7-Oxo-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamides as Selective CB 2 cannabinoid receptor ligands: Structural investigations around a novel class of full agonists

Article abstract of DOI:10.1021/jm300763w

Cannabinoid receptor agonists have gained attention as potential therapeutic targets of inflammatory and neuropathic pain. Here, we report the identification and optimization of a series of 7-oxo-[1,4]oxazino[2,3,4-ij] quinoline-6-carboxamide derivatives as a novel chemotype of selective cannabinoid CB₂ receptor agonists. Structural modifications led to the identification of several compounds as potent and selective cannabinoid receptor agonists (20, hCB₂K_i = 2.5 nM, SI = 166; 21, hCB₂K_i = 0.81 nM, SI = 383; 38, hCB₂K _i = 15.8 nM, SI > 633; 56, hCB₂K_i = 8.12 nM, SI > 1231; (R)-58, hCB₂K_i = 9.24 nM, SI > 1082). The effect of a chiral center on the biological activity was also investigated, and it was found that the (R)-enantiomers exhibited greater affinity at the CB₂ receptor than the (S)-enantiomers. In 3,5-cyclic adenosine monophosphate assays, the novel series behaved as agonists, exhibiting functional activity at the human CB₂ receptor.

Full text of DOI:10.1021/jm300763w

Article
pubs.acs.org/jmc
7-Oxo-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamides as Selective
CB2 Cannabinoid Receptor Ligands: Structural Investigations around
a Novel Class of Full Agonists
Pier Giovanni Baraldi,*,† Giulia Saponaro,† Allan R. Moorman,‡,∥ Romeo Romagnoli,† Delia Preti,†
Stefania Baraldi,† Emanuela Ruggiero,† Katia Varani,§ Martina Targa,§ Fabrizio Vincenzi,§
Pier Andrea Borea,§ and Mojgan Aghazadeh Tabrizi*,†
†Dipartimento di Scienze Farmaceutiche, Universita
̀
di Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara, Italy
‡Research and Development, King Pharmaceuticals, Inc., 4000 CentreGreen Way, Suite 300, Cary, North Carolina 27707,
United States
§Sezione di Farmacologia, Dipartimento di Medicina Clinica e Sperimentale, Universita
̀
di Ferrara, 44121 Ferrara, Italy
S
* Supporting Information
ABSTRACT: Cannabinoid receptor agonists have gained atten-
tion as potential therapeutic targets of inflammatory and neuro-
pathic pain. Here, we report the identification and optimization
of a series of 7-oxo-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide
derivatives as a novel chemotype of selective cannabinoid CB2
receptor agonists. Structural modifications led to the identification
of several compounds as potent and selective cannabinoid recep-
tor agonists (20, hCB2 Ki = 2.5 nM, SI = 166; 21, hCB2 Ki =
0.81 nM, SI = 383; 38, hCB2 Ki = 15.8 nM, SI > 633; 56, hCB2
Ki = 8.12 nM, SI > 1231; (R)-58, hCB2 Ki = 9.24 nM, SI > 1082). The effect of a chiral center on the biological activity was also
investigated, and it was found that the (R)-enantiomers exhibited greater affinity at the CB2 receptor than the (S)-enantiomers. In 3,5-
cyclic adenosine monophosphate assays, the novel series behaved as agonists, exhibiting functional activity at the human CB2 receptor.
targeting CB2 receptors may have considerable therapeutic
potential. Significant drug discovery efforts have been directed
toward developing and characterizing CB2-selective agonists,
both in vitro and in vivo. Indeed, several CB2-selective agonists
have been described previously.11−17 The selective ligands
belong to a number of structural classes. Classical cannabinoids
and cannabimimetic indoles have been the most systematically
studied, and there are sufficient data available to permit some
insight into the structural features of these compounds that lead
to high affinity for the CB2 receptor with little affinity for the
CB1 receptor. The chemical structures of a number of indole-
derived CB2 selective-agonists are shown in Figure 1. Com-
pounds 1 (AM1241)18 and 2 (L768242)19 have condition-
dependent CB2 functional activities, while compound 3
(A-796260),20 developed by Abbott Laboratories, was found
to be a highly selective CB2 agonist with little affinity for CB1.
These compounds have potent analgesic and anti-inflammatory
activity in animal models, being especially effective in models of
neuropathic pain.18
INTRODUCTION
■
Cannabinoids are the pharmacologically active components of
Cannabis sativa, or marijuana, and are known to mediate some
of their actions through the cannabinoid receptors. Two dis-
tinct cannabinoid receptors, named CB1 and CB2, have been
cloned and characterized from mammalian tissues.1,2 The CB1
receptor is abundantly expressed in the central nervous system
(CNS) and is responsible for the psychotropic side effects.3−5
The CB2 receptor is mainly found in cells of the immune
system, though it may be up-regulated in the CNS under patho-
logical conditions.6,7 The main signal transduction pathway
triggered is through Gi proteins, resulting in an inhibition of
adenylate cylase activity and a decrease in cyclic adenosine mono-
phosphate (cAMP) levels.8
At the present time, CB2 receptor agonists have gained
attention as potential therapeutic targets in the management of
neuropathic pain.9 The available data suggest that CB2-selective
agonists show promise for suppressing inflammatory and
neuropathic pain states. Behavioral, electrophysiological, and
neurochemical studies all support a role for CB2 receptor
activation in modulating inflammatory nociception. Effects of
CB2-selective agonists in different inflammatory pain models
(carrageenan, capsaicin, complete Freund’s adjuvant, formalin,
and arachidonic acid) have been demonstrated in many studies.10
These considerations suggest that novel pharmacotherapies
Several recently published studies also disclosed the 4(1H)-
oxo-1,8-naphthyridine-3-carboxamide scaffold16,21 (general struc-
ture 4, Figure 1) and 4-oxo-1,4-dihydroquinoline-3-carboxamide
Received: June 1, 2012
Published: June 28, 2012
© 2012 American Chemical Society
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Ki = 2.52, hCB1 Ki = 420 nM, selectivity index (SI) = 168).
This result led us to initiate a pharmacophore exploration and opti-
mization effort around the oxazinoquinolin-4-one central
scaffold. The novel target compounds are characterized by a
chiral center and were initially obtained as racemates. The
enantiopure forms of 31, 33, 35, 36, 51, 58, and 59 were also
synthesized, starting from the corresponding enantiopure
amino alcohols, to investigate the effect of the chiral center
on the CB2 receptor affinities.
The newly synthesized compounds were tested in competition
binding assays toward both rat CB1 (rCB1) and CB2 (rCB2) re-
ceptors expressed in native tissues as rat brain or spleen and
human CB1 (hCB1) and CB2 (hCB2) receptors expressed in CHO
cells. Affinity data (Ki, nM) were used to calculate the selectivity of
these novel compounds versus CB2 receptors. These ligands were
also examined in cAMP assays on hCB2 CHO cells, with the aim
of evaluating the inhibition of the adenylate cyclase activity and
consequently the potency values (IC50, nM).
CHEMISTRY
■
The target 7-oxo-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxa-
mides 19−59 were prepared following two synthetic routes,
depending upon the availability of the requisite α-halo ketone
or amino alcohol to form the desired substitution patterns at
C-2 and C-3 of the oxazine moiety. Racemic compounds 19−33
and 42−50 were obtained as depicted in Scheme 1. The
benzoxazine25 precursors 9a−g,i,j were prepared in two steps
by deprotonation of 2-nitrophenols 7a−c, followed by
alkylation with the appropriate α-halo ketones to give nitro
ketones 8a−h. In the second step, catalytic hydrogenation of
the nitro ketones 8a−h using 10% palladium-on-carbon in
methanol (or methanol/THF (85:15) for 3-aryl derivatives)
gave the intermediates 9a−g,i,j by a concomitant reduction−
reductive amination sequence. The intermediates 9i and 9j
were apparently formed by a catalytic ring-opening reaction
of the 2-(2-nitrophenoxy)-1-cyclopropylethanone (8g) and
2-(4-methyl-2-nitrophenoxy)-1-cyclopropylethanone (8h), respec-
tively. Subsequent reaction with diethyl (ethoxymethylene)-
malonate (DEEM) at 140 °C furnished the corresponding
methylenemalonate derivatives 10a−g,i,j in 70−90% yields.
A thermally induced cyclization of 10a−g,i,j in poly(phosphoric
acid) (PPA)26 for 1 h afforded the oxazinoquinoline ester
Figure 1. Representive cannabimimetic indoles and naphthyridine
scaffold as CB2-selective agonists.
derivatives (example 5, Figure 2)17,22,23 as structurally diverse
ligands endowed with high affinity and selectivity toward the
CB2 receptor. Some of these analogues were demonstrated to
act as agonists or inverse agonists in functional activity assays,
depending on the nature of the substituents on the different
positions of the heterocyclic scaffold.
Within a research program to identify novel CB2 agonists, we
designed a hybrid chemical structure that incorporated the
structural features of known cannabinoid ligands. Indeed, the
design of the new oxazinoquinoline 20 (Figure 2) was based
on the quinolone compound 516 and the cannabimimetic
indole 6 (WIN55,212-2, Figure 2), which is a potent canna-
binoid agonist and has approximately a 19-fold selectivity for
the CB2 versus CB1 receptor.24 The new N-cyclohexyl-3,7-
dihydro-7-oxo-3-phenyl-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-
carboxamide (20) was synthesized and tested in binding assays,
exhibiting high affinity and selectivity for the CB2 receptor (hCB2
Figure 2. Rational design of novel CB2 ligands.
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a
Scheme 1
a
Reagents and conditions: (i) α-halo ketone, K2CO3, acetone (anhydrous), rt, 16 h; (ii) H2 (4 atm), 10% Pd/C, MeOH, 4 h; (iii) diethyl
(ethoxymethylene)malonate, 140 °C, 2 h; (iv) poly(phosphoric acid), 140 °C, 1 h; (v) 10% NaOH, CH3OH, 80 °C, 1 h; (vi) amine, EDC, HOBt,
DMF, rt, 6 h or amine, DIEA, HBTU, DMF, rt, 16 h.
derivatives 11a−g,i,j in 75−95% yields. After saponification of
the ethyl ester functionality of compounds 11a−g,i,j (sodium
hydroxide), the resulting carboxylic acids 12a−g,i,j were enga-
ged in an amidation reaction with the appropriate amines under
peptide coupling conditions to afford the target amide
compounds 19−33 and 42−50.
The target compounds 55−57 were obtained by displace-
ment of the fluorine substituent at C-10 by treating the amide
53 with different nucleophiles (sodium methoxide, pyrrolidine,
N-methylpiperazine) in alkaline conditions (Scheme 3). The
4-methylpiperazine derivative 57 was converted to the hydro-
chloride salt by treatment with 1,4-dioxane saturated with gaseous
hydrogen chloride.
The synthesis of target compounds 34−41, 51−54, 58, and
59 was accomplished using a procedure similar to that utilized
for preparing optically active ofloxacine (Scheme 2).27 Ethyl 3-
(2,3-difluorophenyl)-, 3-(2,3,4-trifluorophenyl)-, and 3-(2,3,4,5-
tetrafluorophenyl)-3-oxopropanoates (14a−c, respectively)
were prepared from the corresponding benzoic acids (13a−c)
by reaction with carbonyldiimidazole (CDI) in THF to afford
the imidazolide, followed by condensation with the magnesium
salt of monoethyl malonate under essentially neutral conditions
(90% yield).28 Subsequent condensation with triethyl ortho-
formate in refluxing acetic anhydride produced the ethyl 2-
(ethoxymethylene)propionates 15a−c. These intermediates
were reacted with the appropriate amino alcohols in dry methyl-
ene chloride in an addition−elimination sequence to afford the
intermediates 16a−i. Cyclization using potassium carbonate
(K2CO3) in dimethylformamide (DMF) at 130 °C followed by
saponification afforded the carboxylic acids 18a−i that were
coupled with the appropriate amines (in the same manner as
reported in Scheme 1) to afford the final compounds 34−41,
51−54, 58, and 59. In an identical fashion, reaction of inter-
mediate 15a with the appropriate (R)- or (S)-amino alcohol led
to the preparation of the corresponding enantiomerically pure
derivatives itemized in Table 3.
RESULTS AND DISCUSSION
■
Each of the newly synthesized compounds was examined in
[3H]CP-55,940 competition binding experiments for their
affinity and selectivity toward the rat and human recombinant
CB1 and CB2 receptors (Tables 1−3). Among the 51 novel
7-oxooxazinoquinoline-6-carboxamide derivatives, 40 of the
compounds displayed high affinity for the CB2 receptors
(Ki values less than 100 nM).
An initial set of 12 compounds (19−30, Table 1) was syn-
thesized possessing an aromatic moiety (phenyl or 4-tolyl) at
C-3 of the heterocycle. The substituents on the carboxamide
moiety at C-6 were initially selected on the basis of other
cannabinoid pharmacophores, such as those present in the
naphthyridine16,21,29 (4) and quinolone17,22,23 (5) derivatives.
The 3-phenyl analogues bearing a cyclohexyl (20), cyclo-
heptyl (21), adamant-1-yl (22), or 3,5-dimethyladamant-1-yl
(23) carboxamide displayed high affinity at the CB2 and low
affinity at the CB1 receptors. In particular, the oxazinoquino-
linones 21 and 23 were the most potent and selective
compounds in this group (21, h Ki = 0.81 nM, SI = 383; 23, h
Ki = 3.45, SI = 133). The cyclopentyl carboxamide derivative
19 showed lower affinity and poor selectivity at the CB
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a
Scheme 2
a
Reagents and conditions: (i) CDI, THF, (C2H5O)2Mg, monoethyl malonate, rt, 16 h; (ii) CH(OEt)3, Ac2O, 110 °C, 3 h; (iii) appropriate (R)-,
(S)-, or (R,S)-amino alcohol, CH2Cl2, rt, 1 h; (iv) K2CO3, DMF, 130 °C, 7 h; (v) 10% NaOH, CH3OH, 80 °C, 1 h; (vi) amine, EDC, HOBt, DMF,
rt, 6 h or amine, DIEA, HBTU, DMF, rt, 16 h.
a
Introduction of a methyl group at C-2 of the oxazine moiety,
as with compounds 24 and 25, resulted in a 24−27-fold
decrease of affinity (h Ki = 536 and 332 nM, respectively)
relative to that of the analogous compounds lacking the methyl
group (compounds 20 and 22, respectively). Compound 26
with a phenyl group at C-2 was inactive at both CB receptors,
confirming that the affinity of this class of molecules is quite
sensitive to modifications at C-2 of the oxazinoquinoline
nucleus. Interestingly, the introduction of a methyl group at the
para-position of the aryl moiety at C-3 (27 and 28) resulted in
a significant loss of affinity and selectivity for the CB2 and CB1
receptors in comparison with those of the corresponding
compounds 20 and 22 lacking this substituent, suggesting a less
severe steric constraint at this position. Introduction of a
methyl group at C-9 of the heterocyclic nucleus (29, h Ki of
18.4 nM) resulted in a modest decrease in affinity and selec-
tivity compared to those of the analogue lacking the methyl
group (20). The C-9 methyl derivative 30 was not tested due to
its low solubility in DMSO/water.
Scheme 3
The structure−activity relationship (SAR) studies were
extended by replacement of the C-3 phenyl ring with a series
of aliphatic moieties to evaluate the effect of the chain length
and branching (Table 2, compounds 31−51). Introduction of a
methyl group at this position seems to indicate that this may
be a site for enhancement in selectivity, although with an appa-
rent loss in affinity. Derivatives 31−33 each displayed a lower
affinity for both CB2 and CB1 receptors relative to compounds
20−22 bearing a phenyl moiety at this position, although the
selectivity for the CB2 receptor was improved with the
a
Reagents and conditions: (i) CH3ONa, THF, 50 °C, 16 h; (ii)
K2CO3, DMF, pyrrolidine, 100 °C, 10 h; (iii) K2CO3, DMF,
1-methylpiperazine, 100 °C, 10 h, then 1,4-dioxane saturated with
HCl gas, 0 °C, 30 min.
receptors, suggesting the important role of the carboxamide
moiety, as previously reported by Corelli et al.22
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Table 1. Affinity (Ki, nM) and Selectivity Index (SI) of Novel 5-Aryl CB Compounds on Rat and Human CB1 and CB2
a
Receptors and Potency (IC50, nM) of the Novel CB Compounds in hCB2 CHO Cells on cAMP Assays
Ki (nM)
f
hCB2 IC50
(nM)
b
c
d
e
compd R1
R2
R4
R5
R7
rat CB1
rat CB2
hCB1
hCB2
SI
6
(R)-(+)-WIN55,212-2
15.6 1.4
150 16
1150 110
372 36
670 65
545 53
3520 320
2640 225
>10000
7.58 0.72
110 12
12.4 1.3
132 12
420 38
310 29
265 24
460 48
3126 288
2435 231
>10000
4.53 0.42
98 10
2.74
1.35
166
383
19
15.7 1.3
472 43
15.2 1.7
19
20
21
22
23
24
25
26
27
28
29
30
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
4-tolyl
4-tolyl
phenyl
phenyl
cyclopentyl
H
cyclohexyl
cycloheptyl
19.3 2.3
14.7 1.5
18.3 2.4
2.52 0.21
0.81 0.07
14.2 1.5
3.45 0.42
536 58
332 28
>10000
H
5.23 0.42
26
H
adamant-1-yl
3,5-dimethyladamant-1-yl
cyclohexyl
3
H
46
3
133
5.83
7.33
ND
8.78
9.41
73
18.3 1.9
2749 245
1684 152
ND
H
Me
Me
phenyl
H
710 44
443 26
>10000
H
adamant-1-yl
adamant-1-yl
cyclohexyl
H
H
4566 425
4154 410
1568 164
ND
547 32
482 43
4108 380
3877 362
1346 125
ND
468 42
412 40
18.4 1.9
ND
2457 213
2215 211
H
H
adamant-1-yl
cyclohexyl
Me
Me
H
25
3
110
9
H
adamant-1-yl
ND
ND
472 43
a
b
The data are expressed as the mean SEM of n = 4 independent experiments. The affinity values were calculated by using [3H]CP-55,940 as a
c
radioligand on rat brain for CB1 receptors. The affinity values were calculated by using [3H]CP-55,940 as a radioligand on rat spleen for CB2
d
e
receptors. The affinity values were calculated by using [3H]CP-55,940 as a radioligand on human CB1 CHO membranes. The affinity values were
f
calculated by using [3H]CP-55,940 as a radioligand on human CB2 CHO membranes. IC50 values were calculated on cAMP experiments performed
on human CB2 CHO cells.
cycloheptyl and adamant-1-yl amides (32 and 33, respectively).
The addition of a gem-dimethyl group at C-3 (compound 52)
maintained affinity relative to that of analogue 33, while the
selectivity was dramatically reduced (Ki = 56 nM, SI = 4).
Optimal activity with this group of compounds was obtained
with the cycloheptyl moiety on the carboxamide chain
(compound 32, h Ki = 38 nM, SI > 263), in analogy to the
3-phenyl series.
Extending the aliphatic chain to an ethyl group (34−36)
enhanced affinity for both the CB1 and CB2 receptors, although
there was an apparent loss in selectivity. Further extension of
the aliphatic chain at C-3 to a propyl group (42−45) provided
further improvements in receptor affinity without significantly
affecting selectivity relative to those of the corresponding ethyl
derivatives, although the compound with a cycloheptyl amide
lacked sufficient solubility in DMSO/water mixtures to permit
evaluation. In particular, the 3-propyl derivative with a cyclo-
hexyl carboxamide (compound 42) proved to have the greatest
affinity for the CB2 receptor affinity in this series, with an h Ki
value of 0.32 nM. Replacing the n-propyl moiety at C-3 with a
cyclopropyl (48−50) or 2-propyl (compound 51) group led to
a loss in affinity compared to that of the analogous compounds
with the n-propyl moiety.
impacting selectivity. Similarly, the introduction of a methyl at
C-8 (46 and 47) in the 3-propyl series led to a significant
reduction in affinity, particularly at the CB2 receptor, leading
also to reduced selectivity relative to that of the corresponding
compounds lacking the methyl moiety (42 and 44,
respectively). Together, these compounds indicate that the
two receptor subtypes are quite sensitive to modifications in
this portion of the molecular scaffold. Additional substitutions
at C-10 allowed us to explore this sensitivity further. Com-
pounds 55 and 57, in which the fluorine atom of compound 53
is displaced by a methoxy or N-methylpiperazine moiety, retain
high receptor binding affinity, especially compound 55 at the
CB2 receptor, suggesting a difference between the two receptor
subtypes in steric tolerance at this position. Better results were
obtained with compound 56, in which the 10-fluorine atom was
replaced by a pyrrolidine moiety, showing high affinity and
remarkable selectivity at the CB2 receptor (h Ki = 8.12, SI >
1231). Such a result is in accordance with Manera’s findings16
regarding the increased CB2 selectivity for their 7-substituted
quinolones.
Within the 3-ethyl series, we also examined the effect on
receptor affinity and selectivity of structural modifications at the
6-carboxamide side chain (compounds 34−41). As seen with
the 3-phenyl series, the cycloheptyl amide 35 shows greater
affinity and selectivity for the CB2 receptor than the cyclohexyl
amide 34. In contrast, the adamant-1-yl amide 36 retains affi-
nity for the CB2 receptor equivalent to that of the cycloheptyl
derivative while showing a significant increase in selectivity.
Moving the point of attachment of the adamantane ring from
the bridgehead 1-position (36) to the bridging 2-position (37)
In the 3-ethyl series of compounds, additional modifications
on the heterocyclic nucleus were introduced. The introduction
of a fluorine atom at the C-10 position (compound 53) did not
significantly alter affinity at the CB2 receptor relative to that of
compound 36, although the selectivity decreased about 1.5-fold.
The 9,10-difluoro derivative 54 was found to be less active than
the 10-fluoro compound 53 (h Ki = 33 vs 7.34 nM) without
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Table 3. Affinity (Ki, nM) and Selectivity Index (SI) of the Novel Chiral CB Compounds on Rat and Human CB1 and CB2
a
Receptors and Potency (IC50, nM) of the Novel CB Compounds in hCB2 CHO Cells on cAMP Assays
Ki (nM)
b
c
d
e
f
compd
R5
R6
R7
rat CB1
rat CB2
30
250 27
hCB1
hCB2
3
SI
hCB2 IC50 (nM)
90
(R)-31
(S)-31
(R)-33
(S)-33
(R)-35
(S)-35
(R)-36
(S)-36
(R)-51
(S)-51
(R)-58
(R)-59
H
Me
H
cyclohexyl
3560 355
5242 534
>10000
4
2866 245
4652 425
>10000
24
119
25
8
Me
H
cyclohexyl
188 16
724 75
153 14
250 23
17.2 1.6
682 67
18.1 1.7
413 38
9.84 0.91
Me
H
adamant-1-yl
adamant-1-yl
cyclohepty
52
86
5
8
47
78
3
7
>212
>128
25
Me
H
>10000
>10000
ethyl
H
126 11
913 87
242 21
653 58
6.03 0.52
203 19
105
9
4.12 0.38
187 16
ethyl
cycloheptyl
adamant-1-yl
adamant-1-yl
adamant-1-yl
adamant-1-yl
adamant-1-yl
adamant-1-yl
843 77
197 15
578 44
4.5
40
H
ethyl
H
6.17 0.53
4.93 0.45
ethyl
97
8
88
7
7
H
isopropyl
H
95
9
1.75 0.14
19.3 1.9
10.2 1.1
88
7
1.24 0.11
16.2 1.8
9.24 0.84
3.72 0.32
71
isopropyl
864 82
>10000
858 79
>10000
53
55
36
6
3
H
isobutyl
benzyl
>1082
H
121 10
4.65 0.43
105
9
28
17.5 1.8
a
b
The data are expressed as the mean SEM of n = 4 independent experiments. The affinity values were calculated by using [3H]CP-55,940 asa
c
radioligand on rat brain for CB1 receptors. The affinity values were calculated by using [3H]CP-55,940 as a radioligand on rat spleen for CB2
d
e
receptors. The affinity values were calculated by using [3H]CP-55,940 as a radioligand on human CB1 CHO membranes. The affinity values were
f
calculated by using [3H]CP-55,940 as a radioligand on human CB2 CHO membranes. IC50 values were calculated on cAMP experiments performed
on human CB2 CHO cells.
resulted in less affinity and selectivity. Replacing the aliphatic
carbocyclic cycloheptyl amide moiety with a heteroaromatic
4-pyridyl (39) or thiazol-2-yl (40) moiety caused a dramatic loss
of affinity and almost complete loss of selectivity for the CB2
receptor. Similarly, replacing the cycloheptyl amide with the
bulky, noncyclic N,N-diisopropyl amide (41) led to a significant
loss of affinity at the CB2 receptor, although selectivity was
retained or even enhanced. Optimal activity and selectivity in
this series of 3-ethyl derivatives were obtained with the 5-
methylhexan-2-yl carboxamide chain (38, h Ki = 15.8 nM, SI >
633). These data show that affinity and selectivity for the CB2
receptor are quite sensitive to modifications of the 6-carbo-
xamido group.
As noted above, the presence of a gem-dimethyl group
(compound 52, h Ki = 56 nM, SI = 4) does not significantly
alter affinity for the CB2 receptor relative to that of the
monomethyl analogue 33 (h Ki = 47 nM, SI > 212), but has a
profound effect on selectivity. This suggested the possibility of a
difference between the two receptor subtypes in the stereofacial
preference for substituents at this position. To evaluate this
possibility, we prepared a number of compounds (31, 33, 35,
36, and 51) as single enantiomers rather than racemic mixtures
(Table 3). With the two compounds bearing a C-3 methyl
group (31 and 33), a stereochemical preference for the (R)-
enantiomers over the corresponding (S)-enantiomers is ob-
served. Interestingly, the (R)-31 enantiomer (h Ki = 24 nM,
SI = 119) displays a greater affinity for the CB2 receptor than the
racemic mixture (h Ki = 60 nM, SI = 36), but apparently poorer
affinity for the CB1 receptor. As a consequence, (R)-31 also
shows greater selectivity than the racemic mixture. Similarly,
the (R)-enantiomer of 33 binds with greater affinity than the
(S)-enantiomer, although the (R)-enantiomer does not appear
to offer any improvement in affinity or selectivity over the
racemic compound. Extending this analysis to compounds
bearing an ethyl group at C-3, we found that the (R)-enan-
tiomers of 35 and 36 both show approximately a 2-fold en-
hancement in affinity over the racemic compounds. However,
there is also an approximately 2-fold loss in selectivity of the
(R)-enantiomers for the CB2 receptor. This reflects a nearly 4-
fold improvement in affinity of the (R)-enantiomers for the
CB1 receptor. In contrast, the (S)-enantiomers of 35 and 36
displayed an 11−20-fold loss in affinity for the CB2 receptor
with almost complete loss of selectivity. Similarly, with the C-3
isopropyl derivative 51, the (R)-enantiomer bound with greater
affinity to both the CB1 and CB2 receptors than the (S)-
enantiomer or the racemic mixture ((R)-51, hCB1 Ki = 88 nM,
hCB2 Ki = 1.24 nM; (S)-51, hCB1 Ki = 858 nM, hCB2 Ki = 16.2
nM; 51, hCB1 Ki = 323 nM, hCB2 Ki = 3.74 nM). However, the
affinity enhancement was slightly greater for the CB1 receptor,
resulting in an apparent loss in selectivity relative to that of the
racemic mixture.
Recognizing that a significant enhancement in both affinity
and selectivity had been seen only with (R)-31 and (R)-33,
compounds bearing a methyl at C-3, we hypothesized that
introducing a methylene spacer between the oxazine nucleus
and the bulky isopropyl moiety may lead to further enhance-
ments in affinity and selectivity. This led to the synthesis and
evaluation of (R)-58, the enantiopure derivative bearing an
isobutyl moiety at C-3. Indeed, this compound was found to
bind to the CB2 receptor with high affinity and exceptional
selectivity (h Ki = 9.24 nM, SI > 1082). In light of this finding,
we prepared (R)-59 by introduction of a benzyl moiety at C-3,
effectively placing a methylene spacer between the oxazine ring
and the phenyl moiety of compound 22. As expected, the
compound had improved affinity at the CB2 receptor, with a
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Article
Ki of 3.72 nM, and a modest improvement in selectivity (SI = 28)
relative to those of the C-3 phenyl derivative 22.
reduce the potential CB1-associated CNS adverse effects of this
novel series.
The potency of the novel compounds was measured in a
functional assay, evaluating the capability of the compounds to
inhibit forskolin-induced cAMP production in hCB2 CHO cells
(Tables 1−3). The competition binding curves obtained from
receptor binding experiments (Figure 3A) and the dose−response
The potency of the novel compounds was measured in
functional assays, with high potency values (represented by
IC50) that are closely correlated with the high affinity values
(expressed as Ki), revealing that the novel series behaves as CB2
receptor agonists. This novel series of compounds offers an
attractive starting point for further optimization and represents
novel pharmacological tools to evaluate the therapeutic poten-
tial of CB2 agonists in various disease settings, especially
inflammatory pain.
EXPERIMENTAL SECTION
■
Pharmacology. Competition binding experiments were performed
by using [3H]CP-55,940 (specific activity 180 Ci/mmol) that was
obtained from Perkin-Elmer Life and Analytical Sciences (Waltham,
MA). Human CB1 and CB2 receptors expressed in CHO cells were
purchased from Perkin-Elmer Life and Analytical Sciences. All other
reagents were of analytical grade and were obtained from commercial
sources.
Competition Binding Experiments on CB1 and CB2 Receptors. To
study CB1 receptors, rat brain (male Sprague−Dawley rats, Charles
River, Wilmington, MA) was removed, frozen in liquid nitrogen, and
stored at −80 °C. The rat brain tissue was suspended in 50 mM Tris−
HCl buffer, pH 7.4, at 4 °C. The suspension was homogenized with a
Polytron and centrifuged for 10 min at 2000g, and the supernatant was
centrifuged again for 20 min at 40000g. The pellet was resuspended in
a buffer containing 50 mM Tris−HCl, 1 mM EDTA, 3 mM MgCl2,
and 0.5% fatty acid free bovine serum albumin (BSA), pH 7.4, at
30 °C.
Competition binding experiments to rat CB1 receptors were carried
out using [3H]CP-55,940 (1.0 nM), a membrane suspension
containing 40 μg of protein/100 μL and different concentrations
(1 nM to 10 μM) of the examined compounds.30
To investigate CB2 receptors, a [3H]CP-55,940 binding assay was
performed by using rat spleen (male Sprague−Dawley rats, Charles
River) that was homogenized in 50 mM Tris−HCl buffer, pH 7.4, at
4 °C with a Polytron and centrifuged for 10 min at 2000g, and the
supernatant was centrifuged for 20 min at 40000g. The pellet was
resuspended in a buffer containing 50 mM Tris−HCl, 1 mM EDTA,
3 mM MgCl2, and 0.5% fatty acid free BSA, pH 7.4, at 30 °C.
Competition binding experiments to rat CB2 receptors were
performed using [3H]CP-55,940 (0.5 nM), a membrane suspension
containing 80 μg of protein/100 μL and different concentrations
(1 nM to 10 μM) of the examined compounds.30
Human CB1 and CB2 receptors expressed in CHO cells were grown
adherently and maintained in Ham’s F12 containing 10% fetal bovine
serum, penicillin (100 U/mL), streptomycin (100 μg/mL), and
Geneticin (G418; 0.4 mg/mL) at 37 °C in 5% CO2/95% air.24,31,32
For membrane preparation the culture medium was removed, and the
cells were washed with PBS and scraped off T75 flasks in ice-cold
hypotonic buffer (5 mM Tris−HCl, 2 mM EDTA, pH 7.4). The cell
suspension was homogenized with a Polytron and centrifuged for
10 min at 1000g, and the supernatant was then centrifuged for 30 min
at 100000g. The membrane pellet was suspended in 50 mM Tris−HCl
buffer, 0.5% BSA (pH 7.4) containing 5 mM MgCl2, and 2.5 mM
EDTA or 1 mM EDTA for hCB1 or hCB2 receptors, respectively.
Competition binding experiments were performed using 0.5 nM
[3H]CP-55,940 and different concentrations (1 nM to 10 μM) of the
examined compounds or the reference agonist WIN55,212-2 for an
incubation time of 90 or 60 min at 30 °C for CB1 or CB2 receptors,
respectively.
Figure 3. Affinity (Ki, nM) and potency (IC50, nM) of selected novel
CB compounds: (A) competition curves on hCB2 receptors; (B)
inhibition curves of forskolin-stimulated cAMP accumulation in hCB2
CHO cells. Results are given as the mean SEM (n = 4 independent
experiments).
curves (Figure 3B) from cAMP assays are shown for select
compounds (42, 20, 21, (R)-36, and 55). High affinity values,
expressed as Ki values, are seen to be closely associated with
high potency, represented by IC50 values (Tables 1−3 and
Figure 3). The affinity and potency of the novel compounds
were also compared with those of the reference cannabimimetic
compound 6 that is characterized by high affinity and potency
but very low selectivity (Tables 1−3). The functionality of this
new series of oxazinoquinolones was not affected by several
modifications, showing agonistic behavior, not in accordance
with the Stern et al. investigations.23 Interestingly, the affinity
values of the examined compounds at the respective rat and
human receptors were not significantly different, suggesting a
high degree of similarity between the two receptor subtypes
across the two species. As such, these data support the evalua-
tion of in vivo efficacy in rodent animal models of inflammatory
and/or neuropathic pain.
CONCLUSION
■
Bound and free radioactivities were separated by filtering the assay
mixture through Whatman GF/C glass fiber filters using a Brandel cell
We have discovered the 7-oxo-[1,4]oxazino[2,3,4-ij]quinoline-
6-carboxamide chemotype as a novel series possessing sig-
nificant cannabinoid CB2 receptor affinity. A major focus of
the optimization effort was to increase selectivity to avoid or
harvester (Brandel Instruments, Unterfohring, Germany). The filter-
̈
bound radioactivity was counted using a Packard Tri Carb 2810 TR
scintillation counter from Perkin-Elmer Life and Analytical Sciences.
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cAMP Assay to Human CB2 Receptors. CHO cells transfected with
human CB2 receptors were washed with phosphate-buffered saline and
diluted trypsin and centrifuged for 10 min at 200g. The pellet con-
taining CHO cells (1 × 106 cells/assay) was suspended in 0.5 mL of
an incubation mixture, NaCl (150 mM), KCl (2.7 mM), NaH2PO4
(0.37 mM), MgSO4 (1 mM), CaCl2 (1 mM), Hepes (5 mM), MgCl2
(10 mM), glucose (5 mM), pH 7.4, at 37 °C. Then 0.5 mM 4-(3-
butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) as a
phosphodiesterase inhibitor was added and preincubated for 10 min
in a shaking bath at 37 °C.33 The potency of the novel CB compounds
in comparison with a well-known CB agonist (6) was studied in the
presence of forskolin (1 μM). The reaction was terminated by the
addition of cold 6% trichloroacetic acid (TCA). The TCA suspension
was centrifuged at 2000g for 10 min at 4 °C, and the supernatant was
extracted four times with water-saturated diethyl ether. The final
aqueous solution was tested for cAMP levels by a competition protein
binding assay. Samples of cAMP standard (0−10 pmol) were added to
each test tube containing the incubation buffer (Trizma base (0.1 M),
aminophylline (8.0 mM), 2-mercaptoethanol (6.0 mM), pH 7.4) and
[3H]cAMP in a total volume of 0.5 mL. The binding protein pre-
viously prepared from beef adrenals was added to the samples
previously incubated at 4 °C for 150 min, and after the addition of
charcoal, the samples were centrifuged at 2000g for 10 min. The clear
supernatant was counted by using a Perkin-Elmer 2810 TR
scintillation counter (Perkin-Elmer Life and Analytical Sciences).
Data Analysis. The protein concentration was determined
according to a Bio-Rad method (Bradford, 1976) with bovine albumin
as a standard reference. Inhibitory binding constant values, Ki, were
calculated from the IC50 according to the Cheng and Prusoff equa-
tion34 Ki = IC50/(1 + [C*]/KD*), where [C*] is the concentration of
the radioligand and KD* its dissociation constant. A weighted
nonlinear least-squares curve fitting program, LIGAND,35 was used
for computer analysis of inhibition experiments. All the data are
expressed as the mean SEM of n = 4 independent experiments for
the in vitro assays. Statistical analysis of the data was performed using
unpaired two-sided Student’s t test.
Chemistry. Reaction progress and product mixtures were
monitored by thin-layer chromatography (TLC) on silica gel
(precoated F254 Merck plates) and visualized with aqueous potassium
permanganate. 1H NMR data were determined in CDCl3 or DMSO-d6
solutions with a Varian VXR 200 spectrometer or a Varian Mercury
Plus 400 spectrometer. Peak positions are given in parts per million
(δ) downfield, and J values are given in hertz. Light petroleum refers to
the fractions boiling at 40−60 °C. Melting points were determined on
a Buchi-Tottoli instrument and are uncorrected. Chromatography was
performed on Merck 230−400 mesh silica gel. Organic solutions were
dried over anhydrous sodium sulfate. Elemental analyses were per-
formed by the microanalytical laboratory of Dipartimento di Chimica,
University of Ferrara, and were within 0.4% of the theoretical values
for C, H, and N. The mass spectra were obtained on an ESI
Micromass ZMD 2000 mass spectrometer. The chemical purity of the
target compounds was ≥95%.
(CDCl3): δ 8.03−7.98 (m, 2H), 7.90−7.85 (m, 1H), 7.61−7.45
(m, 4H), 7.11−6.95 (m, 2H), 5.45 (s, 2H).
2-(2-Nitrophenoxy)-1-phenylpropan-1-one (8c). Yellow oil. Yield:
1
69%. MS: m/z 272.4 (M + H). H NMR (DMSO-d6): δ 8.08−8.04
(m, 2H), 7.89−7.85 (m, 1H), 7.72−7.59 (m, 3H), 7.15−7.11 (m, 2H),
6.29 (q, J = 6.8 Hz, 1H), 1.57 (d, J = 6.6 Hz, 3H).
2-(2-Nitrophenoxy)-1,2-diphenylethanone (8d). Yellow solid. Mp:
58 °C. Yield: 15%. MS: m/z 334.3 (M + H). 1H NMR (DMSO-d6): δ
8.08−7.86 (m, 2H), 7.30−7.54 (m, 5H), 7.03−7.19 (m, 7H), 6.48
(s, 1H).
2-(5-Methyl-2-nitrophenoxy)-1-phenylethanone (8e). Yellow
1
solid. Mp: 132−134 °C. Yield: 75%. MS: m/z 272.1 (M + H). H
NMR (CDCl3): δ 8.03−7.99 (m, 2H), 7.83−7.54 (m, 4H), 7.16
(s, 1H), 6.96−6.91 (m, 1H), 5.83 (s, 2H), 2.32 (s, 3H).
1-(4-Methylphenyl)-2-(2-nitrophenoxy)ethanone (8f). White
1
solid. Mp: 125 °C. Yield: 73%. MS: m/z 272.8 (M + H). H NMR
(DMSO-d6): δ 7.92−7.86 (m, 3H), 7.58−7.53 (m, 1H), 7.40−7.36
(m, 2H), 7.28−7.23 (m, 1H), 7.16−7.11 (m, 1H), 5.81 (s, 2H), 2.40
(s, 3H).
1-Cyclopropyl-2-(2-nitrophenoxy)ethanone (8g). White solid.
Mp: 58 °C. Yield: 75%. MS: m/z 222.1 (M + H). 1H NMR
(DMSO-d6): δ 7.91−7.86 (m, 1H), 7.61−7.57 (m, 1H), 7.20−7.13
(m, 2H), 5.21 (s, 2H), 2.22 (m, 1H), 1.01−0.90 (m, 4H).
1-Cyclopropyl-2-(4-methyl-2-nitrophenoxy)ethanone (8h).
Brown oil. Yield: 69%. MS: m/z 236.2 (M + H). 1H NMR
(DMSO-d6): δ 7.89 (m, 1H), 7.19−7.12 (m, 2H), 5.20 (s, 2H),
2.32 (s, 3H), 2.21 (m, 1H), 1.01−0.90 (m, 4H).
Synthesis of Benzo[1,4]oxazines 9a−g,i,j). General Procedure. To
a solution of the nitro ketone 8a−h (7 mmol) in methanol (100 mL)
(in the case of 3-aryl in methanol/THF, 85:15) was added 10%
palladium on carbon (0.3 mmol, 0.178 g), and the mixture was shaken
under 4 atm of hydrogen at room temperature for 6 h. The reaction
was monitored by TLC, and when complete, the suspension was
filtered through a pad of Celite to remove the catalyst. The filtrate was
concentrated under vacuum to give a yellow residue that was purified
by column chromatography (SiO2) using ethyl acetate/petroleum
ether (0.5:9.5) to yield the desired compounds. The cyclopropyl
derivative 9g and propyl derivative 9i were separated by column
chromatography using ethyl acetate/petroleum ether (0.2:9.8) as the
eluent.
3,4-Dihydro-3-methyl-2H-benzo[b][1,4]oxazine (9a). Colorless
1
oil. Yield: 80%. MS: m/z 150.1 (M + H). H NMR (DMSO-d6): δ
6.68−6.40 (m, 4H), 5.72 (br s, 1H), 4.15−4.07 (m, 1H), 3.65−3.56
(m, 1H), 3.37−3.36 (m, 1H), 1.07 (d, J = 6.4 Hz, 3H).
3,4-Dihydro-3-phenyl-2H-benzo[b][1,4]oxazine (9b). White solid.
1
Mp: 40 °C. Yield: 76%. MS: m/z 212.2 (M + H). H NMR (DMSO-
d6): δ 7.43−7.37 (m, 5H), 6.85−6.67 (m, 4H), 5.99 (br s, 1H); 4.53−
4.49 (m, 1H),4.34−4.27 (m, 1H), 4.06−3.95 (m, 1H).
3,4-Dihydro-2-methyl-3-phenyl-2H-benzo[b][1,4]oxazine (9c).
Pale yellow oil. Yield: 83%. MS: m/z 226.0 (M + H). 1H NMR
(DMSO-d6): δ 7.34−7.30 (m, 5H), 6.74−6.69 (m, 3H), 6.58−6.43
(m, 1H), 6.38 (br s, 1H), 4.47-.4.44 (m, 2H), 0.98 (d, J = 6.4 Hz, 3H).
3,4-Dihydro-2,3-diphenyl-2H-benzo[b][1,4]oxazine (9d). Orange
solid. Mp: 161−162 °C dec. Yield: 71%. MS: m/z 288.8 (M + H).
1H NMR (CDCl3): δ 7.18−7.13 (m, 7H), 6.99−6.75 (m, 8H), 5.44
(d, J = 3.2 Hz, 1H), 4.70 (d, J = 3 Hz, 1H).
Synthesis of 2-Nitrophenoxy Ketones 8a−h. General Procedure.
To a solution of 2-nitrophenol (14 mmol) in anhydrous acetone (10
mL) were added K2CO3 (15 mmol) and the appropriate halo ketone
(15 mmol). After being stirred vigorously at room temperature until
the starting material disappeared as determined by thin-layer
chromatography (TLC), the reaction mixture was filtered and the
filtrate evaporated under reduced pressure. The residue was dissolved
in ethyl acetate (150 mL) and washed with water (30 mL). The
organic layer was dried over sodium sulfate and concentrated under
vacuum. The residue was purified by column chromatography (SiO2)
using ethyl acetate/petroleum ether as the eluent or by crystallization
to yield the desired compound.
3,4-Dihydro-7-methyl-3-phenyl-2H-benzo[b][1,4]oxazine (9e).
1
White solid. Mp: 88 °C. Yield: 77%. MS: m/z 226.3 (M + H). H
NMR (DMSO-d6): δ 7.41−7.33 (m, 5H), 6.62−6.50 (m, 3H), 6.03
(br s, 1H), 4.48 (m, 1H), 4.17 (m, 1H), 3.91−3.82 (m, 1H), 2.13 (s,
3H).
3,4-Dihydro-3-(4-methylphenyl)-2H-benzo[b][1,4]oxazine (9f).
Colorless oil. Yield: 83%. MS: m/z 226.1 (M + H). 1H NMR
(DMSO-d6): δ 7.29 (dd, J = 8 Hz, 2H), 7.17 (dd, J = 8 Hz, 2H), 6.71−
6.66 (m, 3H), 6.54−6.48 (m, 1H), 6.20 (br s, 1H), 4.39 (m, 1H),
4.21−4.14 (m, 2H), 3.89−3.80 (m, 1H), 2.30 (s, 3H).
3,4-Dihydro-3-cyclopropyl-2H-benzo[b][1,4]oxazine (9g). Yellow
1-(2-Nitrophenoxy)propan-2-one (8a). White solid. Mp: 72 °C.
Yield: 72%. MS: m/z 196.0 (M + H). 1H NMR (DMSO-d6): δ 7.91−
7.86 (m, 1H), 7.61−7.57 (m, 1H), 7.24−7.13 (m, 2H), 5.05 (s, 2H),
2.17 (s, 3H).
1
oil. Yield: 38%. MS: m/z 176.5 (M + H). H NMR (DMSO-d6): δ
2-(2-Nitrophenoxy)-1-phenylethanone (8b). Yellow solid. Mp:
116−117 °C. Yield: 71%. MS: m/z 258.2 (M + H). 1H NMR
6.65−6.59 (m, 3H), 6.45−6.41 (m, 1H), 5.80 (br s, 1H), 4.19−4.15
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(m, 1H), 3.56−3.81 (m, 1H), 2.58−2.57 (m, 1H), 0.78−0.76 (m, 1H)
0.48−0.32 (m, 4H).
3,4-Dihydro-3-propyl-2H-benzo[b][1,4]oxazine (9i). Yellow oil.
Yield: 54%. MS: m/z 178.4 (M + H). 1H NMR (DMSO-d6): δ
6.66−6.58 (m, 3H), 6.46−6.42 (m, 1H), 5.71 (br s, 1H), 4.14−4.10
(m, 1H), 3.72−3.67 (m, 1H), 3.25−3.24 (m, 1H), 1.41−1.37 (m, 4H),
0.91 (t, J = 6.8 Hz, 3H).
3,4-Dihydro-6-methyl-3-propyl-2H-benzo[b][1,4]oxazine (9j).
Yellow oil. Yield: 81%. MS: m/z 192.3 (M + H). 1H NMR
(CDCl3): δ 6.68 (m, 1H), 6.48−6.42 (m, 2H), 4.22−4.16 (m, 1H),
3.86−3.77 (m, 1H), 3.67 (br s, 1H), 3.40−3.35 (m, 1H), 2.21 (s, 3H),
1.49−1.41 (m, 4H), 1.01−0.94 (m, 3H).
Synthesis of Diethyl 2-[(2,3-Dihydro-2/3-substituted-benzo[b]-
[1,4]oxazin-4-yl)methylene]malonates 10a−g,i,j). General Proce-
dure. A mixture of benzo[1,4]oxazine 9a−g,i,j (4.7 mmol) and diethyl
(ethoxymethylene)malonate (4.7 mmol) was heated with stirring at
140 °C for 2 h. The solvent was removed under reduced pressure, and
cold diethyl ether was added to form a precipitate that was filtered and
subsequently purified by column chromatography (SiO2) using ethyl
acetate/petroleum ether (0.5:9.5).
4.30−4.07 (m, 6H), 3.82 (m, 1H), 2.27 (s, 3H), 1.63−1.59 (m, 4H),
1.32−1.22 (m, 6H), 0.92 (t, J = 7 Hz, 3H).
Synthesis of Ethyl 3,7-Dihydro-2/3-substituted-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxylates 11a−g,i,j. General Proce-
dure. A mixture of diethyl ester 10a−g,i,j (0.49 g, 1.5 mmol) and
poly(phosphoric acid) (3 g) was heated at 140 °C for 1 h. The mixture
was poured into ice and water to form a precipitate that was filtered
and washed with cold water.
Ethyl 3,7-Dihydro-3-methyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylate (11a). White solid. Mp: 189 °C dec. Yield:
95%. MS: m/z 274.3 (M + H). 1H NMR (DMSO-d6): δ 8.65 (s, 1H),
7.80−7.75 (m, 1H), 7.40−7.31 (m, 2H), 4.79−4.63 (m, 1H), 4.42−
4.34 (m, 2H), 4.25 (q, J = 7.2 Hz, 2H), 1.41 (d, J = 6.6 Hz, 3H), 1.29
(t, J = 7.2 Hz, 3H).
Ethyl 3,7-Dihydro-3-phenyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylate (11b). White solid. Mp: 239 °C. Yield:
87%. MS: m/z 336.3 (M + H). 1H NMR (DMSO-d6): δ 8.55 (s, 1H),
7.87−7.82 (m, 1H), 7.43−7.28 (m, 5H), 7.13−7.08 (m, 2H), 5.91 (m,
1H), 4.76−4.64 (m, 2H), 4.19 (q, J = 6.2 Hz, 2H), 1.24 (t, J = 7 Hz,
3H).
Ethyl 3,7-Dihydro-2-methyl-7-oxo-3-phenyl-2H-[1,4]oxazino-
Diethyl 2-((2,3-Dihydro-3-methylbenzo[b][1,4]oxazin-4-yl)-
methylene)malonate (10a). Pale yellow oil. Yield: 73%. MS: m/z
320.1 (M + H). 1H NMR (DMSO-d6): δ 7.71 (s, 1H), 7.21−6.95 (m,
4H), 4.21−3.87 (m, 7H), 1.26−1.16 (m, 9H).
[2,3,4-ij]quinoline-6-carboxylate (11c). White solid. Mp: 243−244
1
°C. Yield: 81%. MS: m/z 350.2 (M + H). H NMR (DMSO-d6): δ
8.53 (s, 1H), 7.84−7.83 (m, 1H), 7.43−7.34 (m, 5H), 7.11−7.07 (m,
2H), 5.73 (d, J = 3 Hz, 1H), 4.82−4.78 (m, 1H), 4.21−4.16 (m, 2H),
1.27−1.18 (m, 6H).
Ethyl 3,7-Dihydro-7-oxo-2,3-diphenyl-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylate (11d). White solid. Mp: 289−290 °C dec.
Yield: 75%. MS: m/z 412.1 (M + H). 1H NMR (DMSO-d6): δ 8.63 (s,
1H), 7.93−7.90 (m, 1H), 7.53−7.49 (m, 2H), 7.32−7.13 (m, 8H),
6.66 (d, J = 6.4 Hz, 2H), 6.15−5.95 (m, 2H), 4.22−4.17 (m 2H), 1.25
(t, J = 7.2 Hz, 3H).
Diethyl 2-((2,3-Dihydro-3-phenylbenzo[b][1,4]oxazin-4-yl)-
methylene)malonate (10b). White solid. Mp: 103−104 °C. Yield:
1
81%. MS: m/z 382.2 (M + H). H NMR (CDCl3): δ 8.12 (s, 1H)
7.38−6.87 (m, 9H), 4.55−3.93 (m, 6H) 3.42−3.21 (m, 1H), 1.26
(t, J = 7 Hz, 3H), 0.90 (t, J = 7.2 Hz, 3H).
Diethyl 2-((2,3-Dihydro-2-methyl-3-phenylbenzo[b][1,4]oxazin-
4-yl)methylene)malonate (10c). White solid. Mp: 130 °C. Yield:
89%. MS: m/z 396.2 (M + H). 1H NMR (DMSO-d6): δ 7.85 (s, 1H),
7.33−7.27 (m, 4H), 7.10−6.93 (m, 5H), 5.02 (d, J = 2.6 Hz, 1H),
4.60−4.43 (m, 1H), 4.11−3.95 (m, 3H), 3.64−3.61 (m, 1H), 1.20−
1.13 (m, 6H), 0.93 (t, J = 7.2 Hz, 3H).
Diethyl 2-((2,3-Dihydro-2,3-diphenylbenzo[b][1,4]oxazin-4-yl)-
methylene)malonate (10d). White solid. Mp: 114 °C. Yield: 96%.
MS: m/z 458.2 (M + H). 1H NMR (DMSO-d6): δ 7.96 (s, 1H), 7.42−
7.30 (m, 4H), 7.18−7.04 (m, 8H), 6.43−6.40 (m, 2H), 5.69 (d, J = 2.6
Hz, 1H), 5.34 (d, J = 2.8 Hz, 1H), 4.14−4.10 (m, 2H), 3.98−3.81 (m,
1H), 3.56−3.41 (m, 1H), 1.18 (t, J = 7.2 Hz, 3H), 0.87 (t, J = 7.2 Hz,
3H).
Diethyl 2-((2,3-Dihydro-7-methyl-3-phenylbenzo[b][1,4]oxazin-
4-yl)methylene)malonate (10e). White solid. Mp: 105 °C. Yield:
92%. MS: m/z 396.2 (M + H). 1H NMR (DMSO-d6): δ 7.93 (s, 1H),
7.30−7.15 (m, 6H), 6.87−6.82 (m, 1H), 6.64−6.63 (m, 1H), 5.14 (m,
1H), 4.72−4.65 (m, 1H), 4.36−4.29 (m, 1H), 4.11 (q, J = 6.2 Hz,
2H), 3.88−3.79 (m, 1H), 3.32−3.23 (m, 1H), 2.20 (s, 3H), 1.17 (t, J =
7 Hz, 3H), 0.77 (t, J = 7 Hz, 3H).
Ethyl 3,7-Dihydro-9-methyl-7-oxo-3-phenyl-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxylate (11e). White solid. Mp: 199 °C
1
dec. Yield: 79%. MS: m/z 350.0 (M + H). H NMR (DMSO-d6): δ
8.49 (s, 1H), 7.63−7.62 (m, 1H), 7.36−7.32 (m, 3H), 7.13−7.04 (m,
3H), 5.87 (m, 1H), 4.72−4.59 (m 2H), 4.16 (q, J = 6.4 Hz, 2H), 2.38
(s, 3H), 1.21 (t, J = 7 Hz, 3H).
Ethyl 3,7-Dihydro-7-oxo-3-(4-methylphenyl)-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxylate (11f). White solid. Mp: 183 °C.
Yield: 83%. MS: m/z 350.2 (M + H). 1H NMR (DMSO-d6): δ 8.50 (s,
1H), 7.86−7.81 (m, 1H), 7.43−7.27 (m, 2H), 7.18 (d, J = 7.8 Hz,
2H), 7.00 (d, J = 8 Hz, 2H), 5.84 (m, 1H), 4.72−4.61 (m, 2H), 4.18
(q, J = 7 Hz, 2H), 2.27 (s, 3H), 1.22 (t, J = 7.2 Hz, 3H).
Ethyl 3-Cyclopropyl-3,7-dihydro-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylate (11g). White solid. Mp: 190 °C. Yield:
77%. MS: m/z 300.3 (M + H). 1H NMR (DMSO-d6): δ 8.70 (s, 1H),
7.82−7.76 (m, 1H), 7.37−7.32 (m, 2H), 4.58−4.31 (m, 2H), 4.24 (q, J
= 7.2 Hz, 2H), 3.97 (m, 1H), 1.29 (t, J = 7.2 Hz, 3H), 1.18 (m, 1H),
0.59 (m, 4H).
Diethyl 2-((2,3-Dihydro-3-(4-methylphenyl)benzo[b][1,4]oxazin-
4-yl)methylene)malonate (10f). White solid. Mp: 94 °C. Yield:
87%. MS: m/z 396.2 (M + H). 1H NMR (DMSO-d6): δ 7.94 (s, 1H),
7.33 (m, 1H), 7.08−6.99 (m, 6H), 6.83−6.78 (m, 1H), 5.13 (m, 1H),
4.78−4.62 (m, 1H), 4.38−4.31 (m, 1H), 4.11 (q, J = 6.2 Hz, 2H),
3.97−3.78 (m, 1H), 3.42−3.21 (m, 1H), 2.23 (s, 3H), 1.82 (t, J = 7.4
Hz, 3H), 0.81 (t, J = 7.4 Hz, 3H).
Ethyl 3,7-Dihydro-7-oxo-3-propyl-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylate (11i). White solid. Mp: 133 °C. Yield:
80%. MS: m/z 302.2 (M + H). 1H NMR (DMSO-d6): δ 8.60 (s, 1H);
7.79−7.74 (m, 1H), 7.36−7.30 (m, 2H), 4.60−4.54 (m, 2H), 4.28−
4.21 (m, 3H), 1.82−1.63 (m, 2H), 1.48−1.33 (m, 2H), 1.29 (d, J = 7.2
Hz, 3H), 0.89 (t, J = 7.2 Hz, 3H).
Ethyl 3,7-Dihydro-8-methyl-7-oxo-3-propyl-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxylate (11j). Pale white solid. Mp: 125−
127 °C. Yield: 95%. MS: m/z 316.3 (M + H). 1H NMR (DMSO-d6): δ
8.47 (s, 1H), 7.16−7.07 (m, 2H), 4.79 (m, 1H), 4.53−4.47 (m, 2H),
4.24−4.16 (m, 3H), 2.71 (s, 3H), 1.81−1.68 (m, 4H), 1.31−1.24 (m,
6H), 0.88 (t, J = 7.4 Hz, 3H).
Synthesis of Ethyl 3-(2,3-Difluorophenyl)-3-oxopropanoate, Ethyl
3-(2,3,4-Trifluorophenyl)-3-oxopropanoate, and Ethyl 3-(2,3,4,5-
Tetrafluorophenyl)-3-oxopropanoate (14a−c). Carbonyldiimidazole
(1.78 g, 11 mmol) was added to a solution of the requisite benzoic
acid (13a−c, 10 mmol) in anhydrous THF (50 mL). After the mixture
was stirred at room temperature for 6 h, the magnesium salt of ethyl
malonic acid half-ester (prepared by stirring monoethyl malonate (1.2
mL, 10 mmol) and magnesium ethoxide (0.57 g, 5 mmol) in
anhydrous THF (25 mL) for 1 h at room temperature) was added, and
Diethyl 2-((3-Cyclopropyl-2,3-dihydrobenzo[b][1,4]oxazin-4-yl)-
methylene)malonate (10g). Yellow oil. Yield: 92%. MS: m/z 346.1
1
(M + H). H NMR (DMSO-d6): δ 7.72 (s, 1H), 7.12−6.90 (m, 4H),
4.36−4.35 (m, 1H), 4.20−4.05 (m, 3H), 3.97−3.65 (m, 2H), 3.51−
3.47 (m, 1H), 1.19 (t, J = 7 Hz, 3H), 1.06 (t, J = 7 Hz, 3H), 0.98−0.83
(m, 2H), 0.53−0.43 (m, 3H).
Diethyl 2-((2,3-Dihydro-3-propylbenzo[b][1,4]oxazin-4-yl)-
methylene)malonate (10i). Yellow oil. Yield: 84%. MS: m/z 348.2
1
(M + H). H NMR (DMSO-d6): δ 7.70 (s, 1H), 7.14−6.89 (m, 4H),
4.35 (m, 1H), 4.17−3.78 (m, 6H), 1.48−1.05 (m, 10H), 0.85 (t, J =
7.4 Hz, 3H).
Diethyl 2-(2,3-Dihydro-6-methyl-3-propylbenzo[b][1,4]oxazin-4-
yl)methylene)malonate (10j). Yellow oil. Yield: 78%. MS: m/z 362.3
1
(M + H). H NMR (DMSO-d6): δ 7.84 (s, 1H), 6.88−6.78 (m, 3H),
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the mixture was stirred at room temperature overnight. The solvent
was removed under reduced pressure, and the residue was partitioned
between 5% hydrochloric acid (20 mL) and ether (50 mL). The
aqueous layer was further extracted with ether, and the combined
extracts were washed with aqueous saturated sodium bicarbonate
solution (10 mL) and dried. Evaporation of the solvent in vacuo led to
the desired compounds as clear oils (1.9 g, 79%), which were used
without further purification in the next step.
Synthesis of Ethyl 2-(2,3-Difluorobenzoyl)-3-((2-hydroxy-1-
alkylethyl)amino)acrylates 16a−g, Ethyl 2-(2,3,4-Trifluorobenzo-
yl)-3-((2-hydroxy-1-ethylethyl)amino)acrylate (16h), and Ethyl 2-
(2,3,4,5-Tetrafluorobenzoyl)-3-((2-hydroxy-1-ethylethyl)amino)-
acrylate (16i). A mixture of the appropriate ethyl 3-(2,3-
difluorophenyl)-, 3-(2,3,4-trifluorophenyl)-, or 3-(2,3,4,5-tetrafluoro-
phenyl)-3-oxopropanoate (14a−c, 7.7 mmol), triethyl orthoformate
(2.6 mL, 11.5 mmol), and acetic anhydride (4.6 mL, 45 mmol) was
stirred at 110−120 °C for 3 h. The reaction was concentrated under
reduced pressure to leave an oily residue, which was diluted with
toluene and concentrated. This process was repeated two more times
to afford the desired ethyl 2-(ethoxymethylene)-3-oxo-3-(2,3-difluor-
ophenyl)-, 3-(2,3,4-trifluorophenyl)-, or 3-(2,3,4,5-tetrafluorophenyl)-
propionate (15a−c) as an oil, which was used directly without further
purification.
To a solution of ethyl 2-(ethoxymethylene)-3-oxo-3-(2,3-
difluorophenyl)propionate (15a; 7.7 mmol) in dry methylene chloride
(20 mL) was added a solution of the desired (R,S)-, (R)-, or (S)-2-
amino-1-hydroxyalkane (11 mmol) in methylene chloride (5 mL)
dropwise under cooling with an ice bath. The whole mixture was
stirred at room temperature for 1 h and then concentrated in vacuo to
yield the requisite (R,S)-, (R)-, or (S)-ethyl 2-(2,3-difluorobenzoyl)-3-
((2-hydroxy-1-alkylethyl)amino)acrylates 16a−g as oils, which were
used in the next step without further purification.
In identical fashion, ethyl 2-(ethoxymethylene)-3-oxo-3-(2,3,4-
trifluorophenyl)propionate (15b) or ethyl 2-(ethoxymethylene)-3-
oxo-3-(2,3,4,5-tetrafluorophenyl)propionate (15c) was reacted with 2-
amino-1-hydroxybutane to afford the desired ethyl 2-(2,3,4-trifluor-
obenzoyl)-3-((2-hydroxy-1-ethylethyl)amino)acrylate (16h) and ethyl
2-(2,3,4,5-tetrafluorobenzoyl)-3-((2-hydroxy-1-ethylethyl)amino)-
acrylate (16i) as oils, which were used in the next step without further
purification.
Synthesis of Ethyl 3,7-Dihydro-7-oxo-3-substituted-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxylates 17a−i. General Proce-
dure. A mixture of the requisite ethyl 2-(2,3-difluorobenzoyl)-, 2-
(2,3,4-trifluorobenzoyl)-, or 2-(2,3,4,5-tetrafluorobenzoyl)-3-((2-hy-
droxy-1-substituted-ethyl)amino)acrylate (16a−i, 5.5 mmol), K2CO3
(2.2 g, 16 mmol), and anhydrous DMF (30 mL) was stirred at 130−
140 °C for 7 h. After cooling, ethyl acetate (50 mL) was added to the
reaction mixture. The organic phase was washed with water, dried,
filtered, and concentrated. The residue was purified by flash
chromatography, eluting with ethyl acetate/petroleum ether (7:3) to
afford the desired product as a white solid.
(R)-Ethyl 3,7-Dihydro-3-methyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylate ((R)-17a) and (S)-Ethyl 3,7-Dihydro-3-
methyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylate ((S)-
17a). The NMR data and melting points are identical to those
reported for compound 11a.
(R,S)-Ethyl 3,7-Dihydro-3-ethyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylate (17b), (R)-Ethyl 3,7-Dihydro-3-ethyl-7-
oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylate ((R)-17b),
and (S)-Ethyl 3,7-Dihydro-3-ethyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylate ((S)-17b). White solid. Mp: 157 °C dec.
Yield: 86%. MS: m/z 288.3 (M + H). 1H NMR (DMSO-d6): δ 8.63 (s,
1H), 7.79−7.44 (m, 1H), 7.35−7.30 (m, 2H), 4.61−4.49 (m, 2H),
4.32−4.18 (m, 3H), 1.75−1.72 (m, 2H), 1.28 (t, J = 7 Hz, 3H), 0.93
(t, J = 7.6 Hz, 3H).
8.60 (s, 1H), 7.79−7.74 (m, 1H), 7.39−7.29 (m, 2H), 4.75 (d, J = 10.8
Hz, 1H), 4.32−4.18 (m, 4H), 2.07−1.91 (m, 1H), 1.28 (t, J = 7.2 Hz,
3H), 1.02 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H).
(R)-(+)-Ethyl 3,7-Dihydro-3-isobutyl-7-oxo-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxylate ((R)-17d). Colorless oil. Yield: 54%. MS:
1
m/z 316.3 (M + H). H NMR (CDCl3): δ 8.33 (s, 1H), 8.08−8.04
(m, 1H), 7.32−7.21 (m, 2H), 4.45−4.17 (m, 4H), 1.95−1.51 (m, 4H),
1.22 (m, 3H), 1.05 (t, J = 6.4 Hz, 3H), 0.98 (d, J = 6 Hz, 3H).
(R)-Ethyl 3,7-Dihydro-3-phenyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylate ((R)-17e) and (S)-Ethyl 3,7-Dihydro-3-
phenyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylate ((S)-
17e). The NMR data and melting points are identical to those
reported for compound 11b.
(R)-Ethyl 3,7-Dihydro-3-benzyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylate ((R)-17f). White solid. Mp: 179−180 °C.
Yield: 57%. MS: m/z 350.4 (M + H). 1H NMR (DMSO-d6): δ 8.74 (s,
1H), 8.07−8.03 (m, 1H), 7.74−7.39 (m, 2H), 7.23−7.16 (m, 5H),
5.33−5.29 (m, 2H), 4.25 (q, J = 6.8 Hz, 2H), 3.83 (m, 2H), 3.26 (m,
1H), 1.30 (t, J = 7.2 Hz, 3H).
Ethyl 3,7-Dihydro-3,3-dimethyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylate (17g). Yellow solid. Mp: 216−217 °C.
1
Yield: 28%. MS: m/z 289.3 (M + H). H NMR (CDCl3): δ 8.61 (s,
1H), 8.11−8.07 (m, 1H), 7.36−7.23 (m, 2H), 4.40 (q, J = 7.2 Hz,
2H), 4.12 (s, 2H), 1.62 (s, 6H), 1.42 (t, J = 7.2 Hz, 3H).
Ethyl 3,7-Dihydro-3-ethyl-10-fluoro-7-oxo-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxylate (17h). White solid. Mp: 190−191 °C dec.
Yield: 40%. MS: m/z 306.1 (M + H). 1H NMR (DMSO-d6): δ 8.66 (s,
1H), 7.82−7.70 (m, 1H), 7.45−7.31 (m, 1H), 4.79−4.54 (m 2H),
4.40−4.31 (m, 1H), 4.23 (q, J = 7 Hz, 2H), 1.91−1.83 (m, 2H), 1.28
(t, J = 7 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H).
Ethyl 3,7-Dihydro-3-ethyl-9,10-difluoro-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxylate (17i). White solid. Mp: 190−191
°C dec. Yield: 40%. MS: m/z 324.2 (M + H). 1H NMR (DMSO-d6): δ
8.68 (s, 1H), 7.71−7.57 (m, 1H), 4.79−4.54 (m 2H), 4.40−4.33 (m,
1H), 4.23 (q, J = 7 Hz, 2H), 1.82−1.63 (m, 2H), 1.28 (t, J = 7 Hz,
3H), 0.93 (t, J = 7.4 Hz, 3H).
Synthesis of 3,7-Dihydro-2/3-substituted-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxylic Acids 12a−g,i,j and 18a−i. General
Procedure. A solution of the requisite ethyl 3,7-dihydro-2-substituted-
3-substituted-7-oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylate
(11a−g,i,j, 17a−i, 7.4 mmol), NaOH 10% (10 mL), and methanol (90
mL) was heated at 80 °C for 1 h. The solvent was evaporated under
reduced pressure, and the residue was acidified with 10% HCl at 0 °C.
The precipitate was collected by filtration to give the desired product
as a white solid.
(R,S)-3,7-Dihydro-3-methyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid (12a), (R)-3,7-Dihydro-3-methyl-7-oxo-
2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic Acid ((R)-18a), and
(S)-3,7-Dihydro-3-methyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-
6-carboxylic Acid ((S)-18a). White solid. Mp: 240 °C dec. Yield: 76%.
1
MS: m/z 246.1 (M + H). H NMR (DMSO-d6): δ 15.23 (br s, 1H),
9.05 (s, 1H), 7.95−7.91 (m, 1H), 7.60−7.47 (m, 2H), 4.99−4.84 (m,
1H), 4.50−4.42 (m, 2H), 1.46 (d, J = 7 Hz, 3H).
(R,S)-3,7-Dihydro-3-phenyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid (12b), (R)-3-Phenyl-3,7-dihydro-7-oxo-
2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic Acid ((R)-18e), and
(S)-3-Phenyl-3,7-dihydro-7-oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-
6-carboxylic Acid ((S)-18e). White solid. Mp: 284 °C. Yield: 90%. MS:
1
m/z 308.4 (M + H). H NMR (DMSO-d6): δ 15.10 (br s, 1H), 8.90
(s, 1H), 8.02−7.98 (m, 1H),7.64−7.37 (m, 5H), 7.17−7.12 (m, 2H),
6.10 (m, 1H), 4.85−4.83 (m, 1H), 4.73−4.70 (m, 1H).
3,7-Dihydro-2-methyl-7-oxo-3-phenyl-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid (12c). White solid. Mp: 291−292 °C dec.
1
Yield: 82%. MS: m/z 322.4 (M + H). H NMR (DMSO-d6): δ 15.02
(br s, 1H), 8.93 (s, 1H), 8.02−7.98 (m, 1H), 7.61−7.56 (m, 2H),
7.38−7.34 (m, 3H), 7.11−7.09 (m, 2H), 5.93−5.91 (m, 1H), 4.96−
4.84 (m, 1H), 2.03 (m, 3H).
(R,S)-Ethyl 3,7-Dihydro-3-isopropyl-7-oxo-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxylate (17c), (R)-Ethyl 3,7-Dihydro-3-isopropyl-
7-oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylate ((R)-17c),
and (S)-(−)-Ethyl 3,7-Dihydro-3-isopropyl-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxylate ((S)-17c). White solid. Mp: 142
3,7-Dihydro-7-oxo-2,3-diphenyl-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid (12d). White solid. Mp: >300 °C. Yield:
1
86%. MS: m/z 384.2 (M + H). H NMR (DMSO-d6): δ 15.03 (br s,
1H), 8.73 (s, 1H), 8.02−7.99 (m, 1H), 7.55−7.51 (m, 2H), 7.29−7.12
(m, 8H), 6.71−6.67 (m, 2H), 6.20 (m, 1H), 5.95 (m, 1H).
1
°C. Yield: 71%. MS: m/z 302.2 (M + H). H NMR (DMSO-d6): δ
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3,7-Dihydro-9-methyl-7-oxo-3-phenyl-2H-[1,4]oxazino[2,3,4-ij]-
(br s, 1H), 9.01 (s, 1H), 7.71−7.65 (m, 1H), 4.84−4.76 (m, 2H),
4.44−4.38 (m, 1H), 1.84−1.76 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H).
Synthesis of N-Cycloalkyl- or N,N-Diisopropyl-3,7-dihydro-2/3-
substituted-7-oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxa-
mides 19−21, 24, 27, 29, 31, 32, 34, 35, 41−43, 46, 48, 49, (R)-31,
(S)-31, (R)-35, and (S)-35. General Procedure. To a solution of the
appropriate 3,7-dihydro-7-oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-
carboxylic acid (12a−c,e−g,i,j, (R)-18a, (S)-18a, or 18b, 0.6 mmol)
in DMF (5 mL) were added EDC (0.6 mmol), HOBt (0.6 mmol), and
finally the desired amine (0.9 mmol). The reaction mixture was stirred
at room temperature for 6 h. The solvent was evaporated, and the
residue was purified by column chromatography (SiO2) to yield a
white solid.
N-Cyclopentyl-3,7-dihydro-7-oxo-3-phenyl-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (19). White solid. Mp: 237 °C.
Yield: 64%. MS: m/z 375.2 (M + H). 1H NMR (DMSO-d6): 10.00 (br
d, J = 7.2 Hz, 1H), 8.67 (s, 1H), 7.96−7.91 (m, 1H), 7.46−7.36 (m,
5H), 7.14−7.09 (m, 2H), 6.01 (m, 1H), 4.82−4.59 (m, 2H), 4.25−
4.16 (m, 1H), 1.98−1.30 (m, 8H). Anal. (C23H22N2O3) C, H, N.
N-Cyclohexyl-3,7-dihydro-7-oxo-3-phenyl-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxamide (20). White solid. Mp: 207 °C. Yield:
58%. MS: m/z 389.5 (M + H). 1H NMR (DMSO-d6): δ 10.01 (br d, J
= 7.6 Hz, 1H), 8.69(s, 1H), 7.96−7.91 (m, 1H), 7.49−7.08 (m, 7H),
6.01 (m, 1H), 4.76 (m, 1H), 4.65 (m, 1H), 3.95 (m, 1H), 1.88−1.30
(m, 10H). Anal. (C24H24N2O3) C, H, N.
quinoline-6-carboxylic Acid (12e). White solid. Mp: >300 °C. Yield:
1
86%. MS: m/z 322.5 (M + H). H NMR (DMSO-d6): δ 15.21 (br s,
1H), 8.85 (s, 1H), 7.81−7.79 (m, 1H), 7.39−7.35 (m, 4H), 7.15−7.10
(m, 2H), 6.08 (m, 1H), 4.83−4.69 (m, 2H), 2.48 (s, 3H).
3,7-Dihydro-7-oxo-3-(4-methylphenyl)-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid (12f). White solid. Mp: 294 °C dec.
1
Yield: 90%. MS: m/z 322.2 (M + H). H NMR (DMSO-d6): δ 15.08
(br s, 1H), 8.82 (s, 1H), 8.01−7.96 (m, 1H), 7.63−7.47 (m, 2H), 7.20
(d, J = 8 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.02 (m, 1H), 4.80−4.69
(m, 2H), 2.28 (s, 3H).
3-Cyclopropyl-3,7-dihydro-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid (12g). White solid. Mp: 227−228 °C.
1
Yield: 76%. MS: m/z 272.1 (M + H). H NMR (DMSO-d6): δ 15.17
(br s, 1H), 9.06 (s, 1H), 7.95−7.90 (m, 1H), 7.59−7.46 (m, 2H),
4.64−4.46 (m, 2H), 4.14−4.07 (m, 1H), 1.38−1.11 (m, 1H), 0.79−
0.52 (m, 4H).
3,7-Dihydro-7-oxo-3-propyl-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-
carboxylic Acid (12i). White solid. Mp: 213 °C. Yield: 82%. MS: m/z
1
274.3 (M + H). H NMR (DMSO-d6): δ 15.21 (br s, 1H), 9.00 (s,
1H), 7.95−7.90 (m, 1H), 7.60−7.46 (m, 2H), 4.85 (m, 1H), 4.69−
4.63 (m, 1H), 4.42−4.41 (m, 1H), 1.98−1.11 (m, 4H), 0.88 (t, J = 7
Hz, 3H).
3,7-Dihydro-8-methyl-7-oxo-3-propyl-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid (12j). White solid. Mp: 205 °C dec.
1
Yield: 78%. MS: m/z 289.2 (M + H). H NMR (DMSO-d6): δ 15.53
N-Cycloheptyl-3,7-dihydro-7-oxo-3-phenyl-2H-[1,4]oxazino-
(br s, 1H), 8.91 (s, 1H), 7.36−7.24 (m, 2H), 4.84−4.77 (m, 1H), 4.59
(d, J = 11.4 Hz, 1H), 4.29 (d, J = 10 Hz, 1H), 2.80 (s, 3H), 1.95−1.20
(m, 4H), 0.87 (t, J = 7.4 Hz, 3H).
[2,3,4-ij]quinoline-6-carboxamide (21). White solid. Mp: 169 °C.
1
Yield: 55%. MS: m/z 403.5 (M + H). H NMR (DMSO-d6): δ 10.03
(br d, J = 7.8 Hz, 1H), 8.66 (s, 1H), 7.96−7.92 (m, 1H), 7.46−7.34
(m, 5H), 7.14−7.09 (m, 2H), 6.01 (m, 1H), 4.83−4.58 (m, 2H), 3.95
(m, 1H), 1.95−1.35 (m, 12H). Anal. (C25H26N2O3) C, H, N.
N-Cyclohexyl-3,7-dihydro-2-methyl-7-oxo-3-phenyl-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (24). White solid. Mp: 254
(R,S)-3,7-Dihydro-3-ethyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid (18b), (R)-3,7-Dihydro-3-ethyl-7-oxo-
2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic Acid ((R)-18b), and
(S)-3,7-Dihydro-3-ethyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-
carboxylic Acid ((S)-18b). White solid. Mp: 190 °C. Yield: 78%. MS:
1
°C. Yield: 61%. MS: m/z 403.3 (M + H). H NMR (DMSO-d6): δ
1
m/z 260.1 (M + H). H NMR (DMSO-d6): δ 15.21 (br s, 1H), 9.03
9.97 (br d, J = 7.4 Hz, 1H), 8.68 (s, 1H), 7.97−7.93 (m, 1H), 7.50−
7.35 (m, 5H), 7.10 (m, 2H), 5.88 (m, 1H), 4.89−4.84 (m, 1H), 3.91
(m, 1H), 1.97−1.40 (m, 10H), 1.33 (d, J = 6.2 Hz, 3H). Anal.
(C25H26N2O3) C, H, N.
N-Cyclohexyl-3,7-dihydro-7-oxo-3-p-tolyl-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxamide (27). White solid. Mp: 237 °C. Yield:
64%. MS: m/z 403.4 (M + H). 1H NMR (DMSO-d6): δ 9.99 (br d, J =
7.4 Hz, 1H), 8.62 (s, 1H), 7.95−7.90 (m, 1H), 7.49−7.34 (m, 2H),
7.19 (d, J = 8.00 Hz, 2H), 7.02 (d, J = 8.00 Hz, 2H), 5.94 (m, 1H),
4.72−4.63 (m, 2H), 3.89 (m, 1H), 2.28 (s, 3H), 1.92−1.26 (m, 10H).
Anal. (C25H26N2O3) C, H, N.
(s, 1H), 7.94−7.90 (m, 1H), 7.59−7.45 (m, 2H), 4.81−4.65 (m, 2H),
4.42−4.35 (m, 1H), 1.80−1.75 (m, 2H), 0.93 (t, J = 6.6 Hz, 3H).
(R,S)-3,7-Dihydro-3-isopropyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid (18c), (R)-3,7-Dihydro-3-isopropyl-7-
oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic Acid ((R)-18c),
and (S)-3,7-Dihydro-3-isopropyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid ((S)-18c). White solid. Mp: 210 °C.
1
Yield: 86%. MS: m/z 274.2 (M + H). H NMR (DMSO-d6): δ 14.99
(br s, 1H), 8.99 (s, 1H), 8.31−7.44 (m, 3H), 5.28 (m, 1H), 4.99−3.91
(m, 3H), 2.01 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H), 1.09 (d, J = 6.6 Hz,
3H).
N-Cyclohexyl-3,7-dihydro-9-methyl-7-oxo-3-phenyl-2H-[1,4]-
(R)-3,7-Dihydro-3-isobutyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (29). White solid. Mp: 286
quinoline-6-carboxylic Acid ((R)-18d). White solid. Mp: 131−132 °C.
1
1
°C. Yield: 66%. MS: m/z 403.5 (M + H). H NMR (DMSO-d6): δ
Yield: 80%. MS: m/z 288.3 (M + H). H NMR (DMSO-d6): δ 14.99
10.04 (br d, J = 7.8 Hz, 1H), 8.63 (s, 1H), 7.74 (s, 1H), 7.38−7.35 (m,
3H), 7.21 (s, 1H), 7.011−7.07 (m, 2H), 5.99 (m, 1H), 4.75−4.59 (m,
2H), 3.89 (m, 1H), 2.43 (s, 3H), 1.92−1.24 (m, 10H). Anal.
(C25H26N2O3) C, H, N.
N-Cyclohexyl-3,7-dihydro-3-methyl-7-oxo-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxamide (31). White solid. Mp: 245 °C. Yield:
58%. MS: m/z 327.2 (M + H). 1H NMR (DMSO-d6): δ 10.05 (br d, J
= 7.8 Hz, 1H), 8.82 (s, 1H), 7.89−7.85 (m, 1H), 7.46−7.33 (m, 2H),
4.82 (m, 1H), 4.45−4.36 (m, 2H), 3.81 (m, 1H), 1.88−1.28 (m, 13H).
Anal. (C19H22N2O3) C, H, N.
N-Cycloheptyl-3,7-dihydro-3-methyl-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (32). White solid. Mp: 208 °C.
Yield: 47%. MS: m/z 403.3 (M + H). 1H NMR (CDCl3): δ 10.05 (br
d, J = 7.4 Hz, 1H), 8.71 (s, 1H), 8.09−8.04 (m, 1H), 7.41−7.24 (m,
2H), 4.82 (m, 1H), 4.35−4.26 (m, 2H), 4.21−4.08 (m, 1H), 2.04−
1.97 (m, 2H), 1.71−1.57 (m, 13H). Anal. (C20H24N2O3) C, H, N.
N-Cyclohexyl-3,7-dihydro-3-ethyl-7-oxo-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxamide (34). White solid. Mp: 175 °C. Yield:
69%. MS: m/z 341.3 (M + H). 1H NMR (DMSO-d6): δ 10.05 (br d, J
= 7.8 Hz, 1H), 8.80 (s, 1H), 7.89−7.84 (m, 1H), 7.46−7.32 (m, 2H),
4.67−4.60 (m, 2H), 4.36−4.30 (m, 1H), 3.99 (m, 1H), 1.87−1.28 (m,
12H), 0.92 (t, J = 7.4 Hz, 3H). Anal. (C20H24N2O3) C, H, N.
(br s, 1H), 8.62 (s, 1H), 8.11−8.06 (m, 1H), 7.52−7.26 (m, 2H),
4.82−4.21 (m, 3H), 1.99−1.38 (m, 3H), 1.07−0.95 (m, 6H).
(R)-3-Benzyl-3,7-dihydro-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid ((R)-18f). White solid. Mp: 239−240 °C.
1
Yield: 82%. MS: m/z 323.2 (M + H). H NMR (DMSO-d6): δ 15.08
(br s, 1H), 8.58 (s, 1H), 7.97−7.92 (m, 1H), 7.63−7.54 (m, 2H),
7.31−7.14 (m, 5H), 5.08 (m, 1H), 4.59−4.15 (m, 2H), 3.13−3.05 (m,
2H).
3,7-Dihydro-3,3-dimethyl-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid (18g). White solid. Mp: >300 °C. Yield:
68%. MS: m/z 260.1 (M + H). 1H NMR (CDCl3): δ 15.00 (br s, 1H),
8.88 (s, 1H), 8.15−8.10 (m, 1H), 7.53−7.36 (m, 2H), 4.18 (s, 2H),
1.67 (s, 6H).
3-Ethyl-10-fluoro-3,7-dihydro-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxylic Acid (18h). White solid. Mp: 230 °C. Yield:
1
65%. MS: m/z 278.1 (M + H). H NMR (DMSO-d6): δ 15.00 (br s,
1H), 9.01 (s, 1H), 7.97−7.90 (m, 1H), 7.64−7.54 (m, 1H), 4.84−4.75
(m, 2H), 4.64−4.39 (m, 1H), 1.85−1.73 (m, 2H), 0.94 (t, J = 7.4 Hz,
3H).
9,10-Difluoro-3,7-dihydro-3-ethyl-7-oxo-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxylic Acid (18i). White solid. Mp: 246−248 °C.
1
Yield: 50%. MS: m/z 296.3 (M + H). H NMR (DMSO-d6): δ 15.00
6619
dx.doi.org/10.1021/jm300763w | J. Med. Chem. 2012, 55, 6608−6623

Journal of Medicinal Chemistry
Article
N-Cycloheptyl-3,7-dihydro-3-ethyl-7-oxo-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxamide (35). White solid. Mp: 165 °C. Yield:
51%. MS: m/z 355.3 (M + H). 1H NMR (DMSO-d6): δ 10.09 (br d, J
= 7.8 Hz, 1H), 8.79 (s, 1H), 7.89−7.84 (m, 1H), 7.41−7.35 (m, 2H),
4.70−4.59 (m, 2H), 4.36−4.29 (m, 1H), 4.07−4.01 (m, 1H), 1.87−
1.56 (m, 14H), 0.92 (t, J = 7.2 Hz, 3H). Anal. (C21H26N2O3) C, H, N.
3,7-Dihydro-N,N-diisopropyl-3-ethyl-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (41). White solid. Mp: 187 °C.
7.45−7.32 (m, 2H), 4.70−4.60 (m, 2H), 4.36−4.29 (m, 1H), 4.07−
4.03 (m, 1H), 1.87−1.57 (m, 14H), 0.92 (t, J = 7.2 Hz, 3H). Anal.
(C21H26N2O3) C, H, N. [α]2D5 = −100 (c = 0.01, CHCl3).
Synthesis of N-Adamantyl-, N-Alkyl-, and N-Heteroaryl-3,7-
dihydro-2/3-substituted-7-oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-
carboxamides 22, 23, 25, 26, 28, 30, 33, 36−40, 44, 45, 50−54,
(R)-33, (S)-33, (R)-36, (S)-36, (R)-51, (S)-51, (R)-58, and (S)-59.
General Procedure. To a stirred solution of the respective carboxylic
acid (12a−g, i, j, 18a−i, 0. 2 mmol) in dry DMF
(3 mL) was added diisopropylethylamine (0.8 mmol). The resulting
solution was stirred at room temperature for 10 min before addition
of HBTU (o-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexa-
fluorophosphate; 0.3 mmol) and then stirred for another 3 h.
1-Aminoadamantane, 2-aminoadamantane (for 37), or 1-amino-3,5-
dimethyladamantane (for 45) (0.3 mmol) was then added, and the
solution was stirred for 16 h. DMF was evaporated under reduced
pressure, and the residue was dissolved in ethyl acetate and
successively washed with water-saturated sodium bicarbonate, water,
and brine. The organic phase was dried over anhydrous sodium sulfate,
evaporated, and finally purified by flash chromatography using ethyl
acetate/petroleum ether (8:2) as the eluent.
1
Yield: 36%. MS: m/z 336.2 (M + H). H NMR (DMSO-d6):8.06 (s,
1H), 7.74−7.69 (m, 1H), 7.27−7.23 (m, 2H), 4.63−4.21 (m, 3H),
3.75 (m, 1H), 3.50 (m, 1H), 1.80−1.42 (m, 2H), 1.42 (d, J = 6.4 Hz,
6H), 1.08 (m, 6H), 0.90 (t, J = 7.2 Hz, 3H). Anal. (C20H26N3O3) C,
H, N.
N-Cyclohexyl-3,7-dihydro-7-oxo-3-propyl-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxamide (42). White solid. Mp: 191 °C. Yield:
68%. MS: m/z 355.2 (M + H). 1H NMR (DMSO-d6): δ 10.04 (br d, J
= 8 Hz, 1H), 8.77 (s, 1H), 7.87−7.85 (m, 1H), 7.43−7.33 (m, 2H),
4.75−4.59 (m, 2H), 4.34−4.31 (m, 1H), 3.85 (m, 1H), 1.91−1.29 (m,
14H), 0.87 (t, J = 7.6 Hz, 3H). Anal. (C21H26N2O3) C, H, N.
N-Cycloheptyl-3,7-dihydro-7-oxo-3-propyl-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (43). White solid. Mp: >300 °C.
1
Yield: 46%. MS: m/z 369.6 (M + H). H NMR (DMSO-d6): δ 10.08
N-Adamant-1-yl-3,7-dihydro-7-oxo-3-phenyl-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (22). White solid. Mp: >300 °C.
(br d, J = 8 Hz, 1H), 8.75 (s, 1H), 7.89−7.84 (m, 1H), 7.45−7.31 (m,
2H), 4.74−4.57 (m, 2H), 4.34−4.29 (m, 1H), 4.04 (m, 1H), 3.98 (m,
1H), 1.89−1.29 (m, 15H), 0.88 (t, J = 7.2 Hz, 3H). Anal.
(C22H28N2O3) C, H, N.
1
Yield: 71%. MS: m/z 441.6 (M + H). H NMR (DMSO-d6): δ 9.89
(br s, 1H), 8.59 (s, 1H), 7.95−7.90 (m, 1H), 7.45−7.34 (m, 5H),
7.16−7.11 (m, 2H), 5.96 (m, 1H); 4.92−4.58 (m, 2H), 2.03 (s, 9H),
1.65 (s, 6H). Anal. (C28H28N2O3) C, H, N.
3,7-Dihydro-N-(3,5-dimethyladamant-1-yl)-7-oxo-3-phenyl-2H-
[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide (23). White solid.
Mp: 264 °C. Yield: 63%. MS: m/z 469.5 (M + H). 1H NMR
(DMSO-d6): 9.92 (br s, 1H), 8.56 (s, 1H), 7.95−7.90 (m, 1H), 7.45−
7.37 (m, 5H), 7.17−7.12 (m, 2H), 5.95 (m, 1H), 4.82−4.59 (m, 2H),
2.10−1.59 (m, 7H), 1.39−1.14 (m, 6H), 0.83 (s, 6H). Anal.
(C30H32N2O3) C, H, N.
N-Adamant-1-yl-3,7-dihydro-2-methyl-7-oxo-3-phenyl-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (25). White solid. Mp:
>300 °C. Yield: 71%. MS: m/z 455.6 (M + H). 1H NMR (DMSO-d6):
9.85 (br s, 1H), 8.62 (s, 1H), 7.95−7.91 (m, 1H), 7.52−7.33 (m, 5H),
7.11−7.06 (m, 2H), 5.82 (d, J = 2.8 Hz, 1H), 4.83−4.78 (m, 1H), 2.02
(s, 9H), 1.64 (s, 6H), 1.20 (d, J = 6.2 Hz, 3H). Anal. (C29H30N2O3) C,
H, N.
N-Adamant-1-yl-3,7-dihydro-7-oxo-2,3-diphenyl-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (26). White solid. Mp: 260
°C. Yield: 45%. MS: m/z 517.3 (M + H). 1H NMR (DMSO-d6): 9.86
(br s, 1H), 8.73 (s, 1H), 8.02−7.98 (m, 1H), 7.58−7.54 (m, 2H),
7.33−7.13(m, 8H), 6.71−6.66 (m, 2H), 6.25 (d, J = 2.8 Hz, 1H), 5.96
(d, J = 2.8 Hz, 1H), 2.04 (s, 9H), 1.65 (s, 6H). Anal. (C34H32N2O3) C,
H, N.
N-Cyclohexyl-3,7-dihydro-8-methyl-7-oxo-3-propyl-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (46). White solid. Mp: 213
1
°C. Yield: 55%. MS: m/z 369.4 (M + H). H NMR (DMSO-d6): δ
10.05 (br d, J = 7.8 Hz, 1H); 8.67 (s, 1H), 7.22−7.08 (m, 2H); 4.73−
4.65 (m, 1H); 4.56−4.50 (m, 1H); 4.26−4.19 (m, 1H); 3.82 (m, 1H);
2.78 (s, 3H); 1.88−1.19 (m, 14H); 0.87 (t, J = 7.4 Hz, 3H). Anal.
(C22H28N2O3) C, H, N.
N-Cyclohexyl-3-cyclopropyl-3,7-dihydro-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (48). White solid. Mp: 212 °C.
1
Yield: 63%. MS: m/z 353.2 (M + H). H NMR (DMSO-d6): δ 10.05
(br d, J = 8 Hz, 1H), 8.90 (s, 1H), 7.90−7.86 (m, 1H), 7.43−7.38 (m,
2H), 4.61−4.46 (m, 2H), 4.10−4.05 (m, 1H), 3.85 (m, 1H), 1.99−
1.32 (m, 11H), 0.70.65 (m, 4H). Anal. (C21H24N2O3) C, H, N.
N-Cycloheptyl-3-cyclopropyl-3,7-dihydro-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (49). White solid. Mp: 185 °C.
1
Yield: 52%. MS: m/z 367.6 (M + H). H NMR (DMSO-d6): δ 10.09
(br d, J = 8.4 Hz, 1H), 8.90 (s, 1H), 7.89−7.87 (m, 1H), 7.45−7.35
(m, 2H), 4.55−4.46 (m, 2H), 4.05−4.02 (m, 2H), 1.99−1.90 (m,
2H),1.69−1.41 (m, 10H), 1.30−1.19 (m, 1H), 0.75−0.54 (m, 4H).
Anal. (C22H26N2O3) C, H, N.
(R)-N-Cyclohexyl-3,7-dihydro-3-methyl-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide ((R)-31). White solid. Mp: 245 °C.
1
Yield: 62%. MS: m/z 327.2 (M + H). H NMR (DMSO-d6): δ 10.05
N-Adamant-1-yl-3,7-dihydro-7-oxo-3-(4-methylphenyl)-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (28). White solid. Mp: 171
(br d, J = 7.8 Hz, 1H), 8.82 (s, 1H), 7.89−7.85 (m, 1H), 7.46−7.33
(m, 2H), 4.82 (m, 1H), 4.45−4.36 (m, 2H), 3.81 (m, 1H), 1.88−1.28
(m, 13H). Anal. (C19H22N2O3) C, H, N. [α]2D5 = +81 (c = 0.01,
CH3OH).
1
°C. Yield: 59%. MS: m/z 455.8 (M + H). H NMR (DMSO-d6):
δ 9.89 (br s, 1H), 8.53 (s, 1H), 7.94−7.89 (m, 1H), 7.45−7.34 (m,
2H), 7.21 (d, J = 8.00 Hz, 2H), 7.06 (d, J = 8.00 Hz, 2H), 5.88 (m,
1H), 4.79−4.51 (m, 2H), 2.29 (s, 3H), 2.03 (s, 9H), 1.65 (s, 6H).
Anal. (C29H30N2O3) C, H, N.
(S)-N-Cyclohexyl-3,7-dihydro-3-methyl-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide ((S)-31). White solid. Mp: 245 °C.
1
Yield: 69%. MS: m/z 327.2 (M + H). H NMR (DMSO-d6): δ 10.05
N-Adamant-1-yl-3,7-dihydro-9-methyl-7-oxo-3-phenyl-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (30). White solid. Mp:
>300 °C. Yield: 48%. MS: m/z 455.4 (M + H). 1H NMR (DMSO-d6):
δ 9.93 (br s, 1H), 8.55 (s, 1H), 7.73 (s, 1H), 7.39−7.36 (m, 3H), 7.20
(s, 1H), 7.14−7.10 (m, 2H), 5.94 (m, 1H), 4.82−4.53 (m, 2H), 2.43
(s, 3H), 2.03 (s, 9H), 1.65 (s, 6H). Anal. (C29H30N2O3) C, H, N.
N-Adamant-1-yl-3,7-dihydro-3-methyl-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (33). White solid. Mp: 282 °C.
(br d, J = 7.8 Hz, 1H), 8.82 (s, 1H), 7.89−7.85 (m, 1H), 7.42−7.37
(m, 2H), 4.82 (m, 1H), 4.49−4.37 (m, 2H), 3.81 (m, 1H), 1.88−1.28
(m, 13H). Anal. (C19H22N2O3) C, H, N. [α]2D5 = −81 (c = 0.01,
CH3OH).
(R)-(+)-N-Cycloheptyl-3,7-dihydro-3-ethyl-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide ((R)-35). White solid. Mp:
165 °C. Yield: 72%. MS: m/z 355.3 (M + H). 1H NMR (DMSO-d6): δ
10.09 (br d, J = 7.8 Hz, 1H), 8.79 (s, 1H), 7.89−7.84 (m, 1H), 7.45−
7.35 (m, 2H), 4.71−4.60 (m, 2H), 4.36−4.29 (m, 1H), 4.07−4.01 (m,
1H), 1.87−1.56 (m, 14H), 0.92 (t, J = 7.2 Hz, 3H). Anal.
(C21H26N2O3) C, H, N. [α]2D5 = +100 (c = 0.01, CHCl3).
1
Yield: 55%. MS: m/z 379.3 (M + H). H NMR (DMSO-d6): δ 9.95
(br s, 1H), 8.77 (s, 1H), 7.88−7.84 (m, 1H), 7.45−7.33 (m, 2H), 4.82
(m, 1H), 4.52−4.23 (m, 2H), 2.07 (s, 9H), 1.67 (s, 6H), 1.42 (d, J =
6.8 Hz, 3H). Anal. (C23H26N2O3) C, H, N.
(S)-(−)-N-Cycloheptyl-3,7-dihydro-3-ethyl-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide ((S)-35). White solid. Mp:
165 °C. Yield: 54%. MS: m/z 355.0 (M + H). 1H NMR (DMSO-d6):
δ 10.09 (br d, J = 7.8 Hz, 1H), 8.79 (s, 1H), 7.89−7.84 (m, 1H),
N-Adamant-1-yl-3,7-dihydro-3-ethyl-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (36). White solid. Mp: 219 °C.
1
Yield: 66%. MS: m/z 393.5 (M + H). H NMR (DMSO-d6): δ 9.93
(br s, 1H), 8.74 (s, 1H), 7.88−7.83 (m, 1H), 7.41−7.34 (m, 2H),
6620
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4.65−4.59 (m, 2H), 4.39−4.12 (m, 1H), 2.07 (s, 9H), 1.72 (m, 2H),
1.67 (s, 6H), 0.92 (t, J = 7.4 Hz, 3H). Anal. (C24H28N2O3) C, H, N.
N-Adamant-2-yl-3,7-dihydro-3-ethyl-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (37). White solid. Mp: 240 °C.
Yield: 45%. MS: m/z 393.1 (M + H). 1H NMR (DMSO-d6): 10.61 (d,
J = 8.4 Hz, 1H), 8.81 (s, 1H), 7.92−7.87 (m, 1H), 7.46−7.32 (m, 2H),
4.70−4.60 (m, 2H), 4.36−4.30 (m, 1H), 4.13 (d, J = 8.4 Hz, 1H),
1.98−1.60 (m, 16H), 0.96 (t, J = 7.2 Hz 3H). Anal. (C24H28N2O3) C,
H, N.
3,7-Dihydro-3-ethyl-N-(5-methylhexan-2-yl)-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (38). White solid. Mp: 240
°C. Yield: 24%. MS: m/z 357.1 (M + H). 1H NMR (DMSO-d6): 10.61
(d, J = 8.4 Hz, 1H), 8.80 (s, 1H), 7.88−7.80 (m, 1H), 7.48−7.34 (m,
2H), 4.78−4.66 (m, 2H), 4.40−4.28 (m, 1H), 4.12−3.88 (m, 1H),
1.83−1.40 (m, 5H), 1.18−1.11 (m, 5H), 0.88−0.83 (m, 9H). Anal.
(C21H28N2O3) C, H, N.
4.75−4.64 (m, 2H), 4.39−4.32 (m, 1H), 2.06 (s, 9H), 1.82−1.74 (m,
2H), 1.67 (s, 6H), 0.93 (t, J = 7.6 Hz, 3H). Anal. (C24H27FN2O3) C,
H, N.
N-Adamant-1-yl-9,10-difluoro-3,7-dihydro-3-ethyl-7-oxo-2H-
[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide (54). White solid.
Mp: 262 °C. Yield: 50%. MS: m/z 429.3 (M + H). 1H NMR
(DMSO-d6): δ 9.78 (s, 1H), 8.79 (s, 1H), 7.77−7.68 (m, 1H), 4.82−
4.68 (m, 2H), 4.45−4.38 (m, 1H), 2.06 (s, 9H), 1.82−1.78 (m, 2H),
1.67 (s, 6H), 0.92 (t, J = 7.6 Hz, 3H). Anal. (C24H26FN2O3) C, H, N.
(R)-(+)-N-Adamant-1-yl-3,7-dihydro-3-methyl-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide ((R)-33). White solid. Mp:
282 °C. Yield: 56%. MS: m/z 379.3 (M + H). 1H NMR (DMSO-d6): δ
9.95 (br s, 1H), 8.77 (s, 1H), 7.88−7.84 (m, 1H), 7.45−7.33 (m, 2H),
4.82 (m, 1H), 4.52−4.23 (m, 2H), 2.07 (s, 9H), 1.67 (s, 6H), 1.42 (d,
J = 6.8 Hz, 3H). Anal. (C23H26N2O3) C, H, N. [α]2D5 = +69 (c = 0.01,
CDCl3).
(S)-(−)-N-Adamant-1-yl-3,7-dihydro-3-methyl-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide ((S)-33). White solid. Mp:
282 °C. Yield: 50%. MS: m/z 379.3 (M + H). 1H NMR (DMSO-d6): δ
9.95 (br s, 1H), 8.77 (s, 1H), 7.88−7.84 (m, 1H), 7.45−7.33 (m, 2H),
4.82 (m, 1H), 4.52−4.23 (m, 2H), 2.07 (s, 9H), 1.67 (s, 6H), 1.42 (d,
J = 6.8 Hz, 3H). Anal. (C23H26N2O3) C, H, N. [α]2D5 = −69 (c = 0.01,
CDCl3).
3,7-Dihydro-3-ethyl-7-oxo-N-pyridin-4-yl-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxamide (39). White solid. Mp: 280 °C. Yield:
1
44%. MS: m/z 336.2 (M + H). H NMR (DMSO-d6): 12.68 (br s,
1H), 9.00 (s, 1H), 8.50−8.47 (m, 2H), 7.97−7.93 (m, 1H), 7.74−7.70
(m, 2H), 7.54−7.39 (m, 2H), 4.83−4.64 (m, 2H), 4.42−4.31 (m, 1H),
1.96−1.65 (m, 2H), 0.95 (t, J = 7.6 Hz, 3H). Anal. (C19H17N3O3) C,
H, N.
(R)-(+)-N-Adamant-1-yl-3,7-dihydro-3-ethyl-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide ((R)-36). White solid. Mp:
219 °C. Yield: 69%. MS: m/z 393.1 (M + H). 1H NMR (DMSO-d6): δ
9.94 (br s, 1H), 8.75 (s, 1H), 7.88−7.83 (m, 1H), 7.41−7.34 (m, 2H),
4.65−4.59 (m, 2H), 4.39−4.12 (m, 1H), 2.07 (s, 9H), 1.72 (m, 2H),
1.67 (s, 6H), 0.92 (t, J = 7.4 Hz, 3H). Anal. (C24H28N2O3) C, H, N.
[α]2D5 = +142 (c = 0.01, CHCl3).
(S)-(−)-N-Adamant-1-yl-3,7-dihydro-3-ethyl-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide ((S)-36). White solid. Mp:
219 °C. Yield: 53%. MS: m/z 393.2 (M + H). 1H NMR (DMSO-d6): δ
9.94 (br s, 1H), 8.75 (s, 1H), 7.88−7.83 (m, 1H), 7.45−7.31 (m, 2H),
4.65−4.59 (m, 2H), 4.39−4.12 (m, 1H), 2.07 (s, 9H), 1.77 (m, 2H),
1.68 (s, 6H), 0.92 (t, J = 7.4 Hz, 3H). Anal. (C24H28N2O3) C, H, N.
[α]2D5 = −142 (c = 0.01, CHCl3).
(R)-(+)-N-Adamant-1-yl-3,7-dihydro-3-isopropyl-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide ((R)-51). White solid. Mp:
212 °C. Yield: 47%. MS: m/z 407.5 (M + H). 1H NMR (DMSO-d6): δ
9.90 (br s, 1H), 8.68 (s, 1H), 7.86−7.81 (m, 1H), 7.42−7.28 (m, 2H),
4.79−4.73 (m, 1H), 4.42−4.24 (m, 2H), 2.04 (s, 10H), 1.92 (m, 1H),
1.65 (s, 6H), 0.98 (d, J = 6.6 Hz, 3H), 0.76 (d, J = 6.6 Hz, 3H). Anal.
(C25H30N2O3) C, H, N. [α]2D5 = +42 (c = 0.01, CH3OH).
(S)-(−)-N-Adamant-1-yl-3,7-dihydro-3-isopropyl-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide ((S)-51). White solid. Mp:
220 °C. Yield: 53%. MS: m/z 407.5 (M + H). 1H NMR (DMSO-d6): δ
9.90 (br s, 1H), 8.68 (s, 1H), 7.85−7.81 (m, 1H), 7.42−7.28 (m, 2H),
4.79−4.73 (m, 1H), 4.42−4.24 (m, 2H), 2.04 (s, 10H), 1.92 (m, 1H),
1.65 (s, 6H), 0.99 (d, J = 6.6 Hz, 3H), 0.75 (d, J = 6.6 Hz, 3H). Anal.
(C25H30N2O3) C, H, N. [α]2D5 = −42 (c = 0.01, CH3OH).
(R)-(+)-N-Adamant-1-yl-3,7-dihydro-3-isobutyl-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide ((R)-58). White solid. Mp:
130 °C. Yield: 28%. MS: m/z 421.3 (M + H). 1H NMR (DMSO-d6): δ
9.92 (br s, 1H), 8.67 (s, 1H), 7.88−7.83 (m, 1H), 7.45−7.32 (m, 2H),
4.82−4.75 (m, 1H), 4.63−4.57 (m, 1H), 4.35−4.24 (m, 1H), 2.07 (s,
10H), 1.68 (s, 6H),1.44 (m, 1H), 0.99 (d, J = 5.8 Hz, 3H), 0.90 (d, J =
5.8 Hz, 3H). Anal. (C26H32N2O3) C, H, N.
3,7-Dihydro-3-ethyl-7-oxo-N-thiazol-2-yl-2H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxamide (40). White solid. Mp: 221 °C. Yield:
1
58%. MS: m/z 342.2 (M + H). H NMR (DMSO-d6): 13.62 (s, 1H),
9.05 (s, 1H), 7.96−7.94 (m, 1H), 7.54−7.28 (m, 4H), 4.78−4.65 (m,
2H), 4.43−4.3 (m, 1H), 1.92−1.61 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H).
Anal. (C17H15N3O3S) C, H, N.
N-Adamant-1-yl-3,7-dihydro-7-oxo-3-propyl-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (44). White solid. Mp: 218 °C.
1
Yield: 43%. MS: m/z 407.3 (M + H). H NMR (DMSO-d6): δ 9.93
(br s, 1H), 8.71 (s, 1H), 7.86−7.84 (m, 1H), 7.42−7.32 (m, 2H), 4.71
(m, 1H), 4.61−4.58 (m, 1H), 4.33−4.30 (m, 1H), 2.06 (s, 9H), 1.78
(m, 2H), 1.67 (s, 6H), 1.43−1.21 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H).
Anal. (C25H30N2O3) C, H, N.
3,7-Dihydro-N-(3,5-dimethyladamant-1-yl)-7-oxo-3-propyl-2H-
[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide (45). White solid.
Mp: 219 °C. Yield: 48%. MS: m/z 435.7 (M + H). 1H NMR
(DMSO-d6): 9.96 (br s, 1H), 8.68 (s, 1H), 7.87−7.83 (m, 1H), 7.45−
7.31 (m, 2H), 4.79−4.57 (m, 2H), 4.33 (m, 1H), 2.12−1.15 (m, 17H),
0.92−0.85 (m, 9H). Anal. (C27H34N2O3) C, H, N.
N-Adamant-1-yl-3,7-dihydro-8-methyl-7-oxo-3-propyl-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (47). White solid. Mp: 237
°C. Yield: 53%. MS: m/z 421.3 (M + H). 1H NMR (DMSO-d6): 9.94
(br s, 1H), 8.62 (s, 1H), 7.17−7.05 (m, 2H), 4.85−4.60 (m, 2H), 4.22
(m, 1H), 3.43−3.36 (m, 4H), 2.77 (s, 3H), 2.06 (s, 9H), 1.67 (s, 6H),
0.88 (m, 3H). Anal. (C26H32N2O3) C, H, N.
N-Adamant-1-yl-3-cyclopropyl-3,7-dihydro-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (50). White solid. Mp: 245
1
°C. Yield: 55%. MS: m/z 405.3 (M + H). H NMR (DMSO-d6): δ
9.94 (br s, 1H), 8.88 (s, 1H), 7.89−7.85 (m, 1H), 7.42−7.36 (m, 2H),
4.63−4.18 (m, 2H), 4.04−3.97 (m, 2H), 2.07 (s, 9H), 1.68 (s, 6H),
1.33−1.15 (m, 1H), 0.66−0.51 (m, 4H). Anal. (C25H28N2O3) C, H, N.
N-Adamant-1-yl-3,7-dihydro-3-isopropyl-7-oxo-2H-[1,4]oxazino-
[2,3,4-ij]quinoline-6-carboxamide (51). White solid. Mp: 220 °C.
1
Yield: 62%. MS: m/z 407.5 (M + H). H NMR (DMSO-d6): δ 9.90
(br s, 1H), 8.68 (s, 1H), 7.85−7.81 (m, 1H), 7.42−7.28 (m, 2H),
4.79−4.73 (m, 1H), 4.42−4.24 (m, 2H), 2.04 (s, 10H), 1.92 (m, 1H),
1.65 (s, 6H), 0.99 (d, J = 6.6 Hz, 3H), 0.75 (d, J = 6.6 Hz, 3H). Anal.
(C25H30N2O3) C, H, N.
N-Adamant-1-yl-3,7-dihydro-3,3-dimethyl-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (52). Pale white solid. Mp:
223 °C. Yield: 26%. MS: m/z 393.1 (M + H). 1H NMR (CDCl3): 9.98
(br s, 1H), 8.95 (s, 1H), 8.10−8.05 (m, 1H), 7.41−7.23 (m, 2H), 4.12
(s, 2H), 2.19−2.11 (m, 9H), 1.73 (s, 6H), 1.62 (s, 6H). Anal.
(C24H28N2O3) C, H, N.
(R)-(+)-N-Adamant-1-yl-3-benzyl-3,7-dihydro-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide ((R)-59). White solid. Mp:
153 °C. Yield: 55%. MS: m/z 455.6 (M + H). 1H NMR (DMSO-d6): δ
9.85 (br s, 1H), 8.50 (s, 1H), 7.89−7.85 (m, 1H), 7.43−7.19 (m, 7H),
4.99 (m, 1H), 4.41−4.23 (m, 2H), 3.04 (d, J = 7.4 Hz, 2H), 2.03 (s,
9H), 1.66 (s, 6H). Anal. (C29H30N2O3) C, H, N. [α]2D5 = +190 (c =
0.01, CH3OH).
Synthesis of N-Adamant-1-yl-3,7-dihydro-3-ethyl-10-methoxy-7-
oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide (55). A sol-
ution of N-adamant-1-yl-3,7-dihydro-3-ethyl-10-fluoro-7-oxo-2H-
[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide (53; 58 mg, 0.14
mmol) in dry THF (3 mL) was added to a solution of freshly
prepared CH3ONa (0.28 mmol) under a nitrogen atmosphere. The
N-Adamant-1-yl-3-ethyl-10-fluoro-3,7-dihydro-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (53). White solid. Mp: 232
1
°C. Yield: 65%. MS: m/z 411.2 (M + H). H NMR (DMSO-d6): δ
9.84 (s, 1H), 8.77 (s, 1H), 7.90−7.82 (m, 1H), 7.48−7.38 (m, 1H),
6621
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reaction mixture was heated at 50 °C for 16 h and neutralized using 1
N HCl. The mixture was concentrated in vacuo and the residue
extracted with CH2Cl2. The organic layer was washed with brine, dried
over Na2SO4, filtered, and evaporated to dryness. The crude residue
was purified by chromatography using petroleum ether/ethyl acetate
(1:1) as the eluent, yielding 20 mg (33%) of N-adamant-1-yl-3,7-
dihydro-3-ethyl-10-methoxy-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxamide as a white solid. Mp: 279 °C. MS: m/z 423.1
(M + H). 1H NMR (CDCl3): δ 9.98 (br s, 1H), 8.59 (s, 1H), 8.07 (d,
J = 9 Hz, 1H), 7.12 (d, J = 9 Hz, 1H), 4.65−4.59 (m, 1H), 4.30−4.21
(m, 1H), 4.19 (m, 1H), 4.02 (s, 3H), 2.16−2.10 (m, 9H), 1.91−1.88
(m, 2H), 1.73 (m, 6H), 1.05 (t, J = 7.4 Hz, 3H). Anal. (C25H30N2O3)
C, H, N.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 39-(0)532455921 (P.G.B.); 39-(0)532455926
(M.A.T.). Fax: 39-(0)532455953 (P.G.B.); 39-(0)53245593
(M.A.T.). E-mail: Baraldi@unife.it (P.G.B.); tbj@unife.it
(M.A.T.).
Present Address
∥Alta Vetta Pharmaceutical Consulting, LLC, 3 Skipwith Ct.,
Durham, NC 27707.
Notes
The authors declare no competing financial interest.
Synthesis of N-Adamant-1-yl-3,7-dihydro-3-ethyl-7-oxo-10-pyr-
rolidin-1-yl-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide (56).
K2CO3 (1 mmol) was added to a mixture of N-adamant-1-yl-3,7-
dihydro-3-ethyl-10-fluoro-7-oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-
carboxamide (53; 0.36 mmol), pyrrolidine (2.5 mmol), and anhydrous
DMF (5 mL). The resulting mixture was stirred at 100 °C for 10 h.
The solvent was removed under vacuum, the residue dissolved in ethyl
acetate (30 mL), and the organic layer washed with water (5 mL). The
organic phase was dried over anhydrous sodium sulfate, filtered,
evaporated, and purified by flash chromatography using ethyl acetate/
petroleum ether (3:7) as the eluent. Pale white solid. Mp: 282 °C.
Yield: 50%. MS: m/z 462.4 (M + H). 1H NMR (CDCl3): δ 10.01 (br
s, 1H), 8.50 (s, 1H), 7.71 (d, J = 9 Hz, 1H), 6.80 (d, J = 9.2 Hz, 1H),
4.532−4.38 (m, 2H), 4.14−4.07 (m, 1H), 3.51 (m, 4H), 2.06 (s, 9H),
1.96−1.73 (m, 6H), 1.66 (s, 6H), 0.96 (t, J = 7.4 Hz, 3H). Anal.
(C28H35N3O3) C, H, N.
ACKNOWLEDGMENTS
■
We thank Research and Development, King Pharmaceuticals,
Inc. (Cary, NC), for financial support.
ABBREVIATIONS USED
■
CHO, Chinese hamster ovary; DEEM, diethyl (ethoxymethylene)-
malonate; CDI, 1,1′-carbonyldiimidazole; SI, selectivity index;
EDC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydro-
chloride; HOBt, hydroxybenzotriazole; HBTU, O-benzotriazolyl-
N,N,N′,N′-tetramethyluronium hexafluorophosphate
REFERENCES
■
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cannabinoids in synaptic signaling. Physiol. Rev. 2003, 83, 1017−1066.
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Synthesis of N-Adamant-1-yl-3,7-dihydro-3-ethyl-7-oxo-10-(4-
methylpiperazin-1-yl)-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxa-
mide (57). K2CO3 (1 mmol) was added to a mixture of N-adamant-1-
yl-3,7-dihydro-3-ethyl-10-fluoro-7-oxo-2H-[1,4]oxazino[2,3,4-ij]-
quinoline-6-carboxamide (53; 0.36 mmol), 1-methylpiperazine
(2.5 mmol), and anhydrous DMF (5 mL). The resulting mixture
was stirred at 100 °C for 10 h. The solvent was removed in vacuo, the
residue dissolved in ethyl acetate (30 mL), and the organic layer
washed with water (5 mL). The organic phase was dried over
anhydrous sodium sulfate, filtered, evaporated, and purified by flash
chromatography using ethyl acetate/petroleum ether (3:7) as the
eluent. White solid. Mp: 294 °C dec. Yield: 40%. MS: m/z 491.3 (M +
1
H). H NMR (DMSO-d6): δ 9.95 (s, 1H), 8.61 (s, 1H), 7.78 (d, J =
8.8 Hz, 1H), 7.12 (d, J = 8.6 Hz, 1H), 4.75−4.67 (m, 2H), 4.45−4.38
(m, 1H), 3.25−3.11 (m, 4H), 2.49−2.46 (m, 4H), 2.21 (s, 3H), 2.03
(s, 9H), 1.84−1.69 (m, 2H), 1.65 (s, 6H), 0.93 (t, J = 7.4 Hz, 3H).
Synthesis of N-Adamant-1-yl-3-ethyl-10-(4-methylpiperazin-1-
yl)-7-oxo-3,7-dihydro-2H-[1,4]-oxazino[2,3,4-ij]quinoline-6-carbox-
amide Hydrochloride (57·HCl). A solution of N-adamant-1-yl-3-ethyl-
10-(4-methylpiperazin-1-yl)-7-oxo-3,7-dihydro-7H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxamide (47; 20 mg g, 0.04 mmol) in 1,4-dioxane
saturated with gaseous hydrogen chloride (4 mL) was stirred at 0 °C
for 30 min. The solvent was concentrated in vacuo, and the residue
was suspended in diethyl ether (10 mL), filtered, and washed with cold
diethyl ether. Pale yellow solid. Mp: 240 °C dec. Yield: 69%. MS: m/z
́
́
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Romero, J. Cannabinoid CB2 receptors in human brain inflammation.
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1
527.8 (M + H). H NMR (DMSO-d6): δ 10.48 (br s, 1H), 9.94 (s,
1H), 8.67 (s, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.21 (d, J = 8.8 Hz, 1H),
4.68−4.57 (m, 2H), 4.30−4.25 (m, 1H), 3.70−2.88 (m, 11H), 2.05
(m, 9H), 1.67 (m, 8H), 0.92 (t, J = 7.4 Hz, 3H). Anal.
(C29H39ClN4O3) C, H, N.
(12) Giblin, G. M. P.; O’Shaughnessy, C. T.; Naylor, A. M.; W., L.;
Eatherton, A. J.; Slingsby, B. P.; Rawlings, D. A.; Goldsmith, P.; Brown,
A. J.; Haslam, C. P.; Clayton, N. M.; Wilson, A. W.; Chessell, I. P.;
Wittington, A. R.; Green, R. Discovery of 2-[(2,4-dichlorophenyl)-
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ASSOCIATED CONTENT
■
S
* Supporting Information
Table of elemental analysis of compounds 19−59. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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