Synthesis and biological evaluation of boron-containing polyamines as potential agents for neutron capture therapy of brain tumors

Article abstract of DOI:10.1021/jm980703f

New boron-containing spermidine/spermine (SPD/SPM) analogues have been synthesized: N⁵-[4-(2-aminoethyl-o-carboranyl)butyl] and N⁵-{4-[(2,3- dihydroxypropyl)-o-carboranyl]butyl} SPD/SPM derivatives (ASPD-5, ASPM-5, DHSPD-5, and DHSPM

Full text of DOI:10.1021/jm980703f

1282
J . Med. Chem. 1999, 42, 1282-1292
Syn th esis a n d Biologica l Eva lu a tion of Bor on -Con ta in in g P olya m in es a s
P oten tia l Agen ts for Neu tr on Ca p tu r e Th er a p y of Br a in Tu m or s
J in-Cong Zhuo,*,† J ianping Cai,† Albert H. Soloway,† Rolf F. Barth,‡ Dianne M. Adams,‡ Weihua J i,† and
Werner Tjarks†
College of Pharmacy and Department of Pathology, The Ohio State University, Columbus, Ohio 43210
Received December 14, 1998
New boron-containing spermidine/spermine (SPD/SPM) analogues have been synthesized: N5-
[4-(2-aminoethyl-o-carboranyl)butyl] and N5-{4-[(2,3-dihydroxypropyl)-o-carboranyl]butyl} SPD/
SPM derivatives (ASP D-5, ASP M-5, DHSP D-5, and DHSP M-5) as well as N5-{[4-(dihydroxy-
boryl)phenyl]methyl}spermidine (BBSP D-5). These boronated polyamines retain their ability
to displace ethidium bromide from calf thymus DNA and are rapidly taken up in vitro by F98
rat glioma cells. The in vitro toxicities of ASP D-5, ASP M-5, DHSP D-5, and DHSP M-5 are
lower than those previously reported for N5-[4-(o-carboranyl)butyl] SPD/SPM derivatives (SP D-5
and SP M-5) but similar to those of native SPD and SPM. Very low toxicity was also observed
for BBSP D-5. In vivo studies of ASP D-5 and BBSP D-5 were performed in mice bearing
intracerebral implants of the GL261 glioma and subcutaneous implants of the B16 melanoma.
The biodistribution data found in both tumor models suggest that the polyamines synthesized
to date do not appear to be suitable boron agents for BNCT.
Ch a r t 1
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
(LET) R particles and recoiling 7Li nuclei.1 These
particles have path lengths of 5-9 µm and are capable
of destroying those cells containing sufficient quantities
of 10B required to sustain a lethal 10B(n,R)7Li reaction.
The chemistry and use of BNCT as a cancer treatment
modality has been described in several recent reviews.2-6
A boron delivery agent should be nontoxic, selectively
target tumor cells, and ideally localize within the
nucleus. For BNCT to be effective, there must be ∼20-
30 µg of 10B/g of tumor and a low concentration (<5 µg
of 10B/g of cell) in surrounding normal cells and blood.7,8
Various boron-containing analogues of biologically active
compounds, such as amino acids,9-15 peptides,16-19 and
nucleosides/nucleotides,20-30 have been synthesized. The
basis for their preparation is that such structures might
function in a manner similar to their naturally occurring
counterparts and become selectively incorporated into
either proliferating or more metabolically active tumor
cells. Boron-containing analogues of porphyrins31-34 and
DNA binders35-38 have been also considered as potential
agents for BNCT.
a nonspecific manner.46-48 Their cellular concentration
is increased in tumor cells and specifically within the
nucleus.49-52 In addition, there is a facilitated transport
system that increases the uptake by malignant cells.53
For these reasons, boron-containing polyamines have
been considered as potential BNCT agents.54-56
We have previously designed and synthesized N-
carboranyl-tethered polyamine analogues of spermidine
and spermine54 and have correlated their chemical
structure with their cellular uptake, DNA binding
properties, and in vitro toxicity. The toxicity strongly
depended upon the position of the carboranyl group on
the polyamine scaffold. Polyamines substituted at the
terminal amino groups (SP D-1, SP M-1) had a greater
toxicity than those substituted at one of the central
nitrogen atoms (SP D-5, SP M-5) (Chart 1). Neverthe-
less, even for the latter compounds, the observed in vitro
toxicity was very high. To decrease this toxicity, the
hydrophilic properties of the compounds were increased
by modifying the o-carborane cages in SP D-5 and
SP M-5 by inserting 2-aminoethyl and 2,3-dihydroxy-
propyl groups at the 2-position. These compounds were
designated as ASP D-5, ASP M-5, DHSP D-5, and DH-
SP M-5. Another boronated polyamine with increased
hydrophilicity, [4-(dihydroxyboryl)phenylmethyl]spermi-
dine (BBSP D-5), was also synthesized.
Polyamines such as spermidine (SPD) and spermine
(SPM) are essential for mammalian cell growth and
differentiation,39,40 and their depletion has growth
inhibitory effects on tumors.41,42 This has been the basis
for the preparation of various polyamine synthetase
inhibitors43,44 and synthetic polyamines45 for cancer
treatment. They interact electrostatically with DNA in
† College of Pharmacy.
‡ Department of Pathology.
10.1021/jm980703f CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/23/1999

Polyamines as Potential Agents for BNCT
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1283
Sch em e 1a
r
a
(a) NaH, BnCl; (b) B10H14/EtCN, 30%; (c) BuLi, (CH2)2O, 96%; (d) TsCl, pyridine, 90%; (e) NaN3/DMF, 90%; (f) 1. LiAlH4/THF, 2.
BOC-ON/THF, 93%; (g) H2, Pd-C, MeOH-AcOH, 80%; (h) TsCl, pyridine, CH2Cl2, 82%; (i) NaI, acetone, 95%; (j) BOCNH(CH2)3NH-
(CH2)4NHBOC or BOCNH(CH2)3NH(CH2)4N(BOC)(CH2)3NHBOC, K2CO3/DMF, 8 (52%), 9 (75%); (k) 3 N HCl, ASP D-5 (100%), ASP M-5
(87%).
Ch em ica l Syn th esis
reaction with NaI in acetone (Scheme 2). The prepara-
tion of 13 has been previously described.25 The target
compounds DHSP D-5 and DHSP M-5 were obtained by
alkylation of the protected spermidine (BOC-SPD) or
spermine (BOC-SPM) with the protected dihydroxy
iodide 15 in DMF in the presence of K2CO3, followed by
the removal of the BOC groups as described above.
Since all of the above compounds contain the highly
lipophilic carborane moiety which contributes to the
compounds’ toxicities, a dihydroxyboryl-containing
polyamine, [4-(dihydroxyboryl)phenylmethyl]spermidine
(BBSP D-5), was designed and prepared. Synthesis of
BBSP D-5 is shown in Scheme 3. Reaction of BOC-SPD
with 4-(bromomethyl)phenylboronic acid58 produced 18.
The desired target compound BBSP D-5 was obtained
after removal of the BOC group by acidic hydrolysis.
BBSP D-5 loses one molecule of water easily under
vacuum, forming the dimer Bis-BBSP D-5. This was
determined by NMR in anhydrous aprotic solution
(DMSO-d6) and by elemental microanalysis. Addition
of water to the dimer Bis-BBSP D-5 converted it back
to the monomer BBSP D-5. Concentration of BBSP D-5
from an ethanolic solution yielded Et-BBSP D-5 in
which one hydroxyl group was replaced by an ethoxy
function.
Syntheses of the target compounds ASP D-5 and
ASP M-5 are shown in Scheme 1. (4-Benzyloxybutyl)-
o-carborane (3) was prepared according to previously
described procedures57 by reaction of decaborane(14)
(B10H14) with benzyl 5-hexyn-1-yl ether (2); the latter
compound was obtained by the benzylation of 5-hexyn-
1-ol with benzyl chloride. The monosubstituted carbo-
rane 3 was lithiated with n-butyllithium and converted
to 1-(2-tosylethyl)-2-(4-benzyloxybutyl)-o-carborane (4)
by reaction with ethylene oxide followed by tosylation.
The 1,2-disubstituted-o-carborane 4 was transformed
into the BOC-protected amine 5 by reaction with NaN3,
followed by reduction with LiAlH4 and the protection
of the amino group using BOC-ON [2-(tert-butoxycar-
bonyloxyimino)-2-phenylacetonitrile]. Debenzylation of
5 by catalytic hydrogenation (H2, Pd/C) yielded the
primary carboranyl alcohol which upon treatment with
TsCl/pyridine produced the corresponding carboranyl
tosylate 6. The key iodide 7 was obtained by the reaction
of 6 with NaI in acetone. Alkylation of the protected
spemidine (BOC-SPD) or spermine (BOC-SPM)54 with
the iodide 7 in DMF in the presence of K2CO3 resulted
in the boronation of the corresponding BOC-protected
compounds 8 and 9, respectively (Scheme 1). Removal
of the BOC groups by acidic cleavage afforded the target
compounds ASP D-5 and ASP M-5.
Biologica l Stu d ies
In Scheme 2 are presented the syntheses of the (2,3-
dihydroxypropyl)-o-carboranyl polyamines DHSP D-5
and DHSP M-5. The required iodide 15 was prepared
from compound 13 by the hydrolysis of the acetate with
2 N NaOH. The alcohol was then tosylated with TsCl/
pyridine and the tosyl group replaced with iodine by the
The three major questions regarding these new boron-
containing polyamines were whether (1) their biological
properties were similar to that of SPD/SPM; (2) their
enhanced hydrophilicity diminished their toxicity; and
(3) they possessed the requisite tumor-targeting proper-
ties to be useful BNCT agents. To answer these impor-

1284 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Zhuo et al.
Sch em e 2a
a
(a) AcCl, pyridine, CH2Cl2, 95%; (b) B10H14/EtCN, 35%; (c) Pd2(dba)3, dppe, EtOCO2CH2CHdCH2, THF, 81%; (d) OsO4, pyridine,
NMMPNO, 79%; (e) Me2C(OMe)2, TsOH, 86%; (f) 2 N NaOH, 95%; (g) TsCl, pyridine, CH2Cl2, 84%; (h) NaI, acetone, 87%; (i)
BOCNH(CH2)3NH(CH2)4NHBOC or BOCNH(CH2)3NH(CH2)4N(BOC)(CH2)3NHBOC, K2CO3/DMF, 16 (67%), 17 (74%); (j) 3 N HCl,
DHSP D-5 (98%), DHSP M-5 (100%).
Sch em e 3
Ta ble 1. In Vitro Toxicity of Polyamine Analogues
tant questions, the following biological properties have
been evaluated: (1) their in vitro growth inhibitory
effects using F98 rat glioma cells; (2) their ability to
displace ethidium bromide from calf thymus DNA; (3)
the compound’s in vitro cellular uptake using the F98
glioma cells; and (4) their in vivo biodistribution in mice
bearing intracerebral implants of the GL261 glioma and
subcutaneous implants of the B16 melanoma. The focus
of these latter studies has been to evaluate ASP D-5 and
BBSP D-5 and to compare them with the earlier com-
pounds that have been synthesized and evaluated.54
(1) Eva lu a tion of in Vitr o Toxicity. The assay that
we have employed quantified the number of F98 glioma
cells following a 24-h exposure to the test compounds
by measuring cellular uptake/incorporation of sulfo-
rhodamine B (SRB) and comparing that uptake to cells
that were not exposed to the test compounds.59 The
results are summarized in Table 1. The IC50 (inhibitory
concentration) value is defined as that concentration of
compd
IC50 (µM)
SPD
SP D-5
ASP D-5
DHSP D-5
>100a
22a
>100
>100
SPM
SP M-5
ASP M-5
DHSP M-5
>100a
22a
>100
>100
BBSP D-5
>5000
a
Data taken from ref 54. A 50% reduction in the uptake of
[3H]TdR in F98 glioma cells was measured for SP D-5 and SP M-
5.
the compound that produced a 50% reduction in SRB
absorbance by F98 glioma cells as determined spectro-
photometrically for all compounds compared with the
control cells.60 The in vitro toxicities of carboranyl
polyamines ASP D-5, ASP M-5, DHSP D-5, and DH-

Polyamines as Potential Agents for BNCT
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1285
Ta ble 2. DNA Binding Affinity as Measured by Ethidium
Ta ble 3. In Vitro Cellular Uptake of Boronated Polyamines by
Bromide Displacementa
F98 Glioma Cellsa
compd
IC50 (µM)
Ccellular
Ccellular
(µg of B/g
(µg of B/g
of cells)
SPD
SP D-5
ASP D-5
DHSP D-5
105 ( 8.7
147b
3.8 ( 0.6
66.7 ( 5.7
Cmedia
(µg of B/mL)
of cells),
48 hb
Cmedia
compd
(µg of B/mL) 24 hb 48 hb
SP D-5c
ASP D-5
DHSP D-5
SP M-5c
ASP M-5
DHSP M-5
BBSP D-5
BPAc
0.5
0.5
0.5
0.5
0.5
0.5
43
19
9
0.5
5.4
5.4
65
51
51
43
116
104
BBSP D-5
115.0 ( 7.1
SPM
SP M-5
ASP M-5
DHSP M-5
3.0 ( 1.7
64
15
20
23b
5.3
5.3
5.1
82
60
229
83
4.0 ( 0.9
21.7 ( 3.8
114
a
Ethidium bromide and DNA concentrations were 1.6 and 10
µg, respectively. Data taken from ref 54.
50
570
81
39
b
BSHc
a
F98 cells were incubated with the polyamine analogues at
molar compound concentrations corresponding to the boron amounts
indicated. Cells were washed, counted, and digested. Boron
concentrations Cmedia and Ccellular were determined by DCP-AES
SP M-5, possessing an additional hydrophilic 2-amino-
ethyl or 2,3-dihydroxypropyl group attached to the
carborane moiety, were equivalent (>100 µM) to those
previously reported for the native polyamines, SPD and
SPM.54 They were ∼5 times less toxic than the earlier
structures, SP D-5 and SP M-5,54 and the first dihy-
droxyboryl-containing polyamine, BBSP D-5, tested had
the lowest toxicity of all (IC50 ) 5000 µM).
b
(109 cells = 1 g of cells). Incubating time. c Data taken from ref
54.
Ta ble 4. Biodistribution of ASP D-5 and BBSP D-5 in Mice
Bearing Intracerebral GL261 Gliomas
ASP D-5a
BBSP D-5b
tissue
(µg of B/g)
(µg of B/g)
(2) DNA Bin d in g Affin ity. All of the polyamines
synthesized were evaluated in a DNA binding assay.
The binding of polyamine analogues to DNA was
determined by an ethidium bromide displacement
assay.61-63 The IC50 value was defined as that concen-
tration of polyamine required to decrease the fluores-
cence of the ethidium bromide-calf thymus DNA com-
plex by 50%. The results are summarized in Table 2.
All of the SPD and SPM analogues, possessing 2-ami-
noethyl or 2,3-dihydroxypropyl groups at the 2-position
of the o-carborane cages, had stronger affinity for DNA
than the previously synthesized analogues, SP D-5 and
SP M-5.54 There was a 28-fold enhancement of DNA
binding affinity for ASP D-5 and a 1.6-fold increase for
DHSP D-5 compared with natural SPD. In the case of
BBSP D-5, the values obtained were comparable to
those for SPD. In the case of the spermine analogues,
ASP M-5 showed values comparable to natural SPM,
while DHSP M-5 had diminished DNA binding proper-
ties (21.7 µM). Of all these boron-containing compounds,
the best DNA binder was ASP D-5.
tumor
brain
blood
liver
spleen
kidney
skin
7.0 ( 3.8
1.6 ( 1.0
3.4 ( 2.1
51.3 ( 27.3
30.9 ( 13.2
112.0 ( 53.2
6.3 ( 3.8
5.3 ( 1.9
nm
nm
nm
nm
22.9 ( 10.9
nm
44.0 ( 12.6
nm
nm
nm
muscle
eyes
T/Bl ratio
T/Br ratio
1.7
4.4
a
b
Dose ) 30 µg of compound/g per day. Dose ) 10 µg of boron/g
per day. Five mice were used for each compound, and the values
shown are means ( standard deviations for each set of determina-
tions.
(4) In Vivo Biod istr ibu tion Stu d ies. The data
presented in the proceeding sections showed that both
ASP D-5 and BBSP D-5 possessed low cellular toxicity,
retained the capacity to bind to DNA as other poly-
amines, and had significant in vitro cellular uptake.
These results prompted an in vivo biodistribution study
in tumor-bearing animals of these two compounds that
may be viewed as representative structures of two
different types of boron-containing polyamines. These
studies were performed using two different tumor
models in C57Bl/6 mice: (1) an intracerebrally im-
planted GL261 glioma and (2) a subcutaneously im-
planted B16 melanoma. Animals were placed on a
polyamine-deficient diet and water containing 2% di-
fluoromethylornithine (DFMO), an ornithine decarbox-
ylase inhibitor, 3-4 days prior to the beginning of the
infusion, and this was contined for the duration of the
study. Alzet pumps were implanted, and infusion of the
boronated polyamines was carried out over 3 days.
Following this time period, the pumps were removed,
the animals were euthanized at 0, 12, and 24 h, and
their tissues were removed for boron analysis. These
results are summarized in Table 4. For ASP D-5, the
greatest amount of boron was found in the kidney (112
µg of B/g) and liver (51.3 µg of B/g), while the concentra-
tion in the tumor was only 7.0 µg of B/g. Similarly for
BBSP D-5, the levels in kidney (44.0 µg of B/g) and liver
(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).64 Uptake of the new compounds
was compared with the earlier structures, SP D-5 and
SP M-5, and with two clinically used agents, BSH and
BPA (Table 3). The media concentration in the initial
studies was very low due to the high toxicity of the
earlier carboranyl polyamines. The more recent com-
pounds were much less toxic permitting a 10-fold
increase in media concentration. Nevertheless, the
cellular uptake of these more hydrophilic analogues was
significantly lower than that of the previously described
structures, SP D-5 and SP M-5,54 a matter that will be
considered in the Discussion section. However, all of
these boronated polyamines were sequestered by tumor
cells at a significantly higher level than was observed
for either of the current clinical compounds BSH and
BPA.

1286 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Zhuo et al.
Ta ble 5. Biodistribution of SP D-5 and ASP D-5 in Mice
Ta ble 6. Biodistribution of ASP D-5 in Mice Bearing
Bearing Subcutaneous B16 Melanomas
Subcutaneous B16 Melanomas
SP D-5a
(µg of B/g)
(10 µg of compd/g)
ASP D-5b
(µg of B/g)
(20 µg of compd/g)
dose of 30 µg
of compd/g
(µg of B/g)
dose of 40 µg
of compd/g
(µg of B/g)
tissue
tissue
tumor
brain
blood
liver
kidney
muscle
eyes
9.3 ( 2.5
nm
10.2 ( 3.8
nm
tumor
brain
blood
16.2 ( 5.1
2.0 ( 0.7
18.5 ( 2.8
2.1 ( 1.1
12.5 ( 2.4
74.8 ( 5.6
201.3 ( 43.1
9.8 ( 3.2
nm
3.2 ( 1.1
76.8 ( 12.2
109.6 ( 9.9
8.5 ( 5.2
nm
5.3 ( 3.5
2.9 ( 1.1
liver
76.8 ( 12.2
25.8 ( 14.5
157 ( 36.3
34.2 ( 20.1
4.4 ( 1.3
7.9 ( 3.1
8.1 ( 3.5
87.5 ( 12.3
37.7 ( 14.2
192.5 ( 27.3
101.0 ( 46.7
7.1 ( 1.8
9.2 ( 1.3
6.4 ( 0.5
spleen
kidney
skin by pump
skin
muscle
eyes
T/Bl ratio
0.74
3.2
a
b
Four mice per group; tumor size averaged 150 mg. Five mice
per group; tumor size averaged >500 mg. For both groups, the
boron values shown are means ( standard deviations.
T/Bl ratio
T/Br ratio
3.1
8.1
6.3
8.8
(22.9 µg of B/g) were the highest, while those in tumor
were undetectable.
cal limitation in the synthesis of o-carborane-containing
amines that are very strong bases. This is the case with
the polyamines. This problem can be obviated by mask-
ing the amino function and generating the final products
under acidic conditions as the amine hydrochloride.
These salts are very water-soluble even with the car-
boranyl function, and the resulting aqueous solutions
are stable.
The highly lipophilic properties of the carborane cage
have been a major limitation with this boron moiety
since it can result in nonspecific binding to various
biolipids thereby limiting the tumor specificity of these
boron-containing polyamines. This fact has been the
basis for the incorporation of hydrophilic functions into
the carborane in an attempt to diminish its lipophilicity.
However, no studies were performed to determine the
success of this chemical maneuver. An alternative
approach is to replace the carborane with a dihydroxy-
boryl entity, the same boron component in the clinically
used BPA. This was the rationale for the synthesis of
BBSP D-5. The chemical procedures described and
presented in the Experimental Section are straightfor-
ward, and by-in-large the compounds were formed in
the manner proposed. The question was not the issue
of the novelty of the synthetic chemistry but whether
these compounds would have favorable biological prop-
erties for tumor targeting.
Though the tumor boron levels obtained with ASP D-5
were insufficient to be useful as a single delivery agent
for BNCT, it remains to be determined whether it could
be used as one component in a mixture of compounds
to target different subpopulations of tumor cells. By our
method of in vivo evaluation, BBSP D-5 showed un-
measurable levels of boron in tumor and for this reason
is not considered as an appropriate BNCT agent, despite
the fact that it had the lowest in vitro toxicity and
comparable DNA binding affinity to SPD.
In the evaluation of these compounds as potential
BNCT agents for melanoma, C57Bl/6 mice bearing
subcutaneous implants of the B16 melanoma were used.
In the initial studies, ASP D-5 was compared against
the more toxic analogue SP D-5. Animals were placed
on a polyamine-deficient diet and water containing 0.2%
neomycin together with 2% DFMO for 3 days. The
compounds were then administered intraperitoneally in
two doses per day for 3 days. After the last injection,
the animals were held for 12 h and euthanized, and
their tissues were removed for boron determination. The
results are shown in Table 5. For SP D-554 and ASP D-
5, the largest amounts of boron were found in the kidney
(201 and 110 µg of B/g, respectively) and liver (75-76
µg of B/g), while the values in tumor were only 9-10
µg of B/g. The T/Bl ratio of ASP D-5 was 3.2, and that
of SP D-5 was 0.74.
(2) Biology. The polyamine structure as a scaffold
for targeting malignant cells has such a potential only
if the polyamine transport system is upregulated in
tumor cells compared with normal cells from which they
are derived. This appears to be especially true for brain
tumors44 and has provided the rationale for incorporat-
ing boron moieties into the naturally occurring poly-
amines, SPD and SPM. The in vitro biological results
with carboranyl polyamines show their potential for
selectively delivering boron to tumor cells and thereby
offered the potential of 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 10-100-fold less. Polyamines bind to DNA
nonspecifically, and therefore, boronated polyamines
may be able to target DNA directly once they penetrate
the cell membrane. The hydrophilic properties of these
polyamine salts make them highly water soluble intra-
In a subsequent study, ASP D-5 was administered at
increasing doses using the same tumor model. The
animals were again placed on a polyamine-deficient diet
and water containing 2% DFMO for 3 days prior to the
initiation of and for the duration of the study. Then,
Alzet pumps were implanted, and compound infusion
was carried out over 4 days. At the end of the fourth
day, the animals were euthanized and tissues from each
group of five were removed and analyzed for boron. The
results are summarized in Table 6. The highest amounts
of boron were found in kidney, skin adjacent to the
pump, and liver, which were appreciably greater than
those in the tumor (16-18 µg of B/g).
Discu ssion
(1) Ch em istr y. The o-carborane cage is sufficiently
stable under neutral and acidic conditions for its
incorporation into potential BNCT agents. However, it
undergoes degradation under various basic condi-
tions,65-68 and this property has been the major chemi-

Polyamines as Potential Agents for BNCT
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1287
were measured relative to internal residual protons from the
lock solvent and then referenced to TMS (δ ) 0.0 ppm), and
coupling constants (J ) are reported in Hz. IR (cm-1) were
measured on a Laser Precision Analytical 720-XI spectrometer.
Mass spectra (MS) were carried out at the Campus Chemical
Instrument Center of The Ohio State University on a VG 70-
250S for HR-EI and on a Finnigan MAT-900 for HR-FAB by
Dr. David H. Chang. Elemental analyses were performed by
Robertson Microlit Laboratories, Inc. and Galbraith Labora-
tories, Inc. All compounds gave elemental analyses within
(0.4%. RT (room temperature) is 22-25 °C.
1. Ch em ica l Syn th eses. 6-Ben zyloxyh ex-1-yn e (2).69 To
a suspension of NaH (1.52 g, 60%, 37.8 mmol) in THF (150
mL) was added a solution of hex-5-yn-1-ol (3.38 g, 34.4 mmol)
in THF (20 mL). The mixture was stirred at RT for 1 h. To
the mixture was added dropwise a solution of benzyl bromide
(6.18 g, 36.1 mmol) in THF (20 mL). The resulting mixture
was stirred at RT for 8 h and filtered through a pad of silica
gel. The filtrate was concentrated. The residue was flash
chromatographed (hexane/EtOAc ) 20:1) to give 2 (4.38 g,
68%): 1H NMR (CDCl3) 7.22-7.43 (m, 5H, Ph), 4.49 (s, 2H,
OCH2Ph), 3.49 (t, 2H, J ) 6.1, OCH2CH2), 2.20 (td, 2H, J )
6.8, 2.7, CH2CdCH), 1.94 (t, 1H, J ) 2.7, CCH), 1.60-1.81
(m, 4H); 13C NMR (CDCl3) 138.7 (C, Ph), 128.7 (CH, Ph), 128.3
(2 CH, Ph), 127.5 (2 CH, Ph), 84.3 (C, CCH), 72.9 (PhCH2),
69.8 (OCH2), 68.3 (CH, CCH), 28.8 (CH2), 25.3 (CH2), 18.2
(CH2).
peritoneally or intravenously at suitable concentrations
without the use of solubilizers or cosolvents. However,
the major limitation with the earlier compounds was
their toxicity, especially those compounds with terminal
N-substituted boron moieties (SP D-1, SP M-1).54 The
compounds described in this report address that prob-
lem directly. Although their in vitro uptake is somewhat
reduced when compared with those earlier boronated
polyamines, nevertheless, the cellular concentrations
attained are very substantial, markedly superior to that
of BSH and BPA, and have significantly reduced toxic-
ity.
The most important question obviously is not their
in vitro uptake but what would be their in vivo concen-
tration in tumor-bearing animals? To maximize in vivo
uptake, animals were simultaneously placed on a
polyamine-deficient diet and given an inhibitor of orni-
thine decarboxylase, DFMO, in their drinking water.
The objective of these efforts was to decrease exogenous
levels of naturally occuring polyamines that the animals
received and to reduce the levels of de novo endogenous
polyamines produced. The reasoning was that the
boronated polyamines might be rapidly sequestered by
malignant cells under these conditions.
1-(4-Ben zyloxybu tyl)-o-ca r bor a n e (3). A mixture of com-
pound 2 (3.80 g, 20.2 mmol) and decaborane(14) (2.85 g, 23.3
mmol) in EtCN (6 mL) was refluxed for 2 h. After cooling, the
solvent was evaporated. The residue was chromatographed
(hexane/EtOAc ) 20:1) to give 3 (1.87 g, 30%): IR (neat)
2930w, 2847m, 2570vs, 1346w, 1084m, 716m; 1H NMR (CDCl3)
7.20-7.45 (m, 5H, Ph), 4.49 (s, 2H, OCH2Ph), 3.54 (s, 1H,
B10H10CCH), 3.40-3.52(m, 2H, OCH2CH2), 2.23-2.31 (m, 2H,
B10H10CH2), 1.48-1.63 (m, 4H); 13C NMR (CDCl3) 138.4 (C,
Ph), 128.4 (2CH + CH, Ph), 127.7 (2CH, Ph), 75.4 (C, B10H10C),
73.1 (CH2, PhCH2O), 69.3 (OCH2), 60.9 (CH, B10H10CH), 37.9
(CH2), 29.0 (CH2), 26.2 (CH2); MS (HR-EI) for C13H26B10O calcd
308.2914, found 308.2914. Anal. (C13H26B10O) C, H, N, B.
The in vivo results that we have obtained can be
summarized as not being very promising. The two
compounds that have been evaluated, ASP D-5 and
BBSP D-5, have shown insufficient tumor boron levels
for the former and unmeasurable levels for the latter.
The boron levels in liver, spleen, and kidney were
markedly higher than those observed for the tumor. The
question remains, however, as to why these compounds
did not have better tumor accretion under in vivo
conditions?
Will it not be possible to obtain the necessary tumor
concentrations of boronated polyamines? Were the in
vivo methods used for screening these compounds
inappropriate? To address these questions, it will be
necessary to determine whether labeled SPD and SPM
at the same molar concentrations would have higher
tumor uptake, better tumor-to-normal tissue ratios and
at a tumor cellular concentration necessary for therapy.
Alternatively, it may be that the boronated polyamines,
which we have selected for synthesis, are not the most
useful ones.
1-Tosyleth yl-2-(4-ben zyloxybu tyl)-o-ca r bor a n e (4). To
a solution of 3 (897.5 mg, 2.93 mmol) in THF (45 mL) was
added BuLi (2.2 mL, 1.6 M, 3.51 mmol) at -45 °C. The mixture
was stirred for 40 min while the temperature rose from -45
°C to RT. Then the solution was cooled to -30 °C, and ethylene
oxide (1 mL) was added quickly. After 30 min, an equal amount
of ethylene oxide was added. The reaction mixture was stirred
for an additional 40 min during which time the temperature
rose from -30 °C to RT. The mixture was quenched with a
cold NH4Cl solution and extracted with CH2Cl2 (3 × 50 mL).
The organic layer was washed with brine and dried over
MgSO4. After evaporation of the solvent, the residue was
chromatographed (hexane/EtOAc ) 3:1) to give an intermedi-
ate alcohol (986.6 mg, 2.81 mmol, 96%) as a colorless oil. This
alcohol (870.7 mg, 2.48 mmol) was diluted with CH2Cl2 (50
mL) and cooled to about 0 °C. To this solution was added
pyridine (3 mL) followed by tosyl chloride (947 mg, 4.89 mmol)
divided into six portions and added over 2 h. The mixture was
stirred overnight at RT and washed with 2 N NaOH and water.
After the organic layer was dried over MgSO4 and the solvent
removed, the residue was chromatographed (hexane/EtOAc )
3:1) to give 4 (1.09 g, 2.16 mmol, 87%) as a colorless oil: IR
In conclusion, although we have succeeded in syn-
thesizing boronated polyamines that are appreciably
less toxic than earlier compounds54 and achieve very
adequate in vitro cellular concentrations, their in vivo
uptake by tumors is inadequate as BNCT delivery
agents. It remains to be determined why that is the case
and whether other boronated polyamines can be de-
signed and synthesized that will achieve that objective.
Exp er im en ta l Section
1
(neat) 2937w, 2839w, 2570s, 1354s, 1181s, 1084m, 723m; H
NMR (CDCl3) 7.77 (d, 2H, J ) 6.7, ArH), 7.49-7.21 (m, 7H,
ArH), 4.49 (s, 2H, C6H5CH2-), 4.15 (t, 2H, J ) 6.9, -SO3-
CH2-), 3.49 (t, 2H, J ) 5.8, BnOCH2-), 2.55 (t, 2H, J ) 6.9,
-CH2CB10-), 2.48 (s, 3H, Ar-CH3), 2.25-2.13 (m, 2H, -CH2-
CB10-), 1.72-1.55 (m, 4H, -CH2CH2CH2CH2-); 13C NMR
(CDCl3) 145.4 (C, Ar), 138.4 (C, Ph), 132.7 (C, Ar), 130.0 (2CH,
Ar), 128.4 (CH, Ph), 127.9 (2CH, Ar or Ph), 127.6 (4CH, Ar +
Ph), 80.1 (C, CB10), 75.3 (C, CB10), 73.1 (OCH2Ph), 69.4 (OCH2),
66.9 (OCH2), 35.0 (CH2), 33.9 (CH2), 29.1 (CH2), 26.7 (CH2),
21.6 (CH3); MS (HR-EI) for C22H36B10O4S calcd 506.3282, found
506.3283. Anal. (C22H36B10O4S) C, H, B.
The reagents were purchased from the chemical companies
and used directly without further purification unless otherwise
specified. Anhydrous ethyl ether and tetrahydrofuran (THF)
were distilled from sodium/diphenyl ketone. N,N-Dimethyl-
formamide (DMF) was distilled from BaO. Acetone was
distilled from molecular sieves. Flash column chromatography
was carried out on Merck silica gel 60 (0.040-0.063 mm, 230-
400 mesh). Melting points were determined with a Fisher-
J ohns melting point apparatus and are uncorrected. 1H and
13C NMR spectra were recorded on either a Bruker AC-250,
AC-270, or DPX-400 spectrometer. 1H and 13C chemical shifts

1288 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Zhuo et al.
1-BOC-a m in oet h yl-2-(4-b en zyloxyb u t yl)-o-ca r b or a n e
(5). A mixture of 4 (1.06 g, 2.1 mmol) and NaN3 (367.2 mg,
6.1 mmol) in DMF (6 mL) was stirred for 20 h at RT. Water
and CH2Cl2 were added. The organic layer was separated,
washed with brine, and dried over MgSO4. After evaporation
of the solvent, the intermediate (710 mg, 1.89 mmol, 90%) was
obtained as a colorless oil. This compound (688.6 mg, 1.83
mmol) in THF (15 mL) was added dropwise to a suspension of
LiAlH4 (75.9 mg, 2.0 mmol) in THF (10 mL) at 0 °C. The
mixture was stirred for 1 h from 0 °C to RT. Water was added
carefully to quench the excess LiAlH4. The suspension mixture
was filtered, and the solid was extracted with ether twice. The
organic solutions were combined and dried over MgSO4. After
filtration and removal of solvents, the residue was diluted with
THF (10 mL) and Et3N (0.3 mL) and cooled to 0 °C. A solution
of BOC-ON (531.9 mg, 2.16 mmol) in THF (10 mL) was added
over a few minutes. The solution was then stirred overnight
at RT. The mixture was diluted with ether and washed twice
with 2 N NaOH and water. The organic layer was dried over
MgSO4. After filtration and removal of the solvent, the residue
was chromatographed (hexane/EtOAc ) 5:1) to give 5 (752.6
mg, 1.68 mmol, 93%) as a colorless oil: IR (neat) 2975w, 2570s,
EI) calcd for C13H32B10INO2 471.2410, found 471.2408. Anal.
(C13H32B10INO2) C, H, N.
N1,N10-Bis(BOC)-N5-[4-(2-BOC-a m id oeth yl-o-ca r bor a n -
1-yl)bu tyl]sp er m id in e (8). 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. To a
mixture of K2CO3 (207 mg, 2 mmol) and SPD-BOC (259.1 mg,
0.75 mmol) in DMF (1 mL) was added compound 7 (320 mg,
0.68 mmol) in DMF (1.5 mL) at 60-70 °C. The reaction
mixture was stirred for 1 h. After cooling, ice-water and CH2-
Cl2 were added. The layers were separated, and the aqueous
layer was extracted with CH2Cl2. The combined organic phases
were 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 hexanes-
ethyl acetate (1:1) containing 5% MeOH to give 8 (244.4 mg,
0.36 mmol, 52%) as a colorless oil: IR (neat) 3335w, 2960m,
1
2569m, 1676s, 1504m, 1151s; H NMR (CDCl3) 5.40 (br, 1H,
NH), 5.20 (br, 1H, NH), 4.95 (br, 1H, NH), 3.37-3.00 (m, 6H,
3×-CONCH2-), 2.52-2.11 (m, 12H, N(CH2-)3 + -CH2CH2CH2-
+ -CH2CB10H10CCH2-), 1.70-1.55 (m, 8H, -NCH2CH2CH2-
CH2N- + -CH2CH2CH2CH2-), 1.48 (s, 27H, 3×-C(CH3)3); 13
C
NMR (CDCl3) 156.0 (2C, COO), 155.8 (C, COO), 80.0 and 77.0
(2 C, -B10CCCH2-), 79.6, 78.9 and 78.8 (3×C, OCMe3), 53.7,
53.4 and 52.3 (3×CH2, NCH2), 41.0, 40.4 and 39.9 (3×CH2,
NHCH2), 34.9 and 34.3 (2×CH2, -B10CCCH2-), 28.4 (9×CH3),
27.9 (CH2), 27.6 (CH2), 27.1 (CH2), 26.7 (CH2), 24.4 (CH2); MS
(FAB) calcd for C30H66B10N4O6 688.59, found 688.63. Anal.
(C30H66B10N4O6) C, H, N, B.
1
1691s, 1496m, 1340m, 1151s, 723m; H NMR (CDCl3) 7.45-
7.25 (m, 5H, ArH), 4.69 (s, 1H, NH), 4.49 (s, 2H, ArCH2-), 3.49
(t, 2H, J ) 5.4, -CH2O-), 3.35-3.16 (m, 2H, -CH2NH-), 2.37
(t, 2H, J ) 7.6, -CH2CB10-), 2.30-2.15 (m, 2H, -CH2CB10-),
1.72-1.58 (m, 4H, CH2CH2CH2CH2-), 1.45 (s, 9H, -C(CH3)3);
13C NMR (CDCl3) 155.5 (C, COO), 138.4 (C, Ph), 128.4 (3CH,
Ph), 127.6 (2CH, Ph), 80.1 (C, CB10), 79.9 (C, OCMe3), 77.1
(C, CB10), 73.0 (OCH2Ph), 69.5 (OCH2), 40.0 (CH2N), 34.9
(CH2), 34.5 (CH2), 29.2 (CH2), 28.4 (3CH3), 26.7 (CH2); MS (HR-
EI) calcd for C20H38B10NO3 (M - H) 450.3782, found 450.3897.
Anal. (C20H39B10NO3) C, H, N.
N1,N10,N14-Tr is(BOC)-N5-[4-(2-BOC-a m id oet h yl-o-ca r -
bor a n yl)bu tyl]sp er m in e (9). Compound 9 (678.9 mg, 0.80
mmol, 75%) was obtained from the reaction of SPM-BOC
(629.3 mg, 1.25 mmol) and 7 (504.0 mg, 1.07 mmol): eluting
solvents hexane:ethyl acetate (1:1) containing 5% MeOH; IR
(neat) 3339vs br, 2571vs, 1688vs, 1665vs, 1494vs, 1261vs,
1230vs, 1153vs, 1060s, 1013s, 951m, 850s, 758s, 719vs; 1H
NMR (CDCl3) 3.49-3.05 (m, 10H, 5×-CONCH2-), 2.51-2.39
(m, 10H, N(CH2-)3 + -CH2CB10H10CCH2-), 1.70-1.55 (m, 48H,
2×-CH2CH2CH2- + -NCH2CH2CH2CH2N- + -CH2CH2CH2-
CH2- + 4×-C(CH3)3); 13C NMR (CDCl3) 156.0 (2×C, COO),
155.9 (C, COO), 155.6 C, COO), 80.0, 79.4, 78.9 and 78.8 (4×C,
4×OCMe3), 79.6 and 77.1 (2 C, -B10CC-), 53.8, 53.4 and 52.4
(3×CH2, -N(CH2CH2-)3-), 46.8 and 44.0 (2×CH2, BOCN(CH2-
CH2-)2-), 40.0, 39.7 and 37.8 (3×CH2, BOCNHCH2CH2-), 34.9
and 34.4 (2×CH2, -B10CCCH2-), 28.5 (12×CH3, 4×OC(CH3)3),
27.6 (2×CH2), 27.4 (CH2), 26.8 (CH2), 26.3 (CH2), 24.4 (CH2);
MS (HR-FAB) calcd for C38H81B10N5O8 845.7015, found
845.7194. Anal. (C38H81B10N5O8) C, H, N, B.
1-BOC-a m in oeth yl-2-(4-tosylbu tyl)-o-ca r bor a n e (6). A
mixture of 5 (718.1 mg, 1.6 mmol), Pd/C (10%, 100 mg), and
AcOH (5 mL) in MeOH (20 mL) was stirred under hydrogen
at RT for 10 h. The catalyst was removed, and the solution
was concentrated. The residue was diluted with CH2Cl2,
washed with NaHCO3, and dried over MgSO4. After filtration,
the filtrate was concentrated. The residue was the intermedi-
ate 4-(1-BOC-aminoethyl-o-carboran-2-yl)butanol (460.7 mg,
1.28 mmol, 80%), which was dissolved (460.7 mg, 1.28 mmol)
in CH2Cl2 (25 mL) and cooled to 0 °C; pyridine (1.5 mL) was
added. The tosyl chloride (0.98 g, 5.13 mmol) was added in
several portions. The mixture was then stirred at RT over-
night. The tosylate was purified as previous described for 4 to
give 6 (639 mg, 1.05 mmol, 82%) as a colorless oil: IR (neat)
1-[(2′,2′-Dim eth yl-1′,3′-d ioxocyclop en ta n -4′-yl)m eth yl]-
2-(4-tosylbu tyl)-o-ca r bor a n e (14). Tosyl chloride (5.93 g, 31
mmol) was added in several portions to a cooled (0 °C) mixture
of 4-{2-[(2,2-dimethyl-1,3-dioxocyclopentan-4′-yl)methyl]-o-car-
boran-1-yl}butanol25 (2.85 g, 8.5 mmol) and pyridine (6.0 mL)
in CH2Cl2 (200 mL). The solution was stirred at RT overnight.
Water (10 mL) was added, and the resulting mixture was
stirred for 2 h. The mixture was washed with 1 N HCl (2 × 50
mL), saturated NaHCO3 (2 × 50 mL), and brine (50 mL), dried
over MgSO4, and filtered. After evaporation of the solvent, the
residue was flash chromatographed (EtOAc/hexane ) 1:2) to
afford 14 (3.75 g, 91%): IR (KBr) 2960w, 2569s, 1691s, 1346s,
1151s, 610m; 1H NMR (CDCl3) 7.76-7.81 (m, 2H, ArH), 7.35-
7.39 (m, 2H, ArH), 4.24 (m, 1H, -OCHCH2O-), 4.13 (dd, 1H,
J ) 8.2, 6.0, -OCHCH2O-), 4.03 (t, 2H, J ) 6.0, -SO3CH2-),
3.55 (dd, 1H, J ) 8.2, 6.5, -OCHCH2O-), 2.46 (s, 3H, ArCH3-
), 2.43 (dd, 1H, J ) 15.5, 6.5, -B10CCCH2CH-), 2.38 (dd, 1H,
J ) 15.5, 5.0, -B10CCCH2CH-), 2.23 (ddd, 1H, J ) 15.0, 10.5,
1
2960w, 2569s, 1691s, 1346s, 1151s, 610m; H NMR (CDCl3)
7.70 (d, 2H, J ) 8.3, ArH), 7.37 (d, 2H, J ) 8.3, ArH), 4.80 (s,
1H, -NH), 4.05 (t, 2H, J ) 5.8 -SO3CH2-), 3.34-3.20 (m, 2H,
-CONCH2-), 2.49 (s, 3H, ArCH3), 2.43 (m, 2H, -B10CCH2-),
2.25-2.12 (m, 2H, -B10CCH2-), 1.76-1.5 (m, 4H, -CH2CH2CH2-
CH2-), 1.40 (s, 9H, -C(CH3)3); 13C NMR (CDCl3) 155.6 (C,
COO), 145.0 (C, Ar), 133.2 (C, Ar), 130.0 (2CH, Ar), 127.8 (2CH,
Ar), 79.9 (C, CB10), 79.4 (C, OCMe3), 77.3 (C, CB10), 69.5
(OCH2), 40.1 (CH2N), 34.5 (CH2, -B10CCCH2-), 34.3 (CH2,
-B10CCCH2CH2-), 28.3 and 25.8 (2 CH2, -B10CCCH2CH2CH2-
CH2-), 21.6 (ArCH3); MS (HR-EI) calcd for C13H32B10INO2
515.3496, found 515.3497. Anal. (C13H32B10INO2) C, H, N, B.
1-BOC-a m in oeth yl-2-(4-iod obu tyl)-o-ca r bor a n e (7). To
a solution of 6 (567.4 mg, 1.1 mmol) in dry acetone (20 mL)
was added NaI (331 mg, 2.2 mmol). The mixture was stirred
overnight at RT. After removal of the solid and acetone, the
residue was flash chromatographed (hexane/EtOAc ) 5:1) to
give 7 (490.2 mg, 1.04 mmol, 95%) as a colorless oil: IR (neat)
3342w, 2960w, 2569s, 1691s, 1489m, 1354m, 1151s; 1H NMR
(CDCl3) 4.77 (s, 1H, -NH), 3.40-3.10 (m, 4H, ICH2- +
-CONCH2-), 2.51-2.35 (m, 2H, -B10CCH2-), 2.34-2.15 (m,
2H, -B10CCH2-), 1.97-1.60 (m, 4H, -CH2CH2CH2CH2-), 1.48
(s, 9H, -C(CH3)3); 13C NMR (CDCl3) 156.0 (C, COO), 80.0 and
77.2 (2 C, -B10CCCH2-), 79.5 (C, OCMe3), 40.1 (CH2N), 34.5
(CH2, -B10CCCH2-), 33.8 (CH2, -B10CCCH2CH2-), 32.5 and
30.4 (2 CH2, -B10CCCH2CH2CH2CH2-), 28.4 (3CH3); MS (HR-
6.2, -B10CCCH2CH2-), 2.16 (ddd, 1H, J ) 15.0, 10.0, 6.0, -B10
-
CCCH2CH2-), 1.60-1.70 (m, 2H, -CH2CH2CH2CH2-), 1.46-
1.59 (m, 2H, -CH2CH2CH2CH2-), 1.37 and 1.34 (s, 2 × 3H,
-OC(CH3)2O-); 13C NMR (CDCl3) 145.0 (C, Ar, C-1′), 132.7 (C,
Ar, C-4′), 129.9 (2 CH, Ar), 127.8 (2 CH, Ar), 109.6 (C,
-OCMe2O-), 79.2 and 77.0 (2 C, -B10CCCH2-), 77.4 (CH,
-OCHCH2O-), 69.5 and 69.0 (2 OCH2), 39.4 (CH2, -B10CCCH2-
CH-), 34.3 (CH2, -B10CCCH2CH2-), 28.3 and 25.7 (2 CH2, -B10-
CCCH2CH2CH2CH2-), 26.8 and 25.2 (2 Me, -OC(CH3)2O-), 21.6

Polyamines as Potential Agents for BNCT
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1289
(Me, ArCH3); MS (HR-EI) calcd for C19H36B10O5S 486.3231,
found 486.3232. Anal. (C19H36B10O5S) C, H, B.
3.65-3.42 (m, 2H, -CH2Ar), 3.15-2.84 (m, 4H, 2×-CONC-
H2-), 2.60-2.31 (m, 4H, N(CH2-)2), 1.70-1.30 (m, 6H, -NCH2-
CH2CH2CH2N- + -CH2CH2CH2-), 1.48 (s, 18H, 2×-C(CH3)3);
13C NMR (CDCl3) 156.1 (2×C, COO), 134.8 and 134.3 (2×C,
Ar), 128.4 (4×CH, Ar), 79.1 (2×C, 2×OCMe3), 58.7 (CH2,
NCH2Ar), 53.2 and 51.8 (2×CH2, CH2NCH2), 40.4 and 39.5 (2×
CH2, 2×NHCH2), 28.4 (6×CH3, 2×-C(CH3)3), 27.7 (CH2,
-NCH2CH2CH2-), 26.5d and 23.7 (2×CH2, -NCH2CH2CH2-
CH2N-); MS (HR-EI) calcd for C24H41BN3O6 (M - H) 478.3088,
found 478.3203. Anal. (C24H42BN3O6) C, H, N, B.
Gen er a l P r oced u r e for Dep r otection . Compounds 8, 9,
16, and 17 were stirred overnight in MeOH and 3 N HCl at
40-50 °C. After removal of the solvents, the residue was
dissolved in EtOH and concentrated twice. If the product was
not soluble in EtOH, the compound was refluxed for 2 h in
EtOH, and upon cooling to RT, the solid was collected and
dried under high vacuum.
1-[(2′,2′-Dim eth yl-1′,3′-d ioxocyclop en ta n -4′-yl)m eth yl]-
2-(4-iod obu tyl)-o-ca r bor a n e (15). To a solution of 14 (3.68
g, 7.6 mmol) in dry acetone (50 mL) was added NaI (3.42 g,
22.8 mmol). The mixture was stirred overnight at RT. After
removal of the solid and acetone, the residue was flash
chromatographed (EtOAc/hexane ) 1:2) to give 15 (3.21 g,
96%): IR (KBr) 3342w, 2960w, 2569s, 1691s, 1489m, 1354m,
1151s; 1H NMR (CDCl3) 4.27 (m, 1H, -OCHCH2O-), 4.15 (dd,
1H, J ) 8.3, 6.0, -OCHCH2O-), 3.58 (dd, 1H, J ) 8.3, 6.5,
-OCHCH2O-), 3.19 (t, 2H, J ) 6.8, -ICH2-), 2.46 (dd, 1H, J )
15.5, 6.5, -B10CCCH2CH-), 2.41 (dd, 1H, J ) 15.5, 5.0, -B10
-
CCCH2CH-), 2.19-2.34 (m, 2H, -B10CCCH2CH2-), 1.79-1.87
(m, 2H, -CH2CH2CH2CH2-), 1.60-1.79 (m, 2H, -CH2CH2CH2-
CH2-), 1.40 and 1.36 (s, 2 × 3H, -OC(CH3)2O-); 13C NMR
(CDCl3) 109.6 (C, -OCMe2O-), 79.3 and 76.9 (2 C, -B10CC-
CH2-), 74.4 (CH, -OCHCH2O-), 69.1 (CH2, -OCHCH2O-), 39.5
(CH2, -B10CCCH2CH-), 33.8 (CH2, -B10CCCH2CH2-), 32.5 and
30.4 (2 CH2, -B10CCCH2CH2CH2CH2-), 26.9 and 25.3 (2 Me,
-OC(CH3)2O-). 5.1 (ICH2); MS (HR-EI) calcd for C12H29B10IO2
442.2145, found 442.2192. Anal. (C12H29B10IO2) C, H, B.
N5-[4-(2-Am in oet h yl-o-ca r b or a n yl)b u t yl]sp er m id in e
4HCl (ASP D-5). ASP D-5 (126.8 mg, 0.24 mmol, 100%) was
produced from 8 (162.0 mg, 0.24 mmol): mp 70-3 °C; IR (KBr)
1
3400m, 2980s, 2584s, 1586m, 1459s, 1129m, 716m; H NMR
(CD3OD) 3.40-2.90 (m, 12H, 6×-N+CH2-), 2.85-2.68 (m, 2H,
-CH2CB10), 2.52-2.38 (m, 2H, -CH2CB10), 2.30-2.08 (m, 2H,
-CH2CH2CH2-), 2.00-1.50(m, 8H, -NCH2CH2CH2CH2N- +
-CH2CH2CH2CH2-); 13C NMR (CD3OD) 79.6 and 75.1 (2 C,
-B10CCCH2-), 51.6 (2×CH2, NCH2CH2), 49.1 (CH2, NCH2CH2),
37.9, 37.6 and 35.9 (3×CH2, NH3CH2CH2), 33.3 and 30.3
(2×CH2, -B10CCCH2-), 25.7, 23.3, 22.0, 20.9 and 19.8 (5×CH2);
MS (HR-EI) calcd for C15H42B10N4 (M-4HCl) 388.4340, found
388.4321. Anal. (C15H46B10Cl4N4) C, H, N.
N1,N10-Bis(BOC)-N5-[4-(3,3-d im e t h yl-2,4-d ioxola n yl-
m eth yl-o-ca r bor a n yl)bu tyl]sp er m id in e (16). Compound
16 (538.4 mg, 0.82 mmol, 67%) was obtained from the reaction
of SPD-BOC (461.8 mg, 1.34 mmol) and 15 (535.1 mg, 1.22
mmol): eluting solvents hexane:ethyl acetate (1:1) containing
7% MeOH; IR (neat) 3342w, 2922m, 2569s, 1684s, 1504s,
1
1151s, 1061m; H NMR (CDCl3) 5.25 (br, 1H, NH), 4.75 (br,
1H, NH), 4.30-4.08 (m, 2H, -CH2O-), 3.61-3.50 (m, 1H,
-CHO-), 3.21-3.01 (m, 4H, 2×-CONCH2-), 2.50-2.26 (m, 6H,
N(CH2-)2 + -CHCH2CB10-), 2.26-2.13 (m, 2H, NCH2-), 1.75-
1.45 (m, 12H, -NCH2CH2CH2CH2N- + -NCH2CH2CH2N-
+ -NCH2CH2CH2CH2CB10-), 1.45 (s, 18H, 2×-C(CH3)3), 1.40
(s, 3H, -CH3), 1.33 (s, 3H, -CH3); 13C NMR (CDCl3) 156.0
(2×C, COO), 109.5 (C, -OCMe2O-), 80.1 and 76.8 (2 C,
-B10CC-), 79.0 and 78.9 (2×C, OCMe3), 74.4 (CH, -OCHCH2-
O-), 69.1 (CH2, -OCHCH2O-), 53.7, 53.6 and 52.4 (3×CH2,
-N(CH2CH2-)3-), 40.4 and 40.2 (2×CH2, -NHCH2CH2-), 39.5
and 35.1 (2×CH2, -B10CCCH2-), 28.4 (6×CH3, 2 OCMe3), 28.0
(CH2), 27.6 (CH2), 27.2 (CH3), 26.8 (2×CH2), 25.2 (CH2), 24.4
(CH3); MS (HR-EI) calcd for C29H62B10N3O6 (M - H) 658.5569,
found 658.5684. Anal. (C29H63B10N3O6) C, H, N, B.
N5-[4-(2,3-Dih yd r oxyp r op yl-o-ca r b or a n yl)b u t yl]sp er -
m id in e 3HCl (DHSP D-5). DHSP D-5 (350.6 mg, 0.67 mmol,
98%) was produced from 16 (420.1 mg, 0.68 mmol): IR (KBr)
1
3387s, 2937s, 2562s, 1601m, 1451m, 1016m, 716m; H NMR
(CD3OD) 3.80-2.70 (m, 13H, -CH2O- + -CHO- + 5×-
CONCH2-), 2.60-1.30 (m, 14H, -NCH2CH2CH2N- + -CH2-
CB10H10CCH2- + -NCH2CH2CH2CH2N- + -NCH2CH2CH2-
CH2-); 13C NMR (CD3OD) 81.2 and 80.7 (2×C, -B10CC-
CH2-), 72.2 (CH, -OCH-), 66.9 (CH2, -OCH2), 54.5, 54.3 and
49.0 (3×CH2, -N(CH2CH2-)3-), 40.5 and 40.3 (2×CH2, NH3CH2-
CH2), 39.0 (CH2, -B10CCCH2CH-), 35.5 (CH2, -B10CCCH2-
CH2-), 27.9, 25.8, 24.8, 23.4 and 22.3 (5×CH2). Anal. (C16H46B10-
Cl3N3O2) C, H, N.
N5-[4-(2-Am in oeth yl-o-car bor an yl)bu tyl]sper m in e 5HCl
(ASP M-5). ASP M-5 (430.7 mg, 0.69 mmol, 87%) was produced
from 9 (664.7 mg, 0.79 mmol): mp 175-8 °C; IR (KBr) 3410s,
2945s, 2577s, 1586m, 1451m, 1031m, 716m; 1H NMR (CD3-
OD) 3.38-2.97 (m, 16H, 8×-N+CH2-), 2.70-2.68 (m, 2H,
-CH2CB10), 2.50-2.36 (m, 2H, -CH2CB10), 2.25-2.00(m, 4H,
2×-CH2CH2CH2-), 1.99-1.45 (m, 8H, -NCH2CH2CH2CH2N-
+ -CH2CH2CH2CH2-); 13C NMR (CD3OD) 81.9 and 77.5 (2×C,
-B10CCCH2-), 53.9, 53.7 and 51.4 (3×CH2, -N(CH2CH2-)3-),
48.3 and 45.9 (2×CH2, N(CH2CH2-)2-), 39.8, 38.0 and 35.5
(3×CH2, NH3CH2CH2), 32.5 and 27.8 (2×CH2, -B10CCCH2-),
25.9, 25.2, 23.1 and 22.1 (4×CH2), 24.2 (2×CH2); MS (HR-FAB)
calcd for C18H50B10N5 (M - 5HCl) 445.49, found 449.50. Anal.
(C15H46B10Cl4N4) C, H, N, B.
N1,N10,N14-Tr is(BOC)-N5-[4-(3,3-d im eth yl-2,4-d ioxola n -
ylm eth yl-o-ca r bor a n yl)bu tyl]sp er m in e (17). Compound 17
(706.0 mg, 0.87 mmol, 74%) was obtained from the reaction of
SPM-BOC (616.0 mg, 1.22 mmol) and 15 (513.9 mg, 1.17
mmol): eluting solvents hexane:ethyl acetate (1:1) containing
7% MeOH; IR (neat) 3342w, 2952m, 2569m, 1676s, 1159s; 1H
NMR (CDCl3) 5.25 (br, 1H, NH), 4.25 (br, 1H, NH), 4.29-4.13
(m, 1H, -OCHCH2-), 4.08 (dd, 1H, J ) 8.2, 6.0, -CH2O-), 3.52
(dd, 1H, J ) 8.2, 6.3, -CH2O-), 3.29-2.95 (m, 8H, 4×-
CONCH2-), 2.50-2.10 (m, 10H, N(CH2-)3 + -CH2CB10H10
-
CCH2-), 1.71-1.49 (m, 12H, 2×-CH2CH2CH2- + -NCH2CH2CH2-
CH2N- + -CH2CH2CH2CH2-), 1.38 (s, 9H, -OC(CH3)3), 1.37
(s, 18H, 2×-OC(CH3)3), 1.33 (s, 3H, -CH3), 1.29 (s, 3H, -CH3);
13C NMR (CDCl3) 156.0 (3×C, COO), 109.5 (C, -OCO-), 80.1
and 76.8 (2×C, -B10CC-), 79.5 (2×C, 2×OCMe3), 78.8 (C,
OCMe3), 74.4 (CH, -OCH-), 69.1 (CH2, -OCH2), 53.7, 53.6 and
52.3 (3×CH2, -N(CH2CH2-)3-), 46.9 and 44.9 (2×CH2, BOCN-
(CH2CH2-)2-), 39.7 and 37.8 (2×CH2, BOCNHCH2CH2-), 39.5
(CH2, -B10CCCH2CH-), 35.1 (CH2, -B10CCCH2CH2-), 28.43
(9×CH3, 3×OC(CH3)3), 27.6 and 26.8 (2×Me, -OC(CH3)2O-),
N5-[4-(2,3-Dih yd r oxyp r op yl-o-ca r b or a n yl)b u t yl]sp er -
m in e 4HCl (DHSP M-5). DHSP M-5 (354.6 mg, 0.57 mmol,
100%) was produced from 17 (467.2 mg, 0.57 mmol): mp 72-5
°C; IR (KBr) 3380s, 2945s, 2569s, 1603m, 1451m, 1024m,
716w; 1H NMR (CD3OD) 3.70-2.80 (m, 17H, -CH2O- +
-CHO- + 7×-CONCH2-), 2.60-1.40 (m, 16H, 2×-CH2CH2-
CH2- + -CH2CB10H10CCH2- + -NCH2CH2CH2CH2N- +
-CH2CH2CH2CH2-); 13C NMR (CD3OD) 81.3 and 80.7 (2×C,
-B10CC-), 72.3 (CH, -OCH-), 66.9 (CH2, -OCH2), 54.2, 53.8
and 51.5 (3×CH2, -N+H(CH2CH2-)3-), 48.9 and 48.4 (2×CH2,
N+H2(CH2CH2-)2-), 46.1 and 40.2 (2×CH2, N+H3CH2CH2-), 38.1
(CH2, -B10CCCH2CH-), 35.4 (CH2, -B10CCCH2CH2-), 27.8,
28.6, 28.40, 27.4, 26.9, 26.2 and 24.4 (6×CH2). Anal. (C37H78
-
B10N4O8) C, H, N, B.
N1,N10-Bis(BOC)-N5-[(4-d ih yd r oxybor ylp h en yl)m eth yl]-
sp er m id in e (18). Compound 18 (328.4 mg, 0.68 mmol, 31%)
was obtained from the reaction of SPD-BOC (907.9 mg, 2.63
mmol) and 4-(bromomethyl)phenylboronic acid (470.5 mg, 2.19
mmol) at 140 °C for 2 h: eluting solvents hexane:ethyl acetate
(1:1) containing 3% MeOH; IR (KBr) 3347sbr, 1683vs, 1594m,
1506vs, 1373s, 1351s, 1263s, 1241s, 1160vs; 1H NMR (CDCl3)
8.10-7.92 (m, 2H, ArH), 7.55 (br, 1H, OH), 7.40-7.24 (m, 2H,
ArH), 7.15 (br, 1H, OH), 5.40 (br, 1H, NH), 4.85 (br, 1H, NH),
25.3, 24.4, 24.3, 23.2 and 22.2 (6×CH2). Anal. (C19H54B10
-
Cl4N4O2) C, H, N, B.
Oxy-bis{N5-{[4-(B-h yd r oxybor yl)p h en yl]m eth yl}sp er -
m id in e 3HCl} (Bis-BBSP D-5) a n d N5-{[4-(B,B-Dih yd r oxy-
bor yl)p h en yl]m eth yl}sp er m id in e 3HCl (BBSP D-5). Com-

1290 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Zhuo et al.
pound 18 (960 mg, 2.0 mmol) in MeOH (10 mL) and 3 N HCl
(4 mL) was stirred at RT for 2 days. After removal of the
solvents, the solid was dried under high vacuum (752 mg,
99%). According to 1H and 13C NMR spectral analyses and
elemental microanalyses, this product exists as a dehydro
dimer, Bis-BBSP D-5, in dry (CD3)2SO solution and in the solid
state but as a monomer, BBSP D-5, in D2O or (CD3)2SO/D2O
solution.
has been widely used74 and is based on the incorporation of
sulforhodamine B (SRB) by biosynthetically active (i.e., S
phase) surviving cells following exposure to the test compound.
Ninety-six-well plates (Corning Glass Works, Corning, NY)
were seeded with 10 000 F98 glioma cells/well in Dulbelcco’s
minimal essential medium (DMEM) containing 10% fetal
bovine serum (FBS) (Hyclone, Logan, UT). Plates were incu-
bated overnight at 37 °C in a humidified atmosphere contain-
ing 5% CO2. DMEM was replaced with media containing
varying concentrations of the polyamine analogue and incu-
bated for an additional 24 h at 37 °C. To each well was added
50 µL of cold 50% TCA carefully, and plates were left for 1 h
at 4 °C. Wells were decanted, washed five times with deionized
water, and dried at RT. A 50-µL aliquot of a 0.4% solution of
SRB (Sigma, St. Louis, MO) in 1% acetic acid was added to
each well for a 20-min period at RT. After which, they were
washed five times with distilled water and allowed to dry.
Bound SRB was solubilized in 100 µL of 10 mM unbuffered
Trizma base. Absorbance was read in an OTC 400 automated
ELISA plate reader (Organon Teknika Corp., Durham, CA)
at 492 nm. Mean values from six replicates were used for each
concentration. Values were expressed as the percent of un-
treated control. The concentration required to produce a 50%
reduction in SRB staining (i.e., IC50) was calculated for each
compound.
(2) 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.61-63 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 Instruments, Kana-
bee, 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 fluores-
cence was recorded, and the IC50 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.
Bis-BBSP D-5: IR (KBr) 3300br, 1602s, 1396s, 1359s, 1322s,
1108m, 1005s, 725s, 666s; 1H NMR ((CD3)2SO) 11.19 (br, 2H,
OH), 8.10-8.28 (br, 12H, NH), 7.84 (d, 4H, J ) 8, ArH), 7.63
(d, 4H, J ) 8, ArH), 4.30 (s, 4H, -CH2Ar), 3.05-3.14 (m, 4H,
-N+CH2-), 2.90-3.03 (m, 4H, -N+CH2-), 2.81-2.88 (m, 4H,
-N+CH2-), 2.73-2.81 (m, 4H, -N+CH2-), 1.96-2.18 (m, 4H,
-NCH2CH2CH2N-), 1.79-1.95 (m, 4H, -NCH2CH2-), 1.50-
1.61 (m, 4H, -NCH2CH2-); 1H NMR (CD3OD) 7.80 (d, 2H, J )
8, ArH), 7.62 (d, 2H, J ) 8, ArH), 4.45 (s, 2H, -CH2Ar), 3.30
(t, 2H, J ) 8.5, -N+CH2-), 3.21 (t, 2H, J ) 8.5, -N+CH2-),
3.02 (t, 2H, J ) 7.7, -N+CH2-), 2.99 (t, 2H, J ) 7.4, -N+-
CH2-), 2.35-2.07 (m, 2H, -NCH2CH2CH2N-), 2.07-1.81 (m,
2H, -NCH2CH2-), 1.81-1.58 (m, 2H, -NCH2CH2-); 13C NMR
((CD3)2SO) 135.4 (C, C-B in Ar), 134.5 (2×CH, Ar), 131.3 (C,
Ar), 130.4 (2×CH, Ar), 55.7 (CH2, NCH2Ar), 50.6 and 48.5
(2×CH2, -CH2NCH2-), 38.0 and 36.2 (2×CH2, 2×NCH2-), 24.1
(CH2, NCH2CH2CH2N), 21.0 and 19.7 (CH2, NCH2CH2CH2-
CH2N); 13C NMR (CD3OD) 136.8 (C, C-B in Ar), 135.7 (2×CH,
Ar), 131.9 (C, Ar), 131.4 (2×CH, Ar), 58. 5 (CH2, NCH2Ar),
53.3 and 51.0 (2×CH2, -CH2NCH2-), 40.0 and 38.0 (2×CH2,
2×NCH2-), 25.6 (CH2, NCH2CH2CH2N), 23.1 and 21.9 (CH2,
NCH2CH2CH2CH2N). Anal. (C28H56B2Cl6N6O3) C, H, N, B.
1
BBSP D-5: H NMR ((CD3)2SO/D2O ) 10:1) 7.83 (d, 2H, J
) 8, ArH), 7.53 (d, 2H, J ) 8, ArH), 4.30 (s, 2H, -CH2Ar),
3.08 (t, 2H, J ) 7.7, -N+CH2-), 2.95-3.03 (m, 2H, -N+CH2-),
2.82 (t, 2H, J ) 7.5, -N+CH2-), 2.77 (t, 2H, J ) 7.5, -N+-
CH2-), 1.97-2.09 (m, 2H, -NCH2CH2CH2N-), 1.69-1.83 (m,
2H, -NCH2CH2-), 1.47-1.57 (m, 2H, -NCH2CH2-); 13C NMR
((CD3)2SO/D2O ) 10:1) 135.8 (C, C-B in Ar), 135.1 (2×CH, Ar),
131.6 (C, Ar), 130.8 (2×CH, Ar), 56.4 (CH2, NCH2Ar), 51.4 and
49.2 (2×CH2, -CH2NCH2-), 38.4 and 36.5 (2×CH2, 2×NCH2-),
24.3 (CH2, NCH2CH2CH2N), 21.5 and 20.2 (CH2, NCH2CH2-
CH2CH2N).
N5-{[4-(B-Eth oxy-B-h yd oxybor yl)p h en yl]m eth yl}sp er -
m id in e 3HCl (Et-BBSP D-5). Compound 18 (445 mg, 0.93
mmol) in MeOH (5 mL) and 3 N HCl (2 mL) was stirred at RT
for 2 days. After removal of the solvents, the residue was
dissolved in EtOH and concentrated twice. The solid was
collected and dried under high vacuum to afford Et-BBSP D-5
(367 mg, 0.92 mmol, 99%): IR (neat) 3387s, 3012s, 1594m,
(3) Cellu la r Up ta k e. This 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 (∼5 µg/mL) were
determined by DCP-AES, as described previously.64 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-buffered saline
(PBS), and the wash fluids also were saved for boron deter-
mination. The cells then were desegregated using trypsin-
EDTA (Gibco BRL, Grand Island, NY) and digested in a
mixture of concentrated sulfuric acid and 70% hydrogen
peroxide,75 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 3 and
compared with values previously obtained for sodium un-
decahydromercapto-closo-dodecaborate (BSH) and L-p-bo-
ronophenylalanine (BPA) (Boron Biologicals Inc., Raleigh, NC).
1
1399m, 1324s, 1001m, 633m; H NMR (CD3OD) 7.72 (d, 2H,
J ) 8, ArH), 7.53 (d, 2H, J ) 8, ArH), 4.37 (s, 2H, -CH2Ar),
3.51 (q, 2H, J ) 7, -OCH2CH3), 3.22 (t, 2H, J ) 8.3, -N+-
CH2-), 3.13 (t, 2H, J ) 8.2, -N+CH2-), 2.94 (t, 2H, J ) 7.6,
-N+CH2-), 2.91 (t, 2H, J ) 7.4, -N+CH2-), 2.23-2.08 (m, 2H,
-NCH2CH2CH2N-), 1.92-1.77 (m, 2H, -NCH2CH2-), 1.69-
1.55 (m, 2H, -NCH2CH2-), 1.08 (t, 3H, J ) 7.0, -OCH2CH3);
13C NMR (CD3OD) 135.6 (C, Ar), 131.8 (C, Ar), 131.4 (4×CH,
Ar), 58.6 (CH2, NCH2Ar or OCH2), 58.3 (CH2, OCH2 or NCH2-
Ar), 53.4 and 51.2 (2×CH2, -CH2NCH2-), 40.1 and 38.0 (2×CH2,
2×NCH2-), 25.6 (CH2, NCH2CH2CH2N), 23.2 and 21.9 (CH2,
NCH2CH2CH2CH2N), 18.3 (CH3,-OCH2CH3). Anal. (C16H33
-
BCl3N3O3) C, H, N, B.
2. Biologica l Stu d ies. The variation for all the biological
test data was ∼(10%. All tests were repeated 2-3 times. For
in vitro toxicity studies, six replicates were used for each
sample and at each time point.
(1) In Vitr o Eva lu a tion of Toxic P r op er ties. The F98
glioma cell line was derived from an undifferentiated 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 morphology and in vitro
characteristics have been described in detail,70,71 and it has
been used by us to evaluate a variety of boron compounds as
potential delivery agents for BNCT.59,72,73 The assay that was
employed to detect the toxicity and/or growth inhibitory effects
(4) In Vivo Biod istr ibu tion Stu d ies. a . Su bcu ta n eou s
Tu m or s. Above 250 000 B16 melanoma cells were implanted
subcutaneously in the left flank of C57Bl/6 mice (Charles River
Laboratories, Wilmington, MA). After 7 days, animals were
placed on a polyamine-deficient diet (Dyets, Inc., Bethlehem,
PA) containing 0.2% neomycin and received water containing
eflornithine HCl (DFMO) (Ilex Onclogy, San Antonio, TX). Ten

Polyamines as Potential Agents for BNCT
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1291
(14) Karnbrock, W.; Musiol, H. J .; Moroder, L. Enantioselective
synthesis of S-o-carboranylalanine via methylated bislactim
ethers of 2,5-diketopiperazines. Tetrahedron 1995, 51, 1187-
1196.
(15) Nemoto, H.; Cai, J .; Asao, N.; Iwamoto, S.; Yamamoto, Y.
Synthesis and biological properties of water-soluble p-borono-
phenylalanine dervatives. Relationship between water solubility,
cytotoxicity, and celluler uptake. J . Med. Chem. 1995, 38, 1673-
1678.
days after tumor implantation, Alzet pumps model 1007D
(Alza Corp., Palo Alto, CA) containing compound were im-
planted subcutaneously between the scapulae, and the com-
pounds were administered over 3-4 days while the polyamine-
deficient diet and DFMO in the drinking water were continued
for this period. ASP D-5 was administered at a dose of 30 or
40 µg/g per day, using 5 mice per group. After 4 days, pumps
were removed, animals were sacrificed, and tissues were
removed for boron analysis, which was carried out by DCP-
AES.
(16) Leush, A.; J ungblut, L.; Moroder, L. Design and synthesis of
carboranyl peptides as carriers of 1,2-dicarbadodecarborane
clusters. Synthesis 1994, 305-308.
b. GL261 Br a in Tu m or s. Sedated C57Bl/6 mice were
placed in a stereotactic head frame (Kopf Inc., Tajunga, CA)
and implanted with 105 GL261 cells suspended in serum-free
DMEM containing 1.5% agarose at a depth of 2.5-3.0 mm into
the brain. On day 16 postimplantation, mice were placed on a
polyamine-deficient diet and water containing 2% DFMO (Ilex
Oncology, San Antonio, TX). On day 19, Alzet pumps model
1003D (Alza Corp., Palo Alto, CA) containing the polyamine
analogues were implanted subcutaneously between the scapu-
lae of the mice for 3 days. The ASP D-5 was administered at
a dose of 30 µg/g per day and the BBSP D-5 at a dose of 10 µg
of boron/g per day. C57Bl mice were implanted intracerebrally
with GL261 brain tumors. Animals were placed on a polyamine-
deficient diet and water containing 2% DFMO for 3 days. Alzet
pumps were implanted, and compound infusion was carried
out for 3 days during which time they were maintained on a
polyamine-deficient diet and DFMO. Animals were euthanized
at the end of this 3-day period, and tissues were removed for
boron analysis by DCP-AES. There were 5 mice per group.
(17) Kane, R. R.; Pak, R. H.; Hawthorne, M. F. Solution-phase
segment synthesis of boron-rich peptides. J . Org. Chem. 1993,
58, 991-992.
(18) Kane, R. R.; Hawthorne, M. F. Novel carboranyl amino acids
and peptides: reagent for antibody modification and subsequent
neutron-capture studies. Bioconjugate Chem. 1991, 2, 242-253.
(19) Schwyzer, R.; Do, K. Q.; Eberle, A. N.; Fauche`re, J . L. Synthesis
and biological properties of enkephalin-like peptides containing
carboranylalanine in place of phenylalanine. Helv. Chim. Acta
1981, 64, 2078-2083.
(20) Soloway, A. H.; Zhuo, J .-C.; Rong, F.-G.; Lunato, A. J .; Ives, D.
H.; Barth, R. F.; Anisuzzaman, A. K. M.; Barth, C. D.; Barnum,
B. A. Identification, development, synthesis and evaluation of
boron-containing nucleosides for neutron capture therapy. J .
Organomet. Chem., in press.
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Ack n ow led gm en t. This work was supported by
U.S. Public Health Service Grants R01 CA-71033 and
P30 CA-16058 and the U.S. Department of Energy
Grants DE-AC02-76CH00016 and DE-FG02-98ER62595.
We wish to thank Callery for providing the decaborane
used in the synthesis.
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