Design, synthesis, and evaluation of tetrasubstituted pyridines as potent 5-HT2C receptor agonists

Article abstract of DOI:10.1021/ml500507v

A series of pyrido[3,4-d]azepines that are potent and selective 5-HT_2C receptor agonists is disclosed. Compound 7 (PF-04781340) is identified as a suitable lead owing to good 5-HT_2C potency, selectivity over 5-HT_2B agonism, and in vitro ADME properties commensurate with an orally available and CNS penetrant profile. The synthesis of a novel bicyclic tetrasubstituted pyridine core template is outlined, including rationale to account for the unexpected formation of aminopyridine 13 resulting from an ammonia cascade cyclization.

Full text of DOI:10.1021/ml500507v

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Letter
The design, synthesis and evaluation of tetra-
substituted pyridines as potent 5-HT2C receptor agonists
guy rouquet, Dianna E Moore, Malcolm Spain, Daniel Martin Allwood, Claudio
Battilocchio, David C Blakemore, Paul Vincent Fish, Stephen Jenkinson,
Alan S. Jessiman, Steven V Ley, Gordon McMurray, and R Ian Storer
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 20 Jan 2015
Downloaded from http://pubs.acs.org on January 21, 2015
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ACS Medicinal Chemistry Letters
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The design, synthesis and evaluation of tetra-substituted pyridines
as potent 5-HT receptor agonists
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e
c
a
e
e
Guy Rouquet, Dianna E. Moore, Malcolm Spain, Daniel M. Allwood, Claudio Battilocchio,
a,#
a
d
a
e
David C. Blakemore, Paul V. Fish, Stephen Jenkinson, Alan S. Jessiman, Steven V. Ley,
b,#
a,#,∗
Gordon McMurray, and R. Ian Storer
a
b
Worldwide Medicinal Chemistry, Discovery Biology, Pfizer Global Research and Development, Sandwich Labora-
tories, Sandwich, Kent CT13 9NJ, UK.
c
Worldwide Medicinal Chemistry, Pfizer Global Research and Development, Groton Laboratories, Eastern Point
Road, Groton, CT 06340, USA.
d
Global Safety Pharmacology, Pfizer Global Research and Development, 10646 Science Center Drive, San Diego, CA
92121, USA.
e
The University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
KEYWORDS: Tetra-substituted pyridines, Pyrido[3,4-d]azepine, 5-HT2 receptor agonist, CNS penetration.
C
ABSTRACT: A series of pyrido[3,4-d]azepines that are potent and selective 5-HT2C receptor agonists is disclosed. Com-
pound 7 (PF-04781340) is identified as a suitable lead owing to good 5-HT2C potency, selectivity over 5-HT2 agonism and
B
in vitro ADME properties commensurate with an orally available and CNS penetrant profile. The synthesis of a novel bi-
cyclic tetra-substituted pyridine core template is outlined, including rationale to account for the unexpected formation of
aminopyridine 13 resulting from an ammonia cascade cyclisation.
Serotonin (5-hydroxytryptamine, 5-HT 1) acts as a neuro-
transmitter agonist of at least 14 different receptors classi-
fied into seven major families, 5-HT . The 5-HT class of
1
-7
2
GPCR receptors comprises three members 5-HT , 5-HT
2B
2A
and 5-HT . Agonism of 5-HT in the CNS has been rec-
2C
2C
ognised to have potential for the treatment of obesity,
urinary incontinence, psychiatric disorders and sexual
dysfunction. However, it has been established that selec-
Figure 1. Selected 5-HT2C agonists.
1
9-
Previously Pfizer disclosed several 5-HT agonist series,
tivity over agonism of structurally related receptors 5-
HT2A and 5-HT2B is required. Poorly selective agonists
have been linked to clinical adverse events in humans.
These include hallucinations and cardiovascular effects
2C
1
4
including a pyrimidine-fused azepine template that led
to the discovery of PF-03246799 (4) which offered good
1
4-15
levels of in vitro and in vivo potency.
However com-
2-3
pound 4, despite offering excellent selectivity over both 5-
due to 5-HT2A agonism and chronic cardiac valvulopa-
H
T
s
t
i
l
l
s
h
o
w
e
d
w
e
a
k
b
u
t
m
e
a
s
u
r
a
b
l
e
a
g
o
n
i
s
m
o
f
5
-
H
T
thy and pulmonary hypertension caused by 5-HT2 ago-
nism. Notably the anti-obesity treatment Fen-Phen was
withdrawn in 1997 for causing irreversible valvulopathy
which has been attributed to chronic 5-HT2B agonism.
2
A
2
B
B
4
at 10 ꢀM in both recombinant cell systems and native
1
4
human tissue.
It was later discovered that 4-
methylamino substitution 5 could offer an enhancement
to 5-HT2C agonist potency and simultaneously offer supe-
The resulting search for selective 5-HT2C agonists identi-
fied vabicaserin (2) (SCA-136) as a potential therapy for
schizophrenia and lorcaserin (3) (APD-356) which was
approved in 2012 as Belviq® for treatment of obesity (Fig-
13
rior selectivity over 5-HT . However, these structural
2B
changes rendered amino-substituted pyrimidine com-
pound substrate for multidrug resistance P-
5
a
5
glycoprotein (P-gp), identified by a large efflux ratio
ure 1). Numerous other preclinical 5-HT agonists have
2C
6-8
(ER=10) as measured using an in vitro transfected MDCK
also been reported.
1
6
cell line (Figure 2). A previous correlation analysis of all
1
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Page 2 of 6
compounds tested in this MDCK-MDR1 assay concluded larity within medicinal chemistry, present formidable
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that compounds with efflux ratios of <2.5 are unlikely to
be significantly effluxed from the CNS by P-gp whereas
compounds with ratios >3.0 are at significant risk of ex-
challenges. Preferred synthetic methods typically com-
prise the selective functionalization of a pre-existing pyri-
dine ring or de novo ring synthesis. However, in this in-
stance, the need for a fused bicyclic tetra-substituted pyr-
idine meant that most known methods were not compat-
ible owing to either not supporting fused ring construc-
1
6
hibiting appreciable CNS impairment. In line with this
result, preclinical in vivo efficacy studies of compound 5
showed prohibitive levels of CNS restriction limiting ther-
1
6
20
apeutic efficacy even at high plasma concentrations.
tion or providing the wrong substitution pattern. As a
result, it was necessary to develop suitable chemistry to
access amino-pyridine fused azepine template 7. A route
was proposed based on limited precedent for biaryl ring
synthesis via ammonia cyclisation of an alkyne 8 to give
0
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2
3
4
5
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7
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9
0
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2
3
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9
0
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9
0
1
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9
0
1
2
3
4
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7
8
9
0
21-22
isoquinolone 9 (Scheme 1).
Scheme 1
a
Reagents and conditions: (a) Tf O, NaOtBu, CH Cl , 23 °C,
2
2
2
0
.5 h, then Tf O, 23 °C, 2 h; (b) BnCCH, DIPEA, CuI,
2
Pd(PPh ) Cl , DMF, 23 °C, 2 h; (c) NH , MeOH, 80 °C, 15 h.
3
2
2
3
Carboxybenzyl protected azepine β-ketoester 10 was con-
verted to corresponding vinyl triflate 11 in 81% yield by
treatment with triflic anhydride under basic conditions
(Scheme 1). Sonogashira coupling with benzylacetylene
then provided the desired yne-ene-ester 12 in preparation
for the key cascade cyclisation to the corresponding pyri-
dinone. Treatment of 12 with excess ammonia in metha-
nol at 80 °C led to conversion of starting material to a
single product. Rather than being the anticipated pyridi-
none, the product was instead determined to be amino-
pyridine 13.
Figure 2. P-gp efflux and CNS exposure.
To retain the high 5-HT2C potency and selectivity of com-
pound 5 but with improved CNS penetration, compounds
were sought to provide reduced P-gp efflux. Literature
pharmacophore models for P-gp have highlighted the role
of aromatic hydrophobic interactions and intramolecular
hydrogen bond Acc-Acc distances of ~2.5 Å and ~4.6 Å as
1
7
P-gp recognition features. As illustrated in Figure 3,
compound 5 has Acc-Acc distances of 2.4 Å, 4.1 Å and 4.6
Å suggesting close similarity to this P-gp pharmacophore
1
8-19
pattern of hydrogen bonds.
This unexpected result was repeated to provide gram
This pointed to N-1 in compound 5 being potentially in-
strumental to P-gp recognition when combined with a 4-
amino substituent. Furthermore SAR from related tem-
plates suggested that the N-1 interaction would not be
required for 5-HT2C activity. To test this hypothesis, sev-
eral compounds were designed to reduce the propensity
for N-1 to interact with P-gp. This led to compounds such
as chiral methyl azepine compound 6 that retained good
quantities of aminopyridine 13 and a sample was crystal-
lized from CD OD, enabling an X-ray structure to be ob-
3
tained to further confirm structure assignment (CCDC
1
024393 and supporting information).
23
In order to discount a metal-mediated reaction, the
ammonia cyclisation was also carried out using yne-ene-
ester 12 that had been pre-treated overnight with various
metal scavenger resins (QPTU, QMTU, QSMP; 1g resin
per 0.25mmol of 12). However, these pre-treatments did
not alter yield or product distribution of the cyclisation.
5
-HT2C potency, selectivity and reduced P-gp efflux
1
3, 15
(
ER=2.7) that translated to improved in vivo efficacy.
It
was further proposed that removing N-1 altogether, to
give fused aminopyridine azepine 7, would offer good 5-
HT2C agonist potency without significant P-gp efflux lia-
bility.
To investigate the mechanism of the cyclisation cascade
and further establish the general applicability of this reac-
tion, several related alkyne systems were tested under the
same reaction conditions (Table 1).
The controlled syntheses of tri- and tetra-substituted pyr-
idines, despite their favourable characteristics and popu-
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ACS Medicinal Chemistry Letters
with no evidence for formation of the aminopyridine 16f.
Table 1. Ammonia-mediated cyclisations
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
However, if pre-formed primary amide 17f was exposed to
the reaction conditions the anticipated pyridinone prod-
uct did not form, resulting in a mixture favouring amino-
pyridine 16f. This suggests that an alternative mechanistic
pathway predominates for substrate 14f (Scheme 2).
Crude product
a
Isolated
yield (16)
Scheme 2
ratio
Cmpd
12
Reactant
(15)
(16)
O
OEt
O
NH3
R3
R2
R3
R2
R3
EtO
H
O
2
H N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
100
78%
Ph
Ph
H+
R2
Ph
H+
slow
2
H O
H
14ii
NH3
8
6%
18
trace
NH2
NH
H3N:
NH
NH2
14a
0
<
5%
H2O
Rec. SM
R3
R2
R3
R2
R3
R2
R3
N
H2N
O
O
O
Ph
H+
H+
Bn
R2 Bn
H+
trace
90%
1
6ii
19
fast
Bn
14b
14c
0
<
5%
Rec. SM
6-exo-trig
6-exo-dig
O
O
EtO
NH3
HN
15f, 66% yield
0
0
0
0
100
100
100
100
91%
40%
64%
71%
MeOH
80 °C, 48 h
Ph
Ph
1
4f
O
O
O
1
4d
EtO
EtO
H N
2
6-exo-trig
HN
Bn
NH3
fast
Bn
14f
O
15f
Bn
1
4e
7e
H2N
6
-endo-dig
slow
Bn
H+
17f
1
O
O
NH2
NH3
MeOH
a
1
H2N
HN
N
Product ratio determined by crude H NMR integration. Rec. SM dentoes
yield of recovered starting material
Ph
Ph
80 °C, 48 h
Ph
1
5f, 21% yield
16f, 39% yield
1
7f
n
Interestingly if R =Bn was replaced by R =Ph 14a or R = Bu
1
1
1
It is postulated that in this case the ammonia undergoes
nucleophilic conjugate addition to the alkyne, as opposed
to facilitating allene formation, followed by 6-exo-trig
ring closure to directly give pyridinone 15f. However, if
primary amide 17f is pre-formed this would necessitate 6-
endo-dig closure to give pyridinone 15f, for which orbital
overlap is suboptimal, rationalising the observed mixture
of pyridinone 15f and aminopyridine 16f products. Fur-
thermore, when pyridinone 15f was treated with ammonia
under the same reaction conditions no reaction occurred,
ruling out the formation of 16f via 15f.
1
4b then the reaction did not proceed, instead returning
mostly unreacted starting material. However, when the
cyclisation reactant contained a benzylic R , and aliphatic
1
R and R (12, 14c-e), then cyclisation proceeded to con-
sistently give the corresponding aminopyridines 13, 16c-e
in good yields. To rationalize these results it is proposed
that systems where R =Bn 14ii undergo rapid rearrange-
ment to allenes on treatment with ammonia, driven by
extended conjugative stabilization of the allene with the
Bn aromatic ring (Scheme 2). The allene system likely
reacts with excess ammonia to form primary amide 18,
either directly or via transient cyclisation of the ester car-
bonyl to form an activated electrophilic oxonium. Amide
2
3
1
Aminopyridine 13 proved to be a versatile intermediate
(
Scheme 3). Reductive amination with aldehydes yielded
mono-alkylated products 21a-f in moderate to good
yields. Also, alkylation using iodomethane provided di-
methylated compound 21g. Finally, the application of
Sandmeyer conditions enabled conversion of amino-
pyridine 13 to chloropyridine 20h. The chlorine was then
reduced to give trisubstituted pyridine 21h (Scheme 3).
Compounds 7 and 21a-h were investigated for their ability
to inhibit the binding of a Cy3B™ conjugated analogue of
serotonin to human 5-HT2C receptor utilizing fluores-
cence polarisation technology and cellular membrane
1
8 then cyclizes onto the allene via a 6-exo-dig ring clo-
sure preferentially through oxygen due to superior orbital
overlap versus the nitrogen with the exo-allene π* orbital
to form a reactive hemi-aminal. An ammonia mediated
ring opening to form keto-amidine 19 is then followed by
a 6-exo-trig closure to provide the product aminopyridine
1
6ii. Further support for this mechanism comes from the
reaction of preformed primary amide 17e with ammonia
to successfully provide aminopyridine 16e, suggesting
amide 17e to be an intermediate on the reaction cascade.
preparations generated from recombinant Swiss 3T3 cells
In contrast, aromatic alkyne-ester 14f, under identical
reaction conditions, provided pyridinone 15f exclusively,
24
(
Table 2, K values).
i
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Table 2. 5-HT2C activity, physicochemistry and in vitro PK data for compounds 7 & 21a-h
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5-HT2C
5-HT2B
HLM Clint
RRCK
MDCK-MDR1 MDCK-
AB Papp (×10 MDR1 ER
-6
-
1
-6
Cmpd
7
R
logD
EC50
a,b
a
EC50
(nM)
a,c
a
(mL min
mg )
(×10
cm/s)
a
E
max
K
i
(nM)
a
E
max
K
b
(nM)
-1
cm/s)
(BA/AB)
(nM)
NHMe
NHEt
0.4
0.6
1.7
9
99%
79%
95%
100%
nt
3
3
1484
18
69%
28%
nt
-
-
19
13
8
7
2.5
1.8
nt
2.8
2.3
nt
2
1a
11
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21b
21c
NHCH
NHCH
2
iPr
cPr
36
21
12
0.5
4
nt
22
27
33
-
-
nt
27
11
nt
2
2
1.2
51%
38%
32%
-
-
0.8
1.5
nt
6.1
3.5
nt
2
1d
1e
1f
1g
1h
NHnPr
NHiPr
NHBn
1.0
1.0
1.7
nt
nt
158
nt
nt
-
4
2
nt
13
22
12
35
-
nt
35
43
<8
12
2
2
37%
nt
121
-
0.6
2.1
3.9
3.6
2.2
1.5
2
NMe
H
2
0.6
0.5
30
27
38%
53%
8
2
nt
-
18
a
b
Values are geometric means of up to five experiments. Differences of <2-fold should not be considered significant; % activation by maximum asymptote
c
at 10 ꢀM relative to 5-HT; % activation by maximum asymptote at 30 ꢀM relative to 5-HT; nt denotes not tested
a
Scheme 3
appeared less ligand and lipophilic efficient, providing no
appreciable improvements in 5-HT2C potency. Further-
more, although this series generally showed similar levels
of 5-HT2B potency (EC ), the compounds were either
50
weak partial agonists at 5-HT , characterized by low E
max
2B
values, or showed antagonism (compound 21f). Overall,
compounds also tended to exhibit good metabolic stabil-
ity in human liver microsomes (HLM) and moderate to
good passive permeability in RRCK cells.
Methyamino-substituted pyridine compound 7 looked the
most promising on balance of physicochemistry, potency,
selectivity and metabolic stability. In accordance with the
original design hypothesis, compound 7 also exhibited a
low efflux ratio in the MDCK-MDR1 P-gp assay (P-gp
ER=2.8), a pronounced improvement over the equivalent
pyrimidine compound 5 (P-gp ER=10). This level of P-gp
efflux (ER=2.8) correlates well with other examples from
the broader azepine series such as pyrimidine compound
a
Reagents and conditions: (a) aldehyde or ketone, DCE,
AcOH, 23 °C, 30 min, then PS-BH CN, 55 °C, 18-40 h; (b)
Pd/C, H , EtOH, 45 psi, 23 °C, 3-24 h; (c) MeI, K CO , DMF,
80 °C, 22 h; (d) NaNO , HCl, H O, MeCN, 23 °C, 1 h; (e) Pd/C,
3
2
2
3
6
(ER=2.7) that previously achieved good CNS exposure
2
2
1
3
and efficacy in preclinical in vivo studies.
HCOONH , EtOH, 75 °C, 2 h.
4
In summary, the rational design and synthesis of a series
of pyridine-fused azepines with potent 5-HT2C agonist
activity and low P-gp efflux ratios has been described to
deliver lead compound 7 (PF-04781340). Chemistry was
developed and rationalized to access this template, in-
cluding an ammonia-mediated cascade synthesis of ami-
nopyridine 13. These methods have also been extended to
the synthesis of poly-substituted and fused bicyclic ami-
nopyridines, illustrating potential for broader application.
The 5-HT2C and 5-HT2B functional agonist activities of
selected compounds were evaluated relative to 5-HT (1)
by measuring ability to induce G-protein activation via
recruitment of GTPγS and mobilization of intracellular
calcium for 5-HT2 and 5-HT2 respectively (Table 2, EC
C
B
50
13, 24
and Emax).
Previous studies within Pfizer have shown
compound K at the 5-HT receptor to be the most pre-
i
2C
dictive indicator of free brain exposure required to elicit
25
5
-HT2C related pharmacological effects in vivo (see SI for
cell culture and assay protocols).
ASSOCIATED CONTENT
Compounds 7 and 21a-h exhibited excellent 5-HT2C bind-
ing potency and agonist efficacy (Table 2). Varying the 2-
amino substituent sampled a range of molecular weight
and lipophilicity. However, despite larger and more lipo-
philic substituents being generally well tolerated they
Supporting Information Available
1
13
Experimental procedures and H NMR, C NMR spectra of
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
ACS Medicinal Chemistry Letters
Werner, W.; Wenzel, Z.; Giragossian, C.; Chen, H.; Coffey, S. B.
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Orally active and brain permeable proline amides as highly
selective 5-HT2C agonists for the treatment of obesity. Bioorg.
Med. Chem. Lett. 2010, 20, 2365-2369.
Corresponding Author
Phone: +44(0)1304641854. Email: ian.storer@pfizer.com
*
(13) Storer, R. I.; Brennan, P. E.; Brown, A. D.; Bungay, P. J.;
Present Addresses
Conlon, K. M.; Corbett, M. S.; DePianta, R. P.; Fish, P. V.;
Heifetz, A.; Ho, D. K. H.; Jessiman, A. S.; McMurray, G.; de
Oliveira, C. A. F.; Roberts, L. R.; Root, J. A.; Shanmugasundaram,
V.; Shapiro, M. J.; Sherten, M.; Westbrook, D.; Wheeler, S.;
Whitlock, G. A.; Wright, J. Multiparameter optimization in CNS
drug discovery: design of pyrimido[4,5-d]azepines as potent 5-
HT2C receptor agonists with exquisite functional selectivity over
5-HT2A and 5-HT2B receptors. J. Med. Chem. 2014, 57, 5258-5269.
(14) Andrews, M. D.; Fish, P. V.; Blagg, J.; Brabham, T. K.;
Brennan, P. E.; Bridgeland, A.; Brown, A. D.; Bungay, P. J.;
Conlon, K. M.; Edmunds, N. J.; af Forselles, K.; Gibbons, C. P.;
Green, M. P.; Hanton, G.; Holbrook, M.; Jessiman, A. S.;
McIntosh, K.; McMurray, G.; Nichols, C. L.; Root, J. A.; Storer, R.
I.; Sutton, M. R.; Ward, R. V.; Westbrook, D.; Whitlock, G. A.
Pyrimido[4,5-d]azepines as potent and selective 5-HT2C receptor
agonists: Design, synthesis, and evaluation of PF-3246799 as a
treatment for urinary incontinence. Bioorg. Med. Chem. Lett.
†
R.I.S., D.C.B.: Pfizer Neusentis, The Portway Building, Gran-
ta Park, Cambridge, CB21 6GS, United Kingdom.
ACKNOWLEDGMENT
We thank the Primary Pharmacology Group for screening
data, Jianmin Sun for spectra, Dr J. E. Davies for X-ray crys-
tallography and Asser Bassyouni for 5-HT2B selectivity data.
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ABBREVIATIONS
CCDC, Cambridge Crystallographic Data Centre; CNS, cen-
tral nervous system; HLM, human liver microsomes; MDCK,
Madin-Darby canine kidney; MDR1, multidrug resistance
gene; P-gp, P-glycoprotein; RRCK, Ralph Russ canine kidney
cell line; SM, starting material; Z, carboxybenzyl.
2011, 21, 2715-2720.
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For Table of Contents Use Only
The design, synthesis and evaluation of tetra-substituted pyridines as potent 5-HT re-
ceptor agonists
2C
Guy Rouquet, Dianna E. Moore, Malcolm Spain, Daniel M. Allwood, Claudio Battilocchio, David C. Blakemore, Paul V.
∗
Fish, Stephen Jenkinson, Alan S. Jessiman, Steven V. Ley, Gordon McMurray, and R. Ian Storer
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