Synthesis of novel thiazolothiazepine based HIV-1 integrase inhibitors.

Article abstract of DOI:10.1016/j.bmc.2004.05.037

Thiazolothiazepines are among the smallest and most constrained inhibitors of human immunodeficiency virus type-1 integrase (HIV-1 IN) inhibitors (J. Med. Chem. 1999, 42, 3334). Previously, we identified two thiazolothiazepines lead IN inhibitors with antiviral activity in cell-based assays. Structural optimization of these molecules necessitated the design of easily synthesizable analogs. In order to design similar molecules with least number of substituent, herein we report the synthesis of 10 novel analogs. One of the new compounds (1) exhibited similar potency as the reference compounds, confirming that a thiazepinedione fused to a naphthalene ring system is the best combination for the molecule to accommodate into the IN active site. Thus, the replacement of sulfur in the thiazole ring with an oxygen does not seem considerably affect potency. On the other hand, the introduction of an extra methyl group at position 1 of the polycyclic system or the shift from a thiazepine to an oxazepine skeleton decreased potency. In order to understand their mode of interactions with IN active site, we docked all the compounds onto the previously reported X-ray crystal structure of IN. We observed that compounds 7-9 occupied an area close to D64 and Mg(2+) and surrounded by amino acid residues K159, K156, N155, E152, D116, H67, and T66. The oxygen atom of the oxazolo ring of 7 and 8 could chelate Mg(2+). These results indicate that the new analogs potentially interact with the highly conserved residues important for IN catalytic activities.

Full text of DOI:10.1016/j.bmc.2004.05.037

Bioorganic & Medicinal Chemistry 12 (2004) 4459–4466
Synthesis of novel thiazolothiazepine based HIV-1 integrase
inhibitors
Francesca Aiello,a Antonella Brizzi,b Antonio Garofalo,a,* Fedora Grande,a
Gaetano Ragno,a Raveendra Dayamc and Nouri Neamatic,*
aDipartimento di Scienze Farmaceutiche, Universitꢀa della Calabria, 87036 Arcavacata di Rende (Cs), Italy
bDipartimento Farmaco Chimico Tecnologico, Universitꢀa di Siena, Via A, Moro, 53100 Siena, Italy
cDepartment of Pharmaceutical Sciences, School of Pharmacy, PSC 304, University of Southern California, 1985 Zonal Avenue,
Los Angeles, CA 90089, USA
Received 14 April 2004; revised 26 May 2004; accepted 28 May 2004
Available online 26 June 2004
Abstract—Thiazolothiazepines are among the smallest and most constrained inhibitors of human immunodeficiency virus type-1
integrase (HIV-1 IN) inhibitors (J. Med. Chem. 1999, 42, 3334). Previously, we identified two thiazolothiazepines lead IN inhibitors
with antiviral activity in cell-based assays. Structural optimization of these molecules necessitated the design of easily synthesizable
analogs. In order to design similar molecules with least number of substituent, herein we report the synthesis of 10 novel analogs.
One of the new compounds (1) exhibited similar potency as the reference compounds, confirming that a thiazepinedione fused to a
naphthalene ring system is the best combination for the molecule to accommodate into the IN active site. Thus, the replacement of
sulfur in the thiazole ring with an oxygen does not seem considerably affect potency. On the other hand, the introduction of an extra
methyl group at position 1 of the polycyclic system or the shift from a thiazepine to an oxazepine skeleton decreased potency. In
order to understand their mode of interactions with IN active site, we docked all the compounds onto the previously reported X-ray
crystal structure of IN. We observed that compounds 7–9 occupied an area close to D64 and Mg2þ and surrounded by amino acid
residues K159, K156, N155, E152, D116, H67, and T66. The oxygen atom of the oxazolo ring of 7 and 8 could chelate Mg2þ. These
results indicate that the new analogs potentially interact with the highly conserved residues important for IN catalytic activities.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
two distinct processes referred to as 30-processing and
strand transfer, respectively.2;3
Among the three viral enzymes encoded by the pol gene
of HIV-1, namely reverse transcriptase (RT), protease
(PR), and integrase (IN), only the latter still lacks FDA
approved drugs. Currently adopted cocktails of RT and
PR inhibitors although highly effective, suffer from
several unwanted effects like toxicity, patient adherence,
and emergence of drug-resistant viral phenotypes.1
Therefore, IN represents an attractive and validated
target because it is essential for viral replication cycle
and has no cellular homologue. IN mediates the inte-
gration of retroviral DNA into host chromosomes by
The availability of in vitro assays using recombinant IN
has allowed the discovery of numerous compounds
belonging to different chemical classes. The two most
predominant classes of inhibitors are the hydroxylated
aromatics and the b-diketoacid containing compounds
(for recent reviews, see Refs. 4–6). Currently, two exam-
ples of latter class are under clinical investigations.7;8
Previously, we discovered several novel thiazolothiaze-
pines as IN inhibitors with antiviral activity in cell-based
assays.9 Moreover, unlike many other classes of inhibi-
tors, they retain activity in the presence of Mg2þ as a
cofactor. Due to their low cytotoxicity, low molecular
weight, drug-like properties, and structural novelty we
embarked on a study to perform structural optimiza-
tion. In order to perform an extensive SAR studies, we
decided to design simpler molecules based on the parent
Keywords: HIV-1 integrase inhibitors; Structure-based drug design;
Docking; Thiazolothiazepines; Oxazepines.
* Corresponding authors. Tel.: +39-984-493118; fax: +39-984-493298
(A.G.); tel.: +1-323-442-2341; fax: +1-323-442-1390 (N.N.); e-mail
addresses: garofalo@unical.it; neamati@usc.edu
0968-0896/$ - see front matternbsp;Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.05.037

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F. Aiello et al. / Bioorg. Med. Chem. 12 (2004) 4459–4466
active starting aminoacids were used. Only in the case of
-threonine, optically active final compounds were ob-
tained, because racemization was prevented by the
presence of the methyl group adjacent to the a-carbon of
the amino acid.
O
S
L
b
a
c
N
O
Figure 1. Common structural motif of novel compounds presented in
this study.
2.2. Inhibition of IN by thiazepinedione and oxazepined-
iones
compounds that are easier to synthesize than the pre-
viously reported compounds.
Previously, we identified thiazolothiazepines (Tz-19 and
Tz-20, Table 1) as representatives of a novel class of
HIV-1 IN inhibitors with moderate activities in cell-
based assays.9 In an attempt to establish a coherent
structure–activity relationship amongst thiazolothiaze-
pines we wanted to know whether the sulfur atom could
be replaced by an oxygen and whether a naphthalene
ring could be replaced by fused rings. We also wanted to
employ structure-based optimization methods by
determining all the amino acid residues on the active site
of IN that are important for ligand binding. Our results
presented in Table 1 show that (1) sulfur is preferred
over oxygen, (2) addition of a methyl group at position 1
lowers potency, and (3) fused ring structures can be
tolerated without a significant loss of potency. However,
additional compounds must be tested to establish a
quantitative structure–activity relationship.
The structural motif common to these compounds
consists of a central thiazepinedione moiety (a) fused to
a carbocyclic aromatic system (benzo or naphtho) (b),
and to a 1,3-thiazolidine ring (c), as depicted in Figure 1.
Several structural modifications on the reference com-
pounds have been planned in an attempt to identify
novel compounds with similar potency. Herein, we re-
port the preparation and preliminary biological evalu-
ation of derivatives obtained by: (1) the replacement of
sulfur atom(s) by oxygen or (2) the introduction of a
methyl group on position 1 of the polycyclic system, and
(3) a combination of both features, while maintaining a
fused benzo or naphtho moiety with a various geometry
(Table 1). Such alterations should cause negligible vari-
ation in both lipophilicity and overall topology of rings,
with potential to coordinate a metal cofactor on the IN
active site.
2.3. Docking studies
In order to identify the biologically active conformation
of the compounds 1–10, we docked all the compounds
onto the active site of IN using GOLD. The GOLD fit-
ness scores along with structures are given in Table 1
while the H-bonding interactions and the active site
amino acid residues that interact with compounds are
given in Table 2. Compounds 1–6 and 10 occupied a wide
cavity surrounded by the active site amino acid residues
E152, I151, P142, I141, G140, F139, N117, D116, H114,
D64, and Q62. The bound conformation of the com-
pounds 1 and 6 inside the active site of IN is shown in
Figure 2. Compounds 7–9 occupied an area close to D64
and Mg2þ surrounded by amino acid residues K159,
K156, N155, E152, D116, H67, and T66. The oxygen
atom of the oxazolidine ring of 7 and 8 formed a coor-
dination bond with Mg2þ. An H-bonding interaction is
observed between H67 and compounds 8 and 9 (Table 2).
2. Results and discussion
2.1. Chemistry
The synthesis of thiazepinediones 1–3 has been accom-
plished following the general method outlined in Scheme
1 and as previously described using 2,20-dithiodibenzoic
acid 11 or 3,30-dithio-2,20-dinaphthoic acid 12 and an
appropriate cyclic amino acid 15 or 16 (Chart 1), as
starting materials. A modification of the above proce-
dure was developed for the synthesis of oxazepinedione
derivatives.9–15 2-Acetoxybenzoic acid 17 or the proper
acetoxynaphthoic acid 18–20 was converted to the cor-
responding acid chloride,10 which was in turn added to a
cyclic amino acids 13–16 (Chart 1) giving amides of
general formula 21, in good yields. (R)-())-Thiazolidine-
4-carboxylic acid 13 and ( )-thiazolidine-2-carboxylic
acid 14 are commercially available, while oxazolidine-4-
carboxylic acid 15 and (4S,5R)-oxazolidine-5-methyl-4-
carboxylic acid 16 were prepared starting from formal-
3. Conclusions
dehyde and L-serine or L-threonine, respectively, and in
situ N-acylation as described.11 The hydroxyacids 22, in
turn obtained by controlled hydrolysis of 21, were
subjected to lactonization using 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDC)/4-
(dimethylamino)pyridine (DMAP)13 (method A) or
We designed and synthesized novel and simplified ana-
logs of previously reported thiazolothiazepines. Some of
the new analogs showed similar potency as reference
compounds and amenable to further structural modifi-
cations for enhanced potency and selectivity. Further-
more, our docking studies demonstrate that these
compounds interact with key residues as well as divalent
metal ion important for the catalytic activities of IN.
Further structural modifications will be reported in due
course.
2-chloro-1-methylpyridinium
iodide/triethylamine
(TEA)14 (method B), or acetic anhydride (method C) as
dehydrating agents, leading to polycyclic compounds
4–10 (Scheme 2). It is worth noting that racemization
occurred during synthetic elaboration when optically

F. Aiello et al. / Bioorg. Med. Chem. 12 (2004) 4459–4466
4461
Table 1. Inhibition of HIV-1 integrase catalytic activities and docking scores of thiazepinediones and oxazepinediones
No
Structure
IC50 (lM)
Strand transfer
GOLD score
34.18
30-Processing
40
O
O
S
Tz-19
47
90
S
N
N
O
S
1
95
31.60
O
3
O
O
H
S
CH
2
910
800
904
450
92
900
700
873
400
100
90
36.24
37.78
33.85
32.21
35.28
37.17
35.30
34.07
32.23
N
O
O
O
H
S
CH
3
3
N
O
O
N
O
O
4
S
O
O
O
5
S
N
O
O
O
O
S
Tz-20
N
S
S
O
O
6
7
8
9
100
150
200
350
N
O
O
100
200
300
N
O
O
O
O
H
CH
3
N
O
O
O
O
N
N
S
S
O
O
O
10
300
300
34.42
O

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F. Aiello et al. / Bioorg. Med. Chem. 12 (2004) 4459–4466
400 mesh) was used for flash chromatography columns.
Elemental analyses were performed on a Perkin–Elmer
240C elemental analyzer, and the results are within
0.4% of the theoretical values. Yields refer to purified
products and are not optimized.
R
S
R
S
CO2H
R''
2
2
a,b
R'
CO2H
R'
CO
N
O
11 R = R' = H
12 R-R' = CH=CH-CH=CH
R'' = H, Me
c,d
4.2. ( )-1,13a-Dihydro-3H,5H,13H-naphtho[2,3-f]oxaz-
olo[4,3-c][1,4]thiazepine-5,13-dione (1)
1-3
This compound was prepared starting from oxazolidine-
4-carboxylic acid 15 and 3,30-dithio-2,20-dinaphthoic
acid 12, essentially as described.9 Compound 1 was
obtained as a colorless solid (21% for the entire process
from 15): mp 154–156 °C (dichloromethane/petroleum
Scheme 1. Reagents and conditions: (a) SOCl2/reflux; (b) 15 or 16/
Na2CO3/THF/H2O/rt; (c) NaBH4/EtOH/reflux; (d) CDI/THF/reflux.
1
ether); IR (KBr) 1695, 1635 cmꢁ1; H NMR (CDCl3) d
CO2H
CO2H
CO2H
S
CO2H
Me
8.57 (1H, s), 8.02 (1H, s), 7.97 (1H, m), 7.85 (1H, m),
7.63 (2H, m), 5.34 (1H, 0.5 of ABq, J ¼ 5:4 Hz), 5.23
(1H, 0.5 of ABq, J ¼ 5:4 Hz), 4.85 (1H, d, J ¼ 8:6 Hz),
4.34 (1H, d, J ¼ 5:8 Hz), 3.98 (1H, dd, J ¼ 8:6 and
5.8 Hz); MS (EI) m=z 285 (Mþ). Anal. (C15H11NO3S) C,
H, N.
HN
HN
HN
HN
S
O
O
13
14
15
16
Chart 1.
4. Experimental
4.3. (1R,11aS)-1,11a-Dihydro-1-methyl-3H,5H,11H-ox-
azolo[4,3-c][1,4]benzothiazepin-5,11-dione (2)
4.1. Chemistry
Starting from (4S,5R)-oxazolidine-5-methyl-4-carbox-
ylic acid 16 and 2,20-dithiodibenzoic acid 11, compound
2 was obtained as colorless crystals (32% for the entire
All reactions were carried out under a nitrogen atmo-
sphere. Progress of the reaction was monitored by TLC
on silica gel plates (Riedel-de-Haen, Art. 37341). Or-
ganic solutions were dried over MgSO4 and evaporated
on a rotary evaporator under reduced pressure. Melting
points were measured using an Electrothermal 8103
process from 11): mp 94–96 °C (diethyl ether/light
21
petroleum); ½aꢀ )95.5 (c 1, CHCl3); IR (KBr) 1705,
D
1
1645 cmꢁ1; H NMR (CDCl3) d 8.01 (1H, m), 7.47 (3H,
m), 5.51 (1H, 0.5 of ABq, J ¼ 5:8 Hz), 5.02 (1H, 0.5 of
ABq, J ¼ 5:8 Hz), 4.95 (1H, m), 3.83 (1H, d, J ¼ 3:7),
1.31 (3H, d, J ¼ 6:2); MS (EI) m=z 249 (Mþ). Anal.
(C12H11NO3S) C, H, N.
apparatus and are uncorrected. Specific rotations ½aꢀ
D
were determined to the stated temperature and condi-
tions by a Perkin–Elmer 343 polarimeter. IR spectra
were recorded as thin films on Perkin–Elmer 398 and FT
1600 spectrophotometers. 1H NMR spectra were re-
€
corded on a Bruker 200-MHz spectrometer with TMS as
4.4. (1R,13aS)-1,13a-Dihydro-1-methyl-3H,5H,13H-
naphtho[2,3-f]oxazolo[4,3-c][1,4]thiazepine-5,13-dione (3)
an internal standard: chemical shifts are expressed in d
values (ppm) and coupling constants ðJÞ in Hz. Mass
spectral data were determined at 70 eV with a VG70
spectrometer. Merck silica gel (Kieselgel 60/230–
Starting from (4S,5R)-oxazolidine-5-methyl-4-carbox-
ylic acid 16 and 3,30-dithio-2,20-dinaphthoic acid 12,
R
R
R'
OAc
R'
OAc
CO2H
a,b
R''
CO2H
N
R''
CO
R'''
R'''
21
17 R = R' = R'' = R''' = H
18 R = R''' = H, R'-R'' = CH=CH-CH=CH
19 R = R' = H, R''-R''' = CH=CH-CH=CH
c
20 R'' = R''' = H, R'-R'' = CH=CH-CH=CH
R
R'
OH
CO
22
CO2H
d
4-10
N
R''
R'''
Scheme 2. Reagents and conditions: (a) (COCl)2/toluene/DMF; (b) cyclic aminoacid/THF–H2O/pH 8/rt; (c) Na2CO3/H2O/rt; (d) cyclization (see
Experimental).

F. Aiello et al. / Bioorg. Med. Chem. 12 (2004) 4459–4466
4463
Table 2. H-bonding interactions and the active site amino acids residues interact with compounds 1–10
ꢁ
H-bonding interactions (A)
Compound
Interacting amino acid residues
1
––
––
E152, I151, P142, D116, H114, D64, Q62
E152, I151, P142, D116, H114, D64, Q62
I151, P142, I141, F139, N117, D116, H114, Q62
E152, I151, P142, F139, H114, D116
2
3
––
N–C@Oꢂ ꢂ ꢂHN Q62 (3.13)
4
5
––
––
E152, I151, Q148, P142, P141, H114, D116, D64
E152, I151, P142, D116, D64, Q62
K159, K156, N155, T66, D64
6
7a
8a
9
––
O–C@Oꢂ ꢂ ꢂHN H67 (2.66)
O–C@Oꢂ ꢂ ꢂHN H67 (2.08)b
––
K159, K156, N155, E152, D116, T66, D64
K159, K156, N155, E152, T66, D64
E152, I151, P142, G140, D116, H114, D64, Q62
10
a
2þ
The oxygen atoms of oxazolidine ring of 7 and 8 coordinate with active site Mg2þ ion; Oꢂ ꢂ ꢂMg2þ (2.06 A), Oꢂ ꢂ ꢂMg (1.94 A).
ꢁ
ꢁ
b Backbone NH of H67.
Figure 2. The bound conformation of molecules Tz-19 (A), Tz-20 (B), 1 (C), and 6 (D) inside the active site of IN.
compound 3 was obtained as colorless needles (37% for
the entire process from 12): mp 163–165 °C (diethyl
s), 8.00 (1H, m), 7.80 (1H, m), 7.60 (2H, m), 5.56 and
5.09 (2H, ABq, J ¼ 5:5 Hz), 4.99 (1H, dq, J ¼ 6:5 and
3.4 Hz), 3.93 (1H, d, J ¼ 3:4), 1.29 (3H, d, J ¼ 6:5); MS
(CI) m=z 300 (MHþ). Anal. (C16H13NO3S) C, H, N.
21
ether); ½aꢀ )121.0 (c 1, CHCl3); IR (KBr) 1700,
D
1
1645 cmꢁ1; H NMR (CDCl3) d 8.60 (1H, s), 8.12 (1H,

4464
F. Aiello et al. / Bioorg. Med. Chem. 12 (2004) 4459–4466
4.5. General procedure for the synthesis of compounds 21
of the appropriate hydroxyacid 22 (0.4 mmol) in dry
dichloromethane (15 mL). After heating overnight under
reflux, the mixture was worked up as described for
method A.
The preparation of N-(3-acetoxy-2-naphthoyl)oxazol-
idin-4-carboxylic acid is described as a representative
example. A mixture of formaldehyde (37% solution in
water, 14.0 mmol, 1.0 mL),
L-serine (1.4 g, 13.3 mmol),
and 2 N NaOH (6.5 mL) was stirred overnight at 0 °C.
Sodium carbonate (0.7 g, 6.6 mmol) was added in one
portion to the cold solution. A solution of 3-acetoxy-2-
naphthoic acid chloride (3.3 g, 13.3 mmol) in dry THF
(20 mL) was then added dropwise, while a weakly alkaline
pH was maintained by the addition of solid sodium car-
bonate. The mixture was stirred overnight, then concen-
trated and made acidic (pH 3–4) by adding concentrated
HCl. The solid formed was extracted into ethyl acetate,
and the resulting solution was washed with water, dried,
and evaporated. The resulting residue was purified by
column chromatography on silica gel (CHCl3/CH3OH/
HCO2H, 86/14/1) to give a waxy yellow solid (3.0 g, 70%):
4.9. Method C
A
suspension of the appropriate compound 22
(0.4 mmol) in acetic anhydride (2 mL) was stirred over-
night at room temperature. The resulting solution was
then diluted with chilled water and left for several hours
at 4 °C. The aqueous solution was extracted with chlo-
roform and the organic layer was shaken successively
with a 5% sodium bicarbonate solution and brine. A
pure solid was obtained after evaporation of the solvent.
4.10. ( )-1,11a-Dihydro-3H,5H,11H-thiazolo[4,3-c]-
[1,4]benzoxazepin-5,11-dione (4)
1
IR (KBr) 2700 (br), 1715, 1690, 1635 cmꢁ1; H NMR
(CDCl3) d 9.57 (1H, br, exch. with D2O), 7.78–7.65 (3H,
m), 7.48 (1H, m), 7.32 (2H, m), 5.30 (2H, s), 5.01 (1H, t,
J ¼ 6:7 Hz), 4.40 (1H, m), 4.09 (1H, m), 2.31 (3H, s); MS
(EI) m=z 329 (Mþ). Anal. (C17H15NO6) C, H, N.
Compound 4 was prepared following the method A
(33 mg, 35%); mp 143–145 °C (diethyl ether/light petro-
1
leum); IR (KBr) 1695, 1635 cmꢁ1; H NMR (CDCl3) d
7.94–7.20 (4H, m), 4.86 and 4.72 (2H, ABq,
J ¼ 10:6 Hz), 4.46 (1H, dd, J ¼ 6:8 and 3.3 Hz), 3.76
(1H, dd, J ¼ 12:3 and 3.3 Hz), 3.32 (1H, dd, J ¼ 12:3
and 6.8 Hz); MS (CI) m=z 236 (MHþ). Anal.
(C11H9NO3S) C, H, N.
4.6. General procedure for the synthesis of compounds 22
The preparation of N-(3-hydroxy-2-naphthoyl)oxazol-
idin-4-carboxylic acid is described as a representative
example. N-(3-Acetoxy-2-naphthoyl)oxazolidin-4-car-
boxylic acid (2.0 g, 6.0 mmol) was dissolved in a solution
of sodium carbonate (0.8 g, 7.5 mmol) in water (20 mL)
and stirred overnight at room temperature, then made
acidic (pH 3–4) by adding concentrated HCl at 0 °C. The
solid formed was extracted with ethyl acetate, and the
resulting solution was washed with water, dried, and
evaporated. The resulting residue was purified by crys-
tallization to give a solid (1.5 g, 85%): mp 182–184 °C
(acetone/diethyl ether); IR (KBr) 2800 (br), 1695,
4.11. ( )-2,3-Dihydro-5H,11aH-thiazolo[2,3-c][1,4]ben-
zoxazepin-5,11-dione (5)
Compound 5 was prepared following the method B
(13 mg, 14%); mp 154–156 °C (benzene); IR (KBr) 1700,
1635 cmꢁ1; 1H NMR (CDCl3) d 7.95–7.22 (4H, m), 5.27
(1H, s), 4.20 (1H, m), 3.88 (1H, m), 3.37 (1H, m), 3.16
(1H, m); MS (CI) m=z 236 (MHþ). Anal. (C11H9NO3S)
C, H, N.
1
1635 cmꢁ1; H NMR (CD3OD) d 9.00 (2H, br), 7.82
(1H, s), 7.76 (1H, m), 7.64 (1H, m), 7.40 (1H, m), 7.28
(1H, m), 7.20 (1H, s), 5.21 (2H, s), 4.99 (1H, t,
J ¼ 6:7 Hz), 4.47 (1H, m), 4.11 (1H, m); MS (EI) m=z
287 (Mþ). Anal. (C15H13NO5) C, H, N.
4.12. ( )-1,13a-Dihydro-3H,5H,13H-naphtho[2,3-f]thiaz-
olo[4,3-c][1,4]oxazepine-5,13-dione (6)
This compound was prepared following the method C
(46 mg, 38%); mp 217 °C (dichloromethane/petroleum
1
4.7. Cyclization reaction: method A
ether); IR (KBr) 1705, 1630 cmꢁ1; H NMR (CDCl3) d
8.55 (1H, m), 7.98 (1H, d, J ¼ 8:3 Hz), 7.86 (1H, d,
J ¼ 8:3 Hz), 7.60 (3H, m), 4.91 (1H, 0.5 of ABq,
J ¼ 10:7 Hz), 4.56 (1H, 0.5 of ABq, J ¼ 10:7 Hz), 4.55
(1H, dd, J ¼ 6:8 and 3.4 Hz), 3.80 (1H, dd, J ¼ 12:5 and
3.4 Hz), 3.34 (1H, dd, J ¼ 12:5 and 6.8 Hz); MS (CI) m=z
286 (MHþ). Anal. (C15H11NO3S) C, H, N.
A mixture of the appropriate hydroxyacid 22 (0.4 mmol),
EDC (0.085 g, 0.44 mmol), and DMAP (cat.) in dry di-
chloromethane (20 mL) was stirred at room temperature
for 40 h. The solution was then shaken with a saturated
solution of NH4Cl and water, successively. After drying
and solvent evaporation, a pale yellow oil was obtained
and purified by column chromatography on a silica gel
(15% ethyl acetate in hexanes).
4.13. ( )-1,13a-Dihydro-3H,5H,13H-naphtho[2,3-f]oxaz-
olo[4,3-c][1,4]oxazepine-5,13-dione (7)
4.8. Method B
This compound was prepared following the method A
(56 mg, 52%); mp 205 °C (ethyl ether); IR (KBr) 1695,
1
2-Chloro-1-methylpyridinium iodide (0.12 g, 0.48 mmol)
and TEA (110 lL, 0.8 mmol) were added to a suspension
1635 cmꢁ1; H NMR (CDCl3) d 8.52 (1H, s), 7.95 (1H,
d, J ¼ 7:9 Hz), 7.83 (1H, d, J ¼ 7:9 Hz), 7.60 (3H, m),

F. Aiello et al. / Bioorg. Med. Chem. 12 (2004) 4459–4466
4465
5.28 and 5.21 (2H, ABq, J ¼ 5:3 Hz), 4.82 (1H, dd,
J ¼ 8:8 and 2.4 Hz), 4.28 (2H, m); MS (EI) m=z 269
(Mþ). Anal. (C15H11NO4) C, H, N.
D116 considering the geometry of the Mg2þ ion that was
present in the subunit A of the IN in PDB 1BIS and
subunit A in IN-5CITEP complex X-ray structure (PDB
1SQ4) as recently described.17 All the water molecules
present in protein were removed and hydrogen atoms
were added to the protein considering appropriate ion-
ization states for both the acidic and basic amino acid
residues. Docking was performed using version 1.2 of
the GOLD: Genetic Optimization for Ligand Docking
(Cambridge Crystallographic Data Centre) software
4.14. (1R,13aS)-1,13a-Dihydro-1-methyl-3H,5H,13H-
naphtho[2,3-f]oxazolo[4,3-c][1,4]oxazepine-5,13-dione (8)
Method C was followed for the preparation of com-
20
pound 8 (49 mg, 43%): mp 197–198 °C (benzene); ½aꢀ
D
1H
)197.0 (c 1, CHCl3); IR (KBr) 1700, 1640 cmꢁ1
;
package. A 12 A radius active site was defined consid-
ꢁ
NMR (CDCl3) d 8.60 (1H, s), 7.98 (1H, d, J ¼ 7:5 Hz),
7.86 (1H, d, J ¼ 7:9 Hz), 7.70 (1H, s), 7.65 (2H, m), 5.62
and 5.03 (2H, ABq, J ¼ 5:5 Hz), 4.88 (1H, m), 3.82 (1H,
d, J ¼ 6:9), 1.45 (3H, d, J ¼ 6:0); MS (CI) m=z 284
(MHþ). Anal. (C16H13NO4) C, H, N.
ering the carboxylate oxygen atom (OD1) of amino acid
residue D64 as the center of the active site. All con-
formers of the compounds were docked into the active
site of the IN. Based on the GOLD fitness score, for
each molecule a bound conformation with high fitness
score was considered as the best-bound conformation.
All docking runs were carried out using standard default
settings with a population size of 100, a maximum
number of 100,000 operations, a mutation and crossover
rate of 95.18–20 The fitness function that was imple-
mented in GOLD consisted basically of H-bonding,
complex energy, and ligand internal energy terms.
4.15. ( )-1,13a-Dihydro-3H,5H,13H-naphtho[1,2-f]thiaz-
olo[4,3-c][1,4]oxazepine-5,13-dione (9)
This compound was prepared following the method C
(52 mg, 43%); mp 237 °C (dichloromethane/light petro-
1
leum); IR (KBr) 1725, 1640 cmꢁ1; H NMR (CDCl3) d
8.64 (1H, d, J ¼ 8:1 Hz), 8.03 (1H, d, J ¼ 8:6 Hz), 7.91
(1H, d, J ¼ 7:2 Hz), 7.65 (2H, m), 7.33 (1H, d,
J ¼ 8:6 Hz), 5.00 (1H, 0.5 of ABq, J ¼ 10:5 Hz), 4.90
(1H, 0.5 of ABq, J ¼ 10:5 Hz), 4.64 (1H, dd, J ¼ 6:5 and
1.7 Hz), 3.79 (1H, dd, J ¼ 12:0 and 1.7 Hz), 3.39 (1H,
dd, J ¼ 12:0 and 6.5 Hz); MS (CI) m=z 286 (MHþ).
Anal. (C15H11NO3S) C, H, N.
4.18. Biological materials, chemicals, and enzymes
All compounds were dissolved in DMSO and the stock
solutions were stored at )20 °C. The c[32P]-ATP was
purchased from either Amersham Biosciences or ICN.
The expression systems for the wild-type IN and soluble
mutant INF185KC280S were generous gifts of Dr. Robert
Craigie, Laboratory of Molecular Biology, NIDDK,
NIH, Bethesda, MD.
4.16. ( )-1,13a-Dihydro-3H,5H,13H-naphtho[2,1-f]thiaz-
olo[4,3-c][1,4]oxazepine-5,13-dione (10)
This compound was prepared following the method C
(26 mg, 23%); mp 200 °C (dichloromethane/light petro-
4.19. Preparation of oligonucleotide substrates
leum); IR (KBr) 1740, 1645 cmꢁ1; H NMR (CDCl3) d
The oligonucleotides 21top, 50-GTGTGGAAAATCTC-
TAGCAGT-30 and 21bot, 50-ACTGCTAGAGATTTT-
CCACAC-30 were purchased from Norris Cancer
Center Core Facility (University of Southern California)
and purified by UV shadowing on polyacrylamide gel.
To analyze the extent of 30-processing and strand
transfer using 50-end labeled substrates, 21top was 50-
end labeled using T4 polynucleotide kinase (Epicentre,
Madison, WI) and c[32P]-ATP (Amersham Biosciences
or ICN). The kinase was heat-inactivated and 21bot was
added in 1.5 M excess. The mixture was heated at 95 °C,
allowed to cool slowly to room temperature, and run
through a spin 25 mini-column (USA Scientific) to
separate annealed double-stranded oligonucleotide from
unincorporated material.
1
8.41 (1H, m), 7.90 (2H, m), 7.82 (1H, d, J ¼ 8:4 Hz),
7.70 (2H, m), 4.95 (1H, 0.5 of ABq, J ¼ 10:6 Hz), 4.85
(1H, 0.5 of ABq, J ¼ 10:6 Hz), 4.55 (1H, dd, J ¼ 6:5 and
1.8 Hz), 3.82 (1H, dd, J ¼ 11:8 and 1.8 Hz), 3.37 (1H,
dd, J ¼ 11:8 and 6.5 Hz); MS (CI) m=z 286 (MHþ).
Anal. (C15H11NO3S) C, H, N.
4.17. Molecular modeling
The structures of all the compounds (Table 1) were built
and minimized using Catalyst (Accelrys, Inc.)16 running
on a multiprocessor linux PC and a 24-processor Silicon
Graphics Onyx workstation as described.17 The poling
algorithm implemented within Catalyst was used to
generate conformations for all the compounds. For each
compound all feasible unique conformations were gen-
erated over a 20 kcal/mol range of energies using the best
flexible conformation generation method in Catalyst.
The subunit B of the core domain X-ray structure of IN
(PDB 1BIS) in which all the active site amino acid res-
idues were resolved was chosen for docking purpose. A
Mg2þ ion was placed in the active site between carbox-
ylate oxygen atoms of amino acid residues D64 and
4.20. Integrase assays
To determine the extent of 30-processing and strand
transfer, wild-type IN was preincubated at a final con-
centration of 200 nM with the inhibitor in reaction
buffer (50 mM NaCl, 1 mM HEPES, pH 7.5, 50 lM
EDTA, 50 lM dithiothreitol, 10% glycerol (w/v),
7.5 mM MnCl2, 0.1 mg/mL bovine serum albumin,

4466
F. Aiello et al. / Bioorg. Med. Chem. 12 (2004) 4459–4466
strand transfer that prevent integration and inhibit
HIV-1 replication in cells. Science 2000, 287, 646–
650.
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 50-end 32P-labeled linear oligonucleotide substrate
was added, and incubation was continued for an addi-
tional one hour. Reactions were quenched by the addi-
tion of an equal volume (16 lL) of loading dye (98%
deionized formamide, 10 mM EDTA, 0.025% xylene
cyanol, and 0.025% bromophenol blue). An aliquot
(5 lL) was electrophoresed on a denaturing 20% poly-
acrylamide gel (0.09 M tris-borate pH 8.3, 2 mM EDTA,
20% acrylamide, 8 M urea).
8. Yoshinaga, T.; Sato, A.; Fujishita, T.; Fujiwara, T.
In vitro activity of a new HIV-1 integrase inhibitor in
clinical development. 9th Conference on Retroviruses and
Opportunistic Infections; Seattle, 2002.
9. Neamati, N.; Turpin, J. A.; Winslow, H. E.; Christensen,
J. L.; Williamson, K.; Orr, A.; Rice, W. G.; Pommier, Y.;
Garofalo, A.; Brizzi, A.; Campiani, G.; Fiorini, I.; Nacci,
V. Thiazolothiazepine inhibitors of HIV-1 integrase.
J. Med. Chem. 1999, 42, 3334–3341.
10. Bergeron, R. J.; Wiegand, J.; Wollenweber, M.; McManis,
J. S.; Algee, S. E.; Ratliff-Thompson, K. Synthesis
and biological evaluation of naphthyldesferrithiocin
iron chelators. J. Med. Chem. 1996, 39, 1575–1581.
11. Wolfe, S.; Militello, G.; Ferrari, C.; Hasan, S. K.; Lee, S.
L. Conversion of amino hydroxyacids to oxazolidine-4-
carboxylic acids, oxazolidine-5-carboxylic acids and tetra-
hydrooxazine-4-carboxylic acids. Tetrahedron Lett. 1979,
20, 3913–3916.
Gels were dried, exposed in a PhosphorImager cassette,
and analyzed using a Typhoon 8610 Variable Mode
Imager (Amersham Biosciences) and quantitated using
ImageQuant 5.2. Percent inhibition (% I) was calculated
using the following equation:
% I ¼ 100 ꢃ ½1 ꢁ ðD ꢁ CÞ=ðN ꢁ CÞꢀ;
where C, N, and D are the fractions of 21-mer substrate
converted to 19-mer (30-processing product) or strand
transfer products for DNA alone, DNA plus IN, and IN
plus drug, respectively. The IC50 values were determined
by plotting the logarithm of drug concentration versus
percent inhibition to obtain concentration that pro-
duced 50% inhibition.
12. Falorni, M.; Conti, S.; Giacomelli, G.; Cossu, S.; Socco-
lini, F. Optically active 4-oxaproline derivatives: new
useful chiral synthons derived from serine and threonine.
Tetrahedron: Asymmetry 1995, 6, 287–294.
13. Magnus, P.; Kreisberg, J. D. Synthesis of benzofuranones
related to diazonamide via an intramolecular Pummerer
reaction.
454.
Tetrahedron
Lett.
1999,
40,
451–
14. Saigo, K.; Usui, M.; Kikuchi, K.; Shimada, E.; Mukaiy-
ama, T. New method for the preparation of carboxylic
esters. Bull. Chem. Soc. Jpn. 1977, 50, 1863–1866.
15. Narasaka, K.; Masui, T.; Mukaiyama, T. The facile
synthesis of macrocyclic lactones by the use of 2-chloro-3-
methoxymethyl-1-methylpyridinium iodide. Chem. Lett.
1977, 763–766.
References and notes
1. Richman, D. D. HIV chemotherapy. Nature 2001, 410,
995–1001.
2. Brown, P. O. Integration; Cold Spring Harbor Press: Cold
Spring Harbor, 1999.
16. Accelrys, I. Catalyst; V.4.8: San Diego.
3. Asante-Appiah, E.; Skalka, A. M. HIV-1 integrase:
structural organization, conformational changes, and
catalysis. Adv. Virus Res. 1999, 52, 351–369.
4. Neamati, N. Structure-based HIV-1 integrase inhibitor
design: a future perspective. Expert Opin. Invest. Drugs
2001, 10, 281–296.
17. Long, Y. Q.; Jiang, X. H.; Dayam, R.; Sanchez, T.;
Shoemaker, R.; Sei, S.; Neamati, N. Rational design and
synthesis of novel dimeric diketoacid-containing inhibitors
of HIV-1 integrase: implication for binding to two metal
ions on the active site of integrase. J. Med. Chem. 2004, 47,
2561–2573.
5. Neamati, N. Patented small molecule inhibitors of HIV-1
integrase: a ten-year saga. Expert Opin. Ther. Pat. 2002,
12, 709–724.
6. Dayam, R.; Neamati, N. Small-molecule HIV-1 integrase
inhibitors: the 2001–2002 update. Curr. Pharm. Des. 2003,
9, 1789–1802.
7. Hazuda, D. J.; Felock, P.; Witmer, M.; Wolfe, A.;
Stillmock, K.; Grobler, J. A.; Espeseth, A.; Gabryelski,
L.; Schleif, W.; Blau, C.; Miller, M. D. Inhibitors of
18. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor,
R. Development and validation of a genetic algorithm for
flexible docking. J. Mol. Biol. 1997, 267, 727–748.
19. Nissink, J. W.; Murray, C.; Hartshorn, M.; Verdonk, M.
L.; Cole, J. C.; Taylor, R. A new test set for validating
predictions of protein–ligand interaction. Proteins 2002,
49, 457–471.
20. GOLD Cambridge Crystallographic Data Centre
(CCDC), V1.2, UK, 2001.