Taura et al. 2018 Photocontrol of movement disorders

Supplementary InformationRemote control of movement disorders using a photoactive adenosine A2A receptor

antagonist

Jaume Taura, Ernest G. Nolen, Gisela Cabré, Jordi Hernando, Marc López-Cano, Lucia Squarcialupi, Víctor Fernández-Dueñas, Kenneth A. Jacobson and Francisco Ciruela

1. Supplementary Materials and methods

1.1. General experimental methods for chemical synthesis

All reagents and solvents (regular and anhydrous) were of analytical grade and obtained from

commercial sources. All reactions using anhydrous solvents were carried out under a nitrogen

atmosphere using oven-dried glassware Thin-layer chromatography was performed using

commercially prepared silica gel plates with a fluorescence indicator, and visualization was

achieved with UV light (254 and 365 nm). Preparative chromatographic separations were

performed on silica gel (0.040–0.063 mm). All NMR spectral assignments were determined by

1H (400 MHz), 13C (100 MHz) in CDCl3. Peaks were referenced to residual chloroform signals

(H 7.26 ppm, or C 77.0 ppm). High resolution mass spectra were recorded on an ESI-TOF

instrument. SCH442416 (Tocris Bioscience, Ellisville, MO) and 7-diethylamino-4-

hydroxymethylcoumarin (Indofine Chemical Co., Hillsborough, NJ) were obtained from the

commercial sources specified in the parenthesis. 2-[4-(2-

Aminoethylaminocarbonylethyl)phenylethylamino]-5’-N ethylcarboxamidoadenosine (APEC)

bistrifluoroacetic acid was provided by the NIMH Chemical Synthesis and Drug Supply Program

(https://nimh-repository.rti.org). Alexa Fluor-647 O-succinimidyl ester tris(triethylammonium)

salt was purchased from ThermoFisher Scientific (Waltham, MA).

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Taura et al. 2018 Photocontrol of movement disorders

1.2. Synthesis of (7-(diethylamino)-2-oxo-2H-chromen-4-yl)methyl (4-nitrophenyl) carbonate (2)

The procedure was modified from Liu et al1. To an ice water-cooled solution of coumarin (23

mg, 0.091 mmol) in DCM (1 mL) was added N,N-diisopropylethylamine (1, 80 L, 0.46 mmol,

5 equivalents) and 4-nitrophenyl chloroformate (37 mg, 0.18 mmol, 2 equivalents)

(Supplementary Fig. 1). The ice-bath was removed, and the reaction was stirred overnight at rt.

After 18 h, the solution was concentrated and taken up in EtOAc. This solution was washed with

saturated aq. NaHCO3 (3 ×), brine, and then dried (Na2SO4). Following solvent evaporation, an

ether-hexanes (1:1) solution was added to dissolve impurities leaving behind a yellow precipitate

that was filtered and dried to give 27 mg (73%) of crude product 2 (Supplementary Fig. 1),

which was used directly. 1H NMR (CDCl3) 8.34 (d, J = 9.2 Hz, 2H phenyl), 7.46 (d, J = 9.2 Hz,

2H phenyl), 7.35 (d, J = 9.0 Hz, 1H coumarin), 6.64 (dd, J = 9.0, 2.6 Hz, 1H coumarin), 6.57 (d,

J = 2.6 Hz, 1H coumarin), .6.26 (s, 1H coumarin), 3.46 (q, J = 7.1 Hz, 4H), 1.25 (t, J = 7.1 Hz,

6H). ESI MS (m/z): [M + H]+ calcd for C21H21N2O7, 413.1; found 413.1.

1. Liu, Z.; Liu, T.; Lin, Q.; Bao, C.; Zhu, L. Photoreleasable thiol chemistry for facile and

efficient bioconjugation. Chem. Commun. 2014, 50, 1256‒1258 and associated Supporting

Information.

1.3. Synthesis of (7-(diethylamino)-2-oxo-2H-chromen-4-yl)methyl (2-(furan-2-yl)-7-(3-(4-

methoxyphenyl)-propyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl)carbamate (4,

MRS7145)

To a solution of SCH442416 (3, 6.0 mg, 15 mmol) and 18-crown-6 (4.4 mg, 20 mmol) at rt in

0.5 mL DMF was added 60% NaH (3.5 mg, 87 mmol, 5.7 equivalents) (Supplementary Fig. 1).

After 15 min a solution of the coumarin nitrophenyl carbonate (2, 8.2 mg, 20 mmol, 1.3

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Taura et al. 2018 Photocontrol of movement disorders

equivalents) in 0.5 mL DMF was added. The reaction mixture changed from yellow to greenish.

After 1h, the DMF was co-evaporated onto SiO2 with toluene (3 ×) and chromatographed with

20 to 100% EtOAc/hexanes to afford 6.4 mg (63%) of 4 (Supplementary Fig. 1) as an off-white

crystalline solid.

1H NMR (CDCl3) 8.89 (s, 1H), 8.35 (s, 1H), 7.70 (dd, J = 1.7, 0.7 Hz, 1H furan), 7.39 (d, J =

9.0 Hz, 1H coumarin), 7.35 (dd, J = 3.5, 0.7 Hz, 1H furan), 7.12 (d, J = 8.6 Hz, 1H), 6.79 (d, J =

8.6 Hz, 1H), 6.67 (dd, J = 3.5, 1.7 Hz, 1H furan), 6.65 (dd, J = 9.0, 2.6 Hz, 1H coumarin), 6.59

(d, J = 2.5 Hz, 1H coumarin), .6.41 (s, 1H coumarin), 5.52 (d, J = 1.0 Hz, 2H), 4.58 (t, J = 6.8

Hz, 2H), 3.75 (s, 3H), 3.46 (q, J = 7.1 Hz, 4H), 2.63 (t, J = 7.5 Hz, 2H), 2.35 (quintet, J = 7.5 Hz,

2H), 1.26 (t, J = 7.1 Hz, 6H). 13C NMR (CDCl3) 161.6, 157.8, 156.7, 156.4, 150.8, 149.4,

148.9, 148.2, 146.4, 145.1, 145.0, 137.5, 133.0, 132.0, 129.4, 124.4, 113.7, 113.3, 112.2, 108.8,

107.0, 105.7, 99.1, 97.9, 63.7, 55.2, 47.6, 44.8, 31.9, 31.1, 12.4. HRMS (ESI-TOF) m/z: [M + H]

+ calcd for C35H35N8O6 663.2679; found 663.2680 (Supplementary Fig. 2).

Supplementary Fig. 1. Synthesis of (7-(diethylamino)-2-oxo-2H-chromen-4-yl)methyl (4-nitrophenyl) carbonate (2) and (7-(diethylamino)-2-oxo-2H-chromen-4-yl)methyl (2-(furan-2-

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Taura et al. 2018 Photocontrol of movement disorders

yl)-7-(3-(4-methoxyphenyl)-propyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl)carbamate (4, MRS7145).

Supplementary Fig. 2. 1H NMR (400 MHz) spectra of: 4, 1.3 mM in DMSO-d6

1.4. Synthesis of Alexa-647 conjugate of the A2AR agonist 2-[[2-[4-[2-(2-aminoethyl)-

aminocarbonyl]ethyl]phenyl]ethylamino]-5-N-ethyl-carboxamidoadenosine (7, MRS5842)

A solution of Alexa Fluor-647 O-succinimidyl ester tris(triethylammonium) salt (6, 1 mg, 0.904

mmol) in N,N-dimethylformamide DMF (200 µL) was added to a solution of APEC

bis(trifluoroacetate) (5, 0.98 mg, 1.27 mmol) in DMF (100µL) and triethylamine (0.71 µL, 5.0

mmol). The resulting solution was stirred for 48 h at room temperature under a nitrogen

atmosphere. The reaction was monitored by analytical HPLC. The homogeneous product was

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Taura et al. 2018 Photocontrol of movement disorders

purified by Agilent HPLC with a Luna 5µ RP-C18 semipreparative column (250 x 10.0 mm;

Phenomenex, Torrance, CA) under the following condition: flow rate 1ml/min; H2O-acetonitrile

from 96:4 (v/v) to 0:100 (v/v) in 28 min. The compound eluted at a retention time of 21.28 min.

After lyophilization, 0.804 mg (5.82 µmol) of chromatographically homogeneous product 7 was

recovered for a 64% yield (Supplementary Fig. 3). The analytical purity was >99%, as tested

using a Hewlett-Packard HPLC equipped with a ZORBAX SB-Aq Analytical 4.6 x 50 mm

(retention time 7.94 min) under the following condition: flow rate 0.5ml/min: 5 mM TBAP

(tetrabutylammonium dihydrogenphosphate) in H2O solution in a mixture with acetonitrile from

90:10 (v/v) to 0:100 (v/v) in 14 min. Peaks were detected by UV absorption with a diode array

detector at 254, 326, and 647 nm. ESI-HMRS calculated for C 61H76N11O18S43- (M+H)+1379.58;

found

1380.448.

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Taura et al. 2018 Photocontrol of movement disorders

Supplementary Fig. 3. Synthesis of MRS5842 (7), an Alexa-647 conjugate of the A2AR agonist 2-[[2-[4-[2-(2-aminoethyl)-aminocarbonyl]ethyl]phenyl]ethylamino]-5¢-N-ethyl-carboxamidoadenosine (APEC647).

2. Supplementary Results

2.1. Photochemical evaluation of reference compounds

No clear spectral changes were observed when irradiating the reference compounds (i.e.

SCH442416 and 7-diethylamino-4-hydroxymethylcoumarin) at the same conditions used to

induce MRS7145 photo-uncaging (405 nm, 3 mW, 37 ºC, 30 min, Supplementary Fig. 3).

Therefore, the spectral changes observed for MRS7145 cannot be attributed to photodegradation

of its building blocks.

Supplementary Fig. 4. Time-course of spectral changes registered for SCH442416 (A) and 7-diethylamino-4-hydroxymethylcoumarin (B) in PBS:DMSO 1:1 upon irradiation at 405 nm and 37 ºC during 30 min.

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2.3. Flow cytometry measurements of MRS4842 (APEC647) binding to A2AR

Supplementary Fig. 5. Binding of MRS4842 (APEC647) to A2AR expressed in HEK-293T cells as determined by flow cytometry. HEK-293 cells permenantly expressing A2ARCFP were incubated with increasing concentrations of MRS5842 and analyzed by flow cytometry. In the inset panel a representative histogram of MRS5842 (100 nM) total binding (black line) and nonspecific binding (red line, in the presence of 1 M of ZM241385) is shown. The specific fluorescence detection (FL-1) for each MRS5842 concentration tested was determined by M1 cursor (see inset). Thus, specific events within the M1 region were plotted against MRS5842 concentration.

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0 50 100 150 200 250 3000

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[MRS5842] (nM)

M1

Eve

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Taura et al. 2018 Photocontrol of movement disorders

2.4. Effect of SCH442416 and 7-diethylamino-4-hydroxymethylcoumarin on spontaneous

locomotor activity in mice

Supplementary Fig. 6. Dose-response effect of SCH442416 and coumarin on spontaneous locomotor activity. CD-1 mice were administered with vehicle (Veh, saline), SCH442416 (SCH, 0.3-3 mg/kg) or 7-diethylamino-4-hydroxymethylcoumarin (Coumarin, 0.6-6 mg/kg) 20 min before the horizontal locomotor activity was measured as the total distance travelled for 30 min in an open field arena. Data are represented as mean ± SEM of 5-10 animals per group. ** P < 0.01, ***P < 0.001, one-way ANOVA with a Tukey’s multiple comparison test using the Veh condition as a control.

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2.5. Cartoon showing the 6-OHDA lesion and fiberoptic placement within the mouse brain

Supplementary Fig. 7. Scheme of brain coordinates corresponding to the 6-OHDA administration at the medial forebrain bundle (MFB) (A and C) and the mono ferrule cannule (MFC)-based optic fiber implant at the caudate putamen (CPu or striatum) (B and C). Coronal (A and B) and horizontal (C) diagrams are represented following the Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates atlas.

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