New bicyclam-AZT conjugates: Design, synthesis, anti-HIV evaluation, and their interaction with CXCR-4 coreceptor

Article abstract of DOI:10.1021/jm980358u

We report the synthesis of mono- and bis-tetraazamacrocycle-AZT conjugates. All new compounds were screened for their ability to inhibit HIV- 1 replication in MT4 cell line and were compared to AZT alone. It appears that N-protected covalent prodrugs are

Full text of DOI:10.1021/jm980358u

J . Med. Chem. 1999, 42, 229-241
229
New Bicycla m -AZT Con ju ga tes: Design , Syn th esis, An ti-HIV Eva lu a tion , a n d
Th eir In ter a ction w ith CXCR-4 Cor ecep tor
J ean Dessolin,†,‡ Pascale Galea,‡ Patrick Vlieghe,†,‡ J ean-Claude Chermann,‡ and J ean-Louis Kraus*,†,‡
Laboratoire de Chimie Biomole´culaire, Faculte´ des Sciences de Luminy, case 901, Universite´ de la Me´diterrane´e,
163 avenue de Luminy, 13288 Marseille Cedex 9, France, and INSERM U322 “Re´trovirus et Maladies Associe´es”,
Campus Universitaire de Luminy, BP33, 13273 Marseille Cedex 9, France
Received J une 10, 1998
We report the synthesis of mono- and bis-tetraazamacrocycle-AZT conjugates. All new
compounds were screened for their ability to inhibit HIV-1 replication in MT4 cell line and
were compared to AZT alone. It appears that N-protected covalent prodrugs are equipotent to
AZT as inhibitor of HIV replication, while N-deprotected analogues exhibit both higher activity
and selectivity against HIV-infected cells. The most active antiviral compounds 27, 28, 34,
and 35 were then tested for their binding capability to CXCR-4 receptor. N-Boc analogues 27
and 34 were only weakly effective; in contrast, N-deprotected conjugates 28 and 35 were
antagonists to 12G5 mAb binding until 0.05 and 5 µg/mL, respectively. The stability of compound
28 in human plasma was evaluated, and half-life was found to be approximately 8 h in the
described conditions. All these results seem to demonstrate the confidence of our prodrug
approach, with analogue 28 emerging as the best candidate as lead compound in HIV-1
polytherapy perspective.
In tr od u ction
The search for an effective chemotherapeutic treat-
ment against human immunodeficiency virus (HIV)
infection has led to the development of agents that
target specific and critical events in the HIV replicative
cycle. The most extensively studied of these agents are
the 2′,3′-dideoxynucleoside analogues which terminate
DNA synthesis during the reverse transcription reac-
tion, the non-nucleoside reverse transcriptase (RT)
inhibitors which interact at a specific site on HIV-1 RT,
and the inhibitors of HIV protease, an essential pro-
teolytic enzyme required for the assembly of fully
infectious viral particles.1 More recently, it has been
pointed out that virus-induced cell fusion was a major
hallmark of HIV infection both in vivo and in vitro; it
represents a major mechanism of virus-induced cell
killing.2 These observations have led to the discovery
of a novel class of highly potent antiviral compounds
such as bis-tetraazamacrocycles3-8 (1, 2) or some tet-
rakis(ω-amino alkyl) tetraazamacrocycles9 (3) (Figure
1).
First, it was suggested that bicyclams such as J M
2763 (1) or J M 3100 (2) targeted the virus uncoating
associated process.10 Uncoating has to be preceded by
the removal of the viral envelope and followed by the
fusion process and then decapsidation (removal of capsid
F igu r e 1. Structures of bicyclam J M 2763 1, J M 3100 2, and
tetrakis(ω-amino alkyl) tetraazamacrocycle 3 analogues.
proteins). In recent papers,11-13 it has been shown that
bicyclams 1 and 2 were the first low molecular weight
agents which interfered with the cellular coreceptor for
T-cell tropic viruses, hence preventing cell infection.
At first, our interests were to explore the feasibility
of increasing the delivery of various anti-HIV dideoxy-
nucleosides to HIV-infected cells. For this purpose, we
developed synthetic schemes in order to synthesize new
cyclam- or bicyclam-nucleoside conjugates. Several
observations supported the importance of the synthesis
of these new covalent bicyclam-nucleoside conjugates:
(i) We have found that prodrugs of anti-RT nucleoside
(2′,3′-dideoxy-3′-thiacytidine) bearing various polyami-
nated sidearms at the 5′-O or 4-N positions led to potent
enhancement of the observed anti-HIV activity.14 As it
has been reported that nucleosides such as AZT cross
* To whom correspondence should be addressed. E-mail: kraus@
luminy.univ-mrs.fr. Tel: (33) 491 82 91 41. Fax: (33) 491 82 92 50.
† Universite´ de la Me´diterrane´e.
‡ INSERM U322.
10.1021/jm980358u CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/07/1999

230 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Dessolin et al.
the cell membrane by non-facilitated diffusion and that
its uptake was insensitive to inhibitors of nucleoside
transport,15,16 we have suggested that these new polyam-
inated prodrugs could interact with early events of the
retrovirus replicative cycle.
(ii) Using a three-dimensional method of analysis,3 it
has been shown that when bicyclam 1 (J M 2763) was
assayed against HIV-1 (IIIB) in combination with AZT,
the compounds appeared to act in additive fashion at
the concentrations used. This result was of interest since
physical combination (mixture) of two or three drugs
can act in several ways: additive combinations, which
are distinct from synergistic combinations (e.g., TIBO
plus AZT),3 or from antagonistic combinations (e.g., AZT
plus Ribavirin).3 This study showed that AZT and
bicyclam were not antagonistic drugs.
products were isolated in small quantities (7: 7% yield;
8: 3% yield). These four functionalized compounds were
clearly identified by NMR 1H COSY experiments.
Depending on their relative position from the nitrogen
atoms (R or â) and the substituents of these nitrogen
atoms, the methylene groups of the azamacrocycles
appear at different chemical shifts:
(iii) Covalent conjugate combination of two bicyclam-
nucleoside drugs would differ subtly from physical
combination of a mixture of two drugs in that the
conjugate, transported initially as such into the cells,
could present a specific antiviral activity profile. Indeed,
an enzymic labile conjugate upon hydrolysis could
gradually release the anti-HIV parent nucleoside to
make its own antiviral combination superimposed on
that of the bicyclam.
(iv) Several publications have shown the importance
of chemokine receptors for HIV entry into target cells12
and, in particular, the role of CXCR-4 receptor for the
specific binding of T-tropic HIV strains.17 In parallel,
bicyclams were reported to interfere in HIV fusion
events by targeting CXCR-4 receptors.11
From COSY experiments, it can be observed that the
protons of the methylene group in position 6 (â from
nitrogen 4 and 8, see Scheme 1) is only coupled with
the methylene 5 and 7 which are in R position from N4
and N8 Boc-substituted nitrogen atoms in compound 5,
while in compound 6 the corresponding protons in
position 6 are conjugated with the methylene 5 and 7
which are, respectively, in R position from N4 Boc and
N8 free nitrogen atoms. The same NMR argument was
put forward to elucidate the structure of dialkyl com-
pounds 7 and 8.
Indeed, it was of great interest to study whether the
new bicyclam-AZT conjugates could selectively target
the CXCR-4 receptor and inhibit HIV infection of cell.
This paper describes the synthesis of cyclam- and
bicyclam-AZT conjugates and their in vitro anti-HIV
activities correlated to some of their biophysical proper-
ties and to their selective antagonization of the chemok-
ine receptor CXCR-4.
Tetr a a za m a cr ocycle Con ju ga tes. Starting from
crucial synthon 4, two series of mono-tetraazamacro-
cycle-nucleoside conjugates were prepared, and their
synthesis are summarized in Scheme 2.
Fir st Ser ies: N-Alkyl Tetr aazam acr ocycle-Dide-
oxyn u cleosid e Con ju ga tes. Overnight condensation
of the intermediate 4 with ethyl 5-bromo valerate
afforded the corresponding alkylated polyazamacrocycle.
Hydrolysis of the obtained ester was achieved in quan-
titative yield under heterogeneous basic conditions in
tetrahydrofuran, which gave an overall yield of 28% for
compound 9. Condensations of the cyclam carboxylic
acid derivative 9 with AZT were performed following
different methods. Several assays with DCC/HOBt
(N,N-dicyclohexyl carbodiimide/1-hydroxybenzotriazole)
coupling reagent proved to be carboxylic acid analogue
9 consuming because of the concomitant formation of
N-acyl urea derivatives as byproducts.21,22 The use of
PyBOP (benzotriazol-1-yloxy-tris(pyrrolidino) phospho-
nium hexafluorophosphate) as coupling reagent allowed
the obtention of desired products in good yields. Final
deprotection of the N-Boc-protected tetraazamacro-
cycle-nucleoside conjugate was possible under various
experimental conditions. However, the obtention of solid
chlorhydrate 11 without hydrolysis of the ester link
favored the use of gaseous HCl in anhydrous diethyl
ether.
Ch em istr y
All series of compounds required the synthesis of
common intermediates as illustrated in Scheme 1. Tri-
Boc compound (synthon 4) and a mixture of di-Boc
compounds were obtained in the same step, starting
from the commercially available 1,4,8,11-tetraazacy-
clotetradecane (cyclam), following previously reported
procedures.18,19 Indeed, N-Boc protection of cyclam,
being more suitable, was preferred to N-tosyl protection
used in the preparation of bicyclam.20 It should be
emphasized that if the separation of the tri-Boc com-
pound 4 was easily performed from the mixture of di-
Boc compounds, the isolation and purification of N1,N11
-
di-Boc- and N1,N8-di-Boc-protected tetraazamacrocycle,
in contrast, was difficult. This was particularly due to
the polarity of the substrates. To avoid this time-
consuming purification step, the crude mixture of di-
Boc compounds was directly used in the next step
consisting of an alkylation by ω-bromo ester in refluxing
acetonitrile in the presence of potassium carbonate
(Scheme 1). The resulting compounds were then easily
purified. Compound 5 (monoalkylated N4,N8-di-Boc) was
Secon d Ser ies: N-Acyl Tetr a a za m a cr ocycle-
Did eoxyn u cleosid e Con ju ga tes. Acylation of com-
pound 4 using glutaryl dichloride (Scheme 2) in Schot-
ten-Baumann conditions afforded the expected acyl
chloride 12, along with the bis-polyazamacrocycle 15
resulting from the dimerization of compound 4 as a
isolated in 65% yield, while the monoalkylated N4,N11
-
di-Boc 6 was obtained in 15% yield and the dialkylated

New Bicyclam-AZT Conjugates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 231
Sch em e 1
minor product (15% yield). The condensation of AZT
with compound 12 using 4-DMAP (4-(dimethylamino)-
pyridine) in dichloromethane afforded the desired ester
in low yields (30%). Note that syntheses of compounds
4, 9, 10, 12, and 13 were already described elsewhere.22
Bis-tetr a a za m a cr ocycle Con ju ga tes. The prepara-
tion of the different series of bis-tetraazamacrocycle-
nucleoside conjugates was achieved using the following
general procedure (Scheme 3): compounds 4 and 5 were
condensed with an appropriate bis-electrophile (glutaryl
dichloride for the first series, R,R′-dibromo-p-xylene and
terephthaloyl dichloride for the second and the third
series, respectively). The use of two different mono-
polyazamacrocycles in condensation reaction with a bis-
functionalized compound implied the possible formation
of three different bis-polyazamacrocycles. We observed,
for each of the three series, the concomitant formation
of three different products detected by TLC. Due to the
close Rf values, the purification of the obtained mixture
appeared quite difficult. This problem was solved by
isolation of the mixture of the three bis-tetraazamac-
rocycles (confirmed by NMR) from the crude reaction,
followed by their saponification in tetrahydrofuran. The
resulting compounds, possessing at this stage suf-
ficiently different Rf values, allowed convenient and
rapid purification of the desired unsymmetrical acids
(18, 25, 32), symmetrical diacids (19, 26, 33), and the
dimers (15, 22, 29). By analogy with the synthesis of
the conjugate 10, PyBOP coupling reagent in the
presence of triethylamine in dichloromethane was used
to prepare the bis-macrocycle-AZT conjugates (20, 27,
and 34). These compounds proved to be as resistant

232 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Dessolin et al.
Sch em e 2
versus tert-butyl carbamate deprotection as the corre-
sponding monocyclic conjugates. Thus compounds 21,
28, and 35 were obtained from the respective N-Boc-
protected esters 20, 27, and 34. It should be noted that
release of dideoxynucleoside did not occur in the de-
scribed conditions.
Dimeric compounds such as 15, 22, and 29 were Boc-
deprotected to give the symmetric bis-polyazamacro-
cycles 36, 37, and 38, respectively. These latter were
used as anti-HIV references since dimer 37 was found
to be an anti-HIV compound.4
conditions, AZT alone inhibited syncytia formation with
EC50 ) 0.05 µM.
Monocyclam-AZT conjugates were found to be slightly
less active than the corresponding bicyclam-AZT ana-
logues. Analogues 27 and 28 appeared to be 1 order of
magnitude more active than the parent drug AZT, while
compounds 34 and 35 were equipotent to AZT. The
lower EC50 values for 27 and 28 compared to that of
the parent molecule AZT could be tentatively attributed
to an increase in cellular uptake followed by intracel-
lular release of AZT. The prodrugs appeared to be
lipophilic enough to cross the cellular membrane by
simple diffusion as demonstrated by the antiviral activi-
ties.
Bin d in g Ca p a bility of th e New Dr u gs to th e
CXCR-4 Recep tor a n d Cor r ela tion w ith Th eir
Biop h ysica l P r op er ties. Compounds 27, 28, 34, 35,
and reference compound 2 were studied for their ability
to bind to the CXCR-4 receptor as already reported.24
SDF-1R was also included in the study as the natural
biological ligand of the CXCR-4 receptor. By using
cytometry of flux, drugs were tested for their power to
inhibit the fixation of 12G5 mAb, which specifically
recognizes cellular CXCR-4 receptor. As shown in Figure
2, J M 3100 (2) and N-deprotected analogues 28 and 35
prevented 12G5 mAb fixation to CXCR-4 receptor as
attested by the extinction of the specific fluorescent
signal. Fixation of control mAb B1G6 was not affected
by drug treatment of cells, indicating a specific action
of compounds 28, 35, and J M 3100 on CXCR-4 mol-
ecules. Compounds 2 and 28 were the most efficient
compounds to compete with 12G5 mAb fixation, as they
decreased mAb fixation from 81% and 69%, respectively,
Resu lts
The first objective of this project was to design and
synthesize covalently linked bicyclam-AZT conjugates
with improved anti-HIV activities. The design of these
new conjugates was based on the following finding:
bicyclam moiety are known to interact specifically with
the CXCR-4 coreceptor12,23 used by T-tropic viruses to
infect target cells and then they could help to achieve
higher AZT concentration in infected cells. In this
perspective, the effects of the new bicyclam-AZT con-
jugates on HIV cytopathicity, their interaction with
CXCR-4 chemokine receptor, and the correlation of their
antiviral activities with lipophilicity and biological
stability properties were studied.
An tivir a l Activity Mea su r em en ts. AZT, cyclam/
bicyclam intermediates, and cyclam- or bicyclam-AZT
conjugates were evaluated for their inhibitory effects on
HIV replication in MT4 cell culture (Table 1). Under
assay conditions, all the tested cyclam- or bicyclam-
AZT prodrugs elicited anti-HIV activity with EC50
values ranging from 0.005 to 0.1 µM. In the same testing

New Bicyclam-AZT Conjugates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 233
Sch em e 3
at a concentration of 5 µg/mL; the inhibitory effect was
dose-dependent: at 0.05 µg/mL the inhibitory effect was
maintained at 82% for J M 3100 versus 47% for com-
pound 28. Compound 35, whose structure includes a
phthaloyl linker between the two cyclam rings, was also
efficient in competing for the binding to the CXCR-4
receptor but at higher concentrations, as 52% of inhibi-
tion was observed at 25 µg/mL versus 26% at 5 µg/mL.

234 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Dessolin et al.
Ta ble 1. Antiviral Evaluation of Cyclam/Bicyclam-AZT Conjugates

New Bicyclam-AZT Conjugates
Ta ble 1 (Continued)
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 235
a
b
EC50: concentration in µM required to inhibit syncytia formation by 50% on MT4 cells. CC50: concentration in µM required to cause
50% death of uninfected MT4 cells. c SI: selective index ) CC50/EC50
Chemistry Development, Inc.)/log P 1.0 base calculations.
.
log P determinations were performed using ACD (Advanced
d
In contrast, N-Boc-protected analogue 27 was totally
ineffective whereas compound 34 weakly inhibited 12G5
mAb binding (30% of inhibition at 25 µg/mL).
the cyclam series and from 5.85 to 2.69 for conjugates
belonging to the bicyclam series. As expected, these
values were greater than the those obtained for the
corresponding deprotected cyclam or bicyclam conju-
gates. For example, in the case of AZT-conjugate
congeners, log P values ranged from 1.81 to -1.35, while
the log P value for AZT itself was -0.88. It has been
reported21 that prodrugs of AZT in which the 5′-hydroxyl
group was esterified with various terminal carboxylic
ligands had anti-HIV activity in peripheral blood lym-
phocytes, greater than that of AZT alone. Most of these
AZT prodrugs that had higher partition coefficients than
that of AZT diffused into the cells at a higher level than
AZT itself, suggesting an important influence of parti-
tion coefficients on the diffusion of AZT analogues into
the cells. In the case of cyclam/bicyclam-AZT prodrugs,
conjugates having greater log P values (13, 14, 10, 11,
34, 27, 28, 20, 21) as well as those having lower log P
values (35) than the one of AZT elicited higher or at
least equal anti-HIV activity compared to that of AZT
alone. Therefore, from the results presented in Table
1, lipophilicity is probably not the only factor that
influences the antiviral activity of these enzyme-labile
prodrugs. Starting from the hypothesis that enzymatic
These results suggest that selective competition for
the binding to the CXCR-4 receptor requires a prototype
molecule presenting required structural parameters: (i)
the number of free nitrogen atoms in the bicyclam
moiety should be maximum, and (ii) the preferred linker
between the two bicyclams should be a xylyl or alkyl
group, the phthaloyl linker being less effective. The
presence of only one sidearm bearing the AZT moiety
slightly decreased the binding affinity to the CXCR-4
receptor (compound 28 versus reference compound 2).
Lipoph ilicity, Stability, an d Decom position Stu d-
ies. Previous studies have reported that AZT crossed
the cell membrane by nonfacilitated diffusion and that
its uptake was insensitive to inhibitors of nucleoside
transport.16 This indicates that the partition coefficient
of AZT analogues might have a significant role in their
diffusion. In this perspective, partition coefficients (log
P) were determined for all conjugates using ACD/LogP
software from ChemCAD.
From the results shown in Table 1, log P values for
Boc-protected derivatives ranged from 2.68 to 2.34 in

236 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Dessolin et al.
F igu r e 2. Effect of N-deprotected and N-Boc-protected drugs on CXCR-4 expression. After a 20 min incubation of drugs with
MT4 cells, cells were incubated with anti-CXCR-4 mAb and then washed and counterstained with PE-GAM as described in Material
and Methods. The histograms show the surface expression of CXCR-4 receptor on MT4 cells incubated (A) with 5 µg/mL of
N-deprotected compounds 28 and 35 in comparison with control compound 2 and natural ligand SDF1-R used at 5 µg/mL, and (B)
with 25 µg/mL of Boc-protected compounds 27 and 34 in comparison with control compound 2 and natural ligand SDF1-R used
at 5 µg/mL. The vertical line indicates the cutoff at which fluorescence was less than 1% of cells stained with a negative control
mAb. In parentheses are expressed the percentages of positive cells. Data from one representative experiment out of three are
shown. Figure 2 was treated with Adobe Photoshop software in order to increase the contrast.
hydrolysis sensibilities and stabilities of the prodrugs
are likely to be crucial, we have performed stability and
decomposition studies.
in comparison with other AZT prodrugs, suggests that
their intracellular hydrolysis under in vitro conditions
could limit release of AZT inside infected cells. In this
case, one can expect a decrease in anti-HIV activity of
the AZT prodrugs in comparison to AZT itself. In order
to explain the increased anti-HIV activity of bicyclam-
AZT conjugates 27 and 28 which blocked syncytium
formation at concentrations 2- to 10-fold lower than the
corresponding concentration required when AZT is used
alone, several facts can be highlighted:
(i) It has been reported that bicyclams are inhibitory
to syncytium formation at concentrations that were
about 10- to 100-fold higher than their EC50 in viral
replication assays.4 Thus, the bicyclam moiety could
partake weakly in the inhibition of syncytium formation
in addition to that of AZT.
The usefulness of cyclam/bicyclam-AZT prodrugs
depends not only on the stability of the prodrug for its
transport across cell membrane but also upon its
intracellular reversion to the parent compound, espe-
cially in HIV-infected cells.21 The half-life (t1/2) of
hydrolysis of two selected AZT conjugates, 14 and 28
was determined in human plasma, as well as under
acidic and basic aqueous conditions. Prodrugs 14 and
28 were found chemically stable in basic or acidic
aqueous media (pH 8.3 and 2.2, respectively) up to 24
h at 37 °C. In contrast, in human serum, the stability
determined by HPLC showed that the half-life values
of compounds 14 and 28 were, respectively, 7 and 8 h.
These results suggest that cyclam/bicyclam-AZT pro-
drugs are only slightly sensitive to plasma esterases.
Therefore, these prodrugs may have increased plasma
t1/2 under in vivo conditions.
(ii) It has been found that linear polyamine covalently
coupled to nucleoside anti-reverse transcriptase drugs
potentialized syncytium formation inhibition, specifi-
cally in the case of macrophage-tropic strains of HIV.14
Thus, the bicyclam moiety could enhance the transport
and/or the internalization of AZT in target cells.
Discu ssion
The new synthesized cyclam/bicyclam-AZT prodrugs
differ noticeably from known 5′-O-AZT prodrugs in
several ways: no apparent correlation appears between
lipophilicity and antiviral activity, the highly lipophilic
N-Boc-protected analogues (13, 10, 34, 27, 20) and the
corresponding hydrophilic analogues (14, 11, 35, 28, 21)
eliciting similar antiviral activities. This result suggests
that passive diffusion of the prodrugs into the cells is
not the rate-limiting step. Since esters have been shown
to undergo extensive first-pass metabolism,12 the stabil-
ity of the new prodrugs of AZT was studied. The relative
good enzymatic stability found for the tested prodrugs,
(iii) The bicyclam moiety of compound 28 interacts
with the CXCR-4 receptor which mediates entry of
T-tropic HIV strains, while the released AZT (intra- or
extracellulary) interacts with the enzyme reverse tran-
scriptase as chain terminator. At concentrations that
are effective in inhibiting viral cytopathicity, bicyclam
has been reported to fail to inhibit the formation of giant
cells in a direct syncytium formation assay.11 In these
hypotheses, only the inhibition of syncytium formation
caused by AZT (inhibition of replication) could be
observed, while the possible effect of the bicyclam moiety
on the inhibition of virus infection3 would not. This

New Bicyclam-AZT Conjugates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 237
Company) from freshly opened containers. Preparation and
structural data of compounds 4, 9, 10, 12, and 13 are available
elsewhere.22
Cell Lin es, Vir u s, a n d Cell Cu ltu r e. The CEM cell line
and the T-leukaemia virus type one (HTLV-I) CD4-positive
T-cell line, MT4, were cultured in RPMI/10% FCS and refeeded
twice a week.
The laboratory-adapted strain HIVLAV clade B stock was
prepared from the supernatant of the infected CEM cell line,
and aliquots were kept frozen at -80 °C until use.25
An ti-HIV Activity Assa y. Anti-HIV activity was monitored
by the efficiency of drug compounds to inhibit syncytia forma-
tion after HIV infection of MT4 as already described.26,27
Briefly, 3 × 105 MT4 cells were first preincubated with 100
µL of various concentrations of drug compounds dissolved in
DMSO or in H2O and then diluted in phosphate buffer saline
solution for 1 h at 37 °C. Then 100 µL of an appropriate virus
dilution was added to the mixture, and another 1 h incubation
period at 37 °C was done. After three washes, cells were
resuspended in culture medium in the presence or not of drug
compounds. Cultures were then continued for 7 days at 37 °C
under 5% CO2 atmosphere and refeeded at day 3 postinfection
with culture medium supplemented or not with drug com-
pounds. Each culture well was done in duplicate. The appear-
ance of syncytia was followed each day with an inverted optical
microscope. Typically, the virus dilution used in this assay
(multiplicity of infection of 0.1 TCID50/cell) allowed syncytia
formation at day 5 postinfection. The inhibitory concentration
of drug compounds was expressed as the concentration that
caused 50% inhibition of syncytia formation (EC50) without
means that conjugate 28 behaves as a prodrug of AZT,
delivering the nucleoside to infected cells at higher
concentration.
Other antiviral activity assays, such as viral antigen
expression have to be performed in order to evaluate
the EC50 for inhibiting viral cytopathicity caused by the
bicyclam moiety.
Moreover, the cytotoxicity of AZT and the prodrugs,
determined against MT4 cells by the Trypan blue dye
exclusion method for cell viability, revealed that N-
protected prodrugs were more cytotoxic than AZT,
suggesting that this increase in toxicity may be due to
the partial release of the terminal carboxylic N-Boc
cyclam or bicyclam moiety.
Con clu sion
This is the first time to our knowledge that the
synthesis and antiviral properties of covalently bounded
(AZT)-cyclam/bicyclam prodrug conjugates are re-
ported. From the obtained results, the relative stability
under acidic and basic conditions, as well as in serum,
indicates that these prodrugs might enhance AZT
bioavailability.
Among the cyclam- or bicyclam-AZT conjugate
series, derivatives 27 and 28 emerged as the two most
active antiviral compounds, but 28 had the highest
selective index. Furthermore, compound 28 elicited the
greatest binding affinity to the CXCR-4 coreceptor (with
an extinction coefficient of 69% at 5 µg/mL) whereas
compound 27 did not inhibit binding to CXCR-4. This
indicates that although both compounds were effective
in inhibiting viral replication, they did not use the same
ways to enter into the cells. Hence, compound 28, by
targeting the CXCR-4 receptor at the cell surface,
probably selectively drove AZT compounds into the cells,
increasing its efficiency of action (less cytotoxic for the
same EC50).
direct toxicity for the cells. The cytotoxic concentration (CC50
)
of drug compounds was monitored on growth of noninfected
cells by Trypan blue exclusion assay and corresponded to the
concentration required to cause 50% of cell death. It should
be emphasized that when compounds required the addition of
DMSO to be solubilized in water (compounds 10, 13, 15, 20,
22, 27, 29, 34), the concentration in volume of DMSO used
was in many cases inferior to 10% with respect to water (the
final concentration of DMSO in MT4 cells incubation medium
being less than 2 µM). As far as DMSO could affect the
antiviral activity of the tested drugs,28 antiviral assays in
which solutions containing equal concentration of DMSO in
water were performed and used as standard assays for each
tested drugs. EC50 and CC50 reported values were then
calculated from these standard assays. In any case, final
DMSO concentrations (1/1000) are very far from the percent-
ages28 which induced an enhancement of in vitro HIV-1
infection of T-cells.
CXCR-4 Im m u n oflu or escen ce Cell Sta in in g. Mon o-
clon a l An tibod ies (m Ab) a n d SDF -1r Used . Anti-CXCR-4
mAb 12G5 was from R&D Systems (England). Positive control
mAb B1G6 directed against the â2-microglobulin, irrelevant
mAb (1G11), and secondary antibody (PE-conjugated goat anti
mouse IgG) were purchased from Immunotech-Coultronics
(France). SDF-1R, the natural ligand for CXCR-4, was pur-
chased from R&D Systems (England).
Cell Sta in in g. MT4 cells were preincubated with various
concentrations of drug products (prepared as already stated
for antiviral assays) or in medium alone for 20 min at room
temperature. Then anti-CXCR-4, positive control B1G6, and
irrelevant mAbs were added to the cells, and incubation was
performed at 4 °C for 30 min. After 2 washes, cells were then
incubated with PE-conjugated secondary antibody for another
30 min at 4 °C. After being washed, cells were fixed in 0.1 mL
of 1% paraformaldehyde PBS solution. Cell surface molecule
expression was then analyzed by flow cytometry (ELITE-
Coultronics).
Results are expressed as percentage of positive cells for the
studied molecules. Fixation of the drug compound on the
CXCR-4 receptor was followed by the extinction of the specific
fluorescence signal in comparison to CXCR-4 expression on
nontreated cells. The specificity of drug compound action on
CXCR-4 was evaluated in comparison to the constant B1G6
mAb fixation to cells in the presence or not of drugs.
The presented new bipharmacophore drugs could
represent the first prototype drug associating in a single
molecule an anti-reverse transcriptase inhibitor co-
valently linked to a fusion inhibitor. Antiviral activity
of these new prototypes on AZT-resistant virus strains
is under investigation. Improvement of the selective
index of such a prototype could have clinical potential
in AIDS combinotherapy.
Exp er im en ta l Section
Nuclear magnetic resonance spectra were recorded with a
Bruker AC-250 spectrometer (1H NMR); chemical shifts are
expressed as δ units (part per million) downfield from TMS.
Fast atom bombardment mass spectral analysis were obtained
by Dr. Astier (Laboratoire de Mesures Physiques-RMN, USTL,
Montpellier, France) on a J EOL DX-100 using a cesium ion
source and glycerol/thioglycerol (1:1) or m-nitrobenzyl alcohol
(NOBA) as matrix. Mass calibration was performed using
cesium iodide. Microanalyses were carried out by Service
Central d’Analyses du CNRS (Venaison, France) and were
within 0.4% of the theoretical values. Thin-layer chromatog-
raphy (TLC) and preparative layer chromatography (PLC)
were performed using silica gel plates 0.2, 1, or 2 mm thick
(60F254 Merck). Preparative flash column chromatography was
carried out on silica gel (230-240 mesh, G60 Merck). Analyti-
cal HPLC was performed on a Waters 600E instrument with
a M991 detector using the following conditions: 4.6 × 150 mm
column (Waters Spherisorb S5 ODS2, 5 µM); mobile phases:
A ) 0.1% TFA in H2O, B ) 0.1% TFA in CH3CN; flow rate 1.5
mL/min. All reagents were of commercial quality (Aldrich

238 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Dessolin et al.
Hyd r olysis of th e Com p ou n d s 14 a n d 28 in Acid
Solu tion , in Ba sic Solu tion , a n d in Hu m a n Ser u m . A
solution (0.5 mL) of one of the esters (14: 13 mg/mL; 28: 18
mg/mL in H2O) was added to 2 mL of normal human serum
(NHS), or a saturated aqueous NaHCO3 solution (pH 8.9), or
a 5% aqueous citric acid solution (pH 2.2), and the mixture
was incubated at 37 °C in a water bath. The samples (100 µL)
were withdrawn and added immediately to ice-cold water (400
µL) during the first 8 h every 60 min and then every 12 h until
48 h. The resulting samples were centrifuged, and the super-
natants were filtered through nylon filters (0.45 µm) and then
analyzed by HPLC using the following method: 0% f 100% of
solvent B in 15 min.
As previous experiments demonstrated 267 nm to cor-
respond to the λmax of each compound, this wavelength was
used for the detection. The peak retention time for the three
compounds was shown to be: 8.8 min (AZT), 9.6 min (14), and
10.4 min (28).
Area variation during the time of experiment allowed us to
estimate t1/2 for compounds 14 and 28 as being, respectively,
7 and 8 h in normal human serum.
Ch em ica l Syn th esis. 1,4,8-Tr is(ter t-bu toxyca r bon yl)-
1,4,8,11-tetr a a za cyclotetr a d eca n e 4. To a solution of 1.00
g of cyclam 1 (5.00 mmol, 1 equiv) in ice-cold CH2Cl2 (125 mL)
was added 2.00 g of di-tert-butyl dicarbonate (9.00 mmol, 1.8
equiv). The solution was stirred for 4 h at room temperature.
After solvent evaporation, the crude yellow oil was purified
by flash column chromatography (MeOH/CH2Cl2 5:95) to give
the desired product (“tri-Boc”) as a white-yellow foam (0.60
g, 24% yield) and a mixture of products (0.77 g) identified by
NMR as “di-Boc” compounds (relative to the integration of the
t-Bu signal).
5-[4,8-Bis(ter t-bu toxycar bon yl)-1,4,8,11-tetr aazacyclotet-
r a d eca n yl] P en ta n oic Eth yl Ester 5. To the previously
obtained mixture of products (0.77 g, 1.93 mmol) and an excess
of K2CO3 (0.80 g, 5.77 mmol, 3 equiv) in CH3CN (40 mL) was
added 0.5 equiv of ethyl 5-bromo valerate (0.20 g, 0.96 mmol).
The reaction mixture was refluxed overnight while stirring and
then allowed to cool, and the solid carbonate was removed by
filtration. The solvent was evaporated, and the residue was
partitioned between EtOAc and brine. The organic layer was
separated, washed twice with brine, dried (Na2SO4), and
concentrated to give a brown-yellow oil. Purification by flash
column chromatography (MeOH/CH2Cl2 5:95) afforded com-
pound 5 as a light yellow oil (0.66 g, 65% yield) and its isomer
6 as minor product. Rf ) 0.41 (MeOH/CH2Cl2 1:9). 1H NMR
(CDCl3): 1.25 (t, 3H, J ) 7.10 Hz, -O-CH2-CH3), 1.46 (brs,
20H, t-Bu and -CH2-(CH2)2-CO2Et), 1.49-1.77 (m, 4H, HN-
CH2-CH2-CH2-N and -CH2-CH2-CO2Et), 1.85 (m, 2H,
BocN-CH2-CH2-CH2-NBoc), 2.29 (t, 2H, J ) 7.35 Hz,
-CH2-CO2Et), 2.39 (t, 2H, J ) 7.25 Hz, N-CH2-(CH2)3-CO2-
Et), 2.44-2.60 (m, 4H, N-(CH2)2), 2.78 (t, 2H, J ) 5.25 Hz,
HN-CH2-CH2-CH2-N), 2.85 (t, 2H, J ) 5.10 Hz, HN-CH2-
CH2-NBoc), 3.10-3.30 (m, 4H, -CH2-NBoc), 3.34 (t, 2H, J
) 8.00 Hz, BocN-CH2-CH2-CH2-NBoc), 3.42 (t, 2H, J ) 4.90
Hz, N-CH2-CH2-NBoc), 4.12 (q, 2H, J ) 7.10 Hz, -O-CH2-
CH3). MS (FAB+): 529 (M + H)+.
and appears as bright yellow oil (0.09 g, 7% yield). Rf ) 0.62
(MeOH/CH2Cl2 1:9). 1H NMR (CDCl3): 1.25 (t, 3H, J ) 7.10
Hz, -O-CH2-CH3), 1.46 (brs, 22H, t-Bu and -CH2-(CH2)2-
CO2Et), 1.53-1.74 (m, 6H, N-CH2-CH2-CH2-N and -CH2-
CH2-CO2Et), 1.86 (qt, 2H, BocN-CH2-CH2-CH2-NBoc),
2.30 (t, 2H, J ) 7.20 Hz, -CH2-CO2Et), 2.33-2.61 (m, 8H,
-CH2-N-CH2-(CH2)3-CO2Et), 2.57 (m, 4H, N-CH2-CH2-
NBoc), 3.14-3.35 (m, 8H, -CH2-NBoc), 4.11 (q, 2H, J ) 7.10
Hz, -O-CH2-CH3). MS (FAB+): 657 (M + H)+.
5-[4,11-Bis(ter t-bu toxyca r bon yl)-8-p en ta n oic Eth yl Es-
ter -1,4,8,11-tetr a a za cyclotetr a d eca n yl] P en ta n oic Eth yl
Ester 8. Compound 8 is a byproduct (di-alkylated compound)
obtained during alkylation of the mixture of di-Boc compounds
and appears as bright yellow oil (0.04 g, 3% yield). Rf ) 0.69
(MeOH/CH2Cl2 1:9). 1H NMR (CDCl3): 1.25 (t, 3H, J ) 7.10
Hz, -O-CH2-CH3), 1.43 (brs, 22H, t-Bu and -CH2-(CH2)2-
CO2Et), 1.65 (m, 4H, -CH2-CH2-CO2Et), 1.79 (m, 4H, BocN-
CH2-CH2-CH2-N), 2.32 (t, 4H, J ) 7.30 Hz, -CH2-CO2Et),
2.35-2.63 (m, 8H, -CH2-N-CH2-(CH2)3-CO2Et), 2.60 (m,
4H, N-CH2-CH2-NBoc), 3.11-3.33 (m, 8H, -CH2-NBoc),
4.10 (q, 2H, J ) 7.10 Hz, -O-CH2-CH3). MS (FAB+): 657
(M + H)+.
Gen er al P r ocedu r e A for th e Cou plin g of Tetr aazam ac-
r ocycle Acid s w ith Nu cleosid es. To a solution of tet-
raazamacrocycle acid (1 equiv) in CH2Cl2 and a catalytic
amount of DMAP, were added a large excess of NEt3 (3 equiv),
then 1 equiv of nucleoside dissolved in a mixture of DMF/CH2-
Cl2, and 1.2 equiv of PyBOP reagent. The reaction mixture
was allowed to stir for 8 h at room temperature under nitrogen
atmosphere. The solvent was then removed, and the residue
was dissolved in EtOAc. The organic layer was successively
washed with 5% aqueous citric acid and water and dried over
Na2SO4. Evaporation of the solvent under reduced pressure
gave a crude product which was then purified by flash column
chromatography or PLC, using MeOH/CH2Cl2 as eluent.
Gen er a l P r oced u r e B for th e Dep r otection of N-Boc
Con ju ga tes. Anhydrous 1 N HCl in Et2O was added (10 mL/
mmol) to the mono-tetraazamacrocycle or bis-tetraazamacro-
cycle conjugate, and the resulting solution was stirred until
complete disappearance of the starting conjugate. The solution
was then concentrated to give the hydrochloride conjugate.
3′-Azid o-3′-d eoxy-5′-O-{5-[1,4,8,11-tetr a h yd r och lor id e-
1,4,8,11-t e t r a a za cyclot e t r a d e ca n yl]p e n t a n oyl}t h ym i-
d in e 11. According to the general carbamate deprotection
procedure B, the reaction of compound 10 (0.05 g, 0.06 mmol)
afforded the title compound 11 as a slightly yellow solid, in
quantitative yield (0.03 g, 96% yield). Rf ) 0.10 (MeOH/CH2-
Cl2 1:9). 1H NMR (D2O): 1.64-2.03 (m, 7H, -CH2-CH2-CH2-
CO2Nu and CH3Thy), 2.18 (m, 4H, N-CH2-CH2-CH2-N),
2.27-2.65 (m, 4H, -CH2-CO2Nu and H-2′), 3.10-3.88 (m,
18H, -CH2-N), 3.95-4.07 (m, 1H, H-4′), 4.10-4.48 (m, 3H,
H-3′ and H-5′), 6.17 (m, 1H, H-1′), 7.42 (brs, 1H, H-6Thy). MS
(FAB+): 550, (M + H)+.
Gen er a l P r oced u r e C for th e Cou p lin g of Com p ou n d
12 w ith Nu cleosid es. Compound 12 (0.17 g, 0.26 mmol, 1
equiv) was dissolved in CH2Cl2 (10 mL) giving a greenish
solution, and DMAP (0.06 g, 0.52 mmol, 2 equiv) and 1 equiv
of nucleoside were then added. The reaction mixture was
stirred for 8 h at room temperature. The resulting solution
was successively washed with 5% aqueous citric acid and water
and dried over Na2SO4 and concentrated. Purification was
performed by flash column chromatography or PLC using
MeOH/CH2Cl2 as eluent.
5-[4,11-Bis(ter t-b u t oxyca r b on yl)-1,4,8,11-t et r a a za cy-
clotetr a d eca n yl] P en ta n oic Eth yl Ester 6. Compound 6
was obtained as a slightly yellow oil during alkylation of the
mixture of di-Boc compounds (0.15 g, 15% yield). Rf ) 0.51
(MeOH/CH2Cl2 1:9). 1H NMR (CDCl3): 1.25 (t, 3H, J ) 7.10
Hz, -O-CH2-CH3), 1.44 (brs, 20H, t-Bu and -CH2-(CH2)2-
CO2Et), 1.48-1.70 (m, 4H, HN-CH2-CH2-CH2-NBoc and
-CH2-CH2-CO2Et), 1.80 (qt, 2H, BocN-CH2-CH2-CH2-N),
2.29 (t, 2H, J ) 7.35 Hz, -CH2-CO2Et), 2.35-2.60 (m, 6H,
-(CH2)2-N-CH2-(CH2)3-CO2Et), 2.64 (t, 2H, J ) 5.65 Hz,
HN-CH2-CH2-CH2-NBoc), 2.79 (t, 2H, J ) 5.05 Hz, HN-
CH2-CH2-NBoc), 3.19-3.47 (m, 8H, -CH2-NBoc), 4.12 (q,
2H, J ) 7.10 Hz, -O-CH2-CH3). MS (FAB+): 529 (M + H)+.
3′-Azido-3′-deoxy-5′-O-{5-oxo-5-[4,8,11-tr ih ydr och lor ide-
1,4,8,11-t e t r a a za cyclot e t r a d e ca n yl]p e n t a n oyl}t h ym i-
d in e 14. According to the general carbamate deprotection
procedure B, reaction of compound 13 (0.04 g, 0.05 mmol)
afforded the title compound 14 as a yellow solid in quantitative
yield (0.02 g, 94% yield). Rf ) 0.10 (MeOH/CH2Cl2 1:9). 1H
NMR (D2O): 1.72-1.98 (m, 2H, CO-CH2-CH2-CH2-CO2Nu),
2.00-2.36 (m, 7H, N-CH2-CH2-CH2-N and CH3Thy), 2.39-
2.57 (m, 6H CO-CH2-CH2-CH2-CO2Nu and H-2′), 3.08-3.96
(m, 16H, -CH2-N), 4.14 (m, 1H, H-4′), 4.43-4.62 (m, 3H, H-3′
5-[4,8-Bis(ter t-bu toxyca r bon yl)-11-p en ta n oic Eth yl Es-
ter -1,4,8,11-tetr a a za cyclotetr a d eca n yl] P en ta n oic Eth yl
Ester 7. Compound 7 is a byproduct (di-alkylated compound)
obtained during alkylation of the mixture of di-Boc compounds

New Bicyclam-AZT Conjugates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 239
and H-5′), 6.21 (m, 1H, H-1′), 7.47 (brs, 1H, H-6Thy). MS
(FAB+): 564 (M + H)+.
pen tan oyl)th ym idin e 21. According to the general carbamate
deprotection procedure B, reaction of compound 20 (0.04 g, 0.03
mmol) afforded the title compound 21 as a white solid in
quantitative yield (0.03 g, 95% yield). Rf ) 0.10 (MeOH/CH2-
Cl2 1:9). 1H NMR (D2O): 1.57-1.91 (m, 9H, -CH2-CH2-CH2-
CO2Nu and CH3Thy and CO-CH2-CH2-CH2-CO), 1.91-2.23
(m, 8H, N-CH2-CH2-CH2-N), 2.27-2.59 (m, 8H, CO-CH2-
CH2-CH2-CO and -CH2-CO2Nu and H-2′), 3.07-3.79 (m,
34H, -CH2-N), 4.03 (m, 1H, H-4′), 4.21-4.36 (m, 3H, H-3′
and H-5′), 6.09 (m, 1H, H-1′), 7.37 (brs, 1H, H-6Thy). MS
(FAB+): 846 (M + H)+.
1,1′-Xylyl-bis[4,8,11-tr is(ter t-bu toxyca r bon yl)-1,4,8,11-
tetr a a za cyclotetr a d eca n e] 22. Compound 22 was obtained
as a byproduct during the synthesis of ester 23 and was
purified after the saponification of the whole mixture to give
a white solid (0.17 g, 20% yield). Rf ) 0.84 (MeOH/CH2Cl2 1:9).
1H NMR (CDCl3): 1.46 (brs, 54H, t-Bu), 1.67 (m, 4H, BocN-
CH2-CH2-CH2-N-Ph), 1.91 (m, 4H, BocN-CH2-CH2-CH2-
NBoc), 2.37 (m, 4H, Ph-N-CH2-CH2-CH2-NBoc), 2.61 (m,
4H, Ph-N-CH2-CH2-NBoc), 3.13-357 (m, 24H, BocN-
CH2-), 3.51 (brs, 4H, Ph-CH2-), 7.17 (s, 4H, -Ph-). MS
(FAB+): 1103 (M + H)+.
1,1′-(1,5-Dioxo-p en ta n e)-bis[4,8,11-tr is(ter t-bu toxyca r -
bon yl)-1,4,8,11-tetr a a za cyclotetr a d eca n e] 15. Compound
15 was obtained as a byproduct during the synthesis of ester
16 and was purified after the saponification of the whole
mixture (0.08 g, 17% yield). This compound was also obtained
during the synthesis of acyl chloride 12. Rf ) 0.53 (MeOH/
CH2Cl2 1:9). 1H NMR (CDCl3): 1.39 (s, 54H, t-Bu), 1.69 (m,
8H, N-CH2-CH2-CH2-N), 1.89 (m, 2H, CO-CH2-CH2-
CH2-CO), 2.34 (m, 4H, CO-CH2-CH2-CH2-CO), 3.21-3.50
(m, 32H, -CH2-N-CO). MS (FAB+): 1097 (M + H)+.
5-[4,8-Bis(ter t-bu toxyca r bon yl)-11-{5-oxo-5-[4,8,11-tr is-
(ter t-bu toxyca r bon yl)-1,4,8,11-tetr a a za cyclotetr a d eca n -
yl]p en ta n oyl}-1,4,8,11-tetr a a za cyclotetr a d eca n yl] P en -
ta n oic Acid 18. In a mixture of compound 4 (0.22 g, 0.44
mmol, 1 equiv) and 5 (0.23 g, 0.44 mmol, 1 equiv) in the
biphasic CH2Cl2/NaHCO3 (30 mL: 15 mL), was added glutaryl
dichloride (0.07 g, 0.44 mmol, 1 equiv). The reaction mixture
was stirred for 30 min until disappearance of the starting
tetraazamacrocycles. The aqueous layer was partitioned and
extracted three times with CH2Cl2, and the combined organic
phases were dried over Na2SO4 and concentrated. The NMR
spectrum of the obtained crude white foam confirmed the
existence of at least two different products. This mixture was
submitted to an aqueous 5 N NaOH treatment in THF in the
presence of Triton B (catalytic amount) during 4 h. This last
reaction allowed us to separate the three different compounds
by flash chromatography: compound 15 was eluted with
MeOH/CH2Cl2 5:95, compound 18 was eluted with MeOH/CH2-
Cl2 10:90, and compound 19 was eluted with MeOH/CH2Cl2
20:80. Acid 18 was finally obtained as a white foam (0.29 g,
61% yield). Rf ) 0.36 (MeOH/CH2Cl2 5:95). RMN 1H (CDCl3)
δ: 1.44 (brs, 47H, t-Bu and -CH2-(CH2)2-CO2H), 1.58-1.75
(m, 6H, -CH2-CH2-CO2H and CO-CH2-CH2-CH2-CO and
BocN-CH2-CH2-CH2-N-(CH2)4-CO2H), 1.82-2.00 (m, 6H,
BocN-CH2-CH2-CH2-NBoc), 2.19 (t, J ) 7.30 Hz, 2H,
-CH2-CO2H), 2.25-2.68 (m, 10H, -(CH2)2-N-CH2-(CH2)3-
CO2H and N-CO-CH2-), 3.00-3.40 (m, 28H, -CH2-N-CO
and -CH2-NBoc), 10.25 (brs, 1H, -OH). MS (FAB+): 1097
(M + H)+. MS (FAB-): 1095 (M - H)-.
1,1′-(1,5-Dioxo-p en ta n e)-bis[4,8-bis(ter t-bu toxyca r bo-
n yl)-11-p en t a n oic Acid -1,4,8,11-t et r a a za cyclot et r a d e-
ca n e] 19. Diester 19 was obtained as a byproduct during the
synthesis of ester 16. After saponification of the whole mixture,
the diacid 19 was obtained as a yellow foam (0.10 g, 20% yield).
Rf ) 0.12 (MeOH/CH2Cl2 5:95). RMN 1H (CDCl3) δ: 1.41 (m,
40H, t-Bu and -CH2-(CH2)2-CO2H), 1.58-1.77 (m, 8H,
-CH2-CH2-CO2H and CO-N-CH2-CH2-CH2-N), 1.79-
2.05 (m, 6H, BocN-CH2-CH2-CH2-NBoc and CO-CH2-
CH2-CH2-CO), 2.26 (m, 4H, -CH2-CO2H), 2.29-2.62 (m,
16H, N-CH2- and CO-CH2-CH2-CH2-CO), 3.07-3.60 (m,
24H, -CH2-N-CO and -CH2-NBoc). MS (FAB+): 1097 (M
+ H)+. MS (FAB-): 1095 (M - H)-.
5-[4,8-Bis(ter t-bu toxyca r bon yl)-11-(4-{[4,8,11-tr is(ter t-
b u t oxyca r b on yl)-1,4,8,11-t et r a a za cyclot et r a d eca n yl]-
m eth yl}ben zyl)-1,4,8,11-tetr a a za cyclotetr a d eca n yl] P en -
ta n oic Acid 25. In a mixture of compounds 4 (0.40 g, 0.80
mmol, 1 equiv) and 5 (0.43 g, 0.80 mmol, 1 equiv) in CH3CN
were added K2CO3 (0.33 g, 2.41 mmol, 3 equiv) and R,R′-
dibromo-p-xylene (0.21 g, 0.80 mmol). The reaction mixture
was refluxed for 8 h under a nitrogen atmosphere and then
allowed to cool to room temperature; the solid carbonate was
removed by filtration. The solvent was evaporated, and the
oily residue was dissolved in EtOAc, then successively washed
with aqueous 5% citric acid and with water, dried over Na2-
SO4, and concentrated. NMR spectrum of the obtained crude
white foam confirmed the presence of at least two different
products. Thus the mixture was submitted to an aqueous 5 N
NaOH treatment in THF in the presence of Triton B (catalytic
amount) during 4 h. This last reaction allowed us to separate
the three different compounds by flash chromatography:
compound 22 was eluted with MeOH/CH2Cl2 5:95, compound
25 was eluted with MeOH/CH2Cl2 10:90, and compound 26
was eluted with MeOH/CH2Cl2 20:80. Acid 25 was finally
obtained as a white foam (0.50 g, 56% yield). Rf ) 0.38 (CH2-
1
Cl2/MeOH 95:5). RMN H (CDCl3) δ: 1.43 (brs, 47H, t-Bu and
-CH2-(CH2)2-CO2H), 1.51-1.74 (m, 6H, -CH2-CH2-CO2H
and Ph-N-CH2-CH2-CH2-N), 1.76-1.96 (m, 4H, BocN-
CH2-CH2-CH2-NBoc), 2.25 (t, 2H, J ) 7.25 Hz, -CH2-
CO2H), 2.27-2.67 (m, 14H, -CH2-N), 3.12-3.42 (m, 20H,
-CH2-NBoc), 3.48 (d, J ) 8.30 Hz, 4H, -CH2-Ph), 7.16 (m,
4H, -Ph-). MS (FAB+): 1103 (M + H)+. (FAB-): 1101 (M -
H)-.
1,1′-Xylyl-bis[4,8-bis(ter t-bu toxyca r bon yl)-11-p en ta n o-
ic Acid -1,4,8,11-tetr a a za cyclotetr a d eca n e] 26. Diester 24
was obtained as a byproduct during the synthesis of ester 23.
After saponification of the whole mixture, diacid 26 was
obtained as a yellow-white foam (0.13 g, 15% yield). Rf ) 0.13
3′-Azid o-3′-d eoxy-5′-O-{5-[4,8-bis(ter t-bu toxyca r bon yl)-
11-{5-oxo-5-[4,8,11-tr is(ter t-bu toxyca r bon yl)-1,4,8,11-tet-
r a a za cyclot et r a d eca n yl]p en t a n oyl}-1,4,8,11-t et r a a za -
cyclotetr a d eca n yl]p en ta n oyl}th ym id in e 20. According to
general procedure A, condensation of AZT (0.02 g, 0.08 mmol)
with acid 18 (0.08 g, 0.08 mmol) afforded after purification
the title product as a white foam (0.04 g, 42% yield). Rf ) 0.46
1
(MeOH/CH2Cl2 5:95). RMN H (CDCl3) δ: 1.39 (brs, 40H, t-Bu
and -CH2-(CH2)2-CO2H), 1.43-1.73 (m, 8H, -CH2-CH2-
CO2H and Ph-N-CH2-CH2-CH2-N), 1.74-2.13 (m, 4H,
BocN-CH2-CH2-CH2-NBoc), 2.23 (m, 8H, -CH2-CO2H),
2.26-2.53 (m, 12H, -CH2-N), 3.08-3.39 (m, 16H, -CH2-
NBoc), 3.46 (brs, 4H, -CH2-Ph), 7.14 (m, 4H, -Ph-). MS
(FAB+): 1103 (M + H)+. MS (FAB-): 1101 (M - H)-.
1
(MeOH/CH2Cl2 1:9). H NMR (CDCl3): 1.40 (s, 27H, t-Bu and
-CH2-CH2-CH2-CO2-Nu), 1.53-2.05 (m, 12H, N-CH2-
CH2-CH2-N and CO-CH2-CH2-CH2-CO and -CH2-CH2-
CO2-Nu), 1.93 (d, J ) 1.00 Hz, 3H, CH3Thy), 2.30-2.57 (m,
14H, CO-CH2-CH2-CH2-CO and -(CH2)2-N-CH2-CH2-
CH2-CH2-CO2-Nu and H-2′), 3.12-3.56 (m, 28H, -CH2-
N-CO and -CH2-NBoc), 4.07 (m, 1H, H-4′), 4.19-4.44 (m,
3H, H-3′ and H-5′), 6.09 (t, J ) 6.20 Hz, 1H, H-1′), 7.21 (d, J
) 1.10 Hz, 1H, H-6Thy), 9.15 (brs, 1H, NH). MS (FAB+): 1346
(M + H)+.
3′-Azid o-3′-d eoxy-5′-O-{5-[4,8-bis(ter t-bu toxyca r bon yl)-
11-(4-{[4,8,11-tr is(ter t-bu toxyca r bon yl)-1,4,8,11-tetr a a za -
cyclot e t r a d e ca n yl]m e t h yl}b e n zyl)-1,4,8,11-t e t r a a za -
cyclotetr a d eca n yl]p en ta n oyl}th ym id in e 27. According to
general procedure A, condensation of AZT (0.04 g, 0.15 mmol)
with acid 25 (0.17 g, 0.15 mmol) afforded, after purification,
the title product as a white solid (0.07 g, 35% yield). Rf ) 0.42
1
3′-Azid o-3′-d eoxy-5′-O-(5-{1,4,8-tr ih yd r och lor id e-11-[5-
oxo-5-(4,8,11-tr ih yd r och lor id e-1,4,8,11-tetr a a za cyclotet-
r adecan yl)pen tan oyl]-1,4,8,11-tetr aazacyclotetr adecan yl}-
(MeOH/CH2Cl2 1:9). H NMR (CDCl3): 1.42 (s, 47H, t-Bu and
-CH2-CH2-CH2-CO2Nu), 1.44-1.72 (m, 6H, -CH2-CH2-
CO2Nu and Ph-CH2-N-CH2-CH2-CH2-N), 1.74-2.02 (m,

240 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Dessolin et al.
7H, CH3Thy and BocN-CH2-CH2-CH2-NBoc), 2.26-2.70 (m,
10H, -(CH2)2-N-CH2-CH2-CH2-CH2-CO2Nu and H-2′),
3.17-3.46 (m, 20H, -CH2-NBoc), 3.49-3.70 (d, J ) 7.60 Hz,
4H, -CH2-Ph-CH2-), 4.03 (m, 1H, H-4′), 4.15-4.34 (m, 3H,
H-3′ and H-5′), 6.04 (t, J ) 6.30 Hz, 1H, H-1′), 7.15 (m, 5H,
-Ph- and H-6Thy), 8.90 (brs, 1H, NH). MS (FAB+): 1352 (M
+ H)+.
3′-Azid o-3′-d eoxy-5′-O-[5-(1,4,8,11-tetr a h yd r och lor id e-
11-{4-[(1,4,8,11-t et r a h yd r och lor id e-1,4,8,11-t et r a a za cy-
clotetr adecan yl)m eth yl]ben zyl}-1,4,8,11-tetr aazacyclotet-
r adecan yl)pen tan oyl]th ym idin e 28. According to the general
carbamate deprotection procedure B, reaction of compound 27
(0.06 g, 0.04 mmol) afforded the title compound 28 as a white
solid, in quantitative yield (0.04 g, 97% yield). Rf ) 0.10
(MeOH/CH2Cl2 1:9). 1H NMR (D2O): 1.59 (m, 2H, -CH2-CH2-
CO2Nu), 1.70 (m, 2H, -CH2-CH2-CH2-CO2Nu), 1.78 (brs,
1H, CH3Thy), 2.11 (m, 8H, -N-CH2-CH2-CH2-N-), 2.29-
2.60 (m, 4H, -CH2-CO2Nu and H-2′), 3.08-3.97 (m, 34H,
-CH2-N), 4.08 (m, 1H, H-4′), 4.21-4.58 (m, 7H, -CH2-Ph-
CH2- and H-3′ and H-5′), 6.08 (m, 1H, H-1′), 7.42 (brs, 1H,
H-6Thy), 7.56 (brs, 4H, -Ph-). MS (FAB+): 852 (M + H)+.
cyclotetr a d eca n yl]ca r bon yl}ben zoyl)-1,4,8,11-tetr a a za -
cyclotetr a d eca n yl]p en ta n oyl}th ym id in e 34. According to
general procedure A, condensation of AZT (0.03 g, 0.11 mmol)
with acid 33 (0.13 g, 0.11 mmol) afforded, after purification,
the title product as a white foam (0.10 g, 66% yield). Rf ) 0.44
1
(MeOH/CH2Cl2 1:9). H NMR (CDCl3): 1.42 (s, 47H, t-Bu and
-CH2-CH2-CH2-CO2Nu), 1.45-1.70 (m, 6H, -CH2-CH2-
CO2-Nu and Ph-CO-N-CH2-CH2-CH2-N), 1.72-2.00 (m,
7H, CH3Thy and BocN-CH2-CH2-CH2-NBoc), 2.12-2.64 (m,
10H, -(CH2)2-N-CH2-CH2-CH2-CH2-CO2Nu and H-2′),
3.03-3.50 (m, 24H, BocN-CH2-CH2-N-CO-Ph and -CH2-
NBoc), 3.52-3.70 (m, 4H, BocN-CH2-CH2-CH2-N-CO-
Ph), 3.99 (m, 1H, H-4′), 4.12-4.44 (m, 3H, H-3′ and H-5′), 6.01
(t, J ) 6.20 Hz, 1H, H-1′), 7.39 (brs, 1H, H-6Thy), 7.39 (brs,
1H, CO-Ph-CO), 8.86 (brs, 1H, NH). MS (FAB+): 1380 (M
+ H)+.
3′-Azid o-3′-d eoxy-5′-O-[5-(1,4,8-tr ih yd r och lor id e-11-{4-
[(4,8,11-tr ih yd r och lor id e-1,4,8,11-tetr a a za cyclotetr a d e-
ca n yl)ca r bon yl]ben zoyl}-1,4,8,11-tetr a a za cyclotetr a d e-
ca n yl)p en t a n oyl]t h ym id in e 35. According to the general
carbamate deprotection procedure B, reaction of compound 34
(0.06 g, 0.05 mmol) afforded the title compound 35 as a white
solid in quantitative yield (0.04 g, 97% yield). Rf ) 0.08 (MeOH/
CH2Cl2 1:9). 1H NMR (D2O): 1.60 (m, 2H, -CH2-CH2-CO2-
Nu), 1.65-1.90 (m, 5H, CH3Thy and -CH2-CH2-CH2-
CO2Nu), 2.01 (m, 4H, N-CH2-CH2-CH2-N), 2.15 (m, 4H,
Ph-CO-N-CH2-CH2-CH2-N), 2.36-2.68 (m, 4H, -CH2-
CO2Nu and H-2′), 3.05-3.99 (m, 34H, -CH2-N), 4.09 (m, 1H,
H-4′), 4.24-4.44 (m, 3H, H-3′ and H-5′), 6.11 (m, 1H, H-1′),
7.42 (brs, 1H, H-6Thy), 7.54 (brs, 4H, CO-Ph-CO). MS
(FAB+): 880 (M + H)+.
1,1′-Ter eph th aloyl-bis[4,8,11-tr is(ter t-bu toxycar bon yl)-
1,4,8,11-tetr a a za cyclotetr a d eca n e] 29. Compound 29 was
obtained as a byproduct during the synthesis of ester 30 and
was purified after the saponification of the whole mixture to
give a brown-white solid (0.21 g, 27% yield). Rf ) 0.56 (MeOH/
1
CH2Cl2 1:9). H NMR (CDCl3): 1.42 (brs, 54H, t-Bu), 1.74 (m,
8H, N-CH2-CH2-CH2-N-), 3.08-3.75 (m, 32H, CO-N-
CH2-), 7.40 (brs, 4H, CO-Ph-CO). MS (FAB+): 1131 (M +
H)+.
5-[4,8-Bis(ter t-bu toxyca r bon yl)-11-(4-{[4,8,11-tr is(ter t-
b u t oxyca r b on yl)-1,4,8,11-t et r a a za cyclot et r a d eca n yl]-
ca r b on yl}b en zoyl)-1,4,8,11-t et r a a za cyclot et r a d eca n yl]
P en ta n oic Acid 32. In a mixture of compound 4 (0.35 g, 0.70
mmol, 1 equiv) and 5 (0.37 g, 0.70 mmol, 1 equiv) in the
biphasic CH2Cl2/NaHCO3 was added terephthaloyl dichloride
(0.14 g, 0.70 mmol, 1 equiv). The reaction mixture was stirred
for 30 min until disappearance of the starting tetraazamac-
rocycles. The aqueous layer was partitioned and extracted
three times with CH2Cl2. The combined organic phases were
dried over Na2SO4 and concentrated. NMR spectrum of the
obtained crude white foam confirmed the presence of at least
two different products. This mixture was submitted to an
aqueous 5 N NaOH treatment in THF in the presence of Triton
B (catalytic amount) during 4 h. This last reaction allowed to
separate the three different compounds by flash chromatog-
raphy: compound 29 was eluted with MeOH/CH2Cl2 5:95,
compound 32 was eluted with MeOH/CH2Cl2 10:90, and
compound 33 was eluted with MeOH/CH2Cl2 20:80. Acid 32
was finally obtained as a white foam (0.38 g, 48% yield). Rf )
1,1′-(1,5-Dioxo-p en t a n e)-b is[4,8,11-t r ih yd r och lor id e-
1,4,8,11-tetr a a za cyclotetr a d eca n e] 36. According to the
general carbamate deprotection procedure B, reaction of
compound 15 (0.06 g, 0.05 mmol) afforded the title compound
36 as a yellow solid in quantitative yield (0.02 g, 97% yield).
Rf ) 0.05 (MeOH/CH2Cl2 1:9). 1H NMR (D2O): 1.82 (m, 2H,
CO-CH2-CH2-CH2-CO), 1.89-2.43 (m, 8H, N-CH2-CH2-
CH2-N), 2.53 (t, J ) 7.20 Hz, 4H, CO-CH2-CH2-CH2-CO),
3.26-4.05 (m, 32H, -CH2-N). MS (FAB+): 497 (M + H)+.
1,1′-Xylyl-bis[4,8,11-tr ih yd r och lor id e-1,4,8,11-tetr a a za -
cyclotetr a d eca n e] 37. According to the general carbamate
deprotection procedure B, reaction of compound 22 (0.08 g, 0.07
mmol) afforded the title compound 37 as a white solid in
quantitative yield (0.03 g, 95% yield). Rf ) 0.05 (MeOH/CH2-
Cl2 1:9). 1H NMR (D2O): 2.12 (brs, 8H, N-CH2-CH2-CH2-
N), 2.97-3.68 (m, 32H, N-CH2-), 4.39 (s, 4H, Ph-CH2-), 7.29
(s, 4H, -Ph-). MS (FAB+): 503 (M + H)+.
1,1′-Ter ep h th a loyl-bis[4,8,11-tr ih yd r och lor id e-1,4,8,11-
tetr a a za cyclotetr a d eca n e] 38. According to the general
carbamate deprotection procedure B, reaction of compound 29
(0.07 g, 0.06 mmol) afforded the title compound 38 as a slightly
yellow solid in quantitative yield (0.03 g, 92% yield). Rf ) 0.05
1
0.29 (CH2Cl2/MeOH 95:5). RMN H (CDCl3) δ: 1.45 (brs, 47H,
t-Bu and -CH2-(CH2)2-CO2H), 1.49-1.78 (m, 6H, -CH2-
CH2-CO2H and Ph-CO-N-CH2-CH2-CH2-N), 1.79-2.05
(m, 4H, BocN-CH2-CH2-CH2-NBoc), 2.23 (m, 4H, -CH2-
CO2H and BocN-CH2-CH2-N-(CH2)4-CO2H), 2.50 (m, 4H,
-CH2-N-CH2-(CH2)3-CO2H), 3.10-3.58 (m, 24H, -CH2-
NBoc and BocN-CH2-CH2-CH2-N-CO-Ph), 3.57-3.83 (m,
4H, BocN-CH2-CH2-N-CO-Ph), 7.44 (m, 4H, CO-Ph-CO).
MS (FAB+): 1131 (M + H)+. MS (FAB-): 1129 (M - H)-.
1
(MeOH/CH2Cl2 1:9). H NMR (CDCl3): 2.05 (m, 4H, N-CH2-
CH2-CH2-N), 2.19 (m, 4H, CO-N-CH2-CH2-CH2-N-CO),
2.82-4.00 (m, 32H, N-CH2-), 7.56 (brs, 4H, CO-Ph-CO).
MS (FAB+): 531 (M + H)+.
Ack n ow led gm en t. We thank E. Doria for her
excellence in HIV screening experiments. INSERM and
ANRS are acknowledged for financial support.
1,1′-Ter ep h th a loyl-bis[4,8-bis(ter t-bu toxyca r bon yl)-11-
p en ta n oic a cid -1,4,8,11-tetr a a za cyclotetr a d eca n e] 33. Di-
ester 31 was obtained as a byproduct during the synthesis of
ester 30. After saponification of the whole mixture, diacid 33
was obtained as a yellow foam (0.13 g, 16% yield). Rf ) 0.13
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