Benzothiazinones: A novel class of adenosine receptor antagonists structurally unrelated to xanthine and adenine derivatives

Article abstract of DOI:10.1021/jm300029s

2-(Acyl)amino-4H-3,1-benzothiazin-4-ones and related thienothiazinones were identified as structurally novel antagonists at adenosine receptors (ARs). 6-Methyl-2-benzoylamino-4H-3,1-benzothiazin-4-one (10d) was found to be a balanced AR antagonist with affinity for all human (h) subtypes (K_i hA₁ 65.6 nM; hA_2A 120 nM; hA_2B 360 nM; hA ₃ 30.4 nM), while in rat (r), 10d was a highly potent A ₁-selective antagonist (rA₁ 7.7 nM; rA_2A 546 nM; rA_2B 679 nM, rA₃ >10000 nM). 2-(4- Methylbenzoylamino)-4H-3,1-benzothiazin-4-one (10g) was found to be a potent antagonist at human A_2A (68.8 nM) and A₃ ARs (23.0 nM) with high selectivity versus the other human AR subtypes. In contrast to A ₁ and A₃ ARs, A_2A and A_2B ARs tolerated bulky 2-acyl substituents. tert-Butyl (4-oxo-4H-3,1-benzothiazin-2- ylcarbamoyl)benzylcarbamate (15g, K_i hA_2B 186 nM; hA _2A 603 nM) and 4-(4-benzylpiperazine-1-carbonyl)-N-(4-oxo-4H-3,1- benzothiazin-2-yl)benzamide (15k, hA_2A 69.5 nM; hA_2B 178 nM) were highly selective versus the other AR subtypes. 2-Acylamino-3,1- benzothiazin-4-ones represent novel scaffolds suitable for the development of potent and selective AR antagonists for each of the four receptor subtypes.

Full text of DOI:10.1021/jm300029s

Article
pubs.acs.org/jmc
Benzothiazinones: A Novel Class of Adenosine Receptor Antagonists
Structurally Unrelated to Xanthine and Adenine Derivatives
Michael Gutschow,* Miriam Schlenk, Jurgen Gab, Minka Paskaleva, Mohamad Wessam Alnouri,
̈
̈
̈
Silvia Scolari, Jamshed Iqbal,† and Christa E. Muller*
̈
PharmaCenter Bonn, University of Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, Bonn, Germany
S
* Supporting Information
ABSTRACT: 2-(Acyl)amino-4H-3,1-benzothiazin-4-ones and re-
lated thienothiazinones were identified as structurally novel
antagonists at adenosine receptors (ARs). 6-Methyl-2-benzoylami-
no-4H-3,1-benzothiazin-4-one (10d) was found to be a balanced
AR antagonist with affinity for all human (h) subtypes (Ki hA1 65.6
nM; hA2A 120 nM; hA2B 360 nM; hA3 30.4 nM), while in rat (r), 10d was a highly potent A1-selective antagonist (rA1 7.7 nM;
rA2A 546 nM; rA2B 679 nM, rA3 >10000 nM). 2-(4-Methylbenzoylamino)-4H-3,1-benzothiazin-4-one (10g) was found to be a
potent antagonist at human A2A (68.8 nM) and A3 ARs (23.0 nM) with high selectivity versus the other human AR subtypes. In
contrast to A1 and A3 ARs, A2A and A2B ARs tolerated bulky 2-acyl substituents. tert-Butyl (4-oxo-4H-3,1-benzothiazin-2-
ylcarbamoyl)benzylcarbamate (15g, Ki hA2B 186 nM; hA2A 603 nM) and 4-(4-benzylpiperazine-1-carbonyl)-N-(4-oxo-4H-3,1-
benzothiazin-2-yl)benzamide (15k, hA2A 69.5 nM; hA2B 178 nM) were highly selective versus the other AR subtypes. 2-
Acylamino-3,1-benzothiazin-4-ones represent novel scaffolds suitable for the development of potent and selective AR antagonists
for each of the four receptor subtypes.
PD.16,17 However, a major drawback of xanthine derivatives as
INTRODUCTION
Adenosine receptors (ARs) have been recognized as novel
■
well as adenine-related AR antagonists is their mostly very low
water solubility.18,19 Only recently, novel structures have been
discovered by high-throughput screening of large compound
libraries.11,14 One prominent example is the benzothiazole
SYN-115 (7), an A2A AR antagonist that was developed from a
screening hit.14,20
(potential) drug targets.1,2 Four different subtypes exist,
designated A1, A2A, A2B, and A3, which show a distinct
expression pattern. A1 and A2A ARs are expressed in high
density in certain areas of the brain, whereas A2B and A3 ARs
show much lower brain expression levels.1 While A1 and A3
receptors are coupled to inhibition of adenylate cyclase (AC),
A2A and A2B receptor stimulation leads to AC activation and
enhanced intracellular cAMP levels. Adenosine (1) itself is used
for the acute treatment of supraventricular paroxysmal
tachycardia, and the A2A-selective agonist regadenoson (2)
along with adenosine are applied as diagnostics for myocardial
perfusion imaging (Figure 1).2 Especially, AR antagonists are
promising new drug candidates for a number of indications,
including heart and renal failure (A1), Parkinson’s disease (PD),
Alzheimer’s disease and depression (A2A), asthma and chronic
obstructive pulmonary disease (A2B, A3), and glaucoma (A3).1,2
The xanthine derivatives caffeine (3) and theophylline (4) are
the prototypic nonselective AR antagonists blocking A1, A2A,
and A2B receptors and thereby mediating central and cardiac
stimulatory, diuretic, and antiasthmatic effects.3 Subtype-
selective ligands have been developed by modifying the
xanthine substitution pattern.3−10 Another class of AR
antagonists are structurally related to adenine (5), the
nucleobase of the physiological agonist 1.1,2,5,11−14 Many
mono-, bi-, and tricyclic heteroaromatic structures bearing an
exocyclic amino group like adenine have been found to possess
AR-antagonistic activity.1,2,11,13−15 An example is the pyrazolo-
triazolopyrimidine preladenant (6), an A2A antagonist which is
currently undergoing phase III clinical trials for the therapy of
In the search for nonxanthine-derived, structurally non-
adenine-related AR antagonists, it was the aim of this study to
evaluate 2-(acyl)amino-4H-3,1-benzothiazin-4-ones, such as
compounds 9a−b and 10a−i (Scheme 1), as possible
candidates. Representatives of this heterocyclic class are
assumed to possess biological activities because they might
provide their heteroatoms as potential hydrogen bond
acceptors and the fused phenyl ring for possible π−π
interactions. Adenosine receptor affinity of 4H-3,1-benzothia-
zin-4-ones has not been reported so far. Analogous 4H-3,1-
benzoxazin-4-ones with a ring oxygen in place of sulfur are
hydrolytically less stable.21 2-Amino-4H-3,1-benzoxazin-4-ones
are inhibitors of human leukocyte elastase,22 cathepsin G,23
chymase,24 C1r serine protease of the complement system,25
and human cytomegalovirus protease.26 4H-3,1-Benzothiazin-4-
ones and heterocyclic-fused analogues exhibit anticancer,
antiviral, and antiproliferative activities.27,28 It is noteworthy
that, among the isomeric 4H-1,3-benzothiazin-4-ones, ex-
tremely effective antimycobacterial nitro derivatives have been
explored and their target enzyme decaprenylphosphoryl-β-D-
ribose 2′-epimerase has been identified.29 Herein, we report on
Received: January 9, 2012
Published: March 12, 2012
© 2012 American Chemical Society
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Figure 1. Structures of selected adenosine receptor ligands.
Scheme 1. Synthesis of 2-Aminobenzothiazinones 9a−b and
(10a−i), depending on the reaction conditions. In compounds
12a−b and 13a−b, the fused benzene ring is exchanged for
thiophene (Scheme 2). This bioisosteric replacement gave rise
a
2-Acylaminobenzothiazinones 10a−i
Scheme 2. Synthesis of 2-Acylaminothienothiazinones 12a−
a
b and 13a−b
a
Reagents and conditions: (i) methyl 2-(3-aroylthioureido)-2-
thiophenecarboxylates, concd H2SO4, RT; (ii) from ethyl 2-(3-
benzoylthioureido)-3-thiophenecarboxylates, (1) concd H2SO4, 90
°C, (2) benzoic anhydride, toluene, reflux.
a
Reagents and conditions: (i) (1) concd H2SO4, 100 °C, (2) NaHCO3
(9a from 2-(3-benzoylthioureido)benzoic acid) or (1) concd H2SO4,
RT, (2) NaHCO3 (9b from methyl 2-(3-acetylthioureido)-4,5-
dimethoxybenzoate; (ii) concd H2SO4, −8 °C−RT (10a and 10b
from methyl 2-(3-acetylthioureido)benzoates) or concd H2SO4, RT
(10c−i from 2-(3-benzoylthioureido)benzoic acids).
to compounds with similar molecular geometry and, due to the
electron-rich nature of thiophene, a somewhat enhanced
electron density of the thiazinone fragment. Whereas the
known thieno[3,2-d]-fused derivatives 12a and 12b31 possess
an unsubstituted fused thiophene ring to allow for a direct
comparison with the benzothiazinones 10c and 10g, the [2,3-
d]-fused compounds 13a and 13b bear two methyl substituents
or a cycloaliphatic bridge. For the latter compounds, the
formation of the NH2-substituted thiazinones followed by
rebenzoylation provided the products in better yields than the
direct formation from the open-chain precursors.
The NH2-substituted compound 9a with preformed
benzothiazinone skeleton was subjected to several acylation
reactions (Scheme 3) in order to introduce further structural
diversity into the acyl portion of the products. Amidations were
either carried out with mixed anhydrides, obtained from the
corresponding carboxylic acid and the Yamaguchi reagent
(2,4,6-trichlorobenzoyl chloride),32 or acyl chlorides, generated
by the treatment of the carboxylic acid with oxalyl chloride.
the synthesis of a series of benzothiazinones and thieno
analogues bearing an acylamino substitutent at position 2 as
well as their thieno analogues. These heterocycles were
investigated as novel ligands of the adenosine receptor subtypes
A1, A2A, A2B, and A3.
RESULTS AND DISCUSSION
■
Chemistry. The preparation of the known 4H-3,1-
benzothiazin-4-ones 9a−b and 10a−g and the products 10h−
i in the course of a cyclocondensation reaction is shown in
Scheme 1.30 The required thioureides 8 were obtained from
anthranilic acids or esters and in situ generated acyl
isothiocyanates. Sulfuric acid-promoted cyclization could be
achieved with loss (9a and 9b) or retention of the acyl residue
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a
Scheme 3. Synthesis of 2-Acylaminobenzothiazinones 15a−h
a
Reagents and conditions: (i) (1) N-methylmorpholine, CH2Cl2, RT, (2) 2,4,6-trichlorobenzoyl chloride, RT; (ii) 9a, pyridine, toluene, reflux (for
15a−b, 15f, 15g); (iii) (COCl)2, DMF (cat.), CH2Cl2, RT; (iv) 9a, pyridine, CH2Cl2, reflux (for 15c−e); (v) HCl, ethyl acetate, RT.
To synthesize 15g, 4-(aminomethyl)benzoic acid had to be
Boc-protected,33 then coupled to the mixed anhydride and
finally reacted with 9a. Acidic carbamate deprotection of 15g
yielded the basic benzothiazinone 15h. The following attempts
have been made to attach further ionizable groups to the 2-
substituent as well as to the fused benzene ring of 2-acylamino-
4H-3,1-benzothiazin-4-ones.
Aldehyde 15f was subjected to an oxidation reaction with
oxone34 to obtain the carboxylic acid 15i without affecting the
heterocyclic portion of the molecule (Scheme 4). Carbodiimi-
dazole-promoted coupling of 15i with N-methyl- or N-
benzylpiperazine afforded the basic carboxamides 15j and
15k, respectively. These reactions were performed in DMF to
overcome limitations due to the poor solubility of the starting
compound. Moreover, we used an imidazole buffer system to
prevent strong basic conditions and thus a possible Dimroth
rearrangement of the benzothiazinone ring system.21,30 The
conditions for the preparation of 15j and 15k were then
successfully applied to further amidation reactions in the course
of this study.
Next, we conceived a synthetic access to 2-benzoylamino-
4H-3,1-benzothiazin-4-ones with a carboxyl group in position 6
(or 7) and, in turn, to basic amides of these carboxylic acids to
increase polarity and water solubility of the compounds
(Scheme 5). Oxidation of 4-nitro-m-xylene (16) with
Scheme 5. Synthesis of 2-Acylaminobenzothiazinones 21 and
a
22a−b
Scheme 4. Synthesis of 2-Acylaminobenzothiazinones 15i−
a
k
a
Reagents and conditions: (i) KMnO4, H2O, reflux; (ii) (COCl)2,
DMF (cat), tert-BuOH, pyridine, ZnCl2 (cat), RT; (iii) H2, 10% Pd/C,
EtOH, RT; (iv) benzoyl isothiocyanate, acetonitrile, RT; (v) concd
H2SO4, RT; (vi) (1) 1,1′-carbonyldiimidazole, DMF, RT, (2) N-
methylpiperazine or N-benzylpiperazine (1.5 equiv), imidazole (1
equiv), HCl (2 equiv), DMF, RT.
a
Reagents and conditions: (i) oxone, DMF, RT; (ii) (1) 1,1′-
carbonyldiimidazole, DMF, RT, (2) N-methylpiperazine or N-
benzylpiperazine (1.5 equiv), imidazole (1 equiv), HCl (2 equiv),
DMF, RT.
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potassium permanganate was performed in boiling water
whereby the oxidant was added twice to improve the yield of
4-nitroisophthalic acid (17).35,36 Compound 17 was trans-
formed via its diacyl chloride to the corresponding bis(tert-
butyl) ester 18, followed by catalytic hydrogenation to bis(tert-
butyl) 4-aminoisophthalate (19). The subsequent reaction with
in situ generated benzoyl isothiocyanate in acetonitrile afforded
bis(tert-butyl) 4-(3-benzoylthioureido)isophthalate (20). The
cyclization step, at which the concomitant deprotection
occurred, was performed with concentrated sulfuric acid to
produce the benzothiazinone-6-carboxylic acid 21. Conversion
with N-methyl- and N-benzylpiperazine gave the basic
carboxamides 22a and 22b, respectively.
concentration of test compounds was only 1 μM because
higher concentrations often led to precipitation of the
radioligand [3H]PSB-603, which possesses only moderate
solubility. Selected potent compounds of the present series
were further investigated for their functional properties, in GTP
shift assays at membrane preparations of cells recombinantly
expressing human A1 receptors, or in cAMP accumulation
studies using living CHO cells recombinantly expressing human
A2B receptors, respectively. Results are presented in Table 1,
and Figures 2 and 3. Data of standard ligands are included for
comparison.
Structure−Activity Relationships. The basic structure 2-
amino-4H-3,1-benzothiazin-4-one (9a) showed moderate
affinity at rat A1 and A2A receptors, with Ki values in the low
micromolar range and virtually no affinity for human A2B and
A3 ARs. However, its A1 and A2A affinity was considerably
higher than that of the nonselective standard antagonist caffeine
(see Table 1). Therefore, we decided to have a closer look at
the structure−activity relationships (SARs) of this new scaffold
in the field of AR ligands. 6,7-Disubstitution of the benzene
ring with methoxy groups (9b) improved affinity for A1 ARs by
4-fold, resulting in a submicromolar Ki of 590 nM, and it also
improved affinity for A3 ARs, while no change was observed at
both A2 AR subtypes.
The preparation of the 7-carboxylic acid 25 and its amides
26a and 26b (Scheme 6) followed a similar route as for the 6-
Scheme 6. Synthesis of 2-Acylaminobenzothiazinones 25 and
a
26a−b
Next, we investigated the acylation of the exocyclic 2-amino
group. Acetylation reduced affinity for the A1 AR without much
change at A2A and A2B ARs (compound 10a, see Table 1). In
contrast, affinity for the human A3 AR was dramatically
increased by >50-fold, resulting in a potent and A3-selective
antagonist with a Ki value of 193 nM. At rat A3 ARs, however,
10a was completely inactive. The same was observed for all
other thiazinone derivatives of the present series which were
investigated at rat A3 ARs. Such large species differences are
well-known for the A3 ARs, and most of the known A3
antagonists only bind to human but not to rodent A3
ARs.38,39 For the other AR subtypes, species differences are
typically much lower due to high sequence homologies of the
orthologues. 6,7-Disubstitution with methoxy groups (10b) led
to a 5-fold reduction in A3 affinity, while A1 affinity was
increased. Comparison of the SAR trends of 9a/9b and 10a/
10b shows that the substituents on the 2-position and those on
the 6- and 7-position are interdependent and not parallel.
The breakthrough with regard to the A1 AR was achieved by
acylation of the 2-amino function with a benzoyl group.
Compound 10c showed a Ki value of 25 nM at the rat A1 AR
and was 24-fold less potent at rat A2A, 62-fold less potent at rat
A2B, and inactive at rat A3 receptors. Thus, the compound
showed high A1 selectivity in rat. However, 10c showed
considerable species differences, especially at A1 (affinity: rat >
human) and A3 ARs (human > rat), and therefore the situation
at the human receptors was quite different (Ki values: 309 nM
(hA1), 25.0 nM (rA1); 91.7 nM (hA2A), 609 nM (rA2A); 950
nM (hA2B), 1560 nM (rA2B), 86 nM (hA3), >10000 nM (rA3)).
Thus, 10c is nonselective in humans with highest affinities at
A2A and A3 ARs. Bioisosteric replacement of the benzoyl group
by a 2-, 3-, or 4-pyridinoyl residue (15a, 15b, 15c) led to much
weaker or inactive compounds. The 3-pyridinoyl group (15b)
was best tolerated, resulting in a moderately potent A1/A3-
antagonist while the isomeric compounds 15a and 15c were
inactive. To probe the size of the binding pocket for the N2-acyl
group, phenyl (in 10c) was replaced by a benzyl (15d) or a 2-
cyclohexylethyl residue (15e). However, both modifications led
to a reduction in affinity compared to 10c.
a
Reagents and conditions: (i) benzoyl isothiocyanate, acetone, RT;
(ii) concd H2SO4, RT; (iii) (1) 1,1′-carbonyldiimidazole, DMF, RT,
(2) N-methylpiperazine or N-benzylpiperazine (1.5 equiv), imidazole
(1 equiv), HCl (2 equiv), DMF, RT.
functionalized derivatives. Thus, 2-aminoterephthalic acid (23)
was suspended in acetone, and benzoyl isothiocyanate was
added. Before the precipitation of the product started, some
undissolved starting compound was removed by filtration to
obtain 2-(3-benzoylthioureido)terephthalic acid (24), which
could be used in the next step without further purification.
Cyclocondensation afforded the benzothiazinone 25, which was
subsequently converted to the basic derivatives 26a and 26b.
Biological Evaluation. The new compounds were initially
investigated in radioligand binding studies at rat brain
adenosine A1 and A2A receptors and at human recombinant
A2B and A3 receptors using cell membrane preparations.37
Selected compounds were additionally investigated at human
recombinant A1 and A2A receptors and at recombinant rat A2B
and A3 receptors in order to obtain information on the affinities
and selectivities of the most interesting compounds in both
species, rat and human. [3H]2-Chloro-N6-cyclopentyladenosine
(CCPA) was used as A1-selective radioligand, [3H]3-(3-
hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxan-
thine (MSX-2) for A2A radioligand binding assays, and [3H]8-
(4-(4-(4-chlorophenyl)piperazine-1-sulfonyl)phenyl)-1-propyl-
xanthine (PSB-603) as A2B radioligand. For binding assays at
human A3 receptors, the A3-selective antagonist radioligand
[3H]2-phenyl-8-ethyl-4-methyl-(8R)-4,5,7,8-tetrahydro-1H-
imidazo[2,1-i]purin-5-one (PSB-11) was applied. Because
[3H]PSB-11 is not suitable for the labeling of rat A3 receptors,
the agonist radioligand [3H]NECA was used for binding studies
at rat A3 receptors. Screening was usually performed at 10 and 1
μM, however, in A2B radioligand binding studies, the highest
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Table 1. Adenosine Receptor Affinities of Benzothiazinone and Thienothiazinone Derivatives and Standard Antagonists
a
Ki SEM (nM) human (h); rat (r)
A3 vs [3H]PSB-11 (h) or vs
compd
R
A1 vs [3H]CCPA
A2A vs [3H]MSX-2
A2B vs [3H]PSB-603
[3H]NECA (r)
b
caffeine (3)
44900 (h) 41000 (r)2,
23400 (h) 32500 (r)
33800 (h)
13300 (h) >100000 (r)
30000 (r)2,
b
b
74000 (h)2,
b
b
b
theophylline (4)
8570 (h) 14000 (r)
3680 (h) 22000 (r)
22300 (h)2, 85000 (r)2,
15100 (r)2,
b
b
b
b
preladenant (6)
>1000 (h)2,
0.9 (h)2,
>1000 (h)2,
>1000 (h)2,
2-Aminobenzothiazinones
c
c
9a
2391 308 (r)
590 40 (r)
1580 409 (r)
2800 670 (r)
>1000 (h) (9%)
>10000 (h) (33%)
c
9b
>1000 (h) (7%)
2190 590 (h)
2-Acylaminobenzothiazinones
c
>10000 (h) (26%)
c
c
c
10a
Me
>10000 (h) (0%)
>1000 (h) (11%)
193 30 (h)
>10000 (r) (18%)
c
>10000 (r) (26%)
2390 350 (r)
c
c
10b
10c
Me
Ph
2660 1160 (r)
≥10000 (r) (42%)
>1000 (h) (9%)
964 356 (h)
309 17 (h)
25.0 5.0 (r)
91.7 16.9 (h)
609 61 (r)
950 150 (h)
1560 260 (r)
86.0 17.0 (h)
>10000 (r) (3%)
c
10d
10e
10f
Ph
65.6 14.0 (h)
7.70 3.72 (r)
120 51 (h)
546 119 (r)
360 100 (h)
679 36 (r)
30.4 6.8 (h)
c
>10000 (r) (11%)
c
c
>10000 (h) (9%)
c
c
c
Ph
>10000 (h) (17%)
19.1 5.3 (r)
>1000 (h) (0%)
≥10000 (h) (46%)
≥10000 (r) (42%)
>10000 (r) (40%)
c
c
c
Ph
233 54 (r)
>1000 (h) (0%)
>10000 (r) (0%)
128 23 (h)
c
c
10g
C6H4(4-Me)
C6H4(4-Me)
≥10000 (h) (43%)
68.8 6.8 (h)
656 114 (r)
>1000 (h) (35%)
29.0 1.2 (h)
68.7 13.2 (r)
17.0 0.5 (r)
c
10h
10i
1297 405 (r)
≥1000 (h) (48%)
>1000 (h) (17%)
69.1 6.1 (h)
503 218 (h)
c
c
c
C6H4(4-CO2Me)
2-pyridyl
>10000 (r) (33%)
>10000 (r) (7%)
c
c
c
c
15a
15b
15c
15d
15e
15f
>10000 (r) (12%)
>10000 (r) (0%)
>1000 (h) (7%)
≥10000 (h) (38%)
268 55 (h)
c
c
3-pyridyl
622 7 (r)
>10000 (r) (30%)
>1000 (h) (8%)
c
c
c
c
4-pyridyl
>10000 (r) (33%)
>10000 (r) (0%)
>1000 (h) (15%)
≥10000 (h) (45%)
558 157 (h)
c
c
Bn
>10000 (r) (23%)
1260 170 (r)
>1000 (h) (40%)
c
c
c
c
(CH2)2cyclohexyl
C6H4(4-CHO)
>10000 (r) (10%)
>10000 (r) (20%)
>1000 (h) (0%)
>10000 (h) (30%)
c
c
415 86 (r)
>10000 (r) (27%)
>1000 (h) (25%)
422 103 (h)
c
c
15g
C6H4-(4-CH2NHCO2t-Bu) >10000 (h) (16%)
>10000 (r) (32%)
603 190 (h)
>10000 (r) (30%)
186 9 (h)
>10000 (h) (38%)
c
c
15h
15i
15j
C6H4(4-CH2NH2) × HCl
C6H4(4-CO2H)
563 25 (r)
555 52 (r)
892 149 (r)
2550 680 (r)
>1000 (h) (0%)
8480 1240 (h)
2620 141 (h)
4560 1620 (h)
c
c
≥10000 (r) (48%)
2750 280 (r)
>1000 (h) (5%)
c
4-(4-methyl-piperazine-1-
carbonyl)phenyl
>1000 (h) (24%)
c
c
15k
4-(4-benzyl-piperazine-1-
carbonyl)phenyl
>10000 (h) (27%)
69.5 14.1 (h)
285 38 (r)
178 41 (h)
>1000 (h) (21%)
c
>1000 (r) (13%)
c
c
c
c
21
Ph
Ph
Ph
Ph
Ph
Ph
>10000 (r) (5%)
>10000 (r) (7%)
>1000 (h) (5%)
>10000 (h) (33%)
c
c
c
22a
22b
25
>10000 (r) (18%)
>10000 (r) (16%)
>10000 (r) (19%)
>10000 (r) (20%)
>1000 (h) (2%)
6160 970 (h)
c
c
c
c
>10000 (r) (23%)
>1000 (h) (8%)
≥10000 (h) (42%)
>10000 (h) (13%)
c
c
c
c
>10000 (r) (8%)
>1000 (h) (1%)
c
c
c
c
26a
26b
>10000 (r) (8%)
>10000 (r) (12%)
>1000 (h) (0%)
>10000 (h) (8%)
c
c
c
>10000 (r) (13%)
>10000 (r) (23%)
>1000 (h) (11%)
3210 770 (h)
2-Acylaminothienothiazinones
c
12a
12b
Ph
42.7 9.9 (r)
396 60 (r)
≥1000 (h) (45%)
400 44 (h)
200 9 (h)
C6H4(4-Me)
176 17 (h)
50.0 17.0 (r)
53.7 10.4 (h)
299 31 (r)
56.2 18.3 (h)
c
c
13a
13b
Ph
Ph
>10000 (h) (18%)
107 35 (r)
286 125 (h)
234 65 (r)
>1000 (h) (9%)
55.7 15.2 (h)
>10000 (r) (0%)
c
c
c
c
>10000 (r) (16%)
>10000 (r) (10%)
>1000 (h) (0%)
406 170 (h)
a
b
c
n ≥ 3 unless otherwise noted. Literature data (obtained with different radioligand). Percent inhibition of radioligand binding at indicated
concentration.
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could be confirmed. A carboxylate function in the 6-position
(compound 21) or in the 7-position (compound 25),
respectively, was introduced to increase polarity but was not
tolerated by the ARs. As an alternative strategy to improve
water solubility, N-methyl- and N-benzyl- piperazine-1-carbonyl
residues were introduced in the 6-position (22a, 22b) or in the
7-position (26a, 26b). But these relatively large substituents
were not well tolerated in both positions by the ARs. 6,7-
Disubstitution with the smaller methoxy residues (compound
10f) was better tolerated by the rat A1 and the human A3 AR
subtypes but was less potent than the parent compound 10c.
As a next step, we investigated substitution in the para-
position of the 2-benzoylamino residue in lead compound 10c.
A methyl residue (10g) had only a moderate effect on the AR
affinities, slightly increasing affinity for the human A3 AR (Ki =
29.0 nM). As a matter of fact, 10g was the most potent A3
antagonist of the present series. The compound showed high
selectivity for the human A3 versus the other human AR
subtypes. Introducing into 10g an additional methyl group at
position 6 of the benzothiazinone ring system as in the very
potent A1 and A3 antagonist 10d led to compound 10h. This
dimethyl substitution was not superior to the corresponding
monomethyl derivatives 10d and 10g. An aldehyde function in
the para-position of the benzoylamino residue (15f) reduced
affinity in comparison with a methyl substituent, and further
oxidation to a carboxylate function (compound 15i) led to a
further reduction in affinity especially at the human A3 AR. The
corresponding methyl ester 10i was again somewhat more
potent at the human A3AR, indicating that the A3AR did not
tolerate a negatively charged residue in that position, but 10i
was inactive at the other AR subtypes.
Figure 2. GTP shift assay at A1 receptors determined in radioligand
binding assays at rat brain cortical membrane preparations using the
antagonist radioligand [3H]DPCPX. (A) Binding curve for the full A1
agonist N6-cyclopentyladenosine (CPA); IC50 in the absence of GTP,
6.31 nM; in the presence of 1 mM GTP, 200 nM (32-fold shift). (B)
Binding curve of the potent A1 antagonist 10d in the absence (IC50,
35.1 nM) and in the presence of 1 mM GTP (IC50, 35.7 nM); no
significant GTP shift was observed.
In a further series of compounds, larger substituents,
including basic moieties, were introduced in the para-position
of the benzoylamino residue of lead compound 10c. The large
tert-butyloxycarbonyl-aminomethyl substitution (compound
15g) was only tolerated by human A2B (Ki 186 nM) and to a
lesser extent by human A2A receptors (Ki 603 nM). Thus 15g
showed high selectivity versus the other AR subtypes. It is
interesting to note that the A2A and perhaps even more so the
A2B AR appear to have place to accommodate large substituents
in the binding pocket for the 2-acylamino substituent. The
aminomethyl- (15h) and the 1-(4-methylpiperazinyl)- (15j)
substituted derivatives, containing basic structures with
improved water solubility at physiologic pH value of 7.4,
were much less potent at A1, A2A, and A2B ARs in comparison
with the lead structure 10c and only weakly potent at A3 ARs.
The larger 1-(4-benzylpiperazinyl) derivative 15k, which was
inactive at A1 and A3 ARs, however, showed clearly higher
affinity for A2A and A2B ARs than the lead structure 10c. The
affinity for both A2 AR subtypes was similar (Ki = 69.5 nM at
human A2A, Ki = 285 nM at rat A2A, Ki = 178 nM at human
A2B). This result confirms the finding that both A2A and A2B
ARs appear to accommodate large substituents in the region
where the acylamino residue binds.
Figure 3. Agonist- (NECA-) dependent increase in cAMP
accumulation in the absence and in the presence of 15g (3 μM) in
CHO cells recombinantly expressing human A2B receptors. The
agonist curve was shifted to right in a parallel fashion by the
antagonist. A Kb value of 1238 291 nM (n = 3) was calculated.
Because a 2-benzoylamino substitution (10c) had provided
by far the best results at all AR subtypes so far, we decided to
keep 10c as the lead structure and to further study the SARs of
the 2-benzoylaminothiazinone derivatives. First, we focused on
effects of substituents in the 6- and/or 7-position of the
benzothiazinone core structure of 10c. The introduction of a
methyl group in the 6-position yielded the most potent A1 and
A3 antagonist of the present series (10d). Affinity for the
human A2A and A2B ARs was also improved. 2-Benzoylamino-6-
methyl-4H-3,1-benzothiazin-4-one (10d) is a very potent (Ki =
7.70 nM) and highly selective antagonist for rat A1 ARs.
However, its affinity is lower at human A1 ARs (Ki = 65.6 nM).
In contrast, it is relatively potent at human A2A (Ki = 120 nM)
and human A3 receptors (Ki = 30.4 nM), indicating species
differences not only at A3 but also at A1 ARs and, to a lower
extent, at A2A ARs for this class of compounds. In contrast to
rat, compound 10d is therefore not selective in humans. The
corresponding 6-chloro-substituted derivative 10e was slightly
less potent at rat A1 ARs than the 6-methyl analogue 10d with a
Ki value of 19.1 nM at rat A1, and it was highly A1 selective.
Surprisingly, 10e did not bind to human A1 ARs. Such extreme
species differences at the A1 ARs are unprecedented. To
exclude potential degradation of the compound, we prepared a
fresh stock solution, confirmed the purity by LCMS (see
Supporting Information) and repeated the experiments, which
Finally, we modified the benzothiazinone ring structure of
lead compound 10c by bioisosterically replacing the benzene by
a thiophene ring (compounds 12a−b and 13a−b). The
thieno[3,2-d][1,3]thiazin-4-one derivative 12a was not superior
to the corresponding benzothiazinone derivative 10c. The 2-(p-
methylbenzoylamino) derivative 12b showed quite similar
affinities as its benzothiazinone analogue 10g at all AR
subtypes, but 12b tended to be somewhat more potent at the
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human receptors than the corresponding benzothiazinone 10g.
The isomeric thieno[2,3-d][1,3]thiazin-4-one derivatives 13a
and 13b bear additional substituents on the thiophene ring.
While the dimethyl-substituted thienothiazinone 13a was
comparable in AR affinities to the thieno[3,2-d][1,3]thiazin-4-
ones 12a and 12b, the tricyclic compound 13b was inactive at
A1, A2A, and A3 ARs and showed only low affinity for the A3AR.
Most AR ligands published in the literature have only been
investigated in one species, that is nowadays, due to the
availability of recombinant receptors, in most cases the human
species. However, initial in vivo studies are typically performed
in rodents, either rats or mice. It has been known for a long
time that large species differences exist for A3 antagonists
between human and rodent receptors, most antagonists that are
potent at human A3AR being inactive or only weakly active at
rat A3 ARs.1 In the present study we observed the same species
differences for the new benzothiazinone scaffold. All inves-
tigated compounds showed a large preference for the human
over the rat A3 AR. Surprisingly large species differences were
also observed for the A1 AR but with the opposite preference:
all benzo- and thienothiazinones investigated in both species
showed a preference for the rat receptor, and in some cases the
difference in affinity between the two species was >100-fold
(10g) or even >500-fold (10e). In contrast, species differences
at A2A and A2B receptors were generally smaller, and the
compounds appeared to show a (mostly moderate) preference
for the human receptors. Our results clearly show that AR
antagonists should not only be tested at human but also at
rodent receptors before they may be used as pharmacological
tools in animal models.
CONCLUSIONS
■
In conclusion, we discovered a structurally novel class of AR
antagonists, the 2-(acyl)amino-3,1-benzothiazin-4-ones and
related thienothiazinones. A series of 33 derivatives was
synthesized, and SARs were investigated in radioligand binding
studies at all four AR subtypes. In addition, species differences
were studied for the most potent compounds at human as
compared to rat ARs. Potent antagonists were obtained for each
of the four AR subtypes. Large species differences between
human and rat receptors were observed, not only for the A3 but
also for the A1 receptor subtype, indicating that one should be
extremely cautious in extrapolating from one species to the
other. Benzothiazinones generally showed a large preference for
human over rat A3, while the opposite preference (rat >
human) was observed at the A1 AR. One of the most potent
compounds was 10d, a balanced AR antagonist with good
affinity for all human AR subtypes; in rat, however, 10d proved
to be a highly potent A1-selective antagonist. Compound 10g
was found to be a potent antagonist at human A2A and A3 ARs
with high selectivity versus the other human AR subtypes. In
contrast to the A1 and the A3 receptor subtypes, the A2A and
even more so the A2B receptor tolerated large, bulky 2-acyl
substituents: 15g showed a Ki value of 186 nM at hA2B, 603 nM
at hA2A AR, and was highly selective versus the other AR
subtyes. Similarly, 15k was a potent and highly selective A2A/
A2B antagonist (hA2A 69.5 nM; hA2B 178 nM). The chemical
stability of some of the compounds was investigated by LCMS,
and they were found to be stable at room temperature. Thus, 2-
acylamino-3,1-benzothiazin-4-ones represent novel scaffolds
suitable for the development of potent and selective AR
antagonists for each of the four receptor subtypes.
Functional Characterization of Selected Ligands. The
most potent A1 AR antagonist identified in the series of 2-
acylaminobenzothiazinones was 6-methyl-2-benzoylamino-4H-
3,1-benzothiazin-4-one (10d). Therefore, we selected 10d for
investigating whether it functions as an antagonist (as
expected) or as an agonist. Most AR agonists identified so far
bear a ribose residue like the physiological agonist adenosine
(1), which is lacking in the benzothiazinones. Binding of
agonists is affected by the addition of GTP, which shifts the
curve to the right.40 For these experiments, the antagonist
radioligand [3H]DPCPX was employed. The magnitude of the
shift correlates with the efficacy, full agonists showing larger
shifts than partial agonists, while the binding curves of
antagonists are not affected by GTP. Figure 2 shows that the
full agonist N6-cyclopentyladenosine (CPA) exhibits a large
(32-fold) GTP shift, while the binding curve of 10d remains the
same in the presence as in the absence of GTP, clearly
indicating that the benzothiazinone derivative is an antagonist
at A1 AR receptors.
In addition, we selected one of the most potent and selective
A2B antagonists identified in the present study, benzothiazinone
15g, for functional investigations in cAMP accumulation studies
at human A2B ARs stably expressed in CHO cells. 15g behaved
like a competitive antagonist shifting the concentration−
response curve of the agonist NECA in a parallel manner to
the right. A Kb value of 1238 nM was determined (see Figure
3). Thus, the Kb value was 6.6-fold higher than the Ki value
determined in radioligand binding studies at membranes
preparations of the same cells. The reason for this discrepancy
is unknown because normally a high correlation between Ki and
Kb values at human A2B receptors has been observed.41
EXPERIMENTAL SECTION
Chemistry. Melting points were determined on a Buchi 510
■
̈
melting point apparatus and are uncorrected. Thin layer chromatog-
raphy was performed on Merck aluminum sheets. Preparative column
chromatography was performed on silica gel 60 (Acros Organics)
0.060−0.200 mm. 1H and 13C NMR spectra were acquired on a
1
Bruker Avance DRX 500 spectrometer operating at 500 MHz for H
and 125 MHz for 13C or on a Varian Gemini 2 instrument operating at
1
300 MHz for H and 75 MHz for 13C. IR spectra were recorded on a
Bruker Tensor 27 FT-IR spectrometer. HRMS (ESI) spectra were
recorded on a micrOTOF-Q (Bruker Daltonics) spectrometer.
Elemental analyses were carried out with a Vario EL apparatus.
Compounds 9a, 10c, 10d, 10e,30 9b, 10b,42 10a, 10f,21 10g, 12a, and
12b31 were prepared as described. Synthetic procedures and analytical
data for compounds 10i, 13b, 15a−c, 15e, 15f, 15i, 15k, 21, 22b, 25,
and 26b are listed in the Supporting Information. Purity of the
products was confirmed by TLC, elemental analysis or HRMS, and
NMR spectroscopy. All tested compounds possessed a purity of not
less than 95%.
6-Methyl-2-[(4-methylbenzoyl)amino]-4H-3,1-benzothiazin-4-
one (10h). 2-[3-(4-Methylbenzoyl)thioureido]-5-methylbenzoic acid
(984 mg, 3 mmol), prepared from 5-methylanthranilic acid and 4-
methylbenzoyl isothiocyanate,43 was dissolved in concd H2SO4 (3 mL)
and kept at RT for 2 days. The mixture was poured into ice−water
(200 mL). The precipitate was collected by filtration, extensively
washed with H2O, and dried to afford 10h as a colorless powder (410
mg, 44%), mp 209−211 °C (EtOH). 1H NMR (500 MHz,
[D6]DMSO): δ = 2.38 (s, 3H), 2.43 (s, 3H), 7.33 (d, J = 8.2 Hz,
2H), 7.57 (d, J = 8.3 Hz, 1H), 7.70 (dd, J = 8.3, 2.0 Hz, 1H), 7.85 (br
s, 1H), 7.96 (d, J = 8.2 Hz, 2H), 12.12 (br s1H). 13C NMR (125 MHz,
[D6]DMSO): 20.7, 21.2, 119.3, 123.9, 128.7, 129.2, 130.0, 130.5,
137.1, 137.5, 143.4, 146.0, 153.1, 167.2, 184.8. IR (KBr): ν = 1680,
1630 cm−1 (CO). Anal. Calcd for C17H14N2O2S: C 65.79, H, 4.55,
N 9.03. Found: C 65.75, H 4.62, N 9.30.
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N-(5,6-Dimethyl-4-oxo-4H-thieno[2,3-d][1,3]thiazin-2-yl)-
benzamide (13a). Ethyl 4,5-dimethyl-2-(3-benzoylthioureido)-3-thio-
phenecarboxylate (3.26 g, 10 mmol) was reacted with concd H2SO4 to
obtain 2-amino-5,6-dimethyl-4H-thieno[2,3-d[1,3]thiazin-4-one (1.40
g, 66%),44 mp 263−264 °C. A mixture of 2-amino-5,6-dimethyl-4H-
thieno[2,3-d[1,3]thiazin-4-one (637 mg, 3 mmol), benzoic anhydride
(1.36 g, 6 mmol), and toluene (25 mL) was refluxed for 2 h and kept
at RT overnight. The precipitate was collected by suction filtration and
washed with ethyl acetate to obtain 13a as a brownish solid (460 mg,
49%), mp 248−250 °C (ethyl acetate). 1H NMR (500 MHz,
[D6]DMSO): δ = 2.36 (s, 6H), 7.51−7.54 (m, 2H), 7.62−7.66 (m,
1H), 8.02−8.04 (m, 2H), 12. 40 (s, 1H). 13C NMR (125 MHz,
[D6]DMSO) δ = 12.4, 13.7, 119.4, 128.6, 128.7, 128.7, 128.8, 132.0,
133.2, 157.7, 163.1, 166.9, 177.6. IR (KBr): ν = 1670, 1625 cm−1 (C
O). Anal. Calcd for C15H12N2O2S2: C 56.94, H 3.82, N 8.85. Found:
C, 57.04, H, 3.90, N 8.83.
1H), 8.06 (m, 3H), 8.69 (s, 3H). 13C NMR (125 MHz, [D6]DMSO) δ
= 41.9, 119.6, 124.6, 127.3, 128.5, 128.9, 129.0, 132.2, 136.6, 139.3,
147.1, 154.1, 167.5, 184.8. IR (KBr): ν = 1696, 1675 cm−1 (CO).
HRMS-ESI m/z [M + H]+ calcd for C16H13N3O2S, 312.0801; found,
312.0795.
4-(4-Methylpiperazine-1-carbonyl)-N-(4-oxo-4H-3,1-benzothia-
zin-2-yl)benzamide (15j). 1,1′-Carbonyldiimidazole (357 mg, 2.2
mmol) was added to a mixture of compound 15i (653 mg, 2 mmol)
and DMF (11 mL) and stirred at RT 1 h. To a solution of N-
methylpiperazine (301 mg, 3 mmol), imidazole (136 mg, 2 mmol),
DMF (2 mL), and 1,4-dioxane (2 mL), 1 mL of a 4 N solution of HCl
in 1,4-dioxane was added dropwise. The two mixtures were combined,
kept at RT for 2 h, and poured into satd aqueous NaHCO3 solution
(180 mL). The precipitate was collected by suction filtration, dried,
and recrystallized from EtOH to afford 15j as a yellow powder (110
1
mg, 13%), mp 245−246 °C. H NMR (500 MHz, [D6]DMSO): δ =
N-(4-Oxo-4H-3,1-benzothiazin-2-yl)-2-phenylacetamide (15d).
Oxalyl chloride (1.27 g, 10 mmol) and two drops of DMF were
added to a solution of phenylacetic acid (1.36 g, 10 mmol) in CH2Cl2
(20 mL). After 2 h, the solvent was removed under reduced pressure,
and the residue was dissolved in DMF (12 mL). Pyridine (1.58 g, 20
mmol) and compound 9a (1.39 g, 7.8 mmol) were added and stirred
at RT for 3 h. The mixture was poured into ice-cold 0.5 N HCl (200
mL), and the precipitate was collected by filtration. The crude product
was recrystallized from EtOH to afford 15d as a white solid (840 mg,
2.21 (s, 3H), 2.30 (br s, 2H), 2.38 (br s, 2H), 3.29 (br s, 2H), 3.63 (br
s, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.53 (ddd, J = 7.9, 7.4, 1.0 Hz, 1H),
7.67 (dd, J = 8.2, 1.0 Hz, 1H), 7.89 (ddd, J = 8.3, 6.6, 1.6 Hz, 1H), 8.06
(dd, J = 8.0, 1.3 Hz, 1H), 8.10 (d, J = 8.5 Hz, 2H), 12.35 (s, 1H). 13C
NMR (125 MHz, [D6]DMSO) δ = 41.5, 45.6, 47.0, 54.3, 54.7, 119.6,
124.6, 127.0, 127.2, 128.3, 128.9, 133.4, 136.5, 140.2, 147.1, 154.7,
167.6, 168.1, 184.8. IR (KBr): ν = 1684, 1653, 1635 cm−1 (CO).
HRMS-ESI m/z [M + H]+ calcd for C21H20N4O3S, 409.1329; found,
409.1303.
1
36%), mp 189−192 °C. H NMR (500 MHz, [D6]DMSO): δ = 3.77
N-(6-(4-Methylpiperazine-1-carbonyl)-4-oxo-4H-3,1-benzothia-
zin-2-yl)benzamide (22a). 1,1′-Carbonyldiimidazole (4 mg, 1.6
mmol) was added to a mixture of compound 21 (489 mg, 1.5
mmol), DMF (4 mL), and 1,4-dioxane (2 mL) and stirred at RT 1 h.
To a solution of N-methylpiperazine (301 mg, 3 mmol), imidazole
(136 mg, 2 mmol), DMF (2 mL), and 1,4-dioxane (2 mL), 1 mL of a 4
N solution of HCl in 1,4-dioxane was added dropwise. The two
mixtures were combined, kept at RT for 2 h, and poured into satd
aqueous NaHCO3 solution (150 mL). The precipitate was collected by
suction filtration, dried, and recrystallized from EtOH to afford 22a as
(s, 2H), 7.24−7.28 (m, 1H), 7.31−7.34 (m, 4H), 7.51 (ddd, J = 8.0,
7.1, 1.3 Hz, 1H), 7.60 (dd, J = 8.2, 1.3 Hz, 1H), 7.87 (ddd, J = 8.4, 6.9,
1.6 Hz, 1H), 8.03 (dd, J = 7.9 1.6 Hz, 1H), 12.10 (s, 1H). 13C NMR
(125 MHz, [D6]DMSO) δ = 42.3, 119.6, 124.5, 127.0, 127.3, 128.5,
129.0, 129.5, 134.6, 136.5, 147.7, 153.1, 171.7, 184.5. IR (KBr): ν =
1701, 1653 cm−1 (CO). Anal. Calcd for C16H12N2O2S: C 64.85, H
4.08, N 9.45. Found: C, 65.00, H, 4.07, N 9.08.
tert-Butyl (4-Oxo-4H-3,1-benzothiazin-2-ylcarbamoyl)-
benzylcarbamate (15g). To a solution of 4-(aminomethyl)benzoic
acid (3.02 g, 20 mmol) in THF (60 mL), 1 M NaOH (22 mL) and di-
tert-butyldicarbonate (4.80 g, 22 mmol) were added. The mixture was
stirred at RT for 12 h, the organic solvent was removed under reduced
pressure, the residue was diluted with water (50 mL), and 0.1 M
NaHSO4 was added to obtain pH 2−3. It was extracted with ethyl
acetate (3 × 50 mL), dried, and evaporated to obtain 4-[(tert-
butoxycarbonyl)aminomethyl]benzoic acid (4.52 g, 90%),33 mp 167−
168 °C. N-Methylmorpholine (469 mg, 4 mmol) was added to a
mixture of 4-[(tert-butoxycarbonylamino)methyl]benzoic acid (1.0 g, 4
mmol) and CH2Cl2 (20 mL). After stirring for 10 min, 2,4,6-
trichlorobenzoyl chloride (976 mg, 4 mmol) was added and the
mixture was stirred for additional 2 h. The solvent was removed under
reduced pressure, and the residue was taken up in toluene (40 mL).
Compound 9a (267 mg, 1.5 mmol) were added, the mixture was
refluxed for 4 h, and the hot mixture was filtered. The filtrate was kept
overnight. The precipitate was collected by suction filtration, washed
with diethyl ether, and recrystallized from EtOH to afford 15g as a
1
a colorless powder (240 mg, 39%), mp 264−265 °C. H NMR (500
MHz, [D6]DMSO): δ = 2.22 (s, 3H), 2.35 (br s, 4H), 3.58 (br s, 4H),
7.52−7.56 (m, 2H), 7.65 (t, J = 7.3 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H),
7.87 (dd, J = 8.2, 1.9 Hz, 1H), 8.00 (d, J = 1.9 Hz, 1H), 8.06 (dd, J =
7.6, 1.3 Hz, 2H), 12.33 (br s, 1H). 13C NMR (125 MHz, [D6]DMSO)
δ = 45.6, 54.4, 66.5, 119.2, 123.2, 127.8, 128.6, 128.8, 132.4, 133.2,
134.0, 134.8, 148.0, 155.7, 167.5, 168.1, 184.7. IR (KBr): ν = 1681,
1651, 1623 cm−1 (CO). HRMS-ESI m/z [M + H]+ calcd for
C21H20N4O3S, 409.1329; found, 409.1327.
N-(7-(4-Methylpiperazine-1-carbonyl)-4-oxo-4H-3,1-benzothia-
zin-2-yl)benzamide (26a). 1,1′-Carbonyldiimidazole (195 mg, 1.2
mmol) was added to a mixture of compound 25 (326 mg, 1 mmol),
DMF (4 mL), and 1,4-dioxane (2 mL) and stirred at RT 1 h. To a
solution of N-methylpiperazine (200 mg, 2 mmol), imidazole (136 mg,
2 mmol), DMF (2 mL), and 1,4-dioxane (2 mL), 1 mL of a 4 N
solution of HCl in 1,4-dioxane was added dropwise. The two mixtures
were combined, kept at RT for 2 h, and poured into satd aqueous
NaHCO3 solution (150 mL). The precipitate was collected by suction
filtration, dried, and recrystallized from EtOH to afford 26a as a
1
colorless solid (340 g, 55%), mp 162−164 °C. H NMR (500 MHz,
[D6]DMSO): δ = 1.40 (s, 9H), 4.20 (d, J = 6.0 Hz, 2H), 7.37 (d, J =
8.2 Hz, 2H), 7.46 (t, J = 5.7 Hz, 1H), 7.50−7.54 (m, 1H), 7.66 (d, J =
8.2 Hz, 1H), 7.86−7.90 (m, 1H), 8.00 (d, J = 8.2 Hz, 2H), 8.05 (d, J =
7.6 Hz, 1H), 12.23 (s, 1H). 13C NMR (125 MHz, [D6]DMSO) δ =
28.4, 43.4, 78.1, 119.6, 124.6, 126.9, 127.3, 128.8, 129.1, 130.5, 136.5,
145.8, 147.7, 153,9, 160.0, 176.1, 184.8. IR (KBr): ν = 1684, 1635
cm−1 (CO). Anal. Calcd for C21H21N3O4S: C 61.30, H 5.14, N
10.21. Found: C, 61.40, H 5.32, N 9.83.
1
colorless powder (200 mg, 49%), mp 212−214 °C. H NMR (500
MHz, [D6]DMSO): δ = 2.21 (s, 3H), 2.29 (br s, 2H), 2.41 (br s, 2H),
3.32 (br s, 2H), 3.65 (br s, 2H), 7.47 (dd, J = 8.2, 1.6 Hz, 1H), 7.51−
7.54 (m, 2H), 7.55 (t, J = 1.6 Hz, 1H), 7.62−7.66 (m, 1H), 8.05 (dd, J
= 8.4, 1.3 Hz, 2H), 8.09 (d, J = 8.2 Hz, 1H), 12.33 (br s, 1H). 13C
NMR (125 MHz, [D6]DMSO) δ = 41.5, 45.6, 47.0, 54.2, 54.7, 119.7,
125.2, 125.2, 126.3, 128.6, 128.7, 132.4, 133.1, 143.4, 147.4, 155.4,
167.3, 168.0, 184.6. IR (KBr): ν = 1689, 1653 (br) cm−1 (CO).
Anal. Calcd for C21H20N4O3S: C 61.75, H 4.94, N 13.72. Found: C
61.24, H, 4.83, 13.40.
4-Aminomethyl-N-(4-oxo-4H-3,1-benzothiazin-2-yl)benzamide
hydrochloride (15h). Compound 15g (200 mg, 0.49 mmol) was
suspended in ethyl acetate (10 mL), and a freshly prepared 4 M
solution of HCl in ethyl acetate (15 mL) was added. The mixture was
stirred for 45 min. The product was collected by suction filtration and
washed with small amounts of diethyl ether and ethanol to afford 15h
Biological Assays. Radioligands. Radioligands were obtained
from the following sources: [3H]CCPA from Amersham (58
Ci/mmol), [3H]MSX-2 from Amersham (84 Ci/mmol),
[3H]PSB-603 from Amersham (73 Ci/mmol), [3H]PSB-11
(53 Ci/mmol) from Quotient Biosearch, and [3H]NECA (15.5
Ci/mmol) from Perkin-Elmer. The nonradioactive precursors
1
as a colorless solid (160 mg, 94%), mp 238−240 °C. H NMR (500
MHz, [D6]DMSO): δ = 4.10 (q, J = 5.7 Hz, 2H), 7.53 (ddd, J = 7.6,
7.6, 1.3 Hz, 1H), 7.65−7.69 (m, 3H), 7.89 (ddd, J = 8.4, 7.1, 1.6 Hz,
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of [3H]MSX-2,45 [3H]PSB-603,4 and [3H]PSB-1146 were
synthesized in our laboratory.
ABBREVIATIONS USED
■
AR, adenosine receptor; CCPA, [3H]2-chloro-N6-cyclopenty-
ladenosine; CHO, Chinese hamster ovary; CPA, N6-cyclo-
pentyladenosine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine;
h, human; MSX-2, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-
methoxystyryl)-1-propargylxanthine; NECA, 5′-N-ethylcarbox-
amidoadenosine; r, rat
Membrane Preparations. Membranes from Chinese hamster ovary
(CHO) cells stably transfected with the human A1, human A2A, human
A2B, rat A2B, and human A3 AR were prepared as described.4,46,47 For
assays at rat A3 ARs, commercially available membrane preparations
containing the rat A3 AR expressed in human embryonic kidney
(HEK) cells were obtained from Biotrend (Cologne, Germany).
Frozen rat brains obtained from Pel Freez, Rogers, Arkansas, USA,
were dissected to obtain cortical membrane preparations for A1 assays,
and striatal membrane preparations for A2A assays as de-
scribed.45,48,49,50
Radioligand Binding Assays. Stock solutions of the compounds
were prepared in dimethyl sulfoxide (DMSO); the final concentration
of DMSO was 2.5%. The radioligand concentrations were: [3H]-
CCPA,48 0.5 nM (rat and human A1); [3H]MSX-2,45 1.0 nM (rat and
human A2A); [3H]PSB-603,4 0.3 nM (rat and human A2B); [3H]PSB-
11,46 0.5 nM (human A3), [3H]NECA, 10 nM (rat A3). Binding assays
were performed as described.9,40,45,46,48 About 30−70 μg/mL of
protein were used in the assays. At least three separate experiments
were performed, each in duplicate or triplicate.
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Data Analysis. Data were analyzed using GRAPH PAD PRISM
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̈
ASSOCIATED CONTENT
■
Klotz, K. N.; Muller, C. E. Improving potency, selectivity, and water
̈
S
solubility of adenosine A1 receptor antagonists: xanthines modified at
position 3 and related pyrimido[1,2,3-cd]purinediones. ChemMed-
Chem. 2006, 1, 891−902.
* Supporting Information
Synthesis of additional products, analytical data, selected
concentration−response curves, and results from stability
studies (LCMS measurements). This material is available free
of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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■
Corresponding Author
*Phone: +49-228-73 2301/+49-228-73-2317. Fax: +49-228-73-
2567. E-mail: christa.mueller@uni-bonn.de (C.E.M.);
guetschow@uni-bonn.de (M.G.). Address: Prof. Dr. Christa
́
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̈
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E. Muller and Prof. Dr. Michael Gutschow, Pharma-Zentrum
̈
̈
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R. C.; Ramadori, A. T.; Klotz, K. N.; Trincavelli, M. L.; Martini, C.;
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Bonn, Pharmazeutisches Institut, Pharmazeutische Chemie I,
An der Immenburg 4, D-53121 Bonn, Germany.
Present Address
†Department of Pharmaceutical Sciences, COMSATS Institute
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Nicole Florin, Angelika Fischer, and Dieter Baumert
for skilfull technical assistance. C.E.M. was funded by the
German Federal Ministery for Education and Research (BMBF
01EW0911) in the frame of ERA-NET NEURON.
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