Thiazolothiazepine inhibitors of HIV-1 integrase

Article abstract of DOI:10.1021/jm990047z

A series of thiazolothiazepines were prepared and tested against purified human immunodeficiency virus type-1 integrase (HIV-1 IN) and viral replication. Structure-activity studies reveal that the compounds possessing the pentatomic moiety SC(O)CNC(O) with two carbonyl groups are in general more potent against purified IN than those containing only one carbonyl group. Substitution with electron-donating or -withdrawing groups did not enhance nor abolish potency against purified IN. By contrast, compounds with a naphthalene ring system showed enhanced potency, suggesting that a hydrophobic pocket in the IN active site might accommodate an aromatic system rather than a halogen. The position of sulfur in the thiazole ring appears important for potency against IN, as its replacement with an oxygen or carbon abolished activity. Further extension of the thiazole ring diminished potency. Compounds 1, 19, and 20 showed antiviral activity and inhibited IN within similar concentrations. These compounds inhibited IN when Mn²⁺ or Mg²⁺ was used as cofactor. None of these compounds showed detectable activities against HIV-1 reverse transcriptase, protease, virus attachment, or nucleocapsid protein zinc fingers. Therefore, thiazolothiazepines are potentially important lead compounds for development as inhibitors of IN and HIV replication.

Full text of DOI:10.1021/jm990047z

3334
J . Med. Chem. 1999, 42, 3334-3341
Th ia zoloth ia zep in e In h ibitor s of HIV-1 In tegr a se
Nouri Neamati,*,† J im A. Turpin,‡ Heather E. Winslow,† J ohn L. Christensen,† Karen Williamson,‡ Ann Orr,†
William G. Rice,∇ Yves Pommier,*,† Antonio Garofalo,§ Antonella Brizzi,§ Giuseppe Campiani,§
Isabella Fiorini,§ and Vito Nacci*,§
Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892-4255,
Southern Research Institute, 431 Aviation Way, Frederick, Maryland 21701, Achillion Pharmaceuticals, 1003 West 7th Street,
Suite 401, Frederick, Maryland 21701, and Dipartimento Farmaco Chimico Tecnologico, Universita´ di Siena,
Banchi di Sotto, 55, 53100 Siena, Italy
Received J anuary 27, 1999
A series of thiazolothiazepines were prepared and tested against purified human immunode-
ficiency virus type-1 integrase (HIV-1 IN) and viral replication. Structure-activity studies reveal
that the compounds possessing the pentatomic moiety SC(O)CNC(O) with two carbonyl groups
are in general more potent against purified IN than those containing only one carbonyl group.
Substitution with electron-donating or -withdrawing groups did not enhance nor abolish potency
against purified IN. By contrast, compounds with a naphthalene ring system showed enhanced
potency, suggesting that a hydrophobic pocket in the IN active site might accommodate an
aromatic system rather than a halogen. The position of sulfur in the thiazole ring appears
important for potency against IN, as its replacement with an oxygen or carbon abolished activity.
Further extension of the thiazole ring diminished potency. Compounds 1, 19, and 20 showed
antiviral activity and inhibited IN within similar concentrations. These compounds inhibited
IN when Mn2+ or Mg2+ was used as cofactor. None of these compounds showed detectable
activities against HIV-1 reverse transcriptase, protease, virus attachment, or nucleocapsid
protein zinc fingers. Therefore, thiazolothiazepines are potentially important lead compounds
for development as inhibitors of IN and HIV replication.
In tr od u ction
tive for IN5,6 and useable for therapeutic development,
much information has been gained regarding specific
structural requirements for potential leads. For exam-
ple, a majority of hydroxylated aromatics such as cate-
chol-containing compounds have been demonstrated to
possess potency against the 3′-processing and 3′-strand-
transfer reactions catalyzed by IN.6 However, despite
such promising initial results, compounds containing
this moiety can also cross-link protein,7 chelate metal,8,9
and thus generally lack the desired selectivity.5,6 As
opposed to the catechol-containing compounds, non-
catechol-containing structures are excellent leads to
develop a selective potent IN inhibitor, for they possess
considerably less cytotoxicity (e.g. structures below).
Among these, several sulfonamides,10,11 diaryl sul-
fones,12 and aromatic disulfides13 were found to inhibit
IN function at low micromolar concentrations. However,
only the 2-mercaptobenzenesulfonamides13 and to a
lesser extent the naphthalene disulfonate exhibited
antiviral activity.11,14
In our continuing efforts to identify novel non-cat-
echol-containing inhibitors of IN from compounds shown
to possess antiviral activity in the NCI Antiviral Drug
Screening Program, we discovered benzothiazepine 1 as
a “lead”. Subsequently, a series of compounds were
designed and synthesized to establish a structure-
activity relationship among thiazolothiazepines. Herein,
we present the synthesis and anti-IN activity of 31
thiazepines.
Human immunodeficiency virus type-1 integrase
(HIV-1 IN), one of the three pol gene products, is
required for the efficient insertion of the retroviral
genome into host cell DNA.1 Upon viral adsorption and
reverse transcription, the viral DNA is processed by IN,
which removes the terminal 3′-dinucleotides. Following
nuclear entry, transesterification of phosphodiester
bonds is catalyzed by IN, which cleaves the host DNA
and joins it to the processed 3′-viral termini. These two
steps, known as 3′-processing and 3′-end joining (or
DNA strand transfer), can be reproduced using in vitro
assays, which have sought to identify inhibitors of IN.
Such assays utilize purified IN protein and oligonucle-
otides corresponding to the sequence of the U5 or U3
ends of the HIV long terminal repeat (LTR).2,3 In
addition, IN is able to catalyze an apparent reversal of
the strand-transfer reaction, a process known as disin-
tegration.4 In this reaction, a branched oligonucleotide
substrate consisting of viral and target DNA is resolved
into its constituents. Since IN is unique to retroviruses
and required for viral replication, identifying potent
inhibitors selective for IN should provide novel thera-
peutic strategies for the treatment of AIDS.
Several classes of inhibitors have been reported to
date. Although, none has yet proven to be highly selec-
* Corresponding authors. Dr. Yves Pommier: NCI; phone, (301) 496-
5944; fax, (301) 402-0752; e-mail, pommier@nih.gov. Dr. Vito Nacci:
Universita´ di Siena. Dr. Nouri Neamati: NCI; phone, (301) 435-2463;
fax, (301) 402-0752; e-mail, neamatin@pop.nci.nih.gov.
† National Cancer Institute.
Resu lts a n d Discu ssion s
‡ Southern Research Institute.
Ch em istr y. The synthesis of compounds 1, 5, 11, 13,
14, 21-25, and 28-30 has been accomplished according
∇ Achillion Pharmaceuticals.
§ Universita´ di Siena.
10.1021/jm990047z CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/31/1999

Thiazolothiazepine Inhibitors of HIV-1 Integrase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3335
Sch em e 1a
to procedures reported elsewhere.15-20 Compounds 2-4,
6-10, 12, 19, and 20 have been prepared following
the general method outlined in Scheme 1, described in
ref 15. Schotten-Baumann reaction between the (un)-
substituted 2,2′-dithiobis(benzoic acid chloride) or 3,3′-
dithiobis(2,2′-naphthoic acid chloride), in turn ob-
tained from corresponding acids 32a -g21-25 and thionyl
chloride, and L-thiaproline (Aldrich) (33) or thiazolidine-
2-carboxylic acid (34)26 or 1,3-oxazolidine-4-carboxylic
acid (35)27 gave disulfides 36. NaBH4 reduction of the
crude disulfides gave the corresponding thiophenols in
very good yield. Disulfides 36 and subsequent thio-
phenols were obtained as amorphous solids by ion-
exchange chromatography and were then used without
a thorough characterization. The eventual cyclization
reaction was carried out using N,N′-carbonyldiimida-
zole (CDI) in dry THF leading to optically inactive
tricyclics 2-4, 6-10, 12, and 19, and 20 because of
racemization at C-11a (C-13a in the case of compounds
19 and 20).
a
Reagents: (i) SOCl2/reflux; (ii) 33 or 34 or 35/Na2CO3/THF/
H2O/rt; (iii) NaBH4/EtOH/reflux; (iv) CDI/THF/reflux.
Sch em e 2a
Controlled 3-chloroperbenzoic acid (MCPBA) oxida-
tion of the tricyclic sulfides 4-6 and 10 gave sulfoxides
15-18 (Scheme 2) which were tentatively assigned a
trans configuration on the basis of 1H NMR experiments
and Dreiding stereomodels inspection. This would re-
flect a preferred approach of the electrophile from the
less hindered face of the tricyclic, namely the one taken
up by the C-11a proton. Compound 26 was obtained as
a racemic mixture by reaction of previously described
(()-1,2,3,11a-tetrahydro-5H,11H-pyrrolo[2,1-c][1,4]benzo-
thiazepine-5,11-dione (37)16 with phenylmagnesium
bromide (eq 1).
a
Reagents: (i) MCPBA/CH2Cl2/0 °C.
No effort was made in order to elucidate relative con-
figuration for 26. The basic side chain of compound 27
was installed by means of reaction of 1-methylpipera-
zine on optical active chloro derivative 3815 (eq 2).
In the reaction conditions used, a racemic mixture of
trans-27 was the sole product obtained. Finally, the
synthesis of compound 31 was carried out by Mannich

3336 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Ta ble 1. Anti-HIV-1 Integrase Activities of Thiazepines 1-31
Neamati et al.
integrase assay
IC50 (µM)
cell data (µM)
3′-end
joining
a
b
compd R1
R2 R3 R4
R5
R6
-X-Y-
Z
3′-processing
EC50
CC50
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
H
H
H
H
H
H
H
H
H
Cl
Br
Me
H
Cl
Br
Me
H
OMe
-S-CH2-
-S-CH2-
-S-CH2-
-S-CH2-
-CH2-S-
-CH2-S-
-CH2-S-
-CH2-S-
-CH2-S-
-CH2-S-
-CH2-S-
-CH2-O-
-(CH2)2-
110 ( 12
151; 105
58 ( 15
64 ( 47
208 ( 24
158; 111
87 ( 24
52
90 ( 27
155; 275
670; 630
>333
>1000
406; 495
146 ( 79
120; 60
48 ( 18
55 ( 29
107 >200
>50 >50
227 ( 104 >200 >200
160; 110
74 ( 32
63
100 ( 36
155; 245
333; 330
>333
>100 >100
>200 >200
NRc
35
NO2
H
OMe OMe
>50
>50
>200 >200
>50
>50
H
H
OMe OMe
>1000
343 ( 109 >200 >200
>200 >200
H
H
H
H
H
H
Me
H
Cl
OMe
-(CH2)3-
-S(O)-CH2-
-CH2-S(O)-
-CH2-S(O)-
-CH2-S(O)-
-S-CH2-
-CH2-S-
-(CH2)2-
590 ( 350 590 ( 350 >100 >100
200; 185
260; 215
84.5
215; 222
280; 200
142
40 ( 10
92 ( 30
>1000
>1000
372; 111
>1000
>1000
>1000
>1000
590
47 ( 6
100 ( 40
>1000
>1000
376; 268
>1000
>1000
>1000
>1000
300
60 >316
280 >316
>200 >200
>200 >200
H
H
H
H
H
H
H
H
Ph
H
H
H
H
OAc
OMe
H
H
OH
H
H
H
H
H
-(CH2)2-
-CH2-S-
-(CH2)2-
OMe OMe
>200 >200
>200 >200
H
H
H
H
H
H
-(CH2)2-
OH Ph
H
-(CH2)2-
N(CH2CH2)2NMe
-(CH2)2-
>125 >125
146 >200
H
H
>CO
>CHCO2Et
>CHC6H4-pF
>CHPh
>1000
>1000
>1000
>1000
>1000
>1000
CH2NMe2
CH2N(CH2CH2)2NMe
a
b
EC50: 50% effective concentration. CC50: 50% cytotoxic concentration. c NR: not reached due to cytotoxicity.
reaction on the R-pyrrole position of (()-11-phenyl-5H,-
11H-pyrrolo[2,1-c][1,4]benzothiazepine (39)20 by means
of paraformaldehyde and 1-methylpiperazine dihydro-
chloride in methanol (eq 3).
infected CEM cells. Because of its lack of cytotoxicity,
this compound served as a “lead” for the design and
testing of derivatives. Although 1 was not a potent
antiviral compound, its unique structure and the fact
that it differed from other reported IN inhibitors war-
ranted designing other inhibitors based on this com-
pound. To establish a structure-activity relationship,
30 analogues were prepared and tested in an assay
specific for IN as well as against HIV-1-infected CEM
cells. Several important modifications were made to
determine the importance of each ring and the nature
of the substituents. The first modifications were aimed
at the A and B rings (compounds 1-18). In a second
class of compounds, an extra ring was added and the
importance of the sulfur’s position was analyzed (19 and
20). The third group contains substitutions on all the
rings (21-27), and the last group (28-31) was designed
to determine whether the thiazolothiazepine ring sys-
tem is required for activity (Table 1). The chloro-,
bromo-, and the methyl-substituted derivatives 2-4,
although exhibiting potency similar to that of the parent
compound 1 against purified IN, were inactive against
viral replication and also showed no detectable cytotox-
icity at the highest concentrations tested. Comparable
results were obtained when sulfur was moved to position
Biology. Benzothiazepine 1 was originally identified
as an IN inhibitor (IC50 values for 3′-processing and 3′-
end joining: 110 and 146 µM, respectively) from testing
a series of compounds that were shown to have antiviral
activity in the NCI Antiviral Drug Screening against
CEM cells. Compound 1 with a therapeutic index (TI)
value of >1.8 (50% effective concentration (EC50) value
of 107 ( 26 and 50% cytotoxic concentration (CC50)
value of >200 µM) is moderately active in HIV-1-

Thiazolothiazepine Inhibitors of HIV-1 Integrase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3337
2 as in compounds 5-11 (Table 1). However, the
removal of sulfur (compounds 12-14) abolished anti-
IN and antiviral activities. Oxidation of the sulfur atom
also generally reduced potency (compounds 15-18).
Interestingly, both the naphtho derivatives 19 and 20
showed both antiviral and anti-IN activity. Compound
19 with an IC50 value of 40 µM against IN was more
active than the parent compound 1 against HIV-1
infected cells. Compound 19 with a TI value of > 5 was
the best in this series (EC50: 60 and CC50: > 316 µM).
Additionally, the naphthalene-substituted 20 was also
active against both purified IN (IC50 values of 92 and
100 µM against 3′-processing and 3′-strand transfer,
respectively) and HIV-1-infected cells (EC50: 279-286
and CC50: >316 µM). Other modifications of the ring
system (compounds 21-31) abolished both antiviral and
anti-IN activities.
Role of Diva len t Meta ls. Divalent metal ions, such
as Mn2+ or Mg2+, coordinate with the acidic residues
(D, D, E) of IN’s active site.28-30 Metals are involved in
the catalytic functioning because the enzyme is unable
to perform 3′-processing and 3′-strand transfer without
Mn2+ or Mg2+. Previous studies have implied that the
reported hydroxylated aromatic inhibitors of IN are
potentially metal chelators.8,9,14,31 Thus, chelating met-
als at the active site of IN have been proposed to be
responsible for the inhibition of IN function. However,
hydroxylated aromatics are generally active only when
Mn2+ is used as a cofactor.8 The results of this study
indicate that the thiazolothiazepines are equally active
in Mg2+-based assays. Figure 1 shows a representative
gel comparing the inhibition of IN in the presence of
Mn2+ or Mg2+. Compounds 1, 19, and 20 were active in
the presence of Mg2+ within the same concentration
range as in Mn2+, thus indicating that these compounds
differ from hydroxylated aromatics and perhaps act at
different sites on IN. Inhibition of IN in the presence of
Mg2+ by thiazolothiazepines sets this class of compounds
apart from other IN inhibitors.32,33 The activity in Mg2+
might be related to the antiviral activity of the thiazo-
lothiazepine derivatives because Mg2+ has been pro-
posed to be the metal used in vivo by IN.
F igu r e 1. Inhibition of HIV-1 IN catalytic activities by
thiazolothiazepines. (A) IN assays: A 21-mer blunt-end oli-
gonucleotide corresponding to the U5 end of the HIV-1 proviral
DNA, 5′ end-labeled with 32P, is allowed to react with purified
HIV-1 IN. The initial step involves nucleolytic cleavage of two
bases from the 3′-end, resulting in a 19-mer oligonucleotide.
Subsequently, 3′-ends are then covalently joined to another
identical oligonucleotide, which serves as the target DNA. (B,
C) Concentration-dependent inhibition of HIV-1 IN by thia-
zolothiazepines 1, 19, and 20 using Mn2+ (B) and Mg2+ (C) as
cofactor: lane 1, DNA alone; lanes 2 and 15, DNA and
integrase; lanes 3-6, 7-10, and 11-14, DNA, integrase, and
compounds 1, 19, and 20 at 1000, 333, 111, and 37 µM,
respectively.
Selectivity. The selectivity of compounds 1, 19, and
20 was examined against other sites on the HIV
replication cycle. When tested against reverse tran-
scriptase, protease, virus attachment, or nucleocapsid
protein zinc fingers, none of the compounds exhibited
any detectable activities at 100 µM, suggesting selectiv-
ity against IN. Thus, thiazolothiazepines are potentially
important lead inhibitors of IN.
shape of compounds is as important as their substitu-
ents. The fact that thiazolothiazepines are equally
potent in Mg2+- and Mn2+-based assays suggests that
the IN binding site by these compounds differs from
previously reported inhibitors. Testing these compounds
against other viral proteins (reverse transcriptase,
protease, virus attachment, or nucleocapsid zinc fingers)
suggested selectivity for IN. Another advantage of this
class of compounds is that they are amenable for
preparation of chemical libraries using recent advances
in combinatorial chemistry. Cumulatively, this study
demonstrates that thiazolothiazepines are novel leads
for designing drugs against IN and HIV replication.
Con clu sion s
In an attempt to identify inhibitors of HIV-1 IN, we
tested compounds that were shown to possess antiviral
activity in the NCI Antiviral Drug Screening program.
Compound 1 was identified as a lead in both in vitro
and in vivo studies. In this study we have synthesized
and evaluated a series of thiazolothiazepines as poten-
tial antiviral compounds with activity against purified
IN. On the basis of the structure of tested compounds,
we can conclude that the best substituent for both
antiviral and anti-IN activity is the addition of a
benzene ring to the A ring. Further structure-activity
relationships provided evidence that geometry, size, and
Exp er im en ta l Section
Gen er a l. Where necessary, solvents were dried and purified
according to the recommended procedures. All reactions were
carried out under an argon atmosphere. Progress of the
reaction was monitored by TLC on silica gel plates (Riedel-
de-Haen, Art. 37341). Organic solutions were dried over
MgSO4; evaporation refers to removal of solvent on a rotary

3338 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Neamati et al.
evaporator under reduced pressure. Melting points were
determined using an Electrothermal 8103 apparatus and are
uncorrected. IR spectra were recorded as thin films on Perkin-
Elmer 398 and FT 1600 spectrophotometers. 1H NMR spectra
were recorded on a Bruker 200-MHz spectrometer with TMS
as internal standard. Mass spectral data were determined by
direct insertion at 70 eV with a VG70 spectrometer. Merck
silica gel (Kieselgel 60/230-400 mesh) was used for flash
chromatography columns. Dowex 50 × 2 200 resin (Aldrich)
was used for ion-exchange chromatography. Elemental analy-
ses were performed on a Perkin-Elmer 240C elemental ana-
lyzer, and the results are within (0.4% of the theoretical
values. Yields refer to purified products and are not optimized.
Ch em ica ls. All compounds were dissolved in DMSO, and
all aliquots were also made in DMSO prior to each experiment.
The stock solutions were kept at -20 °C.
P r ep a r a tion of Oligon u cleotid e Su bstr a tes. The HPLC-
purified oligonucleotides AE117, 5′-ACTGCTAGAGATTTTC-
CACAC-3′, and AE118, 5′-GTGTGGAAAATCTCTAGCAGT-3′,
were purchased from Midland Certified Reagent Co. (Midland,
TX). The expression system for the wild-type HIV-1 IN was a
generous gift of Drs. T. J enkins and R. Craigie, Laboratory of
Molecular Biology, NIDDK, NIH, Bethesda, MD. To analyze
the extents of 3′-processing and 3′-strand transfer using 5′-
end labeled substrates, AE118 was 5′-end labeled using T4
polynucleotide kinase (Gibco BRL) and [γ-32P]ATP (DuPont-
NEN). The kinase was heat-inactivated, and AE117 was added
to the same final concentration. The mixture was heated at
95 °C, allowed to cool slowly to room temperature, and run on
a G-25 Sephadex quick spin column (Boehringer Mannheim,
Indianapolis, IN) to separate annealed double-stranded oligo-
nucleotide from unincorporated label.
R efer en ce R ea gen t s for Mech a n ist ic a n d Ta r get -
Ba sed Assa ys. All positive control compounds for individual
assays except AZTTP were obtained from the NCI chemical
repository. The reference reagents for the individual assays
are as follows: attachment, Farmitalia (NSC 65016)34 and
dextran sulfate (NSC 620255); reverse transcriptase inhibition,
rAdT template/primer-AZTEC (Sierra BioResearch, Tuscon,
AZ) and rCdG template/primer-UC3835 (NSC 629243); protease
inhibition, KNI-27236 (NSC 651714); IN inhibitor, ISIS 532037
(NSC 665353) and DIBA-138 (NSC 654077), a NCp7 Zn finger
inhibitor.
IN Assa y. IN was preincubated at a final concentration of
200 nM with the inhibitor in reaction buffer (50 mM NaCl, 1
mM HEPES, pH 7.5, 50 µM EDTA, 50 µM dithiothreitol, 10%
glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/mL bovine serum
albumin, 10 mM 2-mercaptoethanol, 10% dimethyl sulfoxide,
and 25 mM MOPS, pH 7.2) at 30 °C for 30 min. Then, 20 nM
of the 5′-end 32P-labeled linear oligonucleotide substrate was
added, and incubation was continued for an additional 1 h.
Mg2+-based assays were carried essentially as described.39
Reactions were quenched by the addition of 8 µL of loading
dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene
cyanol, 0.025% bromophenol blue). An aliquot (5 µL) was
electrophoresed on a denaturing 20% polyacrylamide gel (0.09
M Tris-borate, pH 8.3, 2 mM EDTA, 20% acrylamide, 8 M
urea). Gels were dried, exposed in a Molecular Dynamics
PhosphorImager cassette, and analyzed using a Molecular
Dynamics PhosphorImager (Sunnyvale, CA). Percent inhibi-
tion was calculated using the following equation:
Hughes, ABL Basic Research, NCI-FCRDC, Frederick, MD)
have been previously described.40 The substrate cleavage of
recombinant HIV-1 protease in the presence of test compounds
was quantified using an HPLC-based methodology with the
artificial substrate Ala-Ser-Glu-Asn-Try-Pro-Ile-Val-amide (Mul-
tiple Peptide Systems, San Diego, CA) and has been previously
described.38,41 The anti-HIV drug testing performed at NCI is
based on a protocol described by Weislow et al.42
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Th ia ze-
p in es 2-4, 6-10, 12, 19, a n d 20. This procedure is illustrated
for the preparation of (()-1,11a-dihydro-7-methoxy-3H,5H,-
11H-thiazolo[4,3-c][1,4]benzothiazepine-5,11-dione (10). A mix-
ture of 2,2′-dithiobis(5-methoxybenzoic acid) (5.5 g, 15 mmol)
(32a )21 and thionyl chloride (40 mL) was refluxed for 1.5 h.
After cooling, the excess of thionyl chloride was removed under
vacuum with the aid of dry benzene (2 × 5 mL). The resulting
solid was dissolved in dry THF (40 mL), and the solution was
added dropwise to a mixture of L-thiaproline (33) (4.0 g, 30.0
mmol) and sodium carbonate (3.2 g, 15.0 mmol) in water (50
mL). Additional sodium carbonate was added from time to time
to maintain a weakly alkaline pH. The mixture was stirred
overnight, then concentrated, and made acidic (pH 3-4) by
adding concentrated HCl. The gummy solid was extracted into
ethyl acetate, and the resulting solution was washed with
water, dried, and evaporated to give a foam. Ion-exchange
column chromatography using methanol as the eluent gave
almost pure disulfide 36a as an amorphous solid. Such a
material (8.95 g, 15 mmol), was dissolved in 85% ethanol (100
mL) containing NaOH (1.2 g, 30 mmol) and to this was added
a solution of NaBH4 (1.14 g, 30 mmol) in ethanol (50 mL). The
mixture was gently refluxed for 0.5 h, then concentrated, and
diluted with chilled water. The cold solution was left at room
temperature for 15 min before being filtered and made acidic
(pH 3-4) by concentrated HCl. The gummy solid was treated
exactly as described before for purification of compound 36a
and then thoroughly dried under vacuum before being sub-
jected to the successive reaction without further manipulation.
Crude thiophenol derivative (6.0 g, 20.0 mmol) was dissolved
into dry THF (80 mL), and N,N′-carbonyldiimidazole (3.24 g,
20.0 mmol) was added in portions. The solution was stirred
for 24 h at room temperature and for 2 h under reflux. The
solvent was evaporated, and the residue was partitioned
between CHCl3 and 0.5 N HCl. The organic solution was
separated and washed with NaHCO3-saturated solution and
water. After drying and evaporation of the solvent, a pasty
residue was obtained and purified by flash chromatography
(8% methanol in EtOAc) to give 4.2 g (74% yield from the
thiophenol precursor) of 10 as a white powder: mp 145-147
1
°C (benzene); IR (KBr) 1700, 1640 cm-1; H NMR (CDCl3) δ
7.46 (d, 1 H, J ) 2.8 Hz), 7.35 (m, 1 H), 7.04 (dd, 1 H, J ) 8.1,
2.8 Hz), 4.88 (half of AB q, 1 H, J ) 10.2 Hz), 4.67 (half of AB
q, 1 H, J ) 10.2 Hz), 4.60 (dd, 1 H, J ) 6.6, 1.5 Hz), 3.87 (s, 3
H), 3.64 (dd, 1 H, J ) 12.7, 1.5 Hz), 3.15 (dd, 1 H, J ) 12.7,
6.6 Hz). Anal. (C12H11NO3S2) C, H, N.
(()-7-C h lo r o -2,3-d ih y d r o -5H -t h ia zo lo [2,3-c][1,4]-
ben zoth ia zep in e-5,11(11a H)-d ion e (2). Starting from 2,2′-
dithiobis(5-chlorobenzoic acid) (32b)22 (5.6 g, 15 mmol), the title
compound 2 was obtained (3.6 g, 63% yield from the thiophenol
precursor) adopting the same procedure as for 10 but using
thiazolidine-2-carboxylic acid (34)26 instead of L-thiaproline
(33): mp 231-232 °C (benzene); IR (KBr) 1695, 1640 cm-1
;
1H NMR (CDCl3) δ 7.98 (d, 1 H, J ) 2.4 Hz), 7.52-7.38 (m, 2
H), 5.35 (s, 1 H), 4.03 (m, 2 H), 3.16 (m, 2 H). Anal. (C11H8-
ClNO2S2) C, H, N.
% I ) 100 × [1 - (D - C)/(N - C)]
where C, N, and D are the fractions of 21-mer substrate
converted to 19-mer (3′-processing product) or 3′-strand-
transfer products for DNA alone, DNA plus IN, and IN plus
drug, respectively. IC50 values were determined by plotting the
drug concentration versus percent inhibition and determining
the concentration that produced 50% inhibition.
HIV-1 Cell a n d Ta r get-Ba sed Assa ys. The cell-based p24
attachment assay has been described in detail elsewhere.38
Assays for activity against HIV-1 reverse transcriptase rAdT
(template/primer) and rCdG (template/primer) using recom-
binant HIV-1 reverse transcriptase (a kind gift from S.
(()-7-B r o m o -2,3-d ih y d r o -5H -t h ia z o lo [2,3-c ][1,4]-
ben zoth ia zep in e-5,11(11a H)-d ion e (3). Starting from 2,2′-
dithiobis(5-bromobenzoic acid) (32c)22 (7.0 g, 15 mmol), the title
compound 3 was obtained (4.0 g, 70% yield from the thiophenol
precursor) adopting the same procedure as for 10 but using
thiazolidine-2-carboxylic acid (34)26 instead of L-thiaproline
(33): mp 219-220 °C (benzene); IR (KBr) 1695, 1640 cm-1
;
1H NMR (CDCl3) δ 8.11 (d, 1 H, J ) 2.2 Hz), 7.62 (dd, 1 H, J
) 8.3, 2.2 Hz), 7.32 (dd, 1 H, J ) 8.3, 2.2 Hz), 5.33 (s, 1 H),
4.00 (m, 2 H), 3.18 (m, 2 H). Anal. (C11H8BrNO2S2) C, H, N.

Thiazolothiazepine Inhibitors of HIV-1 Integrase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3339
(()-2,3-D ih y d r o -7-m e t h y l-5H -t h ia zo lo [2,3-c][1,4]-
ben zoth ia zep in e-5,11(11a H)-d ion e (4). Starting from 2,2′-
dithiobis(5-methylbenzoic acid) (32e)24 (5.0 g, 15.0 mmol), the
title compound 4 was obtained (5.6 g, 76% yield from the
thiophenol precursor) adopting the same procedure as for 10
but using thiazolidine-2-carboxylic acid (34)26 instead of L-
thiaproline (33): mp 197-199 °C (benzene-petroleum ether);
1630 cm-1; 1H NMR (CDCl3) δ 8.52 (s, 1 H), 7.99 (s, 1 H), 7.93
(m, 1 H), 7.84 (m, 1 H,), 7.63 (m, 2 H), 5.40 (s, 1 H), 4.11 (m,
2 H), 3.16 (m, 2 H); MS m/z 301 (M+), 273 (100), 186, 158,
142, 114. Anal. (C15H11NO2S2) C, H, N.
(()-1,13a -Dih yd r o-3H,5H,13H-n a p h th o[2,3-f]th ia zolo-
[4,3-c][1,4]th ia zep in e-5,13-d ion e (20). Starting from 3,3′-
dithiobis(2,2′-naphthoic acid) (32f) (1.22 g, 1.5 mmol), the title
compound 20 (0.34 g, 38% yield from the thiophenol precursor)
was obtained, using the same procedure as for 10 but carrying
out the NaBH4 reduction of the disulfide overnight at room
1
IR (KBr) 1690, 1645 cm-1; H NMR (CDCl3) δ 7.79 (s, 1 H,),
7.29 (m, 2 H), 5.36 (s, 1 H), 4.02 (m, 2 H), 3.12 (m, 2 H), 2.42
(s, 3 H). Anal. (C12H11NO2S2) C, H, N.
1
(()-7-Ch lor o-1,11a -d ih yd r o-3H,5H,11H-th ia zolo[4,3-c]-
[1,4]ben zoth ia zep in e-5,11-d ion e (6). Starting from 2,2′-
dithiobis(5-chlorobenzoic acid) (32b)22 (5.6 g, 15 mmol), the title
compound 6 was obtained (4.0 g, 70% yield from the thiophenol
precursor) using the same procedure as for 10: mp 204-205
temperature: mp 200-203 °C; IR (KBr) 1695, 1630 cm-1; H
NMR (CDCl3) δ 8.51 (s, 1 H), 7.98 (s, 1 H), 7.94 (m, 1 H), 7.84
(m, 1 H), 7.58 (m, 2 H), 4.97 (half of AB q, 1 H, J ) 10.6 Hz),
4.73 (half of AB q, 1 H, J ) 10.6 Hz), 4.66 (dd, 1 H, J ) 6.9,
1.6 Hz), 3.65 (dd, 1 H, J ) 11.8, 1.5 Hz), 3.14 (dd, 1 H, J )
12.1, 6.7 Hz); MS m/z 301 (M+), 273 (100), 186, 158, 142, 114.
Anal. (C15H11NO2S2) C, H, N.
1
°C (benzene); IR (KBr) 1705, 1640 cm-1; H NMR (CDCl3) δ
7.96 (d, 1 H, J ) 2.0 Hz), 7.51-7.38 (m, 2 H), 4.72 (half of AB
q, 1 H, J ) 10.7 Hz), 4.57 (dd, 1 H, J ) 6.9, 1.7 Hz), 3.67 (dd,
1 H, J ) 11.9, 1.7 Hz), 3.19 (dd, 1 H, J ) 11.9, 6.9 Hz). Anal.
(C11H8ClNO2S2) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Su lfoxid es
15-18. This procedure is illustrated for the preparation of
(()-trans-1,11a-dihydro-3H,5H,11H-thiazolo[4,3-c][1,4]ben-
zothiazepine-5,11-dione 2-oxide (16). To a stirred and cooled
(0 °C) solution of compound 515 (0.5 g, 2.0 mmol) in dry
dichloromethane (5 mL) was added ∼80% 3-chloroperbenzoic
acid (0.43 g, ∼2 mmol) in 8 mL of the same solvent dropwise
over about 15 min. After an additional 2 h at 0 °C, the reaction
mixture was filtered and the filter cake was rinsed with
dichloromethane. The combined solution was washed twice
with 5% aqueous K2CO3, dried, and evaporated to give the
crude 16 (0.47 g, 89% yield), which solidified on trituration
with hexane: mp 191-194 °C (benzene); IR (KBr) 1695, 1670
(()-7-Br om o-1,11a -d ih yd r o-3H,5H,11H-th ia zolo[4,3-c]-
[1,4]ben zoth ia zep in e-5,11-d ion e (7). Starting from 2,2′-
dithiobis(5-bromobenzoic acid) (32c)22 (7.0 g, 15 mmol), the title
compound 7 was obtained (4.1 g, 63% yield from the thiophenol
precursor) using the same procedure as for 10: mp 213-214
1
°C (benzene); IR (KBr) 1700, 1635 cm-1; H NMR (CDCl3) δ
8.10 (t, 1 H, J ) 1.0 Hz), 7.63 (dd, 1 H, J ) 8.0, 1.0 Hz), 7.34
(d, 1 H, J ) 8.0 Hz), 4.90 (half of AB q, 1 H, J ) 10.6 Hz), 4.71
(half of AB q, 1 H, J ) 10.6 Hz), 4.55 (dd, 1 H, J ) 6.0, 1.9
Hz), 3.67 (dd, 1 H, J ) 11.8, 1.9 Hz), 3.18 (dd, 1 H, J ) 11.8,
6.0 Hz). Anal. (C11H8BrNO2S2) C, H, N.
cm-1 1H NMR (CDCl3) δ 8.04 (m, 1 H), 7.55 (m, 3 H), 5.75
;
(dd, 1 H, J ) 13.1, 3.0 Hz), 5.14 (t, 1 H, J ) 7.6 Hz), 3.91 (d,
1 H, J ) 13.1 Hz), 3.72 (dd, 1 H, J ) 14.6, 7.6 Hz), 3.23 (ddd,
1 H, J ) 14.6, 7.6, 3.0 Hz). Anal. (C11H9NO3S2) C, H, N.
(()-1,11a -Dih yd r o-7-m eth yl-3H,5H,11H-th ia zolo[4,3-c]-
[1,4]ben zoth ia zep in e-5,11-d ion e (8). Starting from 2,2′-
dithiobis(5-methylbenzoic acid) (32e)24 (5.0 g, 15.0 mmol), the
title compound 8 (6.1 g, 82% yield from the thiophenol
precursor) was obtained as a thick oil, using the same
(()-tr a n s-2,3-Dih yd r o-7-m eth yl-5H-th ia zolo[2,3-c][1,4]-
ben zoth ia zep in e-5,11(11a H)-d ion e 1-Oxid e (15). Starting
from 4 (0.53 g, 2.0 mmol), the title compound 15 (0.39 g, 73%
yield) was obtained using an identical procedure as for 16: mp
procedure as for 10: IR (KBr) 1695, 1640 cm-1 1H NMR
;
(CDCl3) δ 7.74 (s, 1 H,), 7.30 (m, 2 H), 4.87 (half of AB q, 1 H,
J ) 10.2 Hz), 4.66 (half of AB q, 1 H, J ) 10.2 Hz), 4.60 (dd,
1 H, J ) 6.8, 1.7 Hz), 3.62 (dd, 1 H, J ) 12.1, 1.7 Hz), 3.23
(dd, 1 H, J ) 12.1, 6.8 Hz), 2.40 (s, 3 H). Anal. (C12H11NO2S2)
C, H, N.
211-212 °C (benzene); IR (KBr) 1690, 1670 cm-1 1H NMR
;
(CDCl3) δ 7.79 (d, 1 H, J ) 2.5 Hz), 7.38 (m, 2 H), 5.25 (s, 1
H), 4.52 (m, 1 H), 3.40 (m, 1 H), 3.10 (m, 1 H), 2.47 (t, 3 H).
Anal. (C12H11NO3S2) C, H, N.
(()-1,11a -Dih yd r o-8-n it r o-3H ,5H ,11H -t h ia zolo[4,3-c]-
[1,4]ben zoth ia zep in e-5,11-d ion e (9). Starting from 2,2′-
dithiobis(4-nitrobenzoic acid) (32d )23 (5.9 g, 15 mmol), the title
compound 9 was obtained (2.0 g, 34% from the thiophenol
precursor) using the same procedure as for 10: mp 231-232
°C (benzene-petroleum ether); IR (KBr) 1715, 1645 cm-1; 1H
NMR (CDCl3) δ 8.35 (s, 1 H), 8.32 (dd, 1 H, J ) 8.1, 2.0 Hz),
8.16 (d, 1 H, J ) 8.1 Hz), 4.90 (half of AB q, 1 H, J ) 10.5 Hz),
4.77 (half of AB q, 1 H, J ) 10.5 Hz), 4.54 (dd, 1 H, J ) 6.4,
1.5 Hz), 3.70 (dd, 1 H, J ) 11.9, 1.5 Hz), 3.23 (dd, 1 H, J )
11.9, 6.4 Hz). Anal. (C11H8N2O4S2) C, H, N.
(()-tr a n s-7-Ch lor o-1,11a -d ih yd r o-3H,5H,11H-th ia zolo-
[4,3-c][1,4]ben zoth ia zep in e-5,11-d ion e 2-Oxid e (17). Start-
ing from 6 (0.57 g, 2.0 mmol), the title compound 17 (0.41 g,
68% yield) was obtained using an identical procedure as for
1
16: mp 213-215 °C (benzene); IR (KBr) 1695, 1660 cm-1; H
NMR (CDCl3) δ 8.04 (d, 1 H, J ) 2.7 Hz), 7.50 (m, 2 H), 5.75
(dd, 1 H, J ) 12.9, 2.9 Hz), 5.13 (t, 1 H, J ) 7.8 Hz), 3.91 (d,
1 H, J ) 13.0 Hz), 3.73 (dd, 1 H, J ) 14.7, 7.2 Hz), 3.23 (ddd,
1 H, J ) 14.7, 7.8, 2.9 Hz). Anal. (C11H8ClNO3S2) C, H, N.
(()-tr a n s-1,11a-Dih ydr o-7-m eth oxy-3H,5H,11H-th iazolo-
[4,3-c][1,4]ben zoth ia zep in e-5,11-d ion e 2-Oxid e (18). Start-
ing from 10 (0.56 g, 2.0 mmol), the title compound 18 (0.42 g,
71%yield) was obtained using an identical procedure as for
(()-1,11a -D ih y d r o -3H ,5H ,11H -o x a zo lo [4,3-c][1,4]-
ben zoth ia zep in e-5,11-d ion e (12). Starting from commercial
2,2′-dithiodibenzoic acid (32g) (4.6 g, 15.0 mmol), the title
compound 12 was obtained as a thick oil (3.0 g, 42% yield from
the thiophenol precursor) adopting the same procedure as for
10 but using freshly prepared 1,3-oxazolidine-4-carboxylic acid
(32)27 (3.5 g, 30.0 mmol) instead of L-thiaproline (33): IR (KBr)
1
16: mp 172-174 °C (benzene); IR (KBr) 1690, 1660 cm-1; H
NMR (CDCl3) δ 7.55 (d, 1 H, J ) 2.7 Hz), 7.40 (d, 1 H, J ) 8.3
Hz), 7.09 (dd, 1 H, J ) 8.3, 2.7 Hz), 5.76 (dd, 1 H, J ) 12.9,
2.8 Hz), 5.17 (t, 1 H, J ) 7.6 Hz), 3.91 (d, 1 H, J ) 12.9 Hz),
3.89 (s, 3 H), 3.73 (dd, 1 H, J ) 14.7, 7.1 Hz), 3.23 (ddd, 1 H,
J ) 14.7, 7.6, 2.8 Hz). Anal. (C12H11NO4S2) C, H, N.
1
1690, 1635 cm-1; H NMR (CDCl3) δ 7.50 (m, 1 H), 7.22 (m,
3H), 5.27 (half of AB q, 1 H, J ) 5.3 Hz), 5.17 (half of AB q, 1
H, J ) 5.3 Hz), 4.81 (dd, 1 H, J ) 8.8, 1.7 Hz), 4.25 (dd, 1 H,
J ) 5.9, 1.6 Hz), 3.98 (dd, 1 H, J ) 8.9, 5.9 Hz); MS m/z
235 (M+), 207, 177, 150, 136 (100), 108. Anal. (C11H9NO3S) C,
H, N.
(()-2,3-Dih yd r o-5H-n a p h th o[2,3-f]th ia zolo[2,3-c][1,4]-
th ia zep in e-5,13(13a H)-d ion e (19). Starting from 3,3′-dithio-
bis(2,2′-naphthoic acid)25 (32f) (1.22 g, 1.5 mmol), the title
compound 19 (0.27 g, 30% yield from the thiophenol precursor)
was obtained, adopting the same procedure as for 10 but using
thiazolidine-2-carboxylic acid (34) instead of L-thiaproline (33)
and carrying out the NaBH4 reduction of the disulfide over-
night at room temperature: mp 196-198 °C; IR (KBr) 1700,
(()-2,3,11,11a-Tetr ah ydr o-11-h ydr oxy-11-ph en yl-1H,5H-
p yr r olo[2,1-c][1,4]ben zoth ia zep in -5-on e (26). A solution of
compound 3716 (0.88 g, 3.8 mmol) in dry THF (10 mL) was
dropwise added to a solution of PhMgBr in ethyl ether
(obtained from 1.23 g of PhBr, 0.185 g of Mg turnings, and
8 mL of Et2O). The mixture was refluxed for 30 min, then
cooled to room temperature, and quenched by the addition of
NH4Cl-saturated solution. Chloroform extraction and evapora-
tion gave a residue which was purified by column chromatog-
raphy to give the title compound 26 (0.7 g, 59% yield) along
with some other unidentified byproducts. The title compound
was obtained as colorless crystals by crystallization: mp 137-
1
141 °C (ethanol); IR (KBr) 3210 broad, 1620 cm-1; H NMR

3340 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Neamati et al.
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Fabrizi, G.; Fiorini, I.; Lamba, D.; Nacci, V. Thioanalogues of
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11a-tetrahydro-5H,11H- and 5-oxo-2,3,11,11a-tetrahydro-1H,5H-
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derivatives. I. Benzalthio- and bisbenzaldithio-salicylhydrazides.
J . Org. Chem. 1953, 18, 1380-1402.
(CDCl3) δ 8.02 (m, 1 H), 7.85 (m, 1 H), 7.60-7.10 (m, 7 H),
3.87 (m, 1 H), 3.55 (m, 2 H), 3.08 (br s, 1 H), 1.95 (m, 4 H).
Anal. (C18H17NO2S) C, H, N.
(()-tr a n s-2,3,11,11a -Tetr a h yd r o-11-(4-m eth ylp ip er a zi-
n o)-1H,5H-p yr r olo[2,1-c][1,4]ben zoth ia zep in -5-on e (27).
A mixture of (+)-11-chloro-2,3,11,11a-tetrahydro-1H,5H-pyr-
rolo[2,1-c][1,4]benzothiazepin-5-one (38)15 (0.6 g, 2.4 mmol),
freshly distilled 1-methylpiperazine (0.34 mL, 3.1 mmol),
Na2CO3 (1.06 g, 10.0 mmol), and NaI (0.36 g, 2.4 mmol) in dry
CH3CN (30 mL) was gently refluxed for 20 h. The solvent was
evaporated, and the residue was partitioned between CH2Cl2
and water. The organic phase was successively washed with
a 2% solution of Na2S2O3, water, and brine. The residue
obtained after evaporation was chromatographed on silica gel
eluting with 5% methanol in CH2Cl2. The title compound 27
was obtained as a white solid (0.55 g, 73% yield): mp 188-
1
190 °C (2-propanol-isopropyl ether); IR (KBr) 1630 cm-1; H
NMR (CDCl3) δ 7.71 (m, 1 H), 7.50 (m, 2 H), 7.61 (m, 1 H),
7.33 (m, 2 H), 4.36 (d, 1 H, J ) 11.2 Hz), 3.70 (m, 3 H), 2.58
(m, 4 H), 2.38 (m, 4 H), 2.27 (s, 3 H), 2.02 (m, 4 H); MS m/z
317 (100, M+), 217, 180, 139, 99, 70. Anal. (C17H23N3OS) C, H,
N.
(()-3-[(4-Meth ylp ip er a zin -1-yl)m eth yl]-11-p h en yl-5H,-
11H-p yr r olo[2,1-c][1,4]ben zoth ia zep in e (31). A solution of
compound 3920 (0.55 g, 2.0 mmol), paraformaldehyde (0.1 g),
and 1-methylpiperazine dihydrochloride (0.52 g, 3.0 mmol) in
CH3OH (20 mL) was refluxed for 48 h. After cooling, the
mixture was diluted with water and made alkaline (pH 9-10)
by dropwise addition of 1 N NaOH. The oil formed was
extracted with ethyl acetate. The organic layer was washed
with water and dried. After evaporation of the solvent, the
residue obtained was chromatographed (5% CH3OH in
CH2Cl2) to give the title compound 31 as a white solid (0.7 g,
1
91% yield): mp 161-162 °C (2-propanol); H NMR (CDCl3) δ
7.50-7.00 (m, 9 H), 5.97 (s, 1 H), 5.86 (d, 1 H, J ) 3.5 Hz),
5.53 (d, 1 H, J ) 3.5 Hz), 5.44 (half of AB q, 1 H, J ) 14.5 Hz),
5.31 (half of AB q, 1 H, J ) 14.5 Hz), 3.52 (half of AB q, 1 H,
J ) 13.5 Hz), 3.52 (half of AB q, 1 H, J ) 13.5 Hz), 2.52 (m, 8
H), 2.30 (s, 3 H). Anal. (C24H27N3S) C, H, N.
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