliver the Nγ regioisomers 9 in good
yield (>87%; 9:10>10:1). A minor byproduct was found to
be 7, indicating that the 2,4-dioxobutanoate moiety is suscep-
tible to a reverse Claisen reaction in the presence of hydrazine
nucleophiles. The excellent regioselectivity of this condensa-
tion reaction reflects the fact that the two carbonyl groups in 8
have very different electrophilicity. From pyrazolylthiazole 9,
the final diversification in this series was achieved by convert-
ing the ester moiety into the corresponding amide (f11) via
aminolysis in good yield (>73%) or acid (f 12) by saponi-
fication. Thirty-nine Nγ-substituted pyrazolylthiazoles were
ꢀ
sequences of these structural changes vis-a-vis the discovery of
a new CF corrector chemotype, we designed a focused library
based on the pyrazolylthiazole scaffold wherein the substitu-
ents adorning the pyrazole substructure are varied.
Results and Discussion
Chemistry. Pyrazoles can be synthesized through a Knorr
cyclocondensation between a 1,3-diketone and hydrazine or
a substituted hydrazine.10 This general method often suffers
from lack of regioselectivity in the production of substituted
pyrazoles.11 There are only sporadic reports addressing the
synthesis of pyrazolylthiazoles by this condensation reac-
tion.12 However, none of these methods involve diversifica-
tion of this heteroatom-rich bisazole or delineate routes to
acquire both N-regioisomers of the pyrazole from a common
intermediate. Our discovery efforts led us to explore general
and integrated synthetic methods to access a series of func-
tionally and regioisomerically diversified pyrazolylthiazoles;
the results are reported herein.
This synthetic effort began with conversion of the amino
group of 6 into amide 7 (Scheme 1; two variants, pivaloyl and
benzoyl, were prepared based on our bithiazole work)
through a CDI-mediated coupling reaction, which occurred
in greater than 79% yield. Both of these amides demon-
strated corrector activity in prior bithiazole studies.7 Amide
prepared (see Table 1) by varying all three diversity points (two
0
R1 inputs, four R2 inputs, and four NR3R3 inputs). The X-ray
crystal structure determined for one Nγ-substituted analogue
(9b) is shown in Figure 2.
Because the Nβ-substituted pyrazole regioisomer could
not be accessed in practical yield through the direct con-
densation of 8 with monosubstituted hydrazines, an alter-
nate route was explored to synthesize these compounds
(Scheme 2). First, 8 was reacted with hydrazine monohydrate
to deliver pyrazole 13 in 63% yield. The Nβ position of 13
was then selectively alkylated, giving mainly 10 (R = H) in
59-66% yield when R1 = pivaloyl (10:9 = 8:1). The major
byproduct of this reaction is N-alkylation of the amide moiety,
leading to formation of bis-alkylated product (10: R = R2).
This complication was so severe with the benzamide analogue

3774 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Table 1. Nγ-Substituted Pyrazolylthiazole Analogues
Ye et al.
compd
R1
R2
X
compd
R1
R2
X
9a
9b
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C6H5
CH2CHdCH2
C6H4(4-Br)
CH2C6H5
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
11m
11n
11o
11p
11q
11r
11s
11t
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C(CH3)3
C(CH3)3
C(CH3)3
C6H5
C6H5
C6H5
C6H5
CH2CHdCH2
C6H4(4-Br)
CH2C6H5
NH(CH2)OH
NH(CH2)OH
NH(CH2)OH
NH(CH2)OH
NHC6H4(4-OMe)
NHC6H4(4-OMe)
NHC6H4(4-OMe)
NHC6H4(4-OMe)
N(CH2CH2)2O
N(CH2CH2)2O
N(CH2CH2)2O
N(CH2CH2)2O
OH
9c
9d
CH2CH2OH
CH2CHdCH2
C6H4(4-Br)
CH2C6H5
CH2CH2OH
CH2CHdCH2
C6H4(4-Br)
CH2C6H5
9e
9f
9g
C6H5
C6H5
C6H5
9h
CH2CH2OH
CH2CHdCH2
CH2CHdCH2
CH2CHdCH2
C6H4(4-Br)
C6H4(4-Br)
C6H4(4-Br)
CH2C6H5
CH2CH2OH
CH2CHdCH2
C6H4(4-Br)
CH2C6H5
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
NHC6H4(4-OMe)
NHCH2C6H5
11u
11v
11w
11x
12a
12b
12c
12d
12e
12f
N(CH2CH2)2O
NHC6H4(4-OMe)
NHCH2C6H5
CH2CH2OH
CH2CHdCH2
C6H4(4-Br)
CH2C6H5
N(CH2CH2)2O
NHC6H4(4-OMe)
NHCH2C6H5
OH
OH
CH2C6H5
CH2C6H5
CH2CHdCH2
C6H4(4-Br)
CH2C6H5
OH
OH
N(CH2CH2)2O
NHC6H4(4-OMe)
NHCH2C6H5
CH2CH2OH
CH2CH2OH
CH2CH2OH
OH
OH
12g
CH2CH2OH
N(CH2CH2)2O
Scheme 2. Synthesis of Nβ-Substituted Pyrazolylthiazole
Figure 2. X-ray crystallographic structure of Nγ-substituted pyr-
azolylthiazole 9b (note: the S10-C50-C5-N1 dihedral angle is
62.4°).
Reagents: (a) NH2NH2 H2O, EtOH; (b) R2X, K2CO3, acetone,
3
0
60 °C; (c) R3R3 NH, AlMe3, DCM, MW, 100 °C; (d) NH2(CH2)2OH,
EtOH, MW 180 °C; (e) NaOH, THF/H2O.
of 13 (R1 = C6H5) that the major product was, in fact, the
bisalkylation product (10: R = R2); the desired monoalky-
lated product (10: R = H) was obtained in only 20% yield.
Therefore, final diversification on the Nβ-substituted isomers
was focused on pivaloyl amide analogues of 10 and consisted
of hydrolysis or aminolysis to deliver 14 and 15, respectively.
Unlike the relatively easy aminolysis of 9 (Scheme 1) that
proceeds at 0 °C, initial attempts at the aminolysis of 10 (0 °C
in DCM; Scheme 2) failed to deliver 14. X-ray crystallographic
analysis of 10a (R1 =tBu/R2 = allyl; Figure 3) reveals that
the R2 substituent imparts significant steric hindrance at the
carboethoxy and retards amidyl formation at the pyrazole C5
position. This challenge was overcome by employing micro-
wave irradiation in the AlMe3-mediated aminolysis, delivering
various amides and completing pyrazole diversification. In
total, 15 Nβ-substituted pyrazolylthiazoles were prepared for
this focused library (see Table 2).
Structure-Activity Relationships. The Nγ- and Nβ-regio-
isomeric pyrazolylthiazoles were assayed for ΔF508-CFTR
corrector activity. Our established cell-based corrector assay
was used in which I- influx was measured in FRT cells
coexpressing human ΔF508-CFTR and the I--sensitive

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3775
fluorescent sensor YFP-H148Q/I152L.13 Following 24 h
incubation with test compounds, I- influx was determined
from the kinetics of YFP-H148Q/I152L quenching in res-
ponse to I- addition in cells treated with a cAMP agonist and
the potentiator genistein. Out of the 54 compounds tested,
eight had significant corrector activity as judged by concen-
tration-dependent increases in I- influx as exemplified in
Figure 4 for 14g and 14h.
Structures for these compounds as well as their EC50 and
Vmax values from ion influx data are summarized in Table 3.
Their EC50 values range from 0.93 to 8.5 μM while increasing
I- influx up to 7.3 μM/s (note: increased I- influx is a quan-
titative measure of effective ΔF508-CFTR corrector
activity). As illustrated in Table 3, pyrazolylthiazole 14h is
the best corrector among the eight hits and of comparable
potency to our previously reported bithiazole 1. Of the eight
active compounds, 10b, the only ester, and 11d, the only Nγ-
substituted pyrazole, are the least effective pyrazolylthiazole
correctors. Considering that ester-containing pyrazolylthia-
zoles 9 and 10 are all inactive except for 10b, the carboxamide
group at C5 of the pyrazole ring seems to be an impor-
tant determinant of activity. Another observation from
Table 3 is that the majority of active pyrazolylthiazoles are
Nβ-substituted pyrazole isomers; there is only one active
Nγ-substituted corrector (11d) and it has relatively low
activity.
An understanding of the active geometries of our com-
pounds is necessary to develop insights regarding the ΔF508-
CFTR binding site. Molecular models were constructed
based on the X-ray crystal structures of bisazoles 9b
(Figure 2) and 10a (Figure 3) to examine these various
geometries). Image (A) in Figure 5 depicts initial lead
compound 1 in its effective s-cis conformation.8 In this
conformation, the bithiazole rings are nearly coplanar,
which we believe positions the aniline moiety to extend into
an important binding pocket.8 Our least active compound,
11d (image (B)), is forced to adopt a nonplanar geometry
about the pyrazolylthiazole core with a tethering S-C-
C-N dihedral geometry of 62.4°. This conformational
change (planar to nearly orthogonal) relative to 1 is the
consequence of steric interactions between the Nγ 4-bromo-
phenyl substituent on the pyrazole ring and the neighboring
thiazole ring; the X-ray crystal structure of 9b (the ester
precursor to amide 11d; Figure 2) reflects this confor-
mational predisposition. As discussed in other published
work,8 we speculate that a planar arrangement of the
bis-heterocycle core is vital for corrector activity and the
Figure 3. X-ray crystallographic structure of Nβ-substituted pyr-
azolylthiazole 10a (note: the S10-C50-C3-N2 dihedral angle
is 2.6°).
Figure 4. Dose-response relation for increased I- influx in ΔF508-
CFTR cells treated with pyrazolylthiazoles: 14g (in black) and 14h
(in red).
Table 2. Nβ-Substituted Pyrazolylthiazole Analogues
compd
R1
R2
X
compd
R1
R2
X
10a
10b
10c
14a
14b
14c
14d
14e
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
CH2CHdCH2
CH2C6H5
OEt
OEt
OEt
14f
14g
14h
14i
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
CH2C6H5
N(CH2CH2)2O
NHC6H4(4-OMe)
N(CH2CH2)2O
NHCH2C6H5
NH(CH2)2OH
OH
CH2C6H3(2-OMe/4-Cl)
CH2C6H3(2-OMe/4-Cl)
CH2C6H3(2-OMe/4-Cl)
CH2C6H3(2-OMe/4-Cl)
CH2CHdCH2
CH2C6H3(2-OMe/4-Cl)
CH2CHdCH2
CH2CHdCH2
CH2CHdCH2
CH2C6H5
NHC6H4(4-OMe)
NHCH2C6H5
14j
N(CH2CH2)2O
NHC6H4(4-OMe)
NHCH2C6H5
15a
15b
CH2C6H5
OH
CH2C6H5

3776 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Table 3. Pyrazolylthiazole ΔF508-CFTR Corrector Activity
Ye et al.
a Concentration where the increased I- influx is 50% of Vmax b Vmax is maximum increase in I- influx due to compound effect.
.
orthogonal posture of Nγ-substituted analogues may
explain why most of these pyrazolylthiazoles are inactive.
These structural insights are further supported by conforma-
tional analysis of 14g, one of our most potent pyrazolylthia-
zole (image (C)). Nβ substitution on the pyrazole ring of 14g
allows the two heterocycles to adopt a nearly coplanar
conformation without confronting steric congestion. Indeed,
the bisazole dihedral angle in crystalline Nβ-substituted
pyrazole analogue 10a is 2.6° (e.g., nearly planar; see also
the X-ray crystal structure of 10a in Figure 3). Image (C)
shows the s-cis conformation of bisazole 14g in which the Nβ
benzyl moiety can extend into the important binding pocket
discussed above (in the context of 11d).8 In contrast, the
s-trans conformation of 14g (image (D)), obtained by an
∼180° rotation around the tethering thiazole-pyrazole bond,
allows the C5 carboxamide moiety to occupy this binding
pocket. These possibly “dually active conformations” of 14g
may account for some of the corrector activity of bisazole
14g compared to, for example, bisazole 14j.
As various carboxamide-substituted Nβ-substituted pyr-
azoles are active (see 14a/b/e/g/h/j) while the majority of the
corresponding esters are not, the carboxamide group at C5 of
the pyrazole seem to play a significant role in effecting correc-
tor activity. Noting that 14h, our most active pyrazolylthiazole,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3777
Figure 5. Bisazole molecular models. (A) 1: s-cis bithiazole conformation (active conformation; see s-cis-locked bithiazole 2 in Figure 1). (B)
11d: Nγ substituted pyrazole ring nearly orthogonal to the thiazole ring. (C) 14g: Nβ substituted s-cis pyrazolylthiazole f places Nβ benzyl
moiety in the same region of space as the aniline moiety in 1. (D) 14g: Nβ substituted s-trans pyrazolylthiazole f places the C5 amide moiety in
the same region of space as the aniline moiety in 1.
has an amide derived from a secondary amine while all other
active pyrazolylthiazoles incorporate amides derived from
primary amines, suggests that the carboxamide might function
as hydrogen bond acceptor (as opposed to functioning as an H-
bond donor). As discussed above and illustrated in Figure 6a,
we also note that free rotation about the thiazole;pyrazole C,
C-tether of Nβ-substituted pyrazolylthiazoles places the Nβ
benzyl moiety “B” of this heterocycle, in its s-cis configuration,
in nearly the same relative position as the aniline moiety “A1”
of the active8 s-cis conformer bithiazole (see left and center
structures in Figure 6a, respectively). This conformational
homology is not possible in Nγ-substituted pyrazolylthiazoles.
A superimposition of s-cis bithiazole 1 and s-trans con-
former pyrazolylthiazole 14a (see Figure 6b) illustrates an-
other interesting point. Specifically, the s-trans conformer
pyrazolylthiazole can place its C5 amide moiety (A2 on the
pyrazole) in a similar position as the aniline (e.g., A1) moiety
of the s-cis bithiazole conformer. Consequently, Nβ substi-
tuted pyrazoles can also, apparently, be active by placing the
C5 amide moiety (A2) in the binding site addressed by the
aniline moiety (A1) of bithiazoles 1 or 2 (see right and center
structures in Figure 6a, respectively).
It is most interesting to note that Nβ-substituted pyrazoly-
lthiazole 14j, which presents a 5-chloro-2-methoxybenzyl
substituent at Nβ and a C5 amide derived from 2-aminoetha-
nol, retains corrector activity. As illustrated in Figure 6a, this
intriguing result suggests that perhaps the active presenta-
tion of 14j is its s-cis conformation (compare the s-cis pyra-
zolylthiazole and s-cis-locked bithiazole conformations).
In contrast, pyrazolylthiazoles 14a and 14b may be active
through their s-trans conformation (compare the s-trans
pyrazolylthiazole and the s-cis-locked bithiazole con-
formations). The implication here is that pyrazolylthiazoles
Figure 6. (a) Pyrazolylthiazole(s-cis/s-trans) vss-cis-locked bithiazole
conformational interplay where the Nβ benzyl (B) moiety can overlay
the C2 thiazole aniline (A1) moiety or the C5 pyrazole amide (A2)
moiety can overlay the C2 thiazole aniline (A1) moiety. (b) Super-
imposed wireframe models of bithiazole 1 and thiazolylpyrazole 14a.
14e, 14g, and 14h, with aryl-containing benzyl and amide
moieties, may be active through either (or both) their s-cis or
their s-trans conformations. Studies employing additional
pyrazolylthiazole analogues are underway to probe/validate
these initial insights.

3778 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Ye et al.
Experimental Section
All purchasedstarting materials andreagents wereusedwithout
further purification. Product purification was performed either
on an automated flash chromatography system (Combiflash by
Teledyne: 35 min of elution with linear gradient from 100%
hexane to 100% EtOAc solvent) with silica gel columns or on an
HPLC system [Waters: 15 mL/min flow rate, linear gradient
elution with 0.1% TFA-containing H2O/MeCN from 5 to 95%
MeCN in 20 min, Xterra Prep MS C18 OBD column (19 mm ꢀ
100 mm), and dual wavelength absorbance detector]. NMR
spectra (1H at 600 MHz; 13C at 151 MHz) were recorded in
CDCl3 solvent on a Varian 600. Chemical shifts are expressed in
parts per million relative to internal TMS or solvent. Coupling
constants are expressed in units of hertz (Hz). Splitting patterns
are designated as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), and bs (broad singlet). LC/MS (Waters Micromass
ZQ) specifications are as follows: electrospray (þ) ionization,
mass ranging from 100 to 900 Da, 20 V cone voltage. LC: Xterra
MS C18 column (2.1 mm ꢀ 50 mm ꢀ 3.5 μm), 0.2 mL/min water/
acetonitrile (containing 0.1% TFA), 30 min linear gradient
0-100% acetonitrile. The LC/MS UV detector is a diode array
with 200-400 nm wavelength range. Purity is based on the peak
area percentage of the UV diode array signals. Compound
purities by RP-HPLC were g95%.
Δ508-CFTR Corrector Activity Assay. FRT epithelial cells
stably coexpressing human ΔF508-CFTR and the high-sensi-
tivity halide-sensing fluorescent protein YFP-H148Q/I152L18
were used as described previously.6 Cells were grown at 37 °C
(95% air/5% CO2) for 24 h and then incubated for 16-20 h with
50 μL of medium containing the test compound. At the time of
the assay, cells were washed with PBS and then incubated with
PBS containing forskolin (20 μM) and genistein (50 μM) for
20 min. Measurements were carried out using aFLUOstar
fluorescence plate reader (Optima; BMG LABTECH Gmbh)
equipped with 500 ( 10 nm excitation and 535 ( 15 nm emission
filters (Chroma Technology Corp.). Each well was assayed
individually for I- influx by recording fluorescence continu-
ously (200 ms per point) for 2 s (baseline) and then for 12 s after
rapid (<1 s) addition of 165 μL PBS in which 137 mM Cl- was
replaced by I-. Initial I- influx rate was computed exponential
regression. All experiments contained negative control (DMSO
vehicle) and positive control [N-(2-(5-chloro-2-methoxyphenyl-
amino)-40-methyl-4,50-bithiazol-20-yl)benzamide].
Figure 7. Standard calibration curve correlating experimen-
tally determined capacity factor k with log P. Data are shown
for reference compounds ((: A=4-chlorophenol, B=2,4-dichloro-
phenol, C = 3,4,5-trichlorophenol, D = pentachlorophenol, E = p,
p0-DDT), bithiazole 1, and pyrazolylthiazole 11d/14a/14b/14e/14g/
14h/14j (9).
The log P Measurements. The log P represents a com-
pound’s partition coefficient log value determined from
octanol versus water, where a smaller log P correlates with
better water solubility. The log P is a well-established para-
meter for the ADME profiling as it has implications in
solubility, absorption, distribution, metabolism, and excre-
tion14 (which are important for orally administered drugs)
and, according Lipinski’s rule of 5,14 should generally be <5
for good bioavailability.
The log P can be related to the experimentally determined
capacity factor k by measuring the retention time of the
compound using reverse-phase HPLC.15-17 (see Supporting
Information for ClogP values). A standard calibration curve
is constructed using compounds with known log P values
and experimentally determined log k values (see diamond
data points in Figure 7); this standard calibration curve is
then used to correlate log k (determined from measured
retention time; see Supporting Information) with log P for
each pyrazolylthiazole corrector. These data for 11b and
14a/b/e/g/h/j are depicted in Figure 7. Most of the active
pyrazolylthiazole correctors (squares in Figure 7) have log P
values of less than 5, while bithiazole 1 has a log P greater
than 5. The most active pyrazolylthiazole, 14h, has a log P
value of 4.1, which is ∼1.2 log P units better than bithiazole
1, and 14j has a log P of 3.5.
1-(2-Amino-4-methylthiazol-5-yl)ethanone HCl (6). Thiazole
6 was prepared as described in the literature.19
General Procedure A: Preparation of 8 via CDI-Mediated
Amide Formation. Carboxylic acid (1.75 equiv) was dissolved
in DMF (3.3 mL/mmol of carboxylic acid) and carbonyldiimi-
dazole (CDI; 1.75 equiv) was added slowly to manage CO2
evolution. After all the CDI had been added, the solution was
stirred for an additional 15 min, at which point thiazole HCl salt
6 (1 equiv) was added. The reaction mixture was warmed to 85 °C
and stirred for 20 h. After the reaction was complete, the reaction
mixture was cooled to room temperature and poured into water
(17 mL/mmol of carboxylic acid) to precipitate the product,
which was then collected by filtration, washed with water (3 ꢀ
200 mL), and dried under vacuum at 100 °C for 18 h to deliver 7.
N-(5-Acetyl-4-methylthiazol-2-yl)pivalamide (7a). Pivalic acid
Conclusions
Synthetic access to both Nγ- and Nβ-substituted regioiso-
meric pyrazolylthiazoles was achieved by regiodivergent
synthesis from the common intermediate 8. Of the 54 pyr-
azolylthiazoles prepared, eight demonstrated significant
ΔF508-CFTR corrector activity. This new class of bisazole
correctors was designed by replacing one thiazole ring of our
previously reported bithiazole correctors with a pyrazole ring.
The resulting pyrazolylthiazole correctors diversify the bis-
azole corrector family, function as a new chemotype with
corrector activity, and allow for incorporation of additional
functionality to modulate drug properties such as log P. The
work reported here led to the discovery of new correctors with
improved hydrophilicity and with a structural core that
accommodates diversification. Ongoing studies will capitalize
on the structural, conformational, and log P insights reported
here.
(5.0 g, 49 mmol) was reacted with the thiazole HCl 6 (5.4 g,
3
28 mmol) by general procedure A, and 7a was obtained as an off-
white solid (5.3 g, 79%). 1H NMR (600 MHz, CDCl3) δ 9.06 (s,
1H), 2.64 (s, 3H), 2.51 (s, 3H), 1.34 (s, 9H). 13C NMR (151 MHz,
CDCl3) δ 190.66, 176.46, 158.72, 155.21, 125.30, 39.29, 30.44,
27.16 18.08. LC/MS: calcd [M þ Hþ] = 241.10, found 241.12.
(Z)-Ethyl 2-Hydroxy-4-(4-methyl-2-pivalamidothiazol-5-yl)-
4-oxobut-2-enoate (8a). To a solution of LHMDS (1M; 50.3
mL, 50.3 mmol) in THF (50 mL) cooled to -78 °C was slowly
added 7a (5.5 g, 22.9 mmol) in dry THF (100 mL) via a syringe,
and the mixture was stirred for 30 min. Diethyl oxalate (3.7 mL,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3779
27.4 mmol) was added quickly in one portion, and the resulting
mixture was stirred at -78 °C for 2 h before being allowed to
warm to room temperature for another 2 h. When the reaction
was completed, water (50 mL) and 1N aq HCl (50 mL) were
sequentially added to the reaction mixture and the product was
extracted with EtOAc (3 ꢀ 100 mL). The combined organic
extract was washed with brine twice and dried over MgSO4.
Filtration and solvent removal under vacuum delivered the crude
product, which was purified on a silica gel column with auto-
mated flash chromatography (solvent system: gradient hexane/
ethyl acetate) to give 8 as a yellow solid (7.22 g, 92%). 1H NMR
(600 MHz, CDCl3) δ9.08(s, 1H), 6.70 (s, 1H), 4.39 (q, J = 6, 2H),
2.71 (s, 3H), 1.40 (t, J = 6, 3H), 1.35 (s,9H). 13CNMR(151 MHz,
CDCl33) δ 186.24, 176.56, 165.29, 162.01, 160.00, 157.46, 123.06,
101.63, 62.57, 39.33, 27.08, 18.60, 14.12. LC/MS: ESI-MS, calcd
[M þ Hþ] = 341.12, found 341.04.
by filtration. This solid residue was washed with cold ethanol
(ꢀ3), dried under vacuum, and used in the next step without
further purification. A small portion of product remained in the
ethanol filtrate, which was concentrated under reduced pressure
and the resulting solid collected. The two portions of product
were combined and weighed (1.66 g, 83%). 1H NMR (600 MHz,
DMSO þ CDCl3) δ 13.79 and 11.99 (s and s, 1H), 11.77 (s, 1H),
6.97 and 6.85 (s and s, 1H), 4.34 and 4.29 (q and q, J = 7.1, 2H),
2.46 and 2.37 (s and s, 3H), 1.31 (triplet overlapping, J = 7.1,
3H), 1.23 (s, 9H). 13C NMR (151 MHz, DMSO þ CDCl3) δ
176.83(minor) and 176.53, 161.81(minor) and 158.79, 156.90
(minor) and 156.00, 144.96 (minor) and 144.87, 143.11(minor)
and 143.07, 135.30(minor) and 134.57, 116.84, 106.91and 106.22
(minor), 60.93, and 60.15 (minor), 40.05 (minor) and 38.74,
26.59, 16.38 and 15.92 (minor), 14.24 (minor) and 14.15. LC/
MS: calcd [M þ Hþ] = 337.13, found 337.05.
General Procedure B: Preparation of Nγ-Substituted Pyra-
zoles, 9 via Cyclocondensation with 8. A mixture of substituted
hydrazine (or hydrazine hydrochloride; 1.05 equiv) and 8 in
absolute ethanol (3.3 mL/mmol of 8) was stirred at room
temperature for 18 h. After the starting material was consumed
as indicated by TLC, the solvent was removed by rotoevapo-
ration and the concentrate was extracted with ethyl acetate
(3.3 mL/mmol of 8) and washed with water. The ethyl acetate
layer was dried over Na2SO4, filtered, and concentrated under
reduced pressure to deliver the crude Nγ-substituted pyrazole
product, which was then purified by silica gel column chromato-
graphy (Combiflash).
General Procedure D: Preparation of 10 via Alkylation of the
Pyrazolylthiazole 13. Pyrazolylthiazole 13 (1 equiv), K2CO3
(0.75-0.85 equiv), and an alkylating agent were dissolved in
acetone (3.3 mL/mmol of 13). The mixture was then refluxed
(60 °C) for 24 h. When the reaction was complete, the reaction
mixture was cooled to room temperature and concentrated
under reduced pressure. Water was added to the resulting
mixture, which was then extracted with EtOAc (3ꢀ). The
organic extracts were combined, dried over MgSO4, filtered,
and concentrated by rotoevaporation. The resulting crude
product was then purified by silica gel chromatography
(Combiflash; hexane/EtOAc gradient elution).
Ethyl 1-(4-Bromophenyl)-5-(4-methyl-2-pivalamidothiazol-5-
yl)-1H-pyrazole-3-carboxylate (9b). para-Bromophenylhydra-
zine (0.14 g, 0.64 mmol) was reacted with 8a (0.217 g, 0.63
mmol) by general procedure B. After purification, an off-white
product was obtained (0.27 g, 87%). 1H NMR (600 MHz,
CDCl3) δ 8.80 (s, 1H), 7.50 (d, J = 6, 2H), 7.27 (d, J = 6,
2H), 7.02 (s, 1H), 4.46 (q, J = 6, 2H), 2.01 (s, 3H), 1.42 (t, J = 6,
3H), 1.33 (s, 9H). 13C NMR (151 MHz, CDCl3) δ 176.30, 162.19,
158.25, 147.48, 144.95, 138.29, 135.34, 132.52, 126.59, 122.64,
113.14, 112.69, 61.58, 39.34, 27.37, 15.90, 14.61. LC/MS: calcd
[M þ Hþ] and [M þ 2 þ H þ ] = 491.08 and 493.08, found 490.97
and 492.86.
General Procedure C: Preparation of 11a-I via Aminolysis of
Nγ-Substituted Pyrazole Ester 9. Pyrazolylthiazole 9 (1 equiv)
was dissolved in dry DCM (15 mL/mmol of 9) and cooled to 0 °C
for 10 min. AlMe3 (2.0 equiv) in hexane (1.0 M) was added, and
the resulting mixture was stirred at room temperature for 12 h.
When the reaction was complete, water was added, followed by
0.1N aq HCl to neutralized the mixture, which was then
extracted with EtOAc. The EtOAc layer was dried over Na2SO4,
filtered, and concentrated under reduced pressure to give the
crude amide 10, which was then purified by silica gel column
chromatography with Combiflash.
Ethyl 1-Allyl-3-(4-methyl-2-pivalamidothiazol-5-yl)-1H-pyra-
zole-5-carboxylate (10a, R = H, R2 = Allyl). Allyl bromide (0.172
mL, 1.98 mmol) was reacted with pyrazolylthiazole 13 (0.5 g, 1.49
mmol) by general procedure D. Product 10a was obtained after
purification (0.33 g, 59% yield). 1H NMR (600 MHz, CDCl3) δ
8.71 (s, 1H), 6.95 (s, 1H), 6.04 (m, 1H), 5.19 (m, 3H), 5.14 (dd, J =
1.1, 17.0, 1H), 4.37 (q, J = 7.1 2H), 2.52 (s, 3H), 1.39 (t, J = 7.1,
3H), 1.33 (s, 9H). 13C NMR (151 MHz, CDCl3) δ 175.75, 159.36,
155.65, 143.29, 143.19,133.10, 133.09, 118.20, 117.74, 109.61,
61.21, 54.15, 39.11, 27.21, 16.35, 14.23. LC/MS: calcd [M þ
Hþ] = 377.17, found 377.13.
Ethyl 1-Benzyl-3-(4-methyl-2-pivalamidothiazol-5-yl)-1H-pyra-
zole-5-carboxylate (10a, R = H, R2 = Bn). Benzyl chloride (0.12
mL, 0.96 mmol) was reacted with pyrazolylthiazole 13 (0.27g, 0.80
mmol) by general procedure D. Product 10b was obtained after
purification (0.23 g, 66%). 1H NMR (600 MHz, CDCl3) δ 8.74 (s,
1H), 7.31 (m, 5H), 6.96 (s, 1H), 5.76 (s, 2H), 4.33 (q, J = 7.1, 2H),
2.51 (t, J = 7.1 3H), 1.33 (s, 9H). 13C NMR (151 MHz, CDCl3) δ
175.98, 159.62, 155.77, 143.82, 143.61, 137.08, 133.27, 128.89,
128.70, 128.46, 128.06, 128.05, 127.96, 118.51, 109.99, 61.40,
60.59, 55.22, 39.31, 27.43, 21.26, 16.73, 14.41, 0.21. LC/MS: calcd
[M þ Hþ] = 427.18, found 427.08.
Ethyl 1-(5-Chloro-2-methoxybenzyl)-3-(4-methyl-2-pivalami-
dothiazol-5-yl)-1H-pyrazole-5-carboxylate (10a, R = H, R2 =
CH2(2-MeO-5-Clphenyl)). 2-(Bromomethyl)-4-chloro-1-methoxy-
benzene (0.37 g, 1.57 mmol) was reacted with 13 (0.5 g, 1.49 mmol)
by general procedure D. Product 10c was obtained after purifica-
1-(4-Bromophenyl)-N-(4-methoxyphenyl)-5-(4-methyl-2-piva-
lamidothiazol-5-yl)-1H-pyrazole-3-carboxamide (11d). Pyrazo-
lylthiazole 9b (100 mg, 0.20 mmol) was reacted with anisidine
(28 mg, 0.22 mmol) by general procedure C, and an off-white
solid product was obtained (85 mg, 73% yield). 1H NMR (600
MHz, CDCl3) δ 8.84 (s, 1H), 8.64 (s, 1H), 7.61 (d, J = 9.0, 2H),
7.54 (d, J = 8.8, 2H), 7.28 (d, J = 8.8, 2H), 7.11 (s, 1H), 6.91 (d,
1
tion (0.37 g, 51%). H NMR (600 MHz, CDCl3) δ 8.70 (s, 1H),
7.17 (dd, J = 2.6, 8.7, 1H), 7.02 (s, 1H), 6.79 (d, J = 8.7, 1H), 6.59
(d, J = 2.6, 1H), 5.76 (s, 2H), 4.32 (q, J = 7.1, 2H), 3.86 (s, 3H),
2.53 (s, 3H), 1.34 (t, J = 7.1, 3H), 1.32 (s, 9H). 13C NMR (151
MHz, CDCl3) δ 175.90, 159.47, 155.74, 155.25, 144.02, 143.96,
134.06, 128.38, 128.00, 127.27, 125.79, 118.39, 111.59, 110.01, 61.48,
56.01, 50.28, 39.31, 27.45, 16.75, 14.36. LC/MS: purity and calcd
[M þ Hþ] and [M þ 2þH þ ] = 491.15 and 493.14, found 491.10
and 493.08.
General Procedure E: Preparation of 14a-i via Aminolysis of
10 with Nonalcoholic Amines. To a solution of ethyl ester 10
(1 equiv) in dry DCM (10 mL/mmol of 10), which was chilled at
0 °C in a sealed microwave reaction vessel, was added 2.0 M
trimethyl aluminum (1.2 equiv) in hexanes. The resulting mix-
ture was stirred at 0 °C for 10 min, and then the amine reactant
J = 9.0, 2H), 3.82 (s, 3H), 2.05 (d, J = 5.6, 3H), 1.32 (s, 9H). 13
C
NMR (151 MHz, CDCl3) δ 176.29, 159.15, 158.35, 156.63,
147.90, 147.53, 138.28, 136.09, 132.67, 131.03, 126.38, 122.64,
121.74, 114.46, 112.81, 111.50, 77.43, 77.22, 77.01, 55.71, 39.34,
27.36, 15.93. LC/MS: calcd [M þ Hþ] = 568.10, found 568.10.
Synthesis of Ethyl 3-(4-Methyl-2-pivalamidothiazol-5-yl)-1H-
pyrazole-5-carboxylate (13). 2,4-Dioxo-4-(thiazol-5-yl)butano-
ate 8 (2.0 g, 5.9 mmol) and hydrazine hydrate (0.40 mL, 6.4
mmol) were dissolved in absolute ethanol (20 mL), and the
mixture was stirred at room temperature for 20 h. When the
reaction was over, the reaction mixture was concentrated to half
volume by rotoevaporation and the resulting solid was collected

3780 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Ye et al.
(1.2 equiv) in dry DCM (1 mL/mmol of 10) was injected into the
mixture. The reaction tube was then mounted to the microwave
reactor and irradiated with microwave at 100 °C for 40 min.
After cooling, water and 0.1N aq HCl were added to the reaction
mixture sequentially to neutralize the solution. DCM extrac-
tion (3ꢀ), drying over MgSO4, filtration, and rotoevaporation
gave a residue which purified by silica gel chromatography
(CombiFlash).
General Procedure F for Preparation of Amide 11m-p and 14j:
Aminolysis of 9 or 10 with Alcoholic Amine. Pyrazolylthiazole 9
or 13 was dissolved in dry ethanol (4 mL) in a 10 mL microwave
reaction tube. Ethanolamine (20 equivalent) was added to the
solution, the tube was sealed, placed in a microwave reactor, and
the reaction mixture was heated at 180 °C for 30 min. After the
tube had cooled, ethanol was removed under reduced pressure,
aq NH4Cl was added, and the mixture was extracted with
chloroform (ꢀ3). The chloroform extracts were combined and
the solvent removed under reduced pressure to give a crude
product, which was purified with HPLC.
1-(5-Chloro-2-methoxybenzyl)-N-(2-hydroxyethyl)-3-(4-meth-
yl-2-pivalamidothiazol-5-yl)-1H-pyrazole-5-carboxamide (14j).
Following general procedure F, 14j was obtained in 33% yield.
1H NMR (600 MHz, CDCl3) δ 7.19 (dd, J = 2.6, 8.7, 1H), 6.80
(d, J = 8.8, 1H), 6.73 (d, J = 2.6, 1H), 6.72 (s, 1H), 6.52 (s, 1H),
5.76 (s, 2H), 3.85 (s, 3H), 3.82 (t, J = 5.4, 2H), 3.59 (dd, J = 5.5,
10.3, 2H), 2.60 (s, 3H), 1.37 (s, 9H). 13C NMR (151 MHz,
CDCl3) δ 178.26, 160.36, 159.63, 155.31, 140.97, 137.49, 134.35,
128.57, 127.81, 127.17, 125.52, 117.96, 111.65, 104.79, 77.35,
77.22, 77.01, 76.80, 61.75, 55.85, 49.93, 42.12, 39.94, 26.56,
13.43. LC/MS: calcd [M þ Hþ] = 506.16, found 506.15.
1-Allyl-N-(4-methoxyphenyl)-3-(4-methyl-2-pivalamidothiazol-
5-yl)-1H-pyrazole-5-carboxamide (14a). Ethyl ester 10a (100 mg,
0.27 mmol) was reacted with anisidine (40 mg, 33 mmol) by
general procedure E and gave 14a (45 mg, 67%). 1H NMR (600
MHz, CDCl3) δ 8.80 (s, 1H), 7.83 (s, 1H), 7.50 (d, J = 8.7, 2H),
6.91 (d, J = 9.0, 2H), 6.73 (s, 1H), 6.08 (ddd, J = 5.8, 10.9, 16.1,
1H), 5.23-5.10 (m, 4H), 3.81 (s, 3H), 2.51 (s, 3H), 1.32 (s, 9H).
13C NMR (151 MHz, CDCl3) δ 176.02, 157.72, 157.19, 155.66,
143.92, 143.35, 136.48, 133.56, 130.21, 124.50, 122.59, 118.31,
118.09, 114.53, 105.41, 77.46, 77.25, 77.04, 55.74, 54.19, 39.33,
27.44, 16.71. LC/MS: calcd [M þ Hþ] = 454.19, found 454.16.
1-Allyl-N-benzyl-3-(4-methyl-2-pivalamidothiazol-5-yl)-1H-
pyrazole-5-carboxamide (14b). Ethyl ester 10a (100 mg, 0.27
mmol) was reacted with benzylamine (36 μL, 33 mmol) by
general procedure E and gave 14b (85 mg, 91% yield). 1H
NMR (600 MHz, CDCl3) δ 8.93 (s, 1H), 7.30 (m, 4H), 6.87 (t,
J = 5.6, 1H), 6.64 (s, 1H), 6.02 (ddt, J = 5.7, 11.3, 17.0, 1H),
5.12 (m, 4H), 4.59 (d, J = 5.8, 2H), 2.46 (s, 3H), 1.29 (t, J = 2.9,
9H). 13C NMR (151 MHz, CDCl3) δ 176.09, 159.69, 155.66,
143.74, 143.20, 137.89, 136.22, 133.66, 128.98, 127.98, 127.89,
118.34, 117.82, 105.52, 54.04, 43.75, 39.28, 27.38, 16.63. LC/
MS: calcd [M þ Hþ] = 438.20, found 438.13.
Acknowledgment. We thank the Tara K. Telford Fund for
Cystic Fibrosis Research at UC Davis, the National Institutes
of Health (DK072517 and GM076151), and the National
Science Foundation [CHE-0614756, CHE-0443516, CHE-
0449845, CHE-9808183 (NMR spectrometers)] for their gen-
erous support.
Synthesis of N,1-Dibenzyl-3-(4-methyl-2-pivalamidothiazol-5-
yl)-1H-pyrazole-5-carboxamide (14e). Ethyl ester 10b (100 mg,
0.23 mmol), benzylamine (0.51 mL, 4.7 mmol), and sodium
cyanide (5.3 mg, 0.1 mmol) were mixed in MeOH (8 mL) and
refluxed for 18 h. Upon cooling, the methanol was removed
by rotoevaporation and the residue was taken up in EtOAc
(50 mL), washed with water (50 mL; 3ꢀ) and 0.1N aq HCl (50
mL), dried over Na2SO4, filtered, and concentrated under
reduced pressure. The concentrate was purified by silica gel
chromatography (Combiflash), giving pure product of 14e (31.9
mg, 34.41%). 1H NMR (600 MHz, CDCl3) δ 8.71 (s, 1H),
7.37-7.23 (m, 10H), 6.58 (s, 1H), 6.24 (t, J = 5.6, 1H), 5.79
(s, 2H), 4.58 (d, J = 5.8, 2H), 2.50 (s, 3H), 1.32 (s, 9H). 13C NMR
(151 MHz, CDCl3) δ 175.94, 159.67, 155.63, 143.79, 143.36,
137.70, 137.31, 136.12, 129.07, 128.71, 128.33, 128.00, 127.99,
127.94, 118.46, 105.32, 54.98, 43.80, 39.31, 27.44, 16.74. LC/MS:
calcd [M þ Hþ] = 488.21, found 488.20.
Supporting Information Available: CCDC information for 9b
and 10a X-ray crystallographic structures, calculated and ex-
trapolated log P values of active pyrazolylthiazoles 11d/14a/
14b/14e/14g/14h/14j and the bithiazole 1, log k and log P values
of reference compounds (used to establish the log P vs log k
trendline) and pyrazolylthiazole correctors, experimentals for
the preparation of pyrazolylthiazole carboxylic acids of both
regioisomers of N-substituted pyrazoles, and HPLC/mass spec-
tral data for active pyrazolylthiazoles 11d/14a/14b/14e/14g/14h/
14j. This material is available free of charge via the Internet at
http://pubs.acs.org.
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1-(5-Chloro-2-methoxybenzyl)-N-(4-methoxyphenyl)-3-(4-
methyl-2-pivalamidothiazol-5-yl)-1H-pyrazole-5-carboxamide (14g).
Ethyl ester 10c(30 mg, 0.061 mmol) was reacted with anisidine (12
mg, 0.098 mmol) by general procedure E to give 14g (26 mg, 75%).
1H NMR (600 MHz, CDCl3) δ 8.83 (s, 1H), 7.76 (s, 1H), 7.48 (d,
J = 8.5, 2H), 7.16 (dd, J = 2.6, 8.7, 1H), 6.89 (d, J = 9.0, 2H), 6.79
(d, J=2.5, 1H), 6.75(d,J= 8.7, 2H), 5.78 (s, 2H), 3.80 (s, 3H), 3.78
(s, 3H), 2.51 (s, 3H), 1.32 (s, 9H). 13C NMR (151 MHz, CDCl3) δ
176.00, 157.68, 157.18, 155.73, 155.47, 144.06, 143.84, 130.26,
128.52, 128.15, 127.91, 125.76, 122.42, 118.27, 114.53, 111.67,
105.38, 55.97, 55.71, 49.90, 39.32, 27.43, 16.71. LC/MS: calcd [M
þ Hþ] = 568.18, found 568.17.
N-(5-(1-(5-Chloro-2-methoxybenzyl)-5-(morpholine-4-carbonyl)-
1H-pyrazol-3-yl)-4-methylthiazol-2-yl)pivalamide (14h). Ethyl ester
10c (30 mg, 0.061 mmol) was reacted with morpholine (7 μL, 0.091
mmol) by general procedure E to give 14h (28 mg, 87%). 1H NMR
(600 MHz, CDCl3) δ 8.74 (s, 1H), 7.19 (dd, J = 2.6, 8.7, 1H), 6.86
(d, J= 2.6, 1H), 6.77 (d, J= 8.7, 1H), 6.38 (d, J= 9.6, 1H), 5.50 (s,
2H), 3.79 (d, J = 12.0, 3H), 3.66 (d, J = 24.8, 4H), 3.45 (s, 4H),
2.50 (s, 3H), 1.31 (s, 9H). 13C NMR (151 MHz, CDCl3) δ 175.95,
160.92, 155.76, 155.55, 143.95, 143.31, 136.09, 128.96, 128.83,
127.45, 125.67, 118.40, 111.95, 105.67, 66.82, 56.16, 49.29, 39.31,
27.43, 16.72. LC/MS: calcd [M þ Hþ] = 532.18, found 532.14.
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