Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 2. Tadalafil analogs

Article abstract of DOI:10.1016/j.bmcl.2012.01.118

In this Letter we describe our ongoing target repurposing efforts focused on discovery of inhibitors of the essential trypanosomal phosphodiesterase TbrPDEB1. This enzyme has been implicated in virulence of Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). We outline the synthesis and biological evaluation of analogs of tadalafil, a human PDE5 inhibitor currently utilized for treatment of erectile dysfunction, and report that these analogs are weak inhibitors of TbrPDEB1.

Full text of DOI:10.1016/j.bmcl.2012.01.118

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2582–2584
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 2. Tadalafil analogs
Stefan O. Ochiana a, Alden Gustafson b, Nicholas D. Bland b, Cuihua Wang a, Michael J. Russo a,
Robert K. Campbell b, Michael P. Pollastri a,
⇑
a Northeastern University Department of Chemistry and Chemical Biology, 417 Egan Research Center, 360 Huntington Avenue, Boston, MA 02115, United States
b Marine Biological Laboratory, Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, 7 MBL Street, Woods Hole, MA 02543, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 9 February 2012
In this Letter we describe our ongoing target repurposing efforts focused on discovery of inhibitors of the
essential trypanosomal phosphodiesterase TbrPDEB1. This enzyme has been implicated in virulence of
Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). We outline the synthe-
sis and biological evaluation of analogs of tadalafil, a human PDE5 inhibitor currently utilized for treat-
ment of erectile dysfunction, and report that these analogs are weak inhibitors of TbrPDEB1.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Neglected disease
Trypanosoma brucei
Target repurposing
Phosphodiesterase inhibitors
TbrPDEB1
PDE5
Tadalafil
Human African trypanosomiasis (HAT), is a significant ne-
glected tropical disease. Currently, around 50 million people are
at risk of contracting HAT, and annually the incidence of HAT infec-
tions is estimated at 50,000.1,2 The current therapies are severely
limited by cost, route of administration, toxicity to the patient,
and drug resistant trypanosomes.3 The genome of Trypanosoma
brucei codes for five different PDEs, and two of these (TbrPDEB1
and TbrPDEB2) are closely related and demonstrated to be required
for virulence.4,5 These enzymes have been demonstrated to be via-
ble drug targets for trypanosomiasis.4,6,7
metal binding
pocket
O
H
N
solvent-exposed
N
6
N
H
O
NH2
tadalafil
Gln
O
O
O
As a rapid and cost-effective approach to neglected tropical dis-
ease (NTD) drug discovery, we employ a ‘target repurposing’ ap-
proach.8 This approach entails the identification of potential
targets in the parasite that show homology to biological targets
in humans that have already been pursued for drug discovery ef-
forts. Knowledge from these drug discovery efforts can be repur-
posed in the design, synthesis, and optimization of new agents
that inhibit the parasite targets. Furthermore, since the human
homolog is likely to be a potential off-target in an antiparasitic
treatment, previous knowledge about pharmacology of the lead
molecule chemotype can be readily employed.
P-pocket
Figure 1. Putative position of tadalafil in the TbrPDEB1 binding site.
towards the parasitic enzymes. We observed that tadalafil showed
a lack of activity against TbrPDEB1 at 100 lM drug concentrations.
Nevertheless, owing to the particularly extensive history in human
PDE5 inhibitor medicinal chemistry, we were inclined to explore
this chemotype by designing a variety of analogs based on the tad-
alafil scaffold. Furthermore, the tadalafil scaffold has been pursued
by others as antimalarial lead compounds11 and trypanocides.12
To guide our synthetic efforts we first considered the interac-
tions made by tadalafil with human PDE5 that have been reported
by others.13 In PDE5, the benzo[1,3]dioxole moiety points into a
hydrophobic pocket that, based on homology modeling of the
T. brucei enzymes, is predicted to be deeper.7 Such a pocket,
referred to as the P- (or parasite) pocket (Fig. 1), has also been
We describe in this article, and in the preceding article,9 a focus
on the synthesis and assessment of analogs of established PDE5
inhibitors. We7 and others6,10 have shown that some human PDE
inhibitors and their analogs display various degrees of inhibition
⇑
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.2012.01.118

S. O. Ochiana et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2582–2584
2583
CO2Me
NH2
NH2
a, b
R1
CO2Me
O
N
R1
a
b
N
H
N
H
NH
R2
N
H
N
H
1
15
1
1a
R =H
O
1
O
1b
R =Br
R1
R1
CO2Me
O
Scheme 5. Reagents and conditions: (a) Piperonal, CH2Cl2, CF3COOH, 0 °C to rt; (b)
acetyl chloride, Et3N, THF, rt.
O
O
c
N
N
N
N
N
H
Cl
R2
R3
R2
Table 1
2
1
3
Potencies of tricyclic tadalafil analogs against TbrPDEB1
3
4
R =R =H
d
1
3
R =H, R =Me
CONH2
5 R1= Br, R3=H,
NH
R2
Amide config TbrPDEB1 (% inh)a
Scheme 1. Reagents and conditions: (a) R2-CHO, CH2Cl2, CF3COOH, 0 °C to rt; (b)
chloroacetyl chloride, Et3N, CH2Cl2; (c) CH3NH2–HCl, MeOH, Et3N, reflux; (d) NaH,
MeI, THF (Refer to Table 1 for R2).
N
H
Compound R2 config R2
O
7
a
a
19.5
N
HN
Br
O
O
O
O
O
O
8
9
b
a
a
0
N
a
O
N
N
N
H
a
,b
CH3
,b
13.3
N
N
H
22.6b
N
10
a
,b
a
5
6
O
a
O
O
Inhibitor concentration 100.0
Inhibitor concentration 10.0
l
M.
b
O
lM.
Scheme 2. Reagents and conditions: (a) 1H-Pyrazol-5-ylboronic acid, aq Na2CO3,
Pd(dppf)Cl2, dioxane, 90 °C, overnight.
demonstrated by X-ray crystallography to exist in the Leishmania
major homolog, LmjPDEB1.14 The indole N–H interacts with the
glutamine residue that is conserved across all known PDEs. The
western end of tadalafil points towards a small lipophilic sub-
pocket adjacent to the metal binding pocket in the human enzyme;
our homology model of the parasitic enzyme suggests the presence
of some polar amino acids in this region.7
CONH2
a
1
NH
R2
N
H
7
8
A handful of analogs that explored these regions were prepared
(Scheme 1). Treatment of the appropriate tryptophan methyl ester
under Pictet–Spengler conditions in the presence of the appropri-
ate aldehydes provided intermediates 1. Then, acylation with chlo-
roacetyl chloride in the presence of triethylamine gave the
chloroacetyl derivatives 2 in good yield. The piperazinedione deriv-
atives 3 were obtained by reaction with methyl amine in refluxing
methanol.
Preparation of N-methylated analogs of tadalafil and its C6 epi-
mer was achieved by reacting the parent compound with sodium
hydride and methyl iodide, giving 4a and 4b. The brominated ana-
log 5 was prepared by applying the Scheme 1 reaction sequence,
starting with 5-bromotryptophan. Compound 5 could be reacted
under Suzuki conditions to provide the pyrazole analog 6 (Scheme
2). This sidechain was specifically selected among other options for
its small size and H-bond donor–acceptor functionality, which may
interact with the small polar pocket observed to be adjacent to the
metal binding site.
We also wished to explore the solvent-exposed region by
truncation of the diketopiperazine moiety. This was achieved by
treatment of intermediate 1 with ammonium hydroxide to give
the tri-cyclic analogs 7–10 (Scheme 3).
Additional analogs to explore the P-pocket region were
synthesized by the sequence in Scheme 1, using CBZ-glycinal in
the Pictet–Spengler reaction/cyclization sequence to give 3a.
Deprotection
R2=3-piperonyl
9 R2=Me
10 R2=3-pyr
Scheme 3. Reagents and condition: (a) NH4OH, dioxane, rt.
O
O
N
N
11 R=H
b
a
N
12 R=Ac
N
N
H
N
H
O
O
RHN
CbzHN
d
3a
c
O
O
O
H
N
H
N
N
N
N
N
H
H
O
NH
NH
14
13
O
N
NH
N
Scheme 4. Reagents and conditions: (a) H2, Pd/C, EtOH, 3 h; (b)Ac2O, DIEA, THF,
24 h, rt; (c) 3-methyl-1H-pyrazole-4-carboxylic acid, EDC, Et3N, HOBt, CH2Cl2; (d)
nicotinaldehyde, NaBH(OAc)3, THF/DMF, 48 h.

2584
S. O. Ochiana et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2582–2584
Table 2
Compound 15, showed very weak activity (17% inhibition). The
Tadalafil analogs tested against TbrPDEB1
other compounds in Table 2 show low-to-modest potency, includ-
ing those that explored the putative P-pocket. However, compound
6, which was designed to interact with a small sub-pocket adjacent
R1
O
O
to the metal binding region displayed 72% inhibition at 10 lM.
N
Unfortunately, a reliable dose–response experiment was not possi-
ble with this analog due to poor solubility at higher concentrations.
In conclusion, the goal of these experiments was to broadly ex-
plore the structure–activity relationships of the tadalafil scaffold.
Most analogs synthesized displayed a poor inhibition profile with
the notable exception of 6. Given the fairly flat SAR and low poten-
cies observed, we confirm that the tadalafil scaffold does not hold
as strong promise for potent TbrPDEB1 inhibitors as the PDE4
chemotypes previously disclosed.7
N
N
R3
R2
R1
Compound R2
Tadalafil
R3
TbrPDEB1 (% inh)a
H
H
H
0b
O
O
4a
5a
CH3
H
6.9
11.8
Acknowledgments
Br
This work was supported by the National Institutes of Health
(R01AI082577), Boston University and Northeastern University.
We gratefully acknowledge a free academic license to the OpenEye
suite of software.
5
H
Br
11.57
O
O
6
H
1H-Pyrazol-3-yl 71.8
4b
16
CH3
H
H
H
19.7
38.8
Supplementary data
3a
3b
12
H
H
H
H
H
H
21.2
9.4b
0
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 from this overall TbrPDE inhibitor project will be made publi-
cally available as a public data set in the Collaborative Drug Discov-
ery database (www.collaborativedrug.com).
NHCbz
N
NHAc
NH
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.118.
13
H
H
H
H
0
References and notes
O
NH
N
N
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. Brun, R.; Blum, J.; Chappuis, F.; Burri, C. Lancet 2010, 375, 148.
3. Matovu, E.; Seebeck, T.; Enyaru, J. C. K.; Kaminsky, R. Microbes Infect. 2001, 3,
763.
NH
14
6.09
4. Oberholzer, M.; Marti, G.; Baresic, M.; Kunz, S.; Hemphill, A.; Seebeck, T. FASEB
J. 2007, 21, 720.
5. Laxman, S.; Beavo, J. A. Mol. Interventions 2007, 7, 203.
6. Shakur, Y.; de, K. H. P.; Ke, H.; Kambayashi, J.; Seebeck, T. Handb. Exp.
Pharmacol. 2011, 487.
a
Inhibitor concentration 10.0
Inhibitor concentration 100.0
l
l
M.
M.
b
7. 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.
8. Pollastri, M. P.; Campbell, R. K. Future Med. Chem. 2011, 3, 1307.
9. Wang, C.; Ashton, T. D.; Gustafson, A.; Bland, N. D.; Ochiana, S. O.; Campbell, R.
K.; Pollastri, M. P. Bioorg. Med. Chem. Lett. 2011, doi:10.1016/j.bmcl.2012.
01.119.
10. Seebeck, T.; Sterk, G. J.; Ke, H. Future Med. Chem. 2011, 3, 1289.
11. Beghyn, T. B.; Charton, J.; Leroux, F.; Laconde, G.; Bourin, A.; Cos, P.; Maes, L.;
Deprez, B. J. Med. Chem. 2011, 54, 3222.
12. Deprez, B.; Beghyn, T.; Laconde, G.; Charton, J., 2008, WO2008044144A2.
13. Sung, B. J.; Hwang, K. Y.; Jeon, Y. H.; Lee, J. I.; Heo, Y. S.; Kim, J. H.; Moon, J.;
Yoon, J. M.; Hyun, Y. L.; Kim, E.; Eum, S. J.; Park, S. Y.; Lee, J. O.; Lee, T. G.; Ro, S.;
Cho, J. M. Nature 2003, 425, 98.
of 3a (Scheme 4) by catalytic hydrogenation provided the amine
template 11, that could be acylated (to provide 12 or 13) or alkyl-
ated (to provide 14). These analogs were designed in order to both
probe the steric accessibility of the P-pocket, and to interact with
the putative H-bond donors/acceptors present.
Finally, 15 was prepared in an analogous manner to the analogs
shown in Scheme 1, starting from tryptamine (Scheme 5).
All the tadalafil analogs synthesized were screened against
TbrPDEB17 and the data is summarized in Tables 1 and 2. The
tricyclic analogs in Table 1 show weak activity. Although tadalafil
14. Wang, H.; Yan, Z.; Geng, J.; Kunz, S.; Seebeck, T.; Ke, H. Mol. Microbiol. 2007,
1029, 66.
showed no inhibition at 100
lM (Table 2) its C6 epimer 16
displayed almost 40% inhibition at the same concentration.