Synthesis and biological evaluation of clitocine analogues as adenosine kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00454-1

Adenosine kinase (AK) is the primary enzyme responsible for adenosine metabolism. Inhibition of AK effectively increases extracellular adenosine concentrations and represents an alternative approach to enhance the beneficial actions of adenosine as compared to direct-acting receptor agonists. Clitocine (3), isolated from the mushroom Clitocybe inversa, has been found to be a weak inhibitor of AK. We have prepared a number of analogues of clitocine in order to improve its potency and demonstrated that 5′-deoxy-5′-amino-clitocine (7) improved AK inhibitory potency by 50-fold.

Full text of DOI:10.1016/S0960-894X(01)00454-1

Bioorganic& Medicinal Chemistry Letters 11 (2001) 2419–2422
Synthesis and Biological Evaluation of Clitocine Analogues
as Adenosine Kinase Inhibitors
Chih-Hung Lee,* Jerome F. Daanen, Meiqun Jiang, Haixia Yu, Kathy L. Kohlhaas,
Karen Alexander, Michael F. Jarvis, Elizabeth L. Kowaluk and Shripad S. Bhagwaty
Neurological and Urological Diseases Research, Global Pharmaceutical Products Division, Abbott Laboratories,
100 Abbott Park Road, Abbott Park, IL 60064, USA
Received 9 May 2001; accepted 20 June 2001
Abstract—Adenosine kinase (AK) is the primary enzyme responsible for adenosine metabolism. Inhibition of AK effectively
increases extracellular adenosine concentrations and represents an alternative approach to enhance the beneficial actions of aden-
osine as compared to direct-acting receptor agonists. Clitocine (3), isolated from the mushroom Clitocybe inversa, has been found to
be a weak inhibitor of AK. We have prepared a number of analogues of clitocine in order to improve its potency and demonstrated
that 50-deoxy-50-amino-clitocine (7) improved AK inhibitory potency by 50-fold. # 2001 Elsevier Science Ltd. All rights reserved.
Extracellular concentrations of the endogenous neuro-
modulator, adenosine (ADO), are increased under con-
ditions of metabolicstress and trauma (e.g., pain,
inflammation, tissue damage, ischemia, seizure activity,
etc.) and act to limit tissue damage and restore normal
function1 by activating members of the P1 receptor
family, A1, A2A, A2B, and A3 receptors.2 Increased
extracellular ADO concentrations can result in the
inhibition of excitatory amino acid (glutamate) release,
suppression of free radical formation, and neutrophil
adhesion, depending on the phenotype of the tissue
involved.3 Since ADO has a half-life of the order of
seconds in physiological fluids,4 its extracellular actions
are restricted to the tissue and cellular sites where it is
released.5 The effects of extracellular ADO are termi-
nated by its re-uptake and phosphorylation by ADO
kinase (AK; ATP: adenosine 50-phosphotransferase, EC
2.7.1.20) and via deamination by adenosine deaminase
(ADA).6 By preventing ADO phosphorylation, AK
inhibition increases intracellular ADO concentrations,
altering the equilibrium of the bi-directional transport
systems responsible for ADO reuptake with the net
effect of increasing the local concentration of ADO in
the extracellular compartment.7 AK inhibition is a more
effective mechanism for increasing extracellular ADO
levels than inhibition of the catabolic enzyme ADA,7
and is more effective than ADA inhibition in reducing
seizure and nociceptive activity in vivo.8,9 Therefore, we
initiated an effort to identify suitable AK inhibitors for
exploring their potential as therapeutically useful CNS
agents.
A number of agents such as 50-deoxy-50-amino-adeno-
sine (1),10 50-deoxy-5-iodotubercidin (2),11 and clitocine
(3)12 have been found to inhibit AK (Scheme 1).
Although clitocine is a weak AK inhibitor (IC50=2 mM),
we decided to investigate it as a lead structure because
of its structural simplicity.
Clitocine was prepared by the procedure of Kamikawa
et al.13 The 50-modified analogues of clitocine were pre-
pared by 50-hydroxy functional group modification of
the intermediate13 (5) prepared during its synthesis.
Treatment of 5 with the PPh3Br2 gave the 50-bromo
compound 6. Displacement of the 50-bromo group with
potassium phthalimide in DMF followed by sequential
deprotection of the amine and the acetonide groups by
*Corresponding author. Tel.: +1-847-938-1849; fax: +1-847-938-
0072; e-mail: lance.lee@abbott.com
yPresent address: Cellgene Signal Pharmaceuticals.
Scheme 1. Structures of known adenosine kinase inhibitors.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00454-1

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C.-H. Lee et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2419–2422
using hydrazine in refluxing methanol and tri-
fluoroacetic acid, respectively, gave 50-deoxy-50-amino-
clitocine (7) (Scheme 2).
with 15 gave a mixture of anomers, which were purified
by column chromatography to give the pure b-anomer
(16) which is shown in Scheme 4. Amination of 16
followed by deprotection gave 18.
Heating 4 with aniline followed by acetonide deprotec-
tion gave N6-phenyl-clitocine (4a). Deprotection of 6
using trifluoroacetic acid gave 6a. The 50-deoxy analo-
gue of clitocine (3) could not be prepared by the general
dehalogenation of 6 or 6a. Therefore, an alternate syn-
Mitsunobu reaction of 17 with diphenyl phosphoryl-
azide gave the corresponding azido compound (19).
Reduction of the azide to amine followed by deprotec-
tion of the acetonide gave the 50-amino compound (20)
(Scheme 5).
theticroute for preparation of ocmpound
13 was
designed (Scheme 3). Treatment of d-ribono-1,4-lactone
(8) with PPh3/NIS followed by hydrogenation with Pd/
C gave 5-deoxy lactone. Reduction of the lactone to
lactol with DIBAL provided 9 as a mixture of anomers.
The corresponding 50-deoxy compound (22) was pre-
pared by the coupling of 10 with 14 followed by ami-
nation at the 4-position of 21 and deprotection of the
acetonide (Scheme 6). The intermediate 16 was
converted to 16a, 16b, and 16c as shown in Scheme 7.
The conversion of 9 to aminal (10) was accomplished by
treating 9 with AlCl3 followed by NaN3 giving 2-azido
intermediate. Hydrogenation of the resulting 2-azido
intermediate with PtO2 yielded aminal (10). The aminal
(10) was then coupled with the aminochloropyrimidine
(11) in DMF/Et3N and deprotected with trifluoroacetic
acid to give 50-deoxyclitocine (12).
Compounds 26 and 27 were prepared as shown in
Scheme 8. Bromination of 4,6-dihydroxy-pyrimidine
(13) yielded 5-bromo-4,6-dihydroxy-pyrimidine fol-
lowed by 5-bromo displacement with sodium thiophe-
nol to give 5-mercaptophenyl (23). Treatment of 23 with
POCl3 gave the corresponding dichloride and by
sequential oxidation with m-CPBA yielded 24 and 25.
Ribosylation of 24 and 25 followed by aminolysis and
deprotection gave 26 and 27.
In an attempt to find a carboxylate replacement for the
5-nitro group of clitocine, 4,6-dihydroxy-pyrimidine
(13) was formylated and chlorinated in one pot using
Bredereck’s procedure14 and the resulting formylated
product was oxidized to the corresponding carboxylic
acid by KMnO4 in acetone. Esterification of the car-
boxylicacid with diazomethane gave 14. Coupling of 14
Compound 30 with an oxazoline at the 5-position of
pyrimidine was prepared as shown in Scheme 9. 4,6-
Dichloro-pyrimidine-5-carboxylic acid (prepared as
Scheme 2. Reagents: (a) NH3/MeOH; (b) PPh3Br2; (c) potassium
phthalimide; (d) N2H4; (e) TFA/H2O, 12% overall; (f) PhNH2/DMF.
Scheme 4. Reagents: (a) POCl3/DMF; (b) KMnO4/acetone; (c)
CH2N2; (d) Et3N/DMF; (e) NH3/MeOH; (f) TFA/H2O.
Scheme 5. Reagents: (a) (PhO)2PON3/DEAD; (b) PPh3/Py; (c) TFA/
H2O.
Scheme 3. Reagents: (a) PPh3/NIS; (b) DIBAL; (c) AlCl3; (d) NaN3;
(e) PtO2/H2, 44% overall yield; (f) Et3N/DMF/rt; (g) TFA/H2O, 11%
overall yield.
Scheme 6. Reagents: (a) Et3N/DMF; (b) NH3/MeOH; (c) TFA/H2O.

C.-H. Lee et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2419–2422
2421
shown in Scheme 4) was amidated via the acid chloride
to give 28. Conversion of the b-hydroxy amide to the
b-chloro amide followed by cyclization in NaH gave the
oxazoline (29).
group, respectively, led to a slight decrease of in vitro
activity. Replacement by an amino group, on the other
hand, led to a 50-fold improvement in the enzyme inhi-
bitory activity (IC50=35 nM for 7 vs 2 mM for 3).
Replacing the 4-amino group of clitocine by a chloro or
aniline group led to a significant decrease in activity.
Ribosylation of 29, followed by aminolysis and depro-
tection, gave the desired oxazoline 30 and the ring
opened b-hydroxy amide (31).
An attempt was made to find a replacement for the 5-
nitro group of clitocine. It has been proposed that a
crucial H-bond exists between the oxygen of the nitro
group and the NH-group at the 6-position. As a result,
a desirable orientation will form between the ribose and
pyrimidine rings.15 Electron withdrawing functional
groups were chosen keeping this postulate in mind. The
5-methyl carboxylate analogue 18 was found to be
equipotent to clitocine. Increasing the size of the ester
group did not increase the activity (results not shown).
Changing the 50-substituent to an amino group again
led to a large improvement in activity (IC50=2 nM for
20 vs 1 mM for 18). Interestingly, the analogue of this
Adenosine kinase activity of rat brain cytosol was
assayed radiometrically. Assays were carried out at
ambient temperature in a final volume of 100 mL. The
standard reaction mixture contained 64 mM Tris–HCl,
pH 7.5, 40 mM KCl, 0.2 mM MgCl2, 1 mM ATP, 0.2
mM U-[14C]-adenosine (2ꢀ1010 Bq/mmol, Amersham),
an appropriate concentration of the AK inhibitor, and
an aliquot of rat brain cytosol. The reaction was termi-
nated after 15 min by spotting 40 mL of the reaction
mixture onto disks of Whatman DE-81 anion exchange
paper. DE-81 disks were then air-dried, washed for 10
min in 2 mM ammonium formate, rinsed successively
with distilled water, methanol and acetone, and dried.
Disks were then soaked for 5 min in 0.1 N HCl/0.4 M
KCl prior to liquid scintillation.
The structural variation of clitocine was focused initially
at the 50- and 4-positions (Table 1). Replacing the 50-
hydroxy group of clitocine by a 50-bromo or 50-deoxy
Scheme 9. Reagents: (a) SOCl2; (b) NH2CH2CH2OH; (c) NaH; (d)
NH3/MeOH; (e) TFA/H2O.
Table 1. In vitro biological activity of ADO kinase inhibitors
Compd
R1
R2
R3
Adenosine kinase
inhibition IC50 (nM)a
Scheme 7. Reagents: (a) TFA/H2O; (b) Pd/C/H2; (c) (PhO)2PON3/
DEAD; (d) PPh3/py.
1
2
3
4a
6a
7
12
16a
16b
16c
18
20
22
26
27
30
31
15 (ꢁ14)
1.63 (ꢁ1.09)
2000 (ꢁ1000)
>10,000
OH
OH
Br
NH2
H
OH
OH
NH2
OH
NH2
H
OH
OH
OH
OH
NO2
NO2
NO2
NO2
NH2
NHPh
NH2
NH2
NH2
Cl
>10,000
35 (ꢁ12)
NO2
4000 (ꢁ1400)
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
SOPh
>10,000
H
H
600 (ꢁ323)
4 (ꢁ1)
NH2
NH2
NH2
NH2
NH2
NH2
NH2
1000 (ꢁ857)
2 (ꢁ1)
6100 (ꢁ5200)
>10,000
SO2Ph
2-Oxazoline
CONHC2H4OH
>10,000
4000 (ꢁ3420)
>10,000
aAll values are the meanꢁSEM of at least three separate observations
Scheme 8. Reagents: (a) Br2/MeOH; (b) NaSPh; (c) POCl3; (d) m-
CPBA; (e) Et3N/DMF; (f) NH3/MeOH; (g) TFA/H2O.
run in triplicate.

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C.-H. Lee et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2419–2422
compound with no substituent at the 4-position, 16c,
was equally active (IC50=4 nM). Furthermore, the
analogue of 18 with no substituent at the 4-position,
16b, also had activity similar to that of 18 (IC50=0.6
mM vs 1 mM).
References and Notes
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Lett. 1986, 27, 4277.
Replacing the 5-nitro group of clitocine by a phe-
nylsulfoxide or phenylsulfone led to a loss of activity.
This result was disappointing since it was anticipated
that the phenyl group of these compounds would fit in
the site occupied by the iodo-group of 2. The nitro
group of clitocine lacks the bulky hydrophobic nature
of the iodo-group of 2. An oxazoline group at the 5-
position would be an electron withdrawing group with
two heteroatoms. Additionally, it would provide a han-
dle to put a hydrophobicgroup at the 5-position. The
oxazoline analogue 30 was found to be equipotent to
clitocine. More analogues need to be prepared for eval-
uating this substitution further. The hydroxyethyl amide
prepared as a side product was found to be inactive.
13. Kamikawa, T.; Fuijie, S.; Yamagiwa, Y.; Kim, M.;
Kawaguchi, H. I. J. Chem. Soc., Chem. Commun. 1988, 195.
14. Bredereck, H.; Simchen, G.; Wagner, H.; Santos, A. A.
Justus Liebigs Ann. Chem. 1972, 766, 73.
15. Moss, R. J.; Petrie, C. R.; Meyer, R. B.; Nord, L. D.;
Willis, R. C.; Smith, R. A.; Larson, S. B.; Kini, G. D.; Robins,
R. K. J. Med. Chem. 1988, 31, 786.
In conclusion, we have prepared analogues of clitocine
and found that a 50-amino group enhanced the potency
of AK inhibition by more than 50-fold. The ester and
oxazole functionalities were found to replace the 5-nitro
group without loss of activity. The scope of the latter
substitution needs to be explored further.