Exploring the tetrahydroisoquinoline thiohydantoin scaffold blockade the androgen receptor as potent anti-prostate cancer agents

Article abstract of DOI:10.1016/j.ejmech.2017.10.031

Prostate cancer (PC) is a major cause of cancer-related male death in worldwide and the identification of new and improved potent anti-PC molecules is constantly required. A novel scaffold of tetrahydroisoquinoline thiohydantoin was rationally designed based on the enzalutamide structures and our pre-work, leading to the discovery of a series of new antiproliferative compounds. Several new analogues displayed improved androgen receptor (AR) antagonistic activity, while maintaining the higher selective toxicity toward LNCaP cells (AR-rich) versus DU145 cells (AR-deficient) compared to enzalutamide. In fact, compound 55 exhibited promising in vitro antitumor activity by impairing AR unclear translocation. More importantly, 55 showed better pharmacokinetic properties compared to the compound 1 reported in our pre-work. These results demonstrate a step towards the development of novel and improved AR antagonists.

Full text of DOI:10.1016/j.ejmech.2017.10.031

Accepted Manuscript
Exploring the tetrahydroisoquinoline thiohydantoin scaffold blockade the androgen
receptor as potent anti-prostate cancer agents
Xi Xu, Raoling Ge, Lei Li, Jubo Wang, Xiaoyu Lu, Siqi Xue, Xijing Chen, Zhiyu Li,
Jinlei Bian
PII:
S0223-5234(17)30823-1
10.1016/j.ejmech.2017.10.031
EJMECH 9823
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 August 2017
Revised Date: 7 October 2017
Accepted Date: 10 October 2017
Please cite this article as: X. Xu, R. Ge, L. Li, J. Wang, X. Lu, S. Xue, X. Chen, Z. Li, J. Bian, Exploring
the tetrahydroisoquinoline thiohydantoin scaffold blockade the androgen receptor as potent anti-prostate
cancer agents, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.031.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Exploring the tetrahydroisoquinoline thiohydantoin scaffold blockade the
androgen receptor as potent anti-prostate cancer agents
a
a
a
a
b
b
b
Xi Xu , Raoling Ge , Lei Li , Jubo Wang , Xiaoyu Lu , Siqi Xue , Xijing Chen ,
,
a
, a
Zhiyu Li* , Jinlei Bian*
a
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
2
10009, P.R.China
b
School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, P.R.China
*
Corresponding author. Tel.: +86 13951678592
E-mail address: zhiyuli@cpu.edu.cn (Z. Li)
ABSTRACT
Prostate cancer (PC) is a major cause of cancer-related male death in worldwide and the
identification of new and improved potent anti-PC molecules is constantly required. A novel scaffold of
tetrahydroisoquinoline thiohydantoin was rationally designed based on the enzalutamide structures and
our pre-work, leading to the discovery of a series of new antiproliferative compounds. Several new
analogues displayed improved androgen receptor (AR) antagonistic activity, while maintaining the
higher selective toxicity toward LNCaP cells (AR-rich) versus DU145 cells (AR-deficient) compared
to enzalutamide. In fact, compound 55 exhibited promising in vitro antitumor activity by impairing AR
unclear translocation. More importantly, 55 showed better pharmacokinetic properties compared to the
compound 1 reported in our pre-work. These results demonstrate a step towards the development of
novel and improved AR antagonists.
Keywords: Prostate cancer, androgen receptor, antagonist, tetrahydroisoquinoline thiohydantoin
derivatives
1
. Introduction
Prostate cancer (PC) is the second leading cause of cancer-related male death in worldwide and it
is expected to account for 19% of all new cancer diagnoses in 2017 [1]. Initially, most patients with
advanced prostate cancer are treated with androgen-deprivation therapy (ADT) using either
orchiectomy or a luteinising hormone-releasing hormone (LHRH) agonist or antagonist [2, 3].
However, patients invariably develop more aggressive castration-resistant prostate cancer (CRPC)
because of several different mechanisms such as: androgen receptor (AR) aberrations, including gene
amplifications, point mutations as well as AR splice variants [4-9].
AR is a ligand-dependent transcription factor that belongs to the nuclear receptor superfamily and
it plays a critical role in normal prostate development and more over in growth and progression of
prostate cancer [10, 11]. AR is composed of an N-terminal domain (NTD), a DNA-binding domain
1

ACCEPTED MANUSCRIPT
(
DBD), a hinge region and a C-terminal ligand-binding domain (LBD), comprising a Helix-12 (H12).
Activation function (AF-1) and activation function (AF-2) are essential for transcriptional activity,
which are located in the NTD and LBD, separately. With no androgen binding, inactive AR is in the
cytoplasm and binds to heat shock protein 90 (HSP90). After binding, androgen induces a
conformational change of AR, leading to dissocation from the chaperones and the relocation of H12.
The ligand-bound AR homodimer complex translocates to the nucleus and binds to the
androgen-response elements (AREs), then coregulatory proteins, coactivators or corepressors are
recruited to the AR complex to regulate AR target gene transcription [12-15].
Several non-steroidal androgen receptor antagonists have been approved for the treatment of PC.
Flutamide, hydroxyflutamide and bicalutamide (Fig. 1) were first generation non-steroidal AR
antagonists and they decreased androgenic effects by competitively inhibiting the binding of androgen
to AR and induce a conformational change of H12 by steric clashes. However, these antiandrogens
activated AR-LBD point mutants (for example, AR-T877A and AR-W741L, a common mutant
triggered by bicalutamide) with the long time of treatment, as a result of switching these antagonists of
AR to agonists and leading to the relapse of CRPC [16].
First generation AR antagonists
Second generation AR antagonists
Strategy of novel scaffold
for AR antagonists
O
NC
N
O
A
N
N
O2N
CONHCH3
O
F3C
N
NC
S
B
R
F
N
D
F3C
N
F3C
ARN-509
S
H
C
Scaffold 1
Flutamide (R=H)
Hydroxyflutamide (R=OH)
O
B
N
N
C
A
CONHCH3
expand the
D ring
NC
S
NC
F
F
O
F3C
Enzalutamide
O2
S
NC
F3C
N
O
A
H
OH
F3C
N
B
N
Bicalutamide
O
D
OH
S
N
N
C
N
H
Scaffold 2
NC
N
NH
ODM-201
Cl
Electron-deficent Middle linker Hydrophobic ring
ring and side chain
Fig. 1. Structures of non-steroidal AR antagonists and Design of a novel scaffold as AR antagonists.
Enzalutamide, ARN-509 and ODM-201 (Fig. 1) are second generation non-steroidal
antiandrogens with high affinity binding for the AR-LBD. Enzalutamide was approved by FDA in 2012
for the treatment of patients with bicalutamide-resistant CRPC. Enzalutamide, which has higher affinity
to AR-LBD than bicalutamide, can block nuclear translocation of AR and recruitment of coactivator.
ARN-509 is another competitive AR antagonist with potent activity in both preclinical and clinical
trials [17], which shows higher efficacy and is less likely to penetrate the blood-brain barrier than
enzalutamide. SPARTAN (NCT01946204), a Phase III trial of ARN-509, is ongoing with patients of
CRPC [18]. However, Korpal and coworkers found that enzalutamide and ARN-509 could also be
turned into agonists by a mutation of AR-LBD F876L (Phe 876 to Leu). F876L mutant induced the
smaller leucine residue leading that the reposition of the benzamide motif of enzalutamide side chain to
eliminate H12 dislocation caused by steric clashes [19, 20]. ODM-201, a full antagonist, was
2

ACCEPTED MANUSCRIPT
developed for the mutant AR. It shows a similar structure with enzalutamide, but with a longer middle
linker for better fitting into the AR-LBD pocket [21, 22]. Based on the second generation non-steroidal
AR antagonists, we found that they show a common scaffold composed of electron-deficient ring,
middle linker and hydrophobic ring with side chain (Fig. 1).
Previously, based on the SAR of enzalutamide, our group introduced a cyclopentane moiety D
ring fused with thiohydantoin B ring and benzene C ring to the discovery a novel scaffold (Fig. 1) of
indoline thiohydantoin 1 [23], which showed potent antiproliferation against LNCaP-hr cell (AR-rich)
and little inhibitory activity in DU145 cell (AR-deficient) with the IC50 being 15.3 and 143.1 µM. In
addition, 1 exhibited considerable antagonistic activity against AR (58.4% inhibition).The three fused
rings of indoline thiohydantoin (ring B, C, D, Fig. 1) can impose conformational restriction for a
greater steric clashes with residues approach to H12 to induce a larger dislocation in H12 and avoid the
loss of entropic by the rigid structure [23]. This showed that the introduction of D ring into the
enzalutamide is an efficacious and practical method for the finding of novel AR antagonists. Thus, on
one hand, to get more effective AR antagonists and furtherly explore the relationship of the fused rings
scaffold with AR, this paper designed a novel scaffold of tetrahydroisoquinoline thiohydantoin 2
through changing the D ring from cyclopentane into the cyclohexane and synthesize a novel series of
tetrahydroisoquinoline thiohydantoin derivatives (Fig. 1). On the other hand, as a result the
pharmacokinetic properties of 1 was not satisfactory with low plasma exposure and high clearance, we
expected to improve the pharmacokinetic properties of new designed compounds to develop novel and
improved AR antagonists.
2
. Chemistry
Target compounds 13-15 were synthesized as Scheme 1. 2-Fluoro-4-nitrotoluene (3) was oxidized
with potassium permanganate to obtain 2-fluoro-4-nitrobenzoic acid (4). We got 2-fluoro-4-
nitrobenzamide (5) through 4 reacting with aqueous ammonia in the presence of thionyl chloride. 5 was
converted to 4-amino-2-fluorobenzonitrile (7) by the treatment with phosphorus oxychloride and the
reduction with hydrogen and palladium on carbon. Two important intermediates 4-isothiocyanato-2-
fluorobenzonitrile (9) and 4-isothiocyanato-2-trifluoromethylbenzonitrile (10) were readily obtained
from 7 and 8 by reacting with thiophosgen [24]. 1,2,3,4-Tetrahydroisoquinoline-3-formic acid (11) and
3
-methyl-1,2,3,4-tetrahydroisoquinoline-3-formic acid (12) were cyclized upon the treatment with 9
and 10 into the target compounds 13-15 [25]. Next, 2-fluoro-4-methylbenzonitrile (16) was converted
to 4-(2-amino-2-methoxycarbonyl-ethyl)-2-fluoro-benzoic acid methyl ester (20) through the treatment
with N-bromosuccinimide, diethyl acetamidomalonate, hydrolysis and methylation. After protection the
-
NH group of 20 with ethyl chloroformate, we got 4-(2-ethoxycarbonylamino-2-methoxycarbonyl-
2
ethyl)-2-fluoro-benzoic acid methyl ester (21), which was cyclized to obtain 6-fluoro-3,4-dihydro-1H-
isoquinoline-2,3,7-tricarboxylic acid 2-ethyl ester 3,7-dimethyl ester (22). Based on the mechanism of
Pictet-Spengler reaction [26, 27], which was used for the synthesis of isoquinoline 22, we utilized ethyl
chloroformate to decrease the electron cloud density of the -NH group so as to increase the reaction
2
activity of 20. Through the hydrolysis and methylation of 22, we synthesized significant intermediate
6
-fluoro-1,2,3,4-tetrahydro-isoquinoline-3,7-dicarboxylic acid dimethyl ester (24). Compound 24 was
further cyclized with 9 and 10 to yield 25a and 25b, respectively. Target compounds 27a and 27b were
obtained by the hydrolysis with sodium hydroxide and amidation of 25a and 25b.
3

ACCEPTED MANUSCRIPT
O
H3C
F
HOOC
F
NC
NC
F
i
ii
H2N
iii
iv
NO2
NO2
F
NO2
F
NO2
NH2
3
4
5
6
7
NC
R1
R2
O
NC
R1
NC
R1
COOH
v
vi
R2
N
NH
NH2
NCS
N
S
7
, R1 = -F
9, R1 = -F
11, R2 = -H
12, R2 = -CH3
8, R1 = -CF3
10, R1 = -CF3
3
13, R1 = -CF , R2 = -H
1
1
4, R1 = -CF3, R2 = -CH3
5, R1 = -F, R2 = -H
COOEt
COOEt
F
CH3
F
CH Br
F
F
COOH
x
NH2
F
COOCH3
NH2
2
vii
viii
ix
NHAc
NC
NC
NC
HOOC
H COOC
3
1
6
17
18
19
20
F
COOCH3
COOEt
F
COOCH3
F
COOCH3
COOEt
F
COOH
NH
xi
xii
xiii
xiv
N
NH
HN
H3COOC
H COOC
H3COOC
HOOC
3
2
1
22
23
24
NC
R1
NC
R1
NC
R1
O
N
O
O
xv
xvi
xvii
N
N
N
N
N
S
S
S
F
COOCH3
F
F
2
2
5a, R1 = -F
5b, R1 = -CF3
26a, R1 = -F
26b, R1 = -CF3
27a, R1 = -F
COOH
27b, R1 = -CF3
CONHCH3
Scheme 1. Reagents and conditions: (i) KMnO , H O, tert-butanol, reflux, 8 h; (ii) SOCl , toluene, 40
4
2
2
o
o
o
C, 12 h, NH ·H O, DCM, 5 C, 30 min; (iii) POCl , DMF, H O, 25 C, 12 h; (iv) H /Pd-C, MeOH, rt,
3
2
3
2
2
o
1
2 h; (v) CSCl , CHCl , H O, rt, 15 h; (vi) TEA, DMF, 30 C, 1-2 h; (vii) NBS, BPO, CHCl , reflux, 12
2 3 2 3
h; (viii) EtONa, EtOH, diethyl acetamidomalonate, reflux, 18 h; (ix) HCl, reflux, 15 h; (x) SOCl2,
MeOH, reflux, 12 h; (xi) ClCOOC H , pyridine, DCM, rt, 4.5 h; (xii) HCHO, CH COOH, H SO , rt, 5
2
5
3
2
4
h; (xiii) HCl, reflux, 18 h; (xiv) SOCl , MeOH, reflux, 12 h; (xv) 9 or 10, TEA, DMF, rt, 2 h; (xvi)
2
NaOH, THF, H O, rt, 1 h; (xvii) NH CH , DMT-MM, DCM, rt, 3 h.
2
2
3
Scheme 2 outlines the synthesis of compounds 32a-32e and 36a-36i. Starting from commercially
available tyrosine (28), substituents Cl, Br and I were introduced into the ortho of -OH of 28 to obtain
2
9a-29c. Then 29a-29c were cyclized by reacting with formaldehyde into the tetrahydroisoquinoline
derivatives 30a-30c. The methylation of 30a-30c afforded intermediates 31a-31c. After protecting the
NH of 6,8-dibromo-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester (31b)
-
with di-tert-butyl dicarbonate (Boc) O, 33a was treated with different halogenated hydrocarbons to
2
provide the corresponding aromatic ethers 33b-33i in high yields. 33b and 33i were deprotected by
trifluoroacetic acid to yield 31d and 31e, respectively. 31a-31e were converted to the final compounds
3
2a-32e upon the treatment with 10 under basic conditions. 35a-35i, which were synthesized through
the cleavage of the 5,7-dibromo and the N-Boc of 33a-33i, were cyclized with 10 into the final
products 36a-36i.
4

ACCEPTED MANUSCRIPT
X
COOH
COOH
X
COOH
NH
X
COOCH3
NH
i/ii/ iii
iv
v
NH2
NH2
HO
HO
HO
HO
X
X
X
28
29a-29c
30a-30c
31a-31c
Br
COOCH3
Br
HO
COOCH3
Boc
Br
COOCH3
Boc
Br
COOCH3
NH
vi
vii
viii
NH
N
N
HO
R O
1
R1O
Br
Br
Br
33b-33i
Br
31d, 31e
31b
33a
Br
COOCH3
Boc
COOCH3
COOCH3
NH
ix
x
N
N
R O
R O
Boc
R O
2
2
2
Br
3a-33i
3
34a-34i
35a-35i
NC
O
N
X
COOCH3
NH
xi
F3C
N
R O
1
S
X
X
X
OR1
3
3
3
3
3
1a, X = Cl, R = H
1
32a, X = Cl, R = H
1
1b, X = Br, R = H
1
32b, X = Br, R = H
1
1c, X = I, R = H
1
32c, X = I, R = H
1
1d, X = Br, R = -CH
1
3
32d, X = Br, R = -CH3
1
1e, X = Br, R1 = -(CH2)3COOC2H5
3
2e, X = Br, R = -(CH ) COOC H
1 2 3 2 5
NC
O
N
COOCH3
NH
F3C
N
xi
S
R O
2
35a-35i
36a-36i
OR2
3
3
3
3
3
5a, R = H
35f, R = -(CH ) CH
36a, R = H
36f, R = -(CH ) CH
36g, R = -CH CH(CH )
2 2 3 2
36h, R = -cyclohexyl
2
2
2
2 3
3
2
2 2 3 3
5b, R = -CH
35g, R = -CH CH(CH )
36b, R = -CH3
2
3
2
2
3 2
2
5c, R = -CH CH
35h, R = -cyclohexyl
36c, R = -CH CH
2
2
3
2
2
2
3
5d, R = -(CH ) CH 35i, R = -(CH ) COOC H
36d, R = -(CH ) CH 36i, R = -(CH ) COOC H
2
2 2
3
2
2 3
2
5
2 2 2 3
2 2 3 2 5
5e, R = -CH(CH )
36e, R = -CH(CH )
2 3 2
2
3 2
Scheme 2. Reagents and conditions: (i) Cl , MeOH, rt, 12 h; (ii) Br , CH COOH, rt, 4 h; (iii) I ,
2
2
3
2
o
o
3
0%H O , CH COOH, HCl, 60 C, 1 h; (iv) HCHO, 33%HBr, CF COOH, 55 C, 72 h; (v) SOCl ,
2 2 3 3 2
MeOH, reflux, 12 h; (vi) (Boc) O, TEA, 1,4-dioxane, H O, rt, 24 h; (vii) R X, K CO , DMF, rt, 2 h;
2
2
1
2
3
o
(
viii) CF COOH, DCM, rt, 30 min; (ix) TEA, H /Pd-C, MeOH, 55 C, 12 h; (x) CF COOH, DCM, rt,
3
2
3
3
0 min; (xi) 10, TEA, DMF, rt, 1-2 h.
The synthesis of compounds 41a-41d and 44a-44g were shown in Scheme 3. We used
phenylalanine (37) as raw material, which was converted to 38a and 38b by the Pictet-Spengler
reaction and nitrification. However, it was difficult to separate 38a and 38b purely due to the similar
polarity of the two molecules. After trying many methods, we found that decreasing the polarity of the
mixture of 38a and 38b by treating with methylation and (Boc) O could provide 40a and 40b,
2
respectively. Then 40a and 40b were reduced by hydrogen and palladium on carbon to 40c and 40d.
With 40a-40d in hand, it was easy to get tetrahydroisoquinoline derivatives 39a-39d by the treatment
of trifluoroacetic acid. 39a-39d were cyclized with 10 into the final products 41a-41d. Through the
condensation reaction of 40d with different acid chloride or sulfonyl chloride and the deprotection of
5

ACCEPTED MANUSCRIPT
N-Boc, we obtained a series of amide 43a-43g. Finally, target compounds 44a-44g, bearing different
long side chains were synthesized through the reacting of 43a-43g with 10, respectively [28-30].
COOH
NH2
COOH
NH
COOH
i
ii
iii
O N
2
NH
3
8a = 6-NO2
3
7
11
38b = 7-NO2
COOCH3
NH
COOCH3
Boc
COOCH3
iv
vi
O N
O N
O N
2
2
2
N
NH
39a = 6-NO2
9b = 7-NO2
39a, 39b
40a, 40b
3
v
COOCH3
Boc
COOCH3
vi
H N
H2N
2
N
0c = 6-NH2
NH
39c = 6-NH2
4
4
0d = 7-NH2
39d = 7-NH2
NC
F3C
O
COOCH3
NH
9a, R = 6-NO2
NC
vii
N
R1
N
F3C
NCS
S
8
R1
7
10
3
3
3
3
41a, R = 8-NO
2
41b, R = 7-NO
41c, R = 8-NH
41d, R = 7-NH
1
1
9b, R = 7-NO2
1
2
1
9c, R = 6-NH2
1
2
1
9d, R = 7-NH2
1
2
1
COOCH3
COOCH3
COOCH3
viii
ix
N
R2
N
R2
NH
H N
Boc
N
H
Boc
N
2
H
40d
42a-42g
43a-43g
NC
O
N
F3C
N
x
S
NHR2
4
4a, R = -COCH3
44e, R = -COOCH CH
2
2
2
3
4
4
4
4b, R = -COCH CH
44f, R = -SO CH
2
2
3
2
2
3
4c, R = -CO(CH ) CH 44g, R = -SO (CH ) CH
2
2 2
3
2
2
2 3
3
4d, R = -COC(CH )
2
3 3
Scheme 3. Reagents and conditions: (i) HCHO, HCl, reflux, 6 h; (ii) KNO , H SO , rt, 1 h; (iii) SOCl ,
3
2
4
2
MeOH, reflux, 12 h; (iv) (Boc) O, TEA, 1,4-dioxane, H O, rt, 24 h; (v) H /Pd-C, MeOH, rt, 12 h; (vi)
2
2
2
CF COOH, DCM, rt, 30 min; (vii) TEA, DMF, rt, 1 h; (viii) RCl, pyridine, DCM, rt, 3 h; (ix)
3
CF COOH, DCM, rt, 12 h; (x) 10, TEA, DMF, rt, 1-2 h.
3
To expand our SAR study, target compound 55, introducing a fluorine atom into the 44e, was
prepared as depicted in Scheme 4. 1-(Bromomethyl)-3-fluorobenzene (45) was used as raw material.
Due to the reactions of Scheme 4 were similar to Scheme 1 and 3, we did not describe them in this
repeatedly.
The structures of all target compounds were verified using NMR spectroscopy, ESI mass
spectrometry and Infrared spectroscopy. Detailed information about synthesis and characterization of
all compounds is described in the experimental and supporting information sections of this manuscript.
6

ACCEPTED MANUSCRIPT
Scheme 4. Reagents and conditions: (i) EtONa, EtOH, diethyl acetamidomalonate, reflux, 18 h; (ii)
HCl, reflux, 15 h; (iii) HCHO, HCl, reflux, 6 h; (iv) KNO , H SO , rt, 1 h; (v) SOCl , MeOH, reflux,
3
2
4
2
1
2 h; (vi) (Boc) O, TEA, 1,4-dioxane, H O, rt, 24 h; (vii) H /Pd-C, MeOH, rt, 12 h; (viii) ClCOOEt,
2 2 2
pyridine, DCM, rt, 3 h; (ix) CF COOH, DCM, rt, 12 h; (x) 10, TEA, DMF, rt, 1 h.
3
3
. Results and discussion
3
.1. Antiproliferative and androgen receptor antagonist assay
Antiproliferative activity of the newly synthesized compounds was initially evaluated in
LNCaP-hr cell line using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Additionally, in order to verify the probability of synthesized compounds as novel AR antagonists, the
potential cytotoxic effects were also investigated in DU145 cell. LNCaP shows androgen-dependent
cell proliferation whereas DU145 is hormone-independent. Moreover, to clarify whether the
antiproliferative activity is related to any interference with the AR function, the AR antagonist effect
was evaluated using the luciferase reporter gene assay.
The results demonstrated that the novel compound 13 showed a little less potent inhibitory
activity in LNCaP cell than 1 with the IC50 being 31.1 µM vs 15.3 µM (Table 1). However, to our
delight, the antagonistic activity against AR of 13 (89.0% inhibition) is much higher than compound 1
(
58.4% inhibition). This is an encouraging fact that the size of bone skeleton of molecule has a very
important influence on the inhibition activity of AR. Next, we decided to fully and thoroughly study the
SAR of novel scaffold 13 with AR.
Firstly, to simulate the dimethyl moiety of enzalutamide, 14 was synthesized by introducing a
methyl into the position of 10 in compound 13. However, compound 14 did not show a satisfying result
with weak antiproliferative activity in LNCaP (IC50 = 65.3 µM) and antagonistic activity against AR
(
inhibition rate 26.2%, @ 10 µM). This demonstrated that the 10-CH was not necessary for the activity
3
of novel scaffold. Previously, we have found that the electron-withdrawing group -CN of aromatic ring
A is required for binding to AR with the respect to direct interaction with Arg752 [23]. Next, we want
to know whether the trifluoromethyl moiety is necessary for maintaining the activity. Replacement of
the trifluoromethyl with the fluorine atom is associated with decreased activity (i.e. 13 vs 15; 27b vs
2
7a). Especially, compared with 13, the antagonistic activity against AR of 15 was abolished
completely. Modifications of -CF on aromatic ring A do appear to decrease antiproliferative and
3
antagonistic activity.
7

ACCEPTED MANUSCRIPT
Table 1
Antiproliferative and androgen receptor antagonist activity of compounds 1, 13-15, 27a and 27b. All
data are mean values from triplicate experiments.
a
b
IC50/µM
IC50/µM
AR antagonistic
Compound
1
Structure
c
(
LNCaP)
(DU145)
activity % (10 µM)
15.3 ± 0.3
31.1 ± 1.7
65.3 ± 1.9
70.3 ± 0.8
143.1 ± 5.8
58.4 ± 0.6
89.0 ± 3.1
26.2 ± 1.2
N.E
1
1
1
3
4
5
> 200
> 200
37.1 ± 3.2
2
7a
7b
116.5 ± 2.3
28.1 ± 1.1
85.4 ± 2.7
26.7 ± 0.2
47.1 ± 2.3
2
> 200
a
b
The cell proliferation inhibition of LNCaP cell line; See the Experimental Section for details; The cell proliferation inhibition of
c
DU145 cell line; See the Experimental Section for details; Inhibition rate was shown as a ratio to the R1881 control; See the
Experimental Section for details; N.E, no antagonistic effect.
Based on our previous work, structure modifications in the C ring profoundly influence the
activity and drug-like properties of compounds. For the purpose of exploring the SAR of substituent
group in aromatic ring C, we firstly studied the steric hindrance of substituents by designing
compounds 32a-32e and 36a (Table 2). The results indicated that 32a-32e showed a comparable
activity against LNCaP cell line as enzalutamide. However, the five compounds also exhibited potent
inhibitory activity in DU145. Worse more, none of these compounds expressed apparent antagonistic
behavior, especially 32e which had no antagonistic activity. These findings suggested that 32a-32e
might not AR antagonists but cytotoxic agents to exhibit the inhibitory activity in either LNCaP and
DU145. We speculated that the molecular size of compounds 32a-32e might be too large to fit the
receptor cavity. Based on the ideas, we obtained compound 36a by removing the halogen atom of 6 and
8
position to “thin” the molecules 32a-32e. Removing the halogen atoms might make compound 36a
interacting with AR binding pocket more better. To our delighted, 36a showed potent antagonistic
activity against AR (85.7% inhibition) and no inhibitory activity in DU145 (IC50 > 200 µM) which
were consistent with our design. So, compound 36a by removal of halogen atoms showed potent
inhibitory activity toward proliferation of LNCaP through AR antagonistic activity but not cytotoxic
8

ACCEPTED MANUSCRIPT
effect in DU145.
Table 2
Antiproliferative and androgen receptor antagonist activity of compounds 32a-32e and 36a designed
from 13. All data are mean values from triplicate experiments.
a
b
IC50/µM
IC50/µM
AR antagonistic
activity % (10 µM)
36.0 ± 3.9
Compound
X
R
c
(
LNCaP)
(DU145)
17.1 ± 1.5
11.4 ± 0.4
22.1 ± 0.7
33.8 ± 2.1
21.9 ± 1.3
3
2a
2b
-Cl
-Br
-I
-OH
-OH
14.0 ± 0.6
11.1 ± 0.8
16.3 ± 2.1
19.1 ± 1.6
6.3 ± 2.0
3
6.1 ± 0.9
3
2c
-OH
27.3 ± 1.8
3
2d
-Br
-Br
H
-OCH
3
4.6 ± 0.3
3
2e
6a
-O(CH
2
)
3
COOEt
N.E
3
-OH
50.7 ± 0.4
> 200
85.7 ± 2.3
a
b
The cell proliferation inhibition of LNCaP cell line; See the Experimental Section for details; The cell proliferation inhibition of
c
DU145 cell line; See the Experimental Section for details; Inhibition rate was shown as a ratio to the R1881 control; See the
Experimental Section for details; N.E, no antagonistic effect.
Next, to explore the influence of the position of substituents in the ring C on activity, we designed
four compounds 41a-41d with substituents 7- or 8- position (Table 3). Compound 41a and 41c, the
8
-substituted derivatives, did not show significant AR antagonist activity with the inhibition value
being N.E and 14.2%. And their antiproliferative activity against LNCaP cell line were also not high
with the IC50 being 56.4 and 73.0 µM. However, 41b and 41d with the same substituents shifting to
7
-position showed an increasement activity (IC50 values: 43.0 and 18.9 µM). The antagonistic activity
suggested the similar result with the inhibition rate being 59.8% and 37.8%. The results indicated that
compounds with substituents at 7-position were more appropriate than at 8-position for maintaining the
activity.
Previously, we have studied the steric hindrance and the position of substituent group in C ring.
Among the synthesized compounds, 36a exhibited similar AR antagonistic activity compared to
enzalutamide (inhibition: 85.7% vs 86.5%). However, antiproliferative activity against LNCaP of 36a
was not satisfied with the IC50 value being 50.7 µM. Thus, to increase the antiproliferative activity, we
synthesized target compounds 36b-36i through extending the length of side chain (Table 3). Most of
the newly compounds showed increased inhibition activity in LNCaP with IC50 being almost 20 µM.
Compound 36f, bearing four carbon atoms, exhibited a little higher activity than enzalutamide (IC :
50
1
6
0.2 vs 12.5 µM) at the same time maintaining considerable AR antagonistic activity (inhibition:
5.3%). According to the result, we concluded that on the one hand side chain extension indeed
increased the anti-proliferation activity in LNCaP cell, however on the other hand the extension
exhibited a little reduce in AR antagonistic activity. Three or four atom length was best appropriate for
increasing inhibition activity in LNCaP and maintaining AR antagonistic activity.
9

ACCEPTED MANUSCRIPT
Table 3
Antiproliferative and androgen receptor antagonist activity of compounds 41a-41d、36b-36i、44a-44g
and 55. All data are mean values from triplicate experiments.
NC
O
A
F3C
N
B
N D
S
C
R2
8
7
R1
a
b
IC50/µM
IC50/µM
AR antagonistic
activity % (10 µM)
N.E
Compound
R
1
R
2
c
(
LNCaP)
(DU145)
> 200
4
1a
1b
H
-NO
H
-NO
2
2
56.4 ± 2.1
43.0 ± 0.5
73.0 ± 1.6
18.9 ± 3.7
100.1 ± 3.1
22.6 ± 1.4
11.6 ± 0.6
8.6 ± 1.2
4
2
H
> 200
59.8 ± 1.2
14.2 ± 0.5
37.8 ± 0.2
N.E
4
1c
-NH
H
> 200
4
3
1d
6b
-NH
2
89.7 ± 1.1
60.4 ± 4.7
40.9 ± 3.7
62.3 ± 0.5
91.6 ± 3.5
54.2 ± 2.1
52.6 ± 0.8
98.1 ± 1.6
15.4 ± 2.3
> 200
-OCH
-OCH CH
-OCH CH
3
H
3
6c
6d
6e
6f
2
3
H
47.1 ± 3.1
54.0 ± 2.9
45.4 ± 4.2
65.3 ± 2.5
61.5 ± 2.2
61.6 ± 0.4
43.9 ± 4.1
25.0 ± 1.9
4.5 ± 3.0
3
2
2
CH
3
H
3
-OCH(CH
-O(CH CH
-OCH CH(CH
-Ocyclohexyl
-O(CH COOEt
-NHCOCH
-NHCOCH CH
-NHCOCH CH
-NHCOC(CH
-NHCOOCH CH
-NHSO CH
-NHSO (CH CH
-NHCOOCH CH
3
)
2
H
3
2
)
3
3
H
10.2 ± 2.5
10.6 ± 1.9
8.5 ± 0.7
3
6g
6h
2
3
)
2
H
3
H
3
6i
2
)
3
H
18.8 ± 4.1
83.8 ± 1.9
19.5 ± 0.8
10.8 ± 1.6
21.2 ± 4.5
6.3 ± 0.4
4
4a
4b
3
H
4
2
3
H
> 200
4
4c
4d
4e
4f
4g
2
2
CH
3
H
> 200
45.5 ± 4.1
N.E
4
3
)
3
H
83.2 ± 4.4
90.1 ± 3.5
> 200
4
2
3
H
45.4 ± 0.7
13.6 ± 1.9
29.5 ± 2.5
85.1 ± 3.6
86.5 ± 1.1
4
2
3
H
31.1 ± 5.9
3.12 ± 0.3
13.4 ± 1.1
12.5 ± 0.3
4
2
2
)
3
3
H
> 200
5
5
2
3
F
> 200
Enzalutamide
46.1 ± 0.2
a
b
The cell proliferation inhibition of LNCaP cell line; See the Experimental Section for details; The cell proliferation inhibition of
c
DU145 cell line; See the Experimental Section for details; Inhibition rate was shown as a ratio to the R1881 control; See the
Experimental Section for details; N.E, no antagonistic effect.
AR-LBD is more hydrophilic in antagonistic conformation compared with agonistic conformation.
Based on compound 41d, we obtained 44a-44g (Table 3) through introducing hydrophilic moiety to
favor the antagonistic conformation “H12-open.” Most of the compounds had no inhibition in DU145
cell with the IC50 value being more 200 µM, indicating that these compounds had no cytotoxicity. 44f
and 44g, introducing the sulfonamide group, had no effect in AR antagonistic activity with the
inhibition rate decreasing to 13.6% and 29.5%. Amide derivatives 44a-44e exhibited improvement
activity, especially for compound 44e (IC50 = 6.33 µM; inhibition rate = 45.4%), showed the improved
anti-proliferation activity in LNCaP and the AR antagonistic activity compared to 41d (IC = 18.9 µM;
5
0
inhibition rate = 37.8%).
1
0

ACCEPTED MANUSCRIPT
Fig. 2. Dose-dependent androgen receptor antagonist assay for compound 1, 55 and enzalutamide.
Compounds tested at 6 different concentrations. All data are mean values from triplicate experiments.
Finally, to further increase the antagonistic activity and improve pharmacokinetic properties,
introducing a polar fluorine atom in the ortho position of the side chain got target compound 55. The
result indicated that compound 55 showed profoundly AR antagonistic activity compared to 44e
(
inhibition rate: 85.1% vs 58.6%). Moreover, compound 55 showed comparable effect in inhibiting the
LNCaP cell growth and AR antagonistic activity with enzalutamide (the IC50 being 13.4 µM vs 12.5
µM and the inhibition rate being 85.1% vs 86.5%). In addition, 55 exhibited no inhibition activity in
DU145 cell compared to enzalutamide (IC : > 200 µM vs 46.1 µM). A 6-concentrations
50
dose-dependent AR antagonism assay (Fig. 2) showed that 55 possess an antagonistic IC50 in the same
range of enzalutamide and the AR antagonistic activity of 55 was two-fold higher than compound 1. On
the basis of these findings, we summarized the SAR studies for compound 55 in Fig. 3.
No correlation between the inhibitory activity toward LNCaP proliferation and AR antagonism
2
can be identified just as previously report , implying that other modes of action or other parameters
such as: physicochemical property, cell membrane permeability or target activity could have an
significant role in the antiproliferative activity of our novel compounds.
Fig. 3. SAR derived from tetrahydroisoquinoline thiohydantoin derivatives.
3
.2 Immunofluorescence assay of compound 55
The resultant strong antiandrogenic effect of compound 55 can be explained by molecular basis
for its antagonism. To explore molecular basis of 55, the dynamic of AR subcellular distribution was
1
1

ACCEPTED MANUSCRIPT
analyzed by immunofluorescence assay with a confocal microscope (LSM700, Zeiss) [31]. When an
agonist R1881 binds to AR, the R1881-AR complex translocated from the cytoplasm into the nucleus,
causing the localization of the liganded AR proteins in the nucleus (Fig. 4). However, treatment of
enzalutamide and 55 in the presence of R1881 resulted in a distinct distribution pattern in which the
liganded AR proteins were instead dispersed throughout the cytoplasm, indicating that they effectively
interfere with nuclear translocation of the AR proteins. Moreover, the green nuclear AR staining of
compound 55 was much weaker than that of enzalutamide, indicating that 55 can significantly impair
AR nuclear translocation and reduce more levels of nuclear AR than enzalutamide.
Fig. 4. Molecular basis of antiandrogenic effect of compound 55: green staining represents AR, blue
®
staining represents nuclei and merge represents both. AR and nuclei was labeled with Alexa Fluor 488
(
green) goat anti-rabbit IgG (H + L) and DAPI, respectively. LNCaP were treated with R1881 (10 nM)
alone or in combination with compound 55 (10 µM) or enzalutamide (10 µM), and compared to the no
ligand group without treating with R1881.
3
.3 Pharmacokinetic Properties of compound 1, 13, 44e, and 55
Having identified several compounds with excellent in vitro antiproliferative and androgen
receptor antagonist activity, pharmacokinetic evaluations were conducted in rats following gavage
administration at a dose of 5 mg/kg [32], and the results were summarized in Table 4.
Table 4
Pharmacokinetic properties of selected compound 1, 13, 44e, and 55.
compound
AUC0-t(ng·h/mL)
2863
T
1/2(h)
T
max(h)
C
max (ng/mL)
452
CL(mL/h/kg)
1588
1
10.36
5.13
2
2
2
4
1
3
3865
387
1230
4
4e
7034
34.38
24.68
641
570
5
5
29995
2290
146
1
2

ACCEPTED MANUSCRIPT
Abbreviations: AUC0-t, area under the concentration-time curve up to last sampling time; T1/2, elimination half-life; CL, clearance.
Rats dosed at 5 mg/kg gavage administration, n = 3. Data are the mean values.
Compound 55 exhibited favorable plasma exposure (AUC0-t = 29995 ng·h/mL) and relatively low
clearance (CL = 146 mL/h/kg). Compound 1 displayed poor pharmacokinetic properties with low
plasma exposure (AUC0-t = 2863 ng·h/mL) and relatively high clearance (CL = 1588 mL/h/kg),
whereas the hit compound 13 showed a litter higher plasma exposure and a litter decrease of clearance
when compared with that of 1. The results indicated that the pharmacokinetic properties of novel
scaffold of tetrahydroisoquinoline thiohydantoin was better than that of indoline thiohydantoin.
Interestingly, the compound 44e exhibited the longest elimination half-life (T1/2 = 34.38 h) among these
analogues, while its plasma exposure was low (AUC0-t = 7034 ng·h/mL). Based on the
pharmacokinetic properties, therefore, we chose compound 55 for further in vivo studies in our next
work.
3
.4 Molecular Modeling
In order to better elucidate the mechanism of compound 55 antagonism at the molecular level, we
performed molecular modeling calculations and compared them to those of enzalutamide. The
compound 55 and enzalutamide were docked into the active site of AR (PDB code 2OZ7) using
Accelrys Discovery Studio (DS) 3.0 (Accelrys Inc., San Diego, CA, USA). As shown in Fig. 5,
compound 55 could fit into the binding site by the formation of the key hydrogen bonding interaction
with Arg752 similar to that of enzalutamide, while the side chain on ring C was located to different
orientation, formed as a result of the conformational rearrangement of the active site. Enzalutamdie
formed additional hydrogen bond interactions through its fluorine atom on ring C with the side chain of
Leu701. Similarly, 55 kept the interaction between the fluorine atom and the side chain of Asn705, and
formed an additional hydrogen bond between the side chain of Gly708 and the oxygen atom on ring B.
1
3

ACCEPTED MANUSCRIPT
Fig. 5. (A) The predicted binding mode of compound 55 in complex with AR; (B) The 2D diagram of
binding mode of compound 55 in complex with AR; (C) The predicted binding mode of enzalutamide
in complex with AR; (D) The 2D diagram of binding mode of enzalutamide in complex with AR.
4
. Conclusion
Herein we report the synthesis and biological in vitro and vivo evaluation of a novel class of
tetrahydroisoquinoline thiohydantoin derivatives as potent androgen receptor antagonists. Firstly,
compound 13 exhibited much increasement in AR antagonistic activity compared to compound 1
(
inhibition rate: 89.1% vs 58.4%) and on the same time 13 maintained potent antiproliferative activity
in LNCaP cell. Next, we roundly investigated the SAR of novel scaffold tetrahydroisoquinoline
thiohydantoin to obtain compound 55, a potent androgen receptor antagonist. 55 showed comparable
effect in inhibiting the LNCaP cell growth (IC50 = 13.4 µM) and AR antagonistic activity (inhibition
rate = 85.1%) with enzalutamide (IC = 12.5 µM, inhibition rate = 86.5%). In addition, 55 exhibited no
50
inhibition activity in DU145 cell (IC50 > 200 µM) compared to enzalutamide (IC50 = 46.1 µM). More
importantly, 55 showed favorable pharmacokinetic properties with high plasma exposure and relatively
low clearance compared to 1. Finally, the most promising compound 55 has been selected for in vivo
preclinical studies in mouse models, which will be reported in later paper. We believe that it may be a
novel candidate in the treatment of advanced human prostate cancer.
5
. Experimental procedures
5
.1 Chemistry
All reagents and solvents were from commercial sources and were used without further
1
13
purification. With tetramethylsilane (TMS) as internal standard, the H and C NMR spectra were
1
4

ACCEPTED MANUSCRIPT
o
recorded on Bruker AV-300 (300 and 75 MHz) apparatus at 25 C. Samples were prepared as solutions
in deuterated solvent. EI-MS was collected on shimadzu GCMS-2010 instruments. HR-MS spectral
data was obtained on Agilent technologies 6520 Accurate-Mass Q-TOF LC/MS instruments. All target
compounds were purified via silica gel (60 Å, 70-230 mesh) column chromatography. Melting points
were measured by XT-4 melting point apparatus. All target compounds were found to have > 95%
purity.
5
.1.1 2-Fluoro-4-nitro-benzoic acid (4)
To the solution of 2-fluoro-4-nitrotoluene 3 (15.0 g, 96.69 mmol) in tert-butanol/H O (500 mL,
2
v:v = 2:3), tetrabutylammonium chloride (1.5 g, 5.40 mmol) and potassium permanganate (76.4 g, 0.48
mol) were added. After refluxed for 8 h, the reaction mixture was filtered. The filtrate was then
acidified to pH 1 with 1 M HCl and extracted with ethyl acetate (EA). The combined organic layers
were dried over Na SO and concentrated under reduced pressure to give the corresponding product. It
2
4
was obtained as a white solid in 89% yield. 4 was ready for the next step without the further
-
purification. HRMS (ESI): m/z, calculated for C H FNO 184.0079 (M - H) , found 184.0059.
7
4
4
5
.1.2 2-Fluoro-4-nitro-benzamide (5)
To a solution of 4 (10 g, 54.05 mmol) in toluene (100 mL) was added thionyl chloride (12.86 g,
o
0
.1 mol) dropwisely, and the reaction mixture was stirred at 40 C for 12 h. The solvent was removed
under reduced pressure to afford the acid chloride as a viscous oil. This oil was dissolved in CH Cl2
2
o
(
150 mL) and cooled to 0 C. Aqueous ammonia (28%, 200 mL) was slowly added and the reaction
o
was stirred at 4 C for 30 min. The mixture was extracted with EA, and the organic layer was dried
with Na SO , filtered, and concentrated under reduced pressure to afford a yellow solid in 89.33% yield.
2
4
+
HRMS (ESI): m/z, calculated for C H FN O 185.0365 (M + H) , found 185.0388.
7
5
2
3
5
.1.3 2-Fluoro-4-nitro-benzonitrile (6)
To a solution of 5 (10 g, 54.35 mmol) in DMF (100 mL) was added phosphorus oxychloride (35
o
mL) dropwisely. And the mixture was stirred at 25 C for 12 h. This mixture was treated with ice water
(
300 mL) for 30 min. Then the mixture was extracted with EA, and the organic layer was washed with
sat, dried with Na SO , filtered, and concentrated under reduced pressure to afford product 6 (7.82 g,
2
4
+
8
1
6.73%) as a yellow solid. HRMS (ESI): m/z, calculated for C H FN O 167.0261 (M + H) , found
7 3 2 2
67.0283.
5
.1.4 4-Amino-2-fluoro-benzonitrile (7)
A solution of 6 (10.0 g, 60.24 mmol) in methanol (100 mL) was hydrogenated with 10% Pd/C
1.0 g) under hydrogen atmosphere at room temperature for 12 h. After filtration, the filtrate was
(
evaporated to give the corresponding product. It was obtained as a gray solid in 88% yield. 7 was ready
for the next step without the further purification. HRMS (ESI): m/z, calculated for C H FN 137.0487
7
5
2
+
(
M + H) , found 137.0452.
5
.1.5 2-Fluoro-4-isothiocyanato-benzonitrile (9)
Thiophosgen (9.3 g, 80.62 mmol) was added to a suspension of intermediate 7 (10.0 g, 73.53
mmol) in H O/CHCl (450 mL, v:v = 8:1). The mixture was stirred at room temperature for 15 h. After
2
3
the reaction was completed, the reaction mixture was extracted with CHCl (3 × 50 mL). The combined
3
1
5

ACCEPTED MANUSCRIPT
organic layers were dried over Na SO and concentrated under reduced pressure. The crude residue was
2
4
purified by silica gel column chromatography (petroleum ether (PE)/ethyl acetate (EA) 6:1) to give the
corresponding product. It was obtained as a pale yellow oil in 57% yield. HRMS (ESI): m/z, calculated
+
for C H FN S 179.0098 (M + H) , found 179.0028.
8
3
2
5
.1.6 4-Isothiocyanato-2-trifluoromethyl-benzonitrile (10)
The synthesis of intermediate 10 was similar to 9, just replacing the material 7 into 4-amino-2-
o
1
trifluoromethyl-benzonitrile 8. m.p. 41-42 C; H-NMR (300 MHz, CDCl ): δ 7.84 (d, J = 8.25 Hz, 1H,
3
+
Ar-H), 7.59 (s, 1H, Ar-H), 7.48 (d, J = 8.25 Hz, 1H, Ar-H) ppm; MS-EI (m/z): 69, 186, 228 [M] .
5
.1.7 4-(1-Oxo-3-thioxo-4,9-dihydro-10,10a-dihydroimidazol[1,5-b]isoquinolin-2(1H, 3H, 5H)-yl)-2-(t-
rifluoromethyl)benzonitrile (13)
To the solution of 10 (0.23 g, 1 mmol) in DMF (10 mL), 1,2,3,4-tetrahydroisoquinoline-3-carbox-
ylic acid 11 (0.21 g, 1 mmol) and triethylamine (0.15 mol) were added. The mixture was stirred at 30
o
C for 1 h. Then the solution was added into ice water (50 mL), extracted with CH Cl . The organic
2
2
layers were dried over Na SO and concentrated under reduced pressure. The crude residue was
2
4
purified by silica gel column chromatography (PE/EA 6:1) to give the corresponding product. It was
o
1
obtained as a yellow solid in 39% yield. m.p. 215-218 C; H-NMR (300 MHz, CDCl ): δ 7.88 (d, 2H,
3
Ar-H), 7.48 (d, J = 5.94 Hz, 1H, Ar-H), 7.39-7.31 (m, 4H, Ar-H), 5.79 (d, 1H, -CH-), 4.74 (d, J = 6.87
Hz, 1H, -CHH-), 4.51 (q, J = 6.87 Hz, 1H, -CHH-), 3.45 (d, 1H, -CHH-), 3.08 (q, 1H, -CHH-) ppm;
1
3
C-NMR (75 MHz，DMSO-d ): δ 178.3, 172.0, 138.1, 136.3, 133.6, 131.2, 130.8, 129.2, 127.1 (q, J =
6
2
8
67.9 Hz, -CF ), 124.0, 115.0, 108.3, 57.6, 45.6, 29.2 ppm; IR (KBr): 3397, 1742, 1311, 1283, 1134,
3
-1
+
09 cm ; MS-EI (m/z): 104, 130, 387 [M ]; HRMS (ESI): m/z, calculated for C H F N OS 388.0726
19
12
3
3
+
[
M + H] , found 388.0724.
5
5
.1.8 4-(10a-Methyl-1-oxo-3-thioxo-4,9-dihydro-10,10a-dihydroimidazol[1,5-b]isoquinolin-2(1H, 3H,
H)-yl)-2-(trifluoromethyl)benzonitrile (14)
The synthesis of 14 was similar to 13. The product 14 was yellow solid in 35% yield. m.p.
o
1
1
8
1
3
1
1
93-195 C; H-NMR (300 MHz, DMSO-d ): δ 8.38 (d, J = 8.34 Hz, 1H, Ar-H), 8.32 (s, 1H, Ar-H),
6
.08 (d, J = 8.34 Hz, 1H, Ar-H), 7.37 (m, 4H, Ar-H), 5.58 (d, J = 18.09 Hz, 1H, -CHH-), 4.73 (d, J =
8.09 Hz, 1H, -CHH-), 3.55 (d, J = 16.14 Hz, 1H, -CHH-), 3.08 (d, J = 16.14 Hz, 1H, -CHH-), 1.50 (s,
1
3
H, -CH3); C-NMR (75 MHz, DMSO-d ): δ 177.3, 175.0, 138.0, 136.1, 134.0, 131.2, 130.8, 129.9,
6
28.1 (q, J = 268.2 Hz, -CF ), 124.0, 115.0, 108.5, 61.8, 43.6, 19.3 ppm; IR (KBr): 3127.38, 1763.72,
3
-1
+
399.48, 1311.14, 1136.40 cm ; MS-EI (m/z): 104, 130, 144, 401 [M ]; HRMS (ESI): m/z, calculated
+
for C H F N OS 402.0882 [M + H] , found 402.0888.
2
0
14
3
3
5
.1.9 2-Fluoro-4-(1-oxo-3-thioxo-10,10a-ihydroimidazo[1,5-b]isoquinolin-2(1H, 3H, 5H)-yl)benzonitr-
ile (15)
The synthesis of 15 was similar to 13. The product 15 was yellow solid in 56% yield. m.p.
o
1
2
8
10-214 C; H-NMR (300 MHz, DMSO-d ): δ 8.38 (s, 1H, Ar-H), 8.15 (d, J = 8.04 Hz, 1H, Ar-H),
6
.12 (d, J = 3.6 Hz, 1H, Ar-H), 7.74 (m, 1H, Ar-H), 7.65 (d, J = 8.46 Hz, 1H, Ar-H), 7.55 (m, 1H,
Ar-H), 5.49 (t, J = 18.66 Hz, 1H, -CH -), 4.83 (dd, J = 5.43 Hz, 1H, -CH -), 3.47 (t, J = 2.97 Hz, 1H,
2
2
1
3
-
CH-) ppm; C-NMR (75 MHz, DMSO-d ): δ 172.7, 171.4, 160.8, 141.2, 134.0, 130.4, 128.6, 127.5,
6
1
26.9, 126.3, 125.7, 121.3, 110.8, 108.1, 108.0, 72.5, 53.8, 28.1 ppm; IR (KBr): 3125, 2231, 1752,
1
6

ACCEPTED MANUSCRIPT
-1
1
3
617, 1525, 1400, 1347, 1069, 860, 515 cm ; HRMS (ESI): m/z, calculated for C H FN OS
18 12 3
+
60.0577 (M + Na) , found 360.0585.
5
.1.10 4-Bromomethyl-2-fluoro-benzonitrile (17)
To the solution of 2-fluoro-4-methylbenzonitrile 16 (10 g, 74.07 mmol) in CHCl (200 mL),
3
dibenzoyl peroxide (1 g, 4.13 mmol) and N-Bromosuccinimide (19.77 g, 11.11 mmol) were added. The
reaction was stirred at refluxing for 12 h. The mixture was treated with saturated sodium bicarbonate
solution, washed with water and brine. The organic layer was dried over Na SO and concentrated
2
4
under reduced pressure to give the corresponding product 17. It was obtained as a yellow oil in 88%
+
yield. HRMS (ESI): m/z, calculated for C H BrFN 213.9678 (M + H) , found 213.9667.
8
5
5
.1.11 2-Acetylamino-2-(4-cyano-3-fluoro-benzyl)-malonic acid diethyl ester (18)
To the solution of Na (0.76 g, 33.16 mmol) in EtOH (100 mL), diethyl acetamidomalonate (6.45 g,
0.15 mmol) was added. The mixture was stirred for 30 min. Then 17 (6.42 g, 30.15 mmol) was added
3
into the solution. After the reaction mixture was stirred at refluxing for 18 h, the mixture was treated
with ice water (400 mL) and filtered to give the corresponding 18. It was obtained as a white solid in
+
7
1% yield. HRMS (ESI): m/z, calculated for C H N O 350.1321 (M + H) , found 350.1423.
17
19
2
5
5
.1.12 4-(2-Amino-2-carboxy-ethyl)-2-fluoro-benzoic acid (19)
The intermediate 18 (7.50 g, 21.43 mmol) was added into HCl (80 mL). The reaction mixture was
stirred at refluxing for 15 h and TLC analysis indicated that the reaction was completed. The mixture
was filtered to give the white solid 19 in 86% yield. HRMS (ESI): m/z, calculated for C H NO
4
10
10
+
2
28.0603 (M + H) , found 228.0606.
5
.1.13 4-(2-Amino-2-methoxycarbonyl-ethyl)-2-fluoro-benzoic acid methyl ester (20)
To the solution of 19 (10 g, 44.05 mmol) in methanol (100 mL), thionyl chloride (10.48 g, 88.10
o
mmol) was added dropwisely at 0 C. After adding, the mixture was stirred for 30 min maintaining the
o
temperature no more 5 C. Then the solution was stirred at refluxing for 8 h. The reaction mixture was
cooled to room temperature and filtered to give the white solid 20 in 75% yield. HRMS (ESI): m/z,
+
calculated for C H NO 256.0917 (M + H) , found 256.0914.
1
2
14
4
5
.1.14 4-(2-Ethoxycarbonylamino-2-methoxycarbonyl-ethyl)-2-fluoro-benzoic acid methyl ester (21)
To the solution of 20 (4 g, 15.69 mmol) in CH Cl (40 mL), pyridine (4.96 g, 62.74 mmol) was
2
2
added. Then carbonochloridicacid ethylester (1.70 g, 15.69 mmol) was dropped into the solution solwly.
The reaction was stirred at room temperature for 4.5 h. The solvent was removed under reduced
pressure. The the crude was treated with water (100 mL), extracted with EA and washed with brine.
The organic layer was dried over Na SO , filtered and evaporated to give the corresponding product 21.
2
4
It was obtained as a white solid in 60% yield. HRMS (ESI): m/z, calculated for C H NO 328.1119
15
18
6
+
(
M + H) , found 328.1123.
5
.1.15 6-Fluoro-3,4-dihydro-1H-isoquinoline-2,3,7-tricarboxylic acid 2-ethyl ester 3,7-dimethyl ester
(22)
To the solution of 21 (3.83 g, 11.71 mmol) in CH COOH (12 mL) and H SO (4
3
2
4
mL), paraformaldehyde (4.20 g, 58.56 mmol) was added. The reaction mixture was stirred at room
17

ACCEPTED MANUSCRIPT
temperature for 5 h. Then the solution was treated with water, extracted with EA and washed with brine.
The organic layer was dried over Na SO , filtered and evaporated to give the corresponding product 22.
2
4
It was obtained as a white solid in 92% yield. HRMS (ESI): m/z, calculated for C H NO 340.1118
16
18
6
+
(
M + H) , found 340.1113.
5
.1.16 6-Fluoro-1,2,3,4-tetrahydro-isoquinoline-3,7-dicarboxylic acid (23)
The intermediate 22 (5.00 g, 14.74 mmol) was added into HCl (50 mL). The reaction mixture was
stirred at refluxing for 18 h and TLC analysis indicated that the reaction was completed. The mixture
was cooled and filtered to give the white solid 23 in 75% yield. HRMS (ESI): m/z, calculated for
+
C H NO 240.0709 (M + H) , found 240.0706.
11
10
4
5
.1.17 6-Fluoro-1,2,3,4-tetrahydro-isoquinoline-3,7-dicarboxylic acid dimethyl ester (24)
To the solution of 23 (4.22 g, 17.65 mmol) in methanol (50 mL), thionyl chloride (4.20 g, 35.30
o
mmol) was added dropwisely at 0 C. After adding, the mixture was stirred for 30 min maintaining the
o
temperature no more 5 C. Then the solution was stirred at refluxing for 8 h. The reaction mixture was
cooled to room temperature and filtered to give the white solid 24 in 78% yield. HRMS (ESI): m/z,
+
calculated for C H NO 268.0917 (M + H) , found 268.0918.
1
3
14
4
5
.1.18 2-(4-Cyano-3-fluoro-phenyl)-8-fluoro-1-oxo-3-thioxo-1,2,3,5,10,10a-hexahydro-imidazo[1,5-b]
isoquinoline-7-carboxylic acid methyl ester (25a)
To the solution of 9 (0.18 g, 1 mmol) in DMF (10 mL), 24 (0.28 g, 1 mmol) and triethylamine (0.5
o
g, 0.80 mmol) were added. The mixture was stirred at 30 C for 1 h. Then the solution was added into
ice water (50 mL), extracted with EA and washed with brine. The organic layers were dried over
Na SO and concentrated under reduced pressure. The crude residue was purified by silica gel column
2
4
chromatography (PE/EA 6:1) to give the corresponding product 25a. It was obtained as a yellow
o
1
powder in 70% yield. m.p. 228-230 C; H-NMR (300 MHz, DMSO-d ): δ 8.14 (t, J = 5.4 Hz, 1H,
6
Ar-H), 7.95 (d, J = 5.4 Hz, 1H, Ar-H), 7.71 (d, J = 7.8 Hz, 1H, Ar-H), 7.56 (d, J = 8.1 Hz, 1H, Ar-H),
7
.41 (d, J = 8.1 Hz, 1H, Ar-H), 5.41 (m, 1H, -CH-), 4.78 (m, 2H, -CH -), 3.87 (s, 3H, -CH ), 3.37 (q, J
2 3
13
=
6.0 Hz, 2H, -CH -) ppm; C-NMR (75 MHz, DMSO-d ): δ 172.7, 171.4, 165.9, 160.8, 158.5, 142.0,
2 6
1
41.2, 134.0, 130.5, 129.6, 121.3, 115.6, 114.4, 110.8, 108.1, 108.0, 72.5, 53.8, 51.5, 28.1 ppm; IR
-1
(
KBr): 3482, 2236, 1662, 1444, 1367, 1200, 1140, 890, 558 cm ; HRMS (ESI): m/z, calculated for
+
C H F N O S 414.0747 (M + H) , found 414.0713.
2
0
13
2
3
3
5
.1.19 2-(4-Cyano-3-trifluoromethyl-phenyl)-8-fluoro-1-oxo-3-thioxo-1,2,3,5,10,10a-hexahydro-imid-
azo[1,5-b]isoquinoline-7-carboxylic acid methyl ester (25b)
The synthesis of compound 25b was similar to 25a, just replacing the material 9 into 10. It was
o
1
obtained as a yellow powder in 69% yield. m.p. 220-225 C; H-NMR (300 MHz, DMSO-d ): δ 8.38 (d,
6
J = 8.4 Hz, 1H, Ar-H), 8.22 (s, 1H, Ar-H), 8.01 (d, J = 8.4 Hz, 1H, Ar-H), 7.96 (d, J = 8.4 Hz, 1H,
Ar-H), 7.42 (d, J = 7.5 Hz, 1H, Ar-H), 5.43 (s, 1H, -CH-), 4.78 (m, 2H, -CH -), 3.88 (s, 3H, -CH ), 3.40
2
3
13
(
d, J = 4.8 Hz, 1H, -CH -), 3.34 (s, 1H, -CH -) ppm; C-NMR (75 MHz, DMSO-d ): δ 172.7, 171.4,
2 2 6
1
65.9, 158.5, 142.0, 139.9, 134.7, 132.7, 130.5, 129.6 (q, J = 267.3 Hz, -CF ), 119.6, 118.1, 115.8,
3
1
1
4
15.7, 115.6, 114.4, 105.0, 72.5, 53.8, 51.5, 28.1 ppm; IR (KBr): 3409, 2957, 1777, 1628, 1444, 1310,
-1
+
268, 1147, 854, 558 cm ; HRMS (ESI): m/z, calculated for C H F N O S 464.0616 (M + H) , found
21
13
4
3
3
64.0671.
1
8

ACCEPTED MANUSCRIPT
5
.1.20 2-(4-Cyano-3-fluoro-phenyl)-8-fluoro-1-oxo-3-thioxo-1,2,3,5,10,10a-hexahydro-imidazo[1,5-b]
isoquinoline-7-carboxylic acid (26a)
The compound 25a (0.20 g, 0.48 mmol) was dissolved in THF (5 mL) and 1 N aqueous NaOH (5
mL). The mixture was stirred at room temperature for 50 min. The organic solvent was removed under
reduced pressure. The the solution was treated with 1 N HCl to adjust pH being 6 and filtered to give
o
1
the yellow powder 26a in 77% yield. m.p. 246-248 C; H-NMR (300 MHz, DMSO-d ): δ 13.26 (s, 1H,
6
-
COOH), 8.13 (t, J = 8.1 Hz, 1H, Ar-H), 7.91 (m, 1H, Ar-H), 7.71 (d, J = 8.1 Hz, 1H, Ar-H), 7.56 (d, J
7.8 Hz, 1H, Ar-H), 7.35 (d, J = 8.1 Hz, 1H, Ar-H), 5.37 (d, J = 17.4 Hz, 1H, -CH-), 4.84-4.75 (m, 2H,
CH -), 3.38-3.30 (m, 2H, -CH -) ppm; IR (KBr): 3531, 2234, 1760, 1628, 1449, 1376, 1251, 1139, 896,
=
-
2
2
-1
-
5
58 cm ; HRMS (ESI): m/z, calculated for C H F N O S 398.0416 (M - H) , found 398.0436.
19 11 2 3 3
5
.1.21 2-(4-Cyano-3-trifluoromethyl-phenyl)-8-fluoro-1-oxo-3-thioxo-1,2,3,5,10,10a-hexahydro-imid-
azo [1,5-b]isoquinoline-7-carboxylic acid (26b)
The synthesis of compound 26b was similar to 26a, just replacing the material 25a into 25b. It
o
1
was obtained as a yellow powder in 76% yield. m.p. 160-162 C; H-NMR (300 MHz, DMSO-d ): δ
6
1
3.33 (s, 1H, -COOH), 8.40 (t, J = 8.1 Hz, 1H, Ar-H), 8.06 (m, 1H, Ar-H), 7.96 (d, J = 8.1 Hz, 1H,
Ar-H), 7.56 (d, J = 7.8 Hz, 1H, Ar-H), 7.35 (d, J = 8.1 Hz, 1H, Ar-H), 5.39 (d, J = 17.4 Hz, 1H, -CH-),
1
3
4
1
.84-4.75 (m, 2H, -CH -), 3.41-3.36 (m, 2H, -CH -) ppm; C-NMR (75MHz, DMSO-d ): δ 172.7,
2 2 6
71.1, 165.1, 158.9, 142.9, 139.9, 134.7, 132.7, 130.9, 129.6 (q, J = 265.1 Hz, -CF ), 119.6, 118.1,
3
1
1
4
15.8, 115.7, 115.7, 114.4, 105.0, 72.5, 53.8, 28.1 ppm; IR (KBr): 3413, 2235, 1718, 1627, 1443, 1313,
-1
-
253, 1140, 804, 558 cm ; HRMS (ESI): m/z, calculated for C H F N O S 448.0384 (M - H) , found
20
11
4
3
3
48.0384.
5
.1.22 2-(4-Cyano-3-fluoro-phenyl)-8-fluoro-1-oxo-3-thioxo-1,2,3,5,10,10a-hexahydro-imidazol[1,5-b]
isoquinoline-7-carboxylic acid methylamide (27a)
To the solution of 26a (0.1 g, 0.25 mmol) in CH Cl (15 mL), 4-(4,6-dimethoxy-1,3,5-triazin-
2
2
o
2
-yl)-4-methylmpholinium chlide, DMTMM (0.04 g, 0.37 mmol) was added at 0 C, followed by
stirring for 0.5 h. Triethylamine (0.13 g, 1.25 mmol) and methylamine (0.05 g, 0.49 mmol) (33% in
ethanol) were then added into the solution and the mixture was reacted at room temperature for 3 h.
After the reaction was completed, the solvent was removed under reduced pressure. The resulting
slurry was taken up in 1 N HCl and then extracted with EA. The combined organic layer was dried over
Na SO and concentrated under reduced pressure to give the corresponding product 27a. It was
2
4
o
1
obtained as a yellow powder in 78% yield. m.p. 306-309 C; H-NMR (300 MHz, DMSO-d ): δ 8.25 (s,
6
1
H, -NH-), 8.13 (t, J = 8.4 Hz, 1H, Ar-H), 7.69 (m, 2H, Ar-H), 7.55 (d, J = 8.4 Hz, 1H, Ar-H), 7.32 (d,
J = 8.4 Hz, 1H, Ar-H), 5.33 (d, 1H, -CH-), 4.75 (m, 2H, -CH -), 3.33 (s, 2H, -CH -), 2.77 (d, J = 8.4 Hz,
2
2
1
3
3
1
3
H, -CH ) ppm; C-NMR (75 MHz, DMSO-d ): δ 172.7, 171.4, 167.8, 160.8, 156.1, 141.2, 141.1,
3 6
34.0, 129.8, 128.1, 122.1, 121.3, 114.6, 110.8, 108.1, 108.0, 72.5, 53.4, 28.1, 26.7 ppm; IR (KBr):
-1
424, 2244, 1748, 1620, 1450, 1380, 1247, 1168, 819, 512 cm ; HRMS (ESI): m/z, calculated for
+
C H F N O S 413.0811 (M + H) , found 413.0884.
2
0
14
2
4
2
5
.1.23 2-(4-Cyano-3-trifluoromethyl-phenyl)-8-fluoro-1-oxo-3-thioxo-1,2,3,5,10,10a-hexahydro-imid-
azo[1,5-b]isoquinoline-7-carboxylic acid methylamide (27b)
The synthesis of compound 27b was similar to 27a, just replacing the material 26a into 26b. It
1
9

ACCEPTED MANUSCRIPT
o
1
was obtained as a yellow powder in 80% yield. m.p. 329-333 C; H-NMR (300 MHz, DMSO-d ): δ
6
8
7
.38 (d, J = 8.4 Hz, 1H, Ar-H), 8.32 (s, 1H, -NH-), 8.22 (s, 1H, Ar-H), 8.02 (d, J = 8.4 Hz, 1H, Ar-H),
.65 (d, J = 8.4 Hz, 1H, Ar-H), 7.33 (d, J = 7.5 Hz, 1H, Ar-H), 5.39 (d, J = 7.5 Hz, 1H, -CH-), 4.76 (m,
13
J = 7.5 Hz, 2H, -CH -), 3.28 (t, J = 7.5 Hz, 2H, -CH -), 1.12 (t, J = 6.9 Hz, 3H, -CH ) ppm; C-NMR
2
2
3
(
75 MHz, DMSO-d ): δ 172.7, 171.4, 167.8, 156.1, 141.1, 139.9, 134.7, 132.7, 129.8, 128.1 (q, J =
6
2
3
72.0 Hz, -CF ), 122.1, 119.6, 118.1, 115.8, 115.7, 114.6, 105.0, 72.5, 53.4, 28.1, 26.7 ppm; IR (KBr):
3
-1
446, 2963, 1748, 1649, 1487, 1310, 1254, 1140, 856, 561 cm ; HRMS (ESI): m/z, calculated for
+
C H F N O S 463.0854 (M + H) , found 463.0759.
2
1
14
4
4
2
5
.1.24 2-Amino-3-(3,5-dichloro-4-hydroxyphenyl)propanoic acid (29a)
To the solution of tyrosine 28 (5.00 g, 27.6 mmol) in methanol (100 mL), chlorine gas was added
o
at 0 C. The reaction mixture was stirred at room temperature overnight. The solvent was removed
under reduced pressure. The remainder was treated with water and filtered. The filtrate was adjusted pH
being 5 with ammonia water to generate precipitate. Then the precipitate was filtered and washed with
o
1
water to give brown solid 29a in 45% yield. m.p. > 250 C; H-NMR (300 MHz, DMSO-D O): δ 7.17
2
(
s, 2H, Ar-H), 4.30 (m, 1H, -CH-), 3.10 (m, 2H, -CH -).
2
5
.1.25 2-Amino-3-(3,5-dibromo-4-hydroxyphenyl)propanoic acid (29b)
To the solution of tyrosine 28 (10.00 g, 55.2 mmol) in ice acetic acid (100 mL), the solution of
o
bromine (17.3 g, 0.11 mol) in ice acetic acid (30 mL) was dropped at 0 C. The reaction mixture was
stirred at room temperature for 4 h. The solvent was removed under reduced pressure. The crude was
o
1
recrystallized with acetone:PE = 1:1 to give white solid 29b in 67% yield. m.p. > 250 C; H-NMR
(
-
300 MHz, DMSO-D O): δ 9.92 (s, 1H, -COOH), 7.44 (s, 2H, Ar-H), 4.22 (m, 1H, -CH-), 3.00 (m, 2H,
2
CH -).
2
5
.1.26 2-Amino-3-(3,5-diiodo-4-hydroxyphenyl)propanoic acid (29c)
To the solution of tyrosine 28 (2.00 g, 11.0 mmol) in ice acetic acid (14 mL) and HCl (8 mL),
o
iodo (2.8 g, 11.0 mmol) was added. Then 30% H O was dropped into the solution at 65 C. The
2
2
o
mixture was stirred for 30 min maintaining the temperature. The solution was cooled to 15 C and
treated with water (100 mL). The mixture was adjusted pH being 5 with ammonia water to generate
precipitate. Then the precipitate was filtered and washed with water to give brown solid 29c in 80%
o
1
yield. m.p. > 250 C; H-NMR (300 MHz, DMSO-D O): δ 7.46 (s, 2H, Ar-H), 4.10 (t, 1H, -CH-), 2.93
2
(
m, 2H, -CH -).
2
5
.1.27 Typical procedure for the preparation of (30a-30c)
To the solution of 29a-29c (3.60 mmol) in CF COOH (20 mL), 33% HBr in CH COOH was
3
3
dropped. Then paraformaldehyde (0.22 g, 7.34 mmol) was added into the solution. The reaction was
o
stirred at 55 C for 72 h and the TLC indicated that the reaction was completed. The mixture was
o
cooled to 0 C to generate precipitate. Then the precipitate was filtered and washed with EA. The crude
was recrystallized with methanol to give corresponding product 30a-30c. And 30a-30c were ready for
the next step without the further purification.
5
.1.28 Typical procedure for the preparation of (31a-31c)
To the solution of 30a-30c (16.99 mmol) in methanol (150 mL), thionyl chloride (50.97 mmol)
2
0

ACCEPTED MANUSCRIPT
o
was added dropwisely at 0 C. After adding, the mixture was stirred for 30 min maintaining the
o
temperature no more 5 C. Then the solution was stirred at refluxing for 14 h and the TLC indicated
that the reaction was completed. The reaction mixture was cooled to room temperature and filtered to
give corresponding product 31a-31c. And 31a-31c were ready for the next step without the further
purification.
5
.1.29 Typical procedure for the preparation of (32a-32c)
To the solution of compound 10 (1.00 mmol) and TEA (1.00 mmol) in DMF (10 mL), 31a-31c
(
1.00 mmol) was added. The reaction mixture was stirred at room temperature for 1 h and the TLC
indicated that the reaction was completed. The solution was added into ice water and extracted with EA
3 × 20 mL). The combined organic layer was then dried over Na SO and concentrated under reduced
(
2
4
pressure. The crude material was purified by silica gel column chromatography (PE/EA 3:1) to give the
corresponding product.
5
5
.1.30 4-(6,8-Dichloro-7-hydroxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H, 3H,
H)-yl)-2-(trifluoromethyl)benzonitrile (32a)
o
1
It was obtained as a yellow solid in 44.1% yield. m.p. 260-262 C; H-NMR (300 MHz, DMSO-d ):
6
δ 10.27 (s, 1H, -OH), 8.39 (d, J = 8.28 Hz, 1H, Ar-H), 8.23 (s, 1H, Ar-H), 8.02 (d, J = 8.28 Hz, 1H,
Ar-H), 7.48 (s, 1H, Ar-H), 5.35 (s, 1H, -CH-), 4.76 (d, J = 18.18 Hz, 1H, -CHH-), 4.55 (d, J = 18.18 Hz,
1
3
1
1
2
H, -CHH-), 3.32 (s, 2H, -CH -); C-NMR (75 MHz, DMSO-d ): δ 178.6, 171.7, 147.9, 137.8, 136.1,
2 6
33.7, 131.3, 130.8, 128.6 (q, J = 270.2 Hz, -CF ), 127.8, 124.3, 120.8, 120.2, 114.9, 108.4, 57.0, 44.2,
3
-1
+
8.4 ppm; IR (KBr): 3414, 3127, 1762, 1503, 1400, 1314 cm ; MS-EI (m/z): 214, 471 [M ]; HRMS
+
(
ESI): calculated for C H Cl F N O S 471.9896 (M + H) , found 471.9898.
1
9
10
2
3
3
2
5
5
.1.31 4-(6,8-Dibromo-7-hydroxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H, 3H,
H)-yl)-2-(trifluoromethyl)benzonitrile (32b)
o
1
It was obtained as a yellow solid in 39.2% yield. m.p. 242-245 C; H-NMR (300 MHz,
DMSO-d ): δ 10.03 (s, 1H, -OH), 8.39 (d, J = 8.22 Hz, 1H, Ar-H), 8.23 (s, 1H, Ar-H), 8.03 (d, J = 8.22
6
Hz, 1H, Ar-H), 7.64 (s, 1H, Ar-H), 5.28 (d, J = 18.18 Hz, 1H, -CH-), 4.76 (d, J = 17.01 Hz, 1H, -CHH-),
1
3
4
1
1
.48 (d, J = 17.01 Hz, 1H, -CHH-), 3.28 (s, 2H, -CH -); C-NMR (75 MHz, DMSO-d ): δ 178.5, 171.7,
2 6
49.9, 137.8, 136.2, 133.6, 130.7, 127.8 (q, J = 264.8 Hz, -CF ), 126.1, 124.0, 120.5, 115.1, 112.1,
3
-1
08.5, 57.1, 47.2, 28.3, 14.0 ppm; IR (KBr): 3408, 3107, 1760, 1502, 1313 cm ; MS-EI (m/z): 57, 89,
+
+
1
16, 170, 304, 558, 560 [M ]; HRMS (ESI): m/z, calculated for C H Br F N O S 559.8885 (M + H) ,
19
10
2
3
3
2
found 559.8882.
5
5
.1.32 4-(6,8-Diiodo-7-hydroxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H, 3H,
H)-yl)-2-(trifluoromethyl)benzonitrile (32c)
It was obtained as a yellow solid in 36.1% yield. m.p. 239-242 C; H-NMR (300 MHz,
o
1
DMSO-d ): δ 9.66 (s, 1H, -OH), 8.39 (d, J = 8.28 Hz, 1H, Ar-H), 8.22 (s, 1H, Ar-H), 8.03 (d, J = 8.28
6
Hz, 1H, Ar-H), 7.80 (s, 1H, Ar-H), 5.22 (d, 1H, -CH-), 4.74 (d, J = 17.10 Hz, 1H, -CHH-), 4.40 (d, J =
1
3
1
1
5
3
7.10 Hz, 1H, -CHH-), 3.23 (d, J = 8.31 Hz, 2H, -CH -); C-NMR (75 MHz, DMSO-d ): δ 178.6,
2 6
71.7, 154.5, 139.0, 137.8, 136.2, 134.2, 133.8, 127.1 (q, J = 263.4 Hz, -CF ), 114.8, 91.2, 85.2, 57.2,
3
-
1
2.6, 27.8 ppm; IR (KBr): 3127, 1735, 1477, 1400, 1316 cm ; MS-EI (m/z): 89, 116, 144, 170, 272,
+
+
98, 529, 655 [M ]; HRMS (ESI): calculated for C H F I N O S 655.8608 (M + H) , found
19
10 3 2
3
2
2
1

ACCEPTED MANUSCRIPT
6
55.8603.
5
.1.33
2-Tert-butyl-3-methyl-6,8-dibromo-7-hydroxy-3,4-dihydroisoquinoline-2,3(1H)-dicaroxylate
(33a)
To the solution of intermediate 31b (3.60 g, 10.0 mmol) and TEA (1.52 g, 15.0 mmol) in
1
,4-dioxane/ H O (180 mL, v:v = 8:1), the di-tert-butyl dicarbonate (2.4 g, 11.0 mmol) was added
2
o
dropwise slowly at 0 C. The mixture was stirred at room temperature for 24 h and TLC analysis
indicated that the reaction was completed. The solvent was removed under reduced pressure. The crude
residue was treated with water and adjusted pH being 1 with 5 N HCl. Then the solution was extracted
wtih EA and washed with water. The organic layer was dried over Na SO and concentrated under
2
4
reduced pressure to give the corresponding product 33a. 33a was ready for the next step without the
further purification.
5
.1.34 Typical procedure for the preparation of (33b-33i)
To the solution of 33a (2.00 g, 4.43 mmol) and K CO (2.52 g, 17.74 mmol) in DMF (25 mL),
2
3
different alkylation reagents RX (17.74 mmol) was added. The reaction mixture was stirred at room
temperature for 1 h and TLC analysis indicated that the reaction was completed. The solution was
treated with water and extracted with EA. The crude material was purified by silica gel column
chromatography (PE/EA 3:1) to give the corresponding product. 33b-33i was ready for the next step
without the further purification.
5
.1.35 Typical procedure for the preparation of (31d and 31e)
To the solution of 33b or 33i (3.26 mmol) in CH Cl (20 mL), CF COOH (48.3 mmol) was added.
2
2
3
The reaction mixture was stirred at room temperature for 30 min and TLC analysis indicated that the
reaction was completed. The solution was adjusted pH being 7 with saturated aqueous sodium
bicarbonate. The organic layer was washed with water, dried over Na SO and concentrated under
2
4
reduced pressure to give the corresponding product 31d and 31e. 31d and 31e were ready for the next
step without the further purification.
5
5
.1.36 4-(6,8-Dibromo-7-methoxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H, 3H,
H)-yl)-2-(trifluoromethyl)benzonitrile (32d)
The synthesis of 32d was similar to 32a. It was obtained as yellow powder in 30% yield. m.p.
o
1
1
8
1
2
1
3
62-165 C; H-NMR (300 MHz, DMSO-d ): δ 8.39 (d, J = 8.22 Hz, 1H, Ar-H), 8.23 (s, 1H, Ar-H),
6
.02 (d, J = 8.22 Hz, 1H, Ar-H), 7.79 (s, 1H, Ar-H), 5.30 (d, J = 18.09 Hz, 1H, -CH-), 4.76 (m, J =
7.91 Hz, 1H, -CHH-), 4.48 (d, J = 17.91 Hz, 1H, -CHH-), 3.82 (s, 3H, -OCH ), 3.35 (d, J = 5.64 Hz,
3
1
3
H, -CH -); C-NMR (75 MHz, DMSO-d ): δ 178.55, 171.60, 152.33, 137.80, 136.19, 133.72, 131.51,
2
6
27.68 (q, J = 264.0 Hz, -CF ), 117.69, 115.47, 108.46, 60.40, 56.74, 46.91, 28.50; IR (KBr): 3422.75,
3
-1
+
127.97, 1763.71, 1638.01, 1400.37 cm ; MS-EI (m/z): 170, 318, 575 [M ]; HRMS (ESI): calculated
+
for C H Br F N O S 573.9042 (M + H) , found 573.9028.
2
0
12
2
3
3
2
5
.1.37 4-(6,8-Dibromo-7-(4-ethoxy-4-oxobutoxy)-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoqui-
nolin-2(1H, 3H, 5H)-yl)-2-(trifluoromethyl)benzonitrile (32e)
The synthesis of 32e was similar to 32a.It was obtained as yellow powder in 52% yield. m.p.
o
1
1
58-160 C; H-NMR (300 MHz, DMSO-d ): δ 8.41 (d, 1H, Ar-H), 8.23 (s, 1H, Ar-H), 8.04 (d, 1H,
6
2
2

ACCEPTED MANUSCRIPT
Ar-H), 7.80 (s, 1H, Ar-H), 5.32 (d, 2H, -CH -), 4.50 (d, 2H, -CH -), 4.10 (q, 4H, -O-CH -, -COOCH -),
2
2
2
2
1
3
3
1
.98 (t, 1H, -CH-), 2.08 (m, 4H, -CH CH -), 1.20 (t, 3H, -CH ); C-NMR (75 MHz, DMSO-d ) : δ
2 2 3 6
78.3, 172.5, 157.3, 134.0, 136.1, 133.7, 131.9, 130.1, 127.7 (q, J = 272.5 Hz, -CF ), 122.9, 114.0,
3
-1
1
11.9, 66.6, 59.8, 57.9, 30.1, 28.5, 24.1, 14.1 ppm; IR (KBr): 3127.66, 2360.55, 2342.23, 1400.41 cm ;
+
HRMS (ESI): calculated for C H Br F N O S 673.9566 (M + H) , found 673.9554.
2
5
20
2
3
3
4
5
.1.38 Typical procedure for the preparation of (34a-34i)
A solution of 33a-33i (10 mmol) and TEA (10 mmol) in methanol (100 mL) was hydrogenated in
o
the presence of 10% Pd/C and stirred at 55 C for 12 h. The reaction mixture was filtered to remove
Pd/C and the solvent was removed under reduced pressure. The crude residue was treated with CH Cl2
2
and washed with water. The organic layer was dried over Na SO and concentrated under reduced
2
4
pressure to give the corresponding product. 34a-34i were ready for the next step without the further
purification.
5
.1.39 Typical procedure for the preparation of (35a-35i)
To the solution of 34a-34i (3.26 mmol) in CH Cl (20 mL), CF COOH (48.3 mmol) was added.
2
2
3
The reaction mixture was stirred at room temperature for 30 min and TLC analysis indicated that the
reaction was completed. The solution was adjusted pH being 7 with saturated aqueous sodium
bicarbonate. The organic layer was washed with water, dried over Na SO and concentrated under
2
4
reduced pressure to give the corresponding product. 35a-35i were ready for the next step without the
further purification.
5
.1.40 Typical procedure for the preparation of (36a-36i)
To the solution of compound 10 (1.00 mmol) and TEA (1.00 mmol) in DMF (10 mL), 35a-35i
(
1.00 mmol) was added. The reaction mixture was stirred at room temperature for 1 h and the TLC
indicated that the reaction was completed. The solution was added into ice water and extracted with EA
3 x 20 mL). The combined organic layer was then dried over Na SO and concentrated under reduced
(
2
4
pressure. The crude material was purified by silica gel column chromatography (PE/EA 3:1) to give the
corresponding product.
5
.1.41 4-(7-Hydroxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-2-(tri-
fluoromethyl)benzonitrile (36a)
o
1
It was obtained as yellow powder in 39% yield. m.p. 234-237 C; H-NMR (300 MHz, DMSO-d ):
6
δ 9.48 (s, 1H, -OH), 8.39 (d, 1H, Ar-H), 8.36 (s, 1H, Ar-H), 8.22 (d, 1H, Ar-H), 7.13 (d, 1H, Ar-H),
13
6
.73-6.68 (m, 2H, Ar-H), 5.28 (d, 1H, -CH-), 4.74-4.58 (m, 2H, -CH -), 3.17 (d, 2H, -CH -); C-NMR
2 2
(
75 MHz, DMSO-d ): δ 175.7, 169.6, 153.7, 135.5, 133.6, 131.2, 129.1, 128.3 (q, J = 265.7 Hz, -CF ),
6 3
-1
1
25.2, 118.5, 112.3, 110.2, 105.9, 55.6, 43.2, 26.1; IR (KBr): 3397, 3127, 1742, 1503, 1400 cm ;
+
MS-EI (m/z): 66, 73, 84, 120, 146, 403 [M ]; HRMS (ESI): m/z, calculated for C H F N O S
19
13
3
3
2
+
4
04.0675 (M + H) , found 404.0676.
5
.1.42 4-(7-Methoxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-2-(tri-
fluoromethyl)benzonitrile (36b)
o
1
It was obtained as yellow powder in 66% yield. m.p. 234-236 C; H-NMR (300 MHz, DMSO-d ):
6
δ 8.38 (d, 1H, Ar-H), 8.23 (s, 1H, Ar-H), 8.02 (d, 1H, Ar-H), 7.25 (d, 1H, Ar-H), 6.99 (s, 1H, Ar-H),
13
6
.88 (d, 1H, Ar-H), 5.38 (d, 2H, -CH -), 4.71 (m, 2H, -CH -), 3.4-3.2 (m, 4H, -CH , -CH-); C-NMR
2 2 3
2
3

ACCEPTED MANUSCRIPT
(
75 MHz, DMSO-d ): δ 178.4, 172.2, 158.1, 138.2, 136.2, 133.8, 130.2, 122.8 (q, J = 265.3 Hz, -CF ),
6
3
114.9, 113.5, 111.5, 57.9, 55.5, 46.8, 28.5 ppm; IR (KBr): 3126.93, 1745.66, 1502.63, 1400.34,
-1
+
1
135.62 cm ; MS-EI (m/z): 84, 134, 160, 417 [M ]; HRMS (ESI): m/z, calculated for C H F N O S
20 14 3 3 2
+
4
18.0832 (M + H) , found 418.0827.
5
.1.43 4-(7-Ethoxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-2-(trifl-
uoromethyl)benzonitrile (36c)
o
1
It was obtained as yellow powder in 43% yield. m.p. 230-233 C; H-NMR (300 MHz, DMSO-d ):
6
δ 8.38 (d, 1H, Ar-H), 8.24 (s, 1H, Ar-H), 8.02 (d, 1H, Ar-H), 7.24 (d, 1H, Ar-H), 6.96 (s, 1H, Ar-H),
6
1
1
.86 (d, 1H, Ar-H), 5.38 (d, 1H, -CH-), 4.77 (m, 2H, -CH -), 4.06 (q, 2H, -CH -), 3.23 (q, 2H, -CH -),
2 2 2
1
3
.35 (t, 3H, -CH3); C-NMR (75 MHz, DMSO-d ): δ 178.3, 172.1, 157.4, 138.0, 136.1, 133.7, 131.8,
6
30.1, 127.7 (q, J = 268.1 Hz, -CF ), 122.6, 115.0, 113.9, 111.8, 63.1, 57.9, 45.7, 28.5, 14.6 ppm; IR
3
-1
+
(
KBr): 3133.37, 1763.04, 1507.09, 1400.14, 1127.84 cm ; MS-EI (m/z): 91, 120, 146, 174, 431 [M ];
+
HRMS (ESI): m/z, calculated for C H F N O S 432.0988 (M + H) ; found 432.0992.
2
1
16
3
3
2
5
.1.44 4-(7-Propoxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-2-(tri-
fluoromethyl)benzonitrile (36d)
o
1
It was obtained as yellow powder in 34% yield. m.p. 202-205 C; H-NMR (300 MHz, DMSO-d ):
6
δ 8.39 (d, 1H, Ar-H), 8.24 (s, 1H, Ar-H), 8.02 (d, 1H, Ar-H), 7.23 (d, 1H, Ar-H), 6.96 (s, 1H, Ar-H),
6
.84 (d, 1H, Ar-H), 5.34 (d, 2H, -CH -), 4.65 (d, 2H, -CH -), 3.95 (m, 3H, -CH-, CH -), 1.72 (q, 2H,
2 2 2
1
3
-
CH -), 0.98 (t, 3H, -CH ); C-NMR (75 MHz, DMSO-d ): δ 178.4, 172.2, 157.8, 138.1, 136.2, 133.8,
2
3
6
1
30.2, 127.8 (q, J = 272.6 Hz, -CF ), 122.7, 115.1, 113.9, 111.8, 69.1, 57.8, 45.8, 28.5, 21.8, 10.4 ppm;
3
-1
+
IR (KBr): 3127.72, 1766.88, 1507.56, 1400.34 cm ; MS-EI (m/z): 91, 120, 146, 188, 445 [M ]; HRMS
+
(
ESI): m/z, calculated for C H F N O S 446.1145 (M + H) , found 446.1152.
2
2
18
3
3
2
5
.1.45 4-(7-Isopropoxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-2-
(trifluoromethyl)benzonitrile (36e)
o
1
It was obtained as yellow powder in 38% yield. m.p. 220-223 C; H-NMR (300 MHz, DMSO-d ):
6
δ 8.36 (d, 1H, Ar-H), 8.23 (s, 1H, Ar-H), 8.02 (d, 1H, Ar-H), 7.24 (d, 1H, Ar-H), 6.96 (s, 1H, Ar-H),
6
6
1
.83 (d, 1H, Ar-H), 5.37 (d, 1H, -CH-), 4.80-4.58 (m, 3H, -OCH-, -CH -), 3.22 (d, 2H, -CH -), 1.28 (d,
2 2
13
H, -CH(CH ) ); C-NMR (75 MHz, DMSO-d ): δ 178.2, 172.1, 156.3, 138.0, 136.1, 133.7, 131.9,
3
2
6
30.8, 127.6 (q, J = 268.9 Hz, -CF ), 124.0, 115.0, 113.2, 108.3, 69.2, 57.9, 45.7, 28.5, 27.8 ppm; IR
3
-
1
+
(
KBr): 3127.75, 1744.00, 1502.36, 1400.32, 1312.74 cm ; MS-EI (m/z): 91, 120, 146, 445 [M ];
+
HRMS (ESI): m/z, calculated for C H F N O S 446.1145 (M + H) , found 446.115.
2
2
18
3
3
2
5
.1.46 4-(7-Butoxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-2-(trifl-
uoromethyl)benzonitrile (36f)
o
1
It was obtained as yellow powder in 44% yield. m.p. 203-205 C; H-NMR (300 MHz, DMSO-d ):
6
δ 8.39 (d, 1H, Ar-H), 8.23 (s, 1H, Ar-H), 8.02 (d, 1H, Ar-H), 7.24 (d, 1H, Ar-H), 6.97 (s, 1H, Ar-H),
6
.85 (d, 1H, Ar-H), 5.37 (d,1H, -CH-), 4.73-4.68 (m, 2H, -CH -), 3.99 (t, 2H, -OCH -), 3.22 (d, 2H,
2 2
1
3
-
CH -)1.72 (m, 2H, -CH -), 1.47 (m, 2H, -CH -), 0.95 (t, 3H, -CH ); C-NMR (75 MHz, DMSO-d ): δ
2 2 2 3 6
1
1
1
78.2, 172.0, 157.6, 138.0, 136.1, 133.7, 131.8, 130.0, 127.7, 124.0, 122.6, 120.4, 115.2, 114.0, 111.91,
08.4, 67.7, 58.0, 45.8, 30.6, 28.6, 18.7, 13.8 ppm; IR (KBr): 3127.04, 1744.74, 1400.25, 1311.92,
-1
+
173.64 cm ; MS-EI (m/z): 91, 120, 146, 202, 403, 459 [M ]; HRMS (ESI): m/z, calculated for
2
4

ACCEPTED MANUSCRIPT
+
C H F N O S 460.1301 (M + H) , found 460.1308.
2
3
20
3
3
2
5
.1.47 4-(7-Isobutoxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-2-(t-
rifluoromethyl)benzonitrile (36g)
o
1
It was obtained as yellow powder in 38% yield. m.p. 208-210 C; H-NMR (300 MHz, DMSO-d ):
6
δ 8.38 (d, 1H, Ar-H), 8.23 (s, 1H, Ar-H), 8.03 (d, 1H, Ar-H), 7.24 (d, 1H, Ar-H), 6.99 (s, 1H, Ar-H),
6
.89 (d, 1H, Ar-H), 5.37 (d, 1H, -CH-), 4.73-4.62 (m, 2H, -CH -), 3.75 (d, 2H, -OCH -), 3.22 (d, 2H,
2 2
1
3
-
CH -), 2.02 (m, 1H, -CH-), 0.98 (d, 6H, -CH(CH ) ); C-NMR (75 MHz, DMSO-d ): δ 178.1, 172.3,
2
3
2
6
1
5
57.8, 138.1, 136.5, 133.6, 131.8, 130.1, 126.5 (q, J = 264.0 Hz, -CF ), 122.5, 114.8, 113.8, 111.9, 73.8,
3
-1
7.9, 45.6, 28.6, 27.7, 19.1 ppm; IR (KBr): 3127.80, 1750.31, 1508.03, 1400.09, 1312.76 cm ; MS-EI
+
(
m/z): 57, 91, 120, 146, 403, 459 [M ]; HRMS (ESI): m/z, calculated for C H F N O S 460.1301 (M
23
20
3
3
2
+
+
H) , found 460.1304.
5
2
.1.48 4-(7-Cyclohexyloxy-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-
-(trifluoromethyl)benzonitrile (36h)
o
1
It was obtained as yellow powder in 53% yield. m.p. 218-220 C; H-NMR (300 MHz, DMSO-d ):
6
δ 8.38 (d, 1H, Ar-H), 8.24 (s, 1H, Ar-H), 8.02 (d, 1H, Ar-H), 7.23 (d, 1H, Ar-H), 6.99 (s, 1H, Ar-H),
6
2
1
2
8
.86 (d, 1H, Ar-H), 5.37 (d, 1H, -CH-), 4.77 (m, 2H, -CH -), 4.33 (s, 1H, -CH-), 3.21 (d, 2H, -CH -),
2 2
1
3
.01-1.15 (m, 10H, -(CH -) ); C-NMR (75 MHz, DMSO-d ): δ 178.2, 172.2, 156.4, 137.9, 135.9,
2
5
6
33.6, 131.8, 130.1, 127.8 (q, J = 271.4 Hz, -CF ), 122.8, 115.0, 113.4, 74.5, 57.9, 46.9, 31.3, 28.6,
3
-1
4.8, 23.1 ppm; IR (KBr): 3127.55, 2939.85, 1753.82, 1503.09, 1400.35, 1312.91 cm ; MS-EI (m/z):
+
5, 91, 146, 341, 356, 403, 554 [M ]; HRMS (ESI): m/z, calculated for C H F N O S 486.1458 (M +
25
22
3
3
2
+
H) , found 486.1463.
5
.1.49 Ethyl 4-((2-(4-cyano-3-(trifluoromethyl)phenyl)-1-oxo-3-thioxo-1,2,3,5,10,10a-hexahydroimida-
zo[1,5-b]isoquinolin-7-yl)oxy)butanoate (36i)
o
1
It was obtained as yellow powder in 52% yield. m.p. 158-160 C; H-NMR (300 MHz, DMSO-d ):
6
δ 8.39 (d, 1H, Ar-H), 8.19 (s, 1H, Ar-H), 8.02 (d, 1H, Ar-H), 7.23 (d, 1H, Ar-H), 6.99 (s, 1H, Ar-H),
6
1
1
5
.86 (d, 1H, Ar-H), 5.32 (d, 2H, -CH -), 4.50 (d, 2H, -CH -), 4.10 (q, 4H, -O-CH -, -COOCH -), 3.98 (t,
2 2 2 2
13
H, -CH-), 2.08 (m, 4H, -CH CH -), 1.20 (t, 3H, -CH ); C-NMR (75 MHz, DMSO-d ): δ 178.3,
2
2
3
6
72.5, 157.3, 134.0, 136.1, 133.7, 131.9, 130.1, 127.7 (q, J = 263.1 Hz, -CF ), 122.9, 114.0, 111.9, 66.6,
3
-1
9.8, 57.9, 30.1, 28.5, 24.1, 14.1 ppm; IR (KBr): 3127.66, 2360.55, 2342.23, 1400.41 cm ; MS-EI
+
+
(
m/z): 115, 146, 472, 517 [M ]; HRMS (ESI): m/z, calculated for C H F N O S 518.1356 (M + H) ,
25 22 3 3 4
found 518.1358.
5
.1.50 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (11)
To the solution of phenylalanine 37 (16.5 g, 0.1 mol) in HCl (150 ml), 33% formaldehyde solution
70 ml, 1 mol) was added. The reaction mixture was stirred at refluxing for 6 h and TLC analysis
indicated that the reaction was completed. The mixture was filtered to give white solid 11 in 33% yield.
(
o
1
m.p. 245-247 C; H-NMR (300 MHz, DMOS-d ): δ 10.07 (s, 1H, -COOH), 7.27 (s, 4H, Ar-H),
6
4
.43-4.40 (m, 2H, -CH -), 3.81 (s, 1H, -CH-), 3. 45-3.10 (m, 2H, -CH -).
2
2
5
.1.51 Typical procedure for the preparation of (38a and 38b)
o
The compound 11 (2.5 g, 14.1 mmol) was dissolved in H SO (12.5 mL) at -10 C. The solution of
2
4
2
5

ACCEPTED MANUSCRIPT
KNO (1.48 g, 14.7 mmol) in H SO (5.00 mL) was added dropwise slowly into the mixture. The
3
2
4
reaction mixture was stirred at room temperature for 1 h and TLC analysis indicated that the reaction
was completed. The solution was treated with ice water (100 mL) and adjusted pH being 7 with
ammonia water to generate precipitation. Then the mixture was filtered to give yellow solid. 38a and
3
8b were ready for next step without the further purification.
5
.1.52 Typical procedure for the preparation of (39a and 39b)
To the solution of 38a and 38b (10.00 mmol) in methanol (50 mL), thionyl chloride (20.00 mmol)
o
was added dropwisely at 0 C. After adding, the mixture was stirred for 30 min maintaining the
o
temperature no more 5 C. Then the solution was stirred at refluxing for 14 h. The reaction mixture was
cooled to room temperature and filtered to give the responding compound. 39a and 39b were ready for
next step without the further purification.
5
.1.53 Typical procedure for the preparation of (40a and 40b)
To the solution of intermediate 39a and 39b (10.0 mmol) and TEA (1.52 g, 15.0 mmol) in
1
,4-dioxane/H O (180 mL, v:v = 8:1), the di-tert-butyl dicarbonate (2.4 g, 11.0 mmol) was added
2
o
dropwise slowly at 0 C. The mixture was stirred at room temperature for 24 h and TLC analysis
indicated that the reaction was completed. The solvent was removed under reduced pressure. The crude
residue was treated with water and adjusted pH being 1 with 5 N HCl. Then the solution was extracted
wtih EA and washed with water. The organic layer was dried over Na SO and concentrated under
2
4
reduced pressure to give the corresponding product. 40a and 40b were ready for the next step without
the further purification.
5
.1.54 Typical procedure for the preparation of (40c and 40d)
A solution of 40a and 40b (10 mmol) in methanol (100 mL) was hydrogenated in the presence of
0% Pd/C and stirred at room temperature for 12 h. The reaction mixture was filtered to remove Pd/C
1
and the solvent was removed under reduced pressure to give the corresponding product. 40c and 40d
were ready for the next step without the further purification.
5
.1.55 Typical procedure for the preparation of (39c and 39d)
To the solution of 40c and 40d (3.26 mmol) in CH Cl (20 mL), CF COOH (48.3 mmol) was
2
2
3
added. The reaction mixture was stirred at room temperature for 30 min and TLC analysis indicated
that the reaction was completed. The solution was adjusted pH being 7 with saturated aqueous sodium
bicarbonate. The organic layer was washed with water, dried over Na SO and concentrated under
2
4
reduced pressure to give the corresponding product. 39c and 39d were ready for the next step without
the further purification.
5
.1.56 Typical procedure for the preparation of (41a-41d)
To the solution of compound 10 (1.00 mmol) and TEA (1.00 mmol) in DMF (10 mL), 39a-39d
(
1.00 mmol) was added. The reaction mixture was stirred at room temperature for 1 h and the TLC
indicated that the reaction was completed. The solution was added into ice water and extracted with EA
3 × 20 mL). The combined organic layer was then dried over Na SO and concentrated under reduced
(
2
4
pressure. The crude material was purified by silica gel column chromatography (PE/EA 3:1) to give the
corresponding product.
2
6

ACCEPTED MANUSCRIPT
5
.1.57 4-(8-Nitro-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-2-(trifluo-
romethyl)benzonitrile (41a)
o
1
It was obtained as a yellow powder in 34% yield. m.p. 171-173 C; H-NMR (300 MHz,
DMSO-d ): δ 8.40 (d, 1H, Ar-H), 8.32 (s, 1H, Ar-H), 8.25 (s, 1H, Ar-H), 8.15 (d, 1H, Ar-H), 8.04 (d,
6
13
1
H, Ar-H), 7.69 (d, 1H, Ar-H), 5.55 (d, 1H, -CH-), 4.81 (d, 2H, -CH -), 3.45 (m, 2H, -CH -); C-NMR
2 2
(
75 MHz, DMSO-d ): δ 178.2, 172.0, 137.6, 136.0, 133.5, 132.5, 129.1, 128.2 (q, J = 266.3 Hz, -CF ),
6 3
1
27.5, 123.8, 121.9, 114.9, 108.4, 57.3, 45.2, 29.1 ppm; IR (KBr): 3448, 3133, 2371, 1637, 1400, 1313
-1
+
cm ; MS-EI (m/z): 129, 175, 432 [M ]; HRMS (ESI): calculated for C H F N O S 433.0577 (M +
19
11
3
4
3
+
H) , found 433.0583.
5
.1.58 4-(7-Nitro-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-2-(trifluo-
romethyl)benzonitrile (41b)
o
1
It was obtained as a yellow powder in 60% yield. m.p. 219-221 C; H-NMR (300 MHz,
DMSO-d ): δ 8.39 (d, 2H, Ar-H), 8.23 (s, 1H, Ar-H), 8.12 (d, 1H, Ar-H), 8.01 (d, 1H, Ar-H), 7.65 (d,
6
13
1
H, Ar-H), 5.57 (d, 1H, -CH-), 4.86 (q, 2H, -CH -), 3.48-3.41 (m, 2H, -CH -); C-NMR (75 MHz,
2 2
DMSO-d ): δ 178.6, 171.8, 146.4, 139.4, 137.9, 136.2, 133.7, 132.9, 130.6, 127.6 (q, J = 267.1 Hz,
6
-
CF ), 122.0, 56.9, 45.4, 29.4 ppm; IR (KBr): 3439, 2348, 1614, 1312, 1048, 889; MS-EI (m/z): 129,
3
+
+
1
75, 432 [M ]; HRMS (ESI): calculated for C H F N O S 433.0577 (M + H) , found 433.0583.
19 11 3 4 3
5
.1.59 4-(8-Amino-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-2-(trifl-
uoromethyl)benzonitrile (41c)
o
1
It was obtained as a yellow powder in 31% yield. m.p. 225-227 C; H-NMR (300 MHz,
DMSO-d ): δ 8.39 (d, 1H, Ar-H), 8.24 (s, 1H, Ar-H), 8.02 (d, 1H, Ar-H), 7.38 (d, 1H, Ar-H), 7.10 (s,
6
13
2
H, Ar-H), 5.38 (d, 1H, -CH-), 4.72 (m, 2H, -CH -), 3.76 (s, 2H, -NH -), 3.32 (d, 2H, -CH -); C-NMR
2 2 2
(
75 MHz, DMSO-d ): δ 178.2, 172.0, 137.6, 136.0, 133.5, 132.5, 129.1, 128.2 (q, J = 272.8 Hz, -CF ),
6 3
-1
1
27.5, 123.8, 121.9, 114.9, 108.4, 57.3, 45.2, 29.1 ppm; IR (KBr): 3133, 2371, 1400, 1313 cm ; MS-EI
+
+
(
m/z): 119, 145, 402 [M ]; HRMS (ESI): calculated for C H F N OS 403.0835 (M + H) , found
19 13 3 4
4
03.0573.
5
.1.60 4-(7-Amino-1-oxo-3-thioxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-2-(trifl-
uoromethyl)benzonitrile (41d)
o
1
It was obtained as a yellow powder in 31% yield. m.p. 158-160 C; H-NMR (300 MHz, D O): δ
2
8
.40 (s, 1H, Ar-H), 8.28 (s, 1H, Ar-H), 8.08 (s, 2H, Ar-H), 7.44 (d, 2H, Ar-H), 5.42 (s, 1H, -CH-), 4.80
1
3
(
t, 2H, -CH -), 3.36 (t, 2H, -CH -); C-NMR (75 MHz, DMSO-d ): δ 179.2, 178.1, 172.1, 144.2, 137.6,
2 2 6
1
1
37.1, 135.9, 133.5, 131.2, 129.2 (q, J = 269.2 Hz, -CF ), 127.8, 122.1, 120.1, 115.3, 114.2, 108.4,
3
-1
01.8, 57.6, 45.4, 28.8 ppm; IR (KBr): 3127, 1763, 1526, 1400, 1346, 1312, 1140, 1057 cm ; MS-EI
+
+
(
m/z): 91, 119, 145, 402 [M ]; HRMS (ESI): m/z, calculated for C H F N OS 403.0835 (M + H) ,
19 13 3 4
found 403.0839.
5
.1.61 Typical procedure for the preparation of (42a-42g)
To the solutino of 40d (2.00 mmol) and TEA (4.00 mmol) in CH Cl , different acylation and
2
2
sulfonylation reagents (4.00 mmol) were added dropwise slowly. The reaction mixture was stirred at
room temperature for 3 h and TLC analysis indicated that the reaction was completed. The mixture was
treated with water and extracted with CH Cl . The organic layer was washed with saturated sodium
2
2
27

ACCEPTED MANUSCRIPT
bicarbonate solution, washed with brine and drived over Na SO . The solvent was evaporated to give
2
4
corresponding product.
5
.1.62 Typical procedure for the preparation of (43a-43g)
To the solution of 42a-42g (2 mmol) in CH Cl (20 mL), CF COOH (2 mL) was added. The
2
2
3
reaction mixture was stirred at room temperature overnight and TLC analysis indicated that the reaction
was completed. The solution was adjusted pH being 7 with saturated aqueous sodium bicarbonate. The
organic layer was washed with water, dried over Na SO and concentrated under reduced pressure to
2
4
give the corresponding product.
5
.1.63 Typical procedure for the preparation of (44a-44g)
To the solution of compound 10 (1.00 mmol) and TEA (1.00 mmol) in DMF (10 mL), 43a-43g
(
1.00 mmol) was added. The reaction mixture was stirred at room temperature for 1 h and the TLC
indicated that the reaction was completed. The solution was added into ice water and extracted with EA
3 × 20 mL). The combined organic layer was then dried over Na SO and concentrated under reduced
(
2
4
pressure. The crude material was purified by silica gel column chromatography (PE/EA 3:1) to give the
corresponding product.
5
.1.64 N-(2-(4-cyano-3-(trifluoromethyl)phenyl)-1-oxo-3-thioxo-1,2,3,5,10,10a-hexahydroimidazo[1,5
b]isoquinolin-7-yl)acetamide (44a)
It was obtained as yellow powder in 21% yield. m.p. 288-290 C; H-NMR (300 MHz, DMSO-d ):
-
o
1
6
δ 10.00 (s, 1H, -NH-), 8.37 (d, J = 8.1 Hz, 1H, Ar-H), 8.23 (s, 1H, Ar-H), 8.02 (d, J = 8.1 Hz, 1H,
Ar-H),7.60 (s, 1H, Ar-H), 7.44 (d, J = 8.1 Hz, 1H, Ar-H), 7.26 (d, J = 8.4 Hz, 1H, Ar-H), 5.34 (d, J =
1
7.7 Hz, 1H, -CH-), 4.79-4.71 (m, 2H, -CH -), 3.24 (d, J = 6.9 Hz, 2H, -CH -), 2.06 (s, 3H, -CH ) ppm;
2 2 3
1
3
C-NMR (75 MHz, DMSO-d ): δ 178.2, 172.0, 168.3, 137.9, 136.1, 133.7, 131.2, 127.6 (q, J = 267.3
6
Hz, -CF ), 125.4, 123.9, 120.3, 118.1, 116.6, 108.3, 57.7, 45.6, 28.7, 23.9 ppm; IR (KBr): 3363, 3128,
3
-1
2
238, 1753, 1600, 1401, 1348, 1256, 1172, 1052, 888, 851 cm ; HRMS (ESI): m/z, calculated for
+
C H F N O S 467.0760 (M + Na) , found 467.0769.
2
1
15
3
4
2
5
.1.65 N-(2-(4-cyano-3-(trifluoromethyl)phenyl)-1-oxo-3-thioxo-1,2,3,5,10,10a-hexahydroimidazo[1,5
b]isoquinolin-7-yl)propionamide (44b)
It was obtained as yellow powder in 27% yield. m.p. 243-245 C; H-NMR (300 MHz, DMSO-d ):
-
o
1
6
δ 9.92 (s, 1H, -NH-), 8.37 (d, J = 8.1 Hz, 1H, Ar-H), 8.22 (s, 1H, Ar-H), 8.01 (d, J = 8.1 Hz, 1H, Ar-H),
7
1
.62 (s, 1H, Ar-H), 7.44 (d, J = 7.5 Hz, 1H, Ar-H), 7.26 (d, J = 8.1 Hz, 1H, Ar-H), 5.34 (d, J = 17.7 Hz,
H, -CH-), 4.79-4.65 (m, 2H, -CH -), 3.32-3.20 (m, 2H, -CH -), 2.32 (q, J = 7.5 Hz, 2H, -CH -), 1.09 (t,
2
2
2
1
3
J = 7.5 Hz, 3H, -CH ) ppm; C-NMR (75 MHz, DMSO-d ): δ 178.2, 172.0, 138.1, 137.9, 136.1, 133.7,
3
6
1
4
31.2, 130.8, 129.3, 127.6 (q, J = 266.0 Hz, -CF ), 125.3, 123.9, 120.3, 118.1, 116.6, 115.0, 108.3, 57.7,
3
-
1
5.6, 29.4, 9.5 ppm; IR (KBr): 3125, 2238, 1687, 1400, 1312, 1279, 1137, 1067, 851, 830, 560 cm ;
+
HRMS (ESI): m/z, calculated for C H F N O S 459.1097 (M + H) , found 459.1119.
2
2
17
3
4
2
5
.1.66 N-(2-(4-cyano-3-(trifluoromethyl)phenyl)-1-oxo-3-thioxo-1,2,3,5,10,10a-hexahydroimidazo[1,5
b]isoquinolin-7-yl)butyramide (44c)
It was obtained as yellow powder in 46% yield. m.p. 141-143 C; H-NMR (300 MHz, DMSO-d ):
-
o
1
6
δ 9.93 (s, 1H, -NH-), 8.37 (d, J = 8.1 Hz, 1H, Ar-H), 8.23 (s, 1H, Ar-H), 8.02 (d, J = 8.1 Hz, 1H, Ar-H),
2
8