Synthesis and evaluation of 3′-fluorinated 7-deazapurine nucleosides as antikinetoplastid agents

European Journal of Medicinal Chemistry 216 (2021) 1 13290

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European Journal of Medicinal Chemistry

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Synthesis and evaluation of 3⁰-ﬂuorinated 7-deazapurine nucleosides

as antikinetoplastid agents

Jakob Bouton ^a, Arno Furquim d’Almeida ^a, Louis Maes ^b, Guy Caljon ^b,

,

Serge Van Calenbergh ^{a *}, Fabian Hulpia ^a

^aLaboratory for Medicinal Chemistry (Campus Heymans), Ghent University, Ottergemsesteenweg 460, B-9000, Gent, Belgium

^bLaboratory 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:

Kinetoplastid parasites are the causative agents of neglected tropical diseases with an unmet medical

need. These parasites are unable to synthesize the purine ring de novo, and therefore rely on purine

salvage to meet their purine demand. Evaluating purine nucleoside analogs is therefore an attractive

strategy to identify antikinetoplastid agents. Several anti-Trypanosoma cruzi and anti-Trypanosoma brucei

7-deazapurine nucleosides were previously discovered, with the removal of the 3⁰-hydroxyl group

resulting in a signiﬁcant boost in activity. In this work we therefore decided to assess the effect of the

introduction of a 3⁰-ﬂuoro substituent in 7-deazapurine nucleosides on the anti-kinetoplastid activities.

Hence, we synthesized two series of 3⁰-deoxy-3⁰-ﬂuororibofuranosyl and 3⁰-deoxy-3⁰-ﬂuoroxylofur-

anosyl nucleosides comprising 7-deazaadenine and -hypoxanthine bases and assayed these for anti-

parasitic activity. Several analogs with potent activity against T. cruzi and T. brucei were discovered,

indicating that a ﬂuorine atom in the 3⁰-position is a promising modiﬁcation for the discovery of anti-

parasitic nucleosides.

Received 24 November 2020

Received in revised form

26 January 2021

Accepted 8 February 2021

Available online 16 February 2021

Keywords:

Kinetoplastid

3⁰-ﬂuoronucleoside

7-deazapurine

Trypanosoma cruzi

Trypanosoma brucei

1. Introduction

patient monitoring. These factors greatly impede effective treat-

ment of these kinetoplastid diseases [1,8].

The human kinetoplastid protozoan infections are categorized

by the WHO as neglected tropical diseases. Organisms involved are

Trypanosoma brucei rhodesiense (T. b. rhodesiense) and gambiense

(T. b. gambiense), the causative agents of Human African Trypano-

somiasis (HAT) or sleeping sickness. Trypanosoma cruzi (T. cruzi) is

the causative agent of Chagas disease, and Leishmania spp. cause

Leishmaniases. Together these diseases are responsible for 30 000

deaths annually, and nearly one billion people worldwide are at risk

of contracting one of these devasting infections [1].

Currently, no human vaccines are available for any of these three

infectious diseases [2-4], and treatment is therefore focused on

prevention and antiparasitic chemotherapy [5e7]. Most of the

currently available drugs are repurposed from other therapeutic

indications and suffer from several drawbacks such as low efﬁcacy,

suboptimal (non-oral) administration route, toxicity and long

treatment regimens, thereby often requiring hospitalization and

The burden of HAT has ameliorated over the last years due to

successful management strategies, and particularly, due to the

recent approval of fexinidazole, an oral treatment for gambiense

HAT [9,10]. Furthermore, acoziborole, a single-dose oral treatment

option, is currently in advanced clinical trials [1 1,12], setting the

hopes of eliminating HAT by 2030 [9,1 1]. Progress in containing

Leishmaniasis and Chagas disease has been much more limited. The

anti-leishmaniasis drug pipeline has been steadily growing over the

last decade, with multiple candidates with a novel mechanism of

action now entering clinical trials [13,14]. Nevertheless, a large

need for new drug discovery efforts remains, since the current

pipeline has a high failure risk due to its early development stage.

Additionally, combination regimens will likely be needed for

elimination of leishmaniasis [8,15,16]. Lastly, the situation for

Chagas disease is much worse, as the development pipeline is

nearly empty, with only one agent, fexinidazole, currently in clin-

ical trials [1 7]. Although there is consensus that antiparasitic

chemotherapy should be the cornerstone in the treatment of Cha-

gas disease, the proﬁle of the ideal drug is still somewhat debated

[1,18]. In short, despite several successes in the discovery and

* Corresponding author.

E-mail address: serge.vancalenbergh@ugent.be (S. Van Calenbergh).

https://doi.org/10.1016/j.ejmech.2021.113290

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

development of new antikinetoplastid agents, there remains a

pressing need for research efforts into novel chemical entities to

identify new lead candidates for the treatment of these diseases.

Like most other protozoan parasites, the kinetoplastids are pu-

rine auxotroph, meaning that they are unable to synthesize purines

de novo but salvage these to meet their purine demand [19e23].

The purine salvage pathway therefore constitutes an attractive

target for therapeutic intervention [21e23]. Several purine nucle-

oside analogs have already been shown to possess anti-

kinetoplastid activity, either via inhibition of purine salvage

pathway enzymes or by acting as so-called ‘subversive substrates’

that are selectively converted by (purine salvage) enzymes of the

parasite into cytotoxic metabolites [24e26].

Our group has previously identiﬁed several derivatives (e.g.

2e4) from the naturally occurring nucleoside antibiotic tubercidin

(7-deazaadenosine, 1) as potent anti-T. brucei or anti-T. cruzi agents

[27,28] (Fig. 1). Typically, removal of the 3⁰-hydroxyl group of the

ribofuranose ring has resulted in a tremendous boost in anti-

trypanosomal potency [29e31], culminating in the identiﬁcation of

compounds 3 and 4. Compound 3 provided full cure in a stage II

HAT animal model [30], while compound 4 showed promising

in vivo anti-T. cruzi activity [29]. These examples underscore the

potential of modiﬁed purine nucleoside analogs as anti-

kinetoplastid agents.

rationale for investigating 3⁰-deoxy-3⁰-ﬂuoro 7-deazapurine

nucleoside analogs as antikinetoplastid agents.

In this study, we describe the synthesis of 3⁰-deoxy-3⁰-ﬂuoror-

ibofuranosyl and 3⁰-deoxy-3⁰-ﬂuoroxylofuranosyl nucleosides with

purine or 7-deazapurine nucleobases and evaluate their activity

against the three kinetoplastid parasites.

2. Results and discussion

2.1. Chemistry

The synthesis of 3-deoxy-3-ﬂuororibofuranosyl donor 12

roughly matched that reported by Mikhailopulo et al. [43,44]

(Scheme 1). Double acetalization of D-xylose with acetone/H₂SO₄

afforded the bis-acetonide, from which the 3,5-acetal was selec-

tively cleaved under mild acidic conditions to yield 7 [45]. The

primary alcohol was then selectively protected as benzoate ester

and the 1,2-acetonide removed in acidic MeOH, furnishing inter-

mediate 9 as a mixture of a/b anomers. Intermediate 9 was ﬂuo-

rinated with DAST in acceptable yield, following literature

conditions to afford 10 as an anomeric mixture [43,44]. No double

ﬂuorinated nor ribo-2,3-epoxide side products were observed.

Further protecting group manipulations led to glycosyl donor 12,

used to prepare the desired nucleoside analogs (Scheme 2 and 3).

To validate the reported antileishmanial activity of 5 and 6,

these two purine analogs and some closely related analogs were

prepared (Scheme 2). Commercially available nucleobases 6-

chloropurine and 2-amino-6-chloropurine were glycosylated un-

der modiﬁed Vorbrüggen conditions to give the corresponding

nucleosides 13 and 15 in high yield [46]. The 6-amino group was

introduced in a two-step sequence via S_NAr with sodium azide and

Staudinger reduction under conditions reported previously

[47e49]. Deprotection under basic conditions then afforded 3⁰-

deoxy-3⁰-ﬂuoroadenosine 6. Alternatively, deprotection and

concomitant S_NAR with sodium methoxide, followed by substitu-

tion of the methoxy group with NaOH, afforded 3⁰-deoxy-3⁰-ﬂuo-

rinosine 5. The synthesis of the guanosine analogs 16 and 2,6-

diaminopurine derivative 17 was effected in a similar fashion as

for the inosine and adenosine congeners, respectively.

The observation that the 3⁰-hydroxyl group of the ribofuranose

moiety was not required for antitrypanosomal activity and that its

removal often signiﬁcantly increased the activity against trypano-

somes, incited us to explore other 3⁰-modiﬁcations. Since a ﬂuorine

atom is able to serve as both a hydroxyl (similar polarity) [32] or

hydrogen (similar size) [33] isostere, in this study we wanted to

assess the effect of introduction of a 3⁰-ﬂuoro group on the anti-

kinetoplastid activity. The introduction of ﬂuorine atoms in the

ribose moiety of nucleosides has been a very successful strategy for

the discovery of bioactive (antiviral and antiproliferative) nucleo-

side analogs (e.g. gemcitabine [34], sofosbuvir [35]) [36,37]. Fluo-

rine atoms have a profound inﬂuence on the stereoelectronic

properties of the nucleoside and can lock the sugar ring in a

preferred conformation, modulating its biological properties

[32,38,39]. Additionally, the activity of 3⁰-deoxy-3⁰-ﬂuoroinosine 5

and 3⁰-deoxy-3⁰-ﬂuoroadenosine 6 against Leishmania parasites has

been described in the literature [40e42], further strengthening the

The synthesis of 3⁰-deoxy-3⁰-ﬂuoronucleosides bearing a 7-

deazapurine nucleobase is depicted in Scheme 3. Glycosylation of

Fig. 1. Rationale for the 3⁰-ﬂuorinated 7-deazapurine nucleosides.

2

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

Scheme 1. Reagents and conditions: a) H₂SO₄, CuSO₄, acetone; b) 0.2% aq. HCl; c) BzCl, Et₃N, CH₂Cl₂, 0 ^ꢀC; d) H₂SO₄, MeOH, 80 ^ꢀC; e) DAST, MeCN, 0 ^ꢀC to rt; f) BzCl, Et₃N, DMAP,

CH₂Cl₂; g) H₂SO₄, Ac₂O, AcOH, 0 ^ꢀC to rt.

Scheme 2. Reagents and conditions: a) 6-chloropurine (13) or 2-amino-6-chloropurine (15) TMSOTf, DBU, MeCN, 60 ^ꢀC; b) 0.5 M NaOMe in MeOH; c) i. NaN₃, DMF, 65 ^ꢀC, ii. PMe₃,

THF, H₂O, iii. 0.5 M NaOMe in MeOH; d) 2 M NaOH, reﬂux; e) 2 M NaOH, 1,4-dioxane, reﬂux; f) i. NaN₃, DMF, 65 ^ꢀC, ii. PMe₃, THF, H₂O, iii. 0.5 M NaOMe in MeOH.

12 with N-pivaloyl-6-chloro-7-bromo-7-deazaguanine [50] under

the conditions described by Seela et al. [51] afforded an inseparable

mixture of 18 and starting materials, which after treatment with

sodium methoxide allowed to isolate 19. Reductive removal of the

7-bromo atom via Pd(OH)₂-catalyzed hydrogenation afforded 20,

which was further converted to the 7-deazaguanosine analog 21 via

substitution with sodium hydroxide. Various halogenated 7-

deazapurines were introduced via Vorbrüggen glycosylation with

12 under previously reported conditions [52], albeit in substantially

lower glycosylation yields than reported for 1-O-acetyl-tri-O-ben-

zoylribofuranose [52]. Next, the protected nucleosides were con-

verted to either adenosine analogs 30e33 via S_NAr with sodium

azide and Staudinger reduction followed by deprotection, or to

inosine analogs 38e41 via deprotection and subsequent deme-

thylation with TMSI or alternatively, substitution of the 6-OMe

group with NaOH [52]. 25 was reacted with NaN₃in DMF to

afford 29, which was employed as starting material for different

analogs (Scheme 4).

Next, the iodide substituent of 42 was used as a synthetic handle

to introduce different substituents in the 7-position (Scheme 4).

Reaction of 42 with in situ formed CuCF₃in DMF/NMP [28] afforded

43. Staudinger reduction and deprotection then gave access to 44.

Hydrogenation over Pd(OH)₂/C in buffered MeOH simultaneously

removed the iodide substituent and reduced the 6-azide to afford

45, which, after deprotection led to 3⁰-deoxy-3⁰-ﬂuorotubercidin

46, Which upon diazotization with NaNO₂furnished inosine analog

47. A 7-methyl substituent was introduced via a palladium-

catalyzed coupling reaction of 42 with AlMe₃[55], which pro-

ceeded in low yield due to the formation of a signiﬁcant amount of

dehalogenated product. Staudinger reduction and deprotection

then afforded 49. The nitrile substituent of 50 was introduced via

halogen/magnesium exchange with Knochel’s turbo Grignard re-

agent, followed by trapping with tosyl cyanide [56]. The benzoate

groups of 50 were deprotected in methanolic ammonia to afford 51

and the methanimidate 53 in a 1:1 ratio (LC/MS). However, the

imidate was efﬁciently transformed back to nitrile analog 51 by

heating in DMF with a catalytic amount of NaOAc [57]. The car-

boxamide analog 52 was synthesized by reacting 51 with hydrogen

peroxide in the presence of ammonium hydroxide.

Sonogashira reaction on 29 with ethynyltrimethylsilane, fol-

lowed by removal of the TMS group afforded 55. Hydrogenation of

the triple bond then furnished the 7-ethyl-analog 56. A vinyl sub-

stituent was introduced via an aqueous Suzuki reaction with po-

tassium vinyl triﬂuoroborate [58,59]. Four substituted phenyl

analogs (58e61) were prepared via Suzuki reaction under aqueous

conditions [27,29,55]. The 6-oxo-nucleoside 62 was obtained from

58 via diazotation with NaNO₂/AcOH.

The synthesis of the 3-ﬂuoroxylofuranose precursor 67 started

from 1,2-5,6-di-O-isopropylidene-a-D-allofuranose (Scheme 5).

Selective 5,6-acetal removal, followed by periodate cleavage and

reduction of the resulting aldehyde gave 63 [60]. The 5-hydroxyl

group was selectively benzoylated to give 64 [61], after which the

3-ﬂuorine atom was introduced in the xylo-conﬁguration with

DAST [61]. The remaining 1,2-acetonide was hydrolyzed with acidic

methanol and the resulting 2-hydroxyl group benzoylated to afford

66. Acetolysis furnished glycosylation donor 67.

The synthesis of the envisioned 3⁰-deoxy-3⁰-ﬂuoroxylofur-

anosylnucleosides followed similar sequences as for their 3⁰-ribo-

diastereoisomers, involving a Vorbrüggen coupling as the key step

(Scheme 6). Glycosylation of 67 with 6-chloro-7-bromo-7-

deazapurine afforded 68 in a similar yield as obtained for the 3⁰-

ﬂuoro-ribo product 24. Next, nucleophilic aromatic substitution of

the chloride on the 6-position with sodium azide furnished 69,

which, after Staudinger reduction, gave rise to 6-amino-analog 70.

The bromide was removed via catalytic hydrogenation over Pd/C,

and deprotection with sodium methoxide subsequently afforded

3

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

Scheme 3. Reagents and conditions: a) N-pivaloyl-7-bromo-6-chloro-7-deazaguanine [50], BSA, TMSOTf, MeCN, 50 ^ꢀC; b) 0.5 M NaOMe in MeOH, reﬂux; c) Pd(OH)₂/C, H₂, 1 M

NaOAc, MeOH; d) 2 M NaOH, 1,4-dioxane; e) 5-substituted 4-chloropyrrolo [2,3-d]pyrimidine (X ¼ F [53], Cl [54], Br [54], I [54]), BSA, TMSOTf, MeCN, 80 ^ꢀC; f) i. NaN₃, DMF, 65 ^ꢀC, ii.

PMe₃, THF, H₂O; g) 0.5 M NaOMe in MeOH; h) 0.5 M NaOMe in MeOH; i) TMSCl, NaI, MeCN; j) 2 M NaOH, 1,4-dioxane, reﬂux; k) NaN₃, DMF, 65 ^ꢀC.

the 3⁰-deoxy-3⁰-ﬂuoroxylofuranosyl analog of tubercidin (71).

Alternatively, the benzoate groups of 70 were hydrolyzed to yield

72, which was then further transformed into the corresponding

inosine analog 73 with NaNO₂/AcOH. In order to introduce several

substituted phenyl analogs in the 7 position of the purine ring, an

aqueous Suzuki reaction was employed [27,29,55].

cytotoxicity against MRC-5 ﬁbroblasts, as described in literature

[41]. Activity of 6 against L. infantum was, however, limited.

Remarkably, the 7-deaza analog 46 was 16-fold less active against

T. cruzi, but not cytotoxic. Introduction of a halogen at the 7-

position remarkably increased the antiparasitic activity. The 7-

bromo and 7-iodo analogs 32 and 33 were the most potent and

displayed excellent selectivity. In comparison the 7-chloro analog

31 was somewhat less potent against T. cruzi, but markedly more

toxic against PMM cells. The increasing activity trend observed in

this halogen series could be suggestive of a crucial halogen bond

interaction with a parasitic target or transporter. While several

other non-aromatic substituents (e.g. triﬂuoromethyl, cyano or

ethynyl) afforded activity against T. cruzi or L. infantum, these an-

alogs lacked selectivity. Only the 7-vinyl derivative 57 displayed

submicromolar activity against T. cruzi with an acceptable selec-

tivity index (SI ¼ 31). The 7-phenyl-substituted analogs 58e60

displayed good activity against T. cruzi, but were generally less

active than the corresponding ribofuranose and 3⁰-deoxyribofur-

anose analogs [27,29]. The antileishmanial activity of the 3⁰-deoxy-

3⁰-ﬂuororibo adenosine analogs was less promising as none of the

evaluated compounds displayed acceptable selectivity with respect

to host cell toxicity.

2.2. Biological evaluation

All nucleoside analogs were evaluated for their activity against

T. cruzi, L. infantum (Table 1), T. b. brucei and T. b. rhodesiense

(Table 2). Cytotoxicity was assayed against MRC-5 cells (T. cruzi host

cell) and primary mouse macrophages (PMM, L. infantum host cell).

Surprisingly, the previously reported 3⁰-ﬂuororibo inosine

analog 5, reported to be active against L. donovani intracellular

amastigotes [41], was found to be inactive against L. infantum in our

intra-amastigote assay (Table 1a). However, it did display low

micromolar activity against T. cruzi. Unfortunately, when the purine

nucleobase was exchanged for a 7-deazapurine, as in 41, no in vitro

activity could be identiﬁed against either T. cruzi or L. infantum.

Analogs with modiﬁcations at the 7-position (46e62) nor guano-

sine analogs (16e19, Table 1b) were found to exhibit any speciﬁc

antiparasitic activity.

Evaluation of the synthesized 3⁰-deoxy-3⁰-ﬂuoroxylonucleo-

sides (Table 1d) indicated that 3⁰-deoxy-3⁰-ﬂuoroxylofur-

anosyltubercidin 71 was inactive against both protozoa and that its

7-bromo analog 72 did not display selective antiparasitic activity in

The adenosine analogs, on the other hand, showed signiﬁcant

antiparasitic activity (Table 1c). 3⁰-Deoxy-3⁰-ﬂuoroadenosine 6

displayed good activity against T. cruzi, but suffered from

4

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

Scheme 4. Reagents and conditions: a) TMSCF₃, CuI, KF, DMF/NMP 1:1, reﬂux; b) PMe₃, THF, H₂O; c) 0.5 M NaOMe in MeOH; d) Pd(OH)₂/C, H₂, 1 M NaOAc, MeOH; e) NaNO₂, AcOH,

H₂O, 70 ^ꢀC. f) AlMe₃, Pd(PPh₃)₄, reﬂux; g) i. iPrMgCl.LiCl, THF, ꢁ60 ^ꢀC ii. TsCN, ꢁ60 ^ꢀC to rt; h) 7 N NH₃in MeOH; i) H₂O₂(30 % wt.), NH₄OH); j) NaOAc, DMF; k) i. TMS-acetylene,

PdCl₂(PPh₃)₂, CuI, Et₃N, DMF, ii. 7 N NH₃in MeOH; l) Pd/C, H₂, MeOH; m) potassium vinyl triﬂuoroborate, Cs₂CO₃, Pd(OAc)₂, TPPTS, H₂O/MeCN 2:1, 100 ^ꢀC; n) phenylboronic acid,

Na₂CO₃, Pd(OAc)₂, TPPTS, H₂O/MeCN 2:1, 100 ^ꢀC.

Scheme 5. Reagents and conditions: a) i. 70% aq. AcOH, ii. NaIO₄, H₂O, iii. NaBH₄, EtOH; b) BzCl, Et₃N, DCM, ꢁ20 ^ꢀC; c) DAST, pyridine, CH₂Cl₂, - 10 ^ꢀC to rt; d) i. MeOH, conc. HCl,

55 ^ꢀC, ii. BzCl, Et₃N, DMAP, CH₂Cl₂; e) AcOH, Ac₂O, H₂SO₄.

5

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

Scheme 6. Reagents and conditions: a) 7-bromo-6-chloro-7-deazapurine [54], BSA, TMSOTf, MeCN, 80 ^ꢀC; b) NaN₃, DMF, 65 ^ꢀC; c) PMe₃, THF, ii. 1 M aq. AcOH, MeCN, 65 ^ꢀC; d) i. Pd/

C, H₂, aq. NaOAc, EtOH; ii. 0.5 M NaOMe in MeOH; e) 0.5 M NaOMe in MeOH; f) NaNO₂, AcOH, H₂O, 50 ^ꢀCe70 ^ꢀC; g) (substituted) phenylboronic acid, Na₂CO₃, Pd(OAc)₂, TPPTS, H₂O/

MeCN 2:1, 100 ^ꢀC.

contrast to its 3⁰-ﬂuororibo epimer 32. In line with previous ﬁnd-

ings [27,29] the 7-phenyl analogs 74e78 showed potent in vitro

activity against T. cruzi, with 74 emerging as the best analog in this

subset. Interestingly, when equipped with a 7-phenyl substituent,

the 3⁰-ﬂuoroxylo analogs tend to display superior anti-T. cruzi ac-

tivity than their 3⁰-ribo epimers, while an opposing trend is

observed for the 7-halogenated analogs.

cytotoxicity against MRC-5 cells, marking it a promising candidate

for further evaluation. The 7-bromide analog 72 displayed roughly

equal potency, but was found less selective than 71. The corre-

sponding inosine analog 73 was inactive. The 7-phenyl analogs

74e78 were only moderately active with EC₅₀values in the low

micromolar range.

These results indicate that both 3⁰-ﬂuororibo and 3⁰-ﬂuoroxylo

modiﬁcations may provide potent antikinetoplastid nucleosides.

Importantly, the SAR observed in both series was different from

that in the ribonucleoside [27] and 3⁰-deoxyribonucleoside series

[29,30]. Among the 3⁰-ﬂuororibo-nucleosides introduction of a

bromo (32) or iodo (33) substituent in 46 conferred highly potent

and selective anti-T. cruzi activity. Except for the 7-phenyl analogs,

SAR trends in the 3⁰-ﬂuororibo series were similar for T. cruzi and

T. brucei, with 32 and 33 also displaying good activity against T. b.

brucei and T. b. rhodesiense. The 7-phenyl analogs 17e61 displayed

good anti-T. cruzi activity, albeit with lower selectivity compared to

the 3⁰-deoxy analogs [29].

All nucleoside analogs were also evaluated against T. b. brucei

and T. b. rhodesiense (Table 2). The inosine and guanosine analogs

(Tables 2a and 2b) were devoid of activity, except for the weakly

active 14 and 36. 3⁰-Deoxy-3⁰-ﬂuororiboadenosine 6 was found to

be highly active against both T. brucei species, which has not been

previously reported (Table 2c), while the corresponding 7-

deazaadenosine analog 46 was only weakly active. The difference

with the corresponding 3⁰-deoxynucleoside 3 is striking, as it is one

of the most potent antitrypanosomal nucleosides known [30,31].

Similar to what was observed for the T. cruzi activity, introducing a

halogen atom in the 7-position (30e33) led to potent antiparasitic

activity, with the 7-bromo (32) and 7-iodo (33) analogs being the

most active, with EC₅₀values similar to 3⁰-deoxy-3⁰-ﬂuo-

roadenosine 6. These analogs are however still 30-fold less active

than the corresponding 3⁰-deoxy congeners [30,31]. The 7-chloro

analog 31 was tenfold less potent. Other substituents in the 7-

position had less effect on potency, with only the methyl analog

49 and the vinyl analog 57 displaying submicromolar activity. The

7-phenyl analogs 58e60, which displayed good anti-T. cruzi activity,

failed to show selective anti-T. brucei activity, a trend matching

previous observations with ribofuranose [27] and 3⁰-deoxy-

ribofuranose [31] nucleoside analogs.

3⁰-Fluoroxylotubercidin 71 and its 7-bromide derivative 72

displayed highly potent anti-T. brucei activity. Especially 71 shows

promise for further development, considering its exceptional

selectivity versus MRC-5 cells. Also the 7-aryl-substituted analogs

74e78 displayed good anti-T. cruzi activity.

3⁰-ﬂuororibo and 3⁰-ﬂuoroxylo nucleosides are known to exhibit

different sugar puckering, with 3⁰-ﬂuororibo nucleosides mainly

adopting

a

C-2⁰-endo (South) conformation and 3⁰-ﬂuorox-

ylonucleosides preferentially occurring in a C-3⁰-endo (North)

conformation [62e64]. In the absence of the exact mechanism(s) of

action of the present nucleosides, it is premature to link these

conformational preferences to the observed activities.

The anti-T. brucei activity for the 3⁰-deoxy-3⁰-ﬂuoroxylofur-

anosyl nucleosides is depicted in Table 2d. Remarkably, the 3⁰-ﬂu-

oroxylotubercidin analog 71, which was inactive against T. cruzi,

displayed low nanomolar activity against T. b. brucei and T. b. rho-

desiense. This contrasts with the corresponding 3⁰-ﬂuororibofur-

Comparison of the 3⁰-ﬂuororibo and 3⁰-ﬂuoroxylo 7-

deazaadenosine series indicate that for analogs with relatively

small substituents (halogen, CF₃, CN, Me, Et, ethynyl, vinyl …) on

the 7-position, the SAR differed substantially between the 3⁰-ﬂuo-

roribo and the 3⁰-ﬂuoroxylo series. Nevertheless, small substituents

conferred signiﬁcant improvement in activity against T. cruzi, which

anose analog 46 (T. b brucei EC₅₀22.2 2.2

mM, T. b. rhodesiense EC₅₀

1.47 0.91 M). Additionally, derivative 71 did not display any

m

6

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

Table 1

Evaluation of drug sensitivity of 3⁰-deoxy-3⁰-ﬂuoronucleosides against T. cruzi and L. infantum. Cytotoxicity was assayed against human MRC-5 cells and primary mouse

macrophages (PMM). Values represent mean SEM which originate from 2 to 3 independent experiments and are expressed in mM. Values in parentheses represent the values

of the different determinations, where no correct average can be calculated. Values in italics represent the result of a single determination. SI, in vitro selectivity index is the

ratio of the EC₅₀for the host cell (MRC-5 for T. cruzi, PMM for L. infantum) and the EC₅₀of the parasite. Benznidazole was included as a reference for T. cruzi

(EC₅₀¼ 2.02 0.28

m

M) and miltefosine as a reference for L. infantum (EC₅₀¼ 7.47 2.23

mM).

Cpd.

Structure/R ¼

T. cruzi EC₅₀

(

mM)

MRC-5 EC₅₀

(m

M)

SI

T. cruzi

L. infantum EC₅₀

(m

M)

PMM

SI

EC₅₀

(

m

M)

L. infantum

a. 3⁰-ﬂuororibo inosine analogs

5

OH

OMe

H

4.28 0.91

30.1 3.1

> 64.0

>64.0

> 64.0

>15

>2

-

>64.0

> 64.0

>64.0

e

14

47

38

39

40

41

62

36

> 64.0

F

e

Cl

Br

I

Ph

/

b. 3⁰-ﬂuororibo guanosine analogs

16

17

21

20

19

OH

NH₂

H

Br

34.6 4.3

> 64.0

42.0 10.0

> 64.0

1

-

46.9 3.9

> 64.0

>64.0

> 64.0

>1

e

c. 3⁰-ﬂuororibo adenosine analogs

6

/

H

F

Cl

Br

I

CF₃

Me

0.55 0.15

9.09 0.77

0.25 0.1

0.071 0.019

0.041 0.01

0.039 0.007

3.17

13.1 5.8

9.66

7.10

0.04

> 64.0

0.65 0.15

9.14

0.74

4.92 0.04

>64.0

9

27.5 4.9

23.4 14.7

1.75 0.26

0.09 0.01

0.37 0.34

2.33 0.57

27.27

38.4 25.7

2.03

32.5

0.13

> 64.0

39.3 11.5

50.8

12.7

[32.0, >64.0]

>64.0

2.00

>2

e

1

3

9

46

30

31

32

33

44

49

51

52

55

56

57

58

59

61

60

>7

22

194

779

170

4

> 5

< 1

4

< 1

31

> 7

23

19

2

5.49 0.63

13.8 8.9

32.0 8.9

6.65 1.5

7.01

>64.0

1.32

2.38

0.16

0.13

1.02 0.98

22.0 13.3

32.0

>64.0

2.0

32.0

0.13

> 64.0

>64.0

> 64.0

32.0

1

>2

<1

1

CN

CONH₂

Ethynyl

Et

vinyl

Ph

4-Cl-Ph

3,4-diCl-Ph

4-OMe-Ph

1

40.3

e

>1

1

3

20.2 7.7

> 64.0

16.9

4.14

14.1

0.22

8.61

2.83

32.5

8.0

32.0

1

d. 3⁰-ﬂuoroxylo nucleoside analogs

71

72

73

74

76

77

75

78

H

Br

>64.0

7.46 1.81

>64.0

0.17 0.08

[0.21, <0.25]

0.42 0.05

1.45 0.45

0.75 0.25

>64.0

0.75 0.25

>64.0

21.7 0.73

7.38 1.14

18.6 0.82

>64.0

e

>64.0

8.20 3.12

>64.0

20 12

>64.0

8.00

e

2

e

1

2

1

<1

e

4-Cl-Ph

3,4-diCl-Ph

3-F-4-Cl-Ph

4-F-Ph

3-Me-4-Cl-Ph

130

35

44

>44

28

5

1.82

3.87 0.69

5.95 0.87

22.6 9.9

5.34 2.17

32.0

8.00

20.9 1.13

was not observed in the (3⁰-deoxy)ribose series [27,29,30]. Notably,

the anti-T. cruzi activity of the 7-phenyl analogs is more similar

between the 3⁰-ﬂuororibo and the 3⁰-ﬂuoroxylo series, more closely

reﬂecting that of their (3⁰-deoxy)ribose congeners.

None of the synthesized nucleoside analogs was found to exert

speciﬁc antileishmanial activity, which was also observed for the

ribofuranosyl 7-deazapurine analogs [27].

deazaadenosine (tubercidin) analog 71 emerged as a promising

anti-T. brucei agent. The 7-phenyl analogs from both series dis-

played good anti-T. cruzi activity, but were found somewhat less

potent than previously reported 3⁰-deoxy matched analogs. The

presented series of 3⁰-ﬂuoro-modiﬁed nucleosides did not reveal

promising activity against L. infantum. Overall, this study demon-

strates that next to 3⁰-deoxygenation, introduction of a ﬂuorine

atom in the 3⁰-position is tolerated with respect to anti-

kinetoplastid activity, and that a 3⁰-ﬂuorinated ribofuranose pro-

vides an interesting ribose motif for future antiparasitic nucleoside

series.

3. Conclusion

We have described the convergent synthesis of 3⁰-deoxy-3⁰-

ﬂuororibofuranosyl and xylofuranosylnucleosides with

a 7-

deazapurine nucleobase. Common 7-iodo or 7-bromo in-

termediates were used to introduce different modiﬁcations at C7.

All nucleosides were evaluated against the three kinetoplastid

parasites, and signiﬁcant differences in the antiparasitic activity

spectrum were noted amongst the two 3⁰-diastereomeric nucleo-

side series. The 3⁰-ﬂuororibonucleosides 32 (7-Br) and 33 (7-I) were

found to hold promise as anti-T. cruzi agents, and also displayed

good anti-T. brucei activity. In the 3⁰-ﬂuoroxylo series, the 7-

4. Experimental section

4.1. Chemistry

4.1.1. General

All reagents and solvents were obtained from standard com-

mercial sources (analytical grade) and used as received. Moisture-

sensitive reactions were carried out under argon atmosphere.

7

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

Table 2

Evaluation of drug sensitivity of 3⁰-deoxy-3⁰-ﬂuoronucleosides against T. b. brucei and T. b. rhodesiense. Cytotoxicity was assayed against human MRC-5 cells. Values represent

mean SEM which originate from 2 to 3 independent experiments and are expressed in

correct average can be calculated. Values in italics represent the result of a single determination. SI, in vitro selectivity index is the ratio of the EC₅₀for MRC-5 cells and the EC₅₀

for the parasite. Suramin was included as a reference compound (T. b. brucei EC₅₀¼ 0.03 0.01 M, T. b. rhodesiense EC_50¼0.04 0.01 M).

mM. Values in parentheses represent the values of the different determinations, as no

m

Cpd.

R/Structure

T. b. brucei EC₅₀

(m

M)

T. b. rhod. EC₅₀

(

mM)

MRC-5 EC₅₀

(m

M)

SI T. b. brucei

SI T. b. rhod.

a. 3⁰-ﬂuororibo inosine analogs

5

OH

OMe

H

>64.0

> 64.0

-

2

-

7

-

2

-

1

14

47

38

39

40

41

62

36

33.5 0.6

> 64.0

8.64

35.2 1.68

> 64.0

51.6

F

Cl

Br

I

Ph

/

b. 3⁰-ﬂuororibo guanosine analogs

16

17

21

20

19

OH

NH₂

H

Br

42.0 10.0

> 64.0

>64.0

> 64.0

>64.0

> 64.0

-

c. 3⁰-ﬂuororibo adenosine analogs

6

/

H

F

Cl

Br

I

CF₃

Me

0.032 0.0002

22.2 2.2

0.47 0.12

0.13 0.0005

0.031 0.0002

0.032 0.0004

2.02

0.96 0.34

10.5

2.34

0.04

20.5

0.81 0.05

32.0

7.02

0.017 0.0077

1.47 0.91

0.16 0.06

0.031 0.003

0.028 0.004

0.0077 0.0005

2.06

0.21 0.09

0.38

0.12

0.02

21.3

0.34 0.02

32.7

5.18

1.55

25.7

4.92 0.04

>64.0

154

>2

9

108

af

201

3

>67

0

1

4

2

25

>2

2

1

< 1

296

>44

27

448

1152

826

3

>312

3

20

7

2

59

>1

2

46

30

31

32

33

44

49

51

52

55

56

57

58

59

61

60

5.49 0.63

13.8 8.9

32.0 9.0

6.38 2.0

7.01

>64.0

1.32

2.38

0.16

CN

CONH₂

ethynyl

Et

vinyl

Ph

4-Cl-Ph

3,4-diCl-Ph

4-OMe-Ph

40.3

20.2 7.7

> 64.0

16.9

4.14

14.1

2.70

43.6

2

< 1

d. 3⁰-ﬂuoroxylo nucleoside analogs

71

72

73

74

76

77

75

78

H

Br

0.040 0.008

0.032 0.001

16.0 6.8

5.22 0.26

2.34 0.62

4.22 1.88

16.2 5.26

7.23 1.02

0.0063 0.0003

0.0049 0.001

1.57 0.69

2.27 0.35

1.72 0.26

1.43 0.87

14.1 7.27

3.61 3.02

>64.0

0.75 0.25

>64.0

21.7 0.73

7.38 1.1

18.6 0.82

>64.0

>1589

>10 133

23

>4

4

3

4

152

>41

10

4

13

>5

6

4-Cl-Ph

3,4-diCl-Ph

3-F-4-Cl-Ph

4-F-Ph

3-Me-4-Cl-Ph

>4

3

20.9 1.1

Reactions were carried out at ambient temperature, unless other-

wise indicated. Reactions were monitored via analytical TLC or

analytical LC-MS. Analytical TLC was performed on Machery-

Nagel® precoated F254 aluminum plates and were visualized by UV

followed by staining with basic aq. KMnO₄, Cerium-Molybdate, or

sulfuric acid-anisaldehyde spray. Analytical LC-MS was performed

on a Waters AutoPuriﬁcation system (equipped with ACQUITY QDa

(mass; 100e1000 amu)) and 2998 Photodiode Array

Samples were infused in a MeCN/water (1:1) þ 0.1% formic acid

mixture at 100 L/min. NMR spectra were recorded on a Varian

Mercury 300 MHz spectrometer or a Bruker Avance Neo 400 MHz

spectrometer. Chemical shifts ( ) are given in ppm and spectra are

referenced to the residual solvent peak. Coupling constants are

given in Hz. Final compounds were obtained as solids after column

chromatography via lyophilization or concentration in vacuo fol-

lowed by drying under high vacuum. Purity was assessed by means

of LCMS. All obtained ﬁnal compounds had purity >95%, as assayed

by analytical HPLC (UV), unless otherwise indicated.

m

d

(220e400 nm)) using

a

Waters Cortecs® C18 (2.7

mm

100 ꢂ 4.6 mm) column and a gradient system of HCOOH in H₂O

(0.2%, v/v)/MeCN at a ﬂow rate of 1.44 mL/min (95:05 to 00:100 in

6.5 min or 50:50 to 00:100 in 6.5 min). Preparative HPLC was

performed on the same system, using a Phenomenex Luna Omega

4.2. General procedure A: Vorbruggen glycosylation for 7-

deazapurines [52]

Polar column (250 ꢂ 21 mm, 5

mm) and a gradient system of 0.2%

formic acid in water/MeCN at a ﬂow rate of 20 mL/min. Column

chromatography was performed using Machery-Nagel® 60 M silica

The appropriate 7-deazapurine heterocycle (1.0 eq.) was sus-

pended in MeCN (7 mL/mmol). BSA (1.1 eq.) was added, upon which

the solids started to dissolve. After 30 min, when all solids had

dissolved, the glycosyl donor (1.1 eq.) was added, followed by

TMSOTf (1.1 eq.). The reaction mixture was stirred for 30 min at

room temperature, after which it was transferred to a preheated

heating block at 80 ^ꢀC. Heating was continued until TLC analysis

gel (40e63

mm) or made use of a Reveleris X2 (Grace/Büchi)

automated Flash unit employing pre-packed silica columns. Exact

mass measurements were performed on a Waters LCT Premier

XE™ Time of Flight (ToF) mass spectrometer equipped with a

standard electrospray (ESI) and modular Lockspray™ interface.

8

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

indicated completion of the reaction (generally 1h). The reaction

mixture was cooled to room temperature and poured into a sepa-

ration funnel. EtOAc was added, and the organic phase washed with

aq. sat. NaHCO₃and brine. The organic phase was dried over

Na₂SO₄, concentrated in vacuo, and the residue puriﬁed by ﬂash

column chromatography to afford the protected nucleoside.

158 mmol, 79% yield). 1H NMR (300 MHz, CDCl₃) d 1.33 (3 H, s),

1.36e1.41 (3 H, m), 1.43e1.46 (3 H, m), 1.49 (3 H, s), 3.98e4.05 (1 H,

m), 4.09 (2 H, dd, J ¼ 4.5, 1.9 Hz), 4.12e4.21 (1 H, m), 4.27e4.37 (1 H,

m), 4.48e4.56 (1 H, m), 5.91e6.02 (1 H, m) ppm.

Spectral data are in accordance with literature values [65].

1,2-O-isopropylidene-a-D-xylofuranose (7)

To a ﬂask containing 1,2:3,5-di-O-isopropylidene-

a- -xylofur-

D

4.3. General procedure B: Nucleophilic displacement with NaN₃

[47e49]

anose (36.3 g,158 mmol) was added 0.2% hydrochloric acid (250 mL

water, 1.5 mL conc. HCl) The oil rapidly started to dissolve and the

resulting solution was stirred vigorously for 1h30. The mixture was

neutralized with solid NaHCO₃and concentrated under reduced

pressure. CHCl₃(200 mL) was added to the residue, and the inor-

ganic salts were removed by ﬁltration. The solids were washed with

additional CHCl₃and the combined ﬁltrates dried over Na₂SO₄and

concentrated in vacuo to yield 7 (21.6 g, 113 mmol, 72% yield) as a

To a solution of 6-chloro-nucleoside in DMF (10 mL/mmol) was

added NaN₃(2.5 eq.). The resulting suspension was heated to 65 ^ꢀC

overnight and cooled to room temperature. Water (1 mL/mmol)

was added and the mixture poured into a separation funnel. The

mixture was diluted with EtOAc and washed with H₂O (2 x) and

brine. The organic phase is dried over Na₂SO₄and concentrated in

vacuo.

yellow oil. ¹H NMR (300 MHz, CDCl₃)

d 1.33 (3 H, s), 1.50 (3 H, s),

4.00e4.21 (3 H, m), 4.34 (1 H, d, J ¼ 2.6 Hz), 4.54 (1 H, d, J ¼ 3.8 Hz),

6.00 (1 H, d, J ¼ 3.8 Hz) ppm. Spectral data are in accordance with

literature values [65].

4.4. General procedure C: Staudinger reduction and

iminophosphorane hydrolysis [47e49]

1,2-O-isopropylidene-5-O-benzoyl-a-D-xylofuranose (8)

A solution of 7 (21.6 g, 113 mmol) in CH₂Cl₂(500 mL) was cooled

to 0 ^ꢀC. Et₃N (15.8 mL, 113 mmol, 1.0 eq.) was added, followed by

dropwise addition (0.2 mL/min) of BzCl (13.4 mL, 116 mmol, 1.02

eq.). After 4 h, TLC analysis (petroleum ether/EtOAc 50:50) showed

complete conversion of the starting material. The mixture was

transferred to a separation funnel and washed with sat. aq. NaHCO₃

solution (200 mL). the organic phase was dried over Na₂SO₄and

concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (manual, petroleum ether/EtOAc 90:10 and 60:40)

to afford 8 (27.2 g, 92.5 mmol, 82% yield) as a colourless oil. ¹H NMR

The 6-azido-nucleoside was dissolved in THF (5 mL/mmol).

PMe₃(1 M in THF, 2.5 eq.) was added and the mixture stirred for

approximately 1 h. H₂O was added and the mixture stirred further

until TLC analysis indicated full conversion (approx. 5e30 min). The

solution was concentrated in vacuo and co-evaporated with

toluene three times. The residue was either puriﬁed via ﬂash col-

umn chromatography, or used directly in the next reaction (indi-

cated in individual procedures).

4.5. General procedure D: Deprotection e benzoate hydrolysis

(300 MHz, CDCl₃) d 1.33 (3 H, s, CH₃), 1.51 (3 H, s CH₃), 3.11 (br. s, 1H,

OH) 4.20 (1 H, d, J ¼ 2.3 Hz, H-3), 4.36e4.44 (2 H, m, H-4, H-5), 4.60

(1 H, d, J ¼ 3.8 Hz, H-2), 4.80 (1 H, dd, J ¼ 12.6, 9.1 Hz, H-5’), 5.97

(1 H, d, J ¼ 3.5 Hz, H-1), 7.41e7.52 (2 H, m, H_Phe), 7.54e7.64 (1 H, m,

Protected nucleoside was dissolved in MeOH (5 mL/mmol).

NaOMe (5.4 M in MeOH, 0.5 mL/mmol) was added, and the mixture

stirred until TLC analysis indicated full conversion. The mixture was

neutralized to pH 7 via the addition of 4 N HCl, and concentrated in

vacuo. The residue was dissolved in MeOH, adsorbed onto celite

and puriﬁed via ﬂash column chromatography. The residue was

lyophilized to afford the ﬁnal compound.

H

_Phe), 7.95e8.10 (2 H, m, H_Phe) ppm. ¹³C NMR (75 MHz, CDCl₃)

d

26.13 (1C, s), 26.79 (1 C, s), 61.32 (1 C, s), 74.38 (1 C, s), 78.52 (1 C,

s), 85.02 (1 C, s), 104.73 (1 C, s), 111.86 (1 C, s), 128.48 (1 C, s), 129.21

(1 C, s), 129.89 (1 C, s), 133.55 (1 C, s), 167.36 (1 C, s) ppm. HRMS

(ESI): calculated for C₁₅H₁₉O₆([MþH]^þ): 295.1182, found: 295.1190.

1-O-methyl-5-O-benzoyl-a/b-D-xylofuranose (9).

4.6. General procedure E: Suzuki coupling [29]

Conc. H₂SO₄(2 mL) was added to a solution of 8 (19.6 g,

66.5 mmol) in MeOH (200 mL). The reaction mixture was heated to

80 ^ꢀC and stirred for 1 h, TLC analysis (petroleum ether/EtOAc

50:50) indicated full conversion of the reaction. The reaction was

cooled to room temperature, and solid NaHCO₃(20 g) was added.

The suspension was concentrated under reduced pressure, and to

the residue were added EtOAc (300 mL) and a mixture of H₂O/brine

(1:1, 150 mL). The mixture was transferred to a separation funnel,

the phases separated, and the aqueous phase extracted twice more

with EtOAc (200 mL). The combined organic phases were dried

over Na₂SO₄and concentrated in vacuo. The residue was puriﬁed by

ﬂash column chromatography (manual, 40 / 60% EtOAc in pe-

troleum ether) to afford 9 (15.5 g, 57.7 mmol, 87% yield) as a

mixture of anomers, colourless oil. HRMS (ESI): calculated for

7-halonucleoside (1 eq.), boronic acid (1.5 eq.) or tri-

ﬂuoroborate salt (1.5 eq.) Na₂CO₃(when a boronic acid was used, 3

eq.) or Cs₂CO₃(when a triﬂuoroborate salt was used, 3 eq.),

Pd(OAc)₂(0.05 eq.) and TPPTS (0.12 eq.) were added to a 10 mL

round-bottom ﬂask, equipped with a stir bar. Next, the ﬂask was

evacuated and reﬁlled with argon. This procedure was repeated

three times in total. Next, MeCN (2 mL/mmol SM) and H₂O (4 mL/

mmol SM) were added to the solids under argon. After 5 min of

stirring, the mixture was heated to reﬂux. When the starting ma-

terial was fully consumed (usually 1e3 h; as monitored by LC-MS

analysis), the mixture was cooled to ambient temperature, and

neutralized (pH ~ 7) with 4 M aq. HCl. Celite (5 g/mmol) was added

and the mixture was concentrated in vacuo. The residue was pu-

riﬁed by ﬂash column chromatography.

C

₁₃H₁₇O₆([MþH]^þ): 269.1025, found: 269.1022.

1-O-methyl-3-deoxy-3-ﬂuoro-5-O-benzoyl-a/b-D-ribofur-

1,2:3,5-di-O-isopropylidene-

CuSO₄(60.0 g) and H₂SO₄(3.00 mL) were added to a suspension

D-xylose (30.0 g, 200 mmol) in acetone (600 mL). The mixture

a

-

D

-xylofuranose [65]

anose (10)

A solution of 9 (15.5 g, 57.7 mmol) in MeCN (85 mL, 1.5 mL/

mmol) was cooled to 0 ^ꢀC. DAST (13.3 mL, 101 mmol, 1.75 eq.) was

added, and the mixture slowly warmed to room temperature and

stirred overnight. The reaction mixture was poured into a separa-

tion funnel containing H₂O (100 mL) and aq. sat. NaHCO₃solution

(80 mL) added slowly. When gas formation had ceased, the mixture

was extracted with CH₂Cl₂(3 ꢂ 150 mL). The combined organic

phases were dried over Na₂SO₄and concentrated in vacuo. The

of

was stirred for 48 h at room temperature, and ﬁltered. The ﬁltrate

was treated with NH₄OH (28e30% wt., 9.6 mL) and the resulting

precipitate removed by ﬁltration. The ﬁltrate was concentrated in

vacuo and the residue puriﬁed by ﬂash column chromatography

(manual, petroleum ether/EtOAc 80:20 and 70:30) to give 1,2:3,5-

di-O-isopropylidene-a-D-xylofuranose as a yellow oil (36.3 g,

9

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

residue was puriﬁed by ﬂash column chromatography (manual,

petroleum ether/EtOAc 85:15 and 8:2) to afford 10 (9.11 g,

33.4 mmol, 58% yield) as a mixture of anomers, orange oil. HRMS

1-O-acetyl-2,5-di-O-benzoyl-3-deoxy-3-ﬂuoro-

anose (12)

a/b-D-ribofur-

11 (12.5 g, 33.4 mmol) was dissolved in AcOH (100 mL, 3 mL/

mmol) and Ac₂O (12.0 mL, 0.36 mL/mmol) and the resulting solu-

tion cooled to 0 ^ꢀC. Upon solidiﬁcation, H₂SO₄(7.01 mL, 0.21 mL/

mmol) was added dropwise. The reaction mixture liquiﬁed again

and was gradually warmed to room temperature overnight. Solid

NaHCO₃(20 g) was added, followed by very slow addition of H₂O

(100 mL) (careful, gas formation!). When gas formation had ceased,

the mixture was transferred to a separation funnel and extracted

with CHCl₃(3 ꢂ 125 mL). The combined organic fractions were

dried over Na₂SO₄and concentrated in vacuo. The residue was

puriﬁed by ﬂash column chromatography (automated, 0 / 20%

EtOAc in petroleum ether) to afford 12 (10.5 g, 26.2 mmol, 78%

yield) as a mixture of anomers, colourless oil. HRMS (ESI): calcu-

lated for C₂₁H₂₀FO₇([MþNa]^þ): 425.1007, found: 425.1031. NMR

(ESI): calculated for

271.0977. NMR data

C

₁₃H₁₆FO₅([MþH]^þ): 271.0982, found:

a

anomer: ¹H NMR (300 MHz, CDCl₃)

d 2.82

(1 H, d, J ¼ 11.7 Hz, OH), 3.50 (3 H, s, CH₃), 4.12e4.30 (1 H, m, H-2),

4.40e4.66 (3 H, m, H-4, H-5, H-5⁰), 4.91 (1 H, ddd, J ¼ 55.9, 5.6,

1.2 Hz, H-3), 4.98 (1 H, d, J ¼ 5.0 Hz, H-1), 7.42e7.51 (2 H, m, H_Phe),

7.56e7.63 (1 H, m, H_Phe), 7.96e8.04 (2 H, m, H_Phe) ppm. ¹³C NMR

(75 MHz, CDCl₃)

d

55.8 (CH₃), 63.8 (d, J ¼ 10.4 Hz, C-5), 72.3 (d,

J ¼ 17.3 Hz, C-2), 80.5 (d, J ¼ 25.3 Hz, C-4), 90.4 (d, J ¼ 185.4 Hz, C-3),

102.3 (C-1),128.6 (C_Phe),129.4 (C_Phe),129.6 (C_Phe),133.4 (C_Phe),166.0

(C]O) ppm. ¹⁹F NMR (282 MHz, CDCl₃)

d

ꢁ195.4 (1F, ddd, J ¼ 56.5,

26.4, 22.8 Hz) ppm. Observed NOESY couplings: J_OMe-H4.NMR data

anomer: ¹H NMR (300 MHz, CDCl₃)

2.43 (1H, br. s, OH) 3.35 (3 H,

b

d

s, CH₃), 4.27 (1 H, td, J ¼ 4.7, 1.5 Hz, H-2), 4.40e4.64 (3 H, m, H-4, H-

5, H-5’), 4.92 (1 H, t, J ¼ 1.8 Hz, H-1), 5.20 (1 H, dt, J ¼ 53.9, 4.7 Hz, H-

3), 7.37e7.50 (2 H, m, H_Phe), 7.54e7.62 (1 H, m, H_Phe), 7.98e8.16 (2 H,

data

a d 2.17 (3 H, s, CH₃), 4.52

anomer: ¹H NMR (300 MHz, CDCl₃)

(1 H, dd, J ¼ 12.3, 3.8 Hz, H-5), 4.60 (1 H, dd, J ¼ 12.3, 3.8 Hz, H-5⁰),

4.85 (1 H, dtd, J ¼ 25.8, 3.8, 1.8 Hz, H-4), 5.38 (1 H, ddd, J ¼ 56.2, 5.6,

1.5 Hz, H-3), 5.36 (1 H, dt, J ¼ 21.7, 4.9 Hz, H-2), 6.66 (1 H, d,

J ¼ 4.4 Hz, H-1), 7.44e7.53 (4 H, m, H_Phe), 7.56e7.67 (2 H, m, H_Phe),

m, H_Phe) ppm. ¹³C NMR (75 MHz, CDCl₃)

d 55.5 (CH₃), 64.1 (d,

J ¼ 4.6 Hz, C-5), 74.4 (d, J ¼ 16.1 Hz, C-2), 78.4 (d, J ¼ 25.3 Hz, C-4),

92.3 (d, J ¼ 186.6 Hz, C-3), 107.9 (d, J ¼ 4.6 Hz, C-1), 128.4 (C_Phe),

129.6 (C_Phe), 129.7 (C_Phe), 133.2 (C_Phe), 166.2 (C]O) ppm. ¹⁹F NMR

8.03e8.11 (4 H, m, H_Phe) ppm. ¹³C NMR (75 MHz, CDCl₃)

d 21.1 (s,

(282 MHz, CDCl₃)

d

ꢁ213.0 (1F, dddd, J ¼ 55.3,19.2, 4.8, 2.4 Hz) ppm.

CH₃), 63.3 (d, J ¼ 9.2 Hz, C-5), 71.7 (d, J ¼ 15.0 Hz, C-2), 82.4 (d,

J ¼ 25.3 Hz, C-4), 88.4 (d, J ¼ 192.3 Hz, C-3), 94.0 (C-1), 128.6 (C_Phe),

128.6 (C_Phe), 129.1 (C_Phe), 129.6 (C_Phe), 129.9 (C_Phe), 133.5 (C_Phe),

133.7 (C_Phe), 165.1 (C_Phe) 165.4 (C]O, Bz), 165.9 (C]O, Bz), 169.9

Observed NOESY couplings: J_H1-H4, J_OMe-H2, J_OMe-H5

1-O-methyl-2,5-di-O-benzoyl-3-deoxy-3-ﬂuoro-

furanose (11)

A solution of 10 (9.11 g, 33.4 mmol, 1.0 eq.) in CH₂Cl₂(150 mL)

was cooled to 0 ^ꢀC. DMAP (0.410 g, 3.34 mmol, 0.1 eq.) and Et₃N

(8.39 mmol, 60.2 mmol, 1.8 eq.) were added, follow by BzCl

(5.82 mL, 50.2 mmol, 1.5 eq.). After 1h30, TLC analysis (petroleum

ether/EtOAc 80:20) indicated disappearance of the starting mate-

a/b-D-ribo-

(C]O, Ac) ppm. ¹⁹F NMR (282 MHz, CDCl₃)

d

ꢁ195.29 (1F, ddd,

J ¼ 46.9, 25.2, 21.6 Hz) ppm. Observed NOESY couplings: J_H1-H3, J_H2-

_H1NMR data 1.88e2.04 (3 H,

anomer: ¹H NMR (300 MHz, CDCl₃)

b

d

m, CH₃), 4.48 (1 H, dd, J ¼ 12.6, 4.4 Hz, H-5), 4.63e4.80 (2 H, m, H-4,

H-5’), 5.46 (1 H, dt, J ¼ 52.1, 4.4 Hz, H-3), 5.59 (1 H, ddd, J ¼ 8.2, 4.7,

2.1 Hz, H-2), 6.41 (1 H, t, J ¼ 1.9 Hz, H-1), 7.39e7.53 (4 H, m, H_Phe),

7.55e7.70 (2 H, m, H_Phe), 7.99e8.22 (4 H, m, H_Phe) ppm. ¹³C NMR

rial and the presence of two higher running spots (a and b). The

mixture was poured into a separation funnel containing aq. sat.

NaHCO₃solution (80 mL). The phases were separated and the

aqueous phase extracted once more with CH₂Cl₂(150 mL). The

combined organic phases were dried over Na₂SO₄, and concen-

trated in vacuo. The residue was puriﬁed by ﬂash column chro-

matography (manual, 5 / 15% EtOAc in petroleum ether) to afford

11 (12.5 g, 33.4 mmol, quantitative yield) as a mixture of anomers,

colourless oil. Some fractions that contained only one anomer were

collected separately once for analytical purposes. HRMS (ESI):

calculated for C₂₀H₂₀FO₆([M þ NH4]^þ): 392.1504, found: 392.1504.

(75 MHz, CDCl₃)

d

20.8 (CH₃), 63.2 (d, J ¼ 5.8 Hz, C-5), 75.1 (d,

J ¼ 15.0 Hz, C-2), 80.8 (d, J ¼ 24.2 Hz, C-4), 89.3 (d, J ¼ 194.6 Hz, C-3),

98.2 (d, J ¼ 2.3 Hz, C-1), 128.5 (C_Phe), 128.6 (C_Phe), 128.6 (C_Phe), 129.4

(C_Phe), 129.7 (C_Phe), 130.0 (C_Phe), 133.4 (C_Phe), 133.8 (C_Phe), 165.2 (C]

O, Bz), 165.9 (C]O, Bz), 169.1 (C]O, Ac) ppm. ¹⁹F NMR (282 MHz,

CDCl₃)

d

ꢁ209.1 (1F, ddd, J ¼ 51.7, 19.2, 8.4 Hz) ppm. Observed

NOESY couplings: J_acetyl-H-5J_H1dH4

9-(2′,5′-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-

6-chloropurine (13)

b-D-ribofuranosyl)-

NMR data

a

anomer: ¹H NMR (300 MHz, CDCl₃)

d

3.52 (3 H, s, CH₃),

A solution of DBU (0.246 mL, 1.65 mmol, 3.0 eq.) in MeCN

(3.5 mL) was added to a precooled (0 ^ꢀC) mixture of 12 (0.221 g,

0.55 mmol, 1.0 eq.) and 6-chloropurine (0.093 g, 0.60 mmol, 1.1 eq.)

in MeCN (3.5 mL). the solids started to dissolve. TMSOTf (0.398 mL,

2.20 mmol, 4.0 eq.) was added dropwise and the mixture was

heated to 60 ^ꢀC for 4 h. The mixture was cooled to room temper-

ature and poured into a separation funnel containing aq. sat.

NaHCO₃solution (20 mL). The mixture was extracted with EtOAc

(2 ꢂ 25 mL). The combined organic phases were dried over Na₂SO₄

and concentrated in vacuo. The residue was puriﬁed by ﬂash col-

umn chromatography (automated, 0 / 40% EtOAc in petroleum

ether) to afford 13 (210 mg, 0.448 mmol, 88% yield) as a colourless

4.57 (2 H, ddd, J ¼ 24.9, 11.7, 3.5 Hz, H-5, H-5⁰), 4.70 (1 H, dtd,

J ¼ 25.8, 3.8, 3.8, 1.8 Hz, H-4), 5.13 (1 H, ddd, J ¼ 22.8, 5.9, 4.7 Hz, H-

2), 5.29 (1 H, ddd, J ¼ 56.2, 6.2, 2.1 Hz, H-3), 5.33 (1 H, d, J ¼ 4.4 Hz,

H-1), 7.43e7.52 (4 H, m, H_Phe), 7.56e7.65 (2 H, m, H_Phe), 8.04e8.10

(2 H, m, H_Phe), 8.11e8.18 (2 H, m, H_Phe) ppm. ¹³C NMR (75 MHz,

CDCl₃)

d

55.9 (CH₃), 63.6 (d, J ¼ 9.2 Hz, C-5), 72.3 (d, J ¼ 15.0 Hz, C-

4), 80.1 (d, J ¼ 24.2 Hz, C-2), 88.9 (d, J ¼ 192.3 Hz, C-3), 101.7 (C-1),

128.5 (C_Phe), 128.6 (C_Phe), 128.9 (C_Phe), 129.3 (C_Phe), 129.7 (C_Phe),

129.9 (C_Phe), 130.2 (C_Phe), 133.4 (C_Phe), 133.6 (C_Phe), 165.8 (C]O),

166.0 (C]O) ppm. ¹⁹F NMR (282 MHz, CDCl₃)

d

ꢁ194.0 (1F, ddd,

J ¼ 55.3, 25.2, 21.6 Hz) ppm. Observed NOESY couplings: J_H1-H2, J_H1-

NMR data 3.40 (3 H, m,

anomer: ¹H NMR (300 MHz, CDCl₃)

b

d

oil. ¹H NMR (300 MHz, CDCl₃) 4.58e4.67 (1 H, m, H-5⁰), 4.77e4.92

d

H3

CH₃) 4.45e4.74 (3 H, m, H-4, H-5, H-5’), 5.15 (1 H, t, J ¼ 1.6 Hz, H-1),

5.45 (1 H, dt, J ¼ 53.3, 5.0 Hz, H-3), 5.47 (1 H, td, J ¼ 4.8, 1.5 Hz, H-2),

7.43e7.52 (4 H, m, H_Phe), 7.56e7.65 (2 H, m, H_Phe), 8.05e8.14 (4 H,

(2 H, m, H-4, H-5⁰⁰), 5.78 (1 H, ddd, J ¼ 53.0, 4.7, 2.1 Hz, H-3⁰), 6.37

(1 H, dd, J ¼ 17.7, 4.5 Hz, H-2⁰), 6.46 (1 H, dd, J ¼ 7.0, 0.9 Hz, H-1⁰),

7.41e7.54 (4 H, m), 7.55e7.68 (2 H, m), 7.98e8.06 (2 H, m),

8.06e8.13 (2 H, m), 8.25 (1 H, s), 8.50 (1 H, s) ppm. ¹³C NMR

m, H_Phe) ppm. ¹³C NMR (75 MHz, CDCl₃)

d 55.7 (s), 63.9 (d,

J ¼ 4.6 Hz, C-5), 75.3 (d, J ¼ 13.8 Hz, C-2), 79.2 (d, J ¼ 25.3 Hz, C-4),

90.2 (d, J ¼ 194.6 Hz, C-3), 106.1 (d, J ¼ 3.5 Hz, C-1), 128.5 (C_Phe),

129.6 (C_Phe), 129.7 (C_Phe), 129.9 (C_Phe), 133.3 (C_Phe), 133.5 (C_Phe),

165.4 (C]O), 166.1 (C]O) ppm. ¹⁹F NMR (282 MHz, CDCl₃)

(75 MHz, CDCl₃)

d

62.9 (d, J ¼ 9.2 Hz, C-5⁰), 73.3 (d, J ¼ 15.0 Hz, C-2⁰),

81.5 (d, J ¼ 24.2 Hz, C-4⁰), 86.3 (C-1⁰), 89.3 (d, J ¼ 191.2 Hz, C-3’),

128.1 (C_Phe), 128.6 (C_Phe), 128.7 (C_Phe), 129.1 (C_Phe), 129.7 (C_Phe),

130.0 (C_Phe), 132.4 (C_Phe), 133.7 (C_Phe), 134.1 (C_Phe), 144.0 (C-5), 144.1

(C-8), 151.3 (C-6), 151.7 (C-4) 152.2 (C-2), 165.2 (C]O), 165.9 (C]O)

d

ꢁ211.39 (1F, ddd, J ¼ 52.9, 20.4, 4.8 Hz) ppm. Observed NOESY

couplings: J_acetyl-H2, J_acetyl-H1, J_H-1H-4

ppm. ¹⁹F NMR (282 MHz, CDCl₃)

d

ꢁ199.33 (1F, ddd, J ¼ 52.9, 24.0,

10

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

18.0 Hz) ppm. HRMS (ESI): calculated for C₂₄H₁₉ClFN₄O₇([MþH]^þ):

temperature and poured into a separation funnel containing aq. sat.

NaHCO₃solution (20 mL). The mixture was extracted with EtOAc

(2 ꢂ 25 mL). The combined organic phases were dried over Na₂SO₄

and concentrated in vacuo. The residue was puriﬁed by ﬂash col-

umn chromatography (manual, petroleum ether/EtOAc 70:30 and

60:40) to afford 15 (203 mg, 0.397 mmol, 75% yield) as a white

497.1028, found: 497.1024.

9-(3′-deoxy-3′-ﬂuoro-b-D-ribofuranosyl)adenine (6)

13 (0.144 g, 0.289 mmol) was subjected to general procedure B.

The crude residue was subjected to general procedure C (puriﬁca-

tion by column chromatography: automated, 0 / 10% MeOH in

CH₂Cl₂) to afford a residue that was immediately subjected to

general procedure D (reaction time: 1 h; puriﬁcation via ﬂash col-

umn chromatography: automated, 1 / 15% MeOH in CH₂Cl₂) to

afford 6 (13 mg, 0.048 mmol, 17% over 3 steps) as a white solid. ¹H

sticky solid. ¹H NMR (300 MHz, CDCl₃)

d 4.57e4.69 (1 H, m),

4.72e4.93 (2 H, m), 5.70 (1 H, ddd, J ¼ 53.0, 3.8, 1.8 Hz), 6.14e6.34

(2 H, m), 7.40e7.65 (6 H, m), 7.88 (1 H, s), 7.99e8.16 (4 H, m) ppm.

¹³C NMR (75 MHz, CDCl₃)

d

63.1 (d, J ¼ 9.2 Hz), 73.0 (d, J ¼ 15.0 Hz),

NMR (400 MHz, DMSO‑d₆)

d

3.59e3.70 (m, 2H, H-5⁰, H-5⁰⁰), 4.29 (dt,

81.1 (d, J ¼ 24.2 Hz), 86.0, 89.5 (d, J ¼ 191.2 Hz), 125.8, 128.6, 129.0,

J ¼ 27.8, 3.5 Hz, 1 H, H-4⁰), 4.86e4.99 (m, 1 H, H-2⁰), 5.08 (dd,

J ¼ 54.5, 4.3 Hz, 1 H, H-3⁰), 5.76 (dd, J ¼ 6.9, 5.5 Hz, 1 H, OH), 5.93 (d,

J ¼ 8.1 Hz, 1 H, H-1⁰), 5.96 (d, J ¼ 6.0 Hz, 1 H, OH), 7.35e7.44 (m, 2 H,

NH₂), 8.13 (s, 1H, H-2), 8.36 (s, 1 H, OH, H-8) ppm. 13C NMR

129.7, 130.0, 133.7, 134.0, 141.0, 151.8, 153.3, 159.0, 166.1 ppm. ¹⁹F

NMR (282 MHz, CDCl₃)

d

ꢁ199.90 to ꢁ199.50 (1F, m) ppm. HRMS

(ESI): calculated for C₂₄H₂₀ClFN₅O₅([MþH]^þ): 512.1137, found:

512.1124.

(101 MHz, DMSO‑d₆)

d

61.1 (d, J ¼ 11.6 Hz, C-5⁰), 72.1 (d, J ¼ 12.4 Hz,

9-(3′-deoxy-3′-ﬂuoro-b-D-ribofuranosyl)guanine (16).

C-2⁰), 84.1 (d, J ¼ 23.3 Hz, C-4⁰), 87.1 (C-1⁰), 93.3 (d, J ¼ 182.4 Hz, C-

A solution of 15 (209 mg, 0.408 mmol) in 1,4-dioxane (0.4 mL)

and 2 M NaOH (2 mL) was reﬂuxed for 2 h, until TLC analysis (20%

MeOH in CH₂Cl₂) indicated completion of the reaction. The mixture

was cooled to room temperature, neutralized to pH 7 via the

addition of 4 N HCl, and concentrated in vacuo. The residue was

suspended in MeOH, adsorbed onto celite, and puriﬁed via ﬂash

column chromatography (automated, 5 / 25% MeOH in CH₂Cl₂to

afford 16 (39 mg, 0.137 mmol, 34% yield) as a white solid. ¹H NMR

3’), 119.5 (C-5), 140.3 (C-8), 149.2 (C-4), 152.6 (C-2)), 156.3 (C-6)

ppm. ¹⁹F NMR (282 MHz, DMSO‑d₆)

d

ꢁ197.54 to ꢁ197.03 (1F, m)

ppm. HRMS (ESI): calculated for C₁₀H₁₃FN₅O₃([MþH]^þ): 270.1002,

found: 270.1006.

9-(3′-deoxy-3′-ﬂuoro-b-D-ribofuranosyl)-6-methoxypurine

(14)

Compound 13 (186 mg, 0.374 mmol) was subjected to General

procedure D (reaction time: overnight; puriﬁcation by ﬂash column

chromatography: automated, 0 / 8% MeOH in CH₂Cl₂) to afford 14

(87 mg, 0.310 mmol, 83%) as a white solid. ¹H NMR (300 MHz,

(300 MHz, DMSO‑d₆)

d

3.52e3.65 (m, 2 H, H-5⁰, H-5⁰⁰), 4.18 (dt,

J ¼ 27.2, 3.8 Hz, 1 H, H-4⁰), 4.63e4.84 (m, 1 H, H-2⁰), 5.03 (dd,

J ¼ 54.5, 4.1 Hz, 1 H, H-3⁰), 5.24 (t, J ¼ 5.3 Hz, 1 H, OH), 5.73 (d,

J ¼ 8.2 Hz, 1 H, H-1’), 5.89 (d, J ¼ 6.2 Hz, 1 H, OH), 6.51 (br. s., 2 H,

NH₂), 7.94 (s, 1H, H-8), 10.70 (br. s., 1 H, NH) ppm. HRMS (ESI):

calculated for C₁₀H₁₃FN₅O₄([MþH]^þ): 286.0952, found: 286.0959.

Spectral data are in accordance with literature values [46].

DMSO‑d₆)

d

3.57e3.71 (2 H, m, H-5⁰, H-5⁰⁰), 4.10 (3 H, s, CH₃), 4.28

(1 H, dt, J ¼ 26.9, 3.8 Hz, H-4⁰), 4.84e4.99 (1 H, m, H-2⁰), 5.10 (1 H,

dd, J ¼ 54.2, 4.4 Hz, H-3⁰), 5.35 (1 H, t, J ¼ 5.6 Hz, OH), 5.95 (1 H, br.

s., OH), 6.03 (1 H, d, J ¼ 7.9 Hz, H-1⁰), 8.56 (1 H, s, H-8), 8.62 (1 H, s,

H-2) ppm. ¹³C NMR (75 MHz, DMSO‑d₆)

d

54.5 (s, CH₃), 61.3 (d,

2,6-diamino-9-(2′,5′-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-b-D-

ribofuranosyl)purine (17)

J ¼ 11.5 Hz, C-5⁰), 72.8 (d, J ¼ 16.1 Hz, C-4⁰), 84.4 (d, J ¼ 20.7 Hz, C-2⁰),

87.0 (C-1⁰), 93.3 (d, J ¼ 183.1 Hz, C-3’), 121.7 (s),143.0 (C-8),152.3 (C-

2), 152.4 (C-4), 160.9 (C-6) ppm. 1 quaternary carbon missing. ¹⁹F

15 (0.230 g, 0.449 mmol) was subjected to general procedure B.

The crude residue was subjected to general procedure C (puriﬁca-

tion by column chromatography: automated, 0 / 10% MeOH in

CH₂Cl₂) to afford a residue that was immediately subjected to

general procedure D (reaction time: 2 h; puriﬁcation via ﬂash col-

umn chromatography: automated, 5 / 20% MeOH in CH₂Cl₂) to

afford 17 (77 mg, 0.271 mmol, 66% over 3 steps) as a white solid. ¹H

NMR (282 MHz, DMSO‑d₆)

d

ꢁ197.8e197.4 (1F, m) ppm.

HRMS (ESI): calculated for C₁₁H₁₄FN₄O₄([MþH]^þ): 285.0999,

found: 285.1003.

9-(3′-deoxy-3′-ﬂuoro-b-D-ribofuranosyl)hypoxanthine (5).

A solution of 14 (57 mg, 0.201 mmol) in 2 M NaOH (3 mL) was

reﬂuxed for 90 min, after which TLC analysis (20% MeOH in CH₂Cl₂)

showed full conversion. The mixture was cooled to room temper-

ature, neutralized to pH 7 via the addition of 4 N HCl, and

concentrated in vacuo. The residue was suspended in MeOH,

adsorbed onto celite, and puriﬁed via ﬂash column chromatog-

raphy (automated, 4 / 20% MeOH in CH₂Cl₂) to afford 5 (32 mg,

0.118 mmol, 59% yield) as a white solid. ¹H NMR (400 MHz,

NMR (300 MHz, DMSO‑d₆)

d 3.52e3.69 (2 H, m), 4.21 (1 H, dt,

J ¼ 27.5, 3.5 Hz), 4.74e4.91 (1 H, m), 5.02 (1 H, dd, J ¼ 54.8, 4.1 Hz),

5.67e5.79 (3 H, m), 5.84 (1 H, d, J ¼ 6.4 Hz), 6.82 (2 H, br. s), 7.91

(1 H, s) ppm. ¹³C NMR (75 MHz, DMSO‑d₆)

d

61.6 (d, J ¼ 11.5 Hz), 72.1

(d, J ¼ 16.1 Hz), 84.0 (d, J ¼ 21.9 Hz), 86.5, 93.7 (d, J ¼ 182.0 Hz),

114.1, 136.8, 151.9, 156.8, 160.5 ppm. HRMS (ESI): calculated for

C

₁₀H₁₄FN₆O₃([MþH]^þ): 285.1111, found: 285.1124.

DMSO‑d₆)

d

3.59e3.69 (m, 2 H, H-5⁰, H-5⁰⁰), 4.26 (dt, J ¼ 27.3,

5-bromo-4-chloro-2-pivalamido-N7-(2,5-di-O-benzoyl-3′-

4.10 Hz, 1 H, H-4⁰), 4.72e4.89 (m, 1 H, H-2⁰), 5.07 (dd, J ¼ 54.4,

4.1 Hz, 1 H, H-3⁰), 5.26e5.38 (m, 1 H, OH), 5.91 (d, J ¼ 8.1 Hz, 1 H, H-

1⁰), 6.00 (d, J ¼ 6.1 Hz, 1 H, OH), 8.09 (s, 1 H, H-2), 8.35 (s, 1 H, H-8),

deoxy-3′-ﬂuoro-

b-D

-ribofuranosyl)pyrrolo

[2,3-d]pyrimidine

(18)

2-pivalamido-5-bromo-4-chloro

[2,3-d]pyrimidine

[50]

12.43 (br. s, 1 H, NH) ppm. ¹³C NMR (101 MHz, DMSO‑d₆)

d

60.8 (d,

(0.468 g, 1.41 mmol, 1.0 eq.) was suspended in MeCN (8 mL). BSA

(0.267 mL, 1.69 mmol, 1.2 eq.) was added, and after 10 min, TMSOTf

(0.367 mL, 1.97 mmol, 1.4 eq.) was added. 12 (1.018 g, 2.53 mmol, 1.8

eq.) in MeCN (5 mL) was introduced in 3 portions, once every 8 h,

and the mixture was heated to 50 ^ꢀC after addition of the ﬁrst

portion. After 24 h, celite (5 g), was added, the mixture concen-

trated in vacuo and the residue puriﬁed by ﬂash column chroma-

tography (automated,10 / 70% EtOAc in petroleum ether) to afford

a mixture of product and starting materials, that was used as such

in the next reaction.

J ¼ 11.6 Hz, C-5⁰), 72.8 (d, J ¼ 16.0 Hz, C-2⁰), 83.9 (d, J ¼ 21.1 Hz, C-4⁰),

86.2 (C-1⁰) 92.9 (d, J ¼ 182.0 Hz, C-3’), 124.6 (C-5), 138.9 (C-8), 146.2

(C-4), 148.5 (C-2)156.6 (C-6) ppm. ¹⁹F NMR (377 MHz, DMSO‑d₆)

d

ꢁ197.6 to ꢁ197.1 (m) ppm. HRMS (ESI): calculated for

C

₁₀H₁₂FN₄O₄([MþH]^þ): 271.0843, found: 271.0844.

2-amino-9-(2′,5′-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-b-D-ribo-

furanosyl)-6-chloropurine (15)

A solution of DBU (0.237 mL, 1.59 mmol, 3.0 eq.) in MeCN (3 mL)

was added to a precooled (0 ^ꢀC) mixture of 12 (0.213 g, 0.53 mmol,

1.0 eq.) and 2-amino-6-chloropurine (0.099 g, 0.58 mmol, 1.1 eq.) in

MeCN (3 mL). the solids started to dissolve. TMSOTf (0.384 mL,

2.12 mmol, 4.0 eq.) was added dropwise and the mixture was

heated to 60 ^ꢀC for 6 h. The mixture was cooled to room

2-amino-5-bromo-4-methoxy-N7-(3′-deoxy-3′-ﬂuoro-

ribofuranosyl)pyrrolo [2,3-d]pyrimidine (19)

b-D-

18 (used directly from the previous reaction) was dissolved in

0.5 M NaOMe in MeOH (10 mL) and reﬂuxed for 3 h, until TLC

11

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

analysis (10% MeOH in CH₂Cl₂) showed full conversion of the

starting material. The reaction was cooled to room temperature,

neutralized to pH 7 via the addition of 4 N HCl and concentrated in

vacuo. The residue was dissolved in MeOH, adsorbed onto celite

and puriﬁed via ﬂash column chromatography (automated,

0 / 10% MeOH in CH₂Cl₂) to afford 19 (192 mg, 0.51 mmol, 36%

yield over 2 steps) as a light brown solid. HRMS (ESI): calculated for

151.0 (C-4), 151.9 (C-2), 165.3 (C]O), 166.0 (C]O) ppm. C-4a not

found. ¹⁹F NMR (282 MHz, CDCl₃)

d

ꢁ197.77 (1F, ddd, J ¼ 54.1, 26.4,

19.2 Hz, F-3’), ꢁ165.74 (1F, s, F-7) ppm. HRMS (ESI): calculated for

C

₂₅H₁₉ClF₂N₃O₅([MþH]^þ): 514.0981, found: 514.0954.

4,5-dichloro-N7-(2,5-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-

b-D-

ribofuranosyl)pyrrolo [2,3-d]pyrimidine (23)

12 (0.500 g, 1.24 mmol) was subjected to general procedure A,

using 4,5-dichloro-pyrrolo [2,3-d]pyrimidine [54] as the heterocyle

(reaction time: 2h; puriﬁcation: ﬂash column chromatography,

automated, 5 / 25% EtOAc in petroleum ether) to afford 22

(307 mg, 0.597 mmol, 42% yield) as a colourless oil. ¹H NMR

C

₁₂H₁₅BrFN₄O₄([MþH]^þ): 377.0261, found: 377.0245.

2-amino-4-methoxy-N7-(3′-deoxy-3′-ﬂuoro-b-D-ribofur-

anosyl)pyrrolo [2,3-d]pyrimidine (20)

19 (0.115 g, 0.303 mmol) was dissolved in MeOH (4 mL) and 1 M

NaOAc (1 mL). Pd(OH)₂/C (cat.) was added and the mixture was

stirred under hydrogen atmosphere for 72h. The mixture was

ﬁltered over celite, and the ﬁltrate concentrated in vacuo. The

residue was dissolved in MeOH, adsorbed onto celite and puriﬁed

via ﬂash column chromatography (automated, 0 / 10% MeOH in

CH₂Cl₂) to afford 20 (37 mg, 0.124 mmol, 41% yield). ¹H NMR

(300 MHz, CDCl₃)

d

4.56e4.89 (3 H, m), 5.66 (1 H, ddd, J ¼ 53.3, 5.3,

1.8 Hz), 5.97 (1 H, ddd, J ¼ 18.7, 7.2, 4.8 Hz), 6.68 (1 H, d, J ¼ 7.0 Hz),

7.33 (1 H, s), 7.39e7.71 (6 H, m), 7.93e8.15 (4 H, m), 8.55 (1 H, s)

ppm. ¹³C NMR (75 MHz, CDCl₃)

d

63.2 (d, J ¼ 9.2 Hz), 74.0 (d,

J ¼ 15.0 Hz), 81.1 (d, J ¼ 27.6 Hz), 85.5, 89.4 (d, J ¼ 190.3 Hz), 114.9,

123.8,128.6,128.8,129.6,130.0,133.7,134.0,150.5,151.5152.3,165.3,

166.0 ppm. ¹⁹F NMR (377 MHz, CHLOROFORM-d) d ꢁ198.00 (ddd,

J ¼ 52.7, 26.4, 19.4 Hz, 1 F) ppm. HRMS (ESI): calculated for

(300 MHz, CD₃OD) d

3.70e3.91 (2 H, m, H-5, H-5⁰), 4.00 (s, 3H, CH₃),

4.34 (1 H, dt, J ¼ 28.1, 2.6 Hz, H-4⁰), 4.82e4.96 (partially under HOD

peak, 1 H, m, H-2⁰), 5.06 (1 H, dd, J ¼ 54.2, 5.0 Hz, H-3⁰), 5.88 (1 H, d,

J ¼ 8.2 Hz, H-1⁰), 6.34 (1 H, d, J ¼ 3.8 Hz, H-5), 6.98 (1 H, d, J ¼ 3.8 Hz,

C

₂₅H₁₉Cl₂FN₃O₅([MþH]^þ): 530.0686, found: 530.0679.

5-bromo-4-chloro-N7-(2,5-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-

H-6) ppm. ¹³C NMR (75 MHz, CD₃OD)

d

52.4 (CH₃), 61.8 (d,

b-

D-ribofuranosyl)pyrrolo [2,3-d]pyrimidine (24)

J ¼ 11.5 Hz, C-5⁰), 72.4 (d, J ¼ 16.1 Hz, C-2⁰), 83.6 (d, J ¼ 21.9 Hz, C-4⁰),

89.1 (C-1⁰), 93.2 (d, J ¼ 180.8 Hz, C-3’), 98.9 (C-5), 99.6 (C-4a), 121.6

(C-6), 143.2 (C-7a), 153.4 (C-4) ppm. 1 quaternary carbon missing

12 (0.651 g, 1.62 mmol) was subjected to general procedure A,

using 5-bromo-4-chloropyrrolo [2,3-d]pyrimidine as the hetero-

cyle [54] (reaction time: 1h; puriﬁcation: ﬂash column chroma-

tography, automated, 5 / 30% EtOAc in petroleum ether) to afford

24 (0.333 g, 0.58 mmol, 36%) as a white foam. ¹H NMR (300 MHz,

(C-2). ¹⁹F NMR (282 MHz, CD₃OD)

d

ꢁ199.61 to ꢁ199.10 (1F, m)

ppm. HRMS (ESI): calculated for C₁₂H₁₆FN₄O₄([MþH]^þ): 299.1156,

found: 299.1149.

CDCl₃)

d

4.55e4.86 (3 H, m), 5.65 (1 H, ddd, J ¼ 53.3, 4.7, 1.5 Hz),

2-amino-4-oxo-N7-(3′-deoxy-3′-ﬂuoro-

b-D

-ribofuranosyl)

5.96 (1 H, ddd, J ¼ 18.7, 7.2, 4.8 Hz), 6.67 (1 H, d, J ¼ 7.0 Hz), 7.39 (1 H,

pyrrolo [2,3-d]pyrimidine (21)

s), 7.40e7.67 (6 H, m), 7.97e8.16 (4 H, m), 8.53 (1 H, s) ppm. ¹³C

A solution of 20 (33 mg, 0.111 mmol) in 1,4-dioxane (0.4 mL) and

2 M NaOH (2 mL) was reﬂuxed for 2 h, when TLC analysis (20%

MeOH in CH₂Cl₂) indicated completion of the reaction. The mixture

was cooled to room temperature, neutralized to pH 7 via the

addition of 4 N HCl and diluted with DMSO (0.5 mL). The resulting

solution was puriﬁed via reversed phase ﬂash column chroma-

tography (automated, direct injection, 2 / 30% MeOH in H₂O) to

afford 21 (14 mg, 0.049 mmol, 44% yield) as a brown solid. ¹H NMR

NMR (75 MHz, CDCl₃)

d

63.2 (d, J ¼ 9.2 Hz), 74.0 (d, J ¼ 16.1 Hz), 81.1

(d, J ¼ 24.2 Hz), 85.6 (s), 89.3 (dd, J ¼ 188.9, 24.2 Hz), 90.3, 116.0,

128.6, 128.8, 129.7, 130.0, 133.7, 133.9, 150.9, 151.6, 165.3, 166.0 ppm.

¹⁹F NMR (377 MHz, CHLOROFORM-d)

d

ꢁ198.01 (ddd, J ¼ 54.1, 26.4,

19.4 Hz, 1 F) ppm. HRMS (ESI): calculated for C₂₅H₁₉BrClFN₃O₅

([MþH]^þ): 574.0181, found: 574.0220.

4-chloro-5-iodo-N7-(2,5-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-

-ribofuranosyl)pyrrolo [2,3-d]pyrimidine (25)

b-

D

(300 MHz, DMSO‑d₆)

d

3.43e3.65 (2 H, m, H-5⁰, H-5⁰⁰), 4.09 (1 H, dt,

12 (1.29 g, 3.18 mmol) was subjected to general procedure A,

J ¼ 27.8, 4.4 Hz, H-‘), 4.53 (1 H, ddd, J ¼ 25.8, 8.5, 4.4 Hz, H-2⁰), 4.94

(1 H, dd, J ¼ 54.8, 4.4 Hz, H-3⁰), 5.26 (1 H, br. s, OH), 5.69 (1 H, br. s,

OH), 5.88 (1 H, d, J ¼ 8.2 Hz, H-1⁰), 6.26 (1 H, d, J ¼ 3.2 Hz, H-5), 6.36

(2 H, br. s., NH₂), 6.92 (1 H, d, J ¼ 3.5 Hz, H-6), 10.71 (1 H, br. s, NH)

using 4-chloro-5-iodopyrrolo [2,3-d]pyrimidine as the heterocyle

[54] (reaction time: 1h30; puriﬁcation: ﬂash column chromatog-

raphy, automated, 10 / 30% EtOAc in petroleum ether) to afford 24

(776 mg, 1.25 mmol, 39%) as a yellow foam. ¹H NMR (300 MHz,

ppm. ¹³C NMR (75 MHz, DMSO‑d₆)

d

61.5 (d, J ¼ 12.7 Hz, H-5⁰), 72.5

CDCl₃)

d

4.56e4.86 (3 H, m), 5.66 (1 H, ddd, J ¼ 53.0, 4.7, 1.5 Hz),

(d, J ¼ 16.1 Hz, C-2⁰), 83.0 (d, J ¼ 20.7 Hz, C-4⁰), 85.2 (C-1⁰), 93.5 (d,

J ¼ 180.8 Hz, C-3’), 100.7 (C-4a),102.9 (C-5), 117.4 (C-6), 152.1 (C-7a),

153.7 (C-2), 159.7 (C-4) ppm. ¹⁹F NMR (282 MHz, DMSO‑d₆)

5.96 (1 H, ddd, J ¼ 18.7, 7.3, 4.7 Hz), 6.67 (1 H, d, J ¼ 7.0 Hz),

7.37e7.68 (7 H, m), 7.97e8.15 (4 H, m), 8.52 (1 H, s) ppm. ¹³C NMR

(75 MHz, CDCl₃)

d

53.8 (s), 63.2 (d, J ¼ 10.4 Hz), 74.1 (d, J ¼ 16.1 Hz),

d

ꢁ196.68 to ꢁ196.00 (1 F, m) ppm. HRMS (ESI): calculated for

81.1 (d, J ¼ 25.3 Hz), 85.7 (s), 89.6 (d, J ¼ 190.0 Hz), 117.8 (s), 128.2

(s), 128.6 (s), 128.9 (s), 129.7 (s), 130.0 (s), 132.0 (s), 133.7 (s), 133.9

(s), 151.2 (s), 153.1 (s), 165.3 (s), 166.0 (s) ppm. HRMS (ESI): calcu-

lated for C₂₅H₁₉ClFIN₃O₅([MþH]^þ): 622.0042, found: 622.0054.

4-amino-5-ﬂuoro-N7-(2,5-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-

C

₁₁H₁₄FN₄O₄([MþH]^þ): 285.0999, found: 285.0981.

4-chloro-5-ﬂuoro-N7-(2,5-di-O-benzoyl-3-deoxy-3-ﬂuoro-b-

D

-ribofuranosyl)pyrrolo [2,3-d]pyrimidine (22)

12 (0.577 g, 1.43 mmol) was subjected to general procedure A,

using 4-chloro-5-ﬂuoro-pyrrolo [2,3-d]pyrimidine [53] as the het-

erocyle (reaction time: 1h30; puriﬁcation: ﬂash column chroma-

tography, automated, 5 / 25% EtOAc in petroleum ether) to afford

22 (307 mg, 0.597 mmol, 42% yield) as a colourless oil. ¹H NMR

b

-

D

-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (26).

22 (0.100 g, 0.19 mmol) was subjected to general procedure B

and general procedure C (puriﬁcation by column chromatography:

automated, 20 / 70% EtOAc in petroleum ether) to afford 26

(37 mg, 0.075 mmol, 39% yield over 2 steps) as a light yellow oil.

HRMS (ESI): calculated for C₂₅H₂₁F₂N₄O₅([MþH]^þ): 495.1480,

found: 495.1573.

(300 MHz, CDCl₃)

d

4.61 (1 H, dd, J ¼ 12.0, 3.2 Hz, H-5⁰), 4.69e4.90

(2 H, m, H-4, H-5⁰), 5.65 (1 H, ddd, J ¼ 53.3, 4.7, 1.5 Hz, H-3⁰), 5.96

(1 H, ddd, J ¼ 19.0, 7.3, 4.7 Hz, H-2⁰), 6.71 (1 H, d, J ¼ 7.3 Hz, H-1⁰),

7.12 (1 H, d, J ¼ 2.6 Hz, C-6), 7.38e7.70 (6 H, m, H_Phe), 7.96e8.22

(4 H, m, H_Phe), 8.55 (1 H, s, C-2) ppm. ¹³C NMR (75 MHz, CDCl₃)

4-amino-5-chloro-N7-(2,5-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-

b-

D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (27).

d

63.3 (d, J ¼ 9.2 Hz, C-5⁰), 73.8 (d, J ¼ 16.1 Hz, C-2⁰), 80.9 (d,

23 (0.112 g, 0.21 mmol) was subjected to general procedure B

J ¼ 25.3 Hz, C-4⁰), 85.2 (C-1⁰), 89.4 (d, J ¼ 191.2 Hz, C-3⁰), 109.2 (d,

J ¼ 26.5 Hz, C-6), 128.6 (C_Phe), 128.8 (C_Phe), 129.6 (C_Phe), 130.0 (C_Phe),

133.7 (C_Phe), 133.9 (C_Phe), 142.0 (d, J ¼ 255.7 Hz, C-5), 147.6 (C-7a),

and general procedure C (puriﬁcation by column chromatography:

automated, 20 / 70% EtOAc in petroleum ether) to afford 27

(75 mg, 0.147 mmol, 70% yield over 2 steps) as a light yellow oil.

12

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

HRMS (ESI): calculated for C₂₅H₂₁ClFN₄O₅([MþH]^þ): 511.1185,

found: 511.1185.

ppm. ¹⁹F NMR (377 MHz, DMSO‑d₆)

ppm. HRMS (ESI): calculated for

347.0155, found: 347.0163.

d

ꢁ196.59 to ꢁ196.16 (m, 1 F)

C

₁₁H₁₃BrFN₄O₃([MþH]^þ):

4-amino-5-bromo-N7-(2,5-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-

b

-D

-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (28).

4-amino-5-iodo-N7-(3′-deoxy-3′-ﬂuoro-b-D-ribofuranosyl)

24 (0.146 g, 0.254 mmol) was subjected to general procedure B

pyrrolo [2,3-d]pyrimidine (33)

and general procedure C (puriﬁcation by column chromatography:

automated, 10 / 60% EtOAc in petroleum ether) to afford 28

(50 mg, 0.090 mmol, 35% yield over 2 steps) as a yellow oil. HRMS

(ESI): calculated for C₂₅H₂₁BrFN₄O₅([MþH]^þ): 555.0679, found:

555.0674.

JB521 (81 mg, 0.134 mmol) was subjected to general procedure

D (reaction time: 1h30; puriﬁcation: ﬂash column chromatography,

automated, 0 / 12% MeOH in CH₂Cl₂) to afford 33 (40 mg,

0.101 mmol, 76% yield) as an off-white solid. ¹H NMR (300 MHz,

DMSO‑d₆)

d

3.58 (2 H, dd, J ¼ 5.3, 4.1 Hz, H-5, H-5⁰), 4.18 (1 H, dt,

4-amino-5-iodo-N7-(2,5-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-

b-

J ¼ 27.8, 3.8 Hz, H-4⁰), 4.55e4.75 (1 H, m, H-2⁰), 4.99 (1 H, dd,

J ¼ 54.8, 4.1 Hz, H-3⁰), 5.37 (1 H, t, J ¼ 5.6 Hz, OH), 5.78 (1 H, d,

J ¼ 6.7 Hz, OH), 6.05 (1 H, d, J ¼ 8.2 Hz, H-1⁰), 6.73 (2 H, br. s, NH₂),

7.67 (1 H, s, H-6), 8.09 (1 H, s, H-2) ppm. ¹³C NMR (75 MHz,

D

-ribofuranosyl)pyrrolo [2,3-d]pyrimidine (29)

25 (0.126 g, 0.203 mmol) was subjected to general procedure B

and general procedure C (puriﬁcation by column chromatography:

automated, 10 / 60% EtOAc in petroleum ether) to afford 29

(81 mg, 0.134 mmol, 66% yield over 2 steps) as a colourless oil.

HRMS (ESI): calculated for C₂₅H₂₁FIN₄O₅([MþH]^þ): 603.0541,

found: 603.0544.

DMSO‑d₆)

d

61.4 (d, J ¼ 11.5 Hz, C-5⁰), 72.9 (d, J ¼ 16.1 Hz, C-2⁰), 82.1

(C-5), 83.6 (d, J ¼ 21.9 Hz, C-4⁰), 86.0 (C-1⁰), 93.5 (d, J ¼ 182.0 Hz, C-

3’), 103.8 (C-4a), 127.5 (C-6), 150.9 (C-7a), 152.5 (C-2), 157.7 (C-4)

ppm. ¹⁹F NMR (377 MHz, DMSO‑d₆) d ꢁ196.57 to ꢁ196.13 (m, 1 F)

ppm. HRMS (ESI): calculated for C₁₁H₁₃FIN₄O₃([MþH]^þ): 395.0016,

found: 395.0025.

4-amino-5-ﬂuoro-N7-(3′-deoxy-3′-ﬂuoro-

pyrrolo [2,3-d]pyrimidine (30)

b-D-ribofuranosyl)

26 (37 mg, 0.075 mmol) was subjected to general procedure D

(reaction time: 3h; puriﬁcation: ﬂash column chromatography,

automated, 2 / 12% MeOH in CH₂Cl₂) to afford 30 (13 mg,

0.045 mmol, 61% yield) as a white solid. ¹H NMR (300 MHz,

5-ﬂuoro-4-methoxy-N7-(3′-deoxy-3′-ﬂuoro-

anosyl)pyrrolo [2,3-d]pyrimidine (34)

b-D-ribofur-

22 (182 mg, 0.354 mmol) was subjected to general procedure D

(reaction time: 2h; puriﬁcation: ﬂash column chromatography,

automated, 0 / 8% MeOH in CH₂Cl₂) to afford 34 (73 mg,

0.242 mmol, 68%) as a white solid. HRMS (ESI): calculated for

DMSO‑d₆)

d

3.50e3.62 (2 H, m, H-5⁰, H-5⁰⁰), 4.15 (1 H, dt, J ¼ 28.1,

3.8 Hz, H-4⁰), 4.58 (1 H, ddd, J ¼ 26.1, 7.9, 3.8 Hz, H-2⁰), 4.97 (1 H, dd,

J ¼ 54.5, 4.1 Hz, H-3⁰), 6.09 (1 H, d, J ¼ 8.2 Hz, H-1⁰), 7.02 (2 H, br. s.,

NH₂), 7.34 (1 H, d, J ¼ 2.1 Hz, H-6), 8.05 (1 H, s, H-2) ppm. ¹³C NMR

C

₁₂H₁₄F₂N₃O₄([MþH]^þ): 302.0952, found: 302.0946.

5-chloro-4-methoxy-N7-(3′-deoxy-3′-ﬂuoro-b-D-ribofur-

(75 MHz, DMSO‑d₆)

d

61.4 (d, J ¼ 11.5 Hz, C-5⁰), 72.8 (d, J ¼ 17.3 Hz,

anosyl)pyrrolo [2,3-d]pyrimidine (35)

C-2⁰), 83.4 (d, J ¼ 20.7 Hz, C-4⁰), 85.7 (C-1⁰), 93.3 (d, J ¼ 184.3 Hz, C-

3⁰), 93.1 (d, J ¼ 16.1 Hz, C-4a), 104.7 (d, J ¼ 27.6 Hz,C-6), 143.2 (d,

J ¼ 244.2 Hz, C-5), 147.2 (d, J ¼ 2.3 Hz, C-7a), 153.3 (s, C-2), 156.3 (d,

23 (247 mg) was subjected to general procedure D (reaction

time: 2h; puriﬁcation: ﬂash column chromatography, automated,

0 / 8% MeOH in CH₂Cl₂) to afford 35 (99 mg, 0.312 mmol, 67% yield

%) as a white solid. HRMS (ESI): calculated for C₁₂H₁₄ClFN₃O₄

([MþH]^þ): 318.0657, found: 318.0630.

J ¼ 2.3 Hz, C-4) ppm. ¹⁹F NMR (282 MHz, DMSO‑d₆)

d

ꢁ196.7

to ꢁ196.2 (1F, m, F-7), ꢁ167.4 (1F, s, F-3’) ppm. HRMS (ESI):

calculated for C₁₁H₁₃F₂N₄O₃([MþH]^þ): 287.0956, found: 287.0977.

5-bromo-4-methoxy-N7-(3′-deoxy-3′-ﬂuoro-

anosyl)pyrrolo[2,3-d]pyrimidine (36).

b-D-ribofur-

4-amino-5-chloro-N7-(3′-deoxy-3′-ﬂuoro-

anosyl)pyrrolo [2,3-d]pyrimidine (31)

b-D-ribofur-

24 (0.162 g, 0.282 mmol) was subjected to general procedure D

(reaction time: 1h30; puriﬁcation: ﬂash column chromatography,

automated, 0 / 6% MeOH in CH₂Cl₂) to afford 36 (80 mg,

0.221 mmol, 78% yield) as a white solid. ¹H NMR (300 MHz,

27 (37 mg, 0.075 mmol) was subjected to general procedure D

(reaction time: 3h; puriﬁcation: ﬂash column chromatography,

automated, 2 / 12% MeOH in CH₂Cl₂) to afford 31 (26 mg,

0.086 mmol, 58% yield) as a white solid. ¹H NMR (300 MHz,

DMSO‑d₆)

d

3.55e3.67 (2 H, m, H-5⁰, H-5⁰⁰), 4.05 (3 H, CH₃), 4.22

DMSO‑d₆)

d

3.58 (2 H, t, J ¼ 4.5 Hz, H-5, H-5⁰), 4.18 (1 H, dt, J ¼ 28.1,

(1 H, dt, J ¼ 27.5, 3.8 Hz, H-4⁰), 4.54e4.81 (1 H, m, H-2⁰), 5.02 (1 H,

dd, J ¼ 54.5, 4.1 Hz, H-1⁰), 5.28 (1 H, br. s., OH), 5.84 (1 H, br. s., OH),

6.19 (1 H, d, J ¼ 7.9 Hz, H-1⁰), 7.88 (1 H, s, H-6), 8.46 (1 H, s, H-2)

3.8 Hz, H-4⁰), 4.52e4.81 (1 H, m, H-2⁰), 5.00 (1 H, dd, J ¼ 54.2, 4.1 Hz,

H-3⁰), 5.37 (1 H, t, J ¼ 5.6 Hz, OH), 5.80 (1 H, d, J ¼ 6.4 Hz, OH), 6.07

(1 H, d, J ¼ 7.9 Hz, H-1⁰), 6.91 (6 H, br. s., NH₂), 7.59 (1 H, s, H-6), 8.09

ppm. ¹³C NMR (75 MHz, DMSO‑d₆)

d

54.4 (CH₃), 61.3 (d, J ¼ 11.5 Hz,

(1 H, s, H-2) ppm. ¹³C NMR (75 MHz, DMSO‑d₆)

d

61.4 (d, J ¼ 12.7 Hz,

C-5⁰), 73.3 (d, J ¼ 16.1 Hz, C-2⁰), 83.8 (d, J ¼ 20.7 Hz, C-4⁰), 86.0 (C-1⁰),

87.6 (C-5), 93.4 (d, J ¼ 182.0 Hz, C-3’),104.7 (C-4a),124.5 (C-6),152.0

(C-2), 152.1 (C-7a), 162.8 (C-4) ppm. ¹⁹F NMR (282 MHz, DMSO‑d₆)

C-5⁰), 72.9 (d, J ¼ 16.1 Hz, C-2⁰), 83.6 (d, J ¼ 20.7 Hz, C-4⁰), 86.0 (C-1⁰),

93.5 (d, J ¼ 182.0 Hz, C-3’), 100.5 (C-5), 103.6 (C-4a), 119.6 (C-6),

150.1 (C-7a), 153.2 (C-2), 157.3 (C-4) ppm. ¹⁹F NMR (282 MHz,

d

ꢁ196.77 to ꢁ196.31 (1 F, m) ppm. HRMS (ESI): calculated for

DMSO‑d₆)

C

d

ꢁ196.9 to ꢁ196.2 (m) ppm. HRMS (ESI): calculated for

C

₁₂H₁₄BrFN₃O₄([MþH]^þ): 362.0152, found: 362.0151.

₁₁H₁₃ClFN₄O₃([MþH]^þ): 303.0660, found: 303.0644.

5-iodo-4-methoxy-N7-(3′-deoxy-3′-ﬂuoro-b-D-ribofur-

4-amino-5-bromo-N7-(3′-deoxy-3′-ﬂuoro-

b

-D

-ribofur-

anosyl)pyrrolo [2,3-d]pyrimidine (37)

anosyl)pyrrolo [2,3-d]pyrimidine (32)

25 (0.227 g, 0.365 mmol) was subjected to general procedure D

(reaction time: 1h30; puriﬁcation: ﬂash column chromatography,

automated, 0 / 6% MeOH in CH₂Cl₂) to afford 37 (28 mg,

0.068 mmol, 19% yield) as a white solid. HRMS (ESI): calculated for

28 (49 mg, 0.088 mmol) was subjected to general procedure D

(reaction time: 1h; puriﬁcation: ﬂash column chromatography,

automated, 0 / 15% MeOH in CH₂Cl₂) to afford 32 (23 mg,

0.066 mmol, 75% yield) as an off-white solid. ¹H NMR (300 MHz,

C

₁₂H₁₄FIN₃O₄([MþH]^þ): 410.0013, found: 410.0022.

DMSO‑d₆)

d

3.58 (2 H, dd, J ¼ 5.3, 4.1 Hz, H-5, H-5⁰), 4.18 (1 H, dt,

4-oxo-5-ﬂuoro-N7-(3′-deoxy-3′-ﬂuoro-b-D-ribofuranosyl)

pyrrolo [2,3-d]pyrimidine (38)

J ¼ 27.8, 4.1 Hz, H-4⁰), 4.51e4.75 (1 H, m, H-2⁰), 5.00 (1 H, dd,

J ¼ 54.5, 4.1 Hz, H-3⁰), 5.37 (1 H, t, J ¼ 5.6 Hz, OH), 5.79 (1 H, d,

J ¼ 6.7 Hz, OH), 6.07 (1 H, d, J ¼ 8.2 Hz, H-1⁰), 6.65e7.02 (2 H, m,

NH₂), 7.65 (1 H, s, H-6), 8.09 (1 H, s, H-2) ppm. ¹³C NMR (75 MHz,

34 (0.073 g, 0.242 mmol) was dissolved in MeCN (8 mL). NaI

(0.182 g, 1.21 mmol, 5.0 eq.) was added, followed by TMSCl

(0.154 mL, 1.21 mmol, 5.0 eq.). The reaction was stirred for 4 h at

room temperature, neutralized via the addition of aq. sat. NaHCO₃

(1 mL) and 2 M Na₂S₂O₃(1 mL), and diluted with MeOH. Celite ®

(2 g) was added, and the mixture was concentrated in vacuo. The

DMSO‑d₆)

d

61.4 (d, J ¼ 11.5 Hz, C-5⁰), 73.0 (d, J ¼ 16.1 Hz, C-2⁰), 83.7

(d, J ¼ 21.9 Hz, C-4⁰), 86.0 (C-1⁰), 87.7 (C-5), 93.5 (d, J ¼ 180.8 Hz, C-

3’), 101.6 (C-4a), 122.1 (C-6), 150.5 (C-7a), 153.0 (C-2), 157.5 (C-4)

13

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

residue was puriﬁed by ﬂash column chromatography (automated,

2 / 20% MeOH/DCM) to afford 38 (61 mg, 0.212 mmol, 88% yield)

7.95 (s, 1 H, H-2), 12.12 (br. s, 1 H, NH) ppm. ¹³C NMR (101 MHz,

DMSO‑d₆)

d

56.0 (C-5), 60.9 (d, J ¼ 11.6 Hz, C-5⁰), 73.2 (d, J ¼ 13.1 Hz,

as a white solid. ¹H NMR (300 MHz, DMSO‑d₆)

d

3.48e3.74 (2 H, m,

C-2⁰), 83.4 (d, J ¼ 21.1 Hz, C-4⁰), 85.5 (C-1⁰), 93.2 (d, J ¼ 181.7 Hz, C-

3’), 108.3 (C-4a), 125.7 (C-6), 145.0 (C-7a), 148.4 (C-2), 157.8 (C-4)

ppm. HRMS (ESI): calculated for C₁₁H₁₂FIN₃O₄([MþH]^þ): 395.9857,

found: 395.9879.

H-5⁰, H-5⁰⁰), 4.19 (1 H, dt, J ¼ 27.8, 4.1 Hz, H-4⁰), 4.40e4.66 (1 H, m,

H-2⁰), 5.01 (1 H, dd, J ¼ 54.5, 4.4 Hz, H-3⁰), 5.22 (1 H, br. s., OH), 5.84

(1 H, d, J ¼ 6.4 Hz, OH), 6.11 (1 H, d, J ¼ 7.3 Hz, H-1⁰), 7.35 (1 H, d,

J ¼ 1.8 Hz, H-6), 7.95 (1 H, s, H-2), 12.15 (1 H, br. s., NH) ppm. ¹³

C

4-azido-5-iodo-N7-(2,5-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-

D-ribofuranosyl)pyrrolo [2,3-d]pyrimidine (42)

b-

NMR (101 MHz, DMSO‑d₆)

d

60.8 (d, J ¼ 11.6 Hz, C-5⁰), 73.0 (d,

J ¼ 16.0 Hz, C-2⁰), 83.2 (d, J ¼ 21.8 Hz, C-4⁰), 85.2 (C-1⁰), 93.0 (d,

J ¼ 181.7 Hz, C-3⁰), 97.6 (d, J ¼ 12.4 Hz, C-4a),103.5 (d, J ¼ 26.9 Hz, C-

6), 145.1 (d, J ¼ 248.5 Hz, C-5), 144.4 (d, J ¼ 2.9 Hz, C-7a), 145.1 (C-2),

156.3 (d, J ¼ 2.2 Hz, C-4) ppm. ¹⁹F NMR (377 MHz, DMSO‑d₆)

d ppm ꢁ196.6 to ꢁ196.1 (1 F,m, F-3’), ꢁ165.4 (1 F, s, F-5). 143.2 (d,

J ¼ 244.2 Hz, C-5), 147.2 (d, J ¼ 2.3 Hz, C-7a), 153.3 (s, C-2), 156.3 (d,

J ¼ 2.3 Hz, C-4) ppm. HRMS (ESI): calculated for C₁₁H₁₂F₂N₃O₄

([MþH]^þ): 288.0796, found: 288.0788.

25 (2.12 g, 3.40 mmol) was subjected to general procedure B

(puriﬁcation: ﬂash column chromatography, automated, 5 / 50%

EtOAc in petroleum ether) to afford 42 (1.46 g, 2.33 mmol, 68%) as a

white foam. ¹H NMR (300 MHz, DMSO‑d₆) d 4.68 (dd, J ¼ 12.0,

5.3 Hz,1 H, H-5⁰), 4.78 (dd, J ¼ 12.3, 3.8 Hz,1 H, H-5⁰⁰), 4.81e4.94 (m,

1 H, H-2⁰), 5.85 (ddd, J ¼ 53.0, 5.0, 2.9 Hz, 1 H, H-3⁰), 6.11 (ddd,

J ¼ 16.8, 6.6, 5.3 Hz, 1 H, H-4⁰), 6.80 (d, J ¼ 6.4 Hz, 1 H, H-1⁰),

7.49e7.60 (m, 6 H, H_Phe), 7.63e7.74 (m, 3 H, H_Phe), 7.93e8.05 (m,

6 H, H_Phe), 8.18 (s, 1 H, H-6), 9.88 (s, 1 H, H-2) ppm. ¹³C NMR

(75 MHz, DMSO‑d₆) d 56.0 (C-5), 63.3 (d, J ¼ 8.1 Hz, C-5⁰), 74.0 (d,

J ¼ 15.0 Hz, C-2⁰), 80.3 (d, J ¼ 24.2 Hz, C-4⁰), 85.4 (C-1⁰), 89.2 (d,

J ¼ 186.6 Hz, C-4’), 107.2 (C-3a), 128.1 (C_Phe), 128.9 (C_Phe) 129.1

(C_Phe), 129.3 (C_Phe), 129.5 (C_Phe), 130.2 (C_Phe), 133.6 (C_Phe), 134.1

(C_Phe), 134.9 (C-7a), 141.8 (C-2), 145.9 (C-4, 162.3 (C]O), 165.4 (C]

O) ppm. ¹⁹F NMR (282 MHz, DMSO‑d₆) d ꢁ199.12 (ddd, J ¼ 52.9,

25.2, 16.8 Hz, 1 F) ppm. HRMS (ESI): calculated for C₂₅H₁₉FIN₆O₅

([MþH]^þ): 629.0446, found: 629.0449.

4-oxo-5-chloro-N7-(3′-deoxy-3′-ﬂuoro-

pyrrolo [2,3-d]pyrimidine (39)

b-D-ribofuranosyl)

34 (0.099 g, 0.312 mmol) was dissolved in MeCN (8 mL). NaI

(0.231 g, 1.56 mmol, 5.0 eq.) was added, followed by TMSCl

(0.198 mL, 1.56 mmol, 5.0 eq.). The reaction was stirred for 4 h at

room temperature, neutralized via the addition of aq. sat. NaHCO₃

(1 mL) and 2 M Na₂S₂O₃(1 mL), and diluted with MeOH. Celite ®

(2 g) was added, and the mixture was concentrated in vacuo. The

residue was puriﬁed by ﬂash column chromatography twice

(automated, 2 / 20% MeOH/DCM) to afford 39 (46 mg, 0.151 mmol,

49% yield) as a light brown solid. ¹H NMR (300 MHz, DMSO‑d₆)

4-azido-5-triﬂuoromethyl-N7-(2,5-di-O-benzoyl-3′-deoxy-

3′-ﬂuoro-b-D-ribofuranosyl)pyrrolo [2,3-d]pyrimidine (43)

d

3.50e3.68 (m, 2 H), 4.11e4.30 (m, 1 H, H-4⁰), 4.48e4.66 (m, 1 H, H-

TMSCF₃(0.216 mL, 1.46 mmol, 3.0 eq.) was added dropwise over

the course of 1 h to a suspension of CuI (0.278 g, 1.46 mmol, 3.0 eq.)

and KF (0.058 g, 1.46 mmol, 3.0 eq.) in a mixture of dry degassed

DMF/NMP 1:1 (3 mL). when all solids had dissolved, 42 (0.306 g,

0.487 mmol, 1.0 eq.) in dry degassed DMF/NMP 1:1 (3 mL) was

added, and the mixture was heated to reﬂux. After 3 h, LC/MS

analysis showed full conversion of the starting material, and the

reaction was cooled to room temperature. The mixture was diluted

with EtOAc (15 mL) and H₂O (5 mL) and the solids were ﬁltered off

over Celite®. The ﬁlter cake was washed extensively with addi-

tional EtOAc (3 ꢂ 25 mL), and the combined ﬁltrates were trans-

ferred to a separation funnel. Additional water (40 mL) was added,

the phases separated, and the organic phase washed twice more

with water (25 mL). The organic layer was dried over Na₂SO₄and

concentrated in vacuo. The residue was used as such in the next

reaction. HRMS (ESI): calculated for C₂₆H₁₉F₄N₆O₅([MþH]^þ):

571.1353, 571.1342.

2⁰), 5.02 (dd, J ¼ 55.1, 4.1 Hz, 1 H, H-3⁰), 5.18e5.28 (m, 1 H, OH), 5.84

(d, J ¼ 6.7 Hz, 1 H, OH), 6.05 (d, J ¼ 8.2 Hz, 1 H, H-1’), 7.54 (s, 1 H, H-

6), 7.96 (s, 1 H, H-2) ppm. HRMS (ESI): calculated for C₁₁H₁₂ClFN₃O₄

([MþH]^þ): 304.0500, found: 304.0513.

4-oxo-5-bromo-N7-(3′-deoxy-3′-ﬂuoro-

pyrrolo [2,3-d]pyrimidine (40)

b-D-ribofuranosyl)

36 (47 mg, 0.130 mmol) was dissolved in 1,4-dioxane (2 mL). 2 M

NaOH (2 mL) was added and the mixture was reﬂuxed for 1h30. The

mixture was cooled to room temperature, neutralized to pH 7 via

the addition of 4 N HCl, and concentrated in vacuo. The residue was

suspended in MeOH, adsorbed onto celite, and puriﬁed via ﬂash

column chromatography (automated, 0 / 15% MeOH in CH₂Cl₂) to

afford 40 (26 mg, 0.075 mmol, 57% yield) as a white solid. ¹H NMR

(300 MHz, DMSO‑d₆) d

3.54e3.63 (2 H, m, H-5⁰, H-5⁰⁰), 4.18 (1 H, dt,

J ¼ 27.8, 4.1 Hz, H-4⁰), 4.47e4.64 (1 H, m, H-2⁰), 4.99 (1 H, dd,

J ¼ 54.8, 4.4 Hz, H-3⁰), 5.23 (1 H, t, J ¼ 5.4 Hz, OH), 5.83 (1 H, d,

J ¼ 6.7 Hz, OH), 6.04 (1 H, d, J ¼ 7.9 Hz, H-1⁰), 7.56 (1 H, s, H-6), 7.95

(1 H, s, H-2), 12.14 (1 H, br. s., NH) ppm. ¹³C NMR (75 MHz,

4-amino-5-triﬂuoromethyl-N7-(3′-deoxy-3′-ﬂuoro-

b-D-ribo-

furanosyl)pyrrolo[2,3-d]pyrimidine (44).

DMSO‑d₆)

d

61.2 (d, J ¼ 11.5 Hz, C-5⁰), 73.5 (d, J ¼ 16.1 Hz, C-2⁰), 83.8

43 (crude) was subjected to general procedure C (no puriﬁca-

tion). The residue was subjected to general procedure D (reaction

time: 2h; puriﬁcation: ﬂash column chromatography, automated,

0 / 8% MeOH in CH₂Cl₂) to afford 44 (15 mg, 0.044 mmol, 9% yield

over 2 steps) as a white solid. ¹H NMR (300 MHz, DMSO‑d₆)

(d, J ¼ 21.9 Hz, C-4⁰), 85.8 (C-1⁰), 91.3 (C-5), 93.4 (d, J ¼ 180.8 Hz, C-

3’), 106.6 (C-4a), 120.8 (C-6), 145.6 (C-7a), 148.2 (C-2), 157.6 (C-4)

ppm. ¹⁹F NMR (282 MHz, DMSO‑d₆)

ppm. HRMS (ESI): calculated for

347.9995, found: 347.9977.

d

ꢁ196.7 to ꢁ196.3 (1 F, m)

C

₁₁H₁₂BrFN₃O₄([MþH]^þ):

d

3.52e3.79 (2 H, m, H-5⁰, H-5⁰⁰), 4.23 (1 H, dt, J ¼ 27.5, 3.5 Hz, H-4⁰),

4-oxo-5-iodo-N7-(3′-deoxy-3′-ﬂuoro-

rolo[2,3-d]pyrimidine (41).

b-

D-ribofuranosyl)pyr-

4.61e4.83 (1 H, m, H-2⁰), 5.03 (1 H, dd, J ¼ 54.8, 4.1 Hz, H-3⁰), 5.41

(1 H, t, J ¼ 5.6 Hz, OH), 5.84 (1 H, d, J ¼ 6.7 Hz, OH), 6.13 (1 H, d,

J ¼ 7.9 Hz, H-1⁰), 6.63 (2 H, br. s., NH₂), 8.15 (1 H, d, J ¼ 1.5 Hz, H-6),

37 (28 mg, 0.068 mmol) was dissolved in 1,4-dioxane (2 mL).

2 M NaOH (2 mL) was added and the mixture was reﬂuxed for 2h.

The mixture was cooled to room temperature, neutralized to pH 7

via the addition of 4 N HCl, and concentrated in vacuo. The residue

was suspended in MeOH, adsorbed onto celite, and puriﬁed via

ﬂash column chromatography (automated, 2 / 15% MeOH in

CH₂Cl₂) to afford JB529 (17 mg, 0.043 mmol, 63% yield) as a white

8.22 (1 H, s, H-2) ppm. ¹³C NMR (75 MHz, DMSO‑d₆)

d ppm 61.3 (d,

J ¼ 11.5 Hz, c-5⁰), 73.1 (d, J ¼ 16.1 Hz, C-2⁰), 84.0 (d, J ¼ 20.7 Hz, C-4⁰),

86.5 (C-1⁰), 93.3 (d, J ¼ 180.8 Hz, C-3’), 98.6 (C-4a), 104.3 (q,

J ¼ 38.0 Hz, C-5), 124.6 (q, J ¼ 6.9 Hz, C-6), 123.8 (q, J ¼ 264.0 Hz,

CF₃), 152.1 (C-7a), 153.6 (C-6), 156.7 (C-4) ppm. HRMS (ESI):

calculated for C₁₂H₁₃F₄N₄O₃([MþH]^þ): 337.0924, found: 337.0933.

solid. ¹H NMR (300 MHz, DMSO‑d₆)

d

3.49e3.77 (m, 2 H, H-5⁰, H-

4-amino-N7-(2,5-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-

b-D-ribo-

5⁰⁰), 4.20 (dt, J ¼ 27.8, 3.8 Hz, 1 H, H-4⁰), 4.44e4.73 (m, 1 H, H-2⁰),

5.00 (dd, J ¼ 54.8, 4.1 Hz, 1 H, H-3⁰), 5.18e5.35 (m, 1 H, OH), 5.84 (d,

J ¼ 3.8 Hz, 1 H, OH), 6.03 (d, J ¼ 8.2 Hz, 1 H, H-1⁰), 7.59 (s, 1 H, H-6),

furanosyl)pyrrolo [2,3-d]pyrimidine (45)

29 (0.276 g, 0.44 mmol) was dissolved in MeOH (4 mL) and 1 M

NaOAc (1 mL). Pd(OH)₂/C (55 mg, 20% wt.) was added and the

14

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

mixture was stirred under hydrogen atmosphere for 2h. The

mixture was ﬁltered over celite, and the ﬁltrate concentrated in

vacuo. The residue was dissolved in CH₂Cl₂, adsorbed onto celite

and puriﬁed via ﬂash column chromatography (automated,

25e100% EtOAc in petroleum ether to afford 45 (198 mg,

0.42 mmol, 94% yield) as a light brown oil. HRMS (ESI): calculated

for C₂₅H₂₂FN₄O₅([MþH]^þ): 477.1574, found: 477.1542.

48 (crude) was subjected to general procedure C. The residue

was subjected directly to general procedure D (reaction time: 2 h;

puriﬁcation: ﬂash column chromatography, automated, 0 / 10%

MeOH in CH₂Cl₂) to afford pure 49 (10 mg, 0.035 mmol, 8% yield

over 3 steps) as a white solid. ¹H NMR (300 MHz, DMSO‑d₆)

d 2.32

(3 H, d, J ¼ 0.9 Hz, CH₃), 3.46e3.66 (2 H, m, H-5⁰, H-5⁰⁰), 4.14 (1 H, dt,

J ¼ 28.4, 3.5 Hz, H-4⁰), 4.51e4.77 (1 H, m, H-2⁰), 4.99 (1 H, dd,

J ¼ 55.1, 4.4 Hz, H-3⁰), 5.45 (1 H, t, J ¼ 5.6 Hz, OH), 5.69 (1 H, d,

J ¼ 7.0 Hz, OH), 5.96 (1 H, d, J ¼ 8.2 Hz, H-1⁰), 6.61 (2 H, br. s., NH₂),

7.08 (1 H, d, J ¼ 1.2 Hz, H-6), 7.98 (1 H, s, H-2) ppm. ¹³C NMR

4-amino-N7-(3′-deoxy-3′-ﬂuoro-b-D-ribofuranosyl)pyrrolo

[2,3-d]pyrimidine (46)

45 (198 mg, 0.42 mmol) was subjected to general procedure D

(reaction time: 2h; puriﬁcation: ﬂash column chromatography,

automated, 0 / 15% MeOH in CH₂Cl₂) to afford 46 (59 mg,

0.22 mmol, 52% yield) as a white solid. ¹H NMR (300 MHz,

(75 MHz, DMSO‑d₆)

d

12.3 (CH₃), 61.6 (d, J ¼ 11.5 Hz, C-5⁰), 72.5 (d,

J ¼ 16.1 Hz, C-2⁰), 83.3 (d, J ¼ 20.7 Hz, C-4⁰), 86.1 (C-1⁰), 94.1 (d,

J ¼ 180.8 Hz, C-3’),103.5 (C-4a),110.5 (C-5),120.0 (C-6),151.4 (C-7a),

151.9 (C-2), 158.4 (C-4) ppm. ¹⁹F NMR (282 MHz, DMSO‑d₆)

DMSO‑d₆)

d

3.51e3.65 (2 H, m, H-5⁰, H-5⁰⁰), 4.19 (1 H, dt, J ¼ 28.1,

3.8 Hz, H-4⁰), 4.62e4.84 (1 H, m, H-2⁰), 5.00 (1 H, m, J ¼ 55.1, 4.1 Hz,

H-3”), 5.61 (1 H, t, J ¼ 5.7 Hz, OH), 5.74 (1 H, d, J ¼ 7.0 Hz, OH), 6.00

(1 H, d, J ¼ 8.2 Hz, H-1⁰), 6.59 (1 H, d, J ¼ 3.8 Hz, H-5), 7.08 (2 H, br. s),

7.35 (1 H, d, J ¼ 3.5 Hz, H-6), 8.03 (1 H, s, H-2) ppm. ¹³C NMR

d

ꢁ196.50 to ꢁ195.86 (1 F, m) ppm. HRMS (ESI): calculated for

C

₁₂H₁₆FN₄O₃([MþH]^þ): 283.1206, found: 283.1194.

4-azido-5-cyano-N7-(2,5-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-b-

D

-ribofuranosyl)pyrrolo [2,3-d]pyrimidine (50)

A solution of 29 (0.483 g, 0.769 mmol, 1.0 eq.) in anhydrous

(75 MHz, DMSO‑d₆)

d

61.6 (d, J ¼ 11.5 Hz, C-5⁰), 72.6 (d, J ¼ 17.3 Hz,

C-2⁰), 83.5 (d, J ¼ 20.7 Hz, C-4⁰), 87.0 (C-1⁰), 93.8 (d, J ¼ 180.8 Hz, C-

3’), 100.3 (C-4a), 103.7 (C-5), 122.8 (C-6), 150.6 (C-7a), 152.1 (C-2),

158.1 (C-4) ppm. ¹⁹F NMR (377 MHz, DMSO‑d₆) d ppm ꢁ197.3

to ꢁ195.3 (m, 1 F) ppm. HRMS (ESI): calculated for C₁₁H₁₄FN₄O₃

([MþH]^þ): 269.1050, found: 269.1060.

toluene (5 mL) was cooled to e 60 ^ꢀC. iPrMgCl.LiCl complex (1.3 M

in THF, 0.768 mL, 0.922 mmol, 1.2 eq.) was added dropwise.

Halogen-magnesium exchange was checked via TLC analysis of

aliquots (directly quenched with aq. sat. NH₄Cl solution), and after

1 h, TLC analysis indicated full conversion of the starting material.

p-toluenesulfonyl cyanide (0.279 g, 1.538 mmol, 2.0 eq.) was added

and the mixture was slowly warmed to room temperature over-

night. The mixture was transferred to a separation funnel and

diluted with CH₂Cl₂(20 mL) and aq. sat. NH₄Cl solution (20 mL).

The phases were separated and the aqueous phase extracted with

CH₂Cl₂(2 ꢂ 25 mL). The combined organic phases were dried over

Na₂SO₄, concentrated in vacuo, and the residue puriﬁed via ﬂash

column chromatography (automated, 0 / 30% EtOAc in petroleum

ether) to afford 50 (246 mg, 0.466 mmol, 61% yield) as a colourless

oil. HRMS (ESI): calculated for C₂₆H₂₉FN₇O₅([MþH]^þ): 528.1432,

found: 528.1168.

4-oxo-N7-(3′-deoxy-3′-ﬂuoro-

b-D-ribofuranosyl)pyrrolo[2,3-

d]pyrimidine (47).

46 (0.036 g, 0.134 mmol) was dissolved in a mixture of H₂O

(1.5 mL) and AcOH (0.5 mL). The mixture was warmed to 50 ^ꢀC,

NaNO₂(0.046 g, 0.67 mmol, 5 eq.) was added, and the mixture was

further heated at 70 ^ꢀC for 48h, when TLC analysis (10% MeOH in

CH₂Cl₂) indicated completion of the reaction. The mixture was

concentrated in vacuo, and the residue co-evaporated with toluene

three times. The residue was taken up in MeOH, adsorbed onto

celite and puriﬁed by ﬂash column chromatography (automated,

2 / 15% MeOH in CH₂Cl₂) to afford 47 (28 mg, 0.104 mmol, 78%

yield) as a white solid. ¹H NMR (300 MHz, DMSO‑d₆)

d

3.48e3.72

4-amino-5-cyano-N7-(3′-deoxy-3′-ﬂuoro-

anosyl)-pyrrolo[2,3-d]pyrimidine (51) and

-ribofuranosyl)

b

-D-ribofur-

(m, 2 H, H-5⁰, H-5⁰⁰), 4.20 (dt, J ¼ 27.8, 4.0 Hz, 1 H, H-4⁰), 4.54e4.74

(m, 1 H, H-2⁰), 5.02 (dd, J ¼ 54.8, 4.1 Hz, 1 H, H-3⁰), 5.24 (t, J ¼ 4.7 Hz,

1 H, OH), 5.82 (d, J ¼ 6.4 Hz, 1 H, OH), 6.07 (d, J ¼ 8.2 Hz, 1 H, H-1⁰),

6.57 (d, J ¼ 3.8 Hz, 1 H, C-5), 7.40 (d, J ¼ 3.5 Hz, 1 H, C-6), 7.93 (s, 1 H,

4-amino-5-

methylcarbimidate-N7-(3′-deoxy-3′-ﬂuoro-

pyrrolo[2,3-d]pyrimidine (53).

b-D

50 (0.246 g, 0.47 mmol) was subjected to general procedure C.

The residue was stirred in 7 N NH₃in MeOH (10 mL) overnight. The

volatiles were removed in vacuo, and the residue puriﬁed by ﬂash

column chromatography (automated, 0 / 12% MeOH in CH₂Cl₂) to

afford 2 products: 51 (48 mg, 0.164 mmol, 35%), white solid and 53

(52 mg, 0.160 mmol, 34%), white solid. Analytical data 51: ¹H NMR

C-2),12.01 (br. s.,1 H, NH) ppm. ¹³C NMR (101 MHz, DMSO‑d₆)

d 61.0

(d, J ¼ 11.6 Hz, C-5⁰), 73.0 (d, J ¼ 16.0 Hz, C-2⁰), 83.2 (d, J ¼ 21.1 Hz, C-

4⁰), 85.7 (C-1⁰), 93.2 (d, J ¼ 180.9 Hz, C-3’), 102.9 (C-4a), 108.6 (C-5),

121.1 (C-6), 144.1 (C-7a), 148.3 (C-2), 158.3 (C-4) ppm. ¹⁹F NMR

(377 MHz, DMSO‑d₆)

d

ꢁ196.45 to ꢁ196.10 (m, 1 F) ppm. HRMS

(ESI): calculated for C₁₁H₁₃FN₃O₄([MþH]^þ): 270.0890, found:

(300 MHz, DMSO‑d₆) d

3.51e3.68 (2 H, m, H-5⁰, H-5⁰⁰), 4.23 (1 H, dt,

270.0892.

J ¼ 26.9, 3.5 Hz, H-4⁰), 4.54e4.81 (1 H, m, H-2⁰), 5.04 (1 H, dd,

J ¼ 54.2, 4.1 Hz, H-3⁰), 5.39 (1 H, t, J ¼ 5.6 Hz, OH), 5.88 (1 H, d,

J ¼ 6.7 Hz, OH), 6.08 (1 H, d, J ¼ 7.9 Hz, H-1⁰), 6.93 (2 H, br. s., NH₂),

8.21 (1 H, s, H-6), 8.42 (1 H, s, H-2) ppm. ¹³C NMR (75 MHz,

4-azido-5-methyl-N7-(2,5-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-

b

-D

-ribofuranosyl)pyrrolo [2,3-d]pyrimidine (48)

29 (0.267 g, 0.423 mmol, 1.0 eq.) and Pd(PPh₃)₄(0.013 g,

0.011 mmol, 2.5 mol%) were dissolved in dry, degassed THF

(2.5 mL). AlMe₃(2 M in toluene, 0.423 mL, 0.846 mmol, 2.0 eq.) was

added, and the mixture was reﬂuxed for 4h, when LCMS analysis

indicated full conversion of the starting material. The mixture was

cooled to 0 ^ꢀC and carefully quenched via the addition of aq. sat.

NH₄Cl solution (1 mL). The mixture was ﬁltered over celite, and the

ﬁlter cake washed extensively with CHCl₃. The ﬁltrate was trans-

ferred to a separation funnel, and H₂O was added. The phases were

separated, and the aqueous phase extracted with more CHCl₃. The

combined organic phases were dried over Na₂SO₄and concentrated

in vacuo. The obtained residue, a complex mixture of product and

side products (as identiﬁed via LCMS), was used crude in the next

reaction.

DMSO‑d₆)

d

61.2 (d, J ¼ 11.5 Hz, C-5⁰), 73.3 (d, J ¼ 16.1 Hz, C-2⁰), 84.1

(C-5), 84.1 (d, J ¼ 21.9 Hz, C-4⁰), 86.9 (C-1⁰), 93.3 (d, J ¼ 182.0 Hz, C-

4’), 101.7 (C-4a), 110.0 (C-6), 115.5 (CN), 132.8 (C-7a), 151.0 (C-2),

157.5 (C-4) ppm. ¹⁹F NMR (282 MHz, DMSO‑d₆)

d

ꢁ197.36

to ꢁ196.94 (1 F, m) ppm. HRMS (ESI): calculated for C₁₂H₁₃FN₅O₃

([MþH]^þ): 294.1002, found: 294.1010. Analytical data 53: HRMS

(ESI): calculated for C₁₃H₁₇FN₅O₄([MþH]^þ): 326.1265, found:

326.1260.

4.7. Conversion of (53) to (51)

53 (52 mg, 0.160 mmol) was dissolved in DMF (10 mL). a cata-

lytic amount NaOAc (15 mg) was added, and the mixture was

heated at 130 ^ꢀC for 2 h, when HRMS analysis indicated full con-

version to 51. The mixture was concentrated in vacuo, the residue

4-amino-5-methyl-N7-(3′-deoxy-3′-ﬂuoro-

anosyl)pyrrolo [2,3-d]pyrimidine (49)

b-D-ribofur-

15

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

dissolved in MeOH, adsorbed onto celite, and puriﬁed via ﬂash

column chromatography (automated, 0 / 12% MeOH in CH₂Cl₂) to

afford 51 (33 mg, 0.113 mmol, 70% yield) as a white solid.

(1 H, t, J ¼ 5.7 Hz, OH), 5.69 (1 H, d, J ¼ 6.7 Hz, OH), 6.00 (1 H, d,

J ¼ 8.2 Hz, H-1⁰), 6.58 (2 H, s, NH₂), 7.10 (1 H, s, H-6), 7.99 (1 H, s, H-

2) ppm. ¹³C NMR (75 MHz, DMSO‑d₆)

d 15.2 (CH₃), 19.6 (CH₂), 61.6

4-amino-5-carboxamido-N7-(3′-deoxy-3′-ﬂuoro-

furanosyl)pyrrolo [2,3-d]pyrimidine (52)

b-

D-ribo-

(d, J ¼ 11.5 Hz, C-5⁰), 72.4 (d, J ¼ 16.1 Hz, C-2⁰), 83.3 (d, J ¼ 20.7 Hz, C-

4⁰), 86.3 (C-1⁰), 93.7 (d, J ¼ 182.0 Hz, C-3’), 102.8 (C-4a), 117.6 (C-5),

118.9 (C-6), 151.5 (C-7a), 151.8 (C-2), 158.2 (C-4) ppm. ¹⁹F NMR

H₂O₂solution (30 % wt in H₂O, 1 mL) was added to a solution of

51 (48 mg, 0.164 mmol) in NH₄OH solution (28e30%, 4 mL). The

mixture was stirred for 1h15, when TLC analysis (20% MeOH in

CH₂Cl₂) indicated completion of the reaction. The mixture was

concentrated in vacuo, the residue dissolved in MeOH, adsorbed

onto celite and puriﬁed by ﬂash column chromatography (auto-

mated, 2 / 15% MeOH in CH₂Cl₂) to afford 52 (18 mg, 0.058 mmol,

(282 MHz, DMSO‑d₆)

d

ꢁ196.5 to ꢁ195.9 (1 F, m) ppm. HRMS (ESI):

calculated for C₁₃H₁₈FN₄O₃([MþH]^þ): 297.1363, found: 297.1342.

4-amino-5-vinyl-N7-(3′-deoxy-3′-ﬂuoro-

b-D-ribofuranosyl)

pyrrolo [2,3-d]pyrimidine (57)

Compound 33 (0.080 g, 0.203 mmol) was subjected to general

procedure E, using potassium vinyl triﬂuoroborate as the coupling

partner, and Cs₂CO₃as base. Puriﬁcation via ﬂash column chro-

matography (automated, 0 / 12% MeOH in CH₂Cl₂) afforded 57

(20 mg, 0.068 mmol, 33%) as a white solid. ¹H NMR (300 MHz,

35%) as a white solid. ¹H NMR (300 MHz, DMSO‑d₆)

d 3.59 (2 H, t,

J ¼ 4.7 Hz, H-5⁰, H-5⁰⁰), 4.21 (1 H, dt, J ¼ 27.8, 4.1 Hz, H-4⁰), 4.50e4.75

(1 H, m, H-2⁰), 5.04 (1 H, dd, J ¼ 54.8, 4.1 Hz, H-3⁰), 5.42 (1 H, t,

J ¼ 5.9 Hz, OH), 5.87 (1 H, d, J ¼ 6.7 Hz, OH), 6.02 (1 H, d, J ¼ 8.2 Hz,

H-1⁰), 7.39 (1 H, br. s., NH₂), 7.89 (1 H, br. s., NH₂), 8.06 (1 H, s, H-6),

DMSO‑d₆) d

3.53e3.72 (2 H, m, H-5⁰, H-5⁰⁰), 4.06e4.37 (1 H, m, H-4⁰),

4.55e4.81 (1 H, m, H-2⁰), 5.00 (1 H, dd, J ¼ 55.1, 4.4 Hz, H-3⁰), 5.12

(1 H, dd, J ¼ 11.1, 1.8 Hz, CH]CH₂), 5.47 (1 H, t, J ¼ 5.7 Hz, OH), 5.54

(1 H, dd, J ¼ 17.1, 1.6 Hz, CH]CH₂), 5.75 (1 H, d, J ¼ 6.7 Hz, OH), 6.06

(1 H, d, J ¼ 8.2 Hz, H-1⁰), 6.75 (2 H, br. s, NH₂), 7.08 (1 H, dd, J ¼ 17.3,

11.1 Hz, CH]CH₂), 7.65 (1 H, s, H-6), 8.03 (1 H, s, H-2) ppm. ¹³C NMR

8.14 (1 H, s, H-2) ppm. ¹³C NMR (75 MHz, DMSO‑d₆)

d 61.5 (d,

J ¼ 11.5 Hz, C-5⁰), 72.9 (d, J ¼ 16.1 Hz, C-2⁰), 83.8 (d, J ¼ 19.6 Hz, C-4⁰),

86.6 (C-1⁰), 93.3 (d, J ¼ 186.6 Hz, C-3’), 101.7 (C-4a),111.7 (C-5), 126.1

(C-6), 151.5 (C-7a), 153.3 (C-2), 158.6 (C-4), 166.7 (C]O) ppm. ¹⁹F

NMR (282 MHz, DMSO‑d₆)

d

ꢁ197.10 to ꢁ196.40 (126 F, m) ppm.

(75 MHz, DMSO‑d₆)

d

61.5 (d, J ¼ 12.7 Hz, C-5⁰), 72.7 (d, J ¼ 16.1 Hz,

HRMS (ESI): calculated for C₁₂H₁₅FN₅O₄([MþH]^þ): 312.1108, found:

C-2⁰), 83.5 (d, J ¼ 21.9 Hz, C-4⁰), 86.3 (C-1⁰), 94.8 (t, J ¼ 182.0 Hz, C-

3’), 101.4 (C-4a), 113.8 (CH]CH₂), 114.8 (C-5), 119.5 (C-6), 129.4

(CH]CH₂), 151.5 (C-7a), 152.0 (C-2), 158.2 (C-4) ppm. ¹⁹F NMR

312.1085.

4-amino-5-ethynyl-N7-(3′-deoxy-3′-ﬂuoro-

anosyl)pyrrolo [2,3-d]pyrimidine (55)

b-D-ribofur-

(282 MHz, DMSO‑d₆)

d

ꢁ196.6 to ꢁ196.1 (m) ppm. HRMS (ESI):

Compound 33 (0.218 g, 0.55 mmol, 1.0 eq.), PdCl₂(PPh)₃(0.020 g,

0.028 mmol, 0.05 eq.) and CuI (0.011 g, 0.055 mmol, 0.1 eq.) were

added to a 10 mL round bottom ﬂask. The ﬂask was evacuated and

backﬁlled with argon three times. Then, anhydrous, degassed DMF

(2 mL), Et₃N (0.5 mL) and TMS-acetylene (0.762 mL, 5.5 mmol, 10

eq.) were added. The resulting solution was stirred at room tem-

perature for 3 h, when LCMS analysis indicated completion of the

reaction. The mixture was concentrated in vacuo, the residue taken

up in MeOH, adsorbed onto celite, and puriﬁed via ﬂash column

chromatography (automated, 2 / 12% MeOH in CH₂Cl₂). The in-

termediate TMS-ethynyl nucleoside was stirred overnight in 7 N

NH₃in MeOH (10 mL). The mixture was concentrated in vacuo, and

the residue puriﬁed again by ﬂash column chromatography

(automated, 2 / 12% MeOH in CH₂Cl₂) to afford 55 (70 mg,

0.240 mmol, 44% yield) as a white solid. ¹H NMR (300 MHz,

calculated for C₁₃H₁₆FN₄O₃([MþH]^þ): 295.1206, found: 295.1205.

4-amino-5-phenyl-N7-(3′-deoxy-3′-ﬂuoro-

anosyl)pyrrolo [2,3-d]pyrimidine (58)

b-D-ribofur-

33 (0.166 g, 0.42 mmol) was subjected to general procedure E,

using phenylboronic acid as the coupling partner, and Na₂CO₃as

base. Puriﬁcation via ﬂash column chromatography (automated,

0 / 10% MeOH in CH₂Cl₂) afforded 58 (49 mg, 0.142 mmol, 34%) as

a white solid. ¹H NMR (300 MHz, DMSO‑d₆)

d 3.53e3.68 (2 H, m, H-

5⁰, H-5⁰⁰), 4.25 (1 H, dt, J ¼ 28.1, 3.5 Hz, H-4⁰), 4.63e4.86 (1 H, m, H-

2⁰), 5.03 (1 H, dd, J ¼ 54.8, 4.4 Hz, H-3⁰), 5.43 (1 H, t, J ¼ 5.6 Hz, OH),

5.81 (1 H, d, J ¼ 7.0 Hz, OH), 6.15 (1 H, d, J ¼ 8.2 Hz, H-3⁰), 7.32e7.42

(1 H, m, H_Phe), 7.44e7.52 (4 H, m, H_Phe), 7.55 (1 H, s, H-6), 8.14 (1 H, s,

H-2) ppm. ¹³C NMR (75 MHz, DMSO‑d₆)

d

61.5 (d, J ¼ 11.5 Hz, C-5⁰),

72.8 (d, J ¼ 16.1 Hz, C-2⁰), 83.4 (d, J ¼ 21.9 Hz, C-4⁰), 86.3 (C-1⁰), 93.7

(d, J ¼ 180.8 Hz, C-3’), 101.1 (C-4a), 117.1 (C-5), 121.5 (C-6), 127.5

(C_Phe), 128.9 (C_Phe), 129.5 (C_Phe), 134.7 (C_Phe), 151.6 (C-7a), 152.2 (C-

DMSO‑d₆)

d

3.50e3.71 (2 H, m, H-5⁰, H-5⁰⁰), 4.20 (1 H, dt, J ¼ 27.5,

3.2 Hz, H-4⁰), 4.27 (1 H, s, ethynyl-CH), 4.55e4.79 (1 H, m, H-2⁰),

5.00 (2 H, dd, J ¼ 54.5, 4.4 Hz, H-3⁰), 5.42 (1 H, t, J ¼ 5.6 Hz, OH), 5.80

(1 H, d, J ¼ 6.7 Hz, OH), 6.04 (2 H, d, J ¼ 8.2 Hz, H-1⁰), 6.71 (2 H, br. s.,

NH₂), 7.81 (1 H, s, H-6), 8.12 (1 H, s, H-2) ppm. ¹³C NMR (75 MHz,

2), 157.8 (C-4) ppm. ¹⁹F NMR (282 MHz, DMSO‑d₆)

d

ꢁ196.7

to ꢁ196.1 (m) ppm. HRMS (ESI): calculated for C₁₇H₁₈FN₄O₃

([MþH]^þ): 345.1363, found: 345.1364.

4-amino-5-(4-chlorophenyl)-N7-(3′-deoxy-3′-ﬂuoro-

b-D-

DMSO‑d₆)

d

61.4 (d, J ¼ 10.4 Hz, C-5⁰), 73.0 (d, J ¼ 16.1 Hz, C-2⁰), 77.5

ribofuranosyl)pyrrolo [2,3-d]pyrimidine (59)

(ethynyl-C), 83.8 (d, J ¼ 20.7 Hz, C-4⁰), 83.7 (ethynyl-CH), 86.5 (C-

1⁰), 94.0 (d, J ¼ 180.8 Hz, C-3’), 94.8 (C-5), 102.9 (C-4a), 127.9 (C-6),

150.3 (C-7a), 153.3 (C-2), 158.0 (C-4) ppm. ¹⁹F NMR (282 MHz,

33 (0.070 g, 0.178 mmol) was subjected to general procedure E,

using 4-chlorophenylboronic acid as the coupling partner, and

Na₂CO₃as base. Puriﬁcation via ﬂash column chromatography

(automated, 0 / 12% MeOH in CH₂Cl₂) afforded 59 (49 mg,

0.129 mmol, 73% yield) as a white solid. ¹H NMR (300 MHz,

DMSO‑d₆)

d

ꢁ197.36 to ꢁ196.23 (1 F, m) ppm. HRMS (ESI): calcu-

lated for C₁₃H₁₄FN₄O₃([MþH]^þ): 293.1050, found: 293.1043.

4-amino-5-ethyl-N7-(3′-deoxy-3′-ﬂuoro-

b-

D-ribofuranosyl)

DMSO‑d₆)

d

3.49e3.71 (2 H, m, H-5⁰, H-5⁰⁰), 4.21 (1 H, dt, J ¼ 27.5,

pyrrolo [2,3-d]pyrimidine (56)

3.8 Hz, H-4⁰), 4.63e4.84 (1 H, m, H-2⁰), 5.03 (1 H, dd, J ¼ 54.5, 4.1 Hz,

H-3⁰), 5.43 (1 H, t, J ¼ 5.7 Hz, OH), 5.81 (1 H, d, J ¼ 6.7 Hz, OH), 6.15

(1 H, d, J ¼ 8.2 Hz, H-1⁰), 6.25 (2 H, br. s., NH₂), 7.43e7.56 (4 H, m,

55 (52 mg, was dissolved in MeOH (5 mL). A catalytic amount of

Pd/C was added and the mixture was stirred under hydrogen at-

mosphere for 2h, when HRMS analysis indicated completion of the

reaction. The mixture was ﬁltered over celite, and the ﬁltrate

concentrated in vacuo. The residue was dissolved in MeOH,

adsorbed onto celite, and puriﬁed by ﬂash column chromatography

(automated, 0 / 10% MeOH in CH₂Cl₂) to afford 56 (26 mg,

0.088 mmol, 49% yield) as a white solid. ¹H NMR (300 MHz,

H

_Phe), 7.58 (1 H, s, H-6), 8.15 (1 H, s, H-2) ppm. ¹³C NMR (75 MHz,

DMSO‑d₆)

d

61.5 (d, J ¼ 11.5 Hz, C-5⁰), 72.8 (d, J ¼ 16.1 Hz, C-2⁰), 83.6

(d, J ¼ 21.9 Hz, C-4⁰), 86.3 (C-1⁰), 93.6 (d, J ¼ 182.0 Hz, C-3’),100.9 (C-

4a), 116.0 (C-5), 121.8 (C-6), 129.4 (C_Phe), 130.5 (C_Phe), 132.1 (C_Phe),

133.6 (C_Phe), 151.8 (C-7a), 152.3 (C-2), 157.9 (C-4) ppm. ¹⁹F NMR

(377 MHz, DMSO‑d₆) d ꢁ196.55 to ꢁ196.14 (m, 1 F) ppm. HRMS

(ESI): calculated for C₁₇H₁₇ClFN₄O₃([MþH]^þ): 379.0973, found:

379.0976.

DMSO‑d₆)

d

1.18 (3 H, t, J ¼ 7.5 Hz, CH₃), 2.64e2.83 (2 H, m, CH₂),

3.49e3.67 (2 H, m, H-5⁰, H-5⁰⁰), 4.15 (1 H, dt, J ¼ 28.1, 3.8 Hz, H-4⁰),

4.56e4.79 (1 H, m, H-2⁰), 4.97 (1 H, dd, J ¼ 55.1, 4.4 Hz, H-3⁰), 5.50

4-amino-5-(4-methoxyphenyl)-N7-(3′-deoxy-3′-ﬂuoro-b-D-

16

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

ribofuranosyl)pyrrolo [2,3-d]pyrimidine (60)

TLC analysis (50% EA/PE) showed full conversion of the starting

material. Then, the solution was concentrated in vacuo. The residue

was co-evaporated twice with water (~40 mL), followed by co-

evaporation with toluene (~40 mL, 2 times). The resulting oil was

immediately used in the next step. Water (44.0 mL, 2.2 mL/mmol)

was added, and the solution was placed in an ice bath. After stirring

at this temperature for 5e10 min, NaIO₄(5.17 g, 24.0 mmol, 1.2 eq.)

was added. Then, after another 5 min, the ice bath was removed

and the mixture stirred for 30 min at ambient temperature, after

which TLC analysis (100% EA) showed full conversion of the inter-

mediate diol. Then, the mixture was placed in an ice bath and EtOH

(88.0 mL, 4.40 mL/mmol) was added. The resulting suspension was

ﬁltered, and the residue rinsed once with EtOH (44.0 mL, 2.2 mL/

mmol). The ﬁltrate was placed in an ice bath, and NaBH₄(0.770 g,

20.0 mmol, 1 eq.) was added portionwise. After stirring for 30 min,

TLC analysis (100% EA) showed full conversion of the intermediate

aldehyde. Then, solid NH₄Cl (5.14 g, 100 mmol, 5 eq.) was carefully

added and the mixture stirred for ~10 min. The mixture was sub-

sequently evaporated till dryness, co-evaporated with EtOH and

pre-adsorbed onto Celite®. Puriﬁcation by column chromatography

(2.5 / 5% MeOH/DCM) gave 63 as a clear oil (3.72 g, 19.6 mmol),

which solidiﬁed upon standing, in 98% yield. ¹H NMR (300 MHz,

CDCl₃): 1.37 (s, 3H, CH₃), 1.56 (s, 3H, CH₃), 2.57 (br. s, 2H, OH), 3.73

(ddd, J ¼ 12.0, 3.9, 0.9 Hz, 1H, H3), 3.81e3.86 (m, 1H, H4), 3.92e4.02

(m, 2H, H5), 4.58 (t, J ¼ 4.5 Hz, 1H, H2), 5.81 (d, J ¼ 3.6 Hz, 1H, H1).

Spectral data are in accordance with literature values [66].

33 (0.070 g, 0.178 mmol) was subjected to general procedure E,

using 4-methoxyphenylboronic acid as the coupling partner, and

Na₂CO₃as base. Puriﬁcation via ﬂash column chromatography

(automated, 0 / 12% MeOH in CH₂Cl₂) afforded 60 (43 mg,

0.115 mmol, 65% yield) as a white solid. ¹⁹F NMR (377 MHz,

DMSO‑d₆)

d

ꢁ196.48 to ꢁ196.09 (m, 1 F) ppm. ¹H NMR (300 MHz,

d

3.49e3.66 (2 H, m, H-5⁰, H-5⁰⁰), 3.79 (3 H, s, CH₃), 4.21

(1 H, dt, J ¼ 27.8, 3.8 Hz, H-4⁰), 4.62e4.86 (1 H, m, H-2⁰), 5.02 (1 H,

dd, J ¼ 55.1, 4.4 Hz, H-3⁰), 5.44 (1 H, t, J ¼ 5.6 Hz, OH), 5.79 (1 H, d,

J ¼ 6.7 Hz, OH), 6.14 (2 H, d, J ¼ 7.9 Hz, H-1⁰), 6.96e7.10 (2 H, m,

H

_Phe), 7.33e7.42 (2 H, m, H_Phe), 7.46 (1 H, s, H-6), 8.13 (1 H, s, H-2)

ppm. ¹³C NMR (75 MHz, DMSO‑d₆)

d

55.6 (CH₃), 61.5 (d, J ¼ 12.7 Hz,

C-5⁰), 72.8 (d, J ¼ 16.1 Hz, C-2⁰), 83.4 (d, J ¼ 20.7 Hz, C-4⁰), 86.3 (C-1⁰),

93.7 (d, J ¼ 180.8 Hz, C-4’), 101.3 (C-4a), 114.9 (C_Phe), 116.7 (C-5),

120.9 (C-6), 126.9 (C_Phe), 130.1 (C_Phe), 151.4 (C_Phe), 152.2 (C-7a), 157.9

(C-2), 158.9 (C-4) ppm. HRMS (ESI): calculated for C₁₈H₂₀FN₄O₄

([MþH]^þ): 375.1469, found: 375.1447.

4-amino-5-(3,4-dichlorophenyl)-N7-(3′-deoxy-3′-ﬂuoro-

ribofuranosyl)pyrrolo[2,3-d]pyrimidine (61).

b-D-

33 (0.070 g, 0.178 mmol) was subjected to general procedure E,

using 3,4-dichlorophenylboronic acid as the coupling partner, and

Na₂CO₃as base. Puriﬁcation via ﬂash column chromatography

(automated, 0 / 10% MeOH in CH₂Cl₂) afforded 61 (38 mg,

0.092 mmol, 52% yield) as a white solid. ¹H NMR (300 MHz,

DMSO‑d₆)

d

3.49e3.70 (2 H, m, H-5⁰, H-5⁰⁰), 4.19 (1 H, dt, J ¼ 27.5,

3.8 Hz, H-4⁰), 4.59e4.85 (1 H, m, H-2⁰), 5.03 (2 H, dd, J ¼ 54.8, 4.4 Hz,

H-3⁰), 5.39 (2 H, t, J ¼ 5.9 Hz, OH), 5.80 (2 H, d, J ¼ 6.7 Hz, OH), 6.14

(2 H, d, J ¼ 8.2 Hz, H-1⁰), 6.38 (2 H, br. s, NH₂), 7.41 (1 H, s, H_Phe),

7.58e7.80 (3 H, m, H_Phe, H-6), 8.14 (1 H, s, H-2) ppm. ¹³C NMR

1,2-O-Isopropylidene-5-O-benzoyl-

a

-D

-ribofuranose

[61]

(64). 63 (0.38 g, 2.00 mmol, 1 eq.) was dissolved in DCM (10 mL,

5.0 mL/mmol) and cooled to ꢁ20 ^ꢀC. Then, Et₃N (0.31 mL,

2.20 mmol,1.1 eq.) was added and the resulting solution stirred at e

20 ^ꢀC for ~15 min. Next, BzCl (0.24 mL, 2.10 mmol, 1.05 eq.) was

(75 MHz, DMSO‑d₆)

d

61.4 (d, J ¼ 10.4 Hz, C-5⁰), 72.8 (d, J ¼ 16.1 Hz,

C-2⁰), 83.6 (d, J ¼ 20.7 Hz, C-4⁰), 86.2 (C-1⁰), 93.5 (d, J ¼ 180.8 Hz, C-

3’), 100.7 (C-4a), 114.9 (C-5), 122.4 (C-6), 128.9 (C_Phe), 129.7 (C_Phe),

130.4 (C_Phe), 131.3 (C_Phe), 131.8 (C_Phe), 135.4 (C_Phe), 152.0 (C-7a),

added dropwise with a syringe pump (50 mL/min). After complete

addition, the mixture was stirred at ꢁ20 ^ꢀC for 30 min, after which

TLC analysis (10% acetone/DCM) showed near full conversion of the

starting material. Then, water was added and the resulting mixture

transferred to a separatory funnel. The layers were separated and

the water layer extracted twice more with DCM. Organic layers

were combined, dried over Na₂SO₄, ﬁltered and evaporated till

dryness. The residue was puriﬁed by column chromatography (0%

/ 5% acetone/DCM) to give 64 (0.53 g, 1.80 mmol) as a clear oil,

which solidiﬁed upon standing, in 90% yield. ¹H NMR (300 MHz,

CDCl₃): 1.39 (s, 3H, CH₃), 1.59 (s, 3H, CH₃), 2.50 (d, J ¼ 10.5 Hz, 1H,

OH), 3.90e3.98 (m, 1H, H3), 4.09 (ddd, J ¼ 8.7, 5.1, 2.7 Hz, 1H, H4),

4.46 (dd, J ¼ 12.3, 5.1 Hz, 1H, H5), 4.61 (dd, J ¼ 5.1, 3.9 Hz, 1H, H2),

4.70 (dd, J ¼ 12.3, 2.4 Hz, 1H, H5’), 5.86 (d, J ¼ 4.2 Hz, 1H, H1),

7.41e7.47 (m, 2H, OBz_meta), 7.53e7.60 (m, 1H, OBz_para), 8.04e8.08

(m, 2H, OBz_ortho). Spectral values are in accordance with literature

values [61].

152.4 (C-2),157.9 (C-4) ppm. ¹⁹F NMR (282 MHz, DMSO‑d₆)

d

ꢁ196.8

to ꢁ196.2 (1 F, m) ppm. HRMS (ESI): calculated for C₁₇H₁₆Cl₂FN₄O₄

([MþH]^þ): 413.0583, found: 413.0563.

4-oxo-5-phenyl-N7-(3′-deoxy-3′-ﬂuoro-

b-D-ribofuranosyl)

pyrrolo [2,3-d]pyrimidine (62)

58 (0.024 g, 0.070 mmol) was dissolved in a mixture of H₂O

(1.5 mL) and AcOH (0.5 mL). The mixture was warmed to 50 ^ꢀC,

NaNO₂(0.048 g, 0.70 mmol, 10.0 eq.) was added, and the mixture

was further heated at 70 ^ꢀC for 48h, when TLC analysis (10% MeOH

in CH2Cl2) indicated completion of the reaction. The mixture was

concentrated in vacuo, and the residue co-evaporated with toluene

three times. The residue was taken up in MeOH, adsorbed onto

celite and puriﬁed by ﬂash column chromatography (automated,

0 / 10% MeOH in CH₂Cl₂) to afford 62 (19 mg, 0.055 mmol, 79%

yield) as a white solid. ¹H NMR (300 MHz, DMSO‑d₆)

d

3.48e3.77

1,2-O-Isopropylidene-3-deoxy-3-ﬂuoro-5-O-benzoyl-

xylofuranose [61] (65).

a-D-

(2 H, m, H-5⁰, H-5⁰⁰), 4.20 (1 H, dt, J ¼ 27.5, 3.8 Hz, H-4⁰), 4.55e4.80

(1 H, m, H-2⁰), 5.03 (1 H, dd, J ¼ 54.5, 4.4 Hz, H-3⁰), 5.26 (1 H, t,

J ¼ 5.4 Hz, OH), 5.85 (1 H, d, J ¼ 6.7 Hz, OH), 6.14 (1 H, d, J ¼ 7.9 Hz,

H-1⁰), 7.18e7.40 (3 H, m, H_Phe, H-6), 7.70 (1 H, s, H-2), 7.84e7.98

(3 H, m, H_Phe), 12.08 (1 H, br. s, NH) ppm. ¹³C NMR (75 MHz,

Compound 64 (0.29 g, 0.99 mmol, 1eq.) was dissolved in DCM

(5.0 mL, 5.0 mL/mmol) and cooled to ꢁ10 ^ꢀC. After stirring at that

temperature for 5e10 min, pyridine (0.29 mL, 0.29 mL/mmol, 3.6

eq.) was added, followed by dropwise addition of DAST (0.15 mL,

0.15 mL/mmol, 1.1 eq.). Cooling was removed and the mixture

stirred at ambient temperature overnight, after which TLC analysis

(15% EA/PE) showed full conversion of the starting material. Next,

the mixture was cooled into an ice bath, after which sat. aq. NaHCO₃

solution was carefully added. The mixture is transferred to a sep-

aratory funnel and the layers separated. The water layer was

extracted twice more with DCM. Organic layers are combined, dried

over Na₂SO₄, ﬁltered and evaporated. The residue was puriﬁed by

column chromatography 0% / 10% EA/PE to give 65 (0.090 g,

0.30 mmol) as a clear oil in 31% yield. ¹H NMR (300 MHz, CDCl₃):

1.34 (s, 3H, CH₃), 1.51 (s, 3H, CH₃), 4.48e4.66 (m, 3H, H5, H5’, H4),

DMSO‑d₆)

d

61.3 (d, J ¼ 11.5 Hz,C-5⁰), 73.3 (d, J ¼ 16.1 Hz, C-2⁰), 83.6

(d, J ¼ 21.9 Hz, C-4⁰), 85.8 (C-1⁰), 93.4 (d, J ¼ 182.0 Hz, C-3’), 105.8 (C-

4a), 119.0 (C-5), 121.1 (C-6), 126.7 (C_Phe), 128.4 (C_Phe), 128.5 (C_Phe),

133.9 (C_Phe), 144.8 (C-7a), 149.9 (C-2), 158.7 (C-4) ppm. ¹⁹F NMR

(282 MHz, DMSO‑d₆)

d

ꢁ196.8 to ꢁ196.1 (1 F, m) ppm. HRMS (ESI):

calculated for C₁₇H₁₇FN₃O₄([MþH]^þ): 346.1203, found: 346.1215.

1,2-O-Isopropylidene-a-D-ribofuranose [66] (63). 1,2e5,6-Di-

O-isopropylidene-a-D-allofuranose (5.21 g, 20.0 mmol, 1 eq.) was

dissolved in a mixture (7/3 ratio) of glacial acetic acid (36.8 mL,

1.80 mL/mmol) and water (15.8 mL, 0.8 mL/mmol). The resulting

solution was stirred at ambient temperature overnight, after which

17

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

4.74 (dd, J ¼ 10.8, 3.9 Hz, 1H, H2), 5.05 (dd, J ¼ 50.4, 1.8 Hz, 1H, H3),

6.04 (1H, d, J ¼ 3.9 Hz, H1), 7.42e7.48 (m, 2H, OBz_meta), 7.55e7.61

(m, 1H, OBz_para), 8.05e8.09 (m, 2H, OBz_ortho). ¹⁹F NMR (282 MHz,

CDCl₃): ꢁ208.68 to ꢁ208.36 (m, 1F). Spectral data are in accordance

with literature [61].

1 eq.) was suspended in MeCN (11 mL, 7.0 mL/mmol), after which

BSA (0.43 mL, 1.8 mmol, 1.1 eq.) was added. The resulting mixture

was stirred at ambient temperature until a clear solution was ob-

tained (~10min). Then, 67 (0.71 g, 1.8 mmol, 1.1 eq.) was added as a

solution in MeCN (4 mL). Next, TMSOTf (0.34 mL, 1.9 mmol, 1.17 eq.)

was added and the mixture stirred at ambient temperature for

~15 min. Then, the resulting mixture was heated to 80 ^ꢀC for

~40min, after which TLC analysis (20% EA/PE) showed full conver-

sion of the glycosyl donor starting material. The mixture was cooled

to ambient temperature, and diluted with EA. Then, sat. aq. NaHCO₃

solution was added and the layers separated. The water layers was

extracted twice more with EA. Organic layers were combined, dried

over Na₂SO₄, ﬁltered and evaporated. The residue was puriﬁed by

column chromatography 10 / 15% EA/PE to give 68 (0.33 g,

0.56 mmol) as a yellowish oil in 36% yield. ¹H NMR (300 MHz,

CDCl₃): 4.65e4.88 (m, 3H, H5⁰, H5⁰⁰, H4⁰), 5.39 (dd, J ¼ 50.1, 1.8 Hz,

1H, H3⁰), 5.75 (dd, J ¼ 14.4, 1.8 Hz, 1H, H2⁰), 6.78 (d, J ¼ 1.8 Hz, 1H,

H1’), 7.43e7.52 (m, 4H, OBz_meta), 7.60 (s, 1H, H8), 7.56e7.68 (m, 2H,

OBz_para), 8.03e8.10 (m, 4H, OBz_ortho), 8.63 (s, 1H, C2). ¹⁹F NMR

(282 MHz, CDCl₃): ꢁ201.21 (ddd, J ¼ 50.5, 31.3, 15.8 Hz, 1F). HRMS

(ESI): calculated for C₂₅H₁₉BrClFN₃O₅([MþH]^þ): 574.0175, found:

574.0148.

1-O-Methyl-2,5-di-O-benzoyl-3-deoxy-3-ﬂuoro-a/b-D-xylo-

furanose (66)

Compound 65 (0.29 g, 0.98 mmol, 1 eq.) was dissolved in MeOH

(10.5 mL, 10.8 mL/mmol). Next, concentrated HCl (2.6 mL, 2.7 mL/

mmol) was added. The resulting mixture was stirred at 55 ^ꢀC for

~30 min, after which TLC analysis showed near full conversion of

the starting material. After cooling in an ice bath, Et₃N (4.2 mL,

4.3 mL/mmol) was added until pH~7. Next, the mixture was evap-

orated till dryness. The residue was partitioned between EA/water.

The water layer was extracted twice more with EA. Organic layers

were combined, dried over Na₂SO₄, ﬁltered and evaporated till

dryness. The residue was dissolved in DCM (3.9 mL, 4.0 mL/mmol)

and cooled in an ice bath. Then, Et₃N (0.25 mL, 1.8 mmol, 1.8 eq.)

and a catalytic amount of DMAP were added. Next, BzCl (0.17 mL,

1.5 mmol, 1.5 eq.) was added and the resulting mixture stirred at

ambient temperature overnight. Then, water was added, and the

mixture transferred to a separatory funnel. Layers were separated

and the water layer extracted twice more with DCM. Organic layers

were combined, dried over Na₂SO₄and evaporated. The residue

was puriﬁed by column chromatography to give 66 (0.23 g,

0.61 mmol) as a clear oil in 63% yield. ¹H NMR analysis showed a

4-Azido-5-bromo-N7-(2′,5′-Di-O-benzoyl-3′-deoxy-3′-ﬂu-

oro-b-D-xylofuranosyl)pyrrolo [2,3-d]pyrimidine (69)

Compound 68 (0.33 g, 0.57 mmol, 1 eq.) was dissolved in DMF

(5.7 mL, 10 mL/mmol), after which NaN₃(0.074 g, 1.1 mmol, 2 eq.)

was added. The mixture was heated to 65 ^ꢀC for ~30 min, and

subsequently cooled to ambient temperature. Then, EA was added,

followed by half-saturated aq. NaHCO₃solution. The layers were

separated, and the water layer extracted twice more with EA.

Organic layers were combined, dried over Na₂SO₄, ﬁltered and

evaporated till dryness. The residue was puriﬁed by column chro-

matography 10% / 30% EA/PE to give 69 (0.19 g, 0.33 mmol) as a

white-yellow foam in 58% yield. ¹H NMR (300 MHz, DMSO‑d₆):

4.68e4.82 (m, 2H, H5⁰, H5⁰⁰), 4.82e4.97 (m, 1H, H4⁰), 5.77 (dd,

J ¼ 50.4, 3.3 Hz, 1H, H3⁰), 5.92 (dd, J ¼ 16.2, 2.7 Hz, 1H, H2⁰), 6.80 (d,

J ¼ 2.7 Hz, 1H, H1⁰), 7.50e7.58 (m, 4H, OBz_meta), 7.64e7.74 (m, 2H,

OBz_para), 7.98e8.08 (m, 4H, OBz_ortho), 8.00 (s, 1H, H6), 9.98 (s, 1H,

H2). ¹⁹F NMR (282 MHz, DMSO‑d₆): ꢁ201.17 (ddd, J ¼ 58.9, 30.2,

16.9 Hz, 1F). ¹³C NMR (75 MHz, DMSO‑d₆): 61.4 (d, J ¼ 10.4 Hz, 1C,

C5⁰), 79.1 (d, J ¼ 6.9 Hz, 1C, C4⁰), 80.2 (d, J ¼ 30.9 Hz, 1C, C2⁰), 87.5

(C1⁰), 90.1 (C5), 93.8 (d, J ¼ 184.3 Hz, 1C, C3’), 103.5 (C4a), 124.7 (d,

J ¼ 5.8 Hz,1C, C6),128.3 (OBz),128.8 (OBz),129.3 (OBz),129.8 (OBz),

133.6 (OBz), 134.1 (OBz), 135.2 (C2), 140.5 (C7a), 145.4 (C4), 164.4

(C]O), 165.4 (C]O). HRMS (ESI): calculated for C₂₅H₁₉BrFN₆O₅

([MþH]^þ): 581.0579, found: 581.0599.

ratio of

corresponding to H1. ¹H NMR (300 MHz, CDCl₃): 3.38 (s, 3H, CH₃-

3.49 (s, 3H, CH₃- ), 5.51e4.84 (m, 6H, H5- , H4- , H5-

, H5⁰- , H5⁰-

, H4- ), 5.13 (s, 1H, H1- ), 5.37

), 5.26 (dd, J ¼ 50.7, 4.5 Hz, 1H, H3-

(d, J ¼ 4.5 Hz, 1H, H1- ), 5.38 (ddd, J ¼ 26.4, 4.5, 3.6 Hz, 1H, H2- ),

5.53 (ddd, J ¼ 54.0, 4.8, 3.6 Hz, 1H, H3- ), 7.42e7.49 (m, 8H,

OBz_meta, OBz_meta), 7.55e7.64 (m, 4H, OBz_para, OBz_para),

8.01e8.11 (m, 8H, OBz_ortho, OBz_ortho

). ¹⁹F NMR (282 MHz,

a

:b

anomers of 1:1.7, based on the integration of the signal

a),

b

a

b

a

-a

-b

-

a

-b

-a

-b

CDCl₃): ꢁ201.03 to ꢁ202.55 (m, 1F).

1-O-Acetyl-2,5-di-O-benzoyl-3-deoxy-3-ﬂuoro-a/b-D-xylo-

furanose (67)

Compound 66 (0.73 g, 2.0 mmol, 1 eq.) was dissolved in glacial

acetic acid (6.6 mL, 3.4 mL/mmol). Next, Ac₂O (0.81 mL, 4.4 eq.) was

added and the mixture placed in an ice bath. Then, concentrated

H₂SO₄(0.40 mL, 3.8 eq.) was added dropwise. After complete

addition, the ice bath was removed and the mixture stirred at

ambient temperature for ~30min, after which TLC analysis (15% EA/

PE) showed complete conversion of the starting material. Then,

DCM was added, followed by sat. aq. NaHCO₃solution until pH~7.

Then, the layers were separated and the organic layer isolated and

dried over Na₂SO₄, ﬁltered and evaporated till dryness. The residue

was puriﬁed by column chromatography 0 / 15% EA/PE to give 67

(0.71 g, 1.8 mmol) as a clear oil in 91% yield. ¹H NMR analysis

4-Amino-5-bromo-N7-(2′,5′-Di-O-benzoyl-3′-deoxy-3′-ﬂu-

oro-b-D-xylofuranosyl)pyrrolo [2,3-d]pyrimidine (70)

Compound 69 (0.19 g, 0.33 mmol, 1 eq.) was dissolved in THF

(3.3 mL, 10.0 mL/mmol). Then, PMe₃solution (1 M in THF, 0.66 mL,

2 eq.) was added and the resulting mixture stirred at ambient

temperature for ~1h. Then, the mixture was evaporated till dryness

and the residue re-dissolved in MeCN (3.3 mL, 10.0 mL/mmol).

Next, 1 M aq. AcOH solution (1.10 mL, 3.33 eq.) was added. The

mixture was heated at 65 ^ꢀC for 1h, and subsequently cooled to

ambient temperature. Then, DCM and aq. sat. NaHCO₃were added

and the layers separated. The water layer was extracted twice more

with DCM. Organic layers were combined, dried over Na₂SO₄,

ﬁltered and evaporated till dryness. The residue was puriﬁed by

column chromatography 40% / 80% EA/PE to give 70 (0.15 g,

0.26 mmol) as a white foam in 80% yield. ¹H NMR (300 MHz, CDCl₃):

4.63e4.86 (m, 3H, H5⁰, H5⁰⁰, H4⁰), 5.37 (dd, J ¼ 49.8, 1.8 Hz, 1H, H3⁰),

5.72 (dd, J ¼ 15.0, 1.8 Hz, 1H, H2⁰), 6.68 (d, J ¼ 1.8 Hz, 1H, H1’), 6.84

(br. s, 2H, NH₂), 7.38 (s, 1H, H6), 7.43e7.51 (m, 4H, OBz_meta),

7.56e7.67 (m, 2H, OBz_para), 8.03e8.08 (m, 4H, OBz_ortho), 8.22 (s, 1H,

showed a ratio of 1:1.6

a

/

b

anomers (based on the integration of the

-anomer): 2.14 (s, 3H,

signal for H1). ¹H NMR (300 MHz, CDCl₃,

a

CH₃), 4.58e4.88 (m, 3H, H4, H5, H5⁰), 5.29 (dd, J ¼ 50.7, 4.2 Hz, 1H,

H3), 5.63 (d, J ¼ 11.7 Hz, 1H, H2), 6.38 (s, 1H, H1), 7.42e7.50 (m, 4H,

OBz_meta), 7.55e7.65 (m, 2H, OBz_para), 8.01e8.10 (m, 4H, OBz_ortho). ¹⁹

F

NMR (282 MHz, CDCl₃,

a

-anomer): ꢁ203.23 (ddd, J ¼ 50.5, 26.5,

11.8 Hz, 1F). ¹H NMR (300 MHz, CDCl₃,

b

-anomer): 1.99 (s, 3H, CH₃),

4.51e4.82 (m, 3H, H4, H5, H5’), 5.49 (ddd, J ¼ 53.1, 4.8, 3.0 Hz, 1H,

H3), 5.68 (ddd, J ¼ 22.8, 5.1, 3.3 Hz, 1H, H2), 6.67 (d, J ¼ 4.5 Hz, 1H,

H1), 7.44e7.50 (m, 4H, OBz_meta), 7.56e7.65 (m, 2H, OBz_para),

8.01e8.11 (m, 4H, OBz_ortho). ¹⁹F NMR (282 MHz, CDCl₃,

b-

anomer): ꢁ202.74 (dt, J ¼ 53.7, 22.8 Hz, 1F). HRMS (ESI): calculated

for C₁₉H₁₆FO₅([M ꢁ OAc]^þ): 343.0876, found: 343.0878.

4-Chloro-5-bromo-N7-(2′,5′-Di-O-benzoyl-3′-deoxy-3′-ﬂu-

oro-b-D-xylofuranosyl)pyrrolo [2,3-d]pyrimidine (68)

4-Chloro-5-bromo-pyrrolo [2,3-d]pyrimidine (0.37 g, 1.6 mmol,

18

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

H2). ¹⁹F NMR (282 MHz, CDCl₃): ꢁ201.33 to ꢁ200.98 (m, 1F). HRMS

(ESI): calculated for C₂₅H₂₁BrFN₄O₅([MþH]^þ): 555.0674, found:

555.0682.

88.1 (d, J ¼ 2.3 Hz, C1⁰), 95.9 (d, J ¼ 182.0 Hz, C3’), 99.9 (C4a), 115.8

(C5), 120.2 (d, J ¼ 5.8 Hz, C6), 128.9 (2C, Cphenyl), 130.4 (2C,

Cphenyl), 132.6 (Cphenyl), 133.0 (Cphenyl), 151.0 (C7a), 152.1 (C2),

157.3 (C4). HRMS (ESI): calculated for C₁₇H₁₇ClFN₄O₃([MþH]^þ):

379.0968, found: 379.0984.

4-Amino-5-bromo-N7-(3′-deoxy-3′-ﬂuoro-

anosyl)pyrrolo [2,3-d]pyrimidine (72)

b-D-xylofur-

Compound 70 (0.15 g, 0.26 mmol,1 eq.) was suspended in MeOH

(15 mL, 57 mL/mmol) and NaOMe in MeOH (5.4 M, 0.50 mL) was

added. The mixture was stirred at ambient temperature for ~30min,

after which TLC analysis (10% MeOH/DCM) showed full conversion.

The mixture was neutralized by the addition of aq. 1 M HCl solution

until pH~7, and evaporated till dryness. The residue was puriﬁed by

column chromatography 1% / 10% MeOH/DCM to give 72 (0.064 g,

0.18 mmol) as a white solid in 70% yield. Melting point: 232.4 ^ꢀC. ¹H

NMR (300 MHz, DMSO‑d₆): 3.60e3.76 (m, 2H, H5⁰, H5⁰⁰), 4.21

(dddd, J ¼ 28.2, 9.0, 5.7, 3.3 Hz,1H, H4⁰), 4.47e4.55 (m,1H, H2⁰), 4.99

(t, J ¼ 5.7 Hz, 1H, OH5⁰), 5.03 (ddd, J ¼ 52.2, 3.3, 1.5 Hz, 1H, H3⁰), 6.11

(d, J ¼ 3.0 Hz, 1H, H1⁰), 6.17 (d, J ¼ 4.8 Hz, 1H, OH2⁰), 6.81 (br. s, 2H,

NH₂), 7.27 (s, 1H, H6), 8.12 (s, 1H, H2). ¹⁹F NMR (282 MHz,

DMSO‑d₆): ꢁ200.70 (ddd, J ¼ 51.9, 27.6, 16.9 Hz, 1F). ¹³C NMR

(75 MHz, DMSO‑d₆): 58.1 (d, J ¼ 9.2 Hz, C5⁰), 78.1 (d, J ¼ 26.4 Hz,

C2⁰), 81.3 (d, J ¼ 19.4 Hz, C4⁰), 87.3 (C5), 88.1 (d, J ¼ 2.3 Hz, C1⁰), 95.7

(d, J ¼ 182.0 Hz, C3’), 100.7 (C4a), 120.8 (d, J ¼ 6.8 Hz, C6), 149.5

(C7a), 152.7 (C2), 156.9 (C4). HRMS (ESI): calculated for

4-Oxo-5-bromo-N7-(3′-deoxy-3′-ﬂuoro-b-D-xylofuranosyl)

pyrrolo[2,3-d]pyrimidine (73) Compound 72 (0.070 g, 0.20 mmol,

1 eq.) is suspended in water (4.0 mL). Next, AcOH (0.25 mL, 1.3 mL/

mmol) was added, and the resulting mixture heated to 50 ^ꢀC. Then,

NaNO₂(0.10 g, 1.5 mmol, 7.2 eq.) was added and the heating

increased to 70 ^ꢀC. Monitoring of the reaction by analytical LC/MS

showed full conversion of the starting material after ~4h. Then, the

mixture was allowed to cool to ambient temperature and concen-

trated in vacuo. Next, MeOH and Celite® were added, and evapo-

rated. The residue was puriﬁed by column chromatography

0 / 12% MeOH/DCM to give 73 as a slight pink powder (0.046 g,

0.13 mmol) in 66% yield. Melting point: 275.3 ^ꢀC. ¹H NMR

(300 MHz, DMSO‑d₆): 3.62e3.78 (m, 2H, H5⁰, H5⁰⁰), 4.20e4.28 (br. s,

1H, H4⁰), 4.49e4.53 (m, 1H, H2⁰), 4.99e5.11 (br. s, 2H, OH5⁰, H3⁰),

6.06 (d, J ¼ 2.4 Hz,1H, H1⁰), 6.22 (d, J ¼ 4.8 Hz, OH2⁰), 7.18 (s,1H, H6),

7.98 (s, 1H, H2), 12.16 (br. s, 1H, NH). ¹⁹F NMR (376 MHz,

DMSO‑d₆): ꢁ200.63 (ddd, J ¼ 50.5, 27.6, 15.8 Hz, 1F). ¹³C NMR

(100 MHz, DMSO‑d₆): 58.1 (d, J ¼ 10.3 Hz, 1C, C5⁰), 78.3 (d,

J ¼ 27.1 Hz, 1C, C2⁰), 81.5 (d, J ¼ 19.9 Hz, 1C, C4⁰), 88.6 (d, J ¼ 1.7 Hz,

1C, C1⁰), 90.8 (H-5), 95.6 (d, J ¼ 183.0 Hz,1C, C3⁰), 106.0 (C-4a), 120.0

(d, J ¼ 6.9 Hz, 1C, C-6), 145.3 (C-7a), 146.9 (C-2), 157.2 (C-2). HRMS

(ESI): calculated for C₁₁H₁₂BrFN₃O₄([MþH]^þ): 347.9990, found:

348.0006.

C

₁₁H₁₃BrFN₄O₃([MþH]^þ): 347.0150, found: 347.0170.

4-Amino-N7-(3′-deoxy-3′-ﬂuoro-b-D-xylofuranosyl)pyrrolo

[2,3-d]pyrimidine (71)

Compound 70 (0.11 g, 0.21 mmol, 1 eq.) was dissolved in EtOH

(8.0 mL, 38 mL/mmol), and 3 M aq. NaOAc solution (0.50 mL) was

added. Then, the ﬂask was purged with N₂, and Pd/C added. The N₂

atmosphere was exchanged for H₂(balloon) and the mixture stirred

at ambient temperature until full conversion of the starting mate-

rial was noted. Then, the ﬂask was purged with N₂, the mixture

ﬁltered over Celite®. The ﬁltrate was evaporated till dryness and

the residue partitioned between EA/sat. aq. NaHCO₃solution. The

water layer was extracted twice more with EA. Organic layers were

combined, dried over Na₂SO₄, ﬁltered and evaporated. The residue

was dissolved in MeOH (8.0 mL) and NaOMe/MeOH solution (5.4 M,

0.50 mL) was added. After complete deprotection was observed,

1 M aq. HCl was added until pH~7. The mixture was evaporated and

the residue puriﬁed by column chromatography 1% / 10% MeOH/

DCM to give 71 (0.035 g, 0.13 mmol) as a white solid in 62% yield.

Melting point: 222.7 ^ꢀC. ¹H NMR (300 MHz, DMSO‑d₆): 3.60e3.77

(m, 2H, H5⁰, H5⁰⁰), 4.20 (dddd, J ¼ 28.8, 9.0, 5.7, 3.3 Hz, 1H, H4⁰),

4.44e4.53 (m, 1H, H2⁰), 5.01 (t, J ¼ 5.7 Hz, 1H, OH5⁰), 5.03 (ddd,

J ¼ 51.9, 3.0, 1.2 Hz, 1H, H3⁰), 6.12 (d, J ¼ 3.0 Hz, 1H, H1⁰), 6.15 (d,

J ¼ 4.5 Hz, 1H, OH2⁰), 6.62 (d, J ¼ 3.9 Hz, 1H, H5), 7.05 (br. s, 2H,

NH₂), 7.10 (d, J ¼ 3.9 Hz,1H, H6), 8.08 (s,1H, H2). ¹⁹F NMR (282 MHz,

DMSO‑d₆): ꢁ200.43 (ddd, J ¼ 52.7, 28.8, 16.6 Hz, 1F). ¹³C NMR

(75 MHz, DMSO‑d₆): 58.1 (d, J ¼ 10.4 Hz, C5⁰), 78.5 (d, J ¼ 27.5 Hz,

C2⁰), 81.0 (d, J ¼ 19.4 Hz, C4⁰), 88.3 (d, J ¼ 2.3 Hz, C1⁰), 96.0 (d,

J ¼ 182.0 Hz, C3’), 100.4 (C4a), 102.4 (C5), 120.9 (d, J ¼ 5.7 Hz, C6),

150.1 (C7a), 152.0 (C2), 157.5 (C4). HRMS (ESI): calculated for

4-Amino-5-(3,4-dichlorophenyl)-N7-(3′-deoxy-3′-ﬂuoro-

-xylofuranosyl)pyrrolo[2,3-d]pyrimidine (76).

b-

D

76 was prepared according to general procedure E. 72 (0.10 g,

0.29 mmol, 1eq.) gave rise to 76 (0.038 g, 0.091 mmol) as a silver

coloured solid in 32% yield. Melting point: 233.7 ^ꢀC. ¹H NMR

(300 MHz, DMSO‑d₆): 3.62e3.78 (m, 2H, H5⁰, H5⁰⁰), 4.17e4.32 (m,

1H, H4⁰), 4.63 (d, J ¼ 16.8 Hz, 1H, H2⁰), 4.99 (d, J ¼ 5.4 Hz, 1H, OH5⁰),

5.08 (dd, J ¼ 43.2,1.5 Hz,1H, H3⁰), 6.19e6.20 (m, 2H, H1⁰, OH2⁰), 6.38

(br. s, 2H, NH₂), 7.35 (s, 1H, H6), 7.43 (dd, J ¼ 8.4, 2.1 Hz, 1H, phenyl-

H6), 7.70 (d, J ¼ 8.1 Hz, phenyl-H5), 7.70 (d, J ¼ 1.8 Hz, phenyl-H2),

8.19 (s, 1H, H2). ¹⁹F NMR (282 MHz, DMSO‑d₆): ꢁ200.37 (ddd,

J ¼ 51.6, 27.6, 16.9 Hz, 1F). ¹³C NMR (75 MHz, DMSO‑d₆): 58.1 (d,

J ¼ 10.4 Hz, C5⁰), 78.2 (d, J ¼ 26.3 Hz, C2⁰), 81.1 (d, J ¼ 19.4 Hz, C4⁰),

88.1 (d, J ¼ 2.3 Hz, C1⁰), 96.0 (d, J ¼ 182.0 Hz, C3’), 99.7 (C4a), 114.7

(C5),120.9 (d, J ¼ 6.8 Hz, C6),128.5 (Cphenyl),129.3 (Cphenyl),130.0

(Cphenyl), 130.8 (Cphenyl), 131.4 (Cphenyl), 134.9 (Cphenyl), 151.1

(C7a), 152.2 (C2), 157.4 (C4). HRMS (ESI): calculated for

C

₁₇H₁₆Cl₂FN₄O₃([MþH]^þ): 413.0578, found: 413.0582.

4-Amino-5-(3-ﬂuoro-4-chlorophenyl)-N7-(3′-deoxy-3′-ﬂu-

oro-b-D-xylofuranosyl)pyrrolo[2,3-d]pyrimidine (77).

Compound 77 was prepared according to general procedure E.

72 (0.10 g, 0.29 mmol, 1eq.) gave rise to 77 (0.079 g, 0.20 mmol) as a

silver coloured solid in 69% yield. Melting point: 226.8 ^ꢀC. ¹H NMR

(300 MHz, DMSO‑d₆): 3.62e3.78 (m, 2H, H5⁰, H5⁰⁰), 4.17e4.32 (m,

1H, H4⁰), 4.63 (d, J ¼ 17.1 Hz, 1H, H2⁰), 4.97e5.16 (m, 2H, OH5⁰, H3⁰),

6.20 (d, J ¼ 3.0 Hz, 1H, H1⁰), 6.20 (d, J ¼ 5.3 Hz, 1H, OH2⁰), 6.38 (br. s,

2H, NH₂), 7.31 (dd, J ¼ 8.4, 1.5 Hz, 1H, HPhenyl), 7.33 (s, 1H, H6), 7.47

(dd, J ¼ 10.8, 2.1 Hz, 1H, HPhenyl), 7.65 (t, J ¼ 8.1 Hz, 1H, HPhenyl),

8.19 (s,1H, H2). ¹⁹F NMR (282 MHz, DMSO‑d₆): ꢁ115.83 (dd, J ¼ 10.7,

8.5 Hz, 1F, phenyl), ꢁ200.40 (ddd, J ¼ 51.6, 27.6, 16.9 Hz, 1F, FeC3⁰).

¹³C NMR (75 MHz, DMSO‑d₆): 58.1 (d, J ¼ 10.3 Hz, C5⁰), 78.2 (d,

J ¼ 26.3 Hz, C2⁰), 81.2 (d, J ¼ 19.4 Hz, C4⁰), 88.1 (d, J ¼ 2.3 Hz, C1⁰),

96.1 (d, J ¼ 182.0 Hz, C3’), 99.7 (C4a), 115.0 (C5), 116.7 (d, J ¼ 21.8 Hz,

Cphenyl), 117.7 (d, J ¼ 17.2 Hz, Cphenyl), 120.9 (d, J ¼ 5.7 Hz, C6),

125.5 (d, J ¼ 2.3 Hz, Cphenyl), 130.9 (Cphenyl), 135.8 (d, J ¼ 8.0 Hz,

Cphenyl), 151.1 (C7a), 152.2 (C2), 157.3 (d, J ¼ 245.0 Hz, Cphenyl),

157.3 (C4). HRMS (ESI): calculated for C₁₇H₁₆ClF₂N₄O₃([MþH]^þ):

C

₁₁H₁₄FN₄O₃([MþH]^þ): 269.1045, found: 269.1052.

4-Amino-5-(4-chlorophenyl)-N7-(3′-deoxy-3′-ﬂuoro-b-D-

xylofuranosyl)pyrrolo [2,3-d]pyrimidine (74)

74 was prepared according to general procedure E. 72 (0.10 g,

0.29 mmol, 1 eq.) gave rise to 74 (0.072 g, 0.19 mmol) as a silver

powder in 65% yield. Melting point: 229.7 ^ꢀC. ¹H NMR (300 MHz,

DMSO‑d₆): 3.61e3.78 (m, 2H, H5⁰, H5⁰⁰), 4.24 (dddd, J ¼ 28.2, 9.3,

6.0, 3.0 Hz, 1H, H4⁰), 4.61 (m, 1H, H2⁰), 5.00 (t, J ¼ 5.4 Hz, 1H, OH5⁰),

4.97e5.16 (m, 1H, H3⁰), 6.19 (d, J ¼ 5.1 Hz, 1H, OH2⁰), 6.21 (d,

J ¼ 2.4 Hz,1H, H1⁰), 6.24 (br. s, 2H, NH₂), 7.25 (s,1H, H6), 7.50 (m, 4H,

phenyl), 8.19 (s, 1H, H2). ¹⁹F NMR (282 MHz, DMSO‑d₆): ꢁ200.49

(ddd, J ¼ 51.6, 27.6, 16.6 Hz, 1F). ¹³C NMR (75 MHz, DMSO‑d₆): 58.2

(d, J ¼ 9.2 Hz, C5⁰), 78.2 (d, J ¼ 27.5 Hz, C2⁰), 81.1 (d, J ¼ 19.5 Hz, C4⁰),

19

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

397.0874, found: 397.0872.

4-Amino-5-(4-ﬂuorophenyl)-N7-(3′-deoxy-3′-ﬂuoro-

xylofuranosyl)pyrrolo[2,3-d]pyrimidine (75).

100 mL PBS. The change in color was measured spectrophotomet-

rically at 540 nm after 4 h incubation at 37 ^ꢀC. The results were

expressed as % reduction in parasite burdens compared to control

wells and an IC₅₀value was calculated.

b-D-

Compound 75 was prepared according to general procedure E.

72 (0.10 g, 0.29 mmol,1 eq.) gave rise to 75 (0.083 g, 0.23 mmol) as a

silver coloured solid in 79% yield. Melting point 228.4 ^ꢀC. ¹H NMR

(300 MHz, DMSO‑d₆): 3.61e3.78 (m, 2H, H5⁰, H5⁰⁰), 4.17e4.31 (m,

1H, H4⁰), 4.56e5.65 (m, 1H, H2⁰), 4.97e5.16 (m, 2H, OH5⁰, H3⁰), 6.19

(d, J ¼ 3.9 Hz, 1H, OH2⁰), 6.20 (br. s, 2H, NH₂), 6.21 (d, J ¼ 3.0 Hz, 1H,

H1⁰), 7.21 (s,1H, H6), 7.30 (tt, J ¼ 9.0, 2.1 Hz, 2H, Hphenyl), 7.46e7.53

(m, 2H, Hphenyl), 8.18 (s, 1H, H2). ¹⁹F NMR (282 MHz,

DMSO‑d₆): ꢁ115.80 to ꢁ115.09 (m, 1F, F-phenyl), ꢁ200.49 (ddd,

J ¼ 52.7, 28.8, 16.6 Hz, 1F, FeC3⁰). ¹³C NMR (75 MHz, DMSO‑d₆): 58.1

(d, J ¼ 9.2 Hz, C5⁰), 78.2 (d, J ¼ 26.3 Hz, C2⁰), 81.1 (d, J ¼ 19.4 Hz, C4⁰),

88.1 (d, J ¼ 2.3 Hz, C1⁰), 95.9 (d, J ¼ 182.0 Hz, C3’), 100.1 (C4a), 115.6

(C5), 115.9 (Cphenyl), 120.4 (d, J ¼ 5.6 Hz, C5), 130.3 (d, J ¼ 8.0 Hz,

2C, phenyl), 130.5 (d, J ¼ 2.3 Hz, 2C, Cphenyl), 150.8 (C7a), 152.1

(C2), 157.3 (C4), 161.5 (d, J ¼ 241.5 Hz, Cphenyl). HRMS (ESI):

calculated for voor C₁₇H₁₇F₂N₄O₃([MþH]^þ): 363.1263, found:

363.1275.

4.10. MRC-5 cytotoxicity

MRC-5_SV2cells were cultured in MEM þ Earl’s salts-medium,

supplemented with L-glutamine, NaHCO₃and 5% inactivated fetal

calf serum. All cultures and assays were conducted at 37 ^ꢀC under

an atmosphere of 5% CO₂. Assays were performed in sterile 96-well

microtiter plates, each well containing 10

mL of the watery com-

pound dilutions together with 190

mL of MRC-5_SV2inoculum

(1.5 ꢂ 10⁵cells/mL). Cell growth was compared to untreated-

control wells (100% cell growth) and medium-control wells (0%

cell growth). After 3 days incubation, cell viability was assessed

ﬂuorimetrically after addition of 50 mL resazurin per well. After 4 h

at 37 ^ꢀC, ﬂuorescence was measured (l_ex550 nm, l_em590 nm). The

results were expressed as % reduction in cell growth/viability

compared to control wells and an IC₅₀value was determined.

4-Amino-5-(3-methyl-4-chlorophenyl)-N7-(3′-deoxy-3′-ﬂu-

oro-b-D-xylofuranosyl)pyrrolo[2,3-d]pyrimidine (78).

Compound 78 was prepared according to general procedure E.

72 (0.10 g, 0.29 mmol,1 eq.) gave rise to 78 (0.094 g, 0.24 mmol) as a

silver coloured powder in 83% yield. Melting point: 141.2 ^ꢀC. ¹H

NMR (300 MHz, DMSO‑d₆): 2.39 (s, 3H, phenyl-CH₃), 3.61e3.78 (m,

2H, H5⁰, H5⁰⁰), 4.17e4.31 (m, 1H, H4⁰), 4.58e4.64 (m, 1H, H2⁰),

4.97e5.16 (m, 2H, OH5⁰, H3⁰), 6.19 (d, J ¼ 3.9 Hz, 1H, OH2⁰), 6.20 (br.

s, 2H, NH₂), 6.21 (d, J ¼ 3.0 Hz, 1H, H1⁰), 7.23 (s, 1H, H6), 7.29 (dd,

J ¼ 8.1, 2.4 Hz, 1H, phenyl-H6), 7.45 (d, J ¼ 1.8 Hz, 1H, phenyl-H2),

7.50 (d, J ¼ 8.1 Hz, phenyl-H5), 8.18 (s, 1H, H-2). ¹⁹F NMR

4.11. Leishmania infantum

L. infantum [MHOM/MA (BE)/67] was used. This strain was

maintained in the Golden Hamster (Mesocricetus auratus). Amas-

tigotes were collected from the spleen of an infected donor hamster

using three centrifugation puriﬁcation steps (300 rpm, keeping the

supernatans, 2200 rpm, keeping the supernatans and 3500 rpm,

keeping the pellet) and spleen parasite burdens were assessed

using the Stauber technique. Primary peritoneal mouse macro-

phages were used as host cell and were collected 2 days after

peritoneal stimulation with a 2% potato starch suspension. All

cultures and assays were conducted at 37 ^ꢀC under an atmosphere

of 5% CO₂. Assays were performed in 96-well microtiter plates, each

(282 MHz, DMSO‑d₆): ꢁ200.42 (ddd, J ¼ 52.2, 28.8, 16.9 Hz, 1F). ¹³

C

NMR (75 MHz, DMSO‑d₆): 19.7 (CH₃), 58.2 (d, J ¼ 10.4 Hz, C5⁰), 78.2

(d, J ¼ 26.3 Hz, C2⁰), 78.3 (d, J ¼ 26.7 Hz, 1H, H4⁰), 88.1 (d, J ¼ 2.6 Hz,

C1⁰), 95.9 (d, J ¼ 182.0 Hz, C3’), 99.9 (C4a), 115.9 (C5), 120.1 (d,

J ¼ 5.7 Hz, C6), 127.5 (Cphenyl), 129.3 (Cphenyl), 131.1 (Cphenyl),

132.0 (Cphenyl), 133.1 (Cphenyl), 136.0 (Cphenyl), 150.9 (C7a), 152.1

well containing 10

mL of the compound dilutions together with

190

m

L of macrophage/parasite inoculum (3 ꢂ 10⁴cells þ 4.5 ꢂ 10⁵

(C2), 157.3 (C4). HRMS (ESI): calculated for

C

₁₈H₁₉ClFN₄O₃

parasites/well). The inoculum was prepared in RPMI-1640 medium,

([MþH]^þ): 393.1124, found: 393.1127.

supplemented with 200 mM L-glutamine, 16.5 mM NaHCO₃, and 5%

inactivated fetal calf serum. The macrophages were infected after

48 h. The compounds were added after 2 h of infection. Parasite

multiplication was compared to untreated-infected controls (100%

growth) and uninfected controls (0% growth). After 5 days of in-

cubation, parasite burdens (mean number of amastigotes/macro-

phage) were microscopically assessed after staining the cells with a

10% Giemsa solution. The results were expressed as % reduction in

parasite burden compared to untreated control wells and an IC₅₀

value was calculated.

4.8. Biology

Compound stock solutions were prepared in 100% DMSO at

20 mM. The compounds were serially pre-diluted (2-fold or 4-fold)

in DMSO followed by a further (intermediate) dilution in demin-

eralized water to assure a ﬁnal in-test DMSO concentration of <1%.

Compounds were assayed in 10 concentrations of a 4-fold com-

pound dilution series starting at 64 mM.

4.9. Trypanosoma cruzi

4.12. PMM cytotoxicity

Trypanosoma cruzi, Tulahuen CL2, b-galactosidase strain (nifur-

timox-sensitive) was used. The strain was maintained on MRC-5_SV2

(human lung ﬁbroblast) cells in MEM medium, supplemented with

PMM toxicity was assessed during the in vitro susceptibility

assays via microscopic evaluation of cell detachment, lysis, and

granulation. Evaluation was done by semi-quantitative scoring (no

exact counting was performed) of at least 500 cells distributed over

adjacent microscopic ﬁelds. The results were expressed as %

reduction in normal cells compared to untreated control wells and

an CC₅₀value was determined.

200 mM L-glutamine,16.5 mM NaHCO₃, and 5% inactivated fetal calf

serum. All cultures and assays were conducted at 37 ^ꢀC under an

atmosphere of 5% CO₂. Assays were performed in sterile 96-well

microtiter plates, each well containing 10

mL of the watery com-

pound dilutions together with 190

m

L of MRC-5 cell/parasite inoc-

ulum (4 ꢂ 10³cells/well þ 4 ꢂ 10⁴parasites/well). Parasite growth

was compared to untreated-infected controls (100% growth) and

noninfected controls (0% growth) after 7 days incubation at 37 ^ꢀC

and 5% CO₂. Parasite burdens were assessed after adding the sub-

Declaration of competing interest

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.

strate CPRG (chlorophenolred ß-D-galactopyranoside): 50

mL/well

of a stock solution containing 15.2 mg CPRG þ 250

mL Nonidet in

20

J. Bouton, A. Furquim d’Almeida, L. Maes et al.

European Journal of Medicinal Chemistry 216 (2021) 113290

[19] W.E. Gutteridge, M. Gaborak, A Re-examination of purine and pyrimidine

synthesis in the three main forms of trypanosoma cruzi, Int. J. Biochem. 10 (5)

(1979) 415e422, https://doi.org/10.1016/0020-711X(79)90065-X.

[20] J.J. Marr, R.L. Berens, D.J. Nelson, Purine metabolism in Leishmania donovani

and Leishmania braziliensis, Biochim. Biophys. Acta Gen. Subj. 544 (2) (1978)

360e371, https://doi.org/10.1016/0304-4165(78)90104-6.

[21] J.M. Boitz, B. Ullman, A. Jardim, N.S. Carter, Purine salvage in Leishmania:

complex or simple by design? Trends Parasitol. 28 (8) (2012) 345e352,

https://doi.org/10.1016/j.pt.2012.05.005.

Acknowledgements

F.H. is indebted to the Flanders Fund for Scientiﬁc Research

(FWO) for a postdoctoral fellowship (grant number 1226921 N). 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, Natascha Van Pelt, Margot

Vleminckx and Izet Karalic for excellent technical assistance.

[22] M. Berg, P. Van der Veken, A. Goeminne, A. Haemers, K. Augustyns, Inhibitors

of the purine salvage pathway:

a valuable approach for antiprotozoal

chemotherapy? Curr. Med. Chem. 17 (2010) 2456e2481, https://doi.org/

10.2174/092986710791556023.

[23] M.H. El Kouni, Potential chemotherapeutic targets in the purine metabolism

of parasites, Pharmacol. Ther. 99 (3) (2003) 283e309, https://doi.org/10.1016/

S0163-7258(03)00071-8.

Appendix A. Supplementary data

[24] M. Berg, L. Kohl, P. Van Der Veken, J. Joossens, M.I. Al-Salabi, V. Castagna,

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F. Giannese, P. Cos, W. Versees, J. Steyaert, P. Grellier, A. Haemers, M. Degano,

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.1 13290.

L. Maes, H.P. De Koning, K. Augustyns, Evaluation of nucleoside hydrolase

inhibitors for treatment of african Trypanosomiasis, Antimicrob. Agents

Chemother. 54 (5) (2010) 1900e1908, https://doi.org/10.1128/AAC.01787-09.

[25] A.K. Datta, R. Datta, B. Sen, Antiparasitic chemotherapy: in drug Targets in

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[27] F. Hulpia, G.D. Campagnaro, M. Scortichini, K. Van Hecke, L. Maes, H.P. de

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plastid parasites: aromatic substitutions at position 7 improve activity and

reduce toxicity, Eur. J. Med. Chem. 164 (2019) 689e705, https://doi.org/

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Synthesis and evaluation of 3′-fluorinated 7-deazapurine nucleosides as antikinetoplastid agents

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