Small Molecule Agonists for the Neurotensin 1 Receptor (NTR1 Agonists)

Hershberger P, Hedrick M, Peddibhotla S, et al.

Publication Details

A non-high throughput screening (HTS) scaffold-hop program identified the probe candidate ML301 and associated analogs. ML301 met probe nomination criteria, exhibiting full agonist behavior (79 – 93%) with an half maximal effective concentration (EC50) of 2.0 – 4.1 μM against neurotensin 1 receptor (NTR1) in the primary assay. This probe satisfied our secondary assay requirements; namely, the Ca2+ mobilization Fluorescence Imaging Plate Reader (FLIPR) assay (93% efficacy at 298 nM) and good selectivity relative to NTR2 and G protein-coupled receptor (GPR)35. In further profiling, ML301 showed low potential for promiscuity and improved pharmacological data when compared to existing art. A scaffold hop program was initiated in parallel with the originally planned high throughput screening based approach. This approach identified new scaffolds of which the most promising was a quinazoline derivative represented by the singleton MLS-0233108. Medicinal chemistry optimization of MLS-0233108 led to ML314, the most potent molecule in this second series that exhibited full agonist behavior (100 %) on NTR1 (EC50 = 1.9 μM). ML314 showed good selectivity against NTR2 and GPR35, but did not stimulate Ca2+ mobilization. ML314 is potentially a biased agonist operating via the β-arrestin pathway rather than the traditional Gq coupled pathway. Signaling mediated by β-arrestin has distinct biochemical and functional consequences that may lead to physiological advantages as described below. This probe report describes the discovery and properties of ML301 and summarizes the HTS and follow-up campaign, which identified ML314.

Assigned Assay Grant #: 1 R03 MH089653-01

Screening Center Name & PI: Sanford Burnham Center for Chemical Genomics (SBCCG) & John C. Reed (PI)

Chemistry Center Name & PI: Sanford Burnham Center for Chemical Genomics (SBCCG) & John C. Reed (PI)

Assay Submitter & Institution: Lawrence R. Barak, Duke University Medical Center, Durham NC

Co-Assay Submitter & Institution: James B. Thomas, RTI International

PubChem Summary Bioassay Identifier (AID): 493055

Resulting Publications

1.
Peddibhotla S., Hedrick M. P. et al. Discovery of ML314, a Brain Penetrant Nonpeptidic β-Arrestin Biased Agonist of the Neurotensin NTR1 Receptor. ACS Medicinal Chemistry Letters. 2013;4(9):846–851. [PMC free article: PMC3940307] [PubMed: 24611085]

Probe Structure & Characteristics

This Center Probe Report describes two selective agonists of the neurotensin 1 receptor, ML301, which displays full agonist behavior in a β-arrestin pathway based assay as well as a calcium flux assay, and ML314, which displays full agonist behavior in a β-arrestin pathway based assay, but no activity in a calcium mobilization assay. Potency and selectivity characteristics for ML301 and ML314 are described in the summary table (Table 1).

Table 1. Summary of Probe activities.

Table 1

Summary of Probe activities.

Image ml314fu1

1. Recommendations for Scientific Use of the Probe

Despite the fact that this receptor system was identified many years ago, very few non-peptide ligands have been described for the neurotensin 1 receptor (NTR1), particularly small molecule agonists with CNS activity. As discussed in the original grant application, there is evidence from neurotensin peptide research that strongly supports the use of neurotensin agonists in the pharmacologic intervention of addictive behavior. Therefore, a non-peptide agonist exhibiting in vivo properties, either residing in these probes or in analogs designed subsequently, will be of great value to the addiction research community.

Moreover, the presence of neurotensin receptors on dopaminergic neurons in the CNS suggests investigators studying the modulation of dopamine signaling will also utilize these probes. Ultimately, these probes or further successors will be quickly advanced to animal model testing for evaluation and modeling because of the potential they may have in the treatment of methamphetamine addiction. There is growing evidence in the field that agonism of several CNS receptors may lead to more efficacy against addiction. Both probes have some cross-activity to dopamine receptors and the dopamine transporter assays, though ML314 is less selective against additional GPCRs in a panel competitive binding assays than ML304, so together they may provide tools for use in future comparative pharmacological studies to test this hypothesis of increased efficacy with less selective agonists.

2. Materials and Methods

The details of the primary HTS and additional assays can be found in the “Assay Description” section in the PubChem BioAssay view under the AIDs as listed in Table 2.

Table 2. Summary of Assays and AIDs.

Table 2

Summary of Assays and AIDs.

2.1. Assays

Table 2 summarizes the details for the assays that drove this probe project.

2.2. Probe Chemical Characterization

Chemical name of probe compound & structure including stereochemistry

The IUPAC name of the probe is (2S)-2-{[2-(2,6-dimethoxyphenyl)-1-(7-chloro(4-quinolyl))imidazol-4-yl]carbonylamino}-4-methylpentanoic acid (ML301). The IUPAC name of ML314 is 2-cyclopropyl-6,7-dimethoxy-4-(4-(2-methoxyphenyl)- piperazin-1-yl)quinazoline.

The specific batches prepared, tested and submitted to the MLSMR are archived as SID 126723249 corresponding to CID 49837912 (ML301) and SID 134225039 corresponding to CID 53245590 (ML314) and their structures are shown in Figure 1.

Figure 1. Structures of ML301 and ML314.

Figure 1

Structures of ML301 and ML314.

These probes are not commercially available. 25 mg samples of ML301 and ML314 synthesized at SBCCG, along with five analogs of each, have been deposited in the MLSMR (Evotec) (See Probe Submission Tables 4a and 4b.)

Table 4a. Probe and Analog Submissions to MLSMR (Evotec) for NTR1 Agonists.

Table 4a

Probe and Analog Submissions to MLSMR (Evotec) for NTR1 Agonists.

Table 4b. Analog Submissions to MLSMR (Evotec) for NTR1 Agonists.

Table 4b

Analog Submissions to MLSMR (Evotec) for NTR1 Agonists.

Synthetic Routes: Two potential synthetic routes are outlined for the synthesis of ML301 in Scheme 1 (preferred and alternate). Although the alternate method was fewer steps, its low yields and the high cost of 4-aminoquinoline building blocks made the longer method more preferred and practical for most cases of interest.

Scheme 1. Synthesis of ML301.

Scheme 1

Synthesis of ML301. Preferred route: Synthesis of ML301, conditions: a. 1. NaOAc, water, 1,1-dibromo-3,3,3-trifluoroacetone, 100 °C, 30 min. 2. 2,6-dimethoxybenzaldehyde, ammonium hydroxide, water, methanol, 52%; b. NaOH, water, 100 °C, (more...)

In contrast, one route for ML314 is shown in Scheme 2, (following Scheme 1).

Scheme 2. Synthesis of ML314.

Scheme 2

Synthesis of ML314. Conditions: a): 4M HCl (1,4-dioxane), 100 °C, 15h, 65%; POCl3, reflux, 15 h, 84%; c) 1-(2-methoxyphenyl)piperazine hydrochloride (1.5 equiv.), K2CO3 (3 equiv.), 1,4-dioxane, microwave, 80 °C, 1 h, 25%.

Figure 2. 1H NMR, and LC-MS spectra of ML301.
Figure 2. 1H NMR, and LC-MS spectra of ML301.
Figure 2. 1H NMR, and LC-MS spectra of ML301.

Figure 21H NMR, and LC-MS spectra of ML301

A. 1H NMR Spectrum of ML301 (500 MHz, CDCl3). B. LC of LC-MS spectra of ML301. C. MS of LC-MS spectra

Figure 3. 1H NMR, 13C NMR, and LC-MS spectra of ML314.
Figure 3. 1H NMR, 13C NMR, and LC-MS spectra of ML314.
Figure 3. 1H NMR, 13C NMR, and LC-MS spectra of ML314.

Figure 31H NMR, 13C NMR, and LC-MS spectra of ML314

A. 1H NMR Spectrum (500 MHz, CDCl3). B. LC of LC-MS.(Reverse phase C18 column: isocratic). C. MS of LC-MS spectra.

Solubility and Stability of ML301 and ML314 in PBS at room temperature

The solubility of ML301 and ML314 were investigated in aqueous buffers at room temperature. As noted in the Summary of in vitro ADME/T properties, ML301 has good solubility, while ML314 has good to moderate solubility, relative to their respective potency for NTR1 in aqueous buffer at pH 5, 6.2 and 7.4. To evaluate their potential hydrolytic instability 1 μM solutions of ML301 and ML314 were prepared in acetonitrile:PBS (1:1) and incubated at room temperature, and the amount of the parent compounds remaining at various times were analyzed by LC/MS (Figure 4 time course and Table 3 data). The results indicate that at pH 7.4, both ML301 and ML314 are very stable in acetonitrile:PBS (1:1) with no appreciable loss up to 48 h.

Figure 4. Stability of ML301 and ML314.

Figure 4

Stability of ML301 and ML314. Time course.

Table 3. Stability of ML301 and ML314 in 1:1 PBS:acetonitrile at pH 7.4 ambient temperature.

Table 3

Stability of ML301 and ML314 in 1:1 PBS:acetonitrile at pH 7.4 ambient temperature.

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

Samples of the probe ML301 and ML314 (>25 mg), and five analogs of each (>20 mg), synthesized at SBCCG were submitted to MLSMR (Tables 4a and 4b), and 5 mg of the probes were provided to the Assay Provider.

2.3. Probe Preparation

(Compound lettering as per Scheme 1 & Scheme 2)

ML301 Preparation: Two potential synthetic routes are outlined for the synthesis of ML301 in Scheme 1. Although the alternate method was fewer steps, its low yields and the high cost of 4-aminoquinoline building blocks made the longer method more preferred and practical for most cases of interest. In addition to standard Pinner conditions that generally do not work well for cases involving sterically hindered benzonitriles, many amidine-forming conditions were attempted9–14. Conditions (h), although inefficient, were the best we found for this sterically and electronically disfavorable case. For some analogs without the 2,6-dimethoxy moieties, the shorter route was more preferred. Importantly, both routes produced exclusively the key intermediate regioisomer D (shown in the box), and there is ample literature precedent13,15–17 for this assignment via the amidine (alternate) route. While it may appear that the preferred route could be shortened by performing the N-arylation step on the trifluoromethyl intermediate, followed by CF3 hydrolysis and amino acid coupling, this was not possible because the CF3 hydrolysis failed if the imidazole was N-arylated, and the N-arylation was not efficient when performed on the acid intermediate. Thus, it was necessary to first hydrolyze the CF3 group (b), and then esterify (c) prior to N-arylation (d).

Preferred Route: A, Scheme 1, Step a8: A vessel containing sodium acetate (4.90 g, 59.8 mmol) was charged with 14 mL of water and 1,1-dibromo-3,3,3-trifluoroacetone (8.06 g, 3.54 mL, 29.9 mmol). Some heat was evolved, the vessel was purged with nitrogen, and placed in a 100°C oil bath. After 30 min, the solution was allowed to cool. 2,6-dimethoxybenzaldehyde (4.514 g, 27 mmol), concentrated ammonium hydroxide (28 mL) and methanol were combined and transferred into the vessel as a single solution. The vessel was swept with nitrogen, closed with a stopper, and the yellow solution was stirred at room temperature. White solid was apparent within one hour. After 19 hours, the mixture was concentrated and partitioned with 100 mL of water and 100 mL of ethyl acetate. The organics were dried over MgSO4 and concentrated to give 7.08 g of an orange solid, which contained an approximately 1:1 ratio of product and unreacted 2,6-dimethoxybenzaldehyde. Flash chromatography (550 mL silica gel, 25% to 80% gradient of ethyl acetate / hexane) returned 3.836 g (52%) of 2-(2,6-dimethoxyphenyl)-4-(trifluoromethyl)-1H-imidazole (A) as a pale orange solid. 1H NMR (400 MHz, CDCl3) δ 10.02 (br., 1H), 7.46 (s., 1H), 7.35 (t., J = 8.4 Hz, 1H), 6.66 (d., J = 8.4 Hz, 2H), 3.84 (s., 6H). ESI m/z 273.08 [M+H].

B, Scheme 1, Step b8: A vessel containing 2-(2,6-dimethoxyphenyl)-4-(trifluoromethyl)-1H-imidazole (A) (3.734 g, 13.7 mmol) was charged with 18 mL of 3.75 N sodium hydroxide. The vessel was equipped with a condenser and heated in a 100°C oil bath for 22 h. The mixture was cooled, diluted with 20 mL of water and washed once with 20 mL of ethyl acetate. The pH of the aqueous part was adjusted to ca. 3 with 3 M HCl. The turbid solution was placed in a refrigerator for 5 h before collecting a solid precipitate. The filtrate was refrigerated overnight to yield a smaller second crop for a combined recovery of 3.202 g (94%) of 2-(2,6-dimethoxyphenyl)-1H-imidazole-4-carboxylic acid (B) as a pale pink solid. 1H NMR (400 MHz, DMSO-d6) δ 7.75 (br., 1H), 7.41 (t., J = 8.4 Hz, 1H), 6.74 (d., J = 8.4 Hz, 2H), 3.69 (s., 6H). ESI m/z 249.05 [M+H].

C, Scheme 1, Step c: 2-(2,6-dimethoxyphenyl)-1H-imidazole-4-carboxylic acid (B) (318 mg, 1.28 mmol) was mostly dissolved in 10 mL of absolute ethanol. Thionyl chloride (0.5 mL, 6.9 mmol) was added, the vial was connected to nitrogen and placed in a 75°C block. After 17 h, the mixture was concentrated to an olive green solid, which was combined with 70 mL of brine containing 1.5 g of sodium bicarbonate. This mixture was extracted with five 40 mL portions of 2% EtOH/CHCl3. The extracts were dried over MgSO4 and concentrated to give 242 mg (69%) of ethyl 2-(2,6-dimethoxyphenyl)-1H-imidazole-4-carboxylate (C), a pale yellow solid. 1H NMR (500 MHz, CDCl3) δ 7.87 (br. s., 1H), 7.36 (t., J = 8.4 Hz, 1H), 6.68 (d., J = 8.4 Hz, 1H), 4.38 (q., J = 7.1 Hz, 2H), 3.89 (br. s., 6H), 1.40 (t., J = 7.1 Hz, 3H). ESI m/z 277.18 [M+H].

D, Scheme 1, Step d: Ethyl 2-(2,6-dimethoxyphenyl)-1H-imidazole-4-carboxylate (C) (85 mg, 0.31 mmol), 7-chloro-4-iodoquinoline (267 mg, 0.92 mmol), and cesium carbonate (502 mg, 1.53 mmol) were combined as solids in a reaction vial. Butyronitrile (1.5 mL) was added to produce a slurry. The mixture was heated at 110°C for 18 hours. After cooling, the mixture was charged with 25 mL of brine and extracted with two 25 mL portions of CHCl3. The organics were dried over MgSO4 and concentrated to give 290 mg of a crude tan solid which was purified by flash chromatography (25 mL silica gel, 10% – 30% gradient of ethyl acetate in dichloromethane) to return 58.9 mg (43%) of ethyl 1-(7-chloroquinolin-4-yl)-2-(2,6-dimethoxyphenyl)-1H-imidazole-4-carboxylate (D) as a colorless film. 1H NMR (500 MHz, CDCl3) δ 8.85 (s., 1H), 8.19 (s., 1H), 7.99 (s., 1H), 7.77 (d., J = 9.0 Hz, 1H), 7.55 (dd, J = 9.0, 2.0 Hz, 1H), 7.2 (m., 2H), 6.48 (br., 1H), 6.29 (br., 1H), 4.47 (q., J = 7.1 Hz, 2H), 3.80 (br., 3H), 3.27 (br., 3H), 1.45 (t., J = 7.1 Hz, 3H). ESI m/z 438.21, 440.21 [M+H].

E. Scheme 1, Step e: Ethyl 1-(7-chloroquinolin-4-yl)-2-(2,6-dimethoxyphenyl)-1H-imidazole-4-carboxylate (D) (104 mg, 0.24 mmol) was dissolved in 4 mL of ethanol and 4 mL of water. The solution was treated with 53 mg (0.95 mmol) of solid KOH, and was then stirred at room temperature for 17 h. The mixture was diluted with 20 mL of water and washed once with 15 mL of ethyl acetate. The pH of the aqueous part was adjusted to ca. 2–3 with 1 N HCl, and this solution was extracted with three 20 mL portions of CHCl3. The chloroform extracts were dried over MgSO4 and concentrated to give 84.3 mg (86%) of 1-(7-chloroquinolin-4-yl)-2-(2,6-dimethoxyphenyl)-1H-imidazole-4-carboxylic acid (E), which was carried forward without purification. 1H NMR (500 MHz, CDCl3) δ 8.89 (s., 1H), 8.22 (s., 1H), 8.06 (s., 1H), 7.74 (d., J = 9.0 Hz, 1H), 7.58 (dd. J = 9.0, 2.0 Hz, 1H), 7.26 (t., J = 8.5 Hz, 1H), 7.21 (d., J = 4.6 Hz, 1H), 6.4 (br., 2H), 3.8 (br., 3H), 3.3 (br., 3H). ESI m/z 410.18, 412.18 [M+H].

F, Scheme 1, Step f: Ethyl dimethylaminopropylcarbodiimide hydrochloride (27 mg, 0.14 mmol) and 1-hydroxybenzotriazole hydrate (22 mg, 0.14 mmol) were added to a vial containing 1-(7-chloroquinolin-4-yl)-2-(2,6-dimethoxyphenyl)-1H-imidazole-4-carboxylic acid (E) (58 mg, 0.14 mmol). The vial was then charged with N,N-dimethylformamide (2 mL) and triethylamine (0.06 mL, 0.4 mmol). After stirring for 25 min at room temperature, L-leucine tert-butyl ester (32 mg, 0.14 mmol) was added as solid, and the resulting mixture stirred at room temperature for 20 h. The mixture was transferred into 20 mL of water and extracted with two 25 mL portions of ethyl acetate. The organics were dried over MgSO4 and concentrated to give 154 mg of a crude yellow liquid which was purified by flash chromatography (20 mL silica gel, 25% – 75% gradient of ethyl acetate in hexane) to return 67.6 mg (83%) of (S)-tert-butyl 2-(1-(7-chloroquinolin-4-yl)-2-(2,6-dimethoxyphenyl)-1H-imidazole-4-carboxamido)-4-methylpentanoate (F) as a clear film. 1H NMR (500 MHz, CDCl3) δ 8.84 (br. s., 1H), 8.18 (br. s., 1H), 7.94 (s., 1H), 7.78 (m., 1H), 7.55 (m., 1H), 7.23 (t., J = 8.5 Hz, 1H), 7.16 (m., 1H), 6.5 (br., 1H), 6.3 (br., 1H), 4.76 (m., 1H), 3.8 (br., 3H), 3.25 (br., 3H), 1.95 – 1.55 (m., 3H), 1.52 (s., 9H), 1.03 (m., 6H). ESI m/z 579.36, 581.37 [M+H].

G, Scheme 1, Step g: A solution of (S)-tert-butyl 2-(1-(7-chloroquinolin-4-yl)-2-(2,6-dimethoxyphenyl)-1H-imidazole-4-carboxamido)-4-methylpentanoate (F) (61 mg, 10.5 mmol) in 1 mL of trifluoroacetic acid and 1 mL of dichloromethane was stirred at room temperature for 5 h. The mixture was concentrated. The residue was dissolved in 1.5 mL of methanol and purified by preparative reverse phase HPLC, using a stepwise gradient with 0.1% formic acid (% water:% acetonitrile): t = 0 min, 90:10; t = 2.0 min, 80:20; t = 6.0 min, 30:70; t = 7.5 min, 2:98; t = 8.8 min, 2:98; t = 9.0 min, 90:10. This afforded 45 mg (82%) of ML301 as a white powder after lyophilization. This sample was redissolved with a second batch, and lyophilized to return a uniform batch of 80 mg of material for testing and submission. 1H NMR (500 MHz, DMSO-d6) δ 12.68 (m., 1H), 8.95 (s., 1H), 8.12 (s., 1H), 8.15 (s., 2H), 7.71 (m., 1H), 7.65 (m., 1H), 7.35 (br. m., 1H), 7.24 (t., J = 8.4 Hz, 1H), 6.58 (br. m., 1H), 6.43 (br. m., 1H), 4.50 (m., 1H), 3.70 (br. s., 3H), 3.3 (br. s., 3H), 1.85 (m., 1H), 1.70 (m., 1H), 1.59 (m., 1H), 0.93 (m., 6H). 13C NMR (500 MHz, DMSO-d6) δ 174.3, 161.5, 152.0, 148.8, 141.7, 141.5, 136.8, 134.9, 132.2, 128.1, 127.7, 125.1, 124.9, 122.6, 119.2, 106.2, 103.6, 55.6, 55.2, 50.0, 40.1, 24.4, 23.0, 21.2. ESI m/z 523.30, 525.29 [M+H], HRMS (ESI+ve): Calculated for C27H28ClN4O5, [M+H] = 523.1743, observed [M+H] = 523.1723. (ESI+ve): Calculated for C27H27ClN4NaO5, [M+Na] = 545.1562, observed [M+Na] = 545.1576. [αD24] = −3.5°, c = 0.10, methanol.

Alternative Route to ML301, Scheme 1, Step h: 2,6-dimethoxybenzonitrile (501 mg, 3.07 mmol) and 4-amino-7-chloroquinoline (548 mg, 3.07 mmol) were dissolved together in 20 mL of anhydrous tetrahydrofuran. The vessel was swept with nitrogen and chilled in an ice-water bath. Ethereal 3.0 M ethyl magnesium bromide (2.05 mL, 6.14 mmol) was added dropwise over 3 min. After briefly forming an off-white solid suspended in a yellow solution, within minutes the mixture became a dark amber homogenous solution with some off-white solid material remaining. The vessel was placed in a 75°C oil bath for 21 h before diluting with 60 mL of water. The pH was decreased to ca. 14 by addition of 4 pellets of NaOH. The mixture was then extracted with 60 mL of ethyl acetate, using solid NaCl to facilitate partitioning. The organics were dried over MgSO4 and concentrated to 1.27 g of a crude brown oily paste containing mostly unreacted starting materials. Flash chromatography (150 mL silica gel, 50% ethyl acetate / hexane, then neat ethyl acetate, then 10% methanol in ethyl acetate) returned 100 mg (9.6%) of N-(7-chloroquinolin-4-yl)-2,6-dimethoxybenzimidamide (G). 1H NMR (400 MHz, CDCl3) δ 8.4 (br., 1H), 8.3 (br., 2H), 7.9 (br., 1H), 7.4 (br., 2H), 6.6–6.3 (br. m, 4H), 5.25 (br., 1H), 3.6 (br. 6H). ESI m/z 342.09, 344.10 [M+H].

D, Scheme 1, Step i: Sodium bicarbonate (49 mg, 0.58 mmol) was added to a flask containing N-(7-chloroquinolin-4-yl)-2,6-dimethoxybenzimidamide (G) (99 mg, 0.29 mmol). Ethyl bromopyruvate (73 mg, 0.38 mmol) was then transferred to the flask as a solution in 3 mL of ethanol. The vessel was swept with nitrogen and heated in an 85°C oil bath for 22 h. The mixture was partitioned with 20 mL of water and 20 mL of chloroform. The aqueous part was extracted with two additional 20 mL portions of chloroform. The combined organics were dried over MgSO4 and concentrated to give 97 mg of a brown residue. The crude residue was combined with 110 mg of p-toluenesulfonic acid monohydrate and 3 mL of toluene. The vessel was swept with nitrogen and heated for 4 h in a 115°C oil bath. The mixture was partitioned with 25 mL of saturated sodium bicarbonate and 25 mL of chloroform. The aqueous part was extracted with two additional 20 mL portions of chloroform. The combined organics were dried over MgSO4 and concentrated to give 74 mg of a brown oil which was purified by flash chromatography (20 mL silica gel, gradient 50% – 80% ethyl acetate in hexane) to return 22 mg (17%) of ethyl 1-(7-chloroquinolin-4-yl)-2-(2,6-dimethoxyphenyl)-1H-imidazole-4-carboxylate (D), the same intermediate furnished by Step d.

ML314 preparation: The synthetic route to ML314 was adapted from a previously reported procedure18. Methyl 2-amino-4,5-dimethoxybenzoate (1.0 g, 4.73 mmol) and cyclopropyl carbonitrile (0. 95 g, 14.2 mmol) were weighed into a 40 mL vial and 15 mL of 4M HCl in 1,4-dioxane was added, the vial capped and the mixture heated to 100°C for 15 h. The reaction mixture was cooled and poured carefully into cold saturated NaHCO3 solution (100 mL). The precipitate formed was collected by filtration, washed extensively with water and air-dried to afford the product 2-cyclopropyl-6,7-dimethoxyquinazolin-4-ol (A) as a gray solid (0.76 g, 65%) which was used without any further purification. 1H NMR (500 MHz, DMSO) δ 7.42 (s, J = 1.4 Hz, 1H), 7.07 (s, J = 2.9 Hz, 1H), 3.86 (s, 3H), 3.83 (s, 3H), 1.95 – 1.88 (m, 1H), 1.08 – 1.01 (m, 2H), 1.01 – 0.95 (m, 2H); LRMS (ESI+ve): Calculated for C13H14N2O3, [M+H] = 247.11, observed [M+H] = 247.13.

A (0.3 g, 1.22 mmol) was suspended in POCl3 (10 mL) in a 40 mL vial and the mixture was heated at 110°C for 15 h during which time the suspension turned reddish brown The mixture was then allowed to cool to 23°C and POCl3 was removed on a rotary evaporator. The residue was dissolved in 20 mL of dichloromethane and washed with saturated NaHCO3 solution (3x, 10 mL). The organic layer was collected, dried over anhydrous Na2SO4 and the solvent was evaporated to afford the intermediate 4-chloro-2-cyclopropyl-6,7-dimethoxyquinazoline (0.27 g, 84%). LRMS (ESI+ve): Calculated for C13H13ClN2O2, [M+H] = 265.07, observed [M+H] = 265.08. The intermediate was used in the next step without further purification. 1-(2-methoxyphenyl)piperazine hydrochloride (0.35 g, 1.53 mmol) and K2CO3 (0.7 g, 5.1 mmol) were weighed into a 35 mL microwave reaction tube. 4-chloro-2-cyclopropyl-6,7-dimethoxyquinazoline (0.27 g, 1.02 mmol) solution in 1,4-dioxane(10 mL) was added and the mixture was heated in the microwave at 80°C for 1.5 hours. The mixture was diluted with 50 mL water and then extracted with ethyl acetate (3x, 25 mL). The organic layer was dried over anhydrous Na2SO4 and the solvent was evaporated to afford a dark brown residue. The residue was subjected to silica gel flash chromatography (1:3 ethyl acetate/hexanes) to afford MLS-0463110 (0.105 g, 25%) as a pale yellow solid. 1H NMR (500 MHz, CDCl3) δ 7.25 (s, 1H), 7.12 (s, 1H), 7.08 – 7.03 (m, 1H), 7.02 – 6.95 (m, 2H), 6.92 (d, J = 8.0 Hz, 1H), 4.03 (s, 3H), 3.98 (s, 3H), 3.92 (s, 3H), 3.89 – 3.81 (m, 4H), 3.33 – 3.20 (m, 4H), 2.28 – 2.16 (m, 1H), 1.25 – 1.10 (m, 2H), 1.06 – 0.96 (m, 2H); 13C NMR (126 MHz, CDCl3) δ 165.58, 163.99, 154.50, 152.28, 147.47, 140.98, 123.32, 121.05, 118.39, 111.37, 109.26, 106.69, 103.34, 56.19, 56.01, 55.42, 50.56, 49.82, 17.93, 9.54; LRMS (ESI+ve): Calculated for C24H28N4O3, [M+H] = 421.22, observed [M+H] = 421.34; HRMS (ESI+ve): Calculated for C24H28N4O3, [M+H] = 421.2234, observed [M+H] = 421.2213.

3. Results

3.1. Dose Response Curves for Probe

As shown in Figure 5, both probes ML301 (A) and ML314 (B) are near full agonists relative to NT-1 peptide and are selective against GPR35 and NTR2.

Figure 5. Potency and selectivity of (A) ML301 and (B) ML314 for NTR1 vs. NTR2 and GPR35.

Figure 5

Potency and selectivity of (A) ML301 and (B) ML314 for NTR1 vs. NTR2 and GPR35.

3.2. Cellular Activity

ML301 and ML314 are active in cells because the primary, confirmatory and secondary assays were all conducted in cell-based systems. Interestingly, neither probe shows significant cytotoxicity (LD50) relative to its potency (EC50) against a human hepatocyte cell line (seeTable 5 ADME/T properties). ML301 has an LD50/EC50 ratio of greater than 12–25 fold, while ML314 has an LD50/EC50 ratio of ~16-fold.

Table 5. Counter Assay Results for Neurotensin-1 Agonists.

Table 5

Counter Assay Results for Neurotensin-1 Agonists.

The agonist activity for NTR1 in the primary HCS was confirmed in the DiscoveRx β-arrestin assay (Table 5).

The probes tested in counterassays were selective for NTR1 over NTR2 and GPR35 (Table 5). ML301 data were consistent between this assay and the primary HCS. We also tested these compounds for agonist activity in a calcium mobilization assay (“Ca Mobilization” in Table 5). ML301 was active, consistent with its functioning via the Gq-coupled pathway

3.3. Profiling Assays

As a pro forma activity, the SBCCG is committed to profiling all final probe(s) compound(s) and in certain cases key informative analogs in the PanLabs full panel as negotiated by the MLPCN network. Additional commercial profiling services will be considered for funding by SBCCG as deemed appropriate and informative. The nominated probes and related compounds were evaluated in a detailed in vitro pharmacology screen as shown in Table 6.

Table 6. Summary of in vitro ADME/T Properties of NTR1 agonists ML301 (& analogs) and ML314.

Table 6

Summary of in vitro ADME/T Properties of NTR1 agonists ML301 (& analogs) and ML314.

ADME/T Profile of ML301 (Probe 1): ML301 was evaluated in a detailed in vitro pharmacology screen as shown in Table 6 Despite its structural similarity (imidazole vs. pyrazole) to the prior art compound, ML301 exhibited substantial advantages in this testing, especially with regard to plasma and microsomal stability.

ML301 exhibited good solubility due to the presence of the carboxylic acid moiety.

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 is measured using UV spectroscopy. The compounds exhibited good overall permeability inversely related to the pH of the donor compartment. Because these NTR1 agonists are envisioned as predecessors of psychoactive drugs, a preliminary assessment of their potential to cross the blood brain barrier (BBB) was performed. When incubated with an artificial membrane that models the BBB, much lower permeability was observed. These observations are also consistent with the carboxylic acid function in the compounds, and may present an opportunity for future enhancements.

Plasma protein binding is a measure of a drug’s efficiency of binding 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, and thus have relatively low volumes of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues. The imidazole scaffold compounds (ML301 and its MLS-0446079) exhibited substantial protein binding, but significantly lower than that of the prior art pyrazole.

The stability of small molecules and peptides in plasma may strongly influence in vivo efficacy. Drug candidates are susceptible to enzymatic processes such as those mediated by proteases or esterases in plasma. They may also undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. ML301 showed excellent stability in plasma, significantly better than that of either analog.

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability, which influences how long the candidate may remain intact while circulating in plasma. ML301 showed excellent stability in human and modest stability in mouse liver homogenates, which was much better than that observed for the prior art analog MLS-0437103. None of the compounds showed toxicity (>50 μM) toward human hepatocytes.

ADME/T Profile of ML314 (Probe 2): As described previously for ML301, in vitro pharmacology screening was also conducted for ML314. Consistent with its aqueous solubility data, ML314 exhibited high permeability in the PAMPA assay with increasing pH of the donor compartment. When incubated with an artificial membrane that models the blood-brain-barrier (BBB), ML314 was found to be highly permeable. ML314 was highly plasma protein bound and exhibited very high plasma stability. ML314 was metabolized rapidly when incubated in vitro with human and mouse liver homogenates. This result is not completely surprising because of the presence of several unsubstituted aryl and alkyl positions and Ar-OMe ethers which are prone to oxidation, hydrolysis, conjugation and other metabolic reactions. ML314 showed a > 15-fold window for toxicity (LC50 = 30 μM) towards human hepatocytes. Improving the metabolic stability and toxicity profile of ML314 represents a challenge as well as an avenue for further optimization studies in future.

Profiling against other GPCRs

ML301 and the prior art pyrazole (Entry 17, MLS-0437103) were submitted to the Psychoactive Drug Screening Program (PDSP) at the University of North Carolina (Bryan Roth, PI) for testing in a GPCR binding assay panel. The results (Figure 6a) indicate ML301 shows very little potential for promiscuity across a range of GPCRs. Contrarily, the prior art analog MLS-0437103 showed somewhat higher potential for promiscuity. Follow up dose response studies revealed Ki values of >10 μM (DAT) and 10 μM (NTS1) for ML301, and >10 μM (DAT), 5.2 μM (DOR), 3.4 μM (MOR), and 3.3 μM (NTS1) for MLS-0437103.

Figure 6a. Comparison of ML301 and MLS-0437103 in a GPCR Panel of Assays.

Figure 6a

Comparison of ML301 and MLS-0437103 in a GPCR Panel of Assays.

ML314 was also submitted to the Psychoactive Drug Screening Program (PDSP) at the University of North Carolina (Bryan Roth, PI) for testing in a GPCR binding assay panel. ML314 (at 10 μM) was found to be moderately promiscuous, inhibiting (≥ 50 %) about one third of the GPCRs screened in the panel (Figure 6b). Follow up dose response studies revealed Ki values of 0.4 μM (5-HT1A) 1.0 μM (5-HT3), 0.93 μM (5-HT7), > 10 μM (Beta3), > 10 μM (D5), 1.42 μM (H1) 1.54 μM (MOR), 2.54 μM (PBR) and 0.41 μM (sigma1) for ML314. It is not known whether these activities in binding assays are translated into functional antagonism or agonism of these receptors or downstream signaling. We also highlight the “comment from the PDSP program”: The PDSP has a low threshold for "hits" at primary assays. We perform a large number of secondary assays using a relatively low threshold in order not to miss potential high-affinity compounds. The likelihood of an actual, high-affinity, "hit" is still very low. Thus, one should not over-interpret results from primary assays.

Figure 6b. Evaluation of ML314 (MLS-0463110) and MLS-0233108 in a GPCR Panel of Assays.

Figure 6b

Evaluation of ML314 (MLS-0463110) and MLS-0233108 in a GPCR Panel of Assays.

4. Discussion

Currently, small molecule drug-like compounds are not available for treating methamphetamine abuse. Neurotensin receptor 1 (NTR1) peptide agonists produce behaviors that are exactly opposite to the psychostimulant effects observed with methamphetamine abuse, such as hyperactivity, neurotoxicity, psychotic episodes, and cognitive deficits, and repeated administrations of NTR1 agonists do not lead to the development of tolerance1,2. Recent supporting data from the Hanson laboratory3, collaborators on this project, suggesting that NT receptor agonists may have a role in addiction therapy are: (a) in a methamphetamine self-administration rat model the substitution of the peptide NT agonist (Lys(CH2NH)lys-Pro,Trp-tert-Leu-Leu-Oet) for methamphetamine, did not significantly affect motor activity but dramatically reduced lever pressing (b) the peptide agonist was not self-administered, and (c) the effects were associated with nucleus accumbens dopamine D1 receptors. These findings strengthen the hypothesis that neurotensin receptors are valid targets for antagonizing drug seeking behaviors and preventing relapses.

The NTR1 receptors and molecules relevant to them were reviewed in 20094. Previously starting from the potent NTR1 antagonists SR48692 or SR142948 were reported5,6. Additionally, three partial agonists were identified using a Ca mobilization FLIPR assay5,6. Researchers at Wyeth reported two different chemotypes, each of which showed partial agonist activity for NTR1 in the FLIPR assay7.

4.1. Comparison to Existing Art and How the New Probe is an Improvement

ML301 was designed via a scaffold hop approach based on the previously disclosed molecule with a pyrazole moiety. That molecule was designed in part from knowledge of SR48692. Taken together, these advances underscore the value of drug design via iterative / intuitive structural enhancement of an identified scaffold. Despite having substantial structural similarity to the pyrazole analog, the nominated probe ML301 (imidazole) enjoys intriguing differences / advantages in chemical and biological properties. Despite being less potent than the pyrazole analog and comparably potent to a naphthyl analog, ML301 is a more effective agonist than the pyrzaole analog in the calcium mobilization assay. Identification of a full agonist was a primary objective for this program. ML301 also showed a much better pharmacology profile, including superior protein binding, plasma stability, and hepatic microsomal stability. These improvements over the existing art may enable ML301 or future analogs of ML301 to achieve a distribution profile more favorably disposed toward in vivo activity. It is tractable from a synthetic chemistry perspective and appears much more tolerant of substitution on the imidazolium nitrogen, making the future planned studies practical to execute. Finally, it showed minimal and it has a relatively favorable standing with respect to prior art and potential for intellectual property. Overall, ML301 represents a strong platform on which to launch a medicinal chemistry-based program for enhancement.

ML314 represents the first described β-arrestin pathway selective small molecule NTR1 agonist. It also represents a distinct chemical phenotype compared to ML301 as well as the previously described pyrazole analog. In addition, in contrast to this peptide-based reference agonist, ML314 displays much higher blood-brain barrier permeability and thus has a higher probability of efficacy after systemic dosing, although this may be confounded by its poor microsomal stability. However, ML314 may be suitable for acute experiments.

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*

The two authors have contributed equally to the current work.