Synthesis and biological evaluation of 3,5-diaminoindazoles as cyclin-dependent kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.03.002

A novel series of 3,5-diaminoindazoles were prepared and found to be CDK inhibitors. Potent inhibitors against CDK1 and CDK2 were obtained by introduction of 1λ⁶-isothiazolidine-1,1-dioxide at 5-position of indazole. Anti-proliferative activities of compounds were evaluated using EJ, HCT116, SW620, and A549 cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2008.03.002

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2292–2295
Synthesis and biological evaluation of 3,5-diaminoindazoles
as cyclin-dependent kinase inhibitors
Jinho Lee,a,* Hwangeun Choi,b Kyoung-Hee Kim,b Shinwu Jeong,c Jong-Wook Park,d
Chul-Su Baeka and Sei-Hee Leea
aDepartment of Chemistry, Keimyung University, 1000 Sindang-Dong, Dalseo-Gu, Daegu 704-701, South Korea
bLG Life Sciences, Science Town, Taejon 305-380, South Korea
cUSC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, CA 90089, USA
dDepartment of Immunology, Keimyung University School of Medicine, Daegu, South Korea
Received 19 December 2007; revised 29 February 2008; accepted 3 March 2008
Available online 6 March 2008
Abstract—A novel series of 3,5-diaminoindazoles were prepared and found to be CDK inhibitors. Potent inhibitors against CDK1
and CDK2 were obtained by introduction of 1k6-isothiazolidine-1,1-dioxide at 5-position of indazole. Anti-proliferative activities of
compounds were evaluated using EJ, HCT116, SW620, and A549 cancer cell lines.
Ó 2008 Elsevier Ltd. All rights reserved.
Cyclin-dependent kinases (CDKs) are serine/threonine
kinases which control the proliferation of eukaryotic
cell.1 Activities of CDKs are regulated precisely by mul-
tiple mechanisms. CDKs are activated by cyclin binding
and phosphorylation while deactivated by either re-
moval of cyclin or binding with CDK inhibitors
(CDKIs) such as Cip/Kip and INK families.2 Deregula-
tion of CDKs by abnormal high expression of cyclin
and/or downregulation of CDKIs have been found in
various human tumors.3 These results prompted the
search for the inhibitors of CDKs as a possible candi-
date for the development of anti-cancer chemother-
apy.4,5 Researches have been mainly focused on the
searching for ATP competitive inhibitors.
CDK2, 3,5-diaminoindazole was designed to provide
hydrophobicity to 3-aminopyrazole while not bumping
into the gate-keeper residue (Phe80) of the enzyme.7
During the progress of research, 3-aminoindazole8 and
3-aminopyrazole9 derivatives were reported as potent ki-
nase inhibitors including CDK2. Also, three hydrogen
bonding interactions between pyrazole and CDK2 were
confirmed by the crystal structure determination.9
The preparation of 3-aminoindazole 5 is outlined in
Scheme 1. Commercially available 5-nitroanthranilonit-
rile was reacted with hydroxylamine to provide N-
hydroxyamidine 2. It was converted to 3-aminoindazole
by the reaction of 2 with ethyl phenylacetate via the for-
mation of [1,2,4]-oxadiazole.10 The reduction of nitro
group was followed by the derivatization of amino
group which provided 3,5-diaminoindazole 5.11
The efforts to find new scaffold for the inhibitor of
CDK2 were started with the investigation of X-ray
structure of CDK2.6 The docking study with the ATP
binding pocket of CDK2 resulted 3-aminopyrazole as
an initial hit. It was expected to form three hydrogen
bonds with hinge region of CDK2. However, 3-amino-
pyrazole itself did not show inhibitory activity against
CDK2 up to 100 lM. Based on the X-ray structure of
The inhibitory activities of indazoles showed depen-
dency on the substituents at positions 3 and 5 (Table
1). Phenylacetyl type substituents at position 3 showed
better inhibitory activity against CDK2 than alkanoyl
or benzoyl (data not shown). Enzymatic inhibitory
activity depends largely on the substituents at position
5. Bulky substituents were favored but too flexible sub-
stituents were not allowed (5a, b vs 5c, d). 1k6-Isothiaz-
olidine-1,1-dione at position 5, not coplanar to the plane
of the indazole,13 provided high potency.
Keywords: CDK inhibitor; 3,5-Diaminoindazole; Isothiazolidine-1,1-
dioxide.
*
Corresponding author. Tel.: +82 53 580 5183; fax: +82 53 580
5164; e-mail: jinho@kmu.ac.kr
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.002

J. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2292–2295
2293
After 1k6-isothiazolidine-1,1-dione at position 5 was
found, the effect of substituents at position 3 had been
examined. The modification of phenyl group was per-
formed by acylation of 8 followed by deprotection
(Scheme 2).15 Phenylacetyl group was removed using
regioselective cleavage of the Boc-imide.16 In contrast
to the references, Boc-imide formation, imide bond
cleavage and di-Boc formation were performed in one
step by using 1 equivalent of DMAP.17
OH
NH
N
H
N
HN
N
NH
b
a
O
NH2
NH2
O2N
O2N
O2N
1
2
3
H
N
H
N
N
NH
N
e
c, d
O
O
N
Boc
R1
H2N
The effect of substituent at phenyl ring on the inhibitory
activity against CDK1 and CDK2 was not noticeable.
Even hydroxy and amino group (9j and 9k) which were
expected to make hydrogen bond with the c-carboxylate
of Glu8 did not improve the potency at all (Fig. 1). In
general, 3,5-diaminoindazole derivatives were about 2
order of magnitude more potent against CDK2 than
CDK4 while selectivity between CDK1 and CDK2
was less than 10 (Table 2).
4
5
Scheme 1. General synthetic route for 3,5-diaminoindazole. Reagents
and conditions: (a) NH2OHÆHCl, 80% aqueous EtOH, reflux, 95%; (b)
1—NaH, C6H5CH2CO2Et, THF–DMF (5:2) mixture, 48%; (c)
(Boc)2O, NaOH, THF–H2O (6:1) mixture, quantitative; (d) Pd/C,
H2, MeOH, quantitative; (e) 5a,b,c,d: RCHO, NaBH(AcO)3, DCE–
DMF, yields were 20–30%, 5e,f: 1—Br(CH2)nCOCl (n = 3, 4), pyridine;
2—NaH, THF, yields in two steps were 41% (5e) and 34% (5f), 5g: 1—
CbzNHCH2CO2H, EDC, HOBt, DMF; 2—(n-Bu)4NF, THF, 80%
yield in two steps,12 5h: 1—ClCH2CH2CH2SO2Cl, pyridine, DCM,
quantitative; 2—NaH, DMF, quantitative; (e) sat. HCl in EtOAc,
quantitative.
X-ray crystal structures revealed that chair-shaped 3,5-
diaminoindazole compounds bind in the ATP pocket
of CDK2 using three hydrogen bonds (Fig. 1), which
are formed between aminoindazole moiety and the car-
bonyl group of Glu81, carbonyl and amide groups of
Leu83.18 One side of aminoindazole ring is packed
against the side chain of Phe80, the gate-keeping resi-
due. 1k6-Isothiazolidine-1,1-dione at position 5 rotates
approximately 120 deg compared to the aminoindazole
ring, which makes its oxygen atom to form hydrogen
bond with the side chain of Asp145 and its methylene
moiety in close proximity of Gly13 and Val18. These
additional interactions explain the high potency of
1k6-isothiazolidine-1,1-dione compounds. The phenyl
group makes an angle of 100 deg with respect to the
aminoindazole group and is packed against the side
chain of Ile10. In compound 9j, the hydroxy group
forms additional hydrogen bond with the side chain of
Glu8. In compound 9n, the side chain of Glu8 has an
alternative conformation to accommodate the piperi-
dine group.
Table 1. Structure and inhibitory activity for 3,5-diaminoindazoles
against CDK2a
H
N
N
O
NH
R1
b
Compound
R1
CDK2 IC50 (lM)
4
NO2
C2H5NH
(C2H5)2N
2.0
0.5
5a
5b
5c
5d
5e
5f
5g
5h
0.2
1.8
(n-C3H7)2N
(n-C4H9)2N
CO(CH2)3N
CO(CH2)4N
COCH2NHCON
SO2(CH2)3N
>100
0.17
7.1
2.0
0.036
a The CDK2 inhibitory assays were performed as described in Ref. 14
[ATP] = 100 lM.
Even though substituent at phenyl ring slightly affected
the potency against CDKs, the anti-proliferative activity
against several human cancer cell lines showed high
b Values are means of three experiments.
Boc
N
H
N
N
H
N
Boc
N
N
N
N
O
N
O
N
a
b
NH
Cbz
Cbz
O
O
S
O
O
O
O
S
S
N
5h
6
7
H
N
H2N
R
N
N
N
NH
O
c
d
Cbz
O
S
O
O
O
S
N
N
8
9
Scheme 2. General synthetic route for 9. Reagents and conditions: (a) CbzCl, TEA, DCM, 94%; (b) (Boc)2O, TEA, DMAP, DCM, quantitative; (c)
sat. HCl in EtOAc, 47%; (d) 1—corresponding acyl chloride, THF, reflux; 2—2 N NaOH, yields in two steps were 40–70% except 9j, k, l, m, n, t
which were 10–20%.

2294
J. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2292–2295
Table 3. Anti-proliferative activities of 3,5-diaminoindazole deriva-
tives on different human cancer cell lines
Compound
Cytotoxicitya IC50 (lM)
EJ
3.6
HCT116
A549
SW620
b
5h
9e
9g
9i
4.6
1.23
1.4
2.2
0.93
1.57
1.67
1.5
3.87
0.93
2.0
1.77
6.53
0.83
0.22
1.2
2.57
b
1.56
3.83
9j
5.2
b
b
9l
0.35
0.05
1.1
b
b
9m
9n
9o
9p
9q
9r
9s
9t
0.055
0.98
0.28
0.82
48
2.3
2.47
b
0.15
b
35
1.3
1.4
0.93
0.08
0.12
0.052
1.05
0.31
0.18
2.5
0.09
0.055
1.4
0.04
b
a Exponentially growing cells were treated with test compounds at
various concentrations for 48 h, and then the cell numbers were
measured. The compound concentration with 50% growth inhibition
activity was determined.19 Values are means of three experiments.
b Not determined.
dependency on the type of substituents (Table 3). There
were up to 2 order of magnitude differences in cell
growth inhibitory activity between compounds which
have similar enzymatic IC50 values. 3,5-Diaminoindaz-
oles substituted by (4-diethylaminophenyl)acetyl 9m, 2-
naphtylacetyl 9r, and 4-biphenylacetyl 9s at 3-amino
group potently inhibited the proliferation of human can-
cer cell lines such as EJ (bladder carcinoma), HCT116,
SW620 (colon carcinoma), and A549 (lung carcinoma).
Figure 1. Crystal structure of compounds in complex with CDK2. (a)
Complex structure of 9j (green) with CDK2 reveals three hydrogen
bonds with the CDK2 hinge region. (b) Surface representation of ATP
binding pocket in complex with compound 9n (green). The C, N, and
O atoms are indicated as gray, blue, and red colors.
In summary, we have discovered a novel series of 3,5-
diaminoindazole by structure based drug design, which
is based on the initial hit obtained from the docking
study. Introduction of 1k6-isothiazolidine-1,1-dione at
position 5 of indazole provided high potency against
CDK1 and CDK2. It was found that cytotoxicity de-
pended on the structure of substituent at 3-amino group.
Optimization of 3,5-diaminoindazole afforded effective
inhibitors against cyclin-dependent kinases 1 and 2
which had potent anti-proliferative activities on human
cancer cell lines.
Table 2. Inhibitory activities of 3,5-diaminoindazoles against CDK1,
CDK2, and CDK4
a
Compound
R
IC50 (lM)
CDK1 CDK2 CDK4
5h
9a
9b
9c
9d
9e
9f
C6H5
3-F–C6H4–
4-F–C6H4–
0.08
0.03
0.036
0.01
0.02
3.7
0.71
3.0
0.89
1.05
2.0
1.8
2.0
4.0
1.8
1.75
2.2
0.21
>1
0.05
0.08
2-Cl–C6H4–
3-Cl–C6H4–
4-Cl–C6H4–
3-Br–C6H4–
4-Br–C6H4–
3-CH3–C6H4–
4-CH3–C6H4–
4-HO–C6H4–
4-NH2–C6H4–
4-(CH3)2N–C6H4–
4-(C2H5)2N–C6H4–
0.024
0.01
0.01
0.034
0.012
0.047
0.022
0.05
Acknowledgment
0.007
0.007
0.01
9g
9h
9i
This research was supported by the Bisa Research Grant
of Keimyung University.
0.04
0.028
0.06
0.007
0.009
0.016
0.01
9j
9k
9l
0.028
0.06
References and notes
9m
9n
9o
9p
9q
9r
9s
9t
0.04
0.03
4-(1-Piperidinyl)-C6H4– 0.15
0.75
1.3
1.4
3
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a Values are means of three experiments.

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NMR (400 MHz, DMSO-d6) d 1.90 (2H, p, J = 6.8), 2.99
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(1H, dd, J = 1.6, 9.0), 7.00–7.07 (3H, m), 7.10–7.11 (2H,
m), 7.43–7.45 (4H, m), 10.28 (1H, s), 12.32 (1H, s); 13C
NMR (100 MHz, DMSO-d6) d 18.9, 42.8, 48.0, 48.2,
111.3, 114.9, 116.6, 122.7, 126.1, 126.6, 127.9, 128.0, 128.2,
128.3, 130.3, 132.3, 133.5, 134.2, 139.4, 140.7, 169.7; ESI-
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J = 6.4), 3.34 (2H, s), 6.91–6.88 (2H, m), 6.98–7.04 (5H,
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17. Procedure for the synthesis of compound 7. Compound 6
(5.2 g, 11 mmol) was dissolved in CH2Cl2 (100 ml). Then,
DMAP (1.36 g, 11 mmol), TEA (1.55 ml, 11 mmol) and di-
tert-butyldicarbonate (4.85 g, 22 mmol) were added at room
temperature. After stirring for 30 min at room temperature,
the solvent was evaporated and the resulting residue was
purified by silica gel column chromatography using hexane–
EtOAc (1:2) as eluent, to give compound 7 (6.4 g, quanti-
1
tative). H NMR (400 MHz, CDCl3) d 1.38 (18H, s), 2.61
(2H, m), 3.44 (2H, t, J = 7.6), 3.81 (2H, t, J = 6.4), 5.54 (2H,
s), 7.31 (1H, d, J = 2), 7.45 (3H, m), 7.54 (2H, d, J = 7.2),
7.59 (2H, dd, J = 2, 9.2); 13C NMR(100 MHz, CDCl3) d
18.8, 27.7, 47.3, 48.1, 69.7, 84.1, 109.7, 115.8, 122.8, 122.9,
128.7, 128.8, 128.9, 134.5, 134.6, 137.9, 145.6, 149.9, 150.2;
ESI-MS (m/e) = 587 [M+1].
18. The coordinate has been deposited in the Protein Data
Bank: PDB ID 2R64.
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