Fluorinated CRA13 analogues: Synthesis, in vitro evaluation, radiosynthesis,

in silico and in vivo PET study

Ahmed H.E. Hassana,b,1, Kyung Tae Parkc,1, Hye Jin Kimc, Hyo Jong Leec, Yeong Ho

Kwonc, Ji Young Hwangd, Choon-Gon Jangd, Jin Hwa Chunge, Ki Duk Parkf, Sang Joo

Leee, Seung Jun Ohe*, Yong Sup Leea,c,f*

a Medicinal Chemistry Laboratory, Department of Pharmacy, College of Pharmacy, Kyung Hee

University, Seoul 02447, Republic of Korea.

b Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516,

Egypt.

c Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 02447, Republic of

Korea.

d College of Pharmacy, Sungkyunkwan University, Gyeonggi-do 16419, Republic of Korea

e PETcore, Bio-Imaging Center, Asan Institute for Life Science, Asan Medical Center, Seoul 05505,

Republic of Korea

f KHU-KIST Department of Converging Science and Technology, Kyung Hee University, Seoul 02447,

Republic of Korea.

*Corresponding authors: Yong Sup Lee (e-mail: kyslee@khu.ac.kr) and Seung Jun Oh (e-mail:

sjoh@amc.seoul.kr)

1Both of Ahmed H.E. Hassan and Kyung Tae Park contributed equally.

Biological evaluations Protocols

1. Binding to CB1R and CB2R assay

1.1. Materials and conditions

HEPES, MgCl2, CaCl2, NaCl, DMSO, and Polyethyleneimine were purchased from Sigma

Aldrich, USA. CP-55940 was purchased from the Pharmacology Department of the Korean Food

and Drug Administration. BSA was purchased from GenDEPOT, USA. Cell membranes (hCB1,

human cannabinoid receptor 1, hCB2, human cannabinoid receptor 2), radioligands [3H]SR-

141716A , [3H]CP-55940, liquid scintillation cocktail and Ultima GoldTM Cocktail were

purchased from PerkinElmer, USA. FC96 well harvest plates, 96 well disposable punch tips,

Multiscreen Multiple Punch, and MultiScreen Vacuum Manifold were purchased from Millipore,

USA. The beta-scintillation counter used was a PerkinElmer Tri-Carb 2900TR.

The binding buffer was prepared from distilled water by dissolving the following materials to

provide the specified concentrations: 50 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.2%

BSA. The solution was filtered and stored at 4°C. The wash buffer was prepared from distilled

water by dissolving the following materials to provide the specified concentrations: 50 mM

HEPES, pH 7.4, 500 mM NaCl, 0.1% BSA. The solution was filtered and stored at 4°C. The cell

membranes were diluted ten times with binding buffer. The radiolabelled ligands were diluted

with binding buffer to final concentrations of 0, 1, 2.5, 5, 7.5, 10, 20, 40 nM. CP-55940 was

dissolved in DMSO to a final concentrations of 7 µM to 140 µM.

1.2. Saturation Binding Assay

Saturation binding assay for the used cell membrane bound CB1R was determined using the

following protocol. Aliquots of 10 μl of diluted cell membrane bound CB1R, 10 μl of each

diluted concentration of [3H]SR-141716A, 5 μl of DMSO or CP-55940 solution in DMSO, 10 μl

of DMSO, and 65 μl of binding buffer were mixed together and incubated at 30 ° C for 1 hour.

The FC96-well harvest plate was prepared by adding 200 μl aliquots of 0.33%

polyethyleneimine to each well of the plate and incubation for 30 minutes. After coating, the

cells were sucked by the MultiScreen Vacuum Manifold and 200 μl of the wash buffer was

added to each well to rinse the remaining polyethyleneimine. The incubated binding solutions

were transferred to the FC96-well harvest plate and then aspirated into MultiScreen Vacuum

Manifold. Aliquots of 200 μl of wash buffer were added to each well and washing was

performed 7 times. The plate was dried for 1 h then placed on sliding punch carrier plate and

punched by 96-well disposable punch tip into corresponding vial for each well. To each vial, 1.5

ml of distilled water was added and it was shaken for 30 minutes. To each vial, 10 ml of liquid

scintillation cocktail was added and it was shaken for further 30 minutes. Using the beta-

scintillation counter cpm was determined for each concentration and cpm of specific binding was

calculated by subtracting cpm of non-specific binding (CP-55940 treated concentrations) from

cpm of total binding (non-CP-55940 treated concentrations). The computer program Graphpad

Prism 6 was used to calculate Bmax (maximum binding) and Kd values (concentration of the

radiolabelled ligand attached to 50% of the receptor at equilibrium)..

Similarly, saturation binding assay for the used cell membrane bound CB2R was determined

according to the same protocol for human CB1R saturation binding assay by replacing cell

membrane bound CB1R and radioactive ligand [3H]SR-141716A by cell membrane bound CB2R

and radioactive ligand [3H]CP-55940. Similarly Bmax and Kd were calculated using the computer

program Graphpad Prism 6.

1.3. Human cannabinoid receptors competitive binding assay

The competitive binding assay for the used cell membrane bound CB1R was determined using

the following protocol. The radiolabelled ligand [3H]SR-141716A was diluted with binding

buffer to a final concentration equal to the Kd value obtained from the saturation curve. Solutions

of the synthesized compounds in DMSO were prepared so that the final concentration was the

concentration to be tested. Aliquots of 10 μl of binding buffer-diluted cell membrane bound

CB1R, 10 μl of diluted radiolabelled ligand [3H]SR-141716A, 5 μl of DMSO or CP-55940

dissolved in DMSO, 10 μl of DMSO solution of each concentration of the compounds to be

tested, and 65 μl of binding buffer were incubated at 30°C for 1 hour. The FC96-well harvest

plate was prepared and washed as described under human CB1R saturation binding assay. The

incubated binding solutions were transferred to the FC96-well harvest plate and treated as

described under human CB1R saturation binding assay. Similarly, the remainder steps,

measurements of cpm and calculations of specific binding were performed according to the same

protocol under human CB1R saturation binding assay. The computer program Graphpad Prism 6

was used to draw the competitive binding curves and calculate IC50 of potent compounds. For

CB2R competitive binding assay, the same protocol was performed replacing cell membrane

bound CB1R and radioactive ligand [3H]SR-141716A by cell membrane bound CB2R and

radioactive ligand [3H]CP-55940. The computer program Graphpad Prism 6 was used to draw

the competitive binding curves and calculate IC50 of potent compounds.

2. Measurement of physicochemical properties

For determination of physicochemical properties of compound 7c (pKa and Log D), pH-metric

approach was employed using Sirius T3 instrument (Sirius-Analytical) according to

manufacturer’s recommendations. Briefly, 10 mM DMSO stock solution was prepared (100 ~

150 µL) and transferred to the main T3 instrument autotray. For determination of ionization and

pKa, potentiometric acid-base titrations were conducted to generate 45 points (concentration of

2808.5-2991.7 µM in saturated KCl solution using MDM as cosolvent and 0.5 M HCl and 0.5 M

KOH as titrants; pH range = 1–12). The pH of each point was calculated using equations that

contain pKa, and the calculated points were fitted to the measured curve by manipulating the

pKa value (36 points out of 45 were considered). The pKa that provided the best fit was taken to

be the measured pKa. For determination of lipophilicity profile, the titrations were repeated

again in presence of a two-phase water-octanol system. Each point pH was calculated using

equations that contain pKa and P, and the calculated points are fitted to the measured curve by

manipulating the P value. The P that provides the best fit was taken to be the measured P value,

(reported as log P).

3. In silico docking study

The co-crystal structure of CB1R with the potent cannabinoid agonist AM11542 (PDB code:

3XRA) and the ligands were prepared employing the “prepare protein” and “prepare ligands”

protocols implemented in Discovery studio 4.0, then the prepared ligands were docked to the

prepared CB1R using CDocker docking algorithm. The predicted poses were retrieved, visualized

and analyzed.

0.9991.0180.9821.0251.0261.0846.2651.066 1.0441.0412.138 1.0721.081PPM9.08.07.06.05.04.03.02.01.00.0

8.97648.95538.38098.36028.26288.24288.00927.98867.94277.93887.91967.70017.69677.68307.67937.67537.66147.65807.60337.59907.59617.58877.58237.57877.56797.56517.55857.55527.54137.53817.52207.51727.50437.49697.48367.47887.47687.45856.70536.6847 4.83304.81864.80444.71564.70124.68704.33504.31994.30492.40472.39002.37532.36062.34592.33972.32492.31022.29562.2809

1H NMR of Compound 7a

PPM20018016014012010080 60 40 20 0

199.0938158.5596138.8523135.0002134.0120133.0725131.5029129.0608128.9826128.6893128.6232127.5467126.6234126.3722126.2982126.1450126.0430124.7144122.4185103.121481.512480.197364.380164.340030.617030.4570

13C NMR of Compound 7a

HRMS of Compound 7a

0.9991.0070.9841.0221.0151.0676.2831.059 1.0321.0302.121 4.312PPM9.08.07.06.05.04.03.02.01.00.0

8.98768.96648.39408.37428.37258.25668.23658.23468.00867.98827.94337.93977.92057.70027.69697.68307.67947.67577.66197.65847.60197.59647.58847.58147.56777.56477.55847.55527.54117.53847.52007.51837.51577.49867.48087.47867.46056.68016.6596 4.66004.64564.63204.54254.52784.51344.24514.23054.2155 2.13372.11802.10962.10282.10092.09592.08272.06942.05812.04732.04312.03342.02552.01922.00931.99511.98841.98151.97371.96821.9591

1H NMR of Compound 7b

PPM18016014012010080 60 40 20 0

199.0885158.8146138.9292135.1636134.0082133.0888131.5047131.4352129.0491128.7417128.6137128.6049127.5152126.6094126.3572126.2609126.1575126.0972124.7155122.4999103.029884.557083.243968.0985 27.688927.529625.480725.4421

13C NMR of Compound 7b

HRMS of Compound 7b

0.9991.0140.9831.0231.0271.0686.2461.060 1.0131.0132.126 2.1514.342PPM9.08.07.06.05.04.03.02.01.00.0

9.02459.00348.43088.41108.40898.27968.27728.25928.02988.00937.96647.96267.94347.72537.72187.70827.70457.70067.68687.68327.63227.62917.62157.61527.61167.60767.60527.60157.59437.59127.58107.57757.56387.56067.54437.54137.53737.52097.51787.50297.50057.48276.68736.6667 4.61244.59764.58284.49384.47954.46474.22654.21104.1954 2.06442.04822.03032.01111.99551.92491.90791.89261.88621.87751.87161.86331.85721.84841.84401.82771.82411.80991.79591.77971.76561.75811.74981.74021.72761.71831.70431.7007

1H NMR of Compound 7c

PPM18016014012010080 60 40 20 0

199.0889158.9712138.9797135.2522134.0044133.0959131.5090131.3925129.0365128.6050128.5615127.4969126.5982126.3382126.2259126.1706126.1363124.7186122.5635102.990384.799183.488868.445330.460630.304328.980622.433622.3919

13C NMR of Compound 7c

HRMS of Compound 7c

0.9991.0260.9971.0461.0331.0826.2331.069 1.0311.0182.144 2.1462.1715.923PPM9.08.07.06.05.04.03.02.01.00.0

9.02569.00438.43088.41178.40988.27538.27358.25468.03048.00997.96637.96297.94367.72437.72107.70717.70357.69987.68607.68257.63077.62407.61377.61017.60377.59277.58997.58097.57757.56377.56067.54427.54087.53707.52357.51687.50327.48546.69106.6704 4.57724.56214.54714.45904.44384.42884.21894.20324.1876 2.03172.01541.99871.97881.96301.85721.84161.82381.82011.80481.79331.79001.77771.76041.75651.74131.72631.69571.67871.67311.65751.64921.63901.62151.60201.59101.56711.54961.54461.53181.5267

1H NMR of Compound 7d

PPM180160140120100 80 60 40 20 0

199.0912159.0589139.0056135.3243134.0039133.1030131.5123131.3714129.0274128.6025128.5373128.5241127.4867126.5938126.3376126.1954126.1751126.1576124.7203122.5837102.995184.912583.604968.5568 30.680730.525229.245226.164525.329125.2875

13C NMR of Compound 7d

HRMS of Compound 7d

0.9991.0150.9861.0411.0531.1016.240 2.1682.2322.7941.046 2.1222.0923.041PPM9.08.07.06.05.04.03.02.01.00.0

8.98658.96538.38898.36828.25438.23398.00567.98537.93767.91867.69777.69527.68027.67747.65967.65677.60497.59397.58697.57447.55497.53747.53577.51587.49507.47617.45706.65946.6389 4.30734.29154.27564.19964.18444.16943.00422.04612.03532.01842.00031.98141.96601.93121.91451.89671.87721.86111.75861.73801.72891.72021.70701.69981.6830

1H NMR of Compound 9

PPM180160140120100 80 60 40 20 0

198.8544158.5679138.6324134.9524133.7312132.8073131.2223131.2060128.8144128.4319128.3792128.3681127.2716126.3611126.0847126.0166125.8808125.8085124.4659122.2432102.741669.673867.965537.399828.898128.506022.3721

13C NMR of Compound 9

HRMS of Compound 9