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Bioinorg Chem Appl. 2006; 2006: 71938. Published online 2006 March 2. doi: 10.1155/BCA/2006/71938. | PMCID: PMC1779548 |
Antihuman Immunodeficiency Virus Type 1 (HIV-1) Activity of Rare Earth Metal Complexes of 4-Hydroxycoumarins in Cell Culture Ilia Manolov,1 Sevda Raleva,2 Petya Genova,2 Alexey Savov,3 Liliana Froloshka,2 Daniela Dundarova,2 and Radka Argirova2* 1Department of Organic Chemistry, Faculty of Pharmacy, Medical University, 2 Dunav Street, 1000 Sofia, Bulgaria 2Department of Virology, National Center of Infectious and Parasitic Diseases, 44A Stoletov Street, 1233 Sofia, Bulgaria 3Laboratory of Molecular Pathology, Medical University, 2 Zdrave Street, 1606 Sofia, Bulgaria Received December 23, 2004; Revised March 9, 2005; Accepted June 17, 2005. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited. The cerium Ce(III), lanthanum La(III), and neodymium
Nd(III) complexes with
4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one (warfarin)
(W) and 3,3′-benzylidenebis[4-hydroxycoumarin]
(1) were synthesized and studied for the first time
for cytotoxicity (on MT-2 cells) and as anti-HIV agents under
acute and chronic infection. The
complexes were characterized by different physicochemical methods:
mass spectrometry, 1H NMR, 13C NMR, and IR
spectroscopy. The spectra of the complexes were interpreted on the
basis of comparison with the spectrum of the free ligands.
Anti-HIV effect of the complexes/ligands was measured in MT-2
cells by microtiter infection assay. Detection of endogenous
reverse transcriptase (RT) activity and RT processivity by PCR
indicative for proviral DNA synthesis demonstrated that anti-HIV
activity has not been linked to early stages of viral replication.
No effect on late steps of viral replication has been found using
cells chronically producing HIV-1LAI virus.
La(W) demonstrated anti-HIV
activity (IC50=21.4 μM) close to maximal
nontoxic concentration. Nd(W),
Ce(1), and Nd(1) demonstrated
limited anti-HIV potency, so none of the complexes seems
appropriate to be used in clinic. Further targeting of HIV-1
inhibition by La(W) is under progress. The need for new HIV inhibitors
especially directed to so far unexploited primary targets (integrase, RNase H, viral assembly, etc) is
considered to be promising for the development of anti-HIV
drugs. Coumarins and bicoumarins are
widely spread in nature [ 1, 2]. Their biological properties
are also well known and include anticoagulant, antiproliferative, antimicrobial,
spasmolytic, antitumor, antioxidant, and anti-HIV effect reported
recently [ 3]. A large number of structurally novel coumarin derivatives have
been reported to demonstrate anti-HIV activity in vitro and in
vivo [ 4, 5]. Plant-derived and semisynthetic calanolide compound exhibited in vitro activity against HIV-1 and human
cytomegalovirus [ 6] and has been proven to be a
naturally occurring nonnucleoside reverse transcriptase (RT)
inhibitor [ 1]. Tipravirin, a novel HIV-1 protease inhibitor,
has been developed from a nonpeptidic coumarin template [ 7].
Mazumder et al [ 8], investigating the effect of several HIV-1
protease inhibitors containing 4-hydroxycoumarin (4-hc) residues,
have found antiviral, antiprotease and anti-integrase activities.
It has been reported that coumarin and a number of its 4-hc and
7-hc derivatives, including warfarin ( W), display
cytotoxic effect against tumour cell growth and metastases
dissemination [ 9, 10]. Unfortunately, little is known about the complexing ability of
lanthanides—cerium Ce(III), lanthanum La(III),
and neodymium Nd(III)—with coumarins. Earlier it has
been shown that interesting metalorganic compounds of mendiaxon,
warfarin, coumachlor, and niffcoumar with lanthanides displayed
antitumor activity against P3HR1, K-562, and THP-1 cell lines
[ 11– 15]. The complexes had stronger cell
proliferation-inhibiting effects compared to the inorganic salts.
One could expect that lanthanide complexes with selected active
coumarin ligands should retain or even improve their biological
activity similarly to other lanthanide complexes with hc
derivatives. Here we present the coordination ability of
4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one (warfarin)
(W) and 3,3′-benzylidenebis[4-hydroxycoumarin]
(1) in complexation reaction with Ce, La,
and Nd. An increased anti-HIV activity of
La(W) (IC50=21.4 μM) as compared
to (W) (7.1% inhibition at maximal nontoxic
concentration—MNC) has been demonstrated. Further study has
shown limited anti-HIV potency of Nd(W),
Ce(1), and Nd(1), so none of the
complexes seems appropriate to enter clinical trials. Studies
indicated clearly that HIV-1 RT and protease for all complexes
were not the targets of antiviral activity. Source substances for synthesis Merck products p.a.
Ce(NO3)3 · 6H2O, La(NO3)3 · 6H2O, Nd(NO3)3 · 6H2O.
4-Hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one
(W) and 3,3′-benzylidenebis[4-hydroxycoumarin]
(C25H16O6)
(1) were synthesized by us and
were used to prepare the solutions. Ln(III) complexes of (W) were synthesized
using warfarin sodium salt. The synthesis and analysis of the
complexes of ( W) were described previously [ 11]. The complexes of lanthanides(III) with (1) were
synthesized by reaction of lanthanide(III) salts and the ligand
in amounts equal to metal: ligand molar ratio of 1 : 2. The complexes were prepared by adding an aqueous solution of
lanthanide(III) salts to an aqueous solution of the ligand
subsequently raising the pH of the mixture gradually to ca 5.0 by adding dilute solution of sodium hydroxide. The reaction
mixture was stirred with an electromagnetic stirrer at 25°C for an hour. The precipitate formed at the moment of
solution mixing was filtered, washed several times with water, and
dried in a dessicator to constant weight. The complexes were
insoluble in water, slightly soluble in methanol and ethanol, and
well soluble in DMSO. Analytical and spectroscopic measurements The elemental analyses for C, H, Ln, and H2O were performed according to standard microanalytical
procedures. The IR spectra (Nujol and KBr) were recorded on
IR-spectrometer FTIR-8101M Shimadzu (3800–400 cm−1)
and on IR-spectrometer Perkin-Elmer GX Auto image system
(700–200 cm−1). The 1H NMR spectra were recorded at room temperature on Brucker WP 250 (250 MHz) spectrometer in DMSO-d6. The 13C NMR spectra were recorded at ambient temperature on Brucker 250 WM (62.9 MHz)
spectrometer in DMSO-d6. Chemical shifts are given in ppm
downfield from TMS. The mass spectra were recorded on a Jeol JMS D
300 double-focusing mass spectrometer coupled to a JMA 2000 data
system. The compounds were introduced by direct inlet probe,
heated from 50°C to 400°C at a rate of
100°C/min. The ionization current was 300 mA, the
accelerating voltage 3 kV, and the chamber temperature
150°C. Cells, viruses, and assays Cell lines The following cell lines were used: H9/HTLV III B—chronically infected
with HIV-1LAI
cells, kindly provided by Professor
R. Gallo, and MT-2 uninfected human lymphoblastoid cells, HTLV-I
transformed. The cells were maintained in RPMI 1640 plus 10%
FCS. HIV-1 source As a source of HIV-1, the supernatant of H9/HTLV III B cell line was
used. The supernatants were collected and centrifuged to remove the cells, and virus stocks were prepared with known p24 antigen
content, RT activity and infectivity. MT-2 cells were infected
with HIV-1 (multiplicity of infection ≥ 1) in suspension
(37°C/5% CO2) and then grown parallely with
uninfected ones for 72 hours. Cytotoxicity and anti-HIV assays The following parameters were studied: cytotoxic
concentration 50—CC50 (concentration preventing death of 50% of MT-2 cells), maximal nontoxic concentration—MNC (the highest concentration causing no
cytotoxicity), and inhibitory concentration 50–IC50 (concentration
inhibiting by 50% the viral replication). CC50 and MNC were
detected by neutral red uptake assay [ 16]. IC50 was studied
on MT-2 cells by microtiter infection assay using neutral red
uptake [ 16]. Briefly, the experimental procedure under
conditions of acute infection was performed in 96-well microplates
in the following sequence: - 100 μL MT-2 cells (4–5 × 10 4 cells);
- 50 μL HIV-1 (undiluted or in the appropriate
dilution to obtain multiplicity of infection ≥ 1); contact (2
hours at 37°C/5% CO2); when CC50 and MNC were
detected, growth medium instead of virus has been added;
- 50 μL sample of substance tested starting from
4× MNC (because this volume is 1/4 of the whole volume in
the well) and 2- or 10-fold dilutions in growth medium.
The cells were incubated for 72 hours and then 100 μL of
them were removed to another 96-well plate previously treated for
an hour with 50 μg/mL poly-L-lysine (PLL) in volume
100 μL. A solution of 100 μL per well of neutral
red in growth medium (0.016%) was added and the
cells were incubated for 2 hours at 37°C/5%
CO2. Neutral red dye was extracted by acidified alcohol
(50% ethanol in 1% acetic acid) and measured
colorimetrically at 540 nm. Each plate contained 8 cell
control wells (no virus, no substance) for all kinds of
experiments. When IC50 was detected, a row of 8 viral controls
(plus virus, no substance) and rows of 8 experimental wells (plus
virus, different dilutions of the substance) were performed. The
values of optical density at 540 nm (OD540) were averaged for
each row and the mean values of experimental and control rows were
compared. As far as the ligands and their complexes were DMSO
soluble, the appropriate controls of DMSO and DMSO + HIV-1 in
growth medium were always considered. The effect of the compounds was also studied under conditions of
chronic infection using productively HIV-infected cells (H9/HTLV
III B). The production of infectious virions was measured again
using the cytopathic effect of supernatants on MT-2 cells as
described above. Detection of RT activity Endogenous RT activity of supernatants of HIV-1 infected
MT-2 cells treated/untreated with complexes and ligands was tested
by HS-Lenti Kit-RT assay (Cavidi, Sweden). The kit contained
recombinant RT (rRT) as a standard which made possible
quantitation of the RT in the sample. The method is
nonradioactive and colorimetrically detects RT activity product
(DNA) at 405 nm (OD405) [ 3]. RT processivity by polymerase chain reactions The idea was to demonstrate how far the proviral DNA synthesis has
progressed and whether mRNA splicing has occurred within
HIV-infected cells. (PCRs) were performed using primers M661 and
M667 detecting 200 bp product considered expressive for
double-stranded (ds) proviral DNA [ 17]. DNA for PCR was
extracted from MT-2 (negative control), HIV-1 infected MT-2
untreated cells (positive control)/cells treated with IC50 or MNC
(when IC50 has not been reached) and on 4 and 24 hours after HIV-1
infection. The cells were collected in lysis buffer
(pH = 8.3, TRIS-HCl—10 mM, KCl—50 mM,
MgCl 2—2.5 mM, NP40—0.5%,
and between 0.5% and 20% [ 17].
Nucleol-izing buffer
(500 μL) containing 30 μL proteinase K
and 25 μL 10% SDS was added to the cell lysate. DNA was
extracted by
phenol-chloroform-isoamyl alcohol method. The detection of
amplified product was done after 35 cycles in PCR machine and then
visualized in 1.5% agarose gel. Characterization of Ln(III) complexes of (W) The compositions of the complexes of ( W) were confirmed
by elemental analysis, DTA and TGA, and mass-spectral analysis. The
binding mode of ( W) was further elucidated by IR and
1H NMR spectra of the complexes as compared with this of
the free ligand. Some of these data were presented by us
previously [ 11]. Characterization of Ln(III) complexes of (1) The elemental analysis data of the Ln(III) complexes obtained are in agreement with the formula
Ln(L)(OH) · nH 2O, where
L = C 25H 14O 62−. The results of the
elemental analyses were within ±0.4% of the theoretical
values. The suggested formula was further confirmed by
mass-spectral fragmentation analysis. As seen in
Table 1, the first peak in the Ln(III)
complexes spectra (although with low intensity) corresponds to the
mass weight of the complex formation and the second one to that
of the ligand. The results thus obtained are in agreement with
metal:ligand ratio, 1 : 1. | Table 1Data of the mass spectra of the complexes of
(1). |
The binding mode of (1) to Ln(III) was further elucidated by analysis of the IR spectra of (1) and the complex formation. A broadband characteristic
for ν(O−H) of coordinated water was observed in the
spectra of the complexes in the range of 3500–3450 cm−1.
The most notable change in the ligand spectral features when
coordinated to Ln(III) is the observed C=O red
shift. The ν(C=O) band at 1660 cm−1 in the
ligand spectrum exhibited a red shift of 40 cm−1 in the
spectra of the complexes. This finding may be taken as evidence
for participation of the C=O group in coordination to
the metal ion. Further, a comparison between the ligand and
complex IR spectra revealed that the absorption bands associated
with the stretching ν(O−H) of the phenolic groups
(observed at 3074 cm−1 and 3032 cm−1 in the free ligand) disappeared in the Ln(III) complex spectra, indicating a loss of phenolic protons on complexation and thus
forming a metal-oxygen bonds. The δ(COH) IP modes, which appeared at
1345 cm−1 and 1336 cm−1 in the spectrum of the ligand, were not observed in the spectra of the complexes with
Ln(III) and thus supported the suggestion that the ligand
coordinates to the metal through its deprotonated form,
L2−. The Ln(III) complex spectra showed new
bands, in comparison with that of the free ligand, at
570 cm−1 and 410 cm−1, and they were assigned to
metal-oxygen stretching vibrations, in agreement with literature. The Ln(III) complexes and ( 1) were further studied by their 1H and 13C NMR spectra. The changes of
chemical shifts of the 1H NMR spectra were observed in the
complexes ( Table 2) and they were attributed to
coordination of the ligand to Ln(III). The chemical shifts
of the protons vary in the lanthanide complexes, because of the
shift properties of these metals. | Table 21H NMR spectra of the complexes of
(1). |
Due to electron transfer from the hydroxyl and carbonyl oxygen
atoms to Ln(III), chemical shifts to lower ppm were
observed for the neighboring C-4 and C-2 carbon atoms of the complexes and they confirmed the expected coordination of the
ligand through both deprotonated hydroxyl and carbonyl oxygen
atoms ( Table 3). The other carbon atoms were only
slightly affected from the coordination of the metal. On the basis
of the results thus obtained, it was suggested that the ligand
acts as a tetradentate one in the Ln(III) complex
formation. | Table 313C NMR spectra of the complexes of
(1). |
Cytotoxicity and anti-HIV assays In Table 4, the cytotoxicity (expressed as CC50) and
MNCs of both ligands and their lanthanide complexes are shown. As
seen, the Nd complex of (1) demonstrated at least
5 times lower cytotoxicity compared to the other lanthanides and
the ligand (1) itself. MNCs of the ligands differed in
between the complexes but were maximally nontoxic at 10–50 times
higher concentrations for Nd (1) and
Ce (1) compared to La(1) and the ligand
(1). The ligand ( W) and its complexes
demonstrated both equal cytotoxicity and equal MNCs. As far as
intensive research is often being done to reduce the toxicity of a
compound, we demonstrate here that the complexation with rare
earth metals possibly leads to increase of MNCs of some
derivatives. | Table 4Cytotoxicity as CC50 and MNCs of (1) and
(W) and their Ce, La, and Nd
complexes studied by neutral red uptake assay [16]. |
Dose-effect dependence of ligands (1) and ( W)
and their lanthanide complexes in HIV-1 infected MT-2 cells
according to microtiter infection assay [ 16] are listed in
Table 5. | Table 5Dose-effect dependence of ligands (1) and
(W) and their lanthanide complexes in HIV-1LAI
infected MT-2 cells (72 h incubation) according to microtiter
infection assay [16]. The concentrations tested start with MNC for
each ligand/compound (see Table 4 (more ...) |
The results display the effect on HIV replication of all
ligands/compounds in microtiter infection assay, starting from
MNC. As seen, the inhibitory effects in MT-2/HIV-1 system at or
under the MNC values are very low for all but one of the compounds
under study—La(W)—58.4%. It is the only derivative where IC50 could be reached—21.4 μM. All
other compounds exhibit low antiviral potency according to this
assay. Looking for the target of antiviral activity especially of
La( W) as well as of ( W) and
Nd( W), Ce( 1), and Nd( 1), we detected the endogenous RT activity in the supernatants of HIV-1/MT-2 infected cells and compared it to the results from microtiter infection assay at similar
conditions—treatment for 72 h with MNC. The results are
demonstrated in Table 6. Data indicate a discrepancy in anti-HIV effect measured by microtiter infection assay and
endogenous RT assay. This lack of correspondence is especially
well expressed for the only active compound—La( W). The results show that the demonstrated anti-HIV activity cannot be explained by inhibition
of RT activity—that is, RT is not the target of anti-HIV action.
Therefore, another target to explain the effect of
La( W) on HIV replication should be looked for. | Table 6Comparison of inhibition (%) of HIV-1 replication in
MT-2 cells by microtiter infection assay and endogenous RT assay
by some lanthanide complexes of (1) and (W) at
MNC (72 h incubation). |
Additional evidence that RT processivity has not been influenced
by (1) and (W) and their complexes having shown
some anti-HIV effect were provided by PCR.
shows that HIV double-stranded (ds) proviral DNA synthesis was not
impaired by the complexes. One can see that 4 h after
treatment of HIV-infected MT-2 cells ds proviral DNA was already
observed in both treated (lanes 4, 5, 6, 7, 8, 9) and untreated
HIV-infected cells (lane 2), while MT-2 uninfected cells displayed
no ds DNA (lane 10). The same results but with more intense signal
were registered 24 h after treatment of HIV-infected MT-2
cells with (1) and (W) compounds (lanes 14, 15,
16, 17, 18, 19). Therefore, the results confirm that neither the
ligands nor their rare metal ions complexes have an influence on
the reverse transcription. So, once again, the discrepancy in
anti-HIV activity measured by microtiter infection assay and
endogenous RT detection is clearly demonstrated. The conclusion is
that the active La(W) and the other less potent
anti-HIV agents have no impact on the early steps of HIV
replication. To address further the target of HIV inhibition especially by
La( W), a study of this complex for effect on late
stages of HIV-1 life cycle (protease, budding, assembly) was
carried out. To do this, we treated H9/HTLV III B
cells—chronically producing HIV-1—with La( W)
at 3 concentrations, starting from MNC. Also,
10× dilution of Indinavir was used as a reference for
antiprotease effect. Table 7 represents the results
for the effect of La( W) in 3 concentrations on
late stages of HIV replication. | Table 7Data obtained after treatment of H9/HTLV III B cells with
different concentrations of La(W) to target late
stages of HIV-1 replication (protease, budding,
assembly). |
As seen, H9/HTLV III B cells produced almost equivalent RT
activity when treated with La( W) in 3
concentrations. Additionally, the number of infectious virions as
measured by endpoint dilution analysis on MT-2 cells was
equivalent (only OD540 values for undiluted samples are
shown— Table 7, column (c)). The data in
Table 7 clearly demonstrate no effect of
La( W) on late stages of HIV-1 replication in cell
culture. We especially accentuate that the late stages in this
study include at least three steps—protease activity, budding,
and assembly of HIV-1. The fact that no effect on RT and protease (expressing early and
late stages of HIV-1 replication) was registered; one can suggest
that RT and protease are not targets for the antiviral activity of
La( W). HIV-1 replication is too complicated and a
number of targets and steps (except those studied here) should be
taken into consideration (attachment, coreceptor binding, fusion,
RNase H-activity, integration, inhibition of glycosylation or
sialylation, etc). Although not especially being a subject of
attachment studies, the results of RT assays ( Table 6)
and RT processivity () clearly indicate no
effect of the complexes on the earliest steps of replication.
Research is now in progress to look for putative inhibitory effect
of La( W) first on integration and then further to
specify the mechanism of its action. Here we show for the first time both complexation of rare earth
metal ions with selected coumarins and study of their effect on
HIV replication in cell culture. Although the complexes are of low
potency against HIV-1 and should not enter clinical trials, the
experience with them shows once more that the cytotoxicity could
be reduced and the antiviral effect—highly expressed
through
complexation reactions. Lack of protease and RT
activity has been recently reported for a number of HIV-1
integrase inhibitors [ 18] and on the contrary, antiviral
agents inhibiting both integrase and protease have also been
published [ 8]. The unique and variable activities of
plant-derived and semisynthetic coumarins, as well as novel
approaches for synthesis of potent and less toxic derivatives
could facilitate generation of new lead compounds for
chemotherapeutic intervention. The lanthanide complexes with
selected coumarins could offer such an opportunity. Financial supports from Medical Science Council at the
Medical University, Sofia, through Project no 21/2003 and from National
Fund of Science to the Ministry of Science and Education through
Project no 1411/ 2004 are acknowledged. 1. Zhou P, Takaishi Y, Duan H, et al. Coumarins and bicoumarin from Ferula sumbul: anti-HIV activity and inhibition of cytokine release. Phytochemistry. 2000;53(6):689–697. [PubMed] 2. Kostova I, Raleva S, Genova P, Argirova R. Recent advances in the discovery and development of plant-derived natural
coumarins and their analogues as anti human immunodeficiency virus–type 1 (HIV-1) agents. Biotechnology and Biotechnological Equipment. 2005;19(1):16–22. 3. Shao X, Ekstrand DHL, Bhikhabhai R, Kallander CFR, Gronowitz JS. A non-radioactive microtiter plate reverse transcriptase (RT) assay, based on immobilized template, for screening of RT activity inhibitors and evaluation of their mofe of action. Antiviral Chemistry & Chemotherapy. 1997;8(2):149–159. 4. Thaisrivongs S, Watenpaugh KD, Howe WJ, et al. Structure-based design of novel HIV protease inhibitors: carboxamide-containing
4-hydroxycoumarins and 4-hydroxy-2-pyrones as potent nonpeptidic inhibitors. Journal of Medicinal Chemistry. 1995;38(18):3624–3637. [PubMed] 5. Spino C, Dodier M, Sotheeswaran S. Anti-HIV coumarins from Calophyllum seed oil. Bioorganic & Medicinal Chemistry Letters. 1998;8(24):3475–3478. [PubMed] 6. Xu ZQ, Kern ER, Westbrook L, et al. Plant-derived and semi-synthetic calanolide compounds with in vitro activity against both human immunodeficiency virus type 1 and human cytomegalovirus. Antiviral Chemistry & Chemotherapy. 2000;11(1):23–29. [PubMed] 7. Kashman Y, Gustafson KR, Fuller RW, et al. The calanolides, a novel HIV-inhibitory class of coumarin derivatives from the
tropical rainforest tree, Calophyllum lanigerum. Journal of Medicinal Chemistry. 1992;35(15):2735–2743. [PubMed] 8. Mazumder A, Wang S, Neamati N, et al. Antiretroviral agents as inhibitors of both human immunodeficiency virus type 1 integrase and protease. Journal of Medicinal Chemistry. 1996;39(13):2472–2481. [PubMed] 9. Rosskopf F, Kraus J, Franz G. Immunological and antitumor effects of coumarin and some derivatives. Die Pharmazie. 1992;47(2):139–142. [PubMed] 10. McCulloch P, George WD. Warfarin inhibits metastasis of Mtln3 rat mammary carcinoma without affecting primary tumour growth. British Journal of Cancer. 1989;59(2):179–183. [PubMed] 11. Kostova I, Manolov I, Konstantinov S, Karaivanova M., editors. Synthesis, physicochemical characterisation and cytotoxic screening of new complexes of cerium, lanthanum and neodymium with Warfarin and Coumachlor sodium salts. European Journal of Medicinal Chemistry. 1999;34(1):63–68. 12. Kostova IP, Manolov II, Nicolova IN, Danchev ND. New metal complexes of 4-methyl-7-hydroxycoumarin sodium salt and their pharmacological activity. Il Farmaco. 2001;56(9):707–713. [PubMed] 13. Kostova I, Manolov I, Nicolova I, Konstantinov S, Karaivanova M. New lanthanide complexes of 4-methyl-7-hydroxy-coumarin and their pharmacological activity. European Journal of Medicinal Chemistry. 2001;36(4):339–347. [PubMed] 14. Manolov I, Kostova I, Konstantinov S, Karaivanova M. Synthesis, physicochemical characterization and cytotoxic screening of new
complexes of cerium, lanthanum and neodymium with Niffcoumar sodium salt. European Journal of Medicinal Chemistry. 1999;34(10):853–858. 15. Manolov I, Kostova I, Netzeva T, Konstantinov S, Karaivanova M. Cytotoxic activity of cerium complexes with coumarin derivatives. Molecular modeling of the ligands. Archiv der Pharmazie: Pharmaceutical and Medicinal Chemistry. 2000;333(4):93–98. 16. Montefiori DC, Robinson WE, Jr, Schuffman SS, Mitchell WM. Evaluation of antiviral drugs and neutralizing antibodies to human immunodeficiency virus by a rapid and sensitive microtiter infection assay. Journal of Clinical Microbiology. 1988;26(2):231–235. [PubMed] 17. Zack JA, Arrigo SJ, Weitsman SR, Go AS, Haislip A, Chen IS. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell. 1990;61(2):213–222. [PubMed] 18. Hazuda DJ, Felock P, Witmer M, et al. Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells. Science. 2000;287(5453):646–650. [PubMed]
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