Hybrids consisting of the pharmacophores of salmeterol and roflumilast or phthalazinone: Dual β2-adrenoceptor agonists-PDE4 inhibitors for the treatment of COPD

Article abstract of DOI:10.1016/j.bmcl.2012.11.058

A novel class of dual pharmacology bronchodilators targeting both β₂-adrenoceptor and PDE4 was designed and synthesised by combining the pharmacophores of salmeterol and roflumilast or phthalazinone. All the compounds exhibited better β₂-adrenoceptor agonist activities (pEC₅₀ = 8.47-9.20) than the reference compound salmeterol (pEC₅₀ = 8.3) and good inhibitory activity on PDE4B2 (IC ₅₀ = 0.235-1.093 μM).

Full text of DOI:10.1016/j.bmcl.2012.11.058

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1548–1552
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hybrids consisting of the pharmacophores of salmeterol and
roflumilast or phthalazinone: Dual b2-adrenoceptor agonists-PDE4
inhibitors for the treatment of COPD
Anqiu Liu a, Ling Huang a, Zhiren Wang a, Zonghua Luo a, Fei Mao a, Wenjun Shan a, Jiaxing Xie b,
Kefang Lai b, , Xingshu Li a,
⇑
⇑
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, Guangzhou Medical College, Guangzhou 510120, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of dual pharmacology bronchodilators targeting both b2-adrenoceptor and PDE4 was
designed and synthesised by combining the pharmacophores of salmeterol and roflumilast or phthalazi-
none. All the compounds exhibited better b2-adrenoceptor agonist activities (pEC50 = 8.47–9.20) than the
reference compound salmeterol (pEC50 = 8.3) and good inhibitory activity on PDE4B2 (IC50 = 0.235–
Received 11 September 2012
Revised 1 November 2012
Accepted 15 November 2012
Available online 29 November 2012
1.093 lM).
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
COPD
Bronchodilator
b2-Adrenoceptor agonist
PDE4 inhibitor
Chronic obstructive pulmonary disease (COPD) is characterised
by limited expiratory airflow, which is the result of several types of
anatomical lesions, including loss of lung elastic recoil, fibrosis, and
narrowing of small airways.1 It is the fourth leading cause of mor-
tality worldwide; over 600 million individuals worldwide have
COPD, and nearly 3 million die from this disorder annually.2
Although the exact pathogenesis of COPD is unknown, smooth
muscle dysfunction and chronic inflammation are known to play
important roles in the disease. Consequently, both anti-inflamma-
tory and bronchodilator medicines are used extensively for the
treatment of this disorder.
Inflammation in COPD is not present only in the lungs but is in-
stead a significant systemic inflammation.3 It is believed that lung
inflammation spreads to the systemic circulatory system through
the rich lung capillary network causing systemic inflammation.
COPD is postulated to be involved in the pathogenesis of systemic
inflammation.3,4 It is well known that cyclic adenosine monophos-
phate (cAMP) blocks proliferation and chemotaxis of inflammatory
cells, inhibits proinflammatory cell activity, and suppresses
the release of inflammatory and cytotoxic mediators in the lungs.5
Phosphodiesterases can hydrolyse cAMP to 50-adenosine mono-
phosphate, rendering it inactive. Among the eleven members of
the phosphodiesterase enzyme family, phosphodiesterase type 4
(PDE4)6,7 is particularly abundant in inflammatory, immune, and
airway smooth muscle cells.8 The inflammatory response is highly
sensitive to levels of cAMP. PDE4 inhibitors can block cAMP hydro-
lysis and provide anti-inflammatory activity in the airways by
inhibiting the activity of PDE4. In recent years, PDE4 inhibitors
have been evaluated as promising therapies for the treatment of
inflammatory pulmonary disorders, including asthma and COPD.9
Roflumilast, a PDE4 inhibitor, has been approved by the FDA as a
long-term maintenance therapy for COPD.10
b2-Adrenoceptor agonists are well established in the treatment
of both asthma and COPD due to their rapidly evoking bronchodi-
latation effects.11 The mechanism of action of this class of com-
pounds is believed to involve the stimulation of adenylyl cyclase
and subsequent activation of cAMP/cAMP-dependent protein ki-
nase cascade.12,13 Current b2-receptor agonists include short-acting
(e.g., salbutamol and fenoterol) and long-acting (e.g., salmeterol,
formoterol and indacaterol) species.14 Among them, salmeterol
and formoterol are the two most prescribed long-acting b2-recep-
tor agonists, which provide longer lasting bronchodilation, im-
prove quality of life and are associated with a lower frequency of
exacerbation compared with short-acting b2 agonists.
The multifaceted symptoms associated with some diseases
have encouraged active research in the development of multifunc-
tional drugs. For the treatment of COPD and asthma, successful use
of combination products such as CombiventÒ (salmeterol and flu-
ticasone propionate) and SymbicortÒ (formoterol and budesonide),
⇑
Corresponding authors. Tel./fax: +86 20 3994 3050.
E-mail address: lixs@mail.sysu.edu.cn (K. Lai, X. Li).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.058

A. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1548–1552
1549
as well as combination studies with tiotropium and formoterol,
have confirmed the complementary effects of simultaneously tar-
geting multiple mechanisms with two drugs, which can provide
greater improvement in lung function compared to single agent
bronchodilators.15
1,2-dimethoxybenzene and cis-1,2-cyclohexanedicarboxylic anhy-
dride according the literature.16 Compounds 15a–d were reacted
with intermediate 3 to provide 16a–d. Finally, the dual b2-agonists
and PDE4 inhibitors 17a–d were obtained by deprotection of com-
pounds 16a–d in the presence of Pd/C under hydrogen followed by
reaction with NaBH4 then treated with hydrochloric acid.
In previous work, we have developed of a multivalent approach
to synthesise a class of dual-pharmacology bronchodilators that
target both the b2-adrenoceptor and PDE4.16 Here, we report the
synthesis of a new series of hybrids that combine both salmeterol
and the PDE4 inhibitors roflumilast or phthalazinone (a potent
PDE4 inhibitor),17,18 which are expected to provide both b2-adre-
noceptor agonism activity and PDE4 inhibition activity (Fig. 1).
The syntheses of the dual b2-agonists and PDE4 inhibitors (11a–
d and 17a–d) are shown in Schemes 1–3. First, compound 3 was
prepared in several steps according to procedures reported previ-
ously.19 The reduction of 3 with sodium borohydride provided
alcohol 4, which was then protected with TBDMSCl to give com-
pound 5, the synthetic intermediates containing the pharmaco-
phore of the b2-adrenoceptor agonist.
The synthetic intermediate containing the pharmacophore of
the PDE4 inhibitor roflumilast 9a–d was was shown in Scheme 2.
First, the reaction of isoindoline-1,3-dione with dibromides gave
6a–d, which were reacted with 3-hydroxy-4-methoxybenzalde-
hyde to provide intermediates 7a–d. The oxidation of compounds
7a–d with NaClO2 in the presence of H2O2 gave intermediates
8a–d, which reacted in succession with SOCl2, 4-amino-3,5-dichlo-
ropyridine and H2NNH2 to provide 9a–d, the synthetic intermedi-
ates containing the pharmacophore of the PDE4 inhibitor
roflumilast. Intermediates 10a–d were prepared by the coupling
of 5 and 9 in the presence of triethylamine. Finally, target com-
pounds 11a–d were obtained by the deprotection of 10a–d with
the NH4F–EtOH–H2O system followed by reaction with hydrogen
chloride in alcohol.
The b2-adrenoceptor agonist in vitro assays were carried out
using a protocol described previously.20 Isoprenaline and salmeter-
ol, the known b2-adrenoceptor agonists, were used as reference
compounds. The effect of the synthesised compounds on the tra-
cheal rings of guinea pigs pre-contracted by histamine (Fig. 2) indi-
cated that all the tested compounds produced a relaxant effect and
induced a concentration-dependent relaxation. The maximum re-
sponse (Emax) to each was similar to that evoked by the reference
compound isoprenaline. The results in Table 1 indicate that all
eight synthesised compounds exhibited more potent b2-adreno-
ceptor agonist activity (up to a 9.20 pEC50 value, related to pKd)
than the reference compounds isoprenaline (pEC50 = 7.5) and
salmeterol (pEC50 = 8.3). Among them, the series (17a–17d) con-
nected by a 2–6 carbon linker between the PDE4 inhibitor phthala-
zinone and the b2-adrenoceptor agonist moiety salmeterol
exhibited better activities (with the pEC50 values of 9.08–9.20)
than 11a–11d (with the pEC50 values of 8.47–8.71), the hybrids
consisting of salmeterol and a roflumilast moiety. However, it
seems that the length of the linkers have little effect on the activ-
ities of both series of compounds.
Compounds 11a–11d and 17a–17d were also evaluated for
inhibition of cAMP hydrolysis by recombinant human PDE4B2
in vitro by a colorimetric assay method from Biomol (Enzo Life
Science, Inc., NY, USA) following the protocol described by the
manufacturer and using rolipram and roflumilast as reference
PDE4 inhibitors.22 The results in Table 2 show that most of the
target compounds provided good PDE4B2 inhibitory activity (Com-
pounds 11a, 11b, 11c and 11d gave IC50 values of 1.093, 0.601,
Target compounds 17a–d were synthesised according the route
shown in Scheme 3. First, compound 15a–d was prepared from
0.394 and 0.235 lM; compounds 17a, 17b, 17c and 17d gave IC50
Figure 1. Design strategy for compounds 11a–11d, 17a–11d.

1550
A. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1548–1552
Scheme 1. Reagents and conditions: (a) AlCl3, BrCH2COBr, CH2Cl2, reflux, 15 h, 80%; (b) NaBH4, acetic acid, 10 °C, 1 h, 85%; (c) 2,2-dimethoxypropane, CH2Cl2, rt, 10 h, 82%; (d)
NaBH4, methanol, 0 °C to rt, 1 h, 95%; (e) TBDMSCl, imidazole, DMF, rt, 16 h, 57%.
Scheme 2. Reagents and conditions: (a) K2CO3, DMF, Br(CH2)nBr, rt, 16 h, 78–85%; (b) K2CO3, KI, DMF, isovanillin, 65 °C, 18 h, 73–81%; (c) NaClO2, H2O2, acetonitrile, rt, 24 h,
85–91%; (d) SOCl2, reflux, 6 h; (e) NaH, 4-amino-3,5-dichloropyridine, DMF, 20 °C then rt, 1 h; (f) H2NNH2ꢀH2O, EtOH, reflux, 2 h, 17–20% over three steps; (g) DMF, Et3N,
65 °C, 24 h, 36–41%;(h) NH4F, EtOH, H2O, 45 °C, 48 h; (i) 1 M HCl, EtOH, rt, 12 h, 61–67% over two steps.
values of 0.282, 0.278, 0.271 and 0.263
l
M, respectively). Prelimin-
It is worthy to note that there is still a far distance to ideal bal-
ance of two activities potencies in single molecule (The
ary structure–activity relationship analysis revealed that the inhib-
itory potency of the two series is closely related to the length of the
alkyl chain, but the salmeterol–roflumilast series is more
obvious. Compound 11d, with six methylene groups between the
b2-adrenoceptor agonist moiety and PDE4B2 roflumilast moiety
provided the best inhibitory potency (IC50 = 0.235 lM) of all the
tested compounds, while compound 11d, with three methylene
groups between the two pharmacophores, gave the weakest inhib-
itory potency (IC50 = 1.093 lM). Although the trend in inhibitory
a
pIC50 = 6.58 for PDE4, while the pEC50 = 9.20 for b2-adrenoceptor
agonists 17d). The crystal structure of the complex of roflumilast
and PDE4 showed the cyclopropyl pharmacophore of roflumilast
has a good interaction with the hydrophobic groove of PDE4. The
loss of activity for the PDE4 pharmacophore in the hybrids should
be attributed to the bulky and polarity effect of the molecule which
cyclopropyl moiety of roflumilast was replaced by b2-adrenoceptor
agonists. Therefore, to optimize the structures of the PDE4 inhibi-
tor moiety for increasing the inhibitory potency of the PDE4 would
be the emphases in future work.
potency is similar to that of compounds 11a–11d, compounds
17a–17d show less variation in potency when varying the linker
from two methylene groups to six methylene groups (17a, two
In a summary, we have designed, synthesised and evaluated a
series of dual functional molecules that possess the pharmaco-
phores of long-acting b2-receptor agonist salmeterol and PDE4
methylene groups, IC50 = 0.282
IC50 = 0.263 M).
lM; 17d, six methylene groups,
l

A. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1548–1552
1551
Scheme 3. Reagents and condition: (a)cis-1,2-cyclohexanedicarboxylic anhydride, AlCl3, CH2Cl2, reflux, 20 h, 82%; (b) H2NNH2, EtOH, reflux, 3 h, 93%; (c) Br(CH2)nBr, NaH,
DMF, 0 °C to rt, 1 h, 78–84%; (d) benzylamine, rt, 10 h, 66–77%; (e) CH2Cl2, Et3N, rt,12 h, 65–71%; (f) Pd/C, H2, 500 psi, 24–30 h; (g) NaBH4, methanol, 0 °C to rt, 1 h; (h)1 M HCl,
EtOH, rt, 12 h, 66–72% over three steps.
Table 1
pEC50 for b2-adrenergic agonist compounds in the ex vivo guinea pig trachea
bioassaya
b
Compound
n
pEC50
Emax (%)
Isoprenaline
11a
11b
11c
11d
17a
17b
17c
17d
—
3
4
5
6
2
4
5
6
—
7.54
8.47
8.55
8.71
8.50
9.19
9.17
9.08
9.20
8.3c
100
100
103
103
105
102
100
100
102
—
Salmeterol
a
Effect of isoproterenol and new b2-adrenoceptor agonists on isolated guinea pig
tracheal rings pre-contracted by histamine at 30 lM. The maximum relaxant effect
by isoproterenol was considered 100%.
b
pEC50 values are the mean of three experiments.
Ref. 21.
c
Table 2
Inhibition of cAMP hydrolysis by recombinant human PDE4B2 in the presence of 13
Figure 2. Effect of agonists and isoproterenol on isolated guinea pig tracheal rings
pre-contracted by histamine at 30 M. The maximum relaxant effect by isoprote-
and new compoundsa
l
renol was considered 100%. Each point represents the mean effect (n = 3).
Compounds
n
PDE4B2 inhibition IC50 (lM)
(R)-Rolipram
Phthalazinone (13)
11a
11b
11c
11d
17a
17b
—
—
3
4
5
6
2
4
5
0.500 0.032
0.520 0.048
1.093 0.020
0.601 0.011
0.394 0.024
0.235 0.010
0.282 0.08
0.278 0.012
0.271 0.09
0.263 0.07
0.001 (0.0008b)
inhibitors roflumilast or phthalazinone. All the compounds exhib-
ited better b2-adrenoceptor agonist activities on isolated guinea
pig tracheal rings pre-contracted by histamine than the reference
compounds isoprenaline and salmeterol. Meanwhile, most of the
hybrids are more potent than (R)-Rolipram, the first generation
PDE4 inhibitor, It is worthy to note that compound 17d, with six
methylene groups between the b2-adrenoceptor agonist moiety
and PDE4B2 phthalazinone moiety, showed high inhibitory activity
17c
17d
Roflumilast
6
—
a
Data are of average of three determinations SEM.
Ref. 23.
b
toward PDE4B2 (with an IC50 value 0.263 lM) and exhibited the
most potent agonist activity. As a preliminary study, we use alkyl
linkers of varying lengths to join the two pharmacophores, which
lead to a flat SAR. Further investigations including the linkers mod-
ification, and into inhaled COPD candidates (considering the route
of most drugs for the treatment of COPD are inhaled approach)
based on these results are in progress.

1552
A. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1548–1552
8. Gratteri, P.; Bonaccini, G.; Melani, F. J. Med. Chem. 2005, 48, 1657.
Acknowledgments
9. Vignola, A. M. Resp. Med. 2004, 98, 495.
10. Pinner, N. A.; Hamilton, L. A.; Hughes, A. Clin. Ther. 2012, 34, 56.
11. Waldeck, B. Eur. J. Pharmacol. 2002, 445, 1.
12. Giembycz, M. A.; Newton, R. Eur. Respir. J. 2006, 27, 1286.
13. Lotvall, J. Respir. Med. 2000, 94, S6.
14. Hughes, A. D.; Chin, K. H.; Dunham, S. L.; Jasper, J. R.; King, K. E. Bioorg. Med.
Chem. Lett. 2011, 21, 1354.
We thank the Natural Science Foundation of China (20972198)
and State Key Laboratory of Respiratory Diseases (2007DA-
80154F1110) for financial support of this study.
15. van Noord, J. A.; Aumann, J. L.; Janssens, E.; Smeets, J. J.; Zaagsma, J.; Mueller,
A.; Cornelissen, P. J. G. Respir. Med. 2010, 104, 995.
Supplementary data
16. 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.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.11.
058.
17. VanderMey, M.; Hatzelmann, A.; Van Klink, G. P.; Van der Laan, I. J.; Sterk, G. J.;
Thibaut, U.; Ulrich, W. R.; Timmerman, H. J. Med. Chem. 2001, 44, 2523.
18. VanderMey, M.; Boss, H.; Couwenberg, D.; Hatzelmann, A.; Sterk, G. J.; Goubitz,
K.; Schenk, H.; Timmerman, H. J. Med. Chem. 2002, 45, 2526.
19. Liu, J. T.; Zhou, D.; Jia, X.; Huang, L.; Li, X. S.; Chan; Albert, X. S. Tetrahedron:
Asymmetry 1824, 2008, 19.
References and notes
20. 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.
21. Procopiou, P. A.; Barrett, V. J.; Bevan, N. J.; Biggadike, K.; Butchers, P. R.; Coe, D.
M.; Conroy, R.; Edney, D. D.; Field, R. N.; Ford, A. J.; Guntrip, S. B.; Looker, B. E.;
McLay, I. M.; Monteith, M. J.; Morrison, V. S.; Mutch, P. J.; Richards, S. A.; Sasse,
R.; Smith, C. E. J. Med. Chem. 2009, 52, 2280.
1. Saetta, M.; Turato, G.; Maestrelli, P.; Mapp, C. E.; Fabbri, L. M. Am. J. Respir. Crit.
Care Med. 2001, 163, 1304.
2. Mui, T. S. Y.; Man, S. F. P.; Sin, D. D. Expert Rev. Clin. Immunol. 2008, 4, 365.
3. Gan, W. Q.; Man, S. F. P.; Senthilselvan, A.; Sin, D. D. Thorax 2004, 59, 574.
4. Broekhuizen, R.; Grimble, R. F.; Howell, W. M.; Shale, D. J.; Creutzberg, E. C.;
Wouters, E. F.; Schols, A. N. Am. J. Clin. Nutr. 2005, 1059, 82.
5. Schalkwyk, E. V.; Strydom, K.; Williams, Z.; Venter, L.; Leichtl, S.; Christine, S.
W.; DirK, B.; Philip, G. B. J. Allergy Clin. Immunol. 2005, 116, 292.
6. Bender, A. T.; Beavo, J. A. Pharmacol. Rev. 2006, 58, 488.
22. Cyclic nucleotide phosphodiesterase assay kit: AK-800.
23. Hatzelmann, A.; Schudt, C. J. Pharmacol. Exp. Ther. 2001, 297, 267.
7. Lerner, A.; Epstein, P. M. Biochem. J. 2006, 393, 21.