NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

Cover of Probe Reports from the NIH Molecular Libraries Program

Probe Reports from the NIH Molecular Libraries Program [Internet].

Show details

Selective KOP Receptor Antagonists: Probe 1

, , , , , , , , , , , , , , , , , , , and .

Received: ; Last Update: October 4, 2010.

The specific aim of this project is to identify subtype specific small molecule antagonists of the human kappa opioid (KOP) receptor that represent different chemical scaffolds than the many existing potent and selective literature antagonists. Such antagonists have been shown to prevent reinstatement of drug taking behavior in animal paradigms thought to model relapse, however all current antagonists are long-acting and lead to tolerance due to poor pharmacological clearance. On the basis of antagonist assay paradigms utilized in this project (G protein coupling, ERK activation and beta-arrestin recruitment), it is hypothesized that the mode of action of the identified probe, ML140 (CID-3342390), is through reversible binding at the KOR site occupied by opiates. Thus, in this paradigm, opioid agonist-stimulation is blocked by increasing doses of the antagonist probe. These novel scaffolds for KOP receptor antagonists are of interest because they may be short acting and rapidly cleared, and therefore find utility in the treatment of drug abuse, depression, and chronic pain without tolerance and habituation.

Probe project: Selective KOP Receptor antagonists

Assigned Assay Grant #: 1X01MH084153-01

Screening Center Name & PI: Burnham Center for Chemical Genomics (BCCG) & John C. Reed (renamed Conrad Prebys Center for Chemical Genomics at Sanford|Burnham Medical Research Institute in 2010. But still referred to for NIH Roadmap Purposes as BCCG above)

Chemistry Center Name & PI: Kansas Specialized Chemistry Center (KSCC) & Jeffrey Aubé

Assay Submitter & Institution: Lawrence Barak, Duke University Medical Center Collaborating PI: (Laura M. Bohn, The Scripps Research Institute, FL)

PubChem Summary Bioassay Identifier (AID): AID-1785

Probe Structure & Characteristics

Image ml140fu1
CIDTarget NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]SelectivitySecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID3342390 (sulfonamide)

ML140
KOR
κ-opioid receptor
850 nM IC50
SID-87218794
AID-2285
MOR
μ-opioid receptor

DOR
δ-opioid receptor
>32 μM IC50
SID-87218794
AID-2420

>32 μM IC50
SID-87218794
AID-2357
> 40X (Dx/HCS)
>25X (HCS/HCS)

> 40X (Dx/HCS)
>25X (HCS/HCS)
KOR Transfluor 1310 nM IC50 [SID-87218794, [AID-2348]

MOR Transfluor >32000 nM IC50 [SID-87218794, [AID-2420]

DOR Transfluor >32000 nM IC50 [SID-87218794, AID-2357]

Recommendations for the scientific use of this probe

The probe candidate proposed in this report, by selectively inhibiting the human kappa opioid receptor subtype, would provide a scientific tool useful in helping to elucidate individual brain pathways that underlie addictive behavior, thus enabling improved understanding of the molecular basis of dependency and potentially providing a basis for therapeutic development.

1. Scientific Rationale for Project

Specific Aims

The identification of small molecules, each able to block or activate only a distinct receptor underlying an addiction will provide a means to untangle the many pathways resulting in addictive behavior and create detailed pharmacological maps for designing novel targeted treatments. This project proposes screening a G protein-coupled receptor relevant to drug abuse and to the study and treatment of addiction, in a fashion that afford the unique opportunity to discriminate between G protein and arrestin-based signaling modalities. This project will hopefully contribute to understanding and treating addiction by providing chemical probes for dissecting the individual brain pathways that underlie addictive behavior thus enabling improved understanding of the molecular basis of addiction and potentially providing targeted therapeutics for this affliction.

The specific aim of this project is to identify subtype specific small molecule antagonists of the human kappa opioid receptor (KOR). Such antagonists have been shown to prevent reinstatement of drug taking behavior in animal paradigms thought to model relapse. In addition, they have been shown to block aspects of nicotine withdrawal, and have antidepressant effects in animal models. Use of the existing kappa antagonists to explore these effects in vivo has been limited by their very long duration of pharmacological action (3–4 weeks in rhesus monkeys), which appears to be mediated not by pharmacokinetics but by activation of c-Jun N-terminal kinase (JNK) phosphorylation. Known kappa antagonists all appear to share this effect, which may contribute to their long duration of action in vivo. Novel kappa opioid receptor antagonists that do not activate the JNK pathway would be desirable, but their discovery is beyond the scope of the screening project. Therefore, selective kappa antagonists with new chemical structures may represent valuable leads to the discovery of shorter acting compounds. KOR selective antagonists will enable delineation of KOR specific signaling. Subtype selectivity is defined as selective for KOR but not active against μ (MOR) or δ (DOR) subtypes.

Background and Significance

For normal activities that produce rewards, there is a rapid habituation of the circuits involved and the behaviors will wane. However, for addictive drugs habituation does not occur and dopamine release persists despite repetitive trials. Upon withdrawal of the drug, a decrease of dopamine levels in the nucleus accumbens results, and this has been observed for opioids, cannabinoids, alcohol, amphetamines, and nicotine (1). This loss of dopamine accounts for the withdrawal syndromes observed with these drugs. The prototype opioid drug is morphine. It produces many effects typical of most opioids including analgesia, euphoria, nausea, and respiratory depression. Repeated use of opioids produces physical dependence and tolerance. These manifestations of opioid use are due to the three recognized types of opioid receptors that are members of the GPCR family, the mu (μ), delta (δ), and kappa (κ) subtype receptors. While stimulation of the mu and delta receptors increases dopamine release in the nucleus accumbens, κ opioid receptor (KOR) activation by its endogenous ligand dynorphin-A reduces extracellular dopamine. It has been suggested that stimulation of KOR by endogenous opioids like dynorphins will produce an aversive state and thereby counter the effects of rewarding and addictive compounds like alcohol, cocaine and nicotine. Moreover, exogenous KOR agonists have also been observed to attenuate drug-taking behavior (2–6). However, it may be difficult to strike a balance between opposing the sense of reward gained by drugs of abuse and producing an aversive state; therefore, activation of the KOR may not be therapeutically preferable. Although these statements appear contrary, KOP agonists can both alleviate drug self administration in animal models (most likely via dopamine regulation) and also trigger relapse. This conflicting dual action of KOP agonists alludes to the complex physiological role of K receptors and underscores the need for their further investigation.

Intracranial self-stimulation has become a useful means of assessing reward thresholds in rodents and nonhuman primates. In essence, an animal will press a lever to electrically stimulate the brain via implanted probes. This “self stimulation” will be performed to a certain extent in training and that extent is an indication of the animal’s “reward threshold.” Administration of drugs of abuse have been shown to decrease this reward threshold such that the animal will seek less stimulation to achieve the desired effect. This model paradigm has been likened to positive hedonic states produced by drugs of abuse in human addicts. In rodents, the direct activation of KOP using selective agonists increases reward thresholds (mimicking the withdrawal state) and creating a “depressive-like” state (where more self stimulation is required to achieve the desired effect). Treatment with antagonists has been shown to restore reward thresholds in this model (7). The restoration of reward thresholds may be a very important step in drug abuse treatment as drug cessation is strongly negatively reinforced by aversive feelings, which may be due to a increased reward threshold. Therefore, the development of KOR antagonists may be particularly beneficial in “resetting” this threshold. Furthermore, since an increased reward threshold may manifest as a “depressive state,” then KOR antagonists may also be beneficial for the treatment of depressive disorders. Currently, there are currently no approved agents or compounds for treating the altered reward pathways associated with drug addiction (2).

In this probe report, we describe the discovery and optimization of a novel antagonist for the kappa-(κ)opioid receptor (KOR) that is >40-fold selective over the mu-(μ) (MOR) and the delta-(δ) (DOR) opioid receptors. We sent the probe for broad GPCR paneling and these data are discussed below (see Table 6). Importantly, this probe and its analogs represent a novel chemical class compared to current literature antagonists and displays potentially novel pharmacology. Accordingly, this probe and its analogs may serve as interesting tools to advance addiction research. Additionally, this new chemotype is less complicated compared to known KOP antagonist compounds. The structure contains no stereochemical centers and the short, versatile synthetic route enables both the synthesis of potential analogs and the production of the compound on larger scale.

Table 6. Summary of in vitro ADME Properties of Kappa opioid Agonist probe(s).

Table 6

Summary of in vitro ADME Properties of Kappa opioid Agonist probe(s).

Follow up studies subsequent to the probe report to investigate the properties of the new probes will be required and will employ confirmatory assays to validate that the compounds are interacting directly with the KOR. To demonstrate selective subtype specific binding to the KOR in competitive assays we will utilize a set of radiolabeled compounds that are commercially available for this purpose including, [3H]-Diprenorphine (KOR and MOR), DAMGO (MOR) and Naltrindole [5′,7′-3H] (DOR). Of particular interest is whether the probe is long or short acting at the KOR, and studies of this question in cells and animal models may clarify as to whether the underlying mechanism for antagonist anti-addictive behavior requires activation of JNK.

2. Project Description

a. Original goal for probe characteristics

The original goal from the CPDP was to find antagonists structurally distinct from current literature probes, with potencies of less than 1 µM for the kappa-opioid receptor (KOR), with greater than 100-fold selectivity against the mu-opioid receptor (MOR) and 10-fold selectivity against the delta-opioid receptor (DOR), or to the extent achievable by test concentration limitations.

b. Information for each Assay Implemented and Screening Run

i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen, Secondary)

Table 1Summary of Assays and AIDs

PubChemBioAssay NameAIDsProbe TypeAssay TypeAssay FormatAssay Detection & well format
Summary of small molecule antagonists of the kappa opioid receptor via a luminescent beta-arrestin assay [Summary]1785AntagonistSummaryN/AN/A
uHTS identification of small molecule antagonists of the kappa opioid receptor via a luminescent beta-arrestin assay [Confirmatory]1778AntagonistPrimaryCell- basedLuminescence- DiscoveRx β-arrestin & 1536
SAR analysis of small molecule antagonists of the kappa opioid receptor via a luminescent beta-arrestin assay [Confirmatory]2285AntagonistSARCell- basedLuminescence- DiscoveRx β-arrestin & 1536
HTS Dose response counterscreen for assays utilizing the enzyme, b- galactosidase [Confirmatory]1966AntagonistCounter- screenEnzymaticLuminescence &1536
HTS Image-Based Screen for Selective Antagonists of the KOR Receptor [Confirmatory]2136AntagonistAlternateCell- basedHCS – Transfluor & 384
HTS Image-Based Screen for Antagonists of the DOR Receptor [Confirmatory]2356AntagonistSelectivityCell- basedHCS – Transfluor & 384
SAR analysis of Antagonists of the Kappa Opioid Receptor (KOR) using an Image- Based Assay [Confirmatory]2348, 2491AntagonistAlternateCell- basedHCS – Transfluor & 384
SAR Analysis of Antagonists of the MOR Receptor using an Image-Based Assay [Confirmatory]2420AntagonistSelectivityCell- basedHCS – Transfluor & 384
SAR Analysis of Antagonists of the DOR Receptor using an Image-Based Assay [Confirmatory]2357AntagonistSelectivityCell- basedHCS – Transfluor & 384

ii. Assay Rationale & Description

Unlike imaging or other second messenger assays, the DiscoveRx β-arrestin assay allows for a direct measure of GPCR activation by detection of β-arrestin binding to the KOR1 receptor. In this system, β-arrestin is fused to an N-terminal deletion mutant of β-gal (termed the enzyme acceptor of EA) and the GPCR of interest is fused to a smaller (42 amino acids), weakly complementing fragment termed ProLink™. In cells that stably express these fusion proteins, ligand stimulation results in the interaction of β-arrestin and the Prolink-tagged GPCR, forcing the complementation of the two β-gal fragments and resulting in the formation of a functional enzyme that converts substrate to detectable signal.

Assay materials
  1. OPRK1 b-Arrestin (DiscoveRx)
  2. Assay Medium: Opti-MEM Medium supplemented with 1% hiFBS, 1X Pen/Strep/Glu, 125 μg/mL Hygromycin (1/2 recommended), 250 μg/mL Geneticin (1/2 recommended)
  3. Growth Medium: MEM supplemented with 10% hiFBS, 1X Pen/Strep/Glu, 125 μg/mL Hygromycin (1/2 recommended), 250 μg/mL Geneticin (1/2 recommended)

Table 2Reagents used for the uHTS experiments

ReagentVendor
OPRK1 beta-Arrestin Cell LineDiscoveRx
Assay Medium: Opti-MEM Medium supplemented with 1% hiFBS, 1X Pen/Strep/Glu, 125 μg/mL Hygromycin, 250 μg/mL GeneticinInvitrogen
Growth Medium: MEM supplemented with 10% hiFBS, 1X Pen/Strep/Glu, 125 μg/mL Hygromycin, 250 μg/mL GeneticinInvitrogen

The following uHTS protocol was implemented at single point concentration confirmation:

uHTS protocol

Day 1

  1. Harvest cells using Enzyme-Free Dissociation Buffer (Invitrogen Cat#13151-14). Add 500 cells/well in 5 μL of media to each well of a white, 1536 well plate.
  2. Spin cells at 500 rpm for 1 min, then wrap plates in Saran Wrap.
  3. Incubate overnight at 37C with 5% CO2.

Day 2

  1. Using a Highres Biosolutions pintool pin 30 nL to wells. Columns 1–4 should be DMSO only (Control wells), Columns 5–48 contain test compounds (10µM final in well concentration).
  2. Immediately following pintool addition, add 1.0 μL of assay media to columns 1–2 and 1.0 μL of assay media containing 240 nM dynorphin A for a final assay concentration of 40 nM. Centrifuge plates at 500 rpm for 1 min immediately following additions.
  3. Incubate for 1hr and 30 minutes.
  4. During test incubation, prepare Detection Reagent Solution from DiscoveRx (1 part Galacton Star: 5 parts Emerald II and 19 parts Cell Assay Buffer)
  5. Add 2.5μl of detection reagent solution to each well.
  6. Incubate at room temperature for 60 min in the dark
  7. Read plates in a Perkin Elmer Envision using a luminescence protocol
Dose Response protocol

Day 1

  1. Harvest cells using Enzyme-Free Dissociation Buffer (Invitrogen Cat#13151-14). Add 500 cells/well in 5 μL of media to each well of a white, 1536 well plate.
  2. Spin cells at 500 rpm for 1 min, then wrap plates in Saran Wrap.
  3. Incubate overnight at 37C with 5% CO2.

Day 2

  1. Using a Labcyte Echo, DMSO and test compounds are transferred to wells. DMSO only is transferred to columns 1–3 and 46–48(Control wells), while varying volumes of test compounds are transferred to columns 4–45 to achieve the desired test concentrations. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations.
  2. Immediately following Echo transfer, 1.0 μL of assay media is added to columns 1–3 and 1.0 μL of assay media containing 240 nM dynorphin A is added to columns 4–48 for a final assay concentration of 40 nM. Centrifuge plates at 500 rpm for 1 min immediately following additions.
  3. Incubate for 1hr and 30 minutes.
  4. During test incubation, prepare Detection Reagent Solution from DiscoveRx (1 part Galacton Star: 5 parts Emerald II and 19 parts Cell Assay Buffer)
  5. Add 2.5μl of detection reagent solution to each well.
  6. Incubate at room temperature for 60 min in the dark
  7. Read plates in a Perkin Elmer Envision using a luminescence protocol

The average Z′ for the screen was 0.51, the signal to background (S/B) was 4.41, signal to noise (S/N) was 28.9 and signal to window was 4.26.

Rationale for confirmatory, counter and selectivity assays

The initial frontline counterscreen that was performed shortly following dose response confirmations on both the agonist and antagonist KOR1 primary screens was the β-galactosidase dose response assay. Each confirmed hit (EC50 < 10 µM) was run in a β-gal dose response assay. Because the primary screen is based upon the formation of a functional β-gal enzyme upon β-arrestin migration to the GPCR, we wanted to rule out compound interaction, either stimulatory or inhibitory, with the β-gal enzyme in the absence of GPCR interaction.

The High-Content Imaging-based confirmatory (KOR) and selectivity assays (MOR, DOR) which are based upon the translocation of β-arrestin linked to GFP to other receptor subtypes were developed and performed to confirm antagonist activity in the KOR antagonist primary assay, as well as to ascertain the selectivity of compounds for the KOR receptor vs. the MOR and DOR receptor sub-types.

Improved potency for KOR and increased selectivity against MOR and DOR were primary drivers for compound selection and optimization.

Confirmation assays

The initial confirmatory assays were performed in full dose-response for compounds from solvated DPI stock solutions to confirm activity seen first in test agents from screening library in the initial primary screen. Active compounds were then tested in an alternative format for inhibition of GPCR activation, via the imaging-based KOR High-Content Transfluor Antagonist Assay. In the Transfluor assay, GPCR activation is measured indirectly by via the detection of β-Arrestin-GFP redistribution from the cytosolic compartment to the plasma membrane to coated pits and finally endosomal vesicles. The image-based KOR assays allowed for independent confirmation of KOR activation utilizing an alternative technology.

The following are confirmation assays for this project:

Assay 1: HTS identification of small molecule antagonists of the kappa opioid receptor via a luminescent beta-arrestin assay (AID-1778)

Assay 2: SAR analysis of small molecule antagonists of the kappa opioid receptor via a luminescent beta-arrestin assay (AID-2285)

Assay 3: HTS Image-Based Screen for Selective Antagonists of the KOR Receptor (AID-2136)

Assay 4: SAR analysis of Antagonists of the Kappa Opioid Receptor (KOR) using an Image-Based Assay (AID-2359)

Counterscreen assays

The β-Galactosidase Counterscreen Assay was utilized to ascertain possible enzyme inhibition, which might present the opportunity for false positives from the initial primary assay. The inhibition of activity of the b-galactosidase fragment complementation in the primary KOR1 β-Arrestin Assay in the presence of test agent could lead to decreased signal formation and therefore a false positive result. This counterscreen assay would allow for the detection of these artifactual compounds.

Assay 1: HTS Dose response counterscreen for assays utilizing the enzyme, β-galactosidase (AID-1966)

Secondary Assays

The imaging-based MOR and DOR High-Content Transfluor Antagonist Assays provide for the determination of KOR receptor selectivity. The probe criteria specifies the necessity of at least 100-fold selectivity against MOR and DOR, or within the reasonable limitations imposed for testing compounds at high concentrations, i.e. 100 µM selectivity for a 1 µM compound.

Assay 1: HTS Image-Based Screen for Antagonists of the MOR Receptor (AID-2344)

Assay 2: HTS Image-Based Screen for Antagonists of the DOR Receptor (AID-2356)

Assay 3: SAR Analysis of Antagonists of the MOR Receptor using an Image-Based Assay (AID-2420)

Assay 4: SAR Analysis of Antagonists of the DOR Receptor using an Image-Based Assay (AID-2357)

iii. Summary of Results

The following flowchart summarizes the compound triage and decision tree for advancement of compounds:

Primary Screen/Confirmation Triage.

Figure

Primary Screen/Confirmation Triage.

A library of approximately 290,000 compounds was tested in the KOR1 DiscoveRx b-Arrestin primary screen. Upon data analysis, 606 hits with activity >50% at a single concentration point of 10 µM were identified. Liquid samples were then ordered through DPI and 531 compounds were received.

The compound solutions resupplied by the MLSMR were first confirmed in 10 µM single-point duplicate in the KOR1 DiscoveRx b-Arrestin primary assay. Of these, 213 compounds were confirmed to have at least 50% activity at a 10 µM assay concentration (see Critical Path flowchart).

Critical Path Flowchart for KOR Antagonist Project.

Figure

Critical Path Flowchart for KOR Antagonist Project.

The confirmed compounds were further tested in dose response in the KOR1 DiscoveRx b-Arrestin primary assay to obtain EC50 values and were also tested in a b-Galactosidase Counterscreen assay to assess the possibility that these compounds might inhibit the enzyme. The KOR1 antagonist dose response experiments revealed 148 compounds with EC50 potencies at or below 10 µM. Twenty-nine of the compounds were eliminated from future consideration because they were found to inhibit b-galactosidase activity in the enzymatic counterscreen.

The active, confirmed compounds were then tested in the KOR1 High-Content Transfluor Antagonist assay for further confirmation, then in the MOR and DOR High-Content Transfluor Antagonist assays to determine subtype selectivity.

Chemistry and cheminformatics resources were then employed in the selection of both novel and chemically tractable molecules to pursue for a KOR1 selective probe. Structures of interest and analogs thereof were either purchased as commercial dry powders. In total, 32 structures were received from commercial vendors. These constituted the SAR driving chemistries from which the KOR1 antagonist probe candidate and analogs emerged.

SAR testing of re-constituted powders encompassed dose response testing of compounds in four assays: KOR1 DiscoveRx β-Arrestin Antagonist assay, the KOR High-Content Transfluor Antagonist assay, and the MOR and DOR High-Content Transfluor Antagonist assays.

c. Probe Optimization

i. Description of SAR & chemistry strategy (including structure and data) that led to the probe

The screening of the MLPCN compound collection identified three promising chemotypes (Figure 1). The Series 1 chemotype although sufficiently potent, did not confirm as a selective KOP (over DOP and MOP) antagonist series. The Series 2 scaffold data contained an existing compound meeting probe criteria for potency and selectivity and was selected for resynthesis of the compound as well as a set of 32 analogs to probe the SAR of this series. The Series 3 chemotype contained the compound shown in Figure 1 (IC50 = 1.67 μM) and an additional analog (IC50 = 4.32 μM). With this limited yet promising data as a starting point, we investigated a synthetic strategy towards this class of compounds. Concurrently, we purchased ten analogs to establish additional SAR around this chemotype.

Figure 1. Summary of the hit structures for the KOP antagonist project.

Figure 1

Summary of the hit structures for the KOP antagonist project.

SAR Analysis

Screening of the MLPCN compound collection uncovered a sulfonamide containing (Series 2) compound possessing sufficient potency to meet probe criteria. However, only one additional compound in this series with measurable activity (less than 10 μM) was known. In an effort to uncover additional supporting analogs and to broaden the known SAR of this chemotype we synthesized the 51 analog compounds mentioned in Tables #3A3B. In addition, we synthesized and submitted for screening the compound depicted in Figure 1 to confirm the activity of this synthetic material. The lead compound (CID03342390, entry 1 in Table 3A) for this series remained the most potent compound and is therefore nominated as the probe candidate. The R1 = ethyl analog (CID44601476, entry 2 in Table 3A) was found to be slightly less potent than the lead compound where R1 = methyl. The other synthetic analogs had more substantial changes relative to the lead and unfortunately, turned out to be far less potent.

Table 3A. SAR Analysis for Selective κ –opioid receptor antagonists of the sulfonamide sub-scaffold 1.

Table 3A

SAR Analysis for Selective κ –opioid receptor antagonists of the sulfonamide sub-scaffold 1.

Table 3B. SAR Analysis for Selective κ–opioid receptor antagonists of the sulfonamide sub-scaffold 2.

Table 3B

SAR Analysis for Selective κ–opioid receptor antagonists of the sulfonamide sub-scaffold 2.

Interestingly, the constrained analog CID44601478 (entry 7 in Table 3B) showed no significant (in fact showed negative) inhibition at the higher test concentrations. Based on this observation, these compounds (entries 25) were screened for KOR agonism, and CID44601476 alone of the four compounds confirmed as an apparent weak partial agonist. These limited structure-activity relationship studies suggest that both the tether length as well as the substitution on the left hand aromatic ring and right hand nitrogen may be critical to maintain potency and selectivity. Some investigators have suggested that weak partial agonists may also prove of value in the study and potential treatment of polydrug addiction, so these compounds may retain some interest beyond the present probe report. However, their evaluation in this context is beyond the scope of this MLPCN project.

Table 3C shows the results of side chain constraint between the carboxylic acid terminus of the aromatic ring centroid of this series and the terminal aromatic substituent in this direction. As seen, this change entirely deprived these compounds of KOR binding activity and therefore this SAR direction was not pursued further.

Table 3C. SAR Analysis for Selective κ–opioid receptor antagonists of the sulfonamide sub-scaffold 3.

Table 3C

SAR Analysis for Selective κ–opioid receptor antagonists of the sulfonamide sub-scaffold 3.

3. Probe

a. Chemical name of probe compound

N-[2-[benzyl(propan-2-yl)amino]ethyl]-4-[[(4-methylphenyl) sulfonyl-amino]methyl]benzamide [ML140]

b. Probe chemical structure including stereochemistry

Image ml140fu27

c. Structural Verification Information of probe SID

The probe SID is 87544125

Purity

>98% (HPLC)

Mass Spec

HRMS (ESI) m/z calcd for C27H33N3O3S ([M+H]+), 480.2315, found 480.2316.

Image ml140fu28

NMR Purity

>95% pure (1H-NMR): 1H NMR (400 MHz, CDCl3) δ 1.07 (d, J = 6.8 Hz, 6 H), 2.42 (s, 3 H), 2.67 (t, J = 6.0 Hz, 2 H), 3.00 (sept, J = 2.8 Hz, 1 H), 3.34 (q, J = 5.2 Hz, 2 H), 3.55 (s, 2 H), 4.14 (s, 2 H), 5.34 (br s, 1 H), 6.56 (br s, 1 H), 7.21–7.31 (complex, 9 H), 7.45 (d, J = 8.0 Hz, 2 H), 7.77 (d, J = 8.4 Hz, 2 H). 13C (100 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3) 18.0, 21.5, 49.7, 126.9, 127.0, 127.1, 127.7, 128.4, 128.5, 129.7; u (C, CH2) 37.5, 46.8, 47.8, 53.6, 134.0, 136.9, 139.8, 140.7, 143.5, 166.6.

Image ml140fu29

d. PubChem CID (corresponding to the SID)

PubChem CID is CID3342390

e. Availability from a vendor

This probe is commercially available in milligram quantities from Chem Div (catalog # K788-2192), Aurora Screening Library (catalog # kcd-576427) and AKOS Screening Library (catalog # AKG-K788-2192). Nonetheless, KSCC deposited 50 mg of newly synthesized material with the MLSMR (DPI).

f. MLS#'s of probe molecule and five related samples that were submitted to the SMR collection

Table 4Probe and analog submission for Kappa Opioid Antagonists

Probe/AnalogMLS_IDCIDSIDSource (vendor or BCCG syn)Amt (mg)Date ordered/Submitted
Probe ML140MLS0026993320334239087544125KU syn5002/26/2010
Analog 1MLS002699335955123087218786KU syn2002/26/2010
Analog 2MLS0026993362085420887218785KU syn2002/26/2010
Analog 3MLS002699334955028687218766KU syn2002/26/2010
Analog 4MLS0026993332085419587218784KU syn2002/26/2010
Analog 5MLS0026993374460147687334050KU syn2002/26/2010

g. Mode of action for biological activity of probe

This series of antagonists appear to selectively inhibit the activation the kappa opioid receptor in both the primary assay and the HCS image based assay, with a good translation between these two assays. This series of compounds exhibit 25 to 40-fold selectivity over both the mu- and delta- opioid receptors as antagonists as determined by the primary DiscoveRx and follow-up HCS Transfluor assay.

The probe, CID3342390 [ML140] was submitted to the Psychoactive Drug Screening Program at the University of North Carolina (PDSP, Bryan Roth, PI). This program provided screening of the probe against GPCR Panels of 42 CNS-based receptors and the results are summarized in Table 5 below. While 11 receptor profiles are still pending, the probe showed some moderate (310 – 5400 nM) activities against 5HT5a, 5HT7, α2B, α2C, D3, H2, and M5. The activities against the KOR, MOR and DOR were 40.3, 305.6, and 1,088 nM, respectively. These yield selectivity values of 7.6-fold (MOR/KOR) and 27-fold (DOR/KOR) in the PDSP assays which compare less favorably with those obtained by our DiscoveRx (>40-fold for MOR & DOR) and HCS (>25-fold for MOR & DOR) assays. (see “Probe(s) Structure & Characteristics:” table on p.1). Although not required by the CPDP and outside the scope of the MLPCN project, the potency of the probe compound relative to other CNS receptors is solid and provides an attractive starting point for post-probe optimization.

Table 5. Receptor Paneling of KOP agonist [ML140] CID3342390 through the PDSP.

Table 5

Receptor Paneling of KOP agonist [ML140] CID3342390 through the PDSP.

Is should be noted the that the nanomolar binding Kis of CID3342390 (40.3 nM) for KOR are more potent (21 to 33-fold) than those determined by either functional assay (see table on p. 1) for CID3342390 (850 nM DiscoveRx; 1310 nM HCS). Corrrelation of the functional EC50 data to equilibrium ligand binding values (Ki) is not straightforward, but these data further confirm that the chemotype exemplified by the present probe constitutes a highly potent new scaffold for exploring KOR biology.

We believe that the mode of action of the probe antagonist is the reversible binding at the KOR site occupied by opiates on the basis of the antagonist assay paradigms. (G protein coupling, ERK activation and beta-arrestin recruitment) in which, agonist-stimulation is blocked by increasing doses of the probe. KOR antagonists are of interest because they may find utility in the treatment of drug abuse, depression, and chronic pain. Three of the most potent and selective of the KOR antagonists, NorBNI, GNTI, and JDTic have been recognized for their long acting properties at the KOR that may be associated with JNK activation. While they have common structural features their differences are sufficient to cloud the SAR that underlies this long acting physiological behavior that can be blocked by reversible nonselective opioid antagonists. It is therefore important to determine how this selective, sub micromolar probe functions at the KOR with respect to NorBNI, GNTI, and JDTic.

Interestingly, in the β-arrestin assay, one of the weakly active (IC50>20 µM) analog compounds, CID20968607 exhibited only partial inhibition (40–50% I) at the highest tested concentration (data not shown). Another compound CID44601478 exhibited dose-dependent inverse inhibition (see dose response curves below) at the higher concentrations, and when this compound was tested as an agonist, it appeared to behave as a moderately potent (0.6 µM EC50) partial agonist (~20% Emax). CID44601478 did not antagonize U69,593-stimulated G protein coupling nor ERK activation (data not shown), yet it may be of interest to evaluate the intrinsic efficacy of this compound in each assay to determine if it demonstrates a signaling bias towards the β-arrestin pathway. Although confirmation of this is beyond the purview of the MLPCN (and is being pursued as of the submission of the present probe report), such characteristics, if they do confirm, will represent a potentially important new property set for KOR agents.

Image ml140fu32
Image ml140fu33
Image ml140fu34

h. Detailed synthetic pathway for making probe

We have developed a convergent, modular synthetic route that provided rapid access to the tentative probe compound as well as the 32 analogs for screening (Scheme 1). The sulfonyl chloride and diamine fragments were coupled together in the presence of triethylamine in CH2Cl2. The synthesis of the requisite sulfonamide benzoic acid (1) and diamine fragments (2) were readily achieved using precedented methods.

Scheme 1. Synthetic route for probe molecule and supporting analogsa.

Scheme 1

Synthetic route for probe molecule and supporting analogsa. a Condtions: (a) ArSO2Cl, Na2CO3, H2O; (b) SOCl2; (c) H2NCH2CH2NR2(CH2R3), Et3N, CH2Cl2.

Scheme 2. Synthetic route for probe molecule.

Scheme 2Synthetic route for probe molecule

i. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)

The probe molecule has demonstrated potent antagonist activity in both the primary enzyme complementation assay and the image base orthogonal assay for inhibition of β-arrestin mediated signaling of the kappa opioid receptors, with selectivity over for inhibition of activation of both the mu- and delta opioid receptors.

The criteria for probe as defined in the CPDP Chem Update document filed on October 28, 2009 and revised and refiled on November 21, 2009, and finally refiled after corrections requested by NIH on January 12, 2010 with the NIH PT: a potency for KOR of less than 1 µM, and at least 100-fold selectivity over MOR and 10-fold selectivity over DOR or as obtained by the achievable test concentrations, (e.g. solubility limited). This current antagonist is a novel scaffold from those in the literature and exceeds the criteria for DOR selectivity, though it formally misses the 100-fold selectivity of against MOR; however, the compounds cannot achieve a higher than 30–40 µM test concentrations without some precipitation, and in most cases the dose-response curve is completely flat so the >32 µM is an under estimate of the true IC50, which is certainly >>32 µM, so well within a factor of 2–3 for an effective >100 –fold selectivity against MOR. The assay provider concurs that even at this pro forma lower ~25–40-fold estimate of selectivity the compound should be useful.

In Vitro Pharmacology Profiles of Probe CID03342390 [ML140] (See Table 6). The probe, CID03342390, had poor solubility at all pH’s tested.

The PAMPA (Parallel Artificial Membrane Permeability Assay) assay is used as an in vitro model of passive, transcellular permeability. An artificial membrane immobilized on a filter is placed between a donor and acceptor compartment. At the start of the test, drug is introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments are measured using UV spectroscopy. In this assay, the probe, CID03342390, has excellent permeability in the PAMPA assay with 3-fold less (though still significant) permeability in the blood brain barrier PAMPA assay.

Plasma Protein Binding is a measure of a drug's efficiency to bind to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Highly plasma protein bound drugs are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues. The probe, CID03342390, is highly bound (98.9–99.7%) to both human and mouse plasma.

Plasma Stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which strongly can influence the in vivo efficacy of a test compound. Drug candidates are exposed in plasma to enzymatic processes (proteinases, esterases), and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. The probe, CID03342390, shows excellent stability (100%) in both human and mouse plasma; however it is rapidly metabolized by microsomes (below).

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. This assay addresses the pharmacologic question of how long the parent compound will remain circulating in plasma within the body. The probe, CID03342390, is rapidly metabolized in either Human or mouse microsomes. It however, does not have significant toxicity to human hepatocyctes.

j. Probe properties

Table 7Properties Computed from Structure CID3342390 (MLS0026993322)

Calculated PropertyValue
Molecular Weight479.63422 [g/mol]
Molecular FormulaC27H33N3O3S
XLogP3-AA4.4
H-Bond Donor2
H-Bond Acceptor5
Rotatable Bond Count11
Tautomer Count2
Exact Mass479.2243
MonoIsotopic Mass479.2243
Topological Polar Surface Area78.5
Heavy Atom Count34
Formal Charge0
Complexity699
Isotope Atom Count0
Defined Atom StereoCenter Count0
Undefined Atom StereoCenter Count0
Defined Bond StereoCenter Count0
Undefined Bond StereoCenter Count0
Covalently-Bonded Unit Count1

4. Comparative data showing probe specificity for target in biologically relevant assays

As described in the CPDP these studies are now on-going in the assay provider’s and collaborating laboratories, as post-probe nomination research and we hope to publish jointly in the future. Additionally this probe compound was submitted for broad CNS-centric GPCR paneling at the PDSP (see Table 6 above) and were both shown to be fairly selective to the opioid receptors. Furthermore, the relatively good agreement among the GPCR panel, cell-based beta-arrestin mediated enzyme complementation assay and the more downstream image based beta-arrestin mediated G-protein redistribution with these compounds is notable.

As defined in the CPDP and the initial teleconference calls with the National Institute on Drug Abuse, this probe project was unusual as there are already examples of very potent agonists and antagonists of the kappa-opioid receptors with low nanomolar EC50 and IC50s, that are very selective against the mu- and delta- opioid subtypes. As emphasized during those initial discussions, the overarching purpose was to find new chemical scaffolds that are chemically distinct from the rich literature of known agonists and antagonists as starting points for further synthesis and work by the assay provider and their collaborative chemists. Furthermore, as NIDA is ultimately interested in new antagonists that are short acting. While these animal model studies are out of scope of the MLPCN and indeed would take much more compounds and more time than a probe nomination project, NIDA is was clear that new scaffolds would be a key measure of success. As a comparative measure, Table 8 summarizes our antagonist probe against the precedent state-of-art probes. We note that we have conservatively put the 850 nM value obtained in the functional DiscoveRx assay into this table, despite the fact that the Ki values shown for the existing agents are probably more appropriately compared to the Ki value of 4.3 nM obtained for the probe by the PDSP.

Table 8. Comparison of prior art and current probe.

Table 8

Comparison of prior art and current probe.

While this probe does meet most of the potency criteria for a probe as agreed to upon in the CDPD, the potency and selectivity remain to be improved further. However, the assay provider is interested in this structure and finds the SAR intriguing. Clearly this sulfonamide probe provides novel scaffolds distinct from BNI, GNTI and JDTic that might be further developed as was the main goal of this project. This scaffold while chemically distinct is reminiscent structurally with regard to the overall length and the presence of derivitizable aromatic rings at both ends. Again, we consider this probe project to be successful in defining a novel chemical scaffold that may well have different and interesting pharmacological properties than the current literature examples.

5. Bibliography

1.
Cami J, Farre M. Drug addiction. N Engl J Med. 2003;349:975. [PubMed: 12954747]
2.
Prisinzano TE, Tidgewell K, Harding WW. Kappa opioids as potential treatments for stimulant dependence. Aaps J. 2005;7:E592. [PMC free article: PMC2751263] [PubMed: 16353938]
3.
Xuei X, Dick D, Flury-Wetherill L, Tian HJ, Agrawal A, Bierut L, Goate A, Bucholz K, Schuckit M, Nurnberger J Jr, Tischfield J, Kuperman S, Porjesz B, Begleiter H, Foroud T, Edenberg HJ. Association of the kappa-opioid system with alcohol dependence. Mol Psychiatry. 2006;11:1016. [PubMed: 16924269]
4.
Prisinzano TE. Psychopharmacology of the hallucinogenic sage Salvia divinorum. Life Sci. 2005;78:527. [PubMed: 16213533]
5.
Hasebe K, Kawai K, Suzuki T, Kawamura K, Tanaka T, Narita M, Nagase H. Possible pharmacotherapy of the opioid kappa receptor agonist for drug dependence. Ann N Y Acad Sci. 2004;1025:404. [PubMed: 15542743]
6.
Metcalf MD, Coop A. Kappa opioid antagonists: past successes and future prospects. AAPS J. 2005;7:E704. [PMC free article: PMC2751273] [PubMed: 16353947]
7.
Deng X, Mani NS. A facile, environmentally benign sulfonamide synthesis in water. Green Chem. 2006;8:835.
8.
Ruchelman AL, Houghton PJ, Zhou N, Liu A, Liu LF, LaVoie EJ. 5-(2-Aminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones: Variation of N-alkyl substituents modulates sensitivity to efflux transporters associated with multidrug resistance. J. Med. Chem. 2005;48:792. [PubMed: 15689163]

Appendix. Synthetic procedures and compound characterization

Series 2 Compounds

Synthesis of Series 2 Antagonist Compounds:

General procedure for coupling of the sulfonamide acid chlorides with the diamine fragments.

General procedure for coupling of the sulfonamide acid chlorides with the diamine fragments

The acid chloride, Et3N (2.5 equiv) and the diamine fragment (1.0 equiv) in CH2Cl2 (10 mL/mmol) were stirred at rt for 5 h. Aqueous saturated NaHCO3 (0.5 mL) was added, the solvents removed in vacuo and the residue extracted with CH2Cl2 (3 × 2 mL). The combined filtrates were concentrated and purified by mass-directed, reverse phase preparative HPLC to afford the amino amide compounds.

N-(2-(benzyl(ethyl)amino)ethyl)-4-((4-methylphenylsulfonamido)methyl)benzamide (SID-87457826).

N-(2-(benzyl(ethyl)amino)ethyl)-4-((4-methylphenylsulfonamido)methyl)benzamide (SID-87457826)

1H NMR (CDCl3) δ 1.13 (t, J = 7.2 Hz, 3 H), 2.47 (s, 3 H), 2.64 (q, J = 7.2 Hz, 2 H), 2.69 (t, J = 6.0, 2 H), 3.46 (dt, J = 5.1, 6.0 Hz 2 H), 3.63 (s, 2 H), 4.21 (s, 2 H), 4.84–4.97 (br, 1 H), 6.62–6.69 (br, 1 H), 7.25–7.37 (m, 9 H), 7.59 (d, J = 8.4 Hz, 2 H), 7.81 (d, J =8.4 Hz, 2 H);

N-(2-(dibenzylamino)ethyl)-4-((4-methylphenylsulfonamido)methyl)benzamide (SID-87457827).

N-(2-(dibenzylamino)ethyl)-4-((4-methylphenylsulfonamido)methyl)benzamide (SID-87457827)

1HNMR (CDCl3) δ 2.48 (s, 3 H), 2.73 (t, J = 6.0 Hz, 2 H), 3.50 (dt, J = 4.8, 6.0 Hz, 2 H), 3.65 (s, 4 H), 4.23 (s, 2 H), 4.89 (br, 1 H), 6.45 (br, 1 H), 7.25–7.38 (m, 14 H), 7.54 (d, J = 8.4 Hz, 2 H), 7.82 (d, J = 8.4 Hz, 2 H);

N-(2-(dibenzylamino)ethyl)-4-((3-methylphenylsulfonamido)methyl)benzamide (SID-87457834).

N-(2-(dibenzylamino)ethyl)-4-((3-methylphenylsulfonamido)methyl)benzamide (SID-87457834)

1HNMR (CDCl3) δ 2.46 (s, 3 H), 2.73 (t, J = 5.7 Hz, 2 H), 3.50 (dt, J = 5.0, 5.7 Hz, 2 H), 3.65 (s, 4 H), 4.25 (s, 2 H) 6.45 (br, 1 H), 7.24–7.35 (m, 13 H), 7.43–7.45 (m, 2 H), 7.55 (d, J = 8.0 Hz, 2 H), 7.72–7.75 (m, 2 H);

N-(2-(benzyl(ethyl)amino)ethyl)-4-((4-ethylphenylsulfonamido)methyl)benzamide (SID-87457835).

N-(2-(benzyl(ethyl)amino)ethyl)-4-((4-ethylphenylsulfonamido)methyl)benzamide (SID-87457835)

1HNMR (CDCl3) δ 1.13 (t, J = 7.2 Hz, 3 H), 1.30 (t, J = 7.5 Hz, 3 H), 2.65 (q, J = 7.2 Hz, 2 H), 2.70 (t, J = 5.2 Hz, 2 H), 2.76 (q, J = 7.5 Hz, 2 H), 3.46 (dt, J = 5.2, 6.4 Hz, 2 H), 3.63 (s, 2 H), 4.22 (d, J = 6.0 Hz, 2 H), 4.80 (t, J = 6.3 Hz, 1 H), 6.66 (br, 1 H), 7.24–7.40 (m, 9 H), 7.60 (d, J = 7.2 Hz, 2 H), 7.83 (d, J = 8.5 Hz, 2 H);

N-(2-(benzyl(sec-butyl)amino)ethyl)-4-((4-ethylphenylsulfonamido)methyl)benzamide (SID-87457836).

N-(2-(benzyl(sec-butyl)amino)ethyl)-4-((4-ethylphenylsulfonamido)methyl)benzamide (SID-87457836)

1HNMR (CDCl3) δ 0.85 (t, J = 6.8 Hz, 3 H), 0.97 (d, J = 6.0 Hz, 3 H), 1.20 (t, J = 7.5 Hz, 3 H), 1.29 (m, 1 H), 1.52 (m, 1 H), 2.55–2.70 (m, 5 H), 3.24 (m, 1 H), 3.28–3.39 (m, 2 H), 3.62 (d, J = 14.9 Hz, 1 H), 4.12 (d, J = 6.4 Hz, 2 H), 4.69 (br, 1 H), 6.415 (br, 1 H), 7.11–7.31 (m, 9 H), 7.44 (d, J = 7.7 Hz, 2H), 7.73 (d, J = 7.7 Hz, 2 H);

(R)-4-((4-ethylphenylsulfonamido)methyl)-N-(2-(methyl(1-phenylethyl)amino)ethyl)-benzamide (SID-87457837).

(R)-4-((4-ethylphenylsulfonamido)methyl)-N-(2-(methyl(1-phenylethyl)amino)ethyl)-benzamide (SID-87457837)

1HNMR (CDCl3) δ 1.20 (t, J = 7.6 Hz, 3 H), 1.32 (d, J = 6.4 Hz, 3 H), 2.21 (s, 3 H), 2.43 (m, 1 H), 2.53 (m 1 H), 2.66 (q, J = 7.6 Hz, 2 H), 3.36 (m, 2 H), 3.61 (q, J = 6.4 Hz, 1 H), 4.13 (d, J = 6.0 Hz, 2 H), 4.76 (br, 1 H), 6.57 (br, 1 H), 7.10–7.34 (m, 9 H), 7.54 (d, J = 7.6 Hz, 2 H), 7.73 (d, J = 8.2 Hz, 2 H);

N-(2-(dibenzylamino)ethyl)-4-((4-ethylphenylsulfonamido)methyl)benzamide (SID-87457838).

N-(2-(dibenzylamino)ethyl)-4-((4-ethylphenylsulfonamido)methyl)benzamide (SID-87457838)

1HNMR (CDCl3) δ 1.30 (t, J = 7.6 Hz, 3 H), 2.69–2.80 (m, 4 H), 3.50 (dt, J = 4.5, 6.4 Hz, 2 H), 3.65 (s, 4 H), 4.24 (s, 2H), 6.44 (br, 1 H), 7.25–7.35 (m, 13 H), 7.38 (d, J = 7.6 Hz, 2 H), 7.55 (d, J = 7.6 Hz, 2 H), 7.84 (d, J = 7.6 Hz);

Views

  • PubReader
  • Print View
  • Cite this Page
  • PDF version of this page (823K)

Related information

Similar articles in PubMed

See reviews...See all...

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...