Structure-activity relationship study of pyrimido[1,2-c][1,3]benzothiazin- 6-imine derivatives for potent anti-HIV agents

Article abstract of DOI:10.1016/j.bmc.2012.08.030

3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) is an antiretroviral agent with submicromolar inhibitory activity against human immunodeficiency virus-1 (HIV-1) and HIV-2 infection. In the current study, the structure-activity relationships of accessory groups at the 3- and 9-positions of pyrimido[1,2-c][1,3]benzothiazin-6-imine were investigated for the development of more potent anti-HIV agents. Several different derivatives containing a 9-aryl group were designed and synthesized using Suzuki-Miyaura cross-coupling and Ullmann coupling reactions. Modification of the m-methoxyphenyl or benzo[d][1,3]dioxol-5-yl group resulted in improved anti-HIV activity. In addition, the 2,4-diazaspiro[5.5]undec-2-ene-fused benzo[e][1,3]thiazine derivatives were designed and tested for their anti-HIV activities. The most potent 9-(benzo[d][1,3]dioxol-5-yl) derivative was two-threefold more effective against several strains of HIV-1 and HIV-2 than the parent compound, PD 404182.

Full text of DOI:10.1016/j.bmc.2012.08.030

Bioorganic & Medicinal Chemistry 20 (2012) 6434–6441
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship study of pyrimido[1,2-c][1,3]benzothiazin-
6-imine derivatives for potent anti-HIV agents
Tsukasa Mizuhara a, Shinya Oishi a, , Hiroaki Ohno a, Kazuya Shimura b, Masao Matsuoka b,
⇑
Nobutaka Fujii a,
⇑
a Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
b Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) is an antiretroviral agent
with submicromolar inhibitory activity against human immunodeficiency virus-1 (HIV-1) and HIV-2
infection. In the current study, the structure–activity relationships of accessory groups at the 3- and 9-
positions of pyrimido[1,2-c][1,3]benzothiazin-6-imine were investigated for the development of more
potent anti-HIV agents. Several different derivatives containing a 9-aryl group were designed and synthe-
sized using Suzuki–Miyaura cross-coupling and Ullmann coupling reactions. Modification of the m-
methoxyphenyl or benzo[d][1,3]dioxol-5-yl group resulted in improved anti-HIV activity. In addition,
the 2,4-diazaspiro[5.5]undec-2-ene-fused benzo[e][1,3]thiazine derivatives were designed and tested
for their anti-HIV activities. The most potent 9-(benzo[d][1,3]dioxol-5-yl) derivative was two–threefold
more effective against several strains of HIV-1 and HIV-2 than the parent compound, PD 404182.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 31 July 2012
Revised 22 August 2012
Accepted 22 August 2012
Available online 30 August 2012
Keywords:
Anti-HIV agents
PD 404182
Pyrimidobenzothiazine
1. Introduction
type 5 (CCR5) antagonist (maraviroc).10,11 CXC chemokine receptor
type 4 (CXCR4) antagonists,12–16 CD4 mimics,17–20 gp41-binding
Highly active antiretroviral therapy, involving the co-adminis-
tration of nucleoside reverse transcriptase inhibitors (NRTI), non-
nucleoside reverse transcriptase inhibitors (NNRTI), and/or prote-
ase inhibitors, is a standard treatment regimen for human immu-
nodeficiency virus (HIV) infections. This regimen suppresses the
replication of HIV and controls disease progression in HIV-infected
patients.1,2 Unfortunately, however, an increasing number of pa-
tients with HIV infection/AIDS have failed to respond to the current
antiretroviral therapeutics because of serious problems including
the emergence of drug-resistant HIV variants3 and drug-related ad-
verse effects.4 With this in mind, there is therefore a continuous
need to develop novel anti-HIV drugs that are more effective
against drug-resistant viruses and produce fewer adverse effects.
Recently, a series of extensive studies led to the development of
a series of novel antiretrovirals with new mechanisms of action
for anti-HIV therapy, including a fusion inhibitor (enfuvirtide),5–7
an integrase inhibitor (raltegravir),8,9 and a CC chemokine receptor
peptides21–23 and small molecules24–26 represent promising alter-
native anti-HIV agents.
3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine
(PD 404182) (1) was previously reported as an antimicrobial agent
that inhibited 3-deoxy-D-manno-octulosonic acid 8-phosphate
synthase27 and phosphopantetheinyl transferase (Fig. 1).28,29 Fol-
lowing a recent random screening program using a multinuclear
activation of a galactosidase indicator (MAGI) assay, compound 1
was identified as a new antiretroviral candidate with a high thera-
peutic index (CC50/EC50 >200). The MAGI assay allows for the
inhibitory activity of an early-stage HIV infection, including inhibi-
tion of the virus attachment and membrane fusion to host cells, to
be effectively evaluated.30 Compound 1 showed a similar antiviral
profile in HIV-1 infection to DS 500031 (adsorption inhibitor) and
enfuvirtide (fusion inhibitor). The virucidal effects of compound 1
against the human hepatitis C virus, HIV, and simian immunodefi-
ciency virus have also been reported.32,33 The mechanism of action
for compound 1, however, has not yet been fully understood.
In our previous structure–activity relationship (SAR) study of
compound 1,34 a number of PD 404182 derivatives were designed
and synthesized according to a series of facile synthetic proce-
dures,35,36 in which the tricyclic heterocycles related to PD
404182 were easily obtained in a few steps from benzaldehydes
via C–H functionalization or aromatic nucleophilic substitution.
Abbreviations: CCR5, CC chemokine receptor type 5; CXCR4, CXC chemokine
receptor type 4; MAGI, Multinuclear activation of a galactosidase indicator; NNRTI,
Non-nucleoside reverse transcriptase inhibitors; NRTI, Nucleoside reverse trans-
criptase inhibitors.
⇑
Corresponding authors. Tel.: +81 75 753 4551; fax: +81 75 753 4570.
E-mail addresses: soishi@pharm.kyoto-u.ac.jp (S. Oishi), nfujii@pharm.kyoto-
u.ac.jp (N. Fujii).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.030

T. Mizuhara et al. / Bioorg. Med. Chem. 20 (2012) 6434–6441
6435
N
N
S
N
S
a or b
N
N
N
9
1,3-thiazin-2-imine core
Br
S
4
Nt-Bu
Ar
c
NR
NH
5, 6 (R = t-Bu)
PD 404182 (1)
EC50 = 0.44 0.08 µM
CC50 > 100 µM
7, 8 (R = H)
Scheme 1. Synthesis of 9-aryl-pyrimido[1,2-c][1,3]benzothiazin-6-imine deriva-
tives. Reagents and conditions: (a) R-B(OH)2 or R-Bpin, Pd(PPh3)4,
PdCl2(dppf)ꢀCH2Cl2, K2CO3, toluene or 1,4-dioxane, EtOH, H2O, reflux, 29%-quant.;
(b) pyrazole or imidazole, CuCl, K2CO3, acetylacetone, NMP, 130 °C, 51–71% (for 6n
or 6o); (c) TFA, MS4Å, CHCl3, (MeOH), reflux, 34–94%.
Figure 1. Structure of PD 404182.
The 6-6-6 fused pyrimido[1,2-c][1,3]benzothiazine scaffold and the
heteroatom arrangement in the 1,3-thiazin-2-imine moiety are
indispensable for the inhibitory activity of compound 1 against
HIV infection (Fig. 1). Optimization studies indicated that the intro-
duction of a hydrophobic group on the benzene ring and the cyclic
amidine substructures effectively improved the antiviral activity
by generating a potentially favorable interaction(s) with the target
molecule(s). The most potent compounds identified were twofold
more potent than PD 404182 and contained a phenyl group at 9-po-
sition of pyrimido[1,2-c][1,3]benzothiazine (compound 2) or a gem-
inal dimethyl group on the pyrimidine moiety (compound 3) (Fig. 2).
In the current study, further structural optimization was con-
ducted from the lead compounds 2 and 3 according to three ap-
proaches (Fig. 2), including the introduction of substituents on
the 9-phenyl group (I), the substitution of the 9-phenyl group with
fused arenes or heterocycles (II), and the modification of the cyclic
amidine moiety (III). The anti-HIV profiles of the most potent
derivative are also described.
X
X1
d, e
b or c
X2
X2
NC CN
9 (X1= CH2, X2 = Br)
13 (X = CH2)
14 (X = O)
15 (X = N-PMB)
10 (X1 = O, X2 = Cl)
11 (X1 = NH2·HCl, X2 = Cl)
12 (X1 = N-PMB, X2= Cl)
a
X
X
N
N
f
N
N
H
Br
NR
S
Br
F
16 (X = CH2)
17 (X = O)
18 (X = N-PMB)
19 (X = CH2, R = t-Bu)
20 (X = CH2, R = H)
21 (X = O, R = t-Bu)
22 (X = O, R = H)
23a (X = N-PMB, R = t-Bu)
24a (X = N-PMB, R = H)
g
g
g
Scheme 2. Synthesis of spiropyrimidine-fused benzothiazinimine derivatives.
Reagents and conditions: (a) (i) 4-methoxybenzoyl chloride, Et3N, CH2Cl2, rt; (ii)
LiAlH4, Et2O, rt, 75% (2 steps); (b) malononitrile, DBU, DMF, 50 °C, 8–60% (for 13 and
14); (c) malononitrile, K2CO3, DMF, 65 °C, 85% (for 15); (d) BH3, THF, 0 °C to rt; (e) 4-
bromo-2-fluorobenzaldehyde, I2, K2CO3, t-BuOH, 70 °C, 11–62% [2 steps (d,e)]; (f)
NaH, t-BuNCS DMF, rt ꢁ80 °C, 78–94%; (g) TFA, MS4Å, CHCl3, reflux, 66–72%.
2. Results and discussion
2.1. Synthesis of 9-aryl-3,4-dihydro-2H,6H-pyrimido[1,2-
c][1,3]benzothiazin-6-imine derivatives
The 9-aryl-3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothia-
zin-6-imine derivatives (7 and 8) were synthesized using a Suzu-
ki–Miyaura cross-coupling reaction37–39 of N-(tert-butyl)-
protected bromide 4 with aryl boronic acid (pinacol ester) or by
an Ullmann coupling40 with pyrazole or imidazole (Scheme 1).
Subsequent trifluoroacetic acid (TFA)-mediated deprotection of
the tert-butyl groups afforded the desired biaryl-type derivatives.
or 12, Scheme 2). BH3-mediated reduction of the alkylated malon-
onitriles (13–15) followed by oxidative amidination41 with 4-bro-
mo-2-fluorobenzaldehyde
gave
the
2-phenyl-1,4,5,6-
tetrahydropyrimidine derivatives (16–18). Subsequent exposure
of compounds 16–18 to tert-butylisothiocyanate provided the tet-
racyclic compounds 19, 21, and 23a. Deprotection of the tert-butyl
groups in compounds 19, 21, and 23a afforded the desired spiro-
pyrimidine-fused benzothiazinimine derivatives (20, 22, and
24a). The substitution of the p-methoxybenzyl (PMB) group in
compound 24a was also attempted (Scheme 3). The treatment of
compound 23a with methyl chloroformate or acetyl chloride di-
rectly provided derivatives 23b and 23c, respectively. A two-step
procedure, including the removal of the PMB group by treatment
with 1-chloroethyl chloroformate followed by modification with
mesyl chloride (MsCl) or trimethylsilyl isocyanate (TMSNCO) was
used for the synthesis of the derivatives 23d and 23e, respectively,
because the reaction of compound 23a with MsCl and TMSNCO
failed. Deprotection of the tert-butyl group in 23b–e afforded the
respective N-substituted derivatives 24b–e.
2.2. Synthesis of spiropyrimidine-fused benzothiazinimine
derivatives
The synthesis of the spiropyrimidine-fused derivatives started
with the dialkylation of malononitrile with dihaloalkanes (9, 10,
N
S
N
S
I
N
N
9
NH
NH
R
2
II
[EC50 = 0.24 0.04 µM]
N
N
2.3. Structure–activity relationships of 9-phenylpyrimido[1,2-
c][1,3]benzothiazine derivatives
S
NH
Ar
X
We initially examined substituent effects at the para-position of
the 9-phenyl group of compound 2 (Table 1). The introduction of
methoxycarbonyl (7a), cyano (7b), nitro (7c), and trifluoromethyl
(7d) groups slightly reduced the anti-HIV activity (EC50 = 0.44–
N
N
III
N
S
N
S
3
NH
NH
0.81 lM), whereas a significant decrease in the anti-HIV
[EC50 = 0.24 0.04 µM]
activity was observed following the introduction of a carbamoyl
Figure 2. Strategy for the structural optimization of PD 404182 derivatives.
group (7e) with hydrogen-bond donor/acceptor properties

6436
T. Mizuhara et al. / Bioorg. Med. Chem. 20 (2012) 6434–6441
Table 1
N-PMB
N-R
Structure–activity relationships for biphenyl-type derivatives
N
S
N
S
a
or b
N
N
N
N
Br
Nt-Bu
Br
Nt-Bu
23a
23b (R = CO2Me)
23c (R = Ac)
Ar
Ar
S
NH
23d (R = Ms)
a
Compound
EC50 (lM)
23e (R = CONH2)
N-R
R
N
S
c
2
R@H
R@CO2Me
R@CN
R@NO2
R@CF3
R@CONH2
R@OMe
R@SMe
R@OCF3
0.24 0.04
0.81 0.29
0.44 0.10
0.46 0.06
0.55 0.16
8.71 0.82
0.24 0.04
0.20 0.06
0.38 0.06
N
7a
7b
7c
7d
7e
7f
Br
NH
24b (R = CO2Me)
24c (R = Ac)
24d (R = Ms)
24e (R = CONH2)
7g
7h
Scheme 3. Synthesis of derivatives 24b–e from 23a. Reagents and conditions: (a)
ClCO2Me or AcCl, CH2Cl2, 0 °C, 81–96% (for 23b or 23c); (b) (i) 1-chloroethyl
chloroformate, Et3N, CH2Cl2, 0 °C, then MeOH, reflux, (ii) MsCl or TMSNCO, (Et3N),
CH2Cl2, rt, 29–82% (2 steps, for 23d or 23e); (c) TFA, MS4Å, CHCl3, reflux, 65–94%.
R
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
R@CO2Me
R@CN
0.39 0.09
1.17 0.27
1.26 0.13
1.19 0.19
>10
R@NO2
R@CH(OH)CH3
R@NHAc
R@NHMs
R@OH
R@OMe
R@Oi-Pr
R@Ph
(EC50 = 8.71
including methoxy (7f, EC50 = 0.24
EC50 = 0.20 M), and trifluoromethoxy (7h, EC50 = 0.38
l
M). Compounds containing a hydrophobic group,
M), methylthio (7g,
M) groups
>10
2.62 0.26
0.15 0.05
0.32 0.10
1.35 0.26
l
l
l
showed similar levels of anti-HIV activity to that of compound 2.
These results indicated that the hydrophobic and electron donating
properties of these substituents had a positive impacts on improv-
ing the anti-HIV activity.
R
Similar SARs were observed following modifications at the
meta-position of the 9-phenyl group (Table 1). For example, the
addition of the electron-withdrawing methoxycarbonyl (7i), cyano
(7j), and nitro (7k) groups resulted in a slight decrease in the anti-
7s
7t
R@OMe
R@Ph
0.41 0.10
0.32 0.12
MeO
7u
0.27 0.04
MeO
MeO
HIV activity (EC50 = 0.39–1.26
droxy)ethyl (7l, EC50 = 1.19
mesylamido (7n, EC50 >10
lM), whereas the hydrophilic (1-hy-
l
M), acetamido (7m, EC50 >10
l
M),
l
M), and hydroxy (7o, EC50 = 2.62
l
M)
7v
0.25 0.03
MeO
OMe
groups led to a reduction or loss in the levels of anti-HIV activity.
In contrast, the introduction of a methoxy group (7p) at the
meta-position of the 9-phenyl group improved the inhibitory activ-
Cl
7w
7x
0.32 0.04
0.48 0.06
ity (EC50 = 0.15
maintained the anti-HIV activity of compound 2 (EC50 = 0.32
whereas the introduction of a phenyl group (7r) led to a decrease
in the inhibitory activity (EC50 = 1.35 M).
lM). The more hydrophobic isopropoxy group (7q)
MeO
Cl
lM),
OMe
l
Similar anti-HIV activities to that of compound 2 were also
exhibited by the ortho-methoxy (7s) and ortho-phenyl (7t) deriva-
a
EC50 values represent the concentration of compound required to inhibit the
HIV-1 infection by 50% and were obtained from three independent experiments.
tives (EC50 = 0.41 and 0.32 lM, respectively), suggesting that the
twisted conformation of the biaryl axis in the 9-aryl-modified PD
404182 derivatives can be tolerated and can interact with the tar-
get molecule(s).
To develop more potent anti-HIV agents, several compounds
were designed with bis- and tris-modifications on the 9-phenyl
group of compound 2 (Table 1). The introduction of the 3,4-dime-
anti-HIV activity (EC50 = 0.20
congener (8b, EC50 = 0.39 M). Compound 8c, which contained a
benzo[d][1,3]dioxol-5-yl group, displayed inhibitory activity two-
fold greater than that of compound 2 (EC50 = 0.15 M), whereas
lM) than that of the 1-naphthyl
l
l
thoxy
EC50 = 0.25
fied derivatives 7w and 7x exhibited similar levels of potency to
compound 2 (EC50 = 0.32 and 0.48 M, respectively). Taken to-
gether, these results suggest that the hydrophobic property of
the phenyl substituting group may provide the predominant con-
tribution in any potential interaction with the target molecule(s).
We proceeded to investigate the impact of introducing a
bicyclic aromatic group at the 9-position of the pyrimido[1,2-
(7u,
EC50 = 0.27
l
M)
and
3,4,5-trimethoxy
(7v,
the 2,3-dihydrobenzo[b][1,4]dioxin-6-yl derivative 8d and quino-
lin-6-yl derivative 8e exhibited less favorable effects
(EC50 = 0.26 lM and 0.25 lM, respectively). The introduction of tri-
fluoroacetylindolyl groups (8f and 8g) resulted in no anti-HIV
activity, and the compounds also showed unexpected levels of
cytotoxicity.
The substitution of the 9-phenyl group with a variety of
different heterocyclic substructures was also investigated (Table 2).
Pyridine substitution (8h and 8i) led to a slight reduction in the
lM) groups did not alter the bioactivity. The Cl-modi-
l
c][1,3]benzothiazine scaffold (Table 2). Modifications with
a
anti-HIV activity (EC50 = 0.45 lM and 0.54 lM, respectively),
variety of 3,4-fused phenyl groups were investigated because the
whereas the introduction of a furan (8j), benzofuran (8k), thio-
2-naphthyl-modified analog (8a) exhibited slightly more potent
phene (8l), benzothiophene (8m), and pyrazole (8n) was well

T. Mizuhara et al. / Bioorg. Med. Chem. 20 (2012) 6434–6441
6437
Table 2
Structure–activity relationships for biaryl-type derivatives
N
N
Ar
M)
S
NH
a
a
Compound
Ar
EC50
(l
Compound
Ar
EC50 (lM)
N
8a
0.20 0.06
0.39 0.12
8h
0.45 0.07
0.54 0.04
8b
8i
N
O
O
O
8c
0.15 0.03
0.26 0.07
8j
0.26 0.02
0.20 0.03
O
O
S
8d
8k
O
N
S
8e
0.25 0.04
8l
0.22 0.07
0.26 0.06
8m
H
N
8f
>1.00b
F3COC
F3COC
N
N
N
8n
8o
0.42 0.08
5.12 1.02
N
8g
>1.00b
N
H
a
EC50 values represent the concentration of compound required to inhibit the HIV-1 infection by 50% and were obtained from three independent experiments.
Cytotoxicity was observed at 10 lM.
b
tolerated and had little impact on the activity relative to compound
2 (EC50 = 0.20–0.42 M). It is worthy of note that the substitution
of the 9-phenyl group with a basic imidazole moiety led to a signif-
icant reduction in the anti-HIV (8o, EC50 = 5.12 M).
Taken together, these data led to the identification of two highly
potent compounds 7p and 8c (EC50 = 0.15 M), which contained
2.5. Anti-HIV profiles of the most potent derivative 8c
l
A time-of-drug addition study was carried out to further inves-
tigate the anti-HIV profile of the most potent derivative 8c as an
anti-HIV agent (Fig. 3). This assay has been used previously to
l
l
m-methoxyphenyl and benzo[d][1,3]dioxol-5-yl groups, respec-
tively. Furthermore, no cytotoxic effects were observed for these
derivatives at 10 lM in the MAGI assay.
Table 3
Structure–activity relationships for spiropyrimidine-fused derivatives
N
2.4. Structure–activity relationships of spiropyrimidine-fused
benzothiazinimine derivatives
EC50 = 0.24 0.04 µM
N
S
NH
3
Several spiropyrimidine-fused derivatives were designed for
the SAR study based on the geminal dimethylpyrimidine substruc-
ture 3 (Table 3).42 Cyclohexane (20) and N-methoxycarbonylpi-
peridine (24b) derivatives exhibited the similar levels of anti-HIV
activity to that of the parent dimethyl derivative 3. In contrast,
the tetrahydropyran (22) and N-(p-methoxybenzyl)piperidine
(24a) derivatives exerted inhibitory activities that were five–sev-
enfold lower than that of the parent dimethyl derivative 3. The
N-acetyl- (24c), N-methanesulfonyl- (24d), and N-carbamoyl-
(24e) piperidine derivatives also provided reduced levels of antivi-
ral activity. With this in mind, the N-alkoxycarbonyl piperidine
group was identified as a linkage for the introduction of additional
functional group(s) to PD 404182 with potent anti-HIV activity
(24b).
X
N
S
N
Br
NH
a
Compound
X
EC50 (lM)
20
22
CH2
O
N-PMB
N-CO2Me
N-Ac
0.25 0.01
1.73 0.35
1.45 0.05
0.44 0.02
2.74 0.15
1.81 0.43
>10
24a
24b
24c
24d
24e
N-Ms
N-CONH2
a
EC50 values represent the concentration of compound required to inhibit the
HIV-1 infection by 50% and were obtained from three independent experiments.

6438
T. Mizuhara et al. / Bioorg. Med. Chem. 20 (2012) 6434–6441
pyrimido[1,2-c][1,3]benzothiazinimine identified two potent
derivatives containing m-methoxyphenyl (7p) and benzo
[d][1,3]dioxol-5-yl groups (8c) that exhibited threefold higher
anti-HIV activity than that of PD 404182 (1). The common hydro-
phobic biaryl moiety is effective to improve the antiviral activity,
providing potential interaction with the target molecule(s). In
addition, we demonstrated that the most effective derivative, 8c,
inhibited viral infection against all of the HIV strains examined
and acted at the early stage of the HIV infection. The design and
synthesis of chemical probes based on these SAR data are being
investigated to identify the target molecule(s).
4. Experimental
4.1. Synthesis
4.1.1. General methods
1H NMR spectra were recorded using a JEOL AL-400 or a JEOL
ECA-500 spectrometer. Chemical shifts are reported in d (ppm) rel-
ative to Me4Si (CDCl3) or DMSO (DMSO-d6) as internal standards.
13C NMR spectra were referenced to the residual solvent signal. Ex-
act mass (HRMS) spectra were recorded on a JMS-HX/HX 110A
mass spectrometer. Melting points were measured by a hot stage
melting point apparatus (uncorrected). For flash chromatography,
Wakogel C-300E (Wako) or aluminium oxide 90 standardized
(Merck) were employed. For preparative TLC, TLC silica gel 60
F254 (Merck) or TLC aluminium oxide 60 F254 basic (Merck) were
employed. For analytical HPLC, a Cosmosil 5C18-ARII column
(4.6 ꢂ 250 mm, Nacalai Tesque, Inc., Kyoto, Japan) was employed
with method A [a linear gradient of CH3CN containing 0.1% (v/v)
TFA] or method B [a linear gradient of CH3CN containing 0.1% (v/
v) NH3] at a flow rate of 1 mL/min on a Shimadzu LC-10ADvp (Shi-
madzu Corp., Ltd., Kyoto, Japan), and eluting products were de-
tected by UV at 254 nm. The purity of the compounds was
determined by combustion analysis or HPLC analysis as >95%.
Figure 3. Time of drug addition profiles for infection by HIV-1IIIB strain of HeLa-
CD4/CCR5-LTR/b-gal cells.
Table 4
Anti-HIV activity of compounds 1 and 8c against other HIV strains
a
Strains
EC50
(lM)
134
8c
HIV-1NL4–3
HIV-1BaL
HIV-2EHO
HIV-2ROD
0.38 0.06
0.37 0.06
0.31 0.06
0.30 0.06
0.23 0.09
0.13 0.05
0.14 0.02
0.10 0.04
a
EC50 values represent the concentration of compound required to inhibit the
HIV infection by 50% and were obtained from three independent experiments.
4.1.2. General procedure of Suzuki–Miyaura cross coupling for
9-aryl pyrimido[1,2-c][1,3]thiazine derivatives 5 and 6: N-(tert-
butyl)-3,4-dihydro-9-(4-methoxycarbonylphenyl)-2H,6H-
pyrimido[1,2-c][1,3]benzothiazin-6-imine 5a
approximately determine which stage in the replication cycle of
HIV-1 is inhibited by the compound. Two compounds (1 and 8c)
were selected for testing in this assay together with five standard
anti-HIV agents, including DS5000 (adsorption inhibitor),31 enfu-
virtide (fusion inhibitor),6,7 AZT (NRTI),43 nevirapine (NNRTI),44,45
and raltegravir (integrase inhibitor).8,9 The results revealed that
the infection profile in the presence of compound 8c was similar
to that of DS5000 and enfuvirtide, suggesting that 8c exerted its
anti-HIV activity at the early stages of the viral infection, including
the binding and fusion stage. This was similar to PD 404182, indi-
cating that the bioactivity profile was not influenced by the newly
appended functional group(s).
To a solution of bromide 4 (52.8 mg, 0.15 mmol) and 4-
(methoxycarbonyl)phenylboronic acid (32.4 mg, 0.18 mmol) in a
mixture of toluene (1.5 mL), EtOH (0.9 mL) and 1 M aq K2CO3
(1.5 mL) was added Pd(PPh3)4 (6.9 mg, 4 mol %) and
PdCl2(dppf)ꢀCH2Cl2 (3.7 mg, 3 mol %). After being stirred under re-
flux for 1 h, the mixture was extracted with CHCl3. The organic lay-
ers were dried over MgSO4 and concentrated. The residue was
purified by flash chromatography over aluminum oxide with n-
hexane/EtOAc (10:0/9:1) to give the compound 5a as colorless so-
lid (47.3 mg, 77%): mp 201–202 °C (from CHCl3–n-hexane); IR
(neat) cmꢁ1: 1719 (C@O), 1593 (C@N); 1H NMR (400 MHz, CDCl3)
d: 1.40 (s, 9H, 3 ꢂ CH3), 1.90–1.96 (m, 2H, CH2), 3.65 (t, J = 5.5 Hz,
2H, CH2), 3.89 (t, J = 6.1 Hz, 2H, CH2), 3.94 (s, 3H, CH3), 7.36 (d,
J = 1.7 Hz, 1H, Ar), 7.44 (dd, J = 8.5, 1.7 Hz, 1H, Ar), 7.65 (d,
J = 8.2 Hz, 2H, Ar), 8.10 (d, J = 8.2 Hz, 2H, Ar), 8.28 (d, J = 8.5 Hz,
1H, Ar). 13C NMR (100 MHz, CDCl3) d: 21.9, 30.0 (3C), 45.2, 45.4,
52.1, 54.2, 123.0, 124.8, 127.0 (2C), 127.3, 129.1, 129.6, 129.8,
130.2 (2C), 138.0, 141.7, 143.8, 147.5, 166.8; HRMS (FAB): m/z
calcd for C23H26N3O2S [M+H]+ 408.1746; found: 408.1748.
We also evaluated the antiviral activity of compounds 1 and 8c
against several HIV strains such as HIV-1NL4–3, HIV-1BaL, HIV-2EHO
,
and HIV-2ROD. This study enabled us to estimate the impact of the
target molecules on the process of binding and fusion because
these viruses have different susceptibilities46 to different anti-
HIV agents. These results implied that compounds 1 and 8c exhib-
ited their anti-HIV activity through different mechanisms from
those of the known binding and fusion inhibitors including CCR5
antagonists, CXCR4 antagonists, and enfuvirtide. In addition, com-
pound 8c was two–threefold more effective against these HIV-1
and HIV-2 strains than PD 404182 (Table 4).
4.1.3. N-(tert-Butyl)-3,4-dihydro-9-(1H-pyrazol-1-yl)-2H,6H-
pyrimido[1,2-c][1,3]benzothiazin-6-imine (6n)
3. Conclusions
To a solution of bromide 4 (52.8 mg, 0.15 mmol), pyrazole
(12.3 mg, 0.18 mmol), CuCl (1.5 mg, 0.015 mmol) and K2CO3
(21.8 mg, 0.16 mol) in N-methylpyrrolidone (0.3 mL) was added
In conclusion, we have designed and synthesized a series of PD
404182 derivatives for the development of novel anti-HIV agents.
The structural optimization study on the 9-position of

T. Mizuhara et al. / Bioorg. Med. Chem. 20 (2012) 6434–6441
6439
acetylacetone (3.8
l
L, 0.038 mmol) under an Ar atmosphere. After
temperature for 5 h, EtOAc was added. The mixture was washed
with 5% aq NaHCO3, and dried over MgSO4. After concentration,
the residue was purified by flash chromatography over silica gel
with n-hexane/EtOAc (2:1) to give the title compound 15 as yellow
oil (8.13 g, 85%): IR (neat) cmꢁ1: 2248 (C„N); 1H NMR (400 MHz,
CDCl3) d: 2.22 (t, J = 5.4 Hz, 4H, 2 ꢂ CH2), 2.61 (br s, 4H, 2 ꢂ CH2),
3.48 (s, 2H, CH2), 3.80 (s, 3H, CH2), 6.86 (d, J = 8.5 Hz, 2H, Ar),
7.19 (d, J = 8.8 Hz, 2H, Ar). 13C NMR (100 MHz, CDCl3) d: 31.1,
34.1 (2C), 48.5 (2C), 55.2, 61.9, 113.8 (2C), 115.4 (2C), 129.2,
130.1 (2C), 159.0; HRMS (FAB): m/z calcd for C15H18N3O [M+H]+
256.1450; found: 256.1454.
being stirred at 130 °C for 19 h, EtOAc and brine were added. The
organic layers were washed with H2O, and dried over MgSO4. After
concentration, the residue was purified by flash chromatography
over aluminum oxide with n-hexane/EtOAc (7:3) to give the title
compound 6n as colorless solid (39.8 mg, 71%): mp 132–133 °C
(from CHCl3–n-hexane); IR (neat) cmꢁ1: 1597 (C@N); 1H NMR
(400 MHz, CDCl3) d: 1.39 (s, 9H, 3 ꢂ CH3), 1.90–1.96 (m, 2H, CH2),
3.63 (t, J = 5.6 Hz, 2H, CH2), 3.88 (t, J = 6.2 Hz, 2H, CH2), 6.48 (dd,
J = 2.7, 1.8 Hz, 1H, Ar), 7.47 (dd, J = 8.8, 2.2 Hz, 1H, Ar), 7.56 (d,
J = 2.2 Hz, 1H, Ar), 7.73 (d, J = 1.8 Hz, 1H, Ar), 7.94 (d, J = 2.7 Hz,
1H, Ar), 8.28 (d, J = 8.8 Hz, 1H, Ar). 13C NMR (100 MHz, CDCl3) d:
21.8, 30.0 (3C), 45.0, 45.4, 54.2, 108.2, 114.3, 115.9, 125.4, 126.7,
129.9, 130.8, 137.7, 141.0, 141.7, 147.3; HRMS (FAB): m/z calcd
for C18H22N5S [M+H]+ 340.1596; found: 340.1598.
4.1.7. 3-(4-Bromo-2-fluorophenyl)-9-(4-methoxybenzyl)-2,4,9-
triazaspiro[5.5]undec-2-ene (18)
To a solution of nitrile 15 (4.05 g, 15.9 mmol) in THF (39.8 mL)
was added BH3 in THF (79.5 mL, 79.5 mmol, 1.0 M) at 0 °C under an
Ar atmosphere. The mixture was warmed to rt. After being stirred
at 65 °C for 5 h, the reaction mixture was cooled to 0 °C, and 1 N
HCl was added. After being stirred at rt for 1 h, the mixture was
basified with 2 N NaOH. The whole was extracted with CHCl3
and dried over MgSO4. After concentration, the residue was dis-
solved in t-BuOH (159.0 mL) and 4-bromo-2-fluorobenzaldehyde
(3.23 g, 15.9 mmol) was added. After being stirred at 70 °C for
30 min, K2CO3 (6.59 g, 47.7 mmol) and I2 (5.05 g, 19.9 mmol) were
added. After being stirred at same temperature for 3 h, the reaction
mixture was quenched with sat. Na2SO3 until the iodine color al-
most disappeared. The reaction mixture was basified with 2 N
NaOH. The whole was extracted with CHCl3, and dried over MgSO4.
After concentration, the residue was purified by flash chromatogra-
phy over aluminium oxide with EtOAc–MeOH (10:0/95:5) to give
the title compound 18 as colorless solid (752 mg, 11%): mp 179–
181 °C (from CHCl3–n-hexane), IR (neat) cmꢁ1: 1630 (C@N); 1H
NMR (500 MHz, CDCl3) d: 1.45 (t, J = 5.4 Hz, 4H, 2 ꢂ CH2), 2.35 (t,
J = 5.4 Hz, 4H, 2 ꢂ CH2), 3.16 (s, 4H, 2 ꢂ CH2), 3.40 (s, 2H, CH2),
3.73 (s, 3H, CH3), 4.63 (s, 1H, NH), 6.78 (d, J = 8.6 Hz, 2H, Ar),
7.14–7.23 (m, 4H, Ar), 7.62 (t, J = 8.3 Hz, 1H, Ar). 13C NMR
(100 MHz, CDCl3) d: 27.3, 32.8 (2C), 49.1 (2C), 51.4 (2C), 55.2,
62.7, 113.5 (2C), 119.4 (d, J = 27.3 Hz), 122.7 (d, J = 12.4 Hz),
123.7 (d, J = 9.9 Hz), 127.8 (d, J = 3.3 Hz), 130.2, 130.3 (2C), 131.7
(d, J = 4.1 Hz), 150.3 (d, J = 1.7 Hz), 158.6, 159.7 (d, J = 251.6 Hz);
19F NMR (500 MHz, CDCl3) d: ꢁ114.6. HRMS (FAB): m/z calcd for
4.1.4. General procedure of tert-butyl deprotection for
pyrimido[1,2-c][1,3]benzothiazin-6-imines (7, 8, 20, 22, and 24):
3,4-dihydro-9-(4-methoxycarbonylphenyl)-2H,6H-
pyrimido[1,2-c][1,3]benzothiazin-6-imine (7a)
TFA (2.0 mL) was added to a mixture of N-(tert-butyl)-protected
pyrimido[1,2-c][1,3]benzothiazin-6-imine
5a
(38.4 mg,
0.094 mmol) in small amount of CHCl3 and MS4Å (300 mg, powder,
activated by heating with Bunsen burner). After being stirred un-
der reflux for 1 h, the mixture was concentrated. To a stirring mix-
ture of the residue in CHCl3 was added dropwise Et3N at 0 °C to
adjust pH to 8–9. The whole was extracted with EtOAc. The extract
was washed with sat. NaHCO3, brine, and dried over MgSO4. After
concentration, the residue was purified by flash chromatography
over aluminum oxide with n-hexane/EtOAc (9:1/1:1) to give the ti-
tle compound 7a as colorless solid (27.3 mg, 83%): mp 185–186 °C
(from CHCl3–n-hexane); IR (neat) cmꢁ1: 1719 (C@O), 1619 (C@N),
1566 (C@N); 1H NMR (500 MHz, CDCl3) d: 1.97–2.02 (m, 2H, CH2),
3.71 (t, J = 5.7 Hz, 2H, CH2), 3.94 (s, 3H, CH3), 4.04 (t, J = 6.0 Hz, 2H,
CH2), 7.27 (d, J = 1.7 Hz, 1H, Ar), 7.46 (dd, J = 8.0, 1.7 Hz, 1H, Ar),
7.63 (d, J = 8.6 Hz, 2H, Ar), 8.10 (d, J = 8.6 Hz, 2H, Ar), 8.30 (d,
J = 8.0 Hz, 1H, Ar). 13C NMR (125 MHz, CDCl3) d: 21.0, 43.8, 45.0,
52.2, 122.0, 125.1, 126.2, 126.9 (2C), 129.5, 129.6, 129.7, 130.2
(2C), 142.1, 143.4, 146.2, 153.0, 166.7; HRMS (FAB): m/z calcd for
C
19H18N3O2S [M+H]+ 352.1120; found: 352.1119
4.1.5. Bis(2-chloroethyl)-N-(4-methoxybenzyl)amine (12)
To a suspension of bis(2-chloroethyl)amine hydrochloride 11
(8.92 g, 50.0 mmol) in CH2Cl2 (300 mL) were added Et3N
(2.89 mL, 100.0 mmol) and 4-methoxybenzoyl chloride (6.77 mL,
50.0 mmol). After being stirred at rt for 2 h, the reaction mixture
was washed with 1 N HCl, satd NaHCO3, brine, and dried over
MgSO4. After concentration, the residue was dissolved in anhy-
drous Et2O (250 mL) and LiAlH4 (2.1 g, 55.0 mmol) was slowly
added at 0 °C under an Ar atmosphere. After being stirred at rt
overnight, the reaction mixture was quenched by addition of
water, 2 N NaOH, and water. The mixture was dried over MgSO4.
After concentration, the residue was purified by flash chromatogra-
phy over silica gel with n-hexane/EtOAc (19:1) to give the title
compound 12 as colorless oil (9.88 g, 75%): 1H NMR (400 MHz,
CDCl3) d: 2.90 (t, J = 7.1 Hz, 4H, 2 ꢂ CH2), 3.48 (t, J = 7.1 Hz, 4H,
2 ꢂ CH2), 3.67 (s, 2H, CH2), 3.80 (s, 3H, CH3), 6.86 (d, J = 8.5 Hz,
2H, Ar), 7.24 (d, J = 8.5 Hz, 2H, Ar). 13C NMR (100 MHz, CDCl3) d:
42.0 (2C), 55.2, 56.2 (2C), 58.6, 113.8 (2C), 129.7 (2C), 130.7,
158.9; LRMS (FAB): m/z [M+H]+ 262.
C
22H26BrFN3O [M+H]+ 446.1243; found: 446.1237.
4.1.8. General procedure for t-BuNCS mediated cyclization: 9-
bromo-N-(tert-butyl)-10-(4-methoxybenzyl)-2H-
spiro[benzo[e]pyrimido[1,2-c][1,3]thiazine-3,4’-piperidin]-
6(4H)-imine (23a)
To a mixture of fluoride 18 (2.0 g, 4.48 mmol) and NaH
(358.4 mg, 8.96 mmol; 60% oil suspension) in DMF (14.8 mL) was
added t-BuNCS (1.14 mL, 8.96 mmol) under an Ar atmosphere.
After being stirred at rt overnight, the reaction mixture was
warmed to 60 °C. After being stirred at this temperature for 1 h,
EtOAc was added. The resulting solution was washed with sat.
NaHCO3, brine, and dried over MgSO4. After concentration, the res-
idue was purified by flash chromatography over aluminum oxide
with n-hexane/EtOAc (10:0/9:1) to give the title compound 23a
as colorless solid (2.28 g, 94%): mp 89–91 °C (from CHCl3–n-hex-
ane); IR (neat) cmꢁ1: 1577 (C@N); 1H NMR (500 MHz, CDCl3) d:
1.37 (s, 9H, 3 ꢂ CH3), 1.49–1.52 (m, 4H, 2 ꢂ CH2), 2.40–2.46 (m,
4H, 2 ꢂ CH2), 3.41 (s, 2H, CH2), 3.47 (s, 2H, CH2), 3.75 (s, 2H,
CH2), 3.80 (s, 3H, CH3), 6.85 (d, J = 8.6 Hz, 2H, Ar), 7.22 (d,
J = 8.6 Hz, 2H, Ar), 7.28–7.31 (m, 2H, Ar), 8.03 (d, J = 8.6 Hz, 1H,
Ar). 13C NMR (100 MHz, CDCl3) d: 29.7, 29.9 (3C), 32.6 (2C), 49.2
(2C), 51.6, 54.3, 55.2, 55.5, 62.7, 113.6 (2C), 124.5, 126.3, 126.8,
129.2, 130.0, 130.1, 130.4 (2C), 130.9, 137.5, 146.3, 158.7; HRMS
4.1.6. 1-(4-Methoxybenzyl)piperidine-4,4-dicarbonitrile (15)
To a solution of malononitrile (2.49 g, 37.7 mmol) in DMF
(94.3 mL) was added K2CO3 (5.73 mg, 41.5 mmol). After being stir-
red at 65 °C for 2 h, a solution of chloride 12 (9.88 mg, 37.7 mmol)
in DMF (37.7 mL) was added. After being stirred at same

6440
T. Mizuhara et al. / Bioorg. Med. Chem. 20 (2012) 6434–6441
(FAB): m/z calcd for C27H34BrN4OS [M+H]+ 541.1637; found:
541.1633.
EC50 ¼ 10^½logðA=BÞ ꢂ ð50 ꢁ CÞ=ðD ꢁ CÞ þ logðBÞꢃ;
wherein
ð1Þ
A: of the two points on the graph which bracket 50% inhibition,
the higher concentration of the test compound,
B: of the two points on the graph which bracket 50% inhibition,
the lower concentration of the test compound,
C: inhibitory activity (%) at the concentration B,
D: inhibitory activity (%) at the concentration A.
4.1.9. 9-Bromo-N-(tert-butyl)-10-(methoxycarbonyl)-2H-
spiro[benzo[e]pyrimido[1,2-c][1,3]thiazine-3,4’-piperidin]-
6(4H)-imine (23b)
To the solution of N-(4-methoxybenzyl)piperidine 23a
(40.6 mg, 0.075 mmol) in CH2Cl2 (0.38 mL) was added methyl chlo-
roformate (86.4 lL, 1.13 mmol) at 0 °C under an Ar atmosphere.
After being stirred at same temperature for 30 min, the reaction
mixture was concentrated. The residue was purified by flash chro-
matography over silica gel with n-hexane/EtOAc (1:1) to give com-
pound 23b as a colorless solid (29.2 mg, 81%): mp 157–158 °C
(from n-hexane); IR (neat) cmꢁ1: 1699 (C@O), 1577 (C@N); 1H
NMR (400 MHz, CDCl3) d: 1.37 (s, 9H, 3 ꢂ CH3), 1.46 (t, J = 5.6 Hz,
4H, 2 ꢂ CH2), 3.44 (br s, 4H, 2 ꢂ CH2), 3.56 (br s, 2H, CH2), 3.70
(s, 3H, CH3), 3.81 (s, 2H, CH2), 7.29–7.33 (m, 2H, Ar), 8.05 (d,
J = 8.5 Hz, 1H, Ar). 13C NMR (100 MHz, CDCl3) d: 29.9 (3C), 30.1,
32.2 (2C), 39.9 (2C), 50.8, 52.5, 54.3, 55.2, 124.7, 126.1, 126.8,
129.3, 130.0, 130.9, 137.7, 146.3, 155.9; HRMS (FAB): m/z calcd
for C21H28BrN4O2S [M+H]+ 479.1116; found: 479.1115.
Acknowledgments
We are indebted to Dr. Hideki Maeta, Dr. Masahiko Taniguchi,
Mr. Takayuki Kato, Ms. Kumiko Hiyama, Mr. Shuhei Osaka, Dr.
Megumi Okubo, Dr. Daisuke Nakagawa, Mr. Tatsuya Murakami,
and Dr. Kazunobu Takahashi for excellent technical assistance. This
work was supported by Grants-in-Aid for Scientific Research and
Targeted Protein Research Program from MEXT and Health and La-
bor Science Research Grants (Research on HIV/AIDS, Japan). T.M. is
grateful for JSPS Research Fellowships for Young Scientists.
Supplementary data
4.1.10. 9-Bromo-N-(tert-butyl)-10-(methanesulfonyl)-2H-
spiro[benzo[e]pyrimido[1,2-c][1,3]thiazine-3,4’-piperidin]-
6(4H)-imine (23d)
To the solution of N-(4-methoxybenzyl)piperidine 23a
(54.2 mg, 0.10 mmol) in CH2Cl2 (0.5 mL) were added Et3N
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.08.030.
References and notes
(28.9 lL, 0.20 mmol) and 1-chloroethyl chloroformate (21.8 lL,
0.20 mmol) at 0 °C under an Ar atmosphere. After being stirred at
same temperature for 30 min, the reaction mixture was concen-
trated. The residue was dissolved in MeOH (2.0 mL). After being
stirred under reflux for 10 min, the reaction mixture was concen-
trated. The residue was dissolved in CHCl3, and was washed with
sat. NaHCO3, brine, and dried over MgSO4. After concentration,
1. Thompson, M. A.; Aberg, J. A.; Cahn, P.; Montaner, J. S. G.; Rizzardini, G.; Telenti,
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was added at rt under an Ar atmosphere. After being stirred at rt
for 10 min, the reaction mixture was washed with sat. NaHCO3,
brine, and dried over MgSO4. After concentration, the residue
was purified by flash chromatography over aluminum oxide with
n-hexane/EtOAc (6:4) to give compound 23d as a colorless solid
(40.9 mg, 82%): mp 177 °C (from CHCl3–n-hexane); IR (neat)
:
cmꢁ1 1577 (C@N), 1331 (NSO2), 1155 (NSO2); 1H NMR
(400 MHz, CDCl3) d: 1.38 (s, 9H, 3 ꢂ CH3), 1.62 (t, J = 5.5 Hz, 4H,
2 ꢂ CH2), 2.80 (s, 3H, CH3), 3.21–3.27 (m, 2H, CH2), 3.31–3.37 (m,
2H, CH2), 3.46 (s, 2H, CH2), 3.84 (s, 2H, CH2), 7.29–7.33 (m, 2H,
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29.9 (3C), 32.0 (2C), 34.7, 42.0 (2C), 50.1, 54.4, 55.1, 124.8, 125.9,
126.9, 129.4, 130.0, 130.8, 137.9, 146.3; HRMS (FAB): m/z calcd
for C20H28BrN4O2S2 [M+H]+ 499.0837; found: 499.0840.
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