Dimethyl-diphenyl-propanamide derivatives as nonsteroidal dissociated glucocorticoid receptor agonists

Article abstract of DOI:10.1021/jm100957a

A series of 2,2-dimethyl-3,3-diphenyl-propanamides as novel glucocorticoid receptor modulators is reported. SAR exploration led to the identification of 4-hydroxyphenyl propanamide derivatives displaying good agonist activity in GR-mediated transrepressio

Full text of DOI:10.1021/jm100957a

J. Med. Chem. 2010, 53, 8241–8251 8241
DOI: 10.1021/jm100957a
Dimethyl-diphenyl-propanamide Derivatives As Nonsteroidal Dissociated
Glucocorticoid Receptor Agonists
Bingwei V. Yang,* David S. Weinstein, Lidia M. Doweyko, Hua Gong, Wayne Vaccaro, Tram Huynh, Hai-yun Xiao,
Arthur M. Doweyko, Lorraine Mckay, Deborah A. Holloway, John E. Somerville, Sium Habte, Mark Cunningham,
Michele McMahon, Robert Townsend, David Shuster, John H. Dodd, Steven G. Nadler, and Joel C. Barrish
Bristol-Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-4000, United States
Received July 27, 2010
A series of 2,2-dimethyl-3,3-diphenyl-propanamides as novel glucocorticoid receptor modulators is
reported. SAR exploration led to the identification of 4-hydroxyphenyl propanamide derivatives
displaying good agonist activity in GR-mediated transrepression assays and reduced agonist activity
in GR-mediated transactivation assays. Compounds 17 and 30 showed anti-inflammatory activity
comparable to prednisolone in the rat carrageenan-induced paw edema model, with markedly decreased
side effects with regard to increases in blood glucose and expression of hepatic tyrosine aminotransfer-
ase. A hypothetical binding mode accounting for the induction of the functional activity by a 4-hydroxyl
group is proposed.
Introduction
which retain similar anti-inflammatory properties as conven-
tional steroids, but have reduced transactivation activity.6-12
Glucocorticoid receptor (GRa) agonists, such as dexa-
methasone (dex) 1 and prednisolone (pred) 2, have been
used as anti-inflammatory agents for over 60 years. How-
ever, their systemic use is limited by side effects including
diabetes, osteoporosis, obesity, muscle atrophy, and glau-
coma. As there is still significant unmet medical need for
safe and efficacious treatments for inflammatory and auto-
immune diseases, compounds which improve upon the
risk-benefit ratio of steroids are highly desirable.1 The
anti-inflammatory activity of glucocorticoids is largely due
to their ability to reduce the expression of pro-inflamma-
tory genes via the inhibition of the transcription factors
NFκB (nuclear factor κB) and AP-1 (activator protein-1) by
ligand-bound GR, a pathway termed transrepression (TR).
On the other hand, the ligand-bound GR can also induce
transcription of certain genes, a process termed transacti-
vation (TA). Several side effects of glucocorticoid therapy
are thought to be predominantly or at least partially due to
GR mediated transactivation of various genes involved in
metabolism. Insights into the mechanism of glucocorticoid
action have led to the hypothesis that the undesirable side
effects originating from transactivation may be separated
from the anti-inflammatory effects resulting from trans-
repression.2-5 These findings have attracted extensive
attention on the development of ligands termed “dissociated”
glucocorticoid receptor agonists (DGRA) or “selective glu-
cocorticoid receptor modulator/agonists (SGRM/SEGRA)”,
Recently, we described a novel class of dihydro-ethanoan-
thracene carboxamides as potent and selective nonsteroidal
GR ligands.13 Select compounds from this series, such as
compound 3 (Figure 1), had good activity in GR-mediated
transrepression assays and reduced activity in transactivation
assays performed with cultured cells. As part of our continu-
ing efforts toward the optimization of GR agonists, we
embarked on the study of a series of synthetically more
tractable analogues related to 2,2-dimethyl-3,3-diphenyl-pro-
panamides 4. The structure of 4 can be perceived to be derived
from excision of the methyl-substituted bridgehead carbon of
dihydro-ethanoanthracene 3 (Figure 1). Computational mod-
eling also suggested 3 and 4 should share overall similar
binding poses in GR, where the polar end consisting of the
amidothiazole moiety engages in several H-bonds with
Gln642 and Asn564.13 Synthetic versatility of 4 enabled the
*To whom correspondence should be addressed. Phone: 609-252-
3436. Fax: 609-252-6804. E-mail: bingwei.yang@bms.com.
a Abbreviations: GR, glucocorticoid receptor; TAT, tyrosine amino
transferase; AP-1, activator protein-1; CPE, rat carrageenan-induced
paw edema model.
Figure 1
r
2010 American Chemical Society
Published on Web 11/12/2010
pubs.acs.org/jmc

8242 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Yang et al.
Scheme 1a
a Reagents: (a) TiCl4, (CH3)2CdC(OCH3)-OSiMe3 THF, 0 °C then rt, 60-88%; (b) 40% aq KOH, DMSO, MeOH, 75 °C, 86-99%; (c) LDA,
(CH3)2CHCOOH, THF, 0 °C-60 °C, 2 h; mixed with 5 at 0 °C, 18 h, rt, 56%; (d) Et3SiH, BF3 Et2O, CH2Cl2, 0 °C, 90%; (e) 2-thiazolylamine or 1,3,4-
3
thiadiazol-2-amine, HOBT, EDC, CH3CN, iPr2NEt, 85 °C, 70-81%; (f) BBr3, CH2Cl2, 0 °C-rt, 81-99%; (g) SFC chiral separation with various chiral
columns using CO2/MeOH as mobile phase; (h) (CH3)2CdC(Cl)-NMe2, CH2Cl2, 0 °C, 30 min, then iPr2NEt, 1,3,4-thiadiazol-2-amine, rt, 87%; (i) Br2,
HOAc, 96%; (j) CuCN, DMF, μw, 220 °C, 50%; (k) CH3COCl, AlCl3, ClCH2CH2Cl-PhNO2, rt, 1.5 h, 62.3%.
Scheme 2a
a Reagents: (a) LDA, (CH3)2CHCOOH THF, -30 °C ∼55 °C, 1.5 h, mixed with 27 at 0 °C, 2 h, 90%; (b) Et3SiH, BF3 Et2O, 68%; (c) SFC chiral
3
separation with various chiral columns using CO2/MeOH as mobile phase; (d) NH2PhSH, NMP, K2CO3, μW, 205 °C, 70%; (e) 2-thiazolylamine or
1,3,4-thiadiazol-2-amine, HOBT, EDC, CH3CN, iPr2NEt, 85 °C, 12 h, 60-66%; (f) 32 was prepared from 9H-xanthen-9-one following procedures a, b,
and e, 53.3%.
rapid exploration of structure-activity relationships (SAR)
around the chemical space of the phenyl rings. Herein we
report our studies of 4, which led to the identification of new
potent GR agonists with a favorable “dissociated” profile
both in vitro and in vivo.14
Alternatively, construction of the acid 8a could be effected
under Moresch conditions.15a Reaction of lithium enediolate
derived from isobutyric acid with ketone 5 afforded β-hydro-
xy acid 7, which was deoxygenated by the action of etherated
boron trifluoride and triethyl silane reducing system15b to give
acid 8a. Chiral HPLC separation of racemic acids 8 provided
the corresponding enantiomeric acids 9 and 10. Amidation
reactions of acids 8, as well as chiral acids 9 and 10, with
2-thiazolylamine or 1,3,4-thiadiazol-2-amine followed by
demethylation yielded a series of carboxamides, including
4-hydroxyphenyl (13, 14, 22), 3-methyl-4-hydroxyphenyl
(25), and 3-F-4-hydroxyphenyl (17, 18, 23) propanamides.
The demethylation reaction may occur prior to the amidation,
as in the case of 22. The resulting 4-hydroxyphenyl acid could
offer an easy access to various 4-hydroxyphenyl carboxamides
Chemistry
The compounds described in this work were synthesized by
the routes shown in Schemes 1-2. A series of ortho- and meta-
substituted phenolic analogues were prepared (Scheme 1).
Synthesis of the key intermediate acid 8(a-d) was achieved
through a TiCl4-catalyzed addition of (1-methoxy-2-methyl-
prop-1-enyloxy)trimethylsilane to diphenyl-methanol 6 (a-d),
followed by hydrolysis of the methyl ester thus formed.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8243
Table 1. Activities of Diphenyl Propanamidesa
GR
AP-1 repression
E-selectin repression
EC50 nM (eff % dex)b
GAL 4 reporter
TAT induction
compd
R
chirality
Ki (nM)
EC50 nM (eff % dex)b
EC50 nM (eff % dex)b
EC50 nM (eff % dex)b
dex
pred
4
1.1
1.5
2.9
5.7
2.5
1.2
18.7
20.7
0.6
0.7
4.6
0.4
0.9
7.3
1.2
0.7
0.4
2.2
2.5 (100)
15.8 (97)
>10000
>10000
61.4 (63)
40.9 (63)
>10000
>10000
51.1 (77)
15.9 (79)
>10000 (16)
40.1 (83)
506.0 (74)
>10000
20.3 (69)
21.2 (70)
12.7 (87)
28.6 (53)
1.1 (100)
13.6 (97)
4.2 (100)
82.7 (97)
>10000
>10000
863.4 (14)
372.1 (21)
>10000
>10000
216.4 (39)
119.0 (61)
>10000
114.7 (48)
>10000
>10000
380.2 (22)
121.0 (50)
67.2 (54)
555.8 (13)
5.0 (100)
57.4 (97)
1357 (5)
H
achiral
11a
12
13
14
15
16
17
18
19
20
21
22
23
24
25
OCH3
OH
H
(
(
S
>5000
125.2 (50)
29.5 (52)
2579 (20)
250.7 (21)
>10000
>10000
197.0 (41)
36.1 (48)
>5000
H
R
(
(
S
2-F
3-F
F
37.7 (63)
8.6 (68)
F
Br
R
S
>5000 (12)
8.6 (71)
142.2 (29)
>5000 (16)
2325
CN
COCH3
H
S
205.9 (48)
>5000 (16)
27.5 (56)
7.9 (56)
S
S
748.5 (21)
131.8 (42)
46.5 (57)
>5000 (21)
F
Br
S
S
6.2 (75)
119.3 (53)
CH3
S
a Values are means of at least two experiments done in triplicate. b Efficacy represented as a percentage of the maximal response of dex (100%). % dex
is not reported where <5%.
Table 2. Activities of Xanthene Analoguesa
GR
Ki (nM)
AP-1 repression
EC50 (nM) (eff % dex)b
E-selectin repression
EC50 (nM) (eff % dex)b
GAL 4 reporter
EC50 nM (eff % dex)b
TAT induction
EC50 nM (eff % dex)b
compd
dex
29
30
31
32
1.1
0.3
2.5 (100)
17.5 (81)
7.8 (82)
>5000
1.1 (100)
9.3 (68)
3.5 (62)
>5000
>5000
4.2 (100)
60.1 (30)
85.7 (46)
>10000
>10000
5.0 (100)
27.1 (39)
91.3 (45)
0.8
58.7
6.0
>5000
a Values are means of at least two experiments done in triplicate. b Efficacy represented as a percentage of the maximal response of dex (100%). % dex
is not reported where <5%.
with the employment of 1-chloro-N,N,2-trimethylprop-1-en-1-
amine. Bromination of acid 9a or acetylation of N-(thiazol-2-
yl)propanamide derived from 9a proceeded regioselectively
ortho to the methoxy group. An analogous synthetic sequence
to the one described above led to 3-bromo and 3-acetyl 4-hydro-
xyphenyl propanamides 19, 24, and 21. Bromo thiazole 19 was
converted to 3-cyano-4-hydroxyphenyl thiazole 20 by reaction
with copper(I) cyanide under microwave irradiation.
Conformationally constraining the diphenylpropanamides
as their tricyclic xanthene derivatives 29-32 was accom-
plished by a similar sequence to that outlined in Scheme 1
(Scheme 2).
addition, the transactivation potential of each compound was
also evaluated by its ability to induce tyrosine amino transfer-
ase (TAT) in a human liver cell line derived from Huh7.16
Data are provided in Tables 1 and 2.
We began our investigation by exploring phenyl ring sub-
stitution in 4. Previous studies on the dihydroanthracene
series indicated that most modifications on the aminothiazole
moiety resulted in a considerable loss of binding activities.13
For comparative purposes, the aminothiazole moiety was
thus held constant. Preparations of the 2-, 3-, and 4-substi-
tuted phenyl analogues established the preference for substi-
tution at the C-4 position (data not shown). While small alkyl,
halogen, and cyano groups at C-4 of the phenyl displayed
modest GR binding affinity (Ki 26-29 nM), the unsubstituted
and 4-methoxyphenyl analogues, 4 and 11a, exhibited good
GR binding affinity (2.9 and 5.7 nM, respectively). However,
both analogues possessed poor functional activity in the AP-1
transrepression assay (Table 1).
Results and Discussion
The in vitro assays used to characterize biological activities
of the GR ligands including GR binding, transrepression
(AP-1 and E-selectin (NFκB dependent)), and transactivation
(GAL4-reporter) assays have been reported previously.13 In

8244 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Yang et al.
Figure 2. Comparison of GR LBD binding modes for dex (X-ray structure,17 left) and compound 13 (model, right) illustrates a conserved set
of interactions. In both cases, Arg611/Gln570 residues provide a rich source of H-bond donors, interacting with either the A-ring carbonyl in
dex or the phenol OH in 13. Asn564 provides the H3 H-bonding acceptor/donor combination to dex as well as to 13. While Gln642 is positioned
in the dex structure to H-bond to the 17-OH group, we speculated that it may undergo a slight shift in position to H-bond to the backside of the
carbonyl of the amidothiazole.
The published X-ray structure of GR ligand-binding domain
(LBD) bound dexamethasone reveals interactions between the
C-3 carbonyl oxygen and Gln570/Arg611 residues located on
helices 3 and 5, respectively.17 We speculated that the introduc-
tion of a hydroxyl group to the phenyl ring in our nonsteroidal
series might be able to engage in an H-bonding interaction with
these two residues. We prepared 4-hydroxyphenyl propanamide
with 79% dex efficacy in the AP-1 assay and EC50 = 8.6 nM
with 68% dex efficacy in the E-selectin assay) in contrast to
R-isomer 18, which had minimal transrepression activity.
To circumvent the potential P-450 catalyzed oxidative ring-
opening of thiazole,19 a 1,3,4-thiadiazole ring as bioisosteric
replacement of the thiazole was introduced. In general, the
compound pairs in which the only difference was the amide
substituent (aminothiazole vs aminothiadiazole) had similar
binding affinity, in accord with previous observations from
the dihydro-ethanoanthracene series.13 Of the substituents
evaluated at the 3-position of the phenol ring in the S-isomer
series, most showed GR binding affinity ranging from 0.4 to
1.2 nM, with the exception of the electron-donating methyl
group (25, 2.2 nM) and the larger electron-withdrawing acetyl
group (21, 7.3 nM). In the transrepression assays, 3-Br and
3-F analogues provided considerable improvement in func-
tional activities compared to the unsubstituted phenol con-
geners (e.g., 13 vs 17, 19; 22 vs 23, 24). The agonistic efficacy of
3-halogen substituted analogues reached the levels of
70-87% of dex in the AP-1 assay. Methyl-substituted ana-
logue 25 displayed weaker AP-1 efficacy (53% dex), in line
with its somewhat reduced binding affinity. However, cyano-
substituted analogue 20 showed much weaker agonist activity
(EC50 = 206-506 nM in TR assays) in spite of its GR affinity
being 2-fold stronger than 25.
Several compounds in the series displayed a dissociated
pharmacology with a good degree of separation between
efficacies in the TA assays (TAT, GAL4-reporter) and TR
assays (AP-1, E-selectin). Asexemplified by17, its TA efficacy
(48% dex in the TAT assay) was significantly weaker than the
observed TR agonism efficacy (79% dex in the AP-1 assay).
While exhibiting partial agonism (68-79% dex efficacy in TR
assays), 17 thereby demonstrated a marked separation of TR
and TA efficacies. This is in sharp contrast to pred, which dis-
played equal maximal efficacy in the TR and TA assays (97%
dex efficacy in both AP-1 and TAT assays). The markedly
12, which was found to have modest agonist activity (EC50
=
61 nM with 63% dex efficacy) in the AP-1 transrepression assay.
Chiral separation of the stereoisomers of 12 revealed that most
of the binding affinity and all of the agonist activity resided
mainly with the S-enantiomer, 1318 (Table 1).
A detailed illustration of the modeled interactions of 13 in
the GR binding pocket is shown in Figure 2 along with a
comparison of bound dex. The model indicates that the 4-OH
analogue fits well within the GR binding pocket, and a possible
hydrogen-bonding network can be envisioned between the
phenolic OH of 13 and neighboring residues, Gln570 and
Arg611. In addition, Asn564 and Gln642 likely play a role in
binding through a number of H-bonds to the amidothiazole
moiety. Specifically, Asn564 may engage in two H-bonds to the
amide NH and thiazole N, while Gln642 may form an addi-
tional H-bond with the amide carbonyl oxygen. This latter
H-bond would require that Gln642 undergo a shift in position
compared to its location in the dex complex. This model further
indicates that the pocket around the phenol ring is hydrophobic
and large enough to accommodate small functional groups.
The compound with a fluorine substitution ortho to the
hydroxyl of phenol ring had improved binding affinity when
compared to the compounds with substitution at the meta
position (C-2) or with no substitution (16 versus 15 and 4).
The improved binding potency in 16 also brought about
moderate GR-mediated transrepression activity (EC50
=
51 nM with 77% dex efficacy in AP-1 assay). As anticipated
based on the binding model, compound 17, the S-enantiomer
of 16, exhibited good functional activities (EC50 = 16 nM

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8245
Table 3. Selectivity
H
F
Br
CN
F
CH3
(xanthene)
R
Pred
1.5
>6000
13
17
19
20
23
25
29
30
GR Ki nM
PR Ki nM
1.2
273
0.7
53
0.4
31
0.9
144
0.7
55
2.2
140
0.3
22
0.8
53
AR Ki nM
ERR Ki μM
MR agonista
1058
>100
>5
1034
>50
>5
370
>50
>5
>3000
993
>50
>5
>4000
2341
2.3
696
5.5
>5
>5
0.002 (88)
>5
a In A549 cell line, EC50 (μM)/(% maximal efficacy) determination (aldosterone as a positive control).
decreased TA agonist efficacy compared to TR agonist effi-
cacy was also observed with compounds 19, 23, and 24. In
general, an increase in TR efficacy was accompanied with an
increase of TA efficacy; however, a good degree separation of
TR/TA was still retained for these compounds.
We also studied the effect of reintroducing a conformational
constraint to the diphenylpropanamides. The tricyclic xanthene
template was anticipated to share a similar binding conforma-
tion with both 3 and the acyclic diphenylpropanamides. The
enantiopure hydroxylated xanthenes 29 and 30 provided pro-
mising transrepression activity (81-82% dex efficacy in AP-1)
and reduced transactivation activity (39-45% dex efficacy in
TAT) (Table 2). A similar binding pose for 29 and 30 compared
to the corresponding acylic diphenylpropanamides (13 and 22)
is supported by several observations. In analogy to 4, nonhy-
droxylated xanthene 32 maintained a high level of binding
affinity but was devoid of agonist activity. Similarly, in analogy
to 14, compound 31 (the antipode of 30) provided only weak
binding affinity and no transrepression activity. The absolute
configuration of enantiomer 30 (S) proved to be the same as the
corresponding acyclic diphenylpropanamide 22.20
To monitor steroid receptor cross-reactivity, compounds
listed in Tables 1 and 2 were counterscreened for binding against
the progesterone receptor (PR), androgen receptor (AR) and, in
some cases, the estrogen receptor (ERR) in addition to agonist
activity at the mineralocorticoid receptor (MR, activation in
A549 cell) (Table 3). Compounds 13, 17, 19, 23, 25, 29, and 30
showed excellent selectivity between GR and AR (>870-fold)
as well as ERR (>6800-fold), in addition to modest selectivity
against PR (66-227-fold). None of the tested compounds
displayed agonist activity against MR.
In Vivo Evaluation. Compounds 13, 17, and 30 were
evaluated in the rat carrageenan-induced paw edema (CPE)
model for their anti-inflammatory activity. An acute inflam-
matory response was generated in Sprague-Dawley rats after
injections of carrageenan in the hind paws. Paw swelling was
calculated by subtracting the baseline values from the post
challenge measurements. Initial evaluation of 13, following a
single oral dose of 100 mg/kg, revealed a 46% inhibition of
paw edema, nearly identical to that shown by pred adminis-
tered orally at 30 mg/kg in the same study (42%) (Figure 3).
Blood samples were collected at the conclusion of the study to
measure fasting glucose levels. At an equipotent dose, 13 did
not increase blood glucose while pred triggered a significant
increase compared to vehicle control.
The more potent analogues, 17 and 30, were also evaluated
in the rat CPE model. Both compounds produced a dose-
dependent reduction of edema after the single oral dose. The in
vivo potencies (17: ED50 = 45 mg/kg; 30: ED50 = 44 mg/kg)
compared favorably with pred (ED50 = 19 mg/kg) (Figure 4).
As observed with 13, 17 and 30 also gave little or no increase
in blood glucose relative to pred, even at the highest dose (17,
50 mg/kg; 30, 30 mg/kg vs pred, 30 mg/kg).
Figure 3. Compound 13 and pred in the carrageenan-induced paw
edema model after oral administration in rats: (A) paw edema
inhibition 5 h postdose; (B) blood glucose levels 5 h postdose.
Compound 13 dosed at 100 mg/kg and pred at 30 mg/kg.
GR-mediated transactivation effects of 30 were also eval-
uated in normal, nondiseased Sprague-Dawley (SD) rats
via its ability to induce tyrosine amino transferase (TAT) and
elevate serum glucose. TAT is a liver specific gene, the
hepatic expression of which is closely regulated by activated
GR.21 Fasted SD rats showed a significant increase in serum
glucose and induction of TAT (6 h post dose) when orally
dosed with pred at 30 mg/kg. The oral administration of 30 to
SD rats at 7.5, 15, and 30 mg/kg gave a dose-proportional
increase in serum exposure (as measured 6 h postdose) of 59,
157, and 311 nM, respectively. Unlike pred, 30 did not
provoke measurable increases of serum glucose nor liver
TAT activity at all dose levels, even at the highest dose of
30 mg/kg (Figure 5).
Overall, the anti-inflammatory activity of 30 and 17 are
similar to that of pred, whereas the GR-mediated transacti-
vation activity (as measured by induction of hyperglycemia
or TAT) are significantly reduced compared with pred.
Conclusion
We have described a novel class of dimethyl-diphenyl-
propanamides as potent and selective nonsteroidal ligands
of the glucocorticoid receptor. Select compounds from this
series elicit reduced transactivation relative to steroidal ago-
nists whileretaininggood transrepressionactivities in reporter
gene assays. Compounds 30 and 17 achieved equivalent anti-
inflammatory efficacy in vivo to pred in the rat carrageenan
paw edema assay. Most notably, both compounds induced
little or no side effects in rats with regard to blood glucose
elevation and hepatic TAT induction compared with predni-
solone. Taken together, the dissociation between transrepres-
sion and transactivation observed with 30 and 17 in vitro is
reflected in vivo by a steroid-like anti-inflammatory activity
and markedly reduced transactivation-mediated side effects.

8246 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Yang et al.
Figure 4. Compounds 17 and 30 demonstrated efficacy comparable to pred in suppressing paw edema after oral administration in rats but
promoted little or no blood glucose elevation.
Figure 5. Compound 30 did not lead to measurable increase of serum glucose compared to vehicle control (A) and no induction of liver TAT
enzyme activity relative to pred (B) in normal fasted SD rats, in contrast to pred, even at the highest (and equipotent) oral dose of 30 mg/kg.
Although compounds 30 and 17 have provided a major
advance in identifying a dissociated GR agonist in our program,
many opportunities remain for continued improvement. For
example, phenols often suffer from the drawback of reactive
metabolite formation. Optimization to circumvent the forma-
tion of reactive metabolites by exploring bioisosteric replace-
ments of the phenolic OH is expected to improve the overall
ADMET profile and will be the subject of future reports.
(Thermo Scientific, San Jose, CA) interfaced to a Waters Acquity
ultra performance liquid chromatograph (UPLC) (Milford, MA).
The purity of tested compounds determined by analytical HPLC
system was >95% except as noted. Analytical HPLC was per-
formed on a Shimadsu instrument. Column: YMC Combiscreen
ODS-A, 4.6 mm ꢀ 50 mm; gradient elution 0-100% B/A over 4
min with 1 min hold (solvent A: water 90%/MeOH 10%/H3PO4
0.2%; solvent B: MeOH 90%/water 10%/H3PO4 0.2%); flow rate
4 mL/min; 220 or 254 nm detection wavelength.
Method A. Synthesis of 3-(4-Methoxyphenyl)-2,2-dimethyl-3-
phenylpropanoic Acid (8a).15
Experimental Section
a. To a solution of lithium diisopropylamide (LDA, 22.6 mL,
45.2 mmol) in tetrahydrofuran (THF, 10 mL) at 0 °C was added
dropwise a solution of isobutyric acid (2.102 mL, 22.6 mmol) in
THF (10 mL). The reaction solution was stirred at 0 °C for
30 min and 60 °C for 2 h. The flask was recooled to 0 °C, and a
solution of (4-methoxyphenyl)(phenyl)-methanone 5 (4.8 g, 22.6
mmol) in THF (10 mL) was added dropwise. The solution was
allowed to warm to room temperature and stirred overnight.
The reaction was quenched with 1N HCl until pH ∼3 and
extracted with ethyl acetate (EtOAc) 3 times. The combined
Chemistry. All commercially available chemicals and solvents
were used without further purification. Reactions are performed
under an atmosphere of nitrogen. All new compounds gave
satisfactory 1H NMR, ESI-MS, and mass spectrometry analyses.
1H NMR spectra were obtained on a Bruker 400 MHz or a Jeol
500 MHz NMR spectrometer using residual signal of deuterated
NMR solvent as internal reference. Electrospray ionization (ESI)
mass spectra were obtained on a Waters Micromass ESI-MS single
quadrupole mass spectrometer. High-resolution mass spectral
analysis was performed on an LTQ-FT mass spectrometer

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8247
organic layers were washed with 1N aqueous NaOH (3 times).
The combined basic washes were acidified with concentrated
HCl and extracted with EtOAc (3 times). The organic phase was
washed with brine, dried over MgSO4, and concentrated. The
crude product mixture was purified by flash chromatography
(10% EtOAc in hexanes) to give 4.1 g (60% yield) of 3-hydroxy-
3-(4-methoxyphenyl)-2,2-dimethyl-3-phenyl propanoic acid 7
as an amorphous solid. ESI-MS m/z 299.30 ([M - H]-). HPLC
Rt 3.530 min.
(1 H, dd, J = 8.65, 2.54 Hz), 6.63 (1 H, dd, J = 12.21, 2.54 Hz),
4.76 (1 H, s), 1.30 (3 H, br s), 1.23 (3 H, br s). ESI-MS m/z
301.16. ([M - H]-). HPLC Rt: 3.650 min.
Method C. (S)-3-(4-Methoxyphenyl)-2,2-dimethyl-3-phenyl-
propanoic Acid (9a) and (R)-3-(4-Methoxyphenyl)-2,2-dimethyl-
3-phenylpropanoic Acid (10a). The acid 8a was resolved into its
corresponding enantiomers using chiral supercritical fluid chro-
matography (SFC) with the following conditions. Column,
Chiralpak-AD, 3 cm ꢀ 25 cm; mobile phase, CO2/MeOH/
trifluoroacetic acid (TFA) = 85:15:0.1; flow rate, 60 mL/min;
detection, UV (220 nm). Analytical HPLC conditions. Column,
Chiralcel OJ 4.6 mm ꢀ 250 mm; 10 μm; mobile phase, 1:1
MeOH/EtOH with 0.1% TFA; temperature, ambient; flow rate,
1 mL/min; detection, UV (254 and 220 nm). Retention time
(min): first eluting enantiomer, 7.82 (>99% ee); second eluting
enantiomer, 8.84 (>99% ee).
A sample of the second eluting enantiomer 10a was cocrys-
tallized with (S)-(-)β-methylphenethylamine in acetonitrile
(CH3CN). An X-ray crystal structure determination of the
crystalline material thus obtained proved 10a to be of (R)
absolute stereochemistry. The absolute configuration of the
first-eluting enantiomer 9a has been deduced to be (S).
(S) and (R)-3-(3-Fluoro-4-methoxyphenyl)-2,2-dimethyl-3-phenyl-
propanoic Acids (9b) and (10b).Obtained from chiral separation of 8b
according to a procedure similar to method C.
b. To a solution of 7 (2.0 g, 6.66 mmol) in dichloromethane
(25 mL) at 0 °C was added triethylsilane (10.7 mL, 66.6 mmol),
followed by boron trifluoride diethyl etherate (2.5 mL, 19.98
mmol). The solution was allowed to warm to room temperature
and stirred for 5 h, by which time it was quenched with saturated
aqueous Na2CO3 solution. The aqueous phase was acidified with
1N HCl and extracted with EtOAc (3 times). Recrystallization of
the crude product mixture with EtOAc and hexanes yielded 1.52 g
(80% yield) of 8a as a white solid. 1H NMR (400 MHz, CDCl3) δ
ppm 7.29-7.35 (2 H, m), 7.17-7.29 (5 H, m), 6.81 (2 H, d, J =
8.14 Hz), 4.40 (1 H, s), 3.78 (3 H, s), 1.29 (6 H, s). ESI-MS m/z
283.28 ([M - H]-). HPLC Rt: 3.598 min.
Method B. Synthesis of 3-(4-Methoxy-3-methylphenyl)-2,2-
dimethyl-3-phenylpropanoic Acid (8d)
a. To a solution of (phenyl) magnesium bromide in THF (1M,
8 mL. Eight mmol) at 0 °C was added dropwise a solution of
4-methoxy-3-methylbenzaldehyde (0.6 g, 4 mmol) in THF (2 mL).
After being stirred at 0 °C for 10 min and at room temperature for
3 h, the reaction mixture was poured onto ice-cooled saturated
aqueous NH4Cl. The solution was extracted with EtOAc. The
organic layer was washed with brine, dried over MgSO4, and
concentrated to give (4-methoxy-3-methylphenyl)(phenyl)methanol
6d as a colorless viscous liquid (920 mg). ESI-MS m/z 211
([M-OH]þ). HPLC Rt: 3.226 min.
b. To a solution of 6d (920 mg, 4 mmol) and (1-methoxy-2-
methyl-propenyloxy)-trimethyl-silane (3.25 mL, 16 mmol) in
dichloromethane (8 mL) at 0 °C was added a solution of titanium
tetrachloride in dichloromethane (1 M solution, 8.8 mL, 8.8 mmol)
slowly. The reaction mixture was stirred at 0 °C for 10 min and at
room temperature for 2 h. To the reaction mixture was added water.
After stirring for 10 min, the solution was adjust to pH 7 with
Na2CO3 powder and was extracted with EtOAc. The organic layer
was washed with brine, dried (MgSO4), and concentrated to give
the crude product. The crude product mixture was purified by flash
chromatography (0-15% EtOAc in hexanes) to yield 1.154 g
(92.3% for 2 steps) of methyl 3-(4-methoxy-3-methylphenyl)-2,2-
dimethyl-3-phenylpropanoate. ESI-MS m/z 335.21 ([M þ Na]þ).
HPLC Rt: 3.983 min.
(S)-3-(4-Methoxy-3-methylphenyl)-2,2-dimethyl-3-phenylpro-
panoic Acid (9d). Obtained from chiral separation of 8d accord-
ing to a procedure similar to Method C.
Method D. Synthesis of 3-(4-Hydroxyphenyl)-2,2-dimethyl-3-
phenyl-N-(thiazol-2-yl)propanamide (12). a. 3-(4-Methoxyphenyl)-
2,2-dimethyl-3-phenyl-N-(thiazol-2-yl)propanamide (11a). To a so-
lution of 8a (150 mg, 0.42 mmol) in CH3CN (1 mL) were added
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochlor-
ide (EDC) (202 mg, 1.056 mmol) and 1-hydroxy-7-benzotriazole
(HOBt) (143 mg, 1.056 mmol). The solution was stirred for
i
5 min, and thiazol-2-amine (69 mg, 0.69 mmol) and Pr2NEt
(0.322 mL, 1.85 mmol) were added. The reaction was heated at
75 °C for 12 h. The product mixture was filtered, concentrated,
and purified by silica gel flash chromatography eluted with 30%
EtOAc in hexanes to give 11a as a white solid (173 mg, 89%
yield). 1H NMR (400 MHz, MeOD) δ ppm 7.33-7.43 (3 H, m),
7.29 (2 H, d, J = 8.65 Hz), 7.23 (2 H, t, J = 7.38 Hz), 7.13-7.19
(1 H, m), 7.07 (1 H, d, J = 3.05 Hz), 6.80 (2 H, d, J = 8.65 Hz),
4.54 (1 H, s), 3.73(3 H, s), 1.37 (6 H, s). ESI-MS m/z 367.26 ([M þ
H]þ). HPLC Rt: 3.587 min. HRMS m/z calcd for C21H22N2O2S
([M þ H]þ) 367.1475, found: 367.1475.
b. To a solution of 11a (173 mg, 0.472 mmol) in dichlor-
omethane (10 mL) at 0 °C was added a 1 M solution of boron
tribromide in dichloromethane (1.89 mL, 1.89 mmol). The
reaction solution was stirred at 0 °C for 30 min and at room
temperature for 1 h and then was quenched with methanol. The
reaction mixture was partitioned between water and dichloro-
methane. The organic solution was washed (water and brine),
dried (MgSO4), and concentrated. The crude product mixture
was purified by reverse phase preparative HPLC (column,
YMC, C-18 Ballistic, 30 mm ꢀ 100 mm; 10-90% aq CH3OH/
0.1% TFA, 25 mL/min flow rate, 220 nm detection wavelength,
same for compounds hereafter unless noted) to yield 135 mg
(81%) of 12 as a white solid. 1H NMR (500 MHz, MeOD) δ ppm
7.40 (1 H, d, J = 3.61 Hz), 7.36 (2 H, d, J = 7.49 Hz), 7.23 (2 H, t,
J = 7.49 Hz), 7.13-7.20 (3 H, m), 7.07 (1 H, d, J = 3.61 Hz),
6.66 (2 H, d, J = 8.88 Hz), 4.49 (1 H, s), 1.36 (6 H, s). ESI-MS m/z
353.29 ([M þ H]þ). HPLC Rt: 3.158 min.
c. To a solution of the methyl ester (771 mg, 2.467 mmol) in
MeOH (4 mL) and dimethyl sulfoxide (DMSO, 1 mL) was
added a 40% aqueous solution of KOH (5 mL). The reaction
mixture was heated at 75 °C for 15 h. After removal of methanol,
the solution was adjusted to pH 2 and was extracted with ethyl
ether (Et2O). The ether layer was washed with brine, dried
(MgSO4), and evaporated to give 8d as a white foam (577 mg,
78.5% yield). 1H NMR (400 MHz, MeOD) δ ppm 7.33 (2 H, d,
J = 7.12 Hz), 7.23 (2 H, t, J = 7.38 Hz), 7.15 (2 H, t, J = 7.38
Hz), 7.06 (1 H, d, J = 2.03 Hz), 6.78 (1 H, d, J = 8.65 Hz), 4.35
(1 H, s), 3.77 (3 H, s), 2.13 (3 H, s), 1.23 (6 H, s). ESI-MS m/z
297.43 ([M - H]-). HPLC Rt: 3.753 min.
3-(4-Methoxy-3-fluorophenyl)-2,2-dimethyl-3-phenylpropanoic
Acid (8b). Prepared from 4-methoxy-3-fluorobenzaldehyde
according to method B (55% yield). 1H NMR (400 MHz, MeOD)
δ ppm 7.15-7.39 (5 H, m), 7.03-7.14 (2 H, m), 6.97 (1 H, t, J =
8.69 Hz), 4.38 (1 H, s), 3.83 (3 H, s), 1.25 (3 H, br s), 1.24 (3 H, br s).
ESI-MS m/z 301.4. ([M - H]-). HPLC Rt: 3.516 min.
2,2-Dimethyl-3,3-diphenyl-N-(thiazol-2-yl)propanamide (4).
Prepared from benzophenone according to method A and method
D, Step (a). 1H NMR (500 MHz, MeOD) δ ppm 7.33-7.47 (5 H,
m), 7.24 (4 H, t, J= 7.38 Hz), 7.17 (2 H, t, J= 7.12 Hz), 7.07 (1 H, d,
J = 3.05 Hz), 4.62 (1 H, s), 1.39 (6 H, s). ESI-MS m/z 337.32 ([M þ
H]þ). HPLC Rt: 3.643 min. HRMS m/z calcd for C20H21N2O1S
([M þ H]þ) 337.1369; found 337.1370.
3-(4-Methoxy-2-fluorophenyl)-2,2-dimethyl-3-phenylpropanoic
Acid (8c). Prepared from (2-fluoro-4-methoxyphenyl)(phenyl)-
methanone according to method A (91.2% yield). 1H NMR
(400 MHz, MeOD) δ ppm 7.42 (1 H, t, J = 8.65 Hz), 7.27-7.33
(2 H, m), 7.24 (2 H, t, J = 7.38 Hz), 7.14-7.20 (1 H, m), 6.69

8248 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Yang et al.
(S)-3-(4-Hydroxyphenyl)-2,2-dimethyl-3-phenyl-N-(thiazol-2-
yl)propanamide (13). Prepared from 9a according to method D.
1H NMR (500 MHz, MeOD) δ ppm 7.40 (1 H, d, J = 3.61 Hz),
7.36 (2 H, d, J = 7.49 Hz), 7.23 (2 H, t, J = 7.49 Hz), 7.13-7.20
(3 H, m), 7.07 (1 H, d, J = 3.61 Hz), 6.66 (2 H, d, J = 8.88 Hz),
4.49 (1 H, s), 1.36 (6 H, s). ESI-MS m/z 353.3 ([M þ H]þ). HPLC
Rt: 3.095 min. HRMS m/z calcd for C20H20N2O2S ([M þ H]þ)
353.1318; found 353.1321. Chiracel OJ (90% EtOH/MeOH/
0.1%TFA; 10% Hep, 1 mL/min) Rt 4.80 min, ee >99.7%.
(R)-3-(4-Hydroxyphenyl)-2,2-dimethyl-3-phenyl-N-(thiazol-2-yl)-
3.428 min. HRMS m/z calcd for C20H19BrN2O2S ([M þ H]þ)
431.0428; found 431.0427.
(S)-3-(3-Bromo-4-hydroxyphenyl)-2,2-dimethyl-3-phenyl-N-(1,3,
4-thiadiazol-2-yl)propanamide (20). An Emry process vial was
charged with 19 (38 mg, 0.088 mmol) and copper(I) cyanide
(40 mg, 0.45 mmol) in dimethylformamide (DMF). The reaction
mixture was sealed and exposed to microwave irradiation for
60 min at 220 °C. The reaction was cooled, filtered, and purified
by preparative HPLC to give the TFA salt of 20 as an off-white
solid. (16.5 mg, 50% yield). 1H NMR (400 MHz, CDCl3) δ ppm
7.43-7.51 (3 H, m), 7.23-7.26 (4 H, m), 7.21 (1 H, d, J = 6.10
Hz), 7.03 (1 H, d, J = 4.07 Hz), 6.88 (1 H, d, J = 8.65 Hz), 4.72
(1 H, s), 1.42 (6 H, s). ESI-MS m/z 378.27 ([M þ H]þ). HPLC Rt:
3.195 min. HRMS m/z calcd for C21H19N3O2S ([M þ H]þ)
378.1271; found 378.1271.
1
propanamide (14). Prepared from 9b according to method D. H
NMR (500 MHz, MeOD) δ ppm 7.40 (1 H, d, J = 3.61 Hz), 7.36
(2 H, d, J = 7.49 Hz), 7.23 (2 H, t, J = 7.49 Hz), 7.13-7.20 (3 H,
m), 7.07 (1 H, d, J = 3.61 Hz), 6.66 (2 H, d, J = 8.88 Hz), 4.49 (1 H,
s), 1.36 (6 H, s). ESI-MS m/z 353.3 [(M þ 1)þ]. HPLC Rt: 2.991
min. HRMS m/z calcd for C20H20N2O2S ([M þ H]þ) 353.1318;
found 353.1321. Chiracel OJ (90% EtOH/MeOH/0.1%TFA; 10%
Hep, 1 mL/min) Rt 6.53 min, ee >99.7%.
3-(3-Fluoro-4-hydroxyphenyl)-2,2-dimethyl-3-phenyl-N-(thiazol-
2-yl)propanamide (15). Prepared from 10b according to method D.
1H NMR (400 MHz, MeOD) δ ppm 7.43 (1 H, br s), 7.36 (2 H, d,
J = 7.12 Hz), 7.25 (2 H, t, J = 7.38 Hz), 7.18 (1 H, t, J = 7.38 Hz),
7.10 (1 H, br s), 7.06 (1 H, dd, J= 12.97, 1.78 Hz), 6.99 (1 H, d, J =
8.14 Hz), 6.78 (1 H, t, J = 8.65 Hz), 4.51 (1 H, s), 1.37 (3 H, s), 1.36
(3 H, s). ESI-MS m/z 371.12 ([M þ H]þ). HPLC Rt: 3.250 min.
HRMS m/z calcd for C20H19FN2O2S ([M þ H]þ) 371.1224; found
371.1224.
(S)-3-(3-Acetyl-4-hydroxyphenyl)-2,2-dimethyl-3-phenyl-
N-(thiazol-2-yl)propanamide (21). a. To a solution of AlCl3 in
nitrobenzene (1M, 4.1 mL, 4.1 mmol) was added acetyl chloride
(76μL, 1.065 mmol)atroomtemperature. After 10 min, asolution
of 13 (300 mg, 0.819 mmol) in dichloroethane (2 mL) was added
dropwise. The reaction mixture was stirred at ambient tempera-
ture for 1.5 h and was quenched with ice-cooled 10% aqueous
NH4OH. After stirring for 15 min, the solution was extracted with
EtOAc. The organic phase was washed (water and brine), dried
(MgSO4), and concentrated. The crude product was purified by
silica gel flash chromatography (30% EtOAc in hexanes) to give
3-(3-acetyl-4-methoxyphenyl)-2,2-dimethyl-3-phenyl-N-(thiazol-
2-yl)propanamide as a white solid (208 mg, 62.3% yield). ESI-MS
m/z 409.2 ([M þ H]þ). HPLC Rt: 3.386 min.
3-(2-Fluoro-4-hydroxyphenyl)-2,2-dimethyl-3-phenyl-N-(thiazol-
2-yl)propanamide (16). Prepared from 8c according to method D.
1H NMR (500 MHz, MeOD) δ ppm 7.37-7.44 (1 H, m),
7.29-7.37 (3 H, m), 7.23 (2 H, t, J = 7.42 Hz), 7.16 (1 H, t, J =
7.15 Hz), 7.08 (1 H, br s), 6.53 (1 H, d, J = 8.25 Hz), 6.44 (1 H, d,
J = 12.10 Hz), 4.88 (1 H, br s), 1.33 (6H, s), 1.47 (3 H, s). ESI-MS
m/z 371.12 ([M þ H]þ). HPLCRt: 3.275 min. HRMS m/z calcd for
C20H19FN2O2S ([M þ H]þ) 371.1224; found 371.1224.
b. 3-(3-Acetyl-4-methoxy-phenyl)-2,2-dimethyl-3-phenyl-
N-(thiazol-2-yl)-propanamidewas treatedwithborontribromide,
according to method D, step (b) to provide 21 (30% yield). H
1
NMR (400 MHz, CDCl3) δ ppm 12.12 (1 H, s), 7.70 (1 H, d,
J = 2.03 Hz), 7.49 (1 H, dd, J = 8.65, 2.54 Hz), 7.42 (1 H, br s),
7.23-7.36 (5 H, m), 6.99 (1 H, br s), 6.89 (1 H, d, J = 8.65 Hz),
4.39 (1 H, s), 2.52 (3 H, s), 1.43 (3 H, s), 1.39 (3 H, s). ESI-MS m/z
395.26 ([M þ H]þ). HPLC Rt: 3.600 min. HRMS m/z calcd for
C22H122N2O3S ([M þ H]þ) 395.1424; found 395.1424.
(S)-3-(3-Fluoro-4-hydroxyphenyl)-2,2-dimethyl-3-phenyl-
N-(thiazol-2-yl)propanamide (17). Prepared from 9b according to
method D. 1H NMR (400 MHz, MeOD) δ ppm 7.33-7.42 (3 H,
m), 7.25 (2 H, t, J = 7.63 Hz), 7.15-7.20 (1 H, m), 7.02-7.10 (2 H,
m), 6.98 (1 H, d, J = 8.14 Hz), 6.74-6.82 (1 H, m), 4.51 (1 H, s),
1.37 (3 H, s), 1.36 (3 H, s). ESI-MS m/z 371.12 ([M þ H]þ). HPLC
Rt: 3.255 min. HRMS m/z calcd for C20H19FN2O2S ([M þ H]þ)
371.1224; found 371.1224.
(S)-3-(4-Hydroxyphenyl)-2,2-dimethyl-3-phenyl-N-(1,3,4-thiadia-
zol-2-yl)propanamide (22). a. To a solution of 8a (100 mg,
0.352 mmol) in dichloromethane (3 mL) at 0 °C was added a
solution of boron tribromide in dichloromethane (1M, 1.41 mL,
1.41 mmol). The reaction solution was stirred at 0 °C for 30 min
and at room temperature for 1 h. After it was cooled at 0 °C, the
reaction mixture was quenched with 10% NH4OH and extracted
with dichloromethane. The aqueous layer was acidified with
concentrated HCl and extracted with dichloromethane (3 times).
The combined organic phase was washed (water and brine), dried
(MgSO4), and concentrated to afford (S)-3-(4-hydroxyphenyl)-
2,2-dimethyl-3-phenylpropanoic acid as an off-white solid (95 mg,
99% yield). ESI-MS m/z 269.15 ([M - H]-). HPLC Rt: 3.123 min.
b. To a solution of (S)-3-(4-hydroxyphenyl)-2,2-dimethyl-3-
phenylpropanoic acid (15 mg, 0.055 mmol) in dichloromethane
(2 mL) at 0 °C was added 1-chloro-N,N,2-trimethylprop-1-en-1-
amine (15 μL, 0.111 mmol). The reaction was stirred at 0 °C for
30 min, at which time iPr2NEt (24 μL, 0.138 mmol) was added,
followed by 1,3,4-thiadiazol-2-amine (17 mg, 0.165 mmol).
After 5 min, the reaction mixture was allowed to warm to room
temperature and stirred for 1 h. The solvent was removed. The
crude product mixture was purified by reverse phase preparative
HPLC to yield 17 mg (87%) of 22 as an off-white solid. 1H
NMR (400 MHz, MeOD) δ ppm 8.96 (1 H, s), 7.36 (2 H, d, J =
7.63 Hz), 7.06-7.31 (5 H, m), 6.66 (2 H, d, J = 8.14 Hz), 4.54
(1 H, s), 1.38 (6 H, s). ESI-MS m/z 354.1 ([M þ H]þ). HPLC Rt:
3.032 min. HRMS m/z calcd for C19H19N3O2S ([M þ H]þ)
354.1276; found 354.1274. Chiracel OJ (mobile phase: 100% 1:1
EtOH/MeOH with 0.1% TFA; 220 and 254 nm; 1 mL/min) Rt:
5.40 min; ee >99%.
(R)-3-(3-Fluoro-4-hydroxyphenyl)-2,2-dimethyl-3-phenyl-
N-(1,3,4-thiadiazol-2-yl)propanamide (18). Prepared from 10b
1
according to method D. H NMR (400 MHz, MeOD) δ ppm:
same as compound 15. ESI-MS m/z 371.06 ([M þ H]þ). HPLC
Rt: 3.288 min. HRMS m/z calcd for C20H19FN2O2S ([M þ H]þ)
371.1224; found 371.1224.
(S)-3-(3-Bromo-4-hydroxyphenyl)-2,2-dimethyl-3-phenyl-
N-(thiazol-2-yl)propanamide (19)
a. (S)-3-(3-Bromo-4-methoxyphenyl)-2,2-dimethyl-3-phenyl-
propanoic Acid (26). To a suspension of 9a (1 g, 3.52 mmol) and
NaOAc (433 mg, 5.28 mmol) in acetic acid (20 mL) at room
temperature was added bromine (0.271 mL, 5.28 mmol) dropwise.
After stirring for 3 h, the reaction mixture was poured into 10%
aqueous sodium hydrosulfite (Na2S2O4) solution. The solution
was adjusted to pH 7 with concentrated NH4OH and extracted
with Et2O. The organic extract was washed with brine, dried
(MgSO4), and concentrated to afford (S)-3-(3-bromo-4-meth-
oxyphenyl)-2,2-dimethyl-3-phenyl-propanoic acid 26 as a white
solid (1.239 g, 96.9% yield). 1H NMR (400 MHz, CDCl3) δ ppm
7.49 (1 H, d, J = 2.54 Hz), 7.26-7.30 (4 H, m), 7.25 (1 H, d, J =
2.03 Hz), 7.23 (1 H, d, J = 2.03 Hz), 6.80 (1 H, d, J = 8.65 Hz),
4.36 (1 H, s), 3.86 (3 H, s), 1.30 (6 H, s). HPLC Rt: 3.696 min.
b. 19 was prepared from 26 according to method D (18%
yield). 1H NMR (400 MHz, CDCl3) δ ppm 7.46 (1 H, d, J = 2.03
Hz), 7.42 (1 H, d, J = 2.54 Hz), 7.30-7.35 (2 H, m), 7.18-7.30
(4 H, m), 7.01 (1 H, br s), 6.91 (1 H, d, J = 8.65 Hz), 4.60 (1 H, s),
1.43 (6 H, s). ESI-MS m/z 431.16, 433.16 ([M þ H]þ). HPLC Rt:
(S)-3-(3-Fluoro-4-hydroxyphenyl)-2,2-dimethyl-3-phenyl-N-(1,3,
4-thiadiazol-2-yl)propanamide (23). Prepared from 9b according to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8249
method D with the employment of 1,3,4-thiadiazol-2-amine in the
amidation step. 1H NMR (400 MHz, MeOD) δ ppm 8.97 (1 H, s),
7.36 (2 H, d, J = 7.12 Hz), 7.25 (2 H, t, J = 7.63 Hz), 7.14-7.21
(1 H, m), 7.06 (1 H, dd, J = 12.97, 2.29 Hz), 6.99 (1 H, d, J = 8.14
Hz), 6.78 (1 H, t, J = 8.90 Hz), 4.54 (1 H, s), 1.38 (6 H, s). ESI-MS
m/z 372.12 ([M þ H]þ). HPLCRt: 3.030 min. HRMS m/zcalcdfor
C19H18FN3O2S ([M þ H]þ) 372.1177; found 372.1179.
(400 MHz, CDCl3) δ ppm 8.86 (1 H, s), 7.26 (1 H, d), 7.14 (2 H,
dd, J = 7.4, 2.8 Hz), 6.96-7.05 (2 H, m), 6.66 (1 H, d, J = 2.5
Hz), 6.50 (1 H, dd, J = 8.4, 2.8 Hz), 4.41 (1 H, s), 1.20 (6 H, s).
ESI-/MS m/z 368.06 ([M þ H]þ). HPLC Rt: 3.097 min. HRMS
([M þ Na]þ) calcd for C12H16O10N5 390.0892; found 390.0892.
r,r-Dimethyl-N-1,3,4-thiadiazol-2-yl-9H-xanthene-9-aceta-
mide (32)
(S)-3-(3-Bromo-4-hydroxyphenyl)-2,2-dimethyl-3-phenyl-N-(1,3,
4-thiadiazol-2-yl)propanamide (24). Prepared from 26 according to
method D with the employment of 1,3,4-thiadiazol-2-amine in the
amidation step. 1H NMR (400 MHz, CDCl3) δ ppm 8.54 (1 H, s),
7.23 (1 H, d, J = 2.54 Hz), 7.04-7.11 (4 H, m), 7.02 (1 H, d, J =
2.54 Hz), 6.97-7.01 (1 H, m), 6.70 (1H, d, J = 8.65 Hz), 4.27 (1 H,
s), 1.24 (6 H, s). ESI-MS m/z 432.15, 434.15 ([M þ H]þ). HPLC
Rt: 3.272 min. HRMS m/z calcd for C19H18BrN3O2S ([M þ H]þ)
432.0381; found 432.0380.
a. According to method A, R,R-dimethyl-9H-xanthene-9-
acetic acid was prepared from 9H-xanthen-9-one in 65% yield.
1H NMR (400 MHz, CDCl3) δ ppm 7.32-7.26 (m, 4 H), 7.16
(dd, J = 8.6, 1.1 Hz, 2 H), 7.12-7.06 (m, 2 H), 4.41 (s, 1 H), 1.05
(s, 6 H). ESI-MS m/z 267.42 ([M - H]-). HPLC Rt: 3.725 min.
b. According to method D, step (a), 32 was prepared from
R,R-dimethyl-9H-xanthene-9-acetic acid and 1,3,4-thiadiazol-
1
2-amine (82% yield). H NMR (400 MHz, acetone-d6) δ ppm
9.09 (s, 1 H), 7.35-7.32 (m, 2 H), 7.31-7.30 (m, 2 H), 7.25-7.23
(m, 2 H), 7.08-7.04 (m, 2 H), 4.71 (s, 1 H), 1.20 (s, 6 H). ESI-MS
m/z 350.11 ([M - H]-). HPLC Rt: 3.525 min. HRMS ([M þ
Na]þ) calcd for C12H16O9N5 374.0943; found 374.0941.
(S)-3-(4-Hydroxy-3-methylphenyl)-2,2-dimethyl-3-phenyl-N-(1,3,
4-thiadiazol-2-yl)propanamide (25). Prepared from 9d according to
method D with the employment of 1,3,4-thiadiazol-2-amine in the
amidation step 1H NMR (400 MHz, MeOD) δ ppm 8.97 (1 H, s),
7.36 (2 H, d, J = 7.63 Hz), 7.23 (2 H, t, J = 7.63 Hz), 7.11-7.18
(1 H, m), 6.97-7.06 (2 H, m), 6.61 (1 H, d, J = 8.14 Hz), 4.49 (1 H,
s), 2.09(3H, s), 1.37(6H, s). ESI-MSm/z368.2 ([M þ H]þ). HPLC
Rt: 3.216 min. HRMS m/z calcd for C20H21N3O2S ([M þ H]þ)
368.1427; found 368.1428.
Glucocorticoid Receptor Binding Assay. The GR ligand binding
assay was conducted in fluorescence polarization format which
measures the competition between a test compound and a fluor-
escently labeled ligand (GS-red) for binding to the full length or
ligand binding domain of GRR (Invitrogen, cat. no. P2893).
Compounds were tested in concentrations ranging from 5 μM to
85 pM. IC50 values were determined by fitting the fluorescence
polarization signal data using a four parameter logistic equation.
The Ki values were determined by application of the Cheng-
Prusoff equation22 to the IC50 values, where Ki = IC50/(1 þ ligand
concentration/Kd). The Kd value used for GR was 0.3 nM as
supplied by the assay manufacturer (Invitrogen, cat. no. P2893).
Data shown represent the means of two or more experiments.
PR, ER, and AR ligand binding assays were also conducted in
the fluorescence polarization format to the full length or ligand
binding domain of each nuclear hormone receptor.
Transrepression Assays. AP-1 (activator protein-1) activity
was measured using an AP-1 response element (5 copies) cloned
into a luciferase reporter vector. This reporter was stably
transfected into the human A549 lung epithelial cell line. AP-1
activity was induced for by addition of phorbol myristate
acetate (PMA) (15 ng/mL), and inhibition of induction by
compounds was quantitated by measuring decreased luciferase
activity. NFκB was measured using a truncated, NFκB depen-
dent, E-selectin promoter (-383 bp from transcriptional start)
cloned into a luciferase reporter vector. This reporter was stably
transfected into the human A549 lung epithelial cell line. NFκB
activity was induced using IL-1β (0.5 ng/mL), and inhibition
of induction by compounds was quantitated by measuring
decreased luciferase activity.
(S)-2-(3-Hydroxy-9H-xanthen-9-yl)-2-methyl-N-(thiazol-2-yl)-
propanamide (29)
a. According to method A, 2-(3-methoxy-9H-xanthen-9-yl)-
2-methylpropanoic acid 28 was prepared from 3-methoxy-9H-
xanthen-9-one 27 in 61.2% yield. 1H NMR (400 MHz, CDCl3)
δ ppm 7.31-7.25 (m, 2 H), 7.20-7.05 (m, 3 H), 6.71 (d, J =
2.6 Hz, 1 H), 6.67 (dd, J = 8.5, 2.5 Hz, 1 H), 4.36 (s, 1 H), 3.82 (s,
3 H), 1.04 (d, J = 2.2 Hz, 6 H). ESI-MS m/z 299.20 ([M þ H]þ).
HPLC Rt: 3.710 min.
b. According to a procedure similar to method C, chiral
separation of 28 afforded two enantiomers, (S) and (R)-2-(3-
methoxy-9H-xanthen-9-yl)-2-methylpropanoic acids.
c. To a solution of (S)-28 (200 mg, 0.67 mmol) in 1-methyl-2-
pyrrolidinone (NMP, 4 mL) in a microwave vessel was added
2-aminothiophenol (92 mg, 0.74 mmol) and K2CO3 (102 mg,
0.74 mmol). The reaction mixture was irradiated by microwave
at 205 °C for 1.5 h and then partitioned between EtOAc and
water. The organic layer was extracted with 1N aqueous NaOH.
The combined aqueous layers were acidified to ∼pH 2.0 and
were extracted with EtOAc. The organic phase was washed with
brine, dried over Na2SO4, and concentrated. Purification of the
crude product by preparative HPLC provided (S)-2-(3-hydro-
xy-9H-xanthen-9-yl)-2-methylpropanoic acid as an amorphous
solid (133 mg, 70% yield).
Transactivation Assays. a. GAL4DBD-GR(LBD) Reporter
Assay. The direct transcriptional activity was measured using a
chimera between the GAL4 DNA-binding domain (DBD) and the
human GR ligand-binding domain (GR-LBD) cloned into a
GAL4 Luciferase reporter system. This reporter system was stably
transfected into an NP-1 HeLa cell line.23 The hormone-binding
domains of the estrogen and glucocorticoid receptors contain an
inducible transcription activation function. Response to ligand/
compound induced binding was quantitated by measuring lucifer-
ase activity. Direct activation of the GR-LBD by compounds
(agonist) can be measured as increased luciferase activity.
d. According to method D, Step (a), amidation of (S)-2-(3-
hydroxy-9H-xanthen-9-yl)-2-methylpropanoic acid with 2-ami-
nothiazole afforded 29 (60% yield). 1H NMR (400 MHz,
CDCl3) δ ppm 7.51 (d, 1H), 7.23-7.31 (m, 1 H), 7.09-7.17
(m, 3 H), 6.95-7.06 (m, 2 H), 6.65 (d, J = 2.64 Hz, 1 H), 6.51
(dd, J = 8.25, 2.53 Hz, 1 H), 4.45 (s, 1 H), 1.18 (s, 6 H). ESI-MS
m/z 367.21 ([M þ H]þ). HPLC Rt: 3.175 min. HRMS m/z calcd
for C20H19O3N2S ([M þ H]þ) 367.1111; found 367.1113.
(S)-2-(3-Hydroxy-9H-xanthen-9-yl)-2-methyl-N-(1,3,4-thiadiazol-
2-yl)propanamide (30). Prepared from (S)-2-(3-hydroxy-9H-
xanthen-9-yl)-2-methyl-propanoic acid and 1,3,4-thiadiazol-2-
b. TAT Induction Assay.24 An enzymatic assay used to
measure the induction of tyrosine aminotransferase activity in
cultured hepatoma cell line, human 13D3/Huh7 (derivative
isolated under glucose-limiting condition). Briefly, on day one,
the hepatoma cell line 3D3/Huh7 was plated at 20 K cells/well in
a 96-well flat bottom, tissue culture treated assay plate and
incubated overnight at 37 °C/5% CO2. On day 2, the growth
medium was removed from the wells and replaced with assay
medium containing 20 μM forskolin and test compounds, which
was then incubated overnight at 37 °C with 5% CO2. On day 3,
1
amine according to method D, step (a) (66% yield). H NMR
(400 MHz, CDCl3) δppm 8.87 (1 H, s), 7.26 (1 H, d), 7.14 (2 H, dd,
J = 7.4, 2.8 Hz), 6.96-7.05 (2 H, m), 6.66 (1 H, d, J = 2.5 Hz),
6.50 (1 H, dd, J = 8.4, 2.8 Hz), 4.41 (1 H, s), 1.20 (6 H, s). ESI-MS
m/z 368.30 ([M þ H]þ). HPLCRt: 3.090min. HRMSm/z calcd for
C19H18O3N3S ([M þ H]þ) 368.1063; found 367.1066.
(R)-2-(3-Hydroxy-9H-xanthen-9-yl)-2-methyl-N-(1,3,4-thiadiazol-
2-yl)propanamide (31). Prepared from (R)-28 according to the
procedures (c) and (d) of 29 with the employment of 1,3,4-
thiadiazol-2-amine at the amidation step (42% yield). 1H NMR

8250 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Yang et al.
the assay medium was removed and 100 μL/well of lysis buffer
(125 mM potassium phosphate buffer, pH 7.6, 140 mM KCl,
1 mM EDTA, and 0.1% NP-40) was added. The lysates were
assayed for TAT activity. In a new 96-well plate, 136 μL/well of
TAT substrate (200 mM potassium phosphate buffer, pH 7.6,
5.4 nM tyrosine sodium salt, 0.06 mM pyridoxal 50-phosphate,
and 10.8 mM R-ketoglutarate) and 50 μL/well of lysate were
added and mixed. The plates were incubated at 37 °C for 4 h. The
TAT enzymatic reaction was stopped with KOH and incubated
an additional 30 min at 37 °C. The absorbance was measured at
340 nm. Results were expressed as percent activation and EC50s
were calculated.
Carageenan Paw Edema (CPE) Assay. SD male rats were
isolated and fasted overnight and up to six additional hours on
the day of the study. Baseline paw volume measurements were
obtained by immersing each hind paw to the level of the hair line
inawater-basedplethysmometer. Ratswereadministeredasingle
oral dose of test compound(s) in biologically acceptable medium.
Two hours postdosing, while under anesthesia, both hind paws
were injected subcutaneously with 1% carrageenan in PBS. Paw
volume measurements were obtained 3 h post challenge. Paw
swelling was calculated by subtracting the baseline values from
the post challenge measurements. In the same study, a baseline
blood glucose level and glucose levels at 5 h post dosing were
measured, using a glucometer, on blood samples collected by tail
nicks.
Normal (Nondiseased) Rat Glucose Model and TAT Induction.
SD male rats were fasted overnight and up to six additional
hours on the day of the study. Rats were administered a single
oral dose of test compounds in biologically acceptable medium.
At the conclusion of the study (6 h post treatment), blood
samples were collected, via intracardiac puncture following
euthanasia with CO2 gas, into serum separator tubes for glucose
analysis. Quantitative determination of multiple serum analytes
was performed with the Roche/Hitachi 917 fully automated
chemistry analyzer by using hexokinase method.
In the same study, TAT induction was evaluated 6 h after
compound administration by determination of TAT activity in
liver homogenates. Biopsies were taken from the liver and snap
frozen. Biopsies were homogenized in 150 mM KCl solution
using a PT 3100 polytron and centrifuged. Supernatants were
collected and protein levels were determined. Supernatants
(normalized protein amount/well) were assayed in a similar
manner as described above for TAT induction in vitro. TAT
induction in rats, measured as OD at 340 nm, was defined as %
in TAT activity in comparison to the TAT activity in predniso-
lone-treated animals (30 mg/kg).
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Acknowledgment. We are grateful to the following colleagues
for their support of the project and their help in the preparation
of this manuscript: Haiqing Wang, Mary Ellen Cvijic, Ding
Ren Shen, Melissa Yarde, Ravindra Tejwani, Laishun L. Chen,
Mary Malley, Dauh-Rurng Wu, and Dawn Sun.
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