Decrease in desaturation index in a dose-dependent manner is one of the strong in vivo activities of A939572 [1]. SCD1 enzymatic activity is selectively inhibited by a tiny chemical called A939572. On day 5, A939572 showed a statistically significant dose-dependent reduction in the proliferation of Caki1, A498, Caki2, and ACHN (IC50 of 65 nM, 50 nM, 65 nM, and 6 nM, respectively). Compared to the DMSO+BSA control, all five endoplasmic reticulum stress-related gene expression levels were significantly higher in A939572 (SCDi)-treated Caki1 and A498 cells. This enhanced expression could be prevented by adding OA-BSA.

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The optimization began with an examination of substitution on phenoxy ring. With the ortho-substituted analogues, the IC50s steadily decreased from H (4a, 370 nM) and F (4b, 98 nM) to bulkier halogen groups, such as Cl and Br (4c/A939572 and 4d, <4 nM). Consistent with the SAR trend observed for the pyridazine series,21 increased lipophilicity on the phenoxy ring, especially on ortho-position, correlates with improved inhibitory activity. As can be observed, introduction of 2-methyl group resulted in an inhibitor (4e, 17 nM) with >20-fold improvement in potency. The impact was less pronounced with relatively more hydrophilic methoxy analogue (4f, 81 nM). Meta-substitution was also well tolerated as exemplified with compound 4g (20 nM), although not as optimal as ortho-substituted analogue 4c/A939572 (<4 nM). To explore any additional lipophilic interaction, we introduced a fluoride at the 4, 5 or 6-position on the basis of 4c. As shown in Table 1, 2,4-di-substituted analogue 4j was able to maintain most of the potency observed with 4c, whereas 2,6-di-substitution surrendered all the gain generated from 2-mono-substitution (4h, 330 nM vs 4a, 370 nM). The co-presence of F at 5-position was found to be beneficial as 2,5-analogue 4i exhibited about twofold potency improvement in human SCD1 (4i, hSCD1 IC50 = 18 nM vs 4c, hSCD1 IC50 = 37 nM).[1]

Based on the encouraging results associated with compound 4c/A939572, we next investigated the replacement of amide group with a heterocyclic moiety to further improve SCD1 inhibitory activity. This modification yielded a couple of analogues with good retention of IC50 (4l, 8 nM; 4m, 10 nM), although they offered no potency advantage over 4c/A939572. The presence of a functional group on the aryl 3-position seems to be important to possess high potency, as un-substituted analogue showed dramatic loss of activity (4k, 0.6 μM). Fine-tuning of amide derivatives indicated that only primary amide or small group of secondary amide such as ethylamide was favorable with comparable IC50 to 4c. Bulky group was detrimental to the activity as 4q showed no inhibition at 10 μM.

Since SCD1 activity depends on cytochrome b5 and cytochrome b5 reductase, we established a selectivity assay to exclude the possibility that compounds identified as SCD1 inhibitors may actually inhibit the co-enzyme. The lack of inhibition above the concentration of 10 μM supports the direct SCD inhibition by our urea-based chemotype, represented by compound 4c/A939572.

We next investigated the selectivity profiles of our potent inhibitors. Excellent selectivity over kinases was routinely achieved. 4c/A939572 was also tested in a 3H-Dofetilide binding assay and showed no significant hERG channel blockade activity (IC50 > 100 μM)[1].

Tumor volumes (mm3) were measured after 4 weeks of treatment with A939572 (30 mg/kg, po) and Tem, either separately or in combination, in athymic nude mice (nu/nu) bearing A498 ccRCC xenografts. Similar growth responses were seen with A939572 and Tem monotherapy; at study's end, a 20–30% decrease in tumor volume was seen (in comparison to the placebo control); however, data did not approach statistical significance until the last week of treatment. When the research came to an end, the combination treatment group's tumor volume had decreased by over 60% (in comparison to the placebo control group), with notable reductions observed after around a week of therapy [2].

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SCD1 inhibitors 4b and 4c/A939572 were evaluated in a 5-day efficacy study using ob/ob mice. A desaturation index, calculated as the ratio of 16:0/16:1n7 or 18:0/18:1n9, was used as in vivo biomarker for activity. The lipid classes examined include cholesterol ester, diacylglycerol, free fatty acid, free cholesterol, total phospholipids, and triacylglycerol. 4b (30 mpk bid) and 4c/A939572 (10 mpk bid) consistently reduced desaturation indices of all these lipid categories to lean level, or even lower. Figure 2 showed the desaturation index (18:0/18:1n9) lowering effect with the treatment of SCD1 inhibitors 4b and 4c. A dose dependent triglyceride desaturation index reduction by 4c was also observed in ob/ob mice (data not shown). These observations demonstrated a clear correlation between in vitro activity and in vivo desaturation index [1].

SCD1 in vitro enzymatic assay. [1]
SCD1 activity was determined by measuring the production of tritiated water from the desaturation of 3H (9,10) stearoyl-CoA substrate. One version of the assay uses ob/ob mouse liver microsomes as the source of SCD1 enzyme. Another used recombinant human SCD1 expressed in tandem with human cyt b5/cyt b5R as the source of the SCD1 enzyme.
The SCD1 activity assay was carried out in 96-well plates in a 50 L total assay reaction volume appropriate for high throughput screening. Typically a 50 L reaction volume contained: 5 L of a diluted test compound in 10% DMSO and 10 M stearoyl-CoA, 0.24 M 3H (9,10) stearoyl-CoA in 35 L of assay buffer (250 mM sucrose, 10 mM Tris pH 7.5, 5mM MgCl2, 1 mM DTT, Roche Complete-EDTA proteinase inhibitor cocktail, 2 mM NADH and 50 mM NaF). The reaction was then initiated with the addition of either human or mouse microsomes at a protein concentration of between 10-20 g of protein per reaction depending on the specific activity of the particular microsome preparation.
After a 30 min reaction time the reactions were terminated by the addition of a stop solution, typically 30 L of 4N HCl. A set reaction volume, usually 65L, was then transferred from each well to 50 mg of methanol-prewetted Norit A charcoal in a Millipore Multiscreen Plate. Plates were shaken for 1 min and allowed to sit for 5 min to allow for adsorption of the stearoyl-CoA and the oleoyl-CoA. The 3H-H2O was separated from the charcoal adsorbed stearoyl-CoA and oleoyl-CoA by centrifugation into a collection plate. The charcoal filled Multiscreen Plate was removed, and scintillation fluid was added to each well. Radioactivity was counted, typically in a Microbeta TriLux radioactivity counter. SCD1 activity was expressed relative to control reactions with no inhibitor, after background subtraction. Dose-response curve fitting and IC50 values were determined using non-linear regression analysis.

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Transfections and Luciferase Assays [2]
Caki1 and A498 cells were transiently transfected with p5xATF6-GL3 UPR luciferase reporter and pRL-CMV-renilla luciferase plasmid using Lipofectamine2000. Cells (DMSO vs. A939572 NT vs. shSCD780) were harvested after 48hrs using Promega’s Dual Luciferase assay kit per the manufacturer’s protocol and luciferase activity was measured using a Veritas Luminometer; reported as relative luminescence.

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RNA Isolation and Quantitative PCR [2]
RNA isolation, preparation of cDNA, and QPCR was performed as previously described. TaqMan®FAM™ dye-labeled probes including POLR2A (Hs00172187_m1-normalization control), SCD1(Hs01682761_m1), HSPA5(Hs99999174_m1), CEBPβ(CEBPB Hs00270923_s1), GADD45A(Hs00169255_m1), DDIT3(Hs01090850_m1), HERPUD1(Hs01124269_m1), and ATF6(Hs00232586_m1). Fold change value comparisons: normal vs. tumor, NT vs. target lentivirus, and DMSO vs. A939572 treated samples using the ΔΔCt method

In Vivo Analysis [2]
A498 cells were subcutaneously implanted in athymic nu/nu mice at 1×106 cells/mouse in 50%Matrigel. Tumors reached ~50 mm3 prior to 4wk treatment. A939572 was re-suspended in strawberry flavored Kool-Aid® in sterilized H2O (0.2g/mL) vehicle at 30mg/kg in a 50μl dose. Mice were orally fed by using a syringe to administer the 50μl dose twice daily/mouse. This modified method was found to be effective and less stressful on the mice. Temsirolimus was solubilized in 30% ethanol/saline and administered via intraperitoneal injection at 10mg/kg in a 50μl dose once every 72hrs/mouse. Tumor volumes were calculated using the formula 0.5236(L*W*H) and body weight were measured every 3 days.

In summary, this series of SCD1 inhibitors exhibited favorable pharmacokinetic characteristics in rodents, characterized by moderate volume of distribution and high oral bioavailability in mice (4c/A939572, CLp = 0.4 L/h/kg; Vss = 0.4 L/kg; F = 92%).

[1]. Discovery of piperidine-aryl urea-based stearoyl-CoA desaturase 1 inhibitors. Bioorg Med Chem Lett. 2008 Aug 1;18(15):4298-302.

[2]. Stearoyl-CoA desaturase 1 is a novel molecular therapeutic target for clear cell renal cell carcinoma. Clin Cancer Res. 2013 May 1;19(9):2368-80.

Through molecular scaffold modification, we identified a series of novel stearoyl-CoA desaturase 1 (SCD1) inhibitors. Preliminary structure-activity relationship (SAR) studies revealed highly potent and orally bioavailable piperidine-arylurea SCD1 inhibitors. 4-(2-chlorophenoxy)-N-[3-(methylcarbamoyl)phenyl]piperidine-1-carboxamide 4c exhibited significant in vivo activity and a dose-dependent desaturation index reduction effect. [1] Objective: We aimed to identify stearoyl-CoA desaturase 1 (SCD1) as a novel molecular target for clear cell renal cell carcinoma (ccRCC) and to investigate its effects on tumor cell growth and viability in vitro and in vivo, independently and in combination with currently approved FDA treatments. Experimental design: SCD1 expression was detected in normal tissue samples and ccRCC tissue samples and cell lines from patients. This study utilized a gene knockdown model and the small molecule inhibitor A939572 to target and inhibit SCD1, and employed gene chip analysis to analyze the effects of SCD1 on cell growth, apoptosis, and gene expression. Furthermore, we evaluated a synergistic therapeutic model using the tyrosine kinase inhibitors (TKIs) sunitinib and pazopanib, along with the mTOR inhibitor tesimolimus, for pharmacological inhibition of SCD1. Results showed that SCD1 expression was elevated in all stages of clear cell renal cell carcinoma (ccRCC). Both gene knockdown and pharmacological inhibition of SCD1 reduced tumor cell proliferation and induced apoptosis in vitro and in vivo. Through gene chip analysis, quantitative real-time PCR, and protein analysis of A939572-treated or SCD1 lentivirally knocked-down samples, we observed activation of the endoplasmic reticulum stress response signaling pathway, providing new insights into the mechanism of action of SCD1 in ccRCC. Moreover, the combined use of A939572 and tesimolimus synergistically inhibited tumor growth in vitro and in vivo. Conclusion: Increased SCD1 expression supports the survival of ccRCC cells, therefore we propose it as a new molecular target, either alone or in combination with mTOR inhibitors, for patients who cannot be treated surgically, such as those with advanced or metastatic disease. [2]

C20H22CLN3O3

387.8600

387.135

C, 61.93; H, 5.72; Cl, 9.14; N, 10.83; O, 12.37

1032229-33-6

24905400

White to off-white solid powder

1.302 g/cm3

633.5ºC at 760 mmHg

336.9ºC

4.176

2

3

4

27

511

0

ClC1=C([H])C([H])=C([H])C([H])=C1OC1([H])C([H])([H])C([H])([H])N(C(N([H])C2=C([H])C([H])=C([H])C(C(N([H])C([H])([H])[H])=O)=C2[H])=O)C([H])([H])C1([H])[H]

DPYTYQFYDLYWHZ-UHFFFAOYSA-N

InChI=1S/C20H22ClN3O3/c1-22-19(25)14-5-4-6-15(13-14)23-20(26)24-11-9-16(10-12-24)27-18-8-3-2-7-17(18)21/h2-8,13,16H,9-12H2,1H3,(H,22,25)(H,23,26)

4-(2-chlorophenoxy)-N-[3-(methylcarbamoyl)phenyl]piperidine-1-carboxamide

A939572; A-939572; 1032229-33-6; A939,572; 4-(2-chlorophenoxy)-N-[3-(methylcarbamoyl)phenyl]piperidine-1-carboxamide; 4-(2-chlorophenoxy)-N-(3-(methylcarbamoyl)phenyl)piperidine-1-carboxamide; 4-(2-CHLOROPHENOXY)-N-[3-[(METHYLAMINO)CARBONYL]PHENYL]-1-PIPERIDINECARBOXAMIDE; 1-Piperidinecarboxamide,4-(2-chlorophenoxy)-N-[3-[(methylamino)carbonyl]phenyl]-; CHEMBL469169; A 939,572; A 939572; SCD1 Inhibitor; Stearoyl-CoA Desaturase 1 Inhibitor;

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 : ~100 mg/mL (~257.82 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.45 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 25.0 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: ≥ 2.5 mg/mL (6.45 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 25.0 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: ≥ 2.5 mg/mL (6.45 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 25.0 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.)