Synthesis of 7-chloro-2,3-dihydro-2-[1-(pyridinyl)alkyl]-pyridazino[4,5-b]quinoline-1,4, 10(5H)-triones as NMDA glycine-site antagonists

Article abstract of DOI:10.1016/S0960-894X(03)00750-9

Several members of the 7-chloro-2,3-dihydro-2-[1-(pyridinyl)alkyl]-pyridazino[4,5-b]quinoline-1,4, 10(5H)-triones (2) have been identified as being potent and selective NMDA glycine-site antagonists. Increasing size of the alkyl substituent on the alpha-c

Full text of DOI:10.1016/S0960-894X(03)00750-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3553–3556
Synthesis of 7-Chloro-2,3-dihydro-2-[1-(pyridinyl)alkyl]-
pyridazino[4,5-b]quinoline-1,4,10(5H)-triones as NMDA
Glycine-Site Antagonists
Dean G. Brown,* Rebecca A. Urbanek, Thomas M. Bare, Frances M. McLaren,
Carey L. Horchler, Megan Murphy, Gary B. Steelman, James R. Empfield,
Janet M. Forst, Keith J. Herzog, Wenhua Xiao, Martin C. Dyroff, Chi-Ming C. Lee,
Shephali Trivedi, Kathy L. Neilson and Richard A. Keith
AstraZeneca Pharmaceuticals, 1800 Concord Pike, Wilmington, DE 19850, USA
Received 30 April 2003; accepted 3 July 2003
Abstract—Several members of the 7-chloro-2,3-dihydro-2-[1-(pyridinyl)alkyl]-pyridazino[4,5-b]quinoline-1,4,10(5H)-triones (2)
have been identified as being potent and selective NMDA glycine-site antagonists. Increasing size of the alkyl substituent on the
alpha-carbon led to a progressive decrease in binding affinity. Some of these analogues possess improved drug-like properties such
as cellular permeability, solubility and oral absorption.
# 2003 Elsevier Ltd. All rights reserved.
N-Methyl-d-aspartate (NMDA) antagonists which act
at the allosteric glycine co-agonist site are believed to
have potential therapeutic value in treating a range of
disorders including stroke, Parkinson’s disease, depres-
sion, and neuropathic pain.1 It has been recognized that
a major hurdle in designing efficacious NMDA glycine-
site antagonists is overcoming poor physical properties
such as solubility, cell permeability, plasma protein
binding and brain penetration.2 In this respect, devel-
opment of orally active glycine-site antagonists has been
difficult.
<5%) became a large concern. Extensive formulation of
1 still did not lead to improvements in oral absorption
(Fig. 1).
Compound 1 was then used as the starting point to
incorporate chemical modifications to try and improve
the physical properties of these compounds while main-
taining reasonable affinity (e.g., <250 nM) at the
NMDA glycine site. Based on previous knowledge from
the N-phenyl substituted PQT’s, it was known that
solubility was often improved when the planar geometry
of these compounds was distorted. We followed the
same strategy to improve the physical properties of 1, in
this case employing alkyl-substitution on the a-carbon
to give analogues such as 2.
In previous work, it has been shown that 7-chloro-2,3-
dihydro -2-(phenyl)-pyridazino[4,5 -b]quinoline -1,4,10
(5H)-triones (PQT’s) are potent NMDA glycine-site
antagonists.3 We sought to improve on the physical
properties of these compounds while preserving the
potency and selectivity for the NMDA-glycine site.
Compound 1 (Ki=115 nM vs [3H] MDL 105,519) was
initially identified as a potent NMDA-glycine site antago-
nist, however poor drug-like properties (e.g., aqueous
solubility less than 0.1 mg/mL and oral bioavailability
*Corresponding author: Tel.: +1-302-886-7535; fax +1-302-886-
5382; e-mail: dean.brown@astrazeneca.com
Figure 1.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00750-9

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D. G. Brown et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3553–3556
The synthesis of these analogues is shown below in
Scheme 1. Commercially available pyridyl ketones 3
were converted to tert-butoxy hydrazones 4 in the pres-
ence of catalytic acid.4 Alternatively, non- commercially
available ketones were prepared according to the pro-
cedure of Sheldrake which employs alkylation of a ketal
carbon and subsequent deprotection.5 The hydrazones
were then reduced using catalytic hydrogenation to give
the hydrazines 5. Hydrazines 5 were coupled with acid
amide 66 using carbodiimides to give the intermediate
hydrazine amides 7. The final cyclization was accom-
plished with methanesulfonic acid yielding 2a–t as the
methanesulfonic acid salts.
quite high.8 The low yield was a result of the difficulty of
separating the final material from excess t-butyl carba-
zate. Unfortunately, when the amount of t-butyl carba-
zate was reduced from five equivalents to one
equivalent, the enantioselectivity dropped off rather
precipitously. Using only one equivalent led to nearly
racemic material. These results suggest a competitive
SN1 pathway can occur when the concentration of car-
bazate is low. As an alternative, the racemic hydrazine
can be separated by preparatory chiral HPLC. Coupling
of the chiral hydrazines was accomplished using EDCI
in refluxing CH2Cl2 as outlined in Scheme 1 to give rise
to 2c (Table 1). The opposite enantiomer (R)-13 was
prepared in an analogous fashion to give 2d beginning
with the (S)-alcohol 10 also obtained from the chiral
resolution described above (Scheme 3).
We were also interested in the pyridyl N-oxide deriva-
tives of these compounds. In theory, these compounds
may possess better water solubility than the parent
compounds. The N-oxide analogue of 1 is readily pre-
pared by dissolving 1 in methanolic choline hydroxide
solution, followed by the addition of m-CPBA to give
8a, or correspondingly 8b Scheme 2.
Biological and physical data of these compounds are
shown in Table 1. From the data, it is apparent that
affinity at the glycine site decreased with increasing
substitution on the a-carbon.9 In this regard, the chiral
analogues 2c and 2d represent an interesting compar-
ison. The (S)-isomer 2c was reasonably potent, whereas
the (R)-isomer was absent of activity. The pyridyl N-
oxides 8a and 8b were less potent that the unoxidized
analogues (1 and 2n) with a 5- to 10-fold decrease in
binding affinity being observed.
Chiral analogues of 2 were prepared with greater than
98% enantiomeric purity. The chiral acetate 11 served
as a good starting point and was prepared by enyzmatic
resolution of 1-(2-pyridyl)ethanol 9 using Novozyme-
435.7 The acetate was then hydrolyzed and converted to
mesylate 12. The mesylate was displaced with t-butyl-
carbazate to give the desired chiral hydrazine (S)-13.
Although the yield was low, the enantioselectivity was
Despite the drop-off in binding affinity, the drug-like
physical properties of these a-branched compounds
appeared to improve over the unsubstituted counterparts.
For example, all of the unsubstituted analogues (R=H
for 2a, 2k, 1, 8a) possessed poor Caco-2 permeabilities
(A>B<10 Â 10À7). In the 2- and 4-pyridyl series, sub-
stitution of alkyl groups on the a-carbon resulted in
improved Caco-2 permeability. In these two series small
aliphatic substitution (e.g., R=Me) led in some cases to
30-fold improvements in Caco-2 permeabilities (e.g., 2c,
2n) with only 2- to 3-fold loss in binding activity. The
Caco-2 improvements were not seen in the 3-pyridyl series
when R=Me, but did improve with R=Et (2l vs 2m).
However, the ethyl substituted compound was devoid of
binding activity. These improvements in cellular perme-
ability may be due to the increase of hydrophobicity at
the a-carbon. The N-oxide 8a showed little change in
Caco-2 permeability over the parent compound 1.
In general, solubilities of the substituted analogues were
also improved from the lead compound 1. The
improved solubility may be due to distortion of the
planar conformation, resulting in potentially weaker
crystal packing forces. In some cases, this can be sup-
ported by a corresponding lowering of melting points
between the a-alkyl substituted and unsubstituted ana-
logues.10 For example, compound 1 has a melting point
range between 277 and 278 ꢀC as the methanesulfonate
salt. The a-methyl analogue 2n has a melting point
range between 245 and 247 ꢀC for the same salt form.
Similar observations are seen with 2a and 2k and their
corresponding a-alkyl analogues.
Scheme 1. Reagents and conditions: (a) t-BocNHNH2, THF, HCl
(cat.), 60–95%; (b) 10% Pd/C, MeOH, H2, 40 psi, 18 h, 35–98%; (c) 5,
CMC or EDCI, THF or CH2Cl2, DMAP (cat.), 15–78%; (d)
CH3SO3H, THF, rt, 30–85%.
Scheme 2. Reagents and conditions: (a) choline hydroxide, MeOH;
m-CPBA, 20%.
It is also apparent that the improvement in drug-like
physical properties paralleled an improvement in oral

D. G. Brown et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3553–3556
Table 1. Glycine site affinity, cellular permeability, solubility and oral bioavailability
3555
Compda
Pyridyl position
R
[3H] MDL 105519 KiÆSD (nM)b
Caco-2c (A>B) Â 10À7
Sol (mg/mL)d
F (%)e
1
4
2
2
2
2
2
2
2
2
2
2
3
3
3
4
4
4
4
4
4
4
4
4
H
H
Me
115Æ58
146Æ30
2
3
21
91
130
79
495
160
91
98
56
2
8
50
58
0.05
0.28
5Æ5
—
42Æ16
50Æ8
48Æ17
—
—
—
—
—
2a
2b
2c
2d
2e
2f
2g
2h
2i
221Æ81
>0.32
>0.32
>0.33
>0.20
NT
>0.22
>0.13
0.49
Me (S)
Me (R)
Et
i-Pr
n-Bu
i-Bu
c-prop
c-pent
H
Me
Et
Me
Et
207Æ80
>10,000
2360Æ499
2170Æ1146
>10,000
>10,000
996Æ343
>10,000
99Æ9
2j
2k
2l
>0.17
0.17
—
ꢁ12f
272Æ60
>0.27
>0.25
>0.29
>0.20
>0.19
>0.22
0.07
>0.17
0.13
>0.16
NTh
—
—
2m
2n
2o
2p
2q
2r
2s
2t
>10,000
248Æ70
30Æ8
17Æ7
—
—
—
—
—
—
—
1940Æ408
751Æ319
4340Æ2767
5570Æ1312
5230Æ2380
>10,000
439Æ107
2420Æ530
110
63
160
94
110
40
c-prop
n-Pr
n-Bu
i-Bu
Ph
8ag
8bg
H
Me
5
NTh
1
aAll new compounds were fully characterized by H NMR spectroscopy, MS, and CHN.
bExperiments run in duplicate. Displacement of [3H] MDL 105,519 in rat brain membranes. Experiments run in duplicate. Average Ki is reported.
cCaco-2 measurements were done with Caco-2 monolayers. Samples were taken from the apical and basolateral side and quantified by LC/MS/MS.
dAqueous solubility of methanesulfonate salt at pH=7.4.
ePercent rat oral bioavailability as the methanesulfonate salt. Male Sprague–Dawley rats were dosed orally (10 mg/kg, ASV suspension or meglu-
mine solution) and intravenously (10 mg/kg). Plasma concentrations were measured until the drug was not quantifiable.
fHigh interanimal variability. Estimated bioavailability is reported.
gPyridyl N-oxides.
hNT, not tested.
The NMDA glycine-site antagonists presented here
were designed to have improved solubility and cellular
penetration over the parent compound 1. The introduc-
tion of alkyl groups into the a-position led to such
improvements. However, a narrow window of glycine
binding site affinity exists for these analogues. It was
determined that only a methyl groupwas tolerated with-
out substantial loss in binding activity. The chiral analo-
gue 2c represents the best lead from this series. Although
this compound had a 2- to 3-fold loss in binding activity,
the gain of 10-fold improvement in oral bioavailability
Scheme 3. Reagents and conditions: (a) Novozyme-435, Et2O, vinyl
acetate, 5 h, 4 A sieves, rt; (b) K2CO3, MeOH, 98%; (c) MsCl or
Ms2O, DIEA, CH2Cl2 or toluene, 70%; (d) t-BocNHNH2 (5 equiv),
DIEA, DMF, 110 ꢀC, 6 h, 30%, >98% ee.
makes it a superior compound for future in vivo studies.
Acknowledgements
We thank Jim Hall for assistance in NMR spectro-
scopy. We also thank James Hulsizer, Scott Lehr and
Gary Moore for large-scale preparation of 6. We also
thank Mary J. Bock, Alan S. Kirschner, James A.
Osborn, George D. Stratton, Jian Huang, and Vasudha
Garlapati for measurements of biological and physical
properties. We also thank Edward P. Vacek and Vernon
Alford for insightful conversations.
absorption. For the cases studied, the unsubstituted
compounds 1 and 2k possessed the lowest rat oral
bioavailabilities. In contrast, the a-substituted analo-
gues 2b, 2c, 2d, 2n and 2o had the best oral bioavail-
abilities. It should also be noted that smaller
interanimal variability was seen with the a-branched
compounds as compared to 1 and 2k.

3556
D. G. Brown et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3553–3556
6. (a) Bare, T. M.; Empfield, J. R.; Forst, J. M.; Herzog, K. J.;
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7. The absolute configurations of 10 and 11 have previously
been established, see: (a) Uenishi, J.; Hiraoka, T.; Hata, S.;
Nishiwaki, K.; Yonemitsu, O.; Nakamura, K.; Tsukube, H. J.
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using a Chiralcel-OD column using hexane, ethyl alcohol, and
diethyl amine (95:5:0.05) as eluents.
9. Glycine-site affinities were determined by displacement of
[3H]MDL 105,519 in rat brain membranes as described in:
Baron, B. M.; Siegel, B. W.; Harrison, B. L.; Gross, R. S.;
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