Wild type BTK (IC50 = 0.85 nM), C481S-BTK (IC50 = 0.39 nM)[1].

In the TMD8 cell line, ARQ 531 exhibits significant target inhibition. The IC50 values of WT-BTK and C481S-BTK in biochemical experiments were 0.85 nM and 0.39 nM, respectively. Furthermore, ARQ 531 also has significant inhibitory effects on TEK kinase, as demonstrated by IC50 values of 3.6.4 nM (TXK), 5.80 nM (TEC), and 5.23 nM (BMX). 3.86 nM (LCK), 4.22 nM (YES), 9.71 nM (BLK), 18.3 nM (HCK), 18.8 nM (LYNa), 25.9 nM (FGR), 32.2 nM (FYN), 48.0 nM (FRK) and TRK kinases 11.7 nM (TrkB), 13.1 nM (TrkA), and 19.1 nM (TrkC) are the IC50 of ARQ 531 against SRC kinase. ARQ 531 effectively suppresses the growth of several cell lines (TMD8: GI50=0.13 μM, REC1: GI50=0.18 nM) and is effective against cell lines that are dependent on the PI3K/AKT pathway, Src family kinases, and BCR.

ARQ 531 works well in tumor xenograft models, such as TMD-8. After 14 days, ARQ 531 therapy completely reduced the tumor. In models of arthritis caused by collagen, ARQ 531 is also beneficial. In animal studies, ARQ 531 shown strong efficacy against arthritis. ARQ 531 demonstrated remarkable anticancer efficacy and long-lasting effects in a TMD8 xenograft mouse model driven by BTK. In a mouse model of collagen-induced arthritis (CIA), ARQ 531 showed in vivo effectiveness [1].

BTK biochemical and kinase selectivity assay [1]
Biochemical inhibition assay was measured using full length BTK constructs of wild type or C481S mutant. Kinase profiling was performed by CROs. Profiling on 236 kinases identified 45 kinases with >50% inhibition at 200 nM concentration of ARQ 531. Subsequently, the potency of this ATP competitive inhibitor was determined on such kinases at the physiological 1mM ATP concentration.

---

In vitro PD assay [1]
Cells were treated with increasing concentrations of inhibitors in SUDHL-4 for 2 hours, following stimulation with either anti-IgM or growth factors cells were lysed for Western blot analysis.

---

BTK human PBMC functional assay [1]
Fresh blood was collected from donors and PBMC was isolated using Lymphoprep density gradient protocol. CD69 expression on CD20+ B-cells was assayed with increasing concentrations of ARQ 531 followed by IgM stimulation. The percentage of CD69 expression levels on CD20+ B-Cells was determined by flow cytometry analysis to estimate the potency of ARQ 531.

Cell growth assay [1]
Cells were seeded in 96-well tissue-culture treated plates and treated with compounds or vehicle for 72-hours. CellTiter-Glo reagent was added, and plates were incubated with gentle shaking for 2 minutes. The reaction was allowed to proceed for 10 additional minutes, and then plates were read on a Victor® microplate reader. GI50 values were determined using a fourparameter logistic curve fit.

---

Reverse phase protein assay (RPPA) [1]
TMD8 cells were grown in 6-well plates, and treated with 0.3 μM or 3 μM ARQ 531 or DMSO vehicle for 2 hrs. Cells were then lysed following the RPPA procedure provided by Carna Bio Science, and protein concentration was determined by Bradford. Samples were shipped to Carna for analysis of phospho-protein expression. Data were normalized to the control samples and relative values for individual targets were grouped in their assigned pathways.

Animal care [1]
Six week old female CB-17 SCID mice were purchased commercially and allowed to acclimate 1-2 weeks. Mice were housed in sterile micro isolator cages, five mice per cage and receive food and water ad libitum. All experimental procedures were approved in accordance with ArQule’s Institutional Animal Care and Use Committee (IACUC).

---

Tumor model [1]
Female SCID mice were implanted subcutaneously with 8x106 TMD8 cells in 0.2ml sterile Hanks Balanced Salt Solution (HBSS) with 50% standard concentration BD matrigel in the upper right flank area. Mice were monitored and staged on day 14 (post injection of tumor cells) when size reached approximately 400mg. Oral daily dosing with ARQ 531 at 100 mg/kg, vinblastine or vehicle began on stage day. Tumor measurements and body weights were collected three times a week with electronic calipers and balance. Tumor weight (mg) was calculated from the equation, length x (width)2 /2 and the tumor volume was calculated by assuming unit density 1mg equals to 1mm3.

---

In vivo PD analysis [1]
In vivo Target and pathway inhibition was studied in mouse TMD8 xenograft model. Following oral dosing of BTK inhibitors tumor tissues and plasma were collected for Western blot analysis of phosphorylated proteins and ARQ 531 levels in plasma. Percent inhibition relative to the vehicle control was determined using densitometry analysis and the intensity of actin band was used as a loading control and the percentage of vehicle group was designated as 100%.

---

Collagen-induced arthritis model [1]
DBA1/J mice were immunized with collagen to develop the arthritis, following the onset of arthritis, mice were randomized into treatment groups. Treatment was initiated by oral dosing of ARQ 531 at 25, 50 and 75 mg/Kg and continued daily (QD at 24-hour intervals) through arthritis day 14. Clinical scores were assessed for each of the paws on study arthritis days 1–15. Dexamethasone at 3 mg/Kg was used as a control to monitor inhibition of disease symptoms ADME and pharmacokinetics studies CYP450 inhibition and P-gp substrate/inhibition potential for ARQ 531 as well as pharmacokinetics studies in monkeys and dogs were determined and analyzed by Covance Laboratories Inc.

ARQ 531 has high oral bioavailability, good ADME, pharmacokinetic and metabolic properties.
t1/2 = 9.3 hrs (dogs); 13.6 hours (monkeys)
F = 38% (dogs); 72% (monkeys)

[1]. Targeting PCI-32765-Resistant BTK-C481S Mutation with ARQ 531, a Reversible Non-Covalent Inhibitor of BTK. Clinical Lymphoma Myeloma & Leukemia, 2016, 16: S47-S48.

Nemtabrutinib is an orally available reversible inhibitor of Bruton's tyrosine kinase (BTK; Bruton agammaglobulinemia tyrosine kinase), with potential antineoplastic activity. Upon administration, nemtabrutinib non-covalently binds to and inhibits the activity of both the wild-type and the C481S mutated form of BTK, a resistance mutation in the BTK active site in which cysteine is substituted for serine at residue 481. This prevents the activation of the B-cell antigen receptor (BCR) signaling pathway and BTK-mediated activation of downstream survival pathways. This leads to an inhibition of the growth of malignant B-cells that overexpress BTK. Compared to other BTK inhibitors, nemtabrutinib does not require interaction with the BTK C481 site and inhibits the proliferation of cells harboring the BTK C481S mutation. BTK, a member of the Src-related BTK/Tec family of cytoplasmic tyrosine kinases, is overexpressed or mutated in B-cell malignancies; it plays an important role in the development, activation, signaling, proliferation and survival of B-lymphocytes.

---

Drug Indication
Treatment of mature B-cell malignancies

C25H23CLN4O4

478.927524805069

478.14

C, 62.70; H, 4.84; Cl, 7.40; N, 11.70; O, 13.36

2095393-15-8

129045720

White to off-white solid powder

1.4±0.1 g/cm3

731.6±60.0 °C at 760 mmHg

396.2±32.9 °C

0.0±2.5 mmHg at 25°C

1.687

3.87

3

7

7

34

681

2

ClC1C=C(C=CC=1C(C1=CNC2=C1C(=NC=N2)N[C@H]1CO[C@H](CO)CC1)=O)OC1C=CC=CC=1

JSFCZQSJQXFJDS-QAPCUYQASA-N

InChI=1S/C25H23ClN4O4/c26-21-10-17(34-16-4-2-1-3-5-16)8-9-19(21)23(32)20-11-27-24-22(20)25(29-14-28-24)30-15-6-7-18(12-31)33-13-15/h1-5,8-11,14-15,18,31H,6-7,12-13H2,(H2,27,28,29,30)/t15-,18+/m1/s1

(2-chloro-4-phenoxyphenyl)(4-(((3R,6S)-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)methanone

ARQ531; MK1026; ARQ-531; MK-1026; MK-1026; JTZ51LIXN4; (2-Chloro-4-phenoxyphenyl)(4-(((3R,6S)-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-7h-pyrrolo[2,3-d]pyrimidin-5-yl)methanone; ARQ 531;

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 : ≥ 50 mg/mL (~104.40 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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: ≥ 2.08 mg/mL (4.34 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (4.34 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.

---

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