Design and synthesis of 4-benzyl-1-(2H)-phthalazinone derivatives as novel androgen receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2015.08.002

The androgen receptor (AR) plays important roles in multiple physiological functions, including differentiation, growth, and maintenance of male reproductive organs, and also has effects on hair and skin. In this paper, we report the synthesis of nonsteroidal AR antagonists having a 4-benzyl-1-(2H)-phthalazinone skeleton. Among the synthesized compounds, 11c with two ortho-substituents on the phenyl group potently inhibited SC-3 cell proliferation (IC₅₀: 0.18 μM) and showed high wt AR-binding affinity (IC₅₀: 10.9 μM), comparable to that of hydroxyflutamide (3). Compound 11c also inhibited proliferation of LNCaP cells containing T877A-mutated AR. Docking study of 11c with the AR ligand-binding domain indicated that the benzyl group is important for the antagonism. These phthalazinone derivatives may be useful for investigating potential clinical applications of AR antagonists.

Full text of DOI:10.1016/j.ejmech.2015.08.002

Accepted Manuscript
Design and synthesis of 4-benzyl-1-(2H)-phthalazinone derivatives as novel androgen
receptor antagonists
Kazumi Inoue, Ko Urushibara, Misae Kanai, Kei Yura, Shinya Fujii, Mari Ishigami-
Yuasa, Yuichi Hashimoto, Shuichi Mori, Emiko Kawachi, Mio Matsumura, Tomoya
Hirano, Hiroyuki Kagechika, Aya Tanatani
PII:
S0223-5234(15)30186-0
10.1016/j.ejmech.2015.08.002
EJMECH 8038
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 December 2014
Revised Date: 23 June 2015
Accepted Date: 2 August 2015
Please cite this article as: K. Inoue, K. Urushibara, M. Kanai, K. Yura, S. Fujii, M. Ishigami-Yuasa,
Y. Hashimoto, S. Mori, E. Kawachi, M. Matsumura, T. Hirano, H. Kagechika, A. Tanatani, Design
and synthesis of 4-benzyl-1-(2H)-phthalazinone derivatives as novel androgen receptor antagonists,
European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.08.002.
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ACCEPTED MANUSCRIPT
Graphical abstract
Ms Ref. No. EJMECH-D-14-02570
Title: Design and synthesis of 4-benzyl-1-(2H)-phthalazinone derivatives as novel androgen
receptor antagonists
Corresponding author: Aya Tanatani

ACCEPTED MANUSCRIPT
Design and synthesis of 4-benzyl-1-(2H)-phthalazinone derivatives as novel
androgen receptor antagonists
a
a
a
b,c
d,e
Kazumi Inoue, Ko Urushibara, Misae Kanai, Kei Yura,
Shinya Fujii,
Mari
d
e
d
d
a
Ishigami-Yuasa, Yuichi Hashimoto, Shuichi Mori, Emiko Kawachi, Mio Matsumura,
d
d
,a
Tomoya Hirano, Hiroyuki Kagechika, Aya Tanatani*
a
b
Department of Chemistry, Faculty of Science, and Center for Informational Biology,
Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
c
National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan
d
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10
Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
e
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-0032, Japan
Corresponding author (A. Tanatani)
*
TEL/FAX: +81-3-5978-2716. E-mail: tanatani.aya@ocha.ac.jp
Abstract
The androgen receptor (AR) plays important roles in multiple physiological functions,
including differentiation, growth, and maintenance of male reproductive organs, and also has
effects on hair and skin. In this paper, we report the synthesis of nonsteroidal AR antagonists
having a 4-benzyl-1-(2H)-phthalazinone skeleton. Among the synthesized compounds, 11c
with two ortho-substituents on the phenyl group potently inhibited SC-3 cell proliferation
(
IC : 0.18 µM) and showed high wt AR-binding affinity (IC : 10.9 µM), comparable to that
50 50
of hydroxyflutamide (3). Compound 11c also inhibited proliferation of LNCaP cells
containing T877A-mutated AR. Docking study of 11c with the AR ligand-binding domain
indicated that the benzyl group is important for the antagonism. These phthalazinone
derivatives may be useful for investigating potential clinical applications of AR antagonists.
Keywords: Androgen receptor, Antagonist, Phthalazinone, Prostate cancer

ACCEPTED MANUSCRIPT
1
. Introduction
Androgen receptor (AR) is a ligand-inducible transcriptional factor belonging to the
nuclear receptor superfamily, and the endogenous ligands (androgens) are testosterone and its
1
,2
active metabolite, dihydrotestosterone (DHT).
AR modulates multiple physiological
phenomena, including differentiation, growth, and maintenance of male reproductive organs,
3
,4
and also has effects on hair and skin (androgenic effects).
Since androgen plays an
5
important role in progression of prostate cancer, various AR antagonists have been
investigated for treatment of prostate cancer, and some of them have been brought into
6
7
clinical use, including bicalutamide (1, Figure 1) and flutamide (2). Although hormone
therapy using AR antagonist or combined androgen blockade therapy is effective for most
prostate cancer patients, its efficacy decreases after a few years of treatment, with the
8
,9
development of so-called castration-resistant prostate cancer (CRPC). A major cause of
10-12
CRPC is mutations of AR,
such as T877A, which is the most common mutation of AR in
CRPC. Hydroxyflutamide (3), an activated metabolite of flutamide (2), acts as agonists
3
toward T877A AR, and exacerbates the cancer.
Figure 1
AR antagonists can be classified into two types, that is, steroidal and nonsteroidal
compounds. The nonsteroidal AR antagonists in clinical use have the common
pharmacophore of an anilide structure with nitro, cyano, or related functional groups on the
phenyl ring, as in bicalutamide (1) and flutamide (2). Most of the nonsteroidal AR antagonists
so far known are anilide derivatives developed by using the structures of bicalutamide (1) and
flutamide (2) as lead compounds, including second-generation AR antagonists such as
4
MDV3100 (4, Figure 1), which are effective against CRPC. In the structure of MDV3100,
the bulky aryl ring on the heterocyclic linker is important for the antagonism. Although
development of the same pharmacophore as in bicalutamide (1) and flutamide (2) is thus an
effective strategy to develop AR antagonists with high potency, development of antagonists
bearing other pharmacophore(s) (i.e. non anilide-type molecules) is also a promising approach
to overcome CRPC, and some AR antagonists bearing a unique skeleton or pharmacophore
1
5,16
have been reported.
These compounds have a basic amino or phenolic hydroxyl group on
the aromatic ring as the necessary polar group, instead of the cyano- or nitrophenyl group of
the anilide-type antagonists.
For example, we have reported two AR antagonists without the anilide-type
pharmacophore, i.e., (Z)-4-(4-diethylaminophenylmethylene)-3-phenyl-5(4H)-isoxazolone (5,
1
7
18
Figure 2)
and 4-(N-benzyl-N-nitrophenyl)aminopyrrole-2-carboxamide (6).
These
compounds showed high binding affinity for wild-type (wt) AR, and were effective in LNCaP
cells bearing T877A-mutated AR. Compounds 5 and 6 contain a substituted phenyl group, a

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heterocyclic ring (isoxazolone for 5 and pyrrole for 6), and a linking atom (methyne for 5 and
nitrogen for 6). Structure-activity relationship studies showed that the polar functional group
on the aromatic ring is important for binding to AR, and the bulky substituent on isoxazolone
for 5 or on the linking nitrogen atom for 6 is important for the antagonistic activity, serving to
disturb formation of the active conformation of holo-AR, especially the proper positioning
and orientation of helix-12, the key α-helix motif for ligand-dependent receptor activation.
Based on these considerations, we introduced a phthalazinone as the heterocyclic part instead
of phenylisoxazolone for 5 and pyrrolecarbonylpyrrolidine for 6. Phthalizinone is one of the
1
9
useful heterocyclic building blocks for bioactive molecules, and we assumed that the fused
ring structure could improve the activity. Thus, we designed 4-benzyl-1-(2H)-phthalazinone
as a candidate scaffold for AR antagonists. Here, we report the synthesis and biological
activity of 4-benzyl-1-(2H)-phthalazinone and its derivatives (general formula shown in
Figure 2) as candidate new-generation AR antagonists with a novel pharmacophore.
Figure 2
2
. Chemistry
-Benzyl-1-(2H)-phthalazinone (7) was synthesized according to the literature
Scheme 1). Nucleophilic substitution reaction of benzaldehyde and phthalide using sodium
1
9b
4
(
methoxide as a base in ethyl propionate and methanol gave diketone 12. Treatment of 12 with
hydrazine monohydrate afforded compound 7. Then, derivatives 8 and 9 bearing a
nitrogen-containing functional group on the phenyl group, corresponding to the diethylamino
group of 5 and nitro group of 6, were synthesized. Nitration of 7 with potassium nitrate in
trifluoroacetic acid afforded para- (43%) and ortho-nitrated (26%) compounds (Scheme 1).
Each nitro compound was hydrogenated to afford the amino derivative, which was further
converted to N-acyl, N-mesyl, and N-alkylated derivatives (Scheme 2).
Scheme 1
Scheme 2
Various 4-benzyl-1-(2H)-phthalazinone derivatives with ortho-substituents were
similarly synthesized from ortho-substituted benzaldehyde and phthalide in two steps
(Scheme 3). In the case of compounds bearing two ortho-substituents, the reaction of
intermediates 14 with hydrazine afforded the phthalazines in rather low yield.
4
-Benzyl-1-(2H)-phthalazinone derivative 10f bearing an ortho-carboxyl group was
2
0,21
synthesized from homophthalic acid.
Reaction with phthalic anhydride using sodium
acetate as a base at 200°C afforded deoxybenzoin derivative 15 (27%), which was reacted

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with hydrazine monohydrate to give 10f (Scheme 3).
Scheme 3
3
3
. Results and Discussion
.1. Biological Activity
AR agonistic and antagonistic activities of the synthesized compounds were first
2
2
evaluated in terms of growth-inhibitory activity toward SC-3 cells bearing wt AR. These
cells show androgen-dependent cell proliferation. These phthalazinone derivatives alone did
not affect the proliferation of SC-3, which means they did not act as AR agonists. The
antagonistic activity of the test compounds was examined in terms of their effect on 1 nM
DHT-dependent proliferation of SC-3 cells (Figure S2 in Supplementary data); the IC values
5
0
are shown in Table 1. Parent compound 7 without any substituent on the phenyl ring showed
only weak inhibitory activity (IC : 5.37 µM) on SC-3 cell proliferation. Introduction of a
5
0
nitrogen-containing functional group at the para position (compounds 8) caused loss of the
activity, while compounds 9 bearing ortho-substituents showed inhibitory activity.
Compounds 9a with an o-nitro group and 9b with an o-amino group showed IC values of
5
0
0
.53 µM and 0.22 µM, respectively, and their activity is comparable to that of
hydroxyflutamide (3, IC value: 0.18 µM). Modification of the amino group of 9b, such as
5
0
acylation, or mesylation, decreased the activity. Interestingly, N-mono- or N,N-dialkylation of
aniline moiety of 9b also decreased the activity. Among compounds 10 with various
functional groups at the ortho position, 10d bearing a chlorine atom showed potent inhibitory
activity (IC value: 1.26 µM). The requirement of the substituent in the electronic and steric
5
0
properties for high activity is unknown, introduction of the the proper substituent at ortho
position is effective. Next, we examined the activity of compounds 11 with two
ortho-substituents. Compound 11a with o,o-dimethyl groups showed moderate activity (IC50
value: 1.38 µM), stronger than that of compound 10a with an o-monomethyl group (IC50
value: 9.54 µM). Similar increases in activity was observed among o,o-dihalogenated
compounds, and o,o-dichloro derivative 11c (IC50 value: 0.18 µM) showed most potent
activity in this series, being as active as hydroxyflutamide (3). Then, we examined whether
the effect of 11c on SC-3 proliferation depended on the concentration of DHT. Since 10 nM of
DHT afforded maximum response to SC-3 proliferations, we examined the conditions with
0
.3 – 10 nM of DHT. In this concentration range, the effect of 11c, as well as
hydroxyflutamide (3), showed the inhibitory activity toward SC-3 cells depending on the
concentration of DHT (Figure S3 in Supplementary data).
Table 1

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In order to clarify whether the inhibitory activity of phthalazinone derivatives toward
SC-3 cell proliferation is AR-dependent, selected compounds were subjected to competitive
AR binding assay (Table 2). All the phthalazinone derivatives examined showed AR binding
affinity, and the binding potency correlated well with the activity in SC-3 assay. Thus,
compound 11c showed most potent binding affinity to AR, comparable to that of
hydroxyflutamide (3). Further, phthalazinone derivatives did not inhibit the proliferation of
androgen-independent prostate cancer cell line PC-3 (Figure S1 in Supplementary data).
These results indicated that the inhibition of SC-3 cell proliferation by phthalazinone
derivatives would result from their AR agonistic activity.
Table 2
We then investigated the antagonistic activity of selected compounds toward human
2
3,24
prostate cancer cells LNCaP, bearing T877A-mutated AR (Table 3).
All phthalazinone
derivatives examined inhibited DHT-stimulated proliferation of LNCaP cells. Among them,
compound 11c showed a lower IC value than bicalutamide (1). These compounds also
5
0
inhibited PSA production in LNCaP cells, like bicalutamide (1) (Figure 3). Thus, our novel
phthalazinone derivatives acted as AR antagonists not only toward wt AR, but also toward
T877A-mutated AR.
Table 3
Figure 3
3
.2. Conformational analysis
The structure-activity relationships of phthalazinone derivatives (Table 1) indicate that
the substituent effect of the phenyl group of this series is different from that of compounds 5
and 6, as well as anilide-type AR antagonists. In the case of phthalazinone derivatives,
introduction of polar substituents at the para position (diethylamino group for 5 and nitro
group for 6) or electron-withdrawing groups (trifluoromethyl and cyano groups for
bicalutamide, and trifluoromethyl and nitro groups for flutamide) was not effective, and
introduction of ortho-substituents increased the AR antagonistic activity. Since there is no
remarkable electronic effect of an ortho-substituent, it seemed likely that a change of the
molecular shape caused by the ortho-substituents would be significant for the antagonistic
activity. Thus, in order to clarify the mechanism through which phthalazinone derivatives
show AR antagonistic activity, we examined the binding of phthalazinone derivatives to the
AR ligand-binding domain (LBD). First, we examined the crystal structures of phthalazinone
derivatives 10d and 11c (Figure 4), focusing on the dihedral angles between phthalazinone

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and phenyl groups (Table 4). In particular, introduction of chlorine at both ortho positions
increased the torsion angle (90.64°) around the phenyl-CH bond, Thus, a molecular shape
2
with large dihedral and torsion angles due to the steric effect of the ortho-substituent on the
4
-benzyl-1-(2H)-phthalazinone skeleton appears to be important for the AR binding affinity.
Figure 4
Table 4
3
.3. Docking simulation
Next, we considered possible docking structures between compound 11c and the hAR
LBD. To dock the compound to hAR LBD, we employed a newly developed in-house
procedure rather than using conventional methods. Our method fully utilizes the large amount
2
5
of protein-ligand interaction data available in PDB . Briefly, we first gathered all the known
AR LBD-ligand (mainly a steroid molecule) complex structures, computed the distributions
of all atom types of ligands in the AR LBD, and placed all the atoms of compound 11c to
match the empirically obtained atom distributions. By this method, we can indirectly
incorporate an entropic term in the docking procedure through the observed distribution of the
poses of the ligands against the protein. The detail of the method to analyze the distribution of
the atoms was given in ref 26. During this procedure, we allowed the conformation of the
compound to vary by rotating the torsion angles. A docking structure that satisfies the
empirically well-observed locations of nitrogen, oxygen and chloride atoms was then
2
7
generated on the ligand-activated hAR LBD (PDB ID: 1xow ), energy minimization was
28
carried out using GROMACS, and serious steric clash was released. After the energy
minimization, a slight relocation and deformation of helix-12 was observed (Figure 5). One of
the possible factors of the relocation seemed repulsive interactions between a chloride atom of
compound 11c and the sulfur atom of M895 on helix-12. Since the relative location of
2
9
helix-12 is known to be a key factor for enhancing/suppressing the receptor activity, the
interactions found in the docking study are suggestive in relation to the antagonistic
mechanism of compound 11c. The interaction between the benzylic moiety and helix-12 and
that between one of the chlorine atoms on the compound and the sulfur atom of methionine
on helix-12 seem to be critical factors for the antagonist activity. The docking study also
demonstrated that the location of compound 11c is distant (> 6Å) from the resistant mutation
site T877A, and this may be the reason why the effect of compound 11c on AR is rather
independent of this mutation. Thus, the bulky aryl ring on the heterocyclic ring of compound

ACCEPTED MANUSCRIPT
11c appears to be important for the AR antagonism, as may also be the case for MDV3100, an
effective antagonist for CRPC.
Figure 5
4
. Conclusion
-Benzyl-1-(2H)-phthalazinone derivatives were designed and synthesized as novel AR
4
antagonist candidates with a different pharmacophore from that of conventional AR
antagonists. 4-Benzyl-1-(2H)-phthalazinone derivatives that were ortho-substituted on the
phenyl ring showed AR antagonistic activity. Since an ortho-substituent has no marked
electronic effect, distortion of the overall molecular shape by the substituent may be important
for the activity. Among the synthesized compounds, compound 11c potently inhibited SC-3
cell proliferation and showed high binding affinity to wt AR; its potency is similar to that of
hydroxyflutamide (3). Further, compound 11c acted as an AR antagonist toward LNCaP cells
bearing T877A-mutated AR. Docking study indicated that the benzyl group of compound 11c
would disturb the conformation of helix-3 of the hAR LBD, which in turn might result in an
inactive conformation of helix-12, thereby inactivating the whole receptor. Our phthalazinone
derivatives discussed here have a unique pharmacophore that may be available for
development of clinically useful AR antagonists, including drugs to treat CRPC.
5
5
. Experimental
.1 Chemistry
All reagents were purchased from Sigma-Aldrich Chemical Co., Tokyo Kasei Kogyo Co.
Wako Pure Chemical Industries, and Kanto Kagaku Co., Inc. Silica gel for column
1
13
chromatography was purchased from Kanto Kagaku Co., Inc. H and C NMR spectra were
recorded on JEOL ECS 400 spectrometer or Bruker 600 spectrometer. Mass spectral data
was obtained on a Bruker Daltonics microTOF-2focus or the MStation JMS-700 in the
positive ion detection modes. Melting points were determined on a RFS-30 melting point
apparatus (Round Science). Elemental analysis was performed on MT-6 elemental analyzer
(Yanagimoto). Infrared spectra were obtained on FT/IR-6100 spectrometer (JASCO).
Preparation of compounds
,3-Dioxo-2-phenylindane (12): A solution of sodium methoxide in methanol (28%, 2.196 g,
1.4 mmol) was added to a suspension of benzaldehyde (307 mg, 2.89 mmol) and phthalide
387 mg, 2.89 mmol) in ethyl propionate (1.2 mL) at 30°C, and the mixture was heated under
1
1
(
reflux for 1 h. The reaction mixture was diluted with methanol and, was further heated for 1 h.
After concentration in vacuo, water was added to the residue, and was washed with diethyl

ACCEPTED MANUSCRIPT
ether. After acidified with acetic acid, the suspension was stirred for 15 min. The product was
collected by filtration, washed with water, and dried in vacuo to afford 12 (331 mg, 52%) as
1
colorless powder. H NMR (400 M Hz, CDCl ) δ 8.08 (dd, J = 2.9 Hz, 2 H), 7.91 (dd, J = 2.9
3
Hz, 2 H), 7.35-7.26 (m, 3 H), 7.19 (dd, J = 8.8, 2.0 Hz, 2 H), 4.27 (s, 1 H).
4
-Benzyl-1-(2H)-phthalazinone (7): 12 (51 mg, 0.227 mmol) was suspended in hydrazine
hydrate (300 mg, 6.00 mmol) and was heated at 100°C for 6 h. The reaction mixture was
cooled to 20°C. The product was collected by filtration, washed with water, and dried in
1
vacuo to afford 7 (30 mg, 56%) as colorless powder. mp 204-207°C; H NMR (400 MHz,
CDCl ) δ 10.13 (br, 1 H), 8.45 (dt, J = 9.3, 2.0 Hz, 1 H), 7.77-7.70 (m, 3 H), 7.33-7.21 (m, 5
3
1
3
H), 4.30 (s, 2 H); CNMR (150 MHz, CDCl ) δ 160.8, 146.6, 137.7, 133.6, 131.5, 130.0,
3
+
1
28.9, 128.6, 128.5, 127.2, 127.0, 125.6, 39.0; HRMS calcd for C H N O (M+H) 237.1022,
1
5
13
2
found 237.1017; Anal. calcd for C H N O: C, 76.25; H, 5.12; N, 11.86. found: C, 76.16; H,
1
5
12
2
5
4
.35; N, 11.91.
-(4-Nitorophenyl)methyl-1-(2H)-phthalazinone (8a) and 4-(2-Nitorophenyl)methyl-1-(2H)-
phthalazinone (9a): Potassium nitrate (37 mg, 0.363 mmol) was added to a suspension of 7
91 mg, 0.383 mmol) in trifluoroacetic acid (4 mL) at 0°C. and the mixture was stirred at 0°C
(
for 20 h. The reaction mixture was basified with 2 M sodium hydroxide, and was extract with
ethyl acetate. The organic layer was dried over MgSO , and evaporated. The residue was
4
purified by column chromatography (silica gel, ethyl acetate / n-hexane = 1/1 then 2/1) to
1
afford 8a (43%) and 9a (26%). 8a: pale pink powder; mp 213.3-215.6°C; H NMR (400 MHz,
CDCl ) δ 9.77 (br, 1 H), 8.48-8.46 (m, 1 H), 8.18 (d, J = 8.2 Hz, 2 H), 7.79-7.77 (m, 2 H),
3
1
3
7
1
.68-7.66 (m, 1 H), 7.45 (d, J = 8.2 Hz, 2 H), 4.39 (s, 2 H); CNMR (150 MHz, CDCl ) δ
3
60.6, 147.1, 145.2, 145.1, 133.9, 131.9, 129.7, 129.6, 128.5, 127.5, 124.9, 124.1, 38.6;
+
HRMS calcd for C H N O (M+H) 282.0873, found 282.0878; Anal. calcd for
1
5
12
3
3
C H N O : C, 64.05; H, 3.94; N, 14.94. found: C, 63.86; H, 4.26; N, 14.68. 9a: pale pink
15
11
3
3
1
powder; mp 235.2-237.1°C; H NMR (400 MHz, CDCl ) δ 9.61 (br, 1 H), 8.47 (d, J = 7.8 Hz,
3
1
H), 8.10 (dd, J = 7.8, 1.4 Hz, 1 H), 7.88-7.79 (m 3 H), 7.58 (dt, J = 7.3, 1.4 Hz, 1 H), 7.48
13
(
dt, J = 7.8, 1.4 Hz, 1 H), 7.33 (dd, J = 7.8, 1.4 Hz, 1 H), 4.68 (s, 2 H); CNMR (150 MHz,
CDCl ) δ 160.2, 149.4, 144.8, 134.0, 133.5, 132.6, 132.3, 131.9, 129.8, 128.4, 128.3, 127.4,
3
+
1
25.3, 124.6, 35.9; HRMS calcd for C H N O (M+H) 282.0873, found 282.0878; Anal.
15
12
3
3
calcd for C H N O : C, 64.05; H, 3.94; N, 14.94. found: C, 64.00; H, 3.94; N, 14.94.
1
5
11
3
3
4
-(4-Aminophenyl)methyl-1-(2H)-phthalazinone (8b): A solution of 8a (51 mg, 0.182 mmol)

ACCEPTED MANUSCRIPT
in methanol (15 mL) was hydrogenated with 10% Pd/C (9.8 mg) under hydrogen atmosphere
for 1 h at room temperature. After filtration over celite, and the filtrate were evaporated. The
residue was purified by column chromatography (silica gel, ethyl acetate / n-hexane = 4/1, 5/1,
1
then ethyl acetate only) to afford 8b (86%). green powder; mp 231.3-232.0°C; H NMR (400
MHz, DMSO-d ) δ 12.52 (br, 1 H), 8.22 (dd, J = 7.8, 1.4 Hz, 1 H), 7.90 (d, J = 7.8 Hz, 1 H),
6
7
.84 (dt, J = 7.3, 1.4 Hz, 1 H), 7.79 (dt, J = 7.8, 1.4 Hz, 1 H), 6.94 (d, J = 8.2 Hz, 2 H), 6.46
1
3
(
d, J = 8.7 Hz, 2 H), 4.90 (s, 2 H), 4.08 (s, 2 H); C NMR (150 MHz, DMSO-d ) δ 159.9,
6
1
47.6, 146.3, 133.7, 131.7, 129.7, 129.3, 128.4, 126.4, 126.3, 125.4, 114.5, 37.5; HRMS calcd
+
for C H N O (M+H) 252.1131, found 252.1132; Anal. calcd for C H N O: C, 71.70; H,
1
5
14
3
15 13
3
5
4
.21; N, 16.72. found: C, 71.64; H, 5.36; N, 16.95.
-(2-Aminophenyl)methyl-1-(2H)-phthalazinone (9b) was prepared by the same procedure
1
for 8b. Yield: 86%; green powder; mp 208.9-210.5°C; H NMR (400 MHz, CDCl ) δ 9.72 (br,
3
1
7
H), 8.44 (dd, J = 7.8, 1.4 Hz, 1 H), 7.99 (d, J = 7.8 Hz, 1 H), 7.82 (dt, J = 7.3, 1.4 Hz, 1 H),
.76 (dt, J = 7.8, 1.4 Hz, 1 H), 7.16 (d, J = 7.8 Hz, 1 H), 7.08 (d, J = 7.8, 1.4 Hz, 1 H), 6.73
1
3
(
dt, J = 7.3, 1.4 Hz, 1 H), 6.70 (d, J = 7.8 Hz, 1 H), 4.18 (s, 2 H), 4.12 (br, 2 H); C NMR
(
150 MHz, CDCl ) δ 160.8, 146.0, 133.9, 131.8, 130.6, 129.9, 128.4, 127.3, 125.4, 121.4,
3
+
1
18.6, 116.6, 35.6; HRMS calcd for C H N O (M+H) 252.1131, found 252.1128; Anal.
1
5
14
3
calcd for C H N O: C, 71.70; H, 5.21; N, 16.72. found: C, 71.59; H, 5.31; N, 16.54.
1
5
13
3
4
-(4-Acetaminophenyl)methyl-1-(2H)-phthalazinone (8c): Acetyl chloride (40 ꢀL, 0.570
mmol) was added to a solution of 8b (48 mg, 0.189 mmol) and triethylamine (40 mg, 0.390
mmol) in dichloromethane (15 mL) at 0°C, and the mixture was stirred at room temperature
for 1 h. 2 M Hydrochrolic acid was added to the mixture, extracted with ethyl acetate. The
organic layer was dried over MgSO , and evaporated. The residue was purified by column
4
chromatography (silica gel, ethyl acetate / n-hexane = 2/1) to afford 8c (41.7 mg, 75%) as
1
colorless powder. mp 240.3-242.8°C; H NMR (400 MHz, DMSO-d ) δ 12.57 (br, 1 H), 9.86
6
(s, 1 H), 8.24 (d, J = 7.8 Hz, 1 H), 7.90 (d, J = 7.8 Hz, 1 H), 7.86 (dt, J = 7.3, 1.4 Hz, 1 H),
7
.80 (dt, J = 7.8, 1.4 Hz, 1 H), 7.46 (d, J = 8.8 Hz, 2 H), 7.21 (d, J = 8.8 Hz, 2 H), 4.23 (s, 2
13
H), 1.99 (s,3 H); C NMR (150 MHz, DMSO-d ) δ 168.6, 159.9, 145.8, 138.2, 133.9, 133.1,
6
1
31.9, 129.6, 129.2, 128.4, 126.5, 126.2, 119.7, 37.6, 24.4; HRMS calcd for C H N O
17 16 3 2
+
(
M+H) 294.1237, found 294.1246.
4
-(2-Acetaminophenyl)methyl-1-(2H)-phthalazinone (9c) was prepared by the same
1
procedure for 8c. Yield 75%; colorless powder; mp 265.1-265.9°C; H NMR (400 MHz,

ACCEPTED MANUSCRIPT
CDCl ) δ 9.86 (br, 1 H), 8.51 (br, 1 H), 8.46 (d, J = 7.8 Hz, 1 H), 8.04 (d, J = 8.2 Hz, 1 H),
3
7
.94 (d, J = 8.2 Hz, 1 H), 7.88 (dt, J = 7.3, 1.4 Hz, 1 H), 7.81 (dt, J = 7.8, 1.4 Hz, 1 H), 7.35
1
3
(
d, J = 7.3 Hz, 1 H), 7.11 (t, J = 7.8 Hz, 1 H), 4.27 (s, 2 H), 2.24 (s, 3 H); C NMR (150 MHz,
CDCl ) δ 169.1, 160.6, 146.9, 146.8, 137.3, 134.1, 132.3, 130.4, 129.6, 129.2, 128.4, 127.5,
3
+
1
25.4, 125.1, 124.2, 35.5, 24.8; HRMS calcd for C H N O (M+H) 294.1237, found
1
7
16
3
2
2
94.1237.
4
-(4-Trifluoroacetaminophenyl)methyl-1-(2H)-phthalazinone (8d) was prepared by the
similar procedure for 8c using trifluoroacetyl chloride: Yield 59%; colorless powder; mp
1
2
69.5-270.4°C; H NMR (400 MHz, DMSO-d ) δ 12.58 (br, 1 H), 11.19 (br, 1 H), 8.25 (d, J =
6
7
.8 Hz, 1 H), 7.92 (d, J = 7.3 Hz, 1 H), 7.86 (dt, J = 7.3, 1.5 Hz, 1 H), 7.81 (dt, J = 6.8, 0.98
1
3
Hz, 1 H), 7.56 (d, J = 8.3 Hz, 2 H), 7.33 (d, J = 8.8 Hz, 2 H), 4.29 (s, 2 H); C NMR (150
MHz, DMSO-d ) δ 159.5, 154.5, 145.2, 135.7, 134.7, 133.5, 131.6, 129.2, 128.0, 126.1, 125.7,
6
+
1
21.4, 116.8, 114.9, 37.2; HRMS calcd for C H F N O (M+H) 348.0954, found 348.0946;
1
7
13
3
3
2
Anal. calcd for C H F N O : C, 58.79; H, 3.58; N, 12.10. found: C, 58.76; H, 3.61; N,
1
7
12
3
3
2
1
4
2.10.
-(2-Trifluoroacetaminophenyl)methyl-1-(2H)-phthalazinone (9d) was prepared by the
similar procedure for 8c using trifluoroacetyl chloride. Yield 45%; colorless powder; mp
1
2
06.5-207.0°C; H NMR (400 MHz, CDCl ) δ 11.03 (br, 1 H), 10.07 (br, 1 H), 8.50 (d, J =
3
7
.8 Hz, 1 H), 8.13 (d, J = 8.2 Hz, 1 H), 8.00 (t, J = 8.7 Hz, 1 H), 7.97 (dd, J = 7.8, 0.9 Hz, 1
H), 7.89 (dt, J = 7.3, 0.9 Hz, 1 H), 7.45 (d, J = 7.3 Hz, 1 H), 7.35 (t, J = 7.3 Hz, 1 H), 7.21 (dt,
1
3
J = 7.3, 1.4 Hz, 1 H), 4.33 (s, 2 H), 2.24 (s, 3 H); C NMR (150 MHz, CDCl ) δ 160.1, 146.9,
3
1
35.2, 134.2, 132.6, 130.8, 129.2, 128.8, 128.7, 128.3, 127.8, 126.7, 125.0, 124.1, 34.8;
+
HRMS calcd for C H F N O (M+H) 348.0954, found 348.0954.
1
7
13
3
3
2
4
-(4-Trifluoroacetaminophenyl)methyl-1-(2H)-phthalazinone (8e) was prepared by the similar
procedure for 8c using methanesulfonyl chloride: Yield 22%; colorless powder; mp
1
2
58.5-260.7°C; HNMR (400MHz, DMSO-d ) δ 12.57 (br, 1 H), 9.63 (br, 1 H), 8.25 (d, J =
6
7
.8 Hz, 1 H), 7.93 (d, J = 7.8 Hz, 1 H), 7.87 (dt, J = 7.8, 1.5 Hz, 1 H), 7.81 (dt, J = 7.8, 0.98
1
3
Hz, 1 H), 7.27 (d, J = 8.3 Hz, 2 H), 7.33 (d, J = 8.3 Hz, 2 H), 4.25 (s, 2 H), 2.93 (s, 3 H); C
NMR (150 MHz, DMSO-d ) δ 159.4, 145.2, 136.7, 133.7, 133.4, 131.4, 129.4, 129.1, 127.9,
6
+
1
3
4
26.0, 125.6, 120.2, 40.0, 36.9; HRMS calcd for C H N O S (M+H) 330.0907, found
1
6
16
3
3
30.0905; Anal. calcd for C H N O S: C, 58.34; H, 4.59; N, 12.76. found: C, 58.36; H,
1
6
15
3
3
.73; N, 12.46.

ACCEPTED MANUSCRIPT
4
-(2-Trifluoroacetaminophenyl)methyl-1-(2H)-phthalazinone (9e) was prepared by the similar
procedure for 8c using methanesulfonyl chloride: Yield 36%; colorless powder; mp
1
2
28.9-230.1°C; H NMR (400 MHz, CDCl ) δ 9.96 (br, 1 H), 8.46 (d, J = 7.8 Hz, 1 H), 8.16
3
(br, 1 H), 8.02 (d, J = 8.2 Hz, 1 H), 7.89 (dt, J = 8.2, 1.4 Hz, 1 H), 7.81 (dt, J = 7.3, 0.9 Hz, 1
H), 7.54 (dt, J = 8.2, 0.9 Hz, 1 H), 7.40 (dd, J = 7.8, 0.9 Hz, 1 H), 7.29 (dt, J = 7.8, 0.9 Hz, 1
1
3
H), 7.17 (dt, J = 7.3, 1.4 Hz, 1 H), 4.32 (s, 2 H), 3.04 (s, 3 H); C NMR (150 MHz,
DMSO-d ) δ 159.4, 144.9, 135.5, 134.3, 133.4, 131.5, 130.1, 129.4, 127.8, 127.4, 126.4,
6
+
1
3
4
26.2, 126.0, 125.4, 40.1, 33.6; HRMS calcd for C H N O S (M+H) 330.0907, found
1
6
16
3
3
30.0907; Anal. calcd for C H N O S: C, 58.34; H, 4.59; N, 12.76. found: C, 58.31; H,
1
6
15
3
3
.52; N, 12.67.
4
-(4-Diethylaminophenyl)methyl-1-(2H)-phthalazinone
(8f)
and
4
-(4-ethylaminophenyl)methyl-1-(2H)- phthalazinone (8g): Iodoethane (80 ꢀL, 0.96 mmol)
was added to a solution of 8b (59 mg, 0.233 mmol) in DMF (2.0 mL), and the mixture was
stirred for 1 d at 40°C. After the solvent was removed in vacuo, the residue was purified by
column chromatography (silica gel, ethyl acetate / n-hexane = 2/1) to afford 8f (13 mg, 18%)
1
and 8g (12 mg, 19%). 8f: colorless powder; mp 194.0-195.8°C; H NMR (400 MHz,
DMSO-d ) δ 12.53 (br, 1 H), 8.23 (d, J = 7.3 Hz, 1 H), 7.95 (d, J = 7.3 Hz, 1 H), 7.85 (dt, J =
6
7
.3, 1.4 Hz, 1 H), 7.79 (dt, J = 7.3, 0.9 Hz, 1 H), 7.07 (d, J = 8.7 Hz, 2 H), 6.55 (d, J = 8.7 Hz,
1
3
2
H), 4.12 (s, 2H), 3.25 (q, J = 7.3 Hz, 4 H), 1.02 (t, J = 6.9 Hz, 6 H); C NMR (150 MHz,
CDCl ) δ 160.5, 147.4, 146.8, 133.6, 131.4, 130.1, 129.5, 128.5, 127.0, 125.9, 123.9, 112.1,
3
+
4
4.4, 38.0, 12.7; HRMS calcd for C H N O (M+H) 308.1757, found 308.1751. 8g:
1
9
22
3
1
colorless powder; mp 203.8-205.8°C; H NMR (400 MHz, DMSO-d ) δ 12.52 (br, 1 H), 8.23
6
(dd, J = 7.3, 0.9 Hz, 1 H), 7.92 (d, J = 7.8 Hz, 1 H), 7.84 (dt, J = 6.9, 1.4 Hz, 1 H), 7.79 (dt, J
=
7.8, 1.4 Hz, 1 H), 7.00 (d, J = 8.2 Hz, 2 H), 6.45 (d, J = 8.7 Hz, 2 H), 5.37 (t, J = 5.5 Hz, 1
1
3
H), 4.10 (s, 2 H), 2.94 (q, J = 5.5 Hz, 2 H), 1.10 (t, J = 6.9 Hz, 3 H); C NMR (150 MHz,
CDCl ) δδ 160.7, 147.4, 147.2, 133.5, 131.3, 130.1, 129.5, 128.5, 127.0, 126.0, 125.8, 113.1,
3
+
3
8.6, 38.2, 15.0; HRMS calcd for C H N O (M+H) 280.1444, found 280.1438.
1
7
18
3
4
-(4-Dimethylaminophenyl)methyl-1-(2H)-phthalazinone
(8h)
and
4
-(4-methylaminophenyl)methyl-1-(2H)-phthalazinone (8i) were prepared by the similar
1
procedure for 8f and 8g. 8h: Yield 9%; colorless powder; mp 249.3-250.3°C; H NMR (400
MHz, DMSO-d ) δ 12.54 (br, 1 H), 8.23 (d, J = 8.2 Hz, 1 H), 7.93 (d, J = 8.2 Hz, 1 H), 7.85 (t,
6
J = 8.2 Hz, 1 H), 7.79 (t, J = 7.8 Hz Hz, 1 H), 7.11 (d, J = 8.7 Hz, 2 H), 6,63 (d, J = 8.7 Hz, 2

ACCEPTED MANUSCRIPT
1
3
H), 4.15 (s, 2 H), 2.81 (s, 6 H); C NMR (150 MHz, CDCl ) δ 160.5, 149.6, 147.3, 133.6,
3
1
31.4, 130.1, 129.3, 128.5, 127.1, 125.8, 125.2, 113.0, 38.1, 30.8; HRMS calcd for
+
C H N O (M+H) 280.1444, found 280.1436. 8i: Yield 12%; colorless powder; mp
17
18
3
1
1
96.4-198.2°C; H NMR (400 MHz, DMSO-d ) δ 12.53 (br, 1 H), 8.23 (d, J = 6.4 Hz, 1 H),
6
7
.91 (d, J = 7.8 Hz, 1 H), 7.84 (dt, J = 7.3, 1.4 Hz, 1 H), 7.78 (dt, J = 8.2, 0.9 Hz, 1 H), 7.01
(d, J = 8.2 Hz, 2 H), 6.43 (d, J = 8.2 Hz, 2 H), 5.48 (q, J = 5.0 Hz, 1 H), 4.11 (s, 2 H), 2.59 (d,
1
3
J = 5.0 Hz, 3 H); C NMR (150 MHz, CDCl ) δ 160.4, 147.2, 133.6, 131.4, 130.1, 129.7,
3
+
1
2
29.5, 128.5, 127.1, 126.1, 125.8, 112.9, 38.2, 31.0; HRMS calcd for C H N O (M+H)
16 16 3
66.1288, found 266.1284.
4
-(2-Diethylaminophenyl)methyl-1-(2H)-phthalazinone
(9f)
and
4
-(2-ethylaminophenyl)methyl-1-(2H)-phthalazinone (9g) were prepared from 9b by the
1
similar procedure for 8f and 8g. 9f: Yield 3%; colorless powder; H NMR (400 MHz, CDCl )
3
δ 9.97 (br, 1 H), 8.40 (dd, J = 8.2, 1.4 Hz, 1 H), 7.74 (dd, J = 7.3, 1.4 Hz, 1 H), 7.68 (dt, J =
7
.3, 1.4 Hz, 1 H), 7.63 (dt, J = 7.8, 1.4 Hz, 1 H), 7.20-7.11 (m, 3 H), 6.93 (dt, J = 7.8, 1.8 Hz,
1
3
1
H), 4.35 (s, 2 H), 3.08 (q, J = 6.9 Hz, 4 H), 1.09 (t, J = 6.9 Hz, 6 H); C NMR (150 MHz,
CDCl ) δδ 160.4, 149.1, 148.1, 134.5, 133.4, 131.4, 130.2, 129.8, 128.3, 127.2, 126.8, 126.2,
3
+
1
23.8, 122.6, 47.7, 33.5, 12.5; HRMS calcd for C H N O (M+H) 308.1757, found
1
9
22
3
1
3
08.1763. 9g: Yield 23%; colorless powder; mp 184.9-185.7°C; H NMR (400 MHz, CDCl )
3
δ 10.08 (br, 1 H), 8.43 (dd, J = 7.3, 0.92 Hz, 1 H), 8.00 (d, J = 7.8 Hz, 1 H), 7.80 (dt, J = 7.3,
1
.8 Hz, 1 H), 7.75 (dt, J = 7.3, 1.4 Hz, 1 H), 7.18 (dd, J = 7.3, 1.4 Hz, 1 H), 7.16 (dt, J = 7.8,
1
.4 Hz, 1 H), 6.67 (dt, J = 7.3, 0.9 Hz, 1 H), 6.65 (dd, J = 8.2 Hz, 1 H), 4.16 (s, 2 H), 3.15 (q,
1
3
J = 7.3 Hz, 2 H), 1.29 (t, J = 7.3 Hz, 3 H); C NMR (150 MHz, CDCl ) δ 160.4, 147.5, 146.0,
3
1
1
37.7, 133.7, 131.7, 130.5, 130.0, 128.9, 128.7, 128.6, 127.2, 125.7, 120.7, 116.8, 38.5, 36.1,
+
4.9; HRMS calcd for C H N O (M+H) 280.1444, found 280.1443.
1
7
18
3
4
-(2-Dimethylaminophenyl)methyl-1-(2H)-phthalazinone
(9h)
and
4
-(2-methylaminophenyl)methyl-1-(2H)-phthalazinone (9i) were prepared from 9b by the
1
similar procedure for 8f and 8g. 9h: Yield 16%; white powder; mp 234.5-234.9°C; H NMR
(
400 MHz, CDCl ) δ 9.78 (br, 1 H), 8.39 (dd, J = 1.8 Hz, 1 H), 7.78 (dd, J = 2.7 Hz, 1 H),
3
7
.69-7.66 (m, 2 H), 7.20-7.17 (m, 2 H), 7.12 (d, J = 6.9 Hz, 1 H), 6.93 (dt, J = 6.4, 2.3 Hz, 1
13
H), 4.36 (s, 2 H), 2.79 (s, 6 H); C NMR (150 MHz, CDCl ) δδ 160.7, 152.1, 148.0, 133.6,
3
1
32.6, 131.4, 130.2, 129.9, 128.9, 127.8, 126.8, 126.0, 123.7, 119.7, 45.2, 33.4; Anal. calcd
for C H N O: C, 73.10; H, 6.13; N, 15.04. found: C, 73.22; H, 6.09; N, 15.21. 9i: Yield 9%;
1
7
17
3
1
white powder; mp 194.8-195.7°C; H NMR (400 MHz, CDCl ) δ 9.78 (br, 1 H), 8.43 (dd, J =
3

ACCEPTED MANUSCRIPT
7
0
1
1
.8, 1.4 Hz, 1 H), 8.99 (d, J = 7.3 Hz, 1 H), 7.81 (dt, J = 7.3, 1.4 Hz, 1 H), 7.75 (dt, J = 7.3,
.9 Hz, 1 H), 7.21-7.16 (m, 2 H), 6.69 (d, J = 7.3 Hz, 1 H), 6.65 (d, J = 8.2 Hz, 1 H), 4.60 (br,
1
3
H), 4.15 (s, 2 H), 2.87 (s, 3 H); C NMR (150 MHz, CDCl ) δ 160.1, 148.3, 146.0, 133.8,
3
31.8, 130.4, 130.0, 128.6, 128.6, 127.3, 125.6, 121.0, 117.0, 110.6, 35.9, 30.8; HRMS calcd
+
for C H N O (M+H) 266.1288, found 266.1285.
1
6
16
3
Compounds 13a – e and 14a – d were prepared by the same procedure for 12.
1
2
-(2-Methylphenyl)-1,3-dioxoindane (13a): Yield 86%; yellow powder; H NMR (400 MHz,
CDCl ) δ 8.07 (dd, J = 3.2 Hz, 2 H), 7.91 (dd, J = 2.7 Hz, 2 H), 7.24 (d, J = 7.3 Hz, 1 H), 7.21
3
(dt, J = 7.3, 1.4 Hz, 1 H), 7.13 (dt, J = 7.3, 1.8 Hz, 1 H), 6.88 (d, J = 7.3 Hz, 1 H), 4.52 (s, 1
H), 2.33 (s, 3 H).
1
2
-(2-Methoxyphenyl)-1,3-dioxoindane (13b): Yield 85%; yellow powder; H NMR (400 MHz,
CDCl ) δ 8.03 (dd, J = 2.7 Hz, 2 H), 7.87 (dd, J = 2.7 Hz, 2 H), 7.31 (dt, J = 8.2, 2.7 Hz, 1 H),
3
7
.28 (dd, J = 8.2, 2.7 Hz, 1 H), 7.00 (dt, J = 8.2, 2.7 Hz, 1 H), 6.79 (d, J = 8.2 Hz, 1 H), 4.17
(s, 1 H), 3.44 (s, 3 H).
1
2
-(2-Fluorophenyl)-1,3-dioxoindane (13c): Yield 71%; yellow powder; H NMR (400 MHz,
CDCl ) δ 8.07 (dd, J = 3.2 Hz, 2 H), 7.91 (dd, J = 3.2 Hz, 2 H), 7.36-7.30 (m, 1 H), 7.21 (dt, J
3
=
7.8, 2.3 Hz, 1 H), 7.16 (dt, J = 7.3, 0.9 Hz, 1 H), 7.05 (dt, J = 8.7, 0.9 Hz, 1 H), 4.39 (s, 1
H).
1
2
-(2-Chlorophenyl)-1,3-dioxoindane (13d): Yield 36%; yellow powder; H NMR (400 MHz,
CDCl ) δ 8.06 (dd, J = 3.2 Hz, 2 H), 7.90 (dd, J = 3.2 Hz, 2 H), 7.40-7.38 (m, 1 H), 7.31-7.29
3
(m, 2 H), 7.22-7.20 (m, 1 H), 4.56 (s, 1 H).
1
1
,3-Dioxo-2-(2-triflooromethylphenyl)indane (13e): Yield 72%; yellow powder; H NMR
(
400 MHz, CDCl ) δ 8.09 (dd, J = 3.2 Hz, 2 H), 7.92 (dd, J = 3.2 Hz, 2 H), 7.77 (dd, J = 7.3
3
Hz, 1.8 Hz, 1 H), 7.50-7.42 (m, 2 H), 6.94 (d, J = 6.9 Hz, 1 H), 4.76 (s, 1 H).
1
2
-(2,6-Dimethylphenyl)-1,3-dioxoindane (14a): Yield 70%; yellow powder; H NMR (400
MHz, CDCl ) δ 9.58 (br, 1 H), 8.49 (dd, J = 7.3 Hz, 1 H), 7.99 (dd, J = 7.9 Hz, 1 H), 7.93 (dt,
3
J = 6.9, 1.4 Hz, 1 H), 7.83 (dt, J = 8.2 Hz, 1 H), 7.38 (d, J = 8.2 Hz, 2 H), 7.22 (t, J = 8.2 Hz,
1
H), 4.60 (s, 2 H).

ACCEPTED MANUSCRIPT
1
2
-(2,6-Difluorophenyl)-1,3-dioxoindane (14b): Yield quant; yellow powder; H NMR (400
MHz, CDCl ) δ 8.07 (dd, J = 3.2 Hz, 2 H), 7.92 (dd, J = 3.2 Hz, 2 H), 7.35-7.23 (m, 1 H),
3
6
.93 (t, J = 8.7 Hz, 1 H), 4.70 (s, 1 H).
1
2
-(2,6-Dichlorophenyl)-1,3-dioxoindane (14c): Yield 53%; yellow powder; H NMR (400
MHz, CDCl ) δ 8.05 (dd, J = 3.2 Hz, 2 H), 7.89 (dd, J = 3.2 Hz, 2 H), 7.45 (dt, J = 4.1, 1.3 Hz,
3
1
1
H), 7.25 (dd, J = 5.9, 1.3 Hz, 2 H), 5.28 (s, 1 H).
1
,3-Dioxo-2-(2,3,6-trichlorophenyl)indane (14d): Yield 34%; yellow powder; H NMR (400
MHz, CDCl ) δ 8.06 (q, J = 3.2 Hz, 2 H), 7.92 (q, J = 3.2 Hz, 2 H), 7.42 (t, J = 2.3 Hz, 1 H),
3
7
.20 (d, J = 8.7 Hz, 1 H), 5.33 (d, J = 29 Hz, 1 H).
Compounds 10a – e and 11a – d were prepared by the same procedure for 7.
4
2
7
-(2-Toluyl)methyl-1-(2H)-phthalazinone (10a): Yield 27%; colorless powder; mp
1
09.9-210.8°C; H NMR (400 MHz, CDCl ) δ 9.91 (br, 1 H), 8.47 (dd, J = 3.2, 1.4Hz, 1 H),
3
.79-7.76 (m, 2 H), 7.72 (dd, J = 3.2, 1.3 Hz, 1 H), 7.23 (d, J = 6.9 Hz, 1 H), 7.18 (t, J = 7.3
13
Hz, 1 H), 7.11 (t, J = 7.3 Hz, 1 H), 6.99 (d, J = 7.8 Hz, 1 H), 4.27 (s, 1 H), 2.38 (s, 3 H); C
NMR (150 MHz, CDCl ) δ 160.7, 146.2, 136.4, 135.9, 133.7, 131.5, 130.5, 130.2, 129.0,
3
+
1
28.3, 127.2, 127.0, 126.3, 125.2, 36.3, 20.0; HRMS calcd for C H N O (M+H) 251.1179,
1
6
15
2
found 266.1173.
4
1
-(2-Methoxyphenyl)methyl-1-(2H)-phthalazinone (10b): Yield 63%; colorless powder; mp
1
96.3-197.6°C; H NMR (400 MHz, CDCl ) δ 9.80 (br, 1 H), 8.45-8.43 (m, 1 H), 7.85-7.83
3
(m, 1 H), 7.77-7.92 (m, 2 H), 7.23 (t, J = 6.9 Hz, 1 H), 7.08 (d, J = 7.3 Hz, 1 H), 6.93 (d, J =
1
3
8
.2 Hz, 1 H), 6.86 (t, J = 7.3 Hz, 1 H), 4.29 (s, 2 H), 3.90 (s, 3 H); C NMR (150 MHz,
CDCl ) δ 160.5, 156.9, 147.0, 133.6, 131.4, 130.5, 130.0, 128.3, 128.2, 127.0, 126.0, 125.6,
3
+
1
20.8, 110.7, 55.7, 32.1; HRMS calcd for C H N NaO (M+Na) 289.0947, found
1
6
14
2
2
2
89.0946; Anal. calcd for C H N O ; C, 72,16; H, 5.30; N, 10.52. found: C, 72.12; H, 5.24;
16 14 2 2
N, 10.75.
4
2
7
1
1
-(2-Fluorophenyl)methyl-1-(2H)-phthalazinone (10c): Yield 62%; colorless powder; mp
1
34.7-235.4°C; H NMR (400 MHz, CDCl ) δ 9.77 (br, 1 H), 8.45 (dd, J = 6.9, 1.4Hz, 1 H),
3
.82-7.74 (m, 3 H), 7.19 (dt, J = 7.3, 1.4 Hz, 1 H), 7.11 (d, J = 8.7 Hz, 1 H), 7.06 (dt, J = 7.3,
1
3
H), 4.31 (s, 2 H); C NMR (150 MHz, CDCl ) δ 160.7, 145.7, 133.8, 131.7, 130.8, 130.7,
3
29.8, 128.9, 128.8, 128.3, 127.2, 125.1, 124.5, 115.6, 31.2; HRMS calcd for C H FN NaO
1
5
11
2

ACCEPTED MANUSCRIPT
+
(
M+Na) 277.0748, found 277.0743; Anal. calcd for C H FN O : C, 70.86; H, 4.36; N,
15 11 2
11.02. found: C, 70.97; H, 4.48; N, 11.10.
4
2
-(2-Chlorophenyl)methyl-1-(2H)-phthalazinone (10d): Yield 76%; colorless powder; mp
1
22.5-223.4°C; H NMR (400 MHz, CDCl ) δ 10.03 (br, 1 H), 8.47 (dd, J = 6.4, 2.3 Hz, 1
3
H), 7.82-7.73 (m, 3 H), 7.44 (dd, J = 7.7, 1.4 Hz, 1 H), 7.21 (dt, J = 7.2, 1.8 Hz, 1 H), 7.16 (dt,
13
J = 7.7, 1.8 Hz, 1 H), 7.12 (dd, J = 7.2, 1.8 Hz, 1 H), 4.41 (s, 2 H); C NMR (150 MHz,
CDCl ) δ 160.6, 145.7, 135.4, 134.0, 133.8, 131.7, 130.5, 129.9, 129.8, 128.4, 128.4, 127.2,
3
+
1
27.2, 125.2, 35.8; HRMS calcd for C H ClN O (M+H) 271.0633, found 271.0630; Anal.
1
5
12
2
calcd for C H ClN O: C, 66.55; H, 4.10; N, 10.35. found: C, 66.66; H, 4.03; N, 10.30.
1
5
11
2
4
-(2-Trifluoromethylphenyl)methyl-1-(2H)-phthalazinone (10e): Yield 65%; colorless
1
powder; mp 196.9-197.5°C; H NMR (400 MHz, CDCl ) δ 9.83 (br, 1 H), 8.47 (dd, J = 3.6
3
Hz, 1 H), 7.78-7.73 (m, 3 H), 7.64 (dd, J = 3.6 Hz, 1 H), 7.42 (dt, J = 7.3 Hz, 1 H), 7.36 (t, J =
1
3
7
1
1
.3 Hz, 1 H), 7.13 (d, J = 7.3 Hz, 1 H), 4.49 (s, 2 H); C NMR (150 MHz, CDCl ) δ 160.9,
3
45.5, 136.1, 133.9, 132.2, 131.7, 130.6, 129.9, 128.6, 128.4, 127.2, 127.1, 126.4, 126.4,
+
25.1, 35.1; HRMS calcd for C H F N O (M+H) 305.0896, found 305.0899; Anal. calcd
1
6
12
3
2
for C H F N O : C, 63.16; H, 3.64; N, 9.21. found: C, 62.99; H, 3.67; N, 9.30.
1
6
11
3
2
1
4
-(2,6-Dimethylphenyl)methyl-1-(2H)-phthalazinone (11a): Yield 9%; colorless powder; H
NMR (400 MHz, CDCl ) δ 9.60 (br, 1 H), 8.49 (dd, J = 8.2, 0.9 Hz, 1 H), 8.02 (d, J = 7.8 Hz,
3
1
H), 7.92 (dt, J = 7.3, 1.4 Hz, 1 H), 7.83 (dt, J = 7.3, 0.9 Hz, 1 H), 7.16 (dd, J = 8.6, 2.7 Hz, 1
13
H), 7.10 (d, J = 7.8 Hz, 2 H), 4.30 (s, 2 H), 2.25 (s, 6 H); C NMR (150 MHz, CDCl ) δ
3
1
60.0, 144.7, 137.4, 133.9, 133.6, 131.7, 130.3, 128.2, 128.1, 127.3, 127.3, 124.3, 32.0, 20.4;
+
HRMS calcd for C H N O (M+H) 265.1335, found 265.1334.
1
7
17
2
4
2
7
7
1
-(2,6-Difluorophenyl)methyl-1-(2H)-phthalazinone (11b): Yield 20%; colorless powder; mp
1
70.7-271.0°C; H NMR (400 MHz, CDCl ) δ 9.73 (br, 1 H), 8.47 (dd, J = 7.8, 0.92 Hz, 1 H),
3
.95 (d, J = 7.8 Hz, 1 H), 7.88 (dt, J = 7.3, 1.4 Hz, 1 H), 7.80 (dt, J = 8.2, 1.4 Hz, 1 H),
1
3
.31-7.24 (m, 1 H), 6.94 (t, J = 7.7 Hz, 1 H), 4.34 (s, 2 H); C NMR (150 MHz, CDCl ) δ
3
62.6, 161.0, 160.1, 143.9, 133.8, 131.7, 129.9, 129.0, 128.2, 127.3, 124.4, 111.5, 25.6;
+
HRMS calcd for C H F N O (M+H) 273.0834, found 273.0835; Anal. calcd for
1
5
11
2
2
C H F N O: C, 66.17; H, 3.70; N, 10.27. found: C, 66.12; H, 3.78; N, 10.31.
15
10
2
2
4
-(2,6-Dichlorophenyl)methyl-1-(2H)-phthalazinone (11c): Yield 20%; colorless powder; mp

ACCEPTED MANUSCRIPT
1
2
7
76.6-278.4°C; H NMR (400 MHz, CDCl ) δ 9.91 (br, 1 H), 8.47 (dd, J = 3.2, 1.4Hz, 1 H),
3
.79-7.76 (m, 2 H), 7.72 (dd, J = 3.2, 1.3 Hz, 1 H), 7.23 (d, J = 6.9 Hz, 1 H), 7.18 (t, J = 7.3
13
Hz, 1 H), 7.11 (t, J = 7.3 Hz, 1 H), 6.99 (d, J = 7.8 Hz, 1 H), 4.27 (s, 1 H), 2.38 (s, 3 H); C
NMR (150 MHz, CDCl ) δ 171.3, 160.1, 143.4, 136.5, 133.8, 133.4, 131.7, 130.0, 128.9,
3
+
1
28.3, 128.2, 127.4, 124.2, 33.7; HRMS calcd for C H Cl N O (M+H) 305.0243, found
1
5
11
2
2
3
05.0241; Anal. calcd for C H Cl N O; C, 59.04; H, 3.30; N, 9.18. found: C, 58.94; H, 3.60;
15 10 2 2
N, 9.05.
4
-(2,3,6-Trichlorophenyl)methyl-1-(2H)-phthalazinone (11d): Yield 14%; colorless powder;
1
mp 274.8-275.7°C: H NMR (400 MHz, CDCl ) δ 9.56 (br, 1 H), 8.49 (d, J = 7.8 Hz, 1 H),
3
7
.97 (t, J = 8.2 Hz, 1 H), 7.92 (d, J = 7.8 Hz, 1 H), 7.85 (t, J = 7.8 Hz, 1 H), 7.40 (d, J = 8.7
1
3
Hz, 1 H), 7.33 (d, J = 8.7 Hz, 1 H), 4.64 (s, 2 H); C NMR (150 MHz, DMSO-d ) δ 159.2,
6
1
3
41.9, 136.1, 134.1, 134.0, 133.8, 132.1, 130.9, 129.8, 129.2, 129.0, 127.5, 126.2, 124.9,
+
4.6; HRMS calcd for C H Cl N O (M+H) 338.9853, found 338.9857; Anal. calcd for
15
10
3
2
C H Cl N O: C, 53.05; H, 2.67; N, 8.25. found: C, 53.29; H, 2.90; N, 8.25.
15
9
3
2
2-[2-(2-Carboxyphenyl)acetyl]benzoic acid (15): A mixture of phthalic anhydride (604 mg,
4.08 mmol), homophthalic acid (605 mg, 3.36 mmol), and anhydrous sodium acetate (56 mg,
0.686 mmol) was heated at 200-210°C for 5 h. After cooling, orange mass was warmed with
10% aqueous KOH solution (10 mL) until most of them dissolved, and the resulting deep red
solution was filtered. The filtrate was acidified with 2 M hydrochloric acid, and the
precipitates were collected, which was recrystallized from ethyl acetate to afford 15 (311 mg,
1
2
7
7%) as yellow crystal. H NMR (400 MHz, DMSO-d , 105°C) δ 7.85 (d, J = 7.6 Hz, 1 H),
6
.78 (d, J = 7.4 Hz, 1 H), 7.63 (dt, J = 7.4, 0.72 Hz, 1 H), 7.54 (dt, J = 7.6, 0.90 Hz, 1 H), 7.46
(t, J = 6.2 Hz, 2 H), 7.34 (t, J = 7.7, 1 H), 7.31 (d, J = 7.6 Hz, 1 H), 4.43 (br, 2 H); Anal. calcd
for C H O : C, 67.60; H, 4.25. found: C, 67.52; H, 4.53.
1
6
12
5
2
-[[1-(2H)-Phthalazinon-4-yl]methyl]benzoic acid (10f): A suspension of 15 (244 mg, 0.858
mmol) in hydrazine hydrate (1846 mg, 36.9 mmol) was heated at 100°C for 6 h. After cooling,
the solution was diluted with water, washed with ethyl acetate, and acidified with 2 M
hydrochloric acid. The resulting pale yellow suspension was stirred for 10 min, and the
precipitates were collected, which was washed with water to afford 10f (197 mg, 68%) as
1
yellow powder. mp 247.8°C; H NMR (400 MHz, DMSO-d ) δ 12.39 (s, 1 H), 8.26 (d, J = 7.8
6
Hz, 1 H), 8.02 (d, J = 7.9 Hz, 1 H), 7.92 (t, J = 7.8 Hz, 2 H), 7.85 (t, J = 7.8 Hz, 1 H), 7.48 (t,
1
3
J = 7.3 Hz, 1 H), 7.35 (t, J = 7.3, 1 H), 7.28 (d, J = 7.8 Hz, 1 H), 4.69 (s, 2 H); C NMR (150

ACCEPTED MANUSCRIPT
MHz, DMSO) δ 169.5, 160.4, 147.1, 139.0, 134.5, 132.4, 132.1, 131.8, 131.1, 130.2, 127.8,
+
1
3
27.4, 126.6, 125.8, 36.5; HRMS calcd for C H FN NaO (M+Na) 303.0740, found
1
6
12
2
3
03.0740.
5
.2 Biology
SC-3 Growth Inhibition Assay: The assay was examined by the same procedure described in
1
6
our previous report. SC-3 cells were cultured in MEMα (Wako Co.) supplemented with 2%
FBS and 1 nM DHT at 37°C under 5% CO . All experiments were performed in triplicate or
2
more. Cells were trypsinized and diluted to 20,000 cells/mL with MEMα supplemented with
2
1
% charcoal-stripped FBS. This cell suspension was seeded in 96-well plates at a volume of
00 ꢀL and incubated at 24 h. After removal of 10ꢀL of medium from each well, 10ꢀL of the
drug solution, which was supplemented with serial dilutions of the test compounds or DMSO
as a dilution control in the presence of 1 nM DHT, was added. Then the plates were incubated
at 37°C under 5% CO for 3 d, and the cell number was determined using a Cell Counting
2
Kit-8 (DOJINDO). A 10ꢀL aliquot of WST-8 was added to each well of microcultures, and
the cells were incubated for 2 h. The absorbance at 450 nm was measured with a microplate
reader. This parameter is related to the number of living cells in the culture. IC values were
5
0
determined as the concentration that the compound reduces half amount of DHT-induced cell
growth, calculated by linear approximation of two points adjacent to IC values.
5
0
Competitive Binding Assay using hAR-LBD: The assay was examined by the same
1
6
procedure described in our previous report. A hAR-LBD expression plasmid vector which
encodes GST-hARLBD (627-919 aa, EF domain) fusion protein under the lac promoter was
transfected into Escherichia coli strain HB-101. An overnight culture (10 mL) of the bacteria
was added to 1 L of LB medium and incubated at 27°C until its optical density reached
0
.6-0.7 at 600 nm. Following the addition of IPTG to a concentration of 1 mM, incubation
was continued for an additional 4.5 h. Cells were harvested by centrifugation at 4000g at 4°C
for 15 min and stored at -80°C until use. All subsequent operations were performed at 4°C.
The bacterial pellet obtained from 40 mL of culture was resuspended in 1 mL of ice-cold
TEGDM buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 10% glycerol, 10 mM DTT, 10 mM
sodium molybdate). This suspension was subjected to sonication using 10 × 10s bursts on ice,
and crude GST-hARLBD fraction was prepared by centrifugation of the suspension at
1
2,000g for 30 min at 4°C. This crude receptor fraction was diluted with buffer (20 mM
Tris-HCl pH 8.0, 0.3 M KCl, 1 mM EDTA) to a protein concentration of 0.3-0.5 mg/mL and
used in binding assays as hAR-LBD fraction. Aliquots of the hARLBD fraction were

ACCEPTED MANUSCRIPT
incubated in the dark at 4 °C with [3H]-DHT (PerkinElmer, 4 nM final concentration),
triamcinolone acetonide (1 ꢀM final concentration), and reference or test compounds
(dissolved in DMSO). Nonspecific binding was assessed by addition of a 200-fold excess of
nonradioactive DHT. Thus, binding of 4 nM of [3H]-DHT to hAR LBD without non-labeled DHT
reached radioactivity count of 28100 cpm. When the excess amount of non-labeled DHT was added,
the cpm counts decreased in 2800. The subtract count (25300) was considered as specific binding of
labeled DHT to the DHT binding site. After 15 h, a Dextran T-70/γ-globulin-coated-charcoal
suspension was added to the ligand/protein mixture (1% Norit A, 0.05%γ-globulin, 0.05%
Dextran T-70 final concentration each) and the whole was incubated at 4 °C for 10 min. The
charcoal was removed by centrifugation for 10 min at 1,300g, and the radioactivity of the
supernatant was measured in scintillation cocktail (Ultima Gold®, PerkinElmer) by using a
liquid scintillation counter. All experiments were performed in duplicate or more.
LNCaP cell proliferation and PSA assay: The cell proliferation assay was examined by the
1
6
same procedure described in our previous report. The human prostate adenocarcinoma cell
line, LNCaP was routinely cultivated in RPMI-1640 supplemented with 10% FBS at 37 °C in
a 5% CO humidified incubator. All experiments were performed in triplicate or more. Cells
2
were trypsinized and diluted to 20,000 cells/mL with RPMI-1640 supplemented with 10%
charcoal-stripped FBS. This cell suspension was seeded in 96-well plates at a volume of 100
ꢀ
L and incubated at 24 h. 10 ꢀL of the medium was removed and 10 ꢀL of the drug solution,
supplemented with serial dilutions of the test compounds or DMSO as dilute control in the
presence or absence of 10 nM DHT, was added to each well. Cells were incubated for 6 d, and
half of the media was removed and medium with the test compounds or DMSO as dilute
control in the presence or absence of 10 nM DHT was replaced once after 3 d. At the end of
the incubation time, proliferation was evaluated by using the WST-8. 10 ꢀM of WST-8 was
added to wells, and cells were incubated for 2 h. The absorbance at 450 nm was measured.
This parameter relates to the number of living cells in the culture. The PSA assay was
1
8
examined according to procedure described in our previous report. After the incubation, the
supernatant was collected and the amount of PSA contained in the supernatant was quantified
by the use of f-PSA Enzyme immunoassay test kit (IMMUNOSPEC Corp.) according to the
manufacturer’s instructions.
Acknowledgement
This work was partly supported by JSPS KAKENHI Grant Nos. 25460146 (to FS), No

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2
2136013 (to HK) and 24590137 (to AT), JSPS Core-to-Core Program, A. Advanced
Research Networks, and MEXT Platform for Drug Discovery, Informatics, and Structural Life
Science, Japan. A.T. thanks The Naito Foundation for financial support.
Appendix A. Supplementary data
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List of Schemes and Figures
Figure 1. Structures of typical nonsteroidal AR antagonists
Figure 2. Design of novel AR antagonists
Figure 3. Inhibition of PSA production by phthalazinone derivatives
Figure 4. Crystal structures of phthalazinone derivatives (a) 10d and (b) 11c
2
6
Figure 5. Left: Docking model of compound 11c with hAR LBD (PDB ID: 1xow ). Right:
Docking model of hAR LBD without 11c
Scheme 1. Syntheses of compounds 7, 8a,b, and 9a,b
Scheme 2. Syntheses of compounds 8c-i and 9c-i
Scheme 3. Syntheses of compounds 10 and 11

ACCEPTED MANUSCRIPT
Figure 1
Figure 2
Figure 3

ACCEPTED MANUSCRIPT
Figure 4
(a)
(b)
Figure 5

ACCEPTED MANUSCRIPT
Scheme 1
Scheme 2

ACCEPTED MANUSCRIPT
Scheme 3

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Table. 1. Inhibition of SC-3 cell proliferation by phthalazinone derivatives
Compound
IC (µM)
Compound
IC (µM)
5
0
50
Hydroxyflutamide (3) 0.18 + 0.03
7
8
8
8
8
8
8
8
8
8
5.37 + 2.06
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
a
b
c
d
e
f
g
h
i
9a
9b
9c
9d
9e
9f
9g
9h
9i
0.53 + 0.18
0.22 + 0.03
inactive
1.01 + 0.16
inactive
1.66 + 0.82
3.71 + 2.01
1.95 + 0.27
9.02 + 2.96
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
9.54 + 3.24
9.59 + 1.94
10.5 + 1.0
1.26 + 0.65
3.67 + 0.77
inactive
11a
11b
11c
11d
1.38 + 0.25
3.76 + 0.71
0.18 + 0.03
0.92 + 0.31
Table 2. Binding affinity of phthalazinone derivatives to wt AR
Compound
IC (µM)
50
Hydroxyflutamide (3)
7.69
> 100
45.2
85.4
84.3
10.9
56.7
9
9
1
a
b
0d
1
1
1
1a
1c
1d

ACCEPTED MANUSCRIPT
Table 3. Inhibitory activities of phthalazinone derivatives on proliferation of LNCaP cells
Compound
IC (µM)
50
Bicalutamide (1)
0.85 + 0.24
1.46 + 2.83
0.99 + 0.36
0.86 + 0.15
3.42 + 5.12
0.55 + 0.14
0.92 + 0.62
9
9
1
a
b
0d
1
1a
1c
1d
1
1
Table 4. Crystal data for 10d and 11c
1
0d (R = 2-Cl)
11c (R = 2,6-Cl2)
Empirical formula
Crystal system
Space group
C H ClN O
C H Cl N O
15
11
2
15 10
2
2
Triclinic
Triclinic
—
—
P 1
P 1
Unit cell dimensions
a = 7.300(4) Å
b = 8.038(4) Å
c = 11.462(6) Å
α = 82.165(7)°
β = 84.373(6)°
γ = 66.395(6)°
a = 7.9804(5) Å
b = 9.0057(6) Å
c = 11.2287(7) Å
α = 91.1510(10)°
β = 93.6230(10)°
γ = 100.1800(10)°
R = 0.0345, wR = 0.1042
Final R indices [I>2σ(I)]
R = 0.0465, wR = 0.1144
1
2
1
2
Dihedral angle
Ph-phthalazinone
Tosion angle
88.19°
87.93°
N1-C1-C2-C3
C1-C2-C3-C4
114.12
-53.40
3.02
90.64