Radiosynthesis of [11C]BBAC and [11C]BBPC as potential PET tracers for orexin2 receptors

Article abstract of DOI:10.1016/j.bmcl.2012.01.114

Radiosynthesis of [N-methyl-¹¹C](S)-N-([1,1′-biphenyl]-2- yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2- carboxamide ([¹¹C]BBAC or [¹¹C]3) and [N-methyl- ¹¹C] (S)-N-([1,1′-biphenyl]-2-yl

Full text of DOI:10.1016/j.bmcl.2012.01.114

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2172–2174
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Radiosynthesis of [11C]BBAC and [11C]BBPC as potential PET tracers
for orexin2 receptors
Fei Liu a,c, Vattoly J. Majo b,c, Jaya Prabhakaran b,c, John Castrillion a,c, J. John Mann b,c,d,e, Diana Martinez a,c
,
J.S. Dileep Kumar b,c,d,
⇑
a Division of Substance Abuse, Columbia University Medical Center, United States
b Division of Molecular Imaging and Neuropathology, Columbia University Medical Center, United States
c Department of Psychiatry, Columbia University Medical Center, United States
d New York State Psychiatric Institute, Columbia University, New York, NY 10032, United States
e Department of Radiology, Columbia University, New York, NY 10032, United States
a r t i c l e i n f o
a b s t r a c t
Radiosynthesis of [N-methyl-11C](S)-N-([1,10-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-
yl)thio)acetyl)pyrrolidine-2-carboxamide ([11C]BBAC or [11C]3) and [N-methyl-11C] (S)-N-([1,10-biphe-
nyl]-2-yl)-1-(3-(1-methyl-1H-benzo[d]imidazol-2-yl)propanoyl)pyrrolidine-2-carboxamide ([11C]BBPC
or [11C]-4), two potential PET tracers for orexin2 receptors are described. Syntheses of non-radioactive
standards 3, 4 and corresponding desmethyl precursors 1, 2 were achieved from common intermediate
(S)-2-([1,10-biphenyl]-2-yl)-1-(pyrrolidin-2-yl)ethanone. Methylation using [11C]CH3OTf in the presence
of base in acetone afforded [11C]3 and [11C]4 in 30 5% yield (EOS) with >99 % radiochemical purities
Article history:
Received 9 December 2011
Revised 25 January 2012
Accepted 30 January 2012
Available online 4 February 2012
Keywords:
Orexin
Hypocretin
Positron emission tomography
Radiotracer
with a specific activity ranged from 2.5 0.5 Ci/l
mol (EOB). The logP of [11C]3 and [11C]4 were deter-
mined as 3.4 and 2.8, respectively. The total synthesis time was 30 min from EOB. However, PET scans
performed in a rhesus monkey did not show tracer retention or appropriate brain uptake. Hence [11C]3
and [11C]4 cannot be used as PET tracers for imaging orexin2 receptors.
Ó 2012 Elsevier Ltd. All rights reserved.
Orexin or hypocretin receptors are G-protein coupled recep-
tors (GPCR) that mediate the central actions of the endogenous
neurohormones orexin-A and -B (also known as hypocretins-1
and 2) produced in the lateral hypothalamus.1,2 Pre-clinical and
clinical studies have established that orexin pathway plays a crit-
ical role in motivation, arousal and sleep-wake regulation.3,4
Abnormalities in orexin signaling have been implicated in sleep
disorders such as human narcolepsy–cataplexy,5 irregularities in
central vestibular motor control,6 feeding and gastrointestinal dis-
orders7,8 and addiction.9 Based on the binding of endogenous li-
gands, the orexin receptors are divided into two major types:
orexin1 (OX1R, or HCRT1), and orexin2 (OX2R, or HCRT2R) recep-
tors.10 Among these, OX1R have preferential affinity for
orexin-A, whereas OX2R bind with equal affinities for both neuro-
peptides.1 However, the in vivo selectivity, distribution and
involvements of individual receptors in the pathophysiology of
orexin-mediated disorders are not available. Positron emission
tomography (PET) is an excellent tool for the in vivo quantifica-
tion of biological processes. Currently, there are no PET tracers
available for imaging orexin receptors. We have selected OX2R
as an imaging target anticipating a predominant role for this
receptor in the pathophysiology elicited by orexin A and B owing
to its ability to bind to both endogenous ligands with equal
affinity. Candidates belonging to different templates have been
reported as selective OX2R antagonist ligands.10–15 Among these
N-methylated benzimidazole bis amides of proline as a lead
structure had significantly reduced para-glycoprotein (P-gp) sus-
ceptibility, increased receptor selectivity thereby offering ligands
with increased potency and good blood–brain barrier (BBB)
penetration.16,17 We have selected proline bis-amides 3 and 4 as
candidate PET ligands for OX2R imaging owing to nanomolar
affinity (Ki = 0.2 nM for 3 and 0.8 nM for 4), logP favorable to pen-
etrate BBB (3.2 for 3 and 2.5 for 4), and excellent pharmacokinet-
ics, and brain/plasma ratios.17 Moreover, compound 3 inhibit
ADL-orexinB locomotion model in rats in a dose dependent man-
ner.17 Additionally, the availability of suitable sites for incorporat-
ing C-11 isotope encouraged us to test compounds 3 and 4 as
potential PET imaging agents for OX2R. Herein, we report the
radiosynthesis and in vivo evaluation of two OX2R ligands 3and
4 as potential PET imaging agents in rhesus monkey.
Syntheses of nonradioactive standards 3, 4 and desmethyl pre-
cursors 1, 2 were achieved in good yield starting from(S)-2-([1,10-
biphenyl]-2-yl)-1-(pyrrolidin-2-yl)ethanone (5), reported previously
(Scheme 1).17 Compound 5 was subsequently treated with 2-
bromoacetyl bromide in presence of triethylamine and DMAP to
⇑
Corresponding author. Fax: +1 212 543 1163.
E-mail address: dk2038@columbia.edu (J.S.D. Kumar).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.114

F. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2172–2174
2173
Scheme 1. Syntheses of compounds 1 and 2. Reagents and conditions: (a) 2-
bromoacetyl bromide, Et3N, DMAP, CH2Cl2, 0 °C, 1 h, 72%; (b) 1H-benzo[d]imidaz-
ole-2-thiol, Et3N, ethanol, 86%; (c) 3-(1H-benzo[d]imidazol-2-yl)propanoic acid,
EDC, HOBT, Et3N, DMF, 76%.
Figure 1. PET images of [11C]3 (first row) and [11C]4 (bottom row) in rhesus
monkey. The images shown are sagittal, coronal, and transaxial PET scans (left to
right) generated as the sum of all frames acquired over 120 min. The units of the
color bar are MBq/cc.
obtain the bromide 6 in 72% yield. The radiolabeling precursor 1 for
[
11C]3 was synthesized by coupling compound
6 with 1H-
benzo[d]imidazole-2-thiol in 86% yield.18 Similarly, condensation
of 5 with 3-(1H-benzo[d]imidazol-2-yl)-propanoicacid afforded,
compound 2 (76%), the radiolabeling precursor for [11C]4 (Scheme
1).19
One issue could be the affinity and permeability of the radiotracers.
However, both the affinity and the logP suggested that these radio-
tracers were favorable for use as radiotracers. Another potential is-
sue is that the radiotracers may be P-glycoprotein (P-GP) or multi
drug resist (MDR) substrates or the density of orexin receptors in
the rhesus monkey brain may not be high enough to permit brain
PET imaging. Perhaps a high affinity radioligand with higher spe-
cific activity, and which is not a P-GP or MDR substrate may be re-
quired for the quantification of OX2R by PET.
We found that methylation of 1 and 2 with CH3I in the pres-
ence of Cs2CO3 in DMF at room temperature afforded compounds
3 and 4, respectively, in good yield (Scheme 2).20 Therefore,
radiolabeling of [11C]-3 and [11C]-4 were attempted initially with
[
[
11C]CH3I to optimize the reaction conditions. However,
11C]methylation proceeded in better yield by using [11C]CH3OTf
In summary, we have successfully synthesized [11C]3 and [11C]-
4, as potential PET tracer agents for OX2R. The total time required
for the radiosynthesis were 30 min from EOB using [11C]CH3OTf in
acetone. Radioproducts were obtained in 30 + 5% yield (EOS) with
excellent purities and specific activity in the formulation. However,
in vivo PET studies in rhesus monkeys did not show tracer uptake
in brain.
in acetone at room temperature. Accordingly, radiolabeling reac-
tions were performed using 0.5 mg of the respective precursors
in 0.5 ml acetone in the presence of 5 M NaOH at rt using
[
11C]CH3OTf to provide [11C]3 and [11C]4 in 30 5% yield at the
end of synthesis (EOS) (Scheme 2). The radioproducts were puri-
fied via semipreparative HPLC and solid phase extraction and for-
mulated in saline containing 10% ethanol. The specific activity of
the radioproducts were found to be in the range of 2–3 Ci/lmol
References and notes
(n = 8) at the end of bombardment (EOB) with excellent chemical
and radiochemical purities.21 The total time required for the rad-
iosyntheses were ꢀ30 min. The partition coefficient for [11C]3
and [11C]4 obtained by standard shake flask method were 3.4
and 2.8, respectively.22
Subsequently we examined the BBB permeability and in vivo
distribution of the radiotracers, by PET scans in an anesthetized fe-
male rhesus monkey. Anesthesia was performed with isoflurane
(1–2%) and ketamine to induce general anesthesia and the animal
was under constant cardiovascular monitoring. The radiotracers
1. Sakurai, T.; Amemiya, A.; Ishii, M.; Matsuzaki, I.; Chemelli, R. M.; Tanaka, H.;
Williams, S. C.; Richardson, J. A.; Kozlowski, G. P.; Wilson, S.; Arch, J. R.;
Buckingham, R. E.; Haynes, A. C.; Carr, S. A.; Annan, R. S.; McNulty, D. E.; Liu, W.
S.; Terrett, J. A.; Elshourbagy, N. A.; Bergsma, D. J.; Yanagisawa, M. Cell 1998, 92,
573.
2. van den Pol, A. N.; Gao, X. B.; Obrietan, K.; Kilduff, T. S.; Belousov, A. B. J.
Neurosci. 1998, 18, 7962.
3. Brisbare-Roch, C.; Dingemanse, J.; Koberstein, R.; Hoever, P.; Aissaoui, H.;
Flores, S.; Mueller, C.; Nayler, O.; van Gerven, J.; de Haas, S. L.; Hess, P.; Qiu, C.;
Buchmann, S.; Scherz, M.; Weller, T.; Fischli, W.; Clozel, M.; Jenck, F. Nat. Med.
2007, 13, 150.
4. Thompson, J. L.; Borgland, S. L. Behav. Brain Res. 2011, 217, 446.
5. Nishino, S. Handb. Clin. Neurol. 2011, 99, 765.
6. Zhang, J.; Li, B.; Yu, L.; He, Y. C.; Li, H. Z.; Zhu, J. N.; Wang, J. J. Neuron 2011, 69,
793.
7. Okumura, T.; Nozu, T. J Gastroenterol. Hepatol. 2011, 26, 61.
8. Sakurai, T. Regul. Pept. 1999, 85, 25.
9. Sharf, R.; Sarhan, M.; Dileone, R. J. Brain Res. 2010, 130, 1314.
10. Boss, C.; Brisbare-Roch, C.; Jenck, F. J. Med. Chem. 2009, 52, 891.
11. Gatfield, J.; Brisbare-Roch, C.; Jenck, F.; Boss, C. Chem. Med. Chem. 2010, 5, 1197.
12. Hirose, M.; Egashira, S.; Goto, Y.; Hashihayata, T.; Ohtake, N.; Iwaasa, H.; Hata,
M.; Fukami, T.; Kanatani, A.; Yamada, K. Bioorg. Med. Chem. Lett. 2003, 13, 4497.
13. McAtee, L. C.; Sutton, S. W.; Rudolph, D. A.; Li, X.; Aluisio, L. E.; Phuong, V. K.;
Dvorak, C. A.; Lovenberg, T. W.; Carruthers, N. I.; Jones, T. K. Bioorg. Med. Chem.
Lett. 2004, 14, 4225.
were injected as
1.43 Ci/ mol and injected mass: 1.5
1.94 mCi, specific activity: 1.15 Ci/ mol and injected mass: 1.7 lg
a
bolus (dose: 2.03 mCi, specific activity:
l
l
g for [11C]3 and dose:
l
for [11C]4) and emission data was collected for 122 min. The
images from each scan are shown in Figure 1. Although radioactiv-
ity is seen in the area of the pituitary (transaxial scan, Fig. 1, 3rd
column), no brain region inside the BBB had detectable binding
consistent with specific receptor binding to the radiotracers. The
rational for this lack of specific uptake inside the brain is not clear.
14. Aissaoui, H.; Koberstein, R.; Zumbrunn, C.; Gatfield, J.; Brisbare-Roch, C.; Jenck,
F.; Treiber, A.; Boss, C. Bioorg. Med. Chem. Lett. 2008, 18, 5729.
15. Micheli, F.; Antolini, M.; Di Fabio, R.; Pellacani, A.; Pozzan, A. Bioorg. Med. Chem.
Lett. 2010, 20, 6405.
16. Porter, R. A.; Chan, W. N.; Coulton, S.; Johns, A.; Hadley, M. S.; Widdowson, K.;
Jerman, J. C.; Brough, S. J.; Coldwell, M.; Smart, D.; Jewitt, F.; Jeffrey, P.; Austin,
N. Bioorg. Med. Chem. Lett. 1907, 2001, 11.
N
N
O
O
O
N
N
N
HN
O
a or b
X
HN
X
N
H
R
11
C]- : R = [11C]CH3, X = S
1: X = S
3
3
: R = CH3, X = S;
4: R = CH3, X = CH2;
[
17. Bergman, J. M.; Roecker, A. J.; Mercer, S. P.; Bednar, R. A.; Reiss, D. R.; Ransom,
R. W.; Meacham Harrell, C.; Pettibone, D. J.; Lemaire, W.; Murphy, K. L.; Li, C.;
Prueksaritanont, T.; Winrow, C. J.; Renger, J. J.; Koblan, K. S.; Hartman, G. D.;
Coleman, P. J. Bioorg. Med. Chem. Lett. 2008, 18, 1425.
2
: X = CH2
[
11C]-4: R = [11C]CH3, X = CH2
Scheme 2. Synthesis and radiosynthesis of
[ [
11C]3 and 11C]4. Reagents and
conditions: (a) Cs2CO3, DMF, CH3I; (b) (i) NaOH, acetone, [11C]CH3OTf, (ii) HPLC
purification, yield = 30 5% (EOS).
18. Synthesis of (S)-1-(2-((1H-benzo[d]imidazol-2-yl)thio) acetyl)-N-([1,10-
biphenyl]-2-yl)pyrrolidine-2-carboxamide
(1):
2-Mercaptobenimidazole

2174
(90 mg, 0.6 mmol) in 2 ml ethanol was added to
F. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2172–2174
solution of 0.1 ml of
a
over silica gel using a mixture of 95:5 ethyl acetate–hexane to afford 65 mg of
3 (32%) and 90 mg 4 (45%) as light brown solids which were identified by the
1H NMR and mass spectrum. 1H NMR of 3 (CDCl3, 400 MHz): 2.1 (3H, m, CH2),
2.4 (1H, m, CH2), 3.7 (2H, m, CH2), 3.7 (3H, s, CH3), 4.0 (1H, m, CH),4.4 (1H, m,
CH), 4.6 (1H, m, CH), 7.3 (6H, m), 7.4 (6H, m), 8.1 (1H, dd), 8.4 (1H, s, NH), APCI+
calculated for C27H27N4O2S (MH+): 472.19; Found: d472.46. 1H NMR of 4
(CDCl3, 400 MHz): 1.9 (3H, m, CH2), 2.1 (1H, m, CH2), 2.4 (1H, m, CH2), 2.8 (1H,
m, CH2), 3.0 (2H, m, CH2), 3.5 (2H, m, CH), 3.8 (3H, s, CH3), 4.6 (1H, m, CH),7.2
(4H, m), 7.3 (4H, m),7.5 (3H, m), 7.6 (1H, dd), 8.1(1H, dd), 8.5 (1H, s, NH); HRMS
calculated for C28H29N4O2 (MH+): 453.2291; Found: 453.2294.
triethylamine and 210 mg (0.54 mmol) of (S)-2-([1,10-biphenyl]-2-yl)-1-(1-(2-
bromo-acetyl)pyrrolidin-2-yl)ethanone (6) in 2 ml ethanol. The solution was
stirred for 1 h at room temperature. The solvent was removed and 10 ml of the
water was added to the residue. the organic layers were collected, dried over
MgSO4, filtered and concentrated under vacuum. The result residue was
purified by the chromatographed on silica gel using a mixture of 2:1 ethyl
acetate with hexane and 9⁄⁄:1 ethyl acetate with methanol as eluents to
obtain the desmethyl compound 1 (214 mg, 86%) as a brown solid. 1H NMR
(CDCl3, 400 MHz): 2.1 (3H, m, CH2), 2.4 (1H, m, CH2), 3.6 (2H, m, CH2), 3.7 (2H,
t, CH), 4.2 (1H, m, CH),4.5 (1H, t, CH),7.3(4H, m), 7.4 (4H, m),7.5 (4H, m), 8.1
(1H, s, NH), 8.2 (1H, dd), APCI+ calculated for C26H25N4O2S (MH+): 457.17;
Found: 456.86.
21. Radiosynthesis of [11C]3 and [11C]4: Precursor 1 or 2 (0.6 mg) was dissolved in
400 lL of acetone in a capped 1 ml reactivial. Aqueous NaOH (5 lL, 5 N) was
then added to the solution and the reaction mixture was vortexed. [11C]-
methyl triflate was transported by a stream of argon (20–30 ml/min) into the
reactivial over a period of 5 min at room temperature. At the end of the
trapping, the reaction mixture was diluted with water and directly injected
19. Synthesis
of
(S)-1-(3-(1H-benzo[d]imidazol-2-yl)propanoyl)-N-([1,10-
biphenyl]-2-yl)pyrrolidine-2-carboxamide (2): To a solution of (S)-2-([1,10-
biphenyl]-2-yl)-1-(pyrrolidin-2-yl)ethanone (5, 200 mg, 0.75 mmol) and 2-
benzimidazole propionic acid (143 mg, 0.75 mmol) in DMF (3 ml) was added
HOBT (102 mg, 0.75 mmol), EDC (144 mg, 0.75 mmol), and triethylamine
(76 mg, 1.5 mmol). The reaction mixture was heated to 100 °C for 1 h. After
removal of DMF under high vacuum, the residue was extracted with ethyl
acetate (3 ꢁ 10 ml) and water (3 ꢁ 10 ml). The combined ethyl acetate extracts
was dried over anhydrous MgSO4, filtered and evaporated to provide crude
product. This was chromatographed over silica gel using a mixture of 2:1 ethyl
acetate–hexane to afford desmethyl compound 2 (250 mg, 76%) as a white
solid.1H NMR (CDCl3, 400 MHz): 1.9 (2H, m, CH2), 2.1 (1H, m, CH2), 2.3 (1H, m,
CH2), 2.5 (1H, m, CH2), 2.8(1H, m, CH2), 3.0 (1H, m, CH), 3.2 (1H, m, CH), 3.3
(1H, m, CH), 4.2 (1H, m, CH), 4.6 (1H, m, CH), 7.3 (2H, m), 7.4 (10H, m),8.2 (1H,
m), 8.4 (1H, s); APCl+ calculated for C27H27N4O2 (MH+): 439.21; Found: 439.12.
20. Synthesis of 3 and 4: To a solution of 1 or 2 (0.45 mmol) in dry DMF (1.5 ml) a
room temperature was added Cs2CO3 (186 mg, 0.5 mmol) and iodomethane
(30 ml, 0.45 mmol). The reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was diluted with water (5 ml), extracted
with ethyl acetate (3 ꢁ 10 ml) and the combined extract were dried over
anhydrous MgSO4 and evaporated. The crude products were chromatographed
into a semipreparative RP-HPLC (Phenomenex C18, 10 ꢁ 250 mm, 10
lm) and
eluted with acetonitrile: 0.1 M ammonium formate (50:50) at a flow rate of
10 ml/min. The product fractions of [11C]- and [11C]4 with a retention time of
6–7 and 4–5 min based on
deionized water, and passed through
c
-detector was collected, diluted with 100 ml of
a
classic C-18 Sep-pakÒ cartridge,
respectively. Reconstitution of the product in 1 ml of absolute ethanol
afforded [11C]3 in 30 5% yield and [11C]4 in 25 5% (EOS, n = 6). A portion
of the ethanol solution was analyzed by analytical HPLC (Phenomenex, Prodigy
ODS (3) 4.6 ꢁ 250 mm, 5
formate 50:50, flow rate of: 2 ml/min, retention time: 7.1 min for [11C]3 and
4.8 min for
11C]4, respectively) to determine the specific activity and
radiochemical purity. The chemical and radiochemical purities of [11C]3 and
11C]4 were reconfirmed by RP-HPLC (Waters
Bondapak 4.6 ꢁ 300 mm,
10 m, mobile phase, 50:50 acetonitrile/0.1 M ammonium formate flow rate:
lm mobile phase: acetonitrile/0.1 M ammonium
[
[
l
l
2 ml/min, retention time = 5.8 min). The ethanol solution was then diluted
with 9 ml of saline and passed through a sterile filter.
22. Wilson, A. A.; Jin, L.; Garcia, A.; DaSilva, J. N.; Houle, S. Appl. Rad. Isotop. 2001,
54, 203.