Repurposing human PDE4 inhibitors for neglected tropical diseases. evaluation of analogs of the human PDE4 inhibitor GSK-256066 as inhibitors of pdeb1 of trypanosoma brucei

Article abstract of DOI:10.1111/cbdd.12443

Cyclic nucleotide phosphodiesterases (PDEs) have been identified as important enzyme targets for drug development in both humans and Trypanosoma brucei, the causative agent of human African trypanosomiasis. With this in mind, we recently reported the prof

Full text of DOI:10.1111/cbdd.12443

Chem Biol Drug Des 2015; 85: 549–564
Research Article
Repurposing Human PDE4 Inhibitors for Neglected
Tropical Diseases. Evaluation of Analogs of the Human
PDE4 Inhibitor GSK-256066 as Inhibitors of PDEB1 of
Trypanosoma brucei
Stefan O. Ochiana1, Nicholas D. Bland2,
Human African trypanosomiasis (HAT) is a neglected tropi-
cal disease that affects approximately 10 000 patients in
sub-Saharan Africa (1). Caused by the protozoan parasite
Trypanosoma brucei, the advanced stage of HAT consists
Luca Settimo1, Robert K. Campbell2 and
Michael P. Pollastri1*
1Department of Chemistry and Chemical Biology,
of an infection of the central nervous system, a condition
Northeastern University, 417 Egan Research Center, 360
Huntington Avenue, Boston, MA 02115, USA
2Marine Biological Laboratory, Josephine Bay Paul Center
for Comparative Molecular Biology and Evolution, 7 MBL
Street, Woods Hole, MA 02543, USA
that is invariably fatal if untreated. Current treatments for
the CNS stage of HAT are suboptimal, including eflorni-
thine (alone, or now in combination with nifurtimox) (2),
which is only active against the T. brucei gambiense sub-
species, and melarsoprol, which is a toxic organoarsenical
agent whose toxicity is such that approximately 5% of
patients will die from its side-effects (3).
*Corresponding author: Michael P. Pollastri,
m.pollastri@neu.edu
Cyclic nucleotide phosphodiesterases (PDEs) have
been identified as important enzyme targets for drug
development in both humans and Trypanosoma brucei,
the causative agent of human African trypanosomiasis.
With this in mind, we recently reported the profiling of
a range of human phosphodiesterase inhibitors, show-
ing that human PDE4 inhibitors tend to display the
best potency against the trypanosomal phosphodies-
terase TbrPDEB1. Among these was GSK-256066, a
potent inhibitor of human PDE4 and a weak inhibitor of
TbrPDEB1. In this report, we describe the results of a
structure–activity relationship study of this chemotype,
leading to the discovery of analogs with improved
potency against TbrPDEB1 and micromolar inhibition
of T. brucei cellular growth. We rationalize the potency
trends via molecular docking of the new inhibitors into
a recently reported apo structure of TbrPDEB1. The
studies in this article will inform future efforts in repur-
posing human PDE inhibitors as antitrypanosomal
agents.
As HAT affects some of the poorest patients in the world,
there is little financial incentive to pursue costly de novo
drug discovery programmes. With this in mind, we have
applied a target repurposing approach to therapeutics dis-
covery, wherein essential parasite targets and pathways
are matched with druggable human homologs that have
existing chemical matter that targets them (4). These com-
pounds can provide new leads for antiparasitic drug dis-
covery without undertaking
a costly high-throughput
screening campaign. We have pursued this approach with
a variety of kinase (5,6) and phosphodiesterase (PDE)
inhibitors (7–10).
Phosphodiesterase inhibitors have been developed for a
variety of indications, including treatment of erectile dys-
function and pulmonary hypertension (PDE5), and chronic
obstructive pulmonary disease (PDE4). The success of
these efforts is evident in the approval of various selective
PDE inhibitors for clinical use (11–14). Trypanosoma brucei
expresses five PDEs, including the homologs TbrPDEB1
and B2, which have been together demonstrated to be
essential by RNAi, such that both enzymes must be inhib-
ited to affect parasite survival (15). These two enzymes are
highly homologous (88.5%) (15), and we have shown pre-
viously (7) (and within this work) that inhibitors tested
against both enzymes most frequently display similar
potency against both. Furthermore, the essentiality data
and sequence similarity between human and trypanosomal
PDEs led us to believe that target repurposing could be a
fruitful approach for new inhibitor discovery (7).
Key words: GSK-256066, phosphodiesterase inhibitors,
TbrPDEB1, TbrPDEB2, Trypanosoma brucei
Abbreviations: COPD, chronic obstructive pulmonary dis-
ease; HAT, human African trypanosomiasis; hPDE, human
phosphodiesterase; NECT, nifurtimox/eflornithine combination
therapy; TbrPDEB1, TbrPDEB2, Trypanosoma brucei phos-
phodiesterase B1 or B2.
Received 25 July 2014, revised 22 September 2014 and
accepted for publication 24 September 2014
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12443
549

Ochiana et al.
We previously reported the assessment of a range of estab-
lished human PDE chemotypes against TbrPDEB1 and B2,
and reported that these enzymes are susceptible to a num-
ber of chemotypes, primarily derivatives of established human
PDE4 (hPDE4) inhibitors (Figure 1). Besides piclamilast (1)
and cilomilast (2), we also identified GSK-256066 (3)(16), an
investigational compound for chronic obstructive pulmonary
disease (COPD), as a weak inhibitor of TbrPDEB1 (7).
HMI-11 medium supplemented with 10% fetal bovine
serum (FBS, Sigma). Cells in the mid-logarithmic stage of
growth were diluted to a density of 104 cells/mL and were
incubated with a range of concentrations of inhibitor in
DMSO or DMSO alone. The final concentration of DMSO
was 1%. Cell densities were determined after 48 h using
Alamar blue (Invitrogen, Carlsbad, CA, USA) per the manu-
facturer’s instructions. All values are the mean of three or
more independent experiments.
Besides improving potency at the trypanosomal target,
another significant issue for any target repurposing pro-
gramme is to identify divergent structure–activity relation-
ships (SAR) between the host and pathogen enzymes.
Such selectivity is important to reduce potentially trouble-
some side effect profiles, such as emesis, as observed
with most hPDE4 inhibitors, which has been a significant
challenge to date (7,17).
Chemical Synthesis
Unless otherwise noted, reagents were obtained from
Sigma-Aldrich, Inc. (St. Louis, MO, USA), Fisher Scientific,
Frontier Scientific Services, Inc. (Newark, DE, USA), Matrix
Scientific (Columbia, SC, USA), and used as received.
Boronic acids/esters and aniline reagents were purchased,
except for the boronates listed in the Supporting Information
(Appendix S1). Reaction solvents were purified by passage
through alumina columns on a purification system manufac-
tured by Innovative Technology (Newburyport, MA, USA).
Microwave reactions were performed using a Biotage Initia-
tor-8 instrument. NMR spectra were obtained with Varian
Material and Methods
TbrPDEB1 Biochemical assay
Biochemical assays were performed as previously described
(7) and are described in detail in the Supporting Information
(Appendix S1). Biological assay data and chemical struc-
tures are available in Appendix S1 and as a publically shared
data set at www.collaborativedrugdiscovery.com.
1
NMR systems, operating at 400 or 500 MHz for H acquisi-
tions as noted. LCMS analysis was performed using a
Waters Alliance reverse-phase HPLC, with single-wave-
length UV–visible detector and LCT Premier time-of-flight
mass spectrometer (electrospray ionization). All newly syn-
thesized compounds that were submitted for biological test-
ing were deemed >95% pure by LCMS analysis (UV and
ESI-MS detection) prior to submission for biological testing.
Preparative LCMS was performed on a Waters FractionLynx
system with a Waters MicroMass ZQ mass spectrometer
(electrospray ionization) and a single-wavelength UV–visible
detector, using acetonitrile/H2O gradients with 0.1% formic
acid. Fractions were collected on the basis of triggering
using UV and mass detection. Yields reported for products
obtained by preparative HPLC represent the amount of
pure material isolated; impure fractions were not repurified.
Human PDE4B biochemical assay
This assay was performed at Takeda Pharmaceuticals
using methods previously reported (18).
Trypanosome cell culture assays
Bloodstream forms of Trypanosoma brucei brucei strain
427 were grown at 37 °C in a 5% CO2 atmosphere in
CN
MeO
CO2H
Cl
H
N
H
MeO
O
Ethyl 6-iodo-8-methyl-4-oxo-1,4-dihydroquinoline-
3-carboxylate (6)
O
O
N
Cl
piclamilast (2)
TbrPDEB1 IC50: 4.7 µM
TbrPDEB2 IC50: 11.4
Tbb EC50: 9.6 µM
hPDE4B IC50: 3 nM
cilomilast (1)
TbrPDEB1 IC50: 16.4 µM
hPDE4B IC50: 84 nM
To 4-iodo-2-methylaniline (5a) (5.35 g, 22.96 mmol) was
added diethyl 2-(ethoxymethylene)malonate (5.10 mL,
25.3 mmol), and the reaction mixture was heated at
100 °C for 1 h. The heat was removed, and the white solid
that formed was collected, washed with cyclohexane
(70 mL) and ethanol 30 mL (29), and dried in vacuo at
40 °C overnight to give diethyl 2-(((4-iodo-2-methyl-
phenyl)amino)methylene)malonate (Yield: 98%). 1H NMR
(500 MHz, DMSO-d6) d 10.83 (d, J = 13.6 Hz, 1H), 8.42
(d, J = 13.1 Hz, 1H), 7.65 (s, 1H), 7.56–7.61 (m, 1H), 7.25
(d, J = 8.3 Hz, 1H), 4.20 (q, J = 7.3 Hz, 2H), 4.11
(q, J = 7.3 Hz, 2H), 2.25 (s, 3H), 1.24 (td, J = 6.9, 10.5 Hz,
6H). LCMS found 404.16 [M+H]+. Then to the diethyl 2-
((4-iodo-2-methylphenylamino)methylene)malonate (9.0 g,
22.3 mmol) was added diphenyl ether (35.5 mL,
µ
M
D
Me
N
F
F
O
C
S
CONH2
O
O
O
Cl
HN
H
A
N
O
N
B
Me2NOC
Cl
OMe
roflumilast (4)
TbrPDEB1: 5.3% at 100 µM
hPDE4B IC50: 0.84 nM
GSK-256066 (3)
TbrPDEB1 IC50: 24.6 µM
hPDE4B IC50: 0.008 nM
Figure 1: Previously benchmarked human PDE4 inhibitors
(7,16,31,32).
550
Chem Biol Drug Des 2015; 85: 549–564

Repurposing GSK-256066 for TbrPDEB1 Inhibition
223 mmol). The reaction was run for 45 min at 250 °C.
The mixture was cooled, and isohexane was added
(30 mL). The solid formed (light yellow solid) was collected
by filtration and washed further with isohexane (30 mL).
The solid was dried under high vacuum to give the desired
product as a light yellow solid (Yield: 100%). 1H NMR
(400 MHz, DMSO-d6) d 11.74 (br. s., 1H), 8.37 (s, 1H),
8.28 (s, 1H), 7.89 (s, 1H), 4.21 (q, J = 7.3 Hz, 2H), 1.26
(t, J = 7.3 Hz, 3H). LCMS found 357.95 [M+H]+.
(diphenylphosphine) (9.9 mg, 0.018 mmol). The methyl
3-mercaptobenzoate (11.6 mg, 0.069 mmol) was dis-
solved in toluene (1.5 mL) and then added to the solids.
Lastly, KOtBu (0.092 mL, 0.092 mmol) was added. The
mixture was placed in the MW and heated for 40 min at
170 °C. The crude product was filtered through Celite
after adding methanol and further washed with methanol.
The solvent was concentrated. The crude product was
chromatographed using 50–100% EtOAC in hexane to
give the desired product 9a as an orange solid (Yield:
75%). 1H NMR (400 MHz, CD3OD) d 8.91 (s, 1H), 7.91
(d, J = 7.3 Hz, 1H), 7.85 (s, 1H), 7.50 (s, 1H), 7.42–7.46
(m, 2H), 7.33–7.40 (m, 1H), 6.99 (t, J = 8.0 Hz, 1H), 6.49–
6.53 (m, 1H), 6.30–6.36 (m, 2H), 3.91 (s, 3H), 3.66
(s, 3H), 2.69 (s, 3H). LCMS found 474.01, [M+H]+.
4-chloro-6-iodo-8-methylquinoline-3-carboxamide (7)
NaOH (1.96 g, 49.1 mmol) was dissolved in water (40 mL)
and ethanol (20 mL). The resultant solution was added to 6
(7.97 g, 22.32 mmol), and the mixture was heated and ref-
luxed for 1 h with stirring. Then concentrated HCl was
added until a white precipitate formed. The reaction was
stirred overnight at room temperature. After stirring over-
night, the precipitate was filtered, washed with water and
dried in vacuo to give 6-iodo-8-methyl-4-oxo-1,4-dihydro-
quinoline-3-carboxylic acid as a white solid. (LCMS found
329.89 [M+H]+). To 6-iodo-8-methyl-4-oxo-1,4-dihydro-
quinoline-3-carboxylic acid (7.34 g, 22.30 mmol) was
added thionyl chloride (24.42 mL, 335 mmol), then three
drops DMF were added. The mixture was refluxed for 2 h.
The excess thionyl chloride was evaporated in vacuo, and
the residue was azeotroped with toluene (5 mL, 19). The
crude product 4-chloro-6-iodo-8-methylquinoline-3-car-
bonyl chloride was used in the next step without further
characterization. To stirred ammonium hydroxide (34.7 mL,
892 mmol) was added portionwise 4-chloro-6-iodo-8-
methylquinoline-3-carbonyl chloride (8.16 g, 22.3 mmol),
and the mixture was stirred at room temperature overnight.
The solid formed was filtered, washed with water, and
dried under vacuum at 60 °C to give 7 as a white solid
4-((3-methoxyphenyl)amino)-8-methyl-6-
(phenylthio)quinoline-3-carboxamide (9b)
To compound
8 (40.0 mg, 0.092 mmol) were added
Pd2(dba)3 (16.9 mg, 0.018 mmol) and 2,20-oxybis(2,1-phe-
nylene)bis(diphenylphosphine) (19.9 mg, 0.037 mmol). The
benzenethiol (0.014 mL, 0.138 mmol) was dissolved in tol-
uene and added to the solids. Lastly, KOtBu (0.115 mL,
0.185 mmol) was added. The mixture was heated in the
MW for 40 min at 170 °C. To the crude product was
added methanol, and the solution was filtered through Cel-
ite. Then, the solvent was concentrated. The crude prod-
uct was chromatographed 0–100% EtOAc in hexane to
1
give the desired product as a yellow solid. (Yield: 53%). H
NMR (400 MHz, CD3OD) d 8.89 (s, 1H), 7.44 (s, 2H),
7.18–7.29 (m, 5H), 7.08 (t, J = 8.0 Hz, 1H), 6.58–6.64 (m,
1H), 6.34–6.40 (m, 2H), 3.71 (s, 3H), 2.67 (s, 3H). LCMS
found 416.01, [M+H]+.
1
(Yield: 80%). H NMR (400 MHz, DMSO-d6) d 8.89 (s, 1H),
8.46 (s, 1H), 8.20 (br. s., 1H), 8.10 (s, 1H), 7.99 (br. s., 1H),
2.70 (s, 3H). LCMS found 346.87 [M+H]+.
Methyl 3-(3-carbamoyl-4-(3-methoxyphenylamino)-
8-methylquinolin-6-ylsulfonyl)benzoate (10a)
To 9a (103 mg, 0.218 mmol) was added oxone (401 mg,
0.653 mmol) in DMF (5 mL). The reaction was stirred at
room temperature for 5 h. Then, the reaction mixture was
poured into water (40 mL) and extracted with DCM (59),
and the combined organic layers were dried (Na2SO4) and
concentrated. The crude product was chromatographed
using 0–10% MeOH in DCM to give the desired product as
6-iodo-4-(3-methoxyphenylamino)-
8-methylquinoline-3-carboxamide (8)
Compound 7 (174 mg, 0.499 mmol) was dissolved in ace-
tonitrile, and 3-methoxyaniline (0.059 mL, 0.52 mmol) was
added. The mixture was heated and refluxed overnight.
The precipitate formed was filtered, washed with acetoni-
trile, and the solid (light yellow solid) obtained was dried
under high vacuum to give the title compound (Yield:
90%). 1H NMR (400 MHz, DMSO-d6) d 8.78 (s, 1H), 8.39
(br. s., 1H), 8.31 (br. s., 1H), 8.10 (br. s., 1H), 7.74 (br. s.,
1H), 7.28 (t, J = 8.0 Hz, 1H), 6.73–6.86 (m, 3H), 3.72
(s, 3H), 2.65 (s, 3H). LCMS found 434.97 [M+H]+.
1
an orange solid (Yield: 85%). H NMR (500 MHz, CDCl3) d
10.86 (s, 1H), 8.93 (s, 1H), 8.44 (s, 1H), 8.23–8.26 (m, 1H),
8.21 (d, J = 7.8 Hz, 1H), 7.91 (s, 1H), 7.77 (d, J = 7.8 Hz,
1H), 7.54 (t, J = 7.8 Hz, 1H), 7.15 (t, J = 8.0 Hz, 1H), 6.75
(dd, J = 1.95, 8.3 Hz, 1H), 6.57 (d, J = 7.8 Hz, 1H), 6.54
(s, 1H), 5.90–6.10 (br. s.,2H), 3.97 (s, 3H), 3.65 (s, 3H),
2.75 (s, 3H). LCMS found 506.01, [M+H]+.
Methyl 3-(3-carbamoyl-4-(3-methoxyphenylamino)-
8-methylquinolin-6-ylthio)benzoate (9a)
4-((3-methoxyphenyl)amino)-8-methyl-6-
(phenylsulfonyl)quinoline-3-carboxamide (10b)
To compound 9b (13.4 mg, 0.032 mmol) were added ox-
one (59.5 mg, 0.097 mmol) and DMF (3 mL). The reaction
To
8 (20.0 mg, 0.046 mmol) was added Pd2(dba)3
(8.4 mg, 9.23 lmol), then 2,20-oxybis(2,1-phenylene)bis
Chem Biol Drug Des 2015; 85: 549–564
551

Ochiana et al.
was stirred for 4 h at rt. Then, the reaction mixture was
poured into water (20 mL) and extracted with DCM (59),
the combined organics were washed with brine (19) and
dried under sodium sulfate. The crude product was chroma-
tographed using 50–70% EtOAc in hexane to give the
desired product as a light yellow solid. (Yield: 62%).1H NMR
(400 MHz, CD3OD) d 9.03 (s, 1H), 8.38 (s, 1H), 7.93 (s, 1H),
7.72 (d, J = 7.3 Hz, 2H), 7.64 (t, J = 7.3 Hz, 1H), 7.55 (t,
J = 7.7 Hz, 2H), 7.24 (t, J = 8.0 Hz, 1H), 6.82 (dd, J = 1.8,
8.4 Hz, 1H), 6.63 (d, J = 7.3 Hz, 1H), 6.57 (s, 1H), 3.64 (s,
3H), 2.74 (s, 3H). LCMS found 448.01, [M+H]+.
uct was purified via preparative HPLC to give the desired
product as a yellow solid (Yield: 37%). H NMR (400 MHz,
1
DMSO-d6) d 10.77 (s, 1H), 9.08 (s, 1H), 8.71 (d, J = 4.4 Hz,
1H), 8.32–8.35 (m, 1H), 8.29 (br. s., 1H), 8.26 (s, 1H), 8.08
(d, J = 8.0 Hz, 1H), 7.99 (s, 1H), 7.76 (d, J = 7.3 Hz, 2H),
7.68 (t, J = 7.7 Hz, 1H), 7.12 (t, J = 8.0 Hz, 1H), 6.69 (dd,
J = 1.8, 8.4 Hz, 1H), 6.60 (s, 1H), 6.52 (d, J = 7.3 Hz, 1H),
3.62 (s, 3H), 2.81 (d, J = 4.4 Hz, 3H), 2.69 (s, 3H). LCMS
found 505.01, [M+H]+.
Methyl 3-(3-carbamoyl-4-(3-methoxyphenylamino)-
8-methylquinolin-6-ylamino)benzoate (13)
3-(3-carbamoyl-4-(3-methoxyphenylamino)-8-
methylquinolin-6-ylsulfonyl)benzoic acid (11)
To 8 (40.0 mg, 0.092 mmol) was added 2,20-oxybis(2,
1-phenylene)bis (diphenylphosphine) (19.9 mg, 0.037 mmol),
then Pd(dppf)Cl2ꢀDCM (13.5 mg, 0.018 mmol) and methyl
3-aminobenzoate (27.9 mg, 0.185 mmol). Lastly, KOtBu
(0.185 mL, 0.185 mmol) was added. The reaction mixture
A solution of 10a (60.0 mg, 0.119 mmol) in ethanol (2 mL)
was treated with NaOH (0.890 mL, 1.780 mmol), and the
resulting solution was stirred at 45 °C overnight. The sol-
vent was evaporated. The residue was dissolved in water
and acidified with 1 M HCl to pH 4. The resulting precipi-
tate was filtered, washed with water, and dried in vacuo.
The crude product was purified via preparative HPLC to
give the desired product as a yellow solid (Yield: 31%). 1H
NMR (400 MHz, CD3OD) d 9.01 (s, 1H), 8.38 (s, 1H),
8.34 (s, 1H), 8.24 (d, J = 7.3 Hz, 1H), 7.99 (s, 1H),
7.86 (d, J = 8.0 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.18
(t, J = 8.0 Hz, 1H), 6.74–6.79 (m, 1H), 6.53–6.62 (m, 2H),
3.62 (s, 3H), 2.75 (s, 3H). LCMS found 492.01, [M+H]+.
ᵒ
was heated in the MW for 20 min at 160 C. After the
reaction mixture cooled to ambient temperature, the crude
was filtered through Celite washing with MeOH (39).
The solvent was concentrated under reduced pressure.
Then, the crude product was purified via preparative HPLC
to give 13 as a yellow solid (Yield: 12%). 1H NMR
(400 MHz, CD3OD) d 8.76 (s, 1H), 7.55 (s, 1H), 7.46 (d,
J = 8.0 Hz, 1H), 7.30 (d, J = 2.9 Hz, 1H), 7.26 (s, 1H),
7.13 (t, J = 8.0 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.95
(dd, J = 1.5, 8.0 Hz, 1H), 6.62 (dd, J = 1.8, 8.4 Hz, 1H),
6.50 (t, J = 2.2 Hz, 1H), 6.43–6.47 (m, 1H), 3.88 (s, 3H),
3.70 (s, 3H), 2.70 (s, 3H). LCMS found 457.01, [M+H]+.
6-(3-carbamoylphenylsulfonyl)-
4-(3-methoxyphenylamino)-8-methylquinoline-
3-carboxamide (12a)
8-ethyl-6-iodo-4-((3-methoxyphenyl)amino)
quinoline-3-carboxamide (16b)
A solution of 10a (18.0 mg, 0.036 mmol) in dioxane was
added to a solution of ammonium hydroxide (0.761 mL,
19.55 mmol). The mixture was stirred at room temperature
overnight. Then the solvent was concentrated, and the
residue was partitioned between sat. NH4Cl and ethyl ace-
tate. The aqueous layer was extracted with EtOAc (39),
and the combined organics were washed with brine
and dried over Na2SO4. After filtration and evapo-
ration, the crude product was chromatographed using
0–10% MeOH to give the desired product as a yellow solid
(Yield: 18%). 1H NMR (400 MHz, CD3OD) d 9.02 (s, 1H),
8.36 (s, 1H), 8.31 (s, 1H), 8.10 (s, 1H), 7.99 (s, 1H), 7.81
(d, J = 7.3 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H), 7.17
(t, J = 8.4 Hz, 1H), 6.75–6.80 (m, 1H), 6.55–6.60 (m, 2H),
3.64 (s, 3H), 2.75 (s, 3H). LCMS found 491.01, [M+H]+.
To 15b (19,20) (302 mg, 0.838 mmol) were added aceto-
nitrile and 3-methoxyaniline (0.099 mL, 0.879 mmol). The
mixture was heated at 80 °C overnight. Then the solvent
was concentrated, and the crude product was purified via
chromatography (0–6% MeOH in DCM) to give 16b as a
light yellow solid (Yield: 78%). LCMS found 447.99
[M+H]+. The product was taken to the next step without
further characterization.
General procedure A. Synthesis of intermediate 23
was achieved via published methods (16,19,20) and is
summarized in the Supporting Information (Appendix S1).
For example, compound 7 was dissolved in acetonitrile,
and the desired amine (1.1 equiv) was added. The mixture
was generally heated under reflux overnight or run in the
microwave at 145 °C for 25 min unless stated otherwise
in the protocol. Then the precipitate formed was filtered,
washed with acetonitrile, and the solid obtained was dried
under high vacuum to give 23. These analogs were con-
firmed by LCMS and/or NMR and used in the next step
(General procedure B) without further characterization.
4-(3-methoxyphenylamino)-8-methyl-6-
(3-(methylcarbamoyl)phenylsulfonyl)quinoline-
3-carboxamide (12b)
To 11 (8.6 mg, 0.017 mmol) in DMF was added HATU
(7.3 mg, 0.019 mmol). After 5 min, methylamine HCl
(1.14 mg, 0.017 mmol) and DIEA (6.4 lL, 0.037 mmol) were
added. The resulting solution was stirred at room tempera-
ture for 6 h. The solvent was concentrated. The crude prod-
General procedure B. Suzuki coupling protocol. To
iodo-substituted templates 8 and 23 - (1 equiv.) were
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Chem Biol Drug Des 2015; 85: 549–564

Repurposing GSK-256066 for TbrPDEB1 Inhibition
added the desired boronic acid or ester (1.5 equiv.), Pd
(dppf)Cl2ꢀDCM (0.1 equiv.), dioxane, and sodium carbon-
ate (6 equiv.). The reaction mixture was heated in the MW
at 130 °C, 145 °C, or 160 °C as specified in Scheme 2 or
Scheme 3 for 20 min. After the reaction mixture cooled to
ambient temperature, the crude was filtered through Celite
washing with MeOH/DCM (1:9). The filtrate was concen-
trated under reduced pressure. Unless otherwise noted,
the crude products were chromatographed or purified via
preparative HPLC to give the desired products of the gen-
eral templates 17, 19, 22e,f, and 24, which are character-
ized in the Supporting Information (Appendix S1).
Methyl 3-(3-carbamoyl-4-chloro-8-methylquinolin-
6-yl)benzoate (20a), dimethyl 3,30-(3-carbamoyl-8-
methylquinoline-4,6-diyl)dibenzoate (21a), and
methyl 3-(3-carbamoyl-8-methylquinolin-6-yl)
benzoate (21b)
To 7 (200 mg, 0.577 mmol) were added (3-(methoxycar-
bonyl)phenyl)boronic acid (104 mg, 0.577 mmol), Pd(dppf)
Cl2ꢀDCM (84 mg, 0.115 mmol), dioxane (4 mL), and
sodium carbonate (1.73 mL, 3.46 mmol). The reaction mix-
ture was heated in the MW at 150 °C for 10 min. After the
reaction mixture cooled to ambient temperature, the crude
was filtered through Celite washing with MeOH/DCM (1:9).
The filtrate was concentrated under reduced pressure. The
crude mixture was chromatographed using hexanes/EtOAc
to give as the major product 20 as a white solid (Yield:
29%) and a mixture of 21a and 21b. This mixture was sep-
arated using preparative HPLC to give the clean products
21a (white solid, Yield: 5%) and 21b (white solid, Yield:
2%). (20). 1H NMR (500 MHz, DMSO-d6) d 8.90 (s, 1H),
8.30–8.35 (m, 2H), 8.21 (s, 1H), 8.13–8.17 (m, 2H), 8.03
(d, J = 7.8 Hz, 1H), 7.99 (s, 1H), 7.70 (t, J = 7.8 Hz, 1H),
3.91 (s, 3H), 2.82 (s, 3H). LCMS found 354.01, [M+H]+.
General procedure C. To 20 (1 equiv.) was added ace-
tonitrile, and the required amine (5–6 equiv.) was added.
The reaction was run at 80 °C in a sealed vial for 48 h.
The solvent was concentrated, and the crude product was
purified via preparative HPLC or chromatography to afford
the products (22a-d), which are characterized in the Sup-
porting Information (Appendix S1).
1
5-(3-carbamoyl-4-((3-methoxyphenyl)amino)-8-
methylquinolin-6-yl)isophthalic acid (18a)
(21a). H NMR (500 MHz, DMSO-d6) d 9.00 (s, 1H), 8.07–
8.12 (m, 3H), 7.99 (s, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.83–
7.89 (m, 2H), 7.69–7.75 (m, 2H), 7.60 (t, J = 7.8 Hz, 1H),
7.54 (s, 1H), 7.48–7.50 (m, 1H), 3.32 (s, 6H), 2.87 (s, 3H).
LCMS found 455.11, [M+H]+. (21b). 1H NMR (500 MHz,
CDCl3) d 9.31 (s, 1H), 8.69 (d, J = 2.4 Hz, 1H), 8.40
(s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 7.3 Hz, 2H),
7.91 (d, J = 7.8 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 3.98
(s, 3H), 2.91 (s, 3H). LCMS found 321.01, [M+H]+.
To 17e (7.00 mg, 0.014 mmol) was added LiOH
(2.014 mg, 0.084 mmol), and the solids were dissolved in
a mixture of water:THF:MeOH (1:1:1). The solution was
stirred at 90 °C in a sealed vial for 2 h. Then the solvent
was concentrated. The crude product was purified via
PREP HPLC to give 5-(3-carbamoyl-4-((3-methoxyphenyl)
amino)-8-methylquinolin-6-yl)isophthalic acid (18a), (white
solid, Yield: 25%). 1H NMR (500 MHz, DMSO-d6) d 13.25
(br. s., 1H), 10.47 (s, 1H), 8.99 (s, 1H), 8.39 (s, 1H),
8.21 (s, 1H), 8.15 (m, 2H), 7.96 (s, 2H), 7.67 (s, 1H),
7.15 (t, J = 8.0 Hz, 1H), 6.62–6.56 (m, 2H), 6.54
(d, J = 7.0 Hz, 1H), 3.68 (s, 3H), 2.77 (s, 3H). LCMS
found 472.01, [M+H]+.
4-chloro-6-iodo-N,8-dimethylquinoline-3-
carboxamide (25a)
To sodium hydride (60 % dispersion in mineral oil,
10.9 mg, 0.27 mmol) in DMF (2 mL) was added intermedi-
ate 7 (100 mg, 0.289 mmol) in dry DMF (3 mL). The reac-
tion mixture was stirred for 35 min at rt before adding
iodomethane (0.017 mL, 0.274 mmol), after which the
reaction mixture was stirred at rt overnight. Water and
EtOAc were added, and the aqueous layer was extracted
with EtOAc (39). The combined organics were washed
with brine and finally dried under sodium sulfate. The sol-
vent was concentrated, and the product was confirmed by
LCMS, LCMS found 360.01, [M+H]+. The product was
taken to the next step without further characterization.
3-(3-carbamoyl-4-((3-methoxyphenyl)amino)-8-
methylquinolin-6-yl)-5-(methoxycarbonyl)benzoic
acid (18b)
To 17e (11.40 mg, 0.023 mmol) was added LiOH
(0.273 mg, 0.011 mmol), and the solids were dissolved in
a mixture of water:THF:MeOH (1:1:1). The reaction mixture
was stirred at rt for 12 h. Then another 0.5 equiv. of LiOH
was added, and the temperature was raised to 50 °C. The
reaction ran 6 more hours at 50 °C, and then the solvent
was concentrated. The crude product containing the dies-
ter and the monoacid was chromatographed (0–10%
MeOH/DCM) to give 3-(3-carbamoyl-4-((3-methoxyphenyl)
amino)-8-methylquinolin-6-yl)-5-(methoxycarbonyl) benzoic
acid (18b), (white solid, Yield: 12%). 1H NMR (500 MHz,
DMSO-d6) d 10.50 (s, 1H), 9.00 (s, 1H), 8.42 (s, 1H), 8.24
(d, J = 7.3 Hz, 2H), 8.04 (br. s., 1H), 7.95 (s, 2H), 7.69 (br.
s., 1H), 7.17 (t, J = 8.0 Hz, 1H), 6.66 (dd, J = 1.9, 8.3 Hz,
1H), 6.62 (s, 1H), 6.55 (d, J = 7.8 Hz, 1H), 3.90 (s, 3H),
3.68 (s, 3H), 2.77 (s, 3H). LCMS found 486.01, [M+H]+.
4-chloro-6-iodo-N,N,8-trimethylquinoline-3-
carboxamide (25b)
To sodium hydride (60 % dispersion in mineral oil, 23 mg,
0.57 mmol) in DMF (2 mL) was added intermediate 7
(100 mg, 0.289 mmol) in dry DMF (3 mL). The reaction
mixture was stirred for 35 min at rt before adding iodome-
thane (0.020 mL, 0.317 mmol), after which the reaction
mixture was stirred at rt overnight. Then, to the reaction
mixture were added water and EtOAc. The aqueous layer
Chem Biol Drug Des 2015; 85: 549–564
553

Ochiana et al.
was extracted with EtOAc (39). The combined organics
were washed with brine and finally dried under sodium sul-
fate. The solvent was concentrated, and the product was
confirmed by LCMS, LCMS found 374.01, [M+H]+. The
product was taken to the next step without further charac-
terization.
reaction was run at 80 °C in a sealed vial for 48 h. The
solvent was concentrated, and the crude product was
chromatographed using 0–7% MeOH in DCM to give the
desired product as a yellow solid (Yield: 34%).1H NMR
(500 MHz, CDCl3) d 9.50 (d, J = 7.3 Hz, 1H), 8.75 (s, 1H),
8.39 (d, J = 4.8 Hz, 2H), 8.07 (d, J = 7.8 Hz, 1H), 7.87
(s, 1H), 7.76 (s, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.59 (t,
J = 7.6 Hz, 1H), 7.29 (s, 1H), 5.90 (br. s., 2H), 4.55–4.64
(m, 1H), 2.80 (s, 3H), 2.11–2.20 (m, 2H), 1.78–1.90
(m, 4H), 1.68–1.75 (m, 2H). LCMS found 413.01, [M+H]+.
6-iodo-4-((3-methoxyphenyl)amino)-N,8-
dimethylquinoline-3-carboxamide (26a)
To 25a (104 mg, 0.288 mmol) were added acetonitrile and
3-methoxyaniline (0.034 ml, 0.303 mmol). The mixture was
heated under reflux overnight. The solution was filtered,
and the solids were washed with acetonitrile. The filtrate
was concentrated. The product 26a (Yield: 41%) was con-
firmed by LCMS (found 448.04 [M+H]+) and was taken to
the next step without further characterization.
Molecular modeling
Docking for all compounds was performed using POSIT v
1.0.2 (OpenEye Scientific Software: Santa Fe, NM, USA).
The co-ordinates of the apo crystal structure of TbrPDEB1
were downloaded from the Protein Data Bank (PDB), PDB
code: 4I15 (21). The protein was prepared for docking,
and mild ligand–protein clashes were allowed to account
for the average co-ordinate error expected in PDB struc-
tures. The ‘combine-receptors’ option was used to include
the binding mode observed in the human phosphodiester-
ase 4B2B in complex with a quinoline Inhibitor during
docking in TbrPDEB1 (PDB code: 3FRG) (19). Mild clashes
were also used in pose prediction. All the other options in
POSIT were kept as default. The docked poses were
energy minimized using SZYBKI (v 1.7.0 OpenEye Scientific
Software) allowing partial relaxation of the protein residues
in the direct proximity to the ligand. The docking program
FRED v 3.0.0 (OpenEye Scientific Software) was also used
to investigate alternative binding modes (such as for com-
pound 28).
6-iodo-4-((3-methoxyphenyl)amino)-N,N,8-
trimethylquinoline-3-carboxamide (26b)
To 25b (56.0 mg, 0.149 mmol) were added acetonitrile
and 3-methoxyaniline (0.018 ml, 0.157 mmol). The mixture
was heated under reflux overnight. The solution was fil-
tered, and the solids were washed with acetonitrile. The fil-
trate was concentrated. The product (Yield: 61%) was
confirmed by LCMS (462.01, [M+H]+) and was taken to
the next step without further characterization.
4-((3-methoxyphenyl)amino)-N,8-dimethyl-6-(1-
methyl-1H-indazol-6-yl)quinoline-3-carboxamide
(27a) (synthesized using General procedure B from
26a, yellow solid, Yield: 8%)
1H NMR (500 MHz, DMSO-d6) d 10.05 (s, 1H), 8.87 (s, 1H),
8.59 (d, J = 4.4 Hz, 1H), 8.14 (s, 1H), 8.07 (s, 1H), 8.06
(s, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.69 (s, 1H), 7.34–7.37
(m, 1H), 7.21 (t, J = 8.0 Hz, 1H), 6.64–6.68 (m, 2H), 6.58
(d, J = 8.3 Hz, 1H), 4.07 (s, 3H), 3.70 (s, 3H), 2.80 (s, 3H),
2.66 (d, J = 4.4 Hz, 3H). LCMS found 452.01, [M+H]+.
Water molecule predictions were performed using SZMAP v
1.1.0 (OpenEye Scientific Software) on three different pro-
tein–ligand complex structures: (i) the crystal structure of
the human phosphodiesterase 4B2B(15), (ii) the TbrP-
DEB1–19a complex, and (iii) the TbrPDEB1–24g complex.
The conformations of the ligands of the two latter protein–
ligand complexes were obtained with molecular docking.
The protein–ligand complexes were used as input to
SZMAP, which was then used to predict the solvation of
the active binding site in complex with the ligands using
the semi-continuum solvation theory, in combination with
a single explicit water probe. The results were visualized
with VIDA (OpenEye Scientific Software).
4-((3-methoxyphenyl)amino)-N,N,8-trimethyl-6-(1-
methyl-1H-indazol-6-yl)quinoline-3-carboxamide,
formic acid salt (27b), (synthesized using General
procedure B from 26b, yellow solid, Yield: 17%)
1H NMR (500 MHz, DMSO-d6) d 9.11 (s, 1H), 8.55 (s, 1H),
8.51 (s, 1H), 8.13 (s, 2H), 8.07 (s, 1H), 8.04 (s, 1H), 7.86
(d, J = 8.3 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.17
(t, J = 8.5 Hz, 1H), 6.63 (d, J = 8.3 Hz, 1H), 6.55–6.59
(m, 2H), 4.12 (s, 3H), 3.71 (s, 3H), 2.79 (s, 3H), 2.75
(s, 3H), 2.38 (s, 3H). LCMS found 466.01, [M+H]+.
Results
In the interest of exploring the SAR around 3, we divided
the compound into 4 regions, highlighted in Figure 1. First,
we synthesized a set of analogs to determine the preferred
aryl substituent and connection to the quinoline core for
Region A, following Scheme 1. Synthesis commenced
with the condensation of 5a with diethyl ethoxymethylene-
malonate, followed by cyclization to afford 6. The ester
was hydrolyzed to the acid, and the resulting product was
4-(cyclopentylamino)-8-methyl-6-(3-(oxazol-2-yl)
phenyl)quinoline-3-carboxamide (28)
To 20b (13.00 mg, 0.036 mmol) were added acetonitrile
and lastly cyclopentanamine (0.021 mL, 0.214 mmol). The
554
Chem Biol Drug Des 2015; 85: 549–564

Repurposing GSK-256066 for TbrPDEB1 Inhibition
Me
Me
Me
H
N
Me
N
N
f
NH2 a,b
c,d,e
I
CONH2
I
CO2Et
I
CONH2
I
HN
O
Cl
6
7
5a
8
Me
Me
OMe
N
Me
N
N
g
h
j or k
S
CONH2
OMe
O2S
CONH2
O2S
CONH2
HN
HN
HN
R1
R1
9a: R1=CO2Me
9b: R1=H
3R4RNOC
10a: R=CO2Me
10b: R1=H
11:R1=CO2H
OMe
12a: R3=R4=H
i
OMe
12b: R3=H, R4=Me
3: R=R4=Me
Me
N
l
HN
CONH2
OMe
8
HN
MeO2C
13
Scheme 1: Preparation of analog 13, exploring Region A (Reagents and conditions. (a) EtOCH=C(CO2Et)2, 100 °C, 1 h (98%). (b) Ph2O,
250 °C, 45 min (100%). (c) NaOH, EtOH, reflux, 1 h, then conc. HCl, overnight. (d) SOCl2, DMF, 80 °C, 2 h. (e) NH4OH, rt, overnight
(80%). (f) 3-methoxyaniline, MeCN, 80 °C, overnight (90%). (g) 3-substituted (R1) benzenethiols, Pd2(dba)3, DPEphos, KOtBu, toluene,
170 °C, MW, 30 min (70–85%). (h) Oxone, DMF, rt, 12–24 h (70–90%). (i) NaOH, EtOH, 45ᵒC, overnight (31%). (j) 11, Me2NH or
MeNH2ꢀHCl, HATU, DIEA, DMF, rt (for 3 (21%) and 12b (37%). (k) for 10a: NH4OH, dioxane, rt, overnight (12a,18%). (l) methyl
3-aminobenzoate, Pd(dppf)Cl2, (oxybis(2,1-phenylene))bis (diphenylphosphine), KOtBu, dioxane, 160 °C, MW, 20 min (12%)).
R4
R4
R4
R4
H
N
N
N
f
NH2 a,b
c,d,e
CO2Et
I
CONH2
OMe
I
I
CONH2
I
HN
O
Cl
5a: R4=Me
5b: R4=H
5c: R4=Et
6: R4=Me
14a: R4=H
14b: R4=Et
7: R4=Me
15a: R4=H
15b: R4=Et
Me
8: R4=Me
16a: R4=H
16b: R4=Et
h
Me
R4
N
N
N
R1O2C
g
CONH2
8
Ar
CONH2
OMe
N
HN
CONH2
N
HN
HN
CO2R2
17a-v
19a: R4=Me
19b: R4=H
19c: R4=Et
17e: R1=R2=Me
18a:R1=H; R2=Me
18b:R1=R2=H
OMe
i
OCH3
Scheme 2: Preparation of analogs 17-19 (Reagents and conditions. (a) EtOCH=C(CO2Et)2, 100 °C, 1 h (80–98%). (b) Ph2O, 250 °C,
45 min (81–100%). (c) NaOH, EtOH, reflux, 1 h, then conc. HCl, overnight. (d) SOCl2, DMF, 80 °C, 2 h. (e) NH4OH, rt, overnight (61–
92%). (f) 3-methoxyaniline, MeCN, 80 °C, overnight (78–94%). (g) Ar-B(OH)2, Na2CO3, Pd(dppf)Cl2, dioxane, 130 or 145 °C, MW, 20 min
(20–70%). (h) 1-Methyl-1H-indazole-6-boronic acid, Na2CO3, Pd(dppf)Cl2, dioxane, 160 °C, MW, 20 min (13–56%). (i) LiOH, H2O:THF:
MeOH, rt, 12 h).
chlorinated and quenched with aqueous ammonia afford-
ing the primary carboxamide 7. Reaction of 7 with m-anisi-
dine under reflux provided 8 in high yield. This template
was reacted with commercially available aryl thiols using
palladium catalysis to provide the sulfides, 9, which could
be oxidized to the appropriate sulfones 10 using Oxone.
11, and then standard amide coupling with the appropriate
amine provided 3 and 12b. Treatment of 10a with ammo-
nium hydroxide gave analog 12a. To explore a nitrogen
linker to Region A, the reaction of the commercial methyl
3-aminobenzoate with 8 using a modified palladium-cata-
lyzed coupling procedure provided 13 (22,23).
Amide substituents were introduced via hydrolysis of the
Following synthesis, the analogs were tested at a single
ester functionality of 10a under basic conditions to give
concentration (10 lM) against TbrPDEB1, and those
Chem Biol Drug Des 2015; 85: 549–564
555

Ochiana et al.
Table 1: Biochemical potency data for
analogs of 3a
N
NH2
R1
X
NH
O
O
TbrPDEB1
(% inh)
TbrPDEB1
(IC50 lM)
TbrPDEB2
(IC50 lM)
Compound
R1
X
3
9a
CONMe2
CO2Me
H
SO2
S
S
51 ꢁ 3.9
12 ꢁ 6.1
10.0b
24 ꢁ 1.7
29.8 ꢁ 7.5
–
–
9b
10a
10b
11
CO2Me
H
CO2H
CONH2
CONHMe
CO2Me
SO2
SO2
SO2
SO2
SO2
NH
70 ꢁ 7.9
21 ꢁ 16
82 ꢁ 10
64 ꢁ 7
3.5 ꢁ 0.3
–
15.1 ꢁ 3.9
–
2.9 ꢁ 0.8
6.8 ꢁ 0.6
12a
12b
13
–
–
–
–
66 ꢁ 6.1
31 ꢁ 5.8
16 ꢁ 1.7
84 ꢁ 7.7
aAll values are the mean of three or more replicates ꢁ SEM.
bn = 1.
compounds with >65% inhibition, and those that repre-
sented key SAR points, were tested in a dose–response
assay. We noted that removal of N-alkyl groups from the
benzamide of 3 results in little change in percentage inhibi-
tion, the key data in selecting compounds for IC50 deter-
mination (3, 12a-b, Table 1). Replacement of the
carboxamide with an ester (10a) or carboxylic acid (11)
improved activity by approximately 8-fold over 3. The sul-
fone linker between the quinoline and side chain was bet-
ter than either a sulfide (9a) or amine (13).
twofold of the potency of 17a. Additionally, the hydrolysis
of the diester groups of 17e provided the mono- and diac-
ids 18a-b, which showed low (<50%) inhibition at 10 lM.
The 3-methyl ester substitution was preferred over the 2
or 4 position (Table 2, 17a-17c). We noted that several
compounds bearing replacements for the methyl ester of
17a (17m, 17o, 17p, 17t) were approximately equipotent,
although none was significantly better. Given their increase
in complexity and size over 17a, these analogs were less
desirable. The tetrazole analog 17m was more potent than
the corresponding bioisosteric carboxylic acid (17d), which
was not potent enough to advance to IC50 determination.
Having explored the sulfone, sulfide, and amine linker of
the A region of the inhibitor, we designed analogs with no
intervening atom between the core template and the A
region via a biaryl linkage. This was accomplished using
the route shown in Scheme 2. The iodo template 16 was
prepared using a route analogous to the preparation of 8,
followed by reaction with various boronic acids using
Suzuki coupling chemistry.
The potency of the best biaryl compound in Table 2 (19a)
is similar to the best sulfone from Table 1 (11), leading us
to focus upon these biaryl compounds given their ease of
synthesis and purification compared to the sulfone ana-
logs. With this in mind, we next turned our attention to
preparation of Region B variants, keeping Region A con-
stant with either of the two best Region A substituents:
the methyl indazole fragment of 19a or the methylbenzoate
fragment of 17a.
The potency of the biaryl analog 17a on TbrPDEB1
(Table 2) was within twofold of the IC50 of the matched
sulfone analog 10a (Table 1). The observation that the
matched biaryl linker was essentially equivalent to the more
synthetically intensive sulfone linker allowed us to access a
larger set of analogs via one-step synthesis from the iodo
template (8 or 16) and boronates using Suzuki couplings.
We explored the SAR for this region with the biaryl linkage
using various aryl boronic acids or esters obtained from
commercial sources, or via synthesis (Appendix S1, Sup-
porting Information) (24–26). Besides close-in analogs of
17a, we explored small, heterocyclic replacements and
bioisosteres for the methyl ester moiety (Table 2, 17o-17r).
A small set of analogs bearing fused bicyclic heteroaromat-
ic rings was also prepared (17t-v, 19a). Notably, the
potency of the N-methyl indazole analog 19a was within
These analog syntheses were accomplished using one of
the two routes shown in Scheme 3. Reaction of 7 with
(3-(methoxycarbonyl)phenyl)boronic acid using Suzuki
coupling afforded 20, the bis-arylated product 21, and
the dehalogenated byproduct 21b. The final step required
heating of 20 with the desired amine to give 22a-d, and
28. Additionally, reaction of 7 with various amines under
reflux provided intermediates 23 (General procedure A
and Appendix S1, Supporting Information), followed by
reaction of these intermediates via Suzuki coupling with
1-methyl-1H-indazol-6-ylboronic acid to give 24a-g or 3-
(methoxycarbonyl)phenylboronic acid to afford 22e-f.
556
Chem Biol Drug Des 2015; 85: 549–564

Repurposing GSK-256066 for TbrPDEB1 Inhibition
Table 2: continued
Table 2: Analogs exploring the Region A of 3a
N
N
NH2
NH2
R1
R1
NH
NH
O
O
O
O
TbrPDEB1 TbrPDEB1 TbrPDEB2
TbrPDEB1 TbrPDEB1 TbrPDEB2
Compound R1
(% inh)
(IC50 lM)
(IC50 lM)
Compound R1
(% inh)
(IC50 lM)
(IC50 lM)
CO2Me
17a
94 ꢁ 3.5 6.4 ꢁ 1.7
37 ꢁ 14
6.2 ꢁ 0.6
17o
83 ꢁ 1.3
6 ꢁ 1.7
S
N
Me
e
M
O2C
N
17b
17c
17d
17e
17f
O
17p
17q
53 ꢁ 24 5.9 ꢁ 0.4
N
6 ꢁ 1.4
54 ꢁ 8.8
50 ꢁ 6.1
35 ꢁ 10
–
–
–
CO2Me
27 ꢁ 8.3
31 ꢁ 6.3
–
–
O
N
CO2H
CO2Me
Me
N
17r
O
N
MeO2C
N
23 ꢁ 4.0
Me
NH2
Me
17s
68 ꢁ 6.3 12 ꢁ 2.7
17g
17h
17i
65 ꢁ 5.5 ndb
N
O
O
9 ꢁ 7.1
–
–
17t
85 ꢁ 6.5 5.9 ꢁ 1.0 13.5 ꢁ 3.5
OCF3
NH
8.6 ꢁ 5.2
17u
52 ꢁ 8.3
–
NH
O
N
N
H
17j
74 ꢁ 13
10 ꢁ 6.9
9 ꢁ 0.6
N
NH
17v
18a
43 ꢁ 3.3
10 ꢁ 5.3
–
–
17k
–
–
N
CO2H
N
OMe
17l
N
43 ꢁ 16
CO2H
Me
CO2
N
18b
19a
31 ꢁ 14
–
17m
83 ꢁ 7.5 6.7 ꢁ 2.9
4.6 ꢁ 0.1
H
CO2
84 ꢁ 9.1 3.1 ꢁ 0.5
8.0 ꢁ 3.9
N
NH
N
N
N
N
Me
17n
13 ꢁ 8
–
O
O
aAll values are the mean of three or more replicates ꢁ SEM.
bNo IC50 obtained due to limited solubility at higher concentra-
tions.
Chem Biol Drug Des 2015; 85: 549–564
557

Ochiana et al.
Me
this spacing by an additional carbon (e.g., 22a) led to loss
of activity. Removal of this substituent altogether resulted
in a total loss of activity (21b). Finally, comparison of 17a
and the N-methylated analog 22e reveals loss in activity.
N
b
N
CONH2
N
R2
24a-g
Me
Me
Me
N
Synthesis of analogs varying Regions C and D of 19a
is shown in Schemes 2 and 4. Reaction of 7 with NaH
and iodomethane in DMF provided intermediates 25a-b
(Scheme 4). Heating of these intermediates with m-anisi-
dine in acetonitrile afforded 26a-b, which were sub-
a
N
7
I
CONH2
NH2
R2
c
R2
O
O
d
23
CO2Me
22e-f
Me
jected to Suzuki reaction to provide the Region
C
N
N
variants 27a-b.
e
NH2
NH2
R2
O
R2
The variations at Region D were introduced at the begin-
ning of the synthesis when the appropriate aniline 5 was
used (Scheme 2). The methylindazole fragment was intro-
duced in the last step via Suzuki chemistry to provide ana-
logs 19a-c.
R1
R1
20a: R1=CO2 Me;R2=Cl
20b: R1=oxazol-2-yl; R2=Cl
21a: R1=CO2 Me;R2=methyl-3-benzoate
21b: R1=CO2 Me; R2=H
22a-c: R1=CO2 Me; R2=NHCH2Ar
22d: R1=CO2Me
R2=cyclopentanamine
28: R1=3-oxazyl-2-yl;
R2=cyclopentanamine
As shown in Table 4, the primary amide in Region C is
required; substitution with one or two methyl groups leads
to dramatic loss of activity (19a versus 27a and 27b).
Removal of the Region D methyl group leads to a ~4-fold
loss in activity (19a versus 19b), while extension to an
ethyl group (i.e., 19c) is approximately equipotent.
Scheme 3: Preparation of analogs exploring Region B (Reagents
and conditions. (a) Various amines (R2), MeCN, 80 °C, 24 h (61–
80%) or various amines (R2), MeCN, 145 °C, 25 min (59–80%). (b)
1-Methyl-1H-indazole-6-boronic acid, Na2CO3, Pd(dppf)Cl2,
dioxane, 130 or 145 °C, MW, 20 min (9–22%). (c) (3-
(methoxycarbonyl) phenyl)boronic acid, Na2CO3, Pd(dppf)Cl2,
dioxane, 130–145 °C, MW, 20 min (7–12%). (d) (3-
(methoxycarbonyl) phenyl)boronic acid or (3-(oxazol-2-yl)phenyl)
boronic acid, Na2CO3, Pd(dppf)Cl2, dioxane, 150 °C, MW, 10 min
(20a (30%), 20b (14%), (21a (5%), 21b (2%)). (e) Various amines
(R2), MeCN, 80 °C, 48 h (10–40%)).
The range of activity in this SAR study is relatively narrow,
spanning potency differences of about 100-fold, although
we were pleased that we were able to improve compound
potency about eightfold from 3. Inhibition of both
TbrPDEB1 and B2 is required, although we have previ-
ously noted close correlation in TbrPDEB1 and B2 IC50
values (7). Therefore, we periodically obtained TbrPDEB2
IC50 values for compounds and tested against both PDEs
prior to assessment in T. brucei cells. We note that, with
the exception of one compound 10a, those compounds
tested against TbrPDEB2 showed IC50 values within
~2-fold of TbrPDEB1. Testing against human PDE4B
unfortunately confirmed that these compounds remain
extraordinarily potent inhibitors (Table 5). Nonetheless,
testing of some of the most potent TbrPDEB1/B2 inhibi-
tors does show cellular growth inhibition approximating the
biochemical potency. One notable exception is the lead
compound 3; surprisingly, although this compound is a
As shown in Table 3, compound activity is affected by the
regiochemistry of the methoxy substituent on the R2
group: para substitution is not tolerated (24a), whereas the
ortho methoxy analog 24b is only slightly less potent than
19a. We observe that the ethyl analog 24d, which is isos-
teric to 19a, is just as potent. This led us to question the
essentiality of this oxygen atom, although some sort of
substitution at this position seems important (compare 19a
versus 24a or 24g).
Analogs of 17a are sensitive to the spacing between the B
region substituent and the rest of the molecule. Extending
Me
Me
N
N
R1
N
R1
N
Me
N
R1
N
I
R2
b
N
R2
a
c
7
N
O
NH
O
NH
I
R2
O
Cl
25a: R1=CH3, R2=H
25b: R1=R2=CH3
OMe
OMe
27a, R1=CH3, R2=H
27b, R1=R2=CH3
26a, R1=CH3, R2=H
26b, R1=R2=CH3
Scheme 4: Preparation of analogs 27 exploring Region C (Reagents and conditions. (a) NaH, DMF, 30 min, rt, then CH3I, rt, overnight
(52–100%). (b) 3-methoxyaniline, MeCN, 80 °C, overnight (40–60%). (c) 1-Methyl-1H-indazole-6-boronic acid, Na2CO3, Pd(dppf)Cl2,
dioxane, 160 °C, MW 20 min (8–17%)).
558
Chem Biol Drug Des 2015; 85: 549–564

Repurposing GSK-256066 for TbrPDEB1 Inhibition
Table 3: Biochemical data for analogs exploring Region Ba
e
M
e
M
e
M
N
N
N
NH
CO
NH
CO
2
2
R2
R2
NH
CO
2
R2
e
N
N
M
CO2
e
M
17, 21, 22
28
19, 24
N
O
Compound
R2
TbrPDEB1 (% inh)
TbrPDEB1 (IC50 lM)
6.4 ꢁ 1.7
17a
94 ꢁ 3.5
HN
OMe
21a
41 ꢁ 5.8
–
Me
CO2
21b
22a
0
–
–
H
HN
19 ꢁ 6.6
OMe
Me
HN
HN
22b
22c
19 ꢁ 4.2
22 ꢁ 20
–
28 ꢁ 3.4
22d
22e
80 ꢁ 4.8
59 ꢁ 3.6
ndb
NH
–
OMe
N
22f
72 ꢁ 14
26 ꢁ 1.3
3.8 ꢁ 0.4
HN
N
19a
83.7 ꢁ 9.1
HN
OMe
OMe
24a
24b
34 ꢁ 0.3
60 ꢁ 16
–
HN
HN
7 ꢁ 0.9
MeO
24c
24d
82 ꢁ 12
67 ꢁ 7.5
4.2 ꢁ 0.6
3.5 ꢁ 0.5
HN
HN
OEt
Et
Chem Biol Drug Des 2015; 85: 549–564
559

Ochiana et al.
Table 3: continued
e
M
e
M
e
M
N
N
N
NH
CO
NH
CO
2
2
R2
R2
NH
CO
2
R2
e
N
N
M
CO2
e
M
17, 21, 22
28
19, 24
N
O
Compound
R2
TbrPDEB1 (% inh)
TbrPDEB1 (IC50 lM)
3.5 ꢁ 0.6
24e
85 ꢁ 11
HN
Me
OMe
24f
69 ꢁ 16
3.7 ꢁ 0.2
HN
HN
Me
24g
28
21 ꢁ 6.5
76 ꢁ 2.2
–
5.9 ꢁ 0.67
NH
aAll values are the mean of three or more replicates ꢁ SEM.
bNo IC50 obtained due to limited solubility at higher concentrations.
weak TbrPDEB1/B2 inhibitor, it inhibits cell growth with an
EC50 of 7.8 uM. We presume this must be due to other
mechanisms of inhibition operating in the cell.
values could therefore be explained by their favorable
interaction with this dipole. Within the helix, an important
key amino acid change between the human PDE4B and
parasite PDE is Ser454 to Asn717 (Figure 2A, B)(19). The
docking poses of compounds 11, 17a, and 17m are
shown in Figure 2B, highlighting the hydrogen bonds to
the amide NH2 of Asn717. In addition, the linker in the
substituent in Region A appears to play an important role
in placing the electron-rich substituent in contact with the
Asn717 residue in the N-terminal helix. Woodrow et al.
(16) previously described the detrimental effect of replacing
the sulfone linker with a sulfide (such as in the case of 9a
versus 10a), likely due to the ease of the sulfone to preori-
ent the molecule in the proper conformation compared to
the more flexible sulfide. We infer that the C-C linker-con-
taining compounds must be able to preorient similarly to
their sulfone congeners given the similar activity profile
(e.g., 10a and 17a).
We looked toward molecular modeling to help explain our
observations, utilizing the recently published X-ray crystal
structure of TbrPDEB1 to perform docking experiments of
key analogs of 3. The binding mode of a similar quinoline
derivative in complex with the human PDE4B2B was
reported in a crystal structure published by Lunniss and
co-workers (19). This crystal structure reveals that the
quinoline nitrogen of 3 interacts with the amide NH2 of
Gln874 and that the ligand amide NH2 (Region C in Fig-
ure 1) interacts with carbonyl group of Asn825 residue
(Figure 2A). Both residues (Gln874 and Asn825 in TbrP-
DEB1) are conserved between the human and parasite
PDE and are therefore predicted to give similar interac-
tions with this class of ligands. The amide carbonyl
(Region C) is predicted to give an internal hydrogen bond
with the NH group of the substituent occupying area B
(Figures 1 and 2A).
The substituents in Region B are predicted to occupy the
space near the magnesium ion (Figure 2A). Analysis of the
crystal structure of hPDE4B2B shows that the m-methoxy
ꢀ
The Region A substituents are predicted to point toward
the N-terminus of an alpha-helix, which is slightly positively
charged in consequence of the dipole moment (27). The
fact that electron-rich substituents occupying Region A
(e.g., 11, Figure 2A) display generally better potency
substituent is located in the proximity of magnesium (~4 A),
indirectly interacting with the metal via intervening water
molecules (Figure S1A). However, in the case of TbrP-
DEB1, we note the interesting observation that analogs
with ethyl or methyl replacements of the m-methoxy group
560
Chem Biol Drug Des 2015; 85: 549–564

Repurposing GSK-256066 for TbrPDEB1 Inhibition
Table 4: Exploring Regions C and D of 19
retain potency (compare 19a with 24d and 24e), although
an unsubstituted phenyl ring (24g) is detrimental for
potency (Table 3). Water prediction calculations (SZMAP
v1.1.0, OpenEye Scientific Software) suggest that, in
absence of a meta-position substitution in this phenyl
group, there is a small, unoccupied cavity in the protein–
ligand complex (Figure S1C). This unfavorable situation
may explain the lack of activity of 24g.
R3
N
R4
N
N
NH
O
O
We predict the cyclopentyl-substituted compounds 22d
and 28 to orient in an alternative binding mode, accommo-
dated by an alternative rotamer of Asn825 that is able to
interact with the pyridine nitrogen of the ligand (Figure 2C)
(28,29). We prepared these analogs, and while 28 is a 5.9-
lM inhibitor of TbrPDEB1, 22d was not sufficiently soluble
to test in the dose–response experiment, although we
observed the compound to inhibit TbrPDEB1 80% at 5 lM.
TbrPDEB1
(% inh)
TbrPDEB1
(IC50 lM)
Compound
R3
R4
19a
19b
19c
27a
27b
Me
H
Et
Me
Me
NH2
NH2
NH2
NHCH3
NMe2
84 ꢁ 9.1
84 ꢁ 2.4
79 ꢁ 8.9
34 ꢁ 11
22 ꢁ 3.8
3.1 ꢁ 0.5
12 ꢁ 1.3
3.7 ꢁ 0.5
–
–
Methylation of the amide occupying Region C is detrimental
for potency (compare 19a, 27a, and 27b, Table 4). This
is consistent with the hypothesized essential H-bonding
All values are the mean of three or more replicates ꢁ SEM.
Table 5: Biochemical and cellular characterization of the most potent TbrPDEB1 inhibitors
Compound
Reg. number
cLogP
TbrPDEB1 (IC50 lM)a
TbrPDEB2 (IC50 lM)a
hPDE4 (IC50 lM)a
T. brucei cell (EC50, lM)a
3
NEU-355
NEU-356
NEU-433
NEU-489
NEU-462
NEU-528
NEU-542
4.33
5.04
5.73
5.82
5.17
5.62
6.29
24 ꢁ 1.7
3.5 ꢁ 0.3
6.4 ꢁ 1.7
5.9 ꢁ 1.0
3.1 ꢁ 0.5
3.7 ꢁ 0.5
3.5 ꢁ 0.5
29.8 ꢁ 7.5
15.1 ꢁ 3.9
6.2 ꢁ 0.6
13.5 ꢁ 3.5
8.0 ꢁ 3.9
5.6 ꢁ 3.4
12.1 ꢁ 3.2
7.9 9 10ꢂ6
7.8 ꢁ 2.5
7.3 ꢁ 0.97
7.0 ꢁ 3.9
5.6 ꢁ 1.9
6.8 ꢁ 0.82
7.9 ꢁ 2.7
8.7 ꢁ 1.8
10a
17a
17t
19a
19c
24d
–
4.2 9 10ꢂ5
–
8.3 9 10ꢂ5
–
–
aAll values are the mean of three or more replicates ꢁ SEM.
A
B
C
Figure 2: (A). Overlay of the crystal structure of the human phosphodiesterase 4B2B in complex with a quinoline inhibitor (PDB code:
3FRG, cyan carbon atoms) with the docked conformation of 11 in TbrPDEB1 (green carbon atoms). TbrPDEB1 residue numbering is
used, with the corresponding hPDE4B residues in brackets. (B). Overlay of the docking poses of (i) 17a (yellow carbon atoms), (ii) 11
(green carbon atoms), and (iii) 17m (brown carbon atoms) in TbrPDEB1. (C). Docked conformation of 28 in TbrPDEB1. This pose was
generated using FRED. The side chain of Asn825 has been flipped to allow a hydrogen bond of the amidic nitrogen with the pyridine
nitrogen of the ligand. In all these figures, Mg2+ and Zn2+ are shown as green and gray spheres, respectively, and the interactions of the
ligands with Gln 874, Asn 825, and Asn 717 are shown in blue dashed lines. The surface of the active site and the P-pocket can also be
€
seen in B and C. Images generated using the PYMOL Molecular Graphics System, Version 1.5.0.4 Schrodinger, LLC.
Chem Biol Drug Des 2015; 85: 549–564
561

Ochiana et al.
interaction between this amide NH2 and Asn825 (Figure 2A
and B). Finally, the R3 substituent (Region D) is predicted to
occupy an area, adjacent to the ‘parasite-’ or ‘P-pocket’
(Figure 2B) (7,21), defined by the side chain of Met861 and
the alpha-helix located at the back pocket of the active site.
The presence of a methyl or ethyl group in Region D is pre-
dicted to increase potency by partially filling the P-pocket,
and this potency improvement was confirmed experimen-
tally as can be seen by comparing the potency values for
compounds 19a, 19b, and 19c in Table 4.
(e.g., 19a IC50 = 0.083 nM). Although this represents a
slight improvement in overall selectivity, more must be
done to improve selectivity for a new HAT therapeutic. For
us, and for others (17,18), this appears to be a significant
issue that has yet to be overcome, though with recent
structural biology reports of TbrPDEB1 (21), we expect that
understanding about the ligand–target interactions that
drive selectivity will become more clear in the near future.
Importantly, in most cases, the structure–activity relation-
ships we observe are consistent with the expected binding
modality of this chemotype, based on previous structural
biology reports of 3 bound to hPDE4B (16). For example,
the requirement for an unsubstituted 3-quinoline carboxa-
mide moiety is retained. On the other hand, we have made
a surprising observation regarding the importance of the
m-methoxy substituent in Region B: in hPDE4, this func-
tionality provides ~7-fold potency over an alkyl group at
this position (16), presumably by interacting with the cata-
lytic metal ion via intervening water molecules. On the
other hand, in the parasite enzyme, replacement of this
methoxy group with an isosteric ethyl group shows sur-
prisingly good activity (compare 19a with 24d, which have
equivalent TbrPDEB1 and cellular potency, Table 5).
Discussion and Conclusions
We have applied a target repurposing approach to identify
inhibitors of TbrPDEB1, an essential enzyme of the proto-
zoan pathogen T. brucei. One of the advantages of this
approach is the ability to launch optimization studies for
new inhibitors with minimal upfront screening efforts.
Indeed, our efforts to re-optimize chemotypes related to
piclamilast (7,10), cilomilast (30), and now GSK-256066 for
improved potency against TbrPDEB1 were launched by a
broad assessment of 20 existing human PDE inhibitors (7).
In this report, the SAR of the GSK-256066 chemotype
was elaborated in a rapid fashion and provided improved
analogs such as 10a, 17a, 17t, 19a, 19c, and 24d. This
was possible by systematically studying the four regions of
compound 3 (Figure 1) that we set out to explore. Our
work revealed some general requirements needed for
increased activity. First, for Region A, the most favorable
functionalities were the meta substituted (e.g., ester, car-
boxylic acid, small heterocycles) aryls (e.g., 10a, 11, 17m,
17o, 17p) or the methylindazole fragment (19a). This high-
lights that while Region A needs to be filled with lipophilic
aromatic cores, this is not sufficient for activity. More
important is a requirement for hydrogen acceptor groups
that may interact with Asn717 (Figure 2B). Furthermore,
for Region B, we discovered that substituted aryls in the
meta-position with groups such as methyl, ethyl, methoxy,
or ethoxy (i.e., 24e, 24d, 17a and 24c) are preferred for
increased activity, as there is a small unoccupied cavity in
the protein–ligand complex that we posit is now filled by
these lipophilic groups (Figure S1C). For Region C, the
amide functionally was shown to be essential as it inter-
acts with Asn825, which is present in both hPDE4 and
TbrPDEB1 (shown in Figure 2A). Lastly, for Region D, we
show that by occupying the P-pocket by a methyl (19a) or
ethyl substituent (19c), we observe an increase in activity
(Figure 2B). This region would benefit from additional
exploration, although this is synthetically the most chal-
lenging part of the molecule to vary. In the end, our
medicinal chemistry efforts provided compound 19a,
which is 6.5-fold more active in the TbrPDEB1 biochemical
assay, 10.5-fold less potent against hPDE4, and show
modest potency against T. brucei cells.
Acknowledgments
This work was funded by the National Institutes of Health
(R01AI082577). We are grateful to Dr Emanuele Amata for
helpful discussions. A free academic license to OpenEye
Scientific Software and ChemAxon for their suites of
programmes is gratefully acknowledged.
Conflict of Interest
The authors declare that no conflict of interest exists.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Appendix S1. The screening data has been made freely
available as a shared data set at www.collaborativedrug-
discovery.com.
Figure S1. (A). Hydrogen bond network near the head
group region as seen in the crystal structure of the human
phosphodiesterase 4B2B in complex with a quinoline
Inhibitor (PDB code: 3FRG, cyan carbon atoms).
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