Revisiting Pyrazolo[3,4- d]pyrimidine Nucleosides as Anti- Trypanosoma cruzi and Antileishmanial Agents

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Revisiting Pyrazolo[3,4‑d]pyrimidine Nucleosides as Anti-

Trypanosoma cruzi and Antileishmanial Agents

Jakob Bouton, Ludmila Ferreira de Almeida Fiuza, Camila Cardoso Santos, Maria Angela Mazzarella,

Maria de Nazar é Correia Soeiro, Louis Maes, Izet Karalic, Guy Caljon,* and Serge Van Calenbergh *

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ABSTRACT: Chagas disease and visceral leishmaniasis are two

neglected tropical diseases responsible for numerous deaths around

the world. For both, current treatments are largely inadequate, resulting

in a continued need for new drug discovery. As both kinetoplastid

parasites are incapable of de novo purine synthesis, they depend on

purine salvage pathways that allow them to acquire and process purines

from the host to meet their demands. Purine nucleoside analogues

therefore constitute a logical source of potential antiparasitic agents.

Earlier optimization eﬀorts of the natural product tubercidin (7-

deazaadenosine) involving modiﬁcations to the nucleobase 7-position

and the ribofuranose 3′-position led to analogues with potent anti-

Trypanosoma brucei and anti-Trypanosoma cruzi activities. In this work,

we report the design and synthesis of pyrazolo[3,4-d]pyrimidine

nucleosides with 3′- and 7-modiﬁcations and assess their potential as anti-Trypanosoma cruzi and antileishmanial agents. One

compound was selected for in vivo evaluation in an acute Chagas disease mouse model.

Leishmania species, the disease exists in two main clinical

forms: visceral leishmaniasis (VL) and cutaneous leishmaniasis

(CL). The most severe systemic VL form is usually fatal within

two years without treatment¹¹and is responsible for up to

30 000 deaths every year. Most antileishmanial drugs have

been repurposed from other indications. Eﬃcacy varies by

geographical region, and treatment courses are long, require

hospitalization, and are associated with signiﬁcant side

eﬀects.^12,13While several new chemical entities with distinct

mechanisms of action have recently entered clinical trials,¹⁴⁻¹⁹

new drug discovery eﬀorts are required to ﬁll the early-stage

pipeline.²⁰New drug candidates should be suitable for ﬁeld

conditions and allow for global use, oral dosing, and a short

treatment course.^7,20

Unlike their mammalian hosts, both Trypanosoma cruzi and

Leishmania spp. are obligate auxotrophs for purines, meaning

that they lack de novo purine biosynthesis and rely on the

salvage of purines (nucleosides and/or nucleobases) to meet

their purine demand.²¹⁻²³Consequently, they have developed

a complex set of purine salvage enzymes that allows them to

INTRODUCTION

■

Chagas disease and leishmaniasis are two vector-borne

communicable diseases responsible for numerous deaths

every year. Characterized by the WHO as neglected tropical

diseases (NTDs), they occur mainly in populations living in

poverty in developing regions around the world and have a

severe impact on the lives of aﬀected persons and their

families.^1,2Chagas disease, caused by Trypanosoma cruzi, is

endemic to Latin America, where it is still one of the most

prevalent public health problems.³Migration and speciﬁc

transmission modes have enabled spreading beyond these

geographical boundaries so that it is now considered a global

issue.⁴Chagas disease starts with an acute symptomatic phase

characterized by high-grade parasitemia, which progresses into

an asymptomatic chronic state after a few weeks. While most

people stay asymptomatic for life, 30−40% will develop severe

clinical manifestations after 10−30 years.^5,6Treatment options

are limited to nifurtimox and benznidazole, two old drugs that

have only limited eﬃcacy in the chronic disease phase and are

associated with severe adverse reactions.⁴At present, no

vaccine is available and eﬀective antiparasitic chemotherapy is

therefore key in eliminating this NTD. As the current Chagas

disease pipeline is almost empty, there is a pressing need to

develop novel, safe, and eﬃcacious treatments.⁷⁻⁹

Received: January 24, 2021

Published: March 30, 2021

Leishmaniasis is endemic in 60 countries with Brazil, India,

Ethiophia, Somalia, Kenya, South Sudan, and Sudan reporting

more than 90% of all cases.¹⁰Depending on the causative

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Figure 1. (a) Structures of pyrazolo[3,4-d]pyrimidines. (b) Antitrypanosomal 7-deazapurine nucleosides previously reported by our group. (c)

Metabolism of allopurinol in Leishmania spp. and T. cruzi. (c, d) Pyrazolo[3,4-d]pyrimidine nucleosides reported in this study. Throughout the text,

purine numbering is used as shown in Figure 1d, while in the experimental part, systematic numbering is employed.

acquire and process purines from their hosts. Purine

(nucleoside) analogues therefore constitute an interesting

pool of potential antiparasitic agents. Indeed, several analogues

have been reported that act as inhibitors of enzymes of the

purine salvage pathway or as “subversive substrates”, which are

selectively activated by salvage enzymes of the invading

parasite.²⁴⁻²⁶

An example of such a subversive substrate is allopurinol (1)

(Figure 1a). Next to its use as a treatment for gout, allopurinol

inhibits the growth of several Leishmania and Trypanosoma

species²⁷in which its mechanism of action has been well-

investigated.^28,29It is selectively metabolized by the parasite to

inosine- and adenosine-like nucleotide derivatives 5 and 6 and

ultimately to an ATP analogue (i.e., APPR-TP) that is

incorporated in RNA (Figure 1c).²⁸⁻³⁰Allopurinol has been

evaluated in humans for VL treatment with mixed results.

Although it achieved full cures in some patients, it was not

satisfactory in monotherapy as ﬁrst-line treatment due to its

static rather than cidal character.³¹Nevertheless, allopurinol is

still used in certain combination regimens for the treatment of

CL,^11,32,33and it is the treatment of choice for VL and CL in

dogs.³⁴Allopurinol has also been investigated for the treatment

of Chagas disease with mixed outcomes.³⁵⁻³⁷For example, it

proved eﬀective in treating reactivation after heart trans-

plantation.^38,39Both in leishmaniasis^33,40−42and in Chagas

disease,⁴³⁻⁴⁵allopurinol has demonstrated synergism with

currently used drugs, demonstrating that a nucleobase/

nucleoside analogue could be a valuable addition to the

therapeutic arsenal. The in vitro antileishmanial activity of the

ribonucleoside of allopurinol (2) is several times higher than

allopurinol, but 2 was not further evaluated in vivo due to

production diﬃculties and limited beneﬁt over allopuri-

nol.⁴⁶⁻⁴⁸The 6-amino congeners aminopurinol 3 and riboside

derivative 4 (Figure 1a) were generally more active in vitro

than allopurinol and 2,^46,49,50which may be due to faster

conversion to the same active triphosphate. However, 3 and 4

suﬀered from cytotoxicity and selectivity concerns. Yet, Avila et

al. found aminopurinol to be eﬀective in animal models of both

VL and Chagas disease at dosages as much as 300-fold lower

than used for allopurinol and well below the toxic dose in

humans.^46,51Despite these promising data, neither amino-

purinol 3 nor its ribonucleoside 4 was further evaluated for the

treatment of leishmaniasis or Chagas disease.

Our group recently reported several 7-deazapurine nucleo-

sides derived from the natural product tubercidin (7) that

display potent activity against Typanosoma brucei and T. cruzi

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a

Scheme 1. Synthesis of Ribofuranose-Modiﬁed Pyrazolo[3,4-d]pyrimidine Nucleosides

a

Reagents and conditions: (a) NBS, DMF, 60 °C, 91% (for 12), NIS, DMF, 80 °C, 93% (for 13); (b) NBS, water, 90 °C, 87%; (c) 12 (for 18−

20), 14 (for 26−28), or 13 (for 34) BF₃·OEt₂, MeNO₂, reﬂux, 77% (34); (d) 0.5 M NaOMe in MeOH, 70% over 2 steps (21), 19% over 2 steps

(22), 20% over 2 steps (23), 38% over 2 steps (29), 11% over 2 steps (30), 6% over 2 steps (31), 64% (36); (e) Pd/C, H₂, 1 M aq. NaOAc,

MeOH, 61% (4), 82% (24), 66% (25), 54% (2), 83% (32), 80% (33).

(Figure 1b).⁵²⁻⁵⁶The introduction of selected substituents on

the 7-position (as in 8, 9) led to selective anti-T. cruzi and anti-

T. brucei agents.⁵²Further deletion of the hydroxyl group on

position 3′ aﬀorded compound 10, which was able to fully cure

mice with CNS-stage sleeping sickness,⁵⁵and 9, which

displayed high potency against T. cruzi.⁵³A ﬂuorine atom in

position 3′ also proved favorable, with 11 displaying high anti-

T. cruzi activity.⁵⁷Compound 9 was evaluated in a Chagas

mouse model but failed to deliver sterile cure. Furthermore,

none of these 7-deazapurine nucleosides displayed selective

activity against the phylogenetically related Leishmania, which

is remarkable given that several nucleoside analogues display

antileishmanial potential,^{47,48,58−61}and many nucleosides

combine antichagasic and antileishmanial activities.^46,48,61

Nevertheless, known antileishmanial nucleoside analogues

(e.g., the pyrazolo[3,4-d]pyrimidines (vide supra), carbocyclic

inosine,⁶²9-deazainosine,⁵⁸Formycin B⁵⁸) contain nucleobase

surrogates other than 7-deazapurine, leading us to assume that

a 7-deazapurine base is detrimental for antileishmanial activity.

In the search for nucleoside analogues with improved

antileismanial activity, we decided to explore nucleosides

featuring a pyrazolo[3,4-d]pyrimidine (8-aza-7-deazapurine)

nucleobase. Next to 7-deazapurines, pyrazolo[3,4-d]-

pyrimidines are the only purine isosteres that allow

derivatization via 7-modiﬁcations,^63,64such as the ones found

beneﬁcial to increase the potency and selectivity of (3′-

deoxy)tubercidin. Compared to tubercidin 1,⁶⁵aminopurinol

riboside 4 is signiﬁcantly less toxic.⁵¹Although several 7-

modiﬁed and 3′-modiﬁed pyrazolo[3,4-d]pyrimidine nucleo-

sides have been reported,⁶⁶⁻⁶⁸they have not been explored for

antiparasitic activity. One exception is 7-bromo-allopurinol

riboside, which proved more potent than allopurinol.⁶⁹

In this study, we describe the synthesis and initial evaluation

of a library of 7-substituted and ribofuranose-modiﬁed

pyrazolo[3,4-d]pyrimidine nucleosides, comprising both in-

osine-like (allopurinol riboside) and adenosine-like (amino-

purinol riboside) analogues. The structure−activity relation-

ships for anti-T. cruzi and antileishmanial activity are discussed,

and one analogue was evaluated in an acute Chagas disease

mouse model.

RESULTS AND DISCUSSION

■

Chemistry. The required brominated and iodinated

nucleobase analogues 12 and 13 were readily obtained from

aminopurinol 3 via reaction with N-bromosuccinimide (NBS)

or N-iodosuccinimide (NIS) in N,N-dimethylformamide

(DMF) at elevated temperature (Scheme 1). While we were

also interested in the chloro and ﬂuoro derivatives,

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Scheme 2. Reagents and Conditions: (a) NBS, DMF, 50 °C; (b) NaOMe, MeOH, 70 °C, 51% Over 2 steps; (c) 38, KOH,

TDA-1, MeCN, 8%; (d) 7 N NH₃in MeOH, 90 °C, 76%; (e) H₂, Pd(OH)₂/C, NaOAc, MeOH, 46%

chlorination of aminopurinol 3 with N-chlorosuccinimide

(NCS) or ﬂuorination with diethylaminosulfur triﬂuoride

(DAST) failed to deliver the desired halogenated heterocycles.

Bromination of allopurinol with bromine in water at 90 °C

aﬀorded 14.

(∼148 ppm) (systematic numbering) were again very similar.

Similarly, in this case, H-1′−C-7 coupling was again absent in

the ¹H-¹³C HMBC spectra, further conﬁrming N-9 attachment

of the heterocycle.

Glycosylation of 15 with 7-iodoaminopurinol aﬀorded 34 in

high yield, and deprotection in methanolic ammonia furnished

36. Both 34 and 36 were used for further modiﬁcations (vide

infra). Compound 4 could also be obtained from direct

glycosylation of 4-aminopyrazolo[3,4-d]pyrimidine, followed

by deprotection.

7-Bromoallopurinol 14 and 7-bromoaminopurinol 12 were

coupled with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-^D-ribofuranose,

its 3′-deoxy counterpart 17,⁵³and 3′-deoxy-3′-ﬂuoro analogue

16.⁵⁷Coupling of these three donors with 7-bromoaminopur-

inol 12 was performed with BF₃·OEt₂in reﬂuxing nitro-

methane, as described for 18.⁶⁹For the 7-bromoaminopurinol

nucleosides 18−20, a single product was obtained and no

regioisomers were observed. Deprotection with sodium

methoxide resulted in 21−23, and reductive dehalogenation

with H₂and Pd/C in buﬀered methanol aﬀorded 4, 24, and

25. Remarkably, the combined glycosylation-deprotection

yields were much lower (∼20%) for the 3′-modiﬁed analogues

compared to that of the ribofuranose 4 (∼70%). The correct

regiochemistry of the ﬁnal compounds was conﬁrmed via

comparison of the ¹³C NMR spectra of 24 and 25 with that of

the literature compound 4. The chemical shifts of C-7 (∼133

ppm) and C-5 (∼154 ppm) (systematic numbering) were

identical to reported values, whereas in the N-8 regioisomer, C-

7 would be shifted upﬁeld about 10 ppm and C-5 shifted

The 2′-deoxy analogues were prepared as described by Seela

et al. (Scheme 2).^64,73−75Compound 38 was obtained

eﬃciently by bromination of 4-chloro-1H-pyrazolo[3,4-d]-

pyrimidine with NBS, followed by nucleophilic aromatic

substitution with sodium methoxide. Anion glycosylation of

38 with commercially available Hoﬀer’s chlorosugar aﬀorded

39 in 8% yield. Simultaneous deprotection and introduction of

the 6-amino group to aﬀord 40 was achieved by overnight

heating in methanolic ammonia. Reductive dehalogenation via

hydrogenation over Pd/C in buﬀered methanol aﬀorded 41.

Attempted conversion of 39 to the corresponding 6-oxo

congener in dilute NaOH solution, as described by Seela for

the 3-unsubstituted analogue,⁷¹resulted in glycosidic bond

breakage.

Further modiﬁcations focused on the introduction of

substituents on the 7-position of 21. Diﬀerent phenyl rings

were introduced via aqueous Suzuki coupling reactions with

the appropriate arylboronic acids to furnish 42−63 (Scheme

3). Reaction with 4-chloro-3-cyano-phenylboronic acid gave

rise to signiﬁcant amounts of biphenyl product 75, which was

also isolated. Except for the 2-pyridyl and 2-thiophene

substituents in 65 and 67, which were introduced via Stille

coupling, other heterocyclic substituents were also introduced

under aqueous Suzuki coupling conditions to furnish 64, 66,

and the substituted 2-thienyl analogues 68−70. The vinyl- and

isopropenyl-compounds 71 and 72 were synthesized via the

Suzuki reaction with the respective potassium triﬂuoroborate

salts, while trans-2-vinylphenylboronic acid was used to obtain

73. The synthesis of 74 required multiple additions of

downﬁeld about 5−6 ppm.^70,71In the H-¹³C HMBC spectra,

1

H-1′−C-7 coupling was absent, providing further evidence of

the desired N-9 regiochemistry.

7-Bromoallopurinol 14 was glycosylated under the same

conditions, but for each donor three diﬀerent products with

the same mass were observed on thin-layer chromatography

(TLC), corresponding to the N-9, N-8, and N-1 regioisomers.

The higher-running, less polar spot was the major product and

was presumed to be the correct regioisomer⁷²and was isolated.

Glycosylation yields were generally lower (∼50%) than for the

corresponding aminopurinols. The correct regiochemistries of

32 and 33 were veriﬁed via comparison of their ¹³C NMR

spectra with that of 2.⁷²Compared to the 6-aminonucleosides,

the upﬁeld shift of C-7 in the N-8 isomer is reported to be less

pronounced,⁷²but the values of C-7 (∼135 ppm) and C-5

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Scheme 3. Reagents and Conditions: (a) Boronic Acid or Triﬂuoroborate Salt, Pd(OAc)₂, TPPTS, Na₂CO₃(in the Case of a

Boronic Acid) or Cs₂CO₃(When a Triﬂuoroborate Salt Was Used), MeCN/H₂O 1:2, 16−81% (For All Compounds Except for

67 and 65); (b) Tributylstannylated Heterocycle, Pd(PPh₃)₄, CuI, DMF, 24% (67)*, 66% (65)*; (c) H₂, Pd(OH)₂/C, MeOH,

81%

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Scheme 4. Reagents and Conditions: (a) Phenol (for 79) or 4-Chlorophenol (for 80), CuI, N,N-Dimethylglycine, Cs₂CO₃,

DMA, 120 °C, 5% (79), 3% (80); (b) CuCl, aq. NH₄OH (for 81) or aq. NHMe (for 82) or Pyrrolidine, 1,4-Dioxane/H₂O 1:2

(for 83), 120 °C, 19% (81), 5% (82), 16% (83); (c) Me₄NCl, Cu₂O, L-Proline, 2-Methoxyethanol, 120 °C, 7 days; (d) 0.5 M

NaOMe in MeOH, 34% Over 2 Steps

cyclopropylboronic acid, since reaction with potassium cyclo-

propyltriﬂuoroborate proved unsuccessful. Likewise, the 3′-

deoxy and 2′-deoxy bromonucleosides 23 and 40 were

subjected to the Suzuki reaction with 4-chlorophenylboronic

acid to aﬀord 76 and 41. Finally, the isopropenyl group of

previously obtained 72 was reduced via catalytic hydrogenation

to aﬀord 78.

analogue 93 via hydrogenation over Raney nickel. Alter-

natively, hydration of the nitrile in basic hydrogen peroxide

solution furnished 94. Attempted hydrolysis of nitrile 91⁶⁶

failed to deliver the carboxylic acid but resulted in cleavage of

the N-glycosidic bond instead. A triﬂuoromethyl substituent

was introduced via a cross-coupling reaction with in situ-

56,78

formed CuCF₃

to aﬀord 95. Deprotection using sodium

Ullman coupling of 21 with phenol or 4-chlorophenol under

conditions described for other pyrazolo[3,4-d]pyrimidines⁷⁶

aﬀorded 79 and 80, respectively (Scheme 4). The yields were

very low (5 and 3%) but provided suﬃcient amounts of

product for preliminary evaluation. The synthesis of 81 via

Ullman coupling in aqueous ammonia has already been

described,⁶⁹and methylamine and pyrrolidine could be

coupled under similar conditions to furnish 82 and 83. To

introduce a 7-chloro, 18 was subjected to a copper-catalyzed

retro-Finkelstein reaction.⁷⁷Although this reaction was slug-

gish, 85 was obtained in decent yield after deprotection.

The 7-iodo analogue 36 served as a useful precursor for

another set of analogues (Scheme 5). The Sonogashira

reaction with phenylacetylene furnished 86, which was further

reduced to 87 via catalytic hydrogenation. The Sonogashira

reaction with ethynyltrimethylsilane yielded 88, which was

either further reduced to 89 or reacted with azidotrimethylsi-

lane in a copper(I)-catalyzed azide-alkyne cycloaddition to 90.

A nitrile substituent was introduced via a palladium-catalyzed

coupling reaction with Zn(CN)₂to furnish 91.⁶⁶The nitrile

group was further transformed to a tetrazole 92 via a 1,3-

dipolar cycloaddition reaction or reduced to the aminomethyl

methoxide then furnished 96.

To gain access to the methyl-substituted base 98, 5-amino-4-

cyano-3-methyl-1H-pyrazole 97 was synthesized de novo from

malonitrile and acetyl chloride according to the method of

Haneman (Scheme 6).⁷⁹Ring closure of 97 with thioaceta-

mide instead of formamide furnished the bismethylated

heterocycle 99. Glycosylation of 98 aﬀorded 100, which was

deprotected to 101. Unfortunately, attempted glycosylation of

99 under the same conditions was not successful.

Since a carboxylic acid could not be obtained from the cyano

analogue 91, we looked at other methods to introduce a

carbonyl group on the 7-position. Vilsmeier−Haack formyla-

tion of 35 failed and only led to N-formylation of the 6-amino

group. Palladium-catalyzed carbonylation reactions of 34 with

diﬀerent CO equivalents were also unsuccessful.⁸⁰⁻⁸²Finally,

we tried to convert a vinyl substituent into the corresponding

aldehyde (Scheme 7). To minimize side reactions, we chose to

start from the benzoyl-protected iodide precursor 36. Suzuki

reaction with potassium vinyl triﬂuoroborate⁸³aﬀorded 102 in

acceptable yields. Oxidative cleavage of the vinyl group 102

was accomplished via the Lemieux−Johnson oxidation to

aﬀord aldehyde 103. The carboxylic acid analogue 108 could

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Scheme 5. Reagents and Conditions: (a) Phenylacetylene, PdCl₂(PPh₃)₂, CuI, DMF/Et₃N 4:1, 40%; (b) H₂, Pd(OH)₂/C,

MeOH, 89% (87), 74% (89); (c) (i) Ethynyltrimethylsilane, PdCl₂(PPh₃)₂, CuI, DMF/Et₃N 4:1; (ii) 7 N NH₃in MeOH, 19%;

(d) TMSN₃, CuI, DMF/MeOH 9:1, 100 °C, 40%; (e) Zn(CN)₂, Pd₂(dba)₃, dppf, DMF, 150 °C, 28%; (f) NaN₃, NH₄Cl, LiCl,

DMF, 100 °C, 25%; (g) H₂, Raney Nickel, MeOH, 10%; (h) NH₄OH, H₂O₂, 25%; (i) TMSCF₃, CuI, KF, DMF/NMP 1:1,

Reﬂux; (j) 0.5 M NaOMe in MeOH, 14% Over 2 Steps

now eﬃciently be obtained via the Pinnick oxidation of 103.

Alternatively, the aldehyde functionality of 103 was further

elaborated to a methyl-N-morpholino substituent via reductive

amination (104) or to a diﬂuoromethyl substituent via reaction

with DAST (106). Deprotection under basic conditions

aﬀorded 105 and 107. The carboxylic acid functionality was

further derivatized to diﬀerent amides via HCTU-mediated

coupling to aﬀord analogues 113−116 after benzoate

deprotection. Attempted cyclopropanation of 102 with in

situ-generated diﬂuorocarbene (from BrCF₂CO₂Na⁸⁴) or

dichlorocarbene (generated from CHCl₃and NaOH) failed.

Biological Evaluation. The prepared nucleoside analogues

were evaluated in vitro for their activity against T. cruzi and

Leishmania infantum. Cytotoxicity was assayed against MRC-

5_SV2cells (T. cruzi host cell) and primary mouse macrophages

(PMM, L. infantum host cell) (Table 1).

Although the anti-T. cruzi and antileishmanial activities of

allopurinol (1), aminopurinol (3), and their ribonucleosides 2

and 4 are already known, they were included in this study as

reference compounds. In accordance with various literature

reports,^46,51,851 and 3 displayed good activity against T. cruzi

and L. infantum. Yet, the 20-fold higher activity and selectivity

of aminopurinol (3) compared to allopurinol (1) against L.

infantum is striking. As mentioned before, 3 has never been

evaluated in an in vivo VL model although it is known to be

safe at low doses.^46,51Introduction of a bromo substituent in

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Scheme 6. Reagents and Conditions: (a)(i) NaH, AcCl, Tetrahydrofuran (THF), 0 °C to RT, (ii) Dimethyl Sulfate, Reﬂux, (iii)

H₂NNH₂·H₂O, Et₃N, 38% Over 3 Steps; (b) Formamide, 180 °C, 23%; (c) Thioacetamide, Reﬂux, 36%; (d) 98, BF₃·OEt₂,

MeNO₂, Reﬂux; (e) 0.5 M NaOMe in MeOH, 21% Over 2 Steps

position 7 of allopurinol (1) or aminopurinol (3) led to a

drastic drop in activity, possibly due to impeded conversion by

PRTases, which are essential for the activation of nucleobase

analogues. The 4-APP ribonucleoside 4 displayed potent

activity against both T. cruzi and L. infantum intracellular

amastigotes, as could be expected from literature reports on its

activity against T. cruzi epimastigotes and Leishmania

promastigotes.^49,50In both MRC-5_SV2cells and PMM cells, 4

failed to show cytotoxicity up to 64 μM, but similar to 3, it has

never been evaluated in vivo. Introduction of a halogen atom

on the 7-position of 4 (compounds 21 and 36) led to a severe

decrease in activity against T. cruzi and L. infantum. This is

diﬀerent from earlier reported results in T. cruzi epimastigotes

and Leishmania promastigotes, where the activity was more

comparable to the parent compound 4.^49,50Activity of

allopurinol ribonucleoside 2 was comparable to allopurinol.⁸⁵

In our hands, introduction of a bromide on the 7-position of 2

(compound 29) rendered the compound inactive, which

conﬂicts with a report stating it to be more active than 2

against L. tropica intracellular amastigotes.⁶⁹

The combination of aminopurinol (3) and allopurinol (1)

with a 3′-deoxy-3′-ﬂuororibofuranose moiety (24 and 32)

resulted in inactive compounds. Introduction of a bromide on

the 3-position of 24 rendered the compound highly cytotoxic

(22). It did not display any selective activity, contrasting with

the matched 7-deazapurine nucleoside.⁵⁷The same was true

for the 3′-deoxynucleosides 23, 25, 31, and 33. While the

inactivity of 33 was already noted by Moorman et al.,⁸⁶the

inactivity of 25 was more surprising, as removal of the 3′-

hydroxy group resulted in a major increase in activity against T.

brucei or T. cruzi in earlier 7-deazapurine nucleoside series.^53,55

Again, the introduction of a 7-bromo substituent (23) aﬀorded

a cytotoxic compound without any speciﬁc antiparasitic

activity. The 2′-deoxynucleosides 41 and 40 were also inactive,

as was already reported for 41.⁴⁶

Based on the structure−activity relationship (SAR) of earlier

nucleoside series,^52,53,57,78which demonstrated that modiﬁca-

tions of the 7-position could improve activity, we performed an

extensive substituent screen. In a ﬁrst set of analogues, diﬀerent

substituted phenyl rings were introduced on the 7-position

(Table 2). Remarkably, also in this series, a 4-chlorophenyl

(44) proved to confer the best antitrypanosomal activity (IC₅₀

= 0.32 μM) and was about 50-fold more active than the 7-

phenyl analogue 42. The second most potent para-substituted

analogue was the 4-methylphenyl substituted compound 45.

Further substitution of the 4-chlorophenyl substituent with a

3-ﬂuoro (53), methyl (57), or methoxy group (56) failed to

potentiate its activity. The 2,4-substituted analogues 62 and 63

were less active than the 3,4-disubstituted analogues. Strikingly,

the superiority of this 4-chlorophenyl modiﬁcation has also

been observed for other 7-deazapurine nucleosides and

suggests that the chloro substituent is involved in a crucial

interaction with a parasitic target or transporter, rather than

just reducing the electron density of the phenyl ring. Removal

of the 3′-OH group as in 76 was expected to result in increased

anti-T. cruzi activity, based on earlier observations with 7-

deazapurine nucleosides,⁵³but surprisingly led to a compound

that was 3-fold less active than 44. Nevertheless, 44 is more

potent than its 7-deazapurine congener⁵²and displays an

improved selectivity proﬁle, with no in vitro toxicity in MRC-

5_SV2or PMM cells in concentrations up to 64 μM. The 2′-

deoxy analogue 77 displayed reasonably good anti-T. cruzi

activity but was less potent than both 44 and 76. All phenyl-

substituted analogues displayed in Table 2 were inactive

against L. infantum.

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Scheme 7. Reagents and Conditions: (a) Potassium Vinyl Triﬂuoroborate, Pd(OAc)₂, PPh₃, Cs₂CO₃, DMF/H₂O 9:1, 100 °C,

38%; (b) K₂OsO₄·2H₂O, NaIO₄, 2,6-Lutidine, 1,4-Dioxane/H₂O 3:1, 48%; (c) Morpholine, NaBH₃CN, AcOH, MeOH/THF

2:1; (d) 0.5 M NaOMe in MeOH, 64% Over 2 Steps (105), 58% Over 2 Steps (107), 29% Over 2 Steps (113), 28% Over 2

Steps (114), 61% Over 2 Steps (115), 44% Over 2 Steps (116); (e) NaClO₂, NaH₂PO₄, H₂O₂, THF/H₂O 6:1, 94%; (f) DAST,

CH₂Cl₂; (g) Aq. NHMe (109), Pyrrolidine (110), Aniline (111), or Benzylamine (112), HCTU, N,N-Diisopropylethylamine

(DIPEA), DMF

Bioisosteric replacement of the phenyl ring by a thiophene

resulted in a loss of activity (67−70) (Table 3). The 5-

chlorothiophene and 5-methylthiophene analogues 69 and 68

were 5−10-fold less active than their phenyl counterparts and

displayed lower selectivity. Introducing an extra atom or linker

between the nucleobase and the phenyl ring was also not

tolerated, as illustrated by the inactivity of the phenoxy

analogues 79 and 80 and the low activity of elongated phenyl

analogues 86, 73, and 87. None of these analogues showed any

activity against L. infantum. A number of nitrogen-containing

heterocycles were also introduced on the 7-position (64−66,

90, and 92). A 2- or 4-pyridyl substituent (65 and 66), an N-

methylpyrazole (64), and tetrazole (92) did not lead to any

speciﬁc activity against T. cruzi or L. infantum. While 64 and 92

were devoid of MRC-5_SV2cytotoxicity, the triazole analogue 90

was highly cytotoxic and also did not display any speciﬁc

antiparasitic activity.

Among a series of analogues with small substituents on the

7-position, a nitrile (91) led to reasonable activity against both

T. cruzi and L. infantum (Table 4). The chloro analogue 85

was less active against T. cruzi but displayed higher selectivity

toward MRC-5_SV2cells. A triﬂuoromethyl (96) or diﬂuor-

omethyl (107) substituent resulted in cytotoxic compounds

that did not display selective antiparasitic activity. Among the

carbon-based substituents, the methyl analogue 101 and the

ethynyl analogue 88 displayed moderate anti-T. cruzi activity.

A vinyl (71), ethyl (89), or cyclopropyl (74) substituent led to

inactive compounds, while an isopropenyl (72) or isopropyl

(78) group resulted in cytotoxic compounds with no speciﬁc

antiparasitic activity. An amine (81), methylamine (82), or

pyrrolidine group (83) on the 7-position did not lead to any

signiﬁcant antiparasitic activity, as was the case for an

aminomethyl (93) or morpholinomethyl (105) substituent.

The activity of the amide-substituted nucleosides (94, 113−

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a

Table 1. Evaluation of Drug Sensitivity of Ribofuranose-Modiﬁed Nucleoside Analogues against T. cruzi and L. inf.

cpd.

structure

T. cruzi IC₅₀(μM)

MRC-5 CC₅₀(μM)

SI

L. infantum. IC₅₀(μM)

PMM CC₅₀(μM)

SI

Nucleobases

3

12

1

R = H

R = Br

R = H

R = Br

0.57

38.1

9.75 1.75

>64.0

2.46

5

>1

>7

0.18

22.6

3.51 1.77

>64.0

>355

>2

>18

>64.0

14

Ribonucleosides

4

X = H

X = Br

R = I

R = H

R = Br

0.29 0.03

4.37 0.55

12.62

7.18 3.66

>64.0

25.4

>64.0

>219

>15

2

1.06 0.35

29.4 21.4

25.4

6.66 4.66

>64.0

>60

>2

10

21

36

2

9

29

3′-Deoxy-3′-Fluoronucleosides

24

22

32

30

X = H

X = Br

X = H

R = Br

>64.0

1.06

>64.0

0.23

>64.0

0.08

>64.0

0.13

>64.0

0

1

3′-Deoxynucleosides

25

23

33

31

R = H

R = Br

R = H

R = Br

>64.0

38.5

>64.0

2.2

>64.0

2′-Deoxynucleosides

41

40

R = H

R = Br

>64.0

53.2 10.8

>64.0

24.9 14.0

>64.0

a

Cytotoxicity Was Assayed against Human MRC-5_SV2Cells and Primary Mouse Macrophages (PMMs). Values represent mean SEM, which

originate from 2−3 independent experiments and are expressed in μM. Values in italics represent the result of a single determination because of

inactivity or overt cytotoxicity. SI: in vitro selectivity index is the ratio of CC₅₀for the host cell (MRC-5_SV2for T. cruzi, PMM for L. inf.) and IC₅₀of

the parasite. Benznidazole was included as a reference for T. cruzi (IC₅₀= 2.02 0.28 μM) and miltefosine as a reference for L. infantum (IC₅₀

7.47 2.23 μM).

=

116) varied: a carboxamide group (94) again resulted in a

cytotoxic compound, while adding a methyl group on the

amide nitrogen (113) removed all cytotoxic eﬀects and

provided a compound with moderate anti-T. cruzi activity.

Amide analogues with bigger groups (114−116) were inactive.

Overall, the SAR of the pyrazolo[3,4-d]pyrimidine nucleo-

sides for anti-T. cruzi and anti-L. infantum activities turned out

to be completely diﬀerent from previously reported 7-

deazapurine^52,53,57and 1,7-dideazapurine⁷⁸nucleoside series.

As already known from several literature reports, the parent

nucleosides aminopurinol riboside 4 and allopurinol riboside 2

displayed good in vitro activity against both T. cruzi and L.

infantum. Introduction of a halogen atom on the 7-position had

a detrimental eﬀect on the activity against both T. cruzi and L.

infantum and modiﬁcations at the 3′-position of the ribose

moiety, expected to result in more potent compounds based on

SAR studies of earlier nucleoside series, completely abolished

activity, or resulted in cytotoxic compounds. A substituent

screen of the 7-position of 4 revealed highly varying eﬀects on

the antiparasitic activity and cytotoxicity to MRC-5_SV2and

PMM. A 4-chlorophenyl substituent resulted in a compound

with potent anti-T. cruzi activity and devoid of cytotoxic eﬀects

in MRC-5_SV2or PMM at concentrations up to 64 μM.

Contrary to expectations, removal of the 3′-hydroxyl group of

44 resulted in a compound that was 2−3-fold less potent (76).

The insertion of a linker (oxygen, carbon-based, amide)

between the oxygen and the phenyl ring was not tolerated.

Bioisosteric replacement of the phenyl ring with a thiophene

was also not tolerated and resulted in a 10-fold decrease in

activity. Other heterocycles in the 7-position resulted in

compounds with low antiparasitic activity. The eﬀect of other,

smaller substituents varied greatly. Some resulted in moderate

anti-T. cruzi activity (e.g., chloride, methyl, ethynyl), while

other substituents aﬀorded highly cytotoxic nonselective

compounds. Overall, none of the 7-modiﬁed analogues

displayed good activity against L. infantum, suggesting that,

regardless of the nature of the heterocyclic nucleobase,

substituents on this position are not tolerated. 44 was the

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a

Table 2. Evaluation of Drug Sensitivity of 7-Modiﬁed Nucleoside Analogues against T. cruzi and L. infantum

cpd.

structure (R =)

T. cruzi IC₅₀(μM)

MRC-5 CC₅₀(μM)

SI

>5

L. inf. IC₅₀(μM)

PMM CC₅₀(μM)

SI

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

75

77

76

H

13.1

>64.0

[32.0, >64.0]

>64.0

4-OMe

4-Cl

4-Me

4-F

4-NO₂

18.2

>4

>64.0

57.0 9.3

>64.0

0.32 0.02

1.77 0.92

3.36 1.88

10.3 5.8

>64.0

6.34 1.90

15.6 6.2

32.0

2.82 1.94

4.66 2.71

1.19 0.92

4.24 1.55

1.14 1.02

0.81 0.13

43.8

>64.0

1.33 0.51

45.3

6.06 2.69

3.76 0.61

>64.0

2.49 0.42

1.04 0.32

>197

>36

>19

>6

4-t-Bu

4-CF₃

>10

>4

>2

>23

>14

>54

>15

>56

>79

>1

4-OCF₃

4-CN

3,4-diCl

3-Cl-4-F

4-Cl-3-F

3,4-diF

4-Cl-3-OMe

4-Cl-3-Me

4-Cl-3-CF₃

4-Cl-3-CN

4-Cl-3,5-diF

4-Cl-3-OEt

2,4-diCl

4-Cl-2-Me

>17

>1

>11

>17

>64.0

52.8 11.2

>64.0

>1

1

>22

>61

[32.5, >64.0]

>64.0

a

Cytotoxicity was assayed against human MRC-5SV2 cells and primary mouse macrophages (PMMs). Values represent mean SEM, which

originate from 2−3 independent experiments and are expressed in μM. Values in parentheses represent the values of the diﬀerent determinations,

as no correct average can be calculated. Values in italics represent the result of a single determination because of inactivity or overt cytotoxicity. SI:

in vitro selectivity index is the ratio of CC₅₀for the host cell (MRC-5_SV2for T. cruzi, PMM for L. inf.) and IC₅₀of the parasite. Benznidazole was

included as a reference for T. cruzi (IC₅₀= 2.02 0.28 μM) and miltefosine as a reference for L. infantum (IC₅₀= 7.47 2.23 μM).

most potent analogue for T. cruzi and was more active and

more selective than its matched 7-deazapurine nucleoside

congener.⁵²Because of its potent anti-T. cruzi activity and

favorable selectivity proﬁle, 44 was selected for further

evaluation in an acute Chagas disease mouse model.

Metabolic Stability of Compound 44. The in vitro

metabolic stability of 44 was evaluated using male mice and

pooled human liver microsomes (S9 fraction) (Table 5).

Compound 44 was not susceptible to Phase-I and Phase-II

metabolism in both mouse and human microsomes with 100%

of the parent drug remaining after 60 min. These results

favored further evaluation of 44 in an in vivo laboratory rodent

model.

In Vivo Evaluation of Compound 44. To determine its

eﬃcacy in vivo, 44 was evaluated in an acute Chagas disease

model using the Y strain of T. cruzi in Swiss male mice.^92,9344

was evaluated at 0.25, 2.5, or 25 mg/kg b.i.d or in combination

with benznidazole (44 at 2.5 mg/kg b.i.d + benznidazole at 10

mg/kg q.d.). Compounds were administered orally for 5

consecutive days, starting the administration at parasitemia

onset on day 6 postinfection (dpi), which peaked at 8 dpi in

untreated animals. 44 at 25 mg/kg gave 99% reduction in the

parasitemia peak at 8 dpi, which was similar to the optimal q.d.

dose of benznidazole at 100 mg/kg. Lower dosages (0.25 and

2.5 mg/kg) only gave partial reduction of parasitemia (43%).

Coadministration of 44 (2.5 mg/kg) and benznidazole (10

mg/kg) reached 71% reduction, which was slightly better than

benznidazole alone. However, no mice sustained negative

parasitemia, hence failing parasitological cure. In the 2.5 mg/

mk and 25 mg/kg treatment groups, 5 out of 6 mice (83%)

survived until the end of the experiment (34 dpi), similar to

benznidazole at 10 mg/kg. In the benznidazole 100 mg/kg

group, all mice survived. In the untreated control group, all

mice succumbed to the infection by day 27. In the 0.25 mg/kg,

only one mouse survived at 34 dpi (Figure 2).

As the in vitro T. cruzi screening was performed with the

Tulahuen strain (DTU IV) and in vivo assays with the Y strain

(DTU II), additional in vitro screens were conducted with the

latter. The ﬁndings conﬁrmed the high potency of 44 against

intracellular forms (IC₅₀= 0.26 0.03 μM, SI > 1900) present

in cardiomyocytes (Table 6), similar to the values obtained

with the Tulahuen strain intracellular amastigotes. In addition,

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Table 3. Evaluation of Drug Sensitivity of 7-Modiﬁed Pyrazolo[3,4-d]pyrimidine Nucleoside Analogues against T. cruzi and L.

a

infantum

cpd.

T. cruzi IC₅₀(μM)

MRC-5 CC₅₀(μM)

SI

L. infantum. IC₅₀(μM)

PMM CC₅₀(μM)

SI

Thiophenes

67

69

68

70

10.4

32

3

>64.0

[32.0,>64.0]

>64.0

2.56 1.12

7.10 4.2

32

47.3 6.12

>64.0

14

>9

>2

>1

Elongated phenyls

79

80

86

73

87

>64.0

29.4 11.1

16.5

52.3

>64.0

32

>64.0

>2

>64.0

49.4 8.7

50.8

>2

>4

>1

>64.0

Nitrogen-containing heterocycles

65

66

64

90

92

30.1 17.8

21.5

45.3

0.43

>64.0

36.4 4.1

>64.0

0.6

1

[50.8, >64.0, >64.0]

25.4

>64.0

43.1

>64.0

1

>3

>1

1

>1

>64.0

a

Cytotoxicity was assayed against human MRC-5_SV2cells and primary mouse macrophages (PMMs). Values represent mean

SEM, which

originate from 2−3 independent experiments and are expressed in μM. Values in parentheses represent the values of the diﬀerent determinations,

as no correct average can be calculated. Values in italics represent the result of a single determination because of inactivity or overt cytotoxicity. SI:

in vitro selectivity index is the ratio of CC₅₀for the host cell (MRC-5_SV2for T. cruzi, PMM for L. inf.) and IC₅₀of the parasite. Benznidazole was

included as a reference for T. cruzi (IC₅₀= 2.02 0.28 μM) and miltefosine as a reference for L. infantum (IC₅₀= 7.47 2.23 μM).

44 did not exert cardiotoxicity in two-dimensional (2D) and

three-dimensional (3D) cardiac cell cultures, giving CC₅₀

values up to 500 and 200 μM, respectively. After discarding

the potential impact of parasite strain on the in vitro and in vivo

outcomes, we next evaluated the activity of 44 (as well as 4)

against the nondividing and highly infective bloodstream

trypomastigote form. Both compounds proved to be inactive

(IC₅₀> 81 μM), while benznidazole gave an IC₅₀of 5.7 0.6

μM. These results corroborate former studies using pyrrolo-

[2,3-b]pyridine (1,7-dideazapurine) nucleoside analogues that

failed to achieve parasitological cure in treated mice despite

parasitemia suppression and high animal survival rates.⁷⁸The

inability to kill bloodstream trypomastigotes may explain the

parasitemia recrudescence and thus lack of sterile cure, as has

also been reported for other nucleoside analogues.^53,78The

lack or low activity against bloodstream trypomastigotes

resembles that of the azole ergosterol biosynthesis inhibitors

that failed in clinical trials for Chagas disease,^87,88raising the

potential relevance of targeting both the intracellular multi-

plicative amastigotes and the nonreplicative trypomastigote

forms. Recent ﬁndings also highlighted the role of metabolic

heterogeneity in drug eﬃcacy upon recalcitrant T. cruzi

infection.⁸⁹The authors reported that limiting exogenous

glutamine impairs ergosterol biosynthesis inhibitors (azoles) to

act upon intracellular amastigotes. In addition to the

occurrence of nonreplicative forms (like dormant amastigotes

and trypomastigotes), the impact of metabolic and environ-

mental heterogeneity must be considered in the search for

novel anti-T. cruzi agents as these factors can modulate drug

eﬃcacy.

Eﬀect of the T. cruzi Host Cell on Drug Sensitivity to

Compound 44. To investigate whether host cell permeability

could be a limiting factor for the in vivo T. cruzi eﬃcacy or lack

of antileishmanial activity of compound 44, we evaluated the

eﬀect of 44 against T. cruzi in PMM host cells (Table 7).

Surprisingly, 44 was completely inactive against T. cruzi in

PMM cells, while benznidazole retained its activity in both cell

types. To rule out drug eﬄux as the cause of this eﬀect, the

experiment was repeated in the presence of the ABC

transporter inhibitors verapamil, cyclosporine A, and probene-

cid. In all cases, 44 was inactive, indicating that its inactivity in

PMM cells is likely due to permeability issues. These ﬁndings

might oﬀer further explanation as to why 44 was not able to

fully clear T. cruzi infection in vivo. Tissue tropism in Chagas

disease has been demonstrated to play an important role in

persistence,⁹⁰⁻⁹²and limited permeability in certain tissues has

also been implicated in the ineﬀectivity of Posaconazole in

curing T. cruzi infections.⁹³These results could also oﬀer an

explanation as to why several of the herein-reported nucleoside

analogues, as well as others that were previously found to

display potent anti-T. cruzi activity, are inactive when evaluated

against L. infantum intracellular amastigotes in PMM host

cells.^52,94The origins of this lack of permeability in PMM cells

are currently unclear and require further study. Furthermore, it

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Table 4. Evaluation of Drug Sensitivity of 7-Modiﬁed Nucleoside Analogues against T. cruzi and L. infantum

cpd.

T. cruzi IC₅₀(μM)

MRC-5 CC₅₀(μM)

SI

L. infantum. IC₅₀(μM)

PMM CC₅₀(μM)

SI

85

91

96

107

101

88

71

89

74

72

78

81

2.89 0.23

0.95

27

>64.0

34.6

13.3

>22

36

<1

12

>64.0

8

>64.0

0.25

32.7 15.5

10.4 2.3

12.7

>64.0

24.1

>64.0

0.25

40.0 12

[8.00, >64.0]

32

>64.0

32

>64.0

8

>1

>8

8.3

2.42

1

3

4.68 1.33

2.78 0.04

>64.0

29.5

57.5 6.5

[46.5, >64.0]

>64.0

0.35

20

<1

3

1

>64.0

7.64

10.6

23

>64.0

6.82

1

82

83

93

105

94

>64.0

11.1

>64.0

0.25

>64.0

59.3

>64.0

0.25

50.8

>64.0

2

>64.0

8.11

>64.0

8

>64.0

8

>1

5

4

1

113

114

115

116

4

>64.0

16

>64.0

32.5

>64.0

32

>64.0

1

>64.0

a

Cytotoxicity was assayed against human MRC-5_SV2cells and primary mouse macrophages (PMMs). Values represent mean

SEM, which

originate from 2−3 independent experiments and are expressed in μM. Values in parentheses represent the values of the diﬀerent determinations,

as no correct average can be calculated. Values in italics represent the result of a single determination because of inactivity or overt cytotoxicity. SI:

in vitro selectivity index is the ratio of CC₅₀for the host cell (MRC-5_SV2for T. cruzi, PMM for L. inf.) and IC₅₀of the parasite. Benznidazole was

included as a reference for T. cruzi (IC₅₀= 2.02 0.28 μM) and miltefosine as a reference for L. infantum (IC₅₀= 7.47 2.23 μM).

Table 5. In Vitro Metabolic Stability of Compound 44 Using

Male Mouse and Pooled Human S9 Microsomal Fractions

CONCLUSIONS

■

a

We described the design and synthesis of a library of

pyrazolo[3,4-d]pyrimidine nucleosides that were evaluated

for in vitro activity against T. cruzi and L. infantum intracellular

amastigotes. SAR trends were highly diﬀerent from earlier

reported nucleoside series. Modiﬁcations of the 3′-position of

the parent adenosine- and inosine-like analogues aminopurinol

riboside 4 and allopurinol riboside 2 were detrimental to

activity and led to inactive compounds. The introduction of a

halogen atom on the 7-position led to a signiﬁcant decrease in

activity. An extensive screen of substituents on the 7-position 4

revealed varying eﬀects on antiparasitic activity and selectivity

toward MRC-5_SV2and PMM cells. A 4-chlorophenyl

substituent on the 7-position (44) aﬀorded good anti-T.

cruzi activity and selectivity and was also metabolically stable in

human and mouse liver microsomes. 44 was next evaluated in

an acute Chagas disease model, resulting in a rapid, almost

complete reduction in parasitemia. Treatment with 44 led to

the survival of 5 out of 6 mice but failed to induce sterile

parasitological cure. None of the new analogues showed good

in vitro activity against L. infantum, which could potentially be

attributed to limited permeability in the PMM host cell.

mouse % 44

remaining

human % 44

remaining

phase-I/II

time

mean

STDEV

mean

STDEV

CYP450-NADPH

0

15

30

60

0

15

30

60

100

103

108

100

103

115

100

102

101

100

106

104

11.4

12.8

16.1

10.3

7.8

8.5

UGT enzymes

3.2

0.3

3.0

2.8

9.5

7.2

a

The depicted values are the percentage of remaining parent

compound at the various time points of incubation (0−15−30−60

min). Data originate from two independent experiments of two

biological replicates. Diclofenac (susceptible to Phase-I and Phase-II

metabolism) was included as reference to ensure proper assay

performance (data not shown).

would be worthwhile to evaluate if certain nucleoside prodrugs

could lead to improved permeability and/or improved in vivo

eﬃcacy.

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Figure 2. In vivo eﬃcacy of 44 administered orally for 5 days in Swiss mice infected with the Y strain of T. cruzi. Parasitemia curve for 44 at (A)

0.25 mg/kg/b.i.d, (B) 2.5 mg/kg/b.i.d, (C) 25 mg/kg/b.i.d, and (D) coadministration of 44 at 2.5 mg/kg + benznidazole (Bz) at 10 mg/kg/b.i.d.

Mortality rates were up to 35 dpi (E). ** p value ≤ 0.05.

Table 6. In Vitro Eﬃcacy of 4 and 44 against T. cruzi Y-

EXPERIMENTAL SECTION

■

Strain Bloodstream Trypomastigotes and Intracellular

Chemistry. General. All reagents and solvents were obtained from

standard commercial sources and were of analytical grade. Unless

otherwise speciﬁed, they were used as received. All moisture-sensitive

reactions were carried out under an argon atmosphere. Reactions were

carried out at ambient temperature, unless otherwise indicated.

Reactions were monitored via analytical TLC or analytical LCMS.

Analytical TLC was performed on Machery−Nagel precoated F254

aluminum plates and visualized by UV followed by staining with basic

aq. KMnO₄, cerium-molybdate, or sulfuric acid-anisaldehyde spray.

Analytical LCMS was performed on a Waters AutoPuriﬁcation system

a

Amastigotes in Cardiac Cells

T. cruzi Y bloodstream

trypomastigotes IC₅₀

T. cruzi Y intracellular primary cardiac

amastigotes IC₅₀(μM) cells LC₅₀(μM)

cpd.

(μM)

4

44

>81

2.46 0.3

0.26 0.03

>500

500

a

IC₅₀values are depicted as means

determinations, using duplicates.

SEM of two independent

Table 7. Evaluation of Drug Sensitivity of 44 and Benznidazole (Bz) against T. cruzi in PMM Host Cells, Alone or with

Coadministration of Verapamil (8 μM), Probenecid (700 μM), or Cyclosporin A (2 μM)

T. cruzi (MRC-5) IC₅₀T. cruzi (PMM) IC₅₀

T. cruzi (PMM) + verapamil

IC₅₀(μM)

T. cruzi (PMM) + probenecid

IC₅₀(μM)

T. cruzi (PMM) + cyclosporin A

IC₅₀(μM)

cpd.

(μM)

>64.0

1.96 0.25

44

Bz

0.32 0.02

2.02 0.28

>64.0

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(equipped with ACQUITY QDa (mass; 100−1000 amu)) and 2998

Photodiode Array (220−400 nm) using a Waters Cortecs C18 (2.7

μm, 100 mm × 4.6mm) 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−

00:100 in 6.5 min or 50:50−00:100 in 6.5 min), or a Waters Alliance

XE separation module using a Phenomenex Kinetix C8 (2.6 μm, 50

mm × 2.10 mm) column and the same gradient system. Preparative

HPLC was performed on the same system, using a Phenomenex Luna

Omega Polar column (250 mm × 21 mm, 5 μm) and a gradient

system of 0.2% formic acid in water/MeCN at a ﬂow rate of 20 mL/

min (gradients are speciﬁed in the individual procedures). Column

chromatography was performed manually using Machery−Nagel 60

vacuo. The resulting solid was puriﬁed via ﬂash column chromatog-

raphy to aﬀord the ﬁnal product.

General Procedure E: Suzuki Reaction. Compound 21 (1 equiv)

or 23 (in the case of 76, 1 equiv), boronic acid (1.5 equiv) or

triﬂuoroborate salt (1.5 equiv) Na₂CO₃(when a boronic acid was

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

equiv), Pd(OAc)₂(0.05 equiv), and TPPTS (0.12 equiv) 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

material was fully consumed (usually 1−3 h; as monitored by LCMS

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

puriﬁed by ﬂash column chromatography.

General Procedure F: Amide Coupling. The nucleoside carboxylic

acid (1.0 equiv) was dissolved in DMF (10 mL/mmol). DIPEA (3.0

equiv) was added, followed by HCTU (2.5 equiv). After 5 min, the

respective amine (5.0 equiv) was added and the reaction mixture was

stirred overnight. When TLC or LCMS analysis indicated full

conversion, the reaction mixture was diluted with excess EtOAc and

transferred to a separation funnel. The organic phase was washed with

1 N HCl, aq. sat. NaHCO₃, and brine sequentially. The organic phase

was then dried over Na₂SO₄and concentrated in vacuo. The residue

was used directly in the next reaction, without puriﬁcation.

M silica gel (40−63 μm) or on a Reveleris X2 (Grace/Buchi)

̈

automated ﬂash unit employing prepacked 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 a modular Lockspray interface. Samples were infused in a

MeCN/water (1:1) + 0.1% formic acid mixture at 100 μL/min. NMR

spectra were recorded on a Varian Mercury 300 MHz spectrometer or

a Bruker Avance Neo 400 MHz spectrometer. Chemical shifts (δ) are

given in ppm, and spectra are referenced to the residual solvent peak.

Coupling constants are given in Hz. Systematic numbering is

employed for NMR assignments in the individual procedures. Melting

points were determined on a Buchi-545 apparatus and are

̈

uncorrected. Purity was assessed by means of liquid chromatog-

raphy−mass spectrometry (LCMS). All obtained ﬁnal compounds

had purity >95%, as assayed by analytical HPLC (UV), unless

otherwise indicated.

3-Bromo-4-amino-1H-pyrazolo[3,4-d]pyrimidine (12). 4-Amino-

1H-pyrazolo[3,4-d]pyrimidine 3 (8.26 g, 61.1 mmol, 1.0 equiv) was

dissolved in DMF (60 mL). NBS (11.4 g, 64.2 mmol, 1.05 equiv) was

added, and the mixture was heated at 60 °C overnight. The mixture

was cooled to room temperature and poured into ice-cold water (350

mL). The resulting suspension was stirred for 10 min at 0 °C and

ﬁltered overnight. The solids were collected and dried under a high

vacuum overnight to aﬀord 12 (11.9 g, 55.7 mmol, 91% yield) as an

General Procedure A: Large-Scale BF₃·OEt₂-Mediated Glycosy-

lation with Commercially Available 1-O-Acetyl-2,3,5-tri-O-benzoyl-

β-^D-ribofuranose. The respective pyrazolo[3,4-d]pyrimidine (1.0

equiv) and 1-O-acetyl-tri-O-benzoyl-β-^D-ribofuranose (1.5 equiv)

were added to dry nitromethane (2.5 mL/mmol). The mixture was

heated to reﬂux, when BF₃·OEt₂(1.5 equiv) was added, upon which

the solids started to dissolve. After 90 min of heating at reﬂux, the

solvent was removed in vacuo. The resulting oil was dissolved in

CH₂Cl₂(sonicate until fully dissolved) and directly poured on a silica

column (preconditioned with CH₂Cl₂). The column was eluted with

100% CH₂Cl₂until all excess 1-O-acetyl-tri-O-benzoyl-β-D-ribofur-

anose had eluted and then with 5% acetone in CH₂Cl₂to collect the

product.

General Procedure B: Small-Scale BF₃·OEt₂-Mediated Glycosyla-

tion with Protected Ribose Derivative. The respective pyrazolo[3,4-

d]pyrimidine (1.1 equiv) and ribose derivative (1.0 equiv) were added

to dry nitromethane (2.5 mL/mmol). The mixture was heated to

reﬂux, when BF₃·OEt₂(1.0 equiv) was added, upon which the solids

started to dissolve. After 90 min of heating at reﬂux, the solvent was

removed in vacuo. The resulting oil was dissolved in CH₂Cl₂, celite

(1.5 g/g starting material) was added, and the solvent was removed in

vacuo. The resulting solid was puriﬁed by ﬂash column chromatog-

raphy to aﬀord the protected nucleoside.

General Procedure C: Deprotection with NaOMe. Protected

nucleoside (1.0 equiv) was dissolved in CH₂Cl₂(0.5 mL/mmol).

MeOH (5 mL/mmol) was added, followed by NaOMe in MeOH

(5.4 M, 0.2 mL/mmol). The mixture was stirred at room temperature

or 60 °C (for 36) until TLC analysis (20% MeOH in CH₂Cl₂)

indicated completion of the reaction. The reaction was neutralized to

pH 7 via the addition of 4 N HCl, celite (1.5 g/g starting material)

was added, and the solvents were removed in vacuo. The solid residue

was brought onto a silica column and eluted with a mixture of MeOH

and CH₂Cl₂to isolate the ﬁnal product.

General Procedure D: Catalytic Hydrogenation in Buﬀered

MeOH. The nucleoside analogue was dissolved in a mixture of MeOH

(8 mL/mmol) and aq. 1M NaOAc (2 mL/mmol). The ﬂask was

placed under a nitrogen atmosphere, and a catalytic amount of Pd/C

was added. The atmosphere was exchanged for H₂and the mixture

was stirred until TLC analysis (20% MeOH in CH₂Cl₂) indicated

completion of the reaction. The mixture was then ﬁltered over celite,

celite was added to the ﬁltrate, and the solvents were removed in

1

oﬀ-white solid. H NMR (300 MHz, DMSO-d₆) δ 6.91 (1H, br. s,

NH₂′), 7.72 (1H, br. s, NH₂″), 8.16 (1H, s, C-6), 13.75 (1H, br. s,

NH) ppm. HRMS (ESI): calcd for C₅H₅BrN₅([M + H]⁺): 213.9728,

found: 213.9731.

3-Iodo-4-amino-1H-pyrazolo[3,4-d]pyrimidine (13). 4-Amino-

1H-pyrazolo[3,4-d]pyrimidine 3 (5.68 g, 42.1 mmol, 1.0 equiv) was

dissolved in DMF (40 mL). NIS (10.4 g, 46.3 mmol, 1.1 equiv) was

added, and the mixture was heated at 80 °C overnight. The mixture

was cooled to room temperature and poured into ice-cold water (350

mL). The resulting suspension was stirred for 10 min at 0 °C and

ﬁltered overnight. The solids were collected and dried under a high

vacuum overnight to aﬀord 13 (10.2 g, 39.1 mmol, 93% yield) as a

1

white solid. H NMR (300 MHz, DMSO-d₆) δ 8.16 (1H, s, C-6),

13.80 (1H, br. s, NH) ppm. HRMS (ESI): calcd for C₅H₅IN₅([M +

H]⁺): 261.9590, found: 261.9594.

3-Bromo-allopurinol (14). Allopurinol (4.55 g, 33.4 mmol, 1.0

equiv) was suspended in water (300 mL). Bromine (4.29 mL, 83.6

mmol, 2.5 equiv) was added carefully, and a reﬂux cooler with a

septum was placed on top of the ﬂask. The reﬂux cooler was

connected via vacuum tubing to a large ﬂask containing excess aq. 2

M Na₂S₂O₃solution. The reaction mixture was heated at 90 °C

overnight and cooled down to room temperature. A mixture of aq. sat.

NaHCO₃(75 mL) and aq. 2 M Na₂S₂O₃(75 mL) was added through

the reﬂux cooler, after which the reaction mixture turned white. The

suspension was cooled down further to 0 °C, stirred for 10 min, and

ﬁltered. The solids were washed with ice-cold water (3×), collected,

and dried over a high vacuum overnight to aﬀord 14 (6.26 g, 29.1

1

mmol, 87%) as a light-yellow solid. H NMR (300 MHz, DMSO-d₆)

δ 8.04 (1H, d, J = 3.8 Hz), 12.22 (1H, br. s.), 13.98 (1H, br. s) ppm.

¹³C NMR (75 MHz, DMSO-d₆) δ 104.5 (C-3a), 121.9 (C-3), 149.4

(C-7a), 154.6 (C-6), 157.0 (C-4) ppm. HRMS (ESI): calcd for

C₅H₄BrN₄O ([M + H]⁺): 214.9568, found: 214.9572.

3-Bromo-4-amino-1-(2′,3′,5′-tri-O-benzoyl-β-^D-ribofuranosyl)-

pyrazolo[3,4-d]pyrimidine⁶⁹(18). Compound 12 (10.3 g, 48.1

mmol, 1.0 equiv) and 1-O-acetyl-tri-O-benzoyl-β-^D-ribofuranose

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Journal of Medicinal Chemistry

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Article

(36.4 g, 72.2 mmol, 1.5 equiv) were subjected to general procedure A

to aﬀord 18 (20.3 g, 30.8 mmol, 64% yield) as a brown oil. ¹H NMR

(300 MHz, DMSO-d₆) δ 4.51−4.67 (2 H, m, H-5′, H-5″), 4.82−4.89

(1H, m, H-4′), 6.10 (1H, t, J = 5.7 Hz, H-3′), 6.25 (1H, dd, J = 5.4,

3.4 Hz, H-2′), 6.67 (1H, d, J = 3.2 Hz, H-1′), 7.38−7.70 (9H, m,

H_Phe), 7.84−8.05 (6H, m, H_Phe), 8.24 (1H, s, H-6) ppm. ¹³C NMR

(75 MHz, DMSO-d₆) δ 63.6 (C-5′), 71.3 (C-3′), 74.2 (C-2′), 79.5

(C-4′), 86.4 (C-1′), 100.3 (C-3a), 121.2 (C-3) 128.8 (C_Phe), 129.0

(C_Phe), 129.2 (C_Phe), 129.2 (C_Phe), 129.7 (C_Phe), 129.8 (C_Phe), 129.8

(C_Phe), 129.8 (C_Phe), 133.9 (C_Phe), 134.3 (C_Phe), 134.4(C_Phe), 155.5

(C-7a), 157.7 (C-6), 157.8 (C-4), 165.0 (CO), 165.1 (CO),

165.9 (CO) ppm. HRMS (ESI): calcd for C₃₁H₂₅BrN₅O₇([M +

H]⁺): 658.0937, found: 658.0925.

3-Bromo-4-amino-1-(2′,5′-di-O-benzoyl-3′-deoxy-3′-ﬂuoro-β-^D-

ribofuranosyl)pyrazolo[3,4-d]pyrimidine (19). Compound 12 (0.330

g, 1.54 mmol, 1.1 equiv) and compound 16 (0.563 g, 1.40 mmol)

were subjected to general procedure B. Puriﬁcation by ﬂash column

chromatography (automated, 0 → 5% MeOH in CH₂Cl₂) aﬀorded

semipure 19, which was used as such in the next reaction. HRMS

(ESI): calcd for C₂₄H₂₀BrFN₅O₅([M + H]⁺): 556.0632, found:

556.0590.

3-Bromo-4-amino-1-(2′,5′-di-O-benzoyl-3′-deoxy-β-^D-

ribofuranosyl)pyrazolo[3,4-d]pyrimidine (20). Compound 12 (0.395

g, 1.84 mmol, 1.1 equiv) and compound 17 (0.643 g, 1.67 mmol, 1.0

equiv) were subjected to general procedure B. Puriﬁcation by ﬂash

column chromatography (automated, 0 → 5% MeOH in CH₂Cl₂)

aﬀorded semipure 20, which was used as such in the next reaction.

HRMS (ESI): calcd for C₂₄H₂₁BrN₅O₅([M + H]⁺): 538.726, found:

538.0706.

3-Bromo-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]pyrimidine

(21). Compound 18 (12.7 g, 19.4 mmol) was subjected to general

procedure C (reaction time: 2 h). Puriﬁcation was performed via ﬂash

column chromatography (manual, ﬁrst 5% MeOH in CH₂Cl₂to

remove higher-running impurities, and then 15% MeOH in CH₂Cl₂)

to isolate 21 (4.68 g, 13.5 mmol, 70% yield) as a white solid. ¹H NMR

(300 MHz, DMSO-d₆) δ 3.38−3.47 (1H, m, H-5′), 3.50−3.60 (1H,

m, H-5″), 3.89 (1H, dd, J = 10.0, 4.7 Hz, H-4′), 4.16 (1H, dd, J = 9.4,

4.7 Hz, H-3′), 4.55 (1H, dd, J = 10.8, 5.6 Hz, H-2′), 4.81 (1H, t, J =

5.9 Hz, OH), 5.16 (1H, d, J = 5.3 Hz, OH), 5.40 (1H, d, J = 5.9 Hz,

OH), 6.06 (1H, d, J = 5.0 Hz, H-1′), 8.24 (1H, s, H-6) ppm. ¹³C

NMR (75 MHz, DMSO-d₆) δ 62.2 (C-5′), 70.6 (C-3′), 72.9 (C-2′),

85.3 (C-4′), 88.1 (C-1′), 99.7 (C-3a), 119.3 (C-3), 155.0 (C-7a),

157.0 (C-6), 157.4 (C-4) ppm. HRMS (ESI): calcd for

C₁₀H₁₃BrN₅O₄([M + H]⁺): 330.0202, found: 330.0198.

64.4 (C-5′), 74.6, (C-2′) 81.6 (C-4′), 91.0 (C-1′), 99.8 (C-3a), 119.6

(C-3), 155.0 (C-7a), 157.5 (C-6), 157.7 (C-4) ppm. HRMS (ESI):

calcd for C₁₀H₁₃BrN₅O₃([M + H]⁺): 330.0202, found: 330.0195.

4-Amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]pyrimidine⁷⁰(4).

Compound 21 (120 mg, 0.347 mmol) was subjected to general

procedure D (reaction time: 2 h). Flash column chromatography

(automated, 4 → 20% MeOH in CH₂Cl₂) aﬀorded 4 (57 mg, 0.213

mmol, 61% yield) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ

3.38−3.64 (2H, m, H-5′, H-5″), 3.87−3.96 (1H, m, H-4′), 4.22 (1H,

dd, J = 9.7, 4.4 Hz, H-3′), 4.60 (1H, dd, J = 10.0, 5.0 Hz, H-2′), 4.88

(1H, t, J = 5.9 Hz, OH), 5.14 (1H, d, J = 4.7 Hz, OH), 5.36 (1H, d, J

= 6.4 Hz, OH), 6.09 (1H, d, J = 4.7 Hz, H-1′), 8.18 (1H, s, H-3), 8.20

(1H, s, H-6) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ 62.5 (C-5′),

71.0 (C-2′), 73.2 (C-3′), 85.1 (C-4′), 88.6 (C-1′), 100.5 (C-3a),

133.5 (C-3), 154.1 (C-7a), 156.2 (C-6), 158.1 (C-4) ppm. HRMS

(ESI): calcd for C₁₀H₁₄N₅O₄([M + H]⁺): 268.1046, found:

268.1032. Spectral data are in accordance with literature values.⁷⁰

4-Amino-1-(3′-deoxy-3′-ﬂuoro-β-D-ribofuranosyl)pyrazolo[3,4-

d]pyrimidine (24). Compound 22 (0.058 g, 0.167 mmol) was

subjected to general procedure D (reaction time: 15 min).

Puriﬁcation via ﬂash column chromatography (automated, 2 →

15% MeOH in CH₂Cl₂) aﬀorded 24 (37 mg, 0.137 mmol, 82% yield)

1

as a white solid. H NMR (300 MHz, DMSO-d₆) δ 3.47−3.65 (2H,

m, H-5′, H-5″), 4.21 (1H, dt, J = 24.9, 4.7 Hz, H-4′), 4.93−5.31 (3H,

m, H-3′, H-2′, OH), 5.83 (1H, d, J = 6.4 Hz, OH), 6.10 (1H, d, J =

7.0 Hz, H-1′), 7.50−8.07 (2H, m, NH₂), 8.20 (2H, s, H-6, H-3) ppm.

¹³C NMR (75 MHz, DMSO-d₆) δ 61.2 (d, J = 10.4 Hz, C-5′), 71.3

(d, J = 17.3 Hz, C-2′), 83.2 (d, J = 21.9 Hz, C-4′), 87.7 (C-1′), 92.7

(d, J = 182.0 Hz, C-3′), 100.8 (C-3a), 133.6 (C-3), 154.3 (C-7a),

156.2 (C-6), 158.1 (C-4) ppm. ¹⁹F NMR (282 MHz, DMSO-d₆) δ

−198.52 (1F, dt, J = 54.1, 25.2 Hz) ppm. HRMS (ESI): calcd for

C₁₀H₁₃FN₅O₃([M + H]⁺): 270.1002, found: 270.0998.

4-Amino-1-(3′-deoxy-β-^D-ribofuranosyl)pyrazolo[3,4-d]-

pyrimidine (25). Compound 23 (0.050 g, 0.151 mmol) was subjected

to general procedure D (reaction time: 3 h). Puriﬁcation via ﬂash

column chromatography (automated, 2 → 15% MeOH in CH₂Cl₂)

1

aﬀorded 25 (26 mg, 0.100 mmol, 66% yield) as a white solid. H

NMR (300 MHz, DMSO-d₆) δ 1.98 (1H, ddd, J = 12.7, 6.2, 2.2 Hz,

H-3′), 2.33 (1H, ddd, J = 12.7, 9.1, 5.7 Hz, H-3″), 3.36−3.53 (2H, m,

H-5′, H-5″), 4.32 (1H, ddd, J = 11.4, 9.1, 5.9 Hz, H-4′), 4.51−4.61

(1H, m, H-2′), 4.74 (1H, t, J = 5.7 Hz, OH), 5.53 (1H, d, J = 3.8 Hz,

OH), 6.14 (1H, d, J = 1.5 Hz, H-1′), 7.50−7.98 (2H, br. m, NH₂),

8.15 (1H, s, H-3), 8.19 (1H, s, H-6) ppm. ¹³C NMR (75 MHz,

DMSO-d₆) δ 36.1 (C-3′), 64.2 (C-5′), 74.5 (C-2′), 81.0 (C-4′), 90.7

(C-1′), 100.1 (C-3a), 133.2 (C-3a), 153.7 (C-7a), 156.1 (C-6), 158.0

(C-4) ppm. HRMS (ESI): calcd for C₁₀H₁₄N₅O₃([M + H]⁺):

252.1079, found: 252.1075.

3-Bromo-4-oxo-1-(2′,3′,5′-tri-O-benzoyl-β-^D-ribofuranosyl)-

pyrazolo[3,4-d]pyrimidine⁶⁹(26). Compound 14 (6.45 g, 30.0

mmol, 1.0 equiv) and 1-O-acetyl-tri-O-benzoyl-β-^D-ribofuranose

(22.7 g, 45.0 mmol, 1.5 equiv) were subjected to general procedure

A to aﬀord, after recrystallization from MeOH, 26 (6.26 g, 9.49 mmol,

32% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 4.66 (1H,

dd, J = 12.2, 4.6 Hz, H-5′), 4.80 (1H, dd, J = 12.3, 3.8 Hz, H-5″),

4.84−4.89 (1H, m, H-4′), 6.26 (1H, dd, J = 6.1, 5.3 Hz, H-3′), 6.34

(1H, dd, J = 5.3, 3.0 Hz, H-2′), 6.72 (1H, d, J = 2.9 Hz, H-1), 7.34−

7.62 (9H, m, H_Phe), 7.94−8.15 (7H, m, H_Phe, H-6), 11.91 (1H, br. s.,

NH) ppm. ¹³C NMR (101 MHz, CDCl₃) δ 63.5 (C-5′), 71.6 (C-2′),

74.6 (C-3′), 80.4 (C-4′), 87.2 (C-1′), 106.0 (C-3a), 124.5 (C-3),

128.5 (C_Phe), 128.5 (C_Phe), 128.5 (C_Phe), 128.6 (C_Phe), 128.7 (C_Phe),

129.5 (C_Phe), 129.8 (C_Phe), 129.8 (C_Phe), 129.9 (C_Phe), 133.2 (C_Phe),

133.6 (C_Phe), 133.7 (C_Phe), 148.1 (H-7a), 153.9 (H-6), 158.5 (H-4),

165.1 (CO), 165.2 (CO), 166.3 (CO) ppm. HRMS (ESI):

calcd for C₃₁H₂₄BrN₄O₈([M + H]⁺): 659.0778, found: 659.0774.

3-Bromo-4-oxo-1-(2′,5′-tri-O-benzoyl-3′-deoxy-3′-ﬂuoro-β-^D-

ribofuranosyl)pyrazolo[3,4-d]pyrimidine (27). Compounds 14

(0.338 g, 1.57 mmol, 1.1 equiv) and 16 (0.575 g, 1.43 mmol, 1.0

equiv) were subjected to general procedure B. TLC analysis (5%

MeOH in CH₂Cl₂) indicated the presence of a major apolar spot and

two smaller more polar spots. The major apolar spot was presumed to

3-Bromo-4-amino-1-(3′-deoxy-3′-ﬂuoro-β-^D-ribofuranosyl)-

pyrazolo[3,4-d]pyrimidine (22). Compound 19 (used directly from

the previous reaction) was subjected to general procedure C (reaction

time: 90 min). Puriﬁcation via ﬂash column chromatography

(automated, 0 →10% MeOH in CH₂Cl₂) aﬀorded 22 (97 mg,

1

0.279 mmol, 20% yield over 2 steps) as a white solid. H NMR (300

MHz, DMSO-d₆) δ 3.47−3.54 (2H, m, H-5′, H-5″), 4.11−4.27 (1H,

m, H-4′), 4.84−5.18 (3H, m, H-3′, H-2′, OH), 5.87 (1H, d, J = 6.7

Hz, OH), 6.06 (1H, d, J = 7.3 Hz, H-1′) ppm, 6.99 (1H, br. s, OH),

8.09 (1H, br. s, OH), 8.24 (1H, s, H-6). ¹³C NMR (75 MHz, DMSO-

d₆) δ 61.4 (d, J = 10.4 Hz, C-5′), 71.6 (d, J = 16.1 Hz, C-2′), 83.8 (d,

J = 20.7 Hz, C-4′), 87.5 (C-1′), 92.9 (d, J = 182.0 Hz, C-3′), 100.5

(C-3a), 120.2 (C-3), 155.9 (C-7a), 157.6 (C-6), 157.9 (C-4) ppm.

¹⁹F NMR (282 MHz, DMSO-d₆) δ −198.19 (1F, dt, J = 54.1, 25.2

Hz) ppm. HRMS (ESI): calcd for C₁₀H₁₂BrFN₅O₃([M + H]⁺):

348.0108, found: 348.0090.

3-Bromo-4-amino-1-(3′-deoxy-β-^D-ribofuranosyl)pyrazolo[3,4-

d]pyrimidine (23). Compound 20 (used directly from the previous

reaction) was subjected to general procedure C (reaction time: 2 h).

Flash column chromatography (automated, 2 → 20% MeOH in

CH₂Cl₂) aﬀorded 23 (0.106 g, 0.321 mmol, 19% yield over 2 steps).

¹H NMR (300 MHz, DMSO-d₆) δ 1.98 (1H, ddd, J = 12.7, 6.3, 2.1

Hz, H-3′), 2.25 (1H, ddd, J = 12.8, 9.0, 5.7 Hz, H-3″), 3.38−3.50

(2H, m, H-5′, H-5″), 4.31 (1H, ddd, J = 11.6, 9.3, 5.8 Hz, H-4′), 4.55

(1H, dt, J = 5.4, 1.8 Hz, H-2′), 6.11 (1H, d, J = 1.8 Hz, H-1′), 8.24

(1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 36.3 (C-3′),

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be the desired N-1 regioisomer⁷²27 and was isolated via ﬂash

chromatography (automated, 0 → 5% MeOH in CH₂Cl₂) and used as

such in the next reaction. HRMS (ESI): calcd for C₂₄H₁₉BrFN₄O₆

([M + H]⁺): 557.0472, found: 557.0457.

3-Bromo-4-oxo-1-(2′,5′-tri-O-benzoyl-3′-deoxy-β-^D-

ribofuranosyl)pyrazolo[3,4-d]pyrimidine (28). Compounds 14

(0.348 g, 1.62 mmol, 1.1 equiv) and 17 (0.565 g, 1.47 mmol, 1.0

equiv) were subjected to general procedure B. TLC analysis (5%

MeOH in CH₂Cl₂) indicated the presence of a major apolar spot and

two smaller more polar spots. The major apolar spot was presumed to

be the desired N-1 regioisomer⁷²28 and was isolated via ﬂash

chromatography (automated, 0 → 5% MeOH in CH₂Cl₂) and used as

such in the next reaction. HRMS (ESI): calcd for C₂₄H₂₀BrN₄O₆([M

+ H]⁺): 539.0566, found: 539.0552.

269.0886, found: 269.0891. Spectral data are in accordance with

literature values.⁷²

4-Oxo-1-(3′-deoxy-3′-ﬂuoro-β-D-ribofuranosyl)pyrazolo[3,4-d]-

pyrimidine (32). Compound 30 (0.031 g, 0.089 mmol) was subjected

to general procedure D (reaction time: 30 min). Puriﬁcation via ﬂash

column chromatography aﬀorded 32 (20 mg, 0.074 mmol, 83% yield)

1

as a white solid. H NMR (300 MHz, DMSO-d₆) δ 3.47−3.57 (2H,

m, H-5′, H-5″), 4.13−4.29 (1H, m, H-4′), 4.89−5.22 (2H, m, H-3′,

H-2′), 6.09 (1H, d, J = 7.0 Hz, H-1′), 8.15 (1H, s, H-6), 8.22 (1H, s,

H-4), 12.22 (1H, br. s, NH) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ

61.0 (d, J = 10.4 Hz, C-5′), 71.6 (d, J = 16.1 Hz, C-2′), 83.4 (d, J =

20.7 Hz, C-4′), 87.1 (C-1′), 92.5 (d, J = 182.0 Hz, C-3′), 106.6 (C-

3a), 135.8 (C-3), 148.9 (C-7a), 153.5 (C-6), 157.0 (C-4) ppm. ¹⁹F

NMR (282 MHz, DMSO-d₆) δ −198.82 (1F, dt, J = 54.1, 24.8 Hz)

ppm. HRMS (ESI): calcd for C₁₀H₁₂FN₄O₄([M + H]⁺): 271.0843,

found: 271.0834.

3-Bromo-4-oxo-1-β-^D-ribofuranosyl-pyrazolo[3,4-d]pyrimidine⁶⁹

(29). Compound 26 (0.530 g, 0.804 mmol) was subjected to general

procedure C (reaction time: 1 h). Puriﬁcation via ﬂash column

chromatography (automated, 4 → 20% MeOH in CH₂Cl₂) aﬀorded

29 (105 mg, 0.302 mmol, 38% yield) as a white solid. ¹H NMR (300

MHz, DMSO-d₆) δ 3.36−3.47 (1H, m, H-5′), 3.48−3.59 (1H, m, H-

5″), 3.88 (1H, dd, J = 10.3, 4.7 Hz, H-4′), 4.10−4.18 (1H, m, H-3′),

4.44−4.53 (1H, m, H-2′), 4.66−4.80 (1H, m, OH), 5.16 (1H, br. s.,

OH), 5.42 (1H, br. s., OH), 6.00 (1H, d, J = 5.0 Hz, H-1′), 8.15 (1H,

s, H-6), 12.41 (1H, br. s, NH) ppm. HRMS (ESI): calcd for

C₁₀H₁₂BrN₄O₅([M + H]⁺): 346.9991, found: 347.0004. Spectral data

are in accordance with literature values.⁶⁹

4-Oxo-1-(3′-deoxy-β-^D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine

(33). Compound 31 (0.023 g, 0.069 mmol) was subjected to general

procedure D (reaction time: 30 min). Puriﬁcation via ﬂash column

chromatography aﬀorded 33 (14 mg, 0.056 mmol, 80% yield) as a

white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.91−2.05 (1H, m, H-

3′), 2.23−2.37 (1H, m, H-3″), 3.33−3.56 (2H, m, H-5′, H-5″), 4.25−

4.42 (1H, m, H-4′), 4.49−4.63 (1H, m, H-2′), 4.85 (1H, br. s, OH),

5.59 (1H, br. s, OH), 6.12 (1H, s, H-1′), 8.13 (1H, s, H-3), 8.14 (1H,

s, H-6), 12.2 (1H, s, NH) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ

36.0 (C-3′), 64.1 (C-5′), 74.7 (C-2′), 81.4 (C-4′), 90.7 (C-1′), 105.9

(C-3a), 135.4 (C-3), 148.5 (C-7a), 152.4 (C-6), 157.1 (C-4) ppm.

HRMS (ESI): calcd for C₁₀H₁₃N₄O₄([M + H]⁺): 253.0937, found:

253.0921.

3-Iodo-4-amino-1-(2′,3′,5′-tri-O-benzoyl-β-^D-ribofuranosyl)-

pyrazolo[3,4-d]pyrimidine⁹⁵(34). Compound 13 (10.1 g, 39.1

mmol, 1.0 equiv) and 1-O-acetyl-2,3,5-tri-O-benzoyl-β-^D-ribofuranose

(29.6 g, 58.7 mmol, 1.5 equiv) were subjected to general procedure A

to aﬀord 34 (21.2 g, 30.0 mmol, 77% yield) as a yellow oil. ¹H NMR

(300 MHz, CDCl₃) δ 4.56−4.90 (3H, m, H-5′, H-5″, H-4′), 6.20

(1H, t, J = 5.6 Hz, H-3′), 6.36 (1H, dd, J = 5.0, 3.2 Hz, H-2′), 6.76

(1H, d, J = 2.9 Hz, H-2′), 7.35−7.62 (9H, m, H_Phe), 7.91−8.14 (6H,

m, H_Phe), 8.34 (1H, s, H-6) ppm. HRMS (ESI): calcd for

C₃₁H₂₅IN₅O₇([M + H]⁺): 706.0799, found: 706.0824. Spectral

data are in accordance with literature values.⁹⁵

3-Bromo-4-oxo-1-(3′-deoxy-3′-ﬂuoro-β-D-ribofuranosyl)-

pyrazolo[3,4-d]pyrimidine (30). Compound 27 (used directly from

the previous step) was subjected to general procedure C (reaction

time: 3 h). Flash column chromatography (automated, 2 → 12%

MeOH in CH₂Cl₂), followed by an additional puriﬁcation via

preparative RP-HPLC (0.2% formic acid in H₂O/MeCN 98:02 to

0:100 in 18 min) aﬀorded 30 (56 mg, 0.160 mmol, 11% yield over 2

steps) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 3.44−3.62

(2H, m, H-5′, H-5″), 4.21 (1H, dt, J = 25.8, 5.3 Hz, H-4′), 4.90 (1H,

ddd, J = 24.6, 7.0, 4.4 Hz, H-2′), 5.08 (1H, dd, J = 54.2, 4.1 Hz, H-3′),

6.03 (1H, d, J = 7.0 Hz, H-1′), 8.18 (1H, s, H-6), 12.43 (1H, br. s,

NH) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 60.9 (d, J = 10.4 Hz, C-

5′), 71.5 (d, J = 16.1 Hz, C-2′), 83.6 (d, J = 20.7 Hz, C-4′), 87.0 (C-

1′), 92.3 (d, J = 183.1 Hz, C-3′), 105.4 (C-3a), 122.8 (C-3), 150.3

(C-7a), 154.5 (C-6), 156.3 (C-4) ppm. ¹⁹F NMR (282 MHz, DMSO-

d₆) δ −198.39 (1F, dt, J = 54.1, 25.2 Hz) ppm. HRMS (ESI): calcd

for C₁₀H₁₁BrFN₄O₄([M + H]⁺): 348.9948, found: 348.9996.

3-Bromo-4-oxo-1-(3′-deoxy-β-^D-ribofuranosyl)pyrazolo[3,4-d]-

pyrimidine (31). Compound 28 (used directly from the previous

step) was subjected to general procedure C (reaction time: 3 h). Flash

column chromatography (automated, 2 → 15% MeOH in CH₂Cl₂),

followed by an additional puriﬁcation via preparative RP-HPLC (0.2%

formic acid in H₂O/MeCN 98:02 to 30:70 in 18 min) aﬀorded 31

(31 mg, 0.093 mmol, 6% yield over 2 steps) as a white solid. ¹H NMR

(300 MHz, CD₃OD) δ 2.10 (1H, ddd, J = 13.1, 6.4, 1.9 Hz, H-3′),

2.48 (1H, ddd, J = 13.2, 9.4, 5.6 Hz, H-3″), 3.62 (1H, dd, J = 11.7, 5.9

Hz, H-5′), 3.70 (1H, dd, J = 11.7, 4.1 Hz, H-5″), 4.46−4.56 (1H, m,

H-4′), 4.67 (1H, dt, J = 5.6, 1.6 Hz, H-2′), 6.24 (1H, d, J = 1.5 Hz, H-

1′), 8.05 (1H, s, H-6) ppm. ¹³C NMR (75 MHz, CD₃OD) δ 37.0 (C-

3′), 66.1 (C-5′), 77.3 (C-2′), 83.6 (C-4′), 93.3 (C-1′), 107.0 (C-3a),

124.4 (C-3), 150.5 (C-7a), 155.5 (C-6), 159.1 (C-4) ppm. HRMS

(ESI): calcd for C₁₀H₁₂BrN₄O₄([M + H]⁺): 331.0042, found:

331.0047.

4-Amino-1-(2′,3′,5′-tri-O-benzoyl-β-D-ribofuranosyl)pyrazolo-

[3,4-d]pyrimidine⁷⁰(35). 4-Aminopyrazolo[3,4-d]pyrimidine 3 (3.16

g, 23.4 mmol, 1.0 equiv) and 1-O-acetyl-2,3,5-tri-O-benzoyl-β-^D-

ribofuranose (17.7 g, 35.1 mmol, 1.5 equiv) were subjected to general

procedure A to aﬀord 35 (8.06 g, 13.9 mmol, 59% yield) as a colorless

foam. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 4.54 (1H, dd, J = 12.3,

4.4 Hz, H-5′), 4.68 (1H, dd, J = 12.3, 3.5 Hz, H-5″), 4.89−4.95 (1H,

m, H-4′), 6.17−6.33 (2H, m, H-2′, H-3′), 6.75 (1H, d, J = 2.6 Hz, H-

1′), 7.40−7.56 (6H, m, H_Phe), 7.59−7.72 (3H, m, H_Phe), 7.87−8.03

(6H, m, H_Phe), 8.47 (1H, br. s, NH₂), 8.40 (1H, s, H-3), 8.43 (1H, s,

H-6), 9.13 (1H, br. s., NH₂′) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ

63.2 (C-5′), 71.0 (C-2′), 74.2 (C-3′), 79.1 (C-4′), 86.2 (C-1′), 100.3

(-3a), 128.4 (C_Phe), 128.5 (C_Phe), 128.6 (C_Phe), 128.7 (C_Phe), 128.8

(C_Phe), 129.2 (C_Phe), 129.3 (C_Phe), 129.4 (C_Phe), 133.5 (C_Phe), 133.9

(C_Phe), 134.0 (C_Phe), 135.7 (C-3), 152.0 (C-7a), 152.8 (C-6), 154.5

(C-4), 164.6 (CO), 164.7 (CO), 165.4 (CO) ppm. HRMS

(ESI): calcd for C₃₁H₂₆N₅O₇([M + H]⁺): 580.1832, found:

580.1793. Spectral data are in accordance with literature values.⁷⁰

3-Iodo-4-amino-1-β-D-ribofuranosylpyrazolo[3,4-d]pyrimidine⁹⁵

(36). Compound 34 (7.43 g, 10.5 mmol) was subjected to general

procedure C, with the exception that the temperature was raised to 60

°C to aid dissolution, (reaction time: 2 h). Puriﬁcation via ﬂash

column chromatography (manual, 5% MeOH in CH₂Cl₂to remove

higher-running impurities and then 15% MeOH in CH₂Cl₂) aﬀorded

4-Oxo-1-β-^D-ribofuranosylpyrazolo[3,4-d]pyrimidine (Allopuri-

nol Riboside)⁷²(2). Compound 29 (65 mg, 0.187 mmol) was

submitted to general procedure D (reaction time: 1 h). Flash column

chromatography (automated, 10 → 35% MeOH in CH₂Cl₂) aﬀorded

1

2 (27 mg, 0.101 mmol, 54% yield) as a white solid. H NMR (300

1

MHz, DMSO-d₆) δ 3.43 (1H, dd, J = 11.7, 5.9 Hz, H-5′), 3.57 (1H,

dd, J = 11.7, 4.4 Hz, H-5″), 3.91 (1H, dd, J = 10.0, 5.0 Hz, H-4′), 4.21

(1H, t, J = 4.8 Hz, H-3′), 4.54 (1H, m, J = 4.5, 4.5 Hz, H-2′), 4.77

(1H, br. s., OH), 4.96−5.63 (2H, m, OH, OH), 6.07 (1H, d, J = 4.4

Hz, H-1′), 8.13 (1H, s, H-3), 8.17 (1H, s, H-6), 12.37 (1H, br. s,

NH) ppm. HRMS (ESI): calcd for C₁₀H₁₃N₄O₅([M + H]⁺):

36 (2.63 g, 6.69 mmol, 64% yield) as an oﬀ-white solid. H NMR

(300 MHz, DMSO-d₆) δ 3.43 (1H, dd, J = 11.7, 5.6 Hz, H-5′), 3.55

(1H, dd, J = 11.7, 4.4 Hz, H-5″), 3.89 (1H, dd, J = 10.0, 4.4 Hz, H-

4′), 4.16 (1H, t, J = 4.7 Hz, H-3′), 4.53−4.60 (1H, m, H-2′), 4.82

(1H, br. s, OH), 5.15 (1H, br. s, OH), 5.38 (1H, br. s, OH), 6.03

(1H, d, J = 5.0 Hz, H-1′), 8.23 (1H, s, H-6) ppm. ¹³C NMR (75

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MHz, DMSO-d₆) δ 62.7 (C-5′), 71.1 (C-2′), 73.3 (C-3′), 85.7 (C-

4′), 88.7 (C-1′), 103.9 (C-3a), 121.0 (C-3), 154.9 (C-7a), 156.8 (C-

6), 158.2 (C-4) ppm. HRMS (ESI): calcd for C₁₀H₁₃IN₅O₄([M +

H]⁺): 394.0012, found: 394.0021.

3-Bromo-4-chloropyrazolo[3,4-d]pyrimidine (37). 4-

Chloropyrazolo[3,4-d]pyrimidine (5.00 g, 32.4 mmol, 1.0 equiv)

was dissolved in DMF (50 mL). NBS (6.33 g, 35.6 mmol, 1.1 equiv)

was added, and the mixture was heated at 50 °C for 3 h. H₂O (100

mL) was added, and the mixture was extracted with EtOAc (3 ×150

mL). The combined organic phases were dried over Na₂SO₄and

concentrated in vacuo. The residue was used crude in the next

reaction. HRMS (ESI): calcd for C₅H₂BrClN₄([M + H]⁺): 232.9230,

found: 232.9242.

C₁₀H₁₃BrN₅O₃([M + H]⁺): 330.0202, found: 330.0231. Spectral

data are in accordance with literature values.⁶⁴

4-Amino-1-(2′-deoxy-β-D-ribofuranosyl)pyrazolo[3,4-d]-

pyrimidine⁷⁴(41). Compound 40 (72 mg, 0.218 mmol) was

subjected to general procedure D (reaction time: 30 min).

Puriﬁcation via ﬂash column chromatography (automated, 5 →

15% MeOH in CH₂Cl₂) aﬀorded 41 (52 mg, 0.100 mmol, 46% yield)

as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 2.23 (1H, ddd, J =

13.1, 6.7, 3.9 Hz, H-2′), 2.80 (1H, dt, J = 13.0, 6.3 Hz, H-2″), 3.33−

3.41 (1H, m, H-5′, partially under water peak), 3.52 (1H, dd, J = 11.4,

5.3 Hz, H-5″), 3.81 (1H, td, J = 5.6, 3.5 Hz, H-4′), 4.43 (1H, br. s., H-

3′), 4.80 (1H, br. s., OH), 5.24 (1H, br. s., OH), 6.54 (1H, t, J = 6.5

Hz, H-1′), 7.45−7.98 (2H, m, NH₂), 8.15 (1H, s, H-3), 8.19 (1H, s,

H-6) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ 38.0 (C-2′), 62.5 (C-

5′), 71.1 (C-3′), 84.0 (C-4′), 87.6 (C-1′), 100.5 (C-3a), 133.1 (C-3),

153.7 (C-7a), 156.0 (C-6), 158.0 (C-4) ppm. HRMS (ESI): calcd for

C₁₀H₁₄N₅O₃([M + H]⁺): 252.1097, found: 252.1084. Spectral data

are in accordance with literature values.⁹⁶

3-Bromo-4-methoxypyrazolo[3,4-d]pyrimidine⁷³(38). Com-

pound 37 (crude) was dissolved in MeOH (50 mL). NaOMe (5.4

M in MeOH, 20 mL) was added, and the mixture was heated at 70 °C

for 2 h. The reaction was quenched via the addition of aq. sat. NH₄Cl

(200 mL) and H₂O (50 mL). The mixture was extracted with EtOAc

(3 × 250 mL), and the combined organic phases were dried over

Na₂SO₄and concentrated in vacuo. The residue was puriﬁed by ﬂash

column chromatography (manual, petroleum ether/EtOAc 60:40) to

aﬀord 38 (3.76 g, 16.4 mmol, 51% yield over 2 steps) as an oﬀ-white

3-Phenyl-4-amino-1-β-D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine⁹⁷(42). Compound 21 (0.132 g, 0.38 mmol) was

subjected to general procedure E, using phenylboronic acid as the

coupling partner and Na₂CO₃as the base. Puriﬁcation via ﬂash

column chromatography (automated, 2 → 15% MeOH in CH₂Cl₂),

followed by an additional puriﬁcation by preparative RP-HPLC (0.2%

formic acid in H₂O/MeCN 0:100 to 49:51 in 18 min) aﬀorded 42

1

solid. H NMR (400 MHz, DMSO-d₆) δ 4.10 (3H, s, CH₃), 8.56

(1H, s, H-6), 14.27 (1H, br. s., NH) ppm. ¹³C NMR (101 MHz,

DMSO-d₆) δ 54.4 (C-5′), 101.4 (C-3a), 118.1 (C-3), 156.0 (C-7a),

156.4 (C-6), 163.0 (C-4) ppm. HRMS (ESI): calcd for C₆H₆BrN₄O

([M + H]⁺): 228.9725, found: 228.9738. Spectral data are in

accordance with literature values.⁷³

1

(44 mg, 0.118 mmol, 34% yield) as a white solid. H NMR (300

MHz, DMSO-d₆) δ 3.42−3.53 (1H, m, H-5′), 3.55−3.67 (1H, m, H-

5″), 3.93 (1H, dd, J = 10.0, 4.7 Hz, H-4′), 4.22−4.30 (1H, m, H-3′),

4.60−4.70 (1H, m, H-2′), 4.77−4.89 (1H, m, OH), 5.06−5.20 (1H,

m, OH), 5.33−5.49 (1H, m, OH), 6.19 (1H, d, J = 4.7 Hz, H-1′),

7.47−7.62 (3H, m, H_Phe), 7.65−7.72 (2H, m, H_Phe), 8.29 (1H, s, H-6)

ppm. HRMS (ESI): calcd for C₁₆H₁₈N₅O₄([M + H]⁺): 344.1359,

found: 344.1356. Spectral data are in accordance with literature

values.⁹⁷

3-Bromo-4-methoxy-1-(2′-deoxy-3′,5′-di-O-(p-toluoyl)-β-D-

ribofuranosyl)pyrazolo[3,4-d]pyrimidine⁷³(39). Powdered KOH

(1.60 g, 28.6 mmol, 4.0 equiv) and TDA-1 (0.227 mL, 0.71 mmol,

0.1 equiv) were added to a suspension of 38 (1.64 g, 7.14 mmol, 1.0

equiv) in MeCN (250 mL). The mixture was stirred for 20 min before

Hoﬀer’s chlorosugar (2.64 g, 6.78 mmol, 0.95 equiv) was added. The

mixture was stirred for another 30 min and ﬁltered over celite. The

ﬁltrate was concentrated in vacuo, and the residue was adsorbed onto

celite and puriﬁed via ﬂash column chromatography (automated, 5 →

35% EtOAc in petroleum ether) to aﬀord 39 (310 mg, 0.55 mmol, 8%

3-(4-Methoxyphenyl)-4-amino-1-β-D-ribofuranosylpyrazolo[3,4-

d]pyrimidine (43). Compound 21 (0.087 g, 0.25 mmol) was

subjected to general procedure E, using 4-methoxyphenylboronic

acid as the coupling partner and Na₂CO₃as the base. Puriﬁcation via

ﬂash column chromatography (automated, 1 → 12% MeOH in

CH₂Cl₂), followed by an additional puriﬁcation by preparative RP-

HPLC (0.2% formic acid in H₂O/MeCN 98:02 to 41:59 in 10.5 min)

1

yield) as a white solid. H NMR (400 MHz, CDCl₃) δ 2.41 (3H, s,

CH_{3 toluoyl}), 2.44 (3H, s, CH_{3 toluoyl}), 2.67 (1H, ddd, J = 14.3, 6.4, 3.0

Hz, H-2′), 3.52 (1H, dt, J = 14.0, 6.8 Hz, H-2″), 4.19 (3H, s, OCH₃),

4.47−4.68 (3H, m, H-5′, H-5″, H-4′), 5.80−5.92 (1H, m, H-3′), 6.91

(1H, t, J = 6.7 Hz, H-1′), 7.18−7.33 (4H, m, H_toluoyl), 7.88−7.99 (2H,

m, H_toluoyl), 8.00−8.07 (2H, m, H_toluoyl), 8.51−8.63 (1H, s, H-6) ppm.

¹³C NMR (101 MHz, CDCl₃) δ 21.7 (2 × CH_{3 toluoyl}), 35.8 (OCH₃),

54.6 (C-2′), 64.1 (C-5′), 75.4 (C-3′), 82.8 (C-4′), 85.0 (C-1′), 103.8

(C-3a), 120.4 (C-3), 126.6 (C_toluoyl), 127.0 (C_toluoyl), 129.1 (C_toluoyl),

129.2 (C_toluoyl), 129.8 (C_toluoyl), 129.9 (C_toluoyl), 143.7 (C_toluoyl), 144.3

(C_toluoyl), 156.4 (-6), 163.9 (C-4), 165.9 (CO), 166.3 (CO)

ppm. HRMS (ESI): calcd for C₂₇H₂₆BrN₄O₆([M + H]⁺): 581.1036,

found: 581.1009. Spectral data are in accordance with literature

values.⁷³

4-Amino-3-bromo-1-(2′-deoxy-β-D-ribofuranosyl)pyrazolo[3,4-

d]pyrimidine⁶⁴(40). Compound 39 (0.310 g, 0.48 mmol) was stirred

in 7 N NH₃in MeOH (20 mL) in a pressure tube at 90 °C for 24 h.

The reaction vessel was cooled down to room temperature before it

was opened, and its contents were transferred to a pear-shaped ﬂask.

The volatiles were removed in vacuo, and the residue was adsorbed

onto celite and puriﬁed by ﬂash column chromatography (automated,

2 → 10% MeOH in CH₂Cl₂+ 1% NH₄OH) to aﬀord 40 (120 mg,

0.363 mmol, 76% yield) as a white solid. ¹H NMR (400 MHz,

DMSO-d₆) δ 2.25 (1H, m, J = 13.3, 6.8, 4.2 Hz, H-2′), 2.74 (1H, dt, J

= 13.0, 6.3 Hz, H-2″), 3.36 (1H, dt, J = 11.6, 5.9 Hz, H-5′), 3.50 (1H,

dt, J = 11.4, 5.6 Hz, H-5″), 3.80 (1H, td, J = 5.7, 3.6 Hz, H-4′), 4.34−

4.46 (1H, m, H-3′), 4.75 (1H, t, J = 5.8 Hz, OH), 5.27 (1H, d, J = 4.6

Hz, OH), 6.51 (1H, t, J = 6.4 Hz, H-1′), 8.23 (1H, s, H-6) ppm. ¹³C

NMR (101 MHz, DMSO-d₆) δ 37.8 (C-2′), 62.3 (C-5′), 70.8 (C-3′),

83.9 (C-4′), 87.7 (C-1′), 99.8 (C-3a), 119.0 (C-3), 154.5 (C-7a),

157.0 (C-6), 157.4 (C-4) ppm. HRMS (ESI): calcd for

1

aﬀorded 43 (42 mg, 0.112 mmol, 45% yield) as a white solid. H

NMR (300 MHz, DMSO-d₆) δ 3.46 (1H, dd, J = 11.7, 5.6 Hz, H-5′),

3.60 (1H, dd, J = 11.7, 4.1 Hz, H-5″), 3.84 (3H, s, CH₃), 3.93 (1H,

dd, J = 9.6, 4.7 Hz, H-4′), 4.26 (1H, t, J = 4.8 Hz, H-3′), 4.65 (1H, t, J

= 4.8 Hz, H-2′), 6.18 (1H, d, J = 4.7 Hz, H-1′), 7.05−7.19 (2H, m,

H_Phe), 7.55−7.67 (2H, m, H_Phe), 8.27 (1H, s, H-6) ppm. ¹³C NMR

(75 MHz, DMSO-d₆) δ 55.7 (CH₃), 62.8 (C-5′), 71.4 (C-2′), 73.7

(C-3′), 85.6 (C-4′), 88.8 (C-1′), 98.2 (C-3a), 115.1 (C_Phe), 125.3

(C_Phe), 130.0 (C_Phe), 145.2 (C-3), 155.7 (C-7a), 156.4 (C-6), 158.7

(C-4), 160.3 (C_Phe) ppm. HRMS (ESI): calcd for C₁₇H₂₀N₅O₅([M +

H]⁺): 374.1464, found: 374.1455.

3-(4-Chlorophenyl)-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-

d]pyrimidine (44). Compound 21 (0.087 g, 0.25 mmol) was

subjected to general procedure E, using 4-chlorophenylboronic acid

as the coupling partner and Na₂CO₃as the base. Puriﬁcation via ﬂash

column chromatography (automated, 1 → 12% MeOH in CH₂Cl₂),

followed by an additional puriﬁcation by preparative RP-HPLC (0.2%

formic acid in H₂O/MeCN 0:100 to 41:59 in 10.5 min) aﬀorded 44

1

(38 mg, 0.101 mmol, 40% yield) as a white solid. H NMR (300

MHz, DMSO-d₆) δ 3.44 (1H, dd, J = 11.7, 5.6 Hz, H-5′), 3.58 (1H,

dd, J = 12.0, 4.7 Hz, H-5″), 3.91 (1H, dd, J = 10.0, 5.0 Hz, H-4′), 4.25

(1H, t, J = 4.7 Hz, H-3′), 4.63 (1H, t, J = 4.8 Hz, H-2′), 4.86 (1H, br.

s, OH), 5.33 (2H, br. s, OH, OH), 6.17 (1H, d, J = 4.4 Hz, H-1′),

6.99 (2H, br. s, NH₂), 7.57−7.70 (4H, m, H_Phe), 8.26 (1H, s, H-6)

ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.3 (C-5′), 70.8 (C-2′),

73.2 (C-3′), 85.2 (C-4′), 88.4 (C-1′), 97.8 (C-3a), 129.2 (C_Phe),

130.0 (C_Phe), 131.4 (C_Phe), 133.7 (C_Phe), 143.8 (C-3), 155.4 (C-7a),

156.1 (C-6), 158.2 (C-4) ppm. HRMS (ESI): calcd for

C₁₆H₁₇ClN₅O₄([M + H]⁺): 378.0969, found: 378.0981.

4223

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Article

3-(4-Methylphenyl)-4-amino-1-β-D-ribofuranosylpyrazolo[3,4-

d]pyrimidine (45). Compound 21 (0.094 g, 0.27 mmol) was

subjected to general procedure E, using 4-methylphenylboronic acid

as the coupling partner and Na₂CO₃as the base. Puriﬁcation via ﬂash

column chromatography (automated, 2 → 15% MeOH in CH₂Cl₂),

followed by an additional puriﬁcation by preparative RP-HPLC (0.2%

formic acid in H₂O/MeCN 98:02 to 41:59 in 10.5 min) aﬀorded 45

(1H, t, J = 5.0 Hz, H-2′), 4.86 (1H, br. s, OH), 5.17 (1H, br. s, OH),

5.44 (1H, br. s, OH), 6.19 (1H, d, J = 4.4 Hz, H-1′), 7.54−7.68 (4H,

m, H_Phe), 8.28 (1H, s, C-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ

31.0 (CH(CH₃)₃), 34.5 (CH(CH₃)₃), 62.4 (C-5′), 70.9 (C-2′), 73.3

(C-3′), 85.2 (C-4′), 88.4 (C-1′), 97.8 (C-3a), 126.0 (C-3_Phe, C-5_Phe),

127.9 (C-2_Phe,C-6_Phe), 129.8 (C-1_Phe), 144.8 (C-3), 151.4 (C-4_Phe),

155.3 (C-7a), 156.0 (C-6), 158.2 (C-4) ppm. HRMS (ESI): calcd for

C₂₀H₂₆N₅O₄([M + H]⁺): 400.1985, found: 400.1972.

1

(40 mg, 0.112 mmol, 42% yield) as a white solid. H NMR (300

MHz, DMSO-d₆) δ 2.40 (3H, s, CH₃), 3.40−3.52 (1H, m, H-5′),

3.54−3.67 (1H, m, H-5″), 3.93 (1H, dd, J = 9.1, 5.0 Hz, H-4′), 4.27

(1H, q, J = 4.7 Hz, H-3′), 4.65 (1H, q, J = 5.0 Hz, H-2′), 4.85 (1H, t,

J = 5.7 Hz, OH), 5.14 (1H, d, J = 5.3 Hz, OH), 5.41 (1H, d, J = 5.6

Hz, OH), 6.19 (1H, d, J = 4.4 Hz, H-1′), 7.38 (2H, d, J = 7.9 Hz,

H_Phe), 7.57 (2H, d, J = 7.9 Hz, H_Phe), 8.27 (1H, s, H-6) ppm. ¹³C

NMR (75 MHz, DMSO-d₆) δ 20.9 (CH₃), 62.4 (C-5′), 70.9 (C-2′),

73.2 (C-3′), 85.2 (C-4′), 88.4 (C-1′), 97.8 (C-3a), 128.1 (C_Phe),

129.8 (C_Phe), 138.4 (C_Phe), 144.9 (C-3), 155.3 (C-7a), 156.0 (C-6),

158.2 (C-4) ppm. HRMS (ESI): calcd for C₁₇H₂₀N₅O₄([M + H]⁺):

358.1515, found: 358.1500.

3 - ( 4 - T r i ﬂ u o r o m e t h y l p h e n y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (49). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 4-

triﬂuoromethylphenylboronic acid as the coupling partner and

Na₂CO₃as the base. Puriﬁcation via ﬂash column chromatography

(automated, 2 → 15% MeOH in CH₂Cl₂), followed by an additional

puriﬁcation by preparative RP-HPLC (0.2% formic acid in H₂O/

MeCN 98:02 to 33:67 in 12 min) aﬀorded 49 (67 mg, 0.163 mmol,

1

47% yield) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 3.48

(1H, dd, J = 11.7, 5.0 Hz, H-5′), 3.60 (1H, dd, J = 11.4, 3.5 Hz, H-

5″), 3.94 (1H, dd, J = 10.0, 4.7 Hz, H-4′), 4.27 (1H, t, J = 4.5 Hz, H-

3′), 4.66 (1H, t, J = 4.5 Hz, H-2′), 4.84 (1H, br. s., OH), 5.18 (1H, br.

s., OH), 5.45 (1H, br. s., OH), 6.21 (1H, d, J = 4.4 Hz, H-1′), 7.86−

7.96 (4H, m, H_Phe), 8.30 (1H, s, H-6) ppm. ¹³C NMR (75 MHz,

DMSO-d₆) δ 62.8 (C-5′), 71.3 (C-2′), 73.7 (C-3′), 85.7 (C-4′), 88.9

(C-1′), 98.3 (C-3a), 126.5 (q, J = 3.5 Hz, C-3Phe, C-5Phe), 129.4 (q,

J = 32.2 Hz, C-4Phe), 129.5 (C-2Phe, C-6Phe), 137.0 (C-4Phe),

144.0 (C-3), 156.0 (C-7a), 156.6 (C-6), 158.6 (C-4) ppm. 1

quaternary carbon (CF₃) missing. ¹⁹F NMR (282 MHz, DMSO-d₆) δ

−61.06 (1F, s) ppm. HRMS (ESI): calcd for C₁₇H₁₇F₃N₅O₄([M +

H]⁺): 412.1233, found: 412.1225.

3 - ( 4 - T r i ﬂ u o r o m e t h o x y p h e n y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (50). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 4-

triﬂuoromethoxyphenylboronic acid as the coupling partner and

Na₂CO₃as the base. Puriﬁcation via ﬂash column chromatography

(automated, 4 → 15% MeOH in CH₂Cl₂), followed by an additional

puriﬁcation by preparative RP-HPLC (0.2% formic acid in H₂O/

MeCN 98:02 to 0:100 in 18 min) aﬀorded 50 (40 mg, 0.094 mmol,

27% yield) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 3.37−

3.50 (1H, m, H-5′), 3.53−3.66 (1H, m, H-5″), 3.93 (1H, dd, J = 10.0,

5.6 Hz, H-4′), 4.26 (1H, dd, J = 7.0, 5.0 Hz, H-3′), 4.65 (0 H, dd, J =

8.2, 4.1 Hz, H-2′), 4.84 (1H, t, J = 5.3 Hz, OH), 5.18 (1H, br. s.,

OH), 5.45 (1H, d, J = 4.4 Hz, OH), 6.19 (1H, d, J = 4.7 Hz, H-1′),

7.54 (2H, dd, J = 8.8, 0.9 Hz, H_Phe), 7.71−7.89 (2H, m, H_Phe), 8.29

(1H, s, H_Phe) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.8 (C-5′),

71.3 (C-2′), 73.7 (C-3′), 85.7 (C-4′), 88.9 (C-1), 98.3 (C-3a), 120.60

(q, J = 255.0 Hz, CF₃), 122.1 (C-3_Phe, C-5_Phe), 130.7 (C-2_Phe, C-6_Phe),

132.3 (C-1), 144.1 (C-3), 149.1 (C-4_Phe), 155.9 (C-7a), 156.5 (C-6),

158.7 (C-4) ppm. ¹⁹F NMR (282 MHz, DMSO-d₆) δ −56.61 (1F, s)

ppm. HRMS (ESI): calcd for C₁₇H₁₇F₃N₅O₅([M + H]⁺): 428.1182,

found: 428.1204.

3-(4-Fluorophenyl)-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (46). Compound 21 (0.120 g, 0.35 mmol) was subjected

to general procedure E, using 4-ﬂuorophenylboronic acid as the

coupling partner and Na₂CO₃as the base. Puriﬁcation via ﬂash

column chromatography (automated, 2 → 15% MeOH in CH₂Cl₂),

followed by an additional puriﬁcation by preparative RP-HPLC (0.2%

formic acid in H₂O/MeCN 98:02 to 32:68 in 12 min) aﬀorded 46

1

(66 mg, 0.183 mmol, 52% yield) as a white solid. H NMR (300

MHz, DMSO-d₆) δ 3.46 (1H, dd, J = 11.9, 5.7 Hz, H-5′), 3.60 (1H,

dd, J = 11.7, 4.4 Hz, H-5″), 3.93 (1H, dd, J = 10.0, 4.4 Hz, H-4′), 4.26

(1H, t, J = 4.7 Hz, H-3′), 4.65 (1H, t, J = 4.7 Hz, H-2′), 4.85 (1H, br.

s, OH), 5.13 (1H, br. s, OH), 5.40 (1H, br. s, OH), 6.18 (1H, d, J =

4.4 Hz, H-1′), 6.97 (2H, br. s, NH₂), 7.32−7.47 (2H, m, H_Phe), 7.63−

7.76 (2H, m, H_Phe), 8.28 (1H, s, H-6) ppm. ¹³C NMR (75 MHz,

DMSO-d₆) δ 62.8 (C-5′), 71.3 (C-2′), 73.6 (C-3′), 85.6 (C-4′), 88.8

(C-1′), 98.3 (C-3a), 116.6 (d, J = 21.9 Hz, C-3_Phe,C-5_Phe), 129.5 (d, J

= 3.5 Hz, C-1_Phe),130.9 (d, J = 9.2 Hz, C-2_Phe,C-6_Phe), 144.4 (C-3),

155.8 (C-7a), 156.5 (C-6), 158.6 (C-4), 163.0 (d, J = 245.0 Hz, C-

4_Phe) ppm. ¹⁹F NMR (282 MHz, DMSO-d₆) δ −113.07 to −112.95

(1F, m) ppm. HRMS (ESI): calcd for C₁₆H₁₇FN₅O₄([M + H]⁺):

362.1265, found: 362.1265.

3-(4-Nitrophenyl)-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (47). Compound 21 (0.120 g, 0.35 mmol) was subjected

to general procedure E, using 4-nitrophenylboronic acid as the

coupling partner and Na₂CO₃as the base. Puriﬁcation via ﬂash

column chromatography (automated, 2 → 15% MeOH in CH₂Cl₂),

followed by an additional puriﬁcation by preparative RP-HPLC (0.2%

formic acid in H₂O/MeCN 98:02 to 32:68 in 12 min) aﬀorded 47

1

(70 mg, 0.181 mmol, 52% yield) as a light-brown solid. H NMR

(300 MHz, DMSO-d₆) δ 3.47 (1H, dd, J = 11.7, 5.9 Hz, H-5′), 3.61

(1H, dd, J = 11.7, 4.7 Hz, H-5″), 3.95 (1H, dd, J = 10.0, 4.7 Hz, H-

4′), 4.28 (1H, t, J = 4.4 Hz, H-3′), 4.67 (1H, br. s., H-2′), 4.84 (1H,

br. s., OH), 5.16 (1H, br. s., OH), 5.43 (1H, br. s., OH), 6.22 (1H, d,

J = 4.7 Hz, H-1′), 7.94 (2H, d, J = 8.8 Hz, H_Phe), 8.31 (1H, s, H-6),

8.37−8.45 (2H, m, H_Phe) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ

62.3 (C-5′), 70.8 (C-2′), 73.2 (C-3′), 85.3 (C-4′), 88.5 (C-1′), 98.0

(C-3a), 124.3 (C-3_Phe, C-5_Phe), 129.6 (C-2_Phe, C-6_Phe), 138.9 (C-1_Phe),

143.1 (C-3), 147.4 (C-4_Phe), 155.6 (C-7a), 156.2 (C-6), 158.1 (C-4)

ppm. HRMS (ESI): calcd for C₁₆H₁₇N₆O₆([M + H]⁺): 389.1210,

found: 389.1211.

3-(4-Cyanophenyl)-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (51). Compound 21 (0.120 g, 0.35 mmol) was subjected

to general procedure E, using 4-cyanophenylboronic acid as the

coupling partner and Na₂CO₃as the base. Puriﬁcation via ﬂash

column chromatography (automated, 4 → 15% MeOH in CH₂Cl₂),

followed by an additional puriﬁcation by preparative RP-HPLC (0.2%

formic acid in H₂O/MeCN 98:02 to 33:67 in 12 min) aﬀorded 51

1

(56 mg, 0.152 mmol, 43% yield) as a white solid. H NMR (400

MHz, DMSO-d₆) δ 3.46 (1H, dd, J = 10.9, 5.2 Hz, H-5′), 3.60 (1H,

dd, J = 11.6, 3.6 Hz, H-5″), 3.94 (1H, dd, J = 9.9, 4.8 Hz, H-4′), 4.27

(1H, dd, J = 8.6, 3.6 Hz, H-3′), 4.65 (1H, dd, J = 8.6, 4.9 Hz, H-2′),

4.75−4.90 (1H, m, OH), 5.16 (1H, d, J = 4.0 Hz, OH), 5.43 (1H, d, J

= 5.4 Hz, OH), 6.21 (1H, d, J = 4.5 Hz, H-1′), 7.07 (2H, br. s, NH₂),

7.85 (2H, d, J = 8.3 Hz, H-3_Phe, H-5_Phe), 8.02 (2H, d, J = 8.3 Hz, H-

2_Phe, H-6_Phe), 8.30 (1H, s, H-6) ppm. ¹³C NMR (101 MHz, DMSO-

d₆) δ 62.3 (C-5′), 70.9 (C-2′), 73.3 (C-3′), 85.3 (C-4′), 88.5 (C-1′),

97.9 (C-3a), 111.3 (C-4_Phe), 118.8 (CN), 129.1 (C-2_Phe, C-6_Phe),

133.1 (C-3_Phe, C-5_Phe), 137.1 (C-1_Phe), 143.4 (C-3), 155.6 (C-7a),

156.2 (C-6), 158.2 (C-4) ppm. HRMS (ESI): calcd for C₁₇H₁₇N₆O₄

([M + H]⁺): 369.1311, found: 369.1241.

3-(4-tert-Butylphenyl)-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-

d]pyrimidine (48). Compound 21 (0.120 g, 0.35 mmol) was

subjected to general procedure E, using 4-tert-butylphenylboronic

acid as the coupling partner and Na₂CO₃as the base. Puriﬁcation via

ﬂash column chromatography (automated, 2 → 15% MeOH in

CH₂Cl₂), followed by an additional puriﬁcation by preparative RP-

HPLC (0.2% formic acid in H₂O/MeCN 98:02 to 33:67 in 12 min)

1

aﬀorded 48 (55 mg, 0.138 mmol, 39% yield) as a white solid. H

NMR (300 MHz, DMSO-d₆) δ 1.35 (9H, s, 3 × CH₃), 3.45 (1H, dd,

J = 11.7, 5.9 Hz, H-5′), 3.61 (1H, dd, J = 11.7, 4.1 Hz, H-5″), 3.95

(1H, dd, J = 9.7, 4.7 Hz, H-4′), 4.27 (1H, t, J = 4.7 Hz, H-3′), 4.64

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Article

3-(3,4-Dichlorophenyl)-4-amino-1-β-D-ribofuranosylpyrazolo-

[3,4-d]pyrimidine (52). Compound 21 (0.118 g, 0.34 mmol) was

subjected to general procedure E, using 3,4-dichlorophenylboronic

acid as the coupling partner and Na₂CO₃as the base. Puriﬁcation via

ﬂash column chromatography (automated, 0 → 15% MeOH in

CH₂Cl₂), followed by an additional puriﬁcation by preparative RP-

HPLC (0.2% formic acid in H₂O/MeCN 98:02 to 41:59 in 10.5 min)

ﬂash column chromatography (automated, 0 → 15% MeOH in

CH₂Cl₂), followed by an additional puriﬁcation by preparative RP-

HPLC (0.2% formic acid in H₂O/MeCN 98:02 to 33:67 in 12 min)

1

aﬀorded 55 (66 mg, 0.174 mmol, 50% yield) as a white solid. H

NMR (300 MHz, DMSO-d₆) δ 3.40−3.51 (1H, m, H-5′), 3.58 (1H,

d, J = 3.8 Hz, H-5″), 3.93 (1H, dd, J = 10.0, 5.3 Hz, H-4′), 4.26 (1H,

t, J = 4.5 Hz, H-3′), 4.64 (1H, t, J = 4.7 Hz, H-2′), 4.74−4.88 (1H, m,

OH), 5.03−5.29 (1H, m, OH), 5.33−5.51 (1H, m, OH), 6.18 (1H, d,

J = 4.4 Hz, H-1′), 6.90−7.33 (2H, m, H_Phe), 7.40−7.55 (1H, m,

H_Phe), 7.55−7.73 (1H, m, H_Phe), 8.28 (1H, s, H-6) ppm. ¹³C NMR

(75 MHz, DMSO-d₆) δ 62.3 (C-5′), 70.4 (C-2′), 73.3 (C-3′) 85.2

(C-4′), 88.4 (C-1′), 97.8 (C-3a), 117.3−118.7 (C_Phe), 124.9−125.9

(C_Phe), 129.5−130.4 (C_Phe), 143.0 (d, J = 2.3 Hz, C-3), 147.9−148.8

(C_Phe), 151.3 (C-7a), 155.0−155.6 (C_Phe), 156.1 (C-6), 158.1 (C-4)

ppm. ¹⁹F NMR (282 MHz, DMSO-d₆) δ −138.90 to −138.66 (1F,

m), −137.66 to −137.43 (1F, m) ppm. HRMS (ESI): calcd for

C₁₆H₁₆F₂N₅O₄([M + H]⁺): 380.1170, found: 380.1185.

1

aﬀorded 52 (45 mg, 0.109 mmol, 32% yield) as a white solid. H

NMR (300 MHz, DMSO-d₆) δ 3.40−3.52 (1H, m, H-5′), 3.55−3.65

(1H, m, H-5″), 3.93 (1H, dd, J = 9.7, 4.7 Hz, H-4′), 4.26 (1H, t, J =

4.5 Hz, H-3′), 4.65 (1H, t, J = 3.8 Hz, H-2′), 4.83 (1H, br. s., OH),

5.17 (1H, br. s., OH), 5.43 (1H, br. s., OH), 6.18 (1H, d, J = 4.7 Hz,

H-1′), 6.95−7.37 (2H, m, NH₂), 7.63 (1H, dd, J = 8.2, 2.1 Hz, H-

6_Phe), 7.81 (1H, d, J = 8.5 Hz, H-5_Phe), 7.85 (1H, d, J = 2.1 Hz, H-

2_Phe), 8.29 (1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.2

(C-5′), 70.8 (C-2′), 73.2 (C-3′), 85.2 (C-4′), 88.4 (C-1′), 97.8 (C-

3a), 128.5 (C-6_Phe), 130.1 (C-2_Phe), 131.3 (C-5_Phe), 131.6 (C-3_Phe

)

131.7 (C-4_Phe), 133.1 (C-1_Phe), 142.6 (C-3), 155.4 (C-7a), 156.1 (C-

6), 158.2 (C-4) ppm. HRMS (ESI): calcd for C₁₆H₁₆Cl₂N₅O₄([M +

H]⁺): 412.0579, found: 412.0575.

3 - ( 3 - M e t h o x y - 4 - c h l o r o p h e n y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (56). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 3-

methoxy-4-chlorophenylboronic acid as the coupling partner and

Na₂CO₃as the base. Puriﬁcation via ﬂash column chromatography

(automated, 0 → 15% MeOH in CH₂Cl₂), followed by an additional

puriﬁcation by preparative RP-HPLC (0.2% formic acid in H₂O/

MeCN 98:02 to 33:67 in 12 min) aﬀorded 56 (86 mg, 0.211 mmol,

3 - ( 3 - C h l o r o - 4 - ﬂ u o r o p h e n y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (53). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 3-chloro-4-

ﬂuorophenylboronic acid as the coupling partner and Na₂CO₃as the

base. Puriﬁcation via ﬂash column chromatography (automated, 0 →

15% MeOH in CH₂Cl₂), followed by an additional puriﬁcation by

preparative RP-HPLC (0.2% formic acid in H₂O/MeCN 98:02 to

32:68 in 12 min) aﬀorded 53 (75 mg, 0.197 mmol, 56% yield) as a

white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 3.40−3.51 (1H, m, H-

5′), 3.54−3.67 (1H, m, H-5″), 3.93 (1H, dd, J = 10.0, 5.3 Hz, H-4′),

4.26 (1H, dd, J = 10.0, 5.0 Hz, H-3′), 4.64 (1H, dd, J = 10.5, 5.6 Hz,

H-2′), 4.82 (1H, t, J = 5.9 Hz, OH), 5.14 (1H, d, J = 5.6 Hz, OH),

5.40 (1H, d, J = 5.9 Hz, OH), 6.18 (1H, d, J = 4.4 Hz, H-1′), 6.79−

7.38 (2H, br. s, NH₂), 7.52−7.69 (2H, m, H-5_Phe, H-6_Phe), 7.80 (1H,

dd, J = 7.0, 2.1 Hz, H-2_Phe), 8.28 (1H, s, H-6) ppm. ¹³C NMR (75

MHz, DMSO-d₆) δ 62.2 (C-5′), 70.8 (C-2′), 73.2 (C-3′), 85.2 (C-

4′), 88.4 (C-1′), 97.8 (C-3a), 117.5 (d, J = 21.9 Hz, C-5_Phe), 120.1 (d,

J = 20.7 Hz, C-3_Phe), 129.1 (d, J = 8.1 Hz, C-6_Phe), 130.3 (d, J = 3.5

Hz, C-1_Phe), 130.4 (C-2_Phe), 142.8 (C-3), 155.4 (C-7a), 156.1 (C-6),

158.2 (C-4) ppm. 1 quaternary carbon (C-4_Phe) missing. ¹⁹F NMR

(282 MHz, DMSO-d₆) δ −118.16 to −115.34 (142 F, m) ppm.

HRMS (ESI): calcd for C₁₆H₁₆ClFN₅O₄([M + H]⁺): 396.0875,

found: 396.0891.

3 - ( 3 - F l u o r o - 4 - c h l o r o p h e n y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (54). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 3-ﬂuoro-4-

chlorophenylboronic acid as the coupling partner and Na₂CO₃as the

base. Puriﬁcation via ﬂash column chromatography (automated, 0 →

15% MeOH in CH₂Cl₂), followed by an additional puriﬁcation by

preparative RP-HPLC (0.2% formic acid in H₂O/MeCN 98:02 to

33:67 in 12 min) aﬀorded 54 (82 mg, 0.207 mmol, 59% yield) as a

white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 3.39−3.52 (1H, m, H-

5′), 3.59 (1H, dt, J = 12.0, 4.7 Hz, H-5″), 3.93 (1H, q, J = 4.7 Hz, H-

4′), 4.26 (1H, q, J = 5.0 Hz, H-3′), 4.65 (1H, q, J = 5.2 Hz, H-2′),

4.82 (1H, t, J = 5.7 Hz, OH), 5.14 (1H, d, J = 5.6 Hz, OH), 5.41 (1H,

d, J = 5.9 Hz, OH), 6.18 (1H, d, J = 4.4 Hz, H-1′), 7.20 (2H, br. s,

NH₂), 7.51 (1H, ddd, J = 8.2, 2.1, 0.6 Hz, H-5_Phe), 7.63 (1H, dd, J =

10.1, 2.1 Hz, H-2_Phe), 7.76 (1H, t, J = 8.1 Hz, H-6_Phe), 8.28 (1H, s, H-

6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.3 (C-5′), 70.8 (C-2′),

73.3 (C-3′), 84.8 (C-4′), 88.3 (C-1′) 97.8 (C-3a), 116.8 (d, J = 20.7

Hz, C-2_Phe), 120.0 (d, J = 18.4 Hz, C-4_Phe), 125.5 (d, J = 3.5 Hz, C-

5_Phe), 131.3 (C-6_Phe), 133.4 (d, J = 8.1 Hz, C-1_Phe), 142.9 (C-3), 155.4

1

60% yield) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 3.47

(1H, dd, J = 11.0, 5.4 Hz, H-5′), 3.61 (1H, dd, J = 11.0, 4.7 Hz, H-

5″), 3.82−4.04 (4H, m, H-4′, CH₃), 4.27 (1H, t, J = 4.7 Hz, H-3′),

4.66 (1H, t, J = 4.7 Hz, H-2′), 4.85 (1H, br. s., OH), 5.03−5.24 (1H,

m, OH), 5.31−5.57 (1H, m, OH), 6.19 (1H, d, J = 4.7 Hz, H-1′),

7.25 (1H, dd, J = 8.1, 1.9 Hz, H-6_Phe), 7.36 (1H, d, J = 1.8 Hz, H-

2_Phe), 7.59 (1H, d, J = 7.9 Hz, H-5_Phe), 8.28 (1H, s, H-6) ppm. ¹³C

NMR (75 MHz, DMSO-d₆) δ 56.0 (CH₃), 62.3 (C-5′), 70.8 (C-2′),

73.2 (C-3′), 85.2 (C-4′), 88.5 (C-1′), 97.8 (C-3a), 112.8 (C-2_Phe),

121.0 (C-6_Phe), 121.7 (C-4_Phe), 130.5 (C-5_Phe), 132.6 (C-1_Phe), 144.0

(C-3), 154.8 (C-7a), 155.3 (C-3_Phe), 156.0 (C-6), 158.2 (C-4) ppm.

HRMS (ESI): calcd for C₁₇H₁₉ClN₅O₅([M + H]⁺): 408.1075, found:

408.1099.

3 - ( 3 - M e t h y l - 4 - c h l o r o p h e n y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (57). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 3-methyl-

4-chlorophenylboronic acid as the coupling partner and Na₂CO₃as

the base. Puriﬁcation via ﬂash column chromatography (automated, 2

→ 15% MeOH in CH₂Cl₂), followed by an additional puriﬁcation by

preparative RP-HPLC (0.2% formic acid in H₂O/MeCN 98:02 to

33:67 in 12 min) aﬀorded 57 (88 mg, 0.224 mmol, 64% yield) as a

1

white solid. H NMR (300 MHz, DMSO-d₆) δ 2.56 (3H, s, CH₃),

3.54 (1H, dt, J = 11.8, 6.0 Hz, H-5′), 3.68 (1H, dt, J = 12.0, 4.7 Hz,

H-5″), 4.01 (1H, dd, J = 9.4, 4.7 Hz, H-4′), 4.34 (1H, q, J = 5.2 Hz,

H-3′), 4.73 (1H, q, J = 5.2 Hz, H-2′), 4.92 (1H, t, J = 5.9 Hz, OH),

5.22 (1H, d, J = 5.6 Hz, OH), 5.48 (1H, d, J = 5.9 Hz, OH), 6.26

(1H, d, J = 4.7 Hz, H-1′), 6.57−7.45 (2H, m, NH₂), 7.55−7.77 (3H,

m, H_Phe), 8.36 (1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ

19.8 (CH₃), 62.3 (C-5′), 70.8 (C-2′), 73.2 (C-3′), 85.2 (C-4′), 88.4

(C-1′), 97.8 (C-3a), 127.4 (C_Phe), 129.6 (C_Phe), 130.9 (C_Phe), 131.4

(C_Phe), 134.0 (C_Phe), 136.3 (C_Phe), 143.9 (C-3), 155.4 (C-7a), 156.1

(C-6), 158.1 (C-4) ppm. HRMS (ESI): calcd for C₁₇H₁₉ClN₅O₄([M

+ H]⁺): 392.1126, found: 392.1073.

3-(3-Triﬂuoromethyl-4-chlorophenyl)-4-amino-1-β-^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (58). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 3-

triﬂuoromethyl-4-chlorophenylboronic acid as the coupling partner

and Na₂CO₃as the base. Puriﬁcation via ﬂash column chromatog-

raphy (automated, 2 → 15% MeOH in CH₂Cl₂), followed by an

additional puriﬁcation by preparative RP-HPLC (0.2% formic acid in

H₂O/MeCN 98:02 to 33:67 in 12 min) aﬀorded 58 (50 mg, 0.112

mmol, 32% yield) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ

3.46 (1H, dd, J = 11.6, 5.7 Hz, H-5′), 3.60 (1H, dd, J = 11.7, 4.1 Hz,

H-5″), 3.94 (1H, dd, J = 9.7, 5.0 Hz, H-4′), 4.27 (1H, t, J = 4.5 Hz, H-

3′), 4.66 (1H, t, J = 4.4 Hz, H-2′), 4.83 (1H, br. s., OH), 5.16 (1H, br.

(C-7a), 156.1 (C-6), 158.1 (C-4), 157.4 (d, J = 244.5 Hz, C-3_Phe

)

ppm. ¹⁹F NMR (282 MHz, DMSO-d₆) δ −115.13 (dd, J = 10.2, 7.8

Hz) ppm. HRMS (ESI): calcd for C₁₆H₁₆ClFN₅O₄([M + H]⁺):

396.0875, found: 396.0860.

3-(3,4-Diﬂuorophenyl)-4-amino-1-β-^D-ribofuranosylpyrazolo-

[3,4-d]pyrimidine (55). Compound 21 (0.120 g, 0.35 mmol) was

subjected to general procedure E, using 3,4-diﬂuorophenylboronic

acid as the coupling partner and Na₂CO₃as the base. Puriﬁcation via

4225

https://doi.org/10.1021/acs.jmedchem.1c00135

J. Med. Chem. 2021, 64, 4206−4238

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

s., OH), 5.40 (1H, br. s., OH), 6.20 (1H, d, J = 4.7 Hz, H-1′), 7.23

(2H, br. s., NH₂), 7.77−8.10 (3H, m, H_Phe), 8.30 (1H, s, H-6) ppm.

¹³C NMR (75 MHz, DMSO-d₆) δ 62.2 (C-5′), 70.8 (C-2′), 73.2 (C-

3′), 85.3 (C-4′), 88.5 (C-1′), 97.9 (C-3a), 122.7 (q, J = 275.0 Hz,

CF₃), 127.0 (C_Phe), 127.5 (d, J = 4.6 Hz, C_Phe), 131.1 (C_Phe), 132.0

(C_Phe), 132.4 (C_Phe), 133.6 (C_Phe), 142.6 (C-3), 155.5 (C-7a), 156.1

(C-6), 158.2 (C-4) ppm. ¹⁹F NMR (377 MHz, DMSO-d₆) δ −61.4

(s) ppm. HRMS (ESI): calcd for C₁₇H₁₆F₃ClN₅O₄([M + H]⁺):

446.0843, found: 446.0853.

the base. Puriﬁcation via ﬂash column chromatography (automated, 2

→ 15% MeOH in CH₂Cl₂) aﬀorded 61 (108 mg, 0.256 mmol, 73%

yield) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.40 (3H, t,

J = 6.9 Hz, CH₃), 3.60 (1H, ddd, J = 12.0, 10.0, 4.7 Hz, H-5′), 3.94

(1H, dd, J = 10.0, 4.4 Hz, H-5″), 4.20 (2H, q, J = 6.9 Hz, CH₂), 4.27

(1H, dd, J = 10.0, 5.0 Hz, H-4′), 4.66 (1H, dd, J = 10.3, 5.0 Hz, H-3′),

4.85 (1H, dd, J = 6.4, 5.6 Hz, H-2′), 5.14 (1H, d, J = 5.6 Hz, OH),

5.40 (1H, d, J = 5.9 Hz, OH), 6.19 (1H, d, J = 4.4 Hz, OH), 6.69−

7.23 (2H, m, NH₂), 7.24 (1H, dd, J = 8.2, 1.8 Hz, H-6_Phe), 7.34 (1H,

d, J = 1.8 Hz, H-2_Phe), 7.59 (1H, d, J = 8.2 Hz, H-5_Phe), 8.28 (1H, s,

H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 14.6 (CH₃), 62.3 (C-

5′), 64.3 (CH₂), 70.8 (C-2′), 73.2 (C-3′), 85.3 (C-4′), 88.5 (C-5′),

97.8 (C-3a), 113.6 (C-2 _Phe), 120.9 (C-6_Phe), 121.9 (C-4 _Phe), 130.5

(C-5 _Phe), 132.5 (C-1_Phe), 144.1 (C-3), 154.1 (C-3), 155.3 (C-7a),

156.0 (C-6), 158.2 (C-4) ppm. HRMS (ESI): calcd for

C₁₈H₂₁ClN₅O₅([M + H]⁺): 422.1231, found: 422.1141.

3 - ( 3 - C y a n o - 4 - c h l o r o p h e n y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (59). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 3-cyano-4-

chlorophenylboronic acid as the coupling partner and Na₂CO₃as the

base. Puriﬁcation via ﬂash column chromatography (automated, 2 →

15% MeOH in CH₂Cl₂), followed by an additional puriﬁcation by

preparative RP-HPLC (0.2% formic acid in H₂O/MeCN 98:02 to

40:60 in 12 min) aﬀorded 59 (25 mg, 0.062 mmol, 18% yield) as a

white solid and 75 (7 mg, 0.014 mmol, 4% yield) as a white solid.

3-(2,4-Dichlorophenyl)-4-amino-1-β-^D-ribofuranosylpyrazolo-

[3,4-d]pyrimidine (62). Compound 21 (0.120 g, 0.35 mmol) was

subjected to general procedure E, using 2,4-dichlorophenylboronic

acid as the coupling partner and Na₂CO₃as the base. Puriﬁcation via

ﬂash column chromatography (automated, 2 → 15% MeOH in

CH₂Cl₂), followed by an additional puriﬁcation by preparative RP-

HPLC (0.2% formic acid in H₂O/MeCN 98:02 to 33:67 in 12 min)

1

Analytical data 59: H NMR (400 MHz, DMSO-d₆) δ 3.37−3.51

(1H, m, H-5′), 3.59 (1H, ddd, J = 11.5, 9.6, 4.3 Hz, H-5″), 3.93 (1H,

q, J = 4.8 Hz, H-4′), 4.26 (1H, q, J = 4.8 Hz, H-3′), 4.65 (1H, q, J =

5.0 Hz, H-2′), 4.82 (1H, t, J = 5.8 Hz, OH), 5.15 (1H, d, J = 5.4 Hz,

OH), 5.42 (1H, d, J = 5.6 Hz, OH), 6.19 (1H, d, J = 4.5 Hz, H-1′),

6.79−7.65 (2H, m, NH₂), 7.90 (1H, d, J = 8.1 Hz, H_Phe), 7.96 (H, dd,

J = 8.3, 2.4 Hz, H_Phe), 8.17 (1H, d, J = 2.0 Hz, H_Phe), 8.29 (1H, s, H-

6) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ 60.3 (C-5′), 70.8 (C-2′),

73.2 (C-3′), 85.2 (C-4′), 88.4 (C-1′), 97.9 (C-3a), 112.9 (C_Phe),

116.0 (C_Phe), 130.7 (C_Phe), 132.3 (C_Phe), 134.2 (C_Phe), 134.4 (C_Phe),

135.6 (C_Phe), 142.1 (C-3), 155.5 (C-7a), 156.2 (C-6), 158.1 (C-4)

ppm. HRMS (ESI): calcd for C₁₇H₁₆ClN₆O₄([M + H]⁺): 403.0922,

found: 403.0929. Analytical data 75: ¹H NMR (400 MHz, DMSO-d₆)

δ 3.41−3.53 (1H, m, H-5′), 3.61 (1H, dt, J = 11.8, 4.8 Hz, H-5″),

3.95 (1H, dd, J = 10.0, 4.9 Hz, H-4′), 4.29 (1H, dd, J = 10.4, 5.1 Hz,

H-3′), 4.65 (1H, dd, J = 10.3, 5.0 Hz, H-2′), 4.82 (1H, t, J = 5.4 Hz,

OH), 5.16 (1H, d, J = 5.6 Hz, OH), 5.43 (1H, d, J = 5.8 Hz, OH),

6.22 (1H, d, J = 4.4 Hz, H-1′), 7.15 (2H, br. s, NH₂), 7.84−8.23 (5H,

1

aﬀorded 62 (42 mg, 0.102 mmol, 29% yield) as a white solid. H

NMR (300 MHz, DMSO-d₆) δ 3.37−3.48 (1H, m, H-5′), 3.52−3.63

(1H, m, H-5″), 3.92 (1H, dd, J = 10.0, 4.8 Hz, H-4′), 4.22 (1H, dd, J

= 9.7, 5.0 Hz, H-3′), 4.61 (1H, dd, J = 9.7, 4.7 Hz, H-2′), 4.82 (1H, t,

J = 5.9 Hz, OH), 5.14 (1H, d, J = 5.6 Hz, OH), 5.41 (1H, d, J = 5.9

Hz, OH), 6.16 (1H, d, J = 4.4 Hz, H-1′), 7.52 (1H, d, J = 8.5 Hz, H-

3_Phe), 7.57 (1H, dd, J = 8.2, 2.1 Hz, H-5_Phe), 7.79 (1H, d, J = 2.1 Hz,

H-6_Phe), 8.25 (1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ

62.4 (C-5′), 70.9 (C-2′), 73.2 (C-3′), 85.2 (C-4′), 88.6 (C-1′), 99.4

(C-3a), 127.8 (C-3_Phe), 129.7 (C-6_Phe), 130.3 (C-1_Phe), 133.2 (C-

2_Phe), 133.9 (C-5_Phe), 134.7 (C-4_Phe), 141.2 (C-3), 154.7 (C-7a),

156.2 (C-6), 157.8 (C-4) ppm. HRMS (ESI): calcd for

C₁₆H₁₆Cl₂N₅O₄([M + H]⁺): 412.0579, found: 412.0586.

m, H_Phe), 8.30 (1H, d, J = 2.0 Hz, H_Phe), 8.32 (1H, s, H-6) ppm. ¹³

C

3 - ( 2 - M e t h y l - 4 - c h l o r o p h e n y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (63). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 2-methyl-

4-chlorophenylboronic acid as the coupling partner and Na₂CO₃as

the base. Puriﬁcation via ﬂash column chromatography (automated, 2

→ 15% MeOH in CH₂Cl₂) aﬀorded 63 (83 mg, 0.212 mmol, 61%

yield) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 2.18−2.37

(3H, m, CH₃), 3.42 (1H, dd, J = 11.7, 5.9 Hz, H-5′), 3.58 (1H, dd, J =

11.7, 4.4 Hz, H-5″), 3.93 (1H, dd, J = 9.1, 4.7 Hz, H-4′), 4.23 (1H, t,

J = 5.0 Hz, H-3′), 4.60 (1H, t, J = 4.5 Hz, H-2′), 6.19 (1H, d, J = 4.1

Hz, H-1′), 7.30−7.45 (2H, m, NH_2,H_Phe), 7.49 (1H, s, H_Phe), 8.30

(1H, s, H-6) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ 19.9 (CH₃),

62.8 (C-5′), 71.4 (C-2′), 73.9 (C-3′), 85.6 (C-4′), 88.9 (C-1′), 99.4

(C-3a), 126.7 (C_Phe), 131.0 (C_Phe), 132.1 (C_Phe), 134.1 (C_Phe), 139.8

(C_Phe), 143.8 (C-3), 155.0 (C-7a), 156.0 (C-6), 157.9 (C-4) ppm.

HRMS (ESI): calcd for C₁₇H₁₉ClN₅O₄([M + H]⁺): 392.1126, found:

392.1077.

NMR (101 MHz, DMSO-d₆) δ 62.2 (C-5′), 70.8 (C-2′), 73.3 (C-3′),

85.2 (C-4′), 88.4 (C-1′), 98.0 (C-3a), 111.3 (C_Phe), 112.6 (C_Phe),

115.6 (C_Phe), 118.0 (C_Phe), 130.6 (C_Phe), 131.0 (C_Phe), 133.1 (C_Phe),

133.4 (C_Phe), 133.4 (C_Phe), 134.7 (C_Phe), 135.1 (C_Phe), 136.0 (C_Phe),

137.3 (C_Phe), 141.1 (C_Phe), 142.5 (C-3), 155.5 (C-7a), 156.2 (C-6),

158.2 (C-4) ppm. HRMS (ESI): calcd for C₂₄H₁₉ClN₇O₄([M +

H]⁺): 504.1187, found: 504.1199.

3 - ( 3 , 5 - D iﬂ u o r o - 4 - c h l o r o p h e n y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (60). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 3,5-

diﬂuoro-4-chlorophenylboronic acid as the coupling partner and

Na₂CO₃as the base. Puriﬁcation via ﬂash column chromatography

(automated, 2 → 15% MeOH in CH₂Cl₂), followed by an additional

puriﬁcation by preparative RP-HPLC (0.2% formic acid in H₂O/

MeCN 98:02 to 33:67 in 12 min) aﬀorded 60 (70 mg, 0.169 mmol,

48% yield) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 3.40−

3.52 (1H, m, H-5′), 3.60 (1H, dt, J = 11.8, 4.8 Hz, H-5″), 3.93 (1H,

dd, J = 10.0, 4.7 Hz, H-4′), 4.27 (1H, dd, J = 10.0, 5.0 Hz, H-3′), 4.65

(1H, dd, J = 10.5, 5.3 Hz, H-2′), 4.82 (1H, t, J = 5.9 Hz, OH), 5.14

(1H, d, J = 5.6 Hz, OH), 5.41 (1H, d, J = 5.9 Hz, OH), 6.18 (1H, d, J

= 4.7 Hz, H-1′), 7.24 (2H, br. s., NH₂), 7.46−7.65 (2H, m, H_Phe),

8.28 (H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.2 (C-5′), 70.8

(C-2′), 73.2 (C-3′), 85.2 (C-4′), 88.5 (C-1′), 97.9 (C-3a), 108.8 (t, J

= 20.7 Hz, C_Phe), 112.8 (dd, J = 23.0, 2.3 Hz, C_Phe), 133.1 (t, J = 10.4

Hz, C_Phe), 142.1 (d, J = 2.3 Hz, C-3), 155.7 (C-7a), 156.2 (C-6),

156.6 (d, J = 3.5 Hz, C_Phe), 158.0 (C-4), 159.9 (d, J = 4.6 Hz) ppm.

¹⁹F NMR (282 MHz, DMSO-d₆) δ −112.93 (2F, d, J = 7.2 Hz) ppm.

3-(1-Methylpyrazol-4-yl)-4-amino-1-β-^D-ribofuranosylpyrazolo-

[3,4-d]pyrimidine (64). Compound 21 (0.120 g, 0.35 mmol) was

subjected to general procedure E, using 1-methylpyrazole-4-boronic

acid as the coupling partner and Na₂CO₃as the base. Puriﬁcation via

ﬂash column chromatography (automated, 2 → 15% MeOH in

CH₂Cl₂) aﬀorded 64 (65 mg, 0.187 mmol, 53% yield) as a white

1

solid. H NMR (300 MHz, DMSO-d₆) δ 3.45 (1H, dt, J = 11.2, 5.4

Hz, H-5′), 3.59 (1H, dt, J = 12.0, 4.2 Hz, H-5″), 3.80−4.02 (4H, m,

CH₃, H-4′), 4.24 (1H, dd, J = 9.7, 5.0 Hz, H-3′), 4.62 (1H, dd, J =

9.7, 5.1 Hz, H-2′), 4.86 (1H, t, J = 5.2 Hz, OH), 5.12 (1H, d, J = 5.3

Hz, OH), 5.38 (1H, d, J = 5.9 Hz, OH), 6.13 (1H, d, J = 4.7 Hz, H-

1′), 6.67−7.46 (2H, m, NH₂), 7.74 (1H, s, H-5_pyrazole), 8.10 (1H, s, H-

3_pyrazole), 8.24 (1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ

62.4 (C-5′), 70.9 (C-2′), 73.1 (C-3′), 85.2 (C-4′), 88.4 (C-1′), 98.1

(C-3a), 113.3 (C-4_pyrazole), 130.4 (C-5_pyrazole), 137.7 (C-3_pyrazole), 155.0

(C-7a), 156.0 (C-6), 158.3 (C-4) ppm. 1 quaternary carbon (C-3)

HRMS (ESI): calcd for C₁₆H₁₅ClF₂N₅O₄([M + H]⁺): 414.0781,

found: 414.0733.

3 - ( 3 - E t h o x y - 4 - c h l o r o p h e n y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (61). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 3-ethoxy-

4-chlorophenylboronic acid as the coupling partner and Na₂CO₃as

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missing. HRMS (ESI): calcd for C₁₄H₁₈N₇O₄([M + H]⁺): 348.1420,

found: 348.1418.

5′), 71.3 (C-2′), 73.6 (C-3′), 85.8 (C-4′), 88.9 (C-1′), 98.1 (C-3a),

128.3 (C-3_Het), 128.5(C-4_Het), 128.9 (C-5_Het), 134.3 (C-2_Het), 139.4

(C-3), 155.7 (C-7a), 156.6 (C-6), 158.6 (C-4) ppm. HRMS (ESI):

calcd for C₁₄H₁₆N₅O₄S ([M + H]⁺): 350.0923, found: 350.0932.

3 - ( 5 - M e t h y l t h i o p h e n - 2 - y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (68). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 5-

methylthiophene-2-boronic acid as the coupling partner and Na₂CO₃

as the base. Puriﬁcation via ﬂash column chromatography

(automated, 2 → 15% MeOH in CH₂Cl₂) aﬀorded 64 (65 mg,

0.187 mmol, 53% yield) as a white solid. ¹H NMR (300 MHz,

DMSO-d₆) δ 3.34 (3H, s, CH₃), 3.44 (1H, dt, J = 11.9, 6.1 Hz, H-5′),

3.59 (1H, dt, J = 11.4, 4.7 Hz, H-5″), 3.91 (1H, dd, J = 9.7, 4.7 Hz, H-

4′), 4.23 (1H, dd, J = 10.0, 5.0 Hz, H-3′), 4.58 (1H, dd, J = 9.7, 5.0

Hz, H-2′), 4.82 (1H, t, J = 5.9 Hz, OH), 5.13 (1H, d, J = 5.6 Hz,

OH), 5.40 (1H, d, J = 5.9 Hz, OH), 6.13 (1H, d, J = 4.7 Hz, H-1′),

6.82−6.97 (1H, m, C-4_Het), 6.99−7.21 (2H, m, NH₂), 7.27 (1H, d, J

= 3.5 Hz, C-3_Het), 8.25 (1H, s, H-6) ppm. ¹³C NMR (75 MHz,

DMSO-d₆) δ 14.9 (CH₃), 62.5 (C-5′), 70.9 (C-2′), 73.2 (C-3′), 85.3

(C-4′), 88.4 (C-1′), 97.5 (C-3a), 126.8 (C-4_Het), 128.0 (C-5_Het),

131.5 (C-3_Het), 139.1 (C-2_Het), 141.3 (C-3), 155.2 (C-7a), 156.2 (C-

6), 158.1 (C-4) ppm. HRMS (ESI): calcd for C₁₅H₁₈N₅O₄S ([M +

H]⁺): 364.1079, found: 364.1088.

3 - ( 5 - C h l o r o t h i o p h e n - 2 - y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (69). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 5-

chlorothiophene-2-boronic acid as the coupling partner and Na₂CO₃

as the base. Puriﬁcation via ﬂash column chromatography

(automated, 2 → 15% MeOH in CH₂Cl₂), followed by an additional

puriﬁcation by preparative RP-HPLC (0.2% formic acid in H₂O/

MeCN 98:02 to 40:60 in 12 min) aﬀorded 69 (55 mg, 0.143 mmol,

41% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 3.36−

3.51 (1H, m, H-5′), 3.59 (1H, dt, J = 11.6, 5.0 Hz, H-5″), 3.92 (1H,

dd, J = 10.3, 5.1 Hz, H-4′), 4.23 (1H, dd, J = 10.0, 5.1 Hz, H-3′), 4.60

(1H, dd, J = 10.0, 5.0 Hz, H-2′), 4.82 (1H, t, J = 5.8 Hz, OH), 5.15

(1H, d, J = 5.6 Hz, OH), 5.42 (1H, d, J = 5.9 Hz, OH), 6.15 (1H, d, J

= 4.5 Hz, H-1′), 7.25 (2H, d, J = 4.0 Hz, H-4_Het), 7.35 (1H, d, J = 3.9

Hz, H-3_Het), 8.28 (1H, s, H-6) ppm. ¹³C NMR (101 MHz, DMSO-

d₆) δ 62.3 (C-5′), 70.9 (C-2′), 73.2 (C-3′), 85.3 (C-4′), 88.4 (C-1′),

97.5 (C-3a), 128.0 (C-3_Het), 128.2 (C-5_Het), 129.3 (C-4_Het), 132.8

(C-2_Het), 138.1 (C-3), 155.3 (C-7a), 156.3 (C-6), 158.2 (C-4) ppm.

HRMS (ESI): calcd for C₁₄H₁₅ClN₅O₄S ([M + H]⁺): 384.0533,

found: 384.0550.

3-(Pyridin-2-yl)-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine, Formic Acid Salt (65). Compound 21 (0.173 g, 0.50

mmol, 1.0 equiv), Pd(PPh₃)₄(0.087 g, 0.075 mmol, 0.15 equiv), and

CuI (0.010 g, 0.05 mmol, 0.1 equiv) were dissolved in dry degassed

DMF (2 mL) under argon. 2-(Tributylstannyl)pyridine was added,

and the mixture was warmed to 100 °C. After 2 h, LCMS analysis

indicated completion of the reaction. The mixture was cooled to room

temperature, diluted with MeOH (15 mL) and MeCN (15 mL), and

washed with hexanes (2 × 15 mL). The MeOH/MeCN phase was

concentrated in vacuo. The residue was adsorbed onto celite and

puriﬁed by ﬂash column chromatography (automated, 4 → 15%

MeOH in CH₂Cl₂), followed by an additional puriﬁcation by

preparative HPLC (0.2% formic acid in H₂O/MeCN 98:02 to

33:67 in 12 min) to aﬀord 67 (80 mg, 0.232 mmol, 66% yield) as a

white solid. NMR analysis showed the presence of an extra proton,

1

indicating that 65 was isolated as its formic acid salt. H NMR (300

MHz, DMSO-d₆) δ 3.43−3.58 (1H, m, H-5′), 3.60−3.74 (1H, m, H-

5″), 3.97 (1H, dd, J = 9.7, 4.7 Hz, H-4′), 4.27−4.41 (1H, m, H-3′),

4.60−4.74 (1H, m, H-2′), 4.88 (1H, br. s., OH), 5.17 (1H, br. s.,

OH), 5.47 (1H, br. s., OH), 6.21 (1H, d, J = 4.4 Hz, H-1′), 7.51 (1H,

ddd, J = 7.5, 5.1, 1.2 Hz, H-4_pyr), 8.04 (1H, td, J = 7.8, 1.8 Hz, H-5_pyr),

8.11 (1H, d, J = 3.2 Hz), 8.24 (1H, s, H-6), 8.28 (1H, d, J = 7.9 Hz,

H-3_pyr), 8.74 (1H, dd, J = 4.1, 0.9 Hz, H-6_pyr), 9.93 (1H, d, J = 3.8 Hz,

NH) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.4 (C-5′), 71.0 (C-

2′), 73.4 (C-3′), 85.4 (C-4′), 88.6 (C-1′), 98.5 (C-3a), 121.0 (C-

3_pyr), 124.1 (C-5_pyr), 138.3 (C-3), 143.7 (C-4_pyr), 148.5 (C-6_pyr),

150.7 (C-2_pyr), 155.7 (C-7a), 156.6 (C-6), 158.8 (C-4)ppm. HRMS

(ESI): calcd for C₁₅H₁₇N₆O₄([M + H]⁺): 345.1311, found:

345.1312.

3-(Pyridin-4-yl)-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (66). Compound 21 (0.120 g, 0.35 mmol) was subjected

to general procedure E, using pyridine-4-boronic acid as the coupling

partner and Na₂CO₃as the base. Puriﬁcation via ﬂash column

chromatography (automated, 2 → 15% MeOH in CH₂Cl₂) aﬀorded

1

66 (37 mg, 0.056 mmol, 16% yield) as a white solid. H NMR (300

MHz, DMSO-d₆) δ 3.39−3.55 (1H, m, H-5′), 3.55−3.70 (1H, m, H-

5″), 3.94 (1H, dd, J = 9.7, 4.8 Hz, H-4′), 4.28 (1H, dd, J = 10.0, 5.2

Hz, H-3′), 4.66 (1H, dd, J = 9.7, 5.0 Hz, H-2′), 4.84 (1H, t, J = 5.9

Hz, OH), 5.16 (1H, d, J = 5.6 Hz, OH), 5.43 (1H, d, J = 5.6 Hz, OH),

6.21 (1H, d, J = 4.4 Hz, H-1′), 7.18 (2H, br. s., NH₂), 7.67 (2H, d, J =

5.9 Hz, H-3_pyr, H-5_pyr), 8.31 (1H, s, H-6), 8.74 (2H, d, J = 5.3 Hz, H-

2_pyr, H-6_pyr) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.3 (C-5′), 70.8

(C-2′), 73.2 (C-3′), 85.3 (C-4′), 88.6 (C-1′), 97.9 (C-3a), 122.8 (C-

3_pyr, C-5_pyr), 139.8 (C-3), 142.6 (C-4_pyr), 150.3 (C-2_pyr, C-6_pyr), 155.6

(C-7a), 156.2 (C-6), 158.1 (C-4) ppm. HRMS (ESI): calcd for

C₁₅H₁₇N₆O₄([M + H]⁺): 345.1311, found: 345.1317.

3 - ( 4 - M e t h y l t h i o p h e n - 2 - y l ) - 4 - a m i n o - 1 - β - ^D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (70). Compound 21 (0.120

g, 0.35 mmol) was subjected to general procedure E, using 4-

methylthiophene-2-boronic acid as the coupling partner and Na₂CO₃

as the base. Puriﬁcation via ﬂash column chromatography

(automated, 2 → 15% MeOH in CH₂Cl₂), followed by an additional

puriﬁcation by preparative RP-HPLC (0.2% formic acid in H₂O/

MeCN 98:02 to 40:60 in 12 min) aﬀorded 70 (38 mg, 0.105 mmol,

3-(Thiophen-2-yl)-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (67). Compound 21 (0.173 g, 0.50 mmol, 1.0 equiv),

Pd(PPh₃)₄(0.087 g, 0.075 mmol, 0.15 equiv), and CuI (0.010 g, 0.05

mmol, 0.1 equiv) were dissolved in dry degassed DMF (2 mL) under

argon. 2-(Tributylstannyl)thiophene (0.238 mL, 0.75 mmol, 1.5

equiv) was added, and the mixture was warmed to 100 °C. After 2 h,

LCMS analysis indicated completion of the reaction. The mixture was

cooled to room temperature, diluted with MeOH (15 mL) and

MeCN (15 mL), and washed with hexanes (2 × 15 mL). The

MeOH/MeCN phase was concentrated in vacuo. The residue was

adsorbed onto celite and puriﬁed by ﬂash column chromatography

(automated, 4 → 15% MeOH in CH₂Cl₂), followed by an additional

puriﬁcation by preparative HPLC (0.2% formic acid in H₂O/MeCN

98:02 to 33:67 in 12 min) to aﬀord 67 (42 mg, 0.120 mmol, 24%

1

30% yield) as a white solid. H NMR (400 MHz, DMSO-d₆) δ 2.29

(3H, d, J = 0.5 Hz, CH₃), 3.40−3.50 (1H, m, H-5′), 3.59 (1H, dt, J =

11.9, 4.8 Hz, H-5″), 3.92 (1H, dd, J = 10.1, 4.9 Hz, H-4′), 4.24 (1H,

dd, J = 10.3, 5.0 Hz, H-3′), 4.61 (1H, dd, J = 9.9, 5.1 Hz, H-2′), 4.83

(1H, t, J = 5.8 Hz, OH), 5.15 (1H, d, J = 5.6 Hz, OH), 5.41 (1H, d, J

= 5.9 Hz, OH), 6.15 (1H, d, J = 4.5 Hz, H-1′), 7.28 (1H, t, J = 1.1 Hz,

H-3_Het), 7.30 (1H, d, J = 1.1 Hz, H-5_Het), 8.27 (1H, s) ppm. ¹³C

NMR (101 MHz, DMSO-d₆) δ 15.5 (CH₃), 62.4 (C-5′), 70.9 (C-2′),

73.1 (C-3′), 85.3 (C-4′), 88.4 (C-1′), 97.5 (C-3a), 122.8 (C-5_Het),

130.0 (C-3_Het), 133.5 (C-4_Het), 138.5 (C-3), 139.1 (C-2_Het), 155.2

(C-7a), 156.2 (C-6), 158.1 (C-4) ppm. HRMS (ESI): calcd for

C₁₅H₁₈N₅O₄S ([M + H]⁺): 364.1079, found: 364.1081.

1

yield) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 3.46 (1H,

dt, J = 11.4, 5.4 Hz, H-5′), 3.60 (1H, dt, J = 11.7, 3.8 Hz, H-5″), 3.93

(1H, dd, J = 10.0, 4.7 Hz, H-4′), 4.14−4.31 (1H, m, H-3′), 4.62 (1H,

dd, J = 7.3, 3.2 Hz, H-2′), 4.84 (1H, t, J = 6.2 Hz, OH), 5.16 (1H, br.

s., OH), 5.42 (1H, br. s., OH), 6.16 (1H, d, J = 4.7 Hz, H-1′), 6.83−

7.19 (2H, m, NH₂), 7.25 (2H, dd, J = 5.0, 3.5 Hz, C-4_Het), 7.50 (1H,

dd, J = 3.7, 1.0 Hz, C-3_Het), 7.72 (1H, dd, J = 5.1, 1.0 Hz, C-5_Het),

8.28 (1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.9 (C-

3-Vinyl-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]pyrimidine

(71). Compound 21 (0.118 g, 0.34 mmol) was subjected to general

procedure E, using potassium vinyl triﬂuoroborate as the coupling

partner and Cs₂CO₃as the base. Puriﬁcation via ﬂash column

chromatography (automated, 2 → 15% MeOH in CH₂Cl₂), followed

by an additional puriﬁcation by preparative RP-HPLC (0.2% formic

acid in H₂O/MeCN 98:02 to 33:67 in 12 min) aﬀorded 71 (60 mg,

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0.205 mmol, 58% yield) as a white solid. ¹H NMR (400 MHz,

DMSO-d₆) δ 3.41−3.50 (1H, m, H-5′), 3.60 (1H, dt, J = 11.6, 4.8 Hz,

H-5″), 3.92 (1H, dd, J = 9.4, 4.8 Hz, H-4′), 4.24 (1H, dd, J = 9.9, 5.5

Hz, h-3′), 4.57 (1H, dd, J = 10.1, 5.0 Hz, H-2′), 4.87 (1H, t, J = 5.8

Hz, OH), 5.12 (1H, d, J = 5.4 Hz, OH), 5.36 (1H, d, J = 5.9 Hz, OH),

5.46 (1H, dd, J = 11.8, 1.0 Hz, CHCH₂), 6.05 (1H, dd, J = 17.1, 1.0

Hz, CHCH₂), 6.12 (1H, d, J = 4.3 Hz, H-1′), 7.28 (1H, dd, J =

3-(4-Chlorophenyl)-4-amino-1-(3′-deoxy-β-^D-ribofuranosyl)-

pyrazolo[3,4-d]pyrimidine (76). Compound 23 (0.026 g, 0.079

mmol) was subjected to general procedure E, using 4-chlorophe-

nylboronic acid as the coupling partner and Na₂CO₃as the base.

Puriﬁcation via ﬂash column chromatography (automated, 0 → 10%

MeOH in CH₂Cl₂+ 1% NH₄OH) aﬀorded 76 (23 mg, 0.064 mmol,

1

81% yield) as a white solid. H NMR (400 MHz, DMSO-d₆) δ 2.00

(1H, ddd, J = 12.3, 6.2, 1.6 Hz, H-3′), 2.28−2.46 (1H, m, H-3″),

3.39−3.58 (2H, m, H-5′, H-5″), 4.29−4.41 (1H, m, H-2′), 4.62 (1H,

br. s., H-4′), 4.75 (1H, t, J = 5.7 Hz, OH), 5.59 (1H, d, J = 3.8 Hz,

OH), 6.24 (1H, s, H-1′), 7.61 (2H, d, J = 8.4 Hz, H_Phe), 7.67 (2H, d, J

= 8.4 Hz, H_Phe), 8.28 (1H, s, H-6) ppm. ¹³C NMR (101 MHz,

DMSO-d₆) δ 36.1 (C-3′), 64.1 (C-5′), 74.5 (C-2′), 81.2 (C-4′), 90.6

(C-1′), 97.5 (C-3a), 129.2 (C_Phe), 130.0 (C_Phe), 131.5 (C_Phe), 133.6

(C_Phe), 143.7 (C-3), 155.0 (C-7a), 156.1 (C-6), 158.2 (C-4) ppm.

HRMS (ESI): calcd for C₁₆H₁₇ClN₅O₃([M + H]⁺): 362.1020, found:

362.0999.

17.1, 11.0 Hz, CHCH₂), 7.54 (2H, br. s, NH₂), 8.18 (1H, s, H-6)

ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ 62.4 (C-5′), 70.9 (C-2′),

73.2 (C-3′), 85.3 (C-4′), 88.5 (C-1′), 98.1 (C-3a), 118.5 (CH

CH₂), 127.3 (CHCH₂), 141.9 (C-3), 155.0 (C-7a), 155.9 (C-6),

158.1 (C-4) ppm. HRMS (ESI): calcd for C₁₂H₁₆N₅O₄([M + H]⁺):

294.1202, found: 294.1213.

3-Isopropenyl-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (72). Compound 21 (0.250 g, 0.72 mmol) was subjected

to general procedure E, using potassium isopropenyltriﬂuoroborate as

the coupling partner and Cs₂CO₃as the base. Puriﬁcation via ﬂash

column chromatography (automated, 2 → 15% MeOH in CH₂Cl₂)

3-(4-Chlorophenyl)-4-amino-1-(2′-deoxy-β-^D-ribofuranosyl)-

pyrazolo[3,4-d]pyrimidine (77). Compound 23 (0.042 g, 0.127

mmol) was subjected to general procedure E, using 4-chlorophe-

nylboronic acid as the coupling partner and Na₂CO₃as the base.

Puriﬁcation via ﬂash column chromatography (automated, 1 → 8%

MeOH in CH₂Cl₂+ 1% NH₄OH) aﬀorded 76 (28 mg, 0.098 mmol,

1

aﬀorded 72 (170 mg, 0.553 mmol, 77% yield) as a white solid. H

NMR (300 MHz, DMSO-d₆) δ 2.18 (3H, s, CH₃CCH₂), 3.45 (1H,

dt, J = 11.6, 5.9 Hz, H-5′), 3.55−3.65 (1H, m, H-5″), 3.92 (1H, dd, J

= 9.7, 4.4 Hz, H-4′), 4.23 (1H, dd, J = 9.7, 4.7 Hz, H-3′), 4.59 (1H,

dd, J = 9.4, 5.0 Hz, H-2′), 4.84 (1H, t, J = 5.6 Hz, OH), 5.12 (1H, d, J

= 5.3 Hz, OH), 5.29−5.45 (2H, m, OH, CH₃CCH₂), 5.53 (1H, br.

s., CH₃CCH₂), 6.13 (1H, d, J = 4.1 Hz, H-1′), 7.16 (2H, br. s.,

NH₂), 8.23 (1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 21.7

(CH₃CCH₂), 62.4 (C-5′), 70.9 (C-2′), 73.2 (C-3′), 85.2 (C-4′),

88.5 (C-1′), 97.3 (C-3a), 118.6 (CH₃CCH₂), 137.2 (CH₃C

CH₂), 146.2 (C-3), 154.9 (C-7a), 155.9 (C-6), 158.2 (C-4) ppm.

HRMS (ESI): calcd for C₁₃H₁₈N₅O₄([M + H]⁺): 308.1359, found:

308.1367.

1

81% yield) as a white solid. H NMR (400 MHz, DMSO-d₆) δ 2.29

(1H, ddd, J = 13.2, 6.8, 4.3 Hz, H-2′), 2.85 (1H, dt, J = 12.9, 6.0 Hz,

H-2″), 3.40 (1H, dt, J = 11.7, 6.0 Hz, H-5′), 3.55 (1H, dt, J = 11.4, 5.5

Hz, H-5″), 3.84 (1H, td, J = 5.4, 3.6 Hz, H-4′), 4.42−4.51 (1H, m, H-

3′), 4.77 (1H, t, J = 5.8 Hz, OH), 5.27 (1H, d, J = 4.6 Hz, OH), 6.64

(1H, t, J = 6.4 Hz, H-1′), 7.58−7.64 (2H, m, H_Phe), 7.65−7.72 (2H,

m, H_Phe), 8.27 (1H, s, H-6) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ

38.0 (C-2′), 62.4 (C-5′), 71.1 (C-3′), 83.9 (C-4′), 87.7 (C-1′), 97.8

(C-3a), 129.2 (C_Phe), 130.0 (C_Phe), 131.5 (C_Phe), 133.6 (C_Phe), 143.6

(C-3), 155.0 (C-7a), 156.0 (C-6), 158.1 (C-4) ppm. HRMS (ESI):

calcd for C₁₆H₁₇ClN₅O₃([M + H]⁺): 362.1020, found: 362.1004.

3-Isopropyl-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (78). Compound 72 (0.060 g, 0.195 mmol) was dissolved

in MeOH (5 mL). The ﬂask was placed under a nitrogen atmosphere,

and a catalytic amount of Pd(OH)₂/C was added. The atmosphere

was exchanged for H₂, and the mixture was stirred for 1 h until TLC

analysis (10% MeOH in CH₂Cl₂) indicated completion of the

reaction. The mixture was ﬁltered over celite, celite was added to the

ﬁltrate, and the solvents were removed under reduced pressure. The

solid residue was puriﬁed via ﬂash column chromatography

(automated, 4 → 20% MeOH in CH₂Cl₂) to aﬀord 78 (49 mg,

0.158 mmol, 81% yield) as a white solid. ¹H NMR (300 MHz,

DMSO-d₆) δ 1.26 (6H, d, J = 6.7 Hz, CH(CH₃)₂), 3.38−3.65 (3H,

m, H-5′, H-5″, CH(CH₃)₂), 3.90 (1H, dd, J = 9.4, 4.7 Hz, H-4′), 4.25

(1H, dd, J = 10.3, 5.0 Hz, H-3′), 4.56 (1H, dd, J = 10.0, 5.3 Hz, H-2′),

4.85 (1H, dd, J = 6.7, 5.0 Hz, OH), 5.07 (1H, d, J = 5.6 Hz, OH),

5.32 (1H, d, J = 5.9 Hz, OH), 6.06 (1H, d, J = 4.4 Hz, H-1′), 7.30

(2H, br. s, NH₂), 8.15 (1H, s, H-6) ppm. ¹³C NMR (75 MHz,

DMSO-d₆) δ 22.3 (CH(CH₃)₂), 27.4 (CH(CH₃)₂), 63.0 (C-5′), 71.5

(C-2′), 73.7 (C-3′), 85.6 (C-4′), 89.1 (C-1′), 98.6 (C-3a), 151.6 (C-

3), 155.5 (C-7a), 156.2 (C-6), 158.5 (C-4) ppm. HRMS (ESI): calcd

for C₁₃H₂₀N₅O₄([M + H]⁺): 310.1515, found: 310.1494.

3-O-Phenyl-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (79). Compound 21 (0.220 g, 0.64 mmol, 1.0 equiv),

phenol (0.090 g, 0.96 mmol, 1.5 equiv), CuI (0.024 g, 0.13 mmol, 0.2

equiv), N,N-dimethylglycine (0.040 g, 0.38 mmol, 0.6 equiv), and

Cs₂CO₃(0.417 g, 1.28 mmol, 2.0 equiv) were dissolved in dry

degassed DMA (4 mL) under argon. The reaction was heated at 120

°C overnight, cooled down to room temperature, and concentrated in

vacuo. The residue was taken up in MeOH, celite was added, and the

mixture was concentrated under reduced pressure. The solid residue

was puriﬁed by ﬂash column chromatography (automated, 2 → 15%

MeOH in CH₂Cl₂) to aﬀord 79 (12 mg, 0.033 mmol, 5% yield) as a

white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 3.34−3.42 (1H, m, H-

5′, underwater peak), 3.48 (1H, dd, J = 12.0, 4.4 Hz, H-5″), 3.84 (1H,

dd, J = 10.3, 4.7 Hz, H-4′), 4.07 (1H, t, J = 4.8 Hz, H-3′), 4.43 (1H, t,

J = 4.7 Hz, H-2′), 6.02 (1H, d, J = 4.1 Hz, H-1′), 7.14−7.26 (1H, m,

3-(E-Styryl)-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (73). Compound 21 (0.120 g, 0.35 mmol) was subjected

to a slightly modiﬁed general procedure E, using trans-2-vinyl-

phenylboronic acid as the coupling partner and Na₂CO₃as the base.

Puriﬁcation via ﬂash column chromatography (automated, 2 → 15%

MeOH in CH₂Cl₂), followed by an additional puriﬁcation by

preparative RP-HPLC (0.2% formic acid in H₂O/MeCN 98:02 to

33:67 in 12 min) aﬀorded 72 (55 mg, 0.149 mmol, 43% yield) as a

white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 3.48 (1H, dd, J = 11.7,

5.9 Hz, H-5′), 3.62 (1H, dd, J = 11.7, 4.4 Hz, H-5″), 3.92 (1H, dd, J =

10.0, 4.7 Hz, H-4′), 4.26 (1H, t, J = 4.7 Hz, H-3′), 4.65 (1H, t, J = 5.0

Hz, H-2′), 6.12 (1H, d, J = 4.7 Hz, H-1′), 7.26−7.49 (4H, m, H_Phe),

7.53−7.71 (3H, m, NH₂, H_Phe), 7.74−7.83 (2H, m, 2 × H_vinyl), 8.18

(1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.4 (C-5′),

70.9 (C-2′), 73.1 (C-3′), 85.3 (C-4′), 88.6 (C-1′), 98.5 (C-3a), 118.0

(C_vinyl), 127.4 (C_Phe), 128.0 (C_Phe), 128.3 (C_Phe), 128.6 (C_Phe), 132.2

(C_vinyl), 141.8 (C-3), 155.1 (C-7a), 155.9 (C-6), 158.1 (C-4) ppm.

HRMS (ESI): calcd for C₁₈H₂₀N₅O₄([M + H]⁺): 370.1515, found:

370.1509.

3-Cyclopropyl-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (74). Compound 21 (0.120 g, 0.35 mmol) was subjected

to a slightly modiﬁed general procedure E, using cyclopropylboronic

acid as the coupling partner and Na₂CO₃as the base. After 8 h, a

second portion of cyclopropylboronic acid (1.5 equiv) was added, and

the reaction was stirred for another 16 h, before LCMS analysis

indicated full conversion. Puriﬁcation via ﬂash column chromatog-

raphy (automated, 2 → 15% MeOH in CH₂Cl₂) aﬀorded 74 (72 mg,

0.234 mmol, 67% yield) as a white solid. ¹H NMR (300 MHz,

DMSO-d₆) δ 0.77−1.07 (4H, m, 2 × CH_{2 cyclopropyl}), 2.39−2.48 (1H,

m, CH_cyclopropyl), 3.40 (1H, dt, J = 12.0, 6.0 Hz, H-5′), 3.57 (1H, dt, J

= 11.7, 4.7 Hz, H-5″), 3.87 (1H, dd, J = 9.7, 4.7 Hz, H-4′), 4.20 (1H,

dd, J = 10.0, 5.2 Hz, H-3′), 4.49 (1H, dd, J = 10.0, 5.0 Hz, H-2′), 4.81

(1H, dd, J = 6.6, 5.1 Hz, OH), 5.06 (1H, d, J = 5.9 Hz, OH), 5.30

(1H, d, J = 5.9 Hz, OH), 6.01 (1H, d, J = 4.4 Hz, H-1′), 7.41 (2H, br.

s., NH₂), 8.15 (1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ

7.7 (C_cyclopropyl), 7.9 (C_cyclopropyl), 8.5 (C_cyclopropyl), 62.4 (C-5′), 71.0

(C-2′), 73.2 (C-3′), 85.0 (C-4′), 88.4 (C-1′), 99.5 (C-3a), 147.0 (C-

3), 154.9 (C-7a), 155.9 (C-6), 158.3 (C-4) ppm. HRMS (ESI): calcd

for C₁₃H₁₈N₅O₄([M + H]⁺): 308.1359, found: 308.1342.

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H_Phe), 7.33−7.49 (4H, m, H_Phe), 8.22 (1H, br. s, H-6) ppm. ¹³C NMR

(101 MHz, DMSO-d₆) δ 62.3 (C-5′), 70.7 (C-2′), 73.1 (C-3′), 84.9

(C-4′), 88.1 (C-1′), 119.1 (C_Phe), 124.5 (C_Phe), 129.6 (C_Phe), 152.8

(C_Phe), 154.7 (C-7a), 155.0 (C-6), 157.4 (C-4), 157.4 (C-3) ppm.

Two quaternary carbons missing (C-3, C-3a). HRMS (ESI): calcd for

C₁₆H₁₈N₅O₅([M + H]⁺): 360.1308, found: 360.1308.

3-(1-Pyrrolidin-yl)-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (83). A mixture of 21 (150 mg, 0.43 mmol, 1.0 equiv),

CuCl (0.006 g, 0.043 mmol, 0.1 equiv), pyrrolidine (0.706 mL, 8.60

mmol, 20.0 equiv) in 1,4-dioxane (1.5 mL) and H₂O (3 mL) was

heated in at 120 °C over the weekend. The mixture was cooled to

room temperature and diluted with MeOH. Celite was added, and the

mixture was concentrated in vacuo. The solid residue was puriﬁed by

ﬂash column chromatography (4 → 20% MeOH in CH₂Cl₂),

followed by an additional puriﬁcation by preparative RP-HPLC (0.2%

formic acid in H₂O/MeCN 98:02 to 33:67 in 12 min) to aﬀord 83

3-O-(4-Chlorophenyl)-4-amino1-β-^D-ribofuranosylpyrazolo[3,4-

d]pyrimidine (80). Compound 21 (0.220 g, 0.64 mmol, 1.0 equiv), 4-

chlorophenol (0.123 g, 0.96 mmol, 1.5 equiv), CuI (0.024 g, 0.13

mmol, 0.2 equiv), N,N-dimethylglycine (0.040 g, 0.38 mmol, 0.6

equiv), and Cs₂CO₃(0.417 g, 1.28 mmol, 2.0 equiv) were dissolved in

dry degassed DMA (4 mL) under argon. The reaction was heated at

120 °C overnight, cooled down to room temperature, and

concentrated in vacuo. The residue was taken up in MeOH, celite

was added, and the mixture was concentrated under reduced pressure.

The solid residue was puriﬁed by ﬂash column chromatography

(automated, 2 → 15% MeOH in CH₂Cl₂), followed by an additional

puriﬁcation via preparative RP-HPLC (0.2% formic acid in H₂O/

MeCN 98:02 to 33:67 in 12 min) to aﬀord 80 (8 mg, 0.020 mmol, 3%

yield) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 3.34−3.42

(1H, m, H-5′), 3.46−3.54 (1H, m, H-5″.), 3.84 (1H, dd, J = 10.0, 5.3

Hz, H-4′), 4.02−4.16 (1H, m, H-3′), 4.31−4.52 (1H, m, H-2′),

4.64−4.75 (1H, m, OH), 4.96−5.18 (1H, m, OH), 5.26−5.50 (1H,

m, OH), 6.02 (1H, d, J = 4.1 Hz, H-1′), 7.48 (4H, s, H_Phe), 8.21 (1H,

s, H_Phe) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.1 (C-5′), 70.6 (C-

2′), 73.0 (C-3′), 84.8 (C-4′), 88.0 (C-1′), 89.8 (C-3a), 120.9 (C_Phe),

128.2 (_CPhe), 129.3 (C_Phe), 152.3 (C_Phe), 153.4 (C-7a), 154.9 (C-6),

157.3 (C-4), 157.4 (C-3) ppm. HRMS (ESI): calcd for

C₁₆H₁₇ClN₅O₅([M + H]⁺): 394.0918, found: 394.0927.

1

(23 mg, 0.068 mmol, 16% yield) as a white solid. H NMR (300

MHz, DMSO-d₆) δ 1.83−1.96 (4H, m, 2 × CH_{2 pyrrolidine}), 3.29−3.40

(4H, m, 2 × CH_{2 pyrrolidine}), 3.45 (1H, dd, J = 11.7, 5.6 Hz, H-5′), 3.59

(1H, dd, J = 11.7, 4.1 Hz, H-5″), 3.87 (1H, dd, J = 9.7, 4.7 Hz, H-4′),

4.21 (1H, t, J = 4.8 Hz, H-3′), 4.50 (1H, t, J = 4.5 Hz, H-2′), 4.81

(1H, br. s, OH), 5.02 (1H, br. s, OH), 5.28 (1H, br. s, OH), 6.02

(1H, d, J = 4.4 Hz, H-1′), 6.98 (2H, br. s, NH₂), 8.09 (1H, s, H-6)

ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 24.7 (2 × C_pyrrolidine), 50.3 (2

× C_pyrrolidine), 62.6 (C-5′), 71.1 (C-2′), 73.1 (C-3′), 84.9 (C-4′), 87.9

(C-1′), 92.1 (C-3a), 150.7 (C-3), 155.4 (C-7a), 155.8 (C-6), 157.8

(C-4) ppm. HRMS (ESI): calcd for C₁₄H₂₁N₆O₄([M + H]⁺):

337.1624, found: 337.1630.

3-Chloro-4-amino-1-(2′,3′,5′-tri-O-benzoyl-β-^D-ribofuranosyl)-

pyrazolo[3,4-d]pyrimidine (84). Compound 18 (0.223 g, 0.339

mmol, 1.0 equiv), Cu₂O (0.012 g, 0.085 mmol, 0.25 equiv), Me₄NCl

(0.111 g, 1.02 mmol, 3.0 equiv), and ^L-proline (0.020 g, 0.17 mmol,

0.5 equiv) were suspended in dry degassed 2-methoxy-ethanol (2 mL)

under argon. The mixture was heated at 120 °C for 7 days until

LCMS analysis indicated ∼80% conversion and further progression

had ceased. The mixture was cooled to room temperature and

concentrated in vacuo. The residue was used crude in the next

reaction. HRMS (ESI): calcd for C₃₁H₂₅ClN₅O₇([M + H]⁺):

614.1443, found: 614.2593.

3,4-Diamino-1-β-^D-ribofuranosylpyrazolo[3,4-d]pyrimidine⁶⁹

(81). A mixture of 21 (145 mg, 0.42 mmol, 1.0 equiv), CuCl (0.006 g,

0.042 mmol, 0.1 equiv), and aq. NH₄OH (20−30% wt, 30 mL) was

heated in a pressure reactor at 130 °C overnight. The vessel was

cooled to room temperature, and the contents were diluted with

MeOH and transferred to a pear-shaped ﬂask. Celite was added, and

the mixture was concentrated in vacuo slowly (NH₃evolution!). The

solid residue was puriﬁed by ﬂash column chromatography (4 → 20%

MeOH in CH₂Cl₂), followed by an additional puriﬁcation by

preparative RP-HPLC (0.2% formic acid in H₂O/MeCN 98:02 to

40:60 in 12 min) to aﬀord 81 (23 mg, 0.082 mmol, 19% yield) as a

white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 3.32−3.45 (1H, m, H-

5′), 3.54 (1H, dd, J = 11.7, 4.1 Hz, H-5″), 3.81 (1H, dd, J = 9.4, 4.4

Hz, H-4′), 4.11 (1H, t, J = 4.8 Hz, H-3′), 4.44 (1H, t, J = 5.0 Hz, H-

2′), 4.82 (1H, br. s, OH), 5.03 (1H, br. s, OH), 5.23 (1H, br. s, OH),

5.84 (2H, br. s, NH₂), 5.93 (1H, d, J = 4.7 Hz, H-1′), 7.29 (2H, br. s.,

NH₂), 8.02 (1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.7

(C-5′), 71.0 (C-2′), 72.6 (C-3′), 84.5 (C-4′), 87.4 (C-1′), 90.6 (C-

3a), 148.1 (C-3), 155.0 (C-7a), 156.1 (C-6), 157.8 (C-4) ppm.

Spectral data are in accordance with literature values.⁶⁹HRMS (ESI):

calcd for C₁₀H₁₅N₆O₄([M + H]⁺): 283.1155, found: 283.1179.

3-N-Methylamino-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (82). A mixture of 21 (150 mg, 0.43 mmol, 1.0 equiv),

CuCl (0.006 g, 0.043 mmol, 0.1 equiv), and aqueous methylamine

(40%, 30 mL) was heated in a pressure reactor at 130 °C over the

weekend. The vessel was cooled to room temperature, and the

contents were diluted with MeOH and transferred to a pear-shaped

ﬂask. Celite was added, and the mixture was concentrated in vacuo

slowly (NHMe evolution!). The solid residue was puriﬁed by ﬂash

column chromatography (4 → 20% MeOH in CH₂Cl₂), followed by

an additional puriﬁcation by preparative RP-HPLC (0.2% formic acid

in H₂O/MeCN 98:02 to 33:67 in 12 min) to aﬀord 82 (6 mg, 0.020

3-Chloro-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]pyrimidine

(85). The crude 84 from the previous reaction was subjected to

general procedure C (reaction time: 1 h). Flash column

chromatography (automated, 4 → 20% MeOH in CH₂Cl₂), followed

by an additional puriﬁcation via preparative RP-HPLC (0.2% formic

acid in H₂O/MeCN 98:02 to 88:12 in 4 min, then to 77:23 in 5 min,

and then to 33:67 in 3 min) aﬀorded 85 (35 mg, 0.116 mmol, 34%

1

yield) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 3.42 (1H,

dt, J = 12.0, 5.9 Hz, H-5′), 3.55 (1H, dt, J = 11.7, 5.0 Hz, H-5″), 3.89

(1H, dd, J = 9.7, 4.7 Hz, H-4′), 4.16 (1H, dd, J = 9.7, 4.7 Hz, H-3′),

4.54 (1H, dd, J = 10.5, 5.3 Hz, H-2′), 4.80 (1H, t, J = 5.7 Hz, OH),

5.16 (1H, d, J = 5.3 Hz, OH), 5.40 (1H, d, J = 5.9 Hz, OH), 6.05

(1H, d, J = 4.7 Hz, OH), 7.23 (1H, br. s, NH), 8.03 (1H, s, NH), 8.24

(1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.2 (C-5′),

70.6 (C-2′), 72.9 (C-3′), 85.2 (C-4′), 88.0 (C-1′), 97.5 (C-3a), 132.0

(C-3), 155.1 (C-7a), 157.2 (C-6), 157.3 (C-4) ppm. HRMS (ESI):

calcd for C₁₀H₁₃ClN₅O₄([M + H]⁺): 302.0656, found: 302.0652.

3-Phenylethynyl-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (86). Compound 36 (0.250 g, 0.636 mmol, 1.0 equiv),

PdCl₂(PPh₃)₂(0.022 g, 0.032 mmol, 0.05 equiv), and CuI (0.012 g,

0.1 equiv) 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 phenylacetylene (0.105

mL, 0.96 mmol, 1.5 equiv) were added. The resulting solution was

stirred at room temperature for 3 h until LCMS analysis indicated

completion of the reaction. The mixture was concentrated in vacuo,

and the residue was taken up in MeOH, adsorbed onto celite, and

puriﬁed via ﬂash column chromatography (automated, 2 → 12%

MeOH in CH₂Cl₂) to aﬀord 86 (93 mg, 0.253 mmol, 40% yield) as a

white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 3.46 (1H, dt, J = 11.8,

6.0 Hz, H-5′), 3.59 (1H, dt, J = 11.9, 4.9 Hz, H-5″), 3.93 (1H, dd, J =

9.7, 4.7 Hz, H-4′), 4.21 (1H, dd, J = 9.4, 5.0 Hz, H-3′), 4.63 (1H, dd,

J = 10.8, 5.0 Hz, H-2′), 4.86 (1H, t, J = 5.9 Hz, OH), 5.18 (1H, d, J =

5.6 Hz, OH), 5.43 (1H, d, J = 6.2 Hz, OH), 6.13 (1H, d, J = 5.0 Hz,

H-1′), 7.27−7.55 (3H, m, H_Phe), 7.67−7.84 (2H, m, H_Phe), 8.28 (1H,

s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.7 (C-5′), 71.2 (C-

2′), 73.5 (C-3′), 81.1 (C_ethynyl), 85.8 (C-4′), 88.9 (C-1′), 94.1

1

mmol, 5% yield) as a white solid. H NMR (300 MHz, D₂O) δ 2.85

(3H, s, CH₃), 3.76 (1H, dd, J = 12.6, 4.1 Hz, H-5′), 3.82−3.92 (1H,

m, H-5″), 4.17 (1H, dd, J = 7.0, 3.8 Hz, H-4′), 4.45 (1H, dd, J = 5.0,

3.8 Hz, H-3′), 4.71−4.80 (1H, m, H-2′, partially underwater peak),

6.07 (1H, d, J = 5.0 Hz, H-1′), 8.04 (1H, s, H-6) ppm. A qualitative

¹³C NMR spectrum could not be obtained from the amount of

product available. HRMS (ESI): calcd for C₁₁H₁₇N₆O₄([M + H]⁺):

297.1311, found: 297.1318.

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(C_ethynyl), 101.2 (C-3a), 121.5 (C-3), 127.3 (C_Phe), 129.1 (C_Phe),

130.1 (C_Phe), 132.4 (C_Phe), 154.7 (C-7a), 157.2 (C-6), 158.2 (C-4)

ppm. HRMS (ESI): calcd for C₁₈H₁₈N₅O₄([M + H]⁺): 368.1359,

found: 368.1355.

= 5.9 Hz, OH), 6.04 (1H, d, J = 4.7 Hz, H-1′), 7.32 (2H, br. s., NH₂),

8.15 (1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 13.0

(CH₂CH₃), 21.4 (CH₂CH₃), 62.5 (C-5′), 70.9 (C-2′), 73.0 (C-3′),

85.0 (C-4′), 88.3 (C-1′), 98.6 (C-3a), 147.2 (C-3), 155.1 (C-7a),

155.9 (C-6), 158.2 (C-4) ppm. HRMS (ESI): calcd for C₁₂H₁₈N₅O₄

([M + H]⁺): 296.1359, found: 296.1363.

3-Phenylethyl-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (87). Compound 86 (0.051 g, 0.139 mmol) was dissolved

in MeOH (5 mL). The ﬂask was placed under a nitrogen atmosphere,

and a catalytic amount of Pd(OH)₂/C was added. The atmosphere

was exchanged for H₂and the mixture was stirred for 1 h until TLC

analysis (20% MeOH in CH₂Cl₂) indicated completion of the

reaction. The mixture was ﬁltered over celite, celite was added to the

ﬁltrate, and the solvents were removed under reduced pressure. The

solid residue was puriﬁed via ﬂash column chromatography

(automated, 2 → 20% MeOH in CH₂Cl₂) to aﬀord 87 (46 mg,

0.124 mmol, 89% yield) as a white solid.

3-(1H-1,2,3-Triazol-4-yl)-4-amino-1-β-^D-ribofuranosylpyrazolo-

[3,4-d]pyrimidine (90). Compound 88 (0.125 g, 0.429 mmol, 1.0

equiv) and CuI (0.004 g, 0.021 mmol, 0.05 equiv) were dissolved in

MeOH (0.2 mL) and DMF (1.8 mL). TMSN₃(0.085 mL, 0.644

mmol, 1.5 equiv) was added, and the mixture was heated at 100 °C

overnight. The mixture was cooled to room temperature and

concentrated in vacuo. The residue was dissolved in MeOH, celite

was added, and the solvents were removed under reduced pressure.

The solid residue was puriﬁed by ﬂash column chromatography (4 →

20% MeOH in CH₂Cl₂) to aﬀord 90 (57 mg, 0.170 mmol, 40% yield)

¹H NMR (300 MHz, DMSO-d₆) δ 3.02 (2H, dd, J = 8.6, 7.0 Hz,

CH₂), 3.28 (2H, dd, J = 9.2, 7.0 Hz, CH₂), 3.42 (1H, dt, J = 12.2, 6.3

Hz, H-5′), 3.58 (1H, dt, J = 11.7, 4.7 Hz, H-5″), 3.89 (1H, dd, J =

10.3, 4.6 Hz, H-4′), 4.22 (1H, dd, J = 10.5, 5.0 Hz, H-3′), 4.54 (1H,

dd, J = 10.3, 5.0 Hz, H-2′), 4.83 (1H, dd, J = 6.4, 5.3 Hz, OH), 5.08

(1H, d, J = 5.6 Hz, OH), 5.31 (1H, d, J = 5.9 Hz, OH), 6.06 (1H, d, J

= 4.4 Hz, H-1′), 7.15−7.32 (5H, m, H_Phe), 8.16 (1H, s, H-6) ppm.

¹³C NMR (75 MHz, DMSO-d₆) δ 29.4 (CH₂), 33.4 (CH₂), 62.6 (C-

5′), 71.0 (C-2′), 73.2 (C-3′), 85.0 (C-4′), 88.3 (C-1′), 98.8 (C-3a),

125.8 (C_Phe), 128.1 (C_Phe), 128.5 (C_Phe), 141.1 (C_Phe), 145.2 (C-3),

155.0 (C-7a), 155.8 (C-6), 158.3 (C-4) ppm. HRMS (ESI): calcd for

C₁₈H₂₂N₅O₄([M + H]⁺): 372.1672, found: 372.1684.

1

as a light-brown solid. H NMR (300 MHz, DMSO-d₆) δ 3.48 (1H,

dt, J = 12.0, 5.6 Hz, H-5′), 3.62 (1H, dt, J = 12.0, 4.7 Hz, H-5″), 3.93

(1H, dd, J = 9.4, 4.6 Hz, H-4′), 4.26 (1H, dd, J = 10.0, 5.0 Hz, H-3′),

4.66 (1H, dd, J = 10.3, 5.2 Hz, H-2′), 4.88 (1H, t, J = 5.6 Hz, OH),

5.13 (1H, d, J = 5.6 Hz, OH), 5.43 (1H, d, J = 5.9 Hz, OH), 6.14

(1H, d, J = 5.0 Hz, H-1′), 8.14 (1H, br. s., CH_triazole), 8.25 (1H, s, H-

6), 8.54 (1H, br. s, NH) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.4

(C-5′), 70.9 (C-2′), 73.0 (C-3′), 85.4 (C-4′), 88.8 (C-), 98.0 (C-3a),

131.1 (C_triazole), 136.3 (C-3), 140.3 (C_triazole), 155.1 (C-7a), 156.7 (C-

6), 158.4 (C-4) ppm. HRMS (ESI): calcd for C₁₂H₁₅N₈O₄([M +

H]⁺): 335.1216, found: 335.1205.

3-Ethynyl-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (88). Compound 36 (0.250 g, 0.636 mmol, 1.0 equiv),

PdCl₂(PPh)₃(0.022 g, 0.032 mmol, 0.05 equiv), and CuI (0.012 g,

0.1 equiv) 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 ethynyltrimethylsilane

(0.881 mL, 6.36 mmol, 10 equiv) were added. The resulting solution

was stirred at room temperature for 3 h until LCMS analysis indicated

completion of the reaction. The mixture was concentrated in vacuo,

and the residue was taken up in MeOH, adsorbed onto celite, and

puriﬁed via ﬂash column chromatography (automated, 2 → 12%

MeOH in CH₂Cl₂). The intermediate TMS-ethynyl nucleoside was

stirred overnight in 7 N NH₃in MeOH (10 mL). The mixture was

concentrated in vacuo, and the residue was puriﬁed again by ﬂash

column chromatography (automated, 2 → 12% MeOH in CH₂Cl₂) to

aﬀord 88 (35 mg, 0.120 mmol, 19% yield) as a white solid. ¹H NMR

(300 MHz, DMSO-d₆) δ 3.43 (1H, dt, J = 11.7, 6.0 Hz, H-5′), 3.57

(1H, dt, J = 11.7, 5.0 Hz, H-5″), 3.91 (1H, dd, J = 9.7, 4.7 Hz, H-4′),

4.19 (1H, dd, J = 9.7, 4.7 Hz, H-3′), 4.57 (1H, dd, J = 10.7, 5.2 Hz, H-

2′), 4.70 (1H, s, H_ethynyl), 4.83 (1H, t, J = 5.9 Hz, OH), 5.16 (1H, d, J

= 5.3 Hz, OH), 5.41 (1H, d, J = 6.2 Hz, OH), 6.09 (1H, d, J = 4.7 Hz,

H-1′), 6.79 (2H, br. s, NH₂), 8.25 (1H, s, H-6) ppm. ¹³C NMR (75

MHz, DMSO-d₆) δ 62.2 (C-5′), 70.7 (C-2′), 73.0 (C-3′), 74.9

(C_ethynyl), 85.4 (C-4′), 86.7 (C_ethynyl), 88.4 (C-1′), 101.1 (C-3a),

126.3 (C-3), 154.0 (C-7a), 156.8 (C-6), 157.7 (C-4) ppm. HRMS

(ESI): calcd for C₁₂H₁₄N₅O₄([M + H]⁺): 292.1046, found:

292.1049.

3-Ethyl-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]pyrimidine

(89). Compound 88 (47 mg, 0.161 mmol) was dissolved in MeOH (5

mL). The ﬂask was placed under a nitrogen atmosphere, and a

catalytic amount of Pd(OH)₂/C was added. The atmosphere was

exchanged for H₂and the mixture was stirred for 1 h until TLC

analysis (20% MeOH in CH₂Cl₂) indicated completion of the

reaction. The mixture was ﬁltered over celite, celite was added to the

ﬁltrate, and the solvents were removed under reduced pressure. The

solid residue was puriﬁed via ﬂash column chromatography

(automated, 2 → 20% MeOH in CH₂Cl₂) to aﬀord 87 (35 mg,

0.119 mmol, 74% yield) as a white solid. ¹H NMR (300 MHz,

DMSO-d₆) δ 1.22 (3H, t, J = 7.5 Hz, CH₂CH₃), 2.96 (2H, q, J = 7.5

3-Cyano-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine⁶⁶(91). Compound 36 (2.73 g, 6.95 mmol, 1.0 equiv),

Pd₂(dba)₃(0.382 g, 0.417 mmol, 0.06 equiv), dppf (0.771 g, 1.39

mmol, 0.2 equiv), and Zn(CN)₂(0.490 g, 4.17 mmol, 0.6 equiv) were

dissolved in dry degassed DMF (30 mL) in a ﬂame-dried ﬂask under

argon. The mixture was stirred at 150 °C for 90 min until LCMS

analysis indicated completion of the reaction. The mixture was cooled

to room temperature and concentrated in vacuo. The residue was

taken up in MeOH, celite was added, and the solvents were removed

under reduced pressure. The solid residue was puriﬁed ﬁrst by manual

ﬂash column chromatography (5 → 20% MeOH in CH₂Cl₂) and then

by automated ﬂash column chromatography (4 → 20% MeOH in

CH₂Cl₂) to aﬀord 91 (570 mg, 1.95 mmol, 28% yield) as a white

1

solid. H NMR (300 MHz, DMSO-d₆) δ 3.45 (1H, dt, J = 11.7, 5.9

Hz, H-5′), 3.58 (1H, dt, J = 12.0, 5.0 Hz, H-5″), 3.95 (1H, dd, J = 9.7,

4.7 Hz, H-4′), 4.22 (1H, dd, J = 9.7, 5.0 Hz, H-3′), 4.60 (1H, dd, J =

10.3, 5.2 Hz, H-2′), 4.82 (1H, t, J = 5.9 Hz, OH), 5.23 (1H, d, J = 5.6

Hz, OH), 5.50 (1H, d, J = 5.9 Hz, OH), 6.17 (1H, d, J = 5.0 Hz, H-

1′), 8.35 (1H, s, H-6) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ 62.0

(C-5′), 70.6 (C-2′), 73.3 (C-3′), 85.8 (C-4′), 89.1 (C-1′), 101.2 (C-

3), 113.0 (CN), 116.9 (C-3a), 154.4 (C-7a), 157.1 (C-6), 157.4 (C-

4) ppm. HRMS (ESI): calcd for C₁₁H₁₃N₆O₄([M + H]⁺): 293.0998,

found: 293.1002. Spectral data are in accordance with literature

values.⁶⁶

3-(1H-Tetrazol-5-yl)-4-amino-1-β-D-ribofuranosylpyrazolo[3,4-

d]pyrimidine (92). Compound 91 (0.074 g, 0.253 mmol), NaN₃

(0.021 g, 0.329 mmol, 1.3 equiv), NH₄Cl (0.018 g, 0.329 mmol, 1.3

equiv), and a catalytic amount of LiCl were suspended in DMF (3

mL). The mixture was heated at 100 °C overnight, cooled down to

room temperature, and concentrated in vacuo. The residue was taken

up in MeOH, adsorbed onto celite, and puriﬁed by ﬂash column

chromatography (2 → 40% MeOH in CH₂Cl₂+ 0.1% AcOH) to

aﬀord 92 (21 mg, 0.063 mmol, 25% yield) as a white solid. ¹H NMR

(300 MHz, DMSO-d₆) δ 3.50 (1H, dd, J = 11.7, 6.2 Hz, H-5′), 3.65

(1H, dd, J = 11.9, 4.5 Hz, H-5″), 3.97 (1H, dd, J = 10.3, 5.0 Hz, H-

4′), 4.34 (1H, t, J = 4.8 Hz, H-3′), 4.70 (1H, t, J = 4.8 Hz, H-2′), 6.20

(1H, d, J = 4.4 Hz, H-1′), 8.34 (1H, s, H-6), 8.45 (1H, br. s.,

H_tetrazole), 9.12 (1H, br. s., NH_tetrazole) ppm. ¹³C NMR (75 MHz,

DMSO-d₆) δ 62.4 (C-5′), 70.7 (C-2′), 73.1 (C-3′), 85.7 (C-4′), 89.1

(C-1′), 98.6 (C-3a), 131.3 (C-3), 150.4 (C_tetrazole), 155.0 (C-7a),

156.4 (C-6), 157.5 (C-4) ppm. HRMS (ESI): calcd for C₁₁H₁₄N₉O₄

([M + H]⁺): 336.1169, found: 336.1176.

Hz, CH₂CH₃), 3.43 (1H, dt, J = 12.0, 6.0 Hz, H-5′), 3.59 (1H, dt, J =

12.0, 4.7 Hz, H-5″), 3.89 (1H, dd, J = 9.7, 4.5 Hz, H-4′), 4.21 (1H,

dd, J = 10.3, 5.3 Hz, H-3′), 4.57 (1H, dd, J = 10.0, 5.0 Hz, H-2′), 4.86

(1H, t, J = 6.0 Hz, OH), 5.08 (1H, d, J = 5.6 Hz, OH), 5.30 (1H, d, J

4230

https://doi.org/10.1021/acs.jmedchem.1c00135

J. Med. Chem. 2021, 64, 4206−4238

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

3-Aminomethyl-4-amino-1-β-D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (93). 88 (47 mg, 0.161 mmol) was dissolved in MeOH (5

mL). The ﬂask was placed under a nitrogen atmosphere, and Raney

nickel (slurry in H₂O, 1 mL) was added. The atmosphere was

exchanged for H₂and the mixture was stirred for 1 h until TLC

analysis (20% MeOH in CH₂Cl₂) indicated completion of the

reaction. The mixture was ﬁltered over celite, celite was added to the

ﬁltrate, and the solvents were removed under reduced pressure. The

solid residue was puriﬁed via ﬂash column chromatography

(automated, NH₄OH/MeOH/CH₂Cl₂1/0/99 → 1/25/74), followed

by an additional puriﬁcation via preparative RP-HPLC (0.2% formic

acid in H₂O/MeCN 95:05 to 68:32 in 6 min) to aﬀord 93 (5 mg,

0.017 mmol, 10% yield) as a white solid. ¹H NMR (300 MHz,

CD₃OD) δ 2.66 (2H, s, CH₂NH₂), 3.69 (1H, dd, J = 12.3, 4.4 Hz, H-

5′), 3.81 (1H, dd, J = 12.3, 3.2 Hz, H-5″), 4.10 (1H, dd, J = 7.9, 3.5

Hz, H-4′), 4.38−4.51 (1H, m, H-3′), 4.73−4.80 (1H, m, H-2′), 6.23

(1H, d, J = 4.7 Hz, H-1′), 8.20 (1H, s, H-6), 8.50 (4H, br. s., 2 ×

NH₂) ppm. A qualitative ¹³C NMR spectrum could not be obtained

from the amount of product available. HRMS (ESI): calcd for

C₁₁H₁₇N₆O₄([M + H]⁺): 297.1311, found: 297.1314.

(1H, d, J = 4.7 Hz, H-1′), 8.36 (1H, s, H-6) ppm. ¹³C NMR (101

MHz, DMSO-d₆) δ 62.1 (C-5′), 70.7 (C-2′), 73.1 (C-3′), 85.7 (C-

4′), 88.8 (C-1′), 96.6 (C-3a), 120.7 (q, J = 269.0 Hz, CF₃), 132.8 (q, J

= 38.5 Hz, C-3), 155.6 (C-7a), 156.7 (C-6), 157.2 (C-4). ¹⁹F NMR

(282 MHz, DMSO-d₆) δ −59.61 (1F, s) ppm. HRMS (ESI): calcd for

C₁₁H₁₃F₃N₅O₄([M + H]⁺): 336.0920, found: 336.0917.

5-Amino-3-methyl-1H-pyrazole-4-carbonitrile⁹⁸(97). A solution

of malonitrile (1.11 mL, 20.0 mmol, 1.0 equiv) in THF (20 mL) was

cooled to 0 °C. NaH (60% wt in mineral oil, 1.60 g, 40.0 mmol, 2.0

equiv) was added slowly, and the mixture was stirred for 10 min.

Acetyl chloride (1.43 mL, 20.0 mmol, 1.0 equiv) was added, and the

mixture was gradually warmed to room temperature. After 1 h,

dimethyl sulfate (2.28 mL, 24.0 mmol, 1.2 equiv) was added and the

mixture was heated to reﬂux. After 3 h, the mixture was cooled down

to room temperature, and Et₃N (6.97 mL, 50.0 mmol, 2.0 equiv) was

added, followed by hydrazine hydrate (1.0 mL, 20.0 mmol, 1.0 equiv).

The mixture was again heated at reﬂux temperature for 1 h and cooled

down to room temperature. The mixture was concentrated in vacuo,

diluted with H₂O and EtOAc, and transferred to a separation funnel.

The phases were separated, and the aqueous phase was extracted

twice more with EtOAc. The combined organic phases were dried

over Na₂SO₄and concentrated in vacuo. The residue was taken up in

MeCN, celite was added, and the solvent was removed under reduced

pressure. The solid residue was puriﬁed by ﬂash column

chromatography (manual, petroleum ether/EtOAc 1:1 and 3:7) to

aﬀord 97 (0.930 g, 7.61 mmol, 38% yield) as a yellow sticky foam. ¹H

NMR (300 MHz, DMSO-d₆) δ 2.12 (3H, br. s.), 5.72 (2H, br. s),

11.51 (1H, br. s) ppm. HRMS (ESI): calcd for C₅H₇N₄([M + H]⁺):

123.0671, found: 123.0662. Spectral data are in accordance with

literature values.⁹⁸

3-Carboxamido-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine⁶⁶(94). Compound 91 (55 mg, 0.188 mmol) was

dissolved in aq. NH₄OH (28−30% wt 4 mL). Aq. H₂O₂(30% w/

w, 1 mL) was added, and the mixture was stirred for 2 h until LCMS

analysis indicated full conversion. The residue was diluted with

MeOH, celite was added, and the solvents were removed under

reduced pressure. The solid residue was puriﬁed by ﬂash column

chromatography (automated, 2 → 40% MeOH in CH₂Cl₂+ 0.1%

AcOH) to aﬀord 92 (21 mg, 0.063 mmol, 25% yield) as a brown

1

solid. H NMR (300 MHz, DMSO-d₆) δ 3.47 (1H, dt, J = 12.0, 6.2

3-Methyl-4-amino-1H-pyrazolo[3,4-d]pyrimidine⁹⁹(98). Com-

pound 97 (0.441 g, 3.61 mmol, 1.0 equiv) was dissolved in

formamide (2 mL). The mixture was heated at 180 °C for 24 h

and cooled down to room temperature by pouring into ice-cold water

(25 mL). The resulting solids were ﬁltered oﬀ and dried under a high

vacuum overnight to aﬀord 98 (126 mg, 0.84 mmol, 23% yield) as a

Hz, H-5′), 3.62 (1H, dt, J = 12.0, 5.3 Hz, H-5″), 3.93 (1H, dd, J =

10.3, 4.7 Hz, H-4′), 4.30 (1H, dd, J = 10.3, 4.7 Hz, H-3′), 4.68 (1H,

dd, J = 10.5, 5.3 Hz, H-2′), 4.84 (1H, t, J = 5.9 Hz, OH), 5.08 (1H, d,

J = 5.6 Hz, OH), 5.44 (1H, d, J = 5.9 Hz, OH), 6.14 (1H, d, J = 4.7

Hz, H-1′), 7.99 (1H, s, NH₂), 8.07 (1H, br. s., NH₂), 8.19 (1H, br. s.,

NH₂), 8.24 (1H, s, H-6), 8.86 (1H, br. s., NH₂) ppm. ¹³C NMR (101

MHz, DMSO-d₆) δ 62.3 (C-5′), 70.8 (C-2′), 73.1 (C-3′), 85.7 (C-

4′), 88.9 (C-1′), 99.5 (C-3a), 139.0 (C-3), 155.4 (C-7a), 156.9 (C-6),

158.2 (C-4), 163.8 (CO) ppm. HRMS (ESI): calcd for

C₁₁H₁₅N₆O₅([M + H]⁺): 311.1104, found: 311.1110. Spectral data

are in accordance with literature values.⁶⁶

3-Triﬂuoromethyl-4-amino-1-(2′,3′,5′-tri-O-benzoyl)-β-D-

ribofuranosylpyrazolo[3,4-d]pyrimidine (95). TMSCF₃(0.315 mL,

2.13 mmol, 3.0 equiv) was added dropwise over the course of 1 h to a

suspension of CuI (0.406 g, 2.13 mmol, 3.0 equiv) and KF (0.124 g,

2.13 mmol, 3.0 equiv) in a mixture of dry degassed DMF/NMP 1:1

(3 mL). When all solids had dissolved, 34 (0.500 g, 0.709 mmol, 1.0

equiv) 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

water (5 mL), and the solids were ﬁltered oﬀ over Celite. The ﬁlter

cake was washed extensively with additional EtOAc (3 × 25 mL), and

the combined ﬁltrates were transferred to a separation funnel.

Additional water (40 mL) was added, the phases were separated, and

the organic phase was 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): calcd for

C₃₂H₂₅F₃N₅O₇([M + H]⁺): 648.1706, found: 648.1723.

1

brown solid. H NMR (300 MHz, DMSO-d₆) δ 2.52 (3H, s, CH₃),

7.15 (2H, br. s., NH₂), 8.09 (1H, s, H-6), 12.91 (1H, br. s., NH) ppm.

¹³C NMR (75 MHz, DMSO-d₆) δ 14.9 (CH₃), 98.8 (C-3a), 141.3

(C-3), 156.1 (C-7a), 156.3 (C-6), 158.8 (C-4) ppm. HRMS (ESI):

calcd for C₆H₈N₅([M + H]⁺): 150.0780, found: 150.0768. Spectral

data are in accordance with literature values.⁹⁹

3,6-Dimethyl-4-amino-1H-pyrazolo[3,4-d]pyrimidine (99). Com-

pound 97 (0.206 g, 1.69 mmol, 1.0 equiv) was dissolved in 2-

methoxy-ethanol (3 mL). Thioacetamide (0.253 g, 3.37 mmol, 2.0

equiv) was added, and the mixture was heated at reﬂux overnight. The

mixture was cooled down to room temperature, and the solvent was

removed in vacuo. The residue was taken up in MeOH, adsorbed onto

celite, and puriﬁed by ﬂash column chromatography (manual, 10%

MeOH in CH₂Cl₂) to aﬀord 99 (100 mg, 0.61 mmol, 36% yield) as a

1

light-purple solid. H NMR (300 MHz, DMSO-d₆) δ 2.06 (3H, s,

CH₃), 2.36 (3H, s, CH₃), 7.14 (2H, br. s, NH₂), 12.80 (1H, br. s,

NH) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 14.6 (CH₃) 21.8

(CH₃), 97.0 (C-3a), 141.1 (C-3), 157.3 (C-7a), 158.5 (C-4), 164.9

(C-6) ppm. HRMS (ESI): calcd for C₇H₁₀N₅([M + H]⁺): 164.0936,

found: 164.0914.

3-Methyl-4-amino-1-(2′,3′,5′-tri-O-benzoyl-1-β-^D-ribofuranosyl)-

pyrazolo[3,4-d]pyrimidine (100). Compound 98 (0.100 g, 0.67

mmol) and 1-O-acetyl-2,3,5-tri-O-benzoyl-β-^D-ribofuranose (0.406 g,

0.8 mmol, 1.2 equiv) were subjected to general procedure B.

Puriﬁcation by ﬂash column chromatography (automated, 0 → 5%

MeOH in CH₂Cl₂) aﬀorded 100, which was used as such in the next

reaction. HRMS (ESI): calcd for C₃₂H₂₈N₅O₇([M + H]⁺): 594.1989,

found: 594.2010.

3-Methyl-4-amino-1-β-^D-ribofuranosyl-pyrazolo[3,4-d]-

pyrimidine (101). Compound 98 (used directly from the previous

reaction) was subjected to general procedure C (reaction time: 1 h).

Puriﬁcation by ﬂash column chromatography (automated, 4 → 20%

MeOH in CH₂Cl₂) aﬀorded 101 (40 mg, 0.142 mmol, 21% yield over

2 steps) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 2.53 (3H,

3-Triﬂuoromethyl-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (96). Crude 95 was subjected to general procedure C

(reaction time: 1 h). Puriﬁcation by ﬂash column chromatography (4

→ 20% MeOH in CH₂Cl₂), followed by additional puriﬁcation by

preparative RP-HPLC (0.2% formic acid in H₂O/MeCN 98:02 to

33:67 in 12 min) aﬀorded 96 (33 mg, 0.098 mmol, 14% yield over 2

1

steps) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 3.44 (1H,

dt, J = 12.0, 5.6 Hz, H-5′), 3.58 (1H, dt, J = 12.0, 4.7 Hz, H-5″), 3.94

(1H, dd, J = 9.7, 5.0 Hz, H-4′), 4.22 (1H, dd, J = 8.5, 4.1 Hz, H-3′),

4.61 (1H, dd, J = 9.4, 4.7 Hz, H-2′), 4.83 (1H, t, J = 5.6 Hz, OH),

5.23 (1H, d, J = 5.0 Hz, OH), 5.48 (1H, d, J = 5.6 Hz, OH), 6.18

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s, CH₃), 3.42 (1H, dt, J = 11.9, 6.1 Hz, H-5′), 3.56 (1H, dt, J = 11.4,

4.7 Hz, H-5″), 3.87 (1H, dd, J = 9.7, 4.7 Hz, H-4′), 4.17 (1H, dd, J =

10.0, 5.0 Hz, H-3′), 4.56 (1H, dd, J = 10.5, 5.3 Hz, H-2′), 4.86 (1H, t,

J = 5.9 Hz, OH), 5.10 (1H, d, J = 5.3 Hz, OH), 5.30 (1H, d, J = 5.9

Hz, OH), 6.02 (1H, d, J = 4.7 Hz, H-1′), 7.35 (2H, br. s., NH₂), 8.14

(1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 14.5 (CH₃),

62.4 (C-5′), 70.8 (C-2′), 72.8 (C-3′), 84.9 (C-4′), 88.0 (C-1′), 99.3

(C-3a), 141.9 (C-3), 155.1 (C-7a), 156.0 (C-6), 158.4 (C-4) ppm.

HRMS (ESI): calcd for C₁₁H₁₆N₅O₄([M + H]⁺): 282.1202, found:

282.1216.

followed by NaBH₃CN (0.039 g, 0.62 mmol, 3.0 equiv). After 2 h,

LCMS analysis indicated completion of the reaction. The reaction

mixture was diluted with water (15 mL) and extracted with EtOAc (3

× 25 mL). The combined organic phases were dried over Na₂SO₄,

concentrated in vacuo, and used crude in the next reaction. HRMS

(ESI): calcd for C₃₆H₃₅N₆O₈([M + H]⁺): 679.2516, found:

679.2631.

3-(Morpholinomethyl)-4-amino-1-(β-^D-ribofuranosyl)pyrazolo-

[3,4-d]pyrimidine (105). Compound 104 (crude) was subjected to

general procedure C (reaction time: 90 min). Puriﬁcation by ﬂash

column chromatography (automated, 4 → 20% MeOH in CH₂Cl₂)

aﬀorded 105 (48 mg, 0.131 mmol, 64% yield over 2 steps) as a white

3-Vinyl-4-amino-1-(2′,3′,5′-tri-O-benzoyl-β-^D-ribofuranosyl)-

pyrazolo[3,4-d]pyrimidine (102). Compound 34 (5.66 g, 8.03 mmol,

1.0 equiv), Pd(OAc)₂(0.090 g, 0.40 mmol, 0.05 equiv), PPh₃(0.316

g, 1.20 mmol, 0.15 equiv), potassium vinyl triﬂuoroborate (2.92 g,

10.0 mmol, 1.25 equiv), and Cs₂CO₃(7.85 g, 24.1 mmol, 3.0 equiv)

were dissolved in degassed DMF/H₂O 9:1 (20 mL) under argon. The

mixture was stirred at 100 °C for 72 h, cooled down to room

temperature, and transferred to a separation funnel. Water (50 mL)

was added, and the mixture was extracted with CH₂Cl₂(3 × 100 mL).

The combined organic phases were dried over Na₂SO₄and

concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (automated, 40 → 100% EtOAc in petroleum ether)

1

solid. H NMR (300 MHz, DMSO-d₆) δ 2.38−2.58 (4H, m, 2 ×

CH_{2 morpholine}, under DMSO peak), 3.42 (1H, dt, J = 10.5, 5.0 Hz, H-

5′), 3.49−3.69 (5H, m, H-5″, 2 × CH_{2 morpholine}), 3.75 (1H, d, J = 14.4

Hz, CH₂N), 3.80 (1H, d, J = 14.4 Hz, CH₂N), 3.88 (1H, dd, J = 9.4,

4.7 Hz, H-4′), 4.18 (1H, dd, J = 8.2, 4.4 Hz, H-3′), 4.58 (1H, dd, J =

9.7, 3.8 Hz, H-2′), 4.87 (1H, t, J = 6.2 Hz, OH), 5.10 (1H, d, J = 3.5

Hz, OH), 5.31 (1H, d, J = 4.1 Hz, OH), 6.02 (1H, d, J = 5.0 Hz, H-

1′), 7.79 (1H, br. s., NH₂), 8.17 (1H, s, H-6), 8.55 (1H, br. s., NH₂)

ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 52.9 (2 × CH_{2 morpholine}), 56.1

(CH₂N), 62.4 (C-5′), 66.0 (2 × CH_{2 morpholine}), 70.8 (C-2′), 72.8 (C-

3′), 85.1 (C-4′), 88.4 (C-1′), 99.4 (C-3a), 143.5 (C-3), 155.2 (C-7a),

156.2 (C-6), 158.6 (C-4) ppm. HRMS (ESI): calcd for C₁₅H₂₃N₆O₅

([M + H]⁺): 367.1730, found: 367.1727.

1

to aﬀord 102 (1.85 g, 3.05 mmol, 38% yield) as a colorless oil. H

NMR (300 MHz, CDCl₃) δ 4.64 (1H, dd, J = 11.7, 4.7 Hz, H-5′),

4.73−4.89 (2H, m, H-5″, H-4′), 5.66 (1H, dd, J = 11.1, 1.2 Hz,

HC=CH₂), 5.98 (1H, dd, J = 17.7, 1.2 Hz, HC=CH₂), 6.10 (2H, br. s,

NH₂), 6.34 (1H, t, J = 5.7 Hz, H-3′), 6.44 (1H, dd, J = 5.3, 3.2 Hz, H-

2′), 6.87 (1H, d, J = 3.2 Hz, H-1′), 6.87 (1H, dd, J = 17.9, 11.4 Hz,

HC=CH₂), 7.30−7.45 (6H, m, H_Bz), 7.48−7.61 (3H, m, H_Bz), 7.92−

8.03 (4H, m, H_Bz), 8.05−8.14 (2H, m, H_Bz), 8.35 (1H, s, H-6) ppm.

¹³C NMR (75 MHz, CDCl₃) δ 64.0 (C-5′), 72.0 (C-2′), 74.5 (C-3′),

80.0 (C-4′), 86.7 (C-1′), 98.9 (C-3a), 121.8 (HC=CH₂), 128.2

(HC=CH₂), 128.3 (C_Bz), 128.4 (C_Bz), 128.4 (C_Bz), 128.8 (C_Bz),

128.9 (C_Bz), 129.6 (C_Bz), 129.8 (C_Bz), 129.8 (C_Bz), 133.0 (C_Bz), 133.5

(C_Bz), 133.6 (C_Bz), 144.3 (C-3), 155.4 (C-7a), 155.4 (C-6), 157.5 (C-

4), 165.1 (CO), 165.3 (CO), 166.2 (CO) ppm. HRMS

(ESI): calcd for C₃₃H₂₈N₅O₇([M + H]⁺): 606.1989, found:

606.1978.

3-Diﬂuoromethyl-4-amino-1-(2′,3′,5′-tri-O-benzoyl-β-^D-

ribofuranosyl)pyrazolo[3,4-d]pyrimidine (106). Compound 103

(0.143 g, 0.235 mmol, 1.0 equiv) was dissolved in CH₂Cl₂(5 mL).

DAST (0.155 mL, 1.18 mmol, 5.0 equiv) was added, and the mixture

was stirred overnight at room temperature. The reaction was

quenched via the slow addition of aq. sat. NaHCO₃(20 mL) and

extracted with CH₂Cl₂(3 × 25 mL). The combined organic phases

were dried over Na₂SO₄and concentrated in vacuo. The residue was

taken up in CH₂Cl₂, adsorbed onto celite, and puriﬁed via ﬂash

column chromatography (5 → 65% EtOAc in petroleum ether). The

obtained product was used directly in the next reaction. HRMS (ESI):

calcd for C₃₂H₂₆F₂N₅O₇([M + H]⁺): 630.1800, found: 630.1808.

3-Diﬂuoromethyl-4-amino-1-(β-^D-ribofuranosyl)pyrazolo[3,4-d]-

pyrimidine (107). Compound 106 (used directly from the previous

reaction) was subjected to general procedure C (reaction time: 30

min). Puriﬁcation via ﬂash column chromatography (4 → 20%

MeOH in CH₂Cl₂) aﬀorded 107 (43 mg, 0.136 mmol, 58% yield over

2 steps) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 3.44 (1H,

dt, J = 11.8, 6.0 Hz, H-5′), 3.57 (1H, dt, J = 11.7, 5.0 Hz, H-5″), 3.93

(1H, dd, J = 10.0, 4.7 Hz, H-2′), 4.20 (1H, dd, J = 10.0, 5.0 Hz, H-3′),

4.61 (1H, dd, J = 10.5, 5.3 Hz, H-2′), 4.84 (1H, t, J = 5.7 Hz, OH),

5.19 (1H, d, J = 5.3 Hz, OH), 5.43 (1H, d, J = 5.9 Hz, OH), 6.14

(1H, d, J = 4.7 Hz, H-1′), 6.72 (1H, br. s), 7.42 (1H, t, J = 53.3 Hz),

7.94 (1H, br. s), 8.32 (1H, s) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ

62.6 (C-5′), 71.2 (C-2′), 73.4 (C-3′), 85.9 (C-4′), 89.0 (C-1′) 97.4

(C-3a), 111.5 (t, J = 232.6 Hz, CF₂H), 138.5 (t, J = 28.8 Hz, C-3),

155.8 (C-7a), 157.2 (C-6), 157.6 (C-4) ppm. ¹⁹F NMR (282 MHz,

DMSO-d₆) δ −110.91 (2F, d, J = 52.9 Hz) ppm. HRMS (ESI): calcd

for C₁₁H₁₄F₂N₅O₄([M + H]⁺): 318.1014, found: 318.1019.

4-Amino-1-(2′,3′,5′-tri-O-benzoyl-β-^D-ribofuranosyl)pyrazolo-

[3,4-d]pyrimidine-3-carbaldehyde (103). Compound 102 (1.53 g,

2.52 mmol, 1.0 equiv) and K₂OsO₄·2H₂O (0.019 g, 0.05 mmol, 0.02

equiv) were dissolved in a mixture of dioxane (18.9 mL, 7.5 mL/

mmol) and water (6.3 mL, 2.5 mL/mmol). 2.6-Lutidine (0.584 mL,

5.04 mmol, 2.0 equiv) and NaIO₄(2.16 g, 10.1 mmol, 4.0 equiv) were

added, and the mixture was stirred for 3 h until TLC analysis

(petroleum ether/EtOAc 50:50) indicated full conversion. Aq. sat.

Na₂SO₃(25 mL) was added, and the mixture was stirred for 1 more

hour before it was transferred to a separation funnel. The mixture was

extracted with CH₂Cl₂(3 × 60 mL). The combined organic fractions

were dried over Na₂SO₄and concentrated in vacuo. The residue was

puriﬁed by ﬂash column chromatography (automated, 15 → 70%

EtOAc in petroleum ether) to aﬀord 103 (0.731 g, 1.20 mmol, 48%

yield) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 4.62 (1H, dd, J

= 13.2, 5.3 Hz, H-5′), 4.85−4.94 (2H, m, H-5″, H-4′), 6.32 (1H, t, J =

5.6 Hz, H-3′), 6.49 (1H, dd, J = 5.4, 3.7 Hz, H-2′), 6.58 (1H, br. s,

NH₂), 6.91 (1H, d, J = 3.5 Hz, H-1′), 7.34−7.47 (6H, m, H_Bz), 7.52−

7.63 (3H, m, H_Bz), 7.90 (1H, br. s., NH₂), 7.94−8.05 (4H, m, H_Bz),

8.05−8.16 (2H, m, H_Bz), 8.40 (1H, s, H-6), 9.70 (1H, s, CHO) ppm.

¹³C NMR (75 MHz, CDCl₃) δ 63.3 (C-5′), 71.8 (C-2′), 74.3 (C-3′),

4-Amino-1-(2′,3′,5′-tri-O-benzoyl-β-^D-ribofuranosyl)pyrazolo-

[3,4-d]pyrimidine-3-carboxylic Acid (108). Compound 103 (0.588 g,

0.968 mmol, 1.0 eq) and NaH₂PO₄(0.035 g, 0.291 mmol, 0.3 equiv)

were dissolved in a mixture of THF (9 mL) and H₂O (1.5 mL). Aq.

H₂O₂(30% w/w, 0.110 mL, 0.968 mmol, 1.0 equiv) was added,

followed by dropwise addition of a solution of NaClO₂(0.123 g, 1.36

mmol, 1.4 equiv) in H₂O (1.5 mL). The reaction mixture was stirred

overnight, diluted with 0.5 M HCl (20 mL), and extracted with

EtOAc (3 × 50 mL). The combined organic phases were dried over

Na₂SO₄and concentrated in vacuo. The residue was taken up in

CH₂Cl₂and adsorbed onto celite. The solid residue was puriﬁed by

ﬂash column chromatography (0 → 10% MeOH in CH₂Cl₂+ 0.1%

HOAc) to aﬀord 108 (0.570 g, 0.914 mmol, 94%) as a colorless oil.

¹H NMR (300 MHz, CDCl₃) δ 4.72 (1H, dd, J = 13.2, 6.4 Hz, H-5′),

80.8 (C-4′), 87.3 (C-1′), 99.4 (C-3a), 128.4 (C_Phe), 128.5 (C_Phe

)

(C_Phe), 129.6 (C_Phe), 129.8 (C_Phe), 129.8 (C_Phe), 133.3 (C_Phe), 133.7

(C_Phe), 133.8 (C_Phe), 144.9 (C-3), 156.1 (C-7a), 156.7 (C-6), 157.5

(C-4), 165.1 (CO), 165.3 (CO), 166.0 (CO), 187.9 (CHO)

ppm. HRMS (ESI): calcd for C₃₂H₂₆N₅O₈([M + H]⁺): 608.1781,

found: 608.1792.

3-(Morpholinomethyl)-4-amino-1-(2′,3′,5′-tri-O-benzoyl-β-^D-

ribofuranosyl)pyrazolo[3,4-d]pyrimidine (104). Compound 103

(0.125 g, 0.206 mmol, 1.0 equiv) was dissolved in a mixture of

MeOH (2 mL) and THF (1 mL). Morpholine (0.089 mL, 1.03 mmol,

5.0 equiv) and AcOH (0.236 mL, 4.12 mmol, 20.0 equiv) were added,

4.82−4.94 (2H, m, H-5″, H-4′), 6.32 (1H, t, J = 5.6 Hz, H-3′), 6.58

(1H, dd, J = 5.3, 4.1 Hz, H-2′), 6.83 (1H, d, J = 3.8 Hz, H-1′), 7.31−

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7.70 (9H, m, H_Bz), 7.89−8.13 (6H, m, H_Bz), 8.18 (1H, s, H-6), 11.50

(2H, br. s, NH₂), 12.60 (1H, br. s, COOH) ppm. ¹³C NMR (75

MHz, CDCl₃) δ 63.8 (C-5′), 71.8 (C-2′), 74.1 (C-3′), 80.7 (C-4′),

89.1 (C-1′), 101.0 (C-3a), 128.4 (C_Bz), 128.5 (C_Bz), 128.6 (C_Bz),

128.8 (C_Bz), 129.5 (C_Bz), 129.8 (C_Bz), 129.9 (C_Bz), 132.0 (C_Bz), 132.2

(C_Bz), 133.2 (C_Bz), 133.6 (C_Bz), 133.7 (C_Bz), 147.0 (C-3), 152.5 (C-

7a), 154.1 (C-6), 155.7 (C-4), 165.0 (COOH), 165.1 (CO), 166.1

(CO) ppm. HRMS (ESI): calcd for C₃₂H₂₆N₅O₉([M + H]⁺):

624.1731, found: 624.1745.

(1H, d, J = 5.9 Hz, OH), 5.47 (1H, d, J = 5.9 Hz, OH), 6.16 (1H, d, J

= 5.0 Hz, H-1′), 7.19−7.38 (5H, m, H_Phe), 8.11 (1H, d, J = 3.2 Hz,

NH₂), 8.26 (1H, s, H-6), 8.80 (1H, d, J = 3.2 Hz, NH₂′), 9.38 (1H, t,

J = 6.2 Hz, NH) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 42.3

(CH_{2 benzyl}), 62.3 (C-5′), 70.7 (C-2′), 73.0 (C-3′), 85.7 (C-4′), 89.0

(C-1′), 99.4 (C-3a), 126.9 (C-4_Phe), 127.3 (C-2_Phe, C-6_Phe), 128.4 (C-

3_Phe, C-5_Phe), 138.7 (C-1_Phe), 139.1 (C-3), 155.4 (C-7a), 156.9 (C-6),

158.1 (C-4), 161.7 (CO) ppm. HRMS (ESI): calcd for

C₁₇H₁₉N₆O₅([M + H]⁺): 401.1573, found: 401.1571.

Biological Evaluation. All animal experiments were conducted in

compliance with institutional guidelines.

3-Methylamido-4-amino-1-(β-^D-ribofuranosyl)pyrazolo[3,4-d]-

pyrimidine (113). Compound 108 (0.125 g, 0.20 mmol) was

subjected to general procedure F, with methylamine (40% wt in

H₂O) as the coupling partner. The obtained residue was subjected to

general procedure C (reaction time: 1 h). Puriﬁcation via ﬂash

column chromatography (automated, 4 → 20% MeOH in CH₂Cl₂)

aﬀorded 113 (0.019 g, 0.059 mmol, 29% yield over 2 steps) as a white

Drug Sensitivity Assays. Compound stock solutions were

prepared at 20 mM in 100% dimethyl sulfoxide (DMSO). The

compounds were serially prediluted (2-fold or 4-fold) in DMSO

followed by a further (intermediate) dilution in demineralized water

to assure a ﬁnal in-test DMSO concentration of <1%. Compounds

were assayed in 10 concentrations of a 4-fold compound dilution

series starting at 64 μM.

1

solid. H NMR (300 MHz, DMSO-d₆) δ 2.85 (3H, s, CH₃) 3.35−

3.50 (1H, m, H-5′, partially underwater peak), 3.63 (1H, dd, J = 12.0,

4.7 Hz, H-5′), 3.93 (2H, dd, J = 10.3, 4.7 Hz, H-4′), 4.30 (1H, t, J =

4.7 Hz, H-3′), 4.67 (2H, t, J = 4.7 Hz, H-2′), 6.13 (1H, d, J = 4.7 Hz,

H-1′), 8.24 (1H, s, H-6) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 26.0

(CH₃), 62.4 (C-5′), 70.8 (C-2′), 73.1 (C-3′), 85.7 (C-4′), 89.0 (C-

1′), 99.3 (C-3a), 139.0 (C-3), 155.4 (C-7a), 157.0 (C-6), 158.1 (C-

4), 162.1 (CO) ppm. HRMS (ESI): calcd for C₁₂H₁₇N₆O₅([M +

H]⁺): 325.1260, found: 325.1265.

L. infantum. L. infantum [MHOM/MA(BE)/67] was used. This

strain was maintained in golden hamsters (Mesocricetus auratus)

obtained from Janvier (Le Genest Saint Isle, France) following

approval by the Ethical Committee of the University of Antwerp

(ECD2019−10). Amastigotes were collected from the spleens of

infected donor hamsters as described elsewhere,¹⁰⁰and spleen

parasite burdens were assessed using the Stauber technique. Primary

peritoneal mouse macrophages (PMMs) were used as host cells and

obtained from Swiss mice (Janvier; ethical approval ECD2019−10)

after a 2-day 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 well containing 10 μL of the compound

dilutions together with 190 μL of macrophage/parasite inoculum (3 ×

10⁴cells + 4.5 × 10⁵parasites/well). The inoculum was prepared in

RPMI-1640 medium, 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 incubation, parasite burdens (mean number of amastigotes/

macrophage) were microscopically assessed after staining 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.

PMM Cytotoxicity. PMM toxicity was assessed during the in vitro

Leishmania 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 a CC₅₀value was determined.

T. cruzi. The β-galactosidase expressing T. cruzi Tulahuen CL2

strain (nifurtimox-sensitive) was used. This strain was maintained in

MRC-5_SV2(human lung ﬁbroblast) cells in MEM medium,

supplemented with 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.

3-Pyrrolidinamido-4-amino-1-(β-^D-ribofuranosyl)pyrazolo[3,4-

d]pyrimidine (114). Compound 108 (0.080 g, 0.128 mmol) was

subjected to general procedure F, with pyrrolidine as the coupling

partner. The obtained residue was subjected to general procedure C

(reaction time: 1 h). Puriﬁcation via ﬂash column chromatography

(automated, 1 → 15% MeOH in CH₂Cl₂) aﬀorded 114 (0.006 g,

1

0.016 mmol, 28% yield over 2 steps) as a white solid. H NMR (300

MHz, CD₃OD) δ 1.90−2.12 (4H, m, 2 × CH_{2 pyrrolidine}), 3.60−3.74

(3H, m, H-5′, CH_{2 pyrrolidine}), 3.78 (1H, dd, J = 12.3, 3.2 Hz, H-5″),

4.05−4.21 (3H, m, H-4′, CH_{2 pyrrolidine}), 4.50 (1H, t, J = 5.1 Hz, H-3′),

4.75 (1H, t, J = 4.7 Hz, H-2′) 6.34 (1H, d, J = 3.8 Hz, H-1′), 8.20

(1H, s, H-6) ppm. A qualitative ¹³C NMR spectrum could not be

obtained from the amount of product available. HRMS (ESI): calcd

for C₁₅H₂₁N₆O₅([M + H]⁺): 365.1573, found: 365.1567.

3-Anilinamido-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (115). Compound 108 (0.125 g, 0.20 mmol) was

subjected to general procedure F, with aniline as the coupling

partner. The obtained residue was subjected to general procedure C

(reaction time: 1 h). Puriﬁcation via ﬂash column chromatography

(automated, 1 → 15% MeOH in CH₂Cl₂) aﬀorded 115 (0.047 g,

1

0.122 mmol, 61% yield over 2 steps) as a white solid. H NMR (300

MHz, DMSO-d₆) δ 3.52 (1H, dt, J = 11.6, 6.0 Hz, H-5′), 3.68 (1H,

dt, J = 11.7, 5.0 Hz, H-5″), 3.97 (1H, dd, J = 8.8, 4.4 Hz, H-4′), 4.33

(1H, dd, J = 9.1, 4.7 Hz, H-3′), 4.75−5.00 (2H, m, H-2′, OH), 5.15

(1H, d, J = 5.6 Hz, OH), 5.46 (1H, d, J = 5.9 Hz, OH), 6.19 (1H, d, J

= 5.0 Hz, H-1′), 7.20 (1H, t, J = 7.3 Hz, H-4_aniline), 7.41 (2H, t, J = 7.8

Hz, H-2_aniline, H-6_aniline), 7.78 (2H, d, J = 7.9 Hz, H-3_aniline, H-5_aniline),

8.20 (1H, br. s., NH₂′), 8.29 (1H, s, H-6), 8.54 (1H, br. s., NH₂),

10.45 (1H, s, NH) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ 62.1 (C-

5′), 70.7 (C-2′), 72.8 (C-3′), 85.9 (C-4′), 89.4 (C-1′), 99.7 (C-3a),

121.7 (C-2_aniline, C-6_aniline), 124.8 (C-4_aniline), 128.7 (C-3_aniline, C-

5_aniline), 137.4 (C-1aniline), 138.7 (C-3), 155.5 (C-7a), 157.0 (C-6),

158.1 (C-4), 160.4 (CO) ppm. HRMS (ESI): calcd for

C₁₇H₁₉N₆O₅([M + H]⁺): 387.1417, found: 387.1422.

Assays were performed in sterile 96-well microtiter plates, each well

containing 10 μL of the watery compound dilutions together with 190

μL of MRC-5_SV2cell/parasite inoculum (4 × 10³cells/well + 4 × 10⁴

parasites/well). For some assays, PMMs were used as T. cruzi host

cells. For this purpose, 3 × 10⁴cells were plated per well and infected

two days later with 1.5 × 10⁴parasites. To explore the involvement of

ABC transporters, compound exposure was also combined with

established inhibitors verapamil (8 μM), probenecid (700 μM), or

cyclosporine A (2 μM). T. cruzi 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 substrate CPRG (chlor-

ophenolred ß-^D-galactopyranoside): 50 μL/well of a stock solution

containing 15.2 mg of CPRG + 250 μL of Nonidet in 100 mL of PBS.

3-Benzylamido-4-amino-1-β-^D-ribofuranosylpyrazolo[3,4-d]-

pyrimidine (116). Compound 108 (0.125 g, 0.20 mmol) was

subjected to general procedure F, with benzylamine as the coupling

partner. The obtained residue was subjected to general procedure C

(reaction time: 1 h). Puriﬁcation via ﬂash column chromatography

(automated, 1 → 15% MeOH in CH₂Cl₂) aﬀorded 116 (0.035 g,

1

0.087 mmol, 44% yield over 2 steps) as a white solid. H NMR (300

MHz, DMSO-d₆) δ 3.48 (1H, dt, J = 12.3, 6.2 Hz, H-5′), 3.63 (1H,

dt, J = 12.0, 5.3 Hz, H-5″), 3.94 (1H, dd, J = 9.7, 4.7 Hz, H-4′), 4.32

(1H, dd, J = 10.3, 5.0 Hz, H-3′), 4.46−4.63 (2H, m, CH_{2 benzyl}), 4.72

(1H, dd, J = 10.3, 5.0 Hz, H-2′), 4.85 (1H, t, J = 6.2 Hz, OH), 5.10

4233

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J. Med. Chem. 2021, 64, 4206−4238

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The change in color was measured spectrophotometrically 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 IC₅₀

value was calculated.

reported.¹⁰²To prepare the three-dimensional cultures, isolated

cardiac cells were seeded in 96-well U plates (25 × 10³cell/well),

previously coated with 1% agarose. Both cardiac cultures were

sustained in Dulbecco’s modiﬁed Eagle’s medium (DMEM; without

phenol red; Sigma-Aldrich) supplemented with 5% fetal bovine

serum, 2.5 mM CaCl₂, 1 mM L-glutamine, streptomycin, and 2%

chicken embryo extract, at 37 °C. Noninfected cultures were

incubated for 48 h at 37 °C with crescent concentrations of 44

diluted in supplemented DMEM medium. Morphology was evaluated

by light microscopy, and cellular viability was determined using

PrestoBlue.⁵³The results are expressed as the diﬀerence in reduction

between treated and nontreated cultures adopting the manufacturer’s

instructions. The CC₅₀(minimum concentration that reduces 50% of

the cellular viability) was then determined.¹⁰²

MRC-5_SV2Cytotoxicity. MRC-5_SV2cells were cultured in MEM +

Earl’s salts-medium, supplemented with ^L-glutamine, 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 μL of the

watery compound dilutions together with 190 μL of MRC-5_SV2

inoculum (1.5 × 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 μL of resazurin per well. After 4 h

at 37 °C, ﬂuorescence was measured (λ_ex550 nm, λ_em590 nm). The

results were expressed as % reduction in cell growth/viability

compared to control wells, and an IC₅₀value was determined.

Metabolic Stability. Male mouse and pooled human liver

microsomes were purchased from a commercial source (Corning)

and stored at −80 °C. NADPH generating system solutions A and B

and UGT reaction mix solutions A and B (Corning) were kept at −20

°C. The test compound and the reference compound diclofenac were

formulated in DMSO at 10 mM. The microsomal stability assay was

carried out based on the BD Biosciences Guidelines for Use

(TF000017 Rev1.0) with minor adaptations. The metabolic stability

of the compounds was studied through the CYP450 superfamily

(Phase-I metabolism) by fortiﬁcation with reduced nicotinamide

adenine dinucleotide phosphate (NADPH) and through uridine

glucuronosyl-transferase (UGT) enzymes (Phase-II metabolism) by

fortiﬁcation with uridine diphosphate glucuronic acid (UDPGA). For

the CYP450 and other NADPH-dependent enzymes, both com-

pounds were incubated at 5 μM together with 0.5 mg/mL liver

microsomes in potassium phosphate buﬀer in a reaction started by the

addition of 1 mM NADPH and stopped at the above-listed sampling

times. At these time points, 20 μL was withdrawn from the reaction

mixture and 80 μL of cold acetonitrile (ACN), containing the internal

standard tolbutamide, was added to inactivate the enzymes and

precipitate the protein. The mixture was vortexed for 30 s and

centrifuged at 4 °C for 5 min at 15 000 rpm. The supernatant was

stored at −80 °C until analysis. For the UGT enzymes, both

compounds were incubated at 5 μM together with 0.5 mg/mL liver

microsomes in a reaction started by the addition of 2 mM UDPGA

cofactor. The corresponding loss of the parent compound was

determined using liquid chromatography (UPLC) (Waters Aquity)

coupled with tandem quadrupole mass spectrometry (MS²) (Waters

Xevo), equipped with an electrospray ionization (ESI) interface and

operated in the multiple reaction monitoring (MRM) mode. The

optimal MS parameters and control of the chromatographic

separation conditions were tuned in a preceding experiment.

In Vivo Evaluation. Compounds. Bz (2-nitroimidazole; Labo-

́

̂

ratorio Farmaceutico do Estado de Pernambuco [LAFEPE], Brazil)

was used as a reference drug and was formulated using 3% Tween 80

in distilled water. The nucleoside analogue 44 was diluted using 10%

Tween 80 in sterile water.

Mouse Infection and Treatment. Male Swiss Webster mice (18−

20 g; 4−5 weeks of age) were obtained from the animal facilities of

ICTB (Institute of Science and Biomodels Technology/Fiocruz/RJ/

Brazil). Housing of animals was with a maximum of 6 animals per

cage, in a speciﬁc-pathogen-free (SPF) room at 20−24 °C under a 12

h light and 12 h dark cycle. All animals were provided sterilized water

and chow ad libitum. The animals were acclimatized for 7 days before

the experiments. On the day of infection (0 dpi), animals were

infected by i.p. administration of 10⁴bloodstream trypomastigotes (Y

strain) originating from an infected donor mouse. Noninfected

control mice were age-matched and housed under identical

conditions.⁵³Each experimental group consisted of six animals:

untreated (infected vehicle-treated control) and treated (infected and

treated with 44 or with benznidazole). Treatment was initiated at the

onset of parasitemia (6 dpi) only using mice with detectable

parasitemia. 44 was administered by oral gavage for ﬁve consecutive

days at 25, 2.5, and 0.25 mg/kg twice daily. Benznidazole treatments

at 10 mg/kg and at the optimal dose (100 mg/kg p.o.) were run in

parallel. The eﬃcacy of 44 in coadministration with benznidazole was

also evaluated (44 at 2.5 mg/kg b.i.d + benznidazole at 10 mg/kg/

day). All treatments followed a 5-day (6^thto 10^thdpi) dosing regimen,

and all compound formulations were freshly prepared before

administration. Parasitemia levels in T. cruzi assays were individually

checked by light microscopic counting of parasites in 5 μL of blood,

and mortality rates were checked daily until 34 dpi and expressed as a

percentage of cumulative mortality, as described previously.⁹⁵

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00135.

■

sı

T. cruzi Y-Strain Bloodstream Trypomastigote Activity. Blood-

stream trypomastigotes of the Y strain were obtained by cardiac

puncture of infected Swiss Webster mice on the parasitaemia peak,

and drug sensitivity assays were performed as described.⁵³

HPLC traces of selected ﬁnal compounds and copies of

¹H and ¹³C NMR spectra of synthesized compounds

(PDF)

T. cruzi Y-Strain Intracellular Amastigote Activity. After 24 h of

plating, 2D cardiac cell cultures were infected for 24 h at 37 °C with

bloodstream trypomastigotes of T. cruzi (Y strain) employing a

parasite/host cell ratio of 10:1. Then, the cultures were washed to

remove free parasites and treated for 48 h at 37 °C with a serial

dilution of the compound in culture medium. After drug exposure, the

cultures were rinsed using phosphate-buﬀered saline, ﬁxed, and

stained with Giemsa as described previously.^78,101The mean numbers

of infected host cells and of parasites per infected cell were scored in

200 host cells in two independent experiments each run in duplicate.

Only characteristic parasite nuclei and kinetoplasts were considered as

surviving parasites since irregular structures could represent parasites

undergoing cell death. The compound activity was estimated by

calculating the inhibition levels of the inhibition index (II, percentage

of infected cells vs the mean number of parasites per infected cell).

Cytotoxicity on Cardiac Cells. Cardiac 2D and 3D cell cultures

were obtained from the heart of Swiss Webster mice embryos, as

Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

■

Guy Caljon − Laboratory of Microbiology, Parasitology and

Hygiene (LMPH), University of Antwerp, B-2610 Wilrijk,

Belgium; Phone: +32 (0)3 265 26 01;

Email: Guy.Caljon@uantwerpen.be

Serge Van Calenbergh − Laboratory for Medicinal Chemistry

(Campus Heymans), Ghent University, B-9000 Gent,

Belgium; orcid.org/0000-0002-4201-1264; Phone: +32

(0)9 264 81 24; Email: Serge.VanCalenbergh@UGent.be;

Fax: +32 (0)9 264 81 46

4234

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J. Med. Chem. 2021, 64, 4206−4238

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Sacks, D. L.; Scott, P.; Späth, G. F.; Tarleton, R. L.; Spector, J. M.;

Diagana, T. T. Drug Discovery for Kinetoplastid Diseases: Future

Directions. ACS Infect. Dis. 2019, 5, 152−157.

(8) Field, M. C.; Horn, D.; Fairlamb, A. H.; Ferguson, M. A. J.; Gray,

D. W.; Read, K. D.; De Rycker, M.; Torrie, L. S.; Wyatt, P. G.; Wyllie,

S.; Gilbert, I. H. Anti-Trypanosomatid Drug Discovery: An Ongoing

Challenge and a Continuing Need. Nat. Rev. Microbiol. 2017, 15,

217−231.

̃

(9) De Rycker, M.; Baragana, B.; Duce, S. L.; Gilbert, I. H.

Challenges and Recent Progress in Drug Discovery for Tropical

Diseases. Nature 2018, 559, 498−506.

(10) WHO. Regional Strategic Framework for Elimination of Kala-

Azar from the Sout-East Asia Region (2005-2015); New Delhi, 2005.

(11) Burza, S.; Croft, S. L.; Boelaert, M. Leishmaniasis. Lancet 2018,

392, 951−970.

(12) Sundar, S.; Singh, A. Chemotherapeutics of Visceral

Leishmaniasis: Present and Future Developments. Parasitology 2018,

145, 481−489.

(13) Chakravarty, J.; Sundar, S. Current and Emerging Medications

for the Treatment of Leishmaniasis. Expert Opin. Pharmacother. 2019,

20, 1251−1265.

Authors

Jakob Bouton − Laboratory for Medicinal Chemistry

(Campus Heymans), Ghent University, B-9000 Gent,

Belgium

Ludmila Ferreira de Almeida Fiuza − Laboratório de

Biologia Celular, Instituto Oswaldo Cruz (FIOCRUZ),

Fundaca

Camila Cardoso Santos − Laboratório de Biologia Celular,

Instituto Oswaldo Cruz (FIOCRUZ), Fundaca Oswaldo

Cruz, 21040-360 Rio de Janeiro, Brazil

Maria Angela Mazzarella − Department of Pharmaceutical

Sciences, University of Perugia, Perugia 06100, Italy

̧ o Oswaldo Cruz, 21040-360 Rio de Janeiro, Brazil

̃

̧ o

̃

Maria de Nazaré Correia Soeiro − Laboratório de Biologia

Celular, Instituto Oswaldo Cruz (FIOCRUZ), Fundaca̧ o

̃

Oswaldo Cruz, 21040-360 Rio de Janeiro, Brazil

Louis Maes − Laboratory of Microbiology, Parasitology and

Hygiene (LMPH), University of Antwerp, B-2610 Wilrijk,

Belgium

Izet Karalic − Laboratory for Medicinal Chemistry (Campus

Heymans), Ghent University, B-9000 Gent, Belgium

(14) DNDi. 2019 R&D portfolio in review: Leishmaniasis − DNDi.

https://www.dndi.org/2020/media-centre/news-views-stories/news/

leishmaniasis-rnd-portfolio-update/ (accessed Apr 20, 2020).

(15) Nagle, A.; Biggart, A.; Be, C.; Srinivas, H.; Hein, A.; Caridha,

D.; Sciotti, R. J.; Pybus, B.; Kreishman-Deitrick, M.; Bursulaya, B.;

Lai, Y. H.; Gao, M.-Y.; Liang, F.; Mathison, C. J. N.; Liu, X.; Yeh, V.;

Smith, J.; Lerario, I.; Xie, Y.; Chianelli, D.; Gibney, M.; Berman, A.;

Chen, Y.-L.; Jiricek, J.; Davis, L. C.; Liu, X.; Ballard, J.; Khare, S.;

Eggimann, F. K.; Luneau, A.; Groessl, T.; Shapiro, M.; Richmond, W.;

Johnson, K.; Rudewicz, P. J.; Rao, S. P. S.; Thompson, C.; Tuntland,

T.; Spraggon, G.; Glynne, R. J.; Supek, F.; Wiesmann, C.; Molteni, V.

Discovery and Characterization of Clinical Candidate LXE408 as a

Kinetoplastid-Selective Proteasome Inhibitor for the Treatment of

Leishmaniases. J. Med. Chem. 2020, 63, 10773−10781.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00135

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors would like to thank An Matheeussen, Natascha

Van Pelt, Pim-Bart Feijens, and Margot Vleminckx for

excellent technical assistance.

ABBREVIATIONS

■

(16) Thomas, M. G.; De Rycker, M.; Ajakane, M.; Albrecht, S.;

APP(R), aminopyrazolo[3,4-d]pyrimidine (riboside); Bz,

benznidazole; CL, cutaneous leishmaniasis; DTU, discrete

typing unit; PMM, primary mouse macrophage; PRTase,

phosphoribosyltransferase; L. infantum, Leishmania infantum;

SI, selectivity index; T. cruzi, Trypanosoma cruzi; TDA-1,

tris[2-(2-methoxyethoxy)ethyl]amine; TPPTS, triphenylphos-

phine-3,3′,3″-trisulfonic acid trisodium salt; VL, visceral

leishmaniasis

́

Alvarez-Pedraglio, A. I.; Boesche, M.; Brand, S.; Campbell, L.;

Cantizani-Perez, J.; Cleghorn, L. A. T.; Copley, R. C. B.; Crouch, S.

D.; Daugan, A.; Drewes, G.; Ferrer, S.; Ghidelli-Disse, S.; Gonzalez,

S.; Gresham, S. L.; Hill, A. P.; Hindley, S. J.; Lowe, R. M.; MacKenzie,

C. J.; MacLean, L.; Manthri, S.; Martin, F.; Miguel-Siles, J.; Nguyen,

V. L.; Norval, S.; Osuna-Cabello, M.; Woodland, A.; Patterson, S.;

Pena, I.; Quesada-Campos, M. T.; Reid, I. H.; Revill, C.; Riley, J.;

Ruiz-Gomez, J. R.; Shishikura, Y.; Simeons, F. R. C.; Smith, A.; Smith,

V. C.; Spinks, D.; Stojanovski, L.; Thomas, J.; Thompson, S.;

Underwood, T.; Gray, D. W.; Fiandor, J. M.; Gilbert, I. H.; Wyatt, P.

G.; Read, K. D.; Miles, T. J. Identification of GSK3186899/

DDD853651 as a Preclinical Development Candidate for the

Treatment of Visceral Leishmaniasis. J. Med. Chem. 2019, 62,

1180−1202.

(17) Wyllie, S.; Thomas, M.; Patterson, S.; Crouch, S.; De Rycker,

M.; Lowe, R.; Gresham, S.; Urbaniak, M. D.; Otto, T. D.; Stojanovski,

L.; Simeons, F. R. C.; Manthri, S.; MacLean, L. M.; Zuccotto, F.;

Homeyer, N.; Pflaumer, H.; Boesche, M.; Sastry, L.; Connolly, P.;

Albrecht, S.; Berriman, M.; Drewes, G.; Gray, D. W.; Ghidelli-Disse,

S.; Dixon, S.; Fiandor, J. M.; Wyatt, P. G.; Ferguson, M. A. J.;

Fairlamb, A. H.; Miles, T. J.; Read, K. D.; Gilbert, I. H. Cyclin-

Dependent Kinase 12 Is a Drug Target for Visceral Leishmaniasis.

Nature 2018, 560, 192−197.

(18) Khare, S.; Nagle, A. S.; Biggart, A.; Lai, Y. H.; Liang, F.; Davis,

L. C.; Barnes, S. W.; Mathison, C. J. N.; Myburgh, E.; Gao, M.-Y.;

Gillespie, J. R.; Liu, X.; Tan, J. L.; Stinson, M.; Rivera, I. C.; Ballard, J.;

Yeh, V.; Groessl, T.; Federe, G.; Koh, H. X. Y.; Venable, J. D.;

Bursulaya, B.; Shapiro, M.; Mishra, P. K.; Spraggon, G.; Brock, A.;

Mottram, J. C.; Buckner, F. S.; Rao, S. P. S.; Wen, B. G.; Walker, J. R.;

Tuntland, T.; Molteni, V.; Glynne, R. J.; Supek, F. Proteasome

Inhibition for Treatment of Leishmaniasis, Chagas Disease and

Sleeping Sickness. Nature 2016, 537, 229−233.

REFERENCES

■

(1) Molyneux, D. Neglected Tropical Diseases. Community Eye

Health 2013, 26, 21−24.

(2) Houweling, T. A. J.; Karim-Kos, H. E.; Kulik, M. C.; Stolk, W.

A.; Haagsma, J. A.; Lenk, E. J.; Richardus, J. H.; Vlas, S. J. de.

Socioeconomic Inequalities in Neglected Tropical Diseases: A

Systematic Review. PLoS Neglected Trop. Dis. 2016, 10,

No. e0004546.

(3) Lee, B. Y.; Bacon, K. M.; Bottazzi, M. E.; Hotez, P. J. Global

Economic Burden of Chagas Disease: A Computational Simulation

Model. Lancet Infect. Dis. 2013, 13, 342−348.

(4) Lidani, K. C. F.; Andrade, F. A.; Bavia, L.; Damasceno, F. S.;

Beltrame, M. H.; Messias-Reason, I. J.; Sandri, T. L. Chagas Disease:

From Discovery to a Worldwide Health Problem. Front. Public Health

2019, 7, 166.

(5) Chatelain, E.; Konar, N. Translational Challenges of Animal

Models in Chagas Disease Drug Development: A Review. Drug Des.

Dev. Ther. 2015, 9, 4807−4823.

(6) Pérez-Molina, J. A.; Molina, I. Chagas Disease. Lancet 2018, 391,

82−94.

(7) Rao, S. P. S.; Barrett, M. P.; Dranoff, G.; Faraday, C. J.;

Gimpelewicz, C. R.; Hailu, A.; Jones, C. L.; Kelly, J. M.; Lazdins-

Helds, J. K.; Mäser, P.; Mengel, J.; Mottram, J. C.; Mowbray, C. E.;

4235

https://doi.org/10.1021/acs.jmedchem.1c00135

J. Med. Chem. 2021, 64, 4206−4238

Article Doi

Revisiting Pyrazolo[3,4- d]pyrimidine Nucleosides as Anti- Trypanosoma cruzi and Antileishmanial Agents

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