Design, synthesis and evaluation of dual pharmacology β2- adrenoceptor agonists and PDE4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.11.028

A novel series of formoterol-phthalazinone hybrids were synthesised and evaluated as dual pharmacology β₂-adrenoceptor agonists and PDE4 inhibitors. Most of the hybrids displayed high β₂-adrenoceptor agonist and moderate PDE4 inhibitory activities. The most potent compound, (R,R)-11c, exhibited agonist (EC₅₀ = 1.05 nM, pEC₅₀ = 9.0) and potent PDE4B2 inhibitory activities (IC₅₀ = 0.092 μM).

Full text of DOI:10.1016/j.bmcl.2013.11.028

Bioorganic & Medicinal Chemistry Letters 24 (2014) 249–253
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of dual pharmacology
b2-adrenoceptor agonists and PDE4 inhibitors
a,
Ling Huang a, Wenjun Shan c, Qi Zhou a, Jiaxing Xie b, Kefang Lai b, , Xingshu Li
⇑
⇑
a Institute of Drug Synthesis and Pharmaceutical Process, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
b State Key Laboratory of Respiratory Diseases, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, China
c Jiangsu Hansoh Pharmaceutical Research Institute Co., Ltd, Lianyungang 222000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 June 2013
Revised 24 October 2013
Accepted 12 November 2013
Available online 21 November 2013
A novel series of formoterol–phthalazinone hybrids were synthesised and evaluated as dual pharmacol-
ogy b2-adrenoceptor agonists and PDE4 inhibitors. Most of the hybrids displayed high b2-adrenoceptor
agonist and moderate PDE4 inhibitory activities. The most potent compound, (R,R)-11c, exhibited agonist
(EC50 = 1.05 nM, pEC50 = 9.0) and potent PDE4B2 inhibitory activities (IC50 = 0.092 lM).
Ó 2013 Published by Elsevier Ltd.
Keywords:
Chronic obstructive pulmonary disease
(COPD)
Bronchodilator
b2-Adrenoceptor agonist
PDE4 inhibitor
Chronic obstructive pulmonary disease (COPD) is a chronic
respiratory disease that affects millions of people worldwide. COPD
is characterised by limited airflow to and from the lungs, which is
not fully reversible.1 Although the aetiology of COPD remains to be
fully understood, smooth muscle dysfunction and chronic inflam-
mation are known to play important roles in the pathophysiology
of the disease. Smooth-muscle dysfunction results in exaggerated
bronchoconstriction, bronchial hyperresponsiveness, excessive
proliferation (hyperplasia), and excessive growth (hypertrophy)
of the airway smooth-muscle cells2 and release of proinflamma-
tory mediators.3
b2-Adrenoceptor agonists induce bronchodilatory effects medi-
ated by the relaxation of airway smooth muscles by increasing
cAMP. Thus, inhaled b2 adrenoceptor agonists are widely used to
treat asthma and COPD, providing symptomatic relief by inducing
bronchodilation via the relaxation of airway smooth muscle.4
Currently, salbutamol (a typically short-acting agonist with a rapid
onset of action), salmeterol and formoterol (the two most
prescribed inhaled long-acting b2-agonists) are clinically used as
b2-agonists. In addition, a once-daily b2-agonist indacaterol has
been approved in the USA and Europe for the treatment of COPD.
In recent years, PDE4 has been examined as a suitable target for
anti-inflammatory therapy to treat respiratory diseases.5,6 PDE4
inhibitors have been reported to downregulate inflammatory cell
activity in vitro7,8 and exhibit anti-inflammatory and bronchodila-
tory activity in animal models.9 These therapeutic effects could be
used in the development of new agents, such as steroid-sparing
compounds, to treat diseases associated with chronic airway
inflammation, particularly in the management of asthma and
COPD.10
The multifaceted conditions of some diseases have led to the
development of multifunctional drugs. These drugs possess two
or more complementary biological activities and may represent
an important advancement in the treatment of diseases.11–13 Using
a multivalent approach to drug discovery, Hughes et al. designed
and synthesised dual pharmacology molecules that function as
bronchodilators (Fig. 1, 1) and target both the M3 muscarinic ace-
tylcholine and b2-adrenergic receptors.11 Jones et al. designed and
developed of dual pharmacology b2 agonists–M3 antagonists
(Fig. 1, 2) for the treatment of COPD.14
Recently, we used a multivalent approach to synthesise a class
of dual pharmacology bronchodilators (Fig. 1, 3) that target both
the b2-adrenoceptor and PDE4.15 Here, we present the synthesis
and evaluation of a new series of hybrids that in one molecule
combine both formoterol, which is one of the two most prescribed
inhaled long-acting b2-agonists, and the PDE4 inhibitor phthalazi-
none (Fig. 2).
The synthetic route of dual b2-agonists and PDE4 inhibitors
(11a–11c) is shown in Scheme 1. 1,2-Dimethoxybenzene was
reacted with 1,2-cyclohexanedicarboxylic anhydride to afford a
ketone acid 4, which was subsequently reacted with hydrazine to
⁄ Corresponding authors. Tel./fax: +86 20 3994 3050.
E-mail addresses: lixs@mail.sysu.edu.cn, lixsh@mail.sysu.edu.cn (X. Li).
0960-894X/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.11.028

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L. Huang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 249–253
Figure 1. Structures of reported dual pharmacology bronchodilators for the treatment of asthma or COPD.
Epoxide intermediate 9, the intermediate containing the phar-
macophore of the b2-adrenoceptor agonist, was prepared using
commercially available 4-hydroxy-3-nitro-acetophenone in sev-
eral steps according to previously reported procedures
(Scheme 3).18
In order to investigate the relationship between the chiral cen-
ter of the compounds and the activity of the b2-adrenoceptor, an
enantiomer of 11c, (R,R)-11c, was synthesized using enantiopure
secondly amine (R)-7 and enantiopure epoxide (R)-9 as the inter-
mediates according to the synthetic route shown in Scheme 1.
(R)-7 and (R)-9 were prepared according to procedures reported
previously.18
For studying the in vitro b2-adrenoceptor agonist activity of
target compounds, The effects of 11a–11c and (R,R)-11c on the
tracheal rings of guinea pigs were assessed using (R,R)-formoterol
according to a previously described protocol.19,20 The concentra-
tion–response curves are showen in Figure 3 and the EC50 and
pEC50 values are summarized in Table 1. The maximum relaxant
effect exhibited by Isoproterenol was considered to be 100%. The
EC50 values were estimated from the concentration–response
Figure 2. The synthesised b2-adrenoceptor agonists-PDE4 inhibitors.
yield the known PDE4 inhibitor 4-(3,4-dimethoxyphenyl)-
4a,5,8,8a-tetrahydro-2H-phthalazin-1-one (5).16,17 N-Alkyl phtha-
lazinones 6a–6c were obtained by treating compound 5 with
dibromoalkanes and NaH in DMF. The intermediates were then
reacted with 4-(2-(benzylamino)propyl)phenol (7) to provide
compounds 8a–8c. Finally, the target compounds (11a–11c) were
obtained by coupling epoxide 9 with compounds 8a–8c followed
by deprotection via hydrogenation in the presence of Pd/C.
The racemic secondly amine 7 was synthesised in a one-pot
reaction reductive hydrogenation and subsequent O-demethyla-
tion reaction in an overall yield of 72% (Scheme 2).
Scheme 1. Synthetic scheme for synthesis of dual b2-adrenoceptor agonists-PDE4 inhibitors. Reagents and conditions: (a) AlCl3, CH2CH2, reflux, 76%; (b) NH2NH2, EtOH,
reflux, 32%; (c) Br(CH2)nBr, NaH, DMF, rt, 80–88%; (d) 7, K2CO3, DMF, 60 °C, 60–65%; (e) 9, neat, 120 °C, 55–61%; (f) 10% Pd/C, H2, MeOH, rt, 58–65%.

L. Huang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 249–253
251
Scheme 2. Recemic synthesis of secondary amine 7. Reagents and conditions: (a) 5% Pt/C, H2, MeOH, 60 °C, 95%; (b) BBr3, CH2Cl2, 10 °C, 76%.
Scheme 3. Recemic synthesis of epoxide intermediate 9. Reagents and conditions: (a) (chloromethyl)benzene, K2CO3, actone–H2O, 60 °C, 90.5%; (b) bromine, acetic acid,
20 °C, 85%; (c) NaBH4, THF, 0 °C to rt, 91%; (d) Pt/C, Me2S, H2; formaldehyde, acetic anhydride, 0 °C to rt, 83%; (e) K2CO3, THF/MeOH, rt, 98%.
Table 1
EC50 for b2-adrenergic agonist compounds in the function ex vivo guinea pig trachea bioassay
OH
OH
H
N
H
N
H
N
H
HO
HO
O
HO
O
Isoproterenol
(R, R)-Formoterol
OH
OH
H
H
N
H
N
H
N
O
O
N
O
O
N
N
n-1
O
O
O
O
HO
O
N
HO
O
N
n-1
(R,R)-11c: n = 6
11a-11c: n = 4, 5, 6
a
Compounds
n
EC50 (nM)
pEC50
Isoproterenol
(R,R)-Formoterol
11a
11b
11c
—
—
4
5
6
31.64 0.50
0.13 0.01
10.32 0.22
12.59 0.17
5.02 0.03
1.05 0.01
7.5 (7.8b)
9.9 (9.5c)
8.0
7.9
8.3
(R,R)-11c
6
9.0
a
b
c
Data are of average of three determinations SEM.
Ref. 19.
Ref. 21.
curves by means of nonlinear regression analysis with the experi-
mental data.
pKd) of 5.02 nM and 8.3, respectively. This value is higher than that
of isoproterenol (EC50 = 31.64 nM, pEC50 = 7.5). (R,R)-11c was also
examined as a b2-adrenoceptor agonist using an in vitro evalua-
tion, and the results showed that (R,R)-11c (EC50 = 1.05 nM,
The results shown in Figure 3 indicate that all of the hybrids
exhibited relaxant effects on tracheal rings precontracted with his-
tamine. All of the compounds induced concentration-dependent
relaxation, and the maximum response (Emax) to each compound
was similar to that evoked by the reference compound isoprotere-
nol. Among the three target compounds (11a–11c), compound 11c,
which contains a 6-carbon linker between the phthalazinone and
formoterol moieties, displayed the most potent b2-adrenoceptor
agonist activity (Table 1) with EC50 and pEC50 value (related to
pEC50 = 9.0) exhibited about
a fivefold increase in potency
compared to its racemic form (11c, EC50 = 5.02 nM, pEC50 = 8.3).
However, (R,R)-11c was less potent than that of (R,R)-formoterol
(EC50 = 0.13 nM, pEC50 = 9.9).
When isolated guinea pig tracheal rings were precontracted
with histamine and incubated with the well-known b2-AR selective
inverse agonist ICI-118551 (1 Â 10À9 to 1 Â 10À7 M), the dose–re-

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L. Huang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 249–253
Figure 5. Schild plot for the antagonism of ICI-118551 versus (R,R)-11c on isolated
guinea pig trachea strips precontracted with histamine. The pA2 values were
calculated from the formulation pA2 = Log (CR-1)—Log [B]. Values for concentration
ratio (CR) were obtained from EC50 values in the presence or absence of ICI-118551.
Figure 3. Effects of agonists and isoproterenol on isolated guinea pig tracheal rings
precontracted with 30 lM of histamine. The maximum relaxant effect exhibited by
Isoproterenol was considered to be 100%. Each data point represents the mean
effect (n = 3).
sponse curve for compound (R,R)-11c shifted to the right (Fig. 4),
indicating antagonist effects and the pharmacology of compound
(R,R)-11c is partly driven as b2-adrenoceptor agonist. The result
of schild plot (Fig. 5) showed that the pA2 value for the antagonist
activity of ICI-118551 in the presence of (R,R)-11c was 9.36.
It is well known that the PDE4 family consist of four isoforms:
PDE4A, 4B, 4C and 4D. Knockout studies have revealed that PDE4B
could suppress TNF-a production, which suggested that PDE4B
inhibitors are expected to be useful anti-inflammatory agents.22
PDE4B is a hydrolytic enzyme responsible for the degradation of
the second messenger cAMP. As an increase in intracellular levels
of cAMP can reduce the activation of a wide range of inflammatory,
increasing the cAMP concentration by inhibiting PDE4B activity is
thought to be one of the approach to treatment of COPD.23 Consid-
ering PDE4B2 is the predominant PDE4B species and study24 certi-
fied it undergoes differential regulation of gene expression in
human monocytes and neutrophils, we choose PDE4B2 isoform
as assay enzyme.
Figure 4. The antagonistic action for ICI-118551 on guinea pig tracheal muscle
precontracted with histamine and used (R,R)-11c as agonist. j = (R,R)-11c
(1 Â 10À11 ꢀ 1 Â 10À6 M) in absence of ICI-118551; d = (R,R)-11c (1 Â 10À10.5
ꢀ
Compounds 11a–11c were also tested for inhibition of cAMP
hydrolysis by recombinant human PDE4B2 in vitro using a colouri-
metric assay from Biomol (Enzo Life Science) and the protocol
described by the manufacturer. The known PDE4 inhibitors
1 Â 10À6 M) in the presence of ICI-118551 (1 Â 10À9 M); N = (R,R)-11c (1 Â 10À10
ꢀ 1 Â 10À5 M) in the presence of ICI-118551(1 Â 10À8 M); . = (R,R)-11c (1 Â 10À9
1 Â 10À4 M) in the presence of ICI-118551(1 Â 10À7 M).
ꢀ
Table 2
Inhibition of cAMP hydrolysis by recombinant human PDE4B2 in the presence of 11 and new compoundsa
F
F
NC
HO
O
O
O
O
COOH
Cl
H
N
O
O
O
N
NH
Cl
Roflumilast
Cilomilast
(R)-Rolipram
Compounds
n
PDE4B2 inhibition IC50 (lM)
(R)-Rolipram
5
11a
11b
11c
(R,R)-11c
Cilomilast
Roflumilast
—
—
4
5
6
6
—
—
0.500 0.028
0.520 0.042
0.117 0.008
0.118 0.015
0.104 0.010
0.092 0.009
0.120 (0.095b)
0.001 (0.0008c)
a
b
c
Data are of average of three determinations SEM.
Ref. 25.
Ref. 26.

L. Huang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 249–253
253
Table 3
References and notes
Values (MW, HBD, HBA, PSA, and LogP)a of 11a, 11b, 11c
1. Prat, M.; Buil, M. A.; Fernández, M. D.; Castro, J.; Monleón, J. M.; Tort, L.; Casals,
G.; Ferrer, M.; Huerta, J. M.; Espinosa, S.; López, M.; Segarra, V.; Gavaldà, A.;
Miralpeix, M.; Ramos, I.; Vilella, D.; González, M.; Córdoba, M.; Cárdenas, A.;
Antón, F.; Beleta, J.; Ryder, H. Bioorg. Med. Chem. Lett. 2011, 21, 3457.
2. Carroll, N.; Elliot, J.; Morton, A.; James, A. Am. Rev. Respir. Dis. 1993, 47, 405.
3. John, M.; Au, B.-T.; Jose, P. J.; Lim, S.; Saunders, M.; Barnes, P. J.; Mitchell, J. A.;
Belvisi, M. G.; Fan Chung, K. Am. J. Respir. Cell Mol. Biol. 1998, 18, 84.
4. Summerhill, S.; Stroud, T.; Nagendra, R.; Perros-Huguet, C.; Trevethick, M. J.
Pharmacol. Toxicol. Methods 2008, 58, 189.
11a
11b
11c
MW
HBD
HBA
PSA
LogP
AClogP
AlogP
672
4
9
141.95
4.89
4.53
5.08
686
4
9
141.95
5.33
4.99
5.53
700
4
9
141.95
5.78
5.46
5.99
5. Lipworth, B. J. Lancet 2005, 365, 167.
a
6. Pagès, L.; Gavaldà, A.; Lehner, M. D. Expert Opin. Ther. Pat. 2009, 19, 1501.
7. Torphy, T. Am. J. Respir. Crit. Care Med. 1998, 157, 351.
8. David, M. E. Biochem. Pharmacol. 1999, 57, 965.
9. Souness, J. E.; Aldous, D.; Sargent, C. Immunopharmacology 2000, 47, 127.
10. Beattie, D.; Bradley, M.; Brearley, A.; Charlton, S. J.; Cuenoud, B. M.; Fairhurst, R.
A.; Gedeck, P.; Gosling, M.; Janus, D.; Jones, D.; Lewis, C.; McCarthy, C.;
Oakman, H.; Stringer, R.; Taylor, R. J.; Tuffnell, A. Bioorg. Med. Chem. Lett. 2010,
20, 5302.
Abbreviations: MW, molecular weight; HBD, hydrogen-bond donor atoms; HBA,
hydrogen-bond acceptor atoms; PSA, polar surface area; logP, AClogP, and ALogP,
calculated logarithm of the octanol–water partition coefficient. MW, HBD, HBA,
NROT, PSA, and LogP were calculated online by Marvin software.27 AClogP and
ALogP were calculated online by the ALOGPS 2.1 program.28
11. Hughes, A. D.; Chin, K. H.; Dunham, S. L.; Jasper, J. R.; King, K. E.; Lee, T. W.;
Mammen, M.; Martin, J.; Steinfeld, T. Bioorg. Med. Chem. Lett. 2011, 21, 1354.
12. Provins, L.; Christophe, B.; Danhaive, P.; Dulieu, J.; Durieu, V.; Gillard, M.;
Lebon, F.; Lengelé, S.; Quéré, L.; van Keulen, B. Bioorg. Med. Chem. Lett. 2006, 16,
1834.
compound 5 and rolipram were used as standards (Table 2). The
results in Table 2 indicate that all target compounds provided good
PDE4B2 inhibitory activity (Table 2, compounds 11a–11c exhibited
IC50 values of 0.117, 0.118, and 0.104 lM, respectively). The higher
13. Cashman, J. R.; Voelker, T.; Zhang, H.-T.; O’Donnell, J. M. J. Med. Chem. 2009, 52,
1530.
potency of compounds 11a–11c compared to that of the lead com-
pound 5 suggests that N-substitution is favourable for the inhibi-
tion of human PDE4B2. The stereoisomer (R,R)-11c
14. Jones, L. H.; Baldock, H.; Bunnage, M. E.; Burrows, J.; Clarke, N.; Coghlan, M.;
Entwistle, D.; Fairman, D.; Feeder, N.; Fulton, C.; Hilton, L.; James, K.; Jones, R.
M.; Kenyon, A. S.; Marshall, S.; Newman, S. D.; Osborne, R.; Patel, S.; Selby, M.
D.; Stuart, E. F.; Trevethick, M. A.; Wright, K. N.; Price, D. A. Bioorg. Med. Chem.
Lett. 2011, 21, 2759.
(IC50 = 0.092
PDE4B2 with 11c (IC50 = 0.104
l
M) showed a similar inhibitory activity against
M).
l
15. Shan, W.-J.; Huang, L.; Zhou, Q.; Jiang, H.-L.; Luo, Z.-H.; Lai, K.-F.; Li, X.-S. Bioorg.
Med. Chem. Lett. 2012, 22, 1523.
In order to evaluate the Drug-Like properties of target com-
pounds, the important pharmacological parameters of 11a–11c
were calculated online by the computer programs Marvin27 and
ALOGPS 2.1.28 The values were presented in Table 3.
In summary, we have developed a series of dual pharmacology
molecules that behave as both b2-adrenoceptor agonists and PDE4
inhibitors. These compounds displayed moderate to high b2-adre-
noceptor agonist activities on isolated guinea pig tracheal rings
precontracted with histamine. Among them, compound (R,R)-11c
exhibited the most potent agonist activity with a pEC50 value of
approximately 9.0. Moreover, compound (R,R)-11c displayed
16. Van der Mey, M.; Hatzelmann, A.; Van Klink, G. P. M.; Van der Laan, I. J.; Sterk,
G. J.; Thibaut, U.; Ulrich, W. R.; Timmerman, H. J. Med. Chem. 2001, 44, 2523.
17. Van der Mey, M.; Boss, H.; Couwenberg, D.; Hatzelmann, A.; Sterk, G. J.;
Goubitz, K.; Schenk, H.; Timmerman, H. J. Med. Chem. 2002, 45, 2526.
18. Huang, L.; Liu, J.; Shan, W.; Liu, B.; Shi, A.; Li, X. Chirality 2010, 22, 206.
19. Soriano-Ursúa, M. A.; Correa-Basurto, J.; Valencia-Hernández, I.; Amezcua-
Gutiérrez, M. A.; Padilla-Martínez, I. I.; Trujillo-Ferrara, J. G. Bioorg. Med. Chem.
Lett. 2010, 20, 5623.
20. Shan, W. J.; Huang, L.; Zhou, Q.; Jiang, H. L.; Luo, Z. H.; Lai, K. F.; Li, X. S. Bioorg.
Med. Chem. Lett. 2012, 22, 1523.
21. Handley, D. A.; Senanayake, C. H.; Dutczak, W.; Benovic, J. L.; Walle, T.; Penn, R.
B.; Wilkinson, H. S.; Tanoury, G. J.; Andersson, R. G. G.; Johansson, F.; Morley, J.
Pulm. Pharmacol. Ther. 2002, 15, 135.
22. Jin, S.-L. C.; Conti, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7628.
23. Spina, D. Br. J. Pharmacol. 2008, 155, 308.
24. Wang, P.; Wu, P.; Ohleth, K. M.; Egan, R. W.; Billah, M. M. Mol. Pharmacol. 1999,
56, 170.
25. Christensen, S. B.; Guider, A.; Forster, C. J.; Gleason, J. G.; Bender, P. E.;
Karpinski, J. M.; DeWolf, W. E.; Barnette, M. S.; Underwood, D. C.; Griswold, D.
E.; Cieslinski, L. B.; Burman, M.; Bochnowicz, S.; Osborn, R. R.; Manning, C. D.;
Grous, M.; Hillegas, L. M.; Bartus, J. O. L.; Ryan, M. D.; Eggleston, D. S.;
Haltiwanger, R. C.; Torphy, T. J. J. Med. Chem. 1998, 41, 821.
strong PDE4B2 inhibitory activity with an IC50 of 0.092 lM. Further
studies to investigate the binding affinity of compound (R,R)-11c to
the human b2-adrenoceptor and the efficacy in an animal model of
COPD are in progress.
Acknowledgments
We thank the State Key Laboratory of Respiratory Diseases
(2007DA-80154F1110), National Natural Science Foundation of
China (No. 21302235), Ph.D. Programs Foundation of Ministry of
Education of China (20120171120045), and Distinguished Young
Talents in Higher Education of Guangdong (2012LYM_003) for
financial support of this study.
26. Hatzelmann, A.; Schudt, C. J. Pharmacol. Exp. Ther. 2001, 297, 267.
27. Marvin, Calculator Plugin and Chemical Terms Demo. http://
www.chemaxon.com/marvin/sketch/index.jsp.
28. The ALOGPS 2.1 program. http://www.vcclab.org/lab/alogps/start.html.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
11.028.