Evaluation of chelating agents as anti-angiogenic therapy through copper chelation

Article abstract of DOI:10.1016/j.bmc.2004.07.034

A set of novel polyamine hexadentate cis,cis-1,3,5,-triaminocyclohexane (tach) chelating agents were synthesized and evaluated in conjunction with a selection of both linear and macrocyclic polyamines as copper chelators for novel anti-angiogenic therapy in an in vitro endothelial cell proliferation assay to assess their cytotoxicity and selectivity. Macrocyclic polyamine 15 exhibited the greatest selective activity in this assay while the tach based ligands exhibited cytotoxicity, but no selectivity. The evaluation of several sets of polyamine donor chelating agents including a selection of novel hexadentate 1,3,5-cis,cis-triaminocyclohexane (tach) based derivatives were performed in an in vitro endothelial cell proliferation assay to assess their cytotoxicity and selectivity as novel anti-angiogenic agents. The selective nature of the anti-angiogenic agents for human umbilical vein endothelial cells (HUVEC) was compared to a normal fibroblast cell line and a human Glioma cell line to evaluate these compounds. Linear tri- and tetra-polyamines were superior to both macrocyclic and the tach based polyamine chelating agents in terms of selectivity of its inhibitory activity toward the proliferation of HUVEC cells compared to the fibroblast and human Glioma cells. The linear polyamine, triethylenetetramine (22), previously reported to possess anti-angiogenic properties failed to demonstrate any selectivity for inhibiting the proliferation of HUVEC cells compared to the fibroblast and human Glioma cells.

Full text of DOI:10.1016/j.bmc.2004.07.034

Bioorganic & Medicinal Chemistry 12 (2004) 5133–5140
Evaluation of chelating agents as anti-angiogenic therapy
through copper chelation
Kevin Camphausen, Mary Sproull, Steve Tantama, Vincent Venditto, Sandeep Sankineni,
Tamalee Scott and Martin W. Brechbiel*
Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health,
10 Center Drive, Building 10, Room B3B69, Bethesda, MD 20892-1002, USA
Received 11 February 2004; accepted 15 July 2004
Available online 11 August 2004
Abstract—The evaluation of several sets of polyamine donor chelating agents including a selection of novel hexadentate 1,3,5-cis,cis-
triaminocyclohexane (tach) based derivatives were performed in an in vitro endothelial cell proliferation assay to assess their cyto-
toxicity and selectivity as novel anti-angiogenic agents. The selective nature of the anti-angiogenic agents for human umbilical vein
endothelial cells (HUVEC) was compared to a normal fibroblast cell line and a human Glioma cell line to evaluate these com-
pounds. Linear tri- and tetra-polyamines were superior to both macrocyclic and the tach based polyamine chelating agents in terms
of selectivity of its inhibitory activity toward the proliferation of HUVEC cells compared to the fibroblast and human Glioma cells.
The linear polyamine, triethylenetetramine (22), previously reported to possess anti-angiogenic properties failed to demonstrate any
selectivity for inhibiting the proliferation of HUVEC cells compared to the fibroblast and human Glioma cells.
Published by Elsevier Ltd.
1. Introduction
genesis that bind copper with high affinity include fibro-
blast growth factor (FGF) and vascular endothelial
growth factor (VEGF).8,9 Furthermore, in the rabbit
corneal assay frequently employed to assess angiogenic
activity, both CuSO4 and ceruloplasmin (a copper trans-
port containing protein) can stimulate vessel growth
while the noncopper binding form of ceruloplasmin
lacked activity.10,11 The first studies in animal tumor
models, which linked impeded angiogenesis and there-
fore tumor growth with reduced copper levels were con-
ducted using penicillamine, an early and routinely
employed chelation therapy agent, and/or a low copper
diet.12,13 The efficacy of copper binding agents in the
treatment of human disease has been well demonstrated
in the treatment of Wilsonꢀs disease an inherited meta-
bolic disorder resulting in improper copper sequestra-
tion and toxicity in the body. Thus, the precedent for
copper sequestration therapy has significant standing
and clinical experience of patient tolerance undergoing
such therapy is well established.
In the normal adult, the process of angiogenesis is
tightly regulated that occurs during wound healing and
in the proliferating endometrium during menstruation.1
However, in several pathologic conditions including
tumor formation, diabetic retinopathy, and rheumatoid
arthritis angiogenesis plays a key role.1 The proliferating
endothelium can be targeted through anti-angiogenic
drug therapy in an effort to arrest aberrant growth of
vessels.2 Though there are several known targets for
anti-angiogenic therapy, one of the most intriguing is
the proliferating endothelial cellꢀs requirement for cop-
per as a co-factor in its cellular process.3 Therefore,
potential anti-angiogenic agents might be determined
by their ability to decrease the amount of available cop-
per at the endothelial level.4,5
The body of evidence supporting the importance of cop-
per in tumor formation is sizable, but also somewhat
inferential. Biochemical studies have demonstrated
higher levels of copper in tumor tissue as compared to
nontumor tissue.6,7 Also, multiple stimulators of angio-
Current therapies for Wilsonꢀs disease includes the use
of copper binding molecules such as penicillamine and
more recently tetrathiomolybdate (TM) along with also
the use of zinc salts to alter the biological distribution of
+2 metal ions and eliminate Cu(II).14,15 Combining the
anti-tumor observations in animals and the clinical
*
Corresponding author. Fax: +1-301-402-1923; e-mail: martinwb@
mail.nih.gov
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2004.07.034

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K. Camphausen et al. / Bioorg. Med. Chem. 12 (2004) 5133–5140
utility of TM in patients with Wilsonꢀs disease provided
impetus for clinical trials using TM in patients with met-
astatic cancer. Early results have demonstrated reduced
copper levels and that the drug is well tolerated during
the treatment.16
In our previous studies we had focused on the screening
of a small select series of cis,cis-N,N0,N00-1,3,5,-triamino-
cyclohexane (tach) compounds for anti-angiogenic
activity in an in vitro endothelial cell proliferation assay
to assess their cytotoxicity and selectivity.17 These previ-
ously evaluated compounds were primarily polyamino-
pyridyl compounds such as N,N0,N00-tris(2-pyrid-
ylmethyl)-cis,cis-1,3,5,-triaminocyclohexane (tachpyr)
that had shown significant cytotoxicity against cancer
cell lines when evaluated as iron depletion therapy
drugs.18 Due to their metal binding cavity sizes and that
these compounds had also demonstrated significant sta-
bility as Cu(II) complexing agents,19 they and some re-
lated compounds were studied for anti-angiogenic
activity. The results of the previous studies in fact elim-
inated these specific compounds, not on the basis of low
cytotoxicity, but rather on a lack of selective cytotoxic-
ity and thus a lack of anti-angiogenic activity.17 Recent
efforts to determine intracellular metal ion selectivity of
tachpyr compounds has demonstrated that their activity
stems primarily from iron and zinc sequestration, and
not from significant copper complex formation.20 Sur-
prisingly, the compounds that emerged from our previ-
ous study as potential leads were acyclic analogs of
tachpyr that had demonstrated low or no biological
activity as iron sequestration agents.17 Additionally,
N,N0,N00-tri(2-mercaptoethyl)-cis,cis-1,3,5-triaminocyclo-
hexane (tachENSH), the sole tach based thiol donor
compound, emerged as the overall lead compound.17
Continued evaluation of this compound and its direct
analogs are an ongoing study.
Figure 1. Synthesis of novel hexadentate 1,3,5-cis,cis-triaminocyclo-
hexane (tach) based chelating agents for evaluation of their selective
inhibition of the proliferation of HUVEC cells as compared to
fibroblast and human Glioma cells.
basis an examination of permutation of donor number
and arrangement was deemed important to include in
this study.5
Due to the limited number of compounds previously
examined and therefore limited conclusive data, the cur-
rent study focuses principally on polyamine chelating
agents wherein the donor geometry and number are
the variables (Figs. 1 and 2). Donor geometry includes:
(1) a selection of tach based hexadentate polyamine lig-
ands to assess this pre-organized donor geometry in con-
junction with this donor set; (2) a set macrocyclic
ligands wherein both the donor number and binding
cavity is varied to isolate optimal copper chelation
chemistry for this specific application; and (3) a selection
of acyclic polyamines with varying donor number and
spatial arrangement. The first set was prepared in part
for this study (Fig. 1) to provide a unique group of hex-
amine donor ligands wherein an octahedral geometry
within a pseudo-adamantyl carbon cage about the metal
is formed that has been well characterized.19,21 To this
end, seven compounds of this category are included with
varying alkyl and aryl substituents. The second set of
compounds were chosen based on their association with
Cu(II) complexation use in radiopharmaceutical devel-
opment as well as in Cu(II) metal extraction applica-
tions.22–26 The third set of compounds was included
because there had been previous reports of simple linear
polyamines having anti-angiogenic activity and on that
The determining characteristic of a compound that iden-
tifies it as an anti-angiogenic agent is selective cytostasis
of endothelial cells excluding all other cell types.27 To
evaluate the ability of all the chelating agents in this re-
port to function as anti-angiogenic agents, their ability
to selectively inhibit the proliferation of human umbili-
cal vein endothelial cells (HUVEC) in vitro while not
inhibiting the proliferation of nonendothelial cells was
determined. A normal fibroblast cell line (NIH3T3)
and a human glioma tumor cell line (U251) were utilized
as control nonendothelial cell types. Therefore, a com-
pound that demonstrates growth inhibition of the HU-
VEC cell line at a concentration significantly lower
then the concentration needed to inhibit the normal
and tumor cell lines may be a promising lead compound
for further study as an anti-angiogenic agent.
Herein we report the design and synthesis of novel tach
based hexadentate hexamine chelating agents, their bio-
logical evaluation as potential anti-angiogenic agents,
and the biological evaluation of a select set of previously
reported or commercially available acyclic and cyclic
polyamines.

K. Camphausen et al. / Bioorg. Med. Chem. 12 (2004) 5133–5140
5135
were also prepared as their single enantiomers to elimi-
nate issues pertaining to steric hindrance in metal ion
complex formation as the structures of these complexes
tend to form with varying degrees of spiral character
and that an inappropriately arranged substituent would
be expected to disrupt complexation of metal ions.21
A
single polyaminocarboxylate class ligand, 7, was in-
cluded as well as we had previously reported prepara-
tion of its respective Cu(II) complex.28
The macrocyclic and linear polyamines chosen for
assessment were either commercial in origin or had been
previously prepared as reported.21–25,28–31 The macrocy-
clic chelating agents were chosen based upon ring cavity
size and because cyclam derivatives 14 and 15 and
numerous related derivatives had also been investigated
for chelating Cu(II) radionuclides of interest in nuclear
medicine applications wherein exceptionally strong com-
plexation of the metal ions is a requirement. The two
larger polyamine macrocycles 16 and 17 were chosen
to examine what impact size of the ring and number
of metal binding donors would have on biological activ-
ity.
A selection of linear amines was included in this study
since 22 had been previously reported to exert an anti-
angiogenic effect on tumor.5 To examine some of the
variables surrounding this reported activity, this selec-
tion of analogs varied the number donor amines, the
carbon framework spacing between the amines, and in
some respect geometric organization of the amines.
Figure 2. Linear and macrocyclic tri-, tetra-, penta-, and hexa-amine
chelating agents evaluated for their selectivity of in inhibitory activity
of the proliferation of HUVEC cells as compared to fibroblast and
human Glioma cells.
All the chelating agents were evaluated in a cell prolifer-
ation assay whereby normal, tumor, and endothelial
cells were treated with various concentrations of each
respective agent.17 The efficacy of each compound as a
potential anti-angiogenic agent was determined by the
relative level of cytotoxicity exhibited specific to endo-
thelial cells versus normal and/or tumor cells. While the
sequestration of Cu(II) has been hypothesized as the
potential mode of action being evaluated, actual deter-
mination of the metal(s) being complexed has not been
performed. Thus, the possibility that chelation of other
metal ions such as Zn(II) cannot be ruled out as being
involved. However, as application of the experimental
design to exploit differential sensitivity between HUVEC
and other cell lines specifically suggests Cu(II) complex-
ation as the mode of action. In support of this interpre-
tation, the determination of intracellular metal ion
selectivity of tachpyr compounds demonstrated that
their activity originates primarily from iron and zinc
chelation, and not from significant copper complex for-
mation.20 This directly correlates with the results of non-
specific toxicity against all of the cell lines in the assay
including HUVEC.
2. Results and discussion
The synthesis of the novel tach based ligands 3–5 (Fig.
1) was performed analogously to that of the previously
prepared compounds 1, 2, and 6, with some small vari-
ations to adjust for solubility of intermediates during
isolation. Thus, reaction of the free base of tach gener-
ated in situ with the selected carbamate protected amino
acid active ester cleanly provided the tris-amide adducts
that could be isolated either by precipitation of the
product or by an extractive process. The carbamate pro-
tection groups were removed by treatment with acid and
the amides reduced with diborane that after acidic work
up provided the desired tris-substituted ethylenediamine
arm derivates of tach. No attempts were made to optim-
ize yields of methods and all of the tach based ligands
were carried forward to biological evaluation as their
respective hexa-hydrochloride salts.
The tach based ligands therefore provided an assessment
of various donor patterns within the geometric cons-
trainment of the hexadentate octahedral coordination
sphere conferred by these compounds. Thus, as a direct
consequence of being able to take advantage of the
range of available amino acid, a range of mono- and
di-substitution on the ethylene coordinating arm was
available and included H, methyl, phenyl, and benzyl
along with dimethyl. The derivatives that possess
mono-substitution on the ethylene coordinating arm
Somewhat surprisingly, none of the tach based agents
exhibited any differential or selective cytoxicity toward
the HUVEC cells (Table 1). Compound 6 with benzyl
groups was found to be the most active though
thoroughly unselective confirming previous results
with the analogous methyl substituted compound
exhibiting intermediate activity.21 Interestingly, the

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K. Camphausen et al. / Bioorg. Med. Chem. 12 (2004) 5133–5140
Table 1. Hexadentate 1,3,5-cis,cis-triaminocyclohexane (tach) based
chelating agents evaluated for selective activity (comparative activity
values are mM concentration IC50 measurement to determine differ-
ential cytotoxicity for HUVEC vs control cell lines)a
Table 3. Macrocyclic polyamines evaluated for selective activity
(comparative activity values are mM concentration IC50 measurement
to determine differential cytotoxicity for HUVEC vs control cell lines)a
Compound
HUVEC
NIH3T3
U251
Compound
HUVEC
NIH3T3
U251
18
19
20
21
22
23
24
25
26
1.0
1.0
1.0
1
2
3
4
5
6
7
1.0
1.0
1.0
0.44
0.05
0.32
1.0
0.37
0.14
1.0
0.27
0.11
0.69
1.0
0.008
1.0
1.0
0.02
1.0
0.009
1.0
1.0
1.0
1.0
1.0
0.02
0.003
0.01
0.02
0.004
0.04
0.02
0.001
0.01
0.25
0.39
0.05
1.0
0.66
0.05
0.27
1.0
0.45
0.25
1.0
a Error in IC50 measurements does not exceed 3.0%.
a Error in IC50 measurements does not exceed 3.0%.
tween them as this could strongly affect Cu(II) binding
by altering the bite angle in complex formation to either
favorable or disfavorable geometries.32 Three diethylene-
triamine derivatives were evaluated. No significant
activity was expected from any of these as the number
of donors was deemed inadequate for this application
and that at least four amines would be preferable. This
hypothesis held true initially with diethylenetriamine
itself, 18, and with 19, a simple C-functionalized analog.
However, the more geometrically pre-organized diethyl-
enetriamine 20 was found to exhibit considerable selec-
tive cytotoxicity against the HUVEC cell line.
Differences that may account for this activity include
(1) the stereospecific pre-organizational geometry of
the ligand being capable of forming a far more stable
complex with Cu(II) than with either 19 or 18 and (2)
that 20 might also be somewhat more lipophilic in nat-
ure with inclusion of the cyclohexane ring into the struc-
ture. The polyaminocarboxylate formed from this
triamine has been shown to possess considerably
enhanced stability for several medically useful radionuc-
lides as compared to the analogous compound formed
from 19.33 The uniquely arranged polyamine 21 also
was found to possess considerable selectivity against
the HUVEC cell line and again this might be attributa-
ble to a combination of coordination number and geo-
metry. The selection of tetraamines includes the simplest
polyethylene bridged compound, 22, and three related
compounds that vary the lengths of the three carbon
bridges with either a propylene or butyl central bridge.
Surprisingly, in our hand 22 exhibited no selective activ-
ity at all. All three compounds with a 3 carbon central
bridge, 23, 24, and 25, were found to possess activity
in the assay, although 24 with 3 propylene bridges ap-
peared to be selective toward the U251 and not the HU-
VEC cell line. Compounds 23 and 25 were both found to
be selective toward the HUVEC versus the NIH3T3 and
U251 cell lines. However, 23 also exhibited moderate
relative activity toward U251 while 25 exhibited moder-
ate relative activity toward both the NIH3T3 and U251
cell lines. The higher activity found for 25 was somewhat
unexpected in that the butane spacer would result in
seven-membered chelate ring for a 1:1 Cu(II) com-
plex, which was not predicted as favorable for forming
a stable complex.
Table 2. Macrocyclic polyamines evaluated for selective activity
(comparative activity values are mM concentration IC50 measurement
to determine differential cytotoxicity for HUVEC vs control cell lines)a
Compound
HUVEC
NIH3T3
U251
14
15
16
17
0.5
0.5
1.0
0.09
0.004
0.19
0.25
0.008
0.02
0.76
0.004
0.02
a Error in IC50 measurements does not exceed 3.0%.
polyaminocarboxylate ligand 7 was also found to pos-
sess considerable, albeit unselective, cytotoxicity in this
assay.
The macrocyclic ligands 14 and 15 were expected to pos-
sess some selectivity for chelating Cu(II) while the larger
ring compounds 16 and 17 were unknown in their selec-
tivity toward forming Cu(II) complexes (Table 2). As
partially expected, 15 was found to possess significant
selective cytotoxicity toward the HUVEC cell line, how-
ever, 14 failed to exhibit this property. There are some
significant differences between these two cyclam deriva-
tives that may explain this result. Cyclam 14 has a terti-
ary amine functionalized with an aryl carboxylate group
that may in fact hinder formation of the Cu(II) complex.
This group also then provides a zwitterion that may limit
traversal of the cell membrane and thus charge may
play a role in the lack of its activity. Cyclam 15 is also
functionalized, however, this compound is C-functional-
ized, the array of secondary amines is maintained, and
this status may exert influence on both selectivity and
complex stability. Upon examination of the results ob-
tained for the larger ring polyamines, 16 and 17, both
were found to exhibit considerable cytotoxicity with 16
having considerable activity. Yet, in both cases neither
16 nor 17 have any selectivity against HUVEC cells
and thus their metal binding activity in this arena ap-
pears to not be adequately selective for intracellular
Cu(II).
The linear polyamines (Table 3) were evaluated with
some expectation that these reagents would exhibit some
level of activity in this assay as 22 had been previously
reported as an effective anti-angiogenic agent.5 The
amines were selected to address variation in the number
of amines as well as the carbon framework spacing be-
One overall criteria that seems evident from the data
acquired from evaluating these polyamines in this assay is

K. Camphausen et al. / Bioorg. Med. Chem. 12 (2004) 5133–5140
5137
the consistent requirement for optimal arrangement of
the amines to form Cu(II) complexes. Specifically, inclu-
sion of propylene bridges between the amine donors
appears to be a key component since formation of six-
membered chelate rings tends to be favored for the smal-
ler ionic radius transition metals.32 A second criteria
that also appears influential is a combination of charge
and ability to actually access intracellular Cu(II). This
is apparent from the results from the functionalized acy-
clic and 14-membered ring cyclam ligands where func-
tionalization alters either charge or provides altered
and enhanced relative lipophilicy. Lastly, the results ac-
quired for the two functionalized acyclic ligands 19 and
20 demonstrate the importance of pre-organization and
perhaps specific stereochemistry that should be included
in the design of future Cu(II) chelating agents for anti-
angiogenic agents.
dissolved in EtOAc were transferred to a separatory fun-
nel, extracted with 5% NaHCO3 (3 · 100mL), brine
(100mL), 1N HCl (3 · 100mL), and brine (100mL),
dried over MgSO4, filtered, and either rotary evaporated
to dryness or to a generate a precipitate, which after
cooling was collected, washed with hexanes, and vac-
uum dried.
3.1.3. General procedure for preparation of hexamines
3–5.
3.1.3.1. Deprotection––triamides 9, 11. The triamide
was added to anhydrous 1,4-dioxane saturated with
HCl(g) (200mL) and stirred for 18h. The precipitate
was collected, washed with Et2O, and dried under vac-
uum.
3.1.3.2. Deprotection––triamide 13. The phenylala-
nine triamide was treated with trifluoroacetic acid
(50mL) for 6h, which, after solvent removal by rotary
evaporation and vacuum drying, left a white foam.
3. Experimental
3.1. Ligand syntheses
3.1.3.3. Borane reduction. The deprotected material
was washed into a three-necked round bottom flask with
THF (150mL) and 1M BH3. THF was added while
cooling the suspension with an ice bath. The reaction
mixture was then vigorously refluxed for 6d, after which
any excess borane was decomposed with MeOH. The
solution was rotary evaporated to a gummy solid and
taken up in 100% EtOH (150mL). The EtOH solution
was saturated with HCl(g) while cooling with an ice bath
and then the acidic solution was refluxed for 6h during
which a white precipitate formed. The flask was held at
4ꢀC for 8h and the precipitate was collected on a Buch-
ner funnel, washed with Et2, and dried in vacuo.
3.1.1. Materials and methods. All the reagents and
solvents listed below were obtained from Aldrich, Flu-
ka, Kodak, or Sigma and were used without further
purification. Et2O was distilled from Na and used imme-
diately. The cis,cis-1,3,5-triaminocyclohexane trihydro-
bromide (tach (HBr)3) was prepared as reported in the
literature.34 Compounds 1, 2, 6, 7, 14–17, 19–20 were
prepared as previously reported.21–25,28–31
1H and 13C NMR were obtained using a Varian Gemini
300 instrument and chemical shifts are reported in ppm
on the d scale relative to TMS or TSP. Proton chemical
shifts are annotated as follows: ppm (multiplicity, inte-
gral, coupling constant (Hz)). Chemical ionization mass
spectra (CI-MS) were obtained on a Finnnegan 3000
instrument. Fast atom bombardment (FAB-MS) mass
spectra were taken on an Extrel 400 instrument. Optical
rotations were measured using a Perkin–Elmer 341
instrument with a 100mm cell at 20ꢀC. Elemental anal-
yses were performed by Atlantic Microlabs (Atlanta,
Georgia).
3.1.4. Boc-N-methyl-glycine hydroxysuccinimidyl ester
(10). The amino acid (10.00g, 112mmol), Et3N
(24mL, 173mmol), and Boc-ON (27.7g, 112mmol) were
suspended in 50% aqueous dioxane (300mL) and stirred
for 18h. The solution was transferred to a separatory
funnel, H2O (200mL) was added, and the aqueous layer
extracted with EtOAc (3 · 150mL). The aqueous layer
was returned to the flask, cooled in an ice bath, and
acidified to pH2.0 with 3N HCl. The solution was rap-
idly extracted then with EtOAc (3 · 150mL), the com-
bined extracts dried over MgSO4, filtered, and rotary
evaporation to leave the product (18.0g, 85%).
3.1.2. General procedure for preparation of tach amino
acid triamides (9, 11, 13). The tach (HBr)3 (1equiv) was
treated with NaOH (3equiv) in H2O (10mL) in a two-
necked flask at room temperature fitted with a Dean–
Stark trap and condenser. After formation of a clear
homogeneous solution, benzene (200mL) was added,
and the H2O was removed via azeotropic distillation.
The benzene solution was then reduced to ca. 25mL
and cooled to room temperature. After dilution with
DMF (150mL), the appropriate amino acid active ester
(3equiv) in DMF (30mL) was added. The temperature
was raised to ca. 45ꢀC and the solution was stirred for
18h. The DMF solution was concentrated to near dry-
ness (vacuum rotary evaporation), the solid residue
was suspended in EtOAc (200mL) and vigorously stir-
red. Those derivatives that precipitated directly at this
point were collected on a Buchner funnel, washed with
a small portion of hexanes, and dried. Those that cleanly
1H NMR (CDCL3) d 9.00 (br s, 1H), 3.989 (d, 2H,
J = 23.1), 2.941 (s, 3H), 1.457 (d, 9H, J = 11.1); Mass
Spect. (CI/NH3) 190, 207 (M++1, M++18).
The Boc protected acid (11.00g, 58.2mmol), N-hydroxy-
succinimide (6.70g, 58.3mmol), and EDC (11.16g,
58.2mmol) were suspended in EtOAc (200mL) to which
DMF (100mL) was added to form a clear solution.
After stirring for 18h, the reaction solution was diluted
with EtOAc (100mL) and sequentially extracted with
H2O (100mL), 5% NaHCO3 (100mL), and salt solution
(100mL). After drying over Na2SO4, filtration, and
rotary evaporation, a white solid (13.8g) was obtained,
which was further dried under high vacuum.

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K. Camphausen et al. / Bioorg. Med. Chem. 12 (2004) 5133–5140
1H NMR (DMSO-d6) d 4.394 (s, 2H), 2.819 (s, 4H),
1.409 (s, 3H,), 1.377 (s, 9H); Mass Spect. (CI/NH3)
304 (M++18). The active ester was used without further
purification.
1H NMR (DMSO-d6) d 8.158 (d, 1H, J = 8.1), 7.50–7.34
(m, 5H), 5.639 (d, 1H, J = 8.4), 2.774 (s, 4H), 1.409 (s,
9H); Mass Spect. (FAB/glycerol) 371 (M++23). The ac-
tive ester was used without further purification.
3.1.8. cis,cis-1,3,5-Triaminocyclohexane-tris(N-tert-but-
yloxycarbonyl-2-[R]-phenylacetamide) (13). The tach
(HBr)3 (5.00g, 13.4mmol) was treated as described
above with NaOH (1.613g, 40.0mmol) and 12 (14.03g,
40.4mmol) to produce a white solid (8.9g, 77%).
3.1.5. cis,cis-1,3,5-Triaminocyclohexane-tris(N-tert-but-
yloxycarbonyl-N-methylaminoacetamide) (11). The tach
(HBr)3 (3.90g, 10.5mmol) was treated as described
above with NaOH (1.26g, 31.5mmol) and 10 (9.00g,
31.5mmol) to produce a white solid (4.18g, 62%).
1H NMR (DMSO-d6) d 8.225–8.075 (m, 1H), 7.42–7.11
(m, 5H), 5.073 (q, 1H, J = 9.6), 3.576 (m, 1H), 1.90–1.60
(m, 1H), 1.362 (s, 9H); 13C NMR (DMSO-d6) d 172.90,
154.82, 139.02, 128.23, 127.48, 126.91, 78.44, 57.67,
1H NMR (DMSO-d6) d 7.851 (d, 1H, J = 7.8), 3.717,
3.672 (2s, 3H), 3.330 (m, 1H), 2.782 (s, 2H), 1.821 (br
d, 1H, J = 8.7), 1.390 (s, 3H), 1.328 (s, 6H), 1.117 (m,
1H); 13C NMR (DMSO-d6) d 167.67, 155.07, 78.44,
51.63, 44.75, 37.82, 35.59, 27.99; Mass. Spect. (FAB/
glycerol) 643 (M++1). Anal. Calcd for C30H54N6O9: C,
56.04; H, 8.48; N, 13.04. Found: C, 56.25; H, 8.44; N,
12.81.
44.78, 37.25, 28.17, 25.27; Mass Spect. (FAB/glycerol)
23
D
864 (M++1). ½aꢀ ꢁ8.0 (DMSO, c = 0.001); Anal. Calcd
for C48H60N6O9: C, 66.63; H, 7.01; N, 9.72. Found: C,
66.52; H, 6.85; N, 9.54.
3.1.9. cis,cis-1,3,5-Triamino-tris(2-phenyl-2-aminoethyl-
ene)cyclohexane (5). Boc-triamide 13 (10.9g, 1.32mmol)
was deprotected with trifluoroacetic acid as described
above and after isolation and drying, reduced with
1M BH3ÆTHF (160mL). After the acid work-up, the
product was obtained as a white solid (7.8g, 88%).
3.1.6. cis,cis-1,3,5-Triamino-tris(N-methyl-2-aminoethyl-
ene)cyclohexane (4). Boc-triamide 11 (3.25g, 5.06mmol)
was deprotected with HCl(g) as described above and
after isolation and drying, reduced with 1M BH3ÆTHF
(50mL). After the acidic work-up, the product was ob-
tained as a white solid (1.78g, 68%).
1H NMR (D2O) d 7.533 (s, 5H), 4.554 (t, 1H, J = 7.2),
3.436 (br s, 2H), 2.915 (m, 1H), 2.242 (br d, 1H,
J = 10.2), 1.148 (q, 1H, J = 11.1); 13C NMR (D2O) d
136.26, 133.14, 132.61, 130.42, 56.26, 55.93,
1H NMR (D2O) d 3.330 (s, 3H), 3.12 (m, 1H), 2.783 (s,
2H), 2.494 (d, 1H, J = 11.7), 1.390 (q, 1H, J = 11.7); 13C
NMR (D2O) d 55.49, 47.60, 43.72, 36.23, 33.40; Mass
Spect. (CI/NH3) 301 (M++1). Anal. Calcd for
C15H36N6(HCl)6: C, 34.69; H, 8.17; N, 16.18. Found:
C, 34.18; H, 8.38; N, 16.06.
50.44, 36.58; Mass Spect. (FAB/glycerol) 487 (M++1).
23
D
½aꢀ
ꢁ8.0 (DMSO, c = 0.18); Anal. Calcd for
C30H42N6(HCl)6: C, 51.06; H, 6.87; N, 11.91. Found:
C, 50.85; H, 6.58; N, 12.02.
3.1.7. Boc-[R]-phenylglycine hydroxysuccinimidyl ester
(12). The amino acid (15.00g, 99.0mmol), Et3N
(20.6mL, 148mmol), and Boc-ON (24.4g, 99.0mmol)
were suspended in 50% aqueous dioxane (400mL) and
stirred for 18h. The solution was transferred to a separ-
atory funnel, H2O (200mL) was added, and the aqueous
layer extracted with EtOAc (3 · 150mL). The aqueous
layer was returned to the flask, cooled in an ice bath,
and acidified to pH2.0 with 3N HCl. The solution was
rapidly extracted then with EtOAc (3 · 150mL), the
combined extracts dried over MgSO4, filtered, and
rotary evaporation to leave the product (24.3g, 94%).
3.1.10. Boc-2,2-dimethyl-glycine hydroxysuccinimidyl
ester (8). The amino acid (10.30g, 100mmol), Et3N
(20.8mL, 150mmol), and Boc-ON (24.6g, 100mmol)
were suspended in 50% aqueous dioxane (400mL) and
stirred for 18h. The solution was transferred to a separ-
atory funnel, H2O (150mL) was added, and the aqueous
layer extracted with EtOAc (3 · 150mL). The aqueous
layer was returned to the flask, cooled in an ice bath,
and acidified to pH2.0 with 3N HCl. The solution was
rapidly extracted then with EtOAc (3 · 150mL), the
combined extracts dried over MgSO4, filtered, and
rotary evaporation to leave the product (17.5g, 86%).
1H NMR (DMSO-d6) d 8.032 (d, 1H, J = 4.2), 7.48–7.27
(m, 5H), 5.129 (d, 1H, 5.1), 1.434 (s, 3H), 1.211 (s, 6H);
Mass Spect. (CI/NH3) 269 (M++18).
1H NMR (CDCl3) d 1.530 (s, 6H), 1.442 (s, 9H); Mass
Spect. (CI/NH3) 221 (M++18).
The Boc protected acid (22.00g, 87.5mmol), N-hydroxy-
succinimide (10.1g, 87.8mmol), and EDC (16.6g,
87.8mmol) were dissolved in DMF (200mL) was added
to form a clear solution. After stirring for 18h, the reac-
tion solution was diluted with EtOAc (500mL) and se-
quentially extracted with H2O (100mL), 5% NaHCO3
(100mL), and salt solution (100mL). After drying over
Na2SO4, filtration, and rotary evaporation, a white solid
(25.5g) was obtained, which was further dried under
high vacuum.
The Boc protected acid (11.00g, 54.2mmol), N-hydroxy-
succinimide (6.23g, 54.2mmol), and EDC (10.4g,
54.2mmol) were suspended in EtOAc (250mL) to which
DMF (100mL) was added to form a clear solution.
After stirring for 18h, the reaction solution was diluted
with EtOAc (100mL) and sequentially extracted with
H2O (100mL), 5% NaHCO3 (100mL), and salt solution
(100mL). After drying over Na2SO4, filtration, and ro-
tary evaporation, a white solid (13.0g) was obtained,
which was further dried under high vacuum.

K. Camphausen et al. / Bioorg. Med. Chem. 12 (2004) 5133–5140
5139
1H NMR (DMSO-d6) d 2.777 (s, 4H), 1.481 (s, 6H),
References and notes
1.393 (9H); Mass Spect. (CI/NH3) 318 (M++18). The ac-
tive ester was used without further purification.
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9
(2.00g,
2.92mmol) was deprotected as described above and
after isolation and drying, reduced with 1M BH3ÆTHF
(50mL). After the acid work-up, the product was ob-
tained as a white solid (1.34g, 82%).
1H NMR (D2O) d 3.31–3.20 (m, 1H), 3.252 (s, 2H),
2.586 (d, 1H, J = 10.8), 1.553 (q, 1H, J = 11.7), 1.468
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sus the stimulated positive controls and the un-stimu-
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number where the cellular proliferation was halfway be-
tween the stimulated positive control and the unstimu-
lated negative control.

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