Synthesis and biological evaluation of s-triazine substituted polyamines as potential new anti-Trypanosomal drugs

Article abstract of DOI:10.1021/jm010854+

The P2 transporter is a nucleoside transporter which is unique to the protozoan parasite Trypanosoma brucei, the causative organism of Human African Trypanosomasis. The transporter has been shown to bind some structural motifs not recognized by other tran

Full text of DOI:10.1021/jm010854+

3440
J . Med. Chem. 2001, 44, 3440-3452
Syn th esis a n d Biologica l Eva lu a tion of s-Tr ia zin e Su bstitu ted P olya m in es a s
P oten tia l New An ti-Tr yp a n osom a l Dr u gs
Burkhard Klenke,† Mhairi Stewart,‡ Michael P. Barrett,‡ Reto Brun,§ and Ian H. Gilbert*,†
Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, U.K.,
Institute of Biomedical and Life Science, Division of Infection and Immunity, University of Glasgow, Glasgow G12 8QQ, U.K.,
and Swiss Tropical Institute, Socinstrasse, CH-4002 Basel, Switzerland
Received February 26, 2001
The P2 transporter is a nucleoside transporter which is unique to the protozoan parasite
Trypanosoma brucei, the causative organism of Human African Trypanosomasis. The trans-
porter has been shown to bind some structural motifs not recognized by other transporters. In
this paper we describe the use of the melamine motif, a substrate of the P2 transporter, as a
potential tool to selectively deliver polyamine analogues to the parasites. The synthesis of a
number of polyamine analogues attached to a variety of melamine analogues is described. Many
of the compounds were shown to competitively inhibit uptake of adenosine, indicating that
they are recognized by the transporter. Some of the compounds showed good in vitro activity
against the parasites.
In tr od u ction
Parasitic diseases cause huge suffering in many parts
of the world. However, comparatively little research is
done in the field, and available funds for further
investigations reflect by no means the number and
seriousness of cases.1 African sleeping sickness (Human
African Trypanosomiasis, HAT) has become resurgent
at a time when current chemotherapy is failing.2
According to the World Health Organization (WHO),3
more than 60 million people are at risk of HAT with an
estimated number of 0.3-0.5 million infected people and
about 40 000 deaths reported in 1998. The disease is
lethal when untreated. There are no vaccines avail-
able, and the treatment, once the parasite has crossed
the blood-brain barrier, relies on the use of Melarso-
prol and eflornithine when available at this time.2,4
Melarsoprol is an arsenic based drug with low chemo-
therapeutic index,5 which needs to be administered
intravenously under medical observation. Severe side
effects (myocardial damage, hypertension, exfoliative
dermatitis, and reactive encephalopathy)6 as well as
resistance7 has been reported. Therefore, there is clearly
an urgent need for a new generation of drugs.
F igu r e 1. P2 Recognition Motif
toxicity may be brought about by selective drug uptake
through nutrient transporters in trypanosomes.11
Carter and Fairlamb characterized a unique nucleo-
side transporter in trypanosomes by screening about
190 purine and pyrimidine analogues as substrates for
the transporter.12 The recognition motif for the trans-
porter has been elucidated (Figure 1) and is found on
numerous reagents including aromatic amidines and
amino substituted 1,3,5-triazines as well as adenine and
adenosine.13,14 1,3,5-Triazines are generally not recog-
nized by mammalian nucleoside transporters and should
therefore offer excellent selectivity between host cells
and parasites.7c,14 Furthermore, a 2,4-diamino-1,3,5-
triazine derivative was recently reported as a new lead
against trypanosomiasis.15 Since the 1,3,5-triazine de-
rivatives are selectively taken up by T. brucei, it should
be possible to selectively transport or target cytotoxic
agents to the parasite. In the work described here,
polyamines were selected as the cytotoxic agent.
Polyamines have a variety of essential roles within
the cell where they are involved in growth and dif-
ferentiation.16,17 Polyamine analogues show promise as
anticancer agents, antiparasitic agents, antidiarrhoeals,
anti-HIV agents, metal chelators, and as gene delivery
agents. Polyamine metabolism is known to be a drug
target in African trypanosomes as inhibition of the
initial polyamine biosynthesis enzyme, ornithine decar-
boxylase, by DL-R-difluoromethylornithine (DFMO) is
HAT is caused by the two subspecies Trypanosoma
brucei rhodesiense and Trypanosoma brucei gambiense.
They live in body fluids including the blood and lymph
and, in later stages, the cerebrospinal fluid, where they
grow and divide by binary fission.8 The rational ap-
proach toward the development of new potential chemo-
therapeutics focuses on differences in biochemistry and
metabolism between the human host and the causative
agent.9 However, the biochemistry and metabolism in
trypanosomes are complex and only fragmentarily
investigated.10 In this paper we discuss how selective
* To whom correspondence should be addressed. Tel: +44 29 2087
5800. Fax: +44 29 2087 4149. E-mail: gilbertih@cf.ac.uk.
† Cardiff University.
‡ University of Glasgow.
§ Swiss Tropical Institute.
10.1021/jm010854+ CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/18/2001

Synthesis of s-Triazine Substituted Polyamines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3441
mixtures of mono (17%, 28%, 33%) and bis-protected
products (3d -f; 78%, 68%, 64%), whereas compounds
2a -c were cleanly converted into the bis-protected
products 3a , 3b, and 3c in 97%, 85%, and 76% yield. In
a third step, the reduction of the BOC-protected nitriles
3a -f completed the synthesis of the polyamine back-
bones (4a -f).
The catalytic hydrogenation of 3c-f turned out to be
much more complicated than expected.20 However, the
reduction was finally achieved by catalytic hydrogena-
tion over palladium activated Raney-nickel in dioxane/
water with LiOH as base, and the polyamines 4a -f
were isolated in 84-94% yield.
F igu r e 2. Target Compounds: Lead Structure A
To obtain the first set of compounds (5a -f), carrying
only free amino groups on the triazine units, polyamines
4a -f were arylated with 2-chloro-4,6-diamino-1,3,5-
triazine in ethanol/water (1:1) at 80 °C overnight. In the
absence of water, virtually no reaction took place; also,
using polar aprotic solvents (DMSO, DMF, MeCN) the
reaction was found to be impractically slow. Increasing
the reaction temperature above 80 °C resulted in partial
BOC-deprotection, which left the uncertainty of regio-
selectivity during further arylation. Monitoring the
reaction by mass spectroscopy indicated complete con-
version of the starting material (4a -f) when 2.2 equiv
of arylating agent were used; 2-hydroxy-4,6-diamino-
1,3,5-triazine was detected as a byproduct (MS). The
crude arylated, BOC-protected intermediates were di-
rectly deprotected using TFA at room temperature with
anisole as tert-butyl cation scavenger. Purification was
carried out by ion-exchange chromatography on the
strongly acidic resin Dowex 50WX2-200, using a rough
gradient of aqueous HCl-solution as eluent. The target
compounds 5a -f were isolated as tetrahydrochloric salts
in 87-92% yield after recrystallization from ethanol/
ether.
Finding compound 5c to be the most active within the
first set (compare Table 1), we further investigated the
influence of differently substituted 1,3,5-triazine units
in the central core C12-series. For comparison, ana-
logues of the central core C9-series were also prepared
(Scheme 2).
Using the same strategy and similar reaction condi-
tions as for the preparation of 5a -f, we arylated
polyamines 4b and 4c with four different methylamino
substituted 2-chloro-1,3,5-triazines. The arylating agents
were prepared in one or two steps from cyanuric chloride
by slightly modified literature procedures.21 They turned
out to be significantly less reactive than the unsubsti-
tuted 2-chloro-4,6-diamino-1,3,5-triazine, and use of up
to 4 equiv of arylating agent was necessary to complete
the reaction. After BOC-deprotection, ion-exchange
chromatography, and recrystallization (as above), the
methylamino substituted target compounds 6a -h were
isolated as tetra- or pentahydrochloric salts in 32-83%
yield.
toxic to these cells.10,18 DFMO toxicity may be partly
related to the fact that these parasites lack transporters
for polyamines and thus cannot salvage sufficient
quantities of polyamines from the plasma to bypass
inhibition of polyamine synthesis brought about by ODC
inhibition. Furthermore, since the unique thiol try-
panothione, a spermidine bridged bis-glutathione, is
crucial in the parasite’s defense against oxidative stress
and redox maintenance, polyamines have an additional
vital role in trypanosomes.19 Polyamine transporters
would represent ideal portals of entry for other polyamine
analogues with potential cytotoxic activity. However,
their absence from bloodstream form T. brucei10 means
that alternative routes to target polyamine analogues
to these cells are required. The P2 amino-purine trans-
porter offers the potential to target polyamine analogues
to trypanosomes.
Therefore we aimed to design, synthesize, and evalu-
ate substituted polyamines, carrying 1,3,5-triazine units,
as potential anti-trypanosomal drugs. Preliminary re-
sults13 indicated that this route might be successful, and
lead structure A (Figure 2) was used as a starting point
for two series of analogues. In a first series, the influence
of structural changes of the central core unit was
investigated. In a second series, the effect of additional
methyl substituents on the 1,3,5-triazine was studied.
Here we report the synthesis of the envisaged molecules
and their structure-activity relationship with respect
to their interaction with the P2 transporter and their
activity against Trypanosoma brucei sub-species. Activ-
ity was also determined against the related parasitic
organism Trypanosoma cruzi which does not possess a
P2 transporter.
Ch em istr y
The synthesis of polyamines can be accomplished
using very different strategies.16,17 We used a three-step
procedure (Scheme 1) to form the polyamine backbones,
which were then reacted with suitable 1,3,5-triazines
(Schemes 1, 2, and 3). In a first step, commercially
available alkyl diamines (1a -f) were treated with 2
equiv of acrylonitrile in ethanol at room temperature
to undergo a double Michael addition reaction. The
desired bis-adducts 2a -f were isolated in 62-92% yield
after recrystallization, distillation, or column chroma-
tography. No formation of mono Michael adducts was
observed. Compounds 2a -f were then BOC-protected
under standard conditions using BOC-anhydride in
MeOH at room temperature with TEA as additional
base. In the presence of only 2 equiv of BOC-anhydride,
the sterically more demanding substrates 2d -f formed
Utilizing the comparably low reactivity of the aryl-
ating agents used for the preparation of the bistriazine
substituted compounds 6a -h , we further investigated
the synthesis of monotriazine substituted analogues
(7a -e) without the use of additional protecting group
chemistry (Scheme 3). When polyamines 4b and 4c were
reacted with 2 equiv of a dialkylamino substituted
2-chloro-1,3,5-triazine, MS analysis indicated the highly

3442 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Sch em e 1. Variation of Core Units
Klenke et al.
Sch em e 2. Variation of Triazine Units within the C9 and C12 Series, Bis Substitution
Sch em e 3. Variation of Triazine Units within the C9 and C12 Series, Mono Substitution
selective formation of monoadducts within 15-20 h.
After BOC-deprotection, ion-exchange chromatography,
and recrystallization (as above), the monosubstituted
target compounds 7a , 7d , and 7e were isolated as
pentahydrochloric salts in 51-65% yield. Analogue
reactions with 2-chloro-1,3,5-triazines carrying at least
one free NH2 group were much more difficult to control,
and the formation of bis substituted byproducts could
not be avoided. After BOC-deprotection, ion-exchange
chromatography, and recrystallization (as above) the

Synthesis of s-Triazine Substituted Polyamines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3443
Sch em e 4. Variation of Triazine Units within the C9 and C12 Series, Additional Methylene Bridge
Sch em e 5. Non-Polyamine Analogues
monosubstituted compounds 7b and 7c were isolated
as tetrahydrochloric salts in a 85:15 and 90:10 ratio (1H
NMR) together with their bis substituted analogues 6e
and 6f.
While in compounds 5a -f, 6a -h , and 7a -e the 1,3,5-
triazine units are directly linked to the polyamine
backbone, we were also interested in attaching the
triazine units via an additional methylene spacer. We
reacted polyamines 4b and 4c with aminosubstituted
2-bromomethyl-1,3,5-triazines (Scheme 4), which were
prepared from sodium dicyanamide or dicyandiamide
via substituted biguanides adapting known literature
procedures.22 The stoichiometric reaction between the
amino substituted 2-bromomethyl-1,3,5-triazines and
polyamines 4b and 4c led to mixtures of mono, bis, tris,
and tetra alkylated products with bis and tetra adducts
dominating. By addition of 4-5 equiv of alkylating
agent, the reaction proceeded to the BOC-protected tetra
addition adducts. Bis and tris adducts were found only
as minor byproducts according to MS analysis. Use of a
larger excess of alkylating agent led to the formation of
penta- and hexasubstituted ammonium salts. After
BOC-deprotection, ion-exchange chromatography, and
recrystallization (as above), the aminosubstituted
methylene-bridged target compounds 8a -f were iso-
lated as heptahydrochloric salts in 36-88% yield.
the N:Cl ratio. Dependent on their pKa values and the
pH of the surrounding solvent, the polyamine targets
may exist in different protonated forms.
Biologica l Da ta
All compounds were assayed for their ability to inhibit
adenosine uptake by the P2 transporter which gives an
indication of the affinity between the transporter and
these molecules (Table 1). In vitro toxicity against intact
bloodstream form trypomastigotes (the clinically rel-
evant stage) of Trypanosoma brucei brucei and T. b.
rhodesiense was also measured. All compounds were
also assayed for their activity against the related
parasite T. cruzi (amastigotes), which is the causative
agent for Chagas’ disease. Since these parasites are
intracellular, reagents cannot reach their plasma mem-
branes without first traversing the membrane of the
host cells in which they reside, thus precluding the
possibility of selective activity through selective uptake.
Two nonpolyamine substituted triazines (9 and 10)
were prepared as additional controls to ascertain the
necessity of the polyamine functionality (Scheme 5).
Starting from 1,12-diaminododecane (1c) and 1-amino-
octadecane (1g), reaction with 2 equiv of an amino-
substiuted 2-chloro-1,3,5-triazine afforded compounds
9 and 10 in 76% and 36% yield after crystallization from
ethanol/water (9) or column chromatography (10).
All synthesized intermediates (2a -f, 3a -f, 4a -f) and
final products (5a -f, 6a -h , 7a -e, 8a -f, 9, 10) were
characterized by their 1H NMR, 13C NMR, and mass
spectra. Electrospray MS analysis of the target com-
pounds (hydrochloric salts) displayed the M+H+ peak
of the free base, and HRMS was performed as a proof
of identity. Water and solvent residues were strongly
retained by the compounds, which was reflected in the
combustion analysis. The microanalytical data was used
to determine the number of HCl units per molecule via
In t er a ct ion w it h t h e P 2 Tr a n sp or t er in T.
br u cei. All compounds showed dose dependent inhibi-
tion of adenosine uptake via the P2 transporter. Most
target compounds displayed a moderate affinity toward
the P2 transporter with apparent Ki values in the range
of 3-70 µM. Compounds 5d and 5f were an exception,
showing only weak P2 transporter affinity with appar-
ent Ki ) 163 µM and 125 µM. Compounds 5c, 6a , 6e,

3444 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Ta ble 1. Activities of Compounds
Klenke et al.
L-6 rat
P2 affinity
T. b. rhodesiense
T. b. brucei
T. cruzi
myoblast cells
compd
Ki (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
5a (4)a
5b (4)
5c (4)
5d (4)
5e (4)
5f (4)
6a (4)
6b (5)
6c (4)
6d (5)
6e (5)
6f (5)
6g (5)
6h (5)
7a (5)
7b (4)
7c (4)
7d (5)
7e (5)
8a (7)
8b (7)
8c (7)
8d (7)
8e (7)
8f (7)
9b (0)
10b (0)
72
19
0.50
163
55
125
0.75
10
160
128
16
>163
>152
>152
16
>100
>100
>100
>100
>100
>100
53
>160
>140
>13
>163
>152
>152
>135
>123
>130
16
116
9.7
18
0.80
23
353
nd
295
362
338
338
285
274
289
37.7
nd
9.6
17
1.2
9.8
3.5
2.3
0.81
0.94
1.1
0.94
0.58
1.3
4.02
0.265
0.265
0.57
0.44
0.18
13
40
4.0
32
2.0
14
29
0.135
0.24
3.2
2.5
17
0.41
1.0
25
>100
50.9
143
6.8
17
1.7
84
135.2
127
39.9
57.7
14.2
220
177
177
188
105
76.6
239
48.2
>200
>154
20
20
14.7
32
3.0
0.1
21
2.0
2.9
32
31
0.6
82
17
0.71
>88
54.8
76.8
>85
>77
>77
6.0
30
6.7
4.6
2.4
-
14
12
a
b
Number of HCl units used in the calculation. Poor solubility. Controls: T. b. rhodesiense, melarsoprol IC50 ) 6.1 nM; T. cruzi,
benznidazole, IC50 ) 3.3 µM; T. b. rhodesiense, strain STIB 900; T. b. brucei, strain 427; T. cruzi, strain Tulahuen C4.
6f, 7b, and 7e exhibited an exceptionally high affinity
with apparent Ki values significantly below 1 µM.
Changes in the central core unit and on the triazine
rings were reflected by changing Ki values. In un-
methylated compounds (5a -f), an increase in central
chain length correlated with increased apparent affinity
for the transporter. The introduction of methylamino
and dimethylamino substituents on the triazine units
was well tolerated, although increasing the extent of
methylation on the terminal triazine units leads to a
diminuition of apparent affinity.
groups. Introduction of one (6a ) or two (6b and 6c)
methyl groups per triazine unit resulted in an 10-fold
increase in anti-trypanosomal activity. When four meth-
yl groups per triazine unit were introduced (6d ), an 80-
fold increase in activity compared with 5b was observed.
Similarly, NH2 group replacement starting from 5c (n-
dodecyl chain) led to 2-20-fold higher anti-trypano-
somal activity for the methylated derivatives 6e-h
compared with 5c.
Monosubstituted compounds showed a slight increase
in activity against T. b. rhodesiense compared to the
disubstituted compounds (7a /6c; 7b/6e; 7c/6f; 7d /6g)
except for the fully methylated derivative (7e/6h ) which
showed little effect.
Assays, carried out to access in vitro activity against
T. b. brucei strain 427 displayed similar correlations,
although in general T. b. rhodesiense strain STIB 900
was more sensitive to the active compounds. Strain and
subspecies variability in response to most drugs is a
usual feature of trypanosomes. This could be due to any
of a number of biochemical differences, including dif-
ferences in transporter activity responsible for ac-
cumulating drug, differences in levels or biophysical
properties of drug targets, or other changes in the
metabolic milieu which may alter the impact of com-
pounds on viability. We are not currently in a position
to identify which features distinguish the parasites with
respect to their sensitivity to compounds in the studies
reported here.
The most active trypanocidal compounds were ob-
tained when methylamino substituted triazines were
attached to the C9- or C12-polyamine precursor via an
additional CH2 linker (8a -f). Compound 8f showed
especially good activity against T. b. rhodesiense with
an IC50 value of 0.18 µM and good parasite/host selec-
Thus for the nonyl series (5b and 6a -d ), mono-
methylation (6a ) actually caused a 25-fold increase in
affinity compared to 5b. However, subsequent methyl-
ations reduced affinity. A similar trend was observed
with the dodecyl series (5c and 6e-h ). The tetra
substituted compounds with the extra methylene bridge
(8) all showed lower affinity than the corresponding
disubstituted compounds (8a /5b; 8b/6b; 8c/6c; 8d /5c;
8e/6f) except 8f/6g which showed roughly similar af-
finity. Monosubstitution as compared to disubstitution
(7a -e) led to no effect or loss of affinity for the P2
transporter (compare 7a /6c; 7b/6e; 7c/6f; 7d /6g) in all
cases except one, the fully methylated (7e/6h ).
Tr yp a n ocid a l Activity. All compounds, except those
of the first series (5a -f), displayed dose dependent in
vitro activities against T. b. rhodesiense strain STIB 900
with IC50 values ranging from 0.18 µM to 17 µM (Table
1). In the first series (5a -f), where the central core unit
has been varied, 5c, containing the n-dodecyl chain as
core unit, showed weak activity (IC50 ) 16 µM) against
T. b. rhodesiense. The next most promising compound
was the n-nonyl compound 5b. Starting from 5b (n-
nonyl chain), analogues were designed by replacing NH2
groups on the triazine ring with NHMe and NMe2

Synthesis of s-Triazine Substituted Polyamines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3445
tivity. Other analogues in the same series (8b-e)
showed similarly high activity and good parasite/host
selectivity. One analogue carrying methylene-bridged
triazine units without any methylamino substituents
(8a ) was significantly less active than 8f. Comparable
results were found for the activities against T. b. brucei.
The introduction of additional CH2 linkers between the
polyamine backbone and the triazines increased the
anti-trypanosomal activity up to 1000-fold. Target com-
pound 8c was identified as the most active against T.
b. brucei with an IC50 value of 0.1 µM, making it a very
suitable candidate for in vivo testing.
Compounds 9 and 10 were prepared to establish the
necessity of the polyamine chain. However, these com-
pounds showed poor solubility in aqueous solution,
meaning the data for these compounds presented in
Table 1 is very approximate. Therefore, while it is
unknown if the central amino groups are essential for
activity against the molecular target, they are essential
for the solubility properties of the compounds.
tranpsorter, based on their ability to inhibit the uptake
of radiolabeled adenosine by this route. It is not possible,
using this assay, to determine whether these molecules
are actually internalized via this route, only that they
interact with the transporter. Radiolabeled or other
traceable derivatives of the compounds would be re-
quired to verify whether they are internalized via this
route. Notwithstanding, several compounds proved
highly active against both T. brucei rhodesiense and T.
brucei brucei in vitro, although host toxicity precluded
full evaluation of these compounds in vivo.
The compounds described here were designed to
determine preliminary structure activity relationships.
Activities against T. b. brucei and T. b. rhodesiense (see
IC50 values in Table 1) varied little when the central
core unit was modified (5a -f) with the exception of 5c.
An increase in activity was found, however, when
methylamino or dimethylamino substituted triazine
units were used (6a -h ). Analogues of 6a -h carrying
only one instead of two triazine units (7a -e) showed
slightly higher activity. A further increase in activity
was observed when the triazine units were attached via
an additional methylene spacer (8a -f). These results
demonstrate that the triazine moieties play a crucial
role in the mode of action of this family of compounds.
Currently we cannot explain why the addition of me-
thylene spacers enhances trypanocidal activity. Poly-
amines with a straight long alkyl chain as central core
unit seem to be beneficial. Attempts to assay the
substituted 2-chloro- and 2-bromomethyl-1,3,5-triazines
themselves for their antitrypanosomal activity failed
because of their insolubility in aqueous solution.
In Vivo Activity. Compounds 6e, 8b, 8c, and 8e
were tested for toxicity and anti-trypanosomal activity
in mice. However, 1 mg kg-1 intraperitoneal injection
into mice was not curative, and concentrations greater
than 10 mg kg-1 induced severe acute toxicity.
T. cr u zi. T. cruzi is phylogenetically related to T.
brucei and causes Chagas’ disease. Both pathogens
share a number of biochemical features including the
presence of trypanothione. However, there are also key
differences between these two organisms at the level of
host-pathogen interaction. Critically, T. brucei is a
parasite which lives free in the bloodstream and cere-
brospinal fluid of the host, while T. cruzi amastigotes
replicate in the cytosol of a number of mammalian cell
types. To act against intracellular parasites, drugs must
cross host cell membranes too, thus diminishing the
prospects of selective toxicity through selective uptake.
In general, the compounds were less toxic for T. cruzi
than T. brucei, and the most active ones (6h and 7e)
were also those which were among the most cytotoxic
for mammalian cells, indicating that these products may
enter cells via different routes and induce toxicity within
multiple cell types.
There is no obvious correlation between the P2
transporter affinity and the anti-trypanosomal activity
of the compounds, leading to the conclusion that uptake
through the P2 transporter is not the rate-limiting step
in toxicity. We cannot exclude the possibility that these
products actually enter cells via routes other than the
P2 transporter. In the absence of information about the
specific targets of the compounds, nothing can be stated
about the altered toxic activity of the products. Other
factors apart from the uptake, such as metabolic break-
down and interactions with intracellular targets, are
likely to be critical in determining toxicity.
Discu ssion
The actual mode of action for the reported triazine
substituted polyamines also remains unclear. Kuko-
amine A,23 spermine and spermidine derivatives,24
glutathionyl spermidine phosphinates (GSP),25 and
other polyamine analogues26 have all been shown to be
inhibitors of trypanothione biosynthesis, and it might
be speculated that our compounds act in a similar
manner against T. brucei. However, other modes of
action, also including the displacement of polyamines
from their normal physiological sites of action or inter-
actions involving metabolites of the triazine substituted
polyamines, may also play an important role. To un-
derstand and improve the activities of our compounds,
further research has to verify intracellular drug targets
and possible metabolic pathways.
The P2 amino-purine transporter has been shown to
carry a number of compounds into trypanosomes. The
structure-activity relationship between this transporter
and its substrates has been determined,7,13,14 and it is
possible to generate novel trypanocidal compounds
bearing this recognition motif. Addition of the motif to
cytotoxic moieties could endow them with the ability to
be selectively accumulated and thus selectively toxic to
trypanosomes. Polyamine metabolism has been shown
to be a good chemotherapeutic target in T. brucei.
However, these cells lack transporters for polyamines
which means that this logical route of targeting
polyamine analogues to these cells is not available.
Therefore we have developed polyamine analogues
bearing groups (1,3,5-triazines) enabling recognition by
the P2 amino-purine transporter to see if novel, try-
panocidal polyamine analogues could be produced.
The triazine substituted polyamines 5a -f, 6a -h , 7a -
e, and 8a -f have been shown to interact with the P2
Con clu sion
We have reported the design and synthesis of triazine
substituted polyamines as drugs against T. brucei spp.,

3446 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Klenke et al.
which developed during the following 2-4 h was read photo-
metrically at 540 nm. From the sigmoidal inhibition curve, IC50
values were calculated. Cytotoxicity was assessed in the same
assay using noninfected L-6 cells, and the same serial drug
dilution. The MIC was determined microscopically after 4 days.
the causative agent for African sleeping sickness. Four
different types of test compounds (5a -f, 6a -h , 7a -e,
and 8a -f) were prepared using a five-step synthesis
consisting of Michael addition reaction, BOC-protection,
hydrogenation, arylation, and BOC-deprotection. Fur-
thermore, two non-polyamine analogues (9 and 10) were
prepared by arylation of commercially available amines.
The compounds were designed with the intention of
selectively targeting to the interior of T. brucei via the
P2 amino-purine transporter. All molecules were as-
sayed for their affinity for this transporter, and their
activity against three different parasitic protozoa (T. b.
rhodesiense, T. b. brucei, and T. cruzi). Good activities
were found in several cases. Especially the tetra triazine
substituted polyamine 8c showed an excellent toxicity
profile with high activity against T. b. rhodesiense (IC50
) 0.27 µM) and T. b. brucei (IC50 ) 0.10 µM) and a low
cytotoxicity against rat L-6 cells (MIC ) 177 µM).
In Vivo Biologica l Activity. On day one of the trials
groups of five mice were infected with 2 × 105 T. b. brucei
strain 427 by intraperitoneal injection. On days three and four
after injection, the groups of mice were treated by intraperi-
toneal injection with either the test compounds or diminazene
aceturate (as a positive control to clear infection). A negative
control group received no treatment. The parasitaemia of all
mice was checked daily by tail prick examination, and sick
animals were euthanized.
Ch em istr y. Gen er a l. Purification by column chromatog-
raphy was performed on Sorbosil C60A silica gel 40-60 µm
from Merck. Qualitative thin-layer chromatography (TLC) was
done on precoated aluminum sheets silica gel 60 F254 from
Merck. Compounds were detected either with iodine or 254
nm UV light. All given Rf values refer to the purified material
and were obtained under chamber saturation with the applied
solvent mixture. Purification by ion exchange chromatography
was performed on a Dowex 50WX2-200 resign using an HCl
gradient (50 mL of 0 N, 400 mL of 2 N, 300 mL of 5 N in 1:1
H2O/i-PrOH). The progress of elution was monitored by UV
absorbance at 240 nm. UV spectra were recorded at a Heλlios
R UV/VIS spectral photometer from Unicam. Melting points
were determined with a Gallenkamp melting point apparatus
and are not corrected. 1H and 13C NMR data were obtained
on a Bruker 300 MHz NMR spectrometer using the applied
solvent simultaneously as internal standard. Mass spectra
were recorded at a Platform II mass spectrometer (Micromass)
from Fisons. Ionization was achieved in the positive electro-
spray mode using a 1:1 solvent mixture of acetonitrile and
water as mobile phase. High-resolution mass spectra were
recorded by the National Mass Spectrometry Service Centre
in Swansea at a MAT 900 XLT high resolution double focusing
mass spectrometer from Finnigan using the same ionization
procedure. Accurate mass measurement was performed by
peak matching. Combustion analyses were performed by the
analytical and chemical consultancy services MEDAC Ltd.
Solvents and reagents were used as purchased unless other-
wise stated. All yields quoted in this paper were isolated yields.
Compounds 2a ,30,31 2b,32 2c,31 and 3a 33 have been reported in
the literature earlier. The synthesis of 4a -f has been reported
separately.20
Exp er im en ta l Section
P 2 Tr a n sp or ter Assa y.27 A total of 100 µL of T. b. brucei
427 cells at 2 × 108/mL was added to a buffer containing
radiolabeled adenosine and varying concentrations of potential
inhibitor. The buffer also contained 1 mM inosine, to block
adenosine uptake by the P1 transporter. After a 10 s incuba-
tion at room temperature, the cells were spun through a layer
of oil (1-Bromododecane, 97%) to form a pellet, and the entire
reaction tube was flash frozen in liquid nitrogen. The oil layer
containing the pellet was cut off into a scintillation vial, and
the radioactivity present was measured by scintillation counter.
The apparent Ki value was calculated using the competitive
inhibition equation from the Grafit 4.0 software. Each experi-
ment was done in duplicate.
In Vitr o Biologica l Activity. T. b. br u cei. Serial dilutions
of the test compounds from 200 µM were set out in a 96-well
plate in duplicate. A total of 100 µL of cells (T. b. brucei 427)
at 2 × 105/ml was added to each well, leaving an initial dilution
of 100 µM and 2000 cells per well. After 48 h incubation at 37
°C, Alamar Blue (20 µL)28 was added to each well, and the
plates incubated for a further 24 h. Live cells reduce the
Alamar Blue to a pink color by unspecified dehydrogenases,
and a concomitant change in absorption at 600 nm can be
measured spectrophotometrically. The IC50 value was calcu-
lated from the raw data using the Grafit 4.0 software. Values
were checked by visually examining cell viability and motility
in wells.
T. b. r h od esien se. Minimum Essential Medium (50 µL)
supplemented with 2-mercaptoethanol and 15% heat-inacti-
vated horse serum29 was added to each well of a 96-well
microtiter plate. Serial drug dilutions were added to the wells.
Then 50 µL of trypanosome suspension (T. b. rhodesiense STIB
900) was added to each well (final trypanosome concentration
2 × 104/mL), and the plate incubated at 37 °C under a 5%
CO2 atmosphere for 72 h. Alamar Blue (10 µL)28 was then
added to each well, and incubation continued for a further 2-4
h. The plate was then read with a Millipore Cytofluor 2300
using an excitation wavelength of 530 nm and emission
wavelength of 590 nm. Fluorescence development was ex-
pressed as percentage of the control, and IC50 values deter-
mined.
T. cr u zi. Rat skeletal myoblasts (L-6 cells) were seeded in
96-well microtiter plates at 2000 cells/well/100µL in RPMI
1640 medium with 10% FBS and 2 mM L-glutamine. After 24
h, 5000 trypomastigotes of T. cruzi (Tulahuen strain C2C4
containing the galactosidase (Lac Z gene)) were added in 100
µL per well with 2× a serial drug dilution. The plates were
incubated at 37 °C in 5% CO2 for 4 days. After 96 h, the
minimum inhibitory concentration (MIC) was determined
microscopically. For measurement of the IC50, the substrate
CPRG/Nonidet was added to the wells. The color reaction
Mich a el Ad d ition Rea ction , Gen er a l P r oced u r e. A total
of 1 equiv of diamine (1 M solution in absolute EtOH) was
cooled in an ice bath, and 2 equiv of acrylonitrile (1 M solution
in absolute EtOH) was added dropwise at 0 °C. The nearly
colorless solution was allowed to warm to room temperature
and stirred at room temperature for additional 20 h. After the
solvent was removed at reduced pressure, the obtained residue
was purified by column chromatography on SiO2 using 15%
MeOH in CHCl3 as eluent, by high vacuum distillation, or by
crystallization.
3-(4-[(2-C y a n o e t h y l)a m in o ]b u t y la m in o )p r o p a n e -
n itr ile (2a ). Starting from 1,4-diaminobutane (1a , 500 mg,
5.67 mmol) and acrylonitrile (600 mg, 11.3 mmol), 2a was
obtained as pale yellow oil (864 mg, 78%): Rf (SiO2, 15% MeOH
in CHCl3) 0.36; 1H NMR (CDCl3) δ/ppm ) 2.93 (t, 3J ) 6.7
Hz), 2.66 (m, 4H), 2.54 (t, 3J ) 6.7 Hz, 4H), 1.68 (m, 2H, NH),
1.54 (m, 4H); 13C NMR (CDCl3) δ/ppm ) 119.2 (s), 49.3 (t),
45.4 (t), 28.0 (t), 19.1 (t); MS (ES+) m/z ) 217 (M+Na+, 10%),
195 (M+H+, 100%).
3-(9-[(2-C y a n o e t h y l)a m in o ]n o n y la m in o )p r o p a n e -
n itr ile (2b). Starting from 1,9-diaminononane (1b, 500 mg,
3.16 mmol) and acrylonitrile (335 mg, 6.31 mmol), 2b was
obtained as pale yellow oil (770 mg, 92%): Rf (SiO2, 15% MeOH
in CHCl3) 0.47; 1H NMR (CDCl3) δ/ppm ) 2.96 (t, 3J ) 6.6
3
3
Hz, 4H), 2.65 (t, J ) 7.1 Hz, 4H), 2.56 (t, J ) 6.6 Hz, 4H),
1.51 (m, 4H), 1.39 (bs, 2H, NH), 1.37-1.31 (m, 10H); 13C NMR
(CDCl3) δ/ppm ) 119.2 (s), 49.6 (t), 45.5 (t), 30.4 (t), 29.9 (t),

Synthesis of s-Triazine Substituted Polyamines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3447
29.8 (t), 27.6 (t), 19.1 (t); MS (ES+) m/z ) 287 (M+Na+, 8%),
265 (M+H+, 100%).
(t), 48.1 (t), 44.2 (t), 43.8 (t), 29.9 (t), 29.6 (t), 29.1 (t), 28.8 (q),
27.1 (t), 17.9 (t), 17.4 (t); MS (ES+) m/z ) 487 (M+Na+, 80%),
482 (M+NH4+, 100%), 465 (M+H+, 10%).
3-(12-[(2-Cya n oet h yl)a m in o]d od ecyla m in o)p r op a n e-
n itr ile (2c). Starting from 1,12-diaminododecane (1c, 500 mg,
2.5 mmol) and acrylonitrile (264 mg, 4.98 mmol), 2c was
obtained as pale yellow oil, which solidified on standing (650
mg, 85%): Rf (SiO2, 15% MeOH in CHCl3) 0.44; mp 55-56 °C;
1H NMR (CDCl3) δ/ppm ) 2.96 (t, J ) 6.6 Hz, 4H), 2.66 (t, J
) 7.2 Hz, 4H), 2.56 (t, J ) 6.6 Hz, 4H), 1.52 (m, 4H), 1.34 (m,
2H, NH), 1.41-1.26 (m, 16H); 13C NMR (CDCl3) δ/ppm ) 119.2
(s), 49.7 (t), 45.5 (t), 30.4 (t), 30.0 (t), 29.9 (t), 27.6 (t), 19.1 (t);
MS (ES+) m/z ) 307 (M+H+, 100%)
3-(2-[(2-Cya n oeth yl)a m in o]-1-m eth yleth yla m in o)p r o-
p a n en itr ile (2d ). Starting from (()-1,2-diaminopropane (1d ,
20 g, 270 mmol) and acrylonitrile (28.6 g, 35.5 mL, 540 mmol),
2d was obtained as light yellow oil after high vacuum distil-
lation (40.0 g, 82%): Rf (SiO2, 15% MeOH in CHCl3) 0.32; bp
162-168 °C/0.005 Torr; 1H NMR (CDCl3) δ/ppm ) 3.05-2.82
(m, 4H), 2.97-2.65 (m, 2H), 2.55-2.41 (m, 5H), 1.61 (bs, 2H,
NH), 1.06 (d, 3J ) 6.2 Hz, 3H); 13C NMR (CDCl3) δ/ppm )
119.4 (s), 119.3 (s), 55.1 (t), 52.6 (d), 45.5 (t), 43.0 (t), 19.6 (t),
19.3 (t), 19.0 (q); MS (ES+) m/z ) 203 (M+Na+, 15%), 181
(M+H+, 100%)
N4,N17-Di(ter t-bu tyloxyca r bon yl)-3-(12-[(2-cya n oeth yl)-
a m in o]d od ecyla m in o)p r op a n en itr ile (3c). Starting from
2c (650 mg, 2.12 mmol), triethylamine (470 mg, 4.64 mmol),
and di-tert-butyl dicarbonate (1.02 g, 4.67 mmol), 3c was
obtained as colorless oil (820 mg, 76%): Rf (SiO2, 5% MeOH
1
3
in CH2Cl2) 0.61; H NMR (CDCl3) δ/ppm ) 3.46 (t, 4H, J )
6.68), 3.24 (t, 4H, 3J ) 7.59 Hz), 2.68-2.51 (m, 4H), 1.57-
1.43 (m, 4H), 1.46 (s, 18H), 1.26 (m, 16H); 13C NMR (CDCl3)
δ/ppm ) 155.8 (s), 155.1 (s), 118.9 (s), 118.4 (s), 80.8 (s), 80.6
(s), 49.0 (t), 48.2 (t), 44.2 (t), 43.8 (t), 30.0 (t), 29.9 (t), 29.7 (t),
28.8 (q), 27.1 (t), 17.9 (t), 17.4 (t); MS (ES+) m/z ) 545 (M+K+,
15%), 529 (M+Na+, 65%), 524 (M+NH4+, 100%), 507 (M+H+,
10%).
N4,N7-Di(ter t-b u t yloxyca r bon yl)-3-(2-[(2-cya n oet h yl)-
a m in o]-1-m eth yleth yla m in o)p r op a n en itr ile (3d ). Starting
from 2d (5.00 g, 27.7 mmol), triethylamine (6.16 g, 60.9 mmol),
and di-tert-butyl dicarbonate (13.3 g, 60.9 mmol), 3d was
obtained as colorless micro crystalline powder (8.2 g, 78%): Rf
1
(SiO2, 5% MeOH in CH2Cl2) 0.66; mp 173-175 °C; H NMR
(CDCl3) δ/ppm ) 4.24/4.03 (2m, br, 1H), 3.72-3.33 (m, 4H),
3.33-3.04 (m, 2H), 2.65 (m, 2H), 2.57 (m, 2H), 1.49 (s, 18H),
3-(2-[(2-Cya n oeth yl)a m in o]cycloh exyla m in o)p r op a n e-
n itr ile (2e). Starting from trans-1,2-diaminocyclohexane (1e,
10.0 g, 87.5 mmol) and acrylonitrile (9.3 g, 11.5 mL, 175 mmol),
2e was obtained as light yellow viscous oil after high vacuum
distillation (12.0 g, 62%): Rf (SiO2, 15% MeOH in CHCl3) 0.42;
bp 180 °C/0.09 Torr; mp 74-75 °C; 1H NMR (CDCl3) δ/ppm )
3.04/3.02/2.81/2.77 (4t, 3J ) 6.78, 6.78, 6.34, 6.44 Hz, 4H), 2.49
3
1.23 (d, 3H, J ) 6.59 Hz); 13C NMR (CDCl3) δ/ppm ) 155.4
(s), 155.2 (s), 118.8 (s), 118.3 (s), 81.6 (s), 81.4 (s), 50.2 (d),
43.9 (t), 43.9 (t),41.1 (t), 28.8 (q), 17.6 (t), 17.4 (t), 16.5 (q); MS
(ES+) m/z ) 403 (M+Na+, 100%), 390 (M+NH4+, 15%), 381
(M+H+, 20%). A byproduct was isolated as pale yellow oil and
identified as the mono protected material (1.33 g, 17%): Rf
3
1
(t, J ) 6.78 Hz, 4H), 2.15 (m, 2H), 2.04 (m, 2H), 1.81 (s, 2H,
(SiO2, 5% MeOH in CH2Cl2) 0.59 (tailing); H NMR (CDCl3)
NH), 1.72 (m, 2H), 1.22 (m, 2H), 1.02 (m, 2H); 13C NMR
(CDCl3) δ/ppm ) 119.4 (s), 61.4 (d), 42.8 (t), 32.1 (t), 25.2 (t),
19.7 (t); MS (ES+) m/z ) 221 (M+H+, 100%).
3
δ/ppm ) 3.56-3.54 (m, 2H), 3.20 (d, 2H, J ) 5.67 Hz), 3.06-
3
2.78 (m, 3H), 2.66 (m, br, 2H), 2.48 (t, 2H, J ) 6.31), 1.47 (s,
9H), 1.41 (s, br, 1H, NH), 1.05 (d, 3H, 3J ) 6.40 Hz); 13C NMR
(CDCl3) δ/ppm ) 155.7 (s), 119.2 (s), 118.8 (s), 81.2 (s), 54.9
(t), 54.4 (t), 53.1 (d), 52.5 (d), 45.5 (t), 43.1 (t), 28.7 (q), 19.7
(t), 19.0 (q), 17.9 (t), 17.1 (t); MS (ES+) m/z ) 561 (2M+H+,
10%), 303 (M+Na+, 50%), 281 (M+H+, 100%), 225 (M+2H+-
tBu+, 60%).
3-(4-[(2-Cya n oeth yl)a m in o]cycloh exyla m in o)p r op a n e-
n itr ile (2f). Starting from trans-1,4-diaminocyclohexane (1f,
20 g, 175 mmol) and acrylonitrile (18.6 g, 23 mL, 350 mmol),
2f was obtained as off-white microcrystalline powder after
recrystallization from EtOH (31.8 g, 82%): Rf (SiO2, 15%
MeOH in CHCl3) 0.16; mp 115-116 °C; 1H NMR (CDCl3)
N4,N7-Di(ter t-b u t yloxyca r bon yl)-3-(2-[(2-cya n oet h yl)-
am in o]cycloh exylam in o)pr opan en itr ile (3e). Starting from
2e (5.00 g, 22.7 mmol), triethylamine (5.00 g, 49.4 mmol), and
di-tert-butyl dicarbonate (10.8 g, 49.5 mmol), 3e was obtained
as colorless micro crystalline powder (6.53 g, 68%): Rf (SiO2,
5% MeOH in CH2Cl2) 0.68; mp 202-204 °C; 1H NMR (CDCl3)
δ/ppm ) 4.12-3.98 (m, 1H), 3.86-3.67 (m, 1H), 3.65-3.35 (m,
2H), 3.29-3.05 (m, 2H), 2.74-2.38 (m, 4H), 2.03-1.89 (m, 2H),
1.89-1.75 (m, 2H), 1.57-1.23 (m, 4H), 1.50 (s, 18H); 13C NMR
(CDCl3) δ/ppm ) 155.5 (s), 118.3 (s), 81.4 (s), 55.3 (d), 39.6 (t),
31.4 (t), 28.8 (q), 25.6 (t), 19.0 (t); MS (ES+) m/z ) 443 (M+Na+,
100), 438 (M+NH4+, 48%), 421 (M+H+, 30%). A byproduct was
isolated as pale yellow oil and identified as the mono protected
material (2.06 g, 28%): Rf (SiO2, 5% MeOH in CH2Cl2) 0.63;
1H NMR (CDCl3) δ/ppm ) 4.15-3.96/3.75-3.65 (2m, 1H),
3.65-3.41/3.41-3.24 (2m, 2H), 3.08-2.94 (m, 1H), 2.85-2.59
(m, 3H), 2.53-2.33 (m, 2H), 2.12 (m, 1H), 1.87-1.69 (m, 3H),
1.61-0.98 (m, 6H), 1.49 (s, 9H); 13C NMR (CDCl3) δ/ppm )
156.0 (s), 155.5 (s), 119.3 (s), 118.9 (s), 118.3 (s), 81.4 (s), 81.2
(s), 58.4 (d), 55.3 (d), 42.3 (t), 39.6 (t), 33.1 (t), 31.4 (t), 31.0 (t),
28.8 (q), 26.0 (t), 25.6 (t), 25.2 (t), 20.0 (t), 19.0 (t); MS (ES+)
m/z ) 343 (M+Na+, 30%), 321 (M+H+, 100%), 265 (M+2H+-
tBu+, 12%)
N4,N9-Di(ter t-b u t yloxyca r bon yl)-3-(4-[(2-cya n oet h yl)-
am in o]cycloh exylam in o)pr opan en itr ile (3f). Starting from
2f (5.00 g, 22.7 mmol), triethylamine (5.00 g, 49.4 mmol), and
di-tert-butyl dicarbonate (10.8 g, 49.5 mmol), 3f was obtained
as colorless oil, which crystallized on standing at room tem-
perature (6.09 g, 64%): Rf (SiO2, 5% MeOH in CH2Cl2) 0.69;
mp 135-138 °C; 1H NMR (CDCl3) δ/ppm ) 3.90/3.61 (2m, br,
2H), 3.38 (m, 4H), 2.60 (m, 4H), 1.94-1.78 (m, 4H), 1.56 (m,
4H), 1.48 (s, 18H); 13C NMR (CDCl3) δ/ppm ) 155.1 (s), 118.3
(s), 81.2 (s), 56.2 (d), 54.4 (d), 40.7 (t), 39.5 (t), 30.3 (t), 28.8
(q), 19.3 (t), 18.4 (t); MS (ES+) m/z ) 443 (M+Na+, 30%), 438
(M+NH4+, 100%). A byproduct was isolated as pale yellow oil
3
δ/ppm ) 2.89 (td, J ) 6.59, 4.52 Hz, 4H), 2.49-2.37 (m, 2H),
2.47 (t, 3J ) 6.59 Hz, 4H), 1.90 (m, 4H), 1.19 (m, 2H, NH),
1.16-1.05 (m, 4H); 13C NMR (CDCl3) δ/ppm ) 116.8 (s), 53.9
(d), 40.4 (t), 29.9 (t), 17.3 (t); MS (ES+) m/z ) 221 (M+H+, 50%),
151 (100%)
Boc-P r otection , Gen er a l P r oced u r e. A solution of 1
equiv of Michael adduct (0.25 M in MeOH) was cooled to 0 °C,
and 2 equiv of triethylamine followed by 2 equiv of di-tert-
butyl dicarbonate (0.5 M in MeOH) were added. The mixture
was stirred at room temperature for 24 h, the solvent was
removed under reduced pressure, and the residue was purified
by column chromatography on SiO2 using 5% MeOH in CH2Cl2
as eluent.
N4,N9-Di(ter t-bu t yloxyca r b on yl)-3-(4-[(2-cya n oet h yl)-
a m in o]bu tyla m in o)p r op a n en itr ile (3a ). Starting from 2a
(864 mg, 4.45 mmol), triethylamine (990 mg, 9.78 mmol), and
di-tert-butyl dicarbonate (2.14 g, 9.81 mmol), 3a was obtained
as colorless oil, which crystallized on standing at room tem-
perature (1.70 g, 97%): Rf (SiO2, 5% MeOH in CH2Cl2) 0.57;
1
3
mp 102-104 °C; H NMR (CDCl3) δ/ppm ) 3.47 (t, 4H, J )
6.68 Hz), 3.30 (m, 4H), 2.70-2.53 (m, 4H), 1.53 (m, 4H), 1.48
(s, 18H); 13C NMR (CDCl3) δ/ppm ) 155.6 (s), 155.2 (s), 118.8
(s), 118.3 (s), 80.9 (s), 48.7 (t), 47.6 (t), 44.4 (t), 43.9 (t), 28.8
(q), 26.4 (t), 25.9 (t), 18.0 (t), 17.4 (t); MS (ES+) m/z ) 433
(M+K+, 20%), 417 (M+Na+, 100%), 412 (M+NH4+, 30%).
N4,N14-Di(ter t-bu tyloxyca r bon yl)-3-(9-[(2-cya n oeth yl)-
a m in o]n on yla m in o)p r op a n en itr ile (3b). Starting from 2b
(770 mg, 2.91 mmol), triethylamine (645 mg, 6.37 mmol), and
di-tert-butyl dicarbonate (1.40 g, 6.41 mmol), 3b was obtained
as colorless oil (1.15 g, 85%): Rf (SiO2, 5% MeOH in CH2Cl2)
0.63; 1H NMR (CDCl3) δ/ppm ) 3.46 (t, 4H, 3J ) 6.77 Hz),
3
3.25 (t, 4H, J ) 7.59 Hz), 2.68-2.52 (m, 4H), 1.58-1.47 (m,
4H), 1.47 (s, 18H), 1.29 (m, 10H); 13C NMR (CDCl3) δ/ppm )
155.8 (s), 155.1 (s), 118.4 (s), 118.9 (s), 80.8 (s), 80.6 (s), 49.0

3448 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Klenke et al.
and identified as the mono protected material (2.37 g, 33%):
491.3792. Anal.(C21H42N14‚4HCl‚1H2O) C, H, Cl; N: calcd, 30.0;
Rf (SiO2, 5% MeOH/CH2Cl2) 0.24-0.37; 1H NMR (CDCl3)
found, 29.2.
3
N2-(3-[12-(3-[(4,6-Dia m in o-1,3,5-tr ia zin -2-yl)a m in o]p r o-
p yla m in o)d od ecyl]a m in o-p r op yl)-1,3,5-tr ia zin e-2,4,6-tr i-
a m in e Tetr a h yd r och lor id e (5c). Starting from 4c (100 mg,
0.194 mmol), 2-chloro-4,6-diamino-1,3,5-triazine (62 mg, 0.426
mmol), and NaHCO3 (35 mg, 0.417 mmol), 5c was obtained
as off-white powder (115 mg, 87%): Rf (SiO2, 35% aq NH3/
MeOH 1:10) 0.26; mp(sealed tube) dec > 210 °C; 1H NMR
δ/ppm ) 3.92-3.67 (m, 1H), 3.38 (t, 2H, J ) 7.04 Hz), 2.94
3
3
(t, 2H, J ) 6.59 Hz), 2.59 (m, 2H), 2.51 (t, 2H, J ) 6.59 Hz),
2.46-2.41 (m, 1H), 2.01 (m, 2H), 1.80 (m, 2H), 1.60-1.46 (m,
2H), 1.48 (s, 9H), 1.42 (s, 1H, NH), 1.28-1.14 (m, 2H); 13C NMR
(CDCl3) δ/ppm ) 155.1 (s), 119.1 (s), 118.3 (s), 81.1 (s), 55.8
(d), 55.1 (d), 42.7 (t), 39.7 (t), 32.9 (t), 29.7 (t), 28.8 (q), 19.6
(t), 19.3 (t); MS (ES+) m/z ) 641 (2M+H+, 70%), 343 (M+Na+,
30%), 321 (M+H+, 100%)
3
3
(CD3OD) δ/ppm ) 3.51 (t, 4H, J ) 6.59 Hz), 3.07 (t, 4H, J )
7.59 Hz), 3.01 (t, 4H, 3J ) 8.05 Hz), 2.01 (tt, 4H, 3J ) 7.53,
6.77 Hz), 1.71 (m, 4H), 1.35/1.30 (2m, 16H); NH exchanged;
13C NMR (CD3OD) δ/ppm ) 162.4 (s), 161.2 (s), 158.0 (s), 49.1
(t), 46.3 (t), 38.7 (t), 30.4 (t), 30.3 (t), 30.1 (t), 27.5 (t), 27.1 (t);
MS (ES+) m/z ) 533 (M+H+, 100%), 267 ((M+2H+)/2, 10%);
HR-MS (ES+) calcd for (C24H49N14)+: 533.4264; found 533.4263.
Anal. (C24H48N14‚4HCl‚2H2O) C, H, Cl; N: calcd, 27.4; found,
26.4.
Red u ction , Gen er a l P r oced u r e. A total of 1 equiv of
protected material (0.05 M in dioxan/water 4:1) was treated
with 1 equiv of Raney-nickel (50% suspension in water), 0.1
equiv of palladium (10% on charcoal), and 3 equiv of lithium
hydroxide monohydrate. The reaction mixture was stirred
under hydrogen atmosphere at 50 °C for 20 h. The catalyst
was filtered off, the solvents were removed in vacuo, and a
mixture of 50 mL of water and 25 mL of dichloromethane was
added. After phase separation, the aqueous phase was ex-
tracted twice with dichloromethane (15 mL), the combined
organic layers were dried over sodium sulfate, and the solvent
was removed in vacuo. The data for this step (compounds 4a -
f) have been reported.20
N2-(3-[2-(3-[(4,6-Dia m in o-1,3,5-tr ia zin -2-yl)a m in o]p r o-
p yla m in o)p r op yl]a m in op r op yl)-1,3,5-t r ia zin e-2,4,6-t r i-
a m in e Tetr a h yd r och lor id e (5d ). Starting from 4d (100 mg,
0.257 mmol), 2-chloro-4,6-diamino-1,3,5-triazine (85 mg, 0.584
mmol), and NaHCO3 (49 mg, 0.583 mmol), 5d was obtained
as a colorless powder (130 mg, 91%): Rf (SiO2, 35% aq NH3/
MeOH 1:10) 0.30; mp(sealed tube) dec > 240 °C; 1H NMR
(CD3OD) δ/ppm ) 3.89-3.74 (m, 1H), 3.55 (m, 5H), 3.43-3.33
Ar yla tion a n d Dep r otection , Gen er a l P r oced u r e. A
total of 100 mg of reduced material was dissolved in absolute
EtOH (10 mL) and water (10 mL). A total of 1-2 equiv of the
substituted triazine and 1-2 equiv of NaHCO3 were added,
and the mixture was stirred under reflux for 15-20 h. If ES-
MS indicated incomplete reaction, more substituted triazine
and NaHCO3 were added and reflux continued for another 15-
20 h. The solvents were removed in vacuo, and the residue
was washed twice with water (5 mL). Anisole (0.2 mL, 1.8
mmol) and trifluoroacetic acid (2.0 mL, 25.9 mmol) were added
at 0 °C, and the reaction mixture was stirred at room
temperature for 3 h. The trifluoroacetic acid was removed in
vacuo, the residue was washed twice with ether (5 mL) and
dried in vacuo. The crude product was dissolved or suspended
in water (20 mL) and submitted to an ion-exchange column
(10 mL Dowex 50WX2-200, H+-form). The column was
washed with water (50 mL) and HCl (2 M in water, 400 mL)
and was finally eluted with HCl (5 M in 2-propanol/water 1:1,
300 mL). The solvents were removed in vacuo, and the isolated
sticky solids were precipitated from ethanol/ether to yield the
hydrochloric salts as powders.
3
(dd, J ) 13.36 Hz, 4.40 Hz, 1H), 3.20 (m, 4H), 2.10 (m, 4H),
3
1.52 (d, 3H, J ) 6.59 Hz); EtOH signals at 3.59 (q), 1.18 (t);
NH exchanged; 13C NMR (CD3OD) δ/ppm ) 162.9 (s), 158.1
(s), 53.0 (d), 50.6 (t), 47.6 (t), 44.5 (t), 38.8 (t), 38.7 (t), 27.4 (t),
27.2 (t), 15.3 (q); EtOH signals at 53.6 (t), 18.4 (q); MS (ES+)
m/z ) 407 (M+H+, 100%), 204 ((M+2H+)/2, 50%); HR-MS
(ES+) calcd for (C15H31N14)+: 407.2856; found 407.2855. Anal.
(C15H30N14‚4HCl‚1EtOH‚1H2O) C, N; H: calcd, 6.9; found, 6.4;
Cl: calcd, 23.0; found, 23.7.
N2-(3-[2-(3-[(4,6-Dia m in o-1,3,5-tr ia zin -2-yl)a m in o]p r o-
p yla m in o)cycloh exyl]a m in op r op yl)-1,3,5-tr ia zin e-2,4,6-
tr ia m in e Tetr a h yd r och lor id e (5e). Starting from 4e (100
mg, 0.233 mmol), 2-chloro-4,6-diamino-1,3,5-triazine (75 mg,
0.515 mmol), and NaHCO3 (55 mg, 0.655 mmol), 5e was
obtained as a colorless powder (120 mg, 87%): Rf (SiO2, 35%
1
aq NH3/MeOH 1:10) 0.45; mp(sealed tube) dec > 240 °C; H
NMR (CD3OD) δ/ppm ) 3.55 (m, 6H), 3.33 (m, 2H), 3.15 (m,
2H), 2.35 (m, 2H), 2.20 (m, 2H), 2.14 (m, 2H), 1.85 (m, 2H),
1.64 (m, 2H), 1.41 (m, 2H); EtOH signals at 3.59 (q), 1.18 (t);
NH exchanged; 13C NMR (CD3OD) δ/ppm ) 162.7 (s), 158.1
(s), 59.1 (d), 44.9 (t), 38.9 (t), 27.7 (t), 27.2 (t), 23.7 (t); EtOH
signals at 53.6 (t), 18.4 (q); MS (ES+) m/z ) 447 (M+H+, 100%),
224 ((M+2H+)/2, 35%); HR-MS (ES+) calcd for (C18H35N14)+:
447.3169; found 447.3170. Anal. (C18H34N14‚4HCl‚1EtOH‚
1H2O) H, N, Cl; C: calcd, 36.6; found, 37.2.
(a ) Rea ction s w ith 2-Ch lor o-4,6-d ia m in o-1,3,5-tr ia zin e.
N2-(3-[4-(3-[(4,6-Dia m in o-1,3,5-tr ia zin -2-yl)a m in o]p r op yl-
a m in o)bu tyl]a m in op r op yl)-1,3,5-tr ia zin e-2,4,6-tr ia m in e
Tetr a h yd r och lor id e (5a ). Starting from 4a (100 mg, 0.248
mmol), 2-chloro-4,6-diamino-1,3,5-triazine (80 mg, 0.550 mmol),
and NaHCO3 (46 mg, 0.548 mmol), 5a was obtained as a light
yellow powder (130 mg, 92%): Rf (SiO2, 35% aq NH3/MeOH
1:10): 0.05; mp(sealed tube) dec in two steps: 1. >260 °C, 2.
N2-(3-[4-(3-[(4,6-Dia m in o-1,3,5-tr ia zin -2-yl)a m in o]p r o-
p yla m in o)cycloh exyl]a m in op r op yl)-1,3,5-tr ia zin e-2,4,6-
tr ia m in e Tetr a h yd r och lor id e (5f). Starting from 4f (100
mg, 0.233 mmol), 2-chloro-4,6-diamino-1,3,5-triazine (75 mg,
0.515 mmol), and NaHCO3 (45 mg, 0.536 mmol), 5f was
obtained as a colorless powder (125 mg, 90%): Rf (SiO2, 35%
3
>350 °C; 1H NMR (CD3OD/D2O 10:1) δ/ppm ) 3.52 (t, 4H, J
3
) 6.50 Hz), 3.09 (m, 8H), 2.03 (tt, 4H 3J ) 7.38 Hz, J ) 6.68
Hz), 1.84 (m, 4H); NH exchanged; 13C NMR (CD3OD/D2O 10:
1) δ/ppm ) 162.6 (s), 158.0 (s), 48.2 (t), 46.5 (t), 38.7 (t), 27.2
(t), 24.2 (t); MS (ES+) m/z ) 421 (M+H+, 100%), 211 ((M+2H+)/
2, 50%); HR-MS (ES+) calcd for (C16H33N14)+: 421.3012; found
421.3012. Anal. (C16H32N14‚4HCl‚1H2O) H, N; C: calcd, 32.9;
found, 33.4; Cl: calcd, 24.3; found, 24.8.
1
aq NH3/MeOH 1:10) 0.14; mp(sealed tube) dec > 300 °C; H
NMR (CD3OD) δ/ppm ) 3.52 (m, 4H), 3.11 (m, 6H), 2.30 (m,
4H), 2.01 (m, 4H), 1.58 (m, 4H); NH exchanged; 13C NMR
(CD3OD) δ/ppm ) 163.0 (s), 158.4 (s), 57.0 (d), 44.1 (t), 39.1
(t), 28.1 (t), 27.7 (t); MS (ES+) m/z ) 447 (M+H+, 100%),
224 ((M+2H+)/2, 60%); HR-MS (ES+) calcd for (C18H35N14)+:
447.3169; found 447.3167. Anal. (C18H34N14‚4HCl‚2H2O) H, N,
Cl; C: calcd, 34.4; found, 33.9.
N2-(3-[9-(3-[(4,6-Dia m in o-1,3,5-tr ia zin -2-yl)a m in o]p r o-
p yla m in o)n on yl]a m in op r op yl)-1,3,5-t r ia zin e-2,4,6-t r i-
a m in e Tetr a h yd r och lor id e (5b). Starting from 4b (100 mg,
0.212 mmol), 2-chloro-4,6-diamino-1,3,5-triazine (68 mg, 0.467
mmol), and NaHCO3 (40 mg, 0.476 mmol), 5b was obtained
as off-white powder (120 mg, 89%): Rf (SiO2, 35% aq NH3/
MeOH 1:10) 0.16; mp(sealed tube) dec in two steps: 1. >220
°C, 2. >360 °C; 1H NMR (CD3OD) δ/ppm ) 3.52 (m, 4H), 3.06
(m, br, 8H), 2.02 (m, 4H), 1.73 (m, 4H), 1.39 (m, 10H); NH
exchanged; 13C NMR (CD3OD) δ/ppm ) 162.8 (s), 158.0 (s),
48.1 (t), 46.7 (t), 38.8 (t), 30.1 (t), 30.0 (t), 27.5 (t), 27.3 (t),
27.2 (t); MS (ES+) m/z ) 491 (M+H+, 100%), 246 ((M+2H+)/2,
60%); HR-MS (ES+) calcd for (C21H43N14)+: 491.3795; found
(b) Bis Ad d ition s w ith Am in o Su bstitu ted 2-Ch lor o-
1,3,5-tr ia zin es. N2-(3-((9-((3-((4-Am in o-6-(m eth yla m in o)-
1,3,5-t r ia zin -2-yl)a m in o)p r op yl)a m in o)n on yl)a m in o)-
pr opyl)-N4-m eth yl-1,3,5-tr iazin e-4,2,6-tr iam in e Tetr ah ydr o-
ch lor id e (6a ). Starting from 4b (100 mg, 0.212 mmol),
2-chloro-4-amino-6-methylamino-1,3,5-triazine (2 × 75 mg, 2
× 0.470 mmol), and NaHCO3 (2 × 40 mg, 2 × 0.476 mmol),
6a was isolated as light yellow powder (100 mg, 71%): Rf (SiO2,
35% aq NH3/MeOH 1:10) 0.31; mp(sealed tube) > 120 °C slow

Synthesis of s-Triazine Substituted Polyamines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3449
dec (foam), > 300 dec; 1H NMR (CD3OD) δ/ppm ) 3.51 (m,
4H), 3.14-2.88 (m, 14H), 2.03 (m, 4H), 1.72 (m, 4H), 1.38 (m,
10H); EtOH signals at 3.61 (q), 1.18 (t); NH exchanged; 13C
NMR (CD3OD) δ/ppm ) 161.7 (s), 157.5 (s), 49.2 (t), 46.7 (t),
46.5 (t), 39.0 (t), 38.6 (t), 30.1 (t), 30.0 (t), 28.0 (q), 27.7 (q),
27.5 (t), 27.2 (t); EtOH signals at 53.6 (t), 18.4 (q); MS (ES+)
m/z ) 519 (M+H+, 60%), 260 ((M+2H+)/2, 100%); HR-MS
(ES+) calcd for (C23H47N14)+: 519.4108; found 519.4102. Anal.
(C23H46N14‚4HCl‚1.5EtOH‚1H2O) C, H, N; Cl: calcd, 18.8,
found, 19.4.
(t), 30.6 (t), 30.4 (t), 30.2 (t), 28.0 (q), 27.7 (q), 27.6 (t), 27.2 (t),
27.1 (t); EtOH signals at 53.6 (t), 18.4 (q); MS (ES+) m/z )
597 (M+HCl+H+, 25%), 561 (M+H+, 70%), 281 ((M+2H+)/2,
100%); HR-MS (ES+) calcd for (C26H53N14)+: 561.4577; found
561.4586. Anal. (C26H52N14‚4.5HCl‚1EtOH 2H2O) C, H, N, Cl.
N2-[3-(12-[(3-[4-Am in o-6-(d im eth yla m in o)-1,3,5-tr ia zin -
2-yl]a m in op r op yl)a m in o]d od ecyla m in o)p r op yl]-N4,N4-
d im eth yl-1,3,5-tr ia zin e-2,4,6-tr ia m in e P en ta h yd r och lo-
r id e (6f). Starting from 4c (100 mg, 0.194 mmol), 2-chloro-4-
amino-6-(dimethylamino)-1,3,5-triazine (2 × 74 mg, 2 × 0.426
mmol), and NaHCO3 (2 × 36 mg, 2 × 0.429 mmol), 6f was
isolated as white powder (110 mg, 73%): Rf (SiO2, 35% aq NH3/
MeOH 1:10) 0.38; mp(sealed tube) > 200 °C slow dec (foam),
N2-(3-((9-((3-((4-Am in o-6-(d im eth yla m in o)-1,3,5-tr ia zin -
2-yl)a m in o)p r op yl)a m in o)n on yl)a m in o)p r op yl)-N4,N4-
d im eth yl-1,3,5-tr ia zin e-4,2,6-tr ia m in e P en ta h yd r och lo-
r id e (6b). Starting from 4b (100 mg, 0.212 mmol), 2-chloro-
4-amino-6-(dimethylamino)-1,3,5-triazine (2 × 80 mg, 2 ×
0.461 mmol), and NaHCO3 (2 × 40 mg, 2 × 0.476 mmol), 6b
was isolated as light yellow powder (100 mg, 65%): Rf (SiO2,
35% aq NH3/MeOH 1:10) 0.43; mp(sealed tube) > 150 slow
1
> 300 °C dec; H NMR (CD3OD) δ/ppm ) 3.55 (m, 4H), 3.22/
3.18 (2s, 12H), 3.09 (m, 4H), 3.01 (m, 4H), 2.05 (m, 4H), 1.70
(m, 4H), 1.34-1.29 (m, 16H); EtOH signals at 3.61 (q), 1.18
(t); NH exchanged; 13C NMR (CD3OD) δ/ppm ) 162.2 (s), 157.4
(s), 157.2 (s), 49.0 (t), 46.4 (t), 38.8 (t), 37.6 (q), 37.3 (q), 30.3
(t), 30.2 (t), 29.9 (t), 27.3 (t), 27.0 (t), 26.7 (t); EtOH signals at
53.6 (t), 18.4 (q); MS (ES+) m/z ) 625 (M+HCl+H+, 12%), 589
(M+H+, 8%), 295 ((M+2H+)/2, 100%); HR-MS (ES+) calcd for
1
dec (foam), 270-290 °C dec; H NMR (CD3OD) δ/ppm ) 3.56
(m, br, 4H), 3.23/3.20 (2s, 12H), 3.10 (m, 4H), 3.02 (m, 4H),
2.06 (m, 4H), 1.73 (m, 4H), 1.38 (m, 10H); EtOH signals at
3.61 (q), 1.18 (t); NH exchanged; 13C NMR (CD3OD) δ/ppm )
163.0 (s), 157.5 (s), 157.0 (s), 49.2 (t), 46.7 (t), 38.9 (t), 37.4
(q), 37.2 (q), 30.1 (t), 30.0 (t), 27.5 (t), 27.2 (t), 27.0 (t); EtOH
signals at 53.6 (t), 18.4 (q); MS (ES+) m/z ) 547 (M+H+, 95%),
274 ((M+2H+)/2, 100%); HR-MS (ES+) calcd for (C25H51N14)+:
547.4421; found 547.4423. Anal. (C25H50N14‚4.5HCl‚2EtOH‚
1H2O) C, H, N; Cl: calcd, 19.4, found, 20.2.
(C28H57N14)+: 589.4890; found 589.4895. Anal. (C28H56N14
4.5HCl‚1EtOH‚1H2O) C, H, N, Cl.
‚
N2-[3-(12-[(3-[4,6-Di(m et h yla m in o)-1,3,5-t r ia zin -2-yl]-
am in opr opyl)am in o]dodecylam in o)pr opyl]-N4,N6-dim eth -
yl-1,3,5-tr ia zin e-2,4,6-tr ia m in e p en ta h yd r och lor id e (6g).
Starting from 4c (100 mg, 0.194 mmol), 2-chloro-4,6-bis-
(methylamino)-1,3,5-triazine (2 × 75 mg, 2 × 0.432 mmol), and
NaHCO3 (2 × 36 mg, 2 × 0.429 mmol), 6g was isolated as
white powder (65 mg, 43%): Rf (SiO2, 35% aq NH3/MeOH 1:10)
0.40; mp(sealed tube) > 210 °C dec; 1H NMR (CD3OD) δ/ppm
) 3.57 (m, 4H), 3.08 (m, 4H), 3.03 (m, 4H), 2.98/2.92 (2s, 12H),
2.04 (m, 4H), 1.71 (m, 4H), 1.36-1.31 (m, 16H); EtOH signals
at 3.61 (q), 1.18 (t); NH exchanged; 13C NMR (CD3OD) δ/ppm
) 164.4 (s), 156.3 (s), 155.9 (s), 49.2 (t), 49.1 (t), 46.6 (t), 46.3
(t), 45.8 (t), 39.1 (t), 38.9 (t), 38.5 (t), 37.9 (t), 30.5 (t), 30.4 (t),
30.1 (t), 27.7 (q, br), 27.5 (t), 27.2 (t), 27.0 (t), 24.8 (t,); EtOH
signals at 53.6 (t), 18.4 (q); MS (ES+) m/z ) 625 (M+H++HCl,
10%), 589 (M+H+, 30%), 295 ((M+2H+)/2, 100%), 227 (15%),
195 (15%); HR-MS (ES+) calcd for (C28H57N14)+: 589.4890;
found 589.4899. Anal. (C28H56N14‚5HCl‚2.5EtOH) H, N, Cl; C:
calcd, 44.7; found, 45.3.
N2-(3-((9-((3-((4,6-Di(m et h yla m in o)-1,3,5-t r ia zin -2-yl)-
am in o)pr opyl)am in o)n on yl)am in o)pr opyl)-N4,N6-dim eth -
yl-1,3,5-tr ia zin e-6,4,2-tr ia m in e Tetr a h yd r och lor id e (6c).
Starting from 4b (100 mg, 0.212 mmol), 2-chloro-4,6-bis-
(methylamino)-1,3,5-triazine (2 × 81 mg, 2 × 0.467 mmol), and
NaHCO3 (2 × 40 mg, 2 × 0.476 mmol), 6c was isolated as light
yellow powder (90 mg, 61%): Rf (SiO2, 35% aq NH3/MeOH
1:10) 0.41; mp(sealed tube) > 120 °C slow dec (foam), 280-
290 °C dec; 1H NMR (CD3OD) δ/ppm ) 3.57-3.47 (m, 4H),
3.14-2.99 (m, 8H), 2.98/2.92 (2s, 12H), 2.05 (m, 4H), 1.72 (m,
4H), 1.38 (m, 10H); NH exchanged; 13C NMR (CD3OD) δ/ppm
) 156.4 (s), 156.0 (s), 49.1 (t), 46.7 (t), 46.4 (t), 39.1 (t), 38.9
(t), 38.6 (t), 30.1 (t), 30.0 (t), 27.9 (q), 27.7 (q), 27.5 (t), 27.2 (t),
27.1 (t); MS (ES+) m/z ) 547 (M+H+, 5%), 274 ((M+2H+)/2,
100%); HR-MS (ES+) calcd for (C25H51N14)+: 547.4421; found
547.4418. Anal. (C25H50N14‚4HCl‚1EtOH‚2H2O) C, H, N; Cl:
calcd, 18.3; found, 18.8.
N2-[3-(12-[(3-[4,6-Di(d im eth yla m in o)-1,3,5-tr ia zin -2-yl]-
a m in op r op yl)a m in o]d od ecyla m in o)p r op yl]-N4,N4,N6,N6-
t et r a m et h yl-1,3,5-t r ia zin e-2,4,6-t r ia m in e P en t a h yd r o-
ch lor id e (6h ). Starting from 4c (100 mg, 0.194 mmol),
2-chloro-4,6-bis(dimethylamino)-1,3,5-triazine (2 × 86 mg, 2
× 0.426 mmol), and NaHCO3 (2 × 36 mg, 2 × 0.429 mmol),
6h was isolated as white powder (95 mg, 59%): Rf (SiO2, 35%
aq NH3/MeOH 1:10) 0.52; mp(sealed tube) 192-195 °C; 1H
NMR (CD3OD) δ/ppm ) 3.59 (m, 4H), 3.21 (s, br, 24H), 3.10
(m, 4H), 3.00 (m, 4H), 2.07 (m, 4H), 1.71 (m, 4H), 1.36-1.31
(2m, 16H); EtOH signals at 3.61 (q), 1.18 (t); NH exchanged;
13C NMR (CD3OD) δ/ppm ) 163.1 (s), 156.6 (s), 155.2 (s), 49.1
(t), 46.5 (t), 38.9 (t), 37.3 (q, br), 30.5 (t), 30.4 (t), 30.1 (t), 27.5
(t), 27.2 (t), 26.8 (t); EtOH signals at 53.6 (t), 18.4 (q); MS (ES+)
m/z ) 681 (M+H++HCl, 30%), 645 (M+H+, 35%), 323
((M+2H+)/2, 100%); HR-MS (ES+) calcd for (C32H65N14)+:
645.5516; found 645.5515. Anal. (C32H64N14‚4.5HCl‚1.5EtOH)
C, H, N, Cl.
(c) Mon o Ad d ition s w ith Am in o Su bstitu ted 2-Ch lor o-
1,3,5-tr ia zin es. N2-(3-((9-((3-((4,6-Di(m eth yla m in o)-1,3,5-
tr ia zin -2-yl)a m in o)p r op yl)a m in o)n on yl)a m in o)p r op yl)-
N4,N6-d im eth yl-1,3,5-tr ia zin e-6,4,2-tr ia m in e P en ta h yd r o-
ch lor id e (7a ). Starting from 4b (100 mg, 0.212 mmol),
2-chloro-4,6-bis(methylamino)-1,3,5-triazine (81 mg, 0.467
mmol), and NaHCO3 (40 mg, 0.476 mmol), 7a was isolated as
off-white powder (60 mg, 48%): Rf (SiO2, 35% aq NH3/MeOH
1:10) 0.09; mp(sealed tube) > 150 °C slow dec (foam), 220-
225 °C dec; 1H NMR (CD3OD) δ/ppm ) 3.63-3.46 (m, 2H),
3.19-2.88 (m, 16H), 2.20-1.96 (m, 4H), 1.80-1.64 (m, 4H),
1.49-1.27 (m, 10H); EtOH signals at 3.61 (q), 1.18 (t); NH
exchanged; 13C NMR (CD3OD) δ/ppm ) 161.7 (s), 161.3 (s),
49.2 (t), 45.9 (t), 37.9 (t), 30.1 (t), 30.0 (t), 27.7 (q), 27.4 (t),
N2-(3-((9-((3-((4,6-Di(d im eth yla m in o)-1,3,5-tr ia zin -2-yl)-
a m in o)p r op yl)a m in o)n on yl)a m in o)p r op yl)-N4,N4,N6,N6-
t et r a m et h yl-1,3,5-t r ia zin e-6,4,2-t r ia m in e P en t a h yd r o-
ch lor id e (6d ). Starting from 4b (100 mg, 0.212 mmol),
2-chloro-4,6-bis(dimethylamino)-1,3,5-triazine (2 × 94 mg, 2
× 0.466 mmol), and NaHCO3 (2 × 40 mg, 2 × 0.476 mmol),
6d was isolated as light yellow powder (50 mg, 30%): Rf (SiO2,
35% aq NH3/MeOH 1:10) 0.61; mp(sealed tube) > 120 °C slow
1
dec (foam), 270-280 °C dec; H NMR (CD3OD) δ/ppm ) 3.60
(m, 4H), 3.22 (s, br, 24H), 3.12 (m, 4H), 3.03 (m, 4H), 2.08 (m,
4H), 1.73 (m, 4H), 1.39 (m, 10H); NH exchanged; 13C NMR
(CD3OD) δ/ppm ) 163.1 (s), 156.7 (s), 155.2 (s), 49.3 (t), 46.8
(t), 39.0 (t), 37.4 (q), 30.0 (t), 27.5 (t), 27.2 (t), 26.9 (t); MS (ES+)
m/z ) 603 (M+H+, 80%), 302 ((M+2H+/2, 100%); HR-MS (ES+)
calcd for (C29H59N14)+: 603.5047; found 603.5039. Anal.
(C29H58N14‚5HCl‚3H2O) C, H, N; Cl, calcd, 21.1; found, 21.9.
N2-[3-(12-[(3-[4-Am in o-6-(m eth yla m in o)-1,3,5-tr ia zin -2-
yl]a m in op r op yl)a m in o]d od ecyla m in o)p r op yl]-N4-m eth -
yl-1,3,5-tr ia zin e-2,4,6-tr ia m in e P en ta h yd r och lor id e (6e).
Starting from 4c (100 mg, 0.194 mmol), 2-chloro-4-amino-6-
methylamino-1,3,5-triazine (2 × 68 mg, 2 × 0.426 mmol), and
NaHCO3 (2 × 36 mg, 2 × 0.429 mmol) 6e was isolated as white
powder (120 mg, 83%): Rf (SiO2, 35% aq NH3/MeOH 1:10) 0.31;
1
mp(sealed tube) > 190 °C slow dec (foam), > 300 °C dec; H
NMR (CD3OD) δ/ppm ) 3.52 (m, 4H), 3.14-2.91 (m, 8H), 2.98/
2.94 (2s, 6H), 2.03 (m, 4H), 1.72 (m, 4H), 1.36-1.32 (m, 16H);
EtOH signals at 3.61 (q), 1.18 (t); NH exchanged; 13C NMR
(CD3OD) δ/ppm ) 164.5)s), 163.9 (s), 160.7 (s), 158.9 (s), 157.4
(s), 157.0 (s), 49.1 (t), 46.7 (t), 46.4 (t), 39.0 (t), 38.9 (t), 38.6

3450 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Klenke et al.
27.2 (t), 25.4 (t); EtOH signals at 53.6 (t), 18.4 (q); MS (ES+)
m/z ) 410 (M+H+, 20%), 206 ((M+2H+)/2, 100%); HR-MS
(ES+) calcd for (C20H44N9)+: 410.3720; found 410.3724. Anal.
(C20H43N9‚5HCl‚1.5EtOH‚1H2O) C, H, N, Cl.
241 ((M+2H+)/2, 30%); HR-MS (ES+) calcd for (C25H54N9)+:
480.4502; found 480.4501. Anal. (C25H53N9‚5.5HCl‚3EtOH) C,
H, N, Cl.
(d ) Tetr a -Ad d u cts w ith Am in o Su bstitu ted 2-Br om o-
m eth yl-1,3,5-tr ia zin es. N1,N9-Di(3-(d i((4,6-d ia m in o-1,3,5-
t r ia zin -2-yl)m et h yl)a m in o)p r op yl)-1,9-n on a n ed ia m in e
Hep ta h yd r och lor id e (8a ). Starting from 4b (120 mg, 0.254
mmol), 2-bromomethyl-4,6-diamino-1,3,5-triazine (2 × 115 mg,
2 × 0.564 mmol), and NaHCO3 (2 × 50 mg, 2 × 0.595 mmol),
8a was isolated as light yellow powder (210 mg, 81%): Rf (SiO2,
35% aq NH3/MeOH 1:10) baseline; mp(sealed tube) > 120 °C
N 2-[3-(12-[(3-Am in o p r o p y l)a m in o ]d o d e c y la m in o )-
p r op yl]-N4-m eth yl-1,3,5-tr ia zin e-2,4,6-tr ia m in e Tetr a h y-
d r och lor id e (7b). Starting from 4c (100 mg, 0.194 mmol),
2-chloro-4-amino-6-methylamino-1,3,5-triazine (63 mg, 0.395
mmol), and NaHCO3 (33 mg, 0.393 mmol), an 85:15 mixture
of mono-adduct 7b and bis-adduct 6e was isolated as an off-
white powder (100 mg): Rf (SiO2, 35% aq NH3/MeOH 1:10)
1
1
slow dec (foam), 280-300 °C dec; H NMR (CD3OD) δ/ppm )
0.07 (7b) and 0.32 (6e); mp(sealed tube) 280-290 °C dec; H
3.94/3.92 (2s, 4H, Ar-CH2-N), 3.16 (m, 4H), 3.03 (m, 4H), 2.83
(m, 4H), 2.06 (m, 4H), 1.72 (m, 4H), 1.38 (m, 10H); EtOH
signals at 3.61 (q), 1.18 (t); NH exchanged, Ar-CH2-N
partially exchanged; 13C NMR (CD3OD) δ/ppm ) 167.6 (s),
162.3 (s), 58.3 (t), 58.0 (t), 49.0 (t), 46.8 (t), 30.0 (t), 29.9 (t),
27.4 (t), 27.2 (t), 24.6 (t); EtOH signals at 53.6 (t), 18.4 (q);
MS (ES+) m/z ) 765 (M+H+, 5%), 383 ((M+2H+/2, 100%),
256 ((M+3H+/3, 25%); HR-MS (ES+) calcd for (C31H57N24)+:
765.5198; found 765.5199. Anal. (C31H56N24‚7HCl‚2.5EtOH ‚
2H2O) C, H, N, Cl.
N1,N9-Di(3-(((4-a m in o-6-(d im eth yla m in o)-1,3,5-tr ia zin -
2-yl)m eth yl)a m in o)p r op yl)-1,9-n on a n ed ia m in e Hep ta h y-
d r och lor id e (8b). Starting from 4b (100 mg, 0.212 mmol), 2-
bromomethyl-4-amino-6-(dimethylamino)-1,3,5-triazine (100+50
mg, 0.431+0.215 mmol), and NaHCO3 (36+18 mg, 0.429+0.214
mmol), 8b was isolated as light yellow powder (150 mg, 63%):
Rf (SiO2, 35% aq NH3/MeOH 1:10) baseline; mp(sealed tube)
> 150 °C slow dec (foam), 285-295 °C dec; 1H NMR (CD3OD)
δ/ppm ) 4.00 (s, 4H, Ar-CH2-N), 3.29/3.24 (2s, 24H), 3.10
(m, 4H), 3.01 (m, 4H), 2.91 (m, 4H), 2.02 (m, 4H), 1.70 (m,
4H), 1.36 (m, 10H); EtOH signals at 3.61 (q), 1.18 (t); NH
exchanged, Ar-CH2-N partially exchanged; 13C NMR (CD3OD)
δ/ppm ) 166.6 (s), 162.6 (s), 157.3 (s), 58.3 (t), 57.4 (t), 49.1
(t), 46.7 (t), 37.9 (q), 37.8 (q), 29.9 (t), 29.8 (t), 27.3 (t), 27.1 (t),
24.8 (t); EtOH signals at 53.6 (t), 18.4 (q); MS (ES+) m/z )
916 (M+HCl+H+, 95%), 878 (M+H+, 10%), 440 ((M+2H+)/2,
100%); HR-MS (ES+) calcd for (C39H73N24)+: 877.6450; found
877.6460. Anal. (C39H72N24‚7.5HCl‚3EtOH‚2H2O) C, H, N, Cl.
NMR (CD3OD): 7b; δ/ppm ) 3.61-3.46 (m, 2H), 3.19-2.89
(m, 13H), 2.17-1.94 (m, 4H), 1.76-1.61 (m, 4H), 1.43-1.23
(m, 16H); 6e; δ/ppm ) 3.61-3.46 (m, 4H), 3.19-2.89 (m, 14H),
2.17-1.94 (m, 4H), 1.76-1.61 (m, 4H), 1.43-1.23 (m, 16H);
NH exchanged; 13C NMR (CD3OD): 7b; δ/ppm ) 161.3 (s)
160.0 (s) 157.1 (s), 48.8 (t), 45.5 (t), 37.7 (t), 30.0 (t), 29.9 (t),
29.6 (t), 28.1 (q), 27.0 (t), 26.8 (t), 24.9 (t); 6e; δ/ppm ) 161.3
(s), 160.0 (s), 157.1 (s), 49.0 (t), 46.2 (t), 45.9 (t), 38.8 (t), 38.6
(t), 38.4 (t), 30.0 (t), 29.9 (t), 29.6 (t), 28.1 (q), 27.9 (q), 27.0 (t),
26.8 (t), 26.7 (t); MS (ES+) m/z ) 561 (M6e+H+, 50%), 438
(M7b+H+, 40%)
N 2-[3-(12-[(3-Am in o p r o p y l)a m in o ]d o d e c y la m in o )-
p r op yl]-N4,N4-d im eth yl-1,3,5-tr ia zin e-2,4,6-tr ia m in e Tet-
r a h yd r och lor id e (7c). Starting from 4c (100 mg, 0.194
mmol), 2-chloro-4-amino-6-(dimethylamino)-1,3,5-triazine (70
mg, 0.403 mmol), and NaHCO3 (34 mg, 0.405 mmol), a 90:10
mixture of monoadduct 7c and bis-adduct 6f was isolated as
an off-white powder (80 mg): Rf (SiO2, 35% aq NH3/MeOH
1:10) 0.07 (7c) and 0.39 (6f); mp(sealed tube) 280-290 °C dec;
1H NMR (CD3OD): 7c; δ/ppm ) 3.69-3.47 (m, 2H), 3.28-2.95
(m, 16H), 2.24-1.99 (m, 4H), 1.83-1.63 (m, 4H), 1.50-1.24
(m, 16H); 6f; δ/ppm ) 3.69-3.47 (m, 4H), 3.28-2.95 (m, 20H),
2.24-1.99 (m, 4H), 1.83-1.63 (m, 4H), 1.50-1.24 (m, 16H);
NH exchanged; 13C NMR (CD3OD): 7c; δ/ppm ) 162.5 (s)
157.4 (s) 157.2 (s), 49.0 (t), 45.8 (t), 37.8 (t), 37.5 (q), 30.4 (t),
30.3 (t), 30.0 (t), 27.4 (t), 27.1 (t), 25.1 (t); 6f; δ/ppm ) 162.5
(s), 157.4 (s), 157.2 (s), 49.1 (t), 46.5 (t), 45.8 (t), 38.8 (t), 37.8
(t), 37.5 (q), 37.3 (q), 30.4 (t), 30.3 (t), 30.0 (t), 27.4 (t), 27.1 (t),
26.8 (t); MS (ES+) m/z ) 589 (M6f+H+, 40%), 452 (M7c+H+,
25%)
N1,N9-Di(3-(((4,6-d i(m et h yla m in o)-1,3,5-t r ia zin -2-yl)-
m eth yl)a m in o)p r op yl)-1,9-n on a n ed ia m in e Hep ta h yd r o-
ch lor id e (8c). Starting from 4b (100 mg, 0.212 mmol),
2-bromomethyl-4,6-bis(methylamino)-1,3,5-triazine (2 × 100+50
mg, 2 × 0.431+0.215 mmol), and NaHCO3 (2 × 36+18 mg, 2
× 0.429+0.214 mmol), 8c was isolated as light yellow powder
(100 mg, 42%): Rf (SiO2, 35% aq NH3/MeOH 1:10) baseline;
N 2-[3-(12-[(3-Am in o p r o p y l)a m in o ]d o d e c y la m in o )-
pr opyl]-N4,N6-dim eth yl-1,3,5-tr iazin e-2,4,6-tr iam in e P en ta-
h yd r och lor id e (7d ). Starting from 4c (100 mg, 0.194 mmol),
2-chloro-4,6-bis(methylamino)-1,3,5-triazine (70 mg, 0.403
mmol), and NaHCO3 (34 mg, 0.405 mmol), 7d was isolated as
an off-white powder (80 mg, 65%): Rf (SiO2, 35% aq NH3/
1
mp(sealed tube) > 150 °C slow dec (foam), > 245 °C dec; H
NMR (CD3OD) δ/ppm ) 3.87 (s, 4H, Ar-CH2-N), 3.12 (m, 4H),
3.07-2.95 (m, 4H), 3.05/2.99 (2s, 24H), 2.82 (m, 4H), 2.01 (m,
4H), 1.71 (m, 4H), 1.37 (m, 10H); EtOH signals at 3.61 (q),
1.18 (t); NH exchanged, Ar-CH2-N partially exchanged; 13C
NMR (CD3OD) δ/ppm ) 166.2 (s), 160.3 (s), 58.3 (t), 57.4 (t),
49.0 (t), 46.8 (t), 30.0 (t), 29.9 (t), 28.1 (q), 27.4 (t), 27.1 (t),
24.8 (t), 24.7 (t); EtOH signals at 53.6 (t), 18.4 (q); MS (ES+)
m/z ) 916 (M+HCl+H+, 50%), 900 (M+Na+, 20%), 878 (M+H+,
15%), 440 ((M+2H+)/2, 100%); HR-MS (ES+) calcd for
1
MeOH 1:10) 0.11; mp(sealed tube) 300-305 °C dec; H NMR
(CD3OD) δ/ppm ) 3.54 (m, 2H), 3.20-2.89 (m, 16H), 2.13 (m,
4H), 1.72 (m, 4H), 1.35/1.30 (2m, 16H); EtOH signals at 3.52
(q), 1.18 (t); NH exchanged; 13C NMR (CD3OD) δ/ppm ) 156.2
(s), 155.8 (s), 49.1 (t,), 45.7 (t), 37.8 (t), 30.3 (t), 30.2 (t), 30.0
(t), 27.7 (q), 27.3 (t), 27.0 (t), 25.1 (t); EtOH signals at 53.6 (t),
18.4 (q); MS (ES+) m/z ) 488 (M+HCl+H+, 30%), 452 (M+H+,
70%), 227 ((M+2H+)/2, 60%); HR-MS (ES+) calcd for
(C23H50N9)+: 452.4189; found 452.4191. Anal. (C23H49N9‚5HCl‚
2EtOH) C, H; N: calcd, 17.4; found, 16.8; Cl: calcd, 24.4; found,
24.9.
(C39H73N24)+: 877.6450; found 877.6449; Anal. (C39H72N24
7HCl‚2.5EtOH) C, H, N; Cl: calcd, 19.9; found, 19.4.
‚
N1,N12-Di(3-[(4,6-d ia m in o-1,3,5-t r ia zin -2-yl)m et h yl]-
a m in op r op yl)-1,12-d od eca n ed ia m in e Hep ta h yd r och lo-
r id e (8d ). Starting from 4c (100 mg, 0.194 mmol), 2-bromo-
methyl-4,6-diamino-1,3,5-triazine (3 × 80 mg, 3 × 0.392 mmol),
and NaHCO3 (3 × 33 mg, 3 × 0.393 mmol), 8d was isolated
as light yellow powder (146 mg, 71%): Rf (SiO2, 35% aq NH3/
MeOH 1:10) baseline; mp(sealed tube) > 260 °C dec; 1H NMR
N 2-[3-(12-[(3-Am in o p r o p y l)a m in o ]d o d e c y la m in o )-
p r op yl]-N4,N4,N6,N6-t et r a m et h yl-1,3,5-t r ia zin e-2,4,6-t r i-
a m in e P en ta h yd r och lor id e (7e). Starting from 4c (100 mg,
0.194 mmol), 2-chloro-4,6-bis(dimethylamino)-1,3,5-triazine (80
mg, 0.397 mmol), and NaHCO3 (33 mg, 0.393 mmol), 7e was
isolated as a light yellow powder (80 mg, 62%): Rf (SiO2, 35%
aq NH3/MeOH 1:10) 0.11; mp(sealed tube) 310-315 °C dec;
1H NMR (CD3OD) δ/ppm ) 3.58 (m, 2H), 3.27-2.95 (m, 22H),
2.12 (m, 4H), 1.71 (m, 4H), 1.35/1.30 (2m, 16H); EtOH signals
at 3.52 (q), 1.18 (t); EtOH signals at 3.52 (q), 1.18 (t); NH
exchanged; 13C NMR (CD3OD) δ/ppm ) 156.5 (s), 155.1 (s),
49.1 (t), 45.7 (t), 37.8 (t), 37.3 (q), 30.3 (t), 30.2 (t), 29.9 (t),
27.3 (t), 27.0 (t), 25.1 (t); EtOH signals at 53.6 (t), 18.4 (q);
MS (ES+) m/z ) 516 (M+HCl+H+, 10%), 480 (M+H+, 30%),
3
(CD3OD) δ/ppm ) 3.92 (s, 6H, Ar-CH2-N), 3.15 (t, 4H, J )
7.87 Hz), 3.02 (t, 4H, 3J ) 7.87 Hz), 2.81 (t, 4H, 3J ) 7.23 Hz),
2.05 (m, 4H), 1.72 (m, 4H), 1.37-1.32 (m, 16H); EtOH signals
at 3.61 (q), 1.18 (t); NH exchanged, Ar-CH2-N partially
exchanged; 13C NMR (CD3OD) δ/ppm ) 167.7 (s), 162.4 (s, br),
58.1 (t), 53.6 (t), 49.1 (t), 46.8 (t), 30.5 (t), 30.4 (t), 30.2 (t),
27.6 (t), 27.3 (t), 24.7 (t); EtOH signals at 53.6 (t), 18.4 (q);
MS (ES+) m/z ) 843 (M+HCl+H+, 10%), 807 (M+H+, 5%), 404

Synthesis of s-Triazine Substituted Polyamines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3451
((M+2H+)/2, 100%); HR-MS (ES+) calcd for (C34H63N24)+:
807.5667; found 807.5671. Anal. (C34H62N24‚6.5HCl‚1EtOH‚
2H2O) C, H, N, Cl.
1.25 (m, 30H), 0.87 (m, 3H); NH exchanged; 13C NMR (CD3OD/
CDCl3 10:1) δ/ppm ) 166.4 (s, br), 41.5 (t), 33.1 (t), 30.8 (t),
30.5 (t), 28.0 (t), 27.7 (q), 23.8 (t), 14.5 (q); MS (ES+) m/z )
407 (M+H+, 100%); HR-MS (ES+) calcd for (C23H47N6)+:
407.3862; found 407.3866. Anal. (C23H46N6‚0.5H2O) C, H, N.
N1,N12-Di[3-([4-a m in o-6-(d im eth yla m in o)-1,3,5-tr ia zin -
2-yl]m eth yla m in o)p r op yl]-1,12-d od eca n ed ia m in e Hep ta -
h yd r och lor id e (8e). Starting from 4c (100 mg, 0.194 mmol),
2-bromomethyl-4-amino-6-(dimethylamino)-1,3,5-triazine (2 ×
93 mg, 2 × 0.401 mmol), and NaHCO3 (2 × 35 mg, 3 × 0.417
mmol), 8e was isolated as light yellow powder (200 mg, 88%):
Rf (SiO2, 35% aq NH3/MeOH 1:10) baseline; mp(sealed tube)
155-165 °C dec; 1H NMR (CD3OD) δ/ppm ) 3.96 (s, 5H, Ar-
Ack n ow led gm en t. Funding by the Wellcome Trust
to I.H.G. and M.P.B. is gratefully acknowledged. We
thank Elke Gobright, Les Craven, Ron Edwards, and
Huw Griffiths for technical assistance. This investiga-
tion received financial support from the UNDP/World
Bank/WHO Special Programme for Research and Train-
ing in Tropical Diseases (TDR). We would also like to
acknowledge the EPSRC National Mass Spectrometry
Service Centre for accurate mass spectroscopy.
3
CH2-N), 3.22/3.16 (2s, 24H), 3.16 (t, 4H, J ) 7.96 Hz), 3.03
3
3
(t, 4H, J ) 7.78 Hz), 2.83 (t, 4H, J ) 7.04 Hz), 2.07 (m, 4H),
1.72 (m, 4H), 1.37-1.33 (m, 16H); EtOH signals at 3.61 (q),
1.18 (t); NH exchanged, Ar-CH2-N partially exchanged; 13C
NMR (CD3OD) δ/ppm ) 166.3 (s), 162.9 (s), 157.6 (s), 58.2 (t),
53.6 (t), 49.8 (t), 46.7 (t), 37.7 (q), 37.6 (q), 30.6 (t), 30.5 (t),
30.2 (t), 27.6 (t), 27.3 (t), 24.7 (t); EtOH signals at 58.3 (t),
18.4 (q); MS (ES+) m/z ) 955 (M+HCl+H+, 20%), 919 (M+H+,
30%), 460 ((M+2H+)/2, 100%); HR-MS (ES+) calcd for
Refer en ces
(1) (a) Trouiller, P.; Olliaro, P. L. Drug development output: what
proportion for tropical diseases? Lancet 1999, 354, 164. (b) Hirst,
S. I.; Stapley, L. A. Parasitology: The dawn of a new millennium.
Parasitol. Today 2000, 16, 1-3.
(2) Barrett, M. P. Problems for the chemotherapy of human African
trypanosomiasis. Curr. Opin. Infect. Dis. 2000, 13, 647-651.
(3) (a) WHO Expert Committee Control and Surveillance of
African Trypanosomiasis. In WHO Technical Report Series;
World Health Organisation: Geneva, 1998; Vol. 881. (b)
http://www.who.int. (c) http://www.who.int/tdr.
(4) Nichols, A. C.; Yielding, L.; Agbe, S. A. O. A Chlorodiazirine
Analog of Pentamidine with Anti-Trypanosomal Activity. J .
Parasitol. 2000, 86, 177-180.
(5) (a) Keiser, J .; Burri, C. Physico-chemical properties of the
trypanocidal drug melarsoprol. Acta Tropica 2000, 74, 101-104.
(b) Keiser, J .; Ericsson, O.; Burri, C. Investigations of the
Metabolites of the trypanocidal drug melarsoprol. Clin. Phar-
macol. Ther. 2000, 67, 478-488.
(6) Pepin, J .; Milord, F. The treatment of human African trypano-
somiasis. Adv. Parasitol. 1994, 33, 1-47.
(7) (a) Legros, D.; Fournier, C.; Etchegorry, M. G.; Maiso, F.;
Szumilin, E. Thera peutic failure of melarsoprol among patients
treated for late stage of T. b. gambiense human African try-
panosomiasis in Uganda. Bull. Soc. Pathol. Exot. 1999, 92, 171-
172. (b) Legros, D.; Evans, S.; Maiso, F.; Enyaru, J . C. K.;
Mbulamberi, D. Risk factors for treatment failure after melar-
soprol for Trypanosoma brucei gambiense trypanosomiasis in
Uganda. Trans. R. Soc. Trop. Med. Hyg. 1999, 93, 439-442. (c)
Barrett, M. P.; Fairlamb, A. H. The Biochemical Basis of
Arsenical-Diamidine Crossresistance in African Trypanosomes.
Parasitol. Today 1999, 15, 136-140.
(8) (a) Gutteridge, W. E.; Coombs, G. H. Biochemistry of parasitic
protozoa; Macmillan Press LTD: London, 1977. (b) Molyneux,
D. H.; Ashford, R. W. The biology of trypanosoma and leishma-
nia, parasites of man and domestic animals; Taylor & Francis
Ltd.: London, 1983. (c) Ristic, M. Infectious blood diseases of
man and animals, diseases caused by protista; Weinman, Ed.;
Academic Press: New York, 1968; Vol. 2.
(9) van Calenbergh, S.; Herdewijn, P. Rational Development of New
Sleeping Sickness Drugs. Curr. Med. Chem. 1997, 4, 359-384.
(10) Hide, G.; Mottram, J . C.; Coombs, G. H.; Holmes, P. H.
Trypanosomiasis and Leishmaniasis: Biology and Control; CAB
International: Oxon, 1997.
(11) (a) Zilberstein, D. Transport of nutrients and ions across
membranes of trypanosomatid parasites. Adv. Parasitol. 1993,
261-291. (b) Hasne, M. P.; Barrett, M. P. Drug uptake via
nutrient transporters in Trypanosoma brucei. J . Appl. Microbiol.
2000, 89, 697-701.
(12) (a) Carter, N. S.; Fairlamb, A. H. Arsenical-resistant trypano-
somes lack an usual adenosine transporter. Nature 1993, 361,
173-175. (b) A. H. Fairlamb, personal communication of un-
published data.
(13) Tye, C.-K.; Kasinathan, G.; Barrett, M. P.; Brun, R.; Doyle, V.
E.; Fairlamb, A. H.; Weaver, R.; Gilbert, I. H. An Approach To
Use An Unusual Adenosine Transporter To Selectively Deliver
Polyamine Analogues To Trypanosomes. Bioorg. Med. Chem.
Lett. 1998, 8, 811-816.
(14) De Koning, H. P.; J arvis, S. M. Adenosine Transporters in
Bloodstream Forms of Trypanosoma brucei brucei: Substrate
Recognition Motifs and Affinity for Trypanocidal Drugs. Mol.
Pharmacol. 1999, 56, 1162-1170.
(15) (a) Kaminsky, R.; Brun, R. in vitro and in vivo activities of
trybizine hydrochloride against various pathogenic trypanosome
species. Antimicrob. Agents Chemother. 1998, 42, 2858-2862.
(C42H79N24)+: 919.6919; found 919.6920. Anal. (C42H78N24
6.5HCl‚2EtOH‚1H2O) C, H, N, Cl.
‚
N1,N12-Di[3-([4,6-d i(m et h yla m in o)-1,3,5-t r ia zin -2-yl]-
m eth yla m in o)p r op yl]-1,12-d od eca n ed ia m in e Hep ta h y-
d r och lor id e (8f). Starting from 4c (100 mg, 0.194 mmol),
2-bromomethyl-4,6-bis(methylamino)1,3,5-triazine (2 × 93 mg,
2 × 0.401 mmol), and NaHCO3 (2 × 35 mg, 3 × 0.417 mmol),
8f was isolated as light yellow powder (80 mg, 35%): Rf (SiO2,
35% aq NH3/MeOH 1:10) baseline; mp(sealed tube) > 130 °C
slow dec (foam), > 200 °C dec; 1H NMR (CD3OD) δ/ppm ) 3.82
(s, 4H, Ar-CH2-N), 3.15 (m, 4H), 3.05-2.89 (m, 28H), 2.77
(m, 4H), 2.03 (m, 4H), 1.72 (m, 4H), 1.36-1.33 (m, 16H); EtOH
signals at 3.61 (q), 1.18 (t); NH exchanged, Ar-CH2-N
partially exchanged; 13C NMR (CD3OD) δ/ppm ) 167.7 (s),
166.0(s), 57.8 (t), 53.5 (t), 49.0 (t), 46.8 (t), 30.6 (t), 30.4 (t),
30.2 (t), 27.6 (t), 28.5 (q), 27.9 (q), 27.3 (t), 24.7 (t); EtOH
signals at 58.3 (t), 18.4 (q); MS (ES+) m/z ) 955 (M+HCl+H+,
20%), 919 (M+H+, 100%), 460 ((M+2H+)/2, 50%); HR-MS (ES+)
calcd for (C42H79N24)+: 919.6919; found 919.6933. Anal.
(C42H78N24‚6.5HCl‚2EtOH‚1H2O) C, H, N, Cl.
Oth er An a logu es. N2-12-[(4,6-Dia m in o-1,3,5-tr ia zin -2-
yl)a m in o]d od ecyl-1,3,5-tr ia zin e-2,4,6-tr ia m in e (9). 1,12-
Diaminododecane (1c, 200 mg, 0.998 mmol), 2-chloro-4,6-
diamino-1,3,5-triazine (320 mg, 2.20 mmol), and NaHCO3 (185
mg, 2.20 mmol) in a solvent mixture of absolute EtOH (20 mL)
and water (20 mL) were stirred at 80 °C for 19 h. The solvents
were removed in vacuo, and the residue was washed twice with
water (5 mL) and recrystallized from a mixture of hot absolute
EtOH (25 mL) and water (20 mL). Compound 9 was isolated
as white microcrystalline powder (320 mg, 77%): Rf (SiO2, 35%
aq NH3/MeOH 1:10) 0.73; mp(sealed tube) > 150 °C slow dec
(foam), 168-175 °C; 1H NMR (CD3OD/[D6]-DMSO 1:1) δ/ppm
) 3.12 (m, 4H), 1.38 (m, 4H), 1.15 (m, 16H); NH exchanged;
13C NMR (CD3OD/[D6]-DMSO 1:1) δ/ppm ) 167.2 (s), 166.7
(s), 166.3 (s), 40.5 (t), 40.4 (t), 29.9 (t), 29.7 (t), 29.6 (t), 29.5
(t), 27.0 (t); MS (ES+) m/z ) 419 (M+H+, 100%); HR-MS (ES+)
calcd for (C18H35N12)+: 419.3107; found 419.3103. Anal.
(C18H34N12‚0.9H2O) C, H, N.
N2,N4-Dim et h yl-N6-oct a d ecyl-1,3,5-t r ia zin e-2,4,6-t r i-
a m in e (10). 1-Aminooctadecane (1g, 100 mg, 0.371 mmol),
2-chloro-4,6-bis(methylamino)-1,3,5-triazine (130 mg, 0.749
mmol), and NaHCO3 (65 mg, 0.774 mmol) in a solvent mixture
of absolute ethanol (10 mL) and water (10 mL) were stirred
at 80 °C for 15 h. The solvents were removed in vacuo, and
the residue was separated between CH2Cl2 (15 mL) and water
(15 mL). The aqueous phase was extracted three times with
CH2Cl2 (10 mL), and the combined organic phases were dried
over sodium sulfate. The solvent was removed in vacuo, and
the residue (110 mg, off-white solid) was purified by column
chromatography on SiO2 using 5% MeOH in CH2Cl2 as eluent.
Compound 10 was isolated as white powder (55 mg, 36%): Rf
(SiO2, 5% MeOH in CH2Cl2): 0.20: Rf (SiO2, 35% aq NH3/
MeOH 1:10) 0.50; mp(sealed tube) 58-61 °C; 1H NMR (CD3OD/
CDCl3 10:1) δ/ppm ) 3.31 (m, 2H), 2.86 (m, 6H), 1.54 (m, 2H),

3452 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Klenke et al.
(b) Bacchi, C. J .; Vargas, M.; Rattendi, D.; Goldberg, B.; Zhou,
W. Antitrypanosomal Activity of a New Triazine Derivative, SIPI
1029, In Vitro and In Model Infections. Antimicrob. Agents
Chemother. 1998, 42, 2718-2721.
(23) Ponasik, J . A.; Strickland, C.; Faerman, C.; Savvides, S.; Karplus,
P. A.; Ganem, B. Kukoamine A and other hydrophobic acyl-
polyamines: potent and selective inhibitors of Crithidia fascicu-
lata trypanothione reductase. Biochem. J . 1995, 311, 371-375.
(24) Bonnet, B.; Soullez, D.; Davioud-Charvet, E.; Landry, V.; Hor-
vath, D.; Sergheraert, C. New Spermine and Spermidine Deriva-
tives as Potent Inhibitors of Trypanosoma cruzi Trypanothione
Reductase. Bioorg. Med. Chem. 1997, 5, 1249-1256.
(25) Chen, S.; Lin, C.-H.; Walsh, C. T.; Coward, J . K. Novel Inhibitors
of Thrypanothione Biosynthesis: Synthesis and Evaluation of
a Phosphinate Analogue of Glutathionyl Spermidine (GSP), a
Potent, Slow Binding Inhibitor of GSP Synthetase. Bioorg. Med.
Chem. Lett. 1997, 7, 505-510.
(26) O’Sullivan, M. C.; Zhou, Q. Novel Polyamine Derivatives as
Potent Competitive Inhibitors of Trypanosoma Cruzi Trypano-
thione Reductase. Bioorg. Med. Chem. Lett. 1995, 5, 1957-1960.
(27) Carter, N. S.; Berger, B. J .; Fairlamb, A. H. Uptake of Diamidine
Drugs by the P2 Nucleoside Transporter in Melarsen-sensitive
and -resistant Trypanosoma brucei brucei. J . Biol. Chem. 1995,
270, 28153-28157.
(28) Ra¨z, B.; Iten, M.; Grether-Bu¨hler, Y.; Kaminsky, R.; Brun, R.
The Alamar Blue assay to determine drug sensitivity of African
Trypanosomes (T. b. rhodesiense and T. b. gambiense) in vitro.
Acta Tropica 1997, 68, 139-147.
(29) Balz, T.; Baltz, D.; Giroud, C.; Crockett, J . Cultivation in a semi-
defined medium of animal infective forms of Trypanosoma
brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambi-
ense. EMBO J . 1985, 4, 1273-1277.
(30) Koermendi, O.; Horvath, D. Vereinfacthte Synthesen Des Sper-
mins. Acta Chim. Acad. Sci. Hung. 1954, 4, 5-8.
(31) Israel, M.; Rosenfield, J . S., Modest, E. J . Analogs of Spermine
and Spermidine. I. Synthesis of Polymethylenepolyamines by
reduction of cyanoethylated R,ω-alkylenediamines. J . Med.
Chem. 1964, 7, 710-716.
(32) Weinstock, L. T.; Rost, W. J .; Cheng, C. C. Synthesis of new
Polyamine Derivatives for Cancer Chemoterapeutic Studies. J .
Pharm. Sci. 1981, 70, 956-959.
(33) Blessing, T.; Remy, J .-S.; Behr, J .-P. Template oligomerization
of DNA-Bound cations produces calibrated nanometric particles.
J . Am. Chem. Soc. 1998, 120, 8519-8520.
(16) Karigiannis, G.; Papaioannou, D. Structure, Biological Activity
and Synthesis of Polyamine Analogues and Conjugates. Eur. J .
Org. Chem. 2000, 1841-1863.
(17) (a) Kuksa, V.; Buchan, R.; Lin, P. K. T. Synthesis of Polyamines,
Their Derivatives, Analogues and Conjugates. Synthesis 2000,
1189-1207. (b) Tabor, C. W.; Tabor, H. Polyamines. Annu. Rev.
Biochem. 1984, 53, 749-790. (c) Marton, L. J .; Pegg, A. E.
Polyamines as Targets for Therapeutic Intervention. Annu. Rev.
Pharmacol. Toxicol. 1995, 35, 55-91.
(18) Phillips, M. A.; Coffino, P.; Wang, C. C. Trypanosoma Brucei
Ornithine Decarboxylase - Enzyme Purification, Characteriza-
tion, and Expression in Escherichia-Coli. J . Biol. Chem. 1988,
263, 17933-17941.
(19) Flohe, L.; Hecht, H. J .; Steinert, P. Glutathione and Trypano-
thione in Parasitic Hydroperoxide Metabolism. Free Rad. Biol.
Med. 1999, 27, 966-984.
(20) Klenke, B.; Gilbert, I. H. Nitrile reduction in the presence of
BOC-protected amino groups by catalytic hydrogenation over
palladium activated Raney-Nickel. J . Org. Chem. 2001, 66,
2480-2483.
(21) (a) Pearlman, W. M.; Banks, C. K. Substituted Chlorodiamino-
s-triazines. J . Am. Chem. Soc. 1948, 70, 3726-3728. (b) Irikura,
T.; Abe, Y.; Okamura, K.; Higo, K.; Maeda, A.; Morinaga, F.;
Shirai, G.; Hatae, S. New s-triazine derivatives as depressants
for reticuloendothelial hyperfunction induced by bacterial en-
dotoxin. J . Med. Chem. 1970, 13, 1081-1089. (c) Katritzky, A.
R.; Oniciu, D. C.; Ghiviriga, I.; Barcock, R. A. 4,6-bis-(N,N-
Dialkylamino)-s-triazines and 2,4,6-tris-(N,N-Dialkylamino)-s-
triazines - Synthesis, NMR Spectra and Restricted Rotations.
J . Chem. Soc., Perkin Trans. 2, 1995, 785-792.
(22) (a) Eisa, H. M.; Tantawy, A. S.; El-Kerdawy, M. M. Synthesis of
cetain 2-aminoadamantane derivatives as potential antimicro-
bial agents. Pharmazie 1991, 46, 182-185. (b) Kelarev, V. I.;
Remizov, A. S.; Karakhanov, R. A.; Polivin, Y. N.; Oietaio, D.
Synthesis and properties of sym-triazines. 10. Synthesis of 2,4-
diamino-sym-triazines containing a sterically hindered phenol
substituent. Chem. Heterocycl. Compd. Engl. Transl. 1992, 28,
1189-1193. (c) Baker, B. R.; Ho, B.-T. Analogues of tetrahydro-
folic acid. XXX. Inhibition of dihydrofolic reductase by some
6-substituted 2,4-diamino-s-triazines. J . Heterocycl. Chem. 1965,
2, 340-343. (d) Overberger, C. G.; Michelotti, F. W.; Carabateas,
P. M. Preparation of triazines by the reaction of biguanide and
esters. J . Am. Chem. Soc. 1957, 79, 941-943.
J M010854+