QSAR analysis of pyrazolidine-3,5-diones derivatives as Dyrk1A inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.02.062

Individuals with Down syndrome (DS) suffer from mental retardation. Overexpression and the resulting increased specific activity of Dyrk1A kinase located on chromosome 21 cause a learning and memory deficit in Dyrk1A transgenic mice. To search for therape

Full text of DOI:10.1016/j.bmcl.2009.02.062

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2324–2328
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
QSAR analysis of pyrazolidine-3,5-diones derivatives as Dyrk1A inhibitors
Kyung Ah Koo a, Nam Doo Kim b, Yong Sog Chon c, Min-Su Jung d, Burm-Jong Lee e,
Jung Ho Kim c, Woo-Joo Song d,
*
a Biohealth Products Research Center (BPRC), Inje University, Gimhae, Gyongnam 621-749, South Korea
b R&D Center, Drug Design Team, Equispharm Inc., Ltd, 11F Gyeonggi Bio-center, 864-1, Iui-Dong, Yeongtong-Gu, Suwon, Gyeonggi-Do 443-270, South Korea
c Hanwha Chemical R&D Center, #6 Shinsung-Dong, Yusung-Gu, Daejeon 305-804, South Korea
d Graduate Program in Neuroscience, Institute for Brain Science and Technology (IBST), Inje University, Busanjin Gu, Gaegeum 2 Dong 633-146, Busan 614-735, South Korea
e Department of Chemistry, Inje University, Gimhae, Gyeongnam 621-749, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Individuals with Down syndrome (DS) suffer from mental retardation. Overexpression and the resulting
increased specific activity of Dyrk1A kinase located on chromosome 21 cause a learning and memory def-
icit in Dyrk1A transgenic mice. To search for therapeutic agents with Dyrk1A inhibition activity, previ-
ously we obtained HCD160 as a new hit compound for Dyrk1A inhibition. In the present study, we
synthesized 34 HCD160 derivatives to investigate the quantitative structure–activity relationship (QSAR).
This analysis could provide important information for novel drug discovery for treatment of DS related
learning and memory deficits.
Received 28 August 2008
Revised 29 January 2009
Accepted 16 February 2009
Available online 21 February 2009
Keywords:
Down syndrome
Learning and memory deficit
Dyrk1A inhibitor
QSAR
Ó 2009 Elsevier Ltd. All rights reserved.
Pyrazolidine-3,5-diones
Down syndrome (DS) results from an extra copy of human
chromosome 21. With a frequency of one in roughly every 800
births, it is the most common genetic anomaly.1 In addition to
characteristic physical features, individuals with DS show a variety
of phenotypes, including mental retardation, low muscle tone, con-
genital heart defects, gastrointestinal malformations, immune and
endocrine system defects, a high incidence of leukemia, and early
onset of Alzheimer’s disease (AD). Among the phenotypes, mental
retardation, such as learning and memory deficit and cognitive
decline, is a major factor in preventing DS individuals from leading
independent lives in their early to middle-age years.2 After the
third decade of their lives, individuals with DS also develop patho-
logical hallmarks of AD, amyloid plaques and neurofibrillary tan-
gles (NFTs).3 In the process of searching for genes responsible for
these phenotypes, the Dual specificity tyrosine (Y) phosphorylation
Regulated Kinase 1A (Dyrk1A) gene was isolated from human chro-
mosome 21.4 Dyrk1A protein is a serine/threonine kinase known to
play a critical role in neurodevelopment, and is activated by auto-
phosphorylation at the Tyr-321 residue.5 Transgenic mice over-
expressing the Dyrk1A protein showed hippocampal-dependent
learning and memory deficits.6 Several recent reports suggested
that Dyrk1A may also be involved in the pathological mechanisms
of APP and Tau, and may accelerate the formation of amyloid
plaques and NFTs which are insoluble deposits of b-amyloid and
hyperphosphorylated Tau, respectively, causing the early onset of
AD pathogenesis in DS.7 Thus, the development of therapeutic
agents with Dyrk1A inhibition activity will benefit individuals with
DS in treating mental retardation and AD.
Harmine and epigallocatechin-3-gallate (EGCG), a major tea
compound, were reported as potent Dyrk1A inhibitors in the
course of searching for therapeutic agents.8 Previously, as an initial
attempt to develop a mechanism-based drug to treat Dyrk1A asso-
ciated DS learning and memory deficits, we isolated a synthetic hit
compound through a combination of in silico, in vitro, and in cell-
based screening.9 The hit compound, HCD160, appears to be a good
starting template for further optimization in order to increase the
inhibitory potency against Dyrk1A activity (Fig. 1). In the present
study, we further investigated the quantitative structure–activity
relationship (QSAR) for 34 pyrazolidine-3,5-diones derivatives of
HCD160.
The synthetic strategy was designed to maintain the pyrazoli-
dine-3,5-diones as a core scaffold based on the following observa-
tions: (i) the previous result showed that NH and C@O groups of
HCD160 bind tightly to the backbones of Glu 239 and Met 240 of
Dyrk1A; and (ii) amino acid groups in the ATP binding pocket of
Dyrk1A have predominantly negative charges in the surface mod-
els as compared with other protein kinases, such as glycogen
* Corresponding author. Tel.: +82 51 892 4186; fax: +82 51 892 0059.
E-mail address: wjsong@inje.ac.kr (W.-J. Song).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.062

K. A. Koo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2324–2328
2325
This suggests the operation of different inhibition mechanisms be-
tween Dyrk1A autophosphorylation and substrate phosphorylation.
Roscovitine, used as an internal control, showed less inhibition
3-Methoxy-4-hydroxy phenyl
moiety interacts with Leu
294 and Ile 165 of Dyrk1A.
activity, with IC50 values of 5 lM for autophosphorylation and 63%
OCH3
inhibition of kinase activity.8 The QSAR values for the second series
of 15 HCD160 derivatives with 1,4-disubstituted pyrazolidine-3,5-
diones are reported in Table 2. Compounds 21, 30 and 34 exhibited
inhibition activity against Dyrk1A autophosphorylation with IC50
Cl
O
HO
NH
N
Cl
values of 0.6, 1.2 and 2.5 lM, respectively, while the kinase activity
was inhibited by 44–78%. Among the three compounds, compound
21 showed similar inhibition activity to that of compound 18 on
autophosphorylation and kinase reaction. Therefore, we have de-
scribed the synthetic methods of compounds 18 and 21 in the refer-
ences and note paragraph.12
NH and C=O groups
bind tightly to the
backbones of Glu 239
and Met 240 of Dyrk1A.
O
3,4-Dichloro phenyl
moiety interacts with Phe
238 and Val 173 residues
of Dyrk1A.
Most of the compounds tested in the present study showed less
potent inhibition activity than that of HCD160. However, the effect
Figure 1. Schematic representation of the refined docking model of HCD160 with
Dyrk1A using PharmoMap .
TM
of compounds 18 (IC50 = 0.6
phosphorylation were very potent as compared with that of
HCD160 (IC50 = 2.5 M). And also, IC50 for compounds 18 and 21
on the substrate phosphorylation were 1.25 M and 6 M, respec-
lM) and 21 (IC50 = 0.6 lM) on auto-
l
synthase kinase-3 beta (GSK-3b), cycline-dependent kinase-2
(CDK-2) and casein kinase-2 (CK-2) (Fig. 2).9,10 In silico screening
l
l
TM
tively. Therefore, we further characterized these two compounds
18 and 21 by assessing the specificity of the inhibition and cytotox-
icity. To evaluate the specificity, we performed comparative assay
for them with other well-characterized kinases (Table 3). Selected
kinases included a Dyrk family protein (Dyrk2) and neighboring
proteins in the dendrogram constructed by Becker and Joost, such
as CLK3 and GSK3b.13 Other tested proteins represented kinases
from the core panel described by Davies et al. and from kinase
groups described by Vieth et al.14 As expected from its close rela-
tionship to Dyrk1A, compound 18 showed a strong inhibition
(83%) for Dyrk2 while compound 21 was much less inhibitory
(21%). For GSK3b we have modeled the active site of Dyrk1A, com-
pounds 18 and 21 showed no inhibition and 24% inhibition, respec-
for the ATP binding pocket was carried out using the PharmoScan
system, a structure-based in silico screening tool developed by
IDRTech Inc.11 Modifications of the first series were performed to
introduce various substitutions with stronger hydrogen bond
interaction than that in the 3-methoxy-4-hydroxyphenyl moiety
of HCD160 in the Dyrk1A ATP binding site (Table 1). The second
series were modified to obtain various 1,4-disubstituted pyrazoli-
dine-3,5-diones derivatives (Table 2). All the desired derivatives
were prepared for testing via an in vitro Dyrk1A inhibition assay
as previously described.9 In order to evaluate the inhibitory
potency against Dyrk1A activity, two types of inhibition assays
were performed; autophosphorylation of Dyrk1A and kinase reac-
tion on substrate phosphorylation.
tively. For other tested kinases, two compounds showed
a
The assay results of the first series of 19 HCD160 derivatives are
summarized in Table 1. Seven compounds (2, 3, 8, 13, 15, 17 and
18) show inhibitory activity against Dyrk1A autophosphorylation
moderate (less than 30%) inhibition. The CC50 values (50% cytotoxic
concentration) for compounds 18 and 21 obtained by WST-1 assay
are >500 lM and >250 lM, respectively. We also tested if com-
pounds 18 and 21 compete with ATP in the Dyrk1A ATP binding
pocket. We observed that the inhibitory activities of these com-
pounds were decreased when the reactions were performed at
the increased ATP concentrations, indicating that inhibitors behave
as competitors of ATP as we expected from the modeling (data not
shown).
with IC50 values ranging from 0.6 to 20
compound 18, with IC50 values of 0.6 M, shows roughly fourfold in-
creasedinhibitoryactivityonautophosphorylationrelativeto thatof
HCD160, which showed IC50 values of 2.5 M. However, only com-
lM (Table 1). Among them,
l
l
pounds2 and18showedsimilarinhibitionactivityforautophospho-
rylation and kinase reaction, while other compounds exhibited no
inhibition activity for kinase reaction at a concentration of 10 lM.
Figure 2. Surface models of the ATP binding sites of GSK-3b, Dyrk1A, CDK-2 and CK-2 (blue: positive charged region and red: negative charged region).

2326
K. A. Koo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2324–2328
Table 1
Assay results of the first series of 19 HCD160 derivatives
R4
Cl
O
R3
R2
R5
NH
N
Cl
R1
O
Compounds
R1
R2
R3
R4
R5
IC50 for autophosphorylation (
l
M)
% Inhibition for kinase activity at 10
82
No inhibition
73
lM (n = 3, mean SD)
HCD160
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
OH
Cl
OMe
H
H
H
H
H
OMe
OMe
OMe
H
H
H
H
F
OH
OH
OMe
H
H
H
H
OMe
H
H
H
H
OH
OMe
H
OMe
HO
Cl
H
F
NO2
OMe
H
H
H
H
H
H
OMe
NO2
Cl
F
H
H
OMe
H
H
Cl
Cl
H
H
H
2.5
>50
2.5
20
>50
>50
>50
>50
20
>50
>50
>50
>50
20
>50
15
>50
5
0.6
>50
5
6
2
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
H
OMe
HO
H
H
H
16
17
18
19
H
OMe
76
No inhibition
63
4
Roscovitine
2
Table 2
Assay results of the second series with 1,4-disubstituted pyrazolidine-3,5-diones derivatives
R4
O
R3
R2
R5
NH
N
R6
H
O
Compounds
R2
R3
R4
R5
R6
IC50 for autophosphorylation (
lM)
% Inhibition for kinase activity at 10
No inhibition
lM (n = 3, mean SD)
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
H
H
H
H
H
H
H
OMe
OH
H
H
OMe
H
H
H
OMe
H
H
OMe
H
F
Cl
H
H
H
H
H
OMe
H
H
CN
CN
CN
40
0.6
OH
OMe
H
H
H
H
H
H
OMe
OH
OH
H
OMe
OH
OMe
OH
H
OMe
H
78
4
>50
>50
>50
>50
>50
>50
>50
>50
1.2
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
F
Cl
OMe
OH
OH
OMe
OMe
H
55
3
>50
>50
30
No inhibition
No inhibition
No inhibition
H
H
OH
OMe
F
F
2.5
44
5
Table 3
The structural differences between 3-nitro-4-hydroxyphenyl of
compound18 and 4-cyano-phenyl ring of compound21 have impor-
tant implications to hydrogen bonding and hydrophobic interaction,
respectively in the Dyrk1A ATP binding site. According to molecular
modeling and from the present results for compound 18, the phenyl
ring with a para-substituted hydroxyl group contributes to a hydro-
gen bonding interaction with the Asn 244 residue, in addition to
hydrophobic interaction of the 3,4-dichloro phenyl moiety with
Phe 238 and Val 173 residues (Fig. 3). Hence, the inhibition potency
was improved the Dyrk1A inhibition activity by incorporating the
more hydrophilic 3-nitro group and thereby reinforcing the hydro-
gen bonding interaction with the Glu 291 residue. Additionally, the
4-cyano-phenyl group in compound 21 also forms a strong hydro-
Inhibition of protein kinases by compounds 18 and 21
Protein kinase (Km) Compound 18
CDK5/p35 (15 M)
Compound 21
l
91
75
3
3
4
0
7
1
4
1
104 13
CLK3 (90 M)
M)
M)
l
93
78
76
93
110
69
3
6
5
5
0
1
4
DYRK2 (15
GSK3b (15
l
l
17
102
94
JNK3 (10
l
M)
MAPK2 (155
PKA (10 M)
PKC (45 M)
l
M)
105
72
82
l
l
a
77
The concentration of the compound is 10 lM in all assays. The Km value was used
for ATP concentration for each kinase (Km value in parenthesis). Results are pre-
sented as the kinase activity as a percentage of the control incubations (means of
duplicate determinations SD). All kinases are originated from human.

K. A. Koo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2324–2328
2327
Figure 3. (a) Refined docking and (b) surface binding models of HCD160 and compound 18 with Dyrk1A (blue: positive charged region and red: negative charged region).
phobic interaction with Phe 238 and Val 173 residues of Dyrk1A.
Although, the functional groups on both sides of the pyrazolidine-
3,5-diones core scaffold can form a hydrogen bonding interaction
in the ATP binding site, they did not show good inhibition activity
on kinase reaction relative to that of HCD160.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.062.
As shown in Figure 3, the docking model of HCD160 and com-
pound 18 with Dyrk1A indicated that the following interactions
are very important in the ATP binding site: the hydrogen bonding
of Asn 244 and Glu 291 side chains, the backbone hydrogen bond-
ing interaction of Met 240 residue, polar (hydrophilic) interaction
of Lys 188, and the hydrophobic interactions of Phe 238 and Val
173. The Dyrk1A inhibition activity and the specificity of com-
pounds tested in this study seems to be dependent on chemical
features such as hydrophobic, hydrophilic, and hydrogen bonding
interactions, as the present compounds were selected by using
References and notes
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TM
PharmoMap for the pharmacophore-based in silico screening.
In the present study, we demonstrated the necessity of the 3-ni-
tro-4-hydroxyphenyl moiety and the p-substituted cyanide group in
the phenyl ring in both sides of the pyrazolidine-3,5-diones core
scaffold, as shown in Figure 3. The presence of those moieties
enhanced the Dyrk1A inhibition activity by blocking autophospho-
rylation of Dyrk1A, as revealed in an in vitro assay. The inhibition as-
say showed relatively good activity for HCD160 and Roscovitine,
which were used as internal controls. Bain et al. reported harmine
as a very potent Dyrk1A inhibitor (IC50 of 0.08
the substrate phosphorylation assay with harmine using our kinase
assay system for comparison. IC50 for harmine was 1 M which was
l
M).8 We performed
l
about 12-times higher than that of the reported IC50 value. This dis-
crepancy could come from a difference in the Dyrk1A protein used in
the assay. We used the 763 amino acid full-length protein while Bain
et al. used the C-terminal truncated 1–499 amino acid protein.
In conclusion, the basic pyrazolidine-3,5-diones core presents a
good scaffold for the development of novel compounds inhibiting
Dyrk1A activity in vitro assay. A more convergent approach and
the design of chemical models for further optimization are needed
to obtain more potent and specific inhibitors. The pyrazolidine-3,5-
diones derivatives could be utilized for the development of novel
therapeutic agents for the treatment of DS related learning and
memory deficits.
Acknowledgments
8. (a) Bain, J.; Plater, L.; Elliott, M.; Shpiro, N.; Hastie, C. J.; McLauchlan, H.;
Klevernic, I.; Arthur, J. S.; Alessi, D. R.; Cohen, P. Biochem. J. 2007, 408, 297; (b)
Bain, J.; McLauchlan, H.; Elliott, M.; Cohen, P. Biochem. J. 2003, 371, 199.
9. For Dyrktide information: (a) Himpel, S.; Tegge, W.; Frank, R.; Leder, S.; Joost, H.
G.; Becker, W. J. Biol. Chem. 2000, 275, 2431; b Kim, N. D.; Yoon, J.; Kim, J. H.;
Lee, J. T.; Chon, Y. S.; Hwang, M. K.; Ha, I.; Song, W. J. Bioorg. Med. Chem. Lett.
2006, 16, 3772. The experimental procedures for assays were described
previously. Briefly, the full-length (763 amino acid) mouse Dyrk1A protein
This work was supported by the Korea Science and Engineering
Foundation (KOSEF) grant funded by the Korea government (R01-
2007-000-11910-0). This work was also supported by the Korea
Research Foundation Grant funded by the Korean Government
(KRF-2007-331-E00031 and KRF-2008-314-E00180).

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K. A. Koo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2324–2328
was expressed in Escherichia coli and purified with Ni-NTA resin (Qiagen) using
(a) N0-(3,4-Dichlorophenylhydrazinocarbonyl)acetic acid methyl ester (18a):
Et3N (59.0 mM) was added to a solution of hydrazine (29.5 mM) in dried
CH2Cl2 (100 ml) at room temperature. This reaction mixture was added drop
wise to methyl malonyl chloride (32.4 mM) at 0 °C and then stirred at room
temperature for 2 h. After the reaction was completed, mixture was extracted
with CH2Cl2 and was washed with water and brine. Water layer was extracted
with EtOAc. The combined organic layer was dried over MgSO4, filtered and
concentrated. The desired product of 4.3 g was obtained as a solid. 1H NMR
(300 MHz, DMSO-d6) d 10.00 (s, 1H), 8.30 (s, 1H), 7.36 (d, J = 9.0 Hz, 1H), 6.91
(d, J = 3.0 Hz, 1H), 6.70 (dd, J = 13.3 Hz, 1H), 3.66 (s, 3H), 3.35 (d, J = 5.0 Hz, 2H).
Compound 21a: 1H NMR (300 MHz, DMSO-d6) d 10.09 (s, 1H), 8.75 (s, 1H), 7.57
(d, J = 9.0 Hz, 2H), 6.79 (d, J = 11.0 Hz, 2H), 3.67 (s, 3H), 3.38 (d, J = 6.0 Hz, 2H);
(b) 1-(3,4-Dichlorophenyl)pyrazolidine-3,5-dione (18b): LiOH (0.6 g) was
added to a solution of 3 g of N’-(3,4-dichlorophenylhydrazinocarbonyl)acetic
acid methyl ester (18a) in THF/water (60/20 ml) at room temperature. This
reaction mixture was stirred for 1 h. The mixture was evaporated in vacuo,
poured into EtOAc and washed with water and brine. The organic layer was
dried over MgSO4, filtered and concentrated. The desired pyrazolidinedione
product (1.5 g) was obtained as a solid. 1H NMR (300 MHz, DMSO-d6) d 7.93 (s,
1H), 7.67 (m, 2H), 3.67 (s, 2H). Compound 22b: 1H NMR (300 MHz, DMSO-d6) d
7.83–7.87 (m, 4H), 3.69 (s, 2H); (c) 1-(3,4-Dichlorophenyl)-4-(4-hydroxy-3-
nitrobenzyliden)pyrazolidine-3,5-dione (18): To a solution of pyrazolidinedione
(0.2 mM) in ethanol (10 ml) was added 4-hydroxy-3-nitrobenzaldehyde and
catalytic amount pyridine. The reaction mixture was refluxed for 10 h. The
solid was collected and then washed with EtOAc and dried in vacuo. The
desired product was obtained as a pink solid. 1H NMR (300 MHz, DMSO-d6) d
8.62–8.66 (m, 1H), 8.02 (d, J = 17.0 Hz, 1H), 7.86 (d, J = 17.0 Hz, 1H), 7.68–7.90
(m, 2H), 7.26 (s, 1H), 7,23 (s, 1H); (d) Compound 21: 1H NMR (300 MHz, DMSO-
d6) d 8.79 (s, 1H), 7.65–8.10 (m, 6H), 6.96 (d, J = 9.0 Hz, 1H), 3.87 (d, J = 6.0 Hz,
3H).
its endogenous 13-histidine repeat. Based on the fact that autophosphorylation
at Tyrosine residues (e.g., Tyrosine-111, 145, 159 and 321) requires the binding
of ATP at the ATP pocket of Dyrk1A, the autophosphorylation assay was
developed. The phosphatase treated Dyrk1A (0.8
well Cova plate (Nalgen) in a phosphate buffered solution containing 3.2
hydrosuccinimide and 3.2 1-ethyl-3-(3-dimethylaminopropyl)-
l
g/well) was coated on a 96-
lM N-
l
M
carbodiimide at 4 °C overnight. Autophosphorylation reaction in the presence
or the absence of the testing compound was performed at 25 °C for 1 h in a
kinase buffer (25 mM HEPES buffer, 5 mM MnCl2, 5 mM MgCl2, 0.5 mM DTT)
containing 10 nM ATP and 0.5
counting. The kinase assay was performed at the conditions that showed the
linearity of the reaction. Dyrk1A (0.2 M) was incubated for 20 min with
Dyrktide (50 M) in kinase buffer containing ATP at 25 °C. The
l
Ci 32P-ATP, followed by liquid scintillation
l
l
a
5 lM
inhibition of the kinase reaction was measured by quantifying the amount of
the remaining ATP using Kinase-Glo luminescent reagent (Promega).
10. Insight II 2000; Accelrys, Inc., San Diego, CA; http://www.accelrys.com.
TM
TM
11. The IDRTech Inc’s in-house program, PharmoMap , PharmoLib , and
TM
PharmoScan ; http://www.idrtech.com.
12. Synthetic methods of the compounds 18 and 21.
O
R
O
TEA
MC
H
N
R
OMe
+
NH2
N
H
N
H
OMe
Cl
O
O
18a
21a
O
O
NH
NH
aldehyde
pyridine(cat.)/EtOH
LiOH
R4
N
N
THF/H2O
O
O
R3
13. Becker, W.; Joost, H. G. Prog. Nucl. Acid Res. Mol. Biol. 1999, 62, 1.
14. (a) Davies, S. P.; Reddy, H.; Caivano, M.; Cohen, P. Biochem. J. 2000, 351, 95; (b)
Vieth, M.; Higgs, R. E.; Robertson, D. H.; Shapiro, M.; Gragg, E. A.; Hemmerle, G.
H. Biochim. Biophys. Acta 2004, 1697, 243.
18b
21b
R
R
18 : R=3,4-Cl, R3=OH, R4=NO2
21 : R=4-CN, R3=OH, R4=OMe