5-Lipoxygenase-activating protein inhibitors. Part 2: 3-{5-((S)-1-Acetyl-2,3-dihydro-1H-indol-2-ylmethoxy)-3-tert-butylsulfanyl-1-[4-(5-methoxy-pyrimidin-2-yl)-benzyl]-1H-indol-2-yl}-2,2-dimethyl-propionic acid (AM679)-A potent FLAP inhibitor

Article abstract of DOI:10.1016/j.bmcl.2009.10.131

A series of potent 5-lipoxygenase-activating protein (FLAP) inhibitors are herein described. SAR studies focused on the discovery of novel alicyclic moieties appended to an indole core to optimize potency, physical properties and off-target activities. Subsequent SAR on the N-benzyl substituent of the indole led to the discovery of compound 39 (AM679) which showed potent inhibition of leukotrienes in human blood and in a rodent bronchoalvelolar lavage (BAL) challenge model.

Full text of DOI:10.1016/j.bmcl.2009.10.131

Bioorganic & Medicinal Chemistry Letters 20 (2010) 213–217
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Lipoxygenase-activating protein inhibitors. Part 2: 3-{5-((S)-1-Acetyl-2,3-dih-
ydro-1H-indol-2-ylmethoxy)-3-tert-butylsulfanyl-1-[4-(5-methoxy-pyrimidin-
2-yl)-benzyl]-1H-indol-2-yl}-2,2-dimethyl-propionic acid (AM679)—A potent
FLAP inhibitor
*
Nicholas Stock , Christopher Baccei, Gretchen Bain, Alex Broadhead, Charles Chapman, Janice Darlington,
Christopher King, Catherine Lee, Yiwei Li, Daniel S. Lorrain, Pat Prodanovich, Haojing Rong, Angelina Santini,
Jasmine Zunic, Jilly F. Evans, John H. Hutchinson, Peppi Prasit
Amira Pharmaceuticals, 9535 Waples St., Suite 100, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of potent 5-lipoxygenase-activating protein (FLAP) inhibitors are herein described. SAR studies
focused on the discovery of novel alicyclic moieties appended to an indole core to optimize potency,
physical properties and off-target activities. Subsequent SAR on the N-benzyl substituent of the indole
led to the discovery of compound 39 (AM679) which showed potent inhibition of leukotrienes in human
blood and in a rodent bronchoalvelolar lavage (BAL) challenge model.
Received 25 September 2009
Revised 26 October 2009
Accepted 29 October 2009
Available online 31 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
FLAP
Leukotrienes
Inhibitors
5-Lipoxygenase-activating protein (FLAP) works in concert with
5-lipoxygenase (5-LO) to convert membrane derived arachidonic
acid to the pro-inflammatory mediator leukotriene epoxide
LTA4.1 This is rapidly converted to either LTB4 by LTA4 hydrolase
or LTC4 through reaction with LTC4 synthase (Fig. 1). LTB4 binds
to the GPCRs BLT1 and BLT2 eliciting neutrophil and eosinophil
chemotaxis and subsequent activation of downstream inflamma-
tory responses. LTC4 is further converted to LTD4 and LTE4, and col-
lectively these three are termed cysteinyl leukotrienes (CysLTs).
CysLTs are responsible for bronchoconstriction, airway edema
and hypersecretion of mucus via binding to their GPCRs CysLT1
and CysLT2, however the role of the CysLT2 receptor remains
unclear.2 Recent research also indicates the existence of other
CysLT receptors, namely GPR17,3 P2Y12R,4 and CysLTE.5 ZyfloTM, a
marketed 5-LO inhibitor, ablates the production of both LTB4 and
the CysLTs but is not widely prescribed due to poor pharmacoki-
netic parameters and a low but significant incidence of hepatotox-
icity.6 CysLT1 antagonists are also used clinically for the treatment
of asthma, the most widely prescribed of which is SingulairTM. Inhi-
bition of the FLAP protein is therefore an attractive target as it
would prevent the biosynthesis of both LTB4 and CysLTs and as
such prove a valuable therapy for asthma and other diseases where
LT involvement is implicated.
MK-591 1 and DG031 2 (formerly known as BAYX1005) are
both FLAP inhibitors that have progressed to clinical trials in hu-
man (see Fig. 2).7 The former is an inhibitor of CYP450 (IC50’s
3A4 = 5.2 lM, 2C9 = 0.5 lM), is poorly bioavailable as a crystalline
sodium salt and showed an incidence of mild maculopapular rash
* Corresponding author. Tel.: +1 858 228 4666; fax: +1 858 228 4766.
E-mail address: nick.stock@amirapharm.com (N. Stock).
Figure 1. The arachidonic acid/leukotriene pathway. ZyfloTM is a marketed 5-LO
inhibitor. SingulairTM is a marketed CysLT1 receptor antagonist.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.131

214
N. Stock et al. / Bioorg. Med. Chem. Lett. 20 (2010) 213–217
There are currently no FLAP protein inhibitors on the market,
therefore we directed our efforts at addressing the weaknesses of
previous FLAP inhibitors by identifying novel, potent compounds
devoid of major CYP liabilities with good pharmacokinetic param-
eters. The quinoline moiety is a known pharmacophore for the
FLAP protein and only limited SAR studies have focused on replace-
ment of this entity.10 This functional group has also been impli-
cated as a moiety susceptible to bioactivation and covalent
labeling.11 We initiated our SAR studies by replacing this
pharmacophore with a variety of alicyclic groups (Table 1). All
compounds were screened in a FLAP binding assay using human
Figure 2. MK-591 and BAY X1005, published FLAP inhibitors.
in humans.8 The latter, DG031 is only a weak inhibitor of FLAP (IC50
polymorphonuclear leukocyte (PMN) derived membranes,
a
inhibition of LTB4 ꢀ6
lM in calcium ionophore challenged human
whole blood).9
human leukocyte assay (hLA) whereby LTB4 produced in these cells
is measured after calcium ionophore challenge and finally, to
ascertain the degree of shift in the presence of blood proteins,
compounds were tested in a human whole blood (hWB) assay to
measure the inhibition of LTB4 production following ionophore
challenge after 15 min incubation.12
Table 1
In vitro IC50 assay results for compounds 4–13a
Compounds were prepared from the indole–phenol 313 through
reaction with the appropriate alkylating reagent (Scheme 1). Car-
boxylic acids 4 and 5 derive from alkylation of 3 with the tosylate
of either enantiomer of N-Boc-2-hydroxymethylpyrrolidine and
subsequent hydrolysis of the hindered ethyl ester. Deprotection
of the Boc group (4 N HCl in dioxane) afforded the free amine 6.
Acylation (acetic anhydride, Hunig’s base) afforded the acetylated
product 7 or alternate reaction with methanesulfonylchloride gave
the sulfone 8. Compound 9 was prepared in a similar fashion from
the tosylate of 5-hydroxymethyl-pyrrolidin-2-one. Alkylation of 3
with 2-chloro-40-fluoroacetophenone under similar conditions
with subsequent hydrolysis of the ester yielded 11. The corre-
sponding racemic alcohol 12 arose from NaBH4 reduction of 11.
The amide 10 was synthesized using 2-bromoacetamide as the
electrophile. Benzylic acetamide 13 was prepared from reacting 3
with the tosylate of Boc-protected phenylglycinol, deprotection
of the Boc group (4 N HCl in dioxane), acetylation of the amine
(Ac2O, Hunigs base) and subsequent ester hydrolysis.
No.
R
FLAP binding (nM)
1114
hLA (nM)
29
hWB (nM)
17,221*
4
5
6
7
8
1805
>10,000
6
193
2781
16
67,753
ND
Of the initial compounds prepared, the (S)-stereochemistry
proved the more active (the R-isomers of 6, 7, 8, and 9 were also
less potent than their (S)-counterparts, data not shown). The sim-
ple amide 10 showed reasonable FLAP binding and hLA potency but
403#
4299
shifted to 2.1 lM in the human blood assay. The N-acetylpyrrolidine
441
30
9
616
38
108
28
9024*
2141&
10
11
13
769
11,831
12
13
19
7
469
581
72
23,111*
a
FLAP binding and hLA (human leukocyte) data are the average of n P 3. Unless
otherwise noted. hWB data is the average of two experiments (n = 2, each expt.
averages two donors).
*
n = 1.
n = 3.
n = 72.
Scheme 1. Reagents and conditions: (a) Boc2O, CH2Cl2; (b) TsCl, pyridine; (c)
alkylating agent, Cs2CO3, DMF or MeCN, 60 °C; (d) LiOHꢁH2O, MeOH/THF/H2O
(1:1:1), 60 °C.
&
#

N. Stock et al. / Bioorg. Med. Chem. Lett. 20 (2010) 213–217
215
7 showed excellent potency in all three assays, especially in hWB
(IC50 = 403 nM). However, 7showedsignificantinhibitionofCYP3A4
(64% @ 10 lM) and 2C9 (93% @ 10 lM), indicating a potential for
drug–drug interaction. Pharmacokinetic parameters in rat were also
poor, characterized by low bioavailability, moderate clearance and
low AUC (10 mg/kg po as sodium carboxylate salt: F = 10%,
The N-acylated indoline 20 showed excellent activity in all
three in vitro assays, most notably hWB (IC50 = 169 nM). Unfortu-
nately, pharmacokinetic parameters were similar to its pyrrolidine
counterpart 7, with low bioavailability, moderate clearance and
low Cmax in rat (10 mg/kg as sodium carboxylate salt: F = 11%,
Cl = 25 mL/min/kg, Cmax = 270 nM). CYP inhibition also worsened
Cl = 20 mL/min/kg, AUC = 1.2 h
l
g/mL).
against 3A4 (80% @ 10 lM) and 2C9 (96% @ 10 lM). The (R)-Boc-
Maintaining the more potent (S)-stereochemistry, a small num-
ber of alkyl amide derivatives were prepared (Table 2). Synthesis of
these was accomplished through reaction of the ethyl ester of 6
with the appropriate acyl chloride under standard conditions and
subsequent ester hydrolysis (Scheme 2).
enantiomer 21 again proved less active than the (S)-enantiomer
as in the prior pyrrolidine derivatives vide infra.
Removal of the t-butylthio group using AlCl3 and H2O in dichlo-
romethane allowed the preparation of a variety of acyl and alkyl
substituents at the 3-position of the indole.15 Acylation with a
variety of acyl chlorides under Friedel–Crafts type conditions
and the subsequent reduction with NaBH4 in TFA/dichlorometh-
ane (Scheme 4) afforded the derivatives shown (Table 4). The sulf-
oxide 29 and sulfone 30 were also prepared through sequential
reaction of 1 equiv of m-CPBA in dichloromethane with the
thiane 20.
Removal of the t-butylthio group significantly reduced the
potency against FLAP (22, FLAP binding IC50 = 218 nm). Acyl groups
with substituents alpha to the carbonyl (23, 24) also reduced activ-
ity. However, the t-butylacetyl derivative 25 showed good potency
Increasing the steric bulk of the amide had a detrimental effect
on the overall potency of the compounds, the smaller methyl 7 and
ethyl 14 amides showing the greatest potency in all three in vitro
assays. Subsequent to the completion of this work, X-ray crystal
data of the FLAP protein was published indicating a restricted bind-
ing pocket for the quinoline moiety of 1.14 This correlates well with
our findings that increased steric size of the alicyclic group reduces
potency against FLAP. However, all these derivatives still possessed
significant inhibition of CYP3A4 and 2C9 (data not shown).
With a potent alicyclic substituent identified in the N-acylpyrr-
olidine 7, incorporation of a fused phenyl to the pyrrolidine struc-
ture (indoline) was anticipated to improve potency due to it more
closely mimicking the planar structure of quinoline and thereby
increasing binding affinity to the FLAP protein (Table 3). This was
prepared in analogous fashion reacting the tosylate of N-Boc-pro-
tected (S)-indoline-2-methanol with the indole–phenol 3 to afford
18 and subsequent deprotection and acylation to give 19 and 20,
respectively (Scheme 3).
in binding and hLA with
a reasonable blood shift (hWB
IC50 = 521 nM). Due to the more polar nature of the vinylogous acyl
unit we anticipated an improvement in solubility and potentially
bioavailability. However, upon oral dosing of the sodium salt of
25 (10 mg/kg in rat), it showed low bioavailability (F = 7%) and
low Cmax (190 nM). Removal of the carbonyl group yielded com-
pounds 26, 27, and 28 with good in vitro potencies but activity
against CYP3A4 and 2C9 remained (>80% @ 10 lM). The racemic
sulfoxide 29 loses a significant degree of potency which is regained
Table 2
Table 3
In vitro IC50 data for compounds 14–17a
In vitro IC50 data for indolines 18–21a
No.
R
FLAP binding (nM)
hLA (nM)
hWB (nM)
No.
R
FLAP binding (nM)
hLA (nM)
hWB (nM)
7
14
Me
Et
6
4
16
1.5
403#
307*
18
19
20
21*
Boc
H
Ac
1100
85
1.4
106
4.1
0.5
ND
1431
169&
12,254
15
16
50
8.0
51
885
(R)-Boc
>10,000
102
a
186
5663
FLAP binding and hLA data are the average of n P 3. Unless otherwise noted hWB
data is the average of 1 experiment using two different donors.
n = 3.
Compound 21 was derived from alkylation of 3 with the toslyate of N-Boc-pro-
tected (R)-indoline-2-methanol.
&
17
1345
110
22,270
*
a
FLAP binding and hLA data are the average of n P 3. Unless otherwise noted
hWB data is the average of two experiments (n = 2, each expt. averages two
donors).
n = 1.
n = 72.
*
#
Scheme 2. Reagents and conditions: (a) acyl chloride, diisopropylethylamine,
CH2Cl2; (b) LiOHꢁH2O, MeOH, THF, H2O (1:1:1), 60 °C.
Scheme 3. Reagents and conditions: (a) Boc2O, CH2Cl2; (b) TsCl, Et3N, CH2Cl2; (c) 3,
Cs2CO3, MeCN, 60 °C; (d) 4 N HCl, dioxane; (e) Ac2O, diisopropylethylamine, CH2Cl2.

216
N. Stock et al. / Bioorg. Med. Chem. Lett. 20 (2010) 213–217
Table 5
In vitro IC50’s of biaryl indoline derivatives 34–39 (AM679)a
No.
R
FLAP binding (nM)
1.7
hLA (nM)
0.5
hWB (nM)
235
34
35
36
37
38
39
2.5
9.3
3.8
3.2
2.2
0.6
1.8
573
Scheme 4. Reagents and conditions: (a) AlCl3 (4 equiv), H2O (3 equiv), DCM, 0 °C to
rt; (b) R1COCl, AlCl3, DCE, 80 °C; (c) NaBH4, TFA, DCM (1:1), 0 °C to rt; d) LiOHꢁH2O,
THF/MeOH/H2O (1:1:1), 60 °C.
276*
373
Table 4
SAR at the 3-position of the indole: in vitro IC50 data for compounds 22–30a
0.5
12.6
0.6
1109&
154%
No.
R
FLAP binding (nM)
218
hLA (nM)
12.9
hWB (nM)
31,518
a
FLAP binding and hLA data is the average of n P 3. Unless otherwise noted hWB
22
H
data is the average of one experiment using two different donors.
&
*
n = 2.
n = 3.
n = 4.
23
24
20.6
20.6
263
4901
%
>10,000
63,105
one of these compounds 35, were prepared from the common
intermediate shown. Alkylation of the bromo-phenol 31 with the
indoline-tosylate vide infra, deprotection and acylation afforded
the benzylbromide 32. This could then be reacted with the re-
quired heteroaryl boronic acid or boronate ester under standard
Suzuki reaction conditions (Pd(PPh3)4, K2CO3, DME/H2O, 80 °C) to
afford the desired compounds after subsequent hydrolysis of the
ethyl ester. Alternatively, 32 was converted to the pinacolato-bor-
on derivative 33 and reacted through Suzuki coupling with the
appropriate halogenated heteroaryl compound followed by ester
hydrolysis. In the case of compound 39, 2-chloro-5-fluoropyrimi-
dine was used as the Suzuki reaction coupling partner and pro-
longed ester hydrolysis in the presence of methanol lead to the
displacement of the arylfluoride to yield the methoxypyrimidine
39. Compound 38 was prepared through Suzuki reaction of the
bromo-phenol 31 first, then alkylation of the resulting phenol as
previously described.
25
26
27
28
19
1.3
0.5
1.5
4.2
521
200
428
405
35
15.2
4.7
29
30
S(O)t-Bu
SO2t-Bu
74
335
19
23,154
1744&
4.5
a
FLAP binding and hLA data are the average of n P 3. Unless otherwise noted hWB
data is the average of one experiment using two different donors.
n = 2.
All of the biaryl compounds 34 to 39 showed excellent in vitro
inhibition against FLAP. Of particular note was compound 39 with
an excellent hWB IC50 potency of 154 nM. This compound also
&
in the sulfone 30 albeit with a greater degree of shift in the pres-
ence of human blood proteins compared to the parent thiane 20
(120-fold hWB vs FLAP binding for 30 compared to 387-fold vs
FLAP binding for 20).
During our studies in another structurally similar series of FLAP
inhibitors,13 replacement of the chloro substituent on the phenyl
ring greatly reduced their CYP inhibition activity. We therefore
prepared derivatives of the indoline 20 by replacing the 4-chloro-
benzyl unit with a similar series of heteroaromatic biaryls (Table
5). This was accomplished in several ways (Scheme 5). All but
showed an improved CYP inhibition profile (IC50 3A4 = 16.7
2C9 = 3.7 M, 2D6 >30 M), no time dependent inhibition against
CYP3A4 (0.003 minꢂ1 vs 0.057 minꢂ1 for troleandomycin control
@ 10 M) and no CYP3A4 induction. The pharmacokinetic proper-
lM,
l
l
l
ties of 39 had also significantly improved over the chlorobenzyl
Time-dependent inhibition (TDI) measures a compounds ability to irreversibly
bind to the CYP enzyme and hence reduce its metabolic activity and the induction
assay measures a compounds ability to induce the activity of the CYP enzyme.

N. Stock et al. / Bioorg. Med. Chem. Lett. 20 (2010) 213–217
217
(ꢀ10 mg/mL as the sodium salt in water or 0.03 mg/mL in 0.9%
saline, a vehicle suitable for the RSV study) the successful results
of which have recently published.18
In conclusion, we have discovered a series of potent FLAP inhib-
itors, the best of which possesses a novel N-acylated-(S)-indoline
group that shows excellent potency at inhibiting LTB4 in human
blood (39, AM679, 5 h IC50 = 53 nM), reduced CYP inhibitory activ-
ity compared to 1 and shows excellent leukotriene inhibition in
several in vivo models. Further investigation of the use of 39 in dis-
ease states where leukotrienes are implicated is ongoing.
Acknowledgment
The authors would like to thank Dr. Brian Stearns for assistance
in preparation of the tritiated FLAP ligand used in the FLAP binding
studies.
References and notes
1. (a) Dixon, R. A. F.; Diehl, R. E.; Opas, E.; Rands, E.; Vickers, P. J.; Evans, J. F.;
Gillard, J. W.; Miller, D. K. Nature 1990, 343, 282; (b) Evans, J. F.; Ferguson, A. D.;
Mosley, R. T.; Hutchinson, J. H. Trends Pharmacol. Sci. 2008, 29, 72.
2. Heise, C. E.; O’Dowd, B. F.; Figueroa, D. J.; Sawyer, N.; Nguyen, T.; Im, D.-S.;
Stocco, R.; Bellefeuille, J. N.; Abramovitz, M.; Cheng, R.; Williams, D. L.; Zeng, Z.;
Liu, Q.; Ma, L.; Clements, M. K.; Coulombe, N.; Liu, Y.; Austin, C. P.; George, S. R.;
O’Neill, G. P.; Metters, K. M.; Lynch, K. R.; Evans, J. F. J. Biol. Chem. 2000, 275,
30531.
3. Ciana, P.; Fumagalli, M.; Trincavelli, M. L.; Verderio, C.; Rosa, P.; Lecca, D.;
Ferrario, S.; Parravicini, C.; Capra, V.; Gelosa, P.; Guerrini, U.; Belcredito, S.;
Cimino, M.; Sironi, L.; Tremoli, E.; Rovati, G. E.; Martini, C.; Abbracchio, M. P.
EMBO J. 2006, 25, 4615.
4. Nonaka, Y.; Hiramoto, T.; Fujita, N. Biochem. Biophys. Res. Commun. 2005, 337,
281.
5. Maekawa, A.; Kanaoka, Y.; Xing, X.; Austen, K. F. PNAS 2008, 105, 16695.
6. (a) Berger, W.; De Chante, M. T. M.; Cairns, C. B. Int. J. Clin. Pract. 2007, 61, 663; b
Prescribing Information for ZYFLO CR. Cornerstone Therapeutics, Inc.,
Lexington, MA, May 2007.
Scheme 5. Reagents and conditions: (a) (i) ethyl (S)-2-(toluene-4-sulfonyloxym-
ethyl)-2,3-dihydro-indole-1-carboxylate, Cs2CO3, MeCN or DMF, heat; (ii) 4 N HCl in
dioxane, CH2Cl2; (iii) Ac2O, diisopropylethylamine, DCM; (b) K2CO3, DME, H2O,
Pd(PPh3)4 (cat.), 85 °C, heteroarylboronate (from 32) or halogeno-heteroaryl (from
33); c) LiOHꢁH2O, THF/MeOH/H2O (1:1:1), 60 °C; (d) bis(pinacolato)diboron,
Pd(dppf)Cl2ꢁCH2Cl2 (cat.), KOAc, p-dioxane, 85 °C; (e) 3-bromo-6-methoxypyrid-
azine, K2CO3, DME, H2O, Pd(PPh3)4 (cat.), 85 °C.
7. Diamant, Z.; Timmers, M. C.; van der Veen, H.; Friedman, B. S.; De Smet, M.;
Depré, M.; Hilliard, D.; Bel, E. H.; Sterk, P. J. J. Allergy Clin. Immunol. 1995, 95, 42.
8. (a) Depré, M.; Friedman, B.; Van Hecken, A.; De Lepeleire, I.; Tanaka, W.; Dallob,
A.; Shingo, S.; Porras, A.; Lin, C.; De Schepper, P. J. Clin. Pharm. Therapeut. 1994,
56, 22; (b) Uematsu, T.; Kanamaru, M.; Kosuge, K.; Hara, K.; Uchiyama, N.;
Takenaga, N.; Tanaka, W.; Friedman, B. S.; Nakashima, M. Br. J. Clin. Pharmacol.
1995, 40, 59.
9. Brooks, C. D. W.; Stewart, A. O.; Kolasa, T.; basha, A.; Bhatia, P.; Ratajczyk, J. D.;
Craig, R. A.; Gunn, D.; Harris, R. R.; Bouska, J. B.; Malo, P. E.; Bell, R. L.; Carter, G.
W. Pure Appl. Chem. 1998, 70, 271.
10. (a) Musser, J. H.; Kreft, A. F. J. Med. Chem. 1992, 35, 2501; (b) Frenette, R.;
Hutchinson, J. H.; Léger, S.; Thérien, M.; Brideau, C.; Chan, C. C.; Charleson, S.;
Ethier, D.; Guay, J.; Jones, T. R.; McAuliffe, M.; Piechuta, H.; Riendeau, D.; Tagari,
P.; Girard, Y. Bioorg. Med. Chem. Lett. 1999, 9, 2391.
11. Nicoll-Griffith, D. A.; Chauret, N.; Yergey, J. A.; Trimble, L. A.; Favreau, L.;
Zamboni, R.; Grossman, S. J.; Drey, J.; Herold, E. Drug Metab. Dispos. 1993, 21,
861.
derivative 20, showing reasonable bioavailability, low clearance and
improved AUC (10 mg/kg sodium carboxylate salt in rat po F = 29%,
Cl = 11 mL/min/kg, Cmax = 1.6 lM, AUC = 4.6 h lg/mL, T = 6.8 h).
½
Compound 39 (AM679) was profiled in a rodent bronchoalveolar
lavage (BAL) model to measure its ability to inhibit production of
leukotrienes in vivo.16 Oral administration of 39 (10 mg/kg as the so-
dium carboxylate salt) 4 h prior to ionophore challenge reduced LTB4
and CysLT levels in the rodent lung lavage fluid by 98% and 87%,
respectively, with corresponding average rodent plasma levels of
605 nM (3 h post dose, rat blood LTB4 IC50 = 125 nM). Dose response
studies showed an ED50 of 0.8 and 1.4 mg/kg in rat for LTB4 and Cys-
LT, respectively. When dosed at 3 mg/kg po 16 h prior to ionophore
challenge, significant inhibition of leukotrienes was still evident
(77% and 45% inhibition of LTB4 and CysLT, respectively) indicating
a sustained pharmacodynamic effect as average plasma levels at this
time point were measured at 4 nM.
12. Brideau, C.; Chan, C.; Charleson, S.; Denis, D.; Evans, J. F.; Ford-Hutchinson, A.
W.; Hutchinson, J. H.; Jones, T. R.; Léger, S.; Mancini, J. A.; McFarlane, C. S.;
Pickett, C.; Piechuta, H.; Prasit, P.; Riendeau, D.; Rouzer, C. A.; Tagari, P.;
Vickers, P. J.; Young, R. N.; Abraham, W. M. Can. J. Physiol. Pharmacol. 1992, 70,
799.
Incubation of 39 in the hWB assay for an extended time period
(5 h) indicated an increase in potency against LTB4 production in
human blood (IC50 = 53 nM [n = 6, where each n is the average of
two donors]).17 This time-dependent increase also occurs in rat
blood as compound 39 shows an IC50 of 9 nM when assayed after
incubation in rat blood for 4 h. This equilibrium potency in rat
blood helps explain the aforementioned extended pharmacody-
namic effect in the rat BAL model, where the measured 16 h plas-
ma levels correlate well with the 4 h IC50. Indoline 39 was further
profiled in a murine ocular model of respiratory syncytial virus
(RSV) due to its excellent in vitro parameters and solubility profile
13. Hutchinson, J. H.; Li, Y.; Arruda, J. M.; Baccei, C.; Bain, G.; Chapman, C.; Correa,
L.; Darlington, J.; King, C. D.; Lee, C.; Lorrain, D.; Prodanovich, P.; Rong, H.;
Santini, A.; Stock, N.; Prasit, P.; Evans, J. F. J. Med. Chem. 2009, 52, 5803.
14. Ferguson, A. D.; McKeever, B. M.; Xu, S.; Wisniewski, D.; Miller, D. K.; Yamin, T.;
Spencer, R. H.; Chu, L.; Ujjainwalla, F.; Cunningham, B. R.; Evans, J. F.; Becker, J.
W. Science 2007, 27, 510.
15. Prasit, P.; Fortin, R.; Hutchinson, J. H.; Belley, M. L.; Leger, S.; Frenette, R.;
Gillard, J. U.S. Patent 5,272,145, 1993.
16. Smith, W. G.; Shaffer, A. F.; Currie, J. L.; Thompson, J. M.; Kim, S.; Rao, T.;
Isakson, P. C. J. Pharmacol. Exp. Ther. 1995, 275, 1332.
17. The details of this potency increase in blood for FLAP inhibitors will be
published in due course. Bain, G. et al.
18. Musiyenko, A.; Correa, L.; Stock, N.; Hutchinson, J. H.; Lorrain, D.; Evans. J. F.;
Barik, S. Clin. Vaccine Immunol. 2009. doi:10.1128/CVI.00220-09.