Novel monocyclam derivatives as HIV entry inhibitors: Design, synthesis, anti-HIV evaluation, and their interaction with the CXCR4 co-receptor

Article abstract of DOI:10.1002/cmdc.201000124

The CXCR4 receptor has been shown to interact with the human immunodeficiency virus (HIV) envelope glycoprotein gp120, leading to fusion of viral and cell membranes. Therefore, ligands that can attach to this receptor represent an important class of therapeutic agents against HIV, thus inhibiting the first step in the cycle of viral infection: the virus-cell entry/ fusion. Herein we describe the in silico design, synthesis, and biological evaluation of novel monocyclam derivatives as HIV entry inhibitors. In vitro activity testing of these compounds in cell cultures against HIV strains revealed EC50 values in the low micromolar range without cytotoxicity at the concentrations tested. Docking and molecular dynamics simulations were performed to predict the binding interactions between CXCR4 and the novel monocyclam derivatives. A binding mode of these compounds is proposed which is consistent with the main existing site-directed mutagenesis data on the CXCR4 coreceptor. Moreover, molecular modeling comparisons were performed between these novel monocyclams, previously reported non-cyclam compounds from which the monocyclams are derived, and the well-known AMD3100 bicyclam CXCR4 inhibitors. Our results suggest that these three structurally diverse CXCR4 inhibitors bind to overlapping but not identical amino acid residues in the transmembrane regions of the receptor.

Full text of DOI:10.1002/cmdc.201000124

MED
DOI: 10.1002/cmdc.201000124
Novel Monocyclam Derivatives as HIV Entry Inhibitors:
Design, Synthesis, Anti-HIV Evaluation, and Their
Interaction with the CXCR4 Co-receptor
Sofia Pettersson,[a] Violeta I. Pꢀrez-Nueno,[a] Maria Pau Mena,[b] Bonaventura Clotet,[b]
Josꢀ A. Estꢀ,[b] Josꢀ I. Borrell,*[a] and Jordi Teixidꢁ[a, c]
The CXCR4 receptor has been shown to interact with the
human immunodeficiency virus (HIV) envelope glycoprotein
gp120, leading to fusion of viral and cell membranes. There-
fore, ligands that can attach to this receptor represent an im-
portant class of therapeutic agents against HIV, thus inhibiting
the first step in the cycle of viral infection: the virus–cell entry/
fusion. Herein we describe the in silico design, synthesis, and
biological evaluation of novel monocyclam derivatives as HIV
entry inhibitors. In vitro activity testing of these compounds in
cell cultures against HIV strains revealed EC50 values in the low
micromolar range without cytotoxicity at the concentrations
tested. Docking and molecular dynamics simulations were per-
formed to predict the binding interactions between CXCR4
and the novel monocyclam derivatives. A binding mode of
these compounds is proposed which is consistent with the
main existing site-directed mutagenesis data on the CXCR4 co-
receptor. Moreover, molecular modeling comparisons were
performed between these novel monocyclams, previously re-
ported non-cyclam compounds from which the monocyclams
are derived, and the well-known AMD3100 bicyclam CXCR4 in-
hibitors. Our results suggest that these three structurally di-
verse CXCR4 inhibitors bind to overlapping but not identical
amino acid residues in the transmembrane regions of the re-
ceptor.
Introduction
According to the Joint United Nations Programme on HIV/
AIDS (UNAIDS) and World Health Organization (WHO), a total
of 33.2 million [30.6–36.1 million] people were living with
human immunodeficiency virus (HIV) in 2007.[1] Studies in HIV
biology have provided deep knowledge of the molecular
events involved in the HIV life cycle, which consist of several
steps: viral entry, reverse transcription, integration, gene ex-
pression, gene assembly, budding and maturation.[2–4] Current
antiretroviral therapies (ARTs) against HIV are generally based
on the combination of reverse transcriptase and protease in-
hibitors which is known as highly active antiretroviral therapy
(HAART). At the present time there are nucleoside and non-nu-
cleoside reverse transcriptase inhibitors (NRTI, NNRTI), protease
inhibitors (PI), a fusion inhibitor, an entry inhibitor and an inte-
grase inhibitor available for the treatment of HIV infection.[5]
Despite the large number of drugs available, there are several
concerns about antiretroviral regimens. The drugs can have se-
rious side effects, regimens can be complicated requiring pa-
tients to take several pills at various times during the day, drug
resistance, latent viral reservoirs and drug induced toxic effects
that compromise effective viral control can be developed.
Fusion/entry inhibitors have been shown to be effective in pa-
tients harboring resistance to the NRTI, NNRTI, and PI classes.[6]
Therefore, there is considerable interest in developing novel li-
gands that are resistant to the currently used drugs or new
agents belonging to new classes that further heighten the ef-
fectiveness and durability of HIV therapy.[7,8]
cyclam was AMD3100 (EC50 =0.001 mgmLÀ1) in which the two
cyclam moieties are tethered by a 1,4-phenylenebis(methylene)
bridge. However, AMD3100 has been shown to exhibit poor
oral absorption and toxicity, which are related to its high posi-
tive charge at physiological pH. To improve these characteris-
tics, analogous compounds with fewer basic amine groups
have been developed, such as monocyclam derivatives
(AMD3465 and AMD3451) and non-cyclam derivatives
(AMD070 and 1) (Figure 1).[13–15]
Results and Discussion
Rational design of monocyclam derivatives
It has been proven that the eight amino groups present in
AMD3100 are not essential for the interaction with the CXCR4
co-receptor.[13,16] Furthermore, previous work in our group led
[a] Dr. S. Pettersson, Dr. V. I. Pꢀrez-Nueno, Dr. J. I. Borrell, Dr. J. Teixidꢁ
Grup d’Enginyeria Molecular, Institut Quꢂmic de Sarriꢃ
Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona (Spain)
Fax: (+34)932 056 266
E-mail: jose.borrell@iqs.url.edu
[b] Dr. M. P. Mena, Dr. B. Clotet, Dr. J. A. Estꢀ
Retrovirology Laboratory IrsiCaixa
Hospital Universitari Germans Trias i Pujol
Universitat Autꢄnoma de Barcelona, 08916 Badalona (Spain)
[c] Dr. J. Teixidꢁ
Current address: Matarꢁ-Maresme TecnoCampus Foundation
Edifici TecnoCampus v. 1.0, Vallveric
85 1a Planta Local 3i5, 08304 Matarꢁ Barcelona (Spain)
Bicyclams were the first low molecular weight compounds
with a specific interaction with CXCR4.[9–12] The most potent bi-
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Monocyclam HIV Entry Inhibitors
can be obtained in high yield by alkylation of the non-protect-
ed amino group in 12 with 10 in the presence of potassium
carbonate (Scheme 1). These two precursors were obtained by
previously reported methods. Compound 12 was synthesized
by the N1-Boc protection of cyclam 11.[20] Bromoaldehyde 10
was achieved by reduction of the aldehyde 8 with sodium bor-
ohydride in methanol followed by deprotection of the latent
formyl group in hydrochloric acid and bromination with NBS
and triphenylphosphine.[21] Finally a stepwise reductive amina-
tion of the key intermediate 5 with the corresponding primary
amines 6 and 7 followed by cleavage of the tert-butyl carba-
mates in acidic conditions afforded monocyclams 2 and 3
(Scheme 1).
Figure 1. Structure of bicyclam AMD3100, monocyclams AMD3451 and
AMD3465, and non-cyclam CXCR4 inhibitors AMD070 and 1.
Biological evaluation
We determined the anti-HIV activity of these novel monocy-
clams and compared it with the values obtained for the
known CXCR4 inhibitors AMD3465 and 1. EC50 values of mono-
cyclams 2 and 3 were 0.02 and 0.06 mgmLÀ1, respectively
to the discovery of a series of non-cyclam CXCR4 inhibitors
with 1 being the most active thereof, with only four nitrogen
atoms.[17] Based on this evi-
dence, we designed a library of
new monocyclam derivatives
which combine the cyclam ring
with the diamines used in our
previous work.[17] A selection of
the presumably most active
compounds (2 and 3) was ac-
complished with published vir-
tual screening methods.[18] Here,
we report on the synthesis,
binding mode studies and anti-
Figure 2. Scheme showing the building blocks for synthesis of monocyclams 2 and 3: brominated derivative 4 or
aldehyde 5 and the corresponding amine 6 or 7.
HIV activity of these two novel
monocyclam derivatives.
Chemistry
Described synthetic procedures
for monocyclam derivatives use
the alkylation of an amine, gen-
erally a secondary amine, with a
brominated cyclam derivative
(compound 4).[19] However, the
use of 4 in the alkylation of pri-
mary amines 6 and 7 did not
lead to the target monocyclam
derivatives 2 and 3 due to poly-
alkylation side reactions. There-
fore, we designed a new syn-
thetic strategy to obtain the
target substituted monocy-
clams. The retrosynthetic analy-
sis of these compounds afford-
ed the corresponding primary
amine and a novel key inter-
mediate, the aldehyde deriva-
tive 5 (Figure 2). This compound
Scheme 1. Synthetic route for monocyclam derivatives 2 and 3. Reagents and conditions: a) NaBH4, MeOH, room
temperature, 6 h; b) HCl/H2O, room temperature, 2 h; c) NBS, PPh3, CH2Cl2, reflux, 2 h; d) Boc2O, CH2Cl2, reflux,
6.5 h; e) K2CO3, MeCN, reflux, 16 h; f) 6, Ti(OiPr)4, EtOH, room temperature, 36 h, or 7, MeOH, MW, 1008C, 3 h;
g) NaBH4, room temperature, 12 h; h) HCl/Et2O, room temperature, 12 h.
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3 did not interfere with the
staining using mAbs specific for
CD45 or CD4 (Figure 3b). Taken
together, these results suggest
that compounds 1–3 are specific
antagonists of CXCR4.
Table 1. Anti-HIV activity, cytotoxicity, and Gibbs free energy of selected hits.
[a]
[b]
[c]
[d]
Compd
EC50 [mgmLÀ1
]
CC50 [mgmLÀ1
]
DGexp [kcalmolÀ1
]
DGcalcd [kcalmolÀ1
]
1
2
3
0.008Æ0.001
0.02Æ0.006
0.06Æ0.005
0.004
>25
>25
>25
>25
À10.5
À10.3
À9.7
À10.6
À9.7
À9.9
AMD3465[50]
À11.0
À11.4
[a] Concentration required to inhibit HIV-1-induced cell death by 50% as evaluated with the MTT method in
MT-4 cells; values represent the mean ÆSD of at least two independent evaluations done in triplicate. [b] Con-
centration required to induce 50% death of non-infected MT-4 cells as evaluated with the MTT method. [c] Ex-
perimental Gibbs free energy computed as DGꢀRTln(EC50);[51] R=0.001988, T=298.15 K. [d] Theoretical Gibbs
free energy obtained from the AutoDock Vina docking affinity.[52]
Theoretical binding mode
studies: docking and molecular
dynamics refinement
A
theoretical binding mode
study was performed in order to
predict the binding interactions
(Table 1). These compounds showed potent antiviral activity,
but are 10-fold less active than 1 and AMD3465. We expected
higher antiviral because they combine the best from each
family, a cyclam moiety (from AMD3100) and a pipecoline
(from compound 1). Therefore we performed theoretical bind-
ing mode studies that we describe herein.
of the synthesized monocyclams with the CXCR4 receptor
pocket. Compounds 2 and 3 were docked onto the site-direct-
ed mutagenesis (SDM)-defined CXCR4 binding pocket[16,22–30]
using AutoDock Vina.[31,32] The highest-affinity binding modes
obtained are shown in Figure 4a and 4b, respectively. These
affinities are listed in Table 1, as well as how they correlate
with antiviral activities. The Gibbs free energy (DG) is used to
compare theoretical and experimental activities. The key SDM
residues for monocyclam, non-cyclam, and bicyclam interac-
tions[16,30] are shown as orange sticks. Ligand interactions were
analyzed in MOE[33] using hydrogen bond, ionic, and solvent
contacts with a minimum qualifying score of 5%, and non-
bonded contacts within a maximum distance of 4.5 ꢃ. In the
high-affinity binding conformation of 2, a backbone hydrogen
bond donor was found between the protonated pipecoline
and the Tyr7 oxygen and non-bonded interactions were found
between the ligand and Ile6, Tyr7, Thr8, Ser9, Phe36, Leu41,
Ala95, Ala98, Phe104, Gly105, Asn106, Val112, Tyr190, Asp262,
To verify the specificity of 1–3 for CXCR4, their capacity to
interfere with the staining of monoclonal antibodies (mAbs)
against CXCR4, CD45 (control receptor), or CD4 (HIV receptor)
was tested. MT-4 cells were stained with mAbs alone or to-
gether with study compounds or control compounds with
known epitope specificities. Compounds 1, 2, and 3 inhibited
the staining of CXCR4 on cells with mAb 12G5-PE in a dose-de-
pendent manner, with IC50 values of 0.06, 0.03, and
0.11 mgmLÀ1, respectively (Figure 3a and 3b). The staining of
CXCR4 was also inhibited by the CXCR4 antagonist AMD3100
with an IC50 value of 0.02 mgmLÀ1 and by the unstained 12G5
antibody (IC50 =14.7 mgmLÀ1). Conversely, AMD3100, 1, 2, and
Figure 3. a) Inhibition of mAb 12G5-PE (anti-CXCR4) staining on MT-4 cells by various compounds (CXCR4 antagonist AMD3100 at 0.04 mgmLÀ1, 1 at
0.2 mgmLÀ1, 2 at 0.2 mgmLÀ1, and 3 at 1 mgmLÀ1); the marker was defined as 1% of the control isotype. b) Dose–response curves of the inhibition of staining
~
&
*
^
with mAb anti-CXCR4 12G5-PE by the CXCR4 antagonist AMD3100 ( ), 1 ( ), 2 ( ), and 3 ( ). Staining with anti-CD4 and anti-CD45 was not inhibited by the
compounds; data represent the mean ÆSD of three independent experiments.
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Monocyclam HIV Entry Inhibitors
Figure 4. Potential binding modes of 2 and 3. At top, the highest-affinity binding conformations for a) 2–CXCR4 and b) 3–CXCR4 obtained by docking with
AutoDock Vina. c) 2–CXCR4 and d) 3–CXCR4 binding modes obtained by MD simulations using Amber 8. For clarity, all the intracellular and extracellular loops
are omitted, and only polar hydrogen atoms are shown in structures. The key CXCR4 SDM residues are shown as orange sticks. At the bottom, 2D diagram of
the e) CXCR4–2 and f) CXCR4–3 ligand interactions found for the MD binding modes obtained. The diagram depicts the hydrogen bond, ionic, solvent and
non-bonded contacts. Contact distances are also labeled. The CXCR4 SDM residues (Glu288, Asp262, His281, Tyr 45) with the highest level of the effect of the
mutation on the binding of monocyclam compounds (such as AMD3465, AMD8721, AMD8899, AMD3529 and AMD3389) appear in the binding modes ob-
tained. Other CXCR4 SDM residues (His113, Tyr255, Ile284) with lower levels of the effect of the mutation on the binding of monocyclam compounds also
take part in the binding conformations calculated.
Trp283, Tyr284, Ser285, Glu288 and Phe292. On the other
hand, the high-affinity binding conformation of 3 showed two
ionic interactions between the non-cyclam protonated secon-
dary amine and the Asp262 oxygen and the cyclam ring pro-
tonated N4 and the Glu288 oxygen, respectively. Also, non-
bonded interactions were found between the ligand and Ile6,
Tyr7, Thr8, Ser9, Glu31, Phe36, Leu41, Ala95, Asn106, Val112,
Tyr190, Asp262, His281, Lys282, Trp283, Tyr284, Ser285, Glu288
and Phe292. Docking results agree with some of the CXCR4
SDM residues with the highest level of the effect of the muta-
tion on the binding of monocyclam compounds,[16] such as
AMD3465, AMD3529 (both having quite similar structures to
our monocyclam designed compounds), AMD8721, AMD8899
or AMD3389. These compounds seem to interact especially
with Glu288, Asp262, and His281, which are interactions that
also appear in our monocyclam designed compounds.
These high-affinity docked poses obtained were used as the
starting structures for molecular dynamics (MD) refinement
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using Amber 8.[34] The binding modes obtained are shown in
Figure 4c and 4d, respectively. The ligand interactions were
also analyzed in MOE using the same parameters. They are
shown in Figure 4e and 4 f, respectively. In the binding confor-
mation of 2 a side chain hydrogen bond acceptor was found
between the cyclam ring N8 and the Asn106 amino group, an
ionic interaction was found between the pipecoline protonat-
ed nitrogen and the Glu288 oxygen and non-bonded interac-
tions were found between the ligand and Ile6, Tyr7, Thr8,
Tyr21, Phe36, Leu41, Phe104, Gly105, Asn106, Val112, His113,
Tyr190, Asp262, Tyr284, Ser285 and Glu288. In the binding con-
formation of 3, two side chain hydrogen bond donors were
found between the morpholine protonated nitrogen and the
Ser9 oxygen and the non-cyclam protonated secondary amine
and the Asp262 oxygen, a side chain hydrogen bond acceptor
was found between the morpholine oxygen and the Ser285
hydroxy group. Non-bonded interactions were found between
the ligand and Tyr7, Thr8, Ser9, Tyr45, Ala95, Val112, His113,
Tyr190, Asp262, Lys271, His281, Lys282, Ser285, Glu288, and
Phe292.
cyclam protonated secondary amine and Tyr7 oxygen, a side
chain hydrogen bond acceptor between the piperidine nitro-
gen and Ser9 hydroxy group and two ionic interactions be-
tween the non-cyclam protonated secondary amine and
Asp262 oxygen and the cyclam ring protonated N11 and
Glu288 oxygen, respectively. Non-bonded interactions were
found between AMD3465 and Ile6, Tyr7, Thr8, Ser9, Leu41,
Tyr45, Ala95, Ala98, Asn106, Val112, Tyr190, Lys282, Trp283,
Ile284, Ser285, Glu288 and Phe292. The high-affinity binding
conformation of AMD3451 showed an ionic interaction be-
tween the cyclam ring protonated N11 and Glu288. Non-
bonded interactions were found between AMD3451 and Glu2,
Ile6, Tyr7, Thr8, Ser9, Phe36, Leu41, Ala98, Phe104, Gly105,
Asn106, Val112, Tyr190, Asp262, Lys271, Lys282, Trp283, Ile284,
Ser285 and Glu288. In the high-affinity binding conformation
of AMD3100, two ionic interactions were found between the
cyclam ring protonated N11 and Asp262 oxygens. Non-bonded
interactions were found between AMD3100 and Ile6, Tyr7,
Thr8, Ser9, Cys109, Val112, His113, Asn176, Arg188, Tyr190,
Ile284, Ser285 and Glu288. In the case of 1, the high-affinity
binding conformation showed a side chain hydrogen bond
donor between the non-cyclam protonated secondary amine
and Ser285 oxygen. Two ionic interactions were found, one be-
tween the non-cyclam protonated secondary amine and the
Glu288 oxygen and the other between the pipecoline proton-
ated nitrogen and the other Glu288 oxygen. Non-bonded in-
teractions were found between 1 and Glu2, Ile6, Tyr7, Thr8,
Phe36, Leu41, Ala95, Ala98, Asn106, Val112, His113, Tyr190,
Tyr255, Ile284, Ser285, Glu288 and Phe292. Finally, the high-af-
finity binding conformation of AMD070 showed three side
chain hydrogen bond donors: between the protonated pri-
mary amine and Tyr255 oxygen, the protonated primary amine
and one Glu288 oxygen and the benzimidazole NH and the
other Glu288 oxygen. AMD070 also showed a side chain hy-
drogen bond acceptor between the benzimidazole NH and the
Ser285 hydroxy group and two ionic interactions between the
protonated tertiary amine and both Glu288 oxygen atoms, re-
spectively. Non-bonded interactions were found between
AMD070 and Tyr7, Thr8, Phe36, Leu41, Tyr45, Val112, His113,
Tyr190, Tyr255, Lys282, Ile284, Ser285, Glu288 and Phe292.
Notably, all these molecules interact with overlapping but
not identical residues in the binding pocket of the receptor.
Depending on the compound structures (monocyclam, bicy-
clam, or non-cyclam), their interaction with the key CXCR4
SDM residues can vary. However the main interactions seem to
be conserved for monocyclams (Asp262, His281, Glu288), non-
cyclams (Glu288) and bicyclams (Glu288). In this way, some key
SDM residues (Asp262, His281, Ala175, Ile259, Ile284) seem to
affect the binding of monocyclams at a higher level than they
do for non-cyclams or bicyclams.[16,30]
As for the high-affinity docked conformations, the MD re-
finement results agree with some of the CXCR4 SDM residues
with the highest level of the effect of the mutation on the
binding of monocyclam compounds (Glu288, Asp262, His281,
Trp94, Tyr 45, Tyr116, Asp171) and also other SDM residues
with lower levels of the effect of the mutation on the binding
of monocyclam compounds (Ala175, His113, Glu200, Tyr255,
Ile259, His203, Val196, Ile284, and Tyr121, listed from the upper
to lower level).[16,30] The binding conformations obtained are
also consistent with one of the three binding modes proposed
for monocyclam AMD3465 by Wong et al.,[30] in which there is
an aromatic interaction between His281 and the pyridine ring,
Glu288 makes an ionic interaction with the protonated diben-
zylamine, and the double protonated cyclam ring interacts
with Asp262. However, the binding modes obtained cannot ac-
count for all of the amino acid residues that have been shown
to affect the binding of monocyclam inhibitors in SDM studies.
In general, compounds 2 and 3 appear to mimic the general
binding mode of AMD3100, but in a mode where the non-
cyclam part picks up novel interactions especially with residues
located more toward the extracellular end of transmembrane
(TM)-VI and TM-VII (Asp262, Glu288, His281, Tyr255, Ile259,
Ile284), making essential interactions at each end of the main
ligand binding pocket of the CXCR4 receptor.
Comparison of the theoretical binding modes of bicyclam,
monocyclam, and non-cyclam CXCR4 inhibitors
The binding modes of 2 and 3 were compared with the bind-
ing modes of other reported monocyclams (AMD3465,
AMD3451), the bicyclam AMD3100 and non-cyclams 1 (previ-
ously reported by our group and from which 2 is derived), and
AMD070. Docking results are shown in Figure 5a. The ligand
interactions for the high-affinity binding conformations ob-
tained for 2 and 3 have been described in detail previously. In
the case of AMD3465, the high-affinity binding conformation
showed a backbone hydrogen bond donor between the non-
MD-refined binding poses were compared for monocyclams
2, 3, non-cyclam 1, and the bicyclam AMD3100. They are
shown in Figure 5b. Ligand interactions were analyzed in MOE.
Figure 5c, 5d, and 5e compare the ligand interactions of a
monocyclam (compound 3), non-cyclam (compound 1) and bi-
cyclam (AMD3100), respectively. In the binding conformation
of 3, two side chain hydrogen bond donors were found be-
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Monocyclam HIV Entry Inhibitors
Figure 5. Comparison of the theoretical binding modes of bicyclam, monocyclam and non-cyclam CXCR4 inhibitors. a) Highest-affinity docking poses for mon-
ocyclams 2 (blue), 3 (pink), AMD3465 (yellow), and AMD3451 (gray), non-cyclams 1 (green) and AMD070 (white), and bicyclam AMD3100 (red) obtained using
AutoDock Vina. b) MD binding poses for monocyclams 2 (blue) and 3 (pink), non-cyclam 1 (green) and bicyclam AMD3100 (red) obtained using Amber 8. At
right, ligand interaction 2D diagram comparing the c) monocyclam 3, d) non-cyclam 1, and e) bicyclam AMD3100 interactions found. Hydrogen bonds and
ionic interactions are shown in green and purple dots, respectively. Interaction distances are also labeled. The legend for the diagrams is the same as shown
in Figure 4e.
tween the morpholine protonated nitrogen and the Ser9
oxygen, the non-cyclam protonated secondary amine and the
Asp262 oxygen. A side chain hydrogen bond acceptor was
found between the morpholine oxygen and the Ser285 hy-
droxy group. Non-bonded interactions were found between
the ligand and Tyr7, Thr8, Ser9, Tyr45, Ala95, Val112, His113,
Tyr190, Asp262, Lys271, His281, Lys282, Ser285, Glu288 and
Phe292. For non-cyclam 1, the binding conformation showed
two side chain hydrogen bond donors between the non-
cyclam protonated secondary amine and the Glu288 oxygen
and the other non-cyclam protonated secondary amine and
the Ser285 oxygen. Non-bonded interactions were found be-
tween the ligand and Ile6, Tyr7, Thr8, Ser9, Phe36, Leu41,
Ala95, Val112, His113, Asn176, Arg188, Tyr190, Tyr255, Ser285
and Glu288. Finally, for bicyclam AMD3100, a side chain hydro-
gen bond donor was found between the cyclam ring protonat-
ed N11 and the Ser285 oxygen and two ionic interactions be-
tween the cyclam ring N1 and the Glu288 oxygen and the op-
posite cyclam ring protonated N11 and the Glu288 oxygen.
Non-bonded interactions were found between the ligand and
Ile6, Tyr7, Thr8, Arg30, Phe36, Cis109, Val112, His113, Asn176,
Arg188, Tyr190, Tyr255, Lys282, Ile284, Ser285 and Glu288.
Although these are the first molecular modeling ligand inter-
action studies for 1, 2 and 3, it is interesting to note that the
binding interactions of AMD3100, AMD3465 and AMD3451 de-
scribed here agree with those from previous reports.[16,22,26,30]
None of the dockings into CXCR4 (even refined by MD) are
capable of explaining all mutant results by a direct ligand-re-
ceptor interaction. That is to say, none of the binding modes
obtained could account for all of the amino acid residues that
have been shown to affect the binding of the well-known
CXCR4 bicyclam and monocyclam inhibitors.[16,22,26,30] To ac-
commodate all the amino acid residues indicated by the muta-
tional analysis it is necessary to explore different high-affinity
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tet; m, multiplet; br, broad signal; cs, complex signal. Mass spectra
(m/z (%), EI, 70 eV) were obtained using a Hewlett–Packard
HP5988A spectrometer and Bruker Biotof spectrometer (ESI). Ele-
mental microanalyses were obtained on a Carlo–Erba CHNS-O/EA
1108 and EuroVector EuroEA3000 analyzers. Thin-layer chromatog-
raphy (TLC) was performed on pre-coated sheets of silica 60 Poly-
gram SIL N-HR/UV254 (Macherey–Nagel, 804023). Flash chromatog-
raphy was performed using silica gel 35–70 mm (SDS, 2000027).
Compounds 9, 10,[21] and 12[20] were synthesized by previously de-
scribed methods.
docking binding modes for each molecule. However, in all
binding modes obtained there appear interactions with Glu288
and Asp262, which agree with the CXCR4 SDM residues with
the highest level of the effect of the mutation on the binding
of well-known monocyclam and bicyclam compounds. This
result is consistent with the work of Wong et al.,[30] who pro-
pose two possible explanations for this fact: one explanation is
that there are indeed amino acid residues that interact with
small-molecule inhibitors over a longer distance and the other
is that small-molecule inhibitors can bind in several orienta-
tions to the receptor by directly interacting with different sub-
sets of amino acid residues, the results of mutagenesis reflect-
ing a timed average.
1-[4-(Carboxaldehyde)phenylmethyl]-4,8,11-tris-(tert-butoxycar-
bonyl)-1,4,8,11-tetraazacyclotetradecane (5): A solution of 10
(1.22 g, 2.4 mmol) in MeCN (25 mL) was added to a mixture of 12
(0.49 g, 2.4 mmol) and K2CO3 (0.67 g, 4.8 mmol) in MeCN (25 mL).
The mixture was held at reflux for 16 h. It was then cooled to
room temperature, and the solids were filtered off. The solvent
was removed under reduced pressure, and the residue was puri-
fied by column chromatography (CH2Cl2/MeOH) to give 5 (1.33 g,
Conclusions
1
In conclusion, novel monocyclam derivatives have been de-
signed, synthesized, and biologically evaluated as HIV entry in-
hibitors. In vitro activity testing of these compounds in cell cul-
tures against HIV strains has displayed EC50 values of 0.02 and
0.06 mgmLÀ1 without cytotoxicity at the tested concentrations.
Furthermore, the specificity of these monocyclams for CXCR4
has been proven by staining studies of mAbs against CXCR4,
CD4 and CD45. Docking and MD simulations have been per-
formed to predict the binding interactions between CXCR4
and the novel monocyclam derivatives. A binding mode of
these compounds has been proposed which is consistent with
the CXCR4 SDM residues with the highest level of the effect of
the mutation on the binding of well-known monocyclam com-
pounds such as AMD3465, AMD3529, AMD8721, AMD8899 or
AMD3389. Moreover, docking and MD comparisons have been
performed between monocyclams (the novel monocyclams re-
ported herein, as well as other published monocyclam com-
pounds), non-cyclams (non-cyclam compounds previously re-
ported by our group, from which derive the monocyclams, as
well as published non-cyclams), and bicyclams (the well known
AMD3100 bicyclam). Our results suggest that these three struc-
turally diverse CXCR4 inhibitors, monocyclams (2 and 3), non-
cyclams (compound 1) and bicyclams (AMD3100) bind to over-
lapping but not identical amino acid residues in the transmem-
brane regions of the receptor. Therefore, these new monocy-
clams might serve as novel leads for further pharmacological
investigations as therapeutic agents against HIV.
89%) as a white solid. H NMR (300 MHz, CDCl3): d=9.99 (s, 1H),
7.82 (d, J=8.0 Hz, 2H), 7.45 (d, J=8.0 Hz, 2H), 3.61 (s, 2H), 3.30 (m,
12H), 2.63 (br, 2H), 2.39 (br, 2H), 1.90 (br, 2H), 1.69 (m, 2H), 1.47 (s,
18H), 1.43 ppm (s, 9H); 13C NMR (75.5 MHz, CDCl3): d=191.7, 155.4,
146.4, 135.3, 129.6, 129.4, 79.7, 79.6, 59.7, 53.3, 51.9, 47.8, 47.2,
46.2, 28.6, 28.5, 27.1 ppm; IR (film): n=3004, 2975, 2932, 2870,
2817, 1695, 1607, 1478, 1465, 1390, 1366, 1247, 1165, 859 cmÀ1; MS
(IE, 70 eV): m/z (%): 619.7 (5), 618.7 (14) [M]+; Anal. calcd for
C33H54N4O7: C 64.05, H 8.80, N 9.05, O 18.10, found: C 64.04, H 8.77,
N 8.93.
1-[4-(3-(2-Methylpiperidin-1-yl)propyl-1-aminomethyl)phenyl-
methyl]-4,8,11-tris-(tert-butoxycarbonyl)-1,4,8,11-tetraazacyclo-
tetradecane (13): Ti(OiPr)4 (1.19 g, 4.0 mmol) was added to a solu-
tion of 5 (1.26 g, 2.0 mmol) and 6 (0.33 g, 2.0 mmol) in absolute
EtOH (30 mL). The mixture was stirred at room temperature for
36 h. Then NaBH4 (0.08 g, 2.0 mmol) was added, and the mixture
was stirred at room temperature overnight. H2O (20 mL) was
added, and the solids were filtered off. The product was extracted
with CH2Cl2, dried over MgSO4, and the solvent was removed
under reduced pressure. The residue was purified by column chro-
matography (CH2Cl2/MeOH) to give 13 (1.17 g, 76%) as a yellow
1
oil. H NMR (400 MHz, CDCl3): d=7.22 (m, 4H), 3.75 (s, 2H), 3.52 (s,
2H), 3.34–2.23 (m, 12H), 2.87 (m, 1H), 2.73 (m, 1H), 2.64 (t, J=
6.9 Hz, 2H), 2.62 (br, 2H), 2.37 (m, 3H), 2.26 (m, 2H), 2.11 (m, 1H),
1.90 (br, 2H), 1.73–1.59 (m, 9H), 1.67 (br, 2H), 1.47 (s, 18H), 1.44 (s,
9H), 1.28 (m, 2H), 1.05 ppm (d, J=6.3 Hz, 6H); 13C NMR
(100.6 MHz, CDCl3): d=155.8, 139.3, 137.5, 129.4, 128.2, 79.8, 79.6,
59.7, 56.2, 54.0, 53.2, 52.5, 52.3, 51.5, 48.6, 47.6–46.1, 34.9, 31.1,
28.7, 26.4, 26.0, 24.2, 19.3 ppm; IR (film): n=2974, 2931, 2804,
1690, 1477, 1465, 1413, 1366, 1166, 754 cmÀ1; MS (ESI): m/z (%):
759.6 [M]+; Anal. calcd for C42H74N6O6: C 66.46, H 9.83, N 11.07, O
12.65, found: C 66.47, H 10.26, N 10.93.
Experimental Section
1-[4-(3-Morpholinopropyl-1-aminomethyl)phenylmethyl]-4,8,11-
tris-(tert-butoxycarbonyl)-1,4,8,11-tetraazacyclotetradecane (14):
Compound 5 (1.26 g, 2.0 mmol) and 7 (0.44 g, 3.0 mmol) were dis-
solved in MeOH (15 mL) in a microwave vessel, Na2SO4 was added,
and the vessel was sealed. The mixture was heated for 3 h at
1008C in the microwave. Then it was filtered, diluted with MeOH,
cooled to 08C and treated with NaBH4 (0.08 g, 2.0 mmol) and
stirred at room temperature overnight. H2O (20 mL) was then
added, and the product was extracted with CH2Cl2. The organic
layers were combined, washed with brine, dried over MgSO4, and
the solvent was removed under reduced pressure. The residue was
purified by column chromatography (CH2Cl2/MeOH) to give 14
Chemistry
General methods: IR spectra were recorded in a Nicolet Magna
560 FTIR spectrophotometer. H and 13C NMR spectra were record-
1
ed in a Varian Gemini-300 operating at a field strength of 300 and
75.5 MHz, respectively, and Varian 400-MR operating at a field
strength of 400 and 100.6 MHz, respectively. Chemical shifts (d) are
reported in parts per million (ppm), and coupling constants (J) in
1
Hz using, in the case of H NMR, TMS or TSPNa as an internal stan-
dard and setting, in the case of 13C NMR the reference at the signal
of the solvent, 77.0 ppm (CDCl3). Standard and peak multiplicities
are designated as follows: s, singlet; d, doublet; t, triplet; q, quar-
1278
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ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1272 – 1281

Monocyclam HIV Entry Inhibitors
1
(0.48 g, 32%) as a yellow oil. H NMR (300 MHz, CDCl3): d=7.29 (m,
Molecular dynamics simulations
4H), 3.86 (s, 2H), 3.65 (t, J=4.5 Hz, 4H), 3.53 (s, 2H), 3.34–2.25 (m,
12H), 2.84 (t, J=6.5 Hz, 2H), 2.62 (br, 1H), 2.46 (m, 6H), 2.37 (br,
2H), 1.89 (br, 2H), 1.83 (t, J=6.5 Hz, 2H), 1.67 (br, 2H), 1.47 (s,
18H), 1.44 ppm (s, 9H); 13C NMR (75.5 MHz, CDCl3): d=155.4, 137.6,
129.1, 127.9, 79.6, 79.4, 66.9, 59.4, 57.5, 53.7, 53.4, 51.3, 48.0, 47.3,
45.9, 29.7, 28.5, 26.0 ppm; IR (film): n=2971, 2927, 2853, 2808,
The highest-affinity binding conformation obtained for each ligand
(1, 2, 3, AMD3100) in the docking studies was used as the starting
structure for MD simulations. Ligand structures were parameterized
using the program Antechamber and the general Amber force
field (gaff.dat).[43] The topology and starting coordinates of CXCR4
receptor with each of the ligands in the binding cavity were pre-
pared using Leap. The complexes were immersed in a box of
water molecules (TIP3PBOX) and ClÀ counterions were added to
the solvent bulk of the protein/water complexes to maintain neu-
trality of the system using the program Amber 8.[34] Periodic boun-
dary conditions were applied. The Amber force field ff03[44] all
atom parameters (parm99.dat+frcmod.ff03) were used for the pro-
tein and ClÀ ions. The minimization protocol consisted of two
steps: min1 and min2. Min1 consisted of 200 cycles of steepest de-
scent followed by 4800 cycles of conjugate gradient method until
the root-mean square deviation (RMSD) of the Cartesian elements
of the gradient reached a value <0.1 ꢃ. Restraints on all protein
atoms were applied (force constant of 50 kcalmolÀ1 ꢃÀ2). The min2
step used the same minimization parameters but no restraints
were applied, all the system was minimized. The dynamic protocol
consisted of five steps: MD1, MD2, MD3, MD4, and MD5. The initial
temperature for MD1 was set at 50 K, and for MD2, MD3, MD4, and
MD5 were set at 300 K, while the targeted temperature during the
run was 300 K. The Langevin equilibration scheme,[45] with a colli-
sion frequency value of 0.1 psÀ1, was applied to keep the tempera-
ture constant. The time step for all five dynamic procedures was
0.002 ps. For minimization and MD, the primary cutoff distance for
non bonded interaction was set at 8 ꢃ. In all five dynamic steps,
the Shake algorithm[46] was applied to constrain only bonds involv-
ing hydrogen and omit force evaluations for these bonds. Regard-
ing the MD protocol used, the first step (MD1) aimed for the equili-
bration of water molecules and ions of the water-boxed and
charge-neutralized the model. An initial velocity was given to the
system and trajectories were then allowed to evolve in time ac-
cording to Newtonian laws, keeping the protein model fixed (re-
straints on all protein atoms with a force constant of 25 kcal
molÀ1 ꢃÀ2). The number of dynamics steps was 20000, correspond-
ing to 40 ps. Periodic boundary dynamic, with constant volume
and no pressure control, was performed on the system. In the
second step (MD2), 40 ps of constant pressure dynamic of 1 atm,
with isotropic position scaling, was applied with restraints on all
protein atoms (force constant of 10 kcalmolÀ1 ꢃÀ2). In the subse-
quent steps (MD3, MD4, MD5), 40 ps of constant pressure dynamic
of 1 atm were also applied with restraints on backbone atoms, ap-
plying force constants of 10, 5, and 1 kcalmolÀ1 ꢃÀ2, respectively, to
finally assess the stability of the models over time.
1693, 1477,1464, 1365, 1165 cmÀ1
.
1-[4-(3-(2-Methylpiperidin-1-yl)propyl-1-aminomethyl)phenyl-
methyl]-1,4,8,11-tetraazacyclotetradecane hexahydrochloride
(2): HCl (1m in Et2O, 18 mL) was added to 13 (1.07 g, 1.4 mmol)
and stirred at room temperature overnight. Then the solvent was
removed under reduced pressure to give 2 (1.03 g, 90%) as a
1
white solid. H NMR (400 MHz, D2O): d=7.51 (m, 4H), 4.38 (s, 2H),
4.21 (s, 2H), 3.51 (br, 8H), 3.41 (m, 1H), 3.30 (br, 9H), 3.09 (m, 4H),
2.91 (m, 1H), 2.17–1.99 (br, 6H), 1.86 (m, 2H), 1.71 (m, 1H), 1.59 (m,
3
1H), 1.44 (m, 2H), 1.24 (d, JH,H =6.4 Hz, 3H); 13C NMR (100.6 MHz,
D2O): d=132.9, 132.0, 131.1, 130.6, 60.3, 58.7, 56.8, 52.6, 50.9, 49.6,
48.3, 48.0, 44.9, 44.5, 42.0, 41.4, 38.1–37.6, 31.7, 23.2, 21.6, 20.3,
18.5, 17.4 ppm; IR (film): n=2953, 2749, 2678, 1624, 1581,
1455 cmÀ1; MS (ESI): m/z (%)=459.4 (100) [M+H]+; Anal. calcd for
C27H50N6·6HCl·71= H2O: C 39.89, H 8.82, N 10.33, O 14.76, Cl 20.20,
2
found: C 39.91, H 8.41, N 10.00.
1-[4-(3-Morpholinopropyl-1-aminomethyl)phenylmethyl]-
1,4,8,11-tetraazacyclotetradecane hexahydrochloride (3): HCl
(1m in Et2O, 8 mL) was added to 14 (0.46 g, 0.6 mmol) and stirred
at room temperature overnight. The solvent was then removed
under reduced pressure, and the solid was recrystallized from
1
MeOH to give 3 (0.16 g, 37%) as a white solid. H NMR (300 MHz,
D2O): d=7.59 (m, 4H), 4.36 (br, 2H), 4.33 (br, 2H), 4.16 (br, 2H),
4.11 (br, 2H), 3.83 (m, 2H), 3.59–3.55 (m, 10H), 3.37–3.19 (m, 14H),
2.28–2.09 ppm (m, 6H); 13C NMR (75.5 MHz, D2O): d=132.5, 132.0,
131.2, 64.3, 58.1, 56.2, 54.3, 52.2, 51.4, 50.3, 48.7, 46.9, 44.7, 43.2,
40.0, 21.1, 20.0 ppm; IR (KBr): n=2958, 2787, 1583, 1472, 1440,
1107, 772 cmÀ1; MS (ESI): m/z (%): 447.38 (100) [M+H]+; Anal. calcd
for C25H46N6O·6HCl·3H2O: C 41.71, H 8.14, N 11.67, O 8.89, Cl 29.59,
found: C 41.57, H 7.99, N 11.44.
Docking studies
Docking studies were performed using the AutoDock Vina pro-
gram.[31] This version of AutoDock uses, as input files, the 3D coor-
dinates of both ligand and receptor, which must be converted into
the appropriate format by using the ADT program.[32] The 3D struc-
ture of the CXCR4 receptor was first homology modeled with Mod-
eller[35] and Congen[36] by using bovine rhodopsin as a template[37]
as described in Pꢀrez-Nueno et al.[38] Ligand structures were built,
assigned Gasteiger partial charges,[39] and minimized in MOE with
the MMFF94x force field.[33] Finally, the CXCR4 homology model
and the different ligand structures were prepared for docking ex-
periments using the ADT program. For the ligands, non-polar hy-
drogen atoms were deleted, and rotatable bonds were defined.
For the protein, non-polar hydrogen atoms were deleted and
charges were added to the structure. Both structures were saved
in the appropriated format to be used with AutoDock. A cubic grid
of 20 ꢃ on each side (1 ꢃ grid spacing) was used and was centered
on the CXCR4 SDM-defined binding pocket.[24] 100 independent La-
marckian genetic algorithm (LGA) runs were performed and
pseudo-Solis and Wets minimization methods were applied by
using default parameters. Exhaustiveness was set to 8 and energy_
range was set to 4. Key SDM residues[40–42] in the receptor binding
pocket were allowed to be flexible.
Antiviral activity evaluations
The reference HIV-1 NL4–3 strain was titered in MT-4 cells after
acute infection, and infectivity was measured by evaluating the cy-
topathic effect induced after 5 day cultures as described.[47,48] Anti-
HIV activity (EC50) and cytotoxicity (CC50) measurements in MT-4
cells were based on viability of cells that had been infected or not
infected with HIV-1, all exposed to various concentrations of the
test compound. After the MT-4 cells were allowed to proliferate for
five days, the number of viable cells was quantified by a tetrazoli-
um-based colorimetric method (MTT method). Values represent the
mean Æstandard deviation of at least two independent evalua-
tions done in triplicate.
ChemMedChem 2010, 5, 1272 – 1281
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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J. I. Borrell et al.
MED
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Flow cytometry analysis
Staining of chemokine receptor CXCR4, CD45, and CD4 receptor
on MT-4 cell line was performed as reported previously.[48,49] Briefly,
0.1ꢄ106 cells were washed in PBS and then they were incubated
for 20 min at room temperature with mAbs anti-CD45 conjugated
with fluorescein isothiocyanate at 1:200 dilution, 12G5 (anti-
CXCR4)-phycoerythrin (PE) at 1:50 dilution and Leu3a (anti-CD4)-
peridin chlorophyll protein at 1:50 dilution (BD Biosciences, San
Jose, CA, USA), and with or without study drugs and control com-
pounds (AMD3100 and unstained 12G5). The cells were then
washed with PBS and fixed in PBS containing 1% formaldehyde.
Cells were analyzed by flow cytometry in a FacsCalibur system (BD
Biosciences). Data were acquired and analyzed with CellQuest soft-
ware (BD Biosciences). AMD3100, Leu3a, 12G5 and the study drugs
were tested at various concentrations. The compound concentra-
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from the percent mean inhibition results from three independent
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Acknowledgements
This work was supported in part by the Fundaciꢁ Maratꢁ de TV3
project 020930 and the Spanish Ministerio de Educaciꢁn y Cien-
cia project SAF-2007-6322 (J.I.B. and B.C.) and BFU-2009-06958
(J.A.E.). M.P.M. holds a research fellowship from FIS. V.I.P.-N. held
a 2008FI scholarship from Generalitat de Catalunya, and S.P.
from IQS.
Keywords: CXCR4 antagonists · docking · HIV-1 infection ·
medicinal chemistry · molecular dynamics
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Monocyclam HIV Entry Inhibitors
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[51] It is common in the pharmaceutical industry to have assays that gener-
ate IC50 or EC50 values rather than equilibrium constants. Whereas pKi
and DG form a strict linear correlation [DG=RTln(K)], the correlation
between pEC50 and DG cannot be expected to be exactly linear. How-
ever, IC50 or EC50 values can be used to obtain DG in an approximate
manner, as in: R. Zhou, R. A. Friesner, A. Ghosh, R. C. Rizzo, W. L. Jorgen-
sen, R. M. Levy, J. Phys. Chem. B 2001, 105, 10388–10397.
2287–2303. Approaches based on MD such as LIE (see: J. ꢃqvist, C.
Medina, J. E. Samuelson, J. Protein Eng. 1994, 7, 385–391) or MM-PBSA
(see: J. Srinivasan, T. E. Cheatham, P. Cieplak, P. A. Kollman, D. A. Case, J.
Am. Chem. Soc. 1998, 120, 9401–9409) allow predictions that are relia-
ble and in good agreement with experiment, but are much more com-
putationally expensive. The results obtained using AutoDock Vina affini-
ty scoring function are impressive due to the good agreement with ex-
perimental values obtained.
Received: March 25, 2010
Revised: April 29, 2010
[52] Scoring functions can calculate DGbinding values with low computational
cost, predicting free energy values in good qualitative agreement with
experiment; see: R. Wang, Y. Lu, S. Wang, J. Med. Chem. 2003, 46,
Published online on June 8, 2010
ChemMedChem 2010, 5, 1272 – 1281
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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