Boron-containing polyamines as DNA targeting agents for neutron capture therapy of brain tumors: Synthesis and biological evaluation

Article abstract of DOI:10.1021/jm960787x

Three series of new boron-containing spermidine/spermine (SPD/SPM) analogues have been synthesized: N¹- and N⁵-(4-carboranylbutyl) SPD/SPM derivatives (SPD-1, SPD-5, SPM-1, SPM-5); N¹,N¹⁰-diethyl-N⁵-(4- carboranylbutyl)spermidine (DESPD-5), N¹-N¹⁴-diethyl-N⁵-(4- carboranylbutyl)spermine (DESPM-5); and N⁵,N¹⁰-bis(4- carboranylbutyl)spermine (SPM-5, 10). In vitro studies using rat F98 glioma cells have shown that these polyamines retain the ability to displace ethidium bromide from calf thymus DNA and are rapidly taken up by F98 glioma cells. However, their cytotoxicities, especially these with terminal N- substituted (SPD-1, SPM-1) boron compounds, are greater than those of SPD/SPM. Nevertheless, the groundwork has been created for a new class of boron-containing compounds that maybe useful for boron neutron capture therapy of tumors.

Full text of DOI:10.1021/jm960787x

J . Med. Chem. 1997, 40, 3887-3896
3887
Bor on -Con ta in in g P olya m in es a s DNA Ta r getin g Agen ts for Neu tr on Ca p tu r e
Th er a p y of Br a in Tu m or s: Syn th esis a n d Biologica l Eva lu a tion
J ianping Cai,† Albert H. Soloway,*,† Rolf F. Barth,‡ Dianne M. Adams,‡ J oya R. Hariharan,†
Iwona M. Wyzlic,† and Krista Radcliffe†
College of Pharmacy and Department of Pathology, The Ohio State University, Columbus, Ohio 43210
Received November 12, 1996X
Three series of new boron-containing spermidine/spermine (SPD/SPM) analogues have been
synthesized: N1- and N5-(4-carboranylbutyl) SPD/SPM derivatives (SP D-1, SP D-5, SP M-1,
SP M-5); N1,N10-diethyl-N5-(4-carboranylbutyl)spermidine (DE SP D-5), N1,N14-diethyl-N5-(4-
carboranylbutyl)spermine (DESP M-5); and N5,N10-bis(4-carboranylbutyl)spermine (SP M-5,10).
In vitro studies using rat F98 glioma cells have shown that these polyamines retain the ability
to displace ethidium bromide from calf thymus DNA and are rapidly taken up by F98 glioma
cells. However, their cytotoxicities, especially those with terminal N-substituted (SP D-1, SP M-
1) boron compounds, are greater than those of SPD/SPM. Nevertheless, the groundwork has
been created for a new class of boron-containing compounds that maybe useful for boron neutron
capture therapy of tumors.
In tr od u ction
Boron neutron capture therapy (BNCT) is based on
the nuclear reaction that occurs when 10B, a stable
isotope having a relatively high neutron capture cross
section value (σ ) 3838 barns), is irradiated with
thermal neutrons to produce high linear energy transfer
7
(LET) R particles and recoiling Li nuclei (Scheme 1).1
F igu r e 1. Polyamines.
These particles which have path lengths of 5-9 µm and
are capable of destroying those cells containing suf-
ficient quantities of 10B required to sustain a lethal
10B(n,R)7Li reaction. The use of BNCT as a cancer
F igu r e 2. Carboranyl polyamine analogues.
treatment modality has been described in several recent
reviews2-4 and monographs.5,6 For BNCT to be effec-
Sch em e 1. Boron Nuclear Reaction
tive, there must be ∼20-30 µg 10B per gram of tumor,
and low concentration (<5 µg of 10B/g of cell) in sur-
rounding normal cells and blood.7,8 The boron-contain-
ing delivery agent should be nontoxic, should selectively
target tumor cells, and should ideally localize within the
nucleus. A key question is how can tumor cells be
selectively targeted? There has been a concerted effort
to synthesize a variety of boron-containing analogues
of biologically active compounds such as amino acids,9-13
peptides,14 nucleosides/nucleotides,15-19 and porphy-
rins20-22 as well as DNA binders.14,23-25 Such structures
might function in a manner similar to their naturally
occurring counterparts and become selectively incorpo-
rated into either proliferating or more metabolically
active tumor cells. Another potential class of com-
pounds are the polyamines, including putrescine, sper-
midine (SPD), and spermine (SPM) (Figure 1), which
are essential for cell growth and differentiation, espe-
cially in rapidly proliferating cells.26,27 Polyamine
depletion has growth inhibitory effects.28,29 This has
been the basis for the preparation of various polyamine
synthetase inhibitors30,31 and synthetic polyamines32 for
cancer treatment. Since cationic polyamines bind ioni-
cally to DNA33 and retain a high degree of freedom
within the polyamine cation-DNA complex,34,35 incor-
poration of tumoricidal agents into the polyamine scaf-
fold has yielded compounds that have been reported to
be more active in cancer control than the parent
chemotherapeutic agents.36-39 On the basis of these
observations, we have designed and synthesized three
series of N-(carboranyl-tethered)polyamine analogues of
spermidine and spermine as potential delivery agents
for BNCT and have correlated their chemical structure
with their in vitro cytostatic/cytotoxic effects, DNA
binding properties and cellular uptake. A detailed
account of this work is described below.
Design a n d Syn th esis
Two of the naturally occurring polyamines, spermi-
dine (SPD) and spermine (SPM), were used in the
syntheses of these three series of compounds. The boron
moiety in these analogues was inserted (1) on the
internal N atoms; (2) on the internal N atoms in which
the terminal nitrogens carried monosubstituted ethyl
groups; and (3) on the terminal nitrogen atoms. The
o-carborane nucleus was chosen as the boron moiety and
was tethered to polyamines by a chain of four methylene
groups (Figure 2). The target compounds, whose syn-
theses are described, are shown in Schemes 2-6. These
include N1- and N5-(4-carboranylbutyl) SPD/SPM de-
† College of Pharmacy.
rivatives (SP D-1, SP D-5, SP M-1, SP M-5), N1,N10
diethyl-N5-(4-carboranylbutyl)spermidine (DESP D-5),
-
‡ Department of Pathology.
X Abstract published in Advance ACS Abstracts, October 15, 1997.
S0022-2623(96)00787-X CCC: $14.00 © 1997 American Chemical Society

3888 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Cai et al.
Sch em e 2a
a
(a) 1b, K2CO3/DMF, 60-70 °C; (b) 3 N HCl/MeOH, 40-50 °C; (c) BnBr, K2CO3/DMF, 60-70 °C; (d) NaH, EtI/DMF; (e) Pd/C, H2/
MeOH; (f) concentrated HCl/EtOH, refluxing.
Sch em e 3a
a
(a) 1b, K2CO3/DMF, 60-70 °C; (b) 3 N HCl/MeOH, 40-50 °C.
N1,N14-diethyl-N5-(4-carboranylbutyl)spermine (DESP M-
5), and N5,N10-bis(4-carboranylbutyl)spermine (SP M-
5,10).
The possibility that toxic metabolites of polyamines
could arise from those compounds containing primary
amines43 has led to the synthesis of the N-ethylated
derivatives. For this series of compounds, we used the
bis(ethylated) derivatives of 2a as the starting material,
but only intractable mixtures of the products were
obtained.44 However, when the BOC group was re-
placed by a 2-mesitylenesulfonyl (MesSO2) group fol-
lowed by protection of the internal NH with a benzyl
group, the ethylation reactions of 4 and 12 were carried
out in excellent yields (Schemes 2 and 4).45 The bis(N-
ethylated) intermediates, 5 and 13, were then deben-
zylated by catalytic hydrogenation (H2, Pd/C), yielding
the free secondary amines, 6 and 14, respectively.
These two amines were again alkylated with 1b under
the same conditions as described above and formed the
desired target compounds, DESP D-5 and DESP M-5,
after removal of the 2-mesitylenesulfonyl group in 7 and
15, respectively.
To synthesize internally substituted N-carboranylbu-
tylated derivatives, SPD/SPM were initially protected
at both terminal amino groups with the tert-butoxycar-
bonyl (BOC) group.36 Bis(BOC) intermediates 2a and 8
were then alkylated with 4-carboranylbutyl iodide,
1b,40,41 in DMF in the presence of K2CO3, forming the
corresponding BOC-protected compounds 3 (Scheme 2)
and 9 (Scheme 3) in high yield. Target compounds,
SP D-5 and SP M-5,10, were obtained, following removal
of the BOC groups by acidic cleavage. With respect to
the alkylation of 8, it was not possible to form any of
the mono(carboranylbutylated) species, even when the
molar ratio of 8 to 1 was greater than 1:1. We at-
tributed this result to the highly nucleophilic properties
of the secondary amines in 8, precluding the isolation
of the monoalkylated analogue by this procedure. To
ultimately synthesize target compound SP M-5, it was
necessary to synthesize the tris-BOC protected deriva-
tive of SPM, 10a . Its alkylation by 1b generates 11
(Scheme 4), whose subsequent deprotection produces
SP M-5. Previously, our group reported40 the synthesis
of 3 in two steps from 1b and 2a : the secondary amine
of 2a was first protected with the trimethylsilyl (TMS)
group and then the TMS amide reacted with 1 in THF,
generating 3. When this same procedure was used for
the synthesis of 9, extremely poor yields of the product
were observed. The TMS protecting group reduced the
reactivity of the secondary amino group, necessitating
longer reaction times, and under these conditions
degradation of the carborane function was most prob-
able.42
All of the above compounds have the boron moiety
on an internal nitrogen. To determine whether termi-
nally substituted analogues have more desirable biologi-
cal properties, we undertook the syntheses of target
compounds SP D-1 and SP M-1 shown in Schemes 5 and
6. In both cases, internal nitrogen(s) of SPD/SPM were
protected by forming hexahydropyrimidine ring(s) with
formaldehyde.46,47 For the synthesis of SP D-1, the
primary amine of 16a was then protected with BOC and
subsequently reacted with 1b to give 17 in moderate
yield. Deprotection of both the BOC group and the
methylene group of the hexahydropyrimidine ring is
achieved with acid, yielding the desired target com-
pound. In the case of 18, it reacted with 4-carboranyl-
butyl tosylate, 1a ,18 to give mono(N-carboranylbutylat-

DNA Targeting Agents for Cancer Treatment
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3889
Sch em e 4a
a
(a) 1b, K2CO3/DMF, 60-70 °C; (b) 3 N HCl/MeOH, 40-50 °C; (c) BnBr, K2CO3/DMF, 60-70 °C; (d) NaH, EtI/DMF; (e) Pd/C, H2/
MeOH; (f) concentrated HCl/EtOH, refluxing.
Sch em e 5a
a
(a) 1b, K2CO3/DMF, 25 °C; (b) 3 N HCl/MeOH, 40-50 °C.
Sch em e 6a
a
(a) 1a , K2CO3/DMF, 25 °C; (b) 3 N HCl/MeOH, 40-50 °C.
ed) compound 19 together with some bis(N-carbor-
anylbutylated) byproduct. The latter can be readily
separated from 19 by chromatography. Acid hydrolysis
of 19 yielded SP M-1.
Biologica l Stu d ies
Two major questions regarding these boron-contain-
ing polyamines were whether these structures behaved
biologically similar to SPD/SPM, and whether they
possessed the requisite properties for BNCT agents. To
determine this, the following properties have been
studied: (1) their in vitro growth inhibitory effects using
F98 rat glioma cells; (2) their ability to displace ethid-
ium bromide from calf thymus DNA; and (3) the
compound’s in vitro cellular uptake using the F98
glioma cells.
(1) In Vitr o Eva lu a tion of Cytotoxic/Cytosta tic
P r op er ties. The assay that we have employed quanti-
fied the S phase of F98 glioma cells following a 24 h
exposure to the test compounds by measuring cellular
uptake/incorporation of [3H]TdR and comparing these
data to those obtained with cells that were not exposed
to the compounds.48 The results, summarized in Figure
F igu r e 3. Cytotoxicity/cytostaticity of spermidine analogues.
3 for SPD analogues and in Figure 4 for SPM analogues,
are expressed as counts per minute (cpm) obtained with
cells grown in the presence of the test compound
compared to those in the absence of the compound

3890 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Cai et al.
Ta ble 2. DNA Binding Assaya
compd
IC50 (µM)
SP D
SP D-5
SP M
175
245
17
SP M-5
133
a
Polyamine displacement of ethidium bromide from calf thymus
DNA. Ethidium bromide and DNA concentrations were 1.6 and
10 µg, respectively. IC50 values are the concentration of polyamine
required to decrease the fluorescence of the ethidium bromide-
DNA complex by 50%.
Ta ble 3. In Vitro Cellular Uptakea
Cmedia
Ccellular
compd
SP D-5
DESP D-5
SP M-5,10
SP M-5
(µg of B/mL)
(µg of B/g of cells)
(A) 48 h Incubation
0.5
0.5
1.0
0.5
0.5
43
141
101
64
F igu r e 4. Cytotoxicity/cytostaticity of spermine analogues.
Ta ble 1. In Vitro IC50 of Polyamine Analogues
DESP M-5
37
(B) 24 h Incubation
compda
IC (µM)b
compda
IC (µM)b
SP D-5
BSH
BP A
0.5
570
50
65
39
81
SP D
MESP D
SP D-1
>100
>100
3
DESP M
SP M-1
SP M-5
>100
7.5
22
a
(A) F98 cells were incubated for 48 h with 5 µM polyamine
analogue (the actual boron concentration, Cmedia, was determined
by DCP-AES.) Cells were washed, counted, and digested. Boron
was determinated by DCP-AES (109 cells ≈ 1 g of cells). (B) F98
cells were incubated for 24 h with boronated compounds and
treated as in part A.
SP D-5
DESP D-5
SP M
22
10
>100
DESP M-5
SP M-5,10
25
15
a
Spermidine and its analogues are designated as SPD and
b
spermine and its analogues as SPM. The concentration of the
test compound required to produce a 50% reduction in the uptake
of [3H]TdR, by F98 glioma cells. These values have been extrapo-
lated from the curves shown in Figures 3 and 4.
affinity for DNA than did the corresponding SPD
analogues. Insertion of various boron substituents
decreased their DNA affinity compared with SPD/SPM.
Notably, SP M-5 was a better DNA binder than SPD.
Although the carboranylbutyl group is a bulky substitu-
ent and its incorporation reduces the compound’s bind-
ing potential, our results have demonstrated that
polyamine analogues have DNA binding properties that
are similar to those of the naturally occurring com-
pounds.
(3) In Vitr o Cellu la r Up ta k e Stu d ies. The in vitro
uptake of carboranyl polyamines by F98 glioma cells
was determined by measuring cellular boron content by
means of direct current plasma atomic emission spec-
troscopy (DCP-AES) (Table 2).54 Uptake of the tested
compounds was compared with two clinically used
agents, sodium undecahydromercapto-closo-dodecabo-
rate (BSH) and L-p-boronophenylalanine (BPA) (Table
3). On the basis of the boron content in the media,
which was 100-1000-fold less for the polyamines than
for BSH and BPA, cellular uptake of the carboranyl
polyamines was orders of magnitude greater than that
obtained for BSH and BPA.
(×100). If the compound had no cytotoxic/cytostatic
effects, then uptake of [3H]TdR by cells that either were
exposed or not exposed to the test compound would be
similar. The IC50 value was defined as that concentra-
tion required to reduce the uptake of [3H]TdR by 50%
compared to the controls. All of the carboranyl poly-
amines were toxic with an IC50 < 25 µM (Table 1). They
were considerably more toxic than both SPD/SPM and
their ethylated structures, N1-ethylspermidine (MESP D)
and N1,N14-diethylspermine (DESP M). Both classes of
internally N-substituted analogues, SP D-5, DESP D-
5, SP M-5, and DESP M-5, were less toxic than those
terminally N-substituted derivatives, SP D-1 and SP M-
1. Incorporation of the diethyl group increased the
toxicity of the SPD analogue, DESP D-5, while the SPM
analogue, DESP M-5, was slightly less toxic than SP M-
5. Toxicity also was increased by increasing the number
of carborane substituents on the polyamine (SP M-5,10
vs SP M-5). According to these results, as well as those
reported by others,49,50 incorporation of a hydrophobic
group into the polyamine scaffold increased the com-
pound’s toxicity.
Discu ssion
(2) DNA Bin d in g Assa y. Due to the high toxicity
of many of the carboranyl polyamines, only those two
with relatively low toxicity, SP D-5 and SP M-5, were
evaluated in the DNA binding assay. The binding of
polyamine analogues to DNA was determined by an
ethidium bromide displacement assay.51-53 The C50
value was defined as the concentration of the polyamine
required to decrease the fluorescence of the ethidium
bromide-DNA complex by 50%. The results are sum-
marized in Table 2. SPM analogues, possessing four
positively charged ammonium groups, had stronger
The ortho carborane cage is sufficiently stable under
neutral and acidic conditions for its incorporation into
potential BNCT agents. However, it undergoes degra-
dation under various basic conditions,42,55-57 and this
property has been the major chemical limitation in the
synthesis of ortho carborane-containing amines that are
very strong bases. This is the case with the polyamines.
This problem can be obviated by masking the amino
function and generating the final products under acidic
conditions as the amine hydrochloride. These salts are
very water soluble even with the carboranyl function,

DNA Targeting Agents for Cancer Treatment
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3891
and the resulting aqueous solutions are stable. Dif-
ficulties are encountered in alkylating the secondary
amino groups of the polyamines since both the starting
material and the product have the potential for degrad-
ing the cage and generating reduced yields. Using the
trimethylsilyl (TMS) group to protect the secondary
amine reduces its basicity.40 However, this derivative
also reduces the nucleophilicity of the amino group, and
longer reaction times are required to generate the
product. Under these conditions, degradation of the
carborane cage by the free tertiary amine in the alky-
lated product becomes more significant. After consider-
ing all the factors, direct alkylation without the TMS
masking group over a short time period was the optimal
way for producing the product. However, in the case of
alkylation on cyclic secondary amines (17, 18), a reaction
temperature of 60-70 °C was too high. Reactions under
ambient conditions gave better, but not very high yields.
This result might stem from the fact that such cyclic
amines are stronger degrading agents of the carborane
nucleus.42
compounds possessing the requisite properties for BNCT
delivery agents, i.e., high tumor uptake and retention,
low normal tissue and blood content, and sufficiently
nontoxic when administered to tumor bearing hosts.
The purpose of the assays used to evaluate the
cytotoxic/cytostatic properties of the boronated poly-
amines as potential BNCT delivery agents is dia-
metrically opposite to the requirements of the National
Cancer Institute in identifying cytoreductive chemo-
therapeutic agents.58 The most interesting, and poten-
tially useful compounds for BNCT, would be those that
attain high concentration in tumor cells and are mini-
mally toxic to the host and normal cells. In contrast,
the most useful cancer chemotherapeutic agents are
those demonstrating cytotoxic or growth inhibitory
effects on tumor cells, but their cellular uptake and
concentration have not been considered relevant. The
assay that we have employed measures the uptake of
[3H]TdR by F98 glioma cells following their exposure
to various boron-containing polyamines. Although it is
not a true measure of toxicity since it cannot distinguish
between dead and biosynthetically quiescent cells, as
measured by cpm, uptake does provide quantitative
information on the number of biosynthetically active
cells (i.e. S phase) following exposure to the test
compound compared to the results obtained with cells
grown in the absence of the compound. Since the ideal
compounds for BNCT would either have no or minimal
effects on the uptake of [3H]TdR by S phase cells, we
are screening for those compounds that are not cytotoxic.
Previous experience with the assay has shown it to be
a useful in vitro test for identifying nontoxic compounds
that subsequently could be evaluated in vivo.62
The polyamine structure as a scaffold for targeting
malignant cells has such a potential only when the
polyamine transport system is up-regulated in these
cells by contrast with contiguous normal cells. This
appears to be especially true for brain tumor cells31 and
has provided the rationale for incorporating boron
moieties into the naturally occurring polyamines, sper-
midine and spermine. The in vitro biological results
with carboranyl polyamines show their potential for
selectively delivering boron to tumor cells and thereby
offer us a new class of BNCT agents. They possess the
ability to displace ethidium bromide from calf thymus
DNA, as do their natural counterparts, and are rapidly
taken up by F98 glioma cells in vitro. This uptake is
comparable to that of the clinically used compounds,
BSH and BPA, but at a media concentration that is
100-1000-fold less. Accordingly, carboranyl polyamines
possess advantages as potential BNCT agents when
compared to other boronated compounds such as amino
acids,9-13 nucleosides/nucleotides,15-19 and other poten-
tial DNA-targeting structures.14,24,25 The high hydro-
philic properties of these polyamine salts allow for their
direct administration at suitable concentration levels in
water. No cosolvents such as DMSO or various alcohols
are necessary. The latter can contribute to the com-
pound’s toxicity and the inaccuracy of the biological
evaluations. Polyamines bind to DNA nonspecifically,
and therefore, boronated polyamines may be able to
target DNA directly once they penetrate the cell mem-
brane. This is in contrast to amino acids or nucleosides/
nucleotides, which must be first incorporated into
protein or DNA in order to localize in the cell nucleus.
Such incorporation will be definitely limited due to the
modification of naturally occurring amino acids and
nucleosides/nucleotides by insertion of the boron moiety.
However, the major limitation in the use of the present
compounds appears to be their cellular toxicity, espe-
cially those compounds with terminal N-substituted
boron moieties (SP D-1, SP M-1). Studies currently are
in progress (1) to synthesize other boron-containing
polyamine analogues with potentially lower toxicity; (2)
to correlate their chemical structure with their physio-
chemical properties, toxicity and DNA binding; (3) to
use this information for the design and synthesis of
In conclusion, this report is of the synthesis and
evaluation of a group of new boron-containing polyamines
that are prototypic of a class of compounds that might
be useful as delivery agents for BNCT. The current
limitation of these compounds is their toxicity. Re-
cently, however, we have succeeded in synthesizing less
toxic analogues, and their DNA binding potential and
cellular uptake currently are being evaluated.
Exp er im en ta l Section
The reagents were purchased from the chemical companies
and used directly without further purification unless otherwise
specified. Dry DMF was prepared by drying over BaO and
then distilling. Dry THF was prepared by refluxing with
sodium and distilling the product. Dry ethyl ether was
prepared by refluxing over LiAlH4 followed by distillation.
Merck silica gel 60 (230-400 mesh) was used as solid phase
for flash column chromatograpy. NMR spectra were obtained
on a Bruker AC-250 or AC-270 spectrometer. Chemical shifts
are reported downfield from TMS in spectra obtained in CDCl3,
CD3OD, and DMSO-d6 and from DSS in spectra obtained in
D2O. IR spectra were measured on a Laser Precision Analyti-
cal 720-XI spectrophotometer. MS were obtained on a VG 70-
250S instrument for HR-EI and on a Finnigan MAT-900
instrument for LR-FAB. All melting points were determined
on a Fisher-J ohns melting point apparatus and are uncor-
rected. Elemental analyses were performed by Robertson
Microlit Laboratories, Inc., and Galbraith Laboratories, Inc.
All compounds gave elemental analyses within (0.4% unless
otherwise specified. RT (room temperature) is 22-25 °C.
1. Ch em ica l Syn th eses. Compounds 2a , 8, 10a , 16, and
18 were synthesized following the procedures reported in refs
32, 36, 41, and 46.
Gen er a l P r oced u r e for th e P r ep a r a tion of 2-Mesityl-
en esu lfon a m id es: N1,N10-Bis(m esit ylen esu lfon yl)sp er -

3892 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Cai et al.
m id in e (2b). 2-Mesitylenesulfonyl chloride (7.97 g, 36.44
mmol) in CH2Cl2 (40 mL) was added over 4 h to a mixture of
spermidine (2.65 g, 18.24 mmol), 15% NaOH (30 mL), and
CH2Cl2 (30 mL) at -10 °C under vigorous stirring. After the
addition, the reaction mixture was stirred overnight at RT.
Layers were separated, and the water layer was extracted with
CH2Cl2. The organic phase was washed with 1 N NaOH and
dried over MgSO4. After filtering and concentrating, the
residue was purified by flash column chromatography, eluting
with ethyl acetate-MeOH (3:1) to give 2b (6.7 g, 13.14 mmol,
72%) as a colorless oil: IR (neat, cm-1) 3303 m, 2974 m, 2938
m, 1604 m, 1469 m, 1455 m, 1405 m, 1321 s, 1154 s, 1058 m,
N1,N10-Dieth yl-N1,N10-bis(MesSO2)-N5-(4-o-ca r bor a n yl-
bu tyl)sp er m id in e (7). Compound 7 (1.37 g, 1.79 mmol, 69%)
was synthesized from the reaction of 1b (846.9 mg, 2.63 mmol)
with 6 (1.47 g, 2.59 mmol). The product was eluted with ethyl
acetate-hexane (2:1) containing 2% MeOH: IR (neat, cm-1
)
2938 m, 2588 s, 1604 w, 1455 w, 1380 w, 1314 s, 1148 s, 710w;
1H NMR (CDCl3) δ 7.95 (s, 4H, ArH), 3.80 (s, 1H, B10CH),
3.30-3.14 (m, 8H, 2 × SO2NCH2)2), 2.61 (s, 12H, 4 × ArCH3),
2.29 (s, 6H, 2 × ArCH3), 2.35-2.15 (m, 8H, N(CH2)3
+
CH2CB10), 1.71-1.20 (m, 10H, CH2(CH2N)2 + NCH2CH2CH2-
CH2N + CH2CH2CH2CB2), 1.08 (t, J ) 7.1 Hz, 3H, CH2), 1.06
(t, J ) 7.1 Hz, 3H, CH2); 13C NMR (CDCl3) δ 142.1 t, 142.0 t,
139.9 t, 139.8 t, 133.5 q, 131.7 t, 75.5 q, 61.2 t, 53.3 d, 53.0 d,
51.4 d, 45.0 d, 43.6 d, 40.3 d, 39.9 d, 37.7 d, 26.9 d, 26.4 d,
25.5 d, 25.4 d, 24.2 d, 22.6 s, 20.7 s, 12.7 s, 12.6 s; MS (LR-
FAB) m/ e (calcd, found) for C35H67B10N3O4S2: 762 (8, 7), 763
(26, 23), 764 (54, 55), 765 (92, 92), 766 (100, 100), 767 (60,
66), 768 (24, 26), 769 (8, 9); MS (HR-EI) for C35H64B10N3O4S2
(M - 1) calcd 764.5302, found 764.5303. Anal. (C35H65B10N3-
O4S2) C, H, B, N.
1
737 m; H NMR (CDCl3) δ 7.00 (s, 4H, ArH), 3.00 (t, J ) 7.5
Hz, 2H, SO2NCH2), 2.90 (t, J ) 7.5 Hz, 2H, SO2NCH2), 2.80-
2.50 (m, 4H, N(CH2)2), 2.68 (s, 6H, 2 × ArCH3), 2.66 (s, 6H, 2
× ArCH3), 2.41 (s, 6H, 2 × ArCH3), 1.75-1.60 (m, 2H,
CH2(CH2N)2), 1.60-1.48 (m, 4H, NCH2CH2CH2CH2N); 13C
NMR (CDCl3) δ 141.9 t, 141.8 t, 139.1 t, 139.0 t, 134.0 q, 133.7
q, 132.0 t, 131.8 t, 49.0 d, 48.5 d, 42.4 d, 42.3 d, 27.9 d, 27.6 d,
27.6 d, 27.1 d, 22.9 s, 22.8 s, 20.8 s; MS (HR-EI) for
C25H39N3O4S2 calcd 509.2416, found 509.2374.
N1,N14-Bis(BOC)-N5,N10-bis(4-o-ca r bor a n ylbu tyl)sp er -
m in e (9). Compound 9 (385.8 mg, 0.48 mmol, 72%) was
synthesized from 1b (436.2 mg, 1.34 mmol) and 14 (538.2 mg,
1.34 mmol). The product was eluted with ethyl acetate-
N1,N5,N14-Tr is(m esitylen esu lfon yl)sper m in e (10b). Com-
pound 10b (599.4 mg, 0.80 mmol, 37%) was obtained from the
reaction of 2-mesitylenesulfonyl chloride (1.42 g, 6.49 mmol)
and spermine (439.2 mg, 2.17 mmol). Eluting solvents: ethyl
acetate-MeOH (2:1). 10b was pure enough for use in the next
step [IR (neat, cm-1) 3311 m, 2938 m, 1605 m, 1456 m, 1319
s, 1152 s, 712 m; 1H NMR (CDCl3) δ 7.00 (s, 6H, ArH), 3.31 (t,
J ) 7.2 Hz, 2H, SO2NCH2), 3.11 (t, J ) 7.2 Hz, 2H, SO2NCH2),
2.95 (t, J ) 7.2 Hz, 2H, SO2NCH2), 2.85 (t, J ) 7.2 Hz, 2H,
SO2NCH2), 2.62 (s, 12H, 4 × ArCH3), 2.52 (s, 6H, 2 × ArCH3),
2.52-2.37 (m, J ) 7.2, 4H, N(CH2)2), 2.28 (s, 9H, 3 × ArCH3),
1.82-1.22 (m, 8H, 2 × CH2(CH2N)2 + NCH2CH2CH2CH2N);
13C NMR (CDCl3) δ 142.5 t, 141.9 t, 141.7 t, 140.0 t, 139.0 t,
138.9 t, 133.9 q, 133.5 q, 133.2 q, 132.0 t, 131.9 t, 131.8 t, 49.4
d, 49.0 d, 45.5 d, 43.2 d, 42.8 d, 39.4 d, 27.7 d, 27.5 d, 27.0 d,
25.1 d, 22.8 s, 20.9 s].
MeOH (6:1): IR (neat, cm-1
) 3346 w, 2938 s, 2593 s, 1698 s,
1455 s, 1173 s, 722 w; 1H NMR (CDCl3) δ 3.72 (s, H, 2 ×
B10CH), 3.20-3.07 (m, 4H, 2 × CONCH2), 2.50-2.30 (m, 12H,
2 × N(CH2
)3), 2.30-2.16 (m, 4H, 2 × CH2CB10), 1.68-1.52 (m,
(CH2N)2), 1.50-1.30 (m, 12H, NCH2CH2CH2CH2N
4H, 2 × CH2
+ 2 × CH2
CH2CH2CB10), 1.45 (s, 18H, 2 × C(CH3)3); 13C NMR
(CDCl3
) δ 156.0 q, 77.5 q, 75.4 q, 61.2 t, 54.1 d, 53.4 d, 52.9 d,
39.5 d, 37.9 d, 28.5 s, 27.1 d, 26.6 d, 25.0 d. Anal. (C32
B20N4O4) C, H, B, N.
H78-
N 5-(4-o-Ca r b or a n ylb u t yl)-N 1,N 10,N 14-t r is(BOC)sp e r -
m in e (11). Compound 11 (1.44 g, 2.05 mmol, 85%) was
synthesized from 1b (794.3 mg, 2.43 mmol) and 10b (1468.8
mg, 2.92 mmol). The product was eluted with ethyl acetate-
MeOH (8:1): IR (neat, cm-1) 3348 m, 2935 s, 2592 s, 1696 s,
1514 s, 1456 s, 1391 s, 1273 s, 1174 s, 710 w; 1H NMR (CD3OD)
δ 4.49 (s, 1H, B10CH), 3.21-3.08 (m, 4H, 2 × CONCH2), 3.07-
2.89 (m, 4H, CON(CH2)2), 2.46-2.28 (m, 6H, N(CH2)3), 2.27-
2.18 (m, 2H, CH2CB10), 1.68-1.47(m, 12H, 2 × CH2(CH2N)2 +
NCH2CH2CH2CH2N + CH2CH2CH2CB10), 1.46 (s, 9H, C(CH3)3),
1.44 (s, 18H, 2 × C(CH3)3); 13C NMR (CD3OD) δ 158.3 q, 158.2
q, 157.3 q, 80.8 q, 79.8 q, 77.2 q, 63.5 t, 54.8 d, 54.3 d, 52.7 d,
48.1 d, 45.8 d, 39.8 d, 38.9 d, 38.6 d, 30.0 d, 28.9 s, 28.3 d, 27.9
d, 27.1 d, 24.9 d; MS (HR-EI) C31H70B10N4O6 (M + 2H) calcd
704.6226, found 704.6218.
Gen er a l P r oced u r e for th e Alk yla tion of th e Am in o
F u n ction s of P olya m in es. N1,N10-Bis(BOC)-N5-(4-o-ca r -
bor a n ylbu tyl)sp er m id in e (3). Compound 1b (283.8 mg,
0.87 mmol) in DMF (1.5 mL) was added to a mixture of K2CO3
(143.5 mg, 1.04 mmol) and 2a (359.9 mg, 1.04 mmol) in DMF
(1 mL) heated to 60-70 °C. The reaction mixture was stirred
for an additional hour. After cooling, ice-water and CH2Cl2
were added. The layers were separated, and the aqueous layer
was extracted with CH2Cl2. The organic phase was washed
with 1 N NaOH and dried over MgSO4. After filtering and
removal of the solvent, the residue was purified by flash
column chromatography and eluted with ethyl acetate-MeOH
(50:1) to give 3 (421.5 mg, 0.77 mmol, 89%) as a colorless oil.
N5-Ben zyl-N1,N10,N14-tr is(MesSO2)sp er m in e (12). Com-
pound 12 (2.78 g, 3.31 mmol, 75%) was produced from the
reaction of BnBr (1.13 g, 6.61 mmol) and 10b (3.31 g, 4.42
mmol). The product was eluted with ethyl acetate-hexane
It solidified in the refrigerator: mp 106-8 °C; IR (KBr, cm-1
)
3350 m, 2977 s, 2934 s, 2589 s, 1691 s, 1420 s, 1162 s, 723 m;
1H NMR (CDCl3) δ 3.70 (s, 1H, B10CH), 3.21-3.02 (m, 4H, 2
× CONCH2), 2.50-2.20 (m, 6H, N(CH2)3), 2.20-2.05 (m, 2H,
CH2CB10), 1.70-1.49 (m, 10H, CH2(CH2N)2 + NCH2CH2CH2-
CH2N + CH2CH2CH2CB2), 1.48 (s, 18H, 2 × C(CH3)3); 13C NMR
(CDCl3) δ 156.0 q, 155.9 q, 79.0 q, 78.9 q, 75.3 q, 61.0 t, 53.6
d, 53.3 d, 52.3 d, 41.4 d, 39.6 d, 37.9 d, 28.4 s, 28.0 d, 27.1 d,
27.0 d, 26.5 d, 24.4d; MS (HR-EI) for C23H55B10N3O4 (M + 2)
calcd 547.5123, found 547.2114. Anal. (C23H53B10N3O4) C, H,
B, N.
(2:1): IR (neat, cm-1
) 3312 m, 2939 s, 1605 s, 1405 s, 1152 s,
735 s; 1H NMR (CDCl3) δ 7.48-7.12 (m, 5H, C6H5), 7.00-6.81
(m, 6H, ArH), 3.43 (s, 2H, CH2Ar), 3.25 (t, J ) 7.2 Hz, 2H,
SO2NCH2), 3.07 (t, J ) 7.2 Hz, 2H, SO2NCH2), 2.98-2.77 (m,
4H, SO2N(CH2)2), 2.60 (s, 12H, 4 × ArCH3), 2.54 (s, 6H, 2 ×
ArCH3), 2.41-2.29 (m, 4H, N(CH2)2), 2.22 (s, 6H, 2 × ArCH3),
2.20 (s, 3H, ArCH3), 1.73-1.51 (m, 4H, 2 × CH2(CH2N)2), 1.48-
1.19 (m, 4H, NCH2CH2CH2CH2N); 13C NMR (CD3OD) δ 142.3
t, 141.8 t, 141.7 t, 139.8 t, 139.0 t, 138.8 t, 138.5 q, 134.0 q,
133.7 q, 133.2 q, 137.9 t, 131.8 t, 131.7 t, 128.8 t, 128.3 t, 128.2
t, 127.0 t, 58.0 d, 53.2 d, 52.7 d, 45.8 d, 42.8 d, 42.1 d, 27.6 d,
25.8 d, 25.2 d, 23.9 s, 22.7 s, 22.6 s, 20.7 s. Anal. (C44H62N4-
O6S3) C, H, N.
N1,N10-Bis(MesSO2)-N5-b en zylsp er m id in e (4). Com-
pound 4 (0.85 g, 1.42 mmol, 71%) was obtained from the
reaction of BnBr (425 mg, 2.49 mmol) with 2b (1.01 g, 1.98
mmol). The product was eluted with ethyl acetate-hexane
(1:1): IR (neat, cm-1) 3308 m, 2939 m, 1604 m, 1453 m, 1405
N1,N14-Dieth yl-N5-(4-o-ca r bor a n ylbu tyl)-N1,N10,N14-tr is-
(MesSO2) sp er m in e (15). Compound 15 (2.08 g, 2.08 mmol,
78%) was formed from 1b (875.1 mg, 2.68 mmol) and 14 (2.16
g, 2.68 mmol). The product was eluted with ethyl acetate-
hexane (2:1) containing 2% MeOH: IR (neat, cm-1) 2939 m,
2593 m, 1605 w, 1457 w, 1314 s, 1149 s, 910 s, 733 s; 1H NMR
(CDCl3) δ 7.90 (s, 6H, ArH), 3.25 (s, 1H, B10CH), 3.30-2.95
(m, 12H, 6 × SO2NCH2), 2.59 (s, 6H, 2 × ArCH3), 2.56 (s, 6H,
2 × ArCH3), 2.53 (s, 6H, 2 × ArCH3), 2.40-2.12 (m, 17H, 3 ×
ArCH3 + N(CH2)3 + CH2CB10), 1.75-1.18 (m, 12H, 2 ×
CH2(CH2N-)2 + NCH2CH2CH2CH2N + CH2CH2CH2CB10), 1.04
1
m, 1322 s, 1154 s, 1077 m, 700 m; H NMR (CDCl3) δ 7.45-
7.15 (m, 5H, C6H5), 6.95 (m, 4H, ArH), 6.47 (s, 1H, NH), 5.45
(s, 1H, NH), 3.45 (s, 2H, CH2Ar), 3.05-2.75 (m, 4H, 2 ×
SO2NHCH2), 2.70 (s, 6H, 2 × ArCH3), 2.64 (s, 6H, 2 × ArCH3),
2.52-2.36 (m, 4H, N(CH2)2), 2.35 (s, 6H, 2 × ArCH3), 1.70-
1.51 (m, 2H, CH2(CH2N)2), 1.51-1.45 (m, 4H, NCH2CH2-
CH2CH2N); 13C NMR (CDCl3) δ 141.7 t, 139.0 t, 138.3 q, 133.9
q, 133.7 q, 131.8 t, 131.7 t, 128.9 t, 128.1 t, 126.9 t, 58.38 d,
52.9 d, 52.4 d, 42.2 d, 42.0 d, 27.4 d, 25.7 d, 23.7 d, 22.7 s, 20.7
s; MS (HR-EI) for C32H45N3O4S2 calcd 599.2885, found 599.2850.

DNA Targeting Agents for Cancer Treatment
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3893
(t, J ) 7.1 Hz, 3H, CH3), 0.88 (t, J ) 7.1 Hz, 3H, CH3); 13C
NMR (CD3OD) δ 142.3 t, 142.2 t, 140.0 t, 133.6 q, 133.4 q,
131.9 t, 131.8 t, 75.5 q, 61.2 t, 53.4 d, 53.1 d, 51.5 d, 45.8 d,
43.6 d, 43.2 d, 42.8 d, 40.36 d, 40.2 d, 37.9 d, 27.0 d, 26.5 d,
25.6 d, 25.3 d, 24.2 d, 22.7 s, 20.8 s, 12.8 s, 12.6 s; MS (LR-
FAB) m/ e (calcd, found) for C47H82B10N4O6S3 1001 (6, 8), 1002
(20, 24), 1003 (51, 56), 1004 (87, 90), 1005 (100, 100), 1006
(74, 72), 1006 (36, 33), 1007 (5, 4). Anal. (C47H82B10N4O6S3)
C, H, N; B: calcd, 10.77; found, 8.52.
4H, N(CH2)2), 2.26 (s, 9H, 3 × ArCH3), 1.78-1.54 (m, 4H, 2 ×
CH2(CH2N)2), 1.51-1.12 (m, 4H, NCH2CH2CH2CH2N), 1.08 (t,
J ) 7.1 Hz, 3H, CH3), 0.98 (t, J ) 7.1 Hz, 3H, CH3); 13C NMR
(CD3OD) δ 142.3 t, 142.2 t, 141.9 t, 140.0 t, 122.3 q, 133.2 q,
131.9 t, 131.8 t, 49.2 d, 46.9 d, 45.6 d, 43.3 d, 43.1 d, 42.8 d,
40.2 d, 40.1 d, 28.0 d, 27.2 d, 25.6 d, 25.1 d, 22.7 s, 22.6 s, 22.6
s, 20.8 s, 12.7 s, 12.6 s; MS (LR-FAB) C41H64N4O6S3 (M+) 895.
Gen er a l P r oced u r e for Dep r otection of BOC a n d th e
Meth ylen e Gr ou p of Hexa h yd r op yr im id in e. A mixture
of the protected carboranyl SPD/SPM in 3 N HCl (2-3 mL)
and EtOH (5 mL) was stirred for 10-16 h at 50-60 °C. The
reaction mixture was concentrated, and EtOH was added and
removed twice. The solid residue was purified by (A) recrys-
tallization or by (B) boiling in EtOH (1-2 mL) for 30 min and
allowing to stand for 1 day at RT. The solid was collected by
filtration and dried in high vacuum.
N1-(4-BOC-a m id obu tyl)-N3-(4-o-ca r bor a n ylbu tyl)h exa -
h yd r op yr im id in e (17). Compound 17 (184.9 mg, 0.41 mmol,
44%) was formed by reacting 1b (303.3 mg, 0.93 mmol) and
16b (239.3 mg, 0.93 mmol) at 25 °C. The product was eluted
with ethyl acetate-MeOH (4:1): 1H NMR (CD3OD) δ 3.72 (s,
1H, B10CH), 3.19-3.05 (m, 4H, CH2(N)2 + CONCH2), 2.55-
2.41 (m, 4H, CH2(CH2N)2), 2.30-2.40 (m, 4H, 2 × CH2N),
2.29-2.18 (m, 2H, CH2CB10), 1.66 (quint, J ) 3 Hz, 2H,
N
5-(4-o-Ca r b or a n ylb u t yl)sp er m id in e‚3H Cl (SP D-5).
CH2(CH2N)2), 1.52-1.40 (m, 17H, NCH2CH2CH2CH2N
+
Compound SP D-5 (542.4 mg, 1.20 mmol, 73%) was obtained
from 3 (888.8 mg, 1.63 mmol), using method A MeOH-ethyl
CH2CH2CH2CB2 + C(CH2)2); 13C NMR (CDCl2) δ 155.8 q, 78.5
t, 75.8 d, 75.2 q, 60.9 t, 54.4 d, 52.1 d, 52.0 d, 40.1 d, 37.5 d,
28.2 s, 27.7 d, 26.9 d, 26.2 d, 24.1 d, 23.0 d; MS (HR-EI)
C19H45B10N3O2 calcd 457.4442, found 457.4432.
acetate (1:1): mp 255 °C dec; IR (KBr, cm-1
) 3424 m, 2963 s,
2588 s, 1601 m, 1465 m, 1396 m, 1066 m, 722 m; 1H NMR
(CD3OD) δ 4.61 (s, 1H, B10CH), 3.38-3.08 (m, 6H, N(CH2)3),
3.08-2.78 (m, 4H, 2 × NCH2), 2.38-2.25 (m, 2H, CH2CB10),
2.20-2.01 (m, 2H, CH2(CH2N)2), 1.81-1.40 (m, 8H, NCH2CH2-
CH2CH2N + CH2CH2CH2CB10); 13C NMR (CD3OD) δ 76.8 q,
63.7 t, 53.9 d, 53.6 d, 40.0 d, 37.9 d, 27.4 d, 25.5 d, 24.1 d, 23.1
d, 21.9 d, 20.9 d; MS (LR-FAB) m/ e (calcd, found) for
C13H38B10N3: 341 (9, 9), 342 (28, 29), 343 (64, 67), 344 (100,
1-((4-o-Car bor an ylbu tyl)h exah ydr o-3-pyr im idyl)-4-(h ex-
a h yd r o-3-p yr im id yl)bu ta n e (19). Compound 19 (154.5 mg,
0.36 mmol, 36%) was formed by the reaction of 1a (370.5 mg,
1.00 mmol) with 18 (249 mg, 1.10 mmol) at 25 °C. The product
was eluted with MeOH containing 2% concentrated am-
monium hydroxide: 1H NMR (CDCl3) δ 3.66 (s, 1H, B10CH),
3.39 (s, 2H, CH2(N)2), 3.06 (s, 2H, CH2(N)2), 2.90-2.70 (m, 2H,
NCH2), 2.70-2.15 (m, 14H, 6 × CH2N + CH2CB10), 1.81-1.61
(m, 4H, 2 × CH2(CH2N)2), 1.58-1.30 (m, 8H, NCH2CH2CH2-
CH2N + CH2CH2CH2CB10); 13C NMR (CD3OD) δ 76.2 d, 75.1
q, 69.7 d, 60.8 t, 55.1 d, 55.0 d, 54.3 d, 52.8 d, 52.2 d, 44.9 d,
37.7 d, 27.0 d, 26.8 d, 26.3 d, 25.1 d, 23.3 d. Anal. (C18H44B10N4)
H, N; C: calcd, 50.91; found, 49.95.
100), 345 (95, 81), 346 (44, 44), 347 (6, 5). Anal. (C13H40B10
Cl3N3) C, H, N.
-
N1-(4-o-Ca r b or a n ylb u t yl)sp er m id in e‚3H Cl (SP D-1).
Compound SP D-1 (140.6 mg, 0.31 mmol, 77%) was obtained
from 17 (180 mg, 0.40 mmol) using method B: mp 233-5 °C;
1H NMR (CD3OD) δ 4.57 (s, 1H, B10CH), 3.18-2.81 (m, 10H,
5 × NCH2), 2.43-2.20 (m, 2H, CH2CB10), 2.20-2.0 (m, 2H,
CH2(CH2N)2), 1.90-1.40 (m, 8H, NCH2CH2CH2CH2N + CH2-
CH2CH2CB10); 13C NMR (CD3OD) δ 76.8 q, 63.7 t, 48.4 d, 45.8
d, 40 d, 38 d, 27.4 d, 26.5 d, 25.5 d, 24.2 d, 24; MS (LR-FAB)
m/ e (calcd, found) for C13H38B10N3 341 (8, 9), 342 (28, 29), 343
(64, 63), 344 (100, 100), 345 (95, 88), 346 (46, 42), 347 (6, 5).
Anal. (C13H40B10Cl3N3) C, H, N; B: calcd, 23.87; found, 23.04.
N5-(4-o-Car bor an ylbu tyl)sper m in e‚4HCl (SP M-5). Com-
pound SP M-5 (1010.0 mg, 1.85 mmol, 98%) was obtained from
11 (1.32 g, 1.88 mmol) using method B: mp 110-3 °C; IR (KBr,
cm-1) 3429 m, 2961 s, 2597 s, 1604 w, 1468 w, 721 w; 1H NMR
(CD3OD) δ 4.52 (s, 1H, B10CH), 3.25-2.87 (m, 14H, 7 × NCH2),
2.35-2.20 (m, 2H, CH2CB10), 2.15-1.90 (m, 4H, 2 × CH2(CH2-
N)2), 1.88-1.40 (m, 8H, NCH2CH2CH2CH2N + CH2CH2CH2-
CB10); 13C NMR (DMSO-d6) δ 76.2 q, 63.2 t, 51.4 d, 51.2 d,
48.9 d, 45.9 d, 43.8 d, 36.2 d, 36.1 d, 35.8 d, 26.0 d, 23.4 d,
22.5 d, 22.1 d, 21.0 d, 20.1 d; MS (LR-FAB) m/ e (calcd, found)
Gen er a l P r oced u r e for th e Eth yla tion of th e Am id o
N of P olya m in es a n d Deben zyla tion : N1,N10-Dieth yl-
N1,N10-bis(MesSO2)sp er m id in e (6).45 A solution of 4 (2.59
g, 4.32 mmol) in dry DMF (15 mL) was added slowly to a
suspension of NaH (60%, 518.4 mg 12.96 mmol) in dry DMF
(25 mL) at -5 °C. The reaction mixture was stirred for 45
min at RT and then cooled to 0 °C. To the mixture was added
slowly a solution of EtI (2.03 g, 13.08 mmol) in dry DMF (1
mL). The solution was stirred for 30 min at 0 °C and for an
additional 2 h at RT. Ice-water was added, and the layers
were separated. The aqueous phase was extracted with
CH2Cl2, and the organic phase was washed with 1 N NaOH
and dried over MgSO4. After filtering and removal of the
solvent, the residue was purified by flash column chromatog-
raphy and eluted with ethyl acetate-hexane (1:1). The
intermediate, compound 5 (2.7 g, 4.12 mmol, 96%), was
obtained as a colorless oil. A mixture of 5 (2.7 g, 4.12 mmol)
and 10% Pd/C (249 mg) in MeOH (40 mL) was stirred under
a H2 atmosphere overnight at RT. The Pd/C was removed by
filtration and the methanol by a rotary evaporator. The
residue was dried in high vacuum to give 6 (2.31 g, 4.08 mmol,
99%) as a colorless oil: IR (neat, cm-1) 2937 m, 1604 m, 1457
for C16
H45B10N4 398 (8, 8), 399 (27, 24), 400 (63, 62), 401 (100,
100), 402 (97, 85), 403 (49, 39), 404 (8, 7). Anal. (C16H48B10
-
Cl4N4‚H2O) C, H, N.
N1-(4-o-Car bor an ylbu tyl)sper m in e‚4HCl (SP M-1). Com-
pound SP M-1 (174.9 mg, 0.32 mmol, 80%) was obtained from
19 (168.4 mg, 0.39 mmol) using method B: mp 255-7 °C; IR
1
1
(KBr, cm-1) H NMR (D2O) δ 4.24 (s, 1H, B10CH), 3.20-2.87
m, 1382 m, 1315 s, 1148 s, 710 sl H NMR (CD3OD) δ 6.95 (s,
4H, ArH), 3.39-3.11 (m, 8H, 4 × SO2NCH2), 2.60 (s, 12H, 4 ×
ArCH3), 2.52-2.48 (m, 4H, N(CH2)2), 2.40 (s, 6H, 2 × ArCH3),
1.78-1.61 (m, 2H, CH2(CH2N)2), 1.60-1.28 (m, 4H, NCH2CH2-
CH2CH2N), 1.05 (t, J ) 7.1 Hz, 6H, 2 × CH3); 13C NMR
(CD3OD) δ 142.1 t, 142.0 t, 140.0 t, 133.5 q, 131.9 t, 131.6 t,
49.1 d, 46.8 d, 44.9 d, 43.0 d, 40.1 d, 40.0 d, 27.8 d, 27.1 d,
25.3 d, 22.6 s, 20.9 s, 12.8 s, 12.7 s; MS (HR-EI) C29H48N3O4S3
calcd 566.3120, found 566.3125.
(m, 14H, 7 × NCH2), 2.30-2.21 (m, 2H, CH2CB10), 2.19-1.19
(m, 4H, 2 × CH2(CH2N)2), 1.80-1.40 (m, 8H, NCH2CH2CH2-
CH2N + CH2CH2CH2CB10); 13C NMR (D2O, 500 MHz) δ 76.6
q, 63.3 t, 48.3 d, 48.1 d, 48.0 d, 45.5 d, 37.6 d, 37.4 d, 26.9 d,
25.9 d, 24.7 d, 23.7 d, 23.4 d; MS (LR-FAB) m/ e (calcd, found)
for C16H45B10N4 398 (8, 7), 399 (27, 24), 400 (63, 61), 401 (100,
100), 402 (97, 96), 403 (49, 24), 404 (8, 5). Anal. (C16H48
B10Cl4N4) C, H, N.
-
N1,N14-Dieth yl-N1,N10,N14-tr is(MesSO2)sp er m in e (14).
The intermediate, compound 13 (2.75 g, 3.07 mmol, 93%), was
first prepared from 12 (2.78 g, 3.31 mmol), NaH (60%, 397.5
mg, 9.94 mmol), and EtI (1.56 g, 10.0 mmol). Then 13 (2.62
g, 2.93 mmol) was then converted to compound 14 (2.21 g, 2.74
mmol, 94%) by hydrogenation with 10% Pd/C (250 mg) in
MeOH: IR (neat, cm-1) 2937 m, 1606 m, 1457 m, 1316 s, 1149
s, 735 m; 1H NMR (CD3OD) δ 6.90 (s, 6H, ArH), 3.31-3.18
(m, 4H, 2 × SO2NCH2), 3.11-2.96 (m, 8H, 4 × SO2NCH2), 2.58
(s, 6H, 2 × ArCH3), 2.53 (s, 12H, 4 × ArCH3), 2.50-2.33 (m,
N5,N10-Bis(4-o-ca r bor a n ylbu tyl)sp er m in e‚4HCl (SP M-
5,10). Compound SP M-5,10 (260.4 mg, 0.35 mmol, 92%) was
obtained from 9 (300 mg, 0.38 mmol) using method B: mp
151-3 °C; IR (KBr, cm-1) 3423 m, 2960 s, 2590 s, 1607 m,
1
1468 s, 1066 m, 722 m; H NMR (CD3OD) δ 4.52 (s, 2H, 2 ×
B10CH), 3.42-2.90 (m, 16H, 8 × CH2N), 2.38-2.26 (m, 4H, 2
× CH2CB10), 2.22-1.98 (m, 4H, 2 × CH2(CH2N)2), 1.90-1.41
(m, 12H, NCH2CH2CH2CH2N + 2 × CH2CH2CH2CB2); 13C
NMR (CD3OD) δ 76.8 q, 63.8 t, 54.2 d, 53.6 d, 51.4 d, 38.1 d,
27.5 d, 24.3 d, 23.2 d, 22.2 d; MS (LR-FAB) m/ e (calcd, found)

3894 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Cai et al.
for C22H64B20N4 596 (9, 12), 597 (22, 24), 598 (46, 49), 599 (76,
80), 600 (99, 94), 601 (100, 100), 602 (74, 68), 603 (38, 37), 604
(12, 12). Anal. (C22H66B20Cl4N4‚H2O) C, H, N.
IL) per well. After an additional 18-24 h, the plates were
harvested using a Skatron cell harvester (Skatron, Inc.,
Sterling, VA). Samples were counted in a LS 7800 beta
scintillation counter (Beckman Instruments, Inc., Irvine, CA)
using ScintiVerse scintillation fluid (Fisher Scientific, Fair
Lawn, NJ ). Uptake of [3H]TdR was measured by counts per
minute of cells grown in the presence of the test polyamines
analogues compared to those of cells grown in media lacking
the test compound using six replicates per concentration of
compound. Percent uptake was determined by multiplying
these values by 100, and that concentration required to
produce a 50% reduction in [3H]TdR uptake (i.e. IC50) was
calculated for each compound. Results for SPD and its
analogues are shown in Figure 3 and for SPM and its
analogues in Figure 4.
b. Eth id iu m Br om id e Disp la cem en t Assa y. The bind-
ing of spermidine, spermine, and the polyamine analogues to
DNA was determined by an ethidium bromide displacement
assay.51-53 To 10 µg of calf thymus DNA (Sigma, St. Louis,
MO) in 2.8 mL of buffer containing 2 mM HEPES, 10 µM
EDTA, and 9.4 mM NaCl (pH ) 7.0) was added 1.6 µM
ethidium bromide (Sigma, St. Louis, MO). Fluorescence was
measured on a Model 8000 spectrofluorometer (SLM Instru-
ments, Kankakee, IL). Emission and excitation wavelengths
were 598 and 546 nm, respectively. The various polyamine
compounds were individually added in 10 µL aliquots. The
decrease in fluorescence was recorded, and the C50 value was
defined as the concentration of the polyamine compound
required to reduce fluorescence of the ethidium bromide-DNA
complex by 50%. These results and the comparison with SPD
and SPM are presented in Table 2.
c. Cellu la r Up ta k e. Cellular uptake was determined by
culturing F98 glioma cells in the presence of the test polyamines
at a concentration that was not toxic or growth inhibitory. To
each of four T150 tissue culture flasks (Corning Glass Works,
Corning, NY) was added 5 × 106 F98 glioma cells in DMEM,
supplemented as described above in the toxicity/growth inhibi-
tion assay. After 24 h, the tissue culture medium was
decanted from the flasks and replaced with medium containing
a nontoxic concentration (5 µM) of carboranyl polyamines in
DMEM. Boron concentrations in the media (0.5 µg/mL) were
confirmed by DCP-AES, as described previously.54 Cells were
incubated at 37 °C in an atmosphere containing 5% CO2 for
48 h (approximately four doubling times), following which the
media was removed and its boron content was determined.
Cells were washed three times with phosphate buffer saline
(PBS), and the washes also were saved for boron determina-
tion. The cells then were disaggregated using trypsin-EDTA
(Gibco BRL, Grand Island, NY) and digested in concentrated
sulfuric acid-70% hydrogen peroxide,54 and the boron content
was determined. Boron levels are expressed in micrograms
per gram of cells, and as previously determined by direct cell
counting, 109 cells ≈ 1 g. The results of these studies are
presented in Table 3A and compared with the values obtained
for sodium undecahydromercapto-closo-dodecaborate (BSH)
and L-p-boronophenylalanine (BPA) (Boron Biologicals Inc.,
Raleigh, NC) in Table 3B.
Gen er a l P r oced u r e for Dep r otection of Mesitylen e-
su lfon yl Gr ou p . A mixture of the amide in concentrated HCl
(1 mL), AcOH (0.5 mL), and EtOH (5 mL) was refluxed for 24
h. The reaction mixture was concentrated under reduced
pressure, diluted with water (10 mL), and washed with CH2Cl2.
The aqueous solution was neutralized with 15% NaOH and
extracted with CH2Cl2 (3 × 10 mL). The organic phase was
dried over MgSO4, filtered, and extracted with 3 N HCl (3 ×
3 mL). The acidic solution was then concentrated, and EtOH
was added and removed twice. The solid residue was purified
by boiling in EtOH (1-2 mL) for 30 min and standing for 1
day at RT. The solid was collected by filtration and dried in
high vacuum.
N1,N10-Diet h yl-N5-(4-o-ca r b or a n ylb u t yl)sp er m id in e‚
3HCl (DESP D-5). Compound DESP D-5 (406.3 mg, 0.8
mmol, 47%) was obtained from 7 (1.30 g, 1.70 mmol): mp
181-3 °C; IR (KBr, cm-1) 3413 m, 2948 m, 2592 s, 1456 m,
722 w; 1H NMR (CD3OD) δ 4.52 (s, 1H, B10CH), 3.25-2.85 (m,
14H, 7 × NCH2), 2.35-2.22 (m, 2H, CH2CB10), 2.21-1.94 (m,
2H, CH2(CH2N)2), 1.88-1.37 (m, 8H, NCH2CH2CH2CH2N +
CH2CH2CH2CB10), 1.21 (t, J ) 7.1 Hz, 3H, CH3), 1.20 (t, J )
7.1 Hz, 3H, CH3); 13C NMR (CD3OD) δ 76.8 q, 63.8 t, 54.1 d,
53.7 d, 51.2 d, 45.5 d, 44.4 d, 44.2 d, 38.0 d, 27.5 d, 24.4 d,
24.3 d, 22.1 d, 22.0 d, 11.6 s; MS (LR-FAB) m/ e (calcd, found)
for C17H47B10N3 398 (8, 10), 399 (27, 31), 400 (64, 68), 401 (100,
100), 402 (97, 95), 403 (49, 47), 404 (8, 7). Anal. (C17H48B10Cl3N)
C, H, N.
N1,N14-Diet h yl-N5-(4-o-ca r b or a n ylb u t yl)sp er m in e‚4-
HCl (DESP M-5). Compound DESP M-5 (83.4 mg, 0.14 mmol,
53.7%) was obtained from 15 (260.0 mg, 0.26 mmol): mp
145-7 °C; IR (KBr, cm-1) 3428 m, 2949 s, 2591 s, 1459 m,
1403 w, 723 w; 1H NMR (CD3OD) δ 4.56 (s, 1H, B10CH), 3.42-
2.85 (m, 18H, 9 × NCH2), 2.35-2.22 (m, 2H, CH2CB10), 2.21-
1.98 (m, 4H, 2 × CH2(CH2N)2), 1.92-1.33 (m, 8H, NCH2CH2-
CH2CH2N + CH2CH2CH2CB10), 1.25 (t, J ) 7.1 Hz, 3H, CH3),
1.24 (t, J ) 7.1 Hz, 3H, CH3); 13C NMR (CD3OD) δ 76.8 q,
63.7 t, 54.0 d, 53.4 d, 51.1 d, 48.2 d, 45.9 d, 45.4 d, 44.3 d, 44.2
d, 37.9 d, 27.4 d, 24.2 d, 24.1 d, 22.0 d, 11.5 s; MS (LR-FAB)
m/ e (calcd, found) for C20H54B10N4 455 (8, 11), 456 (27, 32),
457 (63, 67), 458 (100, 100), 459 (99, 99), 460 (52, 34), 561 (10,
9). Anal. (C20H56B10Cl4N4) C, H, N.
2. Biologica l Stu d ies. The variations for all of the
biological test data were ∼(10%; therefore no error bars were
inserted in the figures or tables. All tests were repeated two
or three times. For cytotoxicity/growth inhibitory studies, each
time six replicates were used for each sample.
a . In Vitr o Eva lu a tion of Cytotoxic/Cytosta tic P r op -
er ties. The F98 glioma cell line was derived from an undif-
ferentiated brain tumor induced by administering N-ethyl-N-
nitrosourea to a pregnant inbred CD Fisher 344 rat and has
been propagated in vitro and in vivo since 1971. Its morphol-
ogy and in vitro characteristics have been described in
detail,59,60 and it has been used by us to evaluate a variety of
boron compounds as potential delivery agents for BNCT.48,61,62
The assay that was employed to detect the toxicity and/or
growth inhibitory effects has been widely used63 and is based
on the incorporation of tritiated thymidine ([3H]TdR) by
biosynthetically active (i.e. S phase) surviving cells following
an exposure to the test compound. Ninety-six well plates
(Corning Glass Works, Corning, NY) were seeded with 10 000
F98 cells per well and allowed to grow overnight at 37 °C in
Dulbecco’s minimal essential medium (DMEM) supplemented
with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 100
units/mL of penicillin, containing 100 µg/mL streptomycin, 2
mM L-glutamine, and 0.1 mM MEM nonessential amino acids
(Gibco BRL, Grand Island, NY). This was replaced with media
containing varying concentrations (1-100 µΜ) of spermidine,
spermine, or carboranyl polyamines containing 0.1-10 µg/mL
boron. After 24 h incubation at 37 °C in a humidified
atmosphere containing 5% CO2, the polyamine-containing
media was removed and replaced with polyamine-free DMEM
supplemented with FBS, and then pulsed with 0.1 µCi [3H]TdR
(specific activity 46 Ci/mmol) (Amersham, Arlington Heights,
Ack n ow led gm en t. This work was supported by
U.S. Public Health Service Grants (R01 CA-53896/R01
CA-71033) and U.S. Department of Energy Grants and
Contracts (DE-FG02-90ER60972/DE-AC02-76CH00016).
We acknowledge the assistance of Dr. Beverly Barnum
in the manuscript’s preparation.
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