5-Lipoxygenase-activating protein inhibitors. Part 3: 3-{3-tert- Butylsulfanyl-1-[4-(5-methoxy-pyrimidin-2-yl)-benzyl]-5-(5-methyl-pyridin-2- ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid (AM643) - A potent FLAP inhibitor suitable for topical administration

Article abstract of DOI:10.1016/j.bmcl.2010.06.011

AM643 (compound 6, 3-{3-tert-butylsulfanyl-1-[4-(5-methoxy-pyrimidin-2-yl)- benzyl]-5-(5-methyl-pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid) was identified as a potential candidate for formulation as a topical agent for the treatment of skin disorders involving leukotriene production. Dermal application of 6 using a prototypical vehicle in a murine ear arachidonic acid model showed significant reduction in the concentrations of leukotrienes in mouse skin with concomitant reduction in ear swelling.

Full text of DOI:10.1016/j.bmcl.2010.06.011

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4598–4601
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Lipoxygenase-activating protein inhibitors. Part 3:
3-{3-tert-Butylsulfanyl-1-[4-(5-methoxy-pyrimidin-2-yl)-benzyl]-5-(5-methyl-
pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid
(AM643)—A potent FLAP inhibitor suitable for topical administration
*
Nicholas Stock , Christopher Baccei, Gretchen Bain, Charles Chapman, Lucia Correa, Janice Darlington,
Christopher King, Catherine Lee, Daniel S. Lorrain, Pat Prodanovich, Angelina Santini, Kevin Schaab,
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:
AM643 (compound 6, 3-{3-tert-butylsulfanyl-1-[4-(5-methoxy-pyrimidin-2-yl)-benzyl]-5-(5-methyl-
pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid) was identified as a potential can-
didate for formulation as a topical agent for the treatment of skin disorders involving leukotriene
production. Dermal application of 6 using a prototypical vehicle in a murine ear arachidonic acid
model showed significant reduction in the concentrations of leukotrienes in mouse skin with con-
comitant reduction in ear swelling.
Received 24 March 2010
Revised 1 June 2010
Accepted 2 June 2010
Available online 8 June 2010
Keywords:
Leukotrienes
FLAP
Ó 2010 Elsevier Ltd. All rights reserved.
Inhibitor
5-Lipoxygenase-activating protein (FLAP) in conjunction with
5-lipoxygenase (5-LO) converts membrane derived arachidonic
acid (AA) to the leukotriene epoxide LTA4.1 Bifurcation of the met-
abolic pathway through either LTA4 hydrolase or LTC4 synthase
produces the pro-inflammatory mediators LTB4 and the cysteinyl
leukotrienes (LTC4, LTD4 and LTE4; see Fig. 1). LTB4 binds to the
GPCRs BLT1 and BLT2 eliciting neutrophil and eosinophil chemo-
taxis. The CysLTs activate at least two well defined GPCR’s, namely
production in the pathology of various skin diseases, including pso-
riasis,10 atopic dermatitis, urticaria and others. For instance, LTs
have been shown to be elevated in atopic dermatitis patients11
and treatment with either Zileuton,12 a 5-lipoxygenase (5-LO)
inhibitor, or Montelukast,13 a CysLT1 antagonist, have shown ben-
eficial effects. Patients with urticaria have been successfully trea-
ted with both CysLT1 receptor antagonists and Zileuton.14
Zafirlukast, another CysLT1 antagonist, has also demonstrated
CysLT1 and CysLT2,3 the activation of the former leading to con-
2
traction of smooth muscle (bronchoconstriction), edema and
eosinophil migration. The precise role of the CysLT2 receptor re-
mains unclear, but mice deficient in this receptor indicate a role
for CysLT2 in vascular permeability and inflammation.4 Ongoing re-
search also indicates the existence of several other CysLT receptors,
namely GPR17,5 P2Y12R6 and CysLTE, the latter being first discov-
ered in murine skin.7 Dermal application of LTB4 or the CysLTs to
human skin produces a prolonged wheal and flare reaction, indi-
cating a potential pathological role for leukotrienes in skin disor-
ders.8 The discovery that LTE4 elicits a similar response to LTC4
and LTD4 in this setting may point to the role of the recently dis-
covered CysLTE receptor as a therapeutic target that is yet to be
realized.9 Significant evidence exists to implicate abnormal LT
* 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.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.011

N. Stock et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4598–4601
4599
efficacy in capsular contracture, a post-operative fibrotic skin
disorder.15 There is also evidence that inhibition of LTs may be
beneficial in the treatment of acne.16
As part of our ongoing research into the development of FLAP
inhibitors17, we sought to develop a potent FLAP inhibitor suitable
for topical application as a treatment modality for dermal diseases.
AM643 (6), sodium 3-{3-tert-butylsulfanyl-1-[4-(5-methoxy-
pyrimidin-2-yl)-benzyl]-5-(5-methyl-pyridin-2-yl-methoxy)-1H-
indol-2-yl]-2,2-dimethyl-propionate) was identified as a potent
and selective inhibitor of the FLAP protein as part of the discovery
program that led to the identification of AM103 and AM803, two
oral FLAP inhibitors that have entered clinical trials in humans.18
Compound 6 was synthesized according to Scheme 1. Fischer
indolization of the substituted hydrazine 1, with the thioketone 2
followed by demethylation of the indole afforded indole–phenol
3. Conversion of the 4-bromophenyl moiety to the corresponding
pinacol-boronate (Pd(dppf)Cl2, bis(pinacolato)diboron, KOAc,
dioxane) permitted Suzuki coupling of 2-chloro-5-fluoropyrimi-
dine under standard conditions and alkylation of the phenol with
5-methyl-2-chloromethyl pyridine 418 yielded the elaborated in-
dole 5. Hydrolysis of the hindered geminal-dimethyl ester in the
presence of methanol, with simultaneous displacement of the fluo-
rine on the pyrimidine with a methoxy group, followed by conver-
sion to the sodium carboxylate salt gave 6 in 31% overall yield.
Table 1 summarizes the in vitro data for 6. The FLAP binding
assay was performed using human polymorphonuclear leukocyte
(PMN) derived membranes and the human leukocyte (hLA) assay
measures inhibition of LTB4 synthesis in isolated human leuko-
S
MeO
HO
NH2
O
a
N
N
O
OEt
OEt
S
O
2
Br
3
1
Br
S
O
N
O
N
b
OEt
Cl
N
HCl
N
4
5
N
F
S
O
N
O
O-Na+
12
8
N
c
N
N
6
(AM643)
4
MeO
**
65%
Inhibition
Scheme 1. Reagents and conditions: (a) (i) toluene, AcOH, NaOAc, 2, rt (55%); (ii)
AlCl3, t-BuSH, DCM, 0 °C to rt (93%); (b) (i) Bis(pinacolato)diboron, Pd(dppf)Cl2ÁDCM
(cat), KOAc, p-dioxane, 85 °C (88%); (ii) 2-chloro-5-fluoropyrimidine, K2CO3, DME,
H2O, Pd(PPh3)4 (cat), 80 °C (85%); (iii) 4, Cs2CO3, MeCN, 45 °C (84%); (c) (i) LiOHÁH2O,
THF, MeOH, H2O, 65 °C (98%); (ii) NaOH (1 equiv), EtOH, H2O, lyophilization (quant).
0
VEH
AM643
4000
3000
2000
1000
Table 1
Selected in vitro IC50 data for compound 6a
Assay
IC50
**
FLAP binding
hLA
hWB (15 min)
hWB (5 h)
mWB (15 min)
mWB (4 h)
rWB (15 min)
rWB (4 h)
2 nM
57%
Inhibition
0.6 nM
161 nM
81 nM
26 nM
4 nM
0
VEH
AM643
1000
78 nM
11 nM
800
600
400
200
0
CYP 3A4
CYP 2D6
CYP 2C9
8.8
lM
P=0.09
>30
4.5 lM
lM
COX-1/2
>100 lM
33%
Inhibition
FLAP binding: inhibition of 3H-ligand binding to FLAP
a
membranes (n = 6); hLA; inhibition of LTB4 synthesis fol-
lowing ionophore (A23817) challenge in human leuko-
cytes; hWB: inhibition of LTB4 synthesis following
ionophore challenge (A23817) in human blood after either
15 min or 5 h compound pre-incubation (n = 7, whereby
each n is the average from two different donors); mWB
(mouse whole blood) data is from a single experiment. rWB
(15 min) is the average of six experiments and the 4 h data
is the average of 10 experiments.
VEH
AM643
Figure 2. Oral administration of AM643 (30 mg/kg) decreased AA (4 mg/ear)—
induced ear swelling and tissue CysLT biosynthesis. There was a trend toward
decrease in LTB4 but this did not reach statistical significance. Bars represent the
means SEM of n = 3 mice per group **P <0.01 versus vehicle treated mice, two
tailed t-test.

4600
N. Stock et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4598–4601
cytes after calcium ionophore stimulation. To measure the degree
of protein shift in the presence of blood proteins, the whole blood
(xWB, x = h, human; x = m, mouse; x = r, rat) assay measures LTB4
inhibition following ionophore challenge after both a 15 min and
either a 4 or 5 h pre-incubation period in blood.19
The data demonstrates that compound 6 is a potent inhibitor of
LTB4 biosynthesis in blood with a significant potency increase after
prolonged incubation.20 In human blood at 15 min, 6 exhibits an
IC50 of 161 nM against LTB4 production and at equilibrium after
5 h, possesses an IC50 of 81 nM. Compound 6 shows no inhibition
diate erythema and edema response with subsequent increase in
ear weight.21 Each mouse is dosed topically on one ear with AA
in ethanol (4 mg in 40 lL) and the other ear solely with 40 lL eth-
anol as a control. After a 30 min period ear samples are collected
using a 6 mm dermal punch, weighed and assayed for LT concen-
trations. As a benchmark study, oral administration of 6 to mice
(30 mg/kg in 0.5% methocel) 4 h prior to AA treatment, showed sig-
nificant inhibition of ear edema in the AA treated ear with concom-
itant reduction in both LTB4 and CysLT concentrations, although
the LTB4 inhibition did not reach statistical significance (Fig. 2).
Analysis of plasma samples from these mice indicated sufficient
of either the COX-1 or
2 enzymes at high concentrations
(>100 M). Cytochrome P450 inhibition is minimal and is antici-
l
concentration of compound 6 at 4.5 h post-dose (av 1.5 lM,
pated to be of nominal consequence in a dermal application. One
of the differentiating factors of compound 6 from other FLAP inhib-
itors studied was that the sodium salt proved highly soluble in a
prototypical, nonirritating, dermal vehicle consisting of 75% pro-
pylene glycol, 15% transcutol and 10% water, thus allowing facile
dermal application in animal models.
To demonstrate the effectiveness of AM643 (6) in inhibiting LT
biosynthesis in vivo we utilized a murine topical AA model. In this
model the application of AA to the ears of mice produces an imme-
n = 4) to completely inhibit FLAP and hence LT biosynthesis
(mWB LTB4 IC50 at 4 h = 4 nM). A significant component of the ede-
ma and erythema is due to metabolism of the applied arachidonic
acid through the cyclooxygenase-1 (COX-1) pathway thereby pro-
ducing pro-inflammatory prostaglandins. The levels of inhibition
achieved with the FLAP inhibitor 6 are therefore in line with expec-
tations for complete leukotriene synthesis inhibition alone.
Topical administration of 6 showed a dose dependent decrease
in CysLTs and LTB4 in the mouse ear and also a reduction in edema
as measured by the change in ear weight. At the 0.1% dose concen-
tration of 6, CysLTs were reduced by 37%, LTB4 was reduced 55%
and the change in ear weight by 61%, demonstrating a similar
effect to that seen with systemic dosing (see Fig. 3) Higher concen-
trations of compound 6 (1% and 10%) produced no significant
improvement over the 0.1% dose (data not shown).
4
3
2
To assess the potential for dermal irritancy of AM643 (6), we
employed an abraded rat skin model. The study comprised admin-
#
istering 2 mL of a 10 mg/mL solution of 6 (ꢀ40
lM) in the afore-
1
mentioned vehicle to 20% of the body area of healthy male rats.
The shaved skin was abraded prior to compound administration
then occluded for 24 h after which the dosage site was cleaned of
61%
inhibition
0
0
0.01%
0.1%
AM643 topical concentration
6000
4000
2000
100000
IV
PO
10000
1000
100
10
*
37%
inhibition
0
0
0.01%
0.1%
1
AM643 topical concentration
0
4
8
12
16
20
24
600
Time (hr)
100
10
1
400
200
0
*
*
55%
inhibition
Average Rat (n = 3)
0
0.01%
0.1%
AM643 topical concentration
0
12
24
36
48
60
72
Time (hr)
Figure 3. Topical administration of AM643 (6) (0.01% and 0.1%) led to a concen-
tration dependent decreased AA (4 mg/ear)—induced ear swelling and tissue
leukotriene biosynthesis. Bars represent the means SEM of n = 3 mice per group
Figure 4. Pharmacokinetics of 6. Top: 10 mg/kg in 0.5% methocel (po) and 1 mg/kg
in 10% EtOH/40% PEG400/50% water (iv); bottom: plasma concentration from rat
after topical dosing of 6 (see text).
*
P <0.05 versus vehicle treated mice, Dunnett’s post hoc comparisons following
ANOVA; #P <0.05 versus vehicle treated mice, t-test.

N. Stock et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4598–4601
4601
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.
residual material and the animals observed for a period of 72 h.
Dermal irritancy scoring was conducted following the modified
Draize method22 and blood samples were drawn to assess the de-
gree of systemic compound exposure following dermal application.
Topical administration of 6 in this manner showed no findings of
erythema or edema (a Draize score of zero) and no change in body
weight over the 72 h test period. Of note is the finding that small,
but detectable levels of compound were present in the plasma of
the animals at all time points. Compound 6 possesses a terminal
t½ of approximately 2 h in rat following intravenous dosing, and
a comparison of the dermal and oral rat pharmacokinetics (see
Fig. 4) indicate that a continued slow leaching of compound
through the skin occurs in rat. The presence of low concentrations
of 6 in the rat plasma after topical dosing also demonstrates that
the compound in this preliminary vehicle is suitably penetrant.
In conclusion, we have identified a potent FLAP inhibitor that
demonstrates robust efficacy in a murine arachidonic acid ear
swelling model upon topical administration. Furthermore, AM643
(6) possesses no apparent potential for irritancy in an abraded
rat skin study and shows a long residence time in rodent skin after
topical application. Additional studies and optimization of a suit-
able dermal vehicle for clinical development will be published in
due course.
4. Beller, T. C.; Maekawa, A.; Friend, D. S.; Austen, K. F.; Kanaoka, Y. J. Biol. Chem.
2004, 279, 46129.
5. 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 2006, 25, 4615.
6. Nonaka, Y.; Hiramoto, T.; Fujita, N. Biochem. Biophys. Res. Commun. 2005, 337,
281.
7. Maekawa, A.; Kanaoka, Y.; Xing, X.; Austen, K. F. PNAS 2008, 105, 16695.
8. (a) Camp, R. D. R.; Russell, J. R.; Brain, D.; Wollard, P. M. J. Invest. Dermatol. 1984,
82, 202; (b) Soter, N. A.; Lewis, R. A.; Corey, E. J.; Austen, K. F. J. Invest. Dermatol.
1983, 80, 115.
9. Austen, K. F.; Maekawa, A.; Kanaoka, Y.; Boyce, J. A. J. Allergy Clin. Immunol.
2009, 124, 406.
10. For a review on leukotrienes and prostaglandins in psoriasis see: Ikai, K. J.
Dermatol. Sci. 1999, 21, 135.
11. Hilger, R. A.; Neuber, K.; König, W. Immunology 1991, 74, 689.
12. Woodmansee, D. P.; Simon, R. P. Ann. Allergy Asthma Immunol. 1999, 83, 548.
13. Yanase, D. J.; David-Bajar, K. J. Am. Acad. Dermatol. 2001, 44, 89.
14. (a) Ellis, M. H. J. Allergy Clin. Immunol. 1998, 102, 876; (b) Pacor, M. L.; Di
Lorenzo, G.; Corrocher, R. Clin. Exp. Allergy 2001, 31, 1607.
15. Scuderi, N.; Mazzocchi, M.; Fioramonti, P.; Bistoni, G. Aesthet. Plast. Surg. 2006,
30, 513.
16. Zouboulis, C. C.; Nestoris, S.; Adler, Y. D.; Orth, M.; Orfanos, C. E.; Picardo, M.;
Camera, E.; Cunliffe, W. J. Arch. Dermatol. 2003, 139, 668.
17. (a) 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;
(b) Stock, N.; Baccei, C.; Bain, G.; Broadhead, A.; Chapman, C.; Darlington, J.;
King, C.; Lee, C.; Li, Y.; Lorrain, D. S.; Prodanovich, P.; Rong, H.; Santini, A.;
Zunic, J.; Evans, J. F.; Hutchinson, J. H.; Prasit, P. Bioorg. Med. Chem. Lett. 2010,
20, 213.
Acknowledgment
The authors would like to thank Dr. Brian Stearns for assistance
in preparation of the tritiated FLAP ligand used in the FLAP binding
assay.
18. Stock, N. S.; Zunic, J.; Li, Y.; Arruda. J.; Roppe, J.; Bain, G.; Santini, A.; Darlington,
J.; King, C. D.; Baccei, C.; Lee, C.; Rong, H.; Chapman, C.; Broadhead, A.; Lorrain,
D.; Correa, L.; Hutchinson, J. H.; Evans, J. F.; Prasit, P. J. Med. Chem., manuscript
in review.
19. 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.
20. The details of this potency increase in blood for FLAP inhibitors will be
published in due course. Bain, G. et al.
21. Young, J. M.; Spires, D. A.; Bedord, C. J.; Wagner, B.; Ballaron, S. J.; De Young, L.
M. J. Invest. Dermatol. 1984, 82, 367.
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. Lynch, K. R.; O’Neill, G. P.; Liu, Q.; Im, D.-S.; Sawyer, N.; Metters, K. M.;
Coulombe, N.; Abramovitz, M.; Figueroa, D. J.; Zeng, Z.; Connolly, B. M.; Bai, C.;
Austin, C. P.; Chateauneuf, A.; Stocco, R.; Greig, G. M.; Kargman, S.; Hooks, S. B.;
Hosfield, E.; Williams, D. L., Jr; Ford-Hutchinson, A. W.; Caskey, C. T.; Evans, J. F.
Nature 1999, 399, 789.
22. (a) Draize, J. H.; Woodard, G.; Calvery, H. D. J. Pharmacol. Exp. Ther. 1944, 83,
377; b Draize, J. H. Association of food and drug officials of the United States, Texas
State Dept. of Health, 1959.
3. 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., Jr.; Zeng,