Quinolines as a novel structural class of potent and selective PDE4 inhibitors. Optimisation for inhaled administration

Article abstract of DOI:10.1016/j.bmcl.2009.04.012

Crystallography driven optimisation of a lead derived from similarity searching of the GSK compound collection resulted in the discovery of quinoline-3-carboxamides as highly potent and selective inhibitors of phosphodiesterase 4B. This series has been optimized to GSK256066, a potent PDE4B inhibitor which also inhibits LPS induced production of TNF-α from isolated human peripheral blood mononuclear cells with a pIC₅₀ of 11.1. GSK256066 also has a suitable profile for inhaled dosing.

Full text of DOI:10.1016/j.bmcl.2009.04.012

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5261–5265
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Quinolines as a novel structural class of potent and selective PDE4
inhibitors. Optimisation for inhaled administration
g
a
c
d
Michael D. Woodrow a, , Stuart P. Ballantine , Michael D. Barker , Beth J. Clarke , John Dawson ,
Tony W. Dean c, Christopher J. Delves g, Brian Evans c, Sharon L. Gough c, Steven B. Guntrip b, Stuart Holman a,
Duncan S. Holmes a, Michael Kranz f, Mika K. Lindvaal f, Fiona S. Lucas d, Margarete Neu f, Lisa E. Ranshaw e,
Yemisi E. Solanke d, Don O. Somers f, Peter Ward b, Joanne O. Wiseman d
*
a Immuno-Inflammation CEDD Medicinal Chemistry Dept, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
b Respiratory CEDD Medicinal Chemistry Dept, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
c Molecular Discovery Research Medicinal Chemistry Dept, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
d Respiratory Biology Dept, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
e DMPK Dept, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
f Computational and Structural Chemistry Dept, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
g Biological Reagents and Assay Development Dept, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Crystallography driven optimisation of a lead derived from similarity searching of the GSK compound col-
lection resulted in the discovery of quinoline-3-carboxamides as highly potent and selective inhibitors of
phosphodiesterase 4B. This series has been optimized to GSK256066, a potent PDE4B inhibitor which also
Received 20 February 2009
Revised 2 April 2009
Accepted 2 April 2009
Available online 9 April 2009
inhibits LPS induced production of TNF-a from isolated human peripheral blood mononuclear cells with a
pIC50 of 11.1. GSK256066 also has a suitable profile for inhaled dosing.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
PDE4 inhibitors
SAR
Crystallography
Quinoline
Inhibition of phosphodiesterase 4 (PDE4) has been shown clin-
ically to have an anti-inflammatory effect,1 and is well established
as a target with potential to treat conditions such as asthma and
COPD.2 First generation PDE4 inhibitors (e.g., R-Rolipram, 1) cause
emesis at effective anti-inflammatory doses.3 Second generation
inhibitors such as Roflumilast 2 show efficacy against COPD, but
this is limited by nausea and emesis at high doses.4
One hypothesis that we wished to test was that delivery of the
PDE4 inhibitor directly to the site of action in the lung might im-
prove the therapeutic index, in a similar manner to inhaled gluco-
corticoids.5 A lower dose would be required than in an oral drug,
and the compound need not be systemically bioavailable. This
would result in a dramatically lower systemic exposure, and hence
a reduced level of emesis.
Our goal was to design a third generation of PDE4 inhibitors
with an increased therapeutic index, enabling a robust anti-inflam-
matory action without being dose limited by emetic side effects.
We have recently described the discovery of 3-aminocarboxy-
4-anilino quinolines (e.g., 3).6 In this Letter we describe the
optimization of 3 into GSK256066 (4), a highly potent PDE4
inhibitor suitable for inhaled administration. In parallel with this
approach the series was also optimised for oral administration.7
F
F
MeO
O
O
O
Cl
H
N
N
O
N
Cl
HN
N
OMe
CONH2
HN
N
OMe
CONH2
O
1
2
O
Me
O
O
O
S
S
CONMe2
Me
* Corresponding author. Tel.: +44 1438 762515.
E-mail address: michael.d.woodrow@gsk.com (M.D. Woodrow).
3
4
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.012

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M. D. Woodrow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5261–5265
O
Quinoline 3 is a relatively potent and selective PDE4 inhibitor
R6
CO2Et
R6
(PDE4B pIC50 8.4, for other data see Table 5). Our strategy was to
seek to increase the PDE4 potency, whilst keeping the aqueous sol-
ubility and oral bioavailability low. One method of doing this was
to target a relatively high molecular weight lipophilic compound.
Since PDE4 is an intracellular target we aimed to keep permeability
high, and to judge this by cellular activity.
A crystal structure of our lead 3 bound to PDE4B was deter-
mined to 1.7 Å resolution (Fig. 1).7 This showed the core of our
molecule to be a good fit with the binding site, but revealed the
presence of a large solvent filled pocket (A) that could be accessed
from the 6-position of the quinoline, as well as a small pocket (B) at
the 8-position of the molecule that could be exploited to increase
binding interactions. We focused on these areas to increase the
PDE4 activity of this series.
c, d
a, b
N
H
6a-d
NH2
5a-d
OMe
CONH2
HN
Cl
N
R6
R6
CONH2
e
N
7a-d
8a-d
OMe
CONH2
OMe
CONH2
HN
HN
I
PhS
f
We initially looked to modify the 6-position of the quinoline
ring. The R6 substituent was introduced at the start of the synthesis
with the appropriate aniline 5, which was condensed with diethyl
ethoxymethylenemalonate, and then cyclised at 250 °C in diphenyl
ether to give ester 6 (Scheme 1). This was hydrolysed with sodium
hydroxide and the resulting acid dichlorinated with thionyl chlo-
ride, at both the acid moiety and the 4-position, then quenched
with aqueous ammonia to afford the primary carboxamide 7. The
functionality at R4 was introduced by refluxing 7 in acetonitrile
with 3-methoxyaniline to give the desired compounds 8a–d.9 As
can be seen in Table 1, increased steric bulk at this position was
tolerated, in particular aryl sulfones such as 8a. The observed tol-
erance of the phenyl sulfone at the quinoline 6-position of 8a
was rationalized by the phenyl group pointing towards the large
pocket (A) seen on the crystal structure (Fig. 1). Removal of the sul-
fone here was detrimental (8c).
N
8d
N
9
Scheme 1. Reagents and conditions: (a) EtOCH@C(CO2Et)2, 80 °C; (b) Ph2O, 250 °C;
(c) NaOH, EtOH, relux; (d) (i) SOCl2, DMF, 80 °C; (ii) NH4OH, rt; (e) R4NH2, MeCN,
80 °C; (f) PhSH, Pd2(dba)3, DPEphos, KOtBu, toluene, 100–120 °C or PhSH, CuI,
K3PO4, DMPU, N,N-diethylsalicylamide, 100 °C.
Table 1
Initial SAR of 6-position
HN
N
OMe
CONH2
R6
The linear nature of the initial route limited the diversity that
could be introduced at the 6-position. Modification allowed sul-
fone variation to be introduced at a late stage via the iodoquinoline
8d (Scheme 1). Thus 8d (prepared from 5d) was reacted with aryl
thiols under palladium catalysed coupling conditions10 to give the
sulfide 9.
12
Compds
R6–
PDE4B pIC50
R-Rolipram, 1
Roflumilast, 2
3
—
—
7.4 (9)
9.4 (308)
8.4 (7)
8.8 (2)
8.5 (3)
6.7 (3)
6.8 (2)
7.5 (3)
7.6 (3)
9.1 (2)
8.7 (2)
MeSO2–
PhSO2–
cPentylSO2–
H–
PhS–
Me2NC(O)–
MorpholineC(O)–
Me2NSO2–
Morpholine-SO2–
8a
8b
8c
9
10
11
14a
14b
In addition, chemistry was devised to allow the sulfone linker to
be varied to amide or sulfonamide (Scheme 2). Carbonylation of
iodoquinoline 8d gave the corresponding ethyl ester. Basic hydro-
lysis then coupling with an amine under standard conditions gave
amides 10–11. Sulfonamide linked compounds were obtained
starting from iodoquinoline 7d, which was coupled under Stille
t
conditions with BuSSnBu3 to give the tert-butyl sulfide, and oxi-
OMe
CONH2
O
HN
N
OMe
CONH2
HN
N
I
RR'N
a, b, c
Cl
10-11
8d
Cl
Cl
N
I
CONH2
H2NOC
CONH2
CONH2
S S
N
N
d, e, f
Cl
12
7d
R4
HN
O
O
S
ClO2S
CONH2
g,
RR'N
N
N
f
14a-b
13
Scheme 2. Reagents and conditions: (a) CO, Pd(PPh3)2Cl2, Et3N, EtOH, 80 °C; (b)
NaOH, EtOH, rt; (c) TBTU, RR0NH, DMF, rt; (d) tBuSSnBu3, Pd(PPh3)4, toluene, reflux;
(e) MeSiCl3, Ph2SO, TFA, 0–5 °C; (f) Cl2, AcOH, H2O, rt; (g) RR0NH, DIPEA, CH2Cl2, rt;
(h) 3-methoxyaniline, MeCN, 80 °C.
Figure 1. Crystal structure of PDE4B + 3, indicating 2 pockets, A + B, for additional
binding interactions.

M. D. Woodrow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5261–5265
5263
dized with methyltrichlorosilane to the symmetrical disulfide 12.
This was cleaved with chlorine gas to the sulfonyl chloride 13, then
treated with an amine under standard conditions to form a sulfon-
amide and finally refluxed with 3-methoxyaniline to afford 14a–b.
To thoroughly explore the SAR, the substituent was varied from
a sulfone to sulfide (9) and amide (10–11), but none showed
improvements to PDE4B activity (Table 1). Our hypothesis for lack
of affinity of sulfide 9 is that the sulfone (8a) is pre-oriented in the
bio-active conformation11a whereas the sulfide loses entropy in
attaining this conformation. In addition, both aromatic rings con-
OMe
CONH2
HN
OMe
CONH2
HN
ArSO2
I
a, b
N
N
16a-d
8d
OMe
CONH2
OMe
CONH2
HN
N
HN
N
O
S
O
O
O
S
S
c, d
nected to the sulfur in 8a and 9 are involved in face-to-face
p-
stacking interactions which greatly benefit11b from electron-poor
aromatic systems. Sulfonamides (14a–b) maintained a similar level
of PDE4B inhibition to sulfone linked compounds.
17
CONMe2
CO2Me
O
16a
With stocks of phenyl sulfone intermediate 7a (R6 = PhSO2) in
hand a second round of optimization was carried out at the quin-
oline 4-position, using the same chemistry described in Scheme
1, but varying the amine R4NH2 (addition of diisopropylethylamine
as base was required with an aliphatic amine in the final step) to
give 15a–g. Whilst several groups were of similar potency versus
PDE4B (Table 2), none proved superior to the initial 3-methoxy-
phenyl group (8a). Substitution at this position with an aliphatic
group gave a drop in activity (15d). The SAR can be rationalized
from the crystal structure in Figure 1 as the 3-methoxyphenyl is
a very good fit to the protein surface and we were unable to im-
prove activity at this position.
HN
OMe
O
OMe
CONH2
HN
N
CONH2
O
O
S
e
Br
N
Ar
16b
18 (Ar = 3-furyl)
Scheme 3. Reagents and conditions: (a) ArSH, Pd2(dba)3, DPEphos, KOtBu, toluene,
100–120 °C or ArSH, CuI, K3PO4, DMPU, N,N-diethylsalicylamide, 100 °C; (b) oxone,
DMF, rt; (c) NaOH, MeOH, 80 °C; (d) Me2NH, HATU, DIPEA, DMF, rt; (e) 3-
furylboronic acid, Pd(Ph3)4, Na2CO3, DME, 100 °C.
Table 3
We rationalized earlier that the phenylsulfone group pointed
towards the large pocket (A) seen on the crystal structure
(Fig. 1), and we used this to assist in our design of further mole-
cules. It appeared that there was space here for either meta or para
substitution. This functionality was introduced from iodide 8d via
the corresponding sulfide (prepared under either palladium10 or
copper13 catalysed coupling conditions). The sulfide could be
cleanly oxidised with OxoneTM to the appropriate sulfone 16a–d.
Amide substituents were introduced by hydrolysis of the ester
functionality under basic conditions and then standard amide cou-
pling with the appropriate amine to give 17 (Scheme 3).
As is shown in Table 3, para substituents gave up to a 10-fold
increase in PDE4B activity, but meta substituted amide 17 was
preferable, possibly due to improved Van der Waals contact of
the dimethylcarboxamide with Ser-282, and a desolvation effect
by displacing trapped water molecules. A secondary assay measur-
Further SAR of 6-position
HN
N
OMe
CONH2
O
O
S
R
12
14
Compds
Phenyl substituent
PDE4B pIC50
PBMC pIC50
8a
H
8.8 (2)
9.9 (3)
9.2 (2)
11.0 (2)
8.8 (2)
8.2 (8)
8.9 (6)
7.8 (4)
10.0 (8)
7.8 (4)
16c
16d
17
4-OMe
4-tBu
3-CONMe2
4-(3-Furyl)
18
pounds which may be due to their relatively high lipophilicity
(e.g., 16c clog P = 4.8). Further elaboration to explore the SAR even
more widely at this position was exemplified by Suzuki coupling at
the para position of the 6-phenylsulfone (seeking to maximize the
space-filling of the large pocket A shown in Fig. 1) to give com-
pounds such as 18 (Scheme 3) but these showed no potency in-
crease over 8a.
Having made these changes, we looked to increase the potency
of these compounds by making further modifications. We decided
to exploit the small pocket B that we had seen in the crystal struc-
ture (Fig. 1), and found that we could introduce a small methyl
group at the 8-position of the ring, starting from 2-methyl-4-
(phenylsulfonyl)aniline to give 19 by the same route shown in
Scheme 1. As can be seen in Table 4, this led to a dramatic leap
in potency.
ing inhibition of LPS induced TNF-
a production from isolated hu-
man peripheral blood mononuclear cells14 (PBMC) was used to
determine the activity of these compounds in a relevant cellular
system. It was most encouraging that the majority of these com-
pounds maintained good activity in this assay, with no more than
1 log dropoff from the PDE4B enzyme data except for 16d (Table 3).
This shows the good cellular penetration of this series of com-
Table 2
SAR of 4-position
R4
HN
PhSO2
CONH2
Finally, the para-OMe and meta-CONMe2 substituents on the 6-
aryl ring that were preferred in the 8-H series were combined with
the 8-methyl substituent, to give 20 and 4 by the routes described
earlier starting from 4-iodo-2-methylaniline. The PDE4B potencies
of the 6- and 8- substitution were found to be additive and activity
was maintained in the cellular assay. This afforded GSK256066
(Compound 4), which is among the most potent PDE4 inhibitors
described in the literature.
N
12
Compds
R4
PDE4B pIC50
8a
3-OMe phenyl
3-Et phenyl
3-CN phenyl
Phenyl
Cyclohexyl
3-Pyridyl
8.8 (2)
8.0 (2)
8.5 (3)
8.2 (2)
7.4 (2)
7.4 (4)
8.6 (2)
7.9 (2)
15a
15b
15c
15d
15e
15f
15g
6-Benzothiazolyl
GSK256066 (4) is an exceedingly potent PDE4 inhibitor, with
picomolar activity in both enzyme and cellular assays and activity
4-(3-Me) tetrahydrobenzofuryl

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M. D. Woodrow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5261–5265
Table 4
Table 6
SAR of 8-Me compounds
Lung and plasma levels of GSK256066 after intratracheal administration in rats as an
aqueous suspension at a dose of 30 g/kg
l
Time (h)
Lung concentration
(ng/g)
Plasma concentration
(ng/g)
Lung:plasma ratio
HN
N
OMe
CONH2
O
O
S
0.08
6
2831
2649
1.5
0.6
1887
4415
R
R'
12
14
Compds
R
R0
PDE4B pIC50
PBMC pIC50
the quinoline 6- and 8-substituents, there are significant Van Der
Waals contacts, particularly by Ser-282 with the meta-CONMe2
group and by Met-431 with the 8-methyl group.
8a
19
20
4
H
H
H
8.8 (2)
8.2 (8)
9.3 (6)
10.5 (6)
11.0 (18)
Me
Me
Me
10.0 (2)
10.7 (2)
11.1 (6)
4-OMe
3-CONMe2
GSK256066 was optimized for inhaled administration in order
to increase therapeutic index, so a key pharmacokinetic measure
of success was having high lung and low plasma levels after in-
haled administration. Even 6 h after dosing to rats as an intratra-
cheal aqueous suspension there was a significant concentration
of GSK256066 in the lung, and exceedingly low plasma levels (Ta-
ble 6). Whilst this experiment does not describe the precise loca-
tion of the material in the lung it is clear that there are
substantial quantities present at the 6 h timepoint indicating the
potential for a long duration of action.
In conclusion a novel and exceedingly potent series of PDE4
inhibitors has been identified. This has been optimized to afford
GSK256066, a compound with picomolar activity in vitro and prop-
erties suitable for inhaled dosing. More detailed investigations of
the in vitro and in vivo properties of GSK256066, including its
duration of action and therapeutic index, have been carried out15
and a clinical evaluation of this compound in asthma and COPD
is underway.
at 0.1nM concentration in human whole blood.14 It is >1000-fold
selective versus PDEs 1–3 and 5–7.12 The properties of GSK256066
(Table 5) are highly appropriate for administration via the inhaled
route, with negligible oral bioavailability in rat.
The crystal structure8 of GSK256066 bound to PDE4B was ob-
tained to 1.75 Å resolution (Fig. 2). This shows an excellent fit with
the surface of the enzyme, occupying both pockets that were iden-
tified in the crystal structure of compound 3 bound to PDE4B.
Whilst there are no direct hydrogen bonding interactions from
Table 5
In vitro, physicochemical and pharmacokinetic properties of 4 compared with initial
lead molecule 3
Property
Value
4
3
Acknowledgements
12
PDE4B pIC50
11.1
>1000-fold
11.0
8.4
>1000-fold
—
7.4
Selectivity versus PDE3,5,612
The authors would like to thank Aurore Servais, Katherine Trot-
ter and Christopher Foley for synthesis of molecules whilst on
placement at GSK.
14
PBMC TNF-
a
pIC50
14
Human whole blood TNF-
a
pIC50
9.9
Aqueous solubility at pH 6.4 (mg/mL)
<0.001
0.005
Rat pharmacokinetics:
1 mg/kg
Cl (mL/min/kg)
Vd (L/kg)
po F%
39
10
0.3
10
References and notes
0.8
0%
1.1
1. (a) Houslay, M. D.; Schafer, P.; Zhang, K. Y. J. Drug Discovery Today 2005, 10,
1503; (b) Soto, F. J.; Hanania, N. A. Curr. Opin. Pulm. Med. 2005, 11, 129.
2. For recent reviews see: McKenna, J. M.; Muller, G. W. Cyclic Nucleotide
Phosphodiesterases Health Disease 2007, 667; Dyke, H. J. Exp. Opin. Ther. Patents
2007, 17, 1183; Smith, V. B.; Spina, D. Curr. Opin. Invest. Drugs 2005, 6, 1136.
3. Horowski, R.; Sastre-Y-Hernandez, M. Curr. Ther. Res. 1985, 38, 23.
4. Spina, D. Drugs 2003, 63, 2575.
iv t1/2 (h)
1.1
5. (a) Hogger, P. Curr. Opin. Pulm. Med. 2003, 9, 1; (b) Mollmann, H.; Wagner, M.;
Meibohm, B.; Hochhaus, G.; Barth, J.; Stockmann, R.; Krieg, M.; Weisser, H.;
Falcoz, C.; Derendorf, H. Eur. J. Clin. Pharmacol. 1998, 53, 459.
6. Kranz, M.; Lindvall, M. K.; Hamblin, J. N.; Jones, P. S.; Neu, M. Y.; Somers, D. O. in
preparation.
7. Lunniss, C. J.; Cooper, A. W. J.; Eldred, C. D.; Kranz, M.; Lindvall, M.; Lucas, F. S.;
Neu, A.; Preston, A. G. S.; Ranshaw, L. E.; Redgrave, A. J.; Robinson, J. E.; Shipley,
T. J.; Solanke, Y. E.; Somers, D. O.; Wiseman, J. O. Bioorg. Med. Chem. Lett. 2009,
19, 1380.
8. Methods as described in Ref. 15 of Hamblin, J. N.; Angell, T. D. R.; Ballantine, S.
P.; Cook, C. M.; Cooper, A. W. J.; Dawson, J.; Delves, C. J.; Jones, P. S.; Lindvall,
M.; Lucas, F. S.; Mitchell, C. J.; Neu, M. Y.; Ranshaw, L. E.; Solanke, Y. E.; Somers,
D. O.; Wiseman J. O. Bioorg. Med. Chem. Let., 2008, 14, 4237, except compound
4 was soaked at 1 mM for ꢀ1 day. Compound 4 was refined with partial
occupancy due to incomplete substitution of an Arsenate species that normally
occupies the inhibitor binding site and is derived from the crystallisation
conditions (Xu, R. X.; Hassell, A. M.; Vanderwall, D.; Science 2000, 288, 1822).
The final R-factor achieved for complex 4 following structure refinement was
20.5% (Rfree = 25.0%) and the coordinates deposited in the PDB as entry 3GWT.
Note that red crosses in Figures 1 and 2 show the position of water molecules.
9. All compounds were characterized by LC/MS and 1H NMR analysis.
10. Schopfer, U.; Schlapbach, A. Tetrahedron 2001, 57, 3069.
11. (a) Analysis of CCDC X-ray structures for bis-aryl sulfones show a strong
preference for a 90° torsional angle whereas the sulfides show no torsional
preference.; (b) Mignon, P.; Loverix, S.; Geerlings, P. Chem. Phys. Lett. 2005, 401,
40–46.
12. The ability of compounds to inhibit catalytic activity at PDE4B (human
recombinant), PDE3 (from bovine aorta) and PDE5 (human recombinant) was
Figure 2. Crystal structure of PDE4B + 4.

M. D. Woodrow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5261–5265
determined by Scintillation Proximity Assay (SPA) in 96-well format as 14. Inhibition of LPS induced TNF-
5265
a
production from isolated human peripheral
described in WO2004024728. Values are means of at least two experiments
(n shown in parentheses).
13. Kwong, F. Y.; Buchwald, S. L. Org. Lett. 2003, 5, 793. Copper catalysed conditions
were preferred for products with ester functionality, palladium was used in all
other cases.
blood mononuclear cells (PBMC) or human whole blood using IGEN technology
was carried out as described in patent WO2007036733. Values are means of at
least four experiments (n shown in parentheses).
15. (a) Nials, A.T. in preparation.; (b) Tralau-Stewart, C.J. in preparation.