Synthesis and evaluation of a collection of purine-like C-nucleosides as antikinetoplastid agents

Article abstract of DOI:10.1016/j.ejmech.2020.113101

The kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are the causative agents of neglected tropical diseases with a serious burden in several parts of the world. These parasites are incapable of synthesizing purines de nov

Full text of DOI:10.1016/j.ejmech.2020.113101

European Journal of Medicinal Chemistry 212 (2021) 113101
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and evaluation of a collection of purine-like C-nucleosides
as antikinetoplastid agents
,
, *
Jakob Bouton a, Louis Maes b, Izet Karalic a, Guy Caljon b **, Serge Van Calenbergh a
a Laboratory for Medicinal Chemistry (Campus Heymans), Ghent University, Ottergemsesteenweg 460, B-9000, Gent, Belgium
b Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Universiteitsplein 1 (S7), B-2610, Wilrijk, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are the causative
agents of neglected tropical diseases with a serious burden in several parts of the world. These parasites
are incapable of synthesizing purines de novo, and therefore rely on ingenious purine salvage pathways
to acquire and process purines from their host. Purine nucleoside analogs that may interfere with these
pathways therefore constitute a privileged source of new antikinetoplastid agents. In this study, we
synthetized a collection of C-nucleosides employing five different heterocyclic nucleobase surrogates. C-
nucleosides are chemically and enzymatically stable and allow for extensive structural modification.
Inspired by earlier 7-deazaadenosine nucleosides and known antileishmanial C-nucleosides, we intro-
duced different modifications tailored towards antikinetoplastid activity. Both adenosine and inosine
analogs were synthesized with the aim of discovering new antikinetoplastid hits and expanding
knowledge of structure-activity relationships. Several promising hits with potent activity against Try-
panosoma brucei, Trypanosoma cruzi and Leishmania infantum were discovered, and the nature of the
nucleobase surrogate was found to have a profound influence on the selectivity profile of the
compounds.
Received 5 November 2020
Received in revised form
9 December 2020
Accepted 10 December 2020
Available online 29 December 2020
Keywords:
C-nucleosides
Antiparasitic
Trypanosoma
Leishmania
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
by an acute disease stage that progresses into an asymptomatic
chronic phase. An estimated 30e40% of patients ultimately de-
The kinetoplastid diseases human African trypanosomiasis,
Chagas disease and leishmaniasis are recognized by the WHO as
neglected tropical diseases and are responsible for numerous
deaths every year [1]. Human African trypanosomiasis (HAT,
sleeping sickness) is endemic in sub-Saharan Africa and is caused
by Trypanosoma brucei rhodesiense and Trypanosoma brucei gam-
biense parasites. The disease is transmitted by infected tsetse flies
and progresses from a hemo-lymphatic to an encephalitic stage
that is fatal if left untreated [2]. Chagas disease, caused by Trypa-
nosoma cruzi, is endemic to Latin America and is transmitted via the
bite of a triatomine bug. While the prevalence has declined over the
last years, a dramatic increase in the number of cases in non-
endemic countries has been observed, and Chagas disease is now
considered a global epidemic [3,4]. Chagas disease is characterized
velops chronic disease, resulting in progressive cardiac and/or
gastro-intestinal dysfunction that can be life-threatening [3].
Leishmaniasis is endemic in more than 60 countries spread across
three continents, and is transmitted via sand flies. The disease ex-
ists in multiple clinical forms largely determined by the infecting
subspecies, i.e. localized or diffuse cutaneous, mucocutaneous and
visceral leishmaniasis. Visceral leishmaniasis is a progressive dis-
ease and, if left untreated, is typically fatal within 2 years of
infection [5].
Over the last decades efforts by WHO and DNDi have strongly
contributed to combatting HAT. With a decreasing case burden, the
recent approval of fexinidazole and a promising single-dose oral
treatment (acoziborole) in advanced clinical trials, the prospect of
disease elimination is within sight [6,7]. Much less progress has
been made in containing Chagas disease and leishmaniasis.
Currently available drugs are often ineffective and suffer from
several drawbacks such as toxic side-effects, administration diffi-
culties, long treatment courses, and limited efficacy [2,4,8,9]. While
the leishmaniasis pipeline has been steadily growing, with several
* Corresponding author.
** Corresponding author.
E-mail addresses: Guy.Caljon@uantwerpen.be (G. Caljon), serge.vancalenbergh@
ugent.be (S. Van Calenbergh).
https://doi.org/10.1016/j.ejmech.2020.113101
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

J. Bouton, L. Maes, I. Karalic et al.
European Journal of Medicinal Chemistry 212 (2021) 113101
new chemical entities with distinct mechanisms of action in clinical
development [10], the current pipeline for Chagas disease is nearly
empty. As antiparasitics are considered cornerstones for both
Chagas disease and leishmaniasis therapy, new safe and effective
short-course therapies are urgently needed [2,7,9].
studies [23e27], C-nucleosides have recently attracted a lot of
attention during the search for new HCV therapeutics [22,28e32].
The pyrrolo[2,1-f][1,2,4]triazine nucleoside 6 emerged as a prom-
ising lead compound [22]. The related C-nucleoside GS-441542 (7)
displays broad-spectrum antiviral activity [28] and its phoshpor-
amidate prodrug remdesivir 8 [33,34] has been recently approved
for the treatment of COVID-19. Galidesivir (9), an azanucleoside
with a 9-deazapurine nucleobase, is another C-nucleoside that is
currently in clinical trials for several viral disease indications
[35e37].
The goal of this study was two-fold. First, we wanted to syn-
thesize C-nucleoside equivalents of our previously found 7-
deazaadenosine lead compounds (vide infra). By varying the
nucleobase moiety, we hoped to gain more insight in the SAR and
obtain compounds with improved properties. A pyrrolo[2,1-f]
[1,2,4]triazine moiety serves as a promising 7-deazapurine isostere
and is present in remdesivir [28,30]. Hence, corresponding C-nu-
cleosides are expected to also possess good antitrypanosomal ac-
tivity. As exemplified by compounds 7 and 8, C-nucleosides allow
for the introduction of substituents in the 10-position, a modifica-
tion that has not been explored in the search for antiparasitic
nucleoside analogs.
Secondly, we wanted to broaden the activity spectrum of our
nucleobase library towards Leishmania parasites, which are taxo-
nomically related to T. cruzi and T. brucei. While there is a strong
precedent in literature for nucleosides with antileishmanial activ-
ity, the previously discovered 7-deazapurine nucleoside analogs
were generally devoid of any significant antileishmanial activity, or
lacked the required selectivity [18]. Regarding the extensive opti-
mization and SAR exploration efforts already performed around the
7-deazaadenosine nucleosides, we presumed that alternative
nucleobase surrogates might be more successful for obtaining se-
lective antileishmanial activity. The two most potent anti-
leishmanial nucleosides documented are the C-nucleosides 9-
deazainosine (9-DINO, 10) and formycin B (foB, 11) [38]. As both
are inosine analogs, they act as subversive substrates that are
selectively metabolized by the parasite to the corresponding
Protozoan parasites, including the kinetoplastids, lack the ability
to synthesize the purine ring de novo. Instead, they rely on the
acquisition and salvage of preformed purines to meet their purine
demand. Consequently, they have evolved a complex set of purine
salvage enzymes that allows them to efficiently acquire and use
purines from their host [11e14]. Several of these enzymes are
unique in that they have no mammalian counterpart, and have a
broad substrate capacity. Various purine nucleoside analogs have
thus been identified as inhibitors of purine salvage enzymes, or as
so-called subversive substrates, implying enzymatic conversion to
cytotoxic nucleotide derivatives [15e17]. The screening of purine
nucleosides is therefore a rewarding strategy for the discovery of
new compounds with antiparasitic activity.
Recently, our group reported several nucleoside analogs derived
from the natural product tubercidin (7-deazaadenosine, 1) with
potent activity against T. cruzi and T. brucei (Fig. 1). Introduction of a
4-chlorophenyl substituent (2) afforded analogs with potent ac-
tivity against T. cruzi [18]. Further removal of the 30-hydroxyl group,
as in cordycepin, resulted in a ten-fold increase of anti-T. cruzi ac-
tivity (3) [19]. Two other 30-deoxy 7-deazapurine nucleosides, 4
and 5, displayed highly potent activity against T. brucei and com-
pound 4 (30-deoxytubercidin) displayed curative activity in CNS-
stage HAT animal models [20,21].
In the search for new anti-kinetoplastid hits, we became inter-
ested in expanding our nucleoside analog library with C-nucleo-
sides. C-nucleosides are nucleoside analogs in which the C-N
glycosidic bond is replaced by a C-C bond. C-nucleosides are more
resistant to both enzymatic and acid-catalyzed cleavage than their
N-counterparts, and allow for broader structural variation in the
heterocyclic base moiety, which offers the potential for improved
potency and drug-like properties [22,23]. While in the past atten-
tion towards C-nucleosides was mainly focused on synthetic
Fig. 1. a) Structures of 7-deazapurine hits discovered by our group. b) Examples of antiviral C-nucleosides. c) Structures of the antileishmanial C-nucleosides 9-deazainosine (10)
and formycin B (11). Important structural features are highlighted in blue. Throughout the text, purine numbering will be employed (as shown for 1), while in the experimental part,
IUPAC numbering is used.
2

J. Bouton, L. Maes, I. Karalic et al.
European Journal of Medicinal Chemistry 212 (2021) 113101
adenosine and/or guanosine-like triphosphates that are cytotoxic
to the parasite [39e41]. Since inosine analogs are generally not
phosphorylated in mammalian cells, they can be regarded as
parasite-selective prodrugs, and therefore offer a considerable
selectivity advantage over adenosine nucleosides [41e43]. Indeed,
the earlier mentioned 9-deazainosine was able to deliver full cure
in a visceral leishmaniasis monkey model and did not show any
signs of toxicity [38]. To date however, it has not been further
evaluated. Based on these promising results, we believe that other
inosine C-nucleoside analogs are also likely to provide good anti-
leishmanial activity.
The chemistry of C-nucleosides has been extensively reviewed
[23e27]. Their synthesis has been notoriously difficult, since no
universal “glycosylation” procedure equivalent to the Vorbrüggen
glycosylation used for the synthesis of N-nucleosides has been re-
ported, owing to the varied reactivity of different heterocyclic
nucleobase surrogates. Addition of an organometallic heterocyclic
reagent to a ribonolactone derivative has emerged as one of the
more convenient methods for C-nucleoside synthesis, since it leads
20-deoxy regioisomers was obtained (30-deoxy/20-deoxy ratio 9:1
for 21 and 8:2 for 22), which is similar to the results obtained with
adenosine and formycin by Robins and Moffatt [56,58]. Both
products eluted simultaneously when purified via column chro-
matography and 21 and 22 were therefore purified by preparative
RP-HPLC. The 20-deoxy regioisomers could not be obtained in suf-
ficient quantities for further evaluation.
N-7 derivatization of 19 requires prior protection of the 6-amino
group. Attempted conversion of the 6-amino group of either 17 or
19 into a 1,2,4-triazole failed [59e61]. Therefore, 9-deazaadenosine
19 was peracetylated (Scheme 2). We found that adding excess
MeOH 30 min before work-up of the reaction led to clean formation
of tetraacetylated 23. At this point, we attempted to introduce
differently substituted phenyl groups on the N-7 atom via Chan-
Lam coupling with the appropriate aryl boronic acids. Unfortu-
nately, classical Chan-Lam coupling conditions (Cu(OAc)2, arylbor-
onic acid,
4 Å mol. sieves, pyridine, CH2Cl2) at different
temperatures failed to produce any coupling product. However,
when base-free conditions with boric acid as additive were
employed [62], the reaction proceeded smoothly to afford 24e26.
The deprotection reaction had to be performed at elevated tem-
peratures in order to remove the N-acetyl group, and cleanly
afforded the N-phenyl nucleosides 27e29. Regioisomeric assign-
ment of the phenyl ring (attachment to N-9) was validated via
15N-1H HSQC and HMBC experiments.
to the preferential formation of the b-isomer. Nevertheless, it is not
compatible with all heterocycles and often results in low yields
[44e46].
2. Results and discussion
2.1. Chemistry
Intermediate 14 was also used for the synthesis of pyrazolo[1,5-
a][1,3,5]triazine nucleosides (Scheme 3) [63,64]. Treatment with
hydrazine monohydrochloride at elevated temperature afforded a
complex mixture of products, which upon treatment with ethyl N-
cyanoformimidate (31) yielded 32 and its epimer. Deprotection
with boron trichloride, followed by flash column chromatography
Our investigation started with the 9-deazapurine (pyrrolo[3,2-
d]pyrimidine)
nucleosides.
9-deazainosine
10
and
9-
deazaadenosine have been traditionally obtained in a linear
fashion from 50-O-trityl and 20,30-di-O-isopropylidene protected
ribose in 5:2 and 5:3
b/a
ratios, respectively [47,48]. Alternatively,
purification afforded the b-epimer 33. Diazotation of adenosine
9-deazaguanine can be glycosylated directly on C-7 and the
resulting product can be converted to 9-deazaadenosine [49,50].
However, the long reaction sequence and low yield however render
this method unattractive. We investigated the addition of a met-
allated 9-deazapurine to a ribonolactone derivative, but this was
not successful (data not shown), forcing us to turn back to the
original linear route. Based on a 2007 patent from Biocryst phar-
maceuticals, benzyl protected D-ribofuranose 12 (supporting in-
analog 33 with sodium nitrite in aqueous acetic afforded the cor-
responding inosine analog 34.
The synthesis of the 10-cyano-substituted pyrrolo[2,1-f][1,2,4-
triazine] C-nucleosides is depicted in Scheme 4. The first steps of
the synthesis were performed according to the procedures devel-
oped by researchers at Gilead for the synthesis of remdesivir [33,65].
4-Aminopyrrolo[2,1-f][1,2,4-triazine] 35 was iodinated to afford 36.
The iodinated heterocycle 36 was then first treated with PhMgCl and
TMS-Cl to protect the 6-amino group in situ. Halogen-metal ex-
change with iPrMgCl.LiCl followed by addition of 2,3,5-tri-O-benzyl-
formation) was subjected to
a Horner-Wadsworth-Emmons
reaction with diethyl cyanomethylphosphonate to afford 13 as a
mixture of epimers (Scheme 1) [51]. A hydroxymethylene group
could be introduced in a two-step fashion with Bredereck’s reagent
followed by hydrolysis of the resulting enamine [52,53], but we
found a Claisen condensation of the nitrile carbanion with methyl
formate to be more convenient [54]. The pyrrole ring was then
constructed according to known procedures to afford 15 and 16 as
D-ribonolactone then afforded the hemi-aminal intermediate 37 in
good yield. Intermediate 37 was subjected to anomeric reduction
with triethylsilane and boron trifluoride etherate to afford 38 [46].
The benzyl groups were removed via catalytic hydrogenation over
palladium hydroxide to yield nucleoside 40 in high yield [66].
Alternatively, 37 was reacted with TMSCN, TMSOTf and TfOH to
afford 39 as a mixture of epimers. Deprotection with boron tri-
pure
of the carbethoxy group, however, led to epimerization and 17 was
obtained as a mixture of epimers ( 1:3.7) [55]. Protected 9-
b-epimers [47,48]. The basic conditions required for removal
chloride afforded 7 and the a-epimer 41 in an isolated ratio of 3.7:1.
a/
b
Diastereomeric excess was much lower than reported by Siegel et al.
[33], which could be attributed to the quality of the triflic acid, which
was probably present in its hydrated form. Adenosine analog 7 could
be brominated at C-7 in good yield with 1,3-dibromo-5,5-
dimethylhydantoin (DBDMH) to afford 42.
The pyrrolo[2,1-f][1,2,4-triazine] nucleoside 40 was further elab-
orated to various analogs (Scheme 5). Bromination with DBDMH
under controlled conditions (0 ꢀC, in the dark) cleanly afforded
monobrominated 43 in 93% yield. The bromide was then used for the
introduction of phenyl substituents via Suzuki reactions under
aqueous conditions [67]. Alternatively, 43 was subjected to diazo-
tation with NaNO2 in aqueous acetic acid to afford the inosine analog
46. Under the same conditions, 40 was transformed 47. Again,
attempted 30-deoxygenation afforded a mixture of 30-deoxygenated
and 20-deoxygenated products 48 and 49 (8:2 ratio), which were
deazainosine 18, on the other hand, was obtained as a single
epimer. Hydrogenolysis of the benzyl ethers afforded 9-
deazaadenosine 19 and 9-deazainosine 10. At this stage 19 could
be easily separated from its
column chromatography. The
a
b
-isomer 20 via conventional flash
-stereochemistry of the final com-
pounds was verified via NOESY NMR, where cross-peaks are
observed between H-10 and H-40, and between H-20 and H-8 (pu-
rine numbering) (Supporting information).
Both 9-deazaadenosine 19 and 9-deazainosine 10 were sub-
jected to 30-deoxygenation conditions (Scheme 1), using the three-
step procedure of Robins et al. [56] This method was earlier used by
our group for the 30-deoxygenation of 7-deazapurine nucleosides
[20,57], and cleanly afforded the 30-deoxygenated products as
single regiosiomers. In this case however, a mixture of 30-deoxy and
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J. Bouton, L. Maes, I. Karalic et al.
European Journal of Medicinal Chemistry 212 (2021) 113101
Scheme 1. Synthesis of 9-deazapurine nucleosides. Reagents and conditions: a) diethyl cyanomethylphosphonate, NaH, DME, 0 ꢀC to rt, 84%; b) i. n-BuLi, (iPr)2NH, THF, - 78 ꢀC, ii.
methyl formate, - 78 ꢀC, 58%; c) Bredereck’s reagent, DMF; d) TFA, H2O, CHCl3, 63%; e) aminoacetonitrile.HCl (for 15) or ethyl glycinate.HCl (for 16), NaOAc, MeOH/H2O 10:1; f) ethyl
chloroformate, DBU, CH2Cl2; g) DBU, 68% (15), 46% (16); h) i. K2CO3, EtOH, ii. formamidine acetate, EtOH, reflux, 64% (19), 19% (20), 61% (10); k) i.
MeCN, ii. Pd(OH)2/C, H2, 1 M aq. NaOAc, MeOH, iii. 0.5 M NaOMe in MeOH, 18% (21), 19% (22).
a-acetoxyisobutyryl chloride, NaI,
Scheme 2. N-7 modification of 19. Reagents and conditions: a) i. Ac2O, pyridine,
CH2Cl2, 60 ꢀC, 99%; b) arylboronic acid, Cu(OAc)2, B(OH)3, 4 Å mol. Sieves, MeCN, 80 ꢀC;
c) 0.5 M NaOMe in MeOH, 80 ꢀC, 37% over 2 steps (27), 19% over 2 steps (28), 38% over
2 steps (29).
Scheme 3. Synthesis of pyrazole[1,5-a][1,3,5]-triazine nucleosides. Reagents and
conditions: a) H2NNH2.HCl, EtOH/H2O 5:1, reflux; b) ethyl N-cyanoformimidate (31),
toluene, 80 ꢀC; c) BCl3, CH2Cl2, - 78 ꢀCe0 ꢀC, 18% (3 steps); d) NaNO2, AcOH, H2O, 70 ꢀC,
15%.
separated via preparative RP-HPLC. The desired 30-deoxygenated
product 48 was subjected to diazotation conditions to afford 50, or
brominated with DBDMH at room temperature to afford both mono-
brominated 52 and dibrominated 51. Suzuki reaction of 52 with 4-
chlorophenylboronic acid then afforded 53.
The imidazo[2,1-f][1,2,4]-triazine nucleosides were prepared via
similar procedures as the pyrrolo[2,1-f][1,2,4]triazines (Scheme 6).
In situ TMS protection followed by halogen-metal exchange and
mixture of epimers after anomeric reduction. Deprotection with
boron trichloride yielded the imidazo[2,1-f][1,2,4]-triazine nucle-
oside 56 in low yield. The adenosine analog 56 was further trans-
formed to the corresponding inosine analog 57 via diazotation with
sodium nitrite in aqueous acetic acid.
The synthesis of the imidazo[1,2-a]pyrazine nucleosides
(Scheme 7) was slightly different, since the starting material 58 had
addition to 2,3,5-tri-O-benzyl- -ribonolactone afforded 55 as a
D
already
a chlorine atom present in the 6-position (purine
4

J. Bouton, L. Maes, I. Karalic et al.
European Journal of Medicinal Chemistry 212 (2021) 113101
Scheme 4. Synthesis of the pyrrolo[2,1-f][1,2,4-triazine] nucleosides. Reagents and conditions: a) NIS, DMF, 0 ꢀC, 77%; b) i. TMS-Cl, PhMgCl, THF, 0 ꢀC, ii. iPrMgCl.LiCl, THF, ꢁ15 ꢀC, iii.
2,3,5-tri-O-benzyl-D-ribonolactone, THF, 64%; c) Et3SiH, BF3.OEt2, CH2Cl2, 0 ꢀC, 79%; d) TfOH, TMSOTf, TMSCN, ꢁ78 ꢀC; e) Pd(OH)2/C, H2, AcOH, 63%; f) BCl3, CH2Cl2, - 78 ꢀC to 0 ꢀC,
22% over 2 steps (7), 6% over 2 steps (41); g) DBDMH, DMF, 71%.
numbering). 3-Bromo-8-chloroimidazo[1,2-a]pyrazine 58 was
subjected to halogen-metal exchange with iPrMgCl.LiCl, followed
(21) almost completely removed the cytotoxic effects of 19. The ac-
tivity of 9-deazainosine 10 against T. cruzi and L. infantum was similar
to what was reported in literature [41]. Its rather low activity against
the two T. brucei subspecies is surprising, since it has been reported
to be effective in vivo in combination therapy with eflornithine [72].
by addition of 2,3,5-tri-O-benzyl-
anomeric reduction with boron trifluoride etherate and triethylsi-
lane afforded 59 as a single -isomer. The 6-chloride of 59 was
D-ribonolactone. Subsequent
b
substituted with ammonia to yield the protected adenosine analog
60. Benzyl deprotection of 60 with boron trichloride then afforded
the final nucleoside 61. Reaction with 2-acetoxyisobutyryl iodide
and subsequent hydrogenation of the intermediate halogenated
compounds again resulted in a mixture of 30-deoxygenated and 20-
deoxygenated products (9:1) that was resolved via preparative RP-
HPLC to afford the 30-deoxy regioisomer 62. To install a hydroxy-
group in the 6-position, benzylated chloronucleoside 59 was
reacted with sodium hydroxide. The benzyl groups were removed
via catalytic hydrogenation over palladium hydroxide to afford 63,
together with reduced 64 that was also isolated.
Although 10 has an IC50 value of around 2 mM against L. infantum
intracellular amastigotes, it was able to deliver full cure in a visceral
leishmaniasis monkey model [38], illustrating the difficulties of
translating cellular activity against intracellular amastigotes to in vivo
efficacy [2]. The 30-deoxy-9-deazainosine 22 was inactive against
T. cruzi and L. infantum, which is not suprising given the likely
intraparasitic conversion to the adenosine counterpart 21 that also
lacked significant activity against these parasites. The N-phenyl an-
alogs 27, 28 and 29 displayed good activity against T. cruzi, which is in
line with 7-phenyl-substituted 7-deazaadenosines reported earlier
by our group [18,19]. Interestingly, the Chan-Lam coupling used for
the synthesis of 9-deazapurine nucleosides allowed for the intro-
duction of brominated or iodinated phenyl rings, which was not
possible in earlier series due to cross reactivity in the Suzuki reaction.
Nevertheless, the 4-bromophenyl analog 29 was slightly less potent
than the 4-chlorophenyl analog 27, further providing evidence that a
4-chlorophenyl ring is a preferred substituent for anti-T. cruzi activity
[18,21]. Pleasingly, all three compounds were completely devoid of
cytotoxicity both in MRC-5SV2 and PMM cells, which was not the case
for the corresponding 7-deazapurine nucleosides [18].
2.2. Biology
All final compounds (Fig. 2) were assayed against 4 kinetoplastid
parasites: T. brucei brucei, Trypanosoma b. rhodesiense, T. cruzi and
L. infantum. Cytotoxicity was evaluated in MRC-5SV2 cells (T. cruzi
host cell) and primary mouse macrophages (PMM, L. infantum host
cell). The results are depicted in Table 1.
The cytotoxicity of the natural product 9-deazaadenosine [68] 19
has been long recognized. It is incorporated in RNA, leading to in-
hibition of translation and cell lethality [69e71]. Its 30-deoxygenated
analog 21 displayed decent activity against T. b. brucei and T. b. rho-
desiense and was inactive against T. cruzi and L. infantum. Although it
is a close structural analog of 30-deoxytubercidin (4), it was 10-fold
less active against T. b. brucei and 100-fold less active against T. b.
rhodesiense. As was the case for tubercidin (1) [18], 30-deoxygenation
40 was highly cytotoxic, as recently described by Li et al. [66] Its
7-bromoanalog 43 was less cytotoxic, but still mostly aspecific. The
7-(4-chlorophenyl) analog 44 displayed good activity against T. cruzi,
although it was considerably less selective than 43 or the corre-
sponding 7-deazapurine analog [18]. 45 was about five-fold less
active than 44, but also less cytotoxic. As already reported by Li et al.
[66], the 20-deoxy analog of 40, 49, was not cytotoxic anymore,
5

J. Bouton, L. Maes, I. Karalic et al.
European Journal of Medicinal Chemistry 212 (2021) 113101
Scheme 5. Diversification of the pyrrolo[2,1-f][1,2,4-triazine] nucleosides. Reagents and conditions: a) DBDMH, DMF, 0 ꢀC, 93%; b) arylboronic acid, Pd(OAc)2, TPPTS, Na2CO3, H2O/
MeCN 2:1, 100 ꢀC, 40% (44), 8% (45), 84% (53); c) NaNO2, AcOH, H2O, 70 ꢀC, 58% (46), 30% (47), 22% (50); d) i.
-acetoxyisobutyryl chloride, NaI, MeCN, ii. Pd(OH)2/C, H2, aq. 1 M
a
NaOAc, MeOH, iii. 0.5 M NaOMe in MeOH, 44% over 3 steps (48), 11% over 3 steps (49); e) DBDMH, DMF, 58% (52), 7% (51).
Scheme 6. Synthesis of imidazo[2,1-f][1,2,4-triazine] nucleosides. Reagents and con-
ditions: a) i. TMS-Cl, PhMgCl, THF, 0 ꢀC, ii. iPrMgCl.LiCl, THF, ꢁ15 ꢀC, iii. 2,3,5-tri-O-
benzyl-D-ribonolactone, THF; b) Et3SiH, BF3.OEt2, CH2Cl2, 0 ꢀC; c) BCl3, CH2Cl2, -78 ꢀC to
0
ꢀC, 6% (3 steps); d) NaNO2, AcOH, H2O, 70 ꢀC, 28%.
although it was also inactive against L. infantum and T. cruzi. The 30-
deoxynucleosides 48 and 52 were highly active against both T. b.
brucei and T. b. rhodesiense with IC50 values in the low nanomolar
range, reflecting the outstanding activity of their 7-deazapurine
regioisomers [20]. This further illustrates that pyrrolo[2,1-f][1,2,4]
triazines act as bioisosteres of 7-deazapurines. Interestingly, 52 also
displayed good activity against T. cruzi and L. infantum with accept-
able selectivity indices (24 for T. cruzi, 17 for L. infantum, calculated as
Scheme 7. Synthesis of imidazo[1,2-a]pyrazine nucleosides. Reagents and conditions:
a) i. iPrMgCl.LiCl, 0 ꢀC, THF, ii. 2,3,5-tri-O-benzyl-D-ribonolactone, THF; b) Et3SiH,
BF3.OEt2, 19% (2 steps); c) 7 N NH3 in MeOH, 1,4-dioxane, 120 ꢀC, 91%; d) aq. 2 N NaOH,
1,4-dioxane, reflux; e) Pd(OH)2/C, H2, AcOH, 20% over 2 steps (63), 6% over 2 steps (64);
f) BCl3, CH2Cl2, -78 ꢀCe0 ꢀC, 88%; g) i.
Pd(OH)2/C, H2, aq. 1 M NaOAc, MeOH, iii. 0.5 M NaOMe in MeOH, 20% over 3 steps.
a-acetoxyisobutyryl chloride, NaI, MeCN, ii.
6

J. Bouton, L. Maes, I. Karalic et al.
European Journal of Medicinal Chemistry 212 (2021) 113101
Fig. 2. Overview of the synthesized final compounds.
parasite IC50 divided by host cell CC50). Introduction of a second
bromine atom on the 8-position of 52, as in 51 led to drastic activity
loss. The 30-deoxy analog 53 was slightly more potent and selective
against T. cruzi than its ribo-congener 44, but five-fold less potent
than its 7-deazapurine counterpart [19]. Overall, inosine analogs 46,
47 and 50 displayed much weaker activity than their corresponding
adenosine analogs. The 10-cyano analogs 7, 41 and 42 were generally
inactive, indicating that modifications of the 10-position are not
likely to be beneficial for antiparasitic activity.
The pyrazolo[1,5-a][1,3,5]triazine nucleosides 33 and 34, having
a nitrogen atom on the 7-position as in the canonical purine nu-
cleosides did not display significant activity. The closely related
imidazo[2,1-f][1,2,4]-triazines 56 and 57 were more active. 56 was
considerably less cytotoxic than the earlier described adenosine
analogs 19 and 40. The inosine analog 57 was especially interesting,
since it displayed good activity against T. cruzi and L. infantum, and
was devoid of any cytotoxicity, making this a promising candidate
for further evaluation. Its superior activity compared to the other
inosine analogs in this study (except for 9-deazainosine 10) could
potentially be attributed to the higher similarity of the imidazo[2,1-
f][1,2,4]-triazine nucleobase to the canonical purine ring (same
hydrogen bonding pattern, similar basicity of the nitrogen atom on
the 7-position), resulting in more efficient metabolic conversion to
cytotoxic nucleotides by the parasites. The imidazo[1,2-a]pyrazine
nucleosides (61e64), lacking the nitrogen atom in the 3-position,
were inactive, indicating that the presence of the N-3 atom is likely
crucial for antiparasitic activity.
consisting of five different heterocyclic nucleobase surrogates, and
introduced modifications tailored towards antiparasitic activity. We
discovered potent hits for all three kinetoplastid parasites, with good
selectivity profiles towards two mammalian cell lines. Not unex-
pectedly, the two 30-deoxypyrrolo[2,1-f][1,2,4]triazines 48 and 52
showed potent activity against T. b. brucei and T. b. rhodesiense, given
their high similarity to earlier reported 7-deazapurine nucleosides
[20]. The brominated 30-deoxypyrrolo[2,1-f][1,2,4]triazine nucleo-
side 52 also displayed good activity against both T. cruzi and
L. infantum. The 7-phenyl substituted 9-deazapurine (27e29) as well
as their isomeric pyrrolo[2,1-f][1,2,4]-triazine (44e45, 53) nucleo-
sides showed potent anti-T.cruzi activity. A 4-chlorophenyl ring was
further demonstrated to be a preferred 7-substituent for anti-T. cruzi
activity. It is noteworthy that, while anti-T. cruzi activity was roughly
comparable between these constitutional 7-phenyl isomers, the 9-
deazapurines 27e29 clearly displayed a more favorable cytotox-
icity profile. These results demonstrate that altering the nucleobase
moiety can be a good strategy to increase the selectivity of nucleo-
side analogs, while maintaining good potency. Furthermore, we
discovered two new inosine C-nucleoside analogs (47 and 57) with
good antileishmanial activity in vitro. While 47 showed some cyto-
toxicity in MRC-5SV2 cells, 57 was more potent and devoid of cyto-
toxic effects. Several of the discovered hits show potential for further
evaluation as antikinetoplastid agents.
Declaration of competing interest
3. Conclusion
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
In conclusion, we described
a collection of C-nucleosides
7

J. Bouton, L. Maes, I. Karalic et al.
European Journal of Medicinal Chemistry 212 (2021) 113101
Table 1
Evaluation of drug sensitivity of the synthesized C-nucleoside analogs against T. b. brucei, T. b. rhodesiense, T. cruzi and L. infantum. Cytotoxicity was assayed against human
MRC-5SV2 cells and primary mouse macrophages (PMM). Values represent mean SEM which originate from 2 to 3 independent experiments and are expressed in M. Values
in parentheses represent the values of the different determinations, as no correct average can be calculated. Values in italics represent the result of a single determination
because of inactivity or overt cytotoxicity. Suramin was included as a reference compound for T. b. brucei (EC50 ¼ 0.03 0.01 M) and T. b. rhodesiense (EC50 ¼ 0.04 0.01 M).
Benznidazole was included as a reference for T. cruzi (EC50 ¼ 2.02 0.28 M) and miltefosine as a reference for L. infantum (EC50 ¼ 7.47 2.23 M).
m
m
m
m
m
Cpd.
T. b. brucei IC50
(
m
M)
T. b. rhodesiense IC50
(
m
M)
T. cruzi IC50
(
mM)
L. infantum IC50
(
mM)
MRC-5SV2 CC50
(
m
M)
PMM
CC50 (mM)
9-deazapurine nucleosides
19
21
10
22
27
28
29
0.54
0.05
0.05
0.03
32.5
1.94 0.36
>64.0
>64.0
0.07
0.03
32.0
0.25 0.2
1.33 0.45
>64.0
26.9 1.2
40.3 9.7
41.8 14.8
0.02 0.0003
2.35 0.17
>64.0
26.2 2.3
29.0 2.2
28.9 1.4
32.2 0.94
0.75 0.23
>64.0
0.34 0.09
1.04 0.17
0.46 0.14
0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
[50.8; >64.0; >64.0]
Pyrrolo[2,1-f][1,2,4]triazine nucleosides
40
43
44
45
49
48
52
51
53
47
46
50
7
<0.25
<0.25
<0.25
<0.25
<0.25
[19.8; 11.8; >64.0]
10.79
[40.3; >64.0]
>64.0
>64.0
1.1 0.8
33.9 0.78
[32.6; 22.3; >64.0]
49.4
>64.0
>64.0
<0.25
2.0
5.0
>64.0
>64.0
6.00 2.67
4.53 3.47
[32.0; >64.0; >64.0]
24.0 10.7
>64.0
>64.0
>64.0
1.69 1.54
3.89 1.8
6.78 0.18
6.32 1.31
0.0090 0.0002
0.0021 0.00001
0.43 0.06
3.53 0.27
3.53
30.9
>64.0
8.91 0.04
10.3 0.33
13.0
0.14 0.02
3.71 1.15
5.98 0.09
1.68 0.44
0.00085 0.0003
0.0016 0.0004
0.12 0.003
1.65 1.04
7.06
20.0
33.8
0.58 0.06
1.77 0.09
1.58
0.78 0.12
0.54 0.29
2.45 1.55
>64.0
1.13 0.34
2.36 0.33
[40.3; >64.0]
>64.0
5.67 3.25
0.27 0.24
30.5 18.6
23.3 12.2
5.66
>64.0
>64.0
>64.0
>64.0
0
3
6
20.8 5.76
0.041 0.005
5.99 0.42
0.23 0.01
7.67
>64.0
>64.0
>64.0
>64.0
39.3
>64.0
>64.0
11.0
>64.0
>64.0
32.0
41
42
32.5
Pyrazolo[1,5-a][1,3,5]triazine nucleosides
33
34
24.8 12.4
17.2 7.9
33.8 4.2
36.7 5.5
3.81 1.2
10.1 2.7
21.9 0.8
34.2 8.9
9.32 3.8
>64.0
>64.0
>64.0
Imidazo[2,1-f][1,2,4]-triazine nucleosides
56
57
<0.25
0.5 0.05
2.00
3.01 0.44
0.62
0.6 0.01
5.15
3.17
1.41
>64.0
32.0
>64.0
0
Imidazo[1,2-a]pyrazine nucleosides
61
63
62
64
>64.0
>64.0
>64.0
>64.0
0.81 0.1
>64.0
55.7
30.9 3.4
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
https://doi.org/10.1016/S0140-6736(18)31204-2, 10151.
Acknowledgements
€
[6] E.A. Dickie, F. Giordani, M.K. Gould, P. Maser, C. Burri, J.C. Mottram, S.P.S. Rao,
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The present work has received funding from FWO (S.V.C., G.C.,
L.M.; project number G013118 N). S.V.C. thanks the Hercules
Foundation (project AUGE/17/22 “Pharm-NMR”). G.C. is supported
by research funds of the University of Antwerp (TT-ZAPBOF 33049).
The authors would like to thank An Matheeussen and Margot
Desmet for excellent technical assistance.
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