Synthesis of 1,3,5-cis,cis-triaminocyclohexane N-pyridyl derivatives as potential antitumor agents.

Article abstract of DOI:10.1021/jo0204911

Iron deprivation has been previously proven to be a promising strategy in treating tumor cells. A series of cis,cis-1,3,5-triaminocyclohexane N-pyridyl derivatives as iron-depleting antitumor agents were prepared. Cytotoxic activity of these derivatives was evaluated in the HeLa cancer cell line. Among the tested derivatives, N-ethyl-N,N',N"-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane (17) exhibited potent cytotoxicity against this cancer cell line. On the basis of the structure of 17, a bifunctional iron chelator 24 was designed and prepared. Bifunctional agent 24 possessing a maleimide linker that is functional for conjugation to thiolated monoclonal antibodies is a promising lead compound for development of antitumor conjugates for antibody-targeted therapies.

Full text of DOI:10.1021/jo0204911

Syn th esis of 1,3,5-cis,cis-Tr ia m in ocycloh exa n e N-P yr id yl
Der iva tives a s P oten tia l An titu m or Agen ts
Hyun-soon Chong,*,† Frank M. Torti,‡,§ Suzy V. Torti,§,| and Martin W. Brechbiel†
Chemistry Section, National Cancer Institute, NIH, Bethesda, Maryland 20892, and Departments of
Cancer Biology and Biochemistry, and the Comprehensive Cancer Center, Wake Forest University,
Winston Salem, North Carolina 27157
chongjoy@mail.nih.gov
Received J uly 25, 2002
Iron deprivation has been previously proven to be a promising strategy in treating tumor cells. A
series of cis,cis-1,3,5-triaminocyclohexane N-pyridyl derivatives as iron-depleting antitumor agents
were prepared. Cytotoxic activity of these derivatives was evaluated in the HeLa cancer cell line.
Among the tested derivatives, N-ethyl-N,N′,N”-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclo-
hexane (17) exhibited potent cytotoxicity against this cancer cell line. On the basis of the structure
of 17, a bifunctional iron chelator 24 was designed and prepared. Bifunctional agent 24 possessing
a maleimide linker that is functional for conjugation to thiolated monoclonal antibodies is a
promising lead compound for development of antitumor conjugates for antibody-targeted therapies.
In tr od u ction
and investigated its potential as an anti-cancer drug.5
Cell culture experiments have shown that 1 exhibits
significant antiproliferative activity and induces a cyto-
toxic effect by apoptotic cell death.6a,b Moreover, tachpyr
has been shown to be indifferent to activation of the
tumor suppressor p53 for its antiproliferative activity or
cytotoxic effect.6b This finding is of significant importance
in that tumor cell lines without p53 are substantially less
responsive to numerous chemotherapeutic agents than
tumor cell lines with p53, which is true of over 50% of
human cancers.7 Therefore, 1 possesses great promise not
only as a single antitumor agent, but for use in combina-
tion with p53-targeted antibodies or other chemothera-
peutic agents.
This potential of 1 has led us to design tachpyr
analogues 7, 11, and 17 and to investigate their ability
to act as cytotoxic agents. Tachpyr, being a small
molecule, has a limited biological half-life. Accordingly,
we sought to offset this limitation by creating a bifunc-
tional tachpyr analogue that could be linked to other
molecules or proteins, potentially monoclonal antibodies,
specifically to produce a conjugate that might then be
selectively targeted to tumor cells. On the basis of the
observed cytotoxicity of the tachpyr analogues described
herein, we have proceeded to the design and preparation
of the bifunctional tachpyr 24 possessing a maleimide
Iron deprivation has been developed as a therapeutic
strategy of malignant tumor cells.1 In this tumor therapy,
iron chelators,2 gallium nitrate,3 or monoclonal antibodies
(mAbs) against the transferrin receptor4 have been
employed to produce cellular iron depletion. Numerous
studies have shown that these iron depletion agents
exhibit antiproliferative activity and induce apoptosis in
tumor cells.1 In particular, iron chelators such as
desferrioxamine2a and pyridoxyl isonicotonyl hydrazide
derivatives2b are known to possess potent efficacy in
treatment of cancer cells including hepatoma xenografts,
leukemia, neuroblastoma, and lymphoma. Recently, we
have prepared N,N′,N′′-tris(2-pyridylmethyl)-cis,cis-1,3,5-
triaminocyclohexane (tachpyr, 1) as a novel iron chelator
* To whom correspondence should be addressed. Fax: (301) 402-
1923.
† National Cancer Institute.
‡ Department of Cancer Biology, Wake Forest University.
§ Comprehensive Cancer Center, Wake Forest University.
| Department of Biochemistry, Wake Forest University.
(1) (a) Rakba, N.; Loyer, P.; Gilot, D.; Delcros, J . G.; Glaise, D.; Baret,
P.; Pierre, J . L.; Brissot, P.; Lescoat, G. Carcinogenesis 2000, 21, 943.
(b) Tanaka, T.; Satoh, T.; Onozawa, Y.; Kohroki, J .; Itoh, N.; Ishidate,
M., J r.; Muto, N.; Tanaka, K. Cell Biol. Int. 1999, 23, 541. (c)
Richardson, D. R.; Milnes, K. Blood 1997, 89, 3025. (d) Kemp, J . D.;
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Cancer 1992, 70, 2051. (b) Hoffbrand, A. V.; Ganeshaguru, K.; Hooton,
J . W. C.; Tattersall, M. Brit. J . Haematol. 1986, 33, 517. (c) Lederman,
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Park, G.; Planalp, R. P. Blood 1998, 92, 1384. (b) Abeysinghe, R. D.;
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10.1021/jo0204911 CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/23/2002
8072
J . Org. Chem. 2002, 67, 8072-8078

1,3,5-cis,cis-Triaminocyclohexane N-Pyridyl Derivatives
SCHEME 1
SCHEME 2
moiety. Thus, routine coupling of 24 to thiolated mono-
clonal antibodies (mAbs) via Michael addition of thiol to
maleimide is expected to afford a variety of antitumor
conjugates for the targeted delivery of tachpyr cytotox-
icity. Herein, we describe the synthesis of the tach
N-pyridyl derivatives 7, 11, and 17 and the bifunctional
tachpyr 24 either for use as single antitumor agents or
for use in antibody-targeted therapies.
erably lipophilic, separation of product(s) can be ef-
ficiently achieved via routine silica gel chromatography.
Deprotection of tritylated amines in acidic media is also
quite straightforward, and the simple workup neatly
produces deprotected amines.
As the starting material for trityl protection, tach 3
was prepared from commercially available carboxylic acid
2 according to a procedure published previously (Scheme
2).5 When an equimolar amount of 3 and trityl chloride
was refluxed in chloroform in the presence of K2CO3, the
reaction provided both diprotected tach 4 and monopro-
tected tach 5 with yields of 43% and 20%, respectively.
Interestingly, reaction of 3 with trityl chloride in the
absence of base still provided both 4 and 5 albeit with
increased yields of 52% and 28%, respectively. However,
when an equimolar amount of 3 and trityl chloride in
THF was refluxed in the presence of Et3N as a base, the
reaction selectively provided only monoprotected 5. Syn-
thesis of bis(2-pyridylmethyl)tach 7 is shown in Scheme
2. Thus, monoprotected 5 was reacted with pyridine-2-
carboxaldehyde to afford the corresponding imine, which
was subsequently reduced to 6 with NaBH4. Removal of
the trityl group in 6 by treatment with CF3CO2H afforded
7 in 83% yield. The synthesis of N-propylated-N′,N′′-bis-
(2-pyridylmethyl)tach 11 is shown in Scheme 3. The N,N′-
bis(trityl)tach 4 was reacted with propionyl chloride to
Resu lts a n d Discu ssion
As shown in Scheme 1, the tachpyr analogues as iron
chelators have three structural features, i.e., the lipo-
philic cyclohexane ring, the 2-pyridyl unit, and the
cyclohexyl-based amines. In preparing the desired tach-
pyr analogues 7, 11, and 17, selective partial protection
of the primary amines in cis,cis-1,3,5-triaminocyclohex-
ane (tach, 3) is the key step. We employed the sterically
bulky trityl chloride to partially protect either one or two
of the primary amines in 3. The trityl group is an
attractive amine protecting group that is known to be
selective for primary amines over secondary amines and
has the advantages of easy removal and enhanced
liphophilicity.8 Since trityl-protected amines are consid-
(8) Barlos, K.; Papaioannou, D.; Theodoropoulos, D. J . Org. Chem.
1982, 47, 1324.
J . Org. Chem, Vol. 67, No. 23, 2002 8073

Chong et al.
SCHEME 3
SCHEME 4
afford amide 8 in 51% yield. Removal of the trityl groups
in 8 followed by reaction of the deprotected amines with
pyridine-2-carboxaldehyde and subsequent reduction of
the imines with NaBH4 produced 10. Reduction of 10
with LiAlH4 provided the desired amine 11 along with a
mixture of decomposition products as determined by
mass spectral analysis. However, purification of 11 via
column chromatography turned out to be quite difficult.
Attempted reduction of 10 with BH3 in THF also failed
to provide the desired amine 11. Interestingly, this
particular reduction procedure provided only the amide
chain cleavage product 7 in 88% yield. To avoid these
undesirable complications, 4 was first alkylated to 12
using the modified Borch reductive amination9 with
propionaldehyde and NaBH3CN and was then depro-
tected in CF3CO2H/MeOH/CHCl3. Condensation of amine
13 with pyridine-2-carboxaldehyde and subsequent re-
duction with NaBH4 successfully provided 11 in 58%
yield. Starting with the key intermediate 4, we were able
to synthesize the tachpyr analogue 17 having an ethyl
group (Scheme 4). Condensation of 4 with pyridine-2-
carboxaldehyde followed by reduction with NaBH4 pro-
vided 14, which was subsequently reacted with ethyl
sulfate in CH3CN at room temperature to afford 15 in
80% yield. Deprotection of the trityl groups in 15 followed
by reaction with pyridine-2-carboxaldehyde and subse-
quent treatment with NaBH4 provided compound 17 in
86% yield.
Cytotoxicity of the derivatives 7, 11, and 17 was
evaluated using the HeLa cell line (Figure 1).10 Among
the tested derivatives, both 7 and 11 exhibited somewhat
reduced cytotoxicity as compared to tachpyr 1. Thus,
replacement of a pyridyl group in tachpyr 1 by either a
propyl group (11) or hydrogen (7) resulted in a significant
decrease in cytotoxicity. However, introduction of an ethyl
group (17) into tachpyr 1 seemed to have little effect on
cytotoxicity. The novel tachpyr analogue 17 exhibited
reasonably comparable cytotoxicity to tachpyr 1. This was
a gratifying result in that we were very aware that
tachpyr derivatives wherein all three amines alkylated
to tertiary amines by either methyl, ethyl, or propyl
groups essentially eliminated biological activity.11 Thus,
up until this point it was unclear whether monoderiva-
(10) Mosmann, T. J . Immunol. Methods 1983, 65, 55.
(11) Ma, D.; Liu, F.; Overstreet, T.; Milenic, D. E.; Brechbiel, M. W.
Nucl. Med. Biol. 2002, 29, 91.
(9) Lewin, G.; Schaeffer, C. Heterocycles 1998, 48, 171.
8074 J . Org. Chem., Vol. 67, No. 23, 2002

1,3,5-cis,cis-Triaminocyclohexane N-Pyridyl Derivatives
SCHEME 5
readily undergo Michael addition with a nucleophilic thiol
group. We felt that this group would provide adequate
orthogonal nature to the functional groups of tachpyr
while permitting reasonably efficient conjugation to
proteins. Thus, the bifunctional tachpyr might be conju-
gated to a variety of thiolated mAbs via linkage of the
maleimide and an introduced thiol group to afford
antitumor conjugates for targeted therapy.12 Our strategy
in the construction of the desired bifunctional tachpyr
24 was to introduce a side chain having a pendant
primary amino group to the tachpyr moiety, which could
be converted to an aminoalkylmaleimide. The synthetic
strategy for 24 incorporating a maleimide group is
outlined in Scheme 5. As a starting material, compound
14 was reacted with 4-bromobutylphthalimide to provide
18 in 81% yield. Deprotection of the trityl groups in 18
and subsequent reactions with pyridine-2-carboxaldehyde
and NaBH4 provided 20 in 73% yield. Removal of the
phthalimide group in 20 by treating with hydrazine
hydrate failed to cleanly provide the deprotected amine
21. However, hydrolysis of 20 by refluxing in 5M HCl
completely removed the phthalimide group providing
F IGURE 1. Effects of chelators 7 (O), 11 (3), 17 (1), and
tachpyr 1 (b) on cell viability. Cells were incubated for 72 h
with chelators at various concentrations and viability assessed
as described in the Experimental Section. Each point repre-
sents the mean and standard error of octuplicate cultures.
tization of an amine of tachpyr 1 would in fact be
tolerated. The slight decrease in cytotoxicity was felt to
be completely acceptable in light of the goal of a selec-
tively targetable analogue, by which such a property one
might reasonably expect to then compensate for a mild
loss of activity as observed at this point.
The result prompted us to design a bifunctional tach-
pyr based on the structure of 17. We were particularly
interested in the preparation of a bifunctional tachpyr
possessing a pendent maleimide group, which would
(12) (a) Norman, T. J .; Parker, D.; Royle, L.; Harrison, A.; Antoniw,
P.; King, D. J . J . Chem. Soc., Chem. Commun. 1995, 1877. (b) Finn,
F. M.; Yamanouchi, K.; Titus, G.; Hofmann, K. Bioorg. Chem. 1995,
23, 152. (c) Gil, L.; Han, Y. X.; Opas, E. E.; Rodan, G. A.; Ruel, R.;
Seedor, J . G.; Tyler, P. C.; Young, R. N. Bioorg. Med. Chem. 1999, 7,
901. (d) Hill, K. W.; Taunton-Rigby, J .; Carter, J . D.; Kropp, E.; Vagle,
K.; Pieken, W.; McGee, D. P. C.; Husar, G. M.; Leuck, M.; Anziano, D.
J .; Sebesta, D. P. J . Org. Chem. 2001, 16, 5352.
J . Org. Chem, Vol. 67, No. 23, 2002 8075

Chong et al.
(d), 127.8 (d), 128.6 (d), 147.0 (s); HRMS (positive-ion FAB)
calcd for C33H29N3 [M + H]+ m/z 372.2440, found [M + H]+
m/z 372.2440.
amine 21 in 75% yield. Our initial effort to synthesize
N-aminoalkylmaleimide 22 by reaction of 21 directly with
maleic anhydride and subsequent dehydrative cyclization
was unsuccessful. The reaction provided chromatographi-
cally inseparable multiple products no doubt due to the
presence of the competing secondary amines in 21. We
then tried selective reaction of the primary amine in 21
with the succinimidyl active ester reagent 2313 to intro-
duce a formed maleimide group. To our satisfaction,
treatment of 21 with 23 at room-temperature resulted
in almost complete transformation to the desired product
24 as evidenced by both HPLC and NMR analysis.
Purification by silica gel flash column provided the
conjugate product 24 in 88% yield.
The synthetic techniques to the tachpyr derivatives (7,
11, 17, 21, and 24) reported herein might be directly
applied to preparation of numerous metal chelating
agents as potential anti-cancer drugs having substituents
other than pyridyl groups that might then lead to
extensive structure-activity relationship studies. Ad-
ditionally, entry to numerous mixed donor chelating
agents based on the cis,cis-1,3,5-triaminocyclohexane
platform is now clearly available permitting detailed
studies of their fundamental coordination chemistry.
Bifunctional tachpyr 24 possesses significant potential
for use in targeted therapies. Preparation and biological
evaluation of antibody conjugates of 24 are being actively
pursued and will be published in due course.
Meth od 2. To a solution of 3 (1.20 g, 9.3 mmol) in CHCl3
(30 mL) was added a solution of TrCl (2.59 g, 9.3 mmol) in
CHCl3 (20 mL) over 1.5 h. The resulting mixture was stirred
for 0.5 h and refluxed for 2 h, at which time the reaction
mixture was cooled to room temperature and concentrated in
vacuo. The same chromatographic purification procedure
employed in method 1 provided 4 and 5 in 52% and 28%
isolated yields, respectively.
N-Tr ityl-cis,cis-1,3,5-tr ia m in ocycloh exa n e (5). To a so-
lution of 3 (1.20 g, 9.3 mmol) in THF (70 mL) were sequentially
added Et3N (1.43 mL, 10.23 mmol) and TrCl (2.59 g, 9.3 mmol).
The resulting mixture was refluxed for 4 h, at which time the
reaction mixture was cooled to room temperature and concen-
trated in vacuo. The same chromatographic purification
procedure employed in method 1 provided pure 5 (1.1 g, 32%).
Gen er a l P r oced u r e for th e Rea ction of Am in es (4, 5,
13, 16, a n d 19) w ith P yr id in e-2-ca r boxa ld eh yd e a n d
Su bsequ en t Red u ction of Im in es w ith Na BH4. To a
solution of amines (1 mmol) in benzene (10 mL) was added
pyridine-2-carboxaldehyde (2 mmol). The resulting mixture
was refluxed using a Dean-Stark trap for 7 h. The reaction
mixture was cooled to room temperature and evaporated to
dryness to provide pure imine as determined by NMR. The
obtained imine was dissolved in EtOH (15 mL) and reacted
with NaBH4 (2 mmol). The resulting mixture was stirred at
room temperature for 24 h and filtered, and the filtrate was
concentrated in vacuo. The residue was dissolved in CH2Cl2
(15 mL), filtered, and dried (MgSO4), and the filtrate was
concentrated in vacuo.
N,N′-Bis(2-p yr id ylm eth yl)-N′′-tr ityl-cis,cis-1,3,5-tr ia m i-
n ocycloh exa n e (6). Pure 6 (477 mg, 86%) was thereby
obtained as a white solid: 1H NMR (CDCl3) δ 0.64-0.91 (m, 3
H), 1.28 (d, J ) 4.8 Hz, 2 H), 1.90 (d, J ) 6.1 Hz, 1 H), 2.02-
2.43 (m, 6 H), 3.56 (s, 4 H), 6.97-7.15 (m, 13 H), 7.35-7.51
(m, 8 H), 8.38 (d, J ) 3.8 Hz, 2 H); 13C NMR (CDCl3) δ 39.8
(t), 41.9 (t), 49.3 (t), 52.1 (t), 53.8 (t), 71.2 (s), 121.8 (d), 122.2
(d), 126.1 (d), 127.7 (d), 128.6 (d), 136.3 (d), 147.0 (d), 149.1
(d), 159.4 (d); HRMS (positive-ion FAB) calcd for C45H39N5 [M
+ H]+ m/z 554.3283, found [M + H]+ m/z 554.3312.
N-(2-P yr id ylm et h yl)-N′,N′′-b is-t r it yl-cis,cis-1,3,5-t r i-
a m in ocycloh exa n e (14). Pure 14 (656 mg, 93%) was thereby
obtained as a white solid: 1H NMR (CDCl3) δ 0.35-0.58 (m, 6
H), 0.64-0.80 (m, 3 H), 0.88-1.64 (m, 7 H), 1.84-2.08 (m, 4
H), 3.15 (s, 2 H), 6.92 (t, J ) 3.8 Hz, 1 H), 7.0-7.20 (m, 20 H),
7.42 (d, J ) 8.8 Hz, 12 H), 8.32 (d, J ) 3.8 Hz, 1 H); 13C NMR
(CDCl3) δ 41.4 (t), 45.5 (t), 49.4 (d), 51.8 (t), 53.6 (d), 71.1 (s),
121.6 (d), 121.9 (d), 126.0 (d), 127.9 (d), 128.6 (d), 136.0 (d),
147.0 (s), 149.0 (d), 159.7 (s); HRMS (positive-ion FAB) calcd
for C48H48N4 [M + H]+ m/z 705.3957, found [M + H]+ m/z
705.3945.
Exp er im en ta l Section
Gen er a l Meth od s. 1H, 13C, and APT NMR spectra were
obtained at 300 MHz in CDCl3 solution. Fast atom bombard-
ment mass spectra (FAB-MS) were obtained in the positive-
ion detection mode. Elemental microanalysis was performed
by Galbraith Laboratories, Knoxville, TN.
N,N′-Bis-tr ityl-cis,cis-1,3,5-tr iam in ocycloh exan e (4) an d
N-Tr ityl-cis,cis-1,3,5-tr ia m in ocycloh exa n e (5). To a solu-
tion of tach‚3HBr (9.30 g, 25.2 mmol)5 in H2O (150 mL) was
added NaOH (3.02 g, 75 mmol). The resulting solution was
concentrated in vacuo, and EtOH (60 mL) was added into the
residue. The resulting mixture was stirred for 5 min and put
into a freezer for 1 h. The mixture was filtered, and the filtrate
was concentrated in vacuo. The residue was dissolved into
CHCl3 and filtered, and the filtrate was concentrated in vacuo
to provide the free amine 3 (3.0 g, 94%).
Meth od 1. To a mixture of 3 (1.20 g, 9.3 mmol) and K2CO3
(990 mg, 9.3 mmol) in CHCl3 (30 mL) was added a solution of
TrCl (2.59 g, 9.3 mmol) in CHCl3 (20 mL) over 0.5 h. The
resulting mixture was refluxed for 2 h, at which time the
reaction mixture was cooled to room temperature and concen-
trated in vacuo. The residue was purified via column chroma-
tography on silica gel eluting with 10% CH 3OH-CH2Cl2. Pure
4 (1.79 g, 43%) was thereby obtained as a white solid: 1H NMR
(CDCl3) δ 0.86 (q, 2 H), 1.12-1.20 (m, 4 H), 1.70 (d, J ) 4.2
Hz, 2 H), 2.12-2.30 (m, 5 H), 7.22-7.45 (m, 18 H), 7.64 (d, J
) 8.4 Hz, 12 H); 13C NMR (CDCl3) δ 42.8 (t), 44.1 (t), 47.7 (d),
49.3 (d), 71.0 (s), 126.1 (d), 127.6 (d), 128.5 (d), 146.8 (s); HRMS
(positive-ion FAB) calcd for C42H43N3 [M + H]+ m/z 614.3535,
found [M + H]+ m/z 614.3536.
N-Eth yl-N,N′,N′′-tr is(2-p yr id ylm eth yl)-cis,cis-1,3,5-tr i-
a m in ocycloh exa n e (17). Pure 17 (379 mg, 88%) was thereby
obtained as a white solid: 1H NMR (CDCl3) δ 1.03 (t, J ) 9.0
MHz, 3 H), 1.17-1.29 (m, 4 H), 2.18 (d, J ) 4.5 Hz, 2 H), 2.26
(d, J ) 5.8 Hz, 1 H), 2.55-2.70 (m, 6 H), 3.79 (s, 2 H), 3.95 (s,
4 H), 7.10-7.18 (m, 2 H), 7.30 (d, J ) 6.7 Hz, 2 H), 7.54 (d, J
) 5.4 Hz, 1 H), 7.64 (tt, J ) 9.8 Hz, 4 H), 8.49 (d, J ) 2.7 Hz,
1 H), 8.55 (d, J ) 3.2 Hz, 2 H); 13C NMR (CDCl3) δ 14.3 (q),
35.6 (t), 40.4 (t), 44.9 (t), 52.5 (t), 54.2 (t), 56.2 (t), 56.4 (t),
121.4 (d), 121.8 (d), 122.2 (d), 136.2 (d), 136.4 (d), 148.6 (d),
149.1 (d), 159.7 (s), 162.0 (s); HRMS (positive-ion FAB) calcd
+
+
Further elution with CHCl3/MeOH/NH4OH at 12:4:1 pro-
vided pure 5 (690 mg, 20%) as a white solid: 1H NMR (CDCl3)
δ 0.83-1.0 (m, 4 H), 1.42 (d, J ) 6.3 Hz, 2 H), 1.86-2.60 (m,
8 H), 7.20-7.39 (m, 9 H), 7.75 (d, J ) 7.4 Hz, 6 H); 13C NMR
(CDCl3) δ 44.66 (t), 44.72 (t), 47.72 (d), 49.4 (d), 71.3 (s), 126.2
for C26H34N6 Mr m/z 431.2923, found Mr m/z 431.2932.
N,N′-Bis(2-p yr id ylm et h yl)-N′′-p r op yl-cis,cis-1,3,5-t r i-
a m in ocycloh exa n e (11). The residue was purified via flash
column chromatograpy eluting with triethylamine, methanol,
and CH2Cl2 at 3:15:100 to provide 11 (205 mg, 58%) as a
colorless oil: 1H NMR (CDCl3) δ 0.88 (t, J ) 9.2 Hz, 3 H), 0.98-
1.16 (m, 4 H), 1.38-1.57 (m, 2 H), 2.20-2.28 (m, 4 H), 2.50-
2.61 (m, 8 H), 3.91 (s, 4 H), 7.12 (t, J ) 6.0 Hz, 2 H), 7.26 (d,
(13) Wu, C.; Brechbiel, M. W.; Kozak, R. W.; Gansow, O. A. Bioorg.
Med. Chem. Lett. 1994, 4, 449.
8076 J . Org. Chem., Vol. 67, No. 23, 2002

1,3,5-cis,cis-Triaminocyclohexane N-Pyridyl Derivatives
J ) 7.5 Hz, 2 H), 7.61 (t, J ) 8.2 Hz, 2 H), 8.51 (d, J ) 6.0 Hz,
2 H); 13C NMR (CDCl3) δ 11.7 (q), 23.1 (t), 39.8 (t), 40.2 (t),
48.6 (t), 52.4 (t), 53.7 (d), 53.8 (d), 121.8 (d), 122.3 (d), 136.4
(d), 149.2 (d), 159.7 (s); HRMS (positive-ion FAB) calcd for
solution was adjusted to pH 7 with 5 M NaOH, washed with
CHCl3 (10 mL), and adjusted to pH 13 with 5 M NaOH. The
solvent was evaporated under high vacuum, and the residue
was dissolved in ethanol (20 mL), sonicated for 10 min, and
concentrated in vacuo. The residue was dissolved in CHCl3
(150 mL), dried (MgSO4), and filtered, and the filtrate was
concentrated in vacuo.
C
33H31N5 [M + H]+ m/z 354.2657, found [M + H]+ m/z
354.2658.
N-(4-P h t h a loylb u t yl)-N,N′,N′′-t r is(2-p yr id ylm et h yl)-
cis,cis-1,3,5-tr ia m in ocycloh exa n e (20). Pure 20 (537 mg,
89%) was thereby obtained as a white solid: 1H NMR (CDCl3)
δ 0.95-1.50 (m, 6 H), 1.80-2.04 (3 H), 2.20-2.42 (m, 6 H),
3.08 (t, J ) 7.1 Hz, 2 H), 3.58 (s, 2 H), 3.75 (s, 4 H), 4.40 (s, 2
H), 6.90-7.50 (m, 13 H), 8.19 (d, J ) 4.6 Hz, 2 H), 8.32 (d, J
) 5.3 Hz, 2 H); 13C NMR (CDCl3) δ 25.6 (t), 26.8 (t), 35.1 (t),
39.3 (t), 49.9 (t), 51.8 (t), 54.0 (t), 53.4 (d), 56.3 (d), 63.3 (t),
121.4 (d), 121.6 (d), 122.0 (d), 122.2 (d), 129.8 (d), 130.1 (d),
135.7 (s), 136.2 (d), 136.3 (d), 138.6 (s), 148.1 (d), 148.7 (d),
159.1 (s), 161.0 (s), 169.0 (s); HRMS (positive-ion FAB) calcd
for C36H41N7O2 [M + H]+ m/z 604.3486, found [M + H]+ m/z
604.3477.
Gen er a l P r oced u r e for Dep r otection of Tr ityl Gr ou p s
in 6, 15, a n d 18. CF3CO2H (2 mL) was slowly added to a
mixture of 6, 15, or 18 (1 mmol) in CHCl3 (1 mL) and CH3OH
(1 mL) at -5 °C. The resulting mixture was warmed to room
temperature and stirred for 48 h, at which time the mixture
was evaporated to dryness. H2O (10 mL) was added into the
residue, and the resulting mixture was extracted with CHCl3
(2 × 30 mL) to remove the triphenylmethane. The aqueous
solution was adjusted to pH 7 with 5 M NaOH, washed with
CHCl3 (10 mL), adjusted to pH 13, and extracted with CHCl3
(3 × 50 mL). The combined organic layers were dried (MgSO4)
and filtered, and the filtrate was concentrated in vacuo.
N,N′-Bis(2-p yr id ylm eth yl)-cis,cis-1,3,5-tr ia m in ocyclo-
h exa n e (7). Pure 7 (259 mg, 83%) was thereby obtained as a
colorless oil: 1H NMR (CDCl3) δ 0.95-1.20 (m, 6 H), 1.92 (s,
2 H), 2.02-2.20 (m, 3 H), 2.50-2.78 (m, 2 H), 3.87 (s, 4 H),
7.08 (t, J ) 6.0 Hz, 2 H), 7.24 (t, J ) 7.6 Hz, 2 H), 7.54 (t, J )
7.6 Hz, 2 H), 8.48 (d, J ) 6.0 Hz, 2 H); 13C NMR (CDCl3) δ
39.8 (t), 43.5 (t), 47.6 (d), 52.3 (t), 53.6 (d), 121.7 (d), 122.2 (d),
136.3 (d), 149.1 (d), 159.6 (s); HRMS (positive-ion FAB) calcd
for C18H25N5 [M + H]+ m/z 312.2188, found [M + H]+ m/z
312.2179.
N-Eth yl-N,N′N′′-tr is(2-p yr id ylm eth yl)-cis,cis-1,3,5-tr i-
a m in ocycloh exa n e (16). Pure 16 (209 mg, 84%) was thereby
obtained as a colorless oil: 1H NMR (CDCl3) δ 0.85-1.13 (m,
8 H), 1.97 (d, J ) 6.6 Hz, 4 H), 2.54-2.77 (m, 6 H), 3.77 (s, 2
H), 7.12 (t, J ) 6.3 Hz, 1 H), 7.55 (d, J ) 3.8 Hz, 1 H), 7.64 (t,
J ) 6.6 Hz, 1 H), 8.49 (d, J ) 2.5 Hz, 1 H); 13C NMR (CDCl3)
δ 14.2 (q), 38.5 (t), 44.9 (t), 46.8 (t), 48.1 (d), 56.1 (t), 56.4 (d),
121.5 (d), 122.2 (d), 136.2 (d), 148.6 (d), 162.0 (s); HRMS
(positive-ion FAB) calcd for C14H24N4 [M + H]+ m/z 249.2079,
found [M + H]+ m/z 249.2076.
N-(4-P h th a loylbu tyl)-N-(2-p yr id ylm eth yl)-cis,cis-1,3,5-
tr ia m in ocycloh exa n e (19). Pure 19 (236 mg, 56%) was
thereby obtained as a colorless oil: 1H NMR (CDCl3) δ 0.58
(dd, J ) 10.3 Hz, 1 H), 0.79 (dd, J ) 10.3 Hz, 2 H), 1.0-1.18
(m, 5 H), 1.32-1.42 (m, 2 H), 1.65 (d, J ) 11.3 Hz, 3 H), 2.20-
2.43 (m, 5 H), 3.35 (t, J ) 7.2 Hz, 2 H), 3.52 (s, 2 H), 6.75 (t,
J ) 3.1 Hz, 1 H), 7.23 (t, J ) 7.2 Hz), 7.25-7.43 (m, 3 H), 7.52
(d, J ) 9.3 Hz, 2 H), 8.15 (d, J ) 4.1 Hz, 1 H); 13C NMR (CDCl3)
δ 25.6 (t), 25.8 (t), 37.4 (t), 37.7 (t), 45.9 (t), 47.7 (d), 49.7 (t),
56.2 (d), 56.4 (t), 121.2 (d), 121.9 (d), 122.7 (d), 131.6 (s), 133.4
(d), 136.1 (d), 148.1 (d), 161.3 (s), 167.9 (s); HRMS (positive-
ion FAB) calcd for C24H31N5O2 [M + H]+ m/z 422.2556, found
[M + H]+ m/z 422.2549.
Gen er a l P r oced u r e for Dep r otection of Tr ityl Gr ou p s
in Com p ou n d s 8 a n d 12. CF3CO2H (4 mL) was slowly added
to a mixture of 8 or 12 (2 mmol) in CHCl3 (2 mL) and CH3OH
(2 mL) at -5 °C. The resulting mixture was warmed to room
temperature and stirred for 48 h, at which time the mixture
was evaporated to dryness. H2O (10 mL) was added to the
residue, and the resulting mixture was extracted with CHCl3
(2 × 30 mL) to remove the triphenylmethane. The aqueous
N,N′-Bis(2-p yr id yl)-N′′-p r op ion yl-cis,cis-1,3,5-t r ia m i-
n ocycloh exa n e (9). Pure 9 (297 mg, 80%) was thereby
obtained as a colorless oil: 1H NMR (CDCl3) δ 0.95 (dd, J )
10.8 Hz, 4 H), 1.15 (t, J ) 8.4 Hz, 3 H), 1.56 (s, 4 H), 1.97-
2.28 (m, 4 H), 2.78-2.96 (m, 2 H), 3.90-4.00 (m, 1 H), 5.88 (d,
J ) 4.2 Hz, 1 H); 13C NMR (CDCl3) δ 9.78 (q), 29.6 (t), 42.2 (t),
43.9 (t), 45.1 (t), 45.4 (d), 47.2 (t), 172.7 (s); HRMS (positive-
ion FAB) calcd for C9H19N3O [M + H]+ m/z 186.1606, found
[M + H]+ m/z 186.1602.
N-P r op yl-cis,cis-1,3,5-tr ia m in ocycloh exa n e (13). Pure
13 (303 mg, 88%) was thereby obtained as a colorless oil: 1H
NMR (CDCl3) δ 0.62-0.80 (m, 5 H), 0.82-1.42 (m, 8 H), 1.90
(t, J ) 12.0 Hz, 3 H), 2.36-2.52 (m, 3 H), 2.55-2.72 (m, 2 H);
13C NMR (CDCl3) δ 11.7 (q), 23.4 (t), 43.3 (t), 47.0 (t), 47.7 (d),
49.0 (t), 53.9 (d); HRMS (positive-ion FAB) calcd for C9H21N3
[M + H]+ m/z 172.1814, found [M + H]+ m/z 172.1812
N-P r op ion yl-N′,N′′-bistr ityl-cis,cis-1,3,5-tr ia m in ocyclo-
h exa n e (8). To a solution of 4 (1.76 g, 2.88 mmol) and Et3N
(0.4 mL, 2.88 mmol) in CH2Cl2 (30 mL) at -20 °C was slowly
added propionyl chloride (0.25 mL, 2.88 mL). The resulting
mixture was stirred for 10 min at the same temperature and
purified on a silica gel column sequentially eluting with hexane
and 20% EtOAc in hexane. Pure 8 (980 mg, 51%) was thereby
obtained as colorless oil: 1H NMR (CDCl3) δ 0.76 (dd, J ) 10.4
Hz, 4 H), 0.91-1.50 (m, 7 H), 1.78-2.30 (m, 6 H), 7.24-7.41
(m, 18 H), 7.53 (d, J ) 8.4 Hz, 12 H); 13C NMR (CDCl3) δ 10.1
(q), 30.0 (t), 41.3 (t), 43.9 (t), 45.5 (d), 49.4 (d), 71.2 (s), 126.3
(d), 127.9 (d), 128.7 (d), 147.0 (s), 172.2 (s). Anal. Calcd for
C
47H49N3O: C, 84.02; H, 7.35. Found: C, 84.18; H, 7.23.
N,N′-Bis(2-p yr id ylm et h yl)-N′′-p r op ion yl-cis,cis-1,3,5-
tr ia m in ocycloh exa n e (10). To a solution of 9 (223 mg, 1.21
mmol) in EtOH (20 mL) was added pyridine-2-carboxaldehyde
(258 mg, 2.42 mmol). The resulting mixture was refluxed for
4 h. The reaction mixture was cooled to room temperature and
evaporated to dryness to provide pure imine as determined
by NMR. The residue was dissolved in EtOH (20 mL) and was
reacted with NaBH4 (183 mg, 4.84 mmol). The resulting
mixture was stirred at room temperature for 24 h and filtered,
and the filtrate was evaporated to dryness. The residue was
dissolved in CH2Cl2 (10 mL), dried (MgSO4), and filtered. The
filtrate was concentrated in vacuo to provide compound 10 (415
mg, 93%) as a yellow oil: 1H NMR (CDCl3) δ 0.88-1.02 (m, 7
H), 1.65-1.82 (m, 2 H), 1.97-2.10 (m, 5 H), 2.43-2.55 (m, 2
H), 3.75 (s, 4 H), 6.12 (d, J ) 4.2 Hz, 1 H), 6.98 (t, J ) 5.6 Hz,
1 H), 7.10 (d, J ) 4.5 Hz, 1 H), 7.46 (t, J ) 7.5 Hz, 1 H), 8.36
(d, J ) 3.8 Hz, 1 H); 13C NMR (CDCl3) δ 9.6 (q), 29.3 (t), 39.3
(t), 39.5 (t), 44.9 (d), 51.9 (t), 53.0 (d), 121.6 (d), 121.9 (d), 136.1
(d), 148.8 (d), 159.1 (s), 172.7 (s). HRMS (positive-ion FAB)
calcd for C20H29N5O [M + H]+ m/z 368.2450, found [M + H]+
m/z 368.2447.
LiAlH4 Red u ction of 10. To a solution of 10 (210 mg, 0.57
mmol) in THF (6 mL) at 0 °C was added LiAlH4 (43 mg, 1.13
mmol). The resulting mixture was stirred for 0.5 h and warmed
to room temperature. The resulting mixture was refluxed for
18 h and quenched with MeOH (1 mL) at 0 °C. The resulting
mixture was filtered, and the filtrate was concentrated in
vacuo. The residue was purified via column chromatography
on silica gel eluting with Et3N/MeOH/CH2Cl2 at 3:15:100 to
provide pure 11 (12 mg, 6%) as a slightly brown oil.
Bor a n e Red u ction of 10. To a solution of 10 (190 mg, 0.52
mmol) in THF (5 mL) at 0 °C was added 1 M BH3-THF (1
mL, 1.04 mmol) over 20 min. The resulting mixture was stirred
for 2 h at 0 °C and warmed to room temperature. The resulting
mixture was refluxed for 18 h, cooled to room temperature,
and evaporated to dryness. HCl (6 M, 1 mL) was added into
J . Org. Chem, Vol. 67, No. 23, 2002 8077

Chong et al.
the residue, and the resulting solution was refluxed for 1 h
and evaporated to dryness. NaOH (5 M) was added to the
mixture adjusting pH to 13. The resulting solution was
evaporated, and the residue was extracted with CHCl3 (2 ×
30 mL). The combined organic layers were dried (MgSO4),
filtered, and concentrated in vacuo to provide 7 (140 mg, 88%)
as a colorless oil.
N-(4-Am in obu tyl)-N,N′,N′′-tr is(2-pyr idylm eth yl)-cis,cis-
1,3,5-tr ia m in ocycloh exa n e (21). Compound 20 (350 mg, 0.58
mmol) was dissolved in 5 M HCl (5 mL), and the resulting
mixture was refluxed for 5 h, cooled to 5 °C, and filtered. The
filtrate was adjusted to pH 7 with 5 M NaOH and washed with
CHCl3 (10 mL). The aqueous solution was adjusted to pH 13
with 5 M NaOH. The resulting solution was extracted with
CHCl3 (30 mL), dried (MgSO4), and filtered. The filtrate was
concentrated in vacuo to provide 21 (206 mg, 75%) as a
colorless oil: 1H NMR (CDCl3) δ 0.94-1.40 (m, 6 H), 1.75-
2.25 (m, 8 H), 2.40-2.64 (m, 6 H), 3.75 (s, 2 H), 3.92 (s, 4 H),
7.11-7.16 (m, 3 H), 7.41-7.62 (m, 4 H), 8.45-8.56 (m, 5 H);
13C NMR (CDCl3) δ 26.2 (t), 31.3 (t), 35.5 (t), 40.3 (t), 41.9 (t),
50.6 (t), 52.5 (t), 54.5 (d), 56.6 (d), 56.8 (t), 121.6 (t), 121.9 (d),
122.3 (d), 136.3 (d), 136.4 (d), 148.6 (d), 149.2 (d), 159.7 (s),
161.8 (s); HRMS (positive-ion FAB) calcd for C28H39N7 [M +
H]+ m/z 474.3345, found [M + H]+ m/z 474.3330.
Com p ou n d 24. To a solution of amine 21 (31.7 mg, 0.067
mmol) in anhydrous DMF (2 mL) was added 2313 (18.8 mg,
0.067 mmol). The resulting mixture was stirred for 18 h at
room temperature, and solvent was removed via high vacuum
pump at room temperature. The residue was purified via flash
chromatograpy eluting with triethylamine, methanol, and CH2-
Cl2 at 1:38:460 to provide 24 (38 mg, 88%) as a colorless oil:
1H NMR (CDCl3) δ 0.90-1.41 (m, 10 H), 1.70-1.87 (m, 2 H),
1.97-2.20 (m, 4 H), 2.38-2.50 (m, 6 H), 3.05 (q, J ) 5.2 Hz, 2
H), 0.3.41 (t, J ) 5.3 Hz, 2 H), 3.64 (s, 2 H), 3.82 (s, 4 H),
5.90-5.95 (m, 1 H), 6.55 (s, 2 H), 6.98-7.05 (m, 3 H), 7.16 (d,
J ) 6.9 Hz, 2 H), 7.35 (d, J ) 6.9 Hz, 1 H), 7.49-7.54 (m, 3
H), 8.35 (d, J ) 4.6 Hz, 1 H), 8.41 (d, J ) 4.6 Hz, 1 H); 13C
NMR (CDCl3) δ 24.9 (t), 25.9 (t), 27.3 (t), 33.6 (t), 35.3 (t), 37.1
(t), 39.3 (t), 39.9 (t), 50.3 (t), 52.2 (t), 54.2 (t), 56.6 (t), 56.7 (t),
121.7 (t), 122.0 (d), 122.4 (d), 122.5 (d), 134.1 (d), 136.5 (d),
136.6 (d), 148.7 (d), 149.2 (d), 159.2 (d), 161.5 (s), 170.9 (s),
171.6 (s); HRMS (positive-ion FAB) calcd for C36H48N8O3 [M
+ H]+ m/z 638.3801, found [M + H]+ m/z 638.3771.
N-P r op yl-N′,N′′-b is-t r it yl-cis,cis-1,3,5-t r ia m in ocyclo-
h exa n e (12). To a solution of 4 (612 mg, 1 mmol) in MeOH
(25 mL) at room temperature were added propionaldehyde (46
mg, 0.8 mmol) and NaBH3CN (63 mg, 1 mmol). The resulting
mixture was stirred for 14 h. The mixture was dissolved into
water (75 mL), the pH was adjusted to pH 10 with 0.5 N
NaOH, and the solution was extracted with CHCl3 (2 × 100
mL). The combined organic layers was dried (MgSO4), filtered,
and evaporated in vacuo. The residue was purified via silica
gel column chromatograpy eluting with 3% MeOH in CH2Cl2
to provide 12 (438 mg, 68%) as a colorless oil: 1H NMR (CDCl3)
δ 0.46-0.60 (m, 2 H), 0.81-0.91 (m, 4 H), 1.15-1.56 (m, 6 H),
1.74 (t, J ) 10.7 Hz, 1 H), 2.00-2.38 (m, 6 H), 7.24-7.38 (m,
18 H), 7.62 (d, J ) 8.6 Hz, 12 H); 13C NMR (CDCl3) δ 11.5 (q),
22.5 (t), 41.2 (t), 45.5 (t), 48.4 (t), 49.5 (d), 53.8 (d), 71.2 (s),
126.1 (d), 127.7 (d), 128.6 (d), 147.1 (s); HRMS (positive-ion
FAB) calcd for C47H49N3 [M + H]+ m/z 656.4037, found [M +
H]+ m/z 656.4005.
N-Eth yl-N-(2-pyr idylm eth yl)-N′,N′′-bistr ityl-cis,cis-1,3,5-
tr ia m in ocycloh exa n e (15). To a mixture of 14 (170 mg, 0.24
mmol) and Na2CO3 (26 mg, 0.24 mmol) in CH3CN (2 mL) was
added diethyl sulfate (37 mg, 0.24 mmol). The resulting
mixture was stirred at room temperature for 24 h and filtered,
and the filtrate was evaporated in vacuo. The residue was
purified on basic alumina eluting with 15% EtOAc-hexane
and 25% EtOAc-hexane to provide 15 (240 mg, 80%) as a
colorless oil: 1H NMR (CDCl3) δ 0.35-0.60 (m, 5 H), 0.81-
1.04 (m, 3 H), 1.15-1.63 (m, 4 H), 1.80-2.10 (m, 4 H), 3.08 (s,
2 H), 6.95 (t, J ) 5.4 Hz, 1 H), 7.03-7.18 (m, 20 H), 7.42 (d, J
) 8.8 Hz, 12 H), 8.30 (d, J ) 3.8 Hz, 1 H); 13C NMR (CDCl3)
δ 13.9 (q), 37.4 (t), 44.7 (t), 46.1 (t), 50.1 (t), 55.6 (d), 56.2 (d),
71.2 (s), 121.2 (d), 122.1 (d), 126.2 (d), 127.7 (d), 128.6 (d), 136.0
(d), 147.2 (s), 148.4 (s), 162.5 (s); positive-ion FAB calcd for
Cytotoxicity Assa y. HeLa cells were obtained from the
American Type Culture Collection and cultured at 37 °C in a
humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s
medium with high glucose (4.5 g/L) (DMEM) supplemented
with 10% fetal bovine serum (FBS). Cells were seeded at 2000
cells/well in 96-well microtiter plates (Costar, Cambridge, MA)
and allowed to attach overnight. The next day, chelators were
added at the indicated concentrations. After 72 h, viability was
measured using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) dye reduction assay, as previ-
ously described.10
C
52H52N4 [M + H]+ m/z 733.01, found [M + H]+ m/z 733.42.
N-(4-P h t h a loylb u t yl)-N,N′,N′′-t r is(2-p yr id ylm et h yl)-
cis,cis-1,3,5-tr ia m in ocycloh exa n e (18). To a solution of 14
(2.03 g, 2.88 mmol) in CH3CN (10 mL) were added K2CO3 (796
mg, 5.76 mmol) and N-(4-bromobutyl)phthalimide (813 mg,
2.88 mmol). The resulting mixture was refluxed for 36 h, cooled
to room temperature, and filtered. The filtrate was concen-
trated in vacuo, and the residue was purified on silica gel
column sequentially eluting with 20% EtOAc-hexane and 50%
hexanes-EtOAc to provide 18 (2.11 g, 81%) as a colorless oil:
1H NMR (CDCl3) δ 1.03-1.75 (m, 6 H), 1.90-2.25 (m, 3 H),
3.22 (s, 2 H), 3.50 (t, J ) 3.6, 2 H), 4.01-4.14 (m, 2 H), 7.03-
7.28 (m, 40 H), 7.50-7.78 (m, 38 H), 8.38 (d, J ) 3.6 Hz, 1 H);
13C NMR (CDCl3) δ 25.6 (t), 25.9 (t), 36.8 (t), 37.7 (t), 46.3 (t),
49.5 (t), 50.0 (d), 56.2 (t), 56.5 (d), 71.2 (t), 121.2 (d), 121.9 (d),
123.1 (d), 126.2 (d), 127.7 (d), 128.6 (d), 132.0 (s), 133.8 (d),
136.1 (d), 147.1 (s), 148.3 (d), 162.1 (s), 168.2 (s); HRMS
(positive-ion FAB) calcd for C60H59N5O2 [M + H]+ m/z 904.4951,
found [M + H]+ m/z 904.4566.
Ack n ow led gm en t. We thank the structural mass
spectra group (Dr. L. Pannell, NIDDK, Bethesda, MD)
for obtaining HRMS spectra of all the compounds. This
work was supported in part by Grant No. DK 57781
(S.V.T) from the National Institutes of Health.
Su p p or tin g In for m a tion Ava ila ble: Copies of the 1H and
13C NMR spectra for all prepared compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
J O0204911
8078 J . Org. Chem., Vol. 67, No. 23, 2002