Synthesis and in vitro evaluation of derivatives of the β1-adrenergic receptor antagonist HX-CH 44

Article abstract of DOI:10.1016/j.bmcl.2011.06.106

Isopropyl- and fluoroisopropyl-amino derivatives of the β₁-adrenergic receptor antagonist 2-[4-[3-(tert-butyl-amino)-2- hydroxypropoxy]phenyl]-3-methyl-6-methoxy-4(3H)-quinazolinone ((±)HX-CH 44) were synthesized, including a concise and effici

Full text of DOI:10.1016/j.bmcl.2011.06.106

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5506–5509
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro evaluation of derivatives of the b1-adrenergic
receptor antagonist HX-CH 44
⇑
Karin A. Stephenson, Alan A. Wilson, Sylvain Houle, Neil Vasdev
PET Centre, Centre for Addiction and Mental Health, Department of Psychiatry, University of Toronto, 250 College St., Toronto, Ontario, Canada M5T 1R8
a r t i c l e i n f o
a b s t r a c t
Article history:
Isopropyl- and fluoroisopropyl-amino derivatives of the b1-adrenergic receptor antagonist 2-[4-[3-(tert-
butyl-amino)-2-hydroxypropoxy]phenyl]-3-methyl-6-methoxy-4(3H)-quinazolinone (( )HX-CH 44)
were synthesized, including a concise and efficient preparation of the core, 2-(4-hydroxyphenyl)-6-meth-
oxy-3-methylquinazolin-4(3H)-one. In vitro binding assays showed that the fluorinated analog was
selective towards b1-adrenergic receptors over b2-adrenergic and 5-HT1A receptors. An X-ray crystallo-
graphic characterization of the fluorinated analog is also reported.
Received 20 May 2011
Revised 22 June 2011
Accepted 24 June 2011
Available online 30 June 2011
Keywords:
b-Adrenergic receptors
b-Blocker
Ó 2011 Elsevier Ltd. All rights reserved.
Fluorine
HX-CH 44
In vitro
X-ray crystallography
Recent crystal structures of the b1- and b2-adrenergic recep-
tors1–4 represent a major breakthrough in medicinal chemistry as
their elucidation has significantly expanded opportunities for drug
discovery targeting these therapeutically important G-protein-
coupled receptors. Imaging of b-adrenergic receptors (b-ARs) in
the heart or brain with radiolabeled b-adrenergic antagonists or
agonists (b-blockers) using positron emission tomography (PET)
is a longstanding goal of several laboratories,5–7 including ours.8,9
The most common methodology for such radiosyntheses involves
the introduction of positron-emitting isotopes, 11C (half-
life = 20.4 min) or 18F (half-life = 109.7 min), into the isopropyl-
amine side-chain of the 1-isopropylamino-3-phenoxy-2-propanol
moiety, a characteristic feature of several b-blockers. The use of
Despite extensive efforts, there is still no radiopharmaceutical
available for imaging b1-ARs with PET.6 A lead candidate, 2-[4-[3-
(tert-butylamino)-2-hydroxypropoxy]phenyl]-3-methyl-6-meth-
oxy-4(3H)-quinazolinone (( )HX-CH 44), was selected for derivati-
zation in the present study. This compound has been extensively
characterized pharmacologically, both in vitro and in vivo.15,16
Although the original report of ( )HX-CH 44 demonstrated that
this drug is highly potent and selective (low nM affinity towards
b1 receptors in vitro with b1/b2 selectivity ratio of 800),15 a subse-
quent study by the same group showed that these values are
highly dependent on the assay and the b1 affinity can fluctuate
wildly (from 77 nM to 16 lM) with speculation of multiple binding
sites in rodent heart membranes.16 Racemic HX-CH 44 was previ-
ously radiolabeled with carbon-11 (Fig. 1) and evaluated for cardio-
vascular imaging in animal models.17 Although [11C]-( )HX-CH 44
displayed minimal specific binding to myocardial b1-ARs in the
previous work, development of 18F-labeled derivatives of this com-
pound may prove to yield promising radiotracers as 18F-labeling at
the N-fluoroisopropyl moiety in this class of compounds has shown
improved signal to noise compared with the respective 11C-labeled
analogs.6,7 In addition to the possibility of providing a better match
of the pharmacokinetic profile to the lifetime of the radioisotope,
the longer half-life of fluorine-18 allows more time for radiosyn-
thesis, longer scanning times, as well as the opportunity to trans-
port the radiotracer to neighboring PET facilities. Furthermore,
the previous work based their analysis on a racemic radiotracer,
and the (R)-enantiomer of HX-CH 44 is not active.15 Our goal is
to assess HX-CH 44 derivatives as potential 18F-labeled PET imag-
ing probes.
[
11C]-CGP 1217710–12 and its N-isopropyl analog 11C]-CGP
[
12388,13,14 are established for clinical heart imaging studies
( Fig. 1), and are prepared using the technically demanding
synthons
[ [
11C]-phosgene10 and 11C]-acetone,14 respectively.
Neither compound is subtype-selective, and their widespread use
has been restricted as the production of carbon-11 labeled
radiotracers requires an on-site cyclotron. Our laboratory recently
reported a facile 2-step procedure for the stereoselective syntheses
of 18F-labeled b-blockers bearing a fluoroisopropylamino moiety9
which is a common feature of several radiotracers for imaging
b-ARs in vivo. This radiosynthetic method is being applied to dis-
cover new PET imaging agents for b-ARs.
⇑
Corresponding author. Tel.: +1 416 979 4680; fax: +1 416 979 4656.
E-mail address: neil.vasdev@utoronto.ca (N. Vasdev).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.106

K. A. Stephenson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5506–5509
5507
Figure 1. Chemical structures of [11C]-CGP 12177, [11C]-CGP 12388, and [11C]-HX-CH 44. Asterisk denotes site of 11C.
The present study reports: (1) the synthesis of a racemic N-iso-
propyl derivative, as well as racemic and the 2S-enantiomer of
N-fluoroisopropyl derivatives of HX-CH 44; (2) an efficient route
to the core structure, 2-(4-hydroxyphenyl)-6-methoxy-3-methyl-
quinazolin-4(3H)-one; (3) in vitro binding analyses of these com-
pounds to assess their potency towards b1-adrenergic receptors
and selectivity over b2-adrenergic and 5HT1A receptors; and (4)
the X-ray crystal structure of the fluorinated (2S)-derivative.
New derivatives of HX-CH 44 were prepared, incorporating 1-
isopropylamino group ((R, S)-6) and 1-fluoroisopropylamino
groups ((2R, 2S)-7 and (2S)-7), into the respective 3-phenoxy-2-
propanol moiety (Scheme 1). The HX-CH 44 core was prepared
from 4-(benzyloxy)benzoic acid, that was converted to an acid
chloride with thionyl chloride, and reacted directly with 2-ami-
no-5-methoxybenzoic acid, to prepare the corresponding benzoox-
azin-4-one (1; 62%), from which the N-methylbenzamide (2) was
prepared by reaction with methylamine in 97% yield. Ring-closure
of 2, by condensation of amides, was achieved under basic condi-
tions by heating with KOH to afford the 4(3H)-quinazolinone (3)
in 87% yield. Hydrogenolysis of the benzyl ether to liberate the free
phenol, yielding the HX-CH 44 core (4), was nearly quantitative
(97%). The highly efficient and concise synthesis of 4 is generally
applicable for the preparation of arrays of structurally related qui-
nazolinones. The core compound was used to generate both the
racemic and enantiopure epoxides, (R,S)-5 (82%) and (S)-5 (92%),
Figure 2. Chiral HPLC of
hexanes/ethyl acetate/diethylamine, 1.0 mL/min, k = 254 nm).
7
(ChiralcelÒ OD (250 Â 4.6 mm) column, 70/30/0.1
by reactions with epichlorohydrin or (S)-(+)-glycidyl nosylate,
respectively. Compound
6
was efficiently prepared by
a
Scheme 1. Synthesis 4, 6, (2R, 2S)-7 and (2S)-7. Conditions: (i) (1) SOCl2 (2) 2-amino-5-methoxybenzoic acid, NEt3, 62% (ii) methylamine (33 wt % in EtOH), 97% (iii) KOH, 97%
(iv) Pd/C, H2, 97–100% (g); (v) epichlorohydrin, KOH, DMSO, 82% or (vi) (S)-(+)-glycidyl nosylate, K2CO3, CsF, 92% (vii) isopropylamine, 69% (viii) N-benzyl-1-fluoropropan-2-
amine, 38% from (R,S)-5; (2R, 2S)-7, 45% from (2S)-5; (2S)-7, >99% de with respect to carbon-2. Asterisk denotes chiral center.

5508
K. A. Stephenson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5506–5509
Table 1
were also assayed for 5-HT1A affinity. Whereas (R, S)-6 appeared
In vitro binding affinitiesa (Ki values in
towards b1, b2 and 5-HT1A receptors
lM) for (2R, 2S)-6, (2R, 2S)-7 and (2S)-7
unselective towards all three targets in vitro, both the fluorinated
analogs, (2R, 2S)-7 and (2S)-7, appeared to have high selectivity to-
wards b1-ARs. Fluorination of the isopropylamine moiety has been
shown to increase the lipophilicity of b-blockers,23 likely due to de-
creased basicity of the nitrogen,24,25 and may be responsible for
this enhanced biological behavior. Unfortunately, all three com-
b1
b2
5-HT1A
(2R, 2S)-6
(2R, 2S)-7
(2S)-7
2.4 0.4
2.3 0.4
3.0 0.6
7.7 2.2
>10
5.5 1.0
>10
>10
>10
a
pounds have Ki values between 2 and 3 lM towards b1-ARs and
Ki determinations was generously conducted by the National Institute of Mental
Health’s Psychoactive Drug Screening Program, Contract # NO1MH32004 (NIMH
PDSP). The NIMH PDSP is Directed by Bryan L. Roth MD, PhD at the University of
North Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda
MD, USA. For experimental details please refer to the PDSP web site http://
pdsp.med.unc.edu/ and click on ‘Binding Assay’ on the menu bar.
is likely too high to be useful as leads for PET, particularly for brain
imaging where the b-AR density (Bmax) in the frontal cortex of the
human brain is low (18–147 fmol/mg of protein).26,27 Previous
work,6,7 including ours,8 has demonstrated that radiotracers with
much higher affinities (picomolar) are likely required to success-
fully image b-ARs in humans.
ring-opening reaction of the racemic epoxide (R, S)-5, with isopro-
pylamine in 69% yield. As per our recent successes in synthesizing
fluorinated b-blockers, fluoro-(2S)-toliprolol9 and fluoro-(2S)-
exaprolol,8 the fluorinated derivatives in the present work were
similarly prepared by effecting ring-opening of the epoxides, (R,
S)-5 and (S)-5 by N-benzyl protected fluoroisopropylamine.18 This
convergent approach is straightforward and following hydrogenol-
ysis yielded the fluorinated standard compounds (2S)-7 and (2R,
2S)-7 in moderate yields (38% and 45%, respectively). The diaste-
reomeric excess (de) of (2S)-7 at carbon-2 was >99%, as determined
using chiral HPLC
(Fig. 2). Traditionally, the 2-hydroxyl group of the 1-isopropylami-
no-3-phenoxy-2-propanol moiety is on a stereogenic carbon for
which the 2S-enantiomer has higher affinity for the receptor than
the 2R-enantiomer.19,20 Therefore, both racemic and (2S)-N-fluoro-
isopropyl ( )HX-CH 44 derivatives, ((2R, 2S)-7 and (2S)-7) were
synthesized and evaluated biologically. No attempt was made to
assign the stereochemistry at carbon-5 because it has been shown
that b-blockers fluorinated on the N-isopropyl chain do not show
diastereomeric differences in metabolism or binding in vivo.21
Compounds (R, S)-6, (2R, 2S)-7 and (2S)-7 were assayed for their
in vitro binding affinities towards b1-adrenergic, b2-adrenergic
receptors (Table 1). A study by Roth and coworkers22 speculated
concerns of 5-HT1A affinity with a fluorinated b-blocker used in
PET ([18F]-fluoro-(2S)-Carazolol), therefore all of these compounds
Computer-aided design using the high resolution crystal struc-
tures of b-ARs has been an area of active pursuit in drug discov-
ery.28 We report herein, the X-ray crystal structure of (2S)-7
(Fig. 3). Our laboratory has previously determined the X-ray struc-
tures of N-isopropylbenzamide29 and its N-fluoroisopropyl ana-
log.30 The present structure complements our previous work by
reporting the first crystallographic characterization of the 1-fluoro-
isopropylamino-3-phenoxy-2-propanol moiety that is common to
several 18F-labeled b-blockers and may provide insights into devel-
opment of potent and selective b-blockers. Our future work will
continue to synthesize candidate radiopharmaceuticals to image
b-ARs based on compounds with significantly higher affinities to-
wards these targets, and we anticipate that new candidates for
radiolabeling will be unveiled for this important target in the near
future.
In conclusion, N-isopropyl and N-fluoroisopropyl derivatives of
HX-CH 44 were efficiently synthesized and the synthetic strategy
is generally applicable to preparing arrays of 4(3H)-quinazolinon-
es. In vitro binding assays revealed that although the fluorinated
analogs were selective towards b1-adrenergic receptors they were
not potent enough (Ki = 2 À 3
lM) to warrant radiolabeling with
fluorine-18. The X-ray crystal structure of the (2S)-fluorinated
analog may offer new opportunities to develop higher affinity
b-blockers via computer-aided drug design.
Figure 3. X-ray structure of (2S)-7ÁCHCl3. The structure is disordered about the CH3 and CH2F groups with occupancies of 30% and 70%, respectively.

K. A. Stephenson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5506–5509
5509
8. Stephenson, K. A.; van Oosten, E. M.; Wilson, A. A.; Meyer, J. H.; Houle, S.;
Vasdev, N. Neurochem. Int. 2008, 53, 173.
Acknowledgments
9. Stephenson, K. A.; Wilson, A. A.; Meyer, J. H.; Houle, S.; Vasdev, N. J. Med. Chem.
2008, 51, 5093.
10. Boullais, C.; Crouzel, C.; Syrota, A. J. Labelled Compd. Radiopharm. 1986, 23, 565.
11. Nanoff, C.; Freissmuth, M.; Schutz, W. Naunyn Schmiedebergs Arch. Pharmacol.
1987, 336, 519.
12. Schäfers, M.; Dutka, D.; Rhodes, C. G.; Lammertsma, A. A.; Hermansen, F.;
Schober, O.; Camici, P. G. Circ. Res. 1998, 82, 57.
13. de Jong, R. M.; Willemsen, A. T.; Slart, R. H.; Blanksma, P. K.; van Waarde, A.;
Cornel, J. H.; Vaalburg, W.; van Veldhuisen, D. J.; Elsinga, P. H. Eur. J. Nucl. Med.
Mol. Imaging 2005, 32, 443.
14. Elsinga, P. H.; van Waarde, A.; Jaeggi, K. A.; Schreiber, G.; Heldoorn, M.;
Vaalburg, W. J. Med. Chem. 1997, 40, 3829.
15. Daemmgen, J. W.; Engelhardt, G.; Pelzer, H. Arzneim. Forsch. 1985, 35, 383.
16. Mauz, A. B.; Pelzer, H. J. Cardiovasc. Pharmacol. 1990, 15, 421.
17. Valette, H.; Dollé, F.; Guenther, I.; Demphel, S.; Rasetti, C.; Hinnen, F.; Fuseau,
C.; Crouzel, C. Nucl. Med. Biol. 1999, 26, 105.
We gratefully acknowledge Dr. Bryan Roth at the University of
North Carolina at Chapel Hill and the National Institute of Mental
Health’s Psychoactive Drug Screening Program, Contract
#NO1MH32004, for conducting Ki determinations. Financial sup-
port for this work was provided by the Natural Sciences and Engi-
neering Research Council of Canada (NSERC) and the Canadian
Institutes for Health Research in the form of a Collaborative Health
Research Projects Grant (CHRPJ 322787-06; N.V.) and an NSERC
Postdoctoral Fellowship to K.A.S.
Supplementary data
18. Posakony, J.; Tewson, T. Synthesis 2002, 766.
19. Tewson, T. J.; Stekhova, S.; Kinsey, B.; Chen, L.; Wiens, L.; Barber, R. Nucl. Med.
Biol. 1999, 26, 891.
20. Soloviev, D. V.; Matarrese, M.; Moresco, R. M.; Todde, S.; Bonasera, T. A.; Sudati,
F.; Simonelli, P.; Magni, F.; Colombo, D.; Carpinelli, A.; Galli Kienle, M.; Fazio, F.
Neurochem. Int. 2001, 38, 169.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.106.
References and notes
21. Elsinga, P. H.; Vos, M. G.; van Waarde, A.; Braker, A. H.; de Groot, T. J.; Anthonio,
R. L.; Weemaes, A.-M. A.; Brodde, O.-E.; Visser, G. M.; Vaalburg, W. Nucl. Med.
Biol. 1996, 23, 159.
22. Roth, B. L.; Ernsberger, P.; Steinberg, S.; Rao, S.; Rauser, L.; Savage, J.; Hufeisen,
S.; Berridge, M. S.; Muzic, R. F. Psychopharmacology 2001, 157, 111.
23. Doze, P.; Elsinga, P. H.; Maas, B.; van Waarde, A.; Wegman, T.; Vaalburg, W.
Neurochem. Int. 2002, 40, 145.
1. Rasmussen, S. G.; Choi, H. J.; Rosenbaum, D. M.; Kobilka, T. S.; Thian, F. S.;
Edwards, P. C.; Burghammer, M.; Ratnala, V. R.; Sanishvili, R.; Fischetti, R. F.;
Schertler, G. F.; Weis, W. I.; Kobilka, B. K. Nature 2007, 450, 383.
2. Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G.; Thian, F. S.;
Kobilka, T. S.; Choi, H. J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C.
Science 2007, 318, 1258.
24. Upthagrove, A. L.; Nelson, W. L. Drug Metab. Dispos. 2001, 29, 1377.
25. Schmidt, D. E.; Votaw, J. R.; Kessler, R. M.; De Paulis, T. J. Pharm. Sci. 1994, 83,
305.
3. Rosenbaum, D. M.; Cherezov, V.; Hanson, M. A.; Rasmussen, S. G.; Thian, F. S.;
Kobilka, T. S.; Choi, H. J.; Yao, X. J.; Weis, W. I.; Stevens, R. C.; Kobilka, B. K.
Science 2007, 318, 1266.
26. Cash, R.; Ruberg, M.; Raisman, R.; Agid, Y. Brain Res. 1984, 322, 269.
27. Shimohama, S.; Taniguchi, T.; Fujiwara, M.; Kameyama, M. J. Neurochem. 1987,
48, 1215.
28. Topiol, S.; Sabio, M. Bioorg. Med. Chem. Lett. 2008, 18, 1598.
29. van Oosten, E. M.; Lough, A. J.; Vasdev, N. Acta Crystallogr., Sect. E. 2008, 64,
o1005.
4. Warne, T.; Serrano-Vega, M. J.; Baker, J. G.; Moukhametzianov, R.; Edwards, P.
C.; Henderson, R.; Leslie, A. G.; Tate, C. G.; Schertler, G. F. Nature 2008, 454, 486.
5. Elsinga, P. H.; van Waarde, A.; Visser, T. J.; Vaalburg, W. Clin. Positron Imaging
1998, 1, 81.
6. Kopka, K.; Law, M. P.; Breyholz, H. J.; Faust, A.; Holtke, C.; Riemann, B.; Schober,
O.; Schäfers, M.; Wagner, S. Curr. Med. Chem. 2005, 12, 2057.
7. van Waarde, A.; Vaalburg, W.; Doze, P.; Bosker, F. J.; Elsinga, P. H. Curr. Pharm.
Des. 2004, 10, 1519.
30. Vasdev, N.; van Oosten, E. M.; Stephenson, K. A.; Zadikian, N.; Yudin, A. K.;
Lough, A. J.; Houle, S.; Wilson, A. A. Tetrahedron Lett. 2009, 50, 544.