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Synthesis of carbon-11-labeled aminoalkylindole derivatives as new candidates of cannabinoid receptor radioligands for PET imaging of alcohol abuse
Bioorganic & Medicinal Chemistry Letters 24 (2014) 5581–5586
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Synthesis of carbon-11-labeled aminoalkylindole derivatives as new candidates of cannabinoid receptor radioligands for PET imaging of alcohol abuse Mingzhang Gao, Andy Chufan Gao, Min Wang, Qi-Huang Zheng ⇑ Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, Room 202, Indianapolis, IN 46202, USA
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Article history: Received 14 October 2014 Revised 28 October 2014 Accepted 30 October 2014 Available online 6 November 2014 Keywords: Carbon-11-labeled aminoalkylindole derivatives Radiosynthesis Positron emission tomography (PET) Cannabinoid receptor Alcohol abuse
a b s t r a c t Carbon-11-labeled aminoalkylindole derivatives (1-butyl-7-[11C]methoxy-1H-indol-3-yl)(naphthalene1-yl)methanone ([11C]3), 1-butyl-7-[11C]methoxy-3-(naphthalene-1-ylmethyl)-1H-indole ([11C]5), and 1-butyl-7-[11C]methoxy-3-(naphthalene-2-yl)-1H-indole ([11C]8) were prepared by O-[11C]methylation of their corresponding precursors with [11C]CH3OTf under basic condition (2 N NaOH) and isolated by a simplified solid-phase extraction (SPE) method in 50–60% radiochemical yields based on [11C]CO2 and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 23 min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 185–555 GBq/lmol. Ó 2014 Elsevier Ltd. All rights reserved.
Alcohol abuse is a major substance abuse, approximately 2.5 million people die from alcohol use every year, and its annual health- and crime-related costs are estimated around $235 billion.1 There are several therapeutic options available for the treatment of alcohol abuse, but all existing therapies have only modest efficacy.2 The cause of alcohol abuse is still unknown. However, alcohol abuse results in chemical and biological changes in the brain, and it is often associated with substantial psychiatric comorbidity, brain damage and disease.3 Brain imaging techniques such as positron emission tomography (PET) enable scientists to better understand alcohol abuse and alcohol-related diseases, for example, with PET, researchers can compare groups of alcohol-abusing and nonabusing individuals by quantifying differences in their levels of a particular neurotransmitter molecule like dopamine or neurotransmission component such as a receptor or a transporter.4 We are interested in PET imaging of alcohol abuse, and we have extensively used [11C]raclopride-PET and [18F]fallypride-PET to study D2 receptor in alcohol abuse, since alcohol abuse significantly affects the neurotransmitter dopamine and alters its neurotransmission in the central nervous system (CNS).5–10 More and more evidences suggest that endocannabinoid (eCB) system is another neurotransmitter involved in alcohol abuse, and cannabinoid (CB) receptor (CBR) antagonist/agonist might ⇑ Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278 9711. E-mail address: [email protected] (Q.-H. Zheng). http://dx.doi.org/10.1016/j.bmcl.2014.10.097 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
offer a new therapeutic direction for treatment of alcohol abuse.11,12 Recently a new class of aminoalkylindole derivatives has been developed as CBR ligands with dual CB1R antagonist/ CB2R agonist activity with potential for treatment of alcohol abuse.1 CBR has become an interesting target for PET imaging of alcohol abuse.13 In our previous works, we have developed a series of selective CB1R radioligands and CB2R radioligands, as indicated in Figure 1.14–16 In this ongoing study, we report the design and synthesis of carbon-11-labeled aminoalkylindole derivatives as new candidate dual CB1R/CB2R radioligands for PET imaging of alcohol abuse. Aminoalkylindole derivatives (1-butyl-7-methoxy-1H-indol-3-yl) (naphthalene-1-yl)methanone (3), 1-butyl-7-methoxy-3-(naphthalene-1-ylmethyl)-1H-indole (5), and 1-butyl-7-methoxy-3-(naphthalene-2-yl)-1H-indole (8) are dual CB1R/CB2R ligands with high affinity, and the low nanomolar Ki values for CB1R and CB2R are 1.7 and 0.81 nM, 15.4 and 10.9 nM, and 37.3 and 26.5 nM, respectively.1 According to their in vitro Ki values, compound 3 is the most potent ligand for CB1R and CB2R in comparison with compounds 5 and 8. However, the overall biological evaluation including both in vitro and in vivo studies suggested compounds 5 and 8 appeared to have the most promise to be CBR ligands for use as treatment of alcohol abuse.1 These three representative compounds are selected as the reference standards for radiolabeling. As outlined in Scheme 1, commercially available starting material 7-methoxyindole (1) was subjected to mild alkylating
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N
O NC N
N
H3 11CO
NC N
H3 11CO
Cl
Cl
N
O NC
N H
N
N
H3 11CO
N
Cl
N
O
N H Cl
[11C]JHU75528 ([11C]OMAR)
NC
N
O
N H
N
H3 11CO
Br
N
N H Br
[ 11C]JHU75575 CB1R Radioligands X
O
H 311 CO
N H N H OC 5 H 11
n
N H
O
H 3 CO
n=1, X=H n=1, X=Cl n=2, X=H
N H N OC 5 H 11
N H OC 5 H 11
X
O
H 311 CO
O 11CH3
O
n=1 O 11CH3
O N H
n H 3 CO
Cl
n
O
N OC 5 H 11
n=1, X=H n=1, X=Cl n=2, X=H
n
Cl n=1
H 311 CO O S O N
N CB2R Radioligands
Figure 1. CB1R and CB2R radioligands.14–16
O
O i N H
O 1
N
ii
iii
O 2
N
N O
3
OH
4
Scheme 1. Synthesis of aminoalkylindole derivative reference standard 3 and its precursor 4. Reagents, conditions and yields: (i) KOH, 1-bromobutane, DMF, 50 °C, 88%; (ii) Me2AlCl, 1-naphthoyl chloride, CH2Cl2, 0 °C, 52%; (iii) 48% HBr, HOAc, reflux, 70%.
conditions to give 1-butyl-7-methoxy-1H-indole (2) in 88% yield. Intermediate 2 was then subjected to Friedel–Crafts like acylation conditions, using dimethylaluminum chloride and acid chloride at 0 °C to afford the standard 3 in 52% yield. The desmethylation of compound 3 with 48% HBr in the solution of HOAc provided its desmethylated precursor (1-butyl-7-hydroxy1H-indol-3-yl)(naphthalene-1-yl)methanone (4) in 70% yield. Using protic acid (HBr) instead of the literature method Lewis acid (BBr3)1 as the desmethylating reagent, it significantly improved the yield of desmethylation reaction, because protic acid promoted the desmethylation reaction of phenolic methoxy aminoalkylindole
derivatives to easily form phenolic hydroxyl precursor and CH3Br.17,18 As shown in Scheme 2, the reduction of compound 3 with LiAlH4 gave the standard 5 in 49% yield. Careful workup by adjusting appropriate pH to 5 improved the yield of the reduction from 18% to 49%. Likewise, using 48% HBr in HOAc as the desmethylating reagent, compound 5 was desmethylated to produce its desmethylated precursor 1-butyl-7-hydroxy-3-(naphthalene-1ylmethyl)-1H-indole (6) in 43% yield. Using Lewis acid (BBr3)1 as the desmethylating reagent, compound 5 failed to give 6 in desmethylation reaction.
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O ii
i N O
N O
3
N OH
5
6
Scheme 2. Synthesis of aminoalkylindole derivative reference standard 5 and its precursor 6. Reagents, conditions and yields: (i) LiAlH4, AlCl3, THF, 0 °C, 49%; (ii) 48% HBr, HOAc, reflux, 43%.
I i N H
O 1
N
ii
iii
O 7
N
N O
OH
8
9
Scheme 3. Synthesis of aminoalkylindole derivative reference standard 8 and its precursor 9. Reagents, conditions and yields: (i) 1-bromobutane, I2, KOH, DMF, NaH, rt, 75%; (ii) 2-naphthaleneboronic acid, Pd(OAc)2, Cy-JohnPhos, K2CO3, toluene, EtOH, H2O, reflux, 51%; (iii) 48% HBr, HOAc, reflux, 52%.
As indicated in Scheme 3, a one-pot two-step (N-alkylation and 3-indole iodination) reaction of compound 1 afforded an intermediate 1-butyl-3-iodo-7-methoxy-1H-indole (7) in 75% yield. Intermediate 7 was then subjected to Suzuki coupling conditions utilizing boronic acid to provide the standard 8 in 51% yield, which was undergone the same desmethylating conditions (48% HBr in HOAc) to give its desmethylated precursor 1-butyl-7-hydroxy-3(naphthalene-2-yl)-1H-indole (9) in 52% yield. Synthesis of carbon-11-labeled aminoalkylindole derivatives (1-butyl-7-[11C]methoxy-1H-indol-3-yl)(naphthalene-1-yl)metha none ([11C]3), 1-butyl-7-[11C]methoxy-3-(naphthalene-1-ylmethyl)1H-indole ([11C]5), and 1-butyl-7-[11C]methoxy-3-(naphthalene-2yl)-1H-indole ([11C]8), is indicated in Scheme 4. The desmethylated precursors 4, 6, and 9 were labeled by a reactive [11C]methylating agent, [11C]methyl triflate ([11C]CH3OTf)19,20 prepared from [11C]CO2, under basic conditions (2 N NaOH) in acetonitrile through the O-[11C]methylation and isolated by a simplified solid-phase extraction (SPE) method21–25 to provide target tracers [11C]3, [11C]5, and [11C]9 in 50–60% radiochemical yields, decay corrected to end of bombardment (EOB), based on [11C]CO2. [11C]CH3OTf is a proven methylation reagent with greater reactivity than commonly used [11C]methyl iodide ([11C]CH3I),26 and thus, the radiochemical yield of [11C]3, [11C]5, and [11C]8 was relatively high. The large polarity difference between the sodium salt of the phenolic hydroxyl precursor and the corresponding labeled O-methylated ether product permitted the use of SPE technique for purification of the labeled product from the radiolabeling reaction mixture. A C-18 Plus Sep-Pak cartridge was used in SPE purification technique. The crude reaction mixture was treated with aqueous NaHCO3 and loaded onto the cartridge by gas pressure. The pH of freshly prepared 0.1 M NaHCO3 might be too low to effectively deprotonate a phenolic hydroxyl precursor. However, an excess of 2 N NaOH used in the reaction provided a final pH after addition of the 0.1 M NaHCO3 high enough to deprotonate all the phenolic hydroxyl precursor. Any non-reacted precursor was actually converted to the corresponding sodium salt, and any non-reacted [11C]CH3OTf was actually hydrolyzed to [11C] CH3OH, which would not be trapped to the C-18 Sep-Pak. The cartridge was washed with water to remove radiolabeled by-product [11C]CH3OH, sodium salt of phenolic hydroxyl precursor and reaction solvent, and total 6 mL (2 3 mL) volume of water was enough to wash off all impurity. The final labeled product was
O
O i
N
N
OH
O 11CH3 [ 11C]3
4
i N
N
OH
O11 CH 3 [11C]5
6
i N
N
OH
O 11CH3 9
[11C]8
Scheme 4. Synthesis of carbon-11-labeled aminoalkylindole derivative target tracers [11C]3, [11C]5, and [11C]8. Reagents, conditions and yields: (i) [11C]CH3OTf, CH3CN, 2 N NaOH, 80 °C, 3 min, 50–60%.
eluted with ethanol (2 2 mL), concentrated by rotary evaporation and reformulated in saline (10 mL). In our fully automated radiosynthesis module,27–29 it is difficult to directly elute the labeled product from a C-18 Sep-Pak to a vial using either 1 1 mL ethanol or 2 0.5 mL ethanol, due to the back pressure in the C-18 Sep-Pak and dead volume in the transfer tubing. The high back pressure is resulted from the eluent (water, ethanol and saline) change. In order to elute most of the labeled product from the C-18 Sep-Pak, we need to increase the volume of the eluent ethanol. For the radiotracer produced for animal study, we used 2 2 mL ethanol for elution, and rotary evaporation was required before
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reformulation. For the radiotracer produced for human study, we used 2 1 mL ethanol, no evaporation required, and a C-18 Sep-Pak was used for direct reformulation.9,30–32 We have tried to use a C-18 Light Sep-Pak cartridge instead of a C-18 Plus SepPak cartridge to allow smaller volume (1 mL) of ethanol and to avoid laborious rotary evaporation before formulation. However, there is more serious back pressure in the Light Sep-Pak than in the Plus Sep-Pak, in addition, dead volume in the transfer tubing also affects the elution, and thus it is more difficult to efficiently elute the labeled product from a Light Sep-Pak, which resulted in the low radiochemical yield. The reason is that our home-built fully automated module used many PTFE (polytetrafluoroethylene)/silicone liners (septa) and Teflon tubing, and these materials cannot afford too high pressure gas (N2) push. The pressure of N2 gas introduced in our module is set at 207 kPa (30 Psi). C-18 Light Sep-Pak cartridge works well in manual or semi-automated radiosynthesis in our lab, because we can easily introduce high pressure gas push during the purification and reformulation process. Overall synthesis time was 23 min from EOB, including approximately 11 min for [11C]CH3OTf production, 5 min for O-[11C]methylation reaction, and 7 min for SPE purification, evaporation and reformulation. SPE technique is fast, efficient and convenient and works very well for the O-methylated ether tracer purification using the phenolic hydroxyl precursor for radiolabeling.22,24 The radiosynthesis was performed in an automated selfdesigned multi-purpose 11C-radiosynthesis module, allowing measurement of specific activity at EOB during synthesis.27–29 On line determination of specific activity at EOB is accurate when reverse-phase (RP) high performance liquid chromatography (HPLC) is used as purification method. However, the on-the-fly technique to determine specific activity at EOB is not applied when SPE is used as purification method. The specific activity for the 11 C-tracers produced in our PET chemistry facility usually ranges from 370 to 1110 GBq/mol at EOB according to our previous works. The specific activity of carbon-11-labeled aminoalkylindole derivatives was estimated in a range of 185–555 GBq/mol at the end of synthesis (EOS) based on other compounds produced in our facility using the same targetry conditions which have been measured by the on-the-fly technique or the same SPE purification method.33 The actual measurement of specific activity at EOS was performed by analytical HPLC34,35 and calculated. The exact values of the specific activity for the tracers [11C]3, [11C]5, and [11C]8 were 185–555 GBq/mol at EOS, which are in agreement with the estimated values and the ‘on line’ determined values. Specific activity is defined as the radioactivity per unit mass of a radionuclide or a labeled compound. Specific activity (MBq/mg) = 3.13 109/A t1/2, where A is the mass number of the radionuclide, and t1/2 is the half-life in hours of the radionuclide. For carbon-11, carrierfree 11C, maximum (theoretical) 11C specific activity = 340,918 GBq/lmol.36 Actual specific activity of the 11C-tracers in the PET chemistry facility are depended on two parts: (1) carrier from the 11 C-target, and (2) carrier from the 11C-radiosynthesis unit.37 Furthermore, actual specific activity for the 11C-tracers synthesized by 11C-methylation with [11C]CH3OTf in our PET chemistry facility is depended on two parts: (1) carrier from the cyclotron consisted of the 11C gas irradiation target system, and (2) carrier from the [11C]CH3OTf system, 11C radiolabeled precursor or called 11C radiolabeled methylating reagent. If we can eliminate 12C carrier-added as much as possible, then we will be able to achieve the highest specific activity. The 11C gas target we used is the Siemens RDS-111 Eclipse cyclotron 11C gas target. The technical trick to produce high specific activity [11C]CO2 is we will usually do 2–3 times pre-burn with the same beam current and short time like 10 min before production run. This pre-burn will warm up the cyclotron and eliminate significant amount of 12C carrier-added in the cyclotron 11C gas target. The [11C]CH3OTf production system we used is an Eckert
& Ziegler Modular Lab C-11 Methyl Iodide/Triflate module, convenient gas phase bromination of [11C]methane and production of [11C]CH3OTf, a ‘dry’ method using Br2 different with other ‘dry’ method using I2 and ‘wet’ method using LiAlH4 and HI. Our system will have much less 12C carrier-added in comparison with other ‘dry’ method and ‘wet’ method.20 Chemical purity and radiochemical purity were determined by analytical HPLC.35 The chemical purity of the precursors and reference standards was >93%. The radiochemical purity of the target tracers was >98% determined by radio-HPLC through c-ray (PIN diode) flow detector, and the chemical purity of the target tracers was >90% determined by reversed-phase HPLC through UV flow detector. The experimental details and characterization data for compounds 2–9 and for the tracers [11C]3, [11C]5, and [11C]8 are given.38 In summary, a simple and moderate-to-high-yield synthetic route to aminoalkylindole derivative precursors (4, 6 and 9) and reference standards (3, 5 and 8) and carbon-11-labeled aminoalkylindole derivative target tracers ([11C]3, [11C]5, [11C]8) has been developed. An automated self-designed multi-purpose [11C]-radiosynthesis module for the synthesis of [11C]3, [11C]5, and [11C]8 has been built. The target tracers were easily prepared by O-[11C] methylation of their corresponding precursors using a reactive [11C]methylating agent, [11C]CH3OTf, and isolated by a simplified SPE purification procedure in high radiochemical yields, short overall synthesis time, and high specific activity. These methods are efficient and convenient. It is anticipated that the approaches for the design, synthesis and automation of new tracer, authentic standard and radiolabeling precursor, and improvements to increase radiochemical yield and specific activity of the tracer described here can be applied with advantages to the synthesis of other 11C-radiotracers for PET imaging. These chemistry results combined with the reported in vitro biological data1 encourage further in vivo biological evaluation of carbon-11-labeled aminoalkylindole derivatives as new candidate PET dual radioligands for imaging of cannabinoid receptors in alcohol abuse. Acknowledgments This work was partially supported by the United States Indiana State Department of Health (ISDH) Indiana Spinal Cord & Brain Injury Fund (ISDH EDS-A70-2-079612). 1H NMR and 13C NMR spectra were recorded at 500 and 125 MHz, respectively, on a Bruker Avance II 500 MHz NMR spectrometer in the Department of Chemistry and Chemical Biology at Indiana University Purdue University Indianapolis (IUPUI), which is supported by the United States National Science Foundation (NSF) Major Research Instrumentation Program (MRI) grant CHE-0619254. References and notes 1. Vasiljevik, T.; Franks, L. N.; Ford, B. M.; Douglas, J. T.; Prather, P. L.; Fantegrossi, W. E.; Prisinzano, T. E. J. Med. Chem. 2013, 56, 4537. 2. Merrill, J. O.; Duncan, M. H. Med. Clin. North Am. 2014, 98, 1145. 3. Zahr, N. M.; Kaufman, K. L.; Harper, C. G. Nat. Rev. Neurol. 2011, 7, 284. 4. Mann, K.; Agartz, I.; Harper, C.; Shoaf, S.; Rawlings, R. R.; Momenan, R.; Hommer, D. W.; Pfefferbaum, A.; Sullivan, E. V.; Anton, R. F.; Drobes, D. J.; George, M. S.; Bares, R.; Machulla, H. J.; Mundle, G.; Reimold, M.; Heinz, A. Alcohol Clin. Exp. Res. 2001, 25, 104S. 5. Fei, X.; Mock, B. H.; DeGrado, T. R.; Wang, J.-Q.; Glick-Wilson, B. E.; Sullivan, M. L.; Hutchins, G. D.; Zheng, Q.-H. Synth. Commun. 2004, 34, 1897. 6. Morris, E. D.; Yoder, K. K.; Wang, C.; Normandin, M. D.; Zheng, Q.-H.; Mock, B.; Muzic, R. F.; Froehlich, J. C. Mol. Imaging 2005, 4, 473. 7. Yoder, K. K.; Kareken, D. A.; Seyoum, R. A.; O’Connor, S. J.; Wang, C.; Zheng, Q.H.; Mock, B.; Morris, E. D. Alcohol Clin. Exp. Res. 2005, 29, 965. 8. Yoder, K. K.; Albrecht, D. S.; Kareken, D. A.; Federici, L. M.; Perry, K. M.; Patton, E. A.; Zheng, Q.-H.; Mock, B. H.; O’Connor, S.; Herring, C. M. Synapse 2011, 65, 553. 9. Gao, M.; Wang, M.; Mock, B. H.; Glick-Wilson, B. E.; Yoder, K. K.; Hutchins, G. D.; Zheng, Q.-H. Appl. Radiat. Isot. 2010, 68, 1079.
M. Gao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5581–5586 10. Yoder, K. K.; Mock, B. H.; Zheng, Q.-H.; McCarthy, B. P.; Riley, A. A.; Hutchins, G. D. J. Neurosci. Methods 2011, 196, 70. 11. Albrecht, D. S.; Skosnik, P. D.; Vollmer, J. M.; Brumbaugh, M. S.; Perry, K. M.; Mock, B. H.; Zheng, Q.-H.; Federici, L. A.; Patton, E. A.; Herring, C. M.; Yoder, K. K. Drug Alcohol Depend. 2013, 128, 52. 12. Castaneto, M. S.; Gorelick, D. A.; Desrosiers, N. A.; Hartman, R. L.; Pirard, S.; Huestis, M. A. Drug Alcohol Depend. 2014 [Epub ahead of print]. 13. Wang, L. L.; Yang, A. K.; He, S. M.; Liang, J.; Zhou, Z. W.; Li, Y.; Zhou, S. F. Curr. Pharm. Des. 2010, 16, 1313. 14. Gao, M.; Wang, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2012, 22, 3704. 15. Gao, M.; Wang, M.; Miller, K. D.; Hutchins, G. D.; Zheng, Q.-H. Bioorg. Med. Chem. 2010, 18, 2099. 16. Gao, M.; Xu, J.; Wang, M.; Zheng, Q.-H. Appl. Radiat. Isot. 2014, 90, 181. 17. Wang, M.; Gao, M.; Miller, K. D.; Sledge, G. W.; Hutchins, G. D.; Zheng, Q.-H. J. Labelled Compd. Radiopharm. 2008, 51, 6. 18. Wang, J.-Q.; Gao, M.; Miller, K. D.; Sledge, G. W.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2006, 16, 4102. 19. Jewett, D. M. Int. J. Radiat. Appl. Instrum. A 1992, 43, 1383. 20. Mock, B. H.; Mulholland, G. K.; Vavrek, M. T. Nucl. Med. Biol. 1999, 26, 467. 21. Zheng, Q.-H.; Mulholland, G. K. Nucl. Med. Biol. 1996, 23, 981. 22. Gao, M.; Wang, M.; Hutchins, G. D.; Zheng, Q.-H. Appl. Radiat. Isot. 2008, 66, 1891. 23. Gao, M.; Wang, M.; Miller, K. D.; Zheng, Q.-H. Appl. Radiat. Isot. 2012, 70, 1558. 24. Gao, M.; Gao, A. C.; Wang, M.; Zheng, Q.-H. Appl. Radiat. Isot. 2014, 91, 71. 25. Wang, M.; Gao, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2014, 24, 3700. 26. Allard, M.; Fouquet, E.; James, D.; Szlosek-Pinaudm, M. Curr. Med. Chem. 2008, 15, 235. 27. Mock, B. H.; Zheng, Q.-H.; DeGrado, T. R. J. Labelled Compd. Radiopharm. 2005, 48, S225. 28. Mock, B. H.; Glick-Wilson, B. E.; Zheng, Q.-H.; DeGrado, T. R. J. Label. Compd. Radiopharm. 2005, 48, S224. 29. Wang, M.; Gao, M.; Zheng, Q.-H. Appl. Radiat. Isot. 2012, 70, 965. 30. Gao, M.; Wang, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2014, 24, 254. 31. Wang, M.; Gao, M.; Miller, K. D.; Zheng, Q.-H. Steroids 2011, 76, 1331. 32. Wang, M.; Gao, M.; Miller, K. D.; Sledge, G. W.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2012, 22, 1569. 33. Zheng, Q.-H.; Gao, M.; Mock, B. H.; Wang, S.; Hara, T.; Nazih, R.; Miller, M. A.; Receveur, T. J.; Lopshire, J. C.; Groh, W. J.; Zipes, D. P.; Hutchins, G. D.; DeGrado, T. R. Bioorg. Med. Chem. Lett. 2007, 17, 2220. 34. Gao, M.; Yang, Q.; Wang, M.; Miller, K. D.; Sledge, G. W.; Zheng, Q.-H. Appl. Radiat. Isot. 2013, 74, 61. 35. Zheng, Q.-H.; Mock, B. H. Biomed. Chromatogr. 2005, 19, 671. 36. Lapi, S. E.; Welch, M. J. Nucl. Med. Biol. 2013, 40, 314. 37. Wang, M.; Gao, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2013, 23, 5259. 38. (a) General: All commercial reagents and solvents were purchased from SigmaAldrich and Fisher Scientific, and used without further purification. [11C]CH3OTf was prepared according to a literature procedure.20 Melting points were determined on a MEL-TEMP II capillary tube apparatus and were uncorrected. 1H NMR and 13C NMR spectra were recorded at 500 and 125 MHz, respectively, on a Bruker Avance II 500 MHz NMR spectrometer using tetramethylsilane (TMS) as an internal standard. Chemical shift data for the proton resonances were reported in parts per million (ppm, d scale) relative to internal standard TMS (d 0.0), and coupling constants (J) were reported in hertz (Hz). Liquid chromatography–mass spectra (LC–MS) analysis was performed on an Agilent system, consisting of an 1100 series HPLC connected to a diode array detector and a 1946D mass spectrometer configured for positive-ion/ negative-ion electrospray ionization. The high resolution mass spectra (HRMS) were obtained using a Waters/Micromass LCT Classic spectrometer. Chromatographic solvent proportions are indicated as volume:volume ratio. Thinlayer chromatography (TLC) was run using Analtech silica gel GF uniplates (5 10 cm2). Plates were visualized under UV light. Normal phase flash column chromatography was carried out on EM Science silica gel 60 (230–400 mesh) with a forced flow of the indicated solvent system in the proportions described below. All moisture- and air-sensitive reactions were performed under a positive pressure of nitrogen maintained by a direct line from a nitrogen source. Analytical HPLC was performed using a Prodigy (Phenomenex) 5 lm C-18 column, 4.6 250 mm; mobile phase 3:1:1 CH3CN/MeOH/ 20 mM phosphate buffer solution (pH 6.7); flow rate 1.0 mL/min; and UV (254 nm) and c-ray (PIN diode) flow detectors. C18 Plus Sep-Pak cartridges were obtained from Waters Corporation (Milford, MA). Sterile Millex-GS 0.22 lm filter units were obtained from Millipore Corporation (Bedford, MA). (b) 1-Butyl-7-methoxy-1H-indole (2): To a suspension of KOH (16.25 g, 290 mmol) in DMF (120 mL) was added 7-methoxyindole (1, 8.54 g, 58 mmol). After stirring at room temperature (RT) for an hour, 1-bromobutane (11.12 g, 81.2 mmol) was added and the reaction mixture was heated to 50 °C for 12 h. The resulting mixture was poured into water (200 mL) and extracted with EtOAc (3 120 mL). Combined organic layers were washed with water two times, dried over anhydrous Na2SO4, and concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel using EtOAc/hexanes (1:99) as eluent to afford colorless oily product 2 (10.36 g, 88%). Rf = 0.90 (1:2 EtOAc/hexanes). 1H NMR (CDCl3): d 0.91 (t, J = 7.5 Hz, 3H, CH3), 1.27–1.34 (m, 2H, CH2), 1.74–1.80 (m, 2H, CH2), 3.91 (s, 3H, OCH3), 4.35 (t, J = 7.5 Hz, 2H, CH2), 6.39 (d, J = 3.0 Hz, 1H, Ar-H), 6.59 (d, J = 8.0 Hz, 1H, Ar-H), 6.94–6.98 (m, 2H, Ar-H), 7.18 (d, J = 8.0 Hz, 1H, Ar-H). MS (ESI): 204 ([M+H]+, 100%). (c) (1-Butyl-7-methoxy-1H-indol-3-yl)(naphthalen-1-yl)methanone (3): To a solution of compound 2 (8.81 g, 43.32 mmol) in CH2Cl2 (100 mL) at 0 °C under
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N2 atmosphere was added Me2AlCl (69.4 mL, 1 M in hexanes, 69.4 mmol) dropwise, and the solution was allowed to stir at 0 °C for 40 min, then a solution of 1-naphthoyl chloride (9.25 g, 48.52 mmol) in CH2Cl2 (60 mL) was added dropwise. The reaction mixture was stirred for overnight at rt, and carefully poured into 1 N HCl (70 mL) solution prior to extraction with CH2Cl2 (3 100 mL). The combined organic extracts were washed with NaHCO3, brine, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography on silica gel using EtOAc/hexanes (3:97) as eluent to give white solid product 3 (8.04 g, 52%). Rf = 0.38 (1:5 EtOAc/hexanes), mp 108–110 °C. 1H NMR (CDCl3): d 0.88 (t, J = 7.3 Hz, 3H, CH3), 1.22–1.30 (m, 2H, CH2), 1.72–1.77 (m, 2H, CH2), 3.95 (s, 3H, OCH3), 4.28 (t, J = 7.5 Hz, 2H, CH2), 6.76 (d, J = 8.0 Hz, 1H, Ar-H), 7.20 (s, 1H, Ar-H), 7.24 (t, J = 8.0 Hz, 1H, Ar-H), 7.43–7.51 (m, 3H, Ar-H), 7.62 (dd, J = 1.0, 7.0 Hz, 1H, Ar-H), 7.89 (d, J = 8.0 Hz, 1H, Ar-H), 7.94 (d, J = 8.0 Hz, 1H, Ar-H), 8.09 (d, J = 8.0 Hz, 1H, Ar-H), 8.16 (d, J = 8.5 Hz, 1H, Ar-H). MS (ESI): 358 ([M+H]+, 100%). (d) 1-Butyl-7-methoxy-3-(naphthalen-1-ylmethyl)-1H-indole (5): A solution of AlCl3 (16.0 g, 120 mmol) in THF (80 mL) was dropwise added into a solution of LiAlH4 (2.0 M in THF, 20 mL, 40 mmol) at 0 °C. After 40 min, compound 3 (3.57 g, 10.0 mmol) in THF (60 mL) was added to the reaction mixture and allowed to stir at rt for 48 h. Upon completion, the reaction mixture was cooled down in an ice-bath, quenched with water and acidified with 1 N HCl to pH = 5. The organic phase was then separated, while the aqueous phase was extracted with EtOAc (2 100 mL). The combined organic layers were washed with NaHCO3, brine, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography on silica gel using EtOAc/hexanes (2:98) as eluent to give white solid product 5 (1.68 g, 49%). Rf = 0.84 (1:2 EtOAc/hexanes), mp 47-49 °C. 1H NMR (CDCl3): d 0.85 (t, J = 7.3 Hz, 3H, CH3), 1.19–1.24 (m, 2H, CH2), 1.64–1.70 (m, 2H, CH2), 3.92 (s, 3H, OCH3), 4.21 (t, J = 7.3 Hz, 2H, CH2), 4.49 (s, 2H, CH2), 6.48 (s, 1H, Ar-H), 6.62 (d, J = 8.0 Hz, 1H, Ar-H), 6.98 (t, J = 7.5 Hz, 1H, Ar-H), 7.18 (dd, J = 0.5, 8.0 Hz, 1H, Ar-H), 7.33–7.39 (m, 2H, Ar-H), 7.40–7.47 (m, 2H, Ar-H), 7.72 (d, J = 8.0 Hz, 1H, Ar-H), 7.84 (dd, J = 1.2, 8.0 Hz, 1H, Ar-H), 8.08 (d, J = 8.0 Hz, 1H, Ar-H). MS (ESI): 344 ([M+H]+, 100%). (e) 1-Butyl-3-iodo-7-methoxy-1H-indole (7): A solution of 7-methoxyindole (1, 2.95 g, 20.0 mmol) in DMF (80 mL) was stirred with KOH (1.29 g, 23.0 mmol) at RT for 40 min, and then treated with I2 (5.23 g, 20.6 mmol). After 30 min, NaH (60% in mineral oil, 1.04 g, 26.0 mmol) was added portionwise. After an additional 15 min, 1-bromobutane (3.15 g, 23.0 mmol) was added and the reaction mixture was stirred for overnight. Upon completion (TLC monitoring), water was added, the reaction mixture was extracted with EtOAc (3 80 mL). Combined organic layers were washed with water two times, dried over anhydrous Na2SO4, and concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel using EtOAc/hexanes (0.5:99.5) as eluent to afford white solid product 7 (4.94 g, 75%). Rf = 0.67 (1:9 EtOAc/hexanes), mp 42–44 °C. 1H NMR (CDCl3): d 0.92 (t, J = 7.3 Hz, 3H, CH3), 1.27–1.34 (m, 2H, CH2), 1.73–1.79 (m, 2H, CH2), 3.92 (s, 3H, OCH3), 4.35 (t, J = 7.3 Hz, 2H, CH2), 6.65 (d, J = 7.5 Hz, 1H, Ar-H), 7.01 (dd, J = 0.5, 8.0 Hz, 1H, Ar-H), 7.05–7.08 (m, 2H, Ar-H). MS (ESI): 329 ([M]+, 20%), 203 (100%). (f) 1-Butyl-7-methoxy-3-(naphthalen-2-yl)-1H-indole (8): Pd(OAc)2 (45 mg, 0.2 mmol), 2-(dicyclohexylphosphino)biphenyl (Cy-JohnPhos) (88 mg, 0.25 mmol), K2CO3 (2.08 g, 15.0 mmol), 2-naphthaleneboronic acid (1.55 g, 9.0 mmol), and compound 7 (1.65 g, 5.0 mmol) were added into a solution of toluene (100 mL), EtOH (40 mL), and water (10 mL), and the reaction mixture was allowed to stir at reflux under N2 overnight. Reaction was monitored via TLC, and upon completion, the reaction mixture was then concentrated under vacuo, the residue was extracted with EtOAc (3 100 mL). The combined organic layers were washed with water, brine, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography on silica gel using CH2Cl2/ hexanes (2:98) as eluent to afford pinkish oil product 8 (0.84 g, 51%). Rf = 0.68 (1:8 EtOAc/hexanes). 1H NMR (CDCl3): d 0.95 (t, J = 7.5 Hz, 3H, CH3), 1.33–1.41 (m, 2H, CH2), 1.81–1.87 (m, 2H, CH2), 3.95 (s, 3H, OCH3), 4.42 (t, J = 7.2 Hz, 2H, CH2), 6.68 (d, J = 7.5 Hz, 1H, Ar-H), 7.09 (t, J = 8.0 Hz, 1H, Ar-H), 7.39 (td, J = 1.0, 8.0 Hz, 1H, Ar-H), 7.44 (td, J = 1.0, 7.5 Hz, 1H, Ar-H), 7.62 (d, J = 8.0 Hz, 1H, Ar-H), 7.76 (dd, J = 1.5, 8.5 Hz, 1H, Ar-H), 7.81–7.87 (m, 3H, Ar-H), 8.07 (s, 1H, Ar-H). MS (ESI): 330 ([M+H]+, 100%). (g) General procedure for the preparation of precursors 4, 6 and 9 by Odesmethylation of the indole methyl ether derivatives 3, 5, and 8: A solution of 48% HBr (3 mL) was added into a suspension of the indole derivative 3 (5 or 8) (1.2 mmol) in 5 mL of 30% HBr in HOAc, and then the reaction mixture was stirred and heated to reflux for 2–3 h. Upon the completion, the reaction mixture was cooled down and quenched with NaOH (3 N). After adjusted pH to 7 and extracted with EtOAc (3 60 mL), the combined organic extracts were dried over anhydrous Na2SO4, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel using EtOAc/hexanes (4:96) as eluent to give off-brown oil product 4 (6 or 9) in 43-70% yield. (1-Butyl-7-hydroxy-1H-indol-3-yl)(naphthalen-1-yl)methanone (4): Yield 70%. Rf = 0.25 (1:5 EtOAc/hexanes). 1H NMR (acetone-d6): d 0.87 (t, J = 7.3 Hz, 3H, CH3), 1.28–1.32 (m, 2H, CH2), 1.78–1.84 (m, 2H, CH2), 4.44 (t, J = 7.0 Hz, 2H, CH2), 6.77 (d, J = 7.5 Hz, 1H, Ar-H), 7.05 (t, J = 8.0 Hz, 1H, Ar-H), 7.47–7.59 (m, 4H, ArH), 7.67 (dd, J = 1.2, 7.0 Hz, 1H, Ar-H), 7.97–8.03 (m, 3H, Ar-H), 8.11 (d, J = 8.5 Hz, 1H, Ar-H), 8.95 (s, 1H, OH). 13C NMR (acetone-d6): d 13.70, 19.79, 33.83, 50.12, 109.81, 114.79, 117.40, 123.87, 124.68, 125.94, 126.14, 126.26, 126.38, 126.81, 128.25, 129.86, 130.04, 130.94, 133.81, 139.20, 139.75, 143.66, 192.74. MS (ESI):
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344 ([M+H]+, 100%). HRMS (ESI): calcd for C23H21NO2Na ([M+Na]+) 366.1470, found 366.1472. 1-Butyl-3-(naphthalen-1-ylmethyl)-1H-indol-7-ol (6): Yield 43%. Rf = 0.50 (1:5 EtOAc/hexanes). 1H NMR (acetone-d6): d 0.84 (t, J = 7.5 Hz, 3H, CH3), 1.30–1.45 (m, 2H, CH2), 1.73–1.76 (m, 2H, CH2), 4.42 (t, J = 7.5 Hz, 2H, CH2), 4.55 (s, 2H, CH2), 5.83 (s, 1H, Ar-H), 6.52 (dd, J = 1.0, 8.0 Hz, 1H, Ar-H), 6.72 (t, J = 7.5 Hz, 1H, Ar-H), 6.85 (dd, J = 1.0, 8.0 Hz, 1H, Ar-H), 7.27 (d, J = 7.0 Hz, 1H, Ar-H), 7.42 (t, J = 7.5 Hz, 1H, Ar-H), 7.47–7.49 (m, 2H, Ar-H), 7.81 (d, J = 8.5 Hz, 1H, Ar-H), 7.91– 7.93 (m, 1H, Ar-H), 8.09–8.11 (m, 1H, Ar-H), 8.45 (s, 1H, OH). 13C NMR (acetoned6): d 14.30, 20.60, 35.20, 37.23, 45.95, 102.71, 107.16, 112.47, 120.27, 124.91, 126.34, 126.44, 126.48, 126.82, 126.95, 127.70, 128.07, 129.47, 131.68, 133.02, 134.89, 135.97, 139.88, 144.67. MS (ESI): 330 ([M+H]+, 18%); MS (ESI): 328 ([M H] , 100%). Note: This compound is not so stable. 1-Butyl-3-(naphthalen-2-yl)-1H-indol-7-ol (9): Yield 52%. Rf = 0.45 (1:5 EtOAc/ hexanes). 1H NMR (acetone-d6): d 0.90 (t, J = 7.0 Hz, 3H, CH3), 1.32–1.41 (m, 2H, CH2), 1.86–1.91 (m, 2H, CH2), 4.54 (t, J = 7.2 Hz, 2H, CH2), 6.67 (d, J = 7.0 Hz, 1H, Ar-H), 6.94 (t, J = 8.0 Hz, 1H, Ar-H), 7.41 (td, J = 1.0, 8.0 Hz, 1H, Ar-H), 7.47 (td, J = 1.0, 8.0 Hz, 1H, Ar-H), 7.55–7.57 (m, 2H, Ar-H), 7.83–7.86 (m, 2H, Ar-H), 7.90– 7.93 (m, 2H, Ar-H), 8.16 (s, 1H, Ar-H), 8.68 (s, 1H, OH). 13C NMR (acetone-d6): d 14.20, 20.55, 35.29, 49.53, 107.98, 112.24, 116.75, 121.42, 125.09, 125.78, 126.90, 127.20, 128.43, 128.52, 128.75, 128.94, 130.77, 134.80, 135.17, 145.53. MS (ESI): 316 ([M+H]+, 90%). HRMS (ESI): calcd for C22H22NO ([M+H]+) 316.1701, found 316.1715. (h) General procedure for the preparation of the target tracers (1-butyl-7-[11C] methoxy-1H-indol-3-yl)(naphthalene-1-yl)methanone ([11C]3), 1-butyl-7[11C]methoxy-3-(naphthalene-1-ylmethyl)-1H-indole ([11C]5), and 1-butyl-7[11C]methoxy-3-(naphthalene-2-yl)-1H-indole ([11C]8): [11C]CO2 was produced by the 14N(p,a)11C nuclear reaction in the small volume (9.5 cm3) aluminum gas target provided with the Siemens RDS-111 Eclipse cyclotron. The target gas
consisted of 1% oxygen in nitrogen purchased as a specialty gas from Praxair, Indianapolis, IN. Typical irradiations used for the development were 50 lA beam current for 15 min on target. The production run produced approximately 25.9 GBq of [11C]CO2 at EOB. The phenolic hydroxyl precursor 4 (6 or 9) (0.1– 0.3 mg) was dissolved in CH3CN (300 lL). To this solution was added 2 N NaOH (2 lL). The mixture was transferred to a small reaction vial. No-carrier-added (high specific activity) [11C]CH3OTf (13.9 GBq) that was produced by the gasphase production method20 within 11 min from [11C]CO2 (25.9 GBq) through [11C]CH4 (21.8 GBq) and [11C]CH3Br (13.9 GBq) with silver triflate (AgOTf) column was passed into the reaction vial at rt until radioactivity reached a maximum (2 min), and then the reaction vial was isolated and heated at 80 °C for 3 min. The contents of the reaction vial were diluted with NaHCO3 (0.1 M, 1 mL). The reaction vial was connected to a C-18 Plus Sep-Pak cartridge. The labeled product mixture solution was passed onto the cartridge for SPE purification by gas pressure. The cartridge was washed with H2O (2 3 mL), and the aqueous washing was discarded. The product was eluted from the cartridge with EtOH (2 2 mL), and then passed onto a rotatory evaporator. The solvent was removed by evaporation (3 min) under vacuum. The final volume of ethanol after evaporation was 1 mL. The labeled product was reformulated with saline (10 mL), sterile-filtered through a sterile vented Millex-GS 0.22 lm cellulose acetate membrane and collected into a sterile vial. Total radioactivity (4.7–7.1 GBq) was assayed and the total volume (10–11 mL) was noted for tracer dose dispensing. The overall synthesis time including SPE purification and reformulation was 23 min. The radiochemical yields decay corrected to EOB, from [11C]CO2, were 50–60%. The same procedure was used to prepare the target tracers [11C]3, [11C]5 and [11C]8 from their corresponding precursors 4, 6 and 9. Retention times in the analytical HPLC system were: tR 4 = 3.40 min, tR 3 = 5.48 min, tR [11C]3 = 5.61 min; tR 6 = 5.27 min, tR 5 = 7.44 min, tR [11C]5 = 7.51 min; and tR 9 = 3.79 min, tR 8 = 6.56 min, tR [11C]8 = 6.64 min.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis of carbon-11-labeled aminoalkylindole derivatives as new candidates of cannabinoid receptor radioligands for PET imaging of alcohol abuse Mingzhang Gao, Andy Chufan Gao, Min Wang, Qi-Huang Zheng ⇑ Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, Room 202, Indianapolis, IN 46202, USA
a r t i c l e
i n f o
Article history: Received 14 October 2014 Revised 28 October 2014 Accepted 30 October 2014 Available online 6 November 2014 Keywords: Carbon-11-labeled aminoalkylindole derivatives Radiosynthesis Positron emission tomography (PET) Cannabinoid receptor Alcohol abuse
a b s t r a c t Carbon-11-labeled aminoalkylindole derivatives (1-butyl-7-[11C]methoxy-1H-indol-3-yl)(naphthalene1-yl)methanone ([11C]3), 1-butyl-7-[11C]methoxy-3-(naphthalene-1-ylmethyl)-1H-indole ([11C]5), and 1-butyl-7-[11C]methoxy-3-(naphthalene-2-yl)-1H-indole ([11C]8) were prepared by O-[11C]methylation of their corresponding precursors with [11C]CH3OTf under basic condition (2 N NaOH) and isolated by a simplified solid-phase extraction (SPE) method in 50–60% radiochemical yields based on [11C]CO2 and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 23 min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 185–555 GBq/lmol. Ó 2014 Elsevier Ltd. All rights reserved.
Alcohol abuse is a major substance abuse, approximately 2.5 million people die from alcohol use every year, and its annual health- and crime-related costs are estimated around $235 billion.1 There are several therapeutic options available for the treatment of alcohol abuse, but all existing therapies have only modest efficacy.2 The cause of alcohol abuse is still unknown. However, alcohol abuse results in chemical and biological changes in the brain, and it is often associated with substantial psychiatric comorbidity, brain damage and disease.3 Brain imaging techniques such as positron emission tomography (PET) enable scientists to better understand alcohol abuse and alcohol-related diseases, for example, with PET, researchers can compare groups of alcohol-abusing and nonabusing individuals by quantifying differences in their levels of a particular neurotransmitter molecule like dopamine or neurotransmission component such as a receptor or a transporter.4 We are interested in PET imaging of alcohol abuse, and we have extensively used [11C]raclopride-PET and [18F]fallypride-PET to study D2 receptor in alcohol abuse, since alcohol abuse significantly affects the neurotransmitter dopamine and alters its neurotransmission in the central nervous system (CNS).5–10 More and more evidences suggest that endocannabinoid (eCB) system is another neurotransmitter involved in alcohol abuse, and cannabinoid (CB) receptor (CBR) antagonist/agonist might ⇑ Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278 9711. E-mail address: [email protected] (Q.-H. Zheng). http://dx.doi.org/10.1016/j.bmcl.2014.10.097 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
offer a new therapeutic direction for treatment of alcohol abuse.11,12 Recently a new class of aminoalkylindole derivatives has been developed as CBR ligands with dual CB1R antagonist/ CB2R agonist activity with potential for treatment of alcohol abuse.1 CBR has become an interesting target for PET imaging of alcohol abuse.13 In our previous works, we have developed a series of selective CB1R radioligands and CB2R radioligands, as indicated in Figure 1.14–16 In this ongoing study, we report the design and synthesis of carbon-11-labeled aminoalkylindole derivatives as new candidate dual CB1R/CB2R radioligands for PET imaging of alcohol abuse. Aminoalkylindole derivatives (1-butyl-7-methoxy-1H-indol-3-yl) (naphthalene-1-yl)methanone (3), 1-butyl-7-methoxy-3-(naphthalene-1-ylmethyl)-1H-indole (5), and 1-butyl-7-methoxy-3-(naphthalene-2-yl)-1H-indole (8) are dual CB1R/CB2R ligands with high affinity, and the low nanomolar Ki values for CB1R and CB2R are 1.7 and 0.81 nM, 15.4 and 10.9 nM, and 37.3 and 26.5 nM, respectively.1 According to their in vitro Ki values, compound 3 is the most potent ligand for CB1R and CB2R in comparison with compounds 5 and 8. However, the overall biological evaluation including both in vitro and in vivo studies suggested compounds 5 and 8 appeared to have the most promise to be CBR ligands for use as treatment of alcohol abuse.1 These three representative compounds are selected as the reference standards for radiolabeling. As outlined in Scheme 1, commercially available starting material 7-methoxyindole (1) was subjected to mild alkylating
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N
O NC N
N
H3 11CO
NC N
H3 11CO
Cl
Cl
N
O NC
N H
N
N
H3 11CO
N
Cl
N
O
N H Cl
[11C]JHU75528 ([11C]OMAR)
NC
N
O
N H
N
H3 11CO
Br
N
N H Br
[ 11C]JHU75575 CB1R Radioligands X
O
H 311 CO
N H N H OC 5 H 11
n
N H
O
H 3 CO
n=1, X=H n=1, X=Cl n=2, X=H
N H N OC 5 H 11
N H OC 5 H 11
X
O
H 311 CO
O 11CH3
O
n=1 O 11CH3
O N H
n H 3 CO
Cl
n
O
N OC 5 H 11
n=1, X=H n=1, X=Cl n=2, X=H
n
Cl n=1
H 311 CO O S O N
N CB2R Radioligands
Figure 1. CB1R and CB2R radioligands.14–16
O
O i N H
O 1
N
ii
iii
O 2
N
N O
3
OH
4
Scheme 1. Synthesis of aminoalkylindole derivative reference standard 3 and its precursor 4. Reagents, conditions and yields: (i) KOH, 1-bromobutane, DMF, 50 °C, 88%; (ii) Me2AlCl, 1-naphthoyl chloride, CH2Cl2, 0 °C, 52%; (iii) 48% HBr, HOAc, reflux, 70%.
conditions to give 1-butyl-7-methoxy-1H-indole (2) in 88% yield. Intermediate 2 was then subjected to Friedel–Crafts like acylation conditions, using dimethylaluminum chloride and acid chloride at 0 °C to afford the standard 3 in 52% yield. The desmethylation of compound 3 with 48% HBr in the solution of HOAc provided its desmethylated precursor (1-butyl-7-hydroxy1H-indol-3-yl)(naphthalene-1-yl)methanone (4) in 70% yield. Using protic acid (HBr) instead of the literature method Lewis acid (BBr3)1 as the desmethylating reagent, it significantly improved the yield of desmethylation reaction, because protic acid promoted the desmethylation reaction of phenolic methoxy aminoalkylindole
derivatives to easily form phenolic hydroxyl precursor and CH3Br.17,18 As shown in Scheme 2, the reduction of compound 3 with LiAlH4 gave the standard 5 in 49% yield. Careful workup by adjusting appropriate pH to 5 improved the yield of the reduction from 18% to 49%. Likewise, using 48% HBr in HOAc as the desmethylating reagent, compound 5 was desmethylated to produce its desmethylated precursor 1-butyl-7-hydroxy-3-(naphthalene-1ylmethyl)-1H-indole (6) in 43% yield. Using Lewis acid (BBr3)1 as the desmethylating reagent, compound 5 failed to give 6 in desmethylation reaction.
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O ii
i N O
N O
3
N OH
5
6
Scheme 2. Synthesis of aminoalkylindole derivative reference standard 5 and its precursor 6. Reagents, conditions and yields: (i) LiAlH4, AlCl3, THF, 0 °C, 49%; (ii) 48% HBr, HOAc, reflux, 43%.
I i N H
O 1
N
ii
iii
O 7
N
N O
OH
8
9
Scheme 3. Synthesis of aminoalkylindole derivative reference standard 8 and its precursor 9. Reagents, conditions and yields: (i) 1-bromobutane, I2, KOH, DMF, NaH, rt, 75%; (ii) 2-naphthaleneboronic acid, Pd(OAc)2, Cy-JohnPhos, K2CO3, toluene, EtOH, H2O, reflux, 51%; (iii) 48% HBr, HOAc, reflux, 52%.
As indicated in Scheme 3, a one-pot two-step (N-alkylation and 3-indole iodination) reaction of compound 1 afforded an intermediate 1-butyl-3-iodo-7-methoxy-1H-indole (7) in 75% yield. Intermediate 7 was then subjected to Suzuki coupling conditions utilizing boronic acid to provide the standard 8 in 51% yield, which was undergone the same desmethylating conditions (48% HBr in HOAc) to give its desmethylated precursor 1-butyl-7-hydroxy-3(naphthalene-2-yl)-1H-indole (9) in 52% yield. Synthesis of carbon-11-labeled aminoalkylindole derivatives (1-butyl-7-[11C]methoxy-1H-indol-3-yl)(naphthalene-1-yl)metha none ([11C]3), 1-butyl-7-[11C]methoxy-3-(naphthalene-1-ylmethyl)1H-indole ([11C]5), and 1-butyl-7-[11C]methoxy-3-(naphthalene-2yl)-1H-indole ([11C]8), is indicated in Scheme 4. The desmethylated precursors 4, 6, and 9 were labeled by a reactive [11C]methylating agent, [11C]methyl triflate ([11C]CH3OTf)19,20 prepared from [11C]CO2, under basic conditions (2 N NaOH) in acetonitrile through the O-[11C]methylation and isolated by a simplified solid-phase extraction (SPE) method21–25 to provide target tracers [11C]3, [11C]5, and [11C]9 in 50–60% radiochemical yields, decay corrected to end of bombardment (EOB), based on [11C]CO2. [11C]CH3OTf is a proven methylation reagent with greater reactivity than commonly used [11C]methyl iodide ([11C]CH3I),26 and thus, the radiochemical yield of [11C]3, [11C]5, and [11C]8 was relatively high. The large polarity difference between the sodium salt of the phenolic hydroxyl precursor and the corresponding labeled O-methylated ether product permitted the use of SPE technique for purification of the labeled product from the radiolabeling reaction mixture. A C-18 Plus Sep-Pak cartridge was used in SPE purification technique. The crude reaction mixture was treated with aqueous NaHCO3 and loaded onto the cartridge by gas pressure. The pH of freshly prepared 0.1 M NaHCO3 might be too low to effectively deprotonate a phenolic hydroxyl precursor. However, an excess of 2 N NaOH used in the reaction provided a final pH after addition of the 0.1 M NaHCO3 high enough to deprotonate all the phenolic hydroxyl precursor. Any non-reacted precursor was actually converted to the corresponding sodium salt, and any non-reacted [11C]CH3OTf was actually hydrolyzed to [11C] CH3OH, which would not be trapped to the C-18 Sep-Pak. The cartridge was washed with water to remove radiolabeled by-product [11C]CH3OH, sodium salt of phenolic hydroxyl precursor and reaction solvent, and total 6 mL (2 3 mL) volume of water was enough to wash off all impurity. The final labeled product was
O
O i
N
N
OH
O 11CH3 [ 11C]3
4
i N
N
OH
O11 CH 3 [11C]5
6
i N
N
OH
O 11CH3 9
[11C]8
Scheme 4. Synthesis of carbon-11-labeled aminoalkylindole derivative target tracers [11C]3, [11C]5, and [11C]8. Reagents, conditions and yields: (i) [11C]CH3OTf, CH3CN, 2 N NaOH, 80 °C, 3 min, 50–60%.
eluted with ethanol (2 2 mL), concentrated by rotary evaporation and reformulated in saline (10 mL). In our fully automated radiosynthesis module,27–29 it is difficult to directly elute the labeled product from a C-18 Sep-Pak to a vial using either 1 1 mL ethanol or 2 0.5 mL ethanol, due to the back pressure in the C-18 Sep-Pak and dead volume in the transfer tubing. The high back pressure is resulted from the eluent (water, ethanol and saline) change. In order to elute most of the labeled product from the C-18 Sep-Pak, we need to increase the volume of the eluent ethanol. For the radiotracer produced for animal study, we used 2 2 mL ethanol for elution, and rotary evaporation was required before
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reformulation. For the radiotracer produced for human study, we used 2 1 mL ethanol, no evaporation required, and a C-18 Sep-Pak was used for direct reformulation.9,30–32 We have tried to use a C-18 Light Sep-Pak cartridge instead of a C-18 Plus SepPak cartridge to allow smaller volume (1 mL) of ethanol and to avoid laborious rotary evaporation before formulation. However, there is more serious back pressure in the Light Sep-Pak than in the Plus Sep-Pak, in addition, dead volume in the transfer tubing also affects the elution, and thus it is more difficult to efficiently elute the labeled product from a Light Sep-Pak, which resulted in the low radiochemical yield. The reason is that our home-built fully automated module used many PTFE (polytetrafluoroethylene)/silicone liners (septa) and Teflon tubing, and these materials cannot afford too high pressure gas (N2) push. The pressure of N2 gas introduced in our module is set at 207 kPa (30 Psi). C-18 Light Sep-Pak cartridge works well in manual or semi-automated radiosynthesis in our lab, because we can easily introduce high pressure gas push during the purification and reformulation process. Overall synthesis time was 23 min from EOB, including approximately 11 min for [11C]CH3OTf production, 5 min for O-[11C]methylation reaction, and 7 min for SPE purification, evaporation and reformulation. SPE technique is fast, efficient and convenient and works very well for the O-methylated ether tracer purification using the phenolic hydroxyl precursor for radiolabeling.22,24 The radiosynthesis was performed in an automated selfdesigned multi-purpose 11C-radiosynthesis module, allowing measurement of specific activity at EOB during synthesis.27–29 On line determination of specific activity at EOB is accurate when reverse-phase (RP) high performance liquid chromatography (HPLC) is used as purification method. However, the on-the-fly technique to determine specific activity at EOB is not applied when SPE is used as purification method. The specific activity for the 11 C-tracers produced in our PET chemistry facility usually ranges from 370 to 1110 GBq/mol at EOB according to our previous works. The specific activity of carbon-11-labeled aminoalkylindole derivatives was estimated in a range of 185–555 GBq/mol at the end of synthesis (EOS) based on other compounds produced in our facility using the same targetry conditions which have been measured by the on-the-fly technique or the same SPE purification method.33 The actual measurement of specific activity at EOS was performed by analytical HPLC34,35 and calculated. The exact values of the specific activity for the tracers [11C]3, [11C]5, and [11C]8 were 185–555 GBq/mol at EOS, which are in agreement with the estimated values and the ‘on line’ determined values. Specific activity is defined as the radioactivity per unit mass of a radionuclide or a labeled compound. Specific activity (MBq/mg) = 3.13 109/A t1/2, where A is the mass number of the radionuclide, and t1/2 is the half-life in hours of the radionuclide. For carbon-11, carrierfree 11C, maximum (theoretical) 11C specific activity = 340,918 GBq/lmol.36 Actual specific activity of the 11C-tracers in the PET chemistry facility are depended on two parts: (1) carrier from the 11 C-target, and (2) carrier from the 11C-radiosynthesis unit.37 Furthermore, actual specific activity for the 11C-tracers synthesized by 11C-methylation with [11C]CH3OTf in our PET chemistry facility is depended on two parts: (1) carrier from the cyclotron consisted of the 11C gas irradiation target system, and (2) carrier from the [11C]CH3OTf system, 11C radiolabeled precursor or called 11C radiolabeled methylating reagent. If we can eliminate 12C carrier-added as much as possible, then we will be able to achieve the highest specific activity. The 11C gas target we used is the Siemens RDS-111 Eclipse cyclotron 11C gas target. The technical trick to produce high specific activity [11C]CO2 is we will usually do 2–3 times pre-burn with the same beam current and short time like 10 min before production run. This pre-burn will warm up the cyclotron and eliminate significant amount of 12C carrier-added in the cyclotron 11C gas target. The [11C]CH3OTf production system we used is an Eckert
& Ziegler Modular Lab C-11 Methyl Iodide/Triflate module, convenient gas phase bromination of [11C]methane and production of [11C]CH3OTf, a ‘dry’ method using Br2 different with other ‘dry’ method using I2 and ‘wet’ method using LiAlH4 and HI. Our system will have much less 12C carrier-added in comparison with other ‘dry’ method and ‘wet’ method.20 Chemical purity and radiochemical purity were determined by analytical HPLC.35 The chemical purity of the precursors and reference standards was >93%. The radiochemical purity of the target tracers was >98% determined by radio-HPLC through c-ray (PIN diode) flow detector, and the chemical purity of the target tracers was >90% determined by reversed-phase HPLC through UV flow detector. The experimental details and characterization data for compounds 2–9 and for the tracers [11C]3, [11C]5, and [11C]8 are given.38 In summary, a simple and moderate-to-high-yield synthetic route to aminoalkylindole derivative precursors (4, 6 and 9) and reference standards (3, 5 and 8) and carbon-11-labeled aminoalkylindole derivative target tracers ([11C]3, [11C]5, [11C]8) has been developed. An automated self-designed multi-purpose [11C]-radiosynthesis module for the synthesis of [11C]3, [11C]5, and [11C]8 has been built. The target tracers were easily prepared by O-[11C] methylation of their corresponding precursors using a reactive [11C]methylating agent, [11C]CH3OTf, and isolated by a simplified SPE purification procedure in high radiochemical yields, short overall synthesis time, and high specific activity. These methods are efficient and convenient. It is anticipated that the approaches for the design, synthesis and automation of new tracer, authentic standard and radiolabeling precursor, and improvements to increase radiochemical yield and specific activity of the tracer described here can be applied with advantages to the synthesis of other 11C-radiotracers for PET imaging. These chemistry results combined with the reported in vitro biological data1 encourage further in vivo biological evaluation of carbon-11-labeled aminoalkylindole derivatives as new candidate PET dual radioligands for imaging of cannabinoid receptors in alcohol abuse. Acknowledgments This work was partially supported by the United States Indiana State Department of Health (ISDH) Indiana Spinal Cord & Brain Injury Fund (ISDH EDS-A70-2-079612). 1H NMR and 13C NMR spectra were recorded at 500 and 125 MHz, respectively, on a Bruker Avance II 500 MHz NMR spectrometer in the Department of Chemistry and Chemical Biology at Indiana University Purdue University Indianapolis (IUPUI), which is supported by the United States National Science Foundation (NSF) Major Research Instrumentation Program (MRI) grant CHE-0619254. References and notes 1. Vasiljevik, T.; Franks, L. N.; Ford, B. M.; Douglas, J. T.; Prather, P. L.; Fantegrossi, W. E.; Prisinzano, T. E. J. Med. Chem. 2013, 56, 4537. 2. Merrill, J. O.; Duncan, M. H. Med. Clin. North Am. 2014, 98, 1145. 3. Zahr, N. M.; Kaufman, K. L.; Harper, C. G. Nat. Rev. Neurol. 2011, 7, 284. 4. Mann, K.; Agartz, I.; Harper, C.; Shoaf, S.; Rawlings, R. R.; Momenan, R.; Hommer, D. W.; Pfefferbaum, A.; Sullivan, E. V.; Anton, R. F.; Drobes, D. J.; George, M. S.; Bares, R.; Machulla, H. J.; Mundle, G.; Reimold, M.; Heinz, A. Alcohol Clin. Exp. Res. 2001, 25, 104S. 5. Fei, X.; Mock, B. H.; DeGrado, T. R.; Wang, J.-Q.; Glick-Wilson, B. E.; Sullivan, M. L.; Hutchins, G. D.; Zheng, Q.-H. Synth. Commun. 2004, 34, 1897. 6. Morris, E. D.; Yoder, K. K.; Wang, C.; Normandin, M. D.; Zheng, Q.-H.; Mock, B.; Muzic, R. F.; Froehlich, J. C. Mol. Imaging 2005, 4, 473. 7. Yoder, K. K.; Kareken, D. A.; Seyoum, R. A.; O’Connor, S. J.; Wang, C.; Zheng, Q.H.; Mock, B.; Morris, E. D. Alcohol Clin. Exp. Res. 2005, 29, 965. 8. Yoder, K. K.; Albrecht, D. S.; Kareken, D. A.; Federici, L. M.; Perry, K. M.; Patton, E. A.; Zheng, Q.-H.; Mock, B. H.; O’Connor, S.; Herring, C. M. Synapse 2011, 65, 553. 9. Gao, M.; Wang, M.; Mock, B. H.; Glick-Wilson, B. E.; Yoder, K. K.; Hutchins, G. D.; Zheng, Q.-H. Appl. Radiat. Isot. 2010, 68, 1079.
M. Gao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5581–5586 10. Yoder, K. K.; Mock, B. H.; Zheng, Q.-H.; McCarthy, B. P.; Riley, A. A.; Hutchins, G. D. J. Neurosci. Methods 2011, 196, 70. 11. Albrecht, D. S.; Skosnik, P. D.; Vollmer, J. M.; Brumbaugh, M. S.; Perry, K. M.; Mock, B. H.; Zheng, Q.-H.; Federici, L. A.; Patton, E. A.; Herring, C. M.; Yoder, K. K. Drug Alcohol Depend. 2013, 128, 52. 12. Castaneto, M. S.; Gorelick, D. A.; Desrosiers, N. A.; Hartman, R. L.; Pirard, S.; Huestis, M. A. Drug Alcohol Depend. 2014 [Epub ahead of print]. 13. Wang, L. L.; Yang, A. K.; He, S. M.; Liang, J.; Zhou, Z. W.; Li, Y.; Zhou, S. F. Curr. Pharm. Des. 2010, 16, 1313. 14. Gao, M.; Wang, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2012, 22, 3704. 15. Gao, M.; Wang, M.; Miller, K. D.; Hutchins, G. D.; Zheng, Q.-H. Bioorg. Med. Chem. 2010, 18, 2099. 16. Gao, M.; Xu, J.; Wang, M.; Zheng, Q.-H. Appl. Radiat. Isot. 2014, 90, 181. 17. Wang, M.; Gao, M.; Miller, K. D.; Sledge, G. W.; Hutchins, G. D.; Zheng, Q.-H. J. Labelled Compd. Radiopharm. 2008, 51, 6. 18. Wang, J.-Q.; Gao, M.; Miller, K. D.; Sledge, G. W.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2006, 16, 4102. 19. Jewett, D. M. Int. J. Radiat. Appl. Instrum. A 1992, 43, 1383. 20. Mock, B. H.; Mulholland, G. K.; Vavrek, M. T. Nucl. Med. Biol. 1999, 26, 467. 21. Zheng, Q.-H.; Mulholland, G. K. Nucl. Med. Biol. 1996, 23, 981. 22. Gao, M.; Wang, M.; Hutchins, G. D.; Zheng, Q.-H. Appl. Radiat. Isot. 2008, 66, 1891. 23. Gao, M.; Wang, M.; Miller, K. D.; Zheng, Q.-H. Appl. Radiat. Isot. 2012, 70, 1558. 24. Gao, M.; Gao, A. C.; Wang, M.; Zheng, Q.-H. Appl. Radiat. Isot. 2014, 91, 71. 25. Wang, M.; Gao, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2014, 24, 3700. 26. Allard, M.; Fouquet, E.; James, D.; Szlosek-Pinaudm, M. Curr. Med. Chem. 2008, 15, 235. 27. Mock, B. H.; Zheng, Q.-H.; DeGrado, T. R. J. Labelled Compd. Radiopharm. 2005, 48, S225. 28. Mock, B. H.; Glick-Wilson, B. E.; Zheng, Q.-H.; DeGrado, T. R. J. Label. Compd. Radiopharm. 2005, 48, S224. 29. Wang, M.; Gao, M.; Zheng, Q.-H. Appl. Radiat. Isot. 2012, 70, 965. 30. Gao, M.; Wang, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2014, 24, 254. 31. Wang, M.; Gao, M.; Miller, K. D.; Zheng, Q.-H. Steroids 2011, 76, 1331. 32. Wang, M.; Gao, M.; Miller, K. D.; Sledge, G. W.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2012, 22, 1569. 33. Zheng, Q.-H.; Gao, M.; Mock, B. H.; Wang, S.; Hara, T.; Nazih, R.; Miller, M. A.; Receveur, T. J.; Lopshire, J. C.; Groh, W. J.; Zipes, D. P.; Hutchins, G. D.; DeGrado, T. R. Bioorg. Med. Chem. Lett. 2007, 17, 2220. 34. Gao, M.; Yang, Q.; Wang, M.; Miller, K. D.; Sledge, G. W.; Zheng, Q.-H. Appl. Radiat. Isot. 2013, 74, 61. 35. Zheng, Q.-H.; Mock, B. H. Biomed. Chromatogr. 2005, 19, 671. 36. Lapi, S. E.; Welch, M. J. Nucl. Med. Biol. 2013, 40, 314. 37. Wang, M.; Gao, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2013, 23, 5259. 38. (a) General: All commercial reagents and solvents were purchased from SigmaAldrich and Fisher Scientific, and used without further purification. [11C]CH3OTf was prepared according to a literature procedure.20 Melting points were determined on a MEL-TEMP II capillary tube apparatus and were uncorrected. 1H NMR and 13C NMR spectra were recorded at 500 and 125 MHz, respectively, on a Bruker Avance II 500 MHz NMR spectrometer using tetramethylsilane (TMS) as an internal standard. Chemical shift data for the proton resonances were reported in parts per million (ppm, d scale) relative to internal standard TMS (d 0.0), and coupling constants (J) were reported in hertz (Hz). Liquid chromatography–mass spectra (LC–MS) analysis was performed on an Agilent system, consisting of an 1100 series HPLC connected to a diode array detector and a 1946D mass spectrometer configured for positive-ion/ negative-ion electrospray ionization. The high resolution mass spectra (HRMS) were obtained using a Waters/Micromass LCT Classic spectrometer. Chromatographic solvent proportions are indicated as volume:volume ratio. Thinlayer chromatography (TLC) was run using Analtech silica gel GF uniplates (5 10 cm2). Plates were visualized under UV light. Normal phase flash column chromatography was carried out on EM Science silica gel 60 (230–400 mesh) with a forced flow of the indicated solvent system in the proportions described below. All moisture- and air-sensitive reactions were performed under a positive pressure of nitrogen maintained by a direct line from a nitrogen source. Analytical HPLC was performed using a Prodigy (Phenomenex) 5 lm C-18 column, 4.6 250 mm; mobile phase 3:1:1 CH3CN/MeOH/ 20 mM phosphate buffer solution (pH 6.7); flow rate 1.0 mL/min; and UV (254 nm) and c-ray (PIN diode) flow detectors. C18 Plus Sep-Pak cartridges were obtained from Waters Corporation (Milford, MA). Sterile Millex-GS 0.22 lm filter units were obtained from Millipore Corporation (Bedford, MA). (b) 1-Butyl-7-methoxy-1H-indole (2): To a suspension of KOH (16.25 g, 290 mmol) in DMF (120 mL) was added 7-methoxyindole (1, 8.54 g, 58 mmol). After stirring at room temperature (RT) for an hour, 1-bromobutane (11.12 g, 81.2 mmol) was added and the reaction mixture was heated to 50 °C for 12 h. The resulting mixture was poured into water (200 mL) and extracted with EtOAc (3 120 mL). Combined organic layers were washed with water two times, dried over anhydrous Na2SO4, and concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel using EtOAc/hexanes (1:99) as eluent to afford colorless oily product 2 (10.36 g, 88%). Rf = 0.90 (1:2 EtOAc/hexanes). 1H NMR (CDCl3): d 0.91 (t, J = 7.5 Hz, 3H, CH3), 1.27–1.34 (m, 2H, CH2), 1.74–1.80 (m, 2H, CH2), 3.91 (s, 3H, OCH3), 4.35 (t, J = 7.5 Hz, 2H, CH2), 6.39 (d, J = 3.0 Hz, 1H, Ar-H), 6.59 (d, J = 8.0 Hz, 1H, Ar-H), 6.94–6.98 (m, 2H, Ar-H), 7.18 (d, J = 8.0 Hz, 1H, Ar-H). MS (ESI): 204 ([M+H]+, 100%). (c) (1-Butyl-7-methoxy-1H-indol-3-yl)(naphthalen-1-yl)methanone (3): To a solution of compound 2 (8.81 g, 43.32 mmol) in CH2Cl2 (100 mL) at 0 °C under
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N2 atmosphere was added Me2AlCl (69.4 mL, 1 M in hexanes, 69.4 mmol) dropwise, and the solution was allowed to stir at 0 °C for 40 min, then a solution of 1-naphthoyl chloride (9.25 g, 48.52 mmol) in CH2Cl2 (60 mL) was added dropwise. The reaction mixture was stirred for overnight at rt, and carefully poured into 1 N HCl (70 mL) solution prior to extraction with CH2Cl2 (3 100 mL). The combined organic extracts were washed with NaHCO3, brine, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography on silica gel using EtOAc/hexanes (3:97) as eluent to give white solid product 3 (8.04 g, 52%). Rf = 0.38 (1:5 EtOAc/hexanes), mp 108–110 °C. 1H NMR (CDCl3): d 0.88 (t, J = 7.3 Hz, 3H, CH3), 1.22–1.30 (m, 2H, CH2), 1.72–1.77 (m, 2H, CH2), 3.95 (s, 3H, OCH3), 4.28 (t, J = 7.5 Hz, 2H, CH2), 6.76 (d, J = 8.0 Hz, 1H, Ar-H), 7.20 (s, 1H, Ar-H), 7.24 (t, J = 8.0 Hz, 1H, Ar-H), 7.43–7.51 (m, 3H, Ar-H), 7.62 (dd, J = 1.0, 7.0 Hz, 1H, Ar-H), 7.89 (d, J = 8.0 Hz, 1H, Ar-H), 7.94 (d, J = 8.0 Hz, 1H, Ar-H), 8.09 (d, J = 8.0 Hz, 1H, Ar-H), 8.16 (d, J = 8.5 Hz, 1H, Ar-H). MS (ESI): 358 ([M+H]+, 100%). (d) 1-Butyl-7-methoxy-3-(naphthalen-1-ylmethyl)-1H-indole (5): A solution of AlCl3 (16.0 g, 120 mmol) in THF (80 mL) was dropwise added into a solution of LiAlH4 (2.0 M in THF, 20 mL, 40 mmol) at 0 °C. After 40 min, compound 3 (3.57 g, 10.0 mmol) in THF (60 mL) was added to the reaction mixture and allowed to stir at rt for 48 h. Upon completion, the reaction mixture was cooled down in an ice-bath, quenched with water and acidified with 1 N HCl to pH = 5. The organic phase was then separated, while the aqueous phase was extracted with EtOAc (2 100 mL). The combined organic layers were washed with NaHCO3, brine, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography on silica gel using EtOAc/hexanes (2:98) as eluent to give white solid product 5 (1.68 g, 49%). Rf = 0.84 (1:2 EtOAc/hexanes), mp 47-49 °C. 1H NMR (CDCl3): d 0.85 (t, J = 7.3 Hz, 3H, CH3), 1.19–1.24 (m, 2H, CH2), 1.64–1.70 (m, 2H, CH2), 3.92 (s, 3H, OCH3), 4.21 (t, J = 7.3 Hz, 2H, CH2), 4.49 (s, 2H, CH2), 6.48 (s, 1H, Ar-H), 6.62 (d, J = 8.0 Hz, 1H, Ar-H), 6.98 (t, J = 7.5 Hz, 1H, Ar-H), 7.18 (dd, J = 0.5, 8.0 Hz, 1H, Ar-H), 7.33–7.39 (m, 2H, Ar-H), 7.40–7.47 (m, 2H, Ar-H), 7.72 (d, J = 8.0 Hz, 1H, Ar-H), 7.84 (dd, J = 1.2, 8.0 Hz, 1H, Ar-H), 8.08 (d, J = 8.0 Hz, 1H, Ar-H). MS (ESI): 344 ([M+H]+, 100%). (e) 1-Butyl-3-iodo-7-methoxy-1H-indole (7): A solution of 7-methoxyindole (1, 2.95 g, 20.0 mmol) in DMF (80 mL) was stirred with KOH (1.29 g, 23.0 mmol) at RT for 40 min, and then treated with I2 (5.23 g, 20.6 mmol). After 30 min, NaH (60% in mineral oil, 1.04 g, 26.0 mmol) was added portionwise. After an additional 15 min, 1-bromobutane (3.15 g, 23.0 mmol) was added and the reaction mixture was stirred for overnight. Upon completion (TLC monitoring), water was added, the reaction mixture was extracted with EtOAc (3 80 mL). Combined organic layers were washed with water two times, dried over anhydrous Na2SO4, and concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel using EtOAc/hexanes (0.5:99.5) as eluent to afford white solid product 7 (4.94 g, 75%). Rf = 0.67 (1:9 EtOAc/hexanes), mp 42–44 °C. 1H NMR (CDCl3): d 0.92 (t, J = 7.3 Hz, 3H, CH3), 1.27–1.34 (m, 2H, CH2), 1.73–1.79 (m, 2H, CH2), 3.92 (s, 3H, OCH3), 4.35 (t, J = 7.3 Hz, 2H, CH2), 6.65 (d, J = 7.5 Hz, 1H, Ar-H), 7.01 (dd, J = 0.5, 8.0 Hz, 1H, Ar-H), 7.05–7.08 (m, 2H, Ar-H). MS (ESI): 329 ([M]+, 20%), 203 (100%). (f) 1-Butyl-7-methoxy-3-(naphthalen-2-yl)-1H-indole (8): Pd(OAc)2 (45 mg, 0.2 mmol), 2-(dicyclohexylphosphino)biphenyl (Cy-JohnPhos) (88 mg, 0.25 mmol), K2CO3 (2.08 g, 15.0 mmol), 2-naphthaleneboronic acid (1.55 g, 9.0 mmol), and compound 7 (1.65 g, 5.0 mmol) were added into a solution of toluene (100 mL), EtOH (40 mL), and water (10 mL), and the reaction mixture was allowed to stir at reflux under N2 overnight. Reaction was monitored via TLC, and upon completion, the reaction mixture was then concentrated under vacuo, the residue was extracted with EtOAc (3 100 mL). The combined organic layers were washed with water, brine, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography on silica gel using CH2Cl2/ hexanes (2:98) as eluent to afford pinkish oil product 8 (0.84 g, 51%). Rf = 0.68 (1:8 EtOAc/hexanes). 1H NMR (CDCl3): d 0.95 (t, J = 7.5 Hz, 3H, CH3), 1.33–1.41 (m, 2H, CH2), 1.81–1.87 (m, 2H, CH2), 3.95 (s, 3H, OCH3), 4.42 (t, J = 7.2 Hz, 2H, CH2), 6.68 (d, J = 7.5 Hz, 1H, Ar-H), 7.09 (t, J = 8.0 Hz, 1H, Ar-H), 7.39 (td, J = 1.0, 8.0 Hz, 1H, Ar-H), 7.44 (td, J = 1.0, 7.5 Hz, 1H, Ar-H), 7.62 (d, J = 8.0 Hz, 1H, Ar-H), 7.76 (dd, J = 1.5, 8.5 Hz, 1H, Ar-H), 7.81–7.87 (m, 3H, Ar-H), 8.07 (s, 1H, Ar-H). MS (ESI): 330 ([M+H]+, 100%). (g) General procedure for the preparation of precursors 4, 6 and 9 by Odesmethylation of the indole methyl ether derivatives 3, 5, and 8: A solution of 48% HBr (3 mL) was added into a suspension of the indole derivative 3 (5 or 8) (1.2 mmol) in 5 mL of 30% HBr in HOAc, and then the reaction mixture was stirred and heated to reflux for 2–3 h. Upon the completion, the reaction mixture was cooled down and quenched with NaOH (3 N). After adjusted pH to 7 and extracted with EtOAc (3 60 mL), the combined organic extracts were dried over anhydrous Na2SO4, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel using EtOAc/hexanes (4:96) as eluent to give off-brown oil product 4 (6 or 9) in 43-70% yield. (1-Butyl-7-hydroxy-1H-indol-3-yl)(naphthalen-1-yl)methanone (4): Yield 70%. Rf = 0.25 (1:5 EtOAc/hexanes). 1H NMR (acetone-d6): d 0.87 (t, J = 7.3 Hz, 3H, CH3), 1.28–1.32 (m, 2H, CH2), 1.78–1.84 (m, 2H, CH2), 4.44 (t, J = 7.0 Hz, 2H, CH2), 6.77 (d, J = 7.5 Hz, 1H, Ar-H), 7.05 (t, J = 8.0 Hz, 1H, Ar-H), 7.47–7.59 (m, 4H, ArH), 7.67 (dd, J = 1.2, 7.0 Hz, 1H, Ar-H), 7.97–8.03 (m, 3H, Ar-H), 8.11 (d, J = 8.5 Hz, 1H, Ar-H), 8.95 (s, 1H, OH). 13C NMR (acetone-d6): d 13.70, 19.79, 33.83, 50.12, 109.81, 114.79, 117.40, 123.87, 124.68, 125.94, 126.14, 126.26, 126.38, 126.81, 128.25, 129.86, 130.04, 130.94, 133.81, 139.20, 139.75, 143.66, 192.74. MS (ESI):
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344 ([M+H]+, 100%). HRMS (ESI): calcd for C23H21NO2Na ([M+Na]+) 366.1470, found 366.1472. 1-Butyl-3-(naphthalen-1-ylmethyl)-1H-indol-7-ol (6): Yield 43%. Rf = 0.50 (1:5 EtOAc/hexanes). 1H NMR (acetone-d6): d 0.84 (t, J = 7.5 Hz, 3H, CH3), 1.30–1.45 (m, 2H, CH2), 1.73–1.76 (m, 2H, CH2), 4.42 (t, J = 7.5 Hz, 2H, CH2), 4.55 (s, 2H, CH2), 5.83 (s, 1H, Ar-H), 6.52 (dd, J = 1.0, 8.0 Hz, 1H, Ar-H), 6.72 (t, J = 7.5 Hz, 1H, Ar-H), 6.85 (dd, J = 1.0, 8.0 Hz, 1H, Ar-H), 7.27 (d, J = 7.0 Hz, 1H, Ar-H), 7.42 (t, J = 7.5 Hz, 1H, Ar-H), 7.47–7.49 (m, 2H, Ar-H), 7.81 (d, J = 8.5 Hz, 1H, Ar-H), 7.91– 7.93 (m, 1H, Ar-H), 8.09–8.11 (m, 1H, Ar-H), 8.45 (s, 1H, OH). 13C NMR (acetoned6): d 14.30, 20.60, 35.20, 37.23, 45.95, 102.71, 107.16, 112.47, 120.27, 124.91, 126.34, 126.44, 126.48, 126.82, 126.95, 127.70, 128.07, 129.47, 131.68, 133.02, 134.89, 135.97, 139.88, 144.67. MS (ESI): 330 ([M+H]+, 18%); MS (ESI): 328 ([M H] , 100%). Note: This compound is not so stable. 1-Butyl-3-(naphthalen-2-yl)-1H-indol-7-ol (9): Yield 52%. Rf = 0.45 (1:5 EtOAc/ hexanes). 1H NMR (acetone-d6): d 0.90 (t, J = 7.0 Hz, 3H, CH3), 1.32–1.41 (m, 2H, CH2), 1.86–1.91 (m, 2H, CH2), 4.54 (t, J = 7.2 Hz, 2H, CH2), 6.67 (d, J = 7.0 Hz, 1H, Ar-H), 6.94 (t, J = 8.0 Hz, 1H, Ar-H), 7.41 (td, J = 1.0, 8.0 Hz, 1H, Ar-H), 7.47 (td, J = 1.0, 8.0 Hz, 1H, Ar-H), 7.55–7.57 (m, 2H, Ar-H), 7.83–7.86 (m, 2H, Ar-H), 7.90– 7.93 (m, 2H, Ar-H), 8.16 (s, 1H, Ar-H), 8.68 (s, 1H, OH). 13C NMR (acetone-d6): d 14.20, 20.55, 35.29, 49.53, 107.98, 112.24, 116.75, 121.42, 125.09, 125.78, 126.90, 127.20, 128.43, 128.52, 128.75, 128.94, 130.77, 134.80, 135.17, 145.53. MS (ESI): 316 ([M+H]+, 90%). HRMS (ESI): calcd for C22H22NO ([M+H]+) 316.1701, found 316.1715. (h) General procedure for the preparation of the target tracers (1-butyl-7-[11C] methoxy-1H-indol-3-yl)(naphthalene-1-yl)methanone ([11C]3), 1-butyl-7[11C]methoxy-3-(naphthalene-1-ylmethyl)-1H-indole ([11C]5), and 1-butyl-7[11C]methoxy-3-(naphthalene-2-yl)-1H-indole ([11C]8): [11C]CO2 was produced by the 14N(p,a)11C nuclear reaction in the small volume (9.5 cm3) aluminum gas target provided with the Siemens RDS-111 Eclipse cyclotron. The target gas
consisted of 1% oxygen in nitrogen purchased as a specialty gas from Praxair, Indianapolis, IN. Typical irradiations used for the development were 50 lA beam current for 15 min on target. The production run produced approximately 25.9 GBq of [11C]CO2 at EOB. The phenolic hydroxyl precursor 4 (6 or 9) (0.1– 0.3 mg) was dissolved in CH3CN (300 lL). To this solution was added 2 N NaOH (2 lL). The mixture was transferred to a small reaction vial. No-carrier-added (high specific activity) [11C]CH3OTf (13.9 GBq) that was produced by the gasphase production method20 within 11 min from [11C]CO2 (25.9 GBq) through [11C]CH4 (21.8 GBq) and [11C]CH3Br (13.9 GBq) with silver triflate (AgOTf) column was passed into the reaction vial at rt until radioactivity reached a maximum (2 min), and then the reaction vial was isolated and heated at 80 °C for 3 min. The contents of the reaction vial were diluted with NaHCO3 (0.1 M, 1 mL). The reaction vial was connected to a C-18 Plus Sep-Pak cartridge. The labeled product mixture solution was passed onto the cartridge for SPE purification by gas pressure. The cartridge was washed with H2O (2 3 mL), and the aqueous washing was discarded. The product was eluted from the cartridge with EtOH (2 2 mL), and then passed onto a rotatory evaporator. The solvent was removed by evaporation (3 min) under vacuum. The final volume of ethanol after evaporation was 1 mL. The labeled product was reformulated with saline (10 mL), sterile-filtered through a sterile vented Millex-GS 0.22 lm cellulose acetate membrane and collected into a sterile vial. Total radioactivity (4.7–7.1 GBq) was assayed and the total volume (10–11 mL) was noted for tracer dose dispensing. The overall synthesis time including SPE purification and reformulation was 23 min. The radiochemical yields decay corrected to EOB, from [11C]CO2, were 50–60%. The same procedure was used to prepare the target tracers [11C]3, [11C]5 and [11C]8 from their corresponding precursors 4, 6 and 9. Retention times in the analytical HPLC system were: tR 4 = 3.40 min, tR 3 = 5.48 min, tR [11C]3 = 5.61 min; tR 6 = 5.27 min, tR 5 = 7.44 min, tR [11C]5 = 7.51 min; and tR 9 = 3.79 min, tR 8 = 6.56 min, tR [11C]8 = 6.64 min.