Synthesis and Discovery of Arylpiperidinylquinazolines: new inhibitors

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Synthesis and Discovery of Arylpiperidinylquinazolines: new inhibitors of the vesicular monoamine transporter Brian Provencher, Amy J. Eshleman, Robert A Johnson, Xiao Shi, Olga Kryatova, Jared Nelson, Jianhua Tian, Mario Gonzalez, Peter C Meltzer, and Aaron Janowsky J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00542 • Publication Date (Web): 21 Sep 2018 Downloaded from http://pubs.acs.org on September 22, 2018

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Journal of Medicinal Chemistry

Synthesis and Discovery of Arylpiperidinylquinazolines: new inhibitors of the vesicular monoamine transporter Brian A. Provencher,a,b,* Amy J. Eshleman,c,d Robert A. Johnson,c Xiao Shi, c,d Olga Kryatova,a Jared Nelson,a Jianhua Tian,a Mario Gonzalez,a Peter C. Meltzer,a Aaron Janowskyc,d,e a

b

Organix Inc, 240 Salem St., Woburn, MA, 01801

Department of Chemistry and Biochemistry, Merrimack College, North Andover, MA 01845 c

d

Research Service, VA Portland Health Care System

Departments of Psychiatry and Behavioral Neuroscience, Oregon Health and Science University,

e

The Methamphetamine Abuse Research Center, Oregon Health and Science University, Portland OR 97239

KEYWORDS APQ, VMAT2, DHTB, reserpine, vesicular transporter

Abstract: Methamphetamine, a human vesicular monoamine transporter 2 (VMAT2) substrate, releases dopamine, serotonin and norepinephrine from vesicles into the cytosol of presynaptic

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neurons and induces reverse transport by the monoamine transporters to increase extracellular neurotransmitters. Currently available radioligands for VMAT2 have considerable liabilities: The binding of [3H]dihydrotetrabenazine ([3H]DHTB) to a site on VMAT2 is not dependent on ATP and [3H]reserpine binds almost irreversibly to VMAT2. Herein we demonstrate that several arylpiperidinylquinazolines (APQs) are potent inhibitors of [3H]reserpine binding at recombinant human VMAT2 expressed in HEK-293 cells. These compounds are biodiastereoselective and bioenantioselective. The lead radiolabeled APQ is unique because it binds reversibly to VMAT2, but does not bind the [3H]DHTB binding site. Furthermore, experimentation shows that several novel APQ ligands have high potency for inhibition of uptake by both HEK-VMAT2 cells and mouse striatal vesicles, and may be useful tools for characterizing drug-induced effects on human VMAT2 expression and function.

Introduction Methamphetamine is a highly addictive stimulant.1

The abuse of methamphetamine is

prevalent and escalates with time for many individuals.2 Stimulant-induced psychosis and longterm neuronal loss are indications of methamphetamine-induced neurotoxicity.3-5 Currently, there are no approved pharmacotherapies for treatment of methamphetamine addiction and behavioral therapy has had limited success.6 Among the primary initial neuronal targets for methamphetamine is the human vesicular monoamine transporter 2 (VMAT2).7-10 Expressed in monoaminergic presynaptic neurons, as well as in peripheral tissues, VMAT2 transports cytosolic dopamine (DA), serotonin (5-HT) or norepinephrine (NE) into storage vesicles.11 By transporting neurotransmitters into vesicles, VMAT2 participates in the regulation of cytosolic levels and vesicular stores of biogenic amines.


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Methamphetamine competes with the endogenous neurotransmitters for binding sites and uptake into VMAT2. When VMAT2 binds methamphetamine, vesicular degradation resulting in dopamine efflux into the cytosol of presynaptic neurons is observed.12 This can cause reverse transport by the dopamine transporter to increase dopamine in the extracellular space. Therefore, VMAT2 is a possible target for therapeutics to treat methamphetamine abuse.6,8 The VMAT2, while expressed in vivo on the vesicular membranes of neurons, is also functional when expressed in mammalian cell lines, with the energy source for transport provided by the native V-type H+ -ATPase.13 Recent work reveals that in drosophila brain, amphetamine redistributes vesicle contents, diminishes the vesicle pH-gradient needed for biogenic amine uptake, and requires VMAT2 function.14 Current therapy for methamphetamine abuse consists primarily of cognitive behavioral interventions, however, pharmacological interventions could improve the treatment of methamphetamine abuse.

Vocci and Appel, when reviewing possible targets for

methamphetamine pharmacotherapies, identified the VMAT2 as having an obligatory role in methamphetamine activity.15 Some strategies for pharmacotherapy development have focused on altering psychostimulant interaction with the VMAT2 or with altering VMAT2 function. Lobeline, a lipophilic alkaloid of Indian tobacco, alters VMAT2-mediated DA uptake and release and alters methamphetamineinduced DA release,16,17 and lobeline and its analogs are being investigated as possible therapeutics for methamphetamine abuse.18-20 Reserpine, a high affinity VMAT2 blocker, has been used to treat hypertension, but essentially irreversibly binds to VMAT2 which results in depletion of biogenic amines including dopamine and epinephrine. Thus, recovery of biogenic amine storage requires synthesis of new storage vesicles.21 In addition, reserpine binds to both


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the VMAT2 and the peripheral VMAT1, with side effects including sedation, inability to concentrate or perform complex tasks and sometimes, psychotic depression that appears over many weeks or months.22,23

Tetrabenazine (TBZ) and its analogue, dihydrotetrabenazine

(DHTB), inhibit VMAT2 function, bind reversibly to the VMAT2 at an allosteric binding site and have shown effectiveness in treatment of hyperkinesias characterized by abnormal involuntary movements as observed in Huntington’s disease, Tourette’s syndrome and tardive dyskinesia.22,24

However, TBZ has side effects similar to reserpine including sedation,

depression, akathisia and Parkinsonism.24 Ketanserin binds to VMAT2 with relatively high affinity (~6-45 nM),25 but is nonselective; displaying high affinity for the 5-HT2A receptor, and moderate affinities for the histamine H1 and 5-HT2C receptors.26-28 There are number of radioligands and drugs that bind to the VMAT2 and affect its function. Currently, available radioligands for labeling VMAT2, such as [3H]DHTB and [3H]reserpine have considerable liabilities. [3H]DHTB binding is not ATP-dependent and thus mechanistically is less similar to ATP-dependent VMAT2 function, and [3H]reserpine binds almost irreversibly to VMAT2 and does not lend itself to high-throughput screening.29,30 One model of the binding sites on VMAT2 suggests that the reserpine site has high affinity for substrates and is directed toward the cytosol; in contrast, after the binding of the substrate to VMAT2, a conformational change results in the TBZ-binding conformation.31 Furthermore, it was found that TBZ does not require a proton gradient for binding and that TBZ binding locks the transporter into a confirmation which no longer allows the substrate to bind to VMAT2 by inhibiting the cytoplasmic gate.32 With few available radioligands, the search for novel ligands to serve as chemical probes that bind reversibly and specifically to the VMAT2, and are associated with VMAT2 function, can


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provide a springboard for the development of pharmacotherapies for symptoms related to methamphetamine

addiction.

Herein

we

describe

the

discovery

of

new

arylpiperidinylquinazoline (APQ) ligands and their biological activity in VMAT2 assays. Chemistry

Novel APQ ligands (±)-5 were synthesized from commercially available ethyl 1-benzyl-4-oxo3-piperidinecarboxylate hydrochloride 1.

Conversion from the ketone to triflate 2 was

accomplished using N-phenyl bistrifluorsulfonamide. Suzuki coupling followed by reduction afforded substituted aryl-piperidine (4). N-Alkylation with the known quinazoline 6 afforded racemic, syn-APQ ligands (±)-5.33 To ensure water solubility, the APQs were converted to the HCl salt with HCl in ether. The precipitate was filtered, dissolved in water and freeze dried to afford the APQ-HCl salt which was confirmed by elemental analysis. Enantiomers could be resolved by crystalizing 4 with tartaric acid. Once purified, single enantiomers were subjected to the same N-alkylation and salt procedure as stated above.


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Anti-APQ isomer 9, was prepared from intermediate 4b. Protection of the free amine with Boc anhydride yielded 7. Heating 7 in the presence of sodium ethoxide converted the cisdiastereomer to the more stable trans-diastereomer. Deprotection followed by N-alkylation with the known quinazoline afforded (±)-9. Results and Discussion To begin, the binding affinities of 13 APQ derivatives and reference compounds (ketanserin, DHTB, methamphetamine, reserpine, Ro4-1284, serotonin, and lobeline) for VMAT2 were assessed by inhibition of radioligand binding using [3H]reserpine and [3H]DHTB (Table 1). Methamphetamine had micromolar affinity for the reserpine binding site but had 60 fold lower affinity for the DHTB site.

Ketanserin displays some affinity for the reserpine binding site

albeit five-fold lower than for the DHTB binding site (Table 1, Entry 1). Interestingly, APQs displayed a wide range of binding affinities across both sites and were generally more selective for the reserpine site. Syn-APQ (±)-5a bound to the reserpine site with a Ki=70 nM (Table 1, entry 9). The para-fluoro analog (±)-5b, which contains the same aryl moiety of ketanserin, was found to bind with essentially the same Ki at the reserpine site (74 vs 70 nM). Neither compound showed much affinity for the DHTB site. To explore the diastereoselectivity of the APQ ligand on the receptors, anti-isomer (±)-9 was tested. APQ ligand 9 had less affinity for the reserpine site than the syn-analog, (±)-5b. Additionally, (±)-9 was not as selective for the reserpine site and competed at the DHTB site with a Ki=750 nM. TABLE 1. Binding affinities for the VMAT2 binding sites of [3H]Reserpine and [3H]DHTBa


6

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a

The core APQ structure is given in Schemes 1 and 2. Experiments were conducted as detailed in the

methods. N ≥ 3, except when the Ki was greater than 10 µM, in which case n=2. If some experiments yielded IC50 or Ki values less than 10 µM and other experiments yielded IC50 or Ki values greater than 10 µM, the latter experiments were assigned a value of 10 µM and averages calculated. The actual value is greater than that average and no standard error is reported.

APQ compounds were tested at

concentrations ranging from 1 nM to 10 µM. Data are provided as Ki= nM ± sem unless otherwise

noted. With the knowledge that the syn-isomers (5) were more selective for reserpine over DHTB, we explored the structure-activity relationship of the APQ ligands. Compound (±)-5c lost affinity for the reserpine site while 3-trifluorophenyl analog (±)-5d retained some affinity for the reserpine


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site albeit, much lower than (±)-5b. Other analogs in this series, did not bind at the reserpine or DHTB sites (Table 1, entries 15-18). Thiophene analog, (±)-5i and dioxane analog, (±)-5j were found to bind with moderate affinity to the reserpine site Ki= 450 and 652 nM respectively. To explore the enantioselectivity of the ligands, (+)-5b and (-)-5b were isolated and subjected to the reserpine and DHTB binding assays. (+)-5b had higher affinity than (±)-5b in the reserpine binding assay with a Ki=62 nM (p0.05) but lower affinity than the enantiomer (+)5b (p10 M 24.5

3.1

410

100

HEK-VMAT2 [ H]5HT Uptakeb 3

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Mouse striatal vesicular VMAT2 [3H]5HT Uptakeb

180

39

100

35

28.1

4.4

19.80

0.46

3620

560

2740

340

13.2

1.5

1.79

0.39

49.56

5

Ro4-1284

1932

73

154

31

6

5-HT

2290

330

1430

320

7

lobeline

1950

400

4200

8

( )-9

1150

280

85.5

9

( )-5a, Ar= C6H5

287

46

10

( )-5b, Ar= 4-FC6H4

161

11

(+)-5b

93

5HT2A [125I]DOI 10.7 6.7 101

hDAT [125I]RTI-55

2.5 1.4 14

>1 M >10 M

0.79

98

17

11.5

990

381

56

7400

9.8

24.7

6.3

37.3

19.7

6.4

9.1

3.1

70

39.4

5.6

3.46

0.95

13c

16.0

3.3

3.39

0.95

7.9

6.6

2.2

2.0 1500 8.5

>10 M 2380

600

>9.3 M 1610

180

4170

610

>10 M

2440

390

>10 M

9900

1800

12

(-)-5b

290

110

51.1

13

( )-5c, Ar=4-CF3-C6H4

720

120

2900

14

( )-5d, Ar=3-CF3-C6H4

350

21

125

15

( )-5e, Ar=4-CH3-C6H4

452

33

2700

1300

ND

>10 M

16

( )-5f, Ar=4-NMe2-C6H4

1960

390

5800

1000

ND

>10 M

>10 M

17

( )-5g, Ar=4-Ph-C6H4

3770

210

>10 M

ND

>10 M

>10 M

18

( )-5h, Ar=4-CO2Et-C6H4

1660

120

>10 M

ND

>10 M

>10 M

>9300

>7.1 M

>10 M

>10 M

19

( )-5i, Ar= 2-thienyl

1300 18

900

230

101

17

700

220

142

36

ND 12.8

31.5

>10 M 4.5

>10 M

6.4

>10 M >10 M 7300

2100

O 20

( )-5j, Ar=

ND

O a

Experiments were conducted as detailed in the methods. N ≥ 3, except when the Ki or IC50 was greater

than 10 µM, in which case n=2. If some experiments yielded IC50 or Ki values less than 10 µM and other experiments yielded IC50 or Ki values greater than 10 µM, the latter experiments were assigned a value of 10 µM and averages calculated. The actual value is greater than that average and no standard error is reported. APQ compounds were tested at concentrations ranging from 0.01 nM to 10 µM. Data is b

c

reported as Ki= nM ± sem unless otherwise noted. IC50 = nM ± sem Kd= nM ± sem. ND= Not

Determined

The other racemic APQ ligands were tested against [3H](+)-5b as well. APQ ligand (±)-5a, displayed moderate binding affinity albeit only two times less potent than (±)-5b. Other APQ ligands with electron withdrawing substituents displayed moderate binding affinity with (±)-5d


12

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showing the best results out of the group (entries 13, 14 and 18; Table 2). SAR investigations into electron donating groups also resulted in loss of potency in the [3H](±)-5b binding assay (entries 15-17; Table 2).

Figure 4: Concentration-response curves of inhibition of [3H]5-HT uptake into mouse striatal vesicular membranes. Six APQ compounds and three standards are shown. N=3-6. All APQ compounds were more potent than ketanserin in this assay. The potency of the APQ compounds at inhibition of [3H]5-HT uptake via human VMAT2 expressed in HEK cells was assessed. APQ ligands (±)-5a and all stereoisomers of 5b had similar potencies, ranging from 16 to 51 nM (Table 2). The next most potent compounds ((±)5d, (±)-5i, (±)-5j) had potencies ranging from 101 nM to 142 nM. Interestingly, APQ (±)-5d with a meta-trifluormethyl group, had greater than 20 fold higher potency than (±)-5c with a para-trifluormethyl. APQ ligands (±)-5e, (±)-5f, (±)-5g and (±)-5h demonstrated little potency in the [3H]5-HT uptake assay. The potencies of a subset of APQ compounds and standards for inhibition of [3H]5-HT uptake via endogenous VMAT2 in mouse striatal vesicular membranes were assessed (Table 2, Figure 4). The potencies of all standard compounds were higher in the


13


Page 14 of 37

mouse vesicular preparations (Table 2). The subset of APQ ligands tested in this preparation had higher potencies compared to potencies in the HEK-VMAT2 uptake assay. APQ ligands (±)-5a and all stereoisomers of 5b were very potent, with IC50 values below 10 nM (Table 2). (±)-5d, (+)-9, and (±)-5i had moderate potencies ranging from 12.8 to 31.5 nM. All APQ compounds tested completely inhibited specific [3H]5-HT uptake, defined with 1 µM reserpine.

Figure 5: Correlations of binding affinities and uptake potencies for HEK-VMAT2 for select compounds. Drug-induced inhibition of (A) log (Ki) for inhibition of [3H](+)-5b binding versus log (IC50) for inhibition of [3H]5-HT uptake, (B) log (Ki) for inhibition of [3H]reserpine binding versus log (IC50) for [3H]5-HT uptake (C) log (Ki) for inhibition of [3H]DHTB binding versus log (IC50) for [3H]5-HT uptake. 14 APQ compounds and 7 standard compounds were included in the analysis. Data were analyzed using Spearman’s nonparametric correlation analysis. For the 14 APQ compounds and the seven standard compounds, a good correlation was observed between the log IC50 value for [3H]5-HT uptake and the log Ki value for inhibition of [3H](+)-5b binding (Fig 5A, Spearman’s r= 0.63, p7400 nM [3H]-5b Kd = 161 nM


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Synthesis and Discovery of Arylpiperidinylquinazolines: new inhibitors

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