Enantioselectivity in cardioprotection induced by (S)-(-)-2,2-dimethyl-N- (4′-acetamido-benzyl)-4-spiromorpholone-chromane

Article abstract of DOI:10.1021/jm801459f

This work aimed to determine the enantioselectivity in cardioprotection induced by the racemic mixture of the "archetype" 1a of a new class of spirocyclic-benzopyran derivatives. The racemate was resolved by HPLC and the absolute configuration was accompl

Full text of DOI:10.1021/jm801459f

J. Med. Chem. 2009, 52, 1477–1480
1477
Brief Articles
Enantioselectivity in Cardioprotection induced by (S)-
(-)-2,2-Dimethyl-N-(4′-acetamido-benzyl)-4-spiromorpholone-chromane
Simona Rapposelli,*,† Vincenzo Calderone,‡ Roberto Cirilli,§ Maria Digiacomo,† Cristina Faggi,| Francesco La Torre,§
Mariaelisa Manganaro,† Alma Martelli,‡ and Lara Testai‡
Dipartimento di Scienze Farmaceutiche, UniVersita` di Pisa, Via Bonanno 6, I-56126 Pisa, Italy, Dipartimento di Psichiatria, Neurobiologia,
Farmacologia e Biotecnologie, UniVersita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Istituto Superiore di Sanita`, Dipartimento del Farmaco,
Viale Regina Elena 299, I-00161 Rome, Italy, CRIST, Centro Interdipartimentale di Cristallografia Strutturale, UniVersita` degli Studi di
Firenze Polo Scientifico di Sesto Fiorentino, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Fi), Italy
ReceiVed NoVember 19, 2008
This work aimed to determine the enantioselectivity in cardioprotection induced by the racemic mixture of
the “archetype” 1a of a new class of spirocyclic-benzopyran derivatives. The racemate was resolved by
HPLC and the absolute configuration was accomplished by a combined strategy based on single-crystal
X-ray diffraction and circular dichroism methods. The (S)-(-)-1a enantiomer, evaluated for its anti-ischemic
activity, showed significant cardioprotective effects, whereas the (R)-(+)-1a enantiomer was completely
lacking in anti-ischemic effects.
Introduction
antagonized by the selective mito-KATP channel blocker 5-hy-
droxydecanoic acid (5-HD), thus indicating a clear implication
of such a pharmacological target. Furthermore, the anti-ischemic
effects were not associated with significant hypotensive and
vasorelaxing properties, which represent one of the main limiting
factors for the clinical use of nonselective KATP-openers against
myocardial ischemia.
Bearing in mind these encouraging results, we carried out
further investigations into this class of KATP channel openers.
In particular, the objective of the present work was to verify if
the anti-ischemic activity of this new class of 4-spiro-chromane
derivatives might reside in only one or both enantiomers of the
active compounds previously studied as racemic mixture. To
this purpose, we selected the 2,2-dimethyl-N-(4′-acetamido-
benzyl)-4-spiromorpholone-chromane 1a, whose racemate showed
a high degree of cardioprotection in a Langendorff perfused rat
heart subjected to an ischemia/reperfusion cycle, associated with
very modest direct vasorelaxing activity and with almost
completely negligible hypotensive effects in vivo.6,8
This favorable profile was similar to that shown by diazoxide,
a well-known cardioprotective KATP-opener exhibiting prefer-
ential activity toward mitochondrial channels (at the dose
commonly used in this kind of experimental protocols) and quite
different from that of cromakalim, a very potent KATP-channel
activator of both sarcolemmal and mitochondrial channels, thus
possessing anti-ischemic effects associated with strong vasore-
laxing and hypotensive properties.6,8
In recent years, the incidence of myocardial ischemia has
greatly increased. This pathological event may be limited by
the activation of a protective mechanism that involves many
mediators1-3 such as adenosine, opioids, bradikynin, nitric
oxide, calcium ions, and free radical species. This sort of “self-
defence” mechanism, known as “ischemic preconditioning”
(IPCa), gives myocardiocytes an increased resistance against
ischemia-induced cell damage, resulting in a marked reduction
of the infarct size.
Between the several triggers able to activate IPC, mitochon-
drial ATP-sensitive potassium (mito-KATP) channel is considered
as a major effector in anti-ischemic cardiac protection.4,5 As a
consequence, the activation of this kind of channel seems to be
a challenging target for increasing the resistance of the heart to
tolerate a more prolonged ischemic insult and thus to confer a
“pharmacological pre-conditioning”. Unfortunately, almost all
the potassium channel openers (KCOs) exhibit relevant undes-
ired effects mainly due to their unsatisfactory selectivity toward
the mitochondrial subtype of the KATP channel.
Recent papers of ours6-8 reported the synthesis and phar-
macological evaluation of original spiromorpholine- and spiro-
morpholone-benzopyran derivatives, designed in order to obtain
selective activators of mito-KATP channels. Some compounds
of this series, tested as racemic mixtures, showed appreciable
cardioprotective effects on rat isolated and perfused hearts
submitted to ischemia/reperfusion cycles; this activity was
On this basis, we decided to resolve the racemic mixture of
1a, together with that of its methanesulphonamide analogue
brominated in 6 position (2b) (Figure 1), selected for compara-
tive purposes in order to strengthen the data deriving from their
racemic resolution and to assign the absolute configurations to
the single enantiomers. The direct enantioseparation of 1a and
2b was achieved by high performance liquid chromatography
(HPLC) on a chiral stationary phase (CSP). Single enantiomers,
isolated on a semipreparative scale, were submitted to compara-
tive pharmacological and structural investigations. The absolute
* To whom correspondence should be addressed. Phone: +39-050-
2219582. Fax: +39-050-2219605. E-mail: rappsi@farm.unipi.it.
†
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
‡
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec-
nologie, Universita` di Pisa.
§
Istituto Superiore di Sanita`, Dipartimento del Farmaco.
CRIST, Centro Interdipartimentale di Cristallografia Strutturale, Uni-
|
versita` degli Studi di Firenze Polo Scientifico di Sesto Fiorentino.
a Abbreviations: IPC, ischemic preconditioning; RPP, rate pressure
product; CD, circular dichroism; CSP, chiral stationary phase.
10.1021/jm801459f CCC: $40.75
2009 American Chemical Society
Published on Web 02/12/2009

1478 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Brief Articles
Figure 1. Structures of spirocyclic derivatives 1a and 2b endowed of
anti-ischemic activity.
Figure 3. CD spectra of enantiomers of 1a and 2b in ethanol.
Figure 2. (a) Analytical chromatograms of 1a; (b,c) chromatographic
analysis of enantiomers isolated at semipreparative scale. Column,
Chiralpak IA (250 mm × 4.6 mm I.D); eluent, n-hexane-ethanol 75/
25 (v/v); flow-rate, 1.0 mL min-1; detector, UV (black) and CD (gray)
at 225 nm; temperature, 25 °C.
configuration assignment was accomplished by a combined
strategy based on single-crystal X-ray diffraction and circular
dichroism (CD) methods.
Chemistry
The HPLC enantioseparation of compounds 1a and 2b was
carried out on the amylose-based Chiralpak IA CSP using
normal-phase conditions (for details see Supporting Informa-
tion). Typical chromatograms of 1a with simultaneous UV and
CD detection are shown in Figure 2.
The stereochemical correspondences between the couples of
the isolated enantiomers of 1a and 2b were established by CD
analysis (Figure 3). The structures and absolute configurations
of (+)-2b and (-)-2b, and therefore of (+)-1a and (-)-1a, were
readily secured by X-ray crystallography (Figure 4). As il-
lustrated in Figure 3, dextrorotatory and levorotatory enanti-
omers were shown to have (R) and (S) configuration, respectively.
Figure 4. ORTEP drawing of (S)-(-)-2b and (R)-(+)-2b.
on Langendorff perfused rat hearts subjected to ischemia-
reperfusion cycles. Diazoxide and cromakalim, at doses of 40
and 1 mg/kg, respectively, were also tested as reference drugs.
The ischemic damage was determined through the recording
of the functional postischemic parameter of rate-pressure
product (RPP%) recorded during reperfusion, expressed as a
percentage of the RPP value recorded at the last minute of the
preischemic period. At the end of reperfusion, the treatment of
the heart with triphenyltetrazolium chloride (TTC) made it
possible to carry out a morphological comparison of the necrotic
and healthy areas of the left ventricular tissue, colored white
(or pale pink) and red, respectively, and then to calculate the
Results
The potential cardioprotective effects of the two enantiomers
of 1a (preadministered ip at a dose of 40 mg/kg) were evaluated

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1479
Table 1. Functional (Rate Pressure Product RPP-120′) and
Morphological (% Ischemic Area vs Total Area) Parameters of the Two
Isolated Enantiomeric Forms of 1a (Data are Expressed as Mean (
Standard Error for n ) 4-6 different experiments)
performed by using stainless-steel Chiralpak IA (250 mm × 4.6
mm ID and 250 mm × 10 mm ID) (Daicel, Chemical Industries,
Tokyo, Japan) columns. HPLC-grade solvents were supplied by
Carlo Erba (Milan, Italy). HPLC apparatus consisted in a Perkin-
Elmer (Norwalk, CT) 200 lc pump equipped with a Rheodyne
(Cotati, CA) injector, a HPLC Dionex (CA) model TCC-100 oven
and a Jasco (Jasco, Ishikawa-cho, Hachioji City, Tokyo, Japan)
model 2095 Plus UV/CD detector.
The mobile phases were filtered and degassed by sonication
immediately before using. In analytical enantioseparations, standard
solutions were prepared by dissolving about 2 mg of sample into
10 mL of mobile phase. The injection volume was 10-20 µL. In
semipreparative enantioseparation, a 1 mL sample loop was used.
After semipreparative separation, the collected fractions were
analyzed by chiral analytical columns to determine their enantio-
meric excess (ee).
compd
RPP 120′ (%)a
Ai/Atot (%)a
vehicle
23 ( 4
86 ( 12
45 ( 7
59 ( 13
27 ( 5
64 ( 20
38 ( 6
23 ( 3
25 ( 4
22 ( 5
35 ( 2
16 ( 6
cromakalim
diazoxide
(()-1a
(R)-(+)-1a
(S)-(-)-1a
a Data are expressed as mean ( standard error for n ) 4-6 different
experiments.
morphological parameter ischemia-injured area as a % of the
total area (Ai/Atot%). As shown in Table 1, the ischemia/
reperfusion cycle induced a dramatic reduction of the postis-
chemic functional parameter (RPP-120′ ) 23 ( 4%) and a wide
extension of the myocardial injured areas (Ai/Atot ) 38 ( 6%)
in hearts isolated from vehicle-treated rats. As clearly observed
in previous works, the reference drugs diazoxide and cro-
makalim gave myocardium an increased resistance against
ischemia-reperfusion, resulting in an improved recovery of
postischemic cardiac functionality (RPP-120′ ) 45 ( 7% and
86 ( 12%, respectively) and a marked reduction of the
morphological evidence of tissue damage (Ai/Atot ) 25 ( 4%
and 23 ( 2%, respectively). When administered as a racemic
mixture, compound 1a demonstrated its cardioprotective proper-
ties as described in previous studies, with an overall amelioration
of both the postischemic indicators (RPP 120′ ) 59 ( 13%
and Ai/Atot ) 22 ( 5%).
As concerns the two enantiomers of 1a, the (S)-(-)-1a
enantiomer showed significant cardioprotective effect, with a
clear improvement of both the functional and the morphological
markers (RPP-120′ ) 64 ( 20%; Ai/Atot ) 16 ( 6%). Such an
anti-ischemic activity was almost comparable to that exhibited
by the racemic mixture. On the contrary, the (R)-(+)-1a
enantiomer was completely devoid of anti-ischemic effects
because the postischemic functional and morphological param-
eters recorded in hearts isolated from animals pretreated with
(R)-(+)-1a (RPP-120′ ) 27 ( 5%; Ai/Atot ) 35 ( 2%) were
almost superimposable on those observed in the vehicle-treated
group.
The column hold-up time (t0 ) 3.0 min for 250 mm × 4.6 mm
ID column) was determined from the elution of an unretained
marker (toluene), using ethanol as eluent, delivered at a flow-rate
of 1.0 mL/min.
The eluent composition and the corresponding analytical chro-
matographic data for each resolved compound are summarized as
follows. 1a: n-hexane-ethanol 75/25 (v/v), k1 ) 2.27, R ) 1.45, Rs
) 4.55. 2b: n-hexane-ethyl acetate-trifluoroacetic acid 40/60/0.1
(v/v/v), k1 ) 0.88, R ) 1.36, Rs ) 2.92. k1: retention factor of the
first eluted enantiomer, defined as (t1 - t0)/t0, where t0 is the void
time of the column; R: enantioselectivity factor defined as k2/k1;
Rs: resolution factor defined as 2(t2 - t1)/(w1 + w2), where t1 and
t2 are retention times and w1 and w2 are band widths at the baseline
in time units. Other analytical chromatographic conditions: flow-
rate, 1.0 mL/min; temperature, 25 °C; detector, UV and CD at 225
nm (for 1a) and 280 nm (for 2b).
Specific rotations of enantiomers of 1a and 2b, dissolved in
ethanol, were measured at 589 nm by a Perkin-Elmer polarimeter
model 241 equipped with a Na lamp. The volume of the cell was
1 mL and the optical path was 10 cm. The system was at a
temperature of 20 °C by a Neslab RTE 740 cryostat. The circular
dichroism (CD) spectra of stereoisomers of 1a and 2b, dissolved
in ethanol (concentration about 0.2 mg/mL) in a quartz cell (0.1
cm path length) at 25 °C, were measured by using a Jasco model
J-700 spectropolarimeter. The spectra are averages computed over
three instrumental scans, and the intensities are presented in terms
of ellipticity values (mdeg).
RX-analysis was carried out with a Oxford Diffraction KM4
Xcalibur2 goniometer at room temperature.
Graphite-monochromated Mo KR radiation (40 mA/-40 kV) and
a KM4 CCD/SAPPHIRE detector were used for cell parameter
determination and data collection.
Conclusions
This work demonstrated that the interaction of the new class
of anti-ischemic spirocyclic-benzopyran derivatives with their
intracellular target is enantioselective and that the cardiopro-
tective properties of the selected compound 1a resides in the
levorotatory enantiomer while the dextrorotatory one is devoid
of any activity. In addition, this result appears to be of particular
interest because the peculiarity of such mito-KATP activators is
the limited degree of conformational freedom that could let us
to hypothesize a steric model for the interaction with the active
site of the mito-KATP channel and therefore it could contribute
to the rational design and development of a new innovative class
of anti-ischemic drugs with improved potency and selectivity
for the cardiac target. This enantioselectivity in cardioprotection
showed by this spirocyclic-benzopyran derivative is in ac-
cordance with the results obtained for the benzopyran cyan-
oguanidine BMS180447,9 another well-characterized activator
of mito-KATP channels.
The integrated intensities, measured using the ω scan mode, were
corrected for Lorentz and polarization effects.10 The substantial
redundancy in data allows empirical absorption corrections (SAD-
ABS)11 to be applied using multiple measurements of symmetry-
equivalent reflections.
The structure was solved by direct methods of SIR200412 and
refined using the full-matrix least-squares on F2 provided by
SHELXL97.13
The X-ray CIF files for these structures have been deposited at
the Cambridge Crystallographic Data Center with the deposition
numbers CCDC 710382 and CCDC 710383. Copies of the data
can be obtained, free of charge, from CCDC, 12 Union Road,
Cambridge, CB2 1EZ UK (e-mail: deposit@ccdc.cam.ac.uk;
Internet: //www.ccdc.cam.ac.uk).
Acknowledgment. We thank the Ministero dell’Istruzione,
dell’Universita` e della Ricerca (MIUR) for partial support of
this project through a grant.
Supporting Information Available: Experimental methods,
crystallographic information, X-ray structural analysis, pharmaco-
logical procedures. This material is available free of charge via
the Internet at http://pubs.acs.org.
Experimental Section
Compounds 1a and 2b were synthesized and pharmacologically
tested as previously reported.6 HPLC enantioseparations were

1480 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Brief Articles
(8) Breschi, M. C.; Calderone, V.; Digiacomo, M.; Manganaro, M.;
Martelli, A.; Minutolo, F.; Rapposelli, S.; Testai, L.; Tonelli, F.;
Balsamo, A. Spirocyclic benzopyran-based derivatives as new anti-
ischemic activators of mitochondrial ATP-sensitive potassium channel.
J. Med. Chem. 2008, 51, 6945–6954.
References
(1) Lasley, R. D.; Konyn, P. J.; Hegge, J. O.; Mentzer, R. M. Effects of
ischemic and adenosine preconditioning on interstitial fluid adenosine
and myocardial infarct size. Am. J. Physiol. Heart Circ. Physiol. 1995,
38, H1460–H1466.
(9) Atwal, K. S.; Grover, G. J.; Ahmed, S. Z.; Ferrara, F. N.; Harper,
T. W.; Kim, K. S.; Sleph, P. G.; Dzwonczyk, S.; Russell, A. D.;
Moreland, S.; McCullough, J. R.; Normandin, D. E. Cardioselective
anti-ischemic ATP-sensitive potassium channel openers. J. Med. Chem.
1993, 36, 3971–3974.
(2) Zhang, W. M.; Jin, W. Q.; Wong, T. M. Multiplicity of kappa opioid
receptor binding in the rat cardiac sarcolemma. J. Mol. Cell Cardiol.
1996, 28, 1547–1554.
(3) Schulz, R.; Post, H.; Vahlhaus, C.; Heusch, G. Ischemic precondition-
ing in pigs: a graded phenomenon: its relation to adenosine and
bradykinin. Circulation 1998, 98, 1022–1029.
(4) Sato, T.; Sasaki, N.; O’Rourke, B.; Marba´n, E. Nicorandil, a potent
cardioprotective agent, acts by opening mitochondrial ATP-dependent
potassium channels. J. Am. Coll. Cardiol. 2000, 35, 514–518.
(5) Sato, T.; Sasaki, N.; Seharaseyon, J.; O’Rourke, B.; Marba´n, E.
Selective pharmacological agents implicate mitochondrial but not
sarcolemmal K(ATP) channels in ischemic cardioprotection. Circula-
tion 2000, 101, 2418–2423.
(6) Breschi, M. C.; Calderone, V.; Martelli, A.; Minutolo, F.; Rapposelli,
S.; Testai, L.; Tonelli, F.; Balsamo, A. New benzopyran-based openers
of the mitochondrial ATP-sensitive potassium channel with potent anti-
ischemic properties. J. Med. Chem. 2006, 49, 7600–7602.
(7) Balsamo, A.; Calderone, V.; Rapposelli, S. Preparation of 4-spiro-
heterocyclic 2,2-dimethylchromanes as activators of ATP-sensitive
potassium (KATP) channels. PCT Int. Appl. WO2008007210, 2008,
p 61.
(10) Walker, N.; Stuart, D. An empirical method for correcting diffracto-
meter data for absorption effects. Acta Crystallogr., Sect. A: Found.
Crystallogr. 1983, 39, 158–166.
(11) Sheldrick, G. M. SADABS version 2.03, a Program for Empirical
Absorption Correction; Universita¨t Go¨ttingen: Go¨ttingen, Germany,
1997-2001.
(12) Burla, M. C.; Caliandro, R.; Camalli, M.; Carrozzini, B.; Cascarano,
G. L.; De Caro, L.; Giacovazzo, C.; Polidori, G.; Spagna, R. SIR2004:
an improved tool for crystal structure determination and refinement.
J. Appl. Crystallogr. 2005, 38, 381–388.
(13) Sheldrick, G. M. SHELXL97: Program for Crystal Structure Refine-
ment; Institut fu¨r Anorganische Chemie de Universita¨t Go¨ttingen:
Go¨ttingen, Germany, 1997.
JM801459F