Discovery of a potent and selective prostaglandin D2 receptor antagonist, [(3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4- tetrahydrocyclopenta[b]indol-3-yl]-acetic acid (MK-0524)

Article abstract of DOI:10.1021/jm0603668

The discovery of the potent and selective prostaglandin D₂ (PGD₂) receptor (DP) antagonist [(3R)-4-(4-chlorobenzyl)-7-fluoro-5- (methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic acid (13) is presented. Initial lead antagonists 6 and 7 were found to be potent and selective DP antagonists (DP K_i = 2.0 nM for each); however, they both suffered from poor pharmacokinetic profiles, short half-lives and high clearance rates in rats. Rat bile duct cannulation studies revealed that high concentrations of parent drug were present in the biliary fluid (C_max = 1100 μM for 6 and 3900 μM for 7). This pharmacokinetic liability was circumvented by replacing the 7-methylsulfone substituent present in 6 and 7 with a fluorine atom resulting in antagonists with diminished propensity for biliary excretion and with superior pharmacokinetic profiles. Further optimization led to the discovery of the potent and selective DP antagonist 13.

Full text of DOI:10.1021/jm0603668

794
J. Med. Chem. 2007, 50, 794-806
Discovery of a Potent and Selective Prostaglandin D2 Receptor Antagonist, [(3R)-4-(4-Chloro-
benzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic Acid
(MK-0524)†
Claudio F. Sturino,*,‡ Gary O’Neill,*,§ Nicolas Lachance,‡ Michael Boyd,‡ Carl Berthelette,‡ Marc Labelle,‡ Lianhai Li,‡
Bruno Roy,‡ John Scheigetz,‡ Nancy Tsou, Yves Aubin,‡ Kevin P. Bateman,‡ Nathalie Chauret,‡ Stephen H. Day,‡
Jean-Franc¸ois Le´vesque,‡ Carmai Seto,‡ Jose H. Silva,‡ Laird A. Trimble,‡ Marie-Claude Carriere,§ Danielle Denis,§
Gillian Greig,§ Stacia Kargman,§ Sonia Lamontagne,§ Marie-Claude Mathieu,§ Nicole Sawyer,§ Deborah Slipetz,§
William M. Abraham,| Tom Jones,3 Malia McAuliffe,3 Hana Piechuta,3 Deborah A. Nicoll-Griffith,‡ Zhaoyin Wang,‡
Robert Zamboni,‡ Robert N. Young,‡ and Kathleen M. Metters§
Department of Medicinal Chemistry, Merck Frosst Canada & Co., 16711 Trans Canada Highway, Kirkland, Quebec H9H 3L1, Canada,
Department of Biochemistry, Merck Frosst Canada & Co., 16711 Trans Canada Highway, Kirkland, Quebec H9H 3L1, Canada, Department of
Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, New Jersey 07065, Miller School of Medicine, UniVersity of
Miami at Mount Sinai Medical Center, 430 Alton Road, Miami Beach, Florida 33140, and Department of Pharmacology, Merck Frosst Canada
& Co., 16711 Trans Canada Highway, Kirkland, Quebec H9H 3L1, Canada
ReceiVed March 29, 2006
The discovery of the potent and selective prostaglandin D2 (PGD2) receptor (DP) antagonist [(3R)-4-(4-
chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic acid (13) is
presented. Initial lead antagonists 6 and 7 were found to be potent and selective DP antagonists (DP Ki )
2.0 nM for each); however, they both suffered from poor pharmacokinetic profiles, short half-lives and
high clearance rates in rats. Rat bile duct cannulation studies revealed that high concentrations of parent
drug were present in the biliary fluid (Cmax ) 1100 µM for 6 and 3900 µM for 7). This pharmacokinetic
liability was circumvented by replacing the 7-methylsulfone substituent present in 6 and 7 with a fluorine
atom resulting in antagonists with diminished propensity for biliary excretion and with superior
pharmacokinetic profiles. Further optimization led to the discovery of the potent and selective DP antagonist
13.
Introduction
congestion associated with allergic rhinitis by causing vasodi-
lation in the nasal mucosa.4 It has been reported that nasal
challenge with PGD2 in humans results in a dose-dependent
increase in nasal congestion. This PGD2-induced nasal conges-
tion was produced with approximately 10-fold greater potency
than histamine and with 100-fold greater potency than brady-
kinin.5 In addition to the above finding, it has also been reported
that elevated levels of PGD2 were measured in the nasal
washings of allergic patients that underwent a nasal antigen
challenge.6 Also, DPa knockout mice have been shown to exhibit
a reduced allergic response with no obvious abnormalities.7
PGD2 and its receptor (DP receptor) have been implicated in a
number of physiological processes such as regulation of body
temperature, sleep, and pain.8 The implication that PGD2 is
involved in allergic diseases has prompted us to identify a
suitable clinical PGD2 receptor antagonist to treat the inflam-
matory effects exerted through the actions of PGD2.9
We have recently reported on an indole series of DP receptor
antagonists.10a In particular, indoles 6 and 7 were noted to be
potent and selective DP antagonists. Unfortunately, these
antagonists displayed poor pharmacokinetic profiles that ex-
cluded them from being potential clinical candidates. In this
paper, we wish to highlight some of the medicinal chemistry
issues, principally related to pharmacokinetics and metabolism,
that were addressed during the optimization of an indole series
of DP antagonists leading eventually to the identification of the
potent and selective PGD2 receptor antagonist 13.10b
Seasonal allergic rhinitis is an upper-airway disorder that
typically results in nasal congestion, itching, and rhinorrhea.
Histamine, cysteinyl leukotrienes (CysLTs), thromboxane A2
(TxA2), and prostaglandin D2 (PGD2) are considered key
mediators in allergic rhinitis, as well as in other allergic
conditions such as allergic asthma, atopic dermatitis, and allergic
conjunctivitis.1 Release of the above mediators in response to
IgE-dependent stimuli results in an increase in mucus production
and intranasal pressure. Leukotriene D4 receptor antagonists and
antihistamines have shown efficacy in a guinea pig model of
allergic rhinitis and conjunctivitis.2 While antihistamines are able
to control the symptoms of the early allergic response, namely,
sneezing, itching, and rhinorrhea, they are less effective at
treating symptoms of the late allergic reaction such as nasal
congestion. Even though a number of treatment options are
available today to treat allergic rhinitis, there still remains a
medical need for an orally dosed nasal decongestant with a better
side-effect profile than what is currently available.
In response to IgE-dependent stimuli, PGD2 is the major
cyclooxygenase-derived metabolite obtained from the degranu-
lation of mast cells.3 PGD2 is thought to mediate the nasal
† The x-ray crystallographic data for compound 14 has been deposited
with the Cambridge Crystallographic Data Centre (CCDC601363).
* Corresponding authors. C.F.S.: tel (514) 428-2630, fax (514) 428-
4900, e-mail claudio_sturino@merck.com. G.O.: tel (514) 428-7920, fax
(514) 428-4900, e-mail gary_oneill1@merck.com.
‡ Department of Medicinal Chemistry, Merck Frosst Canada & Co.
§ Department of Biochemistry, Merck Frosst Canada & Co.
Department of Medicinal Chemistry, Merck Research Laboratories.
| University of Miami at Mount Sinai Medical Center.
To date, there has been no reported clinical experience with
the use of DP antagonists in the treatment of airway diseases.
a Abbreviations: DP, PGD2 receptor; TP, TxA2 receptor; EP1-4, PGE2
receptor subtypes 1, 2, 3, and 4; FP, PGF2R receptor; IP, PGI2 receptor.
3 Department of Pharmacology, Merck Frosst Canada & Co.
10.1021/jm0603668 CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/20/2007

DiscoVery of MK-0524
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 795
Scheme 1. Synthesis of 7-Methylsulphone Indole Series of DP Receptor Antagonistsa
i
a (a) I2, Ag2SO4, EtOH; (b) (i) (EtO)4Si, PPTS, ethyl 2-(2-oxocyclopentyl)acetate, 130-140 °C, 6 h; (ii) Pr2NEt, Pd(OAc)2, DMF, 120 °C, 2 h; (c) (i)
2 N NaOH, MeOH, THF, 60 °C; (ii) PyrHBr‚Br2, pyr, -25 °C to rt; (iii) Zn, AcOH, 10 °C; (d) (i) CH2N2, Et2O, THF; (ii) NaH, 4-chlorobenzyl bromide,
DMF; (e) (i) 1-ethoxyvinyltributyltin, Pd2(dba)3, Ph3As, DMF, 80 °C; (ii) 2 N HCl; (iii) 2 N NaOH, THF, MeOH; (f) chiral HPLC separation of enatiomers;
(g) (i) NaBH4, MeOH; (ii) chiral HPLC separation.
Recently, the use of the PGD2 receptor antagonist 13 for the
suppression of nicotinic acid-induced vasodilation in humans
has been reported.11
The synthesis of 13 is outlined in Scheme 2 and incorporates
similar transformations as described above. 4-Fluorophenyl-
amine (8) was iodinated,16 and following palladium mediated
indole synthesis, the adduct 9 was obtained in moderate yield.
For large scale preparation of 9, it was more convenient to make
use of the commercially available 2-bromo-4-fluoroaniline as
starting material as outlined in Scheme 2. Bromination of 9,
followed by benzylation, proceeded to deliver acid 10. The
resolution of this material was accomplished by recrystallization
of its (S)-(-)-1-(napthyl)ethylamine salt. This procedure con-
veniently provided the resolved acid 11 after acid workup.
Bromide 11 was then transformed into the corresponding
methylketone 12 as described. For the introduction of the
methylsulfone, as required in 13, acid 11 was first esterified
with diazomethane and then treated with methanesulfinic acid
sodium salt and CuI in N-methylpyrrolidone (NMP) at 150 °C
for 3 h.17 These conditions were found to cleanly convert the
aryl bromide into a methylsulfone in high yields. Standard
hydrolysis thus delivered the DP antagonist 13. It is worth noting
that this sulfonylation reaction tolerates an acid functional group
because treatment of the free acid 11 under identical conditions
delivered 13 with comparable yields. The absolute stereochem-
istry of the acetic acid side chain present in 11, 12, and 13 was
established to be (R) from a single-crystal X-ray analysis of
the acid intermediate 14.18
Chemistry
The synthetic routes developed for the synthesis of the DP
receptor antagonists are outlined in Schemes 1 and 2. Iodination
of 4-methylsulfonylphenylamine (1) with iodine and silver
sulfate in ethanol12 cleanly afforded the 2-iodo adduct in 80%
yield. This aniline was then condensed with 2-(2-oxocyclopen-
tyl)acetate in the presence of tetraethoxysilane as a dehydrating
reagent and with catalytic quantities of pyridinium p-toluene-
sulfonate (PPTS). The resulting imine was then treated with
Pd(OAc)2 and Hunig’s base in DMF to afford the corresponding
indole in 39% overall yield.13 Introduction of the 7-bromo
substituent was accomplished by first hydrolyzing the ester to
the acid followed by reaction with pyridinium perbromide in
pyridine14 (see Figure 1 for numbering of the 1,2,3,4-tetrahy-
drocyclopenta[b]indole ring system). This reaction is believed
to result in the introduction of three bromine atoms to the indole
ring system, namely, at the 3a, 5, and 8b positions. Zinc
treatment of this intermediate serves to eliminate the bromides
at the 3a and 8b positions and delivers the monobromo adduct
3 in 81% overall yield. It is worth noting that attempts at
brominating ester 2 under similar reaction conditions proved
unsuccessful. Acid 3 was subsequently esterified with diaz-
omethane and then treated with NaH in DMF followed by
4-chlorobenzyl bromide to cleanly generate the benzyl derivative
in 89% yield. The 5-bromo substituent in turn was converted
into a methyl ketone by first performing a Stille cross coupling
reaction with 1-ethoxyvinyltributyltin employing Pd2(dba)3/Ph3-
As (dba ) dibenzylideneacetone) as the catalyst system.15 The
resulting vinyl ether was hydrolyzed to the corresponding ketone
5. This acid was resolved into its individual enantiomers by
HPLC separation on chiral support to deliver enantiomer 6.
Standard reduction of 6 employing NaBH4 in MeOH, followed
by HPLC separation on chiral support of the two diasastereo-
meric alcohols delivered 7 as a single diastereomer.
Results and Discussion
We have recently disclosed the in Vitro prostanoid receptor
binding profiles of antagonists 6 and 7. Both of these compounds
are potent DP antagonists with Ki values of 2.6 ( 0.7 and 1.8
( 0.7 nM for 6 and 7, respectively (Table 1).19 The stereo-
chemistry of the acetic acid side chain (stereochemistry (R)) is
critical for activity because the corresponding (S) stereoisomers
are generally greater than 100-fold less active on the DP
receptor. The stereochemistry at the alcohol center of 7 is also
important for DP activity because the corresponding diastero-
meric alcohol was found to be less potent (Ki ) 8.9 ( 1.8 nM,
n ) 4). Antagonists 6 and 7 are also selective for the remaining

796 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Sturino et al.
Scheme 2. Synthesis of 7-Fluoro Indole Series of DP Receptor Antagonistsa
a (a) I2, NaHCO3, H2O, toluene; (b) (i) (EtO)4Si, PPTS, ethyl 2-(2-oxocyclopentyl)acetate; (ii) iPr2NEt, Pd(OAc)2, DMF; (c) (i) 2 N NaOH, MeOH, THF;
(ii) PyrHBr‚Br2, pyr, -25 °C to rt; (iii) Zn, AcOH, 10 °C; (iv) HCl, MeOH; (v) NaH, 4-chlorobenzyl bromide, DMF; (vi) 2 N NaOH, THF, MeOH; (d) (i)
(S)-(-)-1-(napthyl)ethylamine, MeOH; (ii) recrystallize, EtOH; (iii) 1 N HCl; (e) (i) CH2N2, Et2O, THF; (ii) 1-ethoxyvinyltributyltin, Pd2(dba)3, Ph3As,
DMF, 90 °C; (iii) 1 N HCl; (iv) 1 N NaOH, THF, MeOH; (f) (i) CH2N2, Et2O, THF; (ii) MeSO2Na, CuI, NMP, 150 °C, 3 h; (iii) 2 N NaOH, THF, MeOH;
n
(g) (i) ethyl 2-(2-oxocyclopentyl)acetate, toluene, p-TsOH‚H2O, reflux; (ii) Pd(OAc)2, KOAC, Bu4NCl, DMF, 80 °C.
antagonist 7 is not (8%). In addition, the maximum concentration
(Cmax) achieved with 6 is significantly higher than that with 7
(23 vs 0.29 µM at a dose of 10 mg/kg).21 Even though the
bioavailabilities of these antagonists in rats differ, they both
exhibited short half-lives and high clearance rates (Table 3).
Pharmacokinetic studies with 6 and 7 in other species (mouse,
dog, cynomolgus monkey) also indicated that these antagonists
displayed unsuitable pharmacokinetic profiles (short half-life,
Figure 1. 1,2,3,4-Tetrahydrocyclopenta[b]indole numbering system.
high clearance rates) that precluded them from being potential
clinical candidates and underscored the need to identify other
DP antagonists with improved pharmacokinetic profiles.
prostanoid receptors (TP, EP1-4, FP, and IP, see Table 1)20 with
alcohol 7 exhibiting higher selectivity versus the TP receptor
than ketone 6. In addition, these compounds are potent
In an effort to investigate whether the rapid clearance of
antagonists 6 or 7 was the result of drug metabolism, these
compounds were incubated in a rat hepatocyte preparation and,
after a 2 h period, the percent of parent drug remaining was
determined for each compound.22 Interestingly, as shown in
Table 4, antagonists 6 and 7 were, in fact, not extensively
metabolized with 93-100% of parent drug remaining at the
end of the incubation period. The low metabolic turnover of
these antagonists suggested to us that the rapid clearance in rats
was likely not driven by Phase I or II metabolism. At this time,
we elected to perform a rat bile duct cannulation study to
determine what drug-derived compounds were being excreted
into the biliary fluid in a hope that this would shed some light
on the clearance pathways operative for 6 and 7.23 These
experiments proved quite valuable. In these experiments, a
cannula was inserted into the bile duct of anesthetized rats and,
antagonists in DP functional assays where they inhibit the
production of PGD2-induced cyclic adenosine monophosphate
(cAMP) in washed platelets (WP) (IC50 of 2 nM for both 6 and
7) and in platelet-rich plasma (PRP) (IC50 values of 7.9 ( 2.8
and 8.5 ( 4.3 nM for 6 and 7, respectively). The high degree
of selectivity was maintained versus TP in the functional assay
as demonstrated from the TP PRP aggregation functional assay
(Table 2, see Experimental Procedures for details of the
functional assays), in agreement with the receptor binding
profiles for these compounds.
Given the excellent in Vitro profiles of 6 and 7, the
pharmacokinetic properties of these antagonists were evaluated
in Sprague-Dawley rats, and the results are summarized in
Table 3. Several points from Table 3 are worth discussing. While
the bioavailability of 6 in rats is good, the bioavailability for

DiscoVery of MK-0524
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 797
Table 1. Prostanoid Receptor Binding Affinities, Ki (nM)a
compd
DP
TP
EP1
EP2
EP3
EP4
FP
IP
6
7
11
12
13
2.6 ( 0.71
1.8 ( 0.70
1.5 ( 0.57
1.1 ( 0.22
0.57 ( 0.17
1200 ( 470
7100 ( 820
0.84 ( 0.19
14 ( 2.7
>20000
>20000
230 ( 110
390 ( 33
410 ( 320
310 ( 82
136 ( 8.0
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
4400 ( 1600
>9000
470 ( 46
4400 ( 1500
>20000
14000 ( 3100
>20000
400 ( 100
1200 ( 260
892 ( 250
14000 ( 2100
6628 ( 1343
2.95 ( 0.46
1160 ( 420
>15200
9991 ( 5826
a Values are means from at least three experiments.
Table 2. DP and TP Receptor Functional Activity in PRP and WP
Table 5. Rat Biliary Concentrations of DP Antagonistsa
Cmax of
Assaya
tmax of
DP WP
DP PRP
TP PRP
compd
parent, µM
parent, h
compd
IC50, nMb
IC50, nMc
IC50, nMd
6b
7b
1100
3900
29
81
103
0-0.5
0-0.5
1-1.5
2.5-3
0.5-1
6
7
11
12
13
2.0 ( 0.7
2.0 ( 1.4
0.26 ( 0.04
0.29 ( 0.05
0.09 ( 0.02
7.9 ( 2.8
8.6 ( 4.3
23 ( 9.5
15 ( 0.41
4.0 ( 0.9
8400 ( 4700
43000 ( 16000
80 ( 60
1400 ( 1000
770 ( 490
11c
12c
13b
a The corresponding sodium salts were used for these pharmacokinetic
studies. The compounds were administered iv at 5 mg/kg at a dose volume
of 1 mL/kg; n ) 2 for all studies except for 13 with n ) 5. Cmax ) maximum
concentration; tmax ) time to reach maximum concentration. b iv, vehicle
5% dextrose. c iv, vehicle 60% PEG 200.
a Values are means from at least three experiments. IC50 ) concentration
at which process is inhibited by 50%. b DP WP assay involved inhibition
of the accumulation of cAMP in washed platelets challenged with PGD2.
c DP PRP assay involved inhibition of the accumulation of cAMP in platelet-
rich plasma challenged with PGD2. d TP PRP assay involved the inhibition
of U44619-induced platelet aggregation in platelet-rich plasma.
Table 3. Rat Pharmacokinetic Parameters of DP Antagonistsa
Cmax, µM
C6h, µM
Clp
compd F (%) (10 mg/kg) (10 mg/kg)
T
1/2, he (mL/min) Vdss
6b
7b
128
8
41
52
79
21
0.29
10
21
39
0.6
0.03
3.7
7.5
6.3
1.4
<1
12
4
14
50
1.4
1.9
2.4
0.7
0.8
0.4
0.5
0.5
11c
12d
13b
7
a The corresponding sodium salts were used for these pharmacokinetic
studies. Half-lives are determined from the intravenous (iv) data. F denotes
bioavailability; Cmax denotes the maximum concentration reached; C6h
denotes the concentration 6 h after dosing; T1/2 is the half-life in plasma;
Clp is plasma clearance; Vdss is the volume of distribution. b Oral (po) dose
10 mg/kg, vehicle 0.5% methocel, dose volume 10 mL/kg, n ) 2; iv dose
5 mg/kg, vehicle 5% dextrose, dose volume 1 mL/kg, n ) 2. c po dose 5
mg/kg, vehicle 0.5% methocel, dose volume 10 mL/kg, n ) 2; iv dose 5
mg/kg, vehicle 60% PEG 200, n ) 2. d po dose 10 mg/kg, vehicle 60%
PEG 200, dose volume 10 mL/kg, n ) 2; iv dose 5 mg/kg, vehicle 60%
Figure 2. Medicinal chemistry strategy to elucidate biliary excretion
SAR.
the high biliary levels of 6 and 7, we speculate that this process
is occurring via an active transport mechanism.24
To address the pharmacokinetic liability of the above DP
antagonists, SAR studies were focused on identifying an analog
of 6 or 7 that maintained the DP activity of these antagonists
but with diminished propensity for biliary excretion. At the
outset, this objective was derived from the hypothesis that
reduced biliary excretion would result in improved pharmaco-
kinetic (PK) profiles. Figure 2 highlights the medicinal chemistry
strategy that was undertaken to identify compounds with
diminished propensity for biliary excretion. We elected to divide
the molecule into three distinct regions, as illustrated, modify
in turn the different regions of the molecule, and evaluate any
analog that displayed good DP potency for their rat PK profile.
We initially examined the effects of replacing the 4-chlorobenzyl
substituent of the molecule (region 1), and a number of
replacements were examined including differentially substituted
benzyl groups, alkyl substituents, benzothiazole, and quinoline.
In general, these modifications led to compounds with either
significantly diminished DP activity or comparable rat phar-
macokinetic profiles to antagonist 6. We next turned our
attention to modifying region 2 of the molecule. Modifications
at this region revealed that the carboxylic acid, the (R)
stereochemistry, and the acetic acid are all required for DP
activity. Concurrently, we also examined the benzenoid region
of the molecule (region 3). A number of replacements were
evaluated for the methylsulfone and ketone substituents, and
of the various modifications examined, the most dramatic effect
was seen when the methylsulfone group was replaced with a
fluorine atom, leading to antagonists such as 11 and 12.
e
PEG 200, dose volume 1 mL/kg, n ) 2. T1/2 values were determined from
the iv experiments.
Table 4. Metabolic Stability of DP Antagonists in Rat Hepatocyte
Incubationsa
percent parent remaining
in rat hepatocytes
compd
(50 µM, 2 h incubation)
6
7
11
12
13
93
100
42
35
32
a 0.5 mL of 2 × 106 cells/mL, 37 °C; 95/5 O2/CO2.
subsequently, antagonists 6 and 7 were administered intrave-
nously. Bile fluid was collected in 30 min intervals and analyzed
by HPLC-UV. As summarized in Table 5, these experiments
revealed that the major drug-derived compound present in the
bile fluid was, in fact, intact parent. We were particularly
intrigued by the high levels of parent in the biliary fluid, 1100
µM for 6 and 3900 µM for antagonist 7. These high drug levels
confirmed that the major elimination pathway for antagonists
6 and 7 in rats was via biliary excretion. This mechanism is
also consistent with the in Vitro rat hepatocyte data (Table 4)
suggesting that the metabolism was not playing a significant
role in the clearance of these DP antagonists. In addition, given

798 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Sturino et al.
Figure 3. Radiolabeled DP antagonists.
Several properties of 11 and 12 are worth commenting. In
terms of prostanoid receptor binding profiles, the fluoro analogs
11 and 12 remain potent DP antagonists although their selectivity
versus the TP receptor is significantly diminished (see Table
1). This decrease in TP selectivity, as measured by the
competition receptor binding assay, is evident when comparing
the TP selectivity of the 7-fluoro indole 12 (selectivity ) 13)
versus the corresponding methylsulfone analog 6 (selectivity
) 460, Table 1). As expected, this decreased TP selectivity in
the binding assay translates to a decreased selectivity in the
functional assay as well (Table 2). While the TP selectivity of
the fluoro analogs is lower relative to the methylsulfone
antagonists, the advantage of the 7-fluoro series becomes evident
when one considers their pharmacokinetic properties. First, in
terms of metabolic stability, as measured by the in Vitro
hepatocyte assay, the fluoro antagonists 11 and 12 are signifi-
cantly more metabolized (42% and 35% parent remaining,
respectively) than the methylsulfone analog 6 or 7 (93% and
100% parent remaining, respectively). However, the metaboli-
cally more labile fluorine analogs 11 and 12 displayed superior
rat pharmacokinetic profiles as illustrated in Table 3. Antagonists
11 and 12 have good rat half-lives (12 and 4 h for 11 and 12,
respectively), decreased clearance rates, and similar volumes
of distribution relative to the methylsulfone analogs. This
improvement in pharmacokinetic profile results in higher plasma
drug levels at 6 h as noted in Table 3. Rat bile duct cannulation
studies with the fluoro antagonists 11 and 12 revealed that
significantly lower levels of parent compound were present in
the bile fluid (29 and 81 µM for 11 and 12, respectively) than
was observed with 6 or 7 (Table 5). In particular, the difference
in the biliary excretion observed between antagonists 6 and 12
is quite notable. Replacing the methylsulfone substituent in 6
with a fluorine atom decreased the biliary Cmax from 1100 to
81 µM, a 13.6-fold decrease. It is also worth highlighting that
the extent of metabolic turnover in the rat hepatocyte assay was
not predictive of their in ViVo pharmacokinetic profile due to
the extensive biliary excretion of antagonist 6 and 7 versus the
fluoro analogs 11 and 12. At this point, antagonist 12 displayed
an attractive in Vitro and PK profile, and the focus of the
medicinal chemistry effort was directed at trying to improve
its activity in the functional assay (IC50 in PRP assay of 15
nM).
binding assay. It is 5.2-fold selective versus the TP receptor in
the binding assay, 239-fold selective versus EP2 and >1500-
fold selective against the remaining prostanoid receptors (Table
1). In the functional assay, 13 is a potent inhibitor of PGD2-
induced cAMP formation in both washed platelets (IC50 ) 0.09
nM) and platelet-rich plasma (IC50 ) 4.0 nM, approximately
4-fold more potent than 12).
While the in Vitro profile of 13 attracted our interest, the
pharmacokinetic profile of this antagonist remained a potential
concern. Gratifyingly, as is evident from the data in Table 3,
13 displays an excellent pharmacokinetic profile in rats with
good oral bioavailability (F ) 79%) and half-life (t1/2 ) 7 h)
and a low clearance rate (2.4 mL‚min-1‚kg-1). A rat bile duct
cannulation study with 13 revealed that low levels of parent
drug was present in the biliary fluid (Cmax ) 103 µM) similar
to the other fluoro analogs and in contrast to 7-methylsulfone
antagonists 6 or 7. It thus appears that while the 7-methylsul-
fonyl antagonists suffer from extensive biliary excretion in rats,
the same is not true for the 5-methylsulfone analog 13.
Antagonist 13 also displays excellent pharmacokinetic profiles
in other species (mouse, dog, and cynomolgus monkey), and
thus the improved rat PK profile of 13 relative to antagonist 6
translated to other species. The behavior of 13 in the in Vitro
rat hepatocyte assay is also worth noting. In this assay, the
percent parent remaining from 13 (32%) was similar to the other
two fluoro analogs 11 and 12.
Covalent Protein Labeling. Bioactivation of xenobiotics in
ViVo can result in the formation of reactive intermediates
resulting in covalent protein adduct formation.25 This bioacti-
vation-protein modification process has been associated with
organ toxicity in both preclinical species and humans. There is
also the potential concern that the resulting modified proteins
can behave as haptens and induce an unwanted immune
response. Our in-house strategy to address the potential liabilities
associated with the formation of reactive intermediates has been
to incorporate covalent protein labeling studies early in the lead
optimization phase of drug discovery. A target of 50 pmol equiv/
mg of protein binding is viewed as a low propensity for covalent
protein modification.25
Up to this point, we have seen how the indole substituents
can significantly affect the pharmacokinetic and receptor
selectivity profiles of these DP antagonists. Of equal importance,
we have observed during the course of our studies that the
benzenoid substituents can also affect the level of metabolically
activated covalent protein binding. A number of radiolabeled
DP antagonists were synthesized26 (Figure 3) and incubated in
rat and human microsomes and hepatocytes for 1 h, and the
extent of covalent binding was then evaluated.27 The results of
these experiments are summarized in Table 6. An interesting
Additional SAR studies, focusing primarily on the 5-position
of the tetrahydrocyclopenta[b]indole, revealed that indeed it was
possible to further improve the potency of the DP antagonists.
A number of 5-position replacements were evaluated, including
aryl, thiophene, heterocycles, and alkyl groups, and from these
studies, the 5-methylsulfone 13 was identified. Antagonist 13
is a potent DP antagonist with a Ki of 0.57 nM in the receptor

DiscoVery of MK-0524
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 799
Table 6. In Vitro Covalent Protein Binding (pmol equiv/mg of protein
per h)a
rat
human
microsomesb
(+NADPH)
rat
microsomesb
(+NADPH)
human
compd
hepatocytesc
hepatocytesc
6
7
11
12
13
23 ( 2.1
13 ( 4.5
460 ( 7.5
290 ( 50e
33 ( 0.5
8 ( 0.4
9 ( 4.3
18 ( 0.8
9 ( 0.4
33 ( 1.3
46 ( 3.3e
16 ( 0.5
5 ( 0.5
<5 ( 0.4
33 ( 7.0d
16 ( 4.3d
15 ( 1.4
74 ( 49d
45 ( 19d
38 ( 0.4
Figure 5. Proposed structure of reactive intermediate.
extent as the other two fluoro analogs 11 and 12, with
glucuronidation and hydroxylation being the major pathways,
it differs from these two antagonists in two respects. First, the
extent of covalent labeling is significantly lower in both the
microsomal and hepatocyte systems. We speculate that the
powerful electron-withdrawing methylsulfone substituent present
in 13 serves to destabilize the iminium cation 19 and thus inhibit
its formation, thereby decreasing the extent of protein covalent
labeling. Second, the extent of protein covalent labeling was
found to be similar in the microsomal and hepatocyte systems.
For antagonists 11 and 12, the extent of labeling was signifi-
cantly higher in the rat microsomal incubation than in hepato-
cytes. One of the major differences between microsomal and
hepatocyte systems is that the later possesses naturally occurring
trapping agents, such as glutathione, that serve to intercept
reactive intermediates. Thus, the decreased protein labeling in
rat hepatocyte relative to microsomes for antagonists 11 and
12 is likely the result of the interception of any formed reactive
intermediate. The observation that antagonist 13 displays
comparable labeling in microsomes as hepatocytes supports the
hypothesis that the methylsulfone is preventing the formation
of reactive intermediates such as the iminium cation 19. Thus,
the introduction of a methylsulfone at the 5-position of the indole
core served two important functions: it improved the DP
potency in the functional PRP assay and decreased the propen-
sity for covalent protein labeling.
Prostanoid Receptor Selectivity. It is worth commenting
further on the DP versus TP selectivities of the above DP
antagonists as measured by the receptor binding assay versus
the functional assay. This data is summarized in Table 7 and
serves to illustrate further the difference between the 7-meth-
ylsulfone and 7-fluoro series. For the 7-methylsulfone antago-
nists 6 and 7, the TP selectivities measured in the binding assay
were found to be consistent with the selectivities observed in
the functional assay (Table 7). This is not the case for the
7-fluoro DP antagonists. In all three cases, the TP selectivities
of these antagonists were lower in the competition receptor
binding assay than in the functional assay. In particular, the TP
selectivity of 13 is only 5.2-fold in the binding assay and is
192-fold in the functional assay. Thromboxane (TxA2) acting
through the TP receptor is known to induce platelet aggregation;
thus inhibition of the TP receptor may result in an inhibition of
TxA2-mediated platelet aggregation. In order to determine the
true TP selectivity of 13, a number of experiments were initiated
in an effort to resolve this apparent discrepancy. The lower limit
of sensitivity in the competition receptor binding assays is
approximately 0.5 nM; thus selectivity ratios determined with
this assay may be underestimating the selectivity of antagonist
13. To circumvent this limitation, the dissociation constants (Kd)
for 13 from the DP and TP receptors were measured. From these
experiments, the Kd’s for 13 from the DP and TP receptors were
determined to be 0.03 and 10.9 nM, respectively. The TP
selectivity for 13, as determined from the dissociation data, is
now 360-fold, which is in agreement with the selectivity
measured in the functional assay. Thus, the apparent discrepancy
in DP versus TP selectivity of 13 as measured from the
a Unless indicated otherwise, each value is a mean of triplicate tests from
one experiment. b Microsomal incubations: 1 mg/mL liver microsomes; 1
mM NADPH; 10 µM compound (1 µCi/mL); 1 h incubation at 37 °C; 125
mM phosphate buffer, pH 7.4. No covalent labeling was observed in the
absence of NADPH. c Hepatocyte incubations: 0.25 mL of 1 × 106 cells/
mL; preincubation of 20 min at 37 °C under 95/5 O2/CO2; 10 µM of test
compound (1 µCi/mL); 2 h incubation at 37 °C under 95/5 O2/CO2; 125
mM phosphate buffer, pH 7.4. No covalent labeling was observed in the
absence of NADPH. d Values shown are means from two separate experi-
ments, each performed in triplicate. e Values shown are means from three
separate experiments, each performed in triplicate.
Figure 4. Hydroxyl metabolite of antagonists 11-13.
trend emerges from these in Vitro protein binding studies. First,
for 6 and 7, both of these antagonists displayed low levels of
covalent protein binding in both the rat and human microsomal
incubation systems. This low level of protein binding is
consistent, in part, with their metabolic stability in the in Vitro
rat hepatocyte assay (Table 3). In contrast, with the fluoro
analogs 11 and 12, high levels of protein binding were observed
in the rat microsomal assay (460 and 290 pmol equiv/mg of
protein per hour, respectively). The extent of protein binding
with these antagonists was lower in the rat hepatocyte assay,
and only moderate protein binding was observed in the human
incubation systems. With 13, reduced levels of protein binding
were seen in both the rat and human incubation systems relative
to 11 and 12 with modest levels of binding in the rat microsomal
and hepatocyte systems and low levels in the human incubation
systems.
At this point, it is worth commenting on the protein covalent
labeling with respect to the corresponding in Vitro metabolism
data presented in Table 4. Antagonist 6 displayed low levels of
metabolism in rat hepatocytes and was mainly converted to its
corresponding acylglucuronide, along with low levels of a
monohydroxylation product. Antagonist 7 exhibited undetectable
levels of in Vitro metabolism in rat hepatocytes. Both of these
antagonists display low levels of covalent protein labeling as
discussed above. By contrast, the fluoro analogs 11-13 are
metabolized to a greater extent in vitro (Table 4) with hydroxy-
lation and glucuronidation again being the major pathways. The
major site of hydroxylation for these antagonists was assigned
to be at carbon 1 (see Figure 1 for numbering scheme) to give
the corresponding alcohol 18 (Figure 4) based on LC/MS and
NMR studies. For antagonists 11 and 12, the higher levels of
covalent protein labeling, relative to 6 and 7, correlate with the
increased level of in Vitro metabolism. We have tentatively
assigned the structure of the reactive intermediate leading to
covalent protein labeling as the iminium cation 19 (Figure 5).
Interestingly, while antagonist 13 is metabolized to a similar

800 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Sturino et al.
Table 7. DP Receptor Selectivities of DP Antagonistsa
receptor binding
functional
DP Ki
(nM)
TP Ki
(nM)
selectivity
(DP vs TP)
DP PRP
TP PRP
selectivity
(DP vs TP)
compd
IC50b (nM)
IC50c (nM)
6
7
11
12
13
2.6 ( 0.7
1.8 ( 0.7
1.5 ( 0.57
1.1 ( 0.22
0.57 ( 0.17
1200 ( 470
7100 ( 820
0.84 ( 0.19
14 ( 2.7
460
3900
0.56
13
7.9 ( 2.8
8.6 ( 4.3
23 ( 9.5
15 ( 0.41
4.0 ( 0.9
8400 ( 4700
43000 ( 16000
80 ( 60
1400 ( 1000
770 ( 490
1100
5100
3.5
93
192
2.95 ( 0.46
5.2
a Values are means from at least three experiments. b DP PRP assay: inhibition of the accumulation of cAMP in platelet-rich plasma challenged with
PGD2. c TP PRP assay: the inhibition of U44619-induced platelet aggregation in platelet-rich plasma.
Table 8. Effect of 13 in Inhibiting Nasal Airway Resistance Induced by
where it inhibits the PGD2-induced cAMP production in platelet-
rich plasma with an IC50 of 4.0 nM. Antagonist 13 was also
Intranasal Instillation of PGD2 in Conscious Sheepa
inhibition of nasal
airway resistance (%)
plasma levels of
shown to display an excellent rat pharmacokinetic profile.
Antagonist 13 was also efficacious in a sheep model of allergic
rhinitis.
dose (mg/kg)
antagonist 13 (nM)b,c
0.03
0.1
26 ( 10.7
99 ( 0.7
91 (range 37-152 nM)
670 (range 103-1260 nM)
As part of the medicinal chemistry effort leading to the
discovery of 13, the initial lead antagonists 6 and 7 were shown
to display poor pharmacokinetic profiles. Rat bile duct cannu-
lation studies demonstrated that the rapid clearance of these
antagonists was likely a result of active hepatic excretion of
parent drug into the biliary fluid. The pharmacokinetic issues
with the 7-methylsulfonyl antagonists 6 and 7 were effectively
circumvented by replacing the methylsulfone substituent with
a fluorine atom. Reintroduction of the methylsulfone at the
5-position of the indole led to the discovery of 13. Several key
observations noted during the discovery of 13 were that
apparently subtle changes in the substitution pattern of a
molecule can result in significant pharmacokinetic effects. Also,
while in Vitro metabolic studies are invaluable in drug discovery,
it is equally important to augment this data with in ViVo
pharmacokinetic/metabolism studies to help understand the
clearance mechanism of a drug candidate. Thus, based on these
observations, the strategy we employed to circumvent a biliary
excretion/poor pharmacokinetic issue with our lead DP antago-
nist was to introduce modifications at different regions of the
molecule and examine the effect of these changes on the PK
profile directly. In doing so, we were able to identify analogs
with significantly reduced biliary excretion. In addition, it was
observed that the extent of covalent protein binding can be
attenuated, at least in the context of the current indole series,
by the introduction of a powerful electron-withdrawing sub-
stituent such as a methylsulfone on the indole core. The PGD2
receptor antagonist 13 has been studied in a clinical model of
allergic rhinitis, and the results of these trials will be reported
in due course.
a Values are the means of at least three experiments. b Plasma levels
determined at time of intranasal PGD2 challenge. c Sheep DP PRP IC50
)
1.4 ( 0.2 nM (n ) 3).
competition binding assay versus the functional assay was likely
the result of the limitation in the dynamic range of the DP
receptor binding assay. The DP and TP binding affinities for
the enantiomer of 13 (20) were found to be 9.6 and 1050 nM
respectively. Thus while 20 remains a relatively potent DP
antagonist, it is approximately 320-fold less potent than 13.28
In addition, antagonist 13 is also selective for the CRTH2
receptor (a recently identified second PGD2 receptor29), display-
ing a Ki of 745 ( 319 nM.
In WiWo Efficacy of 13. The potent DP antagonist 13 was
evaluated in a conscious sheep model of PGD2-induced conges-
tion. The sheep model was selected as an in vivo model to
evaluate the DP antagonists since sheep have been extensively
used to study lower-airway disorders and this model has recently
been adapted to examine upper-airway disorders such as allergic
rhinitis.30 The potency (IC50) of 13 on the sheep DP receptor in
the platelet-rich plasma (PRP) assay was measured to be 1.4 (
0.2 nM, comparable to the potency on the human receptor.
Having established that antagonist 13 is active on the sheep
DP receptor, it was then evaluated in the sheep allergic rhinitis
model. Thus, antagonist 13 was administered intravenously, and
30 min later the sheep were challenged with an intranasal
instillation of PGD2 and changes in nasal airflow resistance were
measured by rhinomanometry. As summarized in Table 8, while
only a modest level of inhibition (26%) was observed at a dose
of 0.03 mg/kg, a dose of 0.1 mg/kg of antagonist 13 provided
complete (99%) blockade of the PGD2 induced nasal congestion.
The plasma levels of antagonist 13 at the time of PGD2 challenge
are provided in Table 8 and are well above the PRP IC50 value
for antagonist 13. It is worth noting that these plasma drug levels
may not reflect the drug levels at the site of action, that is, the
nasal vasculature. Thus, in addition to being a potent DP receptor
antagonist in Vitro, 13 is also effective in a sheep model of nasal
congestion.
Experimental Procedures
General Experimental Methods. Melting points are uncor-
rected. NMR spectra were recorded in the indicated solvent with
1
the protonated form as the internal standard for H (400 MHz or
500 MHz) and for 13C (100 MHz or 125 MHz). TLC analyses were
performed on Merck Kieselgel 60 F254 plates. All air-sensitive
reactions carried out under a nitrogen atmosphere. Column chro-
matography was performed with silica gel 230-400 mesh. El-
emental analyses were performed by Prevalere Life Sciences, Inc.,
Whitesboro, NY. The LC/MS and LC/MS/MS studies were
performed using a PE Sciex API 2000 instrument. The HPLC-UV
analyses were performed using a Waters 2690 HPLC system
equipped with a photodiode array detector. For the rat pharmaco-
kinetic and bile duct cannulation studies, all aspects of housing,
care, and use of the animals were in accordance with the guidelines
of the Canadian Council on Animal Care and the “Guide to the
Care and Use of Experimental Animals”.
Conclusion
In summary, we have presented the discovery of 1331, a potent
and selective DP receptor antagonist. This antagonist has a high
affinity for the DP receptor with a competition binding affinity
constant (Ki) of 0.57 nM and a dissociation constant (Kd) of
0.03 nM. Antagonist 13 is 192-fold selective for the thrombox-
ane receptor based on a ratio of Kd values at the DP and TP
receptors. Antagonist 13 is also potent in a DP functional assay
Pharmacokinetic Studies. Sprague-Dawley rats were fasted
overnight and prior to dosing with test compound given either by

DiscoVery of MK-0524
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 801
oral gavage or intravenously. At various time points, blood samples
were collected by tail bleed, and this blood was centrifuged to
furnish plasma samples. The plasma was stored at -10 °C until
time of analysis. At this time, plasma proteins were precipitated
by treating 100 µL of the plasma samples with 100 µL of
acetonitrile. The samples thus obtained were vortexed and centri-
fuged. The resulting supernatants were transferred into sample vials
and subsequently analyzed by LC/MS/MS. Reference standard
samples for each test compound were prepared as described above
using plasma derived from the blood of untreated rats and
subsequently spiked with known concentrations of test compound
typically ranging from 0.01 to 10 µg/mL.
Bile Duct Cannulation Studies. The bile duct cannulation
studies were performed as described in ref 23. Male Sprague-
Dawley rats were obtained from Charles River Laboratories (St.
Constant, Quebec, Canada) and were acclimatized for at least 48 h
before use. The rats (∼350 g) were anesthetized with ketamine (75
mg/kg) and xylazine (5 mg/kg) throughout the duct cannulation
procedure. Blank bile fluid was collected for a period of 30 min
prior to drug treatment. The test compounds were then administered
intravenously in the jugular vein at a dose of 5 mg/kg in either 5%
dextrose or 60% PEG 200 with a dose volume of 1 mL/kg. Bile
samples were then collected in 30 min intervals and kept at -10
°C before analysis. The bile samples were processed as follows:
100 µL of bile fluid was treated with 100 µL of CH3CN and 200
µL of water. The samples were then vortexed and centrifuged. The
resulting supernatants were transferred into sample vials and
analyzed by LC-UV. Reference samples were prepared using blank
bile fluid following the procedure described above and spiking with
known concentrations of test compound.
Receptor Binding and DP and TP Functional Assays. The
receptor binding assays were performed as described in ref 19. The
DP functional assays were performed as follows: Blood was
collected from normal human volunteers, free from medication for
two weeks, by venous puncture. The blood was collected in
Vacutainers without additive and transferred immediately into 50
mL tubes containing 10% (v/v) citrate buffer (65 mM citric acid,
85 mM sodium citrate, 2% (v/v) glucose) and then mixed by
inversion. Platelet-rich plasma (PRP) was prepared by centrifugation
at 150 × g for 15 min. The PRP was removed and transferred into
a clean tube. Half of the resulting PRP fraction from each donor
was used to prepare washed platelets (WP). Citrate buffer (30%
v/v) and 50% v/v of 25 mM HEPES, pH 7.4, Hanks’ balanced salt
solution (HBSS) without Ca2+ or Mg2+ were added to PRP, and
the mixture was centrifuged for 10 min at 800 × g. The cell pellet
was resuspended in 1/10th of the original blood volume in 25 mM
HEPES, pH 7.4, HBSS with Ca2+ and Mg2+ containing 10% (v/v)
citrate buffer, centrifuged, and resuspended as above. The cells were
recentrifuged a third time as above and resuspended in 5 mL of 25
mM HEPES, pH 7.4, HBSS without Ca2+ or Mg2+. Platelet cell
counting was determined in a Beckman Z-2 Coulter counter. Platelet
cell counts in the PRP and WP assays were adjusted to 2.5 × 108
cells/mL using 25 mM HEPES, HSBB without Ca2+ or Mg2+. The
WP and PRP assays were conducted as follows: isobutylmethyl-
xanthine (IBMX; 2 mM final concentration) was added to human
WP or PRP to prevent degradation of cAMP. Samples (100 µL) of
either human WP or PRP were then preincubated (10 min at 37
°C) with increasing concentrations of test compound in DMSO [1%
(v/v) final in assay, kept constant in all samples]. Samples were
then challenged with PGD2 (300 nM final) added in DMSO (kept
constant at 1% (v/v) throughout) and incubated for an additional 2
min at 37 °C. PGD2 was not metabolized during the 2 min
incubation period as determined by HPLC-MS analysis. The
reaction was then terminated by the addition of 200 µL of ethanol
to disrupt the cells and extract the cAMP. The samples were mixed
thoroughly and centrifuged at 1400 × g for 10 min at 4 °C.
Supernatant aliquots (100 µL) were removed, and the ethanol was
removed by evaporation. cAMP was measured by [125I]-cAMP
scintillation proximity assay (SPA) (RPA556, Amersham) according
to the manufacturer’s instructions following reconstitution of the
samples in SPA buffer. cAMP accumulation assays were conducted
using matched WP and PRP from individual donors.
The TP functional assays were performed as follows: Test
compounds were assessed for their ability to inhibit platelet
aggregation in human platelet-rich plasma (PRP) using aggrego-
metry. Citrated blood was collected from donors who were
medication free for at least two weeks. After gentle mixing of the
blood and citrate, samples were centrifuged at 150 × g for 15 min
at rt and the upper phase was removed in order to obtain PRP.
Platelet poor plasma (PPP) was prepared by centrifugation of the
lower phase after PRP removal at 2000 × g for 10 min at rt. Platelet
concentrations were adjusted to 2.5 × 108 platelet/mL using PPP.
The adjusted PRP (250 µL) was stirred in a cuvette at 37 °C for 3
min, and 25 µL of the test compound diluted in buffer [0.9% (w/v)
or 0.15 M NaCl containing 0.25% (w/v) BSA] or vehicle (buffer),
as control, was added to 250 µL of stirring platelets for a 10 min
preincubation period. Agonist (20 µL, 1.36 µM U46619,32 final
concentration, diluted buffer) was then added to the stirring PRP
mixture to induce aggregation, and the incubation continued for 5
min. Maximium aggregation was determined at approximately 5
min. The maximum extent of aggregation achieved in the presence
of U46619 was set to 100%.
Ethyl [7-(Methylsulphonyl)-1,2,3,4-tetrahydrocyclopenta[b]-
indol-3-yl]acetate (2). To a vigorously stirred solution 4-(meth-
ylsulfonyl)-phenylamine (100 g, 584 mmol) dissolved in EtOH (5.5
L) at 50 °C was added a mixture of iodine (49.3 g, 194 mmol) and
silver sulfate (110 g, 353 mmol) in EtOH (1 L). The addition of
iodine and silver sulfate was repeated twice more after 1 and 2 h
of stirring, and the resulting mixture was allowed to stir overnight.
The hot solution was then filtered through Celite, and the solvent
was removed under vacuum. The residue was triturated with warm
(50 °C) EtOH (1 L) for 45 min and then cooled to 0 °C. The
resulting product was filtered and collected to provide 2-iodo-4-
1
(methylsulfonyl)phenylamine as a brown solid (140 g, 80%). H
NMR (acetone-d6): δ 8.08 (d, J ) 2.0 Hz, 1H), 7.63 (dd, J ) 8.5
and 2.0 Hz, 1H), 6.93 (d, J ) 8.5 Hz, 1H), 5.78 (br s, 2H), 3.03 (s,
3H). 13C NMR (acetone-d6): δ 153.6, 139.3, 130.6, 129.7, 114.0,
81.0, 44.8. Mp 201-201 °C. MS (+APCI) m/z 298.2 (M + H)+.
Anal. (C7H8NO2SI) C, H, N.
A solution of 2-iodo-4-(methylsulfonyl)phenylamine (150 g, 503
mmol) and PPTS (4.8 g, 19.1 mmol) in DMF (3.0 L) was degassed
and kept under a N2 atmosphere, and tetraethoxysilane (135 g, 640
mmol) and ethyl 2-(2-oxocyclopentyl)acetate (129 g, 758 mmol)
were then added. The final mixture was heated to 130-140 °C
and stirred for 6 h. Then, DMF (300 mL) was added, and the
solution was degassed before Hunig’s base (270 mL, 1550 mmol)
followed by Pd(OAc)2 (3.4 g, 15.1 mmol) were added successively.
The solution was heated to 120 °C for 2 h and then cooled to rt. At
this time, a 1 N HCl (300 mL) solution and isopropyl acetate (200
mL) were added, and the resulting mixture was filtered through
Celite. The phases were separated, and the acidic phase was
extracted twice with isopropyl acetate (200 mL). The organic layers
were combined, washed with brine, dried over anhydrous Na2SO4,
filtered through Celite, and concentrated. The crude material was
further purified by flash chromatography, eluting with 50% EtOAc
in hexanes, to provide ethyl [7-(methylsulphonyl)-1,2,3,4-tetrahy-
drocyclopenta[b]indol-3-yl]acetate as a yellow solid (63 g, 39%).
1H NMR (acetone-d6): δ 10.23 (br s, 1H), 7.98 (s, 1H), 7.50-
7.50 (m, 2H), 4.14 (q, J ) 7.1 Hz, 2H), 3.70-3.58 (m, 1H), 3.04
(s, 3H), 2.90-2.65 (m, 5H), 2.20-2.10 (m, 1H), 1.22 (t, J ) 7.1
Hz, 3H). MS (+APCI) m/z 322.2 (M + H)+.
[5-Bromo-4-(4-chlorobenzyl)-7-(methylsulfonyl)-1,2,3,4-tet-
rahydrocyclopenta[b]indol-3-yl]acetic Acid (3). To a solution of
2 (61.5 g, 192 mmol) in THF (550 mL) at rt, MeOH (275 mL),
followed by 2 N NaOH (275 mL, 550 mmol), was added. The
reaction was heated to 60 °C for 15 min, cooled to rt, and then
poured into a separatory funnel containing EtOAc/1 N HCl. The
phases were separated, and the acidic phase was extracted twice
with EtOAc. The organic layers were combined, washed with brine,
dried over anhydrous Na2SO4, and evaporated to dryness. The crude
solid was triturated in EtOAc/hexanes to give [7-(methylsulfonyl)-

802 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Sturino et al.
1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]acetic acid as a faint
brown solid, 56.0 g (quantitative yield). H NMR (acetone-d6): δ
10.86 (br s, 1H), 10.25 (br s, 1H), 7.98 (s, 1H), 7.58 (m, 2H), 3.62
(m, 1H), 3.04 (s, 3H), 2.89-2.68 (m, 5H), 2.21 (m, 1H); 13C NMR
(DMSO-d6): δ 173.7, 149.6, 143.6, 131.4, 123.6, 119.3, 118.6,
118.5, 112.8, 45.1, 39.4, 36.0, 35.5, 23.4. Mp 208 °C. MS (+APCI)
m/z 294.0 (M + H)+. Anal. (C14H15NO4S) C, H, N.
nyltributyltin (10.8 g, 29.8 mmol). The resulting mixture was
degassed by bubbling N2 through the solution for several minutes.
In a separate flask was placed Pd2(dba)3 (1.04 g, 1.14 mmol) and
Ph3As (2.8 g, 9.2 mmol) along with DMF (100 mL), and this
mixture was sonicated for 10 min to provide a fine suspension.
The resulting catalyst mixture was then introduced into the above
reaction flask, which was heated at 80 °C for 3 h. After the reaction
was allowed to cool to rt, a 3 N HCl solution (100 mL) was added
to the reaction flask, and the mixture was allowed to stir until TLC
analysis indicated the consumption of the vinyl ether adduct. The
reaction mixture was diluted with water, extracted with ethyl acetate,
dried over anhydrous Na2SO4, and concentrated. The resulting
material was further purified by flash chromatography, eluting with
15% ethyl acetate in hexanes followed by recrystallization from
20% ethyl acetate in hexane, to provide methyl [5-acetyl-4-(4-
chlorobenzyl)-7-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]in-
1
Pyridinium tribromide (154 g, 481.5 mmol) was added to a
solution of 2-[7-(methyl-sulfonyl)-1,2,3-trihydrocyclopenta[2,3-b]-
indol-3-yl]acetic acid (50.8 g, 173.1 mmol) in pyridine (345 mL)
at -25 to -30 °C; the solution was warmed to 0 °C for 15 min
and then to rt for 30 min. At this time, a 1:1 solution of THF/ether
(1.25 L) and a 1:1 brine/6 N HCl solution (2.5 L) were added, the
phases were separated, the aqueous layer was washed with 1:1 THF/
ether, and the combined organic layers were dried with Na2SO4.
The organic phase was cooled to 10 °C, and acetic acid (50.5 mL)
was added followed by a slow addition of zinc dust (70.2 g, 1074
mmol) while maintaining the temperature below 15 °C. The reaction
mixture was then stirred for 1 h at rt. To this mixture, 1 N HCl (3
L) and EtOAc (1.25 L) were added; the phases were separated,
and the aqueous layer was extracted with EtOAc (2 L). The
combined organic layers were dried with Na2SO4, and the solvent
was removed under vacuum. The resulting brownish powder was
stirred with a 20% EtOAc/hexanes solution (1000 mL) to furnish
[5-bromo-4-(4-chlorobenzyl)-7-(methylsulfonyl)-1,2,3,4-tetrahydro-
cyclopenta[b]indol-3-yl]acetic acid (52 g, 81%). 1H NMR (acetone-
d6): δ 11.05 (br s, 1H), 10.37 (br s, 1H), 8.00 (s, 1H), 7.76 (d, J
) 1.2 Hz, 1H), 3.66 (m, 1H), 3.13 (s, 3H), 3.00-2.75 (m, 4H),
2.62 (dd, J ) 16.5 and 8.2 Hz, 1H,), 2.27 (m, 1H). 13C NMR
(DMSO-d6): δ 173.3, 150.2, 140.7, 132.5, 124.5, 120.4, 120.3,
117.7, 104.3, 44.3, 38.5, 35.4, 35.2, 23.1. MS (-APCI) m/z 372.2,
370.2 (M - H)-. Anal. (C14H14BrNO4S) C, H, N.
1
dol-3-yl]acetate as a white solid (6.3 g, 66%). H NMR (CDCl3):
δ 8.22 (d, J ) 1.8 Hz, 1H), 7.89 (d, J ) 1.8 Hz, 1H), 7.31-7.28
(m, 2H), 6.77-6.74 (m, 2H), 5.48 (s, 2H), 3.83-3.79 (m, 1H),
3.62 (s, 3H), 3.15 (s, 3H), 3.09-3.00 (m, 1H), 2.96-2.90 (m, 2H),
2.88-2.79 (m, 1H), 2.60 (dd, J ) 16.0 and 9.7 Hz, 1H), 2.41-
2.35 (m, 1H), 2.20 (s, 3H). 13C NMR (acetone-d6): δ 199.3, 171.6,
152.8, 137.5, 136.3, 132.6, 132.0, 128.7, 128.3, 127.4, 126.7, 121.8,
120.6, 119.6, 50.9, 49.8, 44.0, 38.1, 35.6, 35.3, 28.1, 22.6. MS
(+APCI) m/z 476.1, 474.1 (M + H)+.
The above ester (2.08 g, 4.4 mmol) was hydrolyzed following
the procedure outlined for compound 3 to yield [5-acetyl-4-(4-
chlorobenzyl)-7-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]in-
1
dol-3-yl]acetic acid (1.99 g, 99%) as a brownish solid. H NMR
(acetone-d6): δ 10.76 (br s, 1H), 8.19 (d, J ) 1.7 Hz, 1H), 7.87
(d, J ) 1.7 Hz, 1H), 7.27 (d, J ) 8.5 Hz, 2H), 6.73 (d, J ) 8.4 Hz,
2H), 5.48 (s, 2H), 3.79 (m, 1H), 3.12 (s, 3H), 3.05 (m, 1H), 3.00-
2.70 (m, 3H), 2.55 (dd, J ) 16.2 and 9.9 Hz, 1H), 2.38 (m, 1H),
2.19 (s, 3H). 13C NMR (acetone-d6): δ 199.3, 172.3, 153.0, 137.5,
136.4, 132.6, 131.7, 128.7, 128.3, 127.4, 126.7, 121.8, 120.6, 119.6,
49.8, 44.0, 38.1, 35.6, 35.4, 28.1, 22.6. Mp 152-155 °C. MS
(-ESI) m/z 459.8, 457.7 (M - H)-.
Methyl [5-Bromo-4-(4-chlorobenzyl)-7-(methylsulfonyl)-1,2,3,4-
tetrahydrocyclopenta[b]indol-3-yl]acetate (4). Acid 3 (12.0 g,
24.1 mmol) in THF (450 mL) was esterified with an ether solution
of CH2N2. After removal of the solvents, methyl [5-bromo-7-
(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]ace-
1
[(3R)-5-Acetyl-4-(4-chlorobenzyl)-7-(methylsulfonyl)-1,2,3,4-
tetrahydrocyclopenta[b]indol-3-yl]acetic Acid (6). (()-[5-Acetyl-
4-(4-chlorobenzyl)-7-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta-
[b]indol-3-yl]acetic acid (5) dissolved in EtOH was resolved using
normal phase preparative chiral HPLC [ChiralPak AD column, 50
cm × 5 cm, 20 µm; mobile phase hexane/2-propanol/acetic acid
(70:30:0.4); flow 70-75 mL/min; pressure 280-300 psi; UV 265
nm]. Retention times of the two enantiomers were 38 and 58 min.
The title (R) isomer (retention time 58 min) was thus obtained with
a 96.7% ee. Retention time ) 15.3 min [ChiralPak AD column
250 mm × 4.6 mm, hexane/2-propanol/acetic acid (75:25:0.1)].
tate was obtained as a pale brown solid (12.5 g, quantitative). H
NMR (acetone-d6): δ 10.41 (br s, 1H), 8.03 (d, J ) 1.0 Hz, 1H),
7.79 (d, J ) 1.5 Hz, 1H), 3.71 (s, 3H), 3.70-3.68 (m, 1H), 3.27
(s, 3H), 3.14-2.92 (m, 2H), 2.86-2.82 (m, 2H), 2.64 (dd, J )
16.2 and 8.3 Hz, 1H), 2.29-2.23 (m, 1H). 13C NMR (acetone-d6):
δ 172.7, 149.7, 141.1, 133.5, 125.0, 121.6, 121.0, 118.2, 104.3,
51.1, 44.3, 38.3, 35.52, 35.48, 23.1. Mp 128-130 °C. MS (-APCI)
m/z 386.0, 384.0 (M - H)-; Anal. (C15H16BrNO4S) C, H, N.
To a solution of the above ester (8.8 g, 22.8 mmol) in DMF (90
mL) at -78 °C was added NaH (60% in oil, 790 mg, 19.8 mmol).
The resulting suspension was stirred for 20 min at 0 °C, cooled
again to -40 to - 50 °C and treated with 4-chloro-benzyl bromide
(5.60 g, 27.3 mmol). After 5 min, the temperature was warmed to
0 °C, and the solution was stirred for 20 min. At this time, the
reaction mixture was quenched by the addition of AcOH (2.6 mL),
and this mixture was poured into a separatory funnel containing 1
N HCl/EtOAc. The layers were separated, and the organic layer
was washed with brine, dried over anhydrous Na2SO4, and
concentrated. The crude material was further purified by flash
chromatography eluting with 10% EtOAc in toluene to provide
methyl [5-bromo-4-(4-chlorobenzyl)-7-(methylsulfonyl)-1,2,3,4-tet-
rahydrocyclopenta[b]indol-3-yl]acetate as a white solid (10.1 g,
89%). 1H NMR (acetone-d6): δ 8.06 (d, J ) 1.6 Hz, 1H), 7.76 (d,
J ) 1.6 Hz, 1H), 7.36 (d, J ) 8.5 Hz, 2H), 6.98 (d, J ) 8.4 Hz,
2H), 6.00 (d, JAB ) 17.8 Hz, 1H), 5.89 (d, JAB ) 17.8 Hz, 1H),
3.71-3.67 (m, 1H), 3.59 (s, 3H), 3.17 (s, 3H), 3.05-2.97 (m, 1H),
2.92-2.80 (m, 2H), 2.69 (dd, J ) 16.2 and 3.8 Hz, 1H), 2.48 (dd,
J ) 16.2 and 9.8 Hz, 1H), 2.35-2.28 (m, 1H). 13C NMR (acetone-
d6): δ 171.6, 152.3, 138.8, 138.2, 133.8, 132.5, 128.7, 127.2, 126.9,
123.9, 121.3, 118.5, 103.4, 50.9, 48.6, 44.0, 37.9, 35.5, 35.3, 22.5.
Mp 108-111 °C. MS (+APCI) m/z 512.0, 510.0 (M + H)+.
[5-Acetyl-4-(4-chlorobenzyl)-7-(methylsulfonyl)-1,2,3,4-tet-
rahydrocyclopenta[b]indol-3-yl]acetic Acid (5). To a solution of
4 (10.0 g, 29.9 mmol) in DMF (100 mL) was added 1-ethoxyvi-
[R]2D1 ) -10.9° (c 0.45, MeOH). H NMR (acetone-d6): δ 10.80
1
(br s, 1H), 8.22 (d, J ) 1.7 Hz, 1H), 7.89 (d, J ) 1.7 Hz, 1H), 7.30
(d, J ) 8.5 Hz, 2H), 6.76 (d, J ) 8.4 Hz, 2H), 5.50 (s, 2H), 3.83-
3.78 (m, 1H), 3.15 (s, 3H), 3.11-3.03 (m, 1H), 2.97-2.90 (m,
2H), 2.85-2.80 (m, 1H), 2.58 (dd, J ) 16.1 and 9.9 Hz, 1H), 2.45-
2.39 (m, 1H), 2.20 (s, 3H). 13C NMR (acetone-d6): δ 199.3, 173.0,
153.0, 137.3, 136.4, 132.6, 131.7, 128.7, 128.3, 127.4, 126.7, 121.8,
120.6, 119.6, 49.8, 44.9, 38.1, 35.6, 35.4, 28.1, 22.6. Mp 219 °C.
MS (-APCI) m/z 460.5, 458.3 (M - H)-. Anal. (C23H22ClNO5S)
C, H, N, S.
[(3R)-4-(4-Chlorobenzyl)-5-(1-hydroxyethyl)-7-(methylsulfo-
nyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]acetic acid (7). In
a dry flask was placed (-)-[(3R)-5-acetyl-4-(4-chlorobenzyl)-7-
(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]acetic acid
(350 mg, 0.76 mmol) along with MeOH (30 mL). To this stirred
solution was added NaBH4 portion-wise (ca. 50 mg/portion) in 10-
15 min intervals until the consumption of ketone was noted by TLC
analysis. At this time, the reaction mixture was poured into a
separatory funnel containing saturated aqueous NH4Cl solution (100
mL), a 1 N HCl solution (10 mL), and EtOAc (100 mL). The layers
were separated, and the aqueous layer was extracted with EtOAc;
the combined organic layers were dried over anhydrous Na2SO4
and concentrated. The resulting material was purified by chiral

DiscoVery of MK-0524
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 803
HPLC as follows. The mixture of alcohols dissolved in hot EtOH
was resolved using normal phase preparative chiral HPLC [Chiral-
Pak AD column 50 cm × 5 cm, 20 µm; mobile phase hexane/2-
propanol/acetic acid (80:20:0.4); flow 70-75 mL/min; pressure
280-300 psi; UV 245 nm]. Retention times of the two diastereo-
isomers were 33 and 51 min. The title compound (retention time
33 min) was obtained with >99% de; ee ) 99%. Retention time
) 6.0 min [ChiralPak AD column 250 mm × 4.6 mm, hexane/2-
propanol/acetic acid (75:25:0.2)]. [R]2D1 ) +7.6° (c 1.0, MeOH).
1H NMR (acetone-d6): δ 7.97 (s, 1H), 7.86 (s, 1H), 7.35 (d, J )
8.2 Hz, 2H), 6.90 (d, J ) 8.2 Hz, 2H), 5.92 (d, JAB ) 18.1 Hz,
1H), 5.68 (d, JAB ) 18.1 Hz, 1H), (q, J ) 5.19 Hz, 1H), 3.63-
3.55 (m, 1H), 3.09 (s, 3H), 3.08-2.91 (m, 1H), 2.89-2.79 (m,
2H), 2.70-2.65 (m, 1H), 2.46-2.34 (m, 2H), 1.49 (d, J ) 6.3 Hz,
3H). 13C NMR (acetone-d6): δ 173.3, 152.0, 141.1, 139.4, 133.3,
133.2, 132.6, 129.7, 127.8, 126.4, 121.7, 118.8, 117.3, 64.6, 50.3,
45.0, 39.1, 36.5, 36.2, 24.6, 23.5. MS (-APCI) m/z 462.8, 460.5
(M - H)-. Mp 212 °C. Anal. (C23H24ClNO5S) C, H, N, S.
Ethyl (7-Fluoro-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)-
acetate (9). To a mixture of 4-fluoroaniline (36 mL, 380 mmol) in
water (1 L) and toluene (30 mL) was added NaHCO3 (50.4 g, 599
mmol) and iodine (75.4 g, 298 mmol) over a period of 30 min.
The resulting reaction was then stirred at rt for 30 min. At this
time, the reaction was poured into water, and the pH was adjusted
to 6-7 with an HCl solution. The aqueous layer was extracted with
Et2O (2×), washed with 5% Na2S2O3 solution (2×), and washed
with brine. The resulting solution was dried over anhydrous Na2-
SO4, filtered over a plug of silica gel, and concentrated. The
resulting material was distilled under high vacuum (bp 74-76 °C)
to yield 4-fluoro-2-iodoaniline as an orange oil (40.2 g, 57%). This
material was immediately used in the next reaction. 1H NMR
(CDCl3): δ 7.35 (dd, J ) 7.9 and 2.6 Hz, 1H), 6.90-6.85 (m,
1H), 6.68-6.65 (m, 1H), 3.92 (br s, 2H).
9.75 (br s, 1H), 7.34 (dd, J ) 8.8 and 4.6 Hz, 1H), 7.04 (dd, J )
9.9 and 2.4 Hz, 1H), 6.79 (m, 1H), 3.65-3.56 (m, 1H), 2.87-2.61
(m, 5H), 2.21-2.11 (m, 1H). 13C NMR (acetone-d6): δ 174.0, 157.6
(d, JCF ) 231 Hz), 148.2, 137.8, 124.7 (d, JCF ) 9.3 Hz), 118.4 (d,
JCF ) 3.5 Hz), 112.5 (d, JCF ) 9.7 Hz), 107.9 (d, JCF ) 26.1 Hz),
103.0 (d, JCF ) 23.4 Hz), 38.9, 35.7, 35.3, 22.9; MS (-APCI) m/z
232.2 (M - H)-.
Following the procedure described for compound 3, bromination
of the above acid (127 g, 540 mmol) yielded (5-bromo-7-fluoro-
1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)acetic acid as a brown
1
solid (117 g, 69%). H NMR (acetone-d6): δ 10.84 (br s, 1H),
9.84 (br s, 1H), 7.16-7.09 (m, 2H), 3.67-3.60 (m, 1H), 2.91-
2.71 (m, 4H), 2.61 (dd, J ) 16.4 and 8 Hz, 1H), 2.27-2.19 (m,
1H). 13C NMR (acetone-d6): δ 173.6, 157.0 (d, JCF ) 236 Hz),
149.3, 136.0, 125.3 (d, JCF ) 10.3 Hz), 120.1 (d, JCF ) 4.6 Hz),
110.8 (d, JCF ) 29.2 Hz), 103.4 (d, JCF ) 12.8 Hz), 102.9 (d, JCF
) 23.2 Hz), 38.4, 35.5, 35.3, 23.0. MS (-APCI) m/z 311.8, 309.8
(M - H)-; Anal. (C13H11BrNO2) C, H, N.
Esterification of the above acid (74.1 g, 240 mmol) with HCl in
methanol and alkylation with 4-chlorobenzyl bromide (60 g, 290
mmol) followed by ester hydrolysis, as described previously for
compound 4, provided [5-bromo-4-(4-chlorobenzyl)-7-fluoro-
1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]acetic acid as a brown
solid (74.1 g, 53% over the three steps). 1H NMR (acetone-d6): δ
10.70 (br s 1H), 7.33 (d, J ) 8.4 Hz, 2H), 7.20 (dd, J ) 8.9 and
2.3 Hz, 1H), 7.09 (dd, J ) 9.0 and 2.3 Hz, 1H), 6.95 (d, J ) 8.3
Hz, 2H), 5.92 (d, JAB ) 17.8 Hz, 1H), 5.77 (d, JAB ) 17.8 Hz,
1H), 3.65-3.60 (m, 1H), 3.00-2.65 (m, 4H), 2.42 (dd, J ) 16.1
and 10.1 Hz, 1H), 2.32-2 (m, 1H). 13C NMR (acetone-d6): δ 172.1,
156.9 (d, JCF ) 237.2 Hz), 151.9, 138.7, 134.1, 132.3, 128.6, 127.5
(d, JCF ) 10.2 Hz), 127.2, 119.7 (d, JCF ) 4.5 Hz), 113.6 (d, JCF
) 28.9 Hz), 103.4 (d, JCF ) 22.6 Hz), 102.9 (d, JCF ) 12.1 Hz),
48.4, 38.0, 35.5, 35.2, 22.4. MS (-APCI) m/z 436.3, 434.5 (M -
H)-.
Following the procedure outlined for compound 2, the 4-fluoro-
2-iodoaniline (10.0 g, 42.2 mmol) was treated with ethyl-2-(2-
oxocyclopentyl)acetate (6.57 g, 38.6 mmol) to yield ethyl (7-fluoro-
1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)acetate as a yellow solid
[(3R)-5-Bromo-4-(4-chlorobenzyl)-7-fluoro-1,2,3,4-tetrahydro-
cyclopenta[b]indol-3-yl]acetic Acid (11). A suspension of [5-bromo-
4-(4-chlorobenzyl)-7-fluoro-1,2,3,4-tetrahydrocyclopenta[b]indol-
3-yl]acetic acid (35.3 g, 81 mmol), (S)-(-)-1-(1-naphthyl)ethylamine
(13.8 g, 81 mmol), and methanol (650 mL) was heated to reflux in
an oil bath and mechanically stirred for 30 min. The resulting
suspension was filtered while hot to give 35 g of a white solid
consisting of the salt of the desired/undesired enantiomers in 10.5:1
ratio. This material was then heated to reflux in methanol (1 L) for
3 h, and then the suspension was filtered while hot to give 26 g of
the corresponding salt. A sample of this material was neutralized
and was found to have an enantiomeric purity of 96.9% ee by HPLC
analysis [ChiralCel OD column, hexane/2-propanol/acetic acid (95:
5:0.1), retention times were 9.6 and 14.6 min (desired)]. Recrys-
tallization of the filtrate from above in ethanol (2.8 L) afforded
21.8 g of a salt, which was treated with 1 N HCl and extracted
with ethyl acetate to yield the title enantiomer as a white solid (15.0
1
(5.3 g, 53%). H NMR (acetone-d6): δ 9.76 (br s, 1H), 7.34 (dd,
J ) 8.8 and 4.6 Hz, 1H), 7.03 (d, J ) 9.9 Hz, 1H), 6.78 (td, J )
9.5 and 2.3 Hz, 1H), 4.14 (q, J ) 7.1 Hz, 2H), 3.58-3.54 (m, 1H),
2.85-2.55 (m, 5H), 2.16-2.12 (m, 1H), 1.22 (t, J ) 7.1 Hz, 3H).
Alternative Indole Synthesis. To a flask equipped with a Dean-
Stark apparatus was added 2-bromo-4-fluoroaniline (500 g, 2.63
mmol), toluene (5 L), ethyl 2-(2-oxocyclopentyl)acetate (448 g, 2.63
mmol), and p-TsOH‚H2O (3.3 g, 17.3 mmol). The resulting mixture
was stirred with the aid of a mechanical stirrer and heated to reflux
for 20 h. At this time, the reaction was cooled to rt and concentrated.
The resulting material was treated with DMF (5 L), KOAc (774 g,
n
7.89 mmol), Bu4NCl (775 g, 2.63 mmol), and Pd(OAc)2 (17.7 g,
78.9 mmol), evacuated under house vacuum, and flushed with
nitrogen three times. The mixture was then heated to 80 °C for 1
h. The resulting reaction was poured slowly into a mixture of H2O
(20 L) and EtOAc (10 L) and stirred; the layers were separated,
and the organic layer was washed with H2O (2 × 10 L), dried over
anhydrous Na2SO4, and concentrated. The resulting oil was purified
by flash chromatography, eluting with 5-10% EtOAc/hexane.
Concentration of the chromatography fractions that contained the
desired product provided a solid material that was subsequently
stirred with hexanes and filtered, and the residue was washed with
hexanes to yield 210 g of a white solid. The hexane washings were
combined, washed with 1 N HCl, dried over anhydrous Na2SO4,
concentrated, and heated to 75 °C under high vacuum. The resulting
solid was purified by flash chromatography as described above to
provide an additional 42.0 g of a white solid. The desired indole
(ethyl (7-fluoro-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)acetate)
was obtained in a total yield of 252 g (37%) as a white solid.
[5-Bromo-4-(4-chlorobenzyl)-7-fluoro-1,2,3,4-tetrahydrocyclo-
penta[b]indol-3-yl]acetic Acid (10). Following the procedure
described for 3, hydrolysis of ester 9 (200 g, 760 mmol) yielded
the corresponding acid as a brown oil that was used as such in the
next step (165 g, 92%). 1H NMR (acetone-d6): δ 10.50 (br s, 1H),
g, 42%) in 99% ee. [R]2D1 ) +39.2° (c 1.0, MeOH). H NMR
1
(acetone-d6): δ 10.69 (br s, 1H), 7.33 (d, J ) 8.3 Hz, 2H), 7.20
(dd, J ) 8.9 and 2.1 Hz, 1H), 7.09 (dd, J ) 9.0 and 2.2 Hz, 1H),
6.94 (d, J ) 8.2 Hz, 2H), 5.92 (d, JAB ) 17.8 Hz, 1H), 5.77 (d,
JAB ) 17.8 Hz, 1H), 3.65-3.62 (m, 1H), 2.95-2.65 (m, 4H), 2.42
(dd, J ) 16.1 and 10.1 Hz, 1H), 2.32-2.27 (m, 1H). 13C NMR
(acetone-d6): δ 172.2, 156.9 (d, JCF ) 237 Hz), 151.9, 138.7, 134.1,
132.2, 128.6, 127.5 (d, JCF ) 10.2 Hz), 127.2, 119.7 (d, JCF ) 4.8
Hz), 113.6 (d, JCF ) 28.7 Hz), 103.4 (d, JCF ) 22.7 Hz), 102.9 (d,
JCF ) 12.1 Hz), 48.4, 38.1, 35.6, 35.2, 22.4. MS (-APCI) m/z
436.2, 434.0 (M - H)-. Anal. (C20H16BrClNFO2) C, H, N.
[(3R)-4-(4-Chlorobenzyl)-7-fluoro-5-acetyl-1,2,3,4-tetrahydro-
cyclopenta-[b]indol-3-yl]acetic Acid (12). To a DMF (200 mL)
solution of the methyl ester of bromide 11 (10.0 g, 22.2 mmol)
was added 1-ethoxyvinyltributyltin (10.4 g, 28.8 mmol), and then
nitrogen was slowly bubbled through the solution for approximately
5 min. A separate flask was charged with Pd2(dba)3 (1.0 g, 1.09
mmol), Ph3As (2.5 g, 8.14 mmol), and DMF (920 mL); nitrogen
was bubbled through the solution for several minutes, and then the
solution was sonicated for several additional minutes. This catalyst

804 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Sturino et al.
mixture was then transferred to the reaction flask containing
bromide 11 and subsequently heated at 90 °C for 3.5 h. At this
time, the reaction was cooled to 50 °C, treated with a 1 N HCl
solution (20 mL, 20 mmol), and stirred at this temperature for 2 h.
The reaction mixture was then cooled to rt and extracted with
EtOAc; the organic phase was washed with water and brine, dried
over anhydrous Na2SO4, and concentrated. The resulting residue
was purified by flash chromatography eluting with 30-50% EtOAc/
hexanes followed by a second flash purification eluting with 30%
EtOAc/hexanes to provide [(3R)-4-(4-chlorobenzyl)-7-fluoro-5-
acetyl-1,2,3,4-tetrahydrocyclopenta-[b]indol-3-yl]acetate as an off-
2.34-2.27 (m, 1H). 13C NMR (acetone-d6): δ 173.0, 156.5 (d, JCF
) 237 Hz), 153.9, 139.2, 133.7, 133.3, 130.0 (d, JCF ) 8.9 Hz),
129.6, 128.2, 127.5 (d, JCF ) 7.6 Hz), 122.2 (d, JCF ) 4.2 Hz),
112.3 (d, JCF ) 29.4 Hz), 111.0 (d, JCF ) 22.6 Hz), 50.8, 44.7,
38.6, 36.6, 36.5, 23.3. MS (-ESI) m/z 435.8, 433.8 (M - H)-.
Mp 175 °C. Anal. (C21H19ClFNO4S) C, H, N.
The sodium salt was prepared by the treatment of the above acid
(6.45 g, 14.80 mmol) in EtOH (100 mL) with a 1 N NaOH solution
(14.8 mL, 14.8 mmol). The organic solvent was removed under
vacuum, and the crude solid was dissolved in isopropyl alcohol
(1.2 L) under reflux. The final volume was reduced to 500 mL by
distillation of the solvent. The sodium salt was crystallized by
cooling to rt. The crystalline sodium salt was suspended in H2O,
frozen with a dry ice bath, and lyophilized under high vacuum to
give the sodium salt of 13 (6.0 g, 88%) as an amorphous white
solid. 1H NMR (DMSO-d6): δ 7.63 (dd, J ) 8.5 and 2.6 Hz, 1H),
7.47 (dd, J ) 9.7 and 2.6 Hz, 1H), 7.33 (d, J ) 8.4 Hz, 2H), 6.70
1
white solid (5.5 g, 60%). H NMR (acetone-d6): δ 7.97 (dd, J )
8.9 and 2.5 Hz, 1H), 7.26 (d, J ) 8.5 Hz, 2H), 7.21 (dd, J ) 12.4
and 2.5 Hz, 1H), 6.79 (d, J ) 8.5 Hz, 2H), 5.45 (d, JAB ) 18.2 Hz,
1H), 5.37 (d, JAB ) 18.2 Hz, 1H), 3.77-3.72 (m, 1H), 3.62 (s,
3H), 2.98-2.75 (m, 4H), 2.54 (dd, J ) 15.4 and 9.8 Hz, 1H), 2.36-
2.30 (m, 1H), 2.13 (s, 3H). 13C NMR (acetone-d6): δ 199.1 (d, JCF
) 1.7 Hz), 171.7, 156.2 (d, JCF ) 234.9 Hz), 152.2, 137.0, 132.5,
132.4, 128.5, 128.2, 127.7 (d, JCF ) 9.8 Hz), 127.6 (d, JCF ) 8.0
Hz), 119.0 (d, JCF ) 4.6 Hz), 108.9 (d, JCF ) 26.7 Hz), 107.5 (d,
JCF ) 22.8 Hz), 50.9, 49.4, 38.4, 35.5, 35.3, 28.0, 22.6. MS
(+APCI) m/z 416.1, 413.6 (M + H)+; Anal. (C23H21ClFNO3) C,
H, N.
(d, J ) 8.4 Hz, 2H), 6.06 (d, JAB ) 17.9 Hz, 1H), 5.76 (d, JAB
)
17.9 Hz, 1H), 3.37-3.29 (1H, m), 3.08 (3H, s), 2.83-2.77 (1H,
m), 2.71-2.66 (1H, m), 2.60-2.53 (1H, m), 2.23-2.15 (2H, m),
1.93 (dd, J ) 14.4 and 9.7 Hz, 1H). 13C NMR (D2O): δ 180.5,
155.1 (d, JCF ) 238.0 Hz), 154.6, 137.8, 132.3, 132.2, 129.2 (d,
JCF ) 9.0 Hz), 128.6, 126.8, 124.1 (d, JCF ) 7.8 Hz), 120.7 (d, JCF
) 4.3 Hz), 110.9 (d, JCF ) 22.2 Hz), 110.6 (d, JCF ) 17.3 Hz),
50.0, 43.7, 42.1, 36.7, 35.4, 22.3. Purity, min 97.2% (method A tR
) 10 min; method B tR ) 9.5 min).
The above methyl ester (3.04 g, 7.36 mmol) was taken up in
MeOH (50 mL), THF (45 mL), and water (4.5 mL), treated with a
1 N NaOH solution (22 mL, 22 mmol), and stirred at rt for 3 h. At
this time, the reaction was acidified with a 1 N HCl solution and
extracted with EtOAc; the organic phase was washed with water
and brine, dried over anhydrous Na2SO4, and concentrated. The
resulting residue was purified by flash chromatography, eluting with
50-70% EtOAc/hexanes, to provide an off-white solid that was
then triturated twice with hexanes (2 × 100 mL) to furnish [(3R)-
5-acetyl-4-(4-chlorobenzyl)-7-fluoro-1,2,3,4-tetrahydrocyclopenta-
[b]indol-3-yl]acetic acid as an off-white solid (2.5 g, 90%). [R]D21
) +6.4° (c 1.0, MeOH). 1H NMR (acetone-d6): δ 10.9 (br s, 1H),
7.38 (dd, J ) 8.9 and 2.5 Hz, 1H), 7.26 (d, J ) 8.4 Hz, 2H), 7.21
(dd, J ) 9.8 and 2.5 Hz, 1H), 6.71 (d, J ) 8.3 Hz, 2H), 5.42 (s,
2H), 3.77-3.73 (m, 1H), 3.01-2.95 (m, 1H), 2.91-2.78 (m, 3H),
2,53 (dd, J ) 16.1 and 10.0 Hz, 1H), 2.39-2.27 (m, 1H), 2.14 (s,
3H). 13C NMR (acetone-d6): δ 199.8 (d, JCF ) 1.5 Hz), 172.9,
156.8 (d, JCF ) 235 Hz), 153.0, 137.6, 133.2, 133.0, 129.2, 128.9,
Acknowledgment. We thank the Comparative Medicine
group at Merck Frosst for their help with the pharmacokinetic
studies. We also thank Dr. Daniel Dube for providing the
software used in the calculation of the pharmacokinetic param-
eters reported in this publication. We also thank Tony Pereira,
Steve Chang, and Bindhu Karanam for their help with the rat
half-life measurement of 13.
Supporting Information Available: Full combustion analysis
data along with X-ray crystallographic data of 14 (ORTEP drawing
and five tables). This material is available free of charge via the
Internet at http://pubs.acs.org.
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1
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DiscoVery of MK-0524
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JM0603668