Antimalarial 3-arylamino-6-benzylamino-1,2,4,5-tetrazines

Article abstract of DOI:10.1016/j.bmcl.2010.06.036

We report on novel 3-arylamino-6-benzylamino-1,2,4,5-tetrazines with potent activity against Plasmodium falciparum.

Full text of DOI:10.1016/j.bmcl.2010.06.036

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4496–4498
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antimalarial 3-arylamino-6-benzylamino-1,2,4,5-tetrazines
Duong Nhu a, Sandra Duffy b, Vicky M. Avery b, Andrew Hughes c, Jonathan B. Baell a,
*
a Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
b Discovery Biology, Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Queensland, Australia
c La Trobe University, Bundoora, Victoria, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
We report on novel 3-arylamino-6-benzylamino-1,2,4,5-tetrazines with potent activity against Plasmo-
Received 20 April 2010
Revised 4 June 2010
Accepted 7 June 2010
Available online 10 June 2010
dium falciparum.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Plasmodium falciparum
Malaria
Tetrazines
Malaria is one of the most detrimental infectious parasitic dis-
eases which poses a threat to more than two billion lives world-
wide as estimated in 2002.1 Although eradication has been
successful in certain areas,2 malaria is still prevalent in developing
countries, especially in Africa, which has the highest mortality in
children under the age of five.3,4
major groups based on the biochemical pathways underlying their
modes of action. These include nucleic acid synthesis, protein syn-
thesis, oxidative stress and heme metabolism.
Chloroquine has a long history in treatment of malaria; however,
emergence of resistant strains of Plasmodium raises the need for
ongoing research on alternative efficacious drug candidates.11
Although artemisinin analogues have shown improved perfor-
mance against chloroquine and multi drug-resistant strains of Plas-
modium,5,12 besides the aforementioned financial constraint,
resistance to these drugs has been reported in some areas of
South-East Asia.9 Therefore, development of novel antimalarial
agents, satisfying the criteria of being both potent and synthetically
economical, is of interest.
Through screening a set of in-house compounds at the Swiss
Tropical Research Institute against a panel of parasites,13 we found
that compounds 1 and 2 in Figure 1 were potently toxic to P. falci-
parum. As shown in Table 1, compound 1 exhibited submicromolar
activity towards P. falciparum with an EC50 of 440 nM and was
more than 30 times less toxic towards other parasites and mam-
malian cells. The triazine dimer 2 was even more potent towards
In humans, malaria is caused by four strains of single-celled
eukaryotic protozoa of the genus Plasmodium. These strains include
Plasmodium malariae, Plasmodium vivax,Plasmodium ovale and Plas-
modium falciparum, among which P. falciparum is associated largely
with lethal cases.3,5 Individuals with malaria generally experience
fevers and chills as a result of the body’s inflammatory response. In
addition, more severe symptoms, involving multiple organs and
tissues, result from the destruction of red blood cells (RBCs) and
are manifested by lactic acidosis, malarial anaemia and cerebral
malaria.3,6 Genetic conditions such as sickle cell anaemia, however,
confer some protection against the disease and the trait is conse-
quently preserved in regions where malaria becomes endemic.3
Intensive studies have focused on vaccine and drug develop-
ment targeting different stages of the parasite life cycle to combat
the disease.7–9 The search for effective vaccines, either with sub-
unit or whole parasite approaches, is challenging due to the poly-
morphic nature of the parasites.7–10 In addition, cost is also a
concern as malaria is problematic in third world countries.7 Drug
treatments have been adopted and a majority of antimalarial
agents in the clinic act on the intra-erythrocytic stages.5 In the re-
view by Frederich et al.,5 these drugs have been divided into four
N
N
H
N
N
N
N
N
N
N
SMe
N
Cl
N
N
H
MeO
1
2
WEHI-0033352
WEHI-0059743
* Corresponding author.
E-mail address: jbaell@wehi.edu.au (J.B. Baell).
Figure 1. Two in-house heterocycles screened against a panel of parasites and
found to have potent activity against P. falciparum.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.036

D. Nhu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4496–4498
4497
Table 1
part and so utilized this in the synthesis of 1 as shown in Figure 2.
Here, the least nucleophilic amine is reacted first (4-chloroaniline
for 1) with 5 so that the second substitution can proceed without
forcing conditions. This protocol worked well for all cases where,
like 1, R1NH represented an aniline and so the system remained
relatively activated towards the second incoming nucleophile
R2NH2, this also being a more reactive alkylamine. In this way, 1,
6, 7, 10, 15 and 16 were readily made. The reader is referred to
Table 2 for elaboration of the R groups.
Compounds 15 and 16 involved the use of exotic amine
nucleophiles which were made as shown in Figures3 and 4, respec-
tively. For 15, the required aniline was made by alkylating 3-
hydroxyaniline with mesylated N,N-dimethylaminoethanolamine,
giving 17 as shown in Figure 3. This could then be used in step
(iv) in Figure 2 en route to the synthesis of 15.
Antiparasitic activity profile of 1 and 2
Assay
EC50 (l
M)a
1
2
P. falciparumb
L. infantosumc
T. cruzid
0.44
16
15
>32
>32
0.18
8.6
16
5.7
33
T. bruceie
Cytotoxicityf
a
Values are means of three experiments, 50%.
b
Plasmodium falciparum 3D7 strain, erythrocytic stage, chloroquine was used as a
control, EC50 30 g/ml.
Leishmania infantosum, amastigote stage, miltefosine was used as a control, EC50
l
c
2.4
l
g/ml.
d
Trypanosoma cruzi Tulahaen C2C4 strain, amastigote stage, nifurtimox was used
as a control, EC50 0.24
l
g/ml.
Trypanosoma brucei rhodesiense strain STIB 900, bloodstream form. Suramin
(EC50 0.13 g/ml) was used as a control.
Rat skeletal myoblast cell L-6 strain, Tamoxifen was used as a control, EC50
g/ml.
e
Compound 16 required the synthesis of benzylamine 18 as
shown in Figure 4. This involved alkylation of BOC-protected 3-
hydroxybenzylamine followed by deprotection. The resulting
amine, 18, could then be used in step (v) in Figure 2 en route to
the synthesis of 16.
l
f
4.9
l
In some cases, where R1NH was an alkylamine and not an ani-
line, the second incoming amine nucleophile in addition displaced
the first amine nucleophile. Hence, 9 was produced in a 1:1 mix-
ture with the 3,6-bis(n-butylamino) counterpart resulting from
the additional displacement of the N-benzyl group with butyl-
amine (Table 2).
This mixture was inseparable and the single peak in the HPLC
trace was suggestive of a tightly bound complex. In the case of
methylamine, only 5% of the product mixture comprised 8, the
remaining 95% being the 3,6-bis(methylamino) by-product. Never-
theless enough of 8 could be separated and purified for testing.
This unusual propensity for the benzylamino group to be displaced
in this system remains unexplained.
Synthesis of N-alkylated derivatives of 1 was readily under-
taken. As also shown in Figure 2, alkylation of the exocyclic nitrogen
atoms with R3X proceeded smoothly under standard conditions to
give the monoalkylated compounds 11 (R3 = Me), using MeI, or 13
(R3 = CH2CH2NMe2), using ClCH2CH2NMe2, with the alkyl group
preferentially reacting at the most acidic NH, this being the aniline
R1NH. Small amounts of the respective dialkylated by-products 12
and 14 were also obtained.
Compounds were tested for toxicity to P. falciparum and to mam-
malian cells. The biological results are summarized in Table 1. Here
it can be seen that the levels of inhibition of P. falciparum by com-
pound 1 is similar to that obtained previously. Compound 1 is po-
P. falciparum with an EC50 of 180 nM and was also highly selective,
with the next most potent activity being against Trypanosoma bru-
cei with an EC50 of 5.7 lM.
However, routine quality control indicated that triazine 2 was
comprised of mostly a compound with a molecular weight of 252
instead of the expected 236. While this matter was being investi-
gated (to be reported in due course), tetrazine 1 was selected for
a structure–activity relationship (SAR) investigation and these re-
sults are now reported.
A
small group of 3,6-disubstituted 1,2,4,5-tetrazines with
ambiguous activity in a mouse model of malaria (P. berghei) have
been reported by Werbel et al.14 These compounds were restricted
to 3-phenyl-6-methylamino-1,2,4,5-tetrazines and so are quite
distinct to the tetrazines reported here, that are also more
drug-like by virtue of being deactivated through 3,6-diamino sub-
stitution. There is very little literature on the synthesis of asym-
metrically-substituted 3,6-diaminotetrazines. Rusinov et al.
reported a convenient synthesis that utilized a bis pyrazole deriv-
ative15 (5) as shown in Figure 2. Hence reaction of guanidine
hydrochloride with hydrazine gives triaminoguanidine 3 and this
in turn is reacted with acetylacetone to give 4, the oxidation of
which gives 5. We anticipated that bis pyrazole 5 would be more
conveniently handled than the highly activated bis chloro counter-
N
H
tently anti-malarial with an EC50 of 1.1
less toxic to mammalian cells, with 58% cell death only at 29
l
M and is significantly
N
N
lM.
N
(i)
NNH2
(ii)
NH
N
N
H
N
N
NH2NH NHNH2.HCl
H2N NH2.HCl
4
HO
NH2
Me2N
3
O
S
(i)
Me
O
O
NH2
N
N
.HCl
N
OH
O
R1NH
(iv)
N
N
N
N
N
(ii)
(iii)
(vi)
17
N
N
N
N
NHR2
(v)
N
Figure 3. (i) Methanesulfonyl chloride, dry ether, 0 °C to rt; (ii) NaH, N,N-
dimethylformamide, 0 °C to rt.
N
5
1,6-10,15,16
R1R3N
N
N
R1R3N
N
N
N
N
OH
OH (i)
(ii)
BocNH
(iii)
H2N
N
+
N
NR2R3
NHR2
11,13
12,14
O
O
BocNH
NMe2
NH2
NMe2
Figure 2. Synthesis of asymmetrically-substituted tetrazines. (i) hydrazine hydrate,
1,4-dioxane, reflux 2 h (80%); (ii) acetylacetone (2 equiv), water, rt to 70 °C (60–
90%); (iii) MnO2 (10 equiv)/silica, dichloroethane, rt 2 h (57–69%); (iv) R1NH2
(1.5 equiv), acetonitrile, 40–50 °C; (v) R2NH2 (1.5 equiv), acetonitrile, reflux; (vi)
R3X (10 equiv), K2CO3 or Cs2CO3, KI (if X not I), N,N-dimethylacetamide, 80–90 °C.
18
Figure 4. (i) Boc2O, tetrahydrofuran, 0 °C to rt (96%); (ii) NaH, ClCH2CH2NMe2, N,N-
dimethylformamide, 0 °C to rt (73%); (iii) 1.25 M HCl in methanol, 1 h (22%).

4498
D. Nhu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4496–4498
Compounds 6 and 7 are suggestive of very sharp SAR, as exten-
group was better tolerated on the benzyl ring, and compound 16
returned an EC50 of 5 M although it was also reasonably cytotoxic
with an EC50 of 21 M.
sion of the benzyl group by one methylene or incorporation of a
branched carbon, respectively appears to largely destroy activity.
Keeping the N-benzyl group constant, variations in the left hand side
were then investigated. Replacement of the N-phenyl group with an
N-methyl group (compound 8) led to a loss of activity and the simi-
larly poor activity of N-butyl compound 9 indicated that hydropho-
bicity of the substituent alone was insufficient to restore activity.
Remarkably, even the des-chloro N-phenyl compound 10 was com-
pletely inactive, suggesting very sharp negative SAR and that a
chloro in the 4-position was vital for good activity.
One way of improving solubility and permeability and hence
drug-likeness is through decreasing crystallinity and this can often
be achieved with N-alkylation. Hence we made and tested the
N-methyl compound 11 and showed that such modification was
well tolerated with activity being similar to that of 1. However,
cytotoxicity to mammalian cells was significant with an EC50 of
l
l
Also shown in parentheses in Table 2 for compounds 1, 6, 11, 13,
15 and 16 are the biological activities against the chloroquine-
resistant Dd2 strain of P. falciparum. It can be seen that these com-
pounds are essentially equipotent towards this strain, suggesting
that their mode of activity is independent to that of chloroquine.
In summary, we report novel heterocycles based on 3-(4-chlor-
ophenylamino-6-(benzylamino)-1,2,4,5-tetrazines that are active
against P. falciparum. SAR was probed and revealed both the 4-
chloroaniline and benzylamino substituents were essential for
activity. However, the aniline nitrogen atom could be methylated
(11) and a solubilizing group installed on the benzyl ring (16) with
maintenance of reasonable potency in the low micromolar range.
We recommend caution in progressing this class of compounds be-
cause they have a tendency to be toxic towards mammalian cells
and it currently cannot be ruled out that this may be linked to their
observed antimalarial activity.
12 lM. Solubility can be increased further by installation of a sol-
ubilizing group and in this regard we targeted tertiary amines as
they can be membrane permeable and have been shown to im-
prove activity in other antimalarial compounds through accumula-
tion in the acidic food vacuole of the parasite.16
Acknowledgments
To this end we made and tested amine 13, but it exhibited
weaker activity suggesting that while the N-methyl group in 11
was acceptable, further elongation of the N-alkyl group was not.
In order to explore other positions that may not interfere, we made
two compounds where the tertiary amine was attached via a tether
to the meta position of either the aniline ring or the benzyl ring. It
was envisaged that by being on the meta position, there would be
the maximum topographical space in which to move to either lo-
cate a favorable interaction or avoid an unfavorable interaction.
Where this group was on the aniline ring, activity was weaker
Financial support was received from the Victorian State Govern-
ment OISS Grant and NHRMC IRIISS Grant #361646 and for this we
are grateful.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.036.
References and notes
and compound 15 exhibited an EC50 of 15 lM. In contrast, this
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58%@
(1.1)d
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NA@
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l
8
Me
n-Bu
Ph
—
—
—
Me
NA@115
NA@115
NA@115
2.4
l
l
l
M
M
M
NA@115
NA@115
NA@115
12
l
l
l
M
M
M
9e
10
11
4-ClPh
(3.5)d
12
13
4-ClPh
4-ClPh
CH2Ph
CH2Ph
Me
NTf
NTf
38
CH2CH2- 13
NMe2
(20)d
14
15
4-ClPh
CH2Ph
CH2Ph
CH2CH2- NA@115
NMe2
l
M
NA@115
52%@38
lM
3-
—
—
15
lM
(Me2N-
CH2CH2-
O)Ph
(13)d
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16
4-ClPh
CH2Ph(3-
O CH2CH2-
NMe2)
5g
21
(6)d
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a
Values are means of two experiments, 50%.
Plasmodium falciparum 3D7 strain, erythrocytic stage, chloroquine was used as a
b
control, EC50 4 nM.
c
HEK, puromycin was used as a control, EC50 411 nM.
Plasmodium falciparum Dd2 strain (chloroquine-resistant), erythrocytic stage,
d
chloroquine was used as a control, EC50 173 nM.
e
Tested as a 1:1 mixture with inseparable 3,6-bis(n-butyl) by-product.
Not tested.
Approximate: poorly fitting curve.
f
g