Synthesis and antiprotozoal evaluation of new N4-(benzyl) spermidyl-linked bis(1,3,5-thiadiazinane-2-thiones)

Article abstract of DOI:10.1002/ardp.200800011

The synthesis and in-vitro antiprotozoal evaluation of novel N ⁴-(benzyl)spermidyl-linked bis(1,3,5-thiadiazinane-2-thione) (bis-THTT) derivatives from N⁴-(benzyl)spermidine is disclosed. Several of the new bis-THTT have in-vitro act

Full text of DOI:10.1002/ardp.200800011

708
Arch. Pharm. Chem. Life Sci. 2008, 341, 708–713
Full Paper
Synthesis and Antiprotozoal Evaluation of New N4-
(Benzyl)spermidyl-linked bis(1,3,5-thiadiazinane-2-thiones)
Julieta Coro1, Susan Little2, Vanessa Yardley2, Margarita Suꢀrez1, Hortensia Rodrꢁguez1,
Nazario Martꢁn3, and Rolando Perez-Pineiro4
1 Laboratorio de Sꢀntesis Orgꢁnica, Facultad de Quꢀmica, Universidad de la Habana, Habana, Cuba
2 Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London,
UK
3 Departamento de Quꢀmica Orgꢁnica. Facultad de Ciencias Quꢀmicas, Universidad Complutense, Madrid,
Spain
4 Departamento de Quꢀmica Fina, ICIDCA, 11000 Ciudad Habana, Cuba
The synthesis and in-vitro antiprotozoal evaluation of novel N4-(benzyl)spermidyl-linked bis(1,3,5-
thiadiazinane-2-thione) (bis-THTT) derivatives from N4-(benzyl)spermidine is disclosed. Several of
the new bis-THTT have in-vitro activities against L. donovani and T. cruzi that are comparable or
superior to those of currently employed protozoocidal agents.
Keywords: Protozoocidal activity / Spermidine / 1,3,5-Thiadiazinane-2-thione /
Received: January 9, 2008; accepted: May 2, 2008
DOI 10.1002/ardp.200800011
80000 people. Several drugs are available for treating
leishmaniasis [2, 3]. For example, pentavalent antimonial
compounds, such as sodium stibogluconate (pentostam,
Introduction
Trypanosomatids are parasitic protozoa within the order
Kinetoplastida which comprise the causative agents of
African sleeping sickness (Trypanosoma brucei gambiense
and T. b. rhodesiense; Chagas' disease (T. cruzi), and the
different forms of leishmaniasis (Leishmania donovani, L.
major, and L. tropica) [1]. At present, chemotherapy
against all forms of trypanosomiasis is very limited and
unsatisfactory [2].
2,4:29,49-O-(oxydistibylidyne)bis[D-gluconic
acid]Sb,Sb9-
dioxide trisodium salt nonahydrate) (Fig. 1) and meglu-
mine antimoniate (glucantime) are the drugs used in
first-line chemotherapy. As second-line drugs, amphoteri-
cin B and pentamidine (Fig. 1) are used. However, current
treatments against leishmaniasis are usually unsatisfac-
tory due to some limitations including the route of
administration of the drugs, their unaffordable cost and
toxicity. The chemotherapy for Chagas' disease at hand is
still deficient [2]. It is based on two drugs empirically dis-
covered, nifurtimox ((4-([5-nitrofurfurylidene]-amino)-3-
methylthiomorpholine-1,1-dioxide), now discontinued,
and benznidazole (N-benzyl-2-nitro-1-imidazoleaceta-
mide) (Fig. 1). Although both of these compounds are
able to cure at least 50% of recent infections as indicated
by the disappearance of symptoms, and negativization of
parasitemia and serology, they have important draw-
backs. For example, (a) selective drug sensitivity on differ-
ent T. cruzi strains; (b) these agents also produce serious
side effects including vomiting, anorexia, peripheral
neuropathy, allergic dermopathy, etc.; long-term treat-
ment is an additional disadvantage [4]. Moreover, these
Leishmaniasis affects twelve million people around
the world with an annual death rate of approximately
Correspondence: Rolando Perez-Pineiro, National Institute of Nano-
technology, National Research Council (NINT-NRC), and Department of
Chemistry, University of Alberta, 11421 Saskatchewan Drive, Edmonton,
Alberta, T6G 2M9, Canada.
E-mail: perezpin@ualberta.ca
Fax: +1 780 641 1601
Margarita Suꢁrez, Laboratorio de Sꢀntesis Orgꢁnica, Facultad de Quꢀmi-
ca, Universidad de la Habana, 10400 Ciudad Habana, Cuba.
E-mail: msuarez@fg.uh.cu
Fax: +537835737
Abbreviations: 1,3,5-thiadiazinane-2-thione (THTT); therapeutic index
(TI)
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Arch. Pharm. Chem. Life Sci. 2008, 341, 708–713
Synthesis and Bioactivity of Spermidyl-bis-thiadiazinanes
709
Figure 1. Some of the existing drugs clinically employed for the treatment of trypanosomiasis and leishmaniasis.
compounds are not effective in the chronic stage of the
The 1,3,5-thiadiazinane-2-thione (THTT) ring has great
disease. In addition, there are a number of uncertainties potential in medicinal chemistry due to its wide spec-
concerning gentian violet (N-{4-bis[[4-(dimethylamino)- trum of antimicrobial activity [8]. Furthermore, the high
phenyl]methylene]-2,5-cyclohexadien-1-ylidene}N-meth-
lipid solubility and ease of enzymatic hydrolysis [9] gen-
ylmethan-aminium chloride), the only drug available to erally associate with this heterocycle has motivated its
prevent blood transmission of Chagas' disease, because it use as a biolabile prodrug in the design of drug-delivery
is carcinogenic in animals [5]. This drug was empirically systems (DDS). The aforementioned properties and the
discovered for this purpose some decades ago.
possibility to attach several structurally distinct substitu-
On the other hand, Human African trypanosomiasis or ents to the heterocycle ring to modify either the biologi-
sleeping sickness is caused by two kinetoplastid flagel- cal or physico-chemical properties of these compounds,
lates: T. brucei rhodesiense and T. brucei gambiense. The have prompted us to use the thiadiazinane-2-thione as a
number of new cases yearly is around 500000 people, template in our research program aimed at the develop-
while 60 million individuals are at risk. Sleeping sickness ment of new antiparasitic compounds [10].
is fatal if untreated and has re-emerged in recent years.
Interestingly, despite the presence of two THTT cores
Four drugs are clinically available for the treatment of in the same molecular scaffold appear to be very attrac-
this disease: melarsoprol, suramin, pentamidine, and tive from the point of view of drug design, most of the
eflornithine [2]. The first three compounds were intro- works reported so far deal with the synthesis and bioac-
duced more than half a century ago, while eflornithine tivity of mono-THTT derivatives and only a few papers are
was registered in 1990. However, this compound is only devoted to tethered THTT analogs. The preparation of
effective when sleeping sickness is caused by T. b. gam- 3,39-ethylenbis(5-alkyl-1,3,5-thiadiazine-2-thiones)
biense. Most of these compounds are toxic and lack effi- and 2,3-bis(5-alkyl-2-thiono-1,3,5-thiadiazin-3-yl)pro-
cacy. Parasite resistance is another important drawback pionic acid derivatives [12], account for some recent
[11]
for these drugs [6].
examples of the latter.
Associated with this devastating panorama is the lack
Recently, we described the synthesis and antiprotozoal
of financial motivation from pharmaceutical companies evaluation of two series of alkyl-linked bis[2-thioxo-
for carrying out rigorous research programs bearing in (1,3,5)thiadiazinan-3-yl]carboxylic acids from 1,6-diami-
mind that all of these diseases are closely related with nohexane and 2,2-dimethyl-1,3-propanediamine, respec-
populations with low economical resources; therefore, tively [13, 14]. In general, most of the evaluated com-
efforts to develop new drugs should be achieved largely pounds showed high activity against T. b. rhodesiense and
by academic and governmental institutions [7].
those possessing the less hindered alkyl residue bridging
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J. Coro et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 708–713
[15b], and spermine and spermidine derivatives bearing
benzyl, phenylpropyl and naphthyl groups as substitu-
ents [15c] also displayed a potent antiprotozoal activity.
Furthermore, the presence of secondary nitrogen in the
alkylic chain allows for the exploitation of an additional
point of diversity in the overall synthetic scheme. With
all this considerations in mind, the following paper deals
with preliminary results obtained in the synthesis and
protozoocidal effect of novel N4-(benzyl)spermidyl-linked
bis(1,3,5-thiadiazinane-2-thione) derivatives 6.
Results and discussion
Chemistry
The starting N4-benzyl polyamine 1 was previously syn-
thesized according to a reported methodology by O'Sulli-
van et al. [16] via a protection-deprotection strategy using
ethyl trifluoroacetate as the selective protective group
for primary amines in the presence of secondary amines.
The synthetic route leading to derivatives 5 from the ben-
zylated spermidine is depicted in Scheme 1.
Reaction of polyamine 1 with CS2 / OH– provided the
desired bis-dithiocarbamate salt 2. The formation of this
type of intermediate has been confirmed earlier by our
group through the isolation of the 6-(dithiocarboxya-
mino) hexanoic acid upon acidic cleavage of the resin-
bound dithiocarbamate analog [17]. Based on experimen-
tal facts [18], the condensation of 2 with formaldehyde
would likely lead to the corresponding bis(hydroxy-
methyl-dithiocarbamic acid hydroxymethyl ester)
3
excluding the participation of a methylideneiminium
Mannich-type intermediate in the reaction mechanism
and most likely driving the heterocyclization pathway
via the adduct 4 to form the expected bis-THTT deriv-
atives. A computational study about the probable reac-
tion pathway to the thiadiazinane-2-thione ring from
intermediate 3 has been recently disclosed by our group
[19]. In this report, it has been proposed that the hetero-
cyclic ring is formed through an intramolecular hetero-
cyclization of the adduct 4 via an SN2 reaction. Computa-
tional calculations also predicted an active role of water
in the reaction mechanism which promotes the hetero-
cyclization step. Compounds 5a–d were obtained as sol-
ids in moderate yields and showed satisfactory analytical
and spectroscopic data.
Reagents and conditions: (i) H2O / KOH (20%), CS2, r.t.; (ii) HCHO (37%); (iii) H2N-
R1, buffer phosphate (pH = 7.8); (iv) HCl (7%).
Scheme 1. Synthetic route leading to derivatives 5 from the ben-
zylated spermidine.
both heterocycles displayed the most favored activity /
cytotoxicity profiles expressed as therapeutic index (TI).
Encourage by our previous results, we have decided to
explore the feasibility of synthesis of new bis-THTT using
more complex polyamines as linkage other than the ini-
tially reported diamines. Behind this reasoning is the
known fact that structures containing protonated poly-
amines linked to hydrophobic moieties such as Noresper-
midines-based peptides [15a], alkylpolyaminoguanidines
Antiprotozoal activity
The in-vitro antiprotozoal activity of the new compounds
was evaluated against L. donovani, T. cruzi, T. b. rhodesiense
STIB900, and P. falciparum 3D7. Results are shown in
Table 1. Inspection of the data reveals that the former
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Arch. Pharm. Chem. Life Sci. 2008, 341, 708–713
Synthesis and Bioactivity of Spermidyl-bis-thiadiazinanes
711
Table 1. Results for in-vitro anti-protozoal activity of N4-(benzyl)spermidinyl-linked bis-THTT 5 and their hexyl-linked bis-THTT ana-
logs 7.
Compound
IC50 lMa) (TI)b)
T. b. rhodesiense
Toxicityc) LD50
(lM)
L. donovani
T. cruzi
P. falciparum
5a
5b
5c
5d
6a
6b
6c
5.35 (0.53)
6.45 (1.05)
5.85 (0.78)
5.47 (0.32)
7.07 (1.2)
6.74 (2.4)
5.04 (0.56)
8.69 (0.78)
3.64 (1.25)
4.23 (0.41)
– –
0.96 (2.97)
0.95 (7.16)
1.46 (3.12)
1.14 (1.53)
0.73 (11.4)
0.36 (44.4)
2.30 (22.8)
6.32 (0.45)
23.55 (0.29)
18.08 (0.25)
8.73 (0.20)
10.97 (0.8)
16.95 (1.0)
14.19 (3.7)
2.85
6.8
4.56
1.75
8.3
– –
– –
16.09
52.54
7.26 (7.3)
Pentostan
Benznidazole
Pentamidine
Chloroquine
Podophylotoxin
84.42 (20.43 lgSbv/mL)
4.00
2.50610– 3
0.84
0.50610– 3
a)
Compound concentrations of 30.0, 10.0, 3.0, 1.0, 0.3, and 0.1 lg/mL were used in the IC50 determinations. All reported values are
averages of three independent repeats.
TI (Therapeutic Index) is the ratio of cytotoxicity LD50 to the anti-parasite IC50 (LD50/IC50).
Cytotoxicities were assayed against KB cells using podophylotoxin as standard (LD50 in lM). Compound concentrations of 300.0,
30.0, and 0.3 lg/mL were used in the LD50 determinations. All reported values represent the average of three independent
repeats.
b)
c)
two parasites are particularly susceptible to these com- tozoal activity it is possible to assume that structures 5
pounds. In the case of T. cruzy, activities ranged between are more cytotoxic than their alkyl tethers analogs.
3.64–8.69 lM, very similar to the control drug Benznida-
In the case of the thiadiazinane-2-thiones the mecha-
zole, which had an IC50 of 4.00 lM. Results for L. donovani nisms of biological activity and cytotoxicity are closely
were even more encouraging, revealing that all the bis- related. It has been demonstrated that under physiologi-
THTT were considerably more active than sodium stibo- cal conditions the THTT ring undergoes chemical and / or
gluconate (pentostam). For this parasite, activities were enzymatic degradation to generate highly reactive spe-
more than one order of magnitude higher than the con- cies such as isothiocyanates, dithiocarbamic acids and
trol. None of the compounds showed a remarkable activ- their ester derivatives. These degradation products are
ity against T. b. rhodesiense and P. falciparum 3D7, respec- known for being toxic to mammalian cells and also for
tively.
their capacity to interact with the thiol groups located at
In order to compare the protozoocidal profile of the the active site of the cysteine proteinases (CPs), present in
synthesized compounds in relation to the previously most groups of parasitic protozoa [10a, 20] with the con-
reported hexyl-linked bis-THTT analogs 6 the antiproto- sequent inactivation of the enzymatic activity. The latter
zoal results of the latter and the calculated therapeutic mechanism was recently supported by the fact that 3-fur-
index (TI) values for both series were also included in furyl-5-substituted-1,3,5-thiadiazinane-2-thione
deriv-
Table 1. For comparative purposes, structures 5 and 6 dif- atives are more active against intracellular amastigote
fer only in the nature of their connective backbone (R2). than promastigote forms of Leishmania [10b]. In this case,
As could be observed in Table 1 for compounds in both the difference between the activities of both stages could
series possesing the same aminoacid residue (R1), the dis- be correlated with the expression of CPs, which are more
played protozoocidal profile against the three pasasitic abundant in the amastigote stage, where they play an
lines assayed was almost the same. However, higher val- important role [21].
ues of therapeutic indexes were found for compounds
Taking into account the considerations above, the ori-
6a–c. Considering that both series exerted a similar pro- gen of the higher cytotoxicities of compounds 5 does not
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J. Coro et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 708–713
(Bꢀchi Labortechnik, Flawil, Switzerland) and were not cor-
rected. The progress of the reaction and purity of compounds
seems to solely rely on the degradation product of the
THTT ring, since compounds in both series posses the
same type and number of heterocyclic structures. On the
other hand, it is likely that compounds 5 could also inter-
fere with the polyamine metabolism due to the presence
of a N4-(benzyl)spermidyl moiety. Since THTT derivatives
are renown prodrugs, they could be able to traverse the
cell membrane of the mammalian cells via a polyamine
transporter and to exert their cytotoxic effect upon deg-
radation. Since, at this moment, we do not have conclu-
sive evidences about the latter hypothesis, a more exten-
sive activity / toxicity study will be carried out on these
and other polyamine-linked bis-THTT derivatives.
were monitored by TLC analytical silica gel plates (Merck F254
,
Merck, Darmstadt, Germany). All NMR experiments were per-
formed at 298K for a solution of 30 mg of compound dissolved
in 0.7 mL of DMSO-d6 on a Bruker AVANCE-500 instrument
(Bruker Bioscience, Billerica, MA, USA). Microanalysis was per-
formed in a Perkin–Elmer 2400 CHN (Perkin-Elmer, Norwalk,
CT, USA) by the microanalysis service at Complutense University
of Madrid.
General procedure for [5-(3-(benzyl{4-[5-
(carboxyalkyl)-2-thioxo-1,3,5-thiadiazinan-3-
yl]butyl}-aminopropyl)-6-thioxo-1,3,5-thiadiazinan-3-
yl]carboxylic acids 5a–d
1
To a stirred solution of the previously synthesized N-(3-amino-
propyl)-1N-benzyl-1,4-butanediamine 2 (5 mmol) in 25 mL of
water, potassium hydroxide (0.56 g, 10 mmol, as a 20% aqueous
solution) and carbon disulfide (0.6 mL, 10 mmol) were added at
room temperature. The mixture was subsequently stirred for
4 h. Then, formaldehyde solution 37% (1.6 mL, 20 mmol) was
added and the stirring continued for 1 h. The reaction mixture
was added dropwise to a suspension of the corresponding amino
acid (10 mmol) in a dihydrogen phosphate buffer solution pH =
7.8 (10 mL) and stirred for 2 h. The aqueous solution was passed
through a celite pad, cooled in an ice bath and acidified to pH =
3 with 7% hydrochloric acid. The precipitate was filtered,
washed several times with small portions of cold ether and dried
under vacuum over silica gel for 12 h.
Conclusion
To summarize, we have reported the feasibility of synthe-
sis and the structural elucidation of novel N4-(benzyl)
spermidyl-linked bis-(1,3,5-thiadiazinane-2-thione) deriv-
atives from the corresponding N4-(benzyl) spermidine.
The synthesized compounds showed a potent protozooci-
dal activity against T. cruzy and L. Donovani which turns
to be comparable or greater than the currently employed
chemotherapies. Despite this fact, the novel structures
displayed a higher cytotoxicity than previously synthe-
sized alkyl tethers analogs having the same amino acidic
residues attached to position N5 of the heterocyclic ring.
It has been hypothesized that increased cytotoxicity
could be related with an interference of the polyamine
metabolism in mammals.
In light of the results presented here and taking into
account those from earlier reports, we are evaluating
how the chemical nature of N-substituents influences the
overall activity / cytotoxicity profile in the spermidyl-
linked bis(1,3,5-thiadiazinane-2-thione) series. The syn-
thesis of novel bis-THTT derivatives employing other poly-
amines as bridging moieties will be also the theme of
future investigation in our group.
2-(5-{3-(Benzyl[4-(5-carboxymethyl-2-thioxo-1,3,5-
thiadiazinan-3-yl)butyl]aminopropyl}-6-thioxo-1,3,5-
thiadiazinan-3-yl]acetic acid 5a
From glycine (0.95 g, 10 mmol); yield 5a (0.58 g, 20%); m.p.:
1
122–1238C; H-NMR (DMSO-d6) d: 1.54 (m, 4H, H-11, H-12), 1.84
(m, 2H, H-8), 2.56 (br.s, 4H, H-9, H-10), 3.49 (s, 4H, H-19), 3.75 (br.
s, 2H, H-14), 3.86 (m, 6H, H-7, H-13), 4.48 (s, 4H, H-6), 4.51 (s, 4H,
H-4), 7.28 (m, 1H, H-18), 7.34 (t, 2H, J = 7.2 Hz H-17), 7.38 (d, 2H, J =
6.8 Hz, H-16), 12.01 (br. s, 2H, COOH) ppm; 13C-NMR (DMSO-d6) d:
22.39, 22.49, 23.60 (C-8, C-11, C-12), 49.46, 49.87, 50.88, 52.15 (C-
7, C-9, C-10, C-13), 50.78, 50.82 (C-19), 57.07 (C-14), 58.01 (C-6),
69.36, 69.49 (C-4), 127.62 (C-18), 128.35 (C-17), 129.53 (C-16),
136.80 (C-15), 170.60, 170.64 (C-20), 190.09, 190.27 (C-2) ppm;
Anal. Calcd for C24H35N5O4S4: C, 49.21; H, 6.02; N, 11.95; O, 10.92;
S, 21.89. Found: C, 49.39; H, 6.1; N, 10.5.
This investigation received financial support from the UNDP/
World Bank/WHO Special Program for Research and Training
in Tropical Diseases (TDR) and from CTQ2005-02609/BQU. We
also thanks Third World Academy of Sciences (TWAS) for
research grant (No. 03-069 RG/CHE/LA), to R. Perez-Pineiro.
6-[5-(3-(Benzyl{4-[5-(5-carboxypentyl)-2-thioxo-1,3,5-
thiadiazinan-3-yl]butyl}aminopropyl)-6-thioxo-1,3,5-
thiadiazinan-3-yl]hexanoic acid 5b
From 6-amino-n-hexanoic acid (1.30 g, 10 mmol); yield 5b
(0.50 g, 14%); m.p.: 106–1078C; 1H-NMR (DMSO-d6) d: 1.28 (m, 4H,
H-21), 1.48 (m, 12H, H-11, H-12, H-20, H-22), 1.75 (m, 2H, H-8),
2.19 (t, 4H, J = 7.2 Hz, H-23), 2.41 (m, 4H, H-9, H-10), 2.61 (m, 4H,
H-19), 3.51 (br. s, 2H, H-14), 3.85 (m, 4H, H-7, H-13), 4.43 (m, 8H, H-
6, H-4), 7.21 (m, 4H, H-16, H-17), 7.29 (m, 1H, H-18), 12.01 (br. s,
2H, COOH) ppm; 13C-NMR (DMSO-d6) d: 23.48, 23.56, 23.89 (C-8, C-
11, C-12), 24.26 (C-22), 26.01, 26.02 (C-20, C-21), 33.60 (C-23),
40.04, 40.06 (C-19), 49.95, 50.56, 51.08, 52.71 (C-7, C-9, C-10, C13),
57.32, 57.38 (C-6), 57.78 (C-14), 69.38, 69.53 (C-4), 126.73 (C-18),
128.04 (C-17), 128.68 (C-16), 139.53 (C-15), 174.38 (C-24), 190.03,
The authors have declared no conflict of interest.
Experimental
All reagents were of analytical grade and purified when neces-
sary. Melting points were determined in a Bꢀchi 535 apparatus
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Arch. Pharm. Chem. Life Sci. 2008, 341, 708–713
Synthesis and Bioactivity of Spermidyl-bis-thiadiazinanes
713
190.13 (C-2) ppm; Anal. Calcd for C32H51N5O4S4: C, 55.06; H, 7.36;
N, 10.03. Found: C, 55.29; H, 7.53; N, 10.1.
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2-[5-(3-(Benzyl{4-[5-(1-carboxy-3-methylbutyl)-2-thioxo-
1,3,5-thiadiazinan-3-yl]butyl}aminopropyl)-6-thioxo-1,3,5-
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thiadiazinan-3-yl]-4-methylpentanoic acid 5c
From L-leucine (830 mg, 10 mmol); yield 5c (0.55 g, 19%); m.p.:
126–1278C; 1H-NMR (DMSO-d6) d: 0.88 (m, 12H, H-22, H-23), 1.60
(br. s, 10H, H-12, H-20, H-21), 1.74 (br.s, 2H, H-11), 2.10 (br. s, 2H,
H-8), 3.02 (br. s, 4H, H-9, H-10), 3.51 (m, 2H, H-19), 3.92 (m, 4H, H-
7, H-13) 4.31 (br. s, 2H, H-14), 4.56 (m, 8H, H-6, H-4,), 7.46 (m, 3H,
H-17, H-18), 7.60 (m, 2H, H-16), 12.85 (br. s, 2H, COOH) ppm; 13C-
NMR (DMSO-d6) d: 20.15, 20.79, 23.38 (C-8, C-11, C-12), 21.85,
21.94, 23.09, 23.20 (C-22, C-23), 24.75, 24.79 (C-21), 38.59, 38.63
(C-20), 48.84, 48.89, 50.84, 51.20 (C-7, C-9, C-10, C-13), 55.77,
55.82, 55.87 (C-6, C-14), 60.80, 60.90 (C-19), 67.53, 67.61 (C-4),
128.87 (C-17), 129.56 (C-18), 129.83 (C-15), 131.32 (C-16), 173.29,
173.37 (C-24), 190.95, 191.69 (C-2) ppm; Anal. Calcd for
C34H55N5O4S4: C, 56.27; H, 7.55; N, 9.65. Found: C, 56.35; H, 7.63;
N, 9.79.
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2-[5-(3-(Benzyl{4-[5-(1-carboxy-3-methylsulfanylpropyl)-
2-thioxo-1,3,5-thiadiazinan-3-yl]butyl}aminopropyl)-6-
thioxo-1,3,5-thiadiazinan-3-yl]-4-methyl sulfanyl butanoic
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acid 5d
From L-methionine (1.49 g, 10 mmol); yield 5d (0.73 g, 20%);
m.p.: 118–1208C; 1H-NMR (DMSO-d6) d: 1.59 (m, 2H, H-12), 1.68
(m, 2H, H-11), 1.97 (m, 4H, H-20), 2.03, 2.04 (s, 6H, H-22), 2.06 (m,
2H, H-8), 2.43 (m, 4H, H-21), 2.90 (br. s, 4H, H-9, H-10), 3.61 (m, 2H,
H-19), 3.92 (m, 4H, H-7, H-13), 4.18 (br. s, 2H, H-14), 4.55 (m, 8H, H-
6, H-4), 7.42 (m, 3H, H-16, H-18), 7.56 (m, 2H, H-17) ppm; 13C-NMR
(DMSO-d6) d: 14.67, 14.69 (C-22), 20.72, 21.17, 23.49 (C-8, C-11, C-
12), 29.25, 29.29, 29.33 (C-20, C-21), 49.06, 49.18, 50.93, 51.41 (C-
7, C-9, C-10, C-13), 55.67, 55.74 (C-6), 56.09 (C-14), 61.10, 61.23 (C-
19), 67.65, 67.73 (C-4), 128.74 (C-17), 129.09 (C-18), 130.91 (C-16),
172.59, 172.67 (C-23), 190.86, 191.48 (C-2) ppm; Anal. Calcd for
C30H47N5O4S6: C, 49.08; H, 6.45; N, 9.54. Found: C, 49.41; H, 6.29;
N, 9.70.
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Protozoocidal in-vitro assays
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Lett. 2000, 41, 613–616.
For the L. donovani assay, peritoneal exudate macrophages were
infected with L. donovani amastigotes and exposed to the com-
pounds for five days. IC50 values were determined by comparing
the% infection of the test compounds to uninfected controls
[22]. Protozoocidal activity was evaluated by exposing blood-
stream forms of the parasite to compound for 72 h [23] and anti-
plasmodial activity was determined in a 72-h assay, exposing
infected red blood cells to the compounds and evaluating via
uptake of tritiated hypoxanthine [H]3 [24].
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[20] J. Goksoyr, Acta Chem. Scand. 1964, 18, 1341–1352.
[21] G. Hide (Ed.), Trypanomiasis and Leishmaniasis: Biology
and Control, Cab. International, Wallingford, 1997.
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[23] B. Raz, M. Iten, Y. Grether-Buhler, R. Kaminsky, R. Brun,
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