Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 1. Sildenafil analogs

Article abstract of DOI:10.1016/j.bmcl.2012.01.119

Parasitic diseases, such as African sleeping sickness, have a significant impact on the health and well-being in the poorest regions of the world. Pragmatic drug discovery efforts are needed to find new therapeutic agents. In this Letter we describe target repurposing efforts focused on trypanosomal phosphodiesterases. We outline the synthesis and biological evaluation of analogs of sildenafil (1), a human PDE5 inhibitor, for activities against trypanosomal PDEB1 (TbrPDEB1). We find that, while low potency analogs can be prepared, this chemical class is a sub-optimal starting point for further development of TbrPDE inhibitors.

Full text of DOI:10.1016/j.bmcl.2012.01.119

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2579–2581
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor
analogs as trypanosomal PDE inhibitors. Part 1. Sildenafil analogs
Cuihua Wang a, Trent D. Ashton b, Alden Gustafson c, Nicholas D. Bland c, Stefan O. Ochiana a,
Robert K. Campbell c, Michael P. Pollastri a,
⇑
a Northeastern University Department of Chemistry & Chemical Biology, 417 Egan Research Center, 360 Huntington Avenue, Boston, MA 02115, USA
b Boston University Department of Chemistry, 590 Commonwealth Avenue, Boston, MA 12215, USA
c Marine Biological Laboratory , Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, 7 MBL Street, Woods Hole, MA 02543, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Parasitic diseases, such as African sleeping sickness, have a significant impact on the health and well-
being in the poorest regions of the world. Pragmatic drug discovery efforts are needed to find new ther-
apeutic agents. In this Letter we describe target repurposing efforts focused on trypanosomal phospho-
diesterases. We outline the synthesis and biological evaluation of analogs of sildenafil (1), a human
PDE5 inhibitor, for activities against trypanosomal PDEB1 (TbrPDEB1). We find that, while low potency
analogs can be prepared, this chemical class is a sub-optimal starting point for further development of
TbrPDE inhibitors.
Received 12 December 2011
Revised 25 January 2012
Accepted 30 January 2012
Available online 9 February 2012
Keywords:
Neglected disease
Trypanosoma brucei
Target repurposing
Phosphodiesterase inhibitors
TbrPDEB1
Ó 2012 Elsevier Ltd. All rights reserved.
PDE5
Sildenafil
Tropical diseases such as human African trypanosomiasis (HAT),
leishmaniasis, and Chagas’ disease are often referred to as
‘neglected’ diseases, as the low level of funding of drug discovery
research is disproportionate to the total disease burden. For exam-
ple, collectively these three diseases endanger over 73 million peo-
ple globally1 and create a burden of 4.5 million disability-adjusted
life-years.2,3 Yet, few compounds have advanced to clinical studies
for these diseases.4 The primary reason for this disparity is the high
cost of drug discovery and unlikelihood that these research costs
will be recouped by a research organization via drug sales to highly
impoverished populations.
As a result, cost-effective methods for identification of lead
matter and expediting compound optimization efforts are acutely
needed. We apply the method of ‘target repurposing’, wherein
essential parasite enzymes are matched with proven, druggable
human homologs.4 By doing so, the research outputs of large and
expensive programs of historical drug discovery projects can be
used to initiate and drive optimization projects against parasitic
targets.
adenylate and guanylate cyclase, respectively. Humans possess
eleven PDEs, several of which have been fruitfully pursued for drug
discovery. The most well-known of these is PDE5, an enzyme that
is inhibited by erectile dysfunction drugs such as Viagra™ (sildena-
fil, 1), Cialis™ (tadalafil, 2), and Levitra™ (vardenafil, 3), Figure 1.
Other PDEs are of demonstrated relevance to inflammatory condi-
tions and CNS indications, such as schizophrenia.5–7 Trypanosoma
brucei, the causative agent of HAT, expresses five cAMP-specific
PDEs,8 two of which (TbrPDEB1 and TbrPDEB2) have together been
shown by both RNAi9,10 and small molecules10,11 to be essential for
parasite proliferation. While initial benchmarking and early opti-
mization experiments have uncovered a susceptibility of TbrPDEB1
and B2 to human PDE4 inhibitors,11 the two trypanosomal
enzymes are also 31% identical to PDE5 within their respective cat-
alytic domains, and previous reports cited low potency of 1 and 2
against the trypanosomal enzymes; thus, we describe here early
structure–activity relationships (SAR) of (1), and report such work
for 2 in a subsequent article.12
We initially tested 1 for its inhibitory activity against the cata-
lytic domain of TbrPDEB1, and observed low potency (51% at
Phosphodiesterases (PDEs) are
a family of enzymes that
maintain the balance of cAMP and cGMP in the cell, opposed to
100 lM). However, we deemed this chemotype worth further
exploration given the extensive knowledge of SAR and structural
biology around it. We performed activity scoping experiments by
making strategic changes to the structure of 1, to explore the
impact of variation of the N-methyl group and propyl chain on
⇑
Corresponding author.
E-mail address: m.pollastri@neu.edu (M.P. Pollastri).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.119

2580
C. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2579–2581
O
N
X
O
X
N
N
O
N
N
O
HN
H
O
HN
O
O
N
N
N
N
O
a or b
N
N
N
O2S
HN
N
N
R2
H2N
R2
O
O
N
O2S
O2S
N
OEt
N
2
N
O
15
16
R =Me, X=NH2
N
18
X=OH
19 X=NH2
2
c, d
R =Ph, X=OEt
17 R2=H, X=NH2
1
3
2
N
sildenafil
vardenafil
e
tadalafil
O
N
N
Figure 1. Human PDE5 inhibitors marketed for erectile dysfunction.
N
O2S
HN
N
N
O2S
OH
N
R2
O
O
OEt
A
20: R2=H
OEt
N
O
HN
N
21: R2=CH3
2
22:
R =Ph
N
Region A
Region B
"P-pocket"
1
Scheme 2. Synthesis of 20–22. Reagents and conditions: (a) CDI, A,70 °C, EtOAc,
o/n; (b) PyBroP, A, Et3N, DCE, MW 120 °C 20 min; (c) SOCl2; (d) NH3, iPrOH; (e)
NaOEt, EtOH, MW 120 °C 10 min.
O2S
N
N
Figure 2. Strategy for exploration of the sildenafil core.
1719 is acylated with A using either CDI or PyBroP; these reaction
conditions surprisingly resulted in the partial-to-complete hydro-
lysis of the primary amide (of 14 and 15) or ester (of 16). Thus,
the resulting carboxylic acid 18 was converted to the primary
amide 19 via treatment with thionyl chloride, followed by ammo-
nia in isopropanol. Cyclization to the desired products was effected
under basic conditions.
Following synthesis, the analogs were tested in biochemical as-
says11 at a single concentration. Notably, with one exception (7),
none of the analogs that varied the pyrazole N1 substituent (H,
Me, 3-pyridyl, or acetamide) nor the C3 position (H, Me, Pr, Ph)
showed improved potency over 1.
The removal of the N-methylpiperazinyl sulfonamide head
group resulted in compounds with significant loss in solubility,
and as such biochemical screening data was not possible with
some analogs (Table 1, entries 8, 12–14).
A broad exploration of heterocyclic substitutions in Region B
was undertaken by application of parallel synthesis (Scheme 3).
the pyrazolopyrimidinone core (Region A, Fig. 2), and of the spe-
cific functionality on Region B. We were particularly interested in
exploring the western portion of 1, as this region of the molecule
was proposed to project into a parasite-specific pocket (the P-
pocket). This pocket was first observed in the Leishmania major
phosphodiesterase LmjPDEB113 and is predicted to also exist in
TbrPDEB1,11 but, importantly, is absent from all human PDEs.
Compounds that explore Regions A and B were synthesized
using the routes outlined in Schemes 1 and 2. The known amino-
pyrazole 4a14 or 4b15 was acylated with the appropriate benzoic
acid and cyclized under basic conditions to give 5–10. Pyrazole
N-arylation was achieved using copper catalysis16 to prepare 11–
13. Alkylation of 7 with bromoacetamide provided 14.
The preparation of Strategy B analogs of compound 1 is illus-
trated in Scheme 2. The appropriate aminopyrazole 15,17 16,18 or
O
O
H
N
R3
R3 HN
Table 1
N
COOH
N
EtO
Results of biochemical screening of analogs of 1
+
a, b, c
NH
N
R2
H2N
Entry
Compd
R1
R2
R3
R4
TbrPDEB1a (%inh)
R2
R4
2
O
S
N
4a:
4b:
R =Pr
R =Ph
R4
2
O
N
5-10
R1
O
N
R3 HN
1
1
CH3
Pr
OEt
51.5
N
d
N
R2
2
3
4
5
6
7
8
9
10
11
12
13
14
7
13
22
10
20
8
21
6
9
14
11
12
5
H
Ph
Pr
Ph
Pr
H
Pr
CH3
Ph
Pr
Ph
Ph
Ph
Pr
OEt
OEt
OEt
OEt
OEt
H
OEt
OEt
OEt
OEt
OEt
OEt
OEt
70.9
32.4
17.5
16.0
13.6
8.4
R4
3-Pyr
CH3
H
CH3
CH3
CH3
H
CH3
CH2CONH2
Ph
3-Pyr
H
O
N
CONH2
11-13
N
O
HN
N
e
14
NDb
22.9
21.8
7
7
H
H
H
H
H
H
O2S
N
NDb
NDb
NDb
N
Scheme 1. Synthesis of 5–14. Reagents and conditions: (a) PyBroP, TEA, DCE,
120 °C, MW, 10 min; (b) NH4OH, dioxane, rt; (c) NaOEt/EtOH, 120 °C, MW, 10 min;
(d) R1-I, CuI, trans-cyclohexane-1,2-diamine, Cs2CO3, DMF, 110 °C; (e) NaH, 2-
bromoacetamide, 0 °C to rt. Refer to Table 1 for the identity of R-groups.
a
Standard assay conditions: 100
Compound showed lack of solubility, which precluded testing.
l
M, 10% DMSO.
b

C. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2579–2581
2581
O
In conclusion, with the broad interest in repurposing PDE inhib-
itors for neglected diseases11,20,21 and the extensive precedent in
medicinal chemistry for human PDE5 inhibitors, we note our find-
ing that these initial sildenafil analogs do not represent as strong
starting points for further optimization for TbrPDEB inhibition as
the PDE4 chemotypes previously reported.11 This information
may be deemed important for re-prioritizing the focus of these
efforts.
O
N
N
a, b
HN
R5
H2N
H2N
N
N
N
Pr
Pr
24a-d
23
Scheme 3. Synthesis of 24a–d. Reagents and conditions: (a) R5CO2H, imidazole-
HCl, CDI, then 23, 50 °C, o/n; (b) EtOH, NaOEt, 120 °C, 2 h. Refer to Table 2 for the
identity of R-groups.
Acknowledgments
This work was supported by the National Institutes of Health
(R01AI082577), Boston University and Northeastern University.
We gratefully acknowledge a donation of sildenafil 1 from Pfizer,
Inc. and a free academic license to the OpenEye suite of software.
O
Pr
N
Cl
O
R
N
N
O
HN
N
b
a
N
23
N
N
H
N
NH2
Pr
O
26a
: R=iPr
Supplementary data
25
26b: R=Et
26c: R=Me
Full tabulation of all inhibitors prepared and screened in the
context of this project is included in the Supplementary data, along
with spectroscopic characterization of new compounds. Screening
data will be made publically available as a public data set in the
Collaborative Drug Discovery database (www.collaborativedrug.
com).
Scheme 4. Synthesis of 26a–c. Reagents and conditions: (a) 2-Chloronicotinic acid,
PyBroP, Et3N; (b) R-OH, KOtBu.
Table 2
Analogs of sildenafil (Strategy A)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.119.
#
Compound
R5
TbrPDEB1a (% inh)
1
2
3
4
5
6
7
26c
24a
26a
26b
24b
24c
24d
2-Methoxy-3-pyridyl
3-Methyl-2-pyridyl
2-Isopropoxy-3-pyridyl
2-Ethoxy-3-pyridyl
2-Methyl-5-pyrazinyl
2,4-Dimethyloxazol-5-yl
Thiazol-4-yl
77.2
72.1
53.8
41.5
30.0
29.0
25.4
References and notes
1. Stuart, K.; Brun, R.; Croft, S.; Fairlamb, A.; Gurtler, R. E.; McKerrow, J.; Reed, S.;
Tarleton, R. J. Clin. Invest. 2008, 118, 1301.
2. http://www.who.int/healthinfo/global_burden_disease/2004_report_update/
en/index.html, World Health Organization, accessed 23.01.2012.
3. http://www.who.int/healthinfo/global_burden_disease/metrics_daly/en/,
World Health Organization, accessed 23.01.2012.
4. Pollastri, M. P.; Campbell, R. K. Future Med. Chem. 2011, 3, 1307.
5. Menniti, F. S.; Chappie, T. A.; Humphrey, J. M.; Schmidt, C. J. Curr. Opin. Investig.
Drugs 2007, 8, 54.
a
Standard assay conditions: 100 lM, 10% DMSO.
6. Menniti, F. S.; Faraci, W. S.; Schmidt, C. J. Nat. Rev. Drug Disc. 2006, 5, 660.
7. Lugnier, C. Pharmacol. Ther. 2006, 109, 366.
8. Gould, M. K.; de Koning, H. P. FEMS Microbiol. Rev. 2011.
9. Oberholzer, M.; Marti, G.; Baresic, M.; Kunz, S.; Hemphill, A.; Seebeck, T. FASEB
J. 2007, 21, 720.
10. Shakur, Y.; de, K. H. P.; Ke, H.; Kambayashi, J.; Seebeck, T. Handb. Exp.
Pharmacol. 2011, 487.
11. Bland, N. D.; Wang, C.; Tallman, C.; Gustafson, A. E.; Wang, Z.; Ashton, T. D.;
Ochiana, S. O.; McAllister, G.; Cotter, K.; Fang, A. P.; Gechijian, L.; Garceau, N.;
Gangurde, R.; Ortenberg, R.; Ondrechen, M. J.; Campbell, R. K.; Pollastri, M. P. J.
Med. Chem. 2011, 54, 8188.
12. Ochiana, S. O.; Gustafson, A.; Bland, N. D.; Wang, C.; Campbell, R. K.; Pollastri,
M. P. Bioorg. Med. Chem. Lett. 2011, in press, doi:10.1016/j.bmcl.2012.01.118.
13. Wang, H.; Yan, Z.; Geng, J.; Kunz, S.; Seebeck, T.; Ke, H. Mol. Microbiol. 2007,
1029, 66.
14. Baxendale, I. R.; Ley, S. V. Bioorg. Med. Chem. Lett. 1983, 2000, 10.
15. Liu, H.; He, X.; Choi, H.-S.; Yang, K.; Woodmansee, D.; Wang, Z.; Ellis, D. A.; Wu,
B.; He, Y.; Nguyen, T. N., 2006; WO2006047516A2.
16. Antilla, J. C.; Baskin, J. M.; Barder, T. E.; Buchwald, S. L. J. Org. Chem. 2004, 69,
5578.
17. DeWald, H. A.; Beeson, N. W.; Hershenson, F. M.; Wise, L. D.; Downs, D. A.;
Heffner, T. G.; Coughenour, L. L.; Pugsley, T. A. J. Med. Chem. 1988, 31, 454.
18. Yuan, J.; Gulianello, M.; De, L. S.; Brodbeck, R.; Kieltyka, A.; Hodgetts, K. J.
Bioorg. Med. Chem. Lett. 2002, 12, 2133.
This was achieved by condensing the commercially available pyra-
zole amino amide 23 with various monocyclic heteroaromatic car-
boxylic acids that were available in pre-weighed quantities from a
commercial vendor (ASDI, Inc). Following this amidation reaction,
cyclization was achieved by treatment with sodium ethoxide in
ethanol.
A series of 2-alkoxy-3-pyridyl variations were prepared as
shown in Scheme 4. The acylated aminopyrazole 25 was cyclized
under basic conditions, using an appropriate alcoholic solvent,
which yielded the 2-alkoxypyridyl derivatives 26.
Table 2 summarizes the biochemical results for analogs pre-
pared using this parallel methodology that display >25% inhibition
at 100 lM (characterization and biochemical data for all com-
pounds are listed in the Supplementary data). In brief, heterocycles
show improved potency at TbrPDEB1, specifically, 2-methoxy-3-
pyridyl and 3-methyl-2-pyridyl show the best potency (entries
1–2); larger 2-alkoxy groups (entries 3 and 4) lead to reduced
activity. Unfortunately, dose–response experiments with 26c were
precluded by poor solubility at higher concentrations.
While we appreciate that the potencies of the compounds
shown in Tables 1 and 2 are modest, it is clear that 7, 26c and
24a represent qualitative potency improvement over 1. Nonethe-
less, the current exploration of the various regions of the sildenafil
template is not suggestive of promising structure–activity relation-
ship trends.
19. Fletcher, S. R.; Menet, C. J. M.; Dykes, G. J.; Merayo, M. N.; Schmitt, B. A., 2008;
WO2008071650A2.
20. Seebeck, T.; Sterk, G. J.; Ke, H. Future Med. Chem. 2011, 3, 1289.
21. Beghyn, T. B.; Charton, J.; Leroux, F.; Laconde, G.; Bourin, A.; Cos, P.; Maes, L.;
Deprez, B. J. Med. Chem. 2011, 54, 3222.