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

The pharmacokinetic parameters obtained for 3/Neflamapimod in three species are summarized in Table 3. Systemic clearance was slightly higher than hepatic blood flow for rat and dog, and 3 appears to have significant extravascular distribution in all three species studied, as indicated by the volume of distribution at steady state. The oral pharmacokinetic profile of 3 in TPGS/PEG-400/water (2:7:1) was characterized in male BALB/c mice, Sprague–Dawley rats, and beagle dogs. The results of these single dose studies showed that 3 has excellent bioavailability in all three species (87%, 56%, and 69%, respectively). Compound 3 demonstrated a longer half-life following oral administration as compared to IV administration, suggesting an absorption-rate limited elimination process in the three species. Compound 3 is neither a significant inhibitor nor an inducer of human hepatic cytochrome p450 isozymes. The IC50 values for the inhibition of CYPs 3A4, 2D6, 2C19, 2C9, and 1A2 were determined to be >40 μM. The fraction of 3 bound to plasma protein was 98% and 92% in the rat and the dog, 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 is a member of the class of pyrimidopyridazines that is 6H-pyrimido[1,6-b]pyridazin-6-one substituted at positions 2 and 5 by (2,4-difluorophenyl)sulfanyl and 2,6-dichlorophenyl groups respectively It has a role as an EC 2.7.11.24 (mitogen-activated protein kinase) inhibitor, an anti-inflammatory drug and an apoptosis inducer. It is a pyrimidopyridazine, a difluorobenzene, a dichlorobenzene and an aryl sulfide.
Neflamapimod has been used in trials studying the treatment of Alzheimer's Disease and Mild Cognitive Impairment.

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VX-745, a lead anti-inflammatory candidate, small-molecule inhibitor of mitogen-activated protein kinase (MAPK), is under development by Vertex Pharmaceuticals Inc in association with Kissei Pharmaceutical Co Ltd for the potential treatment of rheumatoid arthritis (RA) [214928]. VX-745 was introduced by Vertex as a potential antiinflammatory drug for the treatment of RA in a pilot phase II trial initiated in November 1999 [346067]. In June 2000, phase II trials were still ongoing [371819] and in January 2001, Vertex initiated a randomized, double-blind, placebo-controlled phase II trial in adult patients with RA, with the objective of evaluating clinical response rates, self-reported patient health assessments and pharmacodynamic markers of drug activity [395083]. During the 33rd Annual Meeting of the American Chemical Society in May 2000, VX-745 was reported to be active against several isotypes of p38 MAPK, including p38alpha, p38beta and p38gamma [368149]. The targeting of p38 MAPK by VX-745 was associated with the suppression of the release of inflammatory mediators, including interleukin (IL)-1beta and tumor necrosis factor (TNF)alpha, known to be implicated in exacerbating the pathophysiology of RA [273648], [368149], [371548], [372054], [408713]. [2]

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p38 mitogen-activated protein kinase (MAPK) is a member of the MAPK family which is activated by cytokines and growth factors, but its role in pathogenesis of multiple myeloma (MM) is unknown. In this study, we demonstrate that the specific p38 MAPK inhibitor VX-745 inhibits interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) secretion in bone marrow stromal cells (BMSCs), without affecting their viability. Tumor necrosis factor alpha (TNF-alpha)-induced IL-6 secretion in BMSCs is also inhibited by VX-745. Importantly, VX-745 inhibits both MM cell proliferation and IL-6 secretion in BMSCs triggered by adherence of MM cells to BMSCs, suggesting that it can inhibit paracrine MM cell growth in the BM milieu and overcome cell adhesion-related drug resistance. These studies therefore identify p38 MAPK as a novel therapeutic target to overcome drug resistance and improve patient outcome in MM. [4]

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The synthesis of novel, selective, orally active 2,5-disubstituted 6H-pyrimido[1,6-b]pyridazin-6-one p38α inhibitors is described. Application of structural information from enzyme–ligand complexes guided the selection of screening compounds, leading to the identification of a novel class of p38α inhibitors containing a previously unreported bicyclic heterocycle core. Advancing the SAR of this series led to the eventual discovery of 5-(2,6-dichlorophenyl)-2-(2,4-difluorophenylthio)-6H-pyrimido[1,6-b]pyridazin-6-one (VX-745). VX-745 displays excellent enzyme activity and selectivity, has a favorable pharmacokinetic profile, and demonstrates good in vivo activity in models of inflammation.
To evaluate the in vivo therapeutic potential of compound 3, we employed the murine collagen-induced arthritis model of human rheumatoid arthritis, in which DBA/1J mice were immunized twice with chick type II collagen. Treatment was initiated once an inflammation score of 2 in each paw had been reached, corresponding to focal swelling of the wrist joint. Results showed that mice treated with 2.5, 5, and 10 mg/kg of compound 3 twice daily for 20 days had 27%, 31%, and 44% improvement in the inflammatory scores, respectively, when compared to vehicle-treated mice at the end of treatment (Table 4; see Supporting Information for details). In addition, histological scores showed a 32–39% protection of bone and cartilage erosion.
Compound 3 (VX-745) and other analogues described in this study represent the first known examples of 5-phenyl-2-(phenylthio)-6H-pyrimido[1,6-b]pyridazin-6-ones as p38 inhibitors. These compounds show potent inhibition of the key inflammatory mediators, IL-1β and TNFα, production in vitro in isolated PBMCs and whole blood. Compound 3 has also been shown to demonstrate anti-inflammatory efficacy in an animal model of rheumatoid arthritis and was 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)