Carbocyclic 3′-deoxyadenosine-based highly potent bisubstrate-analog inhibitor of basophilic protein kinases

Article abstract of DOI:10.1016/j.bmcl.2007.08.016

Carbocyclic analogs of 3′-deoxyadenosine were synthesized as racemates and the resulting stereoisomers were separated by chromatography on a chiral column. The conjugation of obtained compounds with hexa-(d-arginine) via 6-aminohexanoic acid linker led to a highly potent inhibitor of several basophilic protein kinases with some selectivity towards cAMP-dependent protein kinase.

Full text of DOI:10.1016/j.bmcl.2007.08.016

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5336–5339
0
Carbocyclic 3 -deoxyadenosine-based highly potent
bisubstrate-analog inhibitor of basophilic protein kinases
a
a
a
b
Erki Enkvist, Gerda Raidaru, Angela Vaasa, T o˜ nis Pehk,
a
a,
*
Darja Lavogina and Asko Uri
a
Institute of Organic and Bioorganic Chemistry, University of Tartu, 2 Jakobi Street, 51014 Tartu, Estonia
b
National Institute of Chemical Physics and Biophysics, 23 Akadeemia Tee, 12618 Tallinn, Estonia
Received 26 June 2007; revised 8 August 2007; accepted 8 August 2007
Available online 15 August 2007
0
Abstract—Carbocyclic analogs of 3 -deoxyadenosine were synthesized as racemates and the resulting stereoisomers were separated by
chromatography on a chiral column. The conjugation of obtained compounds with hexa-(D-arginine) via 6-aminohexanoic acid linker
led to a highly potent inhibitor of several basophilic protein kinases with some selectivity towards cAMP-dependent protein kinase.
ꢀ
2007 Elsevier Ltd. All rights reserved.
Protein kinases (PKs) play a key role in the regulation of
cellular protein functions. More than 400 human dis-
The development of bisubstrate-analog (biligand) inhib-
itors that simultaneously associate with both ATP and
protein-binding domains of PK could give selective
1
eases have been linked to aberrant PK signaling. This
fact and the recent success with PK inhibitor-based
drugs have made PKs the second largest drug target
4
and potent inhibitors of these dual substrate enzymes.
A linker enabling optimal positioning of the active frag-
ments comprising the bisubstrate inhibitor could lead to
a conjugate with kinase-binding energy substantially
2
after G protein-coupled receptors. Due to apparent
ease of development of high-affinity low molecular
weight inhibitors targeted to the well-defined hydropho-
bic adenine-binding cleft, and despite serious selectivity
problems (additionally to 500 coded PKs in human gen-
ome 1500 other proteins are able to bind adenine nucle-
5
exceeding the sum of binding energies of the fragments.
6
Recently we described novel bisubstrate-analog-type
inhibitors of PK that consisted of adenosine and oligo-
(D-arginine) moieties connected via 6-aminohexanoic
acid linker (ARCs, adenosine–oligoarginine conjugates).
The most active of the compounds, ARC-902 (number 5
in Table 1 of Ref. 6), showed low nanomolar inhibitory
potency toward the catalytic subunit (type a) of cAMP-
dependent protein kinase (cAPK Ca, also known as
1
otides) in combination with high concentration of
competing ATP in the cellular milieu, most of the devel-
oped and studied inhibitors of PKs are competitive with
ATP. The high degree of 3D structural conservation of
adenine nucleotide binding site of these proteins makes
the development of specific inhibitors a challenging task.
6
PKA Ca), and high proteolytic stability. Later we dem-
The structure of protein substrate-binding domain of ki-
nases is more variable and the development of highly
selective peptide inhibitors has been described. How-
ever, longer peptidic structures are needed for achieving
nanomolar potency which leads to problems with cellu-
lar transport and stability of the compounds.
onstrated that such highly potent ARCs really behave as
bisubstrate-analog inhibitors and can be displaced from
their complex with cAPK Ca by both ATP- and protein
3
7
substrate-competitive inhibitors.
The further increase of inhibitory potency of ARC-type
inhibitors could be achieved by optimization of struc-
tures of the moieties and their spatial positioning. The
earlier structure–activity studies with adenosine and
adenine nucleotide analogs have shown that contrary
to other modifications of the structure that decrease
the affinity of adenosine analogs, the removal of the
Keywords: Protein kinases; Bisubstrate inhibitors; Carbocyclic nucle-
oside; Oligoarginine.
*
Corresponding author. Tel.: +372 5175593; fax: +372 7375275;
e-mail: Asko.Uri@ut.ee
0
hydroxyl group from the 3 -carbon of the ribose moiety
0
960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.016

E. Enkvist et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5336–5339
5337
Table 1. Inhibition of cAPK Ca
Compound IC50 (0.1 mM ATP)
O
O
EDC, MeOH
O
a
a
IC50 (1 mM ATP)
O
HO
mCPBA
86-94%)
(
53%)
b
O
1
2
1
1
a/1b
a/2b
a
328 lM (3.48 ± 0.20)
93 lM (4.03 ± 0.16)
>1000 lM
—
—
—
—
(
5a
3
4
O
O
O
+
b
168 lM (3.77 ± 0.18)
O
N
ARC-659-1a/1b/ 3.45 nM (8.46 ± 0.22) 24.5 nM (7.61 ± 0.19)
c
5b
N
O
2a/2b
HN
(
12-16%)
ARC-659-1a/1b
ARC-659-2a/2b
ARC-659-1a
—
—
—
16.8 nM (7.77 ± 0.23)
571 nM (6.24 ± 0.31)
97.3 nM (7.01 ± 0.22)
N
Base
ratio ~ 1:1:1:1
OH
ARC-659-1b
2.41 nM (8.62 ± 0.12) 12.9 nM (7.89 ± 0.25)
1a
N
OH
N
2
a
N
N
a
b
c
O
Concentration of the substrate (ATP) in the kinetic assay.
Values in brackets express pIC50-s with 95% confidence intervals.
ARC-659-1a/1b/2a/2b was synthesized from the mixture of racemic
diastereomers 1a/1b and 2a/2b.
N
NH2
N
NH2
O
N
N
O
1
b
6
O
N
2b
'
O
N
4
'
1'
2'
N
of adenosine may increase the affinity of compounds to-
8
wards several PKs up to 5-fold. Additionally, crystal
N
O
5'
O
N
3
'
N
OH
N
N
structures of cAPK Ca with adenosine derivatives reveal
the absence of polar interactions between the 4 -oxygen
OH
0
Scheme 1. Synthesis of the carbocyclic nucleoside analogs.
of the ribose and the kinase. These facts point to the
possibility of the use of chemically and enzymatically
9
more stable carbocyclic (cyclopentane) adenosine mim-
ics for the design of potent ARC-type inhibitors.
ently, the low yield of the synthesis resulted from insta-
bility of the starting epoxides (5a and 5b) in the reaction
medium. The pairs of diastereomers (1a/1b and 2a/2b)
were separated by column chromatography on silica
gel. The overlapping fractions were purified repeatedly.
Structural elucidation of the diastereomers was based
Here we present the synthesis of cyclopentane-based car-
0
0
bocyclic analogs of 3 -deoxyadenosine (3 -deoxyariste-
romycin), chromatographic separation of the formed
stereoisomers, their conjugation with oligo-(D-argi-
nine)-containing peptides, and characterization of the
latter compounds as highly potent inhibitors of baso-
philic protein kinases.
0
0
on different NOE signals between 1 - or 2 -protons
0
and 4 -proton of the isomers. The diastereomer 1a/1b
eluted faster than 2a/2b in case of normal phase chroma-
tography, which is consistent with the previous data
1
4
concerning ribose counterparts of the compounds.
Enantiomers 1a and 1b were separated by HPLC on a
chiral chromatography column (Chiralpak AD-H).
The shortest synthetic scheme was selected to prepare
0
the derivatives of carbocyclic 3 -deoxyadenosine 1a,
b, 2a, and 2b that are applicable for easy conjugation
1
with peptides. 3-Cyclopentene-1-carboxylic acid (3)
was converted to the methyl ester 4 and the obtained es-
ter was oxidized to the appropriate epoxide (5a/5b ratio
Reactions of the enantiomers (1a and 1b) with (R)-a-
methoxy-a-trifluoromethylphenylacetyl chloride in
pyridine gave Mosher esters 8a and 8b (Scheme 2),
3
and 5b) of the epoxide were separated by column chro-
:1) with mCPBA (Scheme 1). Cis and trans isomers (5a
O
N
O
N
N
(a) TEA, H O
2
N
1
0
O
HO
matography as described before. The products were
treated with the excess of adenine in the presence of
N
(quantitative)
N
N
N
OH
OH
1
1
1b
NaH or a phosphazene base. Nucleophilic ring open-
ing of epoxide required increased temperatures (90–
6
b
1
2
(c)
1
10 ꢁC). The reaction led to a mixture of four stereo-
(b)
R-Mosher chloride
Pyridine
56-58%)
13
isomers (two diastereomers as racemates—1a/1b and
a/2b) in almost equal amounts. The molar ratio of
1) HBTU, HOBt
H N-peptide-Resin
2
2
(
2
) TFA
formed stereoisomers was the same irrespective of
whether the starting epoxide existed as a pure single iso-
mer (5a or 5b) or a mixture of isomers (5a/5b). This was
apparently caused by the racemization occurring at the
O
N
NH-(D-Arg)6-C(O)NH2
HN
N
O
ARC-659-1b
O
0
N
most acidic 4 -carbon adjacent to the ester group in ba-
sic medium at higher temperatures (>100 ꢁC). 1a/1b and
N
N
O
N
8b
O
O
O
2
a/2b were obtained as racemates due to the symmetric
N
N
F3C
OH
structure of the starting epoxides 5a and 5b. The appli-
cation of both NaH and the phosphazene base led to
similar yields and relative proportions of the products.
NaH appeared to be the preferred base due to easier
purification of the products. Adenine alkylation reaction
producing a mixture of 1a/1b and 2a/2b was the most
critical step due to low overall yield (12–16%). Appar-
(a)
(b)
(b)
Analogically: 2a/2b
7a/7b
6a
ARC-659-2a/2b
ARC-659-1a
(
a)
1a
(c)
1
a
8a
Scheme 2. Synthesis of the peptide conjugates and Mosher esters.

5338
E. Enkvist et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5336–5339
respectively, which were used for the determination of
the absolute configuration of the compounds by
Table 2. Residual activities of PKs in the presence of the inhibitor
ARC-659-1b
1
5
19
NMR. Methyl esters (1a, 1b and racemates 1a/1b
and 2a/2b) were hydrolyzed in the presence of triethyl-
amine (TEA). The obtained carboxylic acids (6a/6b,
Kinase
Kinase group
Residual activity (%)
0
.01 lM
1 lM
0 ± 0
1
6
PKA/cAPK
ROCK 2
PKBb
MSK1
PRK2
AGC
AGC
7 ± 1
41 ± 2
74 ± 1
37 ± 8
59 ± 7
92 ± 1
81 ± 7
71 ± 7
86 ± 11
70 ± 6
40 ± 4
62 ± 1
6
a, 6b, and 7a/7b ) were coupled to the peptides with
0
1 ± 0
2 ± 0
3 ± 0
0
O-(1H-benzotriazol-1-yl)-N,N,N ,N -tetramethyluronium
tetrafluoroborate (TBTU) and N-hydroxybenzotriazole
AGC
AGC
AGC
(
sin-bound compounds with trifluoroacetic acid (TFA)
HOBt) activation. Cleavage and deprotection of the re-
3 ± 1
6 ± 1
7 ± 0
PIM1
S6K1
PAK5
CAMK
AGC
STE
0
led to the target conjugates of carbocyclic 3 -deoxyaden-
osine and hexa-(D-arginine) (ARC-659). The peptide
conjugates made from different isomers are designated
by adding the number of the isomer (1a, 1b, etc.) to
the common code of the compounds ARC-659.
10 ± 1
10 ± 3
12 ± 1
19 ± 3
19 ± 1
20 ± 1
27 ± 4
28 ± 1
28 ± 8
30 ± 2
32 ± 1
33 ± 4
51 ± 3
63 ± 6
72 ± 10
73 ± 15
77 ± 4
84 ± 5
84 ± 5
88 ± 7
88 ± 5
91 ± 13
96 ± 8
98 ± 4
104 ± 10
111 ± 8
114 ± 5
MELK
AMPK
SGK1
CAMK
CAMK
AGC
CAMK1
PIM2
CAMK
CAMK
CAMK
AGC
103 ± 2
84 ± 7
76 ± 1
91 ± 2
96 ± 1
25 ± 4
83 ± 7
81 ± 4
86 ± 7
76 ± 6
81 ± 5
97 ± 3
82 ± 5
85 ± 9
82 ± 0
86 ± 5
97 ± 4
88 ± 1
98 ± 3
103 ± 9
101 ± 11
103 ± 4
The inhibitory potencies of the synthesized compounds
CHK1
PDK1
RSK2
were evaluated against cAPK Ca with the application
1
7
of fluorometric TLC kinase activity assay, and the re-
sults are expressed as the IC50 values in Table 1. TAM-
RA-kempide (30 lM) and ATP (0.1 or 1.0 mM) were
used as substrates. The application of higher concentra-
tion of ATP (1.0 mM) in the test system increased the
IC50 value and enabled to avoid tight-binding conditions
AGC
AGC
PKCa
PKBa
PKCf
AGC
AGC
DYRK3
Lck
CMGC
TK
1
8
AURORA B
PKD1
CSK
Other
CAMK
TK
of the assay (IC50 6 Ckinase). The racemic nucleoside
analogs 1a/1b and 2a/2b showed expectedly weak inhib-
itory potencies. The IC50 values for these compounds
were comparable to that of adenosine (IC50 = 350 lM).
The diastereomer 2a/2b, being structurally less similar to
the native adenosine than 1a/1b, showed slightly stron-
ger inhibition. The inhibitory potency of the conjugates
of these racemates with hexa-(D-arginine) differed by
more than 30-fold, and ARC-659-1a/1b (structurally
more similar to adenosine) revealed much higher po-
tency. This difference in activity may originate from
the more effective positioning of the 6-aminohexanoic
acid linker and the peptide in complex with the enzyme.
ERK1
Src
CMGC
TK
GSK3b
CK2
CMGC
Other
CAMK
CAMK
AGC
MAPKAP-K3
PRAK
RSK1
JNK3
NEK6
CDK2-Cyclin A
CMGC
Other
CMGC
To make the inhibitory potencies comparable, assays were run at ATP
6
,20
m
concentration which was close to the K value for the given kinase.
The separated enantiomer 1b was ca. 10-fold more po-
tent than 1a. This result indicates the importance of cor-
0
rect positioning of 2 -hydroxy group for inhibitor
tion potency was determined toward Akt/PKB, SGK1,
and CAMK1. The moderately inhibited kinases (>90%
of inhibition at 1 lM concentration of the inhibitor)
were PIM1, S6K1, PAK5, MELK, and AMPK. Some
other kinases retained higher activity in the presence
of ARC-659-1b and several tested kinases were not
inhibited at all. Generally, the inhibition profile of
ARC-659-1b was similar to that of the previously re-
ported conjugate of adenosine and oligo-(D-arginine),
binding to the enzyme. The conjugates with peptides re-
vealed similar one-magnitude difference in activity. To
the best of our knowledge ARC-659-1b is the most
potent bisubstrate-analog inhibitor of protein kinases
described so far.
The selectivity of ARC-659-1b was tested (on the com-
mercial basis at the Division of Signal Transduction
Therapy, University of Dundee) against the panel of
protein kinases. Inhibition efficiency was determined at
two concentrations of ARC-659-1b (1 and 0.01 lM).
6
ARC-902. However, ARC-659-1b has remarkable
cAPK selectivity while its adenosine counterpart ARC-
902 revealed preference for ROCK-II. Several kinases
of the CAMK group like AMPK, PIM-2, and CAMK1
were inhibited more strongly by ARC-659-1b, whereas
ARC-902 was more potent towards the kinases of the
RSK group. PAK5 and MELK kinases (both known
as basophilic kinases) were also strongly inhibited by
ARC-659-1b.
The inhibition data for 34 kinases are presented as the
percent of residual activity of the kinase in the presence
of the inhibitor relative to control incubations where the
inhibitor was omitted (Table 2). The analysis of the data
reveals that ARC-659-1b most potently inhibits cAPK.
The K value of ꢀ0.5 nM was estimated for this PK (Ta-
i
ble 1; calculated according to the Cheng–Prusoff equa-
In conclusion, we have accomplished synthesis of the
0
2
1
tion with Km of 20 lM for ATP). The compound
also showed strong inhibition (ca. 50% at 10 nM concen-
tration) of ROCK-II, MSK1, and PRK2. Similar inhibi-
carbocyclic analog of 3 -deoxyadenosine along with sep-
aration and characterization of its stereoisomers. The
conjugation of these compounds with hexa-(D-arginine)

E. Enkvist et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5336–5339
5339
peptide led to potent inhibitors of basophilic PKs. The
stereochemistry of the nucleoside part of most active
conjugate ARC-659-1b is similar to that of adenosine,
but the novel conjugate does not incorporate a native
nucleoside with a chemically and enzymatically degrad-
able glycosidic bond. The selectivity study revealed that
ARC-659-1b strongly inhibited basophilic kinases and
was almost inactive toward a number of PKs that do
not contain multiple basic amino acids (arginine and/
or lysine) residues in their consensus phosphorylation
sequences.
Corbin, J. D. Eur. J. Biochem. 1984, 140, 289; (c)
Kwiatkowski, A. P.; King, M. M. Biochemistry 1987, 26,
7636; (d) Bhatnagar, D.; Roskoski, R.; Rosendahl, M. S.;
Leonard, N. J. Biochemistry 1983, 22, 6310.
9
1
. Marquez, V.; Lim, M. Med. Res. Rev. 1986, 6, 1.
0. (a) Martinez, L. E.; Nugent, W. A.; Jacobsen, E. N.
J. Org. Chem. 1996, 61, 7963; (b) Legeret, B.; Sarakauska-
ite, Z.; Pradaux, F.; Saito, Y.; Tumkevicius, S.; Agrofo-
glio, L. A. Nucleosides, Nucleotides, Nucleic Acids 2001,
20, 661.
11. 2-tert-Butylimino-2-diethylamino-1,3-dimethylperhydro-
1,3,2-diazaphosphorine (BEMP) or tert-butylimino-tris
(
dimethylamino)-phosphorane (P1-tBu) Enkvist, E.; Rai-
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Acknowledgments
1
2. Examples of reactions between adenine and epoxides: (a)
Meillon, J. C.; Griffe, L.; Storer, R.; Gosselin, G.
Nucleosides, Nucleotides, Nucleic Acids 2005, 24, 695; (b)
Gosselin, G.; Griffe, L.; Meillon, J. C.; Storer, R.
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Tetrahedron Lett. 2005, 46, 1927.
The research was supported by grants from the Estonian
Science Foundation (6710 and 6778) and the Estonian
Ministry of Education and Sciences (0182611s03).
*
*
*
Supplementary data
13. Analytical data of the key compound (+/ꢁ)-(1S ,3R ,4R )-
-(6-amino-purin-9-yl)-4-hydroxy-cyclopentanecarboxylic
acid methyl ester (1a/1b). UVmax 261 nm (MeOH). HRMS:
3
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.08.016.
+
m/z calcd for C12
H
16
5
N O
3
(M+H ): 278.1243. Found:
1
2
ddd, J = 13.6, 10.0 and 6.8 Hz, 3 h), 2.30 (1H, ddd, J = 13.6,
78.1237. H NMR (500 MHz, CD SOCD ) d 1.88 (1H,
3
3
0
0
7
.5 and 6.5 Hz, 3 l), 2.35 (1H, ddd, J = 13.1, 10.4, 9.4 Hz,
0
0
6
m, J = 10, 9.4, 8.1 and 6.5 Hz, 4 ), 3.64 (3H, s, CH ), 4.49
h), 2.46 (1H, ddd, J = 13.1, 8.1 and 7.4 Hz, 6 l), 3.17 (1H,
0
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0
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