Novel compounds containing multiple guanide groups that bind the HIV coreceptor CXCR4

Article abstract of DOI:10.1128/AAC.00709-10

The G-protein-coupled receptor CXCR4 acts as a coreceptor for human immunodeficiency virus type 1 (HIV-1) infection, as well as being involved in signaling cell migration and proliferation. Compounds that block CXCR4 interactions have potential uses as HI

Full text of DOI:10.1128/AAC.00709-10

Novel Compounds Containing Multiple
Guanide Groups That Bind the HIV
Coreceptor CXCR4
Royce A. Wilkinson, Seth H. Pincus, Joyce B. Shepard,
Sarah K. Walton, Edward P. Bergin, Mohamed Labib and
Martin Teintze
Antimicrob. Agents Chemother. 2011, 55(1):255. DOI:
10.1128/AAC.00709-10.
Published Ahead of Print 11 October 2010.
Updated information and services can be found at:
http://aac.asm.org/content/55/1/255
These include:
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2011, p. 255–263
0066-4804/11/$12.00 doi:10.1128/AAC.00709-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 1
Novel Compounds Containing Multiple Guanide Groups That Bind
the HIV Coreceptor CXCR4ᰔ†
Royce A. Wilkinson,1 Seth H. Pincus,2,3 Joyce B. Shepard,1 Sarah K. Walton,1 Edward P. Bergin,1
Mohamed Labib,4 and Martin Teintze1*
Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 597171; Research Institute for Children,
Children’s Hospital, New Orleans, Louisiana 701182; Department of Pediatrics, LSU Health Sciences Center, New Orleans,
Louisiana 701183; and Novaflux Biosciences, Inc., Princeton, New Jersey 085404
Received 24 May 2010/Returned for modification 15 July 2010/Accepted 30 September 2010
The G-protein-coupled receptor CXCR4 acts as a coreceptor for human immunodeficiency virus type 1
(HIV-1) infection, as well as being involved in signaling cell migration and proliferation. Compounds that block
CXCR4 interactions have potential uses as HIV entry inhibitors to complement drugs such as maraviroc that
block the alternate coreceptor CCR5 or in cancer therapy. The peptide T140, which contains five arginine
residues, is the most potent antagonist of CXCR4 developed to date. In a search for nonpeptide CXCR4 ligands
that could inhibit HIV entry, three series of compounds were synthesized from 12 linear and branched
polyamines with 2, 3, 4, 6, or 8 amino groups, which were substituted to produce the corresponding guanidines,
biguanides, or phenylguanides. The resulting compounds were tested for their ability to compete with T140 for
binding to the human CXCR4 receptor expressed on mammalian cells. The most effective compounds bound
CXCR4 with a 50% inhibitory concentration of 200 nM, and all of the compounds had very low cytotoxicity. Two
series of compounds were then tested for their ability to inhibit the infection of TZM-bl cells with X4 and R5
strains of HIV-1. Spermine phenylguanide and spermidine phenylguanide inhibited infection by X4 strains, but
not by R5 strains, at low micromolar concentrations. These results support further investigation and devel-
opment of these compounds as HIV entry inhibitors.
New therapeutics with novel mechanisms of action are
needed for treatment of human immunodeficiency virus type 1
(HIV-1) infections. Maraviroc, a small molecule antagonist of
the CCR5 receptor, was the first drug of its type to be approved,
and it has proven to be effective against “R5” HIV that uses the
CCR5 coreceptor (9). However, most patients that have failed
conventional therapies also harbor “X4” HIV that uses
CXCR4 or dualtropic viruses that can use either CCR5 or
CXCR4. The appearance of the X4 viruses is associated with
progression to AIDS symptoms (4), and drug resistance is
more often linked to CXCR4-utilizing than to CCR5-utilizing
HIV (39). Therefore, there is also a need to develop effective
drugs that block CXCR4. Thus far, none of the CXCR4 an-
tagonists in development have shown promise in HIV clinical
trials, although some are being pursued as cancer therapeutics
because CXCR4 may be involved in metastasis. Some CXCR4
inhibitors, such as the bicyclam AMD3100 and the monocy-
clam AMD3465, bind to a site located in the transmembrane
domain of the receptor (13, 15, 26, 27, 30, 40). Like the CCR5-
specific small-molecule inhibitors, these compounds are be-
lieved to block viral entry and chemokine signaling by allosteric
mechanism(s). In contrast to CCR5 inhibitors, however, many
of the CXCR4-specific inhibitors contain multiple positive
charges that can interact with additional acidic residues in the
extracellular loops of CXCR4 that are not conserved in CCR5
(2, 10, 27, 28, 35).
T140 is a 14-residue peptide antagonist of CXCR4 that
blocks the binding of X4 strains of HIV-1 in vitro and displaces
the natural agonist ligand SDF-1␣ with nanomolar affinity but
does not bind to other chemokine receptors, such as CCR5
(30–32). It is much smaller than SDF-1␣ (14 residues versus 65
residues) but is rapidly degraded by proteolysis in serum (30)
and has not been pursued as an HIV or cancer therapeutic.
Recently, smaller cyclic peptides based on the structure of
T140 (25, 37), as well as nonpeptide analogs (36), have been
reported. However, the latter have 50% inhibitory concentra-
tions (IC50s) at least 2 orders of magnitude higher than that of
T140. The structure of T140 contains five Arg and two Lys resi-
dues; the latter can be substituted with uncharged side chains
without loss of activity (30). ALX40-4C (N-␣-acetyl-nona-D-argi-
nine amide acetate) is another peptide antagonist of CXCR4 (8).
An early clinical trial showed that it was well tolerated (7) and
provided an important proof of principle that CXCR4 activity
can be safely inhibited in humans. KRH-1636 is a CXCR4
antagonist that exhibits strong activity against X4 strains of
HIV, competitively displaces SDF-1 from the receptor, and
can be absorbed from the duodena of rats (17). Like T140 and
ALX40-4C, KRH-1636 contains an Arg residue. An analog of
this compound (KRH-2731) was reported to have enhanced
activity and was claimed to be orally bioavailable in rats (24),
but neither its structure nor any clinical trial results have been
published. AMD3100 (plerixafor) is a bicyclam with eight sec-
ondary and tertiary amine groups (5, 6). Clinical trials for HIV
have been abandoned because this compound showed poor
efficacy and cardiotoxicity and was not orally bioavailable (16).
* Corresponding author. Mailing address: 111A Chemistry and Bio-
chemistry Building, Montana State University, Bozeman, MT 59717.
Phone: (406) 994-4411. Fax: (406) 994-5407. E-mail: mteintze@montana
.edu.
† Supplemental material for this article may be found at http://aac
.asm.org/.
ᰔ Published ahead of print on 11 October 2010.
255

256
WILKINSON ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Compounds synthesized
The structurally similar monocylam AMD3465 has higher af-
finity for CXCR4 and is potent against X4 HIV strains in vitro
(IC50, 1 to 10 nM) (19) but is also not orally bioavailable (14).
AMD070 (also known as AMD11070), which has two aromatic
rings in addition to a primary and a tertiary amine, has an IC50
of 2 to 26 nM against an X4 HIV strain and is orally bioavail-
able (22, 29).
No. of amines derivatizedb
Starting amine (n)a
Guanide
Biguanide
Phenylguanide
Butanediamine (2)
Hexanediamine (2)
Diethylenetriamine (3)
(DETA)
2
2*
2
2
1 or 2
1 or 2
3‡
1 or 2
3†
Previous studies at Novaflux and Drexel University have led
to the identification and characterization of a new HIV-1 in-
hibitor, NB325 (20). This is a biguanide-based, low-molecular-
weight oligomer (ϳ800 to 1,400 Da) that inhibits infection via
specific interactions with CXCR4 and has been shown to be a
potent HIV-1 inhibitor in a number of in vitro assays (3, 34).
NB325 functions by specifically preventing the interaction of
HIV-1 gp120 with the extracellular loop 2 (ECL2) of the
CXCR4 coreceptor. Persistent interactions between NB325
and CXCR4 result in prolonged inhibition of HIV-1 infection
(33). NB325 blocks SDF-1␣ signaling through CXCR4 but not
SDF-1␣ attachment (34). The specificity of this new class of
compounds as CXCR4 antagonists has also been confirmed by
competitive binding with the peptide antagonist T140 (de-
scribed below) and the inhibition of Ca2ϩ flux induced by the
natural chemokine agonist SDF-1␣ (CXCL12; T. Sakmar, The
Rockefeller University [unpublished data]). However, because
NB325 is a polydisperse preparation consisting of a homolo-
gous series of compounds with 4 to 10 biguanide groups, its
approval for clinical use is unlikely. We therefore developed a
series of discrete, small-molecule inhibitors containing mul-
tiple guanide or biguanide groups to test their ability to
inhibit HIV-1 binding to CXCR4. The compounds were
initially screened for their ability to inhibit the binding and
cross-linking of a fluorescently labeled photoactive analog of
T140 to CXCR4. The most effective inhibitors of T140 binding
and cross-linking were subsequently tested for their ability to
inhibit HIV infection. Some of these compounds inhibited
infection by X4, but not R5 or X4/R5, strains of HIV. The
results demonstrate the potential of these compounds in the
development of X4 HIV entry inhibitors, but these compounds
will have to be improved upon substantially to match the in-
hibition of R5 HIV by maraviroc.
Spermidine (3)
2 or 2–3
1–2
2
1
2 or 3
3–4
3
Spermine (4)
2
3
Trisethylaminomelamine (3)
(TEAM)
Trisbutylaminomelamine (3)
(TBAM)
Trishexylaminomelamine (3)
(THAM)
Bisethylaminomelamine
dimer (2) (BEMA dimer)
PAMAM G0 (4)
DNT2300 (6)
3
3
2
1–2
1–3
ND
2 or 3
2
1–2
4*
5–6*
8
3–4
3–6¶
1–5
4**
6
8
DNT2200 (8)
a n, number of reactive amines in each starting material.
b *, ϩ15 mass also (O-Me replace NH2); **, small amount of ϩ5 phenylgua-
nide groups; †, cyanoamines and biguanides; ‡, cyclic by-products; ¶, masses 4
amu too low. ND, synthesis not done.
compound (BEMA dimer). Treatment of the bis addition product before depro-
tection with excess ethylenediamine, followed by deprotection, gave the desired
trisethylamino product, which was purified by reversed-phase HPLC.
(iii) TBAM synthesis. Synthesis of trisbutylaminomelamine (TBAM) was sim-
ilar to process for the TEAM synthesis. The initial bis addition product, after
deprotection, was treated with excess 1,4-butanediamine and refluxed for 2 h in
water. Purification by HPLC under acidic conditions did not result in separation
of the desired product from the excess diamine. However, under basic conditions
the product bound and was not eluted. Subsequent washing with 0.1% TFA in
water eluted the desired product without the contaminating diamine.
Synthesis of guanide derivatives. The reaction of polyamine starting materials
was carried out using 10% excess O-methylisourea sulfate salt (MP Biomedicals)
in water at basic pH. Free amine compounds were added to the O-methylisourea
in water and incubated at 37 to 42°C for 1 to 2 h. Reactions with acidified amine
starting materials (HCl or TFA salts) were neutralized with triethylamine (TEA)
before being allowed to react. Products were purified by reverse phase HPLC
and characterized by matrix-assisted laser desorption ionization (MALDI) mass
spectrometry.
Synthesis of biguanide derivatives. S-Methyl-guanylisothiouronium iodide was
prepared as previously described (11). Briefly, iodomethane (Acros) was added
dropwise to a suspension of 2-imino-4-thiobiuret (Aldrich) in 100% ethanol
while stirring at room temperature. The reaction flask was shaken at 37°C for 2 h.
The solvent was removed by evaporation, and the residue was washed with cold
diethyl ether to give a white, crystalline solid that was collected by filtration and
used without further purification.
Addition of the biguanide reagent (S-methyl-guanylisothiouronium iodide, in
excess) to the amine compounds was done in 100% ethanol as previously de-
scribed (38) at room temperature or 60°C or in water at room temperature, 37°C,
or 65°C. The pH of the reactions were monitored and adjusted with TEA or
sodium carbonate when it fell below a pH of ϳ8. Reaction times varied between
5 h and 5 days. Products were purified and characterized as described above.
Synthesis of phenylguanide derivatives. S-Methyl-N-phenylisothiouronium io-
dide was prepared as described above by addition of methyl iodide to 1-phenyl-
2-thiourea (Acros). Addition to the amine compounds was accomplished by
refluxing 10% excess of the S-methylthiourea compound with the amine in 50%
acetonitrile-water overnight. Reactions with acidified amine starting materials
(HCl or TFA salts) were neutralized with TEA as described above. The products
were purified and characterized as described above.
MATERIALS AND METHODS
Starting material amine compounds. Putrescine dihydrochloride and sperm-
ine tetrahydrochloride were purchased from Sigma (St. Louis, MO). 1,4-diamino-
butane, 1,6-hexanediamine dihydrochloride, 1,6-hexanediamine, diethylenetri-
amine, and spermidine were purchased from Acros (Morris Plains, NJ).
StarburstPAMAM G(0.0) was purchased from Dendritech, Inc. (Midland, MI).
Priostar dendrimers DNT-2200 and DNT-2300 were purchased from Dendritic
Nanotechnologies, Inc. (Mt. Pleasant, MI).
(i) THAM synthesis. Trishexylaminomelamine (THAM) was synthesized ac-
cording to the method of Kaiser et al. (18). Cyanuric chloride (TCI America,
Tokyo, Japan) was reacted with a 3.3 molar ratio of N-BOC-1,6-diaminohexane
(Alfa Aesar, Ward Hill, MA) in refluxing water for 1.25 h. The pH of the solution
was monitored with phenolphthalein, and the pink color was maintained by
gradual addition of a 0.4 M sodium carbonate solution. The reaction was lyoph-
ilized and the BOC groups removed by treating the residue with 100% triflu-
oroacetic acid (TFA) for 2 h at room temperature. The deprotected product was
purified by reversed-phase high-pressure liquid chromatography (HPLC).
(ii) TEAM synthesis. The synthesis of trisethylaminomelamine (TEAM) was
begun as described above using N-BOC-1,2-diaminoethane produced as previ-
ously described (23). The initial reaction gave the bis addition product (after
deprotection) with one unreacted chlorine. Treatment of this compound with a
concentrated ammonia solution in water resulted in the formation of a dimeric
Synthesis of a photoactive, fluorescent T140 peptide. A fluorescent, photoac-
tive cross-linkable derivative of the CXCR4 binding peptide T140 (Ac-RRNal-
CYRBpaD-KPYRCitCR [Nal, naphthylalanine; Cit, citruline; Bpa, 4-benzoyl-
phenylalanine]) (30, 32) was synthesized on a solid support using standard Fmoc
(9-fluorenylmethoxy carbonyl) chemistry. Fmoc amino acids with appropriate
side-chain protecting groups were purchased from Advanced Chemtech (Louis-

VOL. 55, 2011
GUANIDE-BASED CXCR4 INHIBITORS
257
FIG. 1. Synthetic starting materials. The structures of the starting material polyamines used to synthesize guanide, biguanide, and phenylgua-
nide derivatives are shown. Reactive amines are noted in boldface.
ville, KY). After cleavage from the resin and side-chain deprotection, the crude
peptide was purified by reversed-phase HPLC. A dilute solution of the peptide
in 10 mM ammonium bicarbonate (pH 8) was stirred while bubbling air through
the solution at 4°C until oxidation of the intrapeptide disulfide bond was com-
plete as analyzed by MALDI mass spectrometry. The solution was lyophilized,
and the oxidized peptide was purified by reversed-phase HPLC.
The purified peptide was fluorescently labeled on the ε-amine of the D-lysine
residue by reaction with N-hydroxysuccinimide-activated 6-(fluorescein-5-(and-
6)-carboxamido)hexanoic acid (Invitrogen, Carlsbad, CA) in dry dimethylform-
amide with 10% pyridine overnight at 4°C. The labeled peptide was purified by
reversed-phase HPLC.
CXCR4-T140 cross-link inhibition assay. The ability of the guanide, bigua-
nide, and phenylguanide compounds to interact with CXCR4 was tested by a
cross-link inhibition experiment. Serial dilutions of the inhibitor compounds
were incubated with approximately 1.5 ϫ 105 CXCR4-expressing cells (syn-
CXCR4-C9 in Cf2Th) (1) for 30 min on ice. The photoactive, fluorescent T140
peptide was added to a final concentration of 75 nM, followed by incubation on
ice for an additional 30 min. Cross-linking of the T140 peptide to the receptor

258
WILKINSON ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 2. Generic synthetic reaction scheme. Primary and secondary amines treated with the reactive compounds O-methylisourea (compound
1), S-methyl-N-guanylisothiourea (compound 2), or S-methyl-N-phenylisothiourea (compound 3) give the desired guanide, biguanide, or phenyl-
guanide derivatives.
was carried out by irradiation at 365 nm in a Rayonet Photochemical Reaction
chamber (Southern New England UV Company, Hamden, CT) four times for 5
min with 5 min of cooling on ice between irradiations. The cell pellets were spun
down, washed with 1ϫ phosphate-buffered saline (PBS), resuspended in PAGE
(e.g., DNT2300 yielded a mixture of compounds with either
five or six guanide groups per molecule).
Other deviations from the expected products are noted in
Table 1. In three of the guanide addition reactions, additional
ϩ15- and sometimes ϩ30-atomic mass unit (amu) products
were copurified with the isolated products. For these com-
pounds, 20 to 50% of the isolated product was observed to be
the ϩ15 product, with a small amount (Ͻ10%) of the ϩ30
product. These compounds were tested for activity as isolated;
later, it was discovered that these products were the result of
loss of ammonia during the substitution reaction instead of the
expected loss of methanol. Treatment of these compounds
with concentrated ammonium hydroxide can replace the O-
methyl groups and yield the desired products (see Fig. S1 in the
supplemental material), but the effect of this reaction on the
activity of the compound was not tested.
sample buffer with 40 mM dithiothreitol, and run on an SDS-PAGE gel. The gels
were imaged on a Bio-Rad Molecular Imager FX (Hercules, CA), and the
fluorescent CXCR4 bands were quantified with Bio-Rad Quantity One software.
CCR5-maraviroc analog cross-link inhibition assay. A maraviroc analog con-
taining a benzophenone cross-linking group and a fluorescein tag were prepared
(synthesis to be published later). This compound was used to test the guanide,
biguanide, and phenylguanide compounds for interaction with CCR5 in an assay
similar to that used for the CXCR4-T140 inhibition assay above.
Assay for anti-HIV activity. Cell-free virus supernatants were prepared in
PHA blast cultures as described elsewhere (21). TZM-bl cells (41) were obtained
from the NIH AIDS Research and Reference Reagent Program; 4 ϫ 104 cells
were plated per well. CXCR4 inhibitors were added to the cells for 1 h at 37°C.
Predetermined dilutions of the virus supernatants in 15 ␮g of DEAE-dextran
(Sigma)/ml were then added to the wells containing the cells plus inhibitor.
Three days later, the cells were lysed, and the luciferase activity was measured
(Bright-Glo Luciferase assay; Promega, Madison WI).
Toxicity assays. Compound cytotoxicity was evaluated after 48 h of exposure
using a CellTiter 96 aqueous nonradioactive cell proliferation assay (MTS)
according to the manufacturer’s instructions (Promega, Madison, WI) with the
human breast cancer cell line MDA-MB-231 or the TZM-bl cell line used in the
HIV infectivity assay.
The reaction of diethylenetriamine (DETA) with the phe-
nylguanide reagent resulted in some cyclic by-products (see
Fig. S2 in the supplemental material) that can be encountered
when the reactive amine groups are closely spaced. These same
mechanisms are probably involved in the formation of the
products labeled “cyanoamine and biguanides” in the reaction
of DETA with S-methyl-guanylisothiourea. This reaction pro-
ceeds through the loss of urea and the formation of a cyclic
product, as seen with the phenylguanides, or to the formation
of a cyanoamine. However, mass spectrometry is unable to
differentiate between the two by-products. Finally, the addition
of biguanides to the dendrimer DNT2300 resulted in an unex-
plained deficit of 4 amu from the isolated products regardless
of the number of biguanide groups added.
RESULTS
Guanide, biguanide, and phenylguanide synthesis. The re-
sults of the compound syntheses are summarized in Table 1.
Starting material compounds (see Fig. 1 for structures) are
listed in the first column with the number of amines available
for addition of guanide, biguanide, or phenylguanide groups
(see Fig. 2) in parentheses. The results for each column show
the number of amine groups derivatized. In data presented
with “or” in Table 1, two fractions were isolated with each of
the respective products (e.g., both the monophenylguanide and
the bisphenylguanide additions to butanediamine were iso-
lated and tested separately). In data presented with a dash in
Table 1, the designated products were inseparable by HPLC
CXCR4-T140 cross-link inhibition assay. The compounds
were tested for CXCR4 binding by inhibiting the cross-linking
of a photoactive, fluorescent derivative of the known CXCR4-
binding peptide T140. Initially, this assay was tested by using
NB325 as the inhibitor. Figure 3 shows the results for four

VOL. 55, 2011
GUANIDE-BASED CXCR4 INHIBITORS
259
TABLE 2. CXCR4/T-140-Fl inhibition data
IC50 (␮M)a
Starting amine
Starting
amine
Guanide
Biguanide
Phenylguanide
Butanediamine
Hexanediamine
Diethylenetriamine
(DETA)
Ͼ200
Ն200
Ն200
50
Ͼ200
50
Ͼ200
Ͼ200
Ͼ200
Ͼ200
150 (1), 20 (2)
15 (1), 6 (2)
Spermidine
Ͼ200 8 (2), 10 (3)
75
Ͼ200
65 (1–2)
0.2 (2), 0.2 (3)
0.5 (3–4)
Spermine
10 (2)
20
4–8 (2)
Trisethylaminomelamine
(TEAM)
Ͼ200 (1)
1.5 (3)
Trisbutylaminomelamine
(TBAM)
Ն100
Ͼ200
10
2
Ͼ200 (1–2)
6 (2), 20 (3)
Trishexylaminomelamine
(THAM)
45 (1–3)
Ͼ200 (2)
BEMA dimer
PAMAM GO
DNT2300
Ͼ200
Ͼ200
Ͼ200
Ն200
50
4
ND
15 (1), 70 (2)
Ͼ50 (3–4)
7.5 (3–6)
Ն200 (1–5)
3
1.5
3.5
8
DNT2200
15
a For comparison, NB325, assuming an average molecular weight of ϳ1,000,
has an IC50 of 10 ␮M. Numbers in parentheses indicate the number of groups
added. ND, synthesis not done.
FIG. 3. Inhibition of T140-fluorescein cross-linking to CXCR4.
CXCR4-expressing cells were incubated with serial dilutions of the
polybiguanide NB325 and then allowed to bind the fluorescent, pho-
toactive T140 derivative. After irradiation at 365 nm, the samples were
dissolved in SDS, run on an SDS-PAGE gel, and imaged with a fluo-
rescent gel imager. Inhibition curves for four separate experiments
with one reading per data point are shown.
As a general rule, the inhibition activity increased in the
following order: amines Ͻ biguanides Ͻ guanides Ͻ phenyl-
guanides. However, there are a couple of notable exceptions.
The butanediamine was only active as the guanide derivative,
with an IC50 of 50 ␮M. For THAM, the guanide and biguanide
derivatives were active, but interestingly, the phenylguanide
compound showed no inhibition. The derivatives of linear poly-
amines (butanediamine, hexanediamine, diethylenetriamine,
spermidine, and spermine) generally increased in activity as they
increased in size. As a group, the spermidine and spermine
derivatives were the most active, and the most inhibition was
exhibited by spermidine bisphenylguanide and spermidine tris-
phenylguanide, with IC50s of 200 nM.
separate experiments. Although the relative fluorescence den-
sity (signal strength) varies from gel to gel, the IC50s are quite
reproducible, varying between 0.0006 and 0.001%. Assuming a
median molecular weight of 800 to 1,400 for NB325 gives an
IC50 of ϳ10 ␮M.
A representative gel and analysis are shown in Fig. 4 for the
dendrimer derivative DNT2200 octaguanide. The amount of
fluorescence was quantified as the fluorescence density of a
box encompassing the doublet band (CXCR4) in the middle of
the gel.
The results of the CXCR4-T140 cross-link inhibition assay
are shown in Table 2. The IC50s are given for the starting
material amines and the respective guanide, biguanide, and
phenylguanide derivatives. Most of the starting amines had
IC50s greater than or equal to the highest concentration tested
(200 ␮M), with the exception of spermine, which inhibited the
T140 cross-linking with an IC50 of 75 ␮M.
The melamine core guanide derivatives (TEAM, TBAM,
and THAM) increased in activity as the chain length in-
creased (ethyl Ͻ buty Ͻ hexyl). Conversely, the correspond-
ing phenylguanides showed the opposite trend, with the
THAM bisphenylguanide actually showing no inhibition at
the highest concentration tested (200 mM). The dendrimer
derivatives (PAMAM-G0, DNT2300, and DNT2200) were
FIG. 4. Representative inhibition experiment gel and graphical analysis. Serial dilutions of the inhibitor (DNT2300 hexaphenylguanide) were
incubated with CXCR4-expressing cells. The fluorescent, photoactive derivative of T140 was added, followed by irradiation at 365 nm to induce
cross-linking between the peptide and receptor. Samples were dissolved in SDS, run on an SDS-PAGE gel, and imaged with a fluorescent gel
reader. The concentration of inhibitor is marked at the top of each lane. The intensities of the fluorescent CXCR4 bands (doublet indicated by
arrow) were quantified and plotted with Sigma Plot to determine an approximate IC50 (ϳ1 ␮M). A Western blot of the gel incubated with
monoclonal antibody 1D4 was used to confirm the identity of the CXCR4 bands and their uniformity across the gel (not shown).

260
WILKINSON ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
generally equally active as the number of reactive amines
varied from 4 to 6 to 8.
Inhibition of HIV infection. Six of the most active com-
pounds, and the amines from which they were derived, were
selected for preliminary testing as HIV infection inhibitors.
Spermidine, spermine, DNT2300, and their guanide and phe-
nylguanide derivatives were tested for their ability to inhibit
HIV infection at 10 ␮M in the TZM-bl assay. The compounds
were screened against CXCR4-, CCR5-, and R5/X4-tropic vi-
rus strains. The spermidine and spermine phenylguanides were
effective against both X4 strains, with no inhibition of the
R5/X4 strain and limited inhibition of the R5 strain (Fig. 5).
The underivatized amine spermine appeared to be active
against both R5 and X4 strains at this concentration. However,
in a second TZM-bl assay run to investigate the dose-response
of the active phenylguanides and their respective amine start-
ing materials, the phenylguanides were active at ϳ10-fold-
lower concentrations than the corresponding amines against
three X4 strains (Fig. 6 and 7A). None of the compounds
showed significant activity (Ͼ2-fold reduction) against an R5
virus. At 10 ␮M, spermine did not significantly inhibit fluores-
cent T140 binding to CXCR4 (see above) or the binding of a
fluorescent maraviroc analog to CCR5, nor was it cytotoxic to
TZM-bl cells (data not shown). Therefore, the reason for the
observed inhibition of X4 and R5 infection by the underivat-
ized amines is unclear.
Cytotoxicity. The toxicity of the compounds was evaluated
by using an MTS dye reduction assay in a CXCR4 expressing
human breast cancer cell line for all of the compounds (see
Table S1 in the supplemental material) and in TZM-bl cells for
spermidine phenylguanide and the spermidine control (Fig.
7B). The majority of the compounds showed no toxicity at the
highest concentrations tested against the MDA-MB-231 cells.
Spermidine and spermidine phenylguanide both had CC50 val-
ues of ϳ1,000 ␮M against the TZM-bl cells while showing HIV
inhibition at Ͻ5 ␮M (Fig. 7A).
DISCUSSION
FIG. 5. Initial anti-HIV activity screen. The series (amine, guanide,
and phenylguanide) of the most active derivatives (spermidine, sperm-
ine, and DNT2300) in the T140 cross-link inhibition assay were
screened for anti-HIV activity. TZM-bl cells were plated and preincu-
bated with the inhibitors at a final concentration of 10 ␮M. Previously
titered viral supernatants were added, and the cells were incubated for
3 days. Cells were lysed, and the TAT-driven reporter luciferase ac-
tivity was measured using a Bright-Glo luciferase assay. Experiments
were performed in triplicate with errors shown as means Ϯ the stan-
dard error of the mean (SEM). RLU, relative luminescence units.
We have synthesized a novel series of compounds containing
multiple guanide, biguanide, or phenylguanide groups on lin-
ear aliphatic or dendrimer scaffolds. These compounds bear
some resemblance to the polyethylenehexamethylene bigua-
nide NB325, as well as to the CXCR4 antagonist peptide T140,
which has five guanide groups on the side chains of arginine
residues. Among the new compounds, the bis- and trisphenyl-
guanide derivatives of spermidine exhibited the highest affinity
for CXCR4, with an IC50 of 200 nM in competition assays with
T140. This is a 50-fold-higher affinity than found for NB325 in
this assay and is comparable to that of other nonpeptide ana-
logs of T140 that have been reported (36), although it does not
approach the nanomolar affinity of T140 itself.
The best T140 inhibitors were also tested for their ability to
inhibit HIV infection. The spermine and spermidine deriva-
tives and amines were tested against a panel of X4, R5, or
X4/R5 isolates. Spermine phenylguanide and spermidine phe-
nylguanide were effective at inhibiting the infection of TZM-bl
cells by X4, but not R5, HIV strains, a finding consistent with
the observation that they bound to CXCR4 but not CCR5. In
fact, none of the compounds in Table 2 inhibited the binding of
a fluorescent maraviroc analog to CCR5 (data not shown).
Spermidine phenylguanide had an IC50 of 3 ␮M when tested
for inhibition of three X4 strains (NL4-3, 92HT599, and MN).
The HIV inhibition data were also consistent with the T140
inhibition results in that the phenylguanides were more active
than the related guanide or biguanide compounds. However,
the underivatized parent amines appeared to show inhibition
against both X4 and R5 strains of HIV, even though they were
not effective in inhibiting T140 binding and cross-linking to
CXCR4, suggesting that they were acting via another mecha-
nism.

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GUANIDE-BASED CXCR4 INHIBITORS
261
FIG. 6. HIV inhibition dose-response trials. Spermine (F), spermine phenylguanide (E), spermidine (ꢀ), and spermidine phenylguanide (‚)
were tested for dose-response activity in the TZM-bl luciferase anti-HIV assay. Increased activity against CXCR4-using viral strains were observed
with the phenylguanides compared to the underivatized parent amines. Experiments were performed in triplicate with errors shown as means Ϯ
the SEM. RLU, relative luminescence units.
In experiments with primary human CD4ϩ T lymphocytes as
the target cells, NB325 inhibited HIV-1 IIIB infection with
IC50 concentrations of ca. 0.01% (ϳ71 ␮M) (34). This is ϳ50-
fold higher than the concentrations of spermine phenylguanide
and spermidine phenylguanide required to inhibit X4 HIV
infection, which is consistent with the higher affinity for
CXCR4 relative to NB325. Since NB325 is a mixture, it is of
course possible that it contains a component with greater ac-
tivity. We attempted to fractionate NB325 by reversed-phase,
ion exchange, and size exclusion chromatography but were
unable to isolate a fraction with higher affinity for CXCR4
(data not shown).
The optimal structure for a nonpeptide CXCR4 antagonist
cannot be readily predicted from the data available. Attempts
to develop small cyclic peptide and nonpeptide analogs from
T140 (12, 36, 37) have pointed to the importance of both a
guanide functional group (in the form of an arginine side
chain) and an aromatic group (napthyl). This is consistent with
our observation that the phenylguanides were more active than
either the guanides or the biguanides and suggests that using a
larger aromatic moiety such as naphthalene may increase the
observed CXCR4 binding compared to our initial phenylgua-
nide derivatives.
order of magnitude better binding than the best melamine
derivative (THAM trisguanide), which has the aromatic group
in the center of the molecule with the charges surrounding it.
There also appears to be a necessary balance between charge
and hydrophobic character. This is exemplified in the series of
melamine-derived phenylguanides in which increasing the
length of the spacers between the melamine and phenylgua-
nide groups, which increases overall hydrophobicity, led to a
progressive loss of activity.
The spacing between the positively charged groups, at least
in the linear compounds, also appears to be important. Exper-
iments in the development of NB325 indicated that optimal
efficacy was found when an alternating 2:6 (ethyl-, hexyl-) spac-
ing between biguanides was present compared to evenly
spaced ethyl-, butyl-, or hexyl-based polybiguanides (20). In the
experiments reported here, the most active core molecules
(spermine and spermidine) have spacings of three or four carbons
between the reactive amine groups, whereas the less active dieth-
ylenetriamine and hexanediamine cores have two and six carbons,
respectively. However, the flexibility of the polyamine backbones
versus those of the polybiguanides makes comparison of the
distances between the charged groups in the two types of
molecules difficult.
The positioning of the aromatic group also appears to be
important. The most active phenylguanides (spermidine and
spermine derivatives), with the aromatic groups on the ends
and sides of the main chain of the molecule, have at least an
The compounds reported here, especially the phenylgua-
nides, have significant advantages over the polybiguanides in
that they exhibit higher affinity for CXCR4 and are discrete
compounds rather than heterogeneous mixtures; therefore,

262
WILKINSON ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
RR-16455 from the National Center for Research Resources. The
synthesis of the T140 and maraviroc analogs was supported by grant
AI064107 from NIAID to Edward A. Dratz.
We thank Tarin Richards for performing CCR5 inhibition assays,
Junwei Shen and Paul Grieco for the maraviroc analog, and Edward A.
Dratz for the use of his peptide synthesizer. We thank Mary Cloninger
for a gift of PAMAM G0. Cf2Th-CXCR4 cells from Joseph Sodroski
were obtained through the AIDS Research and Reference Reagent
Program, Division of AIDS, NIAID, National Institutes of Health.
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