Optimization of 8-Hydroxyquinolines as Inhibitors of Catechol O-Methyltransferase

Ingrid Buchler; Daniel Akuma; Vinh Au; Gregory Carr; Pablo de León; Michael DePasquale; Glen Ernst; Yifang Huang; Martha Kimos; Anna Kolobova; Michael Poslusney; Huijun Wei; Dominique Swinnen; Florian Montel; Florence Moureau; Emilie Jigorel; Monika-Sarah ED Schulze; Martyn Wood; James C Barrow

doi:10.1021/acs.jmedchem.8b01126

. Author manuscript; available in PMC: 2019 Nov 8.

Published in final edited form as: J Med Chem. 2018 Oct 19;61(21):9647–9665. doi: 10.1021/acs.jmedchem.8b01126

Optimization of 8-Hydroxyquinolines as Inhibitors of Catechol O-Methyltransferase

Ingrid Buchler ^§, Daniel Akuma ^§, Vinh Au ^§, Gregory Carr ^§,^¶, Pablo de León ^§, Michael DePasquale ^§, Glen Ernst ^§, Yifang Huang ^§, Martha Kimos ^§, Anna Kolobova ^§, Michael Poslusney ^§, Huijun Wei ^§,^¶, Dominique Swinnen ^†, Florian Montel ^†, Florence Moureau ^†, Emilie Jigorel ^†, Monika-Sarah ED Schulze ^║, Martyn Wood ^†, James C Barrow ^§,^¶,^*

PMCID: PMC6398604 NIHMSID: NIHMS1013823 PMID: 30272964

Abstract

A series of 8-hydroxy quinolines were identified as potent inhibitors of catechol O-methyltransferase (COMT) with selectivity for the membrane-bound form of the enzyme. Small substituents at the 7-position of the quinoline were found to increase metabolic stability without sacrificing potency. Compounds with good pharmacokinetics and brain penetration were identified and demonstrated in vivo modulation of dopamine metabolites in the brain. An X-ray co-crystal structure of compound 21 in the S-COMT active site shows chelation of the active site magnesium similar to catechol-based inhibitors. These compounds should prove useful for treatment of many neurological and psychiatric conditions associated with compromised cortical dopamine signaling.

Graphical Abstract

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INTRODUCTION

Proper functioning of dopamine signaling in the prefrontal cortex (PFC) is critical for a number of cognitive and behavioral processes that are impaired in a variety of conditions such as ADHD, obsessive-compulsive disorder, traumatic brain injury, and schizophrenia.¹ One approach towards selective modulation of dopamine signaling in the PFC is by inhibition of catechol O-methyltransferase (COMT), which is the predominant means of dopamine clearance in the PFC because of the lack of expression of synaptic dopamine transporters in this region,² in contrast to the striatum.

COMT is a magnesium-containing metalloenzyme that transfers a methyl group from the cofactor S-adenosylmethionine (SAM) to a number of endogenous and exogenous catechols such as dopamine, catecholestrogens, and dietary polyphenols. The COMT enzyme has two isoforms, designated S-COMT for the soluble form and MB-COMT for the membrane-bound form.³ The enzymatic domains of the two forms are identical, with MB-COMT having an extra 50 amino acids at the N-terminus for membrane anchoring that also influences enzymatic activity.⁴ The expression patterns of the two forms are different, with S-COMT predominating in the periphery and MB-COMT being more prevalent in the brain, although this differential expression is especially pronounced in humans compared to rodents.^{5, 6} Since the goal is to achieve central COMT inhibition, identification of a compound with MB-COMT selectivity may be theoretically desirable.

Inhibitors of COMT are widely used for treatment of Parkinson’s disease due to their role in inhibiting peripheral metabolism and thereby increasing levels of exogenously administered L-DOPA.⁷ Notably, the nitrocatechol scaffold has been exploited to provide the clinically used drugs tolcapone 1 and entacapone 2, as well as the recently approved opicapone 3 (Figure 1).⁸ While they are effective in blocking peripheral COMT activity, entacapone and opicapone have negligible brain penetration, and tolcapone has low but measurable levels in the brain.⁹ Compounds with improved brain penetration have greater efficacy for the non-motor symptoms of Parkinson’s disease,¹⁰ as well as have utility for other psychiatric and neurological conditions such as cognitive impairment in schizophrenia.¹¹ Despite the early clinical success achieved with tolcapone, the drug has been associated with hepatic toxicity.¹² Thus, the risk-benefit profile for tolcapone prevents its widespread deployment and new inhibitors of COMT are needed, especially those that are active in the brain as well as those derived from alternate pharmacophores to de-risk any toxicity arising from the nitro-catechol moiety.

Figure 1. — Clinically used COMT inhibitors

Soon after the discovery of COMT by Julius Axelrod in 1957,¹³ Ross and Haljasmaa investigated a number of compounds for their effects on COMT inhibition and identified 8-hydroxyquinoline (4, Figure 2) as a non-catechol-containing inhibitor.¹⁴ Later, Borchardt and colleagues further explored this template with modest success,¹⁵ but the field subsequently shifted focus to nitrocatechols given the exquisite potency and high ligand efficiency of the nitrocatechol pharmacophore.^{7, 16} While 3 was able to overcome the rapid metabolic clearance associated with tolcapone by taking advantage of a slow off rate that extends the pharmacodynamic action beyond its short plasma half-life,¹⁷ nitrocatechols generally suffer from poor pharmacokinetics and limited brain penetration. Herein, we describe further exploration of the 8-hydroxyquinoline template and the discovery of potent inhibitors with excellent pharmacokinetics and brain penetration.

RESULTS AND DISCUSSION

Borchardt’s exploration of 5-substituted 8-hydroxyquinolines showed that sulfonic acid (compound 5, Figure 2) had similar COMT inhibition to other electron withdrawing groups including chloro and nitro.¹⁵ In designing additional binding interactions for the 8-hydroxyquinoline 4 (Figure 2) outside the metal chelating motif, modeling suggested substituents on either the 5 or 6-position would overlay with nitrocatechol pharmacophore 1 (Figure 2). We prepared both sulfone isomers 6 and 7 to guide future trajectories for exploration. While the 6-substituted compound had a modest potency increase, the 5-position analog 7 appeared more promising with an in vitro MB-COMT pIC₅₀ of 7.7.

As shown in Table 1, a series of functional groups were appended at the 5-position, with sulfones 7 and 11 as well as sulfonamides 12 and 13 being preferable to ketone 9, amide 10, or direct aromatics like 8. Additional sulfones and sulfonamides of varying sizes and polarity were prepared such as 13; however, no meaningful increases in potency were achieved. A free hydroxyl is required for optimum hydrogen-bonding and magnesium coordination in the COMT active site, as compound 14 exhibited no appreciable inhibition of MB-COMT. Poor metabolic stability in rat hepatocytes was observed for representative compounds 12 and 13, and the 8-hydroxyl moiety was suspected for phase II metabolism. Therefore, a series of sulfone and sulfonamide analogs were prepared that investigated substituents at the 7-position to explore steric and electronic effects on potency and metabolic stability.

Table 1.

Potency and Hepatocyte Clearance of 5-substituted 8-hydroxyquinolines

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Expanding the SAR around 5-substituted sulfone and sulfonamide quinolines provided many potent and brain penetrant examples as described in Table 2. Both aryl and alkyl substitution were tolerated, as well as extension into larger and more diverse chemical space (data not shown, see patent application WO2016123576 for additional examples).¹⁸ However, building larger and more expansive sulfone and sulfonamides decreased ligand efficiency, increased lipophilicity and diminished physicochemical properties thereby leaving small substituents with the best combination of potency and physiochemical properties. The clearance profile of both sulfonamides and sulfones is driven by phase II metabolism of the 8-hydroxy group; therefore, screening in hepatocytes was used to capture the impact of metabolism in vitro. Poor metabolic stability was observed with 7-unsubstituted 8-hydroxyquinolines as evident by the high hepatocyte clearance for 15, 16, and 17 (Table 3). A tight correlation between rat hepatocytes and in vivo clearance measured by PK studies was not evident; however, the hepatocyte screen proved useful for triaging compounds with high clearance.

Table 2.

Enzyme Potency of 5,7-substituted 8-hydroxyquinolines^a

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Potency determined by MTase Glo™ Methyltransferase assay from recombinant human or rat MB and S-COMT and are the average of three determinations.

Table 3.

ADME and Pharmacokinetic properties of 5,7-substituted 8-hydroxyquinolines

Example	Hepatocyte Stability (Rat) (μL/min/10⁶ cells)	Rat PK^b
Example	Hepatocyte Stability (Rat) (μL/min/10⁶ cells)	%F	T_1/2 (h)	Cl_p (ml/min/kg)	V_ss (L/kg)
11		24	0.18	26	1.8
12	177
15	39
16	228
17	46
18	12	66	3.5	13	3.8
19	4	23	8.9	0.31	0.23
20	15	100	4.5	12	3.5
21	36	34	0.26	27	1.2
22	78	45	0.31	25	0.67
25	19	32	7.7	0.62	0.41
26	18	37	11	0.3	0.3
28	21	73	2.8	11	2.2
29	14	33	8.6	1	0.8
30	103

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Rat pharmacokinetics after a 1mpk iv dose (n=3) and 3 or 10 mpk oral dose (n=3)

Improvements in pharmacokinetic properties were observed when installing small groups at the 7-position of the quinoline, presumably by affecting the phase II metabolism of the 8-hydroxy moiety. Substituents to modulate the susceptibility of hydroxyl metabolism with respect to both steric and electronic effects were explored. As shown in Table 3, halogens generally showed improved in vitro and in vivo clearance while larger, more sterically bulky groups reduced MB-COMT potency (e.g. compound 23). Strikingly, a CF₃ substituent at the 7-position (compound 4) abolished MB-COMT inhibition. A small methyl substituent was tolerated (compound 30) but proved to have no impact on clearance as measured by rat hepatocytes. Both fluoro and chloro substitution yielded the best profiles with respect to metabolic stability, especially 7-chloro substituted compounds 19, 26 and 29 which exhibited very low clearance and long half-life in rat PK studies (Table 3).

Brain penetration was initially assessed in mice by measuring total brain and plasma levels 5 minutes, 15 minutes, and 1 hour after an intraperitoneal (ip) 10 mg/kg dose in two mice at each timepoint (Supplementary Table 1 for all timepoints and Table 4 for the 1 hour timepoint). Brain penetration was generally rapid, so the 1 hour timepoint was chosen for compound comparison in Table 4. Additionally, free fraction in plasma and brain were measured by equilibrium dialysis. Early examples such as 15 and 16, where hydrogen is in the 7-position exhibited low overall exposure consistent with poor pharmacokinetics. Compounds with either a 7-fluoro or chloro in both sulfone and sulfonamide moieties display good total plasma concentrations but modest brain exposures relative to total plasma. However, most examples in Table 2 have plasma protein binding approaching or greater than 99%, thereby limiting free drug concentrations. Additionally, the examples in Table 4 exhibited higher free fraction in rat brain tissue as compared to plasma which has the expected effect of driving down total brain concentrations.¹⁹ Despite the protein/tissue binding differences, when correcting for free drug in both compartments most compounds had reasonable ratios of unbound plasma and unbound brain concentrations. The lower free brain drug concentrations relative to unbound plasma concentrations observed for compounds such as 20 and 22 are outliers to the trend and could not be explained by P-gp efflux as these compounds were not substrates for the transporter (Table 4) and have good apparent permeability (>100 nm/s, data not shown). Despite these two outliers, overall the observations described in Tables 4 and 5 reinforce the notion that optimization of the fraction unbound in the brain does not result in greater brain exposure, rather plasma free fraction is more correlative to drug levels in brain.¹⁹

Table 4.

Mouse Brain Exposure of 5,7-substituted 8-hydroxyquinolines at one hour

Example	P-gp ER^a	Plasma Protein Binding^b	Brain Tissue Binding^c	Mouse Brain Penetration^d
Example	P-gp ER^a	Plasma Protein Binding^b	Brain Tissue Binding^c	Total Brain (ng/g)	Total Plasma (ng/ml)
11	NT	0.5 (r)	2.9	blq	173
12	NT	3.3 (m)	4.4	10.5	68
15	NT	6.9 (m)	10.3	11	250
16	NT	0.5 (m)	0.5	109	235
17	NT	1.4 (m)	NT	BLQ	3538
18	1.1	0.6 (r)	3.6	1094	2542
19	0.8	0.5 (r)	2.6	5655	16650
20	0.7	1.2 (r)	2.4	527	5720
21	NT	1.2 (r)	1.39	518	1744
22	1.1	0.84 (m)	0.5	1686	9780
25	0.5	0.2 (r)	4.2	1914	6160
26	NT	0.5 (r)	1.6	2792	14669
28	1.2	0.6 (r)	8	110	1819
29	0.4	0.5 (r)	3.4	1154	16250
30	NT	0.5 (m)	1.8	55	576

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A:B/B:A efflux ratio from P-glycoprotein expressing MDCK cells. NT=not tested.

Percent free from either mouse (m) or rat (r) plasma as measured by equilibrium dialysis.

Percent free from rat brain tissue.

Total brain and total plasma concentrations 1 hour after ip dosing 10mg/kg to 2 mice.

Table 5.

Effects of COMT inhibitors on rat biomarkers of dopamine metabolism

Example	Free Plasma (nM)^a	Free Brain (nM)^a	Free CSF (nM)^b	CSF (nM)	CSF/ Free Plasma Ratio	CSF/ Free Brain Ratio	Free CSF/ Free Plasma Ratio	HVA (%)^c	DOPAC (%)^c
1	35	BLQ	NA	BLQ	N/A	N/A	N/A	28	304
20	48	7.7	59	127	2.6	16	1.2	86	173
25	19	12.8	139	321	16.5	25	7.3	66	218
26	32	5.6	NA	358	11	64	N/A	97	213
28	23	35	77	157	6.8	4.4	3.3	72	140

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Free plasma and brain levels calculated using ratios listed in Table 4 on plasma and brain samples taken immediately after CSF sampling. Total concentrations are given in Supplementary Table 2.

Non-specific binding to CSF was determined by equilibrium dialysis using pooled rat CSF as described in reference 24. Compound 20 showed 53% binding, Compound 25 showed 57% binding, and Compound 28 showed 51% binding.

HVA and DOPAC changes (% of vehicle) 4 hours after 10mg/kg oral dose compared to vehicle (n=5–10 rats per group), except 1 (15mpk ip). BLQ=Below the limit of quantitation.

Central nervous system inhibition of COMT produces measurable changes in dopamine metabolites dihydroxyphenyl acetic acid (DOPAC, increase) and homovanillic acid (HVA, decrease). Assessment of these biomarkers of dopamine metabolism in rat CSF with accompanying measurement of inhibitor concentrations in plasma, brain homogenate, and CSF provides a rapid readout for compound brain penetration and activity. While the region of interest is the cortex, it has been demonstrated that CSF concentrations of HVA and DOPAC are reflective of total brain levels²⁰ and responsive to brain-penetrant COMT inhibitors.²¹

Table 5 shows the results from a single oral administration (10 mpk) of COMT inhibitors to rats (n=5–10, see Supplementary Table S2 for details). The animals were sacrificed 4 hours post dose where plasma, brain, and CSF were analyzed for dopamine metabolites and drug levels. Values for plasma protein binding and brain tissue binding (Table 4) were used to determine free plasma and brain levels. Changes in HVA and DOPAC in CSF as measured by LC-MS are shown as a difference between compound treated and vehicle treated controls. The positive control, tolcapone (15 mpk, ip injection), exhibited expected biomarker movement where DOPAC was increased and HVA decreased relative to control. Robust increases in DOPAC were observed for all five compounds tested. More modest decreases were observed for HVA, with only 25 and 28 showing a statistically significant difference compared to the vehicle treated group.

All four quinoline analogs produced good peripheral exposure and brain levels as predicted from the mouse brain penetration studies. Measurable drug concentrations for all compounds in brain homogenate extracts exceeded rat MB-COMT IC₅₀ measurements; however, this series has less activity on S-COMT which may contribute to the modest changes in HVA relative to tolcapone. Further studies with these compounds are in progress to obtain a more comprehensive understanding of the effects of COMT inhibition by different classes of inhibitors. An interesting additional observation is the lack of correlation between measured CSF levels and calculated free plasma and free brain levels, which are expected to be similar for highly permeable compounds that are not transporter substrates since the CSF has very low amounts of protein compared to plasma.²² In addition to P-gp (Table 4), compound 28 was also examined for susceptibility to BCRP and MRP2 transporters, as well as for inhibitory activity against P-gp, BCRP, and MRP2. While compound 28 was not found to be a substrate or an inhibitor for these three transporters (data not shown), that does not rule out susceptibility to other transporters involved in blood-brain-CSF homeostasis.²³ This prompted measurement of free CSF concentrations using equilibrium dialysis.²⁴ Compounds 20, 25, and 28 showed remarkably high bound concentrations in rat CSF (51–57% bound @ 2 μM testing concentration). Even though proteins like albumin are in CSF at less than 1% compared to plasma,²⁵ there exists sufficient non-specific binding in CSF to effect the distribution of highly bound compounds.

Confirmation of the proposed binding mode of the 8-hydroxyquinolines was confirmed by an X-ray co-crystal structure with 21 and SAM in the rat S-COMT active site (Figure 3). As expected, the hydroxyl and quinoline nitrogen make a bidentate interaction with the catalytic magnesium. A free hydroxyl was observed to be required from the SAR studies (see Table 1, compound 14), and the structure reveals a tight hydrogen-bonding network around the hydroxyl similar to what is seen with nitrocatechols.²⁶ The sulfone oxygens make no clear contacts with the protein, but orient the aromatic ring between tryptophan-38 and tryptophan-143 and over the thioether sidechain of methionine-40. The MB and S isoforms of COMT are identical except for an additional 50 amino acids at the N-terminus of MB-COMT that are responsible for membrane anchoring.²⁷ Without a structure of compound 21 bound to MB-COMT for comparison, it is difficult to rationalize the selectivity for MB-COMT over S-COMT in this series, but it has been proposed that residues 27–50 of MB-COMT lay back over the active site to provide additional binding interactions.²⁸ The close proximity of the leucine-198 sidechain to the quinoline 7-position explains the tolerance for only small substituents at the 7-position. As judged by the potency differences seen in Table 1 between sulfone 7 and sulfonamide 12 versus ketone 9 and amide 10, other effects of the 5-substituent may be at play since those substituents should also be able to “fit” in similar binding modes albeit at a torsional cost. Also notable is that there is no electron density for the transferable methyl group of SAM, and the structure is modeled with SAH despite high purity SAM used in the crystallization conditions.

Figure 3. — Compound 21 (magenta) bound to the rat S-COMT active site (PDB 6GY1). The hydroxyl and quinoline nitrogen make a bidentate interaction with the catalytic magnesium ion (green sphere). The sulfone oxygens make no clear contacts with the protein, but orient the aromatic ring between trytophan-38 and tryptophan-143 and over the thioether sidechain of methionine-40.

CHEMISTRY

Scheme 1 shows the preparation of unsubstituted and non-fluoro sulfonamides 12, 13 and 16, beginning with chlorosulfonylation of 8-fluoroquinoline 31 followed by treatment of the desired amine and quenching with sodium 2-(trimethylsilyl)ethan-1olate to displace the labile 8-fluoro group to give 33. Removal of the TMSE protecting group followed by treatment with N-chloro succinimide (NCS) gave the desired sulfonamide 29. The corresponding sulfones 7 and 23 were prepared according to Scheme 2 by PMB protection of 5-iodo-8-hydroxyquinoline 35 followed by copper (I) mediated coupling with 4-methylbenzenesulfinate to install the sulfone group.²⁹ The protecting group was removed with TFA at ambient temperature to provide compound 7. Bromination of 7 was achieved with NBS in chloroform to give example 23. Additional 7-chloro sulfone analogs were prepared by chlorination with NCS followed by benzylating 5-bromo-8-hydroxyquinoline 36 (Scheme 3). Palladium catalyzed cross coupling of thiols produced thioethers 35.³⁰ The desired thioethers could be prepared in either one step (compounds 22 and 26) or by use of an intermediary TMS-thioethane (example 19). Oxidation of the sulfur with mCPBA gave sulfones with minimal quinoline N-oxide formation. Acid-catalyzed deprotection of the benzyl group provided the desired analogs 39. Incorporation of a fluorine at the 7-position of the quinoline was accomplished as described in Scheme 4. A Skraup reaction³¹ was used to prepare the 7-fluoro-8-hydroxyquinoline 41 which could be either chlorosulfonylated and quenched with amines to give sulfonamide 28, or iodinated and protected to give key intermediate 42. Palladium catalyzed coupling of thiols followed by oxidation and deprotection gave the desired sulfones 44. Other quinolines shown in Table 1 could be prepared as shown in Scheme 5, via direct Friedel-Crafts acylation to give ketone 9, or introduction of the 8-hydroxyl as a protected benzyl by SnAr on 8-fluoroquinolines 46 or 48 which led to compounds 10 and 6 respectively.

Scheme 2. — Synthetic protocol for 7 and 23. Reagents and conditions: (a) K₂CO₃, PMB-Cl, ACN, 22%; (b) CuI, DMSO, 4-methylbenzenesulfinate, 90 °C, 22%; (c) TFA, rt, 92%; (d) NBS, CHCl₃, 85%.

Scheme 3. — General synthetic procedures for 19, 22, and 26. Reagents and conditions: (a) NCS, CHCl₃, 78%; (b) K₂CO₃, BnBr, ACN, 89%; (c) 2-trimethylsilylethanethiol (example 19) or R₁SH (examples 22 and 26), Xantphos, Cs₂CO₃, Pd₂(dba)₃, dioxane, 100 °C, 93% example 19; 81% example 22, 93% example 26; (d) for example 19, 4-fluoroiodobenzene, TBAF, Xantphos, Cs₂CO₃, Pd2(dba)₃, dioxane, 90 °C, 93%; (e) mCPBA, DCM; (f) 6M aqueous HCl, 100 °C.

Scheme 4. — General synthetic protocol for **18, 20, 21**, 25, and 28. Reagents and conditions: (a) glycerol, sodium-3-nitrobenzenesulfonate, 70% sulfuric acid, 140 °C, 56%; (b) chlorosulfonic acid, CHCl3, 100 °C, 40%; (c) triethylamine, pyrrolidine, DCM, 0 °C, 98%; (d) NIS, CHCl₃, 40 °C, 92%; (e) NaH, BnBr, DMF, 73%; (f) thiol, Xantphos, Cs₂CO₃, Pd2(dba)3, dioxane, 90 °C; (g) mCPBA, DCM; (h) 6M aqueous HCl, 100 °C.

Scheme 5. — Synthetic protocols for ketone 9, amide 10, and sulfone 6. a) AlCl₃, 4-methylbenzoyl chloride, toluene, 110 °C, 5%; b) NaH, BnOH, THF; c) NaOH, THF/H₂O, 63%; d) 1-Chloro-N,N,2-trimethyl-1-propenylamine, DCM then pyrrolidine; e) HBr, acetic acid, 63% over 2 steps; f) Cu(I)I, K₂CO₃, DMEN, DMSO, sodium 4-methylbenzenesulfinate,46%; g) 6M HCl,87%.

CONCLUSIONS

A series of 8-hydroxyquinolines were prepared and evaluated as COMT inhibitors, and several potent MB-COMT inhibitors were identified that demonstrate selectivity over S-COMT. An X-ray co-crystal structure highlights the important chelation of the active site magnesium; however, the interactions responsible for the observed MB-COMT selectivity are not apparent in the S-OMT structure. Small substituents at the 7-position were found to increase metabolic stability without sacrificing potency, and compounds with good pharmacokinetics such as 25 and 28 were identified. This series of compounds generally has good brain penetration, although high plasma protein binding results in high exposures required for efficacy in a rat biomarker assay of COMT activity. Further in vivo characterization of these compounds on central nervous system dopamine metabolism will be reported in due course.

A vial was charged with 8-benzyloxy-5-bromo-quinoline 51 (150 mg, 0.48 mmol), Pd(PPh₃)₄ (55 mg, 0.05 mmol), 4-methylphenylboronic acid (77 mg, 0.57 mmol), NaHCO₃ (0.48 mL, 0.95 mmol) 2M aqueous solution in 1,4-dioxane (5 mL). The vial was sparged with nitrogen for 5 minutes and heated at 90°C overnight. The reaction was concentrated to a residue, resultant residue partitioned between water and ethyl acetate, layers separated, aqueous extracted (2 × 2 mL), organics passed through a phase separator tube and solution concentrated to an oil. Purification was accomplished by automated silica gel chromatography (12 g pre-packed silica cartridge) eluting with 0–100% ethyl acetate/heptane to provide 8-benzyloxy-5-(p-tolyl)quinoline 52 as a yellow glass (150 mg, 97% yield). ^1gH NMR (400 MHz, DMSO-d₆) δ ppm 2.40 (s, 3 H) 5.36 (s, 2 H) 7.33 – 7.40 (m, 4 H) 7.40 – 7.48 (m, 4 H) 7.51 – 7.61 (m, 4 H) 8.17 (dd, J=8.59, 1.52 Hz, 1 H) 8.89 (dd, J=4.04, 1.52 Hz, 1 H). MS (ES+) m/z 326.0 [M+H]⁺.

Step 3.

A solution of 8-benzyloxy-5-(p-tolyl)quinoline 52 (150 mg, 0.46 mmol) dissolved in 1,4-dioxane (2 mL) and 6N aqueous hydrochloric acid (3 mL, 18 mmol) was heated at 100°C for 4 hours. The material was concentrated to a residue and purified by automated reversed phase HPLC to yield 85 mg (52%) of 5-(p-tolyl)quinoline-8-ol 8 as a TFA salt. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.40 (s, 3 H) 7.30 (d, J=8.08 Hz, 1 H) 7.33 – 7.37 (m, 4 H) 7.51 (d, J=8.08 Hz, 1 H) 7.73 (dd, J=8.59, 4.55 Hz, 1 H) 8.48 (d, J=8.34 Hz, 1 H) 8.98 (dd, J=4.55, 1.52 Hz, 1 H). MS (ES+) m/z 236.2 [M+H]⁺.

(8-Hydroxyquinolin-5-yl)(p-tolyl)methanone (9).

To a mixture of 8-hydroxyquinoline 4 (250 mg, 1.7 mmol) dissolved in toluene (3 mL) was added 4-methylbenzoyl chloride (0.23 mL, 1.7 mmol). The resultant solution was cooled to 0°C and aluminum chloride (574 mg, 4.3 mmol) was added in 2 portions. The resultant mixture was allowed to warm to ambient temperature for 2 hours. The material was quenched by pouring over ice and 1M HCl aqueous solution. The acidic solution was extracted with ethyl acetate (2 × 10 mL). The organics were combined, dried over sodium sulfate and concentrated to a residue. Purification was accomplished by automated normal-phase chromatography using 0–5% methanol/dichloromethane as an eluent to provide 24 mg (5% yield) of title compound 9. ¹H NMR (400 MHz, DMSO-d6) δ ppm 8.89 (br. s., 1 H) 8.53 (d, J = 7.83 Hz, 1 H) 8.11 (d, J = 8.08 Hz, 2 H) 7.99 (d, J = 6.82 Hz, 1 H) 7.68 – 7.76 (m, 2 H) 7.64 (d, J = 4.55 Hz, 1 H) 7.46 (d, J = 7.58 Hz, 2 H) 2.45 (s, 3 H). MS (ES+) m/z 264.1 [M+H]⁺.

(8-Hydroxyquinolin-5-yl)(pyrrolidin-1-yl)methanone (10).

Step 1.

To a solution of ethyl 8-hydroxyquinoine-5-carboxylate 45 (270 mg, 1.2 mmol) in dry DMF (10 mL) was added sodium hydride (60 mg, 1.4 mmol) at ambient temperature. After 5 minutes benzyl bromide (0.18 mL, 1.4 mmol) was added in a dropwise manner. The reaction was allowed to stir at ambient temperature. After 15 minutes the reaction was quenched by addition of ice-water. The aqueous mixture was extracted (2 × 20 mL) ethyl acetate, organic washes combined, dried over sodium sulfate and concentrated to a residue. Purification was accomplished automated normal-phase chromatography (40 g silica gel cartridge) eluting 0–100% ethyl acetate/hexanes to provide 259 mg as a 1:1 mixture of ethyl-8-benzyloxyquinoline-5-carboxylate 53 and 8-benzyloxyquinoline. The mixture was taken on in the next step without additional purification. MS (ES+) m/z 308.0 [M+H]⁺.

Step 2.

To a solution of ethyl-8-benzyloxyquinoline-5-carboxylate 53 mixture (250 mg, 0.81 mmol) in THF (10 mL) was added water (10 mL). The mixture was allowed to stir at 40°C overnight. The pH was adjusted to ~4–5 by addition of 4.5 M aqueous hydrochloric acid. Solids formed and collected by filtration, washed with water and dried in a vacuum oven to yield 8-benzyloxyquinoline-5-carboxylate 46 (144 mg, 63% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.99 (br. s., 1 H) 9.42 (dd, J = 8.84, 1.77 Hz, 1 H) 8.91 (dd, J = 4.04, 1.52 Hz, 1 H) 8.26 – 8.31 (m, 1 H) 7.68 (dd, J = 8.59, 4.04 Hz, 1 H) 7.57 (d, J = 6.82 Hz, 2 H) 7.42 – 7.48 (m, 2 H) 7.34 – 7.41 (m, 2 H) 5.39 (s, 2 H). MS (ES+) m/z 280.0 [M+H]⁺.

Step 3.

To a slurry of 8-benzyloxyquinoline-5-carboxylate 46 (61 mg, 0.22 mmol) in dry dichloroethane (2 mL) was added Ghosez reagent (0.29 mL, 2.1 mmol) at ambient temperature. To drive the reaction into solution additional Ghosez reagent was added (totaling 0.7 mL). After 1 hour, pyrrolidine (0.54 mL, 6.5 mmol) was added. The reaction was stirred at ambient temperature for 1 hour then diluted with dichloromethane and extracted (1 × 3 mL) water. The material was passed through a phase separator tube and organic layer concentrated to a residue. Purification was accomplished by automated normal-phase chromatography using 0–10% methanol/dichloromethane as an eluent. The oily (8-benzyloxy-5-quinolyl)-pyrrolidin-1-yl-methanone solidified upon standing (102 mg). The crude material was taken on without additional purification and or characterization.

A mixture of (8-benzyloxy-5-quinolyl)-pyrrolidin-1-yl-methanone (83 mg, 0.29 mmol) dissolved in acetic acid (0.5 mL) was added hydrobromic acid (0.5 mL). The resultant mixture was heated to 100°C for 3 hours. The material was cooled to ambient temperature and neutralized with 6 M sodium hydroxide. The aqueous solution was extracted (3 × 2 mL) dichloromethane, organics combined, filtered through a phase separator tube and concentrated to a residue. Purification was accomplished by automated normal-phase chromatography using 0–10% methanol/dichloromethane as an eluent. The impure material was concentrated to a residue then purified by automated reversed-phase chromatography eluting 5–95% acetonitrile/water, 0.05% trifluoroacetic acid as modifier. A yellow foam of 8-hydroxyquinolin-5-yl)(pyrrolidin-1-yl)methanone 10 (57 mg, 43% yield) was obtained. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.95 (dd, J = 4.17, 1.39 Hz, 1 H) 8.47 (d, J = 8.59 Hz, 1 H) 7.73 (dd, J = 8.34, 4.55 Hz, 1 H) 7.59 (d, J = 8.08 Hz, 1 H) 7.17 (d, J = 8.08 Hz, 1 H) 3.58 (t, J = 6.82 Hz, 2 H) 3.16 (t, J = 6.44 Hz, 2 H) 1.86 – 1.97 (m, 2 H) 1.73 – 1.84 (m, 2 H). MS (ES+) m/z 243.0 [M+H]⁺.

5-(Cyclopentylsulfonyl)quinoline-8-ol (11).

Step 1.

8-Hydroxyquinoline-5-sulfonic acid hydrate (10 g, 41.1 mmol) was dissolved in sodium hydroxide solution (1 N, 103 mL, 103 mmol) and heated to 75°C. A solution of benzyl chloride (10.4 ml, 90 mmol) in THF (100 mL) was added and resultant mixture maintained at 75°C overnight. After cooling to room temperature, the solution was extracted with diethyl ether twice, then placed chilled at 4°C for 4 hours. The solid was collected by filtration and dried to give 8-benzyloxyquinoline-5-sulfonic acid 54 (6.3 g, 48% yield) a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 – 9.17 (m, 1 H) 8.84 (dd, J = 4.2, 1.6 Hz, 1 H) 7.88 (d, J = 8.1 Hz, 1 H) 7.52 – 7.60 (m, 3 H) 7.39 – 7.46 (m, 2 H) 7.32 – 7.39 (m, 1 H) 7.20 (d, J = 8.1 Hz, 1 H) 5.32 (s, 2 H). MS (ES+) m/z 316.0 [M+H]⁺

Step 2.

8-Methoxy-5-(pyrrolidin-1-yulsulfonyl)quinoline (14).

8-fluoroquinoline-5-sulfonyl chloride 32 (50 mg, 0.20 mmol) was dissolved in DCM (1 mL). To this solution was added a solution of pyrrolidine (17 µL, 0.20 mmol) and DIPEA (71 µL, 0.41 mmol) in DCM (0.5 mL). The resulting mixture was stirred at room temperature for 5 min. Added sodium methoxide, 25 wt % in MeOH (200 µL, 3.5 mmol) and stirred at room temperature for 2 h. The reaction mixture was purified directly by automated normal-phase chromatography (20–100% EtOAc/hexanes (7% MeOH additive), 10 g silica gel cartridge). The product-containing fractions were combined and the solvent removed in vacuo to give 8-methoxy-5-pyrrolidin-1-ylsulfonyl-quinoline 14 (47 mg, 79% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.02 (dd, J = 8.72, 1.64 Hz, 1 H) 8.97 (dd, J = 4.17, 1.64 Hz, 1 H) 8.17 (d, J = 8.34 Hz, 1 H) 7.76 (dd, J = 8.84, 4.04 Hz, 1 H) 7.34 (d, J = 8.59 Hz, 1 H) 4.07 (s, 3 H) 3.15 – 3.21 (m, 4 H) 1.69 – 1.75 (m, 4 H). MS (ES+) m/z 293.0 [M+H]⁺.

5-(Ethylsulfonyl)quinoline-8-ol (15).

Step 1.

Step 5.

To a suspension of 5-iodo-7-(trifluoromethyl)quinolin-8-ol 84 (540 mg, 1.5 mmol) in acetonitrile (10 mL) and DMF (5 mL) was added potassium carbonate (242 mg, 1.7 mmol). To this mixture was added 4-methoxybenzyl chloride (0.26 mL, 1.9 mmol) dropwise followed by catalytic sodium iodide. The reaction was allowed to stir at ambient temperature overnight. The reaction was heated to 50°C for 3 hours. The reaction was cooled to ambient temperature and filtered. The filtrate was concentrated to an oil and partitioned between ethyl acetate and brine. The layers were separated and ethyl acetate layer was dried over sodium sulfate. Purification was accomplished by automated normal phase chromatography (25 g silica gel cartridge) eluting 0–35% ethyl acetate/heptane to yield 429 mg (58% yield) of 5-iodo-8-[(4-methoxyphenyl)methoxy]-7-(trifluoromethyl)quinoline 85 as a waxy orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.12 (dd, J = 4.04, 1.52 Hz, 1 H) 8.47 (dd, J = 8.72, 1.64 Hz, 1 H) 8.29 (s, 1 H) 7.87 (dd, J = 8.72, 4.17 Hz, 1 H) 7.40 – 7.50 (m, 2 H) 6.90 – 7.01 (m, 2 H) 5.56 (s, 2 H) 3.72 – 3.80 (m, 3 H). MS (ES+) m/z 482.0 [M+Na]⁺.

Step 6.

8-[(4-Methoxyphenyl)methoxy]-5-(p-tolylsulfanyl)-7-(trifluoromethyl)quinoline 86 was prepared in a similar manner as Compound 18 step 4. The product was prepared from 5-iodo-8-[(4-methoxyphenyl)methoxy]-7-(trifluoromethyl)quinoline 85 (200 mg, 0.43 mmol) to yield 162 mg (81% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.16 (dd, J = 4.17, 1.64 Hz, 1 H) 8.71 (dd, J = 8.72, 1.64 Hz, 1 H) 7.76 – 7.86 (m, 2 H) 7.43 – 7.51 (m, 2 H) 7.13 – 7.23 (m, 4 H) 6.93 – 7.01 (m, 2 H) 5.60 (s, 2 H) 3.74 – 3.81 (m, 3 H) 2.27 (s, 3 H). MS (ES+) m/z 478.0 [M+Na]⁺.

Step 7.

To a mixture of 8-[(4-methoxyphenyl)methoxy]-5-(p-tolylsulfanyl)-7-(trifluoromethyl)quinoline 86 (160 mg, 0.35 mmol) in DCM (5 mL) at ambient temperature was added 3-chlorobenzenecarboperoxoic acid (173 mg, 0.70 mmol). The reaction was stirred for 2 hours. The reaction was quenched by addition of dimethyl sulfide. The mixture was extracted with 10% sodium sulfite aqueous solution. The organic layer was dried over sodium sulfate and concentrated in vacuo. Purification was accomplished by automated reversed phase chromatography eluting 20–95% ACN/water with 0.05% TFA as modifier. Isolated 5-(p-tolylsulfonyl)-7-(trifluoromethyl)quinolin-8-ol 24 (109 mg, 85% yield) as a solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.02 (d, J = 4.29 Hz, 1 H) 8.94 (d, J = 8.84 Hz, 1 H) 8.48 (s, 1 H) 7.86 – 7.96 (m, 3 H) 7.39 (d, J = 8.34 Hz, 2 H) 2.34 (s, 3 H). MS (ES+) m/z 368.0 [M+H]⁺.

5-(Cyclopentylsulfonyl)-7-fluoroquinolin-8-ol (25).

Step 1.

A mixture of 8-benzyloxy-7-fluoro-5-iodo-quinoline 42 (10.4 g, 27.4 mmol), cyclopentanethiol (4.4 mL, 41.1 mmol), tris(dba)dipalladium (1.2 g, 1.3 mmol), Xantphos (0.8 g, 1.3 mmol) and cesium carbonate (26.8 g, 82.2 mmol) in dioxane (100 mL) was degassed with nitrogen for 15 minutes. The resultant mixture was heated at 90°C for 3 hours. The mixture was passed through a plug of silica eluting with ethyl acetate and concentrated to a residue. The residue was purified via silica gel chromatography eluting with ethyl acetate/hexanes (0–30%) to give 8-benzyloxy-5-cyclopentylsulfanyl-7-fluoro-quinoline 87 (9.2 g, 95% yield) as a slightly colored oil. ¹H NMR (400 MHz, CDCl₃) δ ppm 9.01 (dd, J = 4.2, 1.6 Hz, 1 H) 8.72 (dd, J = 8.6, 1.8 Hz, 1 H) 7.52 – 7.60 (m, 2 H) 7.41 – 7.52 (m, 2 H) 7.30 – 7.40 (m, 3 H) 5.48 – 5.55 (m, 2 H) 3.51 – 3.62 (m, 1 H) 1.95 – 2.10 (m, 2 H) 1.74 – 1.89 (m, 2 H) 1.56 – 1.70 (m, 4 H). MS (ES+) m/z 354.0 [M+H]⁺.

Step 2.

To a stirring solution of 8-benzyloxy-5-cyclopentylsulfanyl-7-fluoro-quinoline 87 (9.2 g, 26.1 mmol) in DCM (200 mL), 3-chlorobenzenecarboperoxoic acid (13.5 g, 54.9 mmol) was added. The solution was stirred at ambient temperature for 1 hour. The reaction was quenched by addition of dimethyl sulfide (20 drops). The resultant mixture was stirred for 20 minutes. Sodium sulfite (1 N solution, 200 mL) was added. The organic solution extracted and separated. The organic layer was dried over sodium sulfate and concentrated. The residue was purified by normal phase chromatography (110 g silica gel cartridge) eluting with ethyl acetate/hexanes (0–50%) to give 8-benzyloxy-5-cyclopentylsulfonyl-7-fluoro-quinoline 88 (7.8 g, 77% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ ppm 9.06 – 9.15 (m, 2 H) 8.15 (d, J = 10.9 Hz, 1 H) 7.59 (dd, J = 8.6, 4.3 Hz, 1 H) 7.52 (dd, J = 7.7, 1.4 Hz, 2 H) 7.29 – 7.39 (m, 3H) 5.70 (s, 2 H) 3.61 (quin, J = 7.6 Hz, 1 H) 2.01 – 2.16 (m, 2 H) 1.73 – 1.90 (m, 4 H) 1.56 – 1.68 (m, 2 H). MS (ES+) m/z 386.0 [M+H]⁺.

Step 3.

8-benzyloxy-5-cyclopentylsulfonyl-7-fluoro-quinoline 88 (7.8 g, 20.3 mmol) in hydrochloric acid (6 N, 150 mL, 900 mmol) was stirred at 100°C for two hours. The solution was cooled in ice-water bath for 30 minutes. The yellow precipitate was isolated by filtration and washed with diethyl ether and air-dried to yield a green solid. The solid was free-based by dissolving it in a slightly basic solution (0.05 N NaOH, 20 mL) then the solution was adjusted to slightly acidic by adding hydrochloric acid (1 N). The solution was extracted with dichloromethane (3 × 100 mL). The combined organic solution was washed with brine (2 × 100 mL) and dried over sodium sulfate. The solution was filtered and concentrated to give slightly colored solid. The solid was purified further by crystallization in heptane/chloroform to give 5-cyclopentylsulfonyl-7-fluoro-quinolin-8-ol 25 (4.3 g, 72% yield) as a solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.96 – 9.07 (m, 2 H) 8.09 – 8.18 (m, 1 H) 7.80 (dd, J = 8.7, 4.2 Hz, 1 H) 3.81 – 3.94 (m, 1 H) 1.82 – 1.97 (m, 2 H) 1.61 – 1.82 (m, 4 H) 1.47 – 1.60 (m, 2 H). MS (ES+) m/z 296.0 [M+H]⁺.

7-Chloro-5-(cyclopentylsulfonyl)quinolin-8-ol (26).

Step 1.

Step 3.

To a suspension of 5-pyrrolidin-1-ium-1-ylsulfonylquinolin-8-ol 94 (1.3 g, 4.6 mmol) in chloroform (15 mL) was added N-chlorosuccinimide (620 mg, 4.6 mmol) and the resulting mixture was stirred at room temperature overnight. Volatiles were evaporated under reduced pressure, and the resulting residue was purified by preparative HPLC (10–100% acetonitrile in water with 0.05% trifluoroacetic acid as modifier) to afford 7-Chloro-5-pyrrolidin-1-ylsulfonyl-quinolin-8-ol 29 as an orange solid (350 mg, 24% yield). ¹H NMR (400 MHz, DMSO-d6) δ ppm 9.12–9.20 (m, 1 H) 8.88–8.95 (m, 1 H) 8.27 (s, 1 H) 7.61–7.68 (m, 1 H) 3.33 (s, 4 H) 1.86 (s, 4 H). MS (ES+) m/z 313.0 [M+H]⁺.

7-Methyl-5-(pyrrolidin-1-ylsulfonyl)quinolin-8-ol (30).

Step 1.

8-Fluoro-7-methyl-quinoline 95 was prepared in a similar manner to Compound 24, step 1. The product was prepared from 2-fluoro-3-methyl-aniline (2.0 g, 15.9 mmol) to provide 2.2 g (88% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.92 (dd, J = 4.17, 1.64 Hz, 1 H) 8.39 (dt, J = 8.34, 1.77 Hz, 1 H) 7.73 (d, J = 8.34 Hz, 1 H) 7.57 (dd, J = 8.34, 4.04 Hz, 1 H) 7.51 (dd, J = 8.08, 7.07 Hz, 1 H) 2.46 (d, J = 2.53 Hz, 3 H).

Step 2.

8-Fluoro-7-methyl-quinoline-5-sulfonyl chloride 96 was prepared in a similar manner to Compound 12, step 1. The product was prepared from 8-fluoro-7-methyl-quinoline 95 (500 mg, 3.1 mmol) to provide 509 mg (63% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.48 (dt, J = 8.72, 1.71 Hz, 1 H) 9.09 (dd, J = 4.80, 1.52 Hz, 1 H) 8.01 (d, J = 7.33 Hz, 1 H) 7.90 (dd, J = 8.72, 4.67 Hz, 1 H) 2.51 – 2.52 (m, 3 H). MS (ES+) m/z 260.9 [M+H]⁺.

Step 3.

8-fluoro-7-methyl-quinoline-5-sulfonyl chloride 96 (50 mg, 0.20 mmol) was dissolved in THF (1.5 mL). To this solution was added DIPEA (106 μL, 0.61 mmol), followed by pyrrolidine (17 μL, 0.20 mmol). The resulting mixture was stirred at room temperature for 15 minutes. To the reaction was added potassium t-butoxide (610 μL, 0.61 mmol) and stirred at ambient temperature for 1 hour. The reaction mixture was treated with ~2 mL saturated NaHCO₃ and ~2 mL ethyl acetate, agitated vigorously and the layers separated. Solvent was removed from the organic fraction in vacuo to give a brown residue which was purified by automated normal phase chromatography (0–100%EtOAc/hexanes, 10 g silica gel cartridge) to provide 8-tert-butoxy-7-methyl-5-pyrrolidin-1-ylsulfonyl-quinoline 97, 45 mg (63% yield). ¹HNMR (400 MHz, DMSO-d₆) δ ppm 8.96 – 9.03 (m, 2 H) 8.06 (s, 1 H) 7.61 – 7.70 (m, 1 H) 3.34 (s, 3 H) 3.17 – 3.27 (m, 4 H) 1.72 (dt, J = 6.51, 3.44 Hz, 4 H) 1.44 (s, 9 H). MS (ES+) m/z 349.0 [M+H]⁺.

Step 4.

4 M Hydrogen chloride in dioxane (0.5 mL, 2 mmol) was added to 8-tert-butoxy-7-methyl-5-pyrrolidin-1-ylsulfonyl-quinoline 97 (45 mg, 0.12 mmol) and stirred at ambient temperature for 2 hours. To this reaction was added Et₂O, collected solid by filtration, washed with Et2O and air-dried to give 7-methyl-5-pyrrolidin-1-ylsulfonyl-quinolin-8-ol hydrochloride 30 (35 mg, 92% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.08 (dd, J = 8.59, 1.52 Hz, 1 H) 8.98 (dd, J = 4.29, 1.52 Hz, 1 H) 8.06 (d, J = 0.51 Hz, 1 H) 7.76 (dd, J = 8.84, 4.29 Hz, 1 H) 3.11 – 3.20 (m, 4 H) 2.43 (s, 3 H) 1.65 – 1.73 (m, 4 H). MS (ES+) m/z 293.0 [M+H]⁺.

COMT Enzyme Purification

C-terminal Hexa-His tagged MB-COMT (human and rat) was constructed in a pCDNA3.1 vector (Invitrogen, Carlsbad, CA) and expressed in HEK293 cells (ATCC, Manassas, VA) using 293 Fectin (Invitrogen, Carlsbad, CA). 300 ml of cells at 83% viability were pelleted (15,000 g, 4 °C, 5 minutes) and re-suspended once in PBS and then in membrane buffer (15 mM Tris, pH 7.5, 1 mM EGTA, 0.3 mM EDTA, 2 mM MgCl₂, PIC cocktail (Roche, Diagnostics, Mannheim Germany)). The samples were then homogenized and frozen and defrosted twice in liquid nitrogen. DNase (MilliporeSigma, St. Louis MO) was then added (5 µl per ml) and incubated for 10 minutes at room temperature. The samples were then centrifuged at 40,000 g at 4°C for 25 minutes. The supernatant was discarded, and the pellet was re-suspended in Tris-sucrose buffer (20 mM Tris, pH 7.4, 250 mM sucrose) and homogenized again. The resulting solution was aliquoted and stored at −80 °C until use. MB-COMT protein concentration from the membrane homogenate was estimated using the Bradford Lowry method.

C-terminal Hexa-His S-COMT (human and rat) was constructed in a pTT5 mammalian cell expression vector (Invitrogen, Carlsbad, CA) and expressed in 293–6E cells (ATTC, Manassas, VA) using linear polyethyenimine (PEI, MilliporeSigma, St. Louis, MO). 0.5 L of cells were harvested at 90% viability and pelleted (15,000 g, 4 C, 5 min). Pellets were frozen at −80 °C. The protein was purified from the pellets using Ni-NTA affinity chromatography and size exclusion per the following: Resin: Ni-NTA (Qiagen, Hilden, Germany) equilibrated in Buffer A. Bed volume: 4 mL. Column: 1.0 cm diameter Econo-column (BioRad, Hercules, CA) Buffer A: 50 mM Tris HCl, 150 mM NaCl, 30 mM imidazole, 2 mM MgCl₂, 0.1 mM TCEP, pH 8.0 (4 °C) Buffer B: 50 mM Tris HCl, 150 mM NaCl, 300 mM imidazole, 2 mM MgCl₂, 0.1 mM TCEP, pH 8.0 (4 °C). The target protein in the soluble lysate fraction was batch bound to 4 mL of Ni-NTA resin at 4 °C for 2 hours. The resin was collected in a 1.0 cm diameter Econo-column, was washed with 20 CV of buffer A, and the protein of interest was eluted in Buffer B. Fractions (1 mL) were collected. The final destination buffer was 100 mM potassium phosphate, 1mM DTT, 5 mM MgCl₂, 20% glycerol at pH 7.2.

Compound dilution

All compounds were diluted to a final concentration of 1.5% DMSO from 10 mM stocks in two steps. Stock (7.5 μl) was diluted serially into 15 μl of DMSO. Then 4.5 μl of the previous was diluted into 55.5 μl of COMT assay buffer (50 mM Tris, 5–10 mM MgCl₂, 2.5 mM DTT, pH 6.9). 1 μl of that was then deposited into a Corning® low volume 384 well white flat bottom polystyrene NBS™ microplate in triplicate.

For 0 and 100% inhibition controls, DMSO and Tolcapone (synthesized in house) were added to each assay plate. DMSO was added to 12 wells on the plate in a final concentration of 1.5% and Tolcapone was added to 12 wells on the plate with a final concentration of 10 μM.

Tolcapone was diluted to 666.6 μM and then diluted in two steps according to the procedure of the compounds above. DMSO (MilliporeSigma, St. Louis, MO) was also diluted according to the same scheme.

Enzyme reaction

COMT activity was measured using the MTase Glo Methyltransferase Assay (Promega, Madison, WI) according to manufacturer’s instructions. Assays were carried out in Corning® low volume 384 well white flat bottom polystyrene NBS™ microplates with a final volume of 5μL containing approximately 7 ng of human MB-COMT as estimated by the Bradford Lowry method from the membrane homogenate, 4 ng of rat MB-COMT as estimated by the Bradford Lowry method from the membrane homogenate, 1 ng of Human S-COMT or 1 ng of Rat S-COMT respectively. All reactions contained 20 μM high purity S-adenosyl methionine (SAM, CisBio, Bedford, MA) in COMT assay buffer (50 mM Tris, 5–10 mM MgCl₂, 2.5 mM DTT, pH 6.9). For MB-COMT, the catechol substrate was 7 μM norepinephrine (MilliporeSigma, St. Louis, MO) and for S-COMT the substrate was 10 μM 7,8-dihydroxy-4-methylcoumarin³² (MilliporeSigma, St. Louis, MO).

Reactions were performed in a 37°C incubator for 1 hr. The plate was removed from the incubator and allowed to cool to room temperature for 15min. MTase reagent A (Promega, Madison, WI) was first diluted 1 to 5 into RO water and 1 μl was then added to the well. The plate was spun down, shaken and allowed to incubate for 30 minutes at room temperature avoiding light. Then 5 μl of MTase reagent B (Promega, Madison, WI) were added to all of the wells. The plate was spun down, shaken and allowed to incubate for 30 minutes at room temperature avoiding light. Luminescence was detected with a Tecan Infinite M100 Pro plate reader.

Standard curve

A standard curve was run on every plate. The amount of S-adenosyl homocysteine (SAH, CisBio, Bedford, MA) produced was determined using a standard curve and a linear back-calculation method. The standard curve comprised of varying concentrations of SAH from 500 nM down to 0 nM while maintaining a final SAM/SAH concentration of 50 μM. In order to correct for background levels present in the enzymatic lysate (MB-COMT), enzyme at assay concentration was added to the standard curve as well.

Determination of Inhibition

Percentage inhibition was calculated by using 10 μM Tolcapone value as 100% inhibition value and the DMSO control as the 0% inhibition value. The dose response curves were constrained at 0% inhibition while keeping the percentage inhibition of the highest compound concentration floating. IC₅₀ was determined by non-linear regressions and curve fitting using a 4-parameter fit with a variable slope in the Dotmatics® studies program.

Potency data presented is an average of three separate experiments in which each data point was run in triplicate (unless otherwise noted) and reported as pIC₅₀ ± SEM in Tables 1 and 2.

X-Ray Crystallography

Following the protocol from M. Ellermann et al.³³ 1 µM rat S-COMT was incubated overnight at 4 °C with 10 µM compound 21 and 1 mM SAM (Sigma-Aldrich) in 50 mM Tris-HCl pH 7.6, 50 mM NaCl, 10 mM DTT, 2 mM MgCl₂, concentrated to 13 mg mL⁻¹ and cleared by centrifugation. Crystallization was set up at 18 °C with hanging drop vapour diffusion and crystals grew in 100 mM Bis-Tris propane pH 5.5–7.0, 100 mM NaCl, 1.2–1.8 M (NH₄)₂SO₄. The crystal was cryo-protected in 25 mM Bis-Tris propane pH 7.3, 40% (w/v) PEG3350, 150 mM sodium formate and flash-cooled in liquid nitrogen for data collection. Diffraction data were collected at 100 K using a MicroMax-007 HF rotating anode X-ray generator (Rigaku) and mar345 image plate detector (marXperts). Data were integrated and scaled using MOSFLM³⁴ and SCALA³⁵. The structure was determined by molecular replacement using PHASER³⁶ (CCP4 program suite³⁷) and the published S-COMT structure (PDB accession code: 3S68)³³, without ligand, as search model. Structural refinement was performed with PHENIX³⁸ with altering cycles of manual model building with COOT³⁹. The crystallographic statistics are summarized in Supplementary Table S3. The protein structure was validated using MolProbity⁴⁰. The coordinates and structure factors have been deposited in the Protein Data Bank (entry code 6GY1). Molecular pictures are prepared using program PyMol⁴¹.

Mouse Blood-Brain Barrier (BBB) Assay

Animals

Male, C57BL/6J mice (8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were group-housed (5/cage) and allowed to habituate to the colony room for one week before handling. All mice were handled on alternating days (3X/week) during the following week and again on the day before testing to acclimate the mice to the experimenter. All procedures were approved by the Sobran, Inc. Rangos Facility Animal Care and Use Committee and followed the Guide for the Care and Use of Laboratory Animals.

Drugs

Novel COMT inhibitors (synthesized in-house) were suspended in vehicle (0.1% Tween80, 0.1% 1510 silicone antifoam, 1% methylcellulose 400c/p in water) and administered through intraperitoneal (ip) injection. The administration volume was 10 mL/kg.

BBB Protocol

On the day of testing, mice were moved from the colony room to a holding room where they were weighed. Drug injections were initiated after an hour acclimation period. Each mouse received a single injection of the test compound (10 mg/kg). At the appropriate time point (5, 15, or 60 minutes post injection), mice were moved individually to the procedure table where they were anaesthetized via isoflurane. The chest cavity was opened and blood was collected via cardiac puncture into Lithium-Heparin 1.3mL microtubes (Sarstadt, Numbrecht, Germany). The mouse was then perfused with phosphate buffered saline (PBS) via a 12mL syringe using a 23 gauge needle to clear the brain of any residual blood and the brain was harvested. Blood was then centrifuged at 2000 rpm at 4°C for 15 minutes to separate the plasma. Plasma was then transferred into Thermo Scientific Matrix tubes for storage. Brains were collected in 5mL Thermo Scientific Nunc tubes and homogenized in a 4x (w/v) volume of PBS, pH 7.4. Plasma and brain homogenate samples were stored at −80°C until analysis.

Rat Biomarker Assay

Animals

Male Sprague Dawley rats (176–200 g on arrival; Charles River Laboratories, Wilmington, MA, USA) were used for all studies. Rats were allowed to habituate to the colony room for one week before handling. All rats were handled individually for at least one minute on each of the three days preceding the day of the experiment. All procedures were approved by the Sobran, Inc. Rangos Facility Animal Care and Use Committee and followed the Guide for the Care and Use of Laboratory Animals.

Drugs

Novel COMT inhibitors (synthesized in-house) and tolcapone (synthesized in-house) were suspended in vehicle (0.1% Tween80, 0.1% 1510 silicone antifoam, 1% methylcellulose 400c/p in water) and administered either orally (po) or through intraperitoneal (ip) injection (tolcapone). Administration volumes were 10 mL/kg and 5 mL/kg for po and ip dosing, respectively.

Biomarker protocol

On the day of testing, rats were transferred to a holding room and weighed. After an hour acclimation period, rats received either vehicle or a COMT inhibitor. At the 4 hour time point, rats were moved to a separate procedure room where they were anaesthetized via isoflurane. Once the rats were determined to be unresponsive, fur on their heads was shaved using electric clippers. The rats were positioned in a stereotaxic frame, with their heads pointed down at a 45-degree angle. To collect cerebrospinal fluid (CSF), previously published protocols⁴² were adapted. Briefly, we used a 23G needle connected via PE50 tubing to a collection syringe to access the cisterna magna according to the protocol. Slight negative pressure was used to ensure the CSF flowed evenly. During collection, we observed the CSF as it flowed through the tubing into the syringe looking for discoloration due to blood contamination. If any blood was observed entering the tubing, a cut was made in the tubing above the blood to minimize the contamination of the CSF sample and only the CSF above the cut was further processed. The CSF was then transferred to previously chilled (dry ice) Eppendorf tubes containing 0.05M perchloric acid (4:1 CSF:perchloric acid ratio). The tubes were put back on dry ice until the end of the procedure. Any samples with visible blood contamination (pink/red color) were not analyzed. Next, the chest cavity was opened, and blood was collected by cardiac puncture. The blood was collected in Lithium-Heparin 1.3mL microtubes (Sarstadt, Numbrecht, Germany) and stored on ice. The rat was then transcardially perfused with ice-cold phosphate buffered saline (PBS) via a peristaltic pump set to a flow rate of 20 mL/min to clear the brain of any residual blood and the brains were collected in 5mL Thermo Scientific Nunc tubes. Blood was then centrifuged at 2000 rpm at 4°C for 15 minutes to separate the plasma. Plasma was then transferred into Thermo Scientific Matrix tubes for storage. CSF, plasma, and brains were stored at −80°C until analysis.

Bioanalysis protocol

Artificial CSF (aCSF) consisted of 125mM NaCl, 5mM potassium chloride, 1mM MgCl2, 5mM D-glucose, 1.25mM sodium phosphase di-basic, 2mM calcium chlroride and 24mM NaHCO3. Triplicate standards and QCs of HVA and DOPAC were prepared in 0.01M perchloric acid in aCSF. Triplicate 10µL standards, QCs, and CSF samples were transferred to a 96-well Corning Axygen ® PCR plate (Corning, NY). 10µL of 100mM sodium tetraborate (MilliporeSigma, St. Louis, MO) in water and 2µL of internal standard consisting of 500 ng/mL each of HVA-d5 and DOPAC-d5 (both C/D/N Isotopes Pointe-Claire, Quebec, CA) in 50% methanol in water (v/v) were added to each sample and the plate was mix for 2 minutes at 1000 rpm. To this was added 10µL of 2% benzoyl chloride in acetonitrile (v/v) to each sample, and the plate was mixed for 4 minutes at 1000 rpm. From the mixture, 5µL of each sample was injected for analysis using an Agilent 6540 QTOF with Jet Stream Electrospray Ionization Source (ESI) and Agilent 1290 UHPLC. Solvents were 10mM ammonium formate in water (A) and 100% acetonitrile (B). Chromatographic separation was achieved over 7.5 minutes using a Phenomenex Luna Omega 2.1 × 100mm, 1.6µm, C18 column with a binary gradient starting 21% B. The flow rate was 500 μl min⁻¹. The autosampler was set at 20 °C. The LC gradient was as follows: 0–4 min, 21% B; 4 – 4.5 min, 21–40% B; 4.5 – 5.0min, 40% B. 5.01 – 6.0 min, 95%B, 6.01 min, 21% B. The mass spec acquisition was performed using full scan MS from m/z 50 to 800 with the following source conditions: Drying and Sheath Gas temperatures at 350°C and 400°C, respectively; Both gas flows at 12L/min; Nebulizer-45psig, VCap, Nozzle and Fragmenter voltages at 3000V, 600V and 100V, respectively. The ammonium adducts of the dual benzoylated DOPAC and DOPAC-d5 (m/z 394.1285 and 399.1599, respectively) and the single benzoylated HVA and HVA-d5 (m/z 304.1179 and 309.1493, respectively) were used for data analysis. Analyte peak areas were determined from the extracted ion chromatograms within a +/− 20ppm window. Concentrations were calculated with linear regression analysis using Agilent Masshunter Quantitative Analysis Software (B.06.00 SP01).

Supplementary Material

NIHMS1013823-supplement-1.pdf^{(453.4KB, pdf)}

ACKNOWLEDGEMENTS

The authors greatfully acknowledge David Boucaut (UCB) for assistance with in vivo studies and Eric Gillent (UCB) and Benoit Culot (UCB) for bioanalysis assistance.

Funding of parts of this work from the National Institutes of Mental Health NIMH R01 MH107126 is gratefully acknowledged.

ABBREVIATIONS USED

%F: bioavailability
ACN: acetonitrile
aCSF: artificial cerebrospinal fluid
ADHD: attention deficit hyperactivity disorder
ADME: absorption, distribution, metabolism, and excretion
AlCl₃: aluminum chloride
BCRP: breast cancer resistance protein
Bis-Tris: Bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane
BLQ: below limit of quantitation
BnBr: benzylbromide
BnOH: benzyl alcohol
CHCl₃: chloroform
CL_p: plasma clearance
COMT: catechol O-methyltransferase
Cs₂CO₃: cesium carbonate
CSF: cerebrospinal fluid
CsF: cesium fluoride
DCM: dichloromethane
DIPEA: N,N-diisopropylethylamine
DMDN: N,N-dimethylethylenediamine
DMF: dimethylformamide
DMSO: dimethyl sulfoxide
DNase: deoxyribonuclease
DOPAC: dihydroxylphenyl acetic acid
DTT: dithiothreitol
EDTA: ethylenediaminetetraacetic acid
EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid
ER: efflux ratio
ESI: electrospray ionization
Et: ethyl
EtOAc: ethyl acetate
HBr: hydrobromic acid
HCl: hydrochloric acid
HEK: human embryonic kidney cells 293
HPLC: high-performance liquid chromatography
HVA: homovanillic acid
ip: intraperitoneal
iv: intravenous
K₂CO₃: potassium carbonate
K_{p u,u}: concentration ratio of unbound drug in brain to unbound drug in plasma
LC: liquid chromatography
LC-MS: liquid chromatography−mass spectrometry
L-DOPA: L-3,4-dihydroxyphenylalanine
M: molar
m: mouse
MB-COMT: membrane bound catechol O-methyltransferase
mCPBA: meta-chloroperoxybenzoic acid
MDCK: Madin-Darby canine kidney cells
MeOH: methanol
MgCl₂: magnesium chloride
MgSO₄: magnesium sulfate
mpk: milligram per kilogram
MRP2: multidrug resistance-associated protein 2
MTase: methyltransferase
N: normal
N/A: not applicable
NaCl: sodium chloride
NaH: sodium hydride
NaHCO₃: sodium bicarbonate
NaOH: sodium hydroxide
NBS: N-bromosuccinimide
NCS: N-chlorosuccinimide
Ni-NTA: nickel-nitrilotriacetic acid
NIS: N-iodosuccinimide
NMR: nuclear magnetic resonance
PBS: phosphate buffered saline
PCR: polymerase chain reaction
Pd(PPh₃)₄: Tetrakis(triphenylphosphine)palladium(0)
Pd₂(dba)₃: tris(dibenzylideneacetone)-dipalladium(0)
PDB: Protein Data Bank
PE: polyethylene
PEG: polyethylene glycol
PFC: prefrontal cortex
P-gp: P-glycoprotein 1
Ph: phenyl
PIC: cocktail protease inhibitor cocktail
pIC₅₀: log(IC₅₀) where IC₅₀ represents the compound/substance concentration required for 50% inhibition
PK: pharmacokinetic
PMB: p-methoxybenzyl
PMB-Cl: p-methoxybenzyl chloride
po: oral
ppm: parts per million
QC: quality control standard
QTOF: quadrapole time of flight
r: rat
RO: reverse osmosis
rpm: revolutions per minute
SAH: S-adenosyl-L-homocysteine
SAM: S-adenosyl methionine
SAR: structure−activity relationship
S-COMT: soluble catechol O-methyltransferase
SnAR: neucleophilic aromatic substitution
T1/2: half-life of the product
TBAF: tetrabutylammonium fluoride
TCEP: (tris(2-carboxyethyl)phosphine)
TFA: trifluoroacetic acid
THF: tetrahydrofuran
TM: trademark
TMS: tetramethylsilane
TMSE: 2-(trimethylsilyl)ethyl
Tris: tris(hydroxymethyl)aminomethane
Tris-HCl: tris(hydroxymethyl)aminomethane hydrochloride
UHPLC: ultra high performance liquid chromatography
UV: ultraviolet
V_ss: volume of distribution
Xantphos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

Footnotes

ASSOCIATED CONTENT

Supporting Information

(Tables of raw data for mouse and rat brain penetration, details for the preparation of Rat S-COMT and the crystallographic details, Molecular formula strings (CSV))

Accession Codes

The PDB accession code for the X-ray structure of 21 bound to Rat S-COMT is 6GY1. Authors will release the atomic coordinates and experimental data upon article publication.

Notes

The authors declare no competing financial interest.

REFERENCES

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS1013823-supplement-1.pdf^{(453.4KB, pdf)}

[R1] 1.Lachman HM; Papolos DF; Saito T; Yu YM; Szumlanski CL; Weinshilboum RM, Human catechol-O-methyltransferase pharmacogenetics: Description of a functional polymorphism and its potential application to neuropsychiatric disorders. Pharmacogenetics 1996, 6, 243–250. [DOI] [PubMed] [Google Scholar]

[R2] 2.(a) Kaenmaki M; Tammimaki A; Myohanen T; Pakarinen K; Amberg C; Karayiorgou M; Gogos JA; Mannisto PT, Quantitative role of COMT in dopamine clearance in the prefrontal cortex of freely moving mice. J Neurochem 2010, 114, 1745–1755; [DOI] [PubMed] [Google Scholar]; (b) Yavich L; Forsberg MM; Karayiorgou M; Gogos JA; Mannisto PT, Site-specific role of catechol-O-methyltransferase in dopamine overflow within prefrontal cortex and dorsal striatum. J Neurosci 2007, 27, 10196–10202. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] 3.Mannisto PT; Kaakkola S, Catechol-O-methyltransferase (COMT): Biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors. Pharmacol Rev 1999, 51, 593–628. [PubMed] [Google Scholar]

[R4] 4.Lotta T; Vidgren J; Tilgmann C; Ulmanen I; Melen K; Julkunen I; Taskinen J, Kinetics of human soluble and membrane-bound catechol O-methyltransferase - a revised mechanism and description of the thermolabile variant of the enzyme. Biochemistry 1995, 34, 4202–4210. [DOI] [PubMed] [Google Scholar]

[R5] 5.Papaleo F; Crawley JN; Song J; Lipska BK; Pickel J; Weinberger DR; Chen J, Genetic dissection of the role of catechol-O-methyltransferase in cognition and stress reactivity in mice. J Neurosci 2008, 28, 8709–8723. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] 6.Reenila I; Mannisto PT, Catecholamine metabolism in the brain by membrane-bound and soluble catechol-O-methyltransferase (COMT) estimated by enzyme kinetic values. Medical Hypotheses 2001, 57, 628–632. [DOI] [PubMed] [Google Scholar]

[R7] 7.Learmonth DA; Kiss LE; Soares-da Silva P, The chemistry of catechol O-methyltransferase inhibitors. Int Rev Neurobiol 2010, 95, 119–162. [DOI] [PubMed] [Google Scholar]

[R8] 8.Bonifacio MJ; Palma PN; Almeida L; Soares-Da-Silva P, Catechol-O-methyltransferase and its inhibitors in Parkinson’s disease. CNS Drug Reviews 2007, 13, 352–379. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R9] 9.Russ H; Müller T; Woitalla D; Rahbar A; Hahn J; Kuhn W, Detection of tolcapone in the cerebrospinal fluid of parkinsonian subjects. Naunyn-Schmiedeberg’s Archives of Pharmacology 1999, 360 (6), 719–720. [DOI] [PubMed] [Google Scholar]

[R10] 10.Müller T, Tolcapone addition improves Parkinson’s disease associated nonmotor symptoms. Ther Adv Neurol Diso 2014, 7, 77–82. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Tunbridge EM; Harrison PJ; Weinberger DR, Catechol-O-methyltransferase, cognition, and psychosis: Val(158)Met and beyond. Biol Psychiat 2006, 60, 141–151. [DOI] [PubMed] [Google Scholar]

[R12] 12.Olanow CW; Watkins PB, Tolcapone. Clinical Neuropharmacology 2007, 30, 287–294. [DOI] [PubMed] [Google Scholar]

[R13] 13.Axelrod J, O-Methylation of epinephrine and other catechols in vitro and in vivo. Science 1957, 126, 400–401. [DOI] [PubMed] [Google Scholar]

[R14] 14.Ross SB; Haljasmaa O, Catechol-O-methyl transferase inhibitors - in vitro inhibition of enzyme in mouse brain extract. Acta Pharmacol Tox 1964, 21, 205–214. [DOI] [PubMed] [Google Scholar]

[R15] 15.Borchardt RT; Thakker DR; Warner VD; Mirth DB; Sane JN, Catechol O-methyltransferase .8. Structure-activity-relationships for inhibition by 8-hydroxyquinolines. J Med Chem 1976, 19, 558–560. [DOI] [PubMed] [Google Scholar]

[R16] 16.Barrow JC, Inhibitors of catechol-O-methyltransferase. CNS & Neurological Disorders-Drug Targets 2012, 11, 324–332. [DOI] [PubMed] [Google Scholar]

[R17] 17.Almeida L; Rocha JF; Falca A; Palma PN; Loureiro AI; Pinto R; Bonifacio MJ; Wright LC; Nunes T; Soares-da-Silva P, Pharmacokinetics, pharmacodynamics and tolerability of opicapone, a novel catechol-O-methyltransferase inhibitor, in healthy subjects. Clin Pharmacokinet 2013, 52, 139–151. [DOI] [PubMed] [Google Scholar]

[R18] 18.Barrow J, Ernst G; Huang Y; Buchler I; Weinberger D COMT Inhibiting Methods and Compositions WO2016123576, 2016.

[R19] 19.Liu XR; Chen CP, Free drug hypothesis for CNS drug candidates. In Blood-Brain Barrier in Drug Discovery: Di L; Kerns EH, Eds. Wiley, Hoboken, New Jersey, 2015, 42–65. [Google Scholar]

[R20] 20.Hutson PH; Curzon G, Dopamine metabolites in rat cisternal cerebrospinal-fluid-major contribution from extrastriatal dopamine neurons. J Neurochem 1986, 46, 186–190. [DOI] [PubMed] [Google Scholar]

[R21] 21.Robinson RG; Smith SM; Wolkenberg SE; Kandebo M; Yao LH; Gibson CR; Harrison ST; Polsky-Fisher S; Barrow JC; Manley PJ; Mulhearn JJ; Nanda KK; Schubert JW; Trotter BW; Zhao ZJ; Sanders JM; Smith RF; McLoughlin D; Sharma S; Hall DL; Walker TL; Kershner JL; Bhandari N; Hutson PH; Sachs NA, Characterization of non-nitrocatechol pan and isoform specific catechol-O-methyltransferase inhibitors and substrates. ACS Chem Neurosci 2012, 3, 129–140. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Lin JH, CSF as a surrogate for assessing CNS exposure: An industrial perspective. Curr Drug Metab 2008, 9, 46–59. [DOI] [PubMed] [Google Scholar]

[R23] 23.(a) Banks WA, From blood-brain barrier to blood-brain interface: new opportunities for CNS drug delivery. Nat Rev Drug Discov 2016, 15 (4), 275–292; [DOI] [PubMed] [Google Scholar]; (b) Rankovic Z, CNS drug design: Balancing physicochemical properties for optimal brain exposure. J Med Chem 2015, 58, 2584–2608. [DOI] [PubMed] [Google Scholar]

[R24] 24.Muller N; Lapicque F; Monot C; Payan E; Gillet P; Bannwarth B; Netter P, Protein-binding of indomethacin in human cerebrospinal-fluid. Biochem Pharmacol 1991, 42, 799–804. [DOI] [PubMed] [Google Scholar]

[R25] 25.Ganrot K; Laurell CB, Measurement of IgG and albumin content of cerebrospinal-fluid, and its interpretation. Clin Chem 1974, 20, 571–573. [PubMed] [Google Scholar]

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PERMALINK

Optimization of 8-Hydroxyquinolines as Inhibitors of Catechol O-Methyltransferase

Ingrid Buchler

Daniel Akuma

Vinh Au

Gregory Carr

Pablo de León

Michael DePasquale

Glen Ernst

Yifang Huang

Martha Kimos

Anna Kolobova

Michael Poslusney

Huijun Wei

Dominique Swinnen

Florian Montel

Florence Moureau

Emilie Jigorel

Monika-Sarah ED Schulze

Martyn Wood

James C Barrow

Abstract

Graphical Abstract

INTRODUCTION

Figure 1.

Figure 2.

RESULTS AND DISCUSSION

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Figure 3.

CHEMISTRY

Scheme 1.

Scheme 2.

Scheme 3.

Scheme 4.

Scheme 5.

CONCLUSIONS

EXPERIMENTAL SECTION

General Procedures.

6-Tosylquinolin-8-ol (6).

Step 1.

Step 2.

Step 3.

5-Tosylquinolin-8-ol (7).

Step 1.

Step 2.

Step 3.

5-(p-Tolyl)quinoline-8-ol (8).

Step 1.

Step 2.

Step 3.

(8-Hydroxyquinolin-5-yl)(p-tolyl)methanone (9).

(8-Hydroxyquinolin-5-yl)(pyrrolidin-1-yl)methanone (10).

Step 1.

Step 2.

Step 3.

5-(Cyclopentylsulfonyl)quinoline-8-ol (11).

Step 1.

Step 2.

Step 3.

Step 4.

Step 5.

5-(Pyrrolidin-1-ylsulfonyl)quinoline-8-ol (12).

Step 1.

Step 2.

Step 3.

5-((3-(4-Fluorophenyl)pyrrolidin-1-yl)sulfonyl)quinoline-8-ol (13).

Step 1.

Step 2.

Step 3.

8-Methoxy-5-(pyrrolidin-1-yulsulfonyl)quinoline (14).

5-(Ethylsulfonyl)quinoline-8-ol (15).

Step 1.

Step 2.

Step 3.

5-((2-Benzylpyrrolidin-1-yl)sulfonyl)quinoline-8-ol (16).

Step 1.