Structure-activity relationship study of acridine analogs as haspin and DYRK2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.04.150

Haspin is a serine/threonine kinase required for completion of normal mitosis that is highly expressed during cell proliferation, including in a number of neoplasms. Consequently, it has emerged as a potential therapeutic target in oncology. A high throughput screen of approximately 140,000 compounds identified an acridine analog as a potent haspin kinase inhibitor. Profiling against a panel of 270 kinases revealed that the compound also exhibited potent inhibitory activity for DYRK2, another serine/threonine kinase. An optimization study of the acridine series revealed that the structure-activity relationship (SAR) of the acridine series for haspin and DYRK2 inhibition had many similarities. However, several structural differences were noted that allowed generation of a potent haspin kinase inhibitor (33, IC₅₀ <60 nM) with 180-fold selectivity over DYRK2. In addition, a moderately potent DYRK2 inhibitor (41, IC₅₀ <400 nM) with a 5.4-fold selectivity over haspin was also identified.

Full text of DOI:10.1016/j.bmcl.2010.04.150

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3491–3494
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study of acridine analogs as haspin
and DYRK2 kinase inhibitors
c
d
d
c
Gregory D. Cuny a,b, , Maxime Robin , Natalia P. Ulyanova , Debasis Patnaik , Valerie Pique ,
*
Gilles Casano c, Ji-Feng Liu e, Xiangjie Lin e, Jun Xian b, Marcie A. Glicksman a,b, Ross L. Stein a,b
Jonathan M. G. Higgins d
,
a Laboratory for Drug Discovery in Neurodegeneration, Brigham & Women’s Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA
b Partners Center for Drug Discovery, Brigham & Women’s Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA
c Aix-Marseille Université, Institut des Sciences Moléculaires de Marseille, iSm2-UMR CNRS 6263, Centre Saint Jérôme, Service 552, 13397 Marseille Cedex 20, France
d Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham & Women’s Hospital and Harvard Medical School, Smith 538A, 1 Jimmy Fund Way,
Boston, MA 02115, USA
e Aberjona Laboratories, Inc., 100 Cummings Center, Suite 242-F, Beverly, MA 01915, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Haspin is a serine/threonine kinase required for completion of normal mitosis that is highly expressed
during cell proliferation, including in a number of neoplasms. Consequently, it has emerged as a potential
therapeutic target in oncology. A high throughput screen of approximately 140,000 compounds identified
an acridine analog as a potent haspin kinase inhibitor. Profiling against a panel of 270 kinases revealed
that the compound also exhibited potent inhibitory activity for DYRK2, another serine/threonine kinase.
An optimization study of the acridine series revealed that the structure–activity relationship (SAR) of the
acridine series for haspin and DYRK2 inhibition had many similarities. However, several structural differ-
ences were noted that allowed generation of a potent haspin kinase inhibitor (33, IC50 <60 nM) with 180-
fold selectivity over DYRK2. In addition, a moderately potent DYRK2 inhibitor (41, IC50 <400 nM) with a
5.4-fold selectivity over haspin was also identified.
Received 14 April 2010
Accepted 30 April 2010
Available online 7 May 2010
Keywords:
Haspin
DYRK2
Kinase
Inhibitor
Oncology
Ó 2010 Elsevier Ltd. All rights reserved.
Structure–activity relationship
Acridine
Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase), also
know as Gsg2 (Germ Cell-Specific Gene-2),1 is a serine/threonine ki-
nase expressed in a variety of tissues (e.g., testis, bone narrow, thy-
mus, and spleen) and in proliferating cells.2 It is also highly
expressed in a number of neoplasms, including Burkitt’s lymphoma3
and diffuse large B cell lymphomas.4 Haspin’s kinase activity func-
tions during mitosis, where it has been shown to phosphorylate his-
tone H3 at Thr-3 (H3T3).5,6 This phosphorylation begins in G2/early
prophase, becomes maximal during prometaphase/metaphase and
then diminishes during anaphase.5,7 Depletion of haspin by RNA
interference significantly reduces H3 Thr-3 phosphorylation in cells
and prevents normal completion of mitosis.5,8,9
Human haspin, consisting of 798-amino acids, contains a C-ter-
minal kinase domain that is a divergent member of the eukaryotic
protein kinase (ePK) superfamily.10,11 Sequence comparisons and
recent crystal structures reveal that haspin contains a number of
structural differences from other ePKs, particularly in the C-termi-
nal lobe of the kinase domain. For example, the highly conserved
DFG motif involved in ATP binding and the APE motif involved in
stabilizing the activation loop in many ePKs are altered. Haspin
adopts a constitutively active conformation and conserved serine,
threonine and tyrosine residues are absent from its activation
loop.10,11
DYRKs (Dual-specificity Tyrosine-regulated Kinases) belong to
the CMGC family of ePKs and contain a conserved kinase domain
and adjacent N-terminal DYRK homology box. This group of ki-
nases can be further divided into class 1 kinases (DYRK1A and
1B) that have an N-terminal nuclear localization signal and a C-ter-
minal PEST region and class 2 kinases (DYRK2, 3, and 4), which lack
these motifs and are predominantly cytosolic. Although DYRKs
phosphorylate substrates on serine or threonine residues, their
activity depends upon autophosphorylation of an essential activa-
tion loop tyrosine during synthesis.12 DYRKs appear to contribute
to regulation of an array of signaling pathways, including NFAT sig-
naling in the brain and immune system, Hedgehog signaling, cas-
pase activity during apoptosis, cell cycle progression and mitosis,
and p53 activation in response to DNA damage.13–16
In order to study the role of haspin’s kinase activity in mitosis
(and other cellular processes) and its potential role in cancer, we
sought to identify and optimize inhibitors. Utilizing a recently
developed time-resolved fluorescence resonance energy transfer
* Corresponding author. Tel.: +1 617 768 8640; fax: +1 617 768 8606.
E-mail address: gcuny@rics.bwh.harvard.edu (G.D. Cuny).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.150

3492
G. D. Cuny et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3491–3494
(TR-FRET) high throughput screening (HTS) assay with histone H3
peptide as substrate and a europium-labeled phosphospecific
monoclonal antibody for detecting phosphorylated substrate
(H3T3ph),17 the acridine derivative 1 was discovered as a potent
CO2H
NH
R1
CO2H H2N
b
a
R2
R1
+
R2
Br
inhibitor (Fig. 1; IC50 = 0.010 lM). Kinase profiling of 1 revealed po-
tent DYRK2 inhibitory activity as well. Herein, we describe the
structure–activity relationship (SAR) of the acridine series for both
haspin and DYRK2 inhibition.
2
3
4
S
Cl
The synthesis of many of the acridine analogs was accomplished
using the procedure outlined in Scheme 1. 2-Bromobenzoic acids 2
were coupled to anilines 3 using a copper-mediated procedure to
give 4.18 Cyclization of 4 to 9-chloroacridines 5 was accomplished
using phosphorus oxychloride. Treatment of 5 with P4S10 in the pres-
ence of DMPU gave6. Alternatively, acid 4 was cyclizedto ketone7 in
the presence of polyphosphoric acid (PPA), which was subsequently
treated with Lawesson’s reagent with microwave (MW) heating at
110 °C to produce 6.19 The thioketone 6 could be alkylated with var-
ious amino-protected alkylbromides (BrCH2(CH2)nY; Y = NHBoc,
NMeBoc, or NPhthalimide) in the presence of base (KOH) and the
phase-transfercatalysttetrabutylammonium iodide (TBAI) in a mix-
ture of toluene and water to give 8. Boc-protected analogs of 8
(Y = NHBoc or NMeBoc) upon treatment of 4 N HCl in a mixture of
1,4-dioxane and methanol gave 9 (Z = NH2 or NHMe). Alternatively
for analogs of 9 with Z = NH2, they could also be prepared directly
from 6 via alkylation.
Acridine analogs where the alkylamine groups were connected
through an O or NH were prepared according to the procedure
illustrated in Scheme 2. Ketone 10 was converted to 11 as previ-
ously described. Then a nucleophilic aromatic substitution with a
mono-N-protected diamine followed by removal of the protecting
group gave 12. Ketone 10 was also alkylated with N-Boc-protected
1-amino-3-bromopropane in the presence of KOH and phase-
transfer catalyst (i.e., TBAI) followed by de-protection to give 13.
Acridine analogs where the alkylamine group was connected
through a methylene were prepared using the method described
in Scheme 3. Diphenylamine derivative 14 was condensed with
acetic acid to give acridine analog 15.20 The methyl substituent
in the 9-position was oxidized with selenium dioxide to give alde-
hyde 16.21 Addition of the anion of an N-Boc-protected alkyne gave
alcohol 17. Exposure of 17 to reducing conditions (Pd/C and Et3SiH)
resulted in concomitant reduction of the alkyne and alcohol. Final-
ly, removal of the protecting group on the amine yielded 18.
The synthesis of a tetrahydroacridine analog is outlined in
Scheme 4. b-Ketoester 19 was allowed to react with 4-anisidine,
20, to produce 21. Cyclization of 21 in sulfuric acid gave ketone 22.
This material was converted to the corresponding thioketone 23.
Alkylation with 1-amino-3-bromopropane hydrobromide gave 24.
Finally, a 2-phenylquinoline analog was synthesized accord to
the procedure outlined in Scheme 5. The acetophenone derivative
25 was coupled with benzamide in the presence of a catalytic
amount of CuI to give 26.22 Base-mediated cyclization of 26 gave
27. Conversion of this material to the thiol analog 28 was accom-
plished with Lawesson’s reagent. Then alkylation and de-protec-
tion as previously described for other analogs yielded 29.
R1
R2
R2
R1
c
N
H
N
5
6
O
e
d
f
R2
R1
4
6
N
H
g
7
Z
n
Y
S
N
S
n
h
R2
R1
R2
R1
N
8
9
Scheme 1. Reagents and conditions: (a) Cu, K2CO3, pentanol, 110 °C; (b) POCl3,
120 °C; (c) DMPU, P4S10, 95 °C; (d) PPA, 110 °C; (e) Lawesson’s reagent, THF, MW at
110 °C; (f) BrCH2(CH2)nY, KOH, TBAI (cat.), toluene, H2O, 110 °C; (g)
BrCH2(CH2)nNH2ÁHBr, phenol, 80 °C; (h) 4 N HCl in 1,4-dioxane, MeOH, rt.
O
Cl
MeO
OMe
MeO
OMe
a
N
H
N
11
10
b, c
d, c
HN
NH2
O
NH2
MeO
OMe
MeO
OMe
N
N
13
12
Scheme 2. Reagents and conditions: (a) POCl3, 120 °C; (b) NH2(CH2)3NHBoc; (c) 4 N
HCl in 1,4-dioxane, MeOH, rt; (d) Br(CH2)3NHBoc, KOH, TBAI (cat.), toluene, H2O,
110 °C.
10 lM. The results demonstrated that this compound was quite
selective and only inhibited ten of the other kinases by P90% (Sup-
plementary Table S1 and Figure S1).23,24 IC50 values were deter-
mined for these kinases (Table 1), with only five being potently
Compound 1 was initially profiled for functional inhibitory
activity against a panel of two hundred and seventy kinases at
inhibited (IC50 <1 lM). DYRK2 was the most sensitive of these ki-
nases (IC50 = 2 nM).23,25
For the SAR study, human haspin kinase inhibitory activity of
the various compounds was evaluated using the same assay uti-
lized for the HTS, except in the presence of varying test compound
concentrations.17 DYRK2 kinase inhibitory activity was measured
by 33P-incorporation into Woodtide peptide substrate in the pres-
ence of human DYRK2 containing an N-terminal GST-fusion pro-
S
NH2
1
MeO
OMe
6
3
N
tein and c
33P-ATP.23
1
Only one of the methoxys in 1 appears necessary for potent has-
pin inhibition. When both of the methoxys were removed (30)
inhibitory activity was dramatically reduced (Table 2). However,
Figure 1. Haspin inhibitor identified by HTS. Also shown is the numbering system
for acridines.

G. D. Cuny et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3491–3494
3493
Table 1
IC50 determinations for 1 against profile panel kinases with P90% inhibition at 10
R
MeO
OMe
l
M
MeO
OMe
b
c
a
Kinase
IC50 (lM)
N
H
N
TRKB
91
15 R = Me
CLK1
0.21
0.10
0.002
0.019
1.6
14
DYRK1A
DYRK2
DYRK3
ROS
16 R = CHO
NHBoc
OMe
HO
HIPK1
HIPK2
PIM1
1.4
1.3
0.72
56
NH2
d, e
MeO
OMe
MeO
PIM2
N
N
18
17
Table 2
Scheme 3. Reagents and conditions: (a) InCl3, AcOH, MW at 230 °C; (b) SeO2, 1,4-
dioxane, 80 °C; (c) HC„CCH2NHBoc, n-BuLi, THF, À78 °C; (d) Pd/C, Et3SiH; (e) 4 N
HCl in 1,4-dioxane, MeOH, rt.
IC50 determinations for haspin and DYRK2 kinase inhibition23
X
( )n
S
R1
R2
CO2Et
NH
6
N
4
CO2Et H2N
a
Compd
X
R1
R2
n
IC50
Haspin
(lM)
+
DYRK2
O
OMe
1
30
31
32
33
34
35
36
37
38
39
40
41
42
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NHMe
NMe2
Phth
Phth
2-OMe
H
2-OMe
2-OMe
2-OMe
2-OH
2-OMe, 3-OMe
2-OMe, 3-Cl
2-OMe
2-OMe
2-OMe
2-OMe
2-OMe
2-OMe
7-OMe
H
H
7-Me
7-Cl
7-OH
H
2
2
2
2
2
2
2
2
1
3
2
2
2
2
0.01024
9.8
0.048
7.7
0.19
0.43
9.9
0.17
>10
9.4
0.29
0.036
0.12
0.23
0.39
1.3
OMe
19
20
0.017
0.070
0.055
0.044
ꢀ7
21
X
S
N
NH2
OMe
OMe
d
b
H
0.079
0.20
N
H
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
H
0.040
0.008
0.046
2.1
22 X = O
23 X = S
24
c
8.8
Scheme 4. Reagents and conditions: (a) POCl3, cat. PTSA, 120 °C; (b) H2SO4, 120 °C;
(c) DMPU, P4S10, 95 °C; (d) Br(CH2)3NH2ÁHBr, phenol, 80 °C.
Phth: phthalimide.
between the thioether at the 9-position of the acridine and the pri-
mary amine was examined. Truncation (37) resulted in reduced
activity, while addition of another methylene unit (38) had only a
minimal impact on potency. Next, the contribution of the amine
was examined. A secondary amine (39) was equally potent and a ter-
tiary amine (40) only resulted in a slight decrease in activity. How-
ever, incorporation of the nitrogen into a phthalimide (41 and 42)
resulted in a significant loss of activity. Finally, the thioether was
examined (Table 3). Replacement of the thioether with an amine
(12) or ether (13) was detrimental. But replacement of the sulfur
with a methylene (18) retained potent inhibitory activity.
O
O
O
MeO
MeO
MeO
Me
Me
NH
Ph
b
a
Br
N
H
27
Ph
O
25
26
SH
N
S
N
NH2
MeO
MeO
c
d, e
Ph
Ph
The SAR for DYRK2 inhibition had many similarities to that ob-
served for haspin inhibition with some notable exceptions. Both
methoxy groups appear to be necessary for DYRK2 inhibitory activ-
ity. For example, removal of both methoxy groups (30) was very
detrimental, while removal of one methoxy (31) still resulted in
a significant erosion of potency. Likewise, replacement of one
methoxy with a methyl (32) or a chlorine atom (33) was not as tol-
erated compared to the results observed for haspin inhibition.
Transposition of the 7-methoxy to the 3-position (35) also lead
to loss in activity. Similarly, and unlike in the haspin SAR, removal
of the 7-methoxy and addition of a 3-chloro group (36) was not tol-
erated for DYRK2 inhibition. The three aromatic rings that com-
prise the acridine also appear necessary for DYRK2 inhibition,
with both 24 and 29 lacking activity. The effect of the tether length
between the thioether at the 9-position of the acridine and the pri-
mary amine (37 and 38) was the same as previously observed for
29
28
Scheme 5. Reagents and conditions: (a) PhC(@O)NH2, 10 mol % CuI, (MeNHCH2)2,
K2CO3, toluene, 170 °C; (b) NaOH, 1,4-dioxane, 110 °C; (c) Lawesson’s reagent, THF,
MW at 150 °C; (d) Br(CH2)3NHBoc, KOH, TBAI (cat.), toluene, H2O, 110 °C; (e) 4 N
HCl in 1,4-dioxane, MeOH, rt.
when only one of the methoxys was removed (31) or replaced with a
methyl (32) or chlorine (33) potent activity (IC50 <100 nM) was re-
tained. The methoxy substituents of 1 could also be replaced with
hydroxyls (34). Interestingly, transposition of the 7-methoxy to
the 3-position (35) resulted in loss of activity. However, the 2-meth-
oxy-3-chloro analog (36) was still quite active. The three aromatic
rings that comprise the acridine also appeared necessary. Both 24
and 29 lacked haspin inhibitory activity. Next, the tether length

3494
G. D. Cuny et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3491–3494
Table 3
Supplementary data
IC50 determinations for haspin and DYRK2 kinase inhibition23
NH2
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.150.
X
MeO
OMe
N
References and notes
Compd
X
Haspin IC50
(lM)
DYRK2 IC50 (lM)
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Nishimune, Y. FEBS Lett. 1994, 355, 4.
12
13
18
NH
O
CH2
1.7
NA
0.025
6.5
NA
0.24
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haspin inhibition. For DYRK2 inhibition the primary amine was
better than secondary (39) or tertiary (40) amines. Interestingly,
incorporation of the nitrogen into a phthalimide resulted in a less
dramatic impact on DYRK2 inhibition compared to haspin and pro-
vided a moderately potent analog (41) that was 5.4-fold selective
for DYRK2 verses haspin. Similar to the observations made with
haspin inhibition, replacement of the thioether with an amine
(12) or ether (13) was detrimental to DYRK2 inhibition, while
replacement of the sulfur with a methylene (18) was tolerated, al-
beit with a fivefold reduction in potency.
In conclusion, a SAR study of the acridine derivative 1, identified
utilizing a recently developed HTS assay, was conducted for both
haspin and DYRK2 inhibition. The study revealed that several
structural features of 1, such as the three acridine aromatic rings,
the presence of one or both methoxy groups, a three or four meth-
ylene tether between the thioether and the acridine, and a thioe-
ther or CH2 (but not an amine or ether) link to the acridine were
necessary for both haspin and DYRK2 inhibition. However, several
structural differences were noted that allowed generation of a po-
tent haspin kinase inhibitor LDN-209929 (33, IC50 <60 nM) with
180-fold selectivity verses DYRK2. In addition, a moderately potent
DYRK2 inhibitor LDN-211848 (41, IC50 <400 nM) with a 5.4-fold
selectivity verses haspin was also identified. Additional optimiza-
tion of the acridine series, potentially utilizing co-crystallization
of these inhibitors with haspin and DYRK2, might provide more po-
tent and selective inhibitors for both kinases. In addition, these
inhibitors will serve as valuable molecular probes to study the cel-
lular functions of these kinases and their potential roles in various
diseases.
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23. See Supplementary data for details.
24. Carna Biosciences (http://www.carnabio.com/) determined a haspin inhibition
IC50 = 22 nM for 1. The assay used full-length human haspin fused at the N-
terminus to GST and expressed using baculovirus, histone H3 peptide as
Acknowledgments
substrate and [ATP] of 100 lM.
25. Determined by Carna Biosciences. Full-length human DYRK2 fused at the N-
terminus to GST and expressed using baculovirus, DYRKtide-F as substrate and
The authors thank Partners Healthcare for financial support. This
work was also supported in part by NIH Grant R01CA122608 to
J.M.G.H.
[ATP] of 10 lM were used.