Concise synthesis and anti-HIV activity of pyrimido[1,2-c][1,3] benzothiazin-6-imines and related tricyclic heterocycles

Article abstract of DOI:10.1039/c2ob25904d

3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) is a virucidal heterocyclic compound active against various viruses, including HCV, HIV, and simian immunodeficiency virus. Using facile synthetic approaches that we developed for the synthesis of pyrimido[1,2-c][1,3]benzothiazin-6-imines and related tricyclic derivatives, the parallel structural optimizations of the central 1,3-thiazin-2-imine core, the benzene part, and the cyclic amidine part of PD 404182 were investigated. Replacement of the 6-6-6 pyrimido[1,2-c][1,3] benzothiazin-6-imine framework with 5-6-6 or 6-6-5 derivatives led to a significant loss of anti-HIV activity, and introduction of a hydrophobic group at the 9- or 10-positions improved the potency. In addition, we demonstrated that the PD 404182 derivative exerts anti-HIV effects at an early stage of viral infection.

Full text of DOI:10.1039/c2ob25904d

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PAPER
Concise synthesis and anti-HIV activity of pyrimido[1,2-c][1,3]benzothiazin-
6-imines and related tricyclic heterocycles†
Tsukasa Mizuhara,a Shinya Oishi,*a Hiroaki Ohno,a Kazuya Shimura,b Masao Matsuokab and
Nobutaka Fujii*a
Received 11th May 2012, Accepted 22nd June 2012
DOI: 10.1039/c2ob25904d
3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) is a virucidal heterocyclic
compound active against various viruses, including HCV, HIV, and simian immunodeficiency virus.
Using facile synthetic approaches that we developed for the synthesis of pyrimido[1,2-c][1,3]-
benzothiazin-6-imines and related tricyclic derivatives, the parallel structural optimizations of the central
1,3-thiazin-2-imine core, the benzene part, and the cyclic amidine part of PD 404182 were investigated.
Replacement of the 6-6-6 pyrimido[1,2-c][1,3]benzothiazin-6-imine framework with 5-6-6 or 6-6-5
derivatives led to a significant loss of anti-HIV activity, and introduction of a hydrophobic group at the
9- or 10-positions improved the potency. In addition, we demonstrated that the PD 404182 derivative
exerts anti-HIV effects at an early stage of viral infection.
integrase inhibitor (raltegravir),8 and a CC chemokine receptor
Introduction
type 5 (CCR5) antagonist (maraviroc)9 are examples of new
Since azidothymidine (AZT), a nucleoside reverse transcriptase
inhibitor (NRTI), was approved for the treatment of HIV infec-
tions, a number of anti-HIV drugs have been launched. For
example, saquinavir and nevirapine were the first protease inhibi-
tor and non-nucleoside reverse transcriptase inhibitor (NNRTI),
respectively.1 Highly active antiretroviral therapy (HAART)
using a combination of these antiretrovirals is a standard treat-
ment regimen for HIV infections. The HAART regimen signifi-
cantly reduces viral load in infected patients, leading to
significant therapeutic gains and reductions in morbidity and
mortality.2 However, long-term administration of multiple anti-
retrovirals to maintain life-long latent infection triggers the emer-
gence of drug-resistant variants3 and drug-related adverse
effects.4 For example, high-level viral resistance to NRTI such as
AZT, stavudine, and didanosine is conferred by mutations fre-
quently observed in patients with virologic failure on an NRTI-
containing regimen.5 In addition, lipodystrophy and metabolic
disorders are often observed in patients receiving HIV protease
inhibitors.6 To overcome these problems, several antiretrovirals
with new mechanisms of action have been developed in this
decade. A peptide-based fusion inhibitor (enfuvirtide),7 an
molecular entities used as anti-HIV agents.
Recently, highly potent small-molecule anti-HIV agents have
been reported, which bind to viral envelope proteins (Fig. 1).
2-Thioxo-1,3-thiazolidine derivative 1 shows potent inhibition of
HIV-1 replication at nanomolar levels,10 which are directed at
the deep hydrophobic pocket in the N-terminal heptad repeat
trimer of the viral gp41. Compound 1 blocks HIV-1-mediated
cell–cell fusion and the formation of gp41 six-helix bundles, as
does enfuvirtide.10b The bisindole derivative 2 also exhibits sub-
micromolar inhibition of HIV-1 replication by interaction with
the gp41 hydrophobic pocket in which compound 1 binds.11
Small-molecule CD4 mimics with oxalamide and related sub-
structures are another series of anti-HIVagents.12 The representa-
tive BMS-448043 (3) exhibits subnanomolar anti-HIV activity
by interaction with the CD4 binding pocket in gp120.12d These
small-molecule entry inhibitors with potential oral bioavailability
will provide alternative combination regimen(s) of anti-HIV
agents for the treatment of drug-resistant variants.
In our efforts to develop novel anti-HIV compounds,13 we
have carried out the random screening of small molecules using
multinuclear activation of a galactosidase indicator (MAGI)
assay, in which the inhibitory activity of early-stage HIV infec-
tion, including virus attachment and membrane fusion to host
cells, is evaluated. Among more than 30 000 compounds
screened, 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-
6-imine 4 (PD 404182) was identified as a potent anti-HIV agent
lead (Fig. 1). Compound 4 was reported to be an enzyme inhibi-
tor against 3-deoxy-D-manno-octulosonic acid 8-phosphate
synthase14 and phosphopantetheinyl transferase,15 exerting
aGraduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-
ku, Kyoto 606-8501, Japan. E-mail: soishi@pharm.kyoto-u.ac.jp,
nfujii@pharm.kyoto-u.ac.jp; Fax: +81-75-753-4570;
Tel: +81-75-753-4561
bInstitute for Virus Research, Kyoto University, Sakyo-ku, Kyoto,
606-8507, Japan
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob25904d
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Scheme 1 Our synthetic methods for PD 404182 derivatives.
Fig. 1 Structures of newly reported anti-HIV compounds (1–3) target-
ing HIV-1 envelope proteins, and PD 404182 (4).
antimicrobial effects. In the course of our SAR investigations in
this study, antiviral activities of 4 against HCV, HIV, and simian
immunodeficiency virus were reported.16,17 Although compound
4 exhibits virucidal effects at high concentrations, the mechan-
ism of action and the target molecule remain ambiguous.17
Recently, we have established two independent approaches for
the synthesis of PD 404182 derivatives (Scheme 1): C–H func-
tionalization of 2-phenyl-1,4,5,6-tetrahydropyrimidine with
water or tert-butylcarbamate in the presence of copper(II) acetate
provides pyrimido[1,2-c][1,3]benzoxazine or pyrimido[1,2-c]-
quinazoline in one or two step(s) (eqn (1), Scheme 1).18 Alterna-
tively, addition of 2-(2-halophenyl)-1,4,5,6-tetrahydropyrimidine
to carbon disulfide, isocyanate, or isothiocyanate, and sub-
sequent aromatic nucleophilic substitution (SNAr) affords pyri-
midobenzothiazines and -oxazines, and pyrimidoquinazolines
(eqn (2), Scheme 1).19 The derivatives obtained from these reac-
tions were easily converted to the pyrimido[1,2-c][1,3]benzothia-
zin-6-imine scaffold. Our two synthetic methods provide a
variety of PD 404182 derivatives from the corresponding benz-
aldehydes in a few steps and in good yields, facilitating the lead
optimization process.20 In this article, a SAR study of PD
404182 derivatives using these synthetic approaches is
described.
Fig. 2 Strategy for SAR study of PD 404182 (4).
Results and discussion
Strategy for the SAR study of PD 404182
PD 404182 consists of three components, namely a 1,3-thiazin-
2-imine core, and left-fused benzene and cyclic amidine moieties
(Fig. 2). In order to obtain detailed insights into the relationships
between the compound structure and anti-HIV activity, we
planned to investigate substituent effects on each component: (I)
derivatives with various heteroatom (N, S, and O) arrangements
on the 1,3-thiazin-2-imine core (Fig. 2); (II) pyrimido[1,2-c][1,3]-
thiazin-6-imine derivatives fused with a substituted benzene ring
or a five- or six-membered aromatic heterocycle; and (III) benzo-
[e][1,3]thiazin-2-imine derivatives fused with a cyclic amidine
ring with or without accessory alkyl or aryl groups.
Synthesis of pyrimido[1,2-c][1,3]benzothiazin-6-imines and
related tricyclic heterocycles
Our investigation began with the synthesis of tricyclic hetero-
cycles with different combinations of heteroatoms on the
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1,3-thiazin-2-imine core. Previously, we reported syntheses of
pyrimido[1,2-c][1,3]benzoxazine and pyrimido[1,2-c]quinazo-
line derivatives using copper(II)-mediated C–H functionaliza-
tion;18 this facilitates the introduction of oxygen or nitrogen
functional groups at the ortho-position of 2-phenyl-1,4,5,6-tetra-
hydropyrimidine (5). Using compound 5 as a key starting
material, a divergent approach was used for the preparation of a
series of scaffolds (Scheme 2).
A one-pot reaction for Cu(OAc)2-mediated C–H functionali-
zation of 5 and subsequent treatment with triphosgene provided
a 1,3-oxazin-2-one derivative 7 (Scheme 2). The same one-pot
procedure using thiophosgene produced a trace amount of the
desired thiocarbonyl derivative 8; treatment of the purified inter-
mediate 6 with thiophosgene provided the desired 1,3-oxazine-2-
thione 8 in high yield. 1,3-Oxazin-2-imine 9 was obtained by
the reaction of 6 with BrCN.
The copper-mediated C–N bond formation of compound 5
with tert-butylcarbamate followed by spontaneous intramolecu-
lar cyclization afforded a pyrimido[1,2-c]quinazolin-6-one scaf-
fold 10, as demonstrated in our previous report (Scheme 2).18
Subsequent treatment with Lawesson’s reagent led to formation
of the thiocarbonyl derivative 11. Since no hydrolysis of the car-
bonyl or thiocarbonyl group of compound 10 or 11 for construc-
tion of the 2-aminoquinazoline structure in 12 occurred, an
alternative approach starting from 2-aminobenzyl alcohol 15 was
used for the synthesis of the 2-aminoquinazoline derivative 12
(Scheme 3). After protection and PCC oxidation of 15, oxidative
amidination21 provided 2-(p-tosylamino)phenyltetrahydropyri-
midine 17. Deprotection followed by BrCN-mediated cyclization
of 17 provided the expected 2-aminoquinazoline derivative 12.
For the synthesis of pyrimido[1,2-c][1,3]benzothiazine deriva-
tives, we adapted the C–H functionalization reaction for C–S
bond formation (Scheme 2). After optimization of the reaction
conditions, we found that exposure of compound 5 to CS2 in the
presence of Cu(OAc)2 directly afforded a pyrimido[1,2-c][1,3]-
benzothiazine-6-thione scaffold 13. Hydrolysis of the thiocarbo-
nyl group in 13 followed by treatment with BrCN or triphosgene
provided 6-imino or 6-oxo derivatives (4 or 14), respectively.
Synthesis of pyrimido[1,2-c][1,3]thiazine derivatives with fused
benzene and heterocycles
Pyrimido[1,2-c][1,3]thiazin-6-imine derivatives 28–30 with a
series of fused ring systems were prepared by consecutive
heterocumulene addition and SNAr reactions (Scheme 4).19
These reactions provide easy access to the construction of the
1,3-thiazin-2-imine derivatives and are more efficient than the
diversity-oriented C–H functionalization approach. The oxidative
amidination of aromatic aldehydes 18–20 with an accessory
Scheme 2 Synthesis of PD 404182 derivatives with different combi-
nations of heteroatoms. Reagents and conditions: (a) Cu(OAc)2, H2O,
O2, DMF, 130 °C, 69%; (b) triphosgene, TMEDA, CH2Cl2, 0 °C to rt,
70% [2 steps (a,b)]; (c) thiophosgene, Et3N, CH2Cl2, 0 °C to rt, quant.;
(d) BrCN, CH2Cl2, rt, 34%; (e) Cu(OAc)2, BocNH2, O2, DMF, 130 °C,
53%; (f) Lawesson’s reagent, xylene, reflux, 19%; (g) Cu(OAc)2, CS2,
O2, 1,4-dioxane, 130 °C, 11%; (h) NaOH, MeOH, H2O, reflux; (i)
BrCN, EtOH, reflux, 61% [2 steps (h,i)]; ( j) triphosgene, Et3N, CH2Cl2,
0 °C to rt, 65% [2 steps (h,j)].
Scheme 3 Synthesis of 2-aminoquinazoline derivative 12. Reagents
and conditions: (a) p-TsCl, pyridine, CHCl3, rt; (b) PCC, silica gel,
CH2Cl2, rt, 80% [2 steps (a,b)]; (c) 1,3-propanediamine, I2, K2CO3,
t-BuOH, 70 °C, 98%; (d) conc. H2SO4, 100 °C, then NaOH, H2O; (e)
BrCN, EtOH, reflux, 66% [2 steps (d,e)].
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Scheme 4 Synthesis of pyrimido[1,2-c][1,3]thiazin-6-imine deriva-
tives fused with substituted benzene and heterocycles (28–30). Reagents
and conditions: (a) 1,3-propanediamine, I2, K2CO3, t-BuOH, 70 °C,
58–91%; (b) NaH, CS2, DMF, 80 °C, 67%-quant.; (c) NaH or t-BuOK,
t-BuNCS, DMF or DMAc, −20–80 °C, 28–95%; (d) (i) NaOH, MeOH,
H2O, reflux, (ii) BrCN, EtOH, reflux, 32–68%; (e) TFA, MS4Å, CHCl3,
reflux, 63–92%.
functional group afforded the corresponding 2-phenyltetrahydro-
pyrimidine derivatives 21–23. The pyrimido[1,2-c][1,3]thiazine-
6-thione scaffold 24 was obtained by additions of 21f,g,i or
23s,t,u to carbon disulfide followed by SNAr-type C–S bond for-
mation. The desired 6-imino derivatives 28f,g,i and 30s,t,u were
obtained via hydrolysis of the thiocarbonyl group of 24 followed
by BrCN treatment. Alternatively, reactions of other 2-phenyl-
tetrahydropyrimidines 21–23 with tert-butyl isothiocyanate
afforded N-(t-Bu)-protected thiazinimine derivatives 25–27,
which were treated with TFA to provide the expected products
28–30.
The intermediates 25e, 25k, and 26k were subjected to further
manipulations to obtain the functionalized derivatives
(Scheme 5). The nitro group of 25e was reduced by hydrogen-
ation to form the 9-amino derivative 31. Alkylation of 31
afforded the 9-(N-methylamino) derivative 25b (eqn (1),
Scheme 5). The 9-acetamide derivative 25c was obtained by
treatment of 31 with acetic anhydride (eqn (2), Scheme 5). Sand-
meyer reaction of 31 gave the 9-azide derivative 25p (eqn (3),
Scheme 5). Me2N- and MeO-substituted derivatives (25a, 26a,
and 26f) were obtained by Me2NH-mediated N-arylation22 of
the 9-bromo 25k and 10-bromo derivatives 26k, and NaOMe-
mediated Ullmann coupling23 of 26k, respectively (eqn (4) and
Scheme 5 Synthesis of 9- or 10-substituted pyrimido[1,2-c][1,3]benzo-
thiazin-6-imine derivatives. Reagents and conditions: (a) H2, Pd/C,
EtOH, rt, 88%; (b) NaOMe, (CH2O)n, MeOH, reflux, then NaBH4,
91%; (c) TFA, MS4Å, CHCl3, reflux, 37–95%; (d) Ac2O, DMAP, Et3N,
CH2Cl2, rt, quant.; (e) NaNO2, AcOH, H2O, 0 °C, then NaN3, 70%;
(f) Pd(OAc)2, t-Bu3P, NHMe2, THF, KOt-Bu, toluene, reflux, quant.;
(g) 2-hydroxyethylvinylether, Pd(OAc)2, 1,3-bis(diphenylphosphino)-
propane, K2CO3, H2O, 90 °C, 13% [2 steps (g,c)]; (h) R-B(OH)2 or
R-Bpin, Pd(PPh3)4, PdCl2(dppf)·CH2Cl2, K2CO3, toluene or 1,4-dioxane,
EtOH, H2O, reflux, 62–96%; (i) n-BuB(OH)2, Pd2(dba)3, P(t-Bu)3,
CsCO3, 1,4-dioxane, reflux, 6% (for 25h); ( j) Pd(Pt-Bu)2, NHMe2,
THF, KOt-Bu, toluene, 170 °C, 67% (for 26a); (k) CuBr, NaOMe,
MeOH, DMF, reflux, 40% (for 26f).
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(7), Scheme 5). The 9-acetyl derivative 25d was obtained by
Heck reaction24 of 25k with 2-hydroxyethyl vinyl ether (eqn (5),
Scheme 5). Other derivatives with a variety of functional groups
(25h,l–o,q and 26l,m,q) were synthesized by Suzuki–Miyaura
coupling reactions25 of 25k and 26k with boronic acids or their
pinacol esters (eqn (6) and (7), Scheme 5). Final deprotection of
the tert-butyl group in 25 and 26 afforded the 9- or 10-substi-
tuted pyrimido[1,2-c][1,3]benzothiazine derivatives 28 and 29,
respectively.
Synthesis of benzo[e][1,3]thiazine derivatives with fused cyclic
amidines
Benzo[e][1,3]thiazine derivatives with various ring-sized and/or
modified cyclic amidine moieties 35 were also synthesized by
the consecutive heterocumulene addition and SNAr reactions
(Scheme 6). Oxidative amidination using several diamines 32
proceeded efficiently to form five- or six-membered rings
(33a–d). The same reaction for the seven-membered amidine
(33e) was incomplete, but purification of the Boc-protected
amidine 36 followed by subsequent deprotection of the Boc
group gave the pure seven-membered amidine 33e. The resulting
amidines were converted to cyclic-amidine-fused benzo[e][1,3]-
thiazin-2-imines 34 via tert-butyl isothiocyanate addition and an
SNAr reaction. TFA-mediated deprotection gave the expected
derivatives 35.
Scheme 6 Synthesis of benzo[e][1,3]thiazine derivatives with fused
cyclic amidines. Reagents and conditions: (a) 2-fluorobenzaldehyde or
2-bromobenzaldehyde, I2, K2CO3, t-BuOH, 70 °C, 68–79%; (b) NaH,
t-BuNCS, DMF, rt −80 °C, 18–50%; (c) TFA, MS4Å, CHCl3, reflux,
16–86%; (d) Boc2O, Et3N, DMAP, CH2Cl2, rt, 37% [2 steps (a,d)]; (e)
TFA, CH2Cl2, reflux, 80%.
Structure–activity relationships of the central heterocyclic core
in pyrimido[1,2-c][1,3]benzothiazines
Initially, the structural requirements of the 1,3-thiazin-2-imine
core substructure in 4 (PD 404182) for anti-HIV activity were
investigated (Table 1). The antiviral activities against the
HIV-1IIIB strain were evaluated using the MAGI assay. Substi-
tution of the imino group in 4 with a carbonyl group (14)
Table 1 SARs for 1,3-thiazin-2-imine core
resulted in a significant decrease in anti-HIV activity (EC50
=
8.94 μM). Pyrimido[1,2-c][1,3]benzoxazines (7–9), pyrimido
[1,2-c]quinazolines (10–12), and pyrimido[1,2-c][1,3]benzothia-
zine-6-thione (13), in which the 1-sulfur and/or 2-imino groups
in 4 were modified, showed no activity. These results suggested
that both the 1-sulfur atom and the 2-imino group are indispensa-
ble functional groups for the inhibitory activity against HIV
infection, and may be involved in potential interactions with the
target molecules.
Compound
X
Y
EC50 (μM)a
4
7
8
9
10
11
12
13
14
S
O
O
O
NH
NH
NH
S
NH
O
S
NH
O
S
NH
S
O
0.44 0.08
>10
>10
>10
>10
>10
>10
>10
8.94 1.07
S
Structure–activity relationships of the benzene substructure in
pyrimido[1,2-c][1,3]benzothiazine
a EC50 values were the concentration that blocks HIV-1 infection by
50% and derived from three independent experiments.
A series of derivatives with modification of the benzene sub-
structure in the pyrimido[1,2-c][1,3]benzothiazine were evalu-
ated for anti-HIV activity (Table 2). The addition of positively
charged N,N-dimethylamino (28a) and N-methylamino groups
(28b) at the 9-position significantly decreased the anti-HIV
activity. The 9-acetamide group (28c), which has hydrogen-bond
donor/acceptor abilities, also attenuated the bioactivity. The
acetyl (28d) and nitro (28e) groups, with hydrogen acceptor
properties, induced slight decreases in the anti-HIV activity. In
contrast, derivatives with less-polarized substituents (28f–o and
28q) at this position generally reproduced the potent anti-HIV
activity of 4. In terms of the electron-donating or -withdrawing
properties of the substituent groups on the benzene substructure,
good correlations were not observed. For example, the electron-
donating methoxy (28f), methyl (28g), and n-butyl groups
(28h), and the electron-withdrawing fluoro (28i) and trifluoro-
methyl groups (28j) exhibited similar anti-HIV activities (EC50
=
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Table 2 SARs for benzene part
Compound
EC50 (μM)a
Compound
EC50 (μM)a
30r
0.56 0.13
4
R = H
0.44 0.08
4.74 1.07
>10
28a
28b
28c
R = NMe2
R = NHMe
R = NHAc
>10
30s
2.55 0.26
28d
28e
28f
28g
28h
28i
R = COMe
R = NO2
R = OMe
R = Me
R = n-butyl
R = F
1.44 0.33
1.13 0.18
0.57 0.09
0.49 0.10
0.44 0.09
0.50 0.07
30k
>10
28j
28k
28l
28m
28n
28o
R = CF3
R = Br
R = Ph
R = vinyl
R = styryl
R = pentenyl
0.53 0.12
0.25 0.09
0.24 0.04
0.18 0.05
0.25 0.05
0.24 0.11
30t
>10
28p
28q
R = N3
R = C6H4(4-Bz)
0.43 0.06
0.53 0.12
30i
1.68 0.19
29a
29e
29f
29g
29k
29l
R = NMe2
R = NO2
R = OMe
R = Me
R = Br
2.12 0.26
3.00 0.59
0.53 0.04
0.38 0.04
0.24 0.05
0.24 0.05
R = Ph
30u
>10
29m
29q
R = vinyl
R = C6H4(4-Bz)
0.40 0.09
0.67 0.16
a EC50 values were the concentration that blocks HIV-1 infection by 50% and derived from three independent experiments.
0.44–0.57 μM), indicating that the antiviral activity is indepen-
dent of the electronic state of the 1,3-benzothiazin-2-imine core
in forming potential π-stacking interaction(s) with the target mol-
ecules. Among the hydrophobic substituents at this position,
bromo (28k), phenyl (28l), vinyl (28m), styryl (28n), and
pentenyl groups (28o) induced inhibitory activity two or three
times greater than that of 4 (EC50 = 0.18–0.25 μM). Modification
with photoreactive azido (28p) and benzoylphenyl groups (28q)
maintained the inhibitory activity; these could be used as probe
molecules to identify the target molecule(s) of 4.26
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Table 3 SARs for cyclic amidine
Similar SARs were observed for modification at the 10-posi-
tion of pyrimido[1,2-c][1,3]benzothiazine. Addition of positively
charged N,N-dimethylamino (29a) and polarized nitro groups
(29e) reduced the anti-HIV activity (EC50 = 2.12 and 3.00 μM,
respectively). Hydrophobic groups including methoxy (29f),
methyl (29g), bromo (29k), phenyl (29l), vinyl (29m), and
4-benzoylphenyl (29q) (EC50 = 0.24–0.67 μM) had favorable
effects on the bioactivity, suggesting potential hydrophobic
interactions of these additional functional groups with the target
molecule(s).
Compound
EC50 (μM)a
4
0.44 0.08
Further miscellaneous modifications of benzothiazine sub-
structure were also investigated (Table 2). The naphtho[2,3-e]-
[1,3]thiazine derivative 30r, with a 9,10-fused benzene, exhib-
35a
35b
>10
ited anti-HIV activity equipotent to that of the parent 4 (EC50
=
0.56 μM). A 6-fold decrease in the anti-HIV activity of the pyri-
dine-fused pyrido[3,2-e][1,3]thiazine derivative (30s) was
observed (EC50 = 2.55 μM). In addition, introduction of
8-bromo (30k) and 8,9-fused benzene (30t, naphtho[2,1-e][1,3]-
thiazine) substituents on benzothiazine resulted in a loss of
activity, suggesting that modification at the 8-position was inap-
propriate for favorable interactions with the target molecule(s).
The 11-fluoro derivative 30i and thiophene-fused 30u, which has
a 5-6-6 framework (thieno[2,3-e][1,3]thiazine), exhibited four
times lower and no inhibitory potencies, respectively.
3.78 1.39
35c
35d
0.35 0.09
0.24 0.04
Structure–activity relationships of cyclic amidine part of
pyrimido[1,2-c][1,3]benzothiazine
A SAR study of the top-right cyclic amidine substructure was
carried out. The five-membered dihydroimidazole derivative 35a
had no anti-HIV activity (Table 3), suggesting that the five-mem-
bered ring may impair the critical interactions with the target
molecule(s) via its small-sized ring strain or indirect effects on
the thiazinimine core with a possibly altered conformation. Simi-
larly, compound 35b with the phenyl-fused dihydropyrimidine
substructure showed lower inhibitory activity (EC50 = 3.78 μM).
Appending one or two methyl groups on the six-membered pyri-
midine (35c and 35d) induced 1.5- to 2-fold higher inhibitory
potencies (EC50 = 0.35 and 0.24 μM, respectively) compared
with that of the parent compound 4. In addition, compound 35e
with a seven-membered tetrahydro-1,3-diazepine substructure
exhibited similar anti-HIV activity to that of 4 (EC50 = 0.31 μM).
35e
0.31 0.06
a EC50 values were the concentration that blocks HIV-1 infection by
50% and derived from three independent experiments.
Mechanistic studies of anti-HIV pyrimido[1,2-c][1,3]-
benzothiazin-6-imines and related tricyclic heterocycles
To investigate the mechanism of action of PD 404182 deriva-
tives, a time of drug addition study was carried out (Fig. 3). In
this experiment, the anti-HIV activity profiles of 427 and its
derivatives 29k27 were compared with those of well-known anti-
HIV agents such as an adsorption inhibitor (DS 5000),28 fusion
inhibitor [enfuvirtide (T-20)],5 NRTI (AZT),29 NNRTI (nevira-
pine),30 and integrase inhibitor (raltegravir).6 After inoculation
of HeLa-CD4/CCR5-LTR/β-gal cells with HIV-1IIIB, each anti-
HIV-1 drug was added at a 90% inhibitory effect concentration
at the indicated time points. The inhibitory effects on the infec-
tion were determined by counting the blue cells 48 h later. This
investigation revealed that compound 4 (PD 404182) had an
inhibitory profile in the early stage of viral infection similar to
Fig. 3 Time of drug addition profiles for infection by HIV-1IIIB strain
of HeLa-CD4/CCR5-LTR/β-gal cells.
those of DS 5000 and enfuvirtide (Fig. 3). Identical profiles were
observed for derivative 29k.
6798 | Org. Biomol. Chem., 2012, 10, 6792–6802
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Table 4 Anti-HIV activity of 4 and 35d against other HIV strains
EC50 (μM)a
derivatives prevent the HIV infection process at an early stage.
For iterative molecular design of more effective derivatives
based on binding modes, the identification of the target molecule(s)
of PD 404182 derivatives is being investigated using derivatives
such as 28p and 28q.
Strain
4
35d
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
0.25 0.03
0.16 0.02
0.17 0.03
0.11 0.03
Experimental section
a EC50 values were the concentration that blocks HIV infection by 50%
and derived from three independent experiments.
General
1H NMR spectra were recorded using a JEOL AL-400 or a
JEOL ECA-500 spectrometer. Chemical shifts are reported in δ
(ppm) relative to Me4Si as an internal standard. 13C NMR
spectra were referenced to the residual solvent signal. Exact 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), TLC Aluminium oxide 60 F254 basic (Merck), or
NH2 Silica Gel 60 F254 Plate (Wako) were employed. For
analytical HPLC, a Cosmosil 5C18-ARII column (4.6 ×
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 cm3 min−1 on a Shimadzu
LC-10ADvp (Shimadzu Corp., Ltd., Kyoto, Japan), and eluting
products were detected by UV at 254 nm. The purity of the com-
pounds was determined by combustion analysis or HPLC analy-
sis as >95% unless otherwise stated.
To gain additional insights into the mechanism of action of
PD 404182 derivatives, the antiviral activities against other HIV
subtypes were evaluated (Table 4). Compound 4 was effective
against not only HIV-1IIIB but also other two HIV-1 strains
(HIV-1NL4-3 and HIV-1BaL) with similar potency. Both HIV-1IIIB
and HIV-1NL4-3 strains utilize CXCR4 as a coreceptor for entry,
while HIV-1BaL strain does CCR5, indicating that chemokine
receptors CXCR4 and CCR5 are not the molecular targets of PD
404182 derivatives. The similar level of antiviral activity of 4
against HIV-2 (HIV-2EHO and HIV-2ROD), which is mainly dis-
tributed in West Africa, was observed. Highly potent inhibitory
activities of a derivative 35d27 against these HIV strains were
observed, as in the case of the SAR study of the HIV-1IIIB strain
discussed above. It has been well known that NNRTIs are not
effective against HIV-2, highlighting that PD 404182 derivatives
do not act as NNRTIs. Although PD 404182 derivatives and
enfuvirtide showed similar anti-HIV-1 profile in the time of drug
addition assay, HIV-2EHO and HIV-2ROD infection were affected
by PD 404182 derivatives, in contrast with the less effective
enfuvirtide,31 suggesting that PD 404182 derivatives may not be
directed at the HIV gp41 envelope protein. Recent reports have
suggested that the antiviral activities of compound 4 against
HIV, HCV, and pseudotype lentiviruses were derived from dis-
ruption of the structural integrities of virions.17 Although the
mechanism of action of PD 404182 derivatives is not fully
understood at this stage, the unidentified biomolecule(s) in
viruses or host cells, including envelope protein(s), lipid mem-
branes and/or sugar chain(s), could be promising molecular
targets for this new class of anti-HIVagents.
General procedure of oxidative amidination: synthesis of 2-(3-
bromo-2-fluorophenyl)-1,4,5,6-tetrahydropyrimidine (23k). To a
solution of 3-bromo-2-fluorobenzaldehyde 20k (0.71 g,
3.5 mmol) in t-BuOH (33 cm3) was added propylenediamine
(285.4 mg, 3.9 mmol). The mixture was stirred at 70 °C for
30 min, and then K2CO3 (1.45 g, 10.5 mmol) and I2 (1.11 g,
4.4 mmol) were added. After being stirred at the same tempera-
ture for 3 h, the mixture was quenched with sat. Na2SO3. The
organic layer was separated and concentrated. The resulting solid
was dissolved in H2O, and then pH was adjusted to 12–14 with
2N NaOH. The whole was extracted with CHCl3. The extract
was dried over MgSO4. After concentration, the resulting solid
was recrystallized from CHCl3–n-hexane to give compound 23k
as colorless crystals (0.62 g, 69%): mp 99 °C; IR (neat) νmax
/
cm−1: 1624 (CvN); δH (400 MHz; CDCl3; Me4Si) 1.84–1.89
(2H, m, CH2), 3.50 (4H, t, J = 5.7 Hz, 2 × CH2), 5.13 (1H, br s,
NH), 7.03 (1H, td, J = 8.0, 0.9 Hz, Ar), 7.54 (1H, ddd, J = 8.0,
6.4, 1.3 Hz, Ar) and 7.69 (1H, ddd, J = 8.0, 6.5, 1.3 Hz, Ar). δC
(100 MHz; CDCl3) 20.6, 42.1 (2C), 109.6 (d, J = 22.3 Hz),
125.1 (d, J = 4.1 Hz), 126.3 (d, J = 13.2 Hz), 129.8 (d, J = 3.3 Hz),
134.3, 150.8 and 156.3 (d, J = 248.3 Hz); δF (500 MHz;
CDCl3) −110.7; Anal. Calc. for C10H10BrFN2: C, 46.72; H,
3.92; N, 10.90. Found: C, 46.64; H, 4.10; N, 10.93%.
Conclusion
In conclusion, we have designed and synthesized PD 404182
derivatives for a novel series of anti-HIV agents. Comprehensive
SAR studies demonstrated that the 6-6-6 fused pyrimido[1,2-c]-
[1,3]benzothiazine scaffold and the heteroatom arrangement in
the thiazinimine moiety are indispensable for the inhibitory
activity of 4 (PD 404182) against HIV infection. Optimization
studies of the benzene and cyclic amidine rings indicate that the
introduction of a hydrophobic group on the benzene ring and the
amidine group is more effective in improving the antiviral
activity, giving potential favorable interaction(s) with the target
molecule(s). In addition, PD 404182 derivatives could be prom-
ising agents for treatment of HIV-2 infection. We also revealed,
using time of drug addition experiments, that PD 404182
General procedure of CS2-mediated cyclization for pyrimido-
[1,2-c][1,3]benzothiazine-6-thiones 24: synthesis of 3,4-dihydro-
2H,6H-pyrimido[1,2-c]thieno[2,3-e][1,3]thiazin-6-thione (24u).
To a mixture of 2-(3-bromothiophen-2-yl)-1,4,5,6-tetrahydropyr-
imidine 23u (122.6 mg, 0.50 mmol) and NaH (40.0 mg,
This journal is © The Royal Society of Chemistry 2012
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1.0 mmol; 60% oil suspension) in DMF (1.7 cm3) was added
CS2 (0.060 cm3, 1.0 mmol) under an Ar atmosphere. After being
stirred at 80 °C for 12 h, the mixture was concentrated. The
residue was purified by flash chromatography over silica gel with
n-hexane–EtOAc (8 : 2) to give the compound 24u as a pale
yellow solid (80.5 mg, 67%): mp 167 °C (from CHCl3–
n-hexane); IR (neat) νmax/cm−1: 1624 (CvN); δH (400 MHz;
CDCl3; Me4Si) 2.04–2.10 (2H, m, CH2), 3.68 (2H, t, J = 5.5 Hz,
CH2), 4.42 (2H, t, J = 6.1 Hz, CH2), 6.76 (1H, d, J = 5.4 Hz,
Ar) and 7.49 (1H, d, J = 5.4 Hz, Ar). δC (100 MHz; CDCl3)
21.5, 45.0, 48.5, 122.3, 128.4, 130.8, 131.0, 141.7 and 189.7;
HRMS (FAB): m/z Calc. for C9H9N2S3 [M + H]+ 240.9928;
found: 240.9936.
HRMS (FAB): m/z Calc. for C12H14N3S [M + H]+ 232.0908;
found: 232.0912.
General procedure of tert-butyl deprotection for pyrimido[1,2-
c][1,3]benzothiazin-6-imines 28–30: synthesis of 3,4-dihydro-9-
nitro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine
(28e).
TFA (1.5 cm3) was added to a mixture of N-(tert-butyl)-3,4-
dihydro-9-nitro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine
25e (47.8 mg, 0.15 mmol) in CHCl3 and molecular sieves 4 Å
(225 mg, powder, activated by heating with Bunsen burner).
After being stirred under reflux for 1.5 h, the mixture was con-
centrated. To a mixture of this residue in CHCl3 was added drop-
wise Et3N at 0 °C to adjust the pH to 8–9. The whole was
extracted with EtOAc, and the extract was washed with sat.
NaHCO3, brine, and dried over MgSO4. After concentration, the
residue was purified by flash chromatography over aluminium
oxide with n-hexane–EtOAc (19 : 1 to 1 : 1) to give compound
28e as a pale yellow solid (24.9 mg, 63%): mp 170–172 °C
(from CHCl3–n-hexane); IR (neat) νmax/cm−1: 1620 (CvN),
1587 (NO2), 1568 (CvN), 1523 (NO2); δH (400 MHz; CDCl3;
Me4Si) 1.97–2.03 (2H, m, CH2), 3.74 (2H, t, J = 5.6 Hz, CH2),
4.04 (2H, t, J = 6.2 Hz, CH2), 7.41 (1H, br s, NH), 7.93 (1H, d,
J = 2.2 Hz, Ar), 8.00 (1H, dd, J = 9.0, 2.2 Hz, Ar) and 8.42 (1H,
d, J = 9.0 Hz, Ar). δC (100 MHz; CDCl3) 20.8, 43.8, 45.2,
118.9, 120.5, 130.4, 130.8, 131.7, 145.1, 148.7 and 151.3; Anal.
Calc. for C11H10N4O2S: C, 50.37; H, 3.84; N, 21.36. Found: C,
50.29; H, 4.03; N, 21.08%.
General procedure of t-BuNCS-mediated cyclization for t-Bu
protected pyrimido[1,2-c][1,3]thiazin-6-imines 25–27, and 34:
synthesis of N-(tert-butyl)-3,4-dihydro-9-nitro-2H,6H-pyrimido[1,2-c]-
[1,3]benzothiazin-6-imine (25e). To a mixture of 2-(2-fluoro-
4-nitrophenyl)-1,4,5,6-tetrahydropyrimidine
21e
(2.0
g,
8.96 mmol) and NaH (716.8 mg, 17.92 mmol; 60% oil suspen-
sion) in DMF (29.8 cm3) was added t-BuNCS (2.28 cm3,
17.92 mmol) under an Ar atmosphere. After being stirred at
−20 °C to rt for 2 days, EtOAc was added. The resulting solu-
tion was washed with sat. NaHCO3, brine, and dried over
MgSO4. After concentration, the residue was purified by flash
chromatography over aluminium oxide with n-hexane–EtOAc
(10 : 0 to 9 : 1) to give compound 25e as a pale yellow solid
(1.77 g, 62%): mp 152–153 °C (from CHCl3–n-hexane); IR
(neat) νmax/cm−1: 1604 (CvN), 1591 (NO2), 1581 (CvN),
1523 (NO2); δH (500 MHz; CDCl3; Me4Si) 1.39 (9H, s, 3 ×
CH3), 1.91–1.96 (2H, m, CH2), 3.66 (2H, t, J = 5.2 Hz, CH2),
3.88 (2H, t, J = 5.7 Hz, CH2), 7.97 (2H, dd, J = 9.7, 2.3 Hz,
Ar), 8.01 (2H, d, J = 2.3 Hz, Ar) and 8.39 (1H, d, J = 9.2 Hz,
Ar). δC (125 MHz; CDCl3) 21.7, 30.0, 45.3, 45.5, 54.5, 119.9,
120.3, 130.0, 131.1, 132.8, 136.1, 146.5 and 148.5; HRMS
(FAB): m/z Calc. for C15H19N4O2S [M + H]+ 319.1229; found:
319.1229.
General procedure of Suzuki–Miyaura cross coupling for 9-
aryl pyrimido[1,2-c][1,3]thiazine derivatives: synthesis of N-(tert-
butyl)-3,4-dihydro-9-phenyl-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-
6-imine 25l. To
a solution of 9-bromo-N-(tert-butyl)-3,4-
dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine 25k
(52.8 mg, 0.15 mmol) and phenylboronic acid (21.9 mg,
0.18 mmol) in a mixture of toluene (1.5 cm3), EtOH (0.9 cm3)
and 1 M aq. K2CO3 (1.5 cm3) was added Pd(PPh3)4 (6.9 mg,
4 mol%) and PdCl2(dppf)·CH2Cl2 (3.67 mg, 3 mol%). After
being stirred at reflux for 1 h, the mixture was extracted with
CHCl3. The organic layers were dried over MgSO4 and concen-
trated. The residue was purified by flash chromatography over
aluminium oxide with n-hexane–EtOAc (10 : 0 to 9 : 1) to give
the compound 25l as colorless solid (44.8 mg, 85%): mp
General procedure of BrCN-mediated cyclization for pyrimido-
[1,2-c][1,3]thiazin-6-imines 28 and 30: synthesis of 3,4-dihydro-
9-methyl-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine
(28g).
3,4-Dihydro-9-methyl-2H,6H-pyrimido[1,2-c][1,3]benzothiazine-6-
thione 24g (62.1 mg, 0.25 mmol) was suspended in a 0.1 M
solution of NaOH in MeOH–H2O (9 : 1) (5 cm3). After being
stirred for 12 h under reflux, the mixture was concentrated. The
residue was suspended in anhydrous EtOH (1 cm3) and BrCN
(53.0 mg, 0.50 mmol) was added under Ar atmosphere. After
stirring for 2 h under reflux, the reaction mixture was quenched
with 2 N NaOH. The whole was extracted with CHCl3, and
dried over MgSO4. After concentration, the residue was purified
by flash chromatography over aluminium oxide with n-hexane–
EtOAc (9 : 1) to give the compound 28g as colorless solid
(39.2 mg, 68%): mp 121 °C (from CHCl3–n-hexane); IR (neat)
νmax/cm−1: 1620 (CvN), 1569 (CvN); δH (500 MHz; CDCl3;
Me4Si) 1.94–1.99 (2H, m, CH2), 2.32 (3H, s, CH3), 3.67 (2H, t,
J = 5.7 Hz, CH2), 4.01 (2H, t, J = 6.3 Hz, CH2), 6.84 (1H, s,
Ar), 7.02 (1H, d, J = 8.6 Hz, Ar), 7.16 (1H, br s, NH) and 8.10
(1H, d, J = 8.6 Hz, Ar). δC (125 MHz; CDCl3) 21.1, 21.1, 43.8,
44.9, 123.6, 124.1, 127.4, 128.6, 128.8, 141.1, 146.6 and 153.6;
122.5–124 °C (from CHCl3–n-hexane); IR (neat) νmax/cm−1
:
1592 (CvN); δH (500 MHz; CDCl3; Me4Si) 1.40 (9H, s, 3 ×
CH3), 1.90–1.95 (2H, m, CH2), 3.64 (2H, t, J = 5.4 Hz, CH2),
3.89 (2H, t, J = 6.0 Hz, CH2), 7.33–7.37 (2H, m, Ar), 7.41–7.44
(3H, m, Ar), 7.58 (2H, d, J = 6.9 Hz, Ar) and 8.25 (1H, d, J =
8.6 Hz, Ar). δC (125 MHz; CDCl3) 21.9, 30.0 (3C), 45.1, 45.4,
54.2, 122.7, 124.8, 126.5, 127.0 (2C), 128.0, 128.8 (2C), 128.9,
129.5, 138.3, 139.4, 142.9 and 147.7; HRMS (FAB): m/z Calc.
for C21H24N3S [M + H]+ 350.1691; found: 350.1683.
Determination of anti-HIV activity
The sensitivity of three HIV-1 strains and two HIV-2 strains was
determined by the MAGI assay.32 The target cells (HeLa-CD4/
CCR5-LTR/β-gal; 104 cells per well) were plated in 96 well flat
microtiter culture plates. On the following day, the cells were
inoculated with the HIV-1 (60 MAGI U per well, giving 60 blue
6800 | Org. Biomol. Chem., 2012, 10, 6792–6802
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9 (a) P. Dorr, M. Westby, S. Dobbs, P. Griffin, B. Irvine, M. Macartney,
J. Mori, G. Rickett, C. Smith-Burchnell, C. Napier, R. Webster,
D. Armour, D. Price, B. Stammen, A. Wood and M. Perros, Antimicrob.
Agents Chemother., 2005, 49, 4721; (b) G. Fätkenheuer, A. L. Pozniak,
M. A. Johnson, A. Plettenberg, S. Staszewski, A. I. M. Hoepelman,
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C. Medhurst, J. F. Sullivan, C. Ridgway, S. Abel, I. T. James, M. Youle
and E. Van Der Ryst, Nat. Med., 2005, 11, 1170.
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J. Sivapackiam, K. Pandya, S. Jiang and A. K. Debnath, J. Med. Chem.,
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cells after 48 h of incubation) and cultured in the presence of
various concentrations of the drugs in fresh medium. Forty-eight
hours after viral exposure, all the blue cells strained with X-Gal
(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) were
counted in each well. The activity of test compounds was deter-
mined as the concentration that blocked HIV-1 infection by 50%
(50% effective concentration [EC50]). EC50 was determined by
using the following formula:
EC50 ¼ 10^½logðA=BÞ ꢀ ð50 ꢁ CÞ=ðD ꢁ CÞ þ logðBÞꢂ;
11 G. Zhou, D. Wu, B. Snyder, R. G. Ptak, H. Kaur and M. Gochin, J. Med.
Chem., 2011, 54, 7220.
wherein 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.
12 (a) P.-F. Lin, W. S. Blair, T. Wang, T. P. Spicer, Q. Guo, N. Zhou,
Y.-F. Gong, H.-G. H. Wang, R. Rose, G. Yamanaka, B. Robinson,
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G. A. Yamanaka, Y.-F. Gong, H.-T. Ho, H. Fang, N. Zhou,
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S. Nakamura, H. Nakano, H. Tamamura, Y. Kobayashi, M. Matsuoka and
N. Fujii, Angew. Chem., Int. Ed., 2002, 41, 2937; (b) N. Fujii, S. Oishi,
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S. C. Peiper, Angew. Chem., Int. Ed., 2003, 42, 3251; (c) S. Ueda,
S. Oishi, Z.-X. Wang, T. Araki, H. Tamamura, J. Cluzeau, H. Ohno,
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Chem., 2007, 50, 192; (d) S. Oishi, S. Ito, H. Nishikawa, K. Watanabe,
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Acknowledgements
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 Labor Science Research Grants (Research on
HIV/AIDS, Japan). T. M. is grateful for JSPS Research Fellow-
ships for Young Scientists.
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