Addressing species specific metabolism and solubility issues in a quinoline series of oral PDE4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.11.010

Species specific conversion of the lead PDE4 inhibitor 1 to the quinolone 3 was identified as the major route of metabolism in the cynomolgus monkey. Modification of the template to give the cinnoline 9 retained potency and selectivity, and greatly improved the pharmacokinetic profile in the cynomolgus monkey compared with 1. Additional SAR studies aimed at improving the solubility of 9 are also described.

Full text of DOI:10.1016/j.bmcl.2009.11.010

Bioorganic & Medicinal Chemistry Letters 20 (2010) 137–140
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Addressing species specific metabolism and solubility issues in a quinoline
series of oral PDE4 inhibitors
C. Lunniss , C. Eldred à, N. Aston, A. Craven, K. Gohil, B. Judkins, S. Keeling, L. Ranshaw,
,
*
E. Robinson, T. Shipley, N. Trivedi
GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, England, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Species specific conversion of the lead PDE4 inhibitor 1 to the quinolone 3 was identified as the major
route of metabolism in the cynomolgus monkey. Modification of the template to give the cinnoline 9
retained potency and selectivity, and greatly improved the pharmacokinetic profile in the cynomolgus
monkey compared with 1. Additional SAR studies aimed at improving the solubility of 9 are also
described.
Received 15 October 2009
Revised 4 November 2009
Accepted 5 November 2009
Available online 10 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
PDE4
COPD
Quinoline
Cinnoline
Cynomolgus monkey
PK
Phosphodiesterase 4 (PDE4) is the predominant isozyme in
nearly all immune and inflammatory cells and is a major regulator
of cAMP content in airway smooth muscle. Inhibition of PDE4
down regulates cAMP production, mediating bronchodilation and
cynomolgus monkey, with a much shorter half life (0.6 h) in the
monkey cf. rat (2.3 h) or dog (2.4 h). Drug metabolism studies were
therefore undertaken in rat, dog, monkey and human hepatocytes
to identify major metabolites and understand which species might
best predict human PK (Fig. 1).
reducing production of inflammatory mediators such as TNF-a. Po-
tential indications for a PDE4 inhibitor as a once daily oral anti-
inflammatory agent include chronic obstructive pulmonary disease
(COPD), asthma, rhinitis and rheumatoid arthritis.1,2
CN
We have previously described identification of the potent quin-
oline PDE4 inhibitor 1 (PDE4B pIC50 = 9.4; Roflumilast PDE4B
pIC50 = 9.4).3 Although this compound has excellent PDE4 potency
and selectivity, high in vivo efficacy, and a favourable pharmacoki-
netic profile in rat and dog,3 we have identified a species specific
metabolic issue with this compound in the cynomolgus monkey.
We here disclose details of the metabolic studies with 1, the phar-
macology of the major metabolite, and efforts to circumvent this
metabolic issue culminating in the identification of the candidate
molecule 9.
NH O
MeO2S
NH2
N
1
CN
CN
Pharmacokinetic (PK) studies with the oral PDE4 inhibitor 1
identified species differences in PK profile between rat, dog and
NH O
NH O
MeO2S
MeO2S
NH2
NH2
N+
O
* Corresponding author.
N
O
E-mail addresses: c.lunniss@biota.com.au, chris@lunniss.net (C. Lunniss).
Present address: Biota Holdings Ltd, 585 Blackburn Road, Melbourne, VIC 3185,
Australia.
H
2
3
à
Present address: Colin Eldred Consulting Ltd., Woodridge House, Earlsbrook,
Bacton, Stowmarket, Suffolk, IP14 4UA, UK.
Figure 1. Quinoline 1 and possible metabolites in monkey hepatocytes.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.010

138
C. Lunniss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 137–140
Consistent with the difference in half life, 1 displayed a >20-fold
CO2H
NH2
I
CO2H
(a), (b)
higher rate of metabolism in monkey hepatocytes compared to rat
hepatocytes. The major [M+16] metabolite in the monkey was as-
signed initially as either the N-oxide 2 or the 2-quinolone 3, and is
not seen to any significant extent in rat, dog or human hepatocytes.
The formation of such a potent active metabolite in the monkey
posed potential development issues for 1 since this was the pro-
posed non-rodent species for toxicological evaluation.4
The identity of the 2-quinolone 3 as the major metabolite in the
monkey was confirmed by synthesis as shown in Scheme 1 (N-
oxide 2 could not be prepared, despite trying multiple oxidation
conditions. The main product isolated was the 2-quinolone possi-
bly owing to spontaneous rearrangement of the N-oxide). Prepara-
tion of intermediate 4 under standard conditions followed by base
catalysed cyclisation gave the hydroxyquinolone intermediate 5.
Acidic hydrolysis of the t-butyl ester followed by treatment with
phosphoryl chloride gave the corresponding 2,4-dichloroquinolinyl
acid chloride; treatment with ammonia gave intermediate 6 with-
out displacement of the aryl chlorine groups. The 4-chloro group
was selectively displaced with the sodium salt of 3-aminobenzoni-
trile to give intermediate 7. Selective introduction of the methyl
sulphone at the quinoline 6-position was achieved by palladium
NH
CF3
O
(c),(d)
O
O
CO2Et
I
I
(e)-(g)
N
N
NH2
N
11
10
(h)
O
Cl
N
(i)-(k)
I
CO2Et
I
CONH2
N
N
N
H
12
(a) - (c)
CO2H
NH2
CO2Et
I
H
N
O
(l),(m)
(n)
O
O
4
(d)
Cl
N
N
H
CN
CONH2
S
CONH2
MeO2S
R6
Cl
N
OH
CONH2
Cl
CO2tBu
O
I
I
N
N
(e) - (g)
N
N
H
9
6
5
(o),(p)
R6O2S
(h)
NHR4
CONH2
N
N
CN
CONH2
CN
CONH2
HN
N
HN
(i),(j)
Scheme 2. Synthesis of the cinnoline template and compound 9. Reagents and
conditions: (a) ICl, aq HCl, 80%; (b) (CF3CO)2O, 1,4-dioxane, 70%; (c) MeMgCl
(5 equiv), THF, 0 °C, 70%; (d) NaOH, MeOH, 90%; (e) NaNO2, concd HCl, THF, H2O; (f)
pyrrolidine, aq K2CO3, 80%; (g) NaH, CO(OEt)2, 80%; (h) CF3COOH, 75%; (i) NaOH,
MeOH, 90%; (j) POCl3; (k) NH4OH, dioxane, 80%; (l) 3-aminobenzonitrile, MeCN,
reflux, 70%; (m) MeSO2Na, CuI, diaminocyclohexane, DMSO, 60%; (n) R6SSnBu3,
Pd(PPh3)4, toluene, DMF, 100 °C, typically 70–90%; (o) R4NH2, MeCN, reflux
(pyridine hydrochloride was added for some aromatic heterocyclic amines),
typically 60–80%; (p) oxone, DMF, rt, typically 60–70%.
I
MeO2S
Cl
N
Cl
7
8
(k)
CN
CONH2
HN
catalysed coupling with the non-nucleophilic (methylthio)tribu-
tylstannane reagent,5 followed by oxidation. Finally, microwave
conditions were found most successful for the hydrolysis of the
2-chloroquinoline 8 to give quinolone 3.
MeO2S
N
O
H
Since X-ray crystallography studies suggested that hydrogen
bonding to the quinoline nitrogen of 1 was a key binding interac-
tion, the potent inhibitory activity of the quinolone 3 (PDE4B
pIC50 = 9.0) was interpreted as binding of 3 to the enzyme as its
2-hydroxyquinoline tautomer.8 As the potent metabolite of quino-
line 1, quinolone 3 was further characterised to investigate its po-
tential for development, and showed good rat PK following
3
Scheme 1. Synthesis of quinolone metabolite 3. Reagents and conditions: (a) ICl,
HCl, rt, 80%; (b) EtI, Cs2CO3, CH3CN, reflux, 70%; (c) HO2CCH2CO2C(CH3)3, (COCl)2,
CHCl3, DMF (cat), rt, 75%; (d) NaOMe, EtOH, rt, 60%; (e) TFA, rt, 70%; (f) POCl3,
100 °C; (g) NH4OH/dioxane, 5–10 °C, 60%; (h) 3-aminobenzonitrile, NaH, DMF,
60 °C, 80%; (i) MeSSnBu3, Pd(PPh3)4, toluene, DMF, 100 °C, 70%; (j) oxone, DMF, rt,
70%; (k) AcOH, H2O, 180 °C, microwave, 50%.

C. Lunniss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 137–140
139
Table 1
poration of a second nitrogen to replace the quinoline with a
cinnoline template would prevent 2-quinolone formation, hence
the cinnoline analogue 9 was prepared as shown in Scheme 2.
The key step of the synthesis was a novel acid catalysed cyclisation
of the pyrrolidinyldiazene intermediate 11,6 prepared in three
steps from the aniline 10 via trapping of the corresponding diazo-
nium salt with pyrrolidine. Ester hydrolysis and chlorination to
give the 4-chlorocinnoline 3-acid chloride, followed by ammonia
treatment gave the versatile intermediate 12. The 6-sulphone
group was introduced in one step using a copper catalysed cou-
pling with the sodium salt of methanesulphonic acid.7 Alterna-
tively, in order to facilitate variation of the cinnoline 4-position
in the last step, the 4-chloro-6-iodo intermediate 12 could be
selectively converted to the 4-chloro-6-alkylthio intermediates
using the thioalkyl(tributyl)stannane coupling methodology de-
scribed in Scheme 1, followed by introduction of the 4-arylamino
group and oxidation of sulphide to sulphone as the final step
(Table 1).
Compound 9 retained excellent in vitro potency and >100-fold
selectivity versus other PDE isozymes (PDE1, 2, 3, 5, 6, 7) and
showed a much improved pharmacokinetic profile in the monkey
compared with 1 (see Table 2). Despite its low solubility, good oral
exposure of 9 could be achieved in both rat and monkey via parti-
cle size reduction (wet bead milling). Consequently, cinnoline 9
was selected for further development; its in vivo efficacy will be
reported separately.
hWB9 potency and cynomolgus monkey PK of quinolone metabolite 3 cf. quinoline
lead 1
Fb (%)
Clpb (ml/min/kg)
t1/2 (h)
a
b
Compound
hWB pIC50
1
3
7.9
7.6
33
52 (7)
82
5.9
0.8
4.2
a
Values are means of at least four experiments.
Values are means of three experiments following solution dosing (or suspen-
b
sion dosing using particle size reduced material in parentheses).
Table 2
PDE4B10 and hWB9 potency and cynomolgus monkey pharmacokineticsc for cinnoline
9
a
b
PDE4B pIC50
>9.0
hWB pIC50
8.6
F%
Clp (ml/min/kg)
1.6
t1/2 (h)
64 (26)
7.2
a
b
c
Mean of two determinations.
Mean of >4 determinations.
Values are means of three experiments following solution dosing (or suspen-
sion dosing using particle size reduced material in parentheses).
solution dosing. More encouragingly, the monkey PK (again follow-
ing solution dosing in 10% DMSO, 15% PEG400, 75% water) was
much improved over 1, with good bioavailability and a much long-
er half life. However poor oral exposure following suspension dos-
ing in 1% methyl cellulose (possibly due to very low aqueous
solubility of 1 lg/ml) and poor in vivo efficacy precluded further
work with this compound.
Attention was next turned to modification of the quinoline tem-
plate to overcome this metabolic issue. It was reasoned that incor-
In order to improve upon the poor solubility of 9, SAR studies
were undertaken with the aim of introducing solubilising modifi-
cations onto the cinnoline template (see Table 3). Solubility
enhancements of 10-fold could be achieved without loss of po-
tency by replacing phenylamino groups with heteroarylamino
groups at this position. In particular, 3-pyridylamino groups
Table 3
SAR studies in the cinnoline series
R4
HN
R6SO2
CONH2
N
N
R8
R4
R6
R8
PDE4B pIC50
hWB pIC50
8.6
Solubility (
1
lg/ml)
Rat F%a
17
a
a
Compound
9
Me
Me
>9.0
NC
*
N
13
Me
Me
10.1
8.8
12
50
Cl
*
N
14
15
16
Me
Et
Me
Me
H
>9.3
8.3
8.0
7.5
37
ntb
7
34
41
66
F
*
N
N
9.5 (n = 1)
9.1
*
N
Me
Cl
F
*
N
17
18
tBu
Me
H
F
9.3
7.9
6.8
1
57
nt
*
*
N
9.2 (n = 1)
ntb
F
a
Values are means of at least two experiments except where indicated in parentheses.
nt = not tested.
b

140
C. Lunniss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 137–140
(e.g., compounds 13 and 16) and the 1-ethyl-5-pyrazolylamino
(e.g., compound 15) group gave useful increases in solubility while
maintaining good in vitro potency and rat pharmacokinetic
profiles.
Acknowledgements
The authors would like to thank Don Somers and Maragete Neu
for crystallography, Michael Kranz for molecular modeling, Jo
Wiseman for the PDE4B enzyme data, and Fiona Lucas and Yemisi
Solanke for the hWB data.
In addition to modifications at the 4-position, limited variations
at the cinnoline 6- and 8-positions were also studied. In line with
observations in the related quinoline series,3 larger alkyl groups
were tolerated in the sulphone moiety (compounds 15 and 17),
however this was at the expense of solubility in the case of the
tert-butyl sulphone 17. Removal of the 8-methyl group altogether
results in ca. 10-fold reduction in potency (cf. compounds 13 and
16), and 8-fluoro analogues (e.g., 18) achieve similar potency to
their 8-H counterparts. From these SAR studies, compound 13
stood out as being equipotent with the cinnoline lead 9 in vitro
and in vivo (data not shown), with an improved pharmacokinetic
profile in the rat cf. 9, and improved solubility.
References and notes
1. (a) Houslay, M. D.; Schafer, P.; Zhang, K. Y. J. Drug Discovery Today 2005, 10,
1503; (b) Francisco, J. S.; Hanania, N. A. Curr. Opin. Pulm. Med. 2005, 11, 129.
2. 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. Lett. 2008, 14, 4237.
3. Lunniss, C. J.; Cooper, A. W. J.; Eldred, C. D.; Kranz, M.; Lindvall, M.; Lucas, F. S.;
Neu, M.; 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.
4. Fura, A. Drug Discovery Today 2006, 11, 133.
5. Dickens, M. J.; Gilday, J. P.; Mowlem, T. J.; Widdowson, D. A. Tetrahedron 1991,
47, 8621.
6. Dowlatshahi, H. A. Synth. Commun. 1985, 1512, 1095.
7. Baskin, J. M.; Wang, Z. Org. Lett. 2002, 4, 4423; Zhu, W.; Ma, D. J. Org. Chem.
2005, 70, 2696.
In conclusion, detailed metabolic studies with the lead quino-
line
1 identified the cynomolgus monkey specific quinolone
metabolite 3, which explained the poor pharmacokinetic profile
in the monkey and allowed development of a medicinal chemistry
strategy to overcome this issue. Synthesis of the cinnoline template
led to the discovery of 9 with much improved pharmacokinetics in
the monkey, along with a number of more soluble analogues, such
as 13, with promising in vitro and rat pharmacokinetic profiles.
More detailed pharmacology of these compounds will be reported
elsewhere.
8. Frank, J.; Katritzky, A. J. Chem. Soc., Perkin Trans. 2 1976, 1428; Warshel, A.;
Papazyan, A. Curr. Opin. Struct. Biol. 1998, 8, 211.
9. Inhibition of LPS induced TNF-
a production in human whole blood was carried
out using MSD technology as described in WO2007036733.
10. The ability of compounds to inhibit catalytic activity at PDE4B was determined
by Scintillation Proximity Assay (SPA) as described in patent WO2004103998.