Quiflapon (formerly known as L-686,70 and MK591 free acid) is a novel, potent, selective and specific 5-Lipoxygenase-activating protein (FLAP) inhibitor with an IC50 value of 1.6 nM in a FLAP binding assay. MK-0591 had no effect on rat 5-lipoxygenase. MK-0591 has a high affinity for 5-lipoxygenase activating protein (FLAP) as evidenced by an IC50 value of 1.6 nM in a FLAP binding assay and inhibition of the photoaffinity labelling of FLAP by two different photoaffinity ligands. Inhibition of activation of 5-lipoxygenase was shown through inhibition of the translocation of the enzyme from the cytosol to the membrane in human PMNLs. MK-0591 was a potent inhibitor of LT biosynthesis in vivo, first, following ex vivo challenge of blood obtained from treated rats and squirrel monkeys, second, in a rat pleurisy model, and, third, as monitored by inhibition of the urinary excretion of LTE4 in antigen-challenged allergic sheep. Inhibition of antigen-induced bronchoconstriction by MK-0591 was observed in inbred rats pretreated with methysergide, Ascaris-challenged squirrel monkeys, and Ascaris-challenged sheep (early and late phase response). These results indicate that MK-0591 is a potent inhibitor of LT biosynthesis both in vitro and in vivo indicating that the compound will be suitable for assessing the role of leukotrienes in pathological situations.

Physicochemical Properties

Molecular Formula	C34H35CLN2O3S
Molecular Weight	587.17100
Exact Mass	586.205
Elemental Analysis	C, 69.55; H, 6.01; Cl, 6.04; N, 4.77; O, 8.17; S, 5.46
CAS #	136668-42-3
Related CAS #	Quiflapon sodium;147030-01-1
PubChem CID	60923
Appearance	White to off-white solid powder
Density	1.2±0.1 g/cm3
Boiling Point	751.3±60.0 °C at 760 mmHg
Flash Point	408.2±32.9 °C
Vapour Pressure	0.0±2.6 mmHg at 25°C
Index of Refraction	1.617
LogP	8.31
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	10
Heavy Atom Count	41
Complexity	873
Defined Atom Stereocenter Count	0
SMILES	O=C(O)C(C)(C)CC1=C(SC(C)(C)C)C2=C(C=CC(OCC3=NC4=CC=CC=C4C=C3)=C2)N1CC5=CC=C(Cl)C=C5
InChi Key	NZOONKHCNQFYCI-UHFFFAOYSA-N
InChi Code	InChI=1S/C34H35ClN2O3S/c1-33(2,3)41-31-27-18-26(40-21-25-15-12-23-8-6-7-9-28(23)36-25)16-17-29(27)37(20-22-10-13-24(35)14-11-22)30(31)19-34(4,5)32(38)39/h6-18H,19-21H2,1-5H3,(H,38,39)
Chemical Name	3-(3-(tert-butylthio)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl)-2,2-dimethylpropanoic acid
Synonyms	Quiflapon free acid; MK 591; MK-591; MK591; L 686708; L-686708; L686708; L-686,708; L 686,708; Quiflapon; 136668-42-3; MK-0591; Quiflapon [INN]; Quiflapon free acid; 3-(3-(tert-butylthio)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl)-2,2-dimethylpropanoic acid; MK 591; CHEMBL16596; L686,708;
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	FLAP/5-lipoxygenase-activating protein (IC50 = 1.6 nM)[1]
ln Vitro	Quiflapon is accessible in whole blood from humans, squirrel monkeys, and rats (IC50 values 510, 69, and 9 nM, respectively), as well as in intact human and induced rat polymorphonuclear leukocytes (PMNL) (IC50 values 3.1 and 6.1 nM, respectively). Rat 5-lipoxygenase is not affected by quilapon. Quiflapon is photoaffinity-tagged to inhibit 5-lipoxygenase-activating protein (FLAP) using two distinct photoaffinity ligands. It exhibits a high affinity for FLAP, with an IC50 value of 1.6 nM in the FLAP binding assay. In human PMNL, 5-lipoxygenase translocation from the cytosol to the membrane can be blocked to prevent 5-lipoxygenase from activating [1].
ln Vivo	Quiflapon has been shown to be a strong inhibitor of LT biosynthesis in vivo in three different settings: an ex vivo challenge in blood drawn from treated rats and squirrel monkeys, a rat pleurisy model, and the inhibition of LTE4 excretion in the urine. Test for allergens in sheep with allergies. Quiflapon was found to have an inhibitory effect on antigen-induced bronchoconstriction (both early and late responses) in ascaris-challenged sheep, ascaris-challenged squirrel monkeys, and inbred rats pretreated with methierg [1]. For days 1-4, 5-9, or 10-14, puppies received daily subcutaneous injections of vehicle or Quiflapon at 10, 20, or 40 mg/kg. Lungs were inflated, fixed, and stained for morphometric and histological studies on day 14. Both the untreated hyperoxia group and the Quiflapon-treated hyperoxia group demonstrated distinct signs of aberrant alveolarization without any inflammation [2].
Cell Assay	For each experiment, equal numbers of cells were plated in six-well plates; 24 h later media were removed and fresh media containing either MK-591 (1 μM, 10 μM or 25 μM) or vehicle were added. After incubation for 24 h, supernatants were collected for Aβ and LDH measurement, and cell pellets harvested in lytic buffer for immunoblot analyses as described in the previous paragraphs. For transfection studies, N2A-APPswe cells were transfected with 1 μg Myc-tagged mΔE-Notch-1 complementary DNA overnight by using Lipofectamine 2000 (Invitrogen). The media were removed and fresh media containing MK-591, L685,458 or vehicle were added. After incubation for 24 h, cells lysates were collected NICD expression levels assayed by western blot analysis.[3]
Animal Protocol	MK-0591 was a potent inhibitor of LT biosynthesis in vivo, first, following ex vivo challenge of blood obtained from treated rats and squirrel monkeys, second, in a rat pleurisy model, and, third, as monitored by inhibition of the urinary excretion of LTE4 in antigen-challenged allergic sheep. Inhibition of antigen-induced bronchoconstriction by MK-0591 was observed in inbred rats pretreated with methysergide, Ascaris-challenged squirrel monkeys, and Ascaris-challenged sheep (early and late phase response).[1] Newborn mice were exposed to either room air or hyperoxia for 14 days. Pups were treated with either vehicle or MK-0591 10, 20, or 40 mg/kg subcutaneously daily for days 1-4, 5-9, or 10-14. On day 14, the lungs were inflated, fixed, and stained for histopathological and morphometric analyses. Hyperoxia groups treated with MK-0591 20 or 40 mg/kg during days P1-P4 or P10-P14 showed alveolarization that resembled that of room air controls while untreated hyperoxia groups showed definite evidence of aberrant alveolarization but no inflammation. In a hyperoxia-exposed newborn mice model, a FLAP inhibitor given during critical window periods may prevent aberration of alveolarization in a dose- and time-dependent manner.[2]
References	[1]. Pharmacology of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid), a potent, orally active leukotriene biosynthesis inhibitor. Can J Physiol Pharmacol. 1992 Jun;70(6):799-8. [2]. 5-Lipoxygenase-activating protein (FLAP) inhibitor MK-0591 prevents aberrant alveolarization in newborn mice exposed to 85% oxygen in a dose- and time-dependent manner. Lung. 2011 Feb;189(1):43-50.
Additional Infomation	MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid, previously L-686,708) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human and elicited rat polymorphonuclear leukocytes (PMNLs) (IC50 values 3.1 and 6.1 nM, respectively) and in human, squirrel monkey, and rat whole blood (IC50 values 510, 69, and 9 nM, respectively). MK-0591 had no effect on rat 5-lipoxygenase. MK-0591 has a high affinity for 5-lipoxygenase activating protein (FLAP) as evidenced by an IC50 value of 1.6 nM in a FLAP binding assay and inhibition of the photoaffinity labelling of FLAP by two different photoaffinity ligands. Inhibition of activation of 5-lipoxygenase was shown through inhibition of the translocation of the enzyme from the cytosol to the membrane in human PMNLs. MK-0591 was a potent inhibitor of LT biosynthesis in vivo, first, following ex vivo challenge of blood obtained from treated rats and squirrel monkeys, second, in a rat pleurisy model, and, third, as monitored by inhibition of the urinary excretion of LTE4 in antigen-challenged allergic sheep. Inhibition of antigen-induced bronchoconstriction by MK-0591 was observed in inbred rats pretreated with methysergide, Ascaris-challenged squirrel monkeys, and Ascaris-challenged sheep (early and late phase response). These results indicate that MK-0591 is a potent inhibitor of LT biosynthesis both in vitro and in vivo indicating that the compound will be suitable for assessing the role of leukotrienes in pathological situations.[1] Bronchopulmonary dysplasia is characterized by prolonged oxygen dependency due to compromised gas-exchange capability. This is attributable mainly to inadequate and aberrant alveolarization resulting from insults like hyperoxia. Leukotrienes are associated with hyperoxia-induced inhibition of alveolarization. We hypothesized that a 5-lipoxygenase-activating protein (FLAP) inhibitor given while newborn mice were exposed to 85% oxygen would prevent aberrant alveolarization in a dose- and time-dependent manner. Newborn mice were exposed to either room air or hyperoxia for 14 days. Pups were treated with either vehicle or MK-0591 10, 20, or 40 mg/kg subcutaneously daily for days 1-4, 5-9, or 10-14. On day 14, the lungs were inflated, fixed, and stained for histopathological and morphometric analyses. Hyperoxia groups treated with MK-0591 20 or 40 mg/kg during days P1-P4 or P10-P14 showed alveolarization that resembled that of room air controls while untreated hyperoxia groups showed definite evidence of aberrant alveolarization but no inflammation. In a hyperoxia-exposed newborn mice model, a FLAP inhibitor given during critical window periods may prevent aberration of alveolarization in a dose- and time-dependent manner.[2]

Solubility Data

Solubility (In Vitro)

DMSO : ≥ 50 mg/mL (~85.15 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 2.5 mg/mL (4.26 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.26 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.7031 mL	8.5154 mL	17.0308 mL
	5 mM	0.3406 mL	1.7031 mL	3.4062 mL
	10 mM	0.1703 mL	0.8515 mL	1.7031 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.