p38α (IC50 = 10 nM); p38β (IC50 = 220 nM)

VX-745 selectively inhibits p38α and p38β MAPK with IC50 values of 10 nM and 220 nM, respectively. It does not, however, inhibit p38γ MAPK or a significant number of other kinases with IC50 values greater than 20 M. VX-745 has an IC50 of 56 and 52 nM for IL-1β and TNFα in a human peripheral blood mononuclear cell (PBMC) assay, respectively. The IL-1 and TNFα induced production of IL-6 and IL-8 as well as the LPS and IL-1β-mediated synthesis of COX-2 are both inhibited by VX-745.[1-3] VX-745 (60 nM-20 µM) inhibits bone marrow stromal cells' (BMSCs') production of IL-6 and VEGF without compromising their viability. Additionally, VX-745 prevents BMSCs from secreting IL-6 when TNF-α is present. Inhibiting both multiple myeloma (MM) cell proliferation and IL-6 secretion in BMSCs that is brought on by MM cells adhering to BMSCs suggests that VX-745 can inhibit paracrine MM cell growth in the BM milieu and overcome drug resistance related to cell adhesion.[4]

VX-745 has an ED50 of 5 mg/kg against adjuvant-induced arthritis (AA) in rats. VX-745 inhibits bone resorption by 93% and inflammation by 56% in AA rats, according to histological results. VX-745 shows a dose-responsive decline in severity score in the traditional cartilage-induced arthritis model. [1-3] When compared to mice that received vehicle treatment, VX-745 (2.5, 5, and 10 mg/kg) improved the inflammatory scores in a type II collagen-induced arthritis (CIA) mouse model by 27%, 31%, and 44%, respectively. Additionally, VX-745 exhibits a 32–39% protection against bone and cartilage erosion according to histological scores. [5]

Using a spectrophotometric coupled-enzyme assay, the IC50 for inhibiting p38α and p38β homologs is determined. A fixed concentration of enzyme (15 nM of p38α or p38β) is incubated with VX-745 in DMSO for 10 min. at 30 °C in 0.1 M HEPES buffer, pH 7.5, containing 10% glycerol, 10 mM MgCl2, 2.5 mM phosphoenolpyruvate, 200 µM NADH, 150 µg/mL pyruvate kinase, 50 µg/mL lactate dehydrogenase, and 200 µM EGF receptor peptide (KRELVEPLTPSGEAPNQALLR). For the p38α and p38β assays, 100 µM and 70 µM ATP, respectively, are used to start the reaction. To track the reaction's progress, the decrease in absorbance at 340 nm is measured. As a function of inhibitor concentration, IC50 is calculated from rate data. [5]

In 96-well culture plates, BMSCs (5 × 104 cells/well) or MM cells (3 × 104 cells/well) are incubated for 48 hours at 37 °C in the presence or absence of Neflamapimod/VX-745. The uptake of [3H]-thymidine ([3H]TdR) is used to measure DNA synthesis. During the final eight hours of 48-hour cultures, [3H]TdR (0.5 μCi/well [.0185 MBq]) is pulsed into the cells. By measuring the absorbance of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye, the growth inhibition of both MM cells and BMSCs by VX-745 is also determined.

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DNA synthesis and growth inhibition assay [4]
Proliferation was measured as previously described. MM cells (3 × 104cells/well) or BMSCs (5 × 104cells/well) were incubated in 96-well culture plates in the presence or absence of Neflamapimod/VX-745 for 48 hours at 37°C. DNA synthesis was measured by [3H]-thymidine ([3H]TdR) uptake. Cells were pulsed with [3H]TdR (0.5 μCi/well [.0185 MBq]) during the last 8 hours of 48-hour cultures. Growth inhibition of both MM cells and BMSCs by VX-745 was also assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye absorbance, as previously described.16 All experiments were performed in quadruplicate.

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Effect of Neflamapimod/VX-745 on paracrine MM cell growth in the BM [4]
To evaluate growth stimulation and signaling in MM cells adherent to BMSCs, 3 × 104 MM.1S cells were cultured in BMSC-coated 96-well plates for 48 hours, in the presence or absence of Neflamapimod/VX-745. DNA synthesis was measured as described above. The Duoset enzyme-linked immunosorbent assay (ELISA) was used to measure IL-6 and VEGF in supernatants of 48-hour cultures of BMSCs with or without MM.1S cells, and in the presence or absence of VX-745, as previously described.

Type II collagen-induced arthritis (CIA) mice model (DBA/1J)
2.5, 5, and 10 mg/kg
Oral gavage twice daily

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Type II Collagen-Induced Arthritis (CIA) Model in Male DBA/1 Mice [5]
The CIA model is established in 10-week-old male DBA/1 mice with minor modifications. Mice are immunized via two intradermal injections administered 3 weeks apart, each consisting of 100 μL of an emulsion prepared by mixing chick type II collagen (200 μg in 10 mM acetic acid) 1:1 (v/v) with complete Freund’s adjuvant.
After the booster immunization, mice are monitored for 2–3 weeks until they develop focal carpal (wrist) swelling in both forepaws, corresponding to an arthritic severity score of level 2. At this stage, the mice are randomized into five treatment groups:

1. Water control: 10 mL/kg, oral (p.o.), twice daily (bid), n = 14
2. 100% propylene glycol (PG) vehicle control: 10 mL/kg, p.o., bid, n = 8
3. Neflamapimod in PG: 10 mg/kg, p.o., bid, n = 7
4. Neflamapimod in PG: 5 mg/kg, p.o., bid, n = 10
5. Neflamapimod in PG: 2.5 mg/kg, p.o., bid, n = 11

Arthritic Scoring:
Clinical symptoms are assessed every other day using a 5-level scoring system:

• Level 1: Wrist erythema
• Level 2: Focal wrist swelling
• Level 3: Complete wrist swelling
• Level 4: Wrist and palm swelling
• Level 5: Swelling of wrist, palm, and fingers

Disease progression is plotted using the combined scores from both forepaws.

Histopathological Evaluation:
On day 20, mice are euthanized, and forepaws are collected, sagittally sectioned, and stained with hematoxylin & eosin (H&E). Joint inflammation is graded as follows:
• Level 1: Synovial infiltration into the joint space
• Level 2: Cartilage erosion
• Level 3: Cartilage and bone erosion
• Level 4: Advanced erosion with pannus formation

Table 3 summarizes the pharmacokinetic parameters of compound 3/Neflamapimod in the three animal groups. Systemic clearance was slightly higher in rats and dogs than in hepatic blood flow, and compound 3 exhibited significant extravascular distribution in all three animal groups, as confirmed by the steady-state volume of distribution. The oral pharmacokinetic characteristics of compound 3 in TPGS/PEG-400/water (2:7:1) were characterized in male BALB/c mice, Sprague-Dawley rats, and beagle dogs. Single-dose studies showed that compound 3 had good bioavailability in all three animal groups (87%, 56%, and 69%, respectively). The longer half-life of compound 3 after oral administration compared to intravenous administration suggests that its elimination in the three animals is rate-limited by absorption. Compound 3 is neither a significant inhibitor nor an inducer of human hepatic cytochrome P450 isoenzymes. The IC50 values of compound 3 for inhibition of CYP 3A4, 2D6, 2C19, 2C9, and 1A2 were all greater than 40 μM. Compound 3 showed binding rates of 98% and 92% to plasma proteins in rats and dogs, respectively.

[1]. American Chemical Society, 2000, IDDB3.

[2]. Curr Opin Investig Drugs . 2001 Aug;2(8):1070-6.

[3]. IDrugs . 2000 Sep;3(9):983-9.

[4]. Blood . 2003 Jan 15;101(2):703-5.

[5]. ACS Med Chem Lett, 2011, 2(10), 758-763.

VX-745 belongs to the pyrimidopyridazine class of compounds with the structure 6H-pyrimido[1,6-b]pyridazine-6-one, substituted at positions 2 and 5 with (2,4-difluorophenyl)thio and 2,6-dichlorophenyl, respectively. It exhibits EC 2.7.11.24 (mitogen-activated protein kinase) inhibitor, anti-inflammatory, and apoptosis-inducing effects. It is a pyrimidopyridazine, difluorobenzene, dichlorobenzene, and aryl sulfide. Neflamapimod has been used in research trials for the treatment of Alzheimer's disease and mild cognitive impairment. VX-745 is a leading anti-inflammatory candidate, belonging to the mitogen-activated protein kinase (MAPK) small molecule inhibitor class, developed by Vertex Pharmaceuticals Inc. in collaboration with Kissei Pharmaceutical Co Ltd. for the treatment of rheumatoid arthritis (RA) [214928]. In November 1999, Vertex initiated a Phase II pilot trial of VX-745 as a potential anti-inflammatory drug for the treatment of RA [346067]. The Phase II clinical trial was still ongoing in June 2000 [371819]. In January 2001, Vertex initiated a randomized, double-blind, placebo-controlled Phase II clinical trial in adult patients with rheumatoid arthritis (RA) to evaluate clinical response rates, patient-reported health status, and pharmacodynamic parameters of drug activity [395083]. At the 33rd Annual Meeting of the American Chemical Society in May 2000, it was reported that VX-745 was active against multiple p38 MAPK subtypes, including p38α, p38β, and p38γ [368149]. VX-745 targets p38 MAPK to inhibit the release of inflammatory mediators, including interleukin (IL)-1β and tumor necrosis factor (TNF) α, which are known to be associated with exacerbation of the pathophysiological process of rheumatoid arthritis (RA) [273648], [368149], [371548], [372054], [408713]. [2] p38 mitogen-activated protein kinase (MAPK) is a member of the MAPK family and can be activated by cytokines and growth factors, but its role in the pathogenesis of multiple myeloma (MM) is unclear. This study shows that the specific p38 MAPK inhibitor VX-745 can inhibit the secretion of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in bone marrow stromal cells (BMSCs) without affecting their cell viability. VX-745 can also inhibit the secretion of IL-6 in BMSCs induced by tumor necrosis factor α (TNF-α). More importantly, VX-745 can inhibit MM cell proliferation and IL-6 secretion in BMSCs induced by MM cell adhesion to BMSCs, suggesting that it can inhibit paracrine growth of MM cells in the bone marrow microenvironment and overcome cell adhesion-related drug resistance. Therefore, these studies identify p38 MAPK as a novel therapeutic target for overcoming MM drug resistance and improving patient prognosis. [4] This article describes the synthesis of novel, selective, orally active 2,5-disubstituted 6H-pyrimido[1,6-b]pyridazine-6-one p38α inhibitors. Using the structural information of the enzyme-ligand complex to guide the screening of compounds, a class of novel p38α inhibitors containing previously unreported bicyclic heterocyclic core structures were finally identified. Further investigation of the structure-activity relationship of this series of compounds finally led to the discovery of 5-(2,6-dichlorophenyl)-2-(2,4-difluorophenylthio)-6H-pyrimido[1,6-b]pyridazine-6-one (VX-745). VX-745 exhibits excellent enzyme activity and selectivity, favorable pharmacokinetic characteristics, and good in vivo activity in an inflammation model. To evaluate the in vivo therapeutic potential of compound 3, we used a mouse collagen-induced arthritis model (mimicking human rheumatoid arthritis), in which DBA/1J mice were immunized twice with chicken type II collagen. Treatment began when the inflammation score of each paw reached 2 points, at which point local swelling of the wrist joint was observed. The results showed that, compared with mice treated with the carrier at the end of treatment, mice treated twice daily with 2.5, 5, and 10 mg/kg of compound 3 for 20 days showed improvements in inflammation scores of 27%, 31%, and 44%, respectively (Table 4; see Supplementary Information for details). Furthermore, histological scores showed a protection rate of 32-39% against bone and cartilage erosion. Compound 3 (VX-745) and other analogues described in this study are among the first known examples of 5-phenyl-2-(phenylthio)-6H-pyrimidino[1,6-b]pyridazine-6-one compounds as p38 inhibitors. These compounds have shown potent inhibition of the production of key inflammatory mediators IL-1β and TNFα in PBMCs isolated in vitro and in whole blood. Compound 3 has also shown anti-inflammatory efficacy in animal models of rheumatoid arthritis and has been designated for further development. [5]

C19H9CL2F2N3OS

436.26

434.981

C, 52.31; H, 2.08; Cl, 16.25; F, 8.71; N, 9.63; O, 3.67; S, 7.35

209410-46-8

3038525

Yellow solid powder

1.6±0.1 g/cm3

578.9±60.0 °C at 760 mmHg

303.9±32.9 °C

0.0±1.6 mmHg at 25°C

1.692

4.79

0

5

3

28

762

0

ClC1C([H])=C([H])C([H])=C(C=1C1C(N=C([H])N2C=1C([H])=C([H])C(=N2)SC1C([H])=C([H])C(=C([H])C=1F)F)=O)Cl

VEPKQEUBKLEPRA-UHFFFAOYSA-N

InChI=1S/C19H9Cl2F2N3OS/c20-11-2-1-3-12(21)17(11)18-14-5-7-16(25-26(14)9-24-19(18)27)28-15-6-4-10(22)8-13(15)23/h1-9H

5-(2,6-dichlorophenyl)-2-(2,4-difluorophenyl)sulfanylpyrimido[1,6-b]pyridazin-6-one

VD31745; VX 745; VX745; 209410-46-8; Neflamapimod; 5-(2,6-Dichlorophenyl)-2-((2,4-difluorophenyl)thio)-6H-pyrimido[1,6-b]pyridazin-6-one; Neflamapimod [USAN]; VRT-031,745; VRT 031745; VD 31745; VX-745; VRT-031745, VD-31745; VRT031745

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)