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Discovery of novel HIV-1 integrase inhibitors

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Discovery of novel HIV-1 integrase inhibitors

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Content DISCOVERY OF NOVEL HIV-1 INTEGRASE
INHIBITORS
by
Aline Chih Ling Kuo
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Pharmaceutical Sciences)
August 2002
Copyright 2002 Chih-Ling Kuo
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UMI Number: 1414904
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UNIVERSITY OF SOUTHERN CALIFORNIA
The Graduate School
University Park
LOS ANGELES, CALIFORNIA 900894695
This thesis, w ritten b y
C kk - Ll n^. _R u a ................. ..
U nder th e direction o f h & T r . . . Thesis
C om m ittee, and approved b y a ll its m em bers,
has been p resen ted to and accepted b y The
Graduate School, in p a rtia l fulfillm ent o f
requirem ents fo r th e degree o f
D ate A ugust 6 , 2002
THESIS COMMITTEE
Chairperson
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DEDICATION
To my grandparents, parents, sister, brothers, and all friends who
have given me encouragements, support, and love during my study
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ACKNOWLEGEMENTS
I wish to knowledge my sincere gratitude to my advisor, Dr. Nouri
Neamati, whose invaluable advice and support encouraged me to pursue and
complete my graduate study. I also wish to thank Professor Eric, J. Lien for his
guidance and help during the SAR analysis, and my writing of thesis. I deeply
appreciate Dr. Ian Haworth, Dr. Michel Bolger, and Dr. Stan Louie for agreeing
to be my committee member and taking interest in my project.
In addition, I wish to thank my laboratory colleague, Dr. Wim Pluymers,
Valery Fikkert for their help with my experiment and friendships. At the same
time, I would like to thank all faculties, staffs, and friends in the School of
Pharmacy, especially all colleagues in Dr. Lien’s and Dr. Neamati’s
Laboratories.
I am very grateful to my dear grandparents, parents, sister and brothers
for giving me support and encourage finishing my study.
iii
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TABLE OF CONTENTS
PAGE
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES viii
ABSTRACT ix
Ch.I Introduction 1
1.1 Retroviral Life Cycle 1
1.2 HIV integrase 6
1.3 HIV Integrase Inhibitors 14
1.4 References 19
Ch. II Inhibition of HIV-1 integrase by
9H-2.3.9-triaza-fluorene derivatives 24
2.1 Introduction 24
2.2 Methods 27
2.3 Results and Discussion 29
2.4 Conclusions 38
2.5 References 47
iv
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Ch. Ill Inhibition of HIV-1 integrase by 2-mercaptobenzene-
sulphonamide derivatives 50
3.1 Introduction 50
3.2 Methods 52
3.3 Results and Discussion 53
3.4 Conclusions 64
3.5 References 65
Ch. IV Inhibition of HIV-1 integrase by 3-carbamoyl-bicyclo
[2.2.1]hept-5-ene-2-carboxylic acid derivatives 67
4.1 Introduction 67
4.2 Methods 69
4.3 Results and Discussion 71
4.4 Conclusions 97
4.5 References 100
Total references 104
v
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LIST OF TABLES
TABLES PAGE
Chapter II
Table 1. 5H-pyridazino[4,5-b] indoles 31
Table 2. 5H-pyridazino[4,5-b] indoles-l-one derivatives 33
Table 3. Newly synthesized compounds 35
Table 4. 5-methyl-6H-l ,2,3a,4,6- pentaaza- cyclopenta[c]
fluorine derivatives 37
Table 5. Physicochemical parameters of compounds 39
Chapter III
Table 6. Inner salt form of mercapto compounds 55
Table 7. Mercapto compounds with substituents at N-position of
sulphonamide group 56
Table 8. Various substituents on trizaolo ring and amide 59
Table 9. Substitution on the mercapto group 61
Table 10. Cyclic analogues of 2-mercapto-benzensulfonamide 63
Chapter IV
Table 11. Sulfonamide derivatives 72
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Table 12. Sulfonamide derivatives 75
Table 13. Bicyclo derivatives 79
Table 14. Bicyclo derivatives 81
Table 15. Bicyclo derivatives 83
Table 16. Bicyclo derivatives 84
Table 17. List of physicochemical parameters of all compounds 86
Table 18. QSAR equation of HIV-1 integrase inhibitors 98
Table 19. F test for physicochemical parameters 98
Table 20. Squared correlation matrix of physicochemical parameters 99
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LIST OF FIGURES
FIGURES PAGE
Chapter I
Fig. 1. HIV Virion 3
Fig. 2. Integration A)3 ’-processing B)Strand Transfer 6
Fig. 3. One-step nucleophilic mechanism of 3’-proceesing and
strand transfer 7
Fig. 4. Structure of HIV-1 integrase N-terminal domain 10
Fig. 5. Structure of HIV-1 integrase core domain 12
Fig. 6. Structure of HIV-1 integrase C-terminal domain 13
Chapter II
Fig. 7. Two-dimensional parameter frame-setting of different
combinations of physicochemical constants 41
Fig. 8. Three-dimensional parameter frame setting 46
Chapter IV
Fig. 9. Two-dimensional parameter frame-setting of different
combinations of physicochemical constants 89
Fig. 10. Three-dimensional parameter frame setting 94
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ABSTRACT
The pol gene of human immunodeficiency vims (HIV) encodes three
essential enzymes, reverse transcriptase (RT), protease (PR), and integrase (IN).
Currently available anti-HIV therapeutic agents consist of RT and PR inhibitors.
Presently, there is no FDA approved drug targeting IN. HIV-1 IN is an essential
enzyme in the life cycle of HIV-1 vims, which catalyze insertion of viral DNA
into the host genome. There is no cellular homologue to HIV IN, making IN an
attractive target for development of therapeutics against HIV. There have been
numerous efforts to develop inhibitors of IN resulting in a number of compounds
demonstrating in vitro inhibitory activity. In order to design potential inhibitors,
three classes of compounds have been tested in vitro using purified HIV integrase
and a 21 base-pair oligonucleotide corresponding to viral long terminal repeats
(LTRs) DNA. Results obtained from IN assays and QSAR analyses identified two
main stmctures with good inhibitory activity. Further studies are underway to
elucidate the binding site for these novel inhibitors.
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CHAPTER I
INTRODUCTION
The Human Immunodeficiency Virus (HIV) is the infectious organism leading to
acquired immunodeficiency syndrome (AIDS). HIV is a single stranded RNA virus,
which can infect CD4+ T-cells within the host. Since AIDS was defined in 1981, over
50 million individuals have been infected. Extensive efforts have been made to
develop treatments against HIV. There are several drugs approved by the Food and
Drug Administration for the treatment of HIV. These dmgs currently on the market
either target reverse transcriptase or protease, which are essential retroviral enzymes
required for HIV replication. However, toxicity, drug resistant, and patient
compliance problems prompted us to look for new targets and therapeutic agents.
One such target is HIV-integrase, an essential enzyme required for viral DNA to
integrate into host DNA. In this study, we attempt to develop a novel class of
inhibitors targeting integrase.
1.1 RETROVIRAL LIFE CYCLE
HIV, like other viruses, cannot replicate without a host cell, which is needed to
generate new virus particles. HIV belongs to the retrovirus family, which carry two
single strains of viral RNAs in a viral envelope called a virion (Fig. 1). HIV-1 virions
are composed of a lipid bilayer, which bares viral surface glycoproteins (gpl20 and
1
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gp41) and surrounds a nucleoprotein core. The nucleoprotein core encloses two
single-strain RNAs along with protein products of gag and pol genes. The virus
generates new virion particles in an inffected host cell. Viral mediated cellular lyses,
releasing the new virion to infect other healthy cells. The major replication steps for
HIV are attachment, reverse transcription, integration and assembly.
The first step for HIV to infect CD4+ T-cells requires glycoprotein-120 binding to
the CD4 receptor, which has four extracellular immunoglobulin (Ig)-like domains.
Following CD4 binding, gp41 is exposed facilitating viral fusion onto the T-cell
membrane. With the help of a T-cell chemokine receptor, CCR5 or CXCR4, the
virus can successfully fuse with the cell.
After fusion, viral nucleoprotein core containing viral RNA and reverse transcriptase,
is released into the host cell, and the reverse transcription pathway is activated. The
process of reverse transcription is catalyzed by reverse transcriptase (one of the pol
gene products). Reverse transcriptase is a heterodimer composed of p66 and p51.
The task of this reaction is to converse viral RNA into DNA code. The reaction takes
place in the partially uncoated nucleoprotein core. First, the viral reverse
transcriptase acts as a RNA-dependent polymerase to convert the viral RNA into a
double -stranded RNA:DNA hybrid. Next, the RNA of the hybrid molecule is
degraded while reverse transcriptase plays the role of RNase H. Then, the
DNA-dependent polymerase activity of reverse transcriptase synthesizes the
complement strand DNA along with the previous DNA template to form the
2
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double-stranded viral DNA. The viral DNA has a highly conserved region, referred
as long terminal repeats (LTRs), located at both ends of the viral DNA. The genetic
information of the virus is located within the flanking LTRs.
HIYVtrtan
g p 120
gp41
SSRNA
Lipid layer
P9
Reverse
T ranscriptase
Host proteins
Fig. 1. HIV Virion (Adapted from http://ntri.tamuk.edu/immunology/aids.html)
After reverse transcription, integrase binds to the viral DNA mediating viral DNA
integration into the host genome. Integration is a two-step process. First, the
integrase initiates cleavage the GT residues, which are highly conserved in the viral
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genome, from the 3’-end of the LTRs. This step of the reaction, named 3’-processing,
takes place in the cytoplasm. The DNA-integrase complex, referred as the
preintegration complex (PIC), can transport through the nuclear pore. It is believed
that integrase mediates transport of cDNA via an unknown mechanism. After the
preintegration complex is transported into the nucleus of the host cell, integrase
carries out final step of the integration: insertion of viral DNA into host genome. The
insertion of the viral DNA into host cell is called strand transfer. These two separate
steps are both carried out by the same catalytic core of integrase.
For the postintegration stage, viral proteins, trans-activator protein (Tat) and a
protein regulator of virion expression (Rev), cooperate with the host cell
transcription proteins (RNA polymerase) to express viral genes in the host genome.
The initiation of RNA transcription follows the normal eukaryotic transcriptional
pathway. However, for HIV replication, without the presence of Tat, the transcription
of viral DNA cannot be completed. Tat is essential for the HIV RNA elongation— Tat
binds onto the trans-activation responsive region (TAR) of the HIV DNA to promote
RNA polymerase activation thus lead to transcription of HIV. Rev, like Tat, also is a
regulatory protein. Tat binds to a specific binding site on HIV RNA and transports
the mature and functional RNA from the nucleus to the cytoplasm. The transported
RNA is subsequently submitted to protein synthesis.
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HIV mRNA in the cytoplasm is translated into three polyproteins— ENV gpl60,
GAG p55 and GAG-POL p i60. ENV gpl60 is the combination of gpl20 and gp41.
On the other hand, GAG p55 contains a matrix, capsid and nucleocapsid protein.
GAG p55 contains pol gene products— reverse transcriptase, integrase and a protease.
To complete the replication, a protease is required to released viral proteins from the
polyproteins. HIV protease is composed of two identical subunits with aspartate
residues paired at the interface. HIV protease cleaves precursor polyproteins by
recognizing a praline residue sitting between two hydrophobic residues. The task is
completed by HIV protease, which liberates itself from the polyprotein and
hydrolyzes peptide bonds in the polyproteins that releases HIV encoded functional
proteins and structure proteins. The ENV gpl60 polyprotein releases gpl20 and gp41
as well as the structure proteins— matrix caspid and nucleocapsid proteins. These
proteins accumulate and aggregate at the inner surface of the plasma membrane.
Structure proteins selectively pack unspliced genome-length transcripts into viral
cores. After budding, viral virions with gpl20 and gp41 expressed on the surface are
released to extra-cellular space.
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1.2 HIV INTEGRASE
As stated earlier, integrase catalyzes a two-step integration process. The initial
step is 3’-processing. The enzyme recognizes CAGT sequence where
phosphordiester bond
B
21
.CAST
3-P
h
’ km
19
iCA
V lr a S p N A
D onor
3 ' - e m io n ln
strand transfer
Host Chromosome
A cceptor
S '-p ro e essin g
Fig 2. Integration A )3’-processing B)Strand Transfer
Adapt from Marchand et at. J. Biol. Chem. 277, 12596-12603, 2002
are cleaves between
A-G base pair. The
second step is
DNA-strand transfer;
the 3’ ends of viral DNA
are inserted into the host
DNA (Fig 2). 1 In a
previous study, both the
3 ‘-processing and strand
transfer reaction are
carried out by HIV-1
integrase transfering
(Rp)-phosphorothioate
form to
(Sp)-phosphorthioate
form at the reaction
center. 2 This implies
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that both reactions consist of two one-step nucleophile reactions (Sn2 reaction).
Otherwise, the product of the reaction will contain both R form and S form of the
phosphorthioate. In the transition state of both reactions, the substituents are
arranged to from trigonal bipyramid. The nucleophile and the leaving group occupy
the apical positions respectively. (Fig. 3)
\ / s
C i ' 1
A T
' ■ ■ ' T 1 1
CG.
A T
9
i d H n j ' g ' . ' O -
Fig. 3. One-step nucleophilic mechanism of 3’-proceesing and strand transfer
Adapted from Gerton et al. J. Biol Chem 1999, 274, 33480-33487
7
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In 3’- processing, a water molecule attacks the phosphodiester bond, which resides
between the G and A residues. The adenosine of remnant viral DNA served as the
leaving group and generate 3’-hydroxyl group. In strand transfer, conversely, the
3’-hydroxyl group of the viral DNA is the attacking nucleophile. However, the
attacking target site in the host DNA is not specified, since viral DNA insertion into
the host cell does not have specific region.3 ,4 Integrase not only catalyzes
3’-processing and strand transfer reaction, but also mediates disintegration
reaction— a phosphoryl-transfer reaction.1 In disintegration, the viral DNA segment is
first liberated from a branched DNA substrate that resembles the product of DNA
strand transfer and then the target DNA part of the substrate is resealed.
The ability of retroviral integrase to cut polynucleotides from viral DNA end and the
coordination of a divalent metal ion at the catalytic site indicates that the retroviral
integrase belongs to a superfamily of polynucleotidyl transferases.4 This superfamily
includes the Mu transposase, the nucleases RNase H, and RuvC. Retroviral integrase
also shares a certain degree of structural or sequence similarity with these enzym es.5’
6,7,8’ HIV-1 integrase is a 32 KDa protein, consisting of 288 amino acids. The intact
integrase comprises three independent folding domains; a N-terminal domain - zinc
ion binding site, a catalytic core domain, the metal cofactor binding domain, and a
C-terminal domain, the DNA binding domain. The central core domain is highly
conserved among retroviral integrases9. It contains a triad of highly conserved acidic
residues, which are important for catalysis.1 0 Point mutation of each residue would
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destroy the activity.9 The isolated core domain of HIV integrase alone maintains the
ability to catalyze disintegration, but not the integration. The N-terminal region has
been demonstrated that this too is essential for site-specific cutting as well as
integrase tetramerization. The mutation studies show that C-terminal is required for
DNA binding. 9 Without these two regions, integrase cannot bind and cleave viral
DNA specifically.
N-TERMINAL: The N-terminal domain is the first 50 amino acids in HIV-1
integrase (Fig. 4). Nuclear magnetic resonance (NMR) studies reveal that the
N-terminal folds into helix-turn-helix form. The HIV-1 integrase N-terminal contains
one pair of zinc coordinating cysteines, Cys-X2-Cys and one pair of histidines,
His-X 3-H is.1 1 This set of residues is called HHCC motif. The N-terminal of HIV-1
integrase is not required for DNA-binding, yet this is a highly conserved region
among DNA binding proteins and invariant among retroviral integrases.1 1 Deletion
mutation of this motif reduces integrase activity in site-specific cleavage of viral
D N A .1 1 The isolated N-terminal domain is stabilized and folded by the coordination
of the zinc to His 12, His 16, Cys40 and Cys43 (The zinc ion is tetrahedrally
coordinated by these residues). 1 2 In contrast, the folding of full-length HIV-1
integrase does not greatly change with the presence of zinc ion. However,
zinc-bound integrase is more active than the zinc-free integrase for both
3’-processing and DNA strand transfer. The binding of zinc ions affects the tertiary
structure— domain-domain interaction. In addition, zinc binding also promotes
9
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tetramerization of HIV-1 integrase in vitro— tetramerized at lower protein
concentration.1 3 These properties of the N-terminal have led to the belief that the
N-terminal of HIV-1 integrase play the role of assisting viral DNA end recognition,
as well as protein-protein interaction, tetramerization. n ’1 3
Cys40
Hisl2
CIys43
H isl6
Fig. 4. Structure of HIV-1 integrase N-terminal domain
(Adapted from Cai et al Nat Struct Biol 1997, 4, 576)
CORE DOMAIN: The core domain is the site where the integration reactions mainly
take place, and also is the site that binds with its metal cofactor— Mn2 + or Mg2+(Fig.
5). Although full-length integrase is required to catalyze 3’-processing and
DNA-strand transfer, core domain alone still has the ability to catalyze disintegration.
10
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1 1 The catalytic core domain of HIV-1 integrase consists of 162 amino acids (residue
50-212). These 162 amino acids compose a seven-stranded /3 -sheet surrounded by
six a -helices.1 4 ’1 5 The catalytic core domain consist of three amino acids, which
are highly conserved among retroviral integrase. These three amino acids are Asp 64
(D), Asp 116 (D) and Glu 152 (E), called DDE motif, which also is found in bacterial
insertion sequences and some retrotransposons. 4 This evolutionary conservation
suggests an important role for these residues in integrase. Consistently, any point
mutation of these three amino acids will abolish the activity.1 6 -1 8 Currently, several
crystal structures of the HIV-1 integrase catalytic domain have been studied.5,15,19
However, part of the structure is invisible in X-ray study of integrase crystal. The
missing region is the polypeptide chain, residues 140-153. This loop is believed to
have certain degree of flexibility.1 9 In the crystal structure, Asp 64 and Asp 116 lie
on the adjacent j3- strands. Since, Glu 152 is in the missing region, the orientation
of Glu 152 within the DDE motif is currently unknown. Recent study by Golgur et al.
reveals that Glu 152 resides at helix- a 4 with its side chain point toward Asp 64 and
i c o .
Asp 116. This study also reveals that only Asp 64 and Asp 116 bind to Mg , not
the third catalytic residue, Glu 152, which does not participate in metal binding.
These finding further confirms the important role of the catalytic core domain in
integration.
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Asp(M
•M ?,+
► A s p 1 1 a
Fig. 5. Structure of HIV-1 integrase core domain.
(Adapted Goldgur et al. Proc Natl Acad Sci U S A 1998,95 ,9150)
C-TERMINAL: The C-terminal has lower level of sequence homology among
retroviruses. It has been shown that this domain contributes to integrase-DNA
binding. (10-51) However, it binds DNA non-specifically.9,17,20 Residue 220 to 270
is the minimal region required for DNA binding (Fig. 6). Although deletion mutation
of this region does not reduce the integrase activity of DNA cleavage, it reduces the
specificity of DNA cleavage. 1 1 This region also plays an important role in
mutlimerization. The NMR structure of this region reveals that it is a homodimer.
The structure of the monomer is composed of antiparallel /3 -barrel and
three-residue 3-helix. 2 1 The overall fold is similar to Src homology 3 (SH3) fold,
12
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2 1
which mediates protein-protein interaction . The isolated C-terminal domain of HIV
integrase has the same non-specific DNA-binding activity as the full-length integrase.
a 1 ^ 7 O ft O O 03
The dimer form is formed by hydrophobic interaction of the /3 -strands.
Residue L242, W243, A248, V 2 5 0 ,1257, and V259 in HIV-1 integrase are involved
in the interface interaction. Mutations in these residues reduce tetramerization and
lead to decrease integrase activity. 2 1 ’2 3 Although a lot of research efforts have been
devoted to investigation of this domain, the DNA-binding mode of the C-terminal of
HIV-1 integrase is still not clear yet.
Lvs242
Val259
Ala24S Va1250
Fig. 6. Structure of HIV-1 integrase C-terminal domain.
(Adapted from Chen et al. Proc.Nat.Acad.Sci.USA 2000,97 8233 )
13
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1.3 HIV INTEGRASE INHIBITORS
By applying purified full-length integrase and radio-labeled oligonucleotide, one can
quantitatively measure the IC50 value of 3’-processing and strand transfer in vitro.
Many HIV-1 integrase inhibitors are developed by utilizing this assay to identify
candidate structures. 24-30 Most of the known inhibitors were developed through
modification of the lead compounds, or identified by 3-D pharmacophore searching
in NCI drug information system (DIS) database.31 -3 3 Many inhibitors have similar
characteristics. These structural characteristics, which are required for potent
inhibitors will be described as follows:
(1) THE DIHYDROXYNAPHOQUINONES -Dihydroxynaphoquinone (1) is not
only an integrase inhibitor; it is also one of the required basic structures for many
reported HIV-1 integrase inhibitors. The well-known inhibitors, which contain the
dihydroxynaphoquinone motif are mitoxantrone (2) and doxorubicin (3). 24 These
compounds are believed to act as inhibitors of DNA binding protein. Thus, they not
only are HIV-1 integrase inhibitors, but also topoisomerase inhibitors. The rationale
is that these compounds interact with DNA and lead to the inhibition of integrase.
However, targeting the conserved DNA sequence may bring about nonspecific
binding of host genome. Thus, highly selective viral DNA binder is required for
accomplishing viral chemotherapy. The structure of the natural product,
quercetagetin (4), which is the most potent integrase inhibitor among several natural
products, also contains the dihydroxynapthoquinone motif. Although quercetagetin
14
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does not have the ability to bind to DNA, it can inhibit integrase with the IC50 value
of 0.8 and 0.1 jdVl for 3’-processing and strand transfer, respectively.3 4
OH O
OH 0 NH(CH2)2NH(CH2)2OH
OH 0 NH(CH2)2NH(CH2)2OH
(2) mitoxantrone 1050=8.0 M -M
OH O
(1) dihydroxynaphthoquinone IC50= 2.5 pM
O OH O
OH
HO
OH
OMe O OH O
OH
OH
I.. NH.
'2
OH O
(3) doxorubicin IC50=2.4 pM (4) quercetagetin IC50=0.8 pM
(2) HYDROXYLATED AROMATICS -Hydroxylated aromatic ring system is the
most common characteristic of the HIV-1 integrase inhibitors. The hydroxylated ring
motifs not only include 1,2-dihydroxyl substituents, Caffeic acid phenethylester
(CAPE)(5) and bisarylamide (6), but also include l-methoxyl-2- hydroxyl, curcumin
(7) and mono hydroxyl substituents, chicoric acid (8), on one aromatic ring. The
catechol moieties are believed to chelate with the metal ion in the catalytic core of
integrase. Thus, modifying or deleting one of the hydroxyl groups will reduce the
activity of the compound. However, there are some other classed of inhibitors, which
15
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only require mono-hydroxyl or methoxyl group for activity (7,8). Another common
feature of hydroxylated aromatics compound is that a linker connects two aromatic
ring systems (5, 6, 7, 8)
(5 ) C A P E IC 5 0 = 1 8 .9 p M
O
N
H
OH
OH
(6) bisarylamide IC50= 0.2 pM
HO
OH
MeO
OMe
(7) Curcumin IC50= 40 pM
16
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HO
HO
COOH
COOH
OH
OH
(8) Chicoric acid IC50= 0.007 pM
Cl
H
(9) 5-CITEP 1050=35
(3) DIKETO ACIDS - By modifying curcumin (7), a series of compounds, which
contain the diketo motif, were identified. l-(5-chloroindol-3-yl)-3- hydroxy-3-
(2H-tetrazol-5-yl)-propenone (5-CITEP) (9) has been shown to have IC50 value of 35
pM and 0.65pM for 3’-processing and strand transfer, respectively.3 5 5-CITEP also
has been shown to interact with residues Lys-156, Lys-159 and Gln-148 of the target
35
enzyme, which are the residues near the active site. L-708906 (10), the potent
inhibitors with a diketo acid, specifically inhibits stand transfer with an IC50 value of
0.1 pM. Moreover, its analogous, L-731988 (11), also inhibits strand transfer at low
concentration. L-731988 has been shown to inhibit strand transfer by binding
within the integrase active site and competing with integrase DNA binding. However,
L-708906 does not compete with target DNA complex. The binding mode of
L-708906 has not yet been illustrated.
17
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o o
c o 2h
C O O H
O B n
(1 0 ) L -7 0 8 9 0 6 IC 5 0= 0.1 |iM
F
(11) L-731988 IC 5 0 = 0 .0 5 |x M
The features described above are common in many HIV-1 integrase inhibitors.
Although many compounds with one or more of these motifs have high inhibitory
activity, these motifs are not absolutely essential for activity in some cases. In the
other words, the compound, which has one or more of these motifs, may have higher
activity than others depending on the rest of the molecule. However, for drug design,
the same motif may have the same limitations, metabolism, toxicity, etc. Besides,
most inhibitors, which have been shown to inhibit recombinant integrase in vitro,
failed to inhibit preintegration complex (PIC) and viral replication in HIV infected
cells. Thus, further identification of potential inhibitors and their structural
characteristics are needed for rationale drug design in the future.
18
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1.4 REFERENCES
1. Asante-Appiah,E; Skalka, A. M. HIV-integrase: structural organization,
conformational changes, and catalysis. Adv. Virus Res 1999, 52, 351-369
2. Englman, A., Mizuuchik, C. HIV-1 DNA integration - Mechanism of viral -D N A
cleavage and DNA strand transfer. Cell 1991, 67, 1211-1221
3. Gerton JL, Herschlag D, Brown, P.O. Stereospecificity of reactions catalyzed by
HIV-1 integrase. J. Biol Chem 1999, 274, 33480-33487
4. Rice, P., Craigie, R., Davies, D. Retroviral integrase and their cousins. Curr. Opin.
Struc. Biol. 1996, 6, 76-83
5. Dyda, F., Hickman, A. B. Jenkins, T. M., Engelman, A. Craigie, R. Davies, D. R.
Crystal structure of the catalytic domain of HIV-1 integrase: similarity of other
polynucleotidyl transferases. Science 1994, 266, 1981-1986
6. Lodi, P. J., Ernst, J. A., Kuszewski, J., Hickman, A.B., Engelman, A., Craigier, R.,
Clore, G. M., Gronenbom, A. M. Solution structure of the DNA binding domain
of HIV-1 integrase. Biochemistry, 1995,34, 9826-9833
7. Eijkelenboom A.„ Lutzke, R., Boelems, R., Plasterk, R., Kaptein, R., Hard, K.
The DNA-binding domain of HIV-1 integrase has an SH3-like fold. Nat. Struct.
Biol. 1995, 2 , 807-810
8. Rice P., Mizuuchi, K. Structure of the bacteriophage Mu transposase core: a
common structural motif for DNA transposition and retroviral integration. Cell,
1995, 82, 209-220
9. Comeils, V., Antoinette, A. M., Oude, G., Ronald, Identification of the catalytic
and DNA-binding region of the human immunodeficiency virus type 1 integrase
protein. Nucleic Acid Res. 1993, 21, 1419-1425
19
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10. Kulkosky, J., Jones, K. S., Katz, R. A., Mack, J. R G. and Skalka, A. M. Residues
critical for retroviral integrative recombination in a region that is highly
conserved among retroviral/retrotransposon integrases and bacterial insertion
sequence transposases. Mol. Cell. Biol. 1992, 12, 2331-2338.
11. Burke, C. J., Sanyal, G, Burner, M. W., Ryan, J. A., LaFemina, R. L., Robbins, H.
L., Zeft, A. S., Middaugh, C. R., Cordingley, M. G . Structural implication of
spectroscopic characterization of a putative zinc finger peptide from HIV-1
integrase. J. Biol. Chem. 1992, 267, 9639-9644
12. Cal, M., Zheng, R., Caffrey, M., Craigie, R., Clore, M., Gronenbom, A. M.
Solution structure of the N-terminal zinc binding domain of HIV-1 integrase. Nat.
Struc. Biol. 1997, 4, 567-577
13. Zheng, R., Jenkins, T. M., Craigie, R. Zinc folds the N-terminal domain of HIV-1
integrase, promotes multimerization, and enhance catalytic activity. Proc. Natl.
Acad. S et USA 1996, 93, 13659-12664
14. Jenkins TM, Hickman AB, Dyda F, Ghirlando R, Davies DR, Craigie R:
Catalytic domain of human immunodeficiency virus type-1 integrase:
identification of a soluble mutant by systematic replacement of hydrophobic
residues.Proc. Natl. Acad. Sci. USA 1995, 92, 6057-6061
15. Goldgur, Y., Dyda, F., Hickman, A. B., Jenkin, T. M. Craigie, R. Three new
structures of the core domain of HIV-1 integrase: An active site that binds
magnesium. Proc. Natl. Acad. Sci. USA 1998, 95, 9150-9154
16. Engelman, A., Craigie, R. Identification of conserved amino acid residues critical
for human immunodeficiency virus type 1 integrase function in vitro J. Virol.
1992, 66, 6361-6369
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17. van Gent, D. C., Oude Groeneger, A. A. M., Plasterk, R. H. Mutational analysis
of the integrase protein of human immunodeficiency virus type 2. Proc. Natl.
Acad. Sci. USA 1992, 89, 9598-9602
18. Kulkosky, J., Jones, K. S., Katz, R. A., Mack, J. R G., Skalka, A. M. Residues
critical for retroviral integrative recombination in a region that is highly
conserved among retroviral/retrotransposon integrases and bacterial insertion
sequence transposases, Mol. Cell. Biol. 1992, 12, 2331-2338
19. Bujacz, G., Alexandratos, J., Qing, Z. L., Clement-Mella, C., Woldawer, A. The
catalytic domain of human immunodeficiency virus integrase: Ordered active site
in the F185H mutant. FEBS Lett. 1996, 175-178
20. Woemer AM, Marcussekura CJ., Characterization of a DNA binding domain in
the C-terminus of HIV1 integrase by deletion mutagenesis. Nucleic Acids Res
1993,21: 3507-3511
21. Eijkelenboom A., Sprangers, R., Hard, K., Lutzke, R., Boelems, R., Plasterk, R.,
Boelens, R., Kaptein, R., Refine solution structure of the C-terminal
DNA-binding domain of human immunovirus-1 integrase. Protein, 1999, 36,
556-564
22. Engelman A, Hickman AB, Craigie R,The core and carboxyl-terminal domains of
the integrase protein of human immunodeficiency virus type 1 each contribute to
nonspecific DNA binding. J Virol 1994, 68, 5911-5917.
23. Lutzke, R., Plasterk, R. Structure-based mutational analysis of the C-terminal
DNA-binding domain of human immunodeficiency virus type 1 integrase:
Critical residues for protein oligomerization and DNA binding. J. Vol. 1998, 72,
4841- 4848.
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24. Fesen, M. R., Kohn, K. W., Leteurtre, F., Pommier, Y. Inhibitors of human
immunodeficiency virus integrase. Proc. Natl. Acad. Sci. USA, 1993, 90,
2399-2430.
25. Zhao, H., Neamati, N., Hong, H., Mazumder, A., Wang, S., Sunder, S., Miline, G.,
Pommier, Y. Burke, T., Coumarin-based inhibitor of HIV integrase. J. Med.
Chem., 1997, 40, 242-249.
26. Mazumder, A., Neamati, N., Owen, J., Sunder, S.,Rando, R., Pommier, Y.
Inhibition of the human immunodeficiency virus type 1 integrase by guanosine
quartet structures. Biochemistry, 1996, 35, 13762-13771
27. Eich, E., Pertz, H., Kaloga, M., Schulz, J., Fesen, M., Mazumder, A., Pommier,
Y., (-)-Arctigenin as a lead structure for inhibitors of human immunodeficiency
virus type-1 integrase. J. Med. Chem. 1996, 39, 86-95
28. Mazumder, A., Wang, S., Neamati, N., Nicklaus, M., Sunder, S., Chen, J., Milne,
G., Rice, W., Burke, T., Pommier, Y. Antiretroviral agents as inhibitors of both
human immunodeficiency virus type 1 integrase and protease. J. Med.
Chem.1996, 39, 2472-2481.
29. Neamati, N., Mazumder, A., Sunder, S., Owen, J., Schultz, RJ. Pommier, Y.,
2-Mercaptobenzensulphonamides as novel inhibitors of human
immunodeficiency virus type 1 integrase and replication. Antiviral Chemistry &
Chemotherapy, 1997, 8, 485-495.
30. Neamati, N., Mazumder, A., Zhao, H., Sunder, S., Burke, JR., Schultz, R.,
Pommier, Y., Diarylsulfones, a novel class of human immunodeficiency virus
type 1 inhibitors. Antimirobial agent & chemotherapy, 1997, 41, 385-393.
31. Nicklus, M., Neamati, N., Hong, H., Mazumder, A., Sunder, S., Chen. J., Milne,
G., Pommier, Y. HIV-1 integrase pharmacophore: discovery of inhibitors through
three-dimenstional database searching. J. Med. Chem. 1997, 40, 920-929.
22
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32. Hong, H., Neamati, N., Wang, S., Nicklus, M., Mazumder, A., Zhao, H., Burke.
JR., Pommier, Y., Milne, G., Discovery of HIV-1 integrase inhibitors by
pharmacophore searching. J. Med. Chem. 1997, 40, 930-936.
33. Buolamwini, J., Assefa, H., CoMFA and CoMSIA 3D QSAR and docking studies
on conformationally-restrained cinnamoyl HIV-1 integrase inhibitors: exploration
of a binding mode at the active site. J. Med. Chem.2002, 45, 841-852.
34. Fesen, M. R., Poimmer Y., Leteurtre, F., Hiroguchi, S., Yung, J., Kohn, K. W.,
Inhibition of HIV-1 integrase by flavones, caffeic acid phenethyl ester (CAPE)
and related compounds. Biochem Pharmacol 1994, 48, 595-608
35. Golduur, Y., Craigie, R., Cohen, G., Fujiwara, T., Yoshinaga, T., Fujishita, T.,
Sugimoto, H., Endo, T., Murai, H., Davies, D. Structure of the HIV-1 integrase
catalytic domain complexd with an inhibitor: a platform for antiviral drug design.
Proc. Natl. Acad. Sci. USA, 1999, 96, 13040-13043.
36. Hazuda, D, Felock, P., Witmer, M., Wolfe, A., Stillmock, K., Grobler, J., Espeseth,
A., Gabryelski, L., Schleif, W., Blau, C., Miller, M., Inhibitors of Strand Transfer
That Prevent Integration and Inhibit HIV-1 Replication in Cells. Science, 287,
646-650.
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CHAPTER II
INHIBITION OF HIV-1 INTEGRASE BY
9H-2.3.9-TRIAZA-FLUORENE DERIVATIVES
2.1 INTRODUCTION
Reverse transcription, integration and proteolysis are the essential steps for the
replication of human immunodeficiency virus-1 (HIV-1), the cause of acquired human
immunodeficiency syndrome (AIDS). These three steps are carried out by three
virus-encoded enzymes, reverse transcriptase, integrase, and protease, respectively.
Theoretically, by inhibiting one of these enzymes inhibition of virus replication may be
accomplished.1 These three enzymes are currently targets of the anti-HIV treatments to
suppress the replication of the HIV-1 virus. The first group of anti-HIV drugs is targeted
against reverse transcriptase. Protease inhibitors block protein processing thus lead to
incomplete viral formation. There are already several enzyme inhibitors approved by
FDA, which can either inhibit viral reverse transcriptase or protease. However, patient
compliance, drug toxicity, drug resistance and the presence of persistent reservoirs of
virus replication are the problems emerging from the combination therapies of existing
dmg. Therefore, seeking for an alternative therapy is the main thrust of HIV therapy.
Integrase is considered to be the third target for the combination therapy because of no
24
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homologous protein existing in the cell. Thus, specific integrase inhibitors would only
associate with viral integrase and may have less toxicity toward the host.
Integrase is a 32 kDa viral enzyme that mediates integration reaction. This step is
believed to be essential for viral replication. Integration is a two-step procedure consists
of 3’-processing and strand transfer. During the 3’-processing, integrase will cut the GT
dinucleotide next to the highly conserved CA residues from long terminal repeats
(LTRs) at the both ends of viral DNA. 3’-processing is important for preparing viral
DNA for subsequent insertion reaction.3 A pair of reactions takes place 5 bases apart to
insert both ends of the viral DNA into the host genome. Purified integrase and
radiolabeled oligonucleotide are applied to determine the IC50 value of 3’-processing
and strand transfer. Many inhibitors are discovered by utilizing this assay to examine
candidate structures.
5H-pyridazino[4,5-b] indoles (I) derivatives have been show to have biological abilities
in previous papers.4 -1 1 These compounds are fist mentioned as antihypertensive agents.
1 0 This class of compounds have also been shown to be inhibitors of blood platelet
aggregation, which selectively inhibit adenosine 3’,5’-cyclicphosphate
phosphodiesterase (cAM P-PDE-IV). 6 Hiremath et al claimed that 11H-1, 2, 4-triazolo
[4,3-b] pyridazino [4,5-b] indoles to have the antimicrobial activities. 1 1 There are
several compounds have been described to have anti-HIV integrase activities as well as
antimicorbial activity. In addition, this class of compounds has the planer
pharmacophore feature existing among HIV-1 integrase inhibitors. This suggests that
25
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5H-pyridazino[4,5-b] indoles derivatives may be potential inhibitor of HIV-1 integrase.
Thus, we have examined the inhibitory abilities of this class of compounds. In this study,
thirty compounds were tested for their inhibitory activity in 3’-processing and
DNA-strand transfer. We also calculate several physical parameters of the compounds
for structure-activity relationships studies. These studies were to establish what
substituents are important factors influencing integrase activity. We used an in-vitro
screening method, by the addition of integrase and oligonucleotide with the putative
CAGT sequence, where the ability to inhibit 3’-processing and strand transfer were
evaluated.
H
(0 5H-pyridazino[4,5-b] indoles
26
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2.2 MEHTODS
CHEMICALS. Compounds were dissolved in DMSO, and all aliquots were also diluted
in DMSO prior to each experiment. The stock solutions were kept at -20 °C. The final
concentration of compounds was between 1000 pM and 33 pM.
PREPARATION OF OLIGONUCLEOTIDE SUBSTRATES. The HPLC-purified
oligonucleotides 21top 3-GlGiQGAVVnCICrAXAjT-3 and
21bcttorp 5-ACIGCrAGAGAliiiGCAC^Cwere ordered from u s c Norris microchemical
core facility (Los Angeles, CA). The expression system for the wild-type HIV-1
integrase was a generous gift of Dr. R. Craigie, Laboratory of Molecular Biology,
NIDDK, NIH, Bethesda, MD. To analyze the extents of 3’-processing and strand
transfer using 5’-end-labeled substrates, 21top was 5’-end-labeled using T4
polynucleotide kinase (Epicentre, Medison, WI) and [y-32 P] ATP (Amersherm). The
kinase was heat-inactivated, and 21botton was added to the final solution. The mixture
was heated at 95 °C, allowed to cool slowly to room temperature, and added onto a G-25
Sephadex quick spin column (USA scientific) to separate annealed double-stranded
oligonucleotide from unincorporated label.
INTEGRASE ASSAY. Integrase 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 pM EDTA,
50 pM dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/mL bovine serum
albumin, 10 mM 2-mercaptoethanol, 10% dimethyl sulfoxide (DMSO), and 25 mM
27
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MOPS, pH 7.2) at 30 °C for 30 min. Then, 20 nM of the 5’-end 32 P-labeled linear
oligonucleotide substrate was added, and incubation was continued for an additional 1 h.
Reactions was quenched by the addition of an equal volume (16 pL) of loading dye
(98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol, 0.025%
bromophenol blue). An aliquot (5 pL) was placed on a denaturing 20% polyacrylamide
gel (0.09 M Tris-borate, 2 mM EDTA, 20% acrylamide, 8 M urea, pH 8.3). The
separation was achieved using 2900 volts for 3.5 hrs. Gels were dried, and exposed on a
film.
PARAMETER FRAME-SETTING. The dipole moment (p) was calculated by the use
of Hyperchem program (Hypercube, Inc, Waterloo, Canada), after geometry
optimization and energy minimization were carried out by AMI semi-empirical method.
1 2 The hydrophobic parameters including the calculated n-octanol/water partition
coefficient (clog P) and the calculated molar refractivity (CMR) were calculated by
using clog P software (Claremont, C A ).13 -1 4 Two-dimensional parameter frame-setting
was plotted by using MS-Excel 2000. Three-dimensional parameter frame-setting was
constructed by using Origin software (Microcal Inc., Northampton, MA). 15 -1 6 The
maximum number of hydrogen bond (Hb) was calculated by the sum of hydrogen bond
donors and acceptors.
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2.3 RESULTS AND CONCLUSIONS
Several studies showed that 5H-pyridazino[4,5-b] indoles (I) derivatives have various
of biological activities in vivo and in vitro. 4 -1 1 11H-1, 2, 4-triazolo [4,3-b] pyridazino
[4,5-b] indoles has been shown to have antimicrobial activities11. In previous studies, it
has been shown that antimicrobial agents may be able to inhibit HIV-integrase. Thus,
we proposed that this class of compounds might have ability to inhibit HIV-1 integrase.
Inhibitory abilities of all the available analogues of 5H-pyridazino[4,5-b] indoles were
examined for both 3’-processiong and strand transfer. In order to further dissect the
structure-activity relationship of these compounds, five different physicochemical
parameters are selected as determinants for analysis. These parameters include dipole
moment (p), calculated octanol / water partition coefficient (clog P), molar refraction
(CMR), molecular weight (MW), and total number of hydrogen bonds possible (Hb).
With these physicochemical parameters, some common features of active compounds
can be delineated to build a model for drug design in the future.
The initial compound 2 shows only moderate inhibitory activity (ICso= 700pM)(Table
1). We first studied the effect of the functionality at the C-4 position on the pyridazine
ring of the lead compound 2. The results are presented in Table 1. First, a halogen at the
1-position of 9H-2.3.9-triaza-fluorene (compound 3) was examined. Such modification
successfully increased the inhibition ability by 5 fold (IC50 =170pM). Additionally,
phenyl amine substitution (compound 4) also doubled the potency. (ICso= 315 pM).
However, replacing the phenyl group with hydroxyl group (5) abolish the activity. This
29
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may be due to the different lipophilicity, polarity or electronic property o f the phenyl
ring at this position. The exact rationale still require further delineation. Compound 27,
which was substituted with a 5-amino- l/f-pyrazole-4- carbonitrile group at C-4 position,
can inhibit 50% 3’-processing interaction at 600 pM. Moreover, it can inhibit strand
transfer under 300 pM. Conversely, the activity of its analogues 28 (which replaced
4-carbonitrile with carboxylmethyl-) is diminished in the assay. Comparing the
compounds in table l,w e hypothesize that a high electron density atom at 4-position
may increase the inhibitory ability of compounds. On the contrary, a bulky group at this
position would decrease the activity.
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Table 1 5H-pyridazino[4,5-b] indoles
No. R 3’-proc(pM) ST(pM)
AHF2 H 700 700
AHF 3 Cl 170 ± 26.46 133 ± 57.53
AHF 4
"ipO
315 ±195 2 8 1 ± 151
AHF 5
H OH
>1000 >1000
AHF
20a
\ s ^ Y ° s / C h3
0
>1000 >1000
AHF 26
ch3
h c ^ n
m 3° N
1
>1000 >1000
AHF 27
NC^
1
600 300
AHF 28
H3C \ P
H 2 N " n N
N
1
>1000 >1000
AHF 37
YVO
o
900 >1000
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Table 2 lists a number of 5H-pyridazino[4,5-b] indoles-1-one derivatives with
various functional groups at N-2 and N-5 positions of 5H-pyridazino[4,5-b] indoles ring.
The data indicate that, this series of compounds are not as potent as the compounds in
Table 1. It appears that the keto group at 1-position lower the activity. 5-N-methyl
(compound 7a) and 2-N-methyl (compound 14m) even lose their inhibitory activity.
However, compound 14 (2 ,5-N, N- dimethyl substitution) has moderate potency (IC5o=
600pM). In a previous study, addition of dimethyl amino and diethyl amino groups on
main active structure increased the activity. Thus, we introduced diethyl methyl amino
and propyl morpholino group to the 2 and 5 positions. Comparison of compounds 16,29,
and 46a shows that diethyl methyl amino group at 5-positions increases the activity. On
the other hand, diethyl methyl amino group at 2-positions either had no effect or
decreased the activity. Conversely, the propyl morpholine substitution abolished all the
activities of the compounds (see compounds 14, 22, 23, and 45). This maybe due to
binding to different active binding sites of the enzyme. It is worth to notice that bulky
groups at 2-position decrease the activity. The bulky side chain at this position may
cause steric hindrance, preventing the inhibitor from inserting the active site of the
enzyme. Furthermore, comparing clog P value, dipole moment (p), CMR and molecular
weight of compounds in Table 2(Table 5), it seems that while these physical properties
are important, however, they are not the only parameters affecting the activities, others
like shape and symmetry may also be involved but not easily parameterized.
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Table. 2. 5H-pyridazino[4,5-b] indoles-1-one derivatives
R .
l1
No R1 R2 3’-proc(pM) ST(pM)
AHF 14m c h 3 H >1000 >1000
AHF 14 c h 3 c h 3 600 600
AHF 7a H c h 3 >1000 >1000
AHF 9 H
O
>1000 >1000
AHF 47 c h 3
o
>1000 >1000
AHF 46a c h 3
^ ~ N \
600 600
AHF 16
^ ~ N \
H 267 ± 76.37 278 ± 83.3
AHF 29
^ N \
411 ± 84.04 278 ± 8.33
AHF 22 H >1000 >1000
AHF 23
^ r \
>1000 >1000
AHF 45 c h 3
r ^ o
>1000 600
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According to the results in Table 1 and 2, four compounds are deigned to dissect the
different properties required for HIV-1 integrase inhibition (Table 3). Compound 3 and
16 both show greatly improved activity than the original structures. In order to study the
property and character of diethyl methyl amino group at position 5, diethyl methyl
amino group of 16 was modified to dimethyl propyl amine (63). The activity o f 63 is 2
fold less than the relative compound, 16. The data in Table 1 indicates that introducing a
high-electron density functional group at position 1 is required for enhancing activity.
To address this possibility, a hydrazine was introduced to the 5H-pyridazino[4,5-b]
indoles at position 1 (compound 20d). It shows 50% inhibition at 333 pM, while the
IC50 of compound 2 is 7 0 0 1 1M. It is consistent with our prediction. Compound 49, as
compared with 3, the activity is diminished with the presence of a methyl group at
position 1. In other words, the chloro- group at 1 position gives lower IC 50 than the keto
group in 14m, which further conforms the previous result. Thus, Compound 64 was
synthesized to improve the inhibition of 16. Surprisingly, the activities of compound 64,
which carboxyl group was replaced by a chloro- group, turned out to be not as good as
expected. This may be due to the change of dipole moment or clog P. This result also
reveals that compounds in Table 1 and 2 perhaps have different binding modes with
HIV-1 integrase, although they share the same main structure feature. To clarify which
factors is essential for the activity, more compounds with a wider range of substituents
are required.
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Table 3. Newly synthesized compounds
Stucture No. 3’-proc(pM) ST(pM)
NHNH
AHF 20d 333 200
CH
AHF 49 500 333
AHF 63 500 500
CH,
n h +:
AHF 64 450 350
CH,
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In Table 4, another class of compounds, namely 5-methyl-6H-l,2,3a,4,6- pentaaza-
cyclopenta[c]fluorine derivatives were tested. None of the compounds in this series has
the activity against 3’-procressing and strand transfer, except compound 1.Compare
compound 1 with other similar compounds in Table 3, one sees that 3-position is a
critical site for this series of compounds to interact with integrase. Another possibility of
compound 1 to have better activity is that with the presence of lone pair electrons at
3-position, the entire ring behaves like an aromatic system. There are studies showing
that the negative n electron cloud can interact with positivly charged metal ion to form a
noncovalent bond. 1 7 -1 9 Thus, compound 1 may from a cation-rc interaction with
Mn2 + /Mg2+ ions which reside in the core domain of integrase. Thus, further reducing the
activity of integrase.
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Table 4 5-methyl-6H-l,2,3a,4,6- pentaaza- cyclopenta[c]fluorine derivatives
No. X R 3’-proc(pM) ST(pM)
AHF24 C H >1000 >1000
AHF 25 C c h 3 >1000 >1000
AHF 38 C >1000 >1000
AHF 39 C
- O
>1000 >1000
AHF 42 C =S >1000 >1000
AHF 43a C
W r Q ?
>1000 >1000
AHF 44a C
XS ' ^ s _ N ^
>1000 >1000
AHF 1 N - 157 150
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To further investigate the property of active compounds, two-dimensional parameter
frame-setting was applied to analyze the data (Fig. 7). It shows that the active
compounds (ICso<1000 pM) and inactive (IC5o>1000 pM) compounds do no have
completely different location in their physicochemical parameters space. Moreover, the
three-dimension parameter frame-setting technique was also applied for further analysis.
Consistently, the separations of active and inactive compounds by using available
physicochemical parameters are not completely satisfactory. This may be due to the
lack of well-separated end points and the number of active compounds is limited.
However, in the three-dimension parameter frame-setting, it was observed that active
compounds do not have high number of possible hydrogen bonds (over 10)(Fig. 8).
Although more hydrogen bond forming sites may improve the affinity of binding, it
may also increase the hydrophilicity of the compound. Moreover, more nonspecific
binding with other sited than the active sites of integrase may take place.
2.4 CONCLUSIONS
Sixty-one compounds in this study have been examined. Although most of the
compounds are not active( IC5o>1000 pM), by analyzing the data and the
physicochemical parameters of the compounds, the following findings are obtained.: 1)
The activity of anti HIV integrase can be promoted by substituting a high electron
density functional group at 1 position of 5H-pyridazino[4,5-b] indoles. 2) A bulky
group at position 2 of 5H-pyridazino[4,5-b] indoles would diminish the activity. 3) In
drug or ligand design, excessive possible hydrogen-bond forming group on a compound
38
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may lead to nonspecific bind, reduce the lipophilicity and thus reduce the activity. To
test these working hypotheses, more systematically synthesized compounds are needed
for further study.
Table 5 Physicochemical parameters of compounds
Compound clog P CMR U Hb MW
AHF1 2.93 6.14 8.409 7 224.23
AHF3 2.69 6.03 5.465 4 217.66
AHF 16 1.77 8.6 5.43 6 297.4
AHF4 4.27 8.88 4.417 6 288.36
AHF20d 2.45 6.27 4.267 9 213.24
AHF29 3.86 12.03 5.135 7 399.58
AHF63 2.35 8.44 5.728 8 283.38
AHF49 2.67 6.49 5.842 3 233.7
AHF64 3.85 9.18 3.99 4 318.85
AHF27 1.54 8.58 6.543 9 291.32
AHF 14 1.92 6.68 4.348 5 227.27
AHF46a 2.45 9.36 4.773 6 312.42
AHF 2 1.92 5.54 4.87 4 183.21
AHF37 2.59 9.28 4.442 8 317.35
AHF5 2.06 6.99 1.932 7 240.31
AHF20a 2.83 8.39 2.912 10 301.37
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Table 5. Continued
Compound clog P CMR U Hb MW
AHF26 3.48 8.19 2.73 6 277.33
AHF28 3.55 9.21 5.024 12 338.37
AHF14m 1.98 6.21 4.936 6 213.24
AHF7a 1.88 6.21 4.128 6 213.24
AHF9 3.4 8.26 4.412 6 275.31
AHF47 3.69 9.19 4.562 5 303.37
AHF22 1.95 8.88 5.271 8 312.37
AHF23 1.53 12 3.965 11 425.54
AHF45 1.55 9.34 4.035 8 326.4
A H F24 1.75 6.44 6.748 6 223.24
A H F25 2.56 7.37 6.351 6 251.29
AHF38 3.98 9.08 6.434 6 327.39
AHF39 3.06 8.95 6.381 6 299.34
AHF 42 2.07 7.51 6.652 9 255.3
AHF 43a 2.56 10.38 8.598 11 368.46
AHF 44a 3.57 10.4 7.173 9 354.48
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A
P 4
B
14
12
10
8
6
4
2
0
0.5
♦ ♦
♦ ♦
♦
♦
♦ X ♦
1.5 2 2.5
clop
3.5
* Active
* Inactive
43
B
♦
♦♦
♦
♦ ♦
♦
♦
♦
♦
^ ♦
♦
-
* Active
Inactive
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
clo p
Fig. 7. Two-dimensional parameter frame-setting of different combinations of
physicochemical constants. ♦ represent active compounds (ICso<1000 pM). A
represent inactive compounds (ICso>1000 pM). (See text for the parameters
used).
41
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c
* Active
Inactive
D
Active
Inactive
Fig. 7. Continued
42
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.E
X I
SC
14
12
10
8
6
4
2
0
A
♦ ♦
♦ A ♦
♦
A A # ♦ £ ♦ Ai A
♦ A
♦ ♦ ♦
♦
4 Active
4 Inactive
0 1 2 3 4 5 6 7
DM
9 10
P i
S
u
♦ ♦
♦ /
♦ ♦
♦
♦
♦ ♦ ♦
♦
* Active
* Inactive
DM
10 12 14
Fig. 7 Continued
43
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G
Active
Inactive
H
Active
Inactive
CMR
Fig. 7 Continued
44
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I
* Active
Inactive
450
400
350
300
250
200
150
100
50
0
$ 1
♦
♦
♦
* Active
Inactive
10 12 14
Hb
Fig. 7 Continued
45
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10 ■
O
350^ ° ° 450
N & N
200
150
Fig. 8 . Three-dimensional parameter frame setting.^ represent active compounds
( I C 5 o < 1 0 0 0 pM). O represent inactive compounds ( I C 5 o > 1 0 0 0 pM).
46
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2.5 REFERENCES
1. Moor, J. P., Stevenson, M., New target for inhibitors of HIV-1 replication. Nat Rev
Mol Cell Biol. 2000, 1,40-49
2. Leung, D., Abbenante, G., Fairlie, D., Protesas inhibitors: Current status and future
prospects. J. Med. Chem., 2000, 43, 305-341
3. Asante-Appiah,E; Skalka, A. M. HIV-integrase: structural organization,
conformational changes, and catalysis. Adv. Virus Res 1999, 52, 351-369
4. Font, M., Monge, A., Cuartero, A., Elorriaga, A., Martinez-Irujo, J. J., Alberdi, E.,
Santiago, E., Prieto, I., Lasarte, J. J., Indole and pyridazino[4,5-b]indoles as
nonucleoside analog inhibitors of HIV-1 reverse trancripatse. Eur. J. Med. Chem..,
1995, 30, 963-71.
5. Monge, A., Aldana, I., Alvarez, T., Losa, M. J., Font, M., Cenarruzabeitia, E.,
Lasheras, B., Frechilla, D., Castiella, E., Fernandez-Alvarez, E.,
I-Hydrazino-4-(3,5-dimethyl-l-pyrazolyl)-5H-pyridazino[4,5-b]indole. A new
antihypertensive agent. Eur. J. Med. Chem., 1991, 26, 655-758
6 . Monge, A., Aldana, I., Alvarez, T., Font, M., Santiago, E., Latre, J. A., Bermejillo,
M. J., Lopez-Unzu, M. J., Fernandez-Alvarez. E., New 5H-pyridazino[4,5-b]indole
derivatives. Synthesis and studies as inhibitors of blood platelet aggregation and
inotropics. J. Med. Chem. 1991, 34, 3023-3029.
7. Monge, A., Aldana, I., Fernandez-Alvarez. E., 4-Hydrazino-5H-pyridazino
[4,5-b]indole, anew antihypertensive agent. Eur. J. Med. Chem. 1978,13, 573-575.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
8 . Monge, A., Aldana, I., Losa, M. J., Font, M., Castiella, E., Frechilla, D.,
Cenarrazabeitia, E., A novel class of cardiotonic agents: synthesis and biological
evaluation of pyidazino[4,5-b]indoles with cyclic AMP phosphodiesterases
inhibiting properties. J. Pharm. Sci. 1993, 82, 526-30.
9. Monge, A., Parrado, P., Font, M., Fernandez-Alvarez. E., Selective thromboxane
synthetase inhibitors and antihypertensive agents. New derivatives of
4-hydrazino-5H-pyridazino[4, 5-b]indole, 4-hydrazinotriazino[4,5-a]indole, and
related compounds. J. Med. Chem. 1987, 30, 1029-1035.
10. Monge, A., Aldana, I., Parrado, P., Font, M., Fernandez-Alvarez. E.,
Antihypertensive Agents: Pyridazino[4,5-b]indole derivatives. J. Pharm. Sci. 1982,
71, 1406-1408
11.Hiremath S. P., Ullagaddi, A., Shivaramayya, K., Purohit, M., Synthesis of
llH-l,2,4-triazolo[4,3-b]pyridazino[4,5-b]indoles. Indian J. Heterocycl. Chem.
1994, 3, 145-148
12. Hypercube, Inc. Hyperchem for Windows, Versoin 5.0, publiscation
HC50-00-02-00, 419, Philliph St., Waterloo, Ontario, Canada, 1996.
13. Clog P version4.0,1999. Biobyte Corp. 201 W. 4thSt.#204, Claremont, CA, 91711.
14. Wang. R.B., Ren, S. J., Lien, L., Lien, E., Chen, C. H., Lin, J. Y., Liu, K.,
Quantitative structure-activity relationship analysis of the inhibitory effect of
flavonoids on angiotensin converting enzyme. Chinese Pharma. J. 1999, 51,
149-162
15. Origin version 6.0, Microcal Software, Inc., 22 Industrial Dr. E. Northampton, MA.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
16. Lien, E., Wang, T., Lien, L. Phytochemical and SAR analyses of limonoids in citrus
and Chinese herbs: Their benefits and risks of drug interaction. Chinese Pharma. J.
2 0 0 2 , in press.
17. Kumpf, R., Dougherty, D. A mechanism for ion selectivity in potassium channels:
compoutational studies of cation- % interactions. Science, 1993, 261, 1708-1710
18. Caldwell, J., Kollman, P. Cation- t c interactions: Nonadditive effects are critical in
their accurate representation. J. Am. Chem. Soc. 1995, 117,4177-4178
19. Dougherty, D., Cation- n interaction in chemistry and biology: A new view of
benzene, Phe, Tyr, andTrp. Science, 1996, 271, 163-168
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CHAPTER III
INHIBITION OF HIV-1 INTEGRASE BY
2-MERCAPTOBENZENESULPHONAMIDE
DERIVATIVES
3.1 INTRODUCTION
A major problem with the current HIV therapeuties is development of drug resistance.
The current available drugs either target HIV-1 reverse transcriptase or protease.
Thus, search for new therapeutic targets for the development of anti-HIV treatment is
highly desirable. Currently, the new targets under consideration for designing
inhibitors of HIV replication include viral-cell attachment, virus-entry, uncoating,
nuclear import, etc1 . Integrase is one of the latest targets that is under investigation.
Integrase is a viral enzyme, which is responsible for insertion of viral DNA into host
DNA. Several inhibitors, which successfully inhibit integrase in vitro, have been
discovered2. However, most of the inhibitors are active in vitro and exhibit no
activity in vivo or with preintegration complex (PIC). Only one inhibitor, a diketo
acid derivative, is currently under clinical trail. Therefore, attempts have been made
to design second and third generations inhibitors.
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Substituted sulphonamide compounds were first identified by three-point
pharmacophore searching of the NCI three-dimension database3. The pharmacophore
was derived from caffeic acid phenethyl ester (CAPE) and NSC 115290, which have
been shown to have IC50 value at 10pM and 5pM, respectively3. Forty-three out of
sixty compounds identified by the study carry sulphonamide functional group. It
implies that the sulphonamide group may be required for high activity. Consequently,
a new family of compounds, 2 -mercaptobenzenesulphoamides has been described to
have inhibitory ability against HIV-1 integrase4. The compounds, which were
demonstrated to have high inhibitory activities against HIV-1 integrase, contain a
mercapto group adjacent to a sulphonamide. In the study, when the mercapto group
was replaced by thioethers, the compound totally lost activity in integrase assay. It
shows that the free mercapto group is important for the activity. In addition, a chloro
group at 4-position also increases the activity of the parent compound. Therefore,
4-chloro-2-mercapto-5-methyl- benzenesulphonamide is the structure of the principle
compounds in this study. To optimize the drug efficacy, various 2-mercoapto-
benzenesulphonamide derivatives were examined. In this study, the effect of
substitutents on sulfonamide and 3-position were explored.
Several cyclic analogous of 2-mercoapto-benzenesulphonamide have been tested for
anticaner and anti-HIV activities5'8. Those compounds, which have anti-HIV
activities, were demonstrated to either inhibit protease or reverse transcriptase.
However, this series of compounds have never been tested for anti-HIV-1 integrase
activity. Thus, cyclic analogous of 2-mercoapto-benzene- sulphonamide,
51
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(6-chrolo-3-merccapto-1,1 -dioxo-[ 1,4,2]dithiazin-3-yl)phenyl methanone derivatives
were selected for testing in this study.
3.2 METHODS
CHEMICALS. 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.
The final concentration of compounds was between lOOOpM and 1.3 pM.
PREPARATION OF OLIGONUCLEOTIDE SUBSTRATES. The HPLC-purified
oligonucleotides 21tcp 5431 GlOGAYv^flO Cl A3CAGT-3 and
2 1bcttan, 5-A C IG C T /^G A IT nC C A ^ were ordered from u s c Norri§
microchemical core facility (Los Angeles, CA). The expression system for the
wild-type HIV-1 integrase was a generous gift of Dr. R. Craigie, Laboratory of
Molecular Biology, NIDDK, NIH, Bethesda, MD. To analyze the extents of
3’-processing and strand transfer using 5’-end-labeled substrates, 2 1 top was
5’-end-labeled using T4 polynucleotide kinase (Epicentre, Medison, WI) and [y-3 2 P]
ATP (Amersherm). The kinase was heat-inactivated, and 21botton was added to the
final solution. The mixture was heated at 95 °C, allowed to cool slowly to room
temperature, and added onto a G-25 Sephadex quick spin column (USA scientific) to
separate annealed double-stranded oligonucleotide from unincorporated label.
INTEGRASE ASSAY. Integrase 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
pM EDTA, 50 pM dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCL, 0.1 mg/mL
bovine serum albumin, 10 mM 2-mercaptoethanol, 10% dimethyl sulfoxide (DMSO),
and 25 mM MOPS, pH 7.2) at 30 °C for 30 min. Then, 20 nM of the 5’-end 32
P-labeled linear oligonucleotide substrate was added, and incubation was continued
for an additional 1 h. Reactions was quenched by the addition of an equal volume
52
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(16 |iL) of loading dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene
cyanol, 0.025% bromophenol blue). An aliquot (5 pL) was placed on a denaturing
20% polyacrylamide gel (0.09 M Tris-borate, 2 mM EDTA, 20% acrylamide, 8 M
urea, pH 8.3). The separation was achieved using 2900 volts for 3.5 hrs. Gels were
dried, and exposed on a film.
3.3 RESULTS AND DISCUSSION
Previously, it was show that a free mercapto group in the 2-mercapto-
sulphonamide class of compounds is required for low IC50 value against HIV-1
integrase. However, the role of the mercapto group in enzyme inhibition is not clear.
The predicted pka value of mercapto at 4-chloro-2-mercapto-benzene-
sulphonamides is 4.97+0.5 (obtained using the ACD/I-Lab Web servies (ACD/pka
6.0)). Thus, the compounds bearing a mercapto group are negatively charged when
the compounds are added into reaction solutions (p H ^ 7.4). Thus, in order to clarify
the ambiguous point, compounds with inner salt form of mercapto group were
examined (compound 3-11). The results are presented in Table 6 . In the previous
report, the data indicated that the bulkier group (two-rings and three-rings)
substituted on the N of sulfonamide enhanced the compounds’ inhibitory abilities. 4
The results shown in Table 6 are also consistent with the observation from the
previous report. Compound 3 and 4 are more potent than other compounds in Table 6 .
Moreover, the number of possible hydrogen bonds forming site may also be the
major factor affect the potency. In Table 6 , the compounds, which have higher
number of hydrogen bonding sites, are more active. Compounds 5 and 6 have one or
53
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three hydrogen bonds, respectively, while other compounds have five or more
hydrogen bonds on the substituent of sulfonamide. In spite of the fact compounds 3
and 4 are relative potent, compared with other structures (see Table 8 ) this set of
compounds are not the most active. This may be due to the presence of a positive
charge in these compounds (Table 6 ). The positively charged nitrogen may delocalize
the negative charge mercapto group; consequently, reduce the ability of mercapto
group to interact with the enzyme. Another possibility is that the binding site of this
class of compounds is a negative charge while that of the enzyme is likely to be a
positive charge. Thus, the compound with positive charge substituent may be more
unlikely to interact with HIV-1 integrase.
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Table 6 . Inner salt form of mercapto compounds.
Main structure No R
IC50
3’-pro ST
ci'- Y ^ r /S
9
3 O 60 60
4 s 60 50
% 0 A
9
5
— C = N
700
6
— C - P h
I I
0
2 0 0 1 1 1
7
— C -O -O E t
333 1 1 1
1 C l
u , . a
K A
9 X C
A
8 - 1 1 1 37
C ' Y Y S
M e A C
° 0 J p ° o
9
/ N\
9 1 1 1 1 1 1
1 0 2 0 0 60
11
X X
333 1 1 1
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The data reported before shows that a compound substituted with acetate group at
5-position is more potent than the compound substituted with a methyl group4. This
indicates that substituting bulk group at 5-position may increase the ability of parent
compound to inhibit HIV-1 integrase. Thus, compound 20~23 were synthesized and
tested for their ability to inhibit HIV-1 integrase. Compound 20 and 21 both inhibit
50% of 3’-processing at 6 pM, while the IC50 value of compound 22 and 23 is 80 and
37 pM, respectively.
Table 7. Mercapto compounds with substituents at N-position of sulphonamide group
Main structure No R1 R2
IC50
3’-pro ST
C,\ y^ W SH
1 2
' O ,
- 2 0 1 2
13
7 a.
- 12.3 8
14 - 15 8
0 M
R1 N = r ^
■ ■ ' ' • - a .
2 0 H Cl 6 1 2
2 1 H Me 6 1 0
2 2 F F 80 37
23 Cl F 37 37
56
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To further investigate how the substituents on triazolo ring and amide affect the
activity of compounds, more compounds were systemically synthesized (compound
42-53). Compound 51 is only slightly more potent than 21. The chloro substituent
may increase the inhibitory ability. Conversely, by comparing 23 with 51 and 20 with
50, one sees that compounds substituted with fluoro have lower activities.
Substituted a phenyl group on 4-N of triazolo group enhanced the activity
(Compounds 42, 47, 48 and 49). Substituent at /^-position of phenyl ring of the amide
group may change the activity. Chrolo substituted compound (46) is twice as active
as methoxyl compound (47). Moreover, compound 22, which was substituted with
two fluoro atoms, is less active than compound 50 with just one fluoro atom.
Compare compound 43, 44, 45, 46, and 47, we can conclude that the stronger the
electon-withdrawing substituent, the higher is the activity (Cl‘>CH3>OCH3). When
the IC50 values of the compound 51 was compared with those of their corresponding
alkyl analogue (43 and 46), the presence of the phenyl ring increased the activity
by 10 fold. Although 49 is not as potent as 51, the presence of the phenyl group also
improved the activities of other alkyl analogue (compound 42, 44, 48, and 49).
Surprisingly, the activities of the compounds (52 and 53), in which the mercapto
group was protected, were not abolished. This is inconsistent with our previous
observation 3’ 4. Compound 53 still retains the activity of compound 42. Compound
52 have an IC50 value of 333pM, although it is 5 times higher than that of compound
44. This may be due to the possibility that under the experimental condition the
carboxylic group will be ionized. In addition, the compounds with carboxyl group
57
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(which were described to decrease their inhibitory activity in the previous report)
were not tested previously at the concentration over 200pM4.
58
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Table 8 . Various substituents on trizaolo ring and amide.
No
Ri r 2 r 3
3’-proc(fj.M) ST(pM)
42
H 3 C - ( 3 —
c 3h 7 H 41 ± 5.3 80
43
ci—C 3
c 4h 9 H 35.7 ± 15 43
44 h3C- < 0 C4H9 H 60 37
45
O - A
C4H9 H 90.5 ± 29 70
46 i-C4H9 H 38.5 ± 2.1 40
47
H 3 c o - ^ y
/-C4H9 H 75 ± 7 1 0 0
48 H 3 C ~ 0 Sec C4H9 H 2 0 0 1 1 1
49 h 3 C 0
r O
H 9.3 ± 2 .3 8 , 8
50
O
-O-
H 45.7± 7.5 50, 50
51 C ^ Q - H 4.1 4.1
52 h C4H9 -CH2COOH 333 333
53
h3 c h Q “
c 3h 7 CH2COOH 50 50
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To verify whether the negative charge is important for activity or not, compounds
(Table 9. 18-19 and 25-30 substituted with various functional groups protecting the
mercapto groups) were examined. As expected, all the compounds in Table 9 were
inactive (IC5o>1000pM). Even though, to illustrate the importance of negatively
charged functional group at 2 -position, more compounds are needed for further study.
Moreover, the compounds which were claimed to have IC50 value over 200pM,
would also need to be tested for their inhibitory activity again in order to establish a
better structure-activity relationship (SAR).
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Table 9. Substituted on mercapto group
Compound No R1 R2 R3 R4
IC50(pM)
3’-proc ST
V OX
0 0
18
— C = N
- - - >1000 -
19
V .
- - - >1000 -
O ' 'O I " 2
24 H H - - 200 200
25 Ph H - - >1000 -
26
= C
1
Ph
H - - >1000 -
R2Y T f’Y
R4
27 H Cl Me SMe >1000 -
28 H Cl Me Cl >1000 -
29 -S02M e H H H >1000 -
30
— SO,
< >
no2
H H H >1000 -
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The inhibition data of cyclic analogues (15-17 and 31-37) are presented in Table 10.
Adding an extra chrolo group at m-position (compound 16) did not improve the
activity of the compound with only one chrolo substituent at p-position (compound
15). In addition, a compound with a nitro group at p-position is more potent than the
one with nitro group at m-position. Thus, it appears that the p-position may be a
critical site for interaction with HIV-1 integrase. Substituting a fluoro or a phenyl
group at p-position (compound 33 and 36) increases the activity over the
unsubstituted compound (31) by 4 fold. Although the compounds listed in Table 9
were substituted with various substituents, the IC50 values only varied from 15 to
3pM. To improve the activity of this series of compounds, the correlation between
the physicochemical properties of the available compounds and the observed
activities need to be analyzed. However, to get a highly correlated linear regression
equation in quantitative structure-activity relationships (QSAR) study, the range of
IC50 values need to cover at least two log-units. Thus, for doing QSAR study of this
set of compounds, additional analogous, with a wider range of activities need to be
tested.
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Table 10. Cyclic analogues of 2-mercapto-benzensulphonamide
O
M e'
No R 3’-proc(pM) ST(pM)
15
X X . ,
7.0 7.0
16
o o
7.0 7.0
17
CM
o
15.0 15.0
31
X)
12.3 12.3
32
xi.
12.3 12.3
33
X X ,
3 3
34 12.3 12.3
35
CM
o
0
11.1 11.1
36 3.7 2
37
x o
6 6
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3.4 CONCLUSION
Base on the structure activity relationship, we propose a model for designing new
integrase inhibitors of 2-mercoapto-benzenesulphonamide. Substitute a bulky group
at 5-position and at sulphonamide of 2-mercapto-benzensulphonamide may enhance
the inhibition ability. Furthermore, negative charge at 2-position is important for
activity. Mercapto group at 2-position is required for high activity, but may not be
absolutely required for activity. The anti-HIV abilities of cyclic
2-mercapto-benzene-sulphonamide analogous were established. These series of
compound have been demonstrated to inhibit reverse transcriptase, protease and
integrase. The compounds not only inhibit HIV replication in vitro but also in vivo.
Thus, in this study, two classes of HIV-1 integrase inhibitors are reported—
2-chrolo-4-mercapto-N-phenyl-5-(4-phenyl-4H-[l,2,4]triazol-3-ylsuflamoyl)-
benzene-amide derivatives (compound 20-23 and 42-53) and (6-chrolo-3-
merccapto-l,l-dioxo-[l,4,2]dithiazin- 3-yl)phenyl methanone derivatives (compound
15-17 and 31-37). Chloro and methyl substituted 2-chrolo-4-mercapto-
N-phenyl-5-(4-phenyl-4H-[ 1,2,4]triazol-3-ylsuflamoyl)- benzene-amide derivatives
(20, 21, 51) and (6-chrolo-3- merccapto-l,l-dioxo-[l,4,2]dithiazin-3-yl)phenyl
methanone derivatives are the most potent compounds in this study (ICso<20 pM).
However, how the compounds interact with integrase and inhibit the catalytic ability
of integrase is not clear yet. More detailed studies are needed to illustrate this point.
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3.5 REFERENCE
1. Moor, J. P., Stevenson, M., New target for inhibitors of HIV-1 replication. Nat
Rev M ol Cell Biol. 2000, 1, 40-49
2. Asante-Appiah,E; Skalka, A. M. HIV-integrase: structural organization,
conformational changes, and catalysis. Adv. Virus Res 1999, 52, 351-369
3. Nicklus, M., Neamati, N., Hong, H., Mazumder, A., Sunder, S., Chen. J., Milne,
G., Pommier, Y. HIV-1 integrase pharmacophore: discovery of inhibitors through
three-dimenstional database searching. J. Med. Chem. 1997, 40, 920-929.
4. Neamati, N., Mazumder, A., Sunder, S., Owen, J., Schultz, RJ. Pommier, Y.,
2-Mercaptobenzensulphonamides as novel inhibitors of human
immunodeficiency virus type 1 integrase and replication. Antiviral Chemistry &
Chemotherapy, 1997, 8, 485-495.
5. Brzozowski, Z., Saczewski, F., A new type of Mixed anhydride and its
applications to the synthesis of 7-substituted 8-chloro-5,5-dioxoimidazo[l,2-b]
[l,4,2]benzodithiazines with in virtro antitumor activity. J. Med. Chem. 2002, 45,
430-437
6. Artico, M., Silvestri, R., Massa, S., Loi, A. G., Corrias, S., Piras, G., Colla, P.,
2-sulfonyl-4-chloroanilino moiety: A potent pharmacophore for the anti-human
immunodeficiency virus type 1 of pyrrol aryl sulfones. J. Med. Chem. 1996, 39,
522-530
7. Turner, S. R., Strohbach, W. J., Tommasi, R. A., Aristoff, P. A., Johnson, P. D.,
Skulnick, H. I., Dolak, L. A., Seest, E. P., Tomich, P. K., Bohanon, M. J., Horng,
M., Lynn, J. C., Chong, K. T., Hinshaw, R. R., Watenpaugh, K. D., Janakiraman.
M. N., Taisrivongs, S., A potent, orally. Bioavailable nonpeptidic HIV protease
inhibitor of the 5, 6-dihydro-4-hydroxy-2-pyrone sulfonamide class. J. Med.
Chem. 1998, 41, 3467-3476.
65
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
8. Arraz, E., Diaz, J. A., Ingate, S. T., Witvrouw, M., Pannecouque, C., Balzarini, J.,
Clercq, E., Vega, S. Novel l,l,3-trioxo-2H, 4H-thienol[3,4-e][l,2,4]thadizaine
derivatives as non-nucleoside reverse transcripase inhibitors that inhibit human
immunodeficiency virus type 1 replication. J. Med. Chem. 1998,41, 4109-4117
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CHAPTER IV
INHIBITION OF HIV-1 INTEGRASE BY
3-C ARB AMO YL-BIC Y CLO
[2.2.1]HEPT-5-ENE-2-CARBOXYLIC ACID DERIVATIVES
4.1 INTRODUCTION
Integrase is one of the three essential enzymes of retrovirus, responsible for the
insertion of viral DNA into host genome. The insertion of viral DNA, namely
integration, is required for effective viral replication. The integration includes two
separate steps. First, integrase removes GT dinucleotides from each end of linear
viral DNA to generate a 3’-OH end. Following the cleavage, the 3’-OH end of viral
DNA joins to the 5’-end of the host DNA by integrase.1 The ability of integrase to
cut polynucleotides from viral DNA end and the coordination of divalent metal ion at
the catalytic site indicate that integrase belongs to a superfamily of polynucleotidyl
transferases. Mu transposase, nucleases RNase H and RuvC also are members of the
superfamily. 2 Enzymes in this superfamily share a certain degree of structural or
sequence similarity. However, there is no known cellular homologue of integrase.
Thus, specific integrase inhibitors would only block viral IN and may have less
toxicity toward the host.
67
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By using purified recombinant integrase and a 21-mer duplex oligonucleotides,
which mimic the sequence of U5 end of HIV long terminal repeat, one can
quantitatively measure the activity of HIV-1 integrase and the inhibitory ability of
the tested compounds. Many novel classes of IN inhibitors have been discovered,
however, most of the compounds, which show considerable potency against purified
IN, were not potent enough to get into clinical trial and many failed in cytotoxicity
testing in cell assay.3-14 The active integrase conformation is ambiguous because the
X-ray crystal structure of the full-length IN-DNA complexes is still unavailable.
Moreover, the orientation of glutamate (E) of DDE motif is not clear yet. This is
because the particular residue resided in a highly flexible loop of integrase. 1 5 -1 8
Although there are ongoing studies tracking the flexible loop and are trying to
orientate the position of residue E, the glutamate 152 orientation of wild type
integrase is still not available. 1 6 These reason mentioned above increases the
difficulty for identifying a high affinity ligand for HIV-1 integrase.
Benzesulfonamide derivatives have been demonstrated to have the ability to inhibit
carbonic anhydrase.19,20 As a consequence, benezesulfonamide derivatives may be
used in the treatment of high intracranial pressure and epilepsy. Some substituted
benzesulfonamide compounds, which were identified by three-point pharmacophore
searching in the NCI three-dimension database, have been shown to have potency
inhibiting HIV-1 integrase. 2 1 In the study, the compounds, which have various
substitiuent at 4-position and N of sulfonamide groups, were tested. In order to
further identify the feathures required for inhibitory activities, we have tested more
68
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derivatives, which have various subsitiuents at 4-position. Quantitative
structure-activity relationship (QSAR) was applied to analyze the relationship
between the biological activities and the physicochemical parameters. Moreover, we
also have applied two and three dimensional parameter frame-setting to interpret the
relationship of the biological activities and the physicochemical parameters.
4.2 METHODS
CHEMICALS. 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.
The final concentration of compounds was between lOOOpM and 4.1 pM.
PREPARATION OF OLIGONUCLEOTIDE SUBSTRATES. The HPLC-purified
21 botton, 5-ACTGCTAGAGATTTTCCACAC-3’
oligonucleotides 21top, 5-GTGTGGAAAATCTCTAGCAGT-3' w ere ordered from
USC Norris microchemical core facility (Los Angeles, CA). The expression system
for the wild-type HIV-1 integrase was a generous gift of Dr. R. Craigie, Laboratory
of Molecular Biology, NIDDK, NIH, Bethesda, MD. To analyze the extents of
3’-processing and strand transfer using 5’-end-labeled substrates, 21 top was
5’-end-labeled using T4 polynucleotide kinase (Epicentre, Medison, WI) and [y-32P]
ATP (Amersherm). The kinase was heat-inactivated, and 21botton was added to the
final solution. The mixture was heated at 95 °C, allowed to cool slowly to room
temperature, and added onto a G-25 Sephadex quick spin column (USA scientific) to
separate annealed double-stranded oligonucleotide from unincorporated label.
INTEGRASE ASSAY. Integrase 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
pM EDTA, 50 pM dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/mL
69
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bovine serum albumin, 10 mM 2-mercaptoethanol, 10% dimethyl sulfoxide (DMSO),
and 25 mM MOPS, pH 7.2) at 30 °C for 30 min. Then, 20 nM of the 5’-end 3 2
P-labeled linear oligonucleotide substrate was added, and incubation was continued
for an additional 1 h. Reactions was quenched by the addition of an equal volume
(16 pL) of loading dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene
cyanol, 0.025% bromophenol blue). An aliquot (5 pL) was placed on a denaturing
20% polyacrylamide gel (0.09 M Tris-borate, 2 mM EDTA, 20% acrylamide, 8 M
urea, pH 8.3). The separation was achieved using 2900 volts for 3.5 hrs. Gels were
dried, and exposed on a film.
PARAMETER FRAME-SETTING. The dipole moment (Dm or p) was calculated
by the use of Hyperchem program (Hypercube, Inc, Waterloo, Canada), after
geometry optimization and energy minimization were carried out by AMI
9 ^
semi-empirical method. The hydrophobic parameters including the calculated
n-octanol/water partition coefficient (clog P) and the calculated molar refractivity
(CMR) were calculated by using clog P software (Claremont, CA). 26 -2 7
Two-dimensional parameter frame-setting was plotted by using MS-Excel 2000.
Three-dimensional parameter frame-setting was constructed by using Origin
software (Microcal Inc., Northampton, MA). 28-29 All regression equations were
derived from CQSAR prograem (BioByte Corp. Claremont, CA) by the use of the
parameters stated above. The indicator variable, I, represents the presence of a D
form substituent (1=1) or L form substituent (1=0). The maximum number of
hydrogen bond (Hb) was calculated as the sum of hydrogen bond donors and
acceptors.
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4.3 RESULTS AND DISCUSSION
Initially thirty-five benzesulfonamide derivatives have been tested for their
inhibitory ability of HVI-1 integrase. The results are listed in Tables 11 and 12. None
of these compounds (1-35) tested in this study showed the ability to inhibit integrase,
except a bicyclo derivative— 3-(4-sulfamoyl- benzylcarbamoyl)- bicycol[2.2.1]hept-
5- ene- 2-carboxlic acid, 17(IC5o=266.5). Among the identified HIV-1 integrase
inhibitors, no bicyclo ring system was identified as the main structure or a
substituent. To further investigate the structure-activity relationship of the bicyclo
system, more compounds were synthesized and examined. To establish a model for
drug design, physicochemical parameters were applied for quantitative
structure-activity relationship (QSAR) analysis. Moreover, to investigate what
physicochemical properties are required for inhibitory ability, three-dimensional
parameter frame setting was also applied for visual presentation of the data.
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Table 11 Sulfonamide derivatives.
S 0 2NH2 —^ y— N
R1
NO
Ri r 2
IC5 0 (m M)
3 ’-p ro c ST
1 s
r OH
COOHO
>1000 >1000
2 s
H
--- --- Jj—NH2
COOHO
>1000 >1000
3 s
H
COOH
>1000 >1000
4 s
H
>1000 >1000
5 s
H
N " "SH
COOH
>1000 >1000
6 s
H H |H a +
/ N 'Y 'N ^ ----- ----- C O O -
C O O H
>1000 >1000
7 s
H W 0H
' coo^ O ^ oh >1000 >1000
8 s
H VHs+
JL
^ V - '- ^ - c o o -
COOH
>1000 >1000
72
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Table 11 continued
N O
R i r 2
n
o n
0
1
3 ’ -proc S T
9 S
H I
COOH
> 1 0 0 0 > 1 0 0 0
10 s
H
^ N ^ ^ C O O H > 1 0 0 0 > 1 0 0 0
11 s
H
"V
COOH
> 1 0 0 0 > 1 0 0 0
12 s
H
/ N \ ^ " - O H
COOH
> 1 0 0 0 > 1 0 0 0
13 s
H
/ N\ ^ " " O H
COOH
> 1 0 0 0 > 1 0 0 0
14 s
COOH
> 1 0 0 0 > 1 0 0 0
15 s
H
.--
\ NH
N ^ =/
> 1 0 0 0 > 1 0 0 0
16 s
COOH
> 1 0 0 0 > 1 0 0 0
73
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Table 11 continued
NO
Ri r 2
IC5 0 (m M)
3’-proc ST
17 O
/
HOOC
b
266.5 266.5
18 S
■5
COOH
>1000 >1000
19 s H N = \
" nY ' V ' n h
COOH
>1000 >1000
29 s
-rV ^>
COOHO
>1000 >1000
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Table 12. Sulfonamide derivatives
N O C om pounds
IC5 0 (pM)
3 ’-proc ST
20 /— . o
/ ^ \ I I H
c i A > r v %
M
s o 2 nh2
>1000 >1000
21
s o 2n h2
>1000 >1000
22
H X ^ N
so2 nh2
>1000 >1000
23
F ^v°
F F H N
J O .
so2 nh2
>1000 >1000
24
F F H N
J O .
so2 nh2
>1000 >1000
25
" Z o
so2 nh2
>1000 >1000
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Table 12 continued
NO Compounds
IC50 (liM)
3’-proc ST
26
F F HN
y y a
H2 N02 S ' \ ss= \
s o 2 nh2
>1000 >1000
27
F F HN F
J T V V "
H 2N02 S ' \ s S i\ F
s o 2 nh2
>1000 >1000
28
F F H N r f
Y y a
h2n o 2 s / \ = s\
s o 2nh2
>1000 >1000
30
F^ " V o
F F
Q
so2 nh2
>1000 >1000
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Table 12 continued
NO Compounds
IC50 (pM)
3’-proc ST
31
F F H N .
S02 N H 2
>1000 >1000
32
’ ^ V °
'" Q
so2 nh2
>1000 >1000
33
’" 'p
h2 no2 s
>1000 >1000
34
F F hn'
X s !
h2no2s \ ^
>1000 >1000
35
so2 nh2
>1000 >1000
77
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Compounds with alkyl or hydroxyl groups substituted on N-position of hydrazino or
amino groups were classified in one subset (Table 13). Replacement of the hydroxyl
group of compound 64 by mercapto group (57) increased the IC50 from 222 // M to
500 fiM. Compound 54, which has three hydroxyl groups (1,1 -bishydroxymethyl
ethan-2-ol, substituted) but no carboxyl group, has moderate inhibitory ability
(IC5o=333 pM). Comparing compounds 45 and 64, one may see that the presence of
the phenyl ring would decrease the activity. Moreover, the presence of an additional
methyl group at 2-C (compound 47) also decreased the activity. However, varying
the chain length of N-substituent has no effect on the activity (58 and 63).
Compound 37 shows 50 % inhibition at 14 pM, while compound 45 has IC50 value at
333pM. The only structural different between 37 and 45 is that 37 has a D-form
substituent and 45 has a L-form substituent. Compare the physicochemical
parameters of these two compounds, there is no obvious difference between these
two compounds (Table 17). Thus, it implies that the increased activity may be due to
steric effect of the substituents. Further comparing other subsets of compounds
(Table 13, 14, 15, and 16), the IC50 values of the compounds (37, 38, 39, 40, 41, and
42) with D-form substituents were 20 fold higher than their corresponding L-form
analogues (45, 47, 61, 59 and 66). Thus, we proposed that D-form substituent maybe
required for high activity, and the compounds may have a specific binding position
with integrase.
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Table 13. Bicyclo derivatives
NO R
ICso (m M)
3’-proc ST
37
H O O C (D-form)
14 10.15
45
H 00C (L-form)
333 333
64
H
HOOC OH
222 305.5
54
H OH
- N /
OH
OH
333 333
57
H
HOOC SH
500 500
38
H
HOOC (D -fo im )
28.5 28.5
47
H
HOOC 0H (L-form)
500 500
58
H
> - c
HOOC \
500 500
63
- N
) ----
HOOC
500 500
79
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Table 14 lists the compounds, with carboxyl group or sulfonamide as the substituent
group R-. Compounds 40 and 41 have potent inhibition (IC5o=12.3pM), while their
amino substituent analogues (61 and 59) have only moderate activity (ICso^SOOpM).
As what have been mentioned before, the presence of the D-form substitution is
essential for effective inhibition. Moreover, it is also confirmed that the chain length
(2 -4 C length) does not affect the activity (comparing 40 with 41 and 59 with 61).
Compounds 43, 49 and 50 are sulfonamide derivatives. The sulfonamide containing
compounds have been described to inhibiting HIV-1 integrase at low concentrations.
However, in this study, the sulfonamide-substituted compounds are not as potent as
the sulfonamide inhibitors described before. Comparing 43/17 and 49, it seems that
switching the sulfonamide group from p-position to o-position or deleting the phenyl
ring decreased the activity. This may be due to the polarity or steric effects. It also
implies that the benzenesulfonamide group in this series of compounds may have
less importance in the interacting with the HIV-1 integrase than the previously
described active compounds.
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Table 14. Bicyclo derivatives
OH
O
N O R
IC50 (liM)
3’-proc S T
40
H
_ ___ ^COOH
HOOC
12.3 12.3
61
H
— ^COOH
HOOC
466.5 466.5
41
H
— H y _ _ / — C 00H
HOOC
12.3 12.3
59
H
~~N y _ y — C 00H
HOOC
500 500
62
/ — COOH
— N
^ C O O H
500 500
43 266.5 266.5
49
-n V C
440 440
50
H
- N v , 0
S—NHa
O
566.5 566.5
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Compounds with aromatic substituents as R- are categorized in Table 15. Compound
39 and 52, which have hydrazino goups, still retain the activity. However, compound
42, which has a pyrrole ring (instead of a phenyl ring of 39), lost the activity by 3
fold. The indole substituent slightly increased the activity (comparing 46 and 66).
Compound 52 has 50% inhibition at 12.3 pM, although the 1-carboxyl group was
deleted. Compounds 39 and 37 in Table 13, with an additional hydroxyl group, have
similar inhibitory ability. However, compound 45 (with a p-OH group) in Table 13 is
more than twice as potent as 66. In Table 15, compound 51 shows moderate
inhibition (IC5o=566.5pM). Deletion of the carboxyl group at 1-C increased the
inhibition ability by 2 fold. However, substituting a carboxyl group at N-position of
the free amide (53) would decreased the activity (IC5o=800pM). Compound 65,
which was substituted with a pentylamino group, has 50% inhibition under 85.5pM.
Compound 65 is the most activity compound among the L-form substituent. This
may be due to the flexible long alkyl chain, therefore, the amino group be
accommodated by the appropriate site to inhibit the enzyme activity.
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Table 15. Bicyclo derivatives
NO R
IC50 (hM)
3’-proc ST
39
-VJD
H00C (D-form)
12.5 12.5
42
h o o c (D-form)
37 37
52
H
12.3 12.3
56
x o
225 300
66
H00C (L-form)
750 750
46
V b
H O O C
650 650
83
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Table 16. Bicyclo derivatives
OH
O
NO R
IC50 (pM)
3’-proc ST
51
o
HOOC NH2
566.5 566.5
67
^ n h 2
266.5 266.5
53
0
.N . /COOH
H
800 800
55
H
— N
S
135 200
60
H
— N
n ( ^ N
700 700
65
H
— N
HOOC '------v
nh2
85.5 85.5
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Two and three-dimensional parameter frame-setting were applied to analyze the data
to illustrate the relationship between the activity and the physicochemical properties.
Five different physicochemical parameters were selected as determinants for analysis.
The parameters, which were submitted for analysis, are dipole moment (p),
calculated octanol/ water partition coefficient (clog P), molar refraction (CMR),
molecular weight (MW), and total number of hydrogen bonds possible (Hb) (Table
17). Compounds were classed by their activity. All the two-dimensional plotting
results are shown in Fig. 9. In two-dimensional parameter frame-setting, the best
separation was obtained by using MW-Hb plot (Fig. 9-1). The plots, which applied
MW as one of the parameter for plotting (Fig. 9-C, G, and I), have better separation
than others. The molecular weights of active compounds are all under 400g/mol. In
addition, it was also observed that active compounds do not have high number of
possible hydrogen bonds ( +20)(Fig. 9-D, F, H and I). Three-dimensional parameter
frame setting shows that the best space separation can be obtained by using MW, Hb,
and p as the independent variables (Fig. 10-A), It is apparent that the active
compounds tend to locate at low MW, low Hb and low p region.
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Table 17. List of physicochemical parameters of all compounds
Compound clogP CMR a MW Hb
CS1 -1.03 8.26 3.994 347.37 23
CS2 -1.71 8.47 7.939 346.38 23
CS3 1.19 8.07 5.591 345.44 18
CS4 -0.37 8.41 7.526 317.39 18
CS5 -0.41 8.9 8.081 335.43 21
CS6 -4.37 10.42 - 419.48 26
CS7 -0.11 8.63 4.314 411.45 24
CS8 -5.65 9.46 - 362.38 23
CS9 1.72 7.14 7.997 359.47 18
CS10 -0.79 7.6 5.668 289.93 18
CS11 -0.26 7.76 4.345 303.36 18
CS12 -1.45 8.22 5.332 319.36 21
CS13 -1.14 10.11 5.137 333.39 21
CS14 1.15 8.59 7.35 379.46 18
CS15 -1.52 8.35 8.646 325.41 16
CS16 0.08 8.7 1.833 329.4 17
CS17 -0.24 8.7 4.311 350.39 16
CS18 0.12 10.27 5.991 395.46 21
CS19 -2.12 8.78 11.836 369.42 21
CS20 0.59 7.56 7.074 354.82 17
CS21 0.06 6.77 5.829 374.27 15
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Table 17. Continued
Compound clogP CMR
I L
MW Hb
CS22 0.56 7.14 6.334 389.28 17
CS23 1.65 8.25 4.127 415.72 13
CS24 1.8 8.53 2.714 460.18 13
CS25 1.96 9.06 4.132 507.18 13
CS26 0.24 9.49 6.93 494.8 20
CS27 0.38 9.51 4.61 528.36 20
CS28 0.28 9.98 4.525 529.25 20
CS29 -5.25 11.41 6 A l l 455.51 28
CS30 1.53 8.68 4.803 409.33 13
CS31 1.21 8.22 1.864 395.31 13
CS32 1.43 7.76 5.834 381.28 13
CS33 1.43 7.76 3.997 381.28 13
CS34 1.43 7.76 3.081 381.28 13
CS35 1.19 7.77 5.219 399.27 13
CS36 2.38 9.88 4.025 368.38 18
CS37 1.14 8.79 4.88 345.35 17
CS38 -0.37 6.68 4.322 283.28 17
CS39 1.81 8.57 5.201 329.35 14
CS40 0.23 6.74 1.621 297.26 19
CS41 -0.01 7.2 5.098 311.29 19
CS42 -0.66 7.88 3.616 319.31 19
CS43 -0.24 8.7 2.467 350.39 16
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Table 17. Continued
Compound clogP CMR
1 1
MW Hb
CS45 1.14 8.79 4.88 345.35 17
CS46 1.34 9.6 3.107 368.38 16
CS47 -0.37 6.68 4.322 283.28 17
CS48 -0.05 9.16 6.495 364.42 16
CS49 0.1 8.23 1.563 336.36 16
CS50 -0.79 5.72 4.226 260.27 16
CS51 -0.94 6.93 6.638 296.28 19
CS52 1.01 7.83 3.405 286.33 11
CS53 -1.69 6.47 2.626 282.25 18
CS54 0.33 6.6 4.252 285.29 18
CS55 0.97 7.19 4.6 297.36 16
CS56 1.38 8.11 2.615 315.32 14
CS57 0.36 6.67 4.407 285.32 17
CS58 1.85 7.45 2.891 295.33 14
CS59 -0.01 7.2 5.098 311.29 19
CS60 -0.05 7.16 3.874 275.3 11
CS61 0.23 6.74 1.621 297.26 19
CS62 0.83 6.71 3.577 297.26 18
CS63 0.87 6.09 3.478 253.25 14
CS64 0.68 6.21 2.9764 269.25 17
CS65 -2.18 7.82 4.975 310.35 17
CS66 1.81 8.57 5.201 329.35 14
CS67 -0.66 5.84 2.798 238.24 14
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A
♦ a c tiv e
▲ in a c tiv e
B
♦ a c tiv e
▲ in a c tiv e
Fig. 9. Two-dimensional parameter frame-setting of different combinations of
physicochemical constants. ♦ represent active compounds (IC5o<1000
pM). ▲ represent inactive compounds (IC50>1000 pM). (See text for the
parameters used).
89
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c
600
500
400
♦ a
300
200
100
■ 3 •2 • 1 0 1 2 3
clogP
D
♦ a c t iv e
A in a c t iv e
c lo g P
Fig. 9. Continued
90
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E
1 0
9
Q
A A
♦ A
A A A
* ♦
A *
♦ AA
4 A
A A
♦
♦ active
A inactive
CMR
F
30
20
■o
f f i
10
A
A A
A A A AA
A A
A
♦ ♦ AA AA ♦
AAA A AA A A A A
♦ ♦ A A ♦ AA
♦ AA AA ♦ ♦ ♦
A
♦ ♦ ♦ A A
............................X ---- —
A A A A A
A A
...........................i ............................ i............................ i............
♦ active
A inactive
10 11
CMR
12
Fig. 9. Continued
91
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G
H
600
500
400
300
200
100
♦ A
♦
A A A a a
4 i A 4
. A ♦ ♦ A . A
♦
♦ A
♦ ♦
♦ active
A inactive
2 3 4 5
DM
♦ active
A inactive
Fig. 9. Continued
92
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I
30
25
20
♦ ♦ ♦ ♦♦
15
10
5
0
50 150 200 250 300 350 400 450 500 550 600 650 0 100
M W
Fig. 9. Continued
♦ active
A in active
93
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25
20
Hb
V c
V . * *
* *
★ ★
250
300
350
400
450
500
550
A
Fig. 10. Three-dimensional parameter frame setting. ^ represents active
compounds and O represents inactive compounds. A. The active
compounds locate at the regions of low MW, low Hb and low p.
B. The compounds with D-form substituent (1=1) are more potent than those
with L-form substituent (1=0)
94
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0.8
0.6
0.4
0.2
,o> o °
0.0
B
Fig. 10. Continued
95
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QS AR analysis was applied to further investigate the relationship between IC50 value
and the physicochemical parameters. IC50 value was correlated with clog P, MW,
CMR, p, and Hb (Table 18). Eq. 4 is the best equation that has only low correlation
coefficient (r=0.476, r2=0.227). Thus, I as an indicator variable was added into the
equation. If the compound contains a D-form substituent, I is set as equals to 1. On
the other hand, I equal to 0, when the compound contains a L-form substotuent. / is
highly correlated with the IC50. Highly correlated equations were obtained with the
presence of I. The best correlation equation is Eq 8. Eq 11 was generated by deleting
two outliners— compound 55 and 65. Those two compounds have unique structures.
Compound 55 is the only one carrying thiadiazole -2-thiol, and 65 is the only one
with a pentylamino group. Although high correlation was obtained, the range of 95%
confident intervals of the coefficients were too wide and adding an extra parameter
did not improve the r too much. This implies that the IC50 and the parameters
examined are not significantly correlated. F-test was applied to exam whether the
IC50 value and physicochemical parameters have the significant correlation (Table
19). The results reveal that those physicochemical parameters do not have significant
correlation with IC50. The less than satisfactory results may be due to the limit of
compounds tested, all the compounds with low IC50 value are the D-form compounds
and those IC50 value of L-form compounds group were all around 300-500 pM.
Thus, even though the IC50 values range cover two log-units, the compounds in the
QSAR study can be roughly grouped into two groups— one group are the D-form
compounds with the IC50 values around 12.3pM, and the other are L-form
96
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compounds with the IC50 values around 400pM. In other words, a linear relationship
is dictated by two clusters of data points with no point in between and beyond (Fig.
10-B.).
4.4 CONCLUSIONS
Several benzenesulfonamide derivatives were identified to have inhibition
ability through the pharmacophore three-dimensional database searching. However,
the benzenesulfonamide derivatives tested in this study did not show any HIV-1
integrase inhibition ability. Even though the testing results of benzenesoulfonamide
derivatives are not as good as expected, the other class of bicyclol inhibitors was
identified. The bicyclol structure is not common among the biological activity
compounds. To date, the bicycol[2.2.1]heptene derivatives were tested for their anti
hepatitis C virus (HCV) protease22, anticonvulsant1 9 , and antitumor activities23. The
ability of bicyclo [2.2.1]heptene derivatives to inhibit HIV-1 replication are not
established. Although a good QSAR model cannot be established in this study, we
have successfully interpreted the essential physicochemical requirement (low MW
(<350g/mol), low p (<5) and low Hb (<20), D-form substituent group) of active
compounds.
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73
CD
■ o
- 5
o
Q .
C
o
CD
Q .
$
~o
CD
3
c / )
c / )
o'
o
o
l-H
CD
O
O
Table 8. QSAR equation of HIV-1 integrase inhibitors
Equations n r r2 S
" O
- 5
c q '
1 LOG 1/IC50 = 0.101(0.102)Hb - 4.036(1.717) 28 0.370 0.137 0.535
S ’
l-H
o
2 LOG 1/IC50 = 0.132(0.18 l)clog P + 0.112(0.101)Hb - 4.276(1.712) 28 0.456 0.208 0.523
3
CD
3 LOG 1/IC50 = 0.096(0.212)clog P + 0.072(0.216)CMR + 0.108(0.103)Hb 28 0.473 0.224 0.529
—s
T 1
-4.740(2.218)
C
4. LOG 1/IC50 = 0.097(0.217) clog P + 0.075(0.221)CMR + 0.022(0.151)U 28 0.476 0.227 0.539
CD
CD
+0.105(0.107)Hb - 4.802(2.303)
■ o
o
n
5 LOG 1/IC50 = 1.319(0.243)1 - 2.590(0.103) 28 0.910 0.827 0.239
c
a
6 LOG I/IC50 = 0.036(0.086)CMR + 1.298(0.249)1 - 2.858(0.643) 28 0.912 0.838 0.239
o
o
7 LOG 1/IC50= -0.022(0.098)clog P + 0.047(0.100)CMR + 1.308(0.257)1 28 0.913 0.834 0.249
— i
o
-2.930(0.727)
g;
l-H
CD
8 LOG I/IC50 = -0.022(0.100)clog P + 0.048(0.102)CMR + 0.005(0.069)U 28 0.913 0.834 0.249
Q _
+1.305(0.265)1 - 2.956(0.812)
3
O
c
9 LOG I/IC50 = 0.000(0.089)clog P + 0.003(0.069)U+1.318(0.263)1 28 0.910 0.828 0.249
l-H
■ O
CD
- 2.600(0.285)
3
in
10 LOG I/IC50 = 0.050(0.088)clog P + 0.000(0.081)CMR - 0.017(0.052)U 26 0.958 0.917 0.182
in
o'
o
+1.350(0.196)1 - 2.600(0.636)
11 LOG I/IC50 = 0.000(0.089)clog P + 0.003(0.069)U+1.318(0.263)I 26 0.958 0.917 0.178
- 2.600(0.285)
V ©
00
Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission.
Table 9. F test for physicochemical parameters
Parameter F vi, v2 V i,V 2 P
clog P -2.34 1,27 >0.1
Hb 4.12 1.26 >0.025
CMR 0.483 1.25 >0.1
F
0.016 1.24 >0.1
I 142.68 1.27 <0.001
Table 10. Squared correlation matrix of physicochemical parameters (R2 and N=28)
CLOGP CMR U MW HB I
CLOGP 1 .246 .015 .172 .022 .054
CMR 1 .015 .921 .0 0 0 .038
U 1 .018 .034 .008
MW 1 .043 .022
HB 1 .155
I 1
SO
SO
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Asset Metadata

Creator Kuo, Aline Chih-Ling (author)

Core Title Discovery of novel HIV-1 integrase inhibitors

School Graduate School

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Chemistry, pharmaceutical,Health Sciences, Pharmacy,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Neamati, Nouri (committee chair), Bolger, Michael B. (committee member), Haworth, Ian S. (committee member), Lien, Eric J. (committee member), Louie, Stan (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-302883

Unique identifier UC11341215

Identifier 1414904.pdf (filename),usctheses-c16-302883 (legacy record id)

Legacy Identifier 1414904.pdf

Dmrecord 302883

Document Type Thesis

Rights Kuo, Aline Chih-Ling

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA