Lck (IC50 = 147 nM in the presence of 1 mM ATP, Km = 12 μM) [1]
Src (IC50 = 9.1 μM) [1]
Fgr (IC50 = 14.1 μM) [1]
Fyn (IC50 = 44.1 μM, >300-fold selective against Fyn) [1][2]
Lyn (IC50 = 1.18 μM) [2]
Hck (IC50 = 1.22 μM) [2]
tie-2 (IC50 > 50 μM) [2]
kdr (IC50 > 50 μM) [2]

With IC50 values of 0.147 μM for Lck and 9.1, 14.1, and 44.1 μM for Src, Fgr, and Fyn, respectively, A-770041 selectively inhibits these Src family kinases [1]. With an EC50 value of 80 nM, A-770041 (0-30 μM; 2 hours) dose-dependently suppresses anti-CD3-induced IL-2 production [1].

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A-770041 inhibited anti-CD3-stimulated IL-2 production in human whole blood with an EC50 of approximately 80 nM, and IL-2 was suppressed greater than 90% at all concentrations above 1 μM [1].
A-770041 inhibited anti-CD3 mAb-induced IL-2 production in human whole blood with an IC50 of 0.08 μM [2].
A-770041 inhibited Concanavalin A-stimulated IL-2 production in whole blood with an EC50 of approximately 80 nM [1].

A-770041 (2.5 mg/kg; ig once) suppresses concanavalin A-induced IL-2 in vivo [1]. A-770041 (2.5-20 mg/kg/day; for 14 days) dosage-dependently enhanced survival at doses 5 and 10 mg/kg/day, and at dose 10, reached 100% survival before 20 mg/kg/day[1].

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A-770041 (2.5 mg/kg oral dose) inhibited Concanavalin A-induced IL-2 production in rats in a plasma concentration-dependent manner with an in vivo EC50 of 78 ± 28 nM. The inhibition fit a sigmoidal curve (r² = 0.79) [1].
A-770041 prevented rejection of hearts transplanted heterotopically in rats from Brown Norway donors to Lewis recipients across a major histocompatibility barrier. Doses at or above 10 mg/kg/day prevented rejection for at least 65 days. At 10 mg/kg/day, 100% of transplanted grafts were still beating at 14 days. At 20 mg/kg/day or 10 mg/kg/day Cyclosporin A, grafts had minimal microvascular changes or multifocal mononuclear infiltrates. Mineralization in myocytes from A-770041-treated grafts was less than Cyclosporin A-treated animals [1].
A-770041 at 10 mg/kg/day (bid) resulted in 100% of transplanted grafts beating at 14 days, with compound plasma concentrations of 2.9 μM. All grafts in animals receiving 10 mg/kg/day (bid) A-770041 or 10 mg/kg/day Cyclosporin A survived to 65 days after transplantation [2].
A-770041 at 2.5 mg/kg/day did not lengthen graft survival time. Dose-dependent increase in survival was observed with doses of 5 and 10 mg/kg/day [1][2].

The purified recombinant tyrosine kinase to be tested was mixed with a biotinylated peptide substrate and varying inhibitor concentrations with 1 mM ATP, 10 mM Mg²⁺, and 2 mM Mn²⁺. After incubation for 60 minutes, a europium cryptate-labeled anti-phosphotyrosine and streptavidin-labeled allophycocyanin were added to the well. The ratio of the signal at 620 and 665 nm was used to calculate IC50 [1].
Inhibitors were screened against a non-phosphorylated construct of human Lck, Lck (64-509) in HTRF format using 1 mM ATP and biotinylated Lck peptide as substrates. Fyn served as a counterscreen [2].
For kinase inhibition assays, the ATP concentration was 1 mM [1].

Heparinized human whole blood was stimulated with anti-CD3 monoclonal antibody and phorbol 12-myristate 13-acetate in the presence of A-770041 (0-30 μM). IL-2 release into plasma was determined 2 hours after stimulation by an enzyme-linked immunosorbent assay [1].
Anti-CD3-stimulated IL-2 production in whole blood was determined. IL-2 production was decreased in a dose-dependent manner [1].

Animal/Disease Models: Male Lewis rat [1]
Doses: 2.5 mg/kg
Route of Administration: intragastric (po) (po)administration; 2.5 mg/kg Primary
Experimental Results:demonstrated inhibitory effect on concanavalin A-induced IL-2, in vivo EC50 value is 78 nM.

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Male Lewis rats (200-300 g) were used. A-770041 was dissolved in 5% ethanol in sterile water for all in vivo studies. For the concanavalin A-induced cytokine production study, male Lewis rats were dosed with 2.5 mg/kg A-770041 or vehicle (1 ml/kg). At 2, 6, 10, and 22 hours after dosing, rats were administered 5 mg/kg concanavalin A (0.5 ml in saline) i.v. via the tail vein. Two hours after concanavalin A administration (i.e., 4, 8, 12, and 24 hours after test compound dosing), rats were euthanized with CO₂, blood was collected by cardiac puncture into heparinized tubes, and plasma was separated [1].
For the heterotopic heart transplantation study, hearts were transplanted from Brown Norway (donor) to Lewis (recipient) rats. Recipient animals were treated with compounds beginning on day -1, and the day of surgery was denoted as day 0. The donor and recipient were anesthetized by inhalation of isoflurane. The graft aorta was anastomosed end-to-side to the recipient abdominal aorta, and the graft pulmonary artery to the vena cava with 7-0 synthetic suture. The abdomen was palpated daily to determine whether the graft was beating. Blood was collected either via the tail vein (200 μl) during survival studies or from the vena cava upon scheduled sacrifice, allowed to clot, and serum was separated and kept at -70°C until analysis [1].
For the 14-day survival study, recipient animals were treated with vehicle control or 2.5 to 20 mg/kg/day of A-770041 in equally divided doses 12 hours apart beginning the day before surgery. Animals were treated for 14 days, and graft viability was assessed by abdominal palpation each day [1].
For the 65-day survival study, recipients were treated with 10 or 20 mg/kg/day of A-770041 in equally divided doses 12 hours apart or 10 mg/kg/day of Cyclosporin A once per day for 65 days [1].
For mouse in vivo studies, compounds were administered orally, and inhibition of TCR-stimulated (anti-CD3 mAb) IL-2 production was measured 2.5 hours after dosing [2].
A-770041 was dosed orally in equally divided doses 12 hours apart in rats for transplant studies [2].

A-770041 has an oral bioavailability (F) of 34.1 ± 7.2% at 10 mg/kg in rats [1].
Half-life (t1/2) is 4.1 ± 0.1 hours in rats [1].
Cmax is 2186 ± 410 nM at 5.3 hours at a dose of 10 mg/kg orally in rats [1].
Area under the curve scaled linearly with dose up to 30 mg/kg when given orally in rats [1].
In mice: Cmax = 3871 ng/ml, Tmax = 1.0 h, Vd = 10.3 L/kg, Clp = 1.5 L/h/kg, T1/2 = 4.7 h, F = 27% [2].
In rats: Cmax = 2079 ng/ml, Tmax = 4.7 h, Vd = 1.2 L/kg, Clp = 0.2 L/h/kg, T1/2 = 4.1 h, F = 52% [2].
A-770041 has a 7-fold lower clearance and a 2-fold higher bioavailability in rat compared to mouse [2].
Single-dose pharmacokinetic studies showed that the area under the curve scaled linearly with dose up to 30 mg/kg when the compound was given orally [1].
Pharmacokinetic modeling determined that twice daily oral dosing in rats is required to maintain Cmin blood concentrations above 1 μM [1].
Serum concentrations of A-770041 in the 14-day transplantation study at 10 mg/kg/day: Day 0: 574 ± 114 nM, Day 3: 1467 ± 446 nM, Day 7: 1369 ± 848 nM, Day 14: 1065 ± 224 nM (C12h, just before the next dose) [1].
At 20 mg/kg/day: Day 0: 994 ± 174 nM, Day 3: 3133 ± 1583 nM, Day 7: 3072 ± 1300 nM, Day 14: 2963 ± 1583 nM [1].

[1]. A-770041, a novel and selective small-molecule inhibitor of Lck, prevents heart allograft rejection.J Pharmacol Exp Ther. 2005 Oct;315(1):36-41.

[2]. Discovery of A-770041, a src-family selective orally active lck inhibitor that prevents organ allograft rejection Bioorganic & Medicinal Chemistry Letters Volume 16, Issue 1, 1 January 2006, Pages 118-122.

N-[4-[1-[4-(4-acetyl-1-piperazinyl)cyclohexyl]-4-amino-3-pyrazolo[3,4-d]pyrimidinyl]-2-methoxyphenyl]-1-methyl-2-indolecarboxamide is an aromatic amide.

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Lck inhibition is an attractive target to prevent acute rejection. A-770041 is an orally bioavailable pyrazolo[3,4-d]pyrimidine with increased selectivity for Lck compared with previously reported compounds such as A-420983. A-420983 is a 37 nM Lck inhibitor but lacks selectivity within the Src family (only 8-fold selective against Fyn). A-770041 was designed to have greater selectivity against Src and Fgr to avoid potential detrimental effects implied from knockout mouse studies [1].
A-770041 is an ATP-competitive inhibitor of Lck [1].

C34H39N9O3

621.73196

621.318

869748-10-7

9549184

White to off-white solid powder

5.316

2

8

6

46

1070

0

ZMNWFTYYYCSSTF-UHFFFAOYSA-N

InChI=1S/C34H39N9O3/c1-21(44)41-14-16-42(17-15-41)24-9-11-25(12-10-24)43-33-30(32(35)36-20-37-33)31(39-43)23-8-13-26(29(19-23)46-3)38-34(45)28-18-22-6-4-5-7-27(22)40(28)2/h4-8,13,18-20,24-25H,9-12,14-17H2,1-3H3,(H,38,45)(H2,35,36,37)

N-[4-[1-[4-(4-acetylpiperazin-1-yl)cyclohexyl]-4-aminopyrazolo[3,4-d]pyrimidin-3-yl]-2-methoxyphenyl]-1-methylindole-2-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~40.21 mM)

Solubility in Formulation 1: 2.08 mg/mL (3.35 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.35 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.35 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)