Synthesis and biological evaluation of benzopyran analogues bearing class III antiarrhythmic pharmacophores

Article abstract of DOI:10.1016/j.bmc.2006.05.065

We have synthesized a series of compounds combining the hydroxy-benzopyran ring of vitamin E with the methylsulfonylaminophenyl group of class III antiarrhythmic drugs, connected through tertiary amine moieties. Evaluation of the antiarrhythmic and antioxidant activity of the new compounds was carried out on isolated rat heart preparations using the non-recirculating Langendorff mode. The new analogues were present, at 10 μM concentration, during ischemia and reperfusion. Selected compounds were further studied by a conventional microelectrode method in order to get insight into their cellular mode of action. The most active compound, N-[4-[2-[[2-(3,4-dihydro-6-hydroxy-2,2,7,8-tetramethyl-2H-1-benzopyran-5 -yl)ethyl] methylamine]ethyl]phenyl]methanesulfonamide (19a), reduces premature beats, prolongs QT and QRS intervals during ischemia and reperfusion, and reduces MDA content, leading to a fast recovery of the heart. In addition, it exhibits moderate class III antiarrhythmic action.

Full text of DOI:10.1016/j.bmc.2006.05.065

Bioorganic & Medicinal Chemistry 14 (2006) 6666–6678
Synthesis and biological evaluation of benzopyran analogues
bearing class III antiarrhythmic pharmacophores
Maria Koufaki,a,* Christina Kiziridi,a Panagiota Papazafiri,b Athanasios Vassilopoulos,b
c
c
c
d
`
´
Andras Varro, Zsolt Nagy, Attila Farkas and Alexandros Makriyannis
aInstitute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation,
48 Vas. Constantinou Avenue, 116 35 Athens, Greece
bDepartment of Animal and Human Physiology, School of Biology, University of Athens, Panepistimiopolis, 15784 Athens, Greece
cDepartment of Pharmacology and Pharmacotherapy University of Szeged, Szeged Dom ter 12 H-6720, Hungary
dNortheastern University, Center for Drug Discovery, 360 Huntington Avenue, 116 Mugar Hall, Boston, MA 02115, USA
Received 13 March 2006; revised 25 May 2006; accepted 31 May 2006
Available online 19 June 2006
Abstract—We have synthesized a series of compounds combining the hydroxy-benzopyran ring of vitamin E with the methylsulfo-
nylaminophenyl group of class III antiarrhythmic drugs, connected through tertiary amine moieties. Evaluation of the antiarrhyth-
mic and antioxidant activity of the new compounds was carried out on isolated rat heart preparations using the non-recirculating
Langendorff mode. The new analogues were present, at 10 lM concentration, during ischemia and reperfusion. Selected compounds
were further studied by a conventional microelectrode method in order to get insight into their cellular mode of action. The most
active compound, N-[4-[2-[[2-(3,4-dihydro-6-hydroxy-2,2,7,8-tetramethyl-2H-1-benzopyran-5-yl)ethyl] methylamine]ethyl]phen-
yl]methanesulfonamide (19a), reduces premature beats, prolongs QT and QRS intervals during ischemia and reperfusion, and reduc-
es MDA content, leading to a fast recovery of the heart. In addition, it exhibits moderate class III antiarrhythmic action.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
artery disease which has resulted in prior ischemia is
most important. Myocardial ischemia causes profound
alterations in normal cardiac electrophysiology and
cellular metabolism, precipitating ventricular arrhyth-
mias or fibrillation.3 The establishment of blood flow
to the myocardium, by procedures such as thrombolysis,
angioplasty and coronary bypass surgery, reduces the
mortality of ischemic tissues. However, the reactive oxy-
gen species (ROS), produced upon the readmission of
oxygenated blood into the ischemic myocardium (reper-
fusion),4 affect selective permeability of cell membranes,
leading to the development of life-threatening ventricu-
lar arrhythmias and/or fibrillation.
Arrhythmias account for nearly one quarter of all car-
diovascular-related deaths. The majority of such deaths
is caused by the degeneration of a normal cardiac
rhythm into ventricular tachycardia (VT) followed by
ventricular fibrillation (VF).1 The shape of the action
potential of the heart cells is strongly controlled by the
correct interplay of ion channels.2 The main ion chan-
nels contributing to the action potential are sodium,
potassium, and calcium channels. Changes in the ionic
mechanism responsible for the generation and the prop-
agation of the normal action potential can cause abnor-
malities in the electrical activity of the heart.
Moreover, post-operative atrial fibrillation is a common
complication after open heart surgery; it increases mor-
bidity, hospital stay, and costs.5 Pharmacologic strate-
gies and regimens aimed at preventing post-operative
atrial fibrillation are necessary to patients undergoing
open heart operations. The prevalence of arrhythmia
in the population is increasing as more people survive
for longer with cardiovascular disease.6 Since many pa-
tients experience a decrease in physical performance as
Although cardiac rhythm disturbances may be the result
of a variety of pathophysiological conditions, coronary
Keywords: Benzopyran analogue; Antiarrhythmic agents; Ischemia/
reperfusion; Cellular electrophysiology.
*
Corresponding author. Tel.: +30 210 7273818; fax: +30 210
7273831; e-mail: mkoufa@eie.gr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.065

M. Koufaki et al. / Bioorg. Med. Chem. 14 (2006) 6666–6678
6667
well as a diminished quality of life, there is still a need
for antiarrhythmic drug therapy.
ideal antiarrhythmic agent does not exist, and drug
selection should be highly individualized.15,16
According to the classification of Vaughan Williams
(1970), based on electrophysiological actions, the antiar-
rhythmic drugs can be defined by four classes:7 class I
consist of antiarrhythmic agents that block sodium
channels, reducing the maximum increase rate of depo-
larization (Vmax). Class II are b-blockers. Class III act
through delaying repolarization of cardiac myocytes
and thus cause a lengthening of APD (potassium-chan-
nel blockers). Class IV block calcium currents in cardiac
tissue.
Thus, it is important to develop therapeutic agents
which could improve heart function with minimal side
effects. We have previously synthesized17 a series of hy-
brid compounds combining the pharmacophoric redox
moiety of vitamin E and key features responsible for
the antiarrhythmic properties of the class I antiarrhyth-
mics procainamide and lidocaine. Some of these com-
pounds, at concentrations of 30–100 lM, prolonged
QRS intervals during reperfusion and enhanced the
post-ischemic recovery without inducing ventricular
fibrillations. Moreover, there was no evidence in our
experiments for drug-induced proarrhythmia.
In the case of antiarrhythmic drugs the delicate balance
between drug efficacy and unexpected adverse side ef-
fects is narrower than in any other class of therapeutic
agents. Concerning class I antiarrhythmics, controlled
trials suggested the effectiveness of routine lidocaine
(class Ib) prophylaxis in preventing ventricular fibrilla-
tion due to acute myocardial infarction.8,9 However, in
1980 CAST (Cardiac Arrhythmia Suppression Trial),
with the drugs encainide and flecainide (class Ic), uncov-
ered the inefficacy and even proarrhythmic risk of sodi-
um channel blockers in post-infarction patients. To
circumvent the problems with class I antiarrhythmics,
pharmacological and clinical research shifted toward
the class III agents.10 Amiodarone (combining class I–
IV properties),11a,11b d,l-sotalol (class II, III)11c and
ambasilide (class II, III), azimilide (class I, III, IV) are
complex class III compounds, while ‘pure’ class III
agents are d-sotalol, dofetilide, ibutilide.
Based on this experience in antiarrhythmic field, we were
interested in continuing our work and to focus our
activities on the synthesis of novel cardioprotective
compounds with improved efficacy in the treatment of
life-threatening arrhythmias. Thus, we synthesized series
of molecules that combine the hydroxy-benzopyran ring
of vitamin E with the methylsulfonylaminophenyl moie-
ty of class III antiarrhythmic drugs.
Specifically, the new compounds combine pharmaco-
phores identified for the most active class III antiar-
rhythmics. Thus, they contain two aromatic rings, one
methylsulfonyl amino group and at least one tertiary
amine, such as a 1,4-piperazine or methylamine moiety.
Evaluation of the antiarrhythmic and antioxidant activ-
ity of the new compounds was carried out on isolated rat
heart preparations using the non-recirculating Langen-
dorff mode. The new analogues were present, at 10 lM
concentration, during ischemia and reperfusion.
Selected compounds were further studied by a conven-
tional microelectrode method in order to get insight into
their cellular mode of action.
Sotalol, amiodarone, ibutilide, and dofetilide are moder-
ately effective in patients with chronic atrial fibrillation.
However, amiodarone appears to be most efficacious.
Moreover, amiodarone and dofetilide are safe in pa-
tients who have had a myocardial infarction and those
with heart failure. The safety of commercially available
d,l-sotalol in these patients is poorly understood. Tor-
sades de pointes is the most serious adverse effect of
sotalol and dofetilide. Amiodarone has minimal proar-
rhythmic risk but has numerous noncardiac toxicities
that require frequent monitoring.12a,12b Dronedarone a
noniodinated benzofuran derivative has been shown to
be more effective in vivo than amiodarone in several
arrhythmia models, particularly in preventing ischemia-
and reperfusion-induced ventricular fibrillation and in
reducing mortality. However, further experimental stud-
ies and long-term clinical trials are required to provide
additional evidence of efficacy and safety of this drug.12c
Azimilide statistically reduced the incidence of new atri-
al fibrillation in recent survivors of myocardial infarc-
tion at high risk for sudden cardiac death.13 In
addition, class III antiarrhythmic agents are increasingly
being used as adjunct therapy to decrease the frequency
of ICD discharges in patients with ventricular arrhyth-
mias and implantable cardioverter defibrillators
(ICDs).14 The antiarrhythmic efficacy of most pure class
III drugs is compromised by their inherent property to
induce excessive lengthening of the action potential
and their inability to prolong the action potential when
most needed, namely during tachycardia. Overall, an
2. Chemistry
The synthesis of the disubstituted piperazine derivatives
5a–e is depicted in Scheme 1. Analogues 2a–e were syn-
thesized by alkylation of the appropriate monosubstitut-
ed piperazines (prepared by the appropriate bromides or
acid chloride and 8-fold excess of piperazine) with bro-
mide 118 in the presence of K2CO3 and TBAI in CH3CN
(compounds 2a–c,e) or DMF (compound 2d). Reduc-
tion of the nitro group using NaBH4 and CuCl gave
analogues 3a–e, which in turn were converted to the cor-
responding methanesulfonamides 4a–e using CH3SO2Cl
in pyridine. Deprotection of the chroman hydroxyl
group was achieved using BF3ÆS(CH3)2 in CH2Cl2, to af-
ford the methanesulfonamides 5a–e.
The 1-[(benzopyran-5-yl)ethyl]piperazine analogue 12 is
synthesized as shown in Scheme 2. Wittig reaction of
aldehyde 6 with Ph3P+CH2OCH3ClÀ in the presence
of t-BuOK gave the enol ether 7 which upon hydroly-
sis19 afforded aldehyde 8. Reductive amination of 8 with
1-(4-nitrophenyl)-piperazine
produced
the
nitro

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M. Koufaki et al. / Bioorg. Med. Chem. 14 (2006) 6666–6678
X
X
N
N
NH2
N
N
NO2
b
Br
MeO
MeO
MeO
a
O
O
O
3a X= -CH2-
2a X= -CH2-
1
3b X= -CH2CH2-
3c X= -CH2CH2O-
3d X= -
2b X= -CH2CH2-
2c X= -CH2CH2O-
2d X= -
3e X= -CO-
2e X= -CO-
X
X
N
N
N
NHSO2Me
d
N
NHSO2Me
MeO
HO
c
O
O
5a X= -CH2-
5b X= -CH2CH2-
5c X= -CH2CH2O-
5d X= -
4a X= -CH2-
4b X= -CH2CH2-
4c X= -CH2CH2O-
4d X= -
5e X= -CO-
4e X= -CO-
Scheme 1. Reagents and conditions: (a) 4-substituted piperazine, K2CO3, TBAI, anhyd CH3CN; (b) 1-(4-nitrophenyl)piperazine, K2CO3, TBAI,
anhyd DMF, 80 ꢁC; (c) NaBH4, CuCl, EtOH, 80 ꢁC; (d) CH3SO2Cl, pyridine, anhyd CH2Cl2; (e) BF3ÆS(CH3)2, anhyd CH2Cl2.
CHO
CHO
OMe
O
MeO
MeO
MeO
c
b
a
O
O
8
7
6
N
O
N
NHR1
N
O
N
NO2
RO
MeO
d
9
10 R= Me, R1= H
e
11 R= Me, R1= SO2Me
12 R= H, R1= SO2Me
f
Scheme 2. Reagents and conditions: (a) Ph3P+CH2OCH3ClÀ, t-BuOK, anhyd THF; (b) p-toluenesulfonic acid, dioxane, H2O; (c) 1-(4-
nitrophenyl)piperazine, NaBH3CN, CH3COOH, CH3CN; (d) NaBH4, CuCl, EtOH, 80 ꢁC; (e) CH3SO2Cl, pyridine, anhyd CH2Cl2; (f) BF3ÆS(CH3)2,
anhyd CH2Cl2.
analogue 9 which was converted to methanesulfonamide
12 using the synthetic procedure described above for
compounds 5a–e.
For the synthesis of the constrained derivative 22
(Scheme 4), bromide 1 and 6-nitroindoline were used
as starting materials. Analogue 21 was synthesized by
reductive alkylation of 6-nitroindoline with aldehyde 8
and then converted to the final compound 23.
The synthesis of methylamino derivatives 19a,b is
depicted in Scheme 3. Condensation of aldehyde 6 with
CH3NO2, in the presence of CH3COONH4, followed by
reduction using LiAlH4,20 gave amine 14, which in turn
was alkylated with 4-nitrophenyl or 2-(4-nitro-phen-
oxy)ethyl bromide to afford amines 15a,b. Methylation
of the secondary amines using HCHO 36% and
HCOOH21 afforded nitro compounds 16a,b which were
converted to methanesulfonamides 19a,b.
3. Results and discussion
The antiarrhythmic activity of the new compounds, at a
concentration of 10 lM, is shown in Table 1 and it is ex-
pressed as incidence of premature beats during the first
10 min of reperfusion. The antioxidant activity is

M. Koufaki et al. / Bioorg. Med. Chem. 14 (2006) 6666–6678
6669
X
N
H
NO2
NH2
O
NO2
O
MeO
MeO
MeO
c
b
a
6
O
15a X= -CH2CH2-
15b X= -CH2CH2O-
14
13
X
X
N
NHR1
N
NO2
Me
Me
RO
MeO
e
d
O
O
17a R= Me, R1= H, X= -CH2CH2-
17b R= Me, R1= H, X= -CH2CH2O-
16a X= -CH2CH2-
16b X= -CH2CH2O-
f
18a R= Me, R1= SO2Me, X= -CH2CH2-
18b R= Me, R1= SO2Me, X= -CH2CH2O-
g
19a R= H, R1= SO2Me, X= -CH2CH2-
19b R= H, R1= SO2Me, X= -CH2CH2O-
Scheme 3. Reagents and conditions: (a) CH3NO2, CH3COONH4, 100 ꢁC; (b) LiAlH4, THF; (c) 4-nitrophenethyl or 2-(4-nitrophenoxy)ethylbromide,
K2CO3, TBAI, anhyd CH3CN; (d) HCHO 36%, HCOOH, 100 ꢁC; (e) NaBH4, CuCl, EtOH, 80 ꢁC; (f) CH3SO2Cl, pyridine, anhyd CH2Cl2; (g)
BF3ÆS(CH3)2, anhyd CH2Cl2.
N
MeO
NO2
a
1
O
N
n
c, d, e
20
HO
NHSO2Me
O
N
O
MeO
22 n=1
23 n=2
b
8
NO2
21
Scheme 4. Reagents and conditions: (a) 6-nitroindoline, K2CO3, TBAI, CH3CN; (b) 6-nitroindoline, NaBH3CN, CH3COOH, CH3CN; (c) NaBH4,
CuCl, EtOH, 80 ꢁC; (d) CH3SO2Cl, pyridine, anhyd CH2Cl2; (e) BF3ÆS(CH3)2, anhyd CH2Cl2.
Table 1. Antiarrhythmic and antioxidant activity, at a concentration of 10 lM, of the new compounds
Compound
Premature beats (%)
MDA (nmol/g)
Remarks
None (control)
5a
3
3
0.6
0.28
134 11
137 13
Tachycardia
Bradycardia
5b
5c
3.15 1.06
3.15 1.9
8.2 1.8
141
9
Tachycardia
Bradycardia
146 25
129 35
95 35
5d
5e
Increase of premature beats
Bradycardia
Fibrillation
2.8 1.04
5.35 2.1
3.9 1.68
1.35 0.58*
3.05 1.06
3.85 1.4
2.11 0.75
5.11 3.5
12
18a
139
105
76 15**
110 14
7
7
Suppression of tachycardia
Fast recovery of the heart
Suppression of tachycardia
Fast recovery
19a
19b
22
23
104.5 16
83 2.8**
116 5.6
Fast recovery
Fibrillation
Amiodarone
n = 3–4.
* p < 0.05, versus control.
** p < 0.01, versus control.

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M. Koufaki et al. / Bioorg. Med. Chem. 14 (2006) 6666–6678
expressed as MDA content (nmoles/gr of cardiac tissue)
at the end of reperfusion.
bit ventricular muscles. The results are summarized in
Table 3. The compounds did not change the resting
potential (RP), the action potential amplitude
(APA), and the maximal rate of depolarization (Vmax).
The observed variations of Vmax in some experiments
most likely reflect inconsistency of the impalement of
the microelectrode. These data suggest no major
change of the fast inward sodium current (INa) after
the application of the analogues. Compounds 5a,b,d
did not alter the repolarization process reflected as
no change of the 50% and 90% action potential dura-
tions (APD50 and APD90). This indicates no, or min-
imal effect of the major repolarizing potassium
channels. However, compounds 5c,e and 19a,b pro-
longed APD by 5–14% which suggests moderate inhi-
bition of the repolarizing potassium current most
likely of the rapid delayed rectifier potassium current
(IKr or HERG). The latter effect represents moderate
class III antiarrhythmic actions and may need further
investigations.
In the absence of compound (control) tachycardia was
observed during reperfusion, while the QT and QRS
intervals shorten during ischemia and reperfusion (Table
2).
Compound 5b, which induces tachycardia upon reper-
fusion, and compounds 5d and 12, which increase the
incidence of premature beats, are the less active
analogues.
Piperazine analogue 5a suppresses tachycardia during
reperfusion, prolongs QT interval during ischemia and
reperfusion, while it affects QRS interval only during
ischemia. Compound 5c suppresses also reperfusion
tachycardia, prolongs QT interval during ischemia and
reperfusion, and prolongs QRS interval only during
reperfusion. Compound 5e is the only piperazine deriv-
ative that reduces premature beats and exhibits antioxi-
dant activity.
4. Conclusions
Among the methylamino analogues, compound 19b,
does not reduce the premature beats but suppresses
reperfusion tachycardia. Although this analogue does
not influence the QT interval during ischemia, it causes
a significant QT widening during reperfusion and it
slightly decreases MDA content. Compound 19a, bear-
ing ethylene group instead of ethylenoxy group of com-
pound 19b, and reduces premature beats, prolongs QT
and QRS intervals during ischemia and reperfusion,
reduces MDA content, leading to a fast recovery of
the heart. In addition, analogue 19a exhibits better anti-
arrhythmic and antioxidant activity than its methoxy
derivative 18a.
Among piperazine derivatives, compounds 5c and
5e suppress reperfusion tachycardia, while com-
pound 5e reduces premature beats and MDA con-
tent, combining antiarrhythmic and antioxidant
properties. The presence of phenyl piperazine moie-
ty (analogues 5d and 12) abolishes antiarrhythmic
activity.
Methylamino derivative 19a and its constrained ana-
logue 23 exhibit antioxidant activity, reduce premature
beats, and induce a fast recovery of the heart during
reperfusion.
The two constrained analogues 22 and 23 induced fast
recovery of the heart during reperfusion. It should be
also noted that the presence of compounds 5c, 19a,b,
and 22 facilitated the recovery of QRS and QT intervals
to the normal values.
Compounds 5c, 19a,b, and 22 facilitated the recovery of
QRS and QT intervals during reperfusion, to the normal
values. Moreover, the cardioprotective compounds 5c,e
and 19a,b do not induce excessive lengthening of the ac-
tion potential, exhibiting moderate class III antiarrhyth-
mic actions. Further studies on animal models as well as
on the possible influence on specific potassium channels
such as IKr, IKs, Ito, and IK1 should clarify the mecha-
The effect of compounds 5a–e and 19a,b on the action
potential parameters was investigated at 5 lM, in rab-
Table 2. QRS and QT intervals (ms) of the analogues 5a,e,c, 19a,b, 22, and 23 during ischemia and reperfusion
Compound
Equilibration
QT
Ischemia
QT
61.5 4.3
Reperfusion
QT
QRS
39 3.2
QRS
QRS
None (control)
5a
105 7.3
28 3.6
36 3.1*
31 2.4
24.4 2.1
26.4 2.2
55.4 4.8
121.3 11.7***
119.7 12***
129.15 14.6***
107.6 10.3***
121.3 5.7***
106.16 24.7**
143.44 9.2***
134.1 17.2***
114 9.6***
96 7.4**
5c
5e
42.7 3.2***
41.7 4.3***
30.6 2.7*
29.14
38.5 4.2**
1
97.4 8.4**
89.3 6.8**
63.3 9.3
108.1 12.5**
102.8 3.6***
129.1 18.4***
19a
19b
21.3
4
43.4 1.4***
33.12 4.7*
39.44 1.7***
42.4 2.3***
22
23
32.5 4.4
33.8 2.1
36.3 3.7**
Amiodarone
n = 3-4.
* p < 0.05, versus control.
** p < 0.01, versus control.
*** p < 0.001, versus control.

M. Koufaki et al. / Bioorg. Med. Chem. 14 (2006) 6666–6678
6671
Table 3. Effect of analogues 5a–e and 19a,b, at a concentration of 5 lM, on the action potential parameters in rabbit isolated right ventricular
papillary muscle
Compound
Experiments
1
RP
APA
APD50
APD90
Vmax
Control
5a
À88
À88
122
125
164
167
201
201 (0%)
201
201
Control
5a
2
1
2
1
2
1
2
1
2
1
2
1
2
À89
117
115
124
133
165
173 (4.8%)
171
186
À87
Control
5b
À84
À85
110
118
107
110
139
140 (0%)
186
208
Control
5b
À93
114
115
136
136
169
168 (0%)
156
163
À91
Control
5c
À87
À86
121
122
167
188
205
224 (9.3%)
223
230
Control
5c
À77
98
109
126
159
175
199 (13.7%)
267
267
À84
Control
5d
À90
À88
104
100
157
157
195
171
208
189 (À3.1%)
161
169 (5%)
Control
5d
À89
114
112
112
137
178
216
À89
Control
5e
À84
À84
117
117
127
150
175
191 (9.1%)
305
297
Control
5e
À93
113
112
154
167
200
211 (5.5%)
163
171
À92
Control
19a
À90
À91
113
120
197
214
230
246 (6.9%)
201
230
Control
19a
À82
105
106
118
127
155
170 (9.7%)
193
260
À86
Control
19b
À86
À86
119
117
110
117
150
160 (6.7%)
171
178
Control
19b
À86
117
103
199
215
237
255 (7.6%)
334
201
À83
RP, resting membrane potential.
APA, action potential amplitude.
APD50–90 = 50% and 90% action potential duration.
Vmax = maximal rate of depolarization.
nism and provide additional evidence of efficacy of these
compounds.
5.2. General procedure for the synthesis of disubstituted
piperazines 2a–e (method A)
To a solution of bromide 1 (0.200 g, 0.64 mmol) in
5 mL anhyd CH3CN, were added, at 0 ꢁC, K2CO3
(0.132 g, 0.96 mmol) and a solution of the appropriate
monosubstituted piperazine (0.64 mmol) in 5 mL an-
hyd CH3CN. A catalytic amount of TBAI was then
added and the mixture was stirred at ambient temper-
ature for 24 h. After completion of the reaction, the
solvent was evaporated, the residue was taken up with
AcOEt and washed with water. The organic layer was
dried and concentrated, and the residue was purified
by column chromatography (CH2Cl2/CH3OH 9.5:1.5).
5. Experimental
5.1. Chemistry
Melting points were determined on a Buchi 510 appara-
tus and are uncorrected. NMR spectra were recorded on
a Bruker AC 300 spectrometer operating at 300 MHz for
1H and 75.43 MHz for 13C. 1H NMR spectra are report-
ed in units of d with CHCl3 resonance at 7.26 ppm used
as the chemical shift resonance. 13C NMR spectra are
reported in units of d relative to CDCl3 at 77.00 ppm.
CDCl3 was used as solvent. Silica gel plates Macherey-
Nagel Sil G-25 UV254 were used for thin-layer chroma-
tography. Chromatographic purification was performed
with silica gel (200–400 mesh). Mass spectra were record-
ed on a Varian Saturn 2000 GC–MS instrument in the EI
mode. Elemental analyses were carried out on a Perkin-
Elmer Series II CHNS/O 2400 analyser.
5.2.1.
1-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-1-benzopyran-5-yl)methyl]-4-[(4-nitrophenyl)methyl]-
piperazine (2a). Yield: 85%, yellow viscous oil. 1H
NMR d: 8.14 (d, 2H, J = 9.1 Hz, ArH), 7.49 (d,
2H, J = 9.1 Hz, ArH), 3.66 (s, 3H, –OCH3), 3.55 (s,
2H, ArCH2N–), 3.50 (s, 2H, –NCH2Ar), 2.86 (t,
2H, J = 6.7 Hz, ArCH2), 2.51–2.49 (m, 8H, CH2),

6672
M. Koufaki et al. / Bioorg. Med. Chem. 14 (2006) 6666–6678
2.18 (s, 3H, ArCH3), 2.10 (s, 3H, ArCH3), 1.76 (t,
2H, J = 6.7 Hz, CH2), 1.31 (s, 6H, CH3) 13C NMR
d: 150.6, 148.0, 147.0, 146.7, 129.4, 127.6, 125.9,
125.1, 123.4, 119.0, 72.9, 62.1, 61.5, 53.5, 52.8, 32.9,
26.9, 20.1, 12.8, 12.1. MS m/z 453 (M+1%), 268
(100%), 233, 217. Anal. Calcd for C26H35N3O4: C,
68.85; H, 7.78; N, 9.26. Found: C, 69.09; H, 7.88;
N, 9.59.
5.3. General procedure for the synthesis of 4-substituted
anilines (method B)
To a solution of the appropriate disubstituted piperazine
(0.35 mmol) in 8 mL abs EtOH, CuCl (1.56 mmol) was
added, at 0 ꢁC, followed by NaBH4 (3.18 mmol) and
the mixture was refluxed for 2 h. The mixture was then
filtered through Celite and washed with CH2Cl2. The fil-
trate was washed with sat aqueous NaCl, dried, and
evaporated to dryness.
5.2.2.
1-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-1-benzopyran-5-yl)methyl]-4-[2-(4-nitrophenyl)ethyl]-
piperazine (2b). Yield: 80%, yellow viscous oil. 1H NMR
d: 8.09 (d, 2H, J = 9.1 Hz), 7.33 (d, 2H, J = 9.1 Hz), 3.63
(s, 3H), 3.47 (s, 3H), 2.84–2.82 (m, 4H), 2.58 (t, 2H,
J = 6.7 Hz), 2.50–2.48 (m, 8H), 2.16 (s, 3H), 2.08 (s,
3H), 1.74 (t, 2H, J = 6.7 Hz), 1.28 (s, 6H). 13C NMR
d: 150.6, 148.5, 148.0, 146.4, 129.5, 127.6, 125.9, 125.1,
123.6, 119.1, 72.9, 61.5, 59.4, 53.4, 52.8, 33.5, 32.9,
26.9, 20.1, 12.8, 12.0.
5.3.1. [4-[4-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]methyl]ani-
line (3a). Yield: 95%, yellow viscous oil. H NMR d:
7.09 (d, 2H, J = 8.5 Hz), 6.61 (d, 2H, J = 8.5 Hz), 3.63
(s, 3H), 3.47 (s, 2H), 3.41 (s, 2H), 2.81 (t, 2H,
J = 6.7 Hz), 2.49–2.47 (m, 8H), 2.16 (s, 3H), 2.08 (s,
3H), 1.73 (t, 2H, J = 6.7 Hz), 1.28 (s, 6H).
1
5.3.2. [4-[4-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-1- benzopyran-5-yl)methyl]piperazin-1-yl]ethyl]aniline
(3b). Yield: 88%, yellow viscous oil. 1H NMR d: 7.05 (d,
2H, J = 9.1 Hz), 6.63 (d, 2H, J = 9.1 Hz), 3.66 (s, 3H),
3.47 (s, 3H), 2.85–2.82 (m, 4H), 2.59 (t, 2H,
J = 6.7 Hz), 2.51–2.48 (m, 8H), 2.17 (s, 3H), 2.08 (s,
3H), 1.74 (t, 2H, J = 6.7 Hz), 1.27 (s, 6H).
5.2.3.
1-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-1-benzopyran-5-yl)methyl]-4-[2-(4-nitrophenoxy)ethyl]-
piperazine (2c). Yield: 45%, yellow viscous oil. 1H NMR
d: 8.17 (d, 2H, J = 9.1 Hz), 6.92 (d, 2H, J = 9.1 Hz), 4.17
(t, 2H, J = 5.5 Hz), 3.64 (s, 3H), 3.47 (s, 2H), 2.84–2.79
(m, 4H), 2.52–2.50 (m, 8H), 2.17 (m, 3H), 2.09 (s, 3H),
1.75 (t, 2H, J = 6.7 Hz), 1.29 (s, 6H). 13C NMR d:
163.8, 150.5, 148.0, 141.5, 127.6, 125.8, 125.1, 119.1,
114.5, 114.4, 72.9, 66.9, 65.0, 61.5, 56.8, 54.0, 53.1,
52.7, 32.9, 26.9, 20.1, 12.8, 12.0. Anal. Calcd for
C27H37N3O5: C, 67.06; H, 7.71; N, 8.69. Found: C,
67.12; H, 7.63; N, 8.32.
5.3.3. 4-[2-[4-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetrameth-
yl-2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]ethoxy]-
1
aniline (3c). Yield: 80%, yellow viscous oil. H NMR d:
6.71 (d, 2H, J = 8.5 Hz), 6.61 (d, 2H, J = 8.5 Hz), 4.05 (t,
2H, J = 5.5 Hz), 3.64 (s, 3H), 3.47 (s, 2H), 2.84 (t, 2H,
J = 6.7 Hz), 2.75 (t, 2H, J = 5.5 Hz), 2.52–2.50 (m,
8H), 2.16 (s, 3H), 2.08 (s, 3H), 1.74 (t, 2H,
J = 6.7 Hz), 1.28 (s, 6H).
5.2.4.
2H-1-benzopyran-5-yl)methyl]-4-(4-nitrophenyl)piperazine
(2d). Bromide and 1-(4-nitrophenyl)piperazine
1-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-
1
(0.100 g, 0.48 mmol), in anhyd DMF at 80 ꢁC, were
treated according to method A. Yield: 0.154 g (73%),
orange solid, mp 126–129 ꢁC. 1H NMR d: 8.09 (d,
2H, J = 9.1 Hz), 6.78 (d, 2H, J = 9.1 Hz), 3.64 (s,
3H), 3.52 (s, 2H), 3.36–3.34 (m, 4H), 2.87 (t, 2H,
J = 6.7 Hz), 2.59–2.57 (m, 4H), 2.19 (s, 3H), 2.10 (s,
3H), 1.76 (t, 2H, J = 6.7 Hz), 1.30 (s, 6H). 13C NMR
d: 154.9, 150.5, 127.8, 125.9, 125.2, 119.0, 112.4, 73.0,
61.6, 52.3, 47.2, 32.8, 26.9, 20.1, 12.8, 12.1. MS m/z:
234 (100%), 219. Anal. Calcd for C25H33N3O4: C,
68.31; H, 7.57; N, 9.56. Found: C, 67.92; H, 7.62; N,
9.16.
5.3.4. 4-[4-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]aniline (3d).
Yield: 0.137 g (96%), green viscous oil. 1H NMR d: 6.78
(d, 2H, J = 8.5 Hz), 6.63 (d, 2H, J = 8.5 Hz), 3.66 (s,
3H), 3.55 (s, 2H), 2.99–2.97 (m, 4H), 2.87 (t, 2H,
J = 6.7 Hz), 2.63–2.61 (m, 4H), 2.19 (s, 3H), 2.10 (s,
3H), 1.75 (t, 2H, J = 6.7 Hz), 1.29 (s, 6H).
5.3.5. 4-[[4-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]carbonyl]aniline
1
(3e). Yield: 78%, yellow viscous oil. H NMR d: 7.23
(d, 2H, J = 8.5 Hz), 6.61 (d, 2H, J = 8.5 Hz), 3.84–
3.82 (m, 2H), 3.63 (s, 3H), 3.48–3.47 (m, 2H),
2.84 (t, 2H, J = 6.7 Hz), 2.44–2.42 (m, 4H), 2.16 (s,
3H), 2.08 (s, 3H), 1.75 (t, 2H, J = 6.7 Hz), 1.28 (s,
6H).
5.2.5. 1-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-1-benzopyran-5-yl)methyl]-4-(4-nitrobenzoyl)pipera-
1
zine (2e). Yield: 95%, yellow solid, mp 192–194 ꢁC. H
NMR d: 8.20 (d, 2H, J = 8.5 Hz), 7.52 (d, 2H,
J = 8.5 Hz), 3.74–3.72 (m, 2H), 3.59 (s, 3H), 3.51 (s,
2H), 3.25–3.23 (m, 2H), 2.81 (t, 2H, J = 6.7 Hz),
2.59–2.57 (m, 2H), 2.39–2.37 (m, 2H), 2.12 (s, 3H),
2.05 (s, 3H), 1.74 (t, 2H, J = 6.7 Hz), 1.25 (s, 6H).
13C NMR d: 167.8, 150.5, 148.2, 142.2, 128.0,
127.7, 125.6, 125.0, 123.8, 118.9, 73.0, 65.0, 61.5,
52.7, 47.9, 42.5, 32.8, 26.9, 20.0, 12.7, 12.0. MS m/
z: 234 (100%), 219. Anal. Calcd for C26H33N3O5: C,
66.79; H, 7.11; N, 8.99. Found: C, 66.86; H, 7.05;
N, 8.67.
5.4. General procedure for the synthesis of 4-substituted
phenylmethanesulfonamides (method C)
To a solution of the appropriate aniline (0.5 mmol) in
6 mL CH2Cl2 and 2 mL pyridine was added at 0 ꢁC
CH3SO2Cl (1 mmol) and the mixture was stirred at
ambient temperature. After completion of the reaction,
AcOEt and sat aqueous solution of NH4Cl were added.
The organic layer was further washed by satd aqueous
NaCl, dried, and concentrated in vacuo. The residue

M. Koufaki et al. / Bioorg. Med. Chem. 14 (2006) 6666–6678
6673
was purified by column chromatography (CH2Cl2/
CH3OH 9.5:1.5).
2H), 2.38–2.36 (m, 2H), 2.15 (s, 3H), 2.08 (s, 3H), 1.75
(t, 2H, J = 6.7 Hz), 1.28 (s, 6H). 13C NMR d: 169.8,
150.6, 148.2, 139.0, 131.4, 128.7, 127.8, 119.6, 73.1,
61.5, 52.9, 52.6, 50.6, 39.4, 32.8, 29.7, 26.9, 20.1, 12.8,
12.1. Anal. Calcd for C27H37N3O5S: C, 62.89; H, 7.23;
N, 8.15. Found: C, 62.65; H, 6.96; N, 8.14.
5.4.1.
N-[4-[[4-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]methyl]-
phenyl]methanesulfonamide (4a). Yield: 55%, white solid,
mp 80–82 ꢁC. 1H NMR d: 7.27 (d, 2H, J = 8.5 Hz), 7.15
(d, 2H, J = 8.5 Hz), 3.64 (s, 3H), 3.47 (s, 2H), 3.42 (s,
2H), 2.98 (s, 3H), 2.83 (t, 2H, J = 6.7 Hz), 2.47–2.38
(m, 8H), 2.16 (s, 3H), 2.08 (s, 3H), 1.74 (t, 2H,
J = 6.7 Hz), 1.28 (s, 6H). 13C NMR d: 150.5, 148.0,
135.5, 130.3, 127.6, 125.9, 125.0, 120.9, 119.1, 72.9,
62.2, 61.5, 53.4, 52.8, 39.2, 32.9, 26.9, 20.1, 12.8, 12.0.
MS m/z: 234 (100%), 219, 179. Anal. Calcd for
C27H39N3O4S: C, 64.64; H, 7.84; N, 8.38. Found: C,
64.36; H, 7.56; N, 8.16.
5.5. General procedure for the synthesis of final phen-
ylmethanesulfonamides (method D)
To a solution of the appropriate phenylmethanesulfona-
mide (0.1 mmol) in 4 mL anhyd CH2Cl2 was added, at
0 ꢁC, BF3ÆS(CH3)2 (1 mmol) and the mixture was stirred
at ambient temperature for 24 h. The solvent is then
evaporated under argon, and AcOEt and water were
added. The organic layer was dried and evaporated to
dryness. The residue was purified by column chromatog-
raphy (CH2Cl2/CH3OH 9.5:1.5).
5.4.2. N-[4-[2-[4-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetra-
methyl- 2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]eth-
yl]phenyl]methanesulfonamide (4b). Yield 80%, white
5.5.1.
N-[4-[[4-[(3,4-Dihydro-6-hydroxy-2,2,7,8-tetra-
1
solid, mp 152–155 ꢁC. H NMR d: 7.15–7.13 (m, 4H),
methyl-2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]methyl]-
phenyl]methanesulfonamide (5a). Yield: 24%, white solid,
3.65 (s, 3H), 3.48 (s, 2H), 2.96 (s, 3H, –NHSO2CH3),
2.84 (t, 2H, J = 6.7 Hz), 2.75–2.70 (m, 2H), 2.53–2.51
(m, 10H,), 2.17 (s, 3H), 2.09 (s, 3H), 1.75 (t, 2H,
J = 6.7 Hz), 1.29 (s, 6H). 13C NMR d: 150.5, 148.0,
137.9, 134.7, 129.8, 127.6, 125.9, 125.1, 121.6, 119.1,
72.9, 61.5, 60.2, 53.4, 52.8, 39.1, 32.9, 26.9, 20.1, 12.8,
12.1. MS m/z: 461, 234 (100%), 219, 179. Anal. Calcd
for C28H41N3O4S: C, 65.21; H, 8.01; N, 8.15. Found:
C, 64.82; H, 8.35; N, 7.73.
1
mp 212–215 ꢁC. H NMR d: 7.30 (d, 2H, J = 8.5 Hz),
7.17 (d, 2H, J = 8.5 Hz), 3.63 (s, 2H), 3.50 (s, 2H),
3.00 (s, 3H), 2.61–2.56 (m, 10H), 2.12 (s, 3H), 2.08 (s,
3H), 1.74 (t, 2H, J = 6.7 Hz), 1.25 (s, 6H). 13C NMR
d: 148.7, 144.4, 130.4, 124.9, 120.8, 115.7, 114.8, 72.2,
61.9, 56.3, 52.7, 39.4, 33.0, 29.7, 26.6, 20.7, 11.8, 11.7.
MS m/z: 487 (M+), 438, 257, 219 (100%). Anal. Calcd
for C26H37N3O4S: C, 64.04; H, 7.65; N, 8.62. Found:
C, 63.88; H, 7.35; N, 8.97.
5.4.3. N-[4-[2-[4-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]eth-
oxy]phenyl]methanesulfonamide (4c). Yield: 74%, white
5.5.2. N-[4-[2-[4-[(3,4-Dihydro-6-hydroxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]eth-
yl]phenyl]methanesulfonamide (5b). Yield: 41%, white
solid, mp 185–187 ꢁC. 1H NMR d: 7.24 (d, 2H,
J = 8.5 Hz), 7.07 (d, 2H, J = 8.5 Hz), 3.78 (s, 2H), 3.61–
3.59 (m, 2H), 3.15–3.13 (m, 2H), 3.06–3.03 (m, 8H),
2.93 (s, 3H), 2.58 (t, 2H, J = 6.7 Hz), 2.08 (s, 3H), 2.06
(s, 3H), 1.72 (t, 2H, J = 6.7 Hz), 1.24 (s, 6H). 13C NMR
d: 147.8, 145.1, 136.7, 132.4, 129.7, 126.1, 121.5, 116.3,
113.9, 72.5, 65.0, 58.0, 55.2, 51.8, 48.9, 39.2, 32.8, 29.4,
26.6, 20.8, 12.0, 11.9. Anal. Calcd for C27H39N3O4S: C,
64.64; H, 7.84; N, 8.38. Found: C, 64.78; H, 8.10; N, 8.53.
1
viscous oil. H NMR d: 7.17 (d, 2H, J = 8.5 Hz), 6.83
(d, 2H, J = 8.5 Hz), 4.10 (t, 2H, J = 5.5 Hz), 3.62 (s,
3H), 3.47 (s, 2H), 2.91 (s, 3H), 2.83 (t, 2H, J = 6.7 Hz),
2.77 (t, 2H, J = 5.5 Hz), 2.52–2.50 (m, 8H), 2.16 (s, 3H),
2.08 (s, 3H), 1.73 (t, 2H, J = 6.7 Hz), 1.28 (s, 6H). 13C
NMR d: 160.0, 150.7, 129.4, 124.7, 119.1, 115.5, 73.1,
65.9, 65.0, 61.4, 56.8, 53.4, 52.7, 52.1, 38.9, 32.8, 26.9,
20.2, 12.8, 12.1. Anal. Calcd for C28H41N3O5SÆH2O: C,
61.18; H, 7.88; N, 7.64. Found: C, 61.27; H, 7.58; N, 7.53.
5.4.4.
N-[4-[4-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]phenyl]-
methanesulfonamide (4d). Yield: 82%, white viscous oil.
1H NMR d: 7.13 (d, 2H, J = 9.1 Hz), 6.85 (d, 2H,
J = 9.1 Hz), 3.66 (s, 3H), 3.53 (s, 2H), 3.10–3.08 (m, 4H),
2.91 (s, 3H), 2.87 (t, 2H, J = 6.7 Hz), 2.60–2.58 (m, 4H),
2.19 (s, 3H), 2.10 (s, 3H), 1.77 (t, 2H, J = 6.7 Hz), 1.29
(s, 6H). 13C NMR d: 150.5, 150.0, 148.1, 127.6, 125.3,
124.5, 119.1, 116.5, 73.0, 61.6, 52.7, 49.2, 38.7, 32.9,
26.9, 20.1, 12.8, 12.1. Anal. Calcd for C26H37N3O4S: C,
64.04; H, 7.65; N, 8.62. Found: C, 63.75; H, 7.73; N, 8.66.
5.5.3. N-[4-[2-[4-[(3,4-Dihydro-6-hydroxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]eth-
oxy]phenyl]methanesulfonamide (5c). Yield: 36%,
yellowish solid, mp 202–204 ꢁC. H NMR d: 7.17 (d,
2H, J = 9.1 Hz), 6.87 (d, 2H, J = 9.1 Hz), 4.09–4.07–
4.05 (m, 2H), 3.65 (s, 2H), 2.93 (s, 3H), 2.89–2.86 (m,
2H), 2.71–2.69 (m, 8H), 2.59 (t, 2H, J = 6.7 Hz), 2.12
(s, 3H), 2.09 (s, 3H), 1.75 (t, 2H, J = 6.7 Hz), 1.26 (s,
6H). 13C NMR d: 156.5, 148.2, 144.5, 130.0, 125.0,
124.2, 122.5, 115.9, 115.3, 114.8, 72.3, 65.6, 56.8, 56.1,
53.4, 53.2, 51.8, 38.5, 32.9, 26.5, 20.6, 11.8, 11.6. Anal.
Calcd for C27H39N3O5S: C, 62.64; H, 7.59; N, 8.12.
Found: C, 63.02; H, 7.97; N, 8.36.
1
5.4.5.
N-[4-[[4-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)methyl]piperazin-1-ylcar-
bonyl]phenyl]methanesulfonamide (4e). Yield: 50%,
1
yellowish solid, mp 194–196 ꢁC. H NMR d: 7.34 (d,
5.5.4. N-[4-[4-[(3,4-Dihydro-6-hydroxy-2,2,7,8-tetrameth-
yl-2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]phenyl]-
methanesulfonamide (5d). Yield: 33%, yellowish viscous
2H, J = 8.5 Hz), 7.21 (d, 2H, J = 8.5 Hz), 3.67–3.65
(m, 2H), 3.62 (s, 3H), 3.49 (s, 2H), 3.33–3.31 (m, 2H)
2.97 (s, 3H), 2.83 (t, 2H, J = 6.7 Hz), 2.52–2.50 (m,
1
oil. H NMR d: 7.16 (d, 2H, J = 8.5 Hz), 6.87 (d, 2H,

6674
M. Koufaki et al. / Bioorg. Med. Chem. 14 (2006) 6666–6678
J = 8.5 Hz), 3.72 (s, 2H), 3.22–3.20 (m, 4H) 2.93 (s, 3H),
2.70–2.68 (m, 4H), 2.62 (t, 2H, J = 6.7 Hz) 2.14 (s, 3H),
2.10 (s, 3H), 1.77 (t, 2H, J = 6.7 Hz), 1.28 (s, 6H). 13C
NMR d: 150.3, 148.9, 148.0, 126.9, 125.1, 124.0, 119.3,
115.8, 72.8, 62.1, 52.5, 38.8, 32.9, 26.6, 20.8, 11.9, 11.6.
Anal. Calcd for C25H35N3O4S: C, 63.40; H, 7.45; N,
8.87. Found: C, 63.77; H, 7.06; N, 8.51.
10 min at 0 ꢁC, NaBH3CN (0.018 g, 0.29 mmol) was
added and the mixture was stirred at ambient tempera-
ture for 24 h. After completion of the reaction the mix-
ture was poured into ice and NaOH 2 N was added until
pH 6. The mixture was then extracted with AcOEt, the
organic layer was washed with satd aqueous NaCl, dried
and the solvent evaporated. The residue was purified by
column chromatography (AcOEt/pet. ether 8:2) Yield:
1
5.5.5.
N-[4-[[4-[(3,4-Dihydro-6-hydroxy-2,2,7,8-tetra-
0.035 g (25%), orange viscous oil. H NMR d: 8.12 (d,
methyl-2H-1-benzopyran-5-yl)methyl]piperazin-1-yl]car-
bonyl]phenyl]methanesulfonamide (5e). Yield: 54%, white
solid, mp 124–126 ꢁC. 1H NMR d: 7.34 (d, 2H,
J = 8.5 Hz), 7.22 (d, 2H, J = 8.5 Hz), 3.67–3.65 (m,
2H), 3.65 (s, 2H), 3.47–3.45 (m, 2H), 3.00 (s, 3H),
2.61–2.56 (m, 6H), 2.12 (s, 3H), 2.09 (s, 3H), 1.75 (t,
2H, J = 6.7 Hz), 1.28 (s, 6H). 13C NMR d: 169.6,
148.3, 138.7, 131.6, 128.9, 119.5, 115.8, 114.4, 72.3,
65.0, 56.1, 53.4, 52.3, 39.7, 33.0, 26.6, 20.8, 11.9, 11.7.
Anal. Calcd for C26H35N3O5S: C, 62.25; H, 7.03; N,
8.38. Found: C, 62.27; H, 7.36; N, 8.66.
2H, J = 8.5 Hz), 6.82 (d, 2H, J = 8.5 Hz), 3.67 (s, 3H),
3.49–3.47 (m, 4H), 2.82–2.79 (m, 2H), 2.71–2.52 (m,
8H), 2.17 (s, 3H), 2.07 (s, 3H), 1.76 (t, 2H,
J = 6.7 Hz), 1.27 (s, 6H). Anal. Calcd for C26H35N3O4:
C, 68.85; H, 7.78; N, 9.26. Found: C, 69.11; H, 7.87;
N, 9.64.
5.9. 4-[4-[2-(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-1-benzopyran-5-yl)ethyl]piperazin-1-yl]aniline (10)
This compound was prepared using method B. Yield:
1
0.028 g (98%), yellow viscous oil. H NMR d: 6.82 (d,
5.6. (3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-1-
benzopyran-5)-vinyl-methyl-ether (7)
2H, J = 8.5 Hz), 6.61 (d, 2H, J = 8.5 Hz), 3.75–3.73
(m, 2H), 3.67 (s, 3H), 3.12–3.10 (m, 2H), 2.82–2.80 (m,
2H), 2.73–2.51 (m, 8H), 2.17 (s, 3H), 2.07 (s, 3H), 1.76
(t, 2H, J = 6.7 Hz), 1.25 (s, 6H).
To a solution of Ph3P+CH2OCH3ClÀ (1.35 g, 3.9 mmol)
in 8 mL anhyd THF was added, at 0 ꢁC, t-BuOK
(0.295 g, 2.6 mmol), and the red mixture was stirred at
0 ꢁC for 15 min. A solution of aldehyde 6 (0.320 g,
1.29 mmol) in 8 mL THF was then added and the mix-
ture was stirred at 0 ꢁC for 15 min and at ambient tem-
perature for 24 h. Satd aqueous NaHCO3 was then
added followed by extraction with diethyl ether. The
organic layer was washed with satd aqueous NaCl, dried
and the solvent was evaporated. Purification by column
chromatography (pet ether/AcOEt 8:2) afforded a mix-
ture of cis/trans isomers. Yield: 0.330 g (93%), yellow
5.10. N-[4-[4-[2-(3,4-Dihydro-6-methoxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)ethyl]piperazin-1-ylphe-
nyl]methanesulfonamide (11)
This compound was prepared using method C. Yield:
1
0.029 g (41%) yellowish viscous oil. H NMR d: 7.15
(d, 2H, J = 8.5 Hz), 6.90 (d, 2H, J = 8.5 Hz), 3.67 (s,
3H), 3.26–3.23 (m, 4H), 2.93 (s, 3H), 2.86–2.81 (m,
2H) 2.73–2.68 (m, 6H), 2.56–2.52 (m, 2H), 2.17 (s,
3H), 2.07 (s, 3H), 1.77 (t, 2H, J = 6.7 Hz), 1.29 (s,
6H). 13C NMR d: 149.9, 148.2, 128.2, 124.6, 124.1,
117.0, 116.7, 72.8, 61.2, 58.4, 53.0, 48.9, 38.9, 32.9,
26.9, 23.9, 20.4, 12.8, 11.9. Anal. Calcd for C27H39N3O4-
SÆH2O: C, 62.40; H, 7.95; N, 8.09. Found: C, 61.98, H,
7.58; N, 7.73.
oil. 1H NMR
d (trans isomer): 7.09 (d, 1H,
J = 13.4 Hz, –CH@CH–OCH3), 5.71 (d, 1H, J =
13.4 Hz, –CH@CH–OCH3), 3.72 (s, 3H, –CH@CH–
OCH3), 3.61 (s, 3H), 2.69 (t, 2H, J = 6.7 Hz), 2.20 (s,
3H), 2.11 (s, 3H), 1.77 (t, 2H, J = 6.7 Hz), 1.31 (s, 6H).
5.7. 3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-1-
benzopyran-5-acetaldehyde (8)
5.11. N-[4-[4-[2-(3,4-Dihydro-6-hydroxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)ethyl]piperazin-1-yl]phen-
yl]methanesulfonamide (12)
To a solution of compound 7 (0.330 g, 1.19 mmol) in
25 mL 1,4-dioxane and 13 mL H2O, a catalytic amount
of p-toluenesulfonic acid was added and the mixture is
refluxed for 24 h. After completion of the reaction, the
mixture was extracted with diethyl ether and the organic
layer was washed with satd aqueous NaCl, dried, and
This compound was prepared using method C. Yield:
0.010 g (40%), yellowish solid, mp 202–205 ꢁC. 1H
NMR d: 7.15 (d, 2H, J = 8.5 Hz), 6.91 (d, 2H,
J = 8.5 Hz), 3.28–3.25 (m, 4H), 2.93 (s, 3H), 2.85–2.81
(m, 2H), 2.73–2.68 (m, 6H), 2.56–2.52 (m, 2H), 2.17 (s,
3H), 2.07 (s, 3H), 1.77 (t, 2H, J = 6.7 Hz), 1.28 (s,
6H). 13C NMR d: 149.2, 148.2, 128.0, 124.6, 123.8,
117.0, 116.4, 72.5, 61.0, 58.4, 48.6, 38.9, 32.9, 26.9,
23.7, 20.4, 12.1, 11.9. Anal. Calcd for C26H37N3O4S:
C, 64.04; H, 7.65; N, 8.62. Found: C, 63.77; H, 7.73;
N, 8.66.
1
the solvent was evaporated. Yield: 0.310 g (100%). H
NMR d: 10.49 (s, 1H, –CHO), 3.76 (s, 3H), 3.59 (s,
2H, ArCH2CHO–), 3.09 (t, 2H, J = 6.5 Hz), 2.20 (s,
3H), 2.16 (s, 3H), 1.73 (t, 2H, J = 6.2 Hz), 1.29 (s, 6H).
5.8. 1-[2-(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-1-benzopyran-5-yl)ethyl]-4-(4-nitrophenyl)piperazine (9)
5.12. 3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-5-(2-
nitroethenyl)-2H-1-benzopyran (13)
To a solution of compound 8 (0.094 g, 0.36 mmol) in
2 mL CH3COOH and 2 mL CH3CN, at 0 ꢁC, a solution
of 1-(4-nitrophenyl)piperazine (0.049 g, 0.24 mmol) in
2 mL CH3COOH was added and after stirring for
Aldehyde 6 (0.200 g, 0.8 mmol) was added in a mixture
of 3 mL anhyd CH3NO2 and cat amount of

M. Koufaki et al. / Bioorg. Med. Chem. 14 (2006) 6666–6678
6675
CH3COONH4. The mixture was stirred at 100 ꢁC for
2 h. The solvent is then evaporated and H2O and a mix-
ture of diethyl ether/CH2Cl2 9:1 were added. The organ-
ic layer was washed with H2O (2· 50 mL), HCl 3 N (2·
25 mL), and satd aqueous NaCl, dried, and the solvent
was evaporated. Yield: 0.223 g (96%), yellow solid, mp
101–103 ꢁC. 1H NMR d: 8.24 (d, 1H, J = 13.4 Hz),
7.95 (d, 1H, J = 13.4 Hz), 3.62 (s, 3H), 2.84 (t, 2H,
J = 6.7 Hz), 2.19 (s, 3H), 2.13 (s, 3H), 1.83 (t, 2H,
J = 6.7 Hz), 1.31 (s, 6H) MS m/z: 291 (M+, 100%).
5.15. General procedure for the synthesis of methylamines
16a,b (method F)
To 0.2 mmol of the appropriate secondary amine
were added at 0 ꢁC HCOOH (1 mL) and HCHO
36% in water (0.05 mL) and the mixture was heated
at 100 ꢁC for 2 h. NaOH 3 N was then added until
pH 8 and the mixture was extracted with AcOEt.
The organic layer was washed with satd aqueous
NH4Cl, sat aqueous NaCl, dried, and evaporated to
dryness.
5.13. 2-(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-
1-benzopyran-5-yl)ethylamine (14)
5.15.1. N-Methyl-N-[2-(4-nitrophenyl)ethyl]-2-(3,4-dihy-
dro-6-methoxy-2,2,7,8-tetramethyl-2H-1-benzopyran-5-
yl)ethylamine (16a). Yield: 93%, yellowish viscous oil.
1H NMR d: 8.13 (d, 2H, J = 8.5 Hz), 7.35 (d, 2H,
J = 8.5 Hz), 3.66 (s, 3H), 2.93–2.88 (m, 2H) 2.77–
2.71 (m, 4H), 2.67 (t, 2H, J = 6.7 Hz), 2.57–2.52 (m,
2H), 2.43 (s, 3H, –N(CH3)–) 2.17 (s, 3H), 2.08 (s,
3H), 1.76 (t, 2H, J = 6.7 Hz), 1.28 (s, 6H). 13C
NMR d: 149.7, 148.6, 148.2, 146.4, 129.5, 128.1,
127.7, 124.0, 123.6, 116.8, 72.8, 61.1, 58.5, 57.0,
42.1, 33.7, 32.9, 26.8, 24.3, 20.3, 12.8, 11.9. MS m/z:
246 (100%), 234, 218. Anal. Calcd for C25H34N2O4:
C, 70.39; H, 8.03; N, 6.57. Found: C, 70.82; H,
8.35; N, 6.73.
To a slurry of LiAlH4 (0.087 g, 2.31 mmol) in 20 mL an-
hyd THF was added dropwise, at 0 ꢁC, a solution of
compound 13 (0.223 g, 0.77 mmol) in 20 mL anhyd
THF and the mixture was refluxed for 2 h. Some drops
of THF/H2O (1:1) were then added to destroy the excess
of LiAlH4. The mixture was diluted in AcOEt and
Na2SO4 was added. Filtration through Celite and evap-
oration of the solvent afforded the desired amine. Yield:
0.200 g (98%), yellow oil. 1H NMR d: 3.62 (s, 3H), 2.83–
2.79 (m, 2H), 2.75–2.72 (m, 2H), 2.67 (t, 2H,
J = 6.7 Hz), 2.15 (s, 3H), 2.06 (s, 3H), 1.75 (t, 2H,
J = 6.7 Hz), 1.31 (s, 6H).
5.14. General procedure for the synthesis of secondary
amines 15a,b (method E)
5.15.2.
N-Methyl-N-[2-(4-nitrophenoxy)ethyl]-2-(3,4-
dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-1-benzopyran-
5-yl)ethylamine (16b). Yield: 93%, yellowish viscous oil.
1H NMR d: 8.17 (d, 2H, J = 9.1 Hz), 6.96 (d, 2H,
J = 9.1 Hz), 4.17 (t, 2H, J = 5.5 Hz) 3.65 (s, 3H),
2.93 (t, 2H, J = 5.5 Hz) 2.82–2.77 (m, 2H), 2.68 (t,
2H, J = 6.7 Hz), 2.62–2.56 (m, 2H), 2.48 (s, 3H),
2.17 (s, 3H), 2.07 (s, 3H), 1.76 (t, 2H, J = 6.7 Hz),
1.28 (s, 6H). 13C NMR d: 163.9, 149.7, 148.2, 141.5,
132.0, 128.1, 127.5, 125.8, 124.0, 116.8, 114.5, 72.8,
67.1, 65.0, 61.1, 57.8, 55.7, 50.8, 42.8, 32.9, 26.8,
24.3, 20.3, 12.8, 11.9. Anal. Calcd for C25H34N2O5:
C, 67.85; H, 7.74; N, 6.33. Found: C, 67.92; H,
7.58; N, 6.32.
To a solution of 4-nitrophenethyl- or 2-(4-nitrophen-
oxy)ethylbromide (0.5 mmol) in 5 mL anhyd CH3CN
was added, at 0 ꢁC, K2CO3 (0.75 mmol), a solution of
amine 14 (0.5 mmol) in 5 mL anhyd CH3CN, and a cata-
lytic amount of TBAI, and the mixture was stirred at
50 ꢁC for 24 h. The solvent was then evaporated and the
residue was extracted with AcOEt and H2O. The organic
layer was washed with satd aqueous NaCl, dried, and
evaporated to dryness. The residue was purified by
column chromatography (CH2Cl2/CH3OH 9.5:1.5).
5.14.1. N-[2-(4-Nitrophenyl)ethyl]-2-(3,4-dihydro-6-meth-
oxy-2,2,7,8-tetramethyl-2H-1-benzopyran-5-yl)ethyla-
mine (15a). Yield: 36%, yellow viscous oil. H NMR d:
8.11 (d, 2H, J = 9.1 Hz), 7.31 (d, 2H, J = 9.1 Hz), 3.62
(s, 3H), 2.93–2.90 (m, 4H), 2.78–2.76 (m, 4H), 2.66 (t,
2H, J = 6.7 Hz), 2.15 (s, 3H), 2.07 (s, 3H), 1.75 (t, 2H,
J = 6.7 Hz), 1.27 (s, 6H). 13C NMR d: 149.7, 148.2,
148.0, 146.5, 129.5, 128.1, 127.4, 124.2, 123.6, 117.0,
72.8, 60.9, 50.3, 49.6, 36.1, 32.8, 26.9, 26.8, 20.4, 12.8,
11.9.
5.16. 4-[2-[N-Methyl-2-(3,4-dihydro-6-methoxy-2,2,7,8-
tetramethyl-2H-1-benzopyran-5-yl)ethylamine]ethyl]ani-
line (17a)
1
This compound was synthesized using method B. Yield:
1
85%, yellowish viscous oil. H NMR d: 7.00 (d, 2H,
J = 7.9 Hz), 6.62 (d, 2H, J = 7.9 Hz), 3.67 (s, 3H),
2.83–2.68 (m, 8H), 2.66 (t, 2H, J = 6.7 Hz), 2.45 (s,
3H) 2.18 (s, 3H), 2.08 (s, 3H), 1.78 (t, 2H, J = 6.7 Hz),
1.29 (s, 6H).
5.14.2.
N-[2-(4-Nitrophenoxy)ethyl]-2-(3,4-dihydro-6-
methoxy-2,2,7,8-tetramethyl-2H-1-benzopyran-5-yl)eth-
ylamine (15b). Yield: 42%, yellowish viscous oil. 1H
NMR d: 8.17 (d, 2H, J = 9.1 Hz), 6.93 (d, 2H,
J = 9.1 Hz), 4.16 (t, 2H, J = 4.9 Hz, –CH2CH2O–) 3.65
(s, 3H), 3.08 (t, 2 H, J = 4.9 Hz, –CH2CH2O–) 2.83
(m, 4H, ArCH2CH2NH–), 2.69 (t, 2H, J = 6.7 Hz),
2.16 (s, 3H), 2.07 (s, 3H), 1.76 (t, 2H, J = 6.7 Hz), 1.27
(s, 6H). 13C NMR d: 163.9, 149.7, 148.2, 141.5, 128.1,
127.3, 125.9, 124.2, 117.0, 114.5, 72.8, 68.3, 65.0, 49.9,
48.2, 32.8, 27.1, 26.9, 20.4, 12.8, 11.9.
5.17. 4-[2-[N-Methyl-2-(3,4-dihydro-6-methoxy-2,2,7,8-
tetramethyl- 2H-1-benzopyran-5-yl)ethylamine]ethoxy]-
aniline (17b)
This compound was synthesized using method B. Yield:
1
97%, yellowish viscous oil. H NMR d: 6.75 (d, 2H,
J = 8.5 Hz), 6.63 (d, 2H, J = 8.5 Hz), 4.06 (t, 2H,
J = 5.5 Hz), 3.66 (s, 3H), 2.90–2.62 (m, 8H), 2.49 (s,
3H, –NCH3), 2.17 (s, 3H), 2.07 (s, 3H), 1.76 (t, 2H,
J = 6.7 Hz), 1.28 (s, 6H).

6676
M. Koufaki et al. / Bioorg. Med. Chem. 14 (2006) 6666–6678
5.18. N-[4-[2-[[2-(3,4-Dihydro-6-methoxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)ethyl]methylamine]ethyl]-
phenyl]methanesulfonamide (18a)
5.22. 3,4-Dihydro-6-methoxy-5-[(6-nitro-2,3-dihydro-1H-
indol-1-yl)methyl]-2,2,7,8-tetramethyl-2H-1-benzopyran
(20)
This compound was synthesized using method C. Yield:
70%, yellowish viscous oil. H NMR d: 7.17–7.15 (m,
The synthesis of this compound was carried out follow-
ing method A. Yield: 93%, orange solid, mp 145–147 ꢁC.
1H NMR d: 7.52 (d, 1H, J = 7.93 Hz), 7.34 (s, 1H), 7.08
(d, 1H, J = 7.9 Hz), 4.26 (s, 2H), 3.64 (s, 3H), 3.28 (t,
2H, J = 8.6 Hz), 2.91 (t, 2H, J = 8.6 Hz), 2.74 (t, 2H,
J = 6.7 Hz), 2.20 (s, 3H), 2.12 (s, 3H), 1.75 (t, 2H,
J = 6.7 Hz), 1.29 (s, 6H). 13C NMR d: 156.2, 153.4,
150.4, 148.6, 148.2, 138.2, 128.2, 126.1, 124.0, 118.2,
113.3, 100.2, 73.1, 61.5, 52.4, 43.6, 38.5, 32.7, 28.2,
26.9, 20.0, 12.9, 12.1. MS m/z: 396 (M+), 233, 217. Anal.
Calcd for C23H28N2O4: C, 69.68; H, 7.12; N, 7.07.
Found: C, 69.96; H, 6.92; N, 7.32.
1
4H), 3.66 (s, 3H), 2.97 (s, 3H, –NHSO2CH3), 2.81–
2.55 (m, 10H), 2.45 (s, 3H, –NCH3), 2.17 (s, 3H), 2.07
(s, 3H), 1.76 (t, 2H, J = 6.7 Hz), 1.27 (s, 6H). 13C
NMR d: 149.7, 148.2, 137.9, 134.6, 129.9, 128.1, 127.6,
124.0, 121.5, 116.9, 72.8, 61.1, 59.0, 56.9, 42.0, 39.2,
32.9, 26.9, 24.1, 20.3, 12.8, 11.9. Anal. Calcd for
C26H38N2O4S: C, 65.79; H, 8.07; N, 5.90. Found: C,
65.92; H, 7.69; N, 6.04.
5.19. N-[4-[2-[[2-(3,4-Dihydro-6-methoxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)ethyl]methylamine]ethoxy]-
phenyl]methanesulfonamide (18b)
5.23. 3,4-Dihydro-6-methoxy-5-[(6-nitro-2,3-dihydro-1H-
indol-1-yl)ethyl]-2,2,7,8-tetramethyl-2H-1-benzopyran
(21)
This compound was synthesized using method C. Yield:
66%, yellowish solid, mp 125–127 ꢁC.
Aldehyde 8 was treated with 6-nitroindoline as described
for analogue 9. Yield: 35%, yellowish viscous oil 1H
NMR d: 7.50 (d, 1H, J = 7.9 Hz), 7.26 (s, 1H), 7.07 (d,
1H, J = 7.9 Hz), 3.72 (s, 3H), 3.62 (t, 2H, J = 8.6 Hz),
3.28 (t, 2H, J = 7.9 Hz), 3.05 (t, 2H, J = 8.6 Hz), 2.82
(t, 2H, J = 7.9 Hz), 2.72 (t, 2H, J = 6.7 Hz), 2.21 (s,
3H), 2.09 (s, 3H), 1.79 (t, 2H, J = 6.7 Hz), 1.29 (s,
6H). 13C NMR d: 155.8, 152.9, 150.7, 148.0, 136.3,
128.4, 125.1, 122.3, 118.5, 112.3, 101.2, 72.9, 61.0,
53.5, 44.6, 37.2, 33.5, 31.8, 28.4, 25.9, 20.3, 12.6, 11.9.
1H NMR d: 7.17 (d, 2H, J = 8.5 Hz), 6.86 (d, 2H,
J = 8.5 Hz), 4.07 (t, 2H, J = 5.5 Hz), 3.64 (s, 3H), 2.91
(s, 3H), 2.89–2.85 (m, 2H), 2.81–2.76 (m, 2H), 2.66 (t,
2H, J = 6.7 Hz), 2.60–2.56 (m, 2H), 2.46 (s, 3H) 2.15
(s, 3H), 2.05 (s, 3H), 1.77 (t, 2H, J = 6.7 Hz), 1.26 (s,
6H). 13C NMR d: 157.3, 149.7, 148.2, 129.1, 128.1,
127.5, 124.7, 124.0, 116.9, 115.5, 72.8, 66.4, 61.1, 57.8,
55.9, 53.4, 42.7, 38.9, 32.9, 26.9, 24.1, 20.3, 12.8, 11.9.
MS m/z: 428, 246 (100%), 231. Anal. Calcd for
C26H38N2O5S: C, 63.65; H, 7.81; N, 5.71. Found: C,
63.88; H, 7.58; N, 5.35.
5.24. N-{1-[(3,4-Dihydro-6-hydroxy-2,2,7,8-tetramethyl-
2H-1-benzopyran-5-yl)methyl]-2,3-dihydro-1H-indol-6-
yl}methanesulfonamide (22)
5.20. N-[4-[2-[[2-(3,4-Dihydro-6-hydroxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)ethyl]methylamine]ethyl]-
phenyl]methanesulfonamide (19a)
The synthesis of this compound was carried out using
method D. Yield: 33%, yellowish solid, mp 149–
1
This compound was synthesized using method D. Yield:
152 ꢁC. H NMR d: 7.09 (d, 1H, J = 7.9 Hz), 6.70 (d,
1
53%, yellowish viscous oil. H NMR d: 7.12–7.10 (m,
1H, J = 6.7 Hz), 6.59 (s, 1H), 4.25 (s, 2H), 3.30 (t, 2H,
J = 7.9 Hz), 2.94 (m, 5H), 2.66 (t, 2H, J = 6.7 Hz),
2.14 (s, 3H), 2.09 (s, 3H), 1.78 (t, 2H, J = 6.7 Hz), 1.29
(s, 6H). 13C NMR d: 153.3, 151.0, 149.7, 144.5, 135.6,
124.2, 121.5, 120.0, 118.3, 117.6, 106.2, 100.3, 72.9,
61.5, 53.1, 40.1, 38.5, 31.4, 27.7, 26.9, 20.5, 12.1, 11.8
Anal. Calcd for C23H30N2O4S: C, 64.16; H, 7.02; N,
6.51. Found: C, 63.88; H, 6.97; N, 6.35.
4H), 2.96 (s, 3H), 2.76–2.67 (m, 8H), 2.61 (t, 2H,
J = 6.7 Hz), 2.45 (s, 3H) 2.16 (s, 3H), 2.10 (s, 3H), 1.77
(t, 2H, J = 6.7 Hz), 1.27 (s, 6H). 13C NMR d: 148.1,
145.0, 137.8, 135.0, 129.9, 128.0, 127.5, 123.9, 121.4,
115.7, 72.2, 60.1, 53.4, 42.2, 39.2, 33.2, 26.7, 24.0, 21.1,
12.5, 12.0. Anal. Calcd for C25H36N2O4S: C, 65.19; H,
7.88; N, 6.08. Found: C, 64.82; H, 7.73; N, 6.41.
5.21. N-[4-[2-[[2-(3,4-Dihydro-6-hydroxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran-5-yl)ethyl]methylamine]ethoxy]-
phenyl]methanesulfonamide (19b)
5.25. N-{1-[(3,4-Dihydro-6-hydroxy-2,2,7,8-tetramethyl-
2H-1-benzopyran-5-yl)ethyl]-2,3-dihydro-1H-indol-6-
yl}methanesulfonamide (23)
This compound was synthesized using method D. Yield:
37%, yellowish viscous oil. H NMR d: 7.15 (d, 2H,
This compound was synthesized using method D. Yield:
29%, yellowish viscous oil. H NMR d: 7.01 (d, 1H,
1
1
J = 8.5 Hz), 6.79 (d, 2H, J = 8.5 Hz), 4.08 (t, 2H,
J = 5.5 Hz), 2.93 (s, 3H), 2.90–2.87 (m, 2H), 2.82–2.78
(m, 4H), 2.59 (t, 2H, J = 6.7 Hz), 2.54 (s, 3H) 2.14 (s,
3H), 2.05 (s, 3H), 1.74 (t, 2H, J = 6.7 Hz), 1.25 (s,
6H). 13C NMR d: 151.2, 149.7, 148.2, 129.5, 128.0,
127.3, 125.1, 124.0, 116.7, 115.3, 72.8, 66.4, 61.0, 57.8,
55.5, 42.7, 38.9, 32.9, 26.9, 24.3, 20.3, 12.2, 11.9. Anal.
Calcd for C25H36N3O5S: C, 63.00; H, 7.61; N, 5.88.
Found: C, 62.79; H, 8.01; N, 5.53.
J = 7.9 Hz), 6.52 (d, 1H, J = 6.7 Hz), 6.46 (s, 1H), 3.46
(t, 2H, J = 7.9 Hz), 3.27 (t, 2H, J = 6.7 Hz), 2.93 (m,
7H), 2.70 (t, 2H, J = 6.7 Hz), 2.12 (s, 3H), 2.09 (s,
3H), 1.81 (t, 2H, J = 6.7 Hz), 1.29 (s, 6H). 13C NMR
d: 155.3, 151.0, 148.2, 143.5, 133.1, 124.0, 121.1, 120.3,
118.8, 116.3, 107.5, 101.3, 72.5, 61.3, 52.1, 40.3, 38.5,
31.4, 28.5, 27.1, 26.3, 20.5, 12.4, 11.9 Anal. Calcd for
C24H32N2O4S: C, 64.84; H, 7.25; N, 6.30. Found: C,
64.82; H, 7.41; N, 6.73.

M. Koufaki et al. / Bioorg. Med. Chem. 14 (2006) 6666–6678
6677
5.26. Biology
and glucose 11. The pH of this solution is 7.35–7.45
when gassed with 95% O2 and 5% CO2 at 37 ꢁC. The
tip of the papillary muscles from the right ventricle
was prepared and individually mounted in a tissue
chamber (volume ꢀ 50 mL).
5.26.1. Evaluation of the activity of the new compounds
against reperfusion arrhythmias. Male Sprague–Dawley
rats weighing about 300–350 g were housed under con-
trolled light (12L:12D) and temperature with free access
to food and water in compliance with the prescriptions
for the care and use of laboratory animals. Rats were
anesthetized with pentobarbital (30–40 mg per animal).
After intravenous administration of heparin, the chests
were opened the hearts were rapidly excised and mount-
ed on a non-recirculating Langendorff perfusion appara-
tus. Retrograde perfusion was established at a pressure
of 90 cm H2O with an oxygenated normothermic
Krebs–Hensleit bicarbonate (KHB) buffer (25 mmol LÀ1
NaHCO3, 118 mmol LÀ1 NaCl, 2.5 mmol LÀ1 CaCl2,
4.7 mmol LÀ1 KCl, 1.4 mmol LÀ1 MgSO4, and
1.2 mmol LÀ1 KH2PO4, pH 7.2, at 25 ꢁC) supplemented
with 11 mmol LÀ1 glucose and equilibrated with 95%
O2/5% CO2. The temperature of the hearts and perfu-
sates was maintained at 37 ꢁC by the use of a water-
jacketed apparatus. All hearts were equilibrated for
20 min under these conditions. At the end of the
equilibration period, hearts were made ischemic for
10 min by perfusing them with the ischemic KHB
(KHB with Tris–HCl 10 mM, pH 6.4, instead of glucose
and equilibrated with N2 before use) followed by 15 min
of reperfusion. The new compounds were present during
ischemia and reperfusion at a final concentration of
10 lM.
The preparations were continuously stimulated (by HSE
[Hugo Sachs Electronik] Stimulator type 215/II, March-
Hugstetten, Germany) at a basic cycle length of 1000 ms
using 2 ms long rectangular constant voltage pulses iso-
lated from ground and delivered across bipolar platinum
electrodes in contact with the preparation. At least 1 h
was allowed for each preparation to equilibrate while
they were continuously superfused with Tyrode’s solu-
tion. Temperature of the superfusate was kept constant
at 37 ꢁC.
Transmembrane potentials were recorded using conven-
tional microelectrode techniques. Microelectrodes filled
with 3 M KCl and having tip resistances of 5–20 MX
were connected to the input of a high impedance elec-
trometer (Experimetria Microelectrode Amplifier Type),
which was referenced to the ground. The voltage out-
puts from the amplifier was displayed on a dual beam
memory oscilloscope (Tektonix 2230 100 MHz Digital
Storage Oscilloscope, Beaverton, OR, USA).
The maximal diastolic potential (MDP) (‘resting poten-
tial,’ RP), action potential amplitude (APA), the first
derivative of transmembrane potentials (Vmax) and ac-
tion potential duration measured at 50% and 90% repo-
larization (APD50–90), were obtained using a software
developed in the Department of Pharmacology, Univ,
of Szeged (HSE-APES). After the control measure-
ments, the compounds were added to the tissue bath
at 5 lM concentration and the measurements were pre-
pared after 30–40 min incubation time. When the
impalement was lost during measurement, readjustment
was attempted. If the difference between the original and
the readjusted action potential parameters did not ex-
ceed 15%, the experiment was continued, otherwise it
was terminated.
5.26.2. Evaluation of antiarrhythmic activity. Electrocar-
diograms were recorded during equilibration, ischemia,
and reperfusion. Arrhythmias were scored according
to the Lambeth Convention Guidelines.22 Arrhythmia
scores (AS) were calculated for the first 10 min of reper-
fusion as the percentage of premature beats.
5.26.3. Evaluation of antioxidant activity. At the end of
the perfusions, hearts were ‘freeze-clamped’ between
aluminum tongs, cooled in liquid N2 and after the
removal of the atria, the ventricles were pulverized un-
der liquid N2 and powders were stored at À80 ꢁC. A
portion of the tissue powder was analyzed for malondi-
aldehyde (MDA) content by using the thiobarbituric
acid assay.23 To prevent auto-oxidation of the samples,
homogenization was carried out at 4 ꢁC in nitrogen
equilibrated solution in the presence of 0.04% butylated
hydroxytoluene, 1.6% ethanol. The values were ex-
pressed as nanomoles of TBA reactive substances
(MDA equivalent) per gram of tissue. 1,1,3,3-Tetraeth-
oxypropane (0,0.5,1.0,2.0,4.0,8.0 and 16.0 nmol) served
as external standard. Results are expressed as mean-
Acknowledgments
This work was supported by the Greek General
Secretariat for Research and Technology, grant Bilateral
Collaboration of Greece–Hungary 736, by the National
Institute of Health, DA3801 and by the Hungarian
National Research Foundation, OTKA T-048698.
References and notes
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SEM. Differences between groups were assessed by
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