Novel l-adenosine analogs as cardioprotective agents

Article abstract of DOI:10.1016/j.bmc.2008.12.011

Two l-nucleosides, l-3′-amino-3′-deoxy-N⁶-dimethyladenosine (l-3′-ADMdA) 1, previously synthesized in our laboratory, and the novel l-3′-amino-3′-deoxy-N⁶-methyladenosine-5′-N-methyluronamide (l-3′-AM-MECA) 2 were evaluated in an isc

Full text of DOI:10.1016/j.bmc.2008.12.011

Bioorganic & Medicinal Chemistry 17 (2009) 5347–5352
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel L-adenosine analogs as cardioprotective agents
Harinath Kasiganesan a, Gary L. Wright a, Maria Assunta Chiacchio b, Giuseppe Gumina c,
*
a Department of Pharmaceutical Sciences, College of Pharmacy, Medical University of South Carolina, 280 Calhoun Street, PO Box 250140, Charleston, SC 29425, USA
b Dipartimento di Scienze Chimiche, Università di Catania, Viale Andrea Doria 6, 95125 Catania, Italy
c Department of Pharmaceutical Sciences, South University School of Pharmacy, 709 Mall Boulevard, Savannah, GA 31406, USA
a r t i c l e i n f o
a b s t r a c t
-30-amino-30-deoxy-N6-dimethyladenosine (
L
-30-ADMdA) 1, previously synthesized
Article history:
Two
in our laboratory, and the novel
-30-AM-MECA) 2 were evaluated in an ischemia/reperfusion model on Langendorff perfused mouse
heart.
-30-ADMdA 1 was found to enhance functional recovery from ischemia (32.2 3.7 cm H2O/s % rate
L
-nucleosides,
L
L
-30-amino-30-deoxy-N6-methyladenosine-50-N-methyluronamide
Received 20 October 2008
Revised 2 December 2008
Accepted 7 December 2008
Available online 14 December 2008
(L
L
pressure product, compared to 21.3 1.4 for the control and 30.7 3.4 for adenosine) and increase the
time to onset of ischemic contracture (14.5 0.9 min, compared to 10.5 1.0 min for the control and
13.6 0.6 min for adenosine) comparable to adenosine. Consistent with the functional recovery data,
decreased infarction area was seen in the case of 1 (19.1 8.4, compared to 40.5 7.2% for the control
Keywords:
L
-Nucleoside
Adenosine
Cardioprotection
and 11.5 2.1% for adenosine). In contrast, L
-30-AM-MECA 2 did not show significant functional recovery,
increased onset of contracture, nor decreased infarction area compared to control. Unlike adenosine,
neither 1 nor 2 induced cardiac standstill in mouse heart.
Ó 2009 Published by Elsevier Ltd.
1. Introduction
2-azidoadenosine and N-ethylcarboxamidoadenosine (NECA) with
A2 AR in different animal tissues.13
L
-Adenosine, a plant hormone is
also inactive on animal enzymes such as S-adenosyl- -homocyste-
ine hydrolase,14 and does not interact with mammalian nucleoside
Adenosine is an important mediator of numerous biological
functions both in the nervous system and peripheral tissues. It
exerts its action by interacting with at least four different recep-
tor (AR) subtypes (A1, A2a, A2b and A3).1 Several studies on the ac-
tion of adenosine in different tissues showed the potential
benefits of AR ligands (agonists or antagonists) for the treatment
of several diseases. Thus, cardioprotective action has been dem-
onstrated for A1 AR,2 A2A AR3 and A3 AR2b,4,5 agonists, whereas
A1 AR6 and A2a AR7 antagonists have shown potential anti-Alzhei-
mer properties. A2a AR antagonists are actively studied as anti-
Parkinson agents,8 and have also been found protective against
free radical neuronal damage.9 A3 AR antagonists are under con-
sideration as agents for the treatment of glaucoma.4,10 A selective
A1 AR agonist showed neuroprotective effect in a rat model of
Huntington’s disease.11 Finally, recent studies indicate that A3
AR may be targets for cancer therapy and chemoprotection.12
Such a range of potential therapeutical applications and the need
of fully understanding the pharmacological properties of each AR
subtype have prompted numerous efforts to discover more potent
and selective ligands to each receptor subtype. Among the struc-
L
transporters.15 In the past, however, favorable features of
L-nucleo-
sides, such as low cellular toxicity16 and high metabolic stabil-
ity,16,17 have been exploited in the design of successful antiviral
and promising anticancer agents. The favorable features of
L
-nucleosides and the potential cardioprotective activity of adeno-
sine analogs prompted us to evaluate
-30-amino-30-deoxy-N6-dim-
ethyladenosine (
-30-ADMdA) 1 (Fig. 1), recently synthesized in our
L
L
laboratory,18 in Langendorff perfused hearts as well as in a meta-
bolic essay on a myoblast cell line responsive to A1/A3 stimulation.
At the same time, in order to evaluate the effect of the favorable
50-carboxamide replacement, we prepared and evaluated the novel
analog
namide (
L
-30-amino-30-deoxy-N6-methyladenosine-50-N-methyluro-
L
-30-AM-MECA) 2, enantiomer of compound 3, a simplified
and A3 AR-selective analog of the A1/A3 agonist IB-MECA (Fig. 1).
2. Results and discussion
2.1. Chemistry
tural modifications reported in literature,
rarely considered, probably following early reports of little or no
interactions of the -enantiomers of adenosine, 2-chloroadenosine,
L-nucleosides have been
L
-30-ADMdA 1 was synthesized from
ously reported.18 -30-AM-MECA 2 was prepared from the common
intermediate 4 (Scheme 1), prepared in seven steps from -xylose
as previously described.18 Protected
-3-amino-3-deoxyribose 4
was protected as the tert-butyldiphenylsilyl ether 5, which was sub-
L-xylose in 11 steps as previ-
L
L
L
L
* Corresponding author.
E-mail address: gumina@musc.edu (G. Gumina).
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.12.011

5348
H. Kasiganesan et al. / Bioorg. Med. Chem. 17 (2009) 5347–5352
H3C
N
CH3
H3C
N
H
NH2
N
N
N
N
N
N
N
N
N
N
O
NHCH3
HO
N
OH
N
O
O
O
HO
OH
HO
NH2
HO
NH2
ADO
L-3'-ADMdA, 1
L-3'-AM-MECA, 2
H
I
H
CH3
N
N
N
N
N
N
N
N
N
N
CH3NH
O
H3CHN
O
O
O
HO
OH
H2N
OH
IB-MECA
3
Figure 1. Adenosine (ADO),
L
-30-ADMdA 1,
L
-30-AM-MECA 2, the selective A3AR agonist 3, and IB-MECA.
HO
OH
OTBDPS
AcO
OTBDPS
O
a
b
c
O
O
O
HO
OH
(55%)
(89%)
(50%)
O
O
HO
L-xylose
O
NHFmoc
O
NHFmoc
AcO
NHFmoc
4
5
6
(82%)
d
H3C
H
N
N
Cl
Cl
Cl
N
N
N
N
N
N
N
N
N
N
N
N
N
O
NHCH3
O
OH
OH
OTBDPS
N
N
f or g
(31 or 43%)
h
e
O
O
O
O
(55%)
(93%)
OH NH2
OAc NHFmoc
AcO
NHFmoc
AcO
NHFmoc
2
9
8
7
Scheme 1. Synthesis of L
-30-AM-MECA 2. Reagents and conditions: (a) seven steps, Ref. 18; (b) TBDPSCl, Py, CH2Cl2, 0 °C to rt, 24 h; (c) AcOH, H2SO4, rt, overnight, then Ac2O,
Py, rt, 4 h; (d) silylated 6-chloropurine, TMSOTf, MeCN, 0 °C to rt, overnight; (e) Et3Nꢀ3HF, THF, rt, 24 h; (f) (diacetoxyiodo)benzene, TEMPO 1:1 MeCN/H2O, rt, 48 h; (g) NaIO4,
RuCl3ꢀH2O, H2O/MeCN/CCl4, rt, 24 h; (h) EtOC(O)Cl, Et3N, DMF, 0 °C, 10 min, then 40% MeNH2/H2O, 0 °C to rt, 48 h.
ject to acetolysis to give acetate 6, almost exclusively as the b-ano-
mer, as expected on the basis of the participation of the 2-acetate
and supported by the lack of coupling between H-1 and H-2 in the
1H NMR spectrum. The 1H NMR of the crude reaction mixture
showed other minor signals in the anomeric region, but none was
of significant intensity to attempt purification or spectroscopic char-
acterization. Acetate 6 was coupled to 6-chloropurine in Vorbrüggen
conditions to give protected nucleoside 7. Also in this case, 1H NMR
of the crude reaction mixture showed only one isomer. The expected
b-stereochemistry could be easily confirmed by desilylation of 7 to 8
and conversion of the latter to 1 according to our reported proce-
dure.18 Oxidation of the primary alcohol functionality of 8 was at-
tempted using two methods. Using (diacetoxyiodo)benzene as the
oxidizer and TEMPO as initiator we were able to obtain carboxylic
acid 9 in moderate yield. The ruthenium-mediated method afforded
lower yields and a product contaminated with a dark impurity, pre-
sumably from the catalyst, that could not be purified chromato-
graphically. Conversion of 9 to the mixed anhydride with ethyl
chloroformate and in situ treatment with 40% aqueousmethylamine
afforded the synthesis of the desired amide 2. The conversion of 9 to
2 is the result of four different reactions: amide formation, Fmoc de-
protection, hydrolysis of the 20-O-acetate and replacement of the 6-
chloride. Similarly to what we observed during the synthesis of 1,18
we noticed that these reactions proceed at different rates, that is,
Fmoc deprotection and amide formation are very fast and complete
within a few minutes, whereas acetate hydrolysis usually requires
2–4 h, and 6-substitution is the slowest process, requiring several
hours to complete. These observations are supported by qualitative
tlc and UV data, consistent with the intermediate formation of the 6-
chloropurine analog of 2.
2.2. Cardioprotection in murine isolated perfused heart
The cardioprotective effect of 1 and 2 was evaluated on the
well established Langendorff mouse isolated perfused heart mod-
el.19 Hearts derived from Swiss Webster mice were subject to
40 min of global ischemia and 60 min of reperfusion. Compounds
were perfused for 5 min prior to ischemia to evaluate potential

H. Kasiganesan et al. / Bioorg. Med. Chem. 17 (2009) 5347–5352
5349
protective effects (Fig. 2). Left ventricular developed pressure
(LVDP) was continuously monitored during the ischemia-reperfu-
sion (IR) protocol and the % functional recovery was expressed as
Consistent with it behaving as adenosine receptor agonist,
-30-ADMdA
decreased evidence of cardiac tissue necrosis
L
1
(Fig. 2A), significantly prolonged the time to ischemic contracture
(Fig. 2B) and improved functional recovery (Fig. 2C). In contrast,
*
the final rate pressure product (RPP)/initial RPP 100. To assess
necrosis after the IR injury, hearts were evaluated by perfusing
them with a 1% TTC solution, which stains viable tissue red. High
resolution scans were made of transverse sections of heart and
the infarction area (i.e., not stained red) was determined and ex-
pressed as a % of total tissue area. The cardioprotective effects of
adenosine were reflected in: significantly less evidence of necrosis
as assessed with TTC staining (Fig. 2A); a delayed time until onset
of ischemic contracture (Fig. 2B); and most importantly, signifi-
cantly increased recovery of function upon reperfusion after
40 min of ischemia (Fig. 2C).
treatment of hearts with
effects and did not significantly alter any of these parameters when
comparedtocontrols(Fig.2). Adenosine (50 M)producedatransient
L
-30-AM-MECA 2 provided no salubrious
l
arrest of contractile activity during its perfusion as previously re-
ported.20 Interestingly, unlike adenosine, neither analog caused a ces-
sation of heart beating (i.e., cardiac standstill) during their perfusion.
In summary, we have described a novel L-nucleoside (L
-30-AD-
MdA, 1) endowed with cardioprotective effect. In view of its nature
of adenosine analog, we speculate that the activity of 1 may be due
to agonistic action on A1 and/or A3 AR subtypes, but further studies
will be needed to confirm this speculation. Notably,
is the first -nucleoside that shows biological activity besides anti-
viral or anticancer effect. Furthermore, since the first step in the ac-
tivation of known antiviral and antitumor -nucleosides is the
interaction with cellular or viral kinases,
-30-ADMdA is one of a
few examples of -nucleosides that interact with animal enzymes
other than kinases.21 This discovery opens a new niche in the
search for AR ligands. The nature of -nucleosides is likely to endow
L-L
-30-ADMdA
L
L
L
L
L
potential candidates with favorable features such as lower toxicity
and higher metabolic stability than their D-counterparts. Our syn-
thetic procedures are versatile, thanks to the different reactivity of
the functional groups in 8 and 9, which can allow the synthesis of
analogs with different substitution patterns and a full structure-
activity relationship study. Such studies are currently in progress.
3. Experimental
All the reactions were carried out under a positive pressure of
argon and monitored by TLC on Uniplates (silica gel) purchased
from Analtech Co. All the reagents and anhydrous solvents were
purchased from various commercial sources and used without fur-
ther purification except where noted. Chromatographic purifica-
tions were performed on flash silica gel (particle size 40–63
purchased from Silicycle or TLC grade silica gel (particle size 5–
15 m) purchased from Sorbent Technologies. All solvents for
lm)
l
chromatographic purifications were HPLC grade. Melting points
were determined on a Barnstead Mel-Temp and are uncorrected.
1H NMR spectra were recorded on Varian 400 MHz spectrometer
using Me4Si as an internal standard and signals are represented
as s (singlet), d (doublet), t (triplet), m (multiplet), or combinations
of the above. Mass spectra were obtained on a Finnigan ‘LCQ’ Li-
quid Chromatograph Ion Trap Mass Spectrometer. UV spectra were
obtained on a BECKMAN DU-650 spectrophotometer. Optical rota-
tions were measured on a Rudolph Research Analytical Autopol IV
digital polarimeter. Elemental analyses were performed by Atlantic
Microlabs Inc. Norcross, GA.
3.1. 5-O-tert-Butyldiphenylsilyl-3-deoxy-3-fluorenylmethylcarbon
ylamino-1,2-O-(1-methylethylidene)-a-L-ribofuranose (5)
tert-Butyldiphenylchlorosilane (2.4 mL, 9.38 mmol) was added
to an ice-cold solution of 4 (2.61 g, 6.34 mmol) and anhydrous pyr-
idine (0.80 mL, 9.89 mmol) in anhydrous dichloromethane (50 mL),
and the resulting solution was allowed to warm up to rt and stirred
for 24 h. Since the reaction did not proceed to completion, more
tert-butyldiphenylsilyl chloride (2.4 mL, 9.38 mmol) and pyridine
(0.80 mL, 9.89 mmol) were added, and stirring was continued at
rt for 24 h more. The resulting solution was washed with a 0.1 N
solution of HCl (2 ꢁ 10 mL), water (10 mL), sodium bicarbonate
saturated solution (10 mL), water (10 mL) and brine (10 mL). The
organic layer was then dried over magnesium sulfate, filtered
and concentrated to a crude that was purified by TLC-grade silica
Figure 2. Effects of L L
-30-AM-MECA and -30-ADMdA on mouse isolated perfused
hearts. A. Infarction area following ischemia-reperfusion (IR). Heart tissue was
stained with TTC, which is reduced to the bright red triphenyl formazane by viable
cells. Red areas indicate living tissue, while colorless or pale yellow areas indicate
necrotic tissue. Data is expressed as the percentage of area that lacks significant TTC
staining (19.1 8.4 for 1, compared to 40.5 7.2% for the control and 11.5 2.1% for
adenosine). B. Time until onset of contracture often correlates with the kinetics of
ischemic ATP depletion, and ultimately the damage sustained by the myocardium
(14.5 0.9 min for 1, compared to 10.5 1.0 min for the control and 13.6 0.6 min
for adenosine). C. Functional recovery of contractility, expressed in % recovery of
RPP (32.2 3.7 cm H2O/s for 1, compared to 21.3 1.4 for the control and 30.7 3.4
*
for adenosine). Statistically different from control (IR).

5350
H. Kasiganesan et al. / Bioorg. Med. Chem. 17 (2009) 5347–5352
gel flash chromatography (1:9 to 1:4 ethyl acetate/hexanes) to give
4.45 (m, 2H), 4.25–4.21 (m, 1H), 4.16–4.12 (m, 1H), 4.03–3.99
(m, 1H), 3.88–3.83 (m, 1H), 2.15 (s, 3H), 1.03 (s, 9H); 13C NMR
(CDCl3, 100 MHz) d 169.4, 152.0, 151.1, 150.8, 143.8, 143.5,
143.4, 141.2, 135.5, 135.3, 132.5, 132.2, 132.0, 129.7, 127.6,
127.5, 126.9, 124.7, 124.6, 119.9, 88.0, 83.2, 75.4, 66.8, 62.8, 50.9,
47.0, 26.6, 20.5, 19.0; Mass ([M+H]+) 788, ([M+Na]+) 810,
([2M+Na]+) 1597. Anal. Calcd for C43H42ClN5O6Si: C, 65.51; H,
5.37; N, 8.88. Found: C, 65.24; H, 5.54; N, 8.56.
5 as a white solid (3.65 g, 89%). 5: Rf 0.16 (1:4 ethyl acetate/hex-
anes); mp 61–63 °C; ½a D25
ꢂ
ꢃ36.94 (c 0.32, CHCl3); 1H NMR (CDCl3,
400 MHz) d 7.80–7.76 (m, 2H), 7.71–7.69 (m, 4H), 7.63–7.57 (m,
2H), 7.42–7.30 (m, 10H), 5.86 (d, J = 2.9 Hz, 1H), 5.08 (d,
J = 9.6 Hz, 1H), 4.65–4.63 (m, 1H), 4.41 (d, J = 6.8 Hz, 2H), 4.34–
4.28 (m, 1H), 4.22 (t, J = 6.6 Hz, 1H), 3.92–3.88 (m, 1H), 3.87–3.83
(m, 1H), 3.79–3.73 (m, 1H), 2.18 (s, 3H), 2.05 (s, 3H), 1.04 (s, 9H);
13C NMR (CDCl3, 100 MHz) d 155.7, 143.8, 143.7, 141.2, 135.6,
133.2, 133.1, 129.6, 127.7, 127.6, 127.0, 125.0, 119.9, 112.3,
104.2, 80.3, 78.9, 66.9, 62.4, 53.3, 47.1, 26.7, 26.5, 26.3, 19.1; Mass
([M+Na]+) 672. Anal. Calcd for C39H43NO6Si: C, 72.08; H, 6.67; N,
2.16. Found: C, 71.80; H, 6.79; N, 2.10.
3.4. 9-(2-O-Acetyl-3-deoxy-3-fluorenylmethylcarbonylamino-
b-L
-ribofuranosyl)-6-chloropurine (8)
Triethylamine trihydrofluoride (1.6 mL, 9.82 mmol) was added
to a stirring solution of 7 (1.54 g, 1.95 mmol) in anhydrous tetrahy-
drofuran (20 mL) at rt, and the resulting solution was stirred at rt for
24 h, then tlc-grade silica gel was added and volatiles were removed
under reduced pressure. The residue was loaded on a tlc-grade silica
gel column packed with dichloromethane and eluted with a gradient
ofdichloromethane to1:19methanol/dichloromethanetogive8 asa
white solid, containing minor impurities (1.00 g, 93%). A small sam-
ple was purified by preparative silica gel tlc (1:19 methanol/dichlo-
romethane) to give pure 8 as a white solid. Rf 0.16 (1:19 methanol/
3.2. 5-O-tert -Butyldiphenylsilyl-3-deoxy-1,2-diacetyl-3-fluore-
nylmethylcarbonylamino-b-
L-ribofuranose (6)
Sulfuric acid (60 L, 1.13 mmol) was added to a stirring mixture
l
of 5 (3.65 g, 5.62 mmol) in acetic acid (50 mL), and the reaction
was stirred at rt overnight. Acetic anhydride (7.2 mL, 76.17 mmol)
was then added, followed by pyridine (1.7 mL, 21.02 mmol), and
the mixture was stirred at rt for 4 h. Volatiles were evaporated in
vacuo, and the residue was dissolved in dichloromethane
(100 mL), washed with a saturated solution of sodium bicarbonate
(20 mL), water (20 mL) and brine (20 mL). The organic solution was
dried over magnesium sulfate, filtered and concentrated under re-
duced pressure to a crude that was purified by tlc-grade silica gel
flash chromatography (1:4 to 3:7 ethyl acetate/hexanes) to give 6
as a white solid (1.94 g, 50%). Rf 0.46 (3:7 ethyl acetate/hexanes);
dichloromethane); mp 196–197 °C; ½a D22
ꢂ
+35.78 (c 0.34, CHCl3); UV
(MeOH) kmax 264.0, 299.5, 288.5; 1H NMR (CDCl3, 400 MHz) d 8.77
(s, 1H), 8.42 (s, 1H), 7.79–7.75 (m, 2H), 7.59–7.57 (m, 2H), 7.44–
7.38 (m, 2H), 7.35–7.29 (m, 2H), 6.22 (d, J = 2.2 Hz, 1H), 5.54–5.52
(m, 1H), 5.20 (d, J = 8.0 Hz, 1H), 4.87–4.82 (m, 1H), 4.60–4.49 (m,
2H), 4.29–4.19 (m, 3 H), 4.00 (d, J = 12.0 Hz, 1H), 3.76 (d,
J = 12.0 Hz, 1H), 2.11 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 169.5,
156.1, 151.8, 151.3, 150.5, 144.5, 143.4, 141.2, 132.2, 127.7, 126.9,
124.6, 119.9, 88.7, 84.3, 75.5, 66.7, 61.1, 50.5, 47.0, 20.5; Mass
([M+H]+) 550, ([M+Na]+) 572. Anal. Calcd for C27H24ClN5O6: C,
58.97; H, 4.40; N, 12.73. Found: C, 59.05; H, 4.62; N, 12.33.
mp 70–72 °C; ½a 2D6
ꢂ
ꢃ24.43 (c 1.00, CHCl3); 1H NMR (CDCl3,
400 MHz) d 7.79–7.76 (m, 2H), 7.68–7.66 (m, 4H), 7.59–7.55 (m,
2H), 7.44–7.28 (m, 10H), 6.12 (s, 1H), 5.17–5.15 (m, 1H), 4.81–
4.79 (m, 1H), 4.55–4.42 (m, 3H), 4.22 (t, J = 6.6 Hz, 1H), 4.00–3.97
(m, 1H), 3.86 (dd, J = 11.3, 3.0, 1H), 3.71 (dd, J = 11.3, 4.1, 1H),
2.14 (s, 3H), 1.90 (s, 3H), 1.09 (s, 9H); 13C NMR (CDCl3, 100 MHz)
d 170.5, 169.5, 155.5, 143.7, 143.6, 141.3, 135.6, 135.5, 135.2,
130.0, 127.7, 127.7, 127.0, 124.8, 120.0, 97.7, 83.1, 75.7, 66.9,
63.3, 50.7, 47.1, 26.8, 22.9, 20.7, 19.0; Mass ([M+Na]+) 716,
([2M+Na]+) 1409. Anal. Calcd for C40H43NO8Si: C, 69.24; H, 6.25;
N, 2.13. Found: C, 69.00; H, 6.24; N, 2.05.
3.5. 1-(6-Chloro-9H-purin-9-yl)-2-O -acetyl-1,3-dideoxy-3-flu-
orenylmethylcarbonylamino-b-L-ribofuranoic acid (9)
Method A: (Diacetoxyiodo)benzene (770 mg, 2.39 mmol) was
added to a suspension of 8 (600 mg, 1.09 mmol) and 2,2,6,6-tetra-
methyl-1-piperidinyloxy (TEMPO) (43 mg, 0.28 mmol) in 1:1 ace-
tonitrile/water (50 mL), and the resulting mixture was stirred at
rt for 48 h, then diluted with water (100 mL) and extracted with
dichloromethane (3 ꢁ 150 mL). The combined organic extracts
were washed with brine (30 mL), dried over sodium sulfate, fil-
tered and concentrated to a crude that was purified by tlc-grade
silica gel flash chromatography. Elution with dichloromethane to
1:49 methanol/dichloromethane allowed to recover unreacted 8
(140 mg, 25%); subsequent elution with 1:19 to 2:23 methanol/
dichloromethane gave 9 as a yellow solid, containing minor impu-
rities (190 mg, 31%). A small amount of pure 9 was obtain by pre-
parative silica gel tlc (3:17 methanol/dichloromethane, washed
with 2:23 methanol/dichloromethane) as a white solid. Rf 0.44
3.3. 9-(2-O-Acetyl-5-O-tert -butyldiphenylsilyl-3-deoxy-3-flu-
orenylmethylcarbonylamino-b-L-ribofuranosyl)-6-chloropurine
(7)
A mixture of 6-chloropurine (0.67 g, 4.33 mmol) and ammo-
nium sulfate (30 mg, 0.23 mmol) in 1,1,1,3,3,3-hexamethyldisilaz-
ane (30 mL) was refluxed for 3 h, then the solvent was removed in
vacuo at 35–40 °C. A solution of 6 (1.88 g, 2.71 mmol) in anhydrous
acetonitrile (30 mL) was added to the residual solid. The resulting
solution was cooled to 0 °C and trimethylsilyl triflate (0.78 mL,
4.31 mmol) was added, and the reaction was stirred at rt overnight.
The resulting solution was diluted to 100 mL with dichlorometh-
ane and slowly added to an ice-cold saturated solution of sodium
bicarbonate (200 mL). The organic layer was separated and the
aqueous phase was extracted with dichloromethane (2 ꢁ
100 mL). The combined organic extracts were washed with water
(50 mL), brine (50 mL), dried over magnesium sulfate, filtered
and concentrated to a crude that was purified by tlc-grade silica
gel flash chromatography (1:49 methanol/dichloromethane) to
give 7 as a white solid (1.75 g, 82%). Rf 0.14 (1:49 methanol/dichlo-
(3:17 methanol/dichloromethane); mp 198–200 °C (dec.); ½a D22
ꢂ
ꢃ11.43 (c 0.30, CHCl3); UV (MeOH) kmax 264.0, 299.0, 288.5; 1H
NMR (DMSO-d6, 400 MHz) d 9.87 (br s, 1H), 8.80 (s, 1H), 8.20 (d,
J = 8.8 Hz, 1H), 7.89 (d, 2H, J = 7.4 Hz), 7.75–7.72 (m, 3H), 7.42 (t,
2H, J = 7.4 Hz), 7.34 (d, 2H, J = 7.4 Hz), 6.49 (d, J = 4.5 Hz, 1H),
5.60 (t, 1H, J = 4.9 Hz), 4.55–4.49 (m, 1H), 4.36–4.29 (m, 2H),
4.24–4.16 (m, 2H), 1.96 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) d
173.3, 169.3, 155.9, 151.8, 151.5, 149.1, 146.8, 143.9, 143.8,
140.7, 131.3, 127.7, 127.2, 127.1, 125.4, 125.3, 120.2, 120.1, 86.3,
83.5, 75.7, 65.9, 55.4, 46.6, 20.4; Mass ([M+H]+) 564. Anal. Calcd
for C27H22ClN5O7: C, 57.50; H, 3.93; N, 12.42. Found: C, 57.28; H,
3.79; N, 9.42. Method B: Ruthenium(III) chloride monohydrate
(7.5 mg, 0.04 mmol) was added to a vigorously stirred biphasic
heterogeneous mixture of 8 (90 mg, 0.16 mmol) and sodium
romethane); mp 91–93 °C (dec.); ½a D25
ꢃ17.83 (c 0.42, CHCl3); UV
ꢂ
(MeOH) kmax 264.0, 299.0; 1H NMR (CDCl3, 400 MHz) d 8.70 (s,
1H), 8.34 (s, 1H), 7.81–7.78 (m, 2H), 7.68–7.57 (m, 6H), 7.44–
7.36 (m, 4H), 7.35–7.29 (m, 4H), 7.26–7.22 (m, 2H), 6.15 (s, 1H),
5.67–5.64 (m, 1H), 5.12–5.06 (m, 1H), 4.99–4.95 (m, 1H), 4.55–

H. Kasiganesan et al. / Bioorg. Med. Chem. 17 (2009) 5347–5352
5351
periodate (145 mg, 0.68 mmol) in water (6 mL), acetonitrile (4 mL)
and carbon tetrachloride (4 mL) at rt, and the resulting mixture
was stirred at rt for 72 h. Solvents were then evaporated under
reduced pressure, the residue was dissolved in methanol, tlc-grade
silica gel was added, and the solvent was removed under reduced
pressure. The residue was loaded on a very thin pad of tlc-grade
silica gel column packed with dichloromethane and eluted with a
gradient of dichloromethane to 1:19 methanol/dichloromethane
to give 9 as a dark yellow oil (40 mg, 43%). Although this product
looked clean by tlc, repeated attempts to purify it by preparative
silica gel tlc did not result in the isolation of a solid.
incubated for 15 min at 37 °C. Then the hearts were weighed and
sliced into ꢄ1 mm transverse sections and scanned using
Microteck film scanner (Miroteck International Inc, New York,
USA). The area of infarcted (unstained) and viable (stained) tissue
was measured using Adobe Photoshop image analysis software.
The ratio of infarct area to total cross sectional area of the ventricle
from each slice was determined (percent infarction).
3.9. Statistical analysis
Baseline data and post-ischemic functional recoveries in differ-
ent experimental groups were analyzed by one-way ANOVA with
Bonferroni’s Correction. A P value of <0.05 was considered signifi-
cant. All values are presented as mean SEM.
3.6. L
-30-Deoxy-N6-methyl-30-methylaminoadenosine-50-methyl-
carboxamide (2)
Triethylamine was added to a solution of 9 (200 mg, 0.36 mmol)
and ethyl chloroformate (40 lL, 0.42 mmol) in anhydrous N,N-
Acknowledgment
dimethylformamide (5 mL) at 0 °C, and the resulting mixture was
stirred at 0 °C for 10 min, then treated with a 40% methylamine solu-
tion in water (5 mL). The mixture was allowed to warm up to rt, then
stirred for 48 h. Solvents were evaporated in vacuo and the residue
was purified by flash silica gel chromatography (dichloromethane
to 1:13 methanol/dichloromethane) to give an orange solid that
was further purified by preparative silica gel tlc: Rf 0.20 (3:17 meth-
anol/dichloromethane) to give pure 2 as an off-white solid (60 mg,
55%). Mp 165–167 °C (dec.); UV (MeOH) kmax 265.5; 1H NMR
(CD3OD, 400 MHz) d 8.34 (s, 1H), 8.30 (br s, 1H), 6.07 (d, J = 4.1 Hz,
1H), 4.62 (dd, J = 5.3, 4.1 Hz, 1H), 4.34 (d, J = 5.7 Hz, 1H), 3.80 (t, J =
5.7 Hz, 1H, D2O exchangeable), 3.11 (br s, 3H), 2.83 (s, 3H), 2.40–
2.38 (m, 1H), 1.26 (br s, 4H, D2O exchangeable); 13C NMR (CD3OD,
100 MHz) d 172.9, 156.8, 153.9, 149.0, 121.4, 92.1, 83.6, 74.6, 50.0,
26.3, 25.6; Mass ([M+H]+) 308. Anal. Calcd for C12H17N7O3: C,
46.90; H, 5.58; N, 31.91. Found: C, 47.07; H, 5.98; N, 31.68.
This research was supported by institutional funds from the
Medical University of South Carolina.
References
1. Fredholm, B. B.; Ijzerman, A. P.; Jacobson, K. A.; Klotz, K.-N.; Linden, . Pharmacol.
Rev. 2001, 53, 527.
2. (a) Donato, M.; Gelpi, R. J. Mol. Cell Biochem. 2003, 251, 153; (b) de Jonge, R.; de
Jong, J. W.; Giacometti, D.; Bradamante, S. Eur. J. Pharmacol. 2001, 414, 55; (c)
de Jong, J. W.; de Jonge, R.; Keijzer, E.; Bradamante, S. Pharmacol. Ther. 2000, 87,
141.
3. (a) Glover, D. K.; Riou, L. M.; Ruiz, M.; Sullivan, G. W.; Linden, J.; Rieger, J. M.;
Macdonald, T. L.; Watson, D. D.; Beller, G. A. Am. J. Physiol. Heart Circ. Physiol.
2005, 288, H1851; (b) Hein, T. W.; Wang, W.; Zoghi, B.; Muthuchamy, M.; Kuo,
L. J. Mol. Cell Cardiol. 2001, 33, 271; (c) Jordan, J. E.; Zhao, Z. Q.; Sato, H.; Taft, S.;
Vinten-Johansen, J. J. Pharmacol. Exp. Ther. 1997, 280, 301.
4. Müller, C. E. Curr. Top. Med. Chem. 2003, 3, 445.
5. (a) DeNinno, M. P.; Masamune, H.; Chenard, L. K.; DiRico, K. J.; Eller, C.; Etienne,
J. B.; Tickner, J. E.; Kennedy, S. P.; Knight, D. R.; Kong, J.; Oleynek, J. J.; Tracey, W.
R.; Hill, R. J. J. Med. Chem. 2003, 46, 353; (b) Tracey, W. R.; Magee, W. P.;
Oleynek, J. J.; Hill, R. J.; Smith, A. H.; Flynn, D. M.; Knight, D. R. Am. J. Physiol.
Heart Circ. Physiol. 2003, 285, H2780; (c) Tracey, W. R.; Magee, W.; Masamune,
H.; Kennedy, S. P.; Knight, D. R.; Buchholz, R. A.; Hill, R. J. Cardiovasc. Res. 1997,
33, 410.
3.7. Langendorff perfused heart preparation
Hearts were isolated from 10 weeks old male Swiss Webster
white mice and perfused with Krebs buffer containing (mM): NaCl,
118; NaHCO3, 25; KCl, 4.7; Mg2SO4, 1.2; KH2PO4, 1.2; glucose, 11;
CaCl2, 1.8, as previously described in detail.22 Buffer was equili-
brated with 95% O2, 5% CO2 at water-jacketed reservoirs main-
tained at 37 °C. After thoracotomy, hearts were excised into ice-
cold perfusion buffer, the aorta was cannulated (20 gauge PVC can-
nula) and perfused at 100 cm H2O pressure. A fluid filled silicon
balloon was inserted into the left ventricle via the mitral valve.
The balloon was attached to a pressure transducer connected to
a Power Lab data acquisition system for the recording of left ven-
tricular pressure (ADInstruments, Colorado, USA). Hearts were im-
mersed in perfusate at 37 °C and balloons inflated to an end
diastolic pressure of ꢄ10 cm H2O. Hearts were stabilized for
20 min and baseline functions like left ventricular developed pres-
sure (LVDP) and heart rate (HR) and coronary flow were measured.
Hearts showing baseline values of LVDP lower than 80 cm H2O,
spontaneous heart rates lower than 300 BPM and/or coronary flow
greater than 5 mL/min were disqualified from the study according
to previously published criteria.22a Hearts were untreated or sub-
ject to preconditioning stimuli either with adenosine or with syn-
thetic analogs 1 or 2 for a 5 min of perfusion. After the baseline
period, all the untreated and treated hearts underwent 40 min glo-
bal ischemia followed by 60 min reperfusion. The parameters of
LVDP and HR were reassessed after reperfusion.
6. Angulo, E.; Casadó, V.; Mallol, J.; Canela, E. I.; Viñals, F.; Ferrer, I.; Lluis, C.;
Franco, R. Brain Pathol. 2003, 13, 440.
7. Dall’Igna, O. P.; Porciúncula, L. O.; Souza, D. O.; Cunha, R. A.; Lara, D. R. Br. J.
Pharmacol. 2003, 138, 1207.
8. (a) Pinna, A.; Volpini, R.; Cristalli, G.; Morelli, M. Eur. J. Pharmacol. 2005, 512,
157; (b) Vu, C. B.; Pan, D.; Peng, B.; Kumaravel, G.; Smits, G.; Jin, X.; Phadke, D.;
Engber, T.; Huang, C.; Reilly, J.; Tam, S.; Grant, D.; Hetu, G.; Petter, R. C. J. Med.
Chem. 2005, 48, 2009; (c) Vu, C. B.; Peng, B.; Kumaravel, G.; Smits, G.; Jin, X.;
Phadke, D.; Engber, T.; Huang, C.; Reilly, J.; Tam, S.; Grant, D.; Hetu, G.; Chen, L.;
Zhang, J.; Petter, R. C. J. Med. Chem. 2004, 47, 4291; (d) Dall’Igna, O. P.; Souza, D.
O.; Lara, D. R. Ann. Pharmacother. 2004, 38, 717; (e) Chase, T. N.; Bibbiani, F.;
Bara-Jimenez, W.; Dimitrova, T.; Oh-Lee, J. D. Neurology 2003, 61, S107; (f)
Schwarzschild, M. A.; Xu, K.; Oztas, E.; Petzer, J. P.; Castagnoli, K.; Castagnoli, N.,
Jr.; Chen, J. –F. Neurology 2003, 61, S55; (g) Chen, J. F.; Xu, K.; Petzer, J. P.; Staal,
R.; Xu, Y. H.; Beilstein, M.; Sonsalla, P. K.; Castagnoli, K.; Castagnoli, N., Jr.;
Schwarzschild, M. A. J. Neurosci. 2001, 21, 143.
9. Behan, W. M. H.; Stone, T. W. Br. J. Pharmacol. 2002, 135, 1435.
10. Okamura, T.; Kurogi, Y.; Hashimoto, K.; Sato, S.; Nishikawa, H.; Kiryu, K.;
Nagao, Y. Bioorg. Med. Chem. Lett. 2004, 14, 3775.
11. Blum, D.; Gall, D.; Galas, M. –C.; d’Alcantara, P.; Bantubungi, K.; Schiffmann, S.
N. J. Neurosci. 2002, 22, 9122.
12. (a) Fishman, P.; Bar-Yehuda, S.; Madi, L.; Cohn, I. Anticancer Drugs 2002, 13,
437; (b) Fishman, P.; Bar-Yehuda, S.; Barer, F.; Madi, L.; Multani, A. S.; Pathak, S.
Exp. Cell Res. 2001, 269, 230.
13. (a) Mustafa, S. J.; Askar, A. O. J. Pharmacol. Exp. Ther. 1985, 232, 49; (b)
Burnstock, G.; Hills, J. M.; Hoyle, C. H. Br. J. Pharmacol. 1984, 81, 533; (c) Brown,
C.; Burnstock, G.; Cusack, N. J.; Meghji, P.; Moody, C. J. Br. J. Pharmacol. 1982, 75,
101; (d) Cusack, N. J.; Planker, M. Br. J. Pharmacol. 1979, 67, 153; (e) Cusack, N.
J.; Jickman, M. E.; Born, G. V. Proc. Royal Soc. London B Bio. Sci. 1979, 206, 139.
14. Wang, M.; Zhang, J.; Andrei, D.; Kuczera, K.; Borchardt, R. T.; Wnuk, S. F. J. Med.
Chem. 2005, 48, 3649.
15. (a) Al Safarjalani, O. N.; Naguib, F. N. M.; el Kouni, M. H. Antimicrob. Agents
Chemother. 2003, 47, 3247; (b) Carter, N. S.; Mamoun, C. B.; Liu, W.; Silva, E. O.;
Landfear, S. M.; Goldberg, D. E.; Ullman, B. J. Biol. Chem. 2000, 275, 10683.
16. (a) Gumina, G.; Chong, Y.; Choo, H.; Song, G. –Y.; Chu, C. K. Curr. Top. Med. Chem.
2002, 2, 1065; (b) Gumina, G.; Song, G. –Y.; Chu, C. K. FEMS Microbiol. Lett. 2001,
202, 9.
3.8. Triphenyltetrazolium chloride staining
Hearts were perfused with 1% triphenyltetrazolium chloride
(TTC) immediately after completion of the experiment and
17. Jurovcik, M.; Holy, A.; Sorm, F. FEBS Lett. 1971, 18, 274.

5352
H. Kasiganesan et al. / Bioorg. Med. Chem. 17 (2009) 5347–5352
18. Gilbert, C. L. K.; Lisek, C. R.; White, R. L.; Gumina, G. Tetrahedron 2005, 61,
8339.
21. The other example known to us is the recent report by Schramm and co-
workers describing the inhibitory activity of -Immucillin-H and -DADMe-
L
L
19. (a) Tikh, E. I.; Fenton, R. A.; Dobson, J. G., Jr. Am. J. Physiol. Heart Circ. Physiol.
2006, 290, H348; (b) Peart, J.; Headrick, J. P. Cardiovasc. Res. 2003, 58, 589; (c)
Sutherland, F. J.; Baker, K. E.; Shattock, M. J.; Hearse, D. J. Clin. Exp. Pharmacol.
Physiol. 2003, 30, 867; (d) Sutherland, F. J.; Baker, K. E.; Shattock, M. J.; Hearse,
D. J. Clin. Exp. Pharmacol. Physiol. 2003, 30, 879.
20. (a) Canyon, S. J.; Dobson, G. P. J. Thorac. Cardiovasc. Surg. 2005, 130, 371; (b)
Nussbaum, E. S.; Sebring, L. A.; Ostanny, I.; Nelson, W. B. Neurosurgery 2000, 47,
240; (c) De Giovanni, J. V.; Edgar, R. A.; Cranston, A. Heart 1998, 80, 330; (d)
Shryock, J. C.; Belardinelli, L. Am. J. Cardiol. 1997, 79, 2.
Immucillin-H toward purine nucleoside phosphorylase (a) Rinaldo-Matthis, A.;
Murkin, A. S.; Ramagopal, U. A.; Clinch, K.; Mee, S. P. H.; Evans, G. B.; Tyler, P. C.;
Furneaux, R. H.; Almo, S. C.; Schramm, V. L. J. Am. Chem. Soc. 2008, 130, 842; (b)
Clinch, K.; Evans, G. B.; Fleet, G. W.; Furneaux, R. H.; Johnson, S. W.; Lenz, D. H.;
Mee, S. P. H.; Rands, P. R.; Schramm, V. L.; Taylor Ringia, E. A.; Tyler, P. C. Org.
Biomol. Chem. 2006, 4, 1131.
22. (a) Sutherland, F. J.; Baker, K. E.; Shattock, M. J.; Hearse, D. J. Clin. Exp.
Pharmacol. Physiol. 2003, 30, 867; (b) Sutherland, F. J.; Baker, K. E.; Shattock, M.
J.; Hearse, D. J. Clin. Exp. Pharmacol. Physiol. 2003, 30, 879.