Discovery of ortho-carborane-conjugated triazines as selective topoisomerase I/II inhibitors

Article abstract of DOI:10.1071/CH11295

The cell growth inhibition profile of 2,4-(2-methyl-ortho-carboranyl)-4- (dimethylamino)-1,3,5-triazine (TAZ-6) was found to be similar to that of ICRF-193, a topoisomerase II inhibitor, as revealed by COMPARE analysis (correlation coefficient (r)≤0.724). Various mono-and di-ortho-carborane- substituted 1,3,5-triazines were synthesized based on the structure of TAZ-6 and tested for their ability to inhibit cell growth and the activities of topoisomerases I and II. Among the compounds synthesized, 3c, 4c, and 4f completely inhibited topoisomerase I activity without affecting topoisomerase II activity, whereas 3a and 3d completely inhibited topoisomerase II activity without affecting topoisomerase I activity, at 100μM.

Full text of DOI:10.1071/CH11295

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem. 2011, 64, 1430–1437
Full Paper
http://dx.doi.org/10.1071/CH11295
Discovery of ortho-Carborane-Conjugated Triazines
as Selective Topoisomerase I/II Inhibitors
A
,
E
A
A
Hiroyuki Nakamura, Atsushi Shoji, Ayano Takeuchi, Hyun Seung
A
B
C
D
Ban, Jong-Dae Lee, Takao Yamori, and Sang Ook Kang
A
Department of Chemistry, Faculty of Science, Gakushuin University, 1-5-1 Mejiro,
Toshima-ku, Tokyo 171-8588, Japan.
B
Department of Chemistry, College of Natural Science, Chosun University, Dong-gu,
Kwangju 501-759, South Korea.
C
Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese
Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan.
D
Department of Chemistry, Korea University, Chochiwon, Chung-nam 339-700,
South Korea.
E
Corresponding author. Email: hiroyuyki.nakamura@gakushuin.ac.jp
The cell growth inhibition profile of 2,4-(2-methyl-ortho-carboranyl)-4-(dimethylamino)-1,3,5-triazine (TAZ-6) was
found to be similar to that of ICRF-193, a topoisomerase II inhibitor, as revealed by COMPARE analysis (correlation
coefficient (r) ¼ 0.724). Various mono- and di-ortho-carborane-substituted 1,3,5-triazines were synthesized based on the
structure of TAZ-6 and tested for their ability to inhibit cell growth and the activities of topoisomerases I and II. Among the
compounds synthesized, 3c, 4c, and 4f completely inhibited topoisomerase I activity without affecting topoisomerase II
activity, whereas 3a and 3d completely inhibited topoisomerase II activity without affecting topoisomerase I activity,
at 100 mM.
Manuscript received: 16 July 2011.
Manuscript accepted: 8 August 2011.
Published online: 16 November 2011.
Introduction
platinum-refractory ovarian cancer, further clinical develop-
[
16]
ment was discontinued due to formulation difficulties.
Benzimidazolyl-1,3,5-triazine (ZSTK474) was also developed
The proper regulation of DNA topology is vital for cell viability.
Topological problems are always associated with DNA repli-
cation, transcription, recombination, and chromatin remodelling.
Topoisomerases I and II solve those problems by introducing
temporary single- (topoisomerase I) or double- (topoisomerase
[
17]
as a potent antitumour agent
3-kinase (PI3K) was identified as a molecular target for
and phosphatidylinositol
[
18]
[19,20]
ZSTK474.
[21]
protozoal,
Not only antitumour activity,
but also anti-
[24,25]
antimicrobial,
[
1–5]
[22]
[23]
II) strand breaks in DNA.
Inhibition of the topoisomerases
antimalarial,
antiviral,
[
26]
can cause permanent DNA damage, triggering significant
cellular events, including apoptosis, and finally cell death.
Therefore, they are considered to be an attractive target for the
and anti-immune activities
reported.
In the meanwhile, much attention has been focussed on
of 1,3,5-triazines have been
[
6–8]
[27,28]
design of chemotherapeutic agents.
was isolated from Camptotheca acuminate,
tives topotecan and irinotecan are known as topoisomerase I
Camptothecin, which
carboranes as potential pharmacophores in drug design.
[
9,10]
and its deriva-
Carboranes consist of the carbon and boron atoms of the C2B10
core and these atoms are hexacoordinate to compensate for the
low electron density, forming 20 triangular faces. Due to the
geometric and electronic features of carboranes, their unique
interaction with target biomolecules has been designed and
[
10–12]
inhibitors,
whereas doxorubicin and etoposide are known
as topoisomerase II inhibitors. These compounds are considered
to intercalate DNA.
[
29,30]
[31]
1
,3,5-Triazines are a class of nitrogen-containing cyclic
compounds with remarkable thermal and chemical stabili-
studied including estrogen,
and heat shock protein (Hsp) 60.
dihydrofolate reductases,
32]
[
[
13]
ties,
and have become one of the important scaffolds for
The 1,3,5-triazine ring has three distinct electrophilic centres
that form 2,4,6-mono, di- or tri-substituted, symmetrical and
nonsymmetrical triazines bearing different substituents. Cyanu-
ric chloride is the key reagent for the synthesis of these
compounds owing to the reactivity of each of the cyanyl chloride
the design of pharmacologically active molecules. In particular,
hexamethylmelamine (HMM) was found to be an antitumour
agent andisclinicallyutilizedforlung,ovarian, andbreastcancers
[
14,15]
(
1
Figure 1).
Trimelamol, 2,4,6-tris(N-hydroxymethylamino)-
,3,5-triazine, was developed as a water-soluble analogue of
[
13]
units. We sought to utilize the triazine core as a template for
the production of potential boron neutron capture therapy
HMM. Although phase I and phase II clinical trials have
shown that trimelamol demonstrates promising activity toward
[
33–35]
(BNCT)
agents, and recently reported the synthesis of
Journal compilation Ó CSIRO 2011
www.publish.csiro.au/journals/ajc

Discovery of ortho-Carborane-Conjugated Triazines
1431
Me
N
NMe2
N
O
NMe2
HO
N
N
N
Me
Me
NH
O
N
r ꢀ 0.724
N
N
N
OH
O
N
N
N
Me
HN
Me N
NMe2
2
N
N
ꢀ
ꢀ C
BH
ICRF-193
TAZ-6
O
Me
HMM
OH
Fig. 2. Structures of di-ortho-carboranyl triazine (TAZ-6) and topoisom-
erase II inhibitor (ICRF-193).
Trimelamol
CHF2
N
N
NR12
Cl
(b)
N
N
N
N
N
N
(a)
Cl
N
Cl
Cl
N
NR1
2
N
N
2a: R1 ꢀ CH3
1
O
O
1
b: R ꢀ Et
c: R1
ꢀ
nPr
ZSTK474
Fig. 1. Structures of 1,3,5-triazines with antitumour activity.
d: R1 ꢀ N-morpholinyl
NR12
NR12
H
(c)
R2
[
36,37]
N
N
N
N
ortho-carborane-containing 1,3,5-triazines.
We tested
these compounds for in vitro antiproliferative activity towards
a panel of 39 human cancer cell lines (termed JFCR39), which is
comparable to the panel developed by the National Cancer
NR12
1
2
N
N
NR
ꢀ
ꢀ
BH
C
[
38,39]
Institute.
We compared the cell growth inhibitory activities
3a:
R1 ꢀ CH3
4a: R1 ꢀ CH , R2 ꢀ CH3
3
b: R1 ꢀ Et
c: R1
nPr
d: R1 ꢀ N-morpholinyl
b: R1 ꢀ Et, R2 ꢀ CH3
towards JFCR39 of more than 200 conventional compounds,
including anticancer drugs and various types of inhibitors, by
ꢀ
c: R1 nPr, R2 ꢀ CH3
ꢀ
[
40]
d: R1 ꢀ N-morpholinyl, R ꢀ CH3
2
using COMPARE analysis. This systemcan be used to predict
the molecular target or the mechanism of action of a test
compound by determining the correlation coefficient (r) between
the cell growth inhibition profiles of the test compound and
various reference compounds with known mechanisms of action.
Among the compounds synthesized, the cell growth inhibition
profile of 2,4-(2-methyl-ortho-carboranyl)-4-(dimethylamino)-
e: R1 ꢀ CH , R2 ꢀ Et
3
f: R1 ꢀ Et, R2 ꢀ Et
1
ꢀ
nPr, R2 ꢀ Et
g: R
h: R1 ꢀ N-morpholinyl, R ꢀ Et
2
i: R1 ꢀ CH , R2
ꢀ
nPr
3
j: R1 ꢀ Et, R2 ꢀ nPr
1
nPr, R2 ꢀ nPr
k: R
ꢀ
1
ICRF-193,
,3,5-triazine (TAZ-6) was found to be similar to that of
41]
l: R1 ꢀ N-morpholinyl, R ꢀ nPr
2
[
a topoisomerase II inhibitor, as indicated by
1
2
COMPARE analysis. In this case, r ¼ 0.724. In this study, we
Scheme 1. Reagents and conditions: (a) HNR (2 equiv), THF, 08C; (b)
n
lithiated ortho-carborane, THF, ꢀ108C; (c) BuLi, R I, THF, ꢀ108C.
2
synthesized various mono- and di-ortho-carborane-substituted
,3,5-triazines based on the structure of TAZ-6 and examined
1
the structure-activity relationships of these compounds with
topoisomerases I and II (Figure 2).
alkyl-group-substituted compounds have non-equivalent NMR
resonances as indicated in the experimental section. For
2
1
instance, compound 4a (R ¼ CH ) shows two H N-methyl
3
Results and Discussion
13
singlets at 3.12 and 3.15 ppm and the corresponding C signals
Chemistry
resonated at 35.9 and 36.1 ppm. These non-equivalent NMR
resonances are considered to be caused by their hindered rota-
tion of the triazine-carborane single bond and these phenomena
The synthesis of mono-ortho-carboranyl triazines 3 and 4 is
shown in Scheme 1. Cyanuric chloride 1 was chosen as the
starting material and treated with two equivalents of various
2
are not observed in compound 3a (R ¼ H), which has no sub-
1
secondary amines (R ¼ methyl, ethyl, and n-propyl) in THF at
stituent on the ortho-carborane (Scheme 1).
The synthesis of di-ortho-carboranyl triazines 6 and 7 is
shown in Scheme 2. Cyanuric chloride 1 was treated with one
0
8C to give 2,4-diamino-1,3,5-triazine derivatives 2 in 90–99 %
[
42]
yields. Furthermore, the reaction of 1 with morpholine gave a
mixture of mono- and di-substituted triazines in 60 and 29 %
yields, respectively. The lithiated ortho-carborane prepared
from the reaction of ortho-carborane with n-BuLi in THF
was reacted with 2 at ꢀ108C under argon atmosphere to afford
the corresponding mono-ortho-carborane-substituted 1,3,5-
triazines 3 in moderate yields (29–51 %). Deprotonation at the
carborane carbon with n-BuLi followedby treatment withvarious
o
equivalent of various secondary amines in THF at 0 C to give
2-amino-4,6-dichloro-1,3,5-triazine derivatives 5 in good to
high yields (65–95 %). The reaction of 5 with the lithiated
ortho-carborane at ꢀ108C gave the corresponding di-ortho-
carborane-substituted 1,3,5-triazines 6 in moderate yields
(29–34 %). Similarly, the alkyl groups were introduced into
the carborane carbon to afford 7 in 39–90 % yields. Alternative-
2
2
2
2
n
iodoalkanes (R ¼ methyl, ethyl, and n-propyl) gave alkyl-
ly, 9a (R ¼ CH
3
), 9b (R ¼ Et), and 9b (R ¼ Pr), which were
group-substituted ortho-carborane-containing 1,3,5-triazine
derivatives
prepared from ortho-carborane 8 according to the literature
procedure with modification,
[
43]
n
were treated with BuLi and
4
in 11–89 % yields. Interestingly, these

1
432
H. Nakamura et al.
NR12
NR12
Table 1. Inhibitory activity of the ortho-carborane-conjugated 1,3,5-
triazines 3, 4, 6 and 7a towards cell growth and topoisomerases I/II
(
a)
(b)
H
N
N
H
N
N
1
b
c
Cl
N
Cl
N
Compound
Growth inhibition
a
Topo I
Topo II
(
GI50/mM)
inhibition
inhibition
5
a: R1 ꢀ CH3
a: R1 ꢀ CH3
6
3a
9.2 ꢁ 1.8
1.8 ꢁ 2.1
1.5 ꢁ 0.5
–
þ
þþ
þ
1
b: R ꢀ Et
1
3
3
3
4
b
c
b: R ꢀ Et
c: R1
ꢀ
nPr
c: R1
nPr
þþ
–
–
ꢀ
d: R1 ꢀ N-morpholinyl
d
a
37.1 ꢁ 12.8
14.2 ꢁ 2.1
14.1 ꢁ 3.7
9.6 ꢁ 0.5
14.7 ꢁ 2.4
40.9 ꢁ 1.5
51.0 ꢁ 7.4
35.1 ꢁ 5.5
16.7 ꢁ 0.4
17.5 ꢁ 0.4
40.1 ꢁ 2.2
.100
þþ
–
H
d: R1 ꢀ N-morpholinyl
–
H
(c)
4b
þ
–
4
4
c
þþ
–
–
d
–
NR12
8
4e
4f
–
–
R2
R2
R2
þþ
–
–
N
N
(g)
4
4
4
4
4
g
–
H
(
d-f)
h
i
–
–
N
–
–
j
þ
–
k
nd
–
nd
–
7a: R1 ꢀ CH , R2 ꢀ CH3
9
a: R2 ꢀ CH3
b: R ꢀ Et
c: R2 nPr
3
4l
15.2 ꢁ 0.2
16.1 ꢁ 5.8
16.8 ꢁ 2.8
30.2 ꢁ 15.8
32.8 ꢁ 5.5
.100
2
b: R ꢀ Et, R2 ꢀ CH3
1
6a
6b
6c
6d
þþ
þþ
þþ
þþ
þþ
þþ
–
þþ
þþ
þþ
þþ
þþ
–
c: R1 nPr, R2 ꢀ CH3
ꢀ
ꢀ
d: R1 ꢀ N-morpholinyl, R2 ꢀ CH3
e: R1 ꢀ CH , R2 ꢀ Et
3
f: R1 ꢀ Et, R2 ꢀ Et
7
a (TAZ-6)
1
ꢀ
nPr, R2 ꢀ Et
g: R
Camptothecin
Etoposide
,0.4
h: R1 ꢀ N-morpholinyl, R2 ꢀ Et
2.8 ꢁ 0.2
þþ
i: R1 ꢀ CH , R2
ꢀ
nPr
nPr
nPr
3
j: R ꢀ Et, R2
1
ꢀ
a
The drug concentrations required to inhibit the HeLa cell growth by 50 %
(GI50) were determined from semilogarithmic dose-response plots, and
results represent mean ꢁ sd of triplicate samples.
k: R1
ꢀ ꢀ
nPr, R2
l: R1 ꢀ N-morpholinyl, R2
ꢀ
nPr
b
Enzymatic activities inhibited partially (þ) and completely (þþ) by
1
2
Scheme 2. Reagents and conditions: (a) HNR
(1 equiv), THF, 08C;
n
compounds at 100 mM. ‘ꢀ’ indicates that no inhibition was observed at
100 mM. Camptothecin and etoposide were used as positive controls for
topoisomerases I and II, respectively.
(
b) lithiated ortho-carborane, THF, ꢀ108C; (c) i) BuLi (2.2 equiv);
2 n
ii) R I (2.1 equiv), THF, ꢀ108C; (d) i) BuLi (1 equiv), ꢀ108C; ii) TMSCl
n
2
(
1.1 equiv), THF, ꢀ108C to r.t.; (e) i) BuLi (1.1 equiv), 08C; ii) R I (1.1
n
equiv), THF, 08C to r.t.; (f) TBAF, THF; (g) i) BuLi (1.1 equiv), 08C; ii) 5
1.1 equiv), THF, 08C to r.t.
supercoiled DNA breaks caused by topoisomerase I partially at
0 mM and completely at 100 mM. Etoposide, a topoisomerase II
(
3
inhibitor, inhibited supercoiled DNA breaks caused by topo-
isomerase II completely at 100 mM. Compound 3c exhibited
topoisomerase I inhibition in a concentration-dependent manner
without affecting topoisomerase II activity at 30 and 100 mM.
We systematically evaluated the inhibitory activities of
monocarborane-conjugated 1,3,5-triazines 3a-d and 4a-l, and
dicarborane-conjugated 1,3,5-triazines 6a-d and 7a toward
topoisomerases I and II. The results are summarized in Table 1.
Selective inhibitions of topoisomerases I and II were observed
in several 1,3,5-triazine derivatives. Compounds 3c, 4b, 4c, 4f,
and 4j selectively inhibited supercoiled DNA breaks caused by
topoisomerase I. In particular, compounds 3c, 4c, and 4f
exhibited complete inhibition of topoisomerase I activity at
100 mM and the inhibitory activities of these compounds are
similar to that of camptothecin. Meanwhile, compounds 3a and
3d selectively inhibited supercoiled DNA breaks caused by
topoisomerase II and the inhibitory activities of these com-
pounds are similar to that of etoposide (complete inhibition at
100 mM, see Figure 3b). Interestingly, the inhibition of both
topoisomerases I and II was observed in di-ortho-carborane-
substituted 1,3,5-triazines (6a-d and 7a). It is considered that the
inhibitory activities of di-ortho-carborane-substituted 1,3,5-
triazines towards both enzymes would be owing to the high
hydrophobic character of ortho-carborane, which would aggre-
gate with the enzymes via hydrophobic interactions to render the
enzymes inert, although the detailed binding modes of these
the resulting lithiated ortho-carboranes were reacted 5 in THF
at 08C to give the corresponding di-ortho-carborane-substituted
1
,3,5-triazines 7 in moderate to good yields (Scheme 2).
Biological Evaluation
We first investigated the cell growth inhibitory activities of the
1,3,5-triazine compounds, camptothecin, and etoposide toward
HeLa cells using the MTT assay. The results are shown in
Table 1. Among the compounds synthesized, compounds 3a and
b, which have no substituent at the carborane moiety, showed
3
the most significant cell growth inhibitory activities and their
GI50 (the concentration that inhibits 50 % of cell growth) values
were 1.8 and 1.5 mM, respectively. Compounds 4a-l, which have
2
long alkyl chains (R ) on the carborane moiety, and di-ortho-
carborane-substituted 1,3,5-triazine compounds (6a-d) showed
decreased cell growth inhibitory activities. In the cases of
compounds 4k and 6a-d, their GI50 values could not be obtained
due to their low solubility in water. Other synthesized di-ortho-
carborane-substituted 1,3,5-triazines 7b-f also showed a similar
tendency and their GI50 values were not detectable (.100 mM:
data not shown).
We next investigated the in vitro inhibition of topoisome-
rases I and II by the compounds. Figure 3 shows supercoiled
DNA breaks caused by topoisomerases I and II to form relaxed
[
44]
DNA.
Camptothecin, a topoisomerase I inhibitor, inhibited

Discovery of ortho-Carborane-Conjugated Triazines
1433
(
a) Topoisomerase I
10
30
100 (µM)
Supercoiled DNA
Relaxed DNA
3c
Camptothecin
Topoisomerase I
(
b) Topoisomerase II
10
30
100 (µM)
Supercoiled DNA
Relaxed DNA
3c
Etoposide
Topoisomerase II
Fig. 3. In vitro inhibition of topoisomerases I and II by compound 3c. Camptothecin and etoposide were used as positive controls for topoisomerases I and II,
respectively. Compound 3c partially inhibited (þ) and completely inhibited (þþ) topoisomerase I activity at 30 and 100 mM, respectively, without affecting
topoisomerase II activity.
compounds to topoisomerases I and II are not clear. Synthesized
di-ortho-carborane-substituted 1,3,5-triazines 7b-f also exhib-
ited complete inhibition of both topoisomerases I and II (data not
shown).
2,4-Bis(N,N-dimethylamino)-6-(o-carboran-1-yl)-1,3,5-
triazine 3a
To a stirred solution of o-carborane (0.29 g, 2.0 mmol) in 10 mL
THF at ꢀ108C was added 1.3 mL of n-BuLi (1.6 M, 2.0 mmol)
via a syringe. Compound 2a (0.40 g, 2.0 mmol) dissolved in
THF (10 mL) was slowly added to the reaction mixture at
Conclusion
ꢀ
108C, and the reaction temperature was maintained at ꢀ108C
We have developed ortho-carborane-substituted 1,3,5-triazines
as selective inhibitors of topoisomerases I and II. The sub-
stituents on the ortho-carborane are essential for the selective
inhibition of topoisomerases I and II. The current findings show
the potential of carborane as an alternative pharmacophore in
for 1 h. The reaction mixture was then warmed slowly to room
temperature, stirred for an additional 3 h, and quenched with
saturated aqueous NH Cl solution. The organic layer was
4
washed with brine, dried with anhydrous MgSO , and concen-
4
trated under reduced pressure. Purification by column chroma-
tography on silica gel with EtOAc/hexane (1:50) gave 3a in
[
27,28]
drug design.
The detailed binding modes of these com-
pounds to topoisomerases I and II are under investigation in our
laboratory.
3
1
2 % yield (0.20 g, 0.63 mmol) as a white solid. MP ¼ 167–
ꢀ
max
1
688C. n
(KBr)/cm 2914, 2573, 1589, 1508, 1379. dH
(
400 MHz, CDCl ) 4.57 (s, 1H), 3.11 (s, 12H). d (100 MHz,
3 C
Experimental
CDCl ) 165.8, 164.5, 74.6, 56.1, 36.1, 36.0. d ꢀ2.42 to ꢀ13.07
3
B
General
(br m, 10B). m/z (HR-ESI, positive) Calc. for C H B N
[M-H] : 312.2962, found: 312.2958.
9 23 10 5
1
13
11
þ
H NMR, C NMR, and B NMR spectra were measured on
JEOL JNM-AL 300 (300 MHz) and VARIAN UNITY-INOVA
00 (400 MHz) spectrometers. Chemical shifts are expressed
in parts per million (ppm, d units) relative to internal tetra-
2,4-Bis(N,N-diethylamino)-6-(o-carboran-1-yl)-1,3,5-triazine
3b was synthesized from 2b (0.27 g, 1.0 mmol) using the
procedure described for 3a to give 3b in 43 % yield (0.16 g,
0.43 mmol) as a white solid, mp 110-1118C. nmax (KBr)/cm
2571, 1566, 1504, 1435, 1364, 1310. d (400 MHz, CDCl ) 4.52
(s, 1H), 3.52 (q, J 7.2, 8H, CH CH ), 1.12-1.18 (m, 12H,
4
1
13
ꢀ1
methylsilane ( H NMR and C NMR) or external BF ꢂ Et O
3
2
1
1
(
B NMR), and the coupling constants are expressed in units of
hertz (Hz). IR spectra were measured on a Shimadzu FTIR-
200A spectrometer. High-resolution mass spectra (ESI) were
H
3
2
3
8
CH CH ). d (100 MHz, CDCl ) 158.6, 156.5, 67.7, 48.9, 34.8,
2 3 C 3
recorded on a Bruker Daltonics micro TOF-15 focus. Elemental
analyses were performed by a CE instrument EA1110 CHNS-O
automatic elemental analyzer. Analytical thin-layer chroma-
tography (TLC) was performed on glass plates (Merck Kieselgel
34.7, 6.3, 5.9. d (96.3MHz, CDCl ) ꢀ2.40 to ꢀ13.08 (br m,
B
3
10B). Anal. Calc. for C H N : C 42.72, H 8.55, N 19.16.
13 31 5
Found: C 43.01, H 8.64, N 19.01 %.
2,4-Bis(N,N-di-n-propylamino)-6-(o-carboran-1-yl)-1,3,5-
triazine 3c was synthesized from 2c (0.32 g, 1.0 mmol) using
the procedure described for 3a to give 3c in 62 % yield (0.27 g,
6
0 F254). Column chromatography was performed on silica
gel (Merck Kieselgel 70-230 mesh). All reactions were carried
out under an argon atmosphere using standard Schlenk techni-
ques. Most chemicals were of analytical grade and used without
further purification.
ꢀ1
0.64 mmol) as a white solid, mp 95-968C. nmax (KBr)/cm
2932, 2872, 2561, 1578, 1502, 1429. dH (400 MHz, CDCl3)
4.48 (s, 1H), 3.44-3.37 (m, 8H), 1.65-1.53 (m, 8H), 0.918-0.871

1
434
H. Nakamura et al.
(
4
m, 12H). d (100 MHz, CDCl ) 165.4, 164.0, 74.8, 55.9, 49.4,
C
2,4-Bis(N,N-dimethyamino)-6-(2-ethyl-o-carboran-1-yl)-1,
3,5-triazine 4e was synthesized from 3a (0.10 g, 0.34 mmol)
and iodoethane (35 mL, 0.44 mmol) using the procedure
described for 4a to give 4e in 11 % yield (13 mg, 0.036 mmol)
3
9.3, 21.4, 20.9, 11.5, 11.4. d (96.3MHz, CDCl ) ꢀ2.40 to
B
3
ꢀ
13.0 (br m, 10B). Anal. Calc. for C H N : C 48.43, H 9.32,
1
7 39 5
N 16.61. Found: C 48.51, H 9.45, N 16.51 %.
ꢀ1
2
,4-Bis-(N-morpholinyl)-6-(o-carboran-1-yl)-1,3,5-triazine
d was synthesized from 2d (0.29 g, 1.0 mmol) using the
procedure described for 3a to give 3d in 87 % yield (0.34 g,
as a white solid, mp 141–1428C. nmax (KBr)/cm 2947, 2554,
3
1589, 1504, 1406, 1375. dH (400 MHz, CDCl ) 3.14 (s, 6H),
3.11(s, 6H), 2.18 (q, J 7.6, 2H), 1.05 (t, J 7.6, 3H). d (100 MHz,
3
C
ꢀ1
0
2
.87 mmol) as a white solid, mp 2888C. nmax (KBr)/cm 2856,
CDCl ) 164.9, 164.2, 81.8, 81.7, 36.1, 35.9, 29.1, 13.9. d
3
B
584, 1578, 1504, 1445, 1236. dH (400 MHz, CDCl ) 4.45
(96.3 MHz, CDCl ) ꢀ2.34 to ꢀ9.68 (br m, 10B). Anal. Calc. for
3
3
(s, 1H), 3.84-3.66 (m, 16H). dC (100 MHz, CDCl ) 166.6,
3
C H N : C 39.15, H 8.06, N 20.75. Found: C 39.36, H 8.16, N
11 27 5
20.56 %.
1
64.0, 74.2, 66.5, 56.0, 43.7, 43.4. dB (96.3MHz, CDCl3)
2.42 to ꢀ13.04 (br m, 10B). Anal. Calc. for C H N : C
ꢀ
2,4-Bis(N,N-diethyamino)-6-(2-ethyl-o-carboran-1-yl)-1,3,
5-triazine 4f was synthesized from 3b (0.25 g, 0.68 mmol) and
iodoethane (70 mL, 0.88 mmol) using the procedure described
for 4a to give 4f in 68 % yield (0.18 g, 0.46 mmol) as a white
1
3 27 5
3
9.68, H 6.92, N 17.80. Found: C 39.71, H 6.71, N 17.67 %.
2
1
,4-Bis(N,N-dimethyamino)-6-(2-methyl-o-carboran-1-yl)-
,3,5-triazine 4a
ꢀ1
solid, mp 89–908C. nmax (KBr)/cm 2961, 2561, 1570, 1501,
1
2H), 1.19-1.13 (m, 12H), 1.06 (t, J 7.6, 3H). dC (100 MHz,
435. d (400 MHz, CDCl ) 3.58-3,50 (m, 8H), 2,19 (q, J 7.6,
H 3
To a stirred solution of 3a (0.15 g, 0.49 mmol) in 3 mL THF at
8C was added 0.4 mL of n-BuLi (1.6 M, 0.64 mmol) via a
0
CDCl ) 164.2, 163.9, 82.1, 81.9, 41.8, 41.6, 29.0, 13.9, 13.4,
3
syringe. Iodomethane (50 ml, 0.80 mmol) was slowly added to
the reaction mixture at 08C, and the reaction temperature was
then warmed slowly to room temperature. After the mixture was
12.9. d (96.3MHz, CDCl ) ꢀ2.53 to ꢀ9.66 (br m, 10B). Anal.
B
3
Calc. for C H N : C 45.77, H 9.11, N 17.79. Found: C 45.80,
1
5 35 5
H 8.96, N 17.62 %.
quenched with saturated aqueous NH Cl solution, the organic
4
2,4-Bis(N,N-di-n-propylamino)-6-(2-ethyl-o-carboran-1-yl)-
1,3,5-triazine 4 g was synthesized from 3c (0.18 g, 0.42 mmol)
and iodoethane (55 mL, 0.69mmol) using the procedure
described for 4a to give 4 g in 83% yield (0.16 g, 0.35 mmol)
layer was washed with brine, dried over anhydrous MgSO , and
4
concentrated under reduced pressure. Purification by column
chromatography on silica gel with EtOAc/hexane (1:50) gave
ꢀ1
4
a in 14 % yield (23 mg, 0.070 mmol) as a white solid, mp 170–
718C. nmax (KBr)/cm 2935, 2584, 2548, 1593, 1506, 1373.
as a white solid, mp 52–538C. nmax (KBr)/cm 2966, 2874,
2577, 1578, 1501, 1429. d (400 MHz, CDCl ) 3.46 (t, J 7.6, 4H),
3.40 (t, J 7.6, 4H), 2.18 (q, J 7.6, 2H), 1.67-1.53 (m, 8H), 1.05 (t, J
ꢀ1
1
d (400 MHz, CDCl ) 3.15 (s, 6H), 3.12 (s, 6H), 1.97 (s, 3H).
H
3
H
3
d (100 MHz, CDCl ) 164.9, 164.4, 80.0, 75.7, 36.1, 35.9, 24.0.
C
7.6, 3H), 0.906 (t, J 7.6, 12H). dC (100MHz, CDCl ) 164.3,
3
3
d (96.3MHz, CDCl ) ꢀ0.61 to ꢀ8.98 (br m, 10B). m/z (HR-
164.0, 82.1, 81.9, 49.4, 49.3, 29.0, 21.5, 20.9, 13.9, 11.5, 11.4. dB
(96.3MHz, CDCl ) ꢀ2.68 to ꢀ9.68 (br m, 10B). Anal. Calc. for
B
3
þ
ESI, positive) Calc. for C H B N [M-H] : 326.3119, found:
5
1
0
25 10
3
3
26.3116.
,4-Bis(N,N-diethyamino)-6-(2-methyl-o-carboran-1-yl)-1,
,5-triazine 4b was synthesized from 3b (0.18 g, 0.48 mmol)
C H N : C 50.75, H 9.64, N 15.57. Found: C 50.56, H 9.78, N
1
9 43 5
2
15.43 %.
3
2,4-Bis-(N-morpholinyl)-6-(2-ethyl-o-carboran-1-yl)-1,3,5-
triazine 4 h was synthesized from 3d (0.20 g, 0.50 mmol) and
iodoethane (50 mL, 0.63 mmol) using the procedure described
for 4a to give 4 h in 52 % yield (0.11 g, 0.26 mmol) as a white
using the procedure described for 4a to give 4b in 56 % yield
0.10 g, 0.27 mmol) as a white solid, mp 115-1168C. n (KBr)/
(
cm 2932, 2584, 1501, 1435. d (400 MHz, CDCl ) 3.58-3.51
max
ꢀ1
H
3
ꢀ
1
(m, 8H), 1.98 (s, 3H), 1.19-1.14 (m, 12H). dC (100 MHz,
CDCl ) 164.4, 163.9, 80.3, 75.7, 41.9, 41.6, 24.0, 13.4, 12.9.
solid, mp 1738C. nmax (KBr)/cm 2856, 2579, 1566, 1504,
1232. d (400 MHz, CDCl ) 3.86-3.68 (m, 16H), 2.15 (q, J 7.6,
3
H
3
d (96.3MHz, CDCl ) ꢀ1.93 to ꢀ9.48 (br m, 10B). Anal. Calc.
2H), 1.05 (t, J 7.6, 3H). d (100 MHz, CDCl ) 165.0, 164.4, 81.9,
C 3
81.2, 66.7, 66.5, 43.8, 43.5, 29.2, 13.9. d (96.3MHz, CDCl )
B 3
B
3
for C H N: C 44.30, H 8.76, N 18.45. Found: C 44.45, H 8.83,
1
4
33
N 18.53 %.
2,4-Bis(N,N-n-propylamino)-6-(2-methyl-o-carboran-1-yl)-
,3,5-triazine 4c was synthesized from 3c (0.20 g, 0.48 mmol)
ꢀ1.76 to ꢀ9.60 (br m, 10B). Anal. Calc. for C H N : C 42.74,
1
5 31 5
H 7.41, N 16.61. Found: C 42.94, H 7.40, N 16.39 %.
1
using the procedure described for 4a to give 4c in 90 % yield
2,4-Bis(N,N-dimethyamino)-6-(2-n-propyl-o-carboran-1-yl)-
1,3,5-triazine4iwas synthesized from3a(0.11 g, 0.36mmol) and
iodopropane (39 mL, 0.40mmol) using the procedure described
for 4a to give 4i in 23% yield (29 mg, 0.081 mmol) as a white
(
0.19 g, 0.43 mmol) as a white solid, mp 80–818C. n (KBr)/
max
ꢀ1
cm 2966, 2934, 2577, 1562, 1501, 1429. dH (400 MHz,
CDCl ) 3.46 (t, J 7.6, 4H), 3.40 (t, J 7.6, 4H), 1.97 (s, 3H),
ꢀ1
3
solid, mp 150-1518C. nmax (KBr)/cm 2563, 1589, 1502, 1404.
d (400MHz, CDCl ) 3.14 (s, 6H), 3.12 (s, 6H), 2.10-2.06 (m,
1
1
.60 (sext, J 7.2, 8H), 0.91 (t, J 7.2, 12H). d (100 MHz, CDCl )
C 3
64.3, 164.1, 80.4, 75.7, 49.4, 49.3, 24.0, 21.4, 20.9, 11.5, 11.4.
H
3
2H), 1.53–1.47 (m, 2H), 0.80 (t, J 7.6, 3H). d (100MHz, CDCl )
C 3
d (96.3MHz, CDCl ) ꢀ2.06 to ꢀ9.45 (br m, 10B). Anal. Calc.
164.9, 164.3, 81.5, 81.0, 37.6, 36.1, 35.9, 22.8, 13.7. dB
(96.3MHz, CDCl ) ꢀ2.22 to ꢀ9.47 (br m, 10B). m/z (HR-ESI,
B
3
for C H N : C 49.62, H 9.49, N 16.08. Found: 49.58, H 9.63, N
1
8
41
5
3
þ
1
5.78 %.
,4-Bis-(N-morpholinyl)-6-(2-methyl-o-carboran-1-yl)-1,3,
-triazine 4d was synthesized from 3d (0.20 g, 0.50 mmol) using
positive) Calc. for C H B N [M-H] : 354.3432, found:
1
2 29 10 5
2
354.3433.
5
2,4-Bis(N,N-diethyamino)-6-(2-n-propyl-o-carboran-1-yl)-
1,3,5-triazine 4j was synthesized from 3b (0.18 g, 0.49 mmol)
and iodopropane (49 mL, 0.50 mmol) using the procedure
described for 4a to give 4j in 76 % yield (0.15 g, 0.38 mmol)
the procedure described for 4a to give 4d in 20 % yield (42 mg,
ꢀ
1
0
2
3
1
.10 mmol) as a white solid, mp 266–2678C. nmax (KBr)/cm
856, 2577, 1574, 1504, 1445, 1234. dH (400 MHz, CDCl3)
ꢀ
1
.86–3.68 (m, 16H), 1.96 (s, 3H). dC (100 MHz, CDCl ) 165.2,
3
64.4, 79.6, 75.8, 66.7, 66.5, 43.8, 43.5, 24.0. d (96.3MHz,
B
as a white solid, mp 788C. nmax (KBr)/cm 2972, 2557, 1570,
1506, 1435. d (400 MHz, CDCl ) 3.58-3.51 (m, 8H), 2.11–2.07
H
3
CDCl ) ꢀ1.71 to ꢀ9.30 (br m, 10B). Anal. Calc. for C H N :
(m, 2H), 1.56-1.46 (m, 2H), 1.20-1.13 (m, 12H), 0.814 (t, J 7.2,
3H). d (100 MHz, CDCl ) 164.2, 163.9, 81.9, 81.0, 41.8, 41.6,
3
14 29 5
C 41.26, H 7.17, N 17.19. Found: C 41.03, H 7.30, N 17.17 %.
C
3

Discovery of ortho-Carborane-Conjugated Triazines
1435
3
7.5, 22.9, 13.8, 13.4, 12.9. d (96.3 MHz, CDCl ) ꢀ2.56 to
(96.3 MHz, CDCl ) ꢀ1.84 to ꢀ12.54 (br m, 10B). Anal. Calc.
B
3
3
ꢀ
9.715 (br m, 10B). Anal. Calc. for C H N : C 47.15, H 9.15,
5
for C H N : C 33.66, H 7.81, N 12.06. Found: C 33.45, H
13 36 4
1
6
37
N 17.18. Found: C 47.12, H 9.16, N 16.91 %.
2,4-Bis(N,N-n-propylamino)-6-(2-n-propyl-o-carboran-1-yl)-
,3,5-triazine 4k was synthesized from 3c (0.25g, 0.58 mmol) and
7.85, N 12.12 %.
2,4-Bis(o-carboran-1-yl)-6-(N-morpholinyl)-1,3,5-triazine
6d was synthesized from 5d (0.13 g, 0.54 mmol) using the
procedure described for 6a to give 6d in 38 % yield (92 mg,
1
iodopropane (60mL, 0.62mmol) using the procedure described
for 4a to give 4k in 90 % yield (0.24g, 0.52mmol) as a yellow
liquid. nmax (KBr)/cm 2964, 2359, 1570, 1491, 1327. dH
ꢀ
1
0.20 mmol) as a white solid, mp 2858C. nmax (KBr)/cm 2590,
1576, 1497, 1283. d (400 MHz, CDCl ) 4.40 (s, 2H), 3.87 (t,
J 4.8, 4H), 3.78 (t, J 4.8, 4H). dC (100 MHz, CDCl ) 167.9,
ꢀ1
H
3
(400 MHz, CDCl ) 3.46 (t, J 7.6, 4H), 3.41 (t, J 7.6, 4H), 2.07
3
3
(
7
4
t, J 8.4, 2H), 1.67-1.46 (m, 10H), 0.908 (t, J 7.6, 12H), 0.804 (t, J
162.9, 72.6, 66.2, 56.0, 44.1. d (96.3MHz, CDCl ) ꢀ1.78 to
B
3
.6, 3H). d (100 MHz, CDCl ) 164.3, 164.0, 82.0, 81.0, 49.3,
C
ꢀ12.75 (br m, 10B). Anal. Calc. for C H N : C 29.32, H 6.71,
11 30 4
3
9.3, 37.4, 22.9, 21.5, 20.9, 13.7, 11.5, 11.4. dB (96.3MHz,
N 12.43. Found: C 29.43, H 6.64, N 12.18 %.
CDCl ) ꢀ2.64 to ꢀ9.64 (br m, 10B). m/z (HR-ESI, positive)
3
þ
Calc. for C H B N [M-H] : 466.4684, found: 466.4684.
45 10
20
5
2,4-Bis(2-methyl-o-carboran-1-yl)-6-(N,N-dimethylamino)-
2,4-Bis-(N-morpholinyl)-6-(2-n-propyl-o-carboran-1-yl)-1,
,5-triazine 4l was synthesized from 3d (0.17 g, 0.44 mmol) and
1
,3,5-triazine 7a
To a stirred solution of 9a (87 mg, 0.55 mmol) in 5 mL THF at
108C was added 0.3 mL of n-BuLi (1.6 M, 0.50 mmol) via a
syringe. Compound 5a (48 mg, 0.25 mmol) dissolved in THF
3
iodopropane (43 mL, 0.44 mmol) using the procedure described
for 4a to give 4 L in 46 % yield (89 mg, 0.20 mmol) as a white
ꢀ
ꢀ1
solid, mp 2108C. nmax (KBr)/cm 2575, 1570, 1504, 1232. dH
400 MHz, CDCl ) 3.84-3.67 (m, 16H), 2.04 (t, J 8.4, 2H), 1.53-
(
5 mL) was slowly added to the reaction mixture at ꢀ108C, and
(
3
the reaction temperature was maintained at ꢀ108C for 1 h. The
reaction mixture was then warmed slowly to room temperature,
and stirred for an additional 3 h. The reaction was quenched with
1
.43 (m, 2H), 0.795 (t, J 7.6, 3H). d (100 MHz, CDCl ) 165.1,
C 3
1
64.4, 81.0, 81.0, 66.7, 66.5, 43.8, 43.5, 37.6, 22.9, 13.8. dB
(
96.3 MHz, CDCl ) ꢀ2.30 to ꢀ9.61 (br m, 10B). Anal. Calc. for
3
saturated aqueous NH Cl solution, and the organic layer was
4
C H N : C 44.12, H 7.64, N 16.08. Found: C 44.07, H 7.73 N
16 33 5
washed with brine, dried with anhydrous MgSO , and concen-
4
trated under reduced pressure. Purification by column chroma-
tography on silica gel with EtOAc/hexane (1:100) gave 7a in
1
5.91 %.
7
1 % yield (77 mg, 0.18 mmol) as a white solid, mp 1778C. n
2
,4-Bis(o-carboran-1-yl)-6-(N,N-dimethylamino)-1,3,5-
max
ꢀ
1
(
KBr)/cm
2934, 2561, 1605, 1493, 1414. dH (400 MHz,
CDCl ) 3.27 (s, 6H), 1.98 (s, 6H). d (100 MHz, CDCl ) 166.2,
triazine 6a
3
C
3
To a stirred solution of o-carborane (0.15 g, 1.1 mmol) in 5 mL
THF at ꢀ108C was added 0.6 mL of n-BuLi hexane solution
1
64.2, 77.8, 76.1, 36.7, 24.0. d (96.3MHz, CDCl ) ꢀ0.86 to
B
3
ꢀ
8.99 (br m, 10B). Anal. Calc. for C H N : C 36.56, H 8.18, N
11 32 4
(
0
1.6 M, 1.1 mmol) via a syringe. Compound 5a (0.11 g,
.59 mmol) dissolved in THF (5 mL) was slowly added to the
1
1.37. Found: C 36.70, H 8.23, N 11.08 %.
2
,4-Bis(2-methyl-o-carboran-1-yl)-6-(N,N-diethylamino)-1,
reaction mixture at ꢀ108C, and the reaction temperature was
maintained at ꢀ108C for 1 h. The reaction mixture was then
warmed slowly to room temperature, stirred for an additional
3
organic layer was washed with brine, dried over anhydrous
3
,5-triazine 7b was synthesized from 5b (0.11 g, 0.51 mmol) and
a (0.15 g, 0.94 mmol) using the procedure described for 7a to
give 7b in 49 % yield (0.12 g, 0.25 mmol) as a white solid, mp
9
h, and quenched with saturated aqueous NH Cl solution. The
4
ꢀ1
1
CDCl ) 3.64 (q, J 6.8, 4H), 1.97 (s, 6H), 1.25 (t, J 6.8, 6H). d
458C. nmax (KBr)/cm 2968, 2581, 1593, 1489. d (400 MHz,
H
3
C
MgSO , and concentrated under reduced pressure. Purification
4
(
(
100 MHz, CDCl ) 166.3, 163.2, 77.9, 76.1, 43.0, 24.0, 12.6. d
3
96.3MHz, CDCl ) ꢀ0.89 to ꢀ9.01 (br m, 10B). m/z (HR-ESI,
B
by column chromatography on silica gel with EtOAc/hexane
(1:10) gave 6a in 51 % yield (0.12 g, 0.30 mmol) as a white
3
-
[M-H] : 467.4723, found:
ꢀ1
negative) Calc. for C13
67.4725.
H
36
B N
20
4
solid, mp 2158C. nmax (KBr)/cm 3072, 2581, 1605, 1497. dH
400 MHz, CDCl ) 4.44 (s, 2H), 3.22 (s, 6H). dC (100 MHz,
CDCl ) 167.4, 163.4, 72.8, 56.0, 36.8. d (96.3MHz, CDCl )
4
(
3
3
B
3
2
,4-Bis(2-methyl-o-carboran-1-yl)-6-(N,N-di-n-
ꢀ
1.75 to ꢀ12.71 (br m, 10B). m/z (HR-ESI, negative) Calc. for
ꢀ
propylamino)-1,3,5-triazine 7c
C H B N [M-H] : 411.4097, found: 411.4093.
28 20 4
9
2
,4-Bis(o-carboran-1-yl)-6-(N,N-diethylamino)-1,3,5-triazine
b was synthesized from 5b (0.14 g, 0.63 mmol) using the
procedure described for 6a to give 6b in 30 % yield (83 mg,
To a stirred solution of 6c (0.16 g, 0.34 mmol) in 5 mL THF at
08C was added 0.4 mL of n-BuLi hexane solution (1.6 M,
0.68 mmol) via a syringe. Iodomethane (42 ml, 0.68 mmol) was
slowly added to the reaction mixture at 08C, and the reaction
temperature was then warmed slowly to room temperature.
6
ꢀ
1
0
.19 mmol) as a white solid, mp 2308C. nmax (KBr)/cm 3072,
581, 1601, 1489. d (400 MHz, CDCl ) 4.41 (s, 2H), 3.60 (q,
J 7.2, 4H, CH CH ), 1.22 (t, J 6.8, 6H, CH CH ). d
2
H
3
After the reaction was quenched with saturated aqueous NH Cl
4
2
3
2
3
C
(
(
100 MHz, CDCl ) 167.4, 162.4, 72.8, 55.9, 43.0, 12.5. dB
3
solution, the organic layer was washed with brine, dried over
anhydrous MgSO , and concentrated under reduced pressure.
96.3 MHz, CDCl ) ꢀ1.79 to ꢀ12.62 (br m, 10B). Anal. Calc.
3
4
for C H N : C 30.26, H 7.39, N 12.83. Found: C 30.04, H 7.35,
1
N 12.87 %.
,4-Bis(o-carboran-1-yl)-6-(N,N-di-n-propylamino)-1,3,5-
triazine 6c was synthesized from 5c (0.21 g, 0.84 mmol) using
the procedure described for 6a to give 6c in 62 % yield (0.24 g,
0
1
4
Purification by column chromatography on silica gel with
EtOAc/hexane (1:100) gave 7c in 90 % yield (0.15 g,
1 32 4
ꢀ1
2
0.31 mmol) as a white solid, mp 167-1688C. nmax (KBr)/cm
2968, 2561, 1589, 1497. d (400 MHz, CDCl ) 3.54 (t, J 7.6,
4H), 1.97 (s, 6H), 1.66 (sext, J 7.6, 4H), 0.97 (t, J 7.6, 6H). dC
H
3
ꢀ1
.52 mmol) as a white solid, mp 1578C. nmax (KBr)/cm 2586,
589, 1499. d (400 MHz, CDCl ) 4.39 (s, 2H), 3.50 (t, J 7.6,
H), 1.63 (sext, J 7.6, 4H), 0.942 (t, J 7.6, 6H). d (100 MHz,
(100 MHz, CDCl ) 166.2, 163.7, 77.9, 76.1, 50.1, 24.0, 20.7,
3
11.3. d (96.3 MHz, CDCl ) ꢀ0.77 to ꢀ9.02 (br m, 10B). Anal.
H
3
B
3
Calc. for C H N : C 36.56, H 8.18, N 11.37. Found: C 36.39, H
15 40 4
C
CDCl ) 167.3, 163.0, 72.8, 55.9, 50.1, 20.7, 11.4. dB
3
8.32, N 11.25 %.

1
436
H. Nakamura et al.
2
,4-Bis(2-methyl-o-carboran-1-yl)-6-(N-morpholinyl)-1,3,
to give 7j in 75 % yield (0.12 g, 0.22 mmol) as a white solid, mp
ꢀ
1
5
-triazine 7d was synthesized from 6d (0.13 g, 0.28 mmol)
using the procedure described for 7c to give 7d in 67 % yield
90 mg, 0.19 mmol) as a white solid, mp 2348C. n (KBr)/
1858C. nmax (KBr)/cm 2968, 2574, 1593, 1491. d (400 MHz,
H
CDCl ) 3.65 (q, J 7.2, 4H), 2.03 (t, J 8.4, 4H), 1.54–1.43 (m, 4H),
1.25 (t, J 7.2, 6H), 0.825 (t, J 7.2, 6H). d (100 MHz, CDCl )
3
(
max
C
3
ꢀ
1
cm 2590, 1578, 1497, 1281. d (400 MHz, CDCl ) 3.90 (t, J
4
166.1, 163.3, 81.4, 79.4, 42.9, 37.7, 22.9, 13.7, 12.6. d (96.3MHz,
B
H
3
.8, 4H), 3.80 (t, J 4.8, 4H), 1.97 (s, 6H). d (100 MHz, CDCl )
3
CDCl ) ꢀ1.28 to ꢀ9.30 (br m, 10B). Anal. Calc. for C H N :
C
3
17 44 4
1
66.6, 163.6, 77.6, 76.2, 66.1, 44.1, 24.0. d (96.3MHz, CDCl )
B
C 39.21, H 8.52, N 10.76. Found: C 39.27, H 8.56, N 10.52 %.
3
ꢀ
0.76 to ꢀ8.97 (br m, 10B). Anal. Calc. for C H N : C
2,4-Bis(2-n-propyl-o-carboran-1-yl)-6-(N,N-di-n-propylamino)-
1,3,5-triazine 7k was synthesized from 5c (63 mg, 0.25 mmol) and
9c (70 mg, 0.37 mmol) using the procedure described for 7a to give
7k in 51 % yield (71 mg, 0.13 mmol) as a white solid, mp 1828C.
1
3 34 4
3
2.62 H 7.16, N 11.71. Found: C 32.55, H 7.19, N 11.81 %.
2
,4-Bis(2-ethyl-o-carboran-1-yl)-6-(N,N-dimethylamino)-1,
,5-triazine 7e was synthesized from 6a (0.21 g, 0.51 mmol) and
3
ꢀ
1
iodoethane (80 mL, 1.0 mmol) using the procedure described for
c to give 7e in 62 % yield (0.15 g, 0.32 mmol) as a white solid,
nmax (KBr)/cm 2934, 2565, 1591, 1491. d (400 MHz, CDCl )
H
3
7
3.55 (t, J 7.6, 4H), 2.02 (t, J 8.4, 4H), 1.66 (sext, J 7.6, 4H), 1.52-1.42
(m, 4H), 0.966 (t, J 7.2, 6H), 0.815 (t, J 7.2, 6H). d (100 MHz,
ꢀ
1
mp 2108C. nmax (KBr)/cm 2584, 1609, 1491. d (400 MHz,
H
C
CDCl ) 3.26 (s, 6H), 2.13 (q, J 7.6, 4H), 1.05 (t, J 7.6, 6H). dC
CDCl ) 166.1, 163.9, 81.4, 79.4, 50.0, 37.7, 22.9, 20.8, 13.7, 11.3. d
B
3
3
(
(
100 MHz, CDCl ) 166.1, 164.3, 82.3, 79.4, 36.7, 29.3, 13.9. dB
3
(96.3MHz, CDCl ) ꢀ1.25 to ꢀ9.27 (br m, 10B). Anal. Calc. for
3
96.3MHz, CDCl ) ꢀ1.34 to ꢀ9.32 (br m, 10B). Anal. Calc. for
C H N : C 41.58, H 8.82, N 10.21. Found: C 41.49, H 8.98, N
19 48 4
10.21 %.
3
C H N : C 33.60, H 7.81, N 12.06. Found: C 33.63, H 7.79, N
1
3 36 4
1
1.76 %.
,4-Bis(2-ethyl-o-carboran-1-yl)-6-(N,N-diethylamino)-1,3,
-triazine 7f was synthesized from 5b (0.27 g, 1.2 mmol) and
2,4-Bis(2-n-propyl-o-carboran-1-yl)-6-(N,N-di-n-propylamino)-
1,3,5-triazine 7l was synthesized from 5d (63 mg, 0.27 mmol) and
9c (95 mg, 0.51 mmol) using the procedure described for 7a to give
2
5
9
give 7f in 38 % yield (0.23 g, 0.47 mmol) as a white solid, mp
b (0.38 g, 2.2 mmol) using the procedure described for 7a to
7l in 75 % yield (0.11 g, 0.20 mmol) as a white solid, mp 2088C. n
max
ꢀ
(KBr)/cm 2968, 2567, 1574, 1493. d (400 MHz, CDCl ) 3.92 (t,
H 3
1
ꢀ
1
1
CDCl ) 3.64 (q, J 7.2, 4H), 2.13 (q, J 7.6, 6H), 1.25 (t, J 7.6, 6H),
648C. nmax (KBr)/cm 2984, 2581, 1585, 1489. d (400 MHz,
J 4.8, 4H), 3.81 (t, J 4.8, 4H), 2.03 (t, J 8.4, 4H), 1.54–1.45 (m, 4H),
0.833 (t, J 7.2, 6H). d (100 MHz, CDCl ) 166.6, 163.8, 81.5, 79.1,
H
3
C
3
1
7
.06 (t, J 7.2, 6H). d (100 MHz, CDCl ) 166.1, 163.2, 82.3,
3
66.2, 44.1, 37.7, 23.0, 13.7. d (96.3MHz, CDCl ) ꢀ1.17 to ꢀ9.22
C
B
3
9.6, 43.0, 29.3, 13.9, 12.6. dB (96.3MHz, CDCl ) ꢀ1.35 to
(br m, 10B). Anal. Calc. for C H N : C 38.18, H 7.92, N 10.48.
17 42 4
3
ꢀ
9.20 (br m, 10B). Anal. Calc. for C H N : C 36.56, H 8.18, N
1
Found: C 38.30, H 7.95, N 10.19 %.
5 40 4
1
1.37. Found: C 36.70, H 8.23, N 11.08 %.
,4-Bis(2-ethyl-o-carboran-1-yl)-6-(N,N-di-n-propylamino)-
,3,5-triazine 7 g was synthesized from 5c (75 mg, 0.30 mmol)
Human Cancer Cell Line Panel Screening: To evaluate
drugs for the cell growth inhibition profile, we established a
human cancer cell line panel combined with a database. The
system as a whole was developed according to the method of
2
1
and 9b (0.10 g, 0.58 mmol) using the procedure described for 7a
to give 7 g in 89 % yield (0.14 g, 0.27 mmol) as a white solid, mp
1
CDCl ) 3.53 (t, J 7.6, 4H, CH CH CH ), 2.13 (q, J 7.2, 4H,
[
38,39]
the National Cancer Institute,
with modification. The cell
ꢀ
1
568C. nmax (KBr)/cm 2934, 2573, 1591, 1493. d (400 MHz,
line panel consisted of 39 human cancer cell lines. With this
system, we have examined the antiproliferative effect of more
than 200 standard compounds, including various anticancer
drugs, and established a new database, as described below.
Measurements of Cell Growth Inhibition as Data
Analysis. The details of measuring cell growth inhibition are
H
3
2
2
3
CH CH ), 1.65 (sext, J 7.6, 4H), 1.05 (t, J 7.6, 6H), 0.965 (t, J 7.6,
2
3
6
2
1
H). d (100 MHz, CDCl ) 166.1, 163.8, 82.3, 79.5, 50.1, 29.3,
C 3
0.8, 13.9, 11.3. d (96.3MHz, CDCl ) ꢀ1.59 to ꢀ9.327 (br m,
B
3
0B). Anal. Calc. for C H N : C 39.21, H 8.52, N 10.76.
1
7 44 4
[
39,45]
Found: C 39.40, H 8.58, N 10.58 %.
,4-Bis(2-ethyl-o-carboran-1-yl)-6-(N-morpholinyl)-1,3,5-
triazine 7 h was synthesized from 6d (0.13 g, 0.28 mmol) and
iodoethane (50 mL, 0.63 mmol) using the procedure described
for 7c to give 7 h in 66 % yield (95 mg, 0.19 mmol) as a white
described elsewhere.
Briefly, the cells were plated at
2
proper density in 96-well plates in RPMI 1640 with 5 % foetal
bovine serum and allowed to attach overnight. The cells were
exposed to compounds for 48 h. Then, the cell growth was
determined according to the sulforhodamine B assay, described
[46]
ꢀ1
solid, mp 2168C. nmax (KBr)/cm
2575, 1576, 1497. dH
400 MHz, CDCl ) 3.91 (t, J 4.8, 4H), 3.80 (t, J 4.8, 4H),
by Skehan et al.
Data calculations were made according to
[
40]
(
the method described previously. Absorbance for the control
well (C) and the tests well (T) were measured at 525 nm.
Moreover, at time 0 (addition of compounds), absorbance
3
2
1
.12 (q, J 7.6, 4H), 1.06 (t, J 7.6, 6H). d (100 MHz, CDCl )
C 3
66.5, 163.7, 82.3, 79.2, 66.1, 44.1, 29.3, 13.9. d (96.3MHz,
B
CDCl ) ꢀ1.146 to ꢀ9.297 (br m, 10B). m/z (HR-ESI, negative)
for the test well (T ) was also measured. Using these measure-
0
3
ꢀ
Calc. for C H B N O [M-H] : 509.4828, found: 509.4827.
1
ments, cell growth inhibition (percentage of growth) by each
concentration of compounds was calculated as: % growth ¼
100 ꢃ [(T ꢀ T )/(C ꢀ T )], when T . T and % growth ¼ 100 ꢃ
5 38 20 4
2,4-Bis(2-n-propyl-o-carboran-1-yl)-6-(N,N-dimethylamino)-
,3,5-triazine 7i was synthesized from 6a (0.23 g, 0.57 mmol) and
1
iodpropane (0.11 mL, 1.1mmol) using the procedure described
for 7c to give 7i in 37 % yield (0.10 g, 0.21 mmol) as a white
0
0
0
[(T ꢀ T )/T], when T , T . By using the computer to process %
50
0
0
growth values, the 50 % growth inhibition parameter (GI )
was determined. The GI50 was calculated as 100 ꢃ [(T ꢀ T )/
ꢀ1
solid, mp 1838C. nmax (KBr)/cm 2968, 2579, 1605, 1491. dH
400 MHz, CDCl ) 3.27 (s, 6H), 2.03 (t, J 8.4, 4H), 1.53–1.44 (m,
0
(
(C ꢀ T )] ¼ 50. The mean graph, which shows the differential
3
0
4
7
H), 0.823 (t, J 7.6, 6H). d (100 MHz, CDCl ) 166.0, 164.2, 81.4,
C
growth inhibition of the drug in the cell line panel, was drawn
based on a calculation using a set of GI50.
3
[
38,45]
9.3, 37.7, 36.7, 22.9, 13.7. d (96.3MHz, CDCl ) ꢀ1.35 to ꢀ9.30
To analyze the
correlation between the mean graphs of compounds A and B,
COMPARE computer algorithm was developed to the method
B
3
(br m, 10B). Anal. Calc. for C H N : C 36.56, H 8.18, N 11.37.
1
5 40 4
Found: C 36.50, H 8.19, N 11.07 %.
,4-Bis(2-n-propyl-o-carboran-1-yl)-6-(N,N-diethylamino)-
,3,5-triazine 7j was synthesized from 5b (65 mg, 0.29 mmol)
and 9c (92 mg, 0.49 mmol) using the procedure described for 7a
[
39]
described by Paull et al. Correlation coefficients were calcu-
2
1
lated using the following formula: r ¼ (S(x 2 x )(y 2 y ))/
i
m
i
m
2
2 1/2
(S(x 2 x ) S(y 2 y ) ) , where x and y are log GI of
i
m
i
m
i
i
50

Discovery of ortho-Carborane-Conjugated Triazines
1437
compounds A and B, respectively, against each cell line, and xm
and y are the mean values of x and y , respectively.
Topoisomerase I and II assays: Topoisomerase assays were
performed according to the manufacturer’s instructions of
TopoGen. Briefly, 0.5 Ag supercoiled DNA, one unit topoisom-
erase I, and the compounds were incubated for 30 min at 378C in
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R. Kallenbach, Bioorg. Med. Chem. Lett. 2008, 18, 1308. doi:10.1016/
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m
i
i
[
25] K. Srinivas, U. Srinivas, K. Bhanuprakash, K. Harakishore, U. S. N.
Murthy, V. J. Rao, Eur. J. Med. Chem. 2006, 41, 1240. doi:10.1016/J.
EJMECH.2006.05.013
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Moreau, V. i. Perron, N. Wilb, M. Asselin, A. A. F. M.-E. v. Doucet, D.
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3
5 mM Tris-HCl (pH 8.0), 72 mM KCl, 5 mM MgCl , and 5 mM
2
DTT. The reactions were ceased by the addition of 6 loading dye
solution and then subjected to electrophoresis on 1 % agarose
gels, with or without ethidium bromide. Topoisomerase II
assays were prepared as above, except that 125 ng supercoiled
DNA and four units topoisomerase II, together with the chemi-
cal compounds were incubated for 30 min at 378C in 10 mM
Tris-HCl (pH 7.9), 50 mM NaCl, 50 mM KCl, 5 mM MgCl2,
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[
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[
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0
.1 mM EDTA, and 1 mM ATP.
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