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Article Cite This: J. Med. Chem. 2018, 61, 865−880
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Discovery of the First Potent, Selective, and Orally Bioavailable Signal Peptide Peptidase-Like 2a (SPPL2a) Inhibitor Displaying Pronounced Immunomodulatory Effects In Vivo Juraj Velcicky,*,† Ursula Bodendorf,‡ Pascal Rigollier,† Robert Epple,⊥ Daniel R. Beisner,⊥,¢ Danilo Guerini,‡ Philip Smith,‡ Bo Liu,⊥ Roland Feifel,§ Peter Wipfli,§ Reiner Aichholz,§ Philippe Couttet,∥ Ina Dix,† Toni Widmer,# Ben Wen,⊥ and Trixi Brandl† †
Global Discovery Chemistry, ‡Autoimmunity Transplantation Inflammation, §Pharmacokinetic Sciences, ∥Preclinical Safety, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland ⊥ The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, United States # Chemical and Pharmaceutical Profiling, Global Drug Development, Novartis Pharma AG, CH-4002 Basel, Switzerland S Supporting Information *
ABSTRACT: Signal peptide peptidase-like 2a (SPPL2a) is an aspartic intramembrane protease which has recently been shown to play an important role in the development and function of antigen presenting cells such as B lymphocytes and dendritic cells. In this paper, we describe the discovery of the first selective and orally active SPPL2a inhibitor (S)-2-cyclopropyl-N1-((S)-5,11dioxo-10,11-dihydro-1H,3H,5H-spiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1′-cyclopropan]-10-yl)-N4-(5-fluoro-2-methylpyridin-3-yl)succinamide 40 (SPL-707). This compound shows adequate selectivity against the closely related enzymes γsecretase and SPP and a good pharmacokinetic profile in mouse and rat. Compound 40 significantly inhibited processing of the SPPL2a substrate CD74/p8 fragment in rodents at doses ≤10 mg/kg b.i.d. po. Oral dosing of 40 for 11 days at ≥10 mg/kg b.i.d. recapitulated the phenotype seen in Sppl2a knockout (ko) and ENU mutant mice (reduced number of specific B cells and myeloid dendritic cells). Thus, we believe that SPPL2a represents an interesting and druggable pharmacological target, potentially providing a novel approach for the treatment of autoimmune diseases by targeting B cells and dendritic cells.
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related to presenilins, the catalytic subunit of γ-secretase.5,6 SPPL2a is an aspartyl I-CLiP mainly located in the membrane of endosomes,7 containing nine transmembrane domains (TMD) bearing the two catalytic aspartates on TMD6 and TMD7. It belongs to the signal peptide peptidase/signal peptide peptidase-like (SPP/SPPL) family of aspartic intramembrane proteases which includes SPP, SPPL2a, SPPL2b, SPPL2c, and SPPL3. Whereas the topology of the γ-secretase complex enables cleavage of type I transmembrane proteins, members of the SPP/SPPL family cleave type II transmembrane proteins. In addition, γ-secretase is known as a complex, with presenilin being the catalytically active part and three other proteins (nicastrin, presenilin enhancer 2 (Pen-2)
INTRODUCTION
Regulated intramembrane proteolysis (RIP) is one of the processes controlling communication between cells and the extracellular environment.1 RIP is mediated by a special family of proteases, the intramembrane cleaving proteases (I-CLiPs), which control signaling pathways by cleaving the transmembrane region of their substrates into extracellular and intracellular domains.2 Depending on their catalytic center, metallo, aspartyl, and serine I-CLiPs have been described.3 The most prominent and best characterized member of this enzyme class is γ-secretase, an aspartyl I-CLiP involved in the generation of the Aβ peptide from the amyloid precursor protein (APP), which is found in amyloid plaques in the brain of Alzheimer disease patients and thus is involved in the pathology of Alzheimer’s disease.4 SPPL2a (signal peptide peptidase-like 2a) has recently been described as an enzyme © 2018 American Chemical Society
Received: September 15, 2017 Published: January 23, 2018 865
DOI: 10.1021/acs.jmedchem.7b01371 J. Med. Chem. 2018, 61, 865−880
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cancer subtypes and therefore focus of several clinical investigations of γ-secretase inhibitors for the treatment of cancer.22 However, inhibition of Notch signaling bares the risk of serious side effects including severe intestinal toxicity or development of skin cancer as seen in Alzheimer patients treated with γ-secretase inhibitors.23,24 Recently, it was shown that some γ-secretase inhibitors are only partially selective and inhibit also SPPL-family members.25,26 Therefore, we have tested several literature described γ-secretase inhibitors for their effect on SPPL2a along with 41 (Z-LL2),7 a literature known SPPL2a inhibitor. A reporter gene assay (RGA) was used to evaluate the inhibition of the SPPL2a activity. The assay uses HEK293 cells cotransfected with expression vectors for human SPPL2a, TNFα-NTF fused to the VP16 transactivation domain, Gal4 transcription factor, and a Gal4-driven luciferase reporter plasmid. In the absence of an SPPL2a inhibitor, the Nterminal fragment of the cleaved substrate fused to VP16/Gal4 translocates into the nucleus and drives expression of luciferase which is assessed by luminescent measurement. The same assay format was used also for the assessment of the inhibitory effect on γ-secretase mediated cleavage of Notch1. However, in this case, only a Notch1-based substrate fused to VP16/Gal4 and the luciferase reporter plasmid are cotransfected into HEK293 cells, which bear endogenous γ-secretase activity. While 41 was highly potent on SPPL2a with a selectivity of >1000 against γ-secretase processing of Notch1 (Table 1), its
and anteriorpharynx-defective 1 (Aph-1), while SPPL2a does not seem to require any additional cofactors.8 SPPL2a was initially shown to be engaged in the processing of the TNFα Nterminal fragment (NTF) in vitro, leading to IL-12 production in human dendritic cells.7 SPPL2a cleavage of type II membrane proteins like TNFα requires a prior shedding of their ectodomain by sheddases (mainly metalloproteases such as, e.g., ADAM 10 or ADAM17),7 a process which generates the actual SPPL2a substrates, the remaining membrane bound NTF.8 Several type II-oriented transmembrane proteins including FasL/CD95, 9 Bri2(Itm2b), 1 0 CD74, 1 1 − 1 3 TMEM106B,14 and NRG1 type III,15 and even a type III protein FVenv (viral),16 were shown to be processed by SPPL2a. To date, however, the NTF of CD74 (invariant chain of the major histocompatibility complex II, MHC-II) is the only substrate shown to be cleaved by SPPL2a in vivo.11−13 Prior to cleavage by SPPL2a, CD74 is processed sequentially by several proteases, including cathepsin S, which is necessary for the generation of the CLIP peptide, which plays an important role in MHC-II associated antigen presentation. Processing of fulllength CD74 finally leads to the generation of the N-terminal 8 kDa CD74, p8 (CD74/p8) transmembrane fragment, which in turn is further processed by SPPL2a generating a short cytoplasmic peptide p4 (4 kDa). Studies in SPPL2a deficient mice showed that accumulation of nonphysiological amounts of CD74/p8 leads to an arrest of splenic B cell maturation, resulting in the depletion of mature B cells. In addition, accumulation of p8 leads to a strong reduction in myeloid dendritic cell (mDC) numbers in SPPL2a-deficient mice.11−13 However, in Sppl2a/CD74 double knockout (ko) mice, the development of mature B cells and mDCs was restored,11 indicating that the accumulation of the CD74/p8 fragment has a detrimental effect on the development of antigen presenting cells like B cells and mDCs. The disturbed B cell development observed upon inhibition of SPPL2a correlates with an impaired tonic B cell receptor, and the accumulation of the CD74/p8 negatively impacts the PI3K/Akt signaling pathway, leading to activation of pro-apoptotic genes p21, p27, and Bim in B220+ cells.17 These results suggest that SPPL2a is strongly involved in the regulation of the adaptive immunity, and thus by inhibiting SPPL2a it might be possible to reduce its antigen presenting capacity.18 Such a reduction could be therapeutically beneficial in autoimmune diseases, among others rheumatoid arthritis or multiple sclerosis, where presentation of autoantigens is a strong driver of the pathology.19 To further investigate the effect of SPPL2a inhibition on the immune system, we aimed to develop an orally available, selective, low molecular weight SPPL2a inhibitor, and our results are disclosed in this publication.
Table 1. Potential Starting Points for Optimization of SPPL2a Inhibitors
compd
hSPPL2aa RGA [μM]
γ-secretaseb RGA [μM]
selectivity (γ-secretase/hSPPL2a)
41 42 30
0.006 ± 0.004 0.010 ± 0.005 0.005 ± 0.001
>10 1666 50 against γ-secretase was sufficient for avoiding the Notch-mediated toxic effects in vivo. The
ortho-position to aniline NH was key for achieving selectivity over γ-secretase for this series. The new analogues showed that 874
DOI: 10.1021/acs.jmedchem.7b01371 J. Med. Chem. 2018, 61, 865−880
Journal of Medicinal Chemistry
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(s, 18H), 0.63−0.68 (m, 4H). LCMS (method e) m/z: 619.4 [2 M + Na]+. tR = 1.57 min. Step 3: Hydrobromic acid solution (33 wt % in AcOH, 38.5 mL, 0.22 mol) was added slowly at 0 °C to a solution of di-tert-butyl 5,6-diazaspiro[2.4]heptane-5,6-dicarboxylate (12 g, 40 mmol) in Et2O (200 mL), and the mixture was stirred at rt for 16 h. After cooling to 0 °C, the solid was filtered off, washed with Et2O, and dried in vacuum to give the compound 16 as dihydrobromide (9.5 g, 36 mmol, 91% yield). 1H NMR (DMSO-d6, 400 MHz): δ 7.80 (br s, 4H), 3.06 (s, 4H), 0.76 (s, 4H). LCMS (method e) m/z: 99.2 [M + H]+. tR = 0.26 min. 1H-Spiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1′-cyclopropane]-5,11(3H,10H)-dione (17). Compound 16 dihydrobromide (5.2 g, 20 mmol) was added at rt to a solution of homophthalic anhydride (3.3 g, 20 mmol) in AcOH (35 mL) and pyridine (18 mL). The solution was then stirred at 130 °C for 20 h. After cooling to rt, the mixture was diluted with water and extracted with CH2Cl2. The combined organic layers were washed with 10% aq HCl, 5% aq NaHCO3, water, and brine, dried (Na2SO4), and concentrated. The crude product was purified by column chromatography (20−50% EtOAc in hexane) to afford the compound 17 (3.3 g, 14 mmol, 69% yield). 1H NMR (DMSO-d6, 400 MHz): δ 7.79 (dd, J = 7.7, 1.4 Hz, 1H), 7.54−7.58 (m, 1H), 7.41−7.47 (m, 2H), 4.20 (d, J = 11.0 Hz, 1H), 4.14 (d, J = 13.4 Hz, 1H), 3.88 (d, J = 10.6 Hz, 1H), 3.46−3.57 (m, 2H), 3.23 (d, J = 10.5 Hz, 1H), 0.75−0.79 (m, 4H). LCMS (method e) m/z: 243.2 [M + H]+. tR = 1.11 min. (S)-10-Amino-1H,3H,5H-spiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1′-cyclopropane]-5,11(10H)-dione (HCl Salt) (18). Step 1: 1 M LiHMDS in THF (26 mL, 26 mmol) was added dropwise at 0 °C to a suspension of 17 (4.1 g, 17 mmol) and isopentyl nitrite (3.4 mL, 26 mmol) in THF (34 mL). The reaction mixture was stirred at rt for 2 h. AcOH was added, and the mixture was evaporated twice in vacuum to yield the (E,Z)-10-(hydroxyimino)-1H-spiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1′-cyclopropane]-5,11(3H,10H)dione as an oil which was used in the next step without further purification. LCMS (method e) m/z: 272.2 [M + H]+, tR = 1.10 and 1.13 min. Step 2: 4 M aq HCl (4.3 mL, 17 mmol) was added at rt to a mixture of crude product from step 1 (4.6 g, 17.0 mmol) in AcOH (92 mL). The mixture was cooled to 0 °C, and Zn powder (4.5 g, 68 mmol) was added slowly. After the addition, the reaction mixture was stirred at rt for 1.5 h. The inorganic zinc residues were filtered off and washed with CH2Cl2. The filtrate was concentrated, redissolved in CH2Cl2, washed with 10% aq NaOH and brine, dried (Na2SO4), and concentrated. The residue was purified by column chromatography (0−4% MeOH in CH2Cl2(NH3)) to yield rac-10-amino-1H,3H,5Hspiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1′-cyclopropane]-5,11(10H)-dione (2.4 g, 9.3 mmol, 55%) as a white solid. 1H NMR (DMSO-d6, 400 MHz): δ 7.73−7.78 (m, 2H), 7.62 (td, J = 7.6, 1.4, 1H), 7.40−7.45 (m, 1H), 4.97 (s, 1H), 4.21 (d, J = 11.2 Hz, 1H), 3.88 (d, J = 10.8 Hz, 1H), 3.54 (d, J = 11.2 Hz, 1H), 3.27 (d, J = 10.8 Hz, 1H), 2.28 (s, 2H), 0.70−0.85 (m, 4H). LCMS (method e) m/z: 258.2 [M + H]+. tR = 0.79 min. Step 3: Boc2O (76 g, 347 mmol) was added at rt to the product of step 2 (68 g, 231 mmol) and Na2CO3 (110 g, 1.04 mol) in dioxane/water (1:1 mixture, 1.4 L), and the resulting mixture was stirred at rt for 2 h. The formed precipitate was filtered off, washed twice with water, and dried under vacuum. The solid was dissolved in dioxane containing 3% formic acid, and the enantiomers were separated by preparative chiral HPLC (Thar SFC-200 instrument; mobile phase, scCO2/EtOH 70:30; column, Chiralpak IC, 5 μM, 250 mm × 30 mm) to provide the (S)-enantiomer (42 g, 117 mmol, >99.5% ee, 48%) and the (R)-enantiomer (43 g, 120 mmol, >99.5% ee, 49%). Analytical data for the (S)-enantiomer. 1H NMR (DMSO-d6, 400 MHz): δ 7.81 (d, J = 7.5 Hz, 1H), 7.61−7.71 (m, 2H), 7.45−7.51 (m, 2H), 5.74 (d, J = 8.9 Hz, 1H), 4.21 (d, J = 11.1 Hz, 1H), 3.90 (d, J = 10.7 Hz, 1H), 3.62 (d, J = 11.1 Hz, 1H), 3.27 (d, J = 10.7 Hz, 1H), 1.42 (s, 9H), 0.71−0.82 (m, 4H). LCMS (method a) m/z: 358.2 [M + H]+. tR = 1.05 min. [α]23D −125.0 (c = 1.0, MeOH). Step 4: tert-Butyl (S)-(5,11-dioxo-10,11-dihydro-1H,3H,5H-spiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1′-cyclopropan]-10-yl)carbamate (2.0 g, 5.6 mmol) was dissolved in CH2Cl2 (28 mL) and treated at 0 °C with 4 M aq HCl in dioxane (28 mL, 112 mmol). After
suboptimal pharmacokinetic parameters were optimized based on the results obtained from a metabolic weak spot identification study. In mouse and rat, 40 showed highly significant inhibition of CD74/p8 processing at doses ≤10 mg/ kg b.i.d. po. In addition, after oral dosing of 40 for 11 days at 10 mg/kg b.i.d. to mice, the phenotype seen with ko and ENU mutant mice (reduced number of B cells and mDCs) was entirely recapitulated. Therefore, we believe that our findings show that SPPL2a is a novel target with promising therapeutic application and could be explored as an approach for the treatment of autoimmune diseases, where antigen presentation is an important driver of pathology.
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EXPERIMENTAL SECTION
Chemistry. All reagents and solvents were purchased from commercial suppliers and used without further purification. All reactions were performed under inert conditions (argon) unless otherwise stated. 1H and 13C NMR spectra were recorded on a Bruker 400 MHz or a Bruker 600 MHz NMR spectrometer. Chemical shifts are reported in parts per million (ppm) relative to an internal solvent reference. Significant peaks are tabulated in the order multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; quintet; m, multiplet; br, broad), coupling constants, and number of protons. Final compounds were purified to ≥95% purity as assessed by analytical liquid chromatography. LCMS method a: Waters UPLC Acquity; column, Acquity HSS T3 1.8 μm, 2.1 mm × 50 mm at 60 °C; eluent A, water + 0.05% HCOOH + 3.75 mM NH4OAc; eluent B, MeCN + 0.04% HCOOH; gradient, 10−95% B in 1.5 min; flow, 1.0 mL/min. LCMS method b: Waters UPLC Acquity; column, Acquity HSS T3 1.8 μm, 2.1 mm × 50 mm at 60 °C; eluent A, water + 0.05% HCOOH + 3.75 mM NH4OAc; eluent B, MeCN + 0.04% HCOOH; gradient, 5−98% B in 1.4 min; flow, 1.0 mL/min. LCMS method c: Waters UPLC Acquity; column, Acquity HSS T3, 1.8 μm, 2.1 mm × 50 mm, at 60 °C; eluent A, water + 0.05% HCOOH + 3.75 mM NH4OAc; eluent B, MeCN + 0.04% HCOOH; gradient, 5−98% B in 9.4 min hold 0.4 min; flow, 0.8 mL/min. LCMS method d: Agilent LCMS; column, Waters SunFire C18, 2.5 μm, 3 mm × 30 mm; eluent A, water + 0.1% HCOOH; eluent B, MeCN + 0.1% HCOOH; gradient, 10−98% B in 2.5 min; flow, 1.4 mL/min. LCMS method e: Agilent LCMS; column, Waters Acquity HSS T3, 1.8 μm, 2.1 mm × 50 mm, at 60 °C; eluent A, H2O + 0.05% TFA; eluent B, MeCN + 0.035% TFA; gradient, 10−100% B in 1.35 min; flow, 0.9 mL/min. Synthesis of Compound 40 (SPL-707). 5,6-Diazaspiro[2.4]heptane Dihydrobromide (16). Step 1: A solution of MsCl (57.2 mL, 734 mmol) in CH2Cl2 (160 mL) was added dropwise at 0 °C to a solution of cyclopropane-1,1-diyldimethanol (25 g, 245 mmol) and Et3N (136 mL, 979 mmol) in CH2Cl2 (250 mL). The reaction mixture was stirred at rt for 16 h. Then 1 M aq HCl (900 mL) was added and the mixture was extracted with CH2Cl2. The combined organic layers were washed with brine, dried (Na2SO4), and concentrated to a volume of 100−150 mL. Hexane was added, and the resulting precipitate was filtered off, washed with hexane, and dried in vacuum to give cyclopropane-1,1-diylbis(methylene) dimethanesulfonate (37.5 g, 145 mmol, 59%). 1H NMR (DMSO-d6, 400 MHz): δ 4.14 (s, 4H), 3.19 (s, 6H), 0.77 (s, 4H). Step 2: A solution of di-tert-butyl hydrazine1,2-dicarboxylate (18.6 g, 80 mmol) in dry DMF (65 mL) was added dropwise at 0 °C to a suspension of NaH (60% dispersion in oil, 6.7 g, 168 mmol) in dry DMF (40 mL), and the suspension was stirred at rt for 1 h. After addition of cyclopropane-1,1-diylbis(methylene) dimethanesulfonate (20.7 g, 80 mmol), the reaction mixture was stirred at rt for 16 h. It was then poured onto ice and water (1.3 L). The formed precipitate was filtered off, washed with water, and dried in vacuum to afford di-tert-butyl 5,6-diazaspiro[2.4]heptane-5,6dicarboxylate (22.2 g, 74 mmol, 93% yield). 1H NMR (DMSO-d6, 400 MHz): δ 3.56 (d, J = 10.6 Hz, 2H), 3.17 (d, J = 10.6 Hz, 2H), 1.41 875
DOI: 10.1021/acs.jmedchem.7b01371 J. Med. Chem. 2018, 61, 865−880
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Article
stirring at 0 °C for 1 h, the reaction mixture was concentrated. The residue was treated with CH2Cl2 and evaporated. It was then triturated with Et2O, the precipitate was filtered off and dried in vacuum to yield the product 18 as an HCl salt (1.7 g, 5.6 mmol, quant), white solid. 1H NMR (DMSO-d6, 400 MHz): δ 9.33 (br s, 3H), 7.89 (d, J = 7.4 Hz, 1H), 7.75 (t, J = 7.2 Hz, 1H), 7.60 (t, J = 7.3 Hz, 1H), 7.46 (d, J = 7.9 Hz, 1H), 5.83 (s, 1H), 4.26 (d, J = 11.1 Hz, 1H), 3.91 (d, J = 10.9 Hz, 1H), 3.53 (d, J = 11.1 Hz, 1H), 3.42 (d, J = 10.3 Hz, 1H), 0.74−0.89 (m, 4H). LCMS (method b) m/z: 258.2 [M + H]+. tR = 0.46 min. [α]23D −160.4 (c = 1.0, MeOH). (S)-4-Benzyl-3-(2-cyclopropylacetyl)oxazolidin-2-one (22). EDC (1.7 g, 8.9 mmol) was added at rt to a mixture of (S)-4benzyloxazolidin-2-one (800 mg, 4.5 mmol), 2-cyclopropylacetic acid (600 mg, 6.0 mmol), and DMAP (565 mg, 4.6 mmol) in CH2Cl2 (5 mL), and the mixture was stirred at rt for 16 h. The mixture was diluted with CH2Cl2 and washed with water, 1 M aq HCl, satd aq NaHCO3, satd aq NH4Cl, water, and brine, dried (Na2SO4), and concentrated to yield the compound 22 (1.01 g, 3.9 mmol, 86%) as a colorless oil. 1H NMR (CDCl3, 400 MHz): δ 7.32−7.38 (m, 2H), 7.28−7.32 (m, 1H), 7.23−7.25 (m, 2H), 4.70−4.78 (m, 1H), 4.19− 4.28 (m, 2H), 3.36 (dd, J = 13.4, 3.2 Hz, 1H), 2.97 (dd, J = 17.0, 6.7 Hz, 1H), 2.75−2.89 (m, 2H), 1.14−1.31 (m, 1H), 0.59−0.69 (m, 2H), 0.22−0.31 (m, 2H). LCMS (method b) m/z: 260.2 [M + H]+. tR = 1.05 min. [α]23D +90.6 (c = 1.0, MeOH). tert-Butyl (S)-4-((S)-4-Benzyl-2-oxooxazolidin-3-yl)-3-cyclopropyl4-oxobutanoate (23). First, 1 M NaHMDS in THF (5.9 mL, 5.9 mmol) was added dropwise at −78 °C to a solution of 22 (1.0 g, 3.9 mmol) in THF (30 mL). After stirring at −78 °C for 1 h, tert-butyl 2bromoacetate (1.2 mL, 7.8 mmol) was added and the solution was stirred for additional 1 h at −78 °C. The reaction mixture was treated with satd aq NH4Cl (2 mL) and allowed to warm to rt before it was dried (Na2SO4) and concentrated. The crude product was purified by column chromatography (0−50% EtOAc in hexane) to yield the compound 23 (0.8 g, 2.1 mmol, 54%) as a colorless oil. 1H NMR (CDCl3, 400 MHz): δ 7.27−7.38 (m, 5H), 4.71 (td, J = 6.7, 3.3 Hz, 1H), 4.13−4.22 (m, 2H), 3.70−3.79 (m, 1H), 3.39 (dd, J = 13.4, 3.2 Hz, 1H), 2.97 (dd, J = 16.8, 10.8 Hz, 1H), 2.73 (dd, J = 13.4, 10.3 Hz, 1H), 2.59 (dd, J = 16.8, 4.3 Hz, 1H), 1.43 (s, 9H), 0.89−1.01 (m, 1H), 0.48−0.61 (m, 2H), 0.40−0.48 (m, 1H), 0.18−0.35 (m, 1H). LCMS (method b) m/z: 374.3 [M + H]+. tR = 1.29 min. [α]23D +69.0 (c = 1.0, MeOH). (S)-4-(tert-Butoxy)-2-cyclopropyl-4-oxobutanoic Acid (24). Hydrogen peroxide (30% in water; 0.88 mL, 8.6 mmol) followed by LiOH (0.18 g, 4.3 mmol) in water (1 mL) were added at 0 °C to a solution of 23 (0.8 g, 2.1 mmol) in THF (20 mL), and the reaction mixture was stirred at 0 °C for 2 h. It was then treated with satd aq NaHSO3 (20 mL) and satd aq NaHCO3 (50 mL) at 0 °C. THF was distilled off under reduced pressure, and the aqueous layer was washed with CH2Cl2, then cooled to 0 °C, acidified with 4 M aq HCl to pH 2, and extracted with CH2Cl2. Combined organic extracts were dried (Na2SO4) and concentrated to yield the product 24 (0.34 g, 1.5 mmol, 70%) as a colorless viscous oil. 1H NMR (DMSO-d6, 400 MHz): δ 12.12 (s, 1H), 2.53 (dd, J = 16.3, 9.8 Hz, 1H), 2.40 (dd, J = 16.0, 5.3 Hz, 1H), 1.87−1.95 (m, 1H), 1.37 (s, 9H), 0.75−0.87 (m, 1H), 0.38− 0.49 (m, 2H), 0.29−0.36 (m, 1H), 0.12−0.21 (m, 1H). LCMS (method b) m/z: 213.2 [M − H]−. tR = 0.90 min. [α]23D +51.2 (c = 1.0, MeOH). (S)-3-Cyclopropyl-4-(((S)-5,11-dioxo-10,11-dihydro-1H,3H,5Hspiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1′-cyclopropan]-10yl)amino)-4-oxobutanoic Acid (29). Step 1: DIPEA (12.5 mL, 72 mmol) and HATU (11.0 g, 29 mmol) were added at rt to a mixture of 18 (HCl salt, 7.1 g, 24 mmol) and 24 (5.7 g, 26 mmol) in DMF (120 mL). The mixture was stirred at rt for 48 h. After concentration, the residue was dissolved in EtOAc, washed with water and brine, dried (Na2SO4), and concentrated. It was then purified by column chromatography (10−50% EtOAc in cyclohexane) to provide (S)tert-butyl 3-cyclopropyl-4-(((S)-5,11-dioxo-3,5,10,11-tetrahydro-1Hspiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1′-cyclopropan]-10yl)amino)-4-oxobutanoate (9.0 g, 20 mmol, 84%) as a white solid. 1H NMR (CDCl3, 400 MHz): δ 7.97 (d, J = 7.4 Hz, 1H), 7.48−7.54 (m,
2H), 7.41−7.45 (m, 1H), 7.30 (d, J = 6.8 Hz, 1H), 6.07 (d, J = 6.7 Hz, 1H), 4.47 (d, J = 11.4 Hz, 1H), 4.04 (d, J = 10.9 Hz, 1H), 3.54 (d, J = 11.3 Hz, 1H), 3.33 (d, J = 10.9 Hz, 1H), 2.93 (dd, J = 16.9, 9.4 Hz, 1H), 2.60 (dd, J = 16.9, 4.4 Hz, 1H), 2.14 (td, J = 9.7, 4.4 Hz, 1H), 1.48 (s, 9H), 1.05−1.12 (m, 1H), 0.78−0.97 (m, 4H), 0.62−0.75 (m, 2H), 0.50−0.56 (m, 1H), 0.29−0.35 (m, 1H). LCMS (method b) m/ z: 454.4 [M + H]+. tR = 1.10 min. [α]23D −58.0 (c = 1.0, MeOH). Step 2: TFA (30 mL, 390 mmol) was addded at rt to a solution of product from step 1 (8.8 g, 20 mmol) in CH2Cl2 (195 mL). The mixture was stirred at rt for 2 h and concentrated. The residue was triturated with Et2O, and the precipitate was filtered off and dried in vacuum to yield the compound 29 (7.4 g, 18 mmol, 95%) as a white solid. 1H NMR (CDCl3, 400 MHz): δ 7.98 (dd, J = 7.7, 1.4 Hz, 1H), 7.49−7.55 (m, 2H), 7.43 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 6.08 (d, J = 6.6 Hz, 1H), 4.48 (d, J = 11.4 Hz, 1H), 4.05 (d, J = 11.0 Hz, 1H), 3.55 (d, J = 11.4 Hz, 1H), 3.34 (d, J = 11.0 Hz, 1H), 3.05 (dd, J = 16.8, 9.3 Hz, 1H), 2.77 (dd, J = 16.8, 3.8 Hz, 1H), 2.18 (td, J = 9.8, 3.8 Hz, 1H), 1.09−1.17 (m, 1H), 0.95−1.00 (m, 1H), 0.82−0.92 (m, 4H), 0.71− 0.78 (m, 1H), 0.58−0.64 (m, 1H), 0.36−0.41 (m, 1H). LCMS (method b) m/z: 398.3 [M + H]+. tR = 0.74 min. [α]23D −54.6 (c = 1.0, MeOH). (S)-2-Cyclopropyl-N1-((S)-5,11-dioxo-10,11-dihydro-1H,3H,5Hspiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1′-cyclopropan]-10yl)-N4-(5-fluoro-2-methylpyridin-3-yl)succinamide (40). EDC (6.4 g, 33.5 mmol) was added at rt to a solution of 29 (6.7 g 16.8 mmol) and 5-fluoro-2-methylpyridin-3-amine hydrochloride (3.3 g 20 mmol) in pyridine (84 mL). The mixture was stirred at rt for 16 h. More 5fluoro-2-methylpyridin-3-amine hydrochloride (323 mg, 2 mmol) and EDC (765 mg, 4 mmol) were added, and the mixture was stirred at rt for 16 h. The reaction mixture was concentrated, the residue was dissolved in CH2Cl2 and extracted with satd aq NaHCO3, water, and brine, dried (Na2SO4), and concentrated. The crude material was purified by column chromatography (0−10% MeOH in CH2Cl2) followed by crystallization from MeOH/Et2O to provide the compound 40 (7.2 g, 14.2 mmol, 71%) as a white solid. 1H NMR (DMSO-d6, 400 MHz): δ 9.49 (s, 1H), 8.72 (d, J = 7.8 Hz, 1H), 8.21 (br s, 1H), 7.88 (d, J = 10.5 Hz, 1H), 7.80 (d, J = 7.1 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.42 (t, J = 7.3 Hz, 1H), 7.32−7.39 (m, 1H), 6.00 (d, J = 7.6 Hz, 1H), 4.24−4.26 (m, 1H), 4.24 (d, J = 11.1 Hz, 1H), 3.95 (d, J = 10.8 Hz, 1H), 3.66 (d, J = 11.0 Hz, 1H), 3.26−3.31 (m, 1H), 2.86−2.98 (m, 1H), 2.58−2.72 (m, 2H), 2.29 (s, 3H), 0.86−0.97 (m, 1H), 0.81 (s, 3H), 0.69−0.76 (m, 1H), 0.59−0.69 (m, 1H), 0.49−0.58 (m, 1H), 0.39−0.49 (m, 1H), 0.21−0.33 (m, 1H). 13C NMR (DMSOd6, 101 MHz): δ 173.6, 170.7, 167.0, 155.8, 146.5, 138.8, 133.3, 131.9, 131.7, 131.5, 130.0, 129.4, 127.8, 123.4, 117.2, 117.0, 52.6, 51.9, 45.5, 38.5, 20.4, 20.3, 14.1, 9.6, 9.4, 4.1, 3.6. LCMS (method c) m/z: 506.2 [M + H]+. tR = 3.97 min (100% purity). HRMS m/z: [M + H]+ calcd for C27H29FN5O4 506.2204, found 506.2196. [α]23D −104.4 (c = 1.0, MeOH). X-ray crystal structure confirmed the structure and absolute stereochemistry. X-ray Crystallographic Data. X-ray crystallographic data obtained for 40 has been deposited with the Cambridge Crystallographic Data Center as CCDC 1574028 and are included in Supporting Information. Cellular Assays. Cell Culture. The mouse B cell line A20 (TIB208, purchased from ATCC) was maintained in complete RPMI1640 medium containing 10% FBS. U-2 OS cells (HTB96, purchased from ATCC) were maintained in DMEM/GlutaMaxTM-I (Invitrogen) supplemented with 10% FBS (Invitrogen). HEK293 cells (CRL-1573, purchased from ATCC). All cells were negatively tested for mycoplasma contamination either by the vendor or in-house. SPPL2a and Notch1/γ-Secretase RGA’s in HEK293 Cells. The SPPL2a luciferase RGA is based on a TNFα(aa1-76)-NTF substrate and the γ-secretase assay has been developed using Notch1 as substrate. The latter assay has been described previously.26 In brief, DNA vectors encoding human Notch1-VP16-Gal4 fusion protein and a Gal4-luciferase reporter for the γ-secretase RGA, or human SPPL2a, VP16-TNFa(aa1-76)-NTF substrate, and the Gal4-luciferase reporter plasmid for the SPPL2a RGA, were transiently transfected in HEK293 cells using FuGENE (Promega). After transfection, the cell 876
DOI: 10.1021/acs.jmedchem.7b01371 J. Med. Chem. 2018, 61, 865−880
Journal of Medicinal Chemistry
Article
Metabolite Identification. In Vitro Experiment with Liver Microsomes. Stock solutions of 31 (2 mmol/L) were prepared in DMSO. Alamethicin solution was prepared (0.125 mmol/L) in water. UDPGA solution (24 mmol/L) was prepared in phosphate buffer 100 mM pH 7.4. In a further experiment, 50 mmol/L GSH in phosphate buffer 100 mM, pH 7.4, was added as a second cofactor. In all samples with active enzymes, a 1:1 mixture of GSH and [13C2-15N-glycine]GSH was used. In vitro metabolites were produced by incubation of 31 at 37 °C for up to 60 min with liver microsomes from mouse, rat, and human. Then 3 μL of liver microsomes, containing 20 mg of protein/mL, were mixed with 417 μL of phosphate buffer, 60 μL of alamethicin solution, and 60 μL of UDPGA. To this reaction mixture, 3 μL of stock solution (2 mM in DMSO) of 31 was added and preincubated for 3 min at 37 °C. After preincubation, the final reaction was started by addition of 60 μL of the NADPH-regenerating system, containing isocitrate-dehydrogenase (1 U/mL), NADP (1 mmol/L), and isocitrate (5 mmol/L). After 1 h, the reaction was stopped with 600 μL of ice-cold acetonitrile. Experiments were conducted according to a generic protocol. The reaction mixture was stored at −80 °C. Sample Preparation of In Vitro Samples. Prior to use, the mixture was centrifuged (10000g, 5 min) and 100 μL of supernatant diluted with 400 μL of water. This final solution was centrifuged again, and aliquots of 5−10 μL were used for HPLC/MS analysis. Capillary High-Performance Liquid Chromatography−Mass Spectrometry (HPLC/MS). Capillary HPLC was performed on a system consisting of a Chorus-220 HPLC pump (CTC Analytics, Zwingen, Switzerland), a Hot Dog-5090 column oven (Prolab, Reinach, Switzerland), and a HTS-PAL autosampler with cooled sample stacks (CTC Analytics, Zwingen, Switzerland). Separations were performed on a Reprosil-Pure-C18-AQ HPLC column (150 mm × 0.3 mm i.d., 3.0 μm particle size) from Maisch (AmmerbuchEntringen, Germany). Separations were performed at 45 °C. The flow rate was 4.5 μL/min, and the injection volume was 1 μL per separation. The solvent system used consisted of aqueous ammonium formate (10 mM, with 0.02% TFA, pH 4)/MeCN (95/5, v/v) as solvent A and aqueous ammonium formate (10 mM, with 0.02% TFA, pH 4)/MeCN/MeOH (5/90/5, v/v/v) as solvent B. The metabolites were separated using a linear solvent gradient: 0 min (5% B), 2 min (5% B), 27 min (95% B), and 32 min (95% B, 6.5 μL/min). Reequilibration of the column was performed at 5% B for 5 min. Prior to analysis, 10 μL of sample was diluted 1/100 (v/v) with water/MeCN (90/10, v/v). Samples were kept in the autosampler at 10 °C. A LTQ XL Orbitrap (Linear Quadrupole 2D Ion Trap/Orbitrap, Thermo Scientific, CA, USA) mass spectrometer was used operating in positive mode electrospray ionization (ESI). Settings for the mass spectrometer were 12 (arbitrary units) for sheath gas flow rate (N2) and a capillary temperature of 275 °C. Auxiliary and sweep gas were not used. The settings for capillary voltage and tube lens voltage were 45 and 95 V, respectively. Auto gain control (AGC) target settings were 5 × 105 and 1 × 104 for full MS and MS/MS, respectively. The resolution was set to 30000 in full scan mode. The omnipresent polysiloxane background ion [C2H6SiO]6+ with m/z 445.12003 was used as external lock mass. In Vivo Experiments. All animals used were 5−12 weeks of age and were maintained in the specific pathogen-free facilities. All animal studies were performed in accordance with the animal experimentation guidelines and laws laid down by the Swiss Federal and Cantonal Authorities. Procedures conducted in the US were approved by the Genomics Institute of the Novartis Research Foundation Institutional Animal Care and Use Committee. In Vivo Pharmacokinetic Studies in Rats. First, 96−120 h before administration of the test substance, adult female wild-type Sprague− Dawley rats (Iffa Credo, France) were anesthetized with isoflurane and catheters were surgically implanted under aseptic precautions (use of sterile instruments and surgical material in combination with local antibiotic prophylaxis) into the femoral artery and vein. Then the catheters were exteriorized in the neck region, connected to a Harvard swivel system (Harvard Instruments), and filled with 0.9% saline containing 100 U·mL−1 heparin. After recovery from anesthesia, the animals were housed individually in special cages with free access to
suspensions were diluted and distributed to white solid 384-well plate at 10000 cells/50 μL/well. After 3 h, 200 nL of compound in DMSO was stamped into the wells in concentration response covering final inhibitor concentrations of 10 μM to 0.3 nM in triplicate. Subsequently, plates were incubated for 24 h at 37 °C, 5% CO2 in a humidified incubator before the addition of 30 μL Bright Glo (Promega). After incubation for 5 min at rt, luminescence was measured and IC50 values were determined by plotting compound concentration vs normalized luminescence values. SPPL2a, SPP, SPPL2b, Mouse SPPL2a, and Rat SPPL2a HighContent Imaging Assays (HCA) in U-2 OS Cells. The high-content imaging assays for human SPPL2a, SPPL2b, and SPP using stable U-2 OS cell lines have been described previously.26 In brief, stable U-2 OS cell lines expressing either human SPPL2a or SPPL2b constitutively and a EGFP-labeled TNFα(aa1-76) NTF substrate under a doxycycline-regulated promoter or human SPP constitutively and a EGFP-labeled EnvSigSeq-SEAP fusion protein substrate under a doxycycline-regulated promoter were used for the imaging assays. The imaging assays to monitor mouse SPPL2a and rat SPPL2a activity were established analogous to the human SPPL2a assay but using the sequence of mouse and rat TNFα(aa1-77)-NTF fused to EGFP-NLS, respectively. For all stable cell lines, cells were seeded at 3000 cells/30 μL/384-well in DMEM/GlutaMaxTM-I (Invitrogen) supplemented with tetracycline-free 10% FBS (Amimed) and incubated at 37 °C, 5% CO2 for 3−4 h. Subsequently, 3.3 μL of inhibitors, prediluted in doxycycline-containing medium for 11-point concentration−response curves, were added to each well using a CyBi well liquid handling device (Cybio AG, Jena, Germany) to result in final inhibitor concentrations ranging from 100 μM to 1 nM (final DMSO concentration 0.9% (v/v)) and 5 μg/mL doxycycline). The cells were incubated with inhibitor at 37 °C, 5% CO2 for 24 h. Thereafter cell were fixed in 4% PFA/PBS and in parallel, nuclei were stained with Hoechst (Invitrogen) 1:5000 in PBS for 30 min. The plates were imaged using a Cellomics ArrayScan VTI HCS reader with 10×/ 0.3NA objective (Thermo Fisher Scientific, USA). Six images per well were acquired. Images for the EGFP signal (Ex395, Em509) and the Hoechst nuclear dye (Ex350, Em425) were acquired simultaneously with image analysis using the “Nuclear Translocation” assay algorithm of the Cellomics ArrayScan software. Nuclei were detected based on the Hoechst staining, the nuclear mask was transferred to the EGFP channel, and a 4 pixel wide cytoplasm ring region was defined around the nucleus. The intensity of the EGFP signal was measured both in the nuclear and in the cytoplasm ring region of each individual cell (in general 800−1000 individual cells were analyzed per well), and the difference of the average nuclear versus average cytoplasmic intensity of the EGFP signal was calculated (“CircRingAvgIntenDiffCh2” = “CircAvgIntenCh2” − “RingAvgIntenCh2″). Additionally the number of cells were acquired (feature termed “ValidCellCount”) and used to calculate cell toxicity (CC50). Percent inhibition was calculated relative to the positive (0.5 μM 42 = 100% inhibition) and negative (DMSO = 0% inhibition) controls. The IC50 value was calculated from the plot of percentage of inhibition vs inhibitor concentration using nonlinear regression analysis software, e.g., Origin (OriginLab Corp.). CD74/p8 Mouse Whole Blood Assay. First, 200 μL of mouse whole blood were transferred in a 800 μL V-deep well plate containing 10 μL of 20× conc compound in DMSO/D-PBS (final concentration of DMSO was 0.5%) and incubated for 5.5 h at 37 °C, 5% CO2 under the rotation. The blood was then transferred to 1 mL U-deep well plate containing 600 μL of RBC lysis buffer. After extensive washing with RBC lysis buffer, the cell pellet was resuspended 2−3 times with 2× 300 μL of D-PBS and centrifuged. The cell pellet was lysed with 40 μL of lysis buffer (25 mM Tris/HCl pH 6.8/150 mM NaCl/2 mM EDTA/1% Triton X-100/0.5% deoxycholate + complete protease inhibitors) for 20 min and cleared by centrifugation at 4000 rpm for 30 min at 4 °C. Then 12 μL of the lysate was mixed with 10 μL of SDS denaturing solution. The solution was processed by Western blotting, and the CD74/p8 bands were visualized after incubation with a rat antimouse CD74 Ab (ln−1). Dose response was determined by quantifying the p8 band intensity calibrated with the CD74 fulllength/b-actin band. 877
DOI: 10.1021/acs.jmedchem.7b01371 J. Med. Chem. 2018, 61, 865−880
Journal of Medicinal Chemistry
Article
food and tap water until and throughout the experiment. Analgesic treatment with Temgesic (10 μg/kg sc, application volume 1 mL/kg) was performed the evening following surgery and the next morning. Compound administration in cassette format was in the morning (6−8 AM). Blood samples were collected at various time points from the femoral artery catheter into Eppendorf tubes coated with sodium EDTA. Blood samples were immediately frozen at −20 °C until final processing (maximum storage was 8 days). Intravenous and oral dosing was typically performed in the same animals after a 48 h washout interval between the single dose applications. The test substances were administered intravenously as a solution in 1-methyl-2pyrrolidone and polyethylene glycol 200 (30:70, v/v) at a dose of 1 mg/kg per compound and orally as a homogeneous aqueous suspension in Tween 80 and carboxymethyl cellulose sodium 0.5/ 0.5/99 (w/w) at a dose of 3 mg/kg per compound. In Vivo Rapid PK Studies in Rats and Mice. Total of six animals were used for each compound, three for intravenous and three for oral administration. For mouse rapid PK studies, six blood samples (50 μL each) were taken via tail vein bleed and the blood samples from the three animals were pooled and centrifuged to obtain pooled plasma samples. For rat rapid PK studies, six blood samples (100 μL each) were taken via saphenous vein puncture. After centrifugation, 20 μL of the plasma samples are pooled across the three rats based on time point within a dosing arm. The pooled plasma samples were diluted appropriately using a generic dilution scheme to ensure that concentrations are within the dynamic range of the standard curve (1−5000 ng/mL). Automated sample preparation and protein precipitation were carried out. Liquid chromatography/mass spectrometry (LC/MS/MS) analysis was used with a fast generic gradient elution method together with atmospheric pressure chemical ionization (APCI) or electrospray (ESI) in the positive or negative ion mode on an API-4000 triple quadruple mass spectrometer. Mouse and Rat PK Analysis. The concentrations of compounds in whole blood were quantified using a liquid chromatography/mass spectrometry (LC-MS/MS) assay. To 10 μL of each blood sample, 2 μL (conc 2.5 μg/mL) of an internal standard was added and the samples were precipitated with 120 μL of acetonitrile. The samples were vortexed thoroughly then centrifuged (10 min, 4 °C). The supernatant (120 μL) was transferred to a clean 96-well plate and mixed with 50 μL of Milli-Q water. The samples were injected (2 μL) onto suitable analytical columns, using gradient methods at various flow rates. Mobile phases typically consisting of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) were used. Compounds and the internal standards were eluted at various retention times. The HPLC systems were interfaced to mass spectrometers. MS/MS analyses were carried out using electrospray ionization (ESI), typically in the positive ionization mode. Compounds and the internal standards were monitored using multiple reaction monitoring (MRM). The standard curves employed for sample quantitation ranged over several log units. The lower limit of quantitation (LLOQ) in blood was determined and used as cutoff for the analytical sensitivity. Known amounts of the compounds were spiked into blood to create quality control samples with three to four known concentrations. The accuracy of the in vivo blood sample concentration determination was considered acceptable when the intra-assay accuracies obtained for the quality control samples were within 70% to 130% of the expected concentrations. Subsequently, from the time concentration data, pharmacokinetic parameters were calculated by noncompartmental regression analyses using an in house fitting program. Analysis of CD74/p8 Accumulation PK/PD Models in Mice and Rats (Blood and Spleen). Spleens (rat or mouse) were rinsed in PBS, and the splenocytes were release by mechanical shearing. The cell suspension was centrifuged, and the cell pellet was washed twice in RBC lysis buffer (AMIMED, BioConcept). Aliquots corresponding to 2 × 106 cells splenocytes were lysed in 30 μL of lysis buffer (25 mM Tris/HCl pH 6.8 + 150 mM NaCl + 2 mM EDTA + 1% Triton X-100 + 0.5% deoxycholate + complete protease inhibitors from Boehringer) and spun down, and the supernatant was used for Western blotting. Then 200 μL of blood were mixed with 2000 μL of RBC lysis buffer
(Amimed; no. 3-13F00-H) then spun down. This treatment was repeated two more time with 300 μL of RBC lysis buffer. The final cell pellet was resuspended in 40 μL of lysis buffer (25 mM Tris/HCl pH 6.8 + 150 mM NaCl + 2 mM EDTA + 1% Triton X-100 + 0.5% deoxycholate + complete protease inhibitors from Boehringer. Mouse CD74/p8 was detected with the ln−1 Ab used at a dilution of 1:2000 (BD Bioscience no. 555317). Rat CD74/p8 was detected with newly developed Ab. The rat AB was obtained after immunizing rabbits with two peptides (QLPILGQRARAPESNCNR and DDQRDLISNHEQLPIC) encompassing sequences at the N-Terminus of rat CD74 (Eurogentec SA, Belgium). The affinity purified antibody showed a high specificity for rat rCD74/p8 and was used at 1:200 for the experiments with rat material Analysis of CD74/p8 Accumulation in Mice after Chronic Dosing of Compound 40. Animals were gavaged po with the indicated doses of compound in either 0.5% MC:0.5% Tween (40) or PEG:D5W, 3:1. At the indicated time points, spleens were dissected and placed in a Miltenyi GentleMACS C tube with 3 mL cell culture media (DMEM − 10% FBS) and run through one cycle (1 min) of dissociation using the Ms_Spleen_01 program. Spleens were filtered through a cell 40 μM strainer washed with cell culture medium, and red blood cells were lysed with 3 mL of ACK lysis buffer for 5 min at room temperature. Cells were washed twice with cell culture medium and counted to determine total cell numbers. For CD74, 1 × 106 cells were lysed with 100 μL of cell lysis buffer (Cell Signaling Technologies), containing PMSF and a protease inhibitor cocktail (HALT, Peirce Scientific). Then 18 μg of cell lysate was run on a 10% Tris-Bis Gel (Novex) and blotted onto nitrocellulose. Membranes were blocked with 5% BSA in TBS-tween. Both full length CD74 and CD74 p8 were detected using rat antimouse CD74, clone IN-1 (BD Pharmingen), and an Alexa488 labeled antirat IgG antibody (LiCor). Blots were scanned on the LICor Odyssey. The full length CD74 band was used to control for loading differences. Percent inhibition was calculated compared by comparing experimental ratio (CD74p8/CD74 full length) to reference compound ratio (42 or 31). PK Sampling. First, 50 μL of blood was sampled retro-orbitally into LiHep coated tubes (Sardstedt) at the indicated time points. For chronically dosed mice, a trough exposure sample was obtained prior to AM dosing. Samples were kept on ice until plasma separation. Samples were spun at 2823g for 5 min at rt to separate out plasma. Samples were stored at −20 °C until analysis. Flow Cytometry of Spleen Cells. Aliquots of 1 × 106 splenocytes were stained in FACS buffer (1× HBSS, 2% FBS, 5 mM EDTA) with the following antimouse antibody cocktails for 30 min on ice in the dark (all antibodies purchased from Biolegend B cells (B220 (clone RA3-6B2), CD21 (clone 7E9), CD23 (clone B3B4), IgM (clone 7E9), and IgD (clone 11−26c.2a)). T-cells: (CD4 (clone GK1.5), CD62L (clone MEL-14), CD3 (clone 17A2), CD8 (clone 53-6.7), and CD44 (clone IM7)). Plasma cells/GC B cells: (B220 (clone RA3-6B2), GL7 (cloneGL7), CD95 (clone SA367H8), CD19 (clone 6D5), CD138 (clone 281-2), and CD11c (clone N418)). Dendritic cells: (PDCA1 (clone 927), CD3 (clone 17A2), NK-1 (clone PK136), CD19 (clone 6D5), CD8 (clone 53-6.7), CD11c (clone N418), and MHCII (clone M5/114.15.2)). Samples were acquired on a BD Fortessa flow cytometer (Beckton Dickinson, Mountain View, CA). Data was analyzed using FlowJo software. Hes1 mRNA Analysis; RNA Extraction and Real-Time PCR. Thymi and jejunum were excised from mice and placed into 1 mL of RNALater solution (Ambion). Samples were kept at 4 °C overnight. The next day, samples were washed with H2O (1 mL) and frozen at −80 °C until further analysis. Total RNA samples were extracted from 10 mg of thymus and jejunum by using the MagNA Pure LC RNA isolation high performance kit according to the manufacturer instructions (Roche Diagnostic). Taqman assays were purchased from Applied Biosystems and used for real-time polymerase chain reaction (RT-PCR) analysis as previously described.43 Statistical significance of mRNA measurements was assessed using the twosample unequal variance, two-tailed distribution (heteroscedastic Student’s t-test, Excel). Coefficients of variation with a P value
