Synthesis and anti-HIV activity of trivalent CD4-mimetic miniproteins

Article abstract of DOI:10.1016/j.bmc.2007.03.064

A series of trivalent CD4-mimetic miniproteins was synthesized, in which three CD4M9 miniprotein moieties were tethered on a threefold-symmetric scaffold. The trivalent miniproteins were designed to target the CD4-binding sites displayed in the trimeric g

Full text of DOI:10.1016/j.bmc.2007.03.064

Bioorganic & Medicinal Chemistry 15 (2007) 4220–4228
Synthesis and anti-HIV activity of
trivalent CD4-mimetic miniproteins
Hengguang Li,a Yongjun Guan,a Agnieszka Szczepanska,b Antonio J. Moreno-Vargas,b
Ana T. Carmona,b Inmaculada Robina,b George K. Lewisa and Lai-Xi Wanga,*
aInstitute of Human Virology, University of Maryland, Baltimore, MD 21201, USA
bDepartment of Organic Chemistry, University of Seville, PO Box 1203, E-41071-Seville, Spain
Received 5 February 2007; revised 16 March 2007; accepted 20 March 2007
Available online 24 March 2007
Abstract—A series of trivalent CD4-mimetic miniproteins was synthesized, in which three CD4M9 miniprotein moieties were teth-
ered on a threefold-symmetric scaffold. The trivalent miniproteins were designed to target the CD4-binding sites displayed in the
trimeric gp120 complex of HIV-1. The synthesis takes advantage of the highly efficient ligation between a cysteine-tagged
CD4M9 miniprotein and a suitable trivalent maleimide that varied in the nature and length of spacer. Antiviral assay revealed that
most of the synthetic trivalent miniproteins demonstrated significantly enhanced anti-HIV activities over the monomeric CD4M9
against both R5- and X4-tropic viruses, indicating the beneficial multivalent effects. One compound that possesses a hydrophobic
linker was shown to be 140-fold more active than CD4M9 against HIV-1Bal infection, implicating a positive contribution of the lipid
portion to the antiviral activity. It was also found that most of the trivalent miniproteins showed comparable anti-HIV activities in
comparison with a typical bivalent miniprotein, regardless of the length of the linker. The results implicate a novel mechanism of the
interactions between the multivalent inhibitors and the trimeric gp120 complex.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
ical trivalent target that presents three CD4-binding
sites in the complex. As an effort to develop more po-
tent HIV-1 entry inhibitors, we are interested in creating
multivalent inhibitors that can simultaneously target
two or three CD4-binding sites in the envelope complex.
There are ample examples in the literature demonstrat-
ing that multivalent interactions generated by a multi-
valent ligand and a corresponding multivalent target
could be collectively much stronger than the otherwise
monovalent interactions.21–26 To apply this concept to
HIV-1 inhibitor design, we have previously synthesized
bivalent CD4-mimetic miniproteins that contain two
CD4M9 moieties tethered with a spacer of varied
length.27 CD4M9 is a scorpion toxin-based 28-mer
polypeptide that binds to the Phe43 cavity of gp120
and inhibits HIV-1 infection at micro-molar concentra-
tion.10 We have found that the bivalent CD4M9
miniproteins demonstrated significantly enhanced (4-
to 21-fold) anti-HIV activity over the monomeric
CD4M9. These encouraging results prompted us to test
whether trivalent CD4-mimetic compounds will possess
further enhanced anti-HIV activities, through targeting
the three CD4-binding sites in the complex simulta-
neously. In this paper, we report the synthesis of a series
The initial step of HIV-1 entry is characterized by the
binding of HIV-1 envelope glycoprotein gp120 to the
host receptor CD4.1 It has been shown that the CD4
binding site on HIV-1 gp120 is centered on a conserved,
hydrophobic pocket denoted the ‘Phe43 cavity’.2,3 Com-
pounds that can bind to the conserved CD4-binding
pocket, thus interfering the CD4-gp120 interactions,
constitute an important class of entry inhibitors against
HIV-1 infection. These include soluble CD4, the CD4-
mimetic miniproteins, HIV-neutralizing antibody b12,
and some small-molecule compounds such as BMS-
806.4–14 Recent structural and biochemical studies have
demonstrated that the HIV-1 envelope glycoprotein
gp120 forms a trimeric complex associated with the in-
ner envelope glycoprotein gp41.15–20 Thus, the trimeric
gp120 complex of the envelope spike constitutes a typ-
Keywords: Miniprotein; CD4-mimetics; HIV; Envelope glycoprotein
gp120; Multivalent inhibitor; Entry inhibitor; CD4 binding sites;
Anti-HIV activity.
*
Corresponding author. Tel.: +1 410 706 4982; fax: +1 410 706
5068; e-mail: wangx@umbi.umd.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.064

H. Li et al. / Bioorg. Med. Chem. 15 (2007) 4220–4228
4221
of trivalent CD4-mimetic miniproteins, in which three
CD4M9 moieties were tethered to a threefold-symmet-
ric scaffold with a spacer of varied length and nature.
Antiviral assays have been performed to reveal the
structure–activity relationships of the synthetic trivalent
miniproteins.
incorporated in the trivalent miniproteins. The sche-
matic structure of the trimeric gp120 complex and the
designed trivalent CD4-mimetic miniproteins are shown
in Figure 1.
2.2. Synthesis
To assemble the designed trivalent miniproteins, the
highly efficient chemoselective ligation between malei-
mide and thiol functionality was chosen, which was suc-
cessfully used for the preparation of the bivalent
CD4M9 miniproteins.27 For the purpose, a series of
threefold-symmetric trivalent maleimides were synthe-
sized on a suitable scaffold. The first class of trivalent
maleimides was built on a cyclic triamine scaffold. Thus,
the reaction of maleimide-containing carboxylic acid
(1a–1d) and the 1,5,9-triazacyclododecane (2) in the
presence of the coupling reagent HATU/DIPEA gave
the triazacyclododecane-centered trivalent maleimides
3a–3d, respectively, in which the maleimide moieties
were spaced with varied length of linkers (Scheme 1).
The products were purified by reverse phase HPLC.
2. Results and discussion
2.1. Design
It has been demonstrated that the HIV-1 envelope gly-
coprotein gp120 is present as a trimeric complex on
the surface of virion or infected T-cells.15–20 Although
the trimeric structure has not been solved by X-ray crys-
tallography, modeling studies suggest that the trimeric
gp120 complex is threefold-symmetric.18 The distance
between any two of the CD4-binding pockets (the
Phe43 cavity) was estimated to be 30–60 A. Based on
these structural features, we sought to create threefold-
symmetric trivalent miniproteins on a scaffold. The tri-
valent miniproteins are designed to target the three
CD4-binding pockets in the trimeric gp120 complex
simultaneously. We have previously created bivalent
CD4-mimetic miniproteins using CD4M9 as the model
inhibitor and showed that the bivalent miniproteins
could have a 4- to 21-fold enhancement in the anti-
HIV activity over the monomeric CD4M9.27 Thus, to
match the distance between any two of the CD4-binding
cavities, spacers with varied length and nature will be
27
˚
Another class of trivalent maleimides was assembled on
the Kemp’s triacid core, which provides another rigid
scaffold of threefold-symmetry. Kemp’s triacid is known
in supramolecular chemistry as useful building block to
assemble peptide–peptoids structures.28,29
For the purpose, the mono-N-Boc-protected diamine
(4a–4c) with varied lengths was coupled to the Kemp’s
triacid
(5)
using
1-ethyl-3-[3-dimethylaminoiso-
30-60 A
propoyl]carbodiimide (EDCI) as the coupling agent to
give the Kemp’s triacid-centered Boc-protected triamine
derivatives 6a–6c, respectively. The Boc groups were re-
moved through treatment with aq HCl/MeOH at 0 ꢁC.
Subsequent neutralization with Na/MeOH gave the free
amino groups that were reacted with N-meth-
oxycarbonylmaleimide to provide the Kemp’s triacid-
centered trivalent maleimides (7a–7c), respectively
(Scheme 2). Similarly, the trimesic acid centered triva-
lent maleimide (8) was synthesized using the same reac-
tion sequence as described for the preparation of 7a–c,
starting with trimesic acid as the scaffold.
a
b
CD4M9
CD4-binding site
gp120
CD4M9
CD4M9
gp41
HIV
Figure 1. Schematic depiction of the trimeric gp120 complex (a) and
the template-assembled trivalent miniproteins (b) (Illustration is not to
scale).
O
O
O
N
R
O
N
H
HATU, DIPEA / THF
N
R CO2H
NH
N
+
HN
O
O
O
N
O
N
R
2
N
N
R
1a: R = (CH2)2
1b: R = (CH2)10
O
O
O
1c: R = (CH2)2CONH(CH2)2(OCH2CH2)2
1d: R = (CH2)2CONH(CH2)2(OCH2CH2)12
3a: R = (CH2)2
3b: R = (CH2)10
3c: R = (CH2)2CONH(CH2)2(OCH2CH2)2
3d: R = (CH2)2CONH(CH2)2(OCH2CH2)12
Scheme 1. Synthesis of triazacyclododecane-centered trivalent maleimides.

4222
H. Li et al. / Bioorg. Med. Chem. 15 (2007) 4220–4228
O
NH
O
O
HN
R
R
O
O
HN
O
O
HOOC
COOH
Me
O
EDCI
NH
H
COOH
R
O
N
O
N
H2N
R
+
Me
Me
H
N
O
CH2Cl2
Me
Me
H
Me
6a: R = CH2
6b: R = CH2OCH2CH2
5
4a: R = CH2
4b: R = CH2OCH2CH2
4c: R = CH2(OCH2CH2)2
6c: R = CH2(OCH2CH2)2
O
O
O
N
O
N
i) HCl, MeOH
ii) Na, MeOH, pH = 7.0
R
N
O
R
R
O
O
HN
O
O
NH
O
O
H
O
R
N
O
NH
N
iii)
N
Me
O
OMe
Me
O
H
O
H
N
O
N
R
R
Me
N
Bu4NHSO4, CHCl3
sat. NAHCO3, Et3N
N
7a: R = CH2
7b: R = CH2OCH2CH2
7c: R = CH2(OCH2CH2)2
O
O
O
O
8: R = CH2(OCH2CH2)2
Scheme 2. Synthesis of Kemp’s acid-centered trivalent maleimides.
To ligate the miniprotein CD4M9 onto the trivalent
maleimide scaffold, an extra cysteine residue with a short
spacer (GG) was introduced at the C-terminus of
CD4M9, because the C-terminus residue of CD4M9
does not directly involve in the interaction with gp120
as revealed by the modeling study.10 Our previous exper-
S
S
phosphate buffer
(pH 6.5)-/MeCN
SH
S
S
+ Trivalent maleimides
C-N-L-A-R-C-Q-L-R-C-K-S-L-G-L-L-G-K-C-A-G-S-F-C-A-C-G-P-G-G-C-NH2
Trivalent miniproteins
(1:1, v/v), r.t. (9a-9d, 10a-10c, and 11)
(3a-3d, 7a-7c, and 8)
S
S
CD4M9-SH
S
S
O
N
O
O
O
N
R
O
S
O
R
N
R
O
HN
O
O
O
N
S
NH
Me
O
H
O
O
R
O
N
N
N
N
R
N
N
R
Me
S
O
S
O
O
Me
10a: R = CH2
10b: R = CH2OCH2CH2
10c: R = CH2(OCH2CH2)2
O
9a: R = (CH2)2
9b: R = (CH2)10
9c: R = (CH2)2CONH(CH2)2(OCH2CH2)2
9d: R = (CH2)2CONH(CH2)2(OCH2CH2)12
= CD4M9
O
S
N
R
H
N
R
O
O
N
O
O
H
O
O
H
O
H
N
R
S
N
O
N
N
N
S
N
O
N
S
H
O
O
O
S
O
O
O
O
12
11: R = CH2(OCH2CH2)2
Scheme 3. Synthesis of trivalent CD4-mimetic miniproteins.

H. Li et al. / Bioorg. Med. Chem. 15 (2007) 4220–4228
4223
9b
9a
988.41
+10
1021.89
+ 10
100
50
0
100
50
0
1135.31
898.62
+11
+ 9
928.97
+ 11
1098.03
+9
823.82
+12
1277.14
+ 8
851.79
+ 12
1235.35
+8
1459.45
+ 7
800
900 1000 1100 1200 1300
m/z
800
950
1100
m/z
1250
1400
9c
9d
973.96
+ 12
1036.41
+ 10
100
50
0
100
50
0
899.09
+ 13
1062.34
+ 11
1151.51
+ 9
942.28
+ 11
1167.56
+ 10
835.01
+ 14
1295.08
+ 8
863.82
+ 12
1298.28
+ 9
1479.86
+ 7
1460.52
+ 8
800 900 1000 1100 1200 1300 1400 1500
750
1000
1250
1500
m/z
m/z
1098.51
10a
10b
988.82
+ 10
1002.17
1113.40
+ 9
100
50
0
+ 10
100
50
0
+ 9
1325.72
+ 8
1252.92
+ 8
911.66
+ 11
899.68
+ 11
1411.19
+ 7
1430.57
+ 7
835.26
+ 12
1646.70
+ 6
1669.65
+ 6
750
1000
1250
m/z
1500
1750
750
1000
1250
m/z
1500
1750
125
100
75
50
25
0
10c
11
1015.76
+ 11
1010.69
+ 10
100
50
0
1128.07
+ 9
1122.75
+ 9
923.58
+ 12
918.82
+ 11
1268.28
+ 8
1262.81
842.17
+ 12
+ 8
1449.70
+ 7
846.61
+ 13
1442.28
+ 7
750
1000
1250
m/z
1500
750
1000
1250
1500
m/z
Figure 2. The ESI-MS profiles of the synthetic miniproteins.
imental data also confirmed that addition of a tag
(GGC) at the C-terminus of CD4M9 did not influence
its anti-HIV activity.27 The ligation of the CD4M9-SH
with the synthetic trivalent maleimide scaffolds (3a–3d,
7a–7c, and 8) was performed in a phosphate buffer
(pH 6.5) containing 50% MeCN at rt, and the reaction
was monitored by HPLC. It was found that when an ex-
cess CD4M9-SH (1.3 mol equiv per maleimide moiety)
was used, the ligation reaction went to completion with-
in 1 h to give the corresponding trivalent miniproteins
(9a–9d, 10a–10c, and 11) in excellent yield (Scheme 3).
The products were easily purified by preparative HPLC,
and the identity of the trivalent miniproteins was
confirmed by ESI-MS (Fig. 2).
2.3. Anti-HIV activity of the synthetic trivalent
miniproteins
The anti-HIV activity of the synthetic miniproteins was
examined by measuring their inhibition against HIV-1
infection to PM1 cells. Two types of HIV-1 viruses were
used. One is the HIV-1Bal that uses the chemokine recep-
tor CCR5 as co-receptor for entry, and the other is the
X4-tropic virus HIV-1IIIB, which uses CXCR4 as co-
receptor for entry. The results of anti-HIV activity are
summarized in Table 1. The lengths of the spacers listed
in Table 1 were the maximum lengths calculated based
on the zigzag C–X–C bonds, which provides a rough
estimate but may not represent the actual distances

4224
Table 1. Anti-HIV activity of the synthetic compoundsa
Compound Spacer
ICb50(lM)
HIV-1IIIB
H. Li et al. / Bioorg. Med. Chem. 15 (2007) 4220–4228
scaffolds and the linkers alone without CD4M9 moiety
did not show inhibitory activity against HIV-1 infection
at the concentrations (up to 10 lM) tested. It was also
observed that the synthetic trivalent CD4M9 minipro-
teins, as well as CD4M9 itself, did exert an effect on
the host cell viability, that is, they were non toxic to
the cells at the concentrations (up to 10 lM) tested. In
contrast to the typical R5-tropic virus HIV-1Bal, the
X4-tropic virus HIV-1IIIB was found to be less sensitive
to the trivalent miniproteins, which resulted in only a
few fold enhancement in the antiviral activity for most
of the trivalent compounds. Finally, the present studies
revealed another interesting feature of the multivalent
inhibitors when the anti-HIV activities of bi- and triva-
lent miniproteins are compared. Compound 12 is a typ-
ical bivalent miniprotein that we previously synthesized,
which showed a 21-fold increase in antiviral activity
over CD4M9 in a PBMC-HIV-1IIIB assay system.27 In
the present study, it was observed that bivalent com-
pound 12 demonstrated about 10-fold enhancement in
antiviral activity in the cell line-based assay against
HIV-1Bal and HIV-1IIIB. However, except for the
compound 9b that has a hydrophobic linker, most of
the synthetic trivalent miniproteins synthesized in this
study showed very similar enhancement of anti-HIV
activity as the bivalent compound, and no further in-
crease in the anti-HIV activity was observed when
extending the valency from two to three, regardless of
the length of the spacer. These unexpected observations
suggest that the third CD4M9 moiety may not contrib-
ute much to the affinitive binding of the gp120 complex
once the first two are in action. These results may impli-
cate a novel mechanism of the interactions between the
multivalent inhibitors and the trimeric gp120 complex.
It is likely that the binding of the bi- or tri-valent mini-
proteins to the first two CD4-binding pockets in the
envelope complex causes dramatic conformational
changes or the collapse of the gp120 trimeric complex,
thus making the third CD4-binding site inaccessible to
the third arm of CD4M9 in the trivalent miniproteins.
entity
length (max)
HIV-1Bal
(relative activity) (relative activity)
CD4M9
9a
9b
N/A
˚
9.7 (1)
12 (1)
18 A
˚
1.5 (6.5)
0.068 (142)
1.3 (7.5)
ndc
3.0 (4)
0.40 (30)
2.5 (4.8)
1.9 (6.3)
ndd
36 A
˚
43 A
˚
138 A
9c
9d
˚
10a
10b
10c
11
19 A
0.79 (12)
1.8 (5.4)
1.0 (9.7)
0.33 (29)
0.95 (10)
0.013 (746)
ndd
˚
22 A
ndd
˚
29 A
ndd
˚
30 A
˚
32 A
N/A
12
sCD4
1.3 (9.2)
0.005 (2400)
a Virus inhibition was performed using PHA-stimulated PM1 the host
cells infected by HIV-1BaL or HIV-1IIIB
.
b The IC50 was defined as the concentrations that reach 50% inhibition
of viral production, and the numbers in the parenthesis indicate the
relative activity to CD4M9 based on the measured IC50
.
c nd (not determined), because no dose response was observed for this
compound.
d Data were not determined.
because of conformational variants. The antiviral stud-
ies with the synthetic miniproteins and HIV-1Bal
revealed several interesting features of the structure–
activity relationships for the synthetic compounds. First
of all, most of the trivalent miniproteins showed en-
hanced anti-HIV activity over the monomeric inhibitor
CD4M9, indicating the beneficial effects of multivalent
interactions generated by the trivalent ligands. Second,
it seems that the length of the linker that separates the
CD4M9 moieties in the miniproteins could be relatively
flexible for the anti-HIV activity. For example, no sig-
nificant difference in the anti-HIV activities was ob-
served between compounds 9a and 9c, or those
between 10a, 10b, and 10c, although the length of the
˚
linker varied dramatically from 20 to 40 A. However,
if the spacer is too long, the multivalent effect would
be lost, as in the case of compound 9d (with a spacer
˚
length of 138 A) that did not show enhancement of
anti-HIV activity. Considering the size of the CD4M9,
3. Conclusion
˚
which would extend ca 10 A from the tethering Cys to
˚
the Phe group, a linker of 20–40 A in length will place
any two of the Phe residues in the trivalent CD4M9
The synthesis of a series of trivalent CD4-mimetic
miniproteins was described, in which three CD4M9
miniprotein moieties were tethered on a threefold-sym-
metric scaffold. Antiviral assays revealed that most of
the trivalent miniproteins showed enhanced anti-HIV
activity over the monomer CD4M9, indicating the gain
of affinity through multivalent interactions. The
structure–activity relationship studies suggested that
not only the length, but also the nature (hydrophobic
versus hydrophilic) of the linker was important for the
enhancement of antiviral activities of the trivalent mini-
proteins, with one compound that has a hydrophobic
linker being 140-fold more active than CD4M9 against
HIV-1Bal infection. Moreover, the similarity in activity
enhancement between most corresponding bi- and triva-
lent miniproteins may reveal a novel mechanism of
interactions between the multivalent ligands with the
trimeric gp120 complex. It is likely that the binding
of the bi- or trivalent miniproteins to the first two
˚
miniproteins in the range of 40–60 A. This distance of
separation could match any two sites of the Phe43 cavity
on the trimeric gp120, which were estimated to be sepa-
˚
rated by 30–60 A. Taken together, the experimental data
provide at least a piece of indirect evidence suggesting a
dynamic nature of the trimeric gp120 complex. Third,
compound 9b, which has a hydrophobic linker, was
found to be over 140-fold more active than CD4M9
against HIV-1Bal infection. It was also significantly more
active than other trivalent miniproteins with a hydro-
philic linker of similar length. In addition, compound
11, which has a hydrophobic aromatic core, also showed
a 30-fold enhancement in antiviral activity. These results
suggest that a hydrophobic tail attached to CD4M9 may
offer additional beneficial effects in enhancing the antivi-
ral activity. This is an interesting issue that deserves fur-
ther investigations. It should be pointed out that the

H. Li et al. / Bioorg. Med. Chem. 15 (2007) 4220–4228
4225
CD4-binding sites in the complex causes dramatic con-
formational changes of the gp120 trimeric complex, thus
making the third CD4-binding site inaccessible to the
third CD4M9 moiety in the trivalent miniproteins. A
more detailed structural and mechanistic study is re-
quired to clarify this point.
12H, tricyclicamino-NCH2CH2), 3.89 (t, 6H,
J = 5.0 Hz, maleimido-NCH2), 6.75 (s, 6H, maleimido-
COCH@CH). 13C (125 MHz, CDCl3):
162.18, 134.50, 70.23, 69.79, 39.29, and 34.44. ESI-MS
d 170.32,
calcd M = 624.64; Found: 625.22 (M + H)+.
1
4.2.2. Trivalent maleimide (3b). Yield, 53%; H NMR
(500 MHz, CDCl3): d 1.31 (s, 18H CH2(CH2)3CH2),
1.61 (t, 6H, J = 6.5 Hz, maleimido-NCH2CH2), 1.64 (t,
6H, J = 5.0 Hz, COCH2CH2), 2.04 (t, 6H, J = 7.5 Hz,
tricyclicamino-NCH2CH2), 2.36 (t, 6H, J = 7.5 Hz, tri-
cyclicamino-NCH2CH2), 3.47 (t, 6H, J = 6.0 Hz, N
CH2CH2), 3.55 (t, 6H, J = 7.5 Hz, COCH2CH2), 6.73
(s, 6H, maleimido-COCH@CH). 13C (125 MHz, CDCl3)
d 211.68, 170.93, 134.07, 110.00, 37.95, 33.51, 29.41,
29.09, 28.54, 26.74, and 26.66. ESI-MS calcd
M = 960.63; Found: 961.42 (M + H)+, 481.20
4. Experimental
4.1. General methods
1H and 13C NMR spectra were recorded on Inova 500
NMR in CDCl3 unless otherwise specified. Chemical
shifts are expressed in ppm downfield using external
Me4Si (0 ppm) as the reference. The ESI-MS spectra were
measured on a micromass ZQ-400 single quadrupole
mass spectrometer. FABMS were measured with a Kra-
tos MS-80-RFA instrument; for HR-FABMS a Micro-
mass AutoSpeQ instrument was used. NaI was used as
cationizing agent and, thioglycerol (TG) or nitrobenzy-
lic alcohol (NBA) as matrix. TLC was performed on
glass plates coated with silica gel 60 F254 (E. Merck).
Flash column chromatography was performed on silica
gel 60 (EM Science, 230–400 mesh). Analytical HPLC
was carried out on a Waters 626 HPLC instrument
(M + 2H)2+
.
4.2.3. Trivalent maleimide (3c). Yield, 65%; 1H NMR
(500 MHz, CDCl3): d 2.04 (t, 6H, J = 6.0 Hz, tricycli-
camino-NCH2CH2), 2.61 (t, 6H, J = 7.0 Hz, tricyclica-
mino-NCOCH2), 2.67 (t, 6H, J = 6.0 Hz, maleimido-
NCH2CH2CO), 3.45 (q, 6H, J = 5.0 Hz, CONHCH2),
3.50 (t, 6H, J = 6.0 Hz, tricyclicamino-NCOCH2-
CH2O), 3.56 (t, 12H, J = 4.5 Hz, tricyclicamino-
NCH2CH2), 3.64 (t, 12H, J = 5.0 Hz, OCH2CH2O),
3.85 (t, 6H, J = 6.0 Hz, OCH2CH2CON), 3.89 (t,
6H, J = 7.5 Hz, maleimido-NCH2), 6.74 (s, 6H,
maleimido-CH@CH), 6.84 (s, 3H, NH). 13C
(125 MHz, CDCl3) d 170.56, 134.25, 70.24, 67.50,
39.29, 34.44, and 33.51. ESI-MS calcd M = 1101.52;
Found: 1124.26 (M + Na)+, 1102.27 (M + H)+,
equipped with
a Waters Nova-Pak C18 column
(3.9 · 150 mm) at 40 ꢁC. The column was eluted with
a linear gradient of 0–90% MeCN containing 0.1%
TFA at a flow rate of 1 mL/min over 20 min. Com-
pounds were detected at 214 nm. Preparative HPLC
was performed on a Waters 600 HPLC instrument with
a Waters C18 preparative column (Symmetry 300,
19 · 300 mm). The column was eluted with a suitable
gradient of MeCN containing 0.1% TFA at 10 mL/
min. The maleimide-acids (1a and 1 b) were obtained
from Pierce Biotechnology Inc. (Rockford, IL). The
PEG linked maleimide-acids (3 and 4) were from
Quanta Biodesign (Powell, OH). The miniprotein
CD4M9-SH (CD4M9-GGC) was prepared as previ-
ously described.27 Other chemicals were purchased
from Aldrich/Sigma and used as received.
551.79 (M + 2H)2+
.
1
4.2.4. Trivalent maleimide (3d). Yield, 48%; H NMR
(500 MHz, CDCl3): d1.98 (t, 6H, J = 6.0 Hz, tricyclica-
mino-NCH2CH2), 2.53 (t, 6H, J = 7.0 Hz, tricyclicami-
no-NCOCH2), 2.63 (t, 6H, J = 6.0 Hz, maleimido-
NCH2CH2CO), 3.42 (q, 12H, J = 5.0 Hz, CON-
HCH2CH2), 3.49 (t, 6H, J = 6.0 Hz, tricyclicamino-
NCOCH2CH2O), 3.55 (t, 6H, J = 5.0 Hz, OCH2CH2-
CONH), 3.62 (t, 12H, J = 2.0 Hz, tricyclicamino-
NCH2CH2), 3.64 (m, 120H, OCH2CH2O), 3.78 (t, 6H,
J = 6.5 Hz, maleimido-NCH2), 3.84 (t, 6H, J = 7.0 Hz,
6.45 (s, 3H, NH), 6.71(s, 6H, maleimido-CH@CH).
4.2. Synthesis of the triazacyclododecane-centered
trivalent maleimides (3a–3d)
To a solution of the maleimide acid 1 (0.8 mmol) and
triazacyclododecane (2) (0.2 mmol) in DMF (10 mL)
were added HATU (0.8 mmol) and DIPEA (0.8 mmol).
The mixture was stirred at rt for 2 h and then diluted
with EtOAc (60 mL). The mixture was then washed with
brine and water. The organic layer was dried over
Na2SO4, filtered, and the filtrate was subjected to silica
gel flash column chromatography using CH2Cl2/MeOH
(99:1 to 90:10, v/v) to give the respective trivalent malei-
mide. The products were further purified by preparative
HPLC (gradient: 20–40% MeCN containing 1% TFA in
30 min; flow rate 10 mL/min) to give pure triazacyclod-
odecane-centered trivalent maleimides (3a–3d).
ESI-MS
(M + 2Na)2+, 1212.50 (M + 2H)2+, 808.62 (M + 3H)3+
606.77 (M + 4H)4+, 485.45 (M + 5H)5+
calcd
M = 2423.77;
Found:
1234.05
,
.
4.3. Synthesis of Kemp’s triacid (KTA)-centered
Boc-protected triamine derivatives (6a–6c)
To a suspension of Kemp’s triacid (KTA) (5) (0.5 mmol)
in dry CH2Cl2 at 0 ꢁC, EDCI (1.65 mmol) was
added. Then, the corresponding mono-N-Boc-protected
diamine (4) (1.65 mmol) were added dropwise to the
suspension. The resulting mixture was stirred at rt over-
night. The reaction mixture was washed with water, and
the organic phase was dried over anhydrous Na2SO4, fil-
tered, and the filtrate was concentrated to dryness. The
residue was purified by column chromatography on sil-
ica gel using CH2Cl2 containing MeOH (3% ! 5%) as
1
4.2.1. Trivalent maleimide (3a). Yield, 62%; H NMR
(500 MHz, CDCl3): d 1.99 (m, 6H, tricyclicamino-
NCH2CH2), 2.70 (t, 6H, J = 5.0 Hz, COCH2), 3.47 (m,

4226
H. Li et al. / Bioorg. Med. Chem. 15 (2007) 4220–4228
the eluent to give the respective Boc-protected triamine
derivative (6a–6c).
separated, dried over anhydrous Na2SO4, and filtered.
The filtrate was concentrated and the residue was sub-
ject to column chromatography on silica gel using
CH2Cl2 containing MeOH (3% ! 5%) as the eluent
to provide the KTA-centered trivalent maleimides
(7a–7c).
4.3.1. KTA-(N-t-butoxycarbonyl-ethylenediamine)3 (6a).
Yield, 64% (as a colorless oil). 1H NMR (300 MHz,
CDCl3): d 1.05 (d, 1H, J = 15.6 Hz), 1.21 (s, 3H), 1.43
(s, 9H), 2.86 (d, 1H, J = 15.6 Hz), 3.21 (bs, 4H), 5.84
(bs, 1H), 7.43 (bs, 1H). 13C NMR (75 MHz, CDCl3), d
28.4, 34.4, 40.1, 40.8, 42.2, 43.3, 79.2, 156.0, and
177.4. FABMS (TG): 385 (12) [M ꢀ 3Boc + H]+, 407
(47) [M ꢀ 3Boc + Na]+, 485 (5) [M ꢀ 2Boc + H]+, 585
(23) [M ꢀ Boc + H]+, 685 (12) [M + H]+, 707 (100)
[M + Na]+. HR-FABMS calcd [M + Na]+C33H60N6O9-
Na 707.4319; Found: 707.4329.
4.4.1. KTA-(1-maleimide-ethylenamine)3 (7a). Yield, 47%
1
(as a colorless oil). H NMR (300 MHz, CDCl3), d 1.00
(d, 1H, J = 15.6 Hz), 1.13 (s, 3H), 2.73 (d, 1H,
J = 15.6 Hz), 3.25 (dAB, 2H, JAB = 5.7 Hz), 3.60 (t, 2H,
J = 5.7 Hz), 6.71 (s, 1H), 7.44 (t, 1H, J = 5.7 Hz). 13C
NMR (75 MHz, CDCl3), d 33.5, 37.1, 38.0, 42.1, 43.0,
134.1, 170.6, 177.7. FABMS (NBA): 647 (21)
[M + Na]+. HR-FABMS calcd [M + Na]+C30H36N6O9-
Na 647.2441; Found: 647.2445.
4.3.2. KTA-(1-t-Butoxycarbonylamino-3-oxa-pentan-5-
amine)3 (6b). Yield, 52% (as a colorless oil). H NMR
1
(300 MHz, CDCl3) d: 1.07 (d, 1H, J = 15.6 Hz), 1.24
(s, 3H), 1.43 (s, 9H), 2.95 (d, 1H, J = 15.3 Hz),
3.20–3.41 (m, 4H), 3.47 (t, 2H, J = 5.1Hz), 3.54 (t, 2H,
J = 5.1Hz), 6.49 (bs, 1H), 7.68 (t, 1H, J = 4.3 Hz). 13C
NMR (75 MHz, CDCl3) d: 28.5, 34.5, 40.1, 40.6, 42.3,
43.1, 68.7, 70.1, 78.7, 156.4, 177.2. FABMS (TG): 717
(8) [M ꢀ Boc + H]+, 817 (9) [M + H]+, 839 (23)
[M + Na]+. HR-FABMS calcd [M + Na]+C39H72N6O12-
Na 839.5106; Found: 839.5114.
4.4.2. KTA-(1-maleimide-3-oxa-pentan-5-amine)3 (7b).
Yield, 53% (as a colorless oil). 1H NMR (300 MHz,
CDCl3), d 1.01 (d, 1H, J = 15.6 Hz), 1.18 (s, 3H), 2.82
(d, 1H, J = 15.6 Hz), 3.20 (dAB, 2H, JAB = 6.0Hz), 3.43
(t, 2H, J = 6.0Hz), 3.65 (A2B2 system, 4H), 6.70 (s,
1H), 7.44 (t, 1H, J = 5.4 Hz). 13C NMR (75 MHz,
CDCl3), d 34.0, 37.1, 39.3, 42.1, 43.0, 67.6, 68.6, 134.1,
170.6, 177.1. FABMS (NBA): 779 (11) [M + Na]+, 811
(23)
[M + Na + MeOH]+.
HR-FABMS
calcd
[M + Na]+C36H48N6O12Na 779.3228; Found: 779.3269.
4.3.3. KTA-(1-t-Butoxycarbonylamino-3,6-dioxa-octan-
8-amine)3 (6c). Yield, 68% (as
a
colorless oil).
1.05 (d, 1H,
4.4.3. KTA-(1-maleimide-3,6-dioxa-octan-8-amine)3 (7c).
Yield, 56% (as a yellow oil). 1H NMR (300 MHz,
CDCl3), d 1.04 (d, 1H, J = 15.6 Hz), 1.20 (s, 3H), 2.87
(d, 1H, J = 15.3 Hz), 3.26 (dAB, 2H, JAB = 5.7 Hz), 3.45
(t, 2H, J = 5.7 Hz), 3.58 (A2B2 system, 6H), 3.69 (A2B2
system, 2H), 6.72 (s, 1H), 7.52 (t, 1H, J = 5.4 Hz). 13C
NMR (75 MHz, CDCl3), d 34.2, 37.1, 39.4, 42.2, 43.1,
67.8, 69.2, 69.9, 70.2, 134.2, 170.6, 177.3. FABMS
(NBA): 889 (6) [M + H]+, 911 (91) [M + Na]+. HR-FAB-
MS calcd [M + Na]+C42H60N6O15Na 911.4014; Found:
911.4047.
1H NMR (300 MHz, CDCl3),
d
J = 15.3 Hz), 1.20 (s, 3H), 1.44 (s, 9H), 2.87 (d, 1H,
J = 15.6 Hz), 3.25–3.38 (m, 4H), 3.49 (t, 2H,
J = 6.3 Hz), 3.55 (t, 2H, J = 5.1Hz), 3.61 (bs, 4H),
5.23 (bs, 1H), 7.56 (t, 1H, J = 5.1Hz). 13C NMR
(75 MHz, CDCl3) d: 28.4, 34.2, 39.4, 40.4, 42.2, 43.1,
69.2, 70.2, 70.3, 79.1, 156.0, 177.3. FABMS (TG):
671 (17) [M ꢀ 3Boc + Na]+, 971 (65) [M + Na]+.
HR-FABMS
calcd
[M + Na]+C45H84N6O15Na
971.5892; Found: 971.5869.
4.4. Synthesis of the KTA-centered trivalent maleimides
(7a–7c)
4.5. Synthesis of the trimesic acid-centered trivalent
maleimide (8)
The respective N-Boc protected triamine 6 (0.2 mmol)
was dissolved in MeOH and cooled to 0 ꢁC. To the
solution was added dropwise concd HCl (2.2 mL).
The reaction mixture was stirred at 0 ꢁC until con-
sumption of the starting material (monitored by
TLC). The reaction mixture was concentrated and the
residue was dissolved again in MeOH and neutralized
with Na to pH 7.0. The formed NaCl was filtered off
and the filtrate dried over anhydrous Na2SO4 and con-
centrated to provide the corresponding free triamine,
which was used for the next step without further puri-
fication. The respective triamine (0.20 mmol) was dis-
solved in CHCl3 (10 mL) and cooled to 0 ꢁC. To the
solution were added tetrabutylammonium hydrogen
sulfate (1.38 mmol) and N-methoxycarbonylmaleimide
(1.38 mmol). Then triethylamine (97 ll) was added
slowly, and the resulting mixture was stirred at 0 ꢁC
for 10 min. Aq satd NaHCO3 solution (10 mL) was
then added in one portion and the mixture was stirred
vigorously at rt for 4 h. The organic layer was
The trimesic acid-based trivalent maleimide (8) was
synthesized in the same way as described for the
preparation of the KTA-centered trivalent maleimides
(7a–7c), using trimesic acid as the template. The triva-
lent maleimide (8) was obtained in 48% yield as a yel-
1
low oil. H NMR (300 MHz, CDCl3), d 3.55–3.76 (m,
12H), 6.68 (s, 1H), 7.26 (bm, 1H), 8.47 (s, 1H). 13C
NMR (75 MHz, CDCl3), d 37.1, 40.1, 67.9, 69.7,
70.0, 70.2, 128.5, 134.1, 135.1, 166.0, 170.7. FABMS
(NBA): 841 (5) [M + H]+, 863 (24) [M + Na]+.
HR-FABMS calcd [M + Na]+C39H48N6O15Na 863.3075;
Found: 863.3099.
4.6. Synthesis of the trivalent CD4-mimetic miniproteins
(9a–9d, 10a–10c, and 11)
To a solution of CD4M9-SH (8 lmol) in a degassed
phosphate buffer (50 mM, pH 6.5) containing 50%
MeCN (3 mL) was added a solution of the respective
trivalent maleimide (3a–3d, 7a–7c, or 8) (2 lmol) in

H. Li et al. / Bioorg. Med. Chem. 15 (2007) 4220–4228
4227
MeCN (0.2 mL). The resulting mixture was gently sha-
ken under N2 at rt, and the ligation reaction was moni-
tored with analytical HPLC, which indicated the
completion of ligation within 1 h. The mixture was
lyophilized and the residue was subjected to preparative
HPLC to give the respective trivalent CD4-mimetic
miniproteins (9a–9d, 10a–10c, and 11).
described in the general method); ESI-MS calcd M =
10090.12; Found: 1442.28 (M + 7H)7+
1262.81
1010.69 (M +
,
(M + 8H)8+
,
1122.75 (M + 9H)9+
,
10H)10+, 918.82 (M + 11H)11+, 842.17 (M + 12H)12+
.
4.7. Anti HIV-1 assay
PM1 cells (2 · 104/well) were exposed to 100 TCID50
viruses of HIV-1Bal or HIV-1IIIB strain, which were pre-
treated with serial dilutions of compounds for 1 h at
37 ꢁC. After 2 h incubation in 200 ll/well of complete
RPMI1640 medium, the cells were washed twice and
cultured in complete RPMI1640 medium supplemented
with the appropriate compounds. The p24 concentra-
tions of day 7 supernatants were quantified with a sand-
wich ELISA. The antiviral activities of the trivalent
CD4-mimetic miniproteins and related compounds were
expressed as concentrations that reach the 50% inhibi-
tion (IC50) of viral production.
4.6.1. Trivalent miniprotein (9a). Yield, 90%; tR 13.60
min (Under the analytical HPLC condition described
in the general method); ESI-MS calcd M = 9873.68;
Found: 1646.77 (M + 6H)6+, 14115.32 (M + 7H)7+
,
1235.29 (M + 8H)8+
(M + 10H)10+
898.69
(M + 12H)+12, 760.54 (M + 13H)13+
,
1098.16 (M + 9H)9+
,
988.42
823.88
,
(M + 11H)+11
,
.
4.6.2. Trivalent miniprotein (9b). Yield, 92%; tR
12.40 min (Under the analytical HPLC condition
described in the general method); ESI-MS calcd
M = 10210.76; Found: 1702.56 (M + 6H)6+, 1459.43
(M + 7H)7+, 1277.13 (M + 8H)8+, 1135.35 (M + 9H)7+
,
1021.95 (M + 10H)10+, 929.24 (M + 11H)11+, 851.87
(M + 12H)12+, 786.42 (M + 13H)13+
Acknowledgments
.
The work was supported in part by the European
Commission (project TRIoH, FP6 Program, contract
LSHG-CT-2003-503480 to I.R.), the ‘Ministerio de
4.6.3. Trivalent miniprotein (9c). Yield, 87%; tR
10.37 min (Under the analytical HPLC condition de-
scribed in the general method); ESI-MS calcd
M = 10354.67; Found: 1479.98 (M + 7H)7+, 1295.15
´
Educacion y Ciencia’, Spain (Grant CTQ2004-00649/
BQU to I.R.), and the U.S. Department of Health and
Human Services (NIH Grant AI51235 to L.X.W.).
(M + 8H)8+
,
1151.45 (M + 9H)9+
,
1036.35 (M +
.
10H)10+, 942.34 (M + 11H)11+, 863.82 (M + 12H)12+
4.6.4. Trivalent miniprotein (9d). Yield, 86%; tR
11.41 min (Under the analytical HPLC condition
described in the general method); ESI-MS calcd
M = 11675.25; Found: 1460.39 (M + 8H)8+, 1298.28
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